International Journal of Hygiene and Environmental Health 217 (2014) 492–498

Contents lists available at ScienceDirect

International Journal of Hygiene and Environmental Health journal homepage: www.elsevier.com/locate/ijheh

The sensitivity of biomarkers for genotoxicity and acute cytotoxicity in nasal and buccal cells of welders Georg Wultsch a , Armen Nersesyan a , Michael Kundi b , Robert Jakse c , Alfred Beham d , Karl-Heinz Wagner e , Siegfried Knasmueller a,∗ a

Institute of Cancer Research, Department of Medicine I, Comprehensive Cancer Center, Medical University of Vienna, 1090 Vienna, Austria Institute of Environmental Health, Medical University of Vienna, 1090 Vienna, Austria c Private Clinics Leech, 8010 Graz, Austria d Institute of Pathology, Medical University of Graz, 8036 Graz, Austria e Department of Nutritional Sciences, University of Vienna, 1090 Vienna, Austria b

a r t i c l e

i n f o

Article history: Received 5 February 2013 Received in revised form 25 September 2013 Accepted 26 September 2013 Keywords: Welders Buccal cells Nasal cells Cytome assay Heavy metals Redox status

a b s t r a c t Welders are inhalatively exposed to fumes which contain genotoxic carcinogens and it was found in epidemiological studies that they have increased cancer rates which may be causally related to DNA damage. In order to assess their health risks and to find out which chemicals cause the adverse effects, bioassays can be performed which enable the detection of genetic damage. The aim of the present study was a comparative investigation with exfoliated buccal and nasal cells in regard to induction of chromosomal alterations and acute cytotoxicity in welders and unexposed controls (n = 22 per group). To elucidate the factors which account for genotoxic and cytotoxic effects, additional biochemical parameters were monitored reflecting the redox status as well as concentrations of different metals and 1-hydroxypyrene (1-OHP) in body fluids. We found in the nasal cells significant induction of alterations which are indicative for DNA damage, i.e. of micronuclei (MNi) and nuclear buds, while elevated rates of nuclear anomalies reflecting cytotoxic effects (condensed chromatin, karyorrhexis, karyolylsis) were detected in cells from both organs. The levels of certain metals (Cr, Cu, Mn, Mo, Ni), but not markers of oxidative damage were significantly higher in the body fluids of the welders. Multivariate Poisson regression analyses indicate that exposure to Mo (15% MNi increase by one standard deviation increase of Mo in serum), Ni (9% increase) and Mn (14% increase) are positively associated with the induction of MNi in nasal cells while Ni was associated with cytotoxic effects in both types of cells (12 and 16% increase). Taken together, our findings indicate that epithelial cells from the respiratory tract are suitable for the detection of DNAdamaging and cytotoxic effects in welders and can be used to assess health risks associated with genomic instability. © 2013 Elsevier GmbH. All rights reserved.

Introduction More than 800,000 workers are employed full time as welders worldwide. In 2010, 337,300 individuals worked as welders, cutters, solderers and braziers and over one million individuals in the USA weld as part of their job duties (Antonini, 2003; Meeker et al., 2007). These groups inhale a number of hazardous compounds (in particular toxic metals and polycyclic aromatic hydrocarbons, PAHs) contained in the gases and fumes which are formed during the welding process and it is well documented that this exposure leads to adverse health effects (Antonini, 2003; Wittczak et al.,

∗ Corresponding author at: Institute of Cancer Research, Internal Medicine I, Medical University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria. Tel.: +43 1 40160 57561; fax: +43 1 40160 957500. E-mail address: [email protected] (S. Knasmueller). 1438-4639/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.ijheh.2013.09.005

2012). Welders suffer for example from pulmonary dysfunctions including asthma and have also increased lung cancer rates with odds ratios around 1.4 (for review see Antonini, 2003; Luce and Stucker, 2011; Mannetje et al., 2012). These effects depend largely on the type of welding and on safety precautions (Corbin et al., 2011; Paris et al., 2010). In order to assess the risks in humans which are exposed to DNAdamaging agents, biomonitoring methods have been developed which enable the detection of genotoxic and acute toxic effects in exfoliated epithelial cells from the oral cavity and from the nasal epithelia (Knasmueller et al., 2011; Thomas et al., 2009). It was shown by Bonassi et al. (2011b) that micronuclei (MNi) in lymphocytes, which are formed as a consequence of structural and numerical chromosomal aberrations are a reliable biomarker for human cancer risks. Results of subsequent analyses with buccal cells suggest that also this cell type may be suitable for the detection of human cancer risks (Bonassi et al., 2011a). In the last years,

