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Current Drug Metabolism, 2011, 12, 507-525 507
Xenobiotic Metabolizing Cytocbrome P450 in Pig;a Promising Animai Model I
EmanuelaPuccinelli8 *, Pier Giovanni Gervasi8and Vincenzo Longob o]stituto di Fisiologia Clinica, CNR, via Moruzzi, 1, 56124 Pisa (Italy), b]stituto di Biologia e Biotecnologia Agraria, CNR, via Moruzzi, 1, 56124Pisa, 1taly
Abstract: The pig has been used as an important animai model far human studies becauseof its similarity in size, physiology and disease development However, in contrast to the extensive data available on the cytochrome P450 (CYP) system far humans and rodents, the data related to pig afe limited because or, among others, the presence of intra-species differences (domestic pigs and minipigs) The knowledge of the CYP superfamily in a given experimental animaI is crucial far pharmacological and toxicological tests in developing drugs and far understanding the metaboli~ pathways of toxicants and carcinogens. In addition, information on the CYP system in pigs is Important smce Il plays a domlnant role m the metabolism of veterinary drugs, whose residues rernain in the porcine tissues which afe food far humans The aim of the present review is to examine -in the liver alId extrahepatic tissues of pig -our current knowledge of the xenobiotic- metabolizing CYPs be!onging lO.famili.es 1-4, in terms of drug metabolism, substrate specificity, inhibition, gene expression and recep- tor-dnven regulatlon, m companson wlth human data It is hoped, furthermore, that this review may stimulate research on the porcine drug-metabollzmg enzymes morder to evaluate the hypothesis whereby pig data may better reflect human drug metabolism and toxicity than those obtained from the traditional non-rodent models.
Keywords: Pig, minipig, cytochrome P450 (CYP), liver, extrahepatic tissues, animai model, xenobiotics, porcine nuclear receptors.
INTRODUCTION Cytochrome P450 (CYP) is acomplex and very wide superfa- The extrapolation of biological animai data to humans is often mily of enzymes which play a major role in xenobiotics metabolism difficult, especially if no human data afe available. An increasing as ~ell as in endogenous compounds oxidation. It represents the number ofstudies suggests the use ofthe pig (or minipig) as a new mam phase-1 metabolic system, and it could be involved in drug- animai model for humans, as this species offers many advantages. drug interactions, toxicity, and bioactivation of carcinogens [8]. The pig is considered a good model in biomedical research because Nevertheless, the amount ofporcine CYPs data is stili scarce com- of its anatomical, physiological, and biochemical similarity to hu- pared to that ofhumans or rodents, and only in the last two decades mango Many organs and systems -including beaTI, nasal cavity, some ~fforts bave been focused on these enzymes [9-11]. One of liver, kidneys, brain, reproductive and gastrointestinal system -~e ~am hur.dles for the met~bo~ic characterizati<:>nofthe pig is the show analogies with humans and advantages compared to other slgrnficant dlfference occumng m the varlous anlffial breeds. Many experimental models [I]. The complete pig genome will be avail- breeds of both conventional pigs (e.g. Large White, Landrace or ~ble soon (www.sanger.ac.uk/Projects/S_scrofa/), and the compara- Duroc) and .minipigs. (e.g. G6~ingen, B~a or Yucat.an) afe used tlve maps obtained so far bave shown an extensive conserved ho- for metabollsm studles, creatmg data mlsunderstandmg and am- mology with the human genome. The pig is also recognized as a biguous interpretations. Even a variety of microminipigs (stili un- model for several major human diseases, such as cardiovascular characterized) has been recently developed with the specific aim of diseases (e.g. atherosclerosis), metabolic diseases (e.g. hypercholes- non~clinical ph~acological/t.oxicologiCal use [12]. However it is terolemia) and neural diseases (e.g. Parkinson's and Alzheimer's) pertment to pomt out that pnrnary structure of CYP enzymes in [2,3,4]. Some pig breeds (e.g. G6ttingen minipig) afe predisposed convention~ p!gs and. min~pigs is not expected to ~iffer signifi- to obesity, and this feature could provide ideaI families for the iden- cantly. Takmg m conslderatlon only the CYPs belongmg to the 1-4 tification of genes involved in this pathology [5]. In addition, the families, which afe the most involved in the xenobiotics biotrans- pig .is th~ only.non-rodent s.pecies in which the generation oftrans- formation, it is relevant to no~e that:. i) there is.~ high ~omology l' gernc anlffials IS well establlshed [5]. Another field in which the pig between humans.and ~nventlon~ ~I~S CYPs; Il) the dlfferences represents a tuming point is that of xenotransplantation. The high between convent!onal plgs and mmlplgs CYP sequences (at least similarity found in heart (including coronary arteries) and liver the only few avallable so far) afe less than I % and they should be makes the pig the best candidate for xenotransplantation to humans, r~garded as alleli.c variants (Table 1). Nevertheless, significantly I thus eliminating the major problem of organ scarcity [6]. Bioartifi- d!fferent data afe m many case expected to result from CYP expres- ciallivers (BALs) bave also been proposed and developed for the slon levels, substrate specificity and CYP-dependent drug metabo- extracorporeal circulation of patients with severe liver failure [7]. lism profiles derived from convetional pigs and minipigs. In view Pigs may be introduced as animai model in the safety assessment of ofthis, minipigs present some advantages with respect to commer- pharmaceutical or chemical products and this could bave a positive cial pigs. For instance, the G6ttingen minipig is a genetically de- impact on the so called 3R's: Replacement (i.e. substitution of dog fm~d model (.lffilik~ rout~ely used dogs and monkeys) since the and monkey -the non-rodent species typically used in toxicology), entlre populatlon hlstory IS well documented from the early devel- Refinement (i.e. use of more suitable species) and Reduction (i.e. o~m.e~t up t<:>th~ present [13]. In ~ddition, the smaller size of minor animai number) [I]. For ali these important potential applica- m~lplgs (whlch IS not due to defectlve genes) makes the animals tions, the characterization of the porcine metabolic and toxicoge- easlly manageable. nomic profile is an essential prerequisite. However, the area of Thus far, only a few porcine CYPs bave been cloned and ex- toxicogenomics remains largely unexploited so far. pressed in a recombinant system to characterize the enzymatic func- tions (for 1-4 CYP families, see Table 2). Even the number of the ..CYP (mini)pig isoforms is stili not complete. Furthermore, very CNR,Plsa,Address correspondenceVia MOruZZI, 1,56124to thls authorPlsa,ltaly; at theTel: Istituto +390503152704; di Fisiologia Clinica, little is known about the regulation of these genes : and -likun e Fax: +390503153328; E-mail: [email protected] humans -there IS a lack of studles almed at Identlfymg the regula- tory response elements m the promoter of CYP genes.
1389-2002/11 $58.00+.00 (!;)2011 Bentham Science Publishers
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508 Current Drug Metabolism, 2011, VoL 12, No.6 Puccinelli et aL "" Table 1. Comparison ofthe Nucleotide and Amino Acid SequencesofPorcine and 8uman CYPs.
% ofidentity Porcine CYP Isoform Accession Number Human CYP Isoform
Nucleotide Amino Acid
CYPIA,I NM_214412 CYPIAI 854 812
CYPIA2 CYPIA2 85.0 81.0
CYPIBI CYPIBI 84.0
CYP2AI9 CYP2A6 87.5
CYP2B22 ABO52256 CYP2B6 74.0
CYP2C8 62.0 CYP2C33 NM_214414 CYP2C9 64.0
CYP2CI9 63.0
78.0 CYP2C42 80.0 81.0
CYP2C49 NM_214420
CYP2D21* D89502 CYP2D6
NM_214394
CYP2El ABO52259
CYP2EI* NM_214421 CYP2EI 79.2
ABO06010 CYP3A4 75.0
Z93099 76.5
AF424780 75.3
NM_2I 4422 75.9
NM_001134824 CYP3A4
NM_214425
CYP4A24/25 NM_214424 CYP4AII 73.0 74.0
.Sequenceisolated from minipigs.
This review gives a comprehensiveknowledge on the members tabolism and disposition of drugs and xenobiotics. When available, ofthe flfSt four (mini)pigs CYP families and the related key nuclear the pig data afe compared to those of humans. In Table 3, some receptors (aryl hydrocarbon receptor, AhR, constitutive androstane drugs known to be metabolized in pigs are reported, together with receptor, CAR, pregnane X receptor, PXR, peroxisome proliferator- the CYP isoforrns which afe presumably involved in these reac- activated receptor alpha, PPARa, and hepatic nuclear factor 4 al- tions. pha, HNF4a), which represent the centrepiece in the overall me-
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- I; I i .' I PorcineCytochrome P450 '" CurrentDrug Metabolism,2011, VoL 12,No.6 509
Table 2. Recombinant Porcine CYPs. I
CYP Isoform ExpressionSystem TestedSubstrates References
CYPIAI HEK-293 Chlorzoxazone [90]
CVPIA2 E. coli 2-Aminofluorene,acetanilide, aniline, caffeine, ethoxyresorufin, methoxyresorufin, testos- [16] terone
CYP2AI9 HEK-293 Chlorzoxamne : .[90] ' , c,c-, .c.~"..,""- CYP2C33 HEK-293 Chlorzoxamne .' c','c [90] , CYP2C49 HEK-293 .., Chlorzoxazone '. ; ,.;." [90]
., }'Cc , i
CYP2D21 S. cerevisiae Bufuralol .,. ...c.-:' ." [77] " CYP2D25 S. cerevisiae Tolterodine,vitamin D3 T' [75-76]
CYP2El E. coli, HEK-2~;3, Chlorzoxazone,p-nitrophenol cC- ~., [86,90]
CYP3A29 Sf9 insectcells Nifedipine,testosterone; ': [91,102]
'...' ,':",; CVP4A21 COS-l cells Lauricacid, taurochenodeoxycholic acid -[117]
CVP4A24/25 .S. cerevisiae Lauricacid, palmitic acid, taurochenodeoxycholic acid [118]
content,similar to the expressionofCYPIA2 in human liver (about TOTAL CYTOCHROME P450 10%)[16]. With regard to CYPIAI mRNA expression,no quantita- The average content of total cytochrome P450 in conventional tive data afe available, but it appearsto be lower than CYPIA2 pig liver microsomes (0.57,0.22 and 0.46 nmol/mg prot. for three expression [16], as reported in humans [18]. lo Table 4 the tissue different pig crossbreeds)is comparableto that ofhumans (0.43 and distribution of porcine CYPs and nuclear receptorsis summarized. 0.26 nmoUmg prot. for Caucasian and Japanese,respectively), Another study deepened the presence of pig CYPIAI/IA2 in the whereas Gòttingen minipigs total CYP is 2 to 3-fold higher [IO]. brain [19]. Both the isoforrns were found expressedat mRNA level Furtherrnore,minipigs show more sex differences in the expression in various brain regions including cortex, cerebellum, midbrain, and activity of CYPs compared to humans or conventional pigs hippocampusand blood-brain interfaces (meninges and cortex cap- [14]. illaries), with a particularly high expression in blood-brain inter- faces. A protein immunorelated to CYPIA has been also found in CYPIA SUBFAMILY porcine olfactory gasaI epithelium [20], and this fmding has been G I F t confirrned and extended by a recent study [21] which demonstratep eDera ea ures the presence ofCYPIAI and IA2 mRNA and related activities in lo mammals, the CYPIA subfamily includes CYPIAI and both respiratory and (at a much higher extent) olfactory nasal mu- CYPIA2, two enzymes involved in the bioactivation ofmany car- cosa. cinogens such as aromatic amines, mycotoxins, and xanthines as well as in the metabolism of several drugs. Ethoxyresorufin 0- Substrates and Reactions deethylase (EROD) and methox~~~orufin O-de~ethylase ~OD) Already in the 1990s, different groups detected EROD and 8!e u~ually us.ed.as marker actlvltl~s of these Isoforrns, typlcally MROD activities in liver microsomes from various pig and minipig mduclble by dloxms and polyaromatlc hydrocarbons (PAHs). breeds,although at lower level than in humanones [22-26]. Iodeed, Porcine CYPIAI has been defmitively isolated and sequenced, EROD activity in pigs ranged from 5 to 95 pmol/min .mg protein revealing a high similarity to human CYPIAI (85.4%) [15]. Por- whereas the same activity in humans ranged from 25 to 190 pmol/ cine ~YPIA2 has been recently cloned from domestic pig (Large mio mg protein. MROD activity ranged from 2 to 13 pmol/min .mg White x Landrace hybrid) liver [16]. The nucleotide and deduced protein in pigs and from 24 to 32 pmol/min .mg protein in humans. amino acid sequencesrevealed 85% and 81% identities to those of However, some differences with the human orthologous were human CYPIA2, respectively, and a high degree of similarity was pointed out: i) besides EROD and MROD, ~-naphthoflavone (~NF) also reported for SRSs (substrate recognition sites) of the porcine was able to induce also 7-pentoxyresorufin O-depenthylation and human CYPIA2. (PROD), 7-ethoxy-4-trifluoromethylcou-marin O-dealkylase (EF- COD) and 7-benzyloxyresorufin O-debenzylase (BROD) activities Tissue Distribution which represent known markers of human CYP2B subfamilies; ii) Pig CYPIAi and IA2 mRNAs bave been found in liver, lung, all-tram-retinal oxidation and 17~-estradiol 2-hydroxylation, both heart, and kidney of domestic Large White x Landrace hybrid pigs, linked to human CYPIAs and 3A4, were not increasedindicating a althoUghat different levels [16, 17].10 the porcine liver, the level of difference in substrate specificity between porcine and human CYPIA2 expressionhas been found to be 3-9% of the total CYP CYPIAs [i7].
