Hepatic microsomal mixed-function oxidase activity in ethanol-treated hamsters and its consequences on the bioactivation of aromatic amines to mutagens

Male golden Syrian hamsters were maintained on ethanol-containing liquid diets for 4 weeks, corresponding to an average daily intake of 17 g/kg body wt. The p-hydroxylation of aniline was markedly enhanced by this treatment while minimal effects were seen in benzphetamine N-demethylase and ethoxyresorufin O-deethylase activities; there was no change in the microsomal levels of cytochromes P-450. Hepatic microsomal preparations from the ethanol-treated hamsters were more efficient than controls fed isocaloric diets in converting 2-aminofluorene, 4-aminobiphenyl, benzidine and 2-acetylaminofluorene into mutagens in the Salmonella mutagenicity test. The same treatment had no effect on the metabolic activation of 2-naphthylamine and even inhibited the mutagenicity of 2-aminoanthracene. No increase was seen in the activation of the two polycyclic aromatic hydrocarbons, benzo[a]pyrene and 3-methylcholanthrene to mutagens and an inhibitory effect was seen with the former. The ethanol-induced increase in the mutagenicity of 2-aminofluorene was inhibited by 2-butanol but not by the hydroxyl radical scavenger dimethylsulphoxide. It is concluded that chronic ethanol ingestion modulates the bioactivation of aromatic amines and amides to mutagens, the effect being substrate dependent. This effect of ethanol may be catalysed by unique form(s) of cytochrome P-450 whose synthesis is induced by such treatment.     

Heterogeneity of hepatic mixed function oxidases

The effects of NADH and increasing concentrations of potassium phosphate buffer, potassium chloride and potassium thiocyanate on several hydroxylations catalyzed by rat liver microsomes were studied. All the hydroxylations were stimulated by NADH in the presence of suboptimal concentrations of NADPH. The 7α-hydroxylation of cholesterol, the 12α-hydroxylation of 7α-hydroxy-4-cholesten-3-one and the 16-hydroxylation of palmitic acid were inhibited by increasing concentrations of potassium phosphate buffer, potassium chloride and potassium thiocyanate to a greater extent than any of the other hydroxylations studied. This finding and the previous finding that these three hydroxylations are not stimulated by phenobarbital treatment suggest differences between these hydroxylations and most other microsomal hydroxylations in liver. The possibility is discussed that different types of cytochrome P-450 may be involved.     

Kepone induction of hepatic mixed function oxidases in the male rat.

Effect of preexposure to kepone on hepatic drug metabolizing enzymes and several parameters of mixed function oxidase (MFO) system was investigated. Male rats were exposed to 0, 50, 100 and 150 ppm kepone in the daily ration for 16 days. Gain in body weight was declined after kepone to 86, 62 and 33% of controls for 50, 100 and 150 ppm kepone respectively. Liver weight was unaltered at all three levels of kepone. While hydroxylation of both aniline and pentobarbital was enhanced, the increased in aliphatic hydroxylation (i.e., pentobarbital) were greater at all levels of exposure to kepone. Aminopyrine demethylase was enhanced roughly 3 fold at 50 ppm kepone and was not further increased at higher levels of kepone. Cytochrome P₄₅₀, NADPH-cytochrome c reductase, and aniline binding were all increased suggesting that there were general increases with intermediate steps of the electron transfer and drug oxidation system. Cytochrome b₅ and NADPH dehydrogenase were unaltered after kepone treatment. These results suggest that kepone is an efficient inducer of hepatic MFO system.     

Activation of aromatic amines to mutagens by various animal species including man

2-Acetylaminofluorene, 2-aminofluorene, 4-aminobiphenyl, 2-naphthylamine, 2-aminoanthracene and benzidine were assayed for mutagenicity in the Ames test in the presence of hepatic microsomal preparations derived from mouse, hamster, rat, pig and man. Prior to each mutagenicity assay all activation systems were fully characterized with respect to mono-oxygenase and mixed-function amine oxidase activities. All compounds were metabolically activated to mutagens by all activation systems, but with markedly different efficiencies, hamster being the only species which readily activated all amines. The hamster also exhibited the highest ethoxyresorufin O-deethylase and dimethylaniline N-oxidase activities.     

