Formaldehyde-induced chromosomal aberrations and apoptosis in peripheral blood lymphocytes of personnel working in pathology departments

Mutation Research 698 (2010) 11–17

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Formaldehyde-induced chromosomal aberrations and apoptosis in peripheral blood lymphocytes of personnel working in pathology departments Mátyás G. Jakab a,∗ , Tibor Klupp a , Krisztina Besenyei a , Anna Biró a , Jeno˝ Major a , Anna Tompa a,b a b

Department of Cytogenetics and Immunology, National Institute of Chemical Safety, H-1097, Nagyvárad tér 2., Budapest, Hungary Institute for Public Health, Semmelweis University, Faculty of Medicine, Budapest, Hungary

a r t i c l e

i n f o

Article history: Received 30 March 2009 Received in revised form 13 December 2009 Accepted 14 February 2010 Available online 1 March 2010 Keywords: Pathology personnel Formaldehyde Genotoxicity Apoptosis Lymphocytes Risk assessment

a b s t r a c t Peripheral blood lymphocytes (PBL) of 37 formaldehyde-exposed women from four pathology departments in Hungary were investigated to collect data on the effects of occupational exposures to formaldehyde and to find a possible relationship between in vivo formaldehyde-induced apoptosis and genotoxic effects. The subjects were divided into two groups: 16 donors exposed to formaldehyde together with various organic solvents, and 21 subjects exposed mainly to formaldehyde. The results were compared with 37 controls (all women) without known occupational exposure. Ambient air concentrations of formaldehyde were measured in three work places, and ranged from 0.23 to 1.21 mg/m3 (mean 0.9 mg/m3 ). Measures of genotoxicity included the determination of the frequencies of chromosomal aberrations (CA), sister-chromatid exchange (SCE), HPRT mutations (variant frequency, VF) and the measurement of UV-induced unscheduled DNA-repair synthesis (UDS). The percentages of premature centromere division (PCD) and of cells with a high frequency of SCE (HF/SCE) were also scored. Apoptosis and cell proliferation were determined by flow cytometry. In both formaldehyde-exposed groups, the apoptotic activity and the CA levels in PBLs were significantly higher than in controls. The CA were mostly breaks of the chromatid type. In the second group, which was mainly exposed to formaldehyde, CA were slightly lower in comparison with the group exposed to formaldehyde and solvents, which may be attributed to a different rate of elimination of damaged lymphocytes as a consequence of formaldehydeinduced apoptotic activity. In the second group, a significant decrease of VF and a non-significant increase in HF/SCE were found compared with the control and the other group. In conclusion, the results demonstrate that exposure to formaldehyde induces apoptosis and CA, indicating an excess cancer risk among subjects occupationally exposed to formaldehyde. The results also emphasize the importance of the measurement of occupational air pollutants, such as formaldehyde, in order to avoid genotoxic effects in the workers. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Formaldehyde (FA, CAS Reg. No. 50-00-0) is a naturally occurring substance in the environment and also an important endogenous substance in the human body. FA is most commonly available commercially as a 30–50% aqueous solution, often referred to as formalin. An endogenous FA level is always present in plants and

Abbreviations: BrdU, 5-bromo-2 -deoxyuridine; CA, chromosome aberrations; FA, formaldehyde; HF/SCE, high frequency SCE cells; HPRT, hypoxanthine-guanine phosphoribosyl transferase; LI, labeling index; MN, micronucleus; PBL, peripheral blood lymphocyte; PCD, premature centromere division; PCD (CSG), centromere separation general, mitoses with more than three chromosomes with PCD; PHA, phytohemagglutinin; SCE, sister-chromatid exchange; SE, standard error; UDS, UVinduced unscheduled DNA-repair synthesis; VF, variant frequency. ∗ Corresponding author at: H-1437 Budapest, P.O. Box 837, Hungary. Tel.: +36 1 476 1111; fax: +36 1 476 1227. E-mail address: [email protected] (M.G. Jakab). 1383-5718/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2010.02.015

animals. FA takes part in a number of methylation processes in cells, e.g., methylated amino acids serve as carrier molecules that transfer FA to various enzyme systems. On the other hand, liberation of FA in certain processes can also cause random methylation and formylation of different cell components, e.g., DNA, and FA bound to and transported by methylated amino acids, e.g., hydroxymethyl arginine, inhibits cell proliferation and induces apoptosis in tumour cells [1]. Environmental sources of FA are motor-vehicle emissions, building materials and plastics, paints, food and tobacco smoke, among others. FA is a volatile organic compound (VOC), a highly reactive air pollutant that is widely present in occupational and environmental settings. FA is used in the synthesis of a large number of different chemical compounds, in the production of a variety of resins, molding materials, nitrogen fertilizers, surface coatings, dyes, tanning agents, plastics, etc., and in textile, rubber, leather and cement industries. FA is also used as an antimicrobial agent in the preparation of human and veterinary drugs and in some cosmetic

