Alterations in drug metabolizing activities in acute hepatosteatosis induced by intake of a high-carbohydrate/fat-free diet after food deprivation

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1 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 59 of 69 Altertions in drug metolizing ctivities in cute heptostetosis induced y intke of high-crohydrte/ft-free diet fter food deprivtion Chul Won Ahn 1, Jung Ah Lee 1, Je Hk Prk 2, Young Chul Kim 1 1 College of Phrmcy, Seoul Ntionl University, Sn 56-1 Shinrim-Dong, Kwnk-Ku, Seoul, Kore; 2 College of Veterinry Medicine, Seoul Ntionl University, Sn 56-1 Shinrim-Dong, Kwnk-Ku, Seoul, Kore Corresponding uthor: Young C. Kim, Professor of Toxicology, College of Phrmcy, Seoul Ntionl University, Sn 56-1 Shinrim-Dong, Kwnk-Ku, Seoul , Kore. Sumission dte: Decemer 23, 2015, Acceptnce dte: Jnury 29, 2016: Puliction dte: Jnury 30, 2016 ABSTRACT Bckground: Lipid ccumultion in heptocytes constitutes mjor component in the pthogenesis of chronic liver injury. However, the impct of lipid deposition in liver on drug metolizing cpcity remins uncler. Purpose of the study: The present study ws undertken to evlute the chnges in heptic cytochrome P-450 (CYP) enzymes in cute heptostetosis. Methods: The rt sujects fsted for 48 h, nd then were provided with highcrohydrte/ft-free diet (FH) or norml diet (FN) for 48 h. Results: Heptic lipid ccumultion ws significnt in the FH group compred to the FN group. In the FN group, there ws smll increse in microsoml p-nitrophenol hydroxylse, p- nitronisole O-demethylse, nd erythromycin N-demethylse ctivities. In contrst, minopyrine N-demethylse ctivity significntly decresed. However, the microsoml enzyme ctivities were ll inhiited y FH intke. Immunolotting nlysis reveled tht CYP2E1, CYP1A, CYP3A, nd CYP2B1/2 proteins decresed in the FH group. Expression of corresponding CYP mrnas ws lso down-regulted. A dose of CCl 4, CYP2E1 sustrte, ws dministered to rts fed with different diets. The liver injury ws significntly lower in the FH

2 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 60 of 69 group compred to the FN group, s determined y the elevtion of serum enzyme ctivities nd histopthologicl exmintion. Conclusions: The results reveled tht cute heptostetosis my result in significnt ltertion in heptic CYP-medited metolizing cpcity, which cn precipitte errtic responses of liver to vrious endogenous nd exogenous sustnces. Key words: heptostetosis, cytochrome P450, non-lcoholic ftty liver disese, drug metolizing cpcity INTRODUCTION Heptostetosis, or ftty liver, is the most common histopthologicl chnge in liver disorders. Ftty liver cn result from ingestion of lcohol nd certin drugs, including corticosteroids, some ntiiotics nd nonsteroidl nti-inflmmtory gents [1]. Additionlly, ecuse of its high prevlence in oesity, dietes nd insulin resistnce, non-lcoholic ftty liver disese (NAFLD) is incresingly pprecited s heptic mnifesttion of the metolic syndrome [2,3]. It is suspected tht the ccumultion of lipid in liver, regrdless of its etiology, would modulte the expression nd ctivity of vrious drug metolizing enzymes. The metolic deposition of drugs nd chemicl sustnces my then e influenced due to the incompetency of heptic drug metolizing cpcity, resulting in ltered phrmcologicl efficcy or dverse drug rections. In fct, chnges in phrmcokinetics of vrious drugs were seen within oese ptients [4,5]. CCl 4 is model heptotoxicnt for free rdicl-induced liver injury. CCl 4 is ioctivted to trichloromethyl free rdicl vi CYP-dependent reductive dehlogention, in which CYP2E1 plys mjor role [6-8]. The trichloromethyl free rdicl inds covlently to lipids nd proteins, resulting in memrne dmge nd inhiition of protein functions. Moreover, this rdicl ttcks polyunsturted ftty cids, generting orgnic free rdicls which my rect with oxygen to form peroxides nd other cytotoxic metolites [9]. Therefore, it is ccepted tht CYP2E1 ctivity is key contriuting fctor in inititing the CCl 4 induced liver toxicity. Erlier studies showed tht liver ftty cid synthesis is enhnced y dietry crohydrtes fter short term fsting. For exmple, within rts who were fed high-crohydrte diet fter eing deprived of food for 2 dys, the lipid synthesis nd triglyceride levels in the liver were incresed [10]. Both circulting nd de novo synthesized ftty cids in liver were implicted in

