P450 Reductase Activity Research Articles

The inhibition by flavonoids of 2-amino-3-methylimidazo[4,5- f]quinoline metabolic activation to a mutagen: a structure–activity relationship study

The mutagenicity of 2-amino-3-methylimidazo[4,5- f]quinoline (IQ) in Salmonella typhimurium TA98 is inhibited by flavonoids with distinct structure–antimutagenicity relationships (Edenharder, R., I. von Petersdorff I. and R. Rauscher (1993) Antimutagenic effects of flavonoids, chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and other heterocyclic amine mutagens from cooked food, Mutation Res., 287, 261–274). With respect to the mechanism(s) of antimutagenicity, the following results were obtained here. (1) 7-Methoxy- and 7-ethoxyresorufin- O-dealkylase activities in rat liver microsomes, linked to cytochrome P-450-dependent 1A1 and 1A2 monooxygenases catalyzing oxidation of IQ to N-hydroxy-IQ ( N-OH-IQ), were effectively inhibited by 16 flavonoids (IC 50: 0.4–9.8 μM). Flavones and flavonols are in general more potent enzyme inhibitors than flavanones, isoflavones, and chalcones. Among flavones the presence of hydroxyl or methoxyl groups resulted in minor changes only. However, among flavonols and flavanones the parent compounds exerted the strongest inhibitory effects, which decreased in dependence on number and position of hydroxyl functions. Contrary to the results obtained in the Salmonella assay in the tests with alkoxyresorufins no extraordinary counteracting effects of isoflavones, of hydroxyl groups at carbons 6 or 2′ or of the elimination of ring B (benzylideneacetone) were detected. (2) No effects of flavonoids on NADPH-dependent cytochrome P-450 reductase activity could be detected. (3) The effects of 30 flavonoids on mutagenicity induced by N-OH-IQ in S. typhimurium TA98NR were again structure dependent. The most striking feature was the, in principle, reverse structure–antimutagenicity pattern as compared to IQ: non-polar compounds were inactive and a 50% inhibition was achieved only by some flavones and flavonols (IC 50: 15.0–148 nmol/ml top agar). Within the flavone and flavonol subgroups inhibitory effects increased in dependence on number and position of hydroxyl functions. Isoflavones and flavanones, however, as well as glycosides, were inactive. Hydroxyl groups at carbons 7, 3′, 4′, and 5′ generated antimutagenic compounds, a hydroxyl function at C5 was ineffective, but hydroxyls at C3 and 6 as well as methoxyl groups at C3′ (isorhamnetin) or 4′ (diosmetin) generated comutagenic compounds. 4. Cytosolic activation of IQ to mutagenic metabolites as determined by experiments with the hepatic S105 fraction comprises about 10% of the mutagenicity after activation by the combined microsomal and cytosolic fractions (S9). The pattern of inhibition as produced by 20 flavonoids was closely similar to that observed with the S9 fraction. 5. In various experiments designed for modulation of the mutagenic response, it could be shown that further mechanisms of flavonoid interaction with the overall mutagenic process may exist, such as interactions with biological membranes (luteolin, fisetin) and effects on fixation and expression of DNA damage (flavone, fisetin).

The effects of diabetes on placental aromatase activity

Diabetes complicates 2–3% of all pregnancies and is associated with an increase in both perinatal morbidity and mortality, though reasons for these adverse outcomes are unknown. Estrogen biosynthesis is a critical factor during pregnancy and is carried out in the placenta via aromatase (cytochrome P450 19A1), which catalyzes the conversion of C-19 androgens to C-18 estrogens. Previous studies have shown that hormones such as insulin-like growth factors and insulin regulate aromatase activity when studied in vitro. Interestingly, levels of these hormones are altered in patients with diabetes. Thus, we hypothesized that the presence of maternal diabetes may alter placental aromatase activity and thus estrogen biosynthesis, possibly serving as one factor in the adverse outcomes of babies born to mothers with diabetes. To this end, we measured the production of 19-hydroxyandrostenedione, 19-oxoadrostenedione and estrone in 30 placental tissues from diabetic patients, using [7- 3H]androst-4-ene-3,17-dione as a model substrate for aromatase (P450 19A1). A statistical difference was detected in the percentage of 19-oxoandrostenedione formed between the overt and control groups ( P < 0.05). Additionally, NADPH P450-reductase levels were measured in these same tissues to determine whether alterations in this enzyme necessary for aromatase activity could be affected by diabetes. No differences in reductase levels were detected among the patient groups. However, a statistical correlation was found between NADPH P450-reductase activity and the formation velocities of all three estrogen products ( P < 0.05). Thus, it appears that the presence of diabetes does not affect placental aromatase activity.

