Genotoxicity of formaldehyde in vitro and in vivo. Günter Speit

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1 Genotoxicity of formaldehyde in vitro and in vivo Günter Speit Universität Ulm Institut für Humangenetik D Ulm (Germany) CEFIC-2012 Günter Speit 1

2 Genotoxicity of formaldehyde in vitro and in vivo Outline of the presentation: I. In vitro genotoxicity / mutagenicity and mode of action II. III. In vivo local genotoxicity / mutagenicity (site of contact) In vivo systemic genotoxicity / mutagenicity CEFIC-2012 Günter Speit 2

3 I. In vitro genotoxicity / mutagenicity of formaldehyde Positive results in standard in vitro genotoxicity tests with mammalian cells: Comet Assay (DPX) Sister Chromatid Exchange Test (SCE) Chromosome Aberration Test Micronucleus Test Mouse Lymphoma Assay CEFIC-2012 Günter Speit 3

4 Formaldehyde (FA) induces DNA damage DNA-protein crosslinks (DPX) DNA-DNA crosslinks DNA monoadducts DPX are assumed to be the most relevant primary DNA alterations induced by FA. DPX are generally measured by indirect methods (e.g., HPLC, K-SDS assay, comet assay). CEFIC-2012 Günter Speit 4

5 Detection of FA-induced DPX by the Comet assay DPX inhibit DNA migration Generally, inhibition of DNA migration induced by irradiation is measured. 2 Gy 2 Gy + FA Among the substances studied so far, FA is the most efficient inhibitor of DNA migration in the comet assay. Detection of FA-induced DNA strand breaks by the comet assay is inexplicable (assay variability, preparation artifacts, exposure to other genotoxins). CEFIC-2012 Günter Speit 5

6 Induction of DPX by FA in the Comet assay A comprehensive characterization of genotoxic effects induced by FA in V79 cells clearly demonstrated: Induction of DPX by FA at conc. 25 µm Strong effects at high conc. ( 200 µm) No effect at low concentrations ( 10 µm) Formaldehyde does not induce DNA strand breaks in the comet assay Speit et al., Mutagenesis 22 (2007) CEFIC-2012 Günter Speit 6

7 Formaldehyde induces clastogenic effects Chromosome aberrations Micronuclei Small colony mutations in the Mouse Lymphoma Assay Formaldehyde is a clastogen CEFIC-2012 Günter Speit 7

8 Does formaldehyde induce gene mutations? No induction of HPRT mutations under standard test conditions 1) No induction of large colony mutations in the Mouse Lymphoma Assay 2) Gene mutations in cancer-related genes in nasal tumors of the rat are most likely secondary events and not directly induced by FA 3,4) FA does not efficiently induce true gene mutations 1) Merk & Speit, 1998; 2) Speit & Merk, ) Recio et al., 1992; 4) Meng et al., 2010 CEFIC-2012 Günter Speit 8

9 Does formaldehyde induce aneuploidy? Aneuploidy is caused by damage to the mitotic apparatus (is not due to an interaction with DNA) Aneuploidy is caused by an interaction with redundant targets (has a threshold mode of action) The micronucleus test (MNT) is the standard genotoxicity test for the detection of aneugens (OECD guideline 487) for regulatory purposes. FA induced micronuclei in cultured human lymphocytes and A 549 cells. FISH analysis revealed that the vast majority of induced MN were centromere negative, thus indicating a clastogenic effect. A significant aneugenic activity of FA was excluded. Speit et al., Mutagenesis 26, (2011) CEFIC-2012 Günter Speit 9

10 In vitro genotoxicity of formaldehyde Conclusions: Formaldehyde induces genotoxic effects (DPX, SCE) and mutations in cultured mammalian cells. Formaldehyde mainly induces chromosomal mutations (clastogenic MoA). Chromosomal effects (chromosome aberrations, micronuclei) are particularly suited for the investigation of FA-induced mutagenic effects in vivo. CEFIC-2012 Günter Speit 10

11 II. In vivo local genotoxicity / mutagenicity Two sources of information: Animal experiments Human biomonitoring studies CEFIC-2012 Günter Speit 11

12 Is formaldehyde (FA) a genotoxic carcinogen? Prolonged inhalation of FA induced nasal tumors in rats. Highly non-linear dose-related increase. Increase in tumor incidence at 6 ppm or greater. McGregor et al., CRT 40, (2010) CEFIC-2012 Günter Speit 12

13 Is formaldehyde a genotoxic carcinogen? Genotoxic carcinogens are mutagenic carcinogens: Mutations are the critical key events for the induction of cancer by genotoxic carcinogens. Mutations are the critical biomarkers for cancer risk assessment for genotoxic carcinogens. Are FA-induced mutations the cause for the formation of nasal tumors? CEFIC-2012 Günter Speit 13

14 Protective mechanisms lead to a threshold for the induction of mutations Exposure of cells / tissues pre-lesion protection by: Proteins, membranes Metabolic inactivation Induction of DNA damage Replication post-lesion protection by: DNA repair Fixation of mutations Speit et al., Mutat. Res. 464,149 (2000) CEFIC-2012 Günter Speit 14