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510 Current Drug Metabolism, 2011, VoL 12, No.6 Puccinelli et aL '" I Table 3. Drugs Metabolized by Pig Liver I
CYP Subfamilies Drug Clinical Use References
2B? Benzphetamine Anorecticdrug [137]
2B, 20 Bufuralol ~-adrenergicreceptor blocker ]
Dextromethorphan Coughsuppressant I] Omeprazole Gastricacid blocker [71]
Oiclofenac NSAlO [58]
2C? Tolbutamide Hypoglycemicdrug 8]
2C ?, 28 ? S-rnephenytoin Anticonvulsant 8]
2C ?, 3A ? Taxol Chemoterapicdrug 8]
20 Oapsone Antibacterialagent [145]
Sulfamethoxazole Antibiotic [145]
Tolterodine Antimuscarinicmuscle rei [79]
Bupropion Antidepressant [92] Chlorzoxazone Musclerelaxant [90]
17a-Ethynylestradiol Syntheticestrogen [11.0] Erythromycin Antibiotic [13.7.59]
Lovastatin lipidemicdrug [108]
Midazolam Benzodiazepine [113]
Nifedipine Antihypertensive ,14] Tacrolimus Immunosuppressant {114]
Ethylmorphine Coughsuppressant ] Triacetyloleandornycin Antibiotic ]
Ampicillin Antibiotic ]
Antipyrine Analgesicand antipyretic 39] Azidothymidine ' AntiretroviraIdrug [144]
Hexobalbital 8arbiturateanaesthetic [143]
Naproxen NSAIO [140]
Norfloxacin Antibiotic (142]
Paracetamol Analgesicand antipyretic (139]
Sulfadimidine Antibacterialagent ]
? Vancomycin Antibiotic (139] Note: fieldsmarked by a questionmark indicateno furdterinfonnation available.
Recently, pig CYPIA2 has been cloned, expressed in a het- CYPIA2 [27] but in keeping with human CYPIA2 [28,29], failed erologous systern and characterized [16]. It was confirmed to pos- to oxidize aniline or testosterone(in the 613position). sess a good catalytic ~ctivity towards caffein~, acetanilid.e, and Pig CYPIA2 is also involved, along with CYP2A19, in the ~ethoxyresorufm (the plg CYPIA2 Vrnax for thlS substrate IS even rnetabolisrn of skatole, whose accurnulation in the adipose tissue is hlgher than that of the hurnan enzyrne), alI known rnarkers of hu- responsible for boar taint [30]. rnan CYPIA2. In addition, the purified pig enzyrne, unlike rat I
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Porcine C:vtochromeP450 Current Drug Metabolism, 2011, VoL 12, No.6 511
Table 4. Relative Expression ofPorcine CYPs and Nuclear Transcriptional F~ttors at mRNA Level in Comparison with the Liver. I
CYPs and Re-.. .Respiratory O!factory . Llver Kldney Lung IntestIne Rraln Heart References ceptors Genes Nasal Mucosa Nasa! Mucosa
CYPIAI +++ ,+ + +/:- + ++ +/- +/- [16-17,19-21] , " ',i
CVPIA2 +++ +/- -;" ~ +++ + +1- [16-17,IP-21]
CVPIBI + + + + + ++ + + [17,19,21]
CYP2AI9 ++ + n.a. n.a. n.a. , n.a. n.L n.L [15]
CYP2B22 +++ ,+ ++I +1- . + + +1- +1- [15,21,58-60] , CYP2C33 ~ +t ,+, + + ; + +, + [58,67]
., CYP2C42 +++ --+ +1- ---[58) .- CYP2C49 +++ -+1- + +1- + +1- + [15,58]
CYP2D25 +++ ++ ' + ,., n.L n.L I" +1- + [75,79]
, '" CYP2El +++ + + + n.a. n.L + +1- [15,88-89]
CYP3A22 +++' ~ + ++ + + + + [21,58-60,102] . ., CVP3A29 [21,58-60,102- +++ + + ++ + + + + ..103,105]
CYP3A46 +++c, + + ++ + + + + [21,58-60,102]
CVP4A21 +++ +++ n.a. n.a. n.L n.a. n.L n.a. [117-118]
CVP4A24125 +++ +++ n.L n.L n.L n.a. n.a. n.L [117-118]
AhR +++ + ++ + + ++ + + [17,19,21,129-130]
CAR :::++... + + + +/~ + + +1- [21,58-60,125-126]
HNF4a +++ ++ + + +I. +1- n.a. n.L [21,58-59,132] '", ' " PPARa '+++ ++ +1- + n.a. D.a. +1- + [123,133]
PXR +++ ++ .c' + + l' ++ ++ + +1-..[2158-60125- " 126,128] +++ = high expression; ++ = moderale expression; + = low expression; +/- = barely detectable expression; -= undetectableexpression; n,a, = data noI available,
Inhibitors agonists J3NF and 3-methylcholanthrene (3-MC) -but Bot with phe- nobarbitai (PB), rifampicin (RIF) or dexametazone (DEX), in The known porcme CYP mducers and Inhlbrtors afe summa- agreement with human data -in association with an increase in rized in Table 5. As in humans, a-naphthoflavone and anti-rat mRNA levels [32-33] CYPIA2 afe able to strongly inhibit EROD activity in liver micro- , . somes from Yucatan minipig [31]. In addition, ellipticine is re- .Fenbend.azole, ~ ~thelmmtlcs wldely u~ed In vetennary medl- ported to be a potent inhibitor of EROD activity performed by re- cme, determm~~ a slgrufi,cant an~ concentratlon-dependen.t Incre~e combinant pig CYPIA2 as the human counterpart [16]. of EROD actlvlty m pnmary plg hepatocytes [34]. Thls findmg , should be taken into account in the treatrnent ofpigs with this drogo
Inducibility and Regulation Besides hepatocytes, primary porcine enterocytes also showed EROD and MROD activities bave been found increased in pri- EROD activity, although not inducible by J3NF nor by 3-MC [35]. mary cultures of pig hepatocytes after a treatrnent with the AhR On the o~er han~, Roos. et al. [~6,] .reported that CY~IAl .was strongly mduced m GOttmgen mmlplg duodenum besldes Ilver,
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.' 512 CurrentDrug Metabolism,2011, VoL 12,No.6 Puccinelliet aL ~ Table 5. Known Porcine CYP Inducers and Inhibitors. I
CYP Subfamily Inducers References lnhibitors References
lA ~-Naphthoflavone [17-19,21,32] Ellipticine [31] 3-MethyIcholanthrene [33] a-Naphthoflavone [16] Benzo(a)pyrene [36-37] Omepmzole [38]
18 J}-Naphthoflavone
2A PhenobarbitaI [SO] 8-Methoxypsoralen [46] . Oiethyldithiocarbamate [46,86] Androgens [50-57]
28 PhenobarbitaI Rifampicin
2C Rifampicin Quercetin [58] Phenobarbital Sulfaphenazole [58] Cortisol Ticlopidine [58] Tranylcypromine [IO] J}-Naphthoflavone (~2]
20 Tolterodine [79] Quinine [81] Quinidine [81] PhenobarbitaI [82]
2E Ethanol [89] Propofol [93] Isoniazide [89] Oiethyldithiocarbamate [46,86] Phenobarbital [24] Androstenone [55] Skatole [94] 17~-Estradiol [55] S-adenosylmethionine [146] 4-Methylpyrazole [46]
3A Dexametazone Ketoconazole [31,102] Rifampicin Triacetyloleandomycin [22,102,108] Phenobarbital [32,35,58] Tiamulin [147]
long, kidney, and spleen after the oral adrninistration of soils con- CYPIAI but not CYPIA2 in alI the analysed extrahepatic tissues, tarninated with different kinds ofP AHs. The induction was particu- although to a different extent [17]. The treatrnent with !}NF deter- larly powerful in the duodenum, which representsa key organ for mined the induction ofCYPIAl, but not ofCYPIA2, in pig cortex, contaminant absorption and consequent distribution to tissues, cerebellum, midbrain, hippocampus, and cortex microvessels. In reaching activity levels even higher than in the liver. Benzo(a) parallel, EROD but not MROD activity was increased by !}NF in alI pyrene, a PAH found in tobacco smoke, is able to strongly increase the brain regions [19]. A recent study [21] has revealed the induci- the expression of CYPIAI in porcine urinary bladder epithelial bility by !}NF of CVPIAI but not of CYPIA2 in respiratory and celi s, indicating this enzyme as both indicator and contributor for olfactory nasal mucosa, at both transcriptional and activity levels. benzo(a)pyrenetoxicity [37]. This fmding is particularly interesting Omeprazole is a potent inducer of CYPIAZ activity -interestingly as the consumption of tobacco products is the most relevant risk not through the action of AhR -(measured by EROD) in both hu- factor for the development ofbladder cancer. man and minipig hepatocytes,but not in rat hepatocytesindicating The inducibility of AhR-regulated CYP genes by !}NF has been pig ~ a m~re ~ppropriate model system for the evaluation of CYP investigated in different extrahepatic tissues of conventional Large drug mductlon m humans [38]. White x Landrace hybrid pigs. A study in lung, heart and kidney, EROD activity, related to CYPIAI and lAZ, has shown along with liver, showed a transcriptional and activity induction of marked sex differences (with females having a much higher activityc.
