INFORMATION TO USERS

This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer.

The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction.

In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion.

Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book.

Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order.

University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor, Ml 48106-1346 USA 313/761-4700 800/521-0600

Order Number 9201772

The direct effects of diphenylhydantoin (DPH) and DPH analogues on fibroblast proliferation

von Deutsch, Daniel Albert, Ph.D. The Ohio State University, 1991

UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106

THE DIRECT EFFECTS OF Dl PHENYLH YDANTOIN (DPH) AND DPH ANALOGUES ON f i b r o b l a s t PROLIFERATION.

dissertation

Presented In Partial Fulfillment of the Requirements for the Degree Doctor of philosophy in the Graduate School of The Ohio State University B y Daniel A. von Deutsch, D.D.S., Ph.D.

* t * * *

The Ohio State University

1991

Dissertation Committee: Approved by

Ralph Stephens ^ - Richard Fertel (dviser Norton Neff Department of Pharmacology.

C o - A d v i s ^ / Department of Pharmacology. DEDICATIONS

To my wife Deborah for her help and understanding. Also, this is dedicated to the memories of my mentors, my father Albert L. von Deutsch and to my advisor, Dr. Daniel Couri. They will be missed greatly. I also dedicate this work to my son, Albert Wolfgang and to all my cats. ACKNOWLEDGEMENTS

I would like to thank Dr. Sarah Tjioe for her help, support and guidance, especially after the death of Dr. Couri. Your help will never be forgotten.

My special thanks to Drs. Andrej Rotter, my advisor, and Adrian Frostholm for their help and understanding through the difficult times following the death of Dr. Couri. Thank you for giving me the guidance necessary for completing my project.

To Dr. Ralph Stephens for his help and guidance through out this project, I give my thanks. His help and support were critical to my learning and to the project.

To Dr. Nick Gerber, I give my special thanks. Without his support this work could never have been done. Thank you.

My thanks to Dr. Richard Fertel for his help and .advice through out this study and especially in the preparation of this document.

I want to thank my good friends Dr. Mark Brose, Dr. Carl Miller, Dan Mullett, Neil Smith and Greg Miller for their help, encouragement and many fun times. CURRICULUM VITAE

EDUCATION

1972-1976 Cleveland State University Cleveland, Ohio B.S. in Biology and Chemistry.

1979-1983 The Ohio State University Columbus, Ohio Doctor of Dental Surgery (D.D.S.) College of Dentistry North East Regional Boards for DDS (1983).

1984-1991 The Ohio State University Columbus, Ohio Graduate Research Fellow / Research Associate Department of Pharmacology, Collage of Medicine.

PUBLICATIONS - Journal Abstracts

1) Rieger,M.R; von Deutsch,D.A & Brown,W.T (1982): The Diametrical Tensile Strength of an Undercondensed Amalgam. J.Dental Res. 61 (Special): 84.

2) von Deutsch,D.A; Brose,M.O; Couri,D. and Rieger,M.R (1988): Effects of Implant Materials and Phenytoin Upon Cellular attachment. J.Dental Res. 67 (Special Issue): 384.

3) von Deutsch,D.A.; Rotter,A.; Tjioe,S. and Couri,D. (1990): The Effect of Diphenylhydantoin (DPH) Upon Cultured Fibroblasts. The Pharmacologist: 32,#3: 360, pg 186.

FIELDS OF STUDY

Major Field: Pharmacology.

Toxicology and Instrumental Analysis: Dr. Daniel Couri

Drug Metabolism: Drs. D. Feller, S. Black & D. Couri.

Radioisotope Methodology: Drs. D. Feller and L. Malspeis.

Immunology: Dr. B. Zwilling

Cell Biology and Culture: Dr. Ralph Stephens TABLE OF CONTENTS

Page

DEDICATIONS...... ii

ACKNOWLEDGEMENTS i i i

CURRICULUM VITA...... iv

LIST OF TABLES...... viii

LIST OF FIGURES......

CHAPTER I, INTRODUCTION ...... 1

a. Barbiturates and Phenobarbital...... 1

b. Diphenylhydantoin...... 3

c. Therapeutic uses for DPH...... 5

d. Clinical Toxicology...... 8

e. DPH induction of Gingival Hyperplasia...... 10

f. DPH Metabolism (P-450 & PGS Pathway...... 13

8. The PGS Pathway...... 16

9. DPH-Induced DNA Single Strand Breaks...... 20

10. Statement of the Problem...... 22

11. Hypothesis...... 23

CHAPTER II, MATERIALS...... 25

v CHAPTER III, METHODS 27

1. Media...... 27

A. F-12 Medium...... B. Serum (FBS & Calf)...... C. Stock & defined Medium...... D. Growth Factors...... 1. Fibroblast Growth Factor (FGF)...... 2. Epidermal Growth Factor (EGF)......

2. cells...... 34 A. Normal Human Dermal Fibroblasts (NHDF)...... B. Human Fetal Lung Fibroblasts (WI-38)...... C. BALB/3T3 Clone A31 Fibroblasts......

3. Cell Growth Conditions...... 36

4. Cell Passage...... 36

5. Cell Counting Methods...... 37

6. Cryopreservation...... 38

7. Experimental Design...... 38

8. Synthesis of DPH Analogues...... 41

A. para-Chlorinated Analogue (p-cl-DPH)...... B. para-Brominated Analogue (p-Br-DPH)...... C. Purification & Identification of Product....

9. Drug Treated Medium & Vehicle...... 44

10. General Procedure for Lysing Attached Cells 45

11. Total DNA Content...... 46

12. Total Protein Content...... 47

13. Statistical Testing...... 48

CHAPTER IV, RESULTS...... 49 A. DPH Dose Response: Serum Supplemented Medium 49 1. Treatment Schedule for Quiescent Cultures.... 2. Quiescent Serum Supplemented Cells...... 3. Growth factor Free Studies...... 4. FGF & EGF Dose Response with DPH...... 5. DPH Dose Response with FGF & EGF......

vi B. Defined Media Studies 75 1. FGF & EGF Free Cell Proliferation with DPH.... 75 a. F-12 Medium...... b. Defined B-Medium...... c. B-Medium with 10% FBS...... d. F-14 BC & HCF...... e. DPH Dose Response (Growth Factor Free)....

2. Structure Activity Relationship in GFF Medium. 79 a. Hydantoin...... b. Ethotoin...... c. Mephenytoin...... d. Phenobarbital...... e. para substituted halogenated analogues....

3. Prostaglandin Synthetase Pathway...... 91 1. Hydantoin...... 2. Dexamethasone...... 3. Progesterone...... 4. DPH Dose Response with & without ASA..... 5. ASA Effects on Hydantoin Dose Response....

CHAPTER V, DISCUSSION...... 102 A. Development of Cell Culture System...... 102 B. Pharmacology of DPH’s Proliferatory Activity 108 1. DPH Dose Response with Serum...... 2. DPH Dose Response with EGF & FGF (serum free). 3. DPH Dose Response with Growth Factor Free Medium and Serum Free

C. TOXICOLOGY...... 113 1. Metabolism...... a. Cytochrome P-450...... b. Prostaglandin Synthetase Pathway

D. Structure Activity Relationship 120

E. Mechanism of Action 127

CHAPTER VI, CONCLUSIONS 130

REFERENCES 133

APPENDIX A P-450 Metabolism...... 143 APPENDIX B Hemocytometer...... 150 APPENDIX C Analogues - Instrumental analysis 152 APPENDIX D RU-486 activity with DPH & Dex... 159

vii LIST OF TABLES Table Page

3-1 Comparison of defined media components (salts, vitamins). Comparison of defined media components (amino acids, other). 29

4-1 OPH dose response curve by cell counting. 53

4-2 DPH dose response curve by total DNA content 56

4-3 DPH effect on serum stimulated cell proliferation, (comparison of incubation times) 57

4-4 Effects of serum, growth factors and growth factor free conditions on DPH dose response curve (compar- 74 ison of ED50 & Emax).

4-5 Analogue-induced proliferation by cell counting. 83

4-6 Screen of growth factor free activity for DPH and DPH-analogues 89

4-7 Combined drug & ASA activity compared to ASACtrl. 93

4-8 Combined drug & ASA activity compared to drug Ctrl. 93

4-9 Comparison of growth factor free DPH and Hydantoin 97 dose response curves (ED50 and Emax).

5-1 Substitution groups on hydantoin to show the different analogues. 121

7-1 TLC Rf values for DPH and DPH analogues (p-CH3-DPH, DPH, p-Cl-DPH and p-Br-DPH). 153

7-2 HPLC mean retention times in methanol. 155

7-3 The effect of DPH, Dexamethasone, Progesterone or RU 486 on NHD fibroblasts when combined with FGF 158 in defined, serum free medium.

vi i i LIST OF FIGURES

Figures Page

1-1 Phenobarbital. 2

1-2 Diphenylhydantoin. 3

1-3 Synthesis of DPH. 4

1-4 Hydantoin ring as primary structure for DPH and analogues. 11

1-5 Phase I metabolism by P-450b. 14

1-6 The prostaglandin synthetase pathway and role in metabolism. 17

3-1 Experimental time schedule for quiescent cell studies. 40

4-1 The DPH dose response curve (determined by Coulter counter). 52

4-2 DPH dose response curve by DNA content. 55

4-3 Serum dose response in quiescent NHD Fibroblast proliferation. 60

4-4 DPH induced proligeration in GFF medium 64

4-5 EGF dose response in serum free medium with DPH or Control. 67

4-6 FGF dose response in serum free medium with DPH or Control. 69

4-7 DPH dose response in serum free medium with FGF 71

4-8 DPH dose response in serum free medium with EFG 73

4-9 Screen of growth factor free media. 78

4-10 DPH dose response in growth factor and serum free 81 medium.

ix Figures Page

4-11 Growth Factor Free effects of Hydantoin on Human Fibroblasts. 85

4-12 Structure of Ethotoin. 87

4-13 Structure of Mephenytoin. 88

4-14 Positions on Phenyl ring. 92

4-15 The effect of ASA on drug induced proliferation. 95

4-16 The calculated curve for DPH & DPH + ASA dose response curve. 99

4-17 The calculated curve for Hydantoin and Hydantoin + ASA dose response curve. 100

5-1 Hydantoin ring positions and substitution groups. 121

5-2 Positions on phenyl ring. 122

5-3 Phenobarbital. 125

7-1 Hemocytometer gride. 150

7-2 TLC plate. 152

7-3 Drug (DPH, DEX, Progesterone & RU 486) response with FGF in NHD fibroblasts. 160

x INTRODUCTION

Sodium diphenylhydantoin (DPH or phenytoin) is a commonly prescribed anticonvulsant used for treating epilepsy. The chemical name for DPH is sodium 5,5-diphenyl-2f4-imidazolidinedione and it belongs to the family of drugs known as the hydantoins. However, in this discussion the more descriptive name, diphenylhydantoin, will be used instead.

Treatment of Epilepsy. Many methods have been employed, with varying degrees of effectiveness, for the treatment of seizure disorders. Bromide, phenobarbital, surgery, ketogenic diet and restricted fluid intake are some examples of the drugs and treatments that were available to the physician during the time DPH was introduced. At that time, phenobarbital was the safest and most effective method available, while potassium bromide was the oldest CNS depressant used to treat epilepsy [1].

The Barbiturates and Phenobarbital. Barbituric acid, 2,4,6(1H,3H,5H)- pyrimidinetrione was synthesized by Baeyer [4,5] and later by Dickey and

Gray (appendix A) [2,6,7]. Barbital is an analogue of barbituric acid and is the oldest barbiturate to be used clinically as a hypnotic agent. The second oldest clinically used barbiturate, phenobarbital, has a behavior quite different from that of barbital and barbituric acid. The presence of a phenyl group on phenobarbital reduces the amount of sedation observed in barbital while giving the molecule the capacity to control seizures.

However, neither barbituric acid nor barbital are anticonvulsants [2,3,7,8].

1 Phenobarbital (5-ethyl-5-pheny1-2,4,6-pyrimidinetrione) is most useful in treating generalized tonic-clonic and partial seizures (fig. 1-1). High phenobarbital concentrations cause sodium and potassium conductance to decrease, as with DPH [1,3]. Also, phenobarbital is believed to enhance chloride conductance by binding to the dihydropicrotoxin sites on the GABA receptor. This effect is thought to be associated with phenobarbital's hypnotic action rather than any anticonvulsant effect [1,3].

II 0

Figure 1-1. PHENOBARBITAL

The barbiturates, barbiturate analogues and hydantoins have acidic properties despite the lack of carboxyl groups. Furthermore, all ureides have at least .one nitrogen atom flanked by two carbonyl groups [7,10].

This molecular arrangement causes the hydrogen, located on nitrogen number three, to become acidic. This acidity arises from the fact that both the lactam (-C0-NH-) and lactim forms (-C(OH)rN-) are in equilibrium with one another [2,7]. In the 1930's, physicians still required an antiepileptic agent that lacked sedation and was more effective in controlling partial and tonic- clonic grand mal seizures than the current therapy. Various investigators began searching amongst analogues of phenobarbital for a compound that would protect against maximal electroshock seizures induced in laboratory animals. Experiments conducted by Merritt and Putman, in 1937, led to their publication in 1938 of findings about a new anticonvulsant drug,

DPH.

Diphenv1hvdantoin. DPH (fig. 1-2) was first synthesized from benzyl urea and NaOH in 1908 by Biltz [8,11,12]. However, in 1946, H.R. Henze

c = o / \ -N l H

Figure 1-2. Diphenylhydantoin (DPH).

filed a patent for the synthesis of DPH and other 5,5-diaryl hydantoins from benzophenone, potassium cyanide and ammonium carbonate (fig. 1-3).

The solvent for this synthesis was acetamide [13]. It was not till 1938 that the anticonvulsant activity associated with DPH was discovered by

Merritt and Putnam [14]. Their discovery was an important first in the 4

Amman Ium Po t a i « Ium Carbonat* Cyan Ida

C - 0 + (NHJjCO, + K-CN

8«nzophsnon«

Ac* < ami da ( So Iv«n i )

NH, // H 3 ,N. K 2 HjO cr<> +CO, S J i C N II H 0 HC I

D I phany Ihydan t a I n Figure 1-3. The synthesis of DPH from benzophenone, ammonium carbonate & cyanide described by Henze.

field of pharmacology because the anticonvulsant activity for DPH was

found via a deliberate search.

They conducted a series of systematic experiments with the intent of

finding compounds that had greater potency than phenobarbital, but

produced less sedation [15]. About 80 compounds structurally similar to

phenobarbital were screened for their effectiveness by using an in vivo

system which used maximal electrical shock [14]. The structures included

phenyl derivatives of phenobarbital which were phenyl, cresyl and tolyl

sulfonates, benzoates, ketones and esters. Also, radicals of carbamic, [15]. Of all the compounds screened, only a few were able to prevent the occurrence of convulsions in the test animals, after they received a full current stimulation of 40 to 45 milliamperes from a 45 volt battery

[14,15].

The purpose for screening such a large group of compounds was to determine which ones could provide greater antiseizure activity than phenobarbital while having less sedation associated with their use. Also, by first determining the animals' seizure threshold, investigators were then able to establish the threshold's stability from day to day. They found that the convulsive threshold was quite stable for untreated animals. Finally, Merritt and Putman determined that the most effective anticonvulsive drug at that time was phenobarbital. However, five drugs not previously demonstrating anticonvulsive activity, were found to be more effective than phenobarbital, and among these was DPH.

s Since 1946, other investigators such as the team of Schldgl, Kraupp,

Wessely and Stormann, synthesized over fifty 3,5-di and trisubstituted hydantoins, systematically substituting groups on either the number three nitrogen or number five carbon. In this study, they either introduced a group into the hydantoin ring at positions three or five, or converted one substituted hydantoin into another desired form, then tested for anticonvulsant activity [16]. The hydantoin analogues 3-((3-hydroxyethyl)- hydantoin and 3-hydroxymethyl-hydantoin had the greatest anticonvulsant activity of all the analogues that they tested [16].

Therapeutic Uses. Merritt and Putman showed that DPH was more effective than phenobarbital in protecting laboratory animals from electrically induced convulsions. In addition, Krayer, Kamm & Gruzhit informed them, via a personal communication, that DPH was well tolerated in test animals independent of dosage format. Therefore, a single massive dose or smaller continuous daily doses have the same effect [17]. With this information in hand, they then considered it safe to conduct clinical trials on patients with seizure disorders.

To test the effectiveness of DPH, Merritt and Putman selected patients who suffered from convulsive seizures for many years. Furthermore, these patients were considered severe epileptics that were refractive to the usual treatments (bromide, phenobarbital, ketogenic diet and restricted fluid intake) [17]. At the time of their publication, 198 patients had been tested over a period of three weeks to eleven months. These patients were grouped according to the type of seizures that they presented: 1)

Grand Mai (118 patients); 2) Petit Mai (74 patients) and 3) "Psychomotor

Equivalent" attacks (6 patients).

Grand mal seizures are considered generalized seizures, in which there is no evidence of a localized onset. Of all of the epileptic seizures, the grand mal type are the most dramatic. They are characterized by an initial tonic rigidity of all extremities, eventually followed by severe jerking of the body, which usually lasts from one to two minutes. During these attacks, it is not uncommon for the patient to bite their tongue or cheek, or for them to have urinary incontinence [3,17].

Merritt and Putman indicated that of the 118 patients that suffered from frequent grand mal seizures, complete relief was obtained in 68 patients (58%). Also, the frequency of attacks in 32 patients (27%) was significantly reduced. In 18 patients (15%), there was only a marginal improvement, at most, in the frequency of attacks when compared to the previous treatments with bromide or phenobarbital. From this data, Merritt

& Putman concluded that OPH proved to be more effective than bromide or

phenobarbital in controlling grand mal seizures [17].

Petit mal (absence) seizures are another form of generalized seizures, characterized by both sudden onset and cessation of the attack, usually

lasting from 10 to 45 seconds. These attacks customarily have mild clonic

jerking of the extremities or eyelids, postural changes and automatisms

(see psychomotor equivalent attacks) associated with them [3,17].

Merritt & Putman showed that of the seventy four patients with frequent petit mal seizures, complete relief was gained in twenty six (35%). In

addition, in another 36 patients (49%), a marked reduction in the number of attacks was observed. For twelve patients (16%), either very little to

no effect for OPH was found [17]. Merritt and Putman noted that the effectiveness of OPH in treating petit mal seizures was greatest when

these seizures were associated with those of the grand mal type [17].

DPH was shown by Merritt and Putman to also be effective in treating

"Psychomotor Equivalent" or complex partial seizures. A psychomotor equivalent, as referred to by these authors, has a localized onset,

normally in one of the temporal lobes. However, the discharge pattern

becomes widespread and usually bilateral in character, with involvement of

the limbic system [3,17]. These patients may have a brief warning prior

to periods of unconsciousness where the patient may stagger or fall. A

characteristic event that occurs is automatism, of which the patient has

no memory. Automatism is an integrated motor response such as lip

smacking, twitching, jerking of the head or even taking ones shoes off or

having a conversation with one's self [3,17]. Merritt and Putman showed that of the six patients in this portion of the study, four (67%) had complete relief from these seizures, while the remaining two patients

(33%) showed a marked reduction in the frequencies of their attacks [17].

Thus, it can be seen that DPH is an effective anti epileptic agent; useful in treating grand mal, petit mal and "Psychomotor Equivalent" attacks.

The mechanism of action for DPH's antiepileptic activity is thought to arise primarily from its ability to block both sodium channels and the generation of repetitive action potentials at therapeutic levels (10-20 pg/ml) [3,18]. In addition to sodium channel blockage, DPH also has been found to have a widespread effect upon various physiological systems, which also may play a roll in DPH's antiepileptic activity. Some examples of the types of systems that DPH also affects are as follows: 1) Calcium conductance: DPH blocks calcium uptake in rat brain slices [19]. In addition, DPH and some classical calcium channel blockers (nimodipine, diltiazem and verapamil), have been shown to inhibit voltage gated calcium flux by different yet functionally coupled mechanisms [20].

DPH also affects both sodium and calcium conductance [2,3,71-74], in addition to having effects upon membrane potentials. DPH was also found to affect the concentrations of some amino acids [3] and levels of various neurotransmitters [1,3] such as 6ABA, acetylcholine, serotonin, dopamine and norepinephrine [21-28].

Clinical Toxicitv. With the widespread clinical use of DPH, much has been learned about the character and problems associated with DPH, including the fact that DPH is 90% protein bound in adults, but less so in the young [1]. The many side effects that have been reported include initiating connective tissue disorders and counteracting the toxic effects of cardiac glycosides and glucocorticoids [1] and also alters the maternal plasma corticosterone levels in pregnant females [29]. OPH has also been implicated as a carcinogen [30]. The most common and one of the most dramatic clinical manifestations associated with DPH therapy is the connective tissue disorder gingival hyperplasia, which has been shown to occur in 20 to 50% of patients taking OPH [2]. In addition to gingival hyperplasia, other connective tissue effects associated with DPH include accelerated rates in vascular and clot organization [31], and accelerated wound healing in humans due to periodontal disease [31,32,33,34,35], oral surgery [36] and ulcers [34,37]. However, surgical wound healing experiments conducted by Donoff [38] showed that the rates of healing in guinea pigs treated with OPH showed no difference from those of the controls. In contrast to the guinea pig, rat skin showed an increased healing response to OPH [39].

With respect to effects on bone, OPH has also been shown to cause increased bone formation in experimentally induced mandibular fractures in rabbits [40] and in fibula fractures in rats [41] and mice [42].

Gudmundson and Lidgren showed in mice that both the tensile and breaking strengths of healing fractures were significantly greater in animals treated with DPH than in controls [42]. In addition, the fracture sites contained a greater amount of extractable collagen than those of the controls. This point is important since the composition of the organic portion of the bone matrix is about 90 percent collagen. Thus, DPH may contribute to an increased rate of healing by increasing the rate of collagen synthesis [42]. In man, it should be noted that the extent of hydantoin-induced connective tissue disorders are greatest in children, where cells have increased mitotic activity, compared to adults [1]. For example, in 1972,

LeFebvre, Haining and Labbe described the changes that had taken place in young retarded patients who required many years of anticonvulsant therapy

[43]. In particular, these children show a thickening of the calvarium

(skull) and a somewhat dramatic coarsening of their facial features, primarily due to an enlargement of the lips and nose [43]. To emphasize the extensiveness of DPH's soft tissue effect, LeFebvre provided photographs of a boy who was being treated over a period of 15 years. The changes that had taken place in appearance were striking as the photographs showed a 4 year old boy with normal facial features and that of the same child at the age of 16 with marked enlargement of the nose and lips [43].

DPH Induction of Gingival Hyperplasia. In 1939, a year after the introduction of DPH as an anticonvulsant agent, O.P. Kimball, a Cleveland physician, found that the use of DPH lead to gingival overgrowth in some patients [44]. Kimball stated that the patients treated with DPH had a much better general health, mental state and personality than prior to treatment. In fact, he was so impressed with the results obtained for

DPH, that he decided to continue treatment even though there were some side effects. Some of the side effects that he observed were the development of soreness in the mouths of some patients along with dizziness and a staggering gait. However, the two conditions, soreness and dizziness, were not related. Kimball noted that in some of the patients, definite hyperplasia of the gums had occurred and that it seemed 11

to suggest scurvy. In addition, of the 119 epileptic patients receiving

DPH, the gums of 51 patients were non responsive while 68 showed varying

degrees of hyperplasia. Of these patients, 57% had gingival hyperplasia

and of these patients, 17 (14%) had advanced cases of hyperplasia [44].

Kimball further observed that patients with marked gingival

hyperplasia also showed definite vitamin C deficiency. In contrast to

this, he noted that no patient with normal gums showed any signs of

vitamin C deficiency. From this, he concluded that the degree of

hyperplasia and vitamin C deficiency were parallel. However, the usual

symptoms of severe and prolonged ascorbic acid deficiency including sore

joints, extreme weakness and purpura were absent. Therefore, Kimball was

not observing scurvy, but rather a condition produced with DPH that caused

hyperplasia and decreased serum ascorbate concentration to about 1/10 the

normal level [44].

Primary Structure-Activity Relationship. For DPH and its analogues,

the hydantoin ring serves as the primary or parent structure. The

C o m p o u n d R i R 2 ^ 3

R H I3 u Hydantoin H H H 1 H Phony 1 Phony 1 h ~ C i ' 2 c = o o p "

\\ 4 j / / Mophony t o 1n CH,- CH,-CHZ- Phony 1

c ------N . . . . . H | E t h o t o i n c h ,- ch 2- H P h * fl>'1

p-QH-OPH (HPPH) H Phony 1 OH-Phonyl

Figure 1-4. HYDANTOIN RING 12

heterocyclic hydantoin molecule (2,4-imidazolidinedione) is composed of three carbon (positions 2,4 & 5) and two nitrogen atoms (positions 1 & 3).

In figure (1-4), it can be seen that oxygen is bound to the carbons located at positions 2 and 4 (dione), while both nitrogen atoms and the carbon located at position 5 are saturated. In addition, the hydantoin ring is asymmetrical and carbon #5 is a prochiral center.

DPH is formed by substituting 2 phenyl groups on carbon number 5 of hydantoin. Viewing DPH with respect to its structure and therapeutic activity, the phenyl groups (or other aromatic groups) located on carbon number five are required for activity against: 1) clinical generalized tonic-clonic seizures and 2) abolition of maximal electroshock seizure patterns generated in laboratory animals. For comparison, hydantoin does not possess any antiseizure activity [15].

With alkyl substitution on carbon number five, there would be a marked rise in the amount of sedation that would be produced. Such is the case for phenobarbital, mephenytoin and nirvanol (mephenytoin's major metabolite), where each bear both an ethyl & phenyl group on carbon five.

Since DPH has no alkyl group at this site, sedation only occurs at very high doses. This insight into the relationship that exists between DPH's structure and therapeutic activity, was helpful in developing a better understanding of what structural changes produced antiepileptic activity from those which produced sedation. In addition, using structure activity relationships for DPH and its analogues, may prove useful in determining mechanisms for some of their side effects. These side effects include

OPH-induced gingival hyperplasia, teratogenicity, carcinogenicity, or 13 increased rates for wound healing. How these side effects may relate to

DPH's structure remains somewhat unclear.

The Metabolic Fate of DPH (Phase I Metabolism). Phase I metabolism is made up of a group of enzymes called the Mixed Function Oxidases (MFO);

NADPH cytochrome P-450 reductase and the terminal oxidase, cytochrome P-

450. Cytochrome P-450 dependent oxidation is the primary means of metabolism for mono or di aromatic hydrocarbons, such as OPH and phenobarbital. In the metabolism of DPH and related compounds, the most important reaction is aromatic hydroxylation.

In both rat and man, the major metabolite of DPH is formed by para hydroxylation of a phenyl ring [1,3]. With DPH, the major metabolite produced is para hydroxy DPH [1,3,47]. However, this reaction may not always deactivate the compound and may, in some instances, involve the generation of toxic reactive intermediate metabolites, like the epoxides.

Some of the metabolites formed by N demethylation may not necessarily be inactive, as with mephenytoin's metabolite nirvanol. Nirvanol is finally deactivated by aromatic hydroxylation.

