The mechanisms of action of the carcinogenic nitroso and related compounds

It is suggested that the proximate carcinogenic forms of dialkylnitrosamines are their oxidation products, which retain the alkylnitrosamino moiety, but have acquired a carbonyl function as a result of omega or beta oxidation of an alkyl group. Such metabolites resemble the locally acting carcinogenic “nitrosamides” and probably have become multifunctional. Their functional groups, being in close proximity, could ensure binding in a concerted manner with apposite reactive centres of chromatin to form a firm bridge, for example, between an amino group of nucleic acid base and thiols of protein chains.     

The carcinogenic activities in mice of compounds related to 3-methylcholanthrene

A number of derivatives of 3-methylcholanthrene have been tested for carcinogenic action by injection into C57 black mice. 1- and 2-Hydroxy-3-methylcholanthrene and 3-methylcholanthrene-1- and 2-one were active carcinogens, but cis-1,2-dihydroxy-3-methylcholanthrene showed only weak activity. The hydrocarbon, 3-methylcholanthrylene (1,2-dehydro-3-methylcholanthrene). was an active carcinogen. 11,12-Epoxy-11,12-dihydro-3-methylcholanthrene showed weak carcinogenic activity whereas cis-11,12-dihydro-11,12-dihydroxy-3-methylcholanthrene was inactive.     

Enhancement of the mutagenicity of carcinogenic arylamines by ethinyl estradiol

Ethinyl estradiol, the estrogenic component of oral contraceptives,has been shown to enhance the mutagenicity of 2-aminofluorene,2-acetylaminofIuorene, N-hydroxy-2-acetylaminofluorene, andN-acetoxy-2-acetylaminofluorene with strain TA 98 of Salmonellatyphimurium and various rat liver activating systems. The magnitudeof the enhancement of mutation produced by ethinyl estradiolis dependent upon: (i) the type of mixed-function oxidase inducerof the liver activating system; (ii) the structure and concentrationof the arylamine; (iii) the concentration of ethinyl estradiol;and (iv) metabolism of ethinyl estradiol to its catechol, 2-hydroxyethinylestradiol, by the activating system. Mox-estrol, a biologicallypotent estrogenic derivative of ethinyl estradiol which is notmetabolized effectively to its catechol by the mixed-functionoxidases, does not enhance the mutagenicity of the above arylaminesand related compounds. Both 2-hydroxyethinyl estradiol and 2-hydroxy-moxestrolenhance the mutagenicity of 2-aminofluorene and 2-acetylaminofluorene.Neither the estrogens nor their catechols are mutagenic by themselvesin this system. In the presence of ethinyl estradiol, a markedinhibition of ring hydroxylation of 2-acetylaminofluorene wasdemonstrated. Since ring hydroxylation is a well establisheddetoxification pathway of arylamine and arylamide metabolism,the enhancement of mutagenicity by ethinyl estradiol may bethe result of a net increase in N-hydroxylation of arylaminesand arylamides.     

Arachidonic acid-dependent peroxidative activation of carcinogenic arylamines by extrahepatic human tissue microsomes

Prostaglandin H synthase (PHS), an arachidonic acid-dependent peroxidase, has been implicated in the peroxidative activation of carcinogenic aromatic amines in extrahepatic carcinogen target tissues of experimental animals. We have examined the arachidonic acid-dependent activation of [3H]benzidine to DNA-bound products by microsomal preparations from 75 normal human tissues obtained during necessary surgical procedures. For several samples of urinary bladder epithelium, prostatic epithelium, colonic mucosa, and peripheral lung tissue, an arachidonic acid-dependent, microsomal-catalyzed activation of benzidine was observed; and the activity could be inhibited appreciably by indomethacin, a known inhibitor of PHS. Little or no arachidonic acid-dependent activity was detected in human placenta, breast, or liver microsomes or the majority of colon microsomes. Substrate specificity was also examined with purified ram PHS and with human bladder and with active colon preparations. Purified PHS catalyzed the activation of benzidine much greater than 2-naphthylamine, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole greater than 4-aminobiphenyl greater than 2-amino-3-methylimidazo[4,5-f]quinoline greater than 3-amino-1-methyl-5H-pyrido[4,3-b] indole. In comparison, human bladder and colon microsomes catalyzed the activation of benzidine greater than 4-aminobiphenyl, 2-amino-6-methyldipyrido[1,2-alpha:3',2'-d]imidazole, 2-naphthylamine greater than 2-amino-3-methylimidazo[4,5-f]quinoline, 3-amino-1-methyl-5H-pyrido[4,3-b]indole. To confirm the occurrence of PHS antigen in human extrahepatic tissues, an avidin/biotin-amplified competitive enzyme-linked immunoabsorbent assay was developed with purified ram PHS and a commercially available monoclonal antibody known to cross-react with human platelet PHS. The avidin/biotin-amplified enzyme-linked immunosorbent assay, which detected ng quantities of ram PHS, clearly established the presence of the PHS protein in human bladder, prostate, and lung microsomes. In contrast, PHS antigen was not detected in the liver or placental microsomes. The interindividual and tissue-dependent variability of PHS and its role in aromatic amine carcinogenesis are discussed.     

