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Effect of ethanol on the distribution of cadmium between the cadmium metallothionein- and non-metallothionein-bound cadmium pools in cadmium-exposed rats
Toxicology, 72 (1992) 251-263 Elsevier Scientific Publishers Ireland Ltd.
251
Effect of ethanol on the distribution of cadmium between the cadmium metallothionein- and non-metallothionein-bound cadmium pools in cadmium-exposed rats G e e t a S h a r m a , R a v i n d r a N a t h and K i r a n Dip Gill Department of Biochemistry, Postgraduate Institute of Medical Education and Research. Chandigar h-160012 (India) (Received July 3rd, 1991; accepted February 9th, 1992)
Summary In an attempt to assess the effect of ethanol on c a d m i u m accumulation, metallothionein (MT) synthesis, Cd-binding capacity and lipid peroxidation, rats were administered either Cd, ethanol or their combination for a period of 4 weeks. A significant increase in Cd accumulation was observed in all the organs of rats under study co-exposed to Cd and ethanol as compared to only Cd-treated rats. Increased M T levels in response to Cd were associated with a marked alteration in the distribution of Cd amongst the two pools of intracellular Cd i,e. Cd bound to MT (Cd-MT) and Cd not bound to MT (non-MT-Cd). Higher levels of non-MT-Cd were observed in liver, kidney and heart of Cd + ethanol-exposed rats as compared to only Cd-exposed rats. Lesser binding of 1°gCd to the protein peak was observed in Cd + ethanol-exposed rats than the Cd-treated rats when hepatic supernatants from all the groups were chromatographed on Sephadex G-75 columns, suggesting that ethanol has a redistributing effect on Cd amongst the two pools. A marked increase in lipid peroxidation was observed which was linear to the increase in non-MT-Cd levels. A positive correlation between non-MT-Cd levels and lipid peroxidation was observed in liver, kidney and heart suggesting that non-MT-Cd levels are more crucial and toxicologically more important than total Cd levels.
Key words: Cadmium; Metallothionein; Lipid peroxidation; Rat
Introduction Cadmium has been established as a serious environmental pollutant which accumulates in mammalian tissues [1] with a very slow turnover rate that causes acute disorders at high doses, namely hepatotoxicity, testicular damage etc. [2]. It has been demonstrated that the accumulation of Cd in tissues and the development of pathological lesions attributable to its presence are determined not only by the intake of this metal but also by that of several nutritional factors [3]. Ethanol is one Correspondence to." Kiran Dip Gill, Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
252 such factor, the effect of which on Cd toxicity is not widely reported. Scanty data available on the influence of ethanol indicates that ethanol modifies Cd toxicity either by altering membrane permeability or by affecting its metabolism [4]. It is now widely established that the passage of heavy metals like Cd through a living system is invariably linked to the induction of a specific low molecular weight cysteine rich, metal binding protein, metallothionein (MT). This MT is widely thought to be protective against Cd toxicity and it is also postulated that intracellular Cd bound to MT is non-toxic [5]. Nomiyama and Nomiyama [6] put forward the concept of non-MT-Cd to explain the nephrotoxicity of Cd. It has been suggested that portions of cadmium are bound to both low and high molecular weight proteins and there is another fraction that is non-protein bound. This Cd has now been recognized to be a more accurate reflection of the critical concentration than the total Cd concentration [7-9]. Recently Goyer et al. [10] demonstrated that Cd-induced cellular toxicity is more directly related to that fraction of Cd in the kidney that is not bound to metallothionein, than to total Cd per se. This report suggested that the distribution factor of Cd between MT and non-MT-Cd pool is a determining factor for Cd -induced toxicity. This distribution of intracellular Cd, however, is reported to be influenced by a number of dietary factors such as deficient intake of proteins and calcium [3]. Ethanol consumption can act as a nutritional stress factor and might influence the distribution of Cd between these two pools. Stacey et al. [11] have proposed that intracellular cadmium interacts with cell membranes resulting in lipid peroxidation which has been recognized as the basic deteriorative reaction in cell damage by environmental pollutants [12]. This work was therefore, planned to study the accumulation of Cd in the presence of ethanol, its distribution between bound Cd and free Cd pools and also on Cd-induced lipid peroxidation. An attempt has also been made to find whether there exists a relationship between the non-MT-Cd and lipid peroxidation. Materials and methods
Animals Male albino rats (Wistar strain) weighing 100-120 g were obtained from the Institute animal house. The animals were housed in polypropylene cages with stainless steel lids and fed rat pellet diet (Hindustan Lever, Bombay) and water ad libitum. Experimental design Animals were divided into following groups of 6 - 8 animals each and were treated for 30 days. Controls. Animals were fed pellet diet and water ad libitum. This group served as the control group. Cd-treated. Animals were given 10 mg/kg body weight cadmium as CdCI 2 intragastrically daily for 30 days and were fed the pellet diet and water ad libitum. Ethanol-treated. Animals were given 7 ml of 10% ethanol (v/v) intragastrically. Pellet diet was given ad libitum. Cd + Ethanol-treated. Cadmium as in the Cd-treated group plus ethanol as in the ethanol-treated group was given.
