Reduced inflammatory potential of peritoneal macrophages recruited in mice pretreated with a glycolipid synthesis inhibitor.

Integral components of mammalian cell membranes, glycosphingolipids (GSL) reside in specialized plasma membrane microdomains critical for cell signaling. N-alkylated nojirimycins are compounds developed for GSL substrate deprivation therapy, blocking GSL synthesis by specifically inhibiting an essential enzyme, ceramide glucosyltransferase. Peritoneal macrophages recruited in mice pretreated with an inhibitory N-alkylnojirimycin displayed a reduced capacity to release either TNFalpha or interleukin-6 when re-exposed to whole killed E. coli in vitro. Cell viability and protein content were not affected. A nojirimycin analogue without GSL inhibitory capacity had no effect. The results show inhibition of GSL synthesis in vivo by an N-alkylnojirimycin can reduce the response to an inflammatory stimulus and indicate N-alkylnojirimycins have experimental and potential clinical value for modulating innate immune responses in vivo.

Role of cytochrome P450 1A2 in bilirubin degradation Studies in Cyp1a2 (-/-) mutant mice.

In congenital jaundice, which is due to defects of bilirubin gluruconidation, bilirubin is degraded by an alternative pathway into unidentified products. Previously, it was shown that plasma bilirubin levels can be decreased in rats with this defect by inducers of CYP1A enzymes. Here, liver microsomes from rats or mice treated with beta-naphthoflavone (BNF) or 3-methylcholanthrene (3 MC) had increased activity for bilirubin degradation. The activity was further stimulated by addition of the coplanar molecule 3,4,3',4'-tetrachlorobiphenyl (TCB). There was more stimulation of bilirubin degradation by TCB in microsomes from BNF-treated rats than in microsomes from BNF-treated mice. CYP1A1 to CYP1A2 ratios were greater in rats treated with BNF. In Cyp1a2 (-/-) mutant mice, 3-MC treatment did not increase the rate of bilirubin degradation, but TCB increased this degradation severalfold. Between SWR and C57BL/6 inbred mouse strains that have a 2-fold difference in hepatic constitutive CYP1A2 levels, there was also a 2-fold difference in bilirubin degradation; TCB did not stimulate in either strain. We conclude that CYP1A2 is responsible for microsomal bilirubin degradation in the absence of TCB. TCB was required for bilirubin degradation by CYP1A1. Manipulation of CYP1A2 may be of therapeutic benefit in patients with these diseases of bilirubin conjugation.

Uroporphyria in Hfe mutant mice given 5-aminolevulinate: a new model of Fe-mediated porphyria cutanea tarda.

Porphyria cutanea tarda (PCT), a liver disease with skin lesions caused by excess liver production of uroporphyrin (URO), is associated with consumption of alcoholic beverages or estrogens, and moderate iron overload. Recently, it has been shown that many PCT patients carry mutations in the HFE gene, which is responsible for hereditary hemochromatosis. Mice homozygous for either the null mutation in the Hfe gene or the C282Y missense mutation rapidly accumulate hepatic parenchymal iron similar to patients with hemochromatosis. Here we investigated whether disruption of the murine Hfe gene would result in hepatic uroporphyria. Mice homozygous for the Hfe-null mutation accumulated high levels of hepatic URO when fed 5-aminolevulinate (ALA). Hfe (+/-) mice also accumulated hepatic URO when fed ALA, but at a much slower rate. The amount of accumulated URO in the null mutant mice was similar to that in wild-type mice treated with iron carbonyl in the diet, or injected with iron dextran. Iron in both wild-type and Hfe (+/-) mice was mostly in Kupffer cells. In contrast, Hfe (-/-) mice had considerable parenchymal iron deposition as well, in a pattern similar to that observed in wild-type mice treated with iron carbonyl. URO accumulation was accompanied by 84% and 33% decreases in hepatic uroporphyrinogen decarboxylase activities in Hfe (-/-) and Hfe (+/-) mice, respectively. No increases in CYP1A2 or other cytochrome P450s were detected in the Hfe-null mutant mice. We conclude that this experimental model of uroporphyria is a valid model for further investigations into the mechanism of PCT.

