Tissue and species specificity of the microsomal metabolism of N-nitrosomethylbenzylamine

The microsomal activation of N-nitrosomethylbenzylamine (NMBzA) by oxidation at the methylene carbon atom was examined in various organs of a number of species to determine the role of metabolism in the organ-specificity of tumour induction by NMBzA. In Sprague-Dawley rats, NMBzA was metabolized by microsomes from liver, lung and oesophageal mucosa. In Fischer F-344 rats and in rabbits, metabolic activity was present in both liver and oesophageal mucosa, the only tissues studied in those species. In contrast, in Syrian hamsters and in BALB/cBYJ mice, no NMBzA metabolism was detectable in the oesophagus, but it occurred at relatively high rates in liver, lung and kidney. The forestomach mucosa exhibited undetectable levels of activity in all species except the hamster in which it was present at a very low level. In human oesophageal mucosal microsomes from six patients, rates of metabolism of NMBzA were either undetectable or approximately 70 times lower than those in the Sprague-Dawley rats. A comparison of NMBzA metabolism in the different species with the known carcinogenicity of the nitrosamine in rats and rabbits, and our preliminary data on the acute toxicity of NMBzA in hamsters and mice suggests that, in the oesophagus at least, metabolic activation of NMBzA is necessary to elicit its toxic and/or carcinogenic effect. However, in the liver, which in all species has high metabolic activity but which is also resistant to the toxic and carcinogenic effects of NMBzA, other factors besides metabolic activation must be involved.     

Developmental pattern of 3-methylcholanthrene-inducible mutagenic activation of N-2-fluorenylacetamide, 2-fluorenamine, and 2,4-diaminoanisole in the rabbit

The effects of 3-methylcholanthrene (MCA) on mutagenic activation of the carcinogenic arylamines N-2-fluorenylacetamide (FAA), 2-fluorenamine (FA), and 2,4-diaminoanisole (2,4-DAA) by liver homogenates were studied postnatally in Dutch rabbits. These effects were compared with the developmental profiles of cytochrome P448 and aryl hydrocarbon hydroxylase (AHH) activity. Mutagenic activation of FA and 2,4-DAA was increased by MCA as early as 2 days after birth, whereas induction of FAA mutagenicity appeared 6 days after birth. Thereafter, induction of all three arylamines closely paralleled induction of cytochrome P448, which was maintained into adulthood. In contrast, induction of AHH activity by MCA was highest at 2 days of age and decreased to control levels 20 days after birth.     

Oxidation-dependent inactivation of aryl sulfotransferase IV by primary N-hydroxy arylamines during in vitro assays

The sulfation of primary N-hydroxy arylamines is a critical intermediate step in the bioactivation of many carcinogenic arylamines, arylamides and nitroaromatics. However, the study of this reaction in vitro is often complicated by the chemical instability of these molecules. We have examined the stability of two highly purified N- hydroxy arylamines, N-hydroxyaniline and N-hydroxy-2-aminofluorene, under different oxidative reaction conditions pertinent to the assay of sulfotransferases. Furthermore, these compounds, as well as the products of their oxidative degradation, were examined for their interactions with homogeneous aryl sulfotransferase (AST) IV. Under reaction conditions where oxidative degradation of the N-hydroxy arylamines occurred, N-hydroxyaniline and N-hydroxy-2-aminofluorene produced time-dependent and irreversible inhibition of AST IV. While this inhibition was not dependent upon the presence of 3'- phosphoadenosine 5'-phosphosulfate in the reaction mixture, analysis of the N-hydroxy arylamines by UV spectroscopy showed that the inhibition of AST IV did require non-enzymatic oxidation of the N-hydroxy arylamine. Under reaction conditions that prevented the oxidative degradation of N-hydroxyaniline, this N-hydroxy arylamine was a substrate for AST IV. Likewise, under similar conditions, 4-chloro-N- hydroxyaniline was also a substrate for the enzyme. In contrast, no AST IV catalyzed sulfation of N-hydroxy-2-aminofluorene was detected under conditions that prevented the oxidation of N-hydroxy-2-aminofluorene. Adequate protection of these N-hydroxy arylamines from oxidative degradation required the addition of L-ascorbic acid to reaction mixtures that had also been degassed and purged with argon. The irreversible inhibition of AST IV exhibited by these N-hydroxy arylamines, even in reaction mixtures where attempts were made to limit oxidative degradation by degassing and purging with argon, emphasized the importance of completely preventing such degradation when utilizing in vitro assays to assess the potential for an N-hydroxy arylamine to serve as a substrate for a specific sulfotransferase.      

