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Statins and fibrates (weak PPARalpha agonists) are prescribed for the treatment of lipid disorders. Both drugs cause myopathy, but with a low incidence, 0.1-0.5%. However, combined statin and fibrate therapy can enhance myopathy risk. We tested the myotoxic potential of PPAR subtype selective agonists alone and in combination with statins in a differentiated rat myotube model. A pharmacologically potent experimental PPARalpha agonist, Compound A, induced myotoxicity as assessed by TUNEL staining at a minimum concentration of 1 nM, while other weaker PPARalpha compounds, for example, WY-14643, Gemfibrozil and Bezafibrate increased the percentage of TUNEL-positive nuclei at micromolar concentrations. In contrast, the PPARgamma agonist Rosiglitazone caused little or no cell death at up to 10 muM and the PPARdelta ligand GW-501516 exhibited comparatively less myotoxicity than that seen with Compound A. An experimental statin (Compound B) and Atorvastatin also increased the percentage of TUNEL-positive nuclei and co-treatment with WY-14643, Gemfibrozil or Bezafibrate had less than a full additive effect on statin-induced cell killing. The mechanism of PPARalpha agonist-induced cell death was different from that of statins. Unlike statins, Compound A and WY-14643 did not activate caspase 3/7. In addition, mevalonate and geranylgeraniol reversed the toxicity caused by statins, but did not prevent the cell killing induced by WY-14643. Furthermore, unlike statins, Compound A did not inhibit the isoprenylation of rab4 or rap1a. Interestingly, Compound A and Compound B had differential effects on ATP levels. Taken together, these observations support the hypothesis that in rat myotube cultures, PPARalpha agonism mediates in part the toxicity response to PPARalpha compounds. Furthermore, PPARalpha agonists and statins cause myotoxicity through distinct and independent pathways.

Statins are widely used to treat lipid disorders. These drugs are safe and well tolerated; however, in <1% of patients, myopathy and/or rhabdomyolysis can develop. To better understand the mechanism of statin-induced myopathy, we examined the ability of structurally distinct statins to induce apoptosis in an optimized rat myotube model. Compound A (a lactone) and Cerivastatin (an open acid) induced apoptosis, as measured by TUNEL and active caspase 3 staining, in a concentration- and time-dependent manner. In contrast, an epimer of Compound A (Compound B) exhibited a much weaker apoptotic response. Statin-induced apoptosis was completely prevented by mevalonate or geranylgeraniol, but not by farnesol. Zaragozic acid A, a squalene synthase inhibitor, caused no apoptosis on its own and had no effect on Compound-A-induced myotoxicity, suggesting the apoptosis was not a result of cholesterol synthesis inhibition. The geranylgeranyl transferase inhibitors GGTI-2133 and GGTI-2147 caused apoptosis in myotubes; the farnesyl transferase inhibitor FTI-277 exhibited a much weaker effect. In addition, the prenylation of rap1a, a geranylgeranylated protein, was inhibited by Compound A in myotubes at concentrations that induced apoptosis. A similar statin-induced apoptosis profile was seen in human myotube cultures but primary rat hepatocytes were about 200-fold more resistant to statin-induced apoptosis. Although the statin-induced hepatotoxicity could be attenuated with mevalonate, no effect was found with either geranylgeraniol or farnesol. In studies assessing ubiquinone levels after statin treatment in rat and human myotubes, there was no correlation between ubiquinone levels and apoptosis. Taken together, these observations suggest that statins cause apoptosis in myotube cultures in part by inhibiting the geranylgeranylation of proteins, but not by suppressing ubiquinone concentration. Furthermore, the data from primary hepatocytes suggests a cell-type differential sensitivity to statin-induced toxicity.

