Susceptibility towards formaldehyde-induced genotoxicity. Günter Speit
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1 Susceptibility towards formaldehyde-induced genotoxicity Günter Speit Universität Ulm Institut für Humangenetik D Ulm (Germany) CEFIC-2012 Günter Speit 1
2 Susceptibility towards formaldehyde-induced genotoxicity Outline of the presentation: I. Introduction II. Nasal sensitivity and susceptibility towards genotoxicity III. Potential susceptibility of different study groups IV. Polymorphisms in the FDH gene and susceptibility V. Summary CEFIC-2012 Günter Speit 2
3 I. Introduction Susceptible subgroups are groups of individuals with predisposition to greater response to an exposure: Genetics (e.g., genetic polymorphisms) Gender differences Life stages (e.g. children) Health status (e.g., asthmatics) Lifestyle factors Nutritional state Exposure to other chemicals CEFIC-2012 Günter Speit 3
4 Background SCOEL (2008): Exposure limit should especially consider possible inter-individual differences in susceptibility to irritation by FA. The critical health effect of exposure to FA is the potential to induce cancer in the upper respiratory tract. There is no reason to assume that subgroups with increased susceptibility to irritation may also have a higher risk for the induction of mutations and cancer but data are not available. We investigated the in vitro genotoxic susceptibility of blood cells in relationship to nasal sensitivity. CEFIC-2012 Günter Speit 4
5 Cellular defence against FA-induced mutagenicity endogenous sources methanol exogenous sources sources DPX Repair formaldehyde glutathione Proliferation NAD + NADH +H + ALDH1A1 ALDH2 adduct formation S - hydroxymethyl - glutathione one NAD + carbon FDH pool NADH +H + S- formylglutathione catalase H 2 O 2 H 2 O Mutation glutathione S -formylglutathione hydrolase formate CO 2 + H 2 O Increased susceptibility may be due to: - impaired metabolic inactivation - impaired DNA repair CEFIC-2012 Günter Speit 5
6 II. Nasal sensitivity and susceptibility towards genotoxicity Study design: 41 volunteers (male non-smokers) Definition of hyper- and hyposensitive subgroups with regard to unspecific nasal irritation (CO 2 -sensitivity). Determination of cellular protection by FDH (gene expression) Determination of cellular susceptibility to DNA damage and cellular repair capacity by tests with peripheral blood as a surrogate tissue for studying mutagen sensitivity. CEFIC-2012 Günter Speit 6
7 II. Nasal sensitivity and susceptibility towards genotoxicity Blood samples were exposed to FA in vitro: Comet assay was performed to measure - FA-induced DNA damage (DPX) - removal (repair) of FA-induced DPX SCE-test was performed to measure - persistence of FA-induced DNA damage - FA-induced genotoxicity (SCE) CEFIC-2012 Günter Speit 7
8 II. Nasal sensitivity and susceptibility towards genotoxicity Results: Large differences in CO 2 -sensitivity (nasal irritation) No significant differences between the subgroups - with regard to the induction of genotoxic effects (DPX, SCE) - with regard to the protection against FA (FDH; DPX-repair) Zeller et al. Mutat. Res. 723, (2011) CEFIC-2012 Günter Speit 8
9 II. Nasal sensitivity and susceptibility towards genotoxicity Correlation analysis for all parameters tested: - for all subjects, - for the subgroups of hypersensitive and hyposensitive subjects, - for the subgroups of extremly hypersensitive and hyposensitive subjects. Results: No correlation between CO 2 -sensitivity and the expression of FDH. No correlation between CO 2 -sensitivity and indicators of cellular sensitivity. No subgroup with particular mutagen sensitivity towards FA was identified. Zeller et al. Mutat. Res. 723, (2011) CEFIC-2012 Günter Speit 9
10 III. Potential susceptibility of different study groups Ex-vivo susceptibility towards FA-induced genotoxicity was tested: - in 30 male smokers - in 30 female non-smokers - in 30 school children The comet assay and the SCE-test were performed in accordance with the previous study. The expression of the FDH-gene was measured by quantitative real-time RT-PCR. Polymorphisms in GSTM1 and GSTT1 were considered. Zeller et al. Arch. Toxicol., in press (2012) CEFIC-2012 Günter Speit 10
11 III. Potential susceptibility of different study groups No biologically relevant differences between the three groups. CEFIC-2012 Günter Speit 11
12 III. Potential susceptibility of different study groups DNA damage SCE-test DNA repair FDH CEFIC-2012 Günter Speit 12
13 III. Potential susceptibility of different study groups Induced genotoxic effects (comet assay) are not associated with polymorphisms in GSTM1 and GSTT1. CEFIC-2012 Günter Speit 13
14 III. Potential susceptibility of different study groups Induced SCE frequencies are slightly higher in subjects with GSTM1 null genotype. Induced SCE frequencies are not associated with polymorphisms in GSTT1 and GSTM1/GSTT1. CEFIC-2012 Günter Speit 14
15 III. Potential susceptibility of different study groups Summary No biologically relevant differences were measured between the three study groups with regard to the tested indicators of cellular sensitivity towards FA-induced genotoxic effects and the expression of FDH. There was no clear association between the induced genotoxic effects and polymorphisms in GSTM1 and GSTT1. None of the groups showed particular mutagen sensitivity towards FA-induced genotoxicity. The results suggest that a low scaling factor to address possible human inter-individual differences in FA-induced mutagenicity could be reasonable. CEFIC-2012 Günter Speit 15
16 IV. Polymorphisms in the FDH gene and susceptibility We identified three SNPs in the FDH-gene (ADH5) with a potential impact on gene expression (NCBI database). No variant allel was detected for 2 SNPs in our study population. 89 Subjects with a variant allele were identified for rs13832 (exon 9). Just et al. Toxicol. Lett. 207, (2011) CEFIC-2012 Günter Speit 16
17 IV. Polymorphisms in the FDH gene and susceptibility The variant allele did not cause a difference in FDH expression (mrna level). The variant allele did not cause a difference in the protection of cells against FA-induced genotoxicity (comet assay): - No difference in background DNA effects - No difference in FA-induced DPX No indication for inter-individual differences in the metabolic inactivation of FA. Just et al. Toxicol. Lett. 207, (2011) CEFIC-2012 Günter Speit 17
18 Susceptibility towards FA-induced genotoxicity Summary Our study did not reveal a polymorphism of FDH which might affect gene expression and lead to inter-individual differences in the protection of FA-induced (geno-)toxicity. Altogether, our studies do not point to relevant differences in susceptibility towards FA-induced genotoxicity which need to be considered in risk assessment. CEFIC-2012 Günter Speit 18
19 THANK YOU Acknowledgements: Jasmin Zeller Alexandra Ulrich Clarissa Riegert Walter Just Simone Neuß Christopher Teller Regina Linsenmeyer Josef Högel Gerhard Triebig Jörg U. Müller Thomas Bruckner Financial support: EPF & CEFIC / FormaCare CEFIC-2012 Günter Speit 19
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