Many isozymes of cytochrome P-450 have been identified in man, mouse, rat & rabbit, where each is responsible for metabolizing different groups of compounds. These enzymes are located within the hepatocyte's lipid membrane portion of the smooth endoplasmic reticulum (SER) [1,7].

The metabolism of DPH by P-450. The metabolic scheme for DPH, where the microsomal oxidation of DPH is shown in figure 1-5. The process requires molecular oxygen, cytochrome P-450 reductase, cytochrome P-450 and a NADPH-generating system. 14

Cyl«

DPH

OH OH

p-OH-DPH m-OH-DPH Mo|ir m<1ak

OH OH 0 I (DMO)

R

OH OH

DPH 3,4 Co I a alia I

R

OH CH.OH

0 PM 3-0-Mattiyl Co I aiha I

R

Figure 1-5. Phase I Metabolise of DPH by Mixed Function Oxidase. Cytochroees P-450 Reductase & P-450b pathway for oxidation of Xenob1ot1cs. The major difference in metabolism among various species may be in the percentage of p-OH-DPH produced (as in rat) or in the primary type of metabolite produced. For example, dogs produce meta hydroxy-DPH (m-OH-

DPH) as their major metabolite. However in man, the metabolite m-OH-DPH is thought not to exist or be produced as a minor metabolite. Other metabolites of DPH formed include the dihydrodiol, 3-0-methyl catechol & catechol. Ultimately these metabolites will undergo conjugation with glucuronides in phase II metabolism [49]. However, during the metabolic process, reactive intermediates can be formed, causing necrotic, mutagenic, teratogenic or carcinogenic changes in target tissues. The epoxide (arene oxide) of DPH was suspected to be the toxic or reactive intermediate responsible for the observed teratogenicity, or possibly even the DPH-induced gingival hyperplasia [48,50,51].

Through the process of epoxidation, an arene oxide is formed when the oxygen binds to the aryl portion of the molecule while an epoxide is formed when the oxygen is bound to alkyl portion. The P-450 pathway is thought to be responsible for forming the arene oxide while an alternate pathway, the prostaglandin synthetase (PGS), is thought to produce the epoxide [52-54]. Finally, the epoxide, or possibly even some other reactive metabolite (such as the semiquinone), is thought to cause lipid peroxidation or bind to cellular macromolecules. Examples of macromolecules that these reactive intermediates could covalently bind to may include DNA, RNA, regulatory proteins and hormonal receptors (T3, glucocorticoid), thereby disrupting normal cellular functions.

DPH Metabolism & Teratogenicity by the PQS Pathway. Some investigators believe that DPH's connective tissue side effects, such as gingival 16 hyperplasia or effects upon collagen, may be due to reactive intermediate metabolites, such as the epoxide or arene oxide [48,50-58]. The major problem with this hypothesis is that reactive intermediates are hard to identify due a short life span and trace quantity. To confirm that free radicals can be formed from DPH in the presence of P6S, Electron Spin

Resonance (ESR) spin trapping studies can be conducted [59,60]. These studies involve the use of free radical scavengers, such as #-Phenyl-N-t- butylnitrone (PBN) or caffeic acid, which form free radical adducts. The spin trapped adducts are extracted and the determinations made by ESR.

Results from Well's spin trapping experiments showed that DPH produced reactive intermediates when incubated with 1000 units of P6S, 0.24 nM arachidonic acid and PBN. In the absence of DPH, no ESR signal was observed [59]. Also, in other experiments intended to investigate oxygen consumption during P6S catalyzed oxidation of DPH, DPH was able to increase the rate of arachidonic acid induced oxygen consumption, as did phenylbutazone. These experiments indicate that the PGS pathway is involved in the oxidation of DPH in the lungs, kidney and liver of mice.

Furthermore, when the PGS pathway was blocked, a reduction in the amount of covalent bound labeled DPH was observed [59]. The observed change in covalent binding suggests that the PGS pathway is involved in the production of reactive DPH metabolites and DPH oxidation.

The Prostaglandin Synthetase Pathway. In addition to the cytochromes

P-450, other pathways have been shown to metabolize DPH (fig. 1-6). These pathways include thyroid peroxidase, horseradish peroxidase and prostaglandin synthetase [59]. It has been shown that by blocking the prostaglandin synthetase (PGS) pathway, teratogenic effects due to DPH and 17

MEMBRANE P H O S P H O L I P I D S

PHOSPHOLIPASE

ARACHIDONIC ACID Inhibited by FATTY ACID I n d o m a t h a c i n a n d ASA CYCLOOXYGEBASE DPH PGG STABLE ANT I OX IDANT RAOICAL

GSSG

HYDROPEROXIDASE Rntioxidants

(Inhibited by P RO T E 1NS Methfmazole) DPH F r a • .• r o v i i tnt n iNniMR AND R a d i c a l ------► LIPID PEROXIDATION LIPIDS BPN \ P G H OPH-BPN Altarad Callular Functions ADDUCT Cytotoxicity Taratogansis Prostaglandins Prostacyc I ins Thromboxanes

Figure 1-6. The Prostaglandin Synthetase Pathway Is thought to be responsible for the Metabolise of DPH and glucocorticoids to reactive internediates. the glucocorticoids could be significantly reduced or blocked [50,52-

57,59]. Therefore, the formation of reactive intermediate metabolites and

free radicals could also be blocked through blocking the PGS pathway.

Reactive intermediates produced by P-450 and the PGS pathway have been demonstrated to covalently bind to macromolecules such as ONA and proteins, causing cellular toxicity and lipid peroxidation [50,52-57,59].

The PGS pathway has been fairly well studied with respect to

teratogenicity, but has not been characterized with respect to DPH's effect on human dermal fibroblast proliferation.

ASA (Aspirin, acetylsalicylic acid) functions by irreversibly binding

to the enzyme cyclooxygenase, while indomethacin inhibits cyclooxygenase by reversibly binding to the enzyme. Inhibition of cyclooxygenase by either ASA or indomethacin causes the inhibition of the PGS pathway. In the absence of P-450, inhibition of the PGS pathway would block the formation of reactive metabolites that may arise from DPH.

DPH has been shown to bind to the glucocorticoid IB receptor which mediates anti-inflammatory & teratogenic activity for both glucocorticoids and DPH [55]. The IB receptor is distinguished from the glucocorticoid II receptor by: 1) it binds DPH, the glucocorticoids and a variety of other steroids. 2) it is able to rebind fresh labeled DPH or dexamethasone after

the ligand has been lost during chromatography [55]. DPH and dexamethasone

inhibit the release of arachidonic acid. This anti-inflammatory activity

is thought to function via a glucocorticoid receptor-mediated induction of phospholipase A2 inhibitory proteins (PLIP). PLIP then acts to inhibit the release of arachidonic acid, thus reducing the production of prostaglandins [55]. In contrast, the II glucocorticoid receptor is not 19

Involved with any anti-inflammatory activity, is specific for dexamethasone and is associated with tyrosine aminotransferase activity.

Also, OPH does not inhibit dexamethasone-induced activity of this enzyme nor does it affect basal enzyme levels. Therefore, it can be assumed that

DPH does not bind to this receptor site.

Inhibition of the PGS pathway in CD-1 mice with either ASA or indomethacin produced a reduction (about 50%) in the incidence of fetal cleft palates due to DPH. However, the inhibition of cyclooxygenase had no significant effect upon the incidence of fetal resorptions or mean fetal weights [54]. Additionally, the inclusion of free radical scavengers such as caffeic acid or PBN significantly reduced the amount of DPH- induced cleft palates and fetal resorptions, but had no effect upon fetal weight loss [54]. Finally, pretreatment with ASA reduced the covalent binding of labeled DPH to fetal proteins by 43%, when compared to the controls. In contrast to these observations, there was an enhancement of

DPH covalent binding along with incidences of DPH-induced cleft palates and fetal resorptions when 12-0-Tetradecanoylphorbol-13-acetate (TPA) was used to stimulate the activity of the PGS pathway [52].

The sites on DPH that may be metabolized has been shown to involve the

#3 nitrogen in the hydantoin ring. A structure activity study determined that DPH and related molecules had the #3 site in common as a site for the formation of an epoxide. The drugs trimethadione and dimethadione showed that phenyl rings were not needed for PGS generated cleft palates or covalent binding [53]. However, this still does not rule out phenyl ring involvement in the metabolism of DPH or some other drug by the PGS pathway. 20

If DPH is metabolized by cultured fibroblasts by either P-450b or the

PQS pathway, then it is possible that the production of reactive intermediates could interfere with DPH-induced cell proliferation.

Interference with DPH-induced proliferation could occur directly by competing with the parent drug or by altering cellular functions.

If DPH is metabolized by cultured fibroblasts, then inducing P-450b activity with phenobarbital should increase DPH metabolism and the production of reactive intermediates [61]. An increased production of reactive intermediates would mean an increased amount of covalent binding of reactive DPH intermediates to the surrounding protein targets [61].

Thus, increased metabolism would most likely cause a decrease in drug- induced proliferation. Inhibition of the PGS pathway with either ASA or indomethacin would block the formation of reactive metabolites [59,61], which should allow DPH to induce proliferation to its full potential.

DPH-Induced DNA Single Strand Breaks. DPH has been shown to bind directly to 9-ethyl adenine forming a 2:1 complex (DPH to 9-ethyladenine).

One proton on a DPH hydrogen bonds to ethyladenine in a Watson-Crick scheme, while the second DPH molecule binds at the N(3)-H [62]. The in vitro binding of DPH to an adenine analogue would tend to suggest that this may also occur inside the cell. Furthermore, within the cell, the epoxide of DPH would be the most likely candidate to bind to DNA.

However, it is also possible that DPH, or a metabolite other than an epoxide, may also create similar toxic effects. Based upon this information, it is apparent that metabolism may be a major factor in producing trace amounts of reactive metabolites. The production of reactive intermediates can potentially lead to extensive damage within the cell. Studies conducted in human peripheral mononuclear cells indicated that l.6x10"* M (40 |ig/m1) of DPH caused a slight, but significant, increase in DNA single-strand breaks. DPH's reactive metabolite, the epoxide, is thought to be responsible for causing this damage. It was also suggested that single strand breaks generated by DPH, or a reactive metabolite, could be masked by DPH-induced DNA cross-linking [63]. STATEMENT OF THE PROBLEM.

In general, DPH is an excellent first line drug for the treating epilepsy and for counteracting the toxic effects of cardiac glycosides and glucocorticoids [1]. Also, DPH has successfully been used to treat combat related wounds [73,74] and leg ulcers in man [34] as well as significantly enhancing the activity of AZT in reducing the spread of AIDS infection, by blocking calcium channels. However, any drug with such a broad range of activities is not without problems.

Connective tissue disorders are common side effects associated with the use of DPH, occurring when it is used over a period of time. The effects upon connective tissue can include gingival hyperplasia, a thickening of the calvarium and produce coarsening of facial features, especially in children [43]. However, the mechanism or mechanisms involved with the effects of DPH on gingival hyperplasia, the rate of wound healing in soft tissue [74,75] and bone [40-42] remain somewhat unclear. Also, how DPH induces cell proliferation is still unknown.

The metabolic catalysts P-450 & PGS can produce reactive intermediates that can potentially covalently bind to cellular or fetal proteins such as regulatory proteins or elements responsible for the inhibition or stimulation of cell proliferation and collagen synthesis. The covalent binding of a reactive intermediate to regulatory elements, or other macromolecules with in the cell (such as DNA, RNA, cellular proteins and

22 23 lipids) could potentially cause cytotoxicity or lipid peroxidation. Thus, these effects may account for much of the side effects associated with the use of OPH, including proliferation.

Another consequences of DPH's activity could be its effects upon the metabolism of endogenous steroid hormones. This was demonstrated by the ability of OPH to induce the metabolism of testosterone to its active metabolite, 5a-DHT. Together, 5«-DHT and DPH may account for some of the gingival hyperplasia observed in those responding epileptic patients.

However, to address some of these areas a better model system would be required. At present the cell culture model system needs to be refined in order to better understand what mechanisms or pathways are involved with

DPH-induced activity. In previous studies by other investigators, they used medium supplemented with various percentages of serum and rapidly dividing cells. These would tend to increase the number of variables encountered, thus reducing the reproducibility of the system. By having a culture system stable and sensitive enough to allow for a systematic study of the structure-activity relationship for DPH, a greater insight into the cellular activity of DPH may be gained. With this information the primary structure responsible for proliferation can be determined through the use of DPH analogues. Additionally, through the use of the analogues, a better understanding of DPH's cellular toxicity and growth promoting effects can be ascertained.

The hypotheses of this study are:

1) That DPH can act directly on Normal Human Dermal (NHD) fibroblast, causing proliferation. Additionally, structurally similar compounds such as phenobarbital or DPH analogues are also able to induce proliferation. 24

2) That DPH-1nduced proliferation may be due to an interaction between

DPH and the growth factors FGF & EGF.

3) That metabolism of DPH by either an isozyme of P-450 or the

Prostaglandin Synthetase (PGS) pathway will reduce DPH-induced proliferation.

4) The most likely structural feature of DPH causing proliferation is the hydantoin ring. MATERIALS

Cell Culture Materials. Plastic T-25, T-75 and T-150 culture flasks along with 6 & 24-Well trays were obtained from Corning Laboratory

Supplies, Corning, N.Y. Culture medium; F-12 and B medium were purchased from 6IBC0, Long Island, N.Y.

Biochemicals. Bradford Protein Assay dye reagent concentrate was purchested from Bio-Rad Laboratories, Richmond Calf. Bicinchoninic Acid, fetal bovine serum, calf serum, penicillin, streptomycin, gentamycin, amphotericin B, thymidine, insulin, hydrocortisone, dexamethasone, progesterone, transferrin (human), MEM 100x vitamins, were purchased from

Sigma Chemicals, St. Louis, Mo. Fibroblast Growth Factor (FGF), Epidermal

Growth Factor (EGF) and BSA were purchased from Boehringer Mannheim

Indianapolis, Indiana.

Radiochemicals. [^Hj-thymidine (83 Ci/mmole) and Aquasol-2 were obtained from New England Nuclear.

Chemicals. DPH (free acid and sodium salt) was obtained from Sigma

Chemicals, St. Louis, Mo. The chemicals acetamide, p-CI-benzophenone, p-

CHj-benzophenone and p-Br-benzophenone, ammonium carbonate and potassium cyanide were obtained from the Aldrich Chemical Company, Milwaukee, Wi.

Ethotoin was a gift from Abbott Laboratories in Abbott Park, 111. RU-38

486 was a gift from Roussel Uclaf, Romainville, France.

25 26

Instrumentation for analytical analysis:

1) Qas Chrowatoqraphv: Varian 3700 GC fitted with the following detectors:

a: Electron Capture Detector (ECD1. for the detection of

compounds that contain halogens,

b: Thermionic Specific Detector (TSD1. for the detection of

compounds that contain nitrogen,

c: Flame Ionization Detector (FID), a universal detector

for the general detection of compounds.

Column: 3 ft packed glass column, 3% 0V17. Conditions: initial 150°C with

1 min hold, program rate 104C/min to 280aC with a 0 min final hold.

21 HPLC: Altex Model 420 Microprocessor Controller/Programmer.

Altex Model 110A Solvent Metering Pump.

Altex Model 165 variable UV detector.

Column: Altex Ultrasphere - ODS 5 p, 4.6 mm x 25 cm.

Wavelength 215 nm, run at 35”C using heating mantle.

31 Spectrophotometer. Beckman model 35 spectrophotometer. METHODS

A. Media.

The medium that was used to maintain stock cell cultures was a modified Eagles minimal essential medium (MEM), from Gibco, that contained

10.119 g/L MEM powder with Earle's salt, L-Glutamine, 2x NEAA, 1.5x Amino

Acids and vitamins. The medium was further supplemented with 0.835 g/L

NaCI, 0.11 g/L pyruvate (Na) and 1.5 g/L sodium bicarbonate. The stock medium also received 1% (v/v) gentamicin, penicillin, streptomycin and amphotericin B, while the medium used in the experiments did not receive antibiotics. The medium's final pH was adjusted to 7.2 and the pH

indicator, phenol red, was used to help monitor the metabolic state of the culture. With time, the medium changes from the usual red color (pH 7.2-

7.4) to a yellow color (pH < 7.2) due to normal cell metabolism. To support cell growth and normal cellular functions, serum was added to the medium.

F-12 medium, developed by Ham, was intended to be used as a chemically defined, serum free medium. Ham's F-10 medium was introduced around 1963 and was much richer in inorganic salts, vitamins and miscellaneous components when compared to other media. Examples of some of the inorganic salts found in F-10 are CuS04.5H20, ZnS04.7H^O, FeSO^HjQ and MgSO4.7H^0.

A major problem associated with F-10 medium was that its amino acid concentrations were low. This led Ham, in 1965, to improve upon his F-10 medium and create the more fortified F-12 medium. In F-12 medium, Ham

27 28

increased the concentrations for seven amino acids (isoleucine, lysine,

proline, threonine, tryptophan, tyrosine and valine), along with altering

several of the inorganic salt & vitamin concentrations (see listings of

media components).

We tested Ham’s F-12 medium, supplemented with 400 pg BSA, 5 pg/1

a insulin, 1x10 M hydrocortisone and 50 pg/ml F6F upon newborn human

foreskin fibroblasts. From these experiments, we concluded that F-12

medium was insufficient to maintain the cells and needed to be enriched.

The cells were shaped like very long, thin spindles and appeared as if

they were starving. We used Ham's F-12 as a base component, that was rich with inorganic salts, together with a medium that had high amino acid concentrations (Table 3-1).

The amino acid concentration for F-12 needed to be increased, so we mixed 250 ml of F-12 to 250 ml of B medium, according to Sato [76].

However, we also included selenium, hydrocortisone, insulin, 500pg BSA and

MEM vitamins, to give what we called F-14 basal medium, since it would be the next logical number in the series of F-10 & F-12. The testing of different defined media showed that the modified B-medium (F-16) gave the best overall response with respect to DPH-induced proliferation, when compared to controls.

SERUM provides the needed vitamins, hormones and growth factors, along with a host of attachment factors, to support normal cellular activity.

However, serum may also contain several factors that may alter normal cellular functions or may even prove to be cytotoxic. This is especially true for serum coming from more mature animals, such as from calves or horses, rather than from fetal animals. One of the differences between 2 9

Table 3-1

COMPARISON OF CELL CULTURE MEDIA Contains INORGANIC SALTS Han's 8 RPMI PGF2 Han's (mg/1) F-12 Medium 1640 F-14* F-10 F-16

CaCl 2 388.00 94.00 94.00 CaC12.2H20 44.00 22.00 44.1 22.00 Ca(N0312.4H20 100.0 -...... CuS04.5H20 0.0025 0.0013 0.002 0.0013 FeN02.3H20 0.50 0.250 0.250 FeS04.7H20 0.834 0.420 0.830 0.420 KC1 400.0 285.0 ------KH2P04 - 83.0------MgC12.6H20 122.00 61.00 61.00 MgS04 (Anhydrous) 200.00 100.00 100.00 MgS04.7H20 200.00 100.00 100.00 152.8 100.00 NnS04.H20 ---- NaCl 7559.0 14475 6000.0 11867 7400.0 11867 NaHC03 1176.0 2200.0 2000.0 1688.0 1200.0 1688.0 NaH2P04.H20 ---- 125.0 ---- 62.5 62.5 NaH2P04.7H20 268.0 1512.0 134.0 290.0 134.0 Na2Se03.5H20 3x10-8M 3x10-8M ZnS04. 2H20 134.0 0.03 134.0 ZnS04.7H20 0.860 2.0 0.430 0.430

VITAMINS F-12 Medium 1640 F-14 F-10 F-16 (mg/ml) mg/1 mg/1 mg/1 mg/1 mg/1 mg/1

L-Ascorbate 5x1Q-4M ------Biotin 0.0073 --- 0.20 0.0037 .0200 ---- D-Ca pantothenate 0.48 3.50 0.25 6.99 0.70 1.99 Cholesterol ------Choline Chloride 13.96 3.50 3.0 13.73 0.70 3.73 Folic acid 1.30 3.50 1.0 7.40 1.30 2.40 i-Inositol 18.00 7.00 35.0 22.50 0.50 7.50 Nicotinamide 0.04 3.50 1.0 6.77 0.60 1.77 p-Aminobenzoic acid --- 1.0 --- Pyridoxal HC1 0.06 3.50 6.78 2.05 Pyridoxine HCl 0.06 --- 1.0 0.03 0.20 0.03 Riboflavin 0.04 0.35 0.2 0.69 0.04 0.20 Thiamine HCl 0.34 3.50 1.0 6.92 1.02 1.92 Vitamine 812 1.36 --- 0.005 0.68 1.36 0.63 Table 3-1 continued

AMINO ACIDS Han's 8 RPMI Han's (•g/1) F-12 Medium 1640 F-14 F-10 F-16

L-Al1n1ne 8.96 19.00 13.95 8.96 13.98 L-Arg1n1ne HCl 211.00 441.00 200.00 326.00 211.00 326.00 L-Asparg1n1ne.H20 1S.01 30.00 50.00 22.50 15.01 22.51 L-Aspart1c Acid 13.30 27.00 20.00 20.15 13.35 20.15 L-Cyst1ne --- 84.00 50.00 42.00 --- 42.00 L-Cyste1ne.HCl.H20 35.12 --- 17.56 35.12 17.56 L-61utanic acid 14.70 30.00 20.00 22.35 14.70 22.35 L-Glutanine 146.00 1185.00 300.00 1265.0 146.20 665.50 L-61yc1ne 7.50 15.00 10.00 11.25 7.50 11.25 L-H1st1d1ne.HCl.H20 20.96 147.00 15.00 83.98 20.96 83.98 L-Hydroxyprol1ne ------20.00 ------L-Isoleudne 3.94 183.00 50.00 93.47 2.60 93.47 L-Leudne 13.10 183.00 50.00 98.05 13.10 98.05 L-Lys1ne HCl 36.50 289.00 40.00 162.75 29.30 162.75 L-Phenylalan1ne 114.00 15.00 59.48 4.96 59.48 59.48 L-Prol1ne 34.50 23.00 20.00 28.75 11.50 28.75 L-Ser1ne 10.50 21.00 30.00 15.75 10.50 15.75 L-Threon1ne 11.90 151.00 20.00 81.45 3.60 81.45 L-Tryptophane 2.04 35.00 5.00 18.52 0.60 18.52 L-Tyros1ne 5.40 126.00 20.00 65.70 1.80 65.70 L-Val1ne 11.70 165.00 20.00 88.35 3.50 88.35

OTHER COMPONENTS Han's B RPMI Han's (•9/1) F-12 Medium 1640 F-14 F-10 F-16

0-Glucose 1802 1000 2000 1401.0 1401 1401.0 Lipolc add 0.21 -- --- 0.100 0.200 0.100 Phenol red 12.00 -- 5.00 11.000 11.00 11.00 Llnoleic add 0.08 -- --- 0.040 0.40 0.040 Na-pyruvate 110.0 -- 55.000 55.00 55.00 Putresdne.2HCl 0.16 -- 0.080 --- 0.080 Hypoxanthine 4.10 -- 2.050 4.10 2.050 Thynldine 3x10-6M -- 3.5x10-6M 0.73 1.5x10-6M Coenzyne A --- -- 1.000 --- 61utath1one (Reduced)------1.00 5x10-6M ------Tween 80 -- 5.000 8SA ------0.500 0.500 Hydrocortisone -- IxlO-SM 1x10-8M Insulin (bovine) -- 0.005 --- 0.005 Transferrin —— 0.001 0.001 31 calf and fetal calf serum (FBS) is that the fetal calf has not as yet developed a fully functional immune system. Having a developed immune system could produce protein products that may be cytotoxic. In these studies, we routinely heat inactivated the calf serum at 56°C for a period of 30 minutes prior to being used. Fetal bovine serum, on the other hand, did not require heat inactivation.

Another problem associated with the use of serum is its variability.

In general, serum can vary greatly between species or even by lots. Serum may also differ by the source animal's age, nutritional status or state of health. With fibroblasts, the source of serum used was limited to that of the bovine; either calf or fetal. It should be noted, however, that the variability associated with the use of serum creates many problems when trying to study cellular activities. Examples of some cellular activities that serum may affect are cell proliferation and cell differentiation.

This is especially true when the changes in that activity are small when compared to control responses. Therefore, whenever possible, one should use a defined, serum-free medium, in order to avoid the inconsistencies that have been associated with the use of serum.

The nomenclature used for B medium supplemented with serum is B-0.25

(0.25% serum), B-5 (5% serum) or B-10 (10% serum). In this system, the numbers refer to the percent serum in the medium. For defined medium, the letter F was used to show that Hams F-12 defined medium was present. This was true for all of the F series media except F-16, which was a specially modified B medium, used to investigate drug effects without interference from either serum or growth factors. 32

Fetal Bovine Serum (FBS) was used in various percentages throughout these studies. Stock cultures normally received medium supplemented with

5.0% FBS, while cultures being arrested (growth inhibited or placed into

Gg) received 0.25% FBS. B-10 FBS medium was used for primary cultures or for treating stock cultures that were stressed.

Calf seruia (CS) was used primarily in these studies to augment the use of FBS or for the deactivation of trypsin. Prior to use, calf serum was first heat inactivated for thirty minutes at 56°C, then the serum was added with FBS to give 10% CS/5% FBS. This was useful in reducing the amount of FBS used, while maintaining sufficient nutrients, hormones, growth factors and protein to support the cells. Medium supplemented with

10% CS alone was useful when passing cells.

Stock Medium. The medium used for most stock cultures was B-medium supplemented with 1% (v/v) penicillin, streptomycin, amphotericin B and gentamicin. Fetal bovine serum was used to supplement the stock medium at concentrations of 5 or 10%.

Defined Medium. We have developed two versions of defined (serum free) medium which we refer to as F-14 and F-16. F-14 medium was used for conducting experiments involving the effects of certain drugs (hydantoins, etc) on the ability of growth factors and hormones to induce DNA synthesis and cell division, while F-16 medium was used to study drug induced proliferation without interference from growth factors.

A variation of F-14 medium (F-14 TC), containing 300 ng/ml of PGF^ and

50 ng/ml FQF, was suitable for maintaining stock cultures of Newborn Human

Dermal Fibroblasts (NHDF) for more than two months without the use of serum. During this time period, these cells were passed five times. This 33

medium could prove useful for studying long term effects of drugs, growth factors or hormones upon cell systems, without interference from serum.

For example, serum interference would be critical in determining cellular fibronectin levels from spent medium, since serum contains large amounts of that protein and many other proteins. With this high background, it would be very difficult to determine the drug effects upon fibronectin production.

Growth Factors. The growth factors that were extensively used in these studies were fibroblast growth factor (FGF) and epidermal growth factor (EGF). Prostaglandin F^ (PGFj^) had only limited use. The role for the growth factors, in these studies, was to support normal cell functions and cell proliferation.

Fibroblast Growth Factor (FGF), lot# 618796, was obtained from

Boehringer and made up according to their instructions: 10 pg FGF was dissolved in 10 ml PBS buffer («pH 7.3) containing 1 mg/ml BSA for a final concentration of 1 pg/ml. Aliquots (1 ml) of FGF were stored at -25#C.

