Induction of sister-chromatid exchanges by 2-aminofluorene in cultured human lymphocytes with and without erythrocytes.

2-Aminofluorene (2-AF), an indirect mutagen reported to be metabolically activated by erythrocytes in the Salmonella mutagenicity test, was studied for the induction of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro with (whole-blood cultures) and without erythrocytes (isolated lymphocyte cultures). 2-AF (0.025-0.8 mM) was present in the cultures for the last 48 h of 72-h cultures. In both types of culture, SCEs increased in a dose-dependent manner, with a statistically significant elevation already at the lowest concentration of 2-AF tested and maximum responses of 2.4-fold (whole blood) and 2.1-fold (isolated lymphocytes), in comparison with mean SCEs/cell in control cultures, at 0.4 and 0.2 mM concentrations (respectively). Thus, the induction of SCEs by 2-AF was not dependent on the presence of erythrocytes. Styrene (2 mM), a positive control chemical known to require erythrocytes for efficient SCE induction in vitro, was shown to produce a 4.9-fold increase in SCEs in whole-blood cultures, but only a slight (1.3-fold) effect in isolated lymphocyte cultures. The results suggest that leukocytes, but not erythrocytes, are important in the metabolic activation of 2-AF in the human lymphocyte SCE assay.     

The influence of GSTM1 and GSTT1 genotypes on the induction of sister chromatid exchanges and chromosome aberrations by 1,2:3,4-diepoxybutane

Cytogenetic tests - chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) - are most often applied in biomonitoring of the genotoxicity of potentially carcinogenic chemicals in human cells. One of the extensively studied genotoxins is diepoxybutane (DEB) - reactive biometabolite of butadiene (BD). Several studies showed a high SCE induction in human lymphocytes exposed in vitro to various concentrations of DEB. DEB also proved to be a potent inducer of chromosome aberrations and micronuclei. A bimodal distribution of SCE frequency after in vitro DEB treatment was observed. The aim of the present study was to examine the ability of DEB to induce different individual cytogenetic response measured by SCE and CA frequency. The possible influence of genetic polymorphism has also been taken into account, by including donors representing positive or null GSTM1 and GSTT1 genotypes. Our study supported the earlier results showing that DEB is an effective inducer of SCEs and CAs, causing also the decrease in replication index (RI). DEB bioactivity measured by SCE induction - but not by CA test - was significantly higher in GSTT1 negative than in GSTT1 positive donors. GSTM1 polymorphism had no influence on these endpoints. The donors GSTT1-/GSTM1+ were shown to be slightly more sensitive to DEB than GSTT1-/GSTM1- individuals. There was also observed a unimodal distribution of DEB-induced SCEs and CAs in the group, despite the fact that the experiment was performed on the lymphocytes obtained from both GSTT1 positive and negative donors.     

Influence of GSTT1, mEH, CYP2E1 and RAD51 polymorphisms on diepoxybutane-induced SCE frequency in cultured human lymphocytes

1,3-Butadiene (BD) is a common chemical in the human environment. Diepoxybutane (DEB) is the most reactive epoxide metabolite of BD. The aim of the present study was to evaluate the influence of polymorphisms in enzymes operating in DEB-metabolism (epoxide hydrolase mEH, CYP2E1 and GSTT1), as well as in the DNA-repair enzyme RAD51, on the frequency of sister chromatid exchange (SCE) induced by DEB in lymphocyte cultures from 63 healthy donors. Their genotypes were determined using PCR and restriction fragment length polymorphism (RFLP)-PCR techniques. The analysis of xenobiotic-metabolizing genes revealed that GSTT1 and CYP2E1 polymorphisms have an influence on DEB-induced SCE frequency. Individuals with the GSTT1 null genotype and CYP2E1 c2 variant allele heterozygotes were observed to have significantly higher SCE frequency than individuals with more common genotypes. A correlation between sensitivity to DEB and GSTT1 null genotype indicates that this pathway is a major detoxification step in DEB metabolism in whole-blood lymphocyte cultures, which has been shown in many studies. The analysis of combined polymorphisms indicated that, in the presence of GSTT1, a significantly higher DEB-induced SCE frequency is observed in the CYP2E1 c2 variant allele heterozygotes than in individuals with the most common CYP2E1 genotype. In the absence of GSTT1, however, the CYP2E1 polymorphism has no influence on DEB-induced SCEs. A significant difference was also observed between individuals characterized by low and high mEH activity, but only in subjects with the GSTT1 null genotype. Lack of GSTT1 resulted in higher SCE frequency in individuals with mEH high-activity genotypes than in individuals with mEH low-activity genotype. In the present study no statistically significant difference in DEB-induced SCEs was observed for the RAD51 polymorphism. The influence of GSTT1 genotype on SCE-frequency in RAD51 variant allele carriers was not analysed as all individuals in this group (except one person) had the GSTT1 gene present. Our study shows that the combined analysis of polymorphisms in metabolizing enzymes may lead to a better understanding of their contribution to an individual's susceptibility to DEB.     

