Signalling the end of the line

Cellular senescence is a stable proliferation arrest induced by triggers such as short telomeres, activated oncogenes and genotoxic stress. Two studies show that cellular senescence induced by genotoxic stress depends on chronic DNA-damage signalling from irreparable damage to telomeres. Hence, dysfunctional or damaged telomeres are the initiators of multiple modes of senescence.     

Use of the SOS-chromotest spot assay as a screening system for detecting genotoxic compounds in crude plant extracts

An SOS-chromotest spot assay was used to detect genotoxic compounds in crude plant extracts. The method allows simultaneous testing of extracts from different species in either a liquid or a solid crystalline form. Extracts from two species of the genus Senna, native to the state of Morelos, Mexico, were assayed. Four genotoxic compounds were isolated, and were identified as quercetin and rutin from S. wislizeni, and 5,7-di- O-methylrutin and 5,7-di-O-methylquercetin from S. skinneri. The SOS-chromotest spot assay proved to be useful for activity- guided fractionation at the beginning of screening for genotoxic compounds in crude plant extracts.     

On the distribution of genotoxic factors in various organs of mice treated with cycasin

The distribution of genotoxic factors in various organs of mice treated orally with methylazoxymethanol-beta-D-glycoside (cycasin) was investigated using the DNA-repair host mediated assay. Indicator of genotoxic activity was a pair of streptomycin dependent Escherichia coli strains differing vastly in DNA repair capacity; uvrB/recA vs. uvr+/rec+. The animal-mediated assays were performed by injecting mixtures of the two strains i.v. and orally into mice, which were subsequently treated with the test chemical and from which the differential survival of the indicator bacteria present in several organs was determined. The same strains and selection procedures were also used for assessing the DNA-damaging activity in vitro. In the animal-mediated assays in which cycasin was applied orally, significant effects were observed at doses of 100 and 500 mg/kg body weight. The organ distribution of genotoxic factors in the host animal was as follows: the highest genotoxic activity was observed in the liver, followed by intestine and stomach; a clear effect was also observed in the kidneys and, to a lower extent, in the blood stream and in the lungs at the highest dose administered (500 mg/kg body weight). Under in vitro conditions a marginal genotoxic effect was observed even in the absence of liver homogenate, indicating that the test compound is possible activated (hydrolysed) by the E. coli cells. Therefore the genotoxic activity of cycasin observed in the gastrointestinal tract was not unexpected, since the substance was applied orally, thereby exposing the indicator bacteria in these organs to high levels of unmetabolised compound, especially in the stomach. In the intestine members of the microbial flora probably contribute to the metabolic activation of the test compound. The occurrence of genotoxic factors remote from the gastrointestinal tract shows that the present compound or active metabolites thereof penetrate through the intestinal barrier. The extraordinarily high genotoxic activity observed in the liver suggests that the compound is additionally activated in this organ. In compliance with previous in vitro findings this second activation step might lead to the formation of the highly reactive aldehydic form of methylazoxymethanol (MAMAL) mediated by dehydrogenases. Comparison with carcinogenicity studies indicates a good correlation between the distribution of genotoxic effects as determined in the present studies and the localisation of tumors in various organs of rodents treated with cycasin.     

An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis

The Sea Empress oil spill resulted in the release of vast quantities of potentially genotoxic contaminants into the coastal environment of the county of Pembrokeshire (UK). We are at present attempting to determine the potential genotoxic impact of the incident upon the native marine species of the area. Here we describe the levels of DNA adducts in specimens of the intertidal teleost, Lipophrys pholis, exposed to extensive oil contamination as an indication of exposure to potential genotoxins. We detected elevated levels of adducts in L. pholis specimens from an area that underwent heavy oil contamination as compared to specimens from a clean reference area devoid of oil contamination. These preliminary studies indicated that the oil contamination induced DNA adducts in the L. pholis specimens, which could potentially cause genetic damage in this native marine species. Further studies are now required to assess the full extent of the genotoxic impact of the oil spill upon the Pembrokeshire area's native marine life.     