Comparative study on metabolic formation of N-arylformamides and N-arylacetamides from carcinogenic arylamines in mammalian species

The metabolism of carcinogenic arylamines was examined focusing on their N-acylation in mammalian species. When 4-aminobiphenyl, 2-aminonaphthalene, 2-aminofluorene, or 1-aminopyrene was given orally to rabbits, the corresponding N-arylformamides were isolated from the urine together with the corresponding N-arylacetamides. Identification of these N-arylformamides and N-arylacetamides was performed unequivocally by comparing their mass and UV spectra, and thin-layer chromatographic behaviors with those of authentic samples. Such metabolic conversion of the arylamines to the N-arylformamides and N-arylacetamides was also observed in guinea pigs and rats. In addition, carcinogenic nitro compounds such as 4-nitrobiphenyl and 2-nitronaphthalene, which are metabolically reducible to the arylamines, were metabolized to the corresponding N-arylformamides and N-arylacetamides in rabbits. On the other hand, quantitative experiments showed that only minor amounts of the N-arylformamides and N-arylacetamides were excreted in the urine or feces of rats and rabbits given the arylamines. This seems to be due to almost complete further metabolism of these N-acyl derivatives in vivo. Liver cytosols from several mammalian species exhibited a significant N-formylating activity toward the arylamines in the presence of N-formyl-L-kynurenine and N-acetylating activity in the presence of acetyl-CoA. In rabbits, the N-formylating activity was clearly higher than the N-acetylating activity, while the reverse was the case in guinea pigs and hamsters. The experiments with rat liver preparations showed that the liver cytosolic N-formylating and N-acetylating activities are due to formamidase and arylamine acetyltransferase, respectively. Furthermore, enzymatic transfer of the formyl group from one arylamine to another was demonstrated.     

Inhibition of carcinogenic effects of polycyclic hydrocarbons by benzyl isothiocyanate and related compounds.

Benzyl isothiocyanate and phenethyl isothiocyanate, two compounds found in cruciferous plants, and phenyl isothiocyanate, a synthetic compound, all inhibit 7,12-dimethylbenz[a]anthracene (CMBA)-induced mammary tumor formation in female Sprague-Dawley rats when administered 4 hours prior to the DMBA. Comparable studies in which benzyl isothiocyanate was administered 24 hours before or 4 hours after DMBA showed almost complete loss of inhibition. Additions of benzyl isothiocyanate or phenethyl isothiocyanate to a diet containing CMBA inhibited formation of neoplasms of the forestomach and pulmonary adenomas in female ICR/Ha mice. Addition of benzyl isothiocyanate to a diet containing benzo[a]pyrene also inhibited carcinogenesis of the mouse forestomach due to this carcinogen. The finding of two additional anutrient dietary compounds which inhibit chemical carcinogenesis focuses on the possibility that dietary constituents of this nature may diminish the impact of exposures to chemical carcinogens.     

The carcinogenic activities in mice of compounds related to benz[a]anthracene

The carcinogenic activities of a number of aromatic hydrocarbons have been compared with some metabolic intermediates by subcutaneous injection into C57 black mice. The monohydroxymethyl derivatives of 7,12-dimethylbenz[a]anthracene and some related compounds were found to be active carcinogens, but were much less so than the parent hydrocarbon. Epoxides formed at the K-region ofchrysene, benz[a]anthracene, 7-methylbenz[a]antnracene and dibenz[a,h]anthracene produced tumours when given at highdose levels, but were not as active as the parent hydrocarbons. The epoxide derived from phenanthrene was inactive.     

Inhibitory effect of diet related sulphydryl compounds on the formation of carcinogenic nitrosamines.

