The content and activity of cytochrome P-450 in long-term culture of hepatocytes from male and female rats

The content of cytochrome P-450 and the capacity for O-demethylation have been measured in cultures of hepatocytes from male and female rats for a period of 21 days. The effect of dexamethasone, insulin, glucagon, phenobarbital and hemin was investigated. In hepatocytes from female rats the content of cytochrome P-450 was unchanged after one day of culture. From day 1 to day 3 the content of cytochrome P-450 decreased by 65% and only the combined addition of dexamethasone, phenobarbital and hemin diminished the fall. After the initial fall, addition of 0.1 microM dexamethasone resulted in a stable value. Addition of 1 microM dexamethasone or 1 mM phenobarbital gave rise to an induction of cytochrome P-450 (285%). The high level of cytochrome P-450 was maintained for 3 weeks. In hepatocytes from male rats the content of cytochrome P-450 decreased by 40% after one day of culture. From day 1 to day 3 the content decreased by 45% and the decrease continued irrespective of the presence of hormones and/or phenobarbital. The O-demethylase activity in cultures of hepatocytes from female rats correlated to the cytochrome P-450 content independent of medium composition and age of the cultures, whereas no correlation was found in cultures from male rats. The present study demonstrates that hepatocytes from female rats in cultures retain O-demethylase activity for at least 3 weeks and that, with the experimental conditions used, the response to the hormones and inducers is different for hepatocytes from male and female rats.     

Role of the pituitary and neonatal androgenic imprinting in the hormonal regulation of liver alcohol dehydrogenase activity

Liver alcohol dehydrogenase activity is increased by thyroidectomy, orchidectomy, or hypophysectomy. We investigated the mechanisms of these hormonal effects by examining the effects of testosterone, dexamethasone and thyroid hormone on liver alcohol dehydrogenase activity in hypophysectomized rats and in cultured hepatocytes, and the effect of administration of androgens to neonatal female rats. Testosterone did not lower alcohol dehydrogenase activity in hypophysectomized rats, whereas dexamethasone and thyroxine produced moderate decreases in activity. Triiodothyronine reduced alcohol dehydrogenase activity of cultured hepatocytes from male and hypothyroid female rats in a dose-dependent fashion, confirming that thyroid hormone had pituitary-independent effects on the enzyme activity. Dexamethasone was required for the expression of alcohol dehydrogenase activity in cultured cells, and it increased the enzyme activity when present at supraphysiologic concentrations. Treatment of neonatal female rats with testosterone reduced the activity of the enzyme in adulthood. The difference in alcohol dehydrogenase activity in adult male and female rats appears to be determined in part by neonatal imprinting by androgens and in part by an effect of testosterone that is either mediated by or dependent upon the pituitary. Thyroid hormone reduces alcohol dehydrogenase activity by a direct effect on the liver.     

Influence of epinephrine on alcohol dehydrogenase activity in rat hepatocyte culture

The effects of epinephrine on alcohol dehydrogenase activity and on rates of ethanol elimination were determined in rat hepatocyte culture. Continuous exposure of the hepatocytes to epinephrine (10 microM) in combination with dexamethasone (0.1 microM) enhanced alcohol dehydrogenase activity on days 4-7 of culture, whereas neither hormone alone had an effect. The increased alcohol dehydrogenase activity was associated with an increased rate of ethanol elimination. Acute addition of 10 microM epinephrine to hepatocytes maintained in culture with 0.1 microM dexamethasone did not change alcohol dehydrogenase activity, but resulted in an immediate marked, but transitory, increase in ethanol elimination within the first 5 min after the addition of the hormone. Prazosin, an alpha 1-adrenergic blocker, and antimycin, an inhibitor of mitochondrial respiration, were powerful inhibitors of the transient increase in ethanol elimination, whereas 4-methylpyrazole was only partially inhibitory. These observations indicate that epinephrine has a chronic effect in increasing alcohol dehydrogenase activity and ethanol elimination and, also, an acute transient effect of increasing ethanol elimination which is not limited by alcohol dehydrogenase activity.     

Inducibility of male-specific isoforms of cytochrome p450 by sex-dependent growth hormone profiles in hepatocyte cultures from male but not female rats.

