WP2 Hcr Research Articles

In vitro metabolism of N-nitrodialkylamines.

The in vitro metabolism of N-nitramines was investigated in order to compare it with that of N-nitrosamines and to elucidate the mode of mutagenic action. N-Nitrodibutylamine (NO2DBA) and N-nitrodiethylamine (NO2DEA) were incubated with liver microsomes and hepatocytes prepared from rats treated with phenobarbital, and the products were analyzed by high-performance liquid chromatography and gas-liquid chromatography. The in vitro metabolic pattern of these nitramines was similar to that of the corresponding nitrosamines, except that N-nitro-N-alkylamines (produced via alpha-hydroxylation) were identified after incubation of the nitrodialkylamines. In the case of NO2DBA, besides N-nitro-N-butylamine, several nitramines produced by omega, omega-1, and omega-2 oxidations were identified as metabolites. NO2DBA and NO2DEA were mutagenic to Escherichia coli WP2 hcr- but not to Salmonella typhimurium TA1535. They were mutagenic only in the presence of hepatic microsomes, whereas their metabolites, N-nitro-N-butylamine and N-nitro-N-ethylamine, were direct mutagens. Thus, N-nitrodialkylamines are also metabolically activated to mutagens through alpha-hydroxylation.

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 μg/plate ( SQ: 3,5,3′,5′-tetra- tert-butylstilbenequinone; VI-III: unidentified), 500 μg/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 μg/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.

Aspects of chloroquine mutagenicity

Using the Ames plate reversion and fluctuation tests, the mutagenic activity of chloroquine was tested in the new tester strains of Salmonella typhimurium, TA97, TA102, and Escherichia coli strains WP 2, WP 2hcr, WP 6 and WP 67. The E. coli transconjugants obtained from the mating transfer of R-plasmid(s) in strains TA97 and TA102 respectively to E. coli WP 2, i.e. EE97 and EE102, were also tested. Chloroquine reverted strain TA97 from histidine dependence to independence and also reverted E. coli strains EE97 and EE102 from tryptophan dependence to independence. The E. coli strains WP 2, WP 2hcr; WP 6 and WP 67 and S. typhimurium TA102 were not affected. S. typhimurium TA97 could be reverted with 250 ng/ml of chloroquine (therapeutic blood level of chloroquine is 300 ng/ml). Reversion generally occurred optimally at the relatively lower concentrations of chloroquine i.e. 25, 50 μg/ml than at higher concentrations. From the properties of the reverted tester strains, the results indicated that chloroquine per se mediated frameshift reversion.

Solvolysis of N-nitroso-N-(1-acetoxyalkyl)alkylamines in phosphate buffer: characterization and mutagenicity of N-nitroso-N-(1-phosphonooxyalkyl)alkylamines.

The kinetics of solvolysis of some α-acetoxy nitrosamines in phosphate buffer solution was investigated and their mutagenic products were identified. N-Nitroso-N-(1-acetoxyalkyl)alkylamines were decomposed in two ways in aqueous phosphate buffer solution : O-acyl fission yielded α-hydroxy nitrosamines which were decomposed into aldehydes and alcohols, while O-alkyl fission gave a resonance hybrid of α-N-nitroso carbonium and iminium ions which, when trapped with phosphate, afforded N-nitroso-N-(1-phosphonooxyalkyl)alkylamines. They were stable in neutral and alkaline aqueous solutions, and were mutagenic in Salmonella typhimurium TA1535 and Escherichia coli WP2 and WP2 hcr-.

Repair inhibition of potential mutations of ultraviolet-irradiatedEscherichia coli by chloroquine and quinacrine

The frequency of ultraviolet(UV)-induced mutations drops rapidly whenEscherichia coli Hcr+ cells (strains WP-2 Hcr+; B/r) are incubated on phosphate-buffered agar (PBA), but is reduced only slightly if chloroquine or quinacrine are incorporated into the medium. The excision-deficient WP-2 Hcr− strain shows little reduction in the number of mutants when incubated on PBA. During postirradiation incubation on PBA, cell viability was relatively unaffected by the presence of the chemicals in the PBA (25 μg/ml quinacrine; 50 μg/ml chloroquine). When cells were given optimal doses of photoreactivating light, no further decline in mutations was obtained during subsequent incubation on PBA. Approximately 64% of the mutants seen when cells are treated with UV-PBA-chloroquine and 90% seen with UV-PBA-quinacrine can be repaired if cells are incubated on PBA. When these chemicals were added to the PBA, both excision-proficient strains (WP-2 Hcr+; B/r) demonstrated a marked reduction in the repair of UV-induced mutations to streptomycin resistance. Our results indicate that these chemicals interfere with the excision of UV-induced pyrimidine dimers, a process that normally occurs during postirradiation incubation on PBA.

