P450 IA1 Research Articles

The role of sulfation and/or acetylation in the metabolism of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in Salmonella typhimurium and isolated rat hepatocytes.

Mutagenic activity of the cooked-food mutagen/carcinogen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is highly dependent upon cytochrome P450 activation to the N-hydroxylated intermediate. In the present study the bioactivation pathways of PhIP were investigated in Salmonella typhimurium and isolated rat hepatocyte preparations. In the Ames/S. typhimurium assay, the acetyltransferase and sulfotransferase enzyme inhibitors pentachlorophenol (PCP) and 2,6-dichloro-4-nitrophenol (DCNP) were used to modulate mutagenicity. DCNP, but not PCP, produced a concentration-dependent decrease in mutagenic activity of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-hydroxy-PhIP). In rat hepatocyte preparations, PCP and DCNP, as well as the cytochrome P450 IA1 and IA2 inhibitor alpha-naphthoflavone (ANF), were used to modulate metabolite, protein adduct, and DNA adduct formation. Incubations of [3H]PhIP (100 microM) with Aroclor 1254-induced or uninduced hepatocytes resulted in the formation of several metabolites, including 4'-(2-amino-1-methylimidazo[4,5-b]pyrid-6-yl)phenyl sulfate (4'-PhIP-sulfate), 2-amino-1-methyl-4'-hydroxy-6- phenylimidazo[4,5-b]pyridine (4'-hydroxy-PhIP), a glucuronide conjugate of 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine, and other uncharacterized metabolites. While PCP or DCNP pretreatment produced a significant decline in sulfate-dependent conjugation of 4'-hydroxy-PhIP to 4'-PhIP-sulfate, these inhibitors produced only slight decreases in PhIP-dependent covalent binding to proteins in hepatocytes derived from either Aroclor 1254-induced or uninduced rats. PhIP DNA adduct levels were relatively unchanged by PCP or DCNP pretreatment of Aroclor 1254-induced hepatocytes. DNA adducts from hepatocytes dosed with N-hydroxy-PhIP, however, resulted in a decrease in adduct levels from cells pretreated with PCP or DCNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of m-xylene on rat nasal cytochrome P450 mixed function oxidase activities

The effect of m-xylene on the rat nasal cytochrome P450 (P450) mixed function oxidase system was analyzed in vitro utilizing microsomes isolated 2, 12, and 24 h following intraperitoneal administration of this solvent in vivo. For comparative purposes, pulmonary and hepatic activities were also measured. Benzyloxyresorufin O-deethylation (BROD) and ethoxyresorufin O-deethylation (EROD), catalytic activities linked with P450 isozymes IIB1 and IA1, respectively, were inhibited in nasal tissue at all times following m-xylene administration. Pulmonary tissue mimicked this m-xylene-dependent inhibition of BROD activity but did not display significant inhibition of EROD activity. In contrast, m-xylene caused a dramatic induction of both BROD and EROD activity in hepatic tissue. The metabolism of a third P450 substrate, cyclopentadienyl manganese tricarbonyl (CMT), was also analyzed. m-Xylene caused significant inhibition of CMT metabolism at all time points in both nasal and pulmonary microsomes but was without effect on hepatic microsomal metabolism of this compound. These data show an inhibitory effect of m-xylene on rat nasal and pulmonary but not hepatic cytochrome P450-dependent metabolism.

Developmental expression of cytochrome P450s within intrasplenically transplanted fetal hepatocytes from spontaneously hypertensive rats.

Fetal hepatocytes were harvested at 20 days of gestation from spontaneously hypertensive rats (SHR) and then transplanted into recipient adult SHR spleens. Morphological examination of the recipient spleens revealed that after 4 wk, large masses of hepatocytes were present in the red pulp with apparent cord-like structures. Of major significance was the fact that hepatocyte transplanted spleens were able to express several families of cytochrome P450 (cyto P450) proteins 2-6 wk after transplantation. Thus, the transplanted hepatocyte tissue appeared to grow and develop a cyto P450 metabolic system. Immunochemical determinations revealed that cytos P450 IA1, P450 IIB1, P450 p, P450 HLp, and P450 LA omega were over detected without any prior induction. None of the cyto P450s could be detected in control nontransplanted spleens, however, all were detected in fetal hepatocyte microsomes before transplantation. All were intensely expressed 6 wk after transplantation, however P450 IA1 and P450 IIB1 did not appear to be expressed by 2 wk after transplantation. These results demonstrate that fetal hepatocytes can be successfully transplanted to the spleens of recipient hosts, and that the fetal hepatocytes appear to grow and develop functional cyto P450 metabolizing systems. Therefore, transplantation of fetal hepatocytes to splenic tissue represents a unique in vivo system of extra hepatic metabolizing tissue.