G. Wultsch et al. / International Journal of Hygiene and Environmental Health 217 (2014) 492–498

attempts have been made to standardize and validate cytome studies with these cells (Bolognesi et al., 2013; Thomas et al., 2009). This method has the advantage of non-invasive collection of the cells and it has been postulated that MN assay with these cells may reflect for human cancer risks since up to 90% of all cancers are of epithelial origin (Cairns, 1975). Apart from MNi, a number of additional endpoints can be evaluated in buccal cells (Thomas et al., 2009): binucleated cells (BN) reflect disturbances of mitosis and are possibly indicative for genetic damage (Thomas et al., 2009) while nuclear buds (NB, also termed “broken eggs”) are formed as a consequence of DNA amplification (Fenech, 2002). Other anomalies such as condensed chromatin (CC), karyorrhexis (KR), karyolysis (KL) and pycnosis reflect acute cytotoxic effects (for details see Thomas et al., 2009). Also exfoliated epithelial cells from nasal epithelia can be used to study the consequences of exposure to toxins and identical endpoints as in buccal cells can be scored (Knasmueller et al., 2011; Wultsch et al., 2012). These cells could be in particular useful for inhalation studies as they come into first direct contact with toxins in the air but only a few studies (less than 20) have been published so far (for review see Knasmueller et al., 2011). However, the collection of nasal cells with cytobrushes is more complicated than the sampling of buccal cells and requires experience. In order to assess if health risks (including cancer) are caused by chromosomal instability can be detected in welders, several cytogenetic studies with lymphocytes have been performed. The results are highly controversial and do not allow to draw firm conclusions. A significant increase of the chromosomal aberration rates was seen only in three studies (Husgafvel-Pursiainen et al., 1982; Koshi et al., 1984; Littorin et al., 1983) while four studies yielded negative results (Elias et al., 1989; Halasova et al., 2012; Jelmert et al., 1992; Knudsen et al., 1992). Also in MN assays with lymphocytes controversial results were obtained. Some studies found positive results (Iarmarcovai et al., 2005, 2006; Sellappa et al., 2010) but it is notable that extremely high levels of Cr and Mn were detected in the blood of the workers which were monitored in these investigations. Furthermore, it is noteworthy that consistently negative results were obtained in sister chromatid exchange (SCE) assays with lymphocytes of welders (Dominici et al., 2011; Husgafvel-Pursiainen et al., 1982; Jelmert et al., 1992; Littorin et al., 1983). We conducted the present study in order to find out if effects can be detected in epithelial cells from the respiratory tract which are directly exposed to the fumes. In the last years, the method for the evaluation of nuclear anomalies in oral mucosa cells has been standardized by an international consortium (Bolognesi et al., 2013; Thomas et al., 2009) and the methods and criteria which they defined can be also used for the evaluation of cells from the nasal cavity (Bonassi et al., 2011b; Knasmueller et al., 2011; Wultsch et al., 2012). It is notable that two earlier studies with buccal cells from welders yielded positive results (Danadevi et al., 2004; Sudha et al., 2011), but these findings may be misleading since non-DNAspecific stains were used (Nersesyan et al., 2006). In order to identify the reasons for putative genotoxic and cytotoxic effects, additional chemical and biochemical measurements were carried out in the present investigation. The components of boiler welding are mainly high end steels and stainless steels. These alloys as well as the welding fumes which are formed contain high amounts of molybdenum (Mo), copper (Cu), chromium (Cr), nickel (Ni) and manganese (Mn) (Antonini, 2003). To study the associations between nuclear anomalies and the levels of metals in body fluids of the welders, the concentrations of these metals were measured before and at the end of the working week and also in unexposed controls. Furthermore, we monitored also the urinary excretion of 1-hydroxypyrene (1-OHP) which is a marker for exposure to polycyclic aromatic hydrocarbons (PAHs) (Jongeneelen et al., 1988). Since some studies indicate that welders suffer from chronic inflammations and oxidative stress (Fidan et al., 2005; Li

493

Table 1 Demographic data of the study participants.a Parameters

Welders (n = 22)

Controls (n = 22)

Age (years) Height (cm) Weight (cm) Smokers Alcohol consumers

38.3 ± 11.3 177.8 ± 6.5 83.3 ± 9.1 9 (40.9%) 14 (63.6%)

36.5 ± 7.0 181.1 ± 5.7 87.9 ± 9.7 10 (45.5%) 5 (22.7%)

a Height and body weight were measured before sampling, all other characteristics were collected by questionnaires. Smokers in both groups consume on average 12 cigarettes per day; alcohol consumers in both groups drank ca. 2 L of beer and 0.5 L of wine per week (ca. 140 g ethanol/week).

et al., 2004), we measured additionally a number of biochemical parameters which are indicative for the redox status such as the levels of malondialdehyde (MDA), a marker of lipid peroxidation, the trolox equivalent antioxidant capacity (TEAC) and oxidized low density lipoprotein (oxLDL) (for review see Knasmuller et al., 2008). Materials and methods Study groups The study was performed with 22 welders and 22 jail wardens matched by age, body mass index (BMI) and life style habits (smoking, alcohol consumption, dietary habits, physical activity). All participants had a similar income and social status. Demographic data were collected with questionnaires and are listed in Table 1. All welders were involved in three types of welding processes (approximately 95% tungsten inert gas (TIG) welding, 4.5% electric arc and 0.5% autogenous welding). All welders worked 8 h per day three weeks before and during the collection of the samples. Collection of exfoliated cells and cytogenetic analyses On the last day of a working week, exfoliated buccal and nasal cells were collected as described earlier (Wultsch et al., 2012). Immediately before sampling, the participants were asked to rinse their mouths twice with tap water. Subsequently, buccal cells were collected from both cheeks by use of wooden spatulas (Paul Hartmann AG, Heidenheim, Germany). Nasal cells were collected from both middle turbinates with cytobrushes (Heinz Herenz, Hamburg, Germany). The samples of each group were collected on the same day. The exfoliated cells from both tissues were smeared on microscopic slides with a few drops of distilled water. From each participant two slides were prepared. After 24 h, they were fixed with 80% cold methanol; 10 min later, they were placed in 80 ml glass beakers with 5.0 M HCl at room temperature for 30 min, rinsed with distilled water for 3 min and subsequently stained with Schiff’s reagent (Sigma–Aldrich, Steinheim, Germany) for 90 min, washed 5 min with running water and then counterstained with 0.2% (w/v) Light Green (Sigma–Aldrich, Steinheim, Germany) for 20 s. From each participant, approximately 2000 buccal and 2000 nasal cells were evaluated. Buccal cells are larger than nasal cells, therefore they were scored under 600-fold magnification while nasal cells were scored under 1000-fold magnification with oil immersion (Nikon Photophot-FXA, Tokyo, Japan). In buccal derived samples, MNi were scored in normal differentiated cells as suggested by Thomas et al. (2009). As described in some previous papers (Chapelin et al., 1996; Danel et al., 1996; Knasmueller et al., 2011), nasal smears consist of different cell types; MNi and all other anomalies were scored in all cell types of epithelial origin (i.e. in ciliated and non-ciliated, and also in squamous and basal cells), but not in various types of leucocytes which are also present in nasal smears

