UV-induced Sister-chromatid Exchanges Research Articles

PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38-/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38-/- cells did not show a significant sensitivity to either UV or H2O2, a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38-/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38-/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.

A Novel Histone Crosstalk Pathway Important for Regulation of UV-Induced DNA Damage Repair in Saccharomyces cerevisiae.

Histone post-translational modifications play vital roles in a variety of nuclear processes, including DNA repair. It has been previously shown that histone H3K79 methylation is important for the cellular response to DNA damage caused by ultraviolet (UV) radiation, with evidence that specific methylation states play distinct roles in UV repair. Here, we report that H3K79 methylation is reduced in response to UV exposure in Saccharomyces cerevisiae This reduction is specific to the dimethylated state, as trimethylation levels are minimally altered by UV exposure. Inhibition of this reduction has a deleterious effect on UV-induced sister chromatid exchange, suggesting that H3K79 dimethylation levels play a regulatory role in UV repair. Further evidence implicates an additional role for H3K79 dimethylation levels in error-free translesion synthesis, but not in UV-induced G1/S checkpoint activation or double-stranded break repair. Additionally, we find that H3K79 dimethylation levels are influenced by acetylatable lysines on the histone H4 N-terminal tail, which are hyperacetylated in response to UV exposure. Preclusion of H4 acetylation prevents UV-induced reduction of H3K79 dimethylation, and similarly has a negative effect on UV-induced sister chromatid exchange. These results point to the existence of a novel histone crosstalk pathway that is important for the regulation of UV-induced DNA damage repair.

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.

Histone H3 K79 methylation states play distinct roles in UV-induced sister chromatid exchange and cell cycle checkpoint arrest in Saccharomyces cerevisiae.

Histone post-translational modifications have been shown to contribute to DNA damage repair. Prior studies have suggested that specific H3K79 methylation states play distinct roles in the response to UV-induced DNA damage. To evaluate these observations, we examined the effect of altered H3K79 methylation patterns on UV-induced G1/S checkpoint response and sister chromatid exchange (SCE). We found that the di- and trimethylated states both contribute to activation of the G1/S checkpoint to varying degrees, depending on the synchronization method, although methylation is not required for checkpoint in response to high levels of UV damage. In contrast, UV-induced SCE is largely a product of the trimethylated state, which influences the usage of gene conversion versus popout mechanisms. Regulation of H3K79 methylation by H2BK123 ubiquitylation is important for both checkpoint function and SCE. H3K79 methylation is not required for the repair of double-stranded breaks caused by transient HO endonuclease expression, but does play a modest role in survival from continuous exposure. The overall results provide evidence for the participation of H3K79 methylation in UV-induced recombination repair and checkpoint activation, and further indicate that the di- and trimethylation states play distinct roles in these DNA damage response pathways.

Application of the biotin–dUTP chromosome labelling technique to study the role of 5-bromo-2′-deoxyuridine in the formation of UV-induced sister chromatid exchanges in CHO cells

The role of 5-bromo-2′-deoxyuridine (BrdU) in the formation of sister chromatid exchanges (SCEs) in cells exposed to UV radiation was studied. Cells were unifilarily labelled (labelling of one strand of chromosomal DNA) with BrdU or biotin-16-2′-deoxyuridine (biotin-dU) and irradiated in G 1 phase of the cell cycle either with 254 nm, which is absorbed by all nucleobases including bromouracil (BrU) or with 313 nm radiation, which is predominantly absorbed by the BrU moiety. Elevated SCE frequencies were observed in cells irradiated at 254 nm (1.2 and 3.0 J m −2) which were pre-labelled with BrdU or biotin-dU. Following irradiation at 313 nm (38 and 96 J m −2) a statistically elevated SCE frequency was observed in cells pre-labelled with BrdU but not with biotin-dU. In cells pre-labelled with BrdU, UV-radiation at 254 nm was 50–80 times more effective in inducing SCEs than that at 313 nm. This result can be accounted for by the fact that in BrdU-DNA the cross-section for uracilyl radical and bromine atom formation is ∼100-fold higher at 254 nm than that at 313 nm. Upon irradiation at 254 nm, BrdU had a strong sensitising effect on SCE induction: the SCE frequencies observed in cells pre-labelled with BrdU are ∼6 times higher than in cells pre-labelled with biotin-dU. From this it follows that BrdU-induced damage is responsible for more than 80% of the SCEs formed in UV irradiated cells unifilarily labelled with BrdU. Based on photochemical considerations and the fact that chemical agents which form DNA interstrand cross-links are among the most potent inducers of SCEs, we propose that an interstrand cross-link may be the major lesion leading to SCEs in BrdU-labelled cells.

