Effect of diethylstilbestrol on the frequencies of sister chromatid exchange in vitro and in vivo.

The influence of diethylstilbestrol (DES) on the SCE frequency was studied in various cell systems (V79, HTC, DON, WI-38, human lymphocytes) differing in their ability to metabolize the two promutagens benzo(a)pyrene and cyclophosphamide as well as after incubation with exogenous metabolic systems (S9 mix and peroxidase/H2O2) and in vivo on cells from the bone marrow of the Chinese hamster. DES concentrations from 10(-6) to 10(-4) M were tested. Slight SCE induction was observed only at the highest concentration (10(-4) M) of DES in human lymphocytes treated with DES for the entire culture period of 72 h and in DON cells. No increase of the SCE rate was determined in the other cell lines and in vivo. The combination with S9 mix or peroxidase/H2O2 also had no influence on the SCE frequency. These findings cast doubt on the assumption that DES is metabolized to a DNA-damaging compound subsequently leading to SCE induction. The positive findings in the SCE test are more likely to be products of an indirect and rather unspecific effect.

Induced micronucleus frequencies in peripheral lymphocytes as a screening test for carriers of a BRCA1 mutation in breast cancer families.

Enhanced sensitivity to the chromosome-damaging effects of ionizing radiation is a feature of many cancer-predisposing conditions. It has been suggested that women with breast cancer are deficient in the repair of radiation-induced DNA damage. We have now investigated whether mutagen sensitivity is related to mutations in the breast cancer gene BRCA1. We studied the induction and repair of DNA damage in lymphocytes of women from families with familial breast cancer and breast and ovarian cancer. The mutagens used were gamma-irradiation and hydrogen peroxide and the DNA effects were determined with the micronucleus test and the comet assay. Women with a BRCA1 mutation (n = 12) and relatives without the familial mutation (n = 10) were compared to controls (i.e., healthy women without family history of breast or ovarian cancer; n = 17). Our results indicate a close relationship between the presence of a BRCA1 mutation and sensitivity for the induction of micronuclei. Compared to a concurrent control, 10 of 11 women with a BRCA1 mutation showed elevated radiation sensitivity. Of the 10 related women without the familial mutation, only 2 had clearly enhanced micronucleus frequencies. In addition to the sensitivity toward gamma-irradiation, hypersensitivity toward hydrogen peroxide was also observed, indicating that the mutagen sensitivity is not solely due to a defect in the repair of DNA double strand breaks. In contrast to the results with the micronucleus assay, we found no significant difference between women with and without a BRCA1 mutation with respect to the induction and repair of DNA damage in the comet assay. This finding suggests a normal rate of damage removal and points to a disturbed fidelity of DNA repair as a direct or indirect consequence of a BRCA1 mutation. Our results support the usefulness of induced micronucleus frequencies as a biomarker for cancer predisposition and suggest its application as a screening test for carriers of a BRCA1 mutation in breast cancer families.

Testing strategies in mutagenicity and genetic toxicology: an appraisal of the guidelines of the European Scientific Committee for Cosmetics and Non-Food Products for the evaluation of hair dyes.