494

G. Wultsch et al. / International Journal of Hygiene and Environmental Health 217 (2014) 492–498

(Ballarin et al., 1992; Sarto et al., 1990). The cells were examined under bright light; when MNi were detected, they were confirmed under fluorescent light. The nuclear anomalies were evaluated in both cell types according to the criteria defined by Thomas et al. (2009). The evaluation of the slides was done by one experienced scorer and crosschecked by another experienced scorer. Twenty fields on the slides with nasal smears were examined by a cytopathologist blinded to the origin of the sample and 100 cells were counted from representative sectors to score the percentage of various types of cells in nasal smears. Blood collection and determination of biochemical parameters From each participant, 10 ml blood were collected in heparinized vacutainer tubes (Becton-Dickinson, Plymouth, UK), subsequently, the plasma was isolated by centrifugation (3000 rpm, 10 min, Sorvall, ThermoScientific, Vienna, Austria). Plasma oxLDL concentrations were measured with a commercially available ELISA kit (Mercodia AB, Uppsala, Sweden). Absorbance of samples and standards was determined with a fluorimeter (BMG Lab Technologies, Offenburg, Germany, for details see Wultsch et al., 2012). MDA levels were determined in plasma according to the method of Ramel et al. (2004). The samples were neutralized after heating (60 min, 100 ◦ C) with methanol/NaOH, centrifuged (3 min, 3000 rpm) and MDA was measured with HPLC (excitation:  532 nm, emission:  563 nm, LaChrom Merck Hitachi Chromatography System, Vienna, Austria). Each sample was measured in duplicate. The total plasma antioxidant capacity (TAC) of plasma was measured as the Trolox equivalent antioxidant capacity (TEAC) according to the protocol published by Ramel et al. (2004). 2,2Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, Sigma) is oxidized to a green-blue radical cation initiated by the H2 O2 dependent oxidation of FeIII to FeIV . The ability of antioxidants to delay color formation is expressed relative to Trolox (mM). After 6 min absorbance was read at 734 nm with a spectrophotometer (Lambda 2, Perkin Elmer, Vienna, Austria) (for details see Wultsch et al., 2012). In addition, the concentrations of different metals were measured in blood and urine at the beginning and at the end of a working week. The levels of molybdenum (Mo) were determined in blood serum, copper (Cu) and nickel (Ni) in urine (calculated per 1 g of creatinine), chromium (Cr) and manganese (Mn) in whole blood. All analyses were performed with graphite furnace atomic absorption spectrometry (Perkin Elmer 4100ZL spectrometer, Ohio, USA). Zeeman background correction was employed to compensate for nonspecific absorption. The levels of the metals were measured in the fluids above the sediments of samples which had been deproteinized with ammonium nitrate for Mn and 65% nitric acid for all other metals. In all measurements, atomic absorption standard solutions containing 1000 ␮g/L of each metal were used. All chemicals came from Carl Roth (Karlsruhe, Germany). The concentrations of 1-OHP were measured in urine according to the procedure described in detail by Marczynski et al. (2009). The compound was analyzed in spot urine samples post shift according to the method developed by Lintelmann and Angerer (1999). The limit of detection was 0.02 ␮g/L. Creatinine was determined with the colorimetric method (Jaffé reaction) published by Taussky (1954). Because of its relatively constant excretion rate in urine (which makes its concentration inversely proportional to the urine flow rate), this parameter is used to normalize analyte concentrations for occupational and environmental exposure monitoring (Boeniger et al., 1993). This approach reportedly works well for individual occupational exposure analysis (e.g. for preshift and postshift samples from the same person)

(Barr et al., 2005; Boeniger et al., 1993). Also the 1-OHP concentrations were related to the levels of creatinine in urine samples in many occupational studies (Barr et al., 2005; Duan et al., 2009; Wang et al., 2011). Statistical analyses In a sensitivity analysis we determined the margin for an increase in MN and other cellular abnormalities of the cytome assay that could be detected by comparison of 22 exposed workers with unexposed controls sampled at a 1:1 ratio and considering an overall significance level (for the eight different nuclear abnormalities in each of the two tissues) of 5% and a power of 80%. The determined margin for an increase was for all abnormalities except pyknosis between 18% and 30%. For pyknosis, a two-fold increase could be detected at the specified power level. Counts of nuclear anomalies were analyzed by Poisson regression with total number of cells counted as offset variable. Occupation (welders and wardens) was used in the primary analysis as an independent variable. Furthermore, two multivariate Poisson regression analyses were performed with metal concentrations in body fluids or markers of oxidative stress as predictor variables. In all analyses, smoking and alcohol consumption were included in the model as potential confounders. Overdispersion was tested and did not reach statistical significance in any of the analyses. Comparisons of metal concentrations in body fluids between welders and controls and within welders between the start and the end of a working week were done by non-parametric methods (Mann–Whitney test and Wilcoxon matched pairs test). Markers of oxidative stress were compared between welders and controls by Mann–Whitney tests. These comparisons were done without adjustment for multiple endpoints. Results 74.2 ± 6.8% of the cells found in the slides with nasal smears which were collected from the welders, were of epithelial origin. A similar number (78.2 ± 5.9%) was found in the smears from the control subjects. Also the numbers of ciliated, goblet, squamous and basal cells were similar in both groups, namely 59.5 ± 7.4, 21.2 ± 3.5, 10.2 ± 2.2 and 9.2 ± 1.5 in the welders and 52.9 ± 6.4, 26.2 ± 7.3, 12.2 ± 3.6, and 7.6 ± 1.8 in unexposed individuals, respectively. These rates are in agreement with results obtained in earlier studies (Chapelin et al., 1996; Danel et al., 1996). The number of basal cells is an important parameter for the regenerative capacity of epithelia (Bolognesi et al., 2013; Thomas et al., 2009). It was in the welders 21% higher as in the controls; however the increase did not reach statistical significance. The results of the cytome assays with exfoliated buccal and nasal cells are summarized in Figs. 1 and 2. The frequencies of the different nuclear anomalies in the controls were in the same range as those which were found in previous studies (Nersesyan et al., 2011; Wultsch et al., 2012). The numbers of MN which are the most relevant parameter of DNA damage were in the buccal cells of the welders 23% higher as in non-exposed individuals but the difference was not significant (p = 0.516) while NB and BN were significantly increased by 55% (p = 0.004) and 25% (p = 0.040). In contrast to the findings obtained with the mouth cells, a clear increase of MNed cells was seen in the nasal cells (97%, p = 0.005). Also the levels of other nuclear anomalies, which are indicative for DNA damage such as NB and BN were significantly elevated by 63% (p = 0.033) and 22% (p = 0.047), respectively. It is notable that MNi were detected in nasal smears only in ciliated and squamous cells. The same observation has been made earlier by Italian groups (Ballarin et al., 1992; Sarto et al., 1990).

