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EFFECT OF SELENIUM SUPPLEMENTATION ON RATS EXPOSED TO CADMIUM OR LEAD

Cercetări Agronomice în Moldova Vol. XLVII , No. 4 (160) / 2014

EFFECT OF SELENIUM SUPPLEMENTATION ON , LACTATE AND ACTIVITIES IN RATS EXPOSED TO CADMIUM OR LEAD

B.G. ŞLENCU1*, C. CIOBANU1, Carmen SOLCAN 2, Alina ANTON2, St. CIOBANU2, Gh. SOLCAN2, Rodica CUCIUREANU1

*E-mail: [email protected]

Received June 13, 2014

ABSTRACT. The purpose of the study was Selenium, coadministered with cadmium, to assess the effect of selenium caused a marked increase in serum LDH supplementation on serum amylase, lactate activity, when compared to cadmium alone dehydrogenase (LDH) and alkaline or Control group while practically it had no phosphatase (ALP) activities in rats, during effect on lead induced changes in LDH subacute exposure to toxic doses of activity. Cadmium and lead induced cadmium or lead through the drinking disturbances in serum ALP activity were not water. The experimental groups (n=6) were: influenced by selenium supplementation. Control, Se (Se+4: 0,2 mg/l), Cd (Cd+2: 150 mg/l), Pb (Pb+2: 300 mg/l), Cd+Se (Cd+2: Key words: Rats; Lead; Cadmium; 150 mg/l; Se+4: 0,2 mg/l) and Pb+Se (Pb+2: Selenium; Blood serum . 300 mg/l; Se+4: 0,2 mg/l). The were sacrificed after 56 days. Amylase, LDH and REZUMAT. Efectul suplimentării cu ALP activities were determined from serum. seleniu asupra activităţii amilazei serice, Se and Pb treatments caused an increase in a lactat dehidrogenazei şi a fosfatazei amylase and LDH activities, when alcaline la sobolani, expuşi la cadmiu sau compared to Control group while Cd caused plumb. Scopul acestui studiu a fost de a an increase in amylase activity and a evalua efectul suplimentării cu seleniu decrease in LDH and ALP activities. Cd+Se asupra unor enzime, amilaza, lactat caused a decrease in amylase activity and an dehidrogenaza (LDH) şi fosfataza alcalină increase in LDH activity, when compared to (ALP) la şobolani, în timpul expunerii Cd. Pb+Se caused a decrease in amylase subacute la doze toxice de cadmiu sau activity in comparison to lead. Selenium plumb, prin intermediul apei de bǎut. supplementation alleviated cadmium or lead Loturile experimentale (n=6) au fost: +4 +2 induced changes in serum amylase activity. Martor, Se (Se : 0,2 mg/l), Cd (Cd : 150

1 “Grigore T. Popa” University of and Pharmacy, School of Pharmacy, Iaşi, Romania 2 “ Ionescu de la Brad” University of Agricultural Sciences and Veterinary Medicine, School of Veterinary Medicine, Iaşi, Romania

