Histomorphological changes and cytochrome P450 isoforms expression and activities in precision-cut liver slices from neonatal rats

Precision-cut rat liver slices are a widely accepted in vitro tool for the examination of drug metabolism, enzyme induction or hepatotoxic effects of xenobiotics. After prolonged incubation, however, distinct histopathological changes and increasing losses in function are seen with liver slices from adult animals. Since tissue from neonatal animals is expected to be less vulnerable, in the present study liver slices from 1-day-old rats were examined for morphological changes and for the expression of different cytochrome P450 (CYP) isoforms after incubation for up to 24 h and after a 24 h in vitro exposure to beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or pregnenolone 16alpha-carbonitrile (PCN). In parallel, CYP activities were assessed by different model reactions in slice homogenates and in intact slices. Histopathological changes were less pronounced in liver slices from 1-day-old rats than in those from adult animals. During the 24 h of incubation even a maturation of the tissue occurred, since the proportion of haemopoietic stem cells declined and the glycogen content of the hepatocytes increased. The CYP expression pattern after 2 and 24 h of incubation was similar to that of normal liver specimens from neonatal rats showing a moderate CYP1A1, 2B1 and 3A2 expression. The immunostaining for CYP1A1 and 2B1 was elevated after incubation with BNF. PB enhanced CYP2B1 and 3A2 expression, and DEX and PCN increased CYP3A2 immunostaining. This induction pattern was paralleled by respective effects on the corresponding model reactions. Thus, besides increased viability, slices from neonatal rats are excellently suited for the evaluation of an in vitro induction of CYP enzymes as well.     

Cytochrome p450 expression of cultured rat small hepatocytes after long-term cryopreservation.

Small hepatocytes (SHs) are hepatic progenitor cells that can be cryopreserved for a long time. After thawing, the cells can proliferate and, when treated with Matrigel, they can differentiate into mature hepatocytes (MHs). In this study, we investigated whether cryopreserved SHs could express cytochromes P450 (P450s), whether P450 expression was induced by appropriate inducers, and whether P450 activities were measurable. 3-Methylcholanthrene (3-MC), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and ethanol were used as inducers for CYP1A, 2B, 3A, and 2E, respectively. Immunoblot analysis indicated that cryopreserved SHs constitutively expressed CYP1A1/2, CYP2E1, and CYP3A2 as much as 26 days after plating. Significant expression of CYP1A1/2 and 3A2 in the cells treated with Matrigel was induced by 3-MC and PCN, respectively. Although Matrigel did not up-regulate the enzymatic activity of CYP1A, CYP3A and CYP2E activities increased. Induction of CYP1A and CYP3A activities by each inducer was observed in cryopreserved cells treated with Matrigel. Although the expression of CYP2B1 could be detected in subcultured SHs treated with PB, it was not detected in cryopreserved SHs. The activity of NADPH-cytochrome P450 reductase was measured in both subcultured and cryopreserved SHs, although the activities in both were approximately 30% of that of MHs. Profiles of (14)C-testosterone metabolites were examined in cultured MHs and in cryopreserved SHs by high-performance liquid chromatography. Similar peaks for testosterone metabolites in MHs and SHs were observed in the same elution time. These results indicate that, although induction of CYP3A and 2B in cryopreserved SHs is inferior to that in subcultured ones, SHs can maintain the expression and activities of P450s after long-term cryopreservation.     

Cytochrome P450-Mediated Metabolism and Nephrotoxicity of N-(3,5-Dichlorophenyl)succinimide in Fischer 344 Rats

The agricultural fungicide N-(3,5-dichlorophenyl)succinimide(NDPS) is nephrotoxic in rats. Previous studies have suggestedthat oxidative hepatic biotransformation is required for theinduction of kidney damage. The experiments described in thispaper were designed to further investigate the relationshipbetween NDPS metabolism and nephrotoxicity using various modulatorsof cytochrome P450 activity. Male Fischer 344 rats were pretreatedwith the P450 inducers Aroclor 1254 (ARO), isoniazid (INH),3-methylcholanthrene (3-MC), and phenobarbital (PB), or theP450 inhibitor 1-aminobenzotriazole (ABT). Control animals receivedvehicle only. NDPS metabolism was investigated using hepatocytesisolated from the various treatment groups. Separate experimentswere also conducted to evaluate the effects of these pre-treatmentson NDPS-induced nephrotoxicity in rats. PB and ARO enhancedformation of the known nephrotoxic NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide,N-(3,5-dichloro-phenyl)2-hydroxysuccinamide acid, and iV-(3,5-dichlorophenyl)-3-hydroxysuccinamicacid, by the hepatocytes. In contrast, ABT inhibited formationof the nephrotoxic metabolites, whereas INH and 3-MC did notalter NDPS biotransformation. NDPS-induced renal damage waspotentiated by pretreating the rats with PB or ARO and was attenuatedby ABT. Compared with control animals, toxicity was unaffectedby INH or 3-MC pretreatments. Thus, there was a correlationbetween pretreatments that induce P450-mediated NDPS metabolismand the effects that these compounds have on NDPS-induced nephrotoxicity.The data indicate that specific P450 isozymes metabolize NDPSto its hydroxylated products and suggest that these metabolitesmediate the nephrotoxicity induced by NDPS.     