Although in vivo expression levels of the male-specific hepatic isoforms of cytochrome P450 (P450) (CYP2C11, CYP2C13, CYP2A2, and CYP3A2) are determined by the episodic growth hormone profile secreted by male rats, these isoforms have been completely refractory to growth hormone regulation in hepatocyte culture. By using species-specific rat growth hormone, at subphysiologic in vivo concentrations administered in two daily episodic pulses, we successfully induced CYP2C11 and CYP2A2 to near normal concentrations. Whereas inductive levels of CYP2C13 were subnormal, CYP3A2 was unresponsive to all hormonal treatments, quickly declining to undetectable concentrations. In agreement with in vivo findings, we observed that induction levels of the isoforms were always greatest when the male hepatocytes were exposed to the masculine-like episodic growth hormone profile and least stimulated by the continuous feminine-like hormone profile. When administered alone, dexamethasone consistently increased isoform levels. However, when administered with growth hormone, the glucocorticoid was always antagonistic, suppressing growth hormone induction of CYP2C11, CYP2C13, and CYP2A2. Finally, the P450 isoforms were completely unresponsive to all treatments when the hepatocytes were derived from female rats, supporting earlier findings that expression levels of sexually dimorphic P450 isoforms are inherently irreversible between sexes.     

The in vivo induction of rat hepatic cytochrome P450-dependent enzyme activities and their maintenance in culture

Cytochrome P450-dependent enzyme activities were measured in hepatocytes from adult male rats, induced in vivo with phenobarbitone, beta-naphthoflavone, dexamethasone or isoniazid: the stability of the induced activities in culture was also determined. Each inducer produced a characteristically different pattern of enzyme activities with dexamethasone, isoniazid and beta-naphthoflavone selectively inducing erythromycin N-demethylase, p-nitrophenol hydroxylase and ethoxyresorufin O-dealkylase respectively. In general, the induced activities were maintained for 24 hr in culture. This indicates the feasibility of an in vivo induction-hepatocyte culture system for the study of metabolism-mediated toxicity.     

Activities of several phase I and phase II xenobiotic biotransformation enzymes in cultured hepatocytes from male and female rats

Hepatocytes were isolated from adult male and female rats and maintained in monolayer culture for up to 24 hr. The degree of preservation of representative phase I and phase II xenobiotic biotransformation enzymes was studied in these cells immediately after isolation, after attachment in culture, and after 24 hr in culture. Regarding phase I pathways, hepatocytes during 24 hr lost 50% of cytochrome P-450, but maintained high mixed function oxidase activities; 75% of aryl hydrocarbon hydroxylase and 65% of benzphetamine demethylase activities were preserved in hepatocytes from males, whereas in hepatocytes from females 70 and 50% of these activities, respectively, were maintained. Of phase II pathways, glutathione transferase activity after 24 hr, tested toward 1,2-dichloro-4-nitrobenzene as substrate, was diminished in male hepatocytes to 20% of the initial liver activity and in female cells, to 35%, whereas the activity tested toward 1-chloro-2,4-dinitrobenzene as substrate was stable. UDP-glucuronosyltransferase activities, tested toward p-nitrophenol and phenolphthalein as substrates, were slightly increased during 24 hr of culture of hepatocytes to levels higher than in liver before perfusion. The level of UDP-glucuronic acid, the endogenous substrate for the enzyme, was reduced after isolation to only 6% of the initial liver value, and then increased during culture to a level approximately 60% of normal. Thus, the changes in xenobiotic biotransformation enzymes and associated constituents in cultured hepatocytes were not uniform, although biotransformation capability remained reasonably intact.     

Regulation of mRNA expression of MDR1, MRP1, MRP2 and MRP3 by prototypical microsomal enzyme inducers in primary cultures of human and rat hepatocytes

The mRNA induction of various transporters by rifampicin (Rif), dexamethasone (Dex) and omeprazole (Ome) was investigated in primary cultures of cryopreserved human and rat hepatocytes. Analysis was performed by quantitative real-time RT-PCR using primers and TaqMan probes. In primary cultures of human hepatocytes, mRNA levels of MDR and MRP1 were increased by about 1.5 fold and 1.3 fold, respectively, by exposure to Rif at 2 to 50 microM as compared with 0.1% DMSO-treated controls. MRP2 mRNA levels in the same human hepatocytes were significantly increased by 1.2 to 1.8 fold by exposure to Rif at 50 microM as compared with controls. In primary cultures of rat hepatocytes, Mdr1a and Mdr1b mRNA levels were not increased or only slightly increased at 24 hr by exposure to any of the inducers at 2, 10 or 50 microM. Mrp2 mRNA levels in the same rat hepatocytes were significantly increased by 7 to 45 fold by exposure to Dex at 2 microM as compared with controls. Based on the species differences observed in the present study, primary cultures of cryopreserved hepatocytes from both the human and rat should be useful in preclinical drug development for evaluating candidate drugs for transporter induction.     