The role of chloroquine supplementation in liquid holding recovery and ultraviolet lethality ofEscherichia coli strains

The ultraviolet (UV)-induced lethality in excision-proficientEscherichia coli strains WP-2 HCR+, B/r HCR+, B/r (λ) HCR+, Blon− HCR+, and WP-2 HCR− is increased when chloroquine (300–500 μg/ml) is added to the postirradiation medium. The degree to which chloroquine enhances the lethality of UV radiation varies for each strain, with strains B/r (λ) and B showing a greater degree of repair inhibition than the other bacterial strains. The D10 (UV showing 10% survival) decreased in B/r (λ) HCR+ strain grown in the presence of 500 μg/ml (dose response of 2.5). InE. coli B, a dose response of 4.0 was obtained in the presence of the same concentration of chloroquine.Escherichia coli B/r, WP-2 HCR+, and WP-2 HCR− strains showed less UV-induced lethality in the presence of chloroquine. The drug also inhibited “liquid holding recovery” (LHR) in irradiated HCR+ strains. These results suggest that chloroquine interferes with the excision repair (HCR function) of UV-induced photoproducts in irradiated bacterial populations.

Alpha-hydroxylation and mutagenicity of unsymmetrical N-nitrosodialkylamines with a butyl group.

In order to compare the extents of metabolic alpha-hydroxylation of the two alkyl groups in unsymmetrical N-nitrosodialkylamines, N-nitroso-N-alkylbutylamines (alkyl = methyl, ethyl, propyl, butyl, and amyl) were incubated with liver microsomes prepared from phenobarbital- or polychlorinated biphenyl (PCB)-treated and untreated rats, and aldehydes produced by the alpha-hydroxylation were determined quantitatively. Although an increased production of aldehydes was observed with the induced microsomes compared with the non-induced microsomes, no marked differences were noted between the two alkyl groups of these unsymmetric nitrosodialkylamines in regard to the extent of alpha-hydroxylation with the exception of N-nitroso-N-methylbutylamine (NMBA). The amount of aldehydes produced from these nitrosamines increased with elongation of the alkyl chain, amounting to 10-30% of the compounds incubated, and the recovery in the incubation (total aldehyde + nitrosamine recovered) was found to be more than 90%, indicating that alpha-hydroxylation is the principal oxidative metabolic pathway of the nitrosamines in vitro. The microsomal alpha-hydroxylation was inhibited by dimethyl sulfoxide and the extent of the hydroxylation was shown to be positively correlated with the mutagenic potency of the nitrosamines. Based on the mutagenic behavior of NMBA tested on Salmonella typhimurium TA1535 and Escherichia coli WP2 hcr- and the extent of the formation of formaldehyde and butyraldehyde from NMBA in the presence of the induced microsomes and S9 mix, it was concluded that NMBA acts as both a methylating and a butylating agent.

The Formation of Photoreactivable Damage by Direct Excitation of DNA in X-irradiatedE. ColiCells

The role of the direct excitation process in the formation of photoreactivable damage (pyrimidine dimers) in E. coli WP2 hcr-exr- cells has been studied. The pyrimidine dimers were detected by photoreactivation following anoxic irradiation by X-rays (220 kVp). The dose modifying factor (DMF) is 1.28 +/- 0.09. A biophysical model is used for a theoretical examination of the importance of the direct excitation process in the formation of photoreactivable damage and the experimental data are consistent with this model.

