The geptrong pharmaceutical product increases efficiency of postreplication repair of permutation intermediates in yeast Saccharomyces cerevisiae

Geptrong is a medication from pure defermentated honey. In medical practice, it is used as hepatoprotector. Genotoxicity analysis revealed antimutagenic activity of the preparation. The spontaneous mutation rate at the ADE4-ADE8 and CAN1 loci was several times lower in case that the yeast cells were plated on the geptrong-containing medium, and the mutation rate was scored using the method of ordered plating. If spontaneous mutation rate was measured by means of the fluctuation method of median, no antimutagenic activity was detected. Geptrong had no effect on the yeast cell survival. At the same time, it substantially decreased the frequency of direct mutations at the ADE4-ADE8 locus, induced by UV-and gamma-radiation, and ethylmetansulfonate. The effect of the geptrong antimutagenic activity on the level of UV-induced mutagenesis in the yeast strains defective for the repair systems rad2, rad51, rad54, rad59, msh2, and hsm3 was examined. Antimutagenic activity was detected in the wild type, as well as in the rad2, rad54, rad59, and hsm3 strains, while rad51, pms1, and msh2 mutants lacked this activity. Based on these data, it is suggested that antimutagenic effect of geptrong is associated with activated repair of mismatches, appeared during the postreplicative recombination repair.     

Differential antimutagenic effects of caffeine and the protease inhibitor antipain on mutagenesis by various mutagens in Escherichia coli

The effects of caffeine (2 mg/ml) and the protease inhibitor antipain (1.75 mb/ml) in the plating agar medium on the yields of prototrophic revertants induced by 10 mutagens in E. coli uvrA^? strains were tested. Mutagenesis by 4-nitroquinoline 1-oxide was greatly diminished by both caffeine and antipain. UV mutagenesis was decreased moderately by caffeine, and greatly by antipain. X-Ray mutagenesis was decreased very slightly by both caffeine and antipain. Mutagenesis by N-hydroxyurethan was inhibited moderately by caffeine, and greatly by antipain; that by methyl methanesulfonate was inhibited moderately by both caffeine and antipain, and that by N-methyl-N′-nitro-N-nitrosoguanidine wa was not suppressed by caffeine but was inhibited moderately by antipain. Mutagenesis by ethyl methanesulfonate was inhibited greatly by caffeine, but only slightly by antipain. The antimutagenic effect of caffeine was strong on furylfuramide (AF-2) mutagenesis, moderate on that of mitomycin C (tested with B/r type strain) and negligible on that of N-methyl-N-nitrosourea. These diverse antimutagenesis patterns are briefly discussed in relation to the current idea that antipain antimutagenesis is due to inhibition of inducible error-prone repair.     

Inhibition of Induced Mutagenesis in Salmonella typhimurium by the Protein of Propionibacterium freudenreichii subsp. shermanii

Culture liquid and cells of Propionibacterium freudenreichii subsp. shermanii VKM-103 exerted a strong antimutagenic effect on mutations induced by 4-nitroquinoline-1-oxide, N-methyl-N′-nitro-N′-nitrosoguanidine, sodium azide (base pair substitutions) and 9-aminoacridine (frameshift mutations). No inhibitory effect was observed against mutagenesis induced by 2-nitrofluorene (frameshift mutations). The highest antimutagenic activity was found in the culture liquid of cells grown for 24 h. Acetic and propionic acids of the culture liquid produced by propionibacteria made no observable contribution to the antimutagenicity. Antimutagenic activity of the culture liquid was considerably reduced by protease treatment and by heating at 92°C for 10 min. Upon dialysis, the culture liquid lost almost all of its inhibitory activity. Cell wash solution also contained high antimutagenic activity which was lost upon protease treatment and dialysis. According to the exclusion limit of the dialysis bag, the molecular weight of the antimutagenic factor, presumably a protein, is less than 1.5 kDa. In addition, the cells of P. shermanii were capable of binding or modifying the mutagens, thereby decreasing their mutagenicity.     

