Evaluation of the DNA-repair host-mediated assay. I. Induction of repairable DNA damage in E. coli cells recovered from liver, spleen, lungs, kidneys, and the blood stream of mice treated with methylating carcinogens

The DNA-repair host-mediated assay was further calibrated by determining the genotoxic activities of 4 methylating carcinogens, namely, dimethylnitrosamine (DMNA), 1,2-dimethylhydrazine (SDMH), methyl nitrosourea (MNU) and methyl methanesulphonate (MMS) in various organs of treated mice. The ranking of the animal-mediated genotoxic activities of the compounds was compared with that obtained in DNA repair assays performed in vitro. The differential survival of strain E. coli K-12/343/113 and of its DNA-repair-deficient derivatives recA, polA and uvrB/recA, served as a measure of genotoxic potency. In the in vitro assays and at equimolar exposure concentrations, MMS and MNU are the most active chemicals, followed by DMNA, which shows a slight genotoxic effect only in the presence of mouse liver homogenate; SDMH has no activity under these conditions. In the host-mediated assays, the order of genotoxic potency of the compounds was quite different: those carcinogens which require mammalian metabolic activation, namely, DMNA and SDMH, show strong effects in liver and blood, a lesser effect in the lungs and kidneys and the least effect in the spleen. The activity of MNU, a directly acting compound, is similar in all organs investigated, but it is clearly lower than that of DMNA and SDMH. MMS, also a directly acting carcinogen, causes some (barely significant) effect at the highest dose tested. A similar order of potency was observed when the compounds were tested in intrasanguineous host-mediated assays with gene mutation as an endpoint. DMNA and SDMH induce comparable frequencies of L-valine-resistant mutants in E. coli K-12/343/113 recovered from liver and spleen of treated mice, the effect in the liver being the strongest. MNU is mutagenic only at a higher dose, while MMS shows no effect. The results are discussed with respect to the literature data on organ-specific DNA adduct formation induced by the compounds. It is concluded that qualitatively there is a good correlation between the degree of genotoxic activity found in the DNA repair host-mediated assay and DNA adduct formation in the animal's own cells. This is exemplified by the finding that the relative order of genotoxic activity of the 4 methylating agents in bacteria recovered from various organs (DMNA approximately equal to SDMH greater than MNU greater than MMS) is reflected by the same order of magnitude in DNA alkylation in corresponding mammalian organs. Quantitatively, the indirectly acting agents DMNA and SDMH seem to induce fewer genotoxic effects in bacteria present in the liver than would be expected on the basis of DNA-adduct formation data.     

Evaluation of the DNA-repair host-mediated assay. III. Relationship between metabolic activation of dimethylnitrosamine and organ-specific differential lethality induced in E. coli indicator strains

In the present study the sensitivity of differential lethality as an endpoint for monitoring the presence of organ-specific genotoxic factors within the DNA-repair host-mediated assay (HMA) was determined. The induction of differential lethality in chemically exposed animals was assessed by measuring the recovery ratio Q, i.e., the relative survival of a repair-deficient E. coli K-12 derivative in comparison with its repair-proficient counterpart. Using untreated animals the interindividual fluctuation of the recovery ratio Q was first quantified and then used to determine the level below which it could be considered indicative of chemically induced differential lethality. This Q value was found to be 0.65 or lower. Using this criterion, a significant decrease of the Q value was observed in mice exposed to DMNA at a dose level as low as 15-30 mumole/kg, i.p. Inter-organ transport (liver----extrahepatic organs) of indicator bacteria was studied in reconstruction experiments using the direct-acting methylating agent MNU. These studies showed that inter-organ transport of indicator bacteria did not interfere with MNU-induced differential lethality. Time-related experiments were used to study the effects of inter-organ transport of genotoxic DMNA metabolites. In these studies significant, time-related differences were found in the induction of differential lethality in various organs of mice treated with DMNA. At a dose level of 200 mumole/kg (i.p.) genotoxic factors appeared within 25 min after administration in the liver. In the lungs and kidneys such factors appeared at a substantially slower rate, e.g., 20-120 min after DMNA administration. In persistence experiments differential lethality reached a maximum 30 min after DMNA treatment. No residual effects were detected 60 min after the injection of the carcinogen. These experiments showed that DMNA-derived genotoxic factors diffused from the liver into the bloodstream. The diffusion of these reactive species followed by their transport via the bloodstream to the lungs accounted for maximally 50% of differential lethality observed in bacteria recovered from the latter organ. In contrast, no indications were found for the transport of genotoxic DMNA metabolites from the liver via the bloodstream to the spleen and the kidneys. These results show that organ-specific effects observed in the DNA-repair HMA procedure after DMNA exposure can be primarily attributed to in situ metabolism, rather than diffusion of genotoxic metabolites from the liver to extrahepatic organs.     

