P.R.M. Kerklaan

Methodologies for the determination of various genetic effects in permeable strains of E. coli K-12 differing in DNA repair capacity Quantification of DNA adduct formation, experiments with organ homogenates and hepatocytes, and animal-mediated assays

Derivatives of E. coli K-12 strain 343/113 differing in DNA repair capacity, in permeability to large molecules, and in some metabolizing activities (nitroreductase, glutathione), were constructed for the quantitative determination of the induction of various genetic effects, such as forward and back mutations, lysogenic induction of prophage lambda, and repairable DNA damage. These E. coli strains can be used in assay procedures which allow variation and control over several experimental conditions, such as oxygen tension, time, pH, temperature of incubation and growth phase of the indicator cells. Methods are described for the simultaneous determination of genetic effects and of DNA-adduct formation during mutagen treatment, i.e. by using radio-labeled compounds or by means of an enzyme-linked immunosorbent assay (ELISA). Mammalian biotransformation of xenobiotics can be investigated by including various fractions of mammalian organs in the system. Examples of the relative effectiveness of the activating potential of S9, S100 and isolated hepatocytes for dialkylnitrosamines and other carcinogens are presented. Host-mediated assays, finally, are described which, in addition to gene mutations, can also be used for the determination of repairable DNA damage in bacteria present in different organs, including the liver, spleen, lungs, kidneys, pancreas, and the blood stream of chemically treated mice. It is concluded that quantitative tests in vitro for assessment of induced mutagenic spectrum and genotoxic potency, combined with the host-mediated assay as a monitor, in vivo, of genotoxic factors present in various organs of animals, may become useful in the assessment of genotoxic (and possibly tumor-initiating) properties of chemicals for which long-term in-vivo mutagenicity and/or carcinogenicity data are not yet available.

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 animals 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.

A differential DNA-repair test using mixtures of E. coli K12 strains in liquid suspension and animal-mediated assays

The feasibility of performing tests for repairable DNA damage in animal assay procedures was investigated by using repair-proficient and repair-deficient derivatives of E. coli K12 strain 343/113, including mutations in the uvrB, recA, polA and dam genes. To avoid variations in the relative recovery of viable cells from different samples, the strains were further marked with auxotrophic growth requirements, so that mixtures could be treated and the survival of each strain determined individually on media containing the corresponding growth factors. Spot tests were performed with the various strains to re-assess the necessity of using a combination of repair deficiencies, when genotoxic agents of differing mode of action are to be detected. Liquid suspension tests on mixtures of the different strains, furthermore, confirmed that the survival of the individual strains can be determined separately on selective media after treatment with methyl methanesulfonate (MMS) and methyl nitrosourea (MNU). These tests were also used to demonstrate that dimethyl nitrosamine (DMNA) is activated by Aroclor-1254-induced rat-liver S9 fractions to genotoxic products, as measured by the low survival of a recA derivative compared with the repair-proficient wild-type strain. Intrasanguineous host-mediated assays using the present derivatives of E. coli K12/343/113 showed that the various strains, injected simultaneously into mice, could be recovered in amounts sufficient for the individual determination of the relative survival in liver, spleen, lungs, kidneys, pancreas and the blood stream of the host animals. Using a mixture of the repair-proficient parent and the recA derivative inoculated into mice that were subsequently treated with MMS, NMU or DMNA, we found that these chemicals induce a larger decrease in survival in the recA strain as compared with the wild-type in cells recovered from the liver and the spleen. The order of genotoxic potency so determined was DMNA greater than MNU greater than MMS; this is similar to the ranking of the carcinogenicity of these compounds in rodents and probably also reflects the various degrees of DNA alkylation in cells of the livers of the treated animals. The general usefulness of the host-mediated differential DNA repair assay for detecting genotoxic factors in various organs of animals remains to be assessed by using chemical mutagens of different modes of action.

Biochemical characterization of glutathione-deficient mutants of Escherichia coli K12 and Salmonella strains TA1535 and TA100.

Glutathione-deficient mutants of Escherichia coli K12/343/408 and Salmonella typhimurium TA1535 and TA100 were characterized biochemically by measuring the rate of formation of (14C)gamma-glutamylcysteine and (14C)glutathione in cell-free extracts of the strains. gamma-Glutamylcysteine synthetase activity was found to be absent in the NGR-2 mutant of E. coli and in the Salmonella mutants TA1535/NG-19, TA100/NG-57 and TA100/NG-11, while only low activities were found in the NGR-9 and NG-54 mutant of E. coli and Salmonella respectively. These results correspond with the decreased levels of glutathione found in these strains. Extracts of the parent strains have normal glutathione levels and show high gamma-glutamylcysteine synthetase activities. It is concluded that the present GSH-deficient strains of E. coli and Salmonella are gshA mutants, analogous to those previously described in E. coli. In addition, the present results show that the fluorometric method used for the determination of glutathione, employing o-phthalaldehyde as a reagent, is not specific for glutathione (at pH 8.0), but also sensitively reacts with gamma-glutamylcysteine.