3 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 61 of 69 the disruption of heptic lipid homeostsis, leding to lipid ccumultion in the livers of the rts who fsted efore eing introduced to high-crohydrte/ft-free diet [11]. In this study, we exmined the chnges in the CYP enzyme system nd CCl 4 toxicity in cute heptostetosis. Since cute ftty liver induced y feeding rts with high-crohydrte/ft-free diet fter food deprivtion my void other complictions ssocited with the progression of chronic liver injury, it is nticipted tht the use of this model cn revel clerer picture of the consequences of ft ccumultion in liver. METHODS Animls nd tretments: Mle Wistr rts, weighing g, were purchsed from Orient- Bio Experimentl Animls (Seongnm, Kore). The use of nimls ws in complince with the guidelines estlished y the Animl Cre Committee of this institute. Rts were cclimted to temperture (22 ± 2 C) nd humidity (55 ± 5 %) controlled rooms with 12-h light/drk cycle (light: , drk: ) for 1 wk efore use. Rts were provided with stndrd regulr diet (Crgill Agri Purin, Seongnm, Kore) efore food deprivtion for 48 h. After the fsting period rts were fed regulr diet (FN) or high-crohydrte/ft-free diet (FH; G-Bio, Gwchon, Kore) for 48 h until scrifice for mesurement of the CYP enzyme system. Different groups of rts were chllenged with dose of CCl 4 (4 mmol/kg, ip) t 48 h fter refeeding the rts for mesurement of liver injury. The stndrd regulr diet contined 64.1 % crohydrte, 20 % protein, 6 % fier, 4.5 % ft with pproprite minerl slts nd vitmins. The highcrohydrte/ft free diet ws mde up of 80 % crohydrte (sucrose nd corn strch, 1:1), 16 % protein, 0 % ft, in ddition to pproprite minerl slts nd vitmins. Mesurements of liver injury: The heptotoxicity of CCl 4 ws estimted y elevtion of serum enzyme ctivities. At 24 h fter CCl 4 tretment, lood ws smpled from dominl ort in rts under light ether nesthesi. Serum soritol dehydrogense (SDH) ws determined spectrophotometriclly [12]. Asprtte minotrnsferse (AST) nd lnine minotrnsferse (ALT) ctivities were determined using the method of Reitmn nd Frnkel [13]. Thiorituric cid rective sustnces (TBARS) were mesured to quntify lipid peroxidtion in liver [14]. For evlution of lipid ccumultion in liver, sections of frozen liver were sliced t 10 μm, immersed in propylene glycol for 5 min, nd stined with Oil red O for 7 min. After rinsing with 85 % propylene glycol nd distilled wter, the sections were counterstined with hemtoxylin for

