Oxidative metabolism of cyclophosphamide: identification of the hepatic monooxygenase catalysts of drug activation

Cytochrome P-450-catalyzed activation of cyclophosphamide to alkylating metabolites was studied in isolated rat liver microsomes and purified, reconstituted P-450 enzyme systems in order to identify the major enzymatic catalysts of drug activation in both uninduced and drug-induced liver tissue. P-450 form PB-4 (P-450 gene IIB1) activated cyclophosphamide with high efficiency [Vmax (app) = 18.2 nmol metabolite/min/nmol P-450; Km (app) = 0.16 mM] via the formation of 4-hydroxycyclophosphamide, which was quantitatively trapped as a bisulfite adduct then characterized following its conversion to cyano derivatives. Antibodies to P-450 PB-4 inhibited cyclophosphamide activation catalyzed by phenobarbital-induced adult male rat liver microsomes (specific activity, 5.4 nmol metabolite/min/mg liver microsomes) in a selective and near quantitative (greater than 80%) fashion; little or no inhibition was obtained using antibodies inhibitory towards six other rat hepatic P-450 forms. Cyclophosphamide activation catalyzed by uninduced adult male rat liver microsomes (specific activity, 0.68 nmol/min/mg), although not inhibited by anti-P-450 PB-4 antibodies, was partially inhibited (approximately 60%) by antibodies to P-450 PB-1 (gene IIC6) and more completely inhibited (greater than 95%) by antibodies reactive with both P-450 PB-1 and P-450 2c (gene IIC11). Consistent with these observations, P-450 PB-1 and P-450 2c both activated cyclophosphamide at moderate rates in reconstituted systems (turnover, 1.6-2.7 nmol metabolite/min/nmol P-450), while seven other purified hepatic P-450 forms exhibited significantly lower activities (turnover less than or equal to 0.5 nmol metabolite/min/nmol P-450). Further studies revealed that the changes in liver microsomal cyclophosphamide activation rates with age and sex and in response to in vivo administration of cisplatin primarily reflect changes in the levels of P-450 forms PB-1 and 2c. These studies establish that P-450 forms PB-1, 2c, and PB-4 are the major catalysts of cyclophosphamide activation in rat hepatic tissue and that the modulation of microsomal cyclophosphamide activation with development and in response to drug exposure largely reflects alterations in the levels of these three hepatic P-450 enzymes.     

Mechanisms of cyclophosphamide action on hepatic P-450 expression

Cyclophosphamide was administered to adult male rats (130 mg/kg, single i.p. injection) and its effects on the P-450 enzymes that contribute to the activation of this drug in rat liver were then assessed. P-450-mediated cyclophosphamide 4-hydroxylase activity in isolated rat liver microsomes decreased by approximately 70% over a 9-day period following drug treatment. This decrease was due to the loss of cytochrome P-450 form 2c (IIC11), a major contributor to cyclophosphamide 4-hydroxylation in untreated male rat liver, while the other major hepatic cyclophosphamide 4-hydroxylase, P-450 PB-1 (IIC6), was largely unaffected. The loss of P-450 2c activity did not result from a decrease in P-450 reductase or from direct inactivation of the P-450 protein by cyclophosphamide or its metabolites, but rather was due to a reduction in hepatic P-450 2c protein and mRNA levels. Hepatic P-450 2a (IIIA2) and P-450 RLM2 (IIA2) were also suppressed by cyclophosphamide treatment. Serum testosterone, which contributes to the expression of P-450s 2c, 2a, and RLM2, was severely depleted in the cyclophosphamide-treated rats; however, this loss was not the direct cause of the decreases in these hepatic P-450s, since the decreases were not reversed upon restoration of normal testosterone levels by human chorionic gonadotropin stimulation of testicular androgen production. In contrast to the suppression of these testosterone-dependent P-450s, P-450 3 (IIA1), P-450j (IIE1), and the P-450-independent microsomal enzyme steroid 5 alpha-reductase were each elevated in rat liver following cyclophosphamide administration. In contrast to P-450 3 and steroid 5 alpha-reductase, however, the elevation of P-450j protein was transient and was not accompanied by an increase in P-450j-associated hepatic microsomal aniline hydroxylase activity. In vitro experiments revealed that P-450j was severalfold more susceptible to inactivation by the cyclophosphamide metabolite acrolein as compared with P-450 3. These observations suggest that P-450j protein is induced by cyclophosphamide treatment but that the protein is inactivated by the cyclophosphamide metabolite acrolein. These findings establish that cyclophosphamide treatment can modulate hepatic P-450 activities through multiple mechanisms and in a manner that may alter P-450 metabolism of cyclophosphamide and perhaps other anticancer drugs that undergo bioactivation in the liver.     

