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The Combined Cardiac Effect of the Anabolic Steroid, Nandrolone And

The Combined Cardiac Effect of the Anabolic Steroid, Nandrolone And

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Benjamin D. Phillis, B.Sc. (Hons) Phatmacology Depattment of Clinical & Experimental Ftome Rd. , Medical School Noth Adelaide Univetsity ADEIAIDE SA 5OOO

û.)r.'-*hr/7enveltîù

Foremost, I would like to thank my two supervisors for the direction that they have given this ptojecr. To Rod, for his unfailing troubleshooting abiJity and to Jenny fot her advice and ability to add scientific rigour'

Many thanks to Michael Adams for his technical assistance and especially fot performing the for the ischaemia-reperfusion projects and for his willingness to work late nights and public holidays.

Lastly I would like to thank my v¡ife for her extreme patience during the tumult of the last 5 years. Her love, suppoït, patience and undetstanding have been invaluable in this endeavout.

Beniamin D. Phillis

Octobet,2005

ADE,I-AIDE

ii T*(¿t of Ao,t",tù

DECI.ARATION I

ACKNOWLEDGEMENTS il

TABLE OF CONTENTS UI

ABBREVIATIONS x

ABSTRACT )ilr

CÉIAPTER t-l Inttoduction 1-1

1.1 Background 1,-1,

1.2What ate anabolic stetoids? 7-1

1,3 General pharmacology of Anabolic t-2 '1,-2 1.3.1 Genomic effects of anabolic steroids

1.3.2 Non-genomic effects of anabolic steroids 1-3

1.4 Clinical use of AS 1.-4

1.5 Patterns of AS abuse 1.-4

1.5.1 abuse by 1.-+

1.5.2 Stetoid abuse by sedentary teenagers r-6

1.5.3 Prevalence of abuse 1-6

1.5.4 Abuse ptevalence in 1.-9

1.6 Cardiotoxicity of anabolic steroids r-9

1.6.1 Reduced cotonary flow 1.-1.1,

1,.6.2 Dtect myocatdial eff ects 1-1 5

1.6.3 1-21

1.7 Difficulties associated with research 1.-24 1-25 1.8 The polydrug abuse Phenomenon 1.9 The pharmacology of 1-26

1.10 Pteparations 1-28 1-29 1.11

lll 1-30 1. .7 2 Pharmacokin etic s 1,-31 1. 1 3 Cocaine CardiotoxicitY

1.13.1 Reduced coronary flow 1.-34 1.-40 1,.13.2 Carðtac 1-43 1.13.3 DirecT cardiac effects 1-47 1.14 Conclusion 1.15,\ims 1-48 1.-49 1.16 Hlpotheses 1-50 1.17 Refetences

CHAPTER 2 2-l

General Methods 2'l 2-1 2.1 Animals _ 2-I 2.2Radtotelemetry recording 2.3 Generalanaesthesia 2-2 a') 2.4 StxgScal irnplantation of radiotelemetry devices 2.4.l.Eccimplants 2-2 2.4.2PioodPressure ImPlants 2-3 2-4 2.4.3 P ost- operative Management 2-4 2.5 Analysis of tadiotelem erry data 2-4 2. 6 Sttg¡cú, procedure for is chaemia-rep erfu sion s tu dies 2-6 2.7 Con¡1nuous recording of cardiac patâmeters during ischaemia-teperfusion. - 2-6 2.8 Induction of ischaemia and reperfusion 2-7 2.9 Analysis of ischaemia-reperfusion data heatts' 2.10 DeterminatLon of the extra-neuronal uptake of noradrenaline in isolated perfused

2-1,1,

2-1,1, 2.1,0.1. P re- tteatment

2.10.2 Protocol 2-1,1,

2.70.3 Calculation of results 2-1.2 2-1.3 2.11 Dtugs 2-15 2.12 References

CTIAPTER 3 3-l The effect of rat sttain on the catdiovasculat tesponse to cocaine 3-1 3-1 3.1. Introduction 3-1 3.1.1 Is the cardiovascular tesponse to intraperitoneal cocaine strain dependent? 3-4 3.2. Aim. lv 3-4 3.3. Hypothesis 3-5 3.4. Methods 3.4.1Animals 3-5 3.4.2Protocol 3-5 3.4.3Data Analysis 3-5 3-6 3.5. Results

3.5.1 Time course of the cocaine response 3-6 3.5.2 Between sttain changes in the HR, SI-4, DP and SP fesponse to cocaine - 3-6 3-9 3.6. Discussion

3.7. Conclusion 3-11,

3.8 Refetences 3-r2

CHAPTER 4 4.I The acute effect of and cocaine on heaft late 4-l

4.1 Introduction 4-1, 4-2 4.2 Nm 4-2 4.3 Hypothesis 4-3 4.4 Methodology 4.4.1Ânimals 4-3 4-3 4.4.2

4.4.3 4-3 4-4 4. 4.4 Statistical Analysis 4-5 4.5 Results

4.5.1 Plasma levels of nandtolone 4-5 4-5 4.5.2 P re-tteatment response

4.5.3 Cocatne resPonse 4-5 4-5 4.5.4 E ffect of nandrolone pre-treatment on the tesponse to cocaine 4-8 4.6 Discussion

4.7 Conclusion 4-9 4-1.0 4.8 References

CHAPTER 5 5'1 The effect of inttavenous nandlolone in tats subiected to catdiac ischaemia and teperfusion 5-1 5-1 5.1 Inttoduction 5-3 5.2 Aim

v 5.3 Hypothesis 5-3

5.4 Methodology 5-4 5.4.1Änimals 5-4

5.4.2 Protocol 5-4

5.4.3Data analysis 5-5

5.4.4 5-5

5.5 Results 5-7

5.5.1 Plasma level of nandrolone 5-7

5.5.2 Atea at risk of ischaemia 5-7

5.5.3 Cardiovascular response to pre-ischaemia nandrolone infusions 5-7

5.5.4 Catdiovascular tesponse to ischaernia and reperfusion 5-1 5-8 5.5.5 Suwival - 5.5.6 Arhythmia 5-9

5.6 Discussion 5-19

5.6.1 Potential mechanisms for an acute nandrolone effect during catdiac

ischaemia 5-21,

5.7 Conclusion 5-22

5.8 References 5-23

CTIAPTER 6 6-l The effect of intravenous cocaine in tats subiected to catdiac ischaemia and tepelfusion 6-1

6.1 Introduction 6-1, 6-2 6.2 Aims 6-2 6.3 Hypotheses

6.4 Methodology 6-3 6-3 6.4.1 '\nimals 6.4.2Protocol 6-3 6-3 6.4.3 Data Ânalysis

6.5 Results 6-5 6-5 6.5.1 ,A,rea at dsk of ischaemia 6-5 6.5.2 C añtovas cular tesp orìs e to pre- tteatment 6-6 6.5.3 Cardiovascular resPonses to ischaemia and reperfusion

6.5.4 Survival 6-l 6-8 6.5.5 8 6.6 Discussion 6-1

vr 6.6.1 Btood pressure effects of cocaine 6-1 8

6. 6.2 E,ff ect o f cocain e and is chaemia/ tep erfusion 6-18

6.6.3 Effect of the nandrolone-cocaine combination on arth¡hmia and 6-20

6.6.4Effect of pre-tteatment orì reperfusion atthythmias 6-22

6.7 Conclusion 6-23

6.8 Refetences 6-2+

CHAPTER 7 7-l Effects of chtonic nandlolone administtation 7-l

7.1 Introduction 7 -1,

7.2 7-2 'A.ims 7.3 Hlpothesis 7-2

7.4 Methodology t--) 7.4.1Animals t-J 7.4.2Protocol 7-3 7.4.3Data Analysis 7-+

7.5 Results 7-7

7.5.1 Change in body weþht 7-7

7.5.2 Change in otgan weights t-7

7.5.3 Histopathology 7-8

7.5.4 Cholestetol, tdglycetides, LDL and HDL profìles' 7-8

7.5.5 Setology 7-8

7.5.6 Plasma level of nandrolone 7-8

7.5.7 Cardtovasculat response to chronic nandrolone treatment 7-9

7.5.8 Cardiovascular response to cocaine administration t-9

7.5.9 Cardtovascular response to ischaemia and reperfusion 7 -1.0

7.5.1.0 Zote at risk of infarctron 7-10

7.5.11 Survival 7 -1.1,

7.5.12 Arth¡hmia 7-1,1,

7.6 Discussion 7-26

7.6.1 Bodyweigh 7-26

7.6.2 Changes in organ weight 7-28

7.6.3 Changes in 7-30

7.6.4 Serological Changes 7-32

7.6.5 Plasma nandrolone level 7-32

vrl 7 .6.6 E ffect of treatment on BP and HR 7-32 7.6.7 CaÁtovascular effect of cocaine infusion / -J3

7.6.8 Affect of pre-treatment on the frequency and duration of arhythmia and on 7-34 7.7 Conclusion 1-36

7.8 Refetences 7-37

CHAPTER 8 8-1 3H]-notadtenaline The effect of nandtolone on extraneuronal [ leuptake in lat heatt 8-1

8.1 Introduction 8-1

8.1.1 Summary 8-1

8.1. .2 Bxtraneutonal uPtake 8-1 9.2,\im 8-5

8.3 Hypothesis 8-5

8.4 Method 8-6

8.5 Results 8-7

8.6 Discussion 8-8

8.7 Conclusion 8-9

8.8 References 8-10

CI{APTER 9 9-t Discussion 9-1

9.1 Rat sttain 9-1, g.2Effects of nandtolone alone 9-1 9.2.7 Cardtovasculat effects of nandtolone in non-ischaemic rats 9-1,

9.2.2 CaÅtovascular effects of nandrolone in tats subjected to catdiac ischaemia

and reperfusion 9-2

9.3 Effects of cocaine alone 9-4 9.4 Cocat¡e effects in combination with chtonic nandrolone 9-5

9.5 Cocaine effects in combination with acute nandtolone 9-6

9. 6 Experimental Lirnitations 9-6 9-7 9.6.L SoeciesI- 9.6.2Statn 9-7

9.6.3 Gender 9-7

9.6.4 Exercise 9-8

vrrl 9.6.5 Sttess 9-8

9.6.6 Route of administtation 9-9

9.6.7 CaÅtac ischaemia 9-10

9.7 Future studies 9-1.1,

9.8 Conclusion 9-1 5

9.9 References 9-11

APPENDIXA. A-1 Reported fatal and.non-fatal catdiovasculat outcomes tesulting ftom anabolic stetoid use A-1

APPENDIX B. B-1 Reported histological changes in laboratory animals administered anabolic stetoids 1

APPENDIX C c-1 Pilot study: chtonic effect of nandtolone in telemeteted mts c-1

APPENDIX D. I Pilot study: extfaneulonal notadrenaline feuptake in tat heatt slices D-l

APPENDIX E. E-1 Effect of anabolic steroids in the CNS E-1

APPENDIX F. F-1 F'-1 Effect of nandtolone on Plasma ET-l

APPENDIX G. G-1

lx û'Ht'r¿tt'-"1/,

ACAT Cholesterol Acetylttans ferase Ach Äcetylcholine ACTH AdrenocorticotroPin ADP Adenosine Diphosphate AIDS Acquired Immunodeltciency ALP Alkaline Phosphatase ALT Alanine Aminottansfetase ANF Atrial Nattiuretic Factor ANOVA Analysis of Vadance AS Anabolic-Androgenic Stetoid(s) AS-ODN Antisense Oligodeoxynucleotide AST Aspartate Aminotrans fetase AUC Area Undet the Cuwe A\v -Albino Wistar BMI Body Mass Index BP Pressure CAI) Cotonary '\ttery Disease CBF Cotonaty Blood Flow CFR Coronary Flow Rate chEl Carboxyestetase-1 CI CK l(inase CNS co Carðiac Output COMT Catechol-O-Methyltransfetase CPK Creatine Phosphokinase CT Core Tempetature DA Dopamine DECA Decanoate DOPEG Dihydroxyp h enylglycol ECG Electtocatdiogram EEG Electroencephalogram EMT Extraneuronal Transporter fot Monoamine Transmitters ET-1 Endothelin-1 GC-MS Gas Chromatography - GGT glutamyltransferase HDL High Density Lipoprotein hgH Fluman HPA Hypothalamic Pituitary Adrenal (axis) HIV Fluman Immunodefi ciencY Virus HR Heatt Rate 5-HT 5, Hydroxytryptamine (setotonin) HTGL Hepatic Triglyceride LiPase HVA Homovanillic Acid i.p. Intrapedtoneal IPSP Inhibitory Post-synap tic Potential i.v. Intravenous LAI) Left Antedor Descending (artety) LDto 1070 Lethal Dose LDro 5070 Lethal Dose

x ABBREVIATIONS LDH Lactate Dehydrogenase LDL Low Density Lipoprotein LV Left Ventricular LVH Left Ventricular HypettroPhY MAO Monoamine Oxidase MBP Mean Blood Ptessute MEAP Met-Enkephalin-Arg-Phe MOPEG methoxyhydtoxyph enylglycol fnPOA Medial Preoptic Atea NA Noradrenaline NDMA N -nitrosodirnethylamine NIDA National Institute of Drug Abuse (JSA) NIH National Institute of Health NK-1 Neurokinin-1 NMDA N-methyl-D-aspartic acid ocT2 Organic Cation TransPorter 2 PAG Petiaqueductd. G:-ay PAI-1 Plasminogen'\ctivator Inhibitor-l PDGF Platelet Derived Growth Factor PC Phosphatidylcholine PE Phenylephrine PEG Polyethylene glycol PgI, Ptostacyclin PRP Platelet Rich Plasma PS Phosphatidylserine PVC Premature Ventricular Contractions RDI Recommended Daily Intake SA Arachidonate s.c. subcutaneous SD Sprague Dawley (rat) SHR Spontaneously H1p eft ensive Rat SLA Spontaneous Locomotor ActivitY SNP SP Substance P TC Total Cholestetol TNF Tumour Necrosis Factor TRIG Triglyceride TXB2 Thromboxane VF Venüicular Fibrillation \rMN Ventromedial Nucleus VT Ven tticular T achy c ar dta VTA Ventral Tegmental Area WKY Wistar Ifoto

xl ABBRE,VIATIONS M

Recent pharmacoepidemiological research shows a significant increase in the use of anabolic-steroids (AS) amongst teenagers and young adults and especially amongst sedentary, young maies administering AS for cosmetic reasons. AS are often used in con¡unctíón'with psychostimulants. Survey findings suggest significant co-abuse of AS and coca¡ne. Unfortunately, despite documented evidence that both drugs alone can induce significant cardiovascular effects only very limited basic science research has been conãucted into the potential for cardiotoxicity with this drug combination.

Nandrolone has been shown to be the AS of choice amongst many recreational users. previous work has found rate (HR) increased significantly following cocaine HCI (41mglkg, i.p.) administration in albino Wistar rats (AW) chronically treated with Àan¿roloñe compared to the cocaine effect in vehicle treated controls (Phillis et al, 2000). Subsequent studies in this thesis established that this HR effect could not be attributed to an acute effect of the last nandrolone dose prior to cocaine administration. Dose-response relationships to cocaine (0,15,45m9/kg, i'p') for cardiovascular variables using radiotelemetry were conducted in freely moving, conscious AW and Sprague-Dawley (SD) rats. These studies indicated that the cardiovascular response to cocaine was not strain dependent. This allowed direct comparisons to be made to literature values for cocaine effects in both strains'

In view of the rather moderate effects of nandrolone observed, the effect of AS pre- treatment on the response to cardiac ischaemia was assessed in order to simulate pre- existing cardiac disease. Nandrolone was administered i.v. at doses reflective of the plasmj concentrations possibly achieved by chronic AS users (10-160p9/kg/min). SD rats were subsequentÍy subjected to 15 minutes occlusion of the left anterior descending (lÁD)'coronary artery and 10 minutes reperfusion, A significant decrease in survival time during ischaemia was noted at the highest nandrolone dose (p<0'001) and significant decieases in the fraction of rats surviving ischaemia (40 & 160pg/[g/min, both p<0.05) compared to control, A significant increase in the mm¡ãtn arrhythmia score was seen at the 3 highest nandrolone doses (all, p<0.05)' A significant increase in the duration of VF during ischaemia was noted for the highest nãndrolone dose (160ug/kgimin) compared to the lowest dose (10¡rg/kg/min). This pro-arrhythmic nandroloie-effect could not be attributed to increases in myocardial noradrenaline (NA) by the blockade of e*raneuronal reuptake, since nandrolone was shown to have too low an inhibiting on extraneuronal noradrenaline uptake in isolated perfused rat . The severity of arrhythmia in rats receiving i.v. nandrolone was not increased by cocaine HCI administration (0'smg/kg/min, i.v) but rather was found to protect against the fatal VF induced by nandrolone alone (O¡tglkglmin). In contrast to the acute effect of nandrolone, chronic nandrolone treatmeñt of SD rats for 3, 6 or 9 weeks had no effect on the response to cardiac ischaemia or repefusion. The basis of th : difference between the effects of chronic and acute effects of nandrolone on ischaemia-induced dysrhythmia was not identified' It may relate to the much lower plasma nandrolone concentration achieved with

xu ABSTRACT chronic nandrolone treatment. Alternatively, chronic treatment may result in down- regulation of the mechanism underlying enhanced arrhythmia after acute dosing'

This is the first study to show a significant and dose dependent increase in the Lambeth arrhythmia score during cardiac ischaemia in rats administered i'v. nandrolone. The mechanism of this effect remains unknown. Potential mechanisms include a CNS effect of nandrolone, an uncoupling of the protective effect of adenosine during early ischaemia by nandrolone or an increase in pro-arrhythmic endothelin-l (Ef-lj. This study suggests that nandrolone abuse in patients with a pre-existing èardiac condition may precipitate life threatening cardiac arrhythmia.

xttt ABSTRACT Introduction

1.1 Bacþround There is a gtowing body of epidemiological evidence indicating an increasing prevalence of the abuse of anabolic steroids in both image conscious, sedentary, teenage males and power athletes, In add.ition, anabolic steroid abuse is highly correlated with stjmulant use. This thesis sets out to firstly review the reported cardiac consequences of abuse of anabolic steroids and the popular ' cocaine, in the literature, and then examine the cardiovascular consequences of the co- administration of nandrolone (a commonly used steroid) and cocaine in an animal model. These experiments were performed in the absence of other confounding risk factors such as poor diet and heavy exercise.

1.2 What are anabolic steroidsP Anabolic steroids are synthetic derivatives of . All anabolic steroids have some andtogenic action, as both the anabolic and androgenic effects ate mediated through the same to as anabolic- S.ockhold, 1gg3). Strictly speaking, they should be referred androgenic steroids. For the sake of simplicity throughout this thesis they will be referred to as anaboiic steroids (AS). One of the most popular AS is nandrolone, which has a high anabolic: androgenic ratio of 8:1 resulting in a lower chance of affecting primary and secondary sex characteristics when it is administered to increase muscle mass ffu-Yahiro et a1.,1'989)'

A1l endogenous are based upon the ringed structure of cholestetol (Colby et a1.,1'988). As testosterone is highly subject to hepatic degradation, a number of modifications have been made to the structure of synthetic steroids to minimise this effect...

7. 1 7-p-esterification (eg. testosterone undecanoâte)

2. 1 7-cr-substitution (eg. methyl testosterone) 3. Mod-ifications an{ add.itions to the steroid ring (eg. and )

7-l CHAPTER T Introdaction 1.3 General pharmacologS' of Anabolic steroids

1.3.1 Genotnic effecß of anabolic steroids The high lipophiliciry of mosr AS allows ready passage across the lipid biJayer of the target (Lukas, 1993) (Fig. 1. 1). Bind,ing ro cytoplasmic steroid recePtors and subsequent homodimer formation, elicits a conformational change in the stetoid-receptor homodimer-complex, which the nucleus, exposes DNr\ binding regions (reviewed by Colby et a/. 1.988). Upon translocation to leading to these " finger" sequences allow binding to "hotmone resPonse elements" on DNA, Bahrke et al', a subsequenr inctease in RNÂ transctiption and production (ùVright, 1980;

19e8).

Almost every cell in the body contains androgen receptors. Therefore the anabolic or androgenic the e/ response is determined. more by the location and type of cell than the nâture of ,\S @ahrke

aL.,1,998).

steroid o OO OO cyloplasm Steroid R, "z¡nc fingers" b¡nd "hormone T response elements" on DNA DNA o Tt$t' G U1 AAñA RNA ! Protein

conÍormational change & exposure of "zinc fingers"

steroid Fig. 1. 1 Intercellular events following binding of AS to intercellular receptors. Lipophilic steroid recePtor' Identical receptor-steroid passes readily into the , where it combines with a cytoplasmic exPoses DNÂ binding regions ("zinc complexes dimerise, inducing a conformational change in the homodimer, which nuclear chromatin' \Tithin the nucleus the fingers,,). Translocation to the nucleus is induced by increased affinity for an increase in the number of RNA ,,zinc fingers,, bind DN,\ regions ("hormone response elements") resulting in transcripts generated, and a subsequent increase in ptoteìn production.

1-2 CHAPTER 1' Introdaction 1.3.2 Non-genomic effecß of anabolic steroids Genomic expression cannot explain all the observed effects of ,\S. A limited numbet of studies have examined the direct effect of androgens on coronâry vasculatute and found that the pharmacological effects occurred quickly, and were not consistent with the slow response normally attributed to changes in expression. It has been demonstrated thât testosterone can block the vasodilaror effect of adenosine in isolated perfused rat hearts (Ceballos et a1.,1'999)' This effect was conserved even when testosterone was coupled to a large mactomoleculat complex, which prevented transport into the cytosol. Testosterone was stitl able to exert its inhibitory effect on in the presence of cyclohexamide, demonstrating that its acute effects were not dependent on RNA translation þrotein synthesis). This vasoconstricting effect of testosterone appears to be mediated in part by the prostaglandin metabolic Pathway. Inhibition of cyclo- oxygenâse-l (cox-1) by indomethacin was found to signifìcantly reduce the vasoconstriction produced by testosterone. A reduction in vasoconsffiction was also observed with a thromboxane A, (f)(A.) antagonist.

on platelet Most recently, Togna et al invesngated the combined effect of testosterone and cocaine and endothelial function (fogna et a/., 2003). Co-incubation of platelet rich plasma with to a sub- testosterone and cocaine induced a significant increase in platelet aggtegability in response threshold concentration of sodium arachidonate. Likewise, co-incubation of endothelium-intact, testosterone phenyiephrine pre-contrâcted, rabbit aortic rings, with a subthreshold concentration of and cocaine caused an inhibition of acetylcholine mediated vascular relaxation. Incubation of alone had platelets or aortic rings with these concentratìons of either cocaine alone or testosterone no effect on platelet aggregability or acetylcholine mediated relaxation. In addition, subthteshold of cocaine' concenffations of testosterone potentiated the inhibitory effect of higher concentrations The testosterone , , did not Prevent the effect observed with a concomitant incubation with both testostefone and cocaine, indicative of an androgen fecePtof that independent mechanism in both cases. The lack of involvement suggests effects on testosterone c^nact on platelets and vessels thtough a non-genomic mechanism' The endothelial cell vessels are possibly elicited through changes in membrane fluidity at the vascular of testosterone level. Togn a et al propose that the increase in aggregatson seen with co-incubation and permeability and cocain e m^y be due to modifìcation of platelet membtane surface

changes.

t-3 CHAPTER l lntrodøction 1.4 Clinical use of AS

Steroids are used cJinically to treat a broad spectrum of indications. is used ro trear the muscle wasting and associated with HIV forbes et a1.,1'992; Grinspoon et al., 1.998). Androgens have also been used in the treatment of patients with excessive ptotein or insufficient protein synthesis, such as patients suffering from signifìcant , malìgnancies and trauma (ChlebowsÞj et a1.,1983; I(opera, 1985), AS have been found to increase erythropoiesis and are therefore used to treât avairety of znaemias (I(ibble et a/.,1987; Marshall, 19gg). Likewise, because of a leukopoietic effecr via stimulation of proliferation of granulocytic colony forming cells, ÄS have also been found to provide a benefìcial effect in the treatment of leukaemia (I{opera, 1985). Testosterone are also routinely used to treat children in conjunction with human growth hormone (hgH) therapy (Hoberman et a1.,1'995)' A variety of

,\S are used to pfomote growth in patients suffering osteoPofosis (I(opera' 1985)'

1.5 Patterns of AS abuse

1.5.1 Steroid abuse by athletes Illicit use by athletes often involves rhe use of doses 10-1000 times the recommended dose for

clinical use (Sullivan et al.,1,ggg).In addition, users will often use 2 or more steroids simultaneously et a1.,2000) (Table (yates et a1.,1,992;Masonis et a/.,1995), in a process known as "stacking" (I-otg 1. L). Many arhletes use AS in "cycles" of 4-18 weeks (I(bble et a/. 1'987), decreasing the dose as competition dates approach (l

1989). This is known as "pyramiding", znd is said to reduce behavioural side effects.

7-4 CHAPTER 1. Introdaction Product Generic Name Route Repoted Dose & frequency Name (Reference) (Ferenchick er Anavat Oxandtolone oral 50mg/day x 16 weeks a|.,1992),7.5mg/àay x 18 months OWinwood er a|.,1990) o:ø.l et a|.1.9 Anadrol-50 oth ^r.es (Fineschi et a|.2001), Deca- (19, 1.m. 200mg durabolin nortestosterone) 2O0mg/week (Cabasso, 199 +), 2}}mg/week x L6 weeks (Ferenchick et a/., 1,992); 1OOmg/week x 8 weeks (I\4cllllop et al. 1,986) (Sullivan e/ Depo- Testosterone-cyPlonate 1.m 4OOmg/week x 5 weeks (ates et al., testostefone a/.,1,999),83mg/day 1992) Dianabol methandrostenolone oral & i.m. (Feraudi et al',1985) Dianabol. i.m. Equipoise --undecyclenate im. (Ferenchick et a/.,1992); Halotestin oral gJieminen et al. 1.996) Metandren onI (lrJieminen et al., 199 6) Oreton Methyl a/ al.200I) Oreton Testosterone-proPionate lm 70Omg/week (Fineschi ee6) Primobolan Methenolone-enanthate i.m., oral 1O0mg/week (Palatini et a1.,1 Depot Proviron oral Q.Jieminen et aL.1996) Mestoranum 1.998) Testex Leo Testosterone-cyclopentilpropionate 1.m. (Hausmann et a/., 250 prolongatum \ü/instrol Stanozolol 1m 7Omg/week (Fineschi et a/.2001), (veterinary) 24\mg/week x 5 weeks (Sullivan e/ 'ùTinstrol-V al., 1999), 28Omg/week x 2 yeats (À4ewis et ø/. 1.996), 40mg/ daY x 6 weeks ((ennedy, 1993), l5mg/daY x 1.8 months fWinwood et al.1'990), 30ms/ðay et a1.,1 986)

Table 1. 1Type, route, dose and dosing frequency of As reportedly abused by athletes

1-5 CH.,\PTER 1 Introdaction 1.5.2 Steroid abuse by sedentary teenagers One of the most worrying trends in the abuse of AS has been recent reports of illicit use in sedentary teenagers. The main reasons for abuse have been variously reported as being to obtain a better body or larger muscles (Ianner et al., 1995; Nilsson et a/., 2001), to "become brave"

(I(ndlundh et a1.,1,998; I(ndlundh et a1.,1.999), to become intoxicated (I{ndlundh et a1.,1998), and to enhance performance (Bahrke et al.1'998).

A recent report also suggests that many HIV sufferers self-administer AS at supra-clinical doses in an âttempt to minimise the muscle wasting associated with AIDS $arriale et al.1'999).

Illicit ,\S can be easily purchased on the . Many of the steroids sold in this manner are from veterinary applications ot diverted prescriptions. An extensive variery of steroids manufacrured in foreign laboratories can be purchased on the street (À{arshall, 1988).

1.5.3 Prevalence of abuse An accurate indication of the prevalence of steroid abuse in the general population and in adolescents is hard to derermine and is complicated by self-repo rt data' Thompson et al rcpott that of the drugs reportedly used by survey respondents, only 660/o werc found in (Ihompson a/ al.,1,992;Thomson et a1.,1,993). This is likely to be due to a combination of errors. Exaggeration of drug use is not uncommon in illicit drug users. Many respondents self-administering black-market AS may not have been supplied with the requested AS (DuRant et al., 1.993a)' As there is no obvious pharmacodynamic effect of AS, it is difficult for users to identify genuine drug. In addition, there is the potential in self-report surveys to confuse AS use with and glucocotticoid.s, prescribed for legitimate medical conditions (Handelsmar. et al.1'997).

Pre valen c e in gyrnn a s ì a \Wales An investigation of the prevalence of illicit steroid use in gymnasia across England, and

Scotland,, reported a lifetime steroid use amongst9.1.o/o of men and2.3o/o of women, where 60/o of men and L.4o/o ofwomen v/ere current users (I{orkia, 1996; I(orkia et a/.,1'997). Polydrug use was found ro be common âmongst ttle 1,667 respondents, with 3.7o/o tepotting co-administration of

cocalne,

7-6 CH,\PTER 1 Introda ction Prcvalence Ìn Ifþh Schoo| age students The .,Monitoring the Furure" survey sponsoted by the NIH (uSA) estimated that in 2000 lifetime (,.ever') prevalence of AS use was 3.0, 3.5 anð, 2.5o/o amongst 8'h, L0ù and 12'6 grade students respectively (see also Table 1. 2). AS and MDMA ("ecstasy") wete the orily 2 drugs for which

strong incteases over the 1'999 datawete repotted'

Steroid abuse in teenagers is not necessarily associated with panicþation in school . In a et recent study, 25o/o of male users repoting lifetime steroid use did not patticipate in sport Çanner

aL.,1,995).

t-7 CH,\PTER 1. Intro dø ction Location Sample fue Male Female Total Time-sPan (%) (Author , year) size (%) Richmond County, 1,881 Overall 4.2 Lifetime ("ever") USA (DuRant et al., 9'h grade 5.4 1.5 1993b Modesto, 742 grade 4.7 3.2 3.8 Lifetime Califotnia, USA (Radakovich et al.,

1 Lifetime Richmond County, 1 ,422 grade 4.8 2.9 3.8 USA (DuRant et al., 1,994) USA (DuRant et al., 12,272 Overall 4.08 1..20 Lifetime 2,26 1 ees) 1,4 yrs 15 yrs 1.54 16 yrs 3.48 1.7 yrs 2.41 ) 18 yts 3.35 Rocky Mountain 6,930 Overall 4.0 1.3 2.7 Lifetime region, USr\ et a|.,1,995) Falkenberg,sweden 1',383 1,4-19 yrs 5.8 1.0 Current use 1995) 15-16 yrs 10.0 Nebraska, USA 4,722 Overall 4.5 0.8 2.5 Current use (Scott et a1.,7996) 7'h grade 1.9 8'h grade 2.8 9'h grade 2.0 10'h grade 2.3 11'h grade 3.4 1,2'h gtade 3.2 & current Eastern States, 1,3,355 Overall 3.2 1.2 Lifetime Australia Overall 1.7 0.4 use (Handelsman el al., 1,997) Uppsala, Sweden 2,742 Overall 1.7 0.1 Lifetime (I{indlundh e/ ø/., 16-1,7 yrs 2.2 0.1 19e8) 1B-19 yrs 0.9 1993 in Table 1. 2 Reports of prevalence of AS use appearing in the English literature since High School age students.

1-8 CH,A.PTER 1 Intro daction 1.5.4 Abuse prevalence in Australia

Very few srudies have assessed the use of ÂS by Australian teenagers. In a recent study of drug use in 203 schools (grades 7-i1) in Victoria and New South ìØales, lifetime ("ever") use of ,\S was reported tobe3.2o/o âmongsr males znd1.2o/o amongst girls, with 1"7o/o of boys and 0.4% of gids reporting use within the last month. The 1995 Australian national drug strategy household survey (Commonwealth Department of Health and Family Services, 1996) indicated a 0'2o/o prevalence of illicit ,\S use in the general population, with 28,800 people having used AS in the previous 12 monrhs. The 1998 survey reported a lifetime use of steroids by L0/o of the male population.

1.6 Cardiotoxicity of anabolic steroids With the high doses of AS associared with illicit use a vast plethora of toxicological effects have been reported (Table 1. 3). Despite these reports, very few studies have been conducted to determine the mortali ty r^te amongst illicit users. Patssinen et al invesngated the mortality rate amongsr male power lifters placed 1"-5'h in the weight series 82.5-1,24kgin Finnish championships mortality dudng Ig77-1.gBZ (n=62) compared to population controls (Parssinen et a/.2000). The rate amongst powerlifters v/as 1,2.9o/o during ttre 1.2-year follow-up, with a 4.6 fold higher risk of (95% Cl 2.04-1,0.45; p=9.9902). The age of death than the popularion conrrols ^verage use was amongst powedifters was 43 yeats (36-53 years). It must be cautioned however that AS strong never verified in these powedifters, and although 2 wete from - there is no study can evidence d.irectly linking illicit AS use ro an increased risk of deliberate death. At best this high risk of AS only be considered as finding a much higher mortality rate âmongst Power lifters at known use. Future study should concentrate on fìnding evidence of premature death amongst fotmer and current AS users.

7-9 CHAPTER 1. Introdøction Physiological Advetse Effect Reference System Ifepatic (Cabasso, 1994; Hausmann et a|.,1'998) Hepatoma (Creagh et al.,'t988) Regenerative nodular (X/inwood et al., 1.990) hyperplasia Elevated serum asPartate fWinwood et a|.,1990) amino transaminase (r\ST) Elevated function test (X/inwood et a/., 1'990) values Gastrointestinal oesophageal varices SVinwood et a1.,1990) Endocrine Decreased testostefone (Biflsma et a|.,1'982) [rJeild,1995) Catdiovasculat Reduced high density (Kleiner et a1.,1990) lipoprotein Increased low density (Glazer,1991) lipoprotein Coronary plaque formation (À4ewis et a|.,1996) Hypertension (I(uipers et al., L991) Ventricular tachy cxdra (,\ppleby et at.,7994;Mewis et a/.,1996; Nieminen et a1.,7996) Âtrial fìbrillation (Sullivan et a1.,1999) Ventricular fi brillation (Cambell et a/.,1993) Left ventricular hyPertroPhY (À4cl(llop et al., 7986; Luke, 1990; Dickerman et aÌ., 1995; Nieminen ø a1.,1996) Right ventricular (Luke, 1990; Cambell et a1.,1993) hyperttophy et a1.,2000) QúIochizrr-l

1-10 CHAPTER 1. Introda ction AS use has been associated with many cardiovascular disturbances, including hypertension, myocard-ial infarction, ventricular and atÀal fìbrillation, Ieft ventricular hyperttophy, cardiomyopathy, myocard.ial fibrosis, thrombosis and congestive heart failute (Table 1. 3 & Appendix A.). There are 3 main models that can help explain how AS may cause cardiotoxicity' It is probable that cannor be attributed solely to any one mechanism and is likely to result

from a combination of effects...

1,. Reduced cotonary flow a. VasosPasm b. Thrombosis c. Artedosclerosis

2. Direct myocatdial effects ^. Cardtac remodelling b. Myocatdial cell iniury

3. Flypertension

The following will consider each of these models in more detail and discuss additional factors, which may increase the risk of sudden cardiac death resulting ftom illicit AS use'

1.6.1 Reduced coronarY flow The observation of associated with ,\S abuse (Table 1. 3) suggests that AS

can reduce coronary flow. Evidence for a number of potential mechanisms ate outlined below'

Vasospasm been A number of athletes, known to be current AS users, dying from sudden catdiac death, have found to have coronary arteries free from thrombus or atheroma (I-uke, 1990; I(ennedy et al', 1993b). Likewise, a number of AS abusing athletes, admitted for infarction have displayed angiogaphically normal arreries (Ferenchick et a|.,1,992) (Appendix A).

vasod-ilatory response in the brachial to sodium nitroprusside and methacholine -,\ depressed ^rtery returned to has been repotred in a patient self-administering nandtolone (Gteen et øl',1993), which found to normal following 8 weeks abstinence. Chronic nandrolone treatment of rabbits has been aona by reduce both endothelium dependent and independent relaxation of ex-uiuo thoracic

decreasing guanylyl cyclase (GC) activity (Ferrer et a/.,1994a; Ferrer et al',1'994b)'

7-t't. CHAPTER 1. Introdaction Thrombosis

Several câse reporrs have detailed thrombosis or plarelet hyperaggregability associated with chronic high dose AS use (AdcNutt et aÌ.,1.988; Huie, 1994; Fisher et a/.,1'996; McCarthy et a|.,2000)'

Aggregation of male and female human platelets in tesponse to a pro-aggregation stimulus is significantly gre teï in the presence of androgen flohnson et al.,1'975)' Male rats and guinea pigs have been found to be 10 rimes more sensitive to aggregating stimulus than age matched females this sensitivity, suggesting a role for flohnson et al., L977). Male casrration signifìcantly reduced testosterone in aggregation ptomotion.

Androgens have been shown to affect the synthesis of a number of eicosanoids thereby increasing blood coagulation and. enhancing the risk of thrombus formation. Testosterone has been demonstrated to increase the production of thtomboxane A2 þlatelet zggegator), while at the same rime decreasing the production of prostacyclins (eg PgI), which normally inhibit platelet

aggregation @erenchick,1.9914!leyrich et a1.,1.992; Melchert et a1.,1995).

system. Several 1,7 u-alkylated androgens have been found to influence the coagulation/fibrinolytic in type Dznazol, a steroid favoured by many weight lifters, significantly improves coagulation '\ al', and B haemophilìa by increasing levels of facror VIII and IX (I-aurelt et a1., L979; Gtalnick et

1e83).

c rctnom Disseminated intravascular coagulation often occurs ìn patients with prostatic ' of the presumably, as a result of thromboplastic substances released from tumour cells or activation and intravascular coagulation system. A range of such as ketaconazole, flutamide

(u- ìtt et al., buserilline been found to be protective against coagulation in these patients ^cetatehave 1.987 ; Marttnez et al., 1'9 88).

unifying Níhatever the role of AS in the fotmation of thrombi, it is unlikely that this represents a aetiology for sudden death in steroid users. Many illicit users suffering myocardial infarction were found to have angiographically normal coronary arteries - free from atherom or thrombus. It is more probabie that the pro-coagulant effects of many andtogens increase the risk of an adverse also display cardjac event occurring. This risk is likely to be increased dramatically in patients who levels of significant arheroscletosis. Increased platelet aggteganon induced by AS may also inctease platelet derived growrh factor eDGÐ, which increases cell ptoliferation and may contribute to atherosclerotic lesion (À4elchert et al., 1,99 5).

7-12 CH,,\PTER 1 Introdøction Atterioscletosis resulting in It has not proven possible ro show a defìnite link between AS abuse and atheroscletosis that AS change turn in an adverse cardiovascular event. However, a number of studies have shown signifìcant rhe normal blood lipid balance so that it resembles the pattern observed in patients with elevated low (Glazer, 1,991). A decre¿sed high density lipoprotein (HDL) and density lipoprotein (I-DL) profile has been reported in a number of athletes self-administering '\S

@owman, 1990)

(HTGL) which An association exists berween ,\S and increases in hepatic triglyceride lipase increases in low- increases high-densiry lipoprotein (HDL) catabolism. Reductions in HDL and Lendets et aÌ', density lipoprotein (LDL) have been demonstrated in humans flülebb et al., 1984; cyanomolgus 19gg; Thompson d/ a1.,1.989;Baldo-Enzi eÍ a1.,1,990; Friedl et a1.,1'990; Glazer,1991), subiected to monkeys (Weyrich et at., 1.992), and tats peeds et al., 1'986; Lewanowitsch et al',2001) men, engâged in regular, chronic ÂS treatment. plasma HDL concentrations will often dectease in fatry streaks' intensive, vigorous exercise (I{ibble et at. 1.987). Increases in LDL may result in in advanced lesion' endothelial , platelet aggregation, and increased cell proliferation tesulting strongly as non- It appears that aromatisable AS such as testosterone do not induce HTGL as et al' 1990)' aromatisable steroids such as stanozolol (Ihompson et a/' 1'989; Baldo-E'nzi

A furthet risk of altered plasma l-ipoprotein level is changed myocardial membtane phosphoJipid composed of a composition and an increased risk of cardiac arrhythmia. Myocardial tissue is (PC)' phospholipid bitayer consisting of phosphatidylethanolamine (PE'), phosphatidylcholine Besides de nouo phosphotidylserine (PS), sphinomyelin and phosphatìdylinositol (Laarse,1987)' sourced from receptor synthesis of cholesterol from acetyl-Cor\, membtanous cholesterol is also

mediated uptake of LDL (I-aarse, 1987) (Fig' 1' 2)'

7-13 CHAPTER 1. Introdaction "i:hç åçn1çrç1 e:a r¡-ie¡"' Ltlt ¿ëetyl CûÅ

upta he

r*€ÊF?er synthesis H¡16ËoÁ E{!dui:lr¡se e $u!5

c F*,Eg {:H$I.HSTEROL

*Ët [rêt lô$

AgÅ1 {.y t. neutr¿l hYdroiase

cholestcrol c¡ter

sou¡ce: Fig. 1. 2 pathways involved in cellular cholesterol supply, metabol¡sm and removâ|. cell a receptor mediated mechanism, and is subiected Laarse et a1.,19g7. Low densiry lipoprotein is taken up into the by in free cholesterol exerts to lysosomal breakdown resulting in an increase in intracellular free cholesterol. Ao increase synthesis. Free cholesterol also an inhibitory effect on HMG-CoA reductase slowing the de novo cholesterol Prevents (ACAT) which stimulates the synthesis of membrane bound LDL receptors, and activates cholesterol acyltransferase the conversion of cholesterol to cholesterol esters'

lipid bilayers The exrent of cholesterol incorporation can chânge the physical properties of cellular found that atteinl smooth plasma Q,aarse, 1987). Fot example, Tulenko el al in the ratio of membranes isolated from 2o/o cholesterol fed rabbits showed a progre ssive elevation free cholesterol to phospholipid (fulenko et a1.,1.998). Chen et al fed rabbits cholesterol Q"/ow/w) cells showed for g weeks and demonstrated that plasma membrane from arterial smooth muscle compositìon evidence of changes in membrane bilayer stfuctufal Pafametefs and phospholipid cholesterol content by (Chen et a1.,1.995). Treatment was found to increase the unesterifled (free) width was highly 670/o and the bilayer width from 56-62Ä. Interestingly, this increase in bilayer content of correlated (r=0.992)with memb rane free cholesterol. No overall change in phospholipid made to the the membrane was observed, but the contribution various phospholipid classes Membranes obtained makeup of the membrane changed significantly with cholesterol treatment. from conrrol rabbits exposed to free cholesterol ìn culture displayed similar changes in

7-74 CHAPTER 1. Introdaction phospholipid content of the membrane. Cholesterol enrichment of the plasma membrane was found to signifìcantly decrease the activity of the Na+/I{+ AT?ase pump (transmembtane protein).

Because AS have been shown in both animals and humans to induce athlerosclerotic changes in serum lipoprotein profiles it would be expected that nandrolone and similar AS may altet the compositio n andf or fztty acid content of the phospholipid membrane due to an increased availability of cholesterol for cellular uptâke. However, Liang el al used "P NMR spectral analysis of whole heart phospholipids from male SD rats administered nandrolone decanoate (3.Omg/kg every 7-9 days for 9 injections following an initial loading dose) to fìnd no signifìcant difference in myocardial phospholipid profìles from drug treated rats and controls (ü"tg et al',1'992). Changes in phospholipid composition of the myocardial membrane were only found in rats subiected to exhaustive exetcise. No differences were found between rats subjected to exercise and simultaneously administered nandtolone when compared to rats, which were exercised, but drug free. Unfortunârely, Liang et al made no âttempt to quantify changes in LDL and HDL with nandrolone treatment so it is not known whether their negative results simply reflect a treatment stfategy which does not have a signifìcant effect on plasma lipoptotein.

t. 6. 2 Direct myocardial effecrc Myocardial cells contain androgen receptors and it thetefore seems reasonable that AS should have a direct effect on the cell, A number of autoradiographic studies have confìrmed the presence of androgen receptors in male and female baboon hearts (À4cGilt et al., 1'980; McGill et al., 1'981). Other studies have demonstrated the presence of myocardial androgen receptors in rat (I*ieg et al',

1.978), dog and human hearts (I\4arsh et a1.,1'998).

Catdìac remodellìng It is not unusual for signifìcant increases in left ventficulaf mass to occur in athletes undertaking intensive aerobic exercise. An inctease in heart tissue is matched by an increase in collateral vessel formation thereby increasing the oxygen supply to the new tissue. A number of autopsy studies following sudden c rdi^c death have noted signifìcant left ventdcular hypertrophy (I-VH) in athletes abusing AS (Appendix A). Work conducted using mouse heatts suggest that this increase in heart muscle is not concomitant with new collateral vessel formation in ,\S treated rodents (fagarakis ef al.,2000). '\n echocardiographic study in hypertensive men conducted over 5 years found that elevated left ventricular mass index was the best indicator of future morbidity (Casale et al., 1.986), although LVH has been found to correlate poorly with high blood pressute (BP)

(Papademe triou et al., 1'9 85).

't-15 CHAPTER l lntrodaction Haman Studie¡ Although cardtac hlpertrophy has been associated with AS abuse in a number of autoPsy studies

(I-uke, 1990; Camb ell et a1.,1993; I(ennedy et a1.,1.993b; Dickerman et al.,1995) and detected with ECG and echocardiogâphy in ìsolated cases (À4cll11op et a/., 1'986; Sullivan et a/., 1'999) these obsewations have proved difficult to reproduce with larger numbers of patients.

Salke ¿/ al rcport that weight training alone increases left ventricular (I-\f posterior wall and ventricular septal thickness. No significant differences were found in athletes self administering large doses of AS (Salke et ø/.,1,985). Likewise, Palatini et alnoted no difference in functional and dimensional echocardiographic indexes between steroid using and non-using weight trainers

@alatini et a1.,1996).

While a number of studies have noted overall increased LV mass or wall thickness in AS using subjects, adjustments for body surface area (BMI) or body mass often eliminate these signifìcant d.ifferences. Most recently, Sader et al assessed changes in cardiac morphology and function using echocardiography in a group of 20 ma\e, r\ustralian bodybuilders, who either self administered ,\S

or were drug free (Sader et a1.,2001). While significant increases in LV mass were noted in the drug- when adjusted with the using group Q45+16 vs. 1,99*1,29, p=0.04), this difference was eliminated cap^ciLy, the adiusted LV body mass index 1BMI). ,\fter further adjustment for maximum strength mass wâs quite similar between groups. Dickerman et al tested cardiac dimensions with echocardiography in a ser of fnternal twins, who had both been weightlifting for 20 yearc 1'5-yeat history of AS use. @ickerman et a1.,1.997a). One twin was drug free, but the other had a No d.ifference could be found in the thickness of the LV posterior wall or the ventricular septum, despite an abnorm zl ezrly diastolic fìlling velocity in the drug-using twin. In a further study Dickerman et al studted 16 competitive bodybuilders recruited for an echocardiographic study (Dickerman et al., 1.997b). Eight of these hear.yweight bodybuilders were drug ftee and the remainder self adminisrered AS. Although a signifìcant increâse in LV posterior wall thickness and LV septal thickness was noted in the drug using group compared to drug free subjects, when adjusted for BMI no signifìcant d-ifference could be observed, although a significant decrease in the BMI adjusted left ventricular end diastolic dimension was noted in the drug using Soup' It was concluded that AS may potentiate concentric left ventricular hypertrophy with decreasing ventricular compliance without affectìng cardiac function. Urhausen et al exzmined the echocardiographic characteristics of male, top-ranking bodybuilders who had a history of AS use (n=14) and those without such a history (n=7) (Jrhauserl et al., 1.989} A significant increase in the LV posterior wall thickness was noted in the steroid-abusing group in comparisorì to corìtrol in the

7-16 CHAPTER l lntrodaction absence of a signifìcant increase in BP. These differences became non-significant when adiusted for body weight.

Sachtleben el al examined body composition and myocardial structure in male weight trainers reporredly using or not using ÂS (Sachtleben et at.,1993). Amongst the steroid users a signifìcant increase was found in LV mass (182.8!26.99vs.21.0.6!429, p<0.05) and interventricular sePtum thickness during the peak of a drug taking cycle, compared to the end of zn "off cycle" of at least 8 weeks duration. In add.ition, LV diameter and posterior wall thickness was signifìcantly greater in steroid administering weight trainers during zfl"o(r period" compared to non-using controls.

endurance Stolt ¿¡ al found that physiologica\ adaptive LVH does not increase QT dispersion in athletes, although the eT interval is prolonged due to increased vagal tone (Stolt ar al., 1'999).In subjects undergoing weight training and administering high doses of AS, an observation of increased eT d.ispersion despite short QT intervals was made. This may indicate altered myocardial stfuctufe in hypertrophied heart and may increase the risk of maJignant arrhythmia'

The apparent contrad-iction between various echocardiographic studies of AS users may be related to the type of exercise that the subjects are practising. Isometric exercise results in increases in left ventricular internal dimension and wall thickness, whereas isotonic exetcise has not been associated with this type of change (I(ennedy, 1,992). Athletes will often mix both forms of exercise' Many studies d.id not d-ifferentiate between users who were currently on a "cycle" and users who wete undertaking a drug free period. Sachtleben et al demonstrated that changes in cardiac morphology

are evident berween cycles (Sachtleben et al.,1.993).In addition, the fact that 1-D echocardiographic

measurements were made in some studies, without the use of 2-D controls increases the chance s of to errorreous dimensions ([Jrhausen et a/., 1.989). ,\ number of studies utilising echocardiography meâsure changes in cardiac dimensions have omitted to confìrm the repoted drug use of their subjects using urine-analysis (Salke et al., 1.985; De Piccoh et a/., 1'991)' This omission is most significant in stud.ies comparing wall thickness changes belween athletes on and off their drug

taking cycle (Jrhausen ¿/ a1.,1989).

Animal Stadie¡ and Animal srud.ies regarding increased cardiac mass with AS administration are also confusing conrradictory, The reported morphological and histological effects of AS are shown in Table 1' 4' age the animals' Unfonunately, a number of investigatìons have failed to propedy control for the of It is probable that the rodent and canine myocardium is more sensitive to steroid administration during the immature phase of its lifecycle (I(inson et at.,1991). I(inson and associates found that into male tats câstrated 10 days after birth, incorporated signifìcantly less (Âzevedo et al.)-leucine

'1.-77 CHAPTER l lntroduction c rdiac protein than sham operated controls. This effect was signifìcandy reversed by testosterone administration. At day 60, exogenous testosterone administration had no significant effect upon incoqporation. This suggests rhat the sensitivity of the rodent heart to testosterone is age dependent. Previously, it has been demonstrated that exogenous testosterone administeted to adult female mice induces a significant increase in ventricular u/et weight, without a corresponding increase in DNA (I{oenig et al., 1982) implyrng that cxdiac growth in these animals was due to hypertrophy of existing cells. It is probable that increases in czrdiac mâss with steroid administration dudng the immature phase of life are due to hyperplasia, and increases in adults are due to hypertrophy (I(nson et a/.,1'991)'

Tagarakis et al invesngated the combined influence of various AS and physical exetcise Qaga::J

Marsh and colleagues demonstrated that mRNA encoding andtogen receptors could be detected in myocytes of male and female adult rats, neonatal rat myocytes, rat heart, dog heatt, and infant and adult human heart (NIarsh et at., 1998). In addition, cultuted czrdiac cells from neonatal rat hearts

have been shown to increase the rate of phenylalanine incorporation, and extent of ANP sectetion, in response to testosterone and (Azevedo et a/', 1983; Marsh ef al., 1'998) phenylalanine incorporarion and ,\NP secrerion have both been shown to be indicators of cardsac hypertrophy. Unfortunately, Marsh et al oriy examined ,\NP secretion and amino acid incorporation in neonatzl cardiac myocytes, which ate still in a signifìcant growth phase (I(olbeck-

Ruhmkorff et al., 1.993).

Animal studies suggest that while hypertrophy can be observed in immature, developing rat hearts it is uncertain whether this same effect can be observed in adult hearts. The work of Tagaral

(Iagarakis et al., 2000).

1-18 CHAPTER 1. Introdaction Myocardial CeII IniurY Some of the fìrst investigations into the pathology of chronic AS use were conducted in the late

1970s. Behrendt and Boffin treated male rats with once weekly injections of methandrostenolone the conclusion of the 13-week treatment period the 11.65mg/kS, i.-.) (Behrendt et at.,1,977). At heart muscle cells were examined with electron microscopy. Mitochondtia and myofibrils were found to display changes similar to those associated with early heat failure' The mitochondria were found to be elongated and swollen, with sparse matrix and cristae. The myofibrils either d.isintegrated as a result of treatment, showed widened and twisted Z-bznds, or a complete dissolution of the sacromeric units. Later studies found an increase in the intermediate sized, nonmyofibrillar filamenrs in muscle cells of the left venfficle (Behrendt, 1'977).

Testosterone (104ifQ has been found to cause â signifìcant release of cytoplasmic lactate dehydrogenase (I-DH) from nt primary myocardial cell cultures (Melchert et a/., I992b). as well Transmission electron microscopy revealed a signifìcant decrease in mitochondria number as disruption of the mitochondrial and plasma membranes (À4elchet et ø/.,1'992a).

Tseng et al administered nandrolone decanoate (2}mg/kg, s.c./day/6 wks) to developing, sponraneously hypertensive rats and found evidence of small focal areas of myocardial cellular

damage with inflammzltofy cellular or fibrotic infìltration (Iseng et al',1'994)'

Direct myocardial ðamzge would be expected to result in a loss of aerobic energy production, cell death and eventually result in fibrosis. Areas of extensive myocardial fìbtosis have been demonstrated to signifìcantly increase the risk of fatalventricular arrhythmia.

't-19 CHAPTER 1. Introdaction Author, Species, AS, dose, Exetcise Morphological Histological changes date strain, ftequency status changes

no change ìn heart None reported @hillis e/ Rat, Aì1, Nandrolone sedentary a1.,2000) 12 weeks decanoate, 2\mg/k1, weight/body weight s.c./3xs weekly/15 Íat10 days Voluntarily Absolute heatt weight None reported flX/oodiwiss Rat, SD, Nandrolone et a/.,2000) 75-909 decanoate, Smg/kS, exercised on was found to be i.m./2xsweeÛy/12 training significandy reduced weeks wheels compared to exercised- drug-free conuols and non-exercised, non- treated tats. Evidence of small focal (Iseng e/ Rat, Nandtolone sedentary No change in absolute al.,1994) SHR,6 decanoate, ZOmg/kZ, ventricular weight. areas of myocardial weeks s.c./daily x 6 weeks Signifìcant inctease in cellular damage with ventricular/100g body inflammatory cellulat weight (cp. control), or fìbrotic infiltration. Significant increase in vertical ventricular diameters, and vertical ventricular circumferences. Exetcìsed on Heart dry weight and None teported þía:ng et a/., Rat, SD, Nandrolone 1992) 4 weeks decanoate, a rodent heart weight to fìnal 3.0mg/kg, i.m./7-9 treadmill body weight was not åays/9 injections v/eârlng a significantly different in weight collat treated rats compared to 5 days/week control. fot 10 weeks was not None reported þizng et al., Rat, SD, Nandrolone Exercised on Heart we.ight 1,ee3) 4 weeks decanoate, a rodent significandy changed in 3.0mg/kS, i.m./7-9 treadmill the nandrolone treated days/9 iniections wearing a gtoup compared to weight collar control at the conclusion 5 days/week of 10 wks treatment. for 10 weeks Likewise, the ratio for heart weight to fìnal body weight was not significantly different between steroid treated rats and controls, regatdless of exercise stâtr]s. Swimming Exercising rats receiving No pathologic @awman et Rat, SD, Stanozolol,5mg, was reported al., \988) 6 weeks i.m./2xs weekly x 5 exercise, 3xs nandrolone, with or condition weeks a week. without dietary proteìn for any organ. suPPlementâtion demonstrated a significant increase in relative heart weight compated to non- exercised, non-drug- treated controls. .../continued over

7-20 CHAPTER 1. Introdøction Author, Species, AS, dose, Exercise Morphological Histological changes date sttain, frequency status changes age (Behrendt, Rat, Methandrostenolone, sedentary None repotted Steroid treatment increase 1977) \Vistar 1.65mg/kg, tesulted in an (females), i.m./week x 13 in intermediate sized, 150g weeks nonmyofìbrillar fìlaments in muscle cells of the cardizc . and (Behrendt e/ Rat, Methandrostenolone, sedentary None tepotted Mitochondtia the a/.,1977) \ùØistar 1.65mg/kg, myofìbrils from (females), i.m./week x 13 hearts of treated rats 150g weeks showed changes similar to those observed in earlyheart failure. The mitochondria were swollen and elongated. The myofibrils showed eìther disintegtation and widened and tu¡isted Z-bands or a complete dissolution of the sarcomeric units. Table 1. 4 Morphological and histological myocard¡al changes repoÉed in laboratory an¡mals Subjected to chronic AS treatmeñt. For more details on histological changes see Appendix B.

1.6.3 I{ypertension The abiliry of ÂS to produce hypertension remains conlroversial and confusing' The observation that Conn's syndrome, which pfoduces increased and subsequently results in hypertension, led to the suggestion that anabolic-androgenic steroids mây also produce elevated BP

(Skelton et a/., 1,969). Two early studies appeared to confirm this hypothesis. Methyltestosterone and testosrerone (10mg, 5xs/wk) were found to produce hyPertension within 6 weeks of administration, to rats drinking 1o/o sâline. Fischer and Swain iniected castrated male rats with

testosrerone clpionate (lmg/wk, i.m.) and found that zfter 3 weeks of treatment, BP had increased significantly in steroid pre-treated rats in comparison to vehicle treated controls @ischer et al.' i,g77). More recently Tseng et alfound nandrolone decanoate Q\mg/kg, s'c., daily) âccelerated the development of hypertension in young, spontaneously hyPertensive rats in comparison to vehicle treated conrrols (Iseng et a/., 1,994). An approximately 2OmmHg difference was found between nandrolone tfeated and vehicle treated fâts at the conclusion of the treatment pefiod. It is not known whether normotensive adult male rats would respond in the same manner to nandrolone

treatment.

Despite these studies showing an iricrease in BP with AS treatment, a number of authors have found no evidence of hypertension with AS administtation. 'ùØolinsky found BP was unchanged following 4 weeks trearment of male râts u/ith testosterone clT)ionate flWolinsky, 1972). Brown and

't-21 CH,\PTER 1. Introdøcrion pilch found that exercising male rats administered methylandrostenolone showed no signifìcant change in heart rate (HR) or BP following 6 weeks of treatment (Brown et ø/., 1'972). Likewise, Greenberg and associates found that the BP of both male and female dogs was unchanged 24 hts after the of testostefone (1Omg/kg, i.m) (Greenberg et al.,1973)'

Many human srudies have also failed to fìnd any indication of hypertension with AS use. Olsson ¿/ women with aÌ report that 7 .Smg/day of oxandrolone administered to 25 men and hyperlipoproteinaemia significantly reduced plasma riglycerides but had no effect on rest-ing BP

(Olsson et al., 1,974). Mcl{llop found significant LVH in a male body buildet with an eight-year history of steroid abuse with no associated hypertension (130/80 mmHg) (r\4cl(llop et a1.,1'986). Friedl et al found no change in BP following 12 weeks chronic treatment of non-athletes with methylresrosrerone (40mg/dzy, p.o.), testosterone enanthate QS0mg/kg/week, i.m.), et a/., 1,990)' Thompson et al e1¡mg/kg, i.-.) or testosrerone enânthate and testolactone (Friedl and Urhau sen et al fo¡61d. no change in BP in male power athletes administering a comPetiuve regimen of steroids compared to their steroid free associates (Jrhausen et a/.,1989; Thompson a/ free at.,1,992). Sader et alfound, similar resting BP in AS using bodybuilders compared to their drug colleagues (Sader et a1.,2001).

lifters, Riebe et al measured BP at rest and during a standardised exercise test in AS abusing weight in their drug free associates, and in sedentary controls (Riebe et a1.,1992). They found that the AS Adjustìng rest and abusing Soup had significantty higher systoJic BP at rest and during exercise. exercise Bp for body weight or biceps size eliminated statistica\ significant differences between groups, This suggests thâr the higher rest and exercise BP values noted in AS users may simply reflect their larger body mass or be related to the larget arm citcumference of these subjects. Blood

pressure measurements u/ere made using sphygmomanometry, which is influenced by biceps size

1,3 was found between subiect atm circumferences' The S,iebe et at.,1.992). Variation of up to cm can use of a standard sphygmomanometry cuff in situations of abnormally large arm circumference result in an over-estimate of BP (Jrhausen et aÌ., 1.989). Further studies should include intaartetizl Bp measurement to avoid diffìculties associated with BP measurement via indirect methods.

It is important to note that heavy resistance exercise itself can cause massive elevations in BP. a MacDougall. et al measured Bp in the brachial in fìve experienced body builders performing ^rtery variety of resistance exercises until failute (ÀtlacDougall et at., 1'985)' The greatest peak Pressures were recorded during the double leg ptess where the mean for the gfoup was 320/250mmHg, with subiects pressures in one subject exceeding 450 /350mmHg. It was concluded that in young healthy performing weight-lifting exercises, the mech atical compression of the blood vessels combines BP. While abuse with a potent pressor and Valsalvâ response to produce extreme elevatjons in '\S 7-22 CHAPTER 1. Introdaction itself may not cause hypertensiofi, othef factors such as vasospasm and arteriosclerosis may alter the pressor resPonse to resistance exercise'

It is possible that AS may cause increases in BP through stimulation of the hypothalamic-pituitary- adrenal (HpA) axis. In â recent study Schlussman and associates found that daily treatment of male SD rats with nandrolone decanoate (1Smg/kg, i.m.) resulted in a significant increase in circulating adrenocorticotropin hormone (ACTH) and t hour after the third steroid injection compared to vehicle treated controls (Schlussman et a/., 2000; Schlussman et a/', 2002). Adrenocorticotropin hormone levels did not remain elevated at 24 hours following nandrolone administration. Unfortunarel!, this study did not monitor BP changes over the nandrolone treatmenr period. Conversely, Alen el al measured ,\CTH levels in athletes self administering ,\S for

26 weeks and then during withdrawal over the following 12 weeks and compared these results to a group exercising with the same physical intensity, but who were drug free for the entjre period

(Alen et at.,1,985). After 8 weeks of ,\S treatment r\CTH levels were found to decrease signifìcantly in comparison to the control gtoup and remained lower than control for the entire drug treatment period. Adrenocort-icotropin hormone levels began to increase 6 weeks after cessation of AS administration. Unfortunately this study made no attempt to correlate serum hormone levels and

Bp. Schyve ns et al recently stud.ied the effect of exogenous ACTH 1S00¡rg/kS) on BP in telemetered

mice (Schyvens et aÌ.,2001). was clearly elevated after2 days of treatment and by day 10 had risen to 136+4mmHg which was signifìcantly higher than vehicle treated controls (p<0.01). Unfortunately, plasma levels of ACTH were not progressively measured over the trearment period and therefore it was not possible to equate changes in BP with the concentration

of circulating ACTH. Turner et alused the tail cuff method to record BP in male SD rats and found that systoJic pressure increased signifìcantly over 10 days of treâtment with an ACTH dose of only 1,0g,"g/kg/day from 1,1,7+1, to 741.+2 mmlHg (Iurner et ø/.,2001). Serum corticosterone was increased to 825+70 ng/mL in -,\CTH treated rats. Further studies by Lou et al found that both ACTH (50}Wg/kg/day) and corticostero ne (1.20pg/kg/day) significantly increased BP compared to control (1,65t6 vs 127mmHg, p<0.001 for ACTH; 162+1,1 vs 11,1'+6 for corticosterone, p<0.001)

g,ou et at.,2001).It remains unclear whether high dose chronic treatment with AS increases BP to hlpertensive levels through increased plasma concentrations of ACTH and corticostefone.

Both animal and human studies indicate that hypertension does not always occur after AS administration. Human data indicates that hypertension may occuf in selected male patients or

athletes. There is a lack of both human and animals studies designed to address the occurrence and potential mechanisms of steroid induced hyPertension.

7-23 CHAPTER l lntrodaction 1.7 Diffrculties associated with anabolic steroid research It has proven extremely difficult to gather well-controlled clinical information from patients abusing AS. Competitive athletes are often unwilling to admit AS use Q'Jieminen et a/., 1'996). patients linked with AS abuse commonly presenting with serious cardiac conditions ^re uncooperarive with ongoing research and zte usually lost to follow-up (Sullivan et a1.,1'999).

The dose of ,\S associated with a fatal abusive incident has never been reported (I\{elchert et a/., lgg¿b). This implies rhat clinically relevant doses for investigations of toxiciry are difficult to extrapolate. However, the plasma concentration following a therapeutic dose of testosterorie enanthate in 19-40 year old males has been shown to reach of 4.37x1'0-tM. As it is ^î ^verz;ge known that steroid users administer AS at doses 1O-1000s the recommended therapeutic dose it is reasonable to suggest that plasma concentrations in the 10-7 to 104M range would not be \Welder, unexpected (Àdelchert et a/.,1992t¡; 1992).

Because of legal aspects of the non-therapeutic use of AS it is diffìcult to implement well controlled clinical trials. The unique adminisration partern of many steroid users involving exceptionally high doses of various types and formulations of AS, administered by a vaÅet'¡ of routes makes a human trial mimicking an abuse situation unethical (Parssinen et a/., 2000). In addition the "drug free hoJidays" taken by some athletes immediately prior to comPetitions adds a further layet of complexiry. Studies concerned with the cardiotoxic potential of AS are hampered by strenuous

exercise in athletes and the highly modifìed diet of many weightlifters and body builders.

Many athletes wilt patticipate in a strict "pte-competition" diet in the 2-4 months pdor to competing (I(leiner et al., 1.990). Food diaries from body-builders preparing for competition have et al., revealed d.iets typically high in protein (>3 tìmes the tecommended daily intake, RDI) (I{Tetner

1990), and cholesterol (>2.5 RDI) (I{eitL et a/.,1,996). Reports of male bodybuilders consuming uP to a dozen eggs a day are not uncommon (Faber et a1.,1986). In addition many comPetitors have increased gut motility due to use. In ân attempt to improve muscle definition in the hours prior to competition, many bodybuilders will reduce water intake, administer and undertake dehydrating exercises such as intensive exercise in a sauna (I(leiner et a/.,1990). Hear'y laxative and use can cause disturbance s increasing the risk of cardiac

abnormalities (Hoes et a/.,1.994; Grobbee et at., 1.995). In addition, severely restricted calcium (360/o RDI) and reduced zinc (75o/o RDI) intake has been reported in women body builders (I(leinet et ø/., 1990). Many competitors believe that by "sodium-lozd)ng" they will be able to maximise their fluid loss. This consists of following a severely salt-restricted diet until the week of the competition.

1-24 CHAPTER l lntroduction Large amounts of salty foods are then consumed fot the next 2-3 days before recommencing a sodium fast (I(leiner et al',1990).

In addition ,\S are ofren not only administered in combination with , but also drugs supposedly helpful in assisting muscle building or defìnition, or to lessen the perceived side effects of AS, such as þrevents I(* Ioss), Clomifen (increase levels) and contfaspasmin (has Ê, & Þ, adrenergic effects) (Hausmann et a|.,1'998).

1.8 The polydrug abuse Phenomenon I;sers of illicit drugs normally do not restrict their use to one drug. Poly-drug abuse is common. ,\ number of reports have indicated severe cardiac consequence s from an .,\S and sympathomimetic combination. Reports include combinations of AS and (Capezzuto et al', 1'989;

Appleby et al., 1'994) as well as AS and cocaine (I(ennedy et al',1'993b)'

Because many AS users are athletes znd are greatJy concerned with their body image and health it could be presumed that they may be unique among drug users, and be unlikely to engâge in poly- 60,000 college students for pharmacy @uRant et a1.,1.993b). Meilman el al suweyed approximately use of various legal and illicit drugs. Amongst a subgroup of 1.7 5 AS users it was found that they were significantly more likely to use , maitjuana, cocaine, , , , opiates, and designer drugs than their steroid free colleagues (À4eilman el at., 1.995).In add.ition, DuRanr et al foer;.Ld that cocaine was preferentìally co-abused in AS users found to be arrending U.S. High Schools @uRant eî a1.,1.993b; DuRant et a/.,1'995). Cocaine was the strongesr co-varyin g factor with the use of AS puRant et al., 1993b; DuRant et al., 1995)' The stabiÌity of this relationship over a 4-month period was also demonsttated (DuRant et a/',1'994)'

months The number of 1,2'h grade U.S. High School students reporting cocaine use in the past 12 current peaked in 1,999 6.20 , and in 2001 was estimated at 4.8o/o flohnston et al., 2002)' The ^t "lifetime prevalence" of cocaine use in these students is estimzted at 8'2o/o [ohnston et aÌ.,2002)' Therefore, although it appears that cocaine use has now stabilised, the prevalence of use remains high. The 2000 National Household Survey on Drug Abuse (ÌrJHSDA) estimated that there ate L4 million illicit drug users in the US, of which 1.2 million are current cocaine users. This represents use 0.5o/o of the population older than L2 years. Amongst youths aged betwe en 16-1'7 prevalence of in the past month is estimated at1..1.o/o.In combination with a signifìcant prevalence of abuse of cocaine the reported lifetime prevalence of AS use has increased 1.2o/o to 3.70/o in 1,2'h gtade reporting use in students berween 2000 and 2001 Sohnston et at.,2002). The percentzge of students

7-25 CHAPTER 1. Intro duction 3 past 12 months also increased during 2001 to approximately 2.4o/o. Thus considerable potential rr co-abuse of AS and cocaine exists in the adolescent population.

Based evidence on of a significant potential for co-abuse of stimulants with AS, in add-ition to rePorts suggestìng high a likelihood of abuse of this drug combination in adolescents, ir was decided to further investigate the potential cardiac toxiciry resulting from this drug combination. Despite the diverse pharmacology of ,tS and cocaine it has been observed that these two drugs share a very similar spectrum of cardiotoxic events, associated with both chronic and acute use. ,\ possible synergistic ot additive cardiotoxic interaction between these two drugs requires further investigation.

1.9 The pharmacolory of cocaine

Cocaine þenzylmethylecognine Cr7H21No4) is an alkaloid exrracted from the South American shrub Erytltroxllon coca. Cocatne has both a sympathomimetic and a local anaesthetic effecr, and is thought to have both central and peripheral actions. Cocaine can on the CNS to prevent ^ct the neuronal reuPtake of noradrenaline and to trigger the release of catecholamines from the adrenal medulla (Schindler et a/.,1992). Cocaine also has a peripheral sympathetic effect through blockade of notadrenaline reuptake (Ivetsen, 1963). In addition Cocaine has been shown ro cause

noradrenaline efflux in isolated tatb:*in rissue (Azzarc et a/.,1974).

A numbet of studies have provided evidence for the importance of the CNS in the cardiovascular response to cocaine. \X/ilkerson el a/ demonstrated that the BP response to intravenous cocaine in conscious dogs was decreased by more thzn 50o/o following pre-treatment with the ganglionic blocker, hexamethonium (X/ilkerson, 1988). Similarly, anaesthesia with almosr eliminated the pressor resPonse to cocaine in comparison to the same rats given an identical dose cocaine of on a different day. Chiueh & I(opin demonstrared that cutring the splanchnic nerve reduced the cocaine-induced release of adrenaline and noradrenaline by 75 and 500/0, respectively, in tats in which the noradrenaline metabolising , catechol o-methyltransferase (CoMt had been inhibited (Chiueh eî a/., 1978). Cocaine methiodide, which does not cross the blood-- bafTie4 infused intravenously into conscious rats until death has been found to be 6 times less

potent than cocaine on â molar basis (Iella er a/.,1992).

Many of the cardiac and behavioural effects of cocaine can be attributed to its ability to block the synaptic monoamine teuptake pump - causing the persistence of noradrenaline, dopamine and serotonin in the cleft (Fig. 1. 3). Under notmal physiological conditions when a sympatheric nerve is stimulated, the noradrenaüne not bound to a receptoï on a receiving nerve terminal, is taken back

CHAPTER 1. Introduction 7-26 up into rhe parenr nerve terminal by the monoamine reuptake pumP ffan Dyke et a/.,1'982)' This prevents the accumulation of noradrenaline in the synaptic cleft and prolongation of a sympathetic fesponse. Cocaine binds to the monoamine reuptake pump and prevents the temoval of noradrenaline from the synaptic cleft. This results in a sympathomimetic response to the presence of cocaine. Under conditions of low sympathetjc tone, synaptic noradrenaline effectively controls the rare of its own release by activating presynaptic ot -adrenergic receptors ffamaguchi el a/., i,g77). In the presence of cocaine, sympathetic tone is elevated and this is insufficient to prevent accumulation of noradrenaLine in the synaptic cleft (Cousineau et a/,,1'986).

reuptake pu ts noradrenaline o receptor vesictes r4r 4 C 4 Receiving nerve terminal o O*O

O cocaine r noradrenaline

Fig. 1. 3 Sympathom¡met¡c act¡on of coca¡ne. Adaþtedfron Van D1ke, 1982. Top: as an action Potential is recePtors on the received at the synapse, vesicles of noradrenaline are released across the synaptic cleft to bind at noradrenalne in the surface of the receiving nerve terminal. To prevent the persistence of this signal all unbound cocaine binds to synaptic cleft is taken up by the parent nerve terminal via the monoamine reuptake pump. Bottom: the monoamine reuptake pump and prevents the teuptake of noradtenaline within the synaptic cleft' This allows terminal resulting in a noradrenaline to petsist and continuously activate noradrenalìne recePtors on the receiving nerve sympathomimetic response chatactetised by an inctease in HR and BP'

7-27 CHAPTER l lntrodaction axon membrane + r' Normal Operation of

lnactivation Gate Activation Gate Axoplasm

Sodium Channel Blocked by T Cocaine

O cocaine sodium

Fig. 1. 4 The local anaesthetic action of cocaine. Adapted from Van D1ke, 1982. Topz Cocaine can block sodium ' In the the transmission of sensory nerve impulses by changing the permeability of the axon membrane to sodium ions, which absence of cocaine a sensory impulse is passed along the axon by a change in permeability to charged)' reverse the charge difference between the axoplasm (negatively charged) and the axon membrane þositively the sodium Bottom: In the presence of cocaine the nerve terminal cannot be stimulated because cocaine blocks channel and Ptevents ion flux.