4 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 62 of 69 2 min efore microscopic exmintion. For histopthologic ssessment of liver injury, the left lterl loe of liver ws sliced nd fixed in 10 % neutrl-uffered formlin. Tissues were processed routinely nd emedded in prffin. Sections of 2-3 µm in thickness were sujected to hemtoxylin nd eosin stining efore microscopic exmintion. Chnges in CYP ctivities: Liver ws homogenized in n ice-cold uffer of M KCl/50 mm Tris-HCl with 1 mm EDTA, ph 7.4. The homogente ws centrifuged t 10000g for 20 min, nd the superntnt ws further centrifuged t g for 60 min. The microsoml pellet ws suspended in the homogenizing medium, followed y recentrifugtion t g for 60 min. The finl pellet otined ws resuspended nd used for mesurement of microsoml CYP ctivities. Heptic microsoml CYP ctivities were determined using the method descried elsewhere [15]. P-nitrophenol hydroxylse ctivity ws determined y mesuring the formtion of p-nitroctechol. Rection mixtures (finl volume of 1 ml) contined 0.1 mm p-nitrophenol, 1.0 mm scoric cid, 0.1 ml of microsoml suspension nd 1 mm NADPH in 0.1 M potssium phosphte uffer (ph 6.8). Incution ws conducted t 37 C for 3 min. Aminopyrine N- demethylse, erythromycin N-demethylse nd p-nitronisole O-demethylse ctivities were determined y mesuring the production of formldehyde or p-nitrophenol. Rection mixtures consisted of 0.1 ml microsoml suspension, 1 mm NADPH, the sustrte (5 mm for minopyrine or erythromycin; 0.1 mm for p-nitronisole) nd 0.1 M potssium phosphte uffer (ph 7.4), in totl volume of 1.0 ml. Incution ws conducted t 37 C for 10 min, efore the ddition of cold 20 % trichlorocetic cid to stop the rections. Immunolotting nd gene expression nlysis: Liver microsoml protein (20 μg) ws loded, seprted y gel electrophoresis, nd trnsferred to nitrocellulose memrnes y electrolotting. The memrnes were locked in Tris-uffered sline contining 5 % nonft dry milk nd 0.1 % Tween-20. The lots were incuted with primry ntiodies t 4 C overnight. Secondry ntiodies were then conjugted to horserdish peroxidse t room temperture for 1 h. Monoclonl ntiodies ginst rt CYP1A1/2, CYP2B1/2, nd polyclonl ntiodies ginst CYP2E1, CYP3A (Detroit R&D, Detroit, MI) were used s proes. Blots were detected y enhnced chemiluminescence. Signl intensity ws normlized reltive to tht of β-ctin. Gene expression ws exmined y quntittive rel-time PCR. Totl heptic RNA ws isolted using TRIzol regent (Invitrogen, Crlsd, CA) nd purified y n RNesy Mini Kit

5 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 63 of 69 (Qigen, Hilden, Germny). cdna ws otined from isolted RNA templte using reverse trnscription. Gene expression ws mesured using cdna with corresponding gene-specific primers nd Light-Cycler System (Roche Dignostic, Mnnheim, Germny), following the mnufcturer s stndrd protocol. The reltive expression ws normlized to tht of glycerldehyde-3-phospte dehydrogense (GADPH). The gene-specific primers pplied were s follows: CYP2E1 sense, 5 -TCTGAGATATGGGCTCCTGA-3, nti-sense, 5 - CGGGGAATGACACAGAGTTT-3 ; CYP3A1 sense, 5 -AGAAATTCGATGTGGAGTGC-3, nti-sense, 5 -AGGTTTGCCTTTCTCTTGCC-3 ; CYP3A2 sense, 5 -GCTTTCAGC TC TCACACTGGAA-3, nti-sense, 5 -TCTATGGGTTCCAAGTCGGTAGA-3 ; CYP1A2 sense, 5 -GGGTGTTGAGAGGCACAAGG-3, nti-sense, 5 -CACTAGGGCCTGCTTGAT GG-3 ; CYP2B1/2 sense, 5 -CCCAATGTTTGGTGGAGGAA-3, nti-sense, 5 -CTGTGAT GCACTGGAAGAGGAA-3 ; GAPDH sense, 5 -GACCCCTTCATTGACCTCAAC-3, ntisense, 5 -GGAGATGATGACCCTTTTGGC-3. Sttisticl nlysis: All results were expressed s the men ± SEM. Mens of the different diet groups were compred using one-wy ANOVA followed y Newmn-Keuls multiple rnge test. The cceptle level of significnce ws estlished t P < RESULTS AND DISCUSSION It hs een known tht diet composition following food deprivtion independently mnipultes heptic ft content in rts [16,17]. In prticulr, the feeding of high crohydrte/ft-free diet fter strvtion in rts ws demonstrted to result in the sequentil ccumultion of triglyceride in their liver tissue. 6 h fter refeeding, slight microvcuolr stetosis, minly loclized in zone I, ws followed fterwrds y microvcuolr stetosis, which extended to ll three zones of heptic loules [11]. Within tht study, the uthors proposed tht fsting-refeeding high crohydrte/ft-free diet could constitute good model of dietry-induced stetosis without pprent necroinflmmtion. The present study, which utilized the sme fsting-feeding schedule, reveled tht triglyceride ccumultion in liver incresed significntly within the rts fed FH fter 2 dy fsting when compred with the rts fed FN fter fsting (Fig. 1A). Histopthologicl exmintion of liver smples lso demonstrted similr results (Fig. 1B). Lipid ccumultion in liver ws mrkedly incresed in the FH group, when exmined fter Oil red O stining. But there ppered to e no significnt inflmmtory rections in the liver of rts fed

