Introduction
Lead is a heavy metal, which has been widely used for decades in paint, gasoline, water pipes, storage batteries, and many other products (Lin et al.
2015). In the last decades, our view on lead toxicity has changed, giving more concern to exposures, previously considered safe. Lead can induce many adverse health effects on various body systems including the nervous, hematological, immune, and genitourinary systems. Nevertheless, due to its malleability, resistance to corrosion, and low melting point, lead is still widely used in many industries (Wang et al.
2012).
Ingestion and inhalation are the primary routes of lead entering the body. After absorption, lead is distributed through the bloodstream to various organs, such as brain, liver, and kidneys (Alya et al.
2015). It has been proposed that kidneys play an important role in the toxicokinetics of lead because they serve as the major organ responsible for its excretion. Therefore, kidneys are particularly exposed to lead toxicity. Lead primarily impairs the function and structure of the renal tubules. Renal tubular epithelial cell necrosis, leukocyte infiltration, and tubular epithelial cell pyknosis have been shown to be induced by lead toxicity. Many studies indicated that long‐term exposure to lead increases the risk of nephropathy, which manifests as increased levels of renal dysfunction biomarkers, such as plasma creatinine and uric acid (Alya et al.
2015; Liu et al.
2012).
Several mechanisms have been proposed to explain lead-induced toxicity. One of them implicated oxidative stress as the underlying mechanism of toxicity (Wang et al.
2012). Oxidative stress results from imbalance between the generation and utilization of reactive oxygen species (ROS). Lead ions have been shown to be associated with increased generation of ROS. Besides, lead is able to dysregulate the antioxidant defenses, including the antioxidant enzymes and the non-enzymatic antioxidants, such as uric acid. (Dobrakowski et al.
2014; Soliman et al.
2015; Wang et al.
2012). In our previous study, we reported higher levels of uric acid in workers chronically exposed to lead compounds (Blood lead (PbB) = 40.40 ± 10.05 µg/dl) for, on average, 16.40 ± 10.20 years compared to the unexposed control group (PbB = 6.39 ± 2.47 µg/dl). Besides, we reported increased levels of other non-enzymatic antioxidants, such as bilirubin, albumin, thiol groups, and α-tocopherol (Dobrakowski et al.
2014). However, the possible association between lead toxicity and the non-enzymatic antioxidant defenses is not fully understood and needs further investigation.
Negative effects of lead on human health may be also due to its impact on the immune system function. It has been shown that lead impairs the function of lymphocytes and cytokine production. Some studies indicate lead-induced shift in the balance of T-helper (Th) lymphocyte function toward Th2-mediated immune response at the expense of Th1-mediated response (García-Lestón et al.
2012; Hsiao et al.
2011). Besides, lead exerts pro-inflammatory properties. In experimental and human studies, lead exposure has been shown to induce expression of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) via activation of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB) (Liu et al.
2012). Both MAPKs and NF-κB can be induced synergistically by uric acid (Kanellis et al.
2003; Liang et al.
2015). Moreover, uric acid exerts its pro-inflammatory action via increasing the levels of chemokines, such as interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and RANTES (regulated on activation, normal T cell expressed and secreted) (Liang et al.
2015; Zhou et al.
2012). Similarly, lead has been shown to induce interleukin-8 (IL-8), a potent chemokine (Lin et al.
2015; Yang et al.
2014).
Up-to-date studies indicate lead-induced dysregulation of the immune response in humans. However, in the available literature, there are no conclusive epidemiological studies regarding the immunotoxic effects of lead in occupationally exposed workers. Similarly to mechanisms underlying the associations between lead and the non-enzymatic antioxidant defense, the exact mechanisms of lead interactions with the immune system in humans still remain unclear (García-Lestón et al.
2012). Some authors postulate that the influence of lead on the immune response is dose-dependent (Hsiao et al.
2011; Iavicoli et al.
2006). Duration of exposure seems to be the second factor modifying interactions of lead with the immune system. In light of this, we decided to examine workers subchronically and chronically exposed to lead. We focused on the influence of lead on the non-enzymatic antioxidant defenses, including uric acid, and chemokine levels, which may be related to the immunomodulatory properties of both lead and uric acid.
