4-(4-nitrobenzyl)pyridine tests for alkylating agents following chemical oxidative activation

A chemical activation system (CAS) designed to mimic the mammalian mixed-function oxidase enzymes was found to activate target compounds to reactive electrophiles. Activated compounds were assayed by reaction with 4-(4-nitrobenzyl)pyridine (NBP). A model nucleophile of 7-alkylguanine of nucleic acids, NBP produces a violet color following alkylation. Twenty compounds from several chemical classes were tested. The test generally gave positive and negative responses where expected. Two compounds, trichloroethylene and diethylnitrosamine, exhibited a linear Beer's law relationship in the concentration range tested. A high degree of linear correlation (r>0.97) was obtained for these compounds. Other compounds showed varying degrees of linear correlation from high correlation (r=0.94) to weak correlation (r=0.44). The CAS-NBP assay results were compared to bacterial mutagenicity and animal carcinogenicity test results when information was available. A good correlation (r=0.80) existed between direct alkylating activity and direct mutagenicity. Similar correlations existed between NBP alkylation following activation and mutagenicity following microsomal activation (r=0.73). Also, different correlations were observed between carcinogenicity and NBP alkylation following activation (r=0.69) and without activation (r=0.38).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/48076/1/244_2004_Article_BF00213289.pd

Enzymic N-demethylation reaction catalysed by red blood cell cytosol.

Red blood cell cytosol promotes enzymic N-demethylation reactions which display typical Michaelis-Menten kinetics with respect to N-methylaniline as substrate. The demethylase activity is linked with hemoglobin (Hb) and is enhanced in the presence of NADH and the NADH-methemoglobin reductase system. It has been adduced that Hb in its oxygenated form is involved in the reaction

Activation of some aromatic amines to mutagenic products by human red blood cell cytosol.

The ability of human red blood cell cytosol to activate aromatic amines was evaluated with the Ames test using Salmonella typhimurium TA98 in the liquid preincubation condition. While negative results were obtained with 4-acetylaminofluorene (4AAF) and 1-naphtylamine (1NA), a slight response was observed for 4-aminobiphenyl (4ABP) and 2-naphthylamine (2NA). Human red blood cell cytosol was able to activate 2-aminofluorene (2AF), 2-acetylaminofluorene (2AAF) and 2-aminoanthracene (2AA) to mutagenic intermediates. Extracts of human red blood cell cytosol incubated with 2AF were analyzed by gas chromatography: N-hydroxy-2-aminofluorene was identified as a metabolite

Mutagenicity of some heterocyclic amines in Salmonella typhimurium with metabolic activation by human red blood cell cytosol.

Purified human red blood cell cytosol was used to activate the heterocyclic amines 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) into mutagenic intermediate(s) in the Salmonella test. The liquid preincubation method in the presence of strain TA98 was utilized. In order to understand the mechanism involved in this metabolic activation, some modulators were incorporated in the medium. The results suggest that an oxygenated hemoprotein, probably oxyhemoglobin, is involved in the activation into genotoxic intermediate(s)

Mutagenic activation of aromatic amines by a human hepatoma cell (Hep G2) supernatant tested by means of Salmonella typhimurium strains with different acetyltransferase activities.

The study was carried out to characterize hepatoma cells (Hep G2) as activation system relevant to man and to investigate which are the tester strains most suitable for the mutagenic assay of aromatic amines. A supernatant prepared from the human hepatoma cell line Hep G2 was used to activate benzidine, 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) in the Salmonella typhimurium reversion assay. Activation by Hep G2 supernatant was studied with increasing concentrations of the three compounds, in tester strains TA98, YG1024, DJ400 and DJ460. Benz[alpha]anthracene (BA) pretreatment of cells increases the mutagenicity of benzidine in strains YG1024, DJ460 and DJ400. Activation of 2-AAF and 2-AF was observed in strains YG1024, DJ400 and, at the highest tested dose, in DJ460. These results were compared with those obtained with S9 from control and Aroclor 1254 (Aro)-pretreated rat liver. With strain TA98 comparable responses were obtained except for 2-AF which was better activated using rat liver S9. The use of strain YG1024 greatly increases the sensitivity of the response. Strain DJ460 makes it possible to detect activation of 2-AF and 2-AAF by Aro-induced rat liver. These results indicate that Hep G2 supernatant is a useful metabolic activation system of human origin that can be used to replace rat liver S9. An appropriate choice of the Salmonella strain not only can increase the sensitivity of the response, but may also help to overcome certain metabolic shortcomings of the Hep G2 cell line and rat liver S9

Mutagenicity evaluation of azaperone in the Salmonella/microsome test.

Azaperone was evaluated for its mutagenic potential by the Salmonella/microsome test. No mutagenic activity towards six S. typhimurium strains could be evidenced with azaperone at doses up to 2,000 micrograms/plate, either without or with metabolic activation at usual test conditions. Higher concentrations of liver post-mitochondrial fraction from Aroclor 1254 (ARO)-pretreated rats did not reveal any increase in the number of revertants towards S. typhimurium strains TA1537, TA1538 and TA98. Moreover, a plate-incorporation test with liver post-mitochondrial fractions from mice pretreated with phenobarbital (PB) and a liquid preincubation test with liver post-mitochondrial fractions from rats pretreated with ARO also failed to reveal any mutagenic action of azaperone towards S. typhimurium strain TA98. Thus, none of the tests used provided any indication of azaperone having a mutagenic action

Metabolic activation by a supernatant from human hepatoma cells: a possible alternative in mutagenic tests.

The supernatant from human Hep G2 hepatoma cells was examined for typical enzymatic activities involved in the metabolism of xenobiotics. Neither cytochrome P-450 nor b5 was detectable, but associated enzymatic activities were found especially after induction with hydrocortisone (HC) and benzanthracene (BA) suggesting that this Hep G2 supernatant contains cyt P-450 IA1 and IA2. Other critical enzymes are also present, but, as expected, at lower activities than in Aroclor 1254 rat liver S9, except for NADH and NADPH cytochrome c reductase. Results of the Ames test indicate that the induced Hep G2 supernatant is a suitable activator for the evaluation of genotoxicity of indirect mutagens