Induction of multidrug resistance protein 3 (mrp3) in vivo is independent of constitutive androstane receptor.

We previously demonstrated that multidrug resistance protein 3 (Mrp3/ABCC3) is induced in rat liver by phenobarbital (PB) and several other microsomal enzyme inducers that induce cytochrome P450 2B (CYP2B). CYP2B is induced by constitutive androstane receptor (CAR)-retinoid X receptor (RXR) heterodimer binding to a phenobarbital-responsive promoter element in the CYP2B promoter. Hepatic mRNA levels of CYP2B and Mrp3 were measured in three models of altered CAR activity to determine whether CAR is also involved in the induction of Mrp3. In Wistar Kyoto rats, where males express higher CAR protein levels than females, the induction of CYP2B1/2 was significantly higher in males than in females by PB, diallyl sulfide, and trans-stilbene oxide but not oltipraz. Mrp3 was induced by each of these treatments, but in contrast to CYP2B1/2, to a similar magnitude in males and females. In male hepatocyte-specific RXRalpha-/- mice, CYP2B10 was not induced by diallyl sulfide or oltipraz but remained inducible by PB and trans-stilbene oxide after considering the decrease in basal CYP2B10 expression. Mrp3, however, was induced by PB, diallyl sulfide, trans-stilbene oxide and oltipraz in both wild-type and RXRalpha-/- mice. Additionally, constitutive expression of Mrp3 was significantly reduced in RXRalpha-/- mice. In CAR-/- mice, the robust induction of CYP2B10 by PB was completely absent. However, Mrp3 was equally induced both in wild-type and CAR-/- mice by PB. These data clearly demonstrate that induction of hepatic Mrp3 by PB and other microsomal enzyme inducers is CAR-independent and implies a role for RXRalpha in the constitutive expression of Mrp3.     

Phenobarbital, beta-naphthoflavone, clofibrate, and pregnenolone-16alpha-carbonitrile do not affect hepatic thyroid hormone UDP-glucuronosyl transferase activity, and thyroid gland function in mice

The effects of representative liver enzyme inducers such as clofibrate (CLO), phenobarbital (PB), pregnenolone-16alpha-carbonitrile (PCN), and beta-naphthoflavone (NF) on hepatic microsomal thyroxin (T4)- UDP-glucuronosyl transferase (UGT) and triiodothyronine (T3)- UGT activities and thyroid function were evaluated in OF-1 male mice after a 14-day po administration. CLO, PB, and PCN induced histological liver hypertrophy, increases in liver weights, in microsomal protein and cytochrome P450 contents as well as increases in specific UGT activities. Despite this, no significant changes in T4-UGT and T3-UGT activities occurred after treatment by any of these compounds. Furthermore, no significant changes in serum T4 and T3 levels were observed and thyroid histology was not affected. NF treatment induced microvacuolation of hepatocytes but did not affect any of the other tested parameters. The results show that, in contrast to the widely described effects in rats, liver enzyme inducers do not affect hepatic thyroid hormone metabolism and thyroid function in mice, suggesting that this species should be less sensitive to thyroid tumor promotion by hepatic microsomal enzyme inducers than rats.     

