Radiation-induced mutagenicity and lethality in ames tester strains of salmonella

Mutation and killing induced by X radiation and 60CO gamma radiation were studied in six different histidine-requiring auxotrophs of Salmonella typhimurium. Strain TA100, which is sensitive to base-pair substitutions, and strains TA2637 and TA98, which are sensitive to frameshifts, carry the pKM101 plasmid and exhibit significantly higher radiation-induced mutations compared to their plasmidless parent strains TA1535, TA1537, and TA1538, respectively. Among the plasmid-containing strains, TA98 and TA2637 are much more sensitive to the mutagenic action of radiation than is TA100 based on a comparison with their respective spontaneous mutation rates; however, no uniformity was observed in the responses of the strains to the lethal action of ionizing radiation. The pKM101 plasmid provides partial protection against lethality in TA100 and TA2637, whereas the same plasmid enhances the lethal action of ionizing radiation in TA98. The following conclusions are consistent with these observations: (1) the standard Ames Salmonella assay correctly identifies ionizing radiation as a mutagenic agent; (2) frameshift-sensitive parent strains are more sensitive to the mutagenic effects of ionizing radiation than is the only strain studied that is sensitive to base-pair substitutions; and (3) enhancement of mutagenesis and survival is related to plasmid-mediated repair of DNA damage induced by ionizing radiation and does not involve damage induced by Cerenkov-generated uv radiation which is negligible for our irradiation conditions.     

Assessment of the uptake and mutagenicity of nickel chloride in salmonella tester strains

NiCl2 was examined for mutagenic activity in a number of Salmonella tester strains. Conditions were established where there was substantial uptake of the metal into the bacterial cells. However, even when the metal ion was apparently taken up, as determined by metal association with cells, there was a lack of mutagenic activity. These results suggest that nickel is unable to induce basepair or frameshift mutations in Salmonella tester strains and are discussed in relationship to the low binding affinity of Ni(II) for DNA.     

Forward mutations to arabinose resistance in Salmonella typhimurium strains: a sensitive assay for mutagenicity testing.

The forward-mutation assay using the L-arabinose-sensitive strain SV3 of Salmonella typhimurium has been calibrated against a selected set of mutagens. Strain SV3 is sensitive to chemicals causing base-pair substitutions, frameshift mutations and deletions. New strains deficient for the excision-repair system or the lipopolysaccharide barrier or both have been selected from strain SV3. The additional mutations do not affect the independence of the assay from experimental artifacts due to physiological or lethal damage or differences in plating density. The new strains are more sensitive than SV3 to certain mutagens. Techniques for using this set of strains are presented and their relative advantages discussed.     

Enhanced mutagenicity of anisidine isomers in bacterial strains containing elevated N-acetyltransferase activity.

In previous studies on the mutagenicity of anisidine isomers, the ortho isomer was considered to be mutagenic towards standard Ames tester strains, while the para isomer gave equivocal results. In the present study we show that both para- and ortho-anisidine isomers are mutagenic in a Salmonella typhimurium tester strain containing elevated levels of N-acetyltransferase (YG1029). p-Anisidine gave a positive mutagenic response using either hamster S9 or ram seminal vesicle microsomes (RSVM) as an activating system, while o-anisidine gave a positive response only with the hamster S9 fraction. The mutagenic response from p-anisidine was greater than with o-anisidine in each case. In tests with p-anisidine and RSVM, the addition of arachidonic acid was not necessary to observe a mutagenic response. Catalase produced a dose-dependent decrease in the mutagenic response with p-anisidine and RSVM; this indicates that endogenous hydrogen peroxide from the bacteria acts as a substrate for the peroxidase activity of RSVM prostaglandin H synthase. These results demonstrate that both anisidine isomers are mutagenic and that N-acetyltransferase enzymes play an important role in their metabolism to mutagenic species.     

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.     