Further mutagenicity studies on pesticides in bacterial reversion assay systems

A total of 228 pesticides (88 insecticides, 60 fungicides, 62 herbicides, 12 plant-growth regulators, 3 metabolites and 3 other compounds) was tested for mutagenicity in bacterial reversion-assay systems with 5 strains (TA100, TA98, TA1535, TA1537 and TA1538) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli. 50 pesticides (25 insecticides, 20 fungicides, 3 herbicides, 1 plant-growth regulator and 1 other compound) were found to be mutagenic. 5 of them required metabolic activation (S9 mix) for their activities. Among various chemical groups, organic phosphates, halogenated alkanes and dithiocarbamates showed higher ratios of mutagens. Although 22 of the pesticides tested have been reported to be carcinogenic, 7 of them, i.e., captan, DBCP, EDB, EDC, ETU, HEH and nitrofen, were detected as mutagens in the present assay. Most of the other 15 non-mutagenic carcinogens were organochlorine pesticides such as α-BHC, chlorobenzilate, p, p′- DDT, dieldrin and quintozene.

Mutagenicity of Nifurpirinol (P-7138) in Escherichia coli WP2 and Salmonella typhimirium TA100

Nifurpirinol, 6-hydroxymethyl-2-[2-(5-nitro-2-furyl)vinyl]pyridine, which has been widely used as an antibacterial drug against diseases of fish, was found to be a potent mutagen. The mutagenic effect was dose-related, and the potencies were calculated to be 1.49 × 10 3, 7.24 × 10 4 and 8.49 × 10 5 revertants/1 × 10 8 survivors at the concentration of 1 μg/ml to Escherichia coli B/r WP2, E. coli B/r WP2 hcr − and Salmonella typhimurium TA100, respectively, without metabolic activation. Nifurpirinol is susceptible to photodegradation, and its mutagenic activity to S. typhimurium TA100 was decreased to about one thousandth when the solution of Nifurpirinol was exposed to sunlight at room temperature for 4 h.

Feasibility of testing DNA repair inhibitors for mutagenicity by a simple method

A simple screening methodology for the determination of mutagenicity of DNA repair inhibitors has been tested in this laboratory. Radiation-resistant E. coli B/r and WP2 hcr + and hcr − are suitable strains for mutagenicity testing. In these strains irradiated with 40–60 ergs/mm 2, chemicals which interfere with repair of ultraviolet-induced pre-mutational lesions can be shown to enhance significantly the frequency of mutations to streptomycin resistance. This phenomenon is termed “mutational synergism” [18,20]. We have attempted to apply the procedure for securing data for “mutational synergism” between ultraviolet (UV) radiation and a number of antimalarial drugs including quinine hydrochloride (50 μg/ml), quinine hydrobromide (50 μg/ml), primaquine diphosphate (50 μg/ml), chloroquine (50 μg/ml), quinine (50 μg/ml) and quinacrine dihydrochloride (25 μg/ml). All drugs tested give synergistic effects with UV light. The synergistic activity ranges from 3- to 35-fold. Quinine and quinacrine dihydrochloride have been found to be much more efficient enhancers of the mutagenic effect of UV than caffeine. In general, we have found that the expression of synergistic action occurs at a concentration well below the minimum inhibitory concentration (MIC) with the drugs tested. The implication of these observations in the establishment of a screening method for the evaluation of the mutagenicity of DNA repair inhibitors is discussed.

Mutagenicity screening of feed additives in the microbial system

18 feed additives were tested for DNA-modifying effects by the repair test named “rec-assay” with Bacillus subtillis H17 (rec +) and M45 (rec −), and for mutagenicity with Escherichia coli WP2 hcr and 5 Salmonella typhimurium tester strains with the use of a top-agar overlay method. Carbadox, furazolidone, panazon and zoalene were positive in both assays. The former 3 were mutagenic for TA100, TA98 and WP2 hcr, while zoalene was mutagenic for all strains. These 4 compounds did not require a metabolic activation for their mutagenic activities. Nicarbazin was weakly mutagenic for TA1538 and TA98 with and without S9 mix. Amprolium and caprylohydroxamic acid also showed very weak mutagenicities only for TA100 with S9 mix and for WP2 hcr with and without S9 mix, resp. The mutagenic activities of carbadox, furazolidone and panazon for TA100 were reduced only by the addition of S9 mix, but not by S9 fraction or blood, whereas that of zoalene was decreased by any of the 3 factors.

