Cell survival after DNA damage in the comet assay.

The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H2O2) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H2O2 or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.

Inter-laboratory automation of the in vitro micronucleus assay using imaging flow cytometry and deep learning.

The in vitro micronucleus assay is a globally significant method for DNA damage quantification used for regulatory compound safety testing in addition to inter-individual monitoring of environmental, lifestyle and occupational factors. However, it relies on time-consuming and user-subjective manual scoring. Here we show that imaging flow cytometry and deep learning image classification represents a capable platform for automated, inter-laboratory operation. Images were captured for the cytokinesis-block micronucleus (CBMN) assay across three laboratories using methyl methanesulphonate (1.25-5.0 µg/mL) and/or carbendazim (0.8-1.6 µg/mL) exposures to TK6 cells. Human-scored image sets were assembled and used to train and test the classification abilities of the "DeepFlow" neural network in both intra- and inter-laboratory contexts. Harnessing image diversity across laboratories yielded a network able to score unseen data from an entirely new laboratory without any user configuration. Image classification accuracies of 98%, 95%, 82% and 85% were achieved for 'mononucleates', 'binucleates', 'mononucleates with MN' and 'binucleates with MN', respectively. Successful classifications of 'trinucleates' (90%) and 'tetranucleates' (88%) in addition to 'other or unscorable' phenotypes (96%) were also achieved. Attempts to classify extremely rare, tri- and tetranucleated cells with micronuclei into their own categories were less successful (≤ 57%). Benchmark dose analyses of human or automatically scored micronucleus frequency data yielded quantitation of the same equipotent concentration regardless of scoring method. We conclude that this automated approach offers significant potential to broaden the practical utility of the CBMN method across industry, research and clinical domains. We share our strategy using openly-accessible frameworks.

Inter-laboratory comparison of the in vivo comet assay including three image analysis systems.

To compare the extent of potential inter-laboratory variability and the influence of different comet image analysis systems, in vivo comet experiments were conducted using the genotoxicants ethyl methanesulfonate and methyl methanesulfonate. Tissue samples from the same animals were processed and analyzed-including independent slide evaluation by image analysis-in two laboratories with extensive experience in performing the comet assay. The analysis revealed low inter-laboratory experimental variability. Neither the use of different image analysis systems, nor the staining procedure of DNA (propidium iodide vs. SYBR® Gold), considerably impacted the results or sensitivity of the assay. In addition, relatively high stability of the staining intensity of propidium iodide-stained slides was found in slides that were refrigerated for over 3 months. In conclusion, following a thoroughly defined protocol and standardized routine procedures ensures that the comet assay is robust and generates comparable results between different laboratories.

AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase ß knockout mouse embryonic fibroblasts.

Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase ß (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.

A random set approach to confidence regions with applications to the effective dose with combinations of agents.

The effective dose (ED) is the pharmaceutical dosage required to produce a therapeutic response in a fixed proportion of the patients. When only one drug is considered, the problem is a univariate one and has been well-studied. However, in the multidimensional setting, that is, in the presence of combinations of agents, estimation of the ED becomes more difficult. This study is focused on the plug-in logistic regression estimator of the multidimensional ED. We discuss consistency of such estimators and focus on the problem of simultaneous confidence regions. We develop a bootstrap algorithm to estimate confidence regions for the multidimensional ED. Through simulation, we show that the proposed method gives 95% confidence regions, which have better empirical coverage than the previous method for moderate to large sample sizes. The novel approach is illustrated on a cytotoxicity study on the effect of two toxins in the leukemia cell line HL-60 and a decompression sickness study of the effects of the duration and depth of the dive.

Alkylating agent methyl methanesulfonate (MMS) induces a wave of global protein hyperacetylation: implications in cancer cell death.

Protein acetylation modification has been implicated in many cellular processes but the direct evidence for the involvement of protein acetylation in signal transduction is very limited. In the present study, we found that an alkylating agent methyl methanesulfonate (MMS) induces a robust and reversible hyperacetylation of both cytoplasmic and nuclear proteins during the early phase of the cellular response to MMS. Notably, the acetylation level upon MMS treatment was strongly correlated with the susceptibility of cancer cells, and the enhancement of MMS-induced acetylation by histone deacetylase (HDAC) inhibitors was shown to increase the cellular susceptibility. These results suggest protein acetylation is important for the cell death signal transduction pathway and indicate that the use of HDAC inhibitors for the treatment of cancer is relevant.

Biological significance of DNA adducts investigated by simultaneous analysis of different endpoints of genotoxicity in L5178Y mouse lymphoma cells treated with methyl methanesulfonate.

