Re-evaluation of a reported increased micronucleus frequency in lymphocytes of workers occupationally exposed to formaldehyde

A replicate evaluation of increased micronucleus (MN) frequencies in peripheral lymphocytes of workers occupationally exposed to formaldehyde (FA) was undertaken to verify the observed effect and to determine scoring variability. May-Grünwald-Giemsa-stained slides were obtained from a previously performed cytokinesis-block micronucleus test (CBMNT) with 56 workers in anatomy and pathology laboratories and 85 controls. The first evaluation by one scorer (scorer 1) had led to a highly significant difference between workers and controls (3.96 vs 0.81 MN per 1000 cells). The slides were coded before re-evaluation and the code was broken after the complete re-evaluation of the study. A total of 1000 binucleated cells (BNC) were analysed per subject and the frequency of MN (in ‰) was determined. Slides were distributed equally and randomly between two scorers, so that the scorers had no knowledge of the exposure status. Scorer 2 (32 exposed, 36 controls) measured increased MN frequencies in exposed workers (9.88 vs 6.81). Statistical analysis with the two-sample Wilcoxon test indicated that this difference was not significant (p=0.17). Scorer 3 (20 exposed, 46 controls) obtained a similar result, but slightly higher values for the comparison of exposed and controls (19.0 vs 12.89; p=0.089). Combining the results of the two scorers (13.38 vs 10.22), a significant difference between exposed and controls (p=0.028) was obtained when the stratified Wilcoxon test with the scorers as strata was applied. Interestingly, the re-evaluation of the slides led to clearly higher MN frequencies for exposed and controls compared with the first evaluation. Bland-Altman plots indicated that the agreement between the measurements of the different scorers was very poor, as shown by mean differences of 5.9 between scorer 1 and scorer 2 and 13.0 between scorer 1 and scorer 3. Calculation of the intra-class correlation coefficient (ICC) revealed that all scorer comparisons in this study were far from acceptable for the reliability of this assay. Possible implications for the use of the CBMNT in human biomonitoring studies are discussed.     

Enhanced frequency of chromosome aberrations in workers occupationally exposed to diagnostic X-rays

To estimate the level of radiation exposure of personnel handling diagnostic X-ray machines, the yield of chromosomal aberrations was analysed in peripheral blood lymphocyte cultures. These occupationally exposed individuals showed higher frequencies of dicentrics as well as acentrics than normal controls. Absorbed radiation doses calculated by extrapolating reference in vitro dose-response curve for dicentrics ranged between 0.13 and 0.17 Gy. This implies exposure beyond the permissible limit of 0.05 Gy/year for the whole body. However, no obvious trend of increased aberrations as a function of either duration of employment or age was noticed. The increase in the aberration yields in this personnel underscores the need of adopting measures to avoid or minimise such overexposure.     

Genomic damages in peripheral blood lymphocytes and association with polymorphisms of three glutathione S-transferases in workers exposed to formaldehyde

DNA and chromosome damages in peripheral blood lymphocytes were evaluated in 151 workers occupationally exposed to formaldehyde (FA) and 112 non-FA exposed controls. The effects of polymorphisms in three glutathione-S-transferase (GSTs) genes on the DNA and chromosome damages were assessed as well. Alkaline comet assay and cytokinesis-block micronucleus (CBMN) assay were used to determine DNA and chromosome damages, respectively. The genotypes of GSTP1 (Ile105Val), GSTT1, and GSTM1 were assayed. The mean 8-h time-weighted average (TWA) concentrations of FA in two plywood factories were 0.83 ppm (range: 0.08–6.30 ppm). FA-exposed workers had higher olive tail moment (TM) and CBMN frequency compared with controls (Olive TM, 3.54, 95%CI = 3.19–3.93 vs. 0.93, 95%CI = 0.78–1.10, P < 0.01; CBMN frequency, 5.51 ± 3.37 vs. 2.67 ± 1.32, P < 0.01). Olive TM and the CBMN frequency also had a dose-dependent relation with the personal FA exposure. Significant association between FA exposure history and olive TM and CBMN frequency were also identified. The level of olive TM was slightly higher in FA-exposed workers with GSTM1 null genotype than those with non-null genotype (3.86, 95%CI = 3.31–4.50 vs. 3.27, 95%CI = 2.83–3.78, P = 0.07) with adjustment of covariates. We also found that FA-exposed workers carrying GSTP1 Val allele had a slightly higher CBMN frequency compared with workers carrying only the wild-type allele (6.32 ± 3.78 vs. 5.01 ± 2.98, P = 0.05). Our results suggest that the FA exposure in this occupational population increased DNA and chromosome damages and polymorphisms in GSTs genes may modulate the genotoxic effects of FA exposure.     