15 Is formaldehyde (FA) a good candidate for a mutagen with a threshold mode of action? Naturally occurring presence of endogenous FA levels; background levels of FA-DNA adducts. Rapid metabolic inactivation counteracts the formation of DNA adducts Efficient repair of primary DNA damage counteracts the production of mutations CEFIC-2012 Günter Speit 15

16 Genotoxicity and mutagenicity of FA in nasal epithelial cells DPX can be induced in all cell types of the nasal mucosa exposed to FA. Mutations are only produced in basal cells: - if FA reaches basal cells, - if DPX are induced in basal cells, - if DPX escape repair, - if damaged basal cells proliferate. Exposure Basal cells Different dose-response for the induction of DPX and mutations. CEFIC-2012 Günter Speit 16

17 Transcellular transmission of FA? Co-cultivation experiments show: FA that has entered nasal epithelial cells - is not released into the culture medium even from highly exposed cells - does not induce DNA damage in other cells in close proximity to the epithelial cells. Only nasal epithelial cells at the surface (site of first contact) are significantly exposed to FA. Neuss et al. Mutagenesis 25, (2010) CEFIC-2012 Günter Speit 17

18 Induction of DNA adducts by FA in the nasal mucosa of rats Sensitive detection of N 2 -hydroxymethyl-dg adducts Differentiation between endogenous and exogenous DNA adducts Nonlinear exposure-response Endogenous DNA adducts predominate at low-dose exposure FA-induced mutagenic effects should only occur at high dose levels Swenberg et al., Toxicol. Sci. 120 (S1) S (2011) CEFIC-2012 Günter Speit 18

19 FA does not induce micronuclei in the nasal mucosa of rats Micronuclei were scored in rat nasal epithelial cells after exposure to FA by inhalation for 4 weeks. Exposure (6 ppm and higher) induced cell proliferation and histopathological changes. No induction of micronuclei. Limitations of this study: - Limited experience with the method (assay variability). - Evaluation of the whole nasal epithelium. - No established positive control. Dose (ppm) MNcells ( )* ± ± ± ± p-value** ± ± ± *) Six rats / group; 2000 cells / rat; **) Wilcoxon test Speit et al. Mutation Res. 721, (2011) CEFIC-2012 Günter Speit 19

20 Does FA induce mutations in nasal mucosa cells? Up to now, there is no experimental evidence for the induction of mutations by FA in nasal mucosa cells. There is concern about a mutagenic activity of FA in the nasal mucosa of humans exposed to FA because of positive human biomonitoring studies with the micronucleus assay. These studies have been critically reviewed and their reliability has been commented*. *) Speit and Schmid, Mutation Res. 613, 1-9 (2006) CEFIC-2012 Günter Speit 20

21 FA does not induce micronuclei in buccal mucosa cells after exposure for 10 days with peak exposures up to 1 ppm. Inhalation study performed under GLP-like conditions Defined exposure over 10 consecutive working days 4 h exposure with peak levels up to 1 ppm Bicycle exercises during exposure (3 x 15 min) Several sampling times (up to 3 weeks after exposure) MN [ ] 1,8 1,6 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 co1 co2 day 0 day 7 day 14 day 21 Speit et al., Mutation Res. 627, (2007) CEFIC-2012 Günter Speit 21

22 FA does not induce micronuclei in nasal mucosa cells after exposure for 5 days with peak exposures up to 0.8 ppm. Inhalation study performed under GLP-like conditions Defined exposure (5 days; 4 h; 0. 8 ppm peak levels Bicycle exercises (3 x 15 min) Several sampling times Limitations of the MNT with nasal cells: - Method not well standardized - Low background value (majority of slides with 0 MN) Zeller et al., Mutagenesis 26, (2011) CEFIC-2012 Günter Speit 22

23 In vivo local genotoxicity / mutagenicity - current status Conclusions: Formaldehyde induces genotoxic effects at the site of first contact (nasal mucosa). It is unknown to what extent these DNA alterations lead to the formation of mutations in basal cells. It is unknown to what extent FA-induced mutations contribute to the formation of nasal tumors. CEFIC-2012 Günter Speit 23

24 III. In vivo systemic genotoxicity / mutagenicity Two sources of information: Animal experiments Human biomonitoring studies CEFIC-2012 Günter Speit 24

25 Systemic mutagenicity of formaldehyde in animal experiments Fischer-344 rats were exposed to FA ( ppm) by inhalation for 4 weeks under GLP conditions. Genotoxicity tests were performed in accordance with international guidelines. Results: No induction of DNA damage / DPX in peripheral blood (comet assay). No induction of SCE in peripheral blood. No induction of micronuclei in peripheral blood (i.e., no indication for a clastogenic or aneugenic effect on bone marrow). Speit et al., Mutat. Res. 677, (2009) CEFIC-2012 Günter Speit 25

