Ineffectiveness of Nickel in augmenting the hepatotoxicity in protein deficient rats

P. Sidhu^a, M. L. Garg^b, P. Morgenstern^c, J. Vogt^d, T. Butz^d, D. K. Dhawan^a*

^aInstitute of Physiology and Experimental Pathophysiology ,Friedrich-Alexander University,Erlangen-91054,Germany.
^bDepartment of Biophysics, Panjab University, Chandigarh-160014, India. ^cUmweltforschungszentrum Leipzig-Halle, Leipzig,
Germany für Physik und Geowissenschaften,Universität Leipzig, Leipzig,Germany.^dFakultät für Physik und
Geowissenschaften,Universität Leipzig, Leipzig,Germany. *Department of Biophysics Panjab University Chandigarh, India.. 

Correspondencia: P. Sidhu
E-mail: pardeepsidhu@yahoo.co.ur

Recibido: 18-X-2004. ]]> Aceptado: 6-III-2005.

Introduction

In the developing countries like India protein nergy malnutrition (PEM), is a prominent cause of malnutrition and this may be associated with adverse functional disorders of body metabolism, which is likely to be exaggerated in conditions of heavy metal toxicity. Malnourished people living in the vicinity of nickel (Ni) industrial and smelting units are more prone to nickel toxicity. The protein calorie malnutrition affects the synthesis of the mRNA suggesting that a defect occurs at a pre-transcriptional level that results in a diminution of the concentration of mRNA¹. PEM is known to be associated with many biochemical disturbances in the body. The increases in serum alanine aminotransferease and alkaline phosphatase in animals fed on a protein deficient diet, suggests hepatocellular dysfunctions^{2, 3}.

Persons afflicted with protein malnutrition are almost deficient in a variety of micronutrients. Protein deficiency has been shown to decrease the hepatic levels of zinc, manganese, copper, calcium and magnesium in experimental animals^{4, 5}. Alterations in the levels of trace elements results in number of diseases like hypoalbuminemia, and anemia in malnourished children⁶. Bhaskaram and Hemalatha, 1995⁷ observed that children suffering from severe protein energy malnutrition have very low levels of the thymulin hormone, which is a sensitive indicator of zinc status in body. It is known that dietary protein variation affects the absorption and uptake of  ⁶⁵Zn⁸.

Nickel is a common respirable sized particulate pollutant and nickel compounds of commercial importance are used in the preparation of nickel alloys, ground coated enamels, in cooling of ceramics and glass, electroplating, nickel-iron storage batteries, electronic components and to prepare nickel catalysts⁹. Nickel breaks down the immunity by affecting the T-cell system and suppresses the activity of natural killer cells in rats and mice^{10, 11}.

 Nickel has been shown to interact with number of trace elements including iron, zinc, copper, manganese, sodium and potassium^{12, 13}. Nickel mobilizes and promotes the excretion of copper, zinc and manganese from organs and promotes storage of chromium in organs¹⁴. Nickel affects the iron metabolism and intensifies the erythropoiesis with induced hemolytic anemia¹⁵.

In view of the increasing incidences of cancer, cardiovascular, respiratory and gastrointestinal diseases, due to heavy metal toxicity, the analysis of dietary intake for the toxic and protective elements and their levelsin tissue needs further research. Since, nutrients are essential for all the fundamental cellular processes as they modulate cellular oxidative and antioxidativedefense system in the body, therefore, it becomes a matter of concern to elucidate their role in manifestation of functional disorders of different organs.

Liver, being a major metabolic organ, plays an important role in the regulation of biochemical and trace element metabolism. The present investigations were designed to study the hepatotoxic effects of nickel in rats subjected to protein deficiency.

Materials and methods

 G-1, Normal Control
Animals in this group served as normal controls and were fed with diet containing normal protein contents (18%). Composition of the diet used was as described by Kaur et al, 1992 and given in table below.

G-2, Protein deficient,(PD)
Protein deficiency was induced in the animals of this group by maintaining them on the laboratory prepared protein deficient diet with 8% protein contents. Composition of the diet used was as described by Kaur et al, 1992 (16) and given in table below.

G-3, Nickel treated, (Ni)
Animals in this group were given nickel in the form of NiSO4. 6H2O at a dose level of 800 mg/L in drinking water and the animals had free access to the drinking water containing nickel and the normal diet.

G-4, Ni+PD treated
Animals in this group were given protein deficient diet as given to G-2 animals and in addition were subjected to Ni treatment as mentioned for G-3 animals.

The treatments of rats continued for a period of eight weeks. At the end of the treatment, the animals were weighted and were sacrificed by exsanguination under light anesthesia. Livers were removed immediately and were perfused and rinsed in normal saline (NaCl, 9 g/l/w/v). One lobe was preserved by freezing for the determination of various trace elements and the other was processed immediately for various biochemical studies.