The biological significance of DNA adducts is under continuous discussion because analytical developments allow determination of adducts at ever lower levels. Central questions refer to the biological consequences of adducts and to the relationship between background DNA damage and exposure-related increments. These questions were addressed by measuring the two DNA adducts 7-methylguanine (7-mG) and O(6)-methyl-2'-deoxyguanosine (O(6)-mdGuo) by LC-MS/MS in parallel to two biological endpoints of genotoxicity (comet assay and in vitro micronucleus test), using large batches of L5178Y mouse lymphoma cells treated with methyl methanesulfonate (MMS). The background level of 7-mG was 1440 adducts per 10(9) nucleotides while O(6)-mdGuo was almost 50-fold lower (32 adducts per 10(9) nucleotides). In the comet assay and the micronucleus test, background was in the usual range seen with smaller batches of cells (2.1% Tail DNA and 12 micronuclei-containing cells per 1000 binucleated cells, respectively). For the comparison of the four endpoints for dose-related increments above background in the low-response region we assumed linearity at low dose and used the concept of the "doubling dose", i.e., we estimated the concentration of MMS necessary to double the background measures. Doubling doses of 4.3 and 8.7microM MMS were deduced for 7-mG and O(6)-mdGuo, respectively. For doubling the background measures in the comet assay and the micronucleus test, 5 to 15-fold higher concentrations of MMS were necessary (45 and 66microM, respectively). This means that the contribution of an increase in DNA methylation to biological endpoints of genotoxicity is overestimated. For xenobiotics that generate adducts without background, the difference is even more pronounced because the dose-response curve starts at zero and the limit of detection of an increase is not affected by background variation. Consequences for the question of thresholds in dose-response relationships and for the setting of tolerable exposure levels are discussed.

Differential susceptibility of rat embryos to methyl methanesulfonate during the pregastrulation period.

The effects of exposure of pregnant rats to methyl methanesulfonate (MMS), an alkylating agent, during the pregastrulation period on embryonic and placental development were investigated. SD rats were treated orally with a single dose of MMS (200 mg/kg) in the morning of gestation days 0, 1, 2, 3, 4, 5, or 6 (GD0 to GD6 groups, respectively). The uterine contents including fetuses and placentas of the dams were examined on gestation day 20. The individual fetuses and placentas were weighed, and the fetuses were examined for external, visceral and skeletal anomalies. The progress of ossification was also evaluated. Both pre- and postimplantation embryonic mortalities were higher in the GD0 group than in the control group. The postimplantation loss was also increased for the GD3, GD4 and GD6 groups. Fetal malformations were rare in survivors of all the MMS-treated groups. Intrauterine growth retardation was apparent for fetuses in groups GD5 and GD6. In addition, placental weight was reduced in the GD6 group, but it was increased in the GD0 group. Effects of MMS on embryonic mortality or on fetal or placental growth were absent or minimal in the GD1 and GD2 groups. These results suggest that the susceptibility of rat embryos to MMS varies during the pregastrulation period.

Preliminary evaluation of DNA damage related with the smoking habit measured by the comet assay in whole blood cells.

The alkaline single-cell gel electrophoresis (SCGE) assay, also called the comet assay, is a rapid and simple method for the detection of DNA damage in individual cells. The objective of this study was to establish if the alkaline SCGE assay in whole blood cells gives similar results as the same method in isolated lymphocytes, because whole blood cells are simpler and more economical to use, specifically in human genotoxic biomonitoring. To validate the method, we first used mouse blood cells, because mouse is one of the most commonly used animals in genetic toxicology testing. Groups of seven CF1 male mice were given i.p. injections of relatively low doses of methyl methanesulfonate (25 mg/kg body weight), a direct acting genotoxic agent, or cyclophosphamide (50 mg/kg body weight), which requires metabolic activation. Three, 6, 8, 12, 16, 20, and 65 hours after treatment, 5 microL of blood were collected from each animal and were processed for the alkaline SCGE assay. On the basis of an analysis of tail moment, the results showed that this assay can detect DNA damage induced by both kinds of alkylating mutagens. We then did a preliminary study to assess the status of DNA damage in a young (19 to 23 years old) healthy population of male smokers (n = 6) and nonsmokers (n = 6) using the comet assay in whole blood cells. A significant difference was observed between the two groups, showing that the method is able to detect DNA damage in the smoking group despite the short time that the volunteers had actually been smoking.

[Biological effect of low doses of alkylating agents in drosophila studies]. / Vliianie malykh doz na biologicheskie éffekty alkiliruiushchikh agentov v issledovaniiakh na drozofile.

