Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury

curcumin

The Journal of Applied Toxicology. 2010 Mar 12.

Detoxification and antioxidant effects of curcumin in rats experimentally exposed to mercury.

Agarwal R, Goel SK, Behari JR.

Toxicokinetics Section, Indian Institute of Toxicology Research (formerly: Industrial Toxicology Research Centre), Council of Scientific and Industrial Research, India, Post Box 80, Mahatma Gandhi Marg, Lucknow 226001, India.

Curcumin, a safe nutritional component and a highly promising natural antioxidant with a wide spectrum of biological functions, has been examined in several metal toxicity studies, but its role in protection against mercury toxicity has not been investigated.

Therefore, the detoxification and antioxidant effects of curcumin were examined to determine its prophylactic/therapeutic role in rats experimentally exposed to mercury (in the from of mercuric chloride-HgCl(2), 12 micromol kg(-1) b.w. single intraperitoneal injection).

Curcumin treatment (80 mg kg(-1) b.w. daily for 3 days, orally) was found to have a protective effect on mercury-induced oxidative stress parameters, namely, lipid peroxidation and glutathione levels and superoxide dismutase, glutathione peroxidase and catalase activities in the liver, kidney and brain.

Curcumin treatment was also effective for reversing mercury-induced serum biochemical changes, which are the markers of liver and kidney injury.

Mercury concentration in the tissues was also decreased by the pre/post-treatment with curcumin.

However, histopathological alterations in the liver and kidney were not reversed by curcumin treatment. Mercury exposure resulted in the induction of metallothionein (MT) mRNA expressions in the liver and kidney. Metallothionein mRNA expression levels were found to decrease after the pre-treatment with curcumin, whereas post-treatment with curcumin further increased MT mRNA expression levels.

Our findings suggest that curcumin pretreatment has a protective effect and that curcumin can be used as a therapeutic agent in mercury intoxication. The study indicates that curcumin, an effective antioxidant, may have a protective effect through its routine dietary intake against mercury exposure.

INTRODUCTION:

Although mercury has been recognized as a hazardous environmental pollutant, inorganic mercury is widely used in certain types of batteries and continues to be an essential component of fluorescent light bulbs (Clarkson, 1997).

Unfortunately this study neglects to mention the largest exposure to inorganic mercury. That of dental mercury amalgam fillings. Here is what the EPA says about the transpost of elemental mercury vapor. 

Once absorbed, elemental mercury is readily distributed throughout the body; it crosses both placental and blood-brain barriers. Elemental mercury is oxidized to inorganic divalent mercury by the hydrogen peroxidase-catalase pathway, which is present in most tissues. The oxidation of elemental mercury to the inorganic mercuric cation in the brain can result in retention in the brain. Inorganic mercury has poor lipophilicity and a reduced capacity for penetrating the blood-brain or placental barriers. Once elemental mercury crosses the placental or blood-brain barriers and is oxidized to the mercuric ion, return to the general circulation is impeded, and mercury can retained in brain tissue.

The primary targets for toxicity of mercury and mercury compounds are the nervous system, the kidney, and the developing fetus. Other systems that may be affected include the respiratory, cardiovascular, gastrointestinal, hematologic, immune, and reproductive systems.

– Team Mercury Exposure

Inorganic mercury accumulates preferentially in the kidneys and may cause acute renal failure (Tanaka-Kagawa et al., 1998). The uptake, accumulation and toxicity of inorganic mercury in the kidney have been related to its binding to endogenous thiol-containing molecules (Zalups, 2000). Thiol-containing enzymes have been identified as the targets of inorganic mercury (Emanuelli et al., 1996; Nogueira et al., 2003). Additionally, binding of mercuric ions to sulfhydryl groups may result in decreased glutathione levels, leading to increases in levels of reactive oxygen species, such as superoxide anion radicals, hydrogen peroxide and hydroxyl radicals (Stohs and Bagchi, 1995). Metallothioneins (MTs), which are metalloproteins, are actively involved in the transport and detoxification of mercury (Olanow and Arendash, 1994; Johnson, 2000) by inhibiting sulfur ligands, which inactivates metalloproteins that normally bind metal ions such as copper (Ladner and Lindestron, 1999). This leads to toxic levels of copper in many types of membrane.

