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Protection against mercury-induced renal damage in Swiss albino mice by Ocimum sanctum
Environmental Toxicology and Pharmacology 19 (2005) 161–167
Protection against mercury-induced renal damage in Swiss albino mice by Ocimum sanctum Mukesh Kumar Sharma, Madhu Kumar∗ , Ashok Kumar Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 23 October 2003; accepted 24 June 2004 Available online 24 August 2004
Abstract Mercury is being widely used in the industry, medical, agriculture and other fields. However, mercury deposition affects the nervous, cardiovascular, pulmonary, gastrointestinal and renal systems, as well as the embryo. In most animals’ species, including man, the kidney is one of the main sites of deposition of inorganic mercury and target organ for its toxicity. The present investigation reports protection against mercury-induced toxicity by Ocimum sanctum (a traditional sacred medicinal plant, family: Labiatae). Swiss albino mice were divided into four groups. (i) Control group—only vehicle (0.9% NaCl) was given (ii) HgCl2 -treated group–5.0 mg/kg b.w. HgCl2 administered as i.p. (iii) Ocimum treated group—10 mg/kg b.w. Ocimum leaves extract was administered orally. (iv) Combination group—Ocimum leaves extract was administered 10 days prior to mercuric chloride administration and continued upto 30 days after mercuric chloride administration (5.0 mg/kg b.w.). The animals were autopsied on day 1, 3, 7, 15 and 30 after treatment. Activity of alkaline phosphatase (ALP), acid phosphatase (ACP), lactate dehydrogenase (LDH) and lipid peroxidation (LPO) were measured in kidney homogenates. The results indicated that there was a significant increase in LPO content, ACP activity and decrease in LDH and ALP activity after HgCl2 treatment. The animals treated with Ocimum alone did not show any significant alterations in ACP and ALP activity. However, a significant increase in LDH activity and decrease in LPO level was observed. In combined treatment of Ocimum with HgCl2 , a significant decrease in LPO content and ACP and elevation in LDH and ALP activity was observed as compared to HgCl2 -treated group. Ocimum extract is also effective in reducing the pathological alterations in the kidney. Thus, the results from the present study suggest that pre-and post-treatment of Ocimum sanctum leaves extract can significantly protect the renal damage against mercuric chloride-induced toxicity. © 2004 Elsevier B.V. All rights reserved. Keywords: Kidney; Mercury; Ocimum; LPO; Phosphatase; LDH
1. Introduction Mercury is being widely used in the industrial, medical, agriculture and other fields (ATSDR, 1989). Mercury is a transitional metal; it promotes the formation of reactive oxygen species (ROS) such as hydrogen peroxides. These ROS enhances the subsequent iron- and copper-induced production of lipid peroxides and the highly reactive hydroxyl radical (Miller et al., 1991; Hussain et al., 1999). These lipid peroxides and hydroxyl radical may cause the cell membrane ∗
Corresponding author. Fax: +91 141 2245725. E-mail addresses: [email protected] (M.K. Sharma), [email protected] (M. Kumar), [email protected] (A. Kumar). 1382-6689/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.06.002
damage and thus destroy the cell. Mercury also inhibits the activities of the free radical quenching enzymes catalase, superoxide dismutase and GSH peroxidase (Benov et al., 1990). Inorganic mercury present in the environment is a well established toxicant to human health (WHO, 1991). It is well known that inorganic mercury causes severe kidney damage after acute and chronic exposure (Zalups, 1997). Thus it is important to develop an effective drug to prevent the mercuryinduced renal damage. Several naturally occurring dietary or non-dietary constituents, as well as parts of several species of edible plants having pharmacological activity, influence the antioxidant enzymes and provide protection against free radical induced damage.
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Earlier in our laboratory we have reported that the Ocimum sanctum leaves extract was effective in reducing the mortality rate and liver damages against mercuric chloride-induced toxicity (Kumar et al., 2001; Sharma et al., 2001; Sharma et al., 2002). Ocimum sanctum (Sacred basil; Green Tulsi; Family-Labiatae) is a traditional medicinal plant. The oil of Ocimum sanctum possesses antibacterial, antifungal (Sinha and Gulati, 1990), antistress (Bhargava and Singh, 1981), immunostimulatory (Mediratta et al., 1988), anticarcinogenic, antioxidant (Banerjee et al., 1996), and radioprotective (Devi et al., 1999) effects. The present investigation has been carried out to evaluate the role of Ocimum sanctum leaves extract in protecting the mercury-induced renal damages in Swiss albino mice.
2. Material and methods 2.1. Animals Random-bred, male Swiss albino mice (6–8 weeks old) were obtained from animal facility, IVRI, Izatnagar (India). The animals were provided with standard mice feed (Hindustan Lever Ltd., India) and tap water ad libitum. Once in a fortnight tetracycline water was given as a preventive measure against infection. 2.2. Ocimum sanctum Ocimum sanctum is an annual herb. Fresh leaves of Ocimum collected locally, were air dried, powdered mechanically and extracted with double distilled water at 60 ◦ C for 36 h. The aqueous extract was vacuum evaporated to powdered form. The extract was re-dissolved in double distilled water just before administration. 2.3. Toxicity of mercury Mercury in the form of inorganic mercury (Mercuric chloride) was used for the present study. It was dissolved in 0.9% NaCl. The dose 5.0 mg/kg body weight dose was selected for the present experiment on the basis of our previous report (Sharma et al., 2002). Mercury was administered intraperitoneally.