Role of DNA repair inhibition in lead- and cadmium-induced genotoxicity: a review.

Compounds of lead and cadmium have been shown to be carcinogenic to humans and experimental animals. However, the underlying mechanisms are still not understood. In mammalian cells in culture, lead(II) is weakly mutagenic after long incubation times and generates DNA strand breaks only after treatment with high, toxic doses. Cadmium(II) induces DNA strand breaks and chromosomal aberrations, but its mutagenic potential is rather weak. However, both metals exert pronounced indirect genotoxic effects. Lead(II) is comutagenic towards UV and N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and enhances the number of UV-induced sister chromatid exchanges in V79 Chinese hamster cells. With regard to DNA repair, lead(II) causes an accumulation of DNA strand breaks after UV-irradiation in HeLa cells, indicating an interference with the polymerization or ligation step in excision repair. Cadmium(II) enhances the mutagenicity of UV light in V79 Chinese hamster cells and an increased sensitivity toward UV light is observed in various rodent and human cell lines. Furthermore, an inhibition of unscheduled DNA synthesis after UV-irradiation and a partial inhibition of the removal of UV-induced DNA lesions has been shown. For both metals, the indirect genotoxic effects are observed at low, nontoxic concentrations, suggesting that an interference with DNA repair processes may be predominant at biologically relevant concentrations. This might also explain the conflicting results of epidemiological studies obtained for both metals. Possible mechanisms of repair inhibition are discussed.

Increased UV-induced SCEs but normal repair of DNA damage in p53-deficient mouse cells.

UV-induced sister chromatid exchanges (SCEs) in p53-deficient mouse cells were studied to obtain more evidence regarding the involvement of p53 protein in the DNA repair pathway as a checkpoint protein. After 5 J/m2 UV irradiation, mutant-type homozygous cells for p53-deficiency showed the same number of SCEs as the heterozygous and wild-type homozygous cells. In the heterozygous and wild-type homozygous cells, no further increase of SCEs was observed after 10 J/m2 UV irradiation. In contrast, in mutant-type homozygous cells about twice as many SCEs were induced by 10 J/m2 UV as by 5 J/m2 UV. In mutant-type homozygous cells, fractions of S-phase cells decreased just after 10 J/m2 UV irradiation, but recovered to higher than control levels within a short time, while in heterozygous and wild-type homozygous cells, the decrease in S-phase cells was prolonged by more than 6 hr and no increase above control levels was observed. Although no difference in UV sensitivity and repair of UV-induced DNA damage was found among the 3 genotypes, which were determined by the relative colony-forming ability after UV irradiation and removal of thymine dimers and (6-4) photoproducts from cellular DNA, our data strongly suggest an impaired checkpoint function in p53-deficient cells when DNA is damaged.

Genetic variations in baseline and ultraviolet light-induced sister chromatid exchanges in peritoneal lymphocytes among different mouse strains

The variation in the induction of sister chromatid exchanges (SCEs) in the peritoneal lymphocytes of different mouse strains was investigated. For the baseline SCEs, BALB/c and outbred ICR showed the lowest frequency and DBA/2 and C57BL/6 the highest. BDF 1 (C57BL/6 × DBA/2) was ranked among the highest, while CDF 1 (BALB/c × DBA/2) was intermediate between the parental strains. Regarding UV-induced SCEs, BALB/c was less susceptible as compared to DBA/2 and C57BL/6. Both BDF 1 and CDF 1 showed values significantly higher than BALB/c, but not significantly different from DBA/2 or C57BL/6. ICR was ranked in the susceptible group. For the baseline SCEs of bone marrow cells, the overall ranking among strains was essentially the same as that for the baseline, but different from that for the UV-induced, SCEs in peritoneal lymphocytes. The present results can be explained by assuming that the major genetic factor contributing to the strain-dependent difference in the baseline SCEs is due to a codominant trait of a single, allele, but that the UV-induced SCEs are complicated by other genetic factor(s).

Dysplastic nevus syndrome: intrafamilial identification of carriers by cytogenetics.

