Biomedical subjects

H Norppa

Publications and source records attributed to H Norppa.

At least 55 records · Page 3Linked to original sources

PubMed1997

Exposure to soluble nickel in electrolytic nickel refining.

Past and present exposure to nickel was studied in an electrolytic nickel refinery, where an increased incidence of nasal cancer had been reported, using nickel analyses in air, blood and urine. Genotoxic effects were studied using analysis of micronuclei from acridine orange-stained smears from the buccal mucosa of the workers. Workers used respirators or masks in tasks where the exposure was expected to be high. Inside the mask, nickel concentrations were 0.9-2.4 micrograms m-3 in such tasks. In those tasks where masks were not used, nickel concentrations in the breathing zone were 1.3-21 micrograms m-3. Air-borne nickel concentrations (stationary sampling) varied between 230 and 800 micrograms m-3 in 1966-1988 with no systematic change; thereafter lower concentrations (170-460 micrograms m-3) have been observed. After-shift urinary concentrations of nickel were 0.1-2 mumol l-1; they showed no correlation with nickel concentrations in the air. Concentrations of nickel in the urine were still elevated after a 2-4 week vacation. The frequency of micronucleated epithelial cells in the buccal mucosa of nickel refinery workers was not significantly elevated by comparison with referents. No relationship was observed between micronucleus frequencies and levels of nickel in air, urine or blood.

Air Pollutants, Occupational↗

PubMed1997

Molecular cytogenetic analysis of buccal cells and lymphocytes from benzene-exposed workers.

Benzene is a well-characterized human carcinogen and clastogen still present in both the occupational and general environment. However, the levels of benzene encountered today are, in most cases, relatively low and new methods, more specific and sensitive than classical cytogenetics, are probably needed to assess if current benzene exposures pose a genotoxic risk to human health. Bearing in mind the leukaemogenic action of benzene, blood lymphocytes appear to be a suitable cell system for biomonitoring studies. Buccal epithelium is an alternative source of tissue for monitoring human exposure to inhaled occupational and environmental genotoxicants. New molecular cytogenetic techniques allowing us to specifically study clastogenic or aneugenic events in human cells may provide the additional sensitivity required. In the present study, fluorescence in situ hybridization was used to examine the content of micronuclei (MN) (using the pan-centromeric DNA probe SO-alphaAllCen) in lymphocytes and buccal cells and to detect numerical abnormalities of chromosome 9 (using a chromosome 9 centromere-specific alphoid DNA probe) in buccal cells from a population occupationally exposed to benzene in an Estonian petrochemical plant. Age-matched Estonian volunteers were used as a control group. Individual benzene exposure levels were estimated to be around 1 p.p.m. (8 h time-weighted average). No increases in the frequency of total MN, MN harbouring whole chromosomes or acentric chromosomal fragments or chromosome 9 numerical abnormalities were detected in relation to benzene exposure in the present study. The lack of positive results was consistent in both buccal cells and lymphocytes, indicating that the benzene exposure levels encountered did not induce detectable clastogenic or aneugenic effects in the exposed workers. Other variables and confounding factors, such as age, smoking or alcohol consumption, did not influence any of the multiple cytogenetic biomarkers analysed.

Benzene↗

PubMed1997

GSTT1-dependent induction of centromere-negative and -positive micronuclei by 1,2:3,4-diepoxybutane in cultured human lymphocytes.

