Activation and detoxication of promutagens by toadfish (Opsanus tau) hepatic postmitochondrial fractions in the Salmonella assay

Three groups of experiments were conducted to characterize the hepatic postmitochondrial fraction (S9) from the oyster toadfish (Opsanus tau) as an activation system for promutagens in the Salmonella assay and to provide an initial evaluation of the extent to which data from standard in vitro assays with mammalian activation systems are predictive of possible genotoxic effects in this marine fish. In the first group of experiments the effects of increasing the concentration of S9 from untreated and 3-methylcholanthrene (MC)- or Aroclor 1254 (AC)-pretreated toadfish and Sprague-Dawley rats on the mutagenicities of different concentrations of 2-aminoanthracene (2AA) and benzo[a]pyrene (BAP) were examined in Salmonella (TA98) plate assays. The maximum levels of 2AA mutagenicity attained by S9 from untreated (UI S9) toadfish and rats were comparable, but UI S9 from toadfish was moreeffective than UI S9 from rats in mediating BAP mutagenicity. MC pretreatment decreased maximum levels of 2AA mutagenicity and increased maximum levels of BAP mutagenicity mediated by S9 from both species. MC pretreatment also altered the pattern of dependence of 2AA mutagenicity on the concentration of S9 protein for S9 from both species. A similar alteration in the pattern of dependence of BAP mutagenicity on the concentration of S9 protein was also observed with S9 from MC-pretreated toadfish. Although AC pretreatment of rats effected changes in the mutagenicities of both test chemicals similar to those effected by MC pretreatment, AC pretreatment of toadfish effected little or no change in the mutagenicities of either test chemical. The changes in the pattern of dependence of 2AA and BAP mutagenicities on the concentration of S9 protein effected by MC pretreatment of toadfish were confirmed in a separate group of experiments. A third group of experiments was designed to examine the effects of α-naphthoflavone (ANF) on the mutagenicities of 2AA and BAP mediated by UI and MC S9 from toadfish. Although ANF did not affect the 2AA mutagenicity mediated by UI S9, a significant decrease in 2AA mutagenicity and a significant increase in BAP mutagenicity mediated by MC S9 and a significant decrease in BAP mutagenicity mediated by UI S9 were observed. These results indicate that 2AA and BAP are effectively activated by toadfish S9 and that, as in rats, these two test chemicals are activated and/or detoxicated by different cytochrome P-450-dependent pathways. These results also support the contention that cytochrome P-450-dependent detoxication pathways can be an important determinant of the mutagenic potency of some promutagens in vitro.     

The spectrum of enzymes involved in activation of 2-aminoanthracene varies with the metabolic system applied

The aim of this study was to estimate the involvement of cytochrome P450s (CYPs) in the metabolic activation of 2-aminoanthracene (2AA) by use of metabolic systems such as liver S9 or hepatocytes from untreated and beta-naphthoflavone (BNF)- or phenobarbital (PB)-treated rats. Metabolic activation was determined in the Salmonella reverse mutation assay (Ames test). Unexpectedly, both enzyme inducers, BNF and PB, significantly decreased the mutagenicity of 2AA activated by S9 fractions. 2AA mutagenicity was detected in the presence of cytochrome P450 inhibitors such as alpha-naphthoflavone (ANF), clotrimazole and N-benzylimidazole to study the contribution of CYP isoenzymes to the activation process. ANF significantly decreased the activation of 2AA by S9 from untreated rats. In contrast, ANF significantly increased the metabolic activation of 2AA by S9 from BNF- and PB-treated rats. The enhanced mutagenicity was not altered by co-incubation with clotrimazole and ANF. Pre-incubation of 2AA in the presence of N-benzylimidazole significantly increased the activation of 2AA by S9 from BNF- and PB-treated rats, which suggests that CYPs play minor role in 2AA metabolic activation by rat liver S9 fractions. In contrast with the results described above, BNF treatment of rats significantly enhanced the activation of 2AA by hepatocytes. ANF attenuated the extent of this activation suggesting that different enzymes play a major role in the activation processes in these metabolic systems. Our results indicate that identification of mutagenic hazard by use of the Ames test may depend on the metabolic system applied.     

Some factors determining the concentration of liver proteins for optimal mutagenicity of chemicals in the Salmonella/microsome assay.

