Mutagenesis Advance Access originally published online on June 28, 2005
Mutagenesis 2005 20(5):311-315; doi:10.1093/mutage/gei043
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Lipoperoxidation products and thiol antioxidants in chromium exposed workers
1Department of Genetics, Faculty of Medical Sciences, New University of Lisbon, Rua da Junqueira 96, P 1349-008 Lisbon, Portugal, 2Toxicology Laboratory and 3Pharmaceutical Sciences Study Center, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal, 4Lusófona University, Lisbon, Portugal and 5Faculty of Sciences and Technology, New University of Lisbon, Monte da Caparica, Portugal
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Abstract |
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Hexavalent chromium is an established carcinogenic agent, which is not directly reactive with DNA. Its genotoxicity involves a reduction step, producing reactive oxygen species and radicals, and also lower valence forms which form stable complexes with intracellular macromolecules. The trivalent form of chromium may directly react with the genetic material and has also been shown to generate oxidative damage in vitro. To further evaluate the importance of in vivo oxidative DNA damage in the toxicity of each valence form, we conducted a comparative study on hexavalent and trivalent chromium-exposed workers (manual metal arc stainless steel welders and leather tanning workers), focusing on the total oxidative status by quantifying the level of lipoperoxidation products in urine. Thiol antioxidants are important in response to oxidative stress, and therefore, the concentration of glutathione and cysteine in peripheral blood lymphocytes was also determined. Chromium exposure was evaluated by quantifying total chromium in plasma and urine. Both groups had a signficant increase in lipid peroxidation products expressed as malondialdehyde (MDA) in urine (tanners 1.42 ± 0.61 µmol/g creatinine, welders 1.67 ± 1.13 µmol/g creatinine versus controls 0.81 ± 0.26 µmol/g creatinine, P < 0.005 in both cases) but only welders had a significant decrease in glutathione concentration in lymphocytes. There was a positive correlation between chromium in plasma and urinary MDA in welders, but not in tanners. This work is part of a larger study of which major results have been published previously including cytogenetics and DNA–protein cross-links in workers exposed to the two different forms of chromium. These results are compared with the results of oxidative damage from this study.
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Introduction |
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The redox biochemistry of chromium is rich, involving oxidation states from –2 to +6, from which by far the most stable are the elemental Cr(0), Cr(III) and Cr(VI) valences (1
). Hexavalent chromium compounds are classified by IARC as known human carcinogens, after substantial epidemiological and experimental data proved their mutagenic and carcinogenic properties (2). Nevertheless, chromium is still a widely used industrial metal to which millions of workers are exposed worldwide in industries, such as pigment production, chrome plating, leather tanning, stainless steel production and welding. Welders are estimated to receive some of the highest acute exposures to hexavalent chromium in welding fumes (2). Trivalent chromium is extensively used in leather tanning as a chelating agent to stabilize collagen fibers in the animal skin, providing it with the known thermal- and hydro-resistance of leather. Chrome tanning is economically advantageous and provides good quality leather, and is not likely to be replaced by the existing alternative tanning agents in the near future.
Trivalent chromium is not regarded as having the same toxicity as Cr(VI), apparently owing to its relative difficulty in crossing cell membranes (3). Nevertheless, evidence obtained from in vitro cell-free systems shows that, once inside the cell, Cr(III) readily complexes several intracellular macromolecules, whereas Cr(VI) is relatively inert towards the genetic material in the absence of reducing systems (4,5). During the reduction of Cr(VI) more reactive forms of chromium are generated, namely the short-lived Cr(V) and Cr(IV) and possibly Cr(II), and the stable Cr(III), as well as other reactive species, including singlet oxygen or hydroxyl radicals (6), capable of inflicting oxidative damage to the cell. The deleterious effects known to chromium may result from the reaction and binding of the reduced forms of the metal to intracellular macromolecules including DNA or from oxidative damage initiated by the side products of chromium reduction.
The abundant presence of membrane phospholipids at sites where reactive oxygen species are formed makes them easily accessible endogenous targets for lipoperoxidation (7). Lipoperoxidation occurs as a chain reaction initiated by free radicals, which propagates itself and can result in the formation of many equivalents of lipid peroxides. This process has been implicated in diverse pathological conditions, including atherosclerosis (8), aging (9), rheumatoid arthritis (10) and cancer (11). It is also involved in the toxicity of pesticides (12), solvents (13) and metals (14).
