Article
The effect of occupational lead exposure on lipid peroxidation, protein carbonylation, and plasma viscosity
Toxicology and Industrial Health 1–7 © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713491804 tih.sagepub.com
_ ´ ska2, Sławomir Kasperczyk1, Ludmiła Słowin´ska-Łozyn Aleksandra Kasperczyk1, Tomasz Wielkoszyn´ski3 and Ewa Birkner1 Abstract The aim of the study was to investigate the influence of occupational lead (Pb) exposure on lipid peroxidation, protein carbonylation, and plasma viscosity in workers. The examined group included 283 healthy male employees of manufacturing facilities using zinc and Pb. The mean blood concentrations of Pb and zinc protoporphyrin as well as the mean urine d-aminolevulinic acid levels were used as markers of exposure for the examined group. Taking into account the obtained mean values of blood lead level, the examined group was divided into three subgroups. When comparing the control group with the subgroups, Pb exposure markers were significantly elevated in all the three subgroups. Concentrations of conjugated dienes (CD), lipid hydroperoxides, malondialdehyde (MDA), and protein carbonyl groups were also significantly increased. Conversely, the levels of total protein and protein sulfhydryls were significantly decreased in the subgroups compared with the controls. The plasma viscosity was significantly elevated in the subgroups. A dose–response between Pb levels and plasma viscosity was not observed. Pb supposedly elevates MDA and CD in a dosedependent manner. In conclusion, occupational Pb exposure induces oxidative stress that results in lipid and protein damage. Moreover, Pb-induced oxidative stress is likely the primary factor that elevates plasma viscosity, despite decreased protein levels. Keywords Lead poisoning, oxidative stress, lipid peroxidation, protein carbonylation, plasma viscosity
Introduction There are many sources of lead (Pb) exposure, such as mining, smelting, refining, painting, ceramic glazing, or battery manufacturing (Patocka and Cerny´, 2003). As a result, Pb is a ubiquitous toxicant that induces many physiological, biochemical, and behavioral dysfunctions (Hamed et al., 2010; Malekirad et al., 2010; Tousson et al., 2011). Long-term accumulation of Pb causes anemia, nephropathy, liver dysfunction, encephalopathy, hypertension, coronary disease, deafness, and reduced sperm count (Khan et al., 2008; Mudipalli, 2007). The best-known toxic effect of Pb is the inhibition of d-aminolevulinic acid (ALA) dehydratase and ferrochelatase, which results in decreased bioavailability of heme and d-ALA accumulation. Moreover, Pb
affects adenosine triphosphatases (ATPases), sodium– potassium–ATP pumps, and mitochondrial oxidative phosphorylation; and Pb interacts with divalent essential metals, such as calcium, iron, and zinc. The toxicity of Pb is also related to its affinity for electron-donor
1
Department of Biochemistry, Medical University of Silesia in Katowice, Jordana, Zabrze, Poland 2 Department of Biophysics, Medical University of Silesia, Jordana, Zabrze, Poland 3 Department of Chemistry, Medical University of Silesia in Katowice, Jordana, Zabrze, Poland Corresponding author: Aleksandra Kasperczyk, Department of Biochemistry, Medical University of Silesia, Jordana 19, Zabrze 41-808, Poland. Email:
[email protected]
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Toxicology and Industrial Health
groups, such as sulfhydryl groups (SH-Gs) of glutathione or proteins. By binding to these groups, Pb disturbs numerous enzymatic processes (Mudipalli, 2007; Patocka and Cerny´, 2003). Many studies have reported that Pb-induced pathologies can be attributed to oxidative stress. Evidence indicates that many mechanisms are involved (Gurer and Ercal, 2000). First, Pb promotes the production of reactive oxygen species (ROS), enhancing the levels of hydrogen peroxide as well as hydroxyl and superoxide radicals (Khan et al., 2008). ALA that accumulates during plumbism undergoes auto-oxidation and is believed to be a source of ROS. Additionally, Pb weakens the antioxidant defense. Pb not only depletes glutathione content but also interferes with antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase (Gurer and Ercal, 2000). ROS attack all cellular structures. The polyunsaturated fatty acid residues of phospholipids undergo an intensified process of lipid peroxidation (LPO), which results in the loss of cellular and mitochondrial membrane integrity (Jackie et al., 2011; Khan et al., 2008). LPO is associated with the formation of conjugated dienes (CD), lipid hydroperoxides (LHP), and degradation products (Moore and Roberts, 1998), such as alkanals, hydroxyl alkanals, ketones, and alkenes (Lodi et al., 2011), including malondialdehyde (MDA) (Khan et al., 2008). Nevertheless, protein modifications caused by ROS generate protein carbonyl (PC) derivatives, which are formed when the lysine, arginine, proline, and histidine side chains of proteins are oxidized (Haleagrahara et al., 2010; Jain, 1985). Oxidized proteins and lipid clusters may form crosslinked aggregates (Jung et al., 2007). Plasma viscosity depends on its macromolecular content, particularly plasma proteins (Dikmenog˘lu et al., 2006). Because products of LPO cross-link or induce secondary oxidative damage to plasma proteins, Pb-induced oxidative stress may result in plasma viscosity changes. This possibility inspired us to investigate the influence of Pb on LPO, protein carbonylation, and plasma viscosity in exposed workers. This is the first study to explore the associations between plasma viscosity and Pb exposure in a significant human population.