253 Pairfed groups respective to all the treated groups were also treated simultaneously. The animals of the pairfed groups were given diet restricted in quantity compared to that consumed by their respective treated groups. Ethanol in these animals was replaced by isocalorically substituted sucrose. Pairfeeding was done to rule out the hormonal effect if any, due to intragastric application. After 30 days of treatment, animals were fasted overnight, anaesthetized and killed by decapitation. The liver, kidney, intestine, lung, spleen and heart were removed and rinsed thoroughly in ice-cold normal saline. Tissues were immediately used for various assays as described below.
Total Cd analysis Total Cd was estimated by using the wet ashing method of Evenson and Anderson [13]. For this, a known weight of tissue was digested with a nitric and perchloric acid mixture (5:1) and evaporated to dryness. The residue was dissolved in a known volume of 10 m M HNO3 and Cd was read directly in/zg/ml on an atomic absorption spectrophotometer (AAS) (Perkin-Elmer Model 4000-A) with a deuterium arc background and resonance line of 228.8. The results were expressed as nmoles Cd/g tissue. Estimation of total M T M T was estimated according to the Cd-saturation/haemoglobin method described by Onosaka and Cherian [14]. For this 10 p p m CdCI2 was added to 12 000 x g supernatant of the tissue homogenate. Free Cd was then removed by binding it with haemoglobin and precipitating by heat treatment. A known volume was made and Cd in the solution was determined by AAS. M T was calculated assuming that 7 g atom of Cd were bound to 1 mol of thionein. Results were expressed as nmoles MT/g tissue. Estimation of Cd bound to M T The same procedure was followed as for total MT, except that CdC12 was not added to saturate all the binding sites of MT. Results were expressed as nmoles of Cd bound to MT. Estimation of non-MT cadmium ( Cd not bound to MT) The values of non-MT cadmium were calculated by subtracting Cd bound to MT from total Cd. In vitro 1°9Cdand 65Zn binding studies The livers from all four groups were homogenized, heat -treated for 1 min and centrifuged at 12 000 x g for 30 min. The supernatant so obtained was incubated with l°9Cd and 65Zn and then chromatographed on a Sephadex G-75 column (90 cm x 1.5 cm). Fractions were collected at a flow rate of 21 ml/h and were monitored for l°9Cd and 65Zn in a LKB g a m m a counter. The optical density of all the fractions was also monitored at 254 nm in a Spectronic-21 photometer. Lipid peroxidation Lipid peroxidation (L-px) in the tissue was estimated by the formation of malon-
254 dialdehyde ( M D A ) a n d m e a s u r e d by the t h i o b a r b i t u r i c acid (TBA) method as described by Wills [15]. Since M D A is a d e g r a d a t i o n p r o d u c t of peroxidized lipids, the d e v e l o p m e n t of color with the characteristics (having a b s o r p t i o n maxima at 532 nm) of a T B A - m a l o n d i a l d e h y d e c h r o m o p h o r e is taken as an index of lipid peroxidation.
