Microbiological mutagenicity studies of pesticides in vitro.

14 pesticides were tested as pure compounds for the induction of point mutations in four strains of Salmonella typhimurium--TA1535, TA1536, TA1537 and TA1538--in the presence and in the absence of rat-liver microsomal fractions and for the induction of resistance to low concentrations of streptomycin in the filamentous bacterium, Streptomyces coelicolor. The technique used was essentially the so-called "spot test". The pesticides investigated were: aminotriazole, Benomyl, Captafol, Captan, Dichlorvos, Dalapon-Na, Dinobuton, Dodine, Ioxynil, Mecoprop, Neburon, Picloram, Triallate and Trichlorphon. In Salmonella, Captan and Triallate were mutagenic on the TA1535 strain; Dichlorvos and Trichlorphon were negative in the spot test but mutagenic after incubation in liquid cultures of strain TA1535. By using the S. coelicolor forward-mutation test, aminotriazole, Dichlorvos, Picloram, Trichlorphon and Triallate were mutagenic with the "spot test" technique; Captan showed a weak mutagenic activity with a "plate-incorporated" technique.     

Mutascreen, an automated bacterial mutagenicity assay

Mutascreen^® is an automated instrument for bacterial mutagenicity testing. The biological principles of the Mutascreen assay are the same as those of the bacterial reverse-mutation assays, like the Ames test, but several operational principles are different. The Mutascreen assay takes place in wells containing only 400 μl of liquid medium. Also, the dispensing of the liquid medium, the bacterial tester strains, the metabolic activation system (S9), and the test solutions is all performed by a computer-controlled robot according to the user's preprogrammed instructions. The turbidity in up to 200 wells is monitored intermittently over a 24-h period by a vertical-pathway photometer, thereby avoiding measurement problems caused by sedimentation. The data for the resulting growth curves is stored for analysis. The auxotrophic growth pattern is altered characteristically by test solutions that are toxic or contain endogenous growth factor(s), while prototrophic growth is observed earlier in the 24-h period when revertants have been induced by the test solution. To compare the Mutascreen assay with the conventional plate assay, 36 chemicals including known carcinogens and noncarcinogens were tested. Both assays identified the same chemicals as mutagens and gave quantitatively similar results, thus testifying to the potential usefulness of automated bacterial mutagenicity testing.     

Rec assay and mutagenicity studies on metal compounds

We carried out rec assays on 127 metal compounds with Bacillus subtilis to check their DNA-damaging capacity and mutagenicity. Certain compounds of beryllium, cobalt, cesium, iridium, osmium, platinum, rhodium, antimony, tellurium, thallium and vanadium were newly found to be positive in addition to those of known positive metals such as arsenic, cadmium, chromium, mercury, molybdenum and selenium. Reverse mutation assays with Escherichia coli and Salmonella strains showed that compounds of rhodium (RhCl₃), tellurium (Na₂H₄TeO₆, Na₂TeO₃) and platinum (PtCl₄, (NH₄)₂PtCl₆) are potent mutagens.     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Detection of gentoxicity of benzidine and its derivatives with the Escherichia coli DJ 702 lacZ reversion mutagenicity assay

AIMS: The feasibility of Escherichia coli DJ 702 lacZ mutagenicity assay to detect genotoxicity of benzidine and its derivatives was evaluated. METHODS AND RESULTS: DJ 702 strain was grown overnight at 30 degrees C in Luria-Bertani (LB) medium containing some components, such as chloramphenicol, ampicillin, delta-aminolevulinic acid, isopropyl-beta-d-thiogalactoside, and trace element mix. The mixtures of a bacterial culture and tested chemical at indicated doses were incubated at 30 degrees C for 30 min. Subsequently, 2 ml of molten top agar was added and the resulting mixtures were immediately poured onto a minimal lactose (ML) plate. Plates were incubated at 30 degrees C for 48 h. The number of colonies was determined by visual scoring. In this study, results showed that all the tested chemicals were mutagenic to DJ 702 strain. CONCLUSIONS: E. coli lac mutagenicity assay using DJ 702 strain can detect the mutagenicity of benzidine and its derivatives. SIGNIFICANCE AND IMPACT OF THE STUDY: We detected the mutagenicity of benzidine and its derivatives in E. coli lac mutagenicity assay using DJ 702, indicating that this assay may be used to detect benzidine and its derivatives in a powerful, sensitive, and convenient mutagenesis assay.     