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.     

Correlation between the adaptive response and individual sensitivity to monoepoxybutene in in vitro experiments on human lymphocytes

Individual variations in the susceptibility to mutagenic/carcinogenic chemicals depend on the activity of xenobiotic metabolizing enzymes and on DNA- and chromosome-damage repair systems. Monoepoxybutene (MEB) is a genotoxic metabolite of 1,3-butadiene (BD), which has been classified as a probable carcinogen in humans. The purpose of the present study was to investigate by in vitro experiments on human whole blood lymphocytes (WBL), whether an individual sensitivity to MEB correlates with the adaptive response to the tested agent. In the analyzed group, 8.3% of blood donors were relatively sensitive to MEB. The comparison of SCE induction in cultures pretreated and not pretreated with an adaptive dose (AD) of MEB showed, that there was an adaptive response to MEB. The adaptive response in the group of relatively sensitive donors was similar to that of the relatively resistant ones. This result suggests that individual sensitivity to the tested agent and adaptive response depend on different biological mechanisms.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Cytogenetic study of workers exposed to chromium compounds

The frequency of sister chromatid exchanges (SCEs), high SCE frequency cells (HFCs), and genetic polymorphism of genotypes glutathione S-transferase (GST) M1 and T1 were analyzed in peripheral lymphocytes of 35 workers occupationally exposed to chromium (Cr) and 35 matched control group. Results showed that workers exposed to Cr showed 6.07 SCE/cell, as compared to 4.76 SCE/cell for the control group (p<0.01). Smokers showed a statistically significant higher frequency of SCE than non-smokers in both groups. The work duration of Cr workers was an important factor. Workers exposed for more than 5 years showed a significantly higher level of SCEs (p<0.05). Workers exposed to Cr for 5 or more years had higher HFC rates (51.4%) than those exposed for less than 5 years (22.9%), with an odds ratio of 4.5 times than those exposed for less than 5 years. In HFC analysis, Cr workers who smoked showed a higher level of HFC (60%) than the control group (5.7%) and also had a higher odds ratio (60.4) compared with the control group. Among non-smokers, the odds ratio was 9.0. Another objective of this study is to investigate the relationship between SCE and genetic polymorphisms of GST M1 and T1 in Cr workers. The results showed that the incidence of GSTM1 null genotype was 60% in the control group and 77.1% in Cr workers, and percentages of GSTT1 deletion were 42.9% and 62.9% in control and exposed individuals, respectively. There was a slightly increased frequency of SCE among Cr workers with GSTM1 null genotype as opposed to non-null genotype individuals. A similar result was seen among the control group; however, there were no statistically significant differences. In conclusion, the current study found the positive induction of SCE in workers who smoked or/and were exposed to Cr. However, different GST genotypes did not influence the level of cytogenetic damage between groups. Despite slight variation in numbers, they all appear to be not different.     

Variation in the frequency of sister chromatid exchanges in repeated human lymphocyte cultures

Summary Repeated blood samples from two healthy donors were taken over a period of about one year to determine the temporal variation in human lymphocyte baseline sister chromatid exchange (SCE)-frequencies. The investigations were performed on whole blood cultures and purified lymphocyte cultures using a standardized protocol for blood collection and cultures. Significant differences in the frequencies of SCEs were found between the two cultivation systems and the two blood donors but also between repeated cultures of the same individual. There was no systematic relationship between the proliferation of the cultures and the basal SCE values. The results indicate the necessity of concurrent controls and repeated blood samples whenever SCEs are used as a test for monitoring human exposure to potential mutagens. Temporal variation in human lymphocyte baseline SCE frequencies is a limiting factor for the detection of minor effects of genotoxic agents.     

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.     

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.     

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.     