Interactions of insulin, thyroxine and dexamethasone with growth hormone in the control of steroid metabolism by isolated rat hepatocytes

In vivo studies have established that continuous infusion of male rats with growth hormone (GH) results in the feminisation of hepatic steroid metabolism. In this study, however, no feminising effects were observed in response to GH treatment alone in isolated hepatocytes. Combined incubations with insulin and GH or with dexamethasone and GH showed that GH antagonised the stimulatory effects of insulin and dexamethasone respectively. Pretreatment of hepatocytes over 30 min with thyroxine and GH also resulted in a decrease in enzyme activity as compared to the response with thyroxine alone. Thus no feminisation effects were seen with any of the combinations of hormones noted above. Preincubation of hepatocytes with dexamethasone, thyroxine and GH, however, resulted in the expression of the feminisation effect noted in vivo. It therefore appears from this study that dexamethasone and thyroxine may play a permissive role in mediating the feminising effects of GH in isolated hepatocytes, and that GH may be interacting with these hormones in vivo to produce such effects.     

Onset of enzyme induction with pregnenolone-16alpha-carbonitrile in male and female rats.

Male and female rats treated with a single 20-mg/kg ip dose of pregnenolone-16alpha-carbonitrile (PCN) produced parallel increases in hepatic cytochrome P-450 and aniline hydroxylase activity. However, the onset of increase in aniline hydroxylase activity and cytochrome P-450 content was slower in male than female animals. The maximal levels achieved in male rats were 89% of the values observed in the female animals. Furthermore, the hepatic aniline hydroxylase activity and P-450 content declined more rapidly in male than in female rats.     

Assessment of the covalent binding potential of 2,2,4-trimethylpentane to rat alpha 2u-globulin

Subchronic exposure of male rats to the nephrotoxin 2,2,4-trimethylpentane (TMP) causes an accumulation of protein droplets in the epithelial cells of the renal cortex. Experimental evidence suggests that these droplets contain alpha 2u-globulin, a low-molecular-weight protein found specifically in the urine of male rats. It has been proposed that aldehyde metabolites of TMP form Schiff base adducts with the lysine groups of alpha 2u-globulin and thereby inhibit renal lysosomal processing of the protein. Accordingly, the ability of TMP and its metabolites to covalently bind to alpha 2u-globulin was examined. As a model, a [14C] formaldehyde-alpha 2u-globulin Schiff base was formed. This protein adduct was stabilized by reduction with cyanoborohydride and could be identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein analysis by SDS-PAGE demonstrated that hepatocytes isolated from male Fischer-344 rats produced significant quantities of alpha 2u-globulin in culture, whereas hepatocytes from female rats did not. A 15-hr exposure of metabolically competent, primary cultures of male rat hepatocytes to [14C] TMP (0.1 and 0.5%, v/v), followed by reduction with cyanoborohydride, dialysis, and analysis with SDS-PAGE, revealed no evidence of radiolabeled alpha 2u-globulin. When [14C]TMP was administered to an adult male Fischer-344 rat (300 mg/kg, ig) 22, 16, and 10 hr before sacrifice, 16% of the administered radioactivity was eliminated in the urine as TMP metabolites. Analysis as above showed no TMP-derived radioactivity in fractions containing alpha 2u-globulin from liver, blood, kidney cortex, or urine. The absence of a detectable covalent interaction between TMP and alpha 2u-globulin following in vitro or in vivo exposure suggests that a TMP-alpha 2u-globulin adduct is not responsible for the excessive formation of protein droplets in the renal cortex of exposed male rats.     