Epidermal Growth Factor (EGF). lot# 69692201, also was obtained from

Boehringer. EGF was made up in the same manner as that of FGF, in 10 ml

PBS buffer containing 1 mg/ml BSA, but with a final concentration of 10 pg/ml. One milliliter aliquots of EGF were then stored at -25°C.

PGF^ was made up according to instructions received. One ml of sterile

95% ethanol was added to dissolve the prostaglandin, followed by 19 ml of

PBS containing 1 mg/ml BSA. The final concentration for PGF^ was 50 pg/ml and 5% ethanol. The stock solution, of PGF-^ was divided into 1 ml aliquots, which were stored at -25°C. PGF^ was used in conjunction with 34

50 ng/ml FGF (F-14 TC medium), to test the feasibility of using a serum free system to support cell growth over long periods of time.

CELLS. Primary cultures of Normal Human Dermal Fibroblasts (NHDF) were established from foreskins. The foreskins, which were received through the OSU Cell Culture Service, were first partially digested in trypsin, then minced into small fragments. The tissue fragments were then placed into modified B-10 medium. Primary cultures were first passed when they reached -95% confluency. Periodically, cells were frozen for storage in liquid nitrogen.

WI-38 Human fetal lung fibroblasts (ATCC CCL 75) is an established human cell line obtained from the American Type Culture Collection (ATCC).

The cell line was derived from normal diploid embryonic (third month of gestation) lung tissue by L. Hayflick, from a Caucasian female. The cells retain their diploid karyotype until the very late passages. These cells have a finite lifetime of 50 ± 10 population doublings with a 24 hour doubling time. WI-38 cells grow in a monolayer and form a multilayered membrane when maintained at high confluency [77]. The repository reference seed stock of WI-38 cells have undergone 10 serial subcultures

(about 12 to 14 cell doublings) from the tissue of origin [36]. WI-38 cells have been shown to produce collagen and fibronectin. Additionally, it has been shown that DPH is capable of enhancing serum induced proliferation in WI-38 cells [78].

BALB/3T3 clone A31 fibroblasts (Mouse. Embryo ATCC CCL 163). BALB/c

3T3 cells have been used to investigate the effects of growth factors upon cell proliferation [77]. The BALB/c 3T3A1 cell line was developed by S.A.

Aaronson and G.T. Todaro in 1968 from disaggregated 14-17 day old BALB/c 35 mouse embryos [77]. The nontumorigenic, contact inhibited 3T3 cells were established under a rigid 3 day transfer schedule, in order to limit the amount of cell to cell contact [77]. Petri dishes were inoculated at 3x105 cells/dish in Oulbecco's modified Eagle's medium, supplemented with 10% calf serum. Following a period of rapid proliferation, the proliferation

rate declined over the next 10 to 15 passages and then increased again by

the 20 to 25th passage. When the cells reached their 25th passage, the rate of growth over the three days prior to passage, was more than double

the inoculation density. This rigid three day schedule for cell transfer allowed Aaronson and Todaro to develop and characterize a permanent, contact inhibited and non-tumorigenic cell line [77].

3T3-L1 fibroblasts (House.Eabrvo ATCC CCL 173) and 3T3 Swiss albino fibroblasts (Mouse. Embryo ATCC CCL 92). These two strains of 3T3 cells have been also used by various investigators to study growth factor activity. Of these two, the L1 strain's proliferatory activity is

inhibited by the addition of glucocorticoids while the activity of other strains is not affected [77,79,80].

The 3T3 Swiss albino mouse cell line was developed by H. Green and G.

Todaro in 1962 from disaggregated Swiss mouse embryos. These cells are fibroblast-like, and were developed under conditions that favored the

retention of contact inhibition of cell proliferation. 3T3 Swiss cells are easily transformed by the oncogenic viruses polyoma and SV 40, where

transformation causes the cells to display reduced contact inhibition

[77].

The L1 substrain was developed from the Swiss albino by clonal

isolation. These cells have a morphology that can be described as adipose cell-like and fibroblast-like. This cell line was developed by H. Green, 36

0. Kehinde and M. Meuth in 1974. High serum content has been reported to enhance fat accumulation in these cells. These cells undergo a preadipose to adipose-like conversion as they go from rapidly dividing cells to that of confluent, contact inhibited state [77].

House 3T3 LI cells serve as a reference cell line for normal human fibroblast activity. This is especially true when studying various growth factor induced responses. For example, 3T3 cells were used to characterize the effects of FGF and EGF alone, or in the presence of the hormones insulin and hydrocortisone. Growth factor induced 3T3 cellular responses have become the standard from which growth factor activity is measured and normal fibroblast responses are compared.

CELL GROWTH CONDITIONS. Cells were maintained in a humidified atmosphere at 37°C with 95% Air/5% C02- Stock cultures were inoculated at

10* cells/cm2 and passed when they reach about 90 to 95% confluence. For passage, cells were counted on a Coulter Counter. Vitality testing was performed periodically on the stock cultures with 0.1 g % trypan blue.

CELL PASSAGE. Cultures were fed every other day and routinely passed when they reached about 90 to 95% confluence. At 90% confluence, the number of mitotic cells per field was about 1 to 3, while examining them at a magnification of 10x. The cells were washed twice with cold PBS, then

0.5 ml/25 cm of 0.01% trypsin and 0.02% EDTA were added. The cultures were then incubated for about 5 minutes at 37°C. The trypsin/EDTA mixture was inactivated with B-medium containing 10% calf serum, at a ratio of 10 ml medium per 1 ml of trypsin/EDTA/cells.

The cell suspension was then transferred into conical centrifuge tubes and the cells were pelleted by centrifuging them at 100x g for 5 minutes. 37

The cells were resuspended in fresh medium, then counted by either hemocytometer or cell counter. Cells were inoculated into flasks (T-25 or

T-75) or wells (twenty four well trays 2 cm2/well) at a density of 10* cells/cm2.

CELL COUNTING METHODS. DPH induced proliferation was determined initially by performing cell counts. Cell counts were important because they showed that cell division really took place and that there was an actual increase in cell number. Cell counts were done by electronic

Coulter cell counter.

The principle of operation for the Coulter cell counter is based upon having particles or cells, suspended in an electrolyte, such as normal saline. The cells can be sized and counted by their passage through an aperture in which the current is flowing (82]. As the particles or cells pass through the aperture, they displace an equal volume of electrolyte, thus causing a change in resistance of the current path. This change in resistance then causes a corresponding change in the current and voltage,

Cells/ml - Cell Counts* 40

Where 40 is theDilutionFactor. Cell countswerereaddirectly off theCoulter counter. Equation 3-2. The number of cells is calculated by a Coulter Counter. Multiplying the cells/ml by the total volume of the cell suspension, gives the total number of cells.

with a magnitude of change that is directly proportional to the cell's or particle's volume (size). Counts are recorded from the number of current

(or voltage) impulses that occur per a specific volume of sample suspension which is drawn through the sample aperture. 38

The sample was prepared by diluting 0.5 ml of the cell suspension into

9.5 ml of normal saline solution, resulting in a 20:1 sample dilution.

From the 20:1 dilution, a 500p1 aliquot was drawn up through the lOOp aperture for counting. Counting the 0.5 ml sample, gives a final dilution of 40:1 [82]. Cell counts were calculated by equation 3-2.

CRYOPRESERBATION Fibroblasts and other cell lines were preserved for future use by freezing in liquid nitrogen. Cells were suspended in B- medium, supplemented with antibiotics, 7% dimethyl sulfoxide (DMSO) and

15% serum (5% FBS and 10% calf serum). Cryo vials received 1 ml aliquots of the cell suspension (10 cells/ml), and were subsequently placed into liquid nitrogen vapors prior to their storage in cryotanks.Recovering frozen cells involved an initial rapid thawing, followed by transferring the cells into a T-25 flask with 4.5 ml of B-medium supplemented with 15%

EFBS. Cell viability, as determined by trypan blue exclusion, was about

90%. The medium was changed twelve hours later to remove the DMSO.

Experimental Design. The effect of DPH upon serum stimulated, rapidly dividing fibroblasts was determined first. One reason for using serum with rapidly dividing cells was to compare our results to those obtained by other investigators. Twenty four well trays (each well 2 cm ) were used, and each well was inoculated at a density of 10* cells/cm2. Each well received approximately 2x10* cells. Drug & vehicle free B-medium, supplemented with 10% FBS (B-10), was used to seed the wells initially.

Twelve hours later, all the cells that were going to attach had already done so. The medium was aspirated off and 2 ml of fresh B-10 medium, containing either drug or vehicle, was added. The reason for the delayed dosing was that we wanted all the cells to start off evenly, free from any 39 drug interference. Thus we could measure DPH's proliferatory effect, rather than its combined effects upon proliferation and cell attachment.

The experiment was terminated at the end of 5 days' exposure to DPH or one of its analogues, and the cells were counted. Cell counts were made by either hemocytometer or Coulter cell counter. The problems associated with this system, such as reproducibility, caused us to seek better methods to determine DPH's effect upon proliferation.

Some of the problems, or sources of variability, that we wanted to eliminate from the system were those caused primarily by the variability and high background associated with the use of serum. Attempting to measure a relatively small drug effect upon cell proliferation, on top of the much larger proliferatory effect that is already occurring due to seeding at a low cell density and the presence of serum, proved to be difficult at best. The primary problem was that the drug effect could easily be masked by the other, larger events. With this in mind, we decided to study the drug effect with confluent cultures of quiescent newborn human dermal fibroblasts.

Twenty four well trays were used and each well was inoculated at a

i * density of 10 cells/cm , as before. The cells were feed three days later with fresh B-10 medium, and allowed to become confluent. The cells were then feed fresh B-medium, supplemented with 0.25% FBS to arrest cell growth. Initially, we used 0.5% FBS to starve the cells, however we found that 0.25% FBS gave better results (cells became quiescent faster). This procedure caused the cells to become quiescent, and is referred to as serum starving or arresting cell growth in the Gg phase of the cell cycle.

This procedure also synchronizes the cell population in each well. The 40 schedule for serum stimulated and serum free experiments with quiescent cells is given in figure 3-1.

» ------,------r I I-Synchronize by I I Cell Types I starving cel Is.I I NHDF, 3T3, WI-38 I Cells are fed I I low serum 0.25%I I 1 Week for cells to come I I I to 100% confluence. !-Low serum puts I I-cells into Gg. I Hours Exp ran. 1 I ( 7 days ) I ( 3 days ) I (2.5 days/60 hrs.) 1

I I I I I * 100 % Confluence I Treatment * Feed Cells Terminate •B 10% FBS Experiment Feed Cells B-0.25% FBS + 0.5 uCi [3 H]-TdR Inoculation of cells Serum. Starve + Add DPH,analogue,or vehicle at a density of the cells to -> Serum (10, 5 or 2.5% FBS) 10,000 cells/cm2. arrest growth, or -> Growth factor (FGF & EGF) or -> Growth factor & Serum free

Figure 3-1 SCHEDULE FOR [3 H]-THYMIDINE INCORPORATION IN QUIESCENT FIBROBLASTS.

The cells were held for three days in fresh B-0.25 medium to insure that they were quiescent (in Gg stage of cell cycle). For these experiments, the wells were washed with PBS, and fresh medium was added.

To the medium, either serum (FBS), the growth factors, hormones and/or drugs were added. In earlier experiments, we used the total DNA content as a measure of proliferation, while later, we found it much more convenient to use [fy]-thymidine incorporation. During [^i]-thymidine incorporation, we added the label (0.25 pCi/ml) together with the drugs, growth factors and carrier (1 mM cold thymidine). 41

In order to test the new cell system, we used two reference cell lines, the Balb/C 3T3 mouse cells and the WI-38 human lung cells. With these cell lines, we first verified the effects of serum stimulation, then the effects of the growth factors FGF (50 ng/ml) and EGF (100 ng/ml) upon cell activity. During this time, the effects of the hormones hydrocortisone

(10 M) and insulin (3 ug/ml) were also verified.

Serum free medium was used to study better what interactions, if any, might be taking place between the growth factors and DPH or one of its analogues. For these studies F-14 medium was used along with either a fixed concentration of growth factor or DPH. By using a DPH dose range of

10*13 M to 10 ® H in the presence of 50 ng/ml FGF or 100 ng/ml EGF, we determined the optimal dose for DPH to be 10"'° M. Later, we determined the optimal dose for the growth factors, FGF and EGF, in the presence of

10"10 M DPH. The concentration range used for the growth factors was for

FGF 0, 5, 10, 25, 50 and 100 ng/ml, and for EGF 0, 10, 25, 50, 100 and 200 ng/ml. The concentration ranges used for the growth factors were based, in part, upon those used in the literature [79,80].

Synthesis of DPH analogues o-Cl, p-Br and p-CHj-DPH. The DPH analogues p-Cl-DPH, p-Br-DPH, p-CHj-DPH were used to aid in determining DPH's structure-activity relationship with respect to its proliferatory activity. The synthesis of the analogues was done by a modification of the procedure as outlined by Henze [13]. This part of the study addressed the effects that changes to DPH's structure might have on cell proliferation. DPH structural changescaused changes in the lipophilic properties, molecular size, the reactivity of the phenyl rings and availability of sites to undergo metabolism. 42

Synthesis of the para-chlorinated analogue (p-Cl-DPH) was done by mixing of 2.16 g of p-CI-benzophenone, 12.5 g acetamide, 2.80 g ammonium carbonate and 0.9 g potassium cyanide. These components were heated in a

Parr bomb for 6 hours at 110°C. While the solution was still hot, the reaction mixture was diluted with water, then cooled. The precipitated crude, 5-phenyl-5-(p-Cl-phenyl)-hydantoin, was filtered and treated with a dilute solution of NaOH, in order to dissolve the hydantoin away from the unreacted benzophenone. After filtration, the alkaline extract was then acidified with 6N HC1 to cause separation of the pure solid p-Cl-DPH from the unreacted compounds. The p-Cl-OPH was then filtered & dried. The melting point for p-Cl-DPH was 243°C, with a yield of approximately 2g.

The synthesis of the para-brominated analogue (p-Br-DPH) required the use of 2.60 g of p-bromobenzophenone and 2.80 g of ammonium carbonate, dissolved in 12.5 g of acetamide. To this, 0.9 g of potassium cyanide was added. The reaction was carried out by heating the mixture in a Parr bomb for 8 hours at 100°C. The remainder of the procedure was as outlined for p-Cl-DPH. The melting point for p-Br-DPH was 239#C, with a yield of about

2.40 g. The synthesis of p-CHj-DPH, using p-CHj-benzophenone as the starting material, was performed in a manner similar to those described for p-Cl and p-Br.

Purification and identification of the products involved recrystalization of the impure samples, followed by the instrumental analysis of the purified product. The DPH analogues were further purified by dissolving the dry impure crystals in 20 ml of hot 95% ethanol, making sure that bumping did not take place. The solution was cooled down and 100 mg of decolorizing carbon was added. The solution was again heated to a 43 gentle boil and was then allowed to boil for another 5 to 10 minutes [83].

Following dissolving the impure crystals, the next step performed was hot filtration.

Hot filtration was performed to remove any insoluble particles, such as non-reacted impurities, dust or decolorizing carbon. The hot solution was then filtered via gravity filtration, using a short-stemmed glass funnel and fluted filter paper. Sticks were employed to hold the funnel up off the 125 ml erlenmeyer flask, which was used to collect the filtrate. To the flask, 5 ml of 95% ethanol was added and then heated to a boil, so the hot vapors would prevent crystal formation within the filter paper or on the funnel's surface. If there were particles still present, as there were when the decolorizing carbon was added, then 100 mg of filter aid was added to the solution, and hot filtration was repeated.

The original receiving flask was rinsed out with 5 ml 95% ethanol and the contents filtered. This helped improve recovery.

After filtration, the hot filtrate was allowed to cool slowly to room temperature. The solution was then placed into an ice bath for 15 minutes, so the crystallization procedure could come to completion. If crystallization did not occur, some impure crystals were added to the super saturated solution to initiate (seed) crystal formation. The crystals were collected via suction filtration, using a Buchner funnel and filtration flasks. The filter cake was washed with two portions of cold triple distilled deionized water, then pressed dry with a clean spatula.

The crystals were spread onto a watch glass and covered to dry. After the crystals were dried, they were weighed to check the yield & then the melting points. In addition to melting points, other methods of analysis 44 were also employed to determine the purity of the products. These methods included thin layer chromatography (TLC), Gas Chromatography (GC) and high performance liquid chromatography (HPLC) [13,16,83]. For more details on analysis, see Appendix E.

DRUG TREATED MEDIUM AND VEHICLE. Drug treated medium received either

DPH, one of the DPH analogues: p-Cl-DPH, p-Br-DPH, p-CHj-DPH, p-OH-DPH (p-

HPPH) or m-OH-DPH (m-HPPH), mephenytoin or ethotoin. In addition, phenobarbital, dexamethasone, progesterone and RU-38486 were also investigated. In all cases, these drugs were dissolved in the vehicle, propylene glycol. The amount of propylene glycol added to the medium depended upon the concentration range of that particular drug and if multiple drugs were used. However, for those concentrations ranging from

- Q -1 10 M up to 10 H, the sodium salt of DPH or analogues were used. The free acid form of the drug was dissolved in NaOH, and then used to make up fresh medium from a packet. For later experiments this procedure was not practical, as we purchased premixed medium. In addition, the concentration ranges used in the later experiments were generally quite low and ranged -13 -fl from 10 M to 10 M. At such low concentrations the stability of the drug may be a problem. Therefore it was best to make the drug up in an organic solvent, such as propylene glycol.

Propylene glycol is a commonly used solvent (vehicle) for pharmaceuticals and is considered harmless when taken internally. This lack of toxicity, for propylene glycol, is most likely due to it being metabolized to pyruvic and acetic acid [87]. The formula for propylene glycol is: CHj-CHOH-CH^OH and it has a molecular weight of 76.09 [37]. 45

Other advantages to using propylene glycol, in addition to its being non-toxic, is its general physical properties. Propylene glycol is a relatively viscous liquid that is stable at normal temperatures (boiling point 88 to 94°C). In addition, it is miscible with many common solvents such as water, acetone and chloroform. Propylene glycol can also serve to dissolve many drugs (such as the hydantoins, barbituates or steroids) as well as many essential oils [35]. Another advantage to using propylene glycol as a vehicle, especially when working with cell cultures, is that it will inhibit fermentation and mold growth [35]. This fact can be of great advantage to save an experiment from possible contamination.

Stock concentrations of DPH, analogues and other drugs were made up in

100% propylene glycol (PG) at concentrations of 10*2 M. Propylene glycol was used at a working concentration of 0.02% for those serum supplemented experimental and vehicle control groups which used either higher drug concentrations (up to 10'* M) or multiple drugs. For those experiments that used defined medium and low drug concentrations, the working concentration for propylene glycol was 0.001%. An exception to using propylene glycol as the solvent was with the drug, acetylsalicylic acid

(ASA). ASA (10'® M) was made up in triple distilled, deionized water.

For screening drugs for activity and optimal dose, the concentration ranges used were initially 0.01 to 10 (ig/ml, with 3 pg/ml (1.19x10-5 M) being the reported optimal dose for DPH. Later studies used drug concentration ranges of 10*9 to 10"* M, 10"12 to 10"® M and finally 10~13 to

10 M in defined, serum free, growth factor free medium.

General Procedure for Lvsinq Attached Cells. This method makes use of cells still attached to the bottom of their wells in order to determine 4 6 the total DNA or protein content of the lysate and t^]-thymidine incorporation.

For [^H]-thymidine incorporation/ 20 pi of labelled medium was pipetted into cocktail vials for reference, followed by 15 ml Aquasol-2. The medium was aspirated and the wells were washed twice with PBS. 1 ml cold 10% TCA was added to each well, incubated 15 min and then aspirated. The cells were washed with 1 ml cold 5% TCA. One ml of cold 100% ethanol was added and then allowed to incubate for 3 0 minutes, at room temperature.

The ethanol was then aspirated and the wells were allowed to dry for about 30 min. If necessary, the trays could be sealed and stored at room temperature overnight for use the next day. To each well, 600 pi of 1 N warm NaOH was added and then incubated for 40 min at 40°C. The NaOH was neutralized by adding 600 pi of 1 N HC1, giving a total volume of 1.2 ml.

The lysate was then used for determining DNA, protein or the incorporation of tritiated thymidine.

Total DMA Content. The total cell ONA content for the treatment groups was analyzed by Hoechst 33258 dye binding* The final working dye concentration was 1 ng/ml [88,89], and was made up from a stock solution of 4 ng/ul Hoechst 33258 dye in buffer "B". The buffer (buffer B) contained: 10 mM Hepes, 200 mM Naci and 10 mM MgCl2 and was adjusted to a pH of 7.2.

Stock standards were made up from calf thymus DNA in TNE (Tris-NaCl-

EDTA) buffer. The concentrations used were: 0, 50, 100, 250, 500, 1000 and 2000 ng/ml and were made up in 1 ml of neutralized 1 N NaOH (1:1 IN

NaOH:1N HC1 mix). 1 ml of sample cell lysate, standard or blank, was added to 1 ml of buffer B containing 2 ng/ml of dye, and then vortexed. 47

Samples were incubated at room temperature for 45 min, in the dark. The

standard curve for DNA concentration was generated on a Farrand Ratio 2

spectrofluormeter at an excitation wavelength of 350 nm and read at an

emission wavelength of 490 nm. The quantity of DNA per culture group was

then determined from this standard curve.

Total Protein Content. The Bicinchoninic Acid (BCA) protein assay [90]

was used to determine the total protein content for the treatment groups.

Standards of bovine serum albumin (BSA) were made up in 1 ml of

neutralized 1 N NaOH (1:1 1 N Na0H:1 N HCl mix) at concentrations of 0,

10, 25, 50, 100 & 500 pg/ml. The reagents used for the BCA protein assay were: a) reagent A: consisting of 1% BCA solution, pH 11.25 and b) reagent

B: consisting of 4% CuS0(.5H20 in triple distilled, deionized water.

The BCA working reagent was prepared by mixing 50 parts Reagent A to

1 part Reagent B. Then, 50 or 100 ul of sample or standard was added to 1 or 2 ml of working reagent, and mixed. Standards and samples were read at

562 nm on a Beckman model 35 spectrophotometer. The standard curve for concentration vs absorption was determined by linear regression.

Rate of DNA Synthesis was determined by investigating [3H]-thymidine

incorporation. The cells were cultured in fresh media, supplemented with

[^H]-thymidine (83 Ci/mmole, New England Nuclear, Boston, Mass). The rate of cumulative DNA synthesis is measured in 24-well trays by adding 0.5

pCi/m1 [^J-thymidine/ml at the time that the experiment is set up (See

Experimental Section under Cells). The cells are lysed after 60 hours of

incubation. From the 1.2 ml of lysate, 500 pi was added to the cocktail vials containing 15 ml of Aquisol-2. The vials were counted for 10 minutes on a Beckman 7000 liquid scintillation counter. The experimental culture system utilized cells that were grown out to

100% confluence, in B-5 medium. The cells were inoculated at a density of

10* cells/cm2 into twenty four well culture trays, each well having an area of 2 cm .

STATISTICAL TESTING We used ANOVA to test if there were any significant differences between the different doses of DPH and control.

In addition, the paired T-test was used to test the hypothesis that there were differences between matched sets of drugs, and between those drugs and their controls. The unpaired T-test was used to test if any significant difference existed between treatment groups and their controls over many experiments. RESULTS

Development of the cell culture svstea.

Selection of Method for Analyzing Growth. Three different methods were tested for the determination of DPH-induced cell proliferation. The assays used were cell counting, total DNA content and [^3-thymidine

incorporation. Cell counting was an important first step, as it established that DPH did cause an increase in the cell numbers. Also, cell counting served to validated the other assays as measures of cell proliferation.

Cell Counts. Our initial culture studies used rapidly dividing human newborn dermal fibroblasts, grown in medium supplemented with 10% FBS.

For these experiments, the cells also received either DPH or propylene glycol (vehicle) only. In order to determine DPH's effect upon cell proliferation, the cell number was determined by hemocytometer for the preliminary studies, while later counts were made by Coulter counter.

The purpose for doing cell counts was to test the hypothesis that DPH was able to cause an increase in the number of fibroblasts, when compared to controls. Furthermore, this would serve to confirm that there was an

increase in cell division. Previous studies showed that 3 pg/ml (1.19xlO*5

M), of DPH was the most effective dose for inducing proliferation in serum-stimulated fibroblasts [78,91]. Therefore, the concentrations of

DPH used in this study ranged from 10"S M (0.25 ng/ml) to 10** M (25.2 pg/ml).

49 so

Determinations for DPH-induced increases in cell number were made by

Coulter electronic cell counter. Cells were fed B-medium supplemented with

10% FBS and DPH (10"9 to 10"* M or 0.25 to 25.2 pg/ml) or vehicle. Figure

4-1 shows the corresponding dose response curve for DPH-induced increases

in cell number. The observed response for DPH at 10"* M was 140% of the control cell counts, while at 10"5 M the response was 170% of the control.

The observed maximal effective dose was 10 M DPH, with a response of 277% of the control (exp-1, Table 4-1). Subsequent experiments (exp-2, Table

4-1) used newborn human dermal (NHD) fibroblasts from a second donor.

The magnitude of the DPH-induced response in the second experiment

(exp-2) was less than that observed with the first experiment (exp-1). For .7 example, the observed response at 10 M was 123.8 ± 1.2% of the total control cell number. In contrast to the observed response at 10"7 M, 10"5

H DPH gave a response that was significantly less than of the control. It should be noted that the NHD fibroblasts, used in the second DPH study were from a different donor than the first. These experiments show that

DPH causes an increase in the cell number at concentrations less than 10’5

M for both cell groups. However, at a concentration of 10"5 M, DPH produced a response (86.4 ± 3.5%) less than that of the control (to see the analogue cell counting data, turn to section on structure activity relationship). From these experiments, we can conclude that DPH causes a significant increase in the total cell number, when compared to the controls. High concentrations of DPH can be toxic to some cells (exp-2), dropping the number of cells to below control levels. In addition, these studies are important in supporting further investigations (total DNA content and [ H]-thymidine incorporation) into DPH-induced proliferation. Figure 4-1. DPH dose response in human dermal fibroblasts. Cells were counted on a Coulter electronic cell counter. Rapidly dividing cells were fed B-medium supplemented with 10% FBS. + DPH (Exp-1) p DPH (Exp-2)

51 of Control Cell Densities OJ O CD ™ 200 250 300 100 150 -10

-9 I o (DPH), Molar Log -8 ______

Figure 4-1 -7 I ______

-6 I I ______

-5 □ I I ______

-4 I I _ 53

Table 4-1

Total Nuaber of Cells DPH-induced Proliferation -in Hunan Dermal Fibroblasts

DPH Experiaent-1 DPH Experinent-2 Molar Cone % Ctrl ± S.D. % Ctrl ± S.D. -S’ 10 I M 158.0 * 28.5 10'? M 184.5 ± 9.5 116.7 ± 1.6 10-' M 277.1 4 25.8 123.8 ± 1.2 10' M 187.6 * 22.1 106.9 ± 15.9 10"' M 170.4 ± 16.4 86.4 ± 3.5 10"4 M 142.0 ± 19.6

The cells used in these studies were normal human dermal fibroblasts, but from different sources. This, in part, accounts for the differences that exist between the DPH-induced responses of two experiments. Results are expressed in % of the control cell counts. N » 5 in experiment 1 and N = 4 in experiment 2. In experiment 2, the cell number for 10 M DPH was not determined due to a lab accident, while 10~4 M was omitted because the concentration was too high and would contribute little information.