Identification of fatty acid hydroperoxide cofactors in the cytochrome P450-mediated oxidation of estrogens to quinone metabolites. Role and balance of lipid peroxides during estrogen-induced carcinogenesis.

Lipid hydroperoxide-supported metabolic redox cycling between diethylstilbestrol (DES) or catechol-estrogens and their corresponding quinones has been postulated previously to play a role in estrogen-induced toxicity and carcinogenesis. As part of an examination of this postulate, we investigated (a) the effectiveness of various lipid hydroperoxides as oxidants in microsome-mediated quinone formation, and (b) the dependence of DES oxidation to quinone on lipid hydroperoxide levels in liver and kidney of hamsters. Kinetic analyses of the lipid hydroperoxide-mediated conversion of DES to DES quinone revealed that of the peroxides tested (9Z,11E,13(S))-13-hydroperoxyoctadecadien-1-oic acid was the most powerful oxidant (Vmax = 10.5 nmol min-1; Km = 21.3 microM) followed by (5(S),6E,8Z,11Z,14Z)-5-hydroperoxyeicosatetra en-1-oic acid (Vmax = 1.0 nmol min-1; Km 10.0 microM). The other pure fatty acid hydroperoxide isomers or mixtures had a lower affinity for the enzyme (Km values ranging from 23.1 to 130 microM) and/or lower maximal velocity (Vmax values ranging from 0.2 to 7.0 nmol min-1). Levels of Z,Z-dienestrol, which arises from spontaneous rearrangement of DES quinone, were not affected in liver of hamsters treated with estradiol implants for 9 days and an injection of 20 mg/kg DES but increased in kidney by 120% over levels observed in animals without implants (5.5 and 12.1 nmol/g of wet tissue, respectively). Z,Z-Dienestrol concentrations correlated directly with lipid hydroperoxide concentrations in liver and kidney of control and estradiol-treated hamsters. The increased conversion of DES to quinone in kidney compared with that in liver was catalyzed by comparable activities of cytochrome P450 IA, the enzyme family catalyzing this oxidation. These results demonstrated that the oxidation of DES to quinone was directly dependent on the lipid hydroperoxide levels in the organ of metabolic conversion in addition to the enzyme activity catalyzing this reaction. The elevated lipid hydroperoxide levels in kidney of estrogen-treated hamsters indicate enhanced estrogen quinone formation and therefore enhanced generation of free radicals by metabolic redox cycling of estrogens in this organ prior to the appearance of malignancy.

Liver accumulation of 2,3,7,8-tetrachloro-[ 3H]dibenzofuran in mice: modulation by treatments with polychlorinated biphenyls

The distribution of 2,3,7,8-tetrachloro-[ 3H]dibenzofuran ([ 3H]TCDF; 40 μg/kg) resembled that earlier reported for 2,3,7,8-tetrachlorodibenzo- p-dioxin, with a strong accumulation in the liver and a selective uptake in the nasal olfactory mucosa of adult and fetal mice. Pretreatments with a series of selected congeners of polychlorinated biphenyls (PCBs), i.e.. I (IUPAC)-77, I-105, I-118, I-126, I-153, I-156, I-169, and a commercial preparation, Aroclor 1254 (25–100 mg/kg body wt. i.p.), were found to modulate the hepatic uptake of [ 3H]TCDF (24 h post- 3H-injection). At a short pretreatment time (4 h), non- ortho-chlorinated congeners decreased the uptake of [ 3H]TCDF equivalents in the liver (e.g., I-126 = 3,3′,4,4′,5-pentachlorobiphenyl: 34% of control), while several mono- and di- ortho PCB congeners and Aroclor 1254 increased the hepatic uptake of [ 3H]TCDF (e.g., I-156 = 2,3,3′,4,4′,5-hexachlorobiphenyl: 183% of control). At a longer pretreatment time (48 h), both a non- ortho (I-169 = 3,3′,4,4′,5,5′-hexachlorobiphenyl) and mono- ortho PCB congener(s) (e.g. I-156) markedly increased the hepatic 3H-uptake (190%), a probable effect of an induction of hepatic binding sites for TCDF. Ethoxyresorufin- O-deethylase activities, regarded to mirror the metabolic activity of cytochrome P-450 IA1 (CYP IA1), were strongly and time-dependently induced after I-169, but not after I-156, pretreatment (25 mg/kg). The initial liver concentrations of the two PCB congeners were similar and increased for I-169 but not for I-156 at later time points. In conclusion, the results show a selective uptake of [ 3H]TCDF in the mouse liver and nasal olfactory mucosa of both dam and fetus. The uptake of [ 3H]TCDF in the liver is influenced both by dose and pre-exposure with PCBs. The presence of a PCB-sensitive, but CYP IA1-independent, hepatic binding site for TCDF is suggested. Consequently, pharmacokinetic interactions with PCBs complicate the toxicity assessment of TCDF in complex mixtures.

