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A mechanistic understanding of carcinogenic genotoxicity is necessary to determine consequences of chemical exposure on human populations and improve health risk assessments. Currently, linear dose-responses are assumed for DNA reactive compounds, ignoring cytoprotective processes that may limit permanent damage. To investigate the biological significance of low-dose exposures, human lymphoblastoid cells were treated with alkylating agents that have different mechanisms of action and DNA targets: methylmethane sulfonate (MMS), methylnitrosourea (MNU), ethylmethane sulfonate (EMS), and ethylnitrosourea (ENU). Chromosomal damage and point mutations were quantified with the micronucleus and hypoxanthine phosphoribosyltransferase forward mutation assays. MNU and ENU showed linear dose-responses, whereas MMS and EMS had nonlinear curves containing a range of nonmutagenic low doses. The lowest observed effect level for induction of chromosomal aberrations was 0.85 microg/mL MMS and 1.40 microg/mL EMS; point mutations required 1.25 microg/mL MMS and 1.40 microg/mL EMS before a mutagenic effect was detected. This nonlinearity could be due to homeostatic maintenance by DNA repair, which is efficient at low doses of compounds that primarily alkylate N(7)-G and rarely attack O atoms. A pragmatic threshold for carcinogenicity may therefore exist for such genotoxins.

The DRAG test is a rapid high-throughput screening assay for detection of repairable adducts by growth inhibition of Chinese hamster ovary cells (CHO) characterized by different defects in DNA repair. A more pronounced growth inhibition caused by a certain DNA-reactive substance in a repair-deficient cell line (EM9, UV4 and UV5) as compared to wild-type cells (AA8) is interpreted as a consequence of their inability to repair induced DNA lesions. Thus, the use of such cell lines in the DRAG test may provide information of the type of DNA lesions induced by a certain genotoxic substance. To select optimal assay conditions, as well as to provide a mechanistic basis for interpreting the results, the model compounds benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE), ethyl methanesulfonate (EMS), mitomycin C (MMC) and hydrogen peroxide (H2O2) were used. These agents can induce bulky adducts, alkyl adducts, cross-links and oxidative damage, respectively. The specificity of the DRAG test constitutes an important prerequisite for its practical use in a broader context. To assess this aspect, we have investigated the genotoxic and cytotoxic properties of a selection of metabolites of and isomers from polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), along with a few other halogenated compounds. All these compounds have been detected as pollutants in the external environment, and for most of them there is no convincing evidence of mutagenicity from conventional assays. As could be predicted from their mode of action, BPDE, MMC, and EMS were all found to be more toxic in the repair-deficient cell lines compared with wild-type cells. The results with H2O2 were inconclusive, and the PCB metabolite 4,4'-diOH-CB80 only exhibited borderline activity, while all other halogenated compounds, or their metabolites, were found to be inactive. In conclusion, the DRAG assay could provide a robust and useful tool when screening large numbers of potentially genotoxic agents, while in addition providing mechanistic information. However, the usefulness of the selected cell lines to detect oxidative damage may be limited.

Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S. TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens.

Cells resist the major mutagenic effects of alkylating agents by the action of O6-methylguanine-DNA methyltransferase (MGMT), which transfers the alkyl (R) group of O6-alkylguanine, produced in DNA by these chemicals, to a cysteine residue in its active site (formation of R-MGMT). We demonstrate that cellular R-MGMT (which represents a record or memory within the cells exposed to these chemicals) can be assayed by its sensitivity toward proteolysis by protease V8. The possible use of this assay for monitoring exposure to alkylating carcinogens was investigated by using cultured cells and a preliminary study with the use of human blood from normal subjects and patients undergoing chemotherapy. Cultured cell experiments show that R-MGMT is sufficiently stable for the monitoring purpose and its level bears a dose-response relationship to the concentrations of the alkylating agent used. Interestingly, experiments with blood from patients undergoing chemotherapy show a gradual formation of R-MGMT in 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and an induced MGMT deficiency in cyclophosphamide-treated patients. The use of this methodology, which allows for the possible quantification of active MGMT (cellular DNA repair capacity) and R-MGMT (recent exposure) simultaneously, in monitoring human exposure to alkylating carcinogens is discussed.

