A comparison of sister-chromatid exchange in mouse peripheral blood lymphocytes exposed in vitro and in vivo to phosphoramide mustard and 4-hydroxycyclophosphamide

Cyclophosphamide (CP) and two of its known metabolites, 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), were analyzed for their ability to induce sister-chromatid exchanges (SCEs) in mouse peripheral blood lymphocytes (PBLs) in vitro and in vivo. At equimolar concentrations, CP is a more potent SCE inducer in vivo than PAM and PAM and 4-OHCP induce equal numbers of SCEs in a dose-dependent manner. The present study also shows that these metabolites of CP are more potent SCE inducers than CP itself in vitro. This relationship might be explained by the differences in pharmacokinetics of these compounds.     

Comparison of sister-chromatid exchange frequencies in mouse T- and B-lymphocytes after exposure to 4-hydroxycyclophosphamide or phosphoramide mustard

The present study was designed to investigate the genotoxicity of 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), both reactive metabolites of cyclophosphamide (CP), for possible differences in SCE-inducing activity in mouse T- and B-lymphocytes. Mouse peripheral blood lymphocytes were isolated and stimulated to divide with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (a polyclonal B-cell activator). Significant concentration-dependent increases in SCE frequencies were observed for both 4-OHCP and PAM with both mitogens, with 4-OHCP being almost twice as potent as PAM. There was no difference in SCE response between T- and B-lymphocytes after exposure to either PAM or 4-OHCP. These data do not support the idea that the difference in SCE response in T- and B-lymphocytes by CP in vivo is due to differential responses to either of the proposed putative metabolites of CP.     

Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells.

The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.     

Cytological characterization of repair-deficient CHO cell line 43-3B. I. Induction of chromosomal aberrations and sister-chromatid exchanges by UV and its modulation with 3-aminobenzamide

The induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) by short-wave ultraviolet (UV) and X-irradiation was studied in Chinese hamster ovary (CHO) wild-type (WT) cells and one of its UV-hypersensitive mutants, 43-3B. The results indicate that CHO 43-3B show high levels of spontaneously occurring chromosomal aberrations and SCEs; these levels are, respectively, approximately 4 and 1.7 times those found in WT CHO. Treatment with UV produced a considerable delay in the cell-cycle progression of the mutant cells compared to the WT cells. Doses of UV that had no effect on WT cells, significantly induced chromosomal alterations in the mutant in a dose-dependent manner. An approximately 5-fold increase in the induced frequencies of SCEs was obtained in 43-3B cells after UV treatment. No synergistic effect was observed with UV irradiation and the inhibitor of poly(ADP-ribose) synthetase, 3-aminobenzamide (3AB), in either cell type. The frequency of SCEs in the mutant cell lines was lower than would be expected if the effects of UV and the inhibitor were additive. X-Ray alone in G1 and in combination with 3AB in G2 did not induce increased frequencies of chromosomal aberrations in mutant cells in comparison to the WT cells.     

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.     

Influence of beta-myrcene on sister-chromatid exchanges induced by mutagens in V79 and HTC cells

The influence of beta-myrcene (MC) on sister-chromatid exchanges (SCE) in V79 cells induced by 4 S9 mix-activated indirect mutagens was studied. The mutagens used were cyclophosphamide (CP), benzo[a]pyrene (BP), aflatoxin B1 (AFB) and 9,10-dimethyl-1,2-benz[a]anthracene (DMBA). MC effectively inhibited SCEs induced by CP and AFB in a dose-dependent manner, but it had no effect on SCE induction by BP and DMBA. MC also reduced CP-induced SCE frequencies in a hepatic tumor cell line (HTC). These cells are metabolically competent and activate CP into its biologically active metabolites. Our results support the suggestion that MC modulates the genotoxicity of indirect-acting mutagens by inhibiting certain forms of the cytochrome P-450 enzymes required for activation of premutagens like CP and AFB.     

