Mutagenesis, Vol. 18, No. 1, 19-24,
January 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Elevated levels of DNA–protein crosslinks and micronuclei in peripheral lymphocytes of tannery workers exposed to trivalent chromium
1 Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, R. da Junqueira 96,P 1349-008 Lisbon, 2 Toxicology Laboratory, Faculty of Pharmacy, University of Lisbon, Lisbon, 3 University Lusófona, Lisbon, 4 Faculty of Sciences and Technology, New University of Lisbon, Monte da Caparica, Portugal and 5 Department of Pathology and Laboratory Medicine, Box G-B511, Brown University, Providence, RI 02912, USA
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Abstract |
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DNA–protein crosslinks (DPC) are a promising biomarker of exposure to hexavalent chromium, a known human carcinogen. Although trivalent chromium is considered to have much lower toxicity, the risk involved in chronic exposure is uncertain. DPC may be a useful tool in clarifying this risk, by signaling an exposure of body tissues to biologically active forms of chromium. DPC quantification was carried out in lymphocytes of a group of tannery workers exposed to trivalent chromium, a small group of manual metal arc stainless steel welders exposed to hexavalent chromium and a control group. This biomarker was compared with the frequency of micronuclei in cytokinesis blocked peripheral lymphocytes as a biomarker of cytogenetic lesions and total plasma and urine chromium levels as an index of exposure. The results indicate a significant increase in the formation of DPC in tannery workers compared with controls (0.88 ± 0.19 versus 0.57 ± 0.21%, P < 0.001, Mann–Whitney test) and an even higher level of DPC in welders (2.22 ± 1.12%, P = 0.03). Tanners showed a significant increase in micronucleated cells compared with controls (6.35 ± 2.94 versus 3.58 ± 1.69
, P < 0.01), whereas in welders this increase was not significant (5.40 ± 1.67). Urinary chromium was increased in both groups, with a greater increase observed in tanners compared with controls (2.63 ± 1.62 versus 0.70 ± 0.38 µg/g creatinine, P < 0.001) than in welders (1.90 ± 0.37 µg/g creatinine, P < 0.005). Plasma chromium was also increased in both groups (tanners 2.43 ± 2.11 µg/l, P < 0.001, welders 1.55 ± 0.67 µg/l, P < 0.005 versus controls 0.41 ± 0.11 µg/l). In summary, chronic occupational exposure to trivalent chromium can lead to a detectable increase in lymphocye DNA damage which correlates with a significant exposure of the cells to the metal. |
Introduction |
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Chromium is one of the most widely used industrial metals. Several million workers worldwide are estimated to be exposed to chromium compounds in an array of industries such as pigment production, chrome plating, stainless steel welding and leather tanning, among others (Cohen et al., 1993
; Barceloux, 1999). Additionally, it is one of the major contaminants in various sites worldwide, including the Superfund sites in the USA (US Environmental Protection Agency, 2002), and yet there remain many unanswered questions on the human health effects of this metal (Pellerin and Booker, 2000). Epidemiological studies have found an increased incidence of lung cancer in both bichromate production and chromate pigment manufacture and there is evidence of a similar risk among chromium platers and chromium alloy workers (Léonard and Lauwerys, 1980; IARC, 1990). These findings are the basis of the IARC decision to classify hexavalent chromium compounds as known human carcinogens (IARC, 1990). The trivalent form of chromium, although the predominant form of the metal in biological systems and inside living cells, seems to play a nutritional role in the body and has considerably lower toxicity, apparently due to its poor membrane permeability (Cohen et al., 1993; Cie
lak-Golonka, 1995). Nevertheless, in vitro studies addressing this valence state have produced positive results in several mutagenicity studies (Léonard and Lauwerys, 1980; Friedman et al., 1987; Rajaram et al., 1995) and may be implicated in an epigenetic mechanism of carcinogenesis (Snow, 1991), although no adequate evidence of carcinogenicity has been found in classical epidemiological studies in industries using mainly trivalent chromium, such as the tanning industry (IARC, 1990; Langård, 1990). This industry uses trivalent chromium compounds in the processing of animal hides into leather. Although its carcinogenicity has been known for several years, there is still a considerable lack of knowledge of the mechanism(s) of toxic action of hexavalent chromium and the risks associated with various routes of exposure to both hexavalent and trivalent chromium compounds. Indeed, the US National Toxicology Program has an ongoing rodent study on the toxicology of hexavalent chromium in drinking water (National Toxicology Program, 2002). A major gap relates to the lack of validated biomarkers applicable to humans exposed to this metal.