Acetyl coenzyme A-dependent binding of carcinogenic and mutagenic dinitropyrenes to DNA

Dinitropyrenes are mutagenic and carcinogenic environmental pollutants found in diesel emissions and urban air particulates. In Salmonella typhimurium these compounds appear to be activated to mutagens by sequential nitroreduction and acetylation. We have examined whether or not similar activation pathways occur with mammalian nitroreductases and acetylases. When rat liver cytosol, NADPH and calf thymus DNA were incubated with [4,5,9,10(-3)H]1-nitropyrene, [4,5,9,10(-3)H]1,3-, 1,6- or 1,8-dinitropyrene very low levels of nitrated pyrene binding with DNA were detected. Addition of acetyl coenzyme A (AcCoA) to these incubations increased the binding of dinitropyrenes 20- to 40-fold while the binding of 1-nitropyrene was not affected. The extent of AcCoA-dependent binding of dinitropyrenes reflected the amount of nitroreduction, as measured by aminonitropyrene formation. However, the increase in binding of dinitropyrenes to DNA in the presence of AcCoA did not occur with dog liver cytosol which is known to be deficient in N-acetylases. These results suggest that cytosolic nitroreductases catalyze the formation of N-hydroxy arylamine intermediates which in the case of dinitropyrenes are converted to reactive N-acetoxy arylamines by cytosolic AcCoA-dependent acetylases.     

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.     

The anomalous biological activity of nitroso-2-oxopropyl compounds

The carcinogenic action of a set of N-nitroso compounds containing the 2-oxopropyl group was considered in relation to their metabolism and their activity as alkylating agents for DNA. In contrast with the great carcinogenic potency of methylnitrosourea and ethylnitrosourea, comparable with the corresponding dialkylnitrosamines, 2-oxopropylnitrosourea is a weak carcinogen with a limited range of target organs in rats and hamsters. 2-Oxopropylnitrosochloroethylurea was somewhat weaker than 2-oxopropylnitrosourea and similarly induced spleen hemangiosarcomas in hamsters, but few tumors of any kind in rats. The relatively much more potent carcinogenicity of nitrosobis-(2-oxopropyl)amine, nitroso-(2-hydroxypropyl) (2-oxopropyl) amine and methylnitroso-2-oxopropylamine suggests that the activity of an oxopropylating agent is not involved in carcinogenesis by nitroso-2-oxopropylamines. The nitrosamines are likely to undergo extensive metabolism to form proximate carcinogenic moieties, probably including the methyldiazonium ion, which are responsible for the induction of a broad range of tumors in rats and hamsters. These include tumors of the liver, pancreas ducts, lung and nasal mucosa in hamsters, and esophagus, liver, lung, thyroid, kidney, trachea, bladder and nasal mucosa in rats.     

Metabolism and disposition of bladder carcinogens in rat and guinea pig: possible mechanism of guinea pig resistance to bladder cancer

The metabolism and disposition of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) were studied in rat and guinea pig. Rat is susceptible whereas guinea pig is resistant to FANFT-induced bladder cancer. Rats and guinea pigs were p.o. administered either 2-[14C]ANFT or 2-[14C]FANFT (100 mg/kg), and 18-h urine and feces were collected. Tissue distribution of radiolabel was determined. In both species, the highest concentrations of radioactivity expressed as nmol/g tissue were observed in the urine and intestines. Urinary metabolites were separated by high-performance liquid chromatography and radioactivity determined by radioanalytical detection. FANFT was not detected in urine from either species under any experimental condition. More ANFT was observed in urine following FANFT than ANFT administration. This deformylation-dependent excretion of FANFT was demonstrated in both species and has been previously described as renal metabolic/excretory coupling. Less ANFT, the carcinogen more proximate than FANFT, is excreted in guinea pigs compared with rats. A unique ANFT metabolite was identified in guinea pig but not rat urine. This metabolite represented 80 and 18% of radioactivity recovered in guinea pig urine following ANFT and FANFT administration, respectively. A metabolite produced by guinea pig liver and kidney microsomes in the presence of uridine-5'-diphosphoglucuronic acid coeluted with this unique metabolite. The urinary metabolite was characterized using hydrolytic enzymes, acid hydrolysis, and mass spectrometry and identified as an ANFT-N-glucuronide. A unique UDP-glucuronosyl-transferase appears to be responsible, at least in part, for the reduced amount of free ANFT excreted by guinea pigs compared with rats. Reduced levels of urinary ANFT observed in guinea pigs may partially explain the resistance of this species to FANFT-induced bladder cancer.     