Cocaine can also block the ttansmission of sensory nerve impulses and cause czrdiac rhythm 1. a). d.isrurbances by changing the permeabiJity of the axon membrane to Na* and I{ (Fig.

1.10 Prepa-rations termed Cocaine is available in a number of different forms. Cocaine hydrochloride salt, colloquially .,sno.w" or "Coke", is often intranasally administered ("snorted"), it can also be injected (i.v. or i'p.) or taken ofâlly (Vlanschreck et al., 1,987;Lathers et a1.,1988; Om, 1,992;Dzs, 1'993). There afe two methods for the preparation of alkaloid cocaine. Cocaine hydtochloride salt can be mixed with buffered ammonia. The alkaloidal cocaine is extracted from using ether. The ether is then evaporated to produce a hatd,, crystalline substance known as "freebase" (Strang et al., 1'993;

'Warner, 1,993). Freebase cocaine does not decompose on heating and can be smoked (Om,1'992) which produces an extremely rapid and intense "hgh", similar in onset of effect to intravenous cocaine cocaine (Pierce er al., 1986; Manschreck et al., 1.987;Lathers et at., 1'988). Alternatively, solid hydrochloride can be combined with "baking soda" (sodium bicarbonate) and heated until a forms (Cornish et a1.,1.996). known colloquiaþ as"cf^ck". Ctack ptoduces a distinct "popping" mixed with sound when heated. Both ctack and freebase cocaine can be smoked in pipes or marljuanaor tobacco and smoked inacigarette flx/arner, 1,993).

7-28 CHÂPTER T Introdaction .,Cutting agents" added on the street to increase cocaine bulk complicate understanding the toxicology of cocaine. These include , , tetrâcâine, , , ("angel dust"), amphetamines, quinine, tâlc and strychnine (Garbet el al., 1,987;

Lathers et aL.,1,988).

1.1 I Metabolism Cocaine is detoxifìed by plasma and hepatic cholinesterases to water-soluble , which can then be excreted in urine (I{amendulis et ø1., 1996). The maior toute of cocaine metabolism in humans involves carboxyesterase-L (chE1) catalysed de-esterifìcation to produce benzoylecgonine little cocaine aPpears unchanged in the urine @E) (Stewart et a1,,1977;Brzezinski ¿/ at.,1.997). Very and most is excreted as the BE .

Carboxyest erase-2 (chE2) can also act on cocaine, producing ecgonine. Ecgonine is highly polar and almosr completely metabolically inert (Pindel et a1.,1,997). Ecgonine accounts forl¡eween32o/o and 49o/o of utinary metabolites.

Lt¡ I çO¡tiá* *

x f,'{. rþ cOüt{ CqMt |t

or+ OCOP?I |r ,1 coc¡ti¡€ tv COçt'r X OCçF¡

H

s5r*lûYl.EC6$ir${g u

(Vitti er Fig. 1. 5 Major metabol¡c pathways for cocaine med¡ated by carboryesterases. Source:

a1., \985)

7-29 CHAPTER l lntrodaction Small âmounrs of cocaine are N-demethylated by to produce norcocaine' N- accounts for only about 2-6o/o of cocaine metabolism (Stewart et al., 1'978; Inat].a,

198e).

H M! Må

H H wÛph

H

COCA¡NÊ t

Fig. 1. 6 p-450 mediated n-demethylation of cocaine to produce nor-cocain€. Source: (vitti er a1.,1985).

It is important to note that when is consumed with cocaine a new toxic metabolite is formed, cocâethylene. In the presence of ethanol some cocaine undergoes trânsesterifìcation with ethanol instead of with u/ater forming an active metabolite @rzezinski et a/., 1997; of Laizure et aÌ.,2003). Co-ingestion of cocaine and has been found to reduce the fraction cocaine dose converted to benzoylecgonine, and increase the formation of norcocaine Qan et aÌ', be implicit 1.999a). The same carboxyesterases involved in cocaine metabolism are also thought to in cocaethylene metabolism (Brzezinski ¿/ a/.,1997;Laizure et a1.,2003)'

1.12 presumably, due to the d.iffìculty of obtaining ethical approval for the use of cocaine in human subjects, very few studies have investigated pharmacokineúcs in man' Howevet, Chow and

âssociates report ge bioavailabiliq of 1,32L, t,,, of 48+13 minutes and 2.10L/min elimination ^ver clearance in five subjects given a 32mgintravenous dose of cocaine (Chow et aÌ.,1985)' This same to those obtained d,ose was administered byJavaid and colleagues whose reported values are similar et al', by chow et al (i.e. F=1.61L, tr,r=41.4-18.2 min, cL,u^=2.45L/min) flavaid et a|.,1978;Javaid 1,gg3).It must be noted that both of these srudies used subjects with previous histories of cocaine the kinetic profìle of cocaine administered by abuse. Javird et al also quantified the differences in intranasal the i.v. route compared to intranasal administration Savaid eî a1.,1,978). It was found that administration produced peak plasma cocaine levels in less than 30 minutes, and that plasma concentrarions remained elevated past 60 minures post inhalation. This resulted in a ptolonged chronotropic effect of cocaine, which only declined after approximately 40 minutes. Importantly'

1-30 CHAPTER l lntrodaction kinetic studies using rats have found that i.p. adminisuation of cocaine closely parallels the kinetics of inrranasal administration in humans fX/ilkinson et a1.,1980; Lathers et al.,19BB)'

Because cocaine is highly lipid soluble, peak levels are evident very rapidly in btain tissue, Experiments performed in rats demonstrate that cocaine rapidly crosses the blood brain barrier and d.istributes unequally in different brain regions Savaid et aL.,1,993). This unequal disposition Pattern may account for d.ifferential biochemical effects in different brain regions. Low levels of cocaine were found, in the nucleus accumbens and frontal cortex, whereas very high cocaine concentrations were discovered in the amygdala, caudate putamen and septum'

Oral cocaine adminisuation to humans results in a predictably long lag period until peak plasma levels are reached. In 7 human volunteers who had all previously used cocaine for recreational purposes cocaine wâs not detected in the plasma until 30 minutes aftet the dose was administered recorded 64+6.7 minutes. $Vilkinson et al.,1,980). Peak plasma coricentrations were ú

Co-ingestion of alcohol and cocaine has been found to modify the pharmacokinetics of cocaine. 20o/o Laizure et al found, that both cocaine and cocaethylene clearances are decreased by about when and cocaine in rats has been given with alcohol Q,aizure er at.,2003). Co-administration of ethanol found to increase systemic bioavailablity of cocaine by 37o/o when administered intraperitoneally

Qan et a1.,1.999b).

1.13 Cocaine CardiotoxicitY ,\ usual recreational dose of cocaine by the intranasal toute is about 100mg (R.esnick et a/.,1977; Grad.man, 19gg). The doses of cocaine reported in the literature and associated with fztalttt¡ vary widely. Plasma concentrations as low as O.lmg/ml and as high as 24 mg/ml have been associated with sudden death Cgl.tli et al., 1.985; Vitullo et ø/., 1,989; Simkhovich et a/', 1'994)' Plasma concenffation in post-mortem blood samples has been found to be unreliable in determining a toxic dose. This is because the tapid hydrolysis of cocaine in unpreserved blood and the site- dependent post-mortem release of cocaine from tissue stores results in large variation in concenffarion (Hearn et a1.,1.991). Perimortem blood samples have proven diffìcult to collect due to the rapid onset of death following overdose, further J-imiting âccess to reliable data on blood concentrations of cocaine.

7-37 CHAPTER l lntrodaction Chronic and acute administration of cocaine has been associated with a wide spectrum of adverse cardiac events including sudden catdiac death, cardiomyopatþ, hypertension, myocardial infarction, myocardial fibrosis, myocarditis, venüiculat tachyczrdia and fibrillatìon, catdiac hypertroph¡ atherosclerosis, thrombosis and aortic fuPtufe (Table 1. 5)

The causes of cocaine-induced sudden death are likely to be multifactorial. Clinical and experimental evidence exists for at least thtee mechanisms of cocaine cardiotoxicity in humans.

l. Reducedcotonaryflow ^. VasosPasm b. Thtombosis c. Arterioscletosis

2. Catdiac arrhythmias calcium flooding; ^. Inttacellulat b. Regional diffetences in the râte of rePoladsation'

j. Direct cardiac effecü such as cardiac hypertrophy and pathological effects

t-32 CFIAPTER l lntmdacrion System Advetse Effect Reference(s) Catdiovasculat Sudden cardiac death @auman et a1.,1.994;Shen et al., 19e5) Cardiomyopathy (Besse et a|.,1,997) Hypertension (Hollandet et a/., 1'997) Myocatdial infarctjon (Isner et a|.,1,986;Stmpson et al., 1986; Mittlemzn et a/.,1,987; Virmani er a1.,1,988) Myocardial contraction (Simpson et a1., L986; Osa.wz et band necrosis a|.,1,994) Myocardial fìbrosis (Isner et a1.,1,986;Dtessler et a/., 1990) Myocarditis (Virmani et a1.,1,988) Ventricular tachy cardta (Isner et a/.,1,986) Ventricular fìbrillation Q'{anji et a|.,1984) Catdiac hypetrophy (Brickner et a1.,1991'; Om et al', 1,993; I{arch et al., 199 5) Coronary vasoconstriction (I-ange et aÌ.,1989) Âtherosclerosis (Dressler et a|.,1'990; Om et al., 1,ee2) Thrombosis (Isner et a1.,1'986) Aortic rupture (Adkins et a/., L993; Baumgattner et a|.,1,997) Respiratory arrest CM.tli et a1.,1'985) Cerebtovascular Cerebral aneurysm (Levine et a1.,1990;Levine et al., 1,ee1) Cerebral infarction (I-evine et a/.,1990;Levine et al., Iee1) Central nefvous system Seizures $onsson et a/.,1'983; CamPbell, 1eB8) Cerebral Vasculitis (Ferenchick,1,991) Cerebral oedema QrJanli et a|.,7984) W.tIi et aÌ.,1'985) Delirium CV.tli el a1.,1'985) Paranoia W.tli et a1.,1985) Other hyperthermia CIfl.tli et a|.,1'985; CamPbell, 1 e88) flonsson et aÌ.,1.983;V/etJt et al., 1 e85) Severe weiqh loss (I(arch et al.,1,998) Table 1. 5 spectrum of toxic events attributed to cocaine abuse.

7-33 CH,\PTER 1. Introdøction (Colemzn et al', One of the most documented outcomes of cocaine abuse is myocardial infarction 1986;'\schereta/'' 1982;Howzrd,etal.,j.9|S;Pasternack eta/.,1'985;Simpson eta/',1'986;Nleiss, to explain reduced 19gg; Holla nder et a1.,7997;Wilson, 1998). A number of models ate avallalle cofonary flow potentially resulting in myocardial cell death.

1.13.1 Reduced coronarY flow

Vasosconst¡ictìon The Cocaine c rr câuse both direct and o(-adrenoceptor-mediated vasoconstriction' myocardial oxygen demand' sympathomimetic action of cocaine results in a dtzmztic increase in unmatched' The Under conditions of coronary naffowlng' oxygen demand and supply afe ^:rtery and subsequent resulting trâns-card,iâc oxygen difference increases the risk of cztdtac ztthythmia action of cocaine can involve a significant Pressor infarction @illman, 1990). The sympathomimetic cocaine abuse (Ädkins affect.The literature supports several cases of aortic rupture associated with the ability of cocaine to et a1., L993;Baumgartner el a1.,1997) Numefous case fePofts have noted (Osawa ef al'' 1994)' induce ischaemia in the absence of signifìcant athetoma or thrombus

use suggest that severely Reports of cerebtal ischaemia and vasculitis attributable to chronic cocaine Reduced regional cetebral reduced blood flow is not exclusive to the heart frededcks et a1.,1'991)' blood flow has been demonsttated in the fàtbrzrin (Stein et a1.,1993)'

Direct mechanism human coronary artery Cocaine has been shown to cause vasoconstriction in freshly explanted umbilical cord (Chokshi a/ rings and symparheric innewarion independent constriction, in human decrease in myocardial at.,19g9).Isolated perfused rar hearts demonstrate a phentolamine resistant catdiac tissue has flow in fesponse to cocaine. Gluteraldehyde fixing of cross-sections of this diameter to be made allowed a direct cotrelation between red.uced cofonary flow and cofonary response to concentfations of cocaine (Vitullo et a1.,1989). Decreased cofonâry flow rate (CFR) in dosing, has been thought to closely resemble plasma concenttations achieved with recreational

evidenced in isolated, perfused rabbit hearts (Simkhovich et a1.,1'994).

ol-mediated mechanism

Human ¡tadie¡ of coronary narrowing by cocaine has been The Cf-adrenocePtof mediated mechanism ^Ítery to substantiate a illustrated by a numb er of in uiuo studtes. Despite this, it has proven difficult Eisenberg et ø/ investigated conclusive link betwee n 2rrtervlr narrowing and myocardial infarction. echocardiography ventficulaf function immed.iately after intravenous cocaine using 2-dimensional

t-34 CHAPTER l lntrodaction in and ECG. No evidence for acute myocardial ischaemia or ieft ventricular dysfunction was found these subjects when administered intravenous doses of cocaine similar to those abused in recteationally. (Eisenb erg et al.,1,gg3). Howevet, it must be noted that this study was perfotmed current cocaine users who may exhibit tolerance to the cardiovasculat effects of cocaine' '\s echocardiograms were only recorded zt 2 and 7 minutes after infusion it is possible thzt cardiac complications occurted in the subacute period (greater thanT minutes)'

reductions However, a number of clinical studies have shown a clear ability of cocaine to produce in coronary diameter. A small dose of intranasal cocaine administered to patients undergoing and cardiac catheterisation for the evaluation of chest pain was found to induce a pressor reduction in coronary diameter which chronotropic response, concomitant with an 8-1,2o/o ^rtefy returned signifìcantly reduced coronâry sinus flow (I-ange er a\.,1.989). Phentolamine administration BP, HR and coronary diameter to baseline,

intranasal cocaine pmglkÐ in cotonary disease patients Moliterno et alstudiedthe effects of ^rte:y undergoing angiography, and found decrease in diameter of 7*1'o/o in non-diseased ^fi ^ve:.:age Flores el coronary segments, and an extra2o/o in diseased segments (l\'{oliterno et a/., 1'994t¡)' ^rtery et a/',1990)' alperformed angiography on 1B patients undetgoing evaluation for chest pain frlores cocaine solution was The decrease in luminal d-iameter following administration of an intranasal segments of the left coronary than non-diseased found to be signifì cznùy greater in diseased ^rtery (meantSD, 29*23o/o vs 13*8%o' p<0.05)'

the metaboütes of Brogan et al demonstrâted that the vasocorìstriction can also be attdbuted to of chest pain were cocaine (Brogan et a1.,1.992). Patients undergoing angiogtaphy for the evaluation was administered either intranasal saline or cocaine IHCI (2mglkg). Significant vasoconstriction At 90 noted at 30 minutes post cocaine administration, which returned to baseline by 60 minutes' which corresponded minutes, piasma levels of benzoylecgonine and ethyl methyl ecgonine peaked, effect decrease in epicard.ial coronary diameter. Presumably, the vasoconstrictive to a further ^rtery angiographically of cocaine metabolites can explain the ischaemic effects of cocaine observed Nademanee et al several hours after reported cocaine ingestion (Ascher et a/.,1'988). Interestingly, withdrawal to find undertook 24-hourHolter-monitor recordings from patìents undergoing cocaine et al',1989)' significant ST elevation during the first 2 weeks of the drug-ftee period Q\ademanee is capable of causing AII episodes of sT elevation wefe âsymptomatic, suggesting that cocaine 'silent ischaemia' long after removal of the drug'

1-35 CHAPTER 1. Introdaction Aninal studies myocardial blood flow and circumflex diameter, before, and Hale and colleâgues measured ^rtery after, a high dose of cocaine (7 or 10 mg/kg, i.v. bolus) or saüne in pentobarbital anaesthetised dogs (Hale et a1.,L989). Artery d.iameter was found to decrease by 15+4o/o in response to cocaine, concomirant with a subepicardial tegional myocardial blood flow reduction of 22%o. 'N separate group of animals, also administered, cocaine, showed evidence of decreased left ventricular function (reduction in HR and left ventricular dP/dt, and an increase in left ventricular end-diastolic and pressure). Hayes et al to find a dose-response relationship between cocaine dose ^ttempted minutes cross-sectional atea of the coronary arteries. Constriction was found to be maximal at 60

afterabolus injection of 0,1,3,6,9 mg/kgcocaine, correspondingwith a2*L0,I7+8,45+9,33+B was made in this and, 46t1,0o/o. red,uction in coronary diameter (Hayes et al., 1'991)' No attemPt study to quantify the progression of myocardial ischaemia resulting from vasoconstriction' i'v') Investigation of the coronzry sinus oxygen content following a low dose of cocaine (1 mg/kg,

in conscious dogs determined that while coronary vascular resistance increased by 24-l3o/o, and the arteital myocard.ial oxygen consumption increased by 41!9'/o this was offset by an increase in et a/',1993)' This oxygen cofitent þy 2.8+0.3 vol%) and coronary blood flow (13+3%) (Shannon by supply suggests that while oxygen demand is increased with cocaine, demand is matched 14 which through an increase in afterial oxygen. ,\ signifìcant rise in haemoglobin from 12 to g/dl- observed effect on persisted for 60 minutes after cocaine injection was thought to conttibute to the uncertain arterial oxygen. The mechanism of this haemoglobin effect was not elucidated. It is of whether the observed. increase in a:rcenal oxygen in this study is conserved at higher doses

cocalne.

effect of It is probable that the vasoconstrictive effects of cocaine are t-ime dependent. A biphasic signifìcant intravenous cocaine has been noted in pigs (Zimring et a/.,1,994)' An initial, brief, but increase in coronary blood flow (CBF) was noted, followed by a more sustained decrease

4 minutes following infusion, coronary diameters were found to decrease' (17.0+3.3%o). From ^rtefy initial vasodilat-ion' This suggests thât the vasoconstrictive response to cocaine only occurs aftet an to its diverse It is possible that the dual-effect of cocaine on coronary vasculature can be atttibuted effect of pharmacology. It is probable that the vasod.ilation is caused by the local anaesthetic arteries (Friedrichs cocaine and the vâsocoristriction by the c¡(-adrenergic response of the cotonary and cocaine produced et al., 1,99¡;Benzaquen et at.,2OO1). Friedrichs et al found that both lidocaine coronary blood a significant decrease in coronary perfusion pressure, under conditions of constant (Friedrichs e/ flow, suggesting that this transient vasodilation was due to a Iocal anaesthetic effect

a/.,1,990).

r-36 CHAPTER l lntroduction of Although most studies investigating vasosPâsm have indeed observed small amounts to induce myocardial vasoconstriction it is uncertain whether the extent of constriction is sufficient induced by cocaine ischaemia (Benzaquen er al.,2OO1). It is probable that the degree of constriction denudation of is much gre^terin diseased subjects. Miniature pigs subiected to regional endothe[al cororrary and fed a high cholesterol diet displayed myointimal the left anterior descend.ing ^rtery, Denuded thickening at the denuded site, which increased the constrictive effect of cocaine' native ones (4815%, cofonafy arteries displayed a signifìcantly gteater constriction (5915%) than

P<0.05) @gashira et a1.,1997).

Thrombosis formation A number of case reports on have demonstrated thrombus Stenberg eT al',1'989)' It is not associated with cocaine use (Simp son et a1.,1'986;Virmani et al',1'988; for the coronary arteries of unusual fot sudden catdjac death to be attributed to cocaine abuse, yet that the fatal event may have the victim to be free of thtombus. In some of these cases it is possible (R.ezkalla er al'' 1'990)' been caused by a clot, which subsequently underwent thrombolysis

plasma from cocaine- Rezkalla investigated cocaine induced platelet aggregation in platelet-rich and collagen-induced platelet naïve patients s.ezkalla et a/., 1,993). Spontaneous, adrenaline cocaine treated samples' In tesponse aggtegation tests produced, no difference between control and an increase tn aggreganon in the to adenosine d-iphosphate induced platelet aggregztton, there wâs diphosphate (ADP)' platelets incubated with cocaine in the pfesence of a 1'¡tg dose of adenosine platelets to certain tory stimuli' This suggests that cocaine may increase the sensitivity of ^ggreg clinical events' However, in uitroplatelet studies do not always accurately correlate with

with other The incubation of platelet-rich plasma does not maintain the interaction of platelets i(ugelmass et al blood cells such as leucocytes of erythrocytes (Heesch et al', 2000)' Therefote as a marker of platelet investigated human platelet activation by examining P-selectin expression blood with cocaine (1OpM acrivation in whole blood (I(ugelmass et al., 1,993). Incubation of whole (range of increase, to 13mNf), induced signifìcant increases in platelet associated fibrinogen 1L2t240/o). However, it must be noted 45*1.2o/o to j,25+.40o/o) and P-selectin expressi on (36*150/o to granules, and the most that only a minor provocation causes a release of stimulants from platelet Yirmartt ef al potent platelet stimulators afe othef platelet ptoducts @enzaquen ef al', 2001)' autopsy patients with cocaine demonstrated serum cocaine concentrations of up to 80¡rM in probable that while the higher mM associated deaths (cited I(ugelmass el at.,1.993),It is therefore I(ugelmass et alsttlfprovide compelling cocaine doses used in this study have little clinical relevance

7-37 CHAPTER l lntroduction expression at pM evidence of an increase in platelet associated fìbrinogen and P-selectin concentrations of cocalne

ìnfusions of cocaine A study performed in New zealanà \Øhite rabbits given daily intravenous 6 ot 1'2 weeks of pmg/kS) or saline, examined segmenrs of zorta for histological changes following of aortic segments from cocaine treated rabbits ffeatment @,ichhorn et at., 1992). The endothelium genetating a favourable produced high levels of the potent vâsoconstfictor, thromboxane 'N2, milieu for thrombosis.

using flow cytometry, Rinder In uiuoinvestigations of the affect of cocaine on platelets are limited. P-selectin) in whole blood from et al obsewed the percent of activated platelets (those expressing was found that chronic cocaine usefs comPared to drug free controls S'inder et al', 1994)' It platelets compared to cocaine users had a significantly greatet (p=0'01) pefcent of activated level of P-selectin controls due to a subset of cocaine users who showed an unusually high uitro platelet activation or expression. Plasma from these same subiects showed no effect upon in experiments whefe zggregatton either d.irectly or in concert with platelet ' However, in mean pefcent of activated platelets subjects received a blinded infusion of cocaine or - the c['-gtanule release must was found to dse sharply for both tfeatments. This suggests that in uiuo be observed between zz occuf by an ind.irect mechanism, and highlights the differences that mzy alinvesttgated the platelet affect of cocaine uiuo anð, i1 uitroinvestigations of aggregability. Heesch et crossover study' in 14 healthy, cocaine-naive subiects in a randomised, double blind Quantifìcation intranasally) or placebo, of the platelet specifìc protein in blood following either cocaine (2mg/kg, thromboglobulin at 120 revealed significant cocaine induced increases in platelet factor 4 and B formztion increased minures following cocaine administration (Heesch et at.,2000). Mictoaggregate 40 and go minures following cocaine administration. Bleeding time decreased signifìcantly ^t This suggests that following cocaine, but the change was not observed to be statistically signifìcant' cause platelet activation, c[- exposufe to a low dose of cocaine çzmg/kÐ in naive subiects can Rindet et al ot¡sewed that granule release and microa ggreg te formation, It is intere that in chronic cocaine users activated platelets persisted for almost 6 hours after cocaine administration in naive subiects' (Rinder et al., 1,994). Heesch et al observed a rapid clearing of mictoaggregates system usually involved in This suggests rhat chronic cocaine use mây inhibit the reticuloendothelial temoving microaggtegates (Hees ch et al', 2000)'

thrombosis and The affect of cocaine on the plasma constituents involved in endogenous that cocaine (2mglkg) thrombolysis has not been well characterised. Moliterno et al found produced a significant administered intranasally to patients undergoing catdszc cathetedsation

1-38 CHÄPTER l Inîroduction ation in plasminogen activator inhibitot eAI-1) activiry compared to saline controls (Nlolitetno tt.,1,994a).It is impottant to observe that PAI-1 is not specifìc to platelets, and is also produced

muscle cells (Heesch et a/.,2000). It must ¿ hepatocytes, mesothelial cells, monocytes and smooth lso be noted that this study enrolled patients with suspected coronary disease.

I(olodgie et al investtqated the arteries from patients dying from cocaine-associated sudden death and who displayed acute coronary thrombosis (I(olodgSe et a1.,1,991). A strong correlation (r=0.68) was found between the extent of luminal narrowing to the number of mast cells in coronary àrlery sections with thrombosis. This relationship was significantly stronger than the same correlation in artedes from pat-ients where the cause of sudden death was attributed to signifìcant coronary thrombosis and who had no history of cocaine abuse and a negative toxicology screen. These results suggest that cocaine m y cause mast cell prolifetation, which results in the release of pro- thrombotic mediators.

Both in uiuo and in aitro studies r to support the pro-thrombic effect of cocaine , either through ^ppe a direct action on platelets or through an inditect mediator. Care must be taken in comparing in uiuo data from cocaine-using and cocaine-naïve subiects, as differences in clearance of microaggregates may altet the number of cells remaining in the circulation displaying P-selectin expression, In aggregometer based studies it is expected that platelet-rich plasma in the absence of interactions with leucocyres and erythrocytes will display a different degree of coagulabiliry compared to whole blood.

Aneiosclerosis Ä limited number of studies have reported the prevalence of arteriosclerosis amongst cocaine users. It is presumed that coronâry plaque formation results from eithet a direct effect of cocaine, or from a cocaine related stress that acceletates cofonary artefy disease from other causes Qressler et al.,

1990). For example it is probable that growth factors released from Cr-granules could indirectly acceletate the deveiopment of atherosclerosis.

Many investigations of the coronary effects of cocaine are corrcerned with users that have presented to hospitals for serious medical conditions or who have died. As a result they probably over-estimate the prevalence of coronary plaque formation amongst cocaine abusers Qrcssler et al.,

19e0).

l-39 CHAPTER l lntrodaction patient sustaining simultaneous Stenberg et al descrtbe a fatalmyocard.ial infarction tn a 38-yezr-old intravenous cocaine use acute thrombosis of two major epicard.ial coronary arteries shortly after severe cofonafy (Stenberg et aÌ., 1989). Likewise, Simpson et al repott autopsy fìndings of thrombosis in a 21- obstructive lesions, as a result of chronic intimal ptoliferation and acute platelet et al descrùte a fatal cardtac year-old man, one hour following injection (Simpson et at.,1'986). Isner Äutopsy revealed event tn a 3l-year-old man, with a history of drug abuse (Isner et al', 1'986)' of the I-AD coton a point where it had narrowed by 90o/o through thrombotic occlusion ^ry ^t formation. The right cofonary was reduced in diametet by 50%. plaque ^ftery

necropsy. It was found that Dressler and associates examined hearts from 22 cocaine addicts at the 4majorepicardial 36o/ooffatalitiesdemonstratedplaqueformationgreater thznT5o/oinoneof meaî of 1.8 narrowed afteries per subfect @ressler et al', 1'990)' Om and cofonary arteries, ^ clinical presentation ptompted associates conducted an investigation into patients in whom the 1'992)' Urine toxicology refernlfor coronary angSography by the attending physician (Om et at', these patients 600/o hað cofonary scfeen was positive for cocaìne in all 33 subiects analysed. of had signifìcant cAD, with disease (cAD). Ir was found that 4oo/o of the patients studied ^,rtefy infarction tn1'2 of 2l}o/odiamerer stenosis. In addition there was enzyrnatic evidence of myocardial US hospitals with cocaine 33 patients Q6n.In a more fecent study of 70 patients presenting at subf ects met the criteria for cAD' associated myocatd-ial infatction, it was found that 670/o of these than or equal to 70%' Mittleman and of these patients 95o/o had, at least one stenosls of greater cocaine use (À4ittlemzn et al', \Welti repor t 24 fatzl:ttes where there was toxicological evidence of Forty-two of cases 1987). Sixty-three percent of cases displayed significant arteriosclerosis. Percent estimated to B0o/o,21o/o displayed 90o/o ot more occlusion' d.isplayed stenosis ^t7o

mechanisms It is notable that cocaine cân cause a reductjon in coronary flow by the same vasospâsm. Chronic use of attributable to AS, Both ÄS and cocaine have been found to induce both drugs has been associated with thrombosis and arteriosclerosis'

I. t 3.2 Cardiac a rrh¡thmias pathers et a1.,1988; Osawa Cardiac archythmia is commonlY associated with cocaine administration Littmann et at',2000)' number of hypotheses et a1.,1994;Shen el al., 1.995; Gamouras eî al',2000; '\ have been forwarded to explain cocaine-mediated arrhythmogenesis.

r-40 CHAPTER 1' Introdaction fn tra cellular calcium flooding hyp o thesis levels in fertet Cocaine has been shown to cause significant increases in intracellular calcium release of calcium from the sarcoplasmic papillary muscle @erreault et al., 1990). Significant et al', 1993) It \s reticulum has been shown to result from cocaine administration to rabbits (Iomita ' cr and ptobable that cocaine elicits this "calcium flooding" through stfong stimulation of B inotropic and adrenoceptors (Billman, 1995). Ä number of studies have shown that the Branch and I(nuepfer chronotropic effects of cocaine can be attenuated using CX, and B-antagonists. on the response to cocaine examined the effect of a range of Cf and B adtenoceptof antagonists found to elicit an immediate and sustained (Smg/kg, i.v.) @ranch et al.,1,gg2). Cocaine was þeak) virtually eliminated by alpha- pressor response with a simultaneous decrease in HR. This effect was zlpha-2 adrenoceptofs to the 1 adrenoceptor antagonists, Evidence was found for a contribution of to attenuate the vasoconstrictof effect of cocaine. Beta-l adrenoceptor blockade was found squirrel monkeys (Schindler ø/ bradycardic effect of cocaine. Similar results have been reported in

a/.,1992).

membrane voltage during Large increases in intracellular calcium can produce oscillations of potential reaches threshold diastole (during repolatisation of the action potential). If membrane dudng these oscillarions, a repetitively sustained action potential results (oscillatory in coupled extrasystoles and afterdepolarisation) (Billman ef øt.,1988), This has been found to result ovedoad câuses the initiation and paroxysmal tachyczrdia @ozìer,1943).It is probable that calcium maintenance of ventticular fìbrillation'

arrhythmia formation it Because of the importance of intracellular calcium telease in ^pprlferft the risk of arthythmia forma[ion' would be expected that blockers would dectease fìbrillation in dogs Billman and Hoskins demonstfated that venpzmil can Pfevent ventricular et al',1988)' No arrhythmia was subjected to âfr exefcise plus ischaemia plus cocaine test (Billman feline cardiac myocytes observed in the absence of cocaine. \X/ork conducted in isolated resulting in extrasystoles (Ilmura demonstrates that cocaine can prolong action potential dutation found to be enhanced by catecholamines er a1.,1.992). The frequency of arrhythmia formation was cocaine induced arrhythmia and attenuated by . Nimod.ipine has been shown to pfevent in squirrel monkeys (I\[anger et al.'1'989)'

ovedoad, and has been noted in a Myocard.ial contfaction band necrosis is attributable to calcium hypercontraction of some number of clinical case fepofts, Contraction bands ate formed by Tazalazt et some sâfcomefes ¡}lat arc toln (vitmani et al', 1988)' safcomefes, interspersed with ^pa:it

7-47 CHAPTER 1Introduction myocardial conuaction band al investtgated 30 cases of cocaine associated fatalities and found than controls (fatalities necrosis in93o/o of subjects, a significantly greater frequency of incidence of the contraction bands due to - overdose) Qzzelzar et a1.,1.987). The diffuseness The presence and correlated d.irectly with the amount of cocaine found in the blood at autoPsy' the urìne and blood, the number of contraction bands was independent of other drugs found in resuscitation'Yirmari et al number of sections of myocardium examined and a history of attempted dying from natufal causes found evidence of myocard.ial conttaction bands tn 23o/o of patients et a/',1988)' The same whose toxicology scfeen was positive for cocaine ot its metabolites ffitmani positive for cocaine' parhology was noted in 3 out of t homicide victims with plasma testing

C a te ch oI atnin e in d u c e d anh y thtni a h yp o th e si s the neural efflux The abilìty of cocaine to inhibit the neuronal re-uptake of noradrenalìne, increase results in a signifìcant of noradrenaline and release catecholamines from the adrenal medulla the squirrel monkey that increase in circulating catecholamines. Trouve et al demonstrated in direct result of increased plasma increases in HR and Bp with cocaine administration were the et al., 1'990)' These changes concentrations of dopamine, norad,renaline and adrenaline (Írouve experiments Trouve et a/ wefe accompanied by transient episodes of arrhythmia' In later administratjon to SD rats demonstrated a similar elevation of catecholamines following cocaine (Irouve et al', 1991)' (60mg/kg, i.p.), which induced increases in BP anð' caràiac arthythmia a combination of enalapitlzt Administration of (calcium ) alone or returned catdiac rhythm to (angiotensin converting enzyme) anð' dtazepam decreased BP and with was normal. A similar effect on cafdiac rhythm and cardiovascular variables Nitrendipine administration in observed in cocaine treated squirrel monkeys (Idanger et at.,1989)' time and increase the conjunction with cocaine infusion was found, to significantly inctease survival lethal dose of cocaine in SD rats (Itouve et al',1986)'

Local anaes th esla hYPo th esi s and channels to cocaine can block both sodium (Shakalis et al., 1967; Pitts e¡ at., 1'989) channels has been found to be delay repolarisation. The interaction of cocaine with cardiac sodium pH dependent (Crumb et a1.,1995)'

ability of cocaine to inhibit The eT intewal on the ECG has been used as an indic ator of the could allow for excitabiìity repoiarisation. Region al cardiac differences in the ràte on repolarisation (I(abas et al',1990)' Tissue to recover in one area ofthe heart before others have fully repolarised tissue may fe-excite the fully that has remained in a depolarised state longer than the surtounding

repolarised regions such that a Premâture impulse is generated'

7-42 CHAPTER l lntrodaction cocaine remain contradictory' while a Stud.ies on the heterogeneiry of repolarisation caused by a canine model of cocaine toxicity' small number of studies have shown QT interval lengtheningin most probably related to the small dose human studies have failed to show this same effect. This is of cocaine that clinical studies are ethically restricted to using.

increased the canine QT interval Billman and Lappi found that lmg/kg, i.v. of cocaine signifìcantly 2 studies using doses of 3 and 4 mgf kg' t"v' (Billman et aÌ.,1.993). This outcome was confìrm ed.by et 1'992)' Schwartz et al obsewed respectively in dogs (Iftbas et a/., 1.990; Temesy-Atmos at', a bolus dose of cocaine (Schwartz elevations in the PR, QRS and QT interval of dogs administered et a1.,1989)

dogs, human data remains equivocal' Despite the observation of cocaine induced arrhythmia in administered cocaine caused an increase in Daniel et al found.that althou gt't 2mg/kz or inftanasally (Daniel et al'' 1995)' Lange HR in human subiects, no change in ECG pafametefs were observed administration of the same cocâlne and associates also noted no significant change in ECG with to hospital with a history of cocaine use in dose (Lang e et a1.,1989). A study of 45 patients admitted pain and those without anginal the iast 24 hours found that both subiects presenting with chest et al', 2000)' The frequency of pain had prolonged QT, QTc and QTc dispetsion (Gamoutas pain group than the pain free repolarisation abnormalìties was significantly gfeater in the chest fibrillation wete observed in patients subjects. Additionally, three examples of lethal ventricular with chest pain.

1.13.3 Direct cardiac eÍTects

Ifi s tologic aI o b s ewatí on s use, and found thzt 32o/o of these Dressler examined 22 fatzltttes d.irectly attributed to cocaine '$íelder (Dressler et al', 1990)' found that subjects had 1 of mofe foci of ventficulaf wall necrosis and 10-tl\4) displayed adult primary myocardial cell cultutes exposed to cocaine (10¡M as an hour after exposure, as well as vacuolisation, granulation and pseudopod.ia formation as eady (I-DH) telease' but did not depressed conttactility and significant lactate dehydrogenase et al'' 1'992)' significantly reduce cell viability (Welder, 1992; Welder

Morphological changes

Høman stttdies previously considered' Left ventricular The dangers of cardiac hyperttophy have aheady been om et al undertook hypertrophy in cocaine usefs has been extensively reported' ^fl users (Om et al'' 1'993)' Exclusion cdteria echocardiographic study of 58 normotenslve cocaine work' Mean values fot both men and included valvulat abnormalities, HfV or excessive physical

1,-43 CHAPTER 1' Introdacîion (men 1'1'2!41 vs 83+23 P=0'001; women women wefe gfeater than normotensive controls 8m/m2' was found between the route of administnnon 110+53 vs g2t14 gm/m2,p=0.037). No correlation these findings by performing an autoPsy and the pfesence of hypertrophy. I3rch et al con1rmed studycomparingtheheartweightsinmaletfaumafatalitiestestingpositiveforcocainewith (I(arch et al', 1995)' Cocaine positive fatalities testing negative for cocaine and historical controls higher heart mass than cocaine fatalities (375+82 g) were found to have a significantly þ<0'01) attempt The same changes wefe not evident in women' No negative Q31!54g, p<0.01) controls. doses abused, nor for the individual Pâtterns of was made to control for the magnitude of cocaine cocaine, and its metabolite concentrations and abuse. In a furthef fetfosPective autopsy stud!, of lethal trauma (eg gun shot wound' bufns' ofgan weights wefe compared in cocaine users dying (I

Aninal Staàies (4mg/kg' i'v'' twice daily) for 3 weeks Sutliff et al rcport that rabbits administered cocaine aortic wet weight without a cortesponding demonstrated a significant increase in cardi ac and' alÈo demonstrated that expression of the c-fos increase in body weight (Suttiff et a1.,1,996). They following a single i'v' dose of in the increased significantly 60 mìnutes pfoto-oncogene ^orta induced increases in wet weight of the f tlne :.t' cocaine lamg/kg). Besse et aÌ measured, cocaine et al'' 1'997)' Atrial and changes in expression of encoding catdtzc @esse secretion under conditions of cardiac natriuretic rzctof (ANÐ has been shown to have increased Twenty-eight days tfeatmentwith cocaine hyperttophy (À{ercadier et a1.,1.989; Marsh et a1.,1998). weight' significantly elevated ANF (4Omg/kg, i.p./daV) signifìcantly increased left ventricular the heavy chain compared to saline secretion and caused an increase in expression of B-myosin to elevated BP as well as hypertrophy controls. Although, increases in ANF cân occur in response observed by Besse et al et al., L99O;Pitts ¿/ al', 1998), the increases @oltin et al., 1990; Lange '\NF BP effect of cocaine' et al found were found to occuf in the absence of an appreciable Johansson chronic infusion, as a result of functional and that BP tetufns to normal, 5 days after beginning of normal 1'9gZ)' Chronic cocaine users will often have d.ispositional tolerance flohansso n et ø1.,

systolic pressure @rickner et al',1'991)'

1.-44 CHAPTER 1' Introdøction hypertrophy There are a number of hypotheses linking cocaine abuse and cardiac

1) The O,adrenergic stimulation h¡rothesis: Elevated catecholamine levels have neonatal rat been linked to LVH. Simpson demonstrated that NA administration to cell protein myocard.ial cell cultute resulted. in a significantly gteater accumulation of compared ro untfeared cells (Simpson, 1985). This hypertrophic effect was and prazosin)' significantly teduced by Cr-adrenoceptof blocking agents (terazosin Meidelt el aÌ demonstrated in the same cultufe system that NA increases the rates production of myofibrillar protein without signifìcantly altering degradation using (Nleidell et al., 1.986). Unfortunately, because these studies wete conducted anabolic neonatal cells, it is not known whether adult cells will show the same

effect.

chronic, subhypertensive levels of NA have been shown to cause LVH in dogs blockade on cardiac hypertrophy þzks et at., L973). The affect of O(-adrenoceptof 1989)' induced by aortic banding has been studied in guinea pigs (Iamai et al', no effect' It Bunazosin was found to pfotect against LVH, whereas a B-blocker had a lowering of afterlozd is not understood whether this protective effect results from

ot a reduction in myocardial ptotein synthests'

It is uncertain what role ct-adrenergic stimulation plays in human catdiac is very low hypertrophy 2s the density of c[ receptofs on human myocardial cells 2oo/o of patients presenting (furlapaty et at.,1985). Shub et al obsewed LvH in only abnormally with adtenal medullary rumouf þheochromocytoma), which produced high catecholamine levels (Shub et al',1'986)'

2) The intetmittent BP elevation model: Even small doses of intravenous cocalne 1990; Lange et câfi cause a significant, transient increase in human BP (Foltìn et al', may sefve as a al., 1,990; Pitts et at., 1,998). Such intermittent increases in BP physiological basis for the production of cardiac hypertrophy'

can be found rn Devereaux et al showed that LVH as detected by echocardiography 1990)' a signifìcant number of patients with mild hypertension (Devereux, to exercise Normotensive subiects who have an unusually large systolic fesponse et al',1990)' Out have been found to have a gfeater probabiliry of LVH (Gottdiener

7-45 CH,A.PTER 1' Introdø ction of 22 men v/ith an abnofmal ptessure fesPonse to exercise, 640/' of these were found to have LVH. This suggests that transient increases in BP (such as those and that ptoduced by cocaine) may be suffìcient to cause câfdiac hyperttophy, LVH in resting hypetension is not a prerequisite for LVH. Brickner et al found normotensive cocâine users (BrickneÍ et al',1991)'

3) Increased pfoto-oncogene expfession: It is possible that cocaine induced c-mJtc increases in circulating noradrenaline can directly induce the proto-oncogenes et al anð, c-fos which afe fesponsible for c^rðt:Lc gfowth' I(olbeck-Ruhmkorff fat heafts, observed that mRNA from c-m1c and c-fos was absent in freshly excised in the and baseline activity was only observed after heatts had been perfused with working mode (I(olbeck-Ruhmkorff ef at.,1'993). Hearts, which were perfused and c-fos after 90 noradrenal,ine, had a 3.8-fold increase in the expressiQn of c-m1c has been mìnutes over baseline levels. Chronic cocaine administration to rabbits blotting found to câuse a time-dependent increase tn c-þs in the âofta using'Western noradrenaline to isolated, (Sutliff et a:.,1.996). Zimmer found that administration of aftefload caused perfused, working fat heafts, combined with an increase in pre-and ptonounced, and to the c-fos and c-m1c mRN,\ signal to appeâf earliet, to be mote that the persist for a longer period of time (zimmer, 1'997). This suggests of combination of rwo hypertrophy-inducing stimuü may induce a greàter degree in athletes cardizchypertrophy than either alone. This may be especially important dramatic increases in abusing cocaine, where intensive exercise is already causing pre and afterlozd.

7-46 CHAPTER 1' Introduction 1.14 Conclusion and AS, very few Despite the similarity in the spectrum of cardiotoxic events attributed to cocaine more studies have investigated the catdiac consequences of co-abuse of these drugs' Far than for AS' experimental evidence is available for potential mechanisms of cocaine cardiotoxicity clinical investigation of the cocaine-,\s combination is hampered by confounding factors formulations by multiple associated with ,\S use, such as poof diet, strenuous exetcise, multiple magnitude foutes, "drug free holidays", stfeet contaminants, and uncertainty over the dose should provide the associated with fatalities. Accord.ìngly, experimental studies in animals free of the mechanism for investigating the cardiovascular consequences of this drug combination studies confounding factors in human subiects. However, there have been only three experimental (Íseng et al',7994; which have investi gated apotential add.itive cardiotoxic effect of these two drugs in this thesis were designed to Phillis et a1.,2000;Togna et a1.,2003). For this feason the studies and its affects in combination investigate in detail the card-iovascular affects of the ,\S, nandrolone, perfusion' The general with cocaine treatmerit, in normal rats and in rats with compromised czrdiac such as polypharmacy and aim of this thesis wâs to address some of the issues raised in this chapter and non-genomic pre-existìng cardizcpathology. As AS have been shown to display both genomic chronic mannef' effects it was necessafy to administer nandrolone in both ân âcute and

1.-47 CHAPTER 1' Introdøction 1.15 Aims

\X/istar 1. To determine whether our previously reported catdiovascular effects of cocaine in the strain of rat extend to the Sprague-Dawley strain of rat'

2. To determine if the previously reported enhancement of the cardiovascular effects of cocaine by chronic nandrolone pre-treatment in the conscious rat occurs following acute nandrolone tfeatment.

of 3. a. To determine whether acute or chronic nandrolone treatmerlt exacerbates the effects myocardial ischaemia/reperfusion in the anaesthetised rat.

b. To determine the effects of acute or chronic nandrolone treatment combined with acute tat' cocaine administration in the ischaemic ot reperfused myocardium in the anaesthetised

the extrâneuronal 4. To detetmine, in an isolated rat heart model, the effects of nandrolone on uptake of noradrenaline'

1,-48 CHAPTER 1 Introdaction 1.16 Hypotheses i,. Cocaine has identical dose-related cardiovascular effects in conscious Albino Wistar and Sptague-Dawley rats.

,) Acute nandrolone treâtment potentiates the cardiovascular effects of cocaine in conscious rats'

3. Acute and chronic nandrolone treatment exacerbates the effects of myocardial ^. ischaemia/reperfusion in the anaesthetised tat'

the ischaemic b. Acute and chtonic nandrolone treatment exacerbates the effects of cocaine in ot reperfused myocardium'

isolated r t he rt tî a 4. Nandrolone inhibits the extraneuronal uptake of noradrenaline in the concenttation dePendent mâffier.

1-49 CHAPTER 1. Introdøction 1.17 References Adkins, M. s., Gaines, sø. E., Anderson, w. r\., Laub, G. w., Fetnandez, J. and McGrath, L. B' ,,Cbronic '(JnasaaÌ Ann Thorac (1,993). þp, o Aortic Di¡section: An Comþlication of Cocaine Inhalation." Sutg 56: 977 -9.

in Alen, M., Reinila, M. and Vihko, R. (1985). "Rerponse of Serøm Horrzones to Androgen Administration Power Athletes. " Med Sci Spotts Exerc 77 (3): 354-359 '

and Acøte Schilophrenic Episode."J Clin Annitto, ¡Ø. J. and Layman,\ø. ,\. (1980). "Anabolic Steroids Psychiatry 4l@: 1' a3-4.

Appleby, M., Fisher, M, and Marrin, M. (1994). "Mltocardial Infarction, Hlperkalaemia and Ventricalar f)ig roiaU in a yoøng Mak Bo þ-Bai/der." Int J Card ol 44: 1,7 1, -17 4.

Cardiac Arrest' Ascher, E. K., Stauffer, J. C. and Gaasch, \ø. H. (1988)' "Coronarl Artery Spasm, Acøte Mlocardial Transient Electrocardiographl, p Itrtores and Stanned Mlocardiam in Cocaine-Associated Infarction." A- J Cardiol 61,(1,1): 939 -41. Staþ of Røt Azevedo, I., Bonisch, H,, Osswald, W. and Tredelenburg, U. (1983)' "Aatoradiograpbic Heaús nr,6und with 3b-Isoþrenaline." Naunyn Schmiedeburg Arch Pharmacol 322:1'-5'

of Neuryyal Uptake of Anines Azzaro, A. J., Ziance, R.J. and Rutledge, C. O. (1974). "The Imþortance Brain Tissøe." Pharmacol Exp Ther þr Ampbetanline-Induced Ríhorc of 3h-Nlrepineþbrine from Isolated J 189(1):110-8.

E. and Bremnet, w. "Metabolic Bagatell, c. Heiman, J. R., Matsumoto, A. M., Rivier, J. J. 0994) ' J., Endocrinol Metab ori Brhorioiral Efects o¡ fl4i-Oon, Exogenous Testosterone in Healtbl Men! J CIin 79(2): 561-7.

Abuse and Bahrke, M. s., Yesalis, c, E. and Browet, I( J. (1998). "Anabolic-Androgenic 'Çteroid pet'onnanæ-EnhancingDrugs amongAd.olescents." Child Adolesc Psychiatr Clin N Am 7(4): 821'-38'

G' and Fellin, R' Baldo-Enzi, G., Giada,F.,zuJtzrú, G., Batoni, L., vitale, B.,Enzi, G., Magnanini, ,,Liþid Self-Adrn'n'stration of Anabolìc (1990). and Aþoprotein Mod.fications in Boþ Bwilders During and after S teroids. " Metaboüsm 39 (2) : 203 -208'

Rich , A' L' (1988)' "A Comparison of Boþ and Organ ll/eigltts' ters, in Target Organs of Røts Giuen Combinations of Exercise, and "A-J Sports Med 16(4): 397-402'

R, (1994). "cocaine-Related sadden Bauman, J. L., Graw., J. J., \X/inecoff, ,\. P, and Hariman, J. Clin Pharmacol 3aQ): Cardiac Dearþ: z rypoíhís*" Correlating Basic Science and Clinical obseraations." J 902-1.1..

of REair of Cocaine-Indaced Chronic Tlþt o Aortic Baumgartner, F. J. and Omari, B. O. (1,gg7). "Method Dissection." Ann Thorac Surg 64: 1'517 -28'

Tiss Res 180(303-315)' Behrendt, H. (i.977). "Efrrt of Anabolic Steroids on Rat Heart Mascle Ce/ls." Cell

Horrnone'" Cell Tiss Behrendt, H. and Boffìn, H. (1.977). "Mlocardial Cell Lesions Caused b1 an Anabolic Res 181: 423-426'

1-50 CFIAPTER 1' Introdaction Benzaquen, B. S., Cohen, V. and Eisenberg, M. J. (2001). "Ef,ects of Cocaine on tbe Coronary Arteries." Am Heatt J 142(3): 402-1'0.

Besse, S., Assayag, P., Latour, C., Janmot, C,, Robett, V', Delcayre, C', Nahas' G' and in Rat:." Eur Swyngheáarr-, É. \tOOl¡. "Molecular ðharacteristics of Cocaine-Indaced Cardiomloþath1 J Pharmacol 338(2): 123-9'

(1982). of Nandrolone Bijlsma, J. \ø., Duursma, s. 4., Thiissen, J. H. and Huber, o. "Influence 108-1'2' D}canoati on the Pitaitary-GonadalAxis in Males." Acta Endocrinol (Copenh) 101(1):

FASEB-J 4(B): Billman, G. E, 0990). "Mecbanisms Responsible for the Cardioroxic Efects of Cocaine." 2469-75.

Fanction." Ctit Rev Toxicol Billman, G. E,. 0995). "Cocaine: A Reuiew of lts Toxic Acîìons on Cardiac 25(2):1,1,3-32.

Billman, G. E, and Hoskins, R. S. (1988). "Cocaine-Indaced Ventricutar Fibrittaîion: Protection Af,orded b1 tlte Calcian Antagonist Verap arnil." FASEB J 2: 2990-299 5'

Billman, G. E. and Lappi, M. D. (1gg3). "Efects of Cocaine on Cardiac Vagal Tone beþre and During ."J Cardiovasc Coronary Artery Occlasioi:Zocain, Exacerba.tes tbe Auîonomic Response îo Mlocardial Pharmacol 22(6): 869 -7 6.

Bowman, S. (1990). "Anabolic Steroids and Infarction þetîerl." Br MedJ 300: 750'

738:273-282' Bozler,E,. (1.943). "The Initiation of Inþalses in Cardiac Muscle." AmJ Physiol '[Jnderþing Branch, C. A. and l(nuepfer, M. M. (1992). "Adrenergic Mechanisms Cardiac and Vascalar 7 42-51. Responns to Cocaine in Coniioas Røts." J Pharmacol Exp Ther 263Q):

Brickner, M. E., Willard,J. E., Eichhorn, E.J., Black,J. and Grayburn, P. A' (1991)'"LtftVentricølar H1þertropþt Associated with Chronic Cocaine Abøsa" Circulztion 84(3): 1130-5'

Brogan, \7. C., Lange,R.r\., Glamann, B. and Hillis, L.D. (1,992). "Recørrent coronary vasoconstriction Med 776: 556-561' CatÃed b1 IntranasalZocaine: Possible Role for Merabolites." Ann Int '

Pe(ormances and Brown, B. S. and pilch, A. H. (1972). "Tþe Efects of Exercise and DianabolTJpon Selected PblsioÌogicat Parameters in tbe Male R¿t." Med Sci Sports Exer 4(3): 1'59-65'

cashman, R. and Bosron, w' F' Brzezinski, M. R., Spink, B. J., Dean, R. 4., Berkman, c. 8., J. ,,Høman and Their Metabolites (1,997). Uurr Coøo*l,trstrrase Hce-l: Binding Sþecifcifl þr Cocaine, , and Analogs." Drug Metab Dispos 25(9):1089-96'

Sci Sports Exerc 26(1):2-4' Cabasso, A. (1994). "Peliosis HEatis in aYoungAdatt Boþbaitder. " Med

in a Cambell, S. 8., Farb, A. and Weber, K. T. (1993). "Patbologic Remodelling of the Mlocardium lveþbtlfter Taking Anabolic s teroids." Blood Pressure 2: 21,3-21' 6.

Med-J- Campbell, B. G. (1988). "Cocaine Abase with Hltpertberrnia, Seiqares and Fatal Complications." Aust 149(7) : 387 -9 '

21-Year-OldBoþ Builder Capezzuto,A., Achilli, A. and Serra, N. (1989). "Mltocardial Infarction in a [I-ttt]." A- J Cardiol 63(20): 1'539.

1-51 CH,\PTER 1 Introdøction G' and casale, P. N., Devereux, R. 8., Milner, M., Zullo, G., Harshfield, G. ,\', Pickering, T. Laragh,j. H. (1986). "Valae of Echocardiograpþic Measarement of Left Ventricalar Mass in Predicting corli*orrrlor Morbid Euents in H1þertensiue Men." Ann Intef Med L05: 173-1'78' Perez' Ceballos, G., Figueroa, L,, Rubio, I., Gollo, G', Garciz, L',Mattsnez, A''Yanez' R'' J'' Morato, T. and-bhamorro, G. (1999). "Acate and Nongenonic Ef,ects of Testosterone on Isolated and Perfased Rat Heart."J Catdiovasc Pharmacol 33: 691'-697 '

Fønction of Chen, M. and Mason, R. P. (1995). "Atherosclerosis Abers the Conþosition, Stractare ønd 101-1'12' Arterial Smooth Møscle Cell Plasma Membranes." Biochim Biophys Acta1272: chiueh, c. c. and I(opin, L J. (1g7g). "centralþ MediatedRelease b1 cocaine of Endogenous Epinepltrine (Jnanesthetiryd Røts." øncl Noreþineþbrine fron tbe Slnpathoadrenal Medullary Slstem of J-Pharmacol-Exp- Ther 205(1):1'48-54.

in chlebowski, R.T., Herrold,J., Richardson, B. and Block,J. B. (1983)' "Efects of Deca-Durabolin Patienß with Aduanced Non-Small Cell Lung ." Clin Res 31: 95,\.

Steroid Use'" Choi, p. y. and pope, H. G. (1,gg4). " toaard L%omen and Ilticit Androgenic-Anabolic Änn Clin Psychiatry 6(1):21'-5'

in S. K., Gal, D., Whelton, J. A. and Isner, J. M. (1989). "Euidence That Fetal Distress with of Cocaine (Jsers Is Due to Vascalar Spøsm and Ma1 Be Atteflaated þt Pre-Treøtment . " Circulation Supp 80(Supp II): II-185'

A. Bowsher, D, and Fischman, M. w. (1985). chow, M. J., Ambfe, J.J., Ruo, T. L, Atkinson, J., J. Ther 38(3): 318- ,,Kinetics of"Cocaine Diitri-bation, Eliminarion, and Cbronotroþic Efects." Clin Pharmacol 24.

and Colby, H. D. and Longhufst, P. A. (1988)' Pþsiotog of Androgenic Hormone¡' Drugs, '\thletes Medical: 1'2-1'9. physical performanc.. J. A. Thomas. NY, Plenum

Related to Coleman, D. L., Ross, T. F. and Naughton, J.L. (1,982). "Nþocardiallschenia and Infarclion \ü/estJ Recreøtional Cocaine Use. " Med 136(5): 444-6'

Abuse: An Epidemic with Mary Public Health Cornish, J. \1, and C,p., O. B. (1996), "Crack Cocøine Conseqaences." Annu Rev Public Health 17: 259-73'

Intersfiilal Cousineau, D., Geresky, C. Ä, and Rose, C. P. (1986)' "Decreased BasaÌ Cardiac Norepinephrine Release after Nearonal Inhibition in Dogs." Circ Res 58: 859-866'

b1 Anabolic Steroids in an Creagh, T. M., Rubin, A. and Evans, D. J. (1988). "Heþatic Tømoars Induced Athtete."J Clin Pathol 4l(4): 441 -3.

Pb Dependence of Cocaine Interaction with Cardiac Sodian Crumb, ¡1. J, and Clarkson, C. \ø. (1995). "Tbe 27 4 : 1 228 - 37' C lt a n n e ls. " J -Phztnzc ol- Exp -Th er Q)

Glamann, D' B', Snydet, R' \ø', Willard' J' Daniel, \ø. C., Pirwitz,M. J., Horton, R. P., Landau, C., et at. (1,995). Efects of Intranasal cocaine'" E.,'Wells, P. J., Hìllis, L. D., Lange, R. -,\., "Electrophlsiotogic Am-J-Cardiol 76 (5) : 39 8-400.

,'Cocaine Pharmacol 33 296- Das, G. (1,993). Abuse in North Arnerica: A Milestone in History."J Clin 31.0.

7-52 CHAPTER 1. Introdaction De piccoli, 8., Giada, F., Benetrin, ,\., Sartori, F. and Piccolo, E. (1991). "Anabolic Steroid Use in Bodl Med 12: 408- Bailders: An Echocardiograþbic Staþ of Ltfî Ventricle Morpbotogy and Fanction' " Int J Sports 41,2.

or Vice Versa? Devereux, R. B. (1990). "Does Increased Blood Prusøre Cause Left Ventricalar H1þertroþb1 [Comnent].",\nn Intern Med 112(3): 1'57 -9'

(1997 a). in Dickerman, R. D., McConathy, W. J,, Schaller, F. and Zachzriah, N' Y. "Echocardiographl Fratemal Twin Boþbailders with One AbasingAnabolic Steroids." Cardiology 88(1): 50-1.

in a Dickerman, R. D., Schaller, F., Ptather, I. and McConathy, \ø. J' (1995)' "sadden Cardiac Death 2T-Year-OldBoþbaitder(JsingAnabolic Steroids." Cardiology 86(2): 1'72-3'

Dickerman, R. D., Schaller, F., Zachariah, W. Y. and McConathy, W' J' Q997b)' "Left Ventricalar Med 7: 90-93' SiTg and. Fanction in Elite Boþbuilders UsingAnabolic Steroids." Clin J Sport

of Dressler, F. A., Malekzadeh, S. and Roberts, Sø. C. (1990). "paantitatiue Anaþsis of Amoants Coronary Aøerial Nanowing in Cocaine Addicts." Am J Cardiol 65(5): 303-8.

DuRant, R. H., Ashworth, c. S., Newman, c. and Rickert, v. l, (1,994). "stabilia of the Relationshþs Adolesc Health 75(2): 1'1'1- þehaeen Anabolic Steroid (Jse and Mattþk Substance lJse amongAdolescents."J 6.

Training and DuRant, R. H., Escobedo, L. G. and Hearh, G. Nø. (1995). "Anabolic-Steroid IJse, Srrengrh Multþk DraglJse amongAdolescents in tlte tJnited States." Pediatrics 96 23-28'

DuRant, R, H., Rickert, V. I. and,\shworth, C, S. (1993a). "(Jse of Anabolic Steroids amongAdolescenls [btter]." N EnglJ Med 329(12): 889. of DuRant, R. H., Rickert, v. I., Ashworth, c. s., Newman, c. and Slavens, G. (1993b). "use Engl Med 328(13): Maltþle Drags amyng Adolescents IYbo Use Anabolic Steroids [See Comments]." N J 922-6.

Artery Egashira, I(, Pipers, F. S. and Morgan, J. P. (1991) ' "Flt:rt of Cocaine on Epica-rdial Coronary Viao and Rîactiui4t in Miniøøre Swine afrer Enãothel¡at Injary and Hþh Chotesnrol Feeding. In Anaþsis."J Clin Invest 88(4): 1,307-1'4.

E', Molina' S'' Bartula'L' L" Prince' M' Eichhorn, E. J., Demian, S. E., Alvatez, L' G'' Willard, J' D., Inman, L. R., Graybúrn, P. A. and Myers, S. I. (1992). "Cocøine-InduædAlterations in Prostaglandin Prodaction in Røbbit Aorta." J Am Coll Cardiol 79(3): 696-703'

R' T' and Schiller, N' B' (1993)' "Ltft Eisenberg, M. J., Mendelson, J., Evans, G. T., Jt., J., Jones, paantitatiue Tao-Dinensional Ecbocardiograþhic Ventricular Fønction Inmediateþ'after Intrauenous Ciocoini: A StildJ." J Am Coll Cardiol 22(6):1581-6.

Antbronetric Measarements' and Faber, M., Benade, -,\. J. S. and van Eck, M. (1986)' "Dietary Inta,ke, MedT: 342' Bkod. Lþid valøes n vi¿nt TrainingAtbletes (Boþ Builders)." Int J Sports

Steroid) on Feraudi, M. and \Øeicker, H. (1985). "Efects of Methandrostenolone (an Anabolic Mascle and Mlocardiam'" Energ Metabolism in tbe Gaineø pig Chanfrs in En s in Gøstrocnemius Int J Biochem 17(1 1): 11'91' -205.

There a Connection?" Ferenchick, G. S. (1991). "Anabolicf Androgenic Steroid Abuse and Tbrombosis: Is Med-Hypotheses 35(1): 27 -37.

l-53 CHAPTER l lntroduction Ferenchick, G. S. and,\delman, S. (1992) . "Mlocardiatlnfarction Associated witb Anøbolic Steroid Use in a Preuiousþ Heattþt 37-Year-Otd lYeþht Lfter." AmHeattJ 124(2):507-8.

Ferrer, F., Encabo, r\., Marin, J. and Balfagon, G. (199aQ. "Cbronic Treatment witlt the Anabolic Steroid, Nøndrolone, Inhibit: VasodilatorResþonses in Rabbit Aorta." BurJ Pharmacol2S2:233-241'.

Ferrer, M., Encabo, 4., Marin, J. and Ba\fzgon, G. (1994b). "Treatment with The Anøbolic Steroid' Nandrolone, Reduce¡ Vasoconstrictor Responses in R¿bbit Arteries." Eur J Pharmacol 258(1-2): 1'03-10.

Fineschi, V., Baroldi, G., Monciotti, F., Reattelli, L. and Turlllazzi, E, (2001). "Anabolic Steroid Abuse and Cardiac Sudden Death." Arch Path Lab Med 125: 253-255'

Fischer, G. M. and Swain, M. L. (1,977). "Efttt of Sex Hormones on Blood Pressure and Vascular -21 - Connectiue Tissue in Castrated and Noncastrated Mate Rats." Am J Physi oI 232(6): H61'7

Fisher, M., Appleby, M., Rittoo, D. and Cotter, L. (1996). "Mlocardial Infurction witb Extensiue 50 (4) : 222-3. Intracoronary Tirimbas Indøæd b1 Anabolic S teroids [S ee CornmentsJ ." Br J Clin Pract

Flores, E. D., Lznge, R,-,\., Cigzroa, R. G. and Hillis, L. D. (1990)."Efttt of Cocaine on Coronary Artery Dimensions ii Atherosclerotii : Enhanced Vasoconstriction at Sites of Stgnifcant S tenoses." J A- Coll Cardiol 16(1): 7 4-9'

Foltin, R. W., Fischman, M. W., Nestadt, G., Strombefgef' H., Cornell, E' E' and Peaflson, G' D' (1990). ,,Demonstration of Nataralistic Metbods for Cocaine Smoking b1 Human Volanteers'" Drug Alcohol Depend 26:1'45-1'54.

Forbes, G. 8., Porta, C. R., Her, B. E. and Griggs, R. C' (1992). "seqaenæ of Cbanges in Boþ 267 (3): Conposition In¿aæd b1 Tutosterone and Reuersal of Changes afnr Drag Is Stopped'" J A- Med Assoc 397-399.

Frankle, M. A., Eichberg, R. and Zachzrizh, S. B. (1988). "Anabolic Androgenic Steroids and a Stroke in an Atltlete: Case Reþort."Arch Phys Med Rehabil69(8): 632'3'

Fredericks, R. K., Lefkowitz, D. S., Challa, V. R. and Troost, B. T. (1991)' "Cerebral Vascalitis Associated with Cocaine Abøse." Stroke 22(1,1):1'437-9.

Lþoþrotein Friedl, K. E,, Hannan, C. J., Jones, R. E. and Plymate, S. R. (1990). "High-Densifl 69-7 4' Cholesterol Is Not Decreased If in Aromatiqable Androgen Is Admini:tered." Metabolism 39(1):

Friedrichs, G. S., \X/ei, H. M. and Merrill, G. F. (1990). "Coronary Vasodilation Caøsed b1 Intrauenoøs Cocaine in the Anistbetiryd Beagle." CanJ Physiol Pharmacol 68(7): 893-7 '

Gamouras, G. Â., Monir, G,, Plunkitt, I(., Gursoy, S. and Dreifus, L. S' (2000)' "Cocaine Abase: Repolarisation Abnorwalities and VentricalarAnhltbnial" AmJ Med Scr320(1):9-1'2.

Garber, M. W. and Flahery, D. (1987). "Cocaine and Sødden Death." Am Fam Physician 36$):227- 30.

Lilerature Reuiew G2azer, G. (1991). "Atherogenic Efects of Anabolic Steroids on Serum Lþid Leuels' A 1'925- pabtisbed Enatam Appeor, ln Arrh1nrem-Med / 992 Mar;l 52Q:535J " Atch Intetn Med 151(10): 33.

Ventricular Gottdiener, J. S., Brown, J., Zoksck, J. and Fletcher, R. D. (1990)' "Left Men witb Norynal Blood Prusare: Relation to Exagerated Blood Pressare Resþonse to Exercise [S Ann Intern Med 112(3): 1'61-6. 't-54 CHAPTER l lntrodaction 137-47 Gradman, A. H. (1988). "Cardiac Efects of Cocaine: AReuiew."YaleJ Biol Med 61(2): '

Gralrrick, H. R. and Rick, M. E, (1983). "Danaqol Increases Factor Viii and Factor Ix in Classic Henophilia and Chri:tnas Disease'" N EnglJ Med 308: 1'393-5'

C. and Taylor, R' R' (1'993)' "Anabolic Steroids and Green, D. J., Cable, N. T., Rankin, J. M., Fox, Vascalar Resplnses." Lancet 342l. 863'

of Adrenergic Actiui4t Greenberg, S., Heirz, D. C. and Long, J. P. (1973) . "Testosterone-Induced DEression in tbe Perfased canine Hindlimb." Proc soc Exp Biol Med 7a2Q):883-8.

rüØalsh, M', Hayde, Grinspoon, S., Corcor zn, c.,Askari, H., schoenfeld, D., wolf, L., Burrows, B.' D., pårlman, i<., Arrd.rson, E., et at. (1,998). "Efects of Androgen Adrnìnistration in Men witb the Aids ll/asting Sltndrone." Ann Intern Med l'29: 18-26'

Cardiac Grobbee, D. E. and Hoes, A. W. (1995). "Non-Potassium-SparingDiuretin andNsk of Sudden D eaî|t." J Hypertens 13(1 2): 1'539 -45.

R. A. (19S9)' "Aduerse Efects of Cocaine Hale, S. L., Alker, I( J., Rezkalla, S., Figures, G' and I(loner, an Experimental on Card,iouasralar D1n)mics, Mlocardiat Bkod Flow, and Coronary Artery Diameîer in Model." Am HeartJ 118(5 Pt 1):927-33.

Anabolic-Androgenic Steroid Handelsman, D. J. and Gupta , L. (1997). "Preualence and Nsk Factors for Abase in Aastralian Hþh school stødents." IntJ Androl 20: 1.59-1'64.

Agenß) and Hausmann, R., Hammer, s. and Betz, P. 0998). "Perforznance Enhancing Drags (Doping Med 111(5): 261'-4' Sad.den Death-a Case Report and Reuiew of tbe Literature ." IntJ Legal

Causes Epicardial Hayes, S. N., Moyer, T. P., Morley, D. and Bove, A. ,\' (1991). "Inlrauenoøs Cocaine Coíonory Varoconsiriction in the Intact Dog.",\m Heart J 721: 1'639-1648'

in Hearn, W. L., I(eran, E, E., \X/ei, H. A. and Hime, G. (1991). "Site-Dependent Postmortem Changes Blood Cocaine Concentrations." J Forensic Sci 36(3): 673-84'

a1d'VØagnet, R' (2000)' Heesch, C. M., \Wilhelm, C. R., Ristich, J., Adnane, J., Bontempo, F. A' \í' in ,,Cocaine Axiuates Platelets and. Increases the Formation of Circalating Platekt Containing Minoagregates Hamans." Heart 83(6): 688-95'

yesalis, History of Slntbetic Testosterone." Sci ,\m 272Q):76' Hoberman, J. M. and C, E. (1995). "The 81.

'$ø., Increase lbe Hoes, A. Grobbee, D. E. and Lubsen,J' (,9g4). "Do Non-Potassium SparingDiuretics 47(5):71'1'-33' Wspi¡Suaden Cardiac Deatb in Hlpertensiue Patienfi? RecentEuidence." Drugs

R. S., Harchelroad, F. P', PhilliPs, S', Brent, Kuligl I( and. Hollander, J. 8., Shih, R. D., Hoffman, J', Associated Mlocardial Thode, H. C. (1,997). "Predictors of Coronary Artery Disease in Patients witlt Cocaine- Med 102(2): 158-63' Infarction. Coronr-Airociated Mltocaid*t In¡ontlon (Cani) StildJ Gr,ilþ. ",\m J

Howard, R. E., Huerer, D. C. and Davis, G. J. (1985). "Acute Mlocardial Infarction Followingcocaine 254(1): 95-6' Abase in a YoanglYoman wiîh Norrnal Coronarl Arteries." J Am Med Assoc

in an Anabolic Steroid [Jser'" Med Sci Huie, M, J. (,994). "An Acute Mlocardial Infarction Onarring Sports Exerc 26@): 408-13'

1-55 CH,\PTER 1' Introducrion Inaba, T. (1989). "Cocaine: Pbannaco,kinetics and Biotran:forrnation in Man." Can J Physiol Pharmacol

67 (9): 11,s4-7 .

Notdin, M. R., Subtamanian, R', Miller, Isner, J. M., Estes, N. A. D., Thompson, P. D., Costanzo G., I(ãtsas, G., Sweeney, K. and Slurner, \ø. Q. (1986). "Acute Cardiac Euenls Tenþoralþ Related to Cocaine Abø.r¿. " N EnglJ Med 315(23): 1438-43.

Iversen, L. L. (1,963). "Tbe Uptake of Noradrenatine b1 the Isolated Perfased Røt Heaø." Bt J Pharmacol 2'1,: 523-537.

Disposition in Disnete Regions of Røt Brain." Biopharm Jzvaid,,J. I. and Davis, J. M. (1993). "Cocaìne Drug Dispos 14(4): 357-64.

C. R., Dekirmeniian, H. and Davis, M' (1978)' "Cocaine Javaid.,J. I., Fischman, M. W., Schuster, J. Plasma Concentration: Relation to Phlsiological and Subjutiue Efects in Hamans." science 202(ß6\:227-B'

E. K., Tucker, S. M., Ginn, H. 8., Maftin, B. R. and Aceto, M. D. (1'992)' "Functionaland "DispositionalJohansson, Tolerance Deueloþs During Continuoas Cocøine Exposøre." Eur J Drug Metab Pharmacokinet 77Q):1,s5-62.

(1975). and Age Dffirences in Haman Platelet Johnson, M., Ramey, E. and Ramwell, P. W. "Sex Agregation." Nature 253: 355-357 .

Sensitiui4t in Platekt Johnson, M., Ramey, E. and Ramwell, P. \ø. (1,977). "Androgen-Mediated Agregation." A- J Physiol %2@: H381 -H385.

G. tlte Future: Narional Results Johnston, L. D., O'Malley, P. M. and Bachman, J. QOOT), "Monitoring 'on 36-37 Adolescent Drag Use - Oierview of Ke1 Findings 2001 ." NIH public atson 02-5105 1'4-1'7 ; '

(1983). Cocaine Poisoning' Importance of Treating Jonsson, S., O'Meara, M. and Young, J. B. "Acøte 1'061 -4. S eiqures and Acidosis." A- J Med 75(6) :

I{abas,J. s., Blanchard, S. M., Marsuyama,Y., Long,J. D., Hoffman, G. lø., Ellinwood, E' H', in the Dog A Smith, Þ. ç. und St urrrr, H. C. (1990). "Cocaine-Mediated Inpairwenî of Cardiac Condøction 185-91' Potentiat Mechanisru þr Sadden Deatb øfter Cocaine." J Pharmacol Exp Ther 252(1):

Kamendulis, L. M., Brzezinski, M. R., Pindel, E. v., Boston, \ø. F. and Dean, R. A. (1'996)' ,,Metabolisrn of Cocaine and Heroin Is Cataþryd b1 rbe Same Human Liuer Carboxltlesterases." J Pharmacol Exp Ther 279Q):71'3-7.

