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Current Drug Metabolism, 2011, 12, 507-525 507

Xenobiotic Metabolizing Cytocbrome P450 in Pig;a Promising Animai Model I

Emanuela Puccinelli8 *, Pier Giovanni Gervasi8 and 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 because of its similarity in size, physiology and diseasedevelopment However, in contrast to the extensive data available on the cytochrome P450 (CYP) system far humans and rodents, thedata related to pig afe limited because or, among others, the presence of intra-species differences (domestic pigs and minipigs) Theknowledge of the CYP superfamily in a given experimental animaI is crucial far pharmacological and toxicological tests in developingdrugs and far understanding the metaboli~ pathways of toxicants and carcinogens. In addition, information on the CYP system in pigs isImportant smce Il plays a domlnant role m the metabolism of veterinary drugs, whose residues rernain in the porcine tissues which afefood 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 porcinedrug-metabollzmg enzymes morder to evaluate the hypothesis whereby pig data may better reflect human drug metabolism and toxicitythan 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 thenumber 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 decadesmango Many organs and systems -including beaTI, nasal cavity, some ~fforts bave been focused on these enzymes [9-11]. One ofliver, kidneys, brain, reproductive and gastrointestinal system -~e ~am hur.dles for the met~bo~ic characterizati<:>n ofthe pig is theshow analogies with humans and advantages compared to other slgrnficant dlfference occumng m the varlous anlffial breeds. Manyexperimental 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 usedtlve 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 ofdiseases (e.g. atherosclerosis), metabolic diseases (e.g. hypercholes- non~clinical ph~acological/t.oxicologiCal use [12]. However it isterolemia) 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 1-4tification 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 dlfferencesrepresents a tuming point is that of xenotransplantation. The high between convent!onal plgs and mmlplgs CYP sequences (at leastsimilarity found in heart (including coronary arteries) and liver the only few avallable so far) afe less than I % and they should bemakes the pig the best candidate for xenotransplantation to humans, r~garded as alleli.c variants (Table 1). Nevertheless, significantly Ithus 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 viewPigs 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 theand 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 ofminor 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, veryAddress correspondence to thls author at the Istituto di Fisiologia Clinica, little is known about the regulation of these genes and -likCNR,Plsa, Via MOruZZI, 1,56124 Plsa,ltaly; Tel: +390503152704; : un e

Fax: +390503153328; E-mail: epuccinelli@ifc.cnr.it 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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""Table 1. Comparison ofthe Nucleotide and Amino Acid Sequences ofPorcine and 8uman CYPs.

% ofidentityPorcine 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_2 I 4422 75.9

NM_001134824 CYP3A4

NM_214425

CYP4A24/25 NM_214424 CYP4AII 73.0 74.0

.Sequence isolated from minipigs.

This review gives a comprehensive knowledge 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, somereceptors (aryl hydrocarbon receptor, AhR, constitutive androstane drugs known to be metabolized in pigs are reported, together withreceptor, 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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Porcine Cytochrome P450 '" Current Drug Metabolism, 2011, VoL 12, No.6 509

ITable 2. Recombinant Porcine CYPs.

CYP Isoform Expression System Tested Substrates 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]

cC- ~., CYP2El E. coli, HEK-2~;3, Chlorzoxazone, p-nitrophenol [86,90]

CYP3A29 Sf9 insect cells Nifedipine, testosterone; ': [91,102]

'...' ,':",;CVP4A21 COS-l cells Lauric acid, taurochenodeoxycholic acid -[117]

CVP4A24/25 .S. cerevisiae Lauric acid, palmitic acid, taurochenodeoxycholic acid [118]

content, similar to the expression ofCYPIA2 in human liver (aboutTOTAL 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 appears to be lower than CYPIA2pig 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 tissuedifferent pig crossbreeds) is comparable to that ofhumans (0.43 and distribution of porcine CYPs and nuclear receptors is summarized.0.26 nmoUmg prot. for Caucasian and Japanese, respectively), Another study deepened the presence of pig CYPIAI/IA2 in thewhereas Gòttingen minipigs total CYP is 2 to 3-fold higher [IO]. brain [19]. Both the isoforrns were found expressed at mRNA levelFurtherrnore, 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 hippocampus and 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 inCYPIA SUBFAMILY porcine olfactory gasaI epithelium [20], and this fmding has beenG 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 aswell as in the metabolism of several drugs. Ethoxyresorufin 0- Substrates and Reactionsdeethylase (EROD) and methox~~~orufin O-de~ethylase ~OD) Already in the 1990s, different groups detected EROD and8!e u~ually us.ed.as marker actlvltl~s of these Isoforrns, typlcally MROD activities in liver microsomes from various pig and minipigmduclble by dloxms and polyaromatlc hydrocarbons (PAHs). breeds, although at lower level than in human ones [22-26]. Iodeed,

Porcine CYPIAI has been defmitively isolated and sequenced, EROD activity in pigs ranged from 5 to 95 pmol/min .mg proteinrevealing 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 .mgWhite 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 sequences revealed 85% and 81% identities to those of However, some differences with the human orthologous werehuman 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-depenthylationand human CYPIA2. (PROD), 7-ethoxy-4-trifluoromethylcou-marin O-dealkylase (EF-

COD) and 7-benzyloxyresorufin O-debenzylase (BROD) activitiesTissue 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, bothheart, and kidney of domestic Large White x Landrace hybrid pigs, linked to human CYPIAs and 3A4, were not increased indicating aalthoUgh at different levels [16, 17].10 the porcine liver, the level of difference in substrate specificity between porcine and humanCYPIA2 expression has 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