Ethylmorphine N-demethylase activity as a marker for cytochrome P450 CYP3A activity in rat hepatic microsomes

The purpose of this study was to evaluate the selectivity and sensitivity of ethylmorphine N-demethylase (EMD) as an indicator of chemically-induced cytochrome P450 CYP3A activity in liver microsomes of rats following treatment with selective enzyme inducers. Male and female Sprague–Dawley (CD®) rats were dosed with either pregnenolone-16α-carbonitrile (PCN; 50 mg/kg per day for 5 days), phenobarbital (PB; 100 mg/kg per day for 4 days), beta-naphthoflavone (βNF; 100 mg/kg per day for 3 days), clofibrate (CF; 300 mg/kg per day for 14 days), isoniazid (ISO; 100 mg/kg per day for 3 days), or dexamethasone (DEX; 50 mg/kg per day for 4 days). Microsomes were isolated, frozen and subsequently assayed for protein, cytochrome P450 content and EMD activity. In males, significant elevations (P<0.01) in EMD activity were observed in microsomes from PB-, DEX- and PCN-dosed animals compared with untreated controls. Microsomes from ISO- and βNF-dosed males showed a reduction (P<0.05) in EMD activity when compared with control microsomes, and CF was without effect. In females, EMD activities were significantly increased in microsomes from PCN, DEX and PB-dosed but not βNF, ISO, or CF-dosed animals. As expected on the basis of sex-related differences in gene expression, EMD activities in untreated animals were considerably higher in males than females, attributable to constitutive CYP3A and CYP2C11 activities. The selectivity of EMD for induced CYP3A was confirmed on the basis of inhibition studies with selected steroid substrates of CYP3A, polyclonal anti-CYP3A1 antibodies and triacetyloleandomycin (TAO), a selective inhibitor of CYP3A. In conclusion, for both sexes, the greatest elevations (≈3–13-fold) in EMD activity were observed in microsomes from rats dosed with DEX, a potent archetypal inducer with lesser but significant increases noted for PCN and PB, indicating that EMD is a reliable indicator of induced rat hepatic cytochrome P450 CYP3A activity.     

Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats.

Earlier studies have shown highly exaggerated mechanism-based liver injury of thioacetamide (TA) in rats following moderate diet restriction (DR) and in diabetes. The objective of the present study was to investigate the mechanism of higher liver injury of TA in DR rats. Since both DR and diabetes induce CYP2E1, we hypothesized that hepatic CYP2E1 plays a major role in the bioactivation-based liver injury of TA. When male Sprague-Dawley rats (250-275 g) were maintained on diet restriction (DR, 35% of ad libitum fed rats, 21 days) the total hepatic microsomal cytochrome P450 (CYP450) was increased 2-fold along with a 4.6-fold increase in CYP2E1 protein, which corresponded with a 3-fold increase in CYP2E1 activity as measured by chlorzoxazone hydroxylation. To further test the involvement of CYP2E1, 24 and 18 h after pretreatment with pyridine (PYR) and isoniazid (INZ), specific inducers of CYP2E1, male Sprague-Dawley rats received a single administration of 50 mg of TA/kg (i.p.). TA liver injury was >2.5- and >3-fold higher at 24 h in PYR + TA and INZ + TA groups, respectively, compared with the rats receiving TA alone. Pyridine pretreatment resulted in significantly increased total CYP450 content accompanied by a 2.2-fold increase in CYP2E1 protein and 2-fold increase in enzyme activity concordant with increased liver injury of TA, suggesting mechanism-based bioactivation of TA by CYP2E1. Hepatic injury of TA in DR rats pretreated with diallyl sulfide (DAS), a well known irreversible in vivo inhibitor of CYP2E1, was significantly decreased (60%) at 24 h. CCl(4) (4 ml/kg i.p.), a known substrate of CYP2E1, caused lower liver injury and higher animal survival confirming inhibition of CYP2E1 by DAS pretreatment. The role of flavin-containing monooxygenase (FMO) in TA bioactivation implicated by previous in vitro studies, and consequent increased TA-induced liver injury in DR rats was tested in vivo with a relatively selective inhibitor of FMO, indole-3-carbinol, and then treated with 50 mg of TA/kg. FMO activity and alanine aminotransferase levels measured at different time points revealed that TA liver injury was not decreased although FMO activity was significantly decreased, suggesting that hepatic FMO is unlikely to bioactivate TA. These findings suggest induction of CYP2E1 as the primary mechanism of increased bioactivation-based liver injury of TA in DR rats.     