Metabolic activation of pyrolysate arylamines by human liver microsomes; possible involvement of a P-488-H type cytochrome P-450

Metabolic activating capacity of human livers for carcinogenic heterocyclic arylamines has been studied using a Salmonella mutagenesis test. A large individual variation was observed among 15 liver samples in the capacities of activation of Glu-P-1 (2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole), IQ (2-amino-3-methylimidazo[4,5-f]quinoline) and MeIQx (2-amino-3,8-dimethyl-3 H-imidazo[4,5-f]quinoxaline). The average numbers of revertants induced by the three heterocyclic arylamines were nearly the same or rather higher in the presence of hepatic microsomes from human than those from rat. In high-performance liquid chromatography, formation of N-hydroxy-Glu-P-1 was detected and accounted for more than 80% of the total mutagenicity observed in the human microsomal system with Glu-P-1, indicating that, similarly to experimental animals, N-hydroxylation is a major activating step for heterocyclic arylamines in human. Addition of flavone or 7,8-benzoflavone to human liver microsomes showed effective inhibition of the mutagenic activation of Glu-P-1, although the treatment rather enhanced microsomal benzo[a]pyrene hydroxylation in human livers. Mutagenic activation of Glu-P-1 by human liver microsomes was also decreased by the inclusion of anti-rat P-448-H IgG, and was well correlated with the content of immunoreactive P-448-H in livers, suggesting the involvement of a human cytochrome P-450, which shares immunochemical and catalytic properties with rat P-448-H, in the metabolic activation of heterocyclic arylamines in human livers.     

Glucuronidation of carcinogenic arylamines and their N-hydroxy derivatives by rat and human phenol UDP-glucuronosyltransferases of the UGT1 gene complex

Since carcinogenic arylamines are sequentially oxidized andconjugated with glucuronic acid, differences in glucuronidationmay critically determine the toxic potential of these compounds.Therefore, N-glucuronldation of 1- and 2-naphthylamine (1-NAand 2-NA), 4-aminobiphenyl (4-ABP) and their N-hydroxy derivativeswas investigated using rat and human liver microsomes and V79cell-expressed phenol UDP-glucuronosyltransferases (UGT) ofthe UGTl gene complex. Cell-expressed UGTs included rat andhuman UGT 1.6, which are known to conjugate planar phenols,and human UGT1.7, conjugating both planar and bulky phenols,(i) N-Glucuronidation of 1- and 2-NA and of N-hydroxy-2-NA wasinducible by 3-methylcholanthrene in rat liver microsomes whereasN-glucuronidation of the bulky arylamines 4-ABP and N-hydroxy-4-ABPwas not In support of these findings mutagenicity of N-hydroxy-2-NAin the Ames test was markedly reduced upon addition of UDP-glucuronicacid using liver homogenates from 3-methylcholanthrene-treatedrats, (ii) With cell-expressed rat UGT1.6, non-carcinogenic1-NA was conjugated with the highest rate and with higher affinitythan 2-NA. UGT1.6 showed poor activity towards N-hydroxy-4-ABPand 4-ABP. (iii) Substrate specificity of human UGT1.6 alsoappeared to be limited to planar 1-NA, 2-NA and its N-hydroxyderivative, whereas UGT1.7 showed broader substrate specificity,including the bulky arylamine 4-ABP and its /V-hydroxy derivative.The results suggest marked differences in substrate specificityof different UGT isozymes for arylamines and their N-hydroxyderivatives.     