The role of the glutathione S-transferase T1 gene (GSTT1) in determining genotoxic response to 1,2:3,4-diepoxybutane (DEB), an epoxide metabolite of 1,3-butadiene, was studied by analysis of micronuclei (MN) in cultured human lymphocytes using the cytokinesis block method. Fluorescence in situ hybridization (FISH) with an alphoid satellite DNA probe specific for the centromeres of all human chromosomes was applied to identify MN harboring whole chromosomes. Whole-blood lymphocyte cultures of 11 GSTM1 (glutathione S-transferase M1)-positive individuals (i.e. having at least one GSTM1 allele), of whom six were GSTT1-positive (with at least one GSTT1 allele) and five GSTT1-null (GSTT1 homozygously deleted), were treated for 48 h (starting 24 h after culture initiation) with two different concentrations (2 and 5 muM) [corrected] of DEB. The GSTT1-null individuals were excessively sensitive to DEB, showing, on average, approximately 2.5 times higher induced MN frequency (control frequency subtracted) than the GSTT1-positive donors, both at 2 muM [corrected] (mean/1000 binucleate cells 29.8 versus 11.8, P < 0.05) and 5 muM [corrected] (87.6 versus 34.0, P < 0.001) DEB. In accordance with the known strong clastogenicity of DEB, MN without centromeric FISH signals were particularly increased, the difference between the two GSTT1 genotypes being statistically significant at both concentrations of DEB (mean induced MN/1000 binucleate cells 23.1 versus 9.9, P < 0.05, at 2 muM [corrected]; 69.7 versus 24.2, P < 0.001, at 5 muM) [corrected]. In addition, centromere-positive (C+) MN were induced, suggesting that DEB also has some aneuploidogenic activity. The GSTT1-null genotype showed a significantly (P < 0.05) higher mean frequency of induced C+ MN than the GSTT1-positive genotype, at both 2 (6.7 versus 1.9) and 5 muM [corrected] (17.9 versus 9.8) DEB. At the higher dose mean nuclear division index was lower in the GSTT1-null group (1.80) than in the GSTT1-positive group (2.05, P < 0.01). These findings support earlier results from the analysis of sister chromatid exchange showing that individual sensitivity to the genotoxic and cytotoxic effects of DEB is largely explained by lack of the GSTT1 gene.

Adult↗

PubMed1997

Cytogenetic markers of susceptibility: influence of polymorphic carcinogen-metabolizing enzymes.

Polymorphisms of xenobiotic-metabolizing enzymes, responsible for individual differences in metabolic activation and detoxification reactions, may profoundly modulate the effects of chemical carcinogens. In the case of genotoxic carcinogens, differences in biological effects due to genetic polymorphisms can be evaluated by cytogenetic methods such as the analysis of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), micronuclei (MN), and changes in chromosome number. These techniques can be applied to any exposure known to induce such alterations, without additional method development for each exposing agent. The influence of polymorphic genes on the cytogenetic effects of a carcinogen can quickly be tested in vitro using metabolically competent cells collected from donors representing different genotypes or phenotypes. For instance, erythrocytes from individuals positive for glutathione S-transferase T1 (GSTT1) express GSTT1, whereas GSTT1-null donors, having a homozygous deletion of the GSTT1 gene, completely lack this detoxification enzyme. This deficiency results in highly increased sensitivity to SCE induction in whole-blood lymphocyte cultures by 1,2:3,4-diepoxybutane, a reactive metabolite of 1,3-butadiene. The same cytogenetic techniques can also be applied as effect biomarkers in studies of human populations exposed to genotoxic carcinogens. For example, elevated rates of chromosome damage have been detected among smokers lacking glutathione S-transferase M1 (GSTM1-null genotype), and the baseline level of SCEs seems to be increased in GSTT1-null individuals. Information obtained from cytogenetic studies of genetic polymorphisms can be used, for example, to recognize the genotoxically relevant substrates of the polymorphic enzymes, to identify genotypes that are susceptible to these genotoxins, to improve in vitro genotoxicity tests utilizing human cells, to increase the sensitivity of cytogenetic endpoints as biomarkers of genotoxic effects in humans, and to direct mechanistic studies and cancer epidemiology.

Carcinogens↗

PubMed1996

Induction of sister chromatid exchange by 3,4-expoxybutane-1,2-diol in cultured human lymphocytes of different GSTT1 and GSTM1 genotypes.