c I ,- , PorcineCytochrome P450 CurrentDrug Metabolism,2011, VoL 12, No, 6 513 than males) in GOttingenminipigs and Meishan conventional pigs Substra1"esandReactions I (~ut Bot in L~dr.ace. or L~drace x Yorkshire x Duro~ domestic No l7J3-estradiol-4-hydroxylaseactivity (a marker reaction for pl~S) [14,39], mdlcatlng an Important role ofhorrnones m the regu- humand h CYPlBl). d " has been. th detectedl in porcine...' liver kidney.' long, latlon ofthese enzymes. an eart, m Icatmg el er a ow assay sensltlvlty or a dlfferent In generai, it can be said that CYPlAI is poorly expressed at substrate specificity of porcine CYPlBI compared to the human constitutive level even in the liver but it is very well inducible in isoforrn [17]. both hepatic and extrahepatic tissues. Differently, CYPIA2 is es- sentially expressed at a high level and inducible only in the liver. Inducibility and Regulation This restriction in.the pres~nceand inducibility ofCYPIA2, which A treatment with J3NF-an agonist ofthe nuclear receptorAhR also reflects what Is.fo~nd In hu~ans [40], could be due to the need which regulates CYP isoforrns of l family -enhanced CYPlBI for HNFI-4 factors In Its regulatlon. mRNA in pig liver but Bot in respiratory and olfactory BasaI mu-
P I h' cosa.these Sincedat a mica'AhRd ' mRNAte an . mvohasl vementbeen found o f aalsodd Itlon" in pigal tissue-speci..Basaitissues fiIC ' o ymorp Isms SOfar, no important mutations ofpig CYPlAI or CYPIA2 has factors in the transcriptional regulation ofthis gene [21]. The treat- been reported. Two amino acid changes (R71C and DII0N) bave ment with J3NFwas able to increasethe transcription ofthis isoform been observed in porcine CYPIA2 and R71 is shared with humans, in pig midbrain and, especially, in capillaries [19], although the but the influence ofthis substitution stili has to be examined [16].' enzymatic induction needs to be confirrned at activity level because ofthe possibility of a post-transcriptionalregulation. CYPIB SUBFAMILY Generai Features CYP2A SUBFAMILY Cytochrome P450 lBI (CYPIBl), the only member of the Generai Features ~YPIB su~family present in mammals, is often found in tumour Up tu now, the only porcine member ofthe CYP2A subfamily tlssue and ISsuspected to play a role in oncogenesisand drug resis- identified and cloned is CYP2AI9, which shares 87.5% of similar- tance. Unlike other P450s, CYPIBI is known to be a predomi- ity with the human orthologous CYP2A6 [15]. CYP2A19 was iso- nantly extrahepatic isoforrn [41]. lated from liver ofLandrace x Large White x Duroc crossbred con- CYPIBI has not been fully characterized in pig yet, and the ve~tional.pigs and it revealed an ORF of 1485 bp encoding 494 swine sequence for CYPIBI is not available in GenBank. Chirulli ammo aclds. A study of Soucek et al. [42} revealed that the se- et al. [17] isolated and sequenced a fragment of 448 bp of pig q~e~~ of the first 20 .amino.ac.idsat the N-terrninus of GOttingen CYPIBI from Large White x Landrace hybrid pigs. Alignment ~mlplg C~.2A ~e hlghly slmliar t~ ~um~ CYP2A6 (70% iden- analysis showed.87% similarity (of both aa and nu sequences) ~Ity). In addltlon, SIXsubstrate recognltlon sltesbave been identified compared with the human orthologous. Very recently, pig CYPIBI m the human CYP2A sequences,and they are ali present in the has been isolated and sequenced,revealing an open reading frame porcine protein [43], suggesting similar properties to the human (ORF) of 1635 bp encoding a protein of 543 aa, as the CYPIB 1 of orthologous. other mammals including humans reported in the GeneBank , '" [Messina A., private communication]. The SRSs of porcine Tissue Dlstnbutlon CYPIBI were totally shared (100%) with those ofbovine ortholo- Pig CYP2A19 mRNA has been found in liver and, to a lower gous and, except for SRSl, also with that of the canine counterpart. extent, in kidney ofLandrace x Large White x Duroc crossbredpigs However, the SRSs ofhuman CYPIBI showed a higher homology [15]. However, CYP2A19 mRNA has not been found in spleen, with thoseofpig CYPIBI than with those ofrat CYPIBI. thymus, lung, muscle, small intestine, heart or ovaries from many conventional pig breeds [44]. Tissue Distribution Messina et al. [21] perforrned real time RT-PCR (reverse tran- Substrates and Reactions scriptase-polymerasechain reaction)experiments on liver, olfactory Coumarin 7-hydroxylation is the most utilized marker activity nasal mucosa and respiratory nasal mucosa samples from Large for human CYP2A6, Although in human liver this activity level is White x Landrace hybrid pigs. The results showed a low constitu- higher than in pig liver (300-1114 pmol/min 'mg protein versus 20- tive expression of this isoforrn in the analysed tissues, and in par- 310 pmol/min ' mg protein) [22,23,31,45], it is also possible to use ticular in the hepatic samples. Immunoblotting experiments per- this reaction as a marker for pig CYP2AI9, as a good correlation forrned using anti-rat CYPIBI antibodies bave revealed a protein has been shown between the activity and the immunochemical band in hepatic and pulmonary microsomes but not in renal or car- level, or the mRNA expression[14,33,44]. Furtherrnore, this activ- diac ones [17]. Nannelli et al. [19] demonstrated the expressionof ity was strongly inhibited by anti-human CYP2A6 antibodies. Also CYPIBI mRNA in various brain regions ofthe same pig breed -the forrnation ofcotinine from nicotine, another human CYP2A6 including cortex, cerebellum, midbrain, hippocampus, meninges marker reaction, is catalyzed by porcine CYP2A. At high nicotine and cerebral capillaries -with similar levels compared to humans. concentrations,other isoenzymes probably contribute to the reac- The high expressionnoticed in the blood-brain interfaces (meninges tion, as the inhibitory anti-human CYP2A6 inhibition rate decreases and cortex microvessels) is in agreement with mouse and human from about 90% to 50%. This is in accordance with human data: data and suggested a role of CYPIBI in the protection of brain CYP2A6 is the predominantenzyme with 50 11Msubstrate, whereas from xenobiotics. Further experiments conflrrned the constitutive with 500 11Mnicotine CYP2B6 and CYP2D6 also play an impor- presence of CYPlBI in several pig tissues such as liver, kidney, tant role [43]. Besides liver and kidney, CYP2A-dependent activity small intestine, lung, nasal mucosa, heart, coronary arteries, adrenal has been found in pig (Large White x Landrace)nasal mucosa, and gland, and spleen, with the higher expression in adrenal gland and in particular in the olfactory mucosa where the activity levels were the lesser mRNA expression in liver [Messina A., private commu- even higher than those in liver [20]. nication]. This e,,:pressio~pattem reflects what f~und in humans, A study perforrned with primary porcine hepatocyte cultures where ~YPIBI IS c~nsldered mostly extrahepatlc and well ex- using two CYP2A inhibitors suggested that CYP2A -unlike pressedm steroldogenlcorgans. CYP2El -has a minor role in 3-methylindole (3-MI or skatole) metabolism, whose accumulation in adipose tissue is responsiblei .' 514 Current Drug Metabolism, 2011, VoL 12, No.6 Puccinelli et aL far the boar taint in 10-15% ofnon-castrated male pigs [46]. How- content\as been demonstrated [56]. Recent microarrays experi- ever, other results obtained by Diaz and Squires [47] revealed that ments effected in pigs with extremely high levels of androstenone the production of3-MI metabolites was affected by the presenceof [57] have confmned a down-regulation ofCYP2AI9 in Duroc but inhibitors of CYP2A6 and CYP2E I in the microsomal incubations not in Norwegian Landrace pigs. This finding confirmed the rela- and a significant negative correlation was found between the tionship between CYP2AI9 and androgens(the higher is androgen CYP2AI9 content/activity and 3-MI levels in fat, thus demonstrat- level, the lower is CYP2Al9-dependent activity), but also high- ing that CYP2AI9 is criticai far an adequate clearance of 3-MI. lighted some differences betweenvarious conventionalpig breeds. Furthermore, a particular single base deletion of CYP2AI9, result- . ing in a frame shift in the coding region that produces a non- Polymorphlsms functional enzyme, was associatedwith high levels of skatole in fat In humans, 7-hydroxylation of coumarin shows large inter- tissue [44]. This finding further pointed out the significant role of individuaI differences due to genetic polymorphisms (at least 20 CYP2AI9 in the skatole biodisposition. Also, the oral administra- different polymorphisms bave been identified far CYP2A6, leading tion of dried chicory root determined a decrease of the concentra- to no, decreased or unchanged enzyme activity) tion of skatole in the adipose tissue of entire male pigs (multiple (http://www.cypalleles.ki.se/cyp2a6.htln). domest~cpig crossbreeds)through an ~crease of transcription and Studies on three different polymorphisms identified in minipig translatlon of CYP2A [48,49]. In addltlon, Matal et al. [30] have CYP2A revealed that unlike far human CYP2A6 the differences recently de~onstrated in .a reconstituted syst~m a role o~ porcin,e recorded on CYP2A i 9 activity afe not due to ~enetic polymor- CYP2AI9 m the formatlon of 3-methyloxymdole and mdole-3- phisms but rather to a different transcriptional regulation [43]. carbinol from skatole. . I d 'n; DNA d fi d G , .Interestmg y, two I erent c s were .I enti. le .m vttmgen" . O,,:er the ~ast years, the .lInport.antrole of human CYP2A6 m minipigs: one was completely homologous to conventional pig the bloaCtlvatlon of some ~dustrlal compoun~s (e.g. tert-butyl CYP2AI9, whereasthe other one encoded a truncated protein miss- ~ethyl .ether and. 1,3-butadle.ne)and procarcmog~ns (e.g. N- ing the last SRS (SRS6). The meaning ofthis deletion is not known, mtroso~lInethylamme and N~mtr?sobenzylmeth~lamme)has been but this sequencemay represent a CYP2A7 or CYP2AI3 cDNA- ascertamed [40]. However, m plg ~e meta~olls~ of these sub- like, Indeed,these two human isoforms afe very similar to CYP2A6 strates and the role ofCYP2AI9 remam to be mvestlgated. (96% and 94% respectively)but they afe poorly functional [40].