Investigation into the pharmacodynamics of the carcinogen N-nitrodimethylamine

N-Nitrodimethylamine (NTDMA) was found to be a carcinogen of the nasal mucosa leading to aesthesioneuroepitheliomas in BDVI rats. N-Nitromethylamine (NTMA), a product of the oxidative metabolism of NTDMA, was also carcinogenic, leading to neurogenic tumours of the lumbar region of the spine. The 100,000 X g supernatant of both liver and nasal mucosa contains an enzyme capable of reducing NTDMA to N-nitrosodimethylamine (NDMA). In the microsomal fraction of both organs, NTDMA is oxidized to formaldehyde. The fractions from nasal mucosa have a higher capacity than the corresponding liver fractions to both oxidize and reduce NTDMA. NDMA was detected in blood and urine from rats treated with NTDMA. The elimination of NTDMA from blood occurs biphasically, with an initial half-life of 3.5 min.     

Species differences in the cytotoxic and genotoxic effects of 2-acetylaminofluorene and its primary metabolites 2-aminofluorene and N-OH-2-acetylaminofluorene

2-Acetylaminofluorene (AAF), 2-aminofluorene (AF) and N-hydroxy-2-acetylaminofluorene(N-OH-AAF) could be activated to mutagens in S. typhimuriumusing either 9000 g supernatant (S9) or hepatocytes isolatedfrom rats, mice, hamsters or guinea pigs. Their relative mutagenicpotency was generally N-OH-AAF > AF > AAF. Monolayer culturesof hepatocytes exposed to AAF/AF/N-OH-AAF showed evidence ofDNA damage measured as unscheduled DNA repair synthesis. Theorder of activity in rat and hamster was N-OH-AAF > AAF >AF, in guinea pig and mouse N-OH-AAF > AF > AAF. OnlyN-OH-AAF caused observable cytotoxicity, and the rat hepatocyteswere the far more sensitive species. Neither the resistanceof guinea pig liver nor the greater susceptibility of the ratliver to the carcinogenic effects of AAF and N-OH-AAF couldbe readily explained by the species differences in activatingthese compounds to mutagens in Salmonella or to DNA damagingagents in the hepatocytes. It is possible that cytotoxic effectsof N-OH-AAF may be of some importance for the observed speciesdifferences in the liver carcinogenic effects of AAF and N-OH-AAF.     

Species difference among experimental rodents in induction of P450IA family enzymes by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

Rats, mice, hamsters and guinea pigs were given an i.p. injection of 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP), a protein-derived pyrolysate component present in cooked foods, and inductions of cytochrome P450 (P450) in the liver and kidney of these animals were examined. The activity and amount of P450s corresponding to the rat P450IA1 and P450IA2 were assessed by means of a bacterial mutation test using 3 carcinogenic heterocyclic aromatic amines including PhIP as substrates and by Western blotting with a monoclonal antibody reactive with both P450IA1 and P450IA2. In rats, PhIP induced P450IA1, P450IA2 and a new but unspecified P450 isozyme in the liver, and induced P450IA1 in the kidney. However, PhIP induced none of these P450 isozymes in mice, hamsters and guinea pigs.     

Species Difference among Experimental Rodents in Induction of P450IA Family Enzymes by 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

Rats, mice, hamsters and guinea pigs were given an i.p. injection of 2-amino-1-methyl-6-phenyl-imidazo[4,5- b ]pyridine (PhIP), a protein-derived pyrolysate component present in cooked foods, and inductions of cytochrome P450 (P450) in the liver and kidney of these animals were examined. The activity and amount of P450s corresponding to the rat P4S0IA1 and P450IA2 were assessed by means of a bacterial mutation test using 3 carcinogenic heterocyclic aromatic amines including PhIP as substrates and by Western blotting with a monoclonal antibody reactive with both P4S0IA1 and P450IA2. In rats, PhIP induced P450IA1, P450IA2 and a new but unspecified P450 isozyme in the liver, and induced P450IA1 in the kidney. However, PhIP induced none of these P450 isozymes in mice, hamsters and guinea pigs.     