G. Wultsch et al. / International Journal of Hygiene and Environmental Health 217 (2014) 492–498

495

most pronounced difference in comparison to the controls was seen with KL cells in the nasal epithelium (increase by ca. 90%, p < 0.001); also in oral mucosa cells substantially higher rates of this type of aberration was found in the welders (increase by 70%, p < 0.001). The numbers of KR cells were elevated in both, nasal and buccal mucosa cells of the welders by 45% (oral mucosa cells p = 0.011. nasal cells p < 0.001). Furthermore, also the frequencies of cells with CC were higher in cells from both organs of the welders, but this effect was less pronounced (oral mucosa cells p = 0.026, nasal cells p < 0.001). As mentioned above, smoking and alcohol consumption had no effect on the rates of the different nuclear anomalies between welders and controls. The results of the measurements of the metals and of 1-OHP in the body fluids are listed in Table 2. In the welders, samples were collected at two time points, namely at the beginning and at the end of a 5-days working week. It can be seen that the levels of all metals except of Ni increased (Cr by 69.8%, Mn by 35.7%, Mo by 14.3%, Cu by 13.2%) during the working period. Comparisons of the welders with unexposed controls show that significant differences were detected at the beginning and at the end of the working week with all metals. Also the levels of 1-OHP were higher by 67.6% at the end of the exposure phase, but they were not significantly different compared to those found in the unexposed controls (Table 2). Table 3 shows the results of the measurements of biochemical parameters which are indicative for the redox status. It can be seen that none of them was significantly higher in the welders. Table 4 summarizes the results of multivariate Poisson regression analyses concerning associations between biochemical parameters (MDA, TEAC, oxLDL), the concentrations of the metals and 1-OH-P and the frequencies of nuclear anomalies in nasal and buccal cells. Interestingly, significant associations were found between the levels of anomalies which reflect DNA damage and certain metals, for example between the Mo concentrations and the total number of MNi, NB and BN in buccal cells (increase of 24%, 20% and 8%, respectively, by one standard deviation increase of Mo in serum) and with MNed cells in nasal cells (increase of 15%). Furthermore, significant associations were observed between exposure to Ni and BN in cells from both epithelia (12% buccal and 14% nasal cells) and also between Mn and formation of BN in both cell types (21% buccal and 16% nasal cells).

Fig. 1. Nuclear anomalies in buccal cells of welders and controls (open bars = controls, gray bars = welders); *p < 0.05, **p < 0.01. Bars represent means (95% confidence intervals) of rates obtained in welders and controls (n = 22 per group). From each participant approximately 2000 cells were analyzed.

Discussion

Fig. 2. Nuclear anomalies in nasal cells of welders and controls (open bars = controls, gray bars = welders); *p < 0.05, **p < 0.01. Bars represent means (95% confidence intervals) of rates obtained in welders and controls (n = 22 per group).

This is the first comparative study in which the induction of a variety of nuclear alterations was investigated in nasal and buccal cells of welders. Taken together, our findings show a clear induction of MNi in the nasal cells, while the effect in buccal cells did not reach significance; other endpoints which are indicative for acute cytotoxic effects were increased in both organs. Furthermore, it is notable that we found in the body fluids of the workers increased

The NB in the nasal cells were projections of the main nuclei while their morphology in buccal cells was typical for so-called “broken eggs” (Bolognesi et al., 2013; Thomas et al., 2009). Except for pycnosis, all endpoints which reflect acute cytotoxic effects were increased in cells from both organs in the welders. The

Table 2 Concentrations of metals and 1-OHP in body fluids of welders at the beginning and the end of a working week and controls (means ± SD). Parameter Mo (␮g/dL) Cr (␮g/L) Mn (␮g/L) Cu (␮g/g) Ni (␮g/L) Creatinine (g/L) 1-OHP (␮g/g)

Welders (W1) 0.007 1.36 10.82 7.27 3.95 1.41 0.37

± ± ± ± ± ± ±

Welders (W2) b

0.011 0.91b 2.64b 2.35b 3.22b 0.76 0.29

0.008 2.31 14.68 8.23 3.97 1.74 0.61

± ± ± ± ± ± ±

b

0.020 1.46a , b 5.61a , b 2.09b 3.20b 0.58a , b 0.30a

 W2/W1 (% W1)

Controls (C)

0.001 (14.3) 0.95 (69.8) 3.86 (35.7) 0.96 (13.2) 0.02 (0.5) 0.33 (23.4) 0.25 (67.6)

0.001 0.22 3.38 2.73 0.53 1.06 0.41

± ± ± ± ± ± ±

0.005 0.17 1.93 1.42 0.69 0.41 0.37

W1 – welders at the beginning of the working week, W2 – at the end;  W2/W1 – difference between metal levels at various time points; levels of Mo were measured in blood serum, Cr and Mn in whole blood, Ni in urine. Cu and 1-OHP were measured in the urine samples and normalized by urinary creatinine. a p < 0.05 compared to the level at the beginning of the week. b p < 0.05 compared to controls;

496

G. Wultsch et al. / International Journal of Hygiene and Environmental Health 217 (2014) 492–498

Table 3 Biochemical parameters in welders and controls.a Biochemical parameters

Welders

MDA (␮M/L) TEAC (mM Trolox equivalent) oxLDL(U/L) Creatinine (g/L)

2.55 0.73 61.76 1.41

± ± ± ±

 W/C (%)

Controls 0.64 0.15 8.12 0.76

2.33 0.67 58.38 1.06

± ± ± ±

0.55 0.15 3.76 0.41

0.22 (9.4) 0.06 (9.0) 3.38 (5.8) 0.35 (33.0)

a Samples were collected from welders at the end of working week. Numbers indicate means ± S.D. of values obtained with welders and controls (n = 22/group). Each sample was analyzed in dublicate.

