Antimutagenic activity of vitamin B1 against damages induced by chemical and physical mutagens in Salmonella typhimurium and Escherichia coli.

Thiamine (vitamin B1) is an essential nutrient acting mainly as an enzymatic cofactor on diverse cell processes. It has been reported that vitamin B1 has a significant role in the signaling pathways related to the response to adverse environmental conditions (chemical and physical). The objectives of this study were to evaluate the antimutagenic potential of vitamin B1 in front of DNA-alkylating agents in the presence/absence of ogt and ada repairing genes in Salmonella typhimurium strains and against damage induced by ultraviolet light type C in Escherichia coli strains mutated at the uvrABC system and recBCD enzymes. For S. typhimurium, an antimutagenesis test (Ames test) was performed using strains deficient in one or both genes (YG7100 ada-/ogt+, YG7104 ada+/ogt-, YG7108 ada-/ogt-). For E. coli, mutated strains (K-12 derived strains Hfr H180 uvrB+/recA+, W3110 uvrB+/recA- and ATCC®8739 uvrB-/recA+) were exposed to UV-C light at different time intervals, with and without vitamin B1. Our results showed that thiamine is an antimutagen against methyl-N-nitro-N-nitrosoguanidine or ethyl-N-nitro-N-nitrosoguanidine only when the ogt gene is present. While for E. coli, the presence of vitamin B1 increased the survival rate, implying an antimutagenesis independent of uvrABC repairing system and recBCD enzymes.

Chemical and UV Mutagenesis.

The ability to create mutations is an important step towards understanding bacterial physiology and virulence. While targeted approaches are invaluable, the ability to produce genome-wide random mutations can lead to crucial discoveries. Transposon mutagenesis is a useful approach, but many interesting mutations can be missed by these insertions that interrupt coding and noncoding sequences due to the integration of an entire transposon. Chemical mutagenesis and UV-based random mutagenesis are alternate approaches to isolate mutations of interest with the potential of only single nucleotide changes. Once a standard method, difficulty in identifying mutation sites had decreased the popularity of this technique. However, thanks to the recent emergence of economical whole-genome sequencing, this approach to making mutations can once again become a viable option. Therefore, this chapter provides an overview protocol for random mutagenesis using UV light or DNA-damaging chemicals.

Disrupted OmpC causes osmosis sensitivity of Escherichia coli in alkaline medium.

The Escherichia coli strain DH42 is sensitive to high osmolarity in an alkaline medium. Using mini-Tn5 mutagenesis, construction of mutant strains by homologous recombination and subcloning of DNA fragment techniques, gene ompC was identified as the key factor that, once disrupted, caused osmosis-sensitivity of E. coli strain DH42 grown in an alkaline medium. Through P1 transduction, a mutant strain, D9 (W3110 ompC:kan), was constructed and growth comparison was performed between DH42 and D9 under different pHs and salt concentrations. The result showed that ompC was necessarily required for hyperosmotic adaptation of E. coli in the alkaline medium.

Genome-shuffling improved acid tolerance and L-lactic acid volumetric productivity in Lactobacillus rhamnosus.

Genome shuffling is an efficient approach for the rapid improvement of industrially important microbial phenotypes. Here we improved the acid tolerance and volumetric productivity of an industrial strain Lactobacillus rhamnosus ATCC 11443 by genome shuffling. Five strains with subtle improvements in pH tolerance and volumetric productivity were obtained from the populations generated by ultraviolet irradiation and nitrosoguanidine mutagenesis, and then they were subjected for recursive protoplast fusion. A library that was more likely to yield positive colonies was created by fusing the lethal protoplasts obtained from both ultraviolet irradiation and heat treatments. After three rounds of genome shuffling, four strains that could grow at pH 3.6 were obtained. We observed 3.1- and 2.6-fold increases in lactic acid production and cell growth of the best performing at pH 3.8, respectively. The maximum volumetric productivity was 5.77+/-0.05 g/lh when fermented with 10% glucose under neutralizing condition with CaCO(3), which was 26.5+/-1.5% higher than the wild type.

Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF.

Targeted oncolytic viruses and immunostimulatory therapeutics are being developed as novel cancer treatment platforms. These approaches can be combined through the expression of immunostimulatory cytokines from targeted viruses, including adenoviruses and herpesviruses. Although intratumoral injection of such viruses has been associated with tumor growth inhibition, eradication of distant metastases was not reported. The major limitations for this approach to date have been (1) inefficient intravenous virus delivery to tumors and (2) the lack of predictive, immunocompetent preclinical models. To overcome these hurdles, we developed JX-594, a targeted, thymidine kinase(-) vaccinia virus expressing human GM-CSF (hGM-CSF), for intravenous (i.v.) delivery. We evaluated two immunocompetent liver tumor models: a rabbit model with reproducible, time-dependent metastases to the lungs and a carcinogen-induced rat liver cancer model. Intravenous JX-594 was well tolerated and had highly significant efficacy, including complete responses, against intrahepatic primary tumors in both models. In addition, whereas lung metastases developed in all control rabbits, none of the i.v. JX-594-treated rabbits developed detectable metastases. Tumor-specific virus replication and gene expression, systemically detectable levels of hGM-CSF, and tumor-infiltrating CTLs were also demonstrated. JX-594 holds promise as an i.v.-delivered, targeted virotherapeutic. These two tumor models hold promise for the optimization of this approach.

[Modification of mutation processes in culture of human lymphocytes with tea extracts].

Antimutagenic activity of the extracts of tea leaves from different stages of technological processing has been investigated. Culture of human lymphocyte cells was used as a test-object. Mutations have been induced with gamma-rays, N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) and benz-[a]-pyren. All the extracts showed ability to decrease the frequency of chromosome aberrations with high effectiveness. The effectiveness of green tea leaf extracts was higher in comparison with the effectiveness of the extracts from the late stages of processing.

The mutagenic activity of the S-nitrosoglutathione/glutathione system in Salmonella typhimurium TA1535.

S-nitrosoglutathine (GSNO) and reduced glutathione (GSH) were tested for mutagenicity against strain Salmonella typhimurium TA1535 in the Ames Standard plate incorporation assay. Neither GSNO not GSH were mutagenic when tested alone. In combination, the GSNO/GSH system induced a positive mutagenic response that ranged from 3 to 20 x over background at concentrations of 10 to 50 micromol (micromol)per plate, respectively. This mutagenic response can be attributable to the generation nitric oxide, among the many other reactive products generated by the reaction of GSNO with GSH.

Histamine pharmacokinetics in tumor and host tissues after bolus-dose administration in the rat.

In this study, we estimated interstitial histamine concentrations in normal and malignant tissues after a single intravenous (i.v.) injection of 0.5 mg/kg histamine dihydrochloride in the rat. The microdialysis technique was used to collect interstitial fluid from subcutis, liver and a NGW adenocarcinoma. Histamine was absorbed with equal efficiency to all tissues (t 1/2 AB 3.9-7.7 minutes) but maximum concentration (Cmax; nmol/l) of histamine was higher in liver (2,388 +/- 357) than in subcutis (951 +/- 125) (p < 0.01) and subcutaneous tumor (523 +/- 140) (p = 0.01) and, moreover, Cmax in liver tumor (1,752 +/- 326) was higher than in subcutaneous tumor (p = 0.01). The tl/2 elimination was significantly longer in subcutis and subcutaneous tumor than in liver and liver tumor. Area under the curve (AUC; mmol-min/l) for histamine was significantly lower in subcutaneous tumor (9.8 +/- 2.3) than in liver (17.6 +/- 1.9) (p = 0.03) and liver tumor (15.8 +/- 1.8) (p = 0.03). Local tissue blood flow as assessed by the 14C-ethanol method was not significantly altered by the histamine administration. In conclusion, after an i.v. injection of histamine dihydrochloride a higher maximum concentration and AUC of histamine was reached in liver and liver tumor than in subcutaneous tissues.