Alteration of acetaminophen metabolism by sulfate and steroids in primary monolayer hepatocyte cultures of rats and mice

Sulfotransferase (ST) is considered to be generally not induced by xenobiotics. However, it has been reported that steroids such as dexamethasone (DEX) and pregnenolone-16a-carbonitrile (PCN) are effective ST inducers in rats, and sulfation of xenobiotics is quite different in rats and mice. The present study is primarily aimed at determining the effect of sulfate and steroids on the metabolism of acetaminophen (AA) in vitro using monolayer cultured hepatocytes of Sprague-Dawley rats and ICR mice. Hepatocytes of rats and mice were incubated with inorganic sulfate (0.25, 0.5, 1.0, 2.0, 4.0 mM) and AA in SO4-depleted media. AA sulfation rates increased as the sulfate concentration was raised to 1.0 mM in rats, whereas the addition of inorganic sulfate to the media had a lesser effect in mice hepatocytes. After pretreatment with DEX (0.1, 1.0, 10, 100 microM) and PCN (0.1, 1.0, 10 microM) for 3 d, hepatocytes were incubated with AA in media containing 4.0 mM SO4-. Pretreatment of the hepatocytes with DEX caused an increase in the glucuronidation and sulfation of AA by 2-3 folds in rats, but to a lesser extent in mice. PCN significantly enhanced the formation of AA-glucuronide and AA-sulfate in mice, but had a minimal effect on rat hepatocytes. In summary, sulfate and DEX markedly enhanced the formation of AA-sulfate in rats hepatocytes, and DEX and PCN increased the sulfation of AA in mice hepatocytes. These results partially support the claim that DEX and PCN are effective ST and uridine diphosphate-glucuronyltransferase inducers in vivo.     

Normal levels of hepatic drug-metabolizing enzymes in neonatally induced, growth hormone-deficient adult male and female rats

The effects of chronic growth hormone deficiency on the growth hormone-dependent, sexually dimorphic hepatic drug-metabolizing enzymes were studied in adult rats of both sexes. Neonatal administration of monosodium L-glutamate produced the expected syndrome characterized by stunted growth, obesity, prolonged vaginal estrus, and an inhibition in the growth of the pituitaries, adrenals, gonads, sexual accessory organs, and kidneys. Associated with these abnormalities was a 90% reduction in the concentration of serum growth hormone in male and female rats. In contrast, the activities of hepatic aniline hydroxylase, total cytochrome P-450, and the Michaelis constants and maximal velocities for hexobarbital hydroxylase and UDP-glucuronosyltransferase were unaffected by the profound deficiency in growth hormone.     

Changes in antioxidant defence systems induced by cyclosporine A in cultures of hepatocytes from 2- and 12-month-old rats

The in vitro effect of cyclosporine A (CsA) was studied in reference to the production of reactive oxygen species (peroxides and superoxide anion) and to cell enzyme-mediated antioxidant defence in hepatocytes isolated from rats aged 2 and 12 months. Primary cultures of hepatocytes were incubated in the presence of concentrations of cyclosporine in the range of 0 to 50 microM for 24 hr, and the release of lactate dehydrogenase into the culture medium was evaluated as a parameter of cytotoxicity and membrane lysis. Peroxides were quantified by using 2',7'-dichlorodihydrofluorescein diacetate, and superoxide anion levels were evaluated by the fluorescence of dihydroethidium. Enzyme activity and gene expression of catalase and Mn- and Cu,Zn-superoxide dismutase were also assayed. CsA cytotoxicity was significantly higher in hepatocytes from rats aged 12 months when compared to those aged 2 months. Intracellular peroxide content resulted in a dose-dependent increase, while the anion superoxide intracellular level slightly decreased as CsA increased from 0-50 microM. The progressive increase in intracellular peroxides in cell cultures in the range from 0-50 microM CsA was associated with the loss of cell viability and accompanied by significantly higher levels of Mn- and Cu, Zn-superoxide dismutase enzyme activities and mRNAs, and slight increases in catalase activity and mRNA. We conclude that, in primary hepatocyte cultures, the cytotoxicity of CsA was dose-dependent in both age groups and significantly higher in cultures from 12-month-old rats when compared to those from 2-month-old animals. The non-coordinated regulation of the gene expression of antioxidant enzyme systems, i.e. catalase and Mn- and Cu,Zn-superoxide dismutases, evidenced to a greater extent in hepatocytes from the older group of rats, could be one of the mechanisms involved in CsA toxicity.     