113 B.G. ŞLENCU et al. mg/l), Pb (Pb+2: 300 mg/l), Cd+Se (Cd+2: of glutathione and sulfhydryl groups, 150 mg/l; Se+4: 0,2 mg/l) şi Pb+Se (Pb+2: +4 resulting in the enhanced production 300 mg/l; Se : 0,2 mg/l). Animalele au fost of various reactive species sacrificate dupǎ 56 de zile. Amilaza, LDH şi ALP au fost determinate din ser. (ROS) such as superoxide ion, Administrarea de Se sau Pb a determinat o hydrogen peroxide and hydroxyl creştere a amilazei şi LDH, în comparaţie cu radicals (Liu et al., 2009). lotul de control, în timp ce Cd a determinat Lead (Pb) is a toxic metal, which o creştere a amilazei şi o scǎdere a LDH şi can induce various behavioral, ALP. Cd+Se au cauzat o scǎdere a amilazei biochemical, haematological and şi o creştere a LDH, în comparaţie cu Cd. Pb+Se au produs o reducere amilazei, în histological disorders, both in humans comparaţie cu plumbul. Suplimentarea cu and animals (Ciobanu et al., 2012). seleniu a atenuat modificǎrile induse de The most deleterious effects have cadmiu sau plumb asupra amilazei serice. been observed on the haemopoietic, Seleniul, coadministrat cu cadmiul, a cauzat nervous, reproductive and renal o creştere marcantă a activitǎţii serice a systems (Papanikolaou et al., 2005). LDH, în comparaţie cu Cd sau lotul martor, One of the main toxicodynamic însǎ nu a avut nici un efect asupra modificǎrilor produse de plumb asupra mechanisms involved in Pb toxicity is activitǎţii LDH. Tulburǎrile induse de the generation of reactive oxygen cadmiu şi plumb asupra activitǎţii serice a species (ROS) (Ercal et al., 2001). ALP nu au fost influenţate de suplimentarea Amylase (α-amylase) is a cu seleniu. involved in . Lactate Cuvinte cheie: şobolani; plumb; cadmiu; selenium; enzime serice. dehydrogenase is an with important roles in carbohydrate INTRODUCTION ; in mammals it has five main isoforms. Alkaline phosphatase Selenium (Se) is an essential is a hydrolase involved in microelement with multiple dephosphorylation (Murray et al., physiological roles. It is a component 2003). of several selelenoenzymes with Selenium exerts protective important functions in the body. The effects against cadmium or lead most important selenoenzymes are the toxicity (Reilly, 2006; Newairy et al., glutathione peroxidases, deiodinases 2007; Othman et Missiry, 1998). and thioredoxin reductases (Navarro There is insufficient and/or Alarcon et Cabrera-Vique, 2008; inconclusive data regarding the effects Hatfield et al., 2006). In high doses, of cadmium or lead and the selenium compounds can be toxic concurrent administration of selenium (Reilly, 2006). on serum amylase, lactate Cadmium (Cd) is a toxic metal. dehydrogenase and alkaline It is a potent carcinogen in multiple phosphatase activities. types of tissues. It has been suggested The objective of this study was that Cd toxicity involves the depletion to assess the effect of selenium

114 EFFECT OF SELENIUM SUPPLEMENTATION ON RATS EXPOSED TO CADMIUM OR LEAD supplementation on serum amylase, Selenium (Se+4), cadmium (Cd+2) and alkaline and lead (Pb+2) were added in the drinking phosphatase activities during subacute water by dissolving sodium selenite exposure to toxic doses of cadmium pentahydrate (Sigma Aldrich), lead or lead through the drinking water, acetate trihydrate (Chemical Company, Romania) and cadmium using a rat experimental model. hemipentahydrate (Chemical Company, Romania). The water dispensers were MATERIALS AND METHODS emptied and refilled everyday and the unconsumed volumes of water were All experimental procedures were measured. conducted according to the The experimental groups were: recommendations set up by 86/609/EEC Control, Se (Se+4: 0,2 mg/l), Cd (Cd+2: directive and other applicable regulations. 150 mg/l), Pb (Pb+2: 300 mg/l), Cd+Se The study protocol was approved by The (Cd+2: 150 mg/l; Se+4: 0,2 mg/l) and Ethics Committee for Research of Pb+Se (Pb+2: 300 mg/l; Se+4: 0,2 mg/l). “Grigore T. Popa” University of Medicine The weight of the animals was and Pharmacy of Iaşi, Romania. measured every 7 days. The experiment A total of 36 male Wistar rats lasted for 56 days. The mean intake of (initial body weight: 250-400 g) were elements reported to body weight (Table randomly divided in six groups (n=6) and 1) was approximated based on the daily each group was kept in a single collective water consumption of each group, the cage, maintained in relative constant cumulative body weights and the environmental conditions (temperature 18 concentration of the elements in the o - 25 C, light/dark cycle: 12 h/12 h) and drinking water. had free access to water and dry feed.