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products. Occupational exposure to FA is common in pathology laboratories, where it is used for disinfection and for the preservation and embalming of biological specimens. Concentrations of FA are sometimes quite high, e.g., during the preparation and handling of solutions during embalming of tissue samples. FA was previously considered as a probably carcinogenic (Group 2A) substance to humans [2]. Recently, FA has been classified as carcinogenic (Group 1) to humans, on the basis of causal relationship between exposure to FA among industrial workers manufacturing FA, and among other professionals, e.g., embalmers and pathologists [3] and the occurrence of nasopharyngeal cancer and leukemia. FA is a cytotoxic compound, as was shown in in vitro experiments on cell lines. Lower doses of FA slightly enhanced the viability of cultured cells, whereas higher doses of FA decreased the viability [4]. FA can act as a cell-proliferation retardation factor and mediates the apoptotic process. In cell cultures, FA at concentrations around 10 mM decreased apoptosis and increased cell proliferation, whereas higher doses enhanced apoptosis and decreased cell proliferation [5]. Endogenous FA increased apoptosis and inhibited the mitotic ratio in cells of the human prostate cell line PC-3 treated with the endogenous FA generator 1-methyl-ascorbigen [6]. In cell culture, FA leads to the formation of DNA-protein crosslinks and DNA single-strand breaks [7] and may cause genotoxicity by damaging DNA and by enhancing mutagenesis through inhibition of the repair of DNA lesions [8]. Data regarding the genotoxic potential of FA in occupationally exposed individuals are often conflicting. Several studies have examined groups of FA-exposed pathology staff, in whom no significant differences were found in the yields of chromosomal aberrations (CA) and sister-chromatid exchange (SCE) in peripheral blood lymphocytes (PBL), as reported by Thomson et al. [9], and similar results were found among medical students exposed to FA in an anatomy dissection laboratory, as was observed by Vasudeva and Anand [10]. No significant changes in the frequency of CA, micronuclei (MN) and SCE were scored in PBL of individuals working in different health-care laboratories including a pathology department [11], who were exposed to lower FA concentrations. In contrast to the negative findings [9–11] many cytogenetic data have been accumulated in the scientific literature demonstrating significant changes caused by FA exposures in vitro and in vivo. The chromatid type CA and SCE were significantly increased in human lymphocytes treated with FA in vitro [12]. An increased level of DNA-protein crosslinks and SCE was observed in industry workers exposed to FA [13]. In FA-exposed anatomy students, MN, CA and SCE in PBL showed a significant increase [14]. An increased frequency of MN was found in PBL of pathologists/anatomists [15], and increased frequencies of MN and SCE were also detected among pathology personnel [16]. Increased frequencies of CA, but no increase in the SCE frequencies in lymphocytes from FA-exposed paper-factory workers, were observed in a study of Bauchinger and Schmid [17]. Both SCE and high-frequency SCE (HF/SCE) were increased in PBL of hospital pathology workers in comparison with controls [18]. The frequency of MN was found to be significantly increased in nasal mucosa cells of pathology- and anatomy-laboratory personnel [19] and from plywood-factory workers, with an FA exposure ranging from 0.1 to 0.39 mg/m3 [20]. In the study by Ye et al. [21], higher frequencies of MN in nasal mucosa cells and higher frequency of SCE in PBL of workers with high levels of FA exposure in a formaldehyde factory were observed (with an exposure level of 0.985 ± 0.286 mg/m3 with a peak exposure-concentration of 1.694 mg/m3 ). In students working in a mortuary, an increase of MN in buccal epithelial cells and an increase of MN and a decrease of SCE in PBL, was found by Suruda et al. [22]. Among FA-exposed factory workers, pathology staff and medical students, a pronounced increase in the frequency of CA in PBL and MN in buccal mucosa