Performance of bacterial strains used for distillery waste treatment on different substrates

Six bacterial strains were isolated and acclimatized on distillery waste. The performance of these bacterial strains in respect to growth, reduction in chemical oxygen demand (COD) values, carbon dioxide production and volatile acid production were studied on five different substrates. Glucose and xylose exhibited growth patterns similar to that on spentwash. Glucose, xylose, casein hydrolysate and amino acids led to very good reduction in COD values compared with glycerol. Rate of substrate consumption was maximum in the case of glucose followed by amino acids, casein hydrolysate, xylose and glycerol. Production of volatile acids and carbon dioxide from glucose amounted to ≈ 50% of the theoretical yield based on glycolysis and the tricarboxylic acid cycle. Production of carbon dioxide followed the usual microbiological growth pattern while volatile acids did not show any such pattern. Carbon dioxide and volatile acids appear to be the major degradation products in distillery waste treatment by these bacteria.     

Computerized evaluation procedure for comparing the electrophoretic protein patterns of bacterial strains

Among 46 isolates of Staphylococcus aureus obtained from cattle in the State of Paraíba, Brazil, four were shown to produce antimicrobial substances (AMS). The two best AMS producers carried single plasmids of about 8·0 kbp and 50 kbp, respectively, which were designated pRJ34 and pRJ35. Curing experiments and molecular analysis associated the AMS production with the presence of these plasmids in the cells. The biochemical properties exhibited by the AMS suggested that they might be bacteriocins (Bac). The bacteriocin encoded by pRJ34 showed properties identical to those of the bacteriocins encoded by other small staphylococcal Bac plasmids. However, the bacteriocin encoded by the large plasmid pRJ35 has shown some properties which distinguish it from the other bacteriocins of Staph. aureus described so far, suggesting it may be a new member of the staphylococcal bacteriocin family.     

Safety evaluation of two bacterial strains used in Asian probiotic products

Probiotics, known for their prophylactic and therapeutic properties, are routinely used by the medical community in various regions of the world. In some Asian countries, these products are controlled as pharmaceutical substances and must adhere to strict regulatory guidelines. However, outside of Europe where the European Food Safety Authority has recently adopted a Qualified Presumption of Safety approach for probiotics used in food and feed, current safety requirements do not necessitate screening for the presence of virulence and other risk factors, which may result in the inadvertent use of probiotic strains harboring harmful genes. A safety evaluation was conducted on Enterococcus faecium R0026 and Bacillus subtilis R0179 used in several commercial probiotic products marketed in Asia. Molecular techniques were used to verify the identity of each strain and antibiotic resistance profiles were determined towards clinically relevant antibiotics. Strains were subsequently screened for the presence of enterotoxins and virulence factors and were subjected to 28 days of repeated high-dose oral toxicity testing in rats. No risk factors or aberrant activities were identified using such a detailed approach. Thus, both microbes were deemed to pose low risk to the consumer and, therefore, safe for use as probiotics.     

The response of recombinant inbred strains of mice to bacterial lipopolysaccharides.

Fourteen recombinant inbred strains of mice have been produced by the inbreeding of the F2 generation of a cross between C57BL/6J and C3H/HeJ progenitor mice. The responses of these BXH strains to bacterial lipopolysaccharides (LPS) have been characterized. Four BXH strains are high LPS responders and nine strains are low LPS responders. One BXH strain shows intermediate responsiveness which may reflect residual heterozygosity. F1 hybrid mice from low x high responder strains were intermediate in their response to LPS suggesting additive genetic control. The LPS responses in backcross mice from the F1 x low LPS responders showed segregation consistent with LPS responsiveness being determined by a single gene. In 13/14 BXH strains, there was concordant inheritance of LPS responsiveness and the major urinary protein locus Mup-1b. The association of the expression of the Mup-1 alleles with LPS responsiveness in the BXH strains suggests that the defective LPS response gene in C3H/HeJ mice is located on chromosome 4.     