Tissue-specific induction of the carcinogen-inducible cytochrome P450 isoforms in the gastrointestinal tract

Gastrointestinal tissues are directly exposed to dietary xenobiotics. In spite of this, modulation of cytochrome P450 (CYP) enzymes in the gastrointestinal tract is not well established. CYP induction could facilitate transformation of chemical agents to potentially toxic or carcinogenic metabolites. This might also determine drug efficacy, burden of foreign chemicals on tissues or bioavailability of certain therapeutic agents. In order to assess the induction of the CYP subfamilies 1A1/2, 2B1/2, 2E1 and 3A2 in the gastrointestinal tract, male Wistar rats were treated with phenobarbital/β-naphthoflavone (PB/NF), cyclohexanol/albendazole (CH/ABZ) or toluene (TL). Microsomal fractions were prepared from tissue samples of the esophagus, the stomach, the duodenum, the colon and the liver. Western blot and enzymatic activity analyses revealed an increase in the expression and activity of CYP1A1/2 and CYP3A2 isoenzymes in the esophageal, duodenal and colonic microsomes from animals treated with PB/NF. CYP1A1/2 and CYP3A2 were induced in hepatic and duodenum microsomes by treatment with CH/ABZ. Our results demonstrate differential induction of CYP along the gastrointestinal tract by known CYP hepatic inducers, being the treatment with PB/NF the best induction system of the CYPs.     

Mitochondrial dysfunction by gamma-irradiation accompanies the induction of cytochrome P450 2E1 (CYP2E1) in rat liver

Multiple biological effects are induced by ionizing radiation through dysfunction of cellular organelles, direct interaction with nucleic acids and production of free radical species. The expression of cytochrome P450s was assessed in the livers of 60Co gamma-irradiated rats. Three gray (G) of gamma-irradiation caused CYP2E1 induction with a 3.6-fold increase in the mRNA at 24 h, whereas the expression of CYP1A2 and CYP3A was not changed. Pharmacokinetics of chlorzoxazone, a specific substrate of CYP2E1, was studied in 3 G-irradiated rats. The area under the plasma concentration-time curve from time zero to infinity of 6-hydroxychlorzoxazone and the amount of 6-hydroxychlorzoxazone excreted in 8 h urine were both significantly greater than those in control rats. Hepatic CYP2E1 was not induced in rats exposed to 0.5-1 G of gamma-rays. Rats irradiated at 6-9 G accumulated doses of gamma-rays exhibited smaller increases in the mRNA due to liver injury than those irradiated at a single dose of 3 G gamma-rays. The plasma glucose and insulin levels were not altered in rats with 3 G of gamma-irradiation. As the exposure level of gamma-irradiation increased, the activity of hepatic aconitase, a key enzyme in energy metabolism in mitochondria, was 30-90% decreased. The amount of mitochondrial DNA per gram of wet liver was 50% decreased in rats exposed to 3 G of gamma-rays. These results demonstrated that gamma-ray irradiation at the exposure level inducing organelle dysfunction induced CYP2E1 in the liver, which might be associated with mitochondrial damage, but not with alterations in glucose or insulin levels.     

Increase in bile flow and biliary excretion of glutathione-derived sulfhydryls in rats by drug-metabolizing enzyme inducers is mediated by multidrug resistance protein 2.

Glutathione (GSH) is an important cellular constituent for normal liver homeostasis. Certain drug-metabolizing enzyme inducers (i.e., phenobarbital [PB] and pregnenolone-16alpha-carbonitrile [PCN]) increase biliary excretion of GSH-derived sulfhydryls (SH) as well as bile flow, whereas other drug-metabolizing enzyme inducers (i.e., 3-methylcholanthrene [3MC] and benzo(a)pyrene [BaP]), do not. The purpose of the study was to determine whether rat multidrug resistance protein 2 (Mrp2) is the inducible transporter responsible for increasing biliary SH excretion and bile flow. Sprague-Dawley (SD) rats were injected ip daily for 4 days with PB, PCN, 3MC, BaP, or vehicle; Mrp2-null Eisai hyperbilirubinemic (EHBR) rats were injected ip daily for 4 days with PCN or vehicle. Although no drug-metabolizing enzyme inducer altered hepatic GSH in SD rats, PB and PCN significantly increased the rate of biliary SH excretion and bile flow. Neither 3MC nor BaP affected the biliary SH excretion rate or bile flow. In control EHBR rats, despite elevated hepatic GSH, the rate of biliary SH excretion was almost completely eliminated and bile flow was dramatically reduced compared with SD rats. Furthermore, PCN treatment did not affect bile flow or the biliary SH excretion rate in EHBR rats. PB and PCN also increased Mrp2 protein levels, but 3MC and BaP did not. None of the drug-metabolizing enzyme inducers tested significantly increased Mrp2 mRNA levels. PCN increased Mrp2 protein, but not Mrp2 mRNA, in a time-dependent manner. In conclusion, Mrp2 is the inducible efflux transporter responsible for increased biliary SH excretion and bile flow after administration of some drug-metabolizing enzyme inducers.     