Concentrations of chromium in blood, serum and urine have long been used in biological monitoring of environmentally or occupationally exposed populations, as a biomarker of exposure (Rajaram et al., 1995). Non-invasive urinary sampling has been extensively used in biological monitoring of chromium, but its reliability is controversial. The half-life of the metal in the bloodstream is short, followed by rapid urinary excretion or storage in body tissues such as bone and liver, so urinary levels, as well as plasma determinations, may not provide an indication of low chronic exposure to chromium (Bukowski et al., 1991; Christensen, 1995; Finley et al., 1996). Furthermore, because all chromium excreted in the urine is in the trivalent form, it provides no information on the valence state absorbed. Red blood cell analysis has been applied to monitor occupational exposure to hexavalent chromium on the basis of the easy membrane passage of this form, reduction and binding with cellular components, as compared with the relative membrane impermeability of trivalent chromium, which tends to bind to plasma proteins such as transferrin, without entering the erythrocyte (Wiegand et al., 1988). This technique is invasive and involves a difficult analytical procedure, in addition to producing conflicting results with low dose exposures.
Hexavalent chromium has been shown to be mutagenic in a variety of bacterial and mammalian assay systems. Trivalent chromium is not as active in similar assays, again apparently due to poor cellular uptake. Yet, trivalent chromium has been shown to interact with DNA in vitro (Bridgewater et al., 1994a). Under these conditions, Cr(III) was shown to produce DNA–protein crosslinks (DPC) and to modify the fidelity and kinetics of DNA replication (Snow, 1991, 1994) and cause guanine-specific polymerase arrest (Bridgewater et al., 1994a,b). Binary and ternary Cr(III)–DNA complexes are mutagenic in human cell systems (Voitkun et al., 1998; Zhitkovich et al., 2001; Quievryn et al., 2002) and may be involved in the genotoxic action of the metal. Therefore, trivalent chromium seems to be the ultimate toxicant form inside living cells, where hexavalent chromium is rapidly reduced and becomes trapped in lower valence forms (Zhitkovich et al., 1996). Reduction of hexavalent chromium seems to be driven by ascorbate and low molecular weight thiols, whereas the major DNA adducts formed are ternary complexes of chromium and amino acids and glutathione (GSH) (Zhitkovich et al., 1995).
Considering the limitations of the currently used biological indicators of exposure and of the early biological effects and conflicting results obtained in animal and in vitro assays, we herein report the first evaluation of DPC as a biomarker of the biologically active dose in a population of tannery workers exposed to trivalent chromium. The induction of micronuclei in cytokinesis blocked peripheral lymphocytes was also assessed, as a biomarker of early biological effects. Chromium exposure was evaluated from urine and plasma chromium levels. A small group of welders, exposed to hexavalent chromium, and a control group were also included in this study.
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Material and methods |
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Subjects
Blood and urine samples were obtained from full-time tannery workers (n = 33) directly involved in the chromium tanning process or the finishing department and full-time manual metal arc stainless steel welders (n = 5). We also collected blood (n = 30) and urine (n = 20) from control individuals not known to be exposed to either environmental or occupational carcinogens. All subjects were from Portugal.