paper (Halasova et al., 2012). Also in SCE assays with lymphocytes consistently negative results were obtained in workers which were involved in different forms of welding (Dominici et al., 2011; Husgafvel-Pursiainen et al., 1982; Jelmert et al., 1992; Littorin et al., 1983). In view of these negative results, our findings support the assumption that the use of exfoliated cells from the respiratory tract may be complimentary and possibly more suitable for the detection of effects caused by inhalative exposure to welding fumes than that of blood cells which were used in aforementioned studies but additional investigations are required to support this assumption. As shown in Table 3, a significant increase of the concentrations of two metals (i.e. Cr and Mn) was detected in the blood of the welders at the end of the working week compared to the levels found at the start and also in comparison to unexposed individuals. It is also noteworthy that the concentrations of all metals in the body fluids of the welders were significantly higher than in the controls. These effects were not unexpected since increased levels of metals were found in body fluids of welders in earlier studies as well. For example, enhanced concentrations of Ni and Cr were reported in studies from India (Danadevi et al., 2003, 2004) and in another investigation, which was performed in France (Botta et al., 2006). The levels of Cr were in these investigations by far (i.e. one to two orders of magnitude) higher as those found in the present investigation, but it is noteworthy that the contents of heavy metal (including Cr) in the blood of non-exposed control subjects in these studies were also much higher than in our investigation while similar concentrations were found in recent studies which were carried out in the Czech Republic (Halasova et al., 2012), in Greece (Alexopoulos et al., 2008) and in Germany (Weiss et al., 2013). The relatively low levels of different metals in particular of Cr) and of 1-OHP which were found in the body fluids of the participants of the present study may be due to the specific working conditions and/or use of protective equipment. The participants of the present study are welding in boilers which are on both ends open which allows a constant air flow; furthermore, exhaust ventilators are installed and protective masks (FFP3) are provided. In this regard it is noteworthy that a recent study of the WELDOX Group in Germany showed that ventilation plays an important role in reduction of welding fumes exposure (Lehnert et al., 2013). It is notable,

levels of heavy metals and 1-OHP while the markers which are representative for the redox status were in both groups identical. The most important finding of this study is the positive result in the nasal cells of the workers indicating that the welding fumes leads to chromosomal alterations (Fig. 2). As shown in Fig. 1, an increase of the MNi rates was also found in the mouth cells, but this effect failed to reach statistical significance. As mentioned in the introduction, two earlier studies with welders have been published in which positive results were obtained in MN assays with buccal cells (Danadevi et al., 2004; Sudha et al., 2011). However, in both investigations, Giemsa was used for staining and we showed earlier that stains which are DNA-non-specific (including May-Gruenwald and Giemsa) lead to false positive results (Nersesyan et al., 2006). Therefore, the findings of these investigations do not allow to draw firm conclusions. As mentioned above, we found no impact of smoking and alcohol consumption on the MNi rates in the present study. In this context it is notable that controversial results were obtained in earlier studies with welders. Danadevi et al. (2004) found an impact of smoking on the MNi frequencies in welders while Sellappa et al. (2010) reported that smoking and also alcohol consumption do not lead to alterations of the frequencies of the different endpoints in buccal cells. Furthermore, no smoking effect was found in chromosomal aberration and MNi assays in a number of studies in lymphocytes of welders (Dominici et al., 2011; Elias et al., 1989; Halasova et al., 2012; Iarmarcovai et al., 2006; Jelmert et al., 1992; Littorin et al., 1983). As mentioned in the introduction, highly inconsistent results were obtained in chromosomal aberration studies with peripheral lymphocytes from welders (a significant increase of aberrations rates was seen only in three out of 6 studies in manual metal arc welders on stainless steel (Husgafvel-Pursiainen et al., 1982; Koshi et al., 1984; Littorin et al., 1983) while in three investigations negative results were found (Elias et al., 1989; Jelmert et al., 1992; Knudsen et al., 1992). Two negative results were reported from studies with tungsten inert gas (TIG) welders (Elias et al., 1989; Knudsen et al., 1992). In a recent study which was carried out in the Czech Republic, no increase of the MNi levels in lymphocytes was found, the type of welding is not specified in this

Table 4 Correlations between nuclear anomalies in buccal and nasal cells, metal levels and oxidative stress markers. Buccal cells MNC

TMN

Nasal cells BN

MDA TEAC oxLDL Molybdenum Chrome Manganese Copper Nickel 1-OHP * **

p < 0.05. p < 0.01.

CC

NB

KR

KL

**

**

P

MNC

TMN

BN

CC

**

**

NB

KR

KL

P

*

*

**

*

*

*

* *

**

* *

*

*

**

**

**

*

**

* **

* *

*

* **

**

*

G. Wultsch et al. / International Journal of Hygiene and Environmental Health 217 (2014) 492–498

that the amounts of all metals which we found in the exposed workers are still in the reference range of the overall population (Thomas, 2008). Also the levels of 1-OHP were increased at the end of the exposure phase but no difference of the concentration of this compound was found at the beginning and at the end of the working week in comparison to the controls. These findings indicate that the welders are exposed to PAHs and this assumption is supported by the results of chemical analyses in which carcinogenic PAHs were detected in welding fumes in concentrations between 300 and 600 ng/m2 (Borska et al., 2003). The levels of 1-OHP which we found in our study in the controls were in the same range as those detected in an earlier study in control subjects from Germany (Marczynski et al., 2009). It is notable that recent studies which were conducted in China and Germany indicate that occupational exposure to PAHs leads to increased rates of MNi (Duan et al., 2009; Wang et al., 2011; Yang et al., 2012) but the levels of 1-OHP were in these investigations in the exposed workers much higher (17–20-fold) than in our study. Cytome assays with nasal cells are not as frequently performed as studies with buccal cells and the procedure is less standardized (Knasmueller et al., 2011). However, the criteria which were defined for investigations with exfoliated buccal cells (Bolognesi et al., 2013; Thomas et al., 2009) can be used also in studies with these cells when their specific morphological characteristics are taken into account. Smears with nasal cells contain ciliated as well as non-ciliated cells. The latter ones include goblet and basal cells as well as squamous cells, their characteristics are described in a previous review (Knasmueller et al., 2011). MNi and other nuclear anomalies were scored in all different types of epithelial cells but not in leucocytes which were also found in nasal smears (Sarto et al., 1990; Wultsch et al., 2012). It can be seen in Table 4 that the concentrations of certain metals such as Mn, Mo and Ni correlate with the rates of nuclear anomalies which reflect DNA damage such as cells with MNi and with the total number of MNi, NB and BN. This can be taken as an indication that exposure to these metals may account for the induction of genetic instability in the workers. So far, only few genotoxicity studies have been published with Mo and the results show that it causes induction of MNi in cultured mammalian cells and also in murine bone marrow cells in vivo (Titenko-Holland et al., 1998). Furthermore, it is notable that it was postulated that Mo may account for the increased rates of lung tumors in welders and metal manufacturing workers (Droste et al., 1999). In long-term carcinogenicity studies concerning inhalative exposure of rats and mice to MoO3 , high incidences of alveolar/bronchiolar adenomas and carcinomas were found (Chan et al., 1998). Also for the Mn (Lima et al., 2011) and Ni (IARC, 1990, 1997) data from in vivo and in vitro experiments are available which yielded positive results. In the case of Ni, it is known that inhalative exposure of rodents causes tumors in the nasal tissue and in the lungs (for review see IARC, 1990, 1997). None of the endpoints which reflect the redox status was elevated in the welders. These findings are in contrast to results obtained in a study in Turkey in which increased TBARS levels were found (Fidan et al., 2005); also in a Chinese investigation a positive result (i.e., higher MDA concentrations in blood by 78%) was detected in welders (Li et al., 2004). The relatively low metal concentrations which we found in the present study indicate that the workers which we investigated are exposed to lower levels of genotoxic substances as in other countries and this may be the reason for the failure to detect changes of the redox parameters. As mentioned in the introduction, the results of several epidemiological studies indicate that welders have increased lung cancer rates (for review see Luce and Stucker, 2011; Mannetje et al., 2012). However, their risks depend strongly on the specific working conditions, on safety