Artery Disease in I(arch, S. 8., Green, G. S. and Young, S. (1995). "Mlocardial Hlpertroþþ and Coronary Male Cocaine (Jsers."J Forensic Sci 40(4): 591-5'

Toxici4t-an I(arch, S. 8,, Stephens, B. and FIo, C. H. (i998). "Relating Cocaine Blood Concentralions to Aøtoþs1t StudJt 0f 99 Cases." J Forensic Sci 43(1): 41-5'

I(ashkin, K. B. and lleber, H. D. (1989). "Hoo,þed on Horvnones? An Anabolic Steroid Addiction 262Q2):3166-70' Hlpotbuis [See CommenßJ." J Am Med ,\ssoc

and I(eith, R. 8., Stone, M. H,, Catson, R. E., Lefavi,R' G. and Fleck, S' i' (1996)' "Nutritional Statas Lipid Profks of Trained Steroid-Using Boþbøitders. " Int J Sport Nutr 6(3): 247 -54' MedJ I(ennedy, C. (1993). "Mlocardial Infarction in Association with Misase of Anabolic Steroids." Ulster 62Q):1,74-6.

7-56 CHAPTER 1. Introdaction Med 22$): I(ennedy, M. C. (1992). "Anabolic Steroid Abase and Toxicologt [See Commenls/' " Aust NZ J 374-81..

I(ennedy, M. C., Corrigan, A. B. and Pilbeam, S. T. (19932). "Mlocardial Infarction and Cerebral Hor*oiiog, in a Yoang ni4 naiWtr Tø,king Anabolic S nroids [I-ttt4 ." Aust NZ J Med 23(6): 7 1'3 ' Aust I(ennedy, M. C. and Lawrence, C. (1993b). "Anabolic Steroid Abøse and Cardiac Dearh." Med J 158:346-348.

I(bble, M. \ø. and Ross, M. B. (1987). "Adaerse Efects of Anabolic Steroids in Atbletes'" Clin Pharm 6(9)t 686-92.

Trigered I(imura, S., Bassett, Ä. L., Xi, H. and Myerbufg, R'J. (,992). "Earþ Afterdepolarilations and 85(ó): 2227 -35. Actiuiyt Induæd b1 Cocaine. A Plossible Mechanism of Cocaine Arrblthnogenesis." Circulation

High Ilndtundh, A. M. S., Isacson, D. G, L., Betglund, L' and Nyberg, F. (1998)' "DopingAmongst Side Efects, and Extent of School Sttldenß in Uppsala, Swrdrn: A Presentation of tbe Attitødes, Distribøtion, [-/s¿. " Scand J Soc Med 26(1):71-7 4'

wiTlt Iindlundh, A. M. S., Isacson, D. G. L., Berglund, L. and Nyberg, F' (1999). "Flctors Associated AdolescentUse of DoþingAgents: Anabolic-Androgenic Steroids."Addiction 9a$):543-553.

I(inson, G. A., Layberry, R. A. and Hebert, B. (1991). "Inflaences of Anabolic Androgens on Cardiac Growth and Metabolism in the Røt." cznJ Physiol Pharmacol 69(11): 1'698-704.

I(leiner, S. M., Bazzane, T. L. and Litchford, M. D, (1990). "Metabolic Profles, Diet, and Health 962-7 Practins of Chamþionsbþ Mate and Female Boþbailders."J A- Diet Âssoc 90(7): '

I{oenig, H., Goldstone, ,\. and Lu, c. Y. (1,982). "Testosleron ltism of tbe Rodent Circ Res 50(6): Heart.Ten*icølar Llroro*rr, Mitochondria, and Cetl Growth Are ' 782-7.

I(olbeck-Ruhmkorff, C., Horban, A. and Zimmet, H. G. (1993). "Eîttt of Pressare and Volame Res 27(11): 1998- ouerload. on proto-oniogroin^pnttiotn in the Isolated Il/orkingRøt Heart." Cardiovasc 2004.

E' and Smialek, ('991)' "Increase in. I(olodgie, F. D., Virmani, R., cornhill, -Coroin,J. F., Herderick,.E. J' Mecbanism of Cocaine-Associated Atherosclerosis and Aduentitial Mast Cells in Abasers: An Altematiue 17 (7) : 1 5 5 3-60. C oron arl V as osþ as m an d T b roru b o si s. " J Am Coll Cardiol

witlt Anabolic I{opera, H. (1985). "Tbe History of Anabolic Steroids and a Reuiew of Clìnical Experience S teroids." Acta Endocrinol Suppl 27l: 1'1' -B'

,,tJse Attending Glmnasiams'" Br I{orkia, p. (1996). of Anabolic Steroids Has Been Reported fu 9% of Men MedJ 3L3: 1009.

I(orkia, p. and Stimson, G. V. (1997). "Indications of Preualence, Practice andEfects of Anabolic SteroidUse in Grear Britain." IntJ Sports Med 18: 557-562'

in R¿t Heaø I(rieg, M., smith, I( and Barrsch, \ø, (1978). "Demonstration of a spectfl Andr1gen Receptor Endocrinology 103(5): Marr-i, Relatìonsbip behyeen Brnd.ing Metabolisrn, and Tissue Leuels of Androgens." 1,686-94.

'Ware, of Høman I(ugelmass, A. D., Oda, 4., Monahan, I{., Cabral, C. and A. (1993)' "Actiuation Platetets b1 Cocaine." Circulation 88: 876-883'

1.-57 CFIAPTER 1. Introdaction I(uipets, H., \X/ijnen,J.A., Hartgens, F. and Willems, S. M. (1991)' "Influence of Anabolic Steroids on Sports Med 12: Boþ Conpoiit¡oo,' Btood pressare, Lrpld Pro¡te and Liuer Fanctions in Boþ Builders." Int J 41,3-8.

82(suppl 1): Laarse, Ä. V. (1987). "Cbolesterol and Mlocardial Membrane Fanction." Basic Res Cardiol 137 -145.

Laizure, S. C., Mandtell, T., Gades, N. M, and Parker, R, B. Q003). "Cocaetþlene Metøbolism and 16-20' Interaction with Cocaine and Etbanol: Role of Carboxltlesterases." Drug Metab Dispos 31(1):

Chronic Inføsion of Laks, M. M., Morady, F. and Swan, H. J. Q,973). "Mltocardiat H1þertroþfu Produced b1 sabblpertensiue Doses of Noreþineþhrine in tbe Dog." chest 64(1):75-8.

\Wells, Bedotto, 8., Lange, R. ,\, ., Flo ide, w., I(im, A. S., P. J', J. Beta- Dan'ziger, R. D. (1 of Cocaine-Indaced Coronary Vasoconstriction by Adrenlrgic Bto ntsl'" 112(1'2): 897 -903'

\Willard, 'W'' Lange,R. r\., Cigaffoz, R, G,, Yancy, C. W', J' E", Popm^,J'J', Sills' M' N'' McBride' í'Cocaine,Indøced Comruents]." N fini A S, and Èi[ir, L. D. (f989). Corunary-Aøery Vasoconstriction [See Engl J }l{ed 321Q3): 7557 -62.

Lathers, C. M., Tyau, L. S., Spino, M. M' and Agarwal, I. (1988)' "Cocaine-Indaced SeiTares' Arrblthnias and Sadden Death." J Clin Pharmzcol28(7):584-93'

Laurell, C. and Rannevik, G. (1979). "A Comparison of Plasma Protein Changes Indøced b1 Danaryl, Pregnangt, and Estrogenr. "J Clin Endocrinol Metab a9(5): 71,9-725.

G. and Bredle' D' "Efects Exltn Leeds, E. M., Wilkerson, J, E., Brown, G. D., I(amen, 0986)' ,of Male and Female Rntí" Med Sci and Anabolic Steroids on Totat and Lþoþrotein Cbolesterol Concentrations in Sports Exer L8(6): 663-667.

M., Vos, P' L' M', Hoitsma' .r\' vzn'tLzar' A'. Lenders, J. \ø. M., Denmacker, P' N. J' '\', Jansel, J'' Lþoproteins, Blood Pressare, and and Thien, T. 09BB). "Deleterious Efects of Aoobol¡t Steroids on Seran LiuerFanction in AmatearBoþ Bailders." IntJ Sports Med 9: 19-23.

Blake, D., Fayad, P., Schultz, L. R., Millikzn, c' Levine, S. R., Brust, J. c., Futrell, N., Brass, L. M., of H., Ho, I( L. and'ú.l.ir, K. M. (1991). "A Comþaratiue Staþ of the Cerebrouascular Conþlications 11'73-7 cotcaine: Alkaloicløl versas Hldrocbloride-a Reuiew." Neurology 41(8): '

Fayad, P'' Levine, s, R., Brust,J.C.,Futrell, N., Ho, K. L., Blake, D., Millikan, c. H., Btass, L.M., the Form of Schultz, L. R., Selwa, J.É., * at. (1,990). "Cerebroaascalar Comþlications of the Use of "Crac,k" Atkatoidal Cocaine." N EnglJ Med 323(11):699-704'

on Lewanowitsch, T. and Irvine, R. (2001). "Efects of Teslosterone Propionate andNandrolone Decanoate 6: 55-61' Boþ Comþosition and Lipoprotein Concentration¡ in tbe Røt." Addtction Biol

rù(/' F' Liang,M. T., Glonek, T., Meneses, P., I(opp, S,J', Paulson, D. J', Gierke, L' and Schwartz' ,,Nmr Anabolic steroids Efect." rntJ N. 1óoz;. spectr^s;nþJ staþ of Heaø ttrospnitlpids. An Exercise and Sports Med 13(5): 41'7-23-

Gierke, L' N7' and schwartz, F' Liang,M. T., Paulson, D. J., I(opp, s. J., Glonek, T., Meneses, P', ,,Efects Int Sports Med N. 1ól3¡. of Anaboic Snio¡¿t oid Eodoronce Exercise on Cardiac Perforraance." J 14(6):324-9

1-58 CHAPTER l lntrodaction Litt, M. R., Bell, W. R. and Lepor, H. A. (1987). "Disseminated Intrauascalar Coagølation in Prostatic Carcinoma Reyersed b1 Ketoconaqole." J Am Med Assoc 258(10): 1361-1362.

Litrmann, L., Monroe, M. H. and Svenson, R. H. (2000). "Brugada-þpe Electrocardiograþhic Patterrt Indøced b1 Cocaine." Mayo Clin Proc 75(8): 845-9'

Long, S. F.,'SØilson, M. C. and Davis, ìø. M, (2000). "The Efects of Nandrolone Decanoate on Cocaine- Indøced Kindling in Male Rútr." Neuropharmacology 39: 2442-2447 '

Lou, Y. I{., \ùØen, C., Li, M., Adams, D.J., ìØang, M. X,, Yang, F., Motris, B. J. and \X/hitworth, J. and A, þOOf¡. "Decreased Renal Expression of Slnthase Isoforrns in Adrenocorticotroþin-Indaced 1 1 6 4 0' C orti c o s tero n e -I n d ø ce d H1þ e rte n s i o n." Hypertension 37 (4) : -7

Lukas, S. E. (1993). "Carrent Perspectiues on Anabolic-Androgenic Steroid Abase." Trends Pharmacol Sci 14Q):61-8.

Lifter Using Anabolic Androgenic Luke, J. L. (1990) . "Sødden Cardiac Death During Exercise in a Il/eight Steroids: Pathological and Toxicological Findings."J Forensic Sci 35(6): 1'441'-1'447 .

R. (1985). "Arterial Blood MacDougall, J. D., Tuxen, D., Sale, D. G., Motoz, J. R' and Sutton, J. Pressare Response to HearltResistance Exercise."J Appl Physiol53(3): 785-90'

Manger, ¡ø. M., Nahas, G. G., Tfouve, R., Vinyard, C. and Golberg, S. R' (1989). "Ninodþins ãs an Anti.dote to Cocaine Induced Cardiac Changes in tbe SquirrelMonkey" FASEB 3: 4883,

Manschreck, T. C., ,A.Ilen, D. F. and Neville, M. (1987). "Freebase Pslchosis: Cases fron a Baharnian Epidemic of Cocaine Abuse." Comptehen Psych 28(6):555-564'

K., Green, G. E. and Schiebinger, R' Marsh, J, D., Lehmann, M. H., Ritchie, R. H., Gwathmey,J. J' (1998).'"Androgen Receptors Mediate Hlpertropþ in Cardiac MloEtes." Circulation 98(3): 256-61'.

Marshall, E. (1 988). "The Drug of Championr. " Science 242: 1'83-4'

Martinez,J. F.T.,Redondo, M. D. T., Silva, I. A, and Lopez-Borrasca, A. (1988). "Disseminated Intrauascu'l)r CoagalaÍion in Prostatic Carcinoma Reuersed b1 Antiandrogenic Theraþ." J Ä- Med -Assoc 260(1,7):2507.

Masonis, A. E. and McCatthy, M. P. (1995). "Direct Ef,ects of the Anabolic/Androgenic Steroids' Stanoqolol and. l7 Aþba-Møl4tlteìstusterone, on Benqodiarypine Binding to tbe Gamma-Aninobufiric Acid(a) Receptor." Neurosci Lett 189(1): 35-8'

McCarthy, I(., Tang, A. T. M., Dalrymple-Hay, M,J. R. and Haw, M. P' (2000). "Ventricular 658- Thrombo:is and Slsteãic Enbolism in Boþbailders: Etiolog and Management." Ann Thorac Surg 70: 60.

McGill, H. C., Anselmo, V. C., Buchanan, J. M. and Sheridan, P. J. (1980) ' "Tbe Heart Is a Target Organ þr Androgen." Science 207 $432): 77 5-7 '

McGill, H. C. and Sheridan, P. j. (1981) . "Naclear rJpuke of Sex Steroid Honrtones in the Cardiouascalar Slstem of the Baboon " Circ-Res a\Q):238-44.

Mclç¡lop, G., Todd, I. C. and Ballantyne, D. (1986). "Increased L'eft Ventricalar Mass in a Boþbailder [JsingAnabolic Steroid¡." BrJ Sports Med 20@: 151'-2.

l-59 CHAPTER 1Introdacîion McNutt, R. A., Ferenchick, G. S., I(irlin, P. C. and Hamlin, N. J. (1983). "Acuîe Mlocardiallnfarction in a 22-Year-Otd lYortd Ctass lYeigltt Lifter (JsingAnabolic Steroid." A- J Cardiol 62: 164.

Meidell, R. S., Sen,r\., Hend.erson, S.l\., Slahetka, M' F'. and Chien, K' R' (1986)' "Aþha l- 251'(5 2): Adrenergic Stirnulation of Rat Mlocardial Cells Increases Protein Slnthesis." ,\m J Physiol Pt I{1,076-84.

Meilman, p. \ø., Crace, R. K., Presley, C. ,\. and Lyeila, R. (1995). "Beltond Perfortnance Enbancement: Poþpbarwary arnung Coltegiate (Jsers of Steroids." J Am Coll Health a4Q):98-104.

Melchert, R. 3,., Herron, T. and rùØelder, A. (1,992a). "Testoslerone Induced Ultrastruclaral Changes in Primary NeonatalRøf Mltocardial Cell Caltares." in vitro cell dev biol 28: 1'361''

rwelder, Steroids on Melcherr, R, B., Herron, T. J. and A. A. (1,992t). "The Efect 0f Anabolic-Androgenic Prinøry Mlocardial cell caltares." Med Sci Sports Exerc 24(2):206-1.2.

Med Sci Melchert, R. B. and Welder, ,\. (1995). "Cardiouascalar Ef,ects of Androgenic-Anabolic Steroids." Sports Exer 27: 1'252-62.

M' de la Bastie, D', Lompre' M'' Mercadier, J. J., Samuel, J. L., Michel, J. 8., Zongazo, '\', '\' Wisnewsky, C., Rupp"poit, L., Levy, B. and Schwartz, K. (1989). "Atrial Natriuretic Factor Cene H979-87 Exþression in not Vnt ¡itt Daring Experimental H1þer-tension." .NmJ Physiol 257 (3 Pt 2): '

Mewis, C., Spyridopoulos, I., I(uhlkamp, V. and Seipel, L. (1996). "Manifestations of Seuere Coronary Heart Disease afnrAnabolic DrugAbøsg" cltn cardiol 19: 153-155.

Mittleman, R. E. and Wetli, C. V. (1987). "Cocaine and Sadden "Natural" Deatb."J Forensic Sci 32(1): 11,-9.

Mochizuki, R. M. and Richter, K.J. (1988). "Cardiomlopathl and Cerebrouascalar Anident Associated with Anabolic-Androgenic Steroid Usø. " Physician Sports Med 16(1 1): 1,09-1'1'4.

Lackner, C. and Hillis, L' D' (1'994a)' Moliterno, D. J., Lange, R. 4., Gerard, R. D., Willatd, J. 8., ,,Inflaence of Iitranasit Cocaine on Plasma Constitaents Associated witb Endogenoas Tbrozabosis and Thromboþsis."A-J Med 96: 492-494'

B. H., Boehrer, D., Glamann, 8., Landau, c., Moliterno, D. J., Willard, J. E., Lange, R. ]\., Negus, J. b1 Rossen, D.,"\Øinnif"rájU. D. Hi[is, L. D. (1,994b). "Coronary-Aøery Vasoconstriction Induced J. ^nã Cocaine, Cigarette Smoking orBoth."N EngiJ Med 330: 454-9'

Embolas and Anabolic Steroìd Abøse'" Am Monrine, T. J. and Gaede, J. T. (1992). "Massiue Palmonarl J Med Assoc 267 (1,7) : 2328-2329'

\Wilkins, N., Robertson, H' A', Nademanee, I(., Gotelick, D. 4,, Josephson, M. 1\., Ryan, M. r\., J. Ann Intetn Mody, F. V. and Inrarachot, V. lilAl). "Mlocardial Ischemia Daring Cocaine IYithdrawal." Med 111(11): 876-80.

witb Cocaine Nanji, A. A. and Filipenko, J. D. (1984). "Aslstole and Ventricular Fibrillation Associated Intoxication." Chest 85(1) : 132-133.

Neild, D. (1995), "Glnecomastia in Boþbøilders." Bt J Clinical Przct 49(4): 1'72'

and. Nieminen, M. S., Ramo, M. P., Viitasalo, M', Heikkila, P',I(arialainen, J', Mantysaari, M' ,,Serioøs of Large Doses of Anabolic Steroids in lYeight Lìfurs'" Heikkila, J. (,996). Cardiouascalar Side Efects Eur Heart J 17 : 1.57 6-1'583' 1-60 CH,\PTER 7 Introducrion (Jse in Falkenberg." Eur Clin Nilsson, S. (1995). "Androgenic Anabolic Steroid among Male Adokscents J Pharmacol 48:9-1'1.

Nilsson, S., Baiji, 4., Marklund, B. and Fridlund, B. (2001). "Trends in the Misuse of Androgenic Sweden'" Scand Prim Anabolic Steroids o*oig Boy l6-l z Years old in a Prinary Healtb Care Area in J Health Carc 19: 1'81'-1'82.

and the Olsson, A. G., Oro, L. and Rossner, S. (1974) . "Efects of Oxandrolone on Plasma Lþoþroteins Intrauenoøs Fat Tolerance in Man." Atherosclerosis 79(2): 337 -46.

303(5): 333-9' Om,,\. (1gg2). "Cardiouascalar Cornþlications of Cocaine." ,NmJ Med Sci 'W., om, 4., Ellahham, S., Vetrovec, G. Guard, c., Reese, S. and Nixon, J. V' (1993)' "Ltft (Jsers." 125(5 Pt 1): 1441'-3' Venìhicalør H1þertroþþ in Normoten:iae Cocaine Am Heart J Artery om, 4., !(/atner, M., Sabri, N,, Cecich, L. and vetrovec, G, (1,992). "Freqaenqt of coronary Dis;ase andLeftVentricalarDlsfanction in Cocaine fJsers." AmJ CardioI69:1'549-1'552'

S' (1994)' "Sødden Death Osawa, M., Mitsukud, Y., Saito, T., Yukawa, N., Matoba, R. and Takeichi, of a Cocaine Abuser." TokaiJ E"p Clin Med 19(3-6): L1'5-9'

and Baldo Enzi, G' (1996)' Palatini, P., Giada, F., Garavelli, G., Sinisi, F., Matio, L.' Michieletto, M' ,,Card.iouascatarEfects'of AnaboüíSturoid; in Il/eight-Trained Subjects."J Clin Pharmacol 36(1,2):11'32-40'

arud AÌcohol Interactions in the Røt: Efect of Cocaine and pan, Nl. J. and Hedayz,M. A. (1999a). "Cocaine Sci 88(12): 1266-74' Alcobol pretreatments oi Coroin, Pharmacokinerics and I arrnacoþnamics." J Pharm

\ü/. and Ahohol Interactions in the Rút: Efect on Cocaine pan, J. and Hedaya, M. A. (1999b). "Cocaine Pharwacokinetics and Pharrnacoþnamics."J Pharm Sci 88(4): 459-67 '

. and Freis, E' (1985). "Echoca ntricular Fanction and H1þertropþ in

Doses oJ Parssinen, M., I{adla, T., I(ovanen, V. and Seppala, T' (2000). "Tbe Efect of Saþrapþsiological 21: 406-41'1" Anabolic And.rogenic Steroids on Collagen Metabolism." IntJ Sports Med

and Acate pasternack, P. F., Colvin, S. B. and Bâumann, F. G. (1985). "Cocaine-Induæd Pectoris (6) : 847 Mlocardial Infurction in Patients Yoanger Than 40 Years." ,\m J Cardiol 55 '

ne 0n Petreault, C.L, Hague, N' L., Ransil, B' J' and Morgan, J' P' (1990)- oJ Ferret Bt Intracellølar CaZ+ Hîndfug and Mltoflanent Ca2* Responsiueness Ven J Pharmacol 101(3): 67 9 -85.

Efects of Cocaine and tþe Phìllis, B. D., Irvine, R. J. and I(ennedy, J' .,\' (2000) ' "Combined Cardiac Anabo¡lic Steroid, Nandrolone, in tbe Rat." EurJ Pharmacol39S(2): 263-72'

p. Cocaine Problem in Aastralia Med J pierce, J. and Levy, S. 0986). "The Looming [Editorial]." '\ust 144(1,1):562-3.

Dean, R' Ä' and Pindei, 8.v., I(ed.ishvili, N. Y., Abraham, T, L., Brzezinski, M. R., Zhang,J., Bosron, \ø. F. (1gg7). d cloning of a Broad specifci4t Human ,Liuer 272(23): 1'4769-75. carboxlresterase Tbat cøaþ of cocaini and Heroin."J Biol chem

7-67 CH,\PTER 1. Introductìon pitts, D. I{. and Marwah, J. (1939). "Aatonomic Actions of Cocaìne." CaflJ Physiol Pharmacol 67(9) 1,1,68-76

Pitts, \ü/. R., Vongpatanasin, \W., Cigatro^,J.8., Hillis, L. D. and Lange, R. A. (1998). "Efects of the Intranoronary Irfusin of Cocaine on Left Ventricular S1:tolic and Diastolic Function in Hamans." Circulation 97 (1,3): 1,270-3.

p, AnaboÌic-Androgenic Steroid Use among Radakovich, J., Brodedck, and Pickell, G. (1993) . "Rate of Pract 6: 341-5' Stildentr in Janior High Scbool."J A- Board Fam

in Man: Resnick, R. 8., I(estenbaum, R. S. and Scwart z,L.I{. (1977). "Acate Slstenic Efects of Cocaine 795: 696-698. A Controlled Staþ fu Intranasal and Intrauenows Roatel" science

720(6 Rezkalla, S. H., Hale, S. and I(loner, R. A. (1990). "Cocaine-Induced Heørt ." Am Heart J Pt 1): 1403-8.

Chang, S. H. (1993)' "Ef,ects of Cocaine on Rezkalla, S. H., Mazza,J. J., I{oner, R. -r\., Tillema, V. and Human Ptatekts in Heahhl Sabjects."A-J Cardiol 72Q):243-6'

Riebe, D., Fernhall, B. and Thompson, P. D. (1992). "The Blood Pressure Resþonse to Exercise in Anabolìc Steroid (Jsers." Med Sci Sports Exerc 24(6): 633-7 ' Aþha- Rindet, H. M., Ault, I{. 4., Jatlow, P. I., I(osten, T. R. and Smith, B. R. (1994) ' "Platelet Granale Release in Cocaine (Jsers." Circulation 90: 1'1'62-1'1'67 '

Toxicol Rockhold, R. \ø. (1gg3). "CardioaascularToxiciQ of Anabolic Steroids.",\nnu Rev Pharmacol 33:497-520.

P' (1993)' Sachtleben, T. R., Berg, I( E., Elias, B. l\., Cheatham, J. P', Felix, G. L. and Hofschire, J' ,,The Sci Sports Exerc Efecti of Anabolic"Steroids on Mlocardial Strøctare and CardiouascalarFitness." Med 25(1,1): 1.240-5.

S' (2001)' Sader, M. 4., Griffiths, K. 4., McCredie, R. J., Handelsman, D' J. and Celermaier, D' Cardiol ,,Anirogenic Anabolic Steroids and Arterial Structare and Funclion in Male Boþbailders."J A- Coll 37(1):224-30.

and Fttnction in Boþ Salke, R. C., Rowland, T. !ø. and Burke, E. J. (1985). "Ltfr Ventricølar Si7'e BaiÌders usingAnabolic steroids." Med Sci sports Exer L7(6): 701'-704'

in tbe Cardiouascular Schindler, C. W., Tella, S. R. and Goldberg, S. R. (1992) . "Adrenoceptor Mechanisms Efects of cocaine in conscious sqøinel Monkels." I;tfe Sci 51(9): 653-60.

Sîress Schlussman, S. D., Nybetg, F, and I(reek, M. J. QO02). "Tbe Efects of A'cta Responsiue Íiltpxhapn¡c-p¡øíary Ad,enalAxis and ti, Doparainergic and Endoge ' Psychiatr Scand 106(Suppl' 412): 1'21'-1'24'

Nyberg, F. and I{reek, M. (2000)' schlussman, s. D., zhou,Y., Johansson, P., I(iuru, l\., Ho, l\., J' Hormone' "Efects of the Androgroí, Aiabotic Steroid, 0n a Leuels in Corticosterone ond Proofiomelanocort'in, Corticotrop and (3)' the Hlpoîbalanøs, Pitiitary and Arytgdala of the Rat''

(1939). and Hemoþnømic Schwartz, A. 8., Janzen,D., Jones, R. T. and Boyle, \ø. "EÌectrocardiograþbic 22Q): 1'59-66' Efects (Intrauenoas Cocaine in Awake and Anestheti

1.-62 CH.,\PTER 1 Introduction Schyvens, C. G., Mangos, G. J., Zhang, Y., IVIcI(enz\e, I{' U. and Whitworth, J' A' (2001)' ,,Teiemetric Monitoring o¡Zdnnorølrtrophin-Induced H1þertension in Mice." Clin Exp Pharmacol Physiol 28(9): 758-60.

in Scort, D, M., Wagner, J. C. and Barlow, T, W. (1,996). "Anabolic Steroid Use among Adolescents Nebraska Schools." A-J Health Syst Pharm 53 2068-2072'

Shakalis, A. and Condouris, G. A. (1,967). "Local Action of Cocaine on Nerues from AdrenalectomiTgd Frogs." Arch Int Pharmacodyn Thet 166 (1) : 93-1 0 1 . shannon, R. P., Stambler, B. s,, I(omamura, I(., Ihara, T. and vatner, s' F. (1993)' "Cholinergic. Modulation of tbe Coronary Vasoconstriction Ind.aced b1 Cocaine in Conscioas Dogs [See Comments]'" Circulation 87(3): 939 -49'

and Gersh, B. (1995)' Shen, \X/. I(, Edwards, \Ø. D., Hammill, S. C., Bailey, K. R., Ballard, D' J. J. ,,Sudden tJnexþected. Nontraumatic Deatb in 54 Yoang Adalts: A 37-Year Poþalation-Based Staþ." A- J

Cardiol 7 6 (3) : 1' 48 - 52.

Shub, C., Cueto-Garcia,L., Sheps, S. G., Ilstrup, D. M. and Tajik, A. J' (1986)' "Echocardiograþhic Findings in Pbeocltronogtîoma." A- J Cardiol 57 (971'-975)' simkhovich, B. z.,I(lonet, R. 4., Alker, I( J. and Giaconi, J. (994). "Time Course of Direct Pharmacol 23(3): Cardiotoxic Efects of Hah Cocaine Concentration in Isolated Røbbit Heart."J Cardiovasc 509-16. Simpson, an Aþha I - r Interaction' Adrenergtc' Euidenæþ

\W. (1986). Cocaine-Indaced Ischenic Heart Disease' Simpson, R. W. and Edwards, D. "Pathogenesis -of Arroþtl Findings in a 2l -Year-Otd Man.",\rch Pathol Lab Med 110(6): 479-84'

D' (1969)' Skelton, F. R., Brownie, ]\., Nickerson, P, l\., Molteni, A', Gallant, S' and Colby, H' Circ Res 24(5): Suppl:35- ,,Adrenal Cortical Dlsfunction as a Basis for Experimental H1tþertensiue Disease." 57.

Blood Flow in the Stein, E. A. and Fuller, S. A. (1993). "Cocaine's Time Action Proftt on Regional Cerebral Rat." B:ain Res 626(1 -2) : 1'17 -26'

MaÌs Steinberger, E. and Smith, I( D. (1.977). "Testosterone Enanthate - a PossìbÌe Reuersible 16 261' -268' C o n tra cep ti u e. " Contrzcep tion (3) :

Stenberg, R, G., \Winnifotd, M. D., Hillis, L' D', Dowling, G' P' and Buja' L' M' q?89) Arch ,,S.irnaltaneoas Acute Tbrombosii of Two Major Coronary Arteries Followìng Inlrauenoas Cocaine UsL" Pathol Lab Med 113(5): 521'-4-

of cocaine in tbe Rat and in Isolated Stewarr, D. J., Inab a,T. and l{alow, \ø. (1978). "N-Deyetb¿lation 207 (1): 1'71-1'77 Røt Hepatogttes: Conparison witb Aminoþrine Denetl lation." J Pharm Exp Ther '

\ø. (1977). "H1droþsis of Cocaine in Høman Plasma by Srewart, D. J., Inaba ,T.,'fang,B. I{. and l{alow, 1 5 57 -1' 5 6 4' C h o lin e s te ra s e. " u' lfe S cienc e s 20 : "pt Stolt,4., I{arÌla,T., Viitasalo, M., Mantysaair, M., I(ujala, u. M. andKatizlainen,J, Q'999)' (Jsing Doses of Anabolic Interual and pt Di$ersion in Endørance Atblens and. in Power Athletes l--arge Steroids." A* J Cardiol 84: 354-366. 7-63 CHAPTER l lntroduction Strang,J.,Johns,A. and Caan,W. (i993). "CocaineintberJk-|991."BrJ Psychiatry162 1'-1'3'

Su, T. P., Pagliaro, M., Schmidt, P. J., Pickar, D., Wolkowitz, O. and Rubinow, D. R. (1993)' ,,Neøroþslcbiatlic Efects of Anabolic Steroids ìn Male Normal Volanteers."J A- Med Assoc 269(21): 27 60- 4. sullivan, M. L., Martinez,c. M. and Gallagher, E. J. Q,999). "AtrialFibrillation and AnaboÌic steroids." J Emerg Med 17(5): 851-857.

Sutliff, R. L., cai, G., Gurdal, H., Snyder, D. L., Roberts, J. and Johnson, M. D' 0996). ,,Cardiouascalar H1tþertrophl and Increased Vascalar Contractile Resþonsiueness Following Repeated Cocaine Administration in Røbbits." Life Sci 58(8): 675-82.

Irapair the Tagarakis, C. V. M., Bloch, W., Harrmann, G. and Addicks, I( (2000). "Anabolic Steroids Eiercise-Indaced Growtb of the capitlary Bed." IntJ Sports Med 21: 41'2-418.

shida' H'' Tamai,J., Hori, M., I(agiya, T., Iwakura, I(', Iw M. \(zrrraà1,'T. (1989). "Role of A of Cardiac Inoue, "rd HlpenrEþ in Gainea PigHearts aith Pressure Ouerloa

Tanner, S. M., Miller, D. W. and Alongi, C. (1995). "Anabolic Steroid Use b1 Adolesænts: Preualence' Motiues, and Knowledge of Fvsks." Clin J Sport Med 5: 108-115'

tbe Heart'" Tzzelazr,H. D., I(arch, S. 8., Stephens, B. G. and Billingham, M. E. (1987)' "Cocaine and Hum Pathol 18(2): 1'95-9.

and Tella, S. R., I(orupolu, G. R., Schindler, c. w. and Goldberg, s. R. (1992). "latbopblsìological 262(3): pbarmanlogical Mecbaniins of Acate Cocaine Toxicilt in Conscious Rntr." J Pharmacol Exp Ther 936-46. \ü/ilkerson, of Temesy-Armos, P. N., Fraker, T. D., Bfewstef, P' S' and R' D' (1'992)' "The E'fects (Jnsedared 19(6): 883-91. Cocaine on Cardiac Electropþsiotog in Conscioas, Dogt." J Cardiovasc Pharmacol

Thiblin, I., I(ristiansson, M. and Rajs, J. (1,997). "Anabolic Androgenic Steroids and Behauioaral Pattems amungViolent Ofenderl" J Forensic Psych 8(2):299-310'

a1d Thompson, P. D., Cullinane, E. M., Sady, S. P., Cheneveft, C., Saritelli, A' L', Sady, M' A' Lþoþrotein Leuels'" Herbeit, e. N. ltlAO). "ContrastingEfectiof Testosterone and Stanololol on Serum J Am Med Assoc 267:1'165-1168' G' Thompson, P.D., Sadariantz,A., Cullinane, E. M., Bodziony, K. s., catlin, D, H., Torek-Both, Use Anabolic and Dtuglas, p. S. (1,gg2). "Left Ventrìcalar Fanction Is Not Imþaired in It/eight Llfters IVho S teroids." J A- Coll Cardiol 19: 27 8-282.

Thomson, p. D., Zmuda,J. M. and Catlin, D. H. (1993). "(Jse of Anabolic Steroids amongAdolesceltrts." N EnglJ À.{ed329(1,2): 888-889.

Vascular Togna, G. I., Togna, A. R., Gnziati,M. and Franconi, M. (2003). "Testosterone and Cocaine: roøc14, of T beir c oncomiltant Abuse." Thromb Res 109: 79 5 -201..

Tomita, F., Bassett, A. L,, Myerburg, R. J. and I(imura, S. (1993). "Efects of Cocaine on Sarcoplasmic Reticølum in Skinned RaT Heart Masch. " Am J Physiol 264(3 Pt 2): HB45-50.

1,-64 CHAPTER 1. Introdaction Toyama, M., \Øatanabe, S,, I(obayashi, T., Iida, I(, I(oseki, S., Yamaguchi, I. and Sugishita,Y. í,Tri witb e;g4). Cor6 of Acøn Mlocaidiat Infarction Associated with Aplastic DuringTreatment Anabolic Steroids."Jpn HeartJ 35(3): 369-73.

Trouve, R. and Nahas, G. (1986). "Nitrendþine: An tu Cardiac and Lethal Toxici4t of Cocaine." Ptoc Soc Exp Biol Med 183(3):392-7.

Trouve, R., Nahas, G,, Manget,'\Xl. M., Vinyard, C. and Golberg, S. (1990). "Interactions of Nimodþine 3: 171'-5' ood Coiaio, on Endogenoas Catecholanines in tbe Squirrel MorktJ." Proc Soc Exp Biol Med \ü/. Trouve, R., Nahas, G, G. and Manger, M. (1991). "Catecholamines, Cocaine Toxicifl, and Their Anüdotes in the Røt'" Proc Soc Exp Biol Med 196(2): 184-7 '

'Sø., of Tseng, y. T., Rockhoid, R. Hoskins, B. and Ho, I. K. (1994). "Cardiouascalar Toxicìties 113-21' Nanlrolone and Cocaine in Sþontaneoasþ Hlpertensiue Rats." Fundam Appl ToxicoI22(1):

Tulenko, T. N., Chen, M., Mason, P. E. and Mason, R. P. (1998). "Pblsical Ef,ects of Cholesterol on Bilayr Anerial Snooth Mascle Membranes: Euidence of Immiscible Cbolesterol Domains and Alterations in lf/idtb DaringAtlterogenesis."J Lipid Res 39: 947-956'

in Turlapaty, p. D. M, V. and Akura, B. M. (1985), "Heterugenous Distribation of Adrenergic Receþtors 2: 617 -642' Coron)ry'Arteries and. Tbeir Inflaence on Coronarl Arteriat Tone." Microcirc Endo Lymph

Turner, S. '$(/., Mangos, G. J. and \x/hitworth, J. A. (2001). "Nitric oxide slntbase Actiui4t in 28(11): 881-3. Adrenocoøicotrophin Inlaced Hlpiøension in tbe Rat." Clin Exp Pharmacol Physiol

in IJrhausen, A.; Holpes, R. and I(ind.ermann, \X/. (1989). "One-and Two-Dìrnensionøl Echocardiograþh1 Boþbøilders [.JsingAnabolic steroids." EurJ Appl Physiol 58: 633-640.

Van Dyke, C. and Byck, R. (1982). "Cocaine'" Sci '\m 246(3):128-41"

Varriale, p., Mirzai, M. and Sedighi, A. (1999). "Acate Mlocardial Infarction Associated with Anabolic 19 (7) : 8 8 1 - 8 84. S teroids in a Y o u ng Hiu-Infe cte d P atien t. " Phztmacotherapy

Vitmani, R., Robinowitz, M., Smialek,J. E. and Smyth, D' F' (1988). "CørdiouascttÌarEfects of Cocaine: 115(5): 1'068-76. An Aatipslt Stuþ of a0 Patients [See Comments]." Am HeattJ

Vittj, T. G. and Boni, R. L, (19S5). Metabolism of Cocaine. Pharmacokinetics and of psychoactive Drugs. G. Bàrneti and Chiang, C, N. Cahfonia,Biomedical Publications:427-440'

Engelmann, G. L. and l(hairallah, P' A' (1989)' Vitullo, J. C.,I{ar,Lfl, R.,Mekhail, N.,lx/icker, P., ,,Cocaine-Ind.uced Small Vessel Spasn in Isolated Rat L arts." Â- J Pathol 135(1): 85-91'

'\x/arner, E. A. (1993). "cocaine Abuse." Ann Intern Med 119(3):226-235.

Webb, O. L., Laskarzewski, P. M. and Glueck, C. J. (1984). "settere of Hþh-Densifit Lþoþrotein Cho t Lfters and Boþ Builders þt Self-Administered Exogenous Testosterone aidAnabolic-A tabolism 33(11): 971-5'

Cocaine Abase." Am HeartJ 171'(4):793' lveiss, R. J. (1986) . "Recurrent Mlocardial Infarction Caøsed b1

of Cocaine Welder, A. A. (1,gg2). "A Primarl Cultøre Slstern of Adatt Rat Heart Cett: þr the Ettaluation Toxiciþt." Toxicology 7 2Q): 1'7 5 -87 .

7-65 CH,\PTER 1. Introdaction .ùØelder,.,\.4., Eselin,J.,\., Melchert, R. B', Davis, S. I( andJF, O.D' (1991) "Efects of Cocaine and 36(2): Noreþineþbrine on primary Caltøres of Neonatal Røt Mlocørdial Cells." J Toxicol Environ Health 75-90. 'Wetli, C. V. and Fishbain, D. A. (1985). "Cocøine-Induced Psitchosis and Sudden Death in Recreational Cocaine (Jsers." J Forensic Sci 30(3): 873-80.

Efects of Testosterone on Weyrich, A. S., Rejeski, \ø. J., Btubaker, P. H. and Parks, i. S. (1992). "Tbe Ltp}ds and. Eicosanoids in Clanomolgus Monkels." Mred Sci Sports F;xet 24(3): 333-338'

Wilkerson, R. D. (1988). "Cardiouascalar Efects of Cocaine in Conscious Dogs: Imþortance of Falþ Fanctional Autonorzic and Cenfral Nervoas Slstems. "J Pharmacol Exp Thet 246(2): 466-71"

OrøÌ Cocaine \ùØilkinson, P,, Van Dyke, C., Jatlow, P., Barash, P. and Byck, R. (1980)' "Intranasøl and Kinetics." Clin Pharmacol Ther 27 (3): 386-94'

-Wilson, L. D. (1998). "Rapid Progression of Coronarl Artery Disease in tbe Setling of Cbronic Cocaine Abusa" J Emerg Med 16(4): 631'-634'

p. (1990). Oesophageal Varices A:sociated with Winwood, J., Roberrson, D. A. and \X/right, R. "Bleeding Anabolic Steroid (Jse in an Atblete." Postgrad Med J 66(780): 864-5'

2552- Wolinsky, H. (1972). "Efects of Androgen Treatment on tlte Male Rat Aorta."J-Clin-Invest 51(10): 5. woodiwiss, 4.J., Trifunovic, 8., Phlippides, M. and Norton, G. R. (2000)' "Efects of an Androgenic Steroid on Exerrise-Indaced Cardiac Renodellingin Røts."J APpl Physiol 88(2): 409-15.

Reviews' R' S, \ldght, J. E. (1980) . Anabolic Steroids and Atþletics. Exercise and Sport Sciences Hrrtio.r and Miller, ó. I. Wasttington, American College of Sports . 8: 1'49-202'

Releasefom yamaguchi, N,, de Champlain, J. and Nadeau, R. A. (1977). "Regulation of Norepineþhrine 108-117' Cardiìc Slnpatbtetic Fibers ln øe oog b1 Presltnaptic Aþha-and-Beta Receptors. " Circ Res 41(1):

Users." Biol yates, ¡í. R., perf, p. and Murray, S. (1992). "Agression and Hostili4t in Anabolic 'îteroid Psychiatry 3l: 1,232-7234.

yu-yahiro,J.4., Michael, R. H., Nasrallah, D. V. and Schofìeld, B. (1989). "Morphologic andHistologic 17(5): 686-9 Abnorynalitiu in Female and. Male Røts Treated with Anabolic Steroids." A- J Sports Med '

Expression'" Ztmmer,H. (1997). "Catecbolamine-Indaced Cardiac Hlpertropþ: Signfrcanæ of Proto-Oncogene J Mol Med 75: 849-859'

and B. R. (1994). "IntracoronarSt Versus Intrauenous Zimring,H. J., Fitzgerald, R. L., Engler, R. L. Ito, Efects f Coti*t oo ðoronory Flow and VentricalarFunction." Circulation 89(4): 1819-28.

l-66 CHAPTER 1. Introdaction General methods

2.1 Animals

All rats were purchased from Central Animal Supplies fX/aite Campus, Adelaide Univetsiry). Rats were provided u¡ith standard rat chow and water ad tibitam. Animals wete maintained in a standard

'l,2ho ,r lighfdark cycle, beginning at 7 AM, and subjected to a constant foom temperature of

22+2oC. The experiments were conducted in strict accordance with the guidelines of the ,,princþles of laboratory care" (I.JIH publication No. 85-23, tevised 1985), the Austtalian Code of practice for the care of animals for scientific purposes of the National Health and Medical

Research Council and the Adelaide University Ethics Comrnittee'

2.2 Radiotelemetry recording

Radiotelemetry is designed to broadcast physiol ogrcal ðata from ttansmitters implanted in the abdominal cavity of labotatory animals to a remote receiver. This allows the recording of stress cardiovascular vadables in freely moving, non-anaesthetised, laboratory animals, reducing

responses to a minimum'

The experiments pfesented in this thesis made use of 2 t¡pes of Data Sriences radiotelemetry implant irnplants (Data Sciences, St. Paul, MN) - the ECG implant GA11CTÄ-F40) and the BP

GA11P,A.-C40).

St Radio sþals from the implants were collected by a receivet (R'41020 Receiver, Data Sciences, St Paul' Paul, MN), which was connected to a computef running LabPto softwate (Data Sciences, to the manufactuter's \aNÐ Gig. 2. l). F,ach implant was calibrated for temperature to conform fiIter. configuration settings. The waveform-sampling fate was set at 1000H2 with a 250H:2

2-1 CHÂPTER 2 General Methods +- Telemetry Implant

Receiver Receiver

ooo o oo: BCMl.OO LabPro system Ambient Pressure Reference

Fig. 2.1 Telemetry feCOrding SetUp. The irnplanted telemetry device transmits physiological data to a recelver to a con¡olidation o.r-th" AM ftequency band. The digrtised information fron severai teceivets is passed sirnultaneously data a suitabie format for recording by the LabPro ,\cquisitionrM software' natix @CM-1,00), which presents the in ResultiÀg wavefoims and irru-.t"t. are saved ifl ASCII text formât for easy manipulation.

2.3 General a¡raesthesia

tü/hen implanting radiotelemetry rmplants, rats weïe anaesthetised with a mixture of methohexitone (60mg/rnl), administered sodium [BdetalrM] (1Omg/rnl) and pentobarbital sodium fNembutalrMl intraperitoneally (i.p) in the ratio 10:L at Srnl/kg. In studies involving preparation fot ischaemia- reperfusion, anaesthesia was induced by pentobarbital sodium (60mg/kg). Prior to commencement of surgery, the rats were tested via the pedal reflex and continuously monitored throughout the experiment to ensule an adequate level of anaesthesia was maintained'

2.4 Surgical implantation of radiotelemetry devices

2.4.1 ECGimplanß ECG radiotelemetry implants (Physiotel implant, model TA11CTA-F4},Data Sciences, St Paul, MN) allowed the measurement of electrocardiogram, (ECG), heart tate (HR)' cote temperature (CT) and spontaneous locomotor activity (SI-A) in freely moving, fully conscious rats' E'ach implant (approx. 30mm x 17mm) had two electrodes coveted by silastic rubing, with approximately also coveted 10mm of exposed v¡ire near the end of the electrodes. The tips of each electrode were with silastic tubing to pfevenr local i¡ritation developing (Fig.2.2).

2-2 CH.,\PTER 2 General Methods ECG implants were placed in the peritoneal cavity and stitched to the abdominal wall with non- absorbable suture. The positive electrode was secured subcutaneously just left of the xyphoid process. The negative electrode was tunnelled under the to the atea of the right scapula and stitched in place. Both electrodes were immobilised with non-absorbable suture to prevent migtation and to reduce movement interference'

Íh{¡{moplüslic BalterY hÐusìnq

Selsing leads

€pory encaptulaied Eiocompa I iblå €l€çlroñ¡cs rnodule o.;ter layot

Fig, 2.2 Data Sciences ECG radiotelemetry implant. Implants consist of a pair of electtode s made ftom et al'' hef,caly vzoond stainless steel extending from an epoxy encâpsulated electtonics module. Source: @roclsttay 1ee3).

2.4.2 Blood Pressure ImPlants Radiorelemetry implants (Physiorel implant, model TA11PA-C40, Data Sciences, St PauI, MN) The allowed the measurement of HR, diastolic pfessufe (DP), systolic pfessufe (SP) and SI-A' implants consist of a small, cylindrical, biocompatible plastic housing about 2cm long, which contains the pressure recording electronics. From this protrudes a fluid fìlled catheter (0'7mm temPerature diameter) with an anti-thrombogenic tip (Fig. 2.3).F;ach implant was calibtated for and blood pressure (BP) to conform to the manufacturer's confìguration settings. The waveform- sampling rate was set at 1000H2 with a 250lH2 Flter'

Blood pressure implants were placed in the peritoneal cavity and stitched to the abdominal wall with non-absorbable suture. The sensing catheter was placed in the descending aorta below the renal arteries, pointing "up-stream". The cathetet was lightly glued in place using tissue adhesive' Full details of the rmplants and surgical procedure is described elsewhete (Btockway ú al., 1'99'l';

Guiol ¿r a1.,t992).

2-3 CHAPTER 2 General Methods EOnaor Elrctronlc¡ modulc calhet¡r

Noncomprc ¡r¡blc lluld

Brttcry Thln-wr[ed llP wlth rntlthronrbogcnlc lllm applicd

Fig. 2.3 Data SCienCes bloOd preSsure implant. The implant consists of a gel fdled catheter that refe¡s et a/',1'993)' pressure from the tip of the implant to a ptessure ttansducer in the body of the implant. Soutce: @rockway

2. 4.3 Pos hoperative Management Wound sites were irrigated with 0.570 to reduce postopetative pain' Iniectable post operatively. Rats ffribrissenrM) and topical antibiotic powdet wete administeted wete allowed to recover for a minimum of 10 days befote expetimentation was begun.

2.5 Analysis of radiotelemetry data

Drug responses wefe averaged (mean*S.E.) and plotted as a function of time' To maintain consistency with previously published radiotelemetry data (Philüs et a1.,2000; Phjllis et a/.,2001) the method. area under the curve (AUC) was also detetmined for each parameter using the tapezoidal AUC was calculared from the raw data using Graphpad PRISM 4.02 (Graphpad Software, San Diego, California, USA). Between treâtment group differences wete analysed using either a single- factor ANOVA or a Student's t-test'

2.6 Surgical procedure for ischaemia-reperfusion studies Induction of ischaemia anð teperfusion was conducted using the methodology established by Mclennan (Àdclennan et al., !985;1988). Following induction of anaesthesia in Sprague-Dawley on rats, the û.acheawas cannulated in preparation for zriftcialvenlilation. Animals wete kept warm catheters a heating table maintaineðat37"C. The left femoral afiery and vein were exposed and inserted to allow fot BP meâsurement and intravenous drug administtauon, tespectively' The The femoral femotal arï.ery catheter was attached to a BP transducet Q{arco Bio-Systems P-10008).

2-4 CHAPTER 2 General Methods vein catheter was attached to a sydnge pump. ,A,ll lines were filled with heparinised saline to mainta:¡patency (Fig. 2. 4 right, toþ.

The chest was opened at the left fifth intercostal space. The fouth and fifth ribs wete sectioned approximately 2 and7mm from the sternum and atnficial ventilation was commenced immediately. Animals wete ventilated with room air with a sttoke volume of 1'8rnl/100g and a rate of 62 sttokes/min. The pericard.ium was then opened and the heart extetiorised by the application of gentle pressure on the rþht chest wall. A 6/0 braided siìk ligatute attached to a 12mm attaumatic around the left antetior taper needle ffascular 1,1,53-1.1, Davis and Geck, Australia) was threaded descending coronary artery about 3mm from its origin, and the heatt replaced in the chest cavity. ,\ shorr section (-5mm) of polyethylene tubing was placed over both free ends of the ligature'

Pre-oxidised Needles (21Gx1) wete used as ECG electtodes (lead II ECG) and were insetted subcutaneously on opposite sides of the chest (Frg. 2.4 /eÍ' t0þ)'

2-5 CHÄPTER 2 General Methods Fqg" 2. 4 teft (top): Position of electrodes across chest of,tat- Right (top): Ischaernia-reperfusion setup' Right (bottom): Ðâta was collected using a ElioPac system for recortËng physiological parameters'

2.7 Conrinuous recordiqg of cædiac parametÆs duringisctraenia-reperfrrsion. tardiac pararneters (ffn, gp and ECG) were continuously recorded using Acq-Knowledge 3'5'6 in graphical format for offline analysis soft,*rat-e @iopac), which allowed waveforrns to be stored (Fig. 2. 4 right, ho#art\.

2"8 Induction of ischaemia and reperfrrsion slit in the T,ig¡-rtening the lþture around the LAD coran ry artery and pulling it dovrn a retatning ligature was then side cf the potyethylene turbing f,or a period of 15 rninutes induced ischaernia. The

released and reperfusion initiated for a further 1Û rnrnutes'

?-6 CHAPTF',R 2 Cencral Merho¡lç Following completion of the procedure, the heatt was excised into ice cold saline and cannulated Evan's blue dye onto a 10 ml sydnge. The ligature was te-tightened and the heart wâs perfused with to determinet}re"area at risk of ischaemia" (o/ozf r). The underperfused region þink) was cut out of the heart, weighed, and exptessed r^lo of total weight' ^s ^

2.9 Analysis of ischaemia-reperfusion data Ventricular tachycarðia (VT) was defined as 4 or more consecuúve ventriculat prematute beats changed from beat to beat (Fig.2. S toþ). Venrricular fibdllation ffF) was defìned as a signal that in morphology and tate (Fig. 2.5 bottom),

Constructing a contingency table and appþing the Chi-squared test evaluated differences in the total number of surviving rats responding with VT or VF dudng ischaemia or reperfusion' tats Fischer's Exact test was applied to 2x2 contingency tables as a post-hoc test' The ftaction responding with VT, VF or VT+VF was also calcuìated for each treatment dudng ischaemia and teperfusion and analysed using the same contingency table method described above. Surwival time during ischaemia and reperfusion was presented as mean*SE and analysed using a single-factor ANOV,\ w-ith Dunnett's post-hoc test (in comparison to control). For tats which died of VF the time of death was taken to be the beginning of the period of fatal vF.

ischaemia and tepetfusion Average dutation þ) of VT or VF or total dysthythmia fl/T+VÐ during was calculated fot each tteatment, and presented as meanlsE'Fot fatal VF a maximum of 120s revealed that was recorded for a single episode. Personal communication with Abeywardena et a/ Differences 120s is the maximum period of VF for which a rathas survived under this procedure.

berween treatment groups in the duration of VT, VF and VT+VF wete analysed with a I(ruskall-

Wallis test with Dunn's multiple comparisons test post-hoc'

The severity of dysrh¡hmia obsewed during occlusion and teperfusion was scored using the as mean*SE. The small number of rats Lambeth convention $X/alker et al., 7988) and presented excluded is that died without displaying arrhythmia were excluded ftom the score (number of rats noted in the legend figute). The scoring method utiTsed wâs as follows:

2-7 CHAPTER 2 General Methods Occlusion 0 = 0-49 premanrre ventricular contracdons eVC)

1 =50-499 PVCs or VF 2 = rnore than 499 PVCs andf or 1 episode of spontaneously reverting VT 3 = more than 1 episode of VT andf orVF, less than 60s total combined duration 4 = VT andf orVF, 60-119s total combined duration 5 = VT andf or VF, more than 119s total combined dutation 6 = FatalVF starting at more than 15 min after occlusion 7 = FataIVF starting at between 4 min and 14 min 59s aftet occlusion 8 = Fatal VF starting at between 1 min and 3 min 59s after occlusion 9 = FatalVF starting befote 1 min after occlusion

Repetfusion 0 = 0-49 ventriculat extta beats (VEB)

1, = 50-249 VEBs 2 - >249 VEBs anðf or 1 episode of VT orVF 3 = more than one episode of vT or vF <20s combined duration 4 = VT andf or VF, 20-60s combined duration 5 = VT andf orVF, mote than 60s combined dutation 6 = FatalVF starting mote than 5 min aftet reperfusion 7 -- FatalVF starting between 2 and 5 min after repetfusion 8 = Fatal VF statting between 20s and 2 min after reperfusion 9 = FatalVF starting within 20s of reperfusion'

using a Differences in Lambeth arrhythmia scofe of duration of arrhythmia was determined in "afea risk of I{ruskal-Wallis non-parametric test, with Dunn's post hoc test. Differences ^t post-hoc ischaemia" and total survival were calculated using a oîe-:vr2;y ANOVA with Dunnett's test. In all statistical tests the level for was p<0.05'

Blood pressure and HR was recorded dudng ischaemia and reperfusion' Cardiovascular variables wete were recorded immediately prior to ischaemia and at 300, 600 and 900s. The same variables time, a BP or recorded priot to reperfusion and at 600s. If VT or VF was present at the designated notmal beat HR reading was taken within 60s before or after the designated time. If no period of recorded' All was evident fot 60s befote or ¡ftet the desþated time, a BP ot HR teading was not data was expressed relative to the value at 0s of ischaemia or reperfusion' ,\ll catdiovasculat not apptopriate for variables were presen ted as mean*SE. Because repeated measures '\NOVA is 2-8 CHAPTER 2 Generøl Methods use when data sets contain missing values, between and within group differences were assessed during ischaemia and repetfusion using two-way ,A,NOVÂ. Diffetences in BP and HR during test' ischaemia between groups were calculated using Bonfertoni's multiple comparisons post-hoc post-hoc tests were applied only to 'treatment' (or 'dose') and 'interaction'. Post-hoc tests were calculated using Mictosoft Excel from the following formula Q{eter et a1.,1990):

Fot each comparison t- meant- mea.n2 11 -+-Nr Nz meanl & meanr: mean values for the compârlson N, & Nr: number of values contributing to the mean PRISM 4'02) MS,..,u,",: The mean squâfe for residuals (taken from the ANOVA results Gtaphpad

To determine the signifi.cance level the computed value for the t tatio (above) was compared against the standard values (denoted tx). if t>t* then the comparison was sigruficant at the norninated significance level'

t* =TlNV(probability, deg-freedom)

TINV: MS Excel fotmula for the inverse of the Student's t-distribuuon Ptobability: significance level (a) Deg_freedom: Degrees of freedom for residuals (taken from '\NOV,\ results Graphpad PRISM 4.02).

2-9 CHAPTER 2 General Meîhods 100

50Ë E

0

0.4

U, =o

0

o.2

5s 50

Ð T E E

0

0.4

al, :! o

0

- 0.2

5s

a perìod of \rF during Fig. 2, 5 TOP: ECG trace showìng a petiod of VT dudng cardiac ischaemia BOTTOM: ischaemia

2-L0 CH'\PTER 2 General Methods 2.10 Determination of the exEa-neuronal uptake of noradrenaline in isolated perfirsed hearts.

2.10.1 Pre-lreatment Apptoximat ely 24 hours prior to sacrifìce rats v/ere treated with (2.3mg/kg' i.p.) to inhibit deplete vesicular noradtenaline and 18 houts before with pargyline HCI (75mg/kg, i'p) to was administered 2-3 hours monoamine oxidase çBry^r et al., 1,986). A futther dose of pargyline priot to sacdfice.

2.10.2 Protocol from The protocol for the determination of extraneuronal uptake of noradrenaline was adapted heart excised into ¡,ryan et a/. (1,986). Rats were anaesthetised with , decapitated and the to the chilled, calcium-free-Ilebs. This prevented the hearts from beating until they were attached a Langendotff aortic cannula, and perfusion with Iftebs had begun. Hearts wele cannulated onto with Iftebs appafâtus via the aorta and fetfogfâdely perfused ât a cofìstant flow of 10ml/min

bicatbonate solution at 37"C and gassed with carbogen (95o/o O2f 5o/o CO). Perfusion pressure was line before monitored using a transducer (Statham P23XL) attached to a side arm of the petfusion the I{rebs entered the aottic cannula. Pressure data were monitoted using an eight-channel 5 MaclabrM unit (l\4aclabf8e, AD instruments, Castle Hill, NSW). -An equilibration phase of from the minutes allowed the perfusion pfessufe to stabilise below 60mmHg' The flow of Iftebs maintained during apex of the heart was monitored to ensure that a flow of 10ml/min had been the petfusion.

eLiminate Flearts were then pre-perfused for20 minutes with Iftebs containing cocaine (Z7pW to this time neuronal NA reuptake and U-0521 (50plv! to block COMT mediated NA metabolism. At to the either corticosterone, nandrolone (various concenttations) or vehicle control was also added I(rebs. Hearts u/ere then perfused for 2 minutes with ascotbic-I{tebs (0.58m1vf, cocaine, U-0521', 3H-N,\ 3H-NA perfusion a the inhibitor of inrerest and (0.85¡r\Q. Pdor to comrnencement of the 3H-NA perfusion, hearts -1ml sample of perfusion medium was collected. At the conclusion of the out were blotted dry. Care was taken to ensure that the intraventricular fluid was gently squeezed and blotted. Blotted hearts were then cut open and stored overnight in 7'5m1s of 0.4M perchloric

acid at 4-6"C.

2-11 CHAPTER 2 General Methods The next day hearts were cut into âpproximately 1mm slices, homogerused in the perchloric acid and then cenúifuged at 10 000 x g. One ml of the tesulting suPernatânt was added to 5ml of scintillation fluid (Optr-Phase) and subjected to beta scintillation counting (Beckman, USA).

associated Separate experiments were conducted to determine the amount of tissue radioactivity laC- with the extracellular space. These experiments were identical to those just described except sorbitol was substituted fot 'H-NA. The data from these experiments was used to calculate the 3H-NA intracellular concenttadon of (Equation 2. 1)'

2.10.3 Calculation of resulß The extent of extraneuronal uptake was calculated as the amine content of the heart tissue' corrected for the amine in the extracellular space (calculated from the [1aC]-sorbitol content of the heart) divided by the total perfusion time (2 mrn) (Equation 2. 1). Experiments performed in 3 hearts found that the extracellular space contained the equivalent of 293pmol/g' lr uptake-, = xf-xß00 - SS lx --1- pmol I g lmn [[ ) trme HH = radioactivity associated with 1ml of supernatant from heatt homogenâte (DPI\4 SA = specific activity pPM/nmol) B = blank pPlVÐ Wt = werght (g) 3H-N,{. SS = average âmount of uptake into the exttacellular space þmol/g) Time = petfusion time (min)

Equation 2. 1 Extraneuronal reuptake of noradrenaline from isolated peÉused rat heaft (pmoyg/min). The amount of radioacúvity (DPÀ4) associated with lml of perchloric acid was multiplied by the tìrne (2 minutes)' totul .rolo-. of supernatant (7.5rnl). Uptake was adjusted for weight and the total perfusion

Uptake of NA (mean*SE) was expressed as a log,o function of the inhibitor concentration' was used to GtaphpadrM pdsm 4.0 for!Øindows (GtaphPad Software, San Diego, California, USA) nandrolone fìt the data to a sigmoidal dose response curve, from which the ICro was determined for and cotticosterone.

2-12 CH.,\PTER 2 General Methods 2.11Drup Ausualia) The anaesthetic methohexitone sodium fBrietalrM] was obtained from Eli-Lilly (Sydney, Australia)' and pentobatbital sodium fNembutalrM] from Rhone Metieux Pty Ltd @inkenba, NoradrenaLine bitartrate and activated alumina were obtained from Sigma Pharmaceuticals (St. Louis, MO, USA).

Äntibiotics used during surgery included TribrissenrM injectable antibiotic flnmethoprim 8Omg/rnl powdet & sulfadiazine 400mg/ml) from Jurox (Silverwater, NSVø, Australia) and topical antibiotic (neomycin sulphate Z.Srng/ g, sulphacetemide sodium 100mg/g, tiffofutazone 2mg/ g, benzocaine Smg/g) from Apex (St. Mary's, NSW, ,\ustralia). Bupivacaine 0.5% (1\.{arcainrM) used to manage post-operative pain was obtained from Astta pharmaceuticals Qrlorth Ryde, NSW, Australia)'

was purchased from New England lring-2,5,6-1fIf levo-Noradrenaline (1..48-2.96T8q/mmol) 3H-N,4. Nuclear (Boston, MA, USA). All stocks of were purifìed before use by a batch alumina pfocess previously reported (de la Lande et a/, 1967). An aliquot of the manufacturet's stock solution was added to 30 ml tubes containing 10ml of HCi (ltlvÐ, 500mg of acid-activated alumina, ascorbic acid (57mN!, and EDTA QT1I\,\. The solution in the tube was continuously bubbled with N, and adjusted to pH 8.4 using sodium carbonate (1M and 0.11\4. The pH was maintained at 8.4 for 4 minutes, after which the solution was allowed to settle and the effluent the decanted. The alumina was then washed twice with 10mI of distilled water, before eluung 5 rninutes, catecholamine with . Five ml of acetic acid (300m1\Q was added and left for and both after which time the supernâtant was decanted. A futher 2mls of acetic acid was added fractions of decanted acetic acid were pooled. The acetic acid stock was then frozen in a 100m1 conical flask by in a slurry of dry ice and alcohol, and subjected to freeze dttrtg overnight. Priot to use, the 'H-NA was reconstituted into ascorbic, catbogen bubbled I{rebs'

The fìnal concentÍation of labelled noradrenaline used was 1.06x10iM (specific activity = 51.8pCi/nmol).

D-faCl-sorbitol (7.40-13.OGBq/mmol) was also obtained from New England Nuclear. The final

concentration of labelled sorbitol used was 63x1OrM (specific activity = 256þCt/nmol)'

l,2;mgReserpine (Sigma) was rnixed with 125mg of citric acid, 5mls of benzyl alcohol and 45mls of all tesetpine, saline (0.9%o). Sonication and gentle heating over 45 minutes was requited to dissolve which was subsequently disuibuted into 2ml aliquots' 2-t3 CH.,\PTER 2 General Metltods Nandrolone, corticostetone âcetate and patgyline vtefe purchased ftom Sigma (St' Louis, MO, USA). Nandrolone and corticosterorie wete dissolved in 100% ethanol when used in isolated perfused heart experiments. When nandrolone v/as prepffed for infusion small milligtam quantiries a stock solution were stored i¡ a 1,00o/o ethanol solution, from which it was diluted with saline to ranged from 4'0 containing B.7o/o ethanol. The final ethanol concenttation in nandrolone infusions to 6.30/o (4.0 to 6.3 ¡i/rrn$.

Ascorbic I{reb,s was made with the following composition (in mlr!: NaCl 118, KCI4'7, NaHCO. 25, D-glucose 5.6, KH2PO4 1.2, CaCIr 2.5' MgClr 0.74, EDT,{ 0.012, âscorbic acid 0.29. IGebs solution was filtered through a 1.2-¡trn Millipore fìlter befote use. All I(rebs components were

analyttcalgtade purity and wefe obtained from BDH (I(lsyth, Victoria' Australia)'

Australia). U-0521 Cocaine Hydrochloride was purchased from Fauldings Pty Ltd (Salisbuty, South salt' (Jpiohn). ,{.11 doses of cocaine were administered as the hydrochloride

The vehicle for nandrolone decanoate (DECA-50, Natute Vet Pty Ltd, Agnes Banks, NSSø, BenzyI alcohol was Ausualia) was arachis þeanut) supplemented with 1.0o/o benzyl alcohol. purchased from Sigma Chemicals (St. Louis, MO, US)

2-14 CHAPTER 2 General Methods 2.12 References

in Brockway, B. p. and FIassler, C. R. (1993) . Application of Rødiotelen-ttry to Cardiouascalar Measurements phor.oroiigy and. Toxicologt New Technologies and Concepts for Reducing Drug . H' Salem andBaskin, S. I. Boca Raton, Florida, CRC: 109-132'

Brockway, B. P., Mills, P. A. and Azat, S. H. (1991). "A New Method þr Continaoa¡ Chronic Cltn Measarement and. Record.ing of Btood Pressare, Heart Røn and Actiui|t in the Rat Via Radio-Telemetry-" Exp Hypettens A llt(S): 885-95.

A. M. (1986). "The Efects of T@roxiy Treatment of Rats on Bryan,L. J., O'Donnell, S. R. and Williams, 87: Neuronal ønd Extranearonal (Jptake and Metabolism t Catecholamines in the Heart." Br J Pharmacol 337 -344.

of Guiol, C., Ledouss al, C. and Surge, J. M. (1992). "A Rød'iotelenetry $ttum fy Chronic Measarernent Pharmacol Toxicol Blood. pressare and. Heart Røte in tie Unrestmined Rat Validation of the Method" J Methods 28(2): 99 -1,05.

Mclennan, P. L., Abeywardena, M. Y. and charnock, J. S. (1985). "Inflaence of Dietary Lipids on 1'41f - Arhlthmiai and Infarctioi after Coronary Ar-tery Ligation in Røts." CanJ Physioi Pharmacol 63(1,1): 7.

Mclennan, P. L., Abeywardena, M. Y. and Charnock, J' S. (1988). "Dietary Fhh Oil Preuents 709-71'7 Ventricular Fibrillation Foiloøng Coronary Artery Occlasion and Reperfasion'" Am Heart J 116: '

&.771'' Nerer,J., \Øasserman, W. and I{utner, M. (1990).,A.pplied Linear Statistical Models: 741-744

Phjllis, B. D., Irvine, R. J. and I{ennedy, J. ,A'. (2000). "Corttbined Cardiac Efects of Cocaine and the Anabolic steroid, Nøndrolone, in the Røt." EurJ Pharmacol 398(2):263-72.

"Modfication of D-Amphetamine-Indaced Phllis, B. D., Ong, J., White, J. M. and Bonnielle, C. (2001). Responses bl in Rats." Psychophatmacology 153 277 -284'

l-^ambeth Conuention: Gaifulinesþr the Stadl of Walker, M. J., Curtis, M. J. and Hearse, D. J. (1988) "The ArrþTthrniaiin Ischaemia, Infarctlon andRepefusion." catdiovasc Res 22: 447 -455'

2-t5 CHAPTER 2 General Methods The efTect of rat stain on the cardiouasculat response to cocarne

3.1. Introduction

3.1.1 Is the cardiovascular response to intrapert'tonea] cocaine straitt dependentP The clinical observation that the dose of cocaine associated with adverse cardiac effects (Isner et a/., considerably from patient to 19g6) andwith lethality Q.{anji et aÌ.,1.984; Smart et a1.,L987) varied patient without being necessarily related to the toute of administration, led to the hypothesis that these differences were of genetic origin (Heavner et al., 1998). Despite these putative pharmacogenetic differences in the cardiovasculat effects of cocaine in human subjects no published data is available on the effect of race on the catdiac cocaine resPonse. Sirnilady, vety few researchers have examined the potential strain depe4dency of the cardiovascular response to cocaine in rats. It remains unknown whether a difference in the cardiovasculat response to cocaine exists between the 2 most commonly used laboratoty rat strains (Sprague Dawley, SD and Albino Wistar, AW). As much of the published work on the catdiovascular effects of cocaine has used SD

animals we were obliged to confirm our early data obtained with AW rats in SDs. Many of the pilot studies for this thesis were conducted using AW rats. Because ischaemia-repetfusion in SD rats was used in later shrdies (Chapters 5-7) to furthet investigate the potential cardiotoxicity of nandrolone and cocaine it was necessary to determine whethet the cardiovascular response to cocaine was similar between the 2 strains.

Ishizuka et al found that restrained Wistar l(yoto (WKÐ rats showed a signihcantly lesser mean blood pressure (I\ßP) and HR response to cocaine than when they were freely moving (Ishrzuka et a/.,7989). This same effect was not noted for spontaneously hlpettensive rats (SHR). Flowever' restrained SHRs were found to have a signifìcantly greater change in the initial MBP and HR response to cocaine than WI{Y tats. This effect could not be observed in freely moving rats. This

not only suggests that the cardiovascular response to cocaine was sttain dependent in these rats, but that it was also affected by the extent of restraint. Later studies by Jin et al fou¡d that the Ç and B-^ of the high affmq site on the cocaine ïeceptor were lower in SHRs than in WKY tats in the the of the high-affìnity site was found to be lower striarum Çtn et ø/., i.ggl).In the hippocampus Ç

3-l CII,\PTER 3 The ffict of rat strain on the cardìouascalar response to coca¿ne in SHRs. This suggests that the differences in cocaine binding in NØI{Y and SHRs may provide a neutochemical basis for the different responses of the two strains to cocaine administtation.

S¡¡ et alcompared the cardiac effect of cocaine in tats identified as being genetically slow amygdala kindling rats (slow) with the effect in fast arnygdala kindling tats (fast) (Shi er a/.,7999)- These 2 rat strains were originally developed for use in epilepsy reseatch (Fr ctoss between \Tistar and Long Evans rats) and were found to have a diffetential sensitivity to the cardiotoxicity of cocaine. Animals were halothane anaesthetised, mechanically ventilated and were administered cocaine (3 ot 4 mg/kg, i.v.) until they died. ,\rrh¡hmias developed at much lowet cumulative cocaine doses in

Slow-kindling rats than in Fast-kindling rats (15+1 vetsus 42+3 mg/kg, p<0.01)' The lethal cocaine dose was significantly lov¡er in Slow than in Fast strains. It was found that the difference in sensitivity to atthythmia between the two stfâins was abolished by the non-selective c¿- adrenoceptor blocket, phentolamine, suggesting that genetic diffetences between Fast and Slow rats mây be related to the o-adrenergic receptor or factors acting via the ø-adrenergic receptor' lower -Additional studies showed that isolated, perfused hearts from slow kindling rats requited cocaine doses to develop c rdtac arhythmias and arrest compared to hearts from genetically Fast amygdalakindling rats (Heavnet et al.,1998).

Branch and I{nuepfet have conducted a comprehensive investigation of within strain effects of showed a differential cocaine in SD rats @ranch et a/., 1994b; a). They found that male SD tats response in cardiac ouÞut (CO) in response to cocaine while arterial pressure and HR responses were consistent. Based on the CO response to cocaine (Smg/kg, i.v.) it was found that a population could be split into responders (a rnean dectease in CO of 15o/o or more) and non-responders

(smaller negative ot positive inctease in CO). The decrease in CO was found to be related to the rate of dse of plasma cocaine levels and wâs not necessatily dependent on the absolute dose

(Branch et a/., L992).It was found that the differential cârdiovascular tesponsiveness in these rats

was mediated by central sympathetic excitation, which is dependent on ct1-adrenetgic recePtor

activation, reduced by B adrenergic receptor activaúon and is not mediated by cyclo-oxygenase metabolites. These changes could not be attributed to diffetences between responders and non- responders in cocaine metabolism or in ditect cardiz'c responsiveness to cocaine @ranch et a/., lee4b).

3-2 CFIAPTER 3 The ffict of rat strain on the cardìo ua: cu I ar re sþo n s e to co caine mouse strains Ruth ¿/ al found that the HR tesponse to cocalne (0-15mg/kg, i.p) it four inbred varied signifìcantly (Ruth e/ a/., 1,988). Because HR was measuted in mice held in testtaining tubes by subcutaneous needle electrodes, it is not known whethet the different HR tesponses simply of reflect a strain dependent stïess response. No sttain differerlces were found in the concenttation suggesting no pharmacokinetic fF{-cocaine in the brain following injection of Smg/kg, explanation for the süain differences.