The European Scientific Committee on Cosmetics and Non-Food Products (SCCNFP) guideline for testing of hair dyes for genotoxic/mutagenic/carcinogenic potential has been reviewed. The battery of six in vitro tests recommended therein differs substantially from the batteries of two or three in vitro tests recommended in other guidelines. Our evaluation of the chemical types used in hair dyes and comparison with other guidelines for testing a wide range of chemical substances, lead to the conclusion that potential genotoxic activity may effectively be determined by the application of a limited number of well-validated test systems that are capable of detecting induced gene mutations and structural and numerical chromosomal changes. We conclude that highly effective screening for genotoxicity of hair dyes can be achieved by the use of three assays, namely the bacterial gene mutation assay, the mammalian cell gene mutation assay (mouse lymphoma tk assay preferred) and the in vitro micronucleus assay. These need to be combined with metabolic activation systems optimised for the individual chemical types. Recent published evidence [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggests that our recommended three tests will detect all known genotoxic carcinogens, and that increasing the number of in vitro assays further would merely reduce specificity (increase false positives). Of course there may be occasions when standard tests need to be modified to take account of special situations such as a specific pathway of biotransformation, but this should be considered as part of routine testing. It is clear that individual dyes and any other novel ingredients should be tested in this three-test battery. However, new products are formed on the scalp by reaction between the chemicals present in hair-dye formulations. Ideally, these should also be tested for genotoxicity, but at present such experiences are very limited. There is also the possibility that one component could mask the genotoxicity of another (e.g. by being more toxic), and so it is not practical at this time to recommend routine testing of complete hair-dye formulations as well. The most sensible approach would be to establish whether any reaction products within the hair-dye formulation penetrate the skin under normal conditions of use and test only those that penetrate at toxicologically relevant levels in the three-test in vitro battery. Recently published data [D. Kirkland, M. Aardema, L. Henderson, L. Müller, Evaluation of the ability of a battery of three in vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity, Mutat. Res. 584 (2005) 1-256] suggest the three-test battery will produce a significant number of false as well as real positives. Whilst we are aware of the desire to reduce animal experiments, determining the relevance of positive results in any of the three recommended in vitro assays will most likely have to be determined by use of in vivo assays. The bone marrow micronucleus test using routes of administration such as oral or intraperitoneal may be used where the objective is extended hazard identification. If negative results are obtained in this test, then a second in vivo test should be conducted. This could be an in vivo UDS in rat liver or a Comet assay in a relevant tissue. However, for hazard characterisation, tests using topical application with measurement of genotoxicity in the skin would be more appropriate. Such specific site-of-contact in vivo tests would minimise animal toxicity burden and invasiveness, and, especially for hair dyes, be more relevant to human routes of exposure, but there are not sufficient scientific data available to allow recommendations to be made. The generation of such data is encouraged.

Exercise-induced DNA effects in human leukocytes are not accompanied by increased formation of 8-hydroxy-2'-deoxyguanosine or induction of micronuclei.

The present study examined the effects of a short-distance triathlon on the induction of DNA effects in peripheral leukocytes, urinary excretion of oxidized DNA bases, and frequency of micronuclei in lymphocytes of human volunteers. Induction of DNA effects was measured as increased DNA migration using the alkaline comet assay. Increased DNA migration was found in leukocytes of all individuals at different time points after exercise and revealed a biphasic pattern. Twenty-four hours postexercise, elevated DNA migration was found, whereas lower values were detected 48 h after exercise. Seventy-two hours postexercise, the maximum increase in DNA migration was found and baseline values were still elevated after 120 h. A modified protocol of the comet assay for the detection of oxidized DNA bases revealed no differences in leukocytes before and directly after the triathlon. Urinary excretion of 8-hydroxy-2'-deoxyguanosine remained unaltered during the 5 consecutive days sampled. No differences were found in the micronucleus-frequency in lymphocytes before or 48 and 96 h after exercise. Our data suggest that DNA effects detected with the comet assay in leukocytes of humans after exercise are secondary effects that do not originate from oxidized DNA bases and do not result in chromosome damage.

Chromosomal mutagen sensitivity associated with mutations in BRCA genes.