Inhibitors CYP28 SU8FAMILY Pig CYP2AI9 can be inhibited by 8-methoxypsoralen and di- Generai Features ethyldithiocarbamate, two typical inhibitors of human CYP2A6 ,. . [47,46]. Furthermore, menthofuran, a potent, mechanism-based The literature about CYP~B s~bf~ùy I.n plg ~s IlInlted. Pig inactivator of CYP2A6, can be utilized as CYP2A inhibitor in pigs CYP~B22, the only CYP2B. Identlfied m thlS spec~esso far, has [47] been Isolated from a cDNA IIbrary of adulI female plg (Landrace x .Large White x Duroc) liver, and sequenced [15]. The isolated Inducibility and Regulation cDNA pres.ented.an, ORF of 1482 bp ~d enc.od~d.a 493 amino L ' I . kn b CYP2AI9 I h ' I acids protem. Thls Isoform showed a hlgher slmùanty to human Itt e IS own a out regu atory mec anlsms. n , .. h t ltu CYP2A d d t ti.. ty .CYP2B6 (81.1 Yoand 74 Yofar nucleotlde and amIno acld sequences, pnmary porcIne epatocy e cu res, -epen en ac VI IS ., .. fi I . d b PB . d. ct CAR t t d b respectlvely), rather than to rat or mouse CYP2Bs. slgm Icant y mcrease y , an m Ire ac .Iva or, an y CITCO (6-( 4-chlorophenyl)imidazo[2, l-h ][1 ,3]thiazole-5-carbalde- Tissue Distribution hyde-O-(3,4-dichlorobenzyl)oxime), a human CAR ligand [50]. .. These results show that the induction pro file of CYP2A in vitro The expressionof CYP2~22 .mRNA was confirmed ~ porc~e shares similarity with that ofhuman CAR-regulated CYPs, indicat- liv~r and, at a ~ess~rextent, m kldney by RT-PCR exp~nments In ing an involvement of pig CAR in the regulation of this subfamily. vanous domeStlcplg cros~breeds(Landrace x Large.W~lte x Duroc A study of Myers et al. [25] demonstrated that a co-treatment with or Landrace x Large Whrte) [15,58,59]. The. cons~rtutlve,presence f3NF, PB, and DEX is able to increase CYP2AI9 marker activity, of CY~2B22 mRNA has als.obee~ ascertamed m po~cme !ung, but not to increase the expression of the enzyme, as assessedwith branchi, and ~achea. Interestm?ly,. m I~ng CYP2B22 I~ baslcally Western blot analysis. In porcine hepatocyte cultures, unlike human expressed at hlgher level than m IIver, m agreement Wlth data of hepatocytes [51], CYP2A was not induced by pyrazole [52]. This other mamma!s ~59]. Fu~e~ore, ~YP2~22 m~NA has been fmding indicated a different regulation ofCYP2As in these species, found at constltutlve level m plg small mteStln,eand m the olfactory ..., .and respiratory nasal mucosa [21,58], Nannelll et al. [60] also dem- .In mml~lgs, CYP2~ ex!:'resslon IS strongly ~ender-depe~dent, onstrated the expression of CYP2B22 in various pig brain regions WI~ ~e hlghest actlvl.ty m females. In partlcular, GOttl:ngen including cortex, cerebellum, midbrain, hippocampus, meninges ml~ll:>lgsshow marked dlfferences (the fe~a~e~have 70-fold h~gher and cerebral capillaries. While in meninges and cerebralcapillaries a~tlVlty than males), but. also Yucatan mmlplgs ~d .convent~onal CYP2B22 mRNA levels afe high and comparable to the hepatic plgs (Landrace x Yor~shlre x Duroc) ,sho,ws°':l1e slgmficant dlffer- ones, in the other compartment the basai expression is about or ences [14, 31]. Expe.nmentsef~e~ted In VIVOwrth male (castrated~r below 10% of the corresponding values in liver, in keeping with non-castrated) GOt.tlnge~ ~1~lplgs have shown that CY~2.A IS what has been reported far humans [61]. The high expression of gre~tly -buI reve':5lbly -mhlbrted by ~drogens on a transcrl~tlon~1 CYP2B22 in blood-brain interfaces might have important implica- basl~,demonstratmg a strong modulatlon by se~ ho:rmonesm th~s tions far either pharmacological profiles ofneuroactive drugs or the specles[50]. It has been p,roved that human chono,m.c.go~adotr~pm regulation of brain blood tension, as well as human CYP2B6 is IS able to suppress hepatlc CYP2A and 2E I actlvrtles m varlous involved in the formation of vasoactive arachidonic acid metabo- domestic pig breeds, probably through an increase in the levels of lites [62]. testicular steroids such as androstenone, dehydroepiandrosterone sulphate,oestradiol and oestrone sulphate [53, 54], thereby decreas- Substrates and Reactions in!? the ~etabolism of skatole and leading to its ~ccumulation in Dated reports failed to detect any CYP2B apoprotein or activity adlposetlssue. An°l!t~r study re,:ealed I?at o~ly ~ sllgh~decrease ?f in primary cultures of pig hepatocytes or minipig (GOttingen) liver CYP2A in VltrO.actlvlty o~curs mcubatmg plg ~Iver~Icrosomes In microsomes using an anti-rat CYP2BI antibody or PROD as a pre~en~eofte.stl~u~ar sterolds [55], whereas,usmg plg hepatocyte.s, marker reaction [32,22]. On the contrary, Donato et al. [23] were a slgmficant mhlbltory effect of androstenone on CYP2A protem;'" " PorcineCytochrome P450 CurrentDrug Metabolism,2011, VoL 12, No, 6 515
able to measure another activity often used as marker of CYP2B CYP2C\UBFAMILY subfamily,ti BROD,L Whin .bothd hepatocyte..eDera cultures and liver micro- G I Feat ures somes rom arge ne omestlc plgS, although at much lower levels than in human samples. The BROD activity in porcine mi- ~he CYP2C subfamily is known to be very divergent amongst crosomeswas about I pmol/min .mg protein whereas human activ- speclesand represents,perhaps, one ofthe largest and most compli- ity was known to be about 12 pmol/min ' mg protein. Bogaards et cated subfamilies, mak~g it difficult to fmd a good mammalian al. [31] tested EFCOD, a reaction catalyzed in humans mainly by model for the extrapolatlon of data to humans. CYP2B6 (but also by other P450 enzymes such as CYPIA2) on In humans, the CYP2C subfamily consists of CYP2C8 2C9 Yucatan minipig liver microsomes. The activity rate was sintilar in 2CI9 and the minor CYP2CI8, accounting for about 20% 'or th~ male and remate minipigs, and comparable to that ofhumans (Km= total P450 hepatic content and playing a prominent role in the me- 1.8-2.3 11M;Vmax= 286-353 pmol/min 'mg protein). Furthermore, tabolism of20-30% oralI drugs [40]. ant!-~at CYP2BI antibodi~s were able to moderately inhib.it th!s In 1995, Zaphiropoulos et al. [67] used a couple of primers actlv~ty ~30-?0%),suggestm~ that ~ <;:YP2Balso takes part m thlS designed on rat CYP2C common re ions to c out some RT- reactlon m plgs. Ano~er typlCal actlvlty related to human CYP2B6, PCR experintents on pig ovarian sa:ples. Eleva:; sequenceswere s~ch as S-mephenytom N-demethy~ase~has been measu~d in pig isolated, eight of which were very sintilar to human CYP2Cs lIver and, to a much I~wer extent, m kldn.ey [58]. Turp~men et al. whereas the other three were more similar to rat CYP2C23. All [45] compa;e.db~proplon hydroxylase actlvlty (a selectlve CYP2ij these clones wereYP2C35 subsequentlyCYP2C36classified d asali CYP2C32,I.. CYP2C33ts ' GmarkerA tt' aCtlvlty... m humansd h at 50 Th11Msubstrate ..."an concentration) in CYP2C34 C some e IC varlan . v mgen mmlplgs an umans. e actlvlty rate was higher m ... minipigs (435 pmol/min omg protein on the average)than in hu- In 1998~Nissen ~t al. .[68] Isolat~d two CYP2C-like .clones mans (131 pmol/min omg protein on the average) from a porcme small mtestme cDNA lIbrary: one was the mcom- ' plete CYP2C42 (now available in the full-lenght form) and the .An mterestl~g work by Kawalligashl et al. [63] revealed that other encoded a pseudogene.Kojima and Morozumi [15] cloned plg CYP2B22 I~ able to metabolize. some .herbicides including CYP2C49, the last porcine CYP2C sequenceavailable in literature. chlortoluron, amlprofos-methyl, pendlmethalm, metolachlor, and .. esprocarb. These information could be useful for human health in ~o far: only CYP2C33, 2C42 and 2C49 bave been studled m order to produce herbicide-tolerant transgenic plants into which detall, whlle the other sequencescould be allelic or splice variants bave been introducedparticular CYP enzymes. ofthese genes. The comparative analysis revealed that CYP2C42 and Inducibility and Regulation CYP2C49 shared 80% of aa sequenceidentity between them, about Desille et al. [24] found CYP2B-related activity, rnRNA, and 60% with CYP2C33, and about 77-81% with human CYP2C9 and protein present aild inducible by PB in printary porcine hepatocytes. 2C~9oF~rthermore, porcine CYP2C33 shared only about 62-64% Also Behnia et al. [33] showed that PROD and BROD activities of Identlty versus human CYP2Cs, suggesting differences in the could be induced by PB in porcine hepatocytes after 8 days in cul- substrate specificity of these enzymes. Diversely, CYP2C33 re- ture, but not after 4 days, when a decreaseof many activities was sulted more similar to whale CYP2C78 or cattle CYP2C86 [69], noted. Other evidence ofthe presenceand inducibility ofCYP2B in sharing an identity of82-83% [58]. pigs bave been found by treating commerciai animals (Landrace x o .o o Poland China) with an induction cocktail containing PB, DEX and Tissue DIstnbutlon j3NF [25]. EFCOD, PROD, and BROD activities and an apoprotein CYP2C33 has been found expressedat mRNA level in porcine intmunoreactive with anti-rat CYP2BI, were alI increased in liver ovarian tissue [69]. Porcine CYP2C49 mRNA has been found microsomal fractions obtain~d from the treated.pi.gs. CYP2B22 has highly ex~ressed in pig liver and poorly expressed in pig kidney been also found markedly mduced at transcnptlonal, protein and [15,58]. Pig CYP2C33, 2C42 and 2C49 mRNAs bave also been I activity level in pig liver and kidney -but not in lung, bronchi and found expressed,although at different levels, in small intestine and trachea -after an in vivo treatrnent with RIF, a known inducer of nasal mucosa (both respiratory and olfactory) [58]. A CYP2C I CYP3As through the nuclear receptor PXR [59]. Sintilarly, member, not better identified, has been found expressedin porcine CYP2B22 was induced in pig liver at transcript and activity level coronary arteries,as assessedby RT-PCR and Western blot analysis after an in vivo treatrnentwith PB, whereas in the small intestine the [70]. s~e. treatrnent increased the mRNA level but not the enzymatic . actlvlty [58]. In the olfactory and respiratory porcine nasal mucosa, Substrates and Reactions the lack of induction of CYP2B22 after an in vivo treatrnent with A study of Anzenbacher et al. [22] revealed the presence, in RI~, .j3NF, or PB has been ascertained at both transcriptional and hepatic Gottingen minipig samples, of tolbutamide 4-hydroxylase act!vlty level [21,58]. CYP2B22 has also been found to be resistant activity -a human CYP2C9 marker activity -althoUgh at lower to mduction by RIF in porcine brain [60]. levels with respectto humans (62-136 pmol/min .mg protein versus In generai, in humans the PB-dependentinduction of CYP2B6 l4~-395 pmol/min o.mg 1:'rotein). Sintilarly, experiments with he- is mediated by the activation of the nuclear receptor CAR and its patlc and renal porcm~ mlc~osomesshowed that pigs and minipigs interactions with the PB-responsive enhancermodules (PBREMs) were able to metabollze d!clofenac (another specific substrate of present in the 5'-tlanking region of CYP2B6 [64]. Furthermore, a human CYP2C9)~ but WI~ lower rates c~~pared to humans cross-talk of the nuclear receptor CAR with other nuclear factors [31,58]. Indeed, dlclofenac 4 -hydroxylase actlvlty ranged from 20 (i.e. PXR, RXR, HNF4a and CCAAT/enhancer-binding protein to 60 pmol/min omg protein in pigs and from 1.5 to 1.9 nrnol/min o alpha C/EBPa) has been observed [65,66]. Little information is mg protein. i? .humans. Other reactions usually used as CYP2C available on the specific regulatory mechanisms of porcine marker actlvltles afe 7-methoxy-4-(trifluoromethyl)coumarin 0- CYP2B22. The lack of induction of CYP2B22 in pig airways and demethyla~ ~C
" I 516 CurrentDrug Metabolism,2011, VoL 12,No.6 Puccinelliet al.