Metabolic activation by a supernatant from human hepatoma cells: a possible alternative in mutagenic tests

The supernatant from human Hep G2 hepatoma cells was examined for typical enzymatic activities involved in the metabolism of xenobiotics. Neither cytochrome P-450 nor b5 was detectable, but associated enzymatic activities were found especially after induction with hydrocortisone (HC) and benzanthracene (BA) suggesting that this Hep G2 supernatant contains cyt P-450 IA1 and IA2. Other critical enzymes are also present, but, as expected, at lower activities than in Aroclor 1254 rat liver S9, except for NADH and NADPH cytochrome c reductase. Results of the Ames test indicate that the induced Hep G2 supernatant is a suitable activator for the evaluation of genotoxicity of indirect mutagens.

Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine.

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of 3-methylcholanthrene induction on the distribution and DNA adduction of 2-amino-3-methylimidazo[4,5-ƒ]quinoline (IQ) in F344 rats

3-Methylcholanthrene (3MC) is a potent inducer of the cytochrome P450IA family of enzymes that catalyses the metabolic activation of the food mutagen/carcinogen 2-amino-3-methylimidazo-[4,5-ƒ]quinoline (IQ). We have examined the effect of pretreatment with 3MC on the distribution and DNA adduct formation of IQ in male Fischer F344 rats. 3 hr after a single dose of [ 14C]IQ (10 mg/kg body weight, by gavage), the level of radioactivity in extrahepatic tissues was 30–70% less in 3MC-pretreated rats than in vehicle control rats. Although the level of radioactivity in the liver did not change after 3MC pretreatment, IQ-DNA adduct levels, measured by the 32P-postlabelling method, were 60% lower in the livers of 3MC-pretreated rats than those of control rats, and 83–97% lower in extrahepatic tissues such as the kidneys, colon, small intestine, bladder, heart and lung. IQ-DNA adducts in the testes and brain were found in control rats but were not detected in 3MC-pretreated rats. The rate of removal of IQ-DNA adducts from the livers of control and 3MC-pretreated animals was the same from 3 to 48 hr. At 48 hr, the adduct level in 3MC-pretreated rats remained lower than that seen in the control rats. The data suggest that 3MC induction of the P450 IA family of cytochromes in vivo results in an increased rate of IQ detoxification.

Effect of pregnandiol on caffeine metabolism in female rats.

Three groups of six 5-week-old Sprague Dawley female rats received i.p. injections of pregnandiol, 1.25, 2.50 or 5 mg/kg, respectively, in triolein daily for 7 days. Caffeine metabolism was studied in liver slices on day 8 by HPLC. Only primary metabolites were formed. N-1 demethylation was the most important pathway (theobromine represented 51% of total dimethylxanthines). Unlike in human in vitro or in vivo, 1,3,7-DAU (6-amino-5-(N-formylmethylamino)-1,3-dimethyluracil) was an important metabolite (9.7% of total caffeine metabolites). Pregnandiol inhibited N-1, N-3 and N-7 demethylation in vitro (-33%, -33% and -28%, respectively, at 5 mg/kg/day), but it had no effect on N-1 demethylation at 1.25 or 2.50 mg/kg/day. Pregnandiol at all doses had no effect on 1,3,7-trimethyluric acid and 1,3,7-DAU formation. These results are consistent with the hypothesis that C-8 hydroxylation and demethylation of caffeine are mediated by different isoenzymes. They indicate that pregnandiol is a potent inhibitor of microsomal drug metabolism, specifically of cytochrome P450 IA, which could explain the immaturity of some metabolic pathways of caffeine in neonates.

The expression of cytochrome P-450, epoxide hydrolase, and glutathione S-transferase in hepatocellular carcinoma.

