Relationship between genotoxicity biomarkers in somatic and
germ cells: findings from a biomonitoring study
L.Migliore1,*, R.Colognato1, A.Naccarati2and E.Bergamaschi3
1
Department of Human and Environmental Sciences, Section of Genetics, University of Pisa, Via S. Giuseppe 22, 56100 Pisa, Italy,2Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Videnska 1083, 14200 Prague 4, Czech Republic and3Department of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Via A. Gramsci 14 I-43100 Parma, Italy
A biomonitoring study to evaluate chromosome and DNA damage respectively in somatic and germ cells of a group of male workers exposed to styrene by using biomarkers of genotoxicity was carried out. Styrene-exposed workers from three different areas of Tuscany and healthy subjects, of comparable mean age, sex and lifestyle characteristics, as a control group not exposed to chemicals, have been enrolled. In addition to previous reports [L. Migliore, A. Naccarati, A. Zanello, R. Scarpato, L. Bramanti and M. Mariani (2002) Hum. Reprod., 17, 2912–2918; L. Migliore, A. Naccarati, F. Coppede` et al. (2006) Pharmacogenet. Genomics, 16, 87–99] we present now data on a cross-sectional investigation involving a homo-geneous group of subjects for which data on both somatic and germ cells have been obtained from individuals (42 exposed and 25 controls). Somatic cell genotoxicity was assessed by analysing the frequency of micronucleated binucleated cells (MNBN) in blood lymphocytes. The micronucleus assay was coupled with centromeric fluores-cence in situ hybridization (FISH) analysis. Primary DNA damage in germ cells was evaluated by alkaline single-cell gel electrophoresis (Comet assay) and the percentage of the tail DNA (%TD) was used as parameter of Comet evalu-ation. Moreover, to investigate the frequencies of aneu-ploidy and dianeu-ploidy in sperm, we performed multicolour FISH, using DNA probes specific for the centromeric regions of sex chromosomes and chromosome 2, in decon-densed sperm nuclei of samples with normal semen parameters in a subgroup of individuals. Mandelic and phenylglyoxylic acids (MAPGA) in end of shift samples were determined as biomarkers of internal dose. MAPGA excretion was consistent with an exposure to styrene above the threshold limit value-time weighted average concen-tration of 20 p.p.m. Styrene workers showed significantly higher frequency of MNBN as compared to controls (13.8 6 5.2 versus 6.2 6 5.1; P < 0.001), due to higher proportions of both micronuclei (MN) arising from chromosomal breakage (C MN) and harbouring whole chromosomes (C1MN). DNA damage in sperm cells was also higher among styrene-exposed, the %TD being 11.02 6 2.99 versus 7.42 6 2.30 in controls (P < 0.001). The incidence of aneuploidy and diploidy for the tested chromosomes in sperm did not show a statistically significant difference between workers and controls. However, a positive
correlation was found between genotoxic damage detected in somatic and in germ cells, even after removing the effect of age (r 5 0.475; P < 0.001). Although cytogenetic biomarkers detected both in somatic and germ cells were interrelated, no relationships were apparent with exposure parameters. Styrene exposure may increase the likelihood of both chromosome and DNA damage in somatic and germ cells, thus supporting the hypothesis of an interference on reproductive capacity among exposed workers. This is the first time that a field study shows a correlation between two biomarkers of genotoxicity evaluated at the same time in somatic and germ cells.
Introduction
Population biomonitoring is becoming an extremely powerful approach to determine the effect of environmental mutagens on human populations, mainly because of the improvement of methods in molecular epidemiology combined with reliable biomarkers of exposure. By applying conventional methods, early effects may be highlighted in accessible cell types, such as blood cells, exfoliated buccal or urothelial cells; thus, genetic biomonitoring allows to detect adverse effects of mutagenic chemicals in human somatic cells (1). However, such an approach has not yet been widely used for developing strategies in risk assessment and disease prevention and no direct information can be drawn on germs cells (2).
Extrapolation of findings gathered in somatic to germ cells of exposed individuals has been proposed only by using molecular dosimetry (e.g. DNA adducts) according to the ‘parallelogram approach’, proposed by Sobels (3), and bio-monitoring studies using germ cells have been encouraged since they would provide dose–response relationships, and data on comparative response with other cell types and on effectiveness for health risk assessment.