A number of investigations have suggested that diffetent behavioural responses to cocaine may be due to strain. These strain differences could result ftom pharmacokinetic or phatmacodynamic factors (Ruth et al., 1988). Only a small number of studies have examined whether the catdiovascular response to cocaine is strarn dependent'

George et al found that the SI-l\ response to vatious cocaine doses (0-60mg/kg' i.p.) was signifìcantly diffetent âmong 4 different inbred rat sttains (ACI [ACI/Nih], LE\Ø ILEW/CRLBRI, NBR tats showed a high degree NBR [[NBR/Nih] and F344 F-344ICRLBRI (Geotge et a/.,1991). of locomotor sensitivity to cocaine, compared to the other 3 sttains. Â full dose-response curve could not be completed for ,{.CI rats as a signifìcant numbet of seizures and deaths wete obserwed striatal at the 4gmg/kg dose. No differences between these strains were found in binding to NBR strain dopaminergic transporteÍs or ïeceptor sites. Interestingly, futther studies found that the no signifi'cant showed the lowest LDs. with cocaine (George' 1'gg1). However, over all 4 strains, correlation was found between the Sr-A effects of cocaine and lethality. Sensitivity to cocaine- induced lethality was found to be highly correlated with baseline levels of ambulatory acuvity (r=0.99, p<0.01). Sirnilarly, cailhol and Mormede found that in \øKY, !øKHA and SHRs administered cocaine (}-2}rng/kg, i.p.) female SHRs showed signifìcantly gfe ter locomotor IMIGIA Thts stimulation compared to IVI{Y (p<0.001) and þ<0.001) females (Cailhol et a/', L999)'

same strain effect was not observed fot males'

stralns ln Despite observations of differential behavioural responses or HR in inbted rât or mouse and HR diffetences in the response to cocaine, a diffetential CO response between SD rats, and BP for notmotensive versus hypettensive fâts, none of these fesPonses to cocaine have been assessed study aimed to strain dependency in commonly used laboratory nofmotenslve fat stfains' This moving AW and characterise the card.iovascular response to i'p. cocaine in fully conscious, freely to a strain- sD rats. To avoid changes in HR (Ruth øl a/., 1,988) and BP between stfâins due dependent stfess response; conscious, freely moving rats wefe used for this study.

3-3 CH,\PTER 3 The ffict of rat strain on the cardìouaç calar response to co ca¿ne 3.2.Aim tr To determine whether a sftain related effect in resPonse to cocaine in tetms of HR, BP and SI-A can be observed between SD and,\W tats'

3.3. Hypothesis The HR, SLA, DP and SP response to cocalne will not be statistically significantly different between sttains.

3-4 CITAPIER 3 The ffict of rat strain on the cardiouascalar resþonse to cocatne 3.4. Methods 3.4.1 Animals Albino Wistar or SD rats were obtained at 12 weeks and housed as per 2.1 General Methods' Anknals were implanted with radiotelemetry devices for tecording HR, SI-A, DP and SP as per Ceneral

Methods 2.4.

3.4.2 Protocol home On each test day the animals were admirristeted cocaine HCI i.p. and then returned to their Baseline cages. Simultaneous recordings of HR, SL'\, DP and SP were carried out for 150 mlns. values were determin ed at 290-300 rninutes following &ug administration. To avoid recording the immediate stress response following handling (Irvine et a/.,1'997) and drug iniection, all variables were recorded from 15 rninutes onwards. Test days wefe sepafated by a minimum of 48 houts' Dose response curves weïe genefated for cocaine (0, 15, 45 mg/kg)' Each dose was administered on 3 occasions.

3.4.3 Data.Analysis Results for HR, SL\, DP and SP for each group of triplicates were averaged and plotted as a function of time. In order to maintain consistency with previously published wotk Qhllhs et a/., 2000; phillis et a1.,2001) which examined cardiovâscular tesponses to sympathomimetic drugs, comparisons were made by calculatmg under the cutve (AUC). AUC was between strain ^rea. BP determined by the tapezorðal method using Gtaphpad PRISM 4.02. The baseline fot HR and The was the of the values at 290, 295 and 300 minutes following drug administtation' ^vef^ge saline ot baseline for SI-A was zero. Between strain differences in the AUC fot the response to

cocaine were determined using an unpaited t-test ât each dose'

3-5 CII,\PTER 3 The ffict of rat $rain on the cardiouascalar retþonse to cocatne 3.5. Results

3.5,1 Time course of tlte cocaine response in Both strains showed an inctease in SI.\ and BP in response to cocaine. A small initial decrease HR was observed in compadson to saline at the highest cocaine dose. In both ,A.\X/ and SD tats this effect lasted until 45 minutes post iniection (Fig 3. 1 C.). Heart rate decteased by 1'0.9+3-5o/o

(4g.3115.6 bpm) in ÄW rats and 10.7+3.4Yo (42.I+13.2 bpm) in SD rats in resPonse to cocaine saline 15 minutes post injectron. No significant 1+5mg/kS) in comparison ro the effect of at difference was found in the size of this decrease between the two sttains (unpaired t-test). ,\n initial SI-A dectease in response to cocaine in comparison to the response to saline u/as not observed for

(Fig 3. 1 D-F), DP (Fig 3.2 A-C) or SP (Fig 3. 2 D-F)'

3.5.2 Between st:ain difl'erences in the IfR, SIA', DP attd SP response to cocatne'

Ca¡diovas cular effe cts ,\W rats were found to have a signifìcandy greater HR response to saline [t(1t¡=3.316' p<0'01] compated to the SD strain (Fig 3. 14.). This difference was not evident for either concentration found of cocaine (15 or 45mg/kg) (Fig 3. 1 B & C). No signifìcant difference between sttains was 3' for the magnitude of the AUC for the DP or SP response to saline or cocaine (Fig 3. 2 A-C Fig

2 D-F).

SLA effects AUC for the SLA response to saline compared to SD rats AW rats were found to have ^ gteater the zu.ea under the SI-Â versus time graph was It(tt¡=2.714, p<0.05] (Fig 3. 1D.).Conversely, the tesponse to cocaine found to be greater in SD rats [t(11)=2.446, p<0.05] compared to AW fot (1smg/kg) (Fig 3. 1 E.). No signifìcant difference between strains was observed at 45mg/kg (Fig 3. 1F.).

3-6 CH,A,PTER 3 The ffict of rat strain on the cardi o uas ctt lar re sþo n s e to co cazn e E ¡a 5 f ; A. t

ffi 8¡dn

ú I ÊE Þ o É - 3 t ¡so +sD +AìV *AW s Ès ,û \e {9 + ê .& \o ú êdF lho (mkliæl ^ê4P Tim ûn'nuþ.I

Ê t É È E 3 o B. E a =

gûdlr ¡ E 3è É- I 5 6

s .F .S \ê e "f +rP.f\O^f l"ma(minl6) "ft Tino (n¡ruû6t) "Ðd

¿ f c" F" ffi st-¡¡r

I Ê. Â É 7

,\" s .Ê ê ê rSS É .Ê"f + ç .ê.f lin. (m¡uisù) fm6(min¡ð)^o ^f

Frg S. 1 Lú pnel:The heart rate (HR) nesponse in AW (^) and 5D ( æ¡ rats adm¡niste¡ed n*p.0.01, (c) coca¡ne. (A) 0mglþ [.r=Gï, trnset: AIJC, urpaired t-test. @) tsrîglks In=G7f, Inset: AUC. tthngflrl[n=]51, rnset: ArJC. Right pnel:The spontaneorF bcomoûor activÍty response (sl ) *p<0'05 ln Alt (^) and SD ( x) rats adminlstered cocaine" (D) Omgltg Ír'=Ç4, Inset: 1'tJC, (E) *p.û.05. ,LIJC. On all tisre-coutse graphs the value at Nsfr¡gf kg [n=Gfl, Inser: ÂUC, (F) 4ífff¡glkg fn=]5], Inset: 3ffi minutes is the averaç of the values at 2X), 295 arñ 300 minutes following drug administration'

2.' ATI ÀffiN 4 T¡ tr A. t .* ür

E I I t e Ê À a-o o

+AIY +rlw .ÊÐ \o +e \o û \e,tP TrE(Ei¡ûhsl "ú "tdÞ filE('lûllrþsl

E

Ë B. a ã ffi

O 1 i I E À oa â

,gЀ\o€^êdP e t € \o t "?f Træ(nin ¡b3l f¡nE(tÍn¡!Él

G. F á

D Ê E 1 À 6 a

.Ê .} {È \O s e Ð 46 \e $,f IIÍE(trinl¡bsl "f "44F t¡¡E(rriñE ) "f

F¡g 3. 2 Lû Ft¡d: d¡asblic press¡lre (DP) response in SD (r) and AW (^) rats fuIJC (9 +S'rry/lq' admin'rstercd co6¡ne. G) ¡mgllg W=cry, Inæt: AIJC (B) lsmglþ þ=G4,Insett (SP) in SD ( r¡ and AW ( ræ þ=l51, rnset Auc- Riglrt nnel: sysûolic pressurie r€sporise ) dminifrrg¿ 6gca¡ne. (D) ûn¡glLg Þ=ÇT,In*t: ArJC (E¡ tsms/le flû=Ç'll,In*t: A1JC F) aítry/4 value at 3ffiminutes is the averøge of the values atZW' W=TSLInset:ÂUG MeanÈS.E- Oft ãlI tftne-course grãphs the 295 nd ffi minutes following drug admiristration'

20 ÆrÀffiñ â Tf F . I 3.6. Discussion

Although a significantly greater HR response to saline was observed in AW rats compared to SD, no such difference was observed for cocaine. Likewise, no signifìcant difference between strains was found for the DP and SP response to saline ot cocaine. This suggests that direct comparisons can be made between the cardiovascular responses to cocaine in the 2 most commonly used tat strains in the published literature. This preliminaty study also supports the use of SD tats in later chapters of this thesis to investigate the cardiac effects of cocaine during ischaemia-reperfusion and allows a comparison to be made to previously published work (Phillis et a/',2000). A pronounced and significantly greater SLA response to cocaine (15mg/kg) was found in SD rats compared to ÄW rats. This suggests that while the behavioutal effects of cocaine may be sttain dependent at some doses, the cardiovascular effects are independent of strain.

It is perhaps not surprising that there ate no significant cardiovascular differences between these 2 strains at the 45mg/kg dose considedng that SD rats are not untelated to Wistars. Sprague Dawley ïats believed to have originated from Wistat females crossed to a 'hybrid' male of unknown ^ïe origin (Lindsey, 197 9; Pass et al., 1'993).

Tolerance to the cardiovascular effects of cocaine is unlikely to explain the lack of a strain effect.

Ansah et ø/ reported no toletance to the HR, SI-A and cote temperature (CT) response to cocaine in Wistar rats (Ansah et a/.,1996). Rats were treated with a once daily dose of cocaine (2}mg/kg, i.p.) and HR, SL,\ and CT measured using radiotelemetry implants. The tachycardic response to cocaine did not change between days 1 and 30 of administtation, indicating that the HR resPonse did not undergo sensitisation or display tolerance. The SI-A response was found to show a marginally significant (p=0.0514) increase in cocaine administered animals across the 30 day treatment period, suggesting that cocaine may induce sensitisation to behavioural effects. However,

changes in SLÄ over 30 days were cyclical in nature and it was acknowledged that the "pattern of

response may be due to vadability in animals, rathet than aphysiological effect"' The present study

used an interdosing intewal of 48 hours. Ptevious studies in non-telemetered animals have found sensitisation to the behavioural effects of interrnittent cocaine, and tolerance to continuously administered cocaine, cardiovasculat effects wefe not reported (R-eith et al',1'987)'

The increases in tsP with cocaine evident in this study ate consistent with work by Pan and Hedaya who exarnined the cardiac effects of i.p. cocaine in the milc rat and demonsTtated a very mild but in uiuo model of sustained cardiovascular effect Qan et a/.,1.998). Pan and Hedaya designed a novel drug disposition, which allowed investigation of simultaneous pharmacokinetic and dynamic effects femoral of cocaine in the same animal (male Wistar rat) by multiple routes. Gastric, abdominal and 3-9 CH,A.PTER 3 The ffict of rat strain on the cørdiouascalar response to cocatne vein câtheters allowed administtation of cocaine by the P.o., i.P. or i.v. route without handling the anmat. Mean arterial ptessure following cocaine (3Omg/kg, i'p.) was found to increase by over the 250 approximately 1,3o/o above baseline at its peak, and sustâined a higher level of pressure minutes of the study compared to i.v. administration. Likewise, both i.p. and i.v. cocaine administtaúon produced a decrease in HR of about 25o/o at its lowest point. With ì'p' longet administration the bradycardic effect of cocaine was prolonged for more than 60 minutes this was not than i.v. cocaine. Although an initial decrease in HR was observed in the present study of the magnitude reported by Pan and Hedaya in awake, freely moving rats. T,ater studies in this thesis found that i.v. cocaine administration ptoduced more significant bradycardia (a reduction of in approximately 70bpm from baseline) (Fig. 6.4). However, studies in chapter 6 were conducted to anaesthetised animals. Pan and Hedaya showed the long lasting nature of the cardiac response i.p. cocaine in the rat by demonstrating that the QRS interval in rats administered i.p' cocaine was still prolonged at 250 minutes following injection. In compatison the QRS intewal of rats administered i.v. cocaine had returned to neâr baseline levels by approximately 50 minutes'

Most avatlable literature on the cardiovascular effects of cocaine in rats concerns 1.v' administration. Flowever, the cardiovascular effects of i.p. cocaine temain important. It has been the demonstrated that the distribution kinetics of cocaine following i.p. injection in rats is similar to absorption profile obtained by intranasal administration in humans flavaid et a/., 1'993).Intranasal insufflation (,snorting') and smoking have increased in populatity over the past 15 years (Hatsukami et ø/., 1996). Current estimations of the proportion of current tecreational cocaine users adrninistering cocaine by nasal insufflation range from 30-40o/o (Gossop et a/., 1994; Fe:'.ri et ø/', concentration of cocaine in rats 1999; Goss op et a/., 2000). Javaid et ø/ found that the plasma maintained administered i.p. cocaine reached maximum at about 10 minutes post injection and was in at this level until approximately 60 minutes flavaid et a1.,1.993). Javaid noted a similar profile et a/', patients administered either a 32 or 96mg cocaine dose inttanasally Savaid et a/.' 1'978;Javøtd hour following 1,gg3). The maintenârrce of a constant plasma cocai¡e concentration for the fìrst administration is consistent with the results presented here. In both strains BP temained elevated for the first 60 minures following cocaine administtation. Although HR initially declined in and response to cocaine, peak HR wasn't obtained cr;rnI 120-140 minutes post iniecdon. lØilkerson snorting colleagues observed a "sustained peak" plasma cocaine concentration in male subiects response was doses of cocaine between 0.19 and 0.75 mg/kg (\(/ilkinson et a/', 1'980)' A peak 70 achieved by about 15 minutes post administration which was maintained until approximately minutes post administration.

3-10 CIIÂPTER 3 The ffict of rat strain on the cardiouasca/ar resþznse t0 clcøzlte 3.7. Conclusion The HR and BP response to inttaperitoneal cocaine is not strain dependent. Spottaneous locomotor activity was found to show a strain dependent tesPonse to cocaine at T1mglkg which was significantJy greaterin SD tats and abolished at high concentration (45mg/kg, i'p')' Therefore behavioural effects at low concentrations of cocaine mây be strain dependent, but this does not affect the catdiovascular response.

3-ll CFIAPTER 3 The ffict of røt strain on the cardioaøscu lar resþonse to cocaine 3.8. References

Ansah, T.,'Wade, L. H. and Shockley, D. C. (1996). "Changes inI-,ocomotorActivitL CoreTemþeratare, 1261,-1267 . and. Heart Rate in Response to Repeated Cocaine Administration." Physiol Behav 60(5):

Branch, C. A. and I(nuepfer, M. M. (1992). "Adrenergic Mechanisms [Jndtrþing Cardiac and Vascalar Responrcs to Cocaine in Conscioas Rats." J Pharmacol Exp Thet 263Q): 7 42 51'.

Branch, C. A. and ISuepfer, M. M. (1994a). "Causes of Dffirenlial Cørdiouascular Sensitiuitl to Cocaine. I: Stadie: in Consdou¡ I7aß." J-Phatmacol-Exp -Ther 269(2): 67 4-83'

to Cocaine. Branch, C. -4. and l(nuepfet, M. M. (1994b). "Caases of Dffirential Cardiouascular Sensitiui4t Ii: s1mþathetic, Metabolic aid cardiac Efects." J-Phatmacol-Exp-Ther 277(2): 1'1'03-1'3.

Cailhol, S. and Mormede, P. (1999). "strain and Sl,x Dffirencu in the l-'ocomotor Resþonse and Behavioral Serusitisation to Cocaine in Hlperactiue R1tÍ" Brâln Res 842: 200-205'

Feri, C. p. and Gossop, M. (1999). "Roate of Cocaine Adrnini:trøtion: Patterns of Use and Problemt aruong ø Bra{lian Sanp/t." Addictive Behaviors 24(6):815-821''

George, tr. R. (1991). "Cocaine Toxicilt: Genetic Dffirences in Cocaine-Indaced l-,ethø/i4t in Rnts." Pharmacol Biochem Behav 38(4): 893-5'

George, F. R., Porrino,L.J.,Ritz, M' C. and Goldberg, S' R' (1991)' Comþarìsons IndicaTe Dffirent S;tei of Action for Cocaine and Amþhetamine ttt Efects." Psychophatmacology 10 4 $) : 457 - 62.

Gossop, M., Griffiths, P., Powis, B. and Strang, J. Q,994). "Cocaine: Patîerns of Use, Roate of Adrninìstation ønd Seuerilt of Dependence." BtJ Psych 164: 660-664'

Gossop, M., Marsden,J., Stewart, D. J. and,Trcacy, S. (2000)' "Roates of DragAdministration and Matttpie Drug Miswse: Regional Variations among Clients Seeking Treatrnent at Programmes Throaþoat England." Addiction 95(8): 1,1.97 -1.206.

Harsukami, D. I{. and Fischman, M. W. (1996). "Crack Cocaine and Cocaine Lþdrochloridt'"J Am Med Assoc 27 6(1,9): 1 580-1 588.

G., Wang, M. and Mclntyre, D. C. (1998)' Fleavner, J. E., Shi, 8., Inners-McBride, I(, Asimakis, J' ,,Cocaine Cardionx;cit1 Dffirs Markedþ in Isoløted Heat'ts of Two Strains of Rats Exhibiting Phenofripic Dffirences in Sensitiuifl to Seilaru." Life Sci 63(8): 625-33'

Efect of Restraint on B/ood Pressare'"J Pharm Irvine, R, J., White, J. M. and Chan, R. (1997). "The Toxicol Methods 38 1'57 -1'62.

Ishizuka, Y., Rockhold, R. 'Ví'., I{irchner, I(., Hoskins, B. and Ho, I. K. (1989). "Dffirential Serusitiui\t to Cocaine in Sþontaneoasþ Íþþertensiue andll/istar-þoto Rat¡." Life Sci 45(3):223-32.

Nordin, M. R., Subtamanian, R., Miller, Isner, J. M., Estes, N, A. D., Thompson, P. D., Costanzo G., I{ãtsas, G., Sweeney, I(- and Stirrner, W. Q. (1986). "Acate Cardiac Euents Temþoralþ Reland n Cocaine Abasø. " N EnglJ Med 315(23): 7438-43'

Disþosition in Discrete Regions of RatBrain."Biopharm Javatd,J. L and Davis,J. M. (1993). "Cocaine Drug Dispos 14(4): 357-64'

3-L2 CH,A.PTER 3 The ffict of rat strain on the cardtio uas ca lar re sþ o ns e to co ca¿n e I., Fischman, M. W., Schustef, C. R., Dekirmenjian, H. and Davis, J. M' (1978). "Cocaint "plasmaboncentration:Javaid, J. Relation n Pþtsiotogica/ and Subjtctiue Efect in Hamans." Science 202(ß6\:227-8'

V., Rockhold, R.W., Floskins, B. and Ho, L K. (1991). "Compari:ons of Cocaine Receþtors in Brain -RegionsfroruJin, lf,/k1t and Shr'" Btatn Res Bull 853-856(27): 853-856'

Rat - Biology and Diseaset. H. Baker' Lindsey, J. R. (1979). Historical Foundation¡. The Laboratory J. NY, Academic Press. 1"

with Cocaine Nanji, A. A. and Filipenko, J. D. (1984) . "Atyto/e and Ventricalar Fibrillation As¡ociated Intoxicøtion." Chest 85(1): 132-133. Pan, ì7. J. and Hedaya, M. A. (1998). "An Animal Model for Simultaneoas pharmacokinetic/Phannaco@namic Inuutigations: Applications to Cocaine,"J Pharm Toxicol Methods 39: 1- 8.

Pass, D. and Freeth, G' (1993). "The Rnt."ANZCAART News 6(+):1'-4'

and the Phjllis, B. D., Irvine, R. J. and I(ennedy, J' A. (2000). "Combined Cardiac Efects ( Cocaine Anabolic Steroid, Nandrolone, in the Rat." E*J Pharmacol 398(2):263-72. phillis, B.D., Ong,J., White,J. M. and Bonnielle, C. (2001). "Modfrcation of D-Amphetamine-Induced 153 277 -284' Resþonses fu BacÌofen in Rats."

Reith, M. E. 4., Benuck, M. and Laitha, A. (1987). "Cocaine Disposition in the Brain after Continuoas or Interruiltent Treatment and l--ocomotor Stimalation in Mice." J Pharm Exp Thet 243(1): 281'-287 '

A1aþsis of Cocaine Eft'tt on l-'acotnotor Ruth, J. A., lJllman, E. A. and Collins, A. C. (1988)' "A, 29: 1'57 -162' Acûuir;es and. Heart Rat€ in Four Inbred Mouse Strains." Pharmacol Biochem Behav

C' and Reigel, C' E' Shi, !,., Fleavner, J. E., Liu, J., Wang, M' J., Lutheret, L' O', Mclntyte, D' ,,Two (1999). Geneiicalþ Sekcted Straint o¡ßott E*hibit Lþpercm:itiui4t orResistance to Cocaine-Indaced Fatal Anhlthmias."J Pharmacol Exp Thet 288(2) : 685-92'

Smatt, R. G. and ,\nglin, L. (1987). "Do lYe Know the l-,ethat Dose of CocaineT" J Fotensic Sci 32(2): 303-1,2.

'Wilkinson, and Oral Cocaine P., Van Dyke, C., Jatlow, P., Barash, P. and Byck, R. (1980). "Intranavl Kìnetics." Clin Pharmacol Thet 27(3):386-94.

3-13 CHAPTER 3 The ffict of rat strain on the cørd'ioua¡ cu lar re[Pznse to co caine C/"-F"tu ¿l

TIte effect of acute nartdrolone and cocatne on heart rate

4.1 Innoduction

Recent ïeports suggest that testosterone can have an acute effect on vessels and platelets in uitro, independent of the slow responses usually associated with changes in . Co- incubation of human platelet dch plasma with testostetone and cocaine has been found to induce a significant increase in platelet aggregability in response to a sub-thteshold concentration of sodium atachidonate even in the presence of the androgen antagonist, flutamide (Togna et a/.,2003). Togna

et al. also showed that co-incubation of endothelium-intact, phenylephrine pre-conttzcied, rabbit aortic tings with testosterone, potentiated the cocaine induced inhibiuon of acetylcholine mediated vascular relaxation by an androgen-independent mecharusm. Similatly, Ceballos el al showed that

testosterone can block the vasodilator effect of adenosine in isolated perfused rat hearts (Ceballos a/ at.,1,999).This effect occurred even v¡hen testosterone transport into the cytosol was prevented or protein synthesis inhibited.

In light of recent reports of the acute effects of testosterone (Ceballos et al.' 1999; Togna tt a/',

2003), the previously reported (Phillis eî a/.,2000) intetaction between nandtolone and cocaine in terms of HR was re-investigated. The aim was to determine whether this interaction required chronic exposure to nandrolone or whether it might have tesulted from an elfect of the last dose of nandrolone priot to acute cocaine injection. In otder to determine whether the enhanced chronotropy was sirnply due to a direct, acute effect of nandrolone it was necessâry to observe whether prior exposufe to a single nandrolone dose (2Omg/kg, s.c.) changed the HR response to

cocaine, as per the previously repotted chtonic study Qhillis et a1.,2000).

4-7 CTIAPTER 4 The ffict of acute nandrolone and cocaine on heart rate 4.2 Aim o To evaluate whether a single of nandrolone Q\mg/kg, s.c.) can significantly increase the HR, core temperature (CT) and SI-A resPonse to cocaine (45mg/kg, i.p.) compared to vehicle tteated controls.

To determine the plasma concentration of nandrolone 250 minutes aftet injection of a single subcutaneous dose (2Omg/kg, s.c.)'

4.3 Hypothesis potentiation of the tachycardiac effect of inttaperitoneal cocaine by nandrolone occuts after chronic administtation of nandtolone but not aftet acute administration.

4-2 CFIAPTER 4 Ttte ffict of acute nandrolone ønd cocaine on heart rate 4.4 Methodolory

4.4.1 Animals Eighteen male albino Wistar ïats were purchased at 12 weeks and wete housed, and implanted with ECG radiotelemetry implants (Physiotel implant, model TA11CT,A.-F40,Data Sciences, St. Paul,

Minn., USA) as described in general methods, secttons 2.1,2'3 and 2.4.1, respectively'

4.4.2 Protocol Rats wete divided into 3 equal groups, and were assigned to receive either a single dose of nandrolone (2}rng/kg, s.c.), saline or vehicle. Rats were allowed an accLimatisation petiod of 30 minutes following activation of the radiotelemetry implants. Nandrolone decanoate (2)mg/kg), vehicle or saline were then administered subcutaneously. In accotdance with the ptotocol from the previously reported chronic study (Phillis et a/.,2000), telemetry monitodng was then continued fot 210 minutes before the administration of cocaine (45mg/kg, i.p.). The effect of cocaine was recotded for a further 180 minutes'

Locomotor activity was used as a phatmacodynamic measure of cocaine effect and was found to peak at40 minutes (250 minutes after pre-treatment with nandrolone, saline or vehicle). In ordet to deterrnine whether there were appreciable levels o[ nandrolone present at the onset of the peak s'c', cocaine effect, a sepârate group of animals were administered either nandtolone (2Omg/kg, n=3) or vehicle (n=3). Animals were ânaesthetised with pentobarbital (45mg/kg, i.p.) 15 minutes Blood before blood collection. Blood was collected ftom the abdominal aofia after 250 minutes' Plasma wâs centrifuged for 10 minutes at 7 000 x g and the tesulting plasma frozen in 5ml aìiquots. was sent on ice to the Racing Fotensic Laboratories (Randwick, NS$q where it was assayed for nandrolone using GC/MS. The limit of detection was 2ngf rnl'

4.4.3 Drugs

Vehicle was prepared from 1'Oo/obenzyl alcohol and peanut oil

4-3 CH,\PTER 4 The ffict of acate nandrolone ønd cocaine on heart rate 4. 4. 4 S ta tis ti cal Analysis Area under the curve calculations The mean and S.E. were calculated for each gtoup of rats at each sampling interval fot each vadable recorded. To maintain consistency with previously published work ("hillis et a/., 2000; phillis et a/., 2001) rhar examined HR, cT and SI-a tesponses to sympathomimetic drugs, area under the curve (AUC) was used to compare drug tesponses between tÍeatment groups' Calculation of AUC was by the trapezoidal method. !Øhen analysing both the resPonse to treâtment and the response to cocaine the same baseline was used, which was detetmined to be the avetage of the last

3 values for the HR, or the CT response to treatment with nandrolone, saline or vehicle (i.e. values at200,205 and210 minutes). A baseline of zero was used for SI-A'

The HR and SLA response to treâtment with nandrolone, vehicle ot saline was compâred to the treatmerìtplus cocaineresporìse by calculating theÂUC for the ftst 180 minutes of the treâtment response and comparing this to the 180 minutes ovet which the cocaine response was recorded. The CT respoflse to cocaine was below baseline at a number of time points and the response to pre-treatment with nandrolone, vehicle and saline was largely above baseline (see Fig. 4. 2). As the CT response to cocaine was found to involve 2 phases, an initial dectease (0-40Ð and a subsequent separately fot each of these phases, and the îet increase (45-180s) the AUC was analysed ^tea determined.

Method for detennining whether pte-treatment oÍ Pre-teatment plus cocaine signifrcantþ altered AUC Differences between groups in the HR or CT response to treâtment alone or treâtment plus

cocaine wete determined using a one-way ANOVA.

Method for detenniníng whether cocaine signifrcantþ incteased the AUC resPonse compared to treatment alone To determine whether cocaine had an effect on HR or CT an unpaired t-test wâs used to examine differences in the ÂUC fesponse in the presence and absence of cocaine.

4-4 CFI,A.PTER 4 The ffict of acute nandrolone and cocaine on hear't rate 4.5 Results

4.5.1 Plasma levels of n¿utdrolone Nandrolone was not detected in plasma from vehicle treated rats. ,{. single dose of nandrolone (3+1 ng/rnl) aT250 minutes eOmg/kg, s.c.) produced a plasma nandrolone level of 1,2.1.+3.2nM aftet injection.

4. 5. 2 Pre- neatmett t resPon se No signifìcant difference was obsewed in the HR (Fig. 4. 1 C.), SLA (Fig. 4. 1D') or CT (Fig' a' 8., C.) response to treatment with nandrolone, saline or vehicle (single-factor r\NOVrA.).

4.5.3 Cocaíne response In all treatment groups cocaine was found to significantly inctease the area undet the FIR versus time curve in comparison ro treatment with nandtolone lt(10)=7.137, p<0.001], saline[t(8)=2'916, significantly p<0.051 or vehicle [t(l;=5.6UU, p<0.001] alone (Fig. 4. 1 C.). Likewise, cocaine increased locomotor activity compared to nandtolone alone [t(10¡=3'326, p<0'01], saline alone (Fig. a. 1 D.). ft(10)=3.711, p<0.011 and vehicle alone [t(10)=3.931., p<0.01]

The CT response to cocaine appeared to be biphasic (Fig. 4.2). An lnitral and immediate dectease in CT was observed which lasted for the first 10-20 minutes following cocaine injection' This

decrease was not evident in the absence of cocaine. The initial decrease was followed by an increase which reached maximum at approxirnately 80 minutes post cocaine injection in all pre-tteatment groups. The 2 main parts of the biphasic CT response were analysed separately (ie. 0-40 minutes minutes Cocaine caused a significant dectease in {AUC'40 -i,,.,",} âûd 45-180 {AUC45rso -i.ut".})' treatment with nandtolone p<0.05], saline [t(10;=6.3r0' ,\UC0_40 -;,,r". coflpâred to [t(9)=2.897, increase in the AUC45 p<0 0011 or vehicle [t(8)=4.263, p<0.01] alone (Fig. 4.2B.). A significant tesponse to cocaine in all pre-treatment gfoups i'e' nandtolone 180 minutes was demonsttated in and vehicle p<0.01] pre- II(IO¡=3.132, p<0.051, saline [t(10)=3.053, p<0.05] [t(10¡=3.557, treatment (Fig. a.2 C.).

4.5.4 Effect of nattdrolone pre-lreahnent on response to cocailte to There \Ã/as no significant difference in the HR (Fig. 4. 1 C.) or SI-A (Fig' a' 1 D') response cocaine in tats treated with nandrolone, compated to those treated with vehicle. Similarly, no difference was found in the respoflse to cocaine in the 3 different pre-treatment gtoups fot 0-40s

and 45-180s of the CT tesponse (Fig. a. 2 B.' C')'

4-5 CII,\PTER 4 The efect of acate nandrolone and cocaine on heart rate G A. .lI{ +s åI +v tr!þ1C .= a os.c q fo Ê +VrC E { o, 3ê -o t É, () - I = I i

{i0 0 t50 2ú t'¡ S V l¡rc S+C V+C TÌme{m¡tries} Trealment

B. D

o 'E C' 6 6 o c ë I J û o o tr o o= ] J o -¡ I o C)l t glc 6G 5l) 1lþ 151! 2lx, 2s0 N s v N+c V+C

TTrne (mirutes) Treatment elg. 4.1 Time counee of the HR, (4,) and SLA (8.) respolsc to tr€atment with nand¡olone (rjn satine (o) or yehide (V) alone (sol¡d tines) cornpared with tñe subseguent tleat¡nert wlfr cocaine-(dotffi lilrc) in rats pre-trcarEd with nandrolone (n), saline (o) or veñicle (V). Ðotted vertical lines rcpresent the last x value used for calculating ÂUC. ArcA Under,the CurUe (Arcc ,-f for the HR (C.) an¿ St¡ (D.) vesus time response to trealnrent alone (N = nandrolone' 3É sali¡re, V veñ¡ae) and pre-treaünent plus coca¡ne (N+C =nandrolone+orrca¡ne' S+C = = *p<Û.05, xxp<0'01, ***P'0'0Ûtr Saline + Co6a¡ne, V+C = ve$iCle + @Ca¡ne)' Mean*SE, n=16- sigpifrcantly difttent ft,om t¡eâtment aløre, unþred t-test- Area 2 so 38 A. o a oa Io C)l voÌ l-¡ I 37 ()

T 37

-50 0 50 100 150 200 25C Time (minutes) B. 1

.EE () N+C S+C V+G ø) o NSV ä ** f{ Treat¡rent

-1

c. ,l * å

.E E C' o N S v Ít¡+c s+c v+c l Trcatment

-1

W" 4.2 (A) Time coutse of the aore temperat¡rc (CT) rcsponæ to treatment with na.-nd¡o¡on" (.), saline (o) or vef¡i(fe (V) alone (solid line) cornpared with the subsequent (o) hatment wjth cocaine tilotæc line) in rarts pretreated with nandrolone (o)' saline or ftom the wh¡de (V). Tne decrease in CT in resprrse to cocdne (denoted "ry"^ 1') was malysed separately 2 imcrcase (,aæx t). Àttga under t'lre curve for area I (AUCG4oo,nnn-) (8") and area (AUgn+à"**."Í (C.) fo¡ treaünent alone (fl = nandrolone" S = saline¡ V = vehicle) and ù'eaune¡rt + cocalnó (il+G*= nand¡olone + cocaine, S+C = saline + cocaine, V+G = vehicle unpaired t- + COCaine). Mean+SE, n=4i6. p<û.û5, p<û.tl, p<û.0û1 sigpificartty di rent frorn üeâtrnent alone' fest. 4.6 Discussion

These results indicate that a single dose of nandrolone (2}rng/kg, s.c.) to natve rats, results in a quantifiable plasma concentration, at 250 minutes following iniectron.

Although intraperitoneally administered cocaine was found to significantly increase the

AUC'-ls.minures foï HR and SI-A and the AUCor-rs'nrinures for CT in comparison to the response to treatment alone, the magnitude of this difference was not significantly different between treatment groups. This suggests that while a significant cocaine effect can be observed with a dose of 45mg/kg,pre-rreatment with a single nandrolone dose (20mg/kg, s.c.) does not alter the HR, SI-A or CT response to cocaine. ìØhjle a single dose tesulted in an appreciable plasma nandrolone level, this did not potentiate the HR effect of cocaine as was observed previously v¡ith chronic nandrolone treatment (Phillis et a/.,2000).

This study suggests that the enhanced chronotropic response to cocaine (45mg/kg' i.p.) resulting from chronic nandrolone treatment reported previously, was unlikely to be due to an acute effect of the previous nandrolone injection. Howevet, it cannot be excluded that a direct, acute effect of nandrolone might be observed with a higher single dose

4-8 CIIAPTER 4 The efect of acute nandrolone and coca¿ne on heart rate 4.7 Conclusion A single subcutaneous dose of nandrolone (2Omg/kg) distributes from the site of injection to produce detectable levels of nandrolone at 4 hours or more following injection' A single subcutaneous injection of nandrolone does not change the HR response to cocaine (45mg/kg, i.p.) in comparison to other pre-treatment groups. The SI-A and CT response to cocaine are also unaffected by pre-treatment. Thus the effect of nandrolone upon the HR resPonse to subsequent cocaine administration (observed previously) is a result of the chtonic effects of nandrolone treatment rathet than a direct drug-.

4-9 CITAPTER 4 The ffict of acute nandrolone and cocaine on lteart rate 4.8 References

Ceballos, G., Figueroa, L., Rubio, I., Gollo, G., Garcia,r\., Martinez, A.,Yanez,R.,Petez,J., Motato, T. and Chamorro, G. (1999). "Acute and Nongenomic Efects of Testosterone on I.çolated and c Pharm acol 33 : 69 7 - 697' P efu s e d Rat He art. " J Cardtovas phìllis, B. D., Irvine, R. J. and Kennedy, J. A. (2000). "Combined Cardiac Efects of Cocaine and the Anabolic steroid, Nøndrolone, in the Røt." EurJ Pharmacol 398(2): 263-72. phillis, B. D., Org,J.,'White,J. M. and Bonnielle, C. (2001). "Modfication of D-Ampheîanine-Indaæd 153 2:77 -284' ReEonses þt Baclofen in Røt¡." Psychophatmacology

Togna,G. I., Togna, A. R., Graziari,M. and Franconi, M. (2003). "Testosterone and Cocaine: Vascalar Toxicilt of Their Concomittant Abuse-" Thtomb Res 109: 795-207'

4-10 CI{APTER 4 The efect of acute nandrolone and cocaine on beart rate The eÍlect of innavenous nandrolone in raß subjected to cardiac ischaemia and repet{usion

5.1 Introduction The cardiovascular consequences of AS administtation to râts repotted to date have been moderate. Chronic nandrolone administration to rats has been associated v¡ith a signifìcantly heightened HR response to cocaine (?hillis d at., 2000), with accelerated development of hlpertension in developing, spontaneously hlpertensive rats (Iseng et a/', 1'994) and with left ventriculat hypertrophy (Tseng et a/.,7994; Tdfunovic et a1.,1995) in sedentary tats'

Âs discussed at length in chaptet 1 (1..6.1), a number of human case reports have linked AS to atherosclerosis (Mewis et al., 7996) and thrombosis, @isher et a/., 1996; Nieminen et a/.,1996) both of which signifìcantly increase the risk of catdiac ischaemia. Consequently, it was decided to investigate the effect of nandrolone administtation in the ptesence of reduced perfusion. It is probable that many of the cardiotoxic effects attributed to AS, which have been discussed in chapter 1, including direct myocardial toxicìty (1.6.2), left ventdcular hypetttophy (1.6.2), myocardial infarction (1.6.1 & table 1.3), hypertension (1.6.3) and hlperlipidaemia (1.6.1) can significantly increase the risk of ventricular arrhythmia dudng both ischaemia and teperfusion'

There are more than 50 years experience with the rat ischaemia-reperfusion model. (Heimberget, The model involves occluding a coron ny usually the left anterior descending (IAD) 1,946). ^rtery, artery because of its easy accessibility, and observing the frequency and duration of resulting fìbdllation premature venfficular conttactions (PVC), ventricular tachycardta (VT) and ventticulat (VF). The occlusion is often then removed, so that an examination of teperfusion arthythmia can

be undertaken.

It is important to note that whereas Íetufn to normal sinus rhythm following vF is very fare tn humans, it is common in rats, (Johnston et at., 1,983b) and is most probably related to heart size. a period of VF in arat lasting more Abeywardena et al þetsonal communication,200l) reported than 100s, which reverted to normal thythm. While VF is usually fatal in humans'

and reperfusion 5-1 CII,{PTER 5 The ffict of intrauenout nandrolone in rats vbjected to cardiac ischaeruia electrocatdiogtaphic hndings have been reported from a patient who displayed two transient bouts of VF, which spontaneously reverted to sinus thythm at the end of ân asymptomatic ischaemic episode (I\4aseri et a1.,1'982).

by ligation of the I-AD coron^ry in the anaesthetised rat induces Cardiacischaemia produced ^ftery 3 disunct phases of arrhYthmia. following occlusion of the LAD coron^ty et a/', I. An eafly phase 4-10 minutes ^fiery @ott\ng 1983; Manning et a1.,1984)' 2. An intermediate phase starts between 1.5 and 4 houts following ischaemia. @otanget a/.,

1983;Johnstor. et a/.,7983a; b; Clements-Jewery et al',2002) 3. A late phase is evident in most rats surviving for greater than 24 hours post permanent- occlusion, manifest by multifocal ptemature ventriculaf contfactions ("VC) (Curtts et a/.,

1,e87).

upon the size of the "^rea risk of infarction" or the The degree of arhythmia is dependent ^t ..occluded zoîe". At the conclusion of the protocol the under petfused area of the heart can be determined by retrogradely perfusing the heart via the aorta with Evan's blue dye. The lack of effective coronary collaterals in the rat heart results in reproducible results for the occluded zone.

(Evans et a/.,1.985)

Ischaemia-reperfusion experiments can be performed in isolated perfused heatts. However, it has been observed that the duration and ftequency of arrhythmia in isolated perfused hearts is significantlyless than that elicited in uiuo paugherty et al., 1'981). The present studywas concerned

with an in viuo moðel of the effect of nandrolone infusion'

The selection of anaesthetìc is important in ìn uiuo sndies of ischaemia-reperfusion. Both haiothane and have been found to have varying degrees of antiarrhythmic action. Halothane has been shown to be catdioprotective in terms of ischaemia induced arrhythmia in the rat, compared to conscious controls, without changing the size of the occluded zone' (l\4acleod et a/., 1983) Halothane anaesthetised rats have a gïeater tendency to die during ischaemia from non-arrhythmic

c rdiac output failure. (Au et aÌ., 1.983). Pentobarbitone has not been shown to have the same anti- anh¡hmic action. (Au et al',1983)

previous studies wrth chronic, (and now acute) nandrolone tleâtment have not been found to signiiìcantly modi$r HR or BP in the absence of cocaine in notmal adult rats. It was thetefote decided to investigate if nandrolone pre-treatment will produce a signifìcant cardiovascular outcome in rats subsequently exposed to cardiac ischaemia. The absorption, distdbution and

5-2 CHÂPTER 5 The ffict of intrauenoas nandrolone in rats sabjected to cardiac ischaernia and repefasion elirnination of nandrolone as administered pteviously is complicated by the use of oil-based preparations. In otder to ensure maximum and simplify interptetation of the pharmacodynamic data from acute nandrolone administtation it was decided to intravenously administet nandrolone.

5.2 Aim

To assess whether acute nandrolone administration incteases the ftequency and duration of arrh¡hmia in rats subjected to cardiac ischaemia and reperfusion'

5.3 Hypothesis Intravenous nandrolone will cause a dose related inctease in arrhythmia, and a corresponding dec¡ease in survival time during I-AD coronary artery occlusion and reperfusion in the

anaesthetised rat.

5-3 CH,A.PTER 5 The ffict of intrauenous nandrolone in rats sabjected to cardiac ischaemia and reperføsion 5.4 Methodolory

5.4.1 Animals

Male Sprague Dawley rats weighing 358+49 on the day of experiment were fasted overnight before sufgefy for ischaemi a, andwere tandomly assigned to either of 5 treatment gtoups'

0N: nandrolone vehicle * saline; 10N: 1Opg/kg/min nandrolone * saline;

40N: 40¡rg/kg/min nandrolone * saline;

80N: 8O¡rg/kg/min nandtolone I saline;

160N: 160¡tg/kg/ffnn nandtolone * saline

5.4.2 Protocol

Rats were anaesthetised with pentobatbital sodium (60mg/kg, i.p.) and a ligatute placed atound the I-AD coron as per General Methods 2.6. The left femotal artery and vein were ^ry ^rtery catheterised for BP measufement and i.v. drug administration tespecUvely. A five-minute equilibration period was allowed following the completion of surgery. Cardiac pârameters were recorded continuously from the conclusion of sufgefy (Genetal Methods 2.7) and data was analysed according to General Methods 2.9. Nandrolone ot the nandrolone vehicle was then

administered at70¡ù/mn for 10 minutes in all pre-treatment groups. As cocaine was to be infused prior to ischaemia in subsequent experiments a control infusion of saline was carried out at

100p1/min for a further 10 minutes (Fig. 5. 1).

In a separate group of 6 arlrrrals blood samples wete collected to detetmine plasma nandrolone levels. Rars were anaestherised with pentobarbital (60mg/kg, i.p.) and a catheter inserted into the

femoral vein. A 1g-minute infusion at70¡tI/minwas conducted with nandrolone (40 ot t60pg/kg)' A further 10 rninutes later, approximately 7ml of blood was collected ftom the abdominal descending aorta into heparin gel tubes, centrifuged and the resulting plasma aliquotted into 5ml tubes and frozen. Samples were sent on ice to the Austtalian Racing Fotensic Laboratory fot

analysis of plasma nandrolone levels (GC-MS gas chromatography, mass spectrometry).

5-4 CII,A.PTER 5 The ffict of intrauenoas nøndrolone in raß subjected to cardiac ischaemia and reperfusion 5.4.3 Data analysis Blood pressure and HR during drug infusiorì was recorded and expressed relative to the value at the beginning of infusion (t=0s). Significance of differences between groups in the BP or HR response to nandrolone or vehicle infusion with tespect to time was determined using a two-factor- rep eated-measures-AN OVA.

All cardiovascular tesponses dudng ischaemia and reperfusion were expressed telative to the value at 0s of ischaemia or reperfusion. This normalised data was analysed using a two-factor ANOVA.. As a number of rats in the nandrolone treated groups died ftom VF during ischaemia it was not possible to analyse between group differences using tepeated measutes as teliability of the test decreases significantly with missing values'

Arthythmia data was anaþsed according to Genetal methods 2'9'

5.4.4 Drugs Nandrolone was dissolved in a 100% ethanol solution from which it was diluted to a stock solution with an ethanol concentration of 8.7o/o. The final concentration of ethanol in the nandrolone

infusion was 1,.8-4.60/o (average, 3.0+0.1%). The average amount of ethanol contained in the 700p1

nandrolone or vehicle infusions *¿5 21.Q+0.4p1.

5-5 CIIAPTER 5 The ffict of intrauenoas tandrolone in raß sabjected to cardiac ischaemia and reþerfusion 15 min 10 min 5 min 10 min 10 min

1. 2. J. 4 5 6

Nandtolone ot Saline Ischaemia Reperfusion vehicle infusion

7. Open chest and place ligature loosely around I-AD coron^ry artery 2. Nandrolone or vehicle infusion (0, 10, 40, 80, 160¡tg/kg/rlun, i'v')' 3. Saline infusion. 4. Ischaemia. 5. Repetfusion. 6. Collect blood & excise heart.

Fig. 5. 1 Ischaemia-reperfus¡on protocol. Rats were allowed a 5-minute equilibration period from opening followed immediately by of the chest until the commencement of a 1O-minute nandrolone or vehicle infusion. This v¡as a lo-minute saline infusion before the induction of ischaemia. Ischaemia was maintained for 15 minutes and tfie "atea at reperfusion for 10 rninutes. Ät the conclusion of the procedure the heart v¡as excised in order to derermine risk of infarction".

and reperfasion 5-6 CH,A.PTER 5 The ffict of intrauenous naftdrllzne in røts sabjected to cardiac i¡chaenia 5.5 Results

5.5.1 Plasma üevel of nandroJone The plasma level of nandrolone was measured 10 minutes aftet the conclusion of infusion in a separâte group of 6 rats. In rats administered 160¡tg/kg/nÍn for 10 minutes (tt=3), the resultant plasma concentrarion wâs signi{ìcantly gre tet [t(4)=4.636, p<0.01] than rats infused with a\pg/kg/ÍÍn (n=3) (915+135 vs 281+15nM, respectively)'

5.5.2 Area at risk of Ischaemia No significant difference was found in the 'aïea at risk of ischaemia' between the various pre- treatment groups (Fig. 5. 2)

5. 5.3 Cardiovascular response to pre-iscltaemia n¿ndrolon e infitstot t

The dose of nandrolone infused was not found to significantly change the ASP (Fig. 5' 3 A.), ^DP (Fig. S. 3 B.) or AHR (Fig. 5. 3 C.) ftom 0s of infusion. Flowever, 'time' was found to câuse â significant decrease in þ<0.0001). No overall interaction between nandtolone 'dose' and ^IlR 'time'was evident in ASP, ADP or AHR.

5,5.4 Cardiovascular response to ischaemia attd reperfusion Ischaemia Nandrolone dose was not found to significantly effect the change in SP (Fig. 5. 4), DP (Fig. 5. a) or HR (Fig. 5. 4) from 0s of ischaemia. However, 'dme'was found to signifìcantly effect ÀSP and and 'dose' ADp þoth, p<0.01). No significant, overall 'interaction', was found between 'time' throughout ischaemia for any of the variables measuted'

Reperfusion The ASp (Fig. S. 4 D.), ADP (Fig. 5. 4 E.) and AHR (Fig. 5. 4 F.) between the beginning and end of reperfusion was not found to vary significantly'

5-7 CFIAPTER 5 The ffict of intrauenous nandrolone in rats sabjrcted to cardiac ischaemia and reperføsion 5.5.5 Sunival

Ischaemia Survival rime decreased significantly þ<0.01) during ischaemia in rats administeted the highest no nandrolone dose (160¡19 /kg/rlin) compared to vehicle treated rats þ<0.001) (Fig. 5. 5)' \X/hile other significant decreases in survival time during ischaemia wete evident between treatment groups, the fraction of rats surviving ischaemia was found to be significantly lower (y2=!2.29, p<0.05) in the 40N (p<0.05) and 160N þ<0.05) treâtment groups in comparison to vehicle treated controls (Table 5. 1). Five deaths during ischaemia which were not due to VF and therefore could not be scored using the Lambeth convention were excluded from all survival calculations. These exclusions are noted in Fig. 5. 5.

Reperfusion No signihcant difference between groups was observed in the numbet of rats suwiving teperfusion (Table 5. 1). No statistically relevant decreases in survival time were observed in any group in comparison to control. (Fig. 5. 5). One death during reperfusion which was not due to VF and therefore could not be scoted using the Lambeth convention was excluded from survival calculations (Fig. 5. 5)

5-8 CFIA.PTER 5 The efect of intrauenous nandrolone in rat¡ sabjected to cardiac ischaemia and reperfasion 5.5.6 Arrhythmia Lambeth scoÍes showed a signifìcant increase ftw=13.69, p<0.01) for the 3 highest doses of nandrolone, compared ro vehicle treated rats (all p<0.05) during ischaemia (Table 5. 2). Convetsely, there were no diffetences found in Lambeth scofes between nandrolone tfeatments and vehicle during reperfusion (Table 5. 2).

Ischaemia A significant difference was found between the ftaction of tats responding to ischaemia with VF and those without any VF (X'=13.66, p<0.01) (Table 5. 3). A significandy greater ftaction of rats treated wrth the highest dose of nandrolone 1t60pg/kg/min) responded with VF compared to the of VF wete vehicle conrrol (\¡tg/kg/rrtln) þ<0.05). Likewise, signifìcant increases in the ftequency found in all doses of nandrolone in comparison to the lowest nandrolone dose of 10¡:'g/kg/rnnn $}¡tg/kg/nÍn, p<0.05; }}pg/kg/nÍn, p<0.05; 160þg/kg/rnln, p<0.01)' No other significant changes in the fraction of rats responding wrth VT, ot VT*VF were found during ischaemia (Table 5.3).

Rats displayed arhythmia of longer duration during cardiac ischaemia than in reperfusion. The mean duration of VF during c^rdi^cischaemia was found to increase with nandrolone tteatment in an approximately dose related manner (Fig. 5. 6.8). A statistically significant increase in mean VF nandrolone duration ftw=15.19, p<0.01) was noted in rats treated with the highest dose of (160¡tg/kg/min) compared to the lowest nandrolone dose (10pg/kg/min) (p<0'05). No obvious

dose related effect was evident for mean VT duration, neither wele any between gtoup differences evident (Fig. 5. 6.4). \X4rile increases in mean VT+VF duration displayed a dose related effect'

none of these increases wefe signifìcantly different from control (Fig' 5. 7).

Increased mortality in nandrolone treated tats did not skew calculations of arthythmia dutation. Although the survival time and the fraction of rats surviving cardiac ischaemia was significantly different between groups, no significant diffetence in the dutation of VT, VF and VT*VF was noted in rats sun¡iving ischaemia (data not shown)

5-9 CIIAPTER 5 The ffict of intrauenout nandrolone in rats sabjected to cardìac ischaemia and reperfusion Reperfusion No significant differences were found in the fraction of surviving rats responding with VT, VF or VT+VF during repetfusion (Table 5. 3).

There was very little VT (Fig. 5. 6.C) or \IF (Fig. 5. 6.D) during teperfusion in these tats, with no significant differences in the response between treatment grouPs. Much smaller mean dutations of VT+VF were noted in cardiac reperfusion compared to catdiac ischaemia, with no significant differences between the responses of the vatious tfeatment gtoups (Fig. 5. 7).

5-10 CHAPTER 5 The ffict of intrauenorc nandrolone in raîs søbjected to cardiac i¡chaemia ad reperføsion 100

L 60 sN 40

20

0 ON 1ON 40N 80N 1 60N Treatment

Fig. 5. 2Zone at risk of infarction (olozlr) in rats administered nandrolone (0, 10' 40' 80' L6O pglkglmin). z = Mass of the underperfused heart region (area at risk of infarction), r = total hearl mass (refer General Methods 2.7). Legend:0N=0pg/ kg/ nin ranclrolone * ¡aline (0.1n//nin/10 ninutes), 10N=101t8/ kg/ nin -r k'g/ + øndrolone + ¡alìne,40N=40p¿/ kgf min nantlrolone * raline, S0N=80¡tg/ kgf min nandrolone ¡aline, l60N=160p8/ nin

1 0-1 4' : a li n e. Single-factot ANOVÀ, n o sign if ca nt dffi re n ce ¡' Mean*S' E', n=

5-71 CIIAPTER 5 The ffict of intrøuenoas nøndro/one in rats vbjected to cardiac ischaerlia and reperfasion A.

g) f É E +0 fL ¡rg/kg/min at, +10 pg/kg/min 'r.40 ug/kg/min -1 +80 pg/kg/min *160 pg/kg/min -1 300 600 Time (s) B

IcD E E oo-

-1

-1 300 600 Time (s) c.

-1 -1 E ¡lCL IÉ, -3

300 600 Time (s)

Fig. 5. 3 Change in A. systolic pressure (SP), B. diastolic pressure (DP) and C. heart rate (HR) in rats infused for 1O minutes with nandrolone O, 10, 40, 80' 160 Vglkglmin' Legend: t

* nandrolone, o 160pg/k¿/nin Lpg/kg/nin nandrolone, L 101tg/kg/nin nantlrolone, 40¡tg/kg/nin nandrolone,0 S7¡tg/kg/nin nandrolone. Repeated-measutes-two-factot-ANOVA. SP: DO.'Ë [F(a,56)=6.933, p>0 05], TIME Fç,56)=0'108, p>0.051, INTERACTION [F(4,S0;=6.356, p>0.05]. DP: DO.ÍE [F(a,56)=6.932, P>0.5], TIME V("56)=0.428, TIME 28, p>0.051, INTERACTION [F(a,56)=1 106; p>0.5]. HR: DO-ÎE [F(a,54)=9.655, p>0.5], F("54)=87 p<0.00011, INTERACTION F6,S+¡=2'2 r; p>0 51, , Mean*S'E, n=!0-14'

5-12 CIIÄPTER 5 The ffict of intrauenoas nandrolone in raß sabjected to cardiac ivhaemia and reþefusion A. D ¡

IE) o -1 I E E -1 *opg/kg/min lL o- tt, +10pg/kg/min at, +40p9/kg/min +80pg/kg/min -1

-160p9/kg/min .1 300 600 900 0 10 40 80 160 Time (s) Dose (¡rg/kg/min)

E

o, Et I T E E E È o- -l o o -1

0 f0 160 300 600 900 (¡rg/kg/min) Time (s) Dose

G F

E E CL o ¡¡ Þ ú É. I I

-1

-1 -l 10 40 300 600 900 Dose (pg/kg/min) Time (s)

Fig. 5, 4 Change in A. Systolic (SP), B. diastolic pressure (DP) C. heaÉ rate (HR) during card¡ac ischaemia in rats treated with nandrolone (O' 10,40,80,160 pglkg/m¡n). Legend:t

+ o 1 60¡tg/ kg/ nin Lpg/ kg/ nin nantlrolone, L 10¡tg/ kg/ nin natttlrolone, 40pg/ /eg/ nin nandrolone, Q 80¡tg/ kg/ nin nandrolone, nanrlrolone. Two-factor ANOVA SP: DOSE p(4,134)=6.932, p>0.051, TIME FQJI+¡=6291, p<00i1, p(J3Ð=s.679, p>0.051, TIME p<0.01]' TNTERACTION [F(8,13a)=0.384, p>0.05]. DP: DOSE [F(2,134)=5.225; p>0.051, TIME p>0.05], rNTERÀCTTON: [F(8,134)=0.486, p>0.05]. HR: DOSE f (4,1,25)=1.259, [F(2,125)=0.265; n=4-14 change ¡n D. Systolic INTERACTION: [F(8,125)=0.198, p>0.05]. Mean+S.E. ,n=5-14 (BP), G{R). (Sp) and E. diastolic pressure (DP) and F. heart rate (HR) at the conclus¡on of repedusion in rats treated with nandrolone (0, 10, 40, 80, L6O Wglkgl min). Two-factor ANOVA SP" no signifcant tlffirenæ,DPt no si¿nifcant dffirence, ÉIR: zo signficant dfferezr¿' MeantS'E ,n=5-13

5-13 CFIAPTER 5 The ffict of intrauenous aandrolone in rats søbjected to cardiac ischaemia and repefasìon (r) (21 (21

Maximum surviwl time 900 for ischaemia

750 (1)

MaXimum survival time 6 600 for reperfusion o E 450 F¡- 300 lotl Il oN 150 l+otl 0 80N R m16oN

Fig. 5. 5 Suruival time during card¡ac ischaemia (I) and repetfus¡on (R) in rats treated w¡th nandrolone (0, 10, 40, SOrl6OVglkglrnan) and saline immediately pr¡or to commencement of ischaemia. parentheses..indicate number of rats in each üeatment group excluded because of death unrelated 'r to yF. Legen¿.0N=0pg/ kC/nin nantlrolone 'r ¡aline (0.1n// nin/10 ninutes), l0N=10¡tg/k¿f min nandrolone saline, -r 40N=40p¿/ kg/ min nandrolone -r saline,80N=80pg/ kgf min nandrolone saline, td0N=160pg/ k¿/nìn + ¡zline'Stng\e-factot --'p<0.001 ftom O¡rg/kg/min. ,{NOVA, Dunnen,s post-hoc test. (I) [F(a,51)=3.963, p<0.01]. signiFrcantly different ,,n" excluded becaase of death unrelated to W. (R) no ignficant dffirenn. aalae extlatle¡ the 6 rat¡ in þarentheset indicated in the fgure

MeanlS.E. n=10-13 (ischaemia), î=6-13 (reperfusion)

5-14 CI{APTER 5 The ffiø of intrauenoas nandrolone in raß sabjected to cardiac ischaemia and reþerfasion Fraction of surviving rats dose IR

ON t3/1,3 13/ 13

(1000/Ð (1007Ð

10N 13/1,3 t3/13

(100%Ð (1000/Ð 40N 6/rc* 6/6 (100%Ð (60%)

80N 7 /10 6/7

(70o/ù (860/ù 160N 6/rc* 6/6 (1007Ð (60"/ù

Table 5. 1 Number of rats sulviving cardiac ischaemia (I) or reperfusion (R). Includes only those rats which obeyed the Lambeth scodng system (ie. excludes death unrelated to VF. See captìon for Fig. 5.5).

patentheses: percentage of rats surviving ischaemia and reperfusion. Legend:0N=01t8/ k'8/nin nandrolone f- sa/ine þ'g/nin (0.1n// nin/10 ninatet), l0N=1L¡tg/ kgf min nantlroloxe * ¡aline, ¿0N=40ltg/ kgf min nandro/one * ¡aline,80N=80/t8/ in comparison to nandrolone I saline, 160N=160ltg/ kg/ nin + saline. Chi-sqttared test with Fischet's exacl test Post-hoc *p<0.05 0N. significantly diffe¡ent from 0N (0pg/kg/min)'

Tteatment Type I R

ON 2+0 1+0

10N 2+0 1+0

40N 4+1x 1+L

80N 4+7x 3+1.

160N 5+1* 1,+1,

Table 5. 2 Lambeth arrhythmia score during card¡ac ischaemia (I) or repeÉusion (R) in rats treated w¡th nandrolone (O-160frg/kg/min). Legend:0N=0pS/kC/nin nandrolone * ¡aline (0.1n//nin/10 nandrolone + niaates), tON=lLpS/ kC/min nandrolone * saline, +ON--a0¡4/ kg/ min nandrolone * saline, S0N=80pg/ kgf nin *p<0.05 :aline, 160N=l60pg/kg/nin * saline.Kruskal-Wallis, Dunn's post-hoc comparisons made to 0N. sigruficantly

different frorn}¡tg/kg/min. MeanlS.E. n=10-13 (ischaemia), n=6-13 (reperfusion)

and reþerfasion 5-15 CHAPTER 5 The ffict of intrauenous nandrolone in rats sabiected to cørdiac ischaeruia Treatment fraction resPonding fraction responding fraction responding with with VT with VF VT or VF Ischaemia RePerfusion Ischaemia Reperfusion Ischaemia Reperfusion

ON 11/14 3/13 3/1,4 2/1,3 1,1, /14 4/1,3

(78.6) Q3.1) (21,.4) (1,4.4) (78.6) (30.8)

10N e /1,3 6/1,3 1, /13 1 /13 e/13 6 /13

(6e.2) (46.2) (7.7) Q.7) (6e.2) (46.2) )/7 40N 1,0/1,2 ) /'7 7 /12# 1/7 1,1,/1,2 (28.6) (83.3) Q8.6) (58.3) (r4.3) (el.7) 80N 10/12 s/7 6/1.2# 2/7 10/1,2 5/7

(83.3) (71,.4) (s0.0) (28.6) (83.3) (71.4)

0/6 e 2/6 160N e /10 2/6 7 /1.0*## /r0 (e0.0) (33.3) (0.0) (e0.0) (33.3) (70.0)

Table 5. 3 Fraction of nandrolone (0, 10, 40, 80,160pg/kg/m¡n) treated rats resPond¡ng w¡th w, VF or W+VF during a lS-minute per¡od of cardiac ischaemia (I) and during 1O minUteS Of CardiaC repeÉUs¡On (R). Parentheses.'percent tats resPonding with VT, VF o¡ \¡T+VF. Legend:

0N=0¡tg/ kg/ nin nandmlone t saline (0.1n//nin/10 ninates), l0N=10ltg/kgf min nandrulone * saline,40N=40/tg/ kg/ nin Exact ndnclmlone * nline, 80N=80p5/ kgf mìn naadrolone * nlina, t 60N=160ltg/ kg/ nìn + saline. Chi-sqtnred test, Fischer's -p<0.05 #p<0.05, +lp<0.01 Test post-hoc. sigrufrcantly different from 0N; signihcantly different from 10N. MeantS.E

5-76 CFIAPTER 5 The efect of intrauenoas nandrolone in røt¡ sabjected to cardiac itchaemia and reperføsion (t) A. B

o o 0, n) E E

20

0 0 ON 10N 40N 80N 160N ON 10N 40N 80N 160N Treatment Treatment (R) c. D 1 I

80

tt, ø o o .E .E F t'-

0N loN 40N 80N 160N ON 10N 40N 80N 160N Treatment Treatment

Fig. 5. 6 Average duration of W in card¡ac ischaemia (4.) and card¡ac reperfus¡on (C.) and duration of VF in ischaem¡a (8.) and repefus¡on (D.) in rats infused with nandrolone (Or1Or4Or8Or16opg/kg/min). Legend:0N=0pg/ kg/ nin nandrulone + ¡aline (0./n// nin/10 ninates), toN=lLpg/ kg/ min nandmlone * mline, ¿oN=47pg/ kC/ min nandrolone r saline, t2N=s|pç/ kg/ rzin nandrolone * saline, #p<0.05 160N=l60ttg/ kg/nin + ¡aline. Knskal-rùØallis test with Dunn's multiple comparisons test. sigruficantly d.ifferent from 10pg/kg/min. MeanlS.E. (n=10-14 ischaemia, n=6-13 reperfusion).

5-77 CIIAPTER 5 The ffict of intrauenoas nandrolone in raß subjected to cardiac ischøemiø and reperfusion 150 l0N 125 Il oN l¿otl g.-. 100 80N m16oN E75o F 50

0 R

Fig. 5. 7 Average duration of W+VF during cardiac ischaemia (I) and cardiac repedusion (R) ¡n rats treated with nandrolone (0, 10, 40, 8o,16opg/kglmin) and saline immediately prior to commencement of ischaemia. Legend:0N=0pg/ kg/ nin nandrolone + ¡aline (0.lnl/ nin/10 ninutet), nandrulone * ¡aline, l0N=10/tS/ kgf min nandrolone + ¡aline,40N=40pg/ kgf min nandrolone t sa/ine,80N=80¡tg/kgf min \ùZâllis (teperfusion)' t d¡N=l60ltC/ k¿/ nin + saline. Ktusk¡l test. Mean*S.E. n=L0-14 (ischaemia), n=6-13

5-18 CII'\PTER 5 The ffict of intrauenous nandrolone in rats sabjected to cardiac ischaemia and reþerfasion 5.6 Discussion This is the frst study to investigate changes in haemodynamics following nandrolone infusion in male rats, although BP and ECG assessment in human volunteers administered i.v. testosterone at physiological and super-physiological doses have been reported (\X/hite et a/., 1'999). White et a/ reported no ECG or BP changes. Flowever, a maximum testosterone dose of only six tjmes the endogenous level was used. It is probable that plasma concentrations teached with illicit use are

sevetal ordets of magnitude higher than this.

The absorption, distribution and eli¡nination of nandtolone âs administered previously is complicated by the use of oil-based preparations. In ordet to ensure maximum bioavailability and simplify interpretation of the pharmacodynamic data from acute nandrolone administration it was decided to intravenously administer nandtolone. As mentioned pteviously, thete are no reports in the literature equating the cardiac effect of nandtolone with plasma nandrolone concenttations. This makes selection of doses for i.v. administration exceptionally diffìcult. ,{.s plasma nandrolone concentrations following an abusive cycle have not been reported it is vety diffìcult to detetmine a pharmacologically relevant dose. Reports of endogenous testosterone levels in adult males range ftom 1.2j. to 3.47 X 108M (Strauss et ø/., 1.985; Rowland et al., 1'987; Melchet et al., 1992). A therapeutic dose of testosterone enanthate administered to 19-40 year old males has been 8M. associated with a mean plasma concentration of 4.37 X 10 As illicit use of AS is associated with a much higher dose than would be clinically presctibed (10-1000 times the recommended

therapeuric dose) it was decided that infusion doses of nandrolone of 10-160¡19 /kg/rntn would be used and plasma nandrolone concentrations measuted following infusion' The plasma concentration achieved with an infusion of 4Optg/kg/min o[ nandrolone was 281+15nM 8t\zf (28.1x10 and with the highest dose (160¡19 /kg/niln) was 915*135nM (91.5+13.5x10r1vÐ' This

suggests that the plasma nandrolone concentration achieved with infusion of 160p'g/kg/min was approximately 25-75 fold greater than the reported endogenous male testosterone level (Strauss a/

a/.,1.985;Rowland et aL,1'987; Melchen et a/.,1'992).

'We found no evidence of an haemodynamic effect of acutely administered nandrolone. No significant difference was found in the change in BP or change in HR from baseline between treatment gtoups. Chronic treatment with nandtolone may be necessary to observe the cardiovascular effects commonly attributed to AS abuse. Howevet, 'time' alone was found to cause

a significant decrease in HR during the 1O-minute nandtolone infusion. This most ptobably reflects

bradycardia produced in response to the open chest. To ptevent this effect obscuring a possible

5-19 CFLA.PTER 5 The ffict of intrauenous nandrolone in raß sabjected to cardiac ischaemia and reþerfasion haemodynamic action of nandrolone, recordings of BP and HR were made in chapter 7 before the chest was opened for induction of ischaemia (Fig' 7.1).

It was observed that the BP and HR response to ischaemia was unaffected by the 'dose' of nandrolone used. 'Time' alone was found to have a significant affect on SP and DP during ischaemia. The tjme-dependent affect is most probably due to the adaptation to the ischaemic insult. A signihcant decrease in SP and DP in all treatment groups can be noted evident during the ffust 5 minutes of ischaemia. Heart rate remained approximately the same or decteased. Blood pressure was found to increase again following the 5th minute of ischaemia in all treatment groups.

,A. decrease in BP and HR immed.iately following induction of ischaemia has been noted by several researchers in both conscious (Botting et a/., 1.983; Curtis et a/., 7987) and pentobarbitone sodium

anaestherised rats (Au et a/., 1983; Mclennan et al., 1,985). !Øhile most studies petfotmed in anaesthetised rats have noted a decrease in HR immediately following occlusion' at least one investigator has noted a small increase in conscious animals flohnston et a/', 1983a; b). After the initial decrease the slow increase in pressure and rate during ischaemia most probably reflects. an adaptation to the carðiac insult. The 'tjme' effect may also be conftibuted to by a dectease in

survival as ischaemia progressed and by arrhythmra which made it diffìcult to record accurate BP and HR values. No BP or HR values could be tepotted while a rat was experiencing VT or VF. If the dutation of atthythmiâ was greâtet than 60s before ot 60s after 0s, 300s, 600s or 900s of ischaemia no cardiovascular values could be reported (see General Methods 2.9). The combination of increased f^t^llty and an inctease in the numbet of rats experiencing arrhythmia as ischaemia progressed resulted in a dectease in 'n' between the beginning of ischaemia and the end of reperfusion. This may have contributed to the effect of time on BP observed dudng ischaemia.

Reperfusion induced arhythmias ate best observed following an ischaemic period of 5-10 minutes

(I\danning et a/.,1,984). As we were interested in observing arrhythmia resulting ftom ischaemia we chose a 15 minute I-AD coronzrry occlusion time. Previous studies have indicated that this ^tteny results in a much greatet degree of arrhythmia during ischaemia than in reperfusion QttLcLennan et a/.,7988). This effect was confìrmed in the present study, where very few arrhythmic events were noted during reperfu sion.

The main finding in this study was an increased numbef of fatalities during cardiac ischaemia in rats receiving nandrolone at the three highest doses, teflected in a decreased survival time during ischaemia. This increased mortality cottesponded with an inctease in the duration of VF and a significantly increased Lambeth score for all three doses of nandtolone compared to conttol' These not be attributed to vadation in the risk of infatction. No arrhythmia was effects could ^re ^f

5-20 CFIAPTER 5 The ffict of intrauenoas nandrolone in røß subjected to card,iøc ischaemia and reperfasion thythm or evident in the absence of cardiac insult. Nandrolone had no significant effect on c rdia.c mortality dudng rePerfusion. j,6.1 Potentjal mechanism for an acute nandrolone effect durittg'cardiac ischaemia indicate that Very few researchers have attempted to investigate the acute effects of AS. Our tesults dose related nandrolone alone did not significantly change BP befote or after ischaemia, and that Many mechanisms could incteases in Lambeth score and VF were only observed during ischaemia' ischaemia is explain these results. The increase in arrhythmia and decrease in survival during cardiac release during I,r\D coron^ry occlusion' potentially related to increases in catecholamine ^ttety

in Myocardial ischaemia has been demonstrated to give rise to an accumulation of catecholamines the myocardium by 3 main mechanisms'. ' j,. An increase in plasma catecholamines via stimulation of the adrenal medulla (Daugherty

et a1.,1'986) ; release of 2. reflex increase in cardiac sympathetic nerve activity, accompanied by a local a reversal catecholamines from the sympathetic nerve endings of the myocardium via of uptake-1 (Schomig, 1990; Dart et a/.,I993;Du et a/.,1993; Du et a/.,1.998); 3. local metabolic release mechanisms, independent of central nervous system

involvement (Schomig, 1 990)' te-uptake of noradrenaline Sex steroids such as testosterone have been shown to inhibit acutely the by smooth muscle in isolated, petfused r^t heatts (Iversen et al' 1'970; Salt, 1972)' (Clarke el Histochemical/fluorescence studies have associated this uptake with cardrac muscle cells er al', L996)' It is at., 1.969). rJptake-2 has also been demonstrated in rat cardtac myocytes (Obst and inhibiuon possible that a synergy exists between ischaernia-induced release of catecholamines pedod' of smooth muscle re-uptake by nandrolone, resulting in increased VF dudng an ischaemic the A number of authors have postulated that because fixed cotonary plaques can prevent increased in these extÍaneuronal uptake (uptake-2) of noradrenalìne, the dsk of ischaemia is greatly and have patients (Lathers et a/.,'1.988;I3bas et a/., 1990). Although both testostefone AS can also block been shown to block uptake-2,it is unknown whether nandrolone and similar this mechanism. This question was add¡essed in chapter B'

and reþerfusion s-21 CFIÂPTER 5 The efect of intrauenoas nandrolone in rat¡ subjected to cørdiac ischaemiø 5.7 Conclusion o fnffavenous nandrolone (10 to 160¡rg/kg/min) in the absence of ischaemia did not cause â significant change in BP ot HR' o No arþthmia was observed in the absence of catdiac ischaemia. o Acute administration of i.v. nandrolone caused a dose related increase in ischaemia induced VF resulting in a significantly incteased Lambeth arrhythmia score at the 3 highest doses, and

consequently a dectease in survival'

and reperfasion 5-22 CI{APTER 5 The ffict of intrauenoas nandrolone in rats subjected to cardiac iscbaemia 5.8 References

,{.u, T. L., Collins, G. r\., Macleod, B. Ä. and Walker, M. J. (1983). "Efects of Prosîaglandin 82, and Nitrogþæine with Halothane, or Pentobarbitone Anaesthesia on Arrhlthrniaç and OtherResponses to Ligatìon of a Coronary Artery in Rats." Br J PharmacolT9(4):929-37 '

and \X/alker, M. (1983) . Efect of Modifcation of Botring, J. H., Johnston, I{. M., Macleod, B. A. J. "The Slmpalhetlc ActiuiU on Retponsu to l)gation of a Coronary Artery in the Conscioa¡ Rat'" Bt J Pharmacol 79(1):265-71,.

Linley, P. (1969) . "Histochemical Flaoaresence Stadies on Noradrenaliue Clarke, D. E., Jones, C. J. and '{.. Accamulation þt tJptake-2 in the Isoløted Rat Heart." BtJ Pharmacol3T 1'-9.