Cytochrome P-450, epoxide hydrolase, and glutathione S-transferase (GST) all play a key role in the metabolism of chemical carcinogens, mutagens, and various anti-cancer drugs. All these functionally associated enzymes might be involved in both the development of hepatocellular carcinoma and in determining the anti-cancer drug sensitivity of such tumors. The expression of two forms of cytochrome P-450 (P-450 IA and P-450 IIIA), microsomal epoxide hydrolase, and three classes of cytosolic GST (alpha, mu, and pi) have been studied immunohistochemically in human hepatocellular carcinoma. The hepatocellular carcinomas were characterized by a consistently high expression of epoxide hydrolase and variable expression of the cytochromes P-450 and GST. Cytochrome P-450 IA and IIIA stained in 64.5% and 41.9% of the 31 hepatocellular carcinomas studied, respectively. Epoxide hydrolase was present in all tumors, and GST types alpha, pi, and mu were identified in 48.4%, 38.7%, and 74.2% of the hepatocellular carcinomas, respectively. This study showed that the expression of xenobiotic-metabolizing enzymes in hepatocellular carcinoma is complex and the presence of different xenobiotic enzymes in hepatocellular carcinoma may contribute to the intrinsic drug resistance of these tumors.

On the bioactivation and genotoxic action of fluoranthene

Fluoranthene (FA) was studied with respect to possible mechanisms of its high mutagenicity but low carcinogenicity, in comparison with the corresponding properties of benzo[a]pyrene (BaP), and with regard to the synergism of these two compounds shown by van Duuren and Goldschmidt (J Natl Cancer Inst 56, 1976, 1237). FA and BaP activated by S9 from Aroclor 1254 (PCB)-treated rats induce HPRT mutations in CHO cells with about equal effectiveness at the same exposure doses, which also lead to the same frequencies of repairable DNA adducts, enzyme-induced strand breaks being used as an indirect measure of adducts to DNA. FA was also shown to be an efficient inducer of SCE in human peripheral lymphocytes cocultivated with PCB-treated HepG2 cells or with liver cells from PCB-pretreated rats. For the induction of SCE, FA and BaP were shown to act additively. From metabolic studies with liver microsomes from C57Bl/6 mice it is concluded that, whereas BaP induces the metabolism of BaP to the mutagenic epoxide, neither BaP nor FA is able to induce the metabolism of FA. In mutation experiments with V79 cells (XEM2) constitutive for P450 IA1 activity, BaP 7,8-diol but not FA 2,3-diol provokes a high frequency of HPRT mutations. In cells constitutive for P450 IA2 enzymatic activity FA and BaP are but weakly mutagenic and practically nonmutagenic, respectively. Due to the additivity of the genotoxic effects of FA and BaP, induction of an error-prone condition by the latter compound seems to be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a single oral dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on hepatic and extrahepatic cytochrome P-450 IA induction in the Swiss mouse

The cytochrome P-450 1A1 induction response following TCDD exposure has been characterized in liver; however little is known regarding extrahepatic targets of TCDD. This study was designed to compare the 1A1 induction response of hepatic to extrahepatic tissue after oral TCDD exposure. 1A1 was induced in all tissues, with liver, lung and kidney exhibiting an early maximum and subsequent gradual decrease over 4 weeks, and intestines demonstrating a rapid loss of enzyme activity over time. The magnitude of induction was significantly greater in liver and lung following oral vs. intraperitoneal administration. These studies suggest differences in 1A1 induction in hepatic vs. extrahepatic tissue and with different routes of administration.

Differential cumene hydroperoxide sensitivity of cytochrome P-450 enzymes IA1 and IIB1 determined by their way of membrane incorporation

The cytochrome P-450-dependent O-dealkylation of all alkoxyresorufins was used to study the effect of cumene hydroperoxide on cytochrome P-450 IIB1 and IA1 in microsomal and reconstituted systems. In liver microsomal systems from respectively phenobarbital and 3-methylcholanthrene pretreated male Wistar rats, cytochrome P-450 IIB1-dependent pentoxyresorufin- O-dealkylation appeared to be more sensitive to cumene hydroperoxide treatment than cytochrome P-450 IA1-dependent ethoxyresorufin- O-dealkylation. This phenomenon was also observed when the cumene hydroperoxide sensitivity of P-450 IIB1 and IA1 was studied in an isosafrole pretreated rat liver microsomal system. The decrease in alkoxy- O-dealkylating activities appeared to proceed by destruction of the cytochrome P-450 component of the enzyme system. Purification and reconstitution of the enzyme system components in a system in which the isolated proteins were not incorporated into a membrane resulted in the disappearance of the difference in sensitivity between the two P-450 enzymes. However, in a reconstituted system with membrane incorporated proteins, again cytochrome P-450 IIB1 expressed a higher sensitivity towards cumene hydroperoxide than cytochrome P-450 IA1. From this it was concluded tha the differential cumene hydroperoxide sensitivity of cytochrome P-450 IIB1 and IA1 is not caused by an intrinsic difference in their sensitivity but by a differential effect of membrane incorporated on their cumene hydropeoxide sensitivity.