Methods Study subjects The examined group consisted of 283 male employees of manufacturing facilities using zinc and Pb, localized in
the southern region of Poland. The mean age of the men in the study was 41.0 + 9.23 years. The workers had an average of 16.6 years of exposure to Pb. Only clinically healthy men were included in the investigation. Blood Pb levels (PbB) and zinc protoporphyrin (ZPP) as well as urine ALA levels were used as markers of Pb exposure. These markers were measured, on average, every 3 months during 2 years of observation. Taking into account the obtained mean values of PbB (PbBmean), the examined group was divided into three subgroups based on their exposure to Pb: low exposure (LE), medium exposure (ME), and high exposure (HE). The LE group consisted of 79 workers with PbBmean < 35 mg dl1. The ME group included 121 workers with PbBmean from 35 to 45 mg dl1, and the HE group consisted of 83 workers with PbBmean > 45 mg dl1. In the last blood samples that were collected, the levels of PbB and ZPP were measured. Serum concentrations of total protein, CD, LHP, MDA, PC groups, and protein SH-G (PSH-G) were also determined, along with the plasma viscosity. The control group included 73 male administrative workers with a mean age of 41.5 + 9.23 years and PbBmean of 6.45 + 2.49 mg dl1. None of the individuals from this group had a history of occupational Pb exposure. Workers suffering from any chronic disease were excluded.
Sampling and laboratory procedures By venipuncture, a volume of 15 ml blood was placed into tubes with ethylenediaminetetraacetic disodium acid solution as an anticoagulant to obtain plasma and erythrocytes, while another 10 ml blood was collected into plain tubes to obtain serum. Whole blood was used for the analysis of the levels of PbB and ZPP. The analysis of PbB was performed using graphite furnace atomic absorption spectrophotometry. Unicam 929 and 939OZ atomic absorption spectrometers with GF90 and GF90Z graphite furnaces were used (Unicam, United Kingdom). The data are expressed in micrograms per deciliter. The concentration of ZPP in the blood was measured directly by means of the Aviv Biomedical hematofluorometer, model 206 (Aviv Biomedical, USA). The instrument measures the ratio of ZPP (as a fluorescent substance) to the absorption of light in the sample (hemoglobin (Hb)). The results are expressed in micrograms of ZPP per gram of Hb. The urine levels of d-ALA were measured by the method of Grabecki et al. (1967) and are expressed in milligrams per liter.
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Kasperczyk et al.
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Plasma was separated by centrifugation. The plasma viscosity was measured using a Brookfield DV-IIþ cone-plate viscometer (Brookfield Engineering Laboratories, USA) at a stable temperature of 37 C (900s1). The amount of protein in the serum was measured by the Biuret method. Concentrations of CD were measured according to Corongiu et al. (1989) using a spectrophotometer (Model A160, Shimadzu, Japan). The method of So¨dergren et al. (1998) was used to measure the concentrations of LHPs, while the method of Reznick and Packer (1994) was used to identify the PC groups. A Shimadzu UV-1700 spectrophotometer was used in both methods. The levels of MDA were determined by Ohkawa et al. (1979) using a spectrofluorometer Perkin Elmer LS45 (Perkin Elmer, USA). The levels of PSH-G were measured using a method developed by Koster et al. (1986). The concentrations of CD, LHP, PC, MDA, and PSH-G are expressed in micromoles per liter.