RK bacterial test for independently measuring chemical toxicity and mutagenicity: Short-term forward selection assay

A short-term bacterial assay system for determining the mutagenic potential of environmental substances was developed and validated. Genotoxic activity was demonstrated for selected substances from 10 categories of chemical agents. The RK test results were obtained with one Escherichia coli assay strain that was transiently exposed to, and then removed from the test substance prior to the selection step for mutant cells. The RK test employs a hitherto unused short-term assay technique for selecting forward mutations in the wild-type selector strain cells. The cells of the selector strain are killed upon shifting to 42°C as a consequence of thermal derepression and subsequent expression of the replication genes from an integrated 10-kilobase fragment of phage λ. Cells that acquire mutations in the responsible killing genes are detected by their colony-forming ability at 42°C. A substance is determined to be genotoxic if it is capable of increasing the forward mutation frequency for appearance of these mutant cells. Toxicity of the agent is independently evaluated by examining its effect on the viability of the selector strain at 30°C, when the viral replication genes remain repressed. The flexible assay protocol enables determination of the effect of pH on mutagenic activity, the requirement for metabolic activation, and assays of nearly insoluble or highly toxic substances.     

Effect of pesticides on bacterial membranes

The effect of pure preparation of ordram, fosalon, DDT, methoxychlorine, hydrel, dihydrel, 2,4-D, 2M-4C and of technical preparations of saturn, linuron, ronstar and keltan on the membrane functions (respiration and motility) of Azospirillum brasilense and Chromatium minutissimum cells and on malate and NADH oxidation by the isolated membranes of Micrococcus lysodeikticus was investigated. The effect varied from irreversible impairment to undetectable impairment of the measured activities depending on the type of bacteria and on the chemical used. The ordram induced permeability of the A. brasilense membranes without inhibition of the respiratory chain activity and selective inhibition of malate oxidase accompanied by stimulation of NADH oxidation in the M. lysodeikticus membranes indicates a possibility of ordram application as a regulator of bacterial metabolism.     

Development and validation of the spiral Salmonella assay: an automated approach to bacterial mutagenicity testing

Since its development by Dr. Bruce Ames and his colleagues more than a decade ago, the Salmonella/mammalian microsome mutagenicity assay has become a widely accepted tool to assist in the identification of chemicals with mutagenic and carcinogenic potential. Several automated approaches to Salmonella testing have been proposed in recent years but have failed to gain acceptance in the scientific community due to poor performance or lack of demonstrated usefulness. In this paper we report on an automated system that successfully generates dose-response data and, moreover, reduces the labor, materials, and sample mass required to obtain such information. In the standard plate-incorporation assay, dose-response relationships are defined by testing discrete doses of the test agent on a series of agar plates. In contrast, the spiral Salmonella assay generates dose-response data from a continuous concentration gradient on a single agar plate. Upon analysis, each spiral plate yields a dose-response curve consisting of 13 data points that span a concentration range of about 15:1, which is equivalent to 5 two-fold serial dilutions. The performance of the spiral Salmonella assay was compared to that of the conventional plate-incorporation assay using 13 mutagens and 7 nonmutagens selected from a variety of chemical classes. Concordant qualitative responses were obtained for all compounds tested, and comparable dose-response relationships were generated by all mutagens with the exception of sodium azide and cyclophosphamide, which are highly water-soluble and, thus, are unable to maintain a well-defined concentration gradient on a spiral plate due to rapid diffusion. In general, toxicity was expressed at a lower dose in the spiral assay, and the mutagenic potencies (slopes of the dose-response curves) were greater in the spiral assay relative to the plate-incorporation assay. These differences will be discussed, as will the applicability of the spiral plating technique to routine screening and its relevancy to future mutagenesis testing.     

Antimutagenic effects of diethyldithiocarbamate towards maleic hydrazide- and N-nitrosodiethylamine-induced mutagenicity in theTradescantia mutagenicity assay

InTradescantia, clone 4430, diethyldithiocarbamate (DEDTC) markedly decreased the frequency of somatic mutations induced by maleic hydrazide (MH) and N-nitrosodiethylamine (NDEA). In contrast, DEDTC had no such effect on N-methyl-N-nitrosourea-induced mutagenesis. The putative degradation and conversion products of MH (maleic acid diamide, succinic acid, maleic acid, lactic acid and hydrazine) exhibited no mutagenic activity in theTradescantia mutagenicity assay.     

Further studies on the feasibility of one-day Salmonella detection by enzyme-linked immunosorbent assay.

A model system previously developed for the rapid detection of Salmonella typhimurium in foods was improved and extended to many other Salmonella serotypes. The original protocol, which consisted of an overnight nonselective culture followed by a specific enzyme-linked immunosorbent assay (ELISA), was modified and improved. A sandwich ELISA which used polyclonal antibodies for the capture stage and a cocktail of monoclonal antibodies for the detector stage was developed. The assay recognized a wide range of Salmonella serotypes; S. enteritidis, the most important serotype in the United Kingdom had a detection limit in the ELISA of about 4 x 10(2) cells ml-1. The cultural stage prior to the ELISA was either a single nonselective broth (incubated for 28 h) or a preenrichment broth (incubated for 7 h) plus a selective broth (incubated for 21 h). Antibodies which bind to cells grown in the unfavorable conditions of a selective medium were selected. It was concluded that, in the future, the shortened protocols for the detection of Salmonella spp. in foods described here will be of considerable value.