Mutagenicity of a furocoumarin epoxide, heraclenin, in Chlamydomonas reinhardii

Treatment of arg- or strd mutant cells of Chlamydomonas reinhardii with a furocoumarin epoxide, heraclenin, plus UV-A resulted in a decrease in survival and a UV-A dose-dependent increase in induced Arg+ or Strs revertants. Imperatorin, a furocoumarin with a very similar structure but lacking an epoxide group showed a very similar phototoxic and photomutagenic activity in these mutant strains. Treating the mutant cells with heraclenin or imperatorin in the dark neither influenced survival nor mutation induction. The results are discussed with respect to the involvement of the epoxide moiety of heraclenin in mutagenicity.     

The rate of sister chromatid exchanges parallel to spontaneous chromosome breakage in Fanconi's anemia and to trenimon-induced aberrations in human lymphocytes and fibroblasts.

The incidence of structural chromosome aberrations and the rate of sister chromatid exchanges (SCE) was investigated in lymphocyte cultures from a patient with typical Fanconi's anemia and his parents. The rate of SCEs was found to be normal. In experiments with the alkylating agent Trenimon the SCE rates proved to be a sensitive indicator for the induction of structural aberrations: in presence of an induced aberration rate half as high as the spontaneous rate in the Fanconi's anemia case, the rate of SCEs was found to be quintupled. Dose-effect relationships for the induction of SCE rates by Trenimon were studied over a wide dose range in lymphocyte and fibroblast cultures. The results reflect the same difference in sensitivity earlier observed in the induction of structural chromosome aberrations, fibroblasts being far more sensitive.     

Effects of CCNU therapy on human chromosomes

Peripheral blood lymphocytes of 9 patients under CCNU therapy were examined for frequency of sister-chromatid exchanges (SCEs) and chromosomal aberrations (CAs). 7 out of 9 patients were treated with only CCNU, whereas the remaining 2 were treated with other chemotherapeutic agents in combination with CCNU. Compared to normal individuals, a significantly increased frequency of SCE was observed in the patients before starting anticancer therapy (P less than 0.001). Increased incidences of structural changes in chromosomes were observed in cells from all the treated patients. The most frequent aberrations were of chromatid type. After administration of a single dose of CCNU, an increase in SCE frequencies was observed which remained elevated even after 6 weeks. It was concluded that increases in SCEs and CAs in lymphocytes were caused by CCNU treatment. Further studies are needed to elucidate whether any CAs observed in the present study could participate in the induction of second neoplasm.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations

We carried out a cross-sectional analysis of sister-chromatid exchanges (SCEs) and chromosomal aberrations induced by diepoxybutane (DEB) in lymphocyte cultures from 58 normal blood donors. DEB-induced SCE frequencies were measured in all subjects and chromosomal aberrations in 18. Analysis of variance was used to assess the contributions of exposure to organic solvents, age, smoking history, alcohol and coffee consumption, and red and white blood cell counts to variations in DEB-induced SCEs. In 10 individuals, the epoxide-detoxifying enzyme, glutathione (GSH)-S-transferase mu, was also measured. We observed a bimodal distribution of DEB-induced SCEs in the study population. Approx. 24% of the individuals were twice as sensitive to the induction of SCEs by DEB as the remaining 76%. Lymphocytes from persons sensitive to SCE induction by DEB contained a 4.4-fold increase in the number of DEB-induced chromatid deletions and exchanges. Within sensitive and resistant groups, significant interindividual variations in DEB-induced SCE frequencies were noted. Cigarette smoking was weakly associated with lower SCE frequencies within each group. Genetic deficiency in GSH-S-transferase mu was not correlated with increased sensitivity to SCE induction by DEB. Sensitivity to induction of SCEs by DEB can be rapidly determined and may be a marker of sensitivity to the induction of genotoxicity by certain classes of mutagens.     

Cytogenetic damage in workers exposed to ethylene oxide

Sister-chromatid exchanges (SECs) and chromosomal aberrations (CAs) were detected in the peripheral lymphocytes of 41 sanitary workers exposed to ethylene oxide (EO) in the sterilizing units of 8 hospitals in the Venice Region. The first group (19 workers) was exposed to 10.7 +/- 4.9 ppm EO, expressed as the time-weighted average concentration for an 8-h working day (TWA/8 h conc.), and the second group (22 workers) to 0.35 +/- 0.12 ppm. Each exposed worker was paired with a control of similar age and smoking habits. A highly significant (P less than 0.001) increase in the mean frequency of SCEs was found in the higher exposure group, 14 (74%) exposed subjects having significantly increased levels of SCEs compared to their matched controls. In the lower exposure group, the increase in mean frequency of SCEs was lower, though still significant (P less than 0.05): 7 (33%) exposed subjects had higher and 1 (5%) had a lower SCE level than the matched controls. From the first group, 10 subjects, 7 of whom had increased SCE levels, were reanalysed 12-18 months after their exposure had been lowered or interrupted: in only 2 of them the SCE level was significantly decreased. A statistically significant correlation between SCE frequency and level of EO exposure (TWA/8 h conc.), as well as a multiple correlation between SCE level and EO exposure, smoking and age were found. However, no interaction could be detected between EO exposure and smoking in the induction of SCEs. In controls, SCE frequency was correlated with smoking and age. In the higher exposure group, the number of both chromatid- and chromosome-type aberrations, independent of gaps, was significantly increased, whereas in the lower exposure group only the frequency of chromosome-type aberrations, excluding gaps, was statistically higher than in controls. The level of CAs remained to a great extent unchanged in the 10 subjects re-examined at a later stage after lowering or halting exposure. Taking the group as a whole, the frequency of cells with total CAs was found to be weakly (P = 0.05) correlated with EO exposure, and was not correlated with smoking, age or SCE frequency.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.     