6 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 64 of 69 FH or FN fter 2 dy food deprivtion. The histologicl chnges oserved in the rts fed FH fter food deprivtion suggest tht this rpid dietry mnipultion could serve for suitle model of dietry-induced cute stetosis. Fig. 1. Lipid ccumultion in rt liver. (A) Heptic triglyceride levels in rts. Ech Vlue represents the men ± SE for 6 or 7 rts. Vlues with different letters (, ) re significntly different one from nother (one-wy ANOVA followed y Newmn-Keuls multiple rnge test, P < 0.05). (B) The liver smples were stined with Oil red O for microscopic exmintion. N, norml control; FN, refeeding regulr diet fter fsting; FH, refeeding high crohydrte/ft-free diet fter fsting. Two dys of fsting resulted in smll chnge in the CYP ctivities in liver (Tle 1). Activities of p-nitrophenol hydroxylse, p-nitronisole demethylse, nd erythromycin N- demethylse were induced slightly, significntly within the rts who fed norml diet fter fsting (FN group), rther thn the rts who were fed norml diet without feeding interruption. In contrst, minopyrine N-demethylse ctivity ws decresed in the FN group compred to the group who hd regulr feeding. However, intke of FH fter fsting significntly inhiited ll the CYP enzyme ctivities determined in this study. The results of immunolotting nlysis showed tht expression of CYP proteins ws not ffected y FN intke fter fsting, except for CYP1A which ws decresed slightly (Fig. 2). Nonetheless, in the FH group, the expression of CYP2E1, CYP3A, CYP1A, nd CYP2B1/2 proteins ws significntly lower thn tht found in the FN

7 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 65 of 69 group. Similr chnges were shown in the CYP mrna expression (Fig. 3). Deprivtion of food for 2 dys followed y FN intke did not ffect the expression of CYP2E1, CYP3A2, CYP1A2, or CYP2B1/2 mrnas, ut decresed tht of CYP3A1 mrna. FH intke fter fsting depressed the expression of ll the CYP mrnas mesured mrkedly. Tle 1. Reltive enzyme ctivities of CYP isoforms in the liver of rts fter feeding of experimentl diets p-nitrophenol p-nitronisole Erythromycine Aminopyrine Group Hydroxylse Demethylse N-Demethylse (nmoles/min/g liver) N-Demethylse N 9.17 ± ± ± ± 3.60 FN ± ± ± ± 1.88 FH 7.61 ± ± ± 3.76 c ± 2.84 Ech Vlue represents the men ± SE for 6 or 7 rts. Vlues with different letters (,, c) re significntly different one from nother (one-wy ANOVA followed y Newmn-Keuls multiple rnge test, P < 0.05). Fig. 2. Expression of CYP proteins in liver. Vlues with different letters (,, c) re significntly different one from nother (onewy ANOVA followed y Newmn-Keuls multiple rnge test, P < 0.05). CYPs / GAPDH (% of control) CYP2E1 CYP3A1 CYP3A2 CYP1A2 CYP2B1/2 Fig. 3. Expression of CYP mrna in liver. Ech vlue represents the men ± SE for 6 or 7 rts. Vlues with different letters (, ) re significntly different one from nother (one-wy ANOVA followed y Newmn- Keuls multiple rnge test, P < 0.05). N FN FH

Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 66 of 69 Serum enzyme ctivities (% control) 800 600 400 200 0 C N FN FH c AST ALT SDH c c Fig. 4. Elevtion of serum enzyme ctivities fter CCl 4 dministrtion.

8 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 66 of 69 Serum enzyme ctivities (% control) C N FN FH c AST ALT SDH c c Fig. 4. Elevtion of serum enzyme ctivities fter CCl 4 dministrtion. Ech Vlue represents the men ± SE for 5 rts. Vlues with different letters (,, c) re significntly different one from nother (one-wy ANOVA followed y Newmn-Keuls multiple rnge test, P < 0.05). C, control rts under regulr feeding without CCl 4 dministrtion. Fig. 5. Histopthologicl evlution of liver tissue fter CCl 4 tretment. Hemtoxylin nd eosin stining. See the text for detils. C, control rts under regulr feeding without CCl 4 dministrtion. Numerous studies hve shown tht heptic ft ccumultion results in significnt ltertions in the expression nd ctivity of CYP enzymes. However, the results compiled from different studies show lck of consistency, which could e ttriuted to difference in species, sex, nd

