Pesticide clastogenicity in Chinese hamster ovary cells

Paraquat, alachlor, butachlor, phorate and monocrotophos, several of the most extensively used pesticides in Taiwan, were investigated for their clastogenicity using chromosome aberration (CAb) induction in Chinese hamster ovary (CHO) cells. Significance levels of the binomial trend analysis and binomial mutagenicity data test were two criteria for the summary judgement of the pesticide clastogenicity. Except for phorate, all pesticides tested were clastogenic to CHO cells in the absence of in vitro metabolic activation by S9. 5 microliters/ml rat-liver extract, S9, were used as the source of in vitro metabolic activation. 3 different outcomes were found after the addition of S9. Paraquat: significant decrease in induced CAbs. Monocrotophos: concomitant occurrence of decreased cytotoxicity and increased clastogenicity. Alachlor, butachlor and phorate: increased cytotoxicities with no sign of enhancement in clastogenicity.     

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.     

BCNU-induced sister chromatid exchanges are increased by X irradiation

We have studied the effect on sister chromatid exchange (SCE) induction in 9L rat brain tumor cells caused by combination treatment with BCNU and X rays. Over the dose and concentration ranges used in these experiments, BCNU induced relatively large numbers of SCEs, while X rays induced few SCEs. When cells were X irradiated immediately after BCNU treatment, the number of SCEs induced was greater than the number of SCEs expected by adding the number of SCEs induced by each agent alone; the number of SCEs induced as a result of this BCNU-X-ray interaction increased as the concentration of BCNU and/or dose of X rays increased. When the addition of bromodeoxyuridine was delayed from 0 to 16 hr after BCNU treatment, the number of SCEs induced declined to control levels by 16 hr. If X irradiation was delayed for up to 16 hr after BCNU treatment the same pattern of decrease was observed; the number of SCEs induced at each time point, however, was greater than that induced by BCNU and X rays alone. X irradiation from 0-16 hr before BCNU treatment produced the same number of SCEs as that produced by BCNU alone. Thus the SCE assay is capable of detecting a drug-X-ray interaction in mammalian cells and provides a sensitive means of studying the sequencing and timing that leads to the interaction.     

The fate of U.V.-induced lesions affecting SCEs,chromosome abberrations and survival of CHO cells arrested by deprivation of arginine

CHO cells undergo proliferative arrest when incubated in medium deficient in the amino acid arginine (ADM). Cells arrested in this way can be released and resume mitotic activity after a brief lag period. The incidence of U.V.-induced sister chromatid exchanges (SCEs) induced in cells arrested in ADM was reduced when the cells were incubated in ADM after irradiation and prior to release. Periods of incubation in ADM of 24 and 48 h prior to release reduced the resulting SCE levels (relative to the SCE levels present in cells irradiated immediately prior to release) by an average of 35 and 45% respectively. A similar time-dependent decrease in the incidence of chromosome aberrations induced in CHO cells arrested in ADM was not observed. Despite the decrease in SCEs over time in ADM, the survival of ADM-arrested cells was not enhanced by a period of incubation in ADM after irradiation of 48 h. These observations are consistent with the hypothesis that the U.V.-induced lesions responsible for the induction of SCE are repaired in time in ADM-arrested CHO cells. Repair of those lesions resulting in chromosome aberrations was not detected in ADM-arrested CHO cells. This absence of repair of certain lesions was apparently reflected in the absence of any enhancement of cell survival.     

Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells [H. Nagasawa, Y. Peng, P.F. Wilson, Y.C. Lio, D.J. Chen, J.S. Bedford, J.B. Little, Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations, Radiat. Res. 164 (2005) 141-147]. In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23 to 0.33SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after alpha-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.     

Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.     

The effect of a tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on sister-chromatid exchange formation in cultured Chinese hamster cells.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) does not increase the sister-chromatid exchange (SCE) frequency in either Chinese hamster ovary (CHO) or lung (V 79) cells which are cultured in the presence of 5-bromodeoxyuridine. Moreover, TPA does not alter the induction of SCEs in CHO cells by mitomycin C during the first 3 cycles following addition of the alkylating agent. These SCE induction data do not by themselves support the hypothesis that tumor promotion by TPA depends on the enhancement of mitotic recombination.     