This short article focuses on genotoxic effects found in germ cells of workers occupationally exposed to styrene, and the relationships with changes in somatic cells. The study is as a part of a more complete survey carried out on workers occupationally exposed from two Italian regions (Emilia-Romagna and Tuscany) (2) in the framework of an EU Project (QLK4-CT-1999-01368: Genetic polymorphisms and biomonitoring of styrene). Styrene is a monomer used world-wide for the production of various plastics, synthetic and polyester resins. It is considered possibly carcinogenic to humans (Group 2B) (4), while its main metabolite, styrene-7,8-oxide, been classified as a probable human carcinogen (Group 2A) (5). Genotoxicity studies, mainly carried out in somatic cells, are not fully conclusive, whereas few genotoxi-city data have been so far obtained in germ cells of exposed individuals (1,6).
*To whom correspondence should be addressed. Tel: 139 050 836220; Fax: 139 050 551290; Email: l.migliore@geog.unipi.it
Ó The Author 2006. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.
All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org 149
Mutagenesis vol. 21 no. 2 pp. 149–152, 2006 doi:10.1093/mutage/gel012
Materials and methods Study population
The study population consisted of 42 male workers, aged 31.8 years (SD 5.3 years), who have been occupationally exposed to styrene for 9.6 years (SD 5.8 years) in factories manufacturing glass-fibre reinforced plastics and boats. A group of 25 male unexposed subjects, aged 31.6 years (SD 7.4 years), was recruited as a control group. Workers were enrolled only if employed for more than 2 years in the past 5 years and continuously for 6 months before biological sampling. Exposure was assessed by determining the urinary excre-tion of the main styrene metabolites, mandelic and phenylglyoxylic acids (MAPGA) (7). For ethical and questionnaire details see Miglioreet al. (2). Micronucleus assay in lymphocytes
The micronucleus test was performed according to Miglioreet al. (2). Two thousand binucleated cells were examined per individual for the presence of micronuclei (MN), following the scoring criteria adopted by the HUman MicroNucleus Project (8). The data were expressed as the frequency of micronucleated binucleated cells (MNBN), i.e. the number of binucleated lymphocytes containing one or more MN per 1000 binucleated cells. Fluorescence in situ hybridization analysis in lymphocytes
Fluorescence in situ hybridization (FISH) analysis was performed using MN slides from both exposed workers and unexposed controls. A digoxigenin-labelled a-satellite DNA probe specific for the centromeres of all human chromosomes (Appligene Oncor, Illkirch, France) was used to distinguish centomere-negative (C ) MN, containing acentric fragments, from centromere-positive (C1) MN, harbouring whole chromosomes. FISH protocol was performed according to Miglioreet al. (2).
The evaluation of the slides was performed using the Nikon Eclipse E800 with a magnitude of 3600. A total of 50 unequivocal MN in binucleated cells were examined for the presence/absence of C1 signals for each sample. Comet assay in germ cells
Comet assay procedure was performed according to McKelvey-Martinet al. (9). Coded slides were viewed using a fluorescence microscope (Nikon Eclipse E800). Observations were performed with a magnification of 3200. Fifty randomly selected cells (25 cells from each replicate slides were scored) per experimental point were evaluated using a Comet Image Analysis System (Kinetic Imaging Ltd, Liverpool, UK; Version 5.5). Results were reported as percentage of the tail DNA (%TD), which is indicative of the presence of DNA damage, expressed as mean of the 50 cells scored.
FISH analysis in germ cells
Multicolour FISH was performed using DNA probes specific for the centromeric regions of sex chromosomes (X and Y) and chromosome 2, in decondensed sperm nuclei of samples with normal semen parameters. For detailed materials and methods see Naccaratiet al. (6).
Statistical analysis
Differences between group means were assessed by Student’s t-test on independent samples. To evaluate the correlation between BNMN frequency in peripheral lymphocytes and the percentage of DNA damage in germ cells, multiple regression analysis was used. The correlation between the two values is expressed by means ofR2and Pearson’s correlation coefficient.
