or cadmium in blood, liver and kidneys

Accepted Manuscript Title: Insight into the oxidative stress induced by lead and/or cadmium in blood, liver and kidneys Author: Vesna Matović, Aleksandra Buha, Danijela Ðukić-Ćosić, Zorica Bulat PII: DOI: Reference:

S0278-6915(15)00057-5 http://dx.doi.org/doi:10.1016/j.fct.2015.02.011 FCT 8221

To appear in:

Food and Chemical Toxicology

Received date: Accepted date:

22-12-2014 6-2-2015

Please cite this article as: Vesna Matović, Aleksandra Buha, Danijela Ðukić-Ćosić, Zorica Bulat, Insight into the oxidative stress induced by lead and/or cadmium in blood, liver and kidneys, Food and Chemical Toxicology (2015), http://dx.doi.org/doi:10.1016/j.fct.2015.02.011. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Insight into the oxidative stress induced by lead and/or cadmium in blood, liver and kidneys

Vesna Matović, Aleksandra Buha, Danijela Đukić-Ćosić, Zorica Bulat Department of Toxicology “Akademik Danilo Soldatović”, University of Belgrade-Faculty of Pharmacy, Vojvode Stepe 450, 11000 Belgrade, Serbia

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Corresponding author: Vesna Matović Vojvode Stepe 450 11000 Belgrade Serbia Phone number: +381113951251 Fax:+381113972840 e-mail: [email protected] 1

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Abbreviations: lead (Pb), cadmium (Cd), sulfhydryl groups (–SH), reactive oxygen species (ROS), superoxide -. . anion (O2 ), hydrogen peroxide (H2O2), hydroxyl radicals (OH ), reactive nitrogen species (RNS), nitric oxide (NO), δ-aminolevulinic acid dehydratase (ALAD), δ-aminolevulinic acid (ALA), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glucose-6-phosphate dehydrogenase (G6PD), zinc (Zn), magnesium (Mg), selenium (Se), glutathione (GSH), glutathione reductase (GR), glutathione-S-transferase (GST), metallothioneins (MT), malondialdehyde (MDA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), intraperitoneal (i.p.), 8-hydroxy-2'-deoxyguanosine (8OHdG), mitogen-activated protein kinases (MAPKs), thiobarbituric acid reactive substances (TBARS), inducible NO synthase (iNOS)

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Highlights

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Results on parameters of oxidative stress in blood, liver and kidney in Pb and/or Cd intoxication were presented.

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Both Pb and Cd induce generation of reactive species and depletion of antioxidant defense system.

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Pb and Cd mixture also causes oxidative stress induction indicating both antagonism and synergism between these two metals.

Abstract

Besides being important occupational hazards, lead and cadmium are nowadays metals of great environmental concern. Both metals, without any physiological functions, can induce serious adverse health effects in various organs and tissues. Although Pb and Cd are non-redox metals, one of the important mechanisms underlying their toxicity is oxidative stress induction as a result of the generation of reactive species and/or depletion of the antioxidant defense system. Considering that the co-exposure to both metals is a much more realistic scenario, the effects of these metals on oxidative status when simultaneously present in the organism have become one of the contemporary issues in toxicology. This paper reviews short and long term studies conducted on Pb or Cd-induced oxidative stress in blood, liver and kidneys as the most prominent target organs of the toxicity of these metals and proposes the possible molecular mechanisms of the observed effects. The review is also focused on the results obtained for the effects of the combined treatment with Pb and Cd on oxidative status in target organs and on the mechanisms of their possible interactions. Keywords: lead, cadmium, mixture, intoxication, oxidative stress

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1. Introduction

Lead (Pb) and cadmium (Cd) are toxic metals of great occupational importance, but are nowadays even more significant as environmental pollutants. Both Pb and Cd can seriously affect organs and various systems of an organism and can cause severe acute and especially chronic intoxications. Current European Cd intake is close to the tolerable weekly intake with recent epidemiological evidence showing that environmental exposure to Cd increases total mortality (EFSA, 2011; Nawrot et al., 2010), while the quantity of Pb used in the 20th century, even though the use of Pb has been restricted in many different fields of its applications, exceeds by far the total consumption in all previous years (Hsu et al., 2002). Lead poisoning has been known since ancient times and the problem of intoxication with Pb became an important issue in the 18th century, during the industrial revolution when this metal was one of the most widely used industrial metals due to its qualities. Many prevention measures against Pb exposure have been accepted all over the world ever since: lead gasoline was banned as well as lead paints and lead pigments, resulting in diminished lead concentrations in the blood of the general population in Europe and other countries. However, the main anthropogenic sources of Pb remain, such as mining, smelting, lead batteries, crystal and ceramic industry, which undoubtedly contribute to the Pb-induced adverse effects in humans and the environment. Lead toxicity deserves special attention in the light of the fact that children are extremely vulnerable to this toxic metal. On the other hand, Cd was discovered in 1817 and was recognized in the 20th century as a toxic metal that can induce severe intoxications, not only in persons occupationally exposed (smelting, electroplating, production of nickel-cadmium batteries, fertilizers), but also in the general population through food and cigarette smoking. A well-known example of Cd-induced intoxications with dramatic outcomes was documented in Japan as Itai-itai disease (Nordberg, 2009). Lead is known to induce a broad range of physiological, biochemical and behavioral dysfunctions in laboratory animals and humans, including affecting the central and peripheral nervous system, hematopoietic system, cardiovascular system, kidneys, liver and reproductive system (ATSDR, 2007). On the other hand, the target organ of Cd chronic toxicity is the kidney, in which Cd has an estimated half-life of 30 years. Studies of populations chronically exposed to low doses of Cd have reported a range of other adverse health outcomes including hypertension, type 2 diabetes mellitus, and cancer (Colacino et al., 2014). Animal studies confirmed Cd adverse effects on liver, kidneys, lungs, pancreas, bones, reproductive organs, hematopoietic, nervous and cardiovascular system as reviewed by Matović et al. (2011). Possible endocrine disruption caused by

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Cd because of its estrogenic activity (Silva et al., 2012) and effects on thyroid function (Buha et al., 2013) have been shown. Spleen, as a target organ of Cd toxicity and immunomodulatory effects of Cd, has been recently shown by Demenesku et al. (2014). Cd and Cd compounds are carcinogenic to humans (IARC 1993, 2012). Cd and Pb toxicity have been comprehensively explored in many in vitro and in vivo studies and various molecular, cellular and intracellular mechanisms were proposed to explain toxicological profiles of these two toxic metals. Although the pathogenesis of deleterious health effects from Pb and Cd exposure is multifactorial, the mechanisms underlying their toxicity are not completely understood. Among the confirmed mechanisms for both Pb and Cd toxicity is their binding to sulfhydryl (–SH) groups thus affecting many enzymes and other –SH containing molecules. The other is their interaction with bioelements in the organism thus affecting directly and indirectly many physiological and biochemical processes. Recent investigations indicate their influence on necrosis and apoptosis, on gene expression, damaged DNA repair, etc. Complete understanding of these mechanisms is still far from being achieved and this topic remains controversial and incomplete, but up to date investigations indicate oxidative stress as an important molecular mechanism for Pb and Cd toxicity. Many findings on the oxidative damage to various biological macromolecules caused by exposure to Pb or Cd suggest that even though these metals are non-redox, they can cause disturbances in oxidative status that can significantly contribute to many adverse effects of these two toxic metals. Lipid peroxidation in red blood cell membranes as a consequence of Pb-induced oxidative stress leads to hemolysis and contributes to Pb-induced anemia (Flora et al., 2012). Studies conducted on three different animal species with different dose levels of Cd and via different routes of exposure provide a substantial body of evidence that confirms oxidative stress as one of the important mechanisms of Cd toxicity, with liver as a critical target organ of acute exposure and kidneys as critical target organ of prolonged exposure to Cd (Matović et al., 2013). Free radical-induced damage caused by Pb and Cd are accomplished by two independent, but related mechanisms (Jomova and Valko, 2011). The first mechanism involves generation of reactive oxygen species (ROS), i.e. superoxide anion (O2-.), hydrogen peroxide (H2O2), hydroxyl radicals (OH.) and reactive nitrogen species (RNS), i.e. nitric oxide (NO), while the second mechanism is achieved via depletion of the cellular antioxidant pool. Undoubtedly, these two mechanisms are simultaneous and interrelated, since increase in ROS and RNS leads to depletion of the antioxidant pool while at the same time depletion of this pool leads to the increase of reactive species. Although non-transition metals, Pb and Cd were shown to be able to induce ROS production. One of the

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mechanisms involved in the early stage of Pb-induced oxidative stress is the inhibition of δaminolevulinic acid dehydratase (ALAD), an important enzyme in heme biosynthesis, which leads to the accumulation of δ-aminolevulinic acid (ALA) that induces generation of ROS (Bechara, 1996). Concerning Cd, it has been confirmed that this toxic metal can replace Fe in various cytoplasmic and membrane proteins, such as ferritin and apoferritin, hence increasing the amount of freely available Fe ions that participate in Fenton reactions and generate ROS (Wätjen and Beyersmann, 2004). It is also known that Cd accelerates free radical formation by increasing intracellular calcium levels (Thévenod, 2009). Another mechanism for Pb- and Cd-induced oxidative stress is their effect on the antioxidative defense system of cells. Pb and Cd have high affinity for –SH groups in enzymes of the antioxidative defense system, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD), and subsequently inhibit their activity (Jomova and Valko, 2011; Kasperczyk et al., 2012; Nair et al., 2013). Apart from targeting –SH groups, Pb and Cd, as divalent cations, can also replace divalent bioelements that serve as important co-factors of antioxidant enzymes such as GPx, SOD and CAT, resulting in their inactivation. This is in agreement with the generally proved antagonism between Pb and Cd on one side and bioelements, i.e zinc (Zn), magnesium (Mg), selenium (Se), etc., on the other (Bulat et al., 2008, 2009; Djukic-Cosic et al., 2006; Gałażyn-Sidorczuk et al., 2012; Matović et al., 2012; Othman and El-Missiry, 1998; Soldatovic et al., 2002). It has been also confirmed that both metals affect levels of glutathione (GSH), a tripeptide that contains more that 90% of the non-tissue sulphur in the human body and represents one of the most important components of antioxidant non-enzymatic protection. Generally speaking, both metals strongly bind to –SH groups and initially deplete GSH stores. Moreover, these toxic metals also inhibit enzymes glutathione reductase (GR), GPx and glutathioneS-transferase (GST) that are important for maintenance of GSH levels (Ahamed and Siddiqui, 2007; Badisa et al., 2007; Cuypers et al., 2010, Flora et al., 2012). Toxicity of Pb and especially Cd is dependent on metallothioneins (MT), –SH rich proteins of low molecular weights with the capacity to bind metals and protect organisms against metal toxicity. Metallotioneins are mildly inducible by Pb, and to a much greater extend by Cd (Gonick, 2011). The Cd-MT complex is mainly formed in the liver then slowly released into the circulation and later delivered to the kidneys; the retention of Cd in both organs is MT-dependent. These proteins detoxify Cd by sequestering it into inert Cd-MT complex thus preventing Cd reactions with target molecules (Klaassen et al. 2009). The processes that are thought to produce oxidative stress triggered by Pb and Cd are summarized in Figure 1. Over the last decades, numerous investigations dealt with various aspects of Pb and Cd toxicity. However it is rather difficult to explain how these two metals with rather similar chemical