Repair of Irradiated Transforming Deoxyribonucleic Acid in Wild Type and a Radiation-Sensitive Mutant of Micrococcus radiodurans

The survival of biological activity in irradiated transforming deoxyribonucleic acid (DNA) has been assayed in the wild type and a radiation-sensitive mutant of Micrococcus radiodurans. The frequency of transformation with unirradiated DNA was lower in the mutant to about the same extent as the mutant's increased sensitivity to radiation. However, in both the wild type and the mutant, the irradiated DNA that was incorporated into the bacterial genome was repaired to the same extent as determined by the loss of transforming activity with increasing radiation dose. This applied to DNA irradiated either with ionizing or ultraviolet (UV) radiation. The rate of inactivation of biological activity after UV radiation was the same in any of the DNA preparations tested. For ionizing radiation, the rate of inactivation varied up to 40-fold, depending on the DNA preparation used, but for any one preparation was the same whether assayed in the wild type or the radiation-sensitive mutant. When recipient bacteria were irradiated with ionizing or UV radiation immediately before transformation, the frequency of transformation with unirradiated DNA fell, rapidly and exponentially in the case of the sensitive mutant but in a more complicated fashion in the wild type. The repair of DNA irradiated with ionizing radiation was approximately the same whether assayed in unirradiated or irradiated hosts. Thus, irradiation of the host reduced the integration of DNA but not its repair.     

Antimutagenic effects of para-aminobenzoic acid in experiments with bacterial cells. Noninterference in the mutation process controlled by plasmid pKM101

The dependence of expression of PABA antimutagenic action in bacterial cells on the character of genetic control of the mutagenic process was studied. PABA antimutagenic activity was largely connected with the negative control of SOS repair which is controlled by bacterial cell genes, but not by pKM101 plasmid genes. These results are in agreement with the idea that the systems of repair and mutagenesis specified by cell genome and plasmids are not identical.     

TheisfA mutation inhibits mutator activity and processing of UmuD protein inEscherichia coli recA730 strains

Further studies on theisfA mutation responsible for anti-SOS and antimutagenic activities inEscherichia coli are described. We have previously shown that theisfA mutation inhibits mutagenesis and other SOS-dependent phenomena, possibly by interfering with RecA coprotease activity. TheisfA mutation has now been demonstrated also to suppress mutator activity inE. coli recA730 andrecA730 lexA51(Def) strains that constitutively express RecA coprotease activity. We further show that the antimutator activity of theisfA mutation is related to inhibition of RecA coprotease-dependent processing of UmuD. Expression of UmuD' from plasmid pGW2122 efficiently restores UV-induced mutagenesis in therecA730 isfA strain and partially restores its mutator activity. On the other hand, overproduction of UmuD'C proteins from pGW2123 plasmid markedly enhances UV sensitivity with no restoration of mutability.     

Antimutagenic effect of volatile decomposition products from thermally oxidized linoleate

The antimutagenic effect of volatile decomposition products from thermally oxidized linoleate on mutagenesis by UV irradiation was investigated in Escherichia coli B/r WP2. When added to an agar medium, these products greatly reduced the number of Trp+ revertants. The same antimutagenic effect was observed by acrolein, 2-hexenal, 2-heptenal, 2-nonenal and 2,4-decadienal; these unsaturated aldehydes were components of volatile products.     

Optimization of submerged fermentation conditions for immunosuppressant mycophenolic acid production by Penicillium roqueforti isolated from blue-molded cheeses: enhanced production by ultraviolet and gamma irradiation

Mycophenolic acid (MPA) is a promising drug owing to its immunosuppressive and biological activities. In this study, two strains of Penicillium roqueforti designated as AG101 and LG109 were selected among several strains isolated from Roquefort cheese samples on the basis of their activity for MPA-producing ability. The appropriate fermentation conditions necessary for MPA biosynthesis by the two respective fungal strains were investigated. These conditions included selection of the cultivation medium, agitation rate, incubation temperature, fermentation time, pH value, inoculum size, and fermentation medium volume. Maximum MPA productivities were maintained when the fermentation process was carried out using a medium composed of (g l^?1): Sucrose, 30; peptone, 5.0; KH₂PO₄, 1.0; MgSO₄·7H₂O, 0.5 and KCl, 0.5; pH 6.0, inoculated with an inoculum size of 6.0 % (v/v), and incubated at 25 °C for 10 days at 120 rpm. The potentiality of both P. roqueforti strains for further improvement of MPA production was applied by mutagenesis through exposure to irradiation by ultraviolet rays (UV, 254 nm) for different periods of time and gamma rays at various doses (KGy). The dry cell weight of both irradiated fungal strains showed a greater reduction when irradiated either with UV or gamma rays. However, the MPA yield of both strains was increased by 1.27–1.39 fold when irradiated with UV rays and by 2.11–2.33 fold when irradiated with gamma rays, as compared with the respective controls (non-irradiated cultures). These findings indicate the future possibility to reduce the cost of producing fermentation-based drugs.     