2.5. Experimental protocol The animal Swiss albino mice were divided into the following four groups: Group I (n = 30): Only vehicle (0.9%NaCl) was given to these animals (Control). Group II (n = 30): The animals were administered HgCl2 5.0 mg/kg body weight in 0.9% NaCl intraperitoneally at once. Group III (n = 30): The animals were administered orally, Ocimum sanctum leaves extract (10 mg/kg body weight) for 30 consecutive days. Group IV (n = 30): The animals were administered Ocimum extract 10 mg/kg body weight orally for 10 days prior to mercuric chloride (5.0 mg/kg body weight) upto 30 days after mercuric chloride administration. These animals were autopsied on 1, 3, 7, 15 and 30 days and kidneys were removed and processed for various biochemical and histological studies. 2.6. Biochemical studies 2.6.1. Alkaline phosphatase activity The alkaline phosphatase activity in the kidney was estimated by Fiske and Subbarow (1925) method. The alkaline phosphatase activity is the difference between inorganic phosphatase content of the incubated and control samples expressed as Bodansky unit. One Bodansky unit corresponds to the liberation of 1 mg of inorganic phosphorous from the tissue in 1 mg/(g h)−1 . In this method the protein content of the tissue homogenates was first precipitated with trichloroacetic acid. The filterate was then treated with molybdate solution. This resulted in the formation of phosphomolybdic acid from the phosphate present in the tissue. The phosphomolybdic acid was then reduced to produce a blue colour whose intensity was proportional to the amount of phosphate liberated. 2.6.2. Acid phosphatase activity Acid phosphatase activity in the kidney was also estimated by the method of Fiske and Subbarow (1925) for the determination of phosphate liberation.
2.4. Chemicals Pyridine, TMP, Sodium dodecyl sulphate, Thio barbituric acid, Sodium b-glycerophosphate, Diethyl barbituric acid and ANSA were procured from Sigma Chemical Company, Hyderabad, (INDIA). Ammonium Molybdate, Dinitrophenyl Hydrazine (DNPH) and Sodium pyruvate were procured from SRL Pvt. Ltd, Mumbai, (INDIA). Trichloro acetic acid was procured from Qualigens Mumbai, (INDIA). TMP (1,1,3,3tetramethoxy propane) was procured from Lancaster, England.
2.6.3. Lactate dehydrogenase activity Lactate dehydrogenase activity in the kidney was estimated by “Wroblewski procedure” given in the sigma technical bulletin no. 500 (September) (Wroblewski, 1967). The substrate used was sodium pyruvate; pH of the buffer was maintained at 7.5 at 37 ◦ C. In the presence of enzyme pyruvic acid it was converted into lactic acid. Further pyruvic acid reacts with 2,4-dinitrophenyl hydrazine and forms an intensely coloured brown hydrazone, which has a high OD at 400–500 nm.
M.K. Sharma et al. / Environmental Toxicology and Pharmacology 19 (2005) 161–167
2.6.4. Lipid peroxidation assay Lipid peroxidation level in the kidney was measured by the method of Okhawa et al. (1979) by thiobarbituric acid reactive substances (TBARS). The concentration of TBARS was expressed as n moles of malondialdehyde per mg of tissue using 1,1,3,3-tetramethoxypropane (TMP) as standard. 2.7. Histological studies Kidneys from autopsied animals were excised out and fixed in Bouins fixatives for 24–48 h. The fixed tissue was further processed by standard method. The sections were stained with Haematoxylin and Eosin. 2.8. Statistical analysis Data are expressed as mean ± S.E. Statistical significance of difference between groups was determined by Student’s t-test.
3. Results 3.1. Biochemical estimations 3.1.1. Alkaline phosphatase In HgCl2 -treated group a highly significant (P < 0.001) inhibition was noticed in alkaline phosphatase activity at all autopsy intervals with respect to their control animals. Ocimum treatment alone did not show any significant alteration in alkaline phosphatase activity at all autopsy intervals. Whereas the combined treatment of Ocimum with mercury, results in highly significant elevation in alkaline phosphatase activity at all autopsy intervals with respect to Hg-intoxicated mice. The normalcy was attained upto day 30 (Scheme 1).
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3.1.2. Acid phosphatase A highly significant (P < 0.001) elevation in acid phosphatase activity was observed in Hg-intoxicated mice throughout whole experimentation period. Only Ocimum treatment did not show any significant alteration in acid phosphatase activity at all autopsy intervals. However, the combined treatment of Ocimum and mercury results in gradual recovery in acid phosphatase activity. A highly significant decline in acid phosphatase activity was noticed throughout the whole experimentation period with respect to HgCl2 -treated animals (Scheme 2). 3.1.3. Lactate dehydrogenase (LDH) A highly significant (P < 0.001) decline was observed in LDH activity in Hg-intoxicated mice with respect to control animals. Ocimum treatment alone did not show any significant alteration in LDH activity at all autopsy intervals. Whereas, pre- and post-treatment of Ocimum with mercury results in significant elevation in LDH activity throughout whole experimentation period with respect to Hg-intoxicated mice (Scheme 3). 3.1.4. Lipid peroxidation (LPO) A highly significant (P < 0.001) elevation was observed in LPO level in Hg-intoxicated mice. In Ocimum treatment alone, LPO level shows a significant decline whereas, combined treatment of Ocimum and mercury shows a highly significant depletion in LPO level at all autopsy intervals with respect to Hg-intoxicated mice (Scheme 4). 3.2. Histological investigation Histological investigation showed widespread morphological alteration in kidney from HgCl2 -intoxicated mice, one-day post-administration. These alterations were characterized by widespread proximal tubular necrosis with absence of brush border and desquamated necrotic epithelial
Scheme 1. Variation in kidney alkaline phosphatase activity in different experimental groups.