The induction of sister chromatid exchanges (SCEs) by a 48-h treatment with 3,4-epoxybutane-1,2-diol (EBD), a metabolite of 1,3-butadiene, was studied in whole-blood lymphocyte cultures of 22 human donors with known genotypes of two polymorphic glutathione S-transferases (GSTs), GSTT1 and GSTM1. For both genes, donors representing a homozygous 'null' genotype lacking the respective GST gene and isozyme and a 'positive' genotype with at least one intact gene and GST activity were included. The mean frequencies of SCE/cell were similar in all genotype groups: GSTT1 null (n = 10) (mean 22.0 for 250 microM and 32.9 for 500 [corrected] microM of EBD), GSTT1 positive (n = 14) (21.3 and 34.6, respectively), GSTM1 null (n = 10) (20.3 and 33.5) and GSTM1 positive donors (n = 15) (20.6 and 34.8). At 500 microM concentration of EBD, the lymphocyte cultures of all donors showed a significantly decreased replication index. No differences in EDB-induced SCEs or in replication index could be associated with the GSTM1 and GSTT1 genotypes either separately or in combination. When SCE induction by EBD was compared to that of two other known epoxide metabolites of butadiene, 1,2:3,4-diepoxybutane (DEB) was effective at concentrations over two orders of magnitude lower than EBD or 1,2-epoxy-3-butene (MEB). It is concluded that EBD is an efficient inducer of SEC in cultured human lymphocytes, although not quite as effective as MEB and clearly less effective than DEB. Contrary to previous findings with DEB and MEB, the polymorphic GSTM1 and GSTT1 do not appear to be involved in the detoxification of EBD in human lymphocytes.

Adult↗

PubMed1996

Repeated analysis of sister chromatid exchange induction by diepoxybutane in cultured human lymphocytes: effect of glutathione S-transferase T1 and M1 genotype.

Spontaneous and diepoxybutane (DEB)-induced sister-chromatid exchanges (SCEs) were examined in whole-blood lymphocyte cultures of 3 men and 4 women. A strong increase in mean number of SCEs per cell with increasing DEB concentrations (0, 2, and 4 microM) was observed in cultures of all subjects, but 3 of the donors were clearly more sensitive than the others. The SCE measurements were repeated 2-6 times per donor over a period of 55 months to assess the stability of the individual SCE response. The results showed that SCE induction by DEB was steady in the individuals during the follow-up at each DEB dose, with no significant differences among the repeated experiments. At 4 microM DEB, the DEB-sensitive and -resistant donors could be reliably be differentiated from each other in all trials. As DEB-sensitivity has been suggested to be due to the lack of glutathione S-transferase (GST) T1, the donors were genotyped for the presence of GSTT1 and GSTM1 genes. The 3 individuals found to be DEB-sensitive were all of the GSTT1 null genotype, whereas the 4 DEB-resistant donors were GSTT1 positive, which supported the role of the GSTT1 gene in determining DEB-sensitivity. Three of the DEB-resistant and none of the DEB-sensitive had the GSTM1 null genotype. Thus, the lack of the GSTM1 gene was not associated with the DEB-sensitivity trait. In conclusion, the present findings show that individual SCE responses to treatment of cultured human lymphocytes with DEB can reliably be reproduced in repeated trials. The results confirm that the GSTT1 gene but not the GSTM1 gene is important in determining individual sensitivity to the in vitro genotoxicity of DEB.

Cells, Cultured↗

PubMed1996

Cytogenetic monitoring of occupational exposure to pesticides: characterization of GSTM1, GSTT1, and NAT2 genotypes.

Occupational exposure of floriculturists is characterized by alternating periods of intense pesticide spraying and reduced or no activity. Induction of sister chromatid exchanges (SCE), structural chromosome aberrations (CA) and micronuclei (MN) was investigated in peripheral lymphocytes of a group of 23 Italian floriculturists and 22 matched controls. Blood sampling was performed during and one month after the end of intensive pesticide treatments, in order to cover a period of high and low exposure, respectively. Each donor was genotyped for glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and N-acetyltransferase 2 (NAT2), three polymorphic genes involved in xenobiotic metabolism, to assess their potential role in individual genotoxic response to the pesticide exposure. No effect of the pesticide exposure on the cytogenetic parameters were detected. Smoking, however, was found to increase SCE levels. The only significant influence of phenotype composition on cytogenetic response was an increase in SCE levels in the GSTT1 positive individuals compared with the GSTT1 nulls (P=0.02). This finding was, however, based on only four GSTT1 null donors (n=41 for GSTT1 positive donors). In addition, a possible interaction was observed between smoking and GSTM1 genotype in the CA assay, GSTM1 null smokers, earlier reported to have an elevated risk for lung cancer, showing higher CA frequencies than GSTM1 positive smokers.

