Antimutagenic effect of garlic (Allium sativum L.) on 4NQO-induced mutagenesis in Escherichia coli WP2

Aqueous extract prepared from garlic bulbs markedly suppressed the mutagenesis in both E. coli WP2 trp- and E. coli WP2 trp- uvrA- induced by 4-nitroquinoline 1-oxide (4NQO), but not that induced by UV. Cellular toxicity, inhibition of the expression of the Trp+ phenotype and delay of the first cell division after 4NQO treatment were not observed in the presence of the extract. Since the extract showed identical antimutagenic effects against 4NQO in both test strains but no effect on the mutagenesis of UV, it seems that the extract might act by inactivating the electrophilic group(s) of 4NQO or inhibiting its metabolic activation.     

Evaluation of drug candidates in a battery of short-term genetic toxicology assays: overview

2-Hydroxy-3-methoxybenzaldehyde (omicron-vanillin), the antimutagenic effect of which has been reported on mutagenesis induced by 4-nitroquinoline 1-oxide (4NQO) in Escherichia coli WP2s, enhanced N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis in the same strain. A remarkable enhancement of mutagenesis provoked by N-methyl-N-nitrosourea (MNU) was also observed by the addition of omicron-vanillin. No enhancing effect was observed on mutagenesis induced by other mutagens such as methyl methanesulfonate (MMS), dimethylsulfate, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate, diethylsulfate, 4NQO and furylfuramide (AF-2). On the contrary, omicron-vanillin greatly suppressed AF-2- and 4NQO-induced mutagenesis and showed a slight suppressing effect against mutagenesis induced by MMS, ENNG and ENU. One possible explanation for the enhancing effect of omicron-vanillin on the mutagenesis induced by MNNG or MNU in E. coli WP2s may be inhibition of an inducible adaptive response. Among 7 derivatives of omicron-vanillin, 2-hydroxy-3-ethoxy-benzaldehyde, omicron-hydroxybenzaldehyde and m-methoxybenzaldehyde showed an enhancing effect on MNNG-induced mutagenesis.     

The repair of large DNA adducts in mammalian cells.

This paper describes experiments involving the measurement of DNA damage and repair after treatment with 4-nitroquinoline 1-oxide (4NQO) or aflatoxin B1 (AFB1) epoxide in a number of mammalian cell cultures primarily associated with defects in the excision repair of UV-induced DNA damage. The results with transformed derivatives of XP cells belonging to different complementation groups showed that the extent of repair of 4NQO adducts at the N2 or C8 of guanosine did not correlate to the extent of repair reported by others after UV-irradiation. An examination of 4NQO repair in rodent UV-sensitive cell lines from different ERCC groups indicated that again there was little correlation between the extent of 4NQO and UV repair. However, regardless of complementation group those mutants that were defective in the repair of pyrimidine dimers and 6,4-photoproducts did exhibit a reduced ability to repair the 4NQO N2 guanosine adduct, whereas those mutants defective in pyrimidine dimer repair alone were able to repair this lesion as normal. In all of these cell lines there was a normal capacity to repair the 4NQO C8 guanosine adduct. Less extensive experiments involving AFB1 epoxide showed an XPC-transformed cell line was able to repair 40% of lesions after 6 h, whereas only 20% of repair is seen after UV. The rodent mutant V-C4 which belongs to the same ionising radiation group as irs2, was partially defective in repairing AFB1-induced damage. These experiments highlight the fact that although there are many commonalities between the repair of UV damages and lesions classed as large DNA adducts differences clearly exist, the most striking example here being the repair of the C8 guanosine 4NQO adduct which rarely correlates with a defect in UV repair.     

Comparative analysis of deletion and base-change mutabilities of Escherichia coli B strains differing in DNA repair capacity (wild-type, uvrA-, polA-, recA-) by various mutagens.