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Scheme 2. Variation in kidney acid phosphatase activity in different experimental groups.
Scheme 3. Variation in kidney LDH activity in different experimental groups.
Scheme 4. Variation in kidney LPO content in different experimental groups.
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Fig. 1. Photomicrograph of kidney showing (a) normal kidney histoarchitecture with distinct proximal tubules with tall epithelium and small lumina and distal tubules with a cuboidal epithelium and larger lumina. H.E. (400×). (b) Degeneration in glomerulus, epithelial of proximal and cuboidal epithelium of distal tubules at day 1 in HgCl2 -treated animals. H.E. (400×). (c) Cellular debris in proximal and distal tubules and absence of brush borders in proximal tubules at day 3. H.E. (200×). (d) Severe necrosis of glomerulus alongwith proximal and distal tubules at day 30. H.E. (400×). (e) Reparative changes. Slight degeneration in proximal tubules with intact glomeruli in Ocimum pre- and post-treated animals. H.E. (400×). (f) Nearly nomal histoarchitecture of proximal and distal tubules. H.E. (400×).
cells in the lumen. Cellular debris was more prominent on day 7 in the lumen of both proximal and distal tubules. The pathological alterations became more pronounced. Pyknosis, karyolysis and karyorhexsis were observed in epithelial cells nuclei, indicating that they were in the process of dyeing. On days 15 and 30 it was difficult to distinguish between different tubules and the whole picture appeared necrotic. The glomerulus showed degenerative changes (Fig. 1a–d).
Kidney showed reparative tendencies in Ocimum preand post-treated group. On day one proximal- and distalconvoluted tubules showed slight degeneration at a few places. Proximal tubule cuboidal epithelium showed absence of brush border but the lumen was not occluded. Upto day 30 the sign of reparation was observed with normal epithelial lining and normal nuclei as compared to HgCl2 -treated group. From day 1 upto day 30,
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glomerulus appeared normal without any epithelium damage (Fig. 1e–f).
4. Discussion The kidney appears to be the critical organ of toxicity for the ingestion of mercuric salts. Several widespread alterations were observed in kidney from Hg-intoxicated mice. It is well established that pars recta (straight segment) of the proximal tubule (particularly the portion at the junction of the cortex and outer medulla) is the segment of the nephron that is most vulnerable to the toxic effects of both inorganic and organic forms of mercury (Zalups et al., 1991b; Zalups and Barfuss, 1996b). It is likely that the luminal and/or basolateral transport of mercury into the proximal tubular epithelial cells is through co-transport of mercury with an endogenous ligand such as glutathione, cysteine or albumin, or through some plasma membrane mercury–ligand complex. In contrast to the distal tubule, which is characterized by relatively tight epithelium with high electrical resistance, the proximal tubule is a leaky epithelium favoring the flux of compounds into the proximal tubular cells. Thus, transporting GSH conjugates, heavy metals and organic anions and cations. It is localized mainly to the proximal tubule than other segments, resulting in proximal tubular accumulation and cellular toxicity of these xenobiotics (Zalups, 1995). The present investigation revealed that mercury intoxication causes significant increase in lipid peroxidation and acid phosphatase and significant decrease in alkaline phosphatase and lactate dehydrogenase activity. The enzyme lactate dehydrogenase (LDH) catalyses the interconversion of lactate and pyruvate in the glycolytic pathway and occurs as a tetrameric molecules. The alkaline phosphatase enzyme is associated with transfer of phosphates and is linked with transportation of intermediate compounds in glycogenesis or glycogenolysis. In the present investigation there was a decrease in LDH and alkaline phosphatase activity after the exposure of mercuric chloride. This may be due to loss of brush border of the proximal tubular cells at an early stage of renal epithelial cellular necrosis. Inorganic mercury directly inhibits LDH activity (Smith et al., 1986). The sensitivity of LDH to Hg suggests cellular toxicity (Smith et al., 1986; Mehra and Kanwar, 1986; Chung and Lee, 1987). There was significant increase in acid phosphatase activity after HgCl2 exposure. Acid phosphatase activity is localized in cellular lysosomes. An enhanced peroxidation of lysosomal membranes due to HgCl2 intoxication causes lysis of membrane and oozing out of the enzyme and hence results in an increased acid phosphatase activity (Mehra and Kanwar, 1986). In the present investigation the lipid peroxidation level showed a highly significant decline following Hg exposure. Mercury has high affinity for GSH and causes the irreversible