could be detected [23]. Similarly, a significantly higher frequency of MN was found in exfoliated buccal cells from pathology and anatomy staff [24], with a FA air concentration ranging from 2 to 4 ppm (2.5–5 mg/m3 ). In view of the often contradictory results of FA-induced genotoxic effects in the literature, the aim of our present investigation was to explain these differences with the use of various genotoxic methods in order to detect genotoxicological changes due to occupational exposure to FA, and simultaneously to study the possible effects of FA-induced apoptosis parallel to genotoxicological and cell-proliferation parameters. Here, we present the results of the genotoxicological end-points, apoptosis induction and cellproliferation measurements in PBL of 37 subjects employed in pathology departments. Because the working conditions of the investigated FA-exposed pathology employees were different, they were divided in two exposure groups: the subjects in the first group were exposed to FA together with various organic solvents, and in the second group they were exposed only to FA. A multiple endpoint genotoxicology monitoring system has been developed at the National Institute of Chemical Safety of Hungary and used for risk assessment of human populations exposed to various genotoxic agents and control subjects since the mid 1980s. The monitor is able to detect changes in the CA, SCE, DNA-repair, HPRT mutation and apoptosis in correlation with working conditions [25,26]. Besides the above-mentioned endpoints of the genotoxicological monitor, the analysis of premature centromere division (PCD) [27] and HF/SCEs [25] was also performed. 2. Materials and methods 2.1. Selection of subjects Altogether 37 women from three hospital pathology departments and a medical university pathology department (four persons from Hospital 1; five persons from Hospital 2, 14 persons from Hospital 3 and 14 persons from the university pathology department) were involved in the study, and the results were compared with those of 37 healthy control subjects (all women) from health-service staff, without known previous occupational exposure to genotoxic agents. The investigations were proposed by the occupational health services to clarify the possible genotoxic consequences of occupational exposure to FA and consequently the potential increase of cancer risk among the investigated persons. The pathology employees were divided into two groups: the first group consisted of 16 subjects (four pathologists, 12 technicians) exposed to FA and various organic solvents, while the second group, comprising 21 subjects (one pathologist, 20 technicians) was mainly exposed to FA during work. All donors took part voluntarily with prior informed consent and the donor’s written permission in the study, and were interviewed by a physician to collect data on age, medication, lifestyle (smoking and drinking habits), as well as medical and work histories in relation to known or suspected chemical mutagens and/or to exposure to ionizing radiation, and the use of protective devices during work. The questionnaire also contained data on sensory perception of FA and specific symptoms in relation to FA exposure, e.g., irritation of the eyes. Measurements of the concentration of FA in ambient air, according to the data given by the pathology departments, were made within 3 years in Hospital 2, within 1 year in Hospital 3 and the university pathology department, prior to our investigations, and were similar in all investigated work places in our study. FA concentrations in ambient air were measured at fixed locations in the laboratories, and the concentrations were in the range of 0.23–1.20 mg/m3 for Hospital 2; 0.63–1.10 mg/m3 for Hospital 3 and 0.40–1.21 mg/m3 for the university pathology department, corresponding to an 8-h time-weighted average exposure (TWA-8) of 0.9 mg/m3 , which exceeds the limits of both the short-term exposure (0.6 mg/m3 ) and TWA-8 (0.6 mg/m3 ) in Hungary. Although no ambient air concentrations of FA were measured in the pathology department of Hospital 1, as working conditions were similar to those of the others, a similar range of FA exposure could be expected in this department. All subjects complained about acute toxic effects (eye irritation) and the presence of FA was perceivable by smell in each laboratory. Main components of the organic solvents used in the histology laboratories were ethyl alcohol, acetone and xylene. Data concerning the ambient air concentration measurements for these chemicals were not available to us, but ambient air concentrations for each of these chemicals were much below both the 8-h time-weighted average exposure (TWA-8) and short-term exposure limits in Hungary (personal communications). Only active smokers were considered to be “smokers”. None of the individuals were addicted to alcohol, subjects considered as “drinkers” consumed less than the equivalent of 80 g pure alcohol daily. All subjects took part in a routine clinical checkup, including hematology, and liver and kidney function tests. Blood was obtained by veni-puncture from each of the sub-

M.G. Jakab et al. / Mutation Research 698 (2010) 11–17 jects investigated: a total of 18 ml blood was collected in 2 VACUETTE® Coagulation tubes filled with 1 ml of 0.109 M (3.2%) buffered tri-sodium citrate (Ref. no. 455322, Greiner Bio-One) for the measurements of UV-induced unscheduled DNA synthesis (UDS). A further 27 ml blood was collected in 3 VACUETTE® Heparin tubes coated with the anticoagulant sodium-heparin (Ref. no. 455051, Greiner Bio-One) for the determination of HPRT gene mutations, CA and SCE frequencies, and for the flowcytometry analyses of apoptosis and cell proliferation. The samples were processed immediately after blood collection. 2.2. The determination of HPRT gene mutations and UV-induced unscheduled DNA synthesis (UDS) in PBL For the determination of HPRT gene mutations, PBL were separated from the blood samples on Histopaque 1077 gradients (Sigma–Aldrich) and cultured in RPMI1640 medium (Sigma–Aldrich) supplemented with 20% fetal calf serum (Gibco Invitrogen Corporation) and 0.5% phytohemagglutinin-P (PHA, Gibco Invitrogen Corporation) without antibiotics in a standard thermostat at 37 ◦ C in a humidified atmosphere containing 5% CO2 . The determination of the frequency of gene mutations at the HPRT locus (variant frequency, VF) was performed by autoradiography of the cells cultured in the presence of 3 H-thymidine (3 H-TdR) (Amersham Biosciences UK Ltd.) and a selective agent (10−4 M 6-thioguanine, TG, Sigma–Aldrich), applying the modified method of Strauss and Albertini [28]. The lectin labeling index (LI) and the HPRT mutation frequency were determined from the same samples, as previously described [29,30]. The VF was calculated according to the equation of Strauss and Albertini [28]: VF = LI[PHA+TG] /LI[PHA] . The measurement of UDS was performed according to Bianchi et al. [31], as previously described [32]. Briefly, PBLs were separated on Histopaque 1077 gradients (Sigma–Aldrich) by density centrifugation. After separation, PBLs were irradiated in open petri dishes by UV light (24 J/m2 ) and then incubated for 3 h with 10 ␮Ci/ml 3 H-TdR (activity: 37 MBq/ml, Amersham) in the absence or presence of 2.5 mM hydroxyurea. The degree of ‘de novo’ UDS was measured by scintillometry based on 3 H-TdR incorporation in separated lymphocytes. UDS was calculated as the difference between radioactivities of the incorporated 3 H-TdR in UV-irradiated and control cultures (relative units). 2.3. Determination of CA and SCE frequencies Whole blood samples were processed for studies of CA and SCE. The cell culture methods were identical in both protocols: samples of 0.8 ml heparinized blood were cultured in duplicate at 37 ◦ C, in 5% CO2 atmosphere, in 10 ml RPMI-1640 (Sigma–Aldrich) supplemented with 20% fetal calf serum (Gibco Invitrogen Corporation) and 0.5% PHA (Gibco Invitrogen Corporation) without antibiotics. For CA and SCE analyses, the cultures were incubated for 50 and 72 h, respectively. 5-Bromo2 -deoxyuridine (BrdU, Sigma–Aldrich) used in SCE analysis to identify the first and subsequent metaphases, was added at a concentration of 5 ␮g/ml at 22 h of culture. Culture harvest, slide preparation and staining were conducted following the standard methods using 5% Giemsa stain (Fluka) for CA [33], and according to the Fluorescence-plus-Giemsa method of Perry and Wolff [34] for SCE. All microscopic analyses were performed blind by permanent staff. CA characterization was carried out in 100 metaphases with 46 ± 1 chromosomes per subject according to Carrano and Natarajan [35]. Mitoses with 45 or 47 chromosomes were considered as aneuploid cells. Mitoses containing only achromatic lesions (gaps) and/or aneuploidy were not considered aberrant. Premature (early) centromere division (PCD, i.e. the separation of centromeres during prophase/metaphase of the mitotic cycle) was scored according to Méhes and Bajnóczky [36]. Total PCD and mitoses with more than three chromosomes with PCD (PCD/CSG, centromere separation general) were also scored. Cells with a higher number of SCE per cell than the 95 percentile of the control (≥10), were considered as high-frequency SCE cells (HFC/SCE), according to Tates et al. [37] and Major et al. [25]. 2.4. Flow-cytometric analysis of apoptosis and cell proliferation in PBL For the measurement of the percentage apoptosis and the S-phase, PBL were separated from the blood samples on Histopaque 1077 gradients (Sigma–Aldrich) and cultured in RPMI-1640 medium (Sigma–Aldrich) supplemented with 20% fetal calf serum (Gibco Invitrogen Corporation) and 0.5% phytohemagglutinin-P (PHA, Gibco Invitrogen Corporation) for 50 h without antibiotics in a standard thermostat