Dose-response curves were compared for deletions [ColBR (resistant to colicin B) mutations being more than 80% deletions] and base changes (reversion of argFam to prototrophy argplus) induced in the same set of E. coli strains (wild-type for DNA repair, uvrA-, polA- and recA-) by N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), hydroxylamine (HA), 4-nitroquinoline I-oxide (4NQO), mitomycin C (MTC, UV and X-rays. All these agents induced deletions as well as base changes in the wild-type strain. Thus chemical mutagenesis differed in E. coli and bacteriophages in vitro, for HA, NTG, EMS and perhaps UV produced only point mutations in phage Tr. The patterns of deletion and base-change mutability in E. coli were surprisingly similar. (I) The recombination less recA- strain was mutable by only three (NTG, EMS, HA) of the seven mutagens for either deletions or base changes. (2) The uvrA- strain, unable to excise pyrimidine dimers, was very highly mutable by 4NQO and UV but immutable by MTC for both deletions and base changes. (3) The polA- strain, defective in DNA polymerase I due to a non-suppressible mutation, was very highly mutable by HA and highly mutable by MTC and 4NQO for both deletions and base changes but was highly mutable only for deletions by UV and X-rays, remaining normally mutable by the other agents for both deletions and base changes despite its high sensitivity to their inactivating action. We conclude that errors in the recA-dependent repair of induced DNA damage (after 4NQO, MTC, UV and X-rays) or errors in replication enhanced by damage to the replication system or to the template strands (after NTG, EMS, and HA) give rise to deletions as well as to base changes. From a comparative analysis of 14 dose-response curves for deletions and base changes, we conclude that the order of mutagenic efficiency relative to killing is (EMS, NTG) greater than (UV, 4NQO) greater than HA greater than (X-rays, MTC), and that X-rays, 4NQO, HA and MTC induce more ColBR deletions than Argplus base changes, whereas UV and EMS induce ColBR deletions and Argplus base changes at nearly equal rates and the specificity of NTG is intermediate between these two types.     

Organ-specific DNA damage induced in mice by the organotropic carcinogens 4-nitroquinoline 1-oxide and dimethylnitrosamine.

The extent of DNA fragmentation induced in lung, kidney, and liver of mice injected with the chemical carcinogens 4-nitroquinoline 1-oxide (4NQO), dimethylnitrosamine (DMN) and the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO) was estimated by the alkaline sucrose gradient technique. A floating of minced lung tissue pieces in the alkaline lysing solution on top of the gradients afforded a gentle method of lung DNA extraction. This technique minimized mechanical shearing of lung DNA and permitted comparisons to be made with liver and kidney DNA sedimentation patterns. The extent of DNA damage induced by 4NQO followed the order: lung, kidney, liver, while that induced by DMN followed the order: liver, kidney, lung. The sites of greatest DNA damage appeared to correlate with sites of high levels of DNA repair synthesis and the sites of tumor induction. No DNA damage was induced by the noncarcinogenic 4-aminoquinoline 1-oxide (4AQO).     

Isolation and characterization of DNA repair deficient mutants from Penicillium chrysogenum

Summary Mutants sensitive to far ultraviolet light (UV) and 4-nitroquinoline-1-oxide (4NQO) have been isolated from Penicillium chrysogenum NRRL 1951. Two strains HP500 and HP508 are examined in detail. Their cross sensitivity to and altered mutation by UV and 4NQO suggests that damage caused by both agents is repaired through similar pathways in Penicillium chrysogenum. Strain HP500 is refractive to UV and 4NQO mutagenesis and is likely to be defective in an error-prone mechanism of repair. Mutation by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) in HP500 is also reduced, indicating involvement of an error-prone UV repair process in MNNG mutagenesis in Penicillium chrysogenum. Strain HP508 shows an increase of forward mutation rate up to 4.5 times over that of the wild-type, when compared at similar surviving fractions and is also hypermutable by 4NQO. The repair defect present in strain HP508 has been demonstrated by its inability to remove DNA sites sensitive to single strand specific nuclease during post-irradiation incubation of protoplasts.     

Effects of 4-nitroquinoline 1-oxide on the DNA-protein complex

The effects of 4-nitroquinoline 1-oxide (4NQO), a well known carcinogenic compound, on the DNA-protein complex and DNA Folding Proteins were investigated. FM3A cells were treated with 10(-6) M or 10(-5) M 4NQO for 30 min. Treatment with the 10(-6) M concentration was confirmed to cause the sedimentation of the DNA-protein complex to become slower. DNA Folding Proteins were then isolated from 4NQO-treated and untreated control cells and analyzed by SDS-polyacrylamide gel electrophoresis. No appreciable differences in the amounts of the major components of DNA Folding Proteins could be found due to 4NQO-treatment, but the 92 K protein was induced in DNA-protein complex by treatment with 4NQO.     

The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.     

Excision repair of DNA base damage in human cells treated with the chemical carcinogen 4-nitroquinoline 1-oxide.