excretion of, up to two GSH tripeptides (Zalups and Lash, 1996). The metal-GSH conjugation process is desirable for the excretion of the toxic metal into the bile. However, it depletes the GSH from the cell and thus decreases the antioxidant potential. We have also reported that mercury causes significant depletion of hepatic-GSH (Sharma et al., 2002). Mercury is a transition metal; it also promotes the formation of reactive oxygen species by fenton transition equation, such as hydrogen peroxides and enhances the subsequent ironand copper-induced production of lipid peroxides and the highly reactive hydroxyl radical (Miller et al., 1991; Hussain et al., 1999). Lipid peroxides alter membrane structure and mitochondrial structure. From the present investigation it can be suggested that HgCl2 treatment significantly reduces the GSH content and the antioxidant potential and thus accelerates the lipid peroxidation, resulting in renal cellular damage. It was observed that an extract of Ocimum sanctum when given alone or in combination significantly declines lipid peroxidation and reduces the mercury toxicity which in turn is reflected by a significant decrease in acid phosphatase activity and an increase in alkaline phosphatase and LDH activity. The protective activity of Ocimum sanctum aqueous leaves extract against mercury-induced toxicity, may to some extent, be mediated through the release of intracellular antioxidants which, in turn, will scavenge the free radicals and also may help in repair of biochemical lesions. Some of the compounds found in the Ocimum plant have been reported to possess strong antioxidant activity. Eugenol and ursolic acid from Ocimum sanctum have been reported to induce protection against free radical induced cellular damages (Balanehru and Nagarajan, 1992; Rajkumar and Rao, 1993). The Ocimum flavonoids Orientin and Vicenin have also exhibited strong inhibitory effect on the fenton reaction generated OH radical activity. They have strong antioxidant activity in vitro and anti lipid peroxidative effect in vivo, which strongly suggest free radical scavenging as a major mechanism by which Ocimum products protect the cellular damage (Ganasoundari et al., 1997, 1998; Uma Devi et al., 2000). In vitro studies further indicate that metal chelation may also have a role in the anti-lipid per oxidative effect of Ocimum leaves (Ganasoundari et al., 1997; Uma Devi et al., 2000). In addition, immune stimulation was suggested as a mechanism contributing to the adaptogenic action of plant (Godhwani et al., 1988). Further, it has been reported that Ocimum sanctum aqueous leaves extract when given alone or in combination significantly enhances the GSH content (Sharma et al., 2002) by inducing the antioxidant potential and thus reduces the reactive oxygen species generated cellular damage. Thus, the present study suggests that deleterious reactive oxygen species or lipid peroxides responsible for HgCl2 induced toxicity may be alleviated by aqueous extract of Ocimum leaves, which in turn reflected in significant decline in LPO content and acid phosphatase and significant enhancement in LDH and alkaline phosphatase activity as compared to HgCl2 -treated group in Swiss albino mice.
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5. Conclusion Pre- and post-treatment of Ocimum sanctum aqueous leaves extract significantly protect against mercury-induced renal toxicity.
Acknowledgement Financial assistance provided by CSIR-New Delhi to one of us (MKS) by grant. No. 9/149/247/2000 (EMR) is highly acknowledged.
References ATSDR (Agency for Toxic Substances and Disease Registry), 1989. Toxicological Profile for Mercury. ATSDR/US. Public Health Service. Balanehru, S., Nagarajan, B., 1992. Intervention of adriamycin-induced free radical damage by ursolic acid. Biochem. Int. 28, 735. Banerjee, S., Prashar, R., Kumar, A., Rao, A.R., 1996. Modulatory influence of alcoholic extract of Ocimum leaves on carcinogenmetabolizing enzyme activities and reduced glutathione levels in mouse. Nutri. Cancer. 25, 205. Benov, L.C., Benchev, I.C., Monovich, O.H., 1990. Thiol antidotes effect on lipid peroxidation in mercury-poisoned rats. Chem. Biol. Interact. 76, 321. Bhargava, K.P., Singh, N., 1981. Antistress activity of Ocimum sanctum linn. Indian J. Med. Res. 73, 443. Chung, H.W., Lee, C.K., 1987. Detoxification effect of red ginseng extract on toxicity of methylmercury chloride to LDH in the liver, kidney, and serum of mouse. Korean J. Zool. 30 (3), 231–238. Devi, P.U., Ganasoundari, A., Rao, B.S.S., Srinivasan, K.K., 1999. In vivo radioprotection by Ocimum flavonoids: survival of mice. Radiat. Res. 151, 74. Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorous. J. Biol. Chem. 66, 375–400. Ganasoundari, A., Uma Devi, P., Rao, B.S.S., 1998. Enhancement of bone marrow radiation protection and reduction in WR-2721 toxicity by Ocimum sanctum. Mutat. Res. 397, 303. Ganasoundari, A., Uma Devi, P., Rao, M.N.A., 1997. Protection against radiation induced chromosome damage in mouse bone marrow by Ocimum sanctum. Mutat. Res. 373, 599. Godhwani, S., Godhwani, J.L., Vyas, D.S., 1988. Ocimum sanctum—a preliminary study evaluating its immunostimulatory profile in albino rats. J. Ethnopharmacol. 24, 193. Hussain, S., Atkinson, A., Thompson, S.J., Khan, A.T., 1999. Accumulation of mercury and its effect on antioxidant enzymes in brain, liver and kidneys of mice. J. Environ. Sci. Health B. 34 (4), 645–660.