S-mephenytoin, a substrate metabolized in humans by CYP2C19 this s~family by hormones, and particularly by androgens and [14,31,25]. However, this activity became detectable in liver micro- growth hormones, has also been suggested for human CYP2Cs somes after an in vivo pig treatment with PB suggesting the pres- [73]. ence of a CYP2C19-like isoform also in this species [58]. The oxi- dation of paclitaxel and amodiaquine -two marker reactions of Polymorphisms human CYP2C8 -did not lead to the formation of correspondent Human CYP2C19 is known to be strongly polymorphic, with metabolites in bolli pig or minipig [58, 72]. the population divisible in poor, intermediate or extensive metabo- So far, no porcine CYP2C has been cloned and expressed in a lizers depending on the functionality ofthe enzyme [74]. However, reconstituted system to characterize its enzymatic functions. How- it is not known whether a pig CYP2C enzyme possessesa similar ever, the results on the expression and activities present in literature feature. It is possible that some of the sequencesavailable in Gen- highlight some differences between human and pig CYP2Cs, sug- bank for porcine CYP2Cs constitute polymorphic variants. Further gesting that pig might not be a suitable model for the study of drugs genomic studies afe needed to ascertain the presence of relevant metabolized in humans by this subfamily. polymorphisrns in pigs and to verify the real differences between P. CYP2C49 tr . ali . tr d d . t . l t h the various breeds (domestic pigs, minipigs, microminipigs). 19 , ansgemc y m o uce m o nce p an s, as been demonstrated to be able to metabolize several herbicides in- CYP2D SUBFAMILY cluding chlortoluron, nortlurazon, amiprofos-methyl, alachlor, and isoxaben. This capability could bave interesting consequencest'or Generai Features human health, e.g. for the construction of herbicide-tolerant trans- In humans, CYP2D6 is one of the most important enzymes for genic crops enriched with CYP species[63]. drug metabolism, accounting for the metabolism of about 15% of ali drugs. Its genetic polymorphism is one of the most relevant Inhibitors clinical human polymorphisms ofoxidative drug metabolism, and it Inhibition experiments carried out in Landrace x Large White is known not U>be inducible at ali [40]. hybrid pigs using inhibitors of human CYP2C such as quercetin, In 1997, a cytochrome P450 which catalyzed 25-hydroxylation sulphaphenazole,ticlopidine and tranylcypromine bave shown a ofvitamin D3 has been cloned ftom domestic pigs and expressedin different selectivity toward porcine CYP2Cs [11,58]. Tolbutamide simian COS cells [75]. Even if human CYP2D6 can not catalyze hydroxylation could be inhibited in pigs by tranylcypromine -a vitamin D3 25-hydroxylation, the cloned porcine CYP showed 70- CYP2C19 inhibitor -but not by sulphaphenazole -a CYP2C9 80% identity with the mammalian members of the 2D subfamily inhibitor [11,58]. Quercetin, a human CYP2C8 inhibitor, was able and was subsequentlynamed CYP2D25. Site-directed mutagenesis to significantly inhibit paclitaxel hydroxylation, tolbutamide hy- studies bave revealed that residues in SRS3 resulted crucial for the droxylation (a çYP2C9-related activity) and S-mephenytoin hy- function of the enzyme in vitamin D3 metabolism, but not for tol- droxylation (a CYP2C19-related activity), whereas ticlopidine and terodine metabolism (a substrateofhuman CYP2D6) [76]. In 2004, sulfaphenazole failed to inhibit ali these reactions [58]. Anti-rat the hepatic CYP2D21 has been cloned ftom Gottingen minipig and CYP2Cli was able to inhibit diclofenac 4'-hydroxylase activity -expressed in yeast cells [77]. This CYP2D2l showed 97.8% iden- another marker of human CYP2C9 -in liver pig microsomes al- tity of amino acid sequencewith the domestic pig CYP2D25. There though at lower levels than in human microsomes [31]. were lO amino acid differences, and one was located in the putative SRS3: Gln204 or Leu204 for CYP2D21 or CYP2D25, respectively. Induclblhty and Regulation It remains to ascertainifthese two enzymes afe allelic variants with In primary porcine hepatocytes tolbutamide 4-hydroxylase ac- a distinct substratespecificity. tivity has been found increased by treatrnent with RIF (a PXR ligand) and PB (a CAR activator), and decreased after treatrnent Tissue Distribution with ~NF (an AhR agonist) [32]. The metabolism of tolbutamide Initially, Skaanild and Friis [14] suggested the absence of and diclofenac in Landrace-Poland China pigs has been found CYP2D in pig liver by immunoblotting experiments using mono- slightly increased after treatrnent with a combination of PB, DEX clonal anti-human CYP2D6 antibodies. Later, the lack of response and ~NF [25]. By investigating in detail the inducibility by PB of in this immunoblotting test has been confirmed and indicated to be CYP2C33, CYP2C42, and CYP2C49, a recent work [58] showed due to differences in the structure of pig and human CYP2D [78]. that in conventional pig liver and small intestine (Landrace x Large Northem blot analysisbave shown that CYP2D25 mRNA is present White hybrid pigs) these three isoforms afe inducible at transcrip- in porcine liver and, to a legger extent, in kidney [75]. RT -PCR tional and activity level. In addition, the treatrnentresulted in an up- experimentsbave revealed that CYP2D25 mRNA is also expressed regulation of mRNA levels of CYP2C42 and CYP2C49 (but not of in small amounts in porcine adrenals, brain, heart, intestine, lung, CYP2C33) also in kidney, but not in respiratory and olfactory nasal muscle, spleen,and thymus [79]. mucosa, demonstrating a tissue- and isoform- differential regula- tion. In pig coronary endothelial tissue the expression of a CYP2C Substrates and Reactions mem~er?not b~tter identified, r~sulted ~obe increased b.y.cortisol in Pig CYP2D25 was able to catalyze 25-hydroxylation ofvitamin assoclatlon ~lth an endothehum-de~lved hyperpolarlzmg fa.ctor D3 [75] and multiple hydroxylations of 25-hydroxyvitamin D3 (EDHF) medlated relaxatlon of artenes [70]. These observatlons [80]. Other reactions catalyzed by CYP2D25 afe 25-hydroxylation support the concept that an epoxygenase homologous to human of vitamin D2 and the conversion of tolterodine into the 5- C~P2C~/9 plays a .crucial role. in the generation of arachidonic hydroxymethyl metabolite (a reaction aIso catalyzed by human acld-denved vasoactlve metabohtes. CYP2D6) [79]. The enzyme activity of CYP2D in pigs has been With regard to the regulation mechanisms of CYP2Cs in pig analysedusing three classical human CYP2D6 test substrates (de- very little is known. It has been suggestedthat the PB induction of brisoquin, bufuralol and dextromethorfan), and differences with pig CYP2Cs is primarily due to a tissue-specific activation of the human CYP2D6 bave beenpointed out. No metabolism of debriso- nuclear receptors CAR, PXR and HNF4a, as found in humans [58]. quin could be detected in bolli Gottingen minipigs or Landrace x Sex differences in the activity ofCYP2Cs bave been investigated Yorkshire x Duroc crossbred conventional pigs [14]. Bufuraloll'- using human CYP2C substrates:no sex differences were detected in hydroxylation activity has been found higher in Gottingen or Yuca- conventional pigs whereas male minipigs showed higher activity tan minipig liver microsomes than in those of human origin (770 than the females, in contrast to what had been seen for other CYP pmol/min mg prot versus 182 pmol/min .mg protein, on the aver- subfamilies in minipigs [14,71]. The regulation ofthe members of age) [22,31]. However, correlation analysis between dextromethor-
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, ,- I
Porcine Cytochrome P450 Current Drug Metabolism, 2011, VoL 12, No, 6 517 I
fan or bufuralol microsomal oxidation rates and the protein content Tissue ~sfribution obtained wi~ ~ti-~um~ CYPs poly~lonal an~ibo~ies indicated Porcine CYP2E1 mRNA has been found highly expressed in that these actlvltles m plgs may be attrlb~ted prlmarlly to CYP2B pig liver and, at lower 1evels, in kidney [15]. The presence of the rather than to CYP2D [~8]. The recombm.~t CYP2D21. enzyme protein in the hepatic tissue was confirmed immunochemical1y by h~ .s.hown bufuralo1 1 -hydroxy1ase actlvlty, con~rmmg that various authors [25,26]. Ch10rzoxazone 6-hydroxylase activity has mmlplg possesses a CYP2D enzyme ~ble .to met~ollze a human aIso been detected in some Giittingen minipig extrahepatic tissues CYP2D6 substrate, aIthough the contrlbutlon to thlS enzyme :w~s such as kidney, lung, small intestine, brain, and leukocytes with the smaller than that of other CYPs, such as CYP2B [77]. Thus, It IS highest level- besides liver -in kidney [88 89]. quite clear that pig is not the best model for the study of processes '
involving CYP2D in humans since pig CYP2D(s) afe different from Substrates and Reactions the human counterpart. Both pig and minipig enzymes were able to convert two proto-
Inhibitors typical substrates of human CYP2E1, chlorzoxazone and p- nitrophenol, into the respective metabolites in liver microsomal Tolterodme was able to mhlblt the mlcrosomal 25-hydroxy- fractions or reconstituted systems with rates similar to those ob- lation ?fvitamin D2, ~he~e~s quinidin~ -a human.CYP2D6 inhibi- tained with the human enzyme [13,22,23,31,45,86-90,91]. Indeed, tor -dld.not mar~e~ly. ~hlblt the reactlon [79]. Juruna-Rome! et a/. chlorzoxazone hydroxylase activity ranged across pig breeds from [8~] .notlced ~ 1~~lbltlon of dextromethorfan
I d ' b ' l ' ty d R I ti, tion of chlorzoxazone and p-nitrophenol. Indeed, CYP2A is aIso n UCI Il an egu a on d d .b th ... emonstrate to contrl ute to ese actlvltles [90 , 92] , an d Monshouwer et a/. [32] found dextromethorfan O-demethy- bupropion (metabolized at high concentration by human CYP2E1) lation induced by PB and RIF aIthough these compounds do not seems to be a more specific substrate for porcine CYP2E1 [92]. induce human CYP2D6. An induction cocktail containing PB, DEX However, recent experiments by Kiihler et a/. [89] performed with and pNF had no effect on the expression of CYP2D in domestic diethyldithiocarbamate indicated that chlorzoxazone 6-hydroxy- pigs at protein and activity level [25]. It has been demonstrated that lation was predominantly due to CYP2E1 in Giittingen minipig PB treatment was able to suppress the activity of CYP2D25 (25- liver, kidney and lung microsomes. CYP2E1 has been identified as hydroxylation of vitamin D3) in pig hepatocytes via a direct en- one of the main enzymes involved in the metabolism of skatole, zyme inhibition and transcriptionally down-regulating the gene whose accumulation in non-castrated male pigs is a major cause of promoter via a mechanism involving PXR or CAR [82]. This could boar taint, aIong with CYP2A19 and CYP1A2 [47,46,30]. explain, at least in part, the PB-induced vitamin D deficiency ob- served in humans. Inhibitors
CYP2E SUBFAMILY Neither a-nap~tho.flavone nor ~r.aftlline, i~hib!ted chlorzoxa- zone 6-hydroxylatlon m Yucatan mmlplg hepatlc mlcrosomes [31]. GeneraI Features Nevertheless, ketoconazole and troleandomycin -two CYP3A in- The only known member ofCYP2E subfamily is CYP2E1 -an hibitors -were able to markedly inhibit chlorzoxazone hydroxylase isoform responsible in humans for the metabolic activation of many activity [31]. Also 4-methylpyrazole (a known CYP2E 1 inhibitor) low molecular weight compounds suspected to act as chemical inhibited p-nitrophenol hydroxylase activity in primary cultured carcinogens. CYP2E 1 is one of the most conserved P450 enzymes porcine hepatocytes [46]. Propofol (2,6-diisopropylphenol), a according to the primary structure, exhibiting nearly 80% sequence widely used intravenous anesthetic agent, markedly inhibited identity across species [83], and literature data suggest a species- CYP2E 1-dependent hydroxylation of chlorzoxazone in both human conserved mechanism for oxidative biotrasformation by CYP2E1 (Ki = 48 11M) and porcine (Ki = 19 11M) hepatic microsomes [93]. [84]. It is a major catalyst of the oxidation of ethanol, benzene, The inhibition of chlorzoxazone 6-hydroxylation with dieth- styrene, chloroform and other chlor derivates of methane and eth- yldithiocarbamate (a typical inhibitor of human CYP2E1) gave alle, and it is known to take part in the activation of nitrosamines comparable Ki values for Giittingen minipig and human enzymes, [85]. indicating a similarity between porcine and human CYP2E1 [86].