In plate assays in the presence of S. typhimurium TA100 and various amounts of liver 9000 X g supernatant (S9) from either untreated, phenobarbitone- (PB) or Aroclor-treated rats, the S9 concentration required for optimal mutagenicity of aflatoxin B1 (AFB) depended both on the source of S9 and on the concentration of the test compound. In these assays, the water-soluble procarcinogen, dimethylnitrosamine (DMN) was mutagenic in S. typhimurium TA1530 only in the presence of a 35-fold higher concentration of liver S9 from PB-treated rats than that required for AFB, a lipophilic compound. In liquid assays, a biphasic relationship was observed in the mutagenicities in S. typhimurium TA100 of benzo[a]pyrene (BP) and AFB and the concentration of liver S9. For optimal mutagenesis of BP, the concentration of liver S9 from rats treated with methylcholanthrene (MC) was 4.4% (v/v); for AFB it was 2.2% (v/v) liver S9 from either Aroclor-treated or untreated rats. At higher concentrations of S9 the mutagenicity of BP and of AFB was related inversely to the amount of S9 per assay. The effect of Aroclor treatment on the microsomemediated mutagenicity of AFB was assay-dependent: in the liquid assay, AFB mutagenicity was decreased, whereas in the plate assay it did not change or was increased. As virtually no bacteria-bound microsomes were detected by electron microscopy, after the bacteria had been incubated in a medium containing 1-34% (v/v) MC-treated rat-liver S9, it is concluded that, in mutagenicity assays, mutagenic metabolites generated by microsomal enzymes from certain pro-carcinogens have to diffuse through the assay medium before reaching the bacteria. Thus the mutagenicity of BP was dependent on both the concentration of rat-liver microsomes and that of total cytosolic proteins and other soluble nucleophiles such as glutathione. At a concentration of 4.4% (v/v) liver S9, the mutagenicity of BP was about 3.6 times higher than in assays containing a 4-fold higher concentration of cytosolic fraction. Studies on the glutathione-dependent reduction of BP mutagenicity in plate assays has shown that, in the presence of liver S9 concentrations greater than that required for optimal mutagenicity, the reduction in mutagenicity was related directly to the concentration of liver S9. Thus, in the Salmonella/microsome assay, when the concentration of rat-liver S9 was increased over and above the amount required for the optimal mutagenicity of BP, the mutagenic metabolites of BP were inactivated (by being trapped with cytosolic nucleophiles and/or by enzymic conjugation with glutathione); this effect increased more rapidly than their rate of formation. The concentration of liver S9 for optimal mutagenicity of test compounds requiring activation catalyzed by mono-oxygenases seems, therefore, to be related to the departure from linearity of the relationship between the rate of formation of mutagenic metabolites and the concentration of liver S9.     

Escherichia coli MTC, a NADPH cytochrome P450 reductase competent mutagenicity tester strain for the expression of human cytochrome P450: comparison of three types of expression systems

Currently three different methods have been taken to develop new mutagenicity tester strains containing human cytochrome P450s (CYPs). Each of these use a single expression vector. In this paper we describe a fourth approach, i.e., the coexpression of a CYP and its electron-transfer flavoprotein, NADPH CYP reductase (RED), encoded by two different expression vectors. The Escherichia coli mutagenicity tester strain BMX100 has been expanded to a strain, MTC which stably expresses human RED. This new tester strain permits the biplasmid coexpression of human CYP1A2 and RED (MTC1A2). This novel strain can be used for the determination of the mutagenicity of chemicals known to be procarcinogens and metabolized by CYP1A2. The mutagenicity tester strain MTC1A2 was compared with: (i) BMX100 using the post-mitochondrial rat liver fraction (S9); (ii) BMX100 with expressing CYP1A2 alone (iii) or with expressing CYP1A2 fused to rat RED or (iv) with expressing CYP1A2, bicistronically coexpressed with rat RED. The biplasmid RED/CYP coexpression system generated a strain with the highest methoxy- and ethoxy-resorufin dealkylase activities and the highest mutagenic activities for the procarcinogens 2-aminoanthracene (2AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). Furthermore, the metabolism of 2AA and IQ was detected more efficiently using the MTC1A2 strain than with the BMX100 strain plus the standard rodent liver S9 metabolic system.     

