and Sara Teresinha Olalla Saad2,4. 1Departamento de Farmacologia, 2Hemocentro, 3Departamento de Medicina Preventiva, 4Departamento de Clinica.
1998 Stockton Press All rights reserved 0144 ± 5952/98 $12.00
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Abnormal antioxidant system in erythrocytes of mercury-exposed workers Mary Luci de Souza Queiroz1, Socrates Calvoso Pena1, Tereza Sueko Ide Salles2, Eduardo Melo de Capitani3, and Sara Teresinha Olalla Saad2,4 1
Departamento de Farmacologia, 2Hemocentro, 3Departamento de Medicina Preventiva, 4Departamento de Clinica MeÂdica. Universidade Estadual de Campinas, Campinas, SP, Brazil. To investigate the effects of chronic exposure to mercury we studied the red cell antioxidant system in mercuryexposed workers through the evaluation of reduced glutathione, catalase and superoxide dismutase systems. Of these workers, some were being exposed at the time and had presented urinary mercury levels considered safe for occupational exposure for at least 3 months prior to the initiation of this study, and others had been on leave for at least 6 months because of intoxication symptoms. Reduced
glutathione levels were lower and catalase activity was higher in the workers which were still being exposed, compared to those on leave and controls. No differences were observed between the workers on leave and controls.
Keywords: mercury; occupational exposure; catalase; glutathione; superoxide dismutase; erythrocyte
Introduction Mammalian erythrocytes are exposed to high oxygen tension and the haemoglobin and red cell membrane are susceptible to autoxidation and lipid peroxidation, respectively. As these cells lack a nucleus, they do not repair their damage and are particularly susceptible to oxidative injury.1 Thus, the red blood cell has a variety of mechanisms to protect haemoglobin and membrane function from free radicals. While superoxide dismutase (SOD) and catalase enzymatically remove superoxide and hydrogen peroxide (H2O2), respectively, red cell reducing equivalents, mainly glutathione, NADH and NADPH may react directly with superoxide and H2O2. The activity of the hexose monophosphate shunt is important since it produces NADPH which is necessary for glutathione production by glutathione reductase.2,3 When mercuric chloride is administered in vivo, the cation preferentially accumulates in the proximal tubules in the cortex of the kidney, probably due to very high intracellular levels of reduced glutathione (GSH) to which Hg2+ is bound.4 In the past years, a prominent role of GSH in a variety of physiological and immunological processes has been fully recognised.5 ± 7 In recent reports, we have demonstrated that in mercury-exposed workers, presenting mercury urinary concentrations below the biological limit values, there are several alterations in the immune response, such as, impairment of neutrophil functions,8,9 increased immunoglobulin levels,10,11 Correspondence: STO Saad Received 8 September 1997; revised 5 February 1998; accepted 5 February 1998
reduced numbers of B lymphocytes12 and reversed CD4+/CD8+ ratio.13 In order to investigate further the changes occurring after chronic exposure to levels of mercury considered safe for occupational exposure, we have designed the present study to investigate the red cell antioxidant system in mercury-exposed workers through the evaluation of the glutathione, catalase and superoxide dismutase systems.