Evaluation of the DNA repair host-mediated assay. II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VALr) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3-4-fold increase in VALr mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Investigations on the use of EDTA-permeabilized E. coli cells in liquid suspension and animal-mediated genotoxicity assays

The potential use of EDTA-permeabilized E. coli cells for the investigation of genotoxic effects of compounds with a large molecular configuration in vitro and in animal-mediated differential DNA-repair assays was studied. The indicator for the induction of (repairable) DNA damage was a pair of E. coli K-12 strains (343/765 and 343/753) differing vastly in DNA-repair capacity (uvr+/rec+ vs. uvrB/recA). Investigations on the influence of EDTA treatment on the viability of these strains show that during short-term exposure (3 min), the EDTA level should not exceed 0.5 mmole/l in the pretreatment mix, since at higher concentrations a marginal titer reduction of the repair-deficient strain occurs, thus indicating a weak genotoxic activity of this chelating agent. Comparisons of the results gained in vitro with permeabilized and untreated cells demonstrate that EDTA exposure leads to a substantial enhancement of the sensitivity of the indicator bacteria towards DNA damage induced by B(a)P and N-Ac-2AAF which is essential for the detection of genotoxic activities of these polycyclic aromatic compounds. Experiments to elucidate the possibility of employing EDTA-treated cells in vivo show that following intravenous and oral administration the recovery rates of permeabilized indicator strains from various mouse organs are substantially lower than those found under identical conditions (exposure time 150 min) with untreated strains. Nevertheless enough viable cells can be recovered from liver, spleen, kidneys, lungs and stomach to allow the investigation of organ-specific genotoxicity. It is furthermore noteworthy that exposure of permeabilized indicator cells in control animals (for 150 min) resulted in a marginal reduction of the relative survival of the repair-deficient strain in all organs investigated, whereas with non-treated strains such effects are only detectable after extended exposure periods. The observation of a slightly elevated genotoxic background under in vivo conditions does not prevent the assessment of the organ distribution of genotoxic effects induced by mutagens and/or carcinogens: in the case of B(a)P, intraperitoneal administration to mice in the dose range of 10-50 mg/kg body weight resulted in a pronounced dose-dependent inactivation of the uvrB/recA cells in the liver. Also in the lungs differential killing effects occurred at the highest dose tested, whereas no genotoxic activities were detectable in stomach, kidneys and spleen of the host animals.     

Evaluation of the DNA repair host-mediated assay: II. Presence of genotoxic factors in various organs of mice treated with chemotherapeutic agents

The DNA repair host-mediated assay was further calibrated by testing 7 chemotherapeutic agents known to possess carcinogenic activity, namely bleomycin (BLM), cis-diamminedichloroplatinum-II (cis-Pt), cyclophosphamide (CP), diethylstilboestrol (DES), isonicotinic acid hydrazide (isoniazid, INH), natulan (NAT) and mitomycin C (MMC). Differential survival of wild-type and uvrB/recA E. coli strains served as a measure of genotoxic activity. In in vitro assays, BLM, cis-Pt and MMC exhibited high genotoxic activity. The other 4 compounds had no measurable effect on the survival of the two strains, either with or without mouse liver preparations. In the host-mediated assays BLM, cis-Pt, MMC and also NAT induced strong killing of the DNA repair-deficient bacteria recovered from liver, spleen, lungs, kidneys and the blood of treated mice compared to the wild-type strain. The results are not indicative of large organ-specific differences in genotoxically active amounts of the drugs immediately after their application to the host animals. CP, INH and DES did not show geneotix activity in these assays even at very high exposure levels. To compare the genetic endpoint measured in the DNA repair assays, i.e. induction of repairable DNA damage, with the induction of gene mutations, the ability of the 7 drugs to induce valine-resistant (VAL^r) mutants in E. coli was measured in host-mediated assays under identical treatment conditions. INH showed considerable mutagenic activity in E. coli cells recovered from liver and spleen, while BLM and MMC induced a 3–4-fold increase in VAL^r mutants above spontaneous levels. The other compounds showed no mutagenic activity under these in vivo conditions. From these results it can be concluded that (i) the type of primary DNA lesions produced by these chemotherapeutic agents (cross-links by MMC and cis-Pt, and strand breaks by BLM and possibly by NAT; base alkylation by INH) appears to determine whether a compound will be highly positive in the DNA repair assay as in the case of BLM, cis-Pt, MMC and NAT, and less effective in inducing mutations under similar conditions, or whether the opposite will occur, as in the case of INH; (ii) DES and CP probably do not interact sufficiently with bacterial DNA to show an effect in either of the genetic endpoints; and (iii) the present DNA repair host-mediated assay may represent a sensitive, rapid and economic method for monitoring genotoxic factors in various organs of experimental animals which have been treated with cytostatic drugs.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

