Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests

Ascorbate caused a dose-dependent increase in sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells and in human lymphocytes. Moreover, in the DNA synthesis inhibition test with HeLa cells, ascorbate gave results typical of DNA-damaging chemicals. Catalase reduced SCE induction by ascorbate, prevented its cytotoxicity in CHO cells, and prevented its effect on HeLa DNA synthesis. Ascorbate reduced induction of SCE in CHO cells by N-methylN′-nitrosoguanidine (MNNG) by direct inactivation of MNNG.     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo.

Two organotin pesticides, triphenyltin acetate (TPTA) and triphenyltin hydroxide (TPTH), were evaluated for their ability to induce micronuclei (MN) and sister chromatid exchange (SCE) in vitro using cultured Chinese hamster ovary (CHO) cells and in vivo BALB/c mouse erythrocytes. Both pesticides induced a dose-dependent increase but only TPTH induced a significant increase in MN at the highest dose (150 ng/ml) tested in CHO cells. With adding S9 microsomal fractions, both pesticides induced a meaningful MN induction at 150 ng/ml and a dose-dependent significant increase in SCE. In vivo MN induction in erythrocytes was conducted by treating BALB/c mice orally or intraperitoneally with these pesticides either in a single or triple treatments. Oral gavage (p.o.) of TPTA resulted in a dose-related significant increase of MN induction in peripheral blood and of TPTH induced a significant increase in micronucleated reticulocyte (MNRETs) only in a single treatment. Intraperitoneal administration of TPTA or TPTH, however, resulted in meaningless random increases in MN though these increases might be attributable to toxic effects. The MNRETs levels in the treatment with both pesticides were independent to the sampling time. This study demonstrated that TPTA and TPTH was potential chromosome mutagens.     

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.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

Genotoxic effects of fluoride evaluated by sister-chromatid exchange

The purpose of this investigation was to study the genotoxic potential of fluoride (in the form of sodium fluoride, NaF) using in vitro and in vivo sister-chromatid exchange (SCE) assays with Chinese hamster cells. The NaF concentrations used in cultures of Chinese hamster ovary (CHO) cells ranged from 0 to 6.3 mM, both with and without S9 activation. Fluoride analysis of the culture medium demonstrated that it contained little indigenous fluoride, and the concentration of added fluoride was not affected by the components of the medium or the S9 mix. The CHO cells cultured in 6.3 mM NaF almost vanished, and at the concentration of 5.3 mM NaF in cultures without S9 microsome, only M1 cells were observed. In in vivo studies, Chinese hamsters were intubated with NaF dosages of 0, 0.1, 1.0, 10, 60 and 130 mg/kg, and the bone marrow (CHBM) cells were examined for SCE frequencies. Bone fluoride data showed that the intubated NaF was effectively absorbed. Death occurred in 3 of the 8 animals given 130 mg NaF/kg. The results indicated that NaF, in dosages up to 5.3 mM in CHO cell cultures and 130 mg/kg in in vivo CHBM cells, did not significantly increase the SCE frequencies over those observed in the negative (distilled water) controls. However, examination of the cell cycle revealed an inhibitory effect of NaF on cell proliferation with doses of NaF at or greater than 1.0 mM in cultured CHO cells and at or greater than 60 mg NaF/kg in in vivo CHMB cells. The results of the present study indicated an inhibition of the cell cycle and death of the cells with increasing concentrations of fluoride but not effect of fluoride on SCE frequency in CHO and CHBM cells.     

Induction of sister-chromatid exchanges in human lymphocytes and Chinese hamster cells exposed to aflatoxin B1 and N-methyl-N-nitrosourea.

The effect of a 1-h exposure to aflatoxin B1 (AFB1) in inducing sister-chromatid exchange in Chinese hamster ovary (CHO) cells and human lymphocytes in the presence or absence of mixed function oxidase ("S9 mix") was compared. CHO cells were also exposed to a graded series of doses of N-methyl-N-nitrosourea, a powerful inducer of SCE whose action was independent of the presence or absence of S9 mix. CHO and human cells showed a close correlation in response to SCE induction by AFB1 and in both cell systems the additon of mixed function oxidases in the S9 mix resulted in a marked enhancement of action of AFB1.     

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.     

Induction of sister chromatid exchanges by styrene and its presumed metabolite styrene oxide in the presence of rat liver homogenate

Styrene and its metabolite styrene oxide were tested for their ability to induce sister chromatid exchanges (SCE) in CHO cells. Styrene oxide appeared to be a potent inducer of SCE. Styrene itself did not increase the number of SCE per metaphase, even in the presence of a metabolic activation system. The metabolic activation system decreased the SCE induction caused by styrene oxide. Induction of SCE by styrene in the presence of metabolic activation occurred when cyclohexene oxide was used as an inhibitor of the enzyme epoxide hydrase.     