Genotoxic evaluation of the pyrethroid lambda-cyhalothrin using the micronucleus test in erythrocytes of the fish Cheirodon interruptus interruptus

In order to develop experimental models able to detect genotoxic effects of pollutants in aquatic organisms, the genotoxicity of the pyrethroid lambda-cyhalothrin was studied using the micronucleus test in erythrocytes of Cheirodon interruptus interruptus. The frequency of micronuclei was examined in blood smears obtained from fishes exposed in vivo to three different concentrations (0.05; 0. 01; 0.001 ug/l) of the compound and sacrificed at nine sampling times (24, 48, 72, 96 h and 8, 12, 15, 19 and 23 days). As a positive control fishes were exposed to 5 mg/l of cyclophosphamide. Results obtained demonstrated the genotoxic effects of the pyrethroid in the experimental model employed. The variation in the micronuclei frequencies in the different sampling times could be related to the blood cell kinetics and the erythrocyte replacement. The results could be considered as a validation of the MN test in fishes for the assessment of genotoxic pollutants.     

Genotoxicity of the some selective estrogen receptor modulators: a review

The objective of this article is to review genotoxicological profile of the major selective estrogen receptor modulators, including clomiphene, tamoxifen, toremifene, raloxifene. These drugs have been used for infertility treatment and breast cancer prevention in high risk-women. However, some studies reported that especially tamoxifen is a genotoxic agent and is related with endometrial cancer. Our review indicate that clomiphene and tamoxifen were found as genotoxic agent in majority of the tests. However published reports showed that toremifene is a weakly genotoxic agent. The genotoxic effects of raloxifene are still poorly known. Further genotoxicity studies should be conducted especially for raloxifene.     

Investigating the Different Mechanisms of Genotoxic and Non-Genotoxic Carcinogens by a Gene Set Analysis

Based on the process of carcinogenesis, carcinogens are classified as either genotoxic or non-genotoxic. In contrast to non-genotoxic carcinogens, many genotoxic carcinogens have been reported to cause tumor in carcinogenic bioassays in animals. Thus evaluating the genotoxicity potential of chemicals is important to discriminate genotoxic from non-genotoxic carcinogens for health care and pharmaceutical industry safety. Additionally, investigating the difference between the mechanisms of genotoxic and non-genotoxic carcinogens could provide the foundation for a mechanism-based classification for unknown compounds. In this study, we investigated the gene expression of HepG2 cells treated with genotoxic or non-genotoxic carcinogens and compared their mechanisms of action. To enhance our understanding of the differences in the mechanisms of genotoxic and non-genotoxic carcinogens, we implemented a gene set analysis using 12 compounds for the training set (12, 24, 48 h) and validated significant gene sets using 22 compounds for the test set (24, 48 h). For a direct biological translation, we conducted a gene set analysis using Globaltest and selected significant gene sets. To validate the results, training and test compounds were predicted by the significant gene sets using a prediction analysis for microarrays (PAM). Finally, we obtained 6 gene sets, including sets enriched for genes involved in the adherens junction, bladder cancer, p53 signaling pathway, pathways in cancer, peroxisome and RNA degradation. Among the 6 gene sets, the bladder cancer and p53 signaling pathway sets were significant at 12, 24 and 48 h. We also found that the DDB2, RRM2B and GADD45A, genes related to the repair and damage prevention of DNA, were consistently up-regulated for genotoxic carcinogens. Our results suggest that a gene set analysis could provide a robust tool in the investigation of the different mechanisms of genotoxic and non-genotoxic carcinogens and construct a more detailed understanding of the perturbation of significant pathways.     