9 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 67 of 69 most importntly the mgnitude or severity of liver stetosis [18]. In generl, CYP3A4 expression, s well s its ctivity, decresed in ftty liver [19,20]. Reduction of other CYPs, including CYP1A2, CYP2C11 nd CYP2A1/2, ws lso demonstrted in ssocition with ft ccumultion in liver [21-23]. In contrst, CYP2E1 incresed in rodent non-lcoholic stetoheptitis (NASH) models [24], wheres reduction in CYP2E1 expression ws lso oserved in heptic stetosis with lck of inflmmtion [20]. Therefore, it is speculted tht the induction of CYP2E1 in NASH results from complictions ssocited with the inflmmtory responses, rther thn ft ccumultion itself. The present results indicte tht CYP enzymes nd ctivities re mostly reduced in cute heptostetosis with miniml to no inflmmtory rections. Tretment of the rts with dose of CCl 4, CYP2E1 sustrte, resulted in n elevtion of serum AST, ALT, nd SDH ctivities when determined 24 h lter (Fig. 4). Similr levels of serum enzyme ctivities were noted in the FN group chllenged with CCl 4. However, in the FH group the elevtion of serum enzyme ctivities ws significntly less thn tht shown in the FN group. Histopthologicl exmintion of liver smples showed comprle results (Fig. 5). Mrked llooning degenertion, ftty chnge, nd Councilmn odies were present in heptocytes of the rts fed FN fter fsting nd the rts who hd regulr feeding. There were inflmmtory rections, including migrtion of neutrophils nd infiltrtion of mononucler leukocytes. The inflmmtory rections nd ftty infiltrtion in heptocytes ppered to e more severe in the rts fed FN fter fsting thn in the rts who hd regulr feeding. However, there ws no infiltrtion of leukocytes seen in heptocytes of the rts fed FH fter fsting. Additionlly, mild llooning degenertion ws present in few heptocytes. Menwhile, lipid peroxidtion estimted y TBARS level ws not ltered y intke of the different diets (dt not shown). The results suggest tht the reduction of the CCl 4 heptotoxicity is ttriutle to down-regultion of the CYP2E1 enzyme nd its ctivity ssocited with heptic ft ccumultion. CONCLUSIONS The present results indicte tht refeeding high crohydrte/ft-free diet fter food deprivtion is effective in inducing ftty chnges in heptocyte with miniml or no inflmmtory responses. As result, in the stetotic livers which were induced, expression of the CYP enzymes ws mrkedly reduced. Furthermore, dose of CCl 4 ppered to e less heptotoxic in the rts fed FH fter fsting thn in the rts fed FN fter fsting. Therefore, it cn e concluded tht the prevention of CCl 4 toxicity in cute heptostetosis is ttriuted to the inhiition of metolic

10 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 68 of 69 ctivtion of this solvent, due to the down-regultion of CYP-dependent drug metolizing enzymes. List of revitions: ALT, lnine minotrnsferse; AST, sprtte minotrnsferse; CYP, cytochrome P-450; FN, stndrd regulr diet; FH, high-crohydrte/ft-free diet; NAFLD, non-lcoholic ftty liver disese; SDH, serum soritol dehydrogense; TBARS, thiorituric cid rective sustnces. Competing interests: The uthors declre tht there re no finncil or other conflicts of interest. Acknowledgements nd Funding: This work ws supported y Ntionl Reserch Foundtion (NRF) grnts (No R1A2A1A nd No ) funded y the Ministry of Eduction, Science nd Technology (MEST), Kore. REFERENCES 1. Burt AD, Mutton A, Dy CP: Dignosis nd interprettion of stetosis nd stetoheptitis. Semin Dign Pthol 1998, 15: Frrell GC, Lrter CZ: Nonlcoholic ftty liver disese: from stetosis to cirrhosis. Heptology 2006, 43(2 Suppl 1):S99-S Yeh MM, Brunt EM: Pthology of nonlcoholic ftty liver disese. Am J Clin Pthol 2007, 128: Edelmn AB, Crlson NE, Cherl G, Munr MY, Stouffer RL, Cmeron JL, Stnczyk FZ, Jensen JT: Impct of oesity on orl contrceptive phrmcokinetics nd hypothlmic-pituitry-ovrin ctivity. Contrception 2009, 80: Skouy SO: Hormonl contrception in oesity, the metolic syndrome, nd dietes. Ann NY Acd Sci 2010, 1205: Rucy JL, Krner JC, Lsker JM: Bioctivtion of hlogented hydrocrons y cytochrome P4502E1. Crit Rev Toxicol 1993, 23: Avsrl S, Yng L, Sun Y, Leung AW, Chn WY, Cheung WT, Lee SS: A temporl study on the histopthologicl, iochemicl nd moleculr responses of CCl(4)-induced heptotoxicity in Cyp2e1-null mice. Toxicology 2006, 228: Reckngel RO, Glende EA Jr, Dolk JA, Wller RL: Mechnisms of cron tetrchloride toxicity. Phrmcol Ther 1989, 43: Pl GL; Chlorinted methnes nd liver injury: highlights of the pst 50 yers. Annu Rev Phrmcol Toxicol 2000, 40: Iritni N, Nishimoto N, Ktsurd A, Fukud H: Regultion of heptic lipogenic enzyme gene expression y diet quntity in rts fed ft-free, high crohydrte diet. J Nutr 1992, 122:28-36.