Clements-Jewery, FI., Flearse, D. J. and Curtis, M. J. Q002). "Independent Contrìbation of Catecholamines ø Arr@thmogeoriit Doriog Euoluing Infarction in the Isolated Rat Heatt." Br J Pharmacol 1it5(3): 807-15.

of Arfuthmias in Curtis, M. J., Macleod, B. A. and Walket, M. J. (1987). "Models for the Stu@ Mlocard.ial lichaemia and Infarction: The IJse of the Rtt." J-Mol-Cell-Cardiol l9(4): 399-419.

Dart, ,{.. M. and Du, X. J. (1993). "(Jnexþected Drug Efects or Aatonomic Fanction During Mlocardial Ischaemia." Catdiovasc Res 27(6): 906-1'+.

Daugherty, 4., Frayn, K. N., Redfern, W. S. and Woodward, B. (1986). "The Role of Catecholarnines in the Þrodaition of ltchaemia-Indaced Ventricalar Anhlthmias in the Rat in Viao and in Vilro." Bt J Pharmacol 87 (1): 265-7 7 .

Daugherty,,\., Mohamed, O. Y. andWoodward, B. (1931). "EÍtrt of Potassiun on CoronaryArtery Ligation Indaced. VentricalarArrhltthnias in the IsolatedRøt Heart."J Physiol 340: 66P.

Re/ease in the Anoxic Heart of the Rat." Du, X. J. and Dart, A. M. (1993). "Mechanitm: of Noradrenaline Catdiovasc Res 27(11): 201.1'-5'

Datt, A. M. (1998). "Proleûion of Nearonal Du, X. J.,Iøoodcock, E. Ä., Little, P. J., Esler, M. D. and Upøke-i Inhibitors in Ischemic and. Anoxic Hearts bjt Noreþinephrine-Dependent and -Infupendtnt c 32$) : 621 -8. M e c h a ni s m ;." J Cardiovas Phatmacol

'W. S. (1985). of Evans, R. G., Val-Mejias, J. E., I(ulevich, J., Fischer, V. and Mueller, H. "Eualuation Efects of lbuþrofen and Verapamil." a Røt Model for Atsessíng litervention¡ to Saluage Irhaemic Mlocardiam: Catdiovasc Res 19(3): 1,32-8.

Fisher, M., Appleby, M., Rittoo, D. and Cotter, L. (1996). "Mlocard'ial Infarction with Extensiue 50(4): 222-3. Intrøcoronary Thrimbis Ind.aced bl Anabolìc S terlids [S ee CommenttJ ." Br J Clin Pract

5-23 CFIAPTER 5 The ffict of intrauenoar nandrolone in røß sabjected to cardzac is c ha e rnia a n d reþerfa sio n Heimberger, R. F. (1,946). "Injection into Peicardial Sac and Ligation of Coronary Artery of the Rat." Arch Surg 52: 677-689.

tJptake-2 b1 Snroids in the IsolatedRat Iversen, L. L. and Salt, P. J. Q,g7O). "Inhibition of Catecholamine Heart." Br J Pharmacol 40(3) : 528-30.

K. M., Macleod, B. A. and Walker, M.J. (1983a). "Efects of a1!.Prostaryclin on 'Anhj¡hniasJohnsron, Resultingfron Coronagt Artery Ligation and on Infurct |iry." BrJ Phatmacol 78(1): 29-37 .

M. (1983b) . "Responses to Ugation of a Coronary Artery Johnsron, K. M., Macleod, B. ,\. and Walker, J. 'in 1340-53. Conscioas Røt: and the Actions of Antiarrþtthnics." Can J Physiol Pharmacol 61(1 1):

Hoffman, G"W', Ellinwood' E' H'' I{abas, J. S., Blanchard, S. M., Matsuyama,Y., Long, J' D', Smith, Þ. f. ,.rd Strauss, H. C. (1990). "Cocaine-Mediated Imþairwent of Card,iac Condøction in the Dog A Thet 252(7): 185-91' Potential Mechanisrn for Sad.dtn Death after Cocaine." J Phatmacol Exp

Lathers, c. M., Tyau, L. S., Spino, M. M. and Agarwal, I. (1988). "cocaine-Induced seiïurcL Arhlthrnias and Sndfun Death'" J Clin Pharmacol 28(7): 584-93'

Macleod, B. 4., Augereau, P. and Walker, M. J. (1983) . "Efects of Halotharc Ane$hesia Comþared with Fentanll Anesthe¡ia and No Anesthesia During Coronary Ligation in Rats." Anesthesiology 58(1): 4+-52'

. Reperfaion-Induced Arrhlthmias in Manning, A. S., Coltart, D.J. and Hearse, D. J. (1984) "Is.chlmia 1nd the Røt. n¡trtt of Xønthine Oxidase Inhibition with A//0parino/'" Cuc Res 55(4): 545-8'

Maseri, ,4.., severi, s. and Marzullo, P. (1982). "Role of coronary Arterial spasm in sadden coronary kchernic Death." Ann N Y Acad Sci 382: 204-1'7.

Mclennan, P. L., Abeywatdena, M. Y. and Chamock,J' S. (1985). "Inflaence of Dt1tary Lipid: on Anhlthrnias and.Infarctioi after Coronary Ar*ry Ligation in Rats." CanJ Physiol Pharmacol 63(1'l): 1'471'- 7.

Mclennan, P. L., Abeywardena, M. Y. and Chamock, J' S. (1988). "Dietary Fish Oil Preuents Ventricalar Fibrillation Foito*ng Coronarl Artery Oulasion and Repefa:ion." Arn Heat J 116:709-71'7 '

Melchert, R. 8., Flerro.r, T.J. and Welder, A. A. (1,992). "The Efect of Anabolic-Androgenic Steroid'r on Primary Mlocardial Cell Calturet." Med Sci Sports Exerc 24Q):206-1'2'

Mewis, C., Spyridopoulos, L, I{uhlkamp, V. and Seþel, L. (1996)' "Manfestations of Seuere Coronary Heart Disea¡iafterAnabotic DrugAbun " CLn Cardiol 19: 153-155'

5-24 CFIAPTER 5 The efect of intrauenoas nandrolone in rats stbjected to cardiac ischaemia ønd reperfasion Nieminen, M. S., Ramo, M. P., Vütasalo, M., Heikkila, P., Karialainen, J'' Mantysaari, M' and ,,Serioas Steroids ll/eiþt I-ifierí" Heikkila, j. (1996j. Cørdioaascalar Sidl Efecß of l-ørse Doses of Anabolic in Eur Heart J 17 : 757 6-1583.

in Obst, O. O., Rose, H. and I(ammermeier, FI. (1996). "CharacteriTation of Catecholamine Uptake2 Isolated. Card'iac M1orytes." Mol Cell Biochem 163-164 181'-3'

of Cocaine and the Phllis, B. D., Irvine, R. J. and l(ennedy, J. A. (2000). "Combined Cardiac E'f,exs Anaboilic steroid, Nandrolone, in the Rat." EutJ Pharmacol 398(2):263-72.

Rowland, T.\7., Morris, A. H., I(elleher, J' F', Haag, B'I: and Reiter' E' O' (1987) "Seram 141(8): 881-3' Testosterone Response to Training in Adolescent Ranners." A- J Dis Child

2 in the lyktedRatHeart b1 Steroids, and Salt, P. J. (g72). "Inhibition of Noradrenaline Uptake Methoxlilatìd Phenykt@laninet." E,ut J Pharmacol 20(3): 329-40'

Circulation Schomig, A. (1990). "Calecholaminet in Mlocard,ial Ischemiø. Slrternzc and Cardiac Release." 82(3 Suppl): IÍ13-22.

in Amatear lYre'çtlers and Its Strauss, R. H., Lanese, R. R. and Malarkey, \Ø. B. (1985). "IYeight l-'o¡s Efect on serum Testosterone I_.euek." J Am Med Assoc 254(23):3337-8.

\woodiwiss, Trifunovic, B., Notton, G. R., Duffìeld, M. J., Avraam, P. and ,\. J. (1995) "Ao Physiol 268(3 Pt 2): H1096-105' Androgenic Steroid. Decreases L.eft Ventricular Compiance iru Røts'" ,{^ J

Toxicities of Tseng, Y. T., Rockhold, R. \ø., Floskins, B. and Ho' I' K' (1994)' "Cardiouascular Toicol 22(I): 1'13-21' Nandrolom and. cocaine in ipontaneoasþ H1þertensiue Røts." Fundam,\ppl

Ahlbetg, A. w., Thompson, P' ,ùØhite, c. M., Ferraro-Borigida, M. J., Moyna, N.M., McGill, c. c., D. and Heller, G. V. (19õ9). "Th; Eiîect of Pharwtacokineticalþ Gaided Acute Intraaenous Testosterone Pharmacol 39: 1038-1043' Administration on E,lectromrdiogrø?hic andblood Pre:sun Variabkt'"J Clin

reþerfuion 5-25 CHAPTER 5 The efect of intraaenoas nandrolone in rats sabjected to cørdiac ischaemia and The effect of innavenous cocaine in raß sub¡ècted to catdiac ischaemia and repet{usion

6.1 Introduction incteased risk of severe cardiac cocaine has been shown to induce myocatdial ischaemia leading to Osawa et al'' 1'994; thythm disturbances in human subiects, including fatal Q'{anii et al', 1'984; 1988)' Prenatal cocaine exposute has Gamouras et a/.,2000) and non-fatal atthythmia (Ascher et al., arrhythmia in the neonatal been demonstrated to increase the incidence of atrial and venüicular pedod (Frassica et a|.,7994).

monitoring (Holter tecording) The cardiac effects of cocaine are long lasting. Ambulatory 24hout weeks following withdrawal has revealed evidence of ischaemia in cocaine users up untìl 2

Q'Jademanee et al., 1989)'

models have been A number of putative mechanisms for cocaine-induced arrh¡hmia in animal to cardiac conduction proposed, including colonary aftery vasoconsttrction (refer 1.1j1'1), changes that a single uni$'ing explanation (refer 1.13.2)and calcium mobilisation (refer 1.Íf.2)' It is unlikely and is probable that atrhythmia is can be forwarded to explain the arhythmic effects of cocaine,

due to a numbet of mechanisms acting in concert'

in chaptet 1, anð In view of the suspected interaction between cocaine and nandrolone, discussed in chaptet 5) in view of the potentiating effects of acute nandrolone during ischaemia, (descdbed investigated in this model of the effect of cocaine in the pfesence and absence of nandrolone was

ischaemia-reP etfu sion.

and reperfzsion 6-l CH,\PTER 6 The efect of intrauenous cocaine in rats søbjected to ischaemia 6.2 Aims 1. To determine whether i.v. cocaine increases ischaemia-induced cardiac arrhythmia.

nandrolone on 2. To determine whether i.v. cocaine modifies the potentiating effect of acute ischaemia-induced cardiac arh¡hmia'

6.3 Hypotheses

7. Cocaine will potentiate arrh¡hmia in ischaemic hearts'

of anhythmia 2. Cocaine will potentiate the effects of nandrolone on occuffence and severity in ischaemic heatts.

and reperfasion 6-2 CI{APTER 6 The ffict of intrauenoøs cocaine in rat¡ sabjeded to ischaemia 6.4 Methodology

6.4.1 Animals to either of Ten week old, male Sprague Dawley fats wefe fasted ovetnight and randomly assigned

5 treatment gtouPs

1,. 40N+C: nandtolone $}¡tg/kg/mtn) * cocaine (0'5mg/kg/min); 2. 40N+S: nandrolone (}þg/kg/rn1n) * saline 3. S*C: saline * cocaine (0.5mg/kg/min) 4. ON+C: vehicle * cocaine (0.5mg/kg/min) 5. 0N+S: vehicle * saline

Results ftom chaptet 5 wete used for treatment groups 2 and 5'

6.4.2 Protocol Nandtolone A hve-minute equilibration period was allowed following the completion of sufgery' for (a}p7/ky/ffrn), saline or the nandrolone vehicle was then administered at l}¡i/nin for a further 10 10 minutes. Cocaine (0, 0.5mg/kg/min, i.v.) was then infused at 100¡rl/min

minutes. The total dose of cocaine received was 5'0mg/kg'

is summadsed in Ischaemia and reperfusion was conducted as pef chapter 5. The ptotocol used (Fig. 6.1).

6.4.3 Data analYsis HR was determined using The effect of pre-treatment with nandrolone, saline ot vehicle on BP and BP and HR in a repeated-measufes-two-factor-ANovA. The effect of cocaine or saline on between groups ptetreated fats wzs determined using a two-factot-ANovA' Differences þost- test (with cortection for hoc) were determined for 'treatment' and 'interaction' using Bonferroni's shown in Fig' multiple compafisons - see General Methods 2.9). Post-hoc tests fot'treatrnent'afe G' changes in BP 6.3 anð.Fig. 6. 4. The post-hoc tests for 'interaction' and shown in Appendix of arthythmia etc were and HR during ischaemia and reperfusion and the dutation and frequency BP and HR and this determined as per Genetal Methods 2'9' Because cocaine depressed both effect of ischaemia on effect had not fetufned to baseline by the beginning of ischaemia the tfeated tâts' cardiovascular variables was detetmined sepatately in cocaine and saline

repefusion 6-3 CH,A.PTER 6 The efect of intrauenous cocaine in rat¡ subjected to ischaemia and 15 min 10 min 5 min 10 min 10 min

5 6. 1. 2. J 4

Nandrolone, sa-line or Cocaine infusion Ischaemia Repetfusion vehicle infusion

7. Open chest and place ligature loosely around LAD 2.Nandrolone,1)þg/kg/'.rlln,i.v'),vehicleorsalineinfusion 3, Cocaine (0.5mg/kg/min) ot saline 4. Ischaemia 5. Reperfusion 6. Excise heafi

period from opening Fig. 6. 1 Ischaemia-repeffus¡on protocol. Rats we¡e allowed a S-minute equilibration vehicle infusion' This was followed of the chest until the coûìrnencernent of a lO-minute nandrolone, saline or Ischaemia was maintained for 15 immediately by a lO-minute cocaine infusion before the induction of ischaemia. the blood was collected and the heart minutes and reperfusion for 10 minutes. ,\t the conclusion of the procedure excised.

and reþetfariln 6-4 CFIAPTER 6The ffict of intmuenoas c0catile in raß sabjected to ischaemia 6.5 Results by cocaine in To aid interpretation of the degree of any additional potential atthythmia caused conjunction with nandtolone, the nandrolone alone (0¡tg/kg/ffiln) results ftom the ptevious order demonstrate chapter (40N+S), have been included here and in all statistical analyses' In to chapter 5 have the arrhythmic effect of cocaine alone, the vehicle plus saline results (ON+S) ftom also been included. Tabular results have these gfoups shaded in grey.

6.5.1 Arca at risk of ischaemia gfoups (data not The area at risk of ischaemia wâs not signifìcantly different between pfe-tfeatment

shown).

6.5. 2 Cardiovascular respons e to pre-treatmettt Response to nandrolone, vehicle or saline Pre-treatment 600s of infusion' The change (Ð it BP and HR ftom 0s of infusion was noted at 300s and effect Ä SP (Fig' 6' 2 Treatment with vehicle, saline or nandrolone was not found to significandy upon A.), DP (Fig. 6.2F..) or  HR (Fig. 6. 2 C.). Time was found to have a signihcant effect ^ and 'tfeatment'was observed a Dp (p<0.05) and a HR Gr<0.001). No interaction between 'time' for any of the vadables measuted.

Response to cocâine or saline change (A) from baseline' The SP, DP and HR response to cocalne ot saline was exptessed as where the baseline was the value 40s pdor to the statt of drug infusion.

response to All rats treated with cocaine demonstrated a strong ininal, but transient, pressot administered cocaine cocaine (Fig. 6. 3). Both the aSP and the aDP from baseline in all rats a sharp depressot effect peaked at 40s post administration. ,\t all time points subsequent to 100s in nandrolone ot was obsewed. No significant fluctuation in either ASP or ADP was recorded

vehicle infused tats administeted saline'

separately by Due to the bi-phasic natufe of the BP response to cocaine both phases were analysed response to cocarne repeated-measures-ANOVA. The ÄSP (Fig. 6. 3 A.) and ADP (Fig' 6' 3 B') (Fig' 6' 3 C') did not was divided into pressor (0-100Ð and depressor phases (100-600s)' Heartrate was analysed simultaneously' display a biphasic fesponse to cocaine and the whole 600s of infusion

and reperfuion 6-5 CIIAPTER 6 The ffict of intrauenons cocaine in rats sabjected to ischaernia The Pre¡sor P/tase .Treatmenr' alone was found to have a significant effect on ÂSP þ<0.01) and ADP þ<0'001) during the frst 100s of the 10 minute drug infusion period. Most of the signifìcant difference to between cocaine and saline treated groups was found at 40s of cocaine infusion corresponding the peak of the cocaine effect (see Fig. 6. 3 Inset A & B. for details of post-hoc tests). Pre- üeâtment with nandrolone was not found to alter the BP response to cocaine. 'Time' alone also observed had a significanr effect on ASP and ÀDP (both, p<0.0001). A signifìcant interaction was Fig' berween ,treatmenr' and 'rime' for both ÀSP and ADP (both, p<0.0001). To aid the clarity of

6. 3 these significant differences are noted separately in Appendix G'

The Depressor Phase Treatmenr alone was found to have a signifìcant effect on aSP þ<0.01) and aDP þ<0.0001) differences during 100-600s of infusion. Posr-hoc tests (Bonferroni's) did not detect any significant BP response between gïoups (Fig. 6. 3). Pre-treatment with nandrolone was not found to alter the A to cocaine, Time was also found to significantly effect ASP and ADP (both, p<0'0001)' ADP significant interaction was observed between 'treatment' and 'time' fot ÄSP (p<0.0001) and ftr<0.0001) (see Appendix G for post-hoc tests)'

or HR Changes dñng 1 0 mintttes of dmg inJøsion þocaine nline) decrease in HR producing at the lowest Cocaine was found to câuse a profound Point ^n ^vera,ge HR of 309+21, 31Q+14 and 323+1,1. bpm in fats pfe-tfeated with infused saline, vehicle or nandrolone (O¡tg/kg/rlrn), respectively, compared with pre-cocaine values of 377+10' 399+8 and the effect of 388+7, respectively. Nandrolone pre-treatment wâs not found to significantly altet alone was found to cocaine on HR compared with saline and vehicle pre-treatment. 'Tteatment' of infusion have a significant effect on þ<0.05). This effect was most pfonounced at 300s ^HR compared to where the ÀlIR was found to be significantly gfeater in the 40N*S and ON*S gfoups 40N+C (p<0.01 and p<0.001, tespectively) (Fig. 6. 4). 'Time' alone was also found to vary between 'treatment' and 'time'was significantly þ<0.0001). A signifìcant interaction þ<0.0001) observed for AHR (see Appendix G for post-þoc tests)

6.5.3 Cardiovascular responses to isclnemia and reperfusion absence of ischaemia in Cocaine was found to produce a significant cardio- effect in the reperfusion on terms of BP and HR. Therefore, only the effect of coronary occlusion and presented below' As cafdiovâsculaf fesponses in cocaine tteated gfoups (S+C, 0N+C, 40N+C) afe non- reported in the previous chapter, there was no significant 'treatment' or 'time' effect fot sP, DP ot HR cocaine tteated gfoups (ON+s, 40N+s), neither was an intetaclion obsewed for the

6-6 ch ae an d rep erfasio n CHAPTER 6 The effect of intrauenoas cocaine in raß subjected to is m ia between these 2 groups ïesponse (two-factor-ANovÂ) during ischaemia. No signifìcant diffetence measuted (unpaired t-test)' was evident during reperfusion for any of the cardiovascular variables

Ischaemia .treatment' (Fig. 6' 5 No effect of or 'time'was obsewed for both ASP and ADP dudng ischaemia r\., B.). Likewise, no intetaction was found between 'tfeatment' and 'time' for BP changes'

6' 5 ,Treatment, alone caused a signifìcant difference in the AHR response dudng ischaemia (Fig' at each time point C.). However, fio signihcant difference was found between tfeâtment gfoups signifìcantly change the AIIR fesPonse during ischaemia' @onferroni's). 'Time' alone did not Likewise, no interaction was observed.

Reperfusion 5 D'), ADP (Fig' 6' 5 No significant difference between gfoups was found for the ASP (Fig' 6' E.) or AHR (Fig. 6. 5 F.) response during rePerfuslon'

6.5.4 Suruival Six of these rats Nine out of a total group of 63 rats (1,4"/") dteð ftom causes not telated to vF. teperfusion)' An addrtional 6 were from tfeatment gfoups receiving cocaine (4 in ischaemia and2 in Lambeth convention they deaths could be attributed to VF. As the former rats do not obey the noted in Fig. 6' 6' Survival could not be scored for arrhythmia severity. All exclusions have been convention and those that data has been sepatated into rats which compüed v¡ith the Lambeth didn't (Table 6.1).

Ischaernia was a significant In rats which complied with the Lambeth convention (Fig. 6' 6 top) thete so close to 0'05' post-hoc dectease in survival during ischaemia (p=0'0454)' As the 'p' value v/as survival was 1000/o in rats tests (Tukey,s) were not able to detect where this significance lay. As survival wefe not obeying the Lambeth convention during fepeffusion, statistical tests of of whether they obeyed conducted. .ùøhen aII rats were included in suwival calculations regardless survival during ischaemia the Lambeth convention or not there was no difference in the length of fraction of rats surviving and teperfusion (Fig. 6. 6, bottom). Howevet, when examining the survival was significanrly gteater ischaemia compared to the number dying of VF it was found that to 40N*S (f=13.4g,p<0.01) in the ON+s þ<0.05) and 40N+c þ<0.05) gtoup in comparison (Table 6.1).

and repefusion 6-7 CHr\PTER 6 The ffict of intrauenoas cocaine in rats subjected to ischaemia Reperfusion Survival time None of the 53 rats compþing with the Lambeth convention died during reperfusion. all cause death was and the fraction of surviving rats did not differ between tfeatment g.foups when considered (Fig. 6.6).

6.5.5 Arrhythmia The infusion of cocaine alone produced no VT or VF

Ischaemia with vT, vF During ischaemia, cocaine did not significantly change the number of rats responding 6' 7'B) or (Table 6. 3) neither was the duration of vT (Fig. 6. 7.4), VF (Fig' or vT*vF ^vef^ge VT+VF (Fig. 6. 9) significantly different between groups' No significant diffetence in the diffetence in the Lambeth scoïe was obsewed between gfoups (Table 6. 2). Despite no significant any episodes number of tats experiencing an arrhythmic episode (Table 6' 3), the duration of number of rats were observed (Fig. 6. 7) ot the Lambeth score (Tabl e 6, 2), a signifìcantly gteâtef administered saline (40N+S) observed to die of VF during ischaemia in nandrolone pre-treated rats (Table 6' 1)' SØhen rats compared to the 0N+C þ<0.05) and 40N+C (p<0.05) tfeatment gfoups only a minor change in the dying from ischaemia were excluded from statistical analysis there was the duration of VF was mean duration of VF duting ischaemia for all gfoups (Fig. 6' 8), although to ON+S now found to be signihcantly gteâtef þ<0.05) in the 40N+S gloup comPared þ<0'05)' shown)' No signifìcant differences wefe observed between gfouPs for VT or VT+VF (data not

Reperfusion different between The number of rats responding with vT, vF or VT+Vtr was not signifìcantly of (Fig' 6' 7'C)' VF tfeatment groups during repetfusion (Table 6. 3) neither was the duration VT the Lambeth (Fig. 6. 7.D) or VT+Vtr (Fig. 6. 9) significantly diffetent between gfouPs. similatly, by pre-tr.e tment (Table 6' 2)' score was also unaffected ^îy

6-8 and reperfasion CFIAPTER 6 Th ffict of intrauenoøs cocaine in raîs sabjected to i¡chaemia A"

B T E E g,È rçS+C +oN+c 0N+c +0$¡+S :l 0N+s 3m 6{X} ïime(s!

-Ð E E o- C¡

300 600 Tim{s} c.

E ÂÂ -É,

G@ T[nefs]

Fqg- G 2 erange in A. SP, ¡l' DP, G HR in r# prÞE€aæd wÍÜ vcùir:þ (T0pUminln slturc (TOfrUm¡n) or mtdrclqrc (liQug,llsslmin). Lsg@e X cølìrîe ('lffiN/?Éiø) * aøtøËøe psøglþd/nøìle) ftS+E,

øeþìda f øm&ø[sne øefreale (7l0¡t$øiø) * ewait¡ø [CIN+q' o øan&plow (4CIftg/:kg1øift] * coeøi'øo [4ON+Cj' - nøw&øfune Repeate*rneaourc-sÞ"{No\¡.,{' (Vt¡ti/øøø) r søbøet (liMú/n?tuu) ION+SÏ (+O¡tgf fof ørø) * sø!åøe [,10r]I+E- tsonferroci,s posr-hoc resr" SP: TRETTMENT tF(4,59t=t-97e, p>0.051; T'lIvIE [F(1J$ì=3.567r; p>0t05þ TIME TNTERTCTION tF(459)=7375, p>0-CI51- DP; TRE{TMENT [F(a59,=CI-q65 p>CI-05]" flF(15Ð=5"95e'' TIME p0-0fl. IIIk TRE{TMENT' ¡F(45E4-Wz" P>0-0q[ MeantsE." n=77-14- [F(X,59)=136.5,p<0-ffi11; INTERACIION [F(45Ð=t-546, P0-0q; A. lnset A. l0 ¡cD -ct' E E E o- o- (t, (t -1

-1

0 100 200 300 400 500 600 o20 80 1(x) Time (s) B. lnset B.

CD Ð I I E E E E o. o. + cl c}

-1

0 100 2@ 300 400 500 600 ozo 40 60 80 100 Time (s) Time (s)

Fig. 6, 3 Change in A. systolic pressure (SP), B. diastolic prssure (DP) relative to the value at 4Os prior to the start of infusion of cocaine (0.5m9/kglmin). tregend: X saline (/ 00¡I/ntn) + rocaiøe (0.5ng/ k1/rrn) lS+Q, f nandrolnøe aehicle (zO¡I/nnn) * rocøine [ON+q, o nandrolone ftA4g/ kg/ lilin) * cocaine [40N+q, t nandrolone uehicle (77fl/rmn) * saline (10¡nlrztin) [0N+S], handrolane ft7ttg/kSlntft) * sabne [4ON+S]' Repeated-measures-,ANOVÂ, Bonferroni's post-hoc test. The line at x=100s represents the end of the ptessor xxx response to cocaine. SP (0-100s) see Inset A: TREATMENT p$p95)=3.790, p<0.0I] p<0.001 sig¡rilcantly different from S+C, #p<0.05, +++p<0.û01 significantly different from ON+C, ++p<0.01 sigmficantly different ftor,n

40N+C.; TIME [F(5,295)=24.5U, p<0.0001]; INTERACTION [F(æ,295)=5.866, p<0.0001] ve Appendìx G þr 'interacÍion'þosT-hoc tests- SP (100-600s): TREATMENT [F(4,354)=4.928, p<0.01]; TIlvfE [F(ó,354)=19.tU, p<0:0004]; B: INTER-A"CTION [F(24,354)=ó.35, p<0.0007f see Appendix G for 'interacrion' post-hoc an. DP (0-100s) see Inset TRE,\TMENT p(p95)=5.578, p<0.0011 *xp<0.01, xxxp<0.001 sigpificantly clrfferent ftom S+C, #p<0.05, ###p<0.0O7 sigmficantly different from ON*C, +p<0.05, +++p<0.001 significantly different frorn 40N+C; TtrME

see Appendix G teits. [F(5,295)=25.95, p<0.0001]; INTERÂCTION [F(20,295)=6.749, p<0.0001] for 'inreracrion'þ^st-hoc DP (100-600s): TRE-ATMENT tF(4,354)=7.99, p<0.00011; TIME P(6,354)=30.11, p<0.00011;. INTERITCTION n=17-74. PQal54)=7.067, p<0.000 7] see Appendix G for 'innraction' þost-boc tests. Mean+S.E. I

e -1 #+ gCL IÉ, -50

0 ,I00 200 300 400 500 600 Time (s)

F¡g. G 4 Chaq¡e in lrert ræ (HR), ldaüre b üle v¡l¡rc d ¡lO8 Driù b the slãrt of i¡úusbn d coeine (O.5n¡gfkg/min), IzgwúX sdi¡¡e (îffiNiøi") * rwøine P5"ryl@*i*) IS+q V,nædttøM a*fübtu

îE6ø¡&vløøe uehtuh (7t¡ú/nìb) + wlùr¡p' FANIvø@ # wøiøe pN+ql" o øøøåøfo¡¡e' $AFg/*Sf ,*u) * øøiø¡e [4ûN+Cb .

ftAgryUrøiø) [ûN+S] ftt¡g/kgffrtiø)* søäße [4ONI+SJ-R.epeated-meøsures-AN0Yd,tsø¡fenroúifsposÍ-hc æsr IfR (fÞdXh): TREATMENT ¡n(4,Aretr=3.n't, p<0"05J ++p<0.0tr, +++p<0"0Û1; T'IME, p(11$,391=37.54" p<0-€KFll; INTERT{CTION [F(44,ffiq=5"V62,Ír<:t-CfÆXf see Apwdäx Gf,ør"irøerøtioø'pt-hw tøsts- ]¿Ieam*SiE" n=lll- 14. A. D.

G G t I E E E E -1 o- o U' at, +0N+C *40N+C ++S+C

300 600 OÍUIC s+c Tirme(s|

B. E I 1

1

ED CD E E E o. o oo.

300 s+G Time (s) Treaûnent

c.

o' Ð f I E E E E É. -É. I

6{n 9(X) 0ìl4c s+c Time (s) Treatmeril

Fig. 6. 5 Lerft: Change in A. Systolic (SP), B. diastolic pressurc (DP) and C. HR during card¡ac ischaenfia. Legendi I nãildrolone uehiile (A¡ù/rztia) * cocaine, o nandrolone e7A/ kg/ ?zin) * cocaine,Xsaline (00¡ll/ xzn)

* cocaine (0.5ngl kg/ntn), Two-factor-,{NOVÂ, Bonferroni's post-hoc test. SP: TREATMENT lF(2,81¡=2.567, TREATMENT p>0.05]; TIME [F(2,81)=O.755, p>0.05];. INTERÂCTION F$,8I)=1.a12, p>O-051' DP: pQ.,8t)=2.932, p>0.05J; TIIvfE pQ,87)=0.793, p>0.051; INTER{CTION F(4Sr;=g.tt, p>0'051- IIR: TRE,tTMENT fQ,sz)=3.754, p<0.05j; TIME lFQ,82)=rJ.021, p>0.05j; INTERÂCTiON [F(a,82)=x'4, p>0:05l. Mear.¡S.E., n--tû-72. Right Change in D, SP, E- DP and F. HR at the conclusio¡r of reperfusion.

Legend: ¿N+C = aandrobne uehicte (7¡tt/ nnn) * coccine,40N+C = nandrollne (O¡tg/kg/nin) * cocaine, S+C = wlirue pQ22)=0.693, p>0'051, (100¡tt/nin) * cocaine (û5øg/k¡,l'tîin). Singfe-factor-ANovÂ. SFl- f Qp2)=0.7, p>0'051- DP:.

>0.0 -X0' IIR: [F(223) =0,66 P 5]:' MeantS-E., t=7 Is+c Eotrl+c ffilotl+s 40N+c l¿ot'¡+s

(21 (1) (1) (1) (21 Maximum surui\al tirne 900 for ischaen¡a 800 700 (21 (1) 600 Maximum surviwltime G for reperfusion ; soo E F 400 300 200 100 0 I R

900 Malmum survi\¿l t¡me for ischaemia 800 700

600 Maximum survi\¡al üme Ø for reperfusion o 500 E l- 400 300 200 100

0 R

number of rats in Fig. 6. 6 Suruival time in isChaemia (I) and reperfusion (R). Parentheses.'indicate :aline (100¡t//nin) + comine each pre-treatment gfouP dying from causes untelated to VF Legend: J+C = uehicle + saline (100y// nin), 40N+C = nandro/one (0.5mg/ kg/ nin),ON+C = uehicle (0¡t// nin) * cocaine,0N+-1 = according to Lambeth convention' (a\pg/ kg/nin) * cocaìne,40N+s = nandrolone + ¡aline. TOP: death defined p<0.05] (R) InvaLid to perform ANOVA Between group diffetences..Single-factor ANOVA. (I) [F(a,a8)=2.634, (reperfusion). BOTTOM: death from all causes' when snwival = L000/o. Mean*S.E. n=10-13 (ischaemia), n--6-13 (R) no ignifcanl dffirence' Mean+s'E Betçreen group differences,' Single-factot ANOV-A. (I) no si¿nficant dffirence '

n =12-7 4 (ischaemia), n=7 -!3 (tepetfusion)'

and reperfasion 6-li CFIAPTER 6 The ffict of intrauenots clcøine in rats søbjected to ischøemia Ftaction of surviving tats VF All causes Treatment I R I R

r0/1.0 1,0/1.0 1,0/1,2 1,0 /10 S+C (100%) (100%) (83Yo) (100%)

1,0/1,2 717 1,0/1,3 7/e 0N+c (83Yo) (1007Ð (77Yo) (78"/o)

ß/73* 13/13 13/14 13/13 ON+S (100%Ð (e3w (1007Ð (100%Ð

1,1/1,1,* 11/1,1, 1,1/12 11, /11 40N+C (100%) (92%o) (100%) (1007Ð

6/1,0 6/6 7 /12 6/7 4ON+S (60%) (1007Ð (s8%) (86Yo)

Table 6. 1 Number of rats surv¡ving as a fract¡on of rats al¡ve at the beginning of ¡schaem¡a' Left hand column: fraction of rats suru¡v¡ng which obey the Lambeth convent¡on. Right hand column: fract¡on of all rats sun ¡v¡ng. Parcntheses: percentage of rats surviving ischaemia and

reperfusion. Legend:J+C= sa/ìne(100pt/nin)rcocaine(0.5ng/ kg/nin),01,i+C= uehic/e(0¡t// nin)rcomìne,0ÀI+'1 = nandro/ane -t ¡aline. Chi-squared test uelticle + :aline (100p// nin),40N+C = nandrolone (7¡tg/ kg/ niil) t cocaine,40lv+-f = *p<0.05 with Fischer,s Exact test post-hoc. signifrcantly different from 40N+S.

Treatment I R

s+c 3+0 1+0 0N+c 3tl 1+1 ON+S 2+0 1+0 40N+C 2+0 0t0 4ON+S 4t1 1t1

Table 6. 2 Lambeth convention score. Legend: S+C = sa/ine (100p// min) r ncaine (0.5ng/ kg/ niil)' 0À'I+C = + saline (1001t//nin)' 40N+C nandrolone nandrolone uebicle (¡¡tt/nin) -r cocaine,OÀI+J = nandmlone uebicle (70¡t// nin) = :ignifcant dfferencet' Mean+SE' (aL¡g/ kg/ nn) * cocaine, 40N+S = nandrolone ft\¡tg/ kg/ nin) + saline. K¡uskal-\Vallis , no

n=1 0-1 3 (ischaemia), n= 6-13 (reperfusion).

6-14 CIIÂPTER 6 The ffict of intrauenou clcaine in røts sabjected to ischaemia and reperfasion Treatment Ftaction tesponding Fraction responding Ftaction responding with with VT with VF VT or VF

Ischaemia Repetfusion Ischaemia Reperfusion Ischaemia Reperfusion s+c e/12 2/10 4/1,2 0/10 e /12 2/10

(7s.0) (20,0) (33.3) (0.0) (7s.0) (20.0)

0N+c 7 /13 1/e s/1,3 1/e 8/1,3 1/e

(s3.8) (11.1) (38.s) (1 1 .1) (61.s) (1 1 .1)

40N+C 7 /1,2 1, /11 5/1,2 0/11 8/12 1, /11 (s8.3) (e.1) (41,.7) (0 0) (66.7) (e.1)

Table 6. 3 Fraction of sun ¡ving rats respond¡ng w¡th W, VF or W+VF during 15 minutes of ischaemia and dur¡ng 10 minutes reperfus¡on. Patentheses.'percent of surviving rats responding with 1r1,VFoTVT*VFdwingl0minutesof reperfusion.Legend: J*C= nline(100p1/ nin)*cocaine(0.5ng/ kg/ nin)'

¿l{+C = nandro/one aehicte (Tpt/nin)-t cocaine,0N+J = nandrolone uelticle (1¡tl/ nin) * ¡aline (00p// nin),40N+C = rignifcant diferencel nantlrolone (0¡tg/ kg/ nin) * cocaine,40^I+-ç = nandrolone ft|pg/ kg/ niil) r ¡aline. Chi-squared test, n0

6-15 CHAPTER 6 The efect of intrauenous clcaine in rats wbjected to ischaemia and repefusion (t)

A. B

1 VT VF

Ø a60 o o E 'r:E i= 40 40

S+C oN+C oN+S 40N+G 40N+S S+C oN+C oN+S 40N+C 40N+S Treatment Treatment (R) VT VF 100 c 100 D

80 80

ø U' o o E E 't= i=

0 S+C oN+C oN+S 40N+C 40N+S s+c oN+c oN+s 40N+C 40N+S Treatment Treatment

Fig. 6. 7 Average duration of \fr in ischaemia (4.) and repefus¡on (C.) and duration of VF in ischaemia (B.) and reperfus¡on (D.). Legend: -1+C= ntine (100pt/nin) t mcaine (05ng/kg/nin),0ÀI+C= (100p// nandrolone nantlrolone uebicle (¡¡tl/ nin) * cocaine,OlV+-l = nandrolone uebìcle (0¡t// nin) * ¡aline nin),40N+C =

(T¡tg/ kg/ n;n) -r saline. Kruskal-rVallis test, n0 signifcanÍ diference:' ø¡¡tg/ kg/nin) t cocaine,40-lv+-f = nantlrolone Meanf S.E., n=L2-1- 4 (ischaemia), ¡=7 -13 (teperfusion).

6-76 CFIAPTER 6 The ffict of intrauenoas cocaine in rats sabjected to is ch ae n ia an d reperfusio n 10

80

Ø 6 * o Ê t- 40

20

0 S+G oN+G oN+s 40N+G 40N+S Treatment

.l+C ¡aline Fig. 6. g Average duration of VF in ischaemia in rats surviving ischaemia. Legend: =

(100¡t//nin) tcocaine(0.5ng/ kg/ nin),ON+C= nandroloneuehicle(70¡t// nìn) tcocaine,0N+-ç= nandroloneuehicle(70¡t//nin) (}¡tg/ kS/ nin) * saline' * ¡a/ine (100p// nìn), 40N+C = nantlrolone 60¡tg/ kg/ nitt) t cocaine,40N+-t = nandrolone *p<0'05 kw=1135, p<0'05' MeantS.E., n=10 (S+C), n=10 (ON+C), n=13 (0N+S), n=11 (40N+C), n=10 (40N+S). test. Signiñcantly different from oN+S, Kruskal-!?allis test with Dunn's multiple comPârisons

150 IS+C Ioru+c 12 flillo¡l+s 40N+C 6. 100 o l¿0ru+s Ê 7 F 5

25

0 R

Fig. 6. 9 Average duration of VT+VF in ischaemia (I) and repeÉusion (R)' Legend: S+C = saline -t nmine,O},I+-i nandrolone uehicle (70¡t// nin) ( 00p// nin) t cocaine (0.5ng/ kg/ miil),0ÀI+C = nantlt'olone uehicle (70pt/ nin) = k¿/ nin) -t ¡a/ine' i ¡aüne (00pt/ nin), 40N+C = nantlrolone (T¡tg/ kg/ nin) * ncaine,40lV+-ç = nandrolone ft1pg/ signficanl dffitenrc' K¡uskal-\ùTallis Mean+S.E., n=I2-14 (ischaemia), n=7 -13 (reperfusion).(I) no si¿tficant dffirenæ, (R) no

test, rìo significant diffe¡ences.

6-17 CIIAPTER 6 The ffict of intrauenoas cocaine in rats subjected to ischaernia and reperfasion 6.6 Discussion

6.6.1 Blood pressure effecß of cocaúte a decline in systolic and .ùØhile cocaine displayed an obvious initial pressor effect, thete was tapid little or no initial increase' but diastolic pfessufes at all time points Past 40s. Heatt fate showed from baseline of between 60 and steadily decreased from baselìne, teaching a maximum depression by pfe-tfeâtment wrth 70 bpm. The catdiovascular response to infused cocaine was unaffected nandtolone. No arrhythmia was observed during cocaine infusion'

depressor effect has The initial pfessof fesponse to lntfavenous cocalne, followed by an rmmediate rats under pentobarbital anaesthesia been previously repotted in rats. Cocaine (zmg/kg/nnn, i'v') in followed by a gtadual deptessor (3Omg/kg, i.v.) was found to ptoduce a transient pressof tesponse, and the extent of the depression phase (Iella et a/., L992).The magnitude of the pfessof resPonse Similady, the tesponse to induced in BP was found to be dependent on the extent of anaesthesia' pentobatbital anaesthetised both cocaine (0.16-5mg /kg) and procaine (0.31 & t-25 mg/kù in shown to involve a dose telated decrease in afierial pfessufe 165mg/kS, i.p.) tats has been ø/ a/., 1'987)' A dampened immediateþ following a short pressof phase in the first minute (Pitts anaesthetised canines' A cardiovasculat response to cocaine has been previously repotted in mean BP 44+5'2 mmHg' cocaine dose of 1mg/kg, when administered to conscious dogs, increased pentobatbital (32mg/kg, i'v')' This dose, administered 30 min after dogs wete anaesthetised with i'v') dogs subjected to a increasedBP only 9.5+3.g mmHg. Pentobatbital anaesthetised (25mg/kg, change in HR or BP (Fraket et a/'' 1990)' This cocaine infusion 1+mg/kS) displayed no signifìcant in conscious animals' A sirnilar same dose had elevated BP and HR by 58o/o and 43o/o, tespectively been demonstrated in mice diminished pfessute lesponse to cocaine aftet pentob arbttal has also

CW^.g ù a1.,1'999).

6.6.2 Efiect of cocaine and ischaemia/reperfusion aSP and aDP fesponse during Nandrolone pfe-tfeatment was not found to significantly effect the for HR during ischaemia ischaemia or reperfusion. The significant effect of 'tfeatment' observed Post-hoc tests wefe negauve may be due to the high variability of the S*c gtoup (Fig. 6. 5)' to interpret' making the biological relevance of the signifìcant treatment effect diffìcult

was sufficient to cause Although it was evident that the dose of cocaine used (total dose=5mg/kg) to produce arthythmia' Alternatively' a signifìcant initial pressof ïesponse, it may have been too low associated with slow cocaine infusion a lack of a pro-atrhythmic action may reflect diffetent effects 1mg/kg have been associated with and bolus administration. Bolus doses of cocaine in excess of

6-18 to ischaemia and reperfa:ion CFIAPTER 6 The ffict of intrauenous clcaine in rat'î sabjected dog (Billman, 1993)' vF when combined with coronary occlusion and tteadmill exercise in the conduction slowing in Bolus doses gfeatff than 3mg/kg, i.v. have been associated with ventticular moderately sevete arthythmia in the dog (I{abas et a/.,1990). Bolus iv doses of 2tng/kgproduced infusion of 0'11mg/kg/min (total anaesthetised dogs (À4ehta et at.,2003). Flowevet, a slow cocaine pro-atrhythmrc effects but was dose, 20 mg/kg) in anaesthetised dogs with normal hearts had no to fìbrillation) (Tisdale el found to raise the threshold for VF (ie. reduced venfficular vulnerability class I anti-arrhythmic properties ø/., 1996). Accordingly, it is possible that cocaine may display some citcumstances (refer l'7j'2 for (Shakalis et al., 1967;Pitts ¿/ a1.,1.989;Crumb et al., 1995) under

review).

has been shown to ptoduce a Because cocaine administration simultaneous with alcohol ingestion McCance-I{atz et al'' 1'998; hìghly toxic, hepatic metabolite called cocaethylene fffilson et a/',1'995; et a/''2002;Laizute et al''2003)' Pa¡ et a:.,1999a;b; Ponsoda et a/.,L999;Wilson et a:.,2001; !íilson formation of this the s+c control gfoup was included in this study to control for possible used in this study was dilute metabolite. This was necessary because the vehicle for the nandrolone significantly depress the ethanol. cocaethylen e (11,.25 & 7.5 mg/kg) has been found to in dogs et a/'' myocardium, decreasing stroke volume, me î aÏtefral pressure and dP/dt SXltlson lees).

increase toxlclty' The combination of cocaine and ethanol itself has been found to considerably cocaine, incteasing Alcohol ddnking in tats has been found to alter the pharmacokinetics of notcocaine (1\dcCance-l{atz et bioavailability, elimination half-life and increasing the formation of ethanol have been found to be a/., 1.998; Pan et al., 1.999a; b). Human hepatocytes treated with to cocaine-induced depleted in reduced glutathione (GSH), making them mote susceptible and ethanol to mongtel oxidative damage (Ponsoda et at., 1999). Bolus administtation of cocaine arrhythmias (Xltlson et a/', dogs has been found to result in prolonged cardiotoxicity and ventricular more toxic than either substance 2001). cocaine and ethanol in combination were found to be

alone.

with ethanol containing The pre-ischaemic HR and BP response to cocaine in rats pre-treated pfe-tfeated with saline or vehicle was fiot significantly diffetent from the fesponse in rats the HR ot BP response nandfolone. Ischaemia did not pfomote any signiFrcant diffetence between saline ot nandrolone and of vehicle treated rats administered cocaine and fats pre-treated with frequency of arhythmia was not subsequently infused with cocaine. The extent, duration and is probable that the very low significantly different in the S-lC group in comparison to 0N*C' It cocaethylene formation' doses of ethanol used in this study did not result in appreciable

6-19 ischaemia and reperfasion CHAPTER 6 The ffict of intrauenoas cocaine in rats vbjected to 6.6.3 EfTect of the nandrolone-cocaùrc combination on arrhythmia and sun't'val No signifìcant increase was noted in Lambeth score during ischaemia fot any pre-treatment, neithet were any signifìcant differences between pre-treatment groups noted fot occurrence and duration of VT, VF or VT+VF. Survival time during ischaemia was unaffected by pte-treatment, but the less fraction of rats which obeyed the Lambeth convention and suwived ischaemia was signifìcantly This in rhe 40N+S group compated to the ON+S and 40N+C groups (Table 6. l Left column). irnplies that rats in the 40N+S treâtment group died towatds the end of the ischaemic period and thetefore did not significantly affect averase group survival time' Impoftântly, the enhanced in those rats mortality from fatal VF in ischaemia due to nandrolone pre-treatment was not present against fatal VF in also pretreated with cocaine. However, although cocaine significantly protected score or in the ischaemia this was not reflected in statistically significant reductions in Lambeth tats dying occuffence or duration of VT or \G by the combined tfeatment. To determine whether during ischaemia skewed the arrhythmia dutation results, the duration of arrhythmia was difference determined only in fats surviving ischaemia (Fig. 6' 5). However' the only signifìcant compared observed was a significant decfeâse þ<0.05) in the dutation of VF in the ON+S group to the 40N+S group. The 40N+C combination was not found to confer a statistically signifìcant nandrolone was dectease in VF duration compared to nandrolone alone. An intermediate dose of combined selected in these experiments in order to test for potentiation of arthythmia with choice of a higher treatment. Since cocaine appeared instead to be protective against fatal VF, the in the dose of nandrolone such as 1,60*g/kg/frnn, which produced more signifìcant increases sensitive test occuffence and duration of VF in ischaemia (chaptet 5), may have provided a mote for the combined effect of cocaine on arrhythmia scotes'

may possibly be Cocaine,s apparent protective effect agøtnst fatal VF in nandrolone treated rats

explained by two pteviously descdbed mechanisms:

and I(* channels 1. Anti-arrhythmic effect.,\lthough cocaine's inhibitory effect on cardiac Na* that anti- conüibutes to its pto-arrþthmic effects (Billman, 1990; Iímuta et a/',7992), it is possible Like Tisdale ¿l arrh¡hmic effects may occuf under some citcumstances, as discussed above (6.6.2). However, if al, thecufrent experiments used iv infusion as opposed to iv bolus (Tisdale et a/.,1'996)' in these experiments, it was a membrane stabilising effect underlies the protective effect of cocaine activity in only apparent in the presence of nandrolone. It was difficult to investigate antiarrhythmic use a the current study as the basic levels of arrhythmia were low' Future experiments should anhythmia' higher concentfadon of nandtolone in order to provide a higher basal level of

6-20 CFIAPTER 6 The efect of intrauenoas cocaine in rats subjected to ischøemia and reperfasion important pump normally 2. Blockade of NA release by the neutonal monoamine pumP' This Iversen et transports NA into the nerve ending and is potently inhibited by cocaine (Ivetsen, 7963; that elevated intra- at.,1,969). Although still controvetsial paugherty et at.,1986) there is evidence catecholamine cardiac catecholamines contribute to arrhythmogenesis in ischaemia. Intracardiâc et a/', 1987; concentfations afe known to be elevated in ischaemia (Schomig et a/.,1984; Schomig Schomig, 1990). Initially in yiuo this occurs by enhanced exocytotic telease due to increased Flowever as ischaemia sympathetic nervous system activation pan et a/.'1983;Dart et a/.,7987)' trânsport of NA by the pfogresses, due to accumulation of intracellular Na* there is fevefse Na* neuronal catecholamine uptake tfansPofter, which is dependent on the transmembtane mediated efflux has been gradient (Schomig, 1990; Datt et a/.,7993;Du et al.' 1998). This carder perfused hearts shown to be blocked in tats with I-AD occlusion Qu et at., 1'998) and in isolated such as cocaine, desiptamine' (Schomig et a/.,7984) by inhibitors of the neutonal uptake tfanspoftef et a/',1998)' In this nisoxetine and (+)-oxaptotiline (Schomig et a/.,1984; Schomrg et a/','l'988;Du formation' the setting uptake-l inhibitors such as cocaine are protective against arrhythmra 'üØhen only mechanism available neuronal uptake pumP tesults in reverse tfanspoft duting ischaemia, the (Schomig, 1990)' for local clearance of NA in the heart is extraneuïonal uptake and metabolism hormones Inhibition of extraneutonal uptake is one non-genomic PfoPeffy of a number of steroid extraneuronal including testostefone (Ivetsen et a/., 1970). If nandrolone were able to inhibit result enhanced exttacellular uptâke, this may not be important in the notmoxic heart but could in these extracellular concentrations during ischaemia. Cocaine would be expected to reduce alone on ischaemia- concentfations. In the present expefiments the protective effect of cocaine Howevef, the extent of this induced arrhythmia as desctibed by Du et alwas not reprodu ceð in uiuo' and duration of ptotection may depend on a number of factors including cocaine concentfation cocaine to be more pronounced exposure. Furthermore, one would expect this particular effect of where nofl-neuronal metabolism of NÀ is blocked'

contrâst with the The effects of combined nandrolone and cocaine, in the setting of ischaemiâ, ischaemia' in which combined effects pteviously demonstrated in the absence of cardiac In the latter case, nandrolone pïe-tfeatment enhanced the positive chronotropic effect of cocaine' nandtolone' A single enhancement of HR response only occutred after chronic treatment with not be excluded that acute subcutaneous dose of nandtolone had no effect; although it could effect of cocaine since nandrolone at higher doses might have influenced the positive chronouopic levels as those achieved a single subcutaneous dose of cocaine did not achieve the same plasma high plasma levels of after chronic dosing or arteriv infusion. In the present expetiments, although to an interaction nandrolone were achieved, the presence of anaesthesia limited the assessment with the decreased HR response to cocaine'

and reperfasion 6-21 CH,\PTER 6 The ffict of intrauenous cocaine in rats sabjected to ischaemia In light of the more prominent effects of chronic nandtolone in the absence of cardiac ischaemia, model, both in the the effects of chronic treatment with nandrolone in the ischaemia/repetfusion next chapter' pfesence and absence of cocaine, wete investigated and ate described in the

6.6.4 EÍfect ofpre-lreatnent on reperfusion anhythmias In tats obeying the Lambeth convention, reperfusion produced no significant dysthythmia' chaptet 5 where Consequently, survival was 10070 for all rats. This is similar to tesults from longet pedod of ischaemia atrhythmia activity was low dudng reperfusion, ptesumably due to the

(À4clennan et al., 1988)'

and repefusion 6-22 CH,\PTER 6 The ffict of intrauenous cocaine in røts sabjected to ischaemia 6.7 Conclusion caused a o In the anaesthetised tat, iv cocaine infusion at 0.5mg/kg/rrnn Fmg/kg total dose) increase, sustained fa1 in HR and a biphasic change in BP, consisting of an initial transient followed by a sustained dectease in BP. These haemodynamic effects of cocaine were unaffected by nandrolone pre-treatment'

in rats with normal a Intravenous cocaine infusion at 0.Smg/kg/min was not pfo-arfhythmic cardiac cofonafy flow, nor did it enhance the incidence of arrhythmia in tats subiected to

ischaemia.

from fatal VF during a Intravenous cocaine infusion signihcantly tevetsed the increased mortality Flowevet' this protective c fdi^cischaemia which was induced by iv nandrolone pre-treatment. score nor effect of cocaine was not reflected in a statistically signifìcant reduction in arrhythmia

the occutrence and dutation of VF or VT during ischaemia'

in reperfusion a No effects of pre-treatment wele detected for mortality ot arrhythmia

and reperfusion 6-23 CHAPTER 6 The ffict of intraaenous clcaine ¡n rats sabjected to ischaemia 6.8 References

ArresÍ, Ascher, E. K., Stauffet, J. C. and Gaasch, W. H. (1988). "Coronary Artery Spasm, Cardiac Acute Mlocardial Transient Ekctrocardiograpil, p Vlorrs and. Stunned. Mlocardium iru Cocaine-Associated Infarction." A- J Cardiol 61(1 1): 939-+1',

of Cocaìne." FASE'B-J 4(8) Billman, G. E. (1990) . "Mechanisms Responsible for the Cardiotoxic Ef,ect: 2469-75

Venticølar Billman, G. E. (1993). "IntracelÌalar Caldam Chelator, Baþta-Am, Preuents Cocaine-Indaced Fibrillation." A- J Physiol 265 (5 Pt 2): H1529 -35'

of Cocaine Interaction with Cardiac Sodium Crumb, W. J. and Clarkson, C. \Ø. (1995). "The Ph Dependence 27 a : 1228-37' C h an n e ls. " J -Pharmacol-Exp -Thet Q)

and Dart, ,\. M., Dietz, R., I(ubler, W., Schomig, A. and Stt_asser, R. (1983). "EfeXs of Cocaine the Rat Heart'" Br Des$ranine on the Neaiatþ Euoked. Ouerflow oi Endogtroot Noradrenaline fron J Plratmacol 7 9 (1) : 7 1' -4.

During Mlocardial Datt, A. M. and Du, X. J. (1993). "(Jnexpected Drug Efects on Aatonornic Function Ischaemia." Cardiovasc Res 27(6): 906-1'4'

Darr, A. M., Riemersma, R. 4., Schomig, A. and Ungat, A. (1987) "Metabolic ReTlymeøsforRekase Phatmacol 90: 43-50' o¡EiAogrooi, Norad.renøline During Mlocardial Ischaemia and Anoxia." Bt J

\X/oodward, of Catecholaminet in Daugherty, r\., Ftayn, K. N., Redfetn, N7. S. and B' (1986)' "The Ro/e Viuo and in Vitro'" Bt the Prodaction of Ischaenia-Indaced Ventricalar Arrhlthnias in the Rnt in J Phatmacol 87 (1): 265-7 7 .

Esler, M. D. and Datt, -,\. M. (1998). "Protectìo-n of Neuronal Du, X. J., Woodcock, E. 4., Little, P. J., Upiakei Inhibitor: in Ischernic and. Anoxic Hearts fu Noreþineþhrine-Dependent and -Indeþenfunt Me chanisms." J Cardiovasc Pharmacol 32@: 621' -8'

(1990)' "Mechanisn of Fraker, T. D., Temesy-Armos, P. N., Brewstet, P. S. and !Øilkerson, R' D. 1012-6. Cocaini-Induced. Aþocardial Deþression in Dogs." Circulation 81(3):

Prenatalþ Nøalsh, E. P. and Lipshultz, S. E'_(1994). "Arrhlthmias ìn Children Frassica, J. J., Orav, E,. J., Exposed io tocaine.",\tch Pediatt Adolesc Med 148(11): 1'1'63-9 '

S' (2000) "Cocaine Abase: Gamouras, G. Â., Monir, G., Plunkitt, I(., Gurso!, S. and Dreifus, L' ' Sci 320(1): 9-1'2' Repolarisation Abnorwalities and. Ventricular Affhlthmias'" A- J Med

Heart'" Br Pharmacol Iversen, L. L. (1963). "The (Jptake of Noradrenaline b1 the l¡olated Pefused Rat J 2l:523-537.

and reperfasion 6-24 CII,A.PTER 6 The ffict of intrauenoas cocaine in rats sttbjected to ischaeruia and Metabolisn of Ivefsen, L. L. and Langer, S. Z. (1,969). "Efects of PhenoxTtbenllyine on the Uptake 37(3): 627 -31 Norad.renaline in the Rat lleart and ." Br J Pharmacol '

p. lJptake-2 b1 Snroid^s in the Isolated kat Ivetsen, L. L. and Salt, J. (1970). "Inhibition of Cancholanìne Heart." Br J Pharmacol 40(3): 528-30'

D', Hoffman' G' W'' Ellinwood' E' H'' I{abas, J. S., M., J' Condaction in the Dog A Smith, p. I( C. d Impairrnent of Cardiac E'xp Thet 252(1):185-91' Potential Mech De harmacol

Aftndepotrirytions and Trigered I(imuta, S., Bassett, Â. L., Xi, H. and Myerburg, R' J' (1992)' ."Earþ plusible Arfuthrnogentsis." circulation 85(6): 2227 -35 ' Actiuiu rniaced. þt Cocaine. A Mechanism of Cocaine

(2003). "cocaethllene and Latzvre, S. C., Mandrell, T., Gades, N. M. and Parker, R. B. .Metabolisn Metab Dispos 3l(1): 1'6-20 ' Interaction with Cocaine ari E tiorott Role of CarboxJ,lesterases." Dtug

Ux of Cocains and Ahohol Is McCance-I( atz, B. F., I(osten, T. R. and Jatlow, P. (1998)' "Concarrent Maltþk-Dose Sta@'" Biol Psychiatry More potent and potentiatþ Mori Tox¡c Than'rJse of Eithn Alone--a 44(4):250-9.

Fish oil Preuents Mclennan, P. L., Abeywardena, M. Y. and chamock, J. S. (1988). "Dietary Am Heart !16: 109-717 ' Ventricular Fibillation Foitor*g Coionary Artery Occlasion and keperfasion'" J

cocaine: An Mehta,,{..,Jain,,\. c. and Mehta, c. M. (2003). "Elexrocardiographic Efects of Intrauenoas nrpri*rriit Staút iil a Canine Modet." J Cardiovasc Phatmacol4l:25-30.

A'' Wilkins' N'' Robertson' H' A'' Nademanee, I(., Gorelick, D. A', Josephson, M' A', Ryan, M' J' lYithdrawal." Ann Intern Mody, F. V. and Intaraiot, v. (ílalj. "Mlocardial Ischemia Daring Cocairc Med 111(11): 876-80.

Associated with Cocaine Nanji, A. ,\. and Filipenko, J. D. (1984) . "Aystole and Ventricalar Fibrillation

I n toxicatio n." Chest 85 (1): 1'32-733'

Takeichi' s' (1994)' "'ladden Death osawa, M., Mitsukuni, Y., Saito, T., Yukawa, N., Matoba, R. and of a Cocaine Abuser." TokaiJ Exp Clin Med 19(3-6): 1'15-9'

a). and Alcohol Interactions in the Røt: Contribution of Cocaine pan, SØ. J. and Hedaya, M. A. (1999 "Cocaine 468-76' Meíabotiies to the Phartnacotogical Efects'" J Pharm Sci 88(4):

'SØ. and Ahohol Interactìons in the Røt: Efect of Cocaine and Pan, J. and Hedaya, M. A. (1999b). "Cocaine Phatm Sci 88(12): 1266-14' Ahohol pretreatruents on Cocaine Pharznankinetics and Pbatrnacodlnamics.";

and reperfasion 6-25 CHÀPTER 6 The ffiø of intrauenoas cocaine in rats subjected to ischaemia CanJ Physiol Pharmacol 67(9) Pitts, D. I{. and Marwah,J. (1989). "AøtonomicActions of Cocaine'" 1.768-76.

Anesthetiryd and pitts, D. I{., udom, c. E. and Marwah, J. (1987). "Cardioaascular Efexs of comine in "u' Consrioas:Rntr." fe Sci 40: 1099-11'11

of M. and castell, v. (1999) ' Toxicitl Ponsoda, X., Bort, R., Jover, R., Gomez-Lechon, J. J. ,"lncre.ased of Gsh." Biochem Pharmacol 58(10): 1579-85' Cocaine 0n Hu,ftan Hrp)í,ryn, Iniaced. @ Ethanol: Ro;'

and Cardiac Re/ease'" Circulauon Schomig, A. (1990). "Catecholamines in þocard.ial Ischemia. Slstenic 82(3 Suppl): II13-22.

(1984)' "Re./e1e of End'ogenoas Schomig, ,\., Darq A. M., Dietz, R., Mayer, E. and K¡ble1, \ø. Lncalþ Mediate Re/ease'" Circ Res 54: 689- Catecholømine in tfu l:chatnic Nþtocardiam of tie Rat. Part A: 707.

(1937)' "Nonexorytotic Release of Schomig, 4., Fischet, S., I(urz, T., Richardt, G. and Schomig, E' Heart: Me hanisn and Metabolic Reqaìrernents'" End.ogenous Noradrenaline'in the Ischenic and Anoxic Rat Citc Res 60(2): 1'9 4-205.

sodiun and schomig, 4., I{utz, T., Richardt, G. and Schomig, E,. (198Ð. "Neuronal Noradrenaline Rel,,ase in Nornoxic and Axoplasmic Amine C)oncentration Determine Calciam-Ildepend'ent Ischimic Rat Hean." Ci¡c Res 63: 2'l'4-22C

of cocaine on Nerues Shakalis, A. and condouris, G. A. (1,967). "l-'oca/ Anesllettc^Action from 166(1) : 93-101 . AdrenaÌe ctomiry d Frogs." -Arch Int Pharmacodyn Ther

of Cocaine in Conscious Tella, S. R., Schindler, C. W. and Goldberg, S. R. (1gg2)' "C-ardioua¡cularEfects and Peripheral Neuronal Monoarnine Uptake and Røts: Relatiue Signifcan:ce of Central S1*pottriii Stimaktion 262Q): 602-1'0' Releare Mechønilrni." J Pharmacol Exp Thet

Webb' C' (1996)' "f!e Efelt^ of Cocaine on Tisdale, J. E., Shim oy'tm, H', Sabbah, H' N' and etapy 16(3): 429-437 ' Ventricillar Fibillation ihreshotd ¡n the Norrnal Canine Heart." Pharmacoth

\x/ang, Deangelis, Travefs, I(. and Motgan, J. P' (1999)' "Dffirential J. F., Hampton, T. G., "CardiouascularJ., to Cocaine irt Mica" Proc Soc E'xp Orprirrrr, Efects o¡ Croirot Znesthetics in the Response Biol Med 221(3):253-9.

Dotbandt, A' (2001)' "Cocaint, Ethanol, and Cocaethllene Wilson, C. B,., Jeromin, J., Garvey, L. and Acad Emerg Med 8(3): 211'-222' Cmdiotixiciry in'an Animlai Mofut of Conine and Ethanol Abuse."

Flow and Cardiac 'Wilson, L. D. and Ftench, S. (2002). "Cocaethllenls Efects of Coronary AXery Blood Fanction in a Canine Mode/'" Clin Toxicol a0(5): 535-546'

and reþefusion 6-26 CFIÂPTER 6 The eftct of intrøuenous cocaine in rats sabjected to ischaemia 'SØilson, E' and Ead' S' (1995)' "Cocaethllene L. D., Henning, R. J., Suttheimer, C., Lavins, E.'Bduai, Phatmacol Caase¡'Dose-Dependeø leduitions in Card.iac Fanction in AnesthetiT,ed Dogt." J Catdiovasc 26(6):965-73.

and repefusion 6-27 CIIAPTER 6 The ffict of intrauenoas cocaine in rats subjected to ischaemìa Effecß of cltronic nandrolone administation

7.1 Introduction Although an increase in arrhythmia has been observed dudng ischaemia with infused nandrolone administer (chapter 5), this is not the usual form of drug delivery. Recreational AS usets most often effects steroids chtonically by the intramuscular or subcutaneous toute. In this chapter the of chronic tfeatment were examined. Plasma levels of nandrolone following chronic tfeatment wefe measured so that a comparison could be made to the level obtained with inttavenous treâtment'

which was The same chronic dosing regimen (nandtolone decanoate 2\mg/kg, s'c'/3xs weekly) previously used over 3 weeks of treatment (Phillis et at., 2000) was used agatt for up to 9 weeks' This dose of nandrolone has been shown to increase the rate of elevation in BP of developing, changes spontâneously hlpertensive rats and to produce myocardial inflammatory and fibrotic therapeutic dose (Tseng et at.,1.994). A nandrolone dose of 2}mg/kgis apptoximately 60 times the the range of doses used illicitly by athletes (u-Yahiro et a/', Sohansson et a/.,7997), and within 1e8e).

In A number of other potentially toxic effects of chronic steroid treatment were also investigated' review addition to arrhythmia, chtonic steroid tteatmefit has been associated with hypertension (fot 'cardiac remodelling) see 1.6.3) (Hal), et a:.,1982; Tseng et a:.,1.994), catdiac hypeftfophy (see L.6.2 (I{ennedy, 1993; Huie, 1.gg4),hypedipidemia (see 1.6.1'atteriosclerosis') peeds et a/',1'986;Glazer' L999) and 1,991) andcellular damage ro rhe liver (Ishak et a1.,1987; Gtageta et a/.,1993;Boaða et a1., Notton ¿l myocardium (see 1.6.2 'myocatdial cell injury) (Tseng et a1.,1994; Trifunovic et al',1995; a1.,2000).

7-l CI{,A.PTER 7 Efects of chronic nandrolone administralion 7.2 Aims

treatment in pentobarbital 1 To determine the cardiovascular effects of chronic nandrolone

anaesthetised, close-chested tats, particularly during catdiac ischaemia and reperfusion'

tesults changes tn 2 To determine if this chronic regimen of nandtolone tfeatment in cholestetol, blood chernistry and pathology'

7.3 H5pothesis ischaemia' Chronic nandrolone tfeatment v¡ill cause an inctease in the degree of arrh¡hmia during which will increase in severity according to the length of nandrolone pfe-treatment'

7-2 CH¿.PTER 7 Efects of chronic nandrolone administration 7.4 Methodology

7.4.1 .Animals

Male Sprague Dawley rats were putchased at 9-1.0 weeks old, 2 weeks before the commencement of treatment. Rats wete housed in gtoups of 4-6 in wire mesh cages. One hundted and eight

animals wete randornly assigned to orie of 4 treatrnent grouPs'

3 weeks' Group 1, 3N & 3V: Treated with nandrolone Q\mg/kg, s.c., n=14) ot vehicle (n=14) fot Group 2, 6N & 6V: Treated with nandrolone (2Omg/kg, s.c., n=14) or vehicle (n=14) for 6 weeks' Group 3, 9N & 9V: Treated with nandtolone Q\mg/kg, s.c., n=14) or vehicle (n=14) for 9 weeks' for 9 Group 3, 9N+C, 9V*C: Treated with nandrolone (2Omg/kg, s.c., n--1'2) or vehicle (n=12) weeks and subjected to a 10 minute cocaine infusion 1O.5mg/kS/min) immediateiy pdor to induction of ischaemia.

account Although only 1.2 fats wefe requited in each tfeâtment group' 14 were treated in order to for animals, which may be lost due to sutgical preparation for ischaemia. Rats were dosed according to a staggeted tteatment schedule, such thal a maximum of 8 tats wete ready for

ischaemia-teperfusion on any one-day.

nandtolone levels at 3 ,\ separate gfoup of 16 rats wele tfeated in otder to provide data on plasma

and 9 weeks of tteatment. These fats wefe randomly assigned to either of 4 groups'

Gtoup A. Tteated with nandrolone (2Omg/kg, s.c., n-4) for 3 weeks' Gtoup B. Treated with vehicle for 3 weeks (n=4) Gtoup C. Treated with nandrolone (2}mg/kg, s'c', n-4) fot 9 weeks' Group D. Treated with vehicle for 9 weeks (n=4)'

7.4.2 Protocol as per Rats were fasted for 72 houts priot to use. ,A.naesthesia and cannulation were conducted ..genetal methods". Blood pïessure was recorded for 5 minutes prior to the chest being opened, period' and for 5 minutes following completion of preparative surgery and the equilibration

after which Rats in groups 1-3 were maintained under ischaernic conditions for 15 minutes a 10- reperfusion was initiated for a further L0 minutes. Rats in gtoup 4 were challenged with

minute cocaine infusion (0.smg/kg/min) priot to initiation of ischaemia (Fig. 7. 1).

7-3 CII-,A,PTER 7 Efects of chronic nandrolone admini¡tration for At the conclusion of the protocol blood was collected into heparinized eppendotff tubes Na* to altetations in and I{* assây, to determine whether potential changes in cardiac th¡hm wete due analysis of plasma . Approximately 1ml of blood was collected into EDTA tubes for F'van's blue cholestetol. The heart was excised into ice-cold saline, re-occluded and perfused with to dye (refer 2.8). The livet, kidneys, , spleens and testes vrefe femoved and weighed heart and determine the potential toxicological effect of chtonic nandrolone pte-treâtment. The kidneys from 4 rats treâted with nandrolone for detetmination of steroid plasma levels and hearts from four controls were sent to Veterinary Pathology at the Institute of Medical and Veterinary for Science (IMVS), Royal Adelaide Hospital, South Australia for pteparation of sections haernlatoxin and staining and light microscopy. The sections wefe âssessed by an independent veterinary pathologist, blinded to the tteatments'

until required. Serum lipoprotein was determined using a Blood samples were stored ^t -20o COBAS-Bio Cenrifugal Analyser (R.oche Diagnostics, USA). Total plasma cholesterol (TC) and kits (Roche Products triglycedde GRIG) concentration were determined using commercial (HDL) (IJNIMATE 7 (07 3664 3) and UNTMATE 5 (07 3679 1)) while high density lipoprotein Low-density concentrations wefe determined using the polyethylene glycol (PE'G) method' lipoprotein (LDL) levels were calculated using the Friedewald equation (Equation 7' 1)

(Friedewald et a/., 1972).

LDL = TC - (HDL + (TNGx 0.45))

LDL=low densirv Equation 7. 1 Fr¡edewald equation for determ¡n¡ng low density lipoprotein. Iipoprotein, TC=total plasma cholesterol, HDL=high density lipoprotein, TRIG=ttiglyceride. rat. Results Assays for TC, TRIG and HDL were performed in duplicate for the plasma from each

were discatded if the duplicate values varied by more than 10o/o'

plasma from rats treated for 3 & 9 weeks (n=3) and theit respective vehicle controls (n=3) were dehydrogenase analysed fot blood chemistry, total and protein, liver function and lactate (LD) at the Institute of Medical and vetednary Science (Queen Elizabeth Hospital' woodville South, SA).

7.4.3 Data Analysis time (mean+SE') Changes in body weight over the days of treatment were graphed as a function of hoc test' and analysed using repeated-measures-two-factor-ANOVÂ with Bonfetroni's posf were Terminal tissue weights, sefum lipoprotein levels and plasma nandtolone concentrat-ions selected pairs' To analysed using single-factor-ANOVA and Bonfettoni's post-hoc test between 7-4 CFIAPTER 7 Efects of chronic nandrolone admìnistration determine the cardiovascular effect of chronic nândrolone treatment, the SP, DP and HR was determined at 2 points (200s apart) dudng the 5 mtnute recotding period' Än average of these values was determined and a group calculated for each treatment. These values were then ^yeï^ge The analysed using single-factor-ÄNOVA and Bonfertoni's post-hoc test between selected pairs. effect of cocaine infusion in nandrolone and vehicle treated rats on cardiovascular panmeters was Differences expressed as change (À) in SP, DP and HR from 40s prior to the start of infusion. between gfoups were then analysed using a fepeât-measutes-2-factor-ANOV'\' In previous chapters cardiovascular variables were reported as AUC to maintain consistency with the authol's pteviously published work (tefet 2.5 lnalysis of Radiotelemetry Data)' However, ANOV-A' is more widely used as a statistical analysis method for this type of data. Results obtained with ischaemia and reperfusion were ptesented as pef General Methods 2.9.

7-5 CIIÂPTER 7 Efects of chronic nandrolone administration a. Groups 1,2,3

5 min 5 min 5 min 15 min 10 min

6. t 2. -). 4 5

Ischaemia Reperfusion

1. BP recotding in closed chested animal' around I,l\D coron^ry rcady for induction of 2. Open chest and place ligatute ^rtery ischaemia. 3. Equilbration period. 4. Ischaemia. 5. Repetfusion. 6. Collect blood & excise heart'

b. Group 4 only

L0 min 5 min 10 min 5 min 5 min 15 min

6 7 1 2. J 4. 5.