Chromosomal mutagen sensitivity is a common feature of cells from patients with different kinds of cancer. A portion of breast cancer patients also shows an elevated sensitivity to the induction of chromosome damage in cells exposed to ionizing radiation or chemical mutagens. Segregation analysis in families of patients with breast cancer indicated heritability of mutagen sensitivity. It has therefore been suggested that mutations in low-penetrance genes which are possibly involved in DNA repair predispose a substantial portion of breast cancer patients. Chromosomal mutagen sensitivity has been determined with the G2 chromosome aberration test and the G(0) micronucleus test (MNT). However, there seems to be no clear correlation between the results from the two tests, indicating that the inherited defect leading to enhanced G(0) sensitivity is different from that causing G2 sensitivity. Less than 5% of breast cancer patients have a familial form of the disease due to inherited mutations in the breast cancer susceptibility genes BRCA1 or BRCA2. Heterozygous mutations in BRCA1 or BRCA2 in lymphocytes from women with familial breast cancer are also associated with mutagen sensitivity. Differentiation between mutation carriers and controls seems to be much better with the MNT than with the G2 assay. Mutagen sensitivity was detected with the MNT not only after irradiation but also after treatment with chemical mutagens including various cytostatics. The enhanced formation of micronuclei after exposure of lymphocytes to these substances suggests that different DNA repair pathways are affected by a BRCA1 mutation in accordance with the proposed central role of BRCA1 in maintaining genomic integrity. Mutations in BRCA1 and BRCA2 seem to predispose cells to an increased risk of mutagenesis and transformation after exposure to radiation or cytostatics. This raises a question about potentially increased risks by mammography and cancer therapy in women carrying a mutation in one of the BRCA genes. Lymphoblastoid cell lines (LCLs) from breast cancer patients have been used to study the mechanisms and genetic changes associated with tumorigenesis. With respect to mutagen sensitivity, conflicting results have been reported. In particular enhanced induction of micronuclei does not seem to be a general feature of LCLs with a BRCA1 mutation in contrast to lymphocytes with the same mutation. Therefore, LCLs are of limited utility for studying the mechanisms underlying chromosomal mutagen sensitivity.

The effect of defective DNA double-strand break repair on mutations and chromosome aberrations in the Chinese hamster cell mutant XR-V15B.

The radiosensitive Chinese hamster cell line XR-V15B was used to study the effect of decreased rejoining of DNA double-strand breaks (DSBs) on gene mutations and chromosome aberrations. XR-V15B cells are hypersensitive to the cytotoxic effects of neocarzinostatin (NCS) and methyl methanesulfonate (MMS). Both mutagens induced more chromosome aberrations in XR-V15B cells than in the parental cell strain. The clastogenic action of NCS was characterized by the induction of predominantly chromosome-type aberrations in cells of both strains, whereas MMS induced mainly chromatid aberrations. The frequency of induced gene mutations at the hprt locus was not increased compared to the parental V79 cells when considering the same survival level. Molecular analysis by multiplex polymerase chain reaction (PCR) of mutants induced by NCS revealed a high frequency of deletions in cells of both cell lines. Methyl methane-sulfonate induced mainly mutations without visible changes in the PCR pattern, which probably represent point mutations. Our findings suggest a link between a defect in DNA DSB repair and increased cytotoxic and clastogenic effects. However, a decreased ability to rejoin DNA DSBs does not seem to influence the incidence and types of gene mutations at the hprt locus induced by NCS and MMS.

"Unusual SCD" explained by differences in BrdU incorporation during subsequent cell cycles.

In the course of three consecutive cell cycles, V79 cells were labeled with BrdU by different labeling protocols. Cells treated for three cycles with bromodeoxyuridine (BrdU) showed third division metaphases, with the typical appearance after fluorescent plus Giemsa (FPG) staining (i.e., 75% of the chromatids showed light staining, 25% showed dark staining). The same staining pattern is achieved by the second labeling protocol, during which the cells have replicated for two cycles in the presence of BrdU and, during the last cycle, in the absence of BrdU. Cells that have replicated only for one cell cycle in BrdU-containing medium, and the following two cycles in normal medium, depict just the opposite staining pattern (i.e., 75% dark, 25% light). These experiments explain how the variation of BrdU substitution in the DNA leads to altered FPG staining. Unexpected staining patterns ("unusual SCD") are also observed after seemingly permanent BrdU substitution. This phenomenon, which has been found in cancer cells, is due to the decrease in BrdU concentration and not to a peculiarity of the cells investigated.

Comparative investigations of the genotoxic effects of metals in the single cells gel (SCG) assay and the sister chromatid exchange (SCE) test.

Sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) were tested for their ability to induce genotoxic effects in the single cell gel (SCG) assay and the sister chromatid exchange (SCE) test in human blood cultures in vitro. Both metals induced DNA damage in white blood cells that was expressed and detected as DNA migration in the SCG assay. Dose dependent effects were seen for cadmium in concentrations from 5 x 10(-4)-5 x 10(-3) M and for arsenic in concentrations from 2 x 10(-4)-1.5 x 10(-3) M. The distribution of DNA migration among cells, a function of dose, revealed that the majority of exposed cells expressed more DNA damage than cells from control cultures and that with increasing length of DNA migration the variability in migration among cells increased as well. Treatment of cells for 2 hr or 24 hr beginning 48 hr after the start of the blood cultures did not increase the SCE frequency in the case of cadmium but caused a small but significant SCE induction with arsenic at the highest concentration. The metal concentrations which could be investigated in the SCE test were much lower due to a strong toxic effect. Metal concentrations which were toxic in the SCE test were without visible effect in the SCG assay. Thus the two endpoints for the determination of genotoxic effects in vitro differed markedly with respect to the detection of genotoxicity induced by metals. These differences and the biological significance of the findings are discussed.

Effect of arsenic and cadmium on the persistence of mutagen-induced DNA lesions in human cells.

The alkaline single cell gel electrophoresis (SCG test or comet assay) was used to characterize the influence of sodium arsenite (NaAsO2) and cadmium sulphate (CdSO4) on the persistence of mutagen-induced DNA lesions. Human blood and SV4O-transformed fibroblasts (MRC5CV1) were treated for 2 hr with methyl methanesulphonate (MMS) or benzo(a)pyrene (BaP). MMS induced concentration-related DNA damage in white Blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage in white blood cells (WBC) and fibroblasts in similar concentrations. For the induction of DNA damage by BaP, higher concentrations had to be applied to WBC than to the fibroblast cell line. To study the influence of metal ions on the persistence of DNA lesions, treated cells were further incubated for 2 hr in the absence (postincubation) or presence (posttreatment) of NaAsO2 or CdSO4. After postincubation, MMS and BaP-induced DNA effects were reduced in both cell types, indicating that repair of DNA lesions had taken place. When the cells were posttreated with NaAsO2 or CdSO4, BaP- and MMS-induced DNA lesions persisted in both cell types, indicating an inhibition of DNA repair by these metals. The results suggest a strong interaction of arsenic and cadmium with BaP- and MMS-induced DNA repair processes.

Detection of crosslinks with the comet assay in relationship to genotoxicity and cytotoxicity.

The alkaline comet assay is a sensitive test for the detection of a variety of DNA lesions. However, crosslinks are not readily detected under standard test conditions. Recently, modifications have been introduced measuring crosslinks by determining the reduction of induced DNA migration. We used the comet assay to comparatively investigate in V79 cells the effect of three different crosslinkers: formaldehyde (FA), which predominantly induces DNA-protein crosslinks, cisplatin (DDP), which mainly produces DNA-DNA-intrastrand crosslinks, and mitomycin C (MMC), which mainly leads to DNA-DNA-interstrand crosslinks. In the standard alkaline comet assay, only MMC induced a slight increase in DNA migration at high toxic concentrations. FA and DDP did not induce any DNA migration under the test conditions used. In the modified comet assay, all three crosslinkers led to a clear reduction of gamma-ray-induced DNA migration. This reduction was seen in the case of FA parallel to the induction of cytotoxicity and SCE, while for MMC and DDP induction of cytotoxicity, SCE and HPRT gene mutations occurred at much lower concentrations than the effects in the comet assay. The DNA-DNA crosslinkers caused a reduction of induced DNA migration only at cytotoxic concentrations. Our results indicate that the modified comet assay protocol is a sensitive test for the detection of DNA-protein crosslinks. However, the results for MMC and DDP suggest that the modified protocol is not well suited for the evaluation of DNA-DNA crosslinkers. Furthermore, the relationship between crosslinking and genotoxicity seems to be very different for the three different types of crosslinking substances.