Results
Epidemiologic data and blood lead concentrations are presented in Tables
1 and
2.
Table 1
Epidemiologic data and lead exposure markers in the group of workers subchronically exposed to lead (n = 34)
Age (years) | 40 | 13 |
Exposure duration (days) | 40 | 3 |
Weight (kg) | 79.3 | 12.5 |
Height (cm) | 176 | 6.71 |
Smokers (%) | 68 % | – |
PbB before exposure to lead (µg/dl) | 10.7 | 7.83 |
PbB after exposure to lead (µg/dl) | 48.7 | 14.2 |
ZPP (μg/g Hb) | 2.66 | 0.64 |
Table 2
Epidemiologic data and lead exposure markers in the group of workers chronically exposed to lead and in the control group
Age (years) | 39 | 8 | 41 | 8 | 0.224 |
Years of work | 13 | 10 | 17 | 9 | 0.128 |
Height (cm) | 177 | 5.58 | 179 | 8.03 | 0.112 |
Weight (kg) | 84.2 | 14.1 | 88.9 | 12.2 | 0.176 |
Smokers (%) | 37 % | – | 32 % | – | 0.702* |
PbB (µg/dl) | 36.6 | 8.60 | 2.22 | 1.42 | <0.001 |
ZPP (μg/g Hb) | 4.24 | 1.82 | 2.38 | 0.61 | <0.001 |
The levels of uric acid and bilirubin were significantly higher after a subchronic exposure to lead compared to the baseline by 22 and 35 %, respectively. Similarly, the values of TAC, TOS, and OSI increased by 15, 50, and 33 %, respectively. At the same time, the levels of thiol group and albumin decreased by 5 and 8 %, respectively (Table
3). The levels of IL-8 and MIP-1β were significantly higher after a subchronic exposure to lead compared to the baseline by 34 and 20 %, respectively, while the levels of eotaxin, IP-10, MCP-1, and RANTES did not change after a subchronic exposure to lead (Table
4).
Table 3
Levels of uric acid, thiol groups, albumin, and bilirubin and values of total antioxidant capacity (TAC), total antioxidant status (TOS), and oxidative stress index (OSI) before and after subchronic exposure to lead, p value–t test for dependent variables, p* value–Wilcoxon’s test
Uric acid (mg/dl) | 6.23 | 1.00 | 7.57 | 1.88 | 22 | <0.001 |
Thiol groups (µmol/g P) | 3.89 | 0.45 | 3.68 | 0.64 | −5 | 0.022 |
Albumin (g/l) | 6.62 | 0.93 | 6.07 | 1.05 | −8 | 0.013 |
Bilirubin (mg/dl) | 0.45 | 0.24 | 0.60 | 0.51 | 35 | 0.027* |
TAC (mmol/l) | 0.76 | 0.10 | 0.87 | 0.14 | 15 | <0.001 |
TOS (µmol/l) | 9.99 | 7.20 | 14.9 | 10.6 | 50 | 0.008* |
OSI (%) | 1.35 | 0.98 | 1.79 | 1.32 | 33 | 0.022* |
Table 4
Levels of interleukin-8 (IL-8), eotaxin, interferon gamma-induced protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), and RANTES (regulated on activation, normal T cell expressed and secreted) before and after subchronic exposure to lead showed as a median and interquartile range (IQR), p value–Wilcoxon’s test
IL-8 (pg/ml) | 4.30 | 3.53 | 5.77 | 6.37 | 34 | 0.047 |
Eotaxin (pg/ml) | 93.0 | 87.1 | 106 | 66.9 | 14 | 0.437 |
IP-10 (pg/ml) | 751 | 713 | 1052 | 795 | 40 | 0.074 |
MCP-1 (pg/ml) | 33.5 | 29.9 | 27.7 | 38.7 | −17 | 0.248 |
MIP-1β (pg/ml) | 51.7 | 30.8 | 62.1 | 43.7 | 20 | 0.002 |
RANTES (pg/ml) | 21,431 | 3305 | 20,434 | 3309 | −5 | 0.174 |
The level of IL-8 was significantly higher in the group of workers chronically exposed to lead than in the control group by 40 %, while the level of IP-10 was significantly lower by 28 %. The levels of the remaining chemokines did not differ between both groups (Table
5).