Comparative activation of 3,3'-dichlorobenzidine and related benzidines to mutagens in the Salmonella typhimurium assay by hepatic S9 and microsomes from rats pretreated with different inducers of cytochrome P-450

The metabolic basis of the differential activation of 4 benzidines--3,3'-dichlorobenzidine (DCB), benzidine (BZ), o-tolidine (TOL) and o-dianisidine (DIN)--to mutagens was examined in the Ames test, using Salmonella typhimurium TA98. For each benzidine congener, the comparative activation by 3 rat liver enzyme systems--(i) postmitochondrial supernatant (S9), (ii) S9 + acetylcoenzyme A (S9-Ac) and (iii) microsomes--and the effect thereon of animal pretreatment with 3 cytochrome P-450 inducers--DCB, 3-methylcholanthrene (MC) and phenobarbital (PB)--were examined. DCB was the most activated of the benzidines, with activation by the 3 systems being in the order: S9 = S9-Ac greater than microsomes, whereas dianisidine and tolidine were the least activated. Benzidine was activated only in the S9 systems but the S9-catalyzed activation of benzidine, unlike that of DCB, was enhanced by added acetylcoenzyme A. Pretreatment with either DCB, MC or PB enhanced the activation of DCB, decreased that of benzidine, and had no effect on that of tolidine or dianisidine. The enhanced DCB activation was most pronounced with DCB pretreatment and was 2.5-fold, 2-fold, and 3-fold, in S9-Ac, S9, and microsomes, respectively. The microsomal-catalyzed DCB activation was inhibited by the cytochrome P-450 inhibitors 2,4-dichloro-6-phenylphenoxyethylamine and alpha-naphthoflavone by 93% and 48%, respectively. DCB, but not its congeners, elicited NADPH-dependent metabolite complex formation with microsomal cytochrome P-450. The results show that DCB is the most mutagenic of the 4 benzidines under conditions of cytochrome-P-450-catalyzed activation and suggest that the DCB activation may be catalyzed most effectively by cytochrome P-450 species induced specifically by the compound itself.     

Selenium inhibition of benzo[a]pyrene, 3-methylcholanthrene, and 3-methylcholanthrylene mutagenicity in Salmonella typhimurium strains TA98 and TA100

Selenium (Se) decreased the mutagenicity of benzo[a]pyrene (BP), 3-methylcholanthrene (3MC), and 3-methylcholanthrylene (3MCE) in Salmonella typhimurium strains TA98 and TA100. Metabolism of BP, 3MC and 3MCE to mutagens was accomplished with the liver S9 fraction from Aroclor 1254-treated male Sprague-Dawley rats. Exposure of the bacteria to 4 nmoles BP, 10 nmoles 3MC, or 10 nmoles 3MCE in the presence of S9, and up to 200 nmoles Se as Na2SeO3 resulted in decreased mutagenicities up to 39, 66 and 60% of their respective control activities without Se in TA98 and up to 46, 52 and 64% of their respective control activities without Se in TA100. Se (200 nmoles) alone was not mutagenic in strains TA98 or TA100 with or without S9. BP, 3MC and 3MCE were not mutagenic in either strain without S9. None of the tested concentrations of BP, 3MC, 3MCE and Se were cytotoxic. Assays of the aryl hydrocarbon hydroxylase (AHH) activity in the S9 preparation revealed decreased AHH activity with increase in Se concentration. The decreased mutagenicity and AHH activity were Se (as Na2SeO3) dependent and could not be duplicated by sulfur (S as Na2SO3). Inhibition of AHH activity by Se provides an explanation of the mechanism of Se inhibition of BP, 3MC and 3MCE mutagenicities in S. typhimurium TA98 and TA100.     

Enhancement of DNA binding,mutagenicity and carcinogenicity of polycyclic aromatic hydrocarbons after induction of cytochrome P-450 by ellipticines in rats and mice

Pretreatment of rats by ellipticines enhanced the microsomal concentration of cytochrome P-450, benzo[a]pyrene (BP) metabolism and activation and, to a smaller extent, ethoxycoumarin deethylation, but not acetanilide hydroxylation. This increased BP biotransformation was essentially due to the formation of bay-region metabolites, BP 9,10-diol, BP 7,8-diol and 9-hydroxy-BP, or to the formation of BP 7,8-diol-9,10-epoxide- and of 9-hydroxy-BP 4,5-oxide-DNA adducts. In the ellipticine series, 9-fluoroellipticine (9-FE) presents a slight inducing potency compared with the parent and 9-hydroxy molecules. Pretreatment of mice with 9-hydroxyellipticine (9-OHE) led also to an increased mutagenicity of BP and to an augmentation of skin carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA). These results clearly show that 9-OHE induces the biosynthesis of cytochrome P-450 which markedly stimulates the mutagenic and carcinogenic potentialities of polycyclic aromatic hydrocarbons (PAH).     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Comparison of cytochrome P-450-dependent metabolism in different developmental stages of Drosophila melanogaster