Short-term treatment with alcohols causes hepatic steatosis and enhances acetaminophen hepatotoxicity in Cyp2e1(-/-) mice.

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.

Effect of arsenite on the induction of CYP1A4 and CYP1A5 in cultured chick embryo hepatocytes.

We had reported previously that 2.5-5 microM sodium arsenite decreased the phenobarbital-mediated induction of CYP2H activity and protein but not CYP2H1 mRNA in chick-embryo hepatocyte cultures. Induction of a CYP1A activity and protein by 3-methylcholanthrene was also decreased by low arsenite concentrations; however, CYP1A mRNAs were not measured in those studies. We report here that low concentrations of arsenite decreased induction of activities and mRNAs of two chicken cytochromes P450, CYP1A (1A4 and 1A5), by 3-methylcholanthrene in chick-embryo hepatocyte cultures. Arsenite treatment did not affect the turnover of either mRNA, nor did it decrease the superinduction of each mRNA caused by treatment with cycloheximide in addition to 3-methylcholanthrene. Glutathione depletion enhanced the effect of arsenite to decrease induction of CYP1A4. These results indicate the induction of CYP1A4 and 1A5 is inhibited by sodium arsenite at the level of transcription, suggesting that the Ah receptor complex may be involved.

The role of conjugation in hepatotoxicity of troglitazone in human and porcine hepatocyte cultures.

In primary human and porcine hepatocyte cultures, we investigated the relationship between metabolism and cytotoxicity of troglitazone. Treatment of human hepatocytes for 2 h with 10, 20, 25, 35, and 50 microM troglitazone in protein-free medium resulted in concentration-dependent decreases in total protein synthesis. Decreases at 10 and 20 microM were reversible by 24 h, however protein synthesis did not recover at concentrations >/=25 microM. Troglitazone at 50 microM caused cellular death. In porcine hepatocytes, 100 microM troglitazone was lethal, whereas at 50 microM, protein synthesis completely recovered by 24 h. Recovery in protein synthesis was associated with metabolism of parent drug, whereas toxicity correlated (r(2) = 0.82) with accumulation of unmetabolized troglitazone. By 1 h, in human hepatocytes, troglitazone was metabolized to similar amounts of sulfate and quinone metabolites with little glucuronide detected. In contrast, porcine hepatocytes metabolized troglitazone to the similar amounts of glucuronide and the quinone metabolites with little sulfate detected. Exposure of human hepatocytes to a combination of 10 microM troglitazone and 10 microM 2,4-dichloro-4-nitrophenol resulted in a 70% decrease in protein synthesis, associated with 90% inhibition in the formation of troglitazone sulfate, a 4-fold increase in unmetabolized troglitazone, and no effect on formation of the quinone metabolite. Treatment with a combination of acetaminophen or phenobarbital with 20 microM troglitazone resulted in sustained decrease in protein synthesis associated with inhibition of sulfation and accumulation of troglitazone. These results suggest that inhibition of troglitazone sulfation may result in increased hepatotoxicity due to exposure to parent drug, or increased metabolism by alternate pathways.

Acetaminophen hepatotoxicity precipitated by short-term treatment of rats with ethanol and isopentanol: protection by triacetyloleandomycin.

Ethanol and isopentanol are the predominant alcohols in alcoholic beverages. We have reported previously that pretreatment of rats with a liquid diet containing 6.3% ethanol plus 0.5% isopentanol for 7 days results in a synergistic increase in acetaminophen hepatotoxicity, compared with rats treated with either alcohol alone. Here, we investigated the role of CYP3A in acetaminophen hepatotoxicity associated with the combined alcohol treatment. Triacetyloleandomycin, a specific inhibitor of CYP3A, protected rats pretreated with ethanol along with isopentanol from acetaminophen hepatotoxicity. At both 0.25 and 0.5 g acetaminophen/kg, triacetyloleandomycin partially prevented elevations in serum levels of alanine aminotransferase. At 0.25 g acetaminophen/kg, triacetyloleandomycin completely protected 6 of 8 rats from histologically observed liver damage, and partially protected the remaining 2 rats. At 0.5 g acetaminophen/kg, triacetyloleandomycin decreased histologically observed liver damage in 7 of 15 rats. In rats pretreated with ethanol plus isopentanol, CYP3A, measured immunohistochemically, was decreased by acetaminophen treatment. This effect was prevented by triacetyloleandomycin. These results suggest that CYP3A has a major role in acetaminophen hepatotoxicity in animals administered the combined alcohol treatment. We also found that exposure to ethanol along with 0.1% isopentanol for only 3 days resulted in maximal increases in acetaminophen hepatotoxicity by the combined alcohol treatment, suggesting that short-term consumption of alcoholic beverages rich in isopentanol may be a risk for developing liver damage from acetaminophen.