Total DNA Content. The total cellular DNA content was used as a measure of DPH-induced growth in rapidly dividing cells and also served as a means of confirming the cell counting data. Results indicated that as the cell number increased in response to DPH, the total DNA content also increased. Similar to previous experiments, serum stimulated NHD fibroblasts were used.

The maximal effective dose obtained for DPH, as shown in table 4-2, was

10'8 M with a response of 134.6% of control DNA content. The observed .I response at 10 M DPH was 134.3% of control, and was not significantly different from the response observed at 10~8 M. However, 10'8 M DPH produced a significantly greater response than the other concentrations tested, when analyzed by AN0VA & the Bonferroni Post Test (fig. 4-2, Table

4-2). The ED5q from this curve was 2.47x10'" M DPH. Figure 4-2. DPH dose response in human dermal fibroblasts. Cell proliferation was determined by total DNA content. Rapidly dividing cells were fed B-medium supplemented with 10% FBS.

54 150

c a> 1 4 0 c o CJ> 1 3 0

120 o

o 110 CJ> o 100

i -ii -io -9 5 -Log (DPH), Mo lor

Figure k-2 Table 4-2

DPH Dose Response deterMlned by Total DNA Content.

(DPH), Molar % Ctrl ± S.D. Significance ,-i£ 1. 10 105.6 7.3 ns -11 ± 2. 10 114.4 ± 8.2 P<0.010 3. 10-10 126.2 8.0 p<0.001 - 9 ± 4. 10 126.4 ± 8.0 p<0.001 - 8 5. 10 134.6 8.8 p<0.001 - 7 ± 6. 10 134.3 ± 4.8 p<0.001 - 6 7. 10 126.1 ± 3.2 p<0.001

DNA Content (ng/ml) expressed as a percentage of the control In Hunan Dermal Fibroblasts. N«3 experiments. Rapidly dividing cultures of NHD Fibroblasts were fed B-medium supplemented with 10% FBS and either vehicle or varing concentrations of DPH. Significance was determined by ANOVA and the Bonferroni Post Test.

From these experiments, it was clear that DPH did cause a dose-related increase in the total DNA content. The use of total DNA content to measure DPH-induced proliferation was more convenient and sensitive than cell counting. However, in many latter experiments (data not shown) the high background fluorescence may have masked some of the DPH-induced effects, especially since this was a small response. Also, other variables may have contributed to these problems, such as rapidly dividing cells and serum. Thus, the first step would be to use a more sensitive assay to measure DPH-induced proliferation, [* H]-thymidine incorporation.

[ H]-Thymidine Incorporation. In the previous experiments, using either total DNA content or cell counts, DPH was shown to affect fibroblast proliferation in a dose dependent manner. Cell counts showed that there was a definite increase in the cell number for the treated groups, when compared to their controls. Additionally, DPH was shown to induce a dose dependent increase in the total DNA content. Therefore, 57 since OPH was able to affect the total DNA content, the next step was to determine the effect of DPH on the rate of DNA synthesis. This was done by measuring [ 3 H]-thymidine ([ 3H]-TdR) incorporation.

The Effect of DPH on [^J-TdR Incorporation in Rapidly Dividing Cells.

Initially, cells were incubated with either DPH or vehicle control (0.001% v/v propylene glycol), for 6 or 12 hours (Table 4-3). DPH concentrations of 10”11, 10”9 and 10"7 M were used in these experiments, rather than a full dose response curve, since it allowed for a more rapid screening of DPH- induced effects. Both the 6 & 12 hour incubation times produced similar results, except at 10~7 M. At 10'11 M DPH, both incubation groups gave almost identical responses at 6 hrs (125 ± 12%) and 12 hrs (129 ± 16%), while at 10~9 M, both incubation times produced their maximal response.

The 6 hour incubation gave a maximal response of 135% and the 12 hour incubation gave a maximal response of 142% of the control response.

Table 4-3

A comparison of different incubation times on [3 H]-TdR incorporation by DPH treated NHD fibroblasts.

DPH 6 Hour Incubation 12 Hour Incubation Molar CPM ±S.D %Ctrl CPM fS.D %Ctr1

Control 7361 ± 1877 100 6625 ± 674 100

t°:1j 9219 i 885 125 8535 ± 1065 129 1° ? 9962 ± 299 135 9396 ± 1268 142 10 7 9371 ± 1976 127 5863 ± 155 88

N ■ 3 for the 6 and 12 hour incubations. B-mediua + 5% FBS.

However, at 10~7 M DPH the response obtained for the 6 hr incubation was

127 ± 27% of the control, while the 12 hr incubation produced a response less than that of control levels (88 * 3%). Thus, these results suggest 5 8

that in rapidly dividing cells a 6 hour incubation time was better for measuring DPH's effect on [^J-TdR incorporation than the 12 hr incubation.

However, previous experiments showed that 10*9 M DPH produced a response

(105 ± 1.9%) not significantly different from the control, while responses

for both 10 -11 and 10 -7 M were less than control.

These experiments illustrate some of the variability found in this system. Contributing factors to the variability may include the use of rapidly dividing cells and serum. The decision was made to carry out further experiments using quiescent cells.

Treatment Schedule for Quiescent Cultures. The quiescent culture system was modeled after that described by O'Farrell and Jimenez De Asua

[79,80]. The cells used to develop this system were cultures of newborn human dermal fibroblasts, derived from foreskins. Briefly, cultures were grown out to 100% confluency and cells were brought to quiescence by reducing the serum to 0.25% FBS for 3 days. Cells were then stimulated to divide with fresh medium containing serum. Initial experiments used various percentages of serum (0.5, 2.5, 5 & 10% FBS with B medium) to test the feasibility and responsiveness of the system. After treatment, cultures were incubated for 48 or 60 hours prior to harvesting.

Testing the System and Selection of Cell Line. House 3T3 cells were selected first because they have been well characterized with respect to their proliferative response to serum, growth factors (EGF and FGF) and hormones (insulin and hydrocortisone).

The next series of serum tests used NHD fibroblasts. For these studies, duplicate experiments were run over a 40 hour time period. Serum Figure 4-3. Mean serum dose response in NHD fibroblasts. Cells were serum growth arrested for 3 days with 0.25% FBS. Cells were then fed fresh medium containing fTI]-TdR and incubated for 40 hours with various percentages of serum.

59 III!

7500

5000

2500

0.50 2.50 5.00 Percent Fetal Bovine Serum (FBS) 61

responses were well matched between the two experiments. Again, 0.5% FBS

served as the control with working percentages of FBS of 2.5, 5.0 and 10%.

A mean maximal response of 351.5 ± 10.9 percent of the control was the

observed response for 10% FBS (fig. 4-3). When DPH concentrations of 10"11,

10"s and 10”7 H were run in this system, only 10”7 M was able to induce

proliferation to any significant degree. This low level of activity for

DPH was troubling, thus prompting us to further modify the system with the

use of growth factors.

Action of DPH with Growth Factors. Following the experiments testing

the responsiveness of the system to serum-stimulated growth, growth factor

and DPH proliferatory activity was examined. The growth factors FGF

(fibroblast growth factor) and EGF (epidermal growth factor), along with

other trophic factors, are able to stimulate fibroblasts to proliferate

[76,92,93,100]. However, little information exists with regard to possible

interaction between DPH and the growth factors. These experiments were

intended to explore the possibility that DPH-induced proliferation was due

to DPH modifying the activity of either FGF or EGF. We thought that DPH may induce proliferation by enhancing the growth factor activity. This

hypothesis was tested by comparing the responses observed for the

individual "control groups" (50 ng/ml FGF or DPH) to those groups

containing the combination of 50 ng/ml FGF with 10”s and 10'7 M DPH. We

expected to see an increase in the FGF-induced response, due to the

presence of DPH.

The results from these studies showed that quiescent cells will

respond to DPH, but due to the amount of variability encountered and the

small DPH-induced response, the system would have to be further modified in order to accurately determine the effects of DPH. Initially, rapidly dividing cells were eliminated to reduce the amount of variability present in the system. This step helped make the system more manageable, but it was apparent that serum would have to be deleted. Since the serum-induced response is many orders of magnitude larger than that from DPH, DPH's activity would periodically become lost in the background. Also, with different batches of serum, variations in this background activity would occur. It is also possible that serum may contain molecules that could interfere with DPH binding sites. In place of the stock, serum supplemented B-medium, a defined medium containing either the growth factors FGF or EGF or growth factor free would be used for the DPH experiments.

Growth Factor Free Medium Controls. For the growth factor dose response experiments, growth factor free (GFF) medium containing either

DPH (10_1® M) or vehicle (propylene glycol) served as the controls. In these experiments (N=18), quiescent NHD fibroblasts were used. The GFF controls from the FGF and EGF dose response experiments indicated that DPH was able to induce low levels of cell proliferation, independent of serum or the growth factors EGF and FGF (fig. 4-4). The mean response for the

DPH treated control group was 21.30 * 4.78 CPM/ug protein (148.72 * 33.38% of control). The vehicle controls gave a response of 14.32 ± 3.44 CPM/ug

Protein (100 ± 24.02% of control). To determine the significance of DPH's effect, a paired, single tailed T-test was conducted. This effect for DPH was highly significant (p<0.0035). However, there was a need to determine whether DPH could effect growth factor-induced cell proliferation. Figure 4-4. DPH-Induced Proliferation in Growth Factor Free Medium. NHD Fibroblasts were fed serum free medium (F-14) containing either DPH (10 M) or vehicle.

63 CPM/ug protein 30 * p < 0.0035 < p * Vehicle Control Vehicle Growth Factor Free Medium.Free GrowthFactor Figure 4-4 DPH(-10M) * r 65

FGF & EOF Dose Response with DPH. In order to determine the effect of

DPH upon the dose response curves for FGF and EGF, DPH was held constant at 10"18 M and the concentrations of FGF and EGF were varied. The medium used for these experiments was defined and serum free F-14 (BC). The range of concentrations used for both EGF and FGF, was from 1 to 100 ng/ml.

For the EGF dose response curve with DPH, the EMX was 42.61 ng/ml with an EDS0 of 14.21 ng/ml and Hill coefficient of 1.44. The control EGF curve had an EMX of 43.97 ng/ml, an EDS0 of 18.19 ng/ml and a Hill coefficient of 1.25. The FGF dose response curve with DPH had an E#ax of 83.68 ng/ml with an ED50 of 27.30 ng/ml and Hill coefficient of 1.23. The control FGF curve had an Enax of 77.55 ng/ml, an EDS0 of 25.02 ng/ml and a Hill coefficient of 1.16, as shown in figures 4-5, 4-6.

DPH Dose Response with FGF & EGF. In these experiments, normal human dermal fibroblasts were fed defined, serum free medium (F-14) treated with

50 ng/ml FGF or 100 ng/ml EGF. In addition, cells received either vehicle

(0.001%) or DPH (10-13 to 10"8 M).

The E ^ for the DPH dose response curve with 50 ng/ml FGF was 10*18 M

DPH and had a mean response of 115.7 ± 1.45 % of control CPM/pg protein.

The ED5q (N=5) for these experiments was 2.05x10’12 M DPH and was calculated from the rising portion of the curve (fig. 4-7 & Table 4-4). At higher concentrations (10*9 and 10"8 M), the amount of DPH-induced proliferation decreased. With the next series of experiments (N=5), the DPH's dose response curve was determined with 100 ng/ml EGF. As before, the EMX for

DPH was 10~18 H and had a mean maximal response of 178.9* 23.15% of control

CPM/pg protein. The EDS0 for DPH was 3.97x10~12 H and was calculated in the same manner as before (fig. 4-8 & Table 4-4). Figure 4-5. EGF Dose Response with 10 M DPH. NHD Fibroblasts were fed serum free medium (F-14) containing DPH or vehicle. ^ EGF Control Response ^ EGF Dose Response + DPH.

6 6 CPM/ug Protein O EGF Dose Response + DPI!+ (-10 Response M) Dose EGF O ♦ EGF Control Response Control EGF ♦

G ng/ml EGF Figure 4-5 100 Figure 4-6. FGF Dose Response with 10~10 M DPH. NHD Fibroblasts were fed serum free medium (F-14) containing either DPH or vehicle. A FGF Control Response A FGF Dose Response + DPH.

68 CPM//ig Protein 100 25 - FFDs epne+DH (-10 M) DPH + Response Dose FGF O ♦ FGF Control Response Control FGF ♦ 1

FGF ng/ml FGF gur 4-6 re u ig F 10

100

Figure 4-7. DPH Dose Response with 50 ng/ml FGF. NHD Fibroblasts were fed serum free medium (F-14) with 50 ng/ml FGF.

70 CPM/ug Protein I— 1 I— 1 I— 1 I— * CD CD I—1 CD DO CD Percent Percent of the Control. I f— > f— > CO o o o -Log (DPH), Molar

Figure 4 -? U Figure 4-8. DPH Dose Response with 100 ng/ml EGF. NHD Fibroblasts were fed serum free medium (F-14) with ng/ml EGF. o u 200 -U £ -H o CD o 4-> 175 CD O U JZ 4-> CM fcn 150

4-3 £ g CD o 125 O U CD CM 100 I J______I______I______l__

-13 -12 11 -10 -9 -8 -Log (DPH), Molar

F ig u re 4-8 Table 4-4

Effects of OroMth Factors & Serum on DPH Dose Response Curve Newborn Human Dermal Fibroblasts.

DPH DPH METHOD & Growth Factor E*ax EDS0 (M) 1) B medium + 10% FBS

a) Cell Counts 1.73X10 ' J 1 0 -! b) DNA CONTENT 10 8 2.47x10 '''

2) Defined medium (Serum Free [^i]-TdR)

a) EGF 100 ng/ml 3.97X10 b) FGF 50 ng/ml 10 "10 2.05X10 "12

In both studies, the EW)( for the DPH dose response curve was 10~10 M but the overall mean magnitude of responses for the EGF treated groups

(178.90 ± 23.15% of the control) were considerably greater than that of the FGF treated group (115.6 ± 3.9%). Thus, in the presence of EGF, DPH produced a greater overall effect than with FGF. Furthermore, when comparing the two median effective doses, the ED5q for the FGF treated group was about half that obtained with EGF. Whether these differences are really significant or not remains to be seen, but the results suggest that in the presence of FGF, the cells appear to be more responsive to

DPH. However, in the presence of EGF, the magnitude of DPH's response was greater than with FGF.

From these studies, it appears that there may be some interaction taking place between DPH and the growth factors. This was reflected by changes that occurred in the EDS0 for the DPH dose response curve, when co­ incubated with EGF, FGF or when under GFF conditions. Also, DPH was able to induce proliferation independent of the use of serum, growth factors and the hormone, hydrocortisone. 75

Choice of Medium for Study of PPH-Induced Proliferation under Growth Factor and Serum Free (GFF) conditions.

From the growth factor studies, it became apparent that DPH was able to induce proliferation in the absence of serum or growth factors. The next step was to find the optimal medium to support DPH-induced proliferation.

OFF Cell Proliferation with DPH. Various selected media were screened for their ability to support cell activity, in the absence of growth factors or serum. The base component of the defined media was modified HEM

(B-medium or B) & Hams F-12 medium, either individually or in a 1:1 combination. The media used included: 1) F-14 media group: 1:1 B-medium to F-12. 2) B-medium group. 3) F-12 medium. Also, other base components include: BSA (500 MSl/ml)* Insulin (5 pg/ml), Hydrocortisone (1.0x10"® M),

Na2SeO 3 (3.0x10'8 M) and Transferrin (1.0 pg/ml).

The reference medium: B-medium, supplemented with 10% fetal bovine serum (B-10% FBS), serves as the medium for the maintenance of stock cultures. Additionally, B-10 medium was used directly in some of the earlier DPH experiments. Figure 4-9 shows the overall magnitude of the media induced cell division on quiescent fibroblasts. Also, individual controls were matched, or paired, with its particular DPH (10~^ M) experimental group. Also, the results obtained from the different media groups were viewed with respect to overall magnitude of the cellular response and as a percentage of the individual controls.

F-12 medium, which is rich in inorganic salts but low in amino acid concentration, gave the worst response with respect to both the magnitude of the medium and DPH effects. For the F-12 cultures, the cells appeared to be in poor condition. Based upon these results, the combined F-12:B medium (F-16) was selected for use with future DPH experiments. Both defined B-medium and B-medium with 10* FBS gave the greatest overall response among the controls. However, the lowest DPH induced activity

(121.4% of matched control) occured in B-medium and in B-10% FBS (122.4%).

In contrast to good cell condition and high responses observed in B- medium, the condition of the cells cultured in F-12 medium were very poor, especially for the DPH treated group. Thus, the response observed with F-

12 medium control (5.6 CPM/ug) did not reflect the poor condition of the cells. Cells given DPH treated F-12 medium had a response of 2.4 CPM/pg and were in worse condition than the controls. The cells in F-12 medium appeared as long, thin spindle shapes reflecting a starved condition.

Thus, the DPH-induced response for F-12 medium was only 42.9% of the matched control activity. The combination medium, F-12:B, gave a control response (22.7 CPM/pg protein) which was nearly 50% of that observed for the two B medium controls. The DPH treated group gave a response of 49.5

CPM/pg protein or 218% of the matched control's response. This was by far the greatest DPH-induced response obtained for all the media groups tested

& was referred to as F-16 medium.

The next best medium for studying the response due to DPH was F-14 +

PGF^ (F-14 TC) followed by F-14 Basal. The control response obtained for

F-14 TC was 7.8 CPM/pg and for F-14 Basal 7.1 CPM/pg. With the addition of

10'10 M DPH, the response for F-14 TC was 13.6 CPM/pg and for F-14 Basal

10.6 CPM/pg. When DPH's response was viewed as a percent of the matched individual controls, F-14 TC gave the second highest DPH response of

174.4%, while F-14 Basal gave the third greatest response of 149.3%. Figure 4-9. Growth Factor Free Medium was Screened for its ability to support DPH-induced Proliferation. NHD Fibroblasts (n=3) were fed defined GFF medium containing either DPH (10 M) or vehicle (propylene glycol). □ Medium Control, ■ Medium + BFH.

77 fT

F-12 F-14 F-16 B B+10% FBS I I Media Control ■ I Media + DPH (-10M) F-14 BC & HCF (HCF = hydrocortisone free) are matched media which gave responses that were nearly equal for both the overall magnitude of counts per pg protein for the media controls (F-14 BC 4.9*0.2 CPM/pg; F-14 BC HCF

4.9*0.1 CPM/pg) and for the DPH treated media (F-14 BC 6.6*0.3 CPM/pg; F-

14 BC HCF 4.9*0.4 CPM/pg). DPH's effect upon the cells was 134.7% of the control response for F-14 BC and 140.8% for F-14 BC HF (hydrocortisone free). From these experiments, it appears that the inclusion of hydrocortisone seemed to make little difference between these two media.

In fact, it may be that the inclusion of hydrocortisone may have actually decreased the magnitude of DPH's effect. From this study, F-16 medium was observed to give the best overall response for DPH-induced proliferation and was selected for use in future experiments.

DPH Dose Response in Qrowth Factor Free (OFF) Medium. The dose response curve for DPH was determined in growth factor free medium, F-16, in the same manner as was the DPH dose responses with EGF or FGF (fig. 4-10).

The concentration range used was 10”13 to 10’8 M, from which the E—aJt & ED50 were determined. The for DPH treated GFF medium was 10',° M, as it was for FGF & EGF treated medium. The maximal response observed for DPH (135% of the control) was comparable to the responses obtained with FGF (115.7

* 1.5) and EGF (178.9 * 23.1). The ED50 for DPH in GFF media was 5.36x10~11

M, which matched well with the ED5q's determined for DPH with EGF and FGF.

Structure Activity Relationship. In order to better understand the nature of DPH’s involvement in enhancing cell proliferation, a structure- activity relationship study was conducted using various analogues of DPH.

The initial compounds investigated were para-substituted analogues of DPH Figure 4-10. DPH dose response curve in GFF medium. NHD Fibroblasts (N=3) were fed serum and growth factor free medium (F-16) containing various concentrations of DPH. Data is expressed as a percentage of the control.

80 150

140

a -«H o CD 130 u 4-J 4 -J o a o Q* o 120 tn o d P § 110 o 100

J______L

11 -10 -9 (DPH), Molar

Figure 4-10 82 that included p-Cl-DPH, p-Br-DPH and p-CH3-DPH. Also, the major metabolite of DPH, p-OH-DPH (HPPH), was investigated for its effect upon cultured fibroblasts.

The initial structure activity studies were conducted in conjunction with DPH (Table 4-1, Exp-2). For these studies, the DPH response served as a reference by which comparisons in activity could be made. Analogue-

induced increases in cell number were determined by Coulter counter, as in the earily DPH studies. Additionally, the medium used in all of the cell counting experiments (with DPH or analogues) was B-medium, supplemented with 10% FBS. This step was necessary as it confirmed that increases in

[ H]-thymidine incorporation were due to increases in the cell number.

The analogue concentrations used ranged from 10 -fl M to 10 -5 M, as in the corresponding DPH study. The maximal effective dose observed for DPH _7 was 10 M and a maximum response of 124 percent of the control cell density (Table 4-5) was observed. DPH, at a concentration of 10~5 M, gave a cellular response of only 86 percent of the control, while in previous studies, 10~5 M DPH gave responses that were greater than control. The analogues proved somewhat more potent than the parent DPH. The most active analogue was p-Cl-DPH. The maximum effective dose observed for p-Cl-DPH was 10"Sl, with a response of 379% of the control cell density. The second most active analogue, with these cells, was p-OH-DPH, which was thought to have very little activity associated with it. The next most active analogue was p-Br-DPH, while p-CHj-DPH gave a response less than that of

DPH (Table 4-5).

The increased proliferation caused by p-OH-DPH, rose steadily from a minimal response of 133% of the control at 10"8 M, to a plateau with a 83 maximum of 151% of the control at a concentration of 10"5 M. The analogue, p-Br-DPH, was able to induce a maximal response of 152 percent of the control, at a concentration of 10‘® M. The response for p-Br-DPH, at lower doses, was about the same as that for DPH, while being much less than that of p-OH or p-Cl-DPH. The least effective analogue for this study was p-CH3-

DPH, with an of 10"7 M and a maximal response of only 114% of control.

We can conclude from these cell counting studies that the analogues are able to stimulate an increase in the total cell number, as does DPH.

Table 4-5

DPH Analogue-Induced Fibroblast Proliferation.

Concentration DPH (Exp-2) p-OH-DPH p-CH,-DPH (N=4) %Ctrl * S.D. %Ctrl * S.D. %Ctrl * S.D.

10'J M 116.7 * iTi 133.4 * 2T 5 99.0 * 0.1 10': M 123.8 * 1.2 148.0* 2.4 109.1*4.6 10"l M 106.9 * 15.9 149.9 * 8.7 68.2 * 8.0 10'® M 86.4 * 3.5 150.8 * 14.5 122.7 * 4.3

Concentration p-Br-DPH p-Cl-DPH (N=4) %Ctr1 * S.D. %Ctrl * S.D...... 1 0 ; h 118.2 * 3.1 284.7 * 3.3 10"' M 116.1 * 2.7 291.8 * 13.2 10"® M 151.6 * 13.1 378.5 * 47.2 10"® M 150.4 * 3.1 234.9 * 18.5

The cellular responses for DPH and some of its analogues was determined by cell counting (Coulter Counter). For all cell counting experiments, NHD fibroblasts were cultured in B-medium supplemented with 10% FBS. The cells used in DPH experiment Exp-1 (Table 4-1) differed from those used in the other experimental groups (Exp-2 & analogues).

Structure Activity Relationship in Growth Factor Free Media. The parent molecule to DPH, hydantoin, has hydrogens at positions R,, R2 and R3 of its ring, and was able to induce proliferation to a greater extent than DPH. Figure 4-11. The effects of hydantoin (HYD) on cell proliferation. NHD Fibroblasts (N=7) were fed serum and growth factor free medium (F-16) containing either hydantoin (10 M) or vehicle. These experiments show that hydantoin had a significantly greater ability to induce cell proliferation than the vehicle control (p<0.0035). Significance was determined by the paired T-test.

84 CPM/ug protein * < p 0.0035 Vehicle Control Control Vehicle Growth Factor Free Medium. Free Factor Growth Figure 4-li HYD(-10 It/I) /i V 00 86

Hydantoin produced a highly significant response (p<0.0035, N=7) of 42.96

± 2.77 CPM/pg (152 t 9.8% of the matched control). For this experiment, the matched control gave a response that was 28.36 ± 3.30 CPM/pg. When compared to the total mean control, hydantoin gave a response was 162 ±

10.5% of control activity (fig. 4-11).

In man and rat, the major DPH metabolite is p-OH-DPH (HPPH). HPPH was screened for its growth promoting activity in this system. The responses obtained for p-OH-DPH and the matched controls, for five experiments (N=5) were: p-OH-DPH 22.96 * 5.10 CPM/pg and the control: 20.20 ± 5.10 CPM/pg.

Results from these experiments indicated that p-OH-DPH had no significant effect upon fibroblast proliferation, when compared to the controls.

Another metabolite of DPH, m-OH-DPH, was investigated. This metabolite was found to be more active than the DPH molecule. The response for DPH was 34.68 ± 3.27 CPM/pg while m-OH-DPH was 43.04 ± 11.04 CPM/pg protein

(N=7). From this, it appears that 10'1® M m-OH-DPH had an activity about equal to that of hydantoin. The response observed for the matched control was 24.11 ± 4.40 CPM/pg. The effect observed with m-OH-DPH amounted to a response of 179 ± 45.8% of the matched control, with a level of significance of p< 0.035, using a single tailed, paired t-test. When compared to the mean total control, m-OH-DPH*s effect amounted to a response of 162 ± 41.7%, while the response for hydantoin was 162 ± 10.5% and DPH 131 ± 12.4%. Other analogues of DPH were also screened for their activity (Tables 4-6).

The two para halogenated analogues, p-Cl-DPH and p-Br-DPH, were tested in this system for their activity along with the para methylated analogue, p-CHj-DPH. We found that all three analogues were very active, with the 87 chlorinated analogue (270% ± 32.8%) being the best overall for inducing cell proliferation, followed closely by p-Br-DPH (232% ± 25.3%) and finally by the para methyl analogue (188% ± 52.9%).

The clinically available anticonvulsant drugs: Nephenytoin & Ethotoin, were tested for their ability to induce cell proliferation. We found these two compounds to be more active in this system than DPH. Of the two compounds, mephenytoin (57.4 ± 2.2 CPM/pg protein) was more active than ethotoin (45.9 ± 4.05 CPM/pg protein; Table 4-6).