Seven members of a family with dysplastic nevus syndrome (DNS) were examined clinically; skin biopsies of unaffected skin from 6 were taken. Biopsy-derived cultivated fibroblasts were examined by cytogenetic methods, i.e. by measuring the spontaneous and the UVB- and UVC-driven increase in sister chromatid exchange (SCE). A male patient with malignant melanoma, his son and his nephew, both with multiple dysplastic nevi, showed a distinctive elevation of UV-induced SCE, whereas the other, unaffected members of the family showed normal values. These results give evidence that in siblings with DNS the affected members can be identified not only on clinicopathological grounds but also by UV-induced elevated SCE at the cytogenetic level.

Increased UV-induced sister-chromatid exchange in cultured fibroblasts of first-degree relatives of melanoma patients

Cultured fibroblasts of 17 first-degree relatives of familial melanoma patients and six first-degree relatives of cutaneous melanoma (CMM) patients with multiple CMM primaries were tested for in vitro sensitivity to UV light. Fibroblasts of nine familial CMM patients with a known UV-sensitivity and 19 healthy probands served as a control. Sister chromatid exchange (SCE) was used as a parameter to detect UV-induced genotoxic damage. We found significantly (p < 0.001) increased UV-induced SCE levels in familial melanoma patients, as well as in first-degree relatives of familial melanoma patients (p < 0.001) after UV-A,B irradiation (375 J/m 2), compared to the healthy probands without a family history of CMM. A significant (p < 0.001) increase of UV-induced SCE was also observed in the relatives of CMM patients with multiple CMM primaries. In addition, the spontaneous SCE were significantly increased (p < 0.05) in familial CMM patients. This study shows that increased UV sensitivity is a familial phenomenon. It is consistent with the concept of a genetic predisposition to CMM, which is based on increased UV sensitivity and may help to define groups with an elevated risk of developing cutaneous malignant melanoma.

BHK cell lines with increased rates of gene amplification are hypersensitive to ultraviolet light.

Four cell lines (MP1, -4, -5, -7), isolated from baby hamster kidney cells after simultaneous selection with N-(phosphonacetyl)-L-aspartate and methotrexate, have previously been shown to amplify their DNA at an increased rate. We now show that all four lines are hypersensitive to killing by UV light and mitomycin C. At high doses of UV light or mitomycin C, the MP lines survived less than 10% or less than 5% as well as parental cells, respectively. After UV irradiation, inhibition of DNA and RNA synthesis was greater in MP than in parental cells, and recovery was slower or absent. A 2- to 3.5-fold increase in the frequency of UV-induced sister chromatid exchange was also seen in the four cell lines. In MP5, unscheduled DNA replication after treatment with UV light was only approximately 70% as great as in parental cells and the other MP lines. In MP4 and MP7 cells S phase was elongated. Although their individual properties confirm that the four cell lines are independent, their common properties suggest a relationship between tolerance of DNA damage and gene amplification.

Suppressing effect of tannic acid on the frequencies of mutagen-induced sister-chromatid exchanges in mammalian cells

The effects of tannic acid ( m-galloyl gallic acid) and 7 of its analogues on the frequencies of sister-chromatid exchanges (SCEs) were investigated in cultured Chinese hamster cells. SCEs induced by UV-light or mitomycin C (MMC) were suppressed by post-treatment with tannic acid and 5 of its analogues. These effects were independent of the extension of the cell cycle. The compounds which showed an SCE-suppressing effect have a common structure of 3 neighboring hydroxy o methoxy groups substituted on the phenyl group in a benzoic acid or ester. These decreasing effects of tannic acid were observed in the G 1 phase but not in the S or G 2 phase of the cell cycle and a greater decline of the frequencies of UV-induced SCEs during liquid holding was seen in the presence of tannic acid. However, cells irradiated with X-rays were not influenced by tannic acid. In cells from a xeroderma pigmentosum (XP) patient, a Fonconi's anemia (FA) patient, and a normal human embryo, MMC-induced SCEs were also decreased by post-treatment with tannic acid. Tannic acid reduced the SCE frequencies in UV-irradiated FA and normal human cells but not in UV-irradiated XP cells. Our results suggest that tannic acid modifies DNA-excision repair and that the decrease in the amount of unrepaired DNA damage might cause the reduction of induced SCEs.