The activities of several drug metabolizing enzymes were compared in microsomes from larvae and adult Drosophila. The cytochrome P-450 content and the benzo[a]pyrene (BP) hydroxylation, p-nitroanisole demethylation and 3- and 4-hydroxylation of biphenyl were 4-20-fold higher in microsomes from adult flies, while 7-ethoxycoumarin deethylase activity and cytochrome c reductase activity were about the same in the two stages. 2-OH-biphenyl was formed in trace amounts by microsomes from adult flies but not to any detectable amount by microsomes from larvae. Pretreatment with phenobarbital (PB), Aroclor 1254 (PCB) or beta-naphthoflavone (BNF) increased the cytochrome P-450 content and the various cytochrome P-450-mediated reactions up to 7-fold in larvae. The effects of the pretreatments were weaker in adult flies, where the increase never was more than 3-fold, and many reactions were unaffected by the pretreatments. BNF was thus inefficient in enhancing all reactions, except a slight (1.3-fold) increase in the formation of 4-OH-biphenyl. Microsomes from both stages exhibited increases in specific protein bands with apparent molecular weights of 51 000-58 000 in the sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis following treatment with PB, PCB and BNF. Differences were observed between larvae and adults with respect both to the number of and the molecular weights of the increased protein bands.     

Possibility of the involvement of 9H-pyrido[3,4-b]indole (norharman) in carcinogenesis via inhibition of cytochrome P450-related activities and intercalation to DNA

This study investigated the inhibitory effect of 9H-pyrido[3,4-b]indole (norharman), one of the naturally occurring beta-carbolines, on cytochrome P450 (CYP)-related activities and the relationship between its inhibitory effect, its intercalation to DNA, and its comutagenic effect. Norharman reduced the mutagenicities of heterocyclic amines (HCAs) containing 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), aflatoxin B1, benzo[a]pyrene (BP), and some nitrosamines in the presence of 10 microl liver S9 (20.9 microg protein/ml) from polychlorinated biphenyl-treated rats. Norharman inhibited microsomal CYP-related enzyme activities and CO-binding to the CYP heme (50% inhibitory concentration (IC50), 0.07-6.4 microg/ml). It also inhibited the formation of 3-hydroxyamino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (N-OH-Glu-P-1) and was a noncompetitive-inhibitor of CYP1A-related activities, while it enhanced the direct mutagenicity of N-OH-Glu-P-1 (50% effective concentration, 25.0 microg/ml) and inhibited topo I activity (IC50, 31.0 microg/ml). In the presence of norharman, S9 up to 100 microl incrementally enhanced the mutagenicities of HCAs, BP and dimethylnitrosamine. These data clarified that norharman acts as an inhibitor of the CYP-mediated biotransformation of Glu-P-1 via inhibition of O2-binding to CYP heme, and its inhibition of CYP enzymes occurs at much lower concentration than that for its intercalation to DNA. It is indicated that norharman's inhibitory effect on CYP results in the inhibition of excess metabolism by S9 and this is more likely the mechanism for comutagenic action than the intercalation. Norharman's inhibition of CYP and its enhancement of the N-OH-Glu-P-1 mutagenicity suggest that beta-carbolines modulate chemical carcinogenesis by controlling the xenobiotic metabolism and by intercalating to DNA.     

Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes

Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).     

Dependence on Salmonella typhimurium enzymes of mutagenicities of nitrobenzene and its derivatives in the presence of rat-liver S9 and norharman

