CAR/PXR provide directives for Cyp3a41 gene regulation differently from Cyp3a11.

This study reports that Cyp3a41 gene contains 13 exons and is localized on the chromosome 5. CYP3A41 is a female-specific isoform that is predominantly expressed in the liver. Estrogen signaling is not responsible for its female specificity. CYP3A41 expression in kidney and brain is observed only in 50% of mice examined. PXR mediates dexamethasone-dependent suppression of CYP3A41. In contrast to CYP3A11, CYP3A41 expression is not induced by pregnenolone-16alpha-carbonitrile (PCN) in wild-type mice, but is significantly suppressed by PCN in PXR(-/-) mice. Phenobarbital and TCPOBOP induce CYP3A11 expression only in the presence of CAR, but have no effect on CYP3A41 expression. Immunoblot and erythromycin demethylase activity analysis reveal robust CYP3A induction after PCN treatment, which is poorly correlated to CYP3A41. These findings suggest a differential role for CAR/PXR in regulating individual CYP3A isoforms by previously characterized CYP3A inducers.

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones.

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b5. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6beta-, 16alpha-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6beta, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the Vmax/Km values for P450 3A9 using either 21-hydroxprogesterone or 6beta-hydroxyprogesterone as a substrate, 4-pregnen-6beta, 21-diol-3, 20-dione may be formed either by 6beta-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6beta-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.

Chronic intragastric infusion of ethanol-containing diets induces CYP3A9 while decreasing CYP3A2 in male rats.

The CYP3A subfamily is the most abundant of the human hepatic cytochrome P450 enzymes. They mediate the biotransformation of many drugs, including a number of psychotropic, cardiac, analgesic, hormonal, immunosuppressant, antineoplastic, and antihistaminic agents. We studied diet/ethanol interactions using total enteral nutrition in adult male Sprague-Dawley rats with diets containing 16% protein, ethanol (13 g/kg), corn oil (fat; 25-45%), and carbohydrate (CHO; 1-21%). Using this model, chronic ethanol feeding decreased CYP3A activity (testosterone 6 beta-hydroxylation) and apoprotein levels (Western blot) (P <.05) and these effects were independent of the dietary CHO/fat ratio. The CYP3A2 mRNA levels decreased (P <.05) in the rats fed ethanol-containing diets by 73 to 83% compared with rats fed control diets, regardless of the CHO/fat ratio. In contrast, ethanol induced CYP3A9 mRNA levels (P <.05) and this effect was greater (P <.05) in the high-CHO/low-fat group (11.3-fold) than in the low-CHO/high-fat group (2.6-fold). Purified recombinant rat P450 3A9 had a chlorzoxazone 6-hydroxylase activity with a turnover number 1.3 nmol/min/nmol of P450. These results indicate that 1) ethanol differentially affects the expression of CYP3A gene family and this regulation appears to be modulated by dietary CHO/fat ratio; 2) the decrease in testosterone 6 beta-hydroxylase activity and CYP3A apoprotein levels are most likely due to the ethanol-induced decrease in CYP3A2 mRNA levels; and 3) CYP3A9 is induced by ethanol and is a low-affinity, high-K(m) chlorzoxazone hydroxylase.

Recombinant cytochrome P450 2D18 metabolism of dopamine and arachidonic acid.

The function of cytochrome P450 (P450) in the mammalian brain is not well understood. In an effort to further this understanding, this study identifies two endogenous substrates for P450 2D18. Previous reports have shown that this isoform is expressed in the rat brain, and the recombinant enzyme catalyzes the N-demethylation of the antidepressants imipramine and desipramine. By further examining the substrate profile of P450 2D18, inferences can be made as to potential endogenous P450 substrates. Herein we demonstrate the metabolism of the central nervous system-acting compounds chlorpromazine and chlorzoxazone with turnover numbers of 1.8 and 0. 9 nmol/min/nmol, respectively. Because the four aforementioned pharmaceutical substrates work by binding to neurotransmitter receptors, binding assays and oxidation reactions were performed to test whether dopamine is a substrate for P450 2D18. These data indicate a K(S) value of 678 microM and that P450 2D18 can support the oxidation of dopamine to aminochrome through a peroxide-shunt mechanism. We also report the P450 2D18-mediated omega-hydroxylation and epoxygenation of arachidonic acid, primarily leading to the formation of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, compounds that have been shown to have vasoactive properties in brain, kidney, and heart tissues. The data presented herein suggest a possible role for P450 involvement in membrane and receptor regulation via epoxyeicosatrienoic acid formation and a potential involvement of P450 in the oxidation of dopamine to reactive oxygen species under aberrant physiological conditions where the sequestering of dopamine becomes compromised, such as in Parkinson's disease.