Statistical analysis Statistical analysis was d o n e by using one way analysis of variance (ANOVA). The significance was calculated using p r e p l a n n e d o r t h o g o n a l contrasts c o m p a r i n g two groups. F values having a P < 0.05 were considered significant. Since the results of the pairfed groups were n o t significantly different from control groups, the data on pairfed groups are omitted. Results
Figure l(a,b,c) shows the c a d m i u m c o n t e n t of various organs of rats treated with c a d m i u m , ethanol or their co-exposure. As expected, Cd a c c u m u l a t i o n was observed in all the organs o f rats exposed to Cd. The m a x i m u m retention of Cd was observed in liver after Cd t r e a t m e n t alone followed by kidney, intestine, spleen, heart and
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lungs. This accumulation, however, increased to about 2.5 times in the liver and kidney of the Cd + ethanol-exposed rats as compared to only Cd-treated rats. Intestine, spleen and lungs showed around 2.2-, 1.5- and 1.9-fold increases in Cd accumulation following Cd and ethanol co-exposure. An increase in the Cd levels was also observed in liver and intestines of only ethanol-treated rats. In response to Cd accumulation, M T was induced in all organs of rats exposed to Cd (Fig. 2). Maximum M T levels were observed in liver and kidney followed by intestine, spleen, heart and lungs. The levels of MT increased further to 3.0 times in liver, 1.6 times in kidney and 1.7 times in intestine in Cd + ethanol-exposed rats relative to only Cd-treated rats. No such further increase was observed in heart, lungs and spleen of Cd + ethanol-exposed rats. No increase in MT levels was observed in any of the organs of rat following only ethanol exposure. Intracellularly, Cd is usually present bound to native MT, but in certain circumstances this Cd can be present as a non-MT-Cd fraction also. The values of Cd bound to M T and not bound to M T are depicted in Fig. l(a,b,c). In the livers of control animals the whole of the Cd present was bound to MT. However, on Cd treatment with increasing accumulation of Cd in the liver, 51% of Cd was found bound to MT while the remaining was in the non-MT-Cd fraction. Conversely, on Cd and ethanol co-exposure, only 36% of the total Cd was present as Cd-MT while the rest was in the non-MT-Cd fraction. Similarly in the kidneys, while 80% of the i
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257
Cd was present as Cd-MT in Cd-treated group, only 57'¼, of the same was bound to MT in Cd + ethanol-exposed group. In the hearts, however, although there was no further increase in cadmium accumulation on Cd and ethanol co-exposure, a significant increase in the levels of non-MT-Cd was observed (41% as compared to 30% that was present as non-MT-Cd in only Cd-treated rats). In the intestine, spleen and lungs of control animals most of the Cd was present bound to MT. On Cd treatment 59%, 74% and 56% of the total Cd was present as Cd-MT in the intestine, spleen and lungs respectively. Although a significant increase in total Cd concentration was observed in these organs after Cd and ethanol co-exposure, the relative percentage of Cd in both these fractions remained the same. The elution profiles of hepatic supernatants chromatographed on Sephadex G-75 are presented in Fig. 3(a-d). 1°gCd w a s eluted in a single peak (fraction numbers 30-40, Fig. 3a). Significantly higher levels of l ° 9 C d w e r e eluted in this peak in the Cd-treated hepatic supernatant (Fig. 3b). This peak was associated with a simultaneous increase in the optical density (O.D.) at 254 nm suggesting the presence of Cd-thiolate bonds. Also in the Cd and ethanol-treated rat liver supernatant l ° 9 C d w a s eluted in the same single peak but the binding of l ° g C d to this peak was 40% less as compared to the binding that was observed in only Cd-treated rats. (Fig. 3c). The elution profile obtained from ethanol-treated rat liver supernatant was almost similar to that of controls (Fig. 3d). Q
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258 The data presented in Fig. 4 show the extent of lipid peroxidation in terms of M D A formations in various organs of rats treated with Cd, ethanol or both. It was observed that Cd treatment lead to a significant increase in lipid peroxidation in all organs of Cd-treated rats except heart and spleen, when compared to controls. The increase in lipid peroxidation was 1.41-fold in liver, 1.27-fold in kidney, 2.57-fold in lungs and 1.81-fold in intestine. On Cd and ethanol co-exposure there was a further increase in lipid peroxidation in liver and kidney which increased from 1.41 times to 2.37 times in liver and from 1.27 times to 1.6 times in kidney. In hearts of Cd + ethanol-exposed rats a significant increase of 1.46 fold was observed in contrast to only Cd-treated rats hearts where no change was observed. On only ethanol exposure a 1.34-fold increase in lipid peroxidation was observed in liver alone.