Table 5
Levels of interleukin-8 (IL-8), eotaxin, interferon gamma-induced protein-10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), and RANTES (regulated on activation, normal T cell expressed and secreted) in the group of workers chronically exposed to lead and in the control group showed as a median and interquartile range (IQR), p value–Mann–Whitney U test
IL-8 (pg/ml) | 2.11 | 0.50 | 2.95 | 0.90 | 40 | <0.001 |
Eotaxin (pg/ml) | 95.3 | 54.6 | 119 | 76.7 | 25 | 0.083 |
IP-10 (pg/ml) | 1237 | 676 | 887 | 552 | −28 | 0.036 |
MCP-1 (pg/ml) | 14.6 | 9.01 | 15.3 | 14.2 | 5 | 0.178 |
MIP-1β (pg/ml) | 65.4 | 29.8 | 58.2 | 28.2 | −11 | 0.197 |
RANTES (pg/ml) | 21,607 | 1576 | 20,752 | 2059 | −4 | 0.109 |
Multiple regression analysis showed that the duration of lead exposure (subchronic vs. chronic) affects IP-10 and MIP-1β concentrations. Besides, the BMI, age, and smoking habits did not significantly affect the levels of those cytokines. In consistence with these results, comparisons made between the medians of the normalized cytokine levels in subchronically and chronically lead-exposed groups showed significant differences in the normalized values of IP-10, MCP-1, and MIP-1β levels between the two groups (Table
6).
Table 6
Normalized cytokine levels obtained from subchronically and chronically lead-exposed groups showed as a median and interquartile range (IQR), p value–Mann–Whitney U test
IL-8 (%) | 134 | 80–229 | 140 | 114–157 | 0.980 |
Eotaxin (%) | 114 | 66–138 | 125 | 87–168 | 0.180 |
IP-10 (%) | 140 | 83–189 | 72 | 55–100 | <0.001 |
MCP-1 (%) | 83 | 32–148 | 105 | 79–176 | 0.007 |
MIP-1β (%) | 120 | 96–181 | 89 | 70–114 | <0.001 |
RANTES (%) | 95 | 95–101 | 96 | 91–101 | 0.660 |
Discussion
Results of the present study showed that subchronic exposure to lead is able to increase uric acid level in the blood. However, increased uric acid level may be not only due to the subclinical renal function impairment but also due to altered purine metabolism. In our previous study, we showed a positive association between lead exposure and the activity of xanthine oxidase, which is responsible for uric acid formation from purines being degraded (Kasperczyk et al.
2013). In consistence with these results, positive correlations between blood lead levels and uric acid concentrations were observed by Hernández-Serrato et al. (
2006) in subjects environmentally exposed to high doses of lead and by Wang et al. (
2002) and Ehrlich et al. (
1998) in battery factory workers. Besides, Khan et al. (
2008) reported an increased uric acid level in a group of chronically lead-exposed workers (PbB = 29.1 μg/dl). Similarly, a study on workers chronically exposed to high doses of lead reported same results (PbB = 80.9 μg/dl). However, there are human studies showing no association between lead exposure and uric acid levels (Konishi et al.
1994; Roels et al.
1994).
Uric acid acts as a scavenger of ROS and serves as a main antioxidant in human plasma (Gersch et al.
2008). Therefore, the elevation of uric acid level may compensate for the simultaneous decrease in thiol group level. As a result, the TAC value was paradoxically increased due to subchronic lead exposure. However, uric acid has also been shown to act as a pro-oxidant. Uric acid is able to react directly with nitric oxide (NO) to form 6-aminouracil. This irreversible reaction, resulting in depletion of NO, can be partially blocked by glutathione (GSH). Therefore, under oxidative stress conditions, when the GSH pool is depleted, uric acid may induce endothelial dysfunction (Gersch et al.