Significance of formaldehyde-induced DNA-protein crosslinks for mutagenesis.

Formaldehyde (FA) is a genotoxic substance, induces tumors in the nasal epithelium of rats, and is suspected to be a human carcinogen. As a primary DNA lesion, FA induces DNA-protein crosslinks (DPC) and the formation of DPC has been used as a measure of exposure for risk estimation. However, the significance of DPC for mutagenesis and carcinogenesis is at present poorly understood. We therefore performed comparative investigations on the induction of DPC and other genetic endpoints by FA in V79 Chinese hamster cells. The amount of DPC was comparatively determined with the K-SDS assay and the comet assay. Both tests gave similar results but the comet assay was much foster and easier to perform. Our results show that FA significantly induces DPC, sister-chromatid exchanges, and micronuclei in the same range of concentrations, parallel to the induction of cytotoxicity (relative cloning efficiency). In contrast, treatment of V79 cells with FA did not induce gene mutations in the HPRT test even after variations of the treatment protocol. Our results indicate that FA-induced DPC seem to be related to cytotoxicity and clastogenicity but do not lead to the formation of gene mutations in mammalian cells. It is suggested that FA-induced DPC do not cause gene mutations that are involved in FA-induced carcinogenesis.

Comparative biomonitoring study of workers at a waste disposal site using cytogenetic tests and the comet (single-cell gel) assay.

Workers exposed to environmental pollutants at a waste disposal site were studied for genotoxic effects with cytogenetic tests and the comet (alkaline single-cell gel) assay. Analyses were performed on peripheral blood samples of 44 workers at a waste disposal site (DM) and 47 subjects of a control group (VE) matched for gender, age, and smoking habits. Chromosomal aberrations were evaluated in 1,000 lymphocytes per individual, sister chromatid exchanges in 50 cells, and DNA migration (tail moment) was determined in 100 leukocytes. Structural chromosome aberrations were more frequent in DM than in VE, but only the frequency of acentric fragments and the percentage of aberrant cells (excluding gaps) was significantly increased. No significant difference was found for the mean frequency of SCE. A statistically significant difference was also seen with the comet assay. The mean tail moment was higher in DM than in VE. However, no correlation was found between cytogenetic data and the effects in the comet assay. The results of our study indicate that DNA effects in the comet assay represent an independent endpoint which might be useful for the biomonitoring of genotoxic effects in addition to established tests.

DNA-damaging effect of cyclophosphamide on human blood cells in vivo and in vitro studied with the single-cell gel test (comet assay).

The single-cell gel (SCG) test was used to study the induction and persistence of DNA damage by cyclophosphamide (CP) in human blood cells after treatment in vitro and in vivo. S9-mix-activated CP (from 0.1 mM upward) induced DNA effects in a concentration-dependent manner in the in-vitro SCG test. Blood cells from various donors showed considerable intra- and interindividual variability. Incubation of CP-treated blood samples at 37 degrees C caused a rapid decrease in DNA effects, but DNA migration was still significantly increased 1 hr after the end of the CP treatment. Comparative studies with the in vitro sister chromatid exchange (SCE) test were performed that demonstrated that much lower CP concentrations (about 100 times) were required for a significant induction of SCEs. A group of 11 patients who suffered from vasculitis/collagen disease and were treated with low CP doses (50-200 mg/day) exhibited an elevated level of DNA damage in the SCG test with peripheral blood cells, compared with a group of 11 control persons or 5 patients without chemotherapy. Increases in DNA damage were variable and not clearly related to the CP dose. SCE tests could successfully be performed with 5 out of the 11 CP-treated patients; all showed significantly increased SCE frequencies. For six patients no result could be obtained with the SCE test due to a failure of lymphocyte proliferation. Three multiple sclerosis patients who received high doses of CP were investigated with the SCG test before, during, and after the treatment. The results indicate that CP-induced DNA effects that are detectable with the SCG test persist in vivo for a period of several days, but for less than 2 weeks. The results of our study provide information with regard to the use of the SCG test in human monitoring. The advantages and limitations of the test are discussed.