Excision repair of DNA damage produced by 4-nitroquinoline 1-oxide (4NQO), a potent chemical carcinogen, was compared in a normal human amnion FL cell line and a xeroderma pigmentosum (XP) cell line unable to repair ultraviolet-induced pyramidine dimers. The main objective of this study was to investigate, by a direct assay of the loss of damage from DNA, whether DNA damage induced by 4NQO in human cells is repaired by the excision-repair system as in Escherichia coli cells. DNA was extracted from FL and XP cells treated with [³H]4NQO, hydrolyzed and subjected to radiochromatographic analysis in order to quantitate the initial formation of 4NQO damage and subsequent disappearance during post-incubation. Two peaks of stable 4NQO-quanine adducts appeared on the chromatogram, together with one peak of stable 4NQO-adenine adduct and a peak due to 4-aminoquinoline 1-oxide (4AQO) released from a labile fraction of 4NQO-guanine adduct during hydrolysis. The three kinds of stable 4NQO-purine adduct disappeared from DNA of the FL cells at almost the same rate of about 60% during 24-h post-incubation in culture medium, and 4AQO disappeared somewhat faster. In the XP cells, however, the stable adducts did not disappear from DNA, whereas about 40% of the 4AQO-releasing adduct disappeared from DNA. These findings at the molecular level quantitatively parallel the previous findings at the cellular level that the XP cells are several times as sensitive as normal cells to killing by 4NQO. These results lead to the conclusion that in human cells 4NQO-induced lethality is mainly due to the four kinds of 4NQO-purine adduct as it is in E. coli, and that the adducts are excisable by the same excision-repair mechanism that works on pyramidine dimers.     

The formation and repair of single-strand breaks in DNA of cultured mammalian cells treated with UV-light, methylating agents or 4-nitroquinoline-1-oxide.

The technique of sedimentation in alkaline sucrose was used to examine the formation and repair of single-strand (SS) breaks in cultured mammalian cells that were treated with methyl methanesulfonate (MMS), methyl nitrosourea (MNUA), 4-nitroquinoline-1-oxide (4NQO) or UV-light. The SS breaks induced by MMS and 4NQO were largely repaired by HeLa cells during a 5-h post-treatment incubation. The SS breaks induced by MNUA and UV-light were not repaired by HeLa cells. L-cells were not able to repair the SS breaks induced by any of the agents, which correlates with the deficiency of these cells for repair synthesis of DNA. The following conclusions are discussed. MNUA and UV-light produce modifications in DNA which are not repaired but are translated into SS breaks in alkali. MMS produces SS breaks intracellularly but these are not derived from a simple depurination of methylated purines. 4NQO produces a modification in DNA which is translated into an SS break in alkali but which can be removed by an intracellular process.     

Mutagenic and error-free DNA repair in Streptomyces

Summary Two mutants of Streptomyces fradiae defective in DNA repair have been characterized for their responses to the mutagenic and lethal effects of several chemical mutagens and ultraviolet (UV) light. S. fradiae JS2 (mcr-2) was more sensitive than wild type to agents which produce bulky lesions resulting in large distortions of the double helix [i.e. UV-light, 4-nitroquinoline-1-oxide (NQO), and mitomycin C (MC)] but not to agents which produce small lesions [i.e. hydroxylamine (HA), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG)]. JS2 expressed a much higher frequency of mutagenesis induced by UV-light at low doses and thus appeared to be defective in an error-free excision repair pathway for bulky lesions analogous to the uvr ABC pathway of Escherichia coli. S. fradiae JS4 (mcr-4) was defective in repair of damage by most agents which produce small or bulky lesions (i.e., HA, NQO, MMS, MNNG, MC, and UV, but not EMS). JS4 was slightly hypermutable by EMS and MMS but showed reduced mutagenesis by NQO and HA. This unusual phenotype suggests that the mcr-4 ⁺ protein plays some role in error-prone repair in S. fradiae.     

Bio-antimutagenic effects of tannic acid on UV and chemically induced mutagenesis in Escherichia coli B/r

Tannic acid suppressed the mutagenesis in E. coli B/r WP2 trp- induced by UV or 4-nitroquinoline 1-oxide (4NQO), but not that induced by gamma-rays or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The depression of mutations induced by UV was most remarkable in the DNA-repair-proficient strain (WP2). Tannic acid, however, showed no bio-antimutagenic effect in the excision repair-deficient strain (WP2s uvrA- or ZA159 uvrB-) under the test conditions where no cellular toxicity was observed. The effect ceased within 30 min after UV irradiation. The inhibition of the expression of Trp+ phenotype and the delay of the first cell division after UV irradiation were not observed in the presence of tannic acid. From these results we conclude that tannic acid may enhance the excision-repair system probably by activating the repair enzymes or by interacting with DNA.     