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Fig. 4. Lipid peroxidation values of various organs measured in terms of malondialdehyde formation from different groups of rats. r-I Controls (n = 6) received diet and water ad libitum; [], cadmium-treated rats (n = 8) received 10 mg cadmium/kg body wt;[~-~, ethanol-treated group (n = 8) received 5.56 g ethanol/kg body wt; l , cadmium + ethanol-treated group (n = 8) received cadmium (10 mg/kg body wt) and ethanol 5.56 g/kg body wt, Values are mean + S.D. of 6-8 observatoins. *Significantlydifferent from control group. *'+Significantly different from control as well as cadmium-treated group.
Discussion C a d m i u m from the environment enters the b o d y through lungs and gastrointestinal tract. Cd thus absorbed is transported into the blood and first accumulates in the liver. Since liver is comprised mainly o f hepatocytes which have a large cell volume and a higher protein content, it absorbs c a d m i u m [16]. The resultant high accumulation o f Cd in kidneys m a y be explained by a high MT-inducing ability o f kidney proximal tubule cells. Mobilization o f Cd b o u n d to several proteins from destructed liver cells also contribute to this accumulation. F r o m the kidneys where C d - M T is catabolized, c a d m i u m is set free and deposited via circulation to various organs o f the b o d y as is depicted in Fig. l(a,b,c). On Cd and ethanol co-exposure there was a further increase in tissue Cd levels. The increase varied from 2.5 times in liver to 1.6 times in lungs. In a similar kind o f experimental design T a n d o n and Tweari [17] also observed a significantly higher accumulation o f Cd in organs like the liver, kidney and spleen after co-exposure to Cd and ethanol. The data in Fig. 2 show that there is induction o f metallothionein synthesis in response to Cd exposure. M a x i m u m induction is observed in liver followed by kidney. It is reported that when c a d m i u m in the form o f its salts is administered to experimental animals, the highest percentage o f dose gets distributed to the liver
261
[18], hence maximum induction takes place in liver. In other organs too, a significant induction o f metallothionein was observed although it was much less as compared to either liver or kidney. Corresponding to increased accumulation o f Cd in the Cd + ethanol-exposed group, an enhanced M T synthesis in liver, kidney and intestines too was observed in this group. As discussed earlier Cd accumulation leads to the synthesis o f Cd-thionein which binds to Cd apart from Zn and Cu. It has been demonstrated that various dietary factors such as a deficient intake o f protein and calcium influence the intracellular distribution o f Cd between the metallothionein and non-metallothionein fractions [3]. Taking this as a working hypothesis, we observed in our data that in the Cd + ethanol-exposed group there was a significant increase in the percentage of n o n - M T - C d in comparison to the only Cd-treated group. Ravi et al. [19] also demonstrated a significant alteration in the distribution o f intracellular cadmium between M T and the n o n - M T fraction in protein calorie malnourished and calcium deficient monkeys exposed to cadmium as compared to the only Cd-treated monkeys. A particularly interesting feature was observed in hearts where no further increase in either Cd or M T levels was observed on Cd and ethanol co-exposure.
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262 However, the distribution of Cd amongst the two fractions i.e. Cd-MT and non-MTCd was significantly altered on Cd and ethanol co-exposure. A significant increase in non-MT-Cd levels was observed as compared to the only Cd-treated group suggesting that ethanol is affecting the distribution of Cd in both fractions. To confirm the redistributing effect of ethanol, hepatic supernatants from all four groups were chromatographed on Sepahdex G-75 columns and it was observed that a significantly lesser binding of l°9Cd to the peak fractions of the Cd + ethanolexposed hepatic supernatant was observed when compared to the only Cd-treated rat liver supernatant (Fig. 3a-d). Ethanol consumption can thus act as a nutritional stress condition and can alter the distribution of Cd between the Cd-MT and nonMT-Cd pools. Stacey et al. [11] postulated that intracellular Cd interacts with the cell membranes leading to lipid peroxidation. Since non-MT-Cd has been proposed to be the toxicologically reactive entity, an attempt was made to find a relationship between the non-MT-Cd and lipid peroxidation, if any. The data in Fig. 4 show that maximum lipid peroxidation was observed in the liver followed by kidney after Cd exposure and the same further increased significantly when the animals were coexposed to Cd and ethanol. It was also observed that the maximum non-MT-Cd levels were present in the liver and kidney of the same group Fig. l(a). A positive correlation between the levels of non-MT-Cd and lipid peroxidation was observed after statistical analysis of the coefficient of correlation. Furthermore, in the heart, although no increase in lipid peroxidation was observed in the Cd-treated rats, a significant increase relative to controls was observed in Cd + ethanol-exposed rats. This again relates well with the non-MT-Cd levels in heart, which increased significantly in animals co-exposed to Cd and ethanol when compared to the only Cd-treated group. Figure 5 depicts the relationship between non-MT-Cd and lipid peroxidation. A linear increase in L-px was observed with respect to increasing levels of non-MT-Cd. The data thus imply that the levels of non-MT-Cd can be more crucial for the induction of lipid peroxidation which eventually leads to peroxidative injury to the cell membranes and other subcellular organelles. In conclusion the results of this study demonstrate that ethanol not only increases the uptake and retention of cadmium but also significantly alters the distribution of Cd between MT and non-MT fractions.