Ginseng extract exhibits antimutagenic activity against induced mutagenesis in various strains of Salmonella typhimurium

Ginseng has been reported to exhibit antioxidant and antimutagenic activity. The present study was undertaken with a view to confirm whether the antioxidant activity of Ginseng is responsible for its antimutagenic action. The concentrated root extract of Panax ginseng (Ginseng extract I) and its lyophilized powder (Ginseng extract II) obtained from two different manufacturing houses, were tested against mutagenesis using the well-standardized Ames microsomal test system. The extracts exhibited antimutagenic effect against hydrogen peroxide induced mutagenesis in TA100 strain, and against mutagenesis produced by 4-nitroquinoline-N-oxide in both TA98 and TA100 strains of Salmonella typhimurium. Both the extracts failed to show any antimutagenic potential against tert-butyl hydroperoxide (an oxidative mutagen) in TA102 strain, a strain highly sensitive to active oxygen species. The extracts also indicated a weak antioxidant activity in a series of in vitro test systems viz., 1,1-diphenyl picryl hydrazyl (DPPH) assay, hydrogen peroxide scavenging and superoxide anion scavenging. The results indicate that the protective effects shown by ginseng extract(s) against 4-nitroquinoline-n-oxide and hydrogen peroxide induced mutagenesis in TA98 and TA100 could mainly be due to its property to initiate and promote DNA repair rather than free radical scavenging action.     

Plasmid pKM101-dependent repair and mutagenesis in Escherichia coli cells with mutations lexB30 tif and zab-53 in the recA gene

Bacterial survival after UV irradiation was increased in E. coli K12 lexB30 and tif zab-53 mutants harboring plasmid pKM101. Mutagenesis in response to UV was observed in these bacteria which, in absence of pKM101, are not UV-mutable. The mutator effect observed in unirradiated wild-type cells containing pKM101 was higher after incubation at 30°C with adenine than at 37°C. This effect was still enhanced by tif mutation, even in the tif zab-53 strain, but it was abolished by lexB30 mutation. In the tif zab-53 (pKM101) strain, repair and mutagenesis of UV-irradiated phage λ was observed, but not in the lexB30 mutant carrying pKM101. The pKM101 mutant, pGW1, was unable to protect tif zab-53 bacteria against killing by UV, whereas the protection of lexB30 was intermediate; moreover, it did not promote the mutator effect at 30°C or enhance phage repair and mutagenesis in tif zab-53 cells. All UV-induced bacterial mutations in lexB30 (pKM101) strain were suppressors; in contrast, true revertants were found after UV irradiation of the tif zab-53 (pKM101) cells.We suggest that the constitutive activity of RecA protein is enough for the production of UV-promoted suppressor mutations, whereas true reversions require a more active form of this protein which could exert its effects directly or by acting at a regulatory level on other cellular functions.     

The interrelation of the antimutagenic action of mannitol to its effect on cellular metabolic processes]

The mannitol influence on mutagenesis of ionizing radiation and cyclophosphate has been studied in albino mongrel rats using the methods of genetic and biochemical analysis. N correlation is determined between antimutagenic action of this preparation and a decrease of malondialdehyde content in cells and free fractions of matrix lysosomes (beta-galactosidase; N-acetyl-beta-D-glucosaminidase) and firmly membrane-structurized microsomal (glucose-6-phosphatase) enzymes, whose level increases under the influence of mutagens. It is shown that, one of the way of antimutagenic actions of mannitol is connected with mutagenesis correction at the stage of origin of mutagenic products and their transport to chromosome DNA.     

Effects of vanillin and o-vanillin on induction of DNA-repair networks: modulation of mutagenesis in Escherichia coli

Vanillin and its isomer o-vanillin have an effect on the adaptive and SOS responses, as well as mutagenesis, induced in Escherichia coli by N-methyl-N-nitrosourea (MNU) and UV irradiation, potentiating in some cases and suppressing in others. o-Vanillin markedly inhibited the MNU-induced adaptive response, while both vanillins potentiated the UV-induced SOS response. These phenomena appear to be responsible for the comutagenic or antimutagenic role of these chemicals in MNU and UV mutagenesis.     

