Investigations of potential susceptibility toward formaldehyde-induced genotoxicity.

Blood samples were taken from three groups of volunteers (30 male smokers, 30 female non-smokers, and 30 school children) and tested for ex vivo susceptibility toward formaldehyde (FA)-induced genotoxicity. Blood samples were exposed to 150 µM FA for 2 h, and the induction of DNA-protein crosslinks (DPX) in leukocytes was measured by a modification of the alkaline comet assay (i.e., reduction of γ-irradiation induced DNA migration). Removal of DPX was determined by the abolition of FA-induced reduction in DNA migration within 4 h after the end of the exposure. Induction and persistence of FA-induced DNA lesions was also measured by the sister chromatid exchange (SCE) test with cultured lymphocytes after treatment of whole blood cultures with FA (150 µM). Furthermore, the expression (mRNA level) of the GSH-dependent formaldehyde dehydrogenase (FDH, identical to alcohol dehydrogenase 5; ADH5) was measured in leukocytes by quantitative real-time RT-PCR with TaqMan probes. The subjects were also analyzed for the GSTM1 and GSTT1 metabolic gene polymorphisms and a correlation analysis with the investigated genetic endpoints for FA-induced genotoxicity was performed. The results indicate that there are no biologically relevant differences between the three study groups with regard to the various indicators of cellular sensitivity toward FA-induced genotoxic effects and the expression of FDH. The induced genotoxic effects were not associated with polymorphisms in GSTM1 and GSTT1. None of the study groups showed particular mutagen sensitivity toward FA-induced genotoxicity. These results suggest that a low scaling factor to address possible human inter-individual differences in FA-induced genotoxicity could be reasonable.

Genetic polymorphisms in the formaldehyde dehydrogenase gene and their biological significance.

The GSH-dependent formaldehyde dehydrogenase (FDH) is the most important enzyme for the metabolic inactivation of formaldehyde. We studied three polymorphisms of this gene with the intention to elucidate their relevance for inter-individual differences in the protection against the (geno-)toxicity of FA. The first polymorphism (rs11568816) was investigated using real-time PCR and restriction fragment analysis in 150 subjects. However, we did not find the polymorphic sequence in any of the subjects. We studied a second polymorphism (rs17028487), representing a base exchange (c.*114A>G) in exon 9 of the FDH gene. We analyzed 70 subjects with the SNaPshot Primer Extension method and subsequent analysis in a ABI PRISM 3100, but no variant allele was identified. A third polymorphism, rs13832 in exon 9 (c.*493G>T), was studied in a group of 105 subjects by the SNaPshot Primer Extension method. 43 of the subjects were heterozygous for the polymorphism (G/T), 46 homozygous for the T allele, and 16 were homozygous for the G-allele. Real-time RT-PCR measurements of FDH mRNA did not indicate a significant difference in transcript levels between the heterozygous and the homozygous groups. The in vitro comet assay after FA exposure of blood samples obtained from 5 homozygous GG and 3 homozygous TT subjects did not lead to a significant difference between these two groups. Altogether, our study did not identify biologically relevant polymorphisms in transcribed regions of the FDH gene, which may lead to inter-individual differences in the metabolic inactivation of FA.

Is individual nasal sensitivity related to cellular metabolism of formaldehyde and susceptibility towards formaldehyde-induced genotoxicity?

Forty-one volunteers (male non-smokers, aged 32 ± 9.6yrs) were tested for susceptibility towards unspecific nasal irritation (sensitivity towards CO(2)) in order to define subgroups of hypersensitive and hyposensitive subjects. Blood samples were taken and the expression (mRNA level) of the GSH-dependent formaldehyde dehydrogenase gene (FDH, identical to alcohol dehydrogenase 5, ADH5; EC 1.2.1.46) was measured in leukocytes by quantitative real-time RT-PCR with TaqMan probes. FDH is the most important enzyme for the metabolic inactivation of FA. Blood samples were exposed to 150µM formaldehyde (FA) for 2h and the induction of DNA-protein crosslinks (DPX) in leukocytes was measured by means of a modification of the alkaline comet assay (i.e., by assessing the reduction of DNA migration induced by γ-radiation). Removal of DPX was determined by the abolition of FA-induced reduction in DNA migration within 4h after the end of the exposure. Furthermore, the induction of sister chromatid exchange (SCE) in cultured lymphocytes was studied after treatment of whole blood cultures with FA (150µM). A correlation analysis was performed for all parameters tested for the whole study group and for hypersensitive and hyposensitive subgroups. The results indicate that despite large differences in CO(2)-sensitivity, the susceptibility towards nasal irritation was not related to the induction of genotoxic effects (DPX, SCE) in peripheral blood or to the protection of blood cells against FA-induced effects (expression of FDH, repair capacity for FA-induced DPX). There was no correlation between CO(2)-sensitivity and the expression of FDH. There was also no close correlation between the various indicators of cellular sensitivity towards FA-induced genotoxic effects and no subgroups were identified with particular mutagen sensitivity towards FA.

Assessment of genotoxic effects and changes in gene expression in humans exposed to formaldehyde by inhalation under controlled conditions.