Induction of mixed function oxidases on oral administration of dieldrin

The administration of dieldrin (30 mg/kg body weight) caused an increase in the liver weight of rats. The metabolism of aflatoxins B₁ and G₁ by the microsomes obtained from the liver of dieldrin-treated animals was enhanced significantly as compared to the controls showing that dieldrin increased the activity of mixed function hydroxylases. Dieldrin caused an increase in the activity of liver microsomal NADPH oxidase and a decrease in the lipid peroxidation. Dieldrin brought about an increase in the phosphatidylcholine content of rat liver.     

Inhibition of rat hepatic mixed function oxidases by antimalarial drugs: selectivity for cytochromes P-450 and P-448

Intraperitoneal administration of chloroquine, primaquine and quinacrine to rats resulted in inhibition of the hepatic microsomal mixed-function oxidases. The N-demethylation of benzphetamine (cytochrome P-450) was inhibited by chloroquine only while the O-deethylation of ethoxyresorufin (cytochrome P-448) was inhibited by primaquine and quinacrine. When incubated with hepatic microsomes from phenobarbital-pretreated rats, chloroquine and primaquine, but not quinacrine, caused a concentration-dependent inhibition of benzphetamine N-demethylase activity. Incubation of hepatic microsomes from beta-naphthoflavone rats with primaquine and quinacrine, but not chloroquine, resulted in a concentration-dependent inhibition of the O-deethylation of ethoxyresorufin. These observations demonstrate that chloroquine and quinacrine are specific inhibitors of cytochromes P-450 and P-448, respectively.     

Heterocyclic aromatic amines, food-derived mutagens: metabolism and relevance to cancer susceptibility

It is estimated that diet contributes to as much as one-third of cancer incidents. Heterocyclic aromatic amines (HCAs) are well-known mutagens/carcinogens found in thermal-processed meat and fish. HCAs require metabolic activation to exert their carcinogenic potential. First step in HCAs activation--the generation of N-hydroxy-HCA derivatives--is catalyzed by cytochrome P450, mainly isoenzyme CYP1A2. Further activation is carried out by N-acetyltransferases and sulfotransferases, which catalyze esterification of N-hydroxy-HCAs. The products of these reactions are highly genotoxic, capable of direct interaction with DNA by adduct formation. HCA-DNA adducts may cause errors in DNA replication and the generation of mutations, which, when not repaired, may contribute to cancer development. On the other hand, among enzymes involved in HCAs detoxication, UDP-glucuronosyltransferases and glutathione S-transferases can be mentioned. Balance between activation and detoxication processes of HCAs, together with genetically determined differences in HCA metabolism are crucial for the assessment of HCA-dependent cancer risk among individuals.     

Response of the mixed function oxidase system of rat hepatic microsomes to parathion and malathion and their oxygenated analogs

$\text{In}$$\text{vitro}$ inhibition of ethylmorphine metabolism in rat hepatic microsomes by parathion, malathion, malaoxon and paraoxon was not well correlated with their effects on NADPH oxidation, cytochrome C reduction or the reduction of cytochrome P-450. A parallel relationship was observed between inhibition of ethylmorphine metabolism by parathion, malathion and malaoxon and the binding affinity of these agents to microsomal cytochrome P-450 obtained from rats pretreated with an anticholinesterase agent, Bis-[?-nitrophenol] phosphate.     

Selective activation of carcinogenic aromatic amines to bacterial mutagens in the marine mussel Mytilus galloprovincialis

The postmitochondrial fraction of the marine mussel Mytilus galloprovincialis digestive gland activates selectively precarcinogenic aromatic amines, but not precarcinogenic benzo[a]pyrene, to Salmonella typhimurium TA 98 mutagens. This activation potential is NADPH-dependent, is not inducible by exposure to Diesel 2 oil and a polluted environment, and is inhibited by methimazole. The characteristics of this activation potential are consistent with the recent finding of the presence of FAD-containing-, and lack of cytochrome P-450 dependent-, monooxygenase activity in Mytilus edulis. The presence of such selective potential in marine invertebrate(s) may bring new insight into our understanding of the fate and the effects of carcinogens in the marine environment.     