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properties and with proposed rather similar mechanisms of oxidative stress induction, produce quite different and specific effects in the organism. With regard to the fact that thousands of compounds are present in the environment, from natural or anthropogenic sources, human exposure to toxic agents cannot be characterized as exposure to a single agent, but more correctly as an exposure to the mixtures of these toxic agents (CDC, 2009). Due to their non-biodegradability, Pb and Cd accumulate in ecosystems and have been identified as leading constituents at various waste sites (Fay and Mumtaz, 1996). In addition to environmental exposure, individuals can be simultaneously exposed to these metals in the workplace. Even though exposure to high doses of these metals seldom happens, chronic low coexposure can still be regarded as a major health concern and global issue (Wang and Fowler, 2008). Concurrent exposure to these metals may produce additive effects or synergistic/antagonistic interactions or even produce completely new effects which are not seen with exposure to only one of the metals. Evaluation of these interactions, especially at the level of several common mechanisms underlying their toxicity, such as oxidative stress induction, is essential for risk assessment of their co-exposure and subsequent mitigation of assessed health risk. Hence, this review will summarize studies conducted on Pb and Cd-induced oxidative stress in blood, liver and kidneys as the most prominent target organs of their toxicity and also will be focused on the effects of their co-exposure on oxidative status in these organs.

2. Lead

After absorption, Pb enters blood and then accumulates in erythrocytes, while in plasma it is present in concentrations less than 1%. This high Pb percentage in erythrocytes can be explained by high Pb affinity for ALAD (Kelada et al., 2001) since Pb has a 20 times higher affinity for this protein than Zn; consequently it can replace Zn in ALAD and thus inhibit its function. Even though ALAD binds around 80% of erythrocyte Pb, its quantities are limited and if the Pb levels in erythrocytes continues to grow, Pb binds to the pyrimidine 5’-nucleotidase, another structure with high affinity for lead (Bergdahl et al., 1998). Unlike the majority of toxic agents, in the case of Pb, blood represents not only the way of Pb transportation, but also the critical target for its toxicity. Pb distributes to many organs, preferentially to liver and kidneys, and accumulates in bones accounting for more than 90% of the body burden. Hence, blood, liver and kidneys will be thoroughly discussed, in this review, in

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terms of Pb ability to change oxidative status in them and cause various toxic effects mediated through oxidative stress induction.

2.1 Effects on oxidative status in blood

The ability of Pb to cause oxidative stress and to influence various enzymatic and nonenzymatic components of antioxidant defense has been shown in many epidemiological and animal studies. There are several mechanisms that could be responsible for Pb ability to cause oxidative stress in blood. Pb, as many other toxic metals, binds to functional –SH groups of enzymes, ALAD and GR being important ones, and subsequently renders them nonfunctional and depresses their activity (Ahamed et al., 2005; Gurer-Orhan et al., 2004). Inhibition of ALAD increases the levels of its substrate ALA which generates the production of H2O2, O2-. and OH. (Bechara, 1996), while GR inhibition leads to concomitant decrease in GSH levels. Enhanced production of ROS in Pb intoxication may be also induced by Pb interactions with oxyhemoglobin that undergoes autooxidation (Gurer and Ercal, 2000). Lead has also been shown to elevate or suppress blood levels of the antioxidant enzymes SOD and CAT depending mainly on the level and duration of exposure to this toxic metal. Higher levels of exposure to Pb can cause increases in the activity of these enzymes as a consequence of elevated ROS production, while their suppression can be explained by the ability of Pb to replace bioelements that serve as important co-factors of these enzymes (Flora et al., 2012). In a study on lead-exposed populations employed in Zn and Pb production facilities, erythrocyte and leukocyte malondialdehyde (MDA) levels positively correlated with Pb blood levels (Kasperczyk et al., 2014). In a non-occupational study conducted on randomly selected children, with no reported accidental exposure to Pb, a strong correlation between Pb blood levels and biochemical indices of oxidative stress was observed (Ahamed et al., 2005). Higher blood Pb levels in children were accompanied by higher MDA levels and lower GSH contents as well as by higher CAT activity in erythrocytes due to increased levels of H2O2 induced by Pb presence in blood. Experimental studies on rats that were given 0.2% Pb-acetate (2000 ppm lead) in drinking water for 9 weeks also indicated significantly elevated CAT activity in blood, erythrocytes and lymphocytes. This subchronic exposure also caused elevated blood MDA levels and decreased GSH levels in blood of treated animals (Tandon et al., 2002). The ability of Pb to cause oxidative stress in blood has been suggested as the underlying molecular mechanism of some of the Pb-related pathologies. Research aiming to investigate the

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etiology of Pb-induced hypertension on rats revealed that the ability of Pb to cause free radical production and lower antioxidant reserves is directly related to vasoconstriction and consequently hypertension (Vaziri et al., 1999). As recently summed up by Patrick (2006), hypertension caused by lead exposure is partly mediated through a decrease in levels of NO, i.e. endothelium relaxing factor which is oxidized by ROS, observed in rats. The hematological system is an important target of Pb toxicity with oxidative stress induction as one of the proposed mechanisms for these toxic effects. Indeed, erythrocyte hemolysis induced by Pb exposure can partly be the result of lipid peroxidation in erythrocyte membranes (Lawton and Donaldson, 1991).

2.2. Effects on oxidative status in liver

Pb hepatotoxicity has been related to the elevation in the levels of serum liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) (Omobowale et al., 2014; Sivaprasad et al., 2004) and alterations in hepatic cholesterol metabolism (Abdou and Hassan, 2014). Due to its specific role in the organism, the liver is, besides blood, the main target of Pbinduced oxidative stress. Acute intraperitoneal (i.p.) administration of 15 mg Pb-acetate/kg, for 7 days (Mohammadi et al., 2014) and 25 mg Pb-acetate/kg b.w. for 5 days (Abdou and Hassan, 2014) induced decreases in SOD and CAT activity and a two-fold decrease in GPx activity. These changes were followed by a significant decrease in the levels of GSH, partly as a result of biliary excretion of Pb bound to the –SH groups of GSH, as proposed by Abdou and Hassan (2014). In prolonged Pb exposure most of the results also indicate decreases in enzyme activities. In rats receiving 0.2% Pb-acetate in drinking water for 5 weeks, the activities of all investigated enzymes (CAT, SOD, GPx, GR, G6PD, and GST) and GSH content were decreased in liver of exposed animals (Sivaprasad et al., 2004). However, chronic exposure of rats to lower levels of Pb (0.1% Pb-acetate in drinking water) and during a longer period (3 months) produced a significant depletion of hepatic GSH and catalase activity, but significantly elevated GPx activity due to removal of enhanced lipid peroxides through GPx reaction (Flora et a.l, 2003). Investigation of the recovery from Pb-induced oxidative stress in rat liver was undertaken by Omobowale et al. 2014. Animals were given 0.25, 0.5 and 1.0 mg Pb-acetate/mL for 6 weeks and then not treated for another 6 weeks. Increase in liver function test parameters, ALT, AST, ALP, as well as, MDA and H2O2 concentrations in liver, and decrease in antioxidant enzymes SOD, CAT, GPx,

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GST activity and the content of GSH were observed after 6 weeks of Pb treatment. At the end of experiment, only SOD activity was recovered and was within the range of controls, pointing to incomplete recovery from the effects of Pb-induced oxidative stress on other enzymes. These experiments indicate strong susceptibility of antioxidant enzymes in liver to Pb toxicity. It is probably a consequence of lead binding to –SH groups in protein structures, as well as of interactions with biometals (Zn, Cu, Mn, Se) in the active center of enzymes. Moreover, CAT is a heme-containing enzyme and is strongly affected by Pb, although decrease of this enzyme can be explained by interactions of Pb and Fe, as well. The most important consequence of Pb-induced oxidative stress in liver is lipid peroxidation (Flora et al., 2003; Omobowale et al., 2014; Valverde et al., 2001) that causes the alteration of membrane integrity and fatty acid composition (Lawton and Donaldson, 1991) and is associated with the increase in MDA level in liver (Jurczuk et al., 2007; Liu et al., 2012; Xu et al., 2008). Pronounced susceptibility of the hepatocyte cell membrane to Pb can be explained by its effect on polyunsaturated fatty acids, such as arachidonic acid (Lawton and Donaldson, 1991). Furthermore, there is evidence that Pb-induced oxidative stress can result in DNA damage; thus, in the experiment on mice orally treated with Pb-acetate for 4 weeks at the doses of 10, 50 and 100 mg/b.w. every other day, gradual increase of ROS and MDA levels in liver was followed by DNA damage and apoptosis, since the high expression of p53 and imbalance of Bax/Bcl-2 occurred (Xu et al. 2008). Liu et al. (2012) treated animals with an aqueous solution of Pb-acetate at a concentration of 500 mg Pb/L via drinking water for 75 days and showed that levels of 8-hydroxy-2'-deoxyguanosine (8OHdG), correlating with the ROS levels, were markedly increased in the liver of Pb-treated rats, suggesting DNA as a target of ROS-induced damage in liver. Aiming to investigate possible mechanisms of Pb-induced apoptosis and their linkage to oxidative stress induction, Wang et al. (2007) carried out an experiment on mice (0.2% Pb-acetate in drinking water for 42 days) and connected ROS production in mitochondria and ROS attack on phospholipid membrane with damage of mitochondrial membrane and subsequent caspase 3-activation leading to apoptosis. In another experiment, with the objective to investigate the gender, dose and time-dependent manner of Pbinduced oxidative stress, besides observed time and dose-dependent increase in liver MDA levels, correlation between oxidative stress, apoptosis and mitogen-activated protein kinases (MAPKs) in hepatocytes was observed. The authors concluded that apoptosis may be induced via oxidative stress-mediated alterations in MAPKs (Mujaibel and Kilarkaje, 2013). 2.3. Effects on oxidative status in kidney