Two transgenic clones X3 and X15 of Nicotiana tabacum var. Xanthi, heterozygous in two genes (a1 and a2) for chloroplast differentiation and transformed with the E. coli DNA repair gene ada cloned downstream from the 1' direction of the dual mas promoter, differed in the expression of the ada gene, in the number of copies of integrated T-DNA and in the response to the mutagenic action of alkylating and non-alkylating agents. The X3 genome contained four copies and the X15 genome one copy of T-DNA, nevertheless the expression of the ada gene, measured by the activity of O6-alkylguanine DNA alkyltransferase (ATase), was about six times higher in X15 than in X3. ATase activity in both clones was highest in extracts from callus whereas very low (X15) or no (X3) activity was detected in leaf extracts. This may explain the lack of difference between X15 and non-transformed tobacco (NTX) in the frequency of N-methyl-N-nitrosourea (MNU)-induced somatic mutations in leaves. In contrast, the frequency of somatic mutations in X3 was about 2-5 times higher than in NTX and X15 after the same doses of MNU, methyl methanesulfonate, maleic hydrazide and gamma-rays. Alteration of plant gene(s) essential in mutation pathway(s) by insertion of T-DNA or by somaclonal variation may explain the higher sensitivity of the X3 clone.

Genotoxic agents known to modify DNA by alkylation reactions (alkylating agents, AAs), either directly or after metabolic conversion to ultimately reactive intermediates, by no means represent a homogeneous class. For instance, their effectiveness for genotoxic damage, when expressed as the number of events (e.g., mutations) per unit exposure dose, varies over a more than 1-million-fold range in dose. Despite the multiplicity of chemical and biological processes involved between DNA adduct formation and expression of genotoxic damage, the principal aims of studies on structure-activity relationships (SARs) are to (a) dissect the multi-step process of genetic damage formation into its most essential components, (b) use SARs for making predictions and, at a later step (c) as a basis for regulatory measures. The analytical tools available for such a comprehensive analysis in eukaryotic systems include determination of multiple genetic endpoints: molecular mutation spectra, relative clastogenicity (clastogenic events in relation to forward mutation induction) and the quantitative measure of enhanced mutagenicity in repair-deficient conditions. The genetic activity profiles obtained in this way can then be compared with fundamental physico-chemical properties of the AAs under consideration (such as Swain-Scott's s value, a useful indicator of the selectivity of an AA in its reactions with nucleophiles of distinct nucleophilic strength n in DNA, RNA and proteins), their functionality (monofunctional versus cross-linking) and their tumorigenic potency (TD50s compared with measures of initial DNA interaction, i.e., O6-/N7-alkylguanine ratios, s values or the covalent binding index determined in the liver in vivo). The combination of these different methods revealed that carcinogenic potencies of AAs in rodents vary over a 10,000-fold range in dose, with the extremes having the following characteristics: (i) Chemicals of a relatively "high carcinogenic potency", as indicated by a low TD50 in rodents, either have low nucleophilic selectivity (and therefore mainly act through O-alkylation in DNA) or are capable of cross-linking DNA. The monofunctional members of this group, typified by N-ethyl-N-nitrosourea, are active in both spermatogonia and post-spermatogonial stages in the mouse and in Drosophila. Cross-linking agents also have a low TD50 value in rodents but are expected generally not to display genetic action in premeiotic stages (exceptions mitomycin C and chlorambucil). (ii) A relatively low carcinogenic potential is associated with AAs of high Swain-Scott s values, typified by trimethyl phosphate, epichlorohydrin or methyl methanesulphonate.(ABSTRACT TRUNCATED AT 400 WORDS)

Ethenocytosine (epsilon C) is a highly mutagenic exocyclic DNA lesion induced by carcinogens vinyl chloride and urethane. We have examined base incorporation and extension at a site-specific epsilon C residue by a quantitative gel electrophoretic assay using an exonuclease-deficient version of Escherichia coli DNA polymerase I (Klenow fragment) as the model enzyme. The data show that the KM for incorporation of adenine or thymine opposite epsilon C by is about 5 orders of magnitude higher than that for the incorporation of guanine opposite normal cytosine. The KM for base extension past epsilon C:A and epsilon C:T pairs is 1-2 orders of magnitude higher than that observed for a C:G pair. Although adenine misinsertion is favored over that of thymine, base extension occurs more readily when the base incorporated opposite epsilon C is thymine.

Reactive intermediates (ultimate mutagens/carcinogens) generated by alkylating agents are unstable and difficult to characterize by chemical means. We have used a genetic system to distinguish the in vivo interactions of eight carcinogenic methylating agents and five ethylating agents by the patterns of induced mutations at different target sites in Escherichia coli WU3610. For this multiple locus assay, target sites were an amber (TAG) and an ochre (TAA) triplet, DNA encoding five suppressor tRNA anticodons, and one unidentified locus. Most of the mutations could be classified as specific sequence changes at the target loci by suppressor analysis using T4 bacteriophage. Ratios of the slopes of dose-response curves for induced mutations were used to generate a profile of preferred sites for mutagenesis independent of mutagen potency. 'Mutational fingerprints' derived from different methylating and ethylating agents were compared, as evidence for the existence of common intermediates responsible for their biological effects. Six methylating agents thought to act via SN1 mechanisms were found to generate similar mutational patterns, indicative of a common mechanism, while two methylating agents reacting via SN2 mechanisms gave different patterns. The mutational fingerprints of SN1- and SN-type ethylating agents were also distinct. Mutational fingerprints may be useful in distinguishing the interactions of different ultimate mutagens.