We compared the ability of rat and human hepatocytes to respond to fenofibric acid and a novel potent phenylacetic acid peroxisome proliferator-activated receptor (PPAR) alpha agonist (compound 1). Fatty acyl-CoA oxidase (FACO) activity and mRNA were increased after treatment with either fenofibric acid or compound 1 in rat hepatocytes. In addition, apolipoprotein CIII mRNA was decreased by both fenofibric acid and compound 1 in rat hepatocytes. Both agonists decreased apolipoprotein CIII mRNA in human hepatocytes; however, very little change in FACO activity or mRNA was observed. Furthermore, other peroxisome proliferation (PP)-associated genes including peroxisomal 3-oxoacyl-CoA thiolase (THIO), peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), peroxisomal membrane protein-70 (PMP-70) were not regulated by PPAR alpha agonists in human hepatocytes. Moreover, other genes that are regulated by PPAR alpha ligands in human hepatocytes such as mitochondrial HMG-CoA synthase and carnitine palmitoyl transferase-1 (CPT-1) were also regulated in HepG2 cells by PPAR alpha agonists. Several stably transfected HepG2 cell lines were established that overexpressed human PPAR alpha to levels between 6- and 26-fold over normal human hepatocytes. These PPAR alpha-overexpressing cells had higher basal mRNA levels of mitochondrial HMG-CoA synthase and CPT-1; however, basal FACO mRNA levels and other PP-associated genes including THIO, HD, or PMP-70 mRNA were not substantially affected. In addition, FACO, THIO, HD, and PMP-70 mRNA levels did not increase in response to PPAR alpha agonist treatment in the PPAR alpha-overexpressing cells, although mitochondrial HMG-CoA synthase and CPT-1 mRNAs were both induced. These results suggest that other factors besides PPAR alpha levels determine the species-specific response of human and rat hepatocytes to the induction of PP.

A subpopulation of the CF-1 mouse strain is sensitive to neurotoxicity following exposure to avermectins, a family of structurally related antiparasitic agents. This unusual sensitivity is the result of a deficiency in the mdr1a P-glycoprotein that normally contributes to a functional blood-brain barrier. Previous studies demonstrated a correlation between P-glycoprotein levels in the brain, intestine, testis, and placenta with an restriction fragment length polymorphism (RFLP) pattern from DNA isolated from the animals. We have demonstrated that only P-glycoprotein derived from the mdr1a gene is deficient in these mice. In this article, we describe the genetic defect in the subpopulation of CF-1 mice resulting in an absence of P-glycoprotein. The data presented describes a reverse transcription--polymerase chain reaction (RT-PCR) protocol that specifically amplifies mdr1a mRNA from tissue and confirms that the P-glycoprotein defect results from a truncated mRNA with a deleted exon 23. Genomic amplification and sequencing of the intron between exon 22 and 23 in Pgp-deficient animals reveals an insertion of approximately 8.35 kb of DNA at the exon 23 intron--exon junction corresponding to a murine leukemia virus. This insertion results in the aberrant splicing of the mRNA and the loss of exon 23 during RNA processing., (Copyright 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:83-89, 2001)

Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families., (Copyright 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:133-142, 2001)

We hypothesized that the drug efflux protein P-glycoprotein (Pgp), the product of the multidrug resistance gene MDR1, might influence hepatic expression of CYP3A or other cytochromes P-450 (P-450s) because Pgp can transport endogenous regulators of these cytochromes. We began with variants of a CF-1 mouse strain containing a defective mdr1a gene that is inherited in a Mendelian fashion. The amount of CYP3A protein in liver was inversely related to the gene dose of the normal mdr1a allele in these mice. mdr1a knockout mice of either mixed (FVB x 129/Ola) or pure FVB genetic background and housed in Amsterdam display an increased expression of CYP2B and CYP3A proteins. However, because mdr1a ablation causes a compensatory increase in hepatic mdr1b (which can efflux intracellular glucocorticoids), we reasoned that mdr1b might mask the overall effect of mdr1a absence on P-450 gene expression. Targeted inactivation of the mdr1b gene increased P-450 expression, but the effect was modest compared with mdr1a ablation. Mice nullizygous for both mdr1a and mdr1b-type Pgps and kept in Amsterdam had dramatically increased levels of CYP3A protein as well as other P-450s examined and of the electron donor P-450 reductase. Consistent with the protein results, CYP3A catalytic activity measured as midazolam 1'- and 4-hydroxylation in liver microsomes from these knockout mice revealed a rank order of activities with mdr1a/1b > mdr1a > mdr1b > (+/+) mice. In contrast to results in mice housed in Amsterdam, in the genetically identical mdr1a or mdr1a/1b (-/-) male mice housed in the United States, hepatic P-450 expression was unaffected by mdr1 genotype or actually showed a slight decrease in mdr1a (-/-) mice. These results provide a revealing picture of mdr1-type Pgp as an upstream regulator of hepatic P-450 expression, and demonstrate that these pharmacologically relevant phenotypes in knockout mice depend not only on the genetic make-up of the mice but also on the environment.