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Pb-induced renal dysfunction can be classified as acute and chronic nephropathy, and occurs mostly at high levels of Pb exposure (>60 μg/dL blood) but damage at lower levels has also been reported (~10 μg/dL blood) (Grant, 2008). Acute Pb-induced nephropathy is characterized functionally by a generalized deficit of tubular transport mechanisms (Fanconi syndrome), morphologically by the appearance of degenerative changes in the tubular epithelium and the nuclear inclusion bodies containing Pb protein complexes. Although the exact mechanism of renal Pb toxicity is not completely understood, numerous experimental data have shown that oxidative stress has an important role. Evidence on Pb-induced oxidative stress in kidneys has been reported not only in in vitro and in vivo animal experiments, but also in several studies on workers exposed to Pb (Ahamed and Siddiqui 2007; Gurer-Orhan et al. 2004). Literature data on the effects of acute Pb exposure on oxidative stress in the kidney of animals are rare. Sharma and Singh (2014) have recently reported that 24 h exposure to 10 mg/kg and 150 mg/kg of Pb-acetate induced increases of renal thiobarbituric acid reactive substances (TBARS) content as indicator of lipid peroxidation, as well as SOD and CAT activities in kidney of Balbc mice. On the other hand, oxidative stress following prolonged Pb exposure is well documented. Several authors confirmed increased lipid peroxidation in the kidney of Pb-exposed animals. Thus, i.p. injection of 20 mg/kg Pb-acetate for 5 days and i.p. administration of 5 mg/kg Pb-acetate for a longer period (30 days) induced increased renal lipid peroxidation (Abdel-Moniem et al., 2011; Lakshimi et al. 2013). These findings are in agreement with Sharma et al. (2010) who reported a significant increase in the level of renal TBARS in rats orally treated for 40 days with Pb-nitrate at a dose of 50 mg/kg b.w., as well as with the experimental results of Wang et al. (2012) who demonstrated lipid peroxidation enhancement as measured by the levels of MDA in kidney of rats exposed to 500 mg Pb/L via drinking water for 8 weeks. Recent investigations performed on mice (Aziz et al., 2012; Sharma and Singh, 2014) also showed that prolonged Pb exposure induces generation of free radicals and lipid peroxidation in kidney, subsequent loss of membrane integrity and inactivation of tubular cell constituents. Most of the authors agree that Pb produces lipid peroxidation mainly via ROS generation such as H2O2, and OH., although experimental data indicate the involvement of nitrogen species as well: i.p. injection of Pb-acetate (20 mg/kg) for 5 days induced an elevation of renal NO as a result of increased activity of inducible NO synthase (iNOS) (Abdel Moniem et al., 2011). Enhanced levels of ROS can be attributed to Pb-induced disruptions in the antioxidant defence system. Several studies have shown that Pb alters the activity of antioxidant enzymes such

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as SOD, CAT, GPx, GST and G6PD as well as, the content of GSH in animals and humans. Prolonged i.p. or oral Pb exposure in experimental studies induced significant decreases in the activity of antioxidant enzymes SOD, CAT, and GPx in renal tissues (Abdel Moneim et al., 2011; Farmand et al., 2005; Lakshmi et al., 2013; Sharma et al., 2010; Wang et al., 2012). Mechanisms of Pb effects on these enzymes can be complex, since Pb can competitively inhibit bioelements absorption and/or replace them in the active sites of enzymes or bind to –SH groups of proteins (Lakshmi et al., 2013; Wang et al., 2012). Taking into account relations between GST and GSH in the redox system, the simultaneous decrease observed in both GST activity and GSH concentration may suggest that the decrease in renal GSH concentration can be, at least partly, the result of a decrease in GST activity. However, there are implications that prolonged Pb exposure can cause significant upregulation of SOD and CAT activity in the kidney of experimental animals (Farmand et al., 2005; Vaziri et al., 2003). Observed increases in the CuZn-SOD and CAT activities in Pb-exposed rats may represent a compensatory response to oxidative stress, suggesting that prolonged Pb-exposure affects their gene expression (Farmand et al., 2005). In addition to oxidative stress as mechanism of Pb toxicity in the kidney, experimental studies have shown that Pb exposure induces an increase in apoptosis not only in liver but in kidneys as well. Sujatha et al. (2011) have demonstrated that chronic treatment of rats with Pb-acetate over a period of 12 weeks diffusely increased the number of apoptotic bodies in proximal tubular cells. Thus, it can be hypothesized that Pb intoxication influences gene expression of apoptosis proteins. 3. Cadmium After absorption Cd enters blood and binds to erythrocyte membranes, blood albumin or MTs and distributes throughout the body. To a much less extent, it can also bind to –SH groups containing proteins such as glutathione and cysteine (Zalups and Ahmad, 2003). After acute Cd intoxication, Cd mainly enters the liver making it the critical organ of short-term Cd exposure. With long-term exposure, Cd induces MT synthesis in a number of tissues. Cadmium binds to these small proteins and is transported to the kidneys in which it is filtered through the glomerular membrane, reabsorbed into proximal tubular cells and deposited in the kidneys. Hence, kidney is considered as the critical organ of toxicity after long term exposure to Cd (Nordberg and Nordberg, 2000). Thus, the ability of Cd to trigger adverse effects in blood, liver and kidneys via oxidative stress induction will be discussed in detail.

3.1. Effects on oxidative status in blood

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Experimental studies have shown changes in oxidative status in blood after exposure to Cd through different routes of exposure, i.e. oral corresponding to food and water Cd intake and intraperitoneal as similar to Cd parenteral exposure through tobacco smoke. In an acute study conducted on rats, one group of rats was orally treated with a single dose of 30 mg Cd/kg b.w. while another group was injected i.p. with a single Cd dose of 1.5 mg/kg b.w. with the aim to compare these two routes of exposure (Buha et al., 2012). Results of the study revealed decreased SOD activity in both Cd-treated groups in comparison to control values that can be attributed to the observed decrease in blood Zn concentrations under the experimental conditions. The increases in other investigated parameters, i.e. O2-., total oxidative status, MDA levels and content of advanced oxidation protein products, was much more profound in i.p.-treated animals, which is in accordance with higher Cd blood levels observed after i.p. treatment. In another study, i.p. exposure to similar dose of Cd (2 mg Cd/kg b.w.) also caused a significant decrease in SOD activity as well as in CAT activity, while levels of GSH were significantly reduced probably due to Cd interactions with –SH groups (Mladenović et al., 2014). Similar effects on oxidative status in blood were observed after prolonged exposure to Cd. Thus, when applied i.p. at the dose of 1 mg/kg b.w/day during 21 days, Cd depleted SOD activity and GSH levels and these effects were accompanied by increased MDA levels in serum (Ashour and ElShemi, 2014). Furthermore, oral Cd administration at the concentration of 40 mg/L via drinking water for 30 days revealed significant decreases in the activity of SOD, CAT and GPx in serum of rats (ElBoshy et al., 2015). On the other hand, 21 days of administration of CdCl2 via gastric gavage resulted in increased activity of antioxidant enzymes, i.e. SOD, GPx, GR and CAT along with a significant elevation of MDA levels (Dwivedi et al., 2012). Different effects of Cd on components of the enzymatic antioxidant system observed in different studies can be explained not only by differences in duration of exposure and applied Cd doses, but also by the activation of defense systems via increased gene expression that is followed by the increase of antioxidant enzymes. In a study performed by Gałażyn-Sidorczuk et al. (2012), it was revealed that the influence of Cd on serum GPx activity, an enzyme that contains Se in its catalytic site, depends on Cd dose, since exposure to 5 mg Cd/L for 6 months produced increase in blood Se concentrations followed by increase in GPx activity, while exposure to 50 mg Cd/L for the same period of time led to the decrease in GPx activity, probably as a result of Cd-induced decreases in blood Se levels. The ability of Cd to produce enhanced free radical generation can lead to detrimental effects on plasma lipids and lipoproteins via lipid peroxidation and contribute to the ability of Cd to produce

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adverse effects on the cardiovascular system. In a recent study conducted by Olisekodiaka et al. (2011), rats were treated with 1 mg Cd/kg b.w/day i.p. for 4 weeks and increased levels of total cholesterol, low-density lipoprotein-cholesterol and triglycerides were observed. Similar adverse effects of Cd on lipid and lipoprotein plasma profile were reported after subcutaneous (s.c.) administration of 3 mg Cd/kg b.w/day over a period of 21 days (Murugavel and Pari, 2007). Furthermore, acute Cd intoxication was shown to result in erythrocyte hemolysis and anemia in rats partly due to the ability of Cd to induce oxidative stress in rat erythrocyte membranes (El-Demerdash et al., 2004).