Mutagenicity of halogenated and other substituted dinitrobenzenes in Salmonella typhimurium TA100 and derivatives deficient in glutathione (TA100/GSH−) and nitroreductase (TA100NR)

In a previous study, it was shown that 1-chloro-2,4-dinitrobenzene (CDNB) was less mutagenic in a glutathione (GSH)-deficient derivative of Salmonella typhimurium TA100 (TA100/GSH-) than in TA100 itself, suggesting that the mutagenicity of the compound is dependent on GSH, possibly mediated by the action of a bacterial nitroreductase(s) on the CDNB-GSH conjugate. In the present study a series of mutagenicity tests were performed to determine how CDNB could be activated after reaction with GSH. In liquid preincubation assays, strains TA100, TA100/GSH- and TA100NR, a nitroreductase-deficient derivative of TA100, were treated with CDNB and its fluoro and bromo analogues (FDNB and BDNB), further with its GSH conjugate (S-GSH-DNB) and possible metabolic products, such as S-cysteine-dinitrobenzene (S-Cys-DNB) and S-methyl-dinitrobenzene (S-methyl-DNB), and with 2 more analogues, O-methyl-dinitrobenzene (O-methyl-DNB) and dinitrobenzene (DNB). CDNB, FDNB and BDNB were found to be mutagenic in TA100 and TA100NR, while TA100/GSH- was much less sensitive to the mutagenic action of these halogenated dinitrobenzenes. DNB, O-methyl-DNB, S-methyl-DNB and S-Cys-DNB induced equal numbers of His+ revertants in TA100 and TA100/GSH-, but were not mutagenic in TA100NR. S-GSH-DNB showed no mutagenic activity in any of the 3 strains under the present experimental conditions. These results suggest that the halogenated aromatics may react with bacterial DNA and produce pre-mutagenic alterations according to 2 mechanisms: direct attack on the DNA through nucleophilic substitution (SN2) of the halogen atoms; activation through GSH conjugation and subsequent nitroreduction of the conjugate or its metabolic products to more reactive intermediates.

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.

The effect of mixed-function oxidase and amine oxidase inhibitors on the activation of dialkylnitrosamines and 1,2-dimethylhydrazine to bacterial mutagens in mice

SummaryThe effect of the mixed-function oxidase inhibitor phenylimidazole (PI) and the amine oxidase inhibitors iproniazid (IPRO) and aminoacetonitrile (AAN) on the mutagenic activity of various carcinogens was determined in intrasanguineous host-mediated assays, using mice as hosts and E. coli 343/113 as an indicator of mutagenic activity. The carcinogenic compounds dimethyl-, diethyl-, methylethyl-, and diethanolnitrosamine (DMNA, DENA, MENA, and DELNA respectively) and 1,2-dimethylhydrazine (SDMH) were administered i.p. to mice pretreated or not with one of the inhibitors. After 4 h exposure to each of the carcinogens, E. coli cells recovered from the liver of non-pretreated mice showed considerable induction of VALr mutations; after pretreatment of the hosts with the three inhibitors, significant reduction of the amounts of induced mutants in vivo was observed. Particularly, PI proved a very efficient inhibitor of DENA, MENA, DELNA, and SDMH mutagenicity (93%–97% reduction), suggesting that these carcinogens are mainly activated by cytochrome P-450-dependent enzymes. However, since PI might also inhibit the NAD-mediated activation of DELNA by alcohol dehydrogenase (ADH), the present experiments do not rule out an additional role of ADH in the in vivo mutagenic activation of DELNA. AAN and IPRO were less and much less effective, respectively, in reducing the mutagenic activity of all compounds. Surprisingly, PI showed less inhibition of the mutagenic activity of DMNA (60% reduction), as compared to the other carcinogens; this indicates that metabolic routes other than the cytochrome P-450-dependent enzyme system may be important for the activation of DMNA.

The Use of Multiply Marked Escherichia coli K12 Strains in the Host-Mediated Assay

Due to the development in the last decade of rapid and sensitive bacterial genetic test systems for detecting potential mutagens and carcinogens(1–3) there has been a recrudescence of testing of environmental chemicals for genotoxicity. Experiments performed in particular with theSalmonella/mammalian microsome test,(4,5) in which the ability of chemicals to cause reversion in auxotrophicSalmonella strains is determined on selective agar medium under the influence of various mammalian organ fractions, have yielded results on a variety of environmental compounds that show definite mutagenic activity in this assay system and are therefore to be considered as potentially mutagenic and carcinogenic in animals (see also Refs. 6 and 7).