Biotransformation of N,N',N'-triethylenethiophosphoramide: oxidative desulfuration to yield N,N',N'-triethylenephosphoramide associated with suicide inactivation of a phenobarbital-inducible hepatic P-450 monooxygenase

Oxidative metabolism of the polyfunctional alkylating agent N,N',N'-triethylenethiophosphoramide (thio-TEPA) was studied in isolated rat liver microsomes and purified, reconstituted cytochrome P-450 (P-450) enzyme systems in order to elucidate the pathways of drug oxidation and to identify the possible contributions of individual P-450 enzymes to the bioactivation of this chemotherapeutic agent. Rat liver microsomes were found to catalyze conversion of thio-TEPA to its oxo metabolite, N,N',N'-triethylenephosphoramide (TEPA), in a P-450-dependent reaction that was markedly stimulated by prior in vivo treatment with drug inducers of hepatic P-450 subfamily IIB (phenobarbital), but not by pretreatment with inducers of P-450 subfamilies IA (beta-naphthoflavone) or IIE (isoniazid). Thio-TEPA depletion and TEPA formation catalyzed by phenobarbital-induced liver microsomes were both inhibited by greater than 90% by antibodies selectively reactive with P-450 PB-4 (gene product IIB1), the major phenobarbital-inducible rat liver microsomal P-450 form, but not by antibodies inhibitory toward 7 other rat hepatic P-450s. Oxidation of thio-TEPA to TEPA was also catalyzed by purified P-450 PB-4 (Km (app) 19 microM; Vmax (app) = 11 mol thio-TEPA metabolized/min/mol P-450 PB-4) following reconstitution of the cytochrome with NADPH P-450 reductase in a lipid environment. Metabolism of thio-TEPA by P-450 PB-4 was associated with a suicide inactivation of the cytochrome characterized by kinactivation = 0.096 min-1, KI = 24 microM, and a partition ratio of 136 +/- 28 (SD) mol thio-TEPA metabolized/mol P-450 inactivated. The thio-TEPA metabolite TEPA, however, did not inactivate the cytochrome, nor was it subject to further detectable metabolism. In microsomal incubations, metabolism of thio-TEPA led to the inactivation of P-450 PB-4 (steroid 16 beta-hydroxylase) as well as P-450 IIIA-related enzymes (steroid 6 beta-hydroxylase) and the P-450-independent enzyme steroid 17 beta-hydroxysteroid:NADP+ 17-oxidoreductase, as demonstrated by use of the P-450 form-selective steroidal substrate androst-4-ene-3,17-dione. In contrast, little or no inactivation of microsomal P-450 IIA-related enzymes (steroid 7 alpha-hydroxylase) or microsomal NADPH P-450 reductase was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

Species Difference among Experimental Rodents in the Activity and Induction of Cytochrome P-450 Isozymes for Mutagenic Activation of Carcinogenic Aromatic Amines

The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9 H -pyrido[2,3- b ]indole acetate (MeAαC) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeAαC were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.     

Effects of advanced leukemia on hepatic drug-metabolizing activity in the mouse

Summary Mice that had received 10⁶ P388 leukemia cells IV 8 days previously exhibited a decrease in the components of the hepatic microsomal mixed function oxidase, with a 58% decrease in cytochrome P-450, and up to a 60% decrease in hepatic microsomal metabolism of biphenyl. Liver weight was increased by 49% due to infiltration of the liver with leukemic cells. Changes in liver drug-metabolizing activity and liver weight were not seen 6 days after administration of P388 leukemia. There was a small increase in serum liver enzyme but no increase in total serum bilirubin in tumor-bearing mice. In vivo total-body plasma clearance of cyclophosphamide, a drug metabolized by hepatic cytochrome P-450, was decreased to 53 ml/min/kg in mice that had received P388 cells 8 days earlier, as against 97.2 ml/min/kg in control mice. Cytochrome P-450-independent metabolism of [¹⁴C]5-fluorouracil, measured by means of [¹⁴C]CO₂in the breath over 3 h, was decreased to 21% of the dose administered by 8 days after tumor cell administration, compared with 31% of the dose in control mice. P388 leukemia cells growing in the ascitic form in the intraperitoneal cavity of mice did not release an inhibitor of 5-fluorouracil metabolism into the ascitic fluid. Total-body plasma clearance of indocyanine green was decreased to 11 ml/min/kg by 8 days after P388 cell administration, compared with 36 ml/min/kg in control mice. The decrease in indocyanine green clearance might reflect a decrease in hepatic blood flow in the tumor-bearing mice. A possible explanation for the decrease in hepatic drug metabolism caused by P388 leukemia is that the hepatocytes are deprived of oxygen and nutrients by the tumor in the liver, coupled with or caused by a physical obstruction of hepatic blood flow.     