3.2. Effects on oxidative status in liver The mechanism of Cd-mediated acute hepatotoxicity has been in the focus of experimental studies on Cd toxicity and although some uncertainties persist, it can be postulated that acute hepatotoxicity involves two pathways. Mechanisms of primary injury of hepatocytes include binding of Cd to –SH groups, which results in mitochondrial permeability transition and mitochondrial dysfunction, with oxidative stress as a major mechanism of acute Cd toxicity. On the other hand, secondary injury from acute Cd exposure is a result of an inflammatory process mediated via activation of Kupffer cells with the release of proinflammatory cytokines and chemokines. It is also well-documented that hepatic levels of MT and GSH influence the primary injury and the severity of acute Cd hepatotoxicity (Rikans and Yamano, 2000). Increased lipid peroxidation in rat hepatocytes induced by Cd was first demonstrated by Müller (1986). Later on, numerous in vitro and in vivo studies were performed in order to investigate Cd-induced hepatotoxicity and Cd has even become an important experimental hepatotoxicant. Recent studies have also shown inconclusive evidence of disturbed oxidative status in liver as a result of Cd exposure. Thus, single i.p. doses of 2.5 mg Cd/kg b.w. and 20 μmol Cd/kg b.w/day (~2.25 mg Cd/kg b.w/day) induced significant effects on both MDA levels (increase) and components of antioxidant system (GSH-Px, CuZn-SOD, Mn-SOD, CAT, and GSH decrease) within 24h after application, pointing to early manifestation of oxidative stress in liver of exposed animals (Casalino et al., 2002; Liu et al., 2011). Similar results were obtained in a time course oral study on mice: 20 mg Cd/kg b.w. caused a significant increase in MDA and decrease in activities of total SOD, CuZn-SOD, Mn-SOD, as well as GSH levels content in liver even after 6h (Đukić-Ćosić, 2011). Observed lipid peroxidation can be attributed not only to disturbances in antioxidative status, but also to changes in Fe content in liver, since positive correlation between MDA levels and Fe content in liver was shown in this study. As previously proposed by Casalino et al. (1997), Cd may replace Fe from cytoplasmic

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and other membrane proteins and thus, enhance concentration of this Fenton metal resulting in excessive ROS generation. In the study aiming to compare the effects of these two routes of Cd exposure, i.e. i.p. and oral, on parameters of oxidative stress in liver, a single i.p. dose of 1.5 mg Cd/b.w. caused more pronounced disturbances in oxidative stress parameters (O2.-, MDA and GSH levels) compared to a single oral dose of 30 mg Cd/kg b.w., suggesting route-dependent oxidative effects of Cd due to higher Cd content achieved in rats liver after i.p. dosing (Matović et al., 2012). Based on these observations, it could be concluded that acute Cd intoxication induces oxidative stress in liver shortly after Cd administration. Furthermore, experiments on rats indicate long lasting disturbances in oxidative status in liver, since the effects on parameters of oxidative stress in liver were observed even 8 weeks after a single i.p. Cd exposure (Cupertino et al., 2013). Along with ROS induction, experimental studies have confirmed the role of RNS induction, i.e. NO excessive synthesis, in Cd-induced oxidative stress. Intraperitoneal injection of 6.5 mg CdCl2/kg b.w. given to rats for 5 days resulted in elevated levels of NO, which reacts with O2-. and produces peroxynitrite radicals that contribute to further oxidative cell damage (Dkhil et al., 2014). The overexpression of NO can be, as in Pb-induced oxidative stress, attributed to the upregulation of iNOS in the conditions of the inflammation reaction triggered by Cd (Othman et al., 2014). In the light of the fact that chronic exposure to Cd results in the accumulation of this toxic metal in liver and induces hepatic injury, many studies were performed in order to assess the effects of prolonged Cd intoxication on liver oxidative status. Thus, exposure to 50 mg Cd/L as drinking fluid for 12 weeks produced decreases in the activities of liver SOD and CAT accompanied by elevation in MDA concentration in the liver of rats (Jurczuk et al., 2004). The authors explained these observations by possible direct interactions of Cd with bioelements important for the function of these enzymes, as well as by decreased availability of these bioelements as a result of their immobilization by binding to MT, excessively synthesized in the presence of Cd. Similar results, showing negative correlation of Cd content in rat liver with GSH, CuZn-SOD and CAT, were observed after a shorter period of exposure (5 weeks) but with higher doses of Cd (200 mg Cd/L) in a study conducted by Jihen et al. (2011). However, a time-course study performed on rats receiving Cd in drinking water (250 mg/L CdCl2) showed the recovery of certain parameters of oxidative stress after 30 days of exposure (Casalino et al., 2002). Having in mind that these parameters were affected after 10 and 20 days of Cd exposure, the ability of organisms to develop various defense mechanisms against oxidative stress in conditions of prolonged Cd exposure can be suggested as well. A recent study on mice proposed possible involvement of the activation of nuclear factor erythroid 2-related factor 2 as a protective

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mechanism against cadmium-induced oxidative stress in liver through induction of genes involved in antioxidant defense (Wu et al., 2012). Among various types of cellular damage, ROS induction can lead to apoptosis as well. In an in vitro study, rat hepatocytes were treated with Cd for 12 or 24 h and rapid intracellular ROS induction was observed at a very early stage, while an increase in apoptotic cell death was seen at later stages. It was hypothesized that Cd-induced ROS may have triggered apoptosis through induction of mitochondrial membrane lipid peroxidation or by alterations in the expression of mitochondrial antiapoptotic proteins (Wang et al., 2014). Similar results pointing to involvement of mitochondrialdependent pathways in Cd-induced apoptosis were obtained in a study on rat hepatocytes exposed to 1-10μM Cd for different periods of time (Pham et al., 2006). However, although Cd-induced ROS production and resulting mitochondrial dysfunctions can mediate a series of liver cell injuries, there must have been other mechanisms to provoke these effects, Cd direct inhibition of DNA repair being one of proposed (Liu et al., 2012). 3.3. Effects on oxidative status in kidney Cd-induced nephrotoxicity is associated with proximal tubular and glomerular damage followed by low molecular weight proteinuria, aminoaciduria, bicarbonaturia, glycosuria, and phosphaturia. Increased N-acetyl-beta-D-glucosaminidase (NAG) urinary activity and excretion of low molecular weight proteins such as retinol-binding protein, lysozyme, ribonuclease, and increased excretion of high molecular weight proteins, such as albumin and transferrin were observed in epidemiologic studies of workers chronically exposed to Cd (Nordberg et al., 2009). Several mechanisms have been proposed to explain Cd-induced renal toxicity after both acute and prolonged exposure, with clear evidence of the apparent role of oxidative stress. Experimental studies indicate that acute Cd exposure rapidly induces membrane damage in kidney cells through the process of lipid peroxidation. Casalino et al. (2002) have demonstrated that the highest increase of MDA in rat kidney occurs 24 h after i.p. application of Cd (2.5 mg Cd/kg), while a two times higher Cd dose applied via the same route application caused an increase in MDA after 72 h (Yiin et al., 1999). Our recent studies carried out on mice confirmed an increase in renal MDA levels only 6 h after oral application of a single Cd dose of 20 mg/kg t.m. (Djukic-Ćosić, 2011). Overexpression of NO observed in the same study has been recently associated with intensive inflammatory reaction (Dkhil et al., 2014). Nitric oxide reacts with superoxide anions and produces peroxynitrite radicals that cause further cell damage and oxidative stress in kidney. Acute Cd intoxication generally inhibits the activity of antioxidative enzymes SOD, CAT and GPx. A decrease in kidney CuZn-SOD, total SOD and CAT activities was observed in above mentioned

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experiments in rats and mice. A decrease in renal activity of CuZn-SOD and Mn-SOD can occur as a consequence of the competition between Cd and bioelements (Cu, and Zn) for the same metal transport proteins, especially ZIP8 transport proteins in kidney cell membranes. Acute Cd treatment was also documented to affect renal GSH. Diminished GSH kidney content was observed in rats 72 h after i.p. injection of 0.4 mg/kg Cd (Sarkar et al., 1995). However, another acute experiment demonstrated a significantly elevated renal GSH content in mice 6, 12, 24 and 48 h after a single oral administration of Cd (20 mg Cd/kg) (Djukic-Cosic et al., 2007), which maybe a consequence of the defense mechanism activation. Kidney is the target organ of prolonged Cd exposure and numerous experimental data demonstrate renal dysfunction after subacute and chronic Cd exposure. Experimental studies confirmed increased levels of TBARS/MDA, in the kidney of Cd-treated animals exposed to Cd by different routes and over different periods of time (1-12 weeks) (Dkhil et al., 2014; Jurczuk et al., 2004; Matović et al., 2013; Morales et al., 2006; Renugadevi and Prabu, 2009, 2010). However, it should be pointed out that lipid peroxidation in the kidney of Cd-intoxicated animals occurs with a delay. For example in a 2-week experiment, the significant increase in MDA levels was observed only at the end of the experiment (Djukić-Ćosić, 2011). Delayed Cd nephrotoxicity is commonly explained by Cd-MT complex scenario which implies that Cd-MT complex is degraded in kidneys releasing high levels of free Cd responsible for tubular injuries. However, Klaassen et al. (2009), based on experimental data, proposed that Cd nephrotoxicity is due to accumulation of inorganic Cd rather than Cd-MT. The impairment of the antioxidant defense system is also considered as an important event in Cd-induced renal toxicity. Many studies demonstrated decreased levels of enzymatic antioxidants in the kidney of animals exposed to prolonged Cd intoxication. Casalino et al. (2002) reported that oral exposure to Cd via drinking water (250 mg/L) during 10 and 30 days induced significant decreases in renal total SOD activity, SOD isoforms CuZn-SOD and Mn-SOD, as well as CAT. The same effects on renal CAT activity were observed after oral exposure to 5 mg/kg of CdCl2 for 4 weeks (Renugadevi and Prabu, 2010). Other routes of Cd intoxication induced similar effects: i.p. injection of CdCl2 (6.5 mg kg) for 5 days and s.c. injection of Cd (1.2 mg Cd/kg) 5 times/week during 9 weeks resulted in significant decreases in renal SOD, CAT, GR and GPx activities (Dkhil et al., 2014; Morales et al., 2006). These findings can be explained by direct Cd/enzyme interaction, i.e Cd binding to –SH groups, or displacement of metal cofactors from the active enzyme sites, while the decrease in GPx activity could be attributed to competition between GPx and metallothioneins for S-aminoacids (Ognjanovic et al., 2008; Renugadevi and Prabu, 2009). However, several studies showed that

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prolonged Cd exposure induces increase in the activity of antioxidant enzymes, especially in kidneys, as a result of the effect of Cd on gene expression and elevated levels of Cu and Zn as co-factors of CuZn-SOD (Djukić-Ćosić, 2011; Jurczuk et al., 2004; Wätjen and Beyersmann, 2004) Several studies have indicated significant decreases in the levels of non-enzymatic antioxidants GSH and TSH (Dkhil et al., 2014; Renugadevi and Prabu 2009). This could be associated with diminished availability of NADPH required for the transformation of oxidized (GSSG) to reduced form (GSH) (Deneke, 2000). Recent experiments proposed that both apoptosis and necrosis can be induced by Cdinduced increase in accumulation of ROS in kidney (Fleury et al., 2002; Hossain et al., 2009). Among the various signaling pathways involved in Cd nephrotoxicity, MAPK and Ca2+ signaling play an important role (Thévenod, 2009). On the other hand, increased ROS levels in renal cells induce the gene expression of the multidrug resistance transporter gene which protects proximal tubule cells against apoptosis (Nair et al., 2013). Furthermore, there is some new evidence that “Fanconi-like syndrome” is associated with inactivation of Na+/K+-ATPase caused by ROS enhanced production in proximal tubular cells (Nair et al., 2013).