Diethyldithiocarbamate suppresses the plant activation of aromatic amines into mutagens by inhibiting tobacco cell peroxidase.

Diethyldithiocarbamate is an antimutagen and repressed the activation of promutagens by plant systems. Earlier work implicated the involvement of tobacco cell (TX1) peroxidases in the plant cell activation of aromatic amines. We now present data that diethyldithiocarbamate represses the activation of 2-aminofluorene and m-phenylenediamine by inhibiting intracellular TX1 peroxidases under in vivo conditions. Concentrations of diethyldithiocarbamate that caused a 50% repression of TX1 cell activation of 2-aminofluorene and m-phenylenediamine also induced a 50% inhibition of TX1 cell peroxidase activity. Diethyldithiocarbamate in a concentration range between 25 and 500 microM directly inhibited peroxidase activity in TX1 cell homogenates in a concentration-dependent manner. Similar results were observed with purified horseradish peroxidase. The kinetics of peroxidase activity were studied in homogenates from control cells and cells treated with 750 microM and 25 mM diethyldithiocarbamate. There was no significant difference among the Km values among the three groups with a mean (+/- standard error) Km of 2.58 +/- 0.23 mM. However, the Vmax differed from 4.02 to 2.12 nmoles tetraguaiacol/min/micrograms protein, in the control and in the 25 mM diethyldithiocarbamate treatment group, respectively. These data indicate that diethyldithiocarbamate is a non-competitive inhibitor of TX1 cell peroxidase.     

Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair

Several chemical mutagens were found to induce sister chromatid exchanges in Chinese hamster chromosomes. Among them, effects of 4NQO and MMC were very similar to those of UV light in that the exchange frequency increased with increasing dose of chemicals and that it was markedly lowered in the presence of 1 mM caffeine during a post-treatment period. The frequency of proflavin-induced sister chromatid exchanges was also found to be dose dependent, but it was insensitive to the caffeine post-treatment. On the other hand, no appreciable increase was detected in the incidence of sister chromatid exchanges in MNNG-treated cells over a 100-fold range of variation in chemical dose. Caffeine by itself raised the exchange frequency only slightly over a control level. It was found that 4NQO and MMC exerted remarkable delayed effects on the exchange induction, whereas proflavin did not. This seems to suggest that the lesions caused by the former mutagens would be long-lived and repeatedly provoke sister chromatid exchanges. These data imply that there are several possible ways in which the initial DNA lesions ultimately lead to the formation of sister chromatid exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid exchanges would have evolved through a caffeine sensitive repair process, probably related to a post-replication repair of DNA damage.     