Post-replication repair and recombination in uvrA umuC strains of Escherichia coli are enhanced by vanillin, an antimutagenic compound

Effects of vanillin on UV killing of umuC mutant strains of E. coli were investigated in order to analyze the antimutagenic role of vanillin in mutagenesis. UV-irradiated uvrA umuC cells showed higher survival when plated on medium containing vanillin rather than medium without vanillin. This increased survival associated with exposure to vanillin was observed more clearly in uvrA umuC lexA(Ind-) and uvrA umuC recF strains. However, the effect was inhibited by additional recB recC mutations and completely blocked by an additional recA mutation. As far as tested the increased survival of UV-treated cells by vanillin was dependent on a capacity for genetic recombination. The effect of vanillin on recombination frequency between 2 plasmid DNA, pATH4 (Cmr Tcs) and pBMX7 (Apr Tcs), in a uvrA umuC background was investigated. A significantly higher frequency of plasmid recombination was observed when vanillin was present in the culture medium. These findings suggest that the antimutagenic effect of vanillin is the result of enhancement of a recA-dependent, error-free, pathway of post-replication repair.     

Antimutagenic Effects of Autoxidized Linoleic and Oleic Acids on UV-Induced Mutagenesis in Escherichia coli

The antimutagenic effects of autoxidized linoleic and oleic acids on mutagenesis by UV irradiation were investigated in Escherichia coli B/r WP2 and WP2s uvrA. When added to an agar medium, these autoxidized acids greatly reduced the number of Trp⁺ revertants without significant effects on survival in WP2, but no such effect was observed with WP2s uvrA. The presence of autoxidized linoleic acid decreased the survival of WP2s uvrA greatly and CM571 recA somewhat. It thus appears that the autoxidized unsaturated fatty acid has antimutagenic effects on the wild type strain and lethal effects on the genetic repair-deficient strains.     

Antimutagenic effects of cinnamaldehyde on chemical mutagenesis in Escherichia coli

Antimutagenic effects of cinnamaldehyde on mutagenesis by chemical agents were investigated in Escherichia coli WP2 uvrA- trpE-. Cinnamaldehyde, when added to agar medium, greatly reduced the number of Trp+ revertants induced by 4-nitroquinoline 1-oxide (4-NQO) without any decrease of cell viability. This antimutagenic effect could not be explained by inactivation of 4-NQO caused by direct interaction with cinnamaldehyde. Mutagenesis by furylfuramide (AF-2) was also suppressed significantly. Mutations induced by methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) were slightly inhibited. However, cinnamaldehyde was not at all effective on the mutagenesis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Two derivatives of cinnamaldehyde, cinnamyl alcohol and trans-cinnamic acid, did not have as strong antimutagenic effects on 4-NQO mutagenesis as cinnamaldehyde had. Because cinnamaldehyde showed marked antimutagenic effects against mutations induced by UV-mimic mutagens but not those induced by MNNG or EMS, it seems that cinnamaldehyde might act by interfering with an inducible error-prone DNA repair pathway.     

Comparative study of the antimutagenic potential of Vitamin E in different E. coli strains

The antimutagenic potential of Vitamin E due to its antioxidative properties was studied. The new Escherichia coli K12 assay-system designed in our laboratory was employed in order to detect the antimutagenic potential of Vitamin E and to determine its molecular mechanisms of action. The assay is composed of three tests. In Test A, we examine the influence of the antioxidant on induced oxidative mutagenesis in a repair-proficient strain. Spontaneous mutagenesis is monitored in Test B, which is performed with two mutator strains, one mismatch repair-deficient (mutS) and another deficient in 8-oxo-dGTP-ase activity (mutT). In Test M, a repair-proficient strain and its mismatch repair-deficient counterpart (mutH), both carrying a plasmid with microsatellite sequences, are used to measure the level of microsatellite instability. To examine the antimutagenic potential of Vitamin E we also used the WP2 antimutagenicity test. Protective properties of Vitamin E against oxidative mutagenesis were detected in all tests with the E. coli K12 assay-system as well as in the WP2 antimutagenicity test. This study confirms that mismatch repair is essential for repair of oxidative DNA damage. The results obtained indicate that Vitamin E prevents the formation of DNA adducts by lipid peroxidation products rather than those formed by direct oxidation of DNA bases. Moreover, it can reduce microsatellite instability. After further validation, the new E. coli K12 assay-system can be used to test the antimutagenic potential of antioxidants.     