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 (MS1)

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 gamma-rays, methyl methanesulfonate (MMS), and 4-nitroquinoline 1-oxide (4NQO), but not sensitive to UV or caffeine, the frequency of SCEs induced by gamma-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 lesions are closely correlated with SCE induction. Thus, the lesions or mechanisms involved in SCE production are in part different from those responsible for cell lethality or CA production.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

The effect of glutathione depletion on sister-chromatid exchange induction by cytostatic drugs

Using sister-chromatid exchanges (SCEs) as an indicator for DNA damage, we investigated the role of glutathione (GSH) as a determinant of cellular sensitivity to the DNA-damaging effects of the cytostatic drugs adriamycin (AM) and cyclophosphamide (CP). Exposure of V79 cells to buthionine sulfoximine (BSO) resulted in a complete depletion of cellular GSH content without toxicity and without increasing the SCE frequency. Subsequent 3-h treatment of GSH-depleted cells with AM or S9-mix-activated CP caused a potentiation of SCE induction. In Chinese hamster ovary (CHO) cells, which showed a higher GSH level compared to V79 cells, BSO treatment led to a depletion of GSH to about 5% of the control and increased SCE induction by AM and CP. Compared to V79 cells, the effect of AM on SCE frequencies was less distinct in CHO cells, while CP exerted a similar effect in both cell lines. Pretreatment of V79 cells with GSH increased the cellular GSH content, but had no effect on the induction of SCEs by AM, and pretreatment with cysteine influenced neither GSH levels nor SCE induction by AM. The study shows that SCEs are a suitable indicator for testing the modulation of of drug genotoxicity by GSH. The importance of different GSH contents of cell lines for their response to mutagens is discussed.     

Butachlor is cytotoxic and clastogenic and induces apoptosis in mammalian cells

The ability of butachlor to induce cytotoxicity, clastogenicity and DNA damage was assessed using Chinese hamster ovary cells (CHO), Swiss mouse embryo fibroblasts (MEF) and human peripheral blood lymphocytes. A dose and time dependent loss of viability was evident upon treatment of CHO cells with butachlor. Cell killing to an extent of 50% was observed when cells were treated with 16.2 micrograms/ml of butachlor for 24 hr or with 11.5 micrograms/ml for 48 hr. The herbicide induced micronuclei significantly in cultured lymphocytes at 24 and 48 hr of treatment suggesting that it is clastogenic. To understand the mechanism of cell death caused by butachlor, its effect on DNA strand breaks was studied in MEF. A concomitant decrease in cell viability was observed with increase in DNA strand breaks. Agarose gel electrophoresis of DNA from herbicide treated CHO cells and cytochemical staining indicate the induction of apoptosis by butachlor.     

Response of X-ray-sensitive CHO mutant cells (xrs-6c) to radiation. II. Relationship between cell survival and the induction of chromosomal damage with low doses of alpha particles

The induction of cytotoxicity, chromosomal aberrations, and sister chromatid exchanges (SCEs) was measured in CHO K-1c cells and in isogenic X-ray-sensitive mutant xrs-6c cells that had been irradiated with X rays and alpha particles in isoleucine-deficient alpha-minimal essential medium in G1 phase of the cell cycle. There was a noticeable shoulder region on the survival curve for CHO K-1c cells irradiated with very low doses of alpha particles, whereas this feature was absent for xrs-6c cells with alpha-particle doses as low as 0.5 cGy. Higher frequencies of chromatid-type aberrations were induced in G1-phase xrs-6c cells than in G1-phase CHO K-1c cells by both gamma- and alpha-particle irradiation. Induction of nonlethal chromosomal aberrations was observed following exposure to 2-6 cGy of alpha particles, doses yielding 97-100% cell survival. Irradiation with 0.5 cGy of alpha particles induced SCE; nearly 60% of irradiated cells contained significantly increased levels of SCE. However, only 3% of the nuclei of cells exposed to 0.5 cGy of alpha-particle radiation were actually traversed by an alpha particle. The observation that a large fraction of cells apparently survive exposure to very low doses of alpha-particle radiation with persistent genetic damage manifested by both chromosomal aberrations and SCEs may have important implications for the carcinogenic hazards of high-LET radiation.     