Statistical analysis Statistical analysis was performed using Statistica 9.1 PL software (StatSoft, Cracow, Poland). Calculations for statistical analysis included the mean and SD. Using Shapiro–Wilk’s test, the normal distribution of the data was verified, while using Levene’s test, homogeneity of variances was verified. An analysis of variance (ANOVA) or Kruskal–Wallis ANOVA test was used for multiple comparisons of data. Statistical comparisons with the control group were performed using Student’s t test, t test with separate variance estimates, or Mann–Whitney U test. The analysis between the groups (LE, ME, and HE) was made by post hoc analysis (Tukey’s test and a nonparametric post hoc test for multiple comparisons). For quality variables (smoking habits), w2 test was used. A Spearman nonparametric correlation was also calculated. An a value of p < 0.05 was considered to be statistically significant.
Results The mean age, weight, and smoking habits were not significantly different in the subgroups compared with the controls (Table 1). When comparing the control group with the subgroups, the PbBmean levels were significantly elevated, over 4-fold in the LE group, over 6-fold in the ME group, and over 7-fold in the HE group. Similarly, the ZPPmean and ALAmean levels, respectively, were
elevated by 112% and 50% in the LE group, by 284% and 67% in the ME group, and by 329% and 101% in the HE group. Compared with the control group, the total protein was significantly decreased by 8% in the LE group, by 7% in the ME group, and by 9% in the HE group. Similarly, the concentrations of PSH in the subgroups decreased by 17%, 11%, and 22% in the LE, ME, and HE groups, respectively. Compared with the controls, the levels of CD, LHP, MDA, and PC, respectively, were significantly increased by 21%, 352%, 13%, and 112% in the LE group, by 25%, 471%, 36%, and 94% in the ME group, and by 27%, 370%, 41%, and 97% in the HE group. The plasma viscosity was elevated by 18% in the LE and ME groups and by 19% in the HE group. The Spearman correlation indicated that there is a positive correlation among the PbB, ZPP, ALA, and plasma viscosity levels as well as the concentrations of LHP, MDA, CD, and PC groups (Table 2). Moreover, the markers of Pb exposure correlate negatively with PSH-G levels. However, the plasma viscosity correlates positively with the levels of LHP, MDA, CD, and PC, whereas it is negatively correlated with PSH concentrations.
Discussion The mean serum total protein concentration was slightly but significantly decreased in the examined population. This finding may be due to the subclinical liver dysfunction as Pb has been indicated as a hepatotoxicant (Mudipalli, 2007). Decreased serum protein levels were reported by Mikhail et al. (1980), who examined Pb tank welders (PbB ¼ 42.19 + 9.81 mg dl1). Khan et al. (2008) also reported decreased serum protein levels in Pb-exposed workers, whereas Al-Neamy et al. (2001) reported no change in this parameter in workers exposed to high doses of Pb (PbB ¼ 77.5 + 42.8 mg dl1). Markers of oxidative damage to lipids and proteins, such as CD, LHP, MDA, and PC, were elevated in the current study. Increases in MDA levels in the ME and HE groups were over 2.75-fold and 3-fold greater than in the LE group. A similar trend was observed in the CD levels. A positive correlation between PbB levels and MDA and CD confirms this relationship. This result suggests that Pb acts in a dose-dependent manner. Recent reports support our findings. In a recent study that examined Pb-exposed workers (PbB ¼ 54.6 + 17 mg dl1), Gurer-Orhan et al. (2004) indicated that MDA levels were elevated in erythrocytes.
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9.23
10.41 2.49 2.47 0.47 0.51 0.91 0.91 5.56 104 23.8 1.60 0.57 12.0 0.07
41.5
47% 80.9 6.45 6.39 1.93 1.96 2.32 2.26 78.1 512 119 3.62 2.90 36.4 1.22
SD 39.3 15.7 48% 80.3 28.9 30.4 3.62 4.14 3.67 3.39 72.2 425 144 16.4 3.27 77.1 1.44
Mean
12.12 4.39 7.82 2.32 2.35 1.43 1.37 4.73 156 33.3 9.00 1.28 41.4 0.11
9.98 10.55
SD
LE group
0.850 0.740