Data from sperm-FISH evaluation were analysed by Mann–Whitney non-parametric test.
Results
The sum of biomarkers of internal dose (GM of MAPGA 630 mg/g creatinine; GSD 3.5) above the biological exposure index of 400 mg/g creatinine revealed that styrene exposure, on a group basis, was above the occupational exposure limit of 20 p.p.m. (10).
Cytogenetic parameters evaluated in peripheral lymphocytes and in germ cells were consistently higher among styrene workers (Table I). Styrene workers showed significantly higher frequency of MNBN as compared to controls (13.8 6 5.2 versus 6.2 6 5.1; P < 0.001), due to higher proportions of both MN arising from chromosomal breakage (C MN) and harbouring whole chromosomes (C1MN). DNA damage in sperm cells was also higher among styrene-exposed, the %TD being 11.02 6 2.99 versus 7.42 6 2.30 in controls (P < 0.001).
On the whole group, a positive correlation was found between the frequency of MNBN peripheral lymphocytes and DNA damage in sperm cells (R25 0.247; P < 0.0001) (Figure 1); such relationship persisted even after removing the effect of age (partial correlation coefficent 5 0.475;P < 0.001). How-ever, when subgroups were analysed separately, a statistically significant relationship (P < 0.001) was observed only in the control group (R25 30.46%; F-ratio: 10.08; P 5 0.0042) but not in the exposed group (R25 2.76%; F-ratio: 1,18; P 5 n.s.). In a small group (18 styrene-exposed subjects and 13 unex-posed controls of the same age range) sperm-FISH analysis was carried out. Although no differences were seen between exposed workers and controls, parameters showing a malseg-regation (22-X and 22-Y) showed a slight, though statistically significant relationship with the %TD, after adjusting for age (r 5 0.416, P 5 0.041 and r 5 0.50, P 5 0.035). Similar relationships were detectable between the above endpoints and the duration of exposure (r 5 0.527, P 5 0.030 and r 5 0.493, P 5 0.045).
Although other cytogenetic biomarkers detected both in somatic and germ cells were found interrelated (with a slight, though statistically significant correlation coefficient; data not shown), no relationships were apparent with biomarkers of internal dose in styrene workers.
Table I. Cytogenetic parameters evaluated in somatic and germ cells of subjects belonging to the group under study
Endpoint Styrene exposed
(n 5 42) Control subjects (n 5 25) Mean 6 SD Mean 6 SD % TD (sperm cells) 11.02 6 2.99 7.42 6 2.30 MN BN/103peripheral lymphocytes 13.8 6 5.2 6.2 6 5.1 C1MNBN 7.34 6 3.23 3.34 6 2.37 C MNBN 6.09 6 3.04 2.21 6 1.43
Styrene-exposed show higher values and statistically significant differences (P < 0.001) in all investigated biomarkers.
Fig. 1. Correlation between the frequency of micronucleated peripheral lymphocytes and of sperm cells with fragmented DNA in a group of 67 subjects (styrene-exposed and control subjects). The regression line with 95% confidence interval for individual (outer line) and group values (inner line) is shown.
L.Migliore et al.
Discussion
This study deals with the correlation between endpoints of genotoxicity both in somatic and germ cells in a subgroup of subjects belonging to a larger cohort study (2), in which 95 workers occupationally exposed to styrene and 98 unex-posed controls have been studied by using an integrated approach involving biomarkers of exposure, effect and sus-ceptibility. We found a significant increase in the frequency of MNBN in styrene-exposed workers from two Italian cohorts compared with the reference population, along with a strong association between GSTT1 null genotype and increased MNBN frequencies in the exposed workers (2).
The present study was focussed on data from the Tuscany cohort, where genotoxic endpoints in germ cells were also simultaneously assessed. By the Comet assay in spermatozoa we already reported a significant increase in primary DNA damage in exposed workers compared with controls; moreover, we found an age-related decline in sperm DNA integrity (1).
This further analysis showed, for the first time, a significant correlation between the genotoxicity biomarkers detected in somatic cells and those evaluated in germ cells.