Evaluation of transcriptional fusions with green fluorescent protein versus luciferase as reporters in bacterial mutagenicity tests.

A bacterial plasmid was constructed on which the regulatory region of the umuC gene of Escherichia coli was fused to the coding sequence of the green fluorescent protein gene (gfp) from the jellyfish Aequorea victoria. Escherichia coli AB1157 strains carrying the plasmid emitted fluorescence in the presence of mutagens that induce the SOS DNA repair system. Data on tests with nitrosoguanidine, methylmethane sulphonate and UV radiation (254 nm) are presented. Although fluorescent detection using this system was not as rapid or sensitive as a similar luminescent equivalent (umuC-luxAB), the gfp reporter system was more robust. Escherichia coli umu gene induction was also analysed in Salmonella typhimurium TA1537 cells following plasmid transfer and exposure to the same range of mutagens. There was no significant difference in sensitivity between the two species. These preliminary results will provide the basis for development of mutagenicity test systems useful in the testing of complex mixtures, such as environmental samples, and the investigation of physiological parameters influencing spontaneous mutagenesis in bacteria.

[Selection of drug-labelled Bacillus sphaericus and studies on transformation and expression of foreign DNA].

NTG was used to make chemical mutation for Bacillus sphaericus, RifR and SmR labelled strains were selected, which could resist drug as much as 100 u/ml. The resistance to drug was stably inherited. The RifR strain was used as recipient and the plasmid containing the lysostaphin gene was transfered into it by protoplasts. Results showed that the lysostaphin gene could be expressed stably at high level in Bacillus sphaericus and the lysostaphin activity was about 122 u/ml medium after shaking culture.

Genetic evidence for activation of the positive transcriptional regulator Xy1R, a member of the NtrC family of regulators, by effector binding.

The Xy1R protein positively controls expression from the Pseudomonas putida TOL plasmid sigma 54-dependent Pu and Ps promoters, in response to the presence of aromatic effectors such as m-xylene, m-methylbenzyl alcohol, and p-chlorobenzaldehyde in the culture medium. Xy1R also autoregulates its own synthesis. A mutant Xy1R regulator called Xy1R7 was isolated after nitrosoguanidine mutagenesis of the wild-type gene and phenotypic selection for mutants that had acquired the ability to recognize m-nitrotoluene, a nitroarene that is not an effector for the wild-type regulator. The mutant regulator exhibited a single point mutation that resulted in a change in codon 172 (GAA-->AAA), which should result in a Glu-->Lys change in the polypeptide chain. The effector profile of the mutant regulator was determined by measuring beta-galactosidase from a fusion of the Pu promoter to a promoterless lacZ gene. The results showed that the mutant regulator had acquired the ability to recognize m-nitrotoluene, and retained the wild-type regulator's ability to recognize most of the wild-type effectors. Full transcriptional activation of the Pu promoter by Xy1R7, as with the wild-type Xy1R protein, requires its full modular structure, namely the sigma 54 recognition site, the integration host factor binding site, and the upstream activation sequences. The Xy1R7 regulator did not stimulate transcription from the Ps promoter in response to the presence of its effectors, and autoregulated its own synthesis at low levels.

Mutagenic potential of nitroguanidine and nitrosoguanidine in the Drosophila melanogaster sex-linked recessive lethal assay.