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at 37 ◦ C in a humidified atmosphere containing 5% CO2. One hour prior to the termination of the incubations, 5 ␮g/ml BrdU (Sigma–Aldrich) was added to the cultures. Cells were washed twice with PBS, fixed in 1 ml ice-cold 70% ethanol and stored at −20 ◦ C until further processing. DNA denaturation prior to propidium iodide (PI, Sigma–Aldrich) and fluorescein isothiocyanate (FITC)-labeled monoclonal antiBrdU (Becton Dickinson) staining was performed at room temperature with 2 M HCl containing 0.2 mg/ml pepsin (Sigma–Aldrich), according to the method of van Erp et al. [38]. DNA was stained with PI and the incorporated BrdU was detected by use of immuno-cytochemistry with FITC-labeled monoclonal antibody. Flow-cytometric analysis was performed with a FACS Calibur (Beckton Dickinson) flow cytometer. Data for at least 10,000 lymphocytes per sample were acquired; CellQuestPro Software was used for the analysis. Cell proliferation was also characterized by flow-cytometric measurement of the expression of the cell-activation marker CD71 on T-lymphocytes [39]. Briefly, heparinized whole blood was mixed and incubated at room temperature for 20 min with the appropriate amount of fluorescence-labelled monoclonal antibodies against surface antigens. The following monoclonal antibodies were used: peridinin–chlorophyll–protein complex (PerCP)-labelled anti-CD3 (T-cell marker), FITC-labelled anti-CD71 (transferrin receptor) and allophycocyanin (APC)-labelled anti-CD45. The monoclonal antibodies were purchased from Becton Dickinson. The erythrocytes were removed through lysis by addition of FACS Lysing solution (Becton Dickinson). After washing with phosphate-buffered saline (PBS, Sigma–Aldrich), samples were analyzed within 4 h after labeling, or fixed with 2% para-formaldehyde. Flow-cytometric analysis was performed on a Becton Dickinson FACSCalibur flow cytometer. Standard forward and side scatter gating combined with CD45 was used to set the lymphocyte gate. Data for at least 10,000 leukocytes per sample were acquired; CellQuest Pro Software (Becton Dickinson) was used for analysis. Phenotypes are expressed as percentage of positive cells of the given lymphocyte subpopulation, e.g., CD71+/CD3+ denotes the percent of CD71-positives among total T-cells. 2.5. Statistical analysis Statistical analysis was made using the GraphPad Prism 3.02 software (GraphPad Software, Inc.). Differences between the study groups and the control group were tested with Student’s t-test, p < 0.05 was considered as statistically significant. A standard linear correlation analysis was used to determine the relationship between apoptosis, CA and PCD.

3. Results Main demographic data of the investigated donors (all females) are summarized in Table 1. The donors’ age was in the range of 37–56 years and 26–60 years, for the personnel in the first and in the second group, and 22–66 years for the controls, respectively. The donors’ time of exposure in service as pathology personnel were 21.8 years (ranging from 7 to 34 years) and 17.7 years (ranging from 4 to 34 years) for the subjects in the two groups, respectively. The number of current smokers and “drinkers” was slightly elevated among the exposed donors. The results of the flow-cytometric measurements on the percentage of apoptotic cells, cells in S-phase and CD71 expression, together with the mean values of LI, VF and UDS, are summarized in Table 2A. The mean percentage of apoptotic cells was significantly (p = 0.00354 and 0.00104) higher in both groups of investigated subjects (9.05 ± 0.88 and 10.46 ± 1.17) in comparison with the mean values of the controls (5.90 ± 0.36). Interestingly, the percentage apoptotic cells was even higher in the group exposed to FA only, in comparison with the group exposed to FA and organic solvents, although this was not statistically significant (p = 0.33). The mean percentage of cells in S-phase, measured by flow cytom-