The effect of streptomycin resistance, caffeine and acriflavine on ultraviolet light-induced reversion to tryptophan independence in strains of Escherichia coliB/r

When the excision proficient strain E. coli WP2 Hcr+trp- was grown to stationary phase by glucose starvation in M-9 minimal medium before UV -irradiation, the ability of nutrient broth enrichment of minimal medium to enhance trp−⊒Trp+ reversion was greatly reduced. Less than 50% of the Trp+ revertants were found to be ochre suppressors. However, in the WWP2 Hcr- strain, 75–86% of the tested revertants were ochre suppressors. This indicates that, under the cultural conditions employed, many potential suppressor mutations were removed by excision repair in the presenc° of broth enrichment. Broth enhancement of reversion also occurred in the Hcr− strain, which indicates that a less error-prone mode of recombination repair functions under minimal growth conditions. An Hcr+strr derivative of WP2 Hcr+ was more resistant than its str8 parent to the lethal effect of UV light and showed a lower UV-induced Trp+ reversion frequency. The percentage of Trp+ revertants that were due to ochre suppressors was markedly reduced in the strr strain. The Hcr−strr strain also had a lower UV-induced Trp+ reversion frequency than its str8 parent. The excision repair inhibitor caffeine had little effect at lower UV doses on increasing Trp+ reversion in both Hcr+ strains. Acriflavine, however, was effective at lower UV doses in enhancing reversion of the Hcr+ strains and the degree of enhancement increased with the dose. Acriflavine appeared to specifically enhance the number of ochre suppressing Trp+ revertants. In both Hcr− strains (str8 and strr) caffeine (500 µg/ml) had no effect on survival but reduced the UV-induced Trp+ reversion frequency acting as an antimutagen. In contrast, acriflavine (2 µg/ml) decreased survival and increased the Trp+ reversion frequency of the Hcr− strains. The data on spontaneous Trp+ reversion frequencies show that the Hcr+str8 strain had a higher spontaneous reversion frequency than the Hcr−str8 strain on all plating media. Further, caffeine was shown to reduce spontaneous Trp+ reversion in both Hcr+ and Hcr− strains while acriflavine increased the spontaneous reversion frequencies of both strains.

Role of pyrimidine dimer excision in loss of potential streptomycin resistance mutations of ultraviolet-irradiated Escherichia coli on phosphate-buffered agar.

The frequency of ultraviolet (UV)-induced mutations to streptomycin resistance dropped rapidly when starved Escherichia coli strains WP-2 B/r and B/r T- were incubated on phosphate-buffered agar (PBA), but was reduced only slightly in a WP-2 hcr- mutant. During postirradiation, incubation viability remained approximately constant. Cells given an optimal recovery treatment with photo-reactivating light showed no further recovery if subsequently incubated on PBA. At least 70% of the mutations induced to streptomycin resistance by UV could be repaired. The loss of potential streptomycin-resistant mutants was markedly reduced in strain B/r T- when 5 mug of acriflavin or 700 mug of caffeine per ml was added to PBA. The excision of UV-induced thymine-containing dimers from E. coli tb/r T- was investigated. Dimer excision progressed more slowly when the cells were incubated on PBA containing acriflavin or caffeine. There was no congruity between the kinetics of dimer excision and the kinetics of mutant loss. Our results indicate that removal of potential streptomycin-resistant mutants is considerably faster than the excision of pyrimidine dimers.

The effect of streptomycin resistance, caffeine and acriflavine on ultraviolet light-induced reversion to tryptophan independence in strains of Escherichia Coli B/r