Extrahepatic drug metabolizing enzymes.

Drug metabolizing enzymes are known to be present and active in most extrahepatic tissues. In this review, evidence is presented that expression and activity of several extrahepatic drug-metabolizing enzymes are regulated in unique ways, which may be associated with tissue functions and activities. In several instances evidence is offered that hormonal effects may be regulated through tissue specific distribution and/or responses of transcription factors.

Expression, induction and regulation of the cytochrome P450 monooxygenase system in the rat glioma C6 cell line.

The cytochrome P450 monooxygenase system consists of NADPH-cytochrome P450 reductase (P450 reductase) and cytochromes P450, which can catalyze the oxidation of a wide variety of endogenous and exogenous compounds. P450 reductase transfers reducing equivalents from NADPH to P450, which in turn catalyzes metabolic reactions. In previous studies, we have used the rat glioma C6 cell line as an in vitro model system and identified the presence of P450 reductase and of cytochrome P450 1A1, 1A2, 2A1, 2B1/2, 2C7, 2D1-5 and 2E1 by reverse transcription followed by polymerase chain reaction (RT-PCR). In C6 cells, the induction of P450 1A1 and 2B1/2 at mRNA level after BA (benzo(a)anthracene) or PB (phenobarbital) treatments was detected. In this study, analysis of microsomal preparations of glioma C6 cells was utilized to demonstrate the presence of P450 2B and P450 reductase at the protein level. ELISAs showed that PB induced P450 2B proteins 12-fold. These experiments further establish that the rat glioma C6 cell line contains an active cytochrome P450 monooxygenase system that can be induced by P450 inducers. We also found that the mRNAs of P450 1A1 and 2B1/2 from glioma C6 cells do not bind to the oligo(dT)-based separation techniques efficiently, suggesting that they may have very short poly(A) tails. The half-lives of P450 1A1 and 2B1/2 mRNA in glioma C6 cells are 1/10 and 1/3 of that in liver, respectively. This may partly contribute to the low expression level of P450s in glial cells. The induction of P450s by BA or PB did not change their mRNA half-lives, indicating the induction may be due to transcriptional regulation. In summary of this study, we believe the presence of the cytochrome P450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors.

Regulation of CYP3A9 gene expression by estrogen and catalytic studies using cytochrome P450 3A9 expressed in Escherichia coli.

Sexual dimorphism in the expression of CYP3A9, a novel form of CYP3A from rat brain, is shown for the first time in rat brain as well as in rat liver. CYP3A9 expression is female specific in rat liver as judged by its 10-fold higher expression in females than in males. CYP3A9 gene expression was inducible by estrogen treatment both in male and in female rats. Ovariectomy of adult female rats elicited a drastic reduction on the mRNA level of CYP3A9 which could be fully restored by estrogen replacement. These results suggest that estrogen may play an important role in the female-specific expression of the CYP3A9 gene. P450 3A9 recombinant protein was expressed in Escherichia coli by means of the pCWOri+ expression vector and the MALLLAVF amino terminal sequence modification. This construct gave a high level of expression (130 nmol P450 3A9/liter culture) and the recombinant protein of the modified P450 3A9 was purified to electrophoretic homogeneity with a specific content of 10.1 nmol P450/mg protein from solubilized fractions through two chromatographic steps. The purified P450 3A9 protein was active in the metabolism of imipramine, erythromycin, benzphetamine, and ethylmorphine as well as 17beta-estradiol in a reconstituted system containing lipid and rat NADPH-P450 reductase. Of special interest is the finding that P450 3A9 can catalyze the formation of desipramine with a turnover number of 4.9 nmol/min/nmol P450, suggesting the possible involvement of this isoform in the metabolism of imipramine in brain. Optimal reconstitution conditions for P450 3A9 activities required a lipid mixture (1:1:1 mixture of L-alpha-dilauroyl phosphatidylcholine, L-alpha-dioleoyl phosphatidylcholine, and phosphatidylserine) and GSH.

