Miguel A. Comendador

DNA damage, and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters (s-values, O6/N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the Mexr-/Mexr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, kc, reaction at a critical nucleophilic strength, nc. For alkylating agents kc can be expressed as the second-order rate constant for hydrolysis, kH2O, and the substrate constant s:kH2OTD50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, ac, for 2 years exposure of the rodents. The TD*50 in mmol/kg x day) could then be written: [formula: see text] This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most SN2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be impro

Influence of mus201 and mus308 mutations of Drosophila melanogaster on the genotoxicity of model chemicals in somatic cells in vivo measured with the comet assay

To check the possibilities of the recently developed comet assay, to be used in mechanistic studies in Drosophila melanogaster, neuroblast cells of third instar larvae are used to analyse in vivo, the effect of two repair deficient mutations: mus201, deficient on nucleotide excision repair, and mus308, deficient in a mechanism of damage bypass, on the genotoxicity of methyl methanesulphonate (MMS), ethyl methanesulphonate (EMS) and N-ethyl-N-nitrosourea (ENU). The obtained results reveal: (1) MMS-induced breaks are most probably consequence of N-alkylation damage mediated abasic (AP) site breakage; (2) MMS and at least part of the EMS induced damage leading to DNA strand breaks are efficiently repaired by the nucleotide excision repair mechanism; (3) ENU and part of EMS induced damage need a functional Mus308 protein to be processed, otherwise they can lead to DNA strand breaks. In addition, the results of this work confirm the validity of neuroblast cells to conduct the comet assay, and the usefulness of this assay in in vivo mechanistic studies related to DNA repair in D. melanogaster.

Acrolein genotoxicity in Drosophila melanogaster. I. Somatic and germinal mutagenesis under proficient repair conditions

The genotoxicity of acrolein in D. melanogaster was investigated using 2 different SMART assays, the eye spot and wing spot tests, and 2 germinal tests, the sex-linked recessive lethal (SLRLT) and sex chromosome loss (SCLT) tests. For the 2 latter, exposure by feeding as well as injection was used. The results indicate that: (i) acrolein is mutagenic in the SLRLT when injected but not when fed; (ii) the SCLT did not reveal clastogenic effects; (iii) acrolein had genotoxic effects in both SMART assays; (iv) we also had several indications that acrolein is metabolized into a second genotoxic product.

Somatic recombination, gene amplification and cancer

The principle objective of this research programme, to analyse chemical induction of somatic recombination and related endpoints, i.e., mobilization of transposing elements and gene amplification, has been approached by means of several assay systems. These have included Drosophila, Saccharomyces and mammalian cell cultures. 6.1. Screening assays for mitotic recombination. A large number of chemicals have been investigated in the three Drosophila assay systems employed--the multiple wing hair/flare wing spot system developed by Graf et al., 1984, the white-ivory system developed by Green et al., 1986 and the white/white+ eye spot assay developed by Vogel (Vogel and Nivard, 1993). Particularly the screening of 181 chemicals, covering a wide array of chemical classes, by the last mentioned assay has shown that measurement of somatic recombination in Drosophila constitutes a sensitive and efficient short-term test which shows a remarkably good correlation with the agent score of 83 short-term tests analysed by ICPEMC (Mendelsohn et al., 1992; Table 2) as well as the assay performance in international collaborative programmes measuring carcinogen/non-carcinogens (de Serres and Ashby, 1981; Ashby et al., 1985, 1988). Also the wing spot assay has gained wide international recognition as a similarly sensitive test. These two assay systems in Drosophila measure both intrachromosomal events and interchromosomal recombination. The white-ivory system on the other hand is based on the loss of a tandem duplication in the white locus, the mechanism of which is less known, but probably involves intrachromosomal recombination. The difference in the mechanism between this assay and the former two was indicated by the lack of response to methotrexate in the white-ivory assay, while this compound was strongly recombinogenic in both the wing spot and white/white+ assays. The use of different strains of Drosophila with the white/white+ assay demonstrated the importance of the background genotype for the outcome of the test. Up to a 60-fold variation was found between the different genotypes in the response to procarcinogens, evidently dependent on differences in the metabolic activation of procarcinogens. In 1989 Schiestl presented results on intrachromosomal recombination in the strain RS112 of Saccharomyces, which indicated a capability to detect a range of chemical carcinogens, which gave negative results in Ames Salmonella assay. Such a test system, which could identify a larger range of potential carcinogens than conventional short-term tests evidently would be of great value and it therefore seemed of importance to follow up the observations by Schiestl. However, studies within this programme on the same strain of Saccharomyces, as well as the strains D7 (measuring intragenic recombination, intergenic recombination, and point mutation) and JD1 (measuring intragenic recombination at two loci) could not support the observations and interpretation by Schiestl (1989). The Drosophila white-ivory system, which presumably responds primarily by intrachromosomal recombination, did not give positive results with these Salmonella-negative agents either. One system to measure mitotic recombination in mammalian cell cultures was developed in the present programme. It was based on heterozygous mutations in both alleles of the adenosine deaminase gene (ADA). The system selects for proficient recombinants generated by the deficient cells. So far only pilot experiments, indicating that this experimental system operates as planned, have been performed. 6.2 Mechanisms of mitotic recombination The induction of mosaic spots in the wing spot and the white/white+ assays is predominantly dependent on interchromosomal recombination. This is evident from the fact that heterozygous inversions reduce the frequency of spots. A relationship between the length of inversions and the reduction of spots was demonstrated in the white/white+ assay--the long inversion ln(l)sc4L

