Arch Toxicol (2005) 79: 355–362 DOI 10.1007/s00204-004-0639-z
G E N O T O X I C IT Y
B. Marczynski Æ R. Merget Æ T. Mensing Æ S. Rabstein M. Kappler Æ A. Bracht Æ M. G. Haufs Æ H. U. Ka¨fferlein T. Bru¨ning
DNA strand breaks in the lymphocytes of workers exposed to diisocyanates: indications of individual differences in susceptibility after low-dose and short-term exposure Received: 14 September 2004 / Accepted: 17 November 2004 / Published online: 3 February 2005 Springer-Verlag 2005
Abstract Diisocyanates are chemically reactive and induce asthma, but data on genotoxic effects of diisocyanates in humans are limited. The investigation presented here used short term diisocyanate chamber exposure to study DNA strand breaks in lymphocytes of 10 healthy individuals and of 42 workers, with airway symptoms, who had previously been exposed to diisocyanates. The alkaline version of the Comet assay was used to analyse DNA strand breaks in lymphocytes. In addition, blood samples of 10 further control individuals without any exposure to diisocyanates were studied. Substances studied were 4,4¢-methylenediphenyldiisocyanate (MDI, n=25), 2,4-toluenediisocynate and 2,6-toluenediisocyanate (TDI, n=5), and 1,6-hexamethylenediisocyanate (HDI, n=12), at concentrations between 5 and 30 ppb for 2 h. Lymphocytes isolated from the subjects before exposure and 30 min and 19 h after were used to evaluate DNA damage. No significant changes in DNA strand-break frequencies were measured, as Olive tail moment (OTM), either between groups or before and after diisocyanate exposure. OTM was similar in subjects with an asthmatic reaction (MDI, n=5; TDI, n=1; HDI, n=1) and in subjects without such a reaction. However, a small and susceptible group (about 10% of the individuals studied) could be identified with higher frequencies of DNA strand breaks in lymphocytes after chamber exposure. The occurrence of DNA damage in this group may be based on indirect mechanisms such as oxidative stress or apoptosis. B. Marczynski (&) Æ R. Merget Æ T. Mensing S. Rabstein Æ M. Kappler Æ A. Bracht Æ M. G. Haufs H. U. Ka¨fferlein Æ T. Bru¨ning Berufsgenossenschaftliches Forschungsinstitut fu¨r Arbeitsmedizin (BGFA), Ruhr-Universita¨t Bochum, Bu¨rkle-de-la-Camp-Platz 1, 44789 Bochum, Germany E-mail:
[email protected] Tel.: +49-234-3024601 Fax: +49-234-3024610
Keywords Diisocyanates Æ Lymphocytes Æ DNA strand breaks Æ Reactive oxygen species
Introduction Diisocyanates are used for manufacture of polyurethane foam, elastomers, adhesives, paint, coatings, and insecticides, and for rock consolidation (IARC 1986). Inhalation of diisocyanate vapour is associated with numerous pulmonary ailments (Bernstein 1996), but there is limited information about the genotoxic potential of diisocyanates. Previous studies have revealed DNA breakage and chromosome aberrations in human lymphocytes in vitro (Marczynski et al. 1992b; Ma¨kiPaakkanen and Norppa 1987). A case study of a worker exposed to diisocyanate revealed an increased level of white blood cell DNA breakage after inhalation exposure to 4,4¢-methylenediphenyldiisocyanate (MDI; Marczynski et al. 1992a). Additionally, elevated levels of anti-dsDNA autoantibodies were found in workers exposed to diisocyanates (Czuppon et al. 1993). To clarify the changes in the DNA induced by diisocyanates it is important to determine the genotoxic effects of these widely used chemicals. The results published so far do not explain whether possible genotoxic effects of diisocyanates are caused: (i) by exposure to the parent compound (diisocyanates themselves); (ii) by metabolic formation of specific reactive intermediates, e.g., nitrenium ions, which are capable to bind to DNA, or (iii) by indirect effects, e.g., inducing the formation of reactive oxygen species (ROS, oxidative stress) (Bolognesi et al. 2001). The presence of inflammation in the airways is an important biological factor in the development of asthma caused by exposure to diisocyanates. Oxidative stress, with the formation of ROS, is a key component of inflammation (Barnes 1990; Weiss 1996). It contributes to the activation of inflammatory cells which induce a
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respiratory burst resulting in the production of ROS, such as hydrogen peroxide (H2O2) (Horvath et al. 1998). Exhaled H2O2 is significantly elevated in asthmatic patients and correlated with the severity of airway inflammation (Emelyanov et al. 2001). Previous results from our laboratory support the hypothesis that diisocyanates may change the intracellular redox steady-state due to production of H2O2 (Marczynski et al. 2003). The Comet assay (single-cell gel electrophoresis) is a very sensitive method for measuring DNA single strand breaks and alkali-labile sites (McKelvey-Martin et al. 1993). Because of its sensitive detection of genetic damage the assay has been adopted as a tool in shortterm genotoxicity and human biomonitoring studies (Fairbairn et al. 1995; Collins et al. 1997; Marczynski et al. 2002). DNA strand breaks could be a result of both direct and indirect effects due to diisocyanates. Pitarque et al. (1999) used an alkaline Comet assay to evaluate DNA damage in peripheral blood mononuclear leucocytes of shoe workers exposed to a mixture of chemicals including organic solvents and MDI (4,4¢-methylenediphenyldiisocyanate). This study revealed no evidence for increased DNA damage due to occupational exposure to diisocyanates. The aim of the study presented here was to evaluate DNA strand breaks in lymphocytes of workers who were formerly exposed to diisocyanates at the workplace and had work-related airways symptoms. For this purpose the workers were exposed to diisocyanates using a standardized chamber exposure protocol. To simulate workplace conditions, exposure experiments were carried out using characteristic diisocyanates which are used in industrial settings, rather than pure single compounds.