The pharmacokinetics and hepatic metabolism of [3H] ivermectin (IVM) and [3H]cyclosporin A (CSA) were investigated in a subpopulation of the CF-1 mouse stock naturally deficient in mdr1a p-glycoprotein (PGP). A survey of key drug-metabolizing activities in liver fractions from PGP-deficient (-/-) or wild-type (+/+) animals indicated the two subpopulations are not different in hepatic metabolic activity and capacity. Intravenous pharmacokinetics of CSA were identical between the two groups, and results from microsomal incubations indicated similar biotransformation of IVM and CSA in liver. Intestinal excretion of [3H]IVM and [3H]CSA was enhanced in PGP (+/+) animals. Absence of PGP resulted in higher blood concentrations of IVM after oral dosing, suggesting enhanced absorption of IVM in (-/-) mice. Concentrations of [3H]IVM and [3H]CSA were always greater in the brains of (-/-) mice compared with (+/+) mice after either i.v. or oral administration. In contrast, liver concentrations of either compound were not different between (+/+) and (-/-) animals after an i.v. dose. These results show the PGP (-/-) and (+/+) subpopulations of CF-1 mice are useful for studying the role of mdr1a PGP in systemic exposure and tissue disposition of PGP substrates in the absence of metabolism differences.

A subpopulation of the CF-1 mouse strain contains a spontaneous mutation in the P-glycoprotein (Pgp) mdr1a gene, which leads to a lack of mdr1a expression in the placenta as well as brain and intestine. Individual CF-1 mice can be identified according to their Pgp status by a restriction fragment length polymorphism. Male and female mice selected on the basis of Pgp genotype were mated and the pregnant dams exposed during gestation to the known Pgp substrate, L-652,280, the 8,9 Z photoisomer of the naturally occurring avermectin Bla, which is known to produce cleft palate in mice. Fetal examination demonstrated that within individual litters, fetuses deficient in Pgp (-/-) were 100% susceptible to cleft palate, whereas their +/- heterozygote littermates were less sensitive. The homozygous +/+ fetuses with abundant Pgp were totally insensitive at the doses tested. The degree of chemical exposure of fetuses within each litter was inversely related to expression of placental Pgp, which was determined by the fetal genotype. These results demonstrate the importance of placental Pgp in protecting the fetus from potential teratogens and suggest that Pgp inhibitors should be carefully evaluated for their potential to increase susceptibility to chemical-induced teratogenesis.

There is a subpopulation of the CF-1 mouse strain that is very sensitive to the neurotoxicity induced by the avermectins, a class of natural products widely used in veterinary and human medicine as anti-parasitic agents. This sensitivity results from a lack of P-glycoprotein in the intestine and brain of sensitive animals, allowing increased penetration of these compounds in the blood and brain, respectively. We describe a restriction fragment length polymorphism that is able to predict which animals will be deficient in this protein, confirming at the genetic level a heterogeneous population of this mouse strain. Breeding studies demonstrated that the inheritance of the markers follows a normal Mendelian autosomal pattern. Sensitive "-/-" animals are deficient in P-glycoprotein in those tissues known to express primarily mdr1a, but have normal P-glycoprotein levels in tissues known to express primarily mdr1b or mdr2, suggesting that the defect in the sensitive animals is limited to the mdr1a gene. The P-glycoprotein expression in the brain is dependent on the genotype, which also determines the susceptibility to the avermectin-induced neurotoxicity, with the "-/-" animals being most sensitive, and the "+/-" animals having less P-glycoprotein and therefore increased CNS sensitivity compared to the "+/+" animals. The ability to segregate this strain into -/- and +/+ animals may prove useful for examining the physiological role of P-glycoprotein in drug absorption and distribution and related toxicity. These data also provide a warning that experiments carried out with P-glycoprotein substrates in the heterogeneous population of the CF-1 mouse must be interpreted with caution., (Copyright 1997 Academic Press.)