CYP1A2 is essential in murine uroporphyria caused by hexachlorobenzene and iron.

Using Cyp1a2(-/-) mice we previously showed that CYP1A2 is absolutely required for hepatic uroporphyrin accumulation caused by iron and 5-aminolevulinate (ALA) treatment, both in the presence and absence of an inducer of CYP1A2. In this study we have used these mice to investigate whether CYP1A2 has an obligatory role in hepatic uroporphyria caused by hexachlorobenzene (HCBZ), an inducer of CYP2B and CYP3A, as well as CYP1A2. Here we treated mice with HCBZ and iron, with and without the porphyrin precursor, ALA, in the drinking water. In iron-loaded wild-type mice given a single dose of HCBZ and ALA, hepatic uroporphyrin (URO) accumulated to 300 nmol/g liver after 37 days, whereas in Cyp1a2(-/-) mice, there was no hepatic URO, even after an additional dose of HCBZ, and a further 29 days of ALA treatment. A similar requirement for CYP1A2 was found in uroporphyria produced in HCBZ and iron-treated mice in the absence of ALA. As detected by Western immunoblotting, HCBZ induced small increases in CYP2B and CYP3A in the livers of all animals. In the wild-type animals, HCBZ also induced CYP1A2 and associated enzyme activities, including uroporphyrinogen oxidation, by about 2-3-fold. In the Cyp1a2(-/-) mice, HCBZ did not increase hepatic microsomal uroporphyrinogen oxidation. These results indicate that, in mice, CYP1A2 is essential in the process leading to HCBZ-induced uroporphyria. Contributions by other CYP forms induced by HCBZ appear to be minimal.

Relative roles of CYP2E1 and CYP1A2 in mouse uroporphyria caused by acetone.

Porphyria cutanea tarda is a liver disease characterized by excess production of uroporphyrin. We previously reported that acetone, an inducer of CYP2E1, enhances hepatic uroporphyrin accumulation in mice treated with iron dextran (Fe) and 5-aminolevulinic acid (ALA). Cyp2e1(-/-) mice treated with Fe and ALA were used to investigate whether CYP2E1 is required for the acetone effect. Hepatic uroporphyrin accumulation was stimulated by acetone in Cyp2e1(-/-) mice to the same extent as in wild-type mice. In the absence of acetone, uroporphyrin accumulated in Cyp2e1(-/-) mice treated with Fe and ALA, but less than in wildtype mice. However, in Cypla2(-/-) mice, uroporphyrin accumulation caused by Fe and ALA, with or without acetone, was completely prevented. Acetone was not an inducer of hepatic CYP1A2 in the wild-type mice. Although acetone is an inducer of CYP2E1, CYP1A2 appears to have the essential role in acetone-enhancement of uroporphyria.

Effect of sodium arsenite on heme metabolism in cultured chick embryo hepatocytes.