Cytogenetic analysis of nasal mucosa cells and lymphocytes from high-level long-term formaldehyde exposed workers and low-level short-term exposed waiters

The evidence for genotoxic potential of formaldehyde (FA) in humans is insufficient and conflicting. We previously reported a higher frequency of micronuclei in nasal and oral exfoliative cells from students exposed to formaldehyde vapor for short-term. To further evaluate the genetic effects of long-term occupational exposure to FA and short-term exposure to FA of indoor sources, the frequencies of micronuclei (MN) in nasal mucosa cells, sister chromatid exchanges (SCEs) of peripheral lymphocytes, and the lymphocyte subsets were evaluated in 18 non-smoking workers (mean exposure duration was 8.6 years) in an FA factory and 16 non-smoking waiters exposed to FA for 12 weeks in a ballroom. A non-smoking student group without occupational exposure (n=23) to FA was used as control. The 8h time-weighted average (TWA) concentrations of formaldehyde was 0.985+/-0.286 mg/m3 with the ceiling exposure concentration of 1.694 mg/m3 in the workshop, and 0.107+/-0.067 mg/m3 in the ballroom (5 h TWA). Higher frequencies of micronuclei per thousand cells in nasal mucosa cells of workers versus control (2.70+/-1.50 versus 1.25+/-0.65, p<0.05) and higher frequency of SCEs in peripheral lymphocytes of workers group (8.24+/-0.89 versus 6.38+/-0.41, p<0.05) were observed. Increased frequency of micronuclei in nasal mucosa cells or SCE in peripheral lymphocytes was not found among waiters group. The results suggest that the genotoxic potential of high level FA exposure may have occupational risks in long-term exposure groups.     

Micronucleus frequencies in workers exposed to lead, zinc, and cadmium

Micronuclei (MN) in blood lymphocytes were determined in 31 male workers occupationally exposed to lead (Pb), zinc (Zn), and cadmium (Cd) and 20 control workers matched for age and smoking habits. Exposed workers have higher MN mean values than control workers (p<0.01). In exposed workers, blood Pb concentrations were also significantly higher than in control workers (p<0.001), but the mean concentrations of Zn and Cd in the blood were not statistically significant compared to the controls (p>0.05). These results suggest that lead may be genotoxic and the human lymphocyte micronucleus test can be used to assess genotoxic effects that result from occupational exposures.     

Cytogenetic analysis of buccal cells from shoe-workers and pathology and anatomy laboratory workers exposed to n-hexane, toluene, methyl ethyl ketone and formaldehyde.

People employed in the shoe manufacture and repair industry are at an increased risk for cancer, the strongest evidence being for nasal cancer and leukaemia. A possible causal role for formaldehyde is likely for cancer of the buccal cavity and nasopharynx. Exfoliated buccal cells are good source of tissue for monitoring human exposure to inhaled and ingested occupational and environmental genotoxicants. To assess the cytogenetic damage related to occupational exposure to airborne chemicals during shoe-making and the processes in pathology and anatomy laboratories, the micronuclei (MN) count per 3000 cells was measured in buccal smears from shoe-workers (group I, n = 22) exposed to mainly n-hexane, toluene and methyl ethyl ketone (MEK) and from anatomy and pathology staff (group II, n = 28) exposed to formaldehyde (FA). Eighteen male university staff were used as controls. The mean time-weighted average (TWA) concentrations of n-hexane, toluene and MEK in 10 small shoe workshops were 58.07 p.p.m., 26.62 p.p.m. and 11.39 p.p.m., respectively. The measured air concentrations of FA in the breathing zone of the anatomy and pathology laboratory workers were between 2 and 4 p.p.m. Levels of 2,5-hexadione (2,5-HD) and hippuric acid (HA), metabolic markers of n-hexane and toluene exposure, respectively, were significantly higher in the urine of workers in group I than in control subjects (p < 0.001 and p < 0.01, respectively). The mean (+/- SD) MN (0/00) [corrected] frequencies in buccal mucosa cells from workers in group I, group II and controls were 0.62 +/- 0.45%, 0.71 +/- 0.56% and 0.33 +/- 0.30%, respectively (p < 0.05 and p < 0.05 compared with controls for group I and group II, respectively). The effects of smoking, age and duration of exposure on the frequency of micronucleated buccal cells from workers in all three groups studied were also evaluated. Overall, the results suggest that occupational exposure to organic solvents, mainly n-hexane, toluene, MEK and FA, may cause cytogenetic damage in buccal cells and that use of exfoliated buccal cells seems to be appropriate to measure exposure to organic solvents.     