'" ITable 3. Drugs Metabolized by Pig Liver I

CYP Subfamilies Drug Clinical Use References

2B? Benzphetamine Anorecticdrug [137]

2B, 20 Bufuralol ~-adrenergic receptor blocker ]

Dextromethorphan Cough suppressant I]

Omeprazole Gastric acid blocker [71]

Oiclofenac NSAlO [58]

2C? Tolbutamide Hypoglycemic drug 8]

2C ? , 28 ? S-rnephenytoin Anticonvulsant 8]

2C ?, 3A ? Taxol Chemoterapic drug 8]

20 Oapsone Antibacterial agent [145]

Sulfamethoxazole Antibiotic [145]

Tolterodine Antimuscarinic muscle rei [79]

Bupropion Antidepressant [92]

Chlorzoxazone Muscle relaxant [90]

17a-Ethynylestradiol Synthetic estrogen [11.0]

Erythromycin Antibiotic [13.7.59]

Lovastatin lipidemic drug [108]

Midazolam Benzodiazepine [113]

Nifedipine Antihypertensive ,14]

Tacrolimus Immunosuppressant {114]

Ethylmorphine Cough suppressant ]

Triacetyloleandornycin Antibiotic ]

Ampicillin Antibiotic ]

Antipyrine Analgesic and antipyretic 39]Azidothymidine ' AntiretroviraI drug [144]

Hexobalbital 8arbiturate anaesthetic [143]

Naproxen NSAIO [140]

Norfloxacin Antibiotic (142]

Paracetamol Analgesic and antipyretic (139]

Sulfadimidine Antibacterial agent ]

? Vancomycin Antibiotic (139]

Note: fields marked by a question mark indicate no furdter infonnation available.

Recently, pig CYPIA2 has been cloned, expressed in a het- CYPIA2 [27] but in keeping with human CYPIA2 [28,29], failederologous systern and characterized [16]. It was confirmed to pos- to oxidize aniline or testosterone (in the 613 position).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 ishlgher 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

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Porcine C:vtochrome P450 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 +++ ,+ ++ +1- + + +1- +1- [15,21,58-60]I .,

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' ..[2158-60125-+++ ++ + + ++ ++ + +1- "

126,128]

+++ = high expression; ++ = moderale expression; + = low expression; +/- = barely detectable expression; -= undetectable expression; n,a, = data noI available,

Inhibitors agonists J3NF and 3-methylcholanthrene (3-MC) -but Bot with phe-

nobarbitai (PB), rifampicin (RIF) or dexametazone (DEX), inThe 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 Current Drug Metabolism, 2011, VoL 12, No.6 Puccinelli et aL

~ITable 5. Known Porcine CYP Inducers and Inhibitors.

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 represents a 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. Inreaching activity levels even higher than in the liver. Benzo(a) parallel, EROD but not MROD activity was increased by !}NF in alIpyrene, 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 andceli s, indicating this enzyme as both indicator and contributor for olfactory nasal mucosa, at both transcriptional and activity levels.benzo(a)pyrene toxicity [37]. This fmding is particularly interesting Omeprazole is a potent inducer of CYPIAZ activity -interestinglyas 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 hepatocytes indicating

The inducibility of AhR-regulated CYP genes by !}NF has been pig ~ a m~re ~ppropriate model system for the evaluation of CYPinvestigated 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 shownalong with liver, showed a transcriptional and activity induction of marked sex differences (with females having a much higher activityc.

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Porcine Cytochrome P450 Current Drug Metabolism, 2011, VoL 12, No, 6 513

than males) in GOttingen minipigs and Meishan conventional pigs Substra1"esand Reactions I(~ut Bot in L~dr.ace. or L~drace x Yorkshire x Duro~ domestic No l7J3-estradiol-4-hydroxylase activity (a marker reaction forpl~S) [14,39], mdlcatlng an Important role ofhorrnones m the regu- human CYPlBl) has been detected in porcine liver kidney longlatlon ofthese enzymes.

d h . d " . th l ...' .' ,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 humanconstitutive 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 RegulationThis restriction in.the pres~nce and inducibility ofCYPIA2, which A treatment with J3NF -an agonist ofthe nuclear receptor AhRalso reflects what Is.fo~nd In hu~ans [40], could be due to the need which regulates CYP isoforrns of l family -enhanced CYPlBIfor HNFI-4 factors In Its regulatlon. mRNA in pig liver but Bot in respiratory and olfactory BasaI mu-P I h' cosa. Since AhR mRNA has been found also in pig Basai tissueso ymorp Isms

th d t ' d ' te . l f dd " al .. fi ' ese a a mica an mvo vement o a Itlon tissue-speci IC