Cocaine infusion Ischaemia Reperfusion

1,. Close-chest BP & HR recording 2. Cocaine infusion (0'smg/kg/min, i.v.) LAD cotonâry teady fot induction of 3. Open chest and place ligatute atound ^rtely ischaernia 4. Equilibration Pedod 5. Ischaemia 6. Reperfusion 7. Collect blood & excise heart

Fig.7.1 Ischaemia-reperfus¡on protocol. Group 4 is subjected to a cocaine intusion (0.5mg/kglmn) for

10 rninutes priot to cofiunencement of ischaernia'

7-6 CIIr\PTER 7 Efects of chronic nandrolone adntinistration 7.5 Results

7.5.1 Change in body weight treatment Body weights wefe recorded for the days prior to sacrifice (Fig' 7' 2)' The hnal day of nandrolone was not included in this data because rats were fasted ptiot to sacrifice. Treatment with for 33 days was for 72 days was not found to signihcanúy vaty body weight. Howevet, tteatment found to signifìcaniy vary body weight (p<0.05). vehicle tteated fats wete found to have a and day 33 signilrcantly gfeâtef body weight at days 22-26 (alil, p<0'05), days 29-31' (all, p<0'01) (Fig. 7. 2 B). Likewise, vehicle tfeated lats þ<0.001) compated to nandrolone treated animals at were found to have a significantly greatet þ<0.001) body weight in compatison to nandrolone (p<0'001) in rats day 24 (p<0.05), days 26-29 þ<0.01) and at all other time points theteafter effect on body weight in treated for 54 days (Fig. 7 . 2 C.).'Time' was found to have a signifi.cant (p<0'0001)' An interaction rats treated for 1.2 days þ<0.0001), 33 days þ<0.0001) and 54 days and between'time'and'treatment'was noted in rats treated for 12 days þ<0.0001),33 þ<0'0001) this interaction became significant at day 22 54 days þ<0.0001). In rats rreated for 33 days 29-31' (^ll' p<0.01) and day 33 þ<0.05) andwas evident ar days 24-26 G,<0.05), days þ<0'001)' day 24 Similarly, in rats treated for 54 days the interaction became statistically significant at 31-54, (p<0.05) and continued to be significant at all points theteafter (days 26-29, p<0.01); days

p<0.001).

7.5.2 Change in organ weights treated rats, orgân Because of alarge difference between bodyweights of nandrolone and vehicle 7. 1)' weights afe pfesented as'average terminal tissue weight/1,009 terminal bodyweight'(Table period Relative weights were found to change significantly (p<0.0001) over the tfeatment administration such that kidneys from nandtolone treated tats at 3, 6 or 9 weeks of nandrolone changes were found to be signifìcantly larger than the respective control (all, p<0'001)' Signifìcant The were also observed in testicular weight over the dutation of the treatment period þ<0'0001)' treated raTs at 9 relative weight of the testes was found to be significantly greater in nandtolone changes were also weeks in comparison to the corresponding vehicle control (p<0.01). Significant relative adrenal observed in the relarive weight of the adrenals (p<0.0001). Significantly latget control (both' weights wete noted for tats administeted nandrolone for 6 or 9 weeks compared to liver weights p<0.001). Nandrolone tfeatment was not found to change spleen weight, but relative less were significantly changed þ<0.01). The relative weight of the liver was significantly þ<0'05) normalised for body in rats treated for 3 weeks with nandtolone compared to contfol. Heart weight such that the weight was found to be significantly changed by nandrolone tÏeâtment þ<0'0001),

7-7 CIIÂPTER 7 Efect: of chronic nandrolone administration relative heart weight was significantly greater in nandrolone treated rats at 6 and 9 weeks þoth, p<0.0001).

7.53 lfisbpathologY H & E staining and light mictoscopy revealed no significant difference in of hearts and kidneys from rats treated for 9 weeks with nandtolone and cortesponding vehicle conttols.

7.5.4 Cholesterol, tnglycerides, LDL attd I{DL profrles' Total cholestetol (TC) was found to be lower in all nandrolone groups in compadson to the vehicle control (Table 7. 2) butwas only found to be statistically signifìcantly different ftom vehicle at 3 weeks of treatment (p<0.01). Total cholesterol in nandtolone treated râts urâs found to inctease significantly at week 9 compared to week 3 of nandrolone treatment (P<0'001)' Although triglyceride levels were consistently lower in nandrolone treated fats compared to control' only statistically relevant diffetences wefe not detected. High density lipoprotein (HDL) was High decreased in rats treated for 3 weeks with nandrolone in compatison to vehicle þ<0.05)' in density lipoprotein was found to be significantly elevated at 9 weeks of nandrolone treatment were comparison to 3 weeks steroid treated rats þ<0.001). No statistically signifìcant diffetences differences detected between treatment groups in LDL or the HDL: LDL tatio, neither were any within groups observed as treatment progressed'

7.5.5 Serology Itwasfoundthatglucose[t(6¡=6'g'o,p<0.001]andbicarbonate|t(6)=2.449,p<0.05]levelswere elevated at 3 and 9 weeks of nandrolone treatment, respeclively compared to the corresponding vehicle treated controls (Table 7. 3).Sodium ft6¡=3.5rî' p<0'051, phosphate lt(6)=4'64', p<0.05], aminotransferase (-AST) p<0.01], alkaline phosphatase (ALP) $(e¡=2.66U, ^spaft,'te (I-D) p<0'05] were all found to have It(67=2.74t,p<0.051 andlactate dehydrogenase [t(6)=2'539, significantly lower values in rats treated fot 3 weeks with nandrolone in compatison to animals 9 weeks of administered vehicle for the same period of time. Signtficantly lower values at 3 and tfeatment were observed fot creatinine lt(6)=2.976, p<0.05 and t(6)=2'778, p<0'05, respectively], and albumin total protein lt(6¡=6.1rr,p<0.001 and t(6)=1.595, p<0.05, tespectivelyl, lt(6)=2'782, p<0.05 and t(6)=1.800, p<0.01, respectively] compared to control. No statistically signifìcant bilirubin' change between groups was observed for potassium, chloride, utea, calcium, total feras e glutamyltran s feras e (GGÐ and alanine aminotrans (ALT)'

7..1.6 Plasma úevel of nzutdrolone

Nandrolone was not detectable in controls'

7-8 CFIAPTER 7 Efect of chronic nandrolone administuarion Plasma nandrolone concenttation wâs found to increase at 3 weeks (p<0'01) and 9 weeks

(1r<0.001) compared to vehicle control (Table 7' 4)'

7.5.7 Cardiovascular- response to chronic n¿utdrolone treatment Efrfect of nandrolone on DP and SP affect either In rats under pentobarbital anaesthesia nandrolone, pre-treatment did not signifìcantly

DP or SP (Fig.7.3)'

Ertfect of nandrolone on IIR He ft t^te in nandtolone tteated fats was found to vary signifìcantly (p<0'01) in tesponse to chronic nandrolone treatment. Flowever, no signifìcant differences were obserwed when rats rùØhen treated for 3, 6 or 9 weeks were compared to their respective controls. comparing within 9 weeks of treatment groups, a significantly lower HR was noted in nandrolone treated rats at tfeâtment compated to 3 and 6 weeks (p<0.001 and p<0.05 respectively) (Fig' 7' a)'

7. 5. 8 Cardiovas cu]ar respons e to cocaJ'n e adm inis tra ttott with nandtoione In 2 separate groups of 72 rats, the effect of cocaine following 9 weeks treatment or vehicle was investigated. The cardiovascular effect was measured as change (A) from baseline as a L0-minute (40s pdor ro the start of cocaine infusion). Cocaine (0.5mg/kg/min) administered infusion to râts prior to artenal occlusion was found to induce an immediate, sharp elevation in SP and DP and DP in rats treated for 9 weeks with nandrolone or vehicle' Change in SP (ASP) to occut at (ADp) is shown in Fig. Z. 5, A. 8.. Maximal ASP in response to cocaine was found 5, B')' 40s following the start of infusion (Fig. 7. 5, A.).Maximal ADP occurred at 60s (Fig' 7' The ASp and ADP ïesponse to cocaine was flot signifìcantly modifìed by pre-treatment' !Øhile At both Sp and Dp displayed an ea.jry increase, this was followed by a slow decrease over time' below 600s, following the start of infusion, both DP and SP displayed a decrease in pressure

baseline. Consequenrly, 'Time' alone was found to signifìcantly effect ASP and ADP þ<0'0001)' by The time at which maximum SP and DP was obtained in response to cocaine was unaffected for ASP pfetfeatment. No significant interaction þetween 'tfeatment' and 'time') was observed

and ADP (Fig' 7. 5, A, B).

whereas HR Fleart rate in nandrolone treated rats adrninistered cocaine increased slowly untii 160s' in vehicle treated rats reached maximum by 40s and then dec[ned rapidly between 180s and 300s' was found' The change in HR (AHR) is shown in Fig. 7. 5, C. No effect of treatment on AHR signifìcant Flowever, 'tjme' alone was found to have a significant effect on AHR (p<0'0001)' A between'time'and'tfeatment" such that the aHR interaction þ<0.01) in tetms of ÂHRwas found 7-9 CIIAPTER 7 Efects of chronìc nandrolone administration to resporise to cocaine was sþificarÍly greater þ<0.01) in nandrolone treated rats compated vehicle at the final time point of infusion (600$ (Fig' 7' 5' C)'

7.5.9 Cardiotrascular response to ischaemia and reperfùsion (eg. vehlcle Because Sp, Dp and HR was still depressed at the end of the cocaine infusion petiod treated rats receiving cocaine ASP: -9.5+8.69mmHg' ADP: -7.0t7'39mmHg, AHR: -33+j.4.23mmHg) the cardiovascular response to ischaemia and reperfusion in rats receiving

cocaine was analysed separately from rats receiving nandrolone or vehicle only.

fschaemia Nandrolone or vehicle treatment only 600s and The change (À) in Sp, DP and HR was calculated from time zero of ischaemia at 300s, 900s of occlusion (Fig. 7. 6). A significant effect of treatment was only found for AHR þ<0.05) post-hoc detect any signihcant differences in HR at (Fig. 7. 6 C.). rests (Bonferonni's) failed to ^ny ischaemia. No effect of 'time' or intetaction between 'treatment' and 'time' time point during ^ny was found for any of the variables measured'

9 weeks nandrolone or vehicle treatmentt plus cocarne Treatment was found to signifìcantly affect ASP (Fig. 7.7 and ADP (Fig' 7' 7 B') þoth, ^.) p<0.01). post-hoc tests did not reveal where this significancelay. Treatment did not significantly either the affect AHR (Fig. Z. 7 C.). Likewise 'rime' alone was not found to signihcantly change and change in Bp or the change in HR. No signifìcant intetaction was found between 'treatment' 'time' fot the BP or HR changes'

Reperlusion Nandrolone or vehicle treatment only the conclusion of The change in Sp, DP and HR was calculated from time zero of reperfusion at the 10 minute reperfusion pedod (600s). No significant difference was found between treatment groups for the asP (Fig. 7. 6 D.), ÄDP (Fig. 7. 6F,) or AHR (Fig. 7. 6 F.) tesponses.

9 weeks nandrolone or vehicle treatmentr plus cocalne DP (Fig' No significant difference was found between treatment groups for the SP (Fig. 7.7 D'), 7.7 E.) or HR (Fig. 7. 7 F.) response during reperfusion'

7.5.10 Zone at risk of infarctton No The area at dsk of infarction was consistent between tfeâtment gfoups (data not shown)' the srudy significant differences were found between or within tfeatment groups for the dutation of

(single- factor AN OV'A.). 7-70 CIIAPTER 7 Efects of chronic nandrolone administration 7.5.11 Sunival Time Five out of a total of 98 rats (-5"/ù died from complications unrelated to VF (8 died from VF)' survival Severity of arhythmiâ was not scored in these tats neither were they included in above calculations. The number of exclusions from each tteatment group is noted in patentheses Fig. 7.8.

No signifìcant difference was found between the survival times of the various treatment groups 1n the number of ischaemia and reperfusion (Fig. 7. 8), neither wete signifìcant differences found in rats surviving ischaemia and reperfusion (Table 7' 5)'

7.5.12 Arrlryrhmia Ischaemia VF or No significant difference was found between the fraction of rats responding with VT, VT+VF during ischaemia (Table 7. 7). Likewise, no significant differences wete found between the duration of VT (Fig. 7. 9.4), VF (Fig. 7. 9'B) or VT+VF (Fig' 7' 1'0) during ischaemia, 7' 6)' neither were any differences in Lambeth arrhythmia score detected between groups (Table was also To ensure that fatalities duting ischaemia did not skew the results, arrhythmia duratron ischaernia calculated in rats which survived ischaemia. The duration of VT, VF or VT+VF during

was not significantly different in rats which survived occlusion (data not shown)'

Reperfusion No signihcant difference between groups for the ftaction of tats responding wrth arrhythmia was 9'C), VF noted dudng reperfusion (Table 7.7). At no time was the frequency of VT (Fig' 7' different (Fig. Z. 9.D) or VT+Vtr (Fig. 7. 10), or the arrhythmia scote (Table 7. 6) statistically between groups.

7-ll CFIAPTER 7 Efects of chronic nandrolone administration A.

g, 4 .9 ìo

3

3 57 91113 Time (days) B.

52

E) 4

Et (¡)ì

37

5 10 15 20 25 30 35 Time (days)

G

52

ctt 4

ct oì

37

0 10 20 30 40 50 60 Time (days)

Êig.7.2 Body we¡ght in rats treated with nandrolone (20mg/kg, s.c'l3xs weekly) over A' 12 days, B. 33 days or C. 54 days prior to sacrifice. I Nandrolone, O vehicle. Repeated Measures, 2-føcror S2' p<0'0001]' ANOV,A., Bonfer¡oni's post-hoc test. A. TREATMENT [F(1,130)=6.359, p>0.05], TIME [F(5,130)=65 resa/ß). B. TREATMENT 451, p<0'05] INTERACTION [F(5,130)=9.947, p<0.0001] þee n*þr þost-hoc [F(1,36a)=6 gn, 0001]' *p<0.05, *xp<0.01, **xp<0.001 sigruficantly different from vehicle treated rats, TIME [F(14,364)=5g p<0 post-hoc resaltt). C. TREATMENT 10' INTERACTION [F(14,364)=84.38, p<0.000I] þee îext þr [F(1,598)=14 rats, TIME t, p<0.0001] *p<0 05, xr,p<0.01, **p<0.001 sþifrcantly different f¡om vehicle treated [F(23,598)=56 p<0.0001],INTERACTION[F(23,598)=I07.2,p<00001]þeetexlforþost-hoctvsu/t:).n=1'3-1'4'MeantSE.

7-12 CFIAPTER 7 Efects of cltronic nandrnlrne administration (g) ev (g) 3N (g) 3v (g) 6N (g) 6v (g) eN xx 1.87+0.06*** 2.03+0.05***###* 1.52!0.03 1.7410.03*** 1.32+0.03 1,.45fl.02 Kidneys x**### (0.3210.00) (0.43t0.01) **+ (0.33t0.01) (0.50t0.01) (0.53+0.01) xx*### (0.31t0.01) )Õ(x XXX 7.48+0.02 1.64+0.03 1.58+0.02* 1.40+0.08 1,.46+0.02* 1.64fl.02 Testes *+ (0.39+0.01) (0.3310.01) (0.3erO.01) (0.3sr0.02) (0.39r0.01) (0.36f0.00)

0.031+0.001 0.035+0.002 0.030+0.002 0.033 0.001 0.028 0.001 0.02710.001 Ad¡enals x** (o.oo9ro.ooo4) *x* (0.00610.0004) (0.0081f0.0003) (0 0070+0.0002) (o.oo82to.oo03) (0.0062+0.0002)

0 5010.02 0.55+0 02 0.53 0.01 0.52+0.04 0.47+0.01 0.55t0.02 Spleen (0.13+0.01) (0.1110.00) (0.13310.003) (0.128+0.010) (0.125+0.004) (0.121+0.004) 9.65+0.49**x 72.96+0.66 10.50+0.52 1,2.34-10.77 9.66+0.36**x L3.34fl-4ó Liver x Q.60t0.1) (2.6ùt0.10) 'F (3.0e+0.18) (2.60+0 11) (2.e310.10) Q.s2r0.0e)

1.65+0.04 1.70+0.05 1,.79+0.01 1.69+0.05 1.ó110.066 1.71fr.034 FIeart x** (0.45+0.01) x*x (0.3610.01) (0.43t0.01) (0.40r0.01) (0.44t0.01) (0.37t0.01) xxx xxx 45617 381+10xxx 498+14 Bodyweþht 403+9 399+10 373+6***

weight on the day of Table 7. 1 Terminal t¡ssue weight in grams. patentheses: terminal tissue weight/1O0g body weight (oormalise-d). ÀIore.'body weight refers to the body

x-L\p<0.00 from 6N- xo<0.05. up<0.0 1,, 1. Sisnificantlv diffetent from 3V.MeantS.E., n=12-14. \t I Lr Trigþceride High density Low density Total cholesterol T¡eatment (TRIG) lipoprotein (HDL) lipoprotein IJDL/LDL (TC) mmol/l mmol/l mmol/l (LDL) mmol/l

3N 1.22+0.07 (n=8)#n 0.30!{.04 (n=9) 0.15+0.05 (n=8)# 0.34!0.04 (n=8) 2.34!0.2s (n=8)

2.07+0.29 (n=4) 3V 1..76m.D þ=10) 0.43!{.14 (n=6) t.09+0,1 (n=6) 0.s9+0.08 (n=4) 6N 1..4.0.09 (n=7) 0.31!{.07 (n=4) 1.01,!0.07 (n=4) 0.36+0.04 (n=3) 2.83!0.09 (n=))

6V 1.17+0.15 (n=6) 0.46m.1'2 (n=5) 1.07!0.05 (n=7) 0.42!0.1'4 (n=4) 3.56!0,98 þ=a)

9N 1.71+0.06 ¡n=l0)** 0.34!{.05 (n=6) 1.19+0.06 (n=9)**+ 0.48+0.06 (n=4) 2.se!0.32 fu=4)

9V "1.80X0.11 (n=9) 0.52!{.14 (n=9) 112+0.09 (n=9) 0.45+0.11 (n=6) 3.14!0.52 þ=6)

Tabfe 7.2Totat cholesterol (TC), triglycer¡de (TRIG), high density lipoprotein (HDL), low density lipoprotein (LDL) in rats treated w¡th nandrolone for 3, 6 or 9 weeks compared to respeçt¡ve veh¡Cle COntrOlS. Legend:3N=3 weeks nandroloile treatment, 3V = 3 weeks aehicle lreatmeflî, 6N = 6 weele¡

ßandr,l,ne treatment, 6V = 6 wæk¡ yehicle treatment, 9N = 9 aee,k¡ nandrolone tredtnenl, 9V = 9 weel

frorn 3V. xxp<0.01, *xt'p<0.001 signifrcantiy diffe¡ent from 3N. Mean*S.E.

7-74 CHAPTER 7 Ef,ects of chronic nandrllzne adrninistration 3N 3V 9N 9V Refetence Values (mean+S.D.)

Glucose ft l0 s412 28 (fer.l), I 7i2 l8 (t,stcd) (ÀfâGuzâwâ r/ ¿1, 1993). ? 3311 I 7 (10 L2 wkt' 1 46105 8 3+0.3 92!09 7.810 ó I 28 (<6 m o / ¿1, 198ó) (mmol/L) ? ó311 0-t 0s zO rvks) (rævine, 1995), 49t ) 0volrord

(10 20 rvks) ¿¿ 1991), 1 16 s1l 25 (10 12 s'ks), 1'ló 513 25 Sodim | t+l 51.1 3 0râtsuzaNâ r 139+0 141+0 139+1 t-t911 pcvine, 119 (<6 m o d ¿1 198ó) (mmol/L) (18,20 rvks) 1995), 013 30 ) ovolford

('3 Potassium 4 ó010 60 (10 20 rvk$ Ofitsuzâlvà ¿/ ¿1 1993), 6 ?510 (10 1 2 rvks), 5 5010 ?5 +.3+0 z 42+0.2 +.2+0.1 4.1+0 2 7ó (<ó m o / ¿1 1986) (mmol/L) (I-cvioe, l9r5),6 810 ) 0volrord

Bicarbonate psv¡ 198r), 2'7 1 !3 o (s'14,n o) (læN¡ / ¿l' l98I) 11+7 '14!2 18+1# 1+!1 28 rtl 0 (2-5 m o ) t al., (mmol/L)

(10-l 2 \'ks)' 101 0t2 00 Chloride 104 st3 50 (10-20 rvk$ Olâßuzawi / ¿/, 1993), 101 ùt2 00 10Gr2 105+2 105+2 10ó+1 10 + 00 (<6 m o / ø/, 1986) (mmol/L) (1S'20 wkÐ (Iævine, l99s), 013 ) 0\/olford

Creatinine 0 07410 002 0 091+0 005 0 07510 002# 0.085+0.003 0 006s (ùlo¡¡ìes d r'r, 2001) (mmol/L)

(10-12 !vkt, IJrea I 32+1 3ó (led), ó 0311 11 (fìsted) Orlßuzrs'â ¿/ d4 1993), 5 l6t0 54 6 9+0.5 1 6lX1 7.5+0.3 ó 3+0.5 perine, 1411 14 (<6 m o) al 1986) (mmol/L) 5 7110 ?t (I8-l.tNks) 199s),7 flVollord

Calcium 2 +?r0 175 (t0 20 rvks) Olaßuzr\'î ¿ ¿/, 1993), 2 72i0 131 (10 I 10 Nks) 2 4!0.05 2.54+0 01 2.51+0 09 2.ó8+0 05 3 26 1 (<6 fl o / ¿¿ 198ú) (mmol/L) (Levine,l995), 9210 ) 0\/olford

Phosphate tt+ 2 29i0 3ó (10 20 rvks) (l\fxtsuzaNl ¿/ ,1 1993), 2 7110 21 (10 20 rvks), 2 0210 3ó (18- 26 3 69+0 04 204!029 1.89+021 2 48Lr 2 7110 53 (<ó m o er a/,1986) (mmol/L) z0 rvk$ (Levine, 1995), ) ovolío.d

Total (18'20 wkt fl 6215 (10-20 rvks) (Ilatsuzlwr / ¿1 I 993), 69Ì3 s (10-12 rvk$, 701+ 0 50+1 57+0 5'tt2 58+1 protern (|-crine, 1995), ?0+? 1 (<6 m o ) Ovolror¿ / ¿/, 198ó) G/L\ Albumin t1É 351? (10-20 wks) (ÀlâtsvÀ\\'À il al,1993),3'l 5!1 7s (IG12 NkÐ, 37 s12 25 (t8-20 2.1+1 32+1 29+7 32+7 rvk$ Pevine, lees) @/r) Total s l3t0 s55 (10'12 rvk$, 5 I 3t1 71 08 só wkt (Lcvinc' 1995), 5 1312 73ó (<ó nì o ) 1t0 2+0 1+0 2+0 bilirubin 0volford ¿ ¿1 198ú)

ALP 20 s'ks fastcd) (llrtsuztva ø øl 1993) 98+8 128+1 91+1 6 131+21 395 511ó2 3 (10 20 rvks fcd), 164 31?9 2 (10 (D/L)

20 wk$ AST lll 105 9+51 3 Ofâsuzrwr d at, 1993), 0 srlt 25 (lG l2 wks), 72 5i1 3 75 (18 190+15 254+'13 2l(t+19 137+13 pevine, 9?128 4 (<6 m o) ¿ ¿¿ 198ó) (U/L) 1995), 0volford GGT 110 1+0 1t0 110 1t0 5 (t0 20 rvk$ Qrvine, 199s) (U/L) 5 (10 l2 wkt, 10110 (l 8-20 \'kt ALT t 38 2115 2 (10 20 NkÐ Ofxtsuziwn ¿ ¿1 1993), 25t7 25++ 22!9 40+6 48+5 (I-evine, 199s), 38+ta 4 (<6 m o) 0\/oltord 4 r'l 198ó) {J /I) LD 101+71 595+95 225187 5 (10 12 rvks), 225187 5 (18-20 wkÐ 0rvine, l99s) 358+54 629+90 (rJ/L) Table 7. 3 Serology for rats treated with 3 or 9 weeks with nandrolone or veh¡cle' and l¡terature treallnenl, 6V 6 ueek¡ ValUgS. Legend: 3N=3 week¡ natuJr4llile trentment, 3V = 3 weeþ.s uehicle treatmenl, 6N = 6 u)eekr ildndrzllile =

gV g uehic/e treaÍmeilt, 9N+C g weeks rutndrLlLile ,rc(úmeilt * cocaine, uehic/e treahtent, gN = 9 u)eeks ttail(ù.o/ltte hvalmenl, = aeeks = aspartate aminoÚansferase, GGT: 9V+C = 9 weeks uehicle treatnenl * cocaine. Mean*SE, n=4. ALP: alkaline phosphatase,AsT: gammâ glutamylttansferase, AIT: alanine aminotransfetase' LD: lactate dehydrogenase. *** different from 3 Effect of nandrolone treafinent on serology: unpaired t-test p<0 05, P<0'01, p<0.001 signifrcantly

#p<0 oop.O.Ot serology: wks vehicle. 05, significantly different from 9 wks vehicle. Effect of treatment duration on tp<0.05, **p.001, trtp<0.001 ^^^p<0001 unpaired t-test significantly different from 3 wks vehicle ^"p'0'01, litetatuÍe setology significantly d.ifferent from 3 wks flandrolone Note: The âge of rats has been noted in Pârentheses for values (only where the age of rats has been published)' 7-75 CII,A.PTER 7 Efectt of chronic nandrllzne adninistration Nandtolone Plasma nandtolone Plasma nandtolone n Treatment concentration (nM) concentration (ng/ml) 3N 3 9o+18xx 2515** 3V 3 0+0 0+0 9N 3 172+23***# 47+6**x # 9V 3 0+0 0+0

Table 7. 4 plasma nandrolone concentrat¡ons. Gas chromatogtaphy - mass sPectrometry method' 6 weeks nandro/one lrvatîlteilt, 6V 6 pee/er Legend: 3N=j weeks nandro/one treatment, 3V = 3 weeks vehicle tredlmettt, 6N = = wee,k¡ aehic/e treatrtefli, 9N+C 9 weeks nandrolone trcdlnenl + uehicle treatment, 9N = 9 aeeks nandmlone tredtnent, 9V = 9 = Bonfetroni's post-hoc test between selected pairs cocaine, 9V*C = 9 aeeks aehicle îreaîment I cocaine. one-way-ANOVA, ##p<0.05, *i.p<0.01, xxxp<0.001 signifrcantly different from respective vehicle control. [F(3,A¡=33.gr, p<0.0001], sigrufrcantly different ftom 3N, MeantS'E, n=3'

1

E' 1 2

E 100 E o 7 o o o ÈL 25

3N 3V 6N 6V 9N 9V treatment

3, 6 & 9 Fig.7.3 Sp (open bars) and DP (filled bars) in pentobarbital anaesthetised rats at weeks of nandrolone (2omg/kg, s.c.) or veh¡cle administration. l-*gend: 3N=3 wee/

7-16 CFTAPTER 7 Efects of chronic ilandrllzne administration ***

350 c' 300 ct .cr o (ú É 150 100 50

0 3N 3V 6N 6V 9N 9V Treatment (20m9/k9, Fig.l.4 HR in pentobarbital anaesthetised rats at 3, 6 & 9 weeks of nandrolone 6N 6 S.C.) Or VehiCle administfatiOn. Legend: 3N=3 week¡ nandrolone îreatmeill, 3V = i weeks uehide treatne,tt, = treatmenl, 9V 9 aeeks aehic/e tre¿ttment, weeks nandrolone treatment, 6V = 6 aeeks uehicle lreatrl¿nt, 9N = 9 week¡ nandrolone = r cocaìne' Si.ngle-factor ANOVÂ' 9N+C = 9 wee/<: nantlrolone tredtment * mcaine, 9V+C = 9 aeek¡ uehicle treatment *p<005' ***p<0001 Bonfetroni's post hoc test between selected pairs p(5'128)=!g22' p<0'01]' signiFrcantiy different from 9 weeks nandrolooe, Mean*SE., t=12-14'

7-17 CHAPTER 7 Efects of chronic naftdrlllne administration A.

35 I Nandrolone 30 *Vehicle 2 E' I E 1 E 1 o- U'

-10 -15 -20 0 100 200 300 400 500 600 Time (s) B

30

IE'' E 1 E 1 ô0-

-10 -1

100 200 300 400 500 600 Time (s) G

20 G T 1 E E -rl rÉ.

-40

100 200 300 400 s00 600 Time (s)

Fig,7.5 A.5P,8, DP and c HR response to a lo-minute coca¡ne (0.5mg/kg/min) infusion (a). in rats treated for 9 weeks with nandrolone ( r ) (2omg lkg, s.c,l3 X weekly) or vehicle p(,2a)=1'767' p>005]' Between group differences.'Repeat-measutes-2-factor-,\NOVÂ, A. SP: TREATMENT TIME|F(11,242)=Jg.92'p<0.0001],INTERACTION|F(11,242)=t.919,p>0'05],B.DP:TREÀTMENT p<0.0001], INTERACTION 685' p>0'05]' c' HR: lF(\,242)=\.633, p>0.051, TIME [F(11,242)=41.00, [F(11,242)=0 p<0.0001], iNTER{CTION TREATMENT IF(1,r32)=J.178, p>0.05], TIME [F(11,132)=1s.tu, F0'1,132)=2'426' ¡=L7-12' p<0.01] þæ Textfor po:r-boc results). MeanlSE ,

7-18 CHTA.PTER 7 Efects of chronic nandrolone administration 1 A. D

ct) +3N I +6N IE) E -1 +9N E E +3V E È *6V (t, È +9V .J'

-1

-1 900 3N 6N 9N 6V 9V Time (s) Treatment

1 E 1

E) ¡ El E f E E e È >-=..--=-/ fr ô o

-1

-1 3V 6V 300 600 3N Time (s) Treatment

F

2 È cl E ¡¡ ¡tE! É. ú. I T

,|

1 9V 300 0 900 3N 6N 9N 6V Time (s) Troatment

Fig.7.6 Left: Change in A. Sp, B. DP and C. HR during cardiac ischaemia in rats treated for 3, 6 or 9 weeks with nandrolone or veh¡cle. Legend:3N (.) = I øeek¡ nandro/one tteatment,6N (c) - 6 weeks uehicle hvatment, 6V (Z) 6 weele¡ uehicle nandrolone treatnent, 9N (o) = 9 week¡ nandrolone treaîmeilt, )V (o) = ,1 aeek¡ = post-hoc test. SP: TREATMENT tre(úrleftt, 9V (o) = 9 weeks uehicle treatmenl. Two factor-ÀNOVÀ, Bonferroni's p>0.05], INTERT\CTION p>005] DP:' [F(5,181)=2.¡g6, p>0.05], TIME [F(2,181)=0.647, [F(10,181)=0089' p>0.05], INTERACTION TREATMENT [F(5,181)=2.211, p>0.0ï, TIME [F(2,181)=0.535, [F(10,181)=0'108' p>0.05]. HR: TRE,\TMENT p(5,160)!.626, p<0'05], TIME [F(2,160)=6'115, p>0.05], INTERACTION D. sP, E. DP' F. HR from the lF(10,160)=0.¡73, p>0.051. MeanaS.E., n=9-I2. Right: change in beginning of repeÉusion in rats treated for 316 or 9 weeks w¡th nandrolone. Single-factor-

ANOVA. SP: no ignfrcant tlffirence,DPz no ignfimnt dffirence,IdP(: no si¿nifmnt dffirence'MeatIS'F ',n=9-L2

7-19 CII'\PTER 7 Efects of chronic nandrnlzne administration +vehicle A. +nandrolone D

¡Ð ICD E -1 E E ts -1 fL o. at, tt,

-1 ve nandrolone 300 T¡mê (s) Treatment

B.

o) Er ! I E E È -1 o- oo- ô -1

-1

nandrolone 300 600 900 vehicle Time (s) Treatment

c. F

È oE è ¡l ¡) ú, É, 10 I I

-1

nandrolonê 300 600 Time (s) Treatment

Fig.7.7 Left: change in A. sp, B. DP & C. HR during card¡ac ischaemia in rats treated for 9 (ie' weeks with nandrolone or veh¡cle plus a 10 minute coca¡ne infusion (0.5mg/kglmin) 9N+C & 9N+V). Two-factor-ANOVA, Bonferoni's post-hoc test SP: TREA.TMENT [F(1,63)=!'$92, p<0'01]' p>0.05],. DP: TREATMENT TrME [F(2,63)=0.¡r¡, p>0.05], INTERACTION [F(2,63)=0.183, F('63)=9'296' p>o'ós] HR: TRE'{TME'NT p<0.011, TIME [F(2,63)=0.515, p>0'05],' INTE'RACTION FQ'ez¡=s'"'' p>0 05], INTER \CTION [F(2,39)=0 '4223, p>0',051' Mean+S E lF(l,20¡=3.252, p>0.051, TIME [F(2,39)=0.2709, ', rats n=r0-12. Right: Change in D. SP, E. DP, F. HR from the beginning of repeÉusion in treated for 9 weeks w¡th nandrolone or veh¡cle plus a 10 minute coca¡ne infusion (O.5mg/kg/min). Single-factor-ANovA. SPz no signficant dffirence; DP no signficant dffirenæ; }{R: no signifcanî dffi re nce. Mean* SE, ¡=9 -I2.

7-20 CFIAPTER 7 Efects of chronic nandrolone admini¡tration (1) (2) (1) (1) 900 --'-- max survival tlme in ischaemia 800 700 a 600 max. survival time ¡n reperfusion o 500 Igl.l E 4oo 3V 300 ffiotl 200 lov Egl,l 100 ffigv 0 I R Egtl+c 9V+C

Fig. 7. g Survival time during ischaemia (I) and repetfusion (R) ¡n rats treated with nandrolone (2omglkg, s.c./3xs weekly) or vehicle and in rats treated with cocaine (O.5mg/kg/min). parentheses: rotal number of rats in each tteatment gtouP with death not compþing v¡ith dre

uehicle treatrfleilt, 6N 6 week: nandrolone Lambeth convendon. Legend: 3N=3 week¡ nandro/one îreatmettt, iV = 3 ueek¡ = trealrleilt,6V=6week¡uebic/etreatillent,9N=gweeksnantlrolonetreahileßt,9V=9weel

Mean+SElvI, n=9-13.

7-27 CHA.PTER 7 Efects of chronic nandrolone administration Ftaction of surviving rats Treatment GtouP Ischaemia RePerfusion

3N 1.1/13 11, /1,1

3V e /1,0 e/e 6N 10/12 1,0/1,0

6V 1,0 /11, t0 /1,0 9N 1,1./12 tl/1,1, 9V 1,2/1,2 12/1,2

9N+C 10/11, 1,0 /10 9V+C 1,2/1,2 12/12

Table 7. 5 Number of rats sulviving as a fraction of rats alive at the beginning of ischaemia. tredtïlent, 6V 6 weeks Legend: jN=3 week¡ nandro/one lr€atlilettr, 3V = ) aeeks aehic/e treatmeilî, 6N = 6 weeþ'¡ nandrolone = weeks uehicle tredtment, 9N+C 9 aeek¡ nandrolone trealment * uehicle treatment, 9N = 9 wee/es nantlnlone treatment, 9V = 9 = test, no signficant dffirences. MeantSEM' n=9-13' cocaine, 9V*C = 9 wee,þ¡ uehicle treatnent * cocaine. Chi-squated

I R

3N 3+7 1+0

3V 4+1, 2+1,

6N 3+1, 1+1,

6V 3+1, 1+0

9N 2+1, 1+0

9V 2+0 2+0

9N+C 2+1, 1+0

9V+C 2+0 1+0

Table 7. 6 Lambeth convention arrhythmia score in rats treated with nandrolone (20m9/kg' pr¡or s.c, /3xs weekly) or veh¡cle and ¡n rats treated with coca¡ne (O'5mg/kg/m¡n) to uehìcle treatment, 6N ischaemia (I) and repeÉus¡on (R). Legend: JN=3 weeks nandruloue lrealmeilt, 3V = i wæ,þs

=6aeeksnatdrolonetreaîment,6V=6weeksuebicletrelltmeilî,9N=gweeksnandro/onetredtmenl,gV=9weeksuebicletreltîtent' I cocaine. Mean+SEM, n=9-14' I(ruskal- 9N+C = 9 week¡ nandrolone trealmenî I cocaine, 9V+C = 9 weeks uehicle Íreatment

Wâllis test, no signfrcant dffirences.

7-22 CH-,\PTER 7 Efect of chronic nandrlloffi administration Ftaction tesponding Fraction resPonding Fraction responding with with VT with VF VT or VF

R I R I R

NV N Y NV NV N v N V

11, 2 4/1 5le \0 /14 1,'t/12 4/11 5/9 7 /74 I /12 0 /'tl 3 1'r /14 /1 3 wks (so.o) (s8.3) (0 0) (78.6) (e\ 7) (36.4) (s5.s) 01.4) (e1.7) (36.4) (5s.s) Q3.3) o/10 'ro/12 8/ 12 + l10 +/1.0 10 /12 t /12 4/10 4/10 s/12 1 /12 2/10 6 wks (66.i) (40 o) (40 o) (83.3) (s8.3) (40 0) (40.0) (41 1) (s8.3) (20 0) (0 0) (83.3)

6 7 4/11 7 6/72 t 112 4 /11 7 /12 +/12 +/12 1/1.1 2/12 /12 /12 /12 9 wks (s8.3) (36.4) (58 3) (so.o) (58.3) (36.+) (s8.3) (33.3) (33 3) (e.1) (16 7) (s0 o)

0/10 1 6/12 1 3110 4/12 9wks* 6/'12 e /p 3/10 4/12 3 /12 3/'t2 /12 /12 (30 0) (s0.0) (30 o) (33 3) (2s.0) (0 0) (8.3) (50 0) (s8 3) (33.3) cocaine (s0.0) Q5.0)

Tabfe 7.7 Fraction of rats respond¡ng with W or VF or w+vF during 15 minutes ischaemia (I) and 10 minutes repefusion (R) after 316 or 9 weeks of nandrolone treatment or 9 weeks of steroid plus a 1O minute coca¡ne infusion (0.5m9/kg/min)' Patentheses: Petcent of rats Chi-squared tesponding wirh VT, \¡F or VT+\IF during ischaemia and reperfusion. Legend: N=nandrolone, V=uehicle'

test, no ignficanî dffirences.

7-23 CIIAPTER 7 Efects of chronic nandrolone adrztinislration (t)

A. B.

o o o o E E i: F

gN+C gv+C gN+C gV+C 3N 6N 6v 9N 9V 3N 3V 6N 6V 9N 9V 3V

(R)

c D

Ø ø q, o t F 40 t:

gN+C gV+C gN+C gV+C 3N 3V 6N 6V 9N 9V 3N 3V 6N 6V 9N 9V

VF in Fig. 7.9 Average duration of \rr in ischaemia (4.) and repeÉusion (C.) and duration of ischaemia (8.) and reperfusion (D.) ¡n rats treated with nandrolone (20m9/k9' s'c' 3xs weekly for 316 or 9 weeks), respective control or nandrolone (9 weeks treatment) and uehicle treatment, 6N 6 aeek¡ COCaine (g.Smg/kg, i.V.). Legend: 3N=3 week¡ nandrolone tredîneflt, 3V = 3 uæk¡ = treatment, 9V 9 wee/e¡ uehic/e treatwle"t, 9N+C = nandrolone tredtment, 6V = 6 uæk¡ uehicle heatment, 9N = 9 weeks n¿ndrolone = tocaine. Kruskal-lØallis lest, n0 rigilifcant diferenæt' 9 ueek¡ nandrolone treatrlefit * cocaine, 9V+C = 9 weeks uehicle tratmenî I

MeanfS.E., n=9-13.

7-24 CI{APTER 7 Efects of chronic nandrolone admini:tration 15 T3N 3V 125 ffiotl l6v G 100 -eN o ffilgv E75 IgNl+c F 9V+C 50

2

0 R

Fig.7.10 Average duration of W+VF during ischaemia (I) or repeÉusion (R) in rats pre- treated with nandrolone (2omg lkg, s.c. /3xs weekly) or vehicle and in rats treated with peekl 6 ueek's COCaine (O.smg/kg/min). Legend:3N=3 pee,ks nandrolone ireatnefll,3V = 3 uehicle tt'e(ttneftt,6N = trcatmeilt, 9 week¡ uehìc/e Íretttîleilt, 9N+C = nanrlro/one treatïtent, 6V = 6 weeþ.s uehicle tredtmettt, 9N = 9 week¡ nandrolone 9V =

gV+C cocaine. ¡=9-1'4 Kruskal-WalÌis test, 9 aeek¡ nandrolone tredtmefit * cocaine, = 9 aeeks aehitle Írealmenl I Mean*SEM, no signficant dffirences.

7-25 CH,q.PTER 7 Efects of chronic nandrolone admini¡tration 7.6 Discussion

7.6.1 Body weight Nandrolone significantly retarded the increase in body weight observed in vehicle treated controls

(Fig. Z. 2) when treatment was continued for 6 or 9 weeks. Johansson observed the same effect on body weight in several studies in which a daily nandrolone dose (15mg/kg, s.c.) was found to induce a significant decrease in body weight compared to control aftet 14 days of treatment (Hallberg et a/.,21}};Johansson et a1.,2000a, Johansson et a/.,2000b). Doses of 2mg/datly and 2}mg/bi-weekly have both been found to signifìcantly reduce body weight compared to conttol

(T-o.g et a/., \996;Long et a1.,2000)'

factors It appears that the anabolic effect of nandrolone on body weight in rats is telated to 3 main gender, the dose and the extent of exercise.

1,. Gender: Yu-Yahiro et ø/ (1.989) administered nandrolone decanoate (-100mg/kg) ot 0.5cc of physiological saline to 6-week-old male or female Wistar rats, once weekly for six treatments (Yu-Yahiro et at.,1989). \X4rjle nandtolone treated female rats gained weight at a similar râte to their saline treated cage-mates, male conttol rats gained signifìcantly mote weight rhan treated rats (control, 234.4+5.4s versus treated, l+4.5+4.0g).Bisschop et a/,

1997 delivered a low (1.Smg/kg, i.m., weekly) and a high (7.5mg/kg, i.m., weekly) dose of nandrolone to male and female rats over 5 weeks (Bisschop et a/.,1997). While both doses of nandrolone were found to increase body weight ftom week 1 of treatment in female tats, a marked decrease in weight was noted for male rats receiving the highest dose in comparison to saline controls after 5 weeks of treatment. A number of studies have

demonsttate d a tetatded somatic body ovel sevefal weeks of nandtolone treatment compared to control. Trifunovic et al (1995) found a significant suppression of growth rate in male rats treated with nandtolone (5mg/kg, i.m., biweekly) for 8 weeks compared to arachis oil treated controls (Ttifunovic et a/., 1995). Interestingly, this significantly lower weight gain than control could not be attributed to diffetences in food intake, gastrointestinal energy absorption or an altered ratio of fat vs. protein metabolism. also found that nandrolone (2Omg/kg, s.c.) decteased terminal Tseng ø at (1994) ^verz;ge body weight in comparison to vehicle treated conttols (Tseng et a1.,1'994).

Conversely, a small number of publications have repotted no significant change in body werght in male nandrolone treated tats. Liang et øt (1'992) found no change in the body weight of rats adrninistered nandrolone at 3.}rng/kg, i.m., -7-9 days following a loading

7-26 CH,\PTER 7 Efect: of chronic nandrolone administration dose of 3.gmg/kg on the Frst day of treatment (I-iang et a1.,1992). This is probably telated to the use of juvenile rats (4 weeks old) and a lower dose of nandrolone than used in other

studies.

2. Dose: A study by I{ochakian and colleagues (1959) found that body weight gain was stimulated in male rats by low doses of testosterone and inhibited by large doses

(I(ochakian et a/.,1,959).,{. number of studies using low AS concentrarions in non-exetcised rats have not observed the same slowing of weight gain that has been observed with larget nandrolone doses. Lewanowitsch and Iwine (2000) failed to obsewe an appteciable difference in the body weight of male rats administered a daily injection of testosterone (1mg/kg) for 2 weeks (Lewanowitsch et at., 2001). Likewise, Bisschop and co-workers found no change in the body weight of male ïâts treated weekly for 5 weeks with low dose nandrolone (1.5mg/kS, i.m.), but found a significant decrease in weight, compared to control with high dose treatment (7.5mg/kg, i.çl (Bisschop et a1.,1997). More recently, that daily doses of nandtolone decanoate (límg/kg, Johansson and colleagues (2000) found s.c.) resulted in nandrolone rats being signifìcantly smallet than vehicle treated controìs (p<0.0001) after 2 weeks of treatment flohansson et a/.,2000a). Further studies confìrmed

this observation flohans son et al',2000b)'

in male 3. Exetcise: \X/oodiwiss et at Q000), found that body weight sigruficantly decteased Sprague-Dawley rats administered a biweekly injection of nandrolone decanoate (5mg/kg, i.m.) compated to si¡nilar sedentary control treated tats fWoodiwiss el a/', 2000). Rats exetcised according to a voluntary running schedule, but similady treated with nandrolone were found to increase in body weight but this inctease was not signihcantly different from drug-ftee exetcised conttols.

The present investigation suPpofts those previous studies which have found a decrease in body weightinmaleïatstreatedwithhighconcentrationsofAs(I-otg eta/.'1'996;Bisschop eta/.,1997;

et a/.,2000). Trifunovic et al (7995) ptoposed a Johansson et a1.,21¡1a;Johansson et a/.,2000b;Long number of explanations for the observation of lowet body weight obsewed for male rats with high dose, chronic nandrolone treatment compared to vehicle controls seen in a number of studies (Irifunovic et a/., 1.995). These hypotheses apply equally to our own observations.,{s Trifunovic and co-workers found that plasma testosterone concentratiorìs were markedly decteased as a resuit of exogenous steroid adrninistration it was postulated that suppression of endogenous sex steroids or other growth factors may explain some of the obsewed decreases in body weight. Changes in

7-27 CH,\PTER 7 Efects oJ chronic nandrolone administrøtion ot altered teceptof fesponse to exogenous' comPafed with andfogen feceptor expfessloll ^n endogenous androgens, could also possibly explain the observation of decreased somatic growth'

7.6.2 Changes in organ weight Although many studies chronically administering AS to rats have observed changes in organ weight it is difficult to determine whether these changes âre simply related to teduced growth with high

dose AS.

L Changes to heart weight: In the present study it was found thât treâtment for 6 or more weeks sigrufìcantly increased the relative wet weight of the heart compared to vehicle death treated rats. This is consistent with the catdiac hypertrophy noted in sudden cardiac attributed to AS abuse (I-uke, 1990; Cambell ú a/.,1993; I(ennedy,1'993;Dicketman et a/', 1995; Hausmann et at., 1,998). Convetsely, Trifunovic et al (1'995) noted a significant dry decrease in absolute, total wet heart weight, and absolute left ventriculat, wet and weight in nandtolone treated tats (5mg/kg, i.m./bi-weekly/8wks) compared to controls (frifunovic et a/., 1.995).It was acknowledged that the obsewed decrease in the absolute weight of the wet and dry left venüicle may be attributable to the signifìcant decrease in body weight induced by high doses of nandrolone. Following an identical treatment regimen to the above study, Nort on et al (2000) found a significant dectease in absolute dght ventricular mass, but not absolute left ventricular mass Q'Jotton et a/., 2000) aftet 3 months of treatment. Absolute left ventricular mass was unchanged by treatment but was increased sìgnificantly (p<0.01) in comparison to control when adiusted fot body weight. A significant increase in relative ventricular weight (ventdculat/100g body weight) and vertical ventricular diameter was found in spontaneously hypertensive rats treated for 6 weeks with NItnl

et ø/.,1988).

It ,\ more detailed study is requited to determine the hlpertrophic potential of nandrolone' in relative is unclear from the results presented in this thesis whethet the observed increase weight heart weight was simply due to an overall decrease in body weight' -Àbsolute heart

7-28 CHI{.PTER 7 Efects of chronic nandrolone administration was not significantly different between pfe-tfeatment gfouPs. Small atirnal echocardiogr aphy m^y provide â means of detetmining changes in wall thickness and

c rdi^c mass as a result of nandtolone treâtment'

2. Liver: Although a signihcant decrease was obsewed in the present study in the relative weight of the liver with nandrolone at weeks 3 of tteatment - no such difference occurted at 9 weeks. Chronic stanozolol treatrnent has been shown to signifìcandy reduce relative (Bauman et al., 1,988), and absolute (Molano et at., 1999) ìiver weight in exercised rats. Yu-Yahiro and -Although we have reported minimal effect of nandrolone on livet weight, be colleagues (1989) found that decteases in [ver weight due to nandrolone treatmerìt could att¡ibuted to lowet livet lipid content (Yu-Yahiro et a/',1'989)'

3. Kidneys: This thesis reports signifìcant increases in both absolute and telative kidney weights at 3, 6 a¡d 9 weeks of nandrolone treatment in comparison to the respecLive vehicle controls. Increases in kidney weight have been consistently associated with chronic, high dose nand¡olone administration to râts. Tseng et al reporTed chronic nandrolone treâtment, induced increases in absolute and telative kidney weight in sedentary tats (Tseng size in et a/., 1,994). Sirnilady, Yu-Yahiro et al rcport significant incteases in absolute kidney rats treâted for 6 weeks with nandrolone decanoate (Yu-Yahiro et a/., 1989). \ßnktn et al repotted a significant and dose dependant increase in the kidney weight of nandtolone decanoate treated rats (À4inkin et a/., L993), although no mechanism to explain this effect was provide d. Blantz et a/ teated ovariectomised female rats with nandrolone decanoate and found a significant increase in absolute kidney weight after 6 and 76 weeks weeks of treatment compared to vehicle treated controls @lantz et a/.,1,988). This increase in kidney in size at 16 weeks was found to be due to tubular hypertrophy and modest increases glomerular size. Interestingly, these authors also found that although kidney size was signifìcantly increased with nandrolone treatment no kidney abnotmalities v/ere observed with light microscopy. The present study is consistent with this finding' Shukla et al found that testosterone injection into both male and lemale mice produced elevated levels of

glucosylceramide synthase, decreased levels of the hydrolase and increased kidney growth (Shukla et a/., 7992). Injection of 17 beta-oestradiol (which has been found to produce level decreases in mouse kidney size) had the opposite effect on conttolling the of

tissue glucosylcetamide. These authors conclude that testostetone may cause increases in

kidney size thtough the production of kidney glycosphingolipids. It is also possible that the increase in heart and kidney weights observed in nandrolone treated t^ts are due to an increase in protein turnover, such that decreases in body weight teflect a shift of carcass

7-29 CIIAPTER 7 Efects of chronic nandrolone administration protein to the synthesis of new tissue at organ sites (fseng et a/.,7994).It is likely that the increase in kidney size with nandrolone treatment reported in this thesis cannot be attributed to a single mechanism.

4. Other ofgans The effect of nandtolone on testiculat weight in the present study was difficult to evaluate. V4rile absolute weight was signihcandy incteased at week 3 and

decreased at week 6 of nandrolone treatment, relative weights were signifìcantly ìncreased in comparison to control at week 9. Nandtolone \r¡as found to cause a consistent increase in the relative weight of adrenals which was signifìcant 6 and 9 weeks of treatment in comparison to vehicle control. The toxicological signifìcance of this increase in adtenal weight is also difficult to determine. Schlussman et al (2002) found admlnisttation of nandrolone decanoate (15mg/kg X 3 each day) to tats ptoduced signifìcant elevations in circulating levels of adrenal corticotropin hormone (ACTH) and corticosterone after the third day of treatment (Schlussman et a/., 2002) resulting from significantly increased pOMC synthesis in the . It is possible that the increase in adrenal size in

the present study reflects increased stimulation of the adtenal cortex by ACTH.

Both Tseng et al and Minkin et al obsewed that chronic nandrolone treatment had no effect on resricular weight phnkin et a/., 1.993; Tseng et al., 1.994). \X4rile Yu-Yahiro report a

decrease in testicular weight in treated rats they did not report organ weights relative to body weight (Yu-Yahiro et a/.,1989). Consistent with our own tesults Tseng et al found a significant increase in relative adrenal weight with nandrolone administration, but not in absolute weight.

7.6.3 Changes in cholesterol TC, TRIG, HDL and LDL all displayed a tendency to be lower in nandrolone treated rats at 3 and 6 weeks of treatment compared to the vehicle conttol for 3 and 6 weeks of administration' However, a statistically significant decrease in nandtolone treâted râts wâs only observed for TC and HDL at 3 weeks in compadson to control. No significant cholestetol, uiglyceride ot lipoprotein changes were observ eð at 6 or 9 weeks in comparison to control.

Ovetall, it appears that alatge dose of nandrolone administered over a significant period of time failed to induce a signifìcant change in serum lipoprotein concentrations in comparison to control values. There is evidence to suggest that the lack of a profound change in the lipoprotein profile of these rats oveÍ the tfeatment pedod is related to two main factors. . .

7-30 CI{'\PTER 7 Efects of chronic nandrolone adtrtinistration i,. . Oral use of AS ptoduces the classical decline in Hl)L and elevation in LDL, increasing the tisk fot catdiovascular disease more than any other known agent (Ftisch et ø/., lggg). Use of iniectable AS does not appeaf to produce such a detrimental outcome. IJnfortunately because no AS is available fot both oral and parenteral human use it has not been possible to test this hypothesis using the same ,{'S. However, a number of studies have been performed examining the effect of otal and patenteral prepatations of diffetent AS.

Thompson et al (7989) performed a crossover design study of the effect of onl stanozolol

and intramuscular testosterone in 11 weight lifters (Thompson et a/',1'989). They tepott that while stanozolol significantly reduced IHDL (-33o/o), increased LDL (+29'/ù and increased hepatic ttiglyceride lipase (HTGL) activity by 1.23o/o, intramusculat testosterone produced

only a modest 9olo decrease in HDL, a 160/o decline in LDL and a 25o/o increase in HTGL activity. This effect carìnot be attributed to a greater androgenic potential of stanozolol as

testostefone was found to moïe effectively suppress trSH and LH secretion.

2. Aromatisation of nandrolone to esttadiol. The available data suggests that those AS which are not readily converted to oestrogenic metabolites mây signifìcantly deptess HDL by increasing the activity of HTGL, compared to compounds such as nandtolone which are teadily metabolised to (Hannan et a/.,1991; Obasanjo et a1.,1'996). Obasanjo and

associates treated Cyanomolgus macaques with nandrolone ovet 2 years and found a su¡prising elevation in plasma estradiol - thought to be due to conversion of androgen to 17p-estradiol.

17cr- Friedl et ø/ (1,990) compared the ability of testosterone enanthate (oestrogenic activiry), methyltestosterone (little or no oesüogenic activity) and testolactone (atomatase inhibitor) to induce changes in plasma HDL in non-athletic, male subjects (Friedl et al., 1990). Testosterone enanthate (280mg/wk, i.m.) produced significant increases in plasma estradiol

concentrations, which reached a maximum at 8 weeks of treatment. No signifrcant change in HDL was observed over 12 weeks of testostetone enanthate treatment compared to the other testosterone preparations. Convetsely, methyltestosterone and testolactone produced no increase in estradiol over time, but both displayed signihcandy depressed HDL compared to testosterone enanthate, presumably due to an observed significant increase in HTGL activity over time. Methyltestosterone also signifìcantly elevated plasma LDL. These results suggest that a decrease in plasma HDL is related to the inability of the administeted

7-3t CHAPTER 7 Efects of chronìc nandrolone admini¡tration AS to be aromatised to estradiol. AS with 17c-substitutions ate likely to dectease HI)L thtough incteases in HTGL actlvlty.

Bauman et al (1.988) reported changes in triglycerides, cholestetol, LDL and HDL in pooled

plasma from male rats treated for 5 weeks with stanozolol (5mg, equivalent to -15mg/kg twice weekly) (Bauman et al., 1,988). In steroid treated, exercised rats, receiving a 16.60/o w/w protein ,levels of TRIG, TC, HDL and the TC/HDL tatio were all found to decrease in comparison to sedentary, untreated, non-dietaty supplemented tats. These decreases were conserved in sirnilarly treated rats, which teceived no dietary supplement. Our own laboratory pewanowitsch et a1.,2001) found that 2 weeks treatment with testosterone (lmg/kg, s.c) produced a sigruficant decrease in HDL in female rats.

7. 6. 4 Serological Changes Compatisons of sodium, potassium and chloride ion levels to litetature values for veterinary pathology reported by Bauman et al rcvealedvalues which wete within the normal nnge @auman et a/.,1988).

The significant decrease in total plasma protein demonsttated at 3 and 9 weeks of treatment in comparison to control treated rats is consistent with the observations of Hall & Hungerford (1982)' Mononephrectomised, female, Sprague-Dawley râts were administered nandrolone (l,}rng/kg/ òay) which was found to signihcantly decrease plasma protein but leave plasma sodium unchanged after

5 weeks of treatment.

7.6.5 Plasma nattdrolotte let'el To the best of the author's knowledge this is the ftst study to measure plasma concentrations of nandrolone following chronic subcutaneous treâtment in the rat. These results indicate that the nandrolone plasma concentration increased to a maximum of 1'72-123 nM with a dose oF 2)rng/kg, s.c. /3 days weekl at week 9, and that levels of endogenous L9-nortestosterone in vehicle treated ïats were below the limits of detection. Nand-rolone plasma levels at 9 weeks of treatment were significantly increased compared to 3 weeks, suggestìng that 'cycle length' in recteational AS usets is a key determinant of plasma AS concentration. The plasma nandtolone concentÍation following acute intravenous administration of 1,60p.g/kgf mtn nandtolone (chapter 5) was more than 5 times gïeater than the highest concentration achieved with chronic administration. As previously discussed there is a dearth of information regarding plasma levels of AS following chronic treatment, and consequently it is difficult to cortelate c tdi^c consequences to plasma concentrations of AS. Abu-shakra et al only demonsttated apoptic damage of mudne-C2-skeletal-

7-32 CFIAPTER 7 Efficts of chronic nandrolone administration muscle cells at concentfations of >10pM (r\bu-Shakn et ø1., 1'991). This may explain the lack of <0.2¡rM cardiac pathology in the present study where plasma nandtolone concentrations were after chronic treatment.

7.6.6 Efiect of lreatment on BP and IfR No signihcant difference was noted in SP, DP or HR in nandrolone treated râts at the end 3' 6 ot 9 weeks treatment in comparison to controls in close-chested rats which were pentobarbital-

anaesthetised in preparation for ischaernia. Although BP in this study was tested in anaesthetised animals these results are still consistent with eadier pilot studies petformed in freely moving,

conscious rats chronically instrumented with radiotelemetry devices. In this pilot study, each of 5 rats acted as their own control during administration of nandrolone for 9 weeks using the same found treatment protocol as presented in the cuttent study. No effect of nandrolone decanoate was dudng ot between the start and fìnish of nandrolone treatment (Appendix C). A lack of effect on on pressure was also noted by Trifunovic and colleagues (1995) using tail-cuff plethysmogtaphy fars chfonically treated for 3 months with a low dose of nandrolone (Smg/kg, i'm./bi-weekly) (Trifunovic et a/., 7995). Likewise, chronic tteatment of rabbits for 4, I or 1'2 weeks with nandrolone decanoate (10mg/kg/week, i.m.) failed to increase BP measured directly in the ear found arïery in comparison to control (Ferret et a/., 1.994). Flowever, Tseng and associates (1994) rats' that nandrolone accelerated the development of hypetension in spontâneously hypertensive

These results are also consistent with those of Hall and Hungetford (1982) who found that nandrolone administered according to a daily chronic schedule in young male tats drinking 1%o saline did not induce hypertension. Rats sirnilatly tteated with testostetone were found to be hypertensive after 5 weeks of administration. As a tesult it is hypothesised that demethylation of

testosterone at C10 (such as in nandrolone) completely desttoys any effect on BP. The effect of testosterone in the absence of saline drinking water and in adult male rats has not been investigated.

in Consistent with the lack of an effect of nandrolone on BP, no significant diffetence was found 6)' the Sp and DP response to ischaemia and repetfusion in the various treatment groups (Fig. 7' The high variability associated with 900s of ischaemia may explain an appatent treatment effect for HR at this point (Bonferroni's test, negative)'

7.6.7 CardiovasculaÍ effect of cocaine infusion in The pressor effect of cocaine was found to be much more ptonounced in this chtonic study than related to chapter 6 and the extent of bradycardia far less severe. This difference is most ptobably

7-33 CII,\PTER 7 Efects of chronic nandrolone admini¡tration vafiation in methodology. In chapter 6, BP and HR were measured in open-chested rats' In an in attempt to obtain a more acc;r)ï^Te measute of these parameters the chest wâs not opened chronically treated râts until after the 10 rninute cocaine infusion was concluded' The difference in the Bp and HR between the chronic and acute pre-treatments is most probably related to whether these parameters were measured in open or close-chested rats' Interestingly, nandtolone pre- treatment was not found to significantly affect the HR response to cocaine when compared to vehicle treated control (Fig. ?. 5). This contrasts to the signifìcantly incteased HR in conscious, freeþ moving rats administered nandrolone (2}ng/kg, s.c.) for 3 weeks and challenged with i'p' related to the use of anaesthetised rats in this cocaine 1a5mg/kg) phillis et al., 2000). This may be present study and the profile of the cocaine response. ì(/hereas i.p. cocaine causes long lasting BP cardiovascular responses the effect of i.v. cocaine is only of short dutation (i.e. in this study returned to baseline between 3 and 6 minutes of infusion). Future studies should invesligate the effect of i.v. cocaine in conscious animals.

The biological relevance of the combination effect of nandrolone and cocaine on SP and DP dudng ischaemia is difficult to interpret (Fig. 7. ?). Atthough a 'treatment' effect was noted for both Sp and DP dudng ischaemia, the post-hoc tests were all non-signifìcant. Nandtolone pre- treatment appeared to reduce the magnitude of the decreases in SP and DP observed dudng ischaemia, but not reperfusion. Because chronic nandrolone treatment alone did not cause a significant increase in arhythmia dutation or frequency it does not appear that the 'protective' effect of the nandrolone and cocaine combination on BP decteases confers any protection against

arrhythmia.

2.6.8 Afiect of pre-treatment on the frequency nd duratiott of arrh¡dtmia and on sutt'iva| Although tteâtment with nandrolone had profound effects on body and organ weights - no difference was found in the frequency and duration of arhythmia. In addition there were no significant effects of nandrolone pre-treatment on the cardiovascular parameters measuted consistent with previous results obtained with 3 weeks pre-tïeâtment (Phillis et al., 2000)' Furthermore, rats treated with nandrolone * cocaine showed no significant difference from conttol duting ischaemia and reperfusion in terms of the survival measures used and the extent of to an arrhr¡hmia. These results suggest that nandrolone administered chronically at a dose relevant or abusive regimen does not signifìcantly alter the degree of arthythmia produced by ischaemia reperfusion but that acute intravenous administration at a dose of 40pg/kg/min and above does'

We can speculate on 2 possible explanations for the obsewed effects.. '

7-34 CII,A.PTER 7 Efects of chronic nandrolone administration 1.. Down regulation of the mechanism tesponsible fot atrhythmia formation. It is possible that chronic nandrolone treatment caused downregulation of an intetmediate factor or receptor involved in arrh¡hmia fotmation. Although there is no example in the literature of tolerance to nandrolone or other AS, it would be expected that changes in receptor expression would be induced by nandrolone as evidenced by almost all agonists and antagonists in many different organ systems.

2. plasma nandtolone concentrations with chtonic treatment were too low. Significant potentiation of ischaemia-induced arrhythmia was noted in acutely treated rats with intravenous admrnistraúon of nandrolone Ø\¡tg/kg/ntln) which produced a plasma concentration of 281nM, a lower dose had no effect. Nand¡olone treatment for 9 weeks in the present study produced a plasma concentration of I72nM. It is possible that chronic nandrolone treatment did not reach the thteshold concentration required for arrhythmia fotmation.

7-35 CIIÂPTER 7 Efects of chronic nandrolone administration 7.7 Conclusion Treatment with nandrolone for 3 or 9 weeks produced a significant elevation in the plasma concentration of 19, nor-testosterone. A significant catabolic effect of nandrolone was observed at 6 a¡d 9 weeks of treatment but no BP or arrh¡hrnia effect was observed in the absence of ischaemia. Furthermore, no evidence of signi{ìcant effects of nandrolone were noted for the number of rats surviving, the survival time, the Lambeth arrhythmia score, or the duration of VT, score VF and VT+VF in either ischaemia or repetfusion. The dose related increase in VF, Lambeth could not be and decrease in survival with intravenous nandrolone treatment desctibed in chapter 5 replicated with chronic subcutaneous administïation. This is possibly due to the lower plasma steroid level achieved with chronic treatment or a ptogressive down-tegulation of a receptot ot intermediate factor involved in arrhythmia formation with âcute tfeatfüent'

7-36 CFL\PTER 7 Efect of chronic nandrrllne admirti'çtration 7.8 References

Abu-Shakra, S., Alhalabi, M. S., Nachtman, F. C., Schemidt, R. A. and Brusilow, W' S' A' (1997)' ,,Anabolic 186- Steroids Ind.ace Injary andApoptosis of Dffirentiated SkeletalMuscle."J Neutosci Res 47: 797.

Bauman, D.H., Richetson,J. T. and Britt,,{.. L. (1988)' "A Comþarison of Boþ and Organ ll/eights, ters, and Pithologìc Changes in Target Organs of Rats Giuen Combinations of Exercise, and'Protein Sapþlenentation." An'J Sports Med 16(4): 397-402'

Bisschop, ,\., Gayan-Ramirez, G., Rollier, H., Dekhuiizen, P. N., Dom, R', de Bock, v' and D..r^m^.r, M. (1997). "Efects of Nandrolone Decanoate onRespiratory andPeripheralMascles in Male and Female l

Boada,L. D., Zarnbado, M., Torres, S., Lopez, r\., Diaz-chico, B. N., Cabrera, J. J. and Luzatdo, ,,Eualaation bl Anabolic-Androgenic Steroid O. n. if OlO). of Acate and Chronic Hepatotoxic Efects Exerted Stanoqolol in Afult Mak Raß." Arch Toxicol 73: 465-472'

Cambell, S. E., Farb, A. and Weber, K. T. (1993). "Pathologic Renodelling of the Aþocardiam in a

e Pres sure 2: 21. 3 -27 6. IT/ e igh t tif e r T a ki ng A n ab o li c s t ro i d¡. " Blo od

in a Dickerman, R. D., Schaller, F., Ptather, I. and McConathy, W. J' (1995)' "Sudùn Cørdiac Death 2T-Year-o/d. Bo@bwilfur [JsingAnabolic steroids." cardiology 86(2): 1'72-3.

Ferrer, F., Encabo,4., Marin,J. and P,alfagon, G. (1994). "ChronicTreatment with the Anabolic Steroid, 233-241.. Nandrolone, Inhibits Vasodilator Responses in Rabbit Aorta." F,ur J Phatmacol 252:

Fdedewald, W.T., Levy, R. L and Freddckson, D. S. (1972). "Estimation of the Concentration of l-nw- 18: 499- Densigt Llpiprotrl, Chole¡nrot in Plasma, unthoat [Jse of the Preþaratiue ultraæntrifage. " C]in Chem 502.

upoprotein Friedl, K. E., Flannan, c. J., Jones, R. E. and Plymate, S. R. (1990)- "Hzgh-Densiryt 39(1): 69-7 4' Choletterol Is Not Decîeased If àn Aronøtirybk Androgen I: Administercd" Metabolism

Frisch, F. and Sumida, K. D. (1999). "Teruporal Ef,ectt of Testosterone Proþionate Injections on Seram Lipopronin Concentrations in Rats." Med Sci Sports E'xer 31(5): 664-9'

Rtuiew G;azer, G. (1991). "Atherogenic Efects of Anabolic Steroids on Seram Lipid Leuels. A Utemtøre pabtished. Enatam Appears ln Arch-intern Med 1992 Mar;l52(3):5351." Ardn Intern Med 15L(10): L925- 33.

A. (1'993)' Gragera,R., Saborido, r\., Molano, F., Jimenez, L., Muniz, E. and Megias, ,"ultrastractaral Histol Histopathol S: 449-455' Choiges Indaced þt Anabolic Steroids in Liuer of TrainedRats." ønd19- Hall, C. E. andHungerford, S. (1982). "similaritie¡ andDffirences betweenEfects of Testosterone 16(4): 581- Nortesto$erone ¡n Ratî øth Particitar R'eference to Íþþertensogenic Potenry,"J Steroid Biochem 5.

7-37 CHÂPTER 7 Efects of chronic nandrolone administraÍion Hallberg, M.,Johansson, P., I(indlundh, .,\. M. S. and Nybetg, F. (2000). "Anabolic-Androgenic Steroìds Afex øi Content of Substanæ P and Søbstance Pl -7 in the Rat Brain." 2l: 845-852.

Plymate, S' R. (1991) "Prychological and Hannan, C. J., trriedl, K. E., Zolå, A.,I{ettler, T. M. and ' Ssrxtm Homouanillic Acid Changes in Men Adninisnred Androgenic Stsroids." Psychoneutoendoctinology 16(4):335-43.

Flausmann, R., F1ammer, S. and Betz,P. (1998). "Performance EnhancingDrugt (DopingAgenß) and Sadden Deøth-a Case RepoX andRettiew of the Lìteratøre." IntJ Legal Med 111(5): 261-4'

Med Sci Huie, M. J. @9+). "An Acute Mlocardial Infarction Onaring in an Anabolic Steroid U¡¿r" Spotts Exetc 26(4): 408-13.

Ishak, K. G. andZimmetman, FI. J. (1987). "Hepatotoxic Efects of the Anabo/ic/Androgenic Steroids." Sem Livet Dis 7(3): 230-236.

p., Nyberg, F. (2000a). "The Efect 0f Oþilid Peþrid¿r in the Johansson, Halberg, M., Ilndlundh, A. and "Rnt Res Brain, qftetr ChronilTreatment with fhe Anabolic Androgenic Steroid, Nandrolone Decanoate." Brain Bull51(5): 41.3-41.8.

P., Lindqvist, ,{., Nyberg, F. and Fahlke, C. (2000b). "Anabolic Androgenic Steroids Afects "ZlrototJohansson, Intake, Defence Behauiors and Brøin OPiuìd : in the Røt." Phatm Biochem Behav 67: 271' 279.

P., Ray, r\., Zhou, Q., Huang, W., I(adsson, l(. and Nyberg, F. (1997). "Anabolic 'AndrogenicJohansson, Steroid¡ Increase Beta-Endnrphìn l-¿uels in the Ventral Tegmental Area in the Male R¿t Brain." Neurosci Res 27(2): 185-9'

I{enned¡ C. (1993). "Mlocardial Infarction in Association with Misase of Anabolic Steroids'" Ulstet Med J 62(2):174-6.

I(ochakian, C. D. and Endahl, B. R. (1959). "Changes inBoþlØeþht of Norrnal and CastratedRøts b1 Dffirent Doses of ." PSF'RM 100: 520-2'

'Wilkerson, (1986)' "Efects of Exercise Leeds, E. M., J. E., Brown, G. D', I(amen, G. and Bredle' D. Sci and Anabolic Steroidr on Total and Lipoþrotein Cholesterol Concentrations in Male and Fernale R¿Lç. " Med Sports Exer 18(6): 663-667.

Levine, B. S. (1995) . Animal Clinical Pathology. Crc Handbook of Toxicology. M. J. Derelanko and Hollinger, M. -A'. Boca Raton, Florida, CRC Press: 51'7-537.

Lewanowitsch, T. and lrvine, R. (2001). "Efecß of Testosterone Proþionate andNandrolone Decanoate on Bo@t Conþosition and Liþoþrotein Concentrations in the Rat." Addtcnon Riol 6: 55-6L'

Lewi, p. J. andMarsboom, R. P. (1981) . ToxinlogtReference Data - Il/istar RzL Amstetdam, Elsevier North Holland Medical Ptess.

Liang,M. T., Glonek, T., Meneses, P., Kopp, S. J', Paulsotl D' J', Gierke, L' W-' and Schwattz' F' N. (ó92). "Nmr Spectroscopl Staþ of Heart Phosþhotþids. An Exercise and Anabolic Steroids Efect." IntJ Spotts Med 13(5): 41'7-23.

Long, S. F., SØilson, M. C. and Davis, \ø. M. (2000). "The Efects of Nandrolone Decanoate on Cocaine- Indaced Kindling in Male Rølr. " Neurophatmacology 39: 2442 2447 '

7-38 CFIAPTER 7 Efects of chronic nandrolone administration Long, S. F., $i/ilson, M. C., Sufka, I( J. and Davis, \Ø. M. (1996). "The Efects of Cocaine and Nand.rolone Co-Administration on Agression in Male Rats." Prog Neuropsychopharmacol Biol Psychiatry 20(5): 839-56'

in a lf,/eþt Ufter Using Anabolic Androgenic Luke, J. L. (1990). "Sud.d¿n Card.iac Death Daring Exercise Steroids: Pathologicat and Toxicological Findings."J Fotensic Sci 35(6): 1441'-1447 '

Matsuzawa, T., Nomura, M. and lJnno, T. (1993). "Clinical Pathology Referenæ Ranges of l-øboratory Animals."J Vet Med Sci 55 351-362.

Minkin, D. M., Meyer, M. E. and van Flaaten, F, (1993). "Behatioral Efectt of l-ong-Tenn Ad.ruinistration of an A-ruabolic Snroid in Intact and Castrated Mak ll/i¡tar Rats." Phartr' Biochem Behav 44:959-963.

and Molano, F., Saborido, ,\., Delgado, J., Motan, M. and Megias, A. (1999)' "Røt Uuer þsosornaÌ 243- Mitochonrlrial Actiuities Are Mod;fed bl Anabotic-Androgenìc Steroid¡." Med Sci Sports Exet 31(2): 250.

Morales, A. I., Barat,J.D., Bruges, M., Arevalo, M.4., Gonzalez de Buittago,J.M., Pakna,P., Branco, p. and percz-barrtor nÃ,F. (2003). "Acute Renal Toxic Efect of Aniodarone in Rats." Pharm Toxicol 92:39-42.

Norton, G. R., Trifunovic, B. and Woodiwiss, A. J. (2000) . "Attenaated Beta-Adrenoceþtor Mediøed Cardiac Contractile Responses FollowingAndrogenic Steroid Adninistration to Sedentary Rnts'" 8'rr J Appl Physiol 81(4): 310-6.

'W'eaver, Obasanjo, L O., Clarkson, T. B. and D. S. (1996). "Efects of the Anabolic Steroid Nandrolone 45(4): Decønoat€ on Plasma Lipids and Coronary Axeries of Femate C1noruoþas Macaqøel" Metabolism +63-8.

and the phillis, B. D., Irvine, R. J. and I(ennedy, J. A. (2000). "conbined cardiac Efects of cocaine Anøbolic steroid, Nandrolone, in the Røt." EurJ Pharmacol 398(2): 263-72.