Table 1 - Estimation of the mean approximate intake of selenium, cadmium and lead throughout the experiment

Se+4 Cd+2 Pb+2 Group (μg/kg b.w.) (mg/kg b.w.) (mg/kg b.w.) Control - - - Se 15,48±0,42 - - Cd - 10,48±0,41 - Pb - - 25,34±0,68 Cd+Se 9,81±0,30 7,36±0,23 - Pb+Se 15,90±0,43 - 23,84±0,64 Values are mean ± standard deviation

The animals were narcotized with anticoagulant by cardiac puncture. Serum 100 mg/kg b.w. of ketamine (injectable was obtained by centrifugation, after solution for veterinary use, administered coagulation. Serum activities of amylase, intraperitoneally) and euthanized at the alkaline phosphatase (ALP) and lactate end of the experiment. Blood was dehydrogenase (LDH) were determined collected in vacutainers without

115 B.G. ŞLENCU et al. using an Accent 200 biochemical analyzer activity, compared to Control group (Cormay, Poland) and specific assay kits. and, respectively, Cd or Pb (Fig. 1). Microsoft Office Excel 2007 with The amount of amylase from Data Analysis add-on was used for serum reflects the balance between statistical processing of experimental the rates of amylase entry and its data. The results are expressed as mean ± standard error of the mean (SEM). A subsequent removal from the blood. paired Student’s t-test was used (p < 0,05 The pancreas and the salivary glands was considered significant). usually account for almost all of the serum amylase, in normal conditions. RESULTS AND DISCUSSION Hyperamylasemia can be the result of an increased rate of entry into the All three Se, Cd and Pb blood or a decreased rate of clearance. treatments produced an increase in Pancreatic lesions or serum amylase activity, the highest can cause hyperamylasemia. A being observed in the Se group. decreased rate of clearance, due to Selenium supplementation of rats renal impairment, can also cause exposed to cadmium or lead resulted hyperamylasemia (Solcan et al., in a decrease in serum amylase 2005).

Figure 1 - Serum amylase activity. Error bars are SEM; ■ = significantly different compared to Control group (p<0,05); □ = significantly different compared to Se (p<0,05); ● = significantly different compared to Cd (p<0,05); ▲ = significantly different compared to Pb (p<0,05)

Pancreatitis has many specificity in the diagnosis of ethiopathogenic causes. Intoxication , but is routinely used for with various organic or inorganic this purpose, together with amylase substances can be a cause (Khurana et and other markers (Yadav et al., Barkin, 2001; Reed et Gorelick, 2002). 2014). Hyperamylasemia has a low