A comparison of methods used for testing staphylokinase (fibrinolysin) production in Staphylococcus strains

Different nutrient bases, fibrinogen or heated plasma preparations and plasminogen sources were compared in tests for staphylokinase production by Staphylococcus aureus and other staphylococcal species. Media containing a nutrient base, bovine fibrin and dog plasma or serum proved to be more sensitive than the other substrates or combinations of substrates tested. Comparative testing on plates with and without dog plasminogen or with plasmin inhibitors, was found to be nescessary for the differentiation of staphylokinase from protease effects. Staphylokinases were produced by S. aureus, S. hyicus, S. simulans, S. sciuri and S. xylosus strains.     

Plant tissue culture as a model system for mutagenicity testing of chemicals

Two karyotypically stabilized callus strains of Crepis capillaris (2n = 7 and 2n greater than or equal to 12, respectively) were employed to illustrate the utility of plant tissue culture method for screening and analysis of cytogenetic effects of chemicals following long-term treatment. The cytogenetic analysis of callus cells revealed significant differences in the toxic and mutagenic effects of chemicals under study (2,4-D, MH, NMU and kinetin). A certain dose-response relationship (though not necessarily linear) was observed. The maximum cytogenetic activity of chemicals was associated with certain intermediate concentrations (4.5 X 10(-5) M-9 X 10(-5) M), whereas a further increase in the dose either gave rise to the opposite effects (i.e. decrease in the ana- and telo-phase aberration rates and increase in the modal karyotype frequency) or the aberration rate remained unchanged.     

The involvement of dimethyl sulfoxide in a bacteriotoxic response of the Ames assay tester strains TA98 and TA100

Dimethyl sulfoxide is a widely accepted and recommended solvent in which to dissolve compounds to be tested for mutagenicity via the Ames Salmonella/mammalian microsome assay. Using tester strains TA98 and TA100, we observed a bacteriotoxic response with various fractions isolated from beer when dissolved in DMSO but not when dissolved in water. Further characterization of the role of solvent in simple model systems consisting of butanol, DMSO and bacteria strongly suggests a chemical reaction occurs between dimethyl sulfoxide and specific chemical constituents of the test substance, nutrient broth, or the Ames bacterial strains. The result of such an interaction could be misinterpreted as a toxic response to the test substance when, in fact, the bacteriotoxicity could be due to another compound, chemically distinct from the test substance.     

The use of a mutationally unstable X-chromosome in Drosophila melanogaster for mutagenicity testing.

Somatic eye-colour mutations in an unstable genetic system, caused by a transposable element in the white locus of the X-chromosome in Drosophila melanogaster, is suggested as an assay system for mutagenicity testing. The system is evaluated by comparison with a corresponding system in a stable X-chromosome. Its sensitivity is confirmed with X-ray and EMS treatment, and it is found to be confined to the specific segment of the X-chromosome where the transposable element is localized.     

The mutagenicity of 4CMB in a microwell bacterial fluctuation test

4CMB gave a positive test response in the bacterial fluctuation test. The presence of S9 mix caused attenuation of the dose response indicating that 4CMB is a direct-acting bacterial mutagen that may undergo enzymatic detoxication.     

Spermidine acetylation in response to a variety of stresses in Escherichia coli

Heat shock, cold shock, ethanol, and alkaline shift, but not hydrogen peroxide, stimulate the accumulation of monoacetylspermidine in Escherichia coli. Acetylation occurs with nearly equal frequencies at both the N1 and N8 positions of this ubiquitous polycation. Spermidine acetylation does not appear to be associated with known stress regulons, such as htpR, oxyR, and SOS. E. coli, capable of acetylating spermidine, constitutively express a spermidine acetyltransferase activity during all phases of growth, and this activity is unaffected by cold shock. A mutant strain, incapable of acetylating spermidine, does not express this enzyme activity but grows at an identical rate as the parent strain at 37 degrees C. These results demonstrate that the monoacetylation of spermidine in E. coli is regulated by some mechanism other than a stress-inducible acetyltransferase and is not essential for growth of these cells. They suggest that polyamine acetylation is involved in the responses of these organisms to a variety of chemical and physical stresses.