Comparison of in vivo and in vitro SCE induction

In vivo and in vitrod sister-chromatid exchange (SCE) induction and cell replication kinetics were compared in P388 cells exposed to 4 mutagens. While concordance was observed between SCE induction and inhibition of cell replication kinetics, certain mutagens were more potent in vivo while others were more potent in vitro. These results indicate that caution should be applied before equating in vivo and in vitro mutagen exposures.     

Correlation of the sister chromatid exchange level with chromosome aberrations induced by chemical mutagens in vivo

The data on the dose dependencies of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations during exposure of mouse bone marrow cells in vivo to 5 alkylating substances are provided. The efficacy of SCE induction was found to be higher than that of chromosomal aberrations. It was established that SCE induced by chemical mutagens in vivo and in vitro are more sensitive and stable tests than chromosomal aberrations.     

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.     

Differences in sensitivity of murine spermatogonia and somatic cells in vivo to sister-chromatid exchange induction by nitrosoureas

Previously published data indicate that spermatogonia (SPG) are less sensitive to a sister-chromatid exchange (SCE) induction for different mutagens. In an earlier study, we have observed that bromodeoxyuridine (BrdU) substituted murine SPG are less sensitive to SCE induction by gamma ray in cells, than bone marrow (BM) and salivary gland (SG) cells in vivo. This was interpreted to mean that SPG are more efficient in DNA repair or are less prone to SCE induction. That the lower induction of SCE could be due to a reduced accessibility of mutagens to the SPG by virtue of a physiological barrier, was discarded by using gamma radiation. The aim of the present study was to establish whether or not there are differences in SCE induction by nitrosoureas among SPG, SG and BM cells with BrdU substituted or unsubstituted DNA. It was observed that SCE induction by methylnitrosourea (MNU) or by ethylnitrosourea (ENU) in SPG was, respectively, five and two times lower than in SG, and ten and three times lower than in BM. In SPG after BrdU incorporation, there was no increase in efficiency of SCE induction; in fact, there was even a slight decrease by exposure to MNU or ENU. BM and SG cells showed an increased efficiency in SCE induction after BrdU incorporation. This implies that SPG are also less sensitive to SCE induction by nitrosoureas, which cause a different kind of damage from previously assayed mutagens.     

Sensitivity of two Chinese hamster cell lines to SCE induction by a variety of chemical mutagens

SCE induction in Chinese hamster Don (lung) cells was compared with that in CHO (ovary) cells exposed under identical conditions to 14 known mutagens. Test protocols used for comparison were selected following a study of Don and CHO cell responses to aflatoxin B1 and benzo[a]pyrene. In the absence of added metabolizing enzymes 9-aminoacridine, 4-nitroquinoline 1-oxide, N-methyl-N-nitrosourea, dimethylcarbamoyl chloride, beta-propiolactone, daunomycin, aflatoxin B1 and 2-aminoanthracene were directly active in both cell lines; every substance positive in CHO cells was also positive in Don cells. However, the latter detected cyclophosphamide, hydrazine sulphate, benz[c]acridine, 3-methylcholanthrene and benzo[a]pyrene without addition of S9. CHO cells did not respond equivalently to these mutagens, either in the presence or absence of S9. Other differences between the cell lines depended on chemical exposure time, S9 pre-incubation or co-incubation conditions. For example, the ability of CHO cells to detect SCEs due to 2-aminoanthracene was acutely dependent on exposure time. In addition, Don cells exhibited lower background SCE values which were less variable than those of CHO cells under the same culture conditions. Although incapable of detecting 4-dimethylaminoazobenzene (butter yellow) and not as sensitive to cyclophosphamide as certain cell lines of liver origin, the pseudodiploid Don cell line possesses other desirable characteristics required for in vitro SCE assays, particularly with regard to intrinsic metabolic activation of polycyclic aromatic hydrocarbons and related substances.     

In vivo induction of sister-chromatid exchanges in liver and marrow cells by drugs requiring metabolic activation

A highly sensitive method for the detection of in vivo induction of sister-chromatid exchange (SCE) has been developed in mice subjected to partial hepatectomy. SCE induction by either acetylaminofluorene (AAF) or cyclophosphamide, drugs requiring metabolic activation, is significantly greater in both regenerating liver and bone-marrow cells of partial hepatectomized animals than in marrow cells of unhepatectomized mice. These experiments have confirmed the ability of AAF, a well known mutagen-carcinogen, to induce SCE formation, even though the cytogenic effects of this drug on non-hepatectomized mice is very small. The in vivo system described has demonstrated the influence of the liver on drug-induced damage to extra-hepatic tissues. The procedures developed should facilitate the detection of drug-induced cytogenic damage and permit the comparison of inter-tissue differences in SCE induction with tissue-specific differences in drug-activation pathways.     