Subjects and methods Population The study group consisted of 27 males occupationally exposed to mercury vapours (HgO). From these, 11 were workers on temporary leave following medical recommendation because of having symptoms compatible with heavy metal intoxication (temporary leave group), and 16 were working in a mercury-producing plant (exposed group). In this plant, mercury is separated from diatomaceous earth contaminated with mercury in the sulphate form, using a waste-cleaning process of chloro-alkali plants that use mercury electrodes to produce chlorine and soda. This diatomaceous earth is roasted in shaft furnaces and metallic mercury is obtained by subsequent condensation. When exposed, the population on temporary leave were in similar working conditions to those of the exposed group. Control subjects n=(11), of comparable age and race, with no history of mercury exposure, were chosen from the laboratory staff. Informed consent was obtained from all the subjects and this work was approved by the
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Ethical Committee of the University Hospital of Campinas. In the exposed group, the mean age of the workers was 32 years (range 18 ± 48 years) and the mean exposure period to mercury was 3.3 years (range 0.5 ± 8 years). In the temporary leave group the workers had been exposed to mercury for 4 ± 10 years and had been on leave from work for at least 6 months. The mean age of the workers was 46 years (range 42 ± 61). Each worker was examined in a standard fashion by a physician including a complete neurological examination. A complete occupational history was noted and included the occurrence in the preceding 6 months of symptoms possibly related to mercury exposure as well as observations on past episodes of micromercurialism, incidence of infections and alcohol and drug ingestion. Alcoholics, drug addicts and those recovering from infectious diseases and workers with other possible exposures in addition to mercury were excluded from the study. A similar protocol was applied to the control group. Smoking habits were present in seven exposed workers (44%), four workers on leave (36%) and ®ve controls (45%). Fish consumption was low in the dietary habits of all groups. Urine samples from each worker were collected for determination of mercury. Mercury exposure data were based on mercury urinary levels corrected for urinary ¯ow rates since this parameter re¯ects better than the blood Hg levels the average long-term exposure and is more indicated for the assessment of the risk of adverse effects and the need for preventive measures.14,15 To avoid errors arising from inaccurate collection of 24 h urine samples, use was made of spot samples voided during the period of blood sampling. Urinary mercury levels were determined by atomic absorption spectrophotometry (Varian model AA 175 equipped with a Hg hollow-cathode lamp) according to the hybrid generator method16 and the results expressed in terms of the urinary creatinine content of the same sample. Urinary creatinine was determined by Jaffe method using spectrophotometry.17 Venous blood sampling was always performed between 8:00 ± 9:00 a.m., when the subjects had been fasting for at least 12 h. Methods Blood samples were collected with EDTA for blood analysis (Coulter counter S), reticulocytes count and Heinz bodies detection, with ACD for reduced glutathione quantitation and measurement of catalase and G6PD activities and with heparin for determination of superoxide dismutase activity. Plasma and buffy coat were removed and ®ltered through an a-cellulose/microcrystaline cellulose column. Red cells were washed twice with 0.9% NaCl, hemolyzed by adding 15 volumes of distilled
water and one part of the suspension was added to b-mercaptoethanol EDTA stabilizing solution. The antioxidant analysis was performed in samples which were run in duplicate. Catalase was assayed by measurement of decomposition of H2O2, by catalase at 230 nm for 10 min, according to Beutler.18 Ethanol was added to stabilize hemolyzate by breaking down `complex 11' of catalase and H2O2. For each sample 50 ml Tris buffer, 900 ml H202 10 mmol, 20 ml water and 20 ml hemolyzate 1 : 2000 were mixed. Activity was expressed in IU/gHb. Glucose-6-phosphate dehydrogenase(G6PD) was assayed according to Beutler18 at 378C. Activity was expressed in IU/gHb. Superoxide dismutase (SOD) was assayed according to Winterbourn et al.19 A chloroformethanol extract was prepared by adding 0.5 ml hemolysate to 3.5 ml ice-cold distilled water, followed by 1.0 ml ethanol and 0.6 ml chloroform. After centrifugation, SOD activity was determined in the supernatant and measured by the inhibition of the reduction of nitroblue tetrazolium by the superoxide anion produced by the reaction of photoreduced ribo¯avin and oxygen. For each sample to be assayed, six tubes were set up containing 10, 20, 40, 60, 80 and 500 ml red cell extract, plus 0.1 mol EDTA containing 1.5 mg sodium cyanide per 100 ml ± 0.2 ml; 1.5 mmol NBT ± 0.1 ml; 0.12 mmol ribo¯avin ± 0.05 ml; mol/15 phosphate buffer to give a total volume of 3 ml. Three tubes containing no red cell extract were also included with each run. Ribo¯avin was added last. After an exposure to uniform illumination for 12 min, optical densities were measured at 560 nm. Results were expressed as units of SOD per gram of haemoglobin. The amount of extract (V ml) which gives half this inhibition (1 unit), was determined from the graph and enzyme activity was calculated as 100 000 units/g Hb. Reduced glutathione (GSH) was measured according to its capacity to reduce 5,5'-dithiobis (2-nitrobenzoic acid-DTNB) formation, a highly coloured yellow anion at 412 nm.18 Presence of Heinz body and reticulocyte counts were evaluated by standard techniques.20 All values are given as mean+s.d. Statistical analysis was carried out using the Mann ± Whitney U test for unpaired data and the coef®cient of Spearman for correlations. Differences at P50.05 were considered signi®cant. All statistical tests were two-sided.