497

measures and probably also on their individual susceptibility. In order to assess the risks of specific occupational scenarios, valid biomarkers are required, which can be also used in combination with chemical analyses to identify the compound(s) which account for the effects and to judge the efficiency of safety measures. The present study indicates that MN assays with exfoliated cells are such an approach. The most important finding of the present study is that a positive result was obtained in the nasal cells of the workers indicating that exposure to welding fumed causes damage of the genetic material in these cells. Despite the comparatively low concentrations of the metals in the body fluids and of the concentrations of the PAH metabolite and the lack of an increase of the oxidative stress markers, the present results indicate that there is still a need for measures to reduce the exposures of welders to fumes in order to avoid adverse health effects caused by genetic damage. References Alexopoulos, E.C., Cominos, X., Trougakos, I.P., Lourda, M., Gonos, E.S., Makropoulos, V., 2008. Biological monitoring of hexavalent chromium and serum levels of the senescence biomarker apolipoprotein J/Clusterin in welders. Bioinorg. Chem. Appl., 420578. Antonini, J.M., 2003. Health effects of welding. Crit. Rev. Toxicol. 33, 61–103. Ballarin, C., Sarto, F., Giacomelli, L., Bartolucci, G.B., Clonfero, E., 1992. Micronucleated cells in nasal mucosa of formaldehyde-exposed workers. Mutat. Res. 280, 1–7. Barr, D.B., Wilder, L.C., Caudill, S.P., Gonzalez, A.J., Needham, L.L., Pirkle, J.L., 2005. Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements. Environ. Health Perspect. 113, 192–200. Boeniger, M.F., Lowry, L.K., Rosenberg, J., 1993. Interpretation of urine results used to assess chemical exposure with emphasis on creatinine adjustments: a review. Am. Ind. Hyg. Assoc. J. 54, 615–627. Bolognesi, C., Knasmueller, S., Nersesyan, A., Thomas, P., Fenech, M., 2013. The HUMN scoring criteria for different cell types and nuclear anomalies in the buccal micronucleus cytome assay – an update and expanded photogallery. Mutat. Res. (Epub ahead of print). Bonassi, S., Coskun, E., Ceppi, M., Lando, C., Bolognesi, C., Burgaz, S., Holland, N., Kirsh-Volders, M., Knasmueller, S., Zeiger, E., Carnesoltas, D., Cavallo, D., da Silva, J., de Andrade, V.M., Demircigil, G.C., Dominguez Odio, A., Donmez-Altuntas, H., Gattas, G., Giri, A., Giri, S., Gomez-Meda, B., Gomez-Arroyo, S., Hadjidekova, V., Haveric, A., Kamboj, M., Kurteshi, K., Martino-Roth, M.G., Montero Montoya, R., Nersesyan, A., Pastor-Benito, S., Favero Salvadori, D.M., Shaposhnikova, A., Stopper, H., Thomas, P., Torres-Bugarin, O., Yadav, A.S., Zuniga Gonzalez, G., Fenech, M., 2011a. The HUman MicroNucleus project on eXfoLiated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. Mutat. Res. 728, 88–97. Bonassi, S., El-Zein, R., Bolognesi, C., Fenech, M., 2011b. Micronuclei frequency in peripheral blood lymphocytes and cancer risk: evidence from human studies. Mutagenesis 26, 93–100. Borska, L., Fiala, Z., Smejkalova, J., Tejral, J., 2003. Health risk of occupational exposure in welding processes I. Genotoxic risk. Acta Medica (Hradec Kralove) 46, 25–29. Botta, C., Iarmarcovai, G., Chaspoul, F., Sari-Minodier, I., Pompili, J., Orsiere, T., BergeLefranc, J.L., Botta, A., Gallice, P., De Meo, M., 2006. Assessment of occupational exposure to welding fumes by inductively coupled plasma-mass spectroscopy and by the alkaline Comet assay. Environ. Mol. Mutagen. 47, 284–295. Cairns, J., 1975. Mutation selection and the natural history of cancer. Nature 255, 197–200. Chan, P.C., Herbert, R.A., Roycroft, J.H., Haseman, J.K., Grumbein, S.L., Miller, R.A., Chou, B.J., 1998. Lung tumor induction by inhalation exposure to molybdenum trioxide in rats and mice. Toxicol. Sci. 45, 58–65. Chapelin, C., Coste, A., Gilain, L., Poron, F., Verra, F., Escudier, E., 1996. Modified epithelial cell distribution in chronic airways inflammation. Eur. Respir. J. 9, 2474–2478. Corbin, M., McLean, D., Mannetje, A., Dryson, E., Walls, C., McKenzie, F., Maule, M., Cheng, S., Cunningham, C., Kromhout, H., Blair, A., Pearce, N., 2011. Lung cancer and occupation: a New Zealand cancer registry-based case-control study. Am. J. Ind. Med. 54, 89–101. Danadevi, K., Rozati, R., Banu, B.S., Grover, P., 2004. Genotoxic evaluation of welders occupationally exposed to chromium and nickel using the Comet and micronucleus assays. Mutagenesis 19, 35–41. Danadevi, K., Rozati, R., Reddy, P.P., Grover, P., 2003. Semen quality of Indian welders occupationally exposed to nickel and chromium. Reprod. Toxicol. 17, 451–456. Danel, C., Erzurum, S.C., McElvaney, N.G., Crystal, R.G., 1996. Quantitative assessment of the epithelial and inflammatory cell populations in large airways of normals and individuals with cystic fibrosis. Am. J. Respir. Crit. Care Med. 153, 362–368. Dominici, L., Villarini, M., Fatigoni, C., Monarca, S., Moretti, M., 2011. Genotoxic hazard evaluation in welders occupationally exposed to extremely low-frequency magnetic fields (ELF-MF). Int. J. Hyg. Environ. Health 215, 68–75.