Adult↗

PubMed1996

Influence of erythrocyte glutathione S-transferase T1 on sister chromatid exchanges induced by diepoxybutane in cultured human lymphocytes.

Humans can be classified as either DEB-sensitive or DEB-resistant on the basis of the frequency of sister chromatid exchanges (SCEs) induced in whole-blood lymphocyte cultures by 1,2,3,4-diepoxybutane (DEB), an epoxide metabolite of 1,3-butadiene. Sensitivity to in vitro SCE induction by DEB has been explained by a deletion of the glutathione S-transferase T1 gene (GSTT1), resulting in a deficiency of erythrocytic GSTT1 activity among GSTT1 null homozygotes. To verify that the GSTT1 activity of erythrocytes is responsible for differential SCE induction by DEB, SCEs induced by a 48-h exposure to DEB (2 or 5 microM) were analyzed in whole-blood and isolated lymphocyte cultures of four GSTT1 positive and four GSTT1 null individuals. The mean frequency of SCEs/cell was about twice as high among the GSTT1 null donors as compared with the GSTT1 positive donors, at both 2 microM DEB (mean 53.1 versus 25.8) and 5 microM DEB (mean 74.4 versus 38.4) in whole-blood lymphocyte cultures. In isolated lymphocyte cultures, DEB induced higher SCE frequencies than in whole-blood cultures and there was essentially no difference between the response of the two GSTT1 genotypes (mean 68.3 for GSTT1+ and mean 69.2 for GSTT1- at 2 microM DEB and 85.0 and 92.7, respectively, at 5 microM DEB). The isolated lymphocyte cultures were also much more sensitive to the cytotoxic effect of DEB, observed as significantly decreased replication indices at both DEB concentrations and genotypes. The whole-blood lymphocyte cultures showed lower replication indices for the GSTT1 null subjects than for those having the GSTT1 gene at both concentrations of DEB. These results support the role of GSTT1 activity in erythrocytes as a major detoxification pathway for DEB.

Adult↗

PubMed1996

In vivo cytogenetic damage revealed by FISH analysis of micronuclei in uncultured human T lymphocytes.

FISH analysis of micronuclei in uncultured human T lymphocytes provides a convenient new possibility to assess structural and numerical chromosome damage in vivo. In women. T-cell micronuclei mostly contained whole chromosomes (71.6%), especially the X chromosome (28.5%). Cell culture (72 h) enhanced the frequency of micronuclei harboring acentric fragments 2.9 fold and the X chromosome 1.5 fold. X-chromosome-positive micronuclei were particularly prevalent (42.0%) in binucleate cells produced by cytochalasin B, a cytokinesis inhibitor used to identify cells that have divided in vitro. This was explained by a decrease in autosome-containing and an increase in X-positive micronuclei.

Adult↗

PubMed1996

Glycophorin A somatic cell mutation frequencies in Finnish reinforced plastics workers exposed to styrene.