4. Pb and Cd mixture–effects on oxidative status in blood, liver and kidneys

The importance of toxicological investigations on chemical mixtures has been recognized over a number of years (WHO, 1981), although not many in vitro and in vivo experiments, or epidemiological studies have been performed concerning Pb and Cd mixtures. Despite the well known fact that oxidative stress is involved in Pb and Cd toxicity, studies that investigate concomitant toxic effects of Cd and Pb on oxidative stress parameters are sparse. One of the studies on human occupational co-exposure to Pb and Cd was done on workers recruited from a nonferrous metal smelter and showed that this mixture affected oxidative status in blood, shown by changes in MDA, GSH, GST and 8-hydroxy-2’-deoxyguanosine blood levels (Garçon et al., 2004). Conterato et al. (2011) investigated blood levels of Pb and Cd and parameters of oxidative stress in battery-manufacturing workers, painters from an automotive industry and workers who were not occupationally exposed to these metals, who served as controls. Although MDA levels were not increased in the exposed groups, elevated activity of GST was observed in both battery-manufacturing workers and painters. Correlation analyses revealed that Pb and Cd contribute to the elevation of this enzyme activity that was explained by the ability of these metals to

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induce GST gene expression. On the other hand, the increased GPx and SOD activities, observed only in the battery workers, correlated with the increased Pb blood levels, while CAT activity, changed only in painters, inversely correlated to Cd blood levels. These changes suggest an additive effect of Cd and Pb on blood GST levels, while other alterations in oxidative stress parameters were distinctively associated with either Pb or Cd blood levels. Hence, there may be a lack of interaction between Pb and Cd on the level of oxidative status in blood after co-exposure by inhalation. Investigations on animals gave some more data on the effects of this mixture on oxidative stress parameters. In a study conducted on female rats during their pregnancy to parturition or until weaning, Pb-acetate and/or Cd-acetate were administrated via drinking water at concentrations of 300 mg/L and 10 mg/l, respectively (Massó et al., 2007). Blood levels of the metals in the dams were significantly increased in experimental groups treated with one toxic metal only (if compared to controls) while co-exposure resulted in diminished levels of both Pb and Cd in comparison to the groups exposed to a single metal. Although the authors did not investigate parameters of oxidative stress in blood of treated dams, lower levels of these metals observed in co-exposed group suggest a possible antagonistic effect of these metals when applied orally due to interactions at the level of the gastrointestinal tract, possibly because of different affinity of these metals for binding to protein transporters, DMT 1 transporter being the most important one. One of the first investigations dealing with the effects of co-exposure to Pb and Cd on liver functions in vivo and further on oxidative status was conducted by Gupta's group who treated female rats i.p. with the same doses of Pb and/or Cd: 0.05 mg Pb-acetate, 0.05 mg Cd-acetate, or combination of 0.025 mg Pb-acetate and 0.025 mg Cd-acetate for 15 days. Lead induced a decrease in the activity of SOD in liver, while the same dose of Cd caused more pronounced effects on parameters of oxidative stress: the reduction of SOD activity was greater, and furthermore, GSH levels were depleted and CAT activity and TBARS levels were increased. The co-exposure produced effects which were similar to the effects of Cd given alone in all investigated parameters, while the total concentration of metals in liver after combined treatment was not significantly different from the individually treated groups. Hence, the authors concluded that when Pb and Cd were given i.p. in similar concentrations, Cd was the one that predominantly affected oxidative status due to its more reactive nature (Pillai and Gupta, 2005). Pandya et al. (2010) continued with same model of experiment, but using 2-fold lower doses. The accumulation of both metals in liver when given in a mixture was decreased compared with groups exposed to a single metal, and this decrease was more prominent in the case of Cd, indicating interactions between these two metals in liver. In this experiment, more profound effects on parameters of oxidative stress (with the exception of GPx activity) were observed in the group treated with Cd only, indicating possible antagonism between

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Cd and Pb on oxidative stress induction in liver. This antagonism resulted in lower levels of hepatic Cd in the co-treated group inducing less pronounced effects on oxidative status. These findings are in accordance with proposed more reactive nature of Cd in producing oxidative stress in comparison to Pb. Another investigation with the results supporting the possible antagonism between Pb and Cd was carried out on bank voles, animals that are good model for environmental exposure. Animals were exposed for 6 weeks to dietary Cd and Pb (Cd-60; Pb-300; Cd-60 + Pb-300 μg/g dry w.t.concentrations 2-fold higher than those expected in high contaminated areas). No significant changes in liver concentrations of toxic metals, TBARS or GSH levels were observed between groups treated with a single metal and a group treated with a mixture (Salińska et al., 2012). Having in mind reproductive and developmental toxicity of Pb and Cd, investigations on the effects of their co-exposure are of special concern. The effect of Pb and Cd mixture in the period of pregnancy and lactation was examined in female Wistar rats receiving 300 mg Pb/L, 10 mg Cd/L, or 300 mg Pb/L+10 mg Cd/L in drinking water from day 1 of pregnancy to parturition or until weaning (Massó et al, 2007). Results showed that Pb and/or Cd during gestation and lactation produced oxidative damage in pup liver. The authors postulated Cd as being highly hepatotoxic on day 0 and Pb as being highly hepatotoxic on day 21 due to differences in their toxicokinetics. However, they concluded that simultaneous administration of both metals seems to have a less pronounced effect than expected, at least in liver. Similar investigations were undertaken by Pillai et al. (2009) using the model of the same doses of toxic metals, 0.05 mg/kg b.w/day Pb or Cd-acetate and in Pb+Cd group mixture of 0.025 mg Pb-acetate and 0.025 Cd-acetate/kg b.w./day administered s.c. to pregnant rats throughout gestation until postnatal day 21. The effects of Pb and Cd on investigated parameters were assessed on the postnatal day 56. Cadmium induced significant decrease in almost all investigated parameters in both female and male rats (GSH, MDA, CAT, Cu,Zn-SOD, Mn-SOD, and GPx), while Pb induced a decrease in CAT activity in males and GPX activity in female rats. In the group exposed to Pb+Cd, the activities of CAT, Cu,Zn-SOD and GPx were lower than in the control group but not significantly different from groups exposed to a single metal, while other parameters were not statistically different from controls, suggesting possible antagonistic interactions between these two metals on oxidative status in the liver of males and females of the F1 generation. According to these investigations on the effect of Pb and Cd mixture on oxidative stress induction, it can be concluded that antagonism between Pb and Cd in liver was observed independently of the route of exposure thus, implying that various mechanisms, during their absorption and distribution, underlie these interactions with the important role of transporters such as DMT1 and members of ZIP family.

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In an attempt to explain the effect of co-exposure to Pb and Cd, Dai et al. (2013) administered to rats by gavage for 90 days, 3 different doses, in an equitoxic mixture ratio, where the highest dose was 1/10 of the previously established LD50 for a mixture of Pb-nitrate and Cd-chloride. Results indicated a strong, significant increase in MDA and decrease in GSH content in liver and kidneys with decreased activities of antioxidant enzymes SOD, CAT, GPx. However, this experimental study does not give the information on the individual effects of Cd or Pb treatment under the same experimental conditions and therefore it is difficult to discuss possible interactions between these metals. In the same experiment, MT-1 and MT-2 gene expressions were investigated in rat liver and renal cortex and their significant up-regulation in both liver and kidneys was shown. The activation of defense systems was explained by the antioxidant effect of MT, availability of cysteine in MT for GSH synthesis, and by MT mRNA transcriptional response triggered by ROS. They also speculated that these changes could be related to the inhibition of steroid organic enzyme activity, and effects on testosterone levels, sperm count and motility after co-exposure to Pb and Cd. Wang et al. (2010) reported that oral co-exposure to Cd (50 mg/L, CdCl2) and Pb (300 mg/L, Pb(CH3COO)2) administered in drinking water to rats for 8 weeks resulted in increased MDA levels in the renal cortex, compared to controls and groups treated with Cd or Pb only. The significant increase in MDA level in kidney of rats co-exposed to Pb and Cd, when compared to the effects of single metal exposure, suggest a synergistic effect between these metals in kidneys. These observations were confirmed by ultrastructural analyses indicating more severe Pb+Cd mitochondrial damage in kidneys than was observed for each individual metal (Wang et al., 2010). Oxidative stress in the kidney of rats exposed Cd (10 mg/L as CdCl2) and Pb (25 mg/L as Pb(CH3COO)2) mixture in drinking water for a prolonged time has been confirmed by the measurement of carbonyls as well. Kidney carbonyls, as markers of protein oxidation, were increased after 30-days and 90-days of exposure in the mixture group when compared to controls and groups treated with single metals (Fowler et al., 2004). These results suggest that co-exposure to these metals induce oxidative damage of proteins as well. Even though investigations confirmed that both metals given separately or in mixture induce oxidative damage in liver and kidneys, the effects of Pb and Cd co-exposure in kidney studies implicate additivity or synergism between these metals and antagonism in liver. These differences could be, at least partly explained by the role of MT which is present in different tissues, in many isoforms, but is critical in regulating the nephrotoxic interactions of metal mixtures in kidneys following chronic exposure. The induction of MT by Cd could impact the effects of Pb through changes in its toxicokinetics and toxicodynamics.

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Literature data indicate that, similarly to Cd or Pb exposure, co-exposure to these toxic metals induces apoptosis. In a 90-day subchronic oral toxicity study, relatively low and environmentally realistic concentrations of lead and cadmium induced significant hepatic and renal apoptosis that consequently impaired their function (Yuan et al., 2014). The authors explained the observed apoptosis by mitochondrial injury and changes in the levels of apoptogenic proteins, such as Bcl-2 and Bax proteins, that activate caspase-3 resulting in apoptotic processes. With the goal to explore the ability of Pb and Cd to directly interact with DNA, Valverde et al. (2001) investigated the DNA response in lung, liver and kidneys of mice exposed to Pb-acetate and Cd-chloride by inhalation. Lead and/or Cd did not interact directly with DNA to produce single strand breaks. However, the authors suggested ROS generation and lipid peroxidation observed in liver and lungs of rats treated with single Pb or Cd doses via inhalation route of exposure as a possible mechanism of genotoxicity.