Dependence on S. typhimurium enzymes of mutagenicities of nitrobenzene (NB) and o-, p-chloronitrobenzenes (o-, p-CNBs), which are only mutagenic in the presence of S9 and norharman (NOH), was investigated using a nitroreductase-deficient strain TA98NR and an esterifying enzyme-deficient strain TA98/1,8-DNP6. NB exhibited mutagenicity towards TA98 but did not towards TA98NR strain in spite of the presence of S9 in the assay system. The mutagenicity of o-CNB towards TA98NR was significantly lower than that of o-CNB towards TA98. In contrast to NB and o-CNB, synthesized phenylhydroxylamine (PHA) and o-chlorophenylhydroxylamine (o-CPHA) exhibited approximately the same mutagenicity towards both tester strains. These results indicate that the nitroreduction required for the appearance of mutagenicity of the nitrobenzene derivatives in the presence of S9 and NOH is dependent on the nitroreductase of the tester strain. In addition, the mutagenicities of PHA and p-CPHA were significantly higher towards TA98/1,8-DNP6 than towards TA98, suggesting that the esterification of their hydroxylamines produced inactivation rather than activation. From these results, it was concluded that S9 and NOH play a role in metabolic activation other than the reduction of the nitro group to hydroxylamine and subsequent esterification for the mutagenesis of NB and its derivatives.     

Detection of mutagenicity of diphenyl ether herbicides in Salmonella typhimurium YG1026 and YG1021

Three kinds of diphenyl ether herbicides, 4-nitrophenyl 2,4,6-trichlorophenyl ether (CNP, chlornitrofen), 2,4-dichlorophenyl 3-methoxy-4-nitrophenyl ether (chlomethoxynil) and 2,4-dichlorophenyl 3-methoxycarbonyl-4-nitrophenyl ether (bifenox), were tested for mutagenicity in Salmonella typhimurium YG1026 and YG1021, which have high nitroreductase activity, and also in S. typhimurium TA100 and TA98. CNP and chlomethoxynil showed mutagenicity in S. typhimurium YG1026, without S9 mix, inducing 50 and 304 revertants per μg. These mutagenicities were suppressed by the addition of S9 mix. CNP and chlomethoxynil were also mutagenic to YG1021 with and without S9 mix, and their mutagenicities were lower than those to YG1026. On the other hand, bifenox was mutagenic to YG1026 only with S9 mix, inducing 3.0 revertants per μg. These three herbicides showed no mutagenicity in S. typhimurium TA100 and TA98 either with or without S9 mix.     

Effect of Siamese cassia leaves on the activities of chemical carcinogen metabolizing enzymes and on mammary gland carcinogenesis in the rat.

Male Wistar rats were fed AIN-76 semipurified diet or diet containing 5% ground lyophilized Siamese cassia leaves for 2 weeks before sacrifice. Hepatic S9 fractions were prepared and assayed for the level of cytochrome P450 (P450), the activities of monooxygenase, i.e., aniline hydroxylase (ANH), aminopyrine-N-demethylase (AMD) as well as the capacity to metabolically activate the mutagenicities of aflatoxin B(1) (AFB(1)) and benzo(a)pyrene (B(a)P). In addition, the activities of detoxificating enzymes such as glutathione-S-transferase (GST) and UDP-glucuronyltransferase (UGT) were also measured. It was found that feeding of Siamese cassia leaves significantly reduced the activities of hepatic ANH and AMD as well as the capacity to activate the mutagenicity of AFB(1) towards Salmonella typhimurium TA100, being 31, 73 and 41% of control group, respectively. It also slightly decreased, but not significantly, the capacity to activate the mutagenicity of B(a)P towards S. typhimurium YG1029. On the other hand, however, the activities of both GST and UGT were markedly increased in those animals, being 250 and 220% of control animals. The anticarcinogenic potential of Siamese cassia leaves was also investigated in female Sprague Dawley rats treated with 9,10-dimethyl-1,2-benzanthracene (DMBA). The animals were fed control diet or diet containing ground lyophilized Siamese cassia leaves 2 weeks prior to and 1 week after intragastrically administration of DMBA, and then they were placed on a pellet diet for additional 25 weeks. Interestingly, it was found that feeding of diet containing 2.5 and 4% Siamese cassia leaves resulted in a significant decrease in the multiplicity of mammary gland tumors as well as a slight delay of the onset of tumor development. The incidence of tumors in the group fed 4% Siamese cassia leaves, but not in the 2.5% group, was lowered, although not significantly, than that of control group. The results in the present study therefore demonstrated that Siamese cassia leaves possess phase II enzyme inducing property as well as the ability to reduce some phase I enzyme activities in rat liver. This Thai vegetable also exhibit cancer chemopreventive potential, at least against DMBA-induced mammary gland carcinogenesis which may be partly due to phase II inducing capacity as well as phase I inhibitory activity.     