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.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compoudns

Cysteine, cysteamine and glutathione all induce sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells when applied to cell cultures at concentrations between 10(-4) and 10(-2) M. Acute exposure of cells th thiol compound for a period of 2--3 h resulted in a unique dose--response relationship in each instance. This consisted of two peak SCE frequencies, one at either extreme of the concentration range. Each peak corresponded to a 2--3-fold increase over the spontaneous level. A chronic exposure of 24 h, in contrast, resulted in a dose--response relationship consisting of a single peak SCE frequency (representing a 4--5-fold increase over the spontaneous level) at a concentration of approx. 4 x 10(-4) M. The effect of Cu2+ ions included in the medium at a concentration of 10(-5) M was to increase the toxicity and, at some concentrations, the SCE levels occurring after either acute or chronic exposure to thiols. Hydrazine and its derivatives, dimethylhydrazine and isonicotinic acid hydrazide (isoniazid), as well as hydrogen peroxide, also induce SCEs in CHO cells. A 2--3-fold increase over the spontaneous level was observed, depending upon the particular treatment protocol applied. SCE yields after 3 h treatment with dimethylhydrazine and isoniazid were increased if Mn2+, but not Cu2+, was included in the tissue culture medium at a concentration of 10(-5) M. SCE yields after a 24-h treatment with dimethylhydrazine in which Mn2+ was present in, and absent from, the medium were similar. Catalase was observed to reduce the SCE levels resulting from treatment with hydrogen peroxide, dimethylhydrazine and isoniazid. The effect of catalase upon SCEs induced by dimethylhydrazine and isoniazid in the presence of Mn2+ was more evident than when Mn2+ was not included in the culture medium. The significance of these results with respect to the possible active chemical species produced and the mutagenic/carcinogenic risk associated with thiol and hydraizine compounds is discussed.     

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.     

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.     

Sister chromatid induction by β-irradiation from incorporated 3H-thymidine: a paradox explained

Sister chromatid exchanges (SCE's) induced by [3H]thymidine (³HdT) of increasing specific activities incorporated over one cycle and 5-bromodeoxyuridine (BrdUrd) over the two following cycles were investigated in synchronised Chinese hamster ovary (CHO) cells. SCEs induced during the first cycle on a T.T template (SCE 1) show little increase with dose compared with those induced in the second cycle on a ³HT.T template (SCE 2) where the linear increase with dose reflects that seen after X irradiation. During the third cycle, SCEs 3.1 and 3.2 are induced on unlabelled T.B or labelled ³HT.B templates respectively. These templates are theoretically present in a 11 ratio after random segregation at second metaphase. Over practically the entire dose range however, the ratio 3.1/3.2, which dereased with dose, was >1.0 and similar to the high values obtained by other workers. At increasing times after BrdUrd introduction, the ratio decreased from >1.0 to <1.0. Measurements showed that the expected 50% level of labelled chromosomes at metaphase in the samples could vary between 42%–59%. Cells with >50% labelled chromosomes were more delayed in the cell cycle due to the ³H-irradiation than those with <50%. Early fixations therefore favoured SCE 3.1 while late favoured SCE 3.2. SCEs due to BrdUrd in ³HT.B and T.B templates showed no synergistic interaction with irradiation-induced SCEs. When these BrdUrd-induced SCEs were removed from the totals then the ³H-induced SCE levels in ³HT.T, and ³HT.B templates (SCE 2 and 3.2) were similar and increased at a similar rate with dose. This was 2–3 times faster than in SCE 1 and 3.1 where the SCE levels due to irradiation were again similar but lower than for 2 and 3.2. The -irradiation source is therefore most effective in inducing SCEs when present in the replicating fork and considerably less effective when it is just behind the fork (SCE 1) and/or in the surrounding chromosomes in the cell.