Results The mean of urinary mercury concentration for the exposed population was 18.5+8.8 and ranged from 4.7 ± 37.5 mg Hg/g creatinine. This value was based
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on the average intraindividual values registered in the last year of observation. All, except two workers (exposed for 6 and 8 months) had six measurements during that period. The distribution among the workers was as follows: 510 mg/g, two workers; 10 ± 20 mg/g, nine workers; 20 ± 30 mg/g, three workers; 30 ± 40 mg/g, two workers. In the temporary leave group, the average urinary mercury levels for the workers, obtained at the time of this study, was 10+6.7 mg/g creatinine.11 These concentrations ranged from 5.0 ± 19.0 mg Hg/g creatinine and the distribution among the workers was as follows: 510 mg/g, seven workers; 10 ± 20 mg/g, four workers. None of the controls had urinary mercury concentrations above 5 mg/g creatinine, which is considered the safe limit of mercury exposure for non-exposed populations. No differences in health status, including results from neurological examination, were observed among three groups. The results of GSH are shown in Figure 1. GSH levels were lower in the workers exposed to mercury compared to the group on temporary leave (P=0.002) and normal controls (P=0.007). In addition, catalase activity was signi®cantly higher in the workers exposed to mercury compared to the temporary leave group (P=0.016) and normal controls (P=0.048) (Figure 2). There were no signi®cant differences between the workers on temporary leave and normal controls. Regarding the activities of SOD and G6PD, there were no differences between all three groups studied (x+s.d. SOD: E=3371.7+1261.9, L=3235.4+ 835.9,. C=3078+70.0; G6PD: E=11.24+1.57, C=12.2+1.0). Heinz body was absent in the blood smear of all subjects studied and the reticulocyte and blood counts were normal in all individuals. No correlation was observed between the GSH levels,
In the present study, we analyzed three groups of subjects. The ®rst group consisted of workers exposed to mercury for a period of at least 6 months and a maximum of 8 years; the second group was composed by individuals previously exposed to mercury, for a period of 4 ± 10 years, who were on temporary leave for at least 6 months; the third group consisted of normal controls, members of the laboratory staff. In all the workers studied urinary mercury concentrations were below the accepted safety threshold level of 50 mg/g creatinine.20 In the temporary leave group the average urinary mercury concentrations were 10+6.7 mg/g creatinine. These relatively high levels could be explained by data in the literature demonstrating that, in workers chronically exposed to mercury, the half time of elimination is about 3 months21 and urinary Hg levels may still exceed 30 mg/g creatinine 3 months after the removal from exposure.14 We observed, in the mercury-exposed group, a reduction in the levels of red cell GSH and an increased catalase activity. SOD and G6PD activities were normal. In the group on temporary leave and controls we have not detected any alteration in the red cell antioxidant system. In a recent review of the in¯uence of the antioxidant systems in the erythrocyte survival, Kurata et al23 reported, in all mammals, a positive correlation between the red cell life-span and the intracellular levels of SOD, GSH-Px and GSH. However, no correlation was observed for catalase
Figure 1 Levels of reduced glutathione in mercury-exposed workers (n=22) and controls (&) (C) (n=10). In the exposed group, 13 workers are presently being exposed (~) (E) and nine were on leave for at least 6 months because of intoxication symptoms (&) (L). *P50.05 compared to workers on leave and controls. (Mann ± Whitney U test)
Figure 2 Erythrocyte catalase activity in mercury-exposed workers (n=27) and controls (&; n=11). In the exposed group, 16 workers were presently being exposed (~) (E) and 11 were on leave because of intoxication symptoms (&) (L). *P50.05 compared to workers on leave and controls (Mann ± Whitney U test)
enzyme activities and duration of exposure or creatine-corrected urinary mercury concentration.