Table 1 Demographic data of the workers (all female). Groups

Controls Exposure to

Formaldehyde and organic solvents Formaldehyde

n

Mean age year ± SE

Active smokers (%)

Number of smokers/nonsmokers

Smokers: mean number of cigarettes/day mean ± SE

Drinkers (%) Number of drinkers/ non-drinkers

37 16

41.8 ± 2.3 44.5 ± 1.8

16.2 37.5

6/31 6/10

10.5 ± 3.4 11.4 ± 3.7

37.8 43.8

14/23 7/9

– 21.8 ± 2.0

21

43.3 ± 2.0

23.8

5/16

18.6 ± 3.3

57.1

12/9

17.7 ± 1.9

Mean duration of exposure year ± SE

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M.G. Jakab et al. / Mutation Research 698 (2010) 11–17

Table 2A Mean values (±SE) of apoptosis induction (%), cell proliferation (S-phase, CD71 expression of T lymphocytes, LI (PHA), %), HPRT mutation frequency (VF, ×10−6 ) and DNA-repair capacity (UDS, arbitrary units) in cultured peripheral lymphocytes among pathology-unit personnel exposed to formaldehyde (all female). Groups

n

Apoptosis %

S-phase (%)

CD71 + /CD3+ %

LI (PHA) %

VF × 10−6

UDS arbitrary units

Mean ± SE Controls Exposure to

*

Formaldehyde and organic solvents Formaldehyde

37 16

5.90 ± 0.36 9.05 ± 0.88*

21.39 ± 1.48 27.03 ± 2.68

1.55 ± 0.19 1.68 ± 0.49

11.19 ± 1.26 13.71 ± 1.40

7.75 ± 1.02 6.32 ± 2.04

6.47 ± 0.41 5.04 ± 0.62

21

10.46 ± 1.17*

25.24 ± 2.38

1.97 ± 0.29

15.67 ± 1.09*

3.68 ± 0.52*

4.73 ± 0.86

Significantly different from the controls (Student’s t-test; p < 0.05).

etry, as well as the mean values of LI, were slightly increased in the samples of both exposed groups in comparison with the controls. In case of the “FA only” group, LI was significantly (p = 0.010) increased in comparison with the controls. The CD71 expression on T-cells in both exposed groups was also elevated compared with that of the control group, although not significantly. The mean VF value, among the investigated personnel exposed to FA only, was significantly (p = 0.001131) decreased in comparison with the controls. Table 2B summarizes the mean values of the cytogenetic parameters. A significantly increased frequency of CA was observed in both groups of pathology workers (p = 0.000718 and p = 0.042446, respectively), in comparison with the controls. In contrast with the percentage of apoptosis, which showed an elevation (cf Table 2A), the mean percentage of CA decreased in the group mainly exposed to FA in comparison with the workers exposed to both FA and organic solvents, although non-significantly (p = 0.25). CA in all persons involved in the study were mainly of the chromatid type. No correlation was found between apoptosis and CA in all exposed donors (correlation coefficient −0.097). The percentage of gaps was significantly increased (p = 0.006217 and p = 0.002878), and aneuploidy showed a significant decrease (p = 0.000002 and p = 0.000312) in both groups of exposed persons in comparison with the controls. There were no significant differences in SCE between the groups of exposed donors and controls. The mean percentage of HF/SCE showed a non-significant (p = 0.16) increase among subjects in the second group, exposed mainly to FA. The percentages of PCDs were significantly higher (p = 0.000290 and p = 0.002139) in both groups of pathology department workers, in comparison with the controls. Similar to PCD, a statistically significant increase (p = 0.002289 and p = 0.014506) in the mean value of PCD (CSG)s was also observed in the samples of both groups of pathology staff, compared with the controls. The correlation coefficients were 0.368 between PCD and apoptosis and 0.021 between CA and PCD, in all exposed persons. These results indicate a weak correlation between apoptosis and PCD and no correlation between CA and PCD, in all exposed persons. No differences in the mean values of the measured cytogenetic and cell-proliferation end-points were observed between subjects with different smoking and drinking habits, with the exception of the SCE percentages for non-smokers and smokers among the controls (5.86 ± 0.15 and 7.26 ± 0.34 per mitotic cell, respectively, p = 0.024304) and in the second group of exposed workers (6.02 ± 0.27 and 7.44 ± 0.48 per mitosis, respectively, p = 0.037193). The mean HF/SCE percentage of smokers among the controls was also (non-significantly) elevated compared with non-smoking controls (0.57 ± 0.23% and 2.67 ± 1.02%, respectively, p = 0.092232). The differences in the mean values of HF/SCE between non-smokers and smokers in the two subgroups of exposed persons showed the same tendency as in the controls, but the differences were much smaller (3.12 ± 2.29% and 3.33 ± 2.61% in the first group, and 6.25 ± 2.55% and 9.6 ± 4.49% in the second group, respectively). To analyze the possible effect of age as a confounding factor on the parameters measured, the subjects in all groups were divided