Characterization of a BMS-181174-resistant human bladder cancer cell line.

This study was undertaken to elucidate the mechanism of cellular resistance to BMS-181174, a novel analogue of mitomycin C (MMC), in a human bladder cancer cell line. The BMS-181174-resistant variant (J82/BMS) was established by repeated continuous exposures of parental cells (J82) to increasing concentrations of BMS-181174 (9-40 nM) over a period of about 17 months. A 2.6-fold higher concentration of BMS-181174 was required to kill 50% of J82/BMS cell line compared with J82. The J82/BMS cell line exhibited collateral sensitivity to 5-fluorouracil (5-FU), but was significantly more cross-resistant to MMC, melphalan, taxol, doxorubicin and VP-16. NADPH cytochrome P450 reductase and DT-diaphorase activities, which have been implicated in bioreductive activation of MMC, were significantly lower in the J82/BMS cell line than in J82. The cytotoxicity of BMS-181174, however, was not affected in either cell line by pretreatment with dicoumarol, which is an inhibitor of DT-diaphorase activity. These results argue against a role of DT-diaphorase in cellular bioactivation of BMS-181174, a conclusion consistent with that of Rockwell et al (Biochem Pharmacol, 50: 1239-1243, 1995). BMS-181174-induced DNA interstrand cross-link (DNA-ISC) frequency was markedly lower in J82/BMS cell line than in J82 at every drug concentration tested. The results of the present study suggest that cellular resistance to BMS-181174 in J82/BMS cell line may be due to reduced DNA-ISC formation. However, the mechanism of relatively lower BMS-181174 induced DNA-ISC formation in J82/BMS cell line than in parental cells remains to be clarified.

Establishment of a Novel Host, High-Red Yeast That Stably Expresses Hamster NADPH–Cytochrome P450 Oxidoreductase: Usefulness for Examination of the Function of Mammalian Cytochrome P450

A novel strain ofSaccharomyces cerevisiaeuseful for expression studies of mammalian microsomal cytochrome P450s was established and named High-red yeast. Hamster NADPH–cytochrome P450 oxidoreductase (P450 reductase) cDNA to be introduced into yeast was isolated from a hamster liver cDNA library. The cDNA was 2421 bp long and contained an entire coding region for 667 amino acids. The NH2-terminal amino acid sequence deduced from the hamster P450 reductase cDNA was identical with that of the enzyme purified from hamster livers except for deletion of the initial methionine. A δ-sequence derived from yeast retrotransposon Ty was cloned and used as a sequence for homologous recombination in a yeast genome.S. cerevisiaeYPH500 was transformed with a multi-integration cassette containing the expression unit of the hamster P450 reductase and the δ-sequence. The transformant showing the highest activity of the P450 reductase was named High-red yeast. High-red yeast carried more than six copies of the multi-integration cassettes in a single chromosome and retained the multi-integration cassettes over a period of 100 generations under nonselective culture conditions, indicating that this yeast was a mitotically stable transformant. The microsomes prepared from High-red yeast had 20 times the P450 reductase activity of the microsomes prepared from the parental yeast. Due to the high activity of the hamster P450 reductase, the 7-ethoxycoumarin deethylase activity of mouse CYP1A1 expressed in High-red yeast was 250 times higher than the activity of mouse CYP1A1 expressed in the parental yeast.