Other Modifications About Carbon #5: Ethotoin (fig. 4-12) has only a phenyl substitution at carbon #5, rather than the diphenyl groups of DPH or the phenyl and ethyl of Mephenytoin. The results observed for ethotoin

I ^ 'N'c=o \ / C N

ch 2 -ch 3 0 Figure 4-12. Ethotoin.

were 45.9 ± 4.05 CPM/pg, while the matched control gave a response of 25.9

± 11.49 CPM/pg. Ethotoin's effect amounted to a response of 177 ± 15.64% of the matched control, while the percent of total mean control, for ethotoin, was 173 ± 15.3%. The activity of Ethotoin, with respect to 88

DPH's activity, was 132 ± 11.7% of DPH's response. Ethotoin had a response about equal to that of hydantoin (107 ± 9.4% of hydantoin's response).

Structurally, Mephenytoin has a phenyl-ethyl group on carbon number 5, rather than the two phenyl groups of DPH (fig. 4-13). The results obtained for mephenytoin were 57.4 ± 2.20 CPH/pg, and the matched control was 25.9

± 11.49 CPM/pg. Mephenytoin's effect amounted to a response of 222 * 8.49% of the matched control, while the percent of total mean control, for mephenytoin, was 188 ± 52.9%. The activity of mephenytoin with respect to the % of control activity was 217 ± 6.3; and 163 ± 6.5% of DPH activity.

With respect to hydantoin, mephenytoin's activity was 135 * 5.1% of the parent molecule's activity.

C N II I 0 CH, Figure 4-13 Mephenytoin

Phenobarbital fPB) has a ring structure very similar to that of DPH and other hydantoin analogues. These structural similarities led us to 89 screen PB for it's ability to induce cell proliferation. In this culture system, 10-1° M PB was able to induce a significant (p<0.05, N=5) proliferatory response of 33.02 ± 7.06 CPM/pg protein. PB's response was

139.1% of the control (23.7 CPM/pg, Table 4-6). Prior to these studies, PB had not been shown to induce proliferation in fibroblasts.

Structurally, PB most closely resembles mephenytoin. When compared to mephenytoin, PB was significantly less active. The observed decrease in the activity associated with PB, when compared to mephenytoin, may be due to either the additional carbonyl group within the barbital ring or the free site on Nitrogen #3 (fig 4-14).

Table 4-6

Growth Factor Free Effects of DPH & DPH analogues on fibroblast proliferation.

Drug (10"10 M) N CPM/pg ±S.D Significance

Control 15 25.6 8.7 P <

Hydantoin 7 43.0 2.8 0.001 DPH 13 34.7 3.3 0.050 Phenobarbital 5 33.0 7.1 ns p-OH-DPH 5 23.0 4.4 ns m-OH-DPH 7 43.0 11.0 0.001 p-Cl-DPH 3 71.4 8.7 0.001 p-Br-DPH 3 61.4 6.7 0.001 P-CH3-DPH 3 49.9 14.0 0.001

Mephenytoin 3 57.4 2.2 0.001 Ethotoin 3 45.9 4.1 0.010 ns = not significant

NHDFibroblasts were fed F-16 GFF medium containing either vehicle or 10 0 M drug. Significance was determined by ANOVA.

Pb and DPH are occasionally prescribed together for the treatment of moderate to severe cases of epilepsy. In addition, PB is able to increase the rate of metabolism for itself and DPH by inducing the mixed function oxidases (MFO), P-450b in rat. The major site for P-450b activity is the 90

liver, where hepatocytes carry out drug metabolism. However, very little

information exists as to the presence of P-450b or other P-450 isozymes in

fibroblasts [1,3,48,50,92,93].

With increased metabolism, the concentration of active parent DPH or

PB molecule is reduced, while the formation of inactive and reactive metabolites is increased. With increased metabolism, a reduction in pharmacological activity would be observed possibly by reducing the concentration of active parent drug, while increasing the amount of

inactive metabolites. The major inactive metabolite of DPH, p-OH-DPH, was

found also to be inactive with respect to inducing fibroblast proliferation. In addition, reactive (toxic) metabolites are also formed

[48,50,94,95].

Reactive metabolites are thought to arise from the formation of arene oxides or epoxides, which can covalently bind to macromolecules such as

DNA, or various cellular proteins (such as enzymes or regulator proteins)

[48,50,52-54,94,95]. The end results of this action is cellular toxicity, which can lead to altered cell function or even death. Therefore, if a phenobarbital-inducible isoform of P-450 is present in cultured human fibroblasts, then PB should be able to induce DPH metabolism, increased metabolism of DPH (and PB) should result in a proliferative response that could range from control levels to a less than an additive response*

We were interested to see if any interactions occurred between DPH and

PB; These possible interactions may include potentiation, synergism of drug action or result in either an additive or inhibitory effect* The combination of 10"10 H PB with 10~10 H DPH, produced a response that was nearly equal to the sum of the individual responses observed for DPH & PB 91

(67.7 CPM/pg protein). In these studies, the activity observed with the combination of DPH & PB, produced a response of 53.73 ± 21.73 CPM/pg protein. This response was significantly greater (p<0.001, N=4) than that of the control (23.7 ± 2.9 CPM/pg protein). Also, this response was significantly greater than that of DPH (p<0.001), when tested by one way

ANOVA. These results are significant as they suggest that DPH and PB are not being metabolized by a cellular oxidase in these cells since PB did not cause a reduction in DPH's activity.

DPH Metabolism - Effect of ASA on DPH response. Because more than one is site available to undergo metabolism, the active parent molecule could be removed at a faster rate than if there were only a single site available. Both DPH and PB contain an unsubstituted position on the phenyl ring and N #3. It may be possible that the availability of these two sites for metabolism could account for the low observed activity, when compared to the analogues. The para substuted analogues, mephenytoin and

i------> p-OH-DPH 1) phenyl hydroxylation. I DPH or p he nobarbital I 1------> N-OH DPH 2) #3 nitrogen hydroxyl at ion. ethotoin all had one of the two probable sites for metabolism blocked.

However, N-demethylation (or N-deethylation) could be another pathway for metabolism. Molecules that have only one site available for metabolism, may have an advantage in that they may be less likely to form toxic metabolites than those molecules with multiple sites. Also, these molecules (analogues) may possess greater activity because fewer of the active molecules would be removed than with molecules having multiple 92

Pot I i I ont on tho Pfl ffl

M e t a

0 r i h o

R Figure 4-14. Structural comparison of hydantoin's 5 membered ring to phenobarb's 6 membered ring. DPH has a phenyl group at R1 while phenobarbital has an ethyl group.

sites available. Likely phase I metabolic pathways may be either cytochrome P-450 or the prostaglandin synthetase pathway.

Prostaglandin Synthetase Pathway We sought to determine whether the prostaglandin synthetase pathway had any impact upon DPH or hydantoin activity. For these experiments, the concentration of aspirin (ASA) was held at 10~6 M, while DPH, hydantoin, progesterone and dexamethasone were

10 -10 M. Controls contained vehicle and 10 -8 M ASA and gave a response of

25.3 ± 5.38 CPM/pg protein.

From the experiments that were run, the following results were obtained, as shown in figure 4-15. The mean activity for DPH was found to be 64.5 ± 13.4 CPM/M9* while the control was 25.3 ± 5.4 CPM/pg protein.

DPH's activity, when of measured against that of the controls, gave a response that was 255% of the control and had a significance of p< 0.001. 93

Table 4-7

Drug + ASA vs the Matched ASA Control

Sample ANOVA ASA (10 .M) + Size Significance DRUG (10 M) CPM/pg ± S.O. N p < % of Control

1) ASA Control 25.3 t 5.4 12 100.0% 2) DPH 64.5 t 13.4 6 *** 0.001 254.9% 3) Dexamethasone 48.4 ± 5.9 2 * 0.050 191.3% 4) Progesterone 45.7 i 12.9 2 * 0.050 180.6% 5) Hydantoin 46.5 i 1.8 4 ** 0.010 183.8%

The mean activity for hydantoin (46.5 t 1.8 CPM/pg protein, 183.8% of control response) was found to be very significant (p<0.01; N=4) when compared to the control with ASA. However, the combination of hydantoin

Table 4-8

The effect of ASA on DPH, Dexamethasone (DEX) and Hydantoin (HYO) induced proliferation with huaan fibroblasts in growth factor free Medium.

ASA free Control.... Combination of: Drug + ASA Significance Group N CPM/pg S.D. Group N CPM/pg S.D. %CPM/pg p ■

Ctrl 15 26.5 ± 2.3 ASA Ctrl 7 30.7 ± 1.9 116.0 ns

DPH 13 34.7 ± 3.3 ASA + DPH 6 64.5 ± 13.4 185.9 0.0043 HYD 7 42.7 ± 2.7 ASA + HYD 4 46.5 ± 1.8 108.3 ns DEX 4 47.4 ± 2.0 ASA + DEX 2 48.4 ± 5.9 102.0 ns

Concentrations: ASA (10'6 M), DPH (10'•10 M). Significance was determined by the Unpaired T-test. ns = not significant.

and ASA produced a response that was only 72% of that of DPH. Thus, it appears that in the presence of 10~6 M ASA, the response obtained for DPH exceeds that of hydantoin, while hydantoin's response is comparable to those observed in earlier studies with hydantoin alone. This implies that

ASA is potentiating DPH's activity to a far greater extent than that of Figure 4-15. Comparison of ASA's effect on DPH, hydantoin (HYD) and dexamethasone (DEX) activity. NHD Fibroblasts were fed F-16 (GFF) medium containing either drug (10 M), drug plus ASA (10 M) or vehicle. Significance was determined by the Unpaired T-test.® Control (Drug + vehicle^ Q Drug + ASA

94 100

p = 0 .0 0 4 3 Drug Response | | Drug + ASA

B

PU u

DPH HYD DEX CTRL Figure 4-15 96 hydantoin (Table 4-8). Comparing the combined DPH and ASA to that of DPH alone, the combination produced a significantly greater response

(p=O.O043, fig. 4-15) than DPH alone.

The mean activity for Dexamethasone (DEX) was 48.4 * 5.9 CPM/pg protein

(N = 2, p<0.O5), which was 191% of the control's activity. DEX and ASA produced a response that was only 75% of DPH and ASA combined. DEX was able to induce a response that was 104% of the combined hydantoin and ASA.

Similar to DEX, the mean activity for the combination of Progesterone and

ASA was 45.6 ± 12.9 CPM/pg protein, which was significantly greater than that of the control (p<0.05, 180.6% of control). However, in order to better characterize the nature of ASA's effect upon the action of these drugs (progesterone, DEX, hydantoin & DPH), further testing is required.

DPH Dose Response with & without ASA. NHD Fibroblasts were fed F-16 GFF medium containing either DPH and vehicle (control) or DPH and 10"® M ASA

(experimental group). For the experimental group, the observed maximal

.0 effective dose was 10 M, with a mean response of 216.1 ± 0.3% of control

CPM/pg protein. The ED50 for the control DPH dose response curve was

53.57x10"12 M, while the ED5q for the DPH dose response curve with 10"® M

ASA was 41.93X10"12M DPH (Table 4 -9 , fig. 4 -1 6 ).

ASA Effects upon Hydantoin Dose Response. NHD Fibroblasts were fed

F-16 GFF medium containing either hydantoin and vehicle (control) or hydantoin and 10"® M ASA (experimental group). As shown in fig. 4-27, the

Em x for the hydantoin control was 10"10 M (24.6*3.9 CPM/pg; 140.1% of control), while in the presence of 10‘® M ASA the EMX was 10"®M (34.8*1.5

CPM/pg; 173.2% of control). The ED5q for hydantoin was 1.54x10"12M, while in the presence of 10"® M ASA the ED50 was 0.72x10"12 M (Table 4-9). 97

Table 4-9

DPH & Hydantoin (HYD) Activity on Fibroblasts

Medium & Treatment EDS0 (Molar) E * a x (Mol^)

DPH 1) GFF 53.57x10"]i; 10-1! 2) GFF + ASA 41.93x10 10" 8

HYD 1) GFF 1.54x10"]? io"18 2) GFF + ASA 0.72x10 10" 9

Alone or with ASA (10~8 M) Defined medium ([^H] — TdR) GFF ■ Growth Factor Free.

The EBax obtained for both DPH and hydantoin was 10"18 M under control -fi conditions. However, in the presence of 10 M ASA, there was a shift in the the EMX to 10"9 M for hydantoin and to 10~® M for DPH. In addition, there was an increase in the magnitude of the responses for both DPH and hydantoin, when in the presence of ASA. In the presence of 10 M ASA, the observed response for 10"18 M DPH was 1.64 times greater than when DPH was used alone (fig 4-16). In addition, the response for 10 -8 M DPH was increased by more than 2 fold in the presence of ASA. The effect of ASA -fi (10 M) on hydantoin-induced proliferation was not nearly as great as that observed with DPH. At the E ^ for hydantoin (10_1° M) the response with ASA was 1.38 times greater than without ASA. However, with ASA present, there was a shift in both the EBax (to 10'9 H) and an increase in the magnitude of the response by a factor of 1.45 times (fig. 4-17). Figure 4-16. DPH dose response curve with and without ASA. NHD Fibroblasts were fed F-16 (GFF) medium containing either DPH or DPH plus ASA (10'6 M) . ^ Control DPH curve,^ DPH + ASA.

98 Percent of Control -§ § 4-> O CJ PL, O Cn u a 200 150

O DPH + ASA (1x10-6 M) (1x10-6 ASA + DPH O ♦ Control DPH Curve Curve DPH Control ♦ ♦ 13 ar -a

-12 Lg (DPH), Molar -Log

-11 gur 4-16 re u ig F

-10 / O

-9 8 - Figure 4-17. Hydantoin dose response curve with and without ASA. NHD Fibroblasts were fed F-16 (GFF) medium containing either hydantoin or hydantoin plus ASA (10 M) . ^ Control Hydantoin curve, ^ Hydantoin + ASA.

100 % of the Control CPM/ug Protein O Hydantoin + + ASA Hydantoin O Ifydantoin♦ Control -13

Lg (Ifydantoin), Molar -Log -12

1

Figure -11 4-17

-10

-9 DISCUSSION.

1. Development of the cell culture system. Studying cellular effects associated with DPH has proven to be an enigma to many researchers. One of the major problems that has been associated with in vivo models is determining whether DPH causes tissue hyperplasia or hypertrophy. Cell culture experiments indicate that DPH does cause cell proliferation, but results are often quite variable and hard to reproduce [78]. For the present studies, the proliferation caused by DPH was often relatively small when compared to serum induced cell proliferation. With serum supplemented medium, a large background activity of cell proliferation was generated. This background of activity can potentially create many problems when attempting to characterize the pharmacology of DPH's cellular effects. This problem becomes increasingly difficult when the drug effect was small relative to the serum induced background. Typically, medium used by others to study cell proliferation is supplemented with 10%

FBS [78,91].

The initial experiments were intended to determine the pharmacology and then the structure activity relationship behind DPH's proliferative activity. The cells used for these studies were Newborn Human Dermal

Fibroblasts (NHD). The advantage in using these cells was that they are readily available and represent a normal human cell type for studying

DPH's effects. However, the disadvantage of using these cells was that they tend to vary somewhat from one source to another. Periodically a

102 103 reference cell line should be used to check whether the observed effect is specific to those NHD fibroblasts being used or if a generalized cellular response has been observed. One established human fibroblast-like cell line is WI-38 cells, which have been shown to respond to DPH in this lab and by other investigators [78].

Another benefit gained from periodically using established cell lines was the ability to have other investigators confirm one's results.

However, it must be noted that with WI-38 cells or other established cell types, there was also a tendency for cells to differ from their normal counterparts over time. Therefore, these cells may not always give an accurate picture of how a normal cell will respond to a particular stimulus. For this reason, it was usually best to conduct the primary experiments in normal cell types.

In the past, rapidly dividing cells were used to represent the wounded state. With this model system, cells at low density (that is the number of cells per cm ) undergo rapid cell division until they reach a high density, at which time they are thought to become contact inhibited.

However, the addition of fresh, serum supplemented or defined media

(containing growth factors & hormones) caused these cells to undergo further cell division. It was possible that a large portion of the cells became contact inhibited while the remainder were still able to be induced to proliferate. With rapidly dividing cells, many variables were present and could potentially interfere with our investigation of drug effects.

This became more apparent when trying to measure a response that was small compared to the serum induced background. These factors could account for much of the variability and poor reproduciblity encountered when 104

investigating DPH's effect on proliferation [78]. Frequently, DPH's

ability to affect proliferation has been questionable as its effect was

masked by the serum-generated background of cell activity. This problem

was apparently independent of the method used to determine cell

proliferation.

In an attempt to reduce the variability within the system, quiescent

cells were used in favor of those that were rapidly dividing. Briefly,

fibroblasts were grown to 100% confluency over a period of 7 days. Then,

cells were serum-arrested with 0.25% FBS for 3 days prior to conducting

the experiments. The concept for this culture system came from those used

by other investigators for studying the effects of growth factors and

hormones upon resting cells [79,60,93]. The primary advantage to using quiescent cells was to eliminate the background "noise" produced by large

numbers of rapidly dividing cells.

After 3T3 cells were serum arrested, quiescent cells were then treated with [fy] thymidine and varying concentrations of serum (0.5, 2.5, 5, 10

and 20% FBS). The intent behind this part of the study was to determine

the functionality of the culture system prior to investigating the cellular effects of DPH or the growth factors EGF and FGF. In these subsequent studies, cells were fed B medium containing 2.5% FBS, FGF or

DPH and [^ij-thymidine. For the initial series of experiments, the cells

used were the mouse 3T3 cell line.

The 3T3 cell line has been used by Jimenez De Ausa and others to characterize the activity of growth factors and hormones upon cellular functions [79,80,93,96]. When serum and FGF were tested in our system,

the results were as expected. Then DPH was added to the system. 105

The serum-arrested cell system was shown to be useful for studying the larger effects produced from serum and growth factors, but was marginal for investigating drug induced activity. In order to determine whether serum was still masking drug induced effects on proliferation, the serum component was eliminated.

Serum free medium was the next logical step in the development of the culture system. By removing serum, a major source of variability was eliminated [92]. The range of protein content from serum supplemented medium was found to vary from 900 to 1600 pg/ml. This amounted to a large source of variability (700 pg/ml) between lots of medium and could potentially alter the free drug concentration. This variation becomes especially critical when drugs are highly protein bound (DPH is 90% protein bound). Also, other potential variables contained in serum could adversely affect DPH-induced cellular activity. These variables include hormones (hydrocortisone, insulin), growth factors (FGF, EGF, PDGF, PGF^, etc), other prostaglandins, essential amino acids, minerals and nutrients, all of which have effects upon cell proliferation [76]. Therefore, in order to study the cellular activity for DPH and analogues, a serum free medium was utilized. The resulting reduction in background cellular activity, made DPH's effects more apparent.

The defined media used in these studies (F-14) was made up from a

50:50 combination of fortified MEM (B-medium) and Hams F-12 media [76,97].

Also, all the defined media used in these studies were supplemented with nutrients and hormones essential for normal cellular activity. These included 1 pg/ml transferrin, 5 pg/ml insulin, 500 pg/ml BSA, 3x10-8 M -fi NajSeOj.SH^, 10 0 M hydrocortisone and additional vitamins# 106

Cultures of quiescent cells, in 24 well trays, used the same treatment schedule as before. On day 3, cultures received defined medium, [3H]- thymidine, growth factor (50 ng/ml FGF or 100 ng/ml EGF) and DPH or vehicle for the control. Much of the variability that plagued the serum supplemented cultures was eliminated by using this system. Later, DPH's effect became more apparent when both a serum and growth factor free system was adopted. For these studies, several media were screened in order to determine which was the most effective in supporting cell activity and in revealing DPH's proliferatory effect.

F-12 media is deficient in amino acids, when compared to all the other media tested. The essential amino acids leucine, isoleucine & glutamine are present in very low concentrations and the amino acid cystine is absent. However, F-12 does contain normal serum components such as vitamin

B-12 and biotin, which are required for the stimulation of DNA synthesis

[92,93]. On the other hand, Ham's F-12 medium contains an important purine, hypoxanthine, which helps increase the rate of DNA synthesis when cells are stimulated by growth factors [92,93]. Putrescine, pyruvate and linoleic acid are present in F-12 media and support DNA synthesis. F-14 medium, by comparison, contains the same components as F-12, but many of the inorganic salts are at half the concentration. When F-12 medium was used alone, the cells were in a very poor condition. The cells appeared to be starved (faint, long, thin, spindle shaped) when compared to the much more robust appearance of cells in other media. This suggested that fibroblasts require relatively high concentrations of amino acids in order to undergo cell division and maintain normal cell functions [92]. 107

F-14 TC medium, the "richest" of all the defined media used in these studies, was designed to maintain the cells for long periods of time in a serum free environment. Unlike the other defined media that were used, F-

14 TC medium contains as a stock component the growth factor prostaglandin

F^ (PGF^, 0.5 mg/1). Since PGF^ is a growth factor with similar activity to that of EGF [77,79], it makes sense that both the control (7.8 CPM/pg protein) and DPH treated groups (13.6 CPM/pg protein) gave the greatest overall responses among the F-14 media series (Table 4-11).

Cultures of NHD fibroblasts were maintained in F-14 TC medium, supplemented with 50 ng/ml FGF, for 3 months. During this time, the cells were passed successfully several times. This medium could prove potentially useful for long term studies such as in collecting collagen or cellular fibronectin under serum free conditions.

The PGF^-free version of F-14 medium, F-14 BC, was the primary defined medium used to conduct experiments with DPH and the growth factors (FGF &

EGF). A hydrocortisone-free version of F-14 medium, F-14 HCF, was utilized for a few experiments, but the results were nearly identical to that of F-14 BC, for DPH induced activity. F-14 BC was the medium used when the growth factor free activity for DPH was first observed.

Following the observation of DPH's growth factor free activity, several media were screened in order to determine which formulation could best support DPH-induced cell proliferation in the absence of serum or growth factors, in these experiments, paired (matched) culture groups were fed media treated with either DPH ("10 M) or vehicle (table 4-11).

The results showed that the DPH-induced response was greatest in F-16 108 medium, followed by the F-14 media series, B medium and B medium with serum. F-12 medium was the least able to support the cells under growth factor free conditions or DPH-induced cell proliferation.

F-16 medium was made up from a 1:1 combination of F-12 and B media.

As with the F-14 series of media, the high mineral and nutrient content of

F-12 was combined with the amino acid rich B media. Unlike the F-14 series, F-16 medium lacked the rich vitamin supplement and a high sodium content. Why cells fed F-16 media gave the best response to DPH under growth factor free conditions is unclear. It could be that the higher sodium content of the F-14 series interfered with DPH activity, thus accounting for the lower overall responses.

The greatest magnitude of cellular responses was observed with B medium and B medium supplemented with 10% FBS. However, for both B media groups, DPH's activity was only about 22% greater than controls. Thus, B medium, either with or without serum was not very effective for demonstrating DPH-induced proliferation. Additionally, 10% FBS made a significant impact upon the overall magnitude of responses for both control and DPH treated cells, when compared to serum and growth factor free groups. However, the presence of 10% FBS seemed to make little difference with respect to the effect of DPH.

2. Pharmacology of DPH-induced proliferation. The pharmacology of DPH- induced cell proliferation was determined by three different techniques, each of which provided critical pieces of information on DPH's activity.

The three techniques used were cell counts, total DNA content and [3H]- 109

thymidine incorporation. Of these techniques, cell counting is preferred by many investigators as the method of choice for determining cell proliferation [92]. The primary reason for this is cell counting proves beyond a doubt that there is an increase in the cell number. The other methods served to confirm the original observation with cell counting and showed that DPH also affects the rate of DNA synthesis and total DNA * content. The most sensitive method used, however, was [H]-thymidine

incorporation.

DPH Dose Response with Serum. It was reported by other investigators that the optimal concentration for DPH to induce proliferation was 3 ng/ml

(1.19x10'5 M), in human fibroblasts [78,91]. Determinations were made by counting the total number of cells and then taking a ratio of the number of cells per total area. This is referred to as the cell density cells/cm ). Our early studies used NHD fibroblasts in B-10% FBS medium, and the results indicated that a concentration of 0.1 ng/ml (3.97x10-7 M) DPH yielded the best response (190.9% of the control response). Subsequent studies, using a range of 10*9 to 10’* M DPH, showed that 10"7 M yielded the maximal response, and the ED50 was 1.727x10 -8 M. At high doses of DPH (10 -5 and 10~* M), the cell response decreased considerably to levels below that of the control. This was not unexpected, as it has been reported that high doses of DPH (25 nd/ml or about 10’* M) caused cellular toxicity [78].

Cell proliferation was also measured in NHD fibroblasts by determining the total DNA content. The concentrations of DPH used in this study ranged from 10*12 to 10~6 M. Concentrations of 10“5 and 10** M were eliminated because cellular responses were near or just below the control

levels. Results from these studies had an of 10*8 M, while the ED50 was 110

2.47x10'11 M. Comparing these results to those from the cell counting

experiments, there appeared to be no major differences between the values

obtained for the Emax (10"8 vs 10~7 M). However, this was not the case for

determinations of the EDSg. In cell counting experiments, the ED5q was

1.727x10"® M DPH, which was almost 700 times greater than the EDsfl from the

DNA experiments (24.70x10-12 M). The higher value observed for the ED50 from

cell counting experiments may arise from several factors including

differences between the cell donors, the cell passage or differences

between batches of serum. In addition, differences could also arise due to

the types of assays used and that a doubling of DNA does not necessarily

reflect a doubling of cell number. Cell counting, as a means of measuring

cellular proliferation, is not as sensitive a technique as measuring the changes in the total DNA content.

DPH Dose Response with EOF & FGF in serum free system. DPH dose response experiments in serum free medium gave a somewhat different profile than with serum. This difference in dose response may result from the differences that exist with the serum content of nutrients, protein and growth factors. The DPH dose response data was collected in the presence of 50 ng/ml FGF, 100 ng/ml EGF, or under growth factor and serum free conditions. The E ^ observed for DPH was 10-1® M for cells in serum free medium. The presence of the growth factors made no difference in the E ^ , when compared to the response obtained from growth factor free experiments. Also, the median effective doses (EDS0) were very similar for

DPH with EGF or FGF in serum free media. When the DPH dose response curve was run with FGF, the ED50 was found to be 2.05x10~12 M DPH, while the EDS0 for the combination of DPH and EGF was 3.97x10*12M DPH. 111

Growth Factor Dose Response in the Presence & Absence of DPH. Dose response effects of the growth factors FGF and EGF were run in the presence of 10’10 M DPH. The purpose for doing these experiments was to determine the degree, if any, that DPH affected the growth factor's activity and if so to what extent. The doses ranged from 1 to 100 ng/ml for FGF or EGF, with a 0 ng/ml growth factor (blank) control. Blank controls received either vehicle or DPH. From the mean FGF dose curve, it appeared that DPH had no significant effect upon the ED5q or the E ^ .

Additionally, DPH did not have a significant effect upon the magnitude of the mean response to FGF. The results for EGF were similar to those with

FGF. DPH had no significant effect upon the EDgg or the EMX.

DPH (10"10 M) appeared to exert a significant (p<0.01, n=3) effect upon

FGF-induced proliferation when human recombinant FGF was used. Previous experiments made used bovine FGF. It should be noted that the individual experiments for the growth factors curves provided more significant results than those obtained from the pooled experiments.