116 EFFECT OF SELENIUM SUPPLEMENTATION ON RATS EXPOSED TO CADMIUM OR LEAD

Cadmium administered for a Control group. The mechanisms of shorter period of time (1-5 days) and interaction between selenium and at lower doses (1 mg/kg b.w. cadmium or lead which could explain subcutaneously) did not cause a this decrease are not known. marked change in serum amylase Both Se and Pb treatments activity in mice (Shimada et al., caused an increase in LDH serum 2012). Cadmium stimulated in vitro activity when compared to Control release of amylase when added alone group, while Cd treatment apparently in the incubation medium of caused a decrease. Simultaneous pancreatic lobules from guinea intake of cadmium and selenium (Linari et al., 2001). Cadmium resulted in a high increase in LDH administered through the drinking serum activity in comparison to Cd water (in sublethal doses, as CdCl2) (Fig. 2). for 26 weeks to rats caused a slight LDH is involved in the terminal increase in serum amylase step of anaerobic pathway, (Lukacinova et al., 2012). which occurs in the cell cytoplasm. Mayland et al. (1986) reported LDH regulates the byproducts of that concurrent administration of mitochondrial oxidative or reductive selenium and lead to sheep increased stress conditions by maintaining lactic serum amylase activities, as compared acid levels at normal values, to the administration of lead alone. converting it to pyruvate (Haffor and Other parameters also indicated that Alhazza, 2007). the coadministration of lead and LDH is used as a diagnostic selenium was more toxic than lead. marker for various and In the present, study the degree pathological conditions, of pancreatic, renal or other type of haematological conditions or some damage caused by Cd or Pb types of tumours, although it has a treatments was apparently not low specificity in some cases (Huijgen sufficiently intense enough to cause a et al., 1997). marked increase in serum amylase In vitro, addition of cadmium (as activity. Selenium alone apparently CdCl2) in the culture medium of was more harmful than cadmium or mouse hepatocytes significantly lead in this case. The increase in enhanced LDH and ALP leakage and serum amylase activity for the Se, Cd increased and Pb groups was not significant. (ROS) production (Pal et al., 2011). Coadministration of selenium In other studies, cadmium caused with cadmium or lead apparently an increase in plasma or serum LDH reduced their toxicity and even activity in rats (Abbes et al., 2007; decreased serum amylase activity Borges et al., 2008; Pari and below the mean value of the Control Shagirtha, 2012) and mice (Brandao group. This decrease lacks statistical et al., 2009). Coadministration of significance when referring to the diphenyl diselenide (an

117 B.G. ŞLENCU et al. organoselenium compound) with cadmium toxicity would cause an cadmium significantly reduced increase in serum LDH activity. One plasma LDH activity, almost to particular of this experiment is that control values (Borges et al., 2008; the interindividual variance Brandao et al., 2009). It has been (expressed by SEM) of LDH activity previously shown that lead cause an in the Control group was very high. increase in serum or plasma LDH The cause of this variability was not activity (Haffor and Alhazza, 2007; identified. It is possible that the actual Waggas, 2012). theoretical mean of the Control group In the current study, LDH serum should have been lower. activity was abnormally lower in the Coadministration of selenium with Cd group, compared to Control group, cadmium was apparently more toxic but it was not significant statistically. than cadmium alone. Lead and This result does not correlate well selenium, coadministered with lead, with the results of other studies. It apparently did not exert significant would have been expected that toxic effects on LDH activity.

Figure 2 - Serum LDH activity. Error bars are SEM; ■ = significantly different compared to Control group (p<0,05); □ = significantly different compared to Se (p<0,05); ● = significantly different compared to Cd (p<0,05); ▲ = significantly different compared to Pb (p<0,05)

Cadmium caused a significant insignificant increase in serum ALP decrease in serum ALP activity, activity was observed when compared compared to Control group. to Control group (Fig. 3). Concomitant intake of selenium and Changes in serum ALP activity cadmium did not result in a marked of diagnostic importance result from change in serum ALP activity, an increased entry of the enzyme into compared to Cd group. In the case of the circulation. This can be the result Pb and Pb+Se groups a slight, of an increased osteoblastic activity in

118 EFFECT OF SELENIUM SUPPLEMENTATION ON RATS EXPOSED TO CADMIUM OR LEAD the case of bone disease or an the current study are contradictory; increased synthesis of alkaline the exact cause was not identified. phosphatase by hepatocytes in the Lead causes significant increase case of hepatobiliary disease (Solcan in serum ALP activities in comparison et al., 2005). Other researchers to non-treated animals (Shalan et al., showed that cadmium caused an 2005; Mehana et al., 2012). In this increase in ALP serum activity when experiment, neither cadmium nor lead compared to controls (Wen et al., caused toxicity on ALP activity; 2002; Pari and Shagirtha, 2012). selenium coadministration did not From this point of view, the results of affect toxicity.

Figure 3 - Serum ALP activity. Error bars are SEM; ■ = significantly different compared to Control group (p<0,05); □ = significantly different compared to Se (p<0,05); ● = significantly different compared to Cd (p<0,05); ▲ = significantly different compared to Pb (p<0,05)

were not influenced by selenium CONCLUSIONS supplementation.

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