SO far, 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 J3NF was able to increase the transcription ofthis isoformbeen observed in porcine CYPIA2 and R71 is shared with humans, in pig midbrain and, especially, in capillaries [19], although thebut 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-transcriptional regulation.CYPIB SUBFAMILYGenerai 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 subfamilytlssue and IS suspected to play a role in oncogenesis and 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 494swine 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.ids at the N-terrninus of GOttingenCYPIBI 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, SIX substrate recognltlon sltes bave been identifiedcompared with the human orthologous. Very recently, pig CYPIBI m the human CYP2A sequences, and they are ali present in thehas 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 lowergous and, except for SRS l, also with that of the canine counterpart. extent, in kidney ofLandrace x Large White x Duroc crossbred pigsHowever, the SRSs ofhuman CYPIBI showed a higher homology [15]. However, CYP2A19 mRNA has not been found in spleen,with those ofpig 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-polymerase chain reaction) experiments on liver, olfactory Coumarin 7-hydroxylation is the most utilized marker activitynasal mucosa and respiratory nasal mucosa samples from Large for human CYP2A6, Although in human liver this activity level isWhite 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 useticular in the hepatic samples. Immunoblotting experiments per- this reaction as a marker for pig CYP2AI9, as a good correlationforrned using anti-rat CYPIBI antibodies bave revealed a protein has been shown between the activity and the immunochemicalband 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 expression of ity was strongly inhibited by anti-human CYP2A6 antibodies. AlsoCYPIBI mRNA in various brain regions ofthe same pig breed -the forrnation ofcotinine from nicotine, another human CYP2A6including cortex, cerebellum, midbrain, hippocampus, meninges marker reaction, is catalyzed by porcine CYP2A. At high nicotineand cerebral capillaries -with similar levels compared to humans. concentrations, other isoenzymes probably contribute to the reac-The high expression noticed in the blood-brain interfaces (meninges tion, as the inhibitory anti-human CYP2A6 inhibition rate decreasesand 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 predominant enzyme with 50 11M substrate, whereasfrom xenobiotics. Further experiments conflrrned the constitutive with 500 11M nicotine 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 activitysmall intestine, lung, nasal mucosa, heart, coronary arterie s, adrenal has been found in pig (Large White x Landrace) nasal mucosa, andgland, and spleen, with the higher expression in adrenal gland and in particular in the olfactory mucosa where the activity levels werethe 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 cultureswhere ~YPIBI IS c~nsldered mostly extrahepatlc and well ex- using two CYP2A inhibitors suggested that CYP2A -unlikepressed m steroldogenlc organs. CYP2El -has a minor role in 3-methylindole (3-MI or skatole)

metabolism, whose accumulation in adipose tissue is responsiblei

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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 androstenonethe production of3-MI metabolites was affected by the presence of [57] have confmned a down-regulation ofCYP2AI9 in Duroc butinhibitors 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 androgenCYP2AI9 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 between various conventional pig breeds.Furthermore, a particular single base deletion of CYP2AI9, result- .ing in a frame shift in the coding region that produces a non- Polymorphlsmsfunctional enzyme, was associated with 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 20CYP2AI9 in the skatole biodisposition. Also, the oral administra- different polymorphisms bave been identified far CYP2A6, leadingtion 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~c pig crossbreeds) through an ~crease of transcription and Studies on three different polymorphisms identified in minipigtranslatlon of CYP2A [48,49]. In addltlon, Matal et al. [30] have CYP2A revealed that unlike far human CYP2A6 the differencesrecently 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.ant role of human CYP2A6 m minipigs: one was completely homologous to conventional pigthe bloaCtlvatlon of some ~dustrlal compoun~s (e.g. tert-butyl CYP2AI9, whereas the 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 sequence may 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 CYP2A6strates 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 Featuresethyldithiocarbamate, 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. Piginactivator of CYP2A6, can be utilized as CYP2A inhibitor in pigs CYP~B22, the only CYP2B. Identlfied m thlS spec~es so far, has[47] been Isolated from a cDNA IIbrary of adulI female plg (Landrace x

.Large White x Duroc) liver, and sequenced [15]. The isolatedInducibility 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 humanItt e IS own a out regu atory mec anlsms. n ,.. h t ltu CYP2A d d t ti.. ty .CYP2B6 (81.1 Yo and 74 Yo far 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 yCITCO (6-( 4-chlorophenyl)imidazo[2, l-h ][1 ,3]thiazole-5-carbalde- Tissue Distributionhyde-O-(3,4-dichlorobenzyl)oxime), a human CAR ligand [50]. ..These results show that the induction pro file of CYP2A in vitro The expression of CYP2~22 .mRNA was confirmed ~ porc~eshares similarity with that ofhuman CAR-regulated CYPs, indicat- liv~r and, at a ~ess~r extent, m kldney by RT-PCR exp~nments Ining an involvement of pig CAR in the regulation of this subfamily. vanous domeStlc plg cros~breeds (Landrace x Large. W~lte x DurocA study of Myers et al. [25] demonstrated that a co-treatment with or Landrace x Large Whrte) [15,58,59]. The. cons~rtutlve ,presencef3NF, PB, and DEX is able to increase CYP2AI9 marker activity, of CY~2B22 mRNA has als.o bee~ ascertamed m po~cme !ung,but not to increase the expression of the enzyme, as assessed with branchi, and ~achea. Interestm?ly,. m I~ng CYP2B22 I~ baslcallyWestern blot analysis. In porcine hepatocyte cultures, unlike human expressed at hlgher level than m IIver, m agreement Wlth data ofhepatocytes [51], CYP2A was not induced by pyrazole [52]. This other mamma!s ~59]. Fu~e~ore, ~YP2~22 m~NA has beenfmding indicated a different regulation ofCYP2As in these species, found at constltutlve level m plg small mteStln,e and 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, meningesml~ll:>lgs show marked dlfferences (the fe~a~e~ have 70-fold h~gher and cerebral capillaries. While in meninges and cerebral capillariesa~tlVlty than males), but. also Yucatan mmlplgs ~d .convent~onal CYP2B22 mRNA levels afe high and comparable to the hepaticplgs (Landrace x Yor~shlre x Duroc) ,sho,w s°':l1e slgmficant dlffer- ones, in the other compartment the basai expression is about orences [14, 31]. Expe.nments ef~e~ted In VIVO wrth male (castrated ~r below 10% of the corresponding values in liver, in keeping withnon-castrated) GOt.tlnge~ ~1~lplgs have shown that CY~2.A IS what has been reported far humans [61]. The high expression ofgre~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:rmones m th~s tions far either pharmacological profiles ofneuroactive drugs or thespecles [50]. It has been p,roved that human chono,m.c.go~adotr~pm regulation of brain blood tension, as well as human CYP2B6 isIS 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, dehydroepiandrosteronesulphate, oestradiol and oestrone sulphate [53, 54], thereby decreas- Substrates and Reactionsin!? the ~etabolism of skatole and leading to its ~ccumulation in Dated reports failed to detect any CYP2B apoprotein or activityadlpose tlssue. An°l!t~r study re,:ealed I?at o~ly ~ sllgh~ decrease ?f in primary cultures of pig hepatocytes or minipig (GOttingen) liverCYP2A in VltrO. actlvlty o~curs mcubatmg plg ~Iver ~Icrosomes In microsomes using an anti-rat CYP2BI antibody or PROD as apre~en~e ofte.stl~u~ar sterolds [55], whereas, usmg plg hepatocyte.s, marker reaction [32,22]. On the contrary, Donato et al. [23] werea slgmficant mhlbltory effect of androstenone on CYP2A protem;'"