Acknowledgement Financial assistance provided by Indian Council of Medical Research, New Delhi is gratefully acknowledged.
References 1
L. Friberg, M. Piscator, G.F. Nordbergand T. Kjellstrom. Cadmium in the Environment, 2nd Edn., CRC Press, Cleveland, 1974, p. 248. 2 R.L. Singhal, Z. Merali and P.D. Hrdina, Aspects of the biochemical toxicologyof cadmium. Fed. Proc., 35 (1976) 75. 3 M. Webb, The metallothioneins: The Chemistry, Biochemistry and Biologyof Cadmium, in M. Webb (Ed.), Elsevier North Holland, Biomedical Press, NY, 1979, pp. 195-266. 4 C.S. Lieber, Alcohol and the liver: Metabolism of ethanol, metaboic effects and pathogenesis of injury. Acta Med. Scand., 703 (1985) 11.
263 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
G.F. Nordberg, Effect of acute and chronic cadmium exposure with special reference to protective effects of metallothionein. Environ. Physiol. Biochem., 1 (1971) 171. K. Nomiyama and H. Nomiyama, Critical concentration of unbound cadmium in rabbit renal cortex. Experientia, 42 (1986) 49. M. Sato and Y. Nagai, Mode of existence of cadmium in rat liver and kidney after prolonged subcutaneous administration. Toxicol. Appl. Pharmacol., 54 (1980) 90-99. Z.A. Shaikh, Metallothionein as a storage protein for cadmium. Its toxicological implications, in E.C. Foulkes (Ed.), Biological Role of Metallothionein, Elsevier, New York, 1982, pp. 69-76. K. Waku, The chemical form of cadmium in subcellular fractions following cadmium exposure. Environ. Health Perspect., 54 (1984) 3744. R.A. Goyer, C.R. Miller, S. Zhu and W. Victery, Non-metallothionein bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat. Toxicol. Appl. Pharmacol., 101 (1989) 232. N.H. Stacey, L.R. Cantilena Jr. and C.D. Klaassen, Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes. Toxicol. Appl. Pharmacol., 53 (1980) 470. G.L. Plaa and H. Witschi, Chemicals, drugs and lipid peroxidation. Annu. Rev, Pharmacol. Toxicol., 16 (1976) 125. M.A. Everson and C.T. Anderson Jr., Ultramicro analysis for copper, cadmium and zinc in human liver tissue by use of atomic absorption spectrophotometry. Clin. Chem., 21 (1975) 537. S. Onosaka and M.G. Cherian, Comparison of metallothionein determination by polarographic and cadmium saturation methods. Toxicol. Appl. Pharmacol., 23 (1982) 270. E.D. Wills, Mechanism of lipid peroxide formation in animal tissues. Biochem. J., 99 (1966) 667. T.J. Caperna and M.L. Failla, Cadmium metabolism by rat liver endothelial and Kupffer cells. Biochem. J., 221 (1984) 631. S.K. Tandon and P.C. Tewari, Effect ofcoexposure to ethanol and cadmium in rats. Bull. Environ. Contam, Toxicol., 39 (1987) 633. M.G. Cherian and R.A. Goyer, Metallothioneins and their role in the metabolism and toxicity of metals. Life Sci., 23 (1978) 1. K. Ravi, V.K. Paliwal and R. Nath, Induction of Cd-Metallothionein in cadmium-exposed monkeys under different nutritional stresses. Toxicol. Lett., 22 (1984) 21.