Adaptation of human tumor cells to tirapazamine under aerobic conditions: Implications of increased antioxidant enzyme activity to mechanism of aerobic cytotoxicity

Tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-di- N-oxide, SR 4233, WIN 59075) is a bioreductive antitumor agent with a high selective toxicity for hypoxic cells. The selective hypoxic toxicity of TPZ results from the rapid reoxidation of the one-electron reduction product, the TPZ radical, in the presence of molecular oxygen with the concomitant production of superoxide radical. Under hypoxia the TPZ radical kills cells by causing DNA double-strand breaks and chromosome aberrations. However, the mechanism of aerobic cytotoxicity is still a matter of debate. In this study, we investigated the mechanism of aerobic cytotoxicity by adapting human lung adenocarcinoma A549 cells to aerobic TPZ exposure and characterizing the changes associated with drug resistance. The adapted cells were resistant to aerobic TPZ exposures (with dose-modifying factors of up to 9.2), although hypoxic sensitivity was largely unchanged. Relative to the parental A549 cell line, adaptation to continuous aerobic TPZ exposure resulted in increased levels of manganese superoxide dismutase (up to 9.4-fold), moderate increases in glutathione reductase (up to 2.1-fold), and loss of both quinone oxidoreductase (DT-diaphorase) activity and NADPH cytochrome P450 reductase activity. There was essentially no change in the activity of the cytoplasmic form of superoxide dismutase (CuZnSOD), catalase, or glutathione peroxidase. The increased activity of antioxidant enzymes in the resistant cell lines (in particular MnSOD) strongly suggests that reactive oxygen species are, in large part, responsible for the toxicity of TPZ under aerobic conditions, and is consistent with aerobic and hypoxic drug cytotoxicity resulting from different mechanisms.

Expression of CYP71B7, a Cytochrome P450 Expressed Sequence Tag fromArabidopsis thaliana

The systematic sequencing of anonymous cDNA clones (expressed sequence tags or ESTs) from the plantArabidopsis thalianahas identified a number of cDNAs with similarity to known cytochrome P450 sequences. The partial sequence of one of these cDNAs, 5G6, indicated that it was likely to encode a full-length cytochrome P450 monooxygenase (cyt P450) sequence. In this paper we describe the complete sequence of this clone, which has been designatedCYP71B7in accordance with the nomenclature for the cyt P450 gene superfamily. The cDNA was used to determine the pattern of expression of the corresponding gene inA. thaliana.Northern hybridization analysis indicated that maximal expression ofCYP71B7occurred in rosette leaves. Weaker hybridizing bands were also detected by Northern analysis of RNA from roots, leaves, flowers, and siliques. No expression could be detected in stem tissue. Southern analysis indicated that theCYP71B7gene was likely to exist as a single copy in the genome ofA. thaliana. CYP71B7was expressed episomally in yeast, and microsomes prepared from transgenic yeast exhibited a carbon monoxide difference spectrum characteristic of cyt P450. Microsomes from yeast expressingCYP71B7were assayed for enzymatic activity with synthetic model cyt P450 substrates. Microsomes from yeast cells expressingCYP71B7or those from control cells exhibited no detectable NADPH-supported 7-ethoxycoumarin or 7-ethoxyresorufin deethylase activities. However, in the presence of cumene hydroperoxide, activity was observed with microsomes from cells expressing CYP71B7 with 7-ethoxycoumarin as substrate. Organic hydroperoxides are well known to support cyt P450 catalysis in the absence of electrons from NADPH. The yeast microsomes contained high levels of endogenous NADPH–ferricytochrome P450 reductase (CPR) activity. The data suggest that thisA. thalianacyt P450, although expressed in an active form, is incapable of accepting electrons from the endogenous yeast CPR protein.

Changes in three hepatic cytochrome P450 subfamilies during a reproductive cycle in turbot (Scophthalmus maximus L.)