into subgroups containing persons under and above the average age (18 subjects with a mean age of 29.28 ± 1.67 years and 19 persons with a mean age of 53.74 ± 1.91 years in controls, seven subjects with a mean age of 38.43 ± 1.73 years and nine persons with a mean age of 49.22 ± 1.53 years in the first group, and 11 subjects with a mean age of 36.18 ± 1.76 years and 10 persons with a mean age of 51.20 ± 1.48 years in the second group, of FA-exposed subjects, respectively). A significant (p = 0.055065) increase in SCE was found (6.88 ± 0.38 per mitosis) in the second group of the “mainly FA” exposed persons with ages above the mean in comparison with the mean SCE (5.88 ± 0.29 per mitosis) of those with ages below the mean. A non-significant increase in the mean percentages of apoptosis (11.22 ± 1.90%) and CA (3.80 ± 0.88%) was found among the donors with ages below the mean in the second exposed group compared with the mean apoptosis (9.63 ± 1.35%) and CA (2.30 ± 0.84%) of the subjects with ages above the mean age (p = 0.502375 for apoptosis and p = 0.234281 for CA, respectively). To analyze the possible effect of the duration of exposure, both groups of the FA-exposed persons were divided into two subgroups, with duration of exposure under and above the mean in the corresponding group (seven subjects with a mean duration of exposure of 14.14 ± 1.72 years and nine persons with a mean duration of 27.67 ± 1.24 years in the first group, and 11 subjects with a mean duration of 10.18 ± 1.29 years and 10 persons with a mean duration of exposure of 26.00 ± 1.69 years in the second group of FA-exposed subjects, respectively). Interestingly, a significantly (p = 0.026564) decreased mean frequency of CA (1.70 ± 0.56%) was found in the subgroup of the second group of FA-exposed subjects, containing persons with durations of exposure above the mean, compared with the mean CA (4.40 ± 0.95%) in the same group, having shorter durations of exposure than the mean of the whole group. In addition, in both FA-exposed groups, a slight, non-significant increase in the mean values of apoptosis could be observed in the subgroups above the mean duration of exposure (apoptosis was 8.87 ± 1.70% for subjects with durations of exposure below, and 9.24 ± 0.94% for subjects with durations of exposure above the mean of the first group; and 9.87 ± 1.38% for subjects with durations of exposure below, and 11.12 ± 1.96% for subjects with durations of exposure above the mean in the second exposed group, respectively). 4. Discussion The results of the present study revealed cytogenetic changes and induction of apoptosis among FA-exposed pathology staff. Mean ambient air FA concentrations were similar at the three worksites, suggesting similar exposures to the pathology staff in the pathology departments investigated. Nevertheless, a considerable FA concentration was always present in the ambient air of the laboratories, and the presence of FA in the air was always observable by olfactory perception. Safety measures and devices were introduced in the last few years in these departments, and the employees have used these protective devices during work (i.e. safety cabinets, gloves, masks, etc.). None of the pathology workers have worn masks or similar personal safety devices equipped with spe-

8.80 ± 1.07* 13.65 ± 1.59* 7.05 ± 2.19 6.36 ± 0.266.02 ± 0.257.44 ± 0.48 5.40 ± 0.61* 6.00 ± 0.65*

Formaldehyde and organic solvents Formaldehyde

Significantly different from the controls (Student’s t-test; p < 0.05). *

2.35 ± 0.46* 3.05 ± 0.62* 21

0.70 ± 0.26

6.16 ± 0.165.86 ± 0.157.26 ± 0.34 6.14 ± 0.235.99 ± 0.316.30 ± 0.37

Mean ± SE

8.89 ± 0.66 4.44 ± 0.48* 3.59 ± 0.36 5.94 ± 0.69* 1.00 ± 0.20 2.88 ± 0.46* 1.62 ± 0.26 4.00 ± 0.55* Controls Exposure to

37 16

0.62 ± 0.18 1.12 ± 0.31

Aneuploidy Gaps Chromosome type (%) Chromatid type Aberrations Total (CA) n Groups

Table 2B Cytogenetic parameters in cultured peripheral lymphocytes among pathology-unit personnel exposed to formaldehyde (all female).

SCE All/non-smokers/smokers 1/mitoses

HF/SCE

3.76 ± 1.14 3.20 ± 1.66

PCD (%)

7.60 ± 0.84 15.06 ± 1.55*

PCD (CSG)