251
Effect of ethanol on the distribution of cadmium between the cadmium metallothionein- and non-metallothionein-bound cadmium pools in cadmium-exposed rats G e e t a S h a r m a , R a v i n d r a N a t h and K i r a n Dip Gill Department of Biochemistry, Postgraduate Institute of Medical Education and Research. Chandigar h-160012 (India) (Received July 3rd, 1991; accepted February 9th, 1992)
Summary In an attempt to assess the effect of ethanol on c a d m i u m accumulation, metallothionein (MT) synthesis, Cd-binding capacity and lipid peroxidation, rats were administered either Cd, ethanol or their combination for a period of 4 weeks. A significant increase in Cd accumulation was observed in all the organs of rats under study co-exposed to Cd and ethanol as compared to only Cd-treated rats. Increased M T levels in response to Cd were associated with a marked alteration in the distribution of Cd amongst the two pools of intracellular Cd i,e. Cd bound to MT (Cd-MT) and Cd not bound to MT (non-MT-Cd). Higher levels of non-MT-Cd were observed in liver, kidney and heart of Cd + ethanol-exposed rats as compared to only Cd-exposed rats. Lesser binding of 1°gCd to the protein peak was observed in Cd + ethanol-exposed rats than the Cd-treated rats when hepatic supernatants from all the groups were chromatographed on Sephadex G-75 columns, suggesting that ethanol has a redistributing effect on Cd amongst the two pools. A marked increase in lipid peroxidation was observed which was linear to the increase in non-MT-Cd levels. A positive correlation between non-MT-Cd levels and lipid peroxidation was observed in liver, kidney and heart suggesting that non-MT-Cd levels are more crucial and toxicologically more important than total Cd levels.
Key words: Cadmium; Metallothionein; Lipid peroxidation; Rat
Introduction Cadmium has been established as a serious environmental pollutant which accumulates in mammalian tissues [1] with a very slow turnover rate that causes acute disorders at high doses, namely hepatotoxicity, testicular damage etc. [2]. It has been demonstrated that the accumulation of Cd in tissues and the development of pathological lesions attributable to its presence are determined not only by the intake of this metal but also by that of several nutritional factors [3]. Ethanol is one Correspondence to." Kiran Dip Gill, Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh-160012, India. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
252 such factor, the effect of which on Cd toxicity is not widely reported. Scanty data available on the influence of ethanol indicates that ethanol modifies Cd toxicity either by altering membrane permeability or by affecting its metabolism [4]. It is now widely established that the passage of heavy metals like Cd through a living system is invariably linked to the induction of a specific low molecular weight cysteine rich, metal binding protein, metallothionein (MT). This MT is widely thought to be protective against Cd toxicity and it is also postulated that intracellular Cd bound to MT is non-toxic [5]. Nomiyama and Nomiyama [6] put forward the concept of non-MT-Cd to explain the nephrotoxicity of Cd. It has been suggested that portions of cadmium are bound to both low and high molecular weight proteins and there is another fraction that is non-protein bound. This Cd has now been recognized to be a more accurate reflection of the critical concentration than the total Cd concentration [7-9]. Recently Goyer et al. [10] demonstrated that Cd-induced cellular toxicity is more directly related to that fraction of Cd in the kidney that is not bound to metallothionein, than to total Cd per se. This report suggested that the distribution factor of Cd between MT and non-MT-Cd pool is a determining factor for Cd -induced toxicity. This distribution of intracellular Cd, however, is reported to be influenced by a number of dietary factors such as deficient intake of proteins and calcium [3]. Ethanol consumption can act as a nutritional stress factor and might influence the distribution of Cd between these two pools. Stacey et al. [11] have proposed that intracellular cadmium interacts with cell membranes resulting in lipid peroxidation which has been recognized as the basic deteriorative reaction in cell damage by environmental pollutants [12]. This work was therefore, planned to study the accumulation of Cd in the presence of ethanol, its distribution between bound Cd and free Cd pools and also on Cd-induced lipid peroxidation. An attempt has also been made to find whether there exists a relationship between the non-MT-Cd and lipid peroxidation. Materials and methods
Animals Male albino rats (Wistar strain) weighing 100-120 g were obtained from the Institute animal house. The animals were housed in polypropylene cages with stainless steel lids and fed rat pellet diet (Hindustan Lever, Bombay) and water ad libitum. Experimental design Animals were divided into following groups of 6 - 8 animals each and were treated for 30 days. Controls. Animals were fed pellet diet and water ad libitum. This group served as the control group. Cd-treated. Animals were given 10 mg/kg body weight cadmium as CdCI 2 intragastrically daily for 30 days and were fed the pellet diet and water ad libitum. Ethanol-treated. Animals were given 7 ml of 10% ethanol (v/v) intragastrically. Pellet diet was given ad libitum. Cd + Ethanol-treated. Cadmium as in the Cd-treated group plus ethanol as in the ethanol-treated group was given.