The w/w+ SMART assay of Drosophila melanogaster detects the genotoxic effects of reactive oxygen species inducing compounds.

The somatic mutation and recombination w/w+ eye assay has been used for genotoxic evaluation of a broad number of chemicals with different action mechanisms yielding high values of sensitivity, specificity and accuracy. The aim of this work was to determine the utility of this assay in the evaluation of reactive oxygen species inducers. For this, we have tested eight compounds: diquat, paraquat, menadione, juglone, plumbagin, streptonigrin, tert-butyl hydroperoxide and 4-nitroquinoline 1-oxide, using the Drosophila Oregon K strain which had previously shown advantageous conditions to test this type of compounds. Diquat was the only chemical for which the results were clearly negative, probably because its high toxicity, whereas indications of a marginal genotoxicity raised for menadione. The remaining compounds were evaluated as positives. The conclusion of these experiments is that the w/w+ assay is capable to detect genotoxic effects induced by compounds that generate reactive oxygen species through different action mechanisms.

Relationships between cisplatin-induced adducts and DNA strand-breaks, mutation and recombination in vivo in somatic cells of Drosophila melanogaster, under different conditions of nucleotide excision repair

Cisplatin is a chemotherapeutic drug widely used in the treatment of several tumours, but this chemotherapy presents problems in terms of side-effects and patient resistance. The detection and determination of cisplatin-induced adducts and the relationship with the physiological or clinical effects of this drug under different repair conditions could be a good measure to assess patients response to such chemotherapy. A new methodological approach to detect and quantify cisplatin adducts by use of high-performance liquid chromatography with inductively coupled plasma mass-spectrometric detection (HPLC-ICP-MS) and isotope-dilution analysis (IDA), is evaluated for its application in vivo, under different repair conditions. This analysis is combined with the use of the Comet assay, which detects DNA strand-breaks, and the w/w(+) SMART assay, which monitors induction of somatic mutation and recombination in Drosophila melanogaster in vivo under different conditions of nucleotide-excision repair. Results show that (i) cisplatin induces in Drosophila several adducts not detected in mammals. The two most abundant cisplatin-induced adducts, identified by electrospray-mass spectrometry as G monoadduct and G-G intrastrand cross-links, were quantified individually; (ii) cisplatin induces higher levels of G monoadducts and G-G cross-links in NER-proficient than in NER-deficient cells; (iii) the level of adducts correlates with their biological consequences, both in terms of DNA strand-breaks (tail-moment values), and of somatic mutation and recombination (frequency of mosaic eyes and clones in 10(4) cells), when the repair status is considered. This work demonstrates the validity and potential of the adduct detection and quantification methodology in vivo, and its use to correlate adducts with their genetic consequences.

The hypermutability conferred by the mus308 mutation of Drosophila is not specific for cross-linking agents

The hypersensitivity of the mus308 mutant of D. melanogaster to cross-linking agents has been suggested to be the consequence of a possible defect of this mutant in DNA cross-link repair. Moreover, the mus308 mutation has been proposed as an animal model for the study of Fanconis anemia. In order to obtain more information about the function controlled by this locus, we have measured the mutability of the mus308 mutant to several mutagens with different modes of action using the sex-linked recessive lethal test. We show that this mutation confers hypermutability not only to the cross-linking agents tested, hexamethylphosphoramide and hexamethylmelamine, but to the point mutagen N-ethyl-N-nitrosourea as well, whereas the response to methyl methanesulfonate was normal. The results suggest that the mus308 locus is not defective in a repair pathway specific for cross-links but is rather involved in a step of a more general post-replication repair process responsible for the removal of non-excised adducts.