Sexual differentiation of the hepatic cytochrome P450 system was characterized in 2-year-old farmed turbot (Scophthalmus maximus L.) during their first spawning period (January–September). The fish were kept in tanks supplied with continuously flowing seawater (34.5%, ppt) at a constant temperature of 16°C and natural photoperiod (60°N). Sampling of liver samples (n = 4–6) was performed once every month for 9 months. Pronounced sex differences were recorded in the activities of 7-ethoxyresorufin O-deethylase (EROD), 7-ethoxycoumarin O-deethylase (ECOD), and NADPH-cytochrome P450 reductase during spawning (May–July). EROD activity in female fish decreased gradually towards the onset of ovulation in May–July to rise again in the postspawning period. The decrease correlated with increasing gonadosomatic index and estradiol17β (E2) levels in plasma. Immunochemical detection of CYP1A (58 kDa), CYP2K-like (47 and 52 kDa), and CYP3A-like (58 and 60 kDa) proteins in Western blotting, and ELISA showed higher protein levels in male compared to female fish from April/May-June, and significant differences were observed in June (CYP2K-like also in April and May). Analysis of monthly variations within sexes during the reproductive cycle shows significant monthly changes in all parameters in both female and male fish. Both CYP2K- and CYP3A-like protein levels were significantly elevated in male fish during spawning in June. To study the induction response during spawning, β-naphthoflavone (BNF) 75 mg/kg body weight) was administered intraperitoneally to both sexes in June. BNF caused a significant increase in EROD and ECOD activities and CYP1A protein levels but had no effect on NADPH-cytochrome P450 reductase activity or CYP2K-like/CYP3A-like proteins. This study documents, for the first time in any fish species or lower vertebrate, the sexual differentiation in the liver of three different CYP subfamilies during sexual maturation and spawning. J. Exp. Zool. 277:313–325, 1997. © 1997 Wiley-Liss, Inc.

Inhibition of Rat and Human Cytochrome P4502E1 Catalytic Activity and Reactive Oxygen Radical Formation by Nitric Oxide

Nitric oxide (NO) reacts with heme-containing enzymes, including certain isoforms of cytochrome P450. Cytochrome P4502E1 (CYP2E1) is induced by ethanol and plays an important role in the toxicity of ethanol and other hepatotoxins. CYP2E1 is also very effective in generating reactive oxygen intermediates such as superoxide radical and H2O2, oxidizing ethanol to the 1-hydroxyethyl radical, and has a high NADPH oxidase activity. The effect of NO on CYP2E1 catalytic activity and generation of reactive oxygen intermediates was evaluated. Incubating liver microsomes isolated from rats treated with pyrazole to induce high levels of CYP2E1, with gaseous NO or NO released from a variety of NO donors such as SNAP, DEA/NO, spermine/NO, and GSNO, resulted in a loss of CYP2E1 catalytic activity with specific substrates such asp-nitrophenol or dimethylnitrosamine. Trapping of NO with hemoglobin resulted in protection of CYP2E1 activity against the inactivation by NO. There was no effect by analogues of the donors which do not release NO nor was there any effect by NO on NADPH-cytochrome P450 reductase activity. Inactivation of CYP2E1 by NO was not prevented by superoxide dismutase or catalase, suggesting that superoxide, H2O2, or peroxynitrite were not responsible for the actions of NO. The inactivated CYP2E1 was not degraded nor did it lose its epitope sites as shown by Western blot analysis. Associated with loss of CYP2E1 catalytic activity was a decrease in the formation of superoxide radical and H2O2, in microsomal lipid peroxidation catalyzed by low, but not high concentration of iron, and in consumption of NADPH. Oxidation of ethanol to the 1-hydroxyethyl radical was also inhibited by NO. ESR experiments indicated the formation of stable heme–NO complexes with CYP2E1. NO appears to compete with O2and CO for binding to CYP2E1 as incubation with gaseous NO, or NO donors inhibited formation of the characteristic CO binding spectrum of P450. Microsomes isolated from a stably transfected HepG2 cell line expressing only CYP2E1 were also inactivated by NO, validating interaction of NO with this isoform of P450. These results indicate that NO inhibits CYP2E1 catalytic activity and generation of reactive radical intermediates. NO may prevent toxicity of agents which require bioactivation by P450 isoforms such as CYP2E1 and in generation of reactive intermediates by CYP2E1.