Exposure to mercury also results in changes in metalloprotein compounds that affect gene expression (Boot, 1995). Some of the processes affected by this control of gene expression include cellular respiration, metabolism, enzymatic processes, metal-specific homeostasis and adrenal stress response systems (Matts et al., 1991). Considering that oxidative stress and endogenous thiol depletion are involved in inorganic mercury toxicity, it has been suggested that antioxidants could contribute to the treatment of mercury poisoning (Patrick, 2002).

Curcumin from turmeric, a well known biologically active compound, has been shown to ameliorate oxidative stress and it is considered to be a potent antioxidant (Eybl et al., 2006). Curcumin is a potent inducer of detoxifying enzymes and thereby counters the toxicity induced by chemical carcinogens (Singletary et al., 1998). It is already used as a drug to control various diseases, including inflammatory disorders, and is expected to be used as a drug to prevent for carcinogenesis and oxidative stress-induced pathogenesis in the future. Curcumin prevents paraquat-induced lung toxicity and protects the cell membrane (Venkatesan, 2000).

Having a polyphenolic structure and diketone functional groups, curcumin is a stronger antioxidant inhibitor of lipid peroxidation than other flavonoids, which have a single phenolic hydroxyl group (Phan et al., 2001). The effective antioxidant property of curcumin inhibits the utilization of vitamins C and E in the liver, thus maintaining their levels (Rukkumani et al., 2003). Curcumin has been reported to protect hepatocytes against alcohol and polyunsaturated fatty acid (PUFA) induced liver toxicity (Rukkumani et al., 2002). It has been used as an antioxidant in toxicity studies of several metals including cadmium (Daniel et al., 2004), copper (Nair et al., 2005), iron (Manjunatha and Srinivasan, 2006), lead (Dairam et al., 2007) and selenium (Padmaja and Raju, 2004). However, the efficacy of curcumin treatment for mercury toxicity has not been investigated so far. We therefore carried out this work to evaluate the efficacy of curcumin in ameliorating mercury toxicity and the antioxidant potential of curcumin treatment before or after mercury exposure in rats. The effect of mercury toxicity on the expressions of metallothionein (MT-I and MT-II) mRNAs in liver and kidney tissues and the role of pre- or post-treatment with curcumin on metallothionein mRNA expression levels were also studied by real-time RT-PCR analysis.

DISCUSSION

The toxicity of HgCl2 is attributed to the high affinity of Hg(II) to thiol groups owing to which it can deplete cellular GSH and damage proteins and thiol enzymes (Augusti et al., 2007). We found decreases in GSH levels in liver and kidney tissues as a result of mercury exposure (Fig. 1B). GSH level may also decrease during neutralization of oxidative stress generated by HgCl2 (Lund et al., 1993). Hg(II) enhances H2O2 production because of its effect on the inner mitochondrial membrane. H2O2 so produced is speculated to induce LPO, which plays a crucial role in Hg(II)- induced nephrotoxicity (Huang et al., 1996). In this study, we found that HgCl2 enhanced LPO in tissues (Fig. 1A).

Oxidative stress and the production of free radicals have been suggested to be involved in Hg-induced tissue injury (Stohs and Bagchi, 1995; Zalups, 2000), and the high affinity between mercury and endogenous thiol-containing molecules, such as GSH and d-ALA-D, seems to contribute to this processes. Girardi and Elias (1995) suggested that changes in GPx and CAT activities do not underlie HgCl2-induced lipid peroxidation or tissue damage. Our results corroborate this suggestion, since we observed lipid peroxidation along with histopathological damage with alterations in SOD, GPx and CAT activities in the liver, kidney and brain tissues. 

These findings also indicate that free radicals generated by HgCl2 altered endogenous antioxidant activity and induced oxidative damage in tissues. The increases in SOD and GPx activities occurred probably as a defense response against hydrogen peroxide generated by HgCl2, as mercury was also detected in the liver and kidney tissues (Fig. 1F). However, mercury was not detected in the brain tissues, which may be due to the fact that inorganic mercury can hardly penetrate the blood–brain barrier.