Blood sampling
Venous blood (25 ml) was collected from each worker or control subject on the last day of the working week, immediately before the lunch break, in disposable polypropylene tubes containing 15 UI lithium heparin (BBraun, Melsungen, Germany). The samples were transported at low temperature and with minimal vibration and arrived at the laboratory within 2 h. Lymphocytes were immediately isolated by a standard Ficol/sodium diatrizoate protocol using Histopaque-1077 (Sigma, St Louis, MO).
Urine sampling
A spot urine sample was obtained from all subjects (worker and control groups) on the last day of the working week before the lunch break. Additionally, spot urine samples were obtained from 25 tannery workers on the last day of the working week (Friday) in the morning (pre-shift) and at the end of the working day (post-shift) to evaluate absorption during the work shift. Urine samples were collected in sterile polyethylene containers and frozen at –20°C until total chromium and creatinine analyses were performed.
Total chromium analysis
All chromium determinations in blood, plasma and urine were performed by graphite furnace atomic absorption according to Granadillo et al.(1994) using a Perkin-Elmer atomic absorption spectrophotometer. Standard curves were included in each run, obtained by adding known amounts of chromium (Spectrosol® trivalent chromium standard solution; BHD Laboratory Supplies, Poole, UK) to a control biological sample. No matrix modifier was used. Urinary chromium concentration was corrected for each individual according to the creatinine values, determined by the standard picric acid method (Kit 555-A; Sigma Diagnostics, St Louis, MO).
Micronucleus assay
The cytokinesis blocked micronucleus assay was performed as follows. Aliquots of 500 µl of heparinized whole blood were cultured in 4.5 ml Ham's F-10 medium supplemented with 24% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 1% L-glutamine (all from Sigma) and 1% heparin (50 U/ml) (BBraun). Lymphocytes were stimulated with 2% (v/v) phytohemagglutinin (PHA-M form; Gibco BRL Life Technologies, Grand Island, NY) and incubated at 37°C.
Cytochalasin B from Sigma was added at 44 h at a final concentration of 12.5 µM (6 µg/ml, 8.34 mM stock solution prepared in DMSO).
After a total of 72 h culture, cells were harvested by centrifugation (10 min, 120 g), washed twice with RPMI 1640, pH 7.2, supplemented with 2% fetal calf serum and centrifuged for 7 min at 120 g. The supernatants were removed and cell suspensions were subjected to a mild hypotonic treatment, consisting of a mixture of RPMI 1640:deionized water 1:4, supplemented with 2% fetal calf serum, pH 7.2. Cells were then centrifuged for 5 min at 120 g, the supernatants removed and smears of the pellets were performed on dry slides. After air drying the slides were fixed with freshly prepared cold methanol/acetic acid (3:1) for 20 min and stained with 4% Giemsa in 0.01 M phosphate buffer, pH 6.8, for 8 min.
For each subject, 1000 binucleated lymphocytes with well-preserved cytoplasm were scored for micronuclei and 1000 lymphocytes were scored for the cytokinesis blocked proliferation index (CBPI), calculated according to Surrallés et al.(1995): CBPI = [MI + 2MII + 3(MIII + MIV)]/total no. of cells, where MI–MIV are the number of cells with one to four nuclei. Micronuclei (MN) were identified according to the criteria described by Kirsch-Volders et al.(2000).
DNA–protein crosslinks
DPC were assessed in isolated lymphocytes of workers and control subjects according to Zhitkovich et al.(1992), as recently modified (Quievryn and Zhitkovich, 2000). To minimize the inter-batch variation, controls and cases were run in parallel and an internal standard was included in each run.
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Results |
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Study groups
The three groups studied had similar demographic characteristics, such as age [tanners (T) 41 ± 11, welders (W) 40 ± 2, controls (C) 43 ± 13 years], percentage of non-smokers (T 75, W 80, C 58%) and percentage of male subjects (T 97, W 100, C 87%).