5.57 ± 0.66 10.63 ± 1.32*

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cific filters for FA. The two groups of investigated persons served as pathology personnel from 7 to 34 years and 4 to 34 years, respectively, thus we have to take into account a considerable duration of exposure to FA. Subjects in the first exposed group could also be exposed to other organic chemicals, as they worked in the histology laboratories using different solvents and stains. It is hard to find employees with single exposure to formaldehyde in histopathology laboratories, as they are usually exposed to small amounts of other solvents. Therefore, to assess the excess risk among these persons in the first group, we should consider the effects of lowdose exposure to organic solvents as a confounding factor and as a cause of bias on the effects of FA. On the other hand, the persons in the second group were almost exclusively exposed to FA, as they were employed in mortuaries, preparing formalin solutions in large quantities for daily use. Flow-cytometric measurements detected an induction of apoptosis among donors in both groups of pathology staff exposed to FA. Our results are in good agreement with the findings of the in vitro experiments by Tyihák et al. [5], demonstrating that higher doses of FA induce apoptosis. However, cell proliferation, as determined by flow-cytometric measurements of the S-phase and expression of CD71, and from the measures of LI, was not decreased in our study, although in vitro experiments have shown a decrease in the viability [4] and mitotic activity [5] of cells at FA doses inducing apoptosis. These differences between the findings of the in vitro studies and our results may be attributed, among others, to differences in doses, duration of exposure, and differences in cell types. The slight decrease in apoptosis among the workers with ages above the mean in the second group, exposed mainly to FA, may be a consequence of adaptation to FA exposure. Mean CA frequencies were significantly elevated in both groups of pathology personnel, in comparison with the controls. This increase is indicative of exposure to clastogenic agents among these workers. As the mean CA frequencies were also significantly elevated in pathology workers in the second group, who were exposed mainly to FA, the CA-inducing effect found in our study can be attributed to FA as a clastogenic agent. However, the CA frequencies were slightly lower in this group of persons exposed to FA only, in comparison with those in the first group of pathology workers exposed both to FA and various organic solvents. This lower frequency of CA may be attributed to the elimination of damaged lymphocytes as a consequence of the observed slight elevation of apoptotic activity in this group. Furthermore, among the workers in the second group of the “only FA” exposed subjects, the CA level was much lower than expected in the subgroup with longer time of exposure (above the mean), in comparison with the persons with shorter durations of exposure (below the mean). At the same time, in this group with longer duration of exposure, a slight, non-significant increase in the mean value of apoptosis was also observed, together with the significantly decreased CA. This decrease in CA may also be due to the slightly elevated apoptotic effect observed. Similarly, the observed non-significant decrease in the mean values of CA among the older persons in the second group of FA-exposed subjects can also be a consequence of the longer durations of exposure, since the higher the age, the longer the time in service, and consequently a longer the duration of exposure can be expected for the pathology staff. Our findings on the elevated CA frequencies in both groups of workers exposed to a mean ambient air concentration of 0.9 mg/m3 FA, in are in good agreement with most of the literature dealing with FA-induced CA (and/or MN) among industrial or pathology workers. The in vitro experiments of Schmid et al. [12] showed that FA induces (mainly chromatid type) CA in PBL. Similarly, in PBL of anatomy students exposed to a mean concentration of 3.17 mg/m3 FA, increased frequencies of MN and CA were found [14]. Among paper-factory workers, increased frequencies of CA in PBL were

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observed in the study of Bauchinger and Schmid [17]. In students in a mortuary, working in embalming areas, an increase of MN [22] was measured in PBL, and an increase of CA was reported in workers manufacturing nitrogen fertilizer [23]. However, no differences were found by Thomson et al. [9] in the yields of CA and SCE in peripheral blood lymphocytes (PBL) of pathology staff, and the frequency of CA was also not increased among FA-exposed medical students working in an anatomy dissection laboratory, as reported by Vasudeva and Anand [10]. No significant changes in CA, MN and SCE were scored in PBL of FA-exposed individuals working in different health-care laboratories including a pathology department [11], but the observed low CA and MN frequencies in the investigated groups of that study may be attributed to the low level of FA exposure (with a mean level of 0.0147 mg/m3 in the low-, and 0.0562 mg/m3 in the high-exposed groups, respectively). The frequency of HPRT point mutations was not increased in our study in either group of FA-exposed workers, which confirms the weak mutagenicity of FA, as it did not induce gene mutations in the Hprt test in in vitro FA-treated V79 hamster cells, according to Merk and Speit [40]. Interestingly, we found a VF that was significantly lower among subjects in the second group. This phenomenon may be explained by the effect of a higher level of apoptosis, i.e. an elimination of mutant cells as a result of induced apoptosis cannot be excluded. Similarly to the VF, UDS also showed a slight decrease in both exposed groups in comparison with the controls. These findings are in concordance with the report on the negative effect of in vitro FA-treatment on the induction of UDS in human bronchial epithelial cells [41]. The observed slight decrease in UDS among the FA-exposed workers is in line with the findings of Emri et al. [42] on the disturbing effect of in vitro FA-treatment on UV-induced repair, causing a significant increase of UVC-induced chromosomal damages in human keratinocytes and fibroblasts. SCE was not elevated in the PBL of the exposed workers in our study. However, in the second group of the “mainly FA”-exposed persons with ages above the mean, a significantly increased SCE was observed, but this increase could probably be attributed to the effect of smoking, as all the smokers in this group belonged to this subgroup with ages above the mean. In a study by Bauchinger and Schmid [17], no increase in the SCE frequencies in lymphocytes from FA-exposed paper-factory workers was observed. Similarly, in the study by Thomson et al. [9] higher levels of FA did not induce SCE. However, in the study by Shaham et al. [18], increased frequencies of SCE and HF/SCE were observed among FA-exposed hospital pathology workers. Increased SCE and MN were detected by Merk and Speit in experiments with in vitro FA-treated V79 cells [40]. In our study HF/SCE was significantly increased in the second group of FAexposed workers, which is in good concordance with the findings of Shaham et al. [18]. PCD and PCD/CSG, a disturbance in the process of mitotic division of chromosomes, were significantly increased in both groups, indicating an effect of genotoxic exposure inducing PCDs (especially, in the case of long-term exposure), as suggested by Major et al. [27]. Similarly to CA, the frequencies of PCD and PCD/CSG were also slightly decreased in the second group, in comparison with the first group of workers. This result may be explained by the observations made by Major et al. [27] that exposure to single agents, as in the case of the second group of pathology personnel exposed mainly to FA, may lead to lower levels of PCD. The correlation coefficients were 0.368 between apoptosis induction and PCD and 0.021 between CA and PCD in all exposed personnel, indicating a weak correlation between apoptosis and PCD. The relation between exposure to FA and the induction of apoptosis and changes in CA, PCDs and PCD/CSG needs further investigation, and more data from larger groups of FA-exposed indi-