The enhancement of LPO and decrease in GSH level showed that the generation of ROS in the brain is due to mercury toxicity. It is well known that the brain tissues are more sensitive to oxidative damage because of their high concentration of unsaturated lipids and high rate of oxidative metabolism (Goering et al., 2002). 

This is also supported by our findings of the highest MDA level in the brain among the tissues examined in mercury- treated animals and by our previous works (Agarwal and Behari, 2007a, b).

Interestingly, we also found SOD depletion in the kidney tissues, in both curcumin pre- and post-treated mercury- exposed animals compared with the control as well as the GNO + Hg and Hg + GNO exposed animals. SOD inhibition may be related to the covalent attachment of mercury ions to SOD active site reactive cysteine residues (Shimojo et al., 2002) or to a decreased availability of Cu and Zn as a result of their binding to metallothioneins, which are involved in the detoxification of metals such as mercury (Brzoska et al., 2002).

Alternatively, SOD inhibition may also be a consequence of excessive reactive oxygen species generation, which would affect enzyme structure (Salo et al., 1988). SOD catalyzes superoxide anion radical dismutation into hydrogen peroxide. Therefore, regardless of the underlying mechanism, SOD inhibition may contribute to the enhanced oxidation observed in mercury-exposed rats.

A similar pattern was also observed for GPx activity as it was increased in the kidney of the GNO + Hg group, but in pre- or post-treatment with curcumin GPx activity was lower than that of the control group. This may be due to the decreased SOD activity in curcumin treatment alone. The decreased CAT activity comes to normal level in both the pre and post-treatment with curcumin. Similarly, we also found the complete restoration of LPO levels in all the tissues examined after curcumin treatment of the mercury-exposed animals. 

Mercury was also detected in whole blood (Fig. 1F), which indicates the circulation of mercury in the body after mercury administration. ALP is a sensitive biomarker and is related to the transport of various metabolites (El-Demerdash, 2001). A significant decrease was observed in serum ALP activity after mercury treatment, which may be due to a breakdown of the membrane transport system, an inhibitory effect on cell growth and proliferation in mercury toxicity (Lakshmi et al., 1991).

The increased activity of LDH (Fig. 2B) may be due to hepatic damage induced by environmental pollutants (Fernandez et al., 1994). El-Demerdash (2001) also found decrease in ALP activity and an increase in LDH activity after mercury exposure. The increased levels of creatinine and BUN indicate mercury-induced nephrotoxicity (Fig. 2C, D), which is also reported by Rumbeiha et al. (2000). ALP and LDH are the best biomarkers of liver and kidney diseases (Pesce, 1984; Wenger, 1984), whereas alterations in BUN and creatinine levels indicate renal toxicity (Murray, 1984; Kaplan, 1984). Histopathological alterations also supported these findings in the liver and kidney tissues associated with mercury toxicity.

We found hepatocyte damage, loss of nuclei and picnotic nuclei in the liver tissue (Fig. 3), which is due to mercury toxicity as also reported by Carmichael and Fowler (1979) and Al-Saleh et al. (2005). In kidney tissue, the sign of toxicity was also observed (Figs 4 and 5). Rumbeiha et al. (2000), Al-Saleh et al. (2005), Alam et al. (2007) and Augusti et al. (2007) have also reported similar changes due to mercury-induced nephrotoxicity. The high MT-I and MT-II mRNA expression levels in the liver tissues of the GNO + Hg group may be due to the oxidative stress caused by ROS generated by mercury toxicity. This is also shown by alteration in LPO and GSH levels and in the activities of SOD, GPx, CAT, ALP and LDH in the liver tissue. Similarly, the kidney tissue also showed induction in MT-I and MT-II mRNA expressions. However, the expression levels were lower than those in the liver tissue. The reason for this may be that the hepatic burden of mercury is as much as 7–8% of the dose 1 h after exposure (Zalups et al., 1999). 