5. Conclusion and future perspectives

Although distinctively different in their specific toxic effects, the toxicity of Pb and Cd is certainly driven by induction of oxidative stress as an important mechanism of their toxicity. Up-todate studies have shown that both Pb and Cd, regardless of their non-redox nature, can cause oxidative stress in various organs including blood, liver and kidney by generation of free radicals and by affecting antioxidant defence system. The effects of these metals on free radical generation are indirect, but include different mechanisms. Thus, Pb induces inhibition of ALAD which in turns increases the levels of ALA followed by the production of free radicals, while the effects of Cd can be mainly explained by its interaction with Fe which is a Fenton metal. Literature data also indicate the effects of Pb and Cd on antioxidant defence system: Pb directly affects various enzymes by binding to –SH groups whereas Cd can inhibit the enzymes activity even indirectly, as a consequence of interactions with bioelements. Similar effect on GSH levels was observed for both metals and can be explained either by Pb or Cd binding to GSH or by their influence on enzymes of GSH cycles. Furthermore, Cd is more potent inducer of the synthesis of MT and has greater effect on the gene expression of antioxidant enzymes in comparison to Pb. It should be emphasized that the parameters of oxidative stress are significantly affected by experimental conditions (dose, type of animal species, duration, and route of exposure) and type of investigated tissue. However, the exact mechanisms of oxidative stress induction, especially in terms of action at molecular and sub-molecular levels, remain to be further explained and this may be the key in understanding the differences between the

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threats these metals pose to human health as well as to the environment. Furthermore, the precise role of oxidative stress in DNA damage and apoptosis triggered by these two metals needs to be further addressed in future studies. Considering that Pb and Cd are still important occupational agents and are even more important as global pollutants, investigations on the co-exposure to Pb and Cd on oxidative status in various target organs of their toxicity, especially at low doses present in the environment, are rather sparse and inconclusive. Research on mechanisms of joint toxicity of Pb and Cd are in progress and the available studies provide evidence of synergistic or antagonistic interactions depending on duration, dose of exposure and type of investigated organ. Hence, studies on toxicity of mixtures, not only of these two metals but also on mixtures of other metals and mixtures of metals with other toxic agents, continue to be the one of the greatest challenges of contemporary toxicology.

Acknowledgment This study was partly supported by the Ministry of Education, Science and Technological Development, Republic of Serbia (Project III 46009).

References

Abdel Moneim, A.E., Dkhil, M.A., Al-Quraishy, S., 2011. The protective effect of flaxseed oil on lead acetate-induced renal toxicity in rats. J. Hazard. Mater. 194, 250-255. Abdou, H.M., Hassan, M.A., 2014. Protective role of omega-3 polyunsaturated fatty acid against lead acetate-induced toxicity in liver and kidney of female rats. Biomed. Res. Int. 2014:435857. doi: 10.1155/2014/435857. Ahamed, M., Verma, S., Kumar, A., Siddiqui, M.K., 2005. Environmental exposure to lead and its correlation with biochemical indices in children. Sci. Total Environ. 346, 48-55. Ahamed, M., Siddiqui, M.K., 2007. Low level lead exposure and oxidative stress: current opinions. Clin. Chim. Acta 383, 57-64.

23

Page 23 of 31

Agency for Toxic Substances and Disease Registry (ATSDR), 2007. Toxicological Profile for Lead. U.S. Department of health and Human Services, Public Health Service, Atlanta, GA. Ashour, T.H., El-Shemi, A.G., 2014. Caffeic acid phenyl ester prevents cadmium intoxication induced disturbances in erythrocyte indices and blood coagulability, hepatorenal dysfunction and oxidative stress in rats. Acta Haematol. Pol. 45, 272-278. Aziz, F.M., Maulood, I.M., Chawsheen, A.H., 2012. Effects of melatonin, vitamin C and E alone or in combination on lead-induced injury in liver and kidney organs of rats. IOSR J. Pharm. 2, 13-18. Badisa, V.L., Latinwo, L.M., Odewumi, C.O., Ikediobi, C.O., Badisa, R.B., Ayuk-Takem, L.T., Nwoga, J., West, J., 2007. Mechanism of DNA damage by cadmium and interplay of antioxidant enzymes and agents. Environ. Toxicol. 22, 144-151. Bechara, E.J., 1996. Oxidative stress in acute intermittent porphyria and lead poisoning may be triggered by 5-aminolevulinic acid. Braz. J. Med. Biol. Res. 29, 841-851. Bergdahl, I.A., Sheveleva, M., Schütz, A., Artamonova, V.G., Skerfving, S., 1998. Plasma and blood lead in humans: capacity-limited binding to delta-aminolevulinic acid dehydratase and other leadbinding components. Toxicol. Sci. 46, 247-253. Bulat, Z.P., Djukić-Ćosić, D., Maličević, Ž., Bulat, P., Matović, V., 2008. Zinc or magnesium supplementation modulates Cd intoxication in blood, kidney, spleen, and bone of rabbits. Biol. Trace Elem. Res. 124, 110-117. Bulat, Z.P., Đukic-Ćosić, D., Đokić, M., Bulat, P., Matović, V., 2009. Blood and urine cadmium and bioelements profile in nickel-cadmium battery workers in Serbia. Toxicol. Ind. Health 25, 129-135. Buha, A., Bulat, Z., Dukić-Ćosić, D., Matović, V., 2012. Effects of oral and intraperitoneal magnesium treatment against cadmium-induced oxidative stress in plasma of rats. Arh. Hig. Rada Toksikol. 63, 247-254. Buha, A., Antonijević, B., Bulat, Z., Jaćević, V., Milovanović, V., Matović, V., 2013. The impact of prolonged cadmium exposure and co-exposure with polychlorinated biphenyls on thyroid function in rats. Toxicol. Lett. 221, 83-90. Casalino, E., Sblano, C., Landriscina, C., 1997. Enzyme activity alteration by cadmium administration to rats: the possibility of iron involvement in lipid peroxidation. Arch. Biochem. Biophys. 346, 171179. Casalino, E., Calzaretti, G., Sblano, C., Landriscina, C., 2002. Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 179, 37-50. CDC, 2009. Centre for Disease Control and Prevention, U.S. Department of Health and Human Services. Fourth National Report on Human Exposure to Environmental Chemicals. Atlanta, USA. Colacino, J.A., Arthur, A.E., Ferguson, K.K., Rozek, L.S., 2014. Dietary antioxidant and antiinflammatory intake modifies the effect of cadmium exposure on markers of systemic inflammation and oxidative stress. Environ. Res. 131, 6-12. Conterato, G.M., Bulcão, R.P., Sobieski, R., Moro, A.M., Charão, M.F., de Freitas, F.A., de Almeida, F.L., Moreira, A.P., Roehrs, M., Tonello, R., Batista, B.L., Grotto, D., Barbosa, F. Jr, Garcia, S.C., Emanuelli, T. 2013. Blood thioredoxin reductase activity, oxidative stress and hematological

24

Page 24 of 31

parameters in painters and battery workers: relationship with lead and cadmium levels in blood. J. Appl. Toxicol. 33, 142-150. Cuypers, A., Plusquin, M., Remans, T., Jozefczak, M., Keunen, E., Gielen, H., Opdenakker, K., Nair, A.R., Munters, E., Artois, T.J., Nawrot, T., Vangronsveld, J., Smeets, K., 2010. Cadmium stress: an oxidative challenge. Biometals 23, 927-940. Cupertino, M.C., Costa, K.L., Santos, D.C., Novaes, R.D., Condessa, S.S., Neves, A.C., Oliveira, J.A., Matta, S.L., 2013. Long-lasting morphofunctional remodelling of liver parenchyma and stroma after a single exposure to low and moderate doses of cadmium in rats. Int. J. Exp. Pathol. 94, 343-351. Dai, S., Yin, Z., Yuan, G., Lu, H., Jia, R., Xu, J., Song, X., Li, L., Shu, Y., Liang, X., He, C., Lv, C., Zhang, W., 2013. Quantification of metallothionein on the liver and kidney of rats by subchronic lead and cadmium in combination. Environ. Toxicol. Pharmacol. 36, 1207-1216. Demenesku, J., Mirkov, I., Ninkov, M., Popov Aleksandrov, A., Zolotarevski, L., Kataranovski, D., Kataranovski, M., 2014. Acute cadmium administration to rats exerts both immunosuppressive and proinflammatory effects in spleen. Toxicology 326, 96-108. Deneke, S.M., 2000. Thiol-based antioxidants. Curr. Top Cell Regul. 36, 151-180. Djukić-Ćosić, D., Ninković, M., Maličević, Ž., Plamenac-Bulat, Z., Matović, V., 2006. Effect of supplemental magnesium on the kidney levels of cadmium, zinc, and copper of mice exposed to toxic levels of cadmium. Biol. Trace Elem. Res. 114, 281-291. Djukić-Ćosić, D., Ninković, M., Maličević, Ž., Matović, V., Soldatović, D., 2007. Effect of magnesium pretreatment on reduced glutathione levels in tissues of mice exposed to acute and subacute cadmium intoxication: a time course study. Magnes. Res. 20, 177-186. Djukić-Ćosić, D., Ćurčić Jovanović, M., Plamenac Bulat, Z., Ninković, M., Maličević, Ž, Matović, V., 2008. Relation between lipid peroxidation and iron concentration in mouse liver after acute and subacute cadmium intoxication. J. Trace Elem. Med. Biol. 22, 66-72. Djukić-Ćosić, D., 2011. Uticaj magnezijuma na oksidativni stress I ravnotežu bioelemenata pri akutnom I subakutnom trovanju miševa kadmijumom (The Effect of Magnesium on Oxidative stress and Bioelements in Mice Exposed to Acute and Subacute Cadmium Intoxication). Ph. D. thesis, Faculty of Pharmacy, University of Belgrade, Belgrade. Dkhil, M.A., Al-Quraishy, S., Diab, M.M., Othman, M.S., Aref, A.M., Abdel Moneim, A.E., 2014. The potential protective role of Physalis peruviana L. fruit in cadmium-induced hepatotoxicity and nephrotoxicity. Food Chem. Toxicol. 74, 98-106. Dwivedi, V.K., Bhatanagar, A., Chaudhary, M., 2012. Protective role of ceftriaxone plus sulbactam with VRP1034 on oxidative stress, hematological and enzymatic parameters in cadmium toxicity induced rat model. Interdiscip. Toxicol. 5, 192-200. EFSA Panel on Contaminants in the Food Chain (CONTAM), 2011. Scientific Opinion on tolerable weekly intake for cadmium. EFSA Journal. 9, 1975. (19 pp.) Available online: www.efsa.europa.eu/efsajournal El-Boshy, M.E., Risha, E.F., Abdelhamid, F.M., Mubarak, M.S., Hadda, T.B., 2015. Protective effects of selenium against cadmium induced hematological disturbances, immunosuppressive, oxidative stress and hepatorenal damage in rats. J. Trace Elem. Med. Biol. 29, 104-110.