Modulation of Ah receptor and CYP1A1 expression by alpha-naphthoflavone in rainbow trout hepatocytes

The objective of this study was to evaluate whether alpha-naphthoflavone (ANF) modulates aryl hydrocarbon receptor (AhR) signaling in rainbow trout (Oncorhynchus mykiss). AhR and cytochrome P450 1A1 (CYP1A1) protein and mRNA content were used as indictors of AhR signaling. Primary culture of rainbow trout hepatocytes were exposed to different concentrations of ANF (10(-9)-10(-5) M), while beta-naphthoflavone (BNF 10(-10)-10(-6) M) and a combination of ANF and BNF were used to elucidate the impact of ANF on AhR signaling. ANF increased AhR and CYP1A1 protein expression in a concentration-related manner; the maximal induction was about 50% that of BNF. Despite the differences in protein content between ANF and BNF stimulation, the maximal AhR and CYP1A1 mRNA abundance seen with the high concentrations of ANF and BNF were similar. ANF significantly decreased ( approximately 50%) BNF-induced AhR protein expression (only at 10(-9) M), but not CYP1A1 protein and gene expression. In addition, ANF at a sub-maximal concentration (10(-7) M) did not affect BNF-induced AhR protein content, but increased the sensitivity of hepatocytes to BNF-mediated CYP1A1 protein expression. Taken together, the mode of action of ANF appears similar to BNF, including modulation of AhR expression and activation of AhR-mediated signaling in rainbow trout hepatocytes. Overall, ANF is not only a partial AhR agonist, but may also modify BNF-mediated AhR signaling in trout hepatocytes.     

Genetic differences in response to pulmonary cytochrome P-450 inducers and oxygen toxicity

The effects of cytochrome P-450 inducers on O2 toxicity were studied in mice. We first examined three cytochrome P-450 inducers, which differ by their specific tissue affinity: phenobarbital sodium (PB), essentially active in the liver, and 3-methylcholanthrene (3-MC) and beta-naphthoflavone (BNF), which are also active in the lung. Both BNF and 3-MC increased the survival rate and significantly decreased pulmonary edema (pulmonary water and wet-to-dry weight ratio) in C57BL/6J mice exposed to hyperoxia (O2 greater than or equal to 95%), whereas PB had no protective effect. In the second part of this study, we compared the action of BNF in two strains of mice. In one (C57BL/6J), cytochrome P-450 can be induced by aromatic hydrocarbons, whereas in the other (DBA/2J) cytochrome P-450 is not inducible by these compounds. Protection against O2 toxicity was assessed in terms of lethality and pulmonary edema and of lung lipid peroxidation (assessed by measuring malondialdehyde). BNF only protected against O2 toxicity in the inducible strain. This protective effect of BNF on O2 toxicity in C57BL/6J mice was associated mainly with a large increase in the components of the cytochrome P-450 system (cytochrome P-450 and cytochrome b5) in the lung. The activity of pulmonary superoxide dismutase was also slightly increased, but the enhancement was not statistically significant. In contrast, in DBA/2J mice neither the components of the cytochrome P-450 system nor the activity of superoxide dismutase showed any increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of the 4 S polycyclic aromatic hydrocarbon-binding protein with the cytochrome P-450c gene

The induction of cytochrome P-450c, the isozyme most closely associated with aryl hydrocarbon hydroxylase activity in the rat, is mediated through a cytosolic polycyclic aromatic hydrocarbon (PAH)-binding protein(s). We have reported on the purification and characterization of a 4 S protein that interacts in a specific and saturable manner with [3H]benzo[a]pyrene and other PAHs. (W. H. Houser et al. (1985) Biochemistry 24, 7839-7845). We have also reported on the specific and saturable interaction of the 4 S protein with a plasmid containing 1.9 kbp of cloned rat P-450c sequence including exon 1, the 5' half of intron 1, and approximately 882 bp upstream information. Our investigations now show that incubation of this protein with a portion of the rat P-450c gene, followed by digestion with either lambda exonuclease or exonuclease III, tentatively identified two protected regions at -225 and -455 bp 5' from exon 1. To further study the significance of these protected regions, a 3.4-kbp fragment containing cytochrome P-450c promoter and 5'-upstream sequences (-882 to +2545) was fused to the chloramphenicol acetyl transferase (CAT) reporter gene and transfected into either rat epithelial RL-PR-C cells or rat hepatoma H-4-II-E cells. Both cell lines expressed CAT activity in response to induction by 3-methylcholanthrene (3MC), indicating that important regulatory regions responsive to 3MC are present in these constructs. However, neither cell line expressed CAT activity in response to 3MC when transfected with plasmids containing deletions (-95 to -724 or -240 to -720) in the regions shown to be protected by our footprinting studies. These results corroborate previous studies which indicated that the 4 S PAH-binding protein interacts in a specific manner with regions of the rat cytochrome P-450c gene. We conclude that the 4 S protein may play an important role in the regulation of expression of cytochrome P-450c in the rat.     