Structure-activity relationship of kynurenate analogues for interaction with the glycine site on the n-methyl-o-aspartate (NMDA) receptor

Propofol, a widely used anesthetic drug, is known to inhibit cytochrome P450 activities in vitro. The goal of this study was to compare cytochrome P450 activities in vitro and in vivo in presence of propofol. In vitro (liver microsomes and freshly isolated hepatocytes), cytochrome P450 IA2 and IIB1 activities were measured as the production of resorufin from ethoxy- and pentoxyresorufin, respectively, in presence of various concentrations of propofol. In vivo, cytochrome P450 activities were assessed as the production of 13CO2 from 13C-aminopyrine injected intravenously, during acute administration or after chronic treatment. In vitro results confirmed the dose-dependent inhibitory effect of propofol on cytochrome P450 activities, both on liver microsomes and isolated hepatocytes. In vivo, the acute administration of propofol induced a significant decrease of 13C-aminopyrine metabolism. Chronic treatment with propofol induced a significant inhibition of 13C-aminopyrine metabolism only after 2 weeks. No enzyme induction was observed. In conclusion, our results demonstrate that propofol inhibits cytochrome P450 also in vivo. Drug interactions may thus occur during propofol administration.

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the β-naphthoflavone-inducible cytochrome P450 IA family

Diethylstilbestrol (DES) or catecholestrogens are metabolized by microsomal enzymes to quinones, DES Q or catecholestrogen quinones, respectively, which have been shown to bind covalently to DNA and to undergo redox cycling. The isoforms of cytochrome P450 catalyzing this oxidation of estrogens to genotoxic intermediates were not known and have been identified in this study by (a) using microsomes of rats treated with various inducers of cytochrome P450; (b) using purified cytochrome P450 isoforms; and (c) examining the peroxide cofactor concentrations necessary for this oxidation by microsomes or pure isoenzymes. The highest rate of oxidation of DES to DES Q was obtained using β-naphthoflavone-induced microsomes (14.0 nmol DES Q/mg protein/min) or cytochrome P450 IA1 (6.4 pmol DES Q/min/pmol P450). Isosafrole-induced microsomes or cytochrome P450 IA2 oxidized DES to quinone at one-third or one-fifth of that rate, respectively. Low or negligible rates of oxidation were measured when oxidations were catalyzed by microsomal rat liver enzymes induced by phenobarbital, ethanol, or pregnenolone-16α-carbonitrile or by pure cytochromes P450 IIB1, IIB4, IIC3, IIC6, IIE1, IIE2, IIG1, or IIIA6. Cytochrome P450 IA1 also catalyzed the oxidation of 2- or 4-hydroxyestradiol to their corresponding quinones. The β-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest “affinity” for cumene hydroperoxide cofactor (K m = 77 μ m). Cofactor concentrations above 250 μ m resulted in decreased rates of oxidation. The other cytochrome P450 isoforms required much higher cofactor concentrations and were not inactivated at high cofactor concentrations. The data demonstrate that β-naphthoflavone-inducible cytochrome P450 IA family enzymes catalyze most efficiently the oxidation of estrogenic hydroquinones to corresponding quinones. This oxidation may represent a detoxification pathway to keep organic hydroperoxides at minimal concentrations. The resulting quinone metabolites may be detoxified by other pathways. However, in cells with decreased detoxifying enzyme activities, quinones metabolites may accumulate and initiate carcinogenesis or cell death by covalent arylation of DNA or proteins.

