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PubMed1996

The Salmonella sulA-test: a new in vitro system to detect genotoxins.

The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay. The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin). Sodium azide and 5-fluorouracil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.

Bacterial Proteins↗
PubMed1996

Validation of the SOS/umu test using test results of 486 chemicals and comparison with the Ames test and carcinogenicity data.

The present study gives a comprehensive update of all umu genotoxicity assay results published so far. The available data of 486 chemicals investigated with the umu test are compared with the Ames test (274 compounds) as well as rodent carcinogenicity data (179 compounds). On the whole, there is good agreement between the umu test and the Ames test results, with a concordance of about 90%. The umu test was able to detect 86% of the Ames mutagens, while the Ames test (using at least 5 strains) detected 97% of the umu positive compounds. The elimination of TA102 from the set of Ames tester strains reduced the percentage of detectable umu genotoxins from 97 to 86%. The agreement between carcinogenesis and umu response was 65%, which is comparable to earlier studies concerning rodent carcinogenesis and Salmonella mutagenesis. The present compilation of umu results provides a database that can be used for the comparison of the SOS-inducing activity of chemicals and their mutagenicity, respectively, carcinogenicity. The results presented here clearly demonstrate that a chemical which induces the expression of the umu operon can be regarded a rodent carcinogen with a high degree of certainty (93%).

Animals↗
PubMed1996

Mutagenicity of dihydroxybenzenes and dihydroxynaphthalenes for Ames Salmonella tester strains.

The mutagenicity of 3 dihydroxybenzene (DHB) and 9 dihydroxynaphthalene (DHN) isomers was examined by using 5 different Ames Salmonella mutagenicity tester strains in the presence and absence of phenobarbital and 5,6-benzoflavone-treated rat liver S9-mix. Of the 3 DHB isomers, 1,4-DHB (hydroquinone) was mutagenic, and of the 9 DHN isomers, 1,3-DHN (naphthoresorcinol), 1,4-DHN (hydronaphthoquinone), 1,6-DHN and 1,7-DHN were mutagenic. Mutagenicity of all the compounds tested was observed in the absence of S9-mix, while 1,4-DHN and 1,6-DHN were also mutagenic in the presence of S9-mix. The mutagenicity of 1,4-DHB and 1,4-DHN for TA104, which is a strain sensitive to oxidative mutagens, was almost completely or partially inhibited by superoxide dismutase (SOD) and/or catalase, indicating the involvement of activated oxygen species in mutagenesis. Furthermore, from the finding that the 4 DHNs were mutagenic for TA2637, the strain sensitive to frameshift mutagens, it is possible that the mutagenicity of DHNs for S. typhimurium was also attributable to DNA adducts that form with quinones and/or semiquinones through oxidation of DHNs. The mutagenicity of 1,3-DHN, which showed the largest number of revertants in strains TA100, TA98, TA2637 and TA104, was greatly decreased, when their pKM101 plasmid-deficient strains, TA1535, TA1538, TA1537 and TA2659 were used. This observation suggests that an SOS repair system was involved in the mutagenesis of 1,3-DHN for S. typhimurium.

Animals↗
PubMed2002

Green fluorescent protein-based biosensor for detecting SOS-inducing activity of genotoxic compounds.

Increasing levels of environmental pollution demand specific and sensitive methods for detection of genotoxic agents in water, food products and environmental samples. Tests for genotoxicity assessment are often based on biosensor strains that respond to DNA damage induced by chemicals. In the present study, fluorescent reporter Escherichia coli strains have been developed, which contain a plasmid-borne transcriptional fusion between the DNA-damage inducible recA promoter and the green fluorescent protein gene (gfp) or a gene encoding a red-shifted, higher intensity GFP variant (mutant 3). GFP-based biosensors allowed the detection of a dose-dependent response to genotoxic agents such as mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and nalidixic acid (NA). A reporter strain carrying recA'-gfp mutant 3 fusion gave more dramatic and sensitive response than a strain containing the wild-type gfp. These results indicate that recA'-gfp mutant 3-based biosensor is potentially useful for detection of genotoxins.