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Kumar, M., Saxena, P.S., Sharma, M.K., Patni, R., 2001. Modulation of toxic effects of mercury by Ocimum sanctum and Spirulina fusiformis. J. Med. Arom. Plant Sci. 22/4A–23/1A, 98–101. Mediratta, P.K., Dewan, V., Bhattacharya, S.K., Gupta, U.S., Maiti, P.C., 1988. Effect of Ocimum sanctum linn. on humoral immune responses. Indian J. Med. Res. 87, 384. Mehra, M., Kanwar, K.C., 1986. Enzyme changes in the brain, liver, and kidney following repeated administration of mercuric chloride. J. Environ. Pathol. Toxicol. Oncol. 7 (1–2), 65–71. Miller, O.M., Lund, B.O., Woods, J.S., 1991. Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II). J. Biochem. Toxicol. 6, 293. Okhawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Analyt. Biochem. 95, 351–358. Rajkumar, D.V., Rao, M.N.A., 1993. Dehydrozingerone and isoeugenol as inhibiotrs of lipid peroxidation and as free radical scavengers. Biochem. Pharmacol., 46. Sharma, M.K., Kumar, M., Kumar, A., 2001. Modulatory influence of Ocimum sanctum and Spirulina fusiformis against mercury-induced toxicity in liver. Bull. Env. Sci. XIX, 85–91. Sharma, M.K., Kumar, M., Kumar, A., 2002. Ocimum sanctum aqueous leaf extract provides protection against mercury-induced toxicity in Swiss albino mice. Indian J. Exp. Biol. 40, 1079–1082. Sinha, G.K., Gulati, B.C., 1990. Antibacterial and antifungal study of some essential oils and some of their constituents. Indian Perfumer. 34, 126. Smith, M.A., Acosta, D., Bruckner, J.V., 1986. Development of a primary culture system of rat kidney cortical cells to evaluate nephrotoxicity of xenobiotics. Food Chem. Toxicol. 24, 551–556. Uma Devi, P., Ganasoundari, A., Vrinda, B., Srinivasan, K.K., Unnikrishnan, M.K., 2000. Radiation protection by the Ocimum flavonoids orientin and vicenin–mechanisms of actioin. Radiat. Res. 154, 455. WHO, 1991. Environmental health criteria 118: inorganic mercuryenvironmental aspects. World Health Organization, Geneva, Switzerland, 115–119. Wroblewski, F., 1967. Sigma technical bulletin no. 500. Zalups, R.K., Barfuss, D.W., 1996b. Nephrotoxicity of inorganic mercury co-administered with L-cysteine. Toxicology 109, 15–29. Zalups, R.K., Gelein, R.M., Cernichiari, E., 1991b. DMPS as a rescue agent for the nephropathy induced by mercuric chloride. J. Pharmacol. Exp. Ther. 256, 1–10. Zalups, R.K., Lash, L.H., 1996. Interactions between glutathione and mercury in the kidney, liver and blood. In: Chang, L.W. (Ed.), Toxicology of Metals. CRC Press, Boca Raton, pp. 145–163. Zalups, R.K., 1995. Organic anion transport and action of ␥glutamyltranspeptidase in kidney linked mechanistically to renal tubular uptake of inorganic mercury. Toxicol. Appl. Pharmacol. 132, 289–298. Zalups, R.K., 1997. Enhanced renal outer medullary uptake of mercury associated with uninephrectomy: implication of a luminal mechanism. J. Toxicol. Environ. Health 50, 173–194.
Protection against mercury-induced renal damage in Swiss albino mice by Ocimum sanctum Mukesh Kumar Sharma, Madhu Kumar∗ , Ashok Kumar Department of Zoology, University of Rajasthan, Jaipur 302004, India Received 23 October 2003; accepted 24 June 2004 Available online 24 August 2004
Abstract Mercury is being widely used in the industry, medical, agriculture and other fields. However, mercury deposition affects the nervous, cardiovascular, pulmonary, gastrointestinal and renal systems, as well as the embryo. In most animals’ species, including man, the kidney is one of the main sites of deposition of inorganic mercury and target organ for its toxicity. The present investigation reports protection against mercury-induced toxicity by Ocimum sanctum (a traditional sacred medicinal plant, family: Labiatae). Swiss albino mice were divided into four groups. (i) Control group—only vehicle (0.9% NaCl) was given (ii) HgCl2 -treated group–5.0 mg/kg b.w. HgCl2 administered as i.p. (iii) Ocimum treated group—10 mg/kg b.w. Ocimum leaves extract was administered orally. (iv) Combination group—Ocimum leaves extract was administered 10 days prior to mercuric chloride administration and continued upto 30 days after mercuric chloride administration (5.0 mg/kg b.w.). The animals were autopsied on day 1, 3, 7, 15 and 30 after treatment. Activity of alkaline phosphatase (ALP), acid phosphatase (ACP), lactate dehydrogenase (LDH) and lipid peroxidation (LPO) were measured in kidney homogenates. The results indicated that there was a significant increase in LPO content, ACP activity and decrease in LDH and ALP activity after HgCl2 treatment. The animals treated with Ocimum alone did not show any significant alterations in ACP and ALP activity. However, a significant increase in LDH activity and decrease in LPO level was observed. In combined treatment of Ocimum with HgCl2 , a significant decrease in LPO content and ACP and elevation in LDH and ALP activity was observed as compared to HgCl2 -treated group. Ocimum extract is also effective in reducing the pathological alterations in the kidney. Thus, the results from the present study suggest that pre-and post-treatment of Ocimum sanctum leaves extract can significantly protect the renal damage against mercuric chloride-induced toxicity. © 2004 Elsevier B.V. All rights reserved. Keywords: Kidney; Mercury; Ocimum; LPO; Phosphatase; LDH
1. Introduction Mercury is being widely used in the industrial, medical, agriculture and other fields (ATSDR, 1989). Mercury is a transitional metal; it promotes the formation of reactive oxygen species (ROS) such as hydrogen peroxides. These ROS enhances the subsequent iron- and copper-induced production of lipid peroxides and the highly reactive hydroxyl radical (Miller et al., 1991; Hussain et al., 1999). These lipid peroxides and hydroxyl radical may cause the cell membrane ∗
Corresponding author. Fax: +91 141 2245725. E-mail addresses: [email protected] (M.K. Sharma), [email protected] (M. Kumar), [email protected] (A. Kumar). 1382-6689/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2004.06.002
damage and thus destroy the cell. Mercury also inhibits the activities of the free radical quenching enzymes catalase, superoxide dismutase and GSH peroxidase (Benov et al., 1990). Inorganic mercury present in the environment is a well established toxicant to human health (WHO, 1991). It is well known that inorganic mercury causes severe kidney damage after acute and chronic exposure (Zalups, 1997). Thus it is important to develop an effective drug to prevent the mercuryinduced renal damage. Several naturally occurring dietary or non-dietary constituents, as well as parts of several species of edible plants having pharmacological activity, influence the antioxidant enzymes and provide protection against free radical induced damage.