N-Nitroso compounds (NOCs) are known to be strong carcinogens in various animals including primates (Preussman and Stewart, (1984) N-Nitroso Compounds). Human exposure to these compounds can be by ingestion or inhalation of preformed NOCs or by endogenous nitrosation from naturally occurring precursors (Bartsch and Montesano, Carcinogenesis, 5 (1984) 1381-1393; Tannebaum (1979) Naturally Occuring Carcinogens, Mutagens and Modulators of Carcinogenesis; Shephard et al., Food Chem. Toxicol., 25 (1987) 91-108). Several factors present in the diet can modify levels of endogenously formed nitrosamines by acting as catalysts or inhibitors. Compounds in the human diet that alter nitrosamine formation would thus play an important role in carcinogenesis study. Earlier researchers have reported the nitrite scavenging nature of sulphydryl compounds (Williams, Chem. Soc. Rev., 15 (1983) 171-196). We therefore studied the modifying effect of sulphydryl compounds viz., cysteine (CE), cystine (CI), glutathione (GU), cysteamine (CEA), cystamine (CEI), cysteic acid (CIA) and thioglycolic acid (TGA) on the nitrosation of model amines viz., pyrrolidine (PYR), piperidine (NPIP) and morpholine (NMOR). Many of these compounds are present in the food we consume. The present work also describes the inhibitory effect of onion and garlic juices on the nitrosation reactions. Both onion and garlic are known to contain sulphur compounds (Block, Sci. Am., 252 (1985) 114-119). Most of these compounds behave as antinitrosating agents and their inhibitory activity towards formation of carcinogenic nitrosamines, under different conditions is described.     

Selective mutagenic activation by cytochrome P3-450 of carcinogenic arylamines found in foods

Heterocyclic arylamines found in cooked foods including fish and beef are potent mutagens and carcinogens. The purpose of this investigation was to determine the specificity of cytochromes P1-450 and P3-450 toward the metabolic activation of these arylamines. We used a novel mutagenicity test system which combined human cells expressing either recombinant cytochrome P1-450 or P3-450 with Salmonella typhimurium to score mutants. Cytochrome P3-450, a single isoform of the cytochrome P-450 supergene family, bioactivated these food mutagens. Cytochrome P1-450 showed little or no activation of these arylamines but was the isoform predominantly responsible for the activation of the aromatic hydrocarbon benzo[a]pyrene-7,8-diol. This assay system should serve to define the specificities of individual cytochromes P-450 in the metabolic activation of carcinogens.     

A quantitative structure-activity analysis of the mutagenic and carcinogenic action of 43 structurally related heterocyclic compounds

Quantitative structure-activity relationships for the carcinogenicityand mutagenicity (against Salmonella typhimurium his- TA98,TA100 and TA1537 strains) of 43 structurally related heterocycliccompounds were formulated. The compounds investigated belongto the following two series of congeners: (a) benzo(thio)-pyranoquinolinesand (b) benzo(thio)pyranoindoles. Their biologic activitieswere correlated with the following parameters: (a) the minimaltopological difference (describing the fit of the consideredmolecules with a possible receptor, enzyme or DNA) and (b) thelipophilicity constants. The computed regression equations suggestthat the structural requirements for carcinogenicity are differentfrom those responsible for mutagenicity in the Ames test.     

Induction of DNA repair synthesis in human urothelial cells by the N-hydroxy metabolites of carcinogenic arylamines

Urinary N-hydroxy metabolites of carcinogenic arylamines were investigated for their abilities to induce unscheduled DNA synthesis (UDS) in human urothelial cell lines HCV 29, HU 1734, and HU 1752, and in a primary culture of human urothelial cells. N-Hydroxy-2-aminofluorene (CAS: 53-94-1; N-OH-AF), N-hydroxy-2-acetylaminofluorene (CAS: 53-95-2; N-OH-AAF), and the N-glucuronide of N-OH-AF induced UDS in HCV 29, HU 1734, and HU 1752. N-Hydroxy-4-aminobiphenyl (CAS: 6810-26-0; N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (CAS: 4463-22-3; N-OH-AABP), N-hydroxy-2-aminonaphthalene (CAS: 613-47-8; N-OH-AN), N-hydroxy-2-acetylaminonaphthalene (CAS: 2508-23-8; N-OH-AAN), and the N-glucuronide of N-OH-ABP induced UDS in HCV 29. However, the N-glucuronide of N-OH-AN did not. The O-glucuronide of N-OH-AAF induced UDS in HCV 29 only when beta-glucuronidase was present. Paraoxon inhibited the induction of UDS in HCV 29 by N-OH-AAF and N-acetoxy-2-acetylaminofluorene (CAS: 6098-44-8), but not by N-OH-AF. When examined in a primary culture of human urothelial cells, N-OH-AF, N-OH-AAF, N-OH-ABP, and N-OH-AABP were active, but N-OH-AN, N-OH-AAN, 2-aminonaphthalene (CAS: 91-59-8), 2-aminofluorene (CAS: 153-78-6;), and 4-aminobiphenyl (CAS: 92-67-1) were not. These results demonstrate that human urothelial cells are able to activate both acetylated and non-acetylated N-hydroxy metabolites of carcinogenic arylamines, and they suggest that O-glucuronidation may be a detoxification mechanism for N-arylacethydroxamic acids.     