Dichloroacetic Acid Treatment Increases Hepatic CYP2E1 in Male and Female Rats

Previously, we showed that the pretreatment of dichloroacetic acid (DCA) increased CHCl3-induced hepatotoxicityin vivoand CHCl3metabolismin vitroin both male and female rats. The effects of DCA on hepatic cytochrome P450-dependent monooxygenases were studied in this experiment. Male and female Sprague–Dawley rats were treated with DCA (each 2.45 mmol/kg) three times (9AM, 4PM, and 9AM) and hepatic microsomes were prepared 3 hr after the last treatment (the same procedure as previous studies). After DCA treatment, the total content of cytochrome P450 (0.67 nmol/mg protein vs 0.79) and the activity of NADPH-dependent cytochrome P450 reductase (227 nmol/mg protein/min vs 332) were significantly increased in female rats, but they were unchanged in males (0.99 vs 0.98 for P450; 315 vs 311 for reductase). Induction of CYP2E1 was observed in both sexes, evidenced as increased activities of aniline andp-nitrophenol hydroxylases and increased CYP2E1 protein amount determined by immunoblot assay. In contrast, the CYP2B-related activity (dealkylation of pentoxyresorufin) and immunoreactive protein did not increase after DCA treatment in either males or females. The activity of ethoxyresorufin dealkylase was decreased in DCA-treated males compared to their controls (310 pmol/mg protein/min vs 229,p< 0.05), but it was not significantly affected in females. These data demonstrate that DCA treatment of both male and female rats altered the population of hepatic cytochrome P450. The results support the hypothesis that DCA increases CHCl3metabolism, and therefore hepatotoxicity, by inducing CYP2E1.

Quantitation of FAD-Dependent Cytochrome P450 Reductase Activity by Photoreduction

NADPH cytochrome P450 reductase binds two flavin cofactors, FMN and FAD, per molecule of reductase. We have developed an assay to quantitate the reduction activity of FMN-bound flavoprotein. This Tris–light assay system takes advantage of the ability of photoactivated flavins to release electrons to acceptors. In turn, electrons derived from Tris buffer restore the flavin to the unexcited, ground state which can again undergo photoactivation to release another electron. FMN-bound reductase, supplied with reducing equivalents from a Tris–light electron generating system, reduces ferricyanide at a rate of 1.8 μmol/min/nmol reductase. Holoreductase in this system is able to catalyze ferricyanide reduction at a rate of 1.6 μmol/min/nmol reductase, while FAD-bound reductase has no activity. The 8-NH2-FAD and 8-OH-FAD analog-reconstituted FMN-bound reductase catalyzes the reduction of ferricyanide at rates of 0.43 and 0.28 μmol/min/nmol reductase, respectively. The riboflavin-reconstituted FMN-bound reductase catalyzes ferricyanide reduction at a rate of 1.1 μmol/min/nmol reductase. FAD or its analogs at the concentrations used to reconstitute enzymatic activity do not support the reduction of ferricyanide in the Tris–light system in the absence of reductase protein. The free flavins, i.e., FMN, 8-OH-FAD, 8-NH2-FAD, and riboflavin, are able to support ferricyanide reduction at a rate of 0.40, 0.52, 0.87, and 0.16 μmol/min/nmol flavin, respectively. This is the first report of an enzymatic assay specific for FMN-bound NADPH cytochrome P450 reductase activity in the absence of its FAD cofactor. Moreover, this report describes the use of an assay procedure based on the provision of reducing equivalents by a Tris–light system which may be useful for other flavin redox enzymes in the absence of reduced pyridine nucleotides or biopterin cofactors.

Differential effect of interleukin-1α on rat hepatic cytochrome P450 monooxygenases

Administration of recombinant human interleukin-1α to adult male rats caused a significant reduction in the levels of hepatic cytochrome P450 and P450 reductase activity at 24 h after the treatment. The mRNAs for cytochrome P450 1A2 and 2E1 were reduced more than 70% at 12 h after administration of interleukin-1a and remained decreased even after 48 h. By contrast, cytochrome P450 2C11 mRNA was reduced only by 30% at 12 h after the treatment and returned to the control levels by 48 h, suggesting that interleukin-1α has a differential effect on the expression of P450 mRNAs. Aniline hydroxylase, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase activities were significantly decreased at 24 h after interleukin-1α treatment. The proteins for cytochrome P450 1A2 and 2E1 were reduced by about 50% at 24 h after interleukin-1a treatment.

Bile acids produce a generalized reduction of the catalytic activity of cytochromes P450 and other hepatic microsomal enzymes in vitro: relevance to drug metabolism in experimental cholestasis.