The relationship between reaction kinetics and mutagenic action of monofunctional alkylating agents in higher eukaryotic systems. IV. The effects of the excision-defective mei-9L1 and mus(2)201D1 mutants on alkylation-induced genetic damage in Drosophila

Repair-defective mutants of Drosophila melanogaster which identify two major DNA excision repair loci have been examined for their effects on alkylation-induced mutagenesis using the sex-linked recessive lethal assay as a measure of genotoxic endpoint. The alkylating agents (AAs) chosen for comparative analysis were selected on the basis of their reaction kinetics with DNA and included MMS, EMS, MNU, DMN, ENU, DEN and ENNG. Repair-proficient males were treated with the AAs and mated with either excision-defective mei-9L1 or mus(2)201D1 females or appropriate excision-proficient control females. The results of the present work suggest that a qualitative and quantitative relationship exists between the nature and the extent of chemical modification of DNA and the induction of of genetic alterations. The presence of either excision-defective mutant can enhance the frequency of mutation (hypermutability) and this hypermutability can be correlated with the Swain-Scott constant S of specific AAs such that as the SN1 character of the DNA alkylation reaction increases, the difference in response between repair-deficient and repair-proficient females decreases. The order of hypermutability of AAs with mei-9L1 relative to mei-9+ is MMS greater than MNU greater than DMN = EMS greater than iPMS = ENU = DEN = ENNG. When the percentage of lethal mutations induced in mei-9L1 females are plotted against those determined for control females, straight lines of different slopes are obtained. These mei-9L1/mei-9+ indices are: MMS = 7.6, MNU = 5.4, DMN = 2.4, EMS = 2.4 and iPMS = ENU = DEN = ENNG = 1. An identical order of hypermutability with similar indices is obtained for the mus(2)201 mutants: MMS(7.3) greater than MNU (5.4) greater than EMS(2.0) greater than ENU(1.1). Thus, absence of excision repair function has a significant effect on mutation production by AAs efficient in alkylating N-atoms in DNA but no measurable influence on mutation production by AAs most efficient in alkylating O-atoms in DNA. The possible nature of these DNA adducts has been discussed in relation to repair of alkylated DNA. In another series of experiments, the effect on alkylation mutagenesis of mei-9L1 was studied in males, by comparing mutation induction in mei-9L1 males vs. activity in Berlin K (control). Although these experiments suggested the existence of DNA repair in postmeiotic cells during spermatogenesis, no quantitative comparisons could be made.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA damage and prophage induction and toxicity of nitrofurantoin in Escherichia coli and Vibrio cholerae cells

Repair-deficient and repair-proficient strains of E. coli K12 were sensitive to nitrofurantoin treatment to varying degrees with the double mutant strain (uvrA 6, recA 13) being most sensitive. Ultraviolet absorption data and thermal chromatography through a hydroxyapatite column revealed that nitrofurantoin treatment of V. cholerae strain OGAWA 154 produced a maximal amount of 55% reversibly bihelical DNA at a nitrofurantoin dose of 120 micrograms/ml/h, which indicated the formation of inter-strand cross-links in DNA. Nitrofurantoin also produced prophage-lambda induction in E. coli K12 strain GY 5027: envA, uvrB, ampA 1, strA (lambda), in a dose-dependent manner, the maximum induction being highly significant (P less than 0.001). Previously published mutation data coupled with the prophage induction data presented here suggest that the genotoxic properties of nitrofurantoin are mediated through the SOS pathway.     