The main finding is the statistically significant correlation (R2 5 0.247; P < 0.001) between somatic and germ cell damage, indicating that both endpoints are interrelated and that genotoxic effects found in somatic cells may be predictive of germ cell damage on a group and at individual level. Among non-exposed controls we obtained confirmatory data of a positive correlation between the two endpoints. The lack of correlation in exposed group can indicate that the increase of the two endpoints is not proportionally comparable, possibly by the role played by exposure itself, as well as by the interac-tion between exposure and individual characteristics relying on metabolic disposition, as revealed by recent studies (2,11). On the other hand, it cannot be disregarded that the genetic background can be crucial for the outcome, when somatic and germ cells are compared, particularly considering the DNA repair capacity. There is limited information about DNA repair capacity in germ cells, but it seems that it could differ in function of spermatogenesis stages (12). Moreover, in somatic cells genotypes of metabolizing and DNA repair genes have been found to affect the genotoxic response in individuals exposed to styrene (13,14).
The incidence of aneuploidy and diploidy measured by FISH analysis in spermatozoa for the chromosomes X, Y and 2 did not show a statistically significant difference between workers and controls. From data, published and discussed by Naccarati et al. (6), it only emerged that confounding factors (i.e. age and smoking habit) appeared to exert a more important effect than exposure to styrene on numerical chromosome alterations in sperm nuclei of subjects selected for normal semen parameters. No noteworthy statistical correlations were found between the aneuploidy and diploidy frequency for the tested chromosomes in sperm and genotoxicity data in somatic cells, besides the slight statistically significant relationship between parameters showing a malsegregation (22-X and 22-Y) and the %TD, after adjusting for age.
The Frits Sobels parallelogram model, which has been pro-posed to estimate genotoxic risk for human beings, is based on experimental data in somatic cells (peripheral blood) of exposed animals and humans and on data from progeny studies of exposed animals (mice) (3). A recent application of the parallelogram approach was carried out for cyclophosphamide,
1,3-butadiene and urethane, to determine a risk assessment for the germ cells by evaluating heritable studies in rodents (15). However, this approach has been extended by proposing to bridge the gap of extrapolation between rodent and human germ cells by studying sperm samples (16). The comparison in the parallelogram of rodent/human sperm data with data from rodent progeny tests to derive at an estimate of human progeny at risk was then considered more promising. A direct comparison between genotoxicity in human somatic and germ cells owing to the improvement and availability of methodo-logies on human sperm is thus representing a further deve-lopment of the basic model.In vitro comparisons by inducing DNA damage in parallel in human sperm (evaluated by Comet assay) and in lymphocytes (chromosome aberrations analysis) employing an anticancer drug, doxorubicin, have been made with encouraging results (17).
A weakness of our study is that the positive correlation found is between two cytogenetic endpoints that cannot be considered of the same significance and fate. DNA fragmenta-tion detected with the Comet assay represents premutafragmenta-tional lesions that can be (or not) repaired successfully (or not). Micronuclei are considered a direct evidence of a true struc-tural or numerical mutation. For this reason, some authors believe that Comet assay is more linked to exposure and does not represent a true biomarker of effects, but preferably a biomarker of exposure, whereas MN are more consistently considered biomarkers of early effect. However, both end-points are indeed sensitive, and the clear relationship between them found in the present study supports the notion that the increased level of basal DNA damage can be detected success-fully by both biomarkers. To our knowledge, this is the first time that different biomarkers of genotoxicity evaluated at the same time in a biomonitoring study show a positive correlation in somatic and germ cells.
Acknowledgements
This work was, in part, supported by the Italian Ministero del Lavoro e della Previdenza Sociale, research project no. 1096 (Impiego di nuove metodologie per la valutazione del rischio genetico in spermatozoi di lavoratori esposti professionalmente), and by EC, contract QLK4-CT-1999-01368 (Genetic poly-morphisms and biomonitoring of styrene; SUSCEPTSTYRENE).