We have used the glycophorin A (GPA) in vivo somatic cell mutation assay to assess the genotoxic potential of styrene exposure in 47 reinforced plastics workers occupationally exposed to styrene and 47 unexposed controls matched for age, gender, and active smoking status. GPA variant erythrocyte frequencies (Vf), reflecting GPA allele loss (phi/N) and allele loss and duplication (N/N) somatic mutations arising in vivo in the erythroid progenitor cells of individuals of GPA M/N heterozygous genotype, were flow cytometrically determined in peripheral blood samples from these subjects. Measurements of styrene exposure of the workers at the time of blood sampling showed a mean 8-h time-weighted average (TWA8-h) styrene concentration of 155 mg/m3 (37 ppm) in the breathing zone. Mean urinary concentrations of the styrene metabolites mandelic acid (MA) and mandelic acid plus phenyl glyoxylic acid (MA+PGA) were 4.4 mmol/liter (after workshift) and 2.1 mmol/liter (next morning), respectively. Multivariate analysis of covariance on log-transformed GPA Vf data with models allowing adjustment for age, gender, smoking status, and styrene exposure showed that N/N Vf were nearly significantly increased among all of the exposed workers (adjusted geometric mean, 6.3 per million versus 5.0 in the controls; P = 0.058) and were statistically significantly elevated (adjusted geometric mean, 6.8 versus 5.0 in the controls; P = 0.036) among workers classified into a high-exposure group according to personal TWA8-h concentration of styrene in the breathing zone of > or = 85 mg/m3 (20 ppm; Finnish threshold limit value). Women in this high exposure group showed especially elevated N/N Vf (adjusted geometric mean 8.5 versus 5.3 in control women; P = 0.020); this elevation was also significant if urinary MA+PGA of > or = 1.2 mmol/liter was used as the basis of classification (adjusted geometric mean, 8.3; P = 0.030). The occupational exposure could not be shown to influence phi/N Vf. Cigarette smoking was associated with significantly elevated GPA Vf among active smokers (P = 0.042 for phi/N and P = 0.020 for N/N) and among active and ex-smokers combined (P = 0.014 for N/N). Its influence on phi/N Vf was especially clear among active smokers in the control group (P = 0.005). An effect of smoking, nearly statistically significant, was also observed for the phi/N Vf of control ex-smokers (P = 0.055) and of all active and ex-smokers combined (P = 0.050). Thus, the two characterized chemical exposures experienced by this group of workers and controls appear to produce differential effects on the two independent classes of GPA variants enumerated in the assay. This result suggests that the genotoxicity of these agents is mediated, at least in part, by different genetic mechanisms. Styrene exposure is associated with a specific increase in GPA N/N Vf; these allele loss and duplication variants reflect predominantly somatic recombination mechanisms in erythroid progenitor cells. Tobacco smoke exposure in active and ex-smokers is also associated not only with an increase in N/N Vf but also with an increase in phi/N Vf, reflecting the induction of GPA gene-inactivating mutations, including point mutations and deletions. This finding is consistent with a broad mechanistic spectrum of tobacco smoke genotoxicity associated with this complex mixture of chemical mutagens. Finally, there was no detectable effect of age on phi/N Vf; however, a highly significant (P = 0.0002) increase in N/N Vf with age, even after adjustment for other variables, was observed.

Adult↗

PubMed1995

Induction of micronuclei by five pyrethroid insecticides in whole-blood and isolated human lymphocyte cultures.

Five pyrethroid insecticides: cypermethrin, deltamethrin, fenpropathrin, fenvalerate and permethrin, were tested for their ability to induce micronuclei in both whole-blood (WB; three donors) and isolated human lymphocyte (IL, 2 donors) cultures, by using the cytokinesis-block method with 6 micrograms/ml cytochalasin B (Cyt-B). Fenvalerate and permethrin were tested with two different concentrations of Cyt-B (3 and 6 micrograms/ml). At the concentration ranges tested, all the five pyrethroids induced clear dose dependent cytotoxic effects, fenpropathrin being the most toxic. Nuclear division index (NDI) and the newly introduced index of cytotoxicity, the cytokinesis block proliferation index (CBPI), reflected the dose dependency more accurately than the percentage of binucleated cells did. CBPI is similar to NDI except that it estimates the average number of cell divisions that the cell population has gone through, and, therefore, classifies both trinucleate and tetranucleate cells into the same category. Cypermethrin and fenpropathrin slightly increased the number of MN and micronucleated cells in WB lymphocyte cultures from two out of the three donors. Deltamethrin produced a positive response only in WB cultures of one donor and in IL cultures of another donor. Permethrin gave mostly negative results, although it increased the MN frequency in WB cultures of one donor when 6 micrograms/ml Cyt-B was used. Fenvalerate did not significantly induce MN. With certain reservations to the purity and isomer composition of each pesticide, the existing information appears to support the idea that pyrethroid insecticides have a weak (cypermethrin, deltamethrin and fenpropathrin) or nule (fenvalerate and permethrin) genotoxic activity in vitro.