Influence of duration of exposure to styrene oxide on sister chromatid exchanges and cell-cycle kinetics in cultured human blood lymphocytes in vitro

Styrene-7,8-oxide, an intermediate of styrene, is a known alkylating mutagen. The present study was carried out to investigate the influence of duration of exposure to styrene-7,8-oxide (styrene oxide) on induction of sister chromatid exchanges (SCEs) and inhibition of cell-cycle kinetics using cultured human blood lymphocytes in vitro. Phytohemagglutinin-stimulated whole-blood lymphocyte cultures obtained from heparinized whole blood from healthy donors were exposed to 100 μM styrene oxide for 22, 36, 48 and 72 h. A reduction of SCEs induction with increase in duration of exposure to styrene oxide was observed, i.e. a clear significant inverse relationship between exposure time and frequencies of SCEs induction due to styrene oxide was obtained. Styrene oxide induces significant elevations in unscheduled DNA synthesis DNA repair as well as S-phase synthesis in human blood lymphocytes in vitro, depending on the duration of exposure. The decrease in the induction of SCEs due to styrene oxide with increasing duration of its exposure may be principally due to an increased DNA repair and partly due to an increasing metabolic transformation to styrene glycol with increasing duration of its exposure as well as to some extent due to cell death at the maximum period of exposure, i.e. 72 h. Although the proliferations of lymphocytes exposed to 100 μM styrene oxide were significantly inhibited at different durations of exposure, no linear relationship between the replication index and the duration of exposure was noticed (r=0.47, p>0.05). Similarly, there was no relationship between replication index and SCE frequency (r=−0.36, p>0.05), suggesting that these two parameters may reflect two different endpoints for the cytogenotoxic effects of styrene oxide.     

Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.     

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na2SeO3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H2O2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H2O2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.     

A decrease in sister chromatid exchange frequency during the prolonged cultivation of human lymphocytes

Sister chromatid exchange (SCE) frequency at different times of fixation was studied in human lymphocyte cultures obtained from 6 donors. No differences were found in the SCE frequency between human lymphocyte cultures fixed at 72 and 96 hours of incubation (10.61 +/- 0.85 and 10.15 +/- 0.81 SCE per cell, respectively). However, a decreased SCE frequency (8.11 +/- 0.36 SCE per cell) was observed in cultures fixed at 120 hours of incubation. For a more detailed studies, one lymphocyte culture was fixed at different times of incubation (from 56 to 128 hours, at each a 8 hours). A slight increase in SCE frequencies was found at the interval between 56 and 88 hours of incubation, while starting from 104 hours of incubation a marked decrease in the SCE frequency was observed. Time-dependent changes in the SCE frequency may be described by the equation y = -1.8614 + 0.3922x - (2.5183 x 10(-3))x2, where y is the number of SCEs per cell, and x--the duration of culture incubation in hours. The observed phenomenon may be associated with changes in proportion of T and B lymphocytes, or with heterochromatization of chromosomes during a prolonged cultivation, or with an early in vitro stimulation of the in vivo long-lived lymphocytes that may be more damaged than the in vivo short-lived and the in vitro late-stimulating ones.     

Reduction of diepoxybutane-induced sister chromatid exchanges by glutathione peroxidase and erythrocytes in transgenic Big Blue mouse and rat fibroblasts

We have investigated the effect of glutathione peroxidase (GSH-Px) and mammalian erythrocytes (RBCs) on spontaneous and diepoxybutane (DEB)-induced sister chromatid exchange (SCE) in primary Big Blue(R) mouse (BBM1) and Big Blue(R) rat (BBR1) fibroblasts. DEB is the putative carcinogenic metabolite of 1,3-butadiene (BD) for which inhalation exposure yields a high rate of malignancies in mice but not in rats. BD is metabolized differently in mice and rats, producing much higher levels of DEB in mice than in rats, which may partly explain the different carcinogenic responses. However, other factors may contribute to the observed differences in the rodent carcinogenic response to BD. DEB is a highly reactive compound. Upon epoxide hydrolysis, DEB can covalently bind to DNA bases. Likewise, DEB generates reactive oxygen species that, in turn, can either damage DNA or produce H(2)O(2). Reduced glutathione (GSH) is known to play a role in the metabolism and detoxification of DEB; and GSH is reduced by GSH-Px in the presence of H(2)O(2). GSH-Px is a constitutive enzyme that is found at high concentrations in mammalian RBCs. Therefore, we were interested in examining the role of RBCs and GSH-Px on DEB-induced SCE in rat and mouse cells for detection of possible differences in the species response. Transgenic BBM1 and BBR1 fibroblasts were treated with either 0, 2 or 4 microM DEB plus 0, 2 or 20 units of GSH-Px with and without 2x10(8) species-specific RBCs. DEB effectively induced SCEs in both rat and mouse cells. The relative induction of SCEs in both cell types was comparable. Both GSH-Px and RBCs alone and in combination were effective in significantly reducing DEB-induced SCEs in both mouse and rat fibroblasts, although there was more variability in the SCE response in rat cells. The present study suggests that GSH-Px may be important in the detoxification of DEB-induced DNA damage that results in the formation of SCEs.     