Comparison of the effects of the synthetic pyrethroid Metofluthrin and phenobarbital on CYP2B form induction and replicative DNA synthesis in cultured rat and human hepatocytes

High doses of Metofluthrin (MTF) have been shown to produce liver tumours in rats by a mode of action (MOA) involving activation of the constitutive androstane receptor leading to liver hypertrophy, induction of cytochrome P450 (CYP) forms and increased cell proliferation. The aim of this study was to compare the effects of MTF with those of the known rodent liver tumour promoter phenobarbital (PB) on the induction CYP2B forms and replicative DNA synthesis in cultured rat and human hepatocytes. Treatment with 50 μM MTF and 50 μM PB for 72 h increased CYP2B1 mRNA levels in male Wistar rat hepatocytes and CYP2B6 mRNA levels in human hepatocytes. Replicative DNA synthesis was determined by incorporation of 5-bromo-2′-deoxyuridine over the last 24 h of a 48 h treatment period. Treatment with 10–1000 μM MTF and 100–500 μM PB resulted in significant increases in replicative DNA synthesis in rat hepatocytes. While replicative DNA synthesis was increased in human hepatocytes treated with 5–50 ng/ml epidermal growth factor or 5–100 ng/ml hepatocyte growth factor, treatment with MTF and PB had no effect. These results demonstrate that while both MTF and PB induce CYP2B forms in both species, MTF and PB only induced replicative DNA synthesis in rat and not in human hepatocytes. These results provide further evidence that the MOA for MTF-induced rat liver tumour formation is similar to that of PB and some other non-genotoxic CYP2B form inducers and that the key event of increased cell proliferation would not occur in human liver.     

吡喹酮在诱导和未诱导大鼠肝微粒体内的羟化代谢概貌及其羟化物的质谱鉴定

用细胞色素P450（P450）特异诱导剂研?究参与吡喹酮（PQT）代谢的P450同工酶，在未诱导肝微粒体内，PQT代谢仅生成其D环单羟化物. 在β-萘黄酮诱导肝微粒体内，PQT代谢后也生成其D环单羟化物. PQT在苯巴比妥诱导的肝微粒体内代谢生成D环，B环和A环三个单羟化物. 在地塞米松和红霉素诱导的肝微粒体内，PQT代谢生成七个代谢产物. 结果表明参与PQT分子羟化的P450同工酶至少包括CYP1A，CYP2B和CYP3A亚家族，每个亚家族代谢PQT的概貌各不相同，CYP3A优先羟化A环，CYP2B优先羟化D环和B环，CYP1A则几乎仅羟化D环.     

Identification of the cyanopregnenolone-inducible form of hepatic cytochrome P-450 as a catalyst of aldrin epoxidation

In light of recent suggestions that hepatic microsomal aldrin expoxidation activity selectively reflects the phenobarbital (PB)-inducible form(s) of cytochrome P-450 (P-450PB), we tested the effect of pregnenolone-16 alpha-carbonitrile (PCN), a synthetic steroid that induces P-450PCN, a form of the cytochrome biochemically and immunochemically distinguishable from P-450PB. In hepatic microsomes prepared from rats receiving PB, 3-methylcholanthrene (3-MC), or PCN, the latter compound produced a greater increase in aldrin epoxidation activity relative to control than did PB, whereas 3-MC decreased enzyme activity. Moreover, the aldrin epoxidation activity in microsomes prepared from PCN- or PB-pretreated rats was selectively inhibited by form-specific antibodies directed against P-450PCN or P-450PB, respectively, whereas anti-P-450MC antibodies gave no inhibition with microsomes prepared from induced or control animals. We conclude that P-450PCN, P-450PB, and probably other cytochromes P-450 catalyze aldrin epoxidation, precluding use of this enzyme as a specific marker of a single form of the cytochrome.     