11 Functionl Foods in Helth nd Disese 2016; 6(1):59-69 Pge 69 of Delzenne NM, Hernux NA, Tper HS: A new model of cute liver stetosis induced in rts y fsting followed y refeeding high crohydrte-ft free diet. J Heptol 1997, 26: Gerlch U: Soritol dehydrogense. In Methods of Enzymtic Anlysis. Edited y Bergmeyer HU. Weinheim: Verlg Chemie; 1983: Reitmn S, Frnkel SK: A colorimetric method for the determintion of serum glutmic oxlcetic nd glutmic pyruvic trnsminses. Am J Clin Pthol 1957, 28: Germnò MP, De Psqule R, D'Angelo V, Ctni S, Silvri V, Cost C: Evlution of extrcts nd isolted frction from Cppris spinos L. uds s n ntioxidnt source. J Agric Food Chem 2002, 50: Kim YC, Yim HK, Jung YS, Prk JH, Kim SY: Heptic injury induces contrsting response in liver nd kidney to chemicls tht re metoliclly ctivted: role of mle sex hormone. Toxicol Appl Phrmcol 2007, 223: Kim TS, Freke HC: High crohydrte diet nd strvtion regulte lipogenic mrna in rts in tissue-specific mnner. J Nutr 1996, 126: Mittendorfer B, Jeschke MG, Wolf SE, Sidossis LS: Nutritionl heptic stetosis nd mortlity fter urn injury in rts. Clin Nutr 1998, 17: Merrell MD, Cherrington NJ: Drug metolism ltertions in nonlcoholic ftty liver disese. Drug Met Rev 2011, 43: Kolwnkr D, Vupplnchi R, Ethell B, Jones DR, Wrighton SA, Hll SD, Chlsni N: Assocition etween nonlcoholic heptic stetosis nd heptic cytochrome P-450 3A ctivity. Clin Gstroenterol Heptol 2007, 5: Leclercq I, Horsmns Y, Desger JP, Delzenne N, Geuel AP: Reduction in heptic cytochrome P-450 is correlted to the degree of liver ft content in niml models of stetosis in the sence of inflmmtion. J Heptol 1998, 28: Donto MT, Lhoz A, Jiménez N, Pérez G, Serrlt A, Mir J, Cstell JV, Gómez-Lechón MJ: Potentil impct of stetosis on cytochrome P450 enzymes of humn heptocytes isolted from ftty liver grfts. Drug Met Dispos 2006, 34: Murry M, Cntrill E, Meht I, Frrell GC: Impired expression of microsoml cytochrome P450 2C11 in choline-deficient rt liver during the development of cirrhosis. J Phrmcol Exp Ther 1992, 261: Su GM, Sefton RM, Murry M: Down-regultion of rt heptic microsoml cytochromes P-450 in microvesiculr stetosis induced y orotic cid. J Phrmcol Exp Ther 1999, 291: Weltmn MD, Frrell GC, Liddle C: Incresed heptocyte CYP2E1 expression in rt nutritionl model of heptic stetosis with inflmmtion. Gstroenterology 1996, 111:

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