Inhibitory Effect of Dimethyl Sulfoxide on the Mutagenicity of Promutagens in the Ames Test

Abstract

The eŠect of dimethyl sulfoxide (DMSO) on the mutagenicity of 14 promutagens belonging to several chemical classes and one direct mutagen as a reference compound was examined in the Ames preincubation test, to clarify how much the test results were aŠected by its inhibitory activity on metabolic enzymes. The mutagens were assayed by the preincubation method using the TA100 or WP2uvrA(pKM101) bacterial test strains in the presence of 1% and 14% DMSO (concentrations in treatment mixture) with S9 mix for 12 promutagens that are known to be activated by CYP enzymes or without S9 mix for 2 promutagens that are known to be activated by bacterial nitroreductase enzymes, and the direct-mutagen. The data indicate that the mutagenicity of 11 of the 14 promutagens was signiˆcantly reduced in the presence of 14% DMSO as compared with that in the presence of 1% DMSO, while the 3 remaining promutagens and the direct mutagen exhibited mutagenicity of equal degree at both concentrations. The largest inhibitory eŠect of DMSO was found on the nitrosamines in WP2uvrA(pKM101) and TA100, and no cytotoxicity was detected by survival test, with 14% DMSO in WP2uvrA(pKM101), at any amounts of dimethylnitrosamine. Further equivalent or slightly lower cytotoxicity in the presence of 14% DMSO than in the presence of 1% DMSO was detected by decrease in the bacterial background-lawn density, as a whole. These observations suggest that the reduction in the yield of revertant colonies in the presence of 14% DMSO with the 11 chemicals was not due to cytotoxicity of DMSO. The inhibitory eŠect of DMSO on the mutagenicity of the promutagens can be explained by its inhibitory eŠect on the drug-metabolizing enzymes involved in the activation/detoxiˆcation pathways. Use of DMSO at a low concentration, such as 1%, may be suggested for the Ames test, as for other in vitro genotoxicity tests.

Ames test study designs for nitrosamine mutagenicity testing: qualitative and quantitative analysis of key assay parameters

“…The amount of inhibition was vehicle-dependent, with DMSO among the most potent inhibitors of chlorzoxazone metabolism. DMSO has previously been shown to inhibit rat CYP2E1 metabolism at low percentage volume concentrations in non-induced liver microsomes [ 67 ], and can diminish the mutagenicity of pro-mutagens in the Ames test [ 29 ], including N -nitrosodialkylamines [ 23 , 30 ]. Therefore, there have been specific regulatory concerns about the use of DMSO as a vehicle for the assessment of NAs in the Ames test.…”

“…Plate incorporation and pre-incubation Ames test methods were investigated to assess whether test article exposure in the test system (before the addition of agar) affects assay sensitivity. In addition, the effect of a range of vehicles was investigated (water, DMSO, methanol, acetonitrile, NMP, DMF, acetone, and DHF), as there have been concerns that organic solvent vehicles can inhibit cytochrome P450 enzyme activity and reduce the sensitivity of the Ames test [ 29 , 30 ]. Finally, since the metabolic activation of NAs can be species-dependent [ 25 , 31 , 32 ] we investigated the use of rat or hamster-induced liver S9 to compare their effects on mutagenic potency, the mechanism of N-alkyl nitrosamine activation involves CYP2E1 metabolism, and this has been extensively described elsewhere [ 33 ].…”

Evaluation of acetone as a solvent for the Ames test

“…MMS is a direct mutagen that does not require metabolic activation [12,13]. CP, MY, 2-AAF, Quinoline, DBA, 2AA, NP, and BP are promutagens that require CYP enzymes contained in S9 mix for metabolic activation [10,13,14]. SA, 2-NF, 9AA, AF2, and 4NQO are promutagens that are metabolically activated by bacterial nitroreductase enzymes [10,13,14].…”

“…The effects of acetone on the mutagenicity of 2AA at the volumes of 25 μL and 50 μL were different among the strains. Mutagenicity of 2AA is more efficiently detected at a lower amount of S9 fraction than 50 μL (amount commonly used for S9 fraction in a test tube) [10,19], suggesting that when the concentration of S9 fraction is high, detoxification pathways is dominant. The magnitude of mutagenicity at varying volumes of acetone in different bacterial strains, would reflect mutations from different types and amounts of DNA adducts.…”

“…In the preincubation test, DMSO is added at a relatively high concentration (14% = 0.1 ml of DMSO/0.7 mL of the mixture) in the reaction mixture (consisting of phosphate buffer [PB] or S9 mix, bacterial culture, and test chemical dissolved in DMSO). DMSO is well known to produce cytotoxicity and inhibit drug metabolism during the preincubation step, resulting in reduced sensitivity in the detection of mutagens [9,10]. We previously examined the effects of multiple concentrations of DMSO on the cytotoxicity to the bacterial strains and on the mutagenicity of mutagens; in the preincubation test, DMSO did not affect the detection of mutagens in spite of moderate cytotoxicity up to a concentration of 14% [9,10].…”