Discussion
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and glutathione reductase. The authors suggested that the relative strengths of the effectiveness of the cellular antioxidant systems and oxygen radical formation are potential candidates in governing the ageing process and in determining red cell life span in mammalian species. In our study, despite the decreased levels of erythrocyte GSH, mercuryexposed workers have not presented anaemia or haemolysis. On the other hand, catalase levels were signi®cantly increased, supporting the hypothesis that the increased catalase activity probably offsets the decrease in GSH levels. In another study,24 female workers exposed to mercury vapours also presented increased catalase activity. Conversely to our ®ndings, however, in that study, catalase activity positively correlated with urinary mercury. We observed that, although there was a signi®cant difference in catalase levels between the group exposed and that on temporary leave, there was no correlation with mercury in urine. Supporting our results, Girardi and Elias25 showed, in the kidneys of rats receiving mercuric chloride treatment, an increase in catalase activity. Elemental mercury undergoes oxidation via the catalase-hydrogen peroxide pathway to mercuric ion, but the reverse process proceeds at a much slower rate.26 Thus, the increased catalase activity observed after exposure to mercury suggests a mechanism of protection provided by the involvement of the enzyme in the process of mercury biotransformation as well as detoxi®cation of the H2O2 generated by this metal.27,28 It has been demonstrated that the uptake of mercury by erythrocytes from acatalasemic humans was lower than that observed in erythrocytes from normal individuals.29 In this regard, Yamamoto et al30 reported an impaired in vitro accumulation of mercury in erythrocytes of acatalasemic mice and suggested that catalase takes up mercury in the red cells, thus protecting other organs from toxic effects of metallic mercury. Moreover, metallic mercury passes through the blood/placenta and placenta/ foetus barriers in acatalasemic mice more readily than in normal mice and the distribution of mercury is higher in the brain and liver and lower in blood and lungs of these animals when compared to controls.31 Glutathione and various components of its metabolic pathways react easily with many heavy metals, including mercury32 ± 34 and it is evident that glutathione metabolism is severely disturbed during mercury intoxication.35 ± 38 Our ®ndings of decreased GSH in erythrocytes of mercury-exposed workers are supported by the observation that inorganic mercury develops a complex with GSH. This complex may be involved in the renal uptake and toxicity for HgCl2.32,39 Depletion of GSH by Hg2+
has also been observed in rat kidney26,35,36 and supports an oxidative stress-like mechanism for Hg2+ toxicity. In vitro studies suggest that the depletion of GSH might be involved in an impaired detoxi®cation of H2O2 in red blood cells, since the use of H2O2 by GSH/GSH peroxidase system minimizes the formation of catalase H2O2 intermediate (compound-I) and thereby limits the capacity of the cells to oxidize mercury.40 It has also been demonstrated that mercury decreases the GSH concentration in other cells, including lymphocytes and monocytes. In addition, the sensitivity of these cells to the immunotoxic effects of mercuric compounds was related to intracellular GSH levels; cells with low levels of GSH were extremely sensitive to mercury.6,7 In vitro studies demonstrated that erythrocytes treated with heavy metals presented a decrease in G6PD and GSH-reductase activities.41 In addition, the G6PD activity was also reduced in placentas exposed to HgCl2.42,43 In our study, however, we have not found any alteration in the activity of the G6PD in the erythrocytes of mercury-exposed workers. The results of normal SOD activity observed in this study are corroborated by the ®ndings of Barregard et al44 who described a normal concentration of SOD in plasma and blood of mercury exposed-workers. On the other hand, Perrin-Nadif et al24 observed increased SOD activity in workers with higher urinary mercury concentrations. It seems, therefore, that the behaviour of the erythrocyte redox system in mercury-exposed workers is quite similar to that observed in other tissues. However, the modi®cations in this system might be reversible since, in the group of workers on temporary leave, the levels of GSH and catalase activity were normal. It is also possible that, the erythrocytes have a protective role on other tissues exposed to mercury. Adverse effects which may occur at relatively low levels of exposure to mercury are of particular interest in relation to preventive action, since these effects may provide critical information for determining, for example, exposure limits. Our results and those obtained by others24,45 ± 47 also indicate that the erythrocyte antioxidant system may provide a sensitive indicator of heavy metals adverse effects.
Acknowledgements This work was supported by grants from the FundacËaÄ o de Amparo aÁ Pesquisa do Estado de SaÄ o Paulo (FAPESP), and Conselho Nacional de Desenvolvimento Cientifico e Tecnolo gico (CNPq).
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