498

G. Wultsch et al. / International Journal of Hygiene and Environmental Health 217 (2014) 492–498

Droste, J.H., Weyler, J.J., Van Meerbeeck, J.P., Vermeire, P.A., van Sprundel, M.P., 1999. Occupational risk factors of lung cancer: a hospital based case-control study. Occup. Environ. Med. 56, 322–327. Duan, H., Leng, S., Pan, Z., Dai, Y., Niu, Y., Huang, C., Bin, P., Wang, Y., Liu, Q., Chen, W., Zheng, Y., 2009. Biomarkers measured by cytokinesis-block micronucleus cytome assay for evaluating genetic damages induced by polycyclic aromatic hydrocarbons. Mutat. Res. 677, 93–99. Elias, Z., Mur, J.M., Pierre, F., Gilgenkrantz, S., Schneider, O., Baruthio, F., Daniere, M.C., Fontana, J.M., 1989. Chromosome aberrations in peripheral blood lymphocytes of welders and characterization of their exposure by biological samples analysis. J. Occup. Med. 31, 477–483. Fenech, M., 2002. Chromosomal biomarkers of genomic instability relevant to cancer. Drug Discov. Today 7, 1128–1137. Fidan, F., Unlu, M., Koken, T., Tetik, L., Akgun, S., Demirel, R., Serteser, M., 2005. Oxidant–antioxidant status and pulmonary function in welding workers. J. Occup. Health 47, 286–292. Halasova, E., Matakova, T., Musak, L., Polakova, V., Letkova, L., Dobrota, D., Vodicka, P., 2012. Evaluating chromosomal damage in workers exposed to hexavalent chromium and the modulating role of polymorphisms of DNA repair genes. Int. Arch. Occup. Environ. Health 85, 473–481. Husgafvel-Pursiainen, K., Kalliomaki, P.L., Sorsa, M., 1982. A chromosome study among stainless steel welders. J. Occup. Med. 24, 762–766. IARC, 1990. Chromium, Nickel and Welding. IARC, Lyon. IARC, 1997. Nickel and nickel compounds, IARC monographs on the evaluation of carcinogenic risks to humans. Chromium, nickel, and welding, Lyon. Iarmarcovai, G., Sari-Minodier, I., Chaspoul, F., Botta, C., De Meo, M., Orsiere, T., BergeLefranc, J.L., Gallice, P., Botta, A., 2005. Risk assessment of welders using analysis of eight metals by ICP-MS in blood and urine and DNA damage evaluation by the comet and micronucleus assays; influence of XRCC1 and XRCC3 polymorphisms. Mutagenesis 20, 425–432. Iarmarcovai, G., Sari-Minodier, I., Orsiere, T., De Meo, M., Gallice, P., Bideau, C., Iniesta, D., Pompili, J., Berge-Lefranc, J.L., Botta, A., 2006. A combined analysis of XRCC1, XRCC3, GSTM1 and GSTT1 polymorphisms and centromere content of micronuclei in welders. Mutagenesis 21, 159–165. Jelmert, O., Hansteen, I.L., Langard, S., 1992. Enhanced cytogenetic detection of previous in vivo exposure to mutagens in human lymphocytes after treatment with inhibitors of DNA synthesis and DNA repair in vitro. Mutat. Res. 271, 289–298. Jongeneelen, F.J., Anzion, R.B., Scheepers, P.T., Bos, R.P., Henderson, P.T., Nijenhuis, E.H., Veenstra, S.J., Brouns, R.M., Winkes, A., 1988. 1-Hydroxypyrene in urine as a biological indicator of exposure to polycyclic aromatic hydrocarbons in several work environments. Ann. Occup. Hyg. 32, 35–43. Knasmueller, S., Holland, N., Wultsch, G., Jandl, B., Burgaz, S., Misik, M., Nersesyan, A., 2011. Use of nasal cells in micronucleus assays and other genotoxicity studies. Mutagenesis 26, 231–238. Knasmuller, S., Nersesyan, A., Misik, M., Gerner, C., Mikulits, W., Ehrlich, V., Hoelzl, C., Szakmary, A., Wagner, K.H., 2008. Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview. Br. J. Nutr. 99 E (Suppl. 1), ES3–ES52. Knudsen, L.E., Boisen, T., Christensen, J.M., Jelnes, J.E., Jensen, G.E., Jensen, J.C., Lundgren, K., Lundsteen, C., Pedersen, B., Wassermann, K., et al., 1992. Biomonitoring of genotoxic exposure among stainless steel welders. Mutat. Res. 279, 129–143. Koshi, K., Yagami, T., Nakanishi, Y., 1984. Cytogenetic analysis of peripheral blood lymphocytes from stainless steel welders. Ind. Health 22, 305–318. Lehnert, M., Weiss, T., Pesch, B., Lotz, A., Zilch-Schoneweis, S., Heinze, E., Van Gelder, R., Hahn, J.U., Bruning, T., 2013. Reduction in welding fume and metal exposure of stainless steel welders: an example from the WELDOX study. Int. Arch. Occup. Environ. Health (Epub ahead of print). Li, G.J., Zhang, L.L., Lu, L., Wu, P., Zheng, W., 2004. Occupational exposure to welding fume among welders: alterations of manganese, iron, zinc, copper, and lead in body fluids and the oxidative stress status. J. Occup. Environ. Med. 46, 241–248. Lima, P.D., Vasconcellos, M.C., Montenegro, R.C., Bahia, M.O., Costa, E.T., Antunes, L.M., Burbano, R.R., 2011. Genotoxic effects of aluminum, iron and manganese in human cells and experimental systems: a review of the literature. Hum. Exp. Toxicol. 30, 1435–1444. Lintelmann, J., Angerer, J., 1999. PAH metabolites. In: Angerer, J., Schaller, K.H. (Eds.), Analysis of Hazardous Substances in Biological Materials. Wiley-VCH, Weinheim, pp. 163–187.