Differential susceptibility of rat and guinea pig colon mucosa DNA to methylation by methylazoxymethyl acetate in vivo

Methylazoxymethanol (MAM) and methylazoxymethyl acetate (MAMOAc) are powerful colon carcinogens in rats, mice and hamsters. In contrast, these agents are not carcinogenic to the colon of the guinea pig. To probe the mechanism responsible for this species difference, we determined the levels of DNA methylation in the livers and colon mucosae of F344 rats and strain-2 guinea pigs after the s.c. administration of 25 mg/kg MAMOAc. While no significant difference was observed between the two species with respect to the degree of liver DNA methylation, the level of O6-methylguanine in guinea pig colon mucosa DNA was 19 times lower than in rat colon mucosa DNA, and the level of 7-methylguanine was below detection limits. However, significant colon mucosa DNA methylation was observed in the guinea pig after the intrarectal administration of 1.25 mg methylnitrosourea. The methylation of colon mucosa DNA in response to MAMOAc in the two species correlated with the activity of alcohol dehydrogenase, an enzyme believed to be involved in the activation of MAM. Thus the resistance of the guinea pig colon to the carcinogenicity of MAM/MAMOAc may be ascribed to the lack of metabolic activation of MAM in this organ.     

Hepatic microsomal N-glucuronidation and nucleic acid binding of N-hydroxy arylamines in relation to urinary bladder carcinogenesis.

Uridine 5'-diphosphoglucuronic acid-fortified hepatic microsomes from dogs, rats, or humans rapidly metabolized [3H]-N-hydroxy-2-naphthylamine (N-HO-2-NA) to a water-soluble product that yielded 98% of the parent N-hydroxy amine upon treatment with beta-glucuronidase. The metabolite was identified as N-(beta-1-glucosiduronyl)-N-hydroxy-2-naphthylamine from ultraviolet, infrared, and mass spectral analyses of the glucuronide and its nitrone derivative. Incubation of N-hydroxy-1-naphthylamine (N-HO-1-NA), N-hydroxy-4-aminobiphenyl (N-HO-ABP), or the N-hydroxy derivatives of 2-aminofluorene, 4-aminoazobenzene, or N-acetyl-2-aminofluorene with uridine 5'-diphosphoglucuronic acid-fortified hepatic microsomes also yielded water-soluble products. beta-Glucuronidase treatment released 80 to 90% of the [3H]-NHO-1-NA and [3H]-N-HO-ABP conjugates as tritiated ether-extractable derivatives. N-HO-1-NA, N-HO-2-NA, and N-HO-ABP and the glucuronides of these N-hydroxy arylamines were relatively stable and nonreactive near neutral pH. At pH 5, the N-glucuronide of N-HO-2-NA and the presumed N-glucuronides of N-HO-1-NA and N-HO-ABP were rapidly hydrolyzed to the N-hydroxy arylamines that were then converted to reactive derivatives capable of binding covalently to nucleic acids. These data support the concept that arylamine bladder carcinogens are N-oxidized and N-glucuronidated in the liver and that the N-glucuronides are transported to the urinary bladder. The hydrolysis of the glucuronides to N-hydroxy arylamines and the conversion of the latter derivatives to highly reactive electrophilic arylnitrenium ions in the normally acidic urine of dogs and humans may be critical reactions for tumor induction in the urinary bladder.     

The metabolism of tritiated thioacetamide in the rat

A method is described for preparing tritiated thioacetamide. Thioacetamide-³H was fed daily to rats at a level which is known to produce liver tumours after six months' feeding. It was shown that less than 1% was excreted unchanged in the urine. Radioactivity was found in all the organs examined and in the chemical constituents of all cell fractions of the liver suggesting metabolism of thioacetamide to acetate. Measurements of the acetate pool size and of the quantity of thioacetamide entering the pool showed that over 95% of the carcinogenic dose of thioacetamide was converted to acetate in 24 hr. Following incubation of thioacetamide with liver slices it was shown that the pathway of metabolism is through acetamide. Liver slices were seen to be three times more active than kidney slices in converting thioacetamide to acetamide and it is suggested that this metabolic step is a necessary prerequisite to the toxic and carcinogenic effects of thioacetamide in the liver.     