Studies on the genotoxicity of endosulfan in bacterial systems

Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E. coli K12. The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells. The differential sensitivity assay revealed that the recA 13 strain was the most sensitive. Endosulfan induced prophage lambda in E. coli and umu gene expression in S. typhimurium cells; however, the extent of the effects were low. Endosulfan also induced a dose-dependent increase in forward mutations in E. coli K12 cells from ampicillin sensitivity to ampicillin resistance. Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage.     

Survival and the repair of single-stranded DNA breaks in gamma-irradiated Escherichia coli cells adapted to methylmethane sulfonate]

The survival and repair of single-strand breaks of DNA in gamma-ray-irradiated E. coli adapted to MMS (20 mkg/ml during 3 hours) have been investigated. It is shown that the survival of adapted bacteria of radioresistant strains B/r, H/r30, AB1157 and W3110 pol+ increases with DMF (dose modification factor) ranging within 1.4-1.8 and in radiosensitive strains Bs-1, AB1157 recA13 and AB1157 lexA3 with DMF ranging within 1.3-1.4, and does not change in strains with mutation in polA gene P3478 polA1 and 016 res-3. There is no increase in radioresistance during the adaptation to MMS under the action of the protein synthesis inhibitor chloramphenicol. The increase in radioresistance during the adaptation to MMS correlates with the acceleration of repair of gamma-ray-induced single-strand breaks in the radioresistant strains B/r and W3110 pol+ and with the appearance of the ability to repair some part of DNA single-strand breaks in the mutant Bs-1, which beyond the adaptation to MMS does not repair these damages. The incomplete reparability of DNA single-strand breaks in P3478 polA1 strain cells, both adapted and non-adapted to MMS, is equal.     

Plasmid R46-mediated protection against bleomycin is poLA+-dependent

Strains of Escherichia coli deficient in post-replication recombination repair were more sensitive to bleomycin than wild-type, repair-proficient strains. Mutants lacking excision repair functions were no more sensitive to bleomycin than the wild-type strains, indicating that this pathway is not involved in the repair of bleomycin-damaged DNA. Plasmid R46 not only protected repair-proficient strains but also those with recB, recC, uvrA or lig genotypes, suggesting that R46 protection against bleomycin is independent of these host repair functions. However, R46 protection was abolished in recA or polA strains, indicating that these gene functions are necessary for plasmid-mediated protection. It is suggested that protection may be due to a recA+-dependent interaction of a plasmid-encoded product with host DNA polymerase I, resulting in an increase in the DNA repair capacity of cells.     

Habituation to alkali and increased u.v.-resistance in DNA repair-proficient and -deficient strains of Escherichia coli grown at pH 9.0

Repair-deficient strains of Escherichia coli carrying polAI or recA mutations were more alkali-sensitive than was their repair-proficient parent but, like strain 1829 ColV, I-K94, they showed habituation to alkali (induction of increased resistance) when grown at pH 9.0. Occurrence of such increased alkali resistance in the recA mutant implies that habituation to alkali does not depend on induction of SOS-related repair mechanisms. Organisms of repair-proficient and repair-deficient strains also became more resistant to u.v.-irradiation after growth at pH 9.0; this increased u.v.-resistance also appeared to be RecA-independent.     

An evaluation of the E. coli K-12 uvrB/recA DNA repair host-mediated assay. I. In vitro sensitivity of the bacteria to 61 compounds.

A differential DNA repair test was evaluated in vitro, using derivatives of E. coli K-12 343/113 with the genotype uvrB-/recA- and uvrB+/recA+. The aim of this study was to characterize the sensitivity of the assay to different compounds in vitro and thereby provide information on the usefulness of this end-point as an indicator of genotoxicity in a host-mediated assay. Sixty-one compounds from diverse chemical groups were tested and of these 32 gave a positive result. The results obtained were compared with results from the Ames test and were in agreement for 49 out of the 61 compounds tested. Chemicals that were detected in this test but negative in the Ames test were 4-aminophenol, catechol, diethylstilbestrol, thioacetamide and thiourea. Seven of the compounds tested gave a negative result in E. coli but were positive in Salmonella. These were 4-aminobiphenyl, benzo[a]pyrene, cyclophosphamide, 1-naphthylamine, N-nitrosobutylpropylamine, quinoline and 2-toluidine. The performance of the in vitro test and reasons for the discrepant results with the Ames test are discussed. The overall concordance between the two tests was about 80%. On the basis of these results we consider these bacterial strains, and differential DNA repair as an end-point, to be sufficiently accurate as an indicator of genotoxicity in vitro and thereby also in vivo.     