Schlussman, S. D., Nyberg, F. and I(reek, M' J' (2002) ' "The the Stress ms." Acta Responsiue Hlpothakmic-pinltary-Ad.renat Axìs and the Doparninerglc a Psychiatr Scand 106 (Suppl. 41'2) : 1'21' -1'24.

Shukla, A., Shukla, G. S. and Radin, N. S. (1992) . "Control of Kidnel Siry @ Sex Horwones: Possible Inuolwment of Glucos1heranifu." AmJ Physiol 262(1'Pt' 1):F24-9'

Thompson, P. D., Cullinane, E. M., Sady, S. P., Chenevert, C., Saritelli, A' L', Sady, M' Â' and Herbeit, p. N. (1989). "ContrastingEfects of Testosterone and Stanoqolol on Serarn Lipopronin l-'eue/s'" J Am Med Assoc 261 7765-1'168'

Trifunovic, 8., Norton, G. R., Duffìeld, M' J', Avraarn, P. and Woodiwiss, 'A'' J' (1995)' "Ao 2): H1096-105' Androgenic Steroid. Decrease: l-.eft Venticalar Compliance in Røts." A* J Physiol 268(3 Pt

Tseng, Y. T., Rockhold, R. !ø., Floskins, B. and FIo, I. K. (1994). "Cardioaascalar Toxicities of 22(1):1'1'3-21'. Nandrolone and. Cocaine in Sþontaneoasþ H1þerleniue Rats." Fwdam Appl Toxicol

'Wolfotd, R' S. T., Schroer, R' r\., Gohs, F. X., Gallo, P'P', Brodeck, M', Falk, H' B' and Ruhren' (1986). ;,Reference Rønge'Data Bar, þ, Serum Chenistry and Henatology Values in l--aboratory AnimalL" J Toxicol Environ Health 18(2): 161-88.

7-39 CII\PTER 7 Efects ( chronìc nandrolone administration G. R. (2000). "Efects of an Androgenic Woodiwiss, A. J,, Trifunovic, 8., Philippides, M. and Notton, Steroid on Exerise-Infuced Cardiøc Remodelling in Ratr." J Appl Physiol 88(2): 409-15. yu-yahiro,J.11., Michael, R. H., Nasrallah, D. V. and Schofield, B. (1989). "Morþhologic andHistologic 17(5): 686-9. Abnorrnatitiis in Female and. Mah Raß Treated with Anabolic Steroids." A-J Spotts Med

7-40 CIIAPTER 7 Efects of chronic nandrolone administration The efTect of nartdrolone on extarteuronal ['IIJ-noradrenaline reuptake in rat heart

8.1 Introduction

8.1.1 Summary increase the Results to date have demonstrated that infused nandrolone 1a0-160¡rg/kg/min) can thete was a incidence and duration of arthythmia in tats subjected to ischaemia (Ch' 5)' As maximum of 30 minutes between the beginning of nandrolone infusion and the end of ischaemia, and i'v' the mechanism of this effect is most likely to be non-genomic. Both a single s.c. injection adminisration of nandrolone were found to have no significant cardiovasculat effect, suggesting this setting one of that ischaemia is required in order to observe an advetse cardiovasculat effect. In by which nandrolone m^y non-genomically is thtough blockade of the extraneuronal the ways ^ct reuptake (uptake-2) of noradrenaline. The resulting decrease in extracellular NA clearance the cotonary combined with increased release of NA ftom cardiac nerves due to ligation of IAD m^y possibly explain the dose dependent increase in arrhythmia' Cocaine can inhibit ^ftery such as neuronal reuptake (uptake-1) of notadren altne (refer 1.9), whercas endogenous androgens testosterone have been demonstrated to prevent the extrâneuronal reuptake of catecholamine it is not known whether nandrolone significantly inhibits Qversen et a/.,7970; Salt, 1972). Howevel, steroids such as the reuptake of noradrenaline from carüac muscle in the same marlner as other cofflcostefone.

8. 1.2 Evtratteuronal uP take 1965 (Iversen, Iversen frst reported the extraneuronal reuptake mechanism for noradtenaline in of 3H- 1965). He found that when isolated rat hearts were perfused with a high concentration in 3H-catecholarnine uptake noradrenaline or'H-adrenaline taopg/Íl) there was a rapidincrease uptake-1 alone at perfusion concentfations above 0.5pg/rnl which was obviously not related to heatt tissue couldn't be (Fig. g. 1). The large increase in the concentration of catecholamine in the was sevetal otdets explained in terms of simple diffusion as the intracellular concentration of NA of magnitude highet than the petfusing medium'

8-1 CH,\PTER 8 The efeo of nandrolone on extraneuronal [3H]-noradrenaline reaþtake in rat heart Iversen named this new uptake mechanism "uptake-2". It was found to differ ftom neuronal uptake in 2 main asPects'. '

1,. The process was reversible. Noradrenaline disappeated ftom the tissue if heatts were both perfused with catecholamine free solution. This suggested that uptake-2 could act in directions. 2. The uptake-2 ptocess appeared to be completely inoperative at low concentrations'

Initially it was thought that uptake-1 operated only at low catecholamine concentrations and that a/., 1968)' extrâneuronal uptake only occurred at high catecholamine concenttation (Simmonds ¿l using tritiated noradrenaline showed active uptake concentrations Flowever, later experiments ^t ^11 (Lightman et al., 1969). It was demonstrated that at low noradrenaLine concentrâtions the intracellular metabolising enzymes for noradtenaline - catechol-O-methyltransferase (COMT) and monoamine oxidase (1\4AO) are not fully saturated. The metabolism of noradrenaline produces methoxyhydtoxyphenylglycol IMoPEG] and dihydroxyphenylglycol [DOPEG], which diffuse 3H-NA acts like a'leaky- rapidly out of the myocardial cells @iebig ú a/.,1978). Thus, uptake of 3H-NA crosses the myocardial membr the same time that radiolabelled pump, whereby ^fle ^t metabolites are being lost. This implies that in initial expetiments where MAO and COMT were not inhibited, radiolabelled metabolites were able to diffuse out of the myocardial cells into the under-estimate of perfusate. As only the radioactivity in the tissue was measuted, this resulted in an the rate of uptake of noradrenaline by cardiac myocytes'

In 7969 l(alsner demonstrated that 178-oesttadiol, pfogesterone and deoxycorticostefone potentiated the contraction response of sympathomimetic amines in rabbit aortic strips (I(alsner' j,g6gb). This effect was also demonstrated using (I(alsner,1969a). Based on this blockade of observation lversen examined whether this effect could be explained by extraneuronal including noradrenaline reuptake (Iversen et a/.,1970). He tested a faíge of stetoid hormones (not nandrolone) and found that oestrogen and corticosterone displayed potent inhibition' Futher to block the studies tested the ability of a range of steroid hormones including male hormones 3H reuptake of NA in perfused heatts (Salt, 1,972). He found that andtosterone and testosterone inhibition of both at a perfusion concentration of 10pg/ml caused an 88.7+4'0o/o and 82.9*2.2o/o uptake-2 respectively. It is probable that this degree of inhibition was underestimated in these lower rare of experiments due to a lack of COMT and MAO inhibition resulting in an apparently NA reuptake than would otherwise be expected'

8-2 CIIÂPTER 8The efect of nandrolone on extranearonal pHl-noradrenaline reaþtake in rat heart 1.2 co

(EE) 1 .0 (¡, .= !rts(! 0 .8 È) oj; 0.6 Ë-v0)ar ì:(E o4 õg.Er 0 2 E 0.0 0.0 0.2 0.4 0.6 0.8 1.0 Perfusion Concentration (pg adrenaline/ml)

pefusion Fig. g. 1 Initial rates of uptake in the isolated rat hea¡t with increasing concentrations of adrenaline, showing emergence of uptake-2 as the peÉusion conCentratiOn waS raised abOve 0.5p9/ml. Sou¡ce: adapted from (Iversen, 1965)'

Iversen and colleagues also demonstrated that a range of haloalkylamines related to phenoxybenzamtne also cause inhibition of uptake-2 (Iversen et a1.,7972).

Subsequent to lversen's pioneering work a number of othet characteristics of extraneuronal noradtenaline reuptake have become obvious' These include

1,. IJptake-2 has a broad substrate specificity which is not festficted solely to 'H- 3H-phenethylamines, and imidazoline catecholamines or but extends to tritiated

derivatives and also tritiated histamine and serotonin (Grohmann et a/',1984)'

can be 2. Resting membrane potential modulates uptake-2. Vascular smooth muscle depolarised by crr-adrenoceptor agonists (.g. noradtenaline and methoxamine) and in hyperpolaÅzed by nicorandil (a vasodilatot). Trendelenburg utilised these propetties of rabbit main pulmo n^ry to show that nicorandil increased the extraneuronal segments ^rteïy 1 9 87)' accumulation o f f FII -is opren aline (Trendelenburg,

hearts was due to Evidence suggests that the uptake that Iversen observed in isolated, perfused atterioles and uptake by myocatdial cells and cannot be atttibuted solely to uptake by the venules, study by other vasculatute. Earþ support for this was provided by a histochemical/fluotescence be predominantly associated with Clarke et al tn 1969 which showed that noradrenaline appeated to noradtenaline (Clatke et the cardiac muscle cells in fat heafts perfused wrth high concentrations of

reaptake in rat heart 8-3 CHAPTER 8 The ffict of nandrolone on extraneuronal [3H]-noradrenaline a/., i.96g). Mote recently, catecholamine uptake has been charactetised in rat carðjtac myocytes unaffected (Obst ef a/.,1,996).It was found that uptake was largely inhibited by corticosterone and by cocaine and . Babin-Ebell & Gliese demonstrated that O-methylisoprenaline, 3H-NA inhibited extraneuronal reuptake of lry 61,.2+3.80/o rn rat atrium and 16.0+3.50/o tn tat ventricle (Babin-Ebell et a/., 1995).

The transporter for uptake-2 has been recently cloned (Grundemann et a/.,1998)' Ovedap in the sensitivity to inhibitors of catecholamine uptake and antagonists of the renal trânsPort of organic cations by the transporter protein organic cation transporter 2 (OCT2) suggested that the extraneuronal transporter may belong to the family of amphiphilic solute facilitators. Successful cloning of the trânsporter frorn a human kidney carcinoma cell line (Caki-l) previously shown to demonstrate uptake-2 in uitro, tesulted in the naming of the uptake-2 tfanspoftef, EMT (exttaneuronal trânsporter for monoamine transmitters) (Grundemann et a/.,1998).In accotdance with data from transfected perfused ïat hearts it was demonstrated that human embryonic kidney cells (293) stably with the EMT cDNA take up adrenaline three times mote effìciently than noradrenaline human and (Grundemann et a/.,1998). The rat EMT demonstrates 93 a¡d91o/ohomology to its in bovine counrerpârts (Paczkowsl

in rat heart 8-4 CI{,\PTER 8 The ffict of nandrolone on extraneuronal [3H]-noradrenaline reaþtake 8.2 Aim

o To determine whether nandtolone can inhibit extrafieutonal notadrenaline reuptake in tat heatt.

¡ Based on the plasma concentration of nandtolone achieved with acute dosing (Ch. 5) to determine whether nandrolone mediated inhibition of notadrenaline uptake is likely to explain the observed cardiac effects ofnandrolone'

8.3 Hypothesis Nandtolone will significant1y inhibit the teuptake of notadrenaline in uitro at concentrations consistent with those at which it potentiated ischaemia-induced arthythmia'

in rat heart 8-5 CI{APTER 8 The ffict of nandrolone on extraneøronal [3HJ-noradrenaline reaþtake 8.4 Method Atbino Wistar ïats were obtained 1.0-12 weeks of age and housed according to General Methods ^t 2.j,. Theprotocols for the investigation of uptake inhibition and the methodology fot analysis of tesults are contained tn General Methods 2.1'0.

In order to provide a rapid means of determining whether nandrolone could inhibit the reuptake of noradrenaline in rat cardiac tissue, a series of pilot expetirnents were conducted using atrial and ventricular slices. It was hoped that a simple heart slice prepatation could provide a means for the study simultaneous study of several tissue samples obtained from the same heart' The results of this 3H-NA and a brief discussion âfe contâined in Appendix D. Although good uptake of could be demonstrated in tissue slices, this could not be signifìcantly inhibited with well established inhibitors of extraneuronal reuptake (i.e. cotticosterone or O-methyl-isoptenaline)'

8-6 CFIAPTER 8 The efect of nandrolone on extranewronal [3Hl-noradrenaline reaptake in rat heart 8.5 Results

The ICro value for nandrolone to inhibit extfaneufonal uptake of ¡3rq-NA (0.85pN! was mofe than rM 2,000 times gte ter than that of corticosterone. The ICro of nandrolone was found to be 3.95x10 utl't compared to an ICuo for corlicostetone of 1.78x10 lfig. a' Z¡ Ve icle +Gorticosterone oNandrolone 250

F 22 .E 20 ct) 17 5 =o 150 tr CL 12 5 o 10 0 -v *,(E 7 CL J 50 25 0 -7 -6 -5 -4 log [inhibitor]

Fig. g. 2 Uptake of [3H]-NA into isolated, pefused rat hearts. Mean*SE, n=10 (vehicle), n=3-6 (all otler concent¡ations

8-7 CH,\PTER 8 The efect of nandrolone on extranearonal pHl-noradrenaline reuþtake in rat heart 8.6 Discussion

The ICro value obtained for corticosterone (1.78x10-ulv! compares favourably with the tepotted literature values. Iversen and Salt report an ICro value for cordcosterone of 7.87x10-6M (Ivetsen al 6M+1.18x10 uM a/., 1.g70).In a latet study Salt fepofts the ICro value as 2.7x1.0 lsalt, 1972). Neither of these studies inhibited metabolism of notadrenaline.

It is unlikely that blockade of the extraneuronal reuptake of noradrenaline can explain the cardiac on cardiac events during I-AD coron^ry ischaernia ot on effects of inffavenous nandrolone ^rtety the positive chronotropic effect of cocaine in rats pretreated with nandrolone for 3 weeks (Phillis e/ a/.,2000). The mean plasma level of nandrolone obtained in Ch. 7 following 9 weeks of tteatment with nandrolone was I72!22.5nM (1.72x10'+2.25x10t\,0. Even when nandrolone was infused at high concentration (t6}pg/kg/min) the mean plasma concentration only reached 915+135nM

(9.15x10r+t.35x10r\.Q. The ICro for nandrolone was found to be more than 4x103 times greater than this plasma level of nandrolone. It is therefore probable that inhibition of the extraneutonal reuptake of noradrenaline cannot fully explain its cardiac effects.

in rat heart 8-8 CH-,\PTER 8 The ffict of nandrolone on extrønearonal [3HJ-noradrenaline reøþtake 8.7 Conclusion Nandrolone inhibits extraneuronal reuptake of notadrenaline v¡ith low potency compated to corticosterone in isolated perfused nt he^tt. The plasma levels of steroid produced by chronic treatment with a high dose of nandrolone and the plasma levels resulting from inttavenous infusion do not approach the concentration associated with the ICro of nandrolone fot exttaneutonal noradrenaline reuptake. Blockade of the extrâneuronal reuptake of notadtenaline is unlikely to provide a mechanism for the ìn uiuo poterttiating effect of nandtolone on ischaemia induced catðfac arrhythmia.

in rat heart 8-9 CFIAPTER 8 The ffict of nøndrolone on extranearonal [3H]-noradrenaline reuþÍake 8.8 References

Babin-Ebell,J. and Gliese, M. (1995). "Extranearonal tJptake of Noradrenaline in Høman Tissae (Jptake- 2 )." HeartVessels 10: 1 51-1 53.

Studfu on Noradrenaline Clarke, D. E., Jones, C. J. and Linley, P. A. (1969) . "Histochemical Fluouresence Br I-9. Accamulation @ Uptake-2 in the Isolated Røt Heart." J Pharmacol37

Fiebig, E. R. and Trendelenburg, U. (1978). "The Nearonal and Extranearonal Uptake and Metabo/isrn of )h-O-Noradrenaline in the Prfand Rat Heart." Naunyn Schmiedeburgs Arch Phatmacol 303 21'-35.

Grohmann, M. and Trendelenburg, U. (1984). "The Sabstrøte Spectfci4t of tJptake2 in the Rat Heart'" Naunyn Schmiedebergs Atch Phatmacol 328: 164-17 3'

Grundemann, D., Schechinget,8., Rappold, G.A.and Schomig, E. (1998). "Molecalarldentifcation of the Cortico¡terone-senitiue Extranearonal Catecholamine Transporter. " nature l(5): 349-351' '

Iversen, L. L. (1.965). "The (Jptake of Catecholamines at High Perfasion Concentrations in the Rat Isolated Heart: A N oue / Cate cho larrine Uptake Proce ss." Br J Pharm acol 25'. 1 8-33.

Iversen, L. L. and Salt, P. J. (,970). "Inhibition of Catecholanine Uptake-2 b1 Steroids in the IsolatedRat Heart." Br J Phatmacol 40(3): 528-30.

Uptake in the Isolated Røt Iversen, L. L., Salt, P. J. and Wilson, H. A. (1.972). "Inhibìtion of Catecholamine 647 Hearr b1t Haloalkllartinet Re/ated to PhenoxlbenTamine." Br J Pharmacol 46 -657 '

I(alsner, S. (1969a). "Mechønism of Hldronrtisone Potentiation of Responses to Eþineþhrine and Noreþineþhrine in Rabbit Aorta." Ckculation Res 24: 383-395'

I(alsner, S. (1969b). "steroid Potentiation of Responses to Slmpathomimetic Amines in Aortic Striþs." Bt J Phatmacol 36 582-593.

Lightman, S. L. and Ivefsen, L. L. (1,969). "The Ro/e of tJptake, in the Extranearonal Metabolism of Catecholamines in the Isolated Rat Heart'" Br J Pharma col 3'I: 638-649 '

Obst, O. O., Rose, H. and l(ammermeier, FL (1996). "Characteriqation of Catecholamine Uptake2 ìn Isolated Cardiac þorytes." Mol Cell Biochem 163-164: 1'81'-3'

paczkowski, F. Â., Bryan-Lluka,L.J.,Porzgen, P., Btuss, M. and Bonisch, H. (1999). "Comþarison of the Pharmacologìcat PrEerties of Clonett Rat, Human, and Bouine Noreþinephrine Transþorters." J Pharmacoi Exp Thet 290(2):761-7.

phllis, B. D., Irvine, R. J. and l(ennedy, J. A. (2000). "Combined Cardiac Efects of Cocaine and the Anabolic steroid, Nandrolone, in the Rat." EurJ Pharmacol 398(2):263-72.

b1 Steroids, Clonidine and Salt, p. J. (,g72). "Inhibition of Noradrenaline Uptake 2 in thelsolatedRatHeaX Methoxlland Pherytet@laninel" E,ut J Pharmacol 20(3)z 329-40'

Simmonds, M. A. and Gillis, C. N. (1968). "Upta,ke of Nonnetaneþhrine andNorepinQhrine b1 Cocaine- Treatedkat Hear-t." J Pharmacol Exp Ther 759Q): 283-289.

Trendelenburg, U. (1987). "The Membrane Potential of Vascalar Smooth Muscle Appeaxr to Modalate Uptake2 of 3h-Isoprenaline." Naunyn Schmiedeburgs Arch Pharmacol 336: 33-36.

8-10 CIIÂPTER 8 The ffict of nandrolone on extranearonal [3H]-noradrena/ine reaþtake in rat heart e/"-î"{rù (

Discussion

The published literature concerning the cardiovascular effects of AS abuse in humans is types, complicated by a large numbet of confounding vadables including abuse of multiple steroid with administtation loute, dose, age, gendet, diet and exercise' Similar problems ate associated The pÏesent animal studies investigating the cardiovasculat coflsequences of AS admrnistration. nandtolone in study has sought to minimise these variables and investigate the cardiac effects of notmotensive, norì-exef cised, adult male rats'

9.1 Rat strain used in this compatison of the effect of cocaine on the two most common strains comparable' thesis indicated that the cardiovascular fespoflse to cocaine in the two sttains was here was These results reassure us that the use of the SD rat fot the experiments ptesented apptopriate and would allow comparison with the majority of previous studies'

9.2 Effects of na¡rdrolone alone

9.2.1 Effècß of nandrolotte itt tton-iscltaemic raß has been Chronic nandtolone treatment of the rats did not increase body weight' This effect nandtolone previously reported by a number of investigators in adult male rats receiving s.c. et a/'' 2000a; decanoate using a tange of doses (Long et a/.,1'996; Hallberg et a/',2000; Johansson and contrasts with the increased body weight gain seen in Johansson et a1.,2000b; Long et a/.,2000) females or immâture males (I-ewanowitsch et a/',2001)'

of Histological examination of the hearts ftom nandrolone tteated animals showed no evidence with cellular toxicity in comparison to hearts from vehicle treated conttols. This is in accotdance a cardiotoxic effect previous studies where light microscopy has failed to ptovide any indication of or of AS in hearts from stanozolol treated rats (Baumar^ et øl', 1988), in liver from oxandrolone nandrolone treated testostefone tteated rats (Horvath et al',7977), and in testes and muscle from

rats (Yu-Yahiro et a|.,1989).

9-l CI{APTER 9 Discas¡ion Neither acute nor chronic treatment of conscious rats with s.c. nandrolone resulted in significant changes in BP or HR. Thete was also no effect of nandtolone on these cardiovascular variables in anaesthetised rats when the drug was administered i.v.. This is consistent with previous fìndings in the rat (Hal. et a/., 1.982; Trifunovic et a/., 1.995) and rabbit (Feret et a/., 1.994). This suggests that tachycarðia and hypertension reported with the use of '\S in humans is either species specific (occurting in humans but not rats) or additional elements are requited befote it is evident.

9.2.2 Carrliovascular effecß of nandrolone út raß sub¡'ected to cardiac ìscltaemia and reperfusiott In the anaesthetised animals subjected to cardiac ischaemia and teperfusion, arrhythmia and mortality during ischaemia were increased by acute nandtolone pte-treâtment. This suggests that perhaps in some of the reported cases of sudden cardiac death associated with steroid abuse, the steroids may have exacetbated an ischaemic episode.

The mechanism of the observed incteases in arh¡hmia dudng ischaemia following nandtolone infusion is uncertain. The possibilty of an interaction at the level of noradrenaline transpotters v/as

examined in chapter 8. The likelihood of nandrolone inhibiting non-rìeutonal transport of NA was excluded due to its low potency relative to the plasma levels achieved in these in uiuo experiments. The ICro for nandrolone inhibition of extraneuronal teuptake wâs mote than 2x103 times gteater than the ICro for corticosterone, and more thân 4x103 times gte ter than the plasma concenttation of nandrolone achieved with i.v. infusion (1,6\yg/kg/Ínn). This suggests that the contribution that blockade of exttaneutonal reuptake by nandrolone makes to myocardial noradrenaline concentration during an ischaemic period is very low. There are a number of potential mechanisms for the pro-atrhythmic effect of nandtolone during ischaemia that are worthy of futher invesúgation...

1. (Incoupling of the pmtective eÍifect o.f adenosine on NA efrIux during ischaemia It is possible that the increased anhythmia observed is in part due to a loss of a protective effect of adenosine. During earþ ischaemia a reflex increase in c^rdiz.c sympathetic nerve activity is

accompanied by local exocytotic NA release (Schomig, 1990). A concomitant rise in adenosine Schreieck ¿/ câuses pte-synaptic inhibition of NA release (Richatdt et a/.,1.987; Richatdt et a1.,1994;

at., 1.999). Schreieck and Richardt demonstrated in isolated petfused rat hearts that a tange of variously selective adenosine receptor antagonists promoted arthythmia in heats subjected to regional ischaemia (Schreieck et al., 1999). Ä sirnilar arthythmogenic effect was observed when endogenous adenosine was inhibited. Convetsely, when adenosine bteakdown was blocked or

adenosine production promoted, the occutrence of atthythmia was signifìcantly decreased.

9-2 CIIAPTER 9 Di:cassion Testosterone has been shown to block the vasodilator effect of adenosine in isolated petfused rat hearts and was unaffected by cyclohexamide, ot coupling to a large dexttan- complex' The effect was found to be of rapid onset, disappeadng quickly aftet removal of the stetoid (Ceballos al a/.,1.999). These fìndings ind.icate that the effects are likeþ to be mediated by a mechanism which is independent of nucleat binding or protein synthesis, and occurring at the cell sutface. If nandrolone acts in a similar wây as testosterone and is zcttve agatnst adenosine released during myocatdial ischaemia it is possible that the pro-arhythmic effect of nandtolone observed in the present study reflects an effect against physiological mechanisms activated in eatþ ischaemia to prevent NA ovetflow. It is unknown whether nandrolone acts in the same mânneï as testosterone to block the vasodilator effects of adenosine. It is also not known whether the blockade of the vasodilator effect of adenosine by testostetone noted by Ceballos et a/ is due to an interaction at the receptor level, at the receptor lipid :u:rtetface, or in a pathway triggered by adenosine receptor activation. Furthermore, it is unclear how adenosine ptovides its protective effect as Schteiech and Richardt found that the antiatrhythmic effect of adenosine could not be correlated with a decrease in ischaemic injury (Schteieck et a/.,1999).

2. CNS action of nand¡olone It is possible that the increases in arrhythmia observed during ischaemia are related to a CNS effect of nandrolone. In adult male rats, electrophotetic application of testosterone has been found to increase the firing tate ofindividual neurorrs in the antertor within 2-30s (amada, et a/.,1985). Recent 1g7g). -A sirnilar effect has been observed in the lateral hlpothalamus (Orsini work has focussed on examining changes in the expression of receptors fot vatious brain neurotransmittets in response to treatment with nandrolone and other AS. Chtonic nandrolone treatment has been shown to affect the expression of dopamine receptots (I{indlundh et a1.,200I), serotonin receptoïs in the hippocampal CA1, medial globus pallidus and nucleus accumbens shell (I(ndlundh et al., 2003), to change expression of subunits of the NMDA feceptof in the hippocampus and hypothalamus (Le Greves et a/., 1997) and significandy enhance SP immunoreactivity in the amygdala, hlpothalamus, striatum and periaqueductal gtey (PAG) summatised in (Hallberg et a/., 2000). The current literature on these CNS changes and others are Appendix E. It is not known if nandrolone and similat AS act upon btain regions important in maintaining normal cardiovascular homeostasis, patticularþ with respect to cenffal Íesponses activated by cardiac ischaemia.

3. Anhythmia ÍnductÍon thtough endothelin-7 A number of recent studies have associated the potent vasoconstricting peptide, endothelin-1 (ET- during LAD coron ischaemia. Szabo et al demonstrated that 1) with arrhythmogenesis ^ry ^rtery effect the inttacoronary ET-1 infusion (30 or 60pmol/ mln/30 min) has a ditect atrhythmogenic in

9-3 CIIÂPTER 9 Discu¡sion absence of coronary occlusion (Szabo et a/.,2000). Flowever, it is unlikeþ that the concentrations of ET-1 used by Szabo resemble the concentration that is achievable in a physiological situation. Lin and Yuan demonstrated that i.v, administtation of pte-proET-i mRNA antisense oligodeoxynucleotide (AS-ODN) 2 hrs before coronary occlusion in tats resulted in a dose dependent protection against VT and Vtr pin et a/., 2002). Monality was reduced ftorn 76.7o/o following adrninisrration of saline to 0% with the highest dose of AS-ODN (9Onmol/kg). Administration of sense oLigodeoxynucleotide resulted in an incidence of VT and VF similar to control. Ä similar dose dependent effect was demonstrated for VT and mortality in the cat in the same study using intrac orornry injection of the ET^ receptor antagonist, 8Q610. Howevet, the role of ET-1 in the induction and maintenance of ischaemia is not without controvetsy. Iskit and Guc found that the ETA-ETB receptor blocker, was not Pïotective against ischaemia and reperfusion induced arrhythmia in rats (Iskit er a/.,1'996).

Following the conclusion of the ischaemia/teperfusion protocol petformed in chaptet 5' of blood was extfacted from the descending A preliminary investigation approximateþ 5ml ^ort^. of the ET-1 plasma concentration þg/ml) in tats pte-treated with vehicle or nandrolone (80*g/kg/min) showed no signifìcant difference between conttols and nandrolone infused rats (Appendix F). Before it is possible rule out a nandrolone effect on ET-1, it is necessary to know the plasma ET-1 concentration immediately prior to ischaemia, rather than at the end of teperfusion. The experiments conducted in support of this thesis did not involve blood sampling throughout ischaemiaf reperfusion. Future wotk should attempt to equate nandtolone and ET-1 plasma levels during cardiac ischaemia.

Recently evidence has begun to emefge which suggests that testostefone mây increase the vasoconsttictive effect of ET-1 on potcine coronary vasculature (Teoh et al,, 2000). At present there is ¡o data on the affects of nandrolone on ET-1 in tat cotonary arteries or any evidence that can ptovide a direct link between incteased plasma ET-1 tesulting in VF during ischaemia in nandtolone treated tats.

9.3 Effects of cocaine alone

Intrapedtoneal cocaine administered to conscious animals resulted in an inctease in BP, HR and

SI-A as would be expected. Flowever, the responses ate somewhat prolonged and smaller in peak effect when compared to literatuïe values where the drug was administeted i'v.. Fot example, the HR increase in response to i.v. cocaine in vehicle pre-üeated anaesthetised rats (close-chested), was found to peak at about 40s of infusion (0.5mg/kg/min, total dose = 5mg). In comparison, when cocaine (45mg/kg,i.p.) was administered i.p. to fully-conscious SD rats, FIR was found to peak at

9-4 CH,{,PTER 9 Discassion approximately 90 minutes. Despite these large diffetences in the catdiovasculat effect of cocaine by the 2 administration routes, the i.p. route is appropdate to mimic the phatmacokinetic prohle of cocaine after inûanasal administration, which is one of the most popular routes of administration in polydrug users.

\X4ren cocaine was administered acutely by the i.v. toute to anaestheúsed tats pte-treated with either saline or ethanol vehicle, and subjected to ischaemia and rePerfusion, there was no inctease in arh¡hmia in comparison to control. This may teflect a lower plasma cocaine concentradon achieved with infusion as opposed to iv bolus administration (tr4ehta et al., 2003). Moteovet, anaesthetised animals display blunted responses to the sympâthomimetic effects of cocaine (Pitts el 1'999)' al., 1.987 ; Fraker et a/., 1'990; T ell'a et a/', 1'992a; Wang et al',

9.4 Cocaine effects in combination with chronic nandrolone previous work found that chronic pre-treatment with nandtolone, significantly (p<0.05) potentiated the HR response to cocaine (45mg/kg, i.p.) in conscious rats (Phillis et a1.,2000)' In anaesthetised rats, chronic nandtolone pte-treatment did not significantly change the BP or HR response to i.v. cocaine (O.5mg/kg/min, total dose = 5mg). The interaction between 'treatment' and ,time' at 600s of infusion for the HR response to cocaine may be explarned by the high the variability associated with the vehicle fesponse at this point (Fig. 7.8. C.). It is possible that failure to observe a signicantly elevated HR response to i.v. cocaine in chtonically nandrolone found treated rats compared to vehicle controls may be related to the use of anaesthesia. Wilkerson that pentoba¡bital anaesthesia sigruficantly reduced the BP, HR and fate-pfessufe fesponse to may cocaine (1mg/kS, i.v.) in dogs fWilketson, 1988). This suggests that pentobarbital anaesthesia have ,dampened' the cardiovasculat response to i.v. cocaine in the SD tats used in chapter 7. It is difficult to compare the effect of i.p. cocaine in conscious, freely moving rats, with the effect of i'v' cocaine in anaesthetised rats. Therefore, further work examining the cardiovascular effects of i'v'

cocaine in fully conscious, chtonically nandrolone treated rats is wartanted.

may It is possible that the pteviously observed effect of nandrolone on the HR response to cocalne not reflect a pharmacokinetic interaction between nandrolone and cocaine (see 9.7) which wâs interaction pfesent in tats tteated acutely with nandtolone. The possibility of a phatmacokinetic between nandrolone and cocaine requires furthet investigation (Long et a/.,2000) (see 9'7 Futute Studies).

Ä number of models have been proposed to explain a repotted pro-arrhythmrc effect of cocaine the local including increases in intracellular calcium, incteased catecholamine concentratton and

9-5 CIIÂPTER 9 Di.rcassion anaesthetic properties of cocaine. These ate reviewed extensively rn 1.73.2. Likewise, chronic nandïolone treatment has been associated with a number of cardiac conditions which could be (1.6.2), expected to increase the risk of ischaemic events, including, left ventriculat h¡pettrophy hypertension (1.6.3), arteriosclerosis (1.6.1), direct toxicity (1.6.2) and acute myocatdial infarction (1.6.1 & table 1.3). However, in the present study cocaine (i.v) in combination with chronic nandrolone had no effect upon the frequency ot dutation of arrhythmia. This may have been due to a low basal arrhythmic activity following chronic nandrolone tteâtment.

9.5 Cocaine effects in combination with acute nandrolone

Acure nandrolone (a0¡rg/kg/min) administration followed by i.v. cocaine (O'Smg/kg, s'c') did not produce a significantly different BP or HR response profile compated to saline or vehicle pre- treated animals. However, cocaine appeated to pfotect against fatal vF during ischaemia which occurred in the presence of nandtolone.

It is possible that the protective action of cocaine is a centrally mediated effect. Bonci and Williams dopamrne demonstrated that cocaine (lOmg/kg, i.p.) can change the notmal synaptic regulation of cells in the VTA (Bonci et a/., 7996). In the absence of cocaine pre-treatment, D1 receptor activarion increased the amplitude of GABÂ' mediated inhibitory post synaptic potentials (IPSP)' Rats which received cocaine fot 2 weeks showed inhibition of the GABAB IPSP. This inhibition metabolism' was blocked by adenosine A1 receptor antagonists and compounds disrupting cAMP Baldo et al ,¡sed r^rtge of adenosine agonists and antagonists to show that In a similar study ^ cocaine withdrawal induces changes in the exptession of adenosine recePtors in the nucleus mechanisms accumbens (Baldo et a/.,1,999). Although both of these studies were concerned with of brain reward, it is possible that sirnilar changes in synaptic transmission may mediate the cardiovascular response to nandrolone. Testosterone has been shown to have an effect at receptors adenosine receptors (Ceballos et at., 1,999). Therefore, it is possible that centtal adenosine

may provide a site of intetaction of ,\S and cocaine'

9.6 F,>rperimental Limitations

Äs with all studies, a lirnited number of factots could be examined. A number of variables are in wotthy of further study and should be considered in the intetpretation of the tesults presented of this tepott. These vadables include species, strain, gender, exetcise, stfess, drug doses and toutes administration.

9-6 CHAPTER 9 Discassion 9.6.1 Species It is clear that the rat model has limitations and that considerable cate must be exetcised in extrapolating results ftom this model to humans. Howevet, although there ate species differences in the response to AS and cocaine thete are also many similarities. Similat plasma profiles are produced in both rats and humans after i.v. cocaine administration flavaid et a/., 1'978;Jeffcoat et a/.,

1989; Pan et at.,1,998). Both rats and humans show similar pharmacodynamic responses to cocaine characterised by dose related increases in SLA, CT, HR and BP (Foltin et a/., 1988; Lange et a/.,

1989; Lomax et ø/.,1990; Tella et ø/., 1992a; Ansah et al.,!996; Evans et al., 1'996;Horowitz et a/.,

1,997) and similar ECG changes (Opitz et a/., 1995; Littmann et a1.,2000; Phillis et a/',2000).Isolated vessels from both humans and rats show the same vasoconstrictive response to exogenously applied cocaine (Isner el a/.,1989;Vitullo et a/.,1.989; Chokshi et a/.,1990; Benzaquen et al',2001). Likewise, similarities exist between rats and humans in the behavioural effects of AS (Annitto et a/., 1980; Conacher et a/.,1,989; Chot et a/.,1,994;Long et a/.,1996) and the induction of changes in et a/., !989; G\azet,1991; plasma lipid profites flX/ebb et al., 1984; Yu-Yahiro et ø1.,1.989; Ztitartt

Lewanowits ch et al., 2001).

9.6.2 Sa-ain \Øhile a sigruficant numbet of investigators have examined the strain related behavioural effects of cocaine, research on cârdiovascular outcomes is Limited. The possible effect of sttain on the cardiovascular response to cocaine is discussed at length in chapter 3. Evidence ptovided in chaptet the 3 indicates thar the BP and HR response to i.p. cocaine in the 2 sttains (AW and SD) used in pfesent study, was not significantly different. Howevet, wistar tats showed a significantly greater SLA response to saline. The opposite effect was observed with the administtation of cocaine, whereby the 15mg/kg dose caused a signihcantly gteatet SLA response in SD rats. This implies that while there is evidence that suggests a strain related behavioural response to cocaine (George et

a/., 1,991.; Cailhol et a/.,1,999) a similar differential cardiovasculat response is not observed.

9.6.3 Gender ,\lthough the studies in this thesis were confined to male animals, gender may be an important factorin the ïesponse to cocaine and nandrolone. Morishtrna et ø/infæed cocaine Qmg/kg/ ffÍî) until circulatory collapse in male and female rats. It was found that the dose required to produce cardiovascular toxic manifestations was signifìcantly lower in male rats than females (N4orishima el effects a/., 7993). Lukas et al stdted the gender dependency of positive and negative psychological of cocaine and correlated these changes to plasma concentration and HR (Lukas et a/.,1'996).It was found that intranasal cocaine HCI caused a sþificantly highet peak plasma concentration in males (L44.4+17.Sng/rrl) compared to females in the follicular stage of the

9-7 CFIAI'IER 9 Disca¡¡ìon (73.2+9.9ng/ml). \X4ren the same group of women wete administered cocaine during their luteal phase the plasma concentration was lower (54.7+8.7 ng/ml). Despite a significant difference between plasma cocaine levels the HR response to cocaine was not signifìcantly different between groups. This suggests that women may be more sensitive than males to the HR effects of cocaine, but that this effect is not altered by the hormonal fluctuation seen in women. Futute shrdies should consider the importance of gender'

9.6.4 Exercise The addition of anaetobic exercise would have been an intetesting series of expedments to âdd to this project but this is qurte diffìcult to undettake using rats as the experimental animal' Most reports using rats have used an aetobic exetcise regime such as treadmill running which is not appropriate to simulate AS users' regimes but may be acceptable as a form of sttess stimulating the HPA axis. Conlee and colleagues demonsttated that treadmill running caused elevations in plasma catecholamines and corticosterorìe which were futther significantly increased by cocaine

(12.5mg/kg, i.p.) (Conle e et a/., 1991). Noradrenaline in treadmill exetcised rats increased by greater

than 2.3 times in cocaine tteated rats compâred to exetcised, saline conttols. Comparable increases in plasma catecholamines were also observed by Oiuka et a/andlFran et a/:using treadmill exercise and cocaine (2}mg/kg, i.p. and Smg/kg, i.v., respectively) (Han et al., 1996; Ojuka et a/., 1996). Because adrenaline has been found to be synergistic with cocaine in reducing coronary flow tate (CFR) in isolated, perfused hearts (Avakian et a/., 1,990) the additive effect of exercise on the catecholamine increase in tesponse to cocaine may be especially important in potentiating the

of cocaine on the heart.

1¿an et al also found a sudden increase in plasma lactate within 2 minutes of commencing treâdmill exercise in cocaine treated rats, compared to vehicle controls (Han et a/., 1'996). This diffetence remained statistically significant (p<0.05) even following a 20 minute recovery period. It was not determined whether this increase in lactate was due to a vasoconstriction mediated reduction of blood flow resulting in hypoxia andlactaTe production. Similar lactate increases in exetcised rats have been observed in animals subjected to chronic cocaine treatment (I(elly et ø/.,1'995).

9.6.5 Stress

Stress is an important variable in determining the cardiovascular response to cocaine in the rat. It has been demonstrated that cocaine (Smg/kg, i.v.) administration and air-iet stress both cause â bimodal c rdi^c output (CO) response in tats thus allowing the classification of 'respondets'

(decreased CO) and 'non-respondets' (increased CO) (I(nuepfer et a/.,1'993). Heatt rale and arterial pressure did not display the same bi-modal distribution. This suggests that in a sub-population of

CH,A.PTER 9 Discassion 9-8 râts (,responders') the c ïdizrc output in response to cocaine is strongly influenced by stress. Later work has found that a similar response is elicited using cold \Ã/ater stress, and that the diffetences in CO within a population of tats in response to cold water or cocaine could be eliminated by administedng (, 25pg/k1, i.v.) or atropine þeripheral anti-muscarinic, O.Smg/kg, i.v.). This suggests that the haemodynamic resPonse to both cocaine and stress is influenced by calcium channel dependent vasculat smooth muscle contraction and muscârinic inhibition of catecholamine telease.

In the present study in ordeï to minimise the stress component of the response to cocalne, cardiovascular variables in chapters 3 and 4 were collected using tadiotelemetry. Radiotelemetry eliminates the extreme stress associated with restraint during tail-cuff measutements of BP is limited to (Clement et a/.,1.989;Irvine et a/.,1997;McDougall et at.,2000). Handling of the animal

the rime of drug injection.

9.6.6 Route of drug admútislration A small number of human studies have investigated the pharmacokinetics of cocaine via different effects' routes, although resulting plasma concentrations have been tarely equated to cardiovascular radiolabelled cocaine after 3 diffetent administration Jeffcoat et al obsewed the disposition of foutes: i.v. injection, snorting and smoke inhalation (Jeffcoat et a/'' 1989)' The plasma concentfatìon-tjrne ptofile for cocaine and the majot metabolite þenzoyl ecgonine) for i'v' 'Wheteas administration and smoke inhalation were almost identical. i.v' injection and smoking via produced large and :zrpìd lncteases in parent drug and metabolite concentration, administration nasal insufflation (snorting) displayed a long lag time to peak cocaine concentlation of other 4+.7+I7.0 min. Cocaine plasma concentration remained appreciably higher than that via the routes until about 250 minutes post insufflation. Benzoyl ecgonine concentrations reached a maximum within approximately 200 rninutes and temained elevated until the conclusion of the that expedment at 500 minutes post insufflation. Branch and I(nuepfer provide evidence to suggest benzoyl ecgonine can cause a profound simultaneous pÍessor and bradycatdic effect upon infusion of a high cocaine and benzoyl ecgonine ltmg/kg) in rats (Branch et a1.,7994). The maintenance high plasma concentration following nasal insufflatìon explains the observation of a consistently HR (>180% baseline) until gteater than 60 minutes post administtation Savaid et a/., 1978)' Obvious methodological problems prevent adrninisttation of cocaine to râts by nasal insufflation' disposition pâttefn of cocaine after i'p' Fottunately, Javaid and Davis demonstrated that the administration in the rat is similar to that observed in humans after intta¡asal administtation $avaid cocaine et a/., 1993). Most anknal studies concerned with cocaine cardiotoxicity have used i.v.

9-9 CIIÄPTER 9 Discussion administration. However, falal catdiotoxic incidents have been reported for subiects using intranasal administration and smoking CW.tIi et a/.,1.985; Mittleman et a/.,1'987).

9.6. 7 Cardiac isclnemta A number of human case reports have linked ÄS abuse to conditions associated with lowered perfusion and increased risk of cardiac ischaemia. Steroid abuse has been linked to athlerosclerosis

(À4ewis er al., 7996) and thrombosis, (Fisher ú a/., 1996; Nieminen et a/., 1.996) both of which significantly increase the dsk of cardiac ischaemia. Likewise cocaine has been associated w'ith vasoconsüictive action reduced perfusion Q-ange et a/., 1,989; Lange et a/., 1990) through a direct (Vitullo et a1.,1.989; Chokshi et ø/., 1990; Simkhovich et a/., 1994).In the present study, chronic pre- treatment with nandrolone did not alter the cardiovascular response to ischaemia or the magnitude and duration of arh¡hmia elicited by a 1S-minute ligation of the LAD coron^ry artery'

In order to maximise bioavailablity acuteþ, nandtolone was infused i.v. to a group of rats, and found to elicit a dose dependent inctease in VF duting ischaemia, and a signihcant increase in the Lambeth arhythmra score compared to vehicle infused conttols. This discrepancy in the arrh¡hmic response between acute and chronic dosing may be related to the much higher plasma nandrolone concenttation achieved with i.v. infusion. Alternauvely, it is also possible that chronic

treatment caused downregulation of receptor(s) or a transduction cascade intermediate, ot activated

a negative feedback mechanism critical for increasing arrhythmia with i.v' infusion. For example, nandïolone has been demonstrated to have a biphasic and tteatment duration dependent effect on HpA activiry. Chronic nandrolone treatment for 1.4 days at both 15 and 45mg/kg signifìcantly

decreased circulating ACTH, 24 hrs after the last injection, while corticosterone only displayed a significant reducrion at the highest dose at the same time point (Zhou et a/., L998). Short tetm treatment with 15mg/kg, i.m. in a different study caused increases in circulating levels of both

,A.CTH and corticosterone after 3 days treatment, an effect which was lost 24 houts after drug

v¡ith&awal (Schlussman et a\.,2000). Schlussman and associates suggest that short tetm nandrolone treâtment may transiently activate the HPA axis whereas chtonic administration results in inhibition. A sirnilar bimodal effect on corticosterone and ACTH has been noted with acute and et a/., 1.996). \X/hile changes in circulating,{.CTH chronic cocaine adrninistration (Zhou ^te ^rr example of a mediator which is variably affected by acute or chronic nandtolone treatment it may not explain the resulting differences in the arrhythmic propensity of acute or chronic nandrolone treatment in the present study. In fact, infusion of the melanocortin peptide ACTFI-(1,-24) (adrenocorticotropin) during a 5 minute occlusion of the rat LAD corot ry artery hâs been shown

to afford a dose dependent protectiofl against reperfusion arrhythrnias @azzani et al',2001';Bazzant teceptor et a/.,2002). Conversely, the reverse effect was obsewed with the melanocortin MC3/MC4

9-10 CII\PTER 9 Di¡cøssion antâgonist SHU 9119 (IVi"ri et a/.,2004).If it is assumed from the wotk of Schlussman that our i.v. infusion of nandrolone caused an increase in ACTH in a similar manner to acute i.m. adminisuation we would predict an antjt-arch¡hmic effect. This contrasts to the increase in VF frequency and duration that was actually observed. More wotk is tequred to detetmine exact ACTH levels following i.v. administration to determine whethet changes in circulating ACTH evoked by acute or chronic nandrolone administration can explain the difference in the occurrence of vF during ischaemia following these different tfeatment feglmes. pre-ischaemic, i.v. cocaine, administered to anaesthetised rats tegardless of âcute or chtonic pre- treatment with nandtolone failed to significanthy affect the frequency and duration of atrhythmra during ischaemia. It is probable that this effect is telated to the btadycardia seen with cocaine administration in pentobarbital treated rats which itself makes observation of a trte effect of cocaine on cardiac rh¡hm difficult to observe. Pentobarbital was utjlised because it has been shown to d.isplay limited effect on cardtac thythm, whereas urethane, halothane and chlotofotm have been shown to have an ati-arhythmic effect (Curtis et a/', 1987)' Unfottunately, pentobarbital has been demonstrated to have a catdio-depressant effect when co-administeted with

cocaine (pitts at a/.,1.987;TeIla et at.,1.992b) and causes a signifìcant decrease in the peak pressor fesponse to cocaine in dogs (Fraker et al., 1,990) and mice 1rffr.g et a/.' 1'999).

occur It was noted that in rats adrninistered cocaine, there was an incteasing tendency for death to which is unrelated to VF, and prevents scoring according to the Lambeth convention. Without the also diffìcult to assign survival time. As measurements of observation of fatùVFitis ^î^ccvr^Te In cardiac output were not taken, it was not possible to determine the exact cause of these deaths' most rats the Lambeth scoring method is necessary to obtain meaningful results from data which

show a large standatd deviation'

Another of the disadvantages of the model used in this study is that attificial ventilation in open- fatal' chested animals allows animals to survive periods of arrhythmia that would otherwise prove Therefore, it is concluded that the anaesthetised ischaemia-teperfusion rat model is not ideal for the evaluation of the cardiovascular effect of drugs with a signifìcant sympathetic component'

9.7 Futr¡re sûrdies with Future studies must concentrate on the mechanism of the observed increase in arrhythmia infused nandrolone. In the present study it is uncertain whethet the pro-atrhythmic effect of nandrolone dudng ischaemia is steroid-receptor mediated or is due to â non-specifìc toxic effect' pre-ischaemic infusion of a steroid receptor antagonist such as flutamide to detetrnine whether the

9-lt CI{ÂPTER 9 Discassion pro-arrhythmic effects of nandrolone during ischaemia can be reduced or eliminated will help detetmine whether toxicity is receptor mediated.

Nandrolone readily crosses the blood brain barder due to its high lipophilicity. -An assessment of the nandrolone concentration in various brain regions following the drug infusion protocol used in this thesis will help elucidate brain regions potentially affected by nandrolone infusion. Identifying possible brain regions involved in cardiovasculat homeostasis and the effect of nandrolone on neurotransmitter release and receptor expression would greaúy conttibute to an undetstanding of the cardiotoxicity of nandrolone. The effect of nandrolone on neurotransmission in the CNS and perþhery is unknown. Due to the low potency of nandrolone for extraneuronal NA reuptake, it is unlikely that increased myocardial NA can explain the observed arhythmia during ischaemia. No studies have been conducted examining the effect of centrally applied anabo[c steroid on cardiotoxicity. However, a CNS dependent mechanism of catdiotoxicity is not without precedent. Tyrosine, the amino-acid precursor of the catecholamine neurotransmitters is an example of a drug which caî centrally to increase the incidence of atthythmia during cardiac ischaemia. It has been ^ct found that tyrosine injected into the Iateral cerebral ventricle of pigs induces ventriculat fìbrillation inl}Oo/o of pigs compared to 52o/o in drug free, conscious conttols (Intby et a/',1992). This effect was attributed to an increase in CNS catecholamine synthesis. It is possible that nandrolone may

also act cenffally through an as yet unknown pâthway to câuse an increase in catdiac arrhythmla.

The direct effects of nandrolone on the heart remain uncettain. Ceballos et a/ demonstrated that testosterone when confined to the coronary vascular lumen can block the adenosine vasodilator effect in isolated perfused tatheafi (Ceballos et ø/., 1999). Future experiments should investigate whether nandrolone can also show this effect. The likelihood of the importance of an adenosine-

mediated pro-arhythmic effect of nandrolone is indicated by the effect of adenosine antagonists during ischaemia. Both, adenosine antagonism with theophylline, 8-phenyltheophylline ot xanthine

amine congener and inhibition of endogenous adenosine telease ((S-(4-nitrobenzyl)-6-thioinosine)

have been shown to cause an increase in cardiac a:r".hythmiain isolated petfused tat hearts (Gotge et be a/.,1.998;Schreieck et a/.,1999).It is possible that the pro-arhythmic effects of nandrolone may of endogenous adenosine release or of catdiac adenosine related to an inhibition ^n ^nT^gonism receptots.

with To date investigations with nandrolone have provided no evidence of a hlpertensive effect acute of chtonic tfeatment. The reported inctease in BP by chtonic 19, methyl testostefone (l\dolteni et a/., 1.969) or testosterone (Fischer et ø/., 1.977) ueatrnent should be confirmed using radiotelemetry. A, animal handling can induce sttess telated incteases in BP and HR, it is impetative to erìsure that previous studies using tail cuff measutements of BP in response to

9-72 CFIAPTER 9 Discassion signifìcant stress component' It is testosterone $1aL et at., 1,982) have not unintentionally repotted a also important to determine whether the accelerated hypertension in nandrolone treated developrng SHRs observed by Tseng et al can be replicated using radiotelemetry (Tseng et a/.,1'994). Only a small number of conflicting studies have addressed the putative hlpertensive effect of AS in rats and consequently the ability of AS to induce hypettension remains conttoversial and confusing (Brown et a1.,1972; !Øolinsky, 1972; Grcenberg et a/.,1973; Fischer et a/.,1977;Hall, et a/.,1982)' These studies have used r^îge of AS, different treâtment periods and species, and examined both ^ exercising or non-exercising animals all of which alone may have a signifìcant effect upon the cardiovascular tesponse to AS.

Despite the significant increase in relative heaftsize observed in this study in response to 6 and 9 weeks nandrolone treâtment, no pathology was detected in heatts compared to vehicle tteated rats.

Future studies involving chronic nandrolone treatment should use highet resolution mictoscopy. ,\ study by Abu-shakra and associates demonstrated apoptotic cell death in mutine

cells treated with stanozolol (Abu-Shakn et al., 1997). However, the doses of stanozolol that cells were exposed to was exceptionally high (>10plv!. A steroid antagonist, failed to reverse cell death

suggesting a direct toxic effect of stanozolol rather than a receptor mediated effect. It is not known whether AS concentrations of greater than 10prM are achievable in ttiuo- In the present study, plasma concentrations of nandrolone associated v/ith atrhythmia during ischaemia only reached approxirnately 0.92¡rM. Although the nandrolone doses used in the ptesent study are reflective of doses of AS used by recreational drug users (Yu-Yahiro et a/., 1'989; Johansson et a/., 1997) tt rs possible that this concenffatiorr was too low in otder to observe myocardial tissue damage. It is possible that a direct effect of nandrolone was rnissed in this study because of the use of low resolution light microscopy. Horvath et al fasled to fìnd a toxic effect of oxandrolone and testostefone on fat hepatocytes using light microscopy and only observed ptoliferation of the smooth-surfaced endoplasmic reticulum when using electron mictoscopy (Horvath et a|.,1'971)'

It is possible that the significant increase in chronotroPy by i.p. cocaine, reported pteviously in fully-conscious chronically nandrolone treated rats in comparison to vehicle controls, is due to a pharmacokinetic interaction between nandrolone and cocaine. Cytochrome P-450 is integral to the metabolism of testosterone and nandrolone, being tesponsible fot hydroxylaion at sevetal been found to be capable of inducing positions Suchau et al., L976). Testosterone has also cytochrome P450 in female mice (Hong et al., 1.989). Hong et al invesisated the P450281'- dependent renal metabolism of the catcinogen N-nitrosodimethylamine Q'{DMA) after 2 days treatmeflt of female mice wrth testosterone (500mg/kg, s.c.). Renal NDMA activity was found to be elevated 17-fold, correspondingwith a signifìcant elevation tnP450281" A similar

effect has been observed for hepatic cytochrome P450 in female rats administered oxandrolone but

9-L3 CIIAPTER 9 Discussion cytochrome P-450 (N4isra not in mâle rars $Vaskiewicz et al., 1995). Cocaine is N-demethylated by et at., L974). Chronic treatment with cocaine (3Omg/kg/day,i.p.) for 2 weeks has been shown to significantly increase tumour necrosis factor-aIpha (TNF-cr) and cteatine phosphokinase (CPÇ in mice hearts and seta. Pre-treatment u/ith the P450 inhibitors or was found to attenuate or abolish the effect of cocaine on TNF-cr and CPI{. Similady, isolated hepatocytes have been shown to have an increased susceptibility to cocaine-induced cellulat damage when livet cells were isolated from rats in which P450 isoforms had been induced via or that chronic flover et a/., 7993; Poet et a/., 1.996). In addition it has been shown cocaine administration can induce its own metabolism in mice Powers et a/.,1'999). The fact that both nandrolone and cocaine are metabolised to some extent by cytochrome P450 suggests that chronic treatment with either drug could alter the c Ídiz.c response elicited by the other, and requires further attention'

9-14 CH,A.PTER 9 Discu¡sion 9.8 Conclusion no High doses of nandrolone, administered by either chronic s'c. or acute s'c' or i'v' route, had significant cardiovasculat effectin the sedentary rat. In conscious animals, chronic, but not single In dose s.c. nandrolone potentiated the tachycardia resulting ftom i.p. cocaine administration' blunted and anaesthetised rats in which the pressor and chronottopic response to cocaine was transient, thete was no effect of acute nandtolone on cardiovascular responses to i.v. cocaine'

there was In anaesthetised rats pretreated with i.v. nandroìone and subjected to cardiac ischaemia effect of an increase in the duration and frequency of VF and significantly increased motality. No on nandrolone pfetfeatment was evident during catdiac reperfusion. The effect of nandtolofìe to 9 weeks' ischaemia-induced arhythmia wâs not observed after chronic nandrolone dosing for up The mechanism of the potentiating effect of acute i.v. nandtolone on arth¡hmia was not identifìed but is unlikely to be due to decreased clearance of noradrenaline by card:tac cells since nandrolone was shown to be too weak an inhibitor of extraneutonal noradrenaline uptake in the isolated perfused t^t he ït, displaying an ICro several orders of magnitude gre Tef than the plasma levels to associated with potentiation of arrhythmia in uiao. The failute of chtonic s.c. nandtolone chronic dosing potentiate ischaemia induced anhythmia and mortality may be due to failure of the be due to regime to achieve sufficiently high plasma levels of nandtolone, or alternafively it may down tegulation of the effect with chronic exPosute to the drug'

In anaesthetised animals iv cocaine infusion had no significant effect on ischaemia-induced On arrh¡hmia when administered alone, and did not exacerbate the effects of infused nandrolone' the contrary, co-administration of cocaine appeared to fevefse the increased mortality and arthythmia during ischaemia due to nandrolone'

The mechanism of the potentiating effect of acute nandtolone on ischaernia-induced arrhythmia adverse requires further study. However these data suggest that AS administration might precipitate the caïdi,.c events in individuals predisposed to cardiac ischaemia. Similady, the mechanism of to apparently protective effect of cocaine is unclear. Flowever, furthet studies are necessarT increased determine whether a similar effect occurs in conscious animals in which the pressor and cocaine by HR responses to cocaine are more pronounced. Moreover, the administration of iv intetaction with infusion may havelimited its pro-arrh¡hmic effects. It is possible that a diffetent doses of nandrolone or ischaemi a may have been observed if bolus doses of cocaine, or higher cocaine, had been administeted.

9-15 CI{,A.PTER 9 D itcussion drugs may be In conclusion, if the tesults presented here extend to humans, the use of these individuals with tolerated in healthy individuals and not lead to c Ídi^c events' Flowevel, in compromised coronary perfusion they ate likely to ptecipitate fatal consequences.

9-16 CIIÄPTER 9 Discassion 9.9 References

Abu-Shakta, S., Alhalabi, M. S., Nachtman, F. C., Schemidt, R. A. and Brusilow, W. S. Â' (1997)' "Anabolic Steroids Indace Injury andApoptosit of Dffirentiated Skeletal Mwscle." J Neurosci Res 47: 186- 197.

Clin Annirto, W. J. and Layman, W. A. (1980). "Anabolic Steroids andAcate Schiqophrenic Episode."J P sychiatry 4l(4) : 1. 43 - 4.

Ansah, T.,'SØade, L. H. and Shockley, D. C. (1996). "Changes in L.ocomotorActiuitJ, Core Temperatare, and Heaù Rate in Response to Repeated Cocaine Admini:tration." Physiol Behav 60(5): 1'261,-t267 .

Avakian, E. V., LeRoy, M., St John-Allan, I{., Sayre, F. W. and Malone, M. H. (1990). "Efect of Chronic Cocaine Administration and Cocaine Il/ithdrawal on Coronarl Flow Rate and Heart Rate Resþonses to Epineþhrine and Cocaine in Isolated Perfased Rat Htarts." Life Sci 46(22): 1'569-7 +.

Baldo, 8., I(oob, G. and Markou, A. (1999). "Ro/e of Adeno¡ine A2 Receþtors in Brain 'ltinalation Reward. ander Baseline Conditions and Daring Cocaine IYithdrawal in Røt¡'" J Neuroscl l9Q\: 1.101'7- 77026.

Bauman, D. H., Richerson,J. T. and Britt,,{.. L. (1988). "A Comþarison of Bofu and Organ ll/eights, P@siologic Parameters, and Pathologic Changes in Target Organs of Rats Giuen Combinations of ExercisL Anaboic Horrnone, and Protein Supplementation." AmJ Sports Med 16(4): 397 -402.

Bazzani, C., Guarini, S., Botticelli, A. R., Zaffe, D., Tomasi, ,\., Bini, A., Cainazzo' M. M., Fetrazza, G., Mioni, C. and Bertolini, A. (2001). "Protectiue Eftrt of Melanocortia Peptides in Rat Mlocardìal 297 (3) : 1, 082-7 . Is che mia. " J . Pharmacol. Exp. Ther.

Bazzani, C., Mioni, C., Fercazzà, G., Cainazzo, M. M., Beftolini, A. and Guadni, S' Q002)' ,,Inuoluement of the Central Nervoas Slstem in the Protectiue Efect of Melanocortin¡ in Mlocardial

1' e o oJ Resus citation 52(1 : 1 09 -1' 5' I s c h a e m i a / I'ep fu si n I r rJ. " )

Benzaqten,B. S., Cohen, V. and Eisenbetg, M. J. (2001) . "Efects of Cocaine on the Coronaryt Ar-teries." Am Heart J 142(3): 402-L0.

Chønges in S1n@tic Bonci, A. and Williams, J. T. (1996). "A Common Mechanism Mediates L,ong-Tern Transmision after Chronic Cocaine and Motphine. " Neuton 16: 631'-639'

Branch, C. A. and lSuepfer, M. M. (1994). "Cãases of Dffirentiat Cardiouasca/ar Senitiuiry n Cocaint. Ii: Sltnpatbetic, Metabolic and Cardiac Ef,ectl" J-Pharmacol-Exp-Th er 27lQ): 1'103-1'3'

Brown,B.S.andPilch,,{. H.(1.972)."TheEfectsofExerciseandDianabolUponSelectedPerforruancesand Pþsiological Pørameters in the Male Røt." Med Sci Sports Exer 4(3): 159-65.

Cailhol, S. and Mormede, P. (1999). "strain and Sex Dffirences in the I-'ocomotorResþonse andBehavioral Senitisation to Cocaine in tþperactiue Røts." Brain Res 842: 200-205'

Ceballos, G., Figueroa, L., Rubio, I., Gollo, G., Garcia, r\., Mattinez, A.,Yanez,R.,Petez'J', Morato, T. and Chamorro, G. (1999). "Acate and Nongenomic Efects of Testosterone on kolated and PerfasedRøt Heart." J Cardiovasc Phatmacol 33: 691'-697 '

Choi, p. y. and Pope, H. G. (1994). "Violence toward ll/omen and Illicit Androgenic-Anabolic Steroid Use." Ann Clin Psychiatry 6(1):21,-5.

9-17 CFIAPTER 9 Discussion Chokshi, S. K., Gal, D., DeJesus, S. and Isner, J. M. (1990). "Cocaine Produces Vasoconstrìction of Human Coronary Arteries: In Vitro Stødies \Jsing Coronary Arteries Obtainedfrom Freshþ Explanted Haman He arts." JACC l5 (2) : 21 5 A.

Telemetry to Record Boþ Temþerature and Clement, J. G., Mills, P. and Brockway, B. (1989). "(Jse of AcfiuìA in Mica"J Phatm Method 2l:1'29-1'40'

Conachet, G. N. and'Sii'orkman, D. G. (1989). "Violent Crime Possibþ Associated wìth Anabolic Snroid Use." ArnJ Psychiatry 146(5):679.

Conlee, R. K., Barnetr, D. !Ø., I(elly, I{. P. and Han, D. H. (1991). "Ef,ects of Cocaine, Exercise, and Resting Conditions on Plasma Cor-ticosterone and Catecholamine Concentration¡ in the Røt." Metabolism 40(10): 1.043-7.

in Curtis, M. J., Macleod, B. A. and Walker, M. J. (1937). "Models þr the StudJ 0f AffhJthrnia¡ Mltocardial Ischaemia and Infarction: The fJ¡e of the Rat."J-Mol-Cell-Cardiol l9(4): 399-41'9 '

E. (1996), "Ar-terial and Venoas Cocaine Plasma Evans, S. M., Cone, E. -RelationshþJ. and Henningfield, J. Concentration¡ in Harzøns: to Roate of Administration, Cardiouascalar Efects and Søbjectiue Efects."1 Phatm Exp Ther 279:1'345-1'356.

Ferrer, F., Encabo, 11., Marin, J. and Balfagon, G. (1994). "Chronic Treatment with the Anøbolic Steroid, Nandrolone, Inhibits Vasodilator Retþonses in Røbbit Aorta." F;ur J Pharmacol 252: 233-241'.

Fischet, G. M. and Swain, M. L. (1,917). "Elttt of Sex Horrzones on Blood Pressare and Vascalar Connectiae Tissue in Castrated and Noncaslrated Mah Rnts." A- J Physiol 232(6): F{617 -21'.

Fisher, M., Appleby, M., Rittoo, D. and Cotter, L. (1996). "Mlocardial Infarction with Extensiue 222-3. Intrøcoronary Tirmbit Ind.aced bl Anabolic Steroid¡ [See Connents]." BrJ Clin Pract 50(4):

Foltin, R. W., McEntee, M. l\., Capriotti, R. M., Pedroso, J. J. and Fischman, M. W. (1988). "Efects of Cocaine, Alone and. in Conbination-with Task Performanc4 on Heart Røte and Blood Pressare." Pharmacol Biochem Behav 3l(2): 387 -91"

Ftaker, T. D., Temesy-Armos, P. N., Brewstet, P. S. and Wilkerson, R' D' (1990) ' "Mechanism of cocaine-Induced aþocardiat Deþression in Dogs." Circulation 8l(3): 1,01'2-6.

Coruparisont Geotge, F. R., Porrino, L. J., Ritz, M' C. and Goldberg, S. R' (1991)' IndicaTe Dffirent Situ; o¡ Zrtlo, ¡o, Cocaine and Amphetamine nt Efects." Psychopharmacology 104(4) : 457 -62.

Glazer, G. (1991). "Atherogenic Efects of Anabolic Snroids on Serum Lipid I-'eue/s. A Lìteratøre keuiew Med 1992 MarJ 52Q):535]'" Arch Intern Med 151(10): 1925- [Pabtished Enatø* Appton in Arch Intern -)-).

Gorge, 8., I{urz, T., I{atus, H. A. and Richardt, G. (1998). "Endogeno-as A/eyosine Suppresses Norþntpfr;oe-Indaced Ventricalar Arrhlthrnias in Rat Heart." Basic Res Catdiol 93(4): 264-8.

Greenberg, S., Fleitz, D. C. and Long, J. P. (1973). "Testo¡terone-Induced Depression of Adrenergit'Actiui4t in the Perfused Canine Hindlinb." Ptoc Soc Exp Biol Med 142(3):883-8'

Hall, C. E. and Hungerford, S. (1982). "sirnilarities andDffirences between Efects of TeÍosterone and 19- Norte¡tosterone ¡n Røti øth Particalar Reference to Hlpertensogeric Potenry."J Steroid Biochem 16(4): 581- 5.

9-18 CI{,A,PTER 9 Discussion Steroids Hallberg, M., Johansson, P., Ilndlundh, A. M. S. and Nybetg, F. (2000) "Anabolic-Androgenic Afect the Content of Substance P and Substance P|-7 in the Røt Brain " Peptides 2l:, 845-852.

Han, D. H., I{elly, I{. P., Fellingham, G. W. and Conlee, R. K. (1996) . "Cocaine and Exerrise: Temporal Changrc in Plasmø l-,eueb of Catecholarnines, l-øctate, Glucose, and Cocaine." A- J Physiol 270(3 Pt 1): E43B-44.

the Sex-Related Flong, J. y., pan, J. M., Ning, S. M. and Yang, C. S. (1989). "Molecalar Basis for Dffileníes inRenalN-Nitrosodirnethllarrine Demethllase in C3h/Hej Mice." Cancet Res 49(11): 2913-9.

Florowitz, B.2.,Panacek, E. A. andJouriles, N.J. (1997). "seuereRhabdomloþsis withRenalFaìlare afier Intranøsal Cocaine Usa" J Emerg Med 15(6): 833-7 '

Florvath, E., I{ovacs, I{., Blascheck,J. A. and Somogp, A. (1971). "Ultra¡tructural Changu Induced in B Cell Pathol 7(\:3a8-55. the Liuer ofRaß bl Varioøs Steroid Cornþoands,"Virchows 'A.rch

on Blood Pres¡wre."J Pharm Irvine, R. J., \Øhite, J. M. and Chan, R. (1997). "The Efect of Restraint Toxicol Methods 38 1'57 -1'62.

Iskit, A. B. and Guc, M. O. (1996). "ThimerosalAttenaates Ischaemia-Reperfasion Arrhlthnias in Rats: No Pharmacol Moification b1 Anti-Ischaenic Agent Trirneta{dine or Endothelin Receþtor Antagonist Bo¡entan " Res 34(1-2): "17-23.

Isner,J. M. and Chokshi, S. K. (1989). "Cocaine andVasosþasm."N EnglJ };4.ed'321(23):1'604-6'

Disposition in Disnete Regiorc of Rat Brain." Biopharm Javatð,J. I. and Davis, J. M. (1993). "Cocaine Drug Dispos 14(4): 357-64'

I., Fischman, M. W., Schuster, C. R., Dekirmenjian, H. and Davis' J. M. (1978). "Cocaine 'plasmø-Concentration:Javatd,J. Relation to P@siotogica/ ønd Sabjtctiue Efects in Hamans." Science 202(4364):227-8.

R. Perez-Reyes, M., I{ill, M., sadlet, B. M. and cook, c. E. (1989). "cocaine "DispotitionJefrcoat, -4., J. in Hamans after Intrauenoas Injection, Nasa/ Intffiation (Snorting), or Smoking." D-g Metab Dispos fl (2): 153-1'59.

p., Nyberg, tr. (2000a). "The Efect 0f OþiuidPeþtides in the Johansson, Halberg, M.,I{indlundh,,\. and "Røt Res Brain, aftetr ChronilTreatment with the Anabolic Androgenic Steroid, Nandrolone Decanoate." Btain Bull51(5): 413-41'8.

p., and Fahlke, C. (2000b). "Anabolic Androgenic Steroids Afecß Johansson, Lindqvist, ,\., Nyberg, F. "Ahohol 271'- Intake, Defence Behauiors and. Brain Oþiuid P, tides in the Røt." Pharm Biochem Behav 67: 279.

P., Ray, A., Zhot, Q., Huang, W., I{adsson, I{. and Nyberg, F. (1997). "Anabolic "AndrogenicJohansson, Steroid¡ Increa¡e Beta-Endorphin l-,eue/¡ in the Ventral Tegtnental Area in the Male Rat Brain'" Neutosci F.es 27Q): 185-9.

and Castell, V. (1993). "Coc1i1e Hepatotoxiritl: Two Jovet, R., Ponsoda, X., Gomez-Lechon, M. J. J "Dffirent Biochem Toxiciry Mechanisms for Phenobarbital-Indaced and Non-Induæd Røt Heþatogtte.r." Pharmacol 46(1 1): 1'9 67 -7 4.

M. R., Mitkin, D. L. and Zachairah,P. K. (1976). "Interaction: (Varioas 19-¡ vith 'HamanJuchau, Placental Microsomal Cltochrorne P450 (P-450 HPTI)." Chem Biol Interact 15(4): 337-47 '

9-19 CI{,A.PTER 9 Discøssion I{elly, I{. P., FIan, D. H., Fellingham, G. w., \vinder, \ø. w. and conlee, R. K. (1995). "Cocaine and Exerche: Pþ:iotogzcalR,,rplns,,r of Cocaint-ConditionedRatr."Með Sci Spotts Exerc 27(1):65-72.

Kindlundh, A. M., Lindblom, J., Bergstrom, L. and Nyberg, F. (2003). "The Anabolic-Androgenic Steroid Nandrolone Induce¡ Alterøtions in the Densi4t of Serotonergic 5htl b and 5ht2 Receptors in the Male Rat Brain." Neuroscience 119(1): 1'13-20.

I{indlundh, A. M. S., Lindblom,J., Bergstrom, L., Wikberg,J.E. S.and Nyberg,F. (2001). "The Anabolic-And¡ogenic Steroid Nandrolone Decanoate Afects the Densìfl of Doþanine Receþtors in the Male Røt Brain." EutJ Neurosci lil: 291-296.

I(tby, D.A.,Johnson, D.,\., J.,P.,Zhao, S. and Lown, B. (1'992). "Control of Ventriculartribrillation arter Coronaryt Artery Orclasion Via Intracerebrouentricalar Injectìons." A- J Physiol 263: H479-483.

Knuepfer, M. M., Branch, C. 4., Mueller, P. J. and Gan, Q. (1993). "stress and Cocaine Elicit Similar Cardùc Oatþat Responses in Indiuidual Rats." AmJ Physiol 265(2 Pt 2): H779-82'

Lange, R. r\., Cigarroa, R. G., Flores, E. D., McBdde, w., Kim, A. S.; NØells, P.J., Bedotto, J' B', Oaizi.ger,R. S. ;d Flillis, L. D. (1990). "Potentiation of Cocaine-Indaced Coronary VøsoconstricÍion bl Beta-

e l1I2(1 2): 8 9 7-903. A dre nirgi c B lo c ka de [S e e C o m m n ts] . " Ann Intern }.l.ed

Lange, R.4., Cígarroa,R.G., Yancy, C'W', Willard,J. E,., Popm^,J'J'' Sills, M' N', McBdde, W', i'Cocaine-Indaced Kirn: A. S. and Hilir, L. D. (1989) . Coronary-Artery Va¡oconstriction [See Comments]." N Engl J rr/.ed 321(23): 1'557 -62.

F' (1997)' "Efects of Le Greves, P., Fluang, W., Johansson, P., Thornwall, M.,Zhou' Q. and Nybetg, Mrnas an Anabolic-Aod.rogrnl Sttro¡d on tfu Regalation of the Nnda Receþtor Nrl, Nr2a and Nr2b Sabanit in Brain Regions of the Male Røt" Neurosci Lett 226(1): 61'-4'

Lewanowitsch, T. and Irvine, R. (2001). "Efects of Testorterone Propionate andNøndrolone Decanoate on Boþ Comþo:ition and Lipoprotein Coucentrations in the Røt." Addiction Riol 6: 55-61.

and Rats." Lin, L. and yuan, \ø. (2002). "Inuoluement of Endothelin-l in Acate lrchemic Arrhlthmias in Cat.ç Clin Sci 103(Suppl. 48): 228s-232s.