26 Induction of DNA adducts by FA in rats and monkeys Sensitive detection of N2-hydroxymethyl-dG adducts. Differentiation between endogenous and exogenous DNA adducts. Exogenous adducts were not detectable in bone marrow and blood. No indication for systemic genotoxicity. Results strongly support negative genotoxicity tests. Swenberg et al., Toxicol. Sci. 120 (S1) S (2011) CEFIC-2012 Günter Speit 26

27 Systemic mutagenicity of formaldehyde? Conclusions: Appropriately performed animal studies indicate that FA does not induce systemic genotoxic and mutagenic effects. No induction of genotoxic effects in bone marrow and blood. No induction of chromosome aberrations and/or aneuploidy in bone marrow. CEFIC-2012 Günter Speit 27

28 Human biomonitoring: Systemic genotoxic effects of FA in humans? Genetic endpoints studied in peripheral blood Genetic endpoint published positive DNA-protein crosslinks (K-SDS assay) 2 2 DNA strand breaks (Comet assay) 3 2 Sister chromatid exchanges (SCE) 14 6 Chromosome aberrations 10 5 Micronuclei (MN) 9 6?? Associations between exposure and effects repeatedly reported. No mechanism known to explain the exposure of lymphocytes. CEFIC-2012 Günter Speit 28

29 FA did not induce SCE and MN in peripheral blood cells of human volunteers exposed to FA 40 male non-smokers; GLP-like conditions Exposure to FA under strictly controlled conditions Exposure for 5 days with peak exposures up to 0.8 ppm No effect on SCE- and MN-frequencies No effect on cell proliferation (PI / NDI) Zeller et al., Mutagenesis 26, (2011) CEFIC-2012 Günter Speit 29

30 Can exposure to formaldehyde in vivo actually lead to cytogenetic effects in cultured human lymphocytes? Increased frequencies of SCE or MN require that blood samples contain lymphocytes with high levels of DPX (i.e., relevant exposure in vivo). SCE and MN are formed in vitro during proliferation. DPX have to persist until replication / cell division in cultured lymphocytes to cause the formation of SCE and MN. For details see: Schmid & Speit, Mutagenesis 22,69-74 (2007) CEFIC-2012 Günter Speit 30

31 DPX are repaired in cultured human blood before cytogentic effects can be produced relative tail moment Comet assay; mean of 3 different blood samples 100 µm FA 200 µm FA 300 µm FA 0 1 h 4 h 8 h 24 h Persistence of DPX until S-phase requires high damage levels. CEFIC-2012 Günter Speit 31

32 Induction of SCE by FA in human blood cultures The frequency of SCE is only increased in lymphocytes with high damage levels at the start of the culture. SCE / mitosis µm 25 µm 50 µm 100 µm 200 µm formaldehyde ** Induction of SCE is accompanied by reduced cell proliferation (cytotoxicity). proliferation index 2,9% 2,7% 2,5% 2,3% 2,1% 1,9% 1,7% 1,5% 0 µm 25 µm 50 µm 100 µm 200 µm formaldehyde ** CEFIC-2012 Günter Speit 32

33 Induction of MN by FA in human blood cultures The frequency of MN is not increased in lymphocytes with high damage levels at the start of the culture. MN [ ] µm 200 µm 250 µm formaldehyde Lymphocytes with high damage levels proliferate but do not produce MN. NDI 1,8 1,7 1,6 1,5 1,4 1,3 1,2 1,1 1,0 ** ** 0 µm 200 µm 250 µm formaldehyde CEFIC-2012 Günter Speit 33

34 Positive results in human biomonitoring studies with cytogenetic endpoints are not plausible The requirements for a positive cytogenetic test are not met in human biomonitoring of FA-exposed populations: Lymphocytes are generally not in direct contact with FA. DPX do not accumulate in lymphocytes. A sufficient number of cells with sufficient damage levels cannot be obtained. A sufficient amount of damage does not persist during cultivation to lead to the formation of SCE or MN. There is no scientific explanation for positive results Without a scientific explanation, these results should not be used for risk assessment. CEFIC-2012 Günter Speit 34

35 Possible reasons for positive biomonitoring studies with peripheral blood Unidentified action / metabolism of FA Chance findings / assay variability Incorrect interpretation / statistics Co-exposure to other genotoxins Inappropriate study performance and psychological influence CEFIC-2012 Günter Speit 35

36 Genotoxicity of formaldehyde - current status Summary FA is genotoxic and mutagenic in vitro FA mainly induces clastogenic effects FA induces genotoxic effects (DPX) at the site of first contact FA does not induce systemic genotoxic/mutagenic effects in animal experiments Cytogenetic effects in human biomonitoring studies are most likely not related to FA exposure CEFIC-2012 Günter Speit 36

37 THANK YOU Acknowledgements: Petra Schütz Regina Linsenmeyer Stefanie Kühner Jasmin Zeller Simone Neuß Oliver Schmid Josef Högel Heinz-Peter Gelbke Lan Ma-Hock Irmgard Weber Wolfgang Kaufmann Stefan Durrer Gerhard Triebig Jörg U. Müller Financial support: CEFIC / FormaCare CEFIC-2012 Günter Speit 37