Biochemical Estimations.

Protein
Protein assay was done by the method of Lowry y cols., 1951¹⁷.

Estimation of liver marker enzymes in liver
The enzyme activity of Alkaline Phosphatase (ALP) was measured by the method of Wooton¹⁸ and the enzyme activities of aspartate aminotransferases (AST) and Alanine Aminotransferase (ALT) were estimated according to the procedure of Reitman and Frankel¹⁹.

- Sample Preparation for EDXRF
The liver tissues of all the animals were oven dried at 70ºC to a constant weight and then ground with the help of Agate Pestle and Mortar. 300 mg dried powder of the tissue so obtained was weighed and mixed with equivalent amount of Hoechst Wachs (wax) to make self supporting pellets. The pellets were made by using a specially designed pure steel dye and a hydraulic press from Paul weber, Germany. A force of approximately 45 KN (Kilo newtons) was applied at the dye top in order to make pellets of uniform thickness.

- EDXRF Setup
In the present work, the pellets of tissues were analyzed using an EDXRF X-Lab, 2000 to determine the levels of various elements. The X-lab, 2000 spectrometer involved a 0.4 kw Pd anode X-ray tube as source of excitation. The power of the X-ray tube was adjusted on line for each individual measurement by the spectropho-tometer software, to secure optimum acquisition parameters for the current analysis.

Presently, different X-ray energies and excitation modes are being used but the most important mode used was of 40 kV and excitation used was polarized X-Ray. A Si (Li) detector coupled with computer (Pentium, 600 MHz, software package SPECTRO X-LAB^PRO 2.2) was used to collect the flourescent X-ray spectra from the samples. The X-ray tube, secondary exciter, target and the Si (Li) detector were placed in a triaxial geometry mode. This geometry was used to minimize the background due to scattered photons.

Results

The results of all the experiments conducted during the current study are depicted in various tables. All the results of various treatment groups have been compared with their normal controls. Results of nickel + protein deficient (G-4) treated group have been compared with the results of the protein deficient group (G-2) also.

Statistsical Analysis ]]> The statistical significance of the values has been determined by using one way analysis of variance (ANOVA) followed by Student-Newman-Keuls test. The determinations are represented as Mean ± SD.

Body weights
The variations in the body weights of the animals subjected to different treatments are shown in table I. It was observed that the protein deficiency resulted in a significant (p < 0.001) decease in the body weights after eight weeks, when compared to normal control rats. Nickel treatment to normal control rats resulted in some decrease (p < 0.05) in the body weights but nickel treatment to protein deficient rats resulted in appreciable reduction (p < 0.001) in the body weights as compared to normal control rats.

Hepatic protein Contents
The hepatic protein contents in various treatment groups expressed as mg g^-1 tissue are shown in tableI. Protein deficient, Ni treated as well as combined protein deficient and nickel treated rats showed significant (P < 0.001) reductions in the hepatic protein contents as compared to normal control rats.

Alkaline phosphatase
Hepatic alkaline phosphatase activity showed a significant elevation (p < 0.01) in rats subjected to protein deficiency, nickel treatment and combined protein deficiency and nickel treatment as shown in table II.

Aspartate Aminotransferase ]]> As regards to hepatic levels of AST, a significant (p < 0.001) elevation was observed in protein deficient and nickel treated protein deficient animals (table II).

Alanine Aminotransferase
Table II depicts the hepatic observations of ALT where significant (p < 0.001) elevation in ALT levels has been observed in protein deficient, nickel and nickel treated protein deficient animals.

Hepatic concentration of various elements
The concentrations of various elements have been depicted in table III. The nickel administration to normal and protein deficient rats has resulted in a significant (p < 0.001) increase in concentrations of nickel in liver tissue.

The concentration of zinc and copper in liver tissue got decreased significantly (p < 0.001) in protein deficient, nickel treated and nickel treated protein deficient animals.

Tissue iron concentrations were found to be decreased in protein deficient animals, but the concentrations of iron got elevated significantly (p < 0.001) in nickel treated and nickel treated protein deficient animals.

It has been observed that selenium got decreased significantly (p < 0.001) in protein deficient, nickel treated and nickel treated protein deficient animals when compared to normal animals. The elevation of selenium in nickel treated protein deficient animals was also significantly (p < 0.05) higher when compared to protein deficient animals.

Significant (p < 0.001) decrease has been observed in potassium concentration in nickel treated and nickel treated protein deficient animals. On the other hand phosphorus and sulfur concentrations were found to be increased significantly in nickel treated and nickel treated protein deficient animals.