Oxidative DNA damage is a preliminary step during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide

The aim of this study was to investigate oxidative DNA damage during 4-nitroquinoline 1-oxide (4NQO)-induced rat tongue carcinogenesis. For this purpose, male Wistar rats were distributed into three groups of 10 animals each and treated with 50 ppm 4NQO solution through their drinking water for 4, 12, and 20 weeks. Ten animals were used as negative control. The alkaline Comet assay modified with lesion-specific enzymes was used to detect single and double strand breaks, labile sites (SBs), and oxidised purines and pyrimidines. Although no histopathological abnormalities were induced in the epithelium after 4 weeks of carcinogen exposure, oxidative DNA damage was detected in the ‘normal’ oral epithelium. In pre-neoplastic lesions and squamous cell carcinomas induced after 12 and 20 weeks following carcinogen exposure, respectively, oxidative DNA damage was also increased (P < 0.05) when compared to negative control. In conclusion, our results suggest that oxidative DNA damage is an early event during multistep carcinogenesis assay induced by 4NQO. This kind of approach should be considered to persons with high risk of oral cancer, such as in smokers or alcohol consumers.     

Deletion and duplication sequences induced in CHO cells by teniposide (VM-26), a topoisomerase II targeting drug,can be explained by the processing of DNA nicks produced by the drug-topoisomerase interaction

Frameshift mutations induced by acridines in bacteriophage T4 have been shown to be due to the ability of these mutagens to cause DNA cleavage by the type II topoisomerase of T4 and the subsequent processing of the 3′ ends at DNA nicks by DNA polymerase or its associated 3′ exonuclease followed by ligation of the processed end to the original 5′ end. An analysis of the ability of nick-processing models is presented here to test the ability of nick processing to account for the DNA sequences of duplications and deletions induced in the aprt gene of CHO cells by teniposide (VM-26) [Han et al. (1993) J. Mol. Biol., 229, 52]. Although teniposide is not an acridine, it induces topoisomerase II-mediated DNA cutting in aprt sequences in vitro and mutagenesis in vivo. Although the previous study noted a correlation between mutation sites and nearby DNA discontinuities induced by the enzyme in vitro, neither the nick-processing model responsible for T4 mutations, nor double-strand break models alone were able to account for most of the mutant sequences. Thus, no single model explained the correlation between teniposide-induced DNA cleavage and mutagenic specificity. This report describes an expanded analysis of the ways that nick-processing models might be related to mutagenesis and demonstrates that a modified nick-processing model provides a biochemical rationale for the mutant speficities. The successful nick-processing model proposes that either 3′ ends at nicks are elongated by DNA polymerase and/or that 5′ ends of nicks are subject to nuclease activity; 3′-nuclease activity is not implicated. The mutagenesis model for nick-processing of teniposide-induced nicks in CHO cells when compared to the mechanism of nick-processing in bacteriophage T4 at acridine-induced nicks provides a framework for considering whether the differences may be due to cell-specific modes of DNA processing and/or due to the precise characteristics of topoisomerase-DNA intermediates created by teniposide or acridine that lead to mutagenesis.     

Estimates of DNA damage by the comet assay in the direct-developing frog Eleutherodactylus johnstonei (Anura, Eleutherodactylidae)

The aim of this study was to use the Comet assay to assess genetic damage in the direct-developing frog Eleutherodactylus johnstonei. A DNA diffusion assay was used to evaluate the effectiveness of alkaline, enzymatic and alkaline/enzymatic treatments for lysing E. johnstonei blood cells and to determine the amount of DNA strand breakage associated with apoptosis and necrosis. Cell sensitivity to the mutagens bleomycin (BLM) and 4-nitro-quinoline-1-oxide (4NQO) was also assessed using the Comet assay, as was the assay reproducibility. Alkaline treatment did not lyse the cytoplasmic and nuclear membranes of E. johnstonei blood cells, whereas enzymatic digestion with proteinase K (40 μg/mL) yielded naked nuclei. The contribution of apoptosis and necrosis (assessed by the DNA diffusion assay) to DNA damage was estimated to range from 0% to 8%. BLM and 4NQO induced DNA damage in E. johnstonei blood cells at different concentrations and exposure times. Dose-effect curves with both mutagens were highly reproducible and showed consistently low coefficients of variation (CV ≤ 10%). The results are discussed with regard to the potential use of the modified Comet assay for assessing the exposure of E. johnstonei to herbicides in ecotoxicological studies.     

