Potential application of LASER/microbe bioassay technology for determining water-soluble vitamins in foods.

A microbiological technique was developed for quantitating niacin by determining microbial growth rates in response to the amount of vitamin available. Unlike the current official AOAC method, the new procedure for niacin measured the growth rates during the early exponential growth phase rather than during the stationary phase. Lactobacillus plantarum was used to determine niacin to a lower limit of 100 pg/mL. The assay time was approximately 6 h, compared with 16-24 h for the current AOAC method. The extent of microbial growth was determined by differential light scattering of a LASER beam. Multiple photodetectors were integrated with a computer system to collect and analyze the data. The use of differential light scattering to determine 8 water-soluble vitamins under stationary phase conditions demonstrated the potential application of the new technology for microorganisms and foods.

Quantitative bioassay using laser-monitored bacteria to assess toxicology and metabolism of bioactive Group IV compounds.

A new approach has been developed for assessing the toxicity and the metabolism of Group IV compounds and for other toxic substances that may interact with them at the molecular level. The approach is based upon the selective responses of sensitive biodetectors whose molecular interactions with a wide variety of toxic substances have been previously described and reported. Detection and quantitation of carbon, silicon, and tin-containing compounds has been accomplished using a laser-monitored bacterial bioassay. This system uses isogenic Bacillus subtilis strains that are genetically engineered to respond differentially to specific toxicants. The response of these biodetectors is monitored by differential light scattering of a laser that is integrated with a computerized system which collects and analyzes this data. The assay can routinely generate fully analyzed data for fixed time intervals, i.e. 60 or 90 min, or kinetic data so that the mechanisms of interactions can be elucidated.

Metabolic activation of DMH by colonic microsomes: a process influenced by type of dietary fat.

A study was conducted to determine whether 1,2-dimethylhydrazine (DMH), a potent colon carcinogen, is activated to a more potent mutagen by the drug-metabolizing system of the colonic mucosa and to determine the extent to which this metabolism is modified by lipids in the diet. DMH-treated rats fed a diet enriched with 10% corn oil exhibited markedly elevated colonic enzyme levels for mutagen production. This diet also produced the greatest number of animals with colon tumors, when compared with diets containing other levels and sources of lipid. The potential risk of a diet in which unsaturated fat is the sole source of lipid is underlined.

Parameters for detection of mutagenesis of 1,2-dimethylhydrazine using an in vitro Bacillus subtilis system.

Parameters for detection of mutagenesis of 1,2-dimethylhydrazine (DMH) using a Bacillus subtilis microbial assay are defined. His+ and his met mutations were induced in B. subtilis TKJ6321 in the absence of rat liver S-9 Mix. This B subtilis mutant showed a greater sensitivity to this chemical than did Salmonella typhimurium TA1535. Protein-binding studies with bovine serum albumin showed the capacity for DMH to bind non-specifically to protein. S-9 mix from Aroclor pretreated animals appeared to decrease the mutagenic response. Kinetics of mutagen stability were also shown, thereby demonstrating the need for more complete investigation of known chemical carcinogens which give negative results in the Ames bioassay alone.

Mutagenicity and DNA-damaging activity for several pesticides tested with Bacillus subtilis mutants.

The active pure compounds of 4 pesticides were tested for DNA-damaging and mutagenic activity in Bacillus subtilis and Salmonella typhimurium tester strains. Included were zinc ethylenebisdithiocarbamate (dithane), 1,2-dihydropyridazine-3,6-dione (maleic hydrazide), O,O-dimethylphosphorodithioate (malathion), and 1,2-dibromoethane (fumazone). These agents gave either weak or negative mutagenic responses with the Salmonella/microsome tests for mutagenicity, but were all positive when the tester was B. subtilis strain TKJ6321. Of the 4 chemicals, only fumazone required metabolic activation with rat-liver S9 mix. Upon activation, it produced a volatile mutagenic product. Dithane, maleic hydrazide, and malathion were all mutagenic and did not require metabolic activation. Among these agents, dithane was strongly mutagenic while fumazone, maleic hydrazide and malathion were moderately mutagenic. Only dithane gave significant DNA-damaging activity when applied to a battery of repair-deficient B. subtilis mutants. For the chemicals reported, it is concluded that B. subtilis is superior to S. typhimurium in the detection of mutagenic activity. We strongly recommend its use for prescreening procedures in combination with the S. typhimurium testers.

DNA-damaging and mutagenic effects of 1,2-dimethylhydrazine on Bacillus subtilis repair-deficient mutants.

Mutagenic, DNA-damaging, and in vivo alteration of DNA have been demonstrated for 1,2-dimethylhydrazine (DMH), a potent inducer of adenocarcinomas of the large intestine and colon of rats. These activities are pH-dependent, with 6.5 giving optimum response. There was no requirement for metabolic activation with rat-liver S9 mix when the appropriate Bacillus subtilis mutant strains were used. The Rec- strains recA8 and mc-1 were greater than 300-fold more sensitive to the DNA-damaging activity of DMH than was their isogenic wild-type parent. The DNA isolated from DMH-treated mc-1 had altered spectroscopic characteristics, and gave a greatly reduced transformation efficiency. Treatment of B. subtilis strain TKJ6321 with DMH at pH 6.5 induced His+, Met+ mutations in substantial numbers at low concentrations of this chemical. The use of B. subtilis mutants in these studies has therefore made it possible to demonstrate mutagenic and DNA-damaging activity in bacteria for this potent carcinogenic chemical.

Mutation induction by the direct treatment of Bacillus subtilis deoxyribonucleic acid.

A procedure was developed to select for specific mutations obtained by means of transformation with DNA previously exposed to potentially dangerous chemical compounds. The 70% co-transformation of hisB and trpC genes in Bacillus subtilis provided a convenient opportunity to select for new mutations. When purified DNA from wild type bacteria was treated with N(OH) acetyl aminofluorene or Hoechst dye 37 507 and used to transform a recipient bearing of a trpC2 mutaion, a high proportion of the Trp+ transformants had new hisB mutations.

Isolation and characterization of thymineless mutants from ultraviolet-sensitive Bacillus subtilis strains.

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy(-) strains differed drastically from Thy(+) ones in their patterns of [(3)H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.