Big Blue (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacI transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt frequencies (observed hprt frequency minus control frequency) of 5.5 +/- 1.0, 11 +/- 2.0, and 19 +/- 2.6 x 10(-6), respectively (average +/- SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt frequencies of 0.9 +/- 0.6 and 9.1 +/- 1.5 x 10(-6). 32P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours after B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adduct load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 +/- 9 and 134 +/- 10 x 10(-6) (15- to 40-fold higher than the induced hprt frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 x 10(-6)). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified lambda-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacI transgene than at the endogenous hprt locus.

Induced rat liver S-9 is routinely used for metabolic activation in cytogenetic assays. When a compound gives a positive test result only with rat S-9, the significance for humans should be assessed. To evaluate the use of human S-9, we used sister-chromatid exchanges (SCEs) and chromosome aberrations (Abs) in Chinese hamster ovary cells to test five pro-mutagens, each preferentially activated by a different family of P-450: benzo(a)pyrene (BP), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), aflatoxin B1 (AFB), and 2-acetylaminofluorene (2-AAF). We tested two human S-9 preparations, one from a single liver and a second pooled from two livers known to have good activity for several P-450s. Concentrations and ratios of NADP and isocitrate were adjusted to optimize NADPH generation by the S-9. Abs were scored 20 hr, and SCEs 29-45 hr, after the beginning of a 3 hr treatment. P-450 enzyme activities were generally higher in rat than human S-9. With the single-liver human S-9, increase in SCEs were seen with all chemicals; with both human S-9s, increases in Abs were seen with all chemicals except BP. (The level of P-450 1A1, required for BP activation, is very low in human liver.) Compared with rat S-9, generally higher concentrations of human S-9 and of promutagens were required to see positive results. However, human S-9 effectively activated 2-AAF, whereas neither of the two types of rat S-9 produced Abs with 2-AAF. We also compared rat S-9s induced with Aroclor 1254 or phenobarbital/ beta-naphthoflavone (PB/beta NF). Although there were some differences in P-450 enzyme activities, these did not translate into differences in Abs induction. At low doses of AFB and of BP, PB/beta NF induced S-9 appeared more effective than Aroclor 1254 induced S-9.

It has previously been shown that Verlukast is converted to Verlukast dihydrodiol in microsomes from beta-naphthoflavone (BNF)-treated, but not uninduced Swiss Webster mice and Sprague-Dawley rats. We have examined the involvement of CYP1A1 in this reaction in more detail. It is concluded that this reaction is catalyzed exclusively by CYP1A1 in rats, mice, and humans based on the following criteria: 1) the epoxidation of Verlukast is negligible in uninduced rats, which express CYP1A2 but not CYP1A1; 2) Verlukast epoxidation is highly inducible by BNF treatment (60- to 200-fold); 3) Verlukast epoxidation in BNF-treated rat microsomes was inhibited by alpha-naphthoflavone (ANF) treatment, indicating that this activity was mediated by the CYP1A subfamily; 4) > 95% of Verlukast epoxidation in BNF-treated rat microsomes was inhibited by antibodies raised against CYP1A1; and 5) Verlukast was epoxidized by human CYP1A1 but not CYP1A2. Thus, Verlukast epoxidation appears to be specific for rat, mouse, and human CYP1A1. Additional studies showed that Verlukast was metabolized to Verlukast dihydrodiol in microsomes from uninduced rhesus monkeys. This reaction was inhibited by nanomolar concentrations of ANF in rhesus monkey microsomes implicating the involvement of the CYP1A subfamily. In addition, the 8-hydroxylation of R-warfarin, a pathway that is selective for rodent and human CYP1A1 activity, was also catalyzed at significant rates by rhesus monkey microsomes. These findings indicate that, unlike rats, mice, and humans, which have very low constitutive levels of hepatic CYP1A1 activity, the uninduced rhesus monkey is able to catalyze reactions specific to CYP1A1 in rodents and humans.(ABSTRACT TRUNCATED AT 250 WORDS)