In 2004, CYP2E 1 was cloned from Landrace x Large White x Inducibility and Regulation Durocyear later,crossbred CYP2E1 domesticwas aIsopig isolated by Kojima from Giittingenand Morozumi minipig [15]. liver A .An In VIVO study m domestlc plgs has sh~wn a marked mduc-
microsomes and the respective cDNA was cloned and sequenced tlon by PB of CYP2E1 ~RNA and apo.protem levels, whereas no [86]. Both porcine CYP2E 1 were just two residues shorter than the changes bave been fo~nd m .the metab~lIsm of.c~lorzoxazone [24]. human orthologous, and the only difference between minipig and On the contrary, an mductlon co~ktall contammg. pNF, PB. and pig sequence was the presence of Asp346 in the former instead of ~EX had no effect on the ~xpre~slon of CYP~E1 m conventlonal Val346 in the latter. However, the differences in the primary struc- plgs (Landrace x Poland Chma.plgs) [25]. In pl~ hepatocytes, ska- tuTe ofthe pig enzyme with respect to the human counterpart do not tole ~nhanced CYP2E~ at protem l~vel [94]. As m hum~s, ethanol include the key amino acid residues predicted to interact with a feedmg was ~ble to mduce hepatlc CYP2E1, and thlS has been human CYP2E1 typical substrate, chlorzoxazone [86]. A study ~emonstr~~~ m both castrated and non-cas~ated Yucatan and Giit- revealed how the recombinant porcine CYP2E1 is more stable than tmgen mmlplgs [88, 89, 95,]. Ethanol aIso mduced chlorzoxazone the human CYP2E1, and that the human form has a greater com- hydr~xyla.se.ac~ivity in minipig brain whereas it led to a r~pressi?n pressibility in the heme active site than the porcine one [87]. ofthls actlvlty m the lung [89]. The same effect was obtamed wlth isoniazide, a known inducer of human CYP2E1. In particular, in I
~
,. I 518 CurrentDrug Metabolism,2011, VoL 12,No.6 Puccinelliet al. ~ I liver, CYP2El activity was increased by etbanol and by isoniazide Tissue Distribution whereas tbe protein level remained unaffected. Hence, CYP2E l CYP3A22, 3A29 and 3A46 mRNAs bave been found in Lan- also appeared to be regulated by post-translational mechanisms. drace x Large White crossbred pig liver and several extrahepatic Nevertbeless, a very weak correlation has been found betweentbe tissues, such as kidney, lung, bronchi, trachea, intestine, heart, capacity to metabolize chlorzoxazone and tbe content ofprot.ein or spleen,respiratory nasal mucosa and olfactory nasalmucosa [21,58, tbe mRNA expression [14], tbus indicating tbat.tbe regulat!on.of 59,102]. These tbree isoforms bave been also found constitutively CYP2E I expressioncould take piace at ali steps m tbe protem blo- expressed in various brain regions, altbough at much lower levels syntbesis,as found in humans [96]. In addition, a study on hnRNA tban in liver [60]. Interestingly, a CYP3A-related activity (benzy- (heterologous nuclear RNA) and mRNA of CYP2EI in G()ttingen loxyquinoline debenzylase, BQD) was found particularly high in minipigs has suggested tbat splicing and/or modulation of mRNA porcine cortex capillaries, suggesting a protective role of CYP3As stability could be involved in CYP2EI regulation [97]. towards tbe entrance of xenobiotics in tbe brain. In porcine brain, Human chorionic gonadotropin stimulation has been demon- unlike liver, higher levels of CYP3A-related activity bave been strated to suppresshepatic CYP2EI in pubertal male pigs, probably found in mitochondria co~pared to mlc.rosomes [~O]. Thus, an through an increase in testicular steroid levels [54], since it has important role of CYP3As m. tbe metabollsm of br~m endogenous been previously observed tbat androstenone and 17f3-oestradiol substrates,such as neurosterold~has beenhypothes~zed. C.YP3A29 exerted an inhibitory effect on CYP2EI enzymatic activity, aI- mRNA has also. been detected m adrenal gland,.s~m, test~s,uterus tbough witb two ditTerentmechanisms [98,55]. A functional analy- and ovary of dltTerent age groups of Bama mmlature plg [103]. sis of pig CYP2EI gene promoter has revealed two activating ele- CYP3A4 is tbe major form ~fP4~0 expressedin human ente~ocytes ments, ODeof which was able to bind tbe hepatic nuclear factor I representing about 70% of mtestmal CYPs [104], and also m por- (HNF-I) and tbe otber was identified as a binding site for tbe chick cine small intestine CYP~J\2~, ~A29, an~ 3A46 mR~As were ~ell ovalbumin upstream promoter transcription factor I (COUP-TFI) expressed[58,102]. A.dlm~lshmg gradlent expresslon of porc~e [99]. In addition, it has been demonstrated tbat androstenonewas CYP3A29 ~ong tbe. mtestme has be~n o~served [102, 105], m able to inhibit tbe binding on COUP-TFI, witbout atTectingHNF-I agreementWltb expenrnentson human mtestme[106]. binding, tbus providing an explanation for tbe inhibition of A very recent work by Hermann and Skaanild [107] has dem- CYP2E I protein expressionby androstenonein pig hepatocytes. onstrated an interesting similarity between human CYP3A4 and Some ditTerencesbave been pointed out between humans and porcine CYP3A29. The CYP3A4 expression is very low during pigs on CYP2El expression and activity [14]: humans and conven- foetal development and beco~es. tbe dominlmt CYP3A iso~orm in tional pigs did not present sex ditTerences in chlonoxazone hy- adult liver, whereastbe opposlte IS for human CYP3A7. Thelr fmd- droxylase activity, whereas in G()ttingen minipigs tbe same activity ings demonstrated tbat minipig .CYP3A29 had a p~~em of ~xpr~s- was much higherin females tban in males. sion comparable to CYP3A4 m humans. In addltlon, an~lbodles raised against human CYP3A 7 revealed tbe presence of an Isoform L Polymorphisms (CYP3A22?) which followed tbe pattem ofCYP3A7 in foetal and Some functional polymorphisms ofporcine CYP2EI bave been adul~ minipig sam~le~. Also expe':ÌIn;entswi~ ~e. recombinant identified as a possible strategy toward tbe selection of genetic porcme C~3A29 mdlcated tb~t tblS Isof~~ IS slmllar to human markers used to reduçe boar taint witbout atTecting tbe levels of CYP3A4 Wltb regard to exp~sslon and aCtlvlty,.~d ~at ~YP3A~9 sexualhormones [100,101]. gives tbe greatest contrlbutlon of CYP3A actlvlty m plg hepatlc microsomes [102]. CYP3A SUBFAMILY GeDeraI Features Substrates and Reactions . ..Testosterone 6f3-hydroxylation (tbe most wldely used marker CY~3A4 represents m hum~s tbe most Important P450 e~- reaction for human CYP3A4) and nifedipine oxidation bave been zyme Wltb reg.ardto tbe presence (It amounts to ap~roxlma~ely~O~o measured in ditTerent strains of minipigs and conventional pigs. of total hepatlc ~YP content) ~d to tbe metabollc func~lon (Il IS Botb tbe substrateswere metabolized witb levels of activities com- able to ~etaboilze about a tbl!d of ali ~~g~). For tblS rea~o~, parable to humans, altbough witb some ditTerencesdepending on CYP3A IS ODeof tbe most. stu.dl.ed~ubfamliles m humans, and It IS tbe breeds of pigs [14,22,24,25,31]. Testosterone 6f3-hydroxylase also necessaryto charactenze Il m plgs. activity in pigs ranged from 0.45 to 4.45 nrnol/min .mg protein, The first porcine CYP3A sequenced and mapped was which compare well witb tbe average activity in humans (1.7 CYP3A29, isolated from a pig small intestine cDN~ library ~68]. nmol/min .mg protein). Also tbe averageporcine nifedipine oxida- The translation of tbe ORF of CYP3A29 resulted m a protem of tion rate (1.5 nmol/min .mg protein) was comparable to tbat of 503 amino acids, which showed a very high similarity to CYPs humans (1.72 nmol/min .mg protein). These two enzyme activities belonging to tbe CYP3A subfamily, and in particular to human were well correlated amongst tbemselves and witb tbe immuno- CYP3A4 (only monkey CYP3A8 scored better) [91]. In addition, chemical levels of pig CYP3A, as assessed witb anti-human tbe deduced N-terminai amino acid sequence of pig CYP3A29 CYP3A4 antibodies [14]. Anotber drug specific substrateofhuman matched very well witb tbe ODeof human CYP3A4 sharing a 60% CYP3A4 -lovastatin -has been recent\y used to validate Bama similarity (12 of20 amino acids identical) [42]. CYP3A29 was also minipigs as a model for drug evaluation in humans [108]. The re- cloned from Gottingen minipig liver [91]. Pig and minipig sults showed tbat tbe metabolites and tbe enzyme kinetic parame- CYP3A29 were ditTerent in just eight amino acid residues, six of ters were similar in pigs and humans. Apart from testosterone,otber which were conservatives. Besides CYP3A29, tbere are four otber sexualhormones can be metabolized by CYP3A4, such as 17 alpha- porcine CYP3A sequencesavailable in Genbank,named CYP3A22, etbynylestradiol (EE2) [109]. The increase in 2-hydroxylation of 3A39, 3A46, and 3A88. However, CYP3A88 is likely to be an alle- EE2 after tbe exposure of porcine primary hepatocytes to various lic variant of CYP3A46. CYP3A22 has been isolated from a Gl>t- CYP3A inducers has been found to be well correlated witb tbe in- tingen minipig liver cDNA library and showed 81.5, 83.9, and crease in 6f3-hydroxylationoftestosterone and witb tbe increase in 76.5% amino acid sequenceidentities witb pig CYP3A29, 3A39, tbe protein level of CYP3A detected by a monoclonal anti-human and 3A46, respectively [77]. Because oftbe high sequencesimilar- CYP3A4 antibody, tbus conflrrning tbe 2-hydroxylation ofEE2 in ity between CYP3A39 and CYP3A46 (93% nucleotide .sequence pigs as being biotransformed by a CYP3A, as in humans [110]. identity) tbe expressionofCYP3A39 has not yet beenstudled. Otber reactions known to be catalyzed mainly by CYP3As in rat
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PorcineC:vtochrome P450 CurrentDrug Metabo/ism,2011, VoL 12,No.6 519