Modifying effects of components of plant essence of the induction of sister-chromatid exchanges in cultured Chinese hamster ovary cells

The influcence of 21 kinds of components of plant essence on sister-chromatid exchanges (SCEs) induced by mitomycin C was investigated in cultured Chinese hamster CHO K-1 cells. Posttreatment with scopoletin, jasmone, caffeic acid and ferulic acid significantly increased the frequency of SCEs. Further investigation of the SCE-enhancing effect of analogues of caffeic acid and ferulic acid revealed that an α,β-unsaturated carbonyl group may be necessary for SCE-enhancing effects. The influence of caffeic acid and ferulic acid on X-ray- or UV-induced SCEs was also studied. The frequencies of SCEs induced by UV were increased by treatment with these compounds. This increasing effect was observed in the S phase of the cell cycle. On the contrary, X-ray-induced SCEs were reduced by the treatment with these compounds. The decreasing effect was observed in the G 1 phase but not in the S or G 2 phase. To explain these contradictory results, we assumed that caffeic acid and ferulic acid may modify the repair of DNA strand breaks.

Enhancing effects of cinoxate and methyl sinapate on the frequencies of sister-chromatid exchanges and chromosome aberrations in cultured mammalian cells

Sister-chromatid exchanges (SCEs) induced by mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) or UV-light in cultured Chinese hamster ovary cells (CHO K-1 cells) were enhanced by cinoxate (2-ethoxyethyl p-methoxycinnamate) or methyl sinapate (methyl 3,5-dimethoxy 4-hydroxycinnamate). Both substances are cinnamate derivatives and cinoxate is commonly used as cosmetic UV absorber. Methyl sinapate also increased the frequency of cells with chromosome aberrations in the CHO K-1 cells treated with MMC, 4NQO or UV. These increasing effects of methyl sinapate were critical in the G 1 phase of the cell cycle and the decline of the frequencies of UV-induced SCEs and chromosome aberrations during liquid holding was not seen in the presence of methyl sinapate. Both compounds were, however, ineffective in cells treated with X-rays. In cells from a normal human embryo and from a xeroderma pigmentosum (XP) patient, MMC-induced SCEs were also increased by the post-treatment with methyl sinapate. The SCE frequencies in UV-irradiated normal human cells were elevated by methyl sinapate, but no SCE-enhancing effects were observed in UV-irradiated XP cells. Our results suggest that the test substances inhibit DNA excision repair and that the increase in the amount of unrepaired DNA damage might cause the enhancement of induced SCEs and chromosome aberrations.

Characterization of the enhancing effect of caffeine on sister-chromatid exchanges induced by ultraviolet radiation in excision-proficient xeroderma pigmentosum lymphoblastoid cells

• Cells of some excision-proficient xeroderma pigmentosum (XP) cell lines are highly sensitive to post-UV caffeine treatment in terms of sister-chromatid exchange (SCE) induction as well as cell lethality. • In the present study, we conducted a detailed investigation of the enhancing effect of caffeine on SCE frequency induced by UV in excision-proficient XP cells, and obtained the following results. (1) Continuous post-UV treatment with 1 mM caffeine markedly enhances UV-induced SCEs and such enhanced SCEs occur with similar frequency during either the 1st or the 2nd cell cycle in the presence of caffeine and 5-bromodeoxyuridine (BrdUrd). (2) The high sensitivity of the cells to post-UV caffeine treatment persists for at least 2 days after UV when irradiated cells are held in either the proliferating or the nonproliferating state prior to the addition of BrdUrd. (3) Caffeine exerts its effect on cells in S phase. (4) Neither BrdUrd in the medium nor the incorporated 5-bromodeoxyuridine monophosphate (BrdUMP) in DNA plays an appreciable role in the expression of the enhancing effect of caffeine. • The most likely explanation for our findings is as follows. In excision-proficient XP cells, the cause of SCE formation such as UV-induced lesions or resulting perturbations of DNA replication persists until the 2nd round or more of post-UV DNA replication. If caffeine is given as post-UV treatment, such abnormalities may be amplified, resulting in a synergistic increase in SCE frequency.

Effects of radioprotective ginseng protein on UV induced sister chromatid exchanges

To elucidate the reaction mechanism of ginseng protein on its antiradiation activity, its effects were studied on sister chromatid exchanges (SCE) induced by UV irradiation in CHO-Kl cells. When cells were irradiated with 254 nm UV light at the dose of 0 to 80 erg/mm2, the frequencies of SCE were increased more than two fold. However, when radio-protective ginseng protein was added to the cells before and after UV irradiation, SCE frequencies were decreased significantly at all UV doses in both cases with no significant differences. As the amount of ginseng protein was varied from 100 to 500 μg/ml, with UV irradiation at 60 erg/mm2, SCE frequencies dropped sharply at the first two concentrations and then reached a sort of plateau in both cases of pre-and post-treatment. When the ginseng protein was treated alone without UV irradiation, there were no changes in SCE frequencies no matter when the protein was added. These results suggest that the ginseng protein could reduce DNA damages, which may play an important role in the reaction mechanism of radioprotective activity of the protein.