Biosensing Techniques↗
PubMed2001

The effects of microgravity on induced mutation in Escherichia coli and Saccharomyces cerevisiae.

We examined whether microgravity influences the induced-mutation frequencies through in vivo experiments during space flight aboard the space shuttle Discovery (STS-91). We prepared dried samples of repair-deficient strains and parental strains of Escherichia (E.) coli and Saccharomyces (S.) cerevisiae given DNA damage treatment. After culture in space, we measured the induced-mutation frequencies and SOS-responses under microgravity. The experimental findings indicate that almost the same induced-mutation frequencies and SOS-responses of space samples were observed in both strains compared with the ground control samples. It is suggested that microgravity might not influence induced-mutation frequencies and SOS-responses at the stages of DNA replication and/or DNA repair. In addition, we developed a new experimental apparatus for space experiments to culture and freeze stocks of E. coli and S. cerevisiae cells.

Alkylating Agents↗
PubMed1997

Role of SOS and OxyR systems in the repair of Escherichia coli submitted to hydrogen peroxide under low iron conditions.

There are at least two mechanisms by which H2O2 induces DNA lesions in Escherichia coli: one in the presence of physiological iron levels and the other in low iron conditions. The survival as well as the induction of SOS response in different DNA repair mutant strains of E coli was evaluated after H2O2 treatment under low iron conditions (pretreatment with an iron chelator). Our results indicate that, in normal iron conditions RecA protein has a relevant role in recombination repair events, while in low iron conditions RecA protein is important as a positive regulator of the SOS response. On the other hand, the oxy delta R mutant is sensitive to the lethal effects of H2O2 only in low iron conditions and this sensitivity cannot be correlated with DNA strand breaks.

Adaptation, Physiological↗
PubMed1997

Potent suppressing activity of the non-polyphenolic fraction of green tea (Camellia sinensis) against genotoxin-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002)--association with pheophytins a and b.

Antigenotoxic and antimutagenic activities of green tea extract and tea-derived polyphenols have been studied using in vitro and in vivo experiments. However, antigenotoxic substances in the non-polyphenolic fraction of green tea have been poorly elucidated. In the present study, the effect of the non-polyphenolic fraction of green tea on genotoxin-induced umu C gene expression was analyzed using a tester bacteria, and potent antigenotoxic substances in the non-polyphenolic fraction were identified. The non-polyphenolic fraction of green tea showed strong suppressive activities against umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indol (Trp-P-1) or mitomycin C (MMC) in the presence or absence of S9 metabolizing enzyme mixture. The non-polyphenolic fraction of green tea exhibited major two-color bands in a silica gel TLC and they were identified as chlorophyll-related compounds, pheophytins a and b, judged by their specific colors, Rf values in silica gel TLC and absorption spectra. These pigments showed significant suppressive activities against umu C gene expression in tester bacteria induced by Trp-P- and MMC in a dose-dependent manner. These results suggest that the non-polyphenolic fraction of green tea contains pheophytins a and b as potent antigenotoxic substances.

Antimutagenic Agents↗
PubMed1998

Absence of an SOS-like system in Neisseria gonorrhoeae.

The DNA repair capacities of Neisseria gonorrhoeae have not been well characterised, however, it is known that the gonococcus possesses an excision repair system. The fact that genes in this system are part of the SOS regulon in Escherichia coli prompted this investigation into the transcriptional regulation of genes involved in DNA repair in N. gonorrhoeae. Northern (RNA-DNA) dot blot hybridisation was used to investigate potential DNA damage-mediated induction of the gonococcal recA, uvrA and uvrB genes. In contrast to the situation in E. coli, transcription of these genes in N. gonorrhoeae was not induced in response to treatment with methyl methanesulfonate (MMS) and UV light. These data indicated that the gonococcus does not possess an SOS-like system that is induced in response to DNA damage.

Adenosine Triphosphatases↗
PubMed2000

SOS induction of the recA gene by UV-, gamma-irradiation and mitomycin C is mediated by polyamines in Escherichia coli K-12.