Electroencephalographic findings in workers exposed to benzene

Preventive EEG examination was carried out in 40 workers significantly exposed to benzene. The EEG findings were compared with those of a control group of 48 healthy persons, a group of 110 workers significantly exposed to toluene and xylene and a group of 236 workers exposed to vinyl chloride. The individuals exposed to benzene exhibited 22.5% of abnormal and 45% threshold findings, the abnormalities being episodic, diffuse or a combination of the two. The effect of benzene entailed a frequent (32.5%) occurrence of a characteristic frequency lability. Sleep phenomena were found in a total of 60% cases (37.5% cases reached stage 1 B3 while 15% reached stage 2 according to Roth [14]). The rapid onset of deeper sleep stages (in 30% cases) is considered typical for benzene exposure. The photic driving response often had an extended frequency range (a total of 61.1%, to beta frequencies only in 30.55%, to both beta and theta frequencies also in 30.55% of cases). The different EEG features characteristic of the neurotoxic action of various types of organic solvents make possible a more efficient diagnostics of the effects of these chemicals on the CNS.     

Genotoxicity and radioresistance in electroplating workers exposed to chromium

A biomonitoring study was carried out to investigate the genetic risk associated to occupational exposure to chromium. The induction of genetic damage was measured by analysing the frequency of micronuclei (MN) in peripheral blood lymphocytes. In addition to the 40 electroplater exposed workers who participated in the study, a group constituted by 18 volunteer donors, without exposure to chromium, was analysed as a control group. Measures of chromium levels at working place and in erythrocytes and urine were obtained, as indicators of exposure. The results from this study indicate that the blood from exposed workers contained higher levels of chromium, when compared with those obtained in the control group, and that a significant increase in the frequency of both the total number of MN and the number of binucleated cells carrying MN (BNMN) was detected. Furthermore, a good direct relationship was obtained between the amount of chromium present in air, erythrocytes or urine and the frequency of MN. To determine the existence of radioresistance as consequence of chromium exposure, the response of lymphocytes to the in vitro gamma-radiation was studied. The results of this experiment show a lower induction in the increase of the frequency of MN after challenge irradiation in the lymphocytes of chromium exposed workers, which should be indicative of an adaptive response.     

Sperm shape abnormalities in mice exposed to californium-252 radiation

Male mice of the B6C3F1 hybrid strain were whole-body irradiated with different doses of 252Cf/60Co. They were killed 35 days later and spermatozoa from cauda epididymides were stained with eosin-Y. The air-dried smears were examined under light microscope for sperm shape abnormalities. There was an increase in the frequency of abnormal sperm in all the treated groups compared to controls. The RBE for the mixed neutron and gamma radiation of 252Cf was 2.6. The RBE for the neutron component was 3.4. The increased frequency of abnormal sperm was associated with a concomitant decrease in testis weight in the irradiated animals.     

Sister chromatid exchange in pathology staff occupationally exposed to formaldehyde