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Porcine Cytochrome P450 Current Drug Metabolism, 2011, VoL 12, No, 6 515

able to measure another activity often used as marker of CYP2B CYP2C\UBFAMILYsubfamily, BROD, in both hepatocyte cultures and liver micro- G I F tti L Wh. d ..eDera ea uressomes rom arge ne omestlc plgS, although at much lowerlevels than in human samples. The BROD activity in porcine mi- ~he CYP2C subfamily is known to be very divergent amongstcrosomes was about I pmol/min .mg protein whereas human activ- specles and 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 mammalianal. [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 2C9Yucatan 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 primersactlv~ty ~30-?0%), suggestm~ that ~ <;:YP2B also 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:; sequences weres~ch as S-mephenytom N-demethy~ase~ has been measu~d in pig isolated, eight of which were very sintilar to human CYP2CslIver 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.d b~proplon hydroxylase actlvlty (a selectlve CYP2ij these clones were subsequently classified as CYP2C32, CYP2C33marker aCtlvlty m humans at 50 11M substrate concentration) in CYP2C34 C

YP2C35 CYP2C36 d ali I.. ts 'GA tt ' ... d h Th ..."an some e IC varlan .

v mgen mmlplgs an umans. e actlvlty rate was higher m ...minipigs (435 pmol/min o mg protein on the average) than in hu- In 1998~ Nissen ~t al. .[68] Isolat~d two CYP2C-like .clonesmans (131 pmol/min o mg 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] clonedplg CYP2B22 I~ able to metabolize. some .herbicides including CYP2C49, the last porcine CYP2C sequence available 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 morder to produce herbicide-tolerant transgenic plants into which detall, whlle the other sequences could be allelic or splice variantsbave been introduced particular CYP enzymes. ofthese genes.

The comparative analysis revealed that CYP2C42 andInducibility and Regulation CYP2C49 shared 80% of aa sequence identity between them, about

Desille et al. [24] found CYP2B-related activity, rnRNA, and 60% with CYP2C33, and about 77-81% with human CYP2C9 andprotein present aild inducible by PB in printary porcine hepatocytes. 2C~9o F~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 thecould 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 decrease of many activities was sulted more similar to whale CYP2C78 or cattle CYP2C86 [69],noted. Other evidence ofthe presence and inducibility ofCYP2B in sharing an identity of82-83% [58].

pigs bave been found by treating commerciai animals (Landrace x o .o oPoland 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 expressed at mRNA level in porcineintmunoreactive with anti-rat CYP2BI, were alI increased in liver ovarian tissue [69]. Porcine CYP2C49 mRNA has been foundmicrosomal fractions obtain~d from the treated. pi.gs. CYP2B22 has highly ex~ressed in pig liver and poorly expressed in pig kidney

Ibeen also found markedly mduced at transcnptlonal, protein and [15,58]. Pig CYP2C33, 2C42 and 2C49 mRNAs bave also beenactivity level in pig liver and kidney -but not in lung, bronchi and found expressed, although at different levels, in small intestine and

Itrachea -after an in vivo treatrnent with RIF, a known inducer of nasal mucosa (both respiratory and olfactory) [58]. A CYP2CCYP3As through the nuclear receptor PXR [59]. Sintilarly, member, not better identified, has been found expressed in porcineCYP2B22 was induced in pig liver at transcript and activity level coronary arteries, as assessed by RT-PCR and Western blot analysisafter an in vivo treatrnent with 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, inRI~, .j3NF, or PB has been ascertained at both transcriptional and hepatic Gottingen minipig samples, of tolbutamide 4-hydroxylaseact!vlty level [21,58]. CYP2B22 has also been found to be resistant activity -a human CYP2C9 marker activity -althoUgh at lowerto mduction by RIF in porcine brain [60]. levels with respect to humans (62-136 pmol/min .mg protein versus