Discussion

Nickel treatment to normal control and protein deficient rats resulted in marked reduction in the body weights as compared to normal control rats. The reduction in body weights following nickel treatment has also been reported earlier^{22, 23}. The decrease in body weight may not solely be attributed to protein deficiency alone as the Ni treatment alone also has caused significant decline in body weight. The decrease in body weight due to nickel treatment has been connected by researchers to be not due to low intake of diet consumption of the rats following toxic treatment with nickel, vis a vis normal rats, and thus it is anticipated that this effect could possibly be due to the overall increased degeneration of lipids proteins as a result of nickel toxicity^23,24.

Rats in protein deficient, Ni treated and combined PD+Ni treated groups, showed a highly significant (p < 0.001) reduction in the hepatic protein contents as compared to rats of normal control group. Davenport y cols., 1994²⁵ demonstrated that in protein deficient states, the reduction in protein contents are due to depletion in amino acid precursors. Nickel in earlier reports, has also been able to cause significant depression in protein levels^26,27. Nickel diminishes the DNA and RNA polymerase activity and decreases DNA replication fidelity²⁸ which in turn can reduce the protein synthesis.

Hepatic alkaline phosphatase activity folowed a significant elevation due to protein deficiency and nickel treatment. This elevation coul be anticipated to the reason that ALP is bound to the intracellular membranes, and does not leak out with the increased permeability of the cell membranes. Moreover, Hultberg and Disaksson, 1983²⁹ proposed that activated macrophages including the Kupffer cells are the cellular source for the increased levels of ALP in conditions of liver damage. Davenport y cols., 1994²⁵ also postulated tath many hepatic and extrahepatic conditions could also result due to protein-restricted diets that in a way caused increased production of alkaline phosphatase isoenzymes from bone and hepatobiliary source.

The aminotransferases are intracellular enzymes, which are active in operating the reversible exchange of aminoacids between alpha-amino and alpha-ketoacids. As all the naturally occurring amino acids can undergo amino transfer reactions thus this class of intracellular enzymes (aminotransferases) form an important link between protein and carbohydrate metabolism³⁰.

Hepatic levels of AST and ALT got significantly (p<0.001) elevated in protein deficient and nickel treated protein deficient animals. The observed increased activities of hepatic AST and ALT in the animals given protein deficient diet are in conformity with the study of Pond y cols., 1992³¹. They reported that the increase in ALT activity in protein deficient pigs shows altered liver functions, although microscopic anatomy revealed no evidence of excessive fat accumulation or of pathologic changes. Similar observations were noticed by Kumari y cols., 1993³² who estimated ALT and AST activities in the samples of 30 cases of pediatric PEM. These authors explained that during hepatobiliary disorder, amino acids are released form exaggerated tissue breakdown and in order to metabolize these amino acids, the process of transmutation gets enhanced leading to increased activity of the related enzymes AST and ALT. Heavy metals have also been found to cause the increase in AST and ALT levels³³. A earlier report from this laboratory has also shown the alterations in the AST and ALT concentrations due to lead intoxication³⁴.

It is now well authorized that the liver has an important function in the regulation of trace element metabolism^{35, 36}. Further trace elements serve as cofactors for many enzymes in numerous metabolic pathways; therefore, changes in the distribution of these essential elements in the body can have both nutritional and toxicological consequences with regard to the metabolism of other metals³⁷. Those metals which are essential for maintenance of the structural and functional integrity of the living organisms are found in all living systems and are conserved within strict concentration limits in the systems³⁸. However, imbalances in the supply of any of the essential elements in the body can have both nutritional and toxicological consequences with regard to the metabolism of other metals. They can further be responsible for the development of clinical signs of trace element deficiencies or can modify the susceptibility to metal toxicity³⁹. It is well insinuated that metals that have similar chemical and physical properties would often interact biologically and antagonize or embellish each other's function³⁴.

In the present study, nickel concentration has been found to be increased in liver tissue following the administration of nickel to normal and protein deficient rats. Our results are in agreement with earlier reports^{40, 41}.

Although zinc had been known to be essential for the growth of microorganisms for over hundred years, the advances in the knowledge of zinc chemistry and biochemistry have been explored only during the last two decades⁴². Furthermore, a large number of zinccontaining enzymes and proteins have been recognized to participate in the metabolism of proteins, nucleic acids, carbohydrates and lipids. Consequently, zinc deficiency is the most significant pathological and biochemical state involving abnormalities in the metal's metabolism. This can be due to inadequate dietary intake, increased requirements or excretion, conditioned deficiency or genetic disorders^{43, 44}.

In present study, observation of depressed Zn levels in liver of protein deficient rats are in conformity with previous studies^{45, 46}. We have also observed decrease in hepatic zinc concentration following nickel toxicity, which may be because nickel mobilizes and promotes the excretion of copper, zinc and manganese from organs and promotes storage of chromium in organs^{14, 23}. Abnormalities in zinc metabolism leading to its deficiency are generally attributed to various factors like, malabsorption, malnutrition, decreased intestinal zinc binding factors or the increased excretion of the zinc via the gastrointestinal tract or via urine are of common occurrence in chronic liver disorders⁴⁷.