In vitro induction of cytochrome P450 enzymes in hepatocytes isolated from the regenerating rat liver

Partial hepatectomy results in the loss of cytochrome P450 enzymes. During regeneration, the levels of cytochrome P450 activities, apoproteins and mRNA are reduced. Our present study investigated CYP1A, CYP2E1 and CYP3A induction in the cells of rat liver regenerating for 1, 3, 7, or 14 days. Hepatocytes were isolated from regenerating liver of hepatectomized rats and treated with enzyme inducers: 3-methylcholanthrene, imidazole and dexamethasone. CYP1A enzymes of the cells isolated from regenerating liver were inducible by 3-methylcholanthrene. The rate of induction of the cells from 3-day regenerating liver by 3-methylcholanthrene was three times higher than that of the hepatocytes of sham-operated rats. Dexamethasone caused about two- or three-fold stronger elevation of CYP3A in the cells of 1-, 3- and 7-day regenerating liver than in hepatocytes of sham-operated animals. However, the degree of CYP2E1 induction by imidazole was the same (about 2.5-fold) at each regenerating time as it was detected in the hepatocytes of sham-operated animals. In conclusion, the inducibility of the cells was retained at each regenerating time, but the degree of induction showed some differences.     

Oxidation of 1,8-cineole, the monoterpene cyclic ether originated from eucalyptus polybractea, by cytochrome P450 3A enzymes in rat and human liver microsomes.

1,8-Cineole, the monoterpene cyclic ether known as eucalyptol, is one of the components in essential oils from Eucalyptus polybractea. We investigated the metabolism of 1,8-cineole by liver microsomes of rats and humans and by recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human P450 and NADPH-P450 reductase cDNAs had been introduced. 1,8-Cineole was found to be oxidized at high rates to 2-exo-hydroxy-1,8-cineole by rat and human liver microsomal P450 enzymes. In rats, pregenolone-16alpha-carbonitrile (PCN) and phenobarbital induced the 1,8-cineole 2-hydroxylation activities by liver microsomes. Several lines of evidence suggested that CYP3A4 is a major enzyme involved in the oxidation of 1,8-cineole by human liver microsomes: (1), 1,8-cineole 2-hydroxylation activities by liver microsomes were inhibited very significantly by ketoconazole, a CYP3A inhibitor, and anti-CYP3A4 immunoglobulin G; (2), there was a good correlation between CYP3A4 contents and 1,8-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples; and (3), of various recombinant human P450 enzymes examined, CYP3A4 had the highest activities for 1,8-cineole 2-hydroxylation; the rate catalyzed by CYP3A5 was about one-fourth of that catalyzed by CYP3A4. Kinetic analysis showed that K(m) and V(max) values for the oxidation of 1,8-cineole by liver microsomes of human sample HL-104 and rats treated with PCN were 50 microM and 91 nmol/min/nmol P450 and 20 microM and 12 nmol/min/nmol P450, respectively. The rates observed using human liver microsomes and recombinant CYP3A4 were very high among other CYP3A4 substrates reported so far. These results suggest that 1,8-cineole, a monoterpenoid present in nature, is one of the effective substrates for CYP3A enzymes in rat and human liver microsomes.     

Influence of recipient gender on cytochrome P450 isoforms expression in intrasplenic fetal liver tissue transplants in rats

Rat livers display a sex-specific cytochrome P450 (P450) isoforms expression pattern which is regulated by a differential profile of growth hormone (GH) secretion. The aim of the present study was to elucidate whether liver cell transplants at an ectopic site are also subject to this influence. Fetal liver tissue suspensions of mixed gender were transplanted into the spleen of adult male or female syngenic recipients. Four months after grafting transplant recipients and age-matched controls were treated with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the solvents and sacrificed 24 or 48 h thereafter. Livers and intrasplenic transplants were evaluated for the expression of the P450 subtypes 1A1, 2B1, 2E1, 3A2 and 4A1 by means of immunohistochemistry. The livers of both male and female rats displayed nearly no P450 1A1, but a distinct P450 2B1, 2E1, 3A2 and 4A1 expression. Whereas no sex differences were seen in the P450 1A1 expression, the immunostaining for P450 2B1, 3A2 and 4A1 was stronger in males and that for P450 2E1 in females. Similarly, in the intrasplenic liver cell transplants almost no P450 1A1, but a noticeable P450 2B1, 2E1, 3A2 and 4A1 expression was observed. Like in the respective livers, the immunostaining for P450 2B1, 3A2 and 4A1 was stronger in the transplants hosted by male than by female rats, whereas the opposite was the case for the P450 2E1 expression. Both in livers and transplants with some sex-specific differences P450 1A1 and 2E1 expression was induced by BNF, that of P450 2B1 by BNF and PB, and that of P450 3A2 by PB and DEX. These results indicate that the P450 system of ectopically transplanted liver cells is influenced by the gender of the recipient organism like that of the orthotopic livers.