Enhancement of X-ray-induced sister chromatid exchanges in hypoxic cells

In these studies we have used wild-type Chinese hamster ovary cells (AA8) and a mutant cell line (UV-41) deficient in excision repair to compare sister chromatid exchange (SCE) induction after X irradiation under oxic and hypoxic conditions. X irradiation of AA8 cells under oxic conditions induced only a slight increase in SCEs, whereas at each dose tested a significantly greater number of SCEs were induced in hypoxic cells. When AA8 cells were X-irradiated and the addition of bromodeoxyuridine (BrdU) was delayed for 20 h to allow DNA lesions to be repaired, the levels of SCEs detected in both oxic and hypoxic cells returned to background levels. X irradiation of UV-41 cells also induced only a slight increase of SCEs in oxic cells, whereas a significant number of SCEs were induced in hypoxic cells. However, in contrast to results with AA8 cells, when hypoxic UV-41 cells were X-irradiated and the addition of BrdU was delayed for 20 h, the number of SCEs remained significantly above background levels. In combination with previous alkaline elution data, these results are consistent with the possibility that DNA-protein crosslinks are responsible for the SCEs induced by X irradiation of hypoxic cells. Irrespective of the mechanism(s) involved, the data presented suggest that the SCE assay may potentially aid in the detection of hypoxic tumor cells.     

1,3-Butadiene and its epoxides induce sister-chromatid exchanges in human lymphocytes in vitro

Sister-chromatid exchanges (SCEs) were induced in human lymphocytes by 1,3-butadiene and its epoxides 3,4-epoxy-1-butene and 1,2:3,4-diepoxybutane. After a pulse treatment of 2 h, 1,3-butadiene produced a weak but reproducible increase in SCEs both with and without S9 mix. The response was similar in cultures of whole blood and of isolated lymphocytes. The 2 epoxide metabolites of butadiene, studied in whole-blood lymphocyte cultures without exogenous metabolic activation, were highly active SCE inducers. The lowest effective concentrations of butadiene, monoepoxybutene, and diepoxybutane were 2000 microM, 25 microM and 0.5 microM, respectively. A slight but dose-dependent increase in SCEs was also observed without an exogenous metabolic system after a 48-h treatment with 1,3-butadiene. Already the lowest concentration tested (500 microM) was effective. Again, the response was similar in cultures of whole blood and isolated lymphocytes, suggesting that the lymphocytes are capable of metabolically activating 1,3-butadiene.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

Sister chromatid exchanges induced in cultured mammalian cells by chromate

Chromate compounds induced sister chromatoid exchanges (SCEs) and chromosome aberrations in cultured mammalian cells. Similar increases in SCE frequency were observed in human fibroblasts exposed to the compounds K2Cr2O7 and K2CrO4. Marked increases in SCE frequency in cells exposed to chromate for a 48-h period were detected at concentrations between 10(-7) and 10(-6) M. Chromosome aberrations (primarily chromatid breaks) were also produced in human cells exposed to K2CrO4 at concentrations between 8 . 10(-7) and 3 . 10(-6) M. K2CrO4, but not the trivalent compound CrCl3, induced SCEs in Chinese hamster ovary (CHO) cells at low concentrations.     

Linear furocoumarins and other constituents from thamnosma texana

Eight linear furocoumarins and three coumarins were isolated and identified from Thamnosma texana. They were xanthotoxin, imperatorin, bergapten, alloimperatorin methyl ether epoxide, heraclenin, isopimpinellin, psoralen, oxypeucedanin, and the coumarins herniarin, osthol and thamnosmin. The linear furocoumarins appear to be agents that account for the known photosensitizing properties of Thamnosma texana, and consequently its colloquial name, ‘blisterweed.’ This is the first report on the occurrence of imperatorin, heraclenin, oxypeucedanin, herniarin or osthol in any Thamnosma species.     

Sister-chromatid exchange in human B- and T-lymphocytes exposed to bleomycin, cyclophosphamide, and ethyl methanesulfonate.