Regulation of expression of cytochrome P-450 2D mRNA in rat brain with steroid hormones.

Cytochrome P-450 2D is a subfamily of the cytochrome P-450-dependent mixed function oxidase system which is widely distributed in the various tissues of mammals. Sex steroid hormones have been shown to affect the expression of CYP2D in rat brain. Testosterone treatment of ovariectomized female rats elicits a dramatic increase in CYP2D expression, estrogen treatment brings about a modest increase in brain CYP2D expression and reduces the increase in CYP2D expression elicited with testosterone when the two hormones are coadministered. Polymerase chain reaction (PCR) has been used in our laboratory, as well as other laboratories, to measure the low levels of message for various P-450s in brain [Hodgson, A.V., White, T.B., White, J.W., Strobel, H.W., 1993. Expression analysis of the mixed function oxidase system in rat brain by the polymerase chain reaction. Mol. Cell. Biochem. 120, 171-179; Omiecinski, C.J., Redlich, C.A., Costa, P., 1990. Induction and developmental expression of cytochrome P450IA1 messenger RNA in rat and human tissues: detection by the polymerase chain reaction. Cancer Res. 50, 4315-4321]. In this study, competitive PCR (cPCR) approaches have been used to determine effects of progesterone and testosterone on CYP2D expression levels in brains of intact and ovariectomized female rats. When administered for seven treatments, testosterone significantly increases the expression of CYP2D in brain from intact female rats, while repeated treatment with progesterone elicits the opposite effect. Coadministration of testosterone and progesterone causes an intermediate effect such that the net result is an increase in expression only slightly above control levels. Interestingly, when ovariectomized female rats treated with testosterone and progesterone are used as a source of brain tissue for RNA preparation a similar trend toward an intermediate value is seen but the net result is an expression level of CYP2D below the control value. This approach utilizes cPCR to analyze the levels of CYP2D mRNA, semi-quantitatively and quantitatively, in the brains of female intact and ovariectomized Sprague-Dawley rats treated with testosterone, progesterone, a combination of the two or corn oil.

Expression and induction of cytochrome P-450 1A1 and P-450 2D subfamily in the rat glioma C6 cell line.

The cytochrome P-450 (P-450) monooxygenase system can catalyze the oxidation of a wide variety of endogenous and exogenous compounds, including steroid hormones, fatty acids, drugs and pollutants. The functions of this system are as diverse as the substrates. Though this enzyme system has the highest level of activity in the liver, it is present in other tissues, including brain. In this study, we have established the rat glioma C6 cell line as an in vitro model system to examine the expression and induction of P-450 1A1 and the P-450 2D subfamily. Rat glioma C6 cells were treated with P-450 inducers phenobarbital (PB) or benzo[a]anthracene (BA). The presence of P-450 1A1 and 2D1-5 was detected by reverse transcription followed by polymerase chain reaction (RT-PCR) and confirmed by restriction enzyme digestion. The induction of P-450 1A1 and 2D1-5 was quantified using competitive PCR. Although P-450 2D1-5 do not seem to be affected by PB or BA treatment, tenfold induction of P-450 1A1 mRNA after BA treatment was detected. Western blot analysis of microsomal preparations of glioma C6 cells demonstrated the presence of P-450 1A1 at the protein level. ELISAs showed that BA induces P-450 1A1 proteins 7.3-fold. These experiments provide further evidence that the rat glioma C6 cell line contains an active cytochrome P-450 monooxygenase system which can be induced by P-450 inducers. In summary, we believe the presence of the cytochrome P-450 monooxygenase system in glial cells of the brain may be important in chemotherapy and carcinogenesis of brain tumors.

Anatomical distribution of NADPH-cytochrome P450 reductase and cytochrome P4502D forms in rat brain: effects of xenobiotics and sex steroids.