The low dose (0.05-0.1 mM) influence of alkylating agents on germ cell survival and male fertility, the level of embryonic and postembryonic lethality as well as the sex-linked recessive lethal (SLRL) frequency induced by high alkylating agent doses was studied in Drosophila melanogaster. The pretreatment of adult males with low doses of methyl and ethyl methanesulfonate (MMS and EMS) did not change or even enhanced EMS cytotoxicity and mutagenicity in both mature sperm and premeiotic cells. On the contrary, the low EMS dose pretreatment of larvae protected them against higher mutagen doses increasing male fertility, decreasing embryonic and postembryonic lethality in F1, and leading to three-fold reduction in the SLRL frequency in F2. The adaptive response was dependent on the Drosophila developmental stage exposed to challenge mutagen doses, since the protection was maximal in larvae and practically absent when the high dose was administered to adult males. The adaptive response observed does not seem to be associated with DNA repair, but it is rather due to other protective mechanisms.

Age-specific changes in expression, activity, and activation of the c-Jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by methyl methanesulfonate in rats.

The stress-activated protein kinases (SAPKs), c-Jun NH(2)-terminal kinases (JNKs) and p38 mitogen-activated protein kinases, were evaluated in the liver and brain of young and old rats in response to a direct-acting alkylating agent, methyl methanesulfonate (MMS). A slight but statistically significant increase in the baseline expression levels of JNK isoforms was detected in both the liver and brain of old as compared with young rats. In the liver of both young and old rats, no basal activities of JNKs were detected. In the brain, JNK activities were constitutively high and significantly increased in old rats compared with their young counterparts. Upon MMS treatment, JNKs were strongly activated in the liver, but not in the brain, of both young and old animals. The basal activity of p38 significantly increased in both the liver and brain of old rats as compared with young rats. An increase in the basal expression of p38 was detected in the brain but not in the liver of old rats. Upon treatment with MMS, p38 was activated in the liver of both young and old rats. In the brain, p38 was only activated in young but not in old rats. Taken together, these results demonstrate age-specific as well as organ-specific SAPKs signaling pathways in the rat in vivo. The possible implications of these findings in terms of resistance to endogenous and environmentally induced genotoxic stress during aging are discussed.

Micronuclei and other nuclear lesions as genotoxicity indicators in rainbow trout Oncorhynchus mykiss.

The induction of micronuclei and other nuclear abnormalities in renal erythrocytes of rainbow trout Oncorhynchus mykiss by six genotoxic compounds is evaluated. Colchicine, mitomycin, cyclophosphamide, acrylamide, methyl-methanesulfonate, and N-ethyl-N-nitrosourea were intraperitoneally injected in trout. Our results show that cyclophosphamide induces the formation of micronuclei and also the other nuclear abnormalities; N-ethyl-N-nitrosourea, acrylamide, and colchicine induce only micronuclei; mitomycin-C induces only other nuclear abnormalities but not micronuclei. Methyl-methanesulfonate does not induce nuclear abnormalities in rainbow trout at the dose assayed in this work. The possible genotoxic origin for the different nuclear abnormalities is discussed.

The response of Parp knockout mice against DNA damaging agents.

Gene-disruption studies involving poly(ADP-ribose) polymerase (Parp) have identified the various roles of Parp in cellular responses to DNA damage. The partial rescue of V[D]J recombination process in SCID/Parp(-/-) double mutant mice indicates the participation of Parp in the repair of DNA strand break. Parp(-/-) mice are more sensitive to the lethal effects of alkylating agents. Parp is also thought to be involved in base-excision repair after DNA damage caused by alkylating agents. On the other hand, resistance of Parp(-/-) mice to DNA damage induced by reactive oxygen species implicates the contribution of Parp to cell death through NAD depletion. Parp(-/-) mice with two different genetic backgrounds also show enhanced sensitivity to the lethal effects of gamma-irradiation. Parp(-/-) mice show more severe villous atrophy of the small intestine compared to the wild-type counterpart in a genetic background of 129Sv/C57BL6. Other forms of enhanced tissue damage have been identified in Parp(-/-) mice with a genetic background of 129Sv/ICR. For example, Parp(-/-) mice exhibit extensive hemorrhage in the glandular stomach and other tissues, such as the testes, after gamma-irradiation. Severe myelosuppression is also observed in both Parp(+/+) and Parp(-/-) mice, but Parp(+/+) mice show extensive extramedullary hematopoiesis in the spleen during the recovery phase of post-irradiation, whereas the spleen of Parp(-/-) mice exhibits severe atrophy with no extramedullary hematopoiesis. The absence of extramedullary hematopoiesis in the spleen is probably the underlying mechanism of hemorrhagic tendency in various tissues of Parp(-/-) mice. These findings suggest that loss of Parp activity could contribute to post-irradiation tissue hemorrhage.

Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 15). Two-week and 4-week administration study of methyl methanesulfonate (MMS).

This study was conducted to assess whether a 2-week treatment period is as effective as 4-week treatment for detection of drug-induced toxicity on the male rat reproductive organs using methyl methanesulfonate (MMS). A two-week study at dose levels of 20 or 40 mg/kg and a 4-week study with 20 mg/kg were conducted. The results can be summarized as follows. No deaths and no apparent clinical signs were observed. Body weights and food consumption were decreased at 40 mg/kg in the 2-week study along with testis and epididymis weights. In the 4-week study, epididymis weights were decreased at 20 mg/kg. The rats treated with 20 mg/kg in the 4-week study and those treated with 40 mg/kg in the 2-week study showed decrease of germ cells, exfoliation of germ cells, vacuolar degeneration of Sertoli cell and cell debris in epididymal ducts on histopathological observation. MMS impairment of spermatogenesis was confirmed by stage analysis. It was concluded that a treatment period of 2 weeks is sufficient to allow evaluation of toxic effects of MMS on the male reproductive organs.

Study of a rat skin in vivo micronucleus test: data generated by mitomycin C and methyl methanesulfonate.

We have developed an in vivo micronucleus (MN) test that uses rat skin as the target organ. Sample preparation involves cold-treating the epidermis with trypsin, peeling it off with a fine forceps, treating it in hypotonic solution, and staining it with acridine orange (A.O.). We evaluated the assay using mitomycin C (MMC) and methyl methanesulfonate (MMS) as model clastogens, applying them as single and repeat treatments. Both chemicals induced a significant, dose-dependent increase in MN frequency in basal cells. One treatment per day for 3 days was optimal for MN induction.

Adaptive response to low dose of EMS or MMS in human peripheral blood lymphocytes.

Human peripheral blood lymphocytes stimulated in vitro for 6 hr were exposed to a low (conditioning) dose of ethyl methanesulfonate (EMS; 1.5 x 10(-4) M) or methyl methanesulfonate (MMS; 1.5 x 10(-5) M). After 6 hr, the cells were treated with a high (challenging) concentration of the same agent (1.5 x 10(-3) M EMS or 1.5 x 10(-4) M MMS). The cells that received both conditioning and challenging doses became less sensitive to the induction of sister chromatid exchanges (SCEs) than those which did not receive the pretreatment with EMS or MMS. They responded with lower frequencies of SCEs. This suggests that conditioning dose of EMS or MMS has offered the lymphocytes to have decreased SCEs. This led to the realization that pre-exposure of lymphocytes to low dose can cause the induction of repair activity. This is a clear indication of the existence of adaptive response induced by alkylating agents whether it is ethylating or methylating in human lymphocytes in vitro.

In vivo induction of sister chromatid exchanges (SCE) in a tropical fish, Etroplus suratensis (Bloch).

Sister chromatid exchange (SCE) test was applied to a tropical cultured fish, Etroplus suratensis by exposing to known mutagens. Intramuscular injections of methyl methane sulphonate (MMS) and cyclophosphamide (CP) for an exposure period of 96 h resulted in significant increase in the frequency of SCE in gill tissues. MMS induced dose dependent increase in SCE. The findings revealed that the test species in the present study may be used in mutagenicity assays for screening environmental pollutants.

Rat hepatocytes with elevated metallothionein expression are resistant to N-methyl-N'-nitro-N-nitrosoguanidine cytotoxicity.