and/or humans afe BQD and erythromycin demethylase (ErD) CYP4A\UBFAMILY [111,112]. While ErD activity was only detectable ~ pig liver b~. Generai Features cause of its low sensitivity, BQD was also detected In extrahepatlc Isoforms belonging to the CYP4A subfamily bave been identi- tissues of domestic pigs [21,58-60]. fied in different species including humans, rodents, monkeys and Both minipig and pig CYP3A29 bave been pu?fied or ex;- dogs [115]. Enzymes of this subf~ily play a key ~ole in b.ile acid pressed in S19 insect celi s, respectively, and,~alysed In a reconstJ- biosynthes~s and in the metaboilsm of fatty aclds ~malnly 00- tuted system [91,102]. The enzymes exhlblted testosterone 6j3- hydroxylatJon and, to a lesser extent, (oo-I)-hydr~xyl~tJon). Me~- hydroxylation and nifipidine oxidation, as repo~e~ for h~man bers of the CYP4A subfamily also.catalyze th~ oxl~atJon?f arachl- CYP3A4. However, domestic pig CYP3A29 exhlblted a hlgher donic acid leading to the formatJon of phY~lologlcally Important affmity (Km) for testosteronethan minipig CYP3A~9,.thus suggest- metabolites involved in blood flow .regul~tJon. [116]. The most ing that the domestic pig enzyme could be more slmllar to human common marker activity of CYP4As ISlaunc acld l~-hydroxylase, CYP3A4 than the minipig enzyme[102]. which seemsto be generally conservedamongst specles [31],
Inbibitors CYP4A21So far, three4A24 CYP4Aand 4A25. isoenzymes Nevertheless,bave it been is stili identified unclear whetherin pigs: Ketoconazole and triacetyloleandomycin (TAO) (typical human CYP4A25' constitutes a different isoform or is a variant of CYP3A4 inhibitors) were able to inhibit 6j3-hydroxylation oftestos- CYP4A24. In 2001, Lundell et al. [117] cloned CYP4A21 from pig terone and N-oxidation of nifedipine, fairly indicating that ODeor liver. The cDNA encoded a protein of 504 amino acids, which more CYP3A-like enzymes afe also responsible for these two reac- sbared a high similarity (74% of identity) with the human tions in pigs [22,31]. A very recent work [I 02] h~ confirmed the CYP4A Il. Later, pig CYP4A24 and 4A25 were clone~ by ~undell efficiency of these two inhibitors to,:"ard.recombmant CYP3A29 [118]. These enzymes shared an extensive ~equen~e!dent!ty (ap- activity, and ketoconazole has been IdentJfied to be more ~otent prox. 99% nucleotide identity and 97% ammo acld IdentJty) and than TAO. Whi1ethe inhibition oftestosterone 6j3-~ydroxylatJonby about 94% identity with CYP4A21. The amino acid diff~rences ketoconazo1ehas been found to be as strong as m hu~ans. [~1), between the two sequenceswere found in N- and C-terminai re- TAO inhibited nifedipine oxidation with.lo,:"er .potency m mml~lg gions, confined to j3-sheets1 and 4, ~d!cating a poss.ibledifference microsomes compared to human ones, mdlcatmg structure varla- in substrate specificity or regioselectJvlty,The porcme CYP4A21, tions in the active sites ofthe CYP3A enzymes [22]. TAO was also 4A24 and 4A25 bave probably evo1vedfrom a common ancestral able -both in pig and minipig microso~es -to strongl:ydecrease the gene -maybe by gene duplication -in conjunction wi~ speci~s- formation of two metabolites of paciltaxel formed m hum~s by specific habits as indicated also by a study [119] m whlch CYP3A4 [72]. TAO was well able to inhibit the m,eta?oilsm~f CYP4A21 and'4A24 revealed a sequenceidentity that extends be- lovastatin, further confirming the efficiency and speclficlty of thlS yond the exons. inhibitor [108], Tissue Distribution Inducibility and Regulation The expression of CYP4A21 mRNA was found in pig kidney The expressionand activities ofCYP3As bave beenfound to be but not in heart, muscle, intestine, spleen, thymus, 1ung,or adrena1 induced in primary porcine hepatocyte and enterocy~ cultures after gland [117]. CYP4A24 and 4A25 mRNAs bave been found in both treatrnent with DEX, RIF or PB, ali drugs able to mduce human pig liver and kidney [118]. With regard to the abundance, CYP3As [32,35,38,110,113, 114], More recentworks h~ve.revealed CYP4A21 seemed to be the major liver CYP4A, whereas that CYP3A22, 3A29, and 3A46 afe inducible at transcrlptJo?al~d CYP4A24 has been found more abundant in kidney [118]. The activity level by an in vivo pig.treatment with RIF and PB m ilve.r expression of a CYP4A protein, Bot better ident!fied, has b~en but BOt in BasaImucosa (resplratory or olf~cto~), lung, bronchl, demonstrated in porcine coronary artenes wlth Immunoblottmg trachea, and kidney [21,58-60]. In the small mtestme, the treatrnent experiments[121]. with PB resulted in an i~~r~aseofmRNA of,these three genes,but Interestingly, it has been demonstrated that, in pig liver, not of the related actJvltJes [58]. InterestJngly, CYP3A2~ ~d CYP4A21 has a developmental-dependentexpression opposed to CYP3~29 (but not CYP~A4~) mRNAs bave been f~und slgrnfi- CYP8BI (a sterol 12a-hydroxylase which is a key step in the for- cantly mcreased by RIF. m ~Ig cerebral corte:" ~d hlppo~am.pus, mation of cholic acid, tha major bile acid in humans), as assessed but not in cerebellum, m~dbram,and blood-~ram mte,rraces,I?dlcat- b RT-PCR experiments. CYP8B 1 was well expressedin the foetal ing a differential re~ulatJo?ofthese CYP~ ~ the varlous reglons ~f s~ples but no signal was detected in weaned pig liver, determining brain. ~owe,:"er,no mductlon ofBQD actJvlty has beenobserved m the lack of cholic acid formation in adult pigs, On the contrar:;, the the bram reglons [60]. ., .expression of CYP4A21 (an enzyme involved in ~e produ~tJo~of Little is known about the regulatJonmechanlsms of CYP3As m hyocholic acid)was much higher in weaned than m !oetal plg ilver pigs. In humans, the induction ofCYP3.A genes generally occurs at [120]. Certainly, further studies ar~ needed to clarlfy the mecha- transcriptionallevel through the actJvatJonof PXR or CAR and the nism underlying the shift of expresslonof thesetwo enzymes and to contribution of other nuclear factors (e,g. HNF4a). As a g~od ex- understandits biological meaning, pression of these nuclear receptors has been demonstrated m po~- cine liver [59], probably the induction mechanisms of .CYP3.Asm Substrates and Reactions pigs afe similar to those shown in humans. The lack ofm~uctlon of Porcine CYP4A21 has been cloned and expressed in a recom- CYP3A-dependentactivities observed in the above-mentlone?por- binant system [117]. This enzyme was able to hydroxylate ~auro- cine extrahepatic tissues could be d~e to the scarce expressl.<>nof chenodeoxycholic acid in 6a position, leading .to th~ f~rmatJon.of these nuclear receptors or to th~ mvolvement of ~ther tJssu~- hyocholic acid (the species-specificprimary bile acld m the plg). specific transcriptional.f~ctors. As m humans,no sex dlff~ren~es m Despite the high similarity between the porcine CYP4A21 an~ ~e the expressionand actJvlty of CYP3~s bave ?een found m plgs or human CYP4A 11, the former showed no oo-hydroxylaseactJvlty minipigs [14]. However, the castratJonof plgs has been demo,n- toward lauric acid and other fatty acids whereas the latter showed strated to determine a slight increase in CYP3A mRNA, protem, no 6a-hydroxylase activity toward taurocheno~eoxyc~olic a~id. and activity [50]. Mutagenesis experiments indicated that three amm.<>acld substJtu- tions in a region around position 315, which was hlghly conserved .I
I ! .' I 520 CurrentDrug Metabolism,2011, VoL 12,No.6 Puccinelliet aL
in alI previously known CYP4As, could be involved in this specific Pi~HNF4a mRNA has been detected in liver, kidney, lung, I catalytic activity ofCYP4A21 [117]. On the contrary, CYP4A24 bronchi, trachea, nasal mucosa, and small intestine [21, 58, 59, and 4A25, expressed in yeast cells, exhibited (0>-)- and (0>-1)- 132]. The resulting pattem of expression was in agreement with hydroxylase activities towards lauric acid and palmitic acid, but not human data [127], showing the higher expression in liver, kidney, hydroxylase activity towards taurochenodeoxycholic acid, like hu- and small intestine and a very low presencein the other tissues. man CYP4All [118]. In particular, microsomal preparations fro~ Porcine PPARa mRNA has been found highly expressed in C':P4A24/25 tran~formed yeast cells re~ealed an ave.ragelaunc liver and kidney, variably expressed in small and large intestine, acld o>-hydroxyla~l.onr~te of 286 pmo~mm .mg protem, wher~as moderately expressed in heart and skeletal muscle, and barely ex- the avera~epalmluc acld o>-hydroxylauonrate was 113 pmol/mm .pressed in brain, spleen, lung and adipose tissue [133]. Further- mg protem. more, PPARa mRNA has been found expressed in vascular endo- In humans,the CYP4A subfamily plays a role in the regulation thelial cells [131]. The quantification ofPPARa mRNA in the liver of vasai tone participating in the formation of vasoactive metabo- revealed that PPARa expressionwas higher in pigs than in rats and lites of arachidonic acid (in particular in the formation of the vaso- mice which, in tIInI, were reported to bave a higher expressionthan constrictor 20-RETE, 20-hydroxyeicosatetraenoic acid) [122]. In in humans [123]. A study performed using two different breeds of porcine COronary arteries, the formation of 20-RETE has been pigs (Duroc and Norwegian Landrace)revealed an age- and breed- found to be reduced after a treatrnent with N-methylsulfonyl-12, 12- dependentexpression ofPPARa in liver and heart [133]. This find- dibromododec-ll-enamide (DDMS), an inhibitor of CYP4A, sug- ing could reflect a difference between the two breeds with respect gesting that porcine CYP4A could also be involved in the formation to the tissue distribution of fat and the use of fatty acids as an en- of20-RETE [121]. ergy source in piglets.