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Earlier in our laboratory we have reported that the Ocimum sanctum leaves extract was effective in reducing the mortality rate and liver damages against mercuric chloride-induced toxicity (Kumar et al., 2001; Sharma et al., 2001; Sharma et al., 2002). Ocimum sanctum (Sacred basil; Green Tulsi; Family-Labiatae) is a traditional medicinal plant. The oil of Ocimum sanctum possesses antibacterial, antifungal (Sinha and Gulati, 1990), antistress (Bhargava and Singh, 1981), immunostimulatory (Mediratta et al., 1988), anticarcinogenic, antioxidant (Banerjee et al., 1996), and radioprotective (Devi et al., 1999) effects. The present investigation has been carried out to evaluate the role of Ocimum sanctum leaves extract in protecting the mercury-induced renal damages in Swiss albino mice.
2. Material and methods 2.1. Animals Random-bred, male Swiss albino mice (6–8 weeks old) were obtained from animal facility, IVRI, Izatnagar (India). The animals were provided with standard mice feed (Hindustan Lever Ltd., India) and tap water ad libitum. Once in a fortnight tetracycline water was given as a preventive measure against infection. 2.2. Ocimum sanctum Ocimum sanctum is an annual herb. Fresh leaves of Ocimum collected locally, were air dried, powdered mechanically and extracted with double distilled water at 60 ◦ C for 36 h. The aqueous extract was vacuum evaporated to powdered form. The extract was re-dissolved in double distilled water just before administration. 2.3. Toxicity of mercury Mercury in the form of inorganic mercury (Mercuric chloride) was used for the present study. It was dissolved in 0.9% NaCl. The dose 5.0 mg/kg body weight dose was selected for the present experiment on the basis of our previous report (Sharma et al., 2002). Mercury was administered intraperitoneally.
2.5. Experimental protocol The animal Swiss albino mice were divided into the following four groups: Group I (n = 30): Only vehicle (0.9%NaCl) was given to these animals (Control). Group II (n = 30): The animals were administered HgCl2 5.0 mg/kg body weight in 0.9% NaCl intraperitoneally at once. Group III (n = 30): The animals were administered orally, Ocimum sanctum leaves extract (10 mg/kg body weight) for 30 consecutive days. Group IV (n = 30): The animals were administered Ocimum extract 10 mg/kg body weight orally for 10 days prior to mercuric chloride (5.0 mg/kg body weight) upto 30 days after mercuric chloride administration. These animals were autopsied on 1, 3, 7, 15 and 30 days and kidneys were removed and processed for various biochemical and histological studies. 2.6. Biochemical studies 2.6.1. Alkaline phosphatase activity The alkaline phosphatase activity in the kidney was estimated by Fiske and Subbarow (1925) method. The alkaline phosphatase activity is the difference between inorganic phosphatase content of the incubated and control samples expressed as Bodansky unit. One Bodansky unit corresponds to the liberation of 1 mg of inorganic phosphorous from the tissue in 1 mg/(g h)−1 . In this method the protein content of the tissue homogenates was first precipitated with trichloroacetic acid. The filterate was then treated with molybdate solution. This resulted in the formation of phosphomolybdic acid from the phosphate present in the tissue. The phosphomolybdic acid was then reduced to produce a blue colour whose intensity was proportional to the amount of phosphate liberated. 2.6.2. Acid phosphatase activity Acid phosphatase activity in the kidney was also estimated by the method of Fiske and Subbarow (1925) for the determination of phosphate liberation.
2.4. Chemicals Pyridine, TMP, Sodium dodecyl sulphate, Thio barbituric acid, Sodium b-glycerophosphate, Diethyl barbituric acid and ANSA were procured from Sigma Chemical Company, Hyderabad, (INDIA). Ammonium Molybdate, Dinitrophenyl Hydrazine (DNPH) and Sodium pyruvate were procured from SRL Pvt. Ltd, Mumbai, (INDIA). Trichloro acetic acid was procured from Qualigens Mumbai, (INDIA). TMP (1,1,3,3tetramethoxy propane) was procured from Lancaster, England.