Evaluation of mutagenic effects of hyperbaric oxygen (HBO) in vitro.

Hyperbaric oxygen (HBO) treatment as used therapeutically (i.e., exposure to 100% oxygen at a pressure of 1.5 bar for a total of 60 min) has been shown to induce DNA damage in the alkaline comet assay with leukocytes from test subjects. Under these conditions, HBO did not lead to an induction of gene- and chromosome mutations. Due to known toxic effects, exposure of humans to HBO is limited and possible genetic consequences of HBO could not be completely evaluated in vivo. We thus established an in vitro HBO model, where human blood cells or V79 cells were exposed to hyperbaric oxygen (98% O(2) and 2% CO(2) at a pressure of either 1.5 or 3 bar) for up to 3 hr in a temperature-controlled hyperbaric chamber. Using the comet assay, we found exposure-related genotoxic effects in V79 cells, whole blood, and isolated lymphocytes. V79 cells showed the highest sensitivity toward HBO-induced DNA damage, and the exposure conditions applied to blood in vitro, to induce DNA migration, had to be higher than those used in vivo. We could also show that prolonged HBO treatment clearly increased the frequency of micronuclei in V79 cells, whereas it exerted only a marginal effect on the frequency of hprt mutations. These results demonstrate that HBO treatment of cell cultures is a well-suited model for investigating the biological significance of oxidative stress. The relationship between oxygen-induced DNA lesions and the formation of gene- and chromosome mutations is discussed.

Evaluation of the mutagenicity of beta-myrcene in mammalian cells in vitro.

The genotoxicity of the terpene beta-myrcene was evaluated in mammalian cells in vitro. Myrcene is the major constituent of oil of bay and hop which are used in the manufacture of alcoholic beverages. Myrcene is also present in lemon grass (Cymbopogon citratus), a plant widely used in Brazilian folk medicine. Recently, it was shown that myrcene is a very potent analgesic substance and might be an alternative to the already available analgesic drugs. Myrcene was tested up to 1,000 micrograms/ml (limit of solubility) in the presence and absence of S9-mix and did not induce chromosome aberrations and sister chromatid exchanges (SCEs) in human lymphocytes in vitro. Neither the mitotic index nor the proliferation index was influenced by the myrcene treatment. Myrcene did not cause increased mutation frequencies at the hprt-locus in V79-cells. Tests with and without S9-mix revealed negative results. There was no indication for induced cytotoxicity. However, myrcene reduced the SCE-inducing effect of cyclophosphamide in human lymphocytes in a dose dependent manner and also reduced the toxic and mutagenic effect of cyclophosphamide in V79-cells. Under the same test conditions, SCE induction by ethyl methanesulfonate (EMS) and benzo [a]pyrene (BP) was not significantly influenced by simultaneous myrcene treatment. The in vitro results show that myrcene is not mutagenic in mammalian cells, but has antimutagenic properties. The possibility that myrcene exerts its antimutagenic activity by inhibiting certain forms of the cytochrome P-450 isoenzymes required for activation of premutagens and precarcinogenes is discussed.

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.

The contribution of cytotoxicity to DNA-effects in the single cell gel test (comet assay).

We evaluated genotoxic and cytotoxic effects of the three non-mutagenic and non-carcinogenic compounds p-nitrophenol, D-menthol and sodium N-lauroyl sarcosine which have previously been shown to induce DNA double strand breaks (DNA dsb) secondary to induced cytotoxicity. We tested whether genotoxic effects in the alkaline single cell gel test (comet assay) may be confounded by cytotoxicity-induced DNA dsb. Cell viability was determined at the end of the treatment using the fluorescein diacetate/ethidium bromide-assay and plating efficiency was used as an indicator of long-term survivability. Experiments with V79 Chinese hamster cells and human white blood cells revealed negative results in the comet assay despite strong cytotoxic effects. However, cells with extremely fragmented DNA ('clouds') occurred but were excluded from the evaluation under the principle that they represent dead cells. We also noticed a significant loss of cells at cytotoxic concentrations that might be attributed to the induction of highly fragmented DNA which is lost during electrophoresis. Since the comet assay allows the determination of DNA effects on the single cell level, a confounding effect of cytotoxicity on test results can be avoided.