Adult↗

PubMed1995

Effects of indomethacin and arachidonic acid on sister chromatid exchange induction by styrene and styrene-7,8-oxide.

Styrene is converted into styrene-7,8-oxide in human lymphocyte cultures, in a reaction probably mediated by oxyhemoglobin. As a consequence, styrene induces sister-chromatid exchanges (SCEs) in whole-blood lymphocyte cultures without exogenous metabolic activation systems. Another metabolic pathway that could be involved in the metabolism of styrene is cooxidation by prostaglandin-endoperoxide synthase (PES). To study the role of PES in the metabolism of styrene, human whole-blood lymphocyte cultures were treated for the entire culture time of 72-h with styrene (0.5 and 1 mM) or styrene-7,8-oxide (50 and 100 microM), in the presence and absence of 75 or 150 microM indomethacin (an inhibitor of PES) and arachidonic acid (substrate of PES). Indomethacin potentiated SCE induction by both styrene and styrene-7,8-oxide; a slight but statistically significant enhancement (16-32%; p < 0.05-p < 0.001) was observed in all treatments with styrene and at 150 microM indomethacin in the case of styrene-7,8-oxide. At 150 microM, arachidonic acid induced a 15-20% suppression (p < 0.01) in SCE induction by both styrene (1 mM only) and styrene-7,8-oxide (100 microM only). Indomethacin or arachidonic acid did not alone influence the frequency of SCEs. The results suggest that PES acts as an inactivation route for styrene and styrene-7,8-oxide in human whole-blood lymphocyte cultures, possibly through PES-mediated binding to glutathione.

Adult↗

PubMed1995

Effects of indomethacin and arachidonic acid on sister chromatid exchange induction by styrene and styrene-7,8-oxide.

Styrene is converted into styrene-7,8-oxide in human lymphocyte cultures, in a reaction probably mediated by oxyhemoglobin. As a consequence, styrene induces sister-chromatid exchanges (SCEs) in whole-blood lymphocyte cultures without exogenous metabolic activation systems. Another metabolic pathway that could be involved in the metabolism of styrene is cooxidation by prostaglandin-endoperoxide synthase (PES). To study the role of PES in the metabolism of styrene, human whole-blood lymphocyte cultures were treated for the entire culture time of 72 h with styrene (0.5 and 1 mM) or styrene-7,8-oxide (50 and 100 microM), in the presence and absence of 75 or 150 microM indomethacin (an inhibitor of PES) and arachidonic acid (substrate of PES). Indomethacin potentiated SCE induction by both styrene and styrene-7,8-oxide; a slight but statistically significant enhancement (16-32%; p < 0.05-0p < 0.001) was observed in all treatments with styrene and at 150 microM indomethacin in the case of styrene-7,8-oxide. At 150 microM, arachidonic acid induced a 15-20% suppression (p < 0.01) in SCE induction by both styrene (1 mM only) and styrene-7,8-oxide (100 microM only). Indomethacin or arachidonic acid did not alone influence the frequency of SCEs. The results suggest that PES acts as an inactivation route for styrene and styrene-7,8-oxide in human whole-blood lymphocyte cultures, possibly through PES-mediated binding to glutathione.

Adult↗

PubMed1995

Influence of GSTM1 genotype on sister chromatid exchange induction by styrene-7,8-oxide and 1,2-epoxy-3-butene in cultured human lymphocytes.