In bile duct-ligated male rats, there is a reduction of total hepatic microsomal cytochrome P450 (P450) levels and of NADPH-cytochrome P450 reductase (P450-reductase) activity, but the changes in activity of individual microsomal enzymes are nonuniform. We have proposed that the initial effect of cholestasis on microsomal proteins is a non-specific reduction caused by bile acid-mediated destruction, whereas the disproportionate lowering of male-specific P450 enzymes results from secondary down-regulation of some cytochrome P450 (CYP) genes. We report herein the results of experiments to test the first part of this hypothesis, at least as indicated by enzyme inhibition. Hepatic microsomal fractions from normal male rats were incubated at 37 degrees C with increasing concentrations of a range of bile acids selected for their varying physicochemical properties. The endpoints were catalytic activity of three individual CYP proteins, CYP 2A1 (measured as testosterone 7 alpha-hydroxylase activity), 2C11 (testosterone 2 alpha-hydroxylase and 16 alpha-hydroxylase) and 3A2 (testosterone 6 beta-hydroxylase), and the non-CYP enzymes, steroid 17 beta-dehydrogenase and P450-reductase. With 0.25 mmol/L cholic acid, a concentration exceeded in serum following bile duct ligation, there was a significant reduction in the activity of all enzymes at 4 h. Cholic acid-mediated inhibition was dose-dependent and there was no difference in inhibitory activity towards the male sex-dependent CYP 2C11 and 3A2 and the non-sex-dependent CYP 2A1 and other microsomal enzymes. Taurocholic acid was twice as potent an inhibitor as unconjugated cholic acid, the respective apparent I50 values being approximately 0.6 mmol/L compared with approximately 1.2 mmol/L. The dihydroxy bile acids, chenodeoxycholic acid and deoxycholic acid, were also more potent inhibitors than cholic acid, exhibiting I50 values in the range of 0.3-0.5 mmol/L, but the monohydroxy bile acid, lithocholic acid, was the most potent inhibitor (I50 approximately 0.2 mmol/L). Thus, the inhibitory potential of bile acids towards microsomal enzymes was inversely related to their extent of hydroxylation, while taurine conjugation enhanced the inhibitory potential of cholic acid. These data confirm the potential of bile acids to inhibit the activity of microsomal enzymes in livers of bile ductligated rats and indicate that such changes can occur with concentrations of bile acids that are physiologically relevant. Further, the results are consistent with the proposal that the disproportionately greater reduction of the male sex-dependent CYP, 2C11 and 3A2, is not explained by a destructive mechanism.

Effects of dietary heated fats on rat liver enzyme activity.

The objective of this study was to evaluate the effects of dietary heated fats from a commercial deep-fat frying operation on rat liver enzyme activity. The fats, partially hydrogenated soybean oil (PHSBO) used for four days and for 7 days (7-DH) for frying foodstuffs in a commercial restaurant, were fed to rats in either free access to food or by pair-feeding graded doses. All diets were isocaloric and contained 15 g/100 g of diet. Experiments were conducted with control rats fed non-heated (NH) PHSBO diet. Animals fed 7-DH diet in each set of experiments had larger amounts of cytochromes P450 and b5 and greater activity of NADPH-cytochrome P450 reductase when compared to controls. The activities of carnitine palmitoyltransferase-I and isocitrate dehydrogenase were significantly lower in rats fed test diets in comparison to controls. A significantly depressed activity of glucose 6-phosphate dehydrogenase was also noticed for these animals when compared to those fed NH. In addition, liver and microsomal protein concentrations were significantly greater in rats fed the used oils in comparison to controls, and liver glycogen was significantly lower.

Stable Expression of Human Cytochrome P450 3A4 in Conjunction with Human NADPH-Cytochrome P450 Oxidoreductase in V79 Chinese Hamster Cells

V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs was shown by Northern and Western analyses. Activity was tested by 6β-hydroxylation of testosterone for cytochrome P450 3A4 and by cytochrome c reduction for NADPH-cytochrome P450 reductase. Five V79 cell lines were obtained expressing cytochrome P450 3A4, human NADPH-cytochrome P450 oxidoreductase, and both. Cytochrome P450 3A4 activity depended highly on cytochrome P450 reductase activity, with lowest activity when only the parental Chinese hamster cytochrome P450 reductase was present, 5- and 10-fold higher when coexpressed with the human NADPH-cytochrome P450 reductase. Correspondingly, cytotoxic and genotoxic potency of aflatoxin B1 was increased by orders of magnitudes when human cytochrome P450 3A4 was coexpressed with the human NADPH-cytochrome P450 reductase. The effect of NADPH-cytochrome P450 reductase coexpression on cytochrome P450 3A4 activity was also tested by nifedipine oxidation and midazolam hydroxylation. Nifedipine oxidation was increased about 10-fold, 1-hydroxylation of midazolam and 4-hydroxylation of midazolam were increased 15-fold.