Materials and methods Subjects Within routine clinical evaluation of industry workers applying for workers’ compensation, work-place related diisocyanate chamber exposure was carried out. Fortytwo workers who showed work-related airways symptoms (such as shortness of breath) were included. A 1-day chamber exposure was performed with these workers, using MDI, TDI (toluenediisocyanate), or HDI (1,6-hexamethylenediisocyanate) atmospheres. Twentyfive workers were submitted to chamber exposure with MDI, five with TDI, and 12 with HDI. Blood samples from these patients were examined for DNA strand break frequencies. As controls, 10 persons of similar age were recruited from a group of subjects without former exposure to diisocyanates, but with mild asthma symptoms and bronchial hyperresponsiveness. These subjects were exposed to TDI, and nine (missing data for one person) were also exposed to MDI 42–105 days earlier. In addition, 10 healthy subjects were not exposed to diisocyanates, but examined for DNA strand break frequencies at the same times as exposed individuals.
Information about occupational history, smoking habits and medical history was obtained for all persons by means of a questionnaire. All subjects gave their informed consent. The study was approved by the ethics committee of the Ruhr-University, Bochum, and was conducted in accordance with the principles defined by the Helsinki Declaration. Study protocol Workers to be experimentally exposed to diisocyanates (‘‘challenged’’) underwent a complete medical examination including lung function measurements (American Thoracic Society 1995), methacholine testing (American Thoracic Society 2000) and skin prick testing (SPT). Asthma was defined as variable shortness of breath at work in combination with bronchial hyperresponsiveness or a ratio between forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) of 99%) HDI were purchased from Merck–Schuchardt (Hohenbrunn, Germany). All other chemicals (Sigma,
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Deisenhofen, Germany) used for Comet assay were of the highest analytical grade. Processing of lymphocytes Blood samples were collected in heparin-treated tubes for lymphocyte preparation. Lymphocytes were isolated by the standard method of centrifugation on a Ficoll density gradient. Seven millilitres of whole blood from each subject was diluted 1:1 with RPMI 1640 solution (pH 7.3) and kept on ice for 15 min. Lymphocytes were separated by centrifugation over 7 mL Lymphoprep at 200g for 30 min. Buffy coats were removed and washed twice with RPMI 1640. Lymphocytes suspended in the RPMI solution were counted in a hemocytometer and approximately 2·104 cells were used immediately for the Comet assay. Cell viability, determined using the trypan blue exclusion technique, was constantly found to be over 98% (Marczynski et al. 2002). Alkaline single-cell gel electrophoresis (Comet assay) A modification of the original descriptions by O¨stling and Johanson (1984) and Singh et al. (1988) was used for the Comet assay (Pouget et al. 1999; Marczynski et al. 2002). One-hundred microlitres of 1% standard agarose dissolved in PBS buffer was taken and allowed to solidify onto a microscope slide at room temperature. Another 10 lL of the lymphocyte suspension was mixed with 75 lL 1.2% low-melting-point agarose maintained at 37C. Subsequently, the resulting solution was coated on the first layer after removal of the cover glass. All subsequent steps were performed under red light to prevent the occurrence of additional DNA damage. The slides were placed on ice for 15 min to allow the gel to solidify. Cover glasses were removed and the slides were immersed for 80 min at 4C in a lysis buffer (1% Triton X-100, 10% DMSO, 2.5 mmol L 1 NaCl, 100 mmol L 1 Na2EDTA, 10 mmol L 1 Tris, sodium lauroylsarcosinate 10%, pH 10). To perform electrophoresis, slides were transferred to a horizontal electrophoresis tank and kept covered with an alkaline solution (1 mmol L 1 Na2EDTA, 300 mmol L 1 NaOH, pH 13) at 4C for 40 min. Thereafter, electrophoresis was performed at 25 V–300 mA for 45 min at 4C. The slides were then washed three times with 0.4 mol L 1 Tris-HCl (pH 7.4), and the nuclei were stained using 45 lL of 0.5 mg mL 1 ethidium bromide. The slides were covered and placed at 4C in a humidified air-tight container to prevent drying. Under these conditions, slides could be kept for several days before analysis. Final analysis was performed with a fluorescence microscope (Olympus, BX60F-3, Olympus Optical Tokyo, Japan) equipped with both a 515–560 nm excitation filter and a 590 nm barrier filter. Each slide (51 cells per slide, using two different slides prepared for one subject) was examined at 20-fold magnification using the computer image-analysis software Komet Version 4.0 (Kinetic Imaging, Liverpool, UK).