Inducibility and Regulation Strutture and Functions To date, very little is known about the induction and regulation Porcine CAR and PXR bave been cloned from cDNA libraries of CYP4As in pigs. An induction cocktail containing j3NF, PB and and characterized[126]. The porcine CAR gene comprised a 1047 DEX had no effect on the protein level of liver CYP4As, as as- bp coding region that encodes a protein of 348 amino acids. The sessed with anti-rat CYP4Al/3 polyclonal antibodies, although the mRNA sequenceof porcine PXR was composed of 1266 bp puta- metabolism of lauric acid was higher in hepatic S l O preparations tive coding region encoding 421 amino acids. The porcine CAR and from treated pigs compared with controls (study performed using PXR proteins showed a high degree of sequence identity in their Landrace x Poland China crossbred pigs) [25]. Also the pig (York- DNA-binding domain (DBD) and ligand-binding domain (LBD) shire x Landrace) adrninistration of clofibrate, a drug which is with 80%-90% amino acid identity with respectto human ones. In strong inducer of CYP4As in mice and rats through the activation particular, porcine PXR has tumed out to be more similar to human of PPARa, failed to induce the mRNA expression of hepatic PXR than mouse PXR (87% vs. 77% identity) and to contain the CYP4As [123]. The unresponsivenessof porcine CYP4A genes to human residues at four locations (R203, P205, Q404, and Q407) peroxisome proliferator agonists is sirnilar to that shown for which afe keys for human PXR activity [128]. The complete se- CYP4As in human hepatocytes[124]. quence of pig HNF-4a mRNA, which is available in Genbank (ac- cessionn. DQ061106), encodesa protein of 474 aa with about 75% NUCLEAR RECEPTORS homology to that of human HNF-4a [132]. The cDNA containing GeneraI Features the ORF ofPPARa has been isolated from Duroc and Norwegian Most ofCYP isoforms afe mainly regulated in a transcriptional Landrace pi~ liv~r [1~3]. ~e deduced protein sequence of0468 aa way through the action of nuclear receptors acting as transcription showed. a hlg~ Idenuty wlth ~e human counterpart (92.3'lo): .An factors. In humans,members ofCYPI, 2,3, and 4 families afe pri- altemauv~ spllced mRNA lac~mg exon 5 (presu~ably determm~g marily regulated by AhR, CAR, PXR, and PPARa, respectively. a. C-termInai truncated. protem) was detected.~ several. porcIne Rowever, the regulation mechanisms afe complicated by the in- ussues,but ~rth~r studles ar.eneeded to ascertamIts potenual effect volvement of many other nuclear factors [e.g. retinoid X receptor on the functlonallty ofthe WIld-type formo (RXR), HNF4a, etc.] and by the overlap existing for the sets of A study performed with Yorkshire pigs revealed that the activa- target genesofthese receptors. tion responseofporcine CAR by CITCO (a specific agonist ofhu- man CAR) and other nine ligands out oftwelve was sirnilar to the Expression response of human CAR. On the other hand, for the murine CAR The mRNA expressionofboth porcine CAR and PXR has been only five ligands were in common with human CAR [134]. Fur- detected in liver, small intestine, kidney, lung, bronchi, trachea, thermore, a pig treatrnent with TCPOBOP (1,4-Bis-[2-(3,5- nasal mucosa and various regions of pig brain by using RT -PCR, dichloropyridyloxy)]benzene,3,3',5,5'-Tetrachloro-I,4-bis(pyridy- with the greatestexpression in liver followed by kidney [21, 58-60, loxy)benzene) (a specific murine CAR ligand) had no effects on 125,126]. It is worth noticing that high levels ofmRNA expression CAR-regulated activities, thus suggesting that porcine CAR is more bave been found in porcine blood-brain interfaces like meninges sirnilar to human CAR than the murine orthologous [50, 125,134]. and cortex capillaries (25-150% of liver expression), indicating a The inducibility of CAR, PXR, AhR and HNF4a transcripts, along possible role in the regulation of genes involved in physiological with their target genes,has been investigated in many pig tissues by functions of the brain [60], whereas human CAR and PXR bave using classical P450 inducers, such as RIF, PB and j3NF [17, 19,21, tIInIed out to be just detectable in human brain [127]. In addition, 58-60]. In alI these studies (performed with Landrace x Large Northem blot analysis bave shown a fair PXR expression in pig White domestic pigs), the nuclear receptors mRNA levels were not heart, colon, and stomach, while a minor expression has been ob- affected by the treatrnent, in agreementwith human data [135]. served in mandibular lymph node, thymus, adrenal gland, ovary, In generai, in liver, the induction of CAR-, PXR-, and AhR- uterus, bladder, spleen,and mesenchymallymph node [128]. regulated genes after the exposure to the classical inducers reflects Porcine AhR, whose gene has been only partially sequencedso the abundant expression of the nuclear receptors in this organoIn far [17], has been found expressed at transcriptionallevel in liver, the extrahepatic tissues, the regulation of the samegenes is proba- lung, heart, kidney, brain, ovary, thyrocytes, artery endothelial bly more complex and lirnited by either the lower levels of these cells, olfactory and respiratory nasalmucosa [17, 19,21,129-131]. main receptors or by the need of other tissue-specific factors. Also single nucleotide polymorphisms (SNPs) and splice variants (SVs) of nuclear receptors can alter biological function, representing a .'
Porcine Cytochrome P450 Current Drug Metabolism, 2011, VoL 12, No.6 521
new layer of complexity. Porcine PXR gene has been characterized ABBRE\.~ TIONS in several pig breeds, and revealed multiple SVs in the ligand- 3-MC = 3-Methylcholanthrene binding domain, as well as human PXR [128,136]. Ofthe five iden- -. tified porcine PXR SVs, SVI was able to significantly increase the 3-Ml -3-Methylmdole transactivation of the wild-type form [136]. Ali human and porcine 20-HETE = 20-Hydroxyeicosatetraenoic acid PXR SVs were species-specific, suggesting that SVs might act as a aa = Amino acid species-specific mechanism for adaptation to different environ- .= AhR A ry I h y dr ocar bon recep tor mentalone nonsynonymous exogenous compoundsSNP (SI78L) encountered has been byfound each in specles.the pig OnlyPXR BALs = Blo-artlficlal ilvers
ligand-binding domain, but further investigations afe needed to pNF = p-naphthotlavone determine its eventual effect [128]. Skatole has been recently iden- BQD = Benzyloxyquinoline debenzylase tified as a novel inverse agonist for pig PXR, as well as for pig . CAR [136]. Porcine CAR multiple SVs has also been recently iden- bp = Base palrs
tified, each ofwhich generated a truncated protein [134]. These five BROD = 7-benzyloxyresorufin O-debenzylase variants were found present in pig liver samples from about 5 to 9% C/EBPa = CCAA T/enhancer-binding protein alpha of total pig CAR, suggesting that they might play a functional role CAR = C onstltutlve.. an drostane recep tor In VIVO. In partlcular, SV2 has been found to slgruficantly decreasç the activity of the wild-type protein. This may represent another cDNA = Complementary deoxyribonucleic acid
level of transcriptional regulation, by which SVs afe generated to CITCO = (6-(4-chlorophenyl)imidazo[2,1- limit the activity ofthe receptor. b][1,3]thiazole-5-carbaldehyde-O-(3,4-
dichlorobenzyl)oxime) CONCLUSIONS .. COUP-TFI = Chlck ovalbumm upstream promoter tran- From the information collected in this review it appears that scription factor 1 (mini)pig could actually represent a new prime large animai model for future drug metabolism and pharmacological studies. On the CYP = Cytochrome P450 basis of substrates, inducers, inhibitors, tissue distribution and regu- DBD = DNA-binding domain lation data -as summarized in Tables 1-5 -no major qualitative DDMS = N-methylsulfonyl-12 12-dibro-mododec- differences among the CYPIAI, IA2, 2B, 2EI and 3As of pig II-enamide' compared to human orthologues bave been revealed so far. How- - f . h b " d . I ..DEX -exametazoneD ever, a note o cautlon as to e put lorwar m re atlon to a StrlCt functional similarity among these orthologous CYPs. This tentative e.g. = Exempli gratia
conclusion must be balanced by the observation that even a simple EDHF = Endothelium-derived hyperpolarizing substitution of a single amino acid, especially in the active gite of factor these enzymes, might dramatically change the substrate specificity. EE2 = 17 al h -th I tr d. I It should also be kept in mind that: i) several drugs afe metabolised p a e y~y es a IO . to different products by multiple CYPs retlecting their tissue- EFCOD = 7-ethoxy-4-(trltluoromethyl)coumarm 0-
specific expression and specific rates; ii) structurally different deethylase CYPs may catalyse the same reaction, with their own specificity ErD = Erythromycin demethylase and rate. Thus, with this caution, the porcine experimental data EROD = Ethoxyresorufin O-deethylase deriving from the drug biotransformation by the above mentioned . CYPs may be considered useful for humans. On the contrary, drug HNF = Hepatlc nuclear factor
metabolic profiles primarily due to the catalysis of CYP2A, hnRNA = Heterologous nuclear RNA CYP2Cs and CYP2D in pig, may not at ali retlect what occurs in i.e. = id est human and therefore pig appears to be an unsuitable model for the LBD - L . d b . d. d . meta bo I.Ism o fili ese dru gs .m h umano A s to th e cat al ytlc... slml 1arlty.-Igan -m mg omaln between pig and human CYPIB I and CYP4As, too limited evi- MROD = Methoxyresorufin O-demethylase
dence afe available to give a clear indication. MFCOD = 7-methoxy-4-(tritluoromethyl) coumarin
Besides the oxidation of xenobiotics, many enzymes belonging O-demethylase to the cytochrome P450 superfamily afe also involved in the me- mRNA = Messenger ribonucleic acid ta~olism ?f several end~geno:us compounds implicated in the regu- ORF = Open reading frame latlon of 11llportant physlologlcal pathways, such as hormones, ster- ols, vitamins, fatty acids and vasoactive molecules. Since pig is P450 = Cytochrome P450
recognized as a useful model for human pathologies, it becomes P AH = Polyaromatic hydrocarbons significant to extend the characterization of CYPs beyond the first PB = Phenobarbital four families. Furthermore, the detailed knowledge ofthe important . enzymatic system of cytochrome P450 is not sufficient to face ali PBREM = PB-responslve enhancer module
the potential applications of the pig as an animai model. Further PPARa = Peroxisome proliferator-activated receptor studies afe needed to extend the characterization to other phase-1 alpha enzymes (such as tlavin-containing monooxygenases, monoamine PROD = 7-pentoxyresorufm O-depenthylation
oxidases,1 I epoxide hydrolase or aldehyde dehydrogenase), for which PXR - P X .. d ." ... 1 bl . I. AI . d h -regnane receptor on y Imlte mlormatlon IS aval a e m Iterature. so, an m- ept examination of phase-2 enzymes (such as UDP-glucurono- RIF = Rifampicin
syltransferases, glutathione S-transferases or sulfotransferases) and RT-PCR = Reverse transcription polymerase chain drug transporters (the so-called phase-3 ofmetabolism) needs to be reaction carried out in pigs. RXR = Retinoid X receptor .'
522 Current Drug Metabolism, 2011, VoL 11, No.6 Puccinelli et aL
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Rece;ved:January 31, 2011 Rev;sed:Marcb 22,2011 Accepted: Marcb 23,2011