Increased DT-diaphorase expression and cross-resistance to mitomycin C in a series of cisplatin-resistant human ovarian cancer cell lines

In a series of ovarian carcinoma cell lines selected in vitro for resistance to cisplatin by continuous exposure to increasing drug concentrations, the level of resistance is proportional to the expression of γ-glutamylcysteine synthetase (γ-GCS). To determine if other detoxicating genes are coordinately expressed, we measured the activity of DT-diaphorase and cytochrome P450 reductase. The specific activity of DT-diaphorase, but not that of cytochrome P450 reductase, increased with increasing resistance to cisplatin. Steady-state mRNA levels for DT-diaphorase correlated with enzyme activity and hence with cisplatin resistance. Since the activity of DT-diaphorase has been associated with sensitivity to quinones, we studied the cytotoxicity of mitomycin C under oxic conditions. Unexpectedly, resistance to mitomycin C increased proportionally with that to cisplatin ( r = 0.997). Pretreatment with buthionine sulfoximine, which inhibits glutathione (GSH) synthesis, failed to sensitize either the sensitive or the resistant lines to mitomycin C. Thus, the basis for collateral resistance to mitomycin C in the cisplatin-resistant lines under oxic conditions is unrelated to overproduction of GSH. Under hypoxia, the toxicity of mitomycin C to the most sensitive (A2780) cell line was unchanged. However, the most resistant (C200) line was 2-fold more resistant to mitomycin C under hypoxic conditions. The coordinate overexpression of DT-diaphorase and γ-GCS in the resistant cell lines is thus associated with hypoxic cell resistance, and supports the involvement of shared mechanisms of gene regulation in the observed resistant phenotype.

Influence of cholesterol and protein diet on liver cytochrome P-450-dependent monooxygenase system in rats

The influence of the cholesterol and protein diet on the mixed-function oxidases system activity and desaturation of fatty acids were examined. The phenobarbital innducibility of these parameters was analyzed too. Investigations were carried out on Wistar adult male rats. The animals were on the cholesterol (0.25%) and protein diet (32%) during 45 days. The cytochrome P-450 and cytochrome b5 contents, the NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase activities, aniline hydroxylase and 4-aminopyrine N-demethylase activities were measured in the hepatic microsomal fraction. In both sexes, the cholesterol diet decreased all the examined parameters with the exception of NADH-cytochrome b5 reductase activity. The protein diet did not change the examined enzyme activities or levels with the exception of induction of NADH-cytochrome b5 reductase and 4-aminopyrine N-demethylase activities. Simultaneous treatment with phenobarbital had heterogenous effect dependent on the type of diet and examined parameter.

Genetic analysis of factors controlling high-level expression of cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities in LPR house flies,Musca domestica

Genetic analysis of factors controlling high-level expression of cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities in LPR house flies,Musca domestica

Genetic analysis of factors controlling high-level expression of cytochrome P450, CYP6D1, cytochrome b5, P450 reductase, and monooxygenase activities in LPR house flies, Musca domestica.

To understand better the biochemical genetics of cytochrome P450 monooxygenase-mediated insecticide resistance, we examined the microsomal monooxygenases in insecticide-susceptible (aabys) and pyrethroid-resistant (LPR) house fly strains, as well as 15 house fly lines derived from crosses of LPR and aabys. In comparison to the aabys strain, LPR had higher levels of total cytochromes P450, cytochrome b5, P450 reductase, CYP6D1, and three P450 monooxygenase activities: 7-ethoxycoumarin O-deethylase (ECOD), methoxyresorufin O-demethylase (MROD), and aromatic hydrocarbon hydroxylase (AHH). The elevated levels of cytochrome b5 were linked to factors on autosomes 1 and 2. This is similar to previous reports on monooxygenase-mediated resistance and is consistent with the idea that elevated cytochrome b5 levels are involved in monooxygenase-mediated resistance in the LPR strain. Linkage of the elevated P450 reductase is different from that of monooxygenase-mediated resistance. Strains having high levels of CYP6D1 (i.e., like LPR) had high levels of P450 reductase, while strains having intermediate levels of CYP6D1 also had high levels of reductase. Therefore, there is no clear evidence that the elevated P450 reductase in the LPR strain is required for the increased monooxygenase activity. Overexpression of total cytochromes P450, CYP6D1 (mRNA and protein), and CYP6D1-mediated monooxygenase activities (MROD and AHH) in LPR microsomes was linked to a combination of factors on autosomes 1 and 2. This demonstrates that increased expression of CYP6D1 in the LPR strain is both cis regulated by a factor(s) on autosome 1 and trans regulated by a factor(s) on autosome 2. The correlation between the overexpression of CYP6D1 mRNA and protein suggests that CYP6D1 expression is regulated transcriptionally. Monooxygenase-mediated resistance in LPR is controlled by factors on autosomes 1 and 2, which supports previous claims that CYP6D1 is responsible for monooxygenase-mediated resistance in the LPR strain.