Detection of DNA effects in human cells with the comet assay and their relevance for mutagenesis.

The single cell gel test (SCG-test or comet assay) is a rapid and sensitive method for measuring DNA damage and repair in individual cells. A wide variety of mutagens have been shown to cause DNA alterations detectable with the comet assay, but it is not yet clear whether a relationship exists between the DNA effects and the induction of mutations. We are therefore investigating in a cell culture system with human cells (MRC5CV1) the induction of DNA damage by environmental mutagens and the formation of mutations at the HPRT gene. In the present study we investigated benzo[a]pyrene (BP), an environmental mutagenic and carcinogenic polycyclic aromatic hydrocarbon, and its reactive metabolite (+)-anti-benzo[a]pyrene-7,8-diol 9, 10-oxide ((+)-anti-BPDE). S9 mix activated BP and the direct acting mutagen (+)-anti-BPDE caused a concentration-related increase in DNA migration in the comet assay. Postincubation experiments indicated that induced DNA effects are eliminated by DNA repair within 24 h. BP-treatment caused a strong genotoxic effect in the comet assay but had only a marginal effect on the frequency of gene mutations. When cells were treated with BP in the presence of cadmium sulphate, a clear increase in genotoxicity was observed while the effect on mutations was unchanged. Our results indicate that DNA alterations detected with the comet assay do not necessarily relate to mutagenesis. The absence of a close relationship between DNA migration in the comet assay and mutagenesis may be explained by the fact that some effects seen in the comet assay occur as a consequence of an error free DNA repair process.

The influence of temperature during alkaline treatment and electrophoresis on results obtained with the comet assay.

The alkaline comet assay (single-cell gel electrophoresis) is becoming established as a genotoxicity test with many fold applications in vitro and in vivo. While the underlying principles are identical, various modifications of the method are in use which clearly affect the sensitivity and resolving power of the assay. One variable of potential importance that has been disregarded until now is temperature during alkaline treatment and electrophoresis. We therefore performed comet assay experiments with human blood and V79 Chinese hamster cells using two different temperatures (4 and 20 degrees C, i.e. room temperature) during alkaline treatment and electrophoresis. DNA damage was induced by the two standard mutagens gamma irradiation and methyl methanesulfonate (MMS). The results clearly indicate significant differences in the detection of background and mutagen-induced DNA damage at these two temperatures. Under otherwise identical test conditions (including the duration of alkaline treatment and electrophoresis), increased temperature during alkaline treatment and electrophoresis strongly enhances DNA migration. Our findings suggest that the comet assay should be performed under strictly controlled and reproducible temperature conditions. In any case the temperature during alkaline treatment and electrophoresis should be stated in a publication to allow for a critical evaluation of results obtained with the comet assay.

The relationship between the induction of SCEs and mutations in Chinese hamster cells. I. Experiments with hydrogen peroxide and caffeine.

Hydrogen peroxide (H2O2) and caffeine were examined for their capacity for inducing SCEs and mutations at the HPRT locus in V79 Chinese hamster cells. Although, under standard conditions, both substances induced SCEs neither caused gene mutations. The SCE induction by both H2O2 and caffeine is influenced by BrdUrd substitution. Whereas H2O2 also induces lesions leading to SCEs in normal DNA, the SCe induction by caffeine depends on the replication of BrdUrd-substituted DNA. In cells with BrdUrd-substituted DNA, H2O2 induces mutations at the HPRT locus parallel to its SCE induction, whereas caffeine in the presence of BrdUrd only has an influence on the SCE rate. It is shown that the experimental conditions of the two test systems can play a decisive role when contradictory results are obtained.