TRIM8 regulated autophagy modulates the level of cleaved Caspase-3 subunit to inhibit genotoxic stress induced cell death

In cancer patients, treatment modalities like chemotherapy and radiation exert their anticancer effects by inducing DNA damage. The cancer cells can survive under genotoxic stress by inducing DNA damage response (DDR) or can undergo cell death. The process of autophagy is emerging as crucial regulator of cell survival during different stress conditions. Post translational modification through ubiquitin plays an essential role in DDR during genotoxic stress conditions. Ubiquitin ligases regulate autophagy and cell death pathways however their role during genotoxic stress conditions is not understood. In the current study we identified TRIM8, RING E3 Ligase, as a novel regulator of autophagy during DDR. TRIM8 regulates lysosomal biogenesis and autophagy flux. The turnover of TRIM8 is high and is stabilized during genotoxic stress conditions. TRIM8 regulated autophagy is essential for its cytoprotective role during genotoxic stress induced cell death. TRIM8 stabilizes the turnover of XIAP during genotoxic stress and forms complex with XIAP and caspase-3 to inhibit its activation in presence of etoposide. TRIM8 mediated autophagy promotes degradation of cleaved caspase-3 subunits. This study described TRIM8, as a novel regulator of DDR-autophagy crosstalk, which may play role in survival of cancer cells in presence of genotoxic agents.     

Proline-Rich Polypeptide-1 Protects the Cells In Vitro from Genotoxic Effects of Mitomycin C

A new proline-rich polypeptide (PRP-1) has been earlier shown to possess a broad spectrum of biological activities and seems to be a potential medicine. The potential genotoxic properties of PRP-1 and protective effect of PRP-1 against genotoxic action of Mitomycin C (MMC) were analyzed in details in the present work. DNA and chromosome damages were studied in KCL-22 cell line of human myeloid leukemia by the Comet assay and micronucleus induction test, respectively. The results suggest that DNA damages are, at least partly, transient and reparable. PRP-1 at the doses 0.5–2.0 l g/ml does not possess genotoxic activity. Moreover, this peptide expresses both preventive and therapeutic effects against MMCinduced DNA damage. Pre-treatment of cells with PRP-1 also prevents the appearance of daughter cells bearing as heavy MMC-induced DNA/chromosome damages as MNs. Thus, the polypeptide studied is able to protect the cells from genotoxic action of MMC. This defense includes not only DNA but also heritable chromosome damage in postmitotic cells. Possible mechanisms of PRP-1 protective action are discussed.     

Evaluation of the genotoxic activity of 2-nitroanthrafurans

We measured the genotoxic activities in two bacterial tests, the Salmonella/histidine assay (a reverse mutation assay) and the SOS chromotest (an assay for SOS induction in E. coli), of three 2-nitroanthrafurans: 2-nitroanthra[1,2-b]furan (R-7688), the isomeric compound 2-nitroanthra[2,1-b]furan (R-7686) and its 8-methoxylated derivative (R-7707). Their genotoxic activities were compared to that of 7-methoxy-2-nitronaphtho[2,1-b]furan (R-7000) which has been studied in previous works (Arnaise et al., 1986). We found that: (1) for all three 2-nitroanthrafurans, as generally observed for other 2-nitrofuran derivatives, the responses were correlated in the 2 tests and were decreased in the presence of an 'activating mixture' and in nitroreductase-deficient strains; (2) in contrast to what is usually observed with other 2-nitrofuran derivatives for which methoxylation increases genotoxic activity, the genotoxic activity of the methoxylated 2-nitroanthrafuran (R-7707) was comparable and may be even lower than that of the unsubstituted 2-nitroanthrafuran (R-7686); (3) the addition of a third ring that leads from 2-nitronaphthofurans to 2-nitroanthrafurans increased slightly the genotoxic activity of these compounds; (4) compounds with the oxygen heteroatom outside the 'bay region', R-7686 and R-7707, gave higher responses than their isomers with the oxygen heteroatom within the 'bay region', R-7688.     