Glutathione S-transferase M1 (GSTM1), catalyzing the conjugation of various reactive molecules with glutathione (GSH), shows genetic polymorphism in humans. Almost half of all Caucasians lack the GSTM1 gene, being theoretically at a higher risk from the toxic effects of substrates for GSTM1. The purpose of the present study was to investigate whether the GSTM1 genotype of lymphocyte donors influences the in vitro induction of sister chromatid exchanges (SCEs) by styrene-7,8-oxide (SO) and 1,2-epoxy-3-butene (MEB), the epoxide metabolites of styrene and butadiene respectively and potential substrates for GSTM1. SCEs induced after a 48 h treatment (started 24 h after culture initiation) by two different concentrations of SO (50 and 150 microM) and MEB (50 and 250 microM) were analyzed in cultured (72 h) lymphocytes of six GSTM1 null (gene deleted) and six GSTM1-positive (gene present) donors. Both SO and MEB were found to clearly increase SCEs. The GSTM1 genotype had no influence on SCE induction by SO. In contrast, MEB produced a higher level of SCEs among the GSTM1 null than GSTM1-positive samples. At 250 microM MEB, the GSTM1 null donors showed 31% more induced SCEs (on average seven more SCEs per cell) than the GSTM1-positive donors (P = 0.02, acetone treatment as the reference). Furthermore, the GSTM1 null genotype was associated with a slight decrease in mitotic index and replication index, regardless of the treatment. The results suggest that GSTM1-mediated GSH conjugation is an important detoxification pathway for MEB, but not for SO, in cultured human lymphocytes.

Adult↗

PubMed1995

Role of GSTT1 and GSTM1 genotypes in determining individual sensitivity to sister chromatid exchange induction by diepoxybutane in cultured human lymphocytes.

The individual genotoxic response of cultured human lymphocytes to diepoxybutane (DEB), an epoxide metabolite of 1,3-butadiene, shows a bimodal distribution. Blood donors can be classified as either DEB-sensitive or DEB-resistant on the basis of the frequency of sister chromatid exchanges (SCEs) induced by DEB in whole-blood lymphocyte cultures. The genetic basis of this phenomenon has thusfar been unknown. To investigate if differences in the ability of individuals to detoxify DEB could explain the bimodal response, sister chromatid exchanges (SCEs) induced by a 48-h treatment with DEB (2 and 5 microM) were analyzed in whole-blood lymphocyte cultures of 20 human donors with known genotypes of two polymorphic glutathione S-transferases (GSTs), GSTT1 and GSTM1. Both polymorphisms include a homozygous null genotype lacking the respective GST gene and isozyme. The mean frequency of SCEs/cell was 1.6 times higher among GSTT1 null donors (n = 8) than GSTT1 positive donors (n = 12) at both 2 microM DEB (mean 67.3 versus 40.9) and 5 microM DEB (mean 123.2 versus 77.5), with no overlapping in DEB-induced individual SCE frequencies between the two genotypes. Thus, all DEB-sensitive individuals were of the GSTT1 null genotype, while all DEB-resistant persons had a detectable GSTT1 gene. A significant (P < 0.05) negative correlation (r = -0.65 at 5 microM, r = -0.56 at 2 microM) was obtained in the GSTT1 positive donors between DEB-induced individual SCE frequency and RBC GSTT1 activity, measured by formaldehyde formation from dichloromethane; the GSTT1 null individuals showed no GSTT1 activity. At 5 microM DEB, the lymphocyte cultures of the GSTT1 null donors also had a significantly decreased replication index, indicating an impact of GSTT1 genotype on the cytotoxicity of DEB. No influence on DEB-induced SCEs or cytotoxic effects was observed for GSTM1 genotype. It is concluded that sensitivity to in vitro SCE induction by DEB is explained by the lack of GSTT1.

Adult↗

PubMed1995

Comparison of separated erythrocyte preparations and manual smears of bone marrow in showing micronucleus induction by clastogens and aneuploidogens in mouse.