253 Pairfed groups respective to all the treated groups were also treated simultaneously. The animals of the pairfed groups were given diet restricted in quantity compared to that consumed by their respective treated groups. Ethanol in these animals was replaced by isocalorically substituted sucrose. Pairfeeding was done to rule out the hormonal effect if any, due to intragastric application. After 30 days of treatment, animals were fasted overnight, anaesthetized and killed by decapitation. The liver, kidney, intestine, lung, spleen and heart were removed and rinsed thoroughly in ice-cold normal saline. Tissues were immediately used for various assays as described below.
Total Cd analysis Total Cd was estimated by using the wet ashing method of Evenson and Anderson [13]. For this, a known weight of tissue was digested with a nitric and perchloric acid mixture (5:1) and evaporated to dryness. The residue was dissolved in a known volume of 10 m M HNO3 and Cd was read directly in/zg/ml on an atomic absorption spectrophotometer (AAS) (Perkin-Elmer Model 4000-A) with a deuterium arc background and resonance line of 228.8. The results were expressed as nmoles Cd/g tissue. Estimation of total M T M T was estimated according to the Cd-saturation/haemoglobin method described by Onosaka and Cherian [14]. For this 10 p p m CdCI2 was added to 12 000 x g supernatant of the tissue homogenate. Free Cd was then removed by binding it with haemoglobin and precipitating by heat treatment. A known volume was made and Cd in the solution was determined by AAS. M T was calculated assuming that 7 g atom of Cd were bound to 1 mol of thionein. Results were expressed as nmoles MT/g tissue. Estimation of Cd bound to M T The same procedure was followed as for total MT, except that CdC12 was not added to saturate all the binding sites of MT. Results were expressed as nmoles of Cd bound to MT. Estimation of non-MT cadmium ( Cd not bound to MT) The values of non-MT cadmium were calculated by subtracting Cd bound to MT from total Cd. In vitro 1°9Cdand 65Zn binding studies The livers from all four groups were homogenized, heat -treated for 1 min and centrifuged at 12 000 x g for 30 min. The supernatant so obtained was incubated with l°9Cd and 65Zn and then chromatographed on a Sephadex G-75 column (90 cm x 1.5 cm). Fractions were collected at a flow rate of 21 ml/h and were monitored for l°9Cd and 65Zn in a LKB g a m m a counter. The optical density of all the fractions was also monitored at 254 nm in a Spectronic-21 photometer. Lipid peroxidation Lipid peroxidation (L-px) in the tissue was estimated by the formation of malon-
254 dialdehyde ( M D A ) a n d m e a s u r e d by the t h i o b a r b i t u r i c acid (TBA) method as described by Wills [15]. Since M D A is a d e g r a d a t i o n p r o d u c t of peroxidized lipids, the d e v e l o p m e n t of color with the characteristics (having a b s o r p t i o n maxima at 532 nm) of a T B A - m a l o n d i a l d e h y d e c h r o m o p h o r e is taken as an index of lipid peroxidation.