DNA adduct formation in various organs of mice was determined after i.p. injection with the ethylating agents N-ethyl-N-nitrosourea (ENU), ethyl methanesulfonate (EMS), and diethyl sulfate (DES). The potency of the 3 chemicals to react either at the O6 position of guanine or at the N-7 position of guanine was related to their potency to induce mutations in the specific-locus assay of the mouse. ENU, which produces relatively high levels of O-alkylations (O6-ethylguanine), is primarily mutagenic in spermatogonia of the mouse, whereas EMS and DES, which produce relatively high levels of N-alkylations (7-ethylguanine) in DNA, are much more mutagenic in post-meiotic stages of male germ cells. The relationship between exposure to ENU and the dose, determined as O6-ethylguanine per nucleotide in testicular DNA, is non-linear. However, the relationship between dose and mutation induction in spermatogonia by ENU appears to be linear, which is expected if O6-ethylguanine is the major mutagenic lesion. The relatively high mutagenic potency of EMS and DES in the late stages of spermatogenesis is probably due to the accumulation of apurinic sites which generate mutations after fertilization. A comparison of mutation induction by ENU in spermatogonia and mutation induction in cultured mammalian cells indicates that about 10 O6-ethylguanine residues were necessary in the coding region of a gene to generate a mutation.

The metabolism of spirohydantoin mustard (SHM), a central nervous system-directed nitrogen mustard with reported anticancer activity, was studied using both the Salmonella/mammalian microsome mutagenicity assay and radiolabeled drug. SHM had little or no mutagenic activity by itself but was metabolized to a mutagen(s) in the presence of mouse postmitochondrial liver fraction (9000 g supernatant, S9). Metabolism was NADPH-dependent and was enhanced with phenobarbital-induced S9. Both SHM and mutagen(s) were extractable in chloroform. Studies using [14C]SHM, uniformly labeled on the bis(2-chloroethyl)amino group, and thin-layer chromatography (TLC) of chloroform extracts of liver S9 incubation mixtures indicated the formation of a single major metabolite fraction that contained a direct-acting mutagen. Chloroform extracts of both blood and brain from BDF1 mice injected i.p. with SHM (60 mg/kg) were found to be mutagenic in the absence of S9. Also, TLC of chloroform extracts of brain taken 15 min after i.p. injection of [14C]SHM suggested the presence of SHM and the mutagenic metabolite. These results suggest that the mutagenic metabolite may have a significant role in the mechanism of action of SHM.

Reaction of nitrosodiethylamine (NDEA) in a non-enzymatic, chemical activation system consisting of ascorbic acid, ferrous ions, EDTA and molecular oxygen yielded an alkylating intermediate that was detected by its reaction with 4-(p-nitrobenzyl)pyridine (PNBP) to yield a colored product. Reaction conditions for the chemical activation-detection system were optimized, allowing detection of approx. 10 mumol NDEA. The method detected a variety of acyclic and cyclic nitrosamines and other compounds that become alkylating agents only following oxidative metabolism.

The site of alkylation of a nucleic acid, in vivo, is greatly dependent on the type of alkylating agent. Most alkylating agents of low mutagenicity or carcinogenicity (such as dimethylsulfate) react primarily with the ring nitrogens. The carcinogenic N-nitroso compounds have a great affinity for alkylating oxygens and react with all ring oxygens as well as the phosphodiesters and, in the case of RNA, with the 2'-O of ribose. Ethylating agents, though in absolute terms less reactive than the corresponding methylating agents, show even greater affinity toward oxygens. It appears that the ethyl nitroso compounds that are carcinogenic are also the most reactive with oxygens.

A simple method of determination (with a high reproducibility) of concentrations of the alkylating compound in the culture medium is described. Thiophosphamide failed to decompose in the course of 24 hours either in the culture mixture or in the human lymphocyte culture. As to phosphamide, dipine and fotrin, these drugs disintegrated significantly in the culture during 24 hours, but not in the course of the first 6 hours.