Proline-rich Polypeptide-1 Protects the Cells In Vitro from Genotoxic Effects of Mitomycin C

A new proline-rich polypeptide (PRP-1) has been earlier shown to possess a broad spectrum of biological activities and seems to be a potential medicine. The potential genotoxic properties of PRP-1 and protective effect of PRP-1 against genotoxic action of Mitomycin C (MMC) were analyzed in details in the present work. DNA and chromosome damages were studied in KCL-22 cell line of human myeloid leukemia by the Comet assay and micronucleus induction test, respectively. The results suggest that DNA damages are, at least partly, transient and reparable. PRP-1 at the doses 0.5–2.0 μg/ml does not possess genotoxic activity. Moreover, this peptide expresses both preventive and therapeutic effects against MMC-induced DNA damage. Pre-treatment of cells with PRP-1 also prevents the appearance of daughter cells bearing as heavy MMC-induced DNA/chromosome damages as MNs. Thus, the polypeptide studied is able to protect the cells from genotoxic action of MMC. This defense includes not only DNA but also heritable chromosome damage in post-mitotic cells. Possible mechanisms of PRP-1 protective action are discussed.     

Genotoxic activity of organic contamination of the Songhua River in the north-eastern region of the People's Republic of China

The Songhua River is one of the biggest rivers in China and is the major freshwater source for industry and agriculture, as well as the source of the drinking water for millions of residents living along it. Heavy contamination of the Songhua River is due to domestic sewage and industrial wastewater. Thus, we set out to determine the carcinogenic potential of water samples taken from drinking water source of Harbin city in the Songhua River. Short-term genotoxic bioassays using Ames, SCE, and cell transformation assays were employed to examine the genotoxic activity of the ether extracts of water samples taken from the Songhua River. The results of the Ames test indicated that there were frame shift mutagens in the water samples, which were both direct and indirect. A dose–response relationship for the SCE assay was obtained, and the SCE cumulative frequency moved obviously to the right with increasing doses of water samples. Typical transformed foci were formed in NIH3T3 cells induced by ether extracts of water samples and the transformation frequency showed a dose–response relationship. The transformed cells showed the characteristics of malignant cells. All of the results indicated that the ether extracts of water samples taken from the Songhua River showed genotoxic activity.     

Role of impaired DNA repair in genotoxic susceptibility of patients with head and neck cancer

DNA repair is critical for genotoxic susceptibility and cancer development. Forty-seven patients with head and neck squamous cell carcinoma (HNSCC) and 38 healthy controls were enrolled in this study. Among the patients, 16 subjects had metastasis of HNSCC. The extent of DNA damage, including oxidative lesions, and efficiency of repair after genotoxic treatment with hydrogen peroxide were examined using the alkaline comet assay. HNSCC cells were sensitive to genotoxic treatment and displayed impaired DNA repair. In particular, lesions caused by hydrogen peroxide were repaired less effectively in cancer cells from patients with metastasis than in cells from healthy controls. We suggest that impaired DNA repair might play a role in genotoxic susceptibility of patients with head and neck cancer. Finally, as a consequence of this finding we have shown that treatment with DNA-reactive drugs could be considered as an effective therapy strategy for head and neck cancer.     

DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay

Laguna Lake, Philippines, is utilised as a resource for drinking and irrigation water and a major source of animal protein for more than 11 million residents. However, it also serves as a huge sink for anthropogenic pollutants from a variety of direct or indirect discharges. Given their well-known properties to accumulate organic pollutants, Lake Laguna sediments have been investigated by use of a newly developed sediment-contact assay to determine the bioavailable fraction of the total genotoxic hazard potential. Freshly fertilized zebrafish eggs (2 h after fertilization) were allowed to develop while exposed to different concentrations of freeze-dried sediments (exposure to bioavailable compounds) as well as organic extracts (complete genotoxic hazard potential including bioaccumulation) prepared from sediments collected at five sites within the lake. Following a 96 h exposure, single-cell suspensions were prepared from macerated larvae and analyzed for DNA strand-breaks by use of the comet assay. Genotoxicity could be identified in both solid-phase exposure scenarios as well as after exposure to organic sediment extracts. Only the South Bay (SB) site did not show a significant genotoxic burden relative to controls in tests of both freeze-dried sediment and organic extracts. In contrast, the Northwest Bay site displayed pronounced genotoxicity to the zebrafish embryos in both exposure scenarios. Perylene and copper in the sediments could be identified as potential causes of the genotoxic response. To compare the results (maximum induction coefficients) of zebrafish embryos with an established comet protocol, rainbow trout liver cells (RTL-W1) were exposed to the same extracts. The findings correlated well (Spearman correlation r=0.90), proving a good reliability of the results from zebrafish primary cells. In conclusion, the present study demonstrates that the bioavailable fraction of the genotoxic pollutants may pose a threat for both benthic organisms and human health via drinking-water and fish consumption.     

Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo

A moderate genotoxic activity of halothane and isoflurane applied as volatile anaesthetics has already been shown. The aim of this work was to estimate a potential genotoxicity of sevoflurane, introduced to clinical practice later than halothane and isoflurane. A genotoxic activity of all three compounds was estimated by using the comet assay in human peripheral blood lymphocytes (PBL) proliferating in vitro. We demonstrated that in contrast to the previously studied anaesthetics, sevoflurane did not induce any increase in DNA migration in the studied conditions. To estimate a genotoxic effect of a prolonged exposure to halogenated anaesthetics in vivo, PBL taken from operating room personnel (n = 29) were tested for DNA degradation and compared with those from a control non-exposed group (n = 20). No significant differences were detected between the groups. We conclude that sevoflurane does not have genotoxic properties, both in vitro and in vivo.     

Contribution of apoptosis to responses in the comet assay

Apoptosis, a physiological process of selected cell deletion, leads to DNA fragmentation in typical segments of 180 base pairs. DNA strand breaks are also an effect induced by genotoxic compounds. The aim of this study was to compare these two types of damaging potentials by a known genotoxic substance and an apoptosis-inducing agent in HT-29 colon adenocarcinoma cells. The cells were incubated for 24h with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a potent DNA damage-inducing agent, staurosporine, an inhibitor of protein kinase C and apoptosis-inducing agent, and hydrogen peroxide, a source of reactive oxygen species. Apoptosis was measured with the Annexin V affinity assay which detects the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the cytoplasmic membrane, an early event in the apoptotic process. DNA damage as an end point of genotoxicity was detected by single cell microgel electrophoresis, also called "comet assay". The results show that apoptosis does not necessarily need to correlate or coincide with DNA damage observed with genotoxic substances in the comet assay. The representative apoptosis-inducing agent (staurosporine) did not induce strand breaks in the tested concentrations (0.5 and 1.0microM); genotoxic doses of the strand break inducing agent MNNG did not induce apoptosis. Therefore, the comet assay can be used as a specific test for detecting genotoxicity, and the results are not necessarily confounded by concomittant processes leading to apoptosis.     

Genotoxicity of benzo[a]pyrene, 2-nitrofluorene and airborne particulates in the DNA-repair host-mediated assay

The genotoxic activity of benzo[a]pyrene (BAP), 2-nitrofluorene (NF) and airborne particulate matter was evaluated in the DNA-repair host-mediated assay after intraperitoneal or intratracheal administration. Dimethylnitrosamine (DMNA), used as a positive control, showed a genotoxic effect after both intraperitoneal and intratracheal administration, the strongest effect being found in liver, followed by lungs and kidneys, whereas a weak effect was observed in the spleen. In general no difference in genotoxicity was found between the 2 administration routes used. For BAP, although clearly positive in vitro, a moderate dose-dependent effect was found only in the liver after intraperitoneal administration. NF, which was positive in vitro both with and without a metabolizing system, produced no genotoxic effect in any of the organs tested after intraperitoneal administration. Extracts of airborne particulate matter which were genotoxic in vitro failed to cause a genotoxic effect in vivo by either route of administration. Possible explanations for the differences between the data obtained in vitro and in vivo are discussed.     