High mutagenic potency of several polycyclic aromatic hydrocarbons induced by liver postmitochondrial fractions from control and xenobiotic-treated immature carp

The metabolism of carcinogens in fish was examined by measuring the activation of different polycyclic aromatic hydrocarbons (PAH) by carp (Cyprinus carpio L.) liver post-mitochondrial fractions (S9) using the Salmonella typhimurium TA100 reverse mutation assay. For this study, 1 non-carcinogen, anthracene (AN), and 4 carcinogens, chrysene (CHR), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC) and 7,12-dimethylbenzanthracene (DMBA), were chosen. The bioactivating potency of the metabolic systems of carp pretreated with phenobarbital (PB), 3MC or Aroclor 1254 (ARO) were compared to uninduced carp liver. The results show that carp liver has the ability to metabolize carcinogenic PAH into mutagenic metabolites, which is enhanced when carp are pretreated with 3MC or ARO, but not with PB. A positive correlation between the induction of aryl hydrocarbon hydroxylase (AHH) activity in carp liver and the mutagenic potencies of CHR, BaP, DMBA and 3MC, has been observed. The bioactivating ability of carp liver S9 was compared with the ability of the same fractions from female Wistar rats (this study) as well as from Sprague-Dawley rats (literature data). When the mutagenic potencies of selected PAH had been normalized on the activity of BaP, the following order of mutagenic activities with S9 fractions from ARO-treated animals was obtained: (1) BaP (1) greater than DMBA (0.26) greater than 3MC (0.22) greater than CHR (0.05) greater than AN (0) for carp; (2) BaP (1) greater than 3MC (0.48) greater than CHR (0.31) greater than DMBA (0.16) greater than AN (0) for Sprague-Dawley rats; and (3) BaP (1) greater than 3MC (0.17) greater than DMBA (0.11) greater than CHR (0) = AN (0) for female Wistar rats. We conclude that carp and rats are very similar in their ability to activate carcinogenic PAH into mutagenic metabolites, which suggests that carp may be very susceptible to the carcinogenic activity of these compounds. According to our results from the mutagenicity study, as well as from the enzyme induction study, we propose the use of carp as a suitable model system for the study of chemical carcinogens.     

Renal cytochrome P-450's-electrophoretic and electron paramagnetic resonance studies.

The cytochrome P-450's of the microsomal mixed function oxidase systems from the rabbit renal cortex, outer medulla, inner medulla, and the liver were compared. Sodium dodecyl sulfate-(SDS) gel electrophoresis and electron paramagnetic resonance (EPR) studies detected cytochrome P-450 proteins in the liver, renal cortex, and outer medulla but not the inner medulla of normal animals. Two cytochrome P-450 peptides, which had molecular weights of 54,500 and 58,900 and which comigrated with known hepatic cytochrome P-450's on SDS gels, were identified in the cortex and outer medulla. Treatment of animals with 3-methylcholanthrene (MC) enhanced the 54,500 and 58,900 peptides in the liver and cortex but produced little change in outer medulla. MC treatment induced faint cytochrome P-450 bands in the inner medulla. The EPR studies detected low spin heme iron absorption lines at g = 2.42, 2.26, and 1.92 in liver, cortex, and outer medulla from untreated animals. The amplitude of the low spin absorption lines was increased by ethanol, a reverse type I compound, and reduced by chloroform, a type I compound, in these tissues. MC treatment increased the amplitude of the heme absorption lines in these tissues, and it induced a barely detectable heme spectrum in the inner medulla. Differences in exogenous substrate binding between hepatic and renal microsomes from MC-treated animals were detected by EPR and optical difference spectroscopy. Acetone, 1-butanol, and 2-propanol gave evidence of binding to the hepatic cytochrome P-450's but no evidence of binding to renal cortical microsomes. These results, along with previous enzymatic studies, suggest that the liver and each area of the kidney contain different substrate specificities and pathways for the metabolism of organic compounds.