Fifty-one ‘‘comets’’ (102 cells/blood sample) were randomly selected from each slide avoiding the edges and damaged parts of the gel, apparently dead cells (comets without a distinct ‘‘comet head’’) and superimposed comets. The image-analysis program automatically calculated the total area of each tail, its absolute average intensity, and its distance to the centre position of the comet head. From this data the program calculated several indicators of DNA damage, from which the Olive tail moment (OTM) was selected for estimation of DNA strand break frequency, because it expresses the migration of the various DNA fragments forming the tail and estimates the relative amounts of DNA (Rojas et al. 1999). Statistical analysis To study the differences between OTM before and after diisocyanate chamber exposure the non-parametric Friedman test was performed for groups of diisocyanate-exposed subjects and controls. If a Friedman test in a group was significant all pairwise comparisons of time points were subjected to the Wilcoxon signed rank test. Also, Comet assay measurements between groups at each time point were compared by use of the Kruskal– Wallis test for three or more groups and by use of the Wilcoxon rank sum test for two groups. All tests were conducted two-sided with a significance level of a=5% using the statistical software package SAS 8.2 (SAS Institute, Cary, NC, USA).
Results and discussion Characteristics of the study population Table 1 depicts the characteristics of the study population. Women were overrepresented among exposed controls whereas examined workers were slightly older than both control groups (exposed and non-exposed). The median age of workers was 40.5 years (range 20– 61), in controls without chamber exposure 36.5 years (range 32–55), and in controls with chamber exposure 34 years (range 22–58). The fraction of current smokers was about 30% in all groups. Thirty-one workers (73.8%) were no longer exposed to diisocyanates at the time of the study. Median time since last occupational exposure of this group was 15 months (range 1–148). As a result of the recruitment of the study population, the groups differed in frequencies of medication, asthma, and atopy. About 17% of the workers had an asthma reaction during or after diisocyanate chamber exposure whereas no controls had such a reaction. Comparisons of OTM between the time points No significant changes in the DNA strand break frequencies could be observed in the lymphocytes of control
358 Table 1 Description of the study population exposed to 4,4¢-methylenediphenyldiisocyanate (MDI), 2,4-toluenediisocyanate and 2,6toluenediisocyanate (TDI), 1,6-hexamethylenediisocyanate (HDI), and participants without experimental exposure (‘‘challenge’’) Controls
No. of subjects Gender (males) Age (years; median and range) Smoking status (current smokers) No. of subjects with medicationa Duration of occupational exposure to diisocyanates (months; median and range) No. of subjects with exposure cessation Since (months; median and range) No. of subjects with asthma No. of subjects with atopy No. of subjects with positive asthmatic reaction during or after diisocyanate exposure a
Workers with occupational exposure
Without exposure
With exposure
MDI
TDI
HDI
Total
10 7 (70%) 36.5 (32–55) 4 (40%) 0 (0%) –
10 2 (20%) 34 (22–58) 3 (30%) 4 (40%) –
25 21 (84%) 44 (20–60) 8 (32%) 15 (60%) 104 (3–457)
5 4 (80%) 31 (29–40) 1 (20%) 4 (80%) 44 (29–151)
12 11 (91.7%) 39.5 (20–61) 4 (33.3%) 7 (58.3%) 81 (5–417)
42 36 (85.7%) 40.5 (20–61) 13 (31%) 26 (61.9%) 89.5 (3–457)
– – 0 (0%) ndb –
– – 10 (100%) 7 (70%) 0 (0%)
20 (80%) 16 (1–148) 12 (48%) 4 (16%) 5 (20%)
2 4 2 1 1
9 (75%) 22 (2–37) 5 (41.7%) 4 (33.3%) 1 (8.3%)
31 (73.8%) 15 (1–148) 19 (45.2%) 9 (21.4%) 7 (16.7%)
(40%) (1–7) (40%) (20%) (20%)
any medication was considered not done
b
individuals with no exposure to diisocyanates (n=10, P=0.50). A boxplot for this comparison is shown in Fig. 1. Median OTM at baseline time point and 5.5 and 24 h later (which correspond to time points 30 min and 19 h after the exposure) were 1.75, 1.87, and 1.50, respectively. In healthy control individuals exposed to TDI no differences of OTM between the time points could be observed (n=10, P=0.41). Median levels were 1.26, 1.32, and 1.62 before and 30 min and 19 h after exposure. Similar results were observed after exposing these individuals to MDI: median OTM was 1.18, 1.20, and 1.12 at the three time points (n=9, P=0.64, Fig. 2). Workers showed no significant differences among the three time points of exposure to MDI, TDI, or HDI (Figs. 3A, B and C). For MDI median OTM was 1.31, 1.40, and 1.44 (n=25, P=0.76), for TDI OTM levels were determined to be 1.22, 1.83 and 1.27 (n=5, P=0.25). Median OTM after chamber exposure to HDI was 1.44, 1.63, and 1.63 (n=12, P=0.34). Furthermore, OTM was similar in subjects with (MDI, n=5; TDI, Fig. 1 Olive tail moment in lymphocytes of controls without exposure (n=10) at three different time points (P=0.50)