2008; Xie et al.
2015). It is well documented that lead exposure induces GSH depletion (Kasperczyk et al.
2014). In light of this, the role of uric acid in the defense against lead toxicity can be divergent and may depend on the specific redox conditions of the particular microenvironment. What makes this issue more complicated is that uric acid also exerts pro-inflammatory properties (Hayden and Tyagi
2004).
In in vitro studies, both lead and uric acid have been reported to induce the expression of IL-8 via MAPKs and NF-κB signaling pathways (Lin et al.
2015; Liang et al.
2015). IL-8 is secreted by multiple cell types in response to pro-inflammatory stimuli and serves as a strong chemotactic agent for neutrophils. Neutrophils may cause oxidative damage to tissues. Consequently, IL-8 may be involved in many inflammatory diseases, such as rheumatoid arthritis, gouty arthritis, asthma, and acute respiratory distress syndrome. Besides, experimental studies showed that IL-8 plays a role in promotion of angiogenesis and metastasis (Lin et al.
2015; Yan et al.
2015). Since this study showed elevated level of IL-8 due to both subchronic and chronic lead exposure, then IL-8 may also display negative effects in humans exposed to lead toxicity.
Similarly, MCP-1 expression and level have been shown to be increased by lead and uric acid through MAPKs and NF-κB signaling pathways in in vitro and experimental studies (Kanellis et al.
2003; Kumawat et al.
2014; Liang et al.
2015; Soliman et al.
2015; Zhou et al.
2012). In addition, uric acid has been reported to increase the expression and levels of RANTES in vitro (Zhou et al.
2012). MCP-1 is released due to pro-inflammatory stimuli and displays a chemoattractive activity on monocytes, basophils, and lymphocytes, thus plays a role in the allergic reactions (Luster and Rothenberg
1997). RANTES also belongs to the chemokine family and is responsible for recruiting a variety of leukocytes into the inflammation sites, such as lymphocytes, macrophages, eosinophils, and basophils (Aldinucci and Colombatti
2014). Levels of both MCP-1 and RANTES were not significantly affected by subchronic and chronic exposure to lead. However, a comparison made between the normalized values of MCP-1 in subchronically and chronically lead-exposed workers showed that the influence of lead on MCP-1 level might be opposite depending on the exposure period.
As in the case of MCP-1 and RANTES, the present study did not confirm the influence of lead exposure on eotaxin level. Eotaxin acts as a chemokine-stimulating eosinophils chemotaxis. Besides, eotaxin induces the release of eosinophils from the bone marrow, their aggregation, and their respiratory burst activity (Rankin et al.
2000).
Results of the present study indicated that a subchronic exposure to lead, apart from increasing the level of IL-8, might induce inflammation via increasing the level of MIP-1β, a member of the MIP-1 family, which orchestrates acute and chronic inflammatory host responses by recruiting pro-inflammatory cells, especially lymphocytes and monocytes (Maurer and von Stebut
2004). Chronic exposure to lead did not significantly affect the level of MIP-1β but influenced the level of IP-10, which serves as a chemokine as well. The level of IP-10 was significantly lower in chronically lead-exposed workers than in the control group. The secretion of IP-10 by lymphocytes depends on IFN-γ level and is related to the Th1-mediated immune response (Antonelli et al.
2014). Therefore, a decrease in IP-10 level due to chronic lead exposure may be caused by lead-induced skewing toward the Th2-mediated immune response as postulated in some experimental studies (Heo et al.
2007;
1996; Hsiao et al.
2011). Besides, it has been postulated that uric acid is also able to trigger the Th2-mediated immune response (Moon et al.
2010). Additionally, multiple regression analysis and comparisons made between the normalized values of IP-10 and MIP-1β in subchronically and chronically lead-exposed workers confirmed that the influence of lead on their levels is divergent in those two different types of exposures. Iavicoli et al. (
2006) showed that the effect of lead exposure on cytokine levels in mice depends on the blood lead level. However, results of the present study indicate that lead may affect cytokine levels in different ways depending on the exposure duration rather than blood lead level.