Metallothionein (MT) is a small cysteine-rich metal-binding protein involved in Zn and Cu homeostasis as well as in heavy metal detoxication. It is also believed that when MT is overexpressed, it can confer resistance against alkylating agents. However, the mechanisms involved are still poorly understood. The purpose of the present work was to investigate whether metal treatment, which induces MT synthesis, could protect isolated rat hepatocytes against the cytotoxic effects of the alkylating agents methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Exposure to 12.5 microM ZnSO4 for 18 hr raised MT levels approximately 15-fold (as measured by the 109Cd-heme assay). When these cells were exposed to increasing concentrations of MNNG, a significant reduction in cell death (as measured by lactate dehydrogenase leakage into extracellular medium) was observed (LC50 = 468 +/- 20 microM vs 362 +/- 13 microM for control cells). On the other hand, Zn pretreatment was not accompanied by resistance against MMS toxicity. In addition, the synthesis of graded amounts of MT, achieved by incubation with various concentrations of Zn or Cu, led to a high correlation between MT levels and the extent of hepatocyte survival. Cd (another MT inducer) failed to protect hepatocytes from MNNG cytotoxicity. Time-course studies also revealed a good correlation between the onset of MT induction by Zn (> 3 hr) and that of protection against MNNG (> 3 hr). The stability of MT in the presence of MNNG was studied by incubating 109Cd-labeled MT with MNNG and by analyzing the mixture using Sephadex G-75 Chromatography. Direct interaction of MNNG with rabbit liver (Cd,Zn)-MT was demonstrated by the release of 109Cd bound to MT. Similar results were obtained with 109Cd-exposed hepatocytes, 109Cd being redistributed from MT to high-molecular-weight proteins after incubation with MNNG. None of the metals used to induce MT modulated glutathione (GSH) because it remained at control levels after 18 hr. However, within 15 min of incubation, MNNG had completely depleted GSH in both control and Zn-pretreated hepatocytes equally. This was followed by a marked decline in MT levels. Taken together, these results suggest that Zn- and Cu-induced tolerance against killing by MNNG appears to be related to the accumulation of MT. The mechanism of protection might reside in the antioxidant properties of MT and on its ability to scavenge electrophilic species.

Chemoprevention of azoxymethane-induced colon carcinogenesis by dietary feeding of S-methyl methane thiosulfonate in male F344 rats.

Modifying effects of dietary exposure of S-methyl methane thiosulfonate (MMTS) isolated from cauliflower Brassica oleracea L. var. botrytis on rat colon carcinogenesis induced by azoxymethane (AOM) and on the expression of cell proliferation biomarkers were investigated in two experiments. In experiment 1, male F344 rats were given three s.c. injections of AOM (15 mg/kg body weight) and fed 100 ppm MMTS for 5 weeks, starting 1 week before the first dose of AOM. The frequency of colonic aberrant crypt foci was determined at 5 weeks after the start. Feeding of 100 ppm MMTS for 5 weeks significantly decreased the number of aberrant crypt foci/colon. Colonic mucosal ornithine decarboxylase activity and the number of silver-stained nucleolar organizer regions per nucleus in colonic epithelium were significantly decreased by MMTS treatment compared with those of AOM alone. In experiment 2, effects of dietary feeding of MMTS at two doses (20 and 100 ppm) during the postinitiation phase on intestinal tumorigenesis initiated with AOM were investigated by using a long-term experiments in male F344 rats. Incidence of intestinal neoplasms of rats fed MMTS-containing diets after AOM exposure were reduced in a dose-dependent manner. Feeding of MMTS during the postinitiation phase decreased the number of aberrant crypt foci/colon, colonic ornithine decarboxylase activity, 5-bromodeoxyuridine-labeling index in colonic epithelium, and polyamine level in blood compared with those of AOM alone. These results suggest that MMTS might be a possible chemopreventive agent for intestinal neoplasia.

Induction of micronuclei and karyotype aberrations during in vivo mouse skin carcinogenesis.

The induction of chromosome and/or genome mutations during the first steps of skin carcinogenesis was followed in male NMRI mice, treated with a 'two-stage' [9,10-dimethyl-1,2-benzanthracene (DMBA) + phorbol-12-myristate-13-acetate (TPA)], or a 'three-stage' [DMBA+methyl methanesulphonate (MMS) + phorbol-12-retinoate-13-acetate (RPA)] protocol. The scoring of micronuclei (MN) in basal and suprabasal keratinocytes allows a relatively fast in vivo estimation of clastogenic and aneugenic effects of various compounds and treatments. Relevant stages were then further analysed by karyotyping the in vivo treated keratinocytes that were allowed to divide during short in vitro cultivation. DMBA used as initiator in both protocols was able to induce MN. The well-known clastogen MMS had an acute but transient effect on MN induction when used alone or as convertor in the three-stage protocol. Neither the propagator RPA, nor the 'full-promotor' TPA, which can carry out conversion as well as propagation, induced statistically significant numbers of MN when applied on mouse skin. Combined treatments, DMBA+MMS and MMS+RPA, showed higher MN frequencies than when MMS treatments were given alone. The full carcinogenic protocols showed significant frequencies of MN but the time points of appearance differed, indicating that the accumulation of aberrations could be more important than the order of appearance. Karyotypic analysis of those stages where the MN assay detected genome and/or chromosome aberrations revealed no specific loss of chromosomes that might be directly related to the carcinogenic process. When chromosome loss and aberrations were both taken into consideration together, chromosomes 7 and 11 and surely 9, 17 and 18 were more frequently involved than others.