As to the clastogenic-, sister-chromatid exchange inducing-and cytotoxic activity of inosine triphosphate in cultures of human peripheral lymphocytes

The influence of commercial inosine triphosphate (ITP) on the chromosome aberration rate, the mitotic rate, sister-chromatid exchange (SCE) frequency, and the proportion of first (X1), second (X2) and third (X3) division metaphases was investigated in 72h cultures of human peripheral lymphocytes. The blood donors had mild inactive arthrosis and a normal health check-up. All cultures of each volunteer were set-up simultaneously. In contrast to a previous report [Arch. Biochem. Biophys. 278 (1990) 238-244], it was demonstrated in two preliminary studies (number of subjects, n=5 each) that ITP at a final concentration of 100 microM does not induce chromosomal aberrations and, furthermore, that not ITP concentrations higher than 100 microM but ITP doses higher than 3.8mM prohibit culture growth.Based on these results, cultures with a final ITP concentration of 3.6mM (max.) and 1.8mM (max./2) were compared with control cultures (number of subjects n=10; three males and seven females, mean age x=57.6 years). Whereas no increase in the chromosomal breakage rate was observed in cultures with an ITP concentration of 1.8mM and only a marginally significant one (P=0.048) for 3.6mM ITP cultures, a highly significant induction of SCEs, not only at an ITP concentration of 3.6mM (P<0.0001) but also at 1.8mM (P<0.0001) was seen. The increase in the SCE frequency was not linear, but steeper from 0 to 1.8mM than from 1.8 to 3.6mM. Nevertheless, the difference between 1.8 and 3.6mM cultures was significant (P=0.027). The distribution of the number of SCEs per metaphase as well as the distribution of SCEs per chromosome correspond to the expected Poisson values. The investigation of the cytotoxic effect of the studied ITP concentrations revealed a highly significant reduction of the mitotic rate from 0 to 1.8mM as well as from 1.8 to 3.6mM in the aberration studies (all P values are equal to smallest possible one for a sample size of 10, namely, 0.002), and in the SCE studies there is a significant decrease in the X3 frequency when ITP is increased (0-1.8mM: P=0.0061 and 1.8-3.6mM: P<0.0001). The proportion of X1 within all X1 and X2 metaphases changes significantly only at the second dose step (0-1.8mM ITP: P=0.22 and 1.8-3.6mM ITP: P<0.0001). The results are discussed.     

Genetic polymorphisms of DNA repair and xenobiotic-metabolizing enzymes: effects on levels of sister chromatid exchanges and chromosomal aberrations

Elevated levels of chromosomal aberrations (CAs) in peripheral blood lymphocytes, widely used as a cytogenetic biomarker of genotoxic effects, have been linked to cancer predisposition. However, tobacco smoking, occupational carcinogen exposure, or time since CA analysis do not appear to explain the cancer predictivity of CAs. Alternatively, the observed CA-cancer association could reflect unidentified exposures or individual susceptibility. We assessed the effects of genetic polymorphisms of DNA repair proteins and xenobiotic-metabolizing enzymes (XMEs) on the levels of CAs and sister chromatid exchanges (SCEs) in peripheral lymphocytes of 145 (CAs) and 60 (SCEs) healthy Caucasians. Genotypes of DNA repair genes X-ray repair cross-complementation group 1 (XRCC1 codons 194, 280, 399) and 3 (XRCC3 codon 241 [corrected]), and XME genes glutathione-S-transferase (GST) M1 and T1 and N-acetyl transferase 2 (NAT2) were determined using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP)-based methods. After Poisson regression adjustment for age, sex, smoking, country, and genotypes, a higher frequency of chromosome-type breaks was observed for NAT2 slow acetylators (in nonsmokers) and GSTT1 null subjects (in smokers). Individuals carrying variant alleles for XRCC1 codons 280 and 194 showed a decreased level of chromosome-type breaks. The effect of GSTM1 null and XRCC1 codon 399 genotypes on the frequency of CAs was modified by smoking. In linear regression models adjusting for age, sex, smoking, and genotypes, none of the polymorphisms significantly affected SCE frequency, although GSTT1 null subjects had a slightly elevated SCE level. Our results are in line with earlier findings on the influence of NAT2, GSTT1, and GSTM1 polymorphisms on the level of lymphocyte chromosome damage and suggest that also XRCC1 polymorphism affects CA frequencies, thus apparently influencing DNA repair phenotype. It remains to be examined whether these or other genetic polymorphisms could explain the observed cancer risk predictivity of high CA frequency.     