Metabolic oxidation of carcinogenic arylamines by rat, dog, and human hepatic microsomes and by purified flavin-containing and cytochrome P-450 monooxygenases

Hepatic N-oxidation and aryl ring oxidation are generally regarded as critical activation and detoxification pathways for arylamine carcinogenesis. In this study, we examined the in vitro hepatic metabolism of the carcinogens, 2-aminofluorene (2-AF) and 2-naphthylamine (2-NA), and the suspected carcinogen, 1-naphthylamine (1-NA), using high-pressure liquid chromatography. Hepatic microsomes from rats, dogs, and humans were shown to catalyze the N-oxidation of 2-AF and of 2-NA, but not of 1-NA; and the rates of 2-AF N-oxidation were 2- to 3-fold greater than the rates of 2-NA N-oxidation. In each species, rates of 1-hydroxylation of 2-NA and 2-hydroxylation of 1-NA were comparable and were 2- to 5-fold greater than 6-hydroxylation of 2-NA or 5- and 7-hydroxylation of 2-AF. Purified rat hepatic monooxygenases, cytochromes P-450UT-A, P-450UT-H, P-450PB-B, P-450PB-D, P-450BNF-B, and P-450ISF/BNF-G but not P-450PB-C or P-450PB/PCN-E, catalyzed several ring oxidations as well as the N-oxidation of 2-AF. Cytochromes P-450PB-B, P-450BNF-B, and P-450ISF/BNF-G were most active; however, only cytochrome P-450ISF/BNF-G, the isosafrole-induced isozyme, catalyzed the N-oxidation of 2-NA. The purified porcine hepatic flavin-containing monooxygenase, which was known to carry out the N-oxidation of 2-AF, was found to catalyze only ring oxidation of 1-NA and 2-NA. No activity for 1-NA N-oxidation was found with any of the purified enzymes. These data support the hypothesis that 1-NA is probably not carcinogenic. Furthermore, carcinogenic arylamines appear to be metabolized similarly in humans and experimental animals and perhaps selectively by a specific form of hepatic cytochrome P-450. Enzyme mechanisms accounting for the observed product distributions were evaluated by Hückel molecular orbital calculations on neutral, free radical, and cation intermediates. A reaction pathway is proposed that involves two consecutive one-electron oxidations to form a paired substrate cation-enzyme hydroxyl anion intermediate that collapses to ring and N-hydroxy products.     

Polymorphic N-acetylation of sulfamethazine and benzidine by human liver: implication for cancer risk?

Extensive epidemiologic studies have indicated a relationship between bladder cancer in populations exposed to arylamines and the slow phenotype for acetylation of arylamines (e.g., sulfamethazine) and aryl hydrazides (e.g., isoniazid). In human liver preparations, we have examined the association between the capability for sulfamethazine acetylation and that for the human bladder arylamine carcinogen, benzidine. By the usual criteria for polymorphic acetylation, we classified the 10 donor subjects as four rapid and six slow acetylators of sulfamethazine. Concurrent tests of benzidine acetylation in the same liver preparations yielded capacities to acetylate benzidine that were directly and significantly correlated with those for sulfamethazine acetylation (r = 0.672; P less than 0.05). We suggest that acetylation of these two compounds is directly related and knowledge of the human acetylator phenotype may be a useful indicator of possible risk for bladder cancer due to exposure of certain arylamines and, perhaps for other cancers in man.     

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.     

Effect of dimethylnitrosamine concentration on its demethylation by liver microsomes from control and 3-methylcholanthrene pretreated rats, hamsters and guinea pigs

The effect of dimethylnitrosamine concentration on its demethylation by liver microsomes from control and 3-methylcholanthrene pretreated (100 mg/kg body wt. 24 h before sacrifice) rats, hamsters and guinea pigs was investigated. At low substrate concentration (2 mM), liver microsomes from pretreated rats and hamsters showed 30-50% lower demethylation activity than their respective controls. No such difference was found in the guinea pig. At high substrate concentration (100 mM), all 3 pretreated species showed 50-100% higher enzyme activity than their respective controls. Enzyme activities among the 3 species showed the following order of activity: hamster greater than guinea pig greater than rat.