References
1. Migliore,L., Naccarati,A., Coppede`,F. et al. (2006) Cytogenetic biomarkers, urinary metabolites and metabolic gene polymorphisms in workers exposed to styrene.Pharmacogenet. Genomics, 16, 87–99. 2. Migliore,L., Naccarati,A., Zanello,A., Scarpato,R., Bramanti,L. and
Mariani,M. (2002) Assessment of sperm DNA integrity in workers exposed to styrene.Hum. Reprod., 17, 2912–2918.
3. Sobels,F.H. (1993) Approaches to assessing genetic risks from exposure to chemicals.Environ. Health Perspect., 101 (Suppl. 3):327–332.
4. International Agency for Research on Cancer (IARC). (2002) Some Traditional Herbal Medicines, Some Mycotoxins, Napthalene and Styrene. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, Vol. 82. IARC, Lyon, France.
5. International Agency for Research on Cancer (IARC). (1994)Some indus-trial chemicals. IARC Monographs on the Evaluation of the Carcinogenic Risk to Humans, Vol. 60. IARC, Lyon, France.
6. Naccarati,A., Zanello,A., Landi,S., Consigli,R. and Migliore,L. (2003) Sperm-FISH analysis and human monitoring: a study on workers occupa-tionally exposed to styrene.Mutat. Res., 537, 131–140.
7. Poggi,G., Giusiani,M., Palagi,U., Paggiaro,P.L., Loi,A.M., Dazzi,F., Siclari,C. and Baschieri,L. (1982) High-performance liquid chromato-graphy for the quantitative determination of the urinary metabolites of Somatic and germ cells genotoxicity biomarkers
toluene, xylene, and styrene.Int. Arch. Occup. Environ. Health, 50, 25–31. 8. Bonassi,S., Fenech,M., Lando,C.et al. (2001) HUman MicroNucleus pro-ject: international database comparison for results with the cytokinesis block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei. Environ. Mol. Mutagen., 37, 31–45.
9. McKelvey-Martin,V.J., Melia,N., Walsh,I.K., Jonhston,S.R., Hughes,C.M., Lewis,S.E.M. and Thompson,M. (1997) Two potential clinical applications of the alkaline single-cell gel electrophoresis assay: (1). Human bladder washing and transitional cell carcinoma of the bladder; and (2). Human sperm and male infertility.Mutat. Res., 375, 93–104.
10. American Conference of the Governmental Industrial Hygienists (ACGIH). (1999) Styrene, Monomer in ACGIH Documentation of the Threshold Limit Values and Biological Exposure Indices. Science Group ACGIH, Cincinnati, USA (http//www.ACGIH.com).
11. Haufroid,V., Jakubowski,M., Janasik,B.et al. (2002) Interest of genotyping and phenotyping of drug-metabolizing enzymes for the interpretation of biological monitoring of exposure to styrene.Pharmacogenetics, 12, 691–702.
12. Olsen,A.K., Lindeman,B., Wiger,R., Duale,N. and Brunborg,G. (2005) How do male germ cells handle DNA damage? Toxicol. Appl. Pharmacol., 207 (2 Suppl), 521–531.
13. Godderis,L., De Boeck,M., Haufroid,V., Emmery,M., Mateuca,R., Gardinal,S., Kirsch-Volders,M., Veulemans,I. and Lison,D. (2004) Influence of genetic polymorphisms on biomarkers of exposure and genotoxic effects in styrene-exposed workers.Environ. Mol. Mutagen., 44, 293–303.
14. Vodicka,P., Tuimala,J., Stetina,R.et al. (2004) Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers.Environ. Health Perspect., 112, 867–871. 15. Anderson,D. (2005) Male-mediated developmental toxicity.Toxicol. Appl.
Pharmacol., 207, 506–513.
16. Adler,I.D. (1996) Future research directions to study genetic damage in germ cells and estimate genetic risk.Environ. Health Perspect., 104, 619–624.
17. Baumgartner,A., Schmid,T.E., Cemeli,E. and Anderson,D. (2004) Parallel evaluation of doxorubicin-induced genetic damage in human lymphocytes and sperm using the comet assay and spectral karyotyping.Mutagenesis, 19, 313–318.
Received on December 22, 2005; revised and accepted February 14, 2006 L.Migliore et al.