Induction of repair synthesis of DNA in mammary and urinary bladder epithelial cells by N-hydroxy derivatives of carcinogenic arylamines

Unscheduled DNA synthesis (UDS)-inducing activity was used as a parameter to estimate the abilities of rat mammary epithelial cells and urothelial cells from various species to activate carcinogenic aromatic amine derivatives. The N-hydroxy, N-hydroxy-N-acetyl, N-hydroxy-N-glucuronosyl derivatives of 2-aminofluorene (2-AF) and 4-aminobiphenyl (4-ABP) induced UDS in primary cultures of rat mammary epithelial cells, but 2-AF, the O-glucuronide of N-hydroxy-N-acetyl-2-AF (N-OH-AAF) and 4-ABP did not. Neither the activity of N-OH-AAF, N-hydroxy-N-formyl-2-AF, nor N-acetoxy-N-acetyl-2-AF was significantly altered by paraoxon, an inhibitor of microsomal N-deacetylase. Although N-hydroxy-3,2'-dimethyl-4-aminobiphenyl (N-OH-DMABP) also induced UDS, its N-acetyl derivative, which can not be activated by intramolecular, N,O-acetyltransfer, did not. Similarly, rat urothelial cells were responsive to the UDS-inducing activity of this hydroxylamine, but not the hydroxamic acid. In contrast, dog urothelial cells were responsive to both compounds. The UDS-inducing activity of N-OH-AAF was inhibited by paraoxon in the dog, but not in rat urothelial cells. N-Hydroxy-N,N'-diacetylbenzidine induced UDS in the urothelial cells of dog, rat, and rabbit, and a human urothelial cell line, HCV-29, whereas benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine did not. Co-treatment with 12-O-tetradecanoylphorbol-13-acetate did not enable benzidine to induce UDS in dog urothelial cells. Rat mammary epithelial cells activated N-OH-DMABP by acetyl coenzyme A-dependent O-acetylation and N-OH-AAF by N,O-acetyltransfer. They could not N-deacetylate N-OH-AAF. These results suggest that rat mammary and bladder epithelial cells are capable of activating N-arylhydroxylamine metabolites of these carcinogens, probably by N,O-acetyltransfer and O-acetylation, whereas dog urothelial cells are more likely to activate these metabolites by N-deacetylation and a reaction that has yet to be identified.     

Repair synthesis of DNA induced by the urinary N-hydroxy metabolites of carcinogenic arylamines in urothelial cells of susceptible species

Urinary N-hydroxy metabolites of the bladder carcinogens, 2-aminofluorene and 4-aminobiphenyl, were examined for the induction of unscheduled DNA synthesis (UDS) in urothelial cells of several susceptible species. N-Hydroxy-2-aminofluorene, N-hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, and the N-glucuronides of these two hydroxylamines induced UDS in the urothelial cells of dogs, rats, and rabbits. N-Hydroxy-2-aminonaphthalene, N-hydroxy-2-acetylaminonaphthalene, and the N-glucuronide of the hydroxylamine were not active. The induction of UDS in dog cells by N-OH-AAF or N-acetoxy-2-acetylaminofluorene, but not by N-hydroxy-2-aminofluorene, was inhibited by paraoxon. The microsomal fraction of dog urothelial cells catalyzed the binding of N-OH-AAF to transfer ribonucleic acid; the enzyme activity was completely inhibited by paraoxon, suggesting that N-deacetylase, but not N-,O-acetyltransferase, was responsible for the binding. The O-glucuronide of N-OH-AAF did not induce UDS in the urothelial cells of dogs, rats, or rabbits, nor did it bind to tRNA in the presence of dog urothelial enzymes, which suggest that N-OH-AAF is detoxified by O-glucuronidation. These results are consistent with the hypothesis that nonacetylated, N-hydroxylated metabolites play a major role in arylamine-induced bladder carcinogenesis. The importance of arylacethydroxamic acid metabolites in bladder carcinogenesis for various species may be inversely related to the rate of hepatic O-glucuronidation.