The relationship in Drosophila males between chemical reaction pattern of mono-functional alkylating agents (AA), described in terms of primary alkylation pattern with DNA and proteins as well as the Swain--Scott s factor, and their biological effectiveness were investigated. The agents chosen for comparative analysis were the nitrosamides ENU and MNU, the methanesulfonic esters iPMS, EMS and MMS, the dialkylsulfate DMS, and the nitrosamines DEN and DMN. Parameters of their biological activity were mortality (LC50) of treated adult males, induction in post-meiotic stages of X-chromosomal recessive lethal mutations and 2--3 translocations after either adult feeding or injection. Induced frequencies of recessive lethals, determined for each AA with a range of concentrations, served as biological dosimeter for interaction with target DNA in the germ line. The results are interpreted as indicating for these AA a causal connection between the pattern of primary alkylation of DNA and the quality of genetic damage observed. 1. The agent with the lowest s value, ENU, and its pendant DEN, failed to produce translocations at mutation frequencies that reached 44% for ENU. The highest chromosome-breaking activity was attributed to AA with high s, MMS and DMS. For MMS, the proportions of translocations (T) to mutations (M) approximately reached a 1 : 1 ratio in stored spermatozoa, at a recessive-lethal frequency of 14%. Ability to break chromosomes, as indicated by the T : M ratios, decreased in the sequence MMS greater than or equal to DMS, MNU greater than DMN greater than EMS greater than iPMS greater than ENU = DEN. 2. Nearly the reversed sequence in relative mutagenci effectivenss was obtained when the (directly acting) AA were arranged on the basis of their CM4/LC50 ratios (CM4, the exposure condition producing 4% recessive lethals after injection): ENU greater than EMS greater than iPMS, MNU greater than MMS = DMS. 3. Among the AA, EMS had a somewhat unique position, in that it was slightly less effective in the translocation test, and also less cytotoxic but more mutagenic in the recessive-lethal test than one would expect from its s value. This is taken as an indication of the influence on biological effectiveness of factors other than the s value, e.g. methylation versus ethylation and the lipid/water partition ratio. An example of the latter was also provided by DMS which, although having the same s as MMS, with its 5-fold higher lipid/water partition ratio, was more toxic than MMS. 4. For those AA that were clearly active in the translocation tests--MMS, DMS, MNU, DMN and EMS--delayed formation of exchanges was observed. Only in 17 out of 555 translocation tests with positive response translocations were already found in progeny from unstored spermatozoa. Consequently, it was concluded that performance of storage experiments in Drosophila is an absolute necessity for the detection of this type of rearrangement by AA. 5...

In Drosophila the connection between primary alkylation pattern and genetic activity of 8 mono-functional alkylating agents (AA)--ENU, DEN, iPMS, MNU, DMN, EMS, MMS and DMS--was investigated in spermatozoa and spermatids of treated R 1 (2), y B/BS Y y+ males. The induction of ring-X loss, Y-chromosome loss, and Y-rearrangements served as parameters of their effectiveness to produce chromosomal aberrations. (1) The ability to cause chromosomal losses decreased in the sequence MMS = DMS greater than MNU = DMN greater than EMS greater than iPMS greater than ENU = DEN. This ranking is consistent with the sequence in effectiveness in producing translocations. (2) The low frequencies of Y-chromosome rearrangements made any meaningful comparison between the AA impossible. The diagnostic value of such tests is poor, because even at toxic concentrations large numbers of offspring must be scored to obtain statistically significant effects. (3) With MNU, DMN, EMS, MMS and DMS, a time-dependent increase in the frequencies of ring-X losses ("storage effect") was found. (4) A stage-by-stage comparison revealed a differential response of spermatozoa and spermatids to these AA. Mature spermatozoa tended to be more resistant to the induction of breaks by MNU, DMN, EMS and MMS. (5) A general observation with all the AA was a reduced rate in survival of X-bearing sperm after treatment with mutagen. There was, however, no apparent quantitative relationship between the shift in the sex ratios observed and the yield of chromosomal aberrations. It is concluded that differences between these AA in their selectivity to numerous nucleophiles of DNA, as expressed by their s values, account for most of the diversity in their genetic effectiveness. The significance for genetic activity of other parameters than the s value appeared from the lower activity of EMS relative to MNU, indicating that methylation was more effective than ethylation in breaking chromosomes.

The simple eukaryotic organism baker's yeast allows demonstration of primary DNA lesions in parallel with measurement of mutagenicity and lethality after treatment with alkylating chemicals. Several anti-cancer drugs formed cross-linked DNA molecules and were genetically active. The mutagenicity and lethality of these drugs varied substantially and were dependent on the function of some processes of DNA dark-repair.

This is a review of the work on molecular dosimetry carried out by several laboratories within the third Environmental Programme of the Commission of the European Communities. The molecular dosimetry project is a coordinated research project in which several laboratories cooperated on various aspects of quantitative comparative mutagenesis. This involved (i) development of assay systems for measurement of DNA adducts using radioactively labelled mutagens; (ii) development of immunological assays for detection and quantification of DNA adducts; (iii) isolation and characterization of monoclonal antibodies against DNA adducts; (iv) quantitative comparison of genetic effects of the model compounds within one assay system; and (v) extrapolation of genetic effects of the model compounds from in vitro systems to whole animal studies.