In generai, in humans the PB-dependent induction 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~osomes showed that pigs and minipigsinteractions with the PB-responsive enhancer modules (PBREMs) were able to metabollze d!clofenac (another specific substrate ofpresent in the 5'-tlanking region of CYP2B6 [64]. Furthermore, a human CYP2C9)~ but WI~ lower rates c~~pared to humanscross-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 o mg 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 CYP2Cavailable 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?) and omep~azo~e hydroxylase. The former isb.rain either by RIF, PB, or j3NF could be due to the scarce expres- a. very ~ensnlve actl':lty d~tected m plg (Landrace ~ Large White)slon ofthese nuclear receptors or to a more complex tissue-specific lIver, kldney, small mtestme and nasal mucosa mlcrosomes [58],regulation [59,21,58]. whereas the latter (tested in Gottingen minipig) has been reported to

be a good activity in liver microsomes [71]. On the contrary, noactivity could be detected in pig or mitiipig liver microsomes using

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516 CurrentDrug Metabolism, 2011, VoL 12, No.6 Puccinelli et 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 CYP2Cssomes 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 Polymorphismshuman CYP2C8 -did not lead to the formation of correspondent Human CYP2C19 is known to be strongly polymorphic, withmetabolites 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 possesses a similarever, the results on the expression and activities present in literature feature. It is possible that some of the sequences available in Gen-highlight some differences between human and pig CYP2Cs, sug- bank for porcine CYP2Cs constitute polymorphic variants. Furthergesting that pig might not be a suitable model for the study of drugs genomic studies afe needed to ascertain the presence of relevantmetabolized 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 SUBFAMILYcluding chlortoluron, nortlurazon, amiprofos-methyl, alachlor, andisoxaben. This capability could bave interesting consequences t'or Generai Features

human health, e.g. for the construction of herbicide-tolerant trans- In humans, CYP2D6 is one of the most important enzymes forgenic 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 relevantInhibitors 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-hydroxylationsulphaphenazole, ticlopidine and tranylcypromine bave shown a ofvitamin D3 has been cloned ftom domestic pigs and expressed indifferent selectivity toward porcine CYP2Cs [11,58]. Tolbutamide simian COS cells [75]. Even if human CYP2D6 can not catalyzehydroxylation 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 subfamilyinhibitor [11,58]. Quercetin, a human CYP2C8 inhibitor, was able and was subsequently named CYP2D25. Site-directed mutagenesisto significantly inhibit paclitaxel hydroxylation, tolbutamide hy- studies bave revealed that residues in SRS3 resulted crucial for thedroxylation (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 substrate ofhuman CYP2D6) [76]. In 2004,sulfaphenazole failed to inhibit ali these reactions [58]. Anti-rat the hepatic CYP2D21 has been cloned ftom Gottingen minipig andCYP2Cli 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 sequence with the domestic pig CYP2D25. Therethough 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 ascertain ifthese two enzymes afe allelic variants with

In primary porcine hepatocytes tolbutamide 4-hydroxylase ac- a distinct substrate specificity.tivity has been found increased by treatrnent with RIF (a PXRligand) and PB (a CAR activator), and decreased after treatrnent Tissue Distributionwith ~NF (an AhR agonist) [32]. The metabolism of tolbutamide Initially, Skaanild and Friis [14] suggested the absence ofand 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 responseand ~NF [25]. By investigating in detail the inducibility by PB of in this immunoblotting test has been confirmed and indicated to beCYP2C33, 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 analysis bave shown that CYP2D25 mRNA is presentWhite hybrid pigs) these three isoforms afe inducible at transcrip- in porcine liver and, to a legger extent, in kidney [75]. RT -PCRtional and activity level. In addition, the treatrnent resulted in an up- experiments bave revealed that CYP2D25 mRNA is also expressedregulation 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 Reactionsmem~er? not b~tter identified, r~sulted ~o be increased b.y.cortisol in Pig CYP2D25 was able to catalyze 25-hydroxylation ofvitaminassoclatlon ~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-hydroxylationsupport 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 humanacld-denved vasoactlve metabohtes. CYP2D6) [79]. The enzyme activity of CYP2D in pigs has been

With regard to the regulation mechanisms of CYP2Cs in pig analysed using three classical human CYP2D6 test substrates (de-very little is known. It has been suggested that the PB induction of brisoquin, bufuralol and dextromethorfan), and differences withpig CYP2Cs is primarily due to a tissue-specific activation of the human CYP2D6 bave been pointed 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 xSex 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 (770than 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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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 microsomalTolterodme 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 <?-d~~ethyl~tlon by 0.3 to 6.0 nmol/min ' mg protein whereas in humans this activity

qu.mme and qumld~e (k;nown ~uman CYP2D6 ~Ibltors) m York- varies from 1.2 to 3.1 nmol/min' mg protein. Porcme p-nitrophenol

shlfe x Landrace P!g lIv.er mlc.rosomes and prlmary hepat~c~t~s hydroxylase activity varied from 0.2 to 0.7 nmol/min ' mg protein

cultu~es. Orphenad~ne, pllocarpme and:esveratrol (CYP2B mhlbl- while the same activity ranges from 0.1 to 0.4 in humans. Further-

~or~ ~ humans, mlce and rats respectl,:ely). bave b.een sho~ ~o more, p-nitrophenol hydroxylase activity has been found at compa-

I~hlblt .dextromethorphan O-demethylatlon m Giittmgen mmlplg rable levels in porcine and human hepatocytes cultures [23]. Proba-

lIvermlcrosomes [78]. bly, pig CYP2E1 is not the only enzyme involved in the hydroxyla-

I d ' b ' l ' ty d R I ti, tion of chlorzoxazone and p-nitrophenol. Indeed, CYP2A is aIson UCI Il an egu a on d d .b th ...