Genetic damage in CHO cells exposed to enzymically generated active oxygen species

The genetic toxicity of active oxygen species produced during the enzymic oxidation of xanthine has been investigated using Chinese hamster ovary (CHO) cells. Incubation of cells with xanthine plus xanthine oxidase resulted in extensive chromosome breakage and sister-chromatid exchange and gave a small increase in frequency of thioguanine-resistant cells (HGPRT test). Inclusion of superoxide dismutase or catalase in the xanthine/xanthine oxidase system inhibited chromosome breakage, whereas only catalase prevented SCE and mutant induction. It is concluded that hydrogen peroxide is responsible for most of the genetic effects observed in CHO cells exposed to xanthine/xanthine oxidase but that superoxide plays a key role in chromosome breakage.     

Spontaneous and mitomycin-C induced sister-chromatid exchanges : Comparison of in vivo and in vitro systems

Frequencies of sister-chromatid exchanges (SCE) were measured in vitro in mouse fibroblasts and in vivo in mouse bone-marrow cells. SCE levels in these cell systems were measured in response to varying concentrations of bromodeoxyuridine (BrdU) and mitomycin-C (MMC). Although BrdU was found to induce SCE in both cellular systems, baseline SCE levels were 2- to 3-fold higher in vitro than in vivo. SCE induction was found to be a linear function of MMC concentration in vivo and in vitro; however the slope of the in vivo curve was 5-fold higher. The interaction of BrdU substituted DNA and MMC was examined by administering a fixed dose of MMC with increasing concentrations of BrdU. The induced SCE frequencies appeared to be additive. In addition to measuring drug-induced SCE, the BrdU differential staining technique allows concomitant measurement of the inhibition of cellular replication by the test drugs.     

Cytogenetic effects of cigarette smoke condensates in vitro and in vivo

Summary Various cigarette smoke condensates (CSC) were analyzed with respect to the induction of sister-chromatid exchanges (SCE) in human lymphocytes in vitro. CSC from a reference cigarette, from three different tobaccos of the reference cigarette, and from a British cigarette induced similar SCE frequencies. CSC from the reference cigarette did not induce SCE in Chinese hamster bone marrow cells in vivo.     

Ah locus-associated differences in induction of sister-chromatid exchanges and in DNA adducts by benzo[a]pyrene in mice.

The effect of alleles of the Ah locus on the induction of sister-chromatid exchanges (SCE) was studied in C57Bl/6 and in DBA/2 mice treated twice intragastrically with benzo[a]pyrene (BP, 100 or 10 mg/kg b.w.). To measure the changes in the frequency of SCE, 2 protocols were used: in vivo in bone marrow cells after implantation of 5-bromodeoxyuridine (BrdU) tablets and in vivo/in vitro in spleen lymphocytes cultured with BrdU. On day 5 mice were killed and SCEs estimated in bone marrow cells. BP-DNA adducts in bone marrow and spleen were analyzed on day 5 after the same exposure to BP. In the spleen lymphocytes SCE frequencies were analyzed after an additional 48 h of culture. We found that at both doses of BP, the number of SCEs and BP-DNA adducts in bone marrow and in spleen cells was significantly higher in aryl hydrocarbon hydroxylase (AHH)-non-inducible (DBA/2) mice than in AHH-inducible (C57BL/6) mice. Only marginal induction of SCE was noted after the high dose of BP in C57BL/6 mice in bone marrow in vivo, whereas a highly significant increase in the frequency of SCEs was found in splenocytes in the in vivo/in vitro test. The spleen cells contained larger amounts of BP-DNA adducts and demonstrated higher absolute levels of SCEs than bone marrow cells. The sensitivity of both the in vivo/in vitro and the in vivo SCE test is high enough for assessment of Ah locus-linked differences in BP genotoxicity in mice at the prolonged time between treatment and cell preparation. The present data confirm the influence of inducibility of AHH in the intestine on the genotoxicity of BP to distal tissues after oral exposure to BP.     

Induction of chromosomal damage in mammalian cells in vitro and in vivo by sulfapyridine or 5-aminosalicylic acid.

Sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA) are the two primary metabolites of the anti-inflammatory drug salicylazosulfapyridine (SASP). These two metabolites were studied for induction of chromosomal damage in mammalian cells, in vitro and in vivo, in an attempt to understand better the genetic effects produced by SASP in humans and laboratory mice. To this end, SP and 5-ASA were tested for induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Abs) in Chinese hamster ovary (CHO) cells in vitro. In addition, they were tested in vivo for induction of micronuclei (MN) in mouse bone marrow polychromatic erythrocytes (PCE). SP gave positive results in the in vitro SCE test and the in vivo MN test, and negative results in the in vitro Abs test. 5-ASA was negative in all three tests. These results indicate that it is the SP metabolite of SASP that is necessary for the induction of chromosomal damage reported to occur in humans and mice after treatment with SASP.