Effects of phosphoramide mustard and acrolein, cytotoxic metabolites of cyclophosphamide, on mouse limb development in vitro

Phosphoramide mustard and acrolein are toxic and reactive metabolites of the widely used anticancer drug and known teratogen cyclophosphamide. To study the mechanism(s) involved and to determine which of the active metabolites of cyclophosphamide is responsible for the production of limb malformations, the effects of exposure of cultured limb buds to phosphoramide mustard and acrolein were investigated. Fore- and hindlimbs were excised from ICR mice on day 12 of gestation and cultured in roller bottles for 6 days. Limbs were exposed to either phosphoramide mustard or acrolein (10 or 50 micrograms/ml) for the first 20 hours of the culture period. Exposure to phosphoramide mustard produced limb reduction malformations in both the fore- and hindlimbs; total limb bone area was greatly reduced, while the relative contribution of the paw to this area in forelimbs was increased. There was a fourfold reduction in both DNA and RNA; protein content was reduced only by one-half. Alkaline phosphatase activity was significantly decreased in fore- and hindlimbs exposed to phosphoramide mustard, whereas creatine phosphokinase activity was only reduced in hindlimbs in the limbs exposed to the higher concentration of phosphoramide mustard. Exposure to acrolein also produced malformed limbs with a mangled appearance; however, total limb bone area and the relative contribution of the long bones versus paw structures were not altered. Acrolein exposure had little effect on growth parameters such as DNA (decreased only in hindlimbs exposed to 50 micrograms/ml), RNA (increased in hindlimbs exposed to 50 micrograms/ml), or protein content. Alkaline phosphatase and creatine phosphokinase activities were not altered in acrolein-exposed fore- or hindlimbs. Thus, phosphoramide mustard and acrolein have dramatically different effects on developing limbs in vitro; this observation may indicate that they have different targets and/or mechanisms of action as teratogens in the limb. The effects of phosphoramide mustard are very similar to those of "activated" cyclophosphamide (4-hydroperoxycyclophosphamide).     

Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister-chromatid exchanges, detected by immunological methods

We have utilized monoclonal antibody against BrdUrd to detect sister-chromatid exchanges in CHO cells. This technique allows detection of SCEs at very low levels of BrdUrd incorporation. At incorporation level of 0.5%, a frequency of about 2 SCEs/cell/cycle was found. In a UV-sensitive mutant (43-3B) which has an increased spontaneous frequency of SCEs, it is found that this increase is due to incorporated BrdUrd. In MMS- and MMC-treated cells, an influence of BrdUrd on the frequencies of induced SCEs was found only when high concentrations of mutagens were employed.     

Cytogenetic effects of 2-methoxyethanol and its metabolite,methoxyacetaldehyde, in mammalian cells in vitro

Glycol ethers such as 2-methoxyethanol (2-ME) are reproductive toxins. The genotoxicity of 2-ME, especially its metabolites: methoxyacetaldehyde (MALD) and methoxyacetic acid (MAA), is not adequately investigated yet. We have shown previously that MALD induced mutation in the bacterial gpt gene which is inserted in an autosome of CHO-AS52 cell line but not in the hprt gene on the X chromosome of CHO-K1-BH4 cell line. These data suggest that MALD induces major deletion-type mutation. If this prediction is correct we would expect to observe that MALD is an efficient inducer of chromosome aberrations in both CHO cell lines. We have conducted a cytogenetic study using both CHO cell lines and human lymphocytes to investigate this phenomenon. Our results show that human lymphocytes treated with 10–30 mM MALD for 1 h or 0.05–0.5 mM MALD for 24 h induced significant dose-dependent increase of sister-chromatid exchanges (SCE) (p < 0.05). It also induced significant dose-dependent increase (p < 0.05) of chromosome aberrations in human lymphocytes (10–40 mM treated for 1 h, or 0.05–2.5 mM for 24 h) and in both CHO cell lines (1.25–20 mM for 3 h). Treatment of these cells with the parent compound, 2-ME did not induce chromosome aberrations nor SCE unless very high doses of the chemical were used. In conclusion, these results indicate that MALD is clastogenic to different cell types therefore it is potentially carcinogenic. The genotoxic effects of 2-ME in humans will be dependent upon the metabolic capability of individuals to bioactivate 2-ME to MALD.     