[90 92] demonstrate to contrl ute to ese actlvltles , , anMonshouwer 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 humanconserved 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

Duroc crossbred domestic pig by Kojima and Morozumi [15]. A year later, CYP2E1 was aIso isolated from Giittingen minipig liver .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

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518 Current Drug Metabolism, 2011, VoL 12, No.6 Puccinelli et al.

I~liver, CYP2El activity was increased by etbanol and by isoniazide Tissue Distributionwhereas 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 extrahepaticNevertbeless, a very weak correlation has been found between tbe 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 nasal mucosa [21,58,tbe mRNA expression [14], tbus indicating tbat.tbe regulat!on.of 59,102]. These tbree isoforms bave been also found constitutivelyCYP2E I expression could take piace at ali steps m tbe protem blo- expressed in various brain regions, altbough at much lower levelssyntbesis, 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 inminipigs has suggested tbat splicing and/or modulation of mRNA porcine cortex capillaries, suggesting a protective role of CYP3Asstability 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 beenstrated to suppress hepatic CYP2EI in pubertal male pigs, probably found in mitochondria co~pared to mlc.rosomes [~O]. Thus, anthrough an increase in testicular steroid levels [54], since it has important role of CYP3As m. tbe metabollsm of br~m endogenousbeen previously observed tbat androstenone and 17f3-oestradiol substrates, such as neurosterold~ has been hypothes~zed. C.YP3A29exerted an inhibitory effect on CYP2EI enzymatic activity, aI- mRNA has also. been detected m adrenal gland,.s~m, test~s, uterustbough witb two ditTerent mechanisms [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 expressed in human ente~ocytesments, ODe of 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 ~ellovalbumin 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 androstenone was CYP3A29 ~ong tbe. mtestme has be~n o~served [102, 105], mable to inhibit tbe binding on COUP-TFI, witbout atTecting HNF-I agreement Wltb expenrnents on 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 expression by androstenone in pig hepatocytes. onstrated an interesting similarity between human CYP3A4 and

Some ditTerences bave been pointed out between humans and porcine CYP3A29. The CYP3A4 expression is very low duringpigs on CYP2El expression and activity [14]: humans and conven- foetal development and beco~es. tbe dominlmt CYP3A iso~orm intional pigs did not present sex ditTerences in chlonoxazone hy- adult liver, whereas tbe 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 IsoformL Polymorphisms (CYP3A22?) which followed tbe pattem ofCYP3A7 in foetal and

Some functional polymorphisms ofporcine CYP2EI bave been adul~ minipig sam~le~. Also expe':ÌIn;ents wi~ ~e. recombinantidentified as a possible strategy toward tbe selection of genetic porcme C~3A29 mdlcated tb~t tblS Isof~~ IS slmllar to humanmarkers used to reduçe boar taint witbout atTecting tbe levels of CYP3A4 Wltb regard to exp~sslon and aCtlvlty,. ~d ~at ~YP3A~9sexual hormones [100,101]. gives tbe greatest contrlbutlon of CYP3A actlvlty m plg hepatlc

microsomes [102].CYP3A SUBFAMILYG I F Substrates and ReactionseDera eatures .

..Testosterone 6f3-hydroxylation (tbe most wldely used markerCY~3A4 represents m hum~s tbe most Important P450 e~- reaction for human CYP3A4) and nifedipine oxidation bave been

zyme Wltb reg.ard to 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 substrates were 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 ditTerences depending onCYP3A IS ODe of tbe most. stu.dl.ed ~ubfamliles m humans, and It IS tbe breeds of pigs [14,22,24,25,31]. Testosterone 6f3-hydroxylasealso necessary to 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 (1.7CYP3A29, isolated from a pig small intestine cDN~ library ~68]. nmol/min .mg protein). Also tbe average porcine 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 of503 amino acids, which showed a very high similarity to CYPs humans (1.72 nmol/min .mg protein). These two enzyme activitiesbelonging 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-humantbe deduced N-terminai amino acid sequence of pig CYP3A29 CYP3A4 antibodies [14]. Anotber drug specific substrate ofhumanmatched very well witb tbe ODe of human CYP3A4 sharing a 60% CYP3A4 -lovastatin -has been recent\y used to validate Bamasimilarity (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, otberwhich were conservatives. Besides CYP3A29, tbere are four otber sexual hormones can be metabolized by CYP3A4, such as 17 alpha-porcine CYP3A sequences available in Genbank, named CYP3A22, etbynylestradiol (EE2) [109]. The increase in 2-hydroxylation of3A39, 3A46, and 3A88. However, CYP3A88 is likely to be an alle- EE2 after tbe exposure of porcine primary hepatocytes to variouslic 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-hydroxylation oftestosterone and witb tbe increase in76.5% amino acid sequence identities witb pig CYP3A29, 3A39, tbe protein level of CYP3A detected by a monoclonal anti-humanand 3A46, respectively [77]. Because oftbe high sequence similar- CYP3A4 antibody, tbus conflrrning tbe 2-hydroxylation ofEE2 inity between CYP3A39 and CYP3A46 (93% nucleotide .sequence pigs as being biotransformed by a CYP3A, as in humans [110].identity) tbe expression ofCYP3A39 has not yet been studled. Otber reactions known to be catalyzed mainly by CYP3As in rat