viduals from pathology and anatomy departments are also needed to analyze the confounding effects of smoking and drinking. Similarly, as in the present study the investigated pathology workers were only women, further investigations are needed to collect data from male pathology workers, in order to study the possible effect of gender on FA-induced apoptosis and changes in the cytogenetic end-points. In conclusion, the results of this study demonstrate that occupational exposure to FA can induce apoptosis and CA, thus indicating a possible excess cancer risk among exposed subjects. The multiple end-point genotoxicological monitor, developed and run in our laboratory, is able to detect changes in cytogenetic and cellproliferation biomarkers. The results emphasize the importance of personal protection at work places, with possible occupational exposure to FA. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the grants NKFP 1/B-047/2004 and ETT 08-590/2006. The authors would like to thank Mr. Zoltán Fodor for the flow-cytometric measurements of cell-surface antigens and Mrs. Ildikó Bárdi, Mrs. Éva Czifra, Mrs. Margit Tölyhi, Mrs. Irén Rétháti, Mrs. Zsuzsanna Szép-Kiss, Mrs. Andrea Hegedüs and Mrs. Tünde Szeremley-Szabóné for their excellent technical help. References [1] L. Trézl, L. Hullán, Zs.M. Jászay, T. Szarvas, I. Petneházy, B. Szende, J. Bocsi, Z. Takáts, K. Vékey, L. Töke, Antagonistic reactions of arginine and lysine against formaldehyde and their relation to cell proliferation, apoptosis, folate cycle and photosynthesis, Mol. Cell. Biochem. 244 (2003) 167–176. [2] IARC, Wood dust and formaldehyde, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 62, Lyon, France, 1995, pp. 217–362. [3] IARC, Formaldehyde, 2-butoxyethanol and 1-tert-butoxypropan-2-ol, IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, vol. 88, Lyon, France, 2006, pp. 39–325. [4] Z.L. Marcsek, Z. Kocsis, B. Szende, A. Tompa, Effect of formaldehyde and resveratrol on the viability of Vero, HepG2 and MCF-7 cells, Cell Biol. Int. 31 (2007) 1214–1219. [5] E. Tyihák, J. Bocsi, F. Timár, G. Rácz, B. Szende, Formaldehyde promotes and inhibits the proliferation of cultured tumour and endothelial cells, Cell Prolif. 34 (2001) 135–141. [6] B. Szende, E. Tyihák, Gy. Szókán, Gy. Kátay, Possible role of formaldehyde in the apoptotic and mitotic effect of 1-methyl-ascorbigen, Pathol. Oncol. Res. 1 (1995) 38–42. [7] R.C. Grafström, A. Fornace Jr., C.C. Harris, Repair and DNA damage caused by formaldehyde in human cells, Cancer Res. 44 (1984) 4323–4327. [8] R.C. Grafström, R.D. Curren, L.L. Yang, C.C. Harris, Genotoxicity of formaldehyde in cultured bronchial fibroblasts, Science 228 (1985) 89–91. [9] E.J. Thomson, S. Shackleton, J.M. Harrington, Chromosome aberrations and sister-chromatid exchange frequencies in pathology staff occupationally exposed to formaldehyde, Mutat. Res. 141 (1984) 89–93. [10] N. Vasudeva, C. Anand, Cytogenetic evaluation of medical students exposed to formaldehyde vapour in the gross anatomy dissection laboratory, J. Am. Coll. Health 44 (1996) 177–179. [11] M. Pala, D. Ugolini, M. Ceppi, F. Rizzo, L. Maiorana, C. Bolognesi, T. Schilirò, G. Gilli, P. Bigatti, R. Bono, D. Vecchio, Occupational exposure to formaldehyde and biological monitoring of Research Institute workers, Cancer Detect. Prev. 32 (2008) 121–126. [12] E. Schmid, W. Göggelmann, M. Bauchinger, Formaldehyde-induced cytotoxic, genotoxic and mutagenic response in human lymphocytes and Salmonella typhimurium, Mutagenesis 1 (1986) 427–431. [13] J. Shaham, Y. Bomstein, A. Melzer, J. Ribak, DNA-protein crosslinks and sister chromatid exchanges as biomarkers of exposure to formaldehyde, Int. J. Occup. Environ. Health 3 (1997) 95–104. [14] J.L. He, L.F. Jin, H.Y. Jin, Detection of cytogenetic effects in peripheral lymphocytes of students exposed to formaldehyde with cytokinesis-blocked micronucleus assay, Biomed. Environ. Sci. 11 (1998) 87–92. [15] G. Iarmarcovai, S. Bonassi, I. Sari-Minodier, M. Baciuchka-Palmaro, A. Botta, T. Orsière, Exposure to genotoxic agents, host factors, and lifestyle influence the number of centromeric signals in micronuclei: a pooled re-analysis, Mutat. Res. 615 (2007) 18–27.

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