Establishment and toxicological application of a cell line expressing human CYP2E1

Male Jcl:Wistar rats were exposed continuously to either ozone (O3) or nitrogen dioxide (NO2) for 7 and 14 days to examine the effects of these gases on the xenobiotic metabolizing systems of lung microsomes. Exposure to 0.2 and 0.4 ppm O3 increased NADPH-cytochrome P-450 reductase activity and cytochrome P-450 as well as microsomal protein by the 14th day, whereas NADH-cytochrome b5 reductase was not affected. The most marked increase was observed in cytochrome P-450. In parallel to this increment, the activities of benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase of exposed animals increased significantly on the 7th and 14th days of exposure to 0.2 and 0.4 ppm O3.In contrast, exposure to 1.2 and 4 ppm NO2 decreased cytochrome P-450 on the 7th day. Moreover, the 7-ethoxycoumarin O-deethylase activity in exposed animals decreased to 61% (P < 0.05) and 74 % (P < 0.001) of control on the 7th and 14th days of exposure to 4 ppm NO2, respectively. This decrease occurred in a dose-dependent manner with exposure to 0.4–4.0 ppm NO2, whilst benzo(a)pyrene hydroxylase activity was not affected.These results show that O3 at low doses induces xenobiotic metabolizing activities in the lung, whereas NO2 reduces this.

Transforming growth factor-beta in in vivo resistance.

The potential role of transforming growth factor-beta in in vivo resistance was examined by administration of transforming growth factor-beta-neutralizing antibodies to animals bearing the EMT-6/Parent tumor or the antitumor alkylating resistance tumors, EMT-6/CTX or EMT-6/CDDP. Treatment of tumor bearing animals with anti-TGF-beta antibodies by intraperitoneal injection daily on days 0-8 post-tumor cell implantation increased the sensitivity of the EMT-6/Parent tumor to cyclophosphamide (CTX) and cisplatin (CDDP) and markedly increased the sensitivity of the EMT-6/CTX tumor to CTX and the EMT6/CDDP tumor to CDDP, as determined by tumor cell survival assay. Bone marrow granulocyte-macrophage colony-forming units (CFU-GM) survival was determined from these same animals. The increase in the sensitivity in the tumors upon treatment with the anti-TGF-beta antibodies was also observed in increased sensitivity of the bone marrow CFU-GM to CTX and CDDP. Treatment of non-tumor-bearing animals with the anti-TGF-beta regimen did not alter blood ATP or serum glucose level but did decrease serum lactate levels. This treatment also decreased hepatic glutathione, glutathione S-transferase, glutathione reductase, and glutathione peroxidase in non-tumor bearing animals by 40-60% but increased hepatic cytochrome P450 reductase in these normal animals. Animals bearing the EMT-6/CTX and EMT-6/CDDP tumors had higher serum lactate levels than normal or EMT-6/Parent tumor-bearing animals; these were decreased by the anti-TGF-beta regimen. Treatment of animals bearing any of the three tumors with the anti-TGF-beta regimen decreased by 30-50% the activity of hepatic glutathione S-transferase and glutathione peroxidase, and increased by 35-80% the activity of hepatic cytochrome P450 reductase. In conclusion, treatment with transforming growth factor-beta-neutralizing antibodies restored drug sensitivity in the alkylating agent-resistant tumors, altering both the tumor and host metabolic states.