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Current Drug Metabo/ism, 2011, VoL 12, No.6 519Porcine C:vtochrome P450

and/or humans afe BQD and erythromycin demethylase (ErD) CYP4A \UBFAMILY[111,112]. While ErD activity was only detectable ~ pig liver b~. Generai Featurescause 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 acidpressed 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 Importantaffmity (Km) for testosterone than minipig CYP3A~9,.thus suggest- metabolites involved in blood flow .regul~tJon. [116]. The mosting that the domestic pig enzyme could be more slmllar to human common marker activity of CYP4As IS launc acld l~-hydroxylase,CYP3A4 than the minipig enzyme [102]. which seems to be generally conserved amongst specles [31],

So far, three CYP4A isoenzymes bave been identified in pigs:Inbibitors CYP4A21 4A24 and 4A25. Nevertheless, it is stili unclear whether

Ketoconazole and triacetyloleandomycin (TAO) (typical human CYP4A25' constitutes a different isoform or is a variant ofCYP3A4 inhibitors) were able to inhibit 6j3-hydroxylation oftestos- CYP4A24. In 2001, Lundell et al. [117] cloned CYP4A21 from pigterone and N-oxidation of nifedipine, fairly indicating that ODe or liver. The cDNA encoded a protein of 504 amino acids, whichmore CYP3A-like enzymes afe also responsible for these two reac- sbared a high similarity (74% of identity) with the humantions in pigs [22,31]. A very recent work [I 02] h~ confirmed the CYP4A Il. Later, pig CYP4A24 and 4A25 were clone~ by ~undellefficiency 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) andthan TAO. Whi1e the inhibition oftestosterone 6j3-~ydroxylatJon by about 94% identity with CYP4A21. The amino acid diff~rencesketoconazo1e has been found to be as strong as m hu~ans. [~1), between the two sequences were found in N- and C-terminai re-TAO inhibited nifedipine oxidation with.lo,:"er .potency m mml~lg gions, confined to j3-sheets 1 and 4, ~d!cating a poss.ible differencemicrosomes 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 evo1ved from a common ancestralable -both in pig and minipig microso~es -to strongl:y decrease 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 whlchCYP3A4 [72]. TAO was well able to inhibit the m,eta?oilsm ~f CYP4A21 and '4A24 revealed a sequence identity that extends be-lovastatin, further confirming the efficiency and speclficlty of thlS yond the exons.inhibitor [108],

Tissue DistributionInducibility and Regulation The expression of CYP4A21 mRNA was found in pig kidney

The expression and activities ofCYP3As bave been found to be but not in heart, muscle, intestine, spleen, thymus, 1ung, or adrena1induced in primary porcine hepatocyte and enterocy~ cultures after gland [117]. CYP4A24 and 4A25 mRNAs bave been found in bothtreatrnent 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 recent works h~ve.revealed CYP4A21 seemed to be the major liver CYP4A, whereasthat CYP3A22, 3A29, and 3A46 afe inducible at transcrlptJo?al ~d CYP4A24 has been found more abundant in kidney [118]. Theactivity 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~enbut BOt in BasaI mucosa (resplratory or olf~cto~), lung, bronchl, demonstrated in porcine coronary artenes wlth Immunoblottmgtrachea, and kidney [21,58-60]. In the small mtestme, the treatrnent experiments [121].with PB resulted in an i~~r~ase ofmRNA 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-dependent expression opposed toCYP3~29 (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 assessedbut not in cerebellum, m~dbram, and blood-~ram mte,rraces, I?dlcat- b RT -PCR experiments. CYP8B 1 was well expressed in the foetaling a differential re~ulatJo? ofthese CYP~ ~ the varlous reglons ~f s~ples but no signal was detected in weaned pig liver, determiningbrain. ~owe,:"er, no mductlon ofBQD actJvlty has been observed m the lack of cholic acid formation in adult pigs, On the contrar:;, thethe bram reglons [60]. ., .expression of CYP4A21 (an enzyme involved in ~e produ~tJo~ of

Little is known about the regulatJon mechanlsms of CYP3As m hyocholic acid) was much higher in weaned than m !oetal plg ilverpigs. In humans, the induction ofCYP3.A genes generally occurs at [120]. Certainly, further studies ar~ needed to clarlfy the mecha-transcriptionallevel through the actJvatJon of PXR or CAR and the nism underlying the shift of expresslon of these two enzymes and tocontribution of other nuclear factors (e,g. HNF4a). As a g~od ex- understand its biological meaning,pression of these nuclear receptors has been demonstrated m po~-cine liver [59], probably the induction mechanisms of .CYP3.As m Substrates and Reactionspigs afe similar to those shown in humans. The lack ofm~uctlon of Porcine CYP4A21 has been cloned and expressed in a recom-CYP3A-dependent activities 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.<>n of chenodeoxycholic acid in 6a position, leading .to th~ f~rmatJon. ofthese nuclear receptors or to th~ mvolvement of ~ther tJssu~- hyocholic acid (the species-specific primary 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~ ~ethe expression and actJvlty of CYP3~s bave ?een found m plgs or human CYP4A 11, the former showed no oo-hydroxylase actJvltyminipigs [14]. However, the castratJon of plgs has been demo,n- toward lauric acid and other fatty acids whereas the latter showedstrated 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