When the excision proficient strain E. coli WP2 Hcr+ trp- was grown to stationary phase by glucose starvation in M-9 minimal medium before UV -irradiation, the ability of nutrient broth enrichment of minimal medium to enhance trp- leads to Trp+ reversion was greatly reduced. Less than 50% of the Trp+ revertants were found to be ochre suppressors. However, in the WWP2 Hcr- strain, 75-86% of the tested revertants were ochre suppressors. This indicates that, under the cultural conditions employed, many potential suppressor mutations were removed by excision repair in the presence of broth enrichment. Broth enhancement of reversion also occurred in the Hcr- strain, which indicates that a less error-prone mode of recombination repair functions under minimal growth conditions. An Hcr+ strr derivative of WP2 Hcr+ was more resistant than its strs parent to the lethal effect of UV light and showed a lower UV-induced Trp+ reversion frequency. The percentage of Trp+ revertants that were due to ochre suppressors was markedly reduced in the strr strain. The Hcr- strr strain also had a lower UV-induced Trp+ reversion frequency than its strs parent. The excision repair inhibitor caffeine had little effect at lower UV doses on increasing Trp+ reversion in both Hcr+ strains. Acriflavine, however, was effective at lower UV doses in enhancing reversiin of the Hcr+ strains and the degree of enhancement increased with the dose. Acriflavine appeared to specifically enhance the number of ochre suppressing Trp+ revertants. In both Hcr- strains (strs and strr) caffeine (500 mug/ml) had no effect on survival but reduced the UV-induced Trp+ reversion frequency acting as an antimutagen. In contrast, acriflavine (2 mug/ml) decreased survival and increased the Trp+ reversion frequency of the Hcr- strains. The data on spontaneous Trp+ reversion frequencies show that the Hcr+ strs strain had a higher spontaneous reversion frequency than the Hcr- strs strain on all plating media. Further, caffeine was shown to reduce spontaneous Trp+ reversion in both Hcr+ and Hcr- strains while acriflavine increased the spontaneous reversion frequencies of both strains.

The effect of streptomycin resistance, caffeine and acriflavine on ultraviolet light-induced reversion to tryptophan independence in strains of Escherichia coli B/r

When the excision proficient strain E. coli WP2 Hcr + trp - was grown to stationary phase by glucose starvation in M-9 minimal medium before UV -irradiation, the ability of nutrient broth enrichment of minimal medium to enhance trp − ⊒Trp + reversion was greatly reduced. Less than 50% of the Trp + revertants were found to be ochre suppressors. However, in the WWP2 Hcr- strain, 75–86% of the tested revertants were ochre suppressors. This indicates that, under the cultural conditions employed, many potential suppressor mutations were removed by excision repair in the presenc° of broth enrichment. Broth enhancement of reversion also occurred in the Hcr− strain, which indicates that a less error-prone mode of recombination repair functions under minimal growth conditions. An Hcr + str r derivative of WP2 Hcr + was more resistant than its str 8 parent to the lethal effect of UV light and showed a lower UV-induced Trp + reversion frequency. The percentage of Trp + revertants that were due to ochre suppressors was markedly reduced in the str r strain. The Hcr − str r strain also had a lower UV-induced Trp + reversion frequency than its str 8 parent. The excision repair inhibitor caffeine had little effect at lower UV doses on increasing Trp + reversion in both Hcr + strains. Acriflavine, however, was effective at lower UV doses in enhancing reversion of the Hcr + strains and the degree of enhancement increased with the dose. Acriflavine appeared to specifically enhance the number of ochre suppressing Trp + revertants. In both Hcr − strains ( str 8 and str r ) caffeine (500 µg/ml) had no effect on survival but reduced the UV-induced Trp + reversion frequency acting as an antimutagen. In contrast, acriflavine (2 µg/ml) decreased survival and increased the Trp + reversion frequency of the Hcr − strains. The data on spontaneous Trp + reversion frequencies show that the Hcr + str 8 strain had a higher spontaneous reversion frequency than the Hcr − str 8 strain on all plating media. Further, caffeine was shown to reduce spontaneous Trp + reversion in both Hcr + and Hcr − strains while acriflavine increased the spontaneous reversion frequencies of both strains.

Replication and repair of UV-induced mutational lesions in chemostat cultures of Escherichia coli WP2 Hcr

Mutation to resistance to bacteriophage T5 was studied in chemostat cultures of Escherichia coli strain WP2 Hcr exposed to ultraviolet radiation (UV). The results are in generally good agreement with those obtained earlier by Bridges and Munson for UV-induced reversion to tryptophan independence in exponentially growing cultures of the same strain: expressed mutant yields followed a dose-squared response, mutations were not expressed before approximately one generation after exposure to UV, there was a slow disappearance of dimers especially noticeable in slowly growing and stationary cultures, and the first replication gave rise to duplex mutants in both strands. Several new results were also obtained. In addition to expressed mutant yields, induction of mutational capacity was also observed to follow a dose-squared response, indcating that the response is not an artifact of selection or repair. Induction also increased with growth rate, apparently as the square of the number of genes for T5-sensitivity per cell. It is suggested that mutagenesis is proportional to the number of genes per cell, that recombination is also proportional to the number of genes per cell, and that the number of mutational lesions is proportional to the product of the two. These results also provide evidence that DNA replication occurs near the end of the cell cycle in slowly growing cultures. Under all growth conditions, latent mutant concentrations mutational capacity) decreased by a factor of two with each successive division. Latent mutants were, however, photoreversible for only the first two generations. If mutagenesis occurs as a recombinant event between two mutational lesions, then the results also indicate that these lesions are separated, on the average, by no more than a single cistron.