Human fetal liver cultures: basal activities and inducibility of epoxide hydrolases and aryl hydrocarbon hydroxylase

The environmental influence of various drugs on the epoxide hydrolase with styrene oxide (EHSO) or benzo(a)pyrene-4,5-oxide (EHBPox) as substrate and the aryl hydrocarbon hydroxylase (AHH) activity was studied in monolayer cultures of human fetal hepatocytes (HFH) obtained at legal abortions. Hepatocytes were isolated by trypsin treatment of liver fragments and primary HFH cultures were maintained in Eagle's minimum essential medium supplemented with 15% newborn calf serum. The HFH were plated on culture dishes and allowed to 'settle' for one day before adding various drugs (in 1 microliter dimethylsulfoxide/ml) or solvent only and assay 1-2 days later. The basal AHH activity [assayed with 3H-benzo(a)pyrene as substrate] varied between 2 and 8.4 pmoles/min/mg protein and the basal EHSO activity was 0.3-4.9 nmoles/min/mg protein (n = 6) after one or two days' culture. The corresponding activity of EHBPox was 0.23-1.48 nmoles/min/mg protein (n = 5). Exposure of cultures to 2 mM phenobarbital (Pb), 2.5-25.0 microM benzanthracene (BA), 0.1 mM trans-stilbene oxide (TSO), or 5 microM beta-naphtoflavone (beta NF) resulted in a 1.2-3.7-fold induction of EHSO. Induction of EHBPox was also observed with Pb, beta NF, BA and TSO as inducers. Pb gave a dose-dependent induction of both EH at 0.1, 1.0 and 2.0 mM. Our results demonstrate that EH and AHH activities in HFH cultures are inducible by classical in vivo inducers. Although difficult to prove, it is plausible that such induction takes place also in intrauterine life.     

Comparative metabolism of flunarizine in rats, dogs and man: an in vitro study with subcellular liver fractions and isolated hepatocytes.

1. The biotransformation of 3H-flunarizine ((E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine dihydrochloride, FLUN) was studied in subcellular liver fractions (microsomes and 12,000 g fraction) and in suspensions or primary cell cultures of isolated hepatocytes of rats, dogs and man. The major in vitro metabolites were characterized by h.p.l.c. co-chromatography and/or by mass spectrometric analysis. 2. The kinetics of FLUN metabolism was studied in microsomes of dog and man. The metabolism followed linear Michaelis-Menten kinetics over the concentration range 0.1-20 microM FLUN. 3. A striking sex difference was observed for the in vitro metabolism of FLUN in rat. In male rats, oxidative N-dealkylation at one of the piperazine nitrogens, resulting in bis(4-fluorophenyl) methanol, was a major metabolic pathway, whereas aromatic hydroxylation at the phenyl of the cinnamyl moiety, resulting in hydroxy-FLUN, was a major metabolic pathway in female rats. In incubates with hepatocytes, these two metabolites were converted to the corresponding glucuronides. 4. In human subcellular fractions, aromatic hydroxylation to hydroxy-FLUN was the major metabolic pathway. In primary cell cultures of human hepatocytes, oxidative N-dealkylation at the 1- and 4-piperazine nitrogen and glucuronidation of bis(4-fluorophenyl)methanol were observed. The in vitro metabolism of FLUN in humans, resembled more than in female rats and in dogs than that in male rats. 5. The present in vitro results are compared with data of previous in vivo studies in rats and dogs. The use of subcellular fractions and/or isolated hepatocytes for the study of species differences in the biotransformation of xenobiotics is discussed.     

Effect of dexamethasone on cytochrome P-450 mediated metabolism of 2-acetylaminofluorene in cultured rat hepatocytes

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to investigate the effect of dexamethasone on cytochrome P-450 activity in cultured rat hepatocytes. In control hepatocytes the metabolism of AAF to its 1-, 5-, 7-, 9- and N-hydroxylated metabolites rapidly declined in culture over the first 24 hr while 3-hydroxylation remained relatively constant. These activities either remained unchanged or increased slightly during the next 48 hr in culture. The addition of dexamethasone (100 nM) to the culture medium had little effect in arresting the initial decline but by 72 hr the 7-, 5- and 3-hydroxylations increased to values 2.5, 16 and 21 times the respective 24-hr values. The inductive effect of dexamethasone on the 3- and 5-hydroxylations of AAF was maximal at 100 nM whereas the 7-hydroxylation increased linearly as a function of the dexamethasone concentration up to 1 microM. Cortisol and corticosterone and the non-glucocorticoids fluoxymesterone and methyltestosterone induced a pattern of AAF metabolism resembling that in dexamethasone-treated cultures, suggesting that a range of steroids not restricted to glucocorticoids may induce multiple cytochrome P-450 isozymes via related mechanisms. Pregnenolone 16 alpha-carbonitrile induced only the 7-hydroxylation of AAF probably reflecting induction of cytochrome P-450p. While dexamethasone was a strong inducer of the 3- and 5-hydroxylations of AAF in hepatocyte culture, assay of these activities in freshly isolated cells after in vivo treatment with dexamethasone showed a strong induction of 7-hydroxylation but only small effects on 3- and 5-hydroxylations. Indeed the profile of AAF metabolism induced in culture by dexamethasone resembles more closely the profile induced by 3-methylcholanthrene in vivo. These data suggest that factors yet to be identified strongly influence the steroid-induced pattern of cytochrome P-450 gene expression.     