DPH Dose Response with Medium Free of Growth Factors & Serum. To date,

DPH has been viewed as not having any mitogenic activity [78,91]. Resting cells (Gg) require the presence of a mitogen, such as EGF, FGF or serum, in order to initiate DNA synthesis and cell division. When using F-14 medium, free of serum or growth factors, DPH was found to induce resting cells to undergo cell division to a significantly greater extent than that of the vehicle control. The magnitude of DPH's response, in growth factor free medium, was essentially the same as we previously observed when DPH was in the presence of either serum or growth factors. Results indicated that 10"10 M DPH induced quiescent cells to initiate cell division at a 112 rate that was significantly higher than that of the matched vehicle control (p<0.0035, N=18). The mean response for cells treated with DPH was 21.30 ± 4.78 CPM/M9 protein, while the vehicle control gave a response of 14.32 ± 3.44 CPM/M9 protein.

When the OPH dose response curve was run in serum (DNA assay), the ED 50 was 24.70x10"12 M. By comparison, the EDS0 derived from the DPH dose response curve in growth factor free medium (GFF) was 2 times greater, at

53.57x10”12 M DPH. The GFF EDjq was some 14 to 26 times greater than the median effective doses previously observed for DPH with EGF (3.97x10 -19 M) and FGF (2.05x10"12 M).

From the above data, it appears that EGF and FGF caused a slight but significant shift to the left in the ED50, but had no effect upon the E ^

(10-1® M) for DPH. This suggests that there may be an interaction of some sort with the growth factors upon DPH's activity. To further support this observation, results obtained from human recombinant FGF dose response curves also suggested that a possible interaction was taking place between the growth factor and DPH. Based upon these studies, it was apparent that further investigations would be required in order to determine whether DPH and the growth factors do interact. To help clarify whether an interaction is taking place between the growth factors & DPH, and if so, to determine the nature of this interaction, normal human dermal fibroblasts from many different donors should be screened.

Serum appeared to have less of an effect upon the EDS0 than did 50 ng/ml FGF or 100 ng/ml EGF. In the presence of serum, the E ^ for DPH was

10'8 rather than 10”’° M, but with no difference in the magnitude of DPH's response. These data suggests that there may be an interaction taking 113

place between DPH and some of the serum factors (growth factors, hormones,

fibronectin, etc). The observation that DPH may be interacting with serum

factors is supported by other investigators [98,99]. Perhaps the observed

shift in the ED50, reflects the serum growth factor content, while the

shift in Emax may result from DPH binding to serum proteins. The binding

of DPH to serum proteins would effectivily reduce the amount of drug

available to induce proliferation. Therefore, in the presence of 1000

pg/ml or more of serum protein, a higher concentration of DPH would be

required to achieve similar results to those in a low protein content.

3. Toxicology. Reactive intermediates are thought to be responsible for

much of DPH's cellular toxicity and teratogenicity [50,52-54,95]. Also,

the work performed in mononuclear cells demonstrated a possible mechanism

by which reactive intermediates may act [50]. It was suggested that DPH

and/or one of its reactive intermediates could cause single-strand breaks

in DNA. Also, both DPH and the reactive intermediates could cause DNA cross linking. However, cross-linking would mask the true extent of the single strand breaks [63]. Also, as DPH concentrations increase, greater quanities of reactive intermediates could be produced. An increase in the production of reactive intermediates would increase the likelihood of cytotoxicity.

If metabolism of DPH occurs in cultured human fibroblasts, then both

inactive and reactive metabolites would be formed. The reactive metabolites, such as epoxides and arene oxides, can be produced by P-450 as a biproduct of normal metabolism. In addition, the PCS pathway has also been shown to produce reactive metabolites of DPH. Reactive metabolites can covalently bind to cellular macromolecules such as DNA, regulatory 114 proteins and enzymes. Covalent binding to macromolecules can potentially cause altered cellular functions, decreased rates of cell proliferation and even cell necrosis. This has been well documented in many different tissue and cell types [48-51,64-70,94,95,104,105], However, very little has been published about drug metabolism in fibroblasts and it is unclear as to what metabolic pathways may be present.

Other possible sources of DPH toxicity may be associated with DPH's effect upon calcium metabolism in fibroblasts and other cell types [99],

4 C !\ . Modeer demonstrated that DPH caused an increase in the uptake of Ca in gingival fibroblasts, as did the growth factor EGF. However, when EGF and

DPH were combined, DPH was found to interfere with the EGF-induced calcium uptake [99].

In addition to having an effect upon calcium metabolism, DPH is known to bind to hormone receptors, such as the glucocorticoid [55,56,104,113].

The glucocorticoid (hydrocortisone) receptor regulates or modifies the cells response to the growth factors EGF & FGF. Whether DPH is capable of regulating cell function via binding to the glucocorticoid receptor is unclear [122-124]. When DPH's broad range of effects are coupled with variations in free DPH concentrations and metabolism, the picture becomes increasingly more complex.

Metabolism. Metabolism is potentially another means by which DPH can produce cytotoxicity and affect proliferation. In the metabolism of DPH, one atom of molecular oxygen is combined with DPH and the active site of

P-450 while the other is incorporated into cellular water. From this process, toxic metabolites (reactive intermediates) can be formed as well as DPH's major inactive metabolite para hydroxy-DPH. Reactive 115 intermediates are epoxides when the oxygen is bound to the substrate's alkyl portion and are called arene oxides when bound to the aryl portion

[94]. These reactive intermediates can be formed by the prostaglandin synthetase pathway (PGS pathway) and cytochrome P-450 [50,52-54,95].

The toxicity produced by reactive metabolites of DPH could cause altered cellular functions, such as changes in proliferation or cellular attachment. Also, alterations in cell function could potentially lead to chemical carcinogenesis, drug toxicity and mutagenesis [50,94], For example, DPH has been demonstrated to bind directly to an adenine analogue, 9-ethyl adenine [62]. This observation suggested that DPH, or one of its metabolites, might interact directly with the cell's DNA. The observation that DPH can act on DNA was supported by studies conducted in human peripheral mononuclear cells. DPH (40 pg/ml, 1.6x10"* M) was found to cause a slight, but significant, increase in DNA single-strand breaks.

This provided some information as to one possible target for DPH or its arene oxide or epoxide induced toxicity.

A. Cytochrome P-450. Isozymes of P-450 can be characterized, in part, by agents responsible for their induction and by the substrate of that isozyme. For example, the isozyme P-450c (P-448, Pj-450) is responsible for metabolism of poly aromatic hydrocarbons, such as the potent carcinogen benzo(a)pyrene [94,95]. In contrast, DPH is metabolized by another isozyme of P-450, P-450b. Also, DPH induces the isozyme P-450p. This isozyme is responsible for metabolizing hydrocortisone, dexamethasone, digitoxin and theophylline is known as P-450p [1/3],

The phenobarbital (PB) inducible isozyme of P-450, P-450b, is the primary pathway for DPH metabolism [48-50]. The major metabolite of DPH 116 formed in man is p-OH-DPH (pHPPH). Hydroxylation at the para position of the phenyl ring results in the inactivation of DPH and increases its water solubility (less lipophilic). Pretreatment with PB would be expected to induce the synthesis of new P-450b, resulting in an increased rate of metabolism for PB and DPH. DPH and PB are deactivated by para (p) hydroxylation of the phenyl ring. Hydroxylation can occur either directly or through epoxidation at the para or meta positions of the phenyl ring

[50]. In man, para hydroxylation is the primary path for the metabolism of DPH in the liver, while meta hydroxylation is thought not to occur.

Other metabolites that are formed include the dihydroxydiol (DHD), 3,4- catechol and 3-0-methyl catechol [48,50], Furthermore, p-hydroxylation serves to make the molecule more water soluble and ready for excretion

[1,3]. However, as a result of p-hydroxylation short-lived reactive metabolites are produced in small quanities in other systems and can cause cytotoxicity [48,50,94].

With PB's induction of P-450b, there is an increase in the metabolism of both DPH and PB, due to an increase in the P-450 content. As a result of increased metabolism, there is more p-OH-DPH (or p-OH-PB) produced, as well as the reactive metabolites. Thus, if cultured fibroblasts do contain an isoform of P-450 similar to rat P-450b, then PB would be able to induce its own metabolism as well as DPH's [48,50]. Additionally, the increased

DPH & PB metabolism that result from induction would cause a reduction in the concentration of the active parent drug. Hence, induction of P-450b would appear as a decreased proliferatory response [1,3,48,50,94,105-108].

However, a search of the literature has not given any data to support the premise that the P-450b isozyme exists in fibroblasts. 117

Studies conducted with cultured fibroblasts (F-16 medium, growth factor free) indicated that PB (10*1® M) caused an increase in cell proliferation of nearly the same magnitude as 10'1® M DPH. After establishing PB's base line activity, the next step was to determine its effects upon DPH-induced cell proliferation. DPH and PB were combined, each at 10"10 M, in growth factor free (F-16) medium and fed to quiescent human dermal fibroblasts. Cultures were then incubated over a period of three days. If cultured fibroblasts contain "P-450b’\ then PB would be able to induce P-450b activity and increase the metabolism of itself and

DPH [48,50,95]. Since the major metabolite of DPH is relatively inactive, the overall effect due to DPH would be reduced. This would most likely be true for PB as well. Thus, a less than additive response for the combination would be expected if "P-450b" were present. An additive response, or better, would suggest that the isozyme was not present.

DPH produced a response as measured by H-thymidine uptake that was

34.7 ± 3.3 CPM/pg protein compared to the control response of 23.6 ± 8.7

CPM/pg. By comparison, DPH's major metabolite p-OH-DPH gave a response of

23.0 ± 4.4 CPM/pg protein, which was not significantly different from the control. PB's response was comparable to that of DPH, at 33.0 ± 7.1

CPM/pg protein. For example, if the observed response for the combination of DPH & PB were additive, then the expected response would approximately be 42.1CPM/pg protein. The calculations to determine the expected results were: Control...... 25.6 CPM/pg. Response less control. DPH...... 34.7 CPM/pg. 9.1 CPM/pg PB...... 33.0 CPM/pg. 7.4 CPH/pg

16.5 CPM/yg <— Sum.

Ctrl (25.6) + Sum (16.5) = 42.1 CPM/pg 118

By comparison, the observed response for the combination of DPH and PB was

53.7 ± 21.7 CPM/pg. This may suggest that ,,P-450b" was not present in cultured human dermal fibroblasts, since the combined response was additive. However, it must be noted that there may be other explaniations for this observation, such as binding.

B. The prostaglandin synthetase pathway. DPH Metabolism - Aspirin Experiments in GFF Medium. Pathways other than that of P-450 have been shown to metabolize DPH. These pathways include thyroid peroxidase, horseradish peroxidase and prostaglandin synthetase [59]. It has been shown that the by blocking the prostaglandin synthetase (PGS) pathway, teratogenic effects due to DPH and the glucocorticoids could be significantly reduced or blocked [50-57]. These results may be explained if by blocking the PGS pathway, the formation of reactive intermediate metabolites and free radicals could also be blocked. These metabolites, as with those produced by P-450, have been shown to be able to cause lipid peroxidation and can covalently bind to macromolecules such as DNA and proteins, causing cellular toxicity [50-57,59]. The role of the PGS pathway in teratogenicity has been fairly well studied. But the role of the pathway has not been characterized with respect to DPH's effect on cell proliferation. We sought to determine whether there was any link between the PGS pathway and DPH's proliferatory effects.

ASA (10’6 H) was combined with DPH (10~10 M) in growth factor free defined medium (F-16) in order to determine whether the PGS pathway was involved in the proliferatory activity for DPH. Results from these studies suggested that DPH's activity was affected by the PGS pathway. By inhibiting cyclooxygenase activity with ASA, DPH activity increased significantly over that of DPH alone (DPH control, p<0.005). Under control conditions (ASA free), 10'^ M DPH produced a response of 34.7 ± 3.3 CPM/pg protein. The vehicle control had a response of 25.6

* 2.7 CPM/pg. However, in the presence of 10"® M ASA, 10”1® M DPH gave a response that was nearly 2 fold greater than that of the DPH control, at

64.5 ± 13.4 CPM/pg protein. ASA also caused a shift to the left for the

ED5q of the DPH dose response curve and an upward shift in the Eux. The shift in the ED50 for DPH was from 53.6x10"12 M to 41.9x10‘12 M with 10"® M

ASA, was not significant. However, there was a shift in the EMX from 10~1®

M to 10 -fl0 M. Furthermore, since the observed response for 10 -fi M ASA was not significantly different from its matched vehicle control, it can be assumed that ASA was not able to induce proliferation in these cells.

When hydantoin and ASA were combined, no significant change in magnitude was observed for hydantoin's effect on proliferation, when compared to the control. However, when the dose reponse curve was run for hydantoin (10 -1 2 to 10"8 M), with and without 10"® M ASA, hydantoin's EDSg was shifted to the left (from 1.54 x 10”12 to 0.7 x 10"12 M). Likewise, the E|ax was shifted upward; from 10”10 to 10"® M.

An examination of the data showed that ASA increased the culture's tolerance to higher concentrations of DPH & hydantoin. Also, ASA appeared to increase the cell's sensitivity to effects of DPH and hydantoin on proliferation as seen with the shift to the left for the ED5q. Also, the effect of ASA on hydantoin-induced cell proliferation was not nearly as great as that observed with DPH. For example, the shift in the EMX for hydantoin was from 10"1® to 10~9 M, while the shift for DPH was from 10"1® to 10® M. When ASA was tested on dexamethasone-induced cell proliferation, no significant effect was observed. 120

To sum up the ASA effect on drug-induced cell proliferation, ASA appeared to be able to potentiate the activity of DPH and hydantoin. Also,

ASA had a greater effect upon proliferation induced by DPH than with that of hydantoin or dexamethasone. However, the mechanism of action behind

ASA's effect remains somewhat unclear. One of the possible explanations for the ability of ASA to potentiate DPH-induced cell proliferation is its effect upon the PGS pathway.

ASA inhibits the PGS pathway by irreversibly binding to the enzyme cyclooxygenase [1,3]. In the absence of P-450, inhibition of the PGS pathway might block the formation of reactive metabolites that may come from DPH or hydantoin. These reactive intermediates could potentially interfere with DPH- or hydantoin-induced proliferation, possibly by competing with the parent drug or altering cellular functions. With doses greater than 10'1D M in growth factor and serum free medium, cytotoxicity as a result of metabolism becomes more likely. With the use of ASA (10~6

M) the formation of reactive metabolites could be blocked, thus allowing

DPH to induce proliferation to its full potential.

ASA may affect DPH-induced cell proliferation to a greater extent than the proliferatory effects of the other drugs tested, because of the number and/or location of potential metabolic sites on the DPH molecule. When

ASA was combined with DPH, DPH was able to induce a proliferatory response nearly equal to or greater than the DPH analogues.

4. Structure Activity Relationship. The structure activity relationship for some members of the hydantoin family of drugs and phenobarbital (PB) was investigated. The pyrimidine ring of PB and the hydantoin ring differ by a carbonyl group inserted between the #1 N & #5 C. 121

Table 5-1

Substitution Groups on Hydantoin Molecule

COMPOUND R 1 R 2 * 3 1) HYDANTOIN HH H

2) DPH H PHENYL PHENYL 3) p-OH-DPH H PHENYL p-OH- PHENYL 4) m-OH-DPH H PHENYL m-OH- PHENYL 5) p-Cl-DPH H PHENYL p-Cl- PHENYL 6) p-Br-DPH H PHENYL p-Br- PHENYL 7) P-CH3-DPH H PHENYL P-CH3-PHENYL 8) ETHOTOIN CH3CH2- H PHENYL 9) MEPHYTOIN CH3- CH3CH2- PHENYL 10) PHENOBARB H CH3CH2- PHENYL

Phenobarb has an extra carbonyl group between the nitrogen #1 & carbon #5.

RJ H ,N.

\. •/ C N R 1

0 Fig. 5-1. Hydantoin Ring Structure.

Structure Activity of the #5 Carbon of the hydantoin ring (Table 5-1, fig. 5-1). The addition of two phenyl groups to the #5 Carbon of the hydantoin ring, yields the compound diphenylhydantoin (DPH). Since hydantoin may be considered the parent molecule for DPH, hydantoin was studied in our fibroblast system. Hydantoin-induced proliferation was determined with cultured normal human dermal fibroblasts at 10~10 M. Tests 122 indicated that hydantoin was more active than DPH for inducing cell proliferation. This fact was somewhat suprising, since the addition of phenyl rings to hydantoin should increase the molecule's ability to enter the cell. Therefore, replacement of hydantoin's R2 and R3 hydrogens with phenyl groups in the drug DPH reduced the proliferatory activity for the hydantoin ring (fig. 5-2). The level of response for DPH and matched controls over several experiments was: 34.68 ± 3.27 CPM/pg (DPH); 26.50 ±

2.70 CPM/pg (matched control). The significance for the effect was p<

0.002 with N=13.

Pot 11 Ion* on the Para phony I ring. Meta

Ortho

R Fig. 5-2. Positions on the phenyl ring.

With the addition of two phenyl groups to hydantoin, stearic effects may be created, thus effectively altering the character of drug:receptor interactions and the distribution of the drug in the cell. This may account for the reduced response observed with DPH. Another possibility is that the phenyl groups may serve as sites for metabolism and the possible formation of either inactive or reactive intermediate metabolites. With the formation of these metabolites, there could be a reduction in the 123

amount of proliferatory activity associated with the drug. Also, this would effectively decrease the amount of drug able to induce an increase

in the proliferatory response.

When the activity observed for p-OH-DPH was compared to that of the

mean control from all experiments, it gave a response of only 87% ± 16.6

% of control values. Using a two-tailed unpaired t-test, p-OH-DPH was

found to have no significant effect upon the induction of fibroblast

proliferation. This lack of activity found in p-OH-DPH could be due to one of three possibilities: 1) That inactivation of the hydantoin ring,

and also DPH, is due to the presence of a hydroxyl group, independent of

the site on the phenyl ring that the phenyl group occupies; 2) Having any group occupy the para position of the phenyl ring, causes deactivation of the molecule; 3) The specific combination of a group (hydroxyl) and the position (para) of the phenyl ring may be responsible for inactivation of the molecule. Based upon the data obtained from p-OH-DPH and the questions that were raised, another DPH metabolite was selected to aid in answering these questions.

A metabolite of DPH, meta hydroxy-DPH (m-OH-DPH), which is thought not to be formed in man was selected to determine whether the decrease in activity due to para hydroxylation was due to either the hydroxyl group, the para position, or a specific combination of the two. If m-OH proved more active than p-OH, then p-OH-DPH's inactivity is due to something other than a generalized deactivation. Thus, the deactivation of the hydantoin ring and of DPH, by para hydroxylation, may be site specific.

The presence of a hydroxyl group at the meta position resulted in growth promoting activity similar to that of hydantoin itself. Thus, the 124 decrease in activity seen With DPH and p-OH-DPH was most likely due to hydroxylation at the para position. Meta hydroxylation seems to overcome the apparent deactivation of the hydantoin ring, due to the phenyl groups.

Since m-OH-DPH induced a response almost equal to that of hydantoin, it is doubtful that the biphenyl addition to the hydantoin ring would cause stearic hinderance, leading to a decrease in hydantoin ring activity. Rather, these results suggest that metabolism of the phenyl ring may attenuate the growth promoting effects of DPH. Thus, deactivation of DPH by para hydroxylation was due to the position that the hydroxyl group occupied.

Was deactivation at the para position a general action for any molecule or was it specific for the hydroxy group? To answer this question, other groups (Cl, Br & CH3) were tested at the para position. It was found that para substitution with the halides Cl or Br, caused a significant increase in cell proliferation over that observed for both hydantoin and DPH. The response observed for p-Cl-DPH was 71.4 ± 12.3

CPM/pg (270 f 32.8% of the control response). The second highest response occurred in another halogenated analogue, p-Br-DPH, with a response of

61.4 ± 6.71 CPM/pg (232 * 25.3% of the control). The para methyl analogue

(p-CH3-DPH) gave a mean response about equal to that of hydantoin at 188

± 53% of control. However, with such a large variation, it was difficult to conclude that p-CH3-DPH induces cell proliferation to any great extent.

Previous reports indicated that p-CH3-DPH did not induce gingival hyperplasia [31],

Changes About Nitrogen #3. Mephenytoin and ethotoin were both used to investigate the effects of having either a methyl or ethyl group at the N 125

#3 site. The presence of either of these groups would effectively block

the site's availability for metabolism and as a binding site for

macromolecules.

Both mephenytoin and ethotoin gave responses greater than those

observed for DPH and about equal to the response for hydantoin.

Therefore, it cannot be concluded that blocking this site with a methyl or

ethyl group will increase the activity for hydantoin.

H 0 0

0

Fig. 5-3. Phenobarbital

Changes to the Hydantoin Ring - Phenobarbital (PB) (fig. 5-3). Of the drugs tested in these experiments, PB most closely resembles mephenytoin

structurally and more specifically, mephenytoin's major metabolite,

nirvanol. Mephenytoin undergoes N demethylation (by P-450) at position R 1

to form the metabolite, nirvanol. Nirvanol bears only a hydrogen at Rj,

thus differing structurally from PB by the insertion of a carbonyl group between C#5 and N#1 of the hydantoin ring. However, with respect to the ability to induce proliferation, PB's response was most similar to OPH 126

rather than mephenytoin. Like Mephenytoin and nirvanol, PB also has both

a phenyl and ethyl group on carbon #5, (positions R? & R3). However, unlike

mephenytoin, both nirvanol and PB only have a hydrogen on nitrogen #3

(fig. 5-3). The structural differences between hydantoin and that of the

pyrmidine ring may partially account for PB's relatively low response when

compared to mephenytoin. However, the unblocked N #3 site may also be

involved since it would be available for metabolism and the production of

epoxides.

Why the DPH analogues produced a greater degree of cell proliferation

than DPH is unclear. It is possible that the para substitution of the

hydrogen on the phenyl ring with one of the halides (Cl or Br) or a methyl group causes DPH's lipophilic characteristics to be altered. This would modify the drug's ability to enter and exit the cell.

The lipophilic activity of DPH and the analogues may be analyzed by examining their partitioning coefficient (log P values). The log P values represent a relative measure of lipophilicity and can give an indication of a molecule's ability to enter the cell. The major metabolite of DPH, p-OH-DPH (log P = 1.09), was the most polar, followed by PB and DPH

(2.40). The para substituted analogues were more lipophilic than DPH, with responses of (in increasing order): p-CH3-DPH (2.62), p-Cl-DPH (2.72) and p-Br-DPH (2.77). The largest and most lipophilic molecule was p-Br-

DPH, while p-CI-DPH produced the greatest response. It appeared that with an increase in lipophilicity, there was also an increase in the proliferatory activity of the molecule. However, even though p-Br-DPH was more lipophilic than p-Cl-DPH, it was less effective than p-Cl-DPH for

inducing proliferation. This observation was based on the analogue studies 127

(from the early cell counting experiments to the present investigations) in which p-Cl-DPH has consistantly given higher responses than p-Br-DPH.

Differences in the ability of p-Cl & p-Br-DPH to induce proliferation may be related to the differences in molecular size. However, there may exist other possible explanations as to the variations in activity between the para halogenated analogues, DPH, p-CH3-DPH and p-OH-DPH. For example, para halogenation causes differences in the charge on the phenyl ring due to electron withdrawl (from the phenyl ring). This tends to deactivate the phenyl ring and makes it less likely to undergo a reaction (metabolism).

It is also possible that the presence of a bromide group may make the molecule too lipophilic, thus trapping the molecule in the lipid membrane or some other compartment within the cell. This would reduce the number of molecules reaching the target site to induce proliferation.

In contrast, para hydroxylation of DPH makes the molecule more polar and water soluble. This would limit the number of p-OH-DPH molecules able to enter the cell and induce proliferation. Furthermore, para hydroxylation makes the phenyl ring more reactive and perhaps more likely to interact with other elements within the cell. This would also be true for p-CHj-DPH, possibly accounting for the great amount of variability observed for this analogue. With the addition of the methyl group, the molecule is more lipophilic than DPH, thus more drug can get in the cell.

However, once inside the cell, p-CHj-DPH would be free to affect many systems, including proliferation.

S. Mechanism of action Results from the structure activity studies showed that hydantoin is the parent structure responsible for DPH induced proliferation. Modifications to the ring, such as through diphenyl 128 addition on Carbon #5 (DPH), reduced the hydantoin ring activity. However, if one of the phenyl rings is para-halogenated (Cl or Br), the hydantoin ring activity appears to be restored. Other modifications, such as the presence of methyl or ethyl groups on N #3, seem not to cause major alterations in hydantoin activity, as was the case with mephenytoin and ethytoin.

The major metabolite of OPH in man, p-OH-DPH, had a response that was not significantly different from the controls. Thus, para-hydroxylation served to inactivate DPH, while meta-hydroxyl at ion essentially restored hydantoin ring activity. However, since it is very unlikely that m-OH-DPH is produced in man, it appears that metabolism would inhibit DPH's proliferative activity. The question now becomes, is DPH metabolized in cultured fibroblasts and if so, by what pathway? With the co-incubation of

PB & DPH, an additive response was observed, suggesting that cytochrome

"P450b" was not present. P450 is the primary metabolic pathway for DPH.

Another mechanism by which DPH could be metabolized would be via the PGS pathway. ASA, which blocks the PGS pathway, would inhibit DPH metabolism and the production of any reactive intermediates. In the presence of ASA, the activity of DPH increased to a level greater than or equal to hydantoin and the para-substitued analogues. This suggests that DPH is metabolized by the PGS pathway. Therefore, in situ metabolism of DPH could be responsible for DPH cytotoxicity and variablity in its responses, while the parent molecule induces growth.

The specific mechanism behind DPH's proliferatory activity remains unclear. However, many possibilities may exist as to how DPH induces proliferation in normal human dermal fibroblasts. It is possible that hormonal receptors, such as the glucocorticoid recptor, may serve as the target for DPH's activity as well as the triiodothyronine receptor. There is some indication that DPH may act on hormonal sites as it has been shown to bind to the glucocorticoid [55,104,105] and triiodothyronine receptors

[109-112], However, whether this has any effect upon cell proliferation is unknown. Jimenez De Asua showed that the hormones insulin (20 ng/ml) and hydrocrtisone (50 ng/ml) produced a small proliferatory response in resting 3T3 cells. These experiments were conducted in the presence of 6%

FBS, but without the addition of any growth factors. By combining the hormones hydrocortisone (50 ng/ml) and insulin (20 ng/ml), a small but additive effect was produced for inducing resting cells to proliferate

[80]. The glucocorticoids can enhance growth factor activity, as can insulin [76,77,80,92,93,96,97,110,113], At concentrations of 3 pg/ml or above, insulin can act like a growth factor in some cell types [79,80].

DPH may act either directly or indirectly at these sites.

Our studies in cultured fibroblasts showed that both dexamethasone and progesterone could induce proliferation. When the antiprogesterone and antiglucocorticoid drug RU-486 was coincubated with DPH and dexamethasone,

RU-486 significantly reduced proliferation. However, further testing with multiple cell lines from different sources would be necessary to confirm this observation. CONCLUSIONS

1) DPH caused an increase in the rate of cell proliferation as demonstrated by cell counting, [3H]-thymidine incorporation and total DNA content.