The extension of the oxidative catabolism of lipid membranes can be evaluated by several endpoints, including the measurement of exhaled alkanes (mainly ethane and pentane), determination of isoprostanes and of a variety of aldehydes in biological samples [reviewed in (7)]. The most widely used method is the quantification of malondialdehyde (MDA), one of the stable aldehydic products of lipoperoxidation, present in biological samples, such as whole blood, plasma or urine (7,15). Urinary lipid peroxidation products most probably reflect the global oxidative status of the whole body (16,17) and have been shown to be as informative as whole blood or plasma values (18). In the thiobarbituric acid (TBA) assay used in this project, the MDA contained in the sample reacts with TBA to form a complex that can be quantified by spectrophotometry and fluorimetry. Although this reaction is not specific for MDA, since other compounds (other aldehydes or bilirubin, for example) can react with thiobarbituric acid to form similar complexes, and other chromophores present in the biological samples may absorb or fluoresce in the same wavelengths as the MDA–TBA complex, the extraction of the TBA–MDA complex from the reaction mixture with an organic solvent overcomes this limitation. The fluorescent quantification is made on this extract, and the result of the TBA–MDA assay is presented as the concentration of TBA-reactive substances (TBARS), expressed as MDA. Protocols have been developed to apply high pressure liquid chromatography (HPLC) separation prior to quantification of the complex (19), which would allow for an improvement in the specificity of the determination. However, results obtained previously showed no added advantage to the HPLC protocol when groups of exposed individuals are compared, particularly for population screening (H. Borba and M. Monteiro, unpublished data). The fluorescent measurement of MDA eliminates most of the interference occurring in spectrophotometric assays, increasing its sensitivity and correlating strongly with the HPLC methods. It adds the advantages of being an economical, straightforward, fast technique, ideal for medium to large-scale population screening.
Several studies have shown a causal association between chromium exposure and elevated peroxidation products in experimental animals administered an oral solution of sodium dichromate or potassium dichromate, in doses ranging from 2.5 to 25 mg/kg/day (20–22). Fewer studies have been conducted to evaluate the role of lipoperoxidation in occupational exposure to chromium, but in one previous report a strong correlation was found between increased Cr(VI) concentrations in blood and urine owing to occupational exposure and the generation of malondialdehyde (18). In another study, the extent of lipid peroxidation in plasma, as measured by the TBA–MDA test, did not show significant differences between workers exposed to chromium and controls (23).
Oxidative DNA damage has been shown to be accompanied by a significant decrease in intracellular antioxidants (24). The main thiol molecules involved in this process are cysteine (25) and glutathione (26).
Previous studies concerning welding industry workers have reported a decrease in intracellular glutathione (23,27), which provides evidence that this antioxidant may be involved in protection of the cell against chromium-induced toxicity. The evaluation of thiol antioxidants in chromium-exposed populations may be of importance to further clarify the toxicity mechanisms of different valence states.
In the present study, we propose to compare two groups of workers exposed in the workplace to the two biologically relevant forms of chromium, in order to evaluate the relative importance of oxidative damage after chromium exposure. Lipoperoxidation was assessed by the urinary concentration of TBARS expressed as MDA in a group of welders exposed to hexavalent chromium, a group of tanners exposed to trivalent chromium and a control group. The concentration of the thiol antioxidants glutathione and cysteine was determined in peripheral lymphocytes obtained from the same groups. Chromium exposure was assessed by total chromium concentrations in urine and plasma.
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Materials and methods |
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Subjects
Blood and urine samples were obtained from full-time employed tannery workers (n = 33) directly involved in the chromium tannyard process or the finishing department and full or part-time manual metal arc stainless steel welders (n = 16). We also collected blood and urine from control individuals (n = 30), not exposed to known environmental or occupational carcinogens. Individual questionnaires were filled out for all subjects within the Occupational Health Services of the companies involved in this work. The workers and control subjects were informed of the objectives of the work, and gave expressed informed and written consent to participate in this study before the collection of urine or blood. The samples of urine and blood were coded and the anonymity of the workers and control population was guaranteed.
Blood sampling
Venous blood (25 ml) was collected from each worker or control subject on the last day of the workweek, immediately before the lunch break, into disposable polypropylene tubes containing 15 UI of lithium heparin (BBraun, Melsungen, Germany). The samples were transported at low temperature and minimal vibration and arrived to 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 (
60 ml) was obtained from all subjects (worker and control groups) on the last day of the workweek before the lunch break. 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 plasma and urine were performed by graphite furnace atomic absorption according to Granadillo et al. (28) 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).