Thus, the maximal hepatic burden of mercury is reached rapidly after exposure, and hepatic elimination of mercury begins within hours. In the Hg + GNO group, induction of MT-I and MT-II mRNA expressions was also observed in both the liver and kidney tissues. 

However, the kidney tissue showed higher expression levels than liver tissue. This may be due to only about 5–6% of the dose present in the liver by the end of the first day after exposure (Zalups and Cherian, 1992; Zalups, 1995).

Numerous studies (Zalups and Cherian, 1992; Zalups, 1995, 2000) indicate that the kidneys of rats exposed to a low dose of inorganic mercury accumulate approximately one-half the dose during the initial 24 h after exposure. Approximately 75% of mercury accumulated in the kidneys is retained by tubular epithelial cells over the subsequent weeks after exposure (Zalups and Koropatnick, 2000).

Curcumin has a long history as a food additive and herbal medicine in India and is also a potent polyphenolic antioxidant. It is a yellow curry spice derived from turmeric. Curcumin is several times more potent than vitamin E as a free radical scavenger, protects the brain from lipid peroxidation and scavenges NO-based radicals. It is a non-toxic, highly promising natural antioxidant compound with a wide spectrum of biological functions. 

The antioxidant activity of curcumin was reported as early as 1975 (Sharma, 1976). Studies have consistently shown that curcumin is relatively nontoxic. Studies using curcumin at 2000 mg kg-1 b.w. revealed no mortalities in mice. Curcumin was administered to wistar rats, guinea pigs and monkeys of both sexes at a dose of 300 mg kg-1 b.w. No pathological or behavioural abnormalities or lethality was observed. No adverse effects were observed on growth, counts of erythrocytes and leucocytes and levels of blood constituents such as hemoglobin, total serum protein and alkaline phosphatase. Human clinical trials also indicate that curcumin is non toxic when administered at doses of 1–8 and 10 g/day (Chattopadhyay et al. 2004).

Curcumin acts as a scavenger of oxygen free radicals (Subramaniam et al., 1994; Ruby et al., 1995) and can protect hemoglobin from oxidation (Unnikrishnan and Rao, 1995). Curcumin also decreases the production of ROS in vivo (Joe and Lokesh, 1994) and inhibits lipid peroxidation in rat liver microsomes, erythrocyte membranes and brain homogenates (Pulla and Lokesh, 1992). Interestingly, curcumin not only exhibits antioxidative and free radical scavenging properties, but also increases the activities of other antioxidant enzymes, such as SOD, CAT and GPx (Pulla and Lokesh, 1994).

To examine the role of curcumin in mercury toxicity, we carried out curcumin pre- or post-treatments to evaluate the prophylactic or therapeutic effects of curcumin. From our findings, it is clear that curcumin prevents mercury toxicity in terms of attenuated LPO, decreased GSH, creatinine and BUN levels and altered endogenous enzyme activities in the liver, kidney, brain and blood tissues. Groups with pre- or post-treated with curcumin also showed reduced mercury concentration in the liver, kidney and blood compared with their corresponding mercury-treated groups.

However, statistically significant mercury concentration reductions in all the tissues were found only in curcumin post-treatment. Curcumin treatment showed no protective effect against histopathological changes in the liver and kidney tissues. This may be due to the fact that the time required for the repair of damage at the cellular level may be short, during which reversal of histopathological changes is not possible.

We found some induction of MT-I and MT-II mRNA expressions in the liver and kidney tissues after curcumin treatment alone, which may be due to ROS normally present in the animals that might be scavenged from the body by curcumin treatment. This elucidated the antioxidative property of curcumin. Pretreatment with curcumin resulted in complete protection of the liver and kidney tissues. In the curcumin-post-treated group, further induction of metallothionein mRNA expression was observed in the liver and kidney tissues. The kidney tissue showed greater induction than the liver tissue. This may be due to the slow elimination of mercury from the kidney tissue.

Thus we observed that curcumin pretreatment resulted in the complete protection against mercury exposure in terms of oxidative stress. In conclusion, the present work suggests that curcumin intake should be helpful in the prevention of mercury toxicity and that curcumin can be used as a therapeutic agent for mercury intoxication.

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