n=1; HDI, n=1) and without an asthma reaction, but was not analysed statistically because of the small numbers. However, a small susceptible group of the workers (about 10%) was found with elevated OTM (increase ‡1.0) showing much higher frequencies of DNA strand breaks in lymphocytes after exposure. Comparison of OTM between the groups Kruskal–Wallis tests revealed no statistically significant differences for OTM between groups before (P=0.08) and 30 min (P=0.08) and 19 h (P=0.64) after diisocyanate exposure. Also, Wilcoxon tests showed no differences at any time point between workers with and without exposure cessation (before exposure P=0.07, 30 min after exposure P=0.13, 19 h after exposure P=0.57), with and without asthma medication (before P=0.23, 30 min P=0.44, 19 h P=0.81), smokers and non-smokers (before P=0.23, 30 min P=0.45, 19 h
359 Fig. 2 Olive tail moment in lymphocytes of controls who were exposed to MDI (n=9) and TDI (n=10) at three different time points (before exposure and 30 min and 19 h after the end of exposure), P=0.64 for MDI and P=0.41 for TDI. Note that 9 of 10 subjects were exposed to both diisocyanates, with a time interval between 42 and 105 days. The squares represent outliers, apparently because of enhanced individual susceptibility
P=0.27), atopics and non-atopics (before P=0.33, 30 min P=0.79, 19 h P=0.24), and men and women (before P=0.11, 30 min P=0.39, 19 h P=0.51). Similarly, workers OTM was not, at any time point, related to the type of diisocyanate (aromatic MDI and TDI or aliphatic HDI) used throughout the experiments (Kruskal–Wallis test: before exposure P=0.72, 30 min after exposure P=0.34, 19 h after exposure P=0.39). The study shows no significant differences in DNA strand break frequencies, determined by alkaline Comet assay, before and after experimental diisocyanate exposure between 5 and 30 ppb. Also Vock et al. (1998) showed no evidence that MDI induces DNA doublestrand breaks by a genotoxic mechanism based on its theoretical DNA–DNA crosslinking ability. They suggested that MDI could induce cell death rather by necrosis than by apoptosis. In rats, MDI induced tumours only together with histopathological signs of toxicity, but in the absence of detectable DNA adducts in the target organs (Vock et al. 1996). Available evidence indicates that tumour induction by MDI relies more on epigenetic modes of action resulting from cytotoxicity than on genotoxicity resulting from reactivity with DNA (Vock et al. 1998). Although we were not able to exclude DNA–DNA crosslinks in lymphocytes after diisocyanate exposure, the results presented here support the findings presented by Vock et al. (1996, 1998) that diisocyanates, at low doses, do not cause genotoxic effects by directly binding to DNA. Although no differences of statistical significance in DNA strand break frequencies were found within and between groups, in four out of 42 workers (9.5%) and in one out of 10 controls (10%) diisocyanates induced a higher number of DNA strand breaks, defined as an increase of ‡1.0 in OTM from baseline to any time point after chamber exposure. Also, four workers and two controls already had elevated OTM (OTM ‡2.5) before exposure. Because of the small size of the groups we were not able to detect factors which might explain the larger