Quantification of lipoperoxidation products in urine
Thiobarbituric acid reactive substances were measured in the urine of subjects according to Dousset et al. (29), with some modifications. In brief, duplicate aliquots of 0.5 ml urine were pipetted into screw cap, heat-resistant glass tubes, containing 25 µl of 0.6% butylhydroxytoluene in absolute alcohol (to avoid further oxidation of the lipids in the sample). A volume of 750 µl of freshly prepared TBA reagent was added (200 mg 2-thiobarbituric acid solubilized in 9 ml of sodium hydroxide 2 N, pH adjusted to 7.4 with 7% perchloric acid, and deionized water added to 25 ml; two volumes of this solution mixed with one volume of 7% perchloric acid to use as TBA reagent). The caps of the tubes were tightly screwed and the tubes were placed in a boiling bath for 10 min. After cooling down to room temperature, 1.5 ml of n-butanol was added to each tube, thoroughly mixed and centrifuged for 15 min at 3000 g. After centrifugation, the supernatant was used to measure fluorescence in a Shimadzu Spectrofluorimeter (515 nm excitation, 553 nm emission). A standard run was included in each batch, prepared from a solution of 2.5 mM tetraethoxypropane to generate MDA (final concentrations of MDA in the standards 0.5, 1.5, 2.5 and 3.5 µM).
Determination of cysteine and glutathione in lymphocytes
Intracellular concentrations of cysteine (Cys) and glutathione (GSH) were measured by HPLC as described previously (30), with minor modifications. In brief, cells were suspended in a phosphate-buffered saline–diethylenetriaminepentaacetic acid (PBS–DPTA) solution followed by acidification with 50 mM of methanesulfonic acid. The samples were subject to two cycles of freezing/thawing and low molecular weight thiols were recovered in the supernatants after centrifugation at 12 000 g, 10 min at room temperature. The standard derivatization contained 50 µl of cell extract, 50 mM of dithiothreitol (DTT) and 5 mM of monobromobimane. A set of reactions was also performed in the absence of DTT. After 10 min of incubation at room temperature, derivatization reactions were terminated by the addition of 25 mM of methanesulfonic acid. HPLC separation of fluorescent derivatives of GSH and Cys was performed using a Shimadzu LC-10ADvp liquid chromatograph, equipped with SIL-10ADvp auto-sampler, Ultrasphere ODS column (5 µm, 250 mm x 4.6 mm) and a RF-10AxL fluorescence detector. A 20 µl injection volume was used, an excitation wavelength of 390 nm, an emission wavelength of 390 nm, and a flow rate of 1.2 ml/min. The aqueous solvent was 0.25% acetic acid, pH 3.5, and the organic solvent was 100% methanol. A linear gradient of 15–25% methanol was performed over 15 min, followed by 5 min of 100% methanol to wash the column and 3 min of 15% methanol to equilibrate the column for the next run. The Cys and GSH peaks eluted at 6 and 10 min, respectively.
Statistical analysis
The statistical analysis for comparison of controls and exposed workers was performed by using the paired Student's t-test.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The purpose of the present study was to evaluate the role of oxidative damage in occupational exposure to industrial chromium in the trivalent and hexavalent forms. Previously we demonstrated that both groups studied here had a significantly elevated internal dose of chromium, reported by increased concentrations of the metal in plasma and urine
2-fold above control values. In addition, the workers were found to have a significant increase in the levels of DNA–protein cross-links (DPC) in peripheral blood lymphocytes when compared with control values (31). DPC have been proposed as a biomarker for the measurement of the biologically active dose of chromium to reach the target organ cells. Furthermore, the induction of micronuclei in cytokinesis-blocked peripheral lymphocytes was evaluated as a marker of cytogenetic damage and compared with DPC. Tanners showed a significant increase in micronucleated cells compared with controls, whereas in welders this increase was not significant (31). This follow-up study was aimed at assessing the formation of lipoperoxidation products in these individuals, in order to clarify the preponderance of the oxidative pathway in the mechanism of action of Cr(III) and Cr(VI). Both occupationally exposed groups show significantly higher concentration of TBARS, expressed as MDA (Figure 1 and Table I), which indicate an increase in generation of reactive oxygen and/or radical species, followed by oxidative damage of membrane lipids and excretion of some of the products of this damage in urine. The highest concentrations of MDA were found among stainless steel welders, exposed to fumes containing hexavalent chromium, which is possibly because of the strong oxidative properties of the chromate ion, and the generation of reactive oxygen species and radicals during Cr(VI) intracellular metabolism. In addition, a positive correlation was found between urinary MDA and plasmatic total chromium concentrations (Figure 2), which supports previous findings of a correlation between urinary and plasmatic MDA and hexavalent chromium exposure (18) and further contributes to establish a causal relationship between them.