25

Page 25 of 31

El-Demerdash, F.M., Yousef, M.I., Kedwany, F.S., Baghdadi, H.H., 2004. Cadmium-induced changes in lipid peroxidation, blood hematology, biochemical parameters and semen quality of male rats: protective role of vitamin E and beta-carotene. Food Chem. Toxicol. 42, 1563-1571. Farmand, F., Ehdaie, A., Roberts, C.K., Sindhu, R.K., 2005. Lead-induced dysregulation of superoxide dismutases, catalase, glutathione peroxidase, and guanylate cyclase. Environ. Res. 98, 33-39. Fay, R.M., Mumtaz, M.M., 1996. Development of a priority list of chemical mixtures occurring at 1188 hazardous waste sites, using the HazDat database. Food Chem. Toxicol. 34, 1163-1165. Flora, S.J., Pande, M., Mehta, A., 2003. Beneficial effect of combined administration of some naturally occurring antioxidants (vitamins) and thiol chelators in the treatment of chronic lead intoxication. Chem. Biol. Interact. 145, 267-280. Flora, G., Gupta, D., Tiwari, A., 2012. Toxicity of lead: A review with recent updates. Interdiscip. Toxicol. 5, 47-58. Fowler, B.A., Whittaker, M.H., Lipsky, M., Wang, G., Chen, X.Q., 2004. Oxidative stress induced by lead, cadmium and arsenic mixtures: 30-day, 90-day, and 180-day drinking water studies in rats: an overview. Biometals 17, 567-568. Gałażyn-Sidorczuk, M., Brzóska, M.M., Rogalska, J., Roszczenko, A., Jurczuk, M., 2012. Effect of zinc supplementation on glutathione peroxidase activity and selenium concentration in the serum, liver and kidney of rats chronically exposed to cadmium. J. Trace Elem. Med. Biol. 26, 46-52. Garçon, G., Leleu, B., Zerimech, F., Marez, T., Haguenoer, J.M., Furon, D., Shirali, P., 2004. Biologic markers of oxidative stress and nephrotoxicity as studied in biomonitoring of adverse effects of occupational exposure to lead and cadmium. J. Occup. Environ. Med. 46, 1180-1186. Gurer, H., Ercal, N., 2000. Can antioxidants be beneficial in the treatment of lead poisoning? Free Radic. Biol. Med. 29, 927-945. Gurer-Orhan, H., Sabir, H.U., Ozgüneş, H., 2004. Correlation between clinical indicators of lead poisoning and oxidative stress parameters in controls and lead-exposed workers. Toxicology 195, 147-154. Gonick, H.C., 2011. 10.1155/2011/686050.

Lead-binding

proteins:

a

review.

J.

Toxicol.

2011:686050.

doi:

Grant, L.D., 2008. Lead and compounds. Environmental Toxicants (John Wiley & Sons, Inc.). pp. 757809. Hsu, P.C., Guo, Y.L., 2002. Antioxidant nutrients and lead toxicity. Toxicology 180, 33-44. IARC, 1993. Beryllium, cadmium, mercury, and exposures in the glass manufacturing industry. International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risks to Humans. IARC Scientific Publications, Lyon, pp. 1-415. IARC, 2012. Arsenic, Metals, Fibres, and Dusts. International Agency for Research on Cancer Monographs on the Evaluation of Carcinogenic Risks to Humans. IARC Scientific Publications, Lyon, pp. 1-465.

26

Page 26 of 31

Jihen, el H., Sonia, S., Fatima, H., Mohamed Tahar S, Abdelhamid K 2011. Interrelationships between cadmium, zinc and antioxidants in the liver of the rat exposed orally to relatively high doses of cadmium and zinc. Ecotoxicol. Environ. Saf. 74, 2099-2104. Jomova, K., Valko, M., 2011. Advances in metal-induced oxidative stress and human disease. Toxicology 283, 65-87. Jurczuk, M., Brzóska, M.M., Moniuszko-Jakoniuk, J., Gałazyn-Sidorczuk, M., Kulikowska-Karpińska, E., 2004. Antioxidant enzymes activity and lipid peroxidation in liver and kidney of rats exposed to cadmium and ethanol. Food Chem. Toxicol. 42, 429-438. Jurczuk, M., Brzóska, M.M., Moniuszko-Jakoniuk, J., 2007. Hepatic and renal concentrations of vitamins E and C in lead- and ethanol-exposed rats. An assessment of their involvement in the mechanisms of peroxidative damage. Food Chem. Toxicol. 45, 1478-1486. Karaca, S., Eraslan, G., 2013. The effects of flaxseed oil on cadmium-induced oxidative stress in rats. Biol. Trace Elem. Res. 155, 423-430. Kasperczyk, A., Machnik, G., Dobrakowski, M., Sypniewski, D., Birkner, E., Kasperczyk, S., 2012. Gene expression and activity of antioxidant enzymes in the blood cells of workers who were occupationally exposed to lead. Toxicology 301, 79-84. Kasperczyk, S., Dobrakowski, M., Kasperczyk, A., Machnik, G., Birkner, E., 2014. Effect of Nacetylcysteine administration on the expression and activities of antioxidant enzymes and the malondialdehyde level in the blood of lead-exposed workers. Environ. Toxicol. Pharmacol. 37, 638647. Kelada, S.N., Shelton, E., Kaufmann, R.B., Khoury, M.J., 2001. Delta-aminolevulinic acid dehydratase genotype and lead toxicity: a HuGE review. Am. J. Epidemiol. 154, 1-13. Klaassen, C.D., Liu, J., Diwan, B.A., 2009. Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol. 238, 215-220. Lakshmi, B.V., Sudhakar, M., Aparna, M., 2013. Protective potential of Black grapes against lead induced oxidative stress in rats. Environ. Toxicol. Pharmacol. 35, 361-368. Lawton, L.J., Donaldson, W.E., 1991. Lead-induced tissue fatty acid alterations and lipid peroxidation. Biol. Trace Elem. Res. 28, 83-97. Liu, T., He, W., Yan, C., Qi, Y., Zhang, Y., 2011. Roles of reactive oxygen species and mitochondria in cadmium-induced injury of liver cells. Toxicol. Ind. Health. 27, 249-256. Liu, C.M., Ma, J.Q., Sun, Y.Z., 2012. Puerarin protects the rat liver against oxidative stress-mediated DNA damage and apoptosis induced by lead. Exp. Toxicol. Pathol. 64, 575-582. Massó, E.L., Corredor, L., Antonio, M.T., 2007. Oxidative damage in liver after perinatal intoxication with lead and/or cadmium. J. Trace Elem. Med. Biol. 21, 210-216. Matović, V., Buha, A., Bulat, Z., Dukić-Ćosić, D., 2011. Cadmium toxicity revisited: focus on oxidative stress induction and interactions with zinc and magnesium. Arh. Hig. Rada Toksikol. 62, 65-76. Matović, V., Buha, A., Bulat, Z., Ðukić-Ćosić, D., Miljković, M., Ivanišević, J., Kotur-Stevuljević, J., 2012. Route-dependent effects of cadmium/cadmium and magnesium acute treatment on parameters of oxidative stress in rat liver. Food Chem. Toxicol. 50, 552-557.

27

Page 27 of 31

Matović, V. , Dukić-Ćosić, D., Buha, A., Bulat, Z., 2013. Route, dose and duration of exposure to cadmium-relevance to oxidative stress induction. Peroxidases: Biochemical Characteristics, Functions and Potential Applications, pp. 159-175. Mladenović, J., Ognjanović, B., Dorđević, N., Matić, M., Knežević, V., Stajn, A., Saičić, Z., 2014. Protective effects of oestradiol against cadmium-induced changes in blood parameters and oxidative damage in rats. Arh. Hig. Rada Toksikol. 65, 37-46. Mohammad, M., Ghaznavi, R., Keyhanmanesh, R., Sadeghipour, H.R., Naderi, R., Mohammadi, H., 2014. Caloric restriction prevents lead-induced oxidative stress and inflammation in rat liver. Sci. World J. 2014:821524. doi: 10.1155/2014/821524. Morales, A.I., Vicente-Sánchez, C., Sandoval, J.M., Egido, J., Mayoral, P., Arévalo, M.A., FernándezTagarro, M., López-Novoa, J.M., Pérez-Barriocanal, F., 2006. Protective effect of quercetin on experimental chronic cadmium nephrotoxicity in rats is based on its antioxidant properties. Food Chem. Toxicol. 44, 2092-2100. Mujaibel, L.M., Kilarkaje, N., 2013. Mitogen-activated protein kinase signaling and its association with oxidative stress and apoptosis in lead-exposed hepatocytes. Environ. Toxicol. Müller, L., 1986. Consequences of cadmium toxicity in rat hepatocytes: mitochondrial dysfunction and lipid peroxidation. Toxicology 40, 285-295. Murugavel, P., Pari, L., 2007. Diallyl tetrasulfide protects cadmium-induced alterations in lipids and plasma lipoproteins in rats. Nutr. Res. 27, 356-361. Nair, A.R., Degheselle, O., Smeets, K., Van Kerkhove, E., Cuypers, A., 2013. Cadmium-Induced Pathologies: Where Is the Oxidative Balance Lost (or Not)? Int. J. Mol. Sci. 14, 6116-6143. Nawrot, T.S., Staessen, J.A., Roels, H.A., Munters, E., Cuypers, A., Richart, T., Ruttens, A., Smeets, K., Clijsters, H., Vangronsveld, J., 2010. Cadmium exposure in the population: from health risks to strategies of prevention. Biometals 23, 769-782. Nordberg, G.F., 2009. Historical perspectives on cadmium toxicology. Toxicol. Appl. Pharmacol. 238, 192-200. Nordberg, M., Nordberg, G.F., 2000. Toxicological aspects of metallothionein. Cell. Mol. Biol. 46, 451463. Nordberg, G.F., Jin, T., Wu, X., Lu, J., Chen, L., Lei, L., Hong, F., Nordberg, M., 2009. Prevalence of kidney dysfunction in humans - relationship to cadmium dose, metallothionein, immunological and metabolic factors. Biochimie 91, 1282-1285. Ognjanović, B.I., Marković, S.D., Pavlović, S.Z., Zikić, R.V., Stajn, A.S., Saicić, Z.S., 2008. Effect of chronic cadmium exposure on antioxidant defense system in some tissues of rats: protective effect of selenium. Physiol. Res. 57, 403-411. Olisekodiaka, M.J., Igbeneghu, C.A., Onuegbu, A.J., Oduru, R., Lawal, A.O., 2012. Lipid, lipoproteins, total antioxidant status and organ changes in rats administered high doses of cadmium chloride. Med. Princ. Pract. 21, 156-159. Omobowale, T.O., Oyagbemi, A.A., Akinrinde, A.S., Saba, A.B., Daramola, O.T., Ogunpolu, B.S., Olopade, J.O., 2014. Failure of recovery from lead induced hepatoxicity and disruption of erythrocyte antioxidant defence system in Wistar rats. Environ. Toxicol. Pharmacol. 37, 1202-1211.