Chromium concentrations in plasma and urine
Total chromium levels were higher in urine and plasma samples from occupationally exposed individuals when compared with controls (T = 2.63 ± 1.62, P < 0.001, W = 1.90 ± 0.37, P < 0.005, versus C = 0.70 ± 0.38 µg/g creatinine, Mann–Whitney test, Figure 1). Plasma values were also significantly increased in both groups (T = 2.43 ± 2.11, P < 0.001, W = 1.55 ± 0.67, P < 0.005, versus C = 0.41 ± 0.11 µg/l). There was a positive correlation between urine and plasma values in tanners (r = 0.93, Pearson's test, Figure 2), which indicates that absorption into the bloodstream of trivalent chromium occurred in these workers. We also found an average 2.7-fold increase (95% confidence interval 2.0–3.5) in urinary chromium values from pre-shift to post-shift in tanners (Figure 3).
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Micronuclei levels
The tanner group presented the highest number of binucleated cells with one or more micronuclei in the cytokinesis blocked micronucleus assay. These results are significantly different from the control group values (6.35 ± 2.94 versus 3.58 ± 1.69
, P < 0.01, Mann–Whitney test). Welders registered a marginal, but not statistically significant, increase (Figure 4 and Tables I–III).
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DNA–protein crosslinks
DPC assessment, expressed as a percentage of crosslinked DNA, showed a significant increase in tanners (0.88 ± 0.19%) (P < 0.001, Mann–Whitney test) when compared with controls (0.57 ± 0.21%). However, DPC among the welder group was even higher (2.22 ± 1.12%); although the welder group comprises only five individuals the results are significant (P < 0.05, Figure 5
).
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Discussion |
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The extensive use of chromium in industrial settings has elicited concern over the safety of workers and surrounding populations, mainly when hexavalent chromium is present. Questions regarding the low efficiency of trivalent chromium absorption have been discussed as an argument for the lower toxicity of this form, mainly based on the reported per os absorption of trivalent chromium not exceeding 3.0% in animal studies and healthy human volunteers (Finley et al., 1996
). Nevertheless, several factors may affect the rate of absorption of the metal, including the chemical species present, the physical properties of the compound (solubility, crystal form, properties of ligands or complex stereochemistry) and the primary exposure route (Léonard and Bernard, 1993). Previous studies have shown a significant increase in total chromium in urine or plasma of individuals occupationally exposed to chromium in industries using Cr(III) where Cr(VI) contamination was absent or unlikely, such as tanneries (Saner et al., 1984; Randall and Gibson, 1987; Minoia and Cavalleri, 1988). In these settings the main exposure routes are inhalation and dermal absorption, although ingestion of airborne particles cannot be ruled out. The findings of the present study are in agreement with these results, showing a significant increase in chromium concentrations in the urine of tanners over the control subjects (Figure 1
). A positive correlation was found between plasma and urinary chromium levels, showing that despite the short half-life of chromium in the blood compartment, in chronic occupational exposure there are relevant amounts of chromium being absorbed, distributed throughout the organism and being excreted in urine, so that chromium levels in either plasma or urine seem to be a valid measure of exposure in routine occupational biomonitoring. Furthermore, the pre- and post-shift total chromium concentration in the urine of tanners showed an average 2.7-fold increase, which demonstrates substantial chromium intake and absorption during the 8 h work shift.
Cytogenetic analysis of micronuclei in lymphocytes of workers in the tanner group showed average values significantly higher than in controls, whereas welders showed no statistical difference from either group. These findings seem to indicate a higher genotoxic risk in the tannery population, since the formation of micronuclei is related to DNA lesions resulting in acentric fragments or whole chromosomes not being included in the resulting nuclei during mitosis (Högstedt, 1984; Kirsch-Volders et al., 1997, 2000). Previous studies have found a weak but significant increase in chromosomal aberrations in tannery workers directly involved in the chromium tanning process (Cid et al., 1991; Sbrana et al., 1991) and other studies have reported the in vitro induction of micronuclei by trivalent chromium (chromium chloride) in human diploid fibroblasts, showing a predominance of aneugenic over clastogenic effects (Seoane and Dulout, 2001). However, leather processing involves a considerable number of potentially genotoxic substances, such as formaldehyde and benzidine (IARC, 1981), which may be contributing to the cytogenetic lesion reported. Welders have been found to have more micronuclei (Vaglenov et al., 1999) than matched controls, while other studies have found no difference (Littorin et al., 1983; Benova et al., 2002). No statistically significant difference in the frequency of micronuclei between welders and controls is reported here. One possible reason is the variable joint exposure of welders to other compounds such as nickel, a potential suppressor of Cr-dependent cytogenetic damage (Katsifis et al., 1996, 1998).