Forty-one volunteers (male non-smokers) were exposed to formaldehyde (FA) vapours for 4 h/day over a period of five working days under strictly controlled conditions. For each exposure day, different exposure concentrations were used in a random order ranging from 0 up to 0.7 p.p.m. At concentrations of 0.3 and 0.4 p.p.m., four peaks of 0.6 or 0.8 p.p.m. for 15 min each were applied. During exposure, subjects had to perform bicycle exercises (∼80 W) four times for 15 min. Blood samples, exfoliated nasal mucosa cells and nasal biopsies were taken before the first and after the last exposure. Nasal epithelial cells were additionally sampled 1, 2 and 3 weeks after the end of the exposure period. The alkaline comet assay, the sister chromatid exchange test and the cytokinesis-block micronucleus test were performed with blood samples. The micronucleus test was also performed with exfoliated nasal mucosa cells. The expression (mRNA level) of the glutathione (GSH)-dependent formaldehyde dehydrogenase (FDH, identical to alcohol dehydrogenase 5; ADH5; EC 1.2.1.46) was measured in blood samples by quantitative real-time reverse transcription-polymerase chain reaction with TaqMan probes. DNA microarray analyses using a full-genome human microarray were performed on blood samples and nasal biopsies of selected subgroups with the highest FA exposure at different days. Under the experimental conditions of this study, inhalation of FA did not lead to genotoxic effects in peripheral blood cells and nasal mucosa and had no effect on the expression of the FDH gene. Inhalation of FA did also not cause alterations in the expression of genes in a microarray analysis with nasal biopsies and peripheral blood cells.

The in vivo or ex vivo origin of micronuclei measured in human biomonitoring studies.

The micronucleus test (MNT) is a well-established assay in genotoxicity testing and human biomonitoring. The cytokinesis-block micronucleus test (CBMNT) is the preferred method for measuring MN in cultured human lymphocytes from human subjects exposed to genotoxins. However, it is unclear to what extent mutagen exposure either leads to the formation of MN already in vivo or to the formation of MN ex vivo during cell culture as a consequence of persisting DNA damage. MN that were already induced in vivo can be determined by scoring MN in mononuclear lymphocytes 24 h after the start of the lymphocyte culture (i.e. in lymphocytes that did not divide yet). Results obtained for cancer patients after chemotherapy suggest that mutagen exposure in vivo mainly leads to the formation of MN during ex vivo proliferation of lymphocytes as a consequence of mis-repair of persistent damage. If these results also apply to other kinds of mutagen exposure, increased MN frequencies in the CBMNT can only be expected for exposures leading to a sufficient amount of damage that persists during ex vivo lymphocyte culture. For a better understanding of the origin of increased MN frequencies and the correct interpretation of results obtained with the CBMNT, further research is recommended: MN in mononuclear lymphocytes should be additionally scored 24 h after the start of the cultures, comparative investigation with the CBMNT and the MNT with reticulocytes should be performed and the kinetics of MN formation in lymphocyte cultures and the repair capacity of lymphocytes for different kinds of DNA damage should be characterised.

Inhalation of formaldehyde does not induce genotoxic effects in broncho-alveolar lavage (BAL) cells of rats.

Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15ppm. Potential genotoxic effects in the lung were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. Broncho-alveolar lavage (BAL) cells were obtained by lung lavage with physiological saline and counted. From one half of the cells, slides for the micronucleus test (MNT) were prepared by cytocentrifugation; with the other half, the comet assay was performed. DNA migration in the comet assay was measured both directly and after irradiation of the cells with 2Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA-protein cross-links (DPX). For the comet assay, four slides were analysed from each cell sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis and tail intensity (% tail DNA) and tail moment were evaluated. The frequency of micronucleated BAL cells was determined in acridine orange-stained slides by analysing 2000 cells per animal. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28 days study with FA concentrations up to 15ppm does not lead to genotoxic effects in BAL cells of rats. Because detection of DPX by the comet assay is a very sensitive biomarker of FA exposure of cells, our results suggest that there is no genetically relevant exposure of the lung after FA inhalation. The results of our inhalation study, which was performed under GLP conditions, call into question the biological significance of previously reported genotoxic effects in the lung of rats after FA inhalation.

Inhalation of formaldehyde does not induce systemic genotoxic effects in rats.

Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential systemic genotoxic effects were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. For this purpose, peripheral blood samples were obtained by puncturing the retro-orbital venous plexus at the end of the exposure period. Blood sampling was carried out in a randomized sequence and samples were coded by sequence number to ensure blind evaluation. Blood samples were used for the comet assay, the sister chromatid exchange test (SCE test) and the micronucleus test (MNT). DNA migration in the comet assay was measured both directly and after irradiation of the blood samples with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA-protein cross-links (DPX). The following positive control groups were included: one group (six animals) was treated with 50mg/kg methyl methanesulfonate (MMS) once by gavage 4h before blood sampling. Another group (six animals) was treated twice orally with 10mg/kg cyclophosphamide (CP) with an interval of 24 h. The last application of CP was 24h before blood sampling. For the comet assay, four slides were analysed from each blood sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis, and tail intensity (% tail DNA) and tail moment were evaluated. For the SCE test, blood was cultured for 56 h in the presence of BrdU (10 microg/ml for the last 35 h) and SCE were counted in 30 second-division metaphases per sample. The MNT with peripheral blood was performed according to the instructions for the micronucleus analysis kit MICROFLOW (Litron Laboratories). Approximately 20,000 cells per sample were analysed by flow cytometry and the percentage of reticulocytes with micronuclei (MN) was determined. The positive control substances induced a significant effect in the genotoxicity tests and thus demonstrated the sensitivity of the test systems. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28-day study with FA concentrations up to 15 ppm does not lead to systemic genotoxic effects in the blood of rats.