Role of xanthine oxidase in the interferon-mediated depression of the hepatic cytochrome P-450 system in mice

Interferon, interferon inducers, and a variety of other immunomodulators are known to depress the hepatic cytochrome P-450 drug-metabolizing system. Two concepts have been proposed to explain this phenomenon. (a) The steady-state of cytochrome P-450 is altered through decreased synthesis and increased degradation of cytochrome P-450 apoprotein. (b) Interferon induces xanthine oxidase; superoxide generated by interferon-induced xanthine oxidase destroys cytochrome P-450. The current study investigated the second concept. Administered polyribonucleotides [polyriboinosinic acid.polyribocytidylic acid (poly IC), polyriboinosinic acid.polycytidylic acid, polylysine and carboxymethylcellulose, mismatched poly IC], recombinant murine gamma-interferon, and a natural murine alpha/beta-interferon were shown to depress hepatic cytochrome P-450 and selected microsomal cytochrome P-450-dependent monooxygenase reactions and to induce hepatic xanthine oxidase activity. The feeding of tungstate in the drinking water largely depleted xanthine oxidase in mice; cytochrome P-450 levels and monooxygenase activities were not affected by tungstate treatment. Tungstate rendered the level of xanthine oxidase much below that in mice that had not received tungstate regardless of whether or not they had received poly IC or interferon; nevertheless, poly IC and interferon produced losses of cytochrome P-450 and monooxygenase activities in these tungstate-treated mice equivalent to those observed in mice that had not received tungstate. The administration of N-acetylcysteine did not prevent the loss of cytochrome P-450 induced by poly IC, as has been reported, nor did the incubation of microsomal cytochrome P-450 with buttermilk xanthine oxidase and hypoxanthine cause a loss of cytochrome P-450, which has also been reported. It is concluded from these studies that the induction of xanthine oxidase and the loss of cytochrome P-450 generated by interferon are coincidental rather than causally related phenomena.     

Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450

The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused approximately 90% inhibition of the metabolism of NNDM. These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.     

Platinum anticancer drugs modulate P-450 mRNA levels and differentially alter hepatic drug and steroid hormone metabolism in male and female rats.

Treatment of male rats with the anticancer drug cisplatin leads to feminization of the profile of cytochrome P-450 and other microsomal enzymes involved in steroid hormone and drug metabolism (G.A. LeBlanc, and D.J. Waxman, J. Biol. Chem., 263: 15732-15739, 1988). The present study uses the rat model to evaluate the differential effects of cisplatin treatment on liver microsomal enzymes between genders, and also examines whether the modulation of enzyme activities by cisplatin and its analogues involves changes in P-450 gene expression. While cisplatin treatment of male rats caused a severalfold increase in female-predominant hepatic enzymes, including testosterone 5 alpha-reductase and testosterone 7 alpha-hydroxylase (P-450 form 2A1), it partially decreased the expression of these enzymes in females. The reduced expression of these estrogen-dependent enzymes in females may derive from the loss of circulating estradiol that was shown to occur in response to cisplatin treatment. Analysis of mRNA levels of individual P-450 forms revealed that the effects of cisplatin on P-450-catalyzed steroid hydroxylase activities in both male and female rats are primarily operative through the drug's effects on P-450 mRNA expression. P-450-dependent cyclophosphamide activation was significantly compromised in male rats after cisplatin administration; however, this activity was not altered in cisplatin-treated females. This sex-dependent effect of cisplatin was due to its suppression of P-450 form 2C11, a male-specific P-450 that is a major contributor to microsomal cyclophosphamide bioactivation in male rat liver. The clinically active cisplatin analogue iproplatin elicited effects very similar to those of cisplatin, while carboplatin and transplatin did not have significant effects on hepatic P-450 expression. Together, these findings demonstrate that the response of rat liver to cisplatin-induced changes in hepatic P-450 enzyme profiles and cyclophosphamide bioactivation capacity differs between the sexes, and in addition, these effects can be minimized by use of carboplatin in place of cisplatin.     