Evaluation of industrial, hospital and domestic wastewater genotoxicity with the Salmonella fluctuation test and the SOS chromotest

An evaluation of the genotoxic potential of different wastewaters collected in the Rouen area was performed with the SOS chromotest (on Escherichia coli PQ37) and the Salmonella fluctuation test on Salmonella typhimurium strains TA98, TA100 and TA102 with or without metabolic activation. The samples were taken during two 1-week periods, one in January and one in April 2003. Six sites were selected for wastewater sampling in order to allow a comparative study between an area of mixed discharge (industrial, hospital and domestic) and an area of primarily domestic discharge. Out of a total of 71 daytime samples tested, 46 (65%) were positive in at least one assay: 22 samples out of 33 in January (67%), and 24 samples out of 38 in April (63%). The two genotoxicity tests have different sensitivities. Indeed, the Salmonella fluctuation test allowed the detection of 56% of the samples as genotoxic in January (18 out of 33), and 63% in April (24 out of 38) while the SOS chromotest allowed the detection of 18% of the samples as genotoxic, whatever the sampling period. The samples collected in domestic wastewater are at least as genotoxic as the samples collected in mixed wastewater. The possible source of the detected genotoxicity (industrial, hospital or domestic) is discussed. The results of this study show that the different types of wastewaters present a genotoxic risk. Additional studies should be undertaken in the analytical field in order to try to identify and quantify the compounds responsible for the genotoxicity. This difficult task will be necessary in order to identify the sources of toxicants and thus to take preventive and/or curative measures to limit the toxicity of the wastewater.     

Ethylene thiourea (ETU). A review of the genetic toxicity studies

Ethylene thiourea (ETU) is a common contaminant, metabolite and degradation product of the fungicide class of ethylene bisdithiocarbamates (EBDCs); as such, they present possible exposure and toxicological concerns to exposed individuals. ETU has been assayed in many different tests to assess genotoxicity activity. While a great number of negative results are found in the data base, there is evidence that demonstrates ETU is capable of inducing genotoxic endpoints. These include responses for gene mutations (e.g. Salmonella), structural chromosomal alterations (e.g. aberrations in cultured mammalian cells as well as a dominant lethal assay) and other genotoxic effects (e.g. bacterial rec assay and several yeast assays).It is important to consider the magnitude of the positive responses as well as the concentrations/doses used when assessing the genotoxicity of ETU. While ETU induces a variety of genotoxic endpoints, it does not appear to be a potent genotoxic agent. For example, it is a weak bacterial mutagen in the Salmonella assay without activation in strain TA1535 at concentrations generally above 1000 μg/plate. Weak genotoxic activity of this sort is usually observed in most of the assays with positive results. Since ETU does not appear very potent and is not extremely toxic to test cells and organisms, it is not surprising to find that ETU does not produce consistent effects in many of the assays reviewed. Consequently, in many instances, mixed results for the same assay type are reported by different investigators, but as reviewed herein, these results may be dependent upon the test conditions in each individual laboratory. A primary shortcoming with many of the reported negative results is that the concentrations or doses used are not high enough for an adequate test for ETU activity. There are also problems with many of the negative assays generally in protocol or reporting, particularly with the in vivo studies (e.g. inappropriate sample number and/or sampling times; inadequate top dose employed).Overall, while ETU does not appear to be a potent genotoxic agent, it is capable of producing genotoxic effects (e.g. gene mutations, structural chromosomal aberrations). This provides a basis for weak genotoxic activity by ETU. Furthermore, based on a suggestive dominant lethal positive result, there may be a concern for heritable effects. Due to the many problems with the conduct and assessment of the in vivo assays, it is worth repeating in vivo     

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 animal's 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.