Sister chromatid exchange (SCE) was measured in peripheral lymphocytes of 90 workers from 14 hospital pathology departments in Israel who were occupationally exposed to formaldehyde (FA) and of 52 unexposed workers from the administrative section of the same hospitals. The mean exposure period to FA was 15.4 years (range 1-39). The results of SCEs are expressed in two variables: (a) mean number of SCEs per chromosome and (b) proportion of high frequency cells (cells with more than eight SCEs). A high correlation was found between these two variables. The adjusted means of both SCEs variables were significantly higher among the exposed compared with that of the unexposed group (P<0.01). Adjustment was made for age, sex, smoking habits, education workers and origin. Evaluation of the influence of years of exposure on the frequency of SCEs showed that the two variables of SCEs were higher among those who were exposed to FA for 15 or more than among those with less than 15 years of exposure. Concerning levels of exposure, both variables of SCEs were the same in the low and in the high levels of exposure sub-groups. However, among the smokers, both variables of SCEs were higher in the high exposure sub-group than in the low exposure sub-group.Our finding of a significant increase of SCEs frequency in peripheral lymphocytes in pathology staff indicates a potential cytogenetic hazard due to FA exposure. We conclude that our data indicate that FA is mutagenic to humans.     

Cytogenetic damage in workers exposed to ethylene oxide

Sister-chromatid exchanges (SECs) and chromosomal aberrations (CAs) were detected in the peripheral lymphocytes of 41 sanitary workers exposed to ethylene oxide (EO) in the sterilizing units of 8 hospitals in the Venice Region. The first group (19 workers) was exposed to 10.7 +/- 4.9 ppm EO, expressed as the time-weighted average concentration for an 8-h working day (TWA/8 h conc.), and the second group (22 workers) to 0.35 +/- 0.12 ppm. Each exposed worker was paired with a control of similar age and smoking habits. A highly significant (P less than 0.001) increase in the mean frequency of SCEs was found in the higher exposure group, 14 (74%) exposed subjects having significantly increased levels of SCEs compared to their matched controls. In the lower exposure group, the increase in mean frequency of SCEs was lower, though still significant (P less than 0.05): 7 (33%) exposed subjects had higher and 1 (5%) had a lower SCE level than the matched controls. From the first group, 10 subjects, 7 of whom had increased SCE levels, were reanalysed 12-18 months after their exposure had been lowered or interrupted: in only 2 of them the SCE level was significantly decreased. A statistically significant correlation between SCE frequency and level of EO exposure (TWA/8 h conc.), as well as a multiple correlation between SCE level and EO exposure, smoking and age were found. However, no interaction could be detected between EO exposure and smoking in the induction of SCEs. In controls, SCE frequency was correlated with smoking and age. In the higher exposure group, the number of both chromatid- and chromosome-type aberrations, independent of gaps, was significantly increased, whereas in the lower exposure group only the frequency of chromosome-type aberrations, excluding gaps, was statistically higher than in controls. The level of CAs remained to a great extent unchanged in the 10 subjects re-examined at a later stage after lowering or halting exposure. Taking the group as a whole, the frequency of cells with total CAs was found to be weakly (P = 0.05) correlated with EO exposure, and was not correlated with smoking, age or SCE frequency.     

Chromosomic aberrations in female workers exposed to pesticides

The purpose of this work was to determine if the occupational exposure to those pesticides used at banana plantations' packaging plants produces genetic damage to somatic cells of female workers. Chromosomal aberrations were scored in lymphocytes of 20 women, 10 female exposed workers and 10 female controls. Workers were recruited from independent farms from two locations in Costa Rica, during January through June in 1996 and 1997. These females had a minimum of three months of work, had never received chemotherapy or radiotherapy and did some of these labors: sealing, spraying or weighting of bananas. Control unexposed females lived in the same area, were of similar age and neither them nor their husbands/mates had ever worked in pesticide related labors. For each female, 100 mitotic figures were scored. The kind of aberrations detected were acentric fragments, dicentric chromosomes, rings, gaps and breaks. Among workers, 16% of cells (n=1000) had one or more abnormalities, whereas control unexposed females had 6% of cells (n=1000) with comparable anomalies (p < 0.05). In conclusion, the pesticide exposure is a risk factor for chromosome aberrations in female somatic cells.     

Sperm age, sex ratio, and hyperhaploidy frequency in mice.