Androgen-dependent renal microsomal cytochrome P-450 responsible for N-hydroxylation and mutagenic activation of 3-methoxy-4-aminoazobenzene in the BALB/c mouse

A murine renal microsomal enzyme responsible for the mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) was characterized by its catalytic activity for the mutagenic and metabolic conversion of 3-MeO-AAB. Incubation of 3-MeO-AAB with a renal or hepatic microsome fraction from male BALB/c mice in the presence of NADPH and NADH yielded N-hydroxy and 4'-hydroxy metabolites of 3-MeO-AAB as determined by two-dimensional thin layer chromatography, and the enzyme responsible for the N-hydroxylation was named 3-MeO-AAB N-hydroxylase. A mutagenicity test using Salmonella typhimurium TA98 bacteria as a tester strain has revealed that N-hydroxy-3-MeO-AAB is a potent direct mutagen but that 4'-hydroxy-3-MeO-AAB is not mutagenic. Although 3-MeO-AAB N-hydroxylase activity in liver microsomes showed no sex difference, the enzyme activity in the kidney was detected from male mice but not from females. However, administration of testosterone to female mice induced the enzyme in the kidney. Castration of male mice depressed the activity of 3-MeO-AAB N-hydroxylase in renal microsomes but it little affected the hepatic activity, and on administration of testosterone to the castrated mice the depressed renal microsomal activity recovered to a normal level. The activity of 3-MeO-AAB hydroxylase and the amount of cytochrome P-450 in renal microsomes showed a close correlation. Both renal and hepatic microsomes required NADPH as a main cofactor to mutagenize 3-MeO-AAB and to yield N-hydroxy-3-MeO-AAB from 3-MeO-AAB, and the enzyme activity was strongly inhibited by 7,8-benzoflavone. When the activities of renal and hepatic 3-MeO-AAB N-hydroxylase were compared on the basis of the amount of cytochrome P-450, the renal type enzyme showed about 8 times greater activity than hepatic type enzyme. These results indicate that the kidney contains an androgen-dependent microsomal 3-MeO-AAB hydroxylase which is different from an isozyme present in the liver and which is a new type of cytochrome P-450 isozyme.     

The effect of phenobarbital on cyclophosphamide antitumor activity.

We have used the spleen colony assay system and survival duration studies in male DBA/2 mice with P388 leukemia to study the effects of microsomal enzyme induction by phenobarbital on the antileukemic activity and bone marrow toxicity of cyclophosphamide. Phenobarbital drinking water (0.5 mg/ml) was given for 7 days prior to cyclophosphamide (10 to 200 mg/kg i.p.). Average daily phenobarbital intake per mouse was 1.25 mg (equivalent to 4 mg/kg/day human dosage). Dose-response curves with and without phenobarbital pretreatment showed a constant 90% (1-log) reduction in the toxicity of cyclophosphamide to leukemic colony-forming units, whereas enzyme induction had no effect on the toxicity of the drug to normal bone marrow colony-forming units. Parallel survival studies confirmed the 1-log diminution in the antileukemic activity of cyclophosphamide in phenobarbital-pretreated mice. This phenobarbital-induced change in the antitumor activity of cyclophosphamide appears explainable on a pharmacokinetic basis. The Friedman and Boger assay for plasma alkylating metabolites showed that the reduction in the area under the plasma metabolite curve caused by enzyme induction exactly predicted the observed reduction in cyclophosphamide antitumor effect.     