Partial suppression of the LexA phenotype by mutations (rnm) which restore ultraviolet resistance but not ultraviolet mulability to Escherichia coli B/r uvrA lexA

In Escherichia coli, lexA mutations eliminate expression of UV-inducible functions, causing pleiotropic effects which include sensitivity to ultraviolet (UV) light and loss of UV mutability. Selection for UV resistance, after 5-bromouracil (BU) treatment of E. coli B/r uvr A lexA-102, has yielded derivatives more resistant than lexA but still refractory to UV mutagenesis. The mutation responsible for the UV-resistant UV-nonmutable phenotype (rnm) is cotransducible with malB to about the same extent as is lexA-102 and is tightly linked to lexA-102 in at least one strain. The rnm mutation may therefore be an intragenic partial suppressor of the LexA phenotype. In addition to increased UV resistance and lack of UV mutability, rnm strains show improved ability to perform postreplication repair and to control postirradiation DNA degration compared to the lexA parent. We ascribe the properties of rnm mutants to their having reacquired control of Exonuclease V activity without having reacquired UV-inducible error-prone postreplication repair. We relate our results to current interpretations of UV mutagenesis and to models of coordinate regulation of UV-inducible functions.     

Replication of Damaged DNA and the Molecular Mechanism of Ultraviolet Light Mutagenesis

Abstract

On UV irradiation of Escherichia coli cells, DNA replication is transiently arrested to allow removal of DNA damage by DNA repair mechanisms. This is followed by a resumption of DNA replication, a major recovery function whose mechanism is poorly understood. During the post-UV irradiation period the SOS stress response is induced, giving rise to a multiplicity of phenomena, including UV mutagenesis. The prevailing model is that UV mutagenesis occurs by the filling in of single-stranded DNA gaps present opposite UV lesions in the irradiated chromosome. These gaps can be formed by the activity of DNA replication or repair on the damaged DNA. The gap filling involves polymerization through UV lesions (also termed bypass synthesis or error-prone repair) by DNA polymerase III. The primary source of mutations is the incorporation of incorrect nucleotides opposite lesions. UV mutagenesis is a genetically regulated process, and it requires the SOS-inducible proteins RecA, UmuD, and UmuC. It may represent a minor repair pathway or a genetic program to accelerate evolution of cells under environmental stress conditions.     

Temperature-dependent antimutagenic activity of acrolein in Escherichia coli

The effect of temperature on the antimutagenic activity of acrolein was investigated using UV-irradiated E. coli B. When incubated at lower temperatures (30°C or 37°C), acrolein greatly reduced the mutation frequency in WP2 (wild-type strain), but no such effect was observed with WP2s and ZA159 (excision repair-deficient strains). The antimutagenic activity of acrolein increased when cells were incubated at higher temperatures (40°C or 42°C). Particularly in excision repair-deficient strains, the antimutagenic activity was observed only at higher temperatures. In heat shock response-deficient background, however, the antimutagenic activity was observed at 30°C even in the excision repair-deficient strains.     

Antimutagenesis by factors affecting DNA repair in bacteria

The term 'antimutagen' was originally used to describe an agent that reduces the apparent yield of spontaneous and/or induced mutations, regardless of the mechanisms involved. The 'antimutagens' include 'desmutagens' and 'bio-antimutagens'. In this article, our attention was focused on the bio-antimutagens affecting DNA repair in bacteria. Cobaltous chloride reduced the frequency of mutations in Escherichia coli induced by MNNG. The possibility that metal compound inhibits the growth of mutagen-treated cells was examined. The results clearly showed that the antimutagen surely reduces the mutation rate. The target of cobaltous chloride was found to be cellular factors including Rec A. Vanillin and cinnamaldehyde had strong antimutagenic activities against UV, 4NQO and AF-2. They stimulated Rec A-dependent recombination repair functions in the mutagen-treated cells. Among plant materials, tannins possess antimutagenic activity against UV-induced mutations in E. coli. It has been found that tannic acid stimulates the excision repair encoded by the uvrA gene thereby reducing the yield of mutants. Substances which are antimutagenic in bacterial systems also had antimutagenic activity in cultured mammalian cell systems. Vanillin reduced the frequency of mutagen-induced chromosomal aberrations.