We had previously reported that low concentrations of sodium arsenite (1-5 microM) decreased the induction of cytochrome P450 CYP1A and CYP2H in cultured chick embryo hepatocytes in parallel with increases in heme oxygenase. However, in those studies exogenous heme did not prevent the decrease in CYPs. In this study, we investigated the effect of arsenite on the synthesis and degradation of heme. Arsenite had no effect on induction of 5-aminolevulinic acid synthase mRNA or activity. Arsenite, at concentrations from 1 to 25 microM, had no effect on protoporphyrin synthesis from 5-aminolevulinic acid and did not increase the accumulation of other porphyrins, indicating that the enzymes in the pathway between 5-aminolevulinic acid synthase and ferrochelatase were unaffected by arsenite. Synthesis of heme from radioactive 5-aminolevulinic acid was slightly decreased (less than 20%) by 2.5 microM arsenite, a concentration that decreased induction of CYP1A and CYP2H by greater than 50%. Rates of biliverdin formation and degradation of exogenous heme were not different in cultures treated simultaneously with arsenite and heme or with heme alone. However, arsenite treatment increased biliverdin formation from heme synthesized from added 5-aminolevulinic acid by 60% and decreased the endogenous heme content of the cells by 30%. Our results suggest that although 2.5 microM arsenite induced heme oxygenase four- to sixfold, this had no effect on degradation of exogenous heme. Degradation of heme synthesized from 5-aminolevulinic acid was increased but this did not affect the regulatory heme pool.

Role of small differences in CYP1A2 in the development of uroporphyria produced by iron and 5-aminolevulinate in C57BL/6 and SWR strains of mice.

Previous work has implicated CYP1A2 in experimental uroporphyria caused by polyhalogenated aromatic compounds, and in uroporphyria caused by iron and 5-aminolevulinate (ALA) in the absence of inducers of CYP1A2. Here we examined whether the different susceptibilities of SWR and C57BL/6 strains of mice to uroporphyria in the absence of inducers of CYP1A2 are related to different levels of CYP1A2. Enzymological assays (ethoxy- and methoxyresorufin dealkylases, and uroporphyrinogen oxidation) and immunoblots indicated that there was about twice the amount of hepatic CYP1A2 in SWR mice compared with C57BL/6 mice. Immunohistochemistry revealed that CYP1A2 was located centrilobularly in the liver, and the staining was more intense in SWR mice than in C57BL/6 mice. Hepatic non-heme iron was about double in SWR compared with C57BL/6 mice. In SWR mice given iron dextran, hepatic iron was 1.7-fold that of C57BL/6 mice given iron dextran. SWR mice administered ALA in the drinking water accumulated much less hepatic protoporphyrin than did C57BL/6 mice. To confirm the importance of small increases in CYP1A2, C57BL/6 mice were given a low dose of 3-methylcholanthrene (MC) (15 mg/kg), as well as iron and ALA. There was about a 5- to 6-fold increase in hepatic uroporphyrin accumulation after 32 days on ALA compared with animals not given MC. In these animals, CYP1A2 was increased by 10-fold at 2 days, but returned to basal levels by 14 days. We conclude that small and transient differences in CYP1A2 may be important in the development of uroporphyria.

The use of human hepatocyte cultures to study the induction of cytochrome P-450.

We have previously reported that paclitaxel (Taxol) is a potent inducer of cytochrome P-450 (CYP) 3A protein and CYP3A mRNA in human hepatocyte cultures. Here we report that Taxol increased CYP3A-dependent testosterone 6beta-hydroxylation in intact hepatocytes. This effect was concentration-dependent, with maximal increase in enzyme activity being observed at 10 microM Taxol. Treatment of hepatocyte cultures with concentrations of Taxol higher than 10 microM caused a dose-dependent decrease in testosterone 6beta-hydroxylase activity, amount of CYP3A protein, and total protein synthesis. The maximal CYP3A activity detected after treatment with Taxol or rifampicin was similar in six separate human hepatocyte cultures, suggesting that the cultures have achieved a limit of maximally inducible CYP3A. The fold increase in enzyme activity, however, was different and was inversely related to the level of expression in untreated hepatocytes, with the greatest increases being observed in the hepatocytes that expressed the lowest basal level of CYP3A. Pretreatment of hepatocytes with triacetyloleandomycin resulted in a 90% inhibition of testosterone 6beta-hydroxylase activity. Our results demonstrate the use of human hepatocyte cultures to investigate the induction of cytochrome P-450 by xenobiotics in intact cells and stress the importance of large dose-response studies as well as the need to assess toxicity in these investigations. The response to inducers of CYP3A activity were very consistent among different hepatocyte donors. Absolute values of testosterone 6beta-hydroxylase activity did not vary more than 2- and 5-fold in induced and untreated hepatocytes, respectively.

Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.

An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions.

Effect of arsenite on induction of CYP1A, CYP2B, and CYP3A in primary cultures of rat hepatocytes.

In earlier studies, sodium arsenite treatment was shown to decrease induction of enzymatic activities associated with hepatic CYPs in rats. Here we investigated the effect of sodium arsenite on induction of CYP2B, CYP1A, and CYP3A in primary cultures of rat hepatocytes. Arsenite decreased the induction of all three families of CYP, as measured enzymatically and immunochemically. These decreases in CYPs occurred at concentrations of arsenite (2.5-10 microM) at which no toxicity was observed; however, toxicity was observed at 25 microM arsenite. With 3-methylcholanthrene as inducer, 5 microM arsenite caused a 55% decrease in CYP1A1 immunoreactive protein and enzyme activity, but only a 25% decrease in CYP1A1 mRNA. With phenobarbital (PB) as the inducer, 2.5 microM arsenite decreased CYP2B enzyme activity and immunoreactive protein 50%, with only a 25% decrease in CYP2B1 mRNA. 5 microM Arsenite decreased CYP2B enzyme activity and immunoreactive protein 80%, but decreased CYP2B1 mRNA only 50%, while CYP3A protein was decreased greater than 75% with no decrease in CYP3A23 mRNA. With dexamethasone (DEX) as inducer, 5 microM sodium arsenite caused a 50% decrease in immunoreactive CYP3A and a 30% decrease in CYP3A23 mRNA. Although arsenite-mediated increases in heme oxygenase (HO) inversely correlated with decreases in CYP2B or CYP1A activity, inclusion of heme in cultures treated with inducers of CYP1A or CYP2B did not prevent the arsenite-mediated decreases in these CYPs. Even though added heme induced HO to similar levels with and without arsenite, decreases in CYPs were only observed in the presence of arsenite. These results suggest that, in rat hepatocytes, elevated levels of HO alone are not responsible for arsenite-mediated decreases in CYP.

Mutant yields and mutational spectra of the heterocyclic amines MeIQ and PhIP at the S1 locus of human-hamster AL cells with activation by chick embryo liver (CELC) co-cultures.

Cooking meat and fish at high temperature creates heterocyclic amines (HA) including 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Several HA are mutagens in the Ames' S9/Salmonella assay. While PhIP is a substantial Ames' test mutagen, it is 1000-fold less active than the extraordinarily potent MeIQ. In contrast, MeIQ is significantly less mutagenic than PhIP in several mammalian cell assays, especially in repair-deficient Chinese hamster ovary (CHO) cells. HA are suspect human carcinogens on the basis of (i) epidemiological evidence, (ii) induction of tumors in rodents and monkeys, (iii) DNA adduct formation and (iv) mutagenic capacity. In this study, MeIQ and PhIP were significant mutagens at the S1 locus of co-cultivated human/hamster hybrid AL cells following metabolic activation by beta-napthoflavone (betaNF)-induced chick embryonic liver cultures (CELC). MeIQ was more mutagenic than PhIP in the CELC+AL cell assay. The mutant response curves increase with dose and then plateau (PhIP), or decrease (MeIQ). The inflections in these response curves coincide with dose-dependent decreases in cytochrome CYP1A1 activity. Molecular analysis of S1- mutants indicates that a substantial fraction, >65%, of the mutations induced by PhIP are deletions of 4.2 to 133 (Mbp); half are larger than 21 Mbp. Mutations induced by MeIQ were smaller, most (56%) being less than 5.7 Mbp. When appropriate metabolic activation is combined with a target locus, which can detect both small and large chromosomal mutations, both MeIQ and PhIP are significant mutagens and clastogens in repair proficient mammalian cells.

Mechanism of the synergistic induction of CYP2H by isopentanol plus ethanol: comparison to glutethimide and relation to induction of 5-aminolevulinate synthase.