Karyotypic and phenotypic changes were found in human adult endothelial cells (EC) during aging in vitro. A trisomy of chromosome 11 was found in 11 out of 12 EC cultures examined, derived from 9 cell lines from 8 donors. The incidence of this trisomy in some cell lines increased over time from 0% to as much as 100% near the end of their in vitro life span. A number of oncogenes and other important genes are on chromosome 11. These genes might play a role in the changes observed. An increase in the percentage of polyploid cells was also found near the end of the in vitro life span in 6 lines. The cellular levels of two gene products characteristic of the EC, von Willebrand factor (vWF) or Factor VIII, and angiotensin converting enzyme (ACE) were also monitored. vWf was studied in 2 lines and was decreased in both with serial passage. ACE decreased in three out of the four lines examined. These chromosomal and phenotypic changes which occur with increasing age in vitro make the endothelial cell a suitable model to study in vitro culture-related changes, senescence, cardiovascular disease, and tumorigenesis.

The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS)

Human liver cytochrome P-450NF is the form of cytochrome P-450 responsible for the oxidation of the calcium-channel blocker nifedipine, which has been reported to show polymorphism in clinical studies. By screening a bacteriophage lambda gt11 expression cDNA library, we isolated two clones: NF95 with an insert length of 0.8 kilobases which gave a stable fusion protein and NF25 with an insert length of 2.2 kilobases. The two clones were both sequenced and shown to be identical in their overlapping section. The sequence of NF25 is 77% similar to that reported for a rat cytochrome "P-450PCN" cDNA (PCN = pregnenolone-16 alpha-carbonitrile). The similarity decreases to 45-53% when the sequence is compared to human cytochromes P-450 belonging to other families [i.e., "pH P-450(1)," "P1-450," "P3-450," and "P-450MP." The deduced amino acid sequence is 73% similar to that of rat cytochrome P-450PCN, and the first 21 amino acids are identical to those reported for human liver cytochrome "P-450p." Sections of these clones were nick-translated and used as probes for analyses of human mRNA and genomic DNA. The number and size of bands indicate that P-450NF belongs to a multigene family, the so-called pregnenolone-16 alpha-carbonitrile-inducible family.

A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.

Freshly prepared isolated hepatocytes were shown to metabolize dimethylnitrosamine producing an alkylating agent which reacted with cellular DNA giving rise to 7-methylguanine and O6-methylguanine. Alkylation of DNA was prevented by aminoacetonitrile and was proportional to the dimethylnitrosamine concentration over the range of 1--90 microM. Isolated hepatocytes were able to catalyze the removal of O6-methylguanine from their DNA. After formation of this product by reaction with N-methyl-N-nitrosourea or dimethylnitrosamine to give extents of alkylation in the range of 0.1--15.0 mumol O6-methylguanine per mole of guanine in DNA, the loss produced in 3 h incubation was comparable to that seen in vivo from liver DNA alkylated to the same extent. The isolated hepatocytes therefore, provide a useful system in which factors influencing O6-methylguanine persistence in DNA can be studied.

The human liver cytochrome P-450 (P-450) proteins responsible for catalyzing the oxidation of mephenytoin, tolbutamide, and hexobarbital are encoded by a multigene family (CYP2C). Although several cDNA clones and proteins related to this "P-450MP" family have been isolated, assignment of specific catalytic activities remains uncertain. Sulfaphenazole was found to inhibit tolbutamide hydroxylation to a greater extent than mephenytoin or hexobarbital hydroxylation. The inhibition by sulfaphenazole was competitive for tolbutamide and hexobarbital hydroxylation but with much different Ki values (5 vs 480 microM, respectively). Inhibition of mephenytoin hydroxylase was not competitive. The results suggest that different P-450 proteins in the P450MP family may be involved in the metabolism of these compounds. A cDNA clone (MP-8) related to the P-450MP family, isolated from a bacteriophage lambda gt11 human liver library, was expressed in Saccharomyces cerevisiae by using the pAAH5 expression vector. Yeast transformed with pAAH5 containing the MP-8 sequence (pAAH5/MP-8) showed a ferrous-CO spectrum typical of the P-450 proteins. Immunoblotting with anti-P450MP revealed that pAAH5/MP-8 microsomes contained a protein with an Mr similar to that of P-450MP-1 (approximately 48,000) that was not present in microsomes from yeast transformed with pAAH5 alone (1.7 X 10(4) molecules of the expressed P-450 per cell). Microsomes from pAAH5/MP-8 contained no detectable mephenytoin 4'-hydroxylase activity but were more active in tolbutamide hydroxylation, on a nanomoles of P-450 basis, than human liver microsomes. The pAAH5/MP-8 microsomes also contained hexobarbital 3'-hydroxylase activity, although the enrichment compared to liver microsomes was not great with respect to the tolbutamide hydroxylase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).