12(R)-Hydroxyeicosatetraenoic acid synthesis by 3-methylcholanthrene- and clofibrate-inducible cytochrome P450 in porcine ciliary epithelium

Porcine ciliary epithelial microsomes synthesized 12[S]-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12[S]-HETE) from arachidonic acid by a membrane-bound lipoxygenase and 12[R]-isomer by the cytochrome P450-dependent monooxygenase system. The activity to form 12(R)-isomer was markedly enhanced by 3-methylcholanthrene and clofibrate. Both basal and induced levels of 12(R)-HETE synthesizing activity were considerably higher in nonpigmented epithelial cells than in pigmented cells of the ciliary processes. The induced activity was suppressed by polyclonal antibodies raised against purified cytochrome P450 IA1 and NADPH-P450 reductase but not by substrates for clofibrate-inducible omega/omega-1 hydroxylases (P450 IVA-mediated). These results suggest that 12(R)-HETE synthesis by porcine ciliary microsomes may be mediated by a cytochrome P450 of the IA family.

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of α 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.

Uroporphyrinogen oxidation catalyzed by reconstituted cytochrome P450IA2

Previous work suggested that the oxidation of uroporphyrinogen to uroporphyrin is catalyzed by cytochrome P450IA2. Here we determined whether purified reconstituted mouse P450IA1 and IA2 oxidize uroporphyrinogen. Cytochromes P450IA1 and IA2 were purified from hepatic microsomes from 3-methylcholanthrene (MC)-treated C57BL/6 mice, using a combination of affinity chromatography and high performance liquid chromatography. Reconstituted P450IA1 was more active than P450IA2 in catalyzing ethoxyresorufin- O-deethylase (EROD) activity, whereas P450IA2 was more active than P450IA1 in catalyzing uroporphyrinogen oxidation (UROX). Both reactions required NADPH, NADPH-cytochrome P450 reductase, and either P450IA1 or IA2. Ketoconazole competitively inhibited both EROD and UROX activities, in microsomes from MC-treated mice. Ketoconazole also inhibited UROX catalyzed by reconstituted P450IA2. In contrast, ketoconazole did not inhibit UROX catalyzed by xanthine oxidase in the presence of iron-EDTA. Superoxide dismutase, catalase, and mannitol inhibited UROX catalyzed by xanthine oxidase/ iron-EDTA, but did not affect UROX catalyzed by either microsomes or reconstituted P450IA2. These results suggest that UROX catalyzed by P450IA2 in microsomes and reconstituted systems does not involve free reactive oxygen species. Two known substrates of cytochrome P450IA2, 2-amino-3,4-dimethylimidazole[4,5- f]quinoline and phenacetin, were shown to inhibit the microsomal UROX reaction, suggesting that uroporphyrinogen binds to a substrate-binding site on the cytochrome P450.

Interleukin-6 down regulates the expression of transcripts encoding cytochrome P450 IA1, IA2 and IIIA3 in human hepatoma cells

Effects of human interleukin-6 (hIL-6), the major acute phase inducer, on the expression of transcripts encoding cytochrome P450s were examined in human hepatoma-derived cells. Using reverse-transcription polymerase chain reaction, it was demonstrated that three hepatoma cell lines, Hep G2, Hep G2f and Hep 3B, express P450 mRNAs encoding IA1, IA2 and IIIA3, the major P450 isozymes involved in carcinogen metabolism, and that they also show induction responses to treatment with their specific inducers. When hepatoma cells were treated with hIL-6, the levels of IA1, IA2 and IIIA3 mRNAs were markedly suppressed. These findings suggest that significant down regulation of cytochrome P450s may occur during the acute phase reaction, which may result in alterations in drug biotransformation.

Covalent binding of food carcinogens MeIQx, MeIQ and IQ to DNA and protein in microsomal incubations and isolated rat hepatocytes.

The metabolic activation of 14C-labelled food carcinogens 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to macromolecular bound species was studied in microsomal and hepatocellular incubations. Several data indicated that the covalent binding was dependent on P450 enzymes: It was dependent on NADPH, it was induced many times by the P450 IA1 and IA2 upregulators beta-naphthoflavone and polychlorinated biphenyls, and was inhibited by the P450 IA1 and IA2 inhibitor alpha-naphthoflavone. In both hepatocellular and microsomal incubations the three compounds bound with similar efficiency, with IQ being somewhat more potent compared to MeIQx and MeIQ. The binding appeared to follow saturation kinetics with Km values less than 20 microM. In incubations with hepatocytes the compounds bound to both cellular DNA and to bovine serum albumin in the medium. The fact that 13-26% of total adducts were formed with bovine serum albumin, indicates that reactive metabolites of the compounds may be transported and react at distant sites from their formation without any further activation.