Polyamines are involved in a wide range of cellular metabolism. In this study we investigated the effects of polyamines on the SOS induction of the recA gene by exposure to UV-, gamma-irradiation and mitomycin C employing polyamine-deficient mutant and wild type Escherichia. coli strains carrying recA'::'lacZ transcriptional fusion. In the polyamine-deficient mutant, the induction factor of the recA gene by UV-, gamma-irradiation and mitomycin C-treatment are about 3.0-, 2.5- and 4-fold lower, respectively, than those of the wild type. The exogenous addition of polyamines restored the reduced induction of the recA gene to the wild type level. Tri-amine spermidine effectively restored the recA induction to a level similar to the wild type, while being less restored by the di-amine putrescine. The restoration of recA induction by polyamines may be accomplished in a dose- and charge-dependent manner. Our results strongly suggest that polyamines may play an essential role as the SOS inducing mediator following exposure to damaging agents in E. coli and provide important information that tackles an interesting question in how cells respond to chemical and physical stresses.

DNA Repair↗
PubMed2002

Assessment of the potential genotoxic risk of Phyllantus orbicularis HBK aqueous extract using in vitro and in vivo assays.

Phyllanthus orbicularis HBK is an endemic Cuban plant whose aqueous extract has been proposed as an effective drug for the treatment of viral diseases. In addition, antimutagenic properties of this extract have also been reported. In the present study, the genotoxicity of this plant extract was assessed using different in vitro and in vivo assays. Results from SOS gene induction, gene reversion and conversion, and SMART assays clearly show that P. orbicularis aqueous extract does not induce either primary DNA damage or mutation. Additionally, no statistically significant difference was found in the percentage of chromosomal aberrations in Chinese hamster ovarian (CHO) cells treated with the plant extract. On the contrary, micronuclei and abnormal anaphase were induced by this extract in CHO cells. This genotoxic effect was related to a high cytotoxicity. Single spots were detected in the SMART assay. These results point to a possible aneugenic effect of the P. orbicularis aqueous extract at cytotoxic doses which are much higher than those seen by their antiviral and antimutagenic activities.

Anaphase↗
PubMed1999

The detection and comparison of the genotoxic effects of some nitro aromatic compounds by the umu and SOS chromotest systems.

Four nitroarenes were tested in two standard genotoxicity assay systems using three well-known bacterial tester strains. The results were as follows: 4-nitroquinoline l-oxide (4NQO) was positive in Quillardet and Hofnung's SOS chromotest using Escherichia coli strain PQ37 both in the presence and absence of microsomal (S9) supplements and in the Salmonella typhimurium umu tester strains NM2009 and NM3009, which express high levels of O-acetyltransferase (O-AT) and O-AT plus nitroreductase (NR) respectively. m-Nitrocinnamic acid (m-NCA) was weakly positive in strains NM2009 and NM3009, but negative in the SOS chromotest; m-dinitrobenzene (m-DNB) was weakly positive in strain NM2009, intermediate positive in strain NM3009, but negative in the SOS chromotest; 2,4-dinitrotoluene (2,4-DNT) was weakly positive in strain NM3009, but negative in strain NM2009 and in the SOS chromotest. However it still showed a dose-response relationship in strain NM2009. In view of these results, it is suggested that investigators planning to screen miscellaneous nitroarenes for their genotoxicity in the future should consider taking advantage of the increased sensitivity which is conferred on S. typhimurium strains NM2009 and NM3009 by virtue of their capacity to overexpress O-AT or O-AT and NR.

4-Nitroquinoline-1-oxide↗
PubMed2000

Mutagenicity and genotoxicity of sorbic acid-amine reaction products.

Sorbic acid as well as potassium and calcium sorbate (E202 and E203) are legally used as preservatives in numerous processed foods. Owing to its system of conjugated double bonds, sorbic acid is likely to undergo a nucleophilic attack, which may turn it into mutagenic products. The cyclic derivatives resulting from a double addition reaction between sorbic acid and various amines at two different temperatures (50 degrees C and 80 degrees C) have been analysed. A genotoxicity study has been performed with HeLa cells and plasmid DNA. A mutagenesis study has been carried out by using the Ames test. A SOS spot test and a cytotoxicity study have been realised as well. The results showed that the products involved exhibited neither mutagenic nor genotoxic activities.