2.6.3. Lactate dehydrogenase activity Lactate dehydrogenase activity in the kidney was estimated by “Wroblewski procedure” given in the sigma technical bulletin no. 500 (September) (Wroblewski, 1967). The substrate used was sodium pyruvate; pH of the buffer was maintained at 7.5 at 37 ◦ C. In the presence of enzyme pyruvic acid it was converted into lactic acid. Further pyruvic acid reacts with 2,4-dinitrophenyl hydrazine and forms an intensely coloured brown hydrazone, which has a high OD at 400–500 nm.
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2.6.4. Lipid peroxidation assay Lipid peroxidation level in the kidney was measured by the method of Okhawa et al. (1979) by thiobarbituric acid reactive substances (TBARS). The concentration of TBARS was expressed as n moles of malondialdehyde per mg of tissue using 1,1,3,3-tetramethoxypropane (TMP) as standard. 2.7. Histological studies Kidneys from autopsied animals were excised out and fixed in Bouins fixatives for 24–48 h. The fixed tissue was further processed by standard method. The sections were stained with Haematoxylin and Eosin. 2.8. Statistical analysis Data are expressed as mean ± S.E. Statistical significance of difference between groups was determined by Student’s t-test.
3. Results 3.1. Biochemical estimations 3.1.1. Alkaline phosphatase In HgCl2 -treated group a highly significant (P < 0.001) inhibition was noticed in alkaline phosphatase activity at all autopsy intervals with respect to their control animals. Ocimum treatment alone did not show any significant alteration in alkaline phosphatase activity at all autopsy intervals. Whereas the combined treatment of Ocimum with mercury, results in highly significant elevation in alkaline phosphatase activity at all autopsy intervals with respect to Hg-intoxicated mice. The normalcy was attained upto day 30 (Scheme 1).
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3.1.2. Acid phosphatase A highly significant (P < 0.001) elevation in acid phosphatase activity was observed in Hg-intoxicated mice throughout whole experimentation period. Only Ocimum treatment did not show any significant alteration in acid phosphatase activity at all autopsy intervals. However, the combined treatment of Ocimum and mercury results in gradual recovery in acid phosphatase activity. A highly significant decline in acid phosphatase activity was noticed throughout the whole experimentation period with respect to HgCl2 -treated animals (Scheme 2). 3.1.3. Lactate dehydrogenase (LDH) A highly significant (P < 0.001) decline was observed in LDH activity in Hg-intoxicated mice with respect to control animals. Ocimum treatment alone did not show any significant alteration in LDH activity at all autopsy intervals. Whereas, pre- and post-treatment of Ocimum with mercury results in significant elevation in LDH activity throughout whole experimentation period with respect to Hg-intoxicated mice (Scheme 3). 3.1.4. Lipid peroxidation (LPO) A highly significant (P < 0.001) elevation was observed in LPO level in Hg-intoxicated mice. In Ocimum treatment alone, LPO level shows a significant decline whereas, combined treatment of Ocimum and mercury shows a highly significant depletion in LPO level at all autopsy intervals with respect to Hg-intoxicated mice (Scheme 4). 3.2. Histological investigation Histological investigation showed widespread morphological alteration in kidney from HgCl2 -intoxicated mice, one-day post-administration. These alterations were characterized by widespread proximal tubular necrosis with absence of brush border and desquamated necrotic epithelial
Scheme 1. Variation in kidney alkaline phosphatase activity in different experimental groups.
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Scheme 2. Variation in kidney acid phosphatase activity in different experimental groups.
Scheme 3. Variation in kidney LDH activity in different experimental groups.
Scheme 4. Variation in kidney LPO content in different experimental groups.
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Fig. 1. Photomicrograph of kidney showing (a) normal kidney histoarchitecture with distinct proximal tubules with tall epithelium and small lumina and distal tubules with a cuboidal epithelium and larger lumina. H.E. (400×). (b) Degeneration in glomerulus, epithelial of proximal and cuboidal epithelium of distal tubules at day 1 in HgCl2 -treated animals. H.E. (400×). (c) Cellular debris in proximal and distal tubules and absence of brush borders in proximal tubules at day 3. H.E. (200×). (d) Severe necrosis of glomerulus alongwith proximal and distal tubules at day 30. H.E. (400×). (e) Reparative changes. Slight degeneration in proximal tubules with intact glomeruli in Ocimum pre- and post-treated animals. H.E. (400×). (f) Nearly nomal histoarchitecture of proximal and distal tubules. H.E. (400×).
cells in the lumen. Cellular debris was more prominent on day 7 in the lumen of both proximal and distal tubules. The pathological alterations became more pronounced. Pyknosis, karyolysis and karyorhexsis were observed in epithelial cells nuclei, indicating that they were in the process of dyeing. On days 15 and 30 it was difficult to distinguish between different tubules and the whole picture appeared necrotic. The glomerulus showed degenerative changes (Fig. 1a–d).
Kidney showed reparative tendencies in Ocimum preand post-treated group. On day one proximal- and distalconvoluted tubules showed slight degeneration at a few places. Proximal tubule cuboidal epithelium showed absence of brush border but the lumen was not occluded. Upto day 30 the sign of reparation was observed with normal epithelial lining and normal nuclei as compared to HgCl2 -treated group. From day 1 upto day 30,
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glomerulus appeared normal without any epithelium damage (Fig. 1e–f).