Methodological aspects of the white-ivory assay of Drosophila melanogaster

The white-ivory somatic assay of Drosophila melanogaster was developed to detect genotoxic agents which induce loss of a tandem duplication. Although the mechanism of this loss is not known, some suggestions point to intrachromosomal recombination as the main reversion mechanism. Since the few papers published to date on this assay present controversial methodologies, prior to a larger study of chemicals with different mechanisms of action, we have carried out an analysis to optimize some conditions of this assay. For this purpose, we have used three different strains and four well characterized mutagenic chemicals: N-ethyl-N-nitrosourea (ENU), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and hexamethyl phosphoramide (HMPA). The results obtained allow us to conclude that: (i) the best strain for this assay is C(1)DX,y,f/Dp(1:1:1:1)wi,y2, although the use of strain FM6,l(1)66a/Dp(1:1:1:1)wi,y2;st/st could be considered for some mechanistical studies; (ii) developmental reasons make it necessary to use as estimate of reversion frequency the proportion of eyes showing at least one spot; (iii) reversion frequency cannot be used as estimate of mutation efficiency, neither can spot size evaluate time of spot induction; (iv) the four chemicals clearly induce loss of the wi duplication; according to their activities they rank ENU > HMPA > MMS approximately EMS.

Acrolein genotoxicity in Drosophila melanogaster. III. Effects of metabolism modification.

In order to investigate the role of metabolism in acrolein genotoxicity in D. melanogaster, the action of several metabolism modifiers, namely phenobarbital, an inducer of xenobiotic metabolism, phenylimidazole and iproniazid, inhibitors of oxidative activities of cytochrome P450, and diethyl maleate, a glutathione-depleting agent, have been assayed using the sex-linked recessive lethal (SLRL) test, with two different administration routes (feeding and injection). The results support the hypothesis that acrolein is not only a direct mutagen but is also transformed, by oxidative activities of cytochrome P450 after glutathione conjugation, into an active metabolite, possibly glycidaldehyde. Moreover, acrolein is deactivated by an enzymatic activity induced by phenobarbital.

Long-term biomonitoring of breast cancer patients under adjuvant chemotherapy: the comet assay as a possible predictive factor

Most chemotherapy treatments induce DNA damage in the exposed patients. Using the comet assay and peripheral blood mononuclear cells (PBMC), we have quantified this induced DNA damage and studied its relationship with GSTM1 and GSTT1 polymorphisms, and clinical parameters. For this purpose, 29 Caucasian women, breast cancer patients under CMF or CEF adjuvant chemotherapy were included in the study. The clinical parameters considered were (i) therapies side effects, like haematological and biochemical toxicities, (ii) prognostic and predictive factors, like hormonal receptor expression, tumour differentiation degree, sickness stage, and nodal status, and (iii) the effectiveness of the chemotherapy measured as five years relapse probability. The results were also related to the confounding factor age. Comet assay results indicate that 13 patients were characterised by absence of induced DNA strand breaks, and 16 patients presented induced DNA strand breaks along the treatment. Relationships between comet variables and clinical parameters, found with principal component analysis, correlations, one-way ANOVA and multivariate logistic regression analyses revealed that: (1) baseline levels of DNA damage are related to GSTM1 genotype and to hormonal receptor expression; (2) GSTM1 genotype also influences comet results after chemotherapy, as it does the AST level; (3) the tail moment values of the cycle 6.1 and the sickness stage might predict cancer relapse at five years: for the Stage, OR = 13.8 (IIB versus I+IIA), 95% CI 0.80-238.97, and for 6.1 cycle TM, OR = 1.3, 95%, CI 0.97-1.79, with a potential model (10* Stage (I-IIA = 0, IIB = 1) + 6.1 cycle), that has a good predictive capacity, with an area under ROC curve of 0.872 (CI 0.62-1.00). To our knowledge, this is the first time such a predictive value is found for the comet assay. Nevertheless, before the comet assay could be used as a tool for oncologists, this relationship should be confirmed in more patients, and problems of standardisation and data interpretation should be solved.