Biochemical and cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells xrs 5 and xrs 6. V. The correlation of DNA strand breaks and base damage to chromosomal aberrations and sister-chromatid exchanges induced by X-irradiation

The X-ray-sensitive Chinese hamster ovary (CHO) mutant cell lines xrs 5 and xrs 6 were used to study the relation between X-ray-induced DNA lesions and biological effects. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCE) were determined in wild-type CHO-K1 as well as mutants xrs 5 and xrs 6 cells following X-irradiation under aerobic and anaerobic conditions. Furthermore, we used a newly developed immunochemical method (based on the binding of a monoclonal antibody to single-stranded DNA) to assay DNA single-strand breaks (SSBs) induced by gamma-rays in these CHO cells, after a repair time of up to 4 h. For all cell lines tested the frequency of X-ray-induced chromosomal aberrations was strongly increased after irradiation in air compared with hypoxic conditions. When compared to the wild-type line, the xrs mutants known to have a defect in repair of DNA double-strand breaks (DSBs) exhibited a markedly enhanced sensitivity to aerobic irradiation, and a high OER (oxygen enhancement ratio) of 2.8-3.5, compared with 1.8-2 in CHO-K1 cells. The induction of SCE by X-rays was relatively little affected in CHO-K1 irradiated in air compared with hypoxic conditions (OER = 0.8), and in xrs 5 (OER = 0.7). A dose-dependent increase in the frequency of SCEs was obtained in xrs 6 cells treated with X-rays in air, and a further increase by a factor of 2 was evident under hypoxic conditions (OER = 0.4). With the immunochemical assay of SSB following gamma-irradiation, no difference was found between wild-type and mutant strains in the number of SSBs induced. The observed rate of rejoining of SSBs was also the same for all cell lines studied.     

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.     

Cytostatic drug activity in plasma, a bioassay for detecting mutagenicity of directly and indirectly acting chemicals, an evaluation of 20 chemicals

We have utilized in vivo drug metabolism for detecting the mutagenicity of known indirectly-acting chemical mutagens. Exponentially growing Chinese hamster ovary (CHO) cells were incubated with plasma derived from treated rats containing active metabolites of the test chemicals. The genotoxicity was assessed by the 18 of tested chemical mutagens. Plasma from rats treated with known non-mutagens did not increase the frequencies of SCEs. The results indicate that this method could be useful for the demonstration of genotoxicity of chemicals which need metabolic activation to be effective, and especially those which are not effective, when in vitro activation conditions (S9 mixture) are used.     

Cytogenetical characterization of UV-sensitive repair-deficient CHO cell line 43-3B. II. Induction of cell killing, chromosomal aberrations and sister-chromatid exchanges by 4NQO, mono- and bi-functional alkylating agents

An established cell line of Chinese hamster ovary (CHO-9) cells and its UV-sensitive mutant 43-3B have been studied for the induction of cell killing, chromosomal aberrations and sister-chromatid exchanges (SCEs) after exposure to different types of DNA-damaging agents such as 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC), diepoxybutane (DEB), methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU). In comparison with the wild-type CHO cells, 43-3B cells showed very high sensitivity to the UV-mimetic agent 4NQO and the DNA cross-linking agents MMC and DEB. The 43-3B cells responded with higher sensitivity to the monofunctional alkylating agents (MMS, EMS and ENU). The increased cytotoxic effects of all these chemicals correlated well with the elevated increase in the frequency of chromosomal aberrations. In 43-3B cells exposed to 4NQO, MMC or DEB the increase in the frequency of chromosomal aberrations was much higher than the increase in the frequency of SCEs (4-10-fold) when compared to the wild-type CHO cells. This suggests that SCEs are results of fundamentally different cellular events. The responses of 43-3B cells to UV, 4NQO, MMC and DEB resemble those of 2 human syndromes, i.e., xeroderma pigmentosum and Fanconi's anemia. These data suggest that 43-3B cells are defective in excision repair as well as the other pathways involved in the repair of cross-links (MMC, DEB) and bulky DNA adducts (4NQO).     