4. Discussion The kidney appears to be the critical organ of toxicity for the ingestion of mercuric salts. Several widespread alterations were observed in kidney from Hg-intoxicated mice. It is well established that pars recta (straight segment) of the proximal tubule (particularly the portion at the junction of the cortex and outer medulla) is the segment of the nephron that is most vulnerable to the toxic effects of both inorganic and organic forms of mercury (Zalups et al., 1991b; Zalups and Barfuss, 1996b). It is likely that the luminal and/or basolateral transport of mercury into the proximal tubular epithelial cells is through co-transport of mercury with an endogenous ligand such as glutathione, cysteine or albumin, or through some plasma membrane mercury–ligand complex. In contrast to the distal tubule, which is characterized by relatively tight epithelium with high electrical resistance, the proximal tubule is a leaky epithelium favoring the flux of compounds into the proximal tubular cells. Thus, transporting GSH conjugates, heavy metals and organic anions and cations. It is localized mainly to the proximal tubule than other segments, resulting in proximal tubular accumulation and cellular toxicity of these xenobiotics (Zalups, 1995). The present investigation revealed that mercury intoxication causes significant increase in lipid peroxidation and acid phosphatase and significant decrease in alkaline phosphatase and lactate dehydrogenase activity. The enzyme lactate dehydrogenase (LDH) catalyses the interconversion of lactate and pyruvate in the glycolytic pathway and occurs as a tetrameric molecules. The alkaline phosphatase enzyme is associated with transfer of phosphates and is linked with transportation of intermediate compounds in glycogenesis or glycogenolysis. In the present investigation there was a decrease in LDH and alkaline phosphatase activity after the exposure of mercuric chloride. This may be due to loss of brush border of the proximal tubular cells at an early stage of renal epithelial cellular necrosis. Inorganic mercury directly inhibits LDH activity (Smith et al., 1986). The sensitivity of LDH to Hg suggests cellular toxicity (Smith et al., 1986; Mehra and Kanwar, 1986; Chung and Lee, 1987). There was significant increase in acid phosphatase activity after HgCl2 exposure. Acid phosphatase activity is localized in cellular lysosomes. An enhanced peroxidation of lysosomal membranes due to HgCl2 intoxication causes lysis of membrane and oozing out of the enzyme and hence results in an increased acid phosphatase activity (Mehra and Kanwar, 1986). In the present investigation the lipid peroxidation level showed a highly significant decline following Hg exposure. Mercury has high affinity for GSH and causes the irreversible
excretion of, up to two GSH tripeptides (Zalups and Lash, 1996). The metal-GSH conjugation process is desirable for the excretion of the toxic metal into the bile. However, it depletes the GSH from the cell and thus decreases the antioxidant potential. We have also reported that mercury causes significant depletion of hepatic-GSH (Sharma et al., 2002). Mercury is a transition metal; it also promotes the formation of reactive oxygen species by fenton transition equation, such as hydrogen peroxides and enhances the subsequent ironand copper-induced production of lipid peroxides and the highly reactive hydroxyl radical (Miller et al., 1991; Hussain et al., 1999). Lipid peroxides alter membrane structure and mitochondrial structure. From the present investigation it can be suggested that HgCl2 treatment significantly reduces the GSH content and the antioxidant potential and thus accelerates the lipid peroxidation, resulting in renal cellular damage. It was observed that an extract of Ocimum sanctum when given alone or in combination significantly declines lipid peroxidation and reduces the mercury toxicity which in turn is reflected by a significant decrease in acid phosphatase activity and an increase in alkaline phosphatase and LDH activity. The protective activity of Ocimum sanctum aqueous leaves extract against mercury-induced toxicity, may to some extent, be mediated through the release of intracellular antioxidants which, in turn, will scavenge the free radicals and also may help in repair of biochemical lesions. Some of the compounds found in the Ocimum plant have been reported to possess strong antioxidant activity. Eugenol and ursolic acid from Ocimum sanctum have been reported to induce protection against free radical induced cellular damages (Balanehru and Nagarajan, 1992; Rajkumar and Rao, 1993). The Ocimum flavonoids Orientin and Vicenin have also exhibited strong inhibitory effect on the fenton reaction generated OH radical activity. They have strong antioxidant activity in vitro and anti lipid peroxidative effect in vivo, which strongly suggest free radical scavenging as a major mechanism by which Ocimum products protect the cellular damage (Ganasoundari et al., 1997, 1998; Uma Devi et al., 2000). In vitro studies further indicate that metal chelation may also have a role in the anti-lipid per oxidative effect of Ocimum leaves (Ganasoundari et al., 1997; Uma Devi et al., 2000). In addition, immune stimulation was suggested as a mechanism contributing to the adaptogenic action of plant (Godhwani et al., 1988). Further, it has been reported that Ocimum sanctum aqueous leaves extract when given alone or in combination significantly enhances the GSH content (Sharma et al., 2002) by inducing the antioxidant potential and thus reduces the reactive oxygen species generated cellular damage. Thus, the present study suggests that deleterious reactive oxygen species or lipid peroxides responsible for HgCl2 induced toxicity may be alleviated by aqueous extract of Ocimum leaves, which in turn reflected in significant decline in LPO content and acid phosphatase and significant enhancement in LDH and alkaline phosphatase activity as compared to HgCl2 -treated group in Swiss albino mice.
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5. Conclusion Pre- and post-treatment of Ocimum sanctum aqueous leaves extract significantly protect against mercury-induced renal toxicity.
Acknowledgement Financial assistance provided by CSIR-New Delhi to one of us (MKS) by grant. No. 9/149/247/2000 (EMR) is highly acknowledged.
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