Induction of rat hepatic drug metabolizing enzymes by dimethylcyclosiloxanes

Low molecular weight dimethylcyclosiloxanes (DMCS) are important precursors in the synthesis of polydimethysiloxane polymers widely used in industry, and in medical and personal care products. The objective of this study was to characterize the ability of two DMCS, octamethylcyclosiloxane (D4) and decamethylcyclopentasiloxane (D5) to induce drug metabolizing enzymes in rats. Male and female Sprague-Dawley rats were administered 1, 5, 20, or 100 mg/kg D4 or D5 in corn oil daily by gavage for 4 days. Changes in the levels of activity and/or immunoreactivity of CYP1A1/2, CYP2B1/2, CYP3A1/2 and NADPH cytochrome P450 reductase in liver microsomes were examined. Significant increases were observed in the liver to body weight ratio in female rats administered either D4 or D5 at doses > or = 20 mg/kg. Increases in the liver to body weight ratio were observed in male rats treated with > or = 100 mg/kg D5 but not with D4. Relatively large increases in CYP2B1/2 enzymatic activity and immunoreactive protein were observed with increasing concentrations of both D4 and D5. Significant increases in 7-pentoxyresorufin O-depentylase (PROD) activity were also detected in male and female rats given D4 at doses > or = 5 mg/kg. D5 increased PROD activity in male rats at doses > or = 20 mg/kg and in female rats at doses > or = 5 mg/kg. 7-Ethoxyresorufin O-deethylase (EROD) activity was increased in both male and female rats receiving > or = 20 mg/kg D4 or > or = 5 mg/kg D5; however, no changes were detected in CYP1A1/2 immunoreactive protein in rats of either sex. D4 and D5 caused significant increases in CYP3A1/2 immunoreactive protein in only male rats treated with 100 mg/kg of either compound. However, significant increases were detected in CYP3A1/2 immunoreactive protein in female rats at D4 doses > or = 20 mg/kg and D5 doses > or = 5 mg/kg. Induction of NADPH cytochrome P-450 reductase immunoreactive protein was observed with D4 in female rats and in both male and female rats with D5. Induction of CYP2B/1/2, CYP3A1/2 and NADPH cytochrome P450 reductase was observed in rats treated with 50 mg/kg phenobarbital by intraperitoneal injection. Maximal CYP2B induction detected with D4 was approximately 50% of the increase observed with phenobarbital. In summary, D4 and D5 induced CYP2B1/2 in adult rat liver in a manner similar to that observed with phenobarbital; however, differences were observed between D4 and D5 in their ability to induce CYP3A1/2 and NADPH cytochrome P450 reductase. Female rats were more sensitive to the inductive properties of low doses of both DMCS than male rats whereas male rats were more responsive to phenobarbital induction.     

Effects of pretreatment with inducers of hepatic mixed function oxidases on DNA repair elicited by various compounds in hepatocytes from adult and neonatal rats

Studies were conducted to assess the effects of inducers of hepatic mixed function oxidases on DNA repair responses to 13 different genotoxic agents in hepatocytes from adult male mice. Phenobarbital pretreatment increased DNA repair elicited by diethylnitrosamine but had no effect on responses to the other compounds. Pretreatment with p,p-dichlorodiphenyltrichloroethane, 3-methylcholanthrene or -naphthoflavone induced the DNA repair responses to a variety of activation-dependent carcinogens. DNA repair responses to the direct-acting alkylating agents methyl methanesulfonate and N-methyl-N-nitro-N-nitrosoguanidine were not increased by any of the pretreatments, which indicated that the pretreatment-related enhancement of responses to the other compounds was due to induction of their metabolic activation. Taken together, the findings suggest that Aroclor, or other pretreatments, may increase the sensitivity of the hepatocyte DNA repair assay for detecting the genotoxicity of certain compounds; however, the potential benefit may be limited due to specific features of the assay. In contrast, Aroclor pretreatment did not produce any enhancement of in vivo DNA repair elicited by dimethylnitrosamine, diethylnitrosamine, o-aminoazotoluene, 2-acetylaminofluorene, 3-methylcholanthrene or aflatoxin B₁, and thus does not appear to be useful for improving the sensitivity of the in vivo/in vitro assay.Whereas the amount of DNA repair produced by dimethylnitrosamine was not increased by classical inducers of liver microsomal enzymes, pretreatment with pyrazole greatly augmented in vitro and in vivo DNA repair responses to dimethylnitrosamine; responses to diethylnitrosamine were increased to a lesser degree by pyrazole pretreatment. The effects of lactational exposure to enzyme inducing agents on DNA repair in neonatal hepatocytes was also investigated.Abbreviations 2-AAF 2-acetylaminofluorene - 4-AB 4-aminobiphenyl - 6-AC 6-aminochrysene - AFB aflatoxin B₁ - ARO Aroclor 1254 - o-AT o-aminoazotoluene - B(a)P benzo[a]pyrene - B-NF beta-naphthoflavone - BZ benzidine - DDT p,p-dichlorodiphenyltrichloroethane - DDE p,p-dichlorodiphenyldichloroethylene - DEN diethylnitrosamine - DMBA 7,12-dimethylbenzanthracene - DMN dimethylnitrosamine - 3-MC 3-methylcholanthrene - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - 2-NA 2-naphthylamine - NNG net nuclear grains - PB phenobarbital - PYR pyrazole