Since the brain is not a homogeneous organ, but one dependent upon the well orchestrated interaction of numerous parts, pathology in one nucleus may have a large impact upon its overall function. Hence, the anatomical distribution of the P450 monoxygenase system in brain, as well as the regulation of its expression, is important in elucidating its function in that organ. In order to study these issues, female rats-both ovariectomized and not-were treated with a number of xenobiotic compounds and sex steroids. The brains from these animals were dissected into 8 discrete regions and the presence and relative level of message for P4502D and P450 reductase determined using polymerase chain reaction. Results of this investigation indicate the presence of mRNA for reductase and P4502D isoforms throughout the rat brain. In addition, quantitative PCR was utilized to demonstrate the effects of xenobiotics (phenobarbital, beta-naphthoflavone, imipramine) and sex hormones (testosterone, estrogen) on the levels of these messages in the female rat brain. Significant induction of message for P4502D forms was noted with testosterone in the absence of estrogen. The level of mRNA for reductase was not significantly influenced by any of the treatments, however. These results raise the issue of a sexual dimorphism in the rat regarding P4502D expression in brain.

Expression of multiple forms of brain cytochrome P450.

Multiple forms of cytochrome P450 (P450) in brain tissue have been demonstrated to be expressible in brain tissue using polymerase chain reaction (PCR) techniques, Northern blotting, hydroxylation activity assessment and cloning approaches. The antidepressant drug imipramine is metabolized by brain microsomes to multiple products by pathways inhibitable by quinidine, 7,8-benzoflavone, and ketoconazole, well-known inhibitors of P450-catalyzed reactions. Moreover, PCR studies revealed that a number of P450s are expressible in brain tissue and in glioma C6 cells. Quantitative PCR studies further demonstrated the response of many of these forms to induction in agreement with hydroxylation activity results.

Identification of inducible mixed function oxidase system in rat glioma C6 cell line.

The mixed function oxidase system consists of NADPH-cytochrome P450 reductase (P450 reductase) and various isoforms of cytochrome P450 (P450), which can catalyze the oxidation of a broad range of endogenous and exogenous compounds. In this study, we examined the rat glioma C6 cell line for the presence of P450 reductase and three isozymes of cytochrome P450, 1A1, 2B1, and 2B2, by reverse transcription followed by PCR (RT-PCR). Rat glioma C6 cells were treated with hepatic P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). Cytochromes P450 1A1, P450 2B1, and P450 2B2, and P450 reductase, were detected in all the different treatment groups. Restriction digestion was used to confirm the PCR fragments and the expected digestion products were obtained. The induction of P450 1A1 and 2B was quantified using competitive PCR. Ten- and five-fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected by competitive PCR. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 spectra with absorption at 450 nm. Ethoxyresorufin O-deethylase activity (11.5 +/- 1.7 pmol/min/mg of microsomal protein) and pentoxyresorufin O-dealkylation activity (8.9 +/- 1.4 pmol/min/mg of microsomal protein) confirmed the induction of P450 1A and 2B at the protein level in response to BA or PB treatments, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active mixed function oxidase system that can be induced by hepatic P450 inducers.

Probing the putative cytochrome P450- and cytochrome c-binding sites on NADPH-cytochrome P450 reductase by anti-peptide antibodies.

Two regions (amino acid residues 110-130 and 204-218) of NADPH-cytochrome P450 reductase (reductase) have been shown to be the putative binding sites for the interaction with cytochrome P450 or cytochrome c. To obtain further insight into the molecular mechanism of protein-protein interaction between these proteins, three anti-peptide antibodies (1A, 2A, and 3A) were generated against the peptides corresponding to these two regions on rat reductase to study the interaction between the reductase and cytochrome P450 or cytochrome c. All three anti-peptide antibodies have high affinity for their peptide antigens on ELISA (titre > 1 x 10(-6) g/L), and they also bind to rat reductase on ELISA under both denatured and native conditions, suggesting that these regions are on the surface of the protein. 1A and 3A also bind to rabbit and human reductase, though 1A binds to human reductase with lower affinity. Antibody 2A does not bind to rabbit or human reductase. Western blot analysis using these anti-peptide antibodies showed similar results. Antibodies 1A and 3A inhibit both cytochrome P4501A1-dependent ethoxycoumarin hydroxylation activity and P4502B1-dependent pentoxyresorufin dealkylation activity, but the inhibition by 1A and 3A was not additive. Antibodies 1A and 3A also have inhibitory effects on the activity of P4501A1-dependent ethoxycoumarin hydroxylation reconstituted with reductase from rabbit and human. However, none of the three anti-peptide antibodies inhibits cytochrome c reduction by rat reductase. These data suggest that reductases from rat, rabbit, and human share similar structure in at least two regions which appear to be on the surface of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of cytochromes P450 1A2, 2A1, 2C7, 2E1 in rat glioma C6 cell line by RT-PCR and specific restriction enzyme digestion.