Biological and environmental monitoring of occupational exposure to cyclophosphamide in industry and hospitals

The aims of the study were to clarify potential exposure situations to anticancer agents during industrial processing, drug manufacture and hospital administration, using cyclophosphamide (CP) as the model compound. CP is considered an animal and human carcinogen, and it is shown to be an indirect mutagen in various test systems using several genetic endpoints. Environmental monitoring was performed by collecting ambient air samples during the different processing and handling stages. Both stationary and personal sampling was used. CP was analyzed by liquid chromatography (HPLC) and mass spectrometry (MS). The process materials and intermediates were also analyzed for genotoxic activity using the Ames test and SCE induction in CHO cells as endpoints. Biological monitoring studies were performed on 147 persons representing 5 groups of workers, control subjects and patients. In the experimental part of the project, the intermediates in the CP manufacturing process, CP I (nor-nitrogen mustard) and CP II (phosphoroxydichloride mustard) were found directly active in the 2 genotoxicity tests. These findings led to improvements in work hygiene when handling CP I and CP II in the process. The CP measurements showed that the highest potential-exposure sites occurred during specific operations of the process, e.g., during emptying of the drying drum and during tablet mass preparation (the range of CP concentrations in air was 0.16-0.49 mg/m3). The correlation between indirect genotoxicity and chemical analyses of the ambient air samples was good, revealing the activity to be due to cyclophosphamide. However, the air samples were found mutagenic without metabolic activation also in the beginning of the process; this is obviously due to CP II particles in the ambient air, since no CP was detected chemically. The personal protection of workers in the plant collaborating in the study is efficient and the production unit is equipped with the best available techniques to protect both the personnel and the quality of the drug. Both the urine mutagenicity analyses using strain TA1535 of Salmonella typhimurium as indicator and the cytogenetic analyses of peripheral blood lymphocytes using sister-chromatid exchanges or structural chromosomal aberrations as endpoints were negative. However, a statistically nonsignificant trend in increased number of micronuclei was observed in binucleated lymphocytes of the worker groups as compared with controls. The studies on the hospital use of CP were performed in 3 oncological units and 1 pharmacy unit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19

The Chinese hamster ovary (CHO) cell mutant, EM9, is defective in rejoining strand breaks, hypersensitive to chlorodeoxyuridine (CldUrd), and has a high frequency of sister-chromatid exchange (SCE). Somatic cell hybrids constructed from fusion of EM9 cells with normal human lymphocytes and fibroblasts, and selected in CldUrd, extensively segregate human chromosomes but preferentially retain markers of human chromosome 19. The SCE frequency in the hybrid clones is low as in normal CHO cells, but in CldUrd-sensitive subclones, which lose the human chromosome 19 markers, SCE frequencies return to mutant levels. We therefore assign a human gene designated repair complementing defective repair in Chinese-hamster (RCC) to chromosome 19. Since this is the second (of two) human genes complementing repair-deficiency mutations in CHO cells assigned to the 19, the assignment and organization of DNA-repair genes is discussed in the light of hemizygosity in CHO cells and the evolutionary conservation of mammalian linkage groups.     

Cytogenetic response to 1,2-dicarbonyls and hydrogen peroxide in Chinese hamster ovary AUXB1 cells and human peripheral lymphocytes

Mutagenic 1,2-dicarbonyls have been reported to occur in coffee and other beverages and in various foods. We have measured the induction of sister-chromatid exchanges (SCEs) and endoreduplicated cells (ERCs) to determine the genotoxicity of various 1,2-dicarbonyl compounds in Chinese hamster ovary (CHO) AUXB1 cells and human peripheral lymphocytes. The 1,2-dicarbonyls glyoxal, methylglyoxal and kethoxal each induced highly significant increases in both SCEs and ERCs in AUXB1 cells. Glyoxal and kethoxal induced SCEs but not ERCs in human peripheral lymphocytes. In addition, hydrogen peroxide induced highly significant levels of SCEs and ERCs in AUXB1 cells. Bisulfite, which reacts with carbonyl groups to form addition products, significantly reduced the frequency of SCEs and the proportion of ERCs when glyoxal, methylglyoxal, kethoxal and diacetyl were administered to AUXB1 cells. In addition, bisulfite blocked the formation of ERCs, but not SCEs, induced by hydrogen peroxide. These in vitro results suggest that 1,2-dicarbonyls may play an important role in the genotoxicity of some foods and beverages.     