Serratia marcescens rpr gene sensitizes Escherichia coli wild-type, xth, and nfo strains to methyl methanesulphonate

It is reported here that the rpr DNA repair gene of Serratia marcescens does not complement an Escherichia coli xth nfo AP endonuclease mutation for resistance to methyl methanesulphonate (MMS). Rather, rpr sensitized Escherichia coli wild-type, xth, and nfo strains to MMS. Also, it was found that rpr could not complement a triple tag alkA recA mutation in E. coli, indicating that there are limits to rpr complementing capabilities. It was determined that rpr gene dosage was not a factor in recA complementation. MMS sensitization of an E. coli wild-type strain, however, was directly related to rpr copy number. These data indicate that Rpr does not have an associated AP endonuclease activity, and that it is incapable of substituting for Tag I, Tag II, and RecA in a tag alkA recA background.     

Induction of umu gene expression by cross-links and other DNA lesions

The induction of umu gene expression by DNA cross-links was investigated in various strains of E. coli with different DNA-repair capacities. Expression was measured by quantifying enzymatic activity of beta-galactosidase produced under regulation of the umu promoter carried on a plasmid carrying the umuC-lacZ gene fusion. The treatment with MMC induced gene expression more efficiently in a wild-type strain when compared with an excision-repair-deficient strain (uvrA). In contrast, PUVA and cis-Pt treatment induced higher levels of the gene expression in the uvrA strain than in the wild-type strain, as did other DNA-damaging agents including 4NQO, MNNG and MMS. None of these chemicals induced umu expression in either lexA and recA strains. The mechanisms of the induction of umu expression by DNA cross-links in relation to DNA damage and repair are discussed.     

Genotoxic effects of methyl isothiocyanate

Aim of the study was to investigate the genotoxic effects of methyl isothiocyanate (MITC), a compound widely distributed in the environment as a constituent of certain vegetables, a soil fumigant and breakdown product of carbamate pesticides. MITC caused only marginal mutation induction in reversion assays with Salmonella strains TA100 and TA98 and, the maximum effect (<2-fold increase over the background rate) was seen at 100microg/ml. In differential DNA-repair assays with E. coli (strains 343/763 uvrB/recA and 343/765 uvr(+)/rec(+)), a pronounced dose-response effect (induction of repairable DNA-damage) was seen at low concentrations (>/=4microg/ml). In both bacterial assays, addition of activation mix (rat liver S-9) led to a reduction of the genotoxic effects. In micronucleus assay and in single cell gel electrophoresis assay with human hepatoma cells (HepG2), clear cut positive results were obtained at exposure concentrations of <5microg/ml. On the contrary, only marginal effects were seen in differential DNA-repair host-mediated assays where E. coli indicator cells were recovered from different inner organs of mice that had been treated orally with a high dose (90mg/kg bw) of the test compound. Further in vitro experiments showed that MITC is inactivated by body fluids (saliva, gastric juice) and that its DNA-damaging properties are attenuated by non-enzymatic protein binding. Since exposure of HepG2 cells to MITC led to formation of thiobarbituric acid reactive substances, it is likely that its DNA-damaging effects involve lipid peroxidation processes. Overall, our findings show that MITC induces only marginal effects at extremely high (almost lethal) doses in inner organs in vivo, but it causes DNA-damage at low concentrations in vitro.     

Development of mutagenicity test using Escherichia coli K-12 as indicator organism

A derivative of Escherichia coli K-12 (strain $\text{343}\text{113}$) has been developed in which mutations in several genes can be detected simultaneously by plating parts of the bacterial population on different selective media. The mutation types include reversions from differently induced auxotrophies (nad^-, arg^-) aand (forward) mutations leading to resistance against 5-methytryptophan and to gal⁺ phenotype. It is assumed that many types of DNA alteration, including deletions and changes involving gross DNA regions, will lead to viable detectable mutants.The usefulness of strain E. coli$\text{343}\text{113}$ was tested in spot tests, in liquid tests, in tests with extracts of mammalian organs, and in mammalian-mediated tests. It is concluded that strain $\text{343}\text{113}$ is at least as useful in routine mutagenicity testing (especially in mammalian-mediated assays) as other present bacterial strains.     