In our previous study on chronically lead-exposed workers, we reported not only a higher level of uric acid but also a higher bilirubin level. In addition, the present study showed an increased level of bilirubin due to subchronic exposure to lead. Bilirubin is the end product of heme degradation. Heme is converted by heme oxygenase (HO) to biliverdin, which is in turn reduced to bilirubin by biliverdin reductase. Bilirubin has been shown to have a strong antioxidant potential against peroxyl radicals; however, it could also exert toxic effects when present in excess (Fuhua et al.
2012; Annabi Berrahal et al.
2007). Several animal studies on rats showed elevated bilirubin levels as a result of lead exposure (Abdel-Moneim et al.
2011; Annabi Berrahal et al.
2007; Ibrahim et al.
2011). Such elevation of bilirubin level may be beneficial owing to its antioxidant properties. In accordance with this, Noriega et al. (
2003) showed that bilirubin administration to rats increased GSH level, enhanced the activity of antioxidant enzymes, and decreased the toxicity induced by δ-aminolevulinic acid (ALA). ALA accumulates because of the lead-induced inhibition of δ-aminolevulinic acid dehydratase (ALAD) (Wang et al.
2015). Therefore, high bilirubin level may contribute to the elevation of the TAC value. On the other hand, results of human studies on the role of bilirubin in lead toxicity are not as conclusive as those of the experimental studies. Al-Neamy et al. (
2001) and Khan et al. (
2008) did not report any significant association between bilirubin level and chronic lead exposure in male workers. The negative results of these studies may be due to the complexity of the possible interactions between lead and heme metabolism. On the one hand, it is well documented that lead inhibits heme biosynthesis (Dobrakowski et al.
2014). Consequently, the depletion of the heme pool may result in its decreased degradation and less bilirubin synthesis. On the other hand, lead may induce heme degradation via induction of the inducible isoform of heme oxygenase (HO-1) (Vargas et al.
2003). Lead may also increase heme degradation via induction of eryptosis (Aguilar-Dorado et al.
2014).
In contrast to uric acid and bilirubin levels, the level of albumin significantly decreased after a subchronic exposure to lead compared to the baseline. Similarly, in our previous study, we reported lower albumin level in chronically lead-exposed workers when compared to the control group. In consistence with our results, Khan et al. (
2008) reported decreased serum albumin and total protein levels in lead-exposed industrial workers. Koo et al. (
1994) reported decreased albumin mRNA level in rat liver due to lead nitrate administration. This experimental study further supports the results of the human studies.
Albumin is the most abundant plasma protein, which serves to buffer the blood, maintain the osmotic pressure, and as a carrier of many compounds (Guo et al.
2014). Because of their ROS scavenging activity, thiol groups of cysteine residues of albumin determine the plasma redox status (Dobrakowski et al.
2014). Therefore, decreased level of thiol groups, observed in the present study may be secondary to the decrease in albumin level. The second possible explanation for the reduced thiol group level is the well-documented high affinity of lead toward thiol groups (Dobrakowski et al.
2014). In accordance with the human studies, decreased level of thiol groups was also reported in experimental studies in rats (El-Missiry
2000; Tandon et al.
2002). Our previous study on chronically lead-exposed workers also showed decreased thiol group level (Dobrakowski et al.
2014). Interactions between lead and thiol groups may also influence the immune response because it has been proposed that lead may affect lymphocyte functions due to its high affinity for the sulfhydryl groups on T-lymphocyte surface receptors. As a result, antigen processing from monocytes to T lymphocytes may be impaired (García-Lestón et al.
2012).
The development of oxidative stress in chronic lead exposure is well established (Kasperczyk et al.
2014). The decrease in thiol group level and the elevation of TOS and OSI values observed in the present study confirm that a subchronic exposure to lead is also able to induce oxidative stress despite increased TAC value.
The results of this study need to be evaluated within the context of its limitations. A major limitation was a limited groups’ size. Besides, the possible confounding role of other pollutants was not taken into consideration.