The effect of cimetidine on cyclophosphamide metabolism in rabbits

Summary Six female rabbits were given 20 mg/kg cyclophosphamide (containing 100 Ci [³H-chloroethyl]-cyclophosphamide) alone or 1 h following 100 mg/kg cimetidine. Serial plasma and urine specimens were collected and levels of cyclophosphamide and its metabolites (4-hydroxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard, and carboxyphosphamide) were measured. 4-Ketocyclophosphamide was the major metabolite present in rabbit plasma and urine, with lesser amounts of 4-hydroxycyclophosphamide, carboxyphosphamide, and phosphoramide mustard also being identified. Cimetidine pretreatment resulted in prolongation of cyclophosphamide's half-life from 24.3±7.3 to 33.5±9.5 min (mean ± SD;P=0.036) but did not significantly alter the AUC_{0–8 h} for the latter drug. Cimetidine pretreatment resulted in a significantly greater AUC_{0–8 h} for 4-hydroxycyclophosphamide (189.4±77 vs 364.6±126.7 mol min/l^–1;P=0.016) as compared with control values. A higher AUC_{0–8 h} value for phosphoramide mustard (53.7±69.2 vs 95.7±34.7 mol min/l^–1) was also observed after cimetidine dosing but the difference was not significant (P=0.21). Kinetics of 4-ketocyclophosphamide and carboxyphosphamide were not significantly affected by cimetidine treatment. Cimetidine was added to hepatic microsomes isolated from phenobarbital-treated rabbits; it did not inhibit cyclophosphamide's metabolism in vitro, suggesting that its in vivo effect may be mediated through mechanisms other than cytochrome P-450 inhibition. Cimetidine pretreatment increases exposure to cyclophosphamide and its major activated metabolite, 4-hydroxycyclophosphamide. Potentiation rather than inhibition of cyclophosphamide's pharmacodynamic effect is to be predicted when cimetidine is given concomitantly with the former. Alterations in hepatic blood flow or mechanisms other than microsomal inhibition by cimetidine may explain this potentiation.Supported in part by the Department of Veteran Affairs and grant CA-49186 from the National Institutes of Health (NIH)Department of Clinical Pharmacology, Sun Yat-sen University of Medical Sciences, Guangzhou, People's Republic of China     

Hepatic cytochrome P-450 isozyme(s) induced by dietary carcinogenic aromatic amines preferentially in female mice of DBA/2 and other strains

DBA/2, BALB/c or (BALB/cxDBA/2)F₁ (CDF₁) mice of both sexeswere treated for 1 week with a dietary hepatocarcinogenic tryptophanpyrolysate component (Trp P-1 or Trp P-2), and the activityof hepatic microsomal enzyme(s) for mutagenk activations ofTrp P-1 and Trp P-2 were assessed by means of a mutation testwith Salmonella typhimurium TA98. In both Ah-responsive (BALB/cand CDF₁) and Ah-nonresponsive (DBA/2) mice, the dietary treatmentwith Trp P-l or Trp P-2 resulted in a significant increase ofthe enzyme activity for mutagenic activations of Trp P-1 andTrp P-2 in females but not in males, except the case of maleBALB/c mice treated with dietary Trp P-1 Also induction of enzyme(s)in female mice was suppressed by an administration of testosterone.The induced hepatic microsomal enzyme(s) was demonstrated tobe cytochrome P-450 isozyme(s) (mol. wt of 55 000 daltons) byimmunoblots with use of an anti-rat cytochrome P-448 monoclonalantibody and by selective inhibition of the activity by additionof 7,8-benzoflavone into the mutation assay system. These findingsindicate that carcinogic aromatic amines such as Trp P-1 andTrp P-2 are able to induce hepatic cytochrome P-450 isozyme(s)not only in Ah-responsive mice (BALB/c and CDF₁) but also inAh-nonresponsive DBA/2 mice and that the cytochrome P-450 inductionis controlled by androgen(s).     

Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator

Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS)     

BCNU-induced quantitative and qualitative changes in hepatic cytochrome P-450 can be correlated with cholestasis