Physiologically aged and unaged sperm from each of 12 sexually mature B6SJLF1/J mice were used to fertilize oocytes from females of the same strain, with each male serving as its own control. Male genomes in 323 and 307 first-cleavage metaphases obtained by in vivo and in vitro fertilization, respectively, were analyzed cytogenetically, using C-banding for detection of the Y chromosome. The sex (X:Y) ratio among all zygotes resulting from in vivo fertilization was 1.18; in zygotes resulting from in vivo fertilization by aged (14-d mating intervals) sperm, however, the ratio was 1.53, which differed significantly (chi 2 = 6.72, P less than 0.01) from the theoretical value of 1.00. Comparison of the sex ratio in zygotes resulting from in vivo fertilization by unaged sperm (3-d mating intervals), 0.94, with that in zygotes resulting from fertilization by aged sperm (using a 2 x 2 contingency table) showed a significant (chi c2 = 4.19, P less than 0.05) relationship between sex ratio and sperm age. In vitro neither the combined nor the individual 3- and 14-d data deviated significantly from the expected sex ratio of 1.00. The frequency of sperm-derived hyperhaploidy did not differ significantly between the in vivo (3.4%) and in vitro (5.9%) populations, but did between unaged (2.5%) and aged (6.8%) sperm (chi c2 = 5.74, P less than 0.01). All hyperhaploid zygotes had a complement of n + 1 chromosomes, except the 14-d in vitro group, where complements of n + 2 and n + 3 chromosomes were seen. Sperm-derived polyploidy, which was observed only in the in vitro group, was independent of sperm age and occurred in 6.8% of the zygotes. These data provide support for the sperm-aging hypothesis and indicate, for the first time, an influence of sperm aging in the male genital tract on the X:Y ratio of conceptuses resulting from natural matings of chromosomally normal males.     

Sperm morphology and sperm velocity in passerine birds

Sperm velocity is one of the main determinants of the outcome of sperm competition. Since sperm vary considerably in their morphology between and within species, it seems likely that sperm morphology is associated with sperm velocity. Theory predicts that sperm velocity may be increased by enlarged midpiece (energetic component) or flagellum length (kinetic component), or by particular ratios between sperm components, such as between flagellum length and head size. However, such associations have rarely been found in empirical studies. In a comparative framework in passerine birds, we tested these theoretical predictions both across a wide range of species and within a single family, the New World blackbirds (Icteridae). In both study groups, sperm velocity was influenced by sperm morphology in the predicted direction. Consistent with theoretical models, these results show that selection on sperm morphology and velocity are likely to be concomitant evolutionary forces.     

Chromosomal aberrations and sister-chromatid exchanges in workers exposed to styrene

Few studies exist about chromosomal damage in workers occupationally exposed to styrene. In the present study, chromosomal aberrations and SCEs were analyzed from cultures of peripheral lymphocytes of workers employed in 6 different reinforced-plastics industries with styrene air exposure levels ranging from 30 to 400 mg/mc. A control group was selected on the base of sex, age and smoking habit. We examined 50-h cultures (for chromosomal-aberrations) and 72-h cultures (for SCEs) for each individual. All workers exposed to styrene, as compared with controls, showed significantly increased frequencies of chromosomal aberrations, while SCEs were significantly increased at 4 of the 6 plants. High SCE values appeared with styrene air concentrations higher than 200 mg/mc. Apart from the possible presence and role of other interfering chemicals in the various plants, chromosomal aberrations seem to be more sensitive than SCEs for the detection of chromosomal damage caused by exposure to low doses of styrene.     

Sperm morphology: Its relevance to compensable and uncompensable traits in semen

The nature of subfertility due to the male or inseminate is as complex as that of the female. Fertilization failure, and failure in embryogenesis, are both of seminal origin. Males also differ in the number of sperms required to reach their maximum fertilization rate. Males requiring more sperm are considered to have compensable seminal deficiencies. These include a number of known viability and morphology traits (including both abnormal heads and tails) and unknown factors (functional or molecular traits) precluding sperm access to the ovum or ability of the sperm to engage the ovum sufficiently to initiate fertilization and the block to polyspermy. Differences in fertility among males or inseminates independent of sperm dosage are considered uncompensable. These seminal deficiencies are associated with fertilizing sperm that are incompetent to maintain the fertilization process or subsequent embryogenesis (once initiated), with most failures occurring prior to maternal recognition of pregnancy; these sperm would pre-empt fertilization by competent sperm. Evidence now exists supporting the concept that the uncompensable effect is due to chromatin aberrations in morphologically normal or near-normal fertilizing sperm present in abnormal ejaculates (elevated content of abnormal sperm). Thus, sperm morphology may be our best indication for the presence of an uncompensable deficiency, although we have yet to identify the incompetent fertilizing sperm clinically.