Sister-chromatid exchange (SCE) frequencies were investigated in mitogen-stimulated cultures of highly purified human peripheral blood B- and T-lymphocytes exposed to bleomycin (BM), cyclophosphamide (CP), or ethyl methanesulfonate (EMS). In untreated controls, T-lymphocytes showed twice as many SCEs as B-lymphocytes. CP (with metabolic activation) and EMS significantly increased the SCE frequencies. EMS induced a similar, dose-dependent SCE increase in both cell populations, whereas CP induced more SCEs in T- than in B-lymphocytes. No clear SCE increase was found in B- and T-lymphocytes treated with BM.     

Induction of sister-chromatid exchanges in ICR 2A frog cells exposed to 265-313 nm monochromatic ultraviolet wavelengths and photoreactivating light

Exposure of ICR 2A frog cells to 265 nm, 289 nm, 302 nm or 313 nm monochromatic ultraviolet (UV) wavelengths induced the formation of sister-chromatid exchanges (SCEs). However, treatment of cells with photoreactivating light (PRL) following the UV irradiations resulted in a lower level of SCEs compared with cells incubated in the dark. Hence, it can be concluded that pyrimidine dimers are the principal photoproducts responsible for the induction of SCEs in cells exposed to 265-313 nm UV due to the specificity of DNA photolyase for the light-dependent monomerization of dimers in DNA. It was also found that the maximum yield of induced SCEs in 313 nm-irradiated cells was only about 7 SCEs per cell whereas the plateau values for the shorter wavelengths were approximately 15-20 SCEs per cell. In addition, treatment of cells with 313 nm plus 265 nm light resulted in a lower level of SCEs than in cells exposed to 265 nm UV alone. These results can be interpreted in the context of a replication model for SCE, in which the high level of non-dimer damages produced in the DNA of 313 nm-irradiated cells inhibits the induction of SCEs by the pyrimidine dimers that are also produced by this wavelength.     

Quantitative evaluation of the effectiveness of the formation of sister chromatid exchanges and chromosome aberrations as affected by chemical mutagens

Dose curves of five chemicals were studied to compare the efficiency of SCE and chromosomal aberration induction by different chemical mutagens. SCEs were found to increase linearly with the dose, whereas chromosomal aberrations--nonlinearly. Using regression coefficients obtained from the dose curves it was found that the efficiency of the studied chemical mutagens in induction of SCEs is 100-300 times as high as that in the induction of chromosomal aberrations.     

Induction of sister-chromatid exchanges and chromosomal aberrations in hematopoietic tissue of a marine fish following in vivo exposure to genotoxic carcinogens

The development of procedures to assess genetic damage in fish exposed in situ to point sources of aquatic pollution can be expected to contribute to the evaluation of the role of genotoxic contaminants in epizootic neoplasia in fish populations. To this end methods have been developed for assessing the in vivo induction of chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in tissues of a marine teleost, the oyster toadfish, which may be applicable to other species. An alternative to the solid tissue and squash techniques for metaphase preparation permits the resolution of more than 100 SCEs/metaphase in toadfish kidney cells, which have moderately large chromosomes (0.122 pg DNA/chromosome). The bleeding of toadfish which have been injected with 5-bromodeoxyuridine (BrdUrd) and the subsequent use of hematopoietic tissue (kidney) for cytogenetic analysis was shown to increase the metaphase yield and provide a more predictable production of second-division metaphases required for SCE analysis. With these methods linear dose-dependent increases in chromatid-type exchange CAs and SCEs were obtained with i.p. exposure to ethyl methanesulfonate (EMS) and cyclophosphamide (CP). The doses required to double the observed control SCE frequencies (least effective doses) were 170 mg/kg for EMS and 7.4 mg/kg for CP. which are comparable to those reported for rodent bone marrow assays. A BrdUrd-sensitive site for chromatid breakage was observed on a pair of apparently homologous acrocentric chromosomes for the toadfish.     

Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following gamma-irradiation

The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post-treated with novobiocin.     

Hypersensitive character of Bloom syndrome B-lymphoblastoid cell lines usable for sensitive carcinogen detection

Various carcinogens were tested with regard to the induction of sister-chromatid exchanges (SCEs) and chromosome aberrations using 3 types of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) (type I with normal frequency of SCEs and normal karyotype; type II with high frequency of SCEs and normal karyotype; type III with high frequency of SCEs and abnormal karyotypes) in the presence and absence of S9 mix. Three types of BS B-LCLs and normal cells showed different responses to the various carcinogens in the level of SCE induction. BS type I cells had the same SCE response as normal cells to carcinogens. Some carcinogens that require metabolic activation (S9 mix) had little effect on type II cells without S9 mix but had high SCE levels with S9 mix. BS type III cells were highly susceptible to both direct and indirect carcinogens with respect to high SCE increase without S9 mix (ca. 140 SCEs/cell), though some carcinogens produced SCEs rated in the medium (ca. 120 SCEs/cell) range, and had a high rate (more than 10%) of centromere spreading (CS), in addition to quadriradials. Therefore BS type III is a unique cell line which can be used to detect carcinogens.