Amines↗
PubMed2000

Participation of stress-inducible systems and enzymes involved in BER and NER in the protection of Escherichia coli against cumene hydroperoxide.

We studied the participation of the stress-inducible systems, as the OxyR, SoxRS and SOS regulons in the protection of Escherichia coli cells against lethal effects of cumene hydroperoxide (CHP). Moreover, we evaluated the participation of BER and NER in the repair of the DNA damage produced by CHP. Our results suggest that the hypersensitivity observed in the oxyR mutants to the lethal effect of CHP does not appear to be due to SOS inducing DNA lesions, but rather to cell membrane damage. On the other hand, DNA damage induced by CHP appears to be repaired by enzymes involved in BER and NER pathways. In this case, Fpg protein and UvrABC complex could be involved cooperatively in the elimination of a specific DNA lesion. Finally, we have detected the requirement for the uvrA gene function in SOS induction by CHP treatment.

Antimutagenic Agents↗
PubMed2001

The SOS-dependent upregulation of uvrD is not required for efficient nucleotide excision repair of ultraviolet light induced DNA photoproducts in Escherichia coli.

We have shown previously that induction of the SOS response is required for efficient nucleotide excision repair (NER) of the major ultraviolet light (UV) induced DNA lesion, the cyclobutane pyrimidine dimer (CPD), but not for repair of 6-4 photoproducts (6-4PP) or for transcription-coupled repair of CPDs [1]. We have proposed that the upregulation of cellular NER capacity occurs in the early stages of the SOS response and enhances the rate of repair of the abundant yet poorly recognized genomic CPDs. The expression of three NER genes, uvrA, uvrB, and uvrD, is upregulated as part of the SOS response. UvrD differs from the others in that it is not involved in lesion recognition but rather in promoting the post-incision steps of NER, including turnover of the UvrBC incision complex. Since uvrC is not induced during the SOS response, its turnover would seem to be of great importance in promoting efficient NER. Here we show that the constitutive level of UvrD is adequate for carrying out efficient NER of both CPDs and 6-4PPs. Thus, the upregulation of uvrA and uvrB genes during the SOS response is sufficient for inducible NER of CPDs. We also show that cells with a limited NER capacity, in this case due to deletion of the uvrD gene, repair 6-4PPs but cannot perform transcription-coupled repair of CPDs, indicating that the 6-4PP is a better substrate for NER than is a CPD targeted for transcription-coupled repair.

Adenosine Triphosphatases↗
PubMed2001

Transcriptional induction of repair genes during slowing of replication in irradiated Saccharomyces cerevisiae.

We investigated the inhibition of cell-cycle progression and replication and the induction of the transcriptional response in diploid budding yeast populations exposed to two different doses of gamma-rays resulting in 15 and 85% survival respectively. We studied the kinetics of the cellular response to ionizing treatment during the period required for all of the surviving cells to achieve at least one cell division. The length of these periods increased with the dose. Irradiated populations arrested as large-budded cells containing partially replicated chromosomes. The extent of the S-phase was proportional to the amount of damage and lasted 3 or 7h depending on the irradiation dose. In parallel to the division study, we carried out a kinetic analysis of the expression of 126 selected genes by use of dedicated microarrays. About 26 genes were induced by irradiation and displayed various pattern of expression. Interestingly, 10 repair genes (RAD51, RAD54, CDC8, MSH2, RFA2, RFA3, UBC5, SRS2, SPO12 and TOP1), involved in recombination and DNA synthesis, display similar regulation of expression in the two irradiated populations. Their pattern of expression were confirmed by Northern analysis. At the two doses, the expression of this group of genes closely followed the extended replication period, and their expression resumed when replication restarted. These results suggest that the damage-induced response and DNA synthesis are closely regulated during repair. The analysis of the promoter regions indicates a high occurrence of the three MCB, HAP and UASH regulatory boxes in the promoters of this group of genes. The association of the three boxes could confer an irradiation-replication specific regulation.

Blotting, Northern↗
PubMed1998

Influence of the UV-activated SOS response on the gamma-radiation-induced mutation spectrum in the lacI gene.