2) DPH-induced proliferation in normal human dermal fibroblasts was independent of serum and the growth factors (EGF & FGF).

3) The presence of serum introduced variablity and increased baseline responseiveness, thus masking the DPH effect.

4) When the DPH dose response curve was run in medium supplemented with

EGF or FGF, the ED5q was shifted to the left while not altering the maximal response. This suggested that the DPH-induced effect was enhanced by the presence of EGF or FGF in this culture system.

5) The responses observed for the para substituted DPH analogues (p-Cl-

DPH, p-Br-DPH, p-CH3-DPH) were all greater than that of DPH. Of all the analogues, p-Cl-DPH was consistently the most active, followed by p-Br-DPH and then p-CH3-DPH. This data confirmed the in vivo observation by Savini, that p-Cl-DPH induced a significantly higher degree of hyperplasia

(proliferation) than DPH. However, the data did not support Savini's observation that p-CHj-DPH was inactive. The level of activity for p-CHj-

DPH was found to be at least as great as that of DPH.

6) The phenyl hydantoin analogues, mephenytoin & ethotoin were also able to induce proliferation to a greater extent than DPH.

130 131

7) The major metabolite of DPH in man, p-OH-DPH (HPPH), was found to have very little proliferative activity associated with it. Thus, HPPH can be thought of as an inactive metabolite. This observation is in agreement with the findings of other observers. However, a metabolite of

DPH that is produced in mice, m-OH-DPH, was able to induce proliferation in cultured NHD fibroblasts, to a greater extent than DPH.

8) The para methyl analogue was somewhat less active than was expected, suggesting that the activation of the phenyl ring may play a negative role with DPH-induced proliferation. This is supported by the fact that a hydroxyl group, which is a strong activating group, releases electrons, causing activation of the phenyl ring. Methylation also releases electrons and is considered weakly activating. In contrast to p-OH-DPH and p-CHj-DPH, the halogenated analogues produced the greatest amount of proliferation among all the drugs tested. Halogenation of the phenyl ring has an opposite effect to that of hydroxylation or methylation. The presence of a halide on a phenyl ring causes a withdraw 1 of electrons and a deactivation of the phenyl ring. Thus, ring activation may account for the lack of an effect observed with p-OH-DPH.

9) Blockage of the #3 nitrogen with an ethyl (ethotoin) or a methyl group

(mephenytoin) produced responses that were equal to that of hydantoin.

Again, the presence of an ethyl or methyl group at the #3 N may block or slow the rate of metabolism for these compounds. This was domonstrated by

Wells [53] with his teratogenicity studies in fetal rats.

10) ASA (10'6 M) was able to potentiate the proliferatory effects of DPH, and to a lesser degree the effects of hydantoin, upon cultured NHD fibroblasts. However, ASA had no significant proliferatory effect over 132 that of the vehicle controls. It is possible that the magnitude of ASA's effect upon hydantoin may be related to the number of potential sites available to undergo metabolism.

11) Viewing DPH activity with hydantoin as a reference, hydantoin ring activity was reduced by the addition of two phenyl groups (the DPH molecule). In presence of ASA, DPH induces proliferation as much or more than hydantoin, suggesting that the phenyl rings may serve as a primary site for metabolism. This differs from Wells finding [53,54] that the number three nitrogen of the hydantoin ring was the principle site for PGS metabolism of DPH in fetal tissue. It may be that having both sites available for metabolism helps account for DPH's relatively small proliferatory response, when compared to the analogues. Also, this could account for the larger effect that ASA had upon DPH-induced proliferation, when compared to hydantoin.

12) The best system to study the direct effects of drug-induced proliferation was with confluent quiescent cells, fed serum and growth factor free defined medium (GFF). Proliferation is then determined by

[ H]-thymidine incorporation (once it has been established that there is an increase in the total cell number by cell counting). REFERENCES

1. Gilman,A.G.; Goodman,L.S.; Gilman,A.: Goodman and Gilman's THE PHARMACOLOGICAL BASIS OF THERAPEUTICS. 6 th Edition. MacMiIlian Publishing Co.; Rail, T.W. & Schleifer, L.S.; Chapter 20 (448-471).

2. Goodman,L.S. & Gilman,A.G. (1940): The Pharmacological Basis of Therapeutics. Pgs 126-173. Publisher: MacMillian Co. NY,NY.

3. Katzung (1989): Basic and Clinical Pharmacology 4th Edition, a LANGE medical book. Pages 288-303. publisher: Appleton & Lange.

4. Baeyer,A. (1863): Unterschungen Uber die HarnsSuregruppe. Justus Liebig's AnnaTen der Chemie. 127 (199-236).

5. Baeyer.A. (1864): Ueber eine neue Klasse Organischer Stick- stoffverbindungen. Justus Liebig's Annalen der Chemie. 130 (129-49).

6. Dickey,J.B. & Gray,A.R. (1943): Barbituric Acid. Organic Synthesis, Collection Vol II. 2 (60).

7. Wheland,G.W. (1949): Advanced organic chemistry. 2nd Edition. Pages 617-621, 637. Publisher: New York - John Wiley & Sons, Inc. London - Chapman & Hall, Limited.

8. The MERCK INDEX - 10th Edition. Editor Martha Windholz. (1983): Merck & Co., Inc. 39, 1054-1056.

9. Carter,M.K. (1951): The history of barbituric acid. J.Chem.Ed. 28 (524-26).

10. Wi1son,C.0., Gisvo1d,0. and Doerge,R.F. (1966): Textbook of Organic Medicinal and Pharmaceutical Chemistry. 5th Edition. Pages 377-379. Publisher: J.B. Lippincott Company.

11. Blitz,H. (1908): Uber die konstitution der Einwir Kungsprodukte von substituierten Harnstoffen auf Benzil und Uber einige neue Methoden zur Darstel lung der 5,5-Diphenyl-hydantoine. Berichte der Deutschen Chemischen Gesellschaft. 41.1 (1379-93).

12. Blitz, H. & Seydel.K. (1911): Eine neue Darstellung von D1 phenyl -ami do- methan (Benzhydryl-amin). Berichte der Deutschen Chemischen Gesellschaft. 44,1 (411-13).

133 134

13. Henze,H.R. (1946): Method for obtaining hydantoins. U.S. Patent Office, Oct. 22, patent #'s 2,409,754; 2,409,755; 2,409,756. For 5,5-diaryl Hydantoins.

14. Merritt,H.H. & Putnam,T.J (1937): Experimental determination of the anticonvulsant properties of some phenyl derivatives. Science. 85. 2213 (525-26).

15. Merritt,H.H. & Putnam,T.J (1938): A new series of anticonvulsant drugs tested by experiments on animals. Archives of Neurology and Psychiatry. 39 (1003-15).

16. schlogl,K.; Wessely.F.; Kraupp,0. und Stormann,H. (1961): Synthese und phareakologie einiger 3,5-Di-und trisubstituierter hydantoine., J. Medicinal & Pharmaceutical Chemistry. 4,2 (231-258).

17. Merritt,H.H. & Putnam,T.J (1938): Sodium diphenyl-hydantoinate in treatment of convulsive disorders. J. Am. Med. Ass. 111 (1068-73).

18. pincus,J.H.; Grove,I; Marino,B.B. and Glaser,G.E. (1970): DPH blocks sodium in hypoxic nerves and offsets cyanide and quabain tox. Arch. Neurol. 22 (566-71).

19. pincus,J.H. & Lee,S.H. (1973): DPH causes decrease in calcium uptake in rat brain slices and decreases Norepi release from the cells. Arch. Neurol. 29 (239-44).

20. Messing,R.0.; Carpenter,C.L. and Greenberg,D.A. (1985): Mechanism of calcium channel inhibition by phenytoin: comparison with classical calcium channel antagonists. J. Pharmacol. E x p . Ther. 235,2 (407-11).

21. vernillo.A.T. and Schwartz,N.B. (1986): Stimulation of collagen and glycosaminoglycan production by phenytoin 5,5-di phenyl hydantoin in monolayer cultures of mesenchymal cells derived from embronic chick sternae. Archs oral Biol. 31.12 (819-23).

22. i_ew,G.M. (1975): Increased hypothalamic norepinephrine in genetically hypertensive rats following administration of diphenylhydantoin (38407). Proc.Soc.Exp.Biol.Med. 148,1 (30-32).

23. Hadfield,M.G & Weber,N.E. (1975): Effect of fighting and diphenyl hydantoin on the uptake of 3H-1 -norepinephrine in vitro in synaptosomes isolated from retired male breeding mice. Biochem. Pharmacol. 24,16 (1538-40).

24. Hadfield.M.G. & Rigby,W.F.C (1976): Dopamine-adaptive uptake changes in striatal synaptosomes after 3 sec of shock-induced fighting. Biochem. Pharmacol. 25.24 (2752-54).

25. Fry,B.W. & Ciarlone,A.E. (1979): Effects of phenytoin on 5- hydroxytryptamine and norepinephrine levels in brain slices. Pharmaco1oqist. 21.3 (183-85). 135

26 Fry,B.W. & Ciarlone,A.E. (1981): Effects of phenytoin on mouse cerebellar 5-hydroxytryptamine and norepinephrine. Neuropharm. 20.6 (623- 25).

27. Saad.S.F (1972): Effects of diazepam on gamma-aminobutyric acid (GABA) content of mouse brain. J.Pharm. Pharmacol. 24 (839-40).

28. Ayala,G.F; Johnston,D; Lin,S. and Dichter,H.N (1977): The mechanism of action of diphenylhydantoin on invertebrate neurons II. Effects on synaptic mechanisms. Brain Res. 121 (259-70).

29. Hansen,D.K; Ho1son,R.R; Sullivan,P.A. & Grafton,T.F. (1988): Alterations in maternal plasma corticosterone levels following treatment with phenytoin. Toxicology & Applied Pharmacology. 96 (24-32).

30. Second Annual Report on Carcinogens. NTP 81-43, Dec, 1981 (204-5).

31. Savini,E.C; Poitevin,R & Payen.J. (1980): Topical application of hydantoins in gingival disorders. Phenytoin -Induced Teratology and Gingival Pathology. Raven Press.

32. 0tto,G; Ludewig,R. & Kotzschke.H.J. (1977): Zur Spezifischen bezinflussung von parodontopathien durch lokale Phenytoin-Applikation. Stomatol. DDR 27.4 (262-68).

33. Chikhani.P. (1972): Utilization de la phinytoine sodique dans 1e traitement des parodontopathies. Actualities Odontastomat. 98 (265-74).

34. Simpson,G.M.; Kunz.E. and Slafta.J. (1965): Use of sodium diphenylhydantoin in treatment of leg ulcers. NY. J. Med. 65 (886-88).

35. Kellin.E.E. & Gorlin,R.J. (1961)-. Healing qualities of an epilepsy drug. Dental Prog. 1 (126-29).

36. Goebel,R.W. (1972): Sodium diphenylhydantoin association with oral Healing. J. Oral Surg. 30 (191-5).

37. Strean.L.P (1966): Chem.Abst.Biochem.Sec. 11219c (Merck & Co,Inc. Application for patent, Belg, May 12, 1965).

38. Donoff.R.B. (1978): The effects of diphenylhydantoin on open wound healing in guinea pigs. Journal of Surgical Research 24, (41-44).

39. Houck,J.C.; Jacob,R.A. and Maengwyn-Davies.G.D. (1960): The effect of sodium dilantin administration upon the chemistry of the skin. J. Clin. Invest. 39 (1758-62).

40. Sklans,S; Taylor,R.G. & Shk1ar,G (1967): Effect of diphenylhydantoin sodium on healing of experimentally produced fractures in rabbit mandibles. J.Oral Surg. 25 (310-19). 136

41. Frymoyer,J.W. (1976): Fracture healing In rats treated with diphenylhydantoin (Dilantin). Journal of Trauma. 16.5 (368-370).

42. Gudmundson,C. and Lindgren,L. (1973): Does diphenyl-hydantoin accelerate healing of fractures in aice? Acta Orthopaedics Scand. 444 (640-49).

43. LeFebvre,E.B; Jaining,R.G. & Labbe,R.G. (1972): Coarse facies, calvarial thickening & hyperphosphatasia associated with long-term anticonvulsant therapy. New Enal.J.Med. 286 (1301-02).

44. Kimball,0.P (1939): The treatment of epilepsy with sodium diphenylhydantoinate. J .Am.Med.Assoc. 112 (1244-45).

45. Konemann,H; Zelle,R; Busser,F. and Hammers,W. (1979): Determination of log values of chloro-substituted benzenes, toluenes and anilines by high-performance liquid chromatography on ODS-silica. J. Chromatoqr. 178 (559-65).

46. Garst.J.E & Wilson,W.C (1984): Accurate, wide-rang, automated, HPLC method for the estimation of octanol/water partition coefficients I: Effects of chromatographic conditions and procedure variables on accuracy and reproducibility of the method. J. Pharmaceutical Sciences. 73,11 (1616-22).

47. Borg8,0; Garle,M & Gutova.M (1972): Identification of 5-(3,4- dihydroxyphenyl)-5-phenylhydantoin as a metabolite of 5,5- diphenylhydantoin (phenytoin) in rats & man. Pharmacology. 7 (129-37).

48. Rao.G.S. & Wortel.J.P (1980): Gingival metabolism of phenytoin and covalent binding of its reactive metabolite to gingival proteins in the rat. Phenvtoin-Induced Teratology & Gingival Pathology. Ed. Hassell, Johnston, & Dudley. Raven Press, NY. (189-98).

49. Dudley,K.H. (1980): Phenytoin metabolism. Phenvtoin-Induced Teratology and Gingival Pathology. Ed. Hassell, T.M; Johnston,M.C. & Dudley,K.H. Raven Press, NY. (11-23).

50. Wei Is,P.G. & Harbison,R.D (1980): significance of the phenytoin reactive arene oxide intermediate, its oxepin tautomer, and clinical factors modifying their roles in phenytoin-induced teratology. Phenvtoin- Induced Teratology & Gingival Path. Ed. Hassell, Johnston, & Dudley Raven Press, NY. (83-112).

51. Dudley,K.H. (1980): Phenytoin metabolism. Phenvtoin-Induced Teratology and Gingival Pathology. Ed. Hassell, T.M; Johnston,M.C. & Dudley,K.H. Raven Press, NY. (11-23).

52. Wells,P.G. and Vo,H.P.N. (1989): Effects of the tumor promoter 12-0- tetradecanoylphorbol-13-acetate on phenytoin-induced embryopathy in mice. Toxicology & Applied Pharmacology. 97 (398-5). 137

53. Wei Is,P.G; Nagal,M.K. & Greco,G.S. (1989): Inhibition of trimethadione and dimethadione teratogenicity by the cyclooxygenase inhibitor acetylsalicyllc acid: A unifying hypothesis for the teratologic effects of hydantoin anticonvulsants and structurally related compounds. Toxicology & Applied Pharmacology. 97 (406-14).

54. Wells,P.G; Zubovits,J.T; Wong,S.T; Molinari,L.M. and Ali,S. (1989): Modulation of phenytoin teratogenicity and embryonic covalent binding by acetylsalicyllc acid, caffeic acid and *-phenyl-N-t-butylnitrone: implications for bioactivation by prostaglandin synthetase. Toxicology & Applied Pharmacology. 97 (192-202).

55. Katsumata,M.; Gupta,C. and Goldman,A.S. (1985): Glucocorticoid receptor IB: Mediator of anti-inflammatory and teratogenic functions of both glucocorticoids & phenytoin. Arch Biochem Biophvs. 243.2 (385-95).

56. Gupta,C; Katsumata,M. and Goldman,A.S. (1984): H-2 influences phenytoin binding and inhibition of prostaglandin synthesis. Immunogenetics. 20.6 (667-76).

57. Hogan,0.L; Ballesteros.A; Koss.M.A. and Isenberg,J.I. (1989): Cyclooxygenase inhibition with indomethacin increases human duodenal mucosal response to prostaglandin El. Digestive Diseases and Sciences. 34,12 (1855-59).

58. Lindhout,D; Hdppener.R.J.E.A. &Meinardi,H (1984): Teratogenicity of antiepileptic drug combinations with special emphasis on epoxidation (of carbamazepine). Epilepsia. 25,1 (77-83).

59. Kubow.S and Wells,P.G. (1989): In v itro bioactivation of phenytoin to a reactive free radical intermediate by prostaglandin synthetase, horseradish peroxidase and thyroid peroxidase. Molecular Pharmacology. 35 (504-11).

60. Skoog,D.A. & West.D.M. (1971): Principles of Instrumental Analysis. Holt, Rinehart and Winston, Inc. (216-217).

61. Lubet.R.A; Mayer,R.T; Cameron,J.W: Nims.R.W; Burke,M.D; Wolff,T and Guengerich,P.F. (1985): Dealkylation of pentoxyresorufin: A rapid and sensitive assay for measuring induction of cytochrome(s) P-450 by phenobarbital and other xenobiotics in the rat. Archives of Biochem and Biophysics. 238,1 (43-48).

62. Mastropaolo,D; Camerman,A. & Camerman,N (1983): Hydrogen bonding interaction of diphenyl hydantoin and 9-ethyladeine crystal structure of a 2:1 complex. Moleculular Pharmacolacology. 23 (273-77).

63. Margaretten.N.C; Hincks.J.R; Warren,R.P. & Coulombe Jr, R.A (1987): Effects of phenytoin and carbamazepine on human natural killer cell activity and genotoxicity in vitro. Toxicology and Applied Pharmacology. 87 (10-17). 138

64. Nebert,D.W. & Negishi.M. (1962): COMMENTARY. Multiple forms of cytochrome P-450 and the importance of molecular biology and evolution. Biochemical Pharmacology 31,14 (2311-17).

65. Karenlampi,S.O. (1983): Effects of cytochrome P1-450 inducers on the cell surface receptors for epidermal growth factor, phorbol 12,13-dibutyrate, or insulin of cultured mouse hepatoma cells. J.Biological Chemistry 258.17 (10378-83).

66. Hannah,R.R. (1981): Regulatory gene product of the Ah complex. Comparison of 2,3,7,8 Tetrachlorodibenzo-p-dioxin & 3-Methylcholanthrene binding to several moieties in mouse liver cytosol. J. Biol. Chemistry 256,9 (4584-90).

67. Negishi,M. et al (1981): Isolation and characterization of a cloned DNA sequence associated with the murine Ah locus and a 3-MC induced form of cytochrome P-450. P.N.A.S. U.S.A. 78.2 (800-4).

68. Tukey,R.H. et al (1981): Structural gene product of the [Ah] complex - evidence for transcriptional control of cytochrome PI-450 Induction by use of a cloned DNA sequence. J.Biol.Chem. 256,13 (6969-74).

69. Israel,0.I. & Whitlock,J.P.(1983): Induction of mRNA specific for cytochrome PI-450 in wild type and variant mouse hepatoma cells. J.Biol.Chem. 258.17 (10390-94).

70. 0key,A.B. et al (1979): Regulatory gene product of the Ah locus. Characterization of the cytosolic inducer-receptor complex and evidence for its nuclear translocation. J. Biol. Chemistry. 254,22 (11636-48).

71. Harvey,W; Grahame,R and Panayi,G.S. (1976): Effects of steroid hormones on human fibroblasts in vitro. II. Antagonism by androgens of cortisol-induced inhibition. Ann. Rheum. Pis. 35 (148-51).

72. Sooriyamoorthy,M; Harvey,W. & Gower,D.B. (1988): The use of human gingival fibroblasts in culture for studying the effects of phenytoin on testosterone metabolism. Archs oral Biol. 33.5 (353-59).

73. El Zayat.S.G. (1989): Preliminary experience with topical phenytoin in wound healing in a war zone. Military Medicine. 154,4 (178-80).

74. Modaghegh,S; Sa1ehian,B; Tavasso1i,M; Djamshidi.A. and Shiekh Rezai, A. (1989): Use of phenytoin in healing of war and non-war wounds. A pilot study of 25 cases. International J. Dermatology. 28.5 (347-50).

75. Shafer,W.G; Beatty,R.E. and Davis,W.B. (1958): Effect of Dilantin sodium on tensile strength of healing wounds. P.S.E.B.M. 98 (348-50).

76. Serrero.G.R.; McClure,D.B. & Sato.G.H. (1980): Growth of mouse 3T3 fibroblasts in serum-free, hormone-supplemented media. Control Mechanisms in Animal Cells. Ed. Jimenez De Asua et al. Raven Press, NY. (523-30). 139

77. ATCC Catalogue of cell lines and hybridomas - 6th Ed, 1988. Editors: Hay,R; Macy.M; Chen,T.R.; McClintock,P & Reid,Y. ATCC. Rockville, Md. (45,57-59).

78. Keith,D.A; Paz,M.A. and Gallop,P.M (1977): The effect of diphenylhydantoin on fibroblasts in v itro . J.Dent.Res. 56,10 (1279-83).

79. O'Farrell,M.K.; Clingan.D.; Rudland.P.S. and Jimenez De Asua.L. (1979): Stimulation of the initiation of DNA synthesis and cell division in several cultured mouse cell types. Experimental Cell Research 118, (311-21).

80. Jimenez De Asua,L (1980): An ordered sequence of temporal steps regulates rate of initiation of DNA synthesis in cultured mouse cells. Control Mechanisms in Animal Cells. Ed. Jimenez De Asua et al. Raven Press, N.Y. (173-97).

8 1. Paul.J. (1975): Cell and Tissue Culture - 5th Edition. Churchill Livingstone, Edinburgh, London & N.Y. (367-369).

82. Coulter Electronics, Inc. (1977): Instruction Manual for the Coulter Counter Model ZBI. Coulter part No. 6500053. Coulter Electronics, Inc. Hialeah, Fla.

83. Roberts,R.M; Gilbert,J.C; Rodewald,L.B. & Wingrove.A.S. (1974): An introduction to modern experimental organic chemistry. 2nd Ed Chapts 1-3. Holt, Rinehart & Winston, N.Y.

84. Toon,S; Mayer,J. & Rowland, M. (1984): Liquid chromatographic determination of lipophilicity with application to a homologous series of barbiturates. J. Pharmaceutical Sciences. 75,5 (625-7).

85. Ettre.L.S. (1973): Practical Gas Chromatography. For the usere of Perkin-Elmer Gas Chromatographs. Perkin-Elmer Corporation, Norwalk, Conn. Order Number: 990-9389.

86. Varian instrument division (1977): Aerograph 63 Ni electron capture detector manual. Includes ECD electrometer operation. Varian Associates, inc. Pub. #: 85-001139-21.

87. Ruddick,J.A. (1972): Toxicology, metabolism, & biochemistry of 1,2 propanediol. Toxicol. Appl. Pharmacol. 21 (102).

88. Gibson-D'Ambrosio,R.E.; Wani,A.A.; Leong,Y.; D'Ambrosio, S.M. : A sensitive microf luorometric method for monitoring unscheduled and scheduled DNA synthesis. Personal communication.

8 9. Brunk,C.F; Jones,K.C. & James,T.W. (1979): Assay for nanogram quantities of DNA in cellular homogenates. Annalvtical Biochemistry. 92 (497-500). 140

90. Smith,P.K; Krohn,R; Hermanson,G; Ma11ia,A; Gartner,F; Provenzano,M; Fujimoto,E.K; Goeke,N.M; Olson,B.J. & Klenk,D.C. (1985): Measurement of protein using bicinchoninic acid. Anal.Biochem. 150. (76-85).

91. Benveniste.K. & Bitar,M. (1980): Effects of phenytoin on cultured human fibroblasts. Phenytoin-Induced Teratology & Gingival Pathology. Raven Press.

92. Cherington,P.V. (1985): Regulation of fibroblast growth by multiple growth factors in serum-free media. In Vitro Models for Cancer Research. Volume III. Chapter 2, (17-51).

93. Jimenez De Asua,L; 0'Farre11;M; Bennett,0; Clingan.D. & Rudland.P. (1977): Interaction of two hormones and their effect on observed rate of initiation of DNA synthesis in 3T3 cells. Nature 265 (151-53).

94. Nebert,D.W (1980): Genetic differences in drug metabolism: possible importance in teratogenesis. Phenytoin-Induced Teratology & Gingival Pathology. Ed. Hassell, Johnston & Dudley. Raven Press, NY. (113-28).

95. Negishid.M & Nebert.D.W (1981): structural gene products of the Ah complex increases in large mRNA from mouse liver associated with cytochrome P1-450 induction by 3-MC. j.Biol. Chemistry. 256,6 (3085-91). 96. Cho-Chung,Y.S. & Clair,T. (1977): Specific glucocorticoid inhibition of growth promoting effects of prostaglandin F« on 3T3 cells. Nature 265 (450-52). *

97. Cristofalo,V.J.; Wallace, J.M. & Rosner,B.A. (1980): Glucocorticoid enhancement of proliferative activity in WI-38 cells. Control Mechanisms in Animal Cells. Ed. Jimenez De Asua et al. Raven Press, NY. (875-87).

98. Modeer,T; Dahlief,G. & 0tteskog,P. (1988): Potentiation of fibroblasts spreading by extracellular matrix from fibroblasts derived from phenytoin induced gingival overgrowth. Acta-Odontol-Scand. 46,2 (101-4).

99. Modeer,T. & Brunius,G. (1989) Effect of phenytoin on intracellular *5 Ca accumulation in gingival fibroblasts in vitro. J. Oral Pathol. Med. 18,8 (485-489).

100. Modeer.T; Dahl1bf,G; Karsten,J. & otteskog,P. (1988): Potentiation of fibroblast DNA synthesis by a phenytoin-induced mononuclear cell derived factor in vitro. Scand J.Dent.Res. 97 (186-87).

101. Modeer,T; Andersson,G. (1990): Regulation of epidermal growth factor receptor metabolism in gingival fibroblasts by Phenytoin in vitro. J. Oral Pathol. Med. 19.4 (188-191).

102. Nebert,D.W. (1978): Genetic differences in microsomal electron transport: The Ah locus. Methods in Enzvmology. H I (226-40). 141

103. Nebert,D.W. et al (1980): Toxic chemical depression of the bone marrow and possible aplastic anemia explainable on a genetic basis. Clinical Tox. 16,1 (99-112).

104. Goldman,A.S. (1984): Biochemical mechanism of glucocorticoid and phenytoin induced cleft palate. Curr.Top. Oev.Biol. 19. (217-39).

105. Kay.E.D; Goldman,A.S and Daniel,J.C. (1990): Common biochemical pathway of dysmorphogenesis in murine embryos: use the glucocorticoid pathway by phenytoin. Teratogenesis-Carcinog-Mutagen. 10,1 (31-39).

106. Aaron,J.S; Bank,S. & Ackert,G (1985): Diphenylhydantoin -induced hepatotoxicity. Am.J.Gastroenterology. 80,3 (200-2).

107. Bresnick,E. (1983): Induction of cytochrome P-450 by xenobiotics. Pharmacological Reviews. 36,2 (156-63).

108. Lamberts,S.W.J; den Holder,F. & Hofland,L.J (1989): The Interrelationship between the effects of insulin-like growth factor I & somatostatin on growth hormone secretion by normal rat pituitary cells: The role of glucocorticoids. Endocrinology. 124,2 (905-11).