DPC have been assessed in several populations which were either occupationally or environmentally exposed to hexavalent chromium compounds and elevated levels were detected in welders and chrome platers (Costa et al., 1991; Zhitkovich et al., 1998). The classical experimental protocol of alkaline elution poses technical difficulties in application to large-scale biomonitoring: it is a time consuming procedure allowing the processing of only a limited number of samples in a few days. The technique used in this study, involving selective precipitation of crosslinked DNA by SDS proved to be a straightforward method of assessing the same biomarker (Costa et al., 1993). The results show a significant increase in DPC values in tannery workers (0.88 versus 0.57%, P = 0.006, Mann–Whitney test). Furthermore, there was a positive correlation between chromium concentrations and DPC in both urine (r = 0.54, P = 0.005, Pearson's test) and plasma (r = 0.45, P = 0.001, Pearson's test). These results support the causal relationship between chromium exposure and increased lymphocyte DPC levels. The welder group presented a higher DPC value, also significantly different from the control value (2.22 versus 0.57%, P = 0.013, Mann–Whitney test). Both hexavalent and trivalent chromium-exposed groups had a significant excess of chromium in biological samples, reflecting an increased body burden of the metal, and showed higher levels of DPC when compared with control individuals. The obvious difference between the welder and tanner groups (>2.5-fold) may reflect the different cellular uptakes of the main valence states to which these groups are exposed. The results obtained in this work show that exposure to trivalent chromium leads to formation of DPC in circulating lymphocytes, although to a lesser extent than with hexavalent chromium exposure. It should be pointed out that the target cells studied were circulating lymphocytes, which raises the possibility of DPC formation in the lungs of exposed workers, with potential biological effects.
The mechanism(s) of hexavalent chromium-induced carcinogenicity is not completely understood. The toxicity of chromium within the cell may result from damage to cellular components during the hexavalent to trivalent chromium reduction process, by generation of free radicals (Kontenkamp and O'Brien, 1994; Agency for Toxic Substances and Disease Registry, 2000), inducing DNA damage similar to that induced by oxidative agents, or by the formation of stable complexes of the reduced forms with DNA (Voitkun et al., 1998). Recent studies indicate a stronger biological relevance of non-oxidative mechanisms in chromium carcinogenesis (Zhitkovich et al., 2001). Reduction of hexavalent chromium seems to be driven by vitamins and low molecular weight thiols and the major DNA adducts formed are ternary complexes of chromium and amino acids and GSH. Recently, we found a significant depletion of GSH in peripheral lymphocytes of workers exposed to Cr(VI) (Quievryn et al., 2001).
Decreased intracellular levels of GSH are likely to be the cause of the increase in the fraction of Cr(III) available for DNA binding, leading to efficient formation of DPC in welders.
In summary, exposure to trivalent or hexavalent chromium can lead to measurable DPC formation, indicating exposure of the cells to reactive forms of chromium.
The study of DPC as a biomarker of biologically active doses of chromium should be pursued further, including determination of the biological role of these lesions and the influence of different biological reducers on their yield.
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Acknowledgments |
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This work was supported by grant ES 08786 from the US National Institute of Health and PhD fellowship 18317/98 from the Portuguese Foundation for Science and Technology.
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Notes |
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6 To whom correspondence should be addressed. Fax: +351 21 3622018; Email: rueff.gene{at}fcm.unl.pt
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References |
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Received on May 20, 2002; revised on July 8, 2002; accepted on July 11, 2002.
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