Nine forms of cytochrome P-450 have been purified to electrophoretic homogeneity from human-liver microsomes. These include the enzymes involved in debrisoquine 4-hydroxylation, phenacetin O-deethylation and mephenytoin 4-hydroxylation, three reactions which are characterized by genetic polymorphism in humans. Evidence for the involvement of the above enzymes comes from reconstituted immunochemical inhibition studies with human-liver microsomes. These and other lines of evidence are consonant with the view that different forms of cytochrome P-450 are involved in the three reactions. The debrisoquine 4-hydroxylase has been studied most extensively in terms of its substrate specificity. In addition, an analogous rat enzyme shows some homology and serves as a useful model. The use of antibodies raised to the rat-liver enzyme in immuno-inhibition studies with human-liver microsomes provides a means of determining the extent to which this enzyme participates in other reactions. Translation of rat-liver mRNA in vitro yields the intact debrisoquine 4-hydroxylase; studies with human mRNA suggest a lower frequency than in rats. The basis for impaired catalytic activity in phenotypically poor human metabolizers appears to be an altered enzyme in all three cases, as opposed to a decreased level of a single enzyme. Using antibody screening of fusion proteins expressed in a cDNA library, it has been possible to isolate cDNA probes for all three of these cytochromes P-450 for use in screening individuals and ultimately determining the basis of these polymorphisms.

Freshly prepared rat hepatocytes isolated by perfusion with collagenase were able to metabolize microM concentrations of dimethylnitrosamine to a methylating agent. The methylation of hepatocyte DNA in this system was complete within 2 hr, and after this time, the content of O6-methylguanine in the DNA declined, showing that the repair system for this product was active in the isolated hepatocytes. When extracellular calf thymus DNA was added to the incubated hepatocytes, this also became methylated. Methylation of this DNA was not due to cell lysis releasing activating enzymes into the medium, showing that the methylating species formed by the hepatocytes from dimethylnitrosamine is sufficiency stable to pass out of the cell in substantial amounts. These results support the possibility that alkylation of liver cells would not be confined to those cells metabolizing dimethylnitrosamine but could be extended to those cells which are in close proximity to the activating cells. These cells could include nonparenchymal cells which are known to be targets for the carcinogenic action of dimethylnitrosamine.

The activity of the alkyl acceptor protein (AAP) responsible for repair of DNA containing the promutagenic lesion O6-alkylguanine was determined in hepatocytes and nonparenchymal cells (NPC) obtained from livers of control rats and rats exposed to hepatocarcinogens that primarily induce vascular or hepatocellular neoplasms. Basal levels of AAP activity were found to be 4-5 times higher in hepatocytes than in NPC. Exposure to 1,2-dimethylhydrazine or diethylnitrosamine produced a 2- to 3-fold enhancement of this activity in hepatocytes after exposure for as little as 3 days. The enhanced hepatocyte activity persisted throughout a 28-day exposure to 1,2-dimethylhydrazine. In contrast, AAP activity in NPC was decreased during the first week of exposure to 1,2-dimethylhydrazine and subsequently returned to control levels. No enhancement of AAP was apparent in the NPC. These and related data suggest that enhancement of this activity in rat hepatocytes is a response to cell proliferation. In contrast, the data clearly demonstrate that neither increased cell replication nor the presence of O6-alkylguanine was capable of enhancing AAP activity in NPC. Cellular differences in the repair of O6-alkylguanine appear to be a critical mechanism responsible for cell specificity in chemical carcinogenesis by alkylating agents.