Study on mutagenicity and antimutagenicity of BHT and its derivatives in a bacterial assay

The mutagenicity of butylated hydroxytoluene (BHT) and its derivatives was investigated by the Ames method using Salmonella typhimurium TA98 and TA100 with or without S9 mix. The compounds were not mutagenic in either indicator strain at concentrations ranging from 50 to 330 micrograms/plate (SQ: 3,5,3',5'-tetra-tert-butylstilbenequinone; VI-III: unidentified), 500 micrograms/plate (BE: 3,5,3',5'-tetra-tert-4,4'-dihydroxy-1,2-diphenylethylene; VI: 2,6-di-tert-butyl-4-methyl-4-tert-butylperoxy-2,5-cyclohexadienone ; VI-I: unidentified; VI-II: 3-acetyl-2,5-di-tert-cyclopenta-2,4-dienone) and 1000 micrograms/plate (BHT). The antimutagenic effects of BHT and its derivatives on mutagenesis by chemical agents were investigated in Salmonella typhimurium TA98 and TA100 and Escherichia coli WP-2 hcr-. VI-II suppressed the mutagenesis induced in TA98 and TA100 by 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2) and that induced in WP-2 hcr- by 4-nitroquinoline-1-oxide (4NQO) without decreasing cell viability. In WP-2 hcr-, the mutagenesis induced by AF-2 and ethyl methanesulfonate was also suppressed significantly. Mutations induced by methyl methanesulfonate were slightly inhibited. However, VI-II had no effect on the mutagenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine.     

Immunochemical approach for the characterization of DNA damages induced by chemical carcinogens

Mouse monoclonal antibody was elicited with 4-nitroquinoline 1-oxide (4NQO) modified poly(dG-dC).poly(dG-dC) and was characterized using enzyme-linked immunosorbent assay and radioimmunoassay. The antibody reacted specifically for 4NQO-poly(dG-dC).poly(dG-dC) but not for 4NQO modified DNA and synthetic polynucleotides such as poly(dG).poly(dC). The antibody crossreacted slightly with brominated or N-acetoxy-2-acetylaminofluorene modified poly(dG-dC).poly(dG-dC) known to adopt Z-conformation. The antibody may recognize unique conformational change in poly(dG-dC).poly(dG-dC) modified by 4NQO. The antibody should be useful for the detection of conformational change in DNA induced by chemical carcinogens.     

Modification of repair DNA synthesis in mutagen-treated human fibroblasts during adaptive response and the antimutagenic effect of garlic extract

Repair DNA synthesis (RDS) in human fibroblasts during the adaptive responses (ARs) induced by cadmium chloride (CdCl2), gamma-radiation, and 4-nitroquinoline-1-oxide (4NQO) was compared in cells pretreated and not pretreated with garlic extract. The RDS was increased during the ARs induced CdCl2 and gamma-irradiation. Garlic extract stimulated RDS in cells treated by the same mutagens. 3-Aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) polymerase, decreased the RDS rate in cells treated with CdCl2 and gamma-irradiation but had no significant effect on cells treated with 4NQO. It was demonstrated that DNA repair was involved into cell protection in different ways in the cases of antimutagen treatment and AR.     

Studying the synergistic damage effects induced by 1.8 GHz radiofrequency field radiation (RFR) with four chemical mutagens on human lymphocyte DNA using comet assay in vitro

The aim of this investigation was to study the synergistic DNA damage effects in human lymphocytes induced by 1.8 GHz radiofrequency field radiation (RFR, SAR of 3 W/kg) with four chemical mutagens, i.e. mitomycin C (MMC, DNA crosslinker), bleomycin (BLM, radiomimetic agent), methyl methanesulfonate (MMS, alkylating agent), and 4-nitroquinoline-1-oxide (4NQO, UV-mimetic agent). The DNA damage of lymphocytes exposed to RFR and/or with chemical mutagens was detected at two incubation time (0 or 21 h) after treatment with comet assay in vitro. Three combinative exposure ways were used. Cells were exposed to RFR and chemical mutagens for 2 and 3h, respectively. Tail length (TL) and tail moment (TM) were utilized as DNA damage indexes. The results showed no difference of DNA damage indexes between RFR group and control group at 0 and 21 h incubation after exposure (P>0.05). There were significant difference of DNA damage indexes between MMC group and RFR+MMC co-exposure group at 0 and 21 h incubation after treatment (P<0.01). Also the significant difference of DNA damage indexes between 4NQO group and RFR+4NQO co-exposure group at 0 and 21 h incubation after treatment was observed (P<0.05 or P<0.01). The DNA damage in RFR+BLM co-exposure groups and RFR+MMS co-exposure groups was not significantly increased, as compared with corresponding BLM and MMS groups (P>0.05). The experimental results indicated 1.8 GHz RFR (SAR, 3 W/kg) for 2h did not induce the human lymphocyte DNA damage effects in vitro, but could enhance the human lymphocyte DNA damage effects induced by MMC and 4NQO. The synergistic DNA damage effects of 1.8 GHz RFR with BLM or MMS were not obvious.