Replication and repair of UV-induced mutational lesions in chemostat cultures of Escherichia coli WP2 Hcr

Mutation to resistance to bacteriophage T5 was studied in chemostat cultures of Escherichia coli strain WP2 Hcr exposed to ultraviolet radiation (UV). The results are in generally good agreement with those obtained earlier by Bridges and Munson for UV-induced reversion to tryptophan independence in exponentially growing cultures of the same strain: expressed mutant yields followed a dose-squared response, mutations were not expressed before approximately one generation after exposure to UV, there was a slow disappearance of dimers especially noticeable in slowly growing and stationary cultures, and the first replication gave rise to duplex mutants in both strands. Several new results were also obtained. In addition to expressed mutant yields, induction of mutational capacity was also observed to follow a dose-squared response, indcating that the response is not an artifact of selection or repair. Induction also increased with growth rate, apparently as the square of the number of genes for T5-sensitivity per cell. It is suggested that mutagenesis is proportional to the number of genes per cell, that recombination is also proportional to the number of genes per cell, and that the number of mutational lesions is proportional to the product of the two. These results also provide evidence that DNA replication occurs near the end of the cell cycle in slowly growing cultures. Under all growth conditions, latent mutant concentrations mutational capacity) decreased by a factor of two with each successive division. Latent mutants were, however, photoreversible for only the first two generations. If mutagenesis occurs as a recombinant event between two mutational lesions, then the results also indicate that these lesions are separated, on the average, by no more than a single cistron.

Respiration, growth and viability of repair-deficient mutants of Escherichia coli after ultraviolet irradiation.

SummaryEscherichia coli WP2 (a tryptophan-requiring strain of B/r) and three mutants deficient in DNA repair processes were used for a comparative study of the effects of ultraviolet radiations on respiration. An hcr− strain behaved similarly to WP2 hcr+; respiration ceased about an hour after U.V. The doses used for stopping respiration were 55 and 520 erg/mm2 respectively; these doses reduced survival in each case to about 0·5 per cent. WP2 rec− (Rec A) and WP2 exr− strains did not cease respiring after receiving a range of doses up to 520 ergs/mm2. Following irradiation, WP2 hcr+ and WP2 hcr− cells degraded their DNA slightly if at all, but degradation was extensive in the rec− and exr− strains at doses that reduced survival to 1·0 per cent (33 and 150 ergs/mm2 respectively). We conclude that cessation of respiration of irradiated Escherichia coli is dependent on the integrity of their DNA and not on the radiation sensitivity.

Evidence for differing modes of interaction of acriflavine with ultraviolet-induced lesions in an Hcr + bacterial strain.

In buffer suspensions of UV-irradiated Escherichia coli B/r WP2 Hcr+ (auxotrophic for tryptophan) acriflavine binds to DNA, but this treatment has little effect on killing and results in the appearance of fewer prototrophs on tryptophan-supplemented minimal agar. If plates contain a broth supplement, however, the buffer-acriflavine treatment greatly increases the yield of UV-induced prototrophs; but this increase does not depend on complete binding of acriflavine to the DNA as a whole, since it is observed with contact times too short for this to occur (as short as 20 seconds). The incorporation of acriflavine in both kinds of plating medium increases the yields of prototrophs. The maximum yield is observed when irradiated bacteria are exposed to acriflavine in buffer before they are plated on medium containing both acriflavine and a broth supplement. Thus post-irradiation effects of acriflavine cannot be accounted for in terms of a single mechanism of action. Our results support the suggestion that phenomena classed together as “mutation frequency decline” may not represent a single specific repair system.