Littorin, M., Hogstedt, B., Stromback, B., Karlsson, A., Welinder, H., Mitelman, F., Skerfving, S., 1983. No cytogenetic effects in lymphocytes of stainless steel welders. Scand. J. Work. Environ. Health 9, 259–264. Luce, D., Stucker, I., 2011. Investigation of occupational and environmental causes of respiratory cancers (ICARE): a multicenter, population-based case-control study in France. BMC Public Health 11, 928. Mannetje, A., Brennan, P., Zaridze, D., Szeszenia-Dabrowska, N., Rudnai, P., Lissowska, J., Fabianova, E., Cassidy, A., Mates, D., Bencko, V., Foretova, L., Janout, V., Fevotte, J., Fletcher, T., Boffetta, P., 2012. Welding and lung cancer in Central and Eastern Europe and the United Kingdom. Am. J. Epidemiol. 175, 706–714. Marczynski, B., Pesch, B., Wilhelm, M., Rossbach, B., Preuss, R., Hahn, J.U., Rabstein, S., Raulf-Heimsoth, M., Seidel, A., Rihs, H.P., Adams, A., Scherenberg, M., Erkes, A., Engelhardt, B., Straif, K., Kafferlein, H.U., Angerer, J., Bruning, T., 2009. Occupational exposure to polycyclic aromatic hydrocarbons and DNA damage by industry: a nationwide study in Germany. Arch. Toxicol. 83, 947–957. Meeker, J.D., Susi, P., Flynn, M.R., 2007. Manganese and welding fume exposure and control in construction. J. Occup. Environ. Hyg. 4, 943–951. Nersesyan, A., Kundi, M., Atefie, K., Schulte-Hermann, R., Knasmuller, S., 2006. Effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells. Cancer Epidemiol. Biomarkers Prev. 15, 1835–1840. Nersesyan, A., Muradyan, R., Kundi, M., Knasmueller, S., 2011. Impact of smoking on the frequencies of micronuclei and other nuclear abnormalities in exfoliated oral cells: a comparative study with different cigarette types. Mutagenesis 26, 295–301. Paris, C., Clement-Duchene, C., Vignaud, J.M., Gislard, A., Stoufflet, A., Bertrand, O., Thiberville, L., Grosdidier, G., Martinet, Y., Benichou, J., Hainaut, P., 2010. Relationships between lung adenocarcinoma and gender, age, smoking and occupational risk factors: a case-case study. Lung Cancer 68, 146–153. Ramel, A., Wagner, K.H., Elmadfa, I., 2004. Plasma antioxidants and lipid oxidation after submaximal resistance exercise in men. Eur. J. Nutr. 43, 2–6. Sarto, F., Tomanin, R., Giacomelli, L., Iannini, G., Cupiraggi, A.R., 1990. The micronucleus assay in human exfoliated cells of the nose and mouth: application to occupational exposures to chromic acid and ethylene oxide. Mutat. Res. 244, 345–351. Sellappa, S., Prathyumnan, S., Keyan, K.S., Joseph, S., Vasudevan, B.S., Sasikala, K., 2010. Evaluation of DNA damage induction and repair inhibition in welders exposed to hexavalent chromium. Asian Pac. J. Cancer Prev. 11, 95–100. Sudha, S., Kripa, S.K., Shibily, P., Joseph, S., Balachandar, V., 2011. Biomonitoring of genotoxic effects among shielded manual metal arc welders. Asian Pac. J. Cancer Prev. 12, 1041–1044. Taussky, H.H., 1954. A microcolorimetric determination of creatine in urine by the Jaffe reaction. J. Biol. Chem. 208, 853–861. Thomas, L., 2008. Labor und Diagnose. Indikation und Bewertung von Laborbefubden für die medinische Diagnostik. TH-Books, Frankfurt/Main. Thomas, P., HollandF N., Bolognesi, C., Kirsch-Volders, M., Bonassi, S., Zeiger, E., Knasmueller, S., Fenech, M., 2009. Buccal micronucleus cytome assay. Nat. Protoc. 4, 825–837. Titenko-Holland, N., Shao, J., Zhang, L., Xi, L., Ngo, H., Shang, N., Smith, M.T., 1998. Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo. Environ. Mol. Mutagen. 32, 251–259. Wang, J., Luo, X., Xu, B., Wei, J., Zhang, Z., Zhu, H., 2011. Elevated oxidative damage in kitchen workers in Chinese restaurants. J. Occup. Health 53, 327–333. Weiss, T., Pesch, B., Lotz, A., Gutwinski, E., Van Gelder, R., Punkenburg, E., Kendzia, B., Gawrych, K., Lehnert, M., Heinze, E., Hartwig, A., Kafferlein, H.U., Hahn, J.U., Bruning, T., 2013. Levels and predictors of airborne and internal exposure to chromium and nickel among welders – results of the WELDOX study. Int. J. Hyg. Environ. Health 216, 175–183. Wittczak, T., Dudek, W., Walusiak-Skorupa, J., Swierczynska-Machura, D., Cader, W., Kowalczyk, M., Palczynski, C., 2012. Metal-induced asthma and chest X-ray changes in welders. Int. J. Occup. Med. Environ. Health 25, 242–250. Wultsch, G., Nersesyan, A., Misik, M., Kundi, M., Wagner, K.H., Szekeres, T., Zakerska, O., Atefie, K., Knasmueller, S., 2013. Formation of micronuclei and other nuclear anomalies in exfoliated nasal and oral cells: results of a human study with workers in a power plant processing poultry litter. Int. J. Hyg. Environ. Health 216, 82–87. Yang, P., Ma, J., Zhang, B., Duan, H., He, Z., Zeng, J., Zeng, X., Li, D., Wang, Q., Xiao, Y., Liu, C., Xiao, Q., Chen, L., Zhu, X., Xing, X., Li, Z., Zhang, S., Zhang, Z., Ma, L., Wang, E., Zhuang, Z., Zheng, Y., Chen, W., 2012. CpG site-specific hypermethylation of p16INK4alpha in peripheral blood lymphocytes of PAH-exposed workers. Cancer Epidemiol. Biomarkers Prev. 21, 182–190.