Nitroguanidine (NG) and its degradation product nitrosoguanidine (NSG) were evaluated for their mutagenic potential by using Drosophila melanogaster sex-linked recessive lethal (SLRL) assay. Following 72 h of feeding exposure, NG and NSG at concentrations of 4-8 micrograms ml-1 and 15-20 mg ml-1, respectively, were not mutagenic in the test system. The frequencies of mutations for NG and the negative control were 0.188% and 0.096%, respectively. The frequencies of mutations for NSG and the negative control experiments were 0.049% and 0.05%, respectively. The positive control mutation frequencies were 15% and 17.8% for the two assays. The differences between the mutation frequencies of NG and NSG and their negative controls were not significant.

Different effect of hyperthermia and heat shock on the action of quinolone drugs versus some mutagens against chloroplasts of Euglena gracilis.

Hyperthermia (37 degrees C permanently) and heat shock (42 degrees C for 10 min, and then 27 degrees C) retarded the elimination of chloroplasts from the flagellate Euglena gracilis induced by quinolone antibacterial chemotherapeutics (OA, NA, Cnx, Ofx, Cpfx, Enx, Nfx) in comparison with their action at 27 degrees C. In the case of OA, NA, and Cnx those hyperthermic conditions completely blocked their action against chloroplasts. On the other hand, both temperature regimes accelerated the antichloroplast activity of the mutagens/carcinogens nitrosoguanidine and furylfuramide.

[The induction of stomach cancer in monkeys with N-methyl-N'-nitro-N-nitrosoguanidine]. / Induktsiia raka zheludka u obez'ian N-metil-N'-nitro-N-nitrozoguanidinom.

Gastric tumors were detected in 2 out of 9 apes following MNNG treatment with a total dose of 800-848 mg/kg (40 mg/kg, thrice a month, 49-50 weeks, via gastric probe). The characteristics, course, symptoms and histologic patterns of the tumors proved comparable with similar neoplasms in humans.

[Chemical mutagenesis and the use of indirect enzymatic criteria for the selection of virulent clones of Bacillus thuringiensis]. / Khimicheskii mutagenez i ispol'zovanie kosvennykh énzimaticheskikh kriteriev dlia otbora virulentnykh klonov kul'tury Bacillus thuringiensis.

The effect of the mutagene nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine) on the growth of Bacillus thuringiensis subsp. galleriae st. 69/6 was being studied. It depends on the physiological state of the cells, dose of the mutagene, pH of the culture medium and exposition. Nitrosoguanidine was found to have the maximum mutagenic effect on the vegetative cells at pH 6.2 and on the spores at pH 5.6.

Mutagenesis of anaerobic cultures of Salmonella typhimurium by nitrosoguanidine, diethyl sulfate and 9-aminoacridine.

Despite a previous report to the contrary, anaerobic cultures of Salmonella typhimurium strain LT2 hisG46 are revertible (although to a slightly reduced extent) by both N-methyl-N'-nitro-N-nitrosoguanidine and diethyl sulfate, while anaerobic cultures of a strain carrying the frameshift hisC3076 marker are fully revertible by 9-aminoacridine.

Comparison of the carcinogenic effectiveness in mouse skin of methyl- and ethylnitrosourea, nitrosourethane and nitrosonitro-guanidine and the effect of deuterium labeling.

The carcinogenic activities of a number of directly acting methylating and ethylating agents have been compared by mouse skin painting in acetone solution. Nitrosomethylurethane and nitrosoethylurethane failed to induce tumors after greater than 60 weeks treatment. Nitrosomethylurea was somewhat more effective than nitrosoethylurea, as measured by the longer latent period than nitrosoethylurea, as measured Nitrosomethylnitroguanidine, by the same measure, was a weaker carcinogen than nitrosoethylnitroguanidine at both dose levels used (0.02 M and 0.008 M); the latter compound was the most potent skin carcinogen of those examined. There was no significant difference in carcinogenic effectiveness when the alkyl group of the nitrosoureas or the nitronitrosoguanidines contained deuterium instead of hydrogen, which supports the concept that alkylation of cellular macromolecules by the intact alkyl group is responsible for carcinogenesis by these compounds.