Summary Male Sprague-Dawley rats were given single i.p. injections of 1,3-bis(2-chloroethyl)-1-Nitrosourea (BCNU) to investigate changes in hepatic microsomal cytochrome P-450 content and metabolic activity. On day 14 after treatment (20 mg/kg), cytochrome P-450 content had decreased by approximately 25% and ethylmorphineN-demethylase activity (nmol product/nmol P-450/min) had decreased by 36%. In contrast, ethylmorphineO-deethylase and 7-ethoxycoumarinO-deethylase activities were not significantly decreased by BCNU treatment. Hepatic delta-aminolevulinic acid synthetase activity was only 60% of control values, and microsomal heme oxygenase activity was slightly but not statistically elevated. Cytochrome P-450 content in control and BCNU-treated rats increased in a similar manner after phenobarbital or -naphthoflavone induction. Electrophoretic analysis of cytochrome P-450 proteins isolated from hepatic endoplasmic reticular membranes of treated and control rats suggested that alterations in these proteins occurred in BCNU-treated rats. These changes in cytochrome P-450 content and activity are very similar to those reported in isolated systems exposed to bile acids or in rats with experimentally produced cholestasis. BCNU has been shown to produce cholestasis, which precedes its effects on microsomal mixed-function oxygenase activity. Thus, the delayed effects of BCNU on microsomal drug metabolism are probably secondary to its interference with bile formation.Abbreviations ALA delta aminolevulinic acid - BCNU 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine) - BHCNU 1,3-b9s(trans-4-hydroxycyclohexyl)-1-nitrosourea - CCNU 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (lomustine) - EM ethylmorphine - EMND ethylmorphineN-demethylase - EMOD ethylmorphineO-deethylase - ECOD ethoxycoumarinO-deethylase - BAPH benzo[a]pyrene hydroxylase - MFO hepatic microsomal mixedfunction oxygenases Supported in part by NIH BRS grant RR07079 and by gifts from the Mayer and Chiles Foundations of Portland, Oregon     

Reconstitution of rabbit liver microsomal N-nitrosopyrrolidine alpha-hydroxylase activity

The in vitro alpha-hydroxylation of N-nitrosopyrrolidine (NPYR) by both isolated rabbit liver microsomes and purified cytochrome P-450 isozymes was investigated. Microsomes from untreated rabbits catalyzed the alpha-hydroxylation of NPYR at rates similar to those reported previously for rats, mice, and hamsters. The effect of established inducers of microsomal P-450 caused complex changes in apparent rates of alpha-hydroxylation of NPYR which made interpretation of responses to inducer pretreatment difficult and suggested the participation of multiple cytochrome P-450 isozymes in the metabolism of NPYR. Partial inhibition of alpha-hydroxylase activity by antibodies against rabbit isozymes 2, 3a, and 5 indicated the participation of at least these three isozymes in microsomal catalysis. Reconstitution studies using purified rabbit isozymes 2, 3a, 3b, 3c, 4, and 6 indicated that isozymes 2, 3a, 4, and 6 possessed significant alpha-hydroxylase activity with isozymes 3a and 6 exhibiting the highest activity when assayed at 20 mM NPYR. As NPYR concentrations were decreased, the rates of catalysis for the reconstituted systems were differentially decreased such that isozyme 3a exhibited the highest activity at low NPYR concentrations. These data indicate that isozyme 3a is the preferred catalyst for the alpha-hydroxylation of NPYR at low substrate concentrations and suggest that conditions such as chronic ethanol consumption which lead to the induction of isozyme 3a in rabbits or its orthologue in other species can account for enhanced rates of alpha-hydroxylation and metabolic activation of NPYR.     

Metabolic oxidation of the carcinogens 4-aminobiphenyl and 4,4'-methylene-bis(2-chloroaniline) by human hepatic microsomes and by purified rat hepatic cytochrome P-450 monooxygenases

Metabolic N-oxidation and ring-oxidation of carcinogenic arylamines by hepatic cytochromes P-450 are generally regarded as critical activation and detoxification pathways, respectively. Two arylamines with known human exposure, 4-aminobiphenyl (ABP) and 4,4'-methylene-bis(2-chloroaniline) (MOCA), have been examined as substrates for 10 different purified rat hepatic cytochromes P-450 and for human liver microsomal preparations from 22 individuals. Metabolites were analyzed by high-performance liquid chromatography and flow scintillation techniques. As reported for certain other carcinogenic arylamines, the isosafrole-inducible isozyme, P-450ISF-G, had the highest catalytic activity for ABP N-oxidation (13.6 nmol/min/nmol P-450), but P-450BNF-B, P-450UT-A, P-450UT-F, and P-450PB-B also showed appreciable activity. Ring-oxidation of ABP occurred only to a minor extent. In contrast, N-oxidation of MOCA was preferentially catalyzed by the phenobarbital-inducible enzymes, P-450PB-B and P-450PB-D (9.0 and 6.6 nmol/min/nmol P-450, respectively). MOCA ring-oxidation and methylene carbon oxidation showed varied cytochrome P-450 selectivity and accounted for 14 to 79% of total oxidation products. There was a 44-fold variation in rates of ABP N-oxidation in the 22 human liver microsomal preparations, while rates of N-oxidation of MOCA varied only 8-fold. Ring/methylene carbon-oxidation of MOCA accounted for 6-19% of total oxidation products in the case of the human microsomal preparations, whereas ring-oxidation of ABP accounted for less than 7% of total oxidation. In addition, there was a strong correlation (R = 0.90) between rates of ABP N-oxidation and phenacetin O-deethylation, which is considered a human genetic polymorphism. Moreover, both the ABP N-oxidation and phenacetin O-deethylation activities of human liver microsomes showed a good correlation (R = 0.72) with the levels of cytochrome P-450 immunochemically related to rat P-450ISF-G. These data indicate that specific inducible and constitutive cytochromes P-450 are involved in the metabolic activation and detoxification of the carcinogens ABP and MOCA. Therefore, individual profiles of cytochromes P-450, affected by both environmental and genetic factors, may be significant determinants of individual susceptibility to arylamine carcinogenesis.     