numbers of lymphocyte DNA strand breaks in the susceptible persons. However, changes in DNA strand break frequencies in this group may be related to a variety of factors which, in general, affect the interpretation of data. These concern the steady-state between mature and new-born white blood cells, changes in endonuclease and protease activity and DNA repair, the contribution of apoptotic and necrotic lymphocytes, and diet. These factors may also explain why increased DNA strand breaks were not consistently found at all times before and after exposure. Nevertheless, the whole experiment (chamber experiments and analytical measurements) was carried out in a standardised way. The Comet assay was directly carried out after blood sampling to avoid any negative influence of blood transport and storage on the analytical results. Mapp et al. (2002) and Wikman et al. (2002) have discussed whether genetic or other factors could lead to genotoxic effects of diisocyanates. However, it remains unclear which molecular mechanisms cause genotoxicity in persons susceptible to diisocyanates. First, the parent compound or the formation of genotoxic metabolites could be responsible for cross-linked DNA, which in turn leads to DNA double strand breaks. Crosslinked DNA has been observed in previous in-vitro experiments after incubation of TDI with blood (Peel et al. 1997). Another possibility would be induction of oxidative stress, in which DNA damage finally occurs directly by formation of oxidized nucleosides (e.g. 8-oxodeoxyguanosine), which can, in turn, lead to DNA strand breaks. However, oxidative stress can also induce DNA damage in lung cells by an indirect mechanism such as induction of apoptosis (Mantell and Lee 2000). Several apoptotic regulatory proteins are differently expressed and associated with hyperoxic cell death. The importance of oxidative stress in lung injury is also supported by Mapp et al. (2002) who demonstrated that polymorphism in GSTP1 is associated with susceptibility to asthma induced by prolonged exposure to TDI and
360 Fig. 3 Olive tail moment in lymphocytes of workers who were exposed to MDI (A, n=25), TDI (B, n=5), and HDI (C, n=12) at three different time points (before exposure and 30 min and 19 h after the end of exposure). Differences between the three time points were not significant, P=0.76 for MDI, P=0.25 for TDI and P=0.34 for HDI. The squares represent outliers, apparently because of enhanced individual susceptibility
that homozygosity for the Val105-encoding alleles confer protection against the development of the asthma-associated phenotype airway hyperresponsiveness. GST serves as an important protector of cells from oxidative
stress products including lipid and DNA peroxides (Aynacioglu et al. 2004). The results reported by Mapp et al. (2002) are further supported by the fact, that in general, oxidative stress is an important factor in asthmatic
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patients (Bowler 2004). Finally, diisocyanates may also be capable of reacting directly with proteins or altering their expression in lung cells (in the absence of oxidative stress); it has, for example, been reported that even at concentrations of 20 ppb TDI enters pulmonary cells and reacts rapidly with intracellular glutathione (Lantz et al. 2001). Altered signal transduction proteins as already described above may trigger DNA degradation (DNA double strand breaks) and apoptosis. Our results based on alkaline single-cell gel electrophoresis include DNA strand breaks and also base modifications (Collins et al. 1996), because the oxidized purine bases (8-oxo-deoxyguanosine and others) and pyrimidine bases could be partly converted into additional DNA single strand breaks during DNA repair (Boiteux 1993). The Comet assay has also been developed with some modifications to detect specific classes of DNA damage such as oxidative DNA damage. This method uses repair DNA glycosylases/endonucleases with specificity for oxidative base damage to create DNA breaks at sites of damage. Thus, DNA repair enzymes recognize DNA modifications that are typically induced by ROS and can be used as specific tools to identify oxidative stress in the cells. Oxidative DNA damage is mutagenic and carcinogenic per se and may be considered as a biomarker of oxidative stress. As suggested by the present pilot study, the role of oxidative stress in diisocyanate exposed-workers deserves further investigation with modified versions of the Comet assay. In general, our study shows that low doses of diisocyanate do not induce DNA strand breaks. There is, however, a small susceptible subpopulation, in which even low doses of diisocyanates are capable of inducing an increased frequency of DNA strand breaks. The mechanism of DNA damage in these cases may include indirect mechanisms (oxidative stress or apoptosis). These results may be important to our understanding of the complexity of diisocyanate reactions in humans. Acknowledgements We thank A. Du¨ker, B. Engelhardt, and E. Schomberg for technical assistance.
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