Previous studies of our group have shown that intracellular or extracellular gamma-irradiation of the lacI gene results in different mutational spectra. One cause for these differences might be the error-prone SOS response, which is activated in the intracellular situation by gamma-irradiation but not in the extracellular situation. Since UV-radiation is a well-established strong inducer of the SOS response, we used bacterial host cells, pretreated with UV-light to study the influence of the SOS response on the gamma-radiation-induced mutation spectrum in the lacI gene in the extracellular situation. If the SOS response was activated, mutations on A:T base pairs and frameshift mutations accounted for 16% and 12% of all mutations, respectively, but they were hardly detected in the absence of an induced SOS response. G:C to T:A transversions increased from 14% to 24% in the presence of an activated SOS response. We can therefore conclude from this study, that SOS-induction of host cells by UV-light influences the extracellular mutation spectrum in the lacI gene, with respect to mutations on A:T base pairs, G:C to T:A transversions and frameshift mutations. This conclusion is supported by the fact that the previously obtained intracellular gamma-radiation-induced mutation spectrum in the lacI gene, in which the SOS response is also involved, shows great similarities with the extracellular mutation spectrum in the presence of an activated SOS response in this study.

Bacterial Proteins↗
PubMed1999

SOS-dependent A-->G transitions induced by hydroxyl radical generating system hypoxanthine/xanthine oxidase/Fe3+/EDTA are accompanied by the increase of Fapy-adenine content in M13 mp18 phage DNA.

Gas chromatography/isotope dilution-mass spectrometry with selected ion monitoring (GC/IDMS-SIM) was used to measure oxidised bases in hypoxanthine/xanthine oxidase/Fe3+/EDTA modified ss M13 mp18 phage DNA. A dose-dependent increase of oxidised bases content in DNA was observed with the biggest augmentation of FapyGua, thymine glycol and FapyAde. The amount of 8-OH-Gua was relatively high both in non-oxidised and oxidised DNA, and increased to the same extent as FapyAde and ThyGly. DNA oxidation caused a dramatic decrease in phage survival after transfection to E. coli. Survival was improved 2.8-fold after induction of the SOS system by UV irradiation of bacteria and mutation frequency of the lacZ gene in SOS conditions increased 7-fold over that in non-irradiated bacteria. Spectrum of mutations was different from those reported previously and mutations were distributed rather randomly within M13 lacZ sequence, which was in contrast to previous findings, where with non-chelated metal ions other types of mutations were found in several clusters. Thus, conditions of DNA oxidation and accessibility of metal ions for DNA bases might be important factors for generating different DNA damages and mutations. Major base substitutions found both in SOS-induced and non-induced E. coli but with higher mutation frequency in SOS-induced cells were C-->A (approximately 20-fold increase in SOS-conditions), G-->A (9-fold increase) and G-->C (4.5-fold increase). Very few G-->T transitions were found. A particularly large group of A-->G transitions appeared only in SOS-induced bacteria and was accompanied by augmentation of FapyAde content in the phage DNA with undetectable 2-OH-Ade. It is then possible that imidazole ring-opened adenine mimics guanine during DNA replication and pairs with cytosine yielding A-->G transitions in SOS-induced bacteria.

Bacteriophage M13↗
PubMed2002

Inhibition of 4-nitroquinoline-1-oxide genotoxicity by Bacillus strains.

The effect of 16 Bacillus strains from pharmaceutical probiotic preparations (Bacillus spp.) and collection (B. subtilis, B. firmus, B. megaterium, B. pumilus) on genotoxicity induced by the known mutagen 4-nitroquinoline-1-oxide (4-NQO) was studied using the short-term bacterial assay SOS-chromotest. with Escherichia coli PQ37 as the tester organism. It was found that the activity of 0.1 mM 4-NQO was reduced (P < 0.01) after coincubation with Bacillus suspensions (10(8) CFU/ml for 150 min at 37 degrees C). All isolates showed potential for deactivating 4-NQO, and genotoxicity inhibition ranged from 92.9 to 100%. There were no appreciable differences in behaviour observed among probiotic and collection strains or in relation to species. The observed antigenotoxicity was associated with a clear-cut modification of 4-NQO molecular characteristics.

4-Nitroquinoline-1-oxide↗