We had previously found that combined treatment with isopentanol and ethanol synergistically induced CYP2H protein and activity in cultured chick nepatoytes. Here we investigated the mechanism of induction of CYP2H by the alcohols and whether they caused a coordinate induction of 5-aminolevulinate synthase (ALAS) mRNA. Treatment with isopentanol alone or in combination with ethanol resulted in coordinate increases in CYP2H1 and ALAS mRNAs. With isopentanol alone, the amounts of CYP2H1 and ALAS mRNAs at 4 to 6 h were similar to those observed after treatment with the alcohol combination, but declined by 11 h. Readdition of isopentanol at 11 h again increased the expression of both mRNAs, indicating that the decreases at 11 h were due to limiting amounts of inducer. Similar results were observed in cells exposed to low concentrations of glutethimide. In the combined alcohol treatment, increases in CYP2H1 and ALAS mRNAs were sustained from 4 h to 11 h after addition of the alcohols, but decreased to control levels by 24 h. Using pulse labeling to measure de novo synthesis of CYP2H1/2 protein, we found that the increases in CYP2H1/2 protein reflected the increases in CYP2H1 mRNA. The half-life of CYP2H1/2 protein, measured from pulse-chase experiments, was approximately twofold greater than the half-life of CYP2H1 mRNA. Our results indicate that the alcohols and glutethimide coordinately increase ALAS and CYP2H1 mRNA, and that increases in CYP2H1/2 protein arise from increases in its mRNA.

Uroporphyrinogen oxidation catalyzed by human cytochromes P450.

Porphyria cutanea tarda is associated with excess hepatic production of uroporphyrin. Oxidation of uroporphyrinogen to uroporphyrin was previously demonstrated to be specifically catalyzed by cytochrome P450 (CYP) 1A2. Here, we investigated the ability of human CYP1A2 to catalyze uroporphyrinogen oxidation (UROX). UROX activity in human liver microsomes was maximally only 10% of the activity in microsomes from livers of untreated mice. There was a poor correlation of UROX activity with methoxyresorufin demethylation, an activity catalyzed predominantly by CYP1A2 and strongly correlated with immunodetectable CYP1A2. With CYP forms expressed in HepG2 cells, the methoxyresorufin demethylation and (ethoxyresorufin deethylation) activities of murine and human CYP1A2 forms were similar, but UROX activity catalyzed by human CYP1A2 was only 15-20% of the activity catalyzed by murine CYP1A2. Human CYP1A1, CYP1A2, and CYP3A4 expressed in lymphoblastoid cells all catalyzed UROX. In insect cells, CYP1A2 was more active in catalyzing UROX than was CYP1A1, CYP2E, CYP3A4, or CYP3A5. Human CYP1A2 expressed in Escherichia coli as a fusion protein with rat CYP oxidoreductase also catalyzed UROX. Reconstituted human CYP1A2 and CYP3A4 were active in catalyzing UROX, with reconstituted CYP1A2 having the highest specific activity obtained in this study. From inhibitor studies, it was concluded that some of the UROX activity in the insect cell microsomes was attributable to expressed CYP and some to an unidentified source. These results indicate that human CYP1A2 is active in catalyzing UROX but has lower activity than the murine orthologue. The results also indicate that most of the UROX activity found in human liver microsomes is not due to CYP1A2.

Induction of cytochrome P4503A by taxol in primary cultures of human hepatocytes.

In primary cultures of human hepatocytes, paclitaxel (Taxol), at pharmacological concentrations, was demonstrated to induce immunoreactive cytochrome P4503A (CYP3A). The magnitude of the inductive response of the hepatocytes to Taxol varied in five separate cultures. In general, exposure to increasing concentrations of Taxol (0.2 to 10 microM) resulted in increases in immunoreactive CYP3A. In four of the cultures, treatment of hepatocytes with the lowest concentration of Taxol tested (0.2 microM) resulted in approximately two-fold increases in CYP3A. In the other culture, however, a six-fold increase in CYP3A was observed at 0.2 microM. Taxol was almost as effective as rifampicin in inducing CYP3A in two of the cultures, but less effective than rifampicin in two other cultures. CYP3A4 mRNA was increased by Taxol. Increases in CYP3A4 mRNA correlated with increases in the levels of immunoreactive CYP3A. These results demonstrate that Taxol is a potent inducer of CYP3A in human hepatocytes. The clinical significance of these findings is discussed.