109. Franklyn,J.A; Davis,J.R; Ramsden.D.B. and Sheppard, M.C. (1985): Phenytoin and thyroid hormone action. J. Endocrinol. 104,2 (201-4).

110. Topiiss,D.J; Kolliniatis.E; Barlow,J.W; Chen-Fee Lim & Stockigt,J.R. (1989): Uptake of 3,5,3'-triiodothyronine by cultured rat hepatoma cells is inhibitable by nonbile acid cholephils, diphenylhydantoin, and nonsteroidal anti-inflammatory drugs. Endocrinology. 124.2 (980-86).

111. Mann,D.N. & Surks,M.I. (1983): 5,5'-D1pheny1hydantoin decreases specific 3,5,3'-tri1odothyronine (T3) binding by rat hepatic nuclear T3 receptors. Endocrinol. 112 (1723-31).

112. Cavalier,R.R; Gavin,L.A; Wallace,A; Hammond,M.E. and Cruise,K (1979): Serum Thyroxine, free T4, triiodothyronine, and reverse-T3 in diphenylhydantoin-treated patients. Metabolism 28,11 (1161-65).

113. Armelin,H.A; Armelin.M.C.S; Farias,S.E; Gambarini,A.G. & Kimura,E. (1980): Effects of hydrocortisone on the growth control of a mutant derived from swiss mouse 3T3 fibroblasts. Control Mechanisms in Animal Cells. Ed. Jimenez De Asua et al. Raven Press, NY. (269-79).

114. Kay,E.D; Goldman,A.S and Daniel,J.C. (1990): Common biochemical pathway of dysmorphogenesis in murine embryos-, use the glucocorticoid pathway by phenytoin. Teratogenesis-Carcinog-Mutagen. 10.1 (31-39).

115. Houck,J.C; Cheng,R.F. & Waters,M.D (1972): The effect of Dilantin on fibroblast proliferation. P.S.E.B.M 139 (969-71) 142

116. Silvestri,E; Veraldi.S; Piferi.M; Sala.F; Bencini.L; Marini,D. (1988); Gingival hyperplasia due to diphenylhydantoin, cyclosporin A & nifedipine. A histo-pathological comparison. Minerva-Stomatol 37,3 (189- 192).

117. Shaftic.A.A; Widdup.L.L; Abate,M.A. and Jacknowitz,A. (1986): Nifedipine induced gingival hyperplasia. Drug Intel!. Clin! Pharm. July- Aug. 20, 7-8 (602-5).

118. Phi1ibert,D. (1984): RU 38486: An original multifaceted antihormone in vitro. Reprint Form Adrenal Steroid Antagonism. Editor: M.K. Agarwal. Walter de Gruyter & Co., Berlin, New York.

119. Couri,D; Suarez del Villar,C; Toy-Manning,P (1980): Simultaneous quantitative assay of phenobarbital, phenytoin & p-hydroxydiphenyl- hydantoin by HPLC and a comparison with EMIT. J.Analyt.Tox. 4 (227-31).

120. Shimada,K; Wakabayashi,H. & Sato,A. (1985): Gas chromatographic determination of m- & p-hydroxyphenytoin in urine of epileptic patients. J.Chromatography. 339 (331-7).

121. Kapetanovic,I.M. and Kupferberg,H.J. (1984): Analysis of p- hydroxyphenytoin in microsomal reactions by high-performance liquid chromatography with electrochemical detection. J.Chromatography. 310 (418- 23).

122. Ringo1d,G.M. (1985): Steroid hormone regulation of gene expression. Ann.Rev Pharmacol.Toxicol. 25 (529-66).

123. Modeer,T; Andersson,G. (1990): Regulation of epidermal growth factor receptor metabolism in gingival fibroblasts by Phenytoin in vitro. J. Oral Pathol. Med. 19.4 (188-191).

124. Sheridan,P.J. et al (1979): Equilibrium: The intra-cellular distribution of steroid receptors. Nature 282 (579-82). APPENDIX A

The Metabolic Fate of DPH. In phase I metabolise, the lipophilic xenobiotics are made more polar by either the addition or the unmasking of hydroxyl, sulfhydryl or amine functional groups. Therefore, by increasing a xenobiotic's polarity, it becomes increasingly more hydrophilic and soluble in water. With increased water solubility, the newly formed metabolite can now be excreted either via the kidneys or the bile.

Another consequence of metabolism is the alteration of a xenobiotic's activity. This can occur via several different classes of reactions, catalyzed by cytochrome P-450 dependent monooxidations. These reactions include epoxidations; oxidative dealkylations (such as N, 0 & S dealkylations); and aromatic and aliphatic hydroxylations. Aromatic hydroxylation is the most important reaction for the metabolism of OPH and phenobarbital. It inactivates the drug's therapeutic action by hydroxylating a phenyl group at its para position. However, this reaction may not always deactivate the compound and may, in some instances, involve the generation of toxic reactive intermediate metabolites, like the epoxides. Additionally, some of the metabolites formed by N demethylation may not necessarily be inactive, as with mephenytoin's metabolite nirvanol. Nirvanol is finally deactivated by aromatic hydroxylation.

Cytochrome P-450. Phase I metabolism is made up of a group of enzymes called the Mixed Function Oxidases (MFO). These enzymes are located within

143 144 the hepatocyte's lipid membrane portion of the smooth endoplasmic reticulum (SER). When preparing liver homogenates, the SER is found in the microsomal fractions. The MFO system, which is responsible for DPH's metabolism, is made up two enzymes: NADPH cytochrome P-450 reductase and the terminal oxidase, cytochrome P-450. Many isozymes of cytochrome P-450 have been identified in man, mouse, rat & rabbit, where each is responsible for metabolizing different groups of compounds.

Isozymes of P-450 can be characterized, in part, by agents responsible for their induction and by the substrates of that isozyme. For example, the isozyme CYP (IA) is responsible for metabolism of polyaromatic hydrocarbons, such as the potent carcinogen benzo(a)pyrene. It is known as LM4 in the rabbit,while in the rat it is referred to as P-448 or P^ASO in man. In contrast, DPH is metabolized by another isozyme of P-450, IIB1 which is known as P-450b in rat or LM? in rabbit. The isozyme responsible for metabolizing hydrocortisone, dexamethasone, digitoxin and theophylline is known as P-450p in rat and human, or LM3C in rabbit. Additionally, this isozyme is DPH inducible [1,7].

The term P-450 refers to the spectral qualities for the isozyme when carbon monoxide binds to its reduced/ferrous heme group. This results in the isozyme having a maximum absorption at a wavelength near 450 nm. other isozymes of P-450 have different absorption maximum from that of P-450b, such as the 3-methylcholanthrene (3-MC) inducible form of P-450 is also known as P-448. When carbon monoxide is bound to the ferrous heme of the isozyme, P-450c has a characteristic absorption maximum at 448 nm.

Mono, di or polycyclic aromatic hydrocarbons, such as DPH and its analogues, phenobarbital, benzo(a)pyrene and 3-MC, all undergo cytochrome 145

P-450 dependent oxidation as their primary means of metabolism. For example, in both rat and man, the major metabolite of DPH is formed by para hydroxylation of a phenyl ring [1,3]. With DPH, the major metabolite produced is para hydroxy DPH [1,3,47].

Induction and Genetic Control of Cytochrome P-450. Enzymes can be classified into two general categories, those that are constitutive or those that are inducible. Constitutive enzymes are characterized as being formed at constant rates and in constant quantities, independent of the cell's metabolic state. One of the best examples of constitutive enzymes are those found in the glycolytic pathway. However, inducible enzymes are those that are normally found in trace quantities within the cell, but in the presence of a substrate or an inducer, enzyme levels can increase a thousand fold or more [64,65],

The genes responsible for the synthesis of inducible enzymes are usually repressed, thus accounting for the enzymes' low concentration.

This repression is lifted in the presence of substrate or some specific inducing molecule [64,65]. When a single molecule induces more than one enzyme, this is then referred to as coordinate induction. The benefit of such a system is in conserving the cell's amino acid pool and metabolic energy, amounting to an economic system of control [64,65]. Cytochrome P-

450c is an inducible enzyme that metabolizes and is induced by polycyclic hydrocarbons. Among those polycyclic hydrocarbons capable of inducing the activity of P-450c are 6-Naphthoflavone, 3-Methylcholanthrene (3-MC) and tetrachlorodibenzo-p-dioxin (TCDD).

Cytochrome P-450b is another isozyme that is inducible, not by polyaromatic hydrocarbons, but by phenobarbital [1,3,66-68]. Some of 146

P450b's substrates include DPH, phenobarbital and other barbiturates, hydrocortisone, digitoxin and estradiol. DPH and spironolactone, in turn, serve as inducers of P-450p (LM3c). The isozyme, P-450p, is primarily associated with the metabolism of digitoxin, hydrocortisone, dexamethasone, testosterone and theophylline [1,3]. The consequence of inducing P-450 activity can best be seen when lab animals are pretreated with phenobarbital or 3-MC prior to doing metabolic studies (table 1-3).

The operon is the primary focus for induction's control mechanism, made up of a series of structural genes, that contain the messages coded for the enzyme's amino acid sequence and the operator gene. The "operator locus" controls transcription of the various structural genes found within the operon. The regulator gene contains the code for the amino acid sequence for the repressor protein, which binds to the operator locus, inhibiting transcription of the structural genes. Finally, the inducer is a molecule that is normally a substrate of the induced enzymes. The interaction of these components cause a change in gene expression, probably derepression, resulting in enzyme induction.

The repressor protein, when bound in a reversible fashion to the inducer, becomes a non-functional unit, no longer capable of producing inhibition. Once inhibition has been lifted from the operon, the structural genes are then free to be transcribed, through the action of an induced RNA polymerase II, producing the associated mRNA segment. This fact was demonstrated by Nebert and others when they investigated the Ah locus in inbred mice, through using a cloned DNA sequence [64,65,67-69].

Also, the Ah locus was located in many different tissue sites, such as the liver & several non-hepatic tissues. They determined that the major 147 regulatory gene product of the Ah locus was the cytosolic receptor for polycyclic hydrocarbon inducers [64-70].

Phenobarbital has been shown to act in a similar manner to 3-MC for inducing enzyme activity. Within the nucleus, phenobarbital stimulates the activity of RNA polymerase, increases RNA content per nucleus and the stability of the 45s RNA, an rRNA precursor. Also, phenobarbital causes changes within the cell by increasing P-450b content, enhancing initiation and elongation of peptide chains and increasing the production of mRNA

[71]. This process may be expected for DPH induction of cytochrome P-450p.

DPH has been shown to causes an increase in the metabolism of testosterone to the 5g-dihydro-testosterone (5g-DHT) and 4-androstenedione forms in cultured human gingival fibroblasts [72]. This is significant in that both 5#-DHT and DPH are active with respect to inducing gingival hyperplasia and increase the rate of collagen synthesis [71].

Sooriyamoorthy proposed that DPH may induce gingival hyperplasia by enhancing the metabolism of testosterone. Whether this is true or not remains to be seen. However, it has become apparent that there is a link of some sort between DPH-induced proliferatory activity and metabolism.

The metabolism of DPH by P-450b. The metabolic scheme for DPH, where the microsomal oxidation of DPH in man is shown in figure 1-6. The process requires molecular oxygen, cytochrome P-450 reductase, cytochrome

P-450b and a NADPH-generating system.

In other species, the major metabolite that is produced may vary in either the percentage of p-OH-DPH produced (as in rat) or in the primary type of metabolite produced. For example, dogs have been shown to produce meta hydroxy-DPH (m-OH-DPH) as their major metabolite. However in man, the 148 metabolite m-OH-DPH is thought not to exist or be produced as a minor metabolite. Other phase I metabolites of DPH that are formed include the dihydrodiol, 3-0-methyl catechol & catechol. Ultimately these metabolites will undergo conjugation with glucuronides in phase II metabolism [49].

During the metabolic process, reactive intermediates can be formed, causing necrotic, mutagenic, teratogenic or carcinogenic changes in target tissues. The epoxide (arene oxide) of DPH was suspected to be the toxic or reactive intermediate responsible for the observed teratogenicity, or possibly even the DPH-induced gingival hyperplasia [48,50,51]. Through the process of epoxidation, an arene oxide is formed when the oxygen binds to the aryl portion of the molecule while an epoxide is formed when the oxygen is bound to alkyl portion. The P-450 pathway is thought to be responsible for forming the arene oxide while an alternate pathway, the prostaglandin synthetase (PGS), is thought to produce the epoxide [52-54].

Finally, the epoxide, or possibly even some other reactive metabolite

(such as the semiquinone), is thought to cause lipid peroxidation or bind to cellular macromolecules. Examples of macromolecules that these reactive intermediates could covalently bind to may include DNA, RNA, regulatory proteins and hormonal receptors (T3, glucocorticoid), thereby disrupting normal cellular functions. APPENDIX B

Hemocytometer.

Counting cells with a hemocvtometer accomp1ishes two tasks. First, the cell vitality can be determined and second, the cell number can be resolved by counting the number of cells present per field for each of the hemocytometer's two chambers (fig. 7-1). The procedure for doing cell counts by hemocytometer involves making a 1:1 dilution of the cell suspension with a vital stain. Typically, 0.5 ml of cell suspension was mixed with 0.5 ml of 0.1% trypan blue vital stain, and from this an aliquot was removed and loaded into each of the hemocytometer's two chambers. Next, using a standard Zeiss microscope, the hemocytometer was read at 100 x, counting both the total number of cells in fields 1, 3, 5,

7 & 9 along with the number of cells taking up the vital stain, trypan blue. Subtracting out the number of cells taking up the vital stain from the total cell number results in the number of vital cells (equation 7-1).

The percent vitality for the culture could then be calculated by dividing the number of vital cells by the total cell number times 100.

Those cells that take up the vital stain, trypan blue, are considered to be non vital or dead. Dying or dead cells lose their selectivity and permit the up take of the vital stain [81]. However, healthy cells with intact surface membranes, are able to selectively exclude many substances, including vital stains.

149 150

A. N - Total Number of Cells - Number of StainedCells

B. Vital Cells/ml-—— No. of Fields Counted C. Total Vi tal Cell Number-Cells/mlxVolume of Cell Suspension ffhereN- Number of Vital Cel Is per Field. DF- DilutionFactor. Equation 7-1. The calculation of the number of vital cells per ml of medium and total cell number by hemocytometer.

Cell counts, determined by a hemocytometer, were used for assessing cellular viability and plating efficiency. Plating efficiency refers to that proportion of cells that are alive and capable of forming colonies.

Cell counts by hemocytometer were also used to ascertain the extent of

DPH-induced proliferation, with the serum-stimulated cell system in rapidly dividing cells, compared to controls. Further studies using cell counts by hemocytometer, proved to be too cumbersome. For larger studies involving cell counting, a Coulter electronic cell counter was used.

1t 3

4 J ( 1

1f

? 9 , « 1 m

Figure 7-1. HEMOCYTOMETER Areas 1, 3, 5, 7 and 9 represent one chamber's fields in which the cells are counted. Each field has the dimensions of 1 x 1 mm in area x 0.1 mm high, giving a volume of 0.1 mm3. APPENDIX C

Synthesis of DPH Analogues - Instrumental Analysis.

Following recrystallization, the products were first analyzed by doing melting point determinations. This method was a quick way to determine the relative purity of the product. Crystals were placed into a capillary tube, and heated slowly near the expected melting point. The crystals were checked for the temperature at which the they first melted and again when the last of the solid finally melted. These two temperatures account for the crystal's specific melting range. If the melting points were different from that expected, the recrystallization process was repeated until all of the unreacted benzophenones were removed. When satisfactory melting points were obtained, more rigorous checks of purity were used such as Thin Layer Chromatography (TLC) and Gas Chromatography (GC). Upon final purification, the DPH analogues were submitted for analysis by

GC-Mass Spectrometry and Direct Probe Mass Spectrometry to assure the purity and identity of the product.

TLC served as a rapid means with which to determine the relative purity of the reaction products. The separations were carried out on silica gel 20 x 20 cm TLC plates (silica). The total volume for the solvent system was 198.5 ml and was composed of 170 ml ethyl acetate, 20 ml methanol, 6 ml water and 2.5 ml of ammonium hydroxide. At the origin,

20 pi of sample was spotted (fig. 7-2). During a run, the solvent front

151 152

THIN LAVER CHROHAIORGAPHY: DPH A ANALOGUES

<— SOLVENT FRONT

O o O o

ORIGIN

F ig u re 7 -2 . Thin layer chromatography of compounds from the synthesis. Lanes 1) DPH 2) p-Cl-DPH 3) p-8r-DPH 4) p-CHj-DPH 5) p-Cl-Bzp 6) p-Br-Bzp 7) p-CHj-Bzp 8zp=benzophenone.

would migrate to a height of 15 cm, followed by the samples at various distances. These distances that the samples migrated were translated into

Rf values. The for each sample was calculated by dividing the distances the samples traveled by that of the solvent front.

The spots (sample) were located, initially, by irradiating the plate with short wavelength UV light. The spots were marked by circling them with a pencil. The plates were then heated for 10 minutes at 75°C to drive off the ammonium hydroxide. The plates were then sprayed with ninhydrin to check if any primary or secondary amines were present. After ninhydrin, the plates were first irradiated with long wavelength UV light to develop 153

Table 7-1

SAMPLE Rf 1. DPH 0.64 2. p-Cl-DPH 0.63 3. p-Br-DPH 0.62 4. p-CH,--DPH 0.62 5. p-Cl- benz 0.85 6. p-Br- benz 0.84 7. p -c h 3.-benz 0.84

the primary amines. The plates were then heated for 10 minutes to develop the secondary amines. For the hydantoins and the benzophenones, both of these steps were negative.

Spraying the plates with diphenylcarbazone (DPC) gave a positive reaction, turning the spots pink, for both the hydantoins & benzophenones.

If a sample gave more than one spot, the samples were recrystallized.

However, if the samples appeared to be pure, then either HPLC or GC analysis was preformed, to further check the purity.

Reversed phase high pressure liquid chromatography (HPLC) analysis of the products was another means by which we were able to determine the relative purity of the analogues [119,120,121]. Generally, with reversed phase liquid chromatography, the mobile phase is more polar than the octadecyl stationary phase. With this system, the order of elution runs from the most hydrophilic compounds to that of the most lipophilic. That is, the solutes' order of elution is of an increasing hydrophobic nature and is the reverse of normal phase separations. The mobile phase that we used was made up of a phosphate buffer (aqueous phase) and methanol

(organic phase). The phosphate buffer was 4 mM (NaH2P04 and Na^POj) and had a pH of 3.5. The aqueous phase accounted for 35% to 80% of the mobile 154 phase's total composition, while the organic phase (HPLC grade methanol) accounted for 20% to 65% of the total.

In order to calculate the partition coefficients for the analogues by

HPLC, our organic phase was made up of only methanol, rather than the more commonly used combination of methanol and acetonitrile. As a powerful modifier of solvent strength, acetonitrile decreases the retention time

(quicker elution) for the more lipophilic compounds, thus greatly changing the assay's overall linearity. The lack of linearity created by the use of acetonitrile would have complicated calculating the analogues' partitioning coefficient, thus it was not added to the organic phase.

The column used was a 30 cm long x 3.9 mm inside diameter Waters C 18 pBondapak, which was wrapped with a heating mantle. The heating mantle was used so the system could be run at temperatures above ambient, the advantage being that there is an overall decrease in the time required to run an assay, while not interfering, as extensively as acetonitrile, with the linearity. Thus, running at elevated temperatures decreases the samples' retention times. The flow rate was 1.5 ml/minutes.

We used an Altex HPLC system, with a UV-VIS detector set at a wavelength of 215 nm. Sample preparation involved dissolving crystals of purified compound or standards into acetone. The standards with known partition coefficients (Log P) consisted of DPH, p-OH-DPH, phenobarbital, barbital, tegretal and diazepam. Running these standards and unknowns with various percentages of methanol, we were able to determine the capacity factors (K') for each percentage. From the log K'values, we were able to determine the partition coefficients (Log P). The mean retention times for each compound are listed in Table 7-2. 155

Calculation of the capacity factors (K1) or the RQ values for the solute, were important in determining the log P values for the analogues.

The capacity factor was calculated from the ratio of the difference between the retention time for the solute and that of the void volume, divided by the retention time for the void volume. The equation is: K' =

(TrT0)/T0, where T1 = retention time for the solute and T 0 is the retention time for the void volume. On the other hand, the value RQ = (TpTgJ/T^

The advantage associated with the use of log RQ values, rather than log

K', was that log RQ has greater linearity when plotted against the log P values for the standards [84].

The partition coefficients (log P) for the analogues were determined to be: p-OH (1.09); DPH (2.40); p-CHg-DPH (2.62); p-Cl-DPH (2.72);

Table 7-2

Mean Retention Times (min) at specific percents of Methanol

Compounds 45 % 50 % 55 % 60 % 65 %

0 . void volume 3.5*0.01 3.4*0.06 3.4*0.03 3.3*0.01 3.3*0.01 1. barbital 5.2*0.01 4.6*0.04 4.1*0.02 3.7*0.01 ------2. p-OH- DPH 7.9*0.03 6.0*0.06 4.8*0.04 4.1*0.02 3. Phenobarbital 9.0*0.03 7.2*0.04 5.7*0.05 4.8*0.04 ------4. DPH ------12.9*0.14 8.7*0.13 6.4*0.08 5.0*0.01 5. tegretol 15.9*0.08 10.5*0.21 7.5*0.17 5.8*0.01 6. P-CH3-DPH 22.4*0.33 13.6*0.23 9.0*0.16 6.5*0.02 7. p-Cl- DPH 31.8*0.61 18.4*0.32 11.6*0.18 7.9*0.02 8. p-Br- DPH 38.5*0.89 21.6*0.42 13.2*0.22 8.8*0.02 9. diazepam — — — — — — — — 27.0*0.02 16.7*0.11 11.2*0.03 p-Br-DPH (2.77), while phenobarbital was (1.42). Higher numbers indicate increased lipophilicity. Thus, para hydroxylation makes DPH more hydrophilic, while para halogenation or methylation makes it more lipophi1ic. 156

Gas chromatography (GC) was also used to help determine the relative purity of the analogues. In addition, a GC equipped with an electron capture detector (ECO), proved especially helpful with detecting the para halogenated analogues. ECD is a highly specific detector for confirming the presence of halogens. The principle of electron capture is based upon the ability of certain types of molecules to absorb electrons. The carrier gas, for ECD, flows constantly through the detector and is ionization by the radioactive source, 63 Ni. A standing current is applied across the two electrodes, capable of capturing all the ions and electrons supplied by the carrier gas. This standing current is amplified and seen on the recorder as the baseline. The introduction into the detector of any electron capturing compound, that is havingan affinity for electrons, produces negative ions. The negative ions are formed by one of two reactions, which results in a decrease in the ion current.

a) AB + e ~ ------> (AB)' b) AB + e ' ------> A + B'

This decrease in current results in a negative response, which must be inverted in order to see the characteristic peaks seen in the recorder

[85,86]. Such a detector is an ideal means with which to detect halogenated compounds.

We used ECD to detect the presence of the halogenated analogues from their halogenated starting material and any non-halogenated background.

For this, we used a Varian 3700 CG, equipped with an ECD detector. We used a 6 foot, 0V 17 column to separate the compounds. In addition, we programmed the temperatures to go from an initial temperature of 150°C, which was held for one minute, to a final temperature of 2756C, with no final hold time. The rate of temperature increase was 10°C per minute. The injector temperature was set at 2908C while the EC detector was set to

340°C. The attenuation settings that were used, were ECD 10 x 128. The sample size injected was 1 pi of dissolved crystals. The chart speed used was 0.25 & 1.0 cm/min. For example, the para-cl analogue had a retention time of 11.6 minutes while its starting material, p-Cl-benzophenone, had a retention time of 3.2 minutes. Thus, ECD proved to be a very powerful technique, especially when used with HPLC, TLC or GC equipped with other types of detectors, such as a nitrogen detector (TSD) or the universal detector, flame ionized detector (FID). APPENDIX D

Serum Free Screen of RU 486 and Drug/FGF Interaction. Various drugs were screened for their activity in serum free defined medium. Quiescent cells were fed (F-14) containing 50 ng/ml FGF and either vehicle (0.001% -7 v/v) or 10 M DPH, dexamethasone (DEX), progesterone and the anti- progesterone/anti-glucocorticoid RU 486 [118]. In addition, RU 486 (10'7

M) was combined with DPH (10-7 M) or DEX (10'7 M). The control for these experiments was 50 ng/ml FGF (Table 7-3 & fig. 7-3).

Table 7-3

Drug vs FGF (50 ng/ml) Control in Defined, Serum Free Medium.

.7 Drug Concentrations are all at 10 M. Significance Treatment CPM S.D. % CPM to FGF 50 ng/ml

FGF 50 ng/ml 3325.3 404.8 100.0 p = 100 4059.7 342.7 122.1 ns

FGF 50 ng/ml + DPH 3730.7 31.1 112.2 ns Dexamethasone 4103.2 209.9 123.4 0.0203 Progesterone 3010.3 183.6 90.5 ns RU 486 2107.8 84.8 63.4 0.0250

Significance as FGF 50 ng/ml + RU 486 (10~7 M) + compared to drug alone

DPH 2686.9 15.1 80.8 0.0595 Dexamethasone 3721.9 49.0 111.9 ns

Medium ■ F 14 (1:1 B & F-12), n * 6. ns * not significant.

The results suggest that DPH caused a slight, but not a significant 3 increase in [ H]-TdR incorporation (112.2 ± 0.9% of the control). FGF

158 Figure 7-3. The mean drug effects on FGF-induced cell proliferation in serum free medium. All treatment groups contain 50 ng/ml FGF and all the drugs were 1x10 M. Human dermal fibroblasts were used in these studies. DEX = Dexamethasone; PRO = Progesterone; RU = RU 486 (n = 6).

159 5000

4000-

3000

2000

1000

t r F 50 F 100 Tdph 'd e x p r o ' RU^ rWS" RU+DEX Figure 7-3 O n O 161

(100 ng/ml) also produced a slight increase (122.1 ± 10.3% of the control) in proliferation over that of the control response. As with DPH, this response was not significant.

While the combination of FGF (50 ng/ml) & dexamethasone (10~7 M) produced a significant response (p= 0.0203; 123.4 ± 6.3%), RU 486 also produced a significant, but negative response (p= 0.0250; 63.4 ± 2.6%).

Combining the anti-progesterone and antiglucocorticoid RU 486 (10 M) with either DPH or DEX (both at 10~7 M) caused a reduction in DPH and DEX- induced responses. The combination of DPH and RU 486, reduced DPH's response by 27.98%. This reduction of DPH's response was extremely significant (p= 0.0001 ) when compared to DPH alone. However, when this response was compared to the FGF control, then the level of significance is reduced to only marginal significance (p= 0.0595). With the combination of RU 486 and DEX, there was a significant reduction (9.29%; p= 0.0273) in dexamethasone's response. However, when comparing the FGF control response to that of the combination of dexamethasone and RU 486, one finds that there is no significant difference between the treatment group and that of the control. From these experiments, it appears that this serum free system was feasible for conducting DPH studies.