Littmann, L., Monroe, M. H. and Svenson, R. H. (2000). "Brugada'þpe Electrocardiogr@hic Pattern Induced b1t Cocaine." Mayo Clin Proc 75(8): 8a5-9'

Lomax, p. and Daniel, K. A. (1990). "Cocaine and Bodl Temþeratare in the Røt: Ef,ects of Ambient

. T e mþe rature." Pharmacol ogy 40 (2) : 1.03 -9

Long, S. F., Wilson, M. C. and Davis, W. M. (2000). "The Efects of Nandrolone Decanoate on Cocaine' Indøced Kindling in Mak Rzlr. " Neuropharmacology 39: 2442-2447 '

Long, S. F., Wilson, M. C., Sufka, I( J. and Davis, W. M. (1996). "The Efects of Cocaine and. Biol NandroÌone Co-Administrøtion on Aggrrssion in Mak Rats." Ptog Neuropsychophatmacol Psychiatry 20(5): 839-56.

Lukas, s. E., Sholar, M., Lundahl, L. H., Lamas, X., I(ouri, E., wines, J. D., I{ragse, L. and ;'Sex l¿uels and Sabiectiue Efects aJter Acun Cocaine Mendelson, J. H. (1996) . Dffirences in Pla¡ma Cocaine Ad.ministratiin in Haman Volanteers." Psychopharmacology (Bed) 125(a): 346-54.

stres¡: McDougall, S. J., Paull, J. R. 4., \Øiddop, R. E. and Lawfence, A. J. (2000) ' "Restraint Dffirentlat Card.iiuø¡calar'úrporrn in lTistar-þoto and Spontaneousþ H1þertensiue Rats." H:pettension 35: 1.26-729.

9-20 CFIAPTER 9 Di¡cutsion Mehta,,\.,Jain,,\. C. and Mehta, C. M. (2003). "Electrocardiographic Efects of Intrauenous Cocaine: An Experimentàl Snþ in a Canine Modet." J Cardiovasc Phatmacol4l: 25-30.

Mewis, C., Spyddopoulos, I., I{uhlkamp, V. and Seipel, L. (1996). "Manfestations of Seuere Coronary Heart DiseasiafterAnabotic DrugAbase. " Clin Cardiol 19: 153-155.

Mioni, c., Guiliani, D., Cnnazzo, M. M., Leone, s.,Bazzani, c., Grieco, P., Novellino, E., Tomasi, A., Bertolini, A. and Guarini, S. (2004). "Farther Eaidence Thøt Melanocortins Preuent Aþocardial Mc3 Receþtors." Eur Phatmacol 477(3): 227 -34. Repefusion Injøry @ Actiuating Melanocortin J

Misra, A. L., Vadlamani, N. L., Bloch, R., Nayak, R. I{. and Mule, S. J. (,974). "Phltsiokgic Disposition Chem Pathol and Metabolism of Tritiøm l-.abelled. Ecgorine (Cocaine Metabolite) in the Rat." Res Commun Phatmacol S: 55-63.

Mittleman, R. E. and Wetli, C. V. (1987) . "Cocaine and Sudùn "Nataral" Death." J Fotensic Sci 32(1): 1.1.-9.

Disease in the Molteni, 4,., Brownie, A. C. and Skelton, F. R. (1969). "Prodaction of Hyertensiue Va¡culør Rot U Metl¿yhestosterone." Lab Invest 2l: 729-37 '

Morishima, H. o.,,\be, Y., Matsuo, M., Akiba, I(., Masaoka, T. and cooper, T. B' (1993). "Gender- 722(2):1'57-63' Related. Dffirences in Cocaine Toxicilt in the Rat [See Comrzenß]." J LaÏ> Clin Med

Nieminen, M. S., Ramo, M. P., Vütasalo, M., Heikkila, P., I{atjalainen, J.' Manty¡-aari, M' and Ufters." Heikkila, j. (1996i. "serious Card.iouascalar Sidl Ef,ects of l-¿rgt Doses of Anabolic Steroids in lYeight Eut Heaft J fl: 1'57 6-1'583.

and Exercise: ojuka, E. o., Bell, J. D., Fellingham, G. w. and conlee, R. K. (1996). "cocaine 124-32' A'tteration in Cørbo@d|øte Metabolisrn in Adrenodernedøllated Ratr." J Appl Physiol 80(1): r Opitz, C. F., Mitchell, G. F., Pfeffet, M. A' and Pfeffer, J' M' (1995)' ' Cìronary Artery Onlasion in the Rat. Continuow Telemøric Ecg Monitoring in Circulation 92Q): 253-61'.

L. and Waynet, M. (19S5) . "Direct E_ît'tt of Androgens on Orsini, J. C., Barone, F. C., Armstrong, D. J. Brain Res Bull 15: I-^ateral Íþpothalanic Neuronal Actiuigtln the Mak Rat: L A Microionoþhertic Stød1'" 293-297. Pan, fø. J and HedaYa, M' A' (1998) "An Animal Model þr Simaltoneoas 39: 1- n u e s tiga ti o n s : Aþþ li c atio n s t o Cocaine."l Phatm Toxicol Methods P h arm a co ki n e ti c f P h arrn a co d1 n am ì c I 8.

of Cocaine and the Phìllis, B. D., Irvine, R. J. and I{ennedy, J. A. (2000). "Conbined Cardiac E'fects Anabolic steroid, Nandrolone, in the Røt." EurJ Phatmacol 398(2):263-72'

pitts, D. I{., Udom, c. E. and Marwah, J. (1987). "cardiouascølar Efects of cocaine in Anesthetiryd and Consrious Rats." Life Sci 40: 1'099-1'111'.

P4502h and P4503a Poet, T. S., McQueen, C. A. and Halpett, J. R. (1996). "PaXicipation of Cltochromes in Cocaine Toxici4t in Rat Hepatorytes." Drug Metab Dispos 7A(\:7 a-80'

Changu Expression of Moase Liuer powets, J. tr. and Shuster, L. (1,ggg). "sabacate Cocaine Treatment Cytochrorne P450 Isoþrrns." Phannacology 58(2): 87-100'

9-2t CI{APTER 9 Discu¡¡ion Richardt, G., Blessing, R. and Schomig, A. (1994). "Cardiac Noradrenaline Rr./eø¡e Accelerates Adenosine Forruation in the Ischenic Røt Heart: Role of Neuronal Noradrenaline Carier and Adrenergic Receptors."J Mol Cell Cardiol 26(1,0): 1321'-8.

tt'4denosine Richardt, G., Wolfgang, !ø., I{nnzhofet, R., Mayet, E' and Schomig' A' (1987). Inhibit¡ Exogttic'Release of É,odogtrort Noradrenaline in Rat Heart: A Protuctiue Mtchani¡n in Earþ Nþtocardial I¡chemia." Circ Res 6l 117-723.

M' (2000)' Schlussman, S. D., Zhou,Y., Johansson, P., I{iuru, ,\., Ho, t\', Nyberg, F. and I(reek, J' ,,Efects of the Androgenic Anabolic Steroid, Nandrolone Decanoatø on Adrenocorticotroþin Honnone, Clnico¡teione and Proopiornelanocortin, Corticotroþin Re/easing Factor (Crfl and Crf Receptorl Mrna l¿aels in the H1þothalamøs, Pitaitary and Anlgdala of the Rat." Neurosci Lett 284(3): 1.90-4.

Schomig, A. (1990). "Catecholømines in Aþtocardial Ischemia. Slsternic and Cardiac P'/./ease." Circulation 82(3 Suppl):111.3-22.

of Isthenia-Induced Schreieck, J. and Richardt, G. (1999). "Endogenous Adenosine Reduce¡ the Occurrence ventricølar Fibrillation in Røt Heart."J MoI Cell cardiol 3l(1): 1%-34.

Simkhovich, B. 2., I{loner, R. ,{.., Alker, I( J. and Giaconi, J. (,994). "Titne Coarse of Direct 23(3): Carcliotoxic EfeXs of High Cocaine Concentration in Isolated Rnbbit Heart." J Cardiovasc Phatmacol 509-16.

Szabo, T., Geller, L., Merkley, 8., Selmeci, L., Juhasz-Nagy, .A' and Solti, F. (2000)' "Inuestigating the DaalNatureofEnfuthelin-l:IschaemiaorDirectArhlthmogenicEfect?"Iile Sci66(26):2527-2541.

Tella, S. R., I(orupolu, G. R., Schindler, C. ìØ. and Goldbetg, S. R. (1992a). "Pøthoþhlsiological and phannacological Meihanisms of Acute Cocaine Toxicitlt in Conscious Ratr." J Phatmacol Exp Ther 262(3): 936-46.

t ìne in Conscious Tella, S. R., Schindler, C. W' and Goldberg, S. R. (1,992b)' RatiRelatirc Stgnzfcanæ of Central S1mþøthetic Stimalation and ine Uptake and Rel,,ase Mechanisms." J Pharrnacol Exp Thet 262(2): 602-1'0'

of l7beta-Estradiol Teoh, FI., euan, 4., Leung, S. W. S. and Man, R. Y. K. (2000). "Dffirential Efects 1301-1'308' and. Testo.çterone on the Contrictite Resþonses of Porcine Coronary Arteries." Br J Phatmacol 729 'SØoodiwiss, Trifunovic, 8., Norton, G. R., Duffield, M. J., Avtaam, P. and A. J' (1995)' "Ao H1096-105' Androgenic SteroidDecreasesL.eftVentricalarComþliance inRats."A-J Physiol263(3 Pt 2):

Tseng, ¡¿. T., Rockhold, R. !ø., Floskins, B. and Ho, I. K. (1994). "Cardioua¡cular Toxicities of 113-21'. Nanlrolone and. Cocaine in Spontaneousþ HlpeÉeniue Røts." Ft¡dam Appl Toxicol 22(1):

G. L. and l(hairallah, P' A' (1989)' Vitullo, J. C., I{atam, R., Mekhail, N., Wicker, P., Engelmann, "Cocaint-Induced Snalt Vessel Spasm in Isolated Rnt Hearts. " A- J Pathol 1it5(1): 85-91 '

Morgan, P (1999) "Dffirential Wang, J. F., Hampton, T. G', Deangeli,, J', Ttavets, K' and J ' Soc E'xp Depr;;r;nt Efecß of Generat on the Cardiouascular Resþonx to Cocaine in Mice." Proc Biol Med 221(3):253-9.

waskiewicz, M.J., choudhuri, S., vanderbeck, S. M.,zhang, X. J. and Thomas,.P. E. (1995)' Metab ,,Ind.action of "Mate-specifl'Cltochrome P450 Isorymes in Femah Røts @ Oxandrolone-" D*g

Dispos ?3(1 1) z 1'29 1' - 6.

9-22 CFIAPTER 9 Discassion Webb, O. L., Laskarzewski, P. M. and Glueck, C. J. (1984). "Seuere Deprusion of High-Densitl Lipoprotein Chohsterol L.euels in lYeiþt Lifters and Bodlt Bailders @ Self-Adrninistered Exogenoas Testosterone and Anabolic-Androgenic S teroids. " Metabolism 33(1 1): 971'-5 '

Werli, C. V. and Fishbain, D. A. (1985). "Cocaine-Indøced Pychosis and Sadùn Death in Recreational Cocaine (Jsers." J Forensic Sci 30(3): 873-80.

\)íilkerson, R. D. (1988). "Cardiouascalar Efects of Cocaine in Consrioa¡ Dogs: Inportance of Falþ Fanctional Aatonomic and Central Nervous Slstemt." J Phatmacol Exp Thet 246(2): 466-71"

SØolinsþ, H. (1,g72). "Efects of Androgen Treatruent on the Ma/e Røt Aorta."J-Clin-Invest 5l(1'0): 2552- 5.

yarnaða,y. (1,g7g). "Efexs of Testosterone on (Jnit Actiuitl in Røt Hlpothalamas and Septam." Btain Res fl2:1.65-1.68.

yu-yahiro,J.4., Michael, R. H., Nasrallah, D. V. and Schofìeld, B. (1989). "Morphgl9gll andHisologic AbnoTnalitii: in Femak and. Mak Rats Treated with Anabolic Steroids." AmJ Sports Med 17(5): 686-9.

p., Anabolic Androgenic Steroid.ç Alter Brain Zhou,y., J., Ho, ,\., Nyberg, F. and l(reek, M. J. (1998). pomc Mma Expression and Hpa Actìui4t in Male Rzlr. National Institute of Drug Abuse Research Monograph Series. L. S. Hards. Washington, D.C., NIH' 179: 115'

Zho.,t, Y., Spangler, R., LaForge, I( S. L', Maggos, C' E', FIo, A' and I(reek, M' J' (1996)' ,,Mod.ulation i Cr¡Xt Mrna in Røt Anterior Pituitary b1 Dexarnethasone: Conelation with Pomc Mrna." Peptides fl:434-41..

zu?ani,IJ., Bernardini, 8., catapano,A., Campana, M., Cerioli, G. and Spattini, M. (1989). "Efects ofAnaiotic'Steroifu, Testosterone, oia Ugn on Blood Lipids and Echocardiogr@hic Parameters in Bo@ Bailders." IntJ Sports Med 10(1): 62-66.

9-23 CHAPTER 9 Discassion ûff"--/;,rfr û'.

Re fatal and non-faal cardiouascular outcomes resulting from anabolic steroid use

A-L & details of Authore Gender Exercise Status History of steroid Met¿bolites Detected Fatel/Non- Clinical details & zge use Fatal Noo fatzl body compeu¡ofl vith chest Myocædial (Appleby et ø1, Ma.le,31 Bodybuilder Had øken anabolic Drug screen not jugular veûous not raised, steroid aod performed Blood pulse 1 00 regulr, Pressüe 19e4) yo. Patient refused to accept mphetmioe comes hedt soufld ûomd and chest cleæ. elevatioo in leads II, III and aVF' wth tetted T- that he had suffered a over 5-10 yeus (ry'pe On ECG there ws signilcant ST and leads V:-Vo myocardial infæction, of steroids ¡ot waves and ST depression in I, aVL dischrçd himself and reported). elevated (163 U/I). was lost to follow-up. Used 2 Frumil (80mg ignificantlY the ST chaoges without development Frusemide and 10mg resoludoo of T-wave inversion in leads II, ) tablets per there was i¡ferolalenl day to reduce body III, Vs and Vo. fluid and i¡creæe muscle deEnitio¡ Consumed 59 of potassium per day and additional as a health tonic. history o[ severe retrostemal chest pain MaIe,23 Bodybuilder An abuse history Non fatal @owman, A non-smoker with no history of heart disease. 1 eeO) y.o. spanning 5 yeus and on admission showed evidence o[ an acute Cædiac catheterisation most recendy 5 weeks Examination of the ECG recorded found evidence of previously lateral infdcúon Treatrnent with streptokiûase did oot prevent fomation o[ a full thickness infrct septakinesis and atherosclero tic with a rise io cædiac enzymes. signifi cant plaque formation in the left anterior descending artery and large obJigue bønch.

NI Diagnosis & det¡fü of Authore Gende¡ Exercise Status History of stercid Metâbolites Detected Fatal/Non- Clinical det¿fü follow-up @ryrëtu) & ¿ge use Fatal hypertrcphY Fatal an lnstmtaneous bilateral pulmonary Ventricular (Dickermn ø/ Male, 20 Amtew bodybuilder Reportedly abused a Drug screeû oot There was oo past or family history o[ cardiac disease. and pulmonary aL,7995) yo. range of steroids pedormed. veotriculæ hypertrophy and haemorhaç- includiog Primobolan- Heart displayeã evidence of significant concent¡ic left Depot (methenolone weþhed 5509. depot), Testosterona Evidence of mild æte¡iosclerosis. (ve terinuían testos terone eoanthate) ¿nd I¿wbolan (veterinrian nandrolone lauate). At the time of death he had just completed a 3- month cycle of steroids. At peaìr cycle he ws administering approximately 7OOmg/week of aûaboLic steroids. Non f¿ul 45-niun rødiztiilg to the l?fi an wilh mrseø, di¿phonsis infæction (Ferenchick a/ MaIe,37 comPeÍûve A history o[ Drug sdeen rot þatu breaIh. y.o. weighdifter iotemittent stercid performed and sboftzes¡ ø1,7992) Exercise stress testing 4 abuse spanaing 7 yean. months after dischrge At the time oF showed no iscbaemic admission he was changes. completing a 1ó weeks infæction cycle of steroid use. msecs in dundon indicative of myocædizJ in cædiac enzyme actirity wæ reporled (creadoe kinase llclzte He admined to the An incrase ^ad abuse of several dehydroçoæe) and hepruin intramusculdy Treatment was begun with intravenous dssue plasminogen activator administered ste¡oids Recovery ws uneventfirl. angiographically including nandrolone Cædiac catheterisation perfomed 3 days after admissioo showed function. decanoate, boldeoone, normal arteries and nomal left vent¡iculr testostefone c]?ronate and stanozolol. He a.lso used oral oxand¡olooe

ÞI UJ Diagnosis & detâils of Gender Exercise Status flistory of steroid Ne Metabolites Fat¿llNon- details Authore follow-up (tr.p"üee) & âse Detecæd Fatal infuction Admitted to hospitaJ Non fatzl No of note- No cwent medicine físhet et al, Male,43 -{mateu bodybuilder Duaúon of abuse is Past was noma.l (1 7 o 1ee6) y.o unknow¡. complaining of Blood PreSSUfe /70) wefe all oormal. Following treatment with At the time of admission severe central back Blood count, ue4 electrolytes, blood glucose and chest x-ny leads & III. iqt¡avenous hepæin and he ms administèriog pain æsæiated with ECG revealed an acute infe¡ior infæct with ST elevation in II the left oral wzrfæin the patient methaodieoone (orally), weadog aod Echocædiography ¡evealed dilation of all the cadiac chambers and wæ dischrged home nandrolone decanoate voñlng. ventriculæ ejection ftacdon was estimated to be 30yo with no follow-up f.m.) and testosterooe (i.m) autopsy reltâled cddiac of the right Cædiac hlpertrophy et Mùe,23 Amatew Abused a wide noge of eazyme F¿tal a|,1998) yo. performance enhancing immunoassay (EIA) foc¿l induation of the endocudium. the left venricu]u drugs. The following and gas Flistology revealed enlæçment and nucleu pol¡morphism o[ were found in his chromatography- muscle fibres. aptrtment folloving mÆs spectrometry Intentitial myocrdial fibrosis aod disseminated foca.l necrosis. death... (Gc-Ms) Testex Leo 250 metabolites o[ prolongatum mesterolone, (testosterone methandienone, cyclopentilpropionate) testoslerone, Primobolan Depot ¡androlone and 100mg (methenolone were enanthate) detected in the Proviron 25mg ufûe. (meserolone) Thybon 100pg (liothyonin hydrochloride) Adalactone 100mg (spirinolactone) Clomifen 25mg Contraspasmin 0.02mg (clenbuterol

Þ 5I & det¿ils of Authon Gender Exercise Stztm History of stercid Metabolites Detect€d FatallNon- Clinical details & ase use Fatal sevefe altet 7 heary ress10r. (Iluie, 1994). Male, Amteu weighdifter 16 weeks prior to A ufine toxicology Noo fata.l substernal chest which progressed to crushiog PãN. 25 y.o. admission he h¿d been screen ws negative for Began expeneflclog 1.e grandfather 8 months post infæction; adninistering 100mg mphetamines, No signiEcaot Prof medical history Fmily history ofinfrction infarction and uncle swived ÂMI zl the the patieot denied of n¿nd¡olone brbiturates, died LT 40 from myocrdial ^n experienciog any further decanoate weekly benzodizepines, zg?. cædiac symptoms with (im) Hecontinued cocai¡e and Increæes in wdnc etz:.n'es obseryed. exerciæ or aay additiooal this practice For 6 pheocyclidine. The Cholesterol was nomal. hypokinesis. steroid use. weeks. Following a 4- udne was flot tested Echærdiography ¡evealed mild proximal left anærior descending artery Cholesterol levels were week break he fo¡ a¡abolic steroids. Coronary angiography revea-led a læge masued again and contioued nmd¡olone lesion consiste¡t with a th¡ombus. found to be at accePtable administration at leveìs 200mg for 6 weeks 3 the development recurreoÇ dull cent¡al chest Myæudral (Kennedy Male,24 Competitive 2-yer history o[ Drug screen not o¡ latal to his left æm 1.993) y.o. bodybuilder a¡abolic sleroid abuse. performed or lipid disordere. Awaiting coronary Over the 6 weeks prior No fmily history of heart diseæe angiognphy. to admission he had Sooked 30 cigrettes daily, a 10-yeu habit. measuements coofi¡med lateral been taking oral Seríat electroødiographs md cudiac enzyme stanozolol (,{0 ags myocrdial infæcúoo. apical daily), nandrolone (200 Echoødiognphy revea.led a small area of iofrction His blood piesiore remained intemittently elevated (muimum 195/110mmHd' mgs, l.m , twlce weekly) and Sustanon 250 (testosterone esters - 1ml, i.m. weekly). haemorrhage Fata) z history of steroid abuse had suffered zi Cerebml el al, Male,21 Body builder 6-yer history of Gas chromatograptuc $etredy Fe¡ola myocrdial inlrction 7 0 months previously. yo anabolic steroid abuse. md mass uncompLicated in te¡al 1993a) later resumed spectroscopic analysis 3 months following this he ¡esumed weight Iifting ztd 8 months oFurine upoo autopsy s te¡oid use was no detected staoozolol At autopsy the corona¡y æteries were found to uise nomally, there and nandrolone. signifcant atheroma in the coronary, cæotid o¡ cerebral ci¡culations A-right cerebellr haemorrhage was present which had spread into the pons and veotdcultr systems. The¡e ws no evide¡ce of væculi¡is in the cerebra.l or ci¡culation.

(JrI Diagnosis & details of Authos Gender Exercise St¿tus IIistory of stercid Metabolites Detected Fatal/Non- Clinical follow-uP (tr.ppüabb) & zse use F*al surface haemorrhaç presenf oû ¡he epicrdial su¡Face of ¡he H)?ertrophic ((entedy et ø1, Ma]e, 18 Professional footballer Not available Fata.l were nomzJ, equidomioant and free ol atheroma of th¡ombus. crdiomyopathy 1993b) y.o glucuroaide Coronary rtef,es (metaboìite o€ Crdiac valves we¡e nomal orymesterone) Histological analysis revealed myofibre dismay with væiation in hbre size and thickening of the walls oÍ the i¡tramura.l æteries consistent with h)?ertroPhic

Oxlme sterone Fatal Petechial haemorrhaçs present on the posterior surface of the heatt, tnð 2 Myocæditis Ç(ennedy et ø1, MaIe,24 Professional footballer Not available haemorrhages on the vith cædiâc puocture. 1 993b) yo glucuronide The co¡ooary arteries quidomnant- Likewise, there was no elidence ol th¡om nd only mimal atheroscle¡osis' -4.n exteûsive area of scæriog wæ found in the ioteryent¡iculæ septum' There were mulúple foci of fibrosis within the sePtum with adjacenl hyperlrophic myæytes and in olaies a monocytic and lymphocytic i¡filtrate consistent with myocrdilis' while using a bench press al a gym Pæmedics reporting to glm Cardiac h)?erüoPhy and puke, 1990; Male,21 At least 7-yeæ history History of parenteral 1 Fz¡d, lound him in ventriculu Ebrillation regional myocædial Czmbd), al al., y.o of hìgh intensity steroid abuse fo¡ and¡osterone -4.utopsy found mrked left-sided cardiac h)?ertrophy. The coronary arteries shorved fibrosis. 1 ee3) sports. Four veæs in sevenl months before Q3Ctng/nl),19-aor- The cudiac va.lves were un¡emukable. vusity . decease. Police reports etiocholaoolone (52 no evidence of arteriosclerosis. Sectioned myocædium revealed extensive regional ûbrosis with principal (Bolh papers Subsequcntly involved indicate possession oF ng/ml), and 19-oor- of the subepicrdial, and the cental aspects o[ the left ve¡uicle and the nþon dffinnt in teslosterof,e c)?loflatc (32 iovolvemeol interventriculr sePtum and of the right ventricle and atria. aquts of the (200mg/nl), ng/ml) (nandrolone myocædium revealed eridence of extensive left and right ventriculr samc cua) nand¡olone decanoate decanoate metabolites) Sectioned (100mg/rn & hypertrophy and dilation. lormation. 200mg/rnÌ) Blood: fouod to be Mi..or.ópic exminaúon also lound evidence of contraction baod Drugs reported to have unsuitable for analysis. been admioistered twice weeldy The last injecúon occuned approx 1 week before decease Far¿l No pasr or [amily disease Âcute (À,fcNutt e/ al, Male,22 World class weþht I¡tramusculu and om] DrW screen not Non s¡rdies revea.led an elevated creaúoe kinase infæctioo 1988) yo li[ter ÂS lor 6 weeks prior periormed Laboralory ofnew to admission. The ECG disclosed bo¡h elevated ST segmett and the development Q No [ollow-up reported I¡og-lerE abuse nol waves. normal reported. Crdiac catheterisation perFormed 10 days after inlrction demonsraled

m cholestcrol ¿¡ admission in Trventy-four days post-infdction after holesterol was found to have reduced

6\I Diagnosis & details of Authon Gender Exercise St¿tus flistow of stercid Metabolites Detected Fatal/Non- Clinical details follow-up øzp"riabþ) & ace use Fatal Noo faøl tachycædia tachycudia as (Mervis e/ al, Mùq28 Bodvbuilder 2 yer history of Dflg screer not The recorded at sinus rhlthm disclosed a pattem, R reduction ln Vr-V the 6ßt mæifestation of 1996) y.o. stmozolol perfomed ECG QrS- severe underþing CHD- administration and T-wve inve¡sions in V¿-Ve and in II, II, and aVF. diffuse hypokinesis and (28Omglweek). Six Echocudiography revealed a dilated left vent¡icle with Patient was dischrged months prior to f¡actional ter. ions in the rþht with 200mg miodæone admission drug Corooary left anterior in addition to administraúon had coronary therapy including aspirin. been discontinued descending ætery. Lack ofperfusion in the apex was discove¡ed using thaJlium myocrdium No further follow-up is at rest and sluned AS as numbness, & (I4ochizuki el 32 Drug screen ûot aod, zøxlt cudiomegaly. aL,1988) y.o. weighdifær s teroids iDtermittendy perfomed since 16 y.o. Had used A CT scan and an EEG were found to be nomal. hemipresis and dysarthia' No follow-up reported a wide vriety of Dischrçd at 7 days wilh fight presented again because oF the sudden steroid s using multiple Four mónths after dischæç the patien! combinations io hþh onset ofleFt-sided facial drooping and hemipæesis hlpodensity in the am ot the right middle doses concomitantly. À CT scan displayed a læge ischaemic accident (CVA)' Oral compounds used cerebral ætery, which indicated an ischaemic ce¡ebrovascular left ventriculæ performance included An echocardiogram demonst¡ated severely impaired methândrostenolone consistent with cardiomyopathy without thrombus. piaoabol), 24 hour Holter recording showed frequent Premature arÀzl òepolaÀz'ødon aod rre oxaodrolone (Anavar), premature ventdculæ cont¡actions. on admissioo he was found to and stanozolol Àfter stabilisation he was refe¡red fo¡ ¡ehabilitatioa - have flaccid IeFt sided heniparesis md sensory loss, blood pressure of t24/80'hezrt flíinstrol) bruits or cudizc IntramusculælY rate 8ó bpm and regular. There was no evideoce of crotid with ooo-specific ST-T iniected compounds -*o.r, gailopt or rubs. An ECG showed sinus r\thm included nand¡olone wave aboormalides in the inferolatera] leads decanoate (Deca- Chest x-ray showed . Ie[t ventriculæ eiectioo Dmbolin) and Echocudiognm provided evidence of a severely reduced modentely severe nandrolone fraction with akinesi¿ of the anterior wall aod septum, late¡al wall' pheoylpropiooate hypokioesia of the infe¡ior wall and mild hypokinesia of the pwabolio). In Dóppler studies of the cæotid utedes showed uniml intimal thickeûing but no addition he reported stenosis or plaques. use of human chorionic gonadotropin and tmoxife¡.

Þ \¡¡ Diagnosis & deteils of Gender Exercise Status History of stercid Metabolites Detected Fatal/Non- Clinical det¿ils Aufhors follow-uP (r.øriebþ) & age use Fattl was obseryed in leads II, III on ECG exmination. Cædizc ¿l M¿Ie,61 Not reported but initizllv with Not relevant (non- Non faøl infrction wæ diagnosed from the ECG md echæædiographic was perfomed on day 48 lee4) y.o. presumably sedeatary gingivial bleedhg at abuse sinratioo) Acuæ myocudial following hospitalisation. which time frodings. Peak ialue ofcreatinine phosphokinase was 4,794 IU/l on day 2 of hospitalization' PancltopeM wæ Elling defects were obsered No follow-up reported noted. Detai.led No atllerosclerotic lesio¡s were obsewed, bul multiPle ends of these regions exminadons revealed in segments 1 -3 o[ the right corooary artery, and the distal aplastic anaemia for displayed delayed flow. which 45mg of Thãse Elling defects strongly suggested the preseoce of thrombi. methenolone enanthate vas prescribed þresumably on a weekly basis). No coronary risk factors, including or hypertension. PÍesented 3 motths later with severe

1990, and 1991 cathetetisation Female, Not reported, but Visited the outpatielt Not relevant (non- fzrzl Infactioa Qoymetal, infarction on the 24ò day of second 19e4) 59 y.o. presumably sedentary clìaic in August 1977 abue sin:ation) Fint were obseryed in leads II, III ¿nd hospital admission complaining of easy At the time oF her 6rst infæctioo ST elevations fatigability and aVF oo ECG analysis. The peak value of No follow-up reported Acute myæædial infrction of the inferio¡ wa.ll was diagnosed. Severe anæmia wæ serum creatinioe phosphokinase was 1,802IlJ /l- obseryed and the Second infa¡ction The peak value patient wæ admined Relapse of the inferio¡ wall was diagoosed based on ECG findings. She was treated with of serum cre¿tinioe phosphokinase wzs 7 07 llJ /l wa-ll. methenolone Iæft ventriculography revealed akinedc motion of the posterior basa-l left corooary aÍery' e¡anthate (100mg) Coronary arteriography showed no sclerotic lesions in the right coron^ry artery' Treatment vas Th¡ombotic filling defects were obsemed at segmenl 2 of the continued in 1982 with and a complete occlusion was seen beyood these lesions. orymetholone (30mg). No coronary risk factors excepl for steroid-induced glucoæ intolerance.

æI Authors Gender Exe¡cise Status History of stemid Metâbolites Detected Fatal/Non- & ape use Fatal for 7 yem with a CD4 count of400/m1 and a vi¡al Acute myocudial Not reported but 8 month history of Admitted with severe Non faul Patient * (aÅzle el al, Male,39 infæction 1999) y.o. presumably ædentary testosterone enanthate squeezing chest pain load of zero. (100 bPm) æd an (500mg, im.) localized to the Physica GIrv+) dePression and No follow-up reported administration every 2 midsternum o[ 4 hom audible th non-Q-vâYe weeks to prevent duntion. T-wve muscle mting (non- Concomit¿ot infæction. were u'ith pezks of 236 U physician approved) symptoms included Serial cratinine kinase (CK) and CK-Nß enzymes hþh, /l mild shortness o[ and 35 ng/dl, respectively at 24 hours. anæ¡ior wz'll breath, nausea and Two-dimensiooa.l echocudiography showed left ventricular diaphoresis. h1'pokinesia. A ãsting lipid profile on day-2 post admission revealed æflm total cholesterol of 272 mg/dl,low-density lipoproiein 767 mg/dl, hþh-density lipoprotein 't4 mgldl, and triglycerides 306 øg/ðL A subÃximz.l exercise st¡ess thallium scintigra.m revealed a conspicuous perfusion defect of the left veot¡iculæ anledor wall Coronary angiogrm showed a rupnrred plaque aod a læç, non-occlusive, supaimposed tluombus of the mid-left ante¡ior

\cI

a References Äppleby, M., Fishet, M. and Mattin, M. (1994). "Mlocardial Infarctìon, Lþtperkalaemia and Ventricular Tàchlcardia in aYoangMak Bod1-Bøilder'" IntJ Cardiol 44:171'-1'74'

Bowmafi, S. (1990). "Anabolic steroids and Infarction [Lttt]." Br MedJ 300: 750.

'$Øeber, Cambell, S. E., Farb, A. and K. T. (1993). "Pathologic Renodelling of the þocardiartt in a lY/eþtlìfter TakingAnabolic Steroids." Blood Pressure 2: 21'3-21'6'

Dickerman, R. D., Schaller, F., Prathet, I. and McConathy, Nø. J. (1995) . "Sødden Cardiac Death in a 2q-Year' O td BoQtbuitdrr (Jsing Anabolic S teroids. " Cardiology 86(2): 1'7 2-3 '

Ferenchick, G. S. and Adelman, S. (1992). "Mlocardial Infarction As¡odated with Anabolic Steroid Use in a Preuioasþ Heahþt 37-Year-Old lf/eight Lifer." Am FIeart J 72aQ): 507-8'

Fisher, M., Àppleby, M., Ritroo, D. and Cotter, L. (1,996). "þocardial Infarction with Extensiue Intracoronary 222-3. Thrombu¡ Induied b1 Anabotic Steroids [See Conruents]." 8r J Clin Ptact 50(4):

Flausmarin, R., Flammer, S. and Befz,P. (1993). "Perforrnance EnhancingDrags (DopingAgenß) and Sadden Death--a Case RepoX and Reuiew of the Litratare." IfItJ Legal Med 111(5): 261'-4.

Steroid User." Med Sci Sports Exerc Huie, M. J. (,9g4). "An Acate Mlocardial Infarction Occurring in an Anabolic 26(4):408-13.

I(ennedy, C. (1993). "Mlocardiallnførction inAssociation with Misure of Anabolic Steroids'"IJlstet ll/cdJ 62(2): 1.74-6.

I(ennedy, M. C., Cordgan, A. B. and Pilbeam, S. T. (1993a) . "MlocardialInfarction ønd Cerebral Haemorrhage in a YoangBoþ Builfur TakingAnabolic Steroids [.tto]." Aust NZ J Med 23(6):713-

I(ennedy, M. C. and Lawrence, C. (1993b). "Anabolic SnroidAbate and Cardiac Death." MedJ Aust 158: 346- 348.

a lYeìght Lifter [Jsing Anabolic Androgenic Snroids: Luke, J. L. (1990). "Sud¿4a Cardiac Death Daring Exercise in Pathotogical and Toxicological Fìndings." J Forensic Sci 35(6): 14+1-1'441 '

McNutt, R. A., Ferenchick, G. S., I{irlin, P. C. and Flamlin, N. J. (198S). "Acate Mlocardial Infarction in a 22- Year-OldlYorld class lYeþhtLiJterUsingAnabolic steroid." AmJ cardiol62:1'64.

Mewis, C., Spyridopoulos, I., I(uhlkamp, V. and Seipel, L. (1996). "Manifestations of Seuere Coronary Heart Disease afrer Anabolic DragAbuse." Clin Cardiol 19: 153-155'

Mochizuki, R. M. and Richter, K. J. (1983). "Cørdiomlopathlt and Cerebrouascular Accid¿nt Associated with Anabo lic-Androgenic S teroid Ura. " Physician Sports Med 16(1 1) : 1.09 -71' 4.

Toyama,M., Watanabe, S., I{obayashi, T., Iida, I{., I{oseki, S., Yamaguchi, I. and Sugishita, Y. (1994)' "Two Steroids." Cases of Acute Mlocardiat Infarction'Associated with Aplastic Anemìa DaringTreatment with Anabolic Jpn HeartJ 35(3):369-73.

yarnale,p.,IÁ",zar,M. and Sedighi, A. (1999). "Acate Mlocardìat Infarction A:¡ociated with Anabolic Steroids in a Young Hiu-Infected Patient." Phatmacoth erapy 19(): 881-884'

A-L0 Reported histological changes in laboratory animals administered anabolic steroids

B-1. Author/date Tre¿tmcnt Regimc Specics / strain/scx Obscn'rnccs

Rag SD, male Liver ad¡enal weight significantly decreased ln all exercised gfouPS compred to controls. @zummet aL,7988) Cont¡ol: no exercise, protein wâs sigrLihcantly less 1n rats ¿dministered steroid supplementatioq or s teroid Testicular weight to control. 30 min exercise (3xs/week), no protein Heart weight wâs significandy gfeatef n steroid treâted râts compared rats supplementation, no steroid. . Hemtoxylin and eosin-staining of Iiver, kidneys, adremls, heart and testes from treated 30 min exercise (3xs/week), 76.6ok w /w reve¿led no significant pathology. protein supplemmg no steroid J0 min exercise (3xs/week), no ProtÉir supplementaúon, s tanozolol (5mg, i.m./3xs/week). 30 min exercise (3xs/week), 16.6o/o w /w protein supplement, stânozolol (5m& i.m./3xs/week). revealed mcfeâse sized, filaments in 1977) Meùrandrostenaolone (Dianabol@) Rat, Wistzr, female Electron ñcfoscoPy @ehrendq treated fâts compared to control. (1.65mg/kg/week/1 3 weeks). muscle cells of the left cardiac ventricle n steroid Control: sesame seed oil F,lectron ñcfoscoPy found that the mitochondria and myofibrils from heart muscle celìs 1n et aL,1.977 Methandrostenaolone (Dianabol@) Raq Wistar, femle @ehreîdt to the morphology changes observed n (1.65mg/kg/week/3 weeks) steroid treâted râts showed swelling and elongation similar Control: sesame seed oil exly heart faiìure. The mâtfl\ wâs sPafse with significandy reduced cflstae. The myofibrils showed either and widened and twisted Z-ba:nds of ù dissolution of the safcomeflc utuß and Rat, Wistar, roIe and female Herotoxylin and eosin-stained slides of the and gastrocflemlus from m¿le @isschop et aL,7997) Nandrolone dec¿noate (1.5 or 7.5mg/kg significant lncreâse 1fl g?e IIx/b dimensions i.m /rveek/5 weeks). female râ ts demonstrated that nand¡olone caused L in both sexes. Cont¡ol: saline Nandrolone caused an inc¡ease n I fibre dimensions in the acutely treated rats (0.5-4 i.p. fot 12 Rat, SD, male Hepatic cytochrome P450 and b5 content increased significandy for @oadz et a1.,1999) Stanozolol (5mg/kg; /dzy) hous, or 1,2,3,4,60,90 ðays. days) but signifrcandy decreased at days ó0 and 90. . No .if"at *". found on sem transaminâse enzymes in any of the groups, eidrer in âcute or ch¡onic studies. . Light microscopic evaluation of H/E, stai¡ed liver sections f¡om animals zt dlly 3 znd 4 of treatrneit revealed .þht to -od"."te mulúfocal lobular inflammation with acidophlic degeneration and evident Kuppfer cells reactrvity o[ the liver tissue compared to control groups- These changes were not evident in rats t¡eated for shorter periods of time. . Cytoplasmic vacuolizaúon and Iipidic degeneration was evident in the mjority of the chronically t eated rats Increased mitosis and binucleation and variability in the size of cell nuclei was seen in 90 days treâted nts but only in 3 hvers from 60 day treated râts Eleven from short-tem and long-term treated animals showed a subcapsular inflamtory lesion (xanogranulom) and an evident enlargment and hyalinosis of centrolobular vein wall Light mcfoscoPy detected no ffilo1 n the liver et ø1,7971) Oxandrolone or testosterone administered Rag SD, ferole ft{oruath alterations 1n oxand¡olone ât 1mg if, 1ml of water by stomch n¡be, Electron frcfoscoPy detected significant endoplasmic re dculum @R) could hardly be seen' for 3 days. Rats were killed 16 hours after treated rats. In some hepâtocytes, no¡rnal rough-surfâced-ER few ribosomes attached the last dose on day 4. . The cistemâe in the hepatocytes were extremely dilated and had only a to them- ' Oxandrolone caused wide variations in tire size and fo¡m of the mitochondria . Testosterone produced only nild changes in hePatocytes. The rough-surfaced-ER was conspicuous but haà a normal ultrast¡uctufe. The compaftrnent ol the cytoplasm ocopied by the smooth-surfaced-ER rvas than in norm¿l midzonal

EË I Obsen'rnccs Au thor/ tlrtc Trertment Rcgimc strri¡r/ scx wâs stained Hematoxylin md eosn and exmined for (senget aL,7994) Nandrolone (ZOng/kg, s.c. / dzy / l-6 R¿t, SH& Male Vmüicular ùssue grading scale was used by z blinded obserer. One weeks) or com oil control. damge by light microscopy. Â semi-quantaúve this scale O=no obvious pathologr, l=increased diffixe cellularity aromd great vessels of heart valves, 2=7 5 small focal afas of cellula¡ dzmge with inflamtory cellular ot 6brotic infiltratior\ 3=mofe than 5 such afeas, 4=presence of at least one large lesion (greater than one-tÏird of the thickness of the chamber w¿Il at that Point), a¡d 5=more than one large lesron. . Nand¡olone treateã rats were fomd to hâve greater myocardial damaç than cof,trol treated rats .83+0.4 vs. 2.83+0.17 female . Treated males and females showed no obvious peliosis or hepatic dysplasia' (Yu-Yahiro et aL,7989) Either 0.5cc of saline or nandrolone Rag Wisør, Male md . B and Oil Red O staining for lipids showed th¿t male but not fem¿le treated livers decanoate 150mg/ml of approximtely Sudan Bl¿ck 10Orng/kg). had much lowed lipid content than those of control. . ,{11 females had abnormal vacuol.ization, stromal oedema, and peliosis of the uteri' ' No pathologic changes were obsered in testes, sPifle, ovaries or muscle' . Theie were no differences in DN,A, or RNA content in any tissues, as measwed by methyl green/pyronin staining. Z ø'" separate gioup of all mle raß, where half were treated with nand¡olone and half with control, testes and liver were found to be signifrcantly heavier in the control versus the treated grouP. Kidneys were heavier in the keated nts versus the control.

t References

Bauman, D. H., Richerson, J. T. and Britt, A. L. (1988). "A Comparison of Bodl and Organ lØeigbtl phlsiologzc parameters, and Patbologic Changes in Target Organs of Rnts Giaen Combinations of Exercise, Anabolic Hor*oor, and Protein Saþlenentution." ÌrmJ Sports Med 16(4): 397 -402'

Behrendt, H.(Ig77)."EfrrtofAnabolicSteroidsonRatHeartMascleCe//s." CellTissRes180(303-315)'

Behrendt, H. and Boffin, H. (1977). "þtocardial Ce// l-,esions Caused b1 an Anabolic Horrnone." Cell Tiss Res 1.8L: 423-426.

Bisschop, A., Gzyan-Ramitez, G., Rollier, H., Dekhuij zen'P. N'' Dom' R', de Bock, V' and Dectamet, M. (1997). "Efects of Nandrolone Decanoate on Respiratory and Peripheral Muscles in Male and Female Rat.'." J AppI Physiol 82(4): 1712-8.

Boada,L.D., zambado,M., Torres, S., Lopez,r\., Diaz-Chico, B. N., Cabrera,J'J. andluzatdo, o. P' (1999). ,,Eualaøtion of Acate and Chronic ftEànn*lc n¡ects Exerted þtAnabolic-Androgenic Steroid Startololol ìn Adalt Male R.at:." Ârch Toxicol73: 465-472'

Indaced in tbe FIorvath, E., I(ovacs, I(, Blascheck, J. A. and Somogyi, ,\. (1971). "(Jltrastruclaral Changes Liuer of Rnt: b1t Varioas Steroid Compoands." Virchows Arch B Cell Pathol 7 (4): 3a8-55.

Tseng, y.T., Rockhold, R.ìø., Floskins, B. and Ho, I. K. (1994). "CardiouascalarToxicities of Nandro/one and ðocaine in Sponlaneousþ Hyertensiue Rats." Fundam Appl ToxicoI22(1): 1'13-21'

and Schofield, B. (1989). "Morphologic and Histologzc yu-yahiro, J. 4., Michael, R. FI., Nasrallah, D. V. Abnorrnalitìu in Fernale and Male Rats Treated with Anabolic S teroids." A- J Sports Med 1'7 (5): 686-9 .

B,-4 ûfl"*'oh>e e

Pilot study: chronic efTect of nartdrolone in telemetered raß

c-1 C.l Introduction The ability of AS to produce hlpertension remains controvetsial and confusing. Howevet,Tseng et of at (1.994) found that nandrolone decano ate Q\mg/kg, s.c., daily) accelerated the development hypertension in young, spontâneously hypertensive rats in comparison to vehicle treated conttols clpionate (Tseng et a/., 1,994). Fischer and Swain injected casttated male rats with testostetone (1mg/wk, i.m.) and found that after 3 weeks of treatment, BP had increased significandy in steroid subjects, pre-treated rats in comparison to vehicle treated controls (Fischer et a/',1'977).In human abusing Rrebe and associates measured BP at rest and during a standatdised exercise test in AS They found weight liftets, in thei¡ drug free associates and in sedentary conttols (Riebe et a1.,1992)' that the AS abusing group had signifìcantly higher SP at test and during exercise. Despite these number of investigations studies suggesting a causal link between hypertension and '{S a significant et a/.,1'974; Mcl(llop in both tats (Brown et a/.,1.972;Wolinsky, 1972) andhuman subiects (olsson et 1'992) have failed to find any et a1.,1986;Urhausen et a/.,1989;Fdedl et ø1.,1'990; Thompson a/.'

evidence of AS related BP changes.

nandrolone It was decided to use radiotelemetry to investigate whether chronically administeted implants available could induce hypertension in AW rats. Because of the small number of telemetry for monitoring BP each rat served as its own control'

C.2 Aim significant change in To detetmine whether non-anaesthetised, freely moving Aw rats display a HR, BP and SI-A ovef 9 weeks of treatment with nandrolone Q}mg/kg, s.c).

C.3 Hypothesis BP at the chronic tfeâtment with nandrolone decanoate will result in a significant increase in rate and SI,l\ will conclusion of 9 weeks of treatment compared to the Pfe-tfeatment period' Heart

be unchanged by nandrolone tteatment'

c-2 C.4 Method

C.4.1 Animals

Albino-NØist21t r^ts were purchased at 12 weeks and were housed according to General Methods 2.1. Rats were anaesthetised (2.3) and surgically implanted with Data-Sciences blood pressure implants (2.2) as per 2.4.2. Post-operative câre was caried out following the details outlined in 2.5

C.4.2 Treatment

Rats were treated for 9 weeks with nandrolone decanoate Q\mg/kg, s.c.) 3 days a week (Àdonday, Wednesday, Friday).

C.4.3 Protocol

Rats were injected subcutaneously with nandrolone (2}mg/kg, s.c') and returned to thei¡ home

cage for 60 minutes. At the conclusion of this acclimatisation petiod, telemetry recordings of HR, SI-A and BP were made for 60 minutes. This protocol was conducted 3 times weekly (À4onday,

'tJ7ednesday and Friday) and continued fot 9 weeks (61 days)'

C.4.4 DataAnaþsis initiation All parametefs wefe gtaphed as functions of time (d"yÐ. The HR, BP and SI-A before of treatment was compâted to the value of these vadables on the final day of nandrolone administration using a Student's t-test.

c-3 C.5 Results

C.5.1 Changes in bodl weight

Rats weighed 571,.6+22g at the beginning of the treatment period. By the 9'r'week of treatment weight had decreas eð,by 74!139 (Fic. c. 1). Body weight decteased significantly at day 61 compared to day 1 [t(8)=2.595, p<0.05] (rig. c. t inset).

C. 5. 2 C h ange s in cardio uas cu lar þaram e te rs Although Sp and DP appeared to decrease slightly between day 38 and day 61, no statistically significant changes were evident between day 1 and 61 of tteatment (fig. C. Z).

C. 5 3 C hangu in cardioaasca lar þaram eters Iìkewise, no significant changes in SLA were evident between the start and conclusion of nandrolone tfeatment. Spontaneous locomotot activity peaked between day 20 and 30 of treatment (fig. C. 3). This corresponded to a peak in HR, which was also evident between 20 and 30 days (fig. C. 2). No significant changes were evident in HR between the beginning and end of nandrolone treatment.

c-4 * 60

o ¡ .9 57 o 525 = 500 cD J sso 61 c¡) Time (days) o = 500

47 0 10 20 30 40 50 60 Time (days)

Fig. C. 1 Body weight following a 3xs weekly administration of nandrolone (20mg/kg, s.c.). *p<0.05 Iniet: \Øeight ¿t start and conclusion of treatment. significaotly diffetent from day 61, unpaired t-test' Mean+S.E., n=5

17 50

1 5 ct) n E 1 2 0 Ð E o 10 o cr ¡- 75 ! o 7 3 an 50 L.o o- 50 5

0 0 10 20 30 40 50 60 Time (days)

Fig. C. 2 Diastolic (Â) and systolic pressure (l), and heaft rate (V) in rats treated with nandrolone (20mg/kg' s.c.) for 9 weeks. Mean*S'E', n=5

c-5 12

10 oø F +,u, o o

2

0 10 20 30 40 50 60 70 Time (days)

Fig. C. 3 Spontaneous locomotor activity (SLA) response to nandrolone treatment (2Ûmglkg, s.c.) over 61 days. MeantS.E., n=5

c-6 C.6 Key Findings a No signifìcant hypetensive effect of nandrolorìe was obsewed during 9 weeks of high dose treatment.

. The peak in SI-A and HR that developed between day 20 and 30 of nandrolone treatment requires futther investigation. This change was not evident in SP and DP. a Nandrolone sþifìcantly reduced total body weight. It is unknown whether this teflects

changes i¡ water composirion, protein or fat disposition'

C.7 Future work

. In order to propedy chatacterise the peak in SLA and HR between days 20 and 30 of treatment this study needs to be repeated with the apptopdate vehicle control animals.

c-7 C.8 References

Brown, B. S. and Pilch, Â. H. (1.972). "The Efects of Exercise ønd Dianabol Upon Selected Perforrnances and Pþtsiotogical Parømeters in the Male Røt." Med Sci Sports Exet 4(3): 159-65'

Fischer, G. M. and Swain, M. L. (1,977). "Eftrt of Sex Horvtones on Blood Pressare and Vascalar Connectiue Tìssue in Ca¡trated and Noncastrated Male l

Liþoprotein Fdedl, K. E., Flannan, C. J., Jones, R. E. and Plymate, S. R. (1990)' "High'Densii 69-7 4. Cholesterol Is Not Deneared If an Aromatiryble Androgen Is Adninistered." Metabolism 39(1):

Mcl{llop, G., Todd, I. C. and Ballantyne, D. (1986). "Increased l-.eft Ventricalar Mass in a Bodlbuildtr [JsingAnabotic Steroids." BrJ Sports Med 20(4): 1'57-2'

Olsson, Ä. G., Oro, L. and Rossner, S. (1974), "Efects of Oxandrolone on Pla:ma Uþoþroteins and the Intrauenous Fat Tolerance in Man." Âtherosclerosis 19(2): 337 -46'

Riebe, D., Fernhall, B. and Thompson, P. D. (1992). "The Blood Pressure Resþonrc to Exercise in Anabolic Steroid [Jsers." Med Sci Spotts Exerc 24(6): 633-7 '

Thompson, P.D., Sadariantz,A., Cullinane, E. M., Bodziony, I( S., Catlin, D. H., Torek-Both, G' Use Anabolic and Dãughs, p. S. (Ig92). "I-.tft Ventricular Function Is Not Irnpaired in lV/eiþt Ufter: lVho Steroids."J A- Coll Cardiol 19:278-282'

Tseng, Y. T., Rockhold, R. W., Hoskins, B. and FIo, I. K. (1994). "Cardìouascalar Toxicities of Nanlrolone and cocaine in spontaneousþ H1þerteniue -I " Fundam Âppl Toxicol 22(1): 11'3-21'

IJrhausen, 4., Flolpes, R. and l(indermann, W. (19S9). "One-and Two-Dimeusional Echocardiogr@@ in Boþbaitderc (JsingAnabolic Steroidr." E*J Appl Physiol 58: 633-640'

2552- Wolinsþ, H. (1.g72). "Efects of And.rogen Treatmsnt on the Male Rat Aota."J-Clin-Invest 51(10): 5.

c-8 ûff"-r/'h Ð

Pilot study: extarteuronal noradrenaline uptake in rat heart slices

D-1 D.l Intoduction

Bönisch et al derrronstrated good uptake of isoprenaline ftom isolated sections of rat aofia and isolated 20mg suips of venfficle @onisch ø a/.,1,974). Babin-Ebell and Gliese demonstrated that human arrium displayed strong uptake-2, which was sensitive to inhibition by O-methyl- isoptenaline (OMÐ (Babin-Ebell et a/.,1.995). The investigation of Babin-Ebell et alwas the first study to show that uptake-2 occurs in human heatt ttssue'

In search of a simple method for investigating the ability of nandtolone to inhibit extrâneuronâl noradrenaline reuptake it was decided to use slices of isolated tat ventricle and attia. It was hoped that large numbers of slices could be generated ftom each heart, minimising the number of animals used and allowing an efficient generation of data.

D.2 Aim

¡ To establish a simple method using isolated rat ventriculat f atnal slices and known inhibitors of extraneuronal noradrenaline (cotticostetone and O-methylisoptenaline{OMI}) to inhibit the uptake of ffq-NÄ. a To compare the uptake inhibition produced with nandtolone to that induced by corticosterone using this method

D.3 Hypothesis

Nandrolone will inhibit the extraneuronal reuptake of ff¡-NA ln rat ventrìculff slices with a sirnilar potency to corticostetone.

D-2 D.4 Method

Rats wete anaesthetised with halothane, decapitated, the heat exteriorised and excised into ice- cold, pre-carbogen bubbled, calcium-free-I{rebs solution. The tissue of interest was then isolated.

Left ventricles were cut with a scalpel into slices of 20+2mg.,\tria were cut in halves longitudinally. Tissues were then weighed on paraffin film and pre-incubated in 2mls of lSebs containing

nialamide (350pN! for t hour to inhibit monoamine oxidase (À4,q.O). Tissues were then further

incubated in nialamide-free solution, in the presence of cocaine (Z7ttW and'U-0521' (50pN! to inhibit uptake-1 and COMT respectively and in the presence or absence of an uptake-2 inhibitor (corticosterone or O-methyl-isoprenaline) at various concentrations. Finally tissues were incubated 3H-N,{. for 30 minutes in (see results for concentration) still in the ptesence of inhibitors of NA re-

uptake and metabolism.

3H-N4., At the conclusion of the incubation with tissues were tinsed for 5s in ice-cold-I{rebs to remove extracellular 'H-NA before being transfetted to 5ml tubes containing 2nl' of HCI (0.11VÐ. Radioactivity wâs extrâcted overnight at 4-6"C. One ml of supernatant was then transferred to 5ml of HI-S,\FE scintillation fluid for scintillation counting. Uptake was calculated according to the following formula...

uptake = [(sr _a)'zf*!p*otr*g -2 L s,l ) wt.'

ST = radioactivity associated with 1ml of supernatant (DPM - blank) DPM -blank SA = specifìc activity (sA = -ffi|)

Iù(/t = weight (-g)

D-3 D.5 Results

To ensure that the protocol was sufficiently robust to detect noradrenaline uptake it was decided to examine the blockade of uptake-1 by cocaine in rat venüicular slices in the absence of uptake-2 3H-NA inhibition. Reuptake of (l.splvÐ was found to be inhibited by 680/o in ventricuiar slices incubated with cocaine (27¡tN\ in comparison to vehicle (saline) tteated conttols (Fig. D. 1)' 3H-NA Uptake associated with left ventricle incubated with corticosterone (25p\4 and (27¡ll$ was not found to be significantly different from conttol. Sirnilady, there was no difference in the uptake of 3H-NA frorn attia and ventricle incubated with corticosterone (85fVÐ ot vehicle when the 3H-NA incubating concentration of was 1.8¡-rM. No effect of corticosterone was found on ventricular slices, even with a high ratio of inhibitor:NA (eg. 85pM corticosterone: 8.9mM NA)' reduce uptake in nt attial However, corticosterone (85 ¡.r1\4) was found to significantly fffl-Na was 22o/o. Similarly OMI (100¡r$ tissue [t(6)=2.669, p<0.05]. The magnitude of the teduction reduced ff!-NA uptake in atrial tissue by 32o/" [t(6)=3.187, p<0'05]'

D-4 # Tissue Noradtenaline n Inhibitor cocarne Uptake (pmol/mg) (nmol/ml) (nmol/ml) (% inhibition) 7. Venuicle 1.80 4 (+) 1.1010.12xx* 4 c) 3.43t0.26

2. Ventricle 27 + coRT (2s) (+) 1.1.72+0.65 (6.65%)

4 VE,HICLE (+) 12.55+0.81

3. Ventricle 1.80 4 coRT (85) (+) 1.01+0.05 (+t.e3%)

4 VEHICLE, (+) 0.97+0.10

4. Atria 1.80 4 coRT (8s) (+) 0.79+0.11 (11.u%)

4 VEHICLE, (+) 0.89+0,03

a 5. Ventricle 8.9x10-3 4 coRT (8s) (+) 6.87x10 3 + 3. 4x10 (5.48%) t 4 VEHICLE, (+) 6.5x10 + 1..2*L0'

3 6. Atda 8.9x10-3 4 coRT (8s) (+) 6.99x10 + 3.4x10r# (22.00%)

4 VF,HICLE (+) 8.94x103 + 6.5x104

l. ,{úra 8.9x10-3 4 oMr (100) (+) 5,44x1.03 + 4.7x1.0+# (t2%)

4 VEHICLE (+) 8.03x103 * 6.7x104

3H-NA O- Fig. D. I uptake in various isolated rat tissues. Legend: CORT = cordcostetone, OMI = mlhyl-isopr..r"line. ***p<0.001 signihcantly different from control (no cocaine), #p<0.05 sþifrcantly different from vehicle, unpaired t-test.

D-5 D.6 Key findinss a Inhibition of extraneuronal uptake (uptake-2) of ffq-NA was only demonstrated in atia with a high inhibitor concentration and a low noradrenaline concenttation' The magnitude of this inhibition is signifìcantly less than that repotted in isolated perfused rat heatts. Salt et al report exrraneuïonal inhibition of ffq-NA of greater than 85o/o with similar inhibitot concentrations in isolated perfused hearts (Salt, 1'972)' o Although exrraneuronal reuptake of ffq-NA could be detected ushg this method it could not be appteciably inhibited in ventricular slices.

D.7 LIMITATIONS a The difficulty of inhibiting fH]-NA reuptake with common uptake-2 inhibitors makes this an unsuitable model in which to investigate the potential ability of nandrolone to inhibit he extfaneufonal reuptake. Petsonal com¡nunication with H. Bonisch (2001) confumed that had also been able to show good uptake-2 in ventricular slices, but was unable to inhibit

this uptake with comrnon uptake-2 inhibitots. These results were never published.

D-6 D.l References

of Noradrenaline in Haman Tissue (Uptake- Babin-Ebell, J. and Gliese, M. (1995). "Extrønearonal Uptake 2)." HeattVessels 10: 151-153.

Bonisch, H., Uhlig, W. and Trendelenburg, U. (1,974). "Anaþsis of the Coraparttnents lnuolued in the Arch Extraneuronal Sørage and. Metabolitn of Imþrenaline in the Pefused Heød. " Naunyn Schmiedeburgs Pharmacol 283: 223-244.

p. 2 in the InÌatedRat Heart Steroid:, Clonidine and Salt, J. (,g72). "Inhibition of Noradrenaline Uptake fui 20 : 329 - 40' Me t h o x1 late d P h e rE k t ly tan in e s. " F;wr J Pharmacol Q)

D-7. ûff"-r/,tn e

EfTect of anabolic steroids in the CIVS

E-1 Re r Evidence for a CNS effect Mictomolat concentrâtions of stânozolol and 17cr-rnethyltestostetone (17,ct-MT) have been GABA rat shown to signifrcantly inhibit binding of pt! þ' GABA¡ recePtor ligand) to the brain cerebrocortical membtanes (I\4asonis e t a/., 1'99 5)'

17, cr-MT, stanozolol and nandrolone have been found lo induce rapid and reversible modulation of GABAergic cu.rfents in neutons of the vent¡omedial nucleus of the et a/.,2000) hypothalamus O¡MN) and the medial pre-optic area (mPO,\) fiorge-Riveta

Dopamine

activiry could be detected'

and the ventral tegmental area. undecylenate) 14 days treatmeniwith an,\S cocktail (nandrolone, testostefone and boldenone atcuate nucleus sigruFicantly decreased p-endorphin immunoreactivity in the rostral area of the in the rat brain (Nlenatd eî a/.,1995).

treatment of male SD rats v¡ith nandtolone decanoate (5 or -Iímg/kg,i.m./dany)Johansson et alfou¡dthat1,4 days induced a 2}-fold increase in p-endorphin in the ventral tegmental ¡tea the highest dose of nandrolone t et al', 1997) ' ryfá "i flohansso

NMDA

Glucocorticoid

nandrolone (l1rrrg/kg/daÐ to mâle Sprague Dawley Substance P tly enhanced SP immunoreactiviry in the amygdala, erg et a/.,2000). Drug withd¡awal did not revetse lhese changes in the PÄG.

E-2 References

Ahima, R. S. and Harlan, R. E. (1992) . "Regulation of G/ucocorticoid Receptor Immanoreactiuitlt in the Røt Hþþocanþas b1 Androge nic-Anabolic s teroids." Brain Res 585 (1 -2) : 311-4.

Hallberg, M.,Johansson, P., I(indlundh, A. M. S. and Nyberg, F. (2000). "AnabolicAndrogenic Steroid¡ zlfex thtr Contint of Sabstance P and Sabstance P|-7 in the Rat Brain." Peptides 2l: 845-852.

P., Halbetg, M.,I{indlundh, A. and Nyberg, F' (2000)' "The Efect of "RatJohansson, Brain, after ChronilTreatment with the Anabolic Androgenic Steroid, Nandrolone D Bull5l(5): +1,3-478.

P., Ray, A., Zhou, Q', Huang, W', IG1l1on,.I(' and Nyberg, tr' (1997)' "Anabolic 'AndrogeniiJohansson, Snroids fnneise Beta-Endntrphin l-tuets in the Ventral Tegnental Area in the Male Rat Brain'" Neurosci Res 27(2): 185-9'

L. P. (2000) "Anabolic Steroids Indace Region-and Jorge-River^,J.C.,Mclntyre, I( L. and Flenderson, ' "Suian;t-Specfií Rapid Mod.aíation of Gaba-a Receþtor Mediated Cørrents ia tfu Røt Forebrain'" J Neutophysi ol 83: 3299-3309 .

I(indlundh, A. M., Lindblom, J., Bergstrom, L. and Nybetg, F. (2003). "The Anabo Receþtors in SteroidNand.rolone IndøcesAlterations in the Densi[t of Serotonergic 5htlb and 5ht2 Brain." Neuroscience 119(1): 1'73-20.

I{indlundh,4.,M.S., Lindblom,J., Bergstrom, L., Wikbetg,J.E.S.and Nybetg,F. (2001). "The Receptors in the Male Rat Anabolic-Androþù Steroid Nandroline Decanoate Aftcts the Densiry'of Doþamine Brain." Eut J Neuro sct 73: 291'-296'

and Nyberg' F (1997)' "Ef,ects of Le Gteves, P., Fluang, W', Johansson, P., Thornwall, M',Zhot, Q' and Nr2b Søbanit Mrnas an Anabolic-Aort ogrri'Strrid on the Regutation of the Nnda Receptor Nrl, Nr2a in Brain Regions of the Male Rø1" Neutosci Lett 226(1): 61'-4'

Masonis, A. E. and McCatthy, M. P. (1995). "Direct Efects of the A steroids, Acid(a) stonoloút and. 17 Aþha-Methltltistosterone, on Ben:lodialepine Binding to the G Receþtor." Neutosci Lett 189(1): 35-8'

Steroids Menard, C. S., Hebett, T.J., Dohanich, G. P. and Hadan, R. E. (1995) ' "Androgenic-Anabolic Modfu Beta-Endorþhin Imminoreactiuiry in the Rat Brain," Brain Res 669(2): 255-62'

Thiblin, I., Finn, 4., Ross, S. B. and Stenfots, C. (1999). "Increased Doþaninergic and 5- ctiyities in Male Rct Brain Following l-,ong-Terrn Treatment with Anabolic Androgenic ol126(6): 1'301'-6.

E-3 qrf-'/,;rn f

EÍTect of nandrolone on plasma ET-I

F-1 F.l Introduction In yiuo rat models have demonsttated that intracoroî^ry Endothelin-1 (ET-l) can câuse catdiac arrhythmia in the absence of cardiac ischaemia (Szabo et a/., 2000). Conversely, pre-proET-l oligodeoxynucleotide (i.v.) has been shown to be ptotective against VT and VF 'RNA antisense suwival during cardiac arrhythmia induced by I-AD occlusion tesulting in a signifìcant increase in that testosterone may þin et a/., 2002). Recently evidence has begun to emerge which suggests et a/., 2000)' increase the vasoconstrictive effect of ET-1 on porcine coronâry vasculature (Teoh These effects were found to be endothelium and andtogen receptor independent. If nandrolone increase in can also inctease the vasoconstrictive effect of ET-1 this may explain the significant Lambeth arhythmia score during ischaemia in rats treated with nandtolone compared to vehicle

treated animals.

F.2 Aim

To determine the plasma concefltÍation of ET-l in post-repetfusion animals

F.3 Hypothesis plasma ET-1 concentration will be significantly increased in nandtolone treated tats compated to

vehicle treated animals.

F.4 Method from the Following the ischaemia-reperfusion procedure in chapter 5, 3-5ml of blood was collected teperfusion abdominal aorta into heparinised eppendorf tubes from rats which survived cardiac centrifuged and and had been treated with either vehicle or nandrolone (80pg/kg/-i"). Blood was assayed the plasma fractionated off into 0.5m1 aliquots and frozenert-20"C until assay. Plasmawas

for ET-1 using a radioimmunoâssay method as detailed elsewhere (Zhang et a/',1998)'

The significance of differences between the plasma concentration of ET-1 in nandrolone and vehicle treated animals was determined using a Student's unpaired t-test.

F.5 Results No significant difference was found between the plasma concenttation of ET-1 in nandrolone and vehicle treated animals (Fig. F. 1)

F-2 Ivehicle I nandrolone (80¡rg/kg/min)

â E cD CL F-I ul IE E o g 2 o-

vehicle nandrolone

Fig. F. 1 plasma ET-1 in rats administered vehicle or nandrolone (80Ug/kglmin) following thã concf usion of ischaemia/reperfusion. Mean*S.E., ¡=5-6. Between grouP differences: unpaired Student's t-test: no sþificant difference.

F.6 Conclusions & Future Directions Although no significant change in plasma ET-1 concenttation was noted in the ptesent study, it is possible that any increases in ET-1 which occutted during ischaemia had returned to notmal by the time that blood was collected. Collection of blood at time points throughout ischaemia may help whether nandrolone c î to increase ET-1 concenttation and thus cause ân increase determine ^ct in ordet to test whethet nandtolone c tr to enhance in arrhythmia. Further studies are required ^ct the vasoconsuictive effect of ET-1 in isolated rat cotonzrty vessels, in a man¡er similat to that of

testosterone in porcine coronary artery rings (Ieoh et al.' 2000)'

F-3 F.7 References

Lin, L, and Yuan, Nø. (2002). "InuoÌuement of Endothelin-| in Acute Ischemic Arrhlthmias in Cats andRøts." Clin Sci 103(Suppl. 48): 228s-232s.

Szabo, T., Geller, L., Merkley, B., Selmeci, L.,Juhasz-Nagy,A. and Solti, F. (2000). "Inautigøtingthe Dual Nature of Endothe/in-l: Ischaemia or Direct Arrhlthmogenic Efect?" Life Sci 66Q6): 2527-2541'.

Teoh, FI., Quan,,{., Leung, S. Nø. S. and Man, R. Y. K. (2000). "DffirentialEfects of lTbeta-E¡tradiol and.Testo:terone on the Contractile Rr,Eonsrc of Porcirue Coronary Arterie:." BtJ PharmacoIl29:1301-1308.

Zinang, Y., Oliver, J. R. and Horowitz, J. D. (1998). "Endothelin B Recepror-Mediated Va¡oconstriction 39: 665-673' Indand fu Enùthelin a ReceþtorAntagonist." Cardiovasc Res

F-4 ûff--ol,;e f

G-1 A. SP response to cocaine infusion >eS+G +0N+G +40N+C +0N+S -l.-40N+S

f6 gE o- an

100 200 300 400 500 600 Time (s) $ . Pr"""or response over first I 00s of infusion Q. Depressor Response from 100-600s of infusion

Et E' E T E E E E o- o- at, Hry an -1

-1

20 40 60 80 100 00 200 300 400 500 600 Time (s) Time (s)

Fig G. 1 Change in A. systolic pressure (SP) relative to the value at 40s prior to the stalt of infusion of cocaine (0.5mg/kg/min). B. The pressor response to cocaine (first 1O0s of coca¡ne infusion). C. The depressor response to cocaine (100-600s of cocaine infusion). Legend: X ¡aline (00¡tt/ n;n) * nmine (05ng/ kg/ nin) fS+Cl, lnandrolone uehicle (}p//nin) t comine [ON+C], o +nandrolone nantlrolone ft7pg/ kg/nin) * comim [40N+C], . nandrolone uehick (}¡tl/ nin) + saline (00¡t// nin) [ON+S],

(T¡tg/ kg/nn)* ¡aline [40N+S]. The error bars have been removed from figutes B. & C. to tetain clarity fot symbols showing sþificance levels. Repeated-measures--ANOVA, Bonfetroni's post-hoc tesr. The line at x=100s represents the end of the pressor response to cocaine. B. SP (0-100s) see Inset A: TREATMENT [F(a,295)=3.790, p<0.01]; TIME *p<0.05, **p<0.01, x**p<0.001 Fþ,29\=2a.53, p<0.00011; INTERACTION [F(20,295)=5.866, p<0.0001] signifrcantly different from S*C; #p<0.05, ##p<0.01, ###p<0.001 signrficandy different ftom 0N+C; +p<0.05, +++p<0.001 sþificantly different from 40N+C. C. SP (100-600s): TREATMENT [F(4,354)=4.928, p<0.01]; TIME *p<0.05, **p<0.01, **'*p<0.001 [F(6,35a)=19.33, p<0.0001]; INTERÀCTION lF(24)5\=ÇJ5, p<0.00011 signifrcantly d.ifferent from S*C; #p<0.05, ###p<0.001 signrficantly diffetent from ON+C; +p<0.05, ++p<0.01,

+ + +p <0. 00 1 significantly different from 40N + C.

G-2 A. oP response to cocaine infusion 'l

1

TEt E E oo- -1

-1

100 200 300 400 500 600 Time (s)

ll. Pr"""o, response overfirst lOOs of infusion Ç. D"pt"."ot Response from 100-600s of infusion

10 +* Hfl ctt ¿E) I E E g *rif E # À o- + o o -1

-1

20 40 60 80 100 200 300 400 500 600 Time (s) Time (s)

Fig G. 2 Change in A. diastolic pressure relat¡ve to the value at 40s pr¡or to the start of infusion of coca¡ne (0.5mg/kg/min). B. The pressor response to cocaine (first lOOs of coca¡ne infusion). C. The depressor response to cocaine (1OO-6OOs of cocaine infusion). Legend:X ¡aline (00p// nin) * cocaine (0.5ng/ kg/nin) [S+C], Ynandrolone uehicle (70¡t// nin) * coraine [0N+C], o *nandrolone nandmlone ft7pg/ kg/min) t cocaine [40N+C], t nandrolone uehicle (0p// nin) + saline (100¡t// nin) [ON+S],

(1pg/ kg/ nn) t ¡akne [40N+S]. The error bars have been removed from figutes B. & C. to retain clarity for symbols showing sþificance levels. Repeated-measures-ÀNOVA, Bonferroni's post-hoc test. The Iine at x=100s represents the end of the pressor response to cocaine. DP (0-100s) see Inset B: TRE,{TMENT [F(4,295)=5 578, p<0.001]; TIME **p<0.01, x**p<0.001 lF(5,295)=25.95, p<0.00011; INTERACTION [F(20,295)=6.749, p<0.0001]. signifrcantly different from S*C; #p<0.05, ###p<0.001 significantly different from ON*C; +p<0.05, +++p<0.001 significantly different ftom 40N*C. DP (100-600s): TREATMENT [F(a,35a)=7 99, p<0.0001]; TIME [F(6,354)=30.11, **p<0.01, xxxp<0.001 p<0.00011;. INTERACTION [F(2a,35a)=7.067, p<0.0001] significandy different ftom S*C; #p<0.05, ##p<0.01, ###p<0.001 significantly different ftom 0N+C; +p<0.05, ++p<0.01 +++p<0.001 signifrcandy different from 40N-l-C. Mean+S.E. n=11.-14.

G-3 HR response to cocaine infusion

30

+ +++ 1 ###

-10 E dr* CL ### .ct ++ +++ ; -30 I < -50

-70

-90 0 100 200 300 400 500 600 Time (s)

Fig G.3 Change in heaft rate (HR), relative to the value at 40s prior to the staft of infusion -ltre of cocaine (O.smg/kg/min). error bars have been removed from to retain clarity for symbols showìng signifrcance Ievels. Legend:X¡akne (100p1/nin) * cocaine (0.5rn¿/ kg/ nin)[S+Cf, Y nandrolone uehic/e (70p// nin) t cocaine

[ON+C], o nandrlone ft1pg/ kg/niil) t cocaine [40N+C], t nandrolone uehic/e (0p// nin) + saline (00¡tl/ nin) [ON+S], *nandrolone ft|pg/ kS/ niil) * saline [40N+S]. Repeated-measures-ÀNOVÂ, Bonferronì's post-hoc test. HR (0-600s):

TREATMENT [F(4,638)=3.227, p<0.05); TIME [F(11,638)=37.50, p<0.001];. INTERACTION [F(44,638)=5.762, p<0.00011 **p<0.01, *xxp<0.001 significantly different from S*C; #p<0.05, ##p<0.01, ###p<0.001 signifrcantly different from ON+C; +p<0.05, ++p<0.01 +++p<0.001 signifrcantly different from 40N+C. Mean*S.E. n=1'1'-1'4.

G-4

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