Further studies on the lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

Our previous results on the genotoxic effect of 8-methoxypsoralen-induced lesions on pBR322 suggested an important involvement of an inducible error-free repair pathway in the repair of plasmid lesions. We present herein further results obtained in order to explore that possibility, together with a more general report on the subject. pBR322 treated with increasing concentrations of 8-MOP plus fixed UVA light irradiation was used to transform several E. coli strains differing in their repair capacities, and plasmid survival and mutagenesis were determined. Survival results suggested that crosslinks were completely lethal in pBR322 whereas monoadducts were partially removed from plasmid DNA mainly through an error-free excision pathway. A mutagenic repair pathway did not show a significant contribution to the total repair process. Cell preirradiation stimulated plasmid recovery in recA+ strains, including the umuC strain, thus confirming our previous results indicating that an inducible error-free repair had occurred. Globally, our results showed a strong requirement on the excision pathway for the repair of psoralen-damaged plasmid DNA. In contrast, the recA dependent pathway was needed only for SOS induction. After a theoretical correction of the data for estimating the effect only due to 8-MOP adducts, a different pattern of repair mechanisms appeared to be involved.     

The partial recA-lexA dependence of the adaptive response of Escherichia coli to exposure to methylmethane sulfonate

A study was made of the adaptive response to methylmethane sulfonate (MMS) in E. coli. (18 strains of B, WP2, and H/r30 groups, including three strains of bacteria with pKM101 plasmid). The adaptation of wild type cells and uvrA- and uvrB- mutants to non-lethal concentrations of MMS (10-30 mkg/ml during 90-120 min) leads to a significant increase in their resistance to lethal MMS concentrations (10-30 mM for 10-120 min): the dose modifying factor (DMF) being 1.5-1.8. In single recA or lexA mutants (or double recA uvr- and lexA uvr- mutants) the efficiency of adaptive response to MMS was significantly lower: the DMF being 1.1-1.2. In Bs-1 gamma R strain with intragenic suppressor of lexA gene the adaptive response efficiency was the same as in B/r (recA+lexA+) strain. There is no adaptive response to MMS in polA- strains. The adaptive response to MMS in E. coli is different from that to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methylnitrosourea (MNM), because in these two cases it is absolutely lexA-recA dependent. It is supposed that a partial recA-lexA dependence of the adaptive response to MMS in E. coli may be due to a specific MMS-induced lethal damage that induces an adaptive repair non-related to the system of recA-lexA-independent adaptive responses to MNNG and MNM. The presence of a plasmid of drug resistance pKM101 exerts no influence on the value, efficiency and recA-lexA-dependence of the adaptive response of E. coli to MMS.     

Mutational and recombinational events in carcinogen-modified plasmid DNA. Influence of host-cell repair genes

A plasmid containing the STR operon has been modified in vitro (i) by irradiation with UV light, (ii) by reaction with ethyl methanesulphonate (EMS), (iii) by reaction with N-acetoxy-2-acetylaminofluorene (AcO-AAF), (iv) by reaction with (+/-)trans-benzo[a]pyrene-7, 8-dihydrodiol-9,10-epoxide (BPDE), and (v) by heating at 70 degrees C to produce apurinic sites. Suitably modified plasmid DNA was then used to transform both repair-proficient and repair-deficient strains of Escherichia coli, and the mutation frequency in the plasmid-encoded rspL+ gene measured. The influence of host mutations in the uvrB+, recA+, umuC+ and lexA+, genes on the mutation frequency have been investigated. Transformation into a uvrB strain significantly decreased survival and increased the level of mutations observed for UV- and AcO-AAF-modified plasmid DNA, while only a small increase in mutation frequency was seen with EMS-modified DNA and no increase in mutation frequency with plasmid DNA containing apurinic sites. Mutagenesis in UV- and BPDE-modified DNA (and probably also DNA containing apurinic sites) was totally dependent on he recA+ gene product, while EMS and AcO-AAF induced mutagenesis was only partially independent on the recA+ gene. Transformation of UV- or BPDE-modified DNA into a umuC or lexA strain, on the other hand, showed no change in mutation frequency from that observed with wild-type strain. Pre-irradiation of the wild-type host with UV light before transformation led to a significant increase in mutation frequency for UV- and BPDE-modified plasmid DNA. These results are discussed in terms of mutational or recombinational pathways which may be available to act on modified plasmid DNA, and suggest that the majority of the mutational events measured in this system are due to recombination between homologous regions on the plasmid and chromosomal DNA.