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520 Current Drug Metabolism, 2011, VoL 12, No.6 Puccinelli et aL

in alI previously known CYP4As, could be involved in this specific Pi~HNF4a mRNA has been detected in liver, kidney, lung, Icatalytic 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 withhydroxylase 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 presence in the other tissues.man CYP4All [118]. In particular, microsomal preparations fro~ Porcine PPARa mRNA has been found highly expressed inC':P4A24/25 tran~formed yeast cells re~ealed an ave.rage launc liver and kidney, variably expressed in small and large intestine,acld o>-hydroxyla~l.on r~te of 286 pmo~mm .mg protem, wher~as moderately expressed in heart and skeletal muscle, and barely ex-the avera~e palmluc acld o>-hydroxylauon rate 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 liverof vasai tone participating in the formation of vasoactive metabo- revealed that PPARa expression was higher in pigs than in rats andlites of arachidonic acid (in particular in the formation of the vaso- mice which, in tIInI, were reported to bave a higher expression thanconstrictor 20-RETE, 20-hydroxyeicosatetraenoic acid) [122]. In in humans [123]. A study performed using two different breeds ofporcine 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- dependent expression 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 respectgesting 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 librariesof CYP4As in pigs. An induction cocktail containing j3NF, PB and and characterized [126]. The porcine CAR gene comprised a 1047DEX had no effect on the protein level of liver CYP4As, as as- bp coding region that encodes a protein of 348 amino acids. Thesessed with anti-rat CYP4Al/3 polyclonal antibodies, although the mRNA sequence of 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 andfrom treated pigs compared with controls (study performed using PXR proteins showed a high degree of sequence identity in theirLandrace 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 respect to human ones. Instrong inducer of CYP4As in mice and rats through the activation particular, porcine PXR has tumed out to be more similar to humanof PPARa, failed to induce the mRNA expression of hepatic PXR than mouse PXR (87% vs. 77% identity) and to contain theCYP4As [123]. The unresponsiveness of 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-

cession n. DQ061106), encodes a protein of 474 aa with about 75%NUCLEAR RECEPTORS homology to that of human HNF-4a [132]. The cDNA containingGeneraI 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 aaway through the action of nuclear receptors acting as transcription showed. a hlg~ Idenuty wlth ~e human counterpart (92.3 'lo): .Anfactors. In humans, members ofCYPI, 2,3, and 4 families afe pri- altemauv~ spllced mRNA lac~mg exon 5 (presu~ably determm~gmarily regulated by AhR, CAR, PXR, and PPARa, respectively. a. C-termInai truncated. protem) was detected. ~ several. porcIneRowever, the regulation mechanisms afe complicated by the in- ussues, but ~rth~r studles ar.e needed to ascertam Its potenual effectvolvement 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 genes ofthese receptors. tion response ofporcine CAR by CITCO (a specific agonist ofhu-

man CAR) and other nine ligands out oftwelve was sirnilar to theExpression response of human CAR. On the other hand, for the murine CAR

The mRNA expression ofboth 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 greatest expression in liver followed by kidney [21, 58-60, loxy)benzene) (a specific murine CAR ligand) had no effects on125,126]. It is worth noticing that high levels ofmRNA expression CAR-regulated activities, thus suggesting that porcine CAR is morebave 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, alongpossible role in the regulation of genes involved in physiological with their target genes, has been investigated in many pig tissues byfunctions 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 LargeNorthem blot analysis bave shown a fair PXR expression in pig White domestic pigs), the nuclear receptors mRNA levels were notheart, colon, and stomach, while a minor expression has been ob- affected by the treatrnent, in agreement with 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 sequenced so the abundant expression of the nuclear receptors in this organo Infar [17], has been found expressed at transcriptionallevel in liver, the extrahepatic tissues, the regulation of the same genes is proba-lung, heart, kidney, brain, ovary, thyrocytes, artery endothelial bly more complex and lirnited by either the lower levels of thesecells, olfactory and respiratory nasal mucosa [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

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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 I h dr b t.= ry y ocar on recep or

mental exogenous compounds encountered by each specles. Only one nonsynonymous SNP (SI78L) has been found in the pig PXR 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 .. dr t= onstltutlve an ostane recep or

In VIVO. In partlcular, SV2 has been found to slgruficantly decreasçthe 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 modelfor 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- -Df . h b " d . I ..DEX -exametazone

ever, a note o cautlon as to e put lorwar m re atlon to a StrlCtfunctional 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. IIt 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 .b I. fili dru . h A th al ... 1 .-Igan -m mg omaln meta o Ism o ese gs m umano s to e cat ytlc slml arlty

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, epoxide hydrolase or aldehyde dehydrogenase), for which PXR - P X1 I.. d ." ... 1 bl . I. AI . d h -regnane receptoron y Imlte mlormatlon IS aval a e m Iterature. so, an m- eptexamination 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

SNP = Single nucleotide polyrnorphisrns 'z'imes in various brain regions of pig. Toxicology, 2009, 265, 69-SRS = Substrate recognition site 79. .

..[20] Manm, S.; Longo, V.; Mazzaccaro, A.; Gervasi, P.G. XenobioticSV = Spllce varlants metabolizing enzymes in pig nasal and hepatic tissues. Xenobiotica,

TAO = Triacetyloleandornycin 1998}8(10),923-935: ..TCP [21] Messina, A.; Nannelll, A.; Flono, R.; Longo, V.; Gervasi, P.G.

OBOP -1,4-BIs-[2-(3,5-dlchloropyndyloxy)] ben- Expression and inducibility of CYPIAI IA2 IBI by jJ-~ne,3,~',5,5'-Tetrachloro-l,4- naphthoflavone and CYP2B22, 3A22, 3A29 3A46 by rifampicin inblS(pyndyloxy)benzene the respiratory and olfactory mucosa ofpig. Toxicology, 2009, 260,

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Rece;ved: January 31, 2011 Rev;sed: Marcb 22, 2011 Accepted: Marcb 23, 2011