Iron plays an important role, not only in oxygen delivery to the tissues, but also as a cofactor with several enzymes involved in energy metabolism and thermoregulation⁵¹. Further, the enzymes cytochromes P450 and b5 involve the metal in the metabolism of compounds such as steroids and in the degradation of xenobiotics^{52, 53}.

The hepatic Fe contents, in the present study, were found significantly depressed in protein deficient rats as compared to normal control group. Reduction in Fe contents following protein deficiency has previously been reported by Klavins et al, 1962⁵⁴ and Tandon y cols., 1999⁴. They concluded that protein plays an important role in the absorption of iron from the gastrointestinal tract and protein deficiency results in decreased absorption of iron.

During the course of this study, we have also observed increased hepatic iron contents in the animals, given nickel treatment, which has also been reported earlier⁵⁵. Pronounced increase in iron concentration at a higher dose of nickel could be because of nickel functioning as a cofactor that facilitates the intestinal absorption of the iron by enhancing its compellation to a lipophilic molecule⁵⁶. Increased hepatic iron levels in response to the toxic conditions established by nickel also suggests that the body requirement of iron has been so severe that more and more of this metal ion is transported to the liver, as it serves as a cofactor for many key enzymes involved in various energy generation pathways, which could have been adversely affected in stress conditions. When serum iron levels exceed the iron binding capacity of transferrin a β₁- glycoprotein synthesized in the liver, the circulating free iron initially accumulates in Kupffer cells and later in the hepatocytes resulting in the increased concentration of iron in liver tissue. It can also be speculated that the nickel treatment leads to the activation of host defense system which results in the enhancement of hepatic iron content so as to combat the toxic conditions created by nickel administration. Another possible reason for increased liver iron concentrations could be thought of due to impaired hepatic elimination of this metal from the liver. Hepatic iron overload as observed in our study may lead to the development of a severe oxidative stress status in the tissue, thus contributing to the concomitant liver injury as reported by Boisier y cols., 1999⁵⁷ and also observed by us in the form of enhanced ALP, AST and ALT levels.

Selenium, which is an essential trace metal and inactivates sulfhydryl groups in certain enzymes and is also a component of glutathione peroxidase⁵⁸. This ubiquitous enzyme located in both cytosol and mitochondrial matrix uses glutathione to reduce organic hydropero-xides, thereby, prevents oxidative damage to various cell organelles. In the present study, selenium was found to be decreased significantly in protein deficient, nickel treated and nickel treated protein deficient animals. It has been reported that nickel and selenium act antagonistically and the detoxifying effect of selenium on nickel toxicity seems to be due to the formation of a Ni-selenide excretable complex⁵⁹ which seems to have been marginalized due to high dose of nickel in the present study.

Potassium is an essential intracellular ion involved in cellular homeostasis and electrical conduction. We have observed a statistically significant decrease in potassium levels in nickel treated and nickel treated protein deficient animals. Rai y cols.,1990¹² has also observed the loss of K⁺ and Na⁺ by Nickel. As potassium is a major cation of intracellular fluid, and functions in balance with the extracellular ionized sodium to maintain normal osmotic pressure and water balance hence it is possible that nickel could have lead to alterations in the membrane permeability of hepatocytes especially with regard to potassium channels or has caused inhibition of ATPase leading to decreased levels of potassium. Its deficiency affects the activity of muscles and transmission of electrochemical impulses. It has been reported that nickel inhibits the Na-K-ATPase⁶⁰.

Phosphorus, which is an essential part of key cellular nucleoproteins such as DNA, has also been found to increase significantly in nickel treated and nickel treated protein deficient animals. It may possibly be due to increased requirement of phosphorus either due to the inhibitory effects of nickel on DNA synthesis leading to hyperplasia or increased mobilization from bones. Sulfur is an important constituent of many amino acids like methionine, cysteine, cystine, homocysteine, homocystine, and taurine and also of enzymes like S-adenosylmethionine (SAM) and glutathione (GSH)⁶¹. Sulfur concentrations got elevated in nickel and PD+Ni treated animals, which could be to counter the toxic effects of nickel.

Our results in the present findings indicate the disturbance in the marker enzymes of rat liver following protein deficiency (PD) and nickel treatment which may be the consequence of alterations in the levels of essential trace elements as a result of hepatic injury.

Conclusion

The findings indicate that protein deficiency does not enhance the signs of nickel toxicity in rats.

This work was supported by grant from IUC-DAE, Kolkata and ICMR, New Delhi.

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