Liquid-holding experiments with human lymphocytes. III. Experiments with G0 and G1 cells

Liquid holding (LH) experiments were performed with human peripheral lymphocytes treated in the G0 (G0-LH) or the G1 (G1-LH) phase of the cell cycle with diepoxybutane (DEB) or methylnitrosourea (MNU). In the G0-LH system, treatment with DEB but not with MNU led to a lowering of the frequencies of sister-chromatid exchanges (SCE). In the G1-LH system treatment with both chemicals led to a lowering of the SCE frequencies during the LH. These results are concluded to mean that lesions induced by DEB but not by MNU can be repaired in G0 cells and that G1 cells can repair both DEB and MNU induced lesions.     

Biological monitoring of workers in the rubber industry. I. Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of vulcanizers

To evaluate the possible genetic consequences of the industrial exposure among the vulcanizers of a rubber plant we measured the in vivo levels of chromosomal aberrations and sister-chromatid exchanges in peripheral lymphocytes of 34 vulcanizers and in an adequate control population. The observed chromosomal aberration frequencies were 1.9 +/- 1.4 aberrations/100 cells in the exposed group and 2.1 +/- 1.5 aberrations/100 cells in the controls. No difference was found between the two groups for the mean value of sister-chromatid exchanges (5.2 +/- 1.3 in the exposed, 5.2 +/- 0.7 in the control group). Cigarette-smoking was clearly associated with increased sister-chromatid exchange frequencies both in the exposed and in the control groups, while chromosomal aberration frequencies were not correlated with smoking habits.     

Baseline and phosphoramide mustard-induced sister-chromatid exchanges in pharmacists handling anti-cancer drugs.

Determinations of baseline and mutagen-induced sister-chromatid exchanges (SCE) have been used as indicators of previous mutagen exposure in several human populations. Mutagen-induced SCE is based on the premise that a genetic outcome may depend not only on a present exposure, but also on a cell's "memory" of previous exposure. The genotoxicity of some anti-cancer drugs including cyclophosphamide (CP) has been studied by determining baseline and mutagen-induced SCE in peripheral blood lymphocytes in treated cancer patients. This study examined the in vivo genotoxic effects of occupational exposure to anti-cancer drug handling by relating baseline and phosphoramide mustard (PM) -induced SCE levels with duration of anti-cancer drug handling as a surrogate for anti-cancer drug exposure dose. The mean baseline SCE for the population was 5.19 +/- 0.17 and was not correlated with duration of drug handling. However, a strong correlation was demonstrated between inducible SCE values and life-time duration of drug handling with r = 0.63 (p less than 0.0001 for low-dose PM challenge (0.1 mg/ml PM) and r = 0.67 (p less than 0.0001) for high-dose PM challenge (0.25 mg/ml PM). A similar relationship was seen for PM-induced SCE and duration of anti-cancer drug handling for the workers' present job with correlations obtained being r = 0.63 (p less than 0.0001) for low-dose PM and r = 0.59 (p less than 0.0001) for high dose PM. The short-lived nature of the baseline SCE lesion is discussed as a limitation in population surveillance studies, as it reflects primarily recent mutagen exposure and persists only for days to weeks after exposure. The induced SCE measure is postulated to provide an integrating dosimeter of remote previous exposure, improving upon the current limitation of the baseline SCE measure and allowing the "unmasking" of previous exposure in a provocative framework.     

Influence of various mitogens on the yield of sister-chromatid exchanges, induced by chemicals, in human lymphocytes

The fluorochrome-plus-Giemsa (FPG) method of Perry and Wolff was used to compare the frequencies of sister-chromatid exchanges (SCEs) induced by cyclophosphamide (CP) or mitomycin C (MMC) in human lymphocytes stimulated by phytohaemagglutinin (PHA), concanavalin A (Con A), Wistaria floribunda (WFA), or lentil lectin (LcH-A) extracts. These 4 mitogens, differing in lectin valency and/or sugar specificity, are considered as activating primarily thymus-derived (T) lymphocytes. Regardless of the mitogen used, control cultures displayed a mean yield of about 8 SCEs/cell. A contact, of 1 h, with mitomycin alone or with cyclophosphamide and enzymatic activation, resulted in a significant augmentation of SCEs dependent on the mitogen used. An approximately 2-fold, 4-fold, or 6-fold increase in SCEs was observed for the cultures stimulated by PHA, Con A, and WFA or LcH-A respectively. Furthermore, there were mitogen-dependent differences in mitotic indices and cell-cycle kinetics in human lymphocytes harvested 72 h after stimulation.