Reverse transcription (RT)/polymerase chain reaction (PCR) was used to detect the expression of six isoforms of cytochrome P450 which belong to five P450 subfamilies in rat glioma C6 cell line. P450 1A2, 2A1, 2C7, 2E1 were identified by RT-PCR in all samples, including untreated cells as well as cells treated with phenobarbital (PB) or benzo(a)anthracene (BA). P450 3A and 2C11 were not detected in our glioma samples although they were detected in liver tissues in our previous study. To confirm proper PCR products, various restriction enzymes were used to digest the PCR fragments and the expected digestion patterns were obtained. These results demonstrate for the first time that glioma C6 cells, representing a single cell type of rat central nervous system (CNS), contain a P450-dependent metabolism system which seems important for understanding drug metabolism, neurotransmission as well as tumor etiology and chemotherapy in brain.

Catalysis of the oxidation of steroid and stilbene estrogens to estrogen quinone metabolites by the beta-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 beta-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-16 alpha-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 beta-naphthoflavone-induced microsomes and cytochrome P450 IA1 had the highest "affinity" for cumene hydroperoxide cofactor (Km = 77 microM). Cofactor concentrations above 250 microM 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 beta-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.

Ethoxyresorufin O-deethylase activity in intact human cells.

The cytochrome P450IA (P-450) family can be induced in both primary and continuous cell culture, as well as in lymphocytes, by polycyclic aromatic and halogenated hydrocarbons. Ethoxyresorufin O-deethylase (EROD) activity has been correlated with both cytochrome P450IA1 and arylhydrocarbon hydroxylase activity in rat hepatocytes. This activity can be assessed in intact cells in culture after induction with benzo[a]anthracene by measuring the production of resorufin and its subsequent release to the medium. EROD activity was observed in intact cells of LS174T, a human colon tumour cell line, at a level (14 pmol/min/mg protein) comparable with that in microsomal preparations (24 pmol/min/mg) and lysed cells (13 pmol/min/mg). Furthermore, this cell line showed time- and concentration-dependent production of resorufin. alpha-Naphthoflavone (5 mum), a P450IA inhibitor, reduced the resorufin production to 5% of control levels, but metyrapone (10 mum) had no significant effect. Dicumarol (10 mum), an inhibitor of quinone oxidoreductase, had only a small effect on the production of resorufin by the LS174T cell line (111% of control). Another human colon tumour cell line, DiFi, also showed benzo[a]anthracene-inducible resorufin production. This intact cell assay has the advantage of allowing the use of cells for preliminary assays without their destruction, and may be of use in quickly screening large numbers of xenobiotics for the ability to induce P450IA1 in a human cell line.

Characterization of cytochrome P450-dependent dimethylhydrazine metabolism in human colon microsomes.

Polyclonal antibodies to components of the rat liver cytochrome P450 system were used to examine the composition and function of the microsomal cytochrome P450-dependent monooxygenase system of human colonic mucosal cells. Anticytochrome P450 reductase antibody gave a strong band of immunocross-reactivity in human colon microsomes at the same molecular weight level as purified cytochrome P450 reductase from rat liver, as well as hepatic microsomes isolated from untreated or phenobarbital-treated rats. These results demonstrate the presence of cytochrome P450 reductase in human colon cells. Similarly, cytochromes P450 IIB1 and IIA1 also appear to be present in Western blots of human colon microsomes. These antibodies, as well as antibodies to reductase and cytochrome b5, inhibit dimethylhydrazine metabolism in human colon microsomes to varying degrees. These data argue for a functional P450-dependent drug metabolism system in colon capable of activating/metabolizing the colon-specific model carcinogen, 1,2-dimethylhydrazine.

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans.

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line.

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IA1 or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P450IA1 and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P450IA1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.