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Welders, additionally, showed a marked decrease in GSH concentration, but not in Cys, when compared with controls, a finding that confirms previous results (23
,27). This decrease may be related to the antioxidant activity of GSH, with an extensive production of the oxidized (GSSG) form of the molecule, or as an outcome of the increased participation of GSH in stable complexes, such as DPC, previously shown to be significantly elevated in this group (31–33). GSH is present in lymphocytes in substantially higher molar concentrations than Cys in control subjects [(27) and the present study, Table I] which may make it more readily available to react with the absorbed chromium and could explain why intracellular GSH concentrations seem to be more affected by chromium exposure than in the case of Cys. The marked depletion in GSH concentrations apparently caused by exposure to welding fumes is in accordance with the findings of a recent study where A549 Human type II alveolar epithelial cells were exposed to a suspension of welding fume particles (34). A significant reduction of total intracellular GSH was reported 2 h after treatment of the cells with 63 µg/ml of welding fume particles, reporting GSH levels as low as 40% of control. Also at 2 h after treatment, this study found a significant increase in intracellular reactive oxygen species, further supporting a correlation between welding fume exposure, oxidative stress and GSH depletion. Considering that welding fumes may contain up to 60% of hexavalent chromium, the observed effects are hypothesized to be owing to the Cr(VI) salts, both soluble and insoluble, present in the particulate matter of the fumes.
The tanner group, exposed to trivalent chromium, also registered significantly elevated concentrations of urinary MDA when compared with control values (Figure 1 and Table I). Cr(III) is less likely than Cr(VI) to undergo reduction within the cell, owing to a low reduction potential of –0.40 V [Cr(III)–Cr(II)] and –0.74 V [Cr(III)–Cr(0)] versus +1.33 V [Cr(VI)–Cr(III)]. Nevertheless, trivalent chromium has been shown in vitro to be able to generate hydroxyl radicals (35) and induce oxidative DNA damage in the presence of hydrogen peroxide (36,37). Cells exposed to trivalent chromium in a variety of saline forms have shown signs of cytotoxicity and apoptosis. Changes in the cell membrane after long-term exposure to trivalent chromium were pointed out as a mechanism of increasing the membrane's permeability to Cr(III) ions (38), which may be a factor in chronic occupational exposure settings. In experimental animals, medium to long-term exposure to trivalent chromium has resulted in a significant increase in the excretion of lipoperoxidation products. The excess in urinary MDA found in tanners may reflect the same oxidative damage pathway in exposed workers, although in this study no positive correlation was found between urinary MDA and plasma chromium concentrations in tanners. The present data do not allow further clarification of the role of chromium in the increased lipoperoxidation in tanners, owing to the multiexposure to which the workers are subject to and the non-specificity of this endpoint.
Thiol antioxidants GSH and Cys were not significantly altered in the tanner group when compared with control values (Table I and Figure 3), showing that, if they are involved in protection against the oxidative stress found in these workers, their turnover is enough to maintain stable their concentrations in the cell.
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Possible explanations for the disparity in GSH profiles between the tanner and welder group, both showing significantly elevated lipoperoxidation products in urine are: (i) The rate of absorption of the different valence states of chromium through cellular membranes. In the tanner case, the access of trivalent chromium with the intracellular medium may be limited by the rate of passage through the cell membrane, as shown by a lower value of DPC when compared with the hexavalent chromium exposed group (31
). The higher concentration of intracellular chromium in welders, with an increase in DNA–Cr–GSH complexes formed, may be enough to reach the threshold of GSH protection, and be responsible for a detectable depletion of available GSH. (ii) Hexavalent chromium is a strong oxidant that undergoes rapid intracellular reduction generating a number of reactive oxygen and radical species, including the hydroxyl radical. The resulting lower valence forms of chromium can form complexes with GSH and other intracellular macromolecules, but this reaction seems to be favored by the previous reduction step, and therefore would be more extensive in the case of Cr(VI) exposure, contributing to a more marked effect on available GSH levels.
The overall results derived from our former study on the genotoxicity associated with chromium exposure (31) and the lipoperoxidation products evaluated in this work are presented in Table II, in order to enable a comparison between these different endpoints.
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In summary, the two groups of workers studied had a significant increase in oxidative stress apparently related to their occupational exposure, with a significant decrease in GSH concentration in the individuals exposed to welding fumes, containing hexavalent chromium, but not in the tanner group. Our results show that the use of TBARS as a marker of oxidative stress should be complemented with antioxidant parameters both in plasma and in blood cells, namely the GSH redox state. The results support an involvement of the oxidative damage pathway in the mechanism of toxicity of chromium in occupationally exposed individuals.
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* To whom correspondence should be addressed. Tel: +351 21 3610290; Fax: +351 21 3622018; Email: rueff.gene{at}fcm.unl.pt
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Received on March 30, 2005; revised on May 6, 2005; accepted on May 27, 2005.
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