DNA damage induced by 4,4'-methylenedianiline in primary cultures of hepatocytes and thyreocytes from rats and humans

4,4'-Methylenedianiline (MDA), an aromatic amine used in various industrial processes and previously found to induce tumor development in liver and thyroid of mice and rats, was evaluated for its DNA-damaging activity in primary cultures of hepatocytes and thyreocytes from rat and human donors. After exposure for 4 and 20 h to MDA concentrations ranging from 10 to 180 microM, a statistically significant increase in the frequency of DNA lesions was revealed by the Comet assay in primary hepatocytes and thyreocytes from donors of both species, the response being dose dependent up to 56-100 microM MDA. DNA fragmentation was more marked after 4 than after 20 h exposure in all four cell types. DNA was damaged to a lesser extent in human hepatocytes and thyreocytes than in corresponding rat cells and in both species in hepatocytes than in thyreocytes. In both rat and human hepatocytes a 20-h exposure to the same MDA concentrations elicited a modest amount of DNA repair synthesis, as evaluated by autoradiography. Evidence of a partial reduction of DNA damage, and therefore of only partial DNA repair, was observed in rat hepatocytes and in rat and human thyreocytes incubated for 16 h in MDA-free medium after a 4-h MDA treatment. A 4-h exposure to 56, 100, and 180 microM MDA did not induce DNA lesions in primary cultures of cells from three rat organs, kidney, urinary bladder mucosa, and brain, which are resistant to MDA carcinogenic activity. Under the same experimental conditions any evidence of DNA damage was absent in primary kidney and urinary bladder cells from human donors. Taken as a whole the results of this work indicate that MDA is specifically activated to DNA-damaging reactive species by hepatocytes and thyreocytes in both rats and humans and thus suggest that liver and thyroid might be the targets of the carcinogenic activity of MDA also in humans.     

Azoreduction of dimethylaminoazobenzene (DAB) in primary cultures of rat hepatocytes. Effect of hypolipidemic agents

This laboratory has investigated the azoreduction of the hepatocarcinogen, N,N-dimethyl-4-aminoazobenzene (DAB), by hepatic microsomal cytochrome P-450 (P-450) and its specific induction by the hypolipidemic drug, clofibrate. To extend these studies further, a primary hepatocyte culture system was developed as a model. Hepatocytes isolated from male Sprague-Dawley rats were incubated in a basal medium containing fetal calf serum, insulin, and hydrocortisone for up to 96 hr with varying concentrations of clofibrate or nafenopin, a related hypolipidemic agent. Both DAB azoreductase and laurate hydroxylase activities decreased rapidly in control cultures. However, there was gradual marked induction of both activities in medium supplemented with clofibrate: hydrocortisone was required for induction. Nafenopin stabilized and induced DAB azoreductase and laurate hydroxylase activities, respectively. The responses of both activities were dose dependent. DAB azoreductase and laurate hydroxylase activities in control hepatocytes retained their ability to respond to clofibrate for up to 96 hr, although the response gradually diminished after 24 hr. In all cases, maximal induction of both enzyme activities was observed 72 hr after addition of drug. Phenobarbital and beta-naphthoflavone did not induce DAB azoreduction, although the normal induction of other P-450-catalyzed pathways, 7-ethoxycoumarin O-deethylation and ethlymorphine N-demethylation, were seen. Suppression of DAB azoreductase activity by inhibitors of P-450 activity confirmed the involvement of this enzyme in DAB azoreduction. The results demonstrate that a primary culture of rat hepatocytes is a useful model for studying the regulation of DAB azoreductase activity.