Changes in the Quantity and Activity of Cytochrome P-450 Isozymes in Primary Cultured Rat Hepatocytes

Hepatocytes from male Spragne-Dawley rats pretreated with a cytochrome P-450 inducer, 3-methoxy-4-aminoazobenzene (3-MeO-AAB), 3-methylcholanthrene (MC) or phenobarbital (PB), were cultured in vitro , and changes in the quantity and activity of microsomal cytochrome P-450 isozymes in the cells were determined by means of immunochemical methods and a bacterial mutation test, respectively. The results of enzyme-linked immunosorbent assay using monoclonal antibodies against rat P-450 isozymes - test using Salmonella typhimurium TA 98 and carcinogenic aromatic amines. These results indicate that microsomal cytochrome P-450c in primary cultured rat hepatocytes is more stable in culture, in terms of both quantity and activity, than cytochrome P-450d and P-450b/e.     

Participation of cytochrome P-450 in reductive metabolism of 1-nitropyrene by rat liver microsomes

Reductive metabolism of carcinogenic 1-nitropyrene by rat liver microsomes and reconstituted cytochrome P-450 systems was investigated. Under the nitrogen atmosphere, 1-aminopyrene was the only detected metabolite of 1-nitropyrene. The reductase activity in liver 105,000 X g supernatant fraction was ascribed to DT-diaphorase, aldehyde oxidase, and other unknown enzyme(s) from the results of cofactor requirements and inhibition experiments. The microsomal reductase activity was inhibited by oxygen, carbon monoxide, 2,4-dichloro-6-phenylphenoxyethylamine, and n-octylamine. Flavin mononucleotide markedly enhanced the activity, and 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride also enhanced it, but slightly. The microsomal activity was induced by the pretreatment of rats with 3-methylcholanthrene, sodium phenobarbital, or polychlorinated biphenyl, and the increments of the activity correlated well with those of the specific contents of cytochrome P-450 in microsomes. The reductase activity could be reconstituted by NADPH-cytochrome P-450 reductase and forms of cytochrome P-450 purified from liver microsomes of polychlorinated biphenyl-induced rats. Among four forms of cytochrome P-450 examined, an isozyme P-448-IId which showed high activity in hydroxylation of benzo(a)pyrene catalyzed most efficiently the reduction of 1-nitropyrene. The results of this study indicate the central role of cytochrome P-450 in the reductive metabolism of 1-nitropyrene in liver microsomes.     

Role of β-carotene on the changes in activity patterns and levels of biotransforming enzymes in transplantable murine lymphoma

The differential levels of induction of hepatic microsomal cytochrome P-450 (cyt. P-450), UDP-glucuronyl transferase (UDPGT) and cytosolic glutathione-S-transferase (GST) activities were evaluated over various periods of time, following tumor transplantation in male Swiss albino mice in the presence and absence of β-carotene supplementation in their basal diet (100 mg/kg). An increase in the total hepatic microsomal cytochrome P-450 and UDP-glucuronyl transferase and cytosolic GSH-transferase activities (1.5 to 2 fold) occurred during the later stage of tumor progression (22 ± 2 days onwards). However, β-carotene supplementation throughout the study increased or decreased the random activity trends of the above markers significantly (P < 0.05−< 0.01). Finally, β-carotene supplementation could enhance the survival of the host bearing lymphoma by almost 2-fold (50–60 days) over and above the lymphoma controls (30–35 days).