The removal of nucleated cells from bone marrow cell suspensions by cellulose column separation and further purification in a Percoll gradient, coupled with slide preparation by a cytocentrifuge, produces uniform preparations of pure well-spread erythrocytes. The use of separated erythrocytes has been a considerable improvement in the in vivo micronucleus (MN) assay, because the optimal cell morphology and the possibility of scoring a high number of polychromatic erythrocytes (PCEs) in a short time are factors contributing to the sensitivity of the assay. As the separation procedure may selectively retain cells containing MN, a study comparing the performance of the erythrocyte fractionation technique and standard manual smear preparation was conducted in male NMRI mice treated with a single injection of two clastogens (cyclophosphamide, 25 or 50 mg/kg, or methylmethane sulfonate, 40 or 80 mg/kg) or two aneuploidogens (colchicine, 0.5 or 1 mg/kg, or vincristine sulfate, 0.05 or 0.1 mg/kg). Both the preparation methods were clearly able to detect the MN-inducing and toxic effects of the treatments. After treatment with cyclophosphamide and methylmethane sulfonate, the frequency of micronucleated PCEs (MNPCEs) was, respectively, 1.6 and 1.8 times higher in the fractionated erythrocytes than in whole bone marrow. Morphological characterization of the MN indicated that this phenomenon was due to improved identification of small micronuclei in the flat cytospun cells of the fractionated erythrocyte preparations. On the other hand, the purified erythrocyte slides contained only about half the number of MNPCEs observed in standard smears, in samples collected from mice treated with colchicine.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗

PubMed1995

Micronuclei induced by alachlor, mitomycin-C and vinblastine in human lymphocytes: presence of centromeres and kinetochores and influence of staining technique.

Antikinetochore antibodies and fluorescence in situ hybridization with an alphoid centromeric probe were applied to the cytokinesis-block micronucleus (MN) assay to study the suitability of these methodologies to detect clastogenic/aneugenic activity in isolated human lymphocytes. The chemicals selected for this study were the herbicide alachlor, the clastogen mitomycin-C (MMC), and the aneugen vinblastine sulphate (VBL). Furthermore, MN frequencies obtained from slides stained with May-Grünwald-Giemsa (MGG) and with the DNA fluorochrome 4',6'-diamidino-2-phenylindole (DAPI) were compared to check if the DNA-specific DAPI facilitated a more accurate recording of MN than the unspecific MGG. The results showed that the detection of kinetochores (KC) or centromeres (CM) within MN are equally reliable and sensitive techniques to study the mode of action of clastogenic and aneugenic agents. The comparison of CM and KC detection in control cultures suggested that up to 17% of spontaneous chromosome-containing MN may be due to KC disruption, whereas the majority are caused by dysfunction in other components of the mitotic apparatus. Alachlor (7.5-20 micrograms/ml) and MMC (0.6 microM) acted as pure clastogens without aneugenic activity, inducing exclusively KC- and CM-negative MN. VBL produced primarily KC- and CM-positive MN, in accordance with its known mechanism of action. A comparison between CM and KC data in the VBL treatment suggested that some 7% of KC-containing MN may not be detected by the probe. The frequencies of MN were generally higher in slides stained with DAPI than in those stained with MGG, especially in controls and clastogen-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetamides↗

PubMed1995

Age-associated micronuclei containing centromeres and the X chromosome in lymphocytes of women.

Chronological aging of women is clearly associated with an increase in both X-chromosome loss and micronuclei formation in peripheral lymphocytes. It has been suggested that micronucleus formation is an important mechanism of chromosome loss. In the present study, fluorescence in situ hybridization was used to study micronuclei content in two age groups (women below 30 and above 50 years old). A probe for centromeric alphoid consensus sequences (SO-alpha AllCen) and a cloned X-specific centromeric probe (pXBR) were separately used to detect the presence of any chromosomes and the X chromosome, respectively. The presence of centromere-positive micronuclei was significantly higher among the older donors (51.5%) than among the younger donors (34.3%). The X chromosome was highly overrepresented in the micronuclei, the older women showing a higher proportion of X-positive micronuclei (24.0%) than the younger women (14.0%). Assuming that the rest of the centromere-positive micronuclei contained autosomes, a significant age-dependent difference was also noted for micronuclei harboring autosomes (27.5% among the older women and 20.3% among the younger women). These findings suggest that both the X chromosome and autosomes are responsible for the age-dependent increase of micronuclei in women's peripheral lymphocytes.

Adult↗