28

Page 28 of 31

Othman, A.I., El Missiry, M.A., 1998. Role of selenium against lead toxicity in male rats. J. Biochem. Mol. Toxicol. 12, 345-349. Othman, M.S., Safwat, G., Aboulkhair, M., Abdel Moneim, A.E. 2014. The potential effect of berberine in mercury-induced hepatorenal toxicity in albino rats. Food Chem. Toxicol. 69, 175-181. Pandya, C.D., Pillai, P.P., Gupta, S.S., 2010. Lead and cadmium co-exposure mediated toxic insults on hepatic steroid metabolism and antioxidant system of adult male rats. Biol. Trace Elem. Res. 134, 307-317. Patrick, L., 2006. Lead toxicity part II: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. Altern. Med. Rev. 11, 114-127. Pham, T.N., Marion, M., Denizeau, F., Jumarie, C., 2006. Cadmium-induced apoptosis in rat hepatocytes does not necessarily involve caspase-dependent pathways. Toxicol. In Vitro 20, 13311342. Pillai, A., Gupta, S., 2005. Antioxidant enzyme activity and lipid peroxidation in liver of female rats coexposed to lead and cadmium: Effects of vitamin E and Mn2+. Free Radic. Res. 39, 707–712. Pillai, P., Patel, R., Pandya, C., Gupta, S., 2009. Sex-specific effects of gestational and lactational coexposure to lead and cadmium on hepatic phase I and phase II xenobiotic/steroid-metabolizing enzymes and antioxidant status. J. Biochem. Mol. Toxicol. 23, 419-431. Renugadevi, J., Prabu, S.M., 2009. Naringenin protects against cadmium-induced oxidative renal dysfunction in rats. Toxicology 256, 128-134. Renugadevi, J., Prabu, S.M., 2010. Quercetin protects against oxidative stress-related renal dysfunction by cadmium in rats. Exp. Toxicol. Pathol. 62, 471-481. Rikans, L.E., Yamano, T., 2000. Mechanisms of cadmium-mediated acute hepatotoxicity. J. Biochem. Mol. Toxicol. 14, 110-117. Salińska, A., Włostowski, T., Zambrzycka, E., 2012. Effect of dietary cadmium and/or lead on histopathological changes in the kidneys and liver of bank voles Myodes glareolus kept in different group densities. Ecotoxicology 21(8), 2235-2243. Sarkar, S., Yadav, P., Trivedi, R., Bansal, A.K., Bhatnagar, D., 1995. Cadmium-induced lipid peroxidation and the status of the antioxidant system in rat tissues. J. Trace Elem. Med. Biol. 9, 144149. Sharma, S., Sinhg, B., 2014. Effects of acute and chronic lead exposure on kidney lipid peroxidation and antioxidant enzyme activities in BALB-C mice (Mus Musculus). Int. J. Sci. Res. 3, 1564-1566. Sharma, V., Sharma, A., Kansal, L., 2010. The effect of oral administration of Allium sativum extracts on lead nitrate induced toxicity in male mice. Food Chem. Toxicol. 48, 928-936. Silva, N., Peiris-John, R., Wickremasinghe, R., Senanayake, H., Sathiakumar, N., 2012. Cadmium a metalloestrogen: are we convinced? J. Appl. Toxicol. 32, 318-332. Sivaprasad, R., Nagaraj, M., Varalakshmi, P., 2004. Combined efficacies of lipoic acid and 2,3dimercaptosuccinic acid against lead-induced lipid peroxidation in rat liver. J. Nutr. Biochem. 15, 1823.

29

Page 29 of 31

Soldatović, D., Matović, V., Vujanović, D., Guiet-Bara, A., Bara, M., Durlach, J., 2002. Metal pollutants and bioelements: retrospective of interactions between magnesium and toxic metals. Magnes. Res. 15, 67-72. Sujatha, K., Srilatha, C.H., Anjaneyulu, Y., Amaravathi, P., 2011. Lead acetate induced nephrotoxicity in wistar albino rats. A pathological, immunohistochemical and ultrastructural studies. Int. J. Pharm. BioSci. 2, B458-B468. Tandon, S.K., Singh, S., Prasad, S., Srivastava, S., Siddiqui, M.K., 2002. Reversal of lead-induced oxidative stress by chelating agent, antioxidant, or their combination in the rat. Environ. Res. 90, 6166. Thévenod, F., 2009. Cadmium and cellular signaling cascades: to be or not to be? Toxicol. Appl. Pharmacol. 238, 221-239. Valverde, M., Trejo, C., Rojas, E., 2001. Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA-metal interaction? Mutagenesis 16, 265-270. Vaziri, N.D., Ding, Y., Ni, Z., 1999. Nitric oxide synthase expression in the course of lead-induced hypertension. Hypertension 34, 558-562. Vaziri, N.D., Lin, C.Y., Farmand, F., Sindhu, R.K., 2003. Superoxide dismutase, catalase, glutathione peroxidase and NADPH oxidase in lead-induced hypertension. Kidney Int. 63, 186-194. Wang, C., Liang, J., Zhang, C., Bi, Y., Shi, X., Shi, Q., 2007. Effect of ascorbic Acid and thiamine supplementation at different concentrations on lead toxicity in liver. Ann. Occup. Hyg. 51, 563-569. Wang, G., Fowler, B.A., 2008. Roles of biomarkers in evaluating interactions among mixtures of lead, cadmium and arsenic. Toxicol. Appl. Pharmacol. 233, 92-99. Wang, L., Li, J., Li, J., Liu, Z., 2010. Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biol. Trace Elem. Res. 137, 69-78. Wang, J., Yang, Z., Lin, L., Zhao, Z., Liu, Z., Liu, X., 2012. Protective effect of naringenin against leadinduced oxidative stress in rats. Biol. Trace Elem. Res. 146, 354-359. Wang, J., Zhu, H., Liu, X., Liu, Z., 2014. Oxidative stress and Ca(2+) signals involved on cadmiuminduced apoptosis in rat hepatocyte. Biol. Trace Elem. Res. 161, 180-189. Wätjen, W., Beyersmann, D., 2004. Cadmium-induced apoptosis in C6 glioma cells: influence of oxidative stress. Biometals 17, 65-78. WHO, 1981. Health Effects of Combined Exposures in the Work Environment. WHO Technical Report Series No. 662. World Health Organization, Geneva. Wu, K.C., Liu, J.J., Klaassen, C.D., 2012. Nrf2 activation prevents cadmium-induced acute liver injury. Toxicol. Appl. Pharmacol. 263, 14-20. Xu, J., Lian, L.J., Wu, C., Wang, X.F., Fu, W.Y., Xu, L.H., 2008. Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice. Food Chem. Toxicol. 46, 1488-1494. Yiin, S.J., Chern, C.L., Sheu, J.Y., Tseng, W.C., Lin, T.H., 1999. Cadmium-induced renal lipid peroxidation in rats and protection by selenium. J. Toxicol. Environ. Health A. 57, 403-413.

30

Page 30 of 31

Yuan, G., Dai, S., Yin, Z., Lu, H., Jia, R., Xu, J., Song, X., Li, L., Shu, Y., Zhao, X., Chen, Z., Fan, Q., Liang, X., He, C., Yin, L., Lv, C., Lei, Q., Wang, L., Mi, Y., Yu, X., Zhang, M., 2014. Sub-chronic lead and cadmium co-induce apoptosis protein expression in liver and kidney of rats. Int. J. Clin. Exp. Pathol. 15, 2905-2914. Zalups, R.K., Ahmad, S., 2003. Molecular handling of cadmium in transporting epithelia. Toxicol. Appl. Pharmacol. 186, 163-188.

Figure 1 Pathways of Pb- and Cd-induced oxidative stress. Pb and Cd are non-redox metals but can induce oxidative damage of lipids, proteins and DNA. Both Pb and Cd can induce generation of reactive oxygen species (ROS): Pb inhibits the activity of δaminolevulinic acid dehydratase (ALAD) leading to the accumulation of δ-aminolevulinic acid (ALA) that induces generation of ROS (superoxide anion O2•-, hydrogen peroxide H2O2, hydroxil radical OH.), while Cd elevates the level of Fenton metals (Fe3+) which breaks down H2O2 to a reactive OH•. Both metals are capable of mediating production of reactive nitrogen species, i.e. nitric oxide (NO) which in reaction with O2•- gives a reactive peroxynitrite anion (ONOO-). Pb and Cd also impair enzyme activity of antioxidative defence system (superoxide dismutase, SOD; catalase, CAT; glutathione peroxidase, GPx; glutathione-S-transferase, GST; gluathione reductase, GR) that could be explained by the replacement of Zn2+, Fe3+, Se2+ by these toxic metals (Me2+) can and they both have impact on nonenzymatic component glutathione (GSSG, GSH). Cadmium induces synthesis of metallothionein (MT) which binds both metals.

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