Inhibition of protein synthesis does not antagonize induction of UV-induced sister-chromatid exchange in xeroderma pigmentosum cells.

Cycloheximide strongly antagonizes the induction of sister-chromatid exchanges by ethyl methanesulfonate or mitomycin C in human skin fibroblast and xeroderma pigmentosum cells (group A). Analogous behavior has been observed in several other species including Chinese hamster and plant cells. This report documents an exception to that pattern: cycloheximide fails to antagonize UV-induced sister chromatid exchange in xeroderma pigmentosum cells, whereas it does in normal human skin fibroblast cells. A genetic defect in these cells is postulated to alter the UV-mediated DNA recombination process.

Chromosomal instability in mutagen-sensitive mutants isolated from mouse lymphoma L5178Y cells II. Abnormal induction of sister-chromatid exchanges and chromosomal aberrations by mutagens in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1)

To determine the mutual relationships between cell survival and induction of sister-chromatid exchanges (SCEs) as well as chromosomal aberrations (CAs), mutagen-induced SCEs and CAs were analyzed in an ionizing radiation-sensitive mutant (M10) and an alkylating agent-sensitive mutant (MS 1) isolated from mouse lymphoma L5178Y cells. The levels of CA induction in both mutants strictly corresponded to the sensitivity to lethal effects of mutagens, except that caffeine-induced CAs in M10 are considerably lower than those in L5178Y. The results clearly indicate that except for caffeine-induced CAs in M10, mutagen-induced lethal lesions are responsible for CA induction. In contrast, SCE induction in mutants was complicated. In M10, hypersensitive to killing by γ-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeince, the frequency of SCEs induced by γ-rays was barely higher than that in L5178Y, and the frequencies of MMS- and UV-induced SCEs were similar to those in L5178Y, but 4NQO- and caffeine-induced SCEs were markedly lower than those in L5178Y. MS 1, which is hypersensitive to MMS and caffeine, but not sensitive to UV or 4NQO, responded to caffeine with an enhanced frequency of SCEs and had a normal frequency of MMS-induced SCEs, but a reduced frequency of UV- and 4NQO-induced SCEs. Thus, susceptibility to SCE induction by mutagens is not necessarily correlated with sensitivity of mutants to cell killing and/or CA induction by mutagens. Furthermore, the spontaneous levels of SCEs are lower in M10 and higher in MS 1 than that in L5178Y (Tsuji et al., 1987). Based on these results, we speculate that M10 may be partially defective in the processes for the formation of SCEs caused by mutagens. On the other hand, MS 1 may modify SCE formation-related lesions induced by UV and 4NQO to some repair intermediates that do not cause SCE formation. In addition, MMS-induced lethal lesions in MS 1 may not be responsible for SCE induction whereas caffeine-induced lethal are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsble for cell lethality or CA production.

Sister-chromatid exchanges induced by ultraviolet light in Bloom's syndrome fibroblasts

The relationships between the cytotoxic effect of ultraviolet light and the UV-induced sister-chromatid exchanges (SCEs) were compared among fibroblast cell strains from two unrelated Bloom's syndrome (BS) patients, one xeroderma pigmentosum (XP) patient belonging to complementation group A and two unrelated normal controls. The “net” induced SCEs as a function of UV fluence, obtained by subtracting spontaneous SCEs from observed SCEs, were much higher in both BS cells and XP group A cells than in normal cells. The relative efficiency of induced SCE, defined as the “net” induced SCEs as a function of surviving fraction after UV irradiation, was higher in BS cells than in normal and XP cells, and there was essentially no difference between XP and normal cells. These results imply that in addition to the extremely high frequency of spontaneous SCEs, the increased efficiency in UV induction of SCEs may reflect the intrinsic defect(s) in BS cells.

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens

3-Aminoharman (3AH, 3-amino-l-methyl-9 H-pyrido[3,4- b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl- N′-nitro- N-nitrosoguanidine (MNNG), 4-nitriquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5 H-pyrido[4,3- b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.