Implications of an elevated sister-chromatid exchange frequency in rat lymphocytes cultured in the absence of erythrocytes

One important variable in complex culture systems such as whole blood is the interaction of the cell types present. To investigate the effects of erythrocytes (RBCs) and monocytes on the sister-chromatid exchange (SCE) frequency, Ficoll-Hypaque-separated Fischer-344 rat leukocytes were added to 1.9 ml of culture medium containing either 4 micrograms phytohemagglutinin or 4-8 micrograms concanavalin A/ml. Bromodeoxyuridine (BrdU;2 microM) was added at 24 h, and the cultures were harvested at 54 or 72 h. SCE frequencies in the mononuclear leukocyte cultures were consistently about 1.5- to 2-fold higher than in the whole-blood cultures. The titration of rat or human RBCs (0.05-2.5 X 10(9)) into purified rat leukocyte cultures reduced the SCE frequency to that of whole-blood cultures. Monocyte depletion decreased the elevated SCE frequency by approximately 50%. Scintillation counting of [14C]BrdU uptake in isolated RBCs revealed that less than 8% of the total amount of BrdU was sequestered. Also, BrdU induced a concentration-dependent increase in SCE in purified leukocytes, but the absolute increase was no greater than in whole-blood lymphocytes. Thus, BrdU had a minor role in the elevated SCE frequency in purified lymphocytes. Neither anti-oxidant enzymes such as catalase and superoxide dismutase nor the hydroxyl radical scavenger, dimethyl sulfoxide, decreased the SCE frequency. Although purified human lymphocytes had a small, but significant increase in SCE compared to whole blood, the magnitude of the dichotomous response between man and rat may represent a fundamental species difference.     

Lack of association between <Emphasis Type="Italic">GSTT1</Emphasis> polymorphism and endogenous or benzo[a]pyrene-induced sister chromatid exchanges as analyzed in metaphase or G2-phase lymphocytes

The objectives of the present work are (1) to verify whether genetic polymorphism in the detoxification gene GSTT1 influences the endogenous sensitivity in terms of sister chromatid exchanges (SCEs)/cell in healthy donors and (2) to test whether in vitro exposure to B[a]P in terms of SCEs/cell can be associated with polymorphism of GSTT1 gene. The presence or absence of the homozygous deletion in GSTT1 gene was determined in peripheral blood cells using multiplex-PCR. For SCEs quantitation, the cytogenetic method used thus far is based on the analysis in metaphase chromosomes. Consequently, G2-arrested cells are not included in the analysis. To overcome this shortcoming of the conventional method, we applied here SCE analysis in G2-phase prematurely condensed chromosomes (G2-PCCs) induced by calyculin-A, using a modified fluorescence-plus-Giemsa staining protocol. Compared to metaphase, a statistically significant increase in the yield of SCEs was notified in the G2-phase analysis after 48 h exposure of peripheral blood lymphocytes to 0.01–1 mM B[a]P, in both GSTT1-positive and -null donors. Therefore, the analysis of SCEs in the G2-phase using calyculin-A induced PCC methodology was shown to be more sensitive compared to the analysis at the metaphase level. Nevertheless, the results obtained do not show an association between the GSTT1 polymorphism with increased endogenous and/or B[a]P-induced SCE-frequencies in peripheral blood lymphocyte chromosomes in vitro. These results highlight not only the effect of B[a]P on cell cycle kinetics but also they demonstrate that conventional cytogenetic analysis at metaphase underestimates the cytogenetic effects of chemicals that delay cell cycle progression in G2-phase.