Statistical analysis Statistical analysis was d o n e by using one way analysis of variance (ANOVA). The significance was calculated using p r e p l a n n e d o r t h o g o n a l contrasts c o m p a r i n g two groups. F values having a P < 0.05 were considered significant. Since the results of the pairfed groups were n o t significantly different from control groups, the data on pairfed groups are omitted. Results
Figure l(a,b,c) shows the c a d m i u m c o n t e n t of various organs of rats treated with c a d m i u m , ethanol or their co-exposure. As expected, Cd a c c u m u l a t i o n was observed in all the organs o f rats exposed to Cd. The m a x i m u m retention of Cd was observed in liver after Cd t r e a t m e n t alone followed by kidney, intestine, spleen, heart and
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lungs. This accumulation, however, increased to about 2.5 times in the liver and kidney of the Cd + ethanol-exposed rats as compared to only Cd-treated rats. Intestine, spleen and lungs showed around 2.2-, 1.5- and 1.9-fold increases in Cd accumulation following Cd and ethanol co-exposure. An increase in the Cd levels was also observed in liver and intestines of only ethanol-treated rats. In response to Cd accumulation, M T was induced in all organs of rats exposed to Cd (Fig. 2). Maximum M T levels were observed in liver and kidney followed by intestine, spleen, heart and lungs. The levels of MT increased further to 3.0 times in liver, 1.6 times in kidney and 1.7 times in intestine in Cd + ethanol-exposed rats relative to only Cd-treated rats. No such further increase was observed in heart, lungs and spleen of Cd + ethanol-exposed rats. No increase in MT levels was observed in any of the organs of rat following only ethanol exposure. Intracellularly, Cd is usually present bound to native MT, but in certain circumstances this Cd can be present as a non-MT-Cd fraction also. The values of Cd bound to M T and not bound to M T are depicted in Fig. l(a,b,c). In the livers of control animals the whole of the Cd present was bound to MT. However, on Cd treatment with increasing accumulation of Cd in the liver, 51% of Cd was found bound to MT while the remaining was in the non-MT-Cd fraction. Conversely, on Cd and ethanol co-exposure, only 36% of the total Cd was present as Cd-MT while the rest was in the non-MT-Cd fraction. Similarly in the kidneys, while 80% of the i
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257
Cd was present as Cd-MT in Cd-treated group, only 57'¼, of the same was bound to MT in Cd + ethanol-exposed group. In the hearts, however, although there was no further increase in cadmium accumulation on Cd and ethanol co-exposure, a significant increase in the levels of non-MT-Cd was observed (41% as compared to 30% that was present as non-MT-Cd in only Cd-treated rats). In the intestine, spleen and lungs of control animals most of the Cd was present bound to MT. On Cd treatment 59%, 74% and 56% of the total Cd was present as Cd-MT in the intestine, spleen and lungs respectively. Although a significant increase in total Cd concentration was observed in these organs after Cd and ethanol co-exposure, the relative percentage of Cd in both these fractions remained the same. The elution profiles of hepatic supernatants chromatographed on Sephadex G-75 are presented in Fig. 3(a-d). 1°gCd w a s eluted in a single peak (fraction numbers 30-40, Fig. 3a). Significantly higher levels of l ° 9 C d w e r e eluted in this peak in the Cd-treated hepatic supernatant (Fig. 3b). This peak was associated with a simultaneous increase in the optical density (O.D.) at 254 nm suggesting the presence of Cd-thiolate bonds. Also in the Cd and ethanol-treated rat liver supernatant l ° 9 C d w a s eluted in the same single peak but the binding of l ° g C d to this peak was 40% less as compared to the binding that was observed in only Cd-treated rats. (Fig. 3c). The elution profile obtained from ethanol-treated rat liver supernatant was almost similar to that of controls (Fig. 3d). Q
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258 The data presented in Fig. 4 show the extent of lipid peroxidation in terms of M D A formations in various organs of rats treated with Cd, ethanol or both. It was observed that Cd treatment lead to a significant increase in lipid peroxidation in all organs of Cd-treated rats except heart and spleen, when compared to controls. The increase in lipid peroxidation was 1.41-fold in liver, 1.27-fold in kidney, 2.57-fold in lungs and 1.81-fold in intestine. On Cd and ethanol co-exposure there was a further increase in lipid peroxidation in liver and kidney which increased from 1.41 times to 2.37 times in liver and from 1.27 times to 1.6 times in kidney. In hearts of Cd + ethanol-exposed rats a significant increase of 1.46 fold was observed in contrast to only Cd-treated rats hearts where no change was observed. On only ethanol exposure a 1.34-fold increase in lipid peroxidation was observed in liver alone.
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Discussion C a d m i u m from the environment enters the b o d y through lungs and gastrointestinal tract. Cd thus absorbed is transported into the blood and first accumulates in the liver. Since liver is comprised mainly o f hepatocytes which have a large cell volume and a higher protein content, it absorbs c a d m i u m [16]. The resultant high accumulation o f Cd in kidneys m a y be explained by a high MT-inducing ability o f kidney proximal tubule cells. Mobilization o f Cd b o u n d to several proteins from destructed liver cells also contribute to this accumulation. F r o m the kidneys where C d - M T is catabolized, c a d m i u m is set free and deposited via circulation to various organs o f the b o d y as is depicted in Fig. l(a,b,c). On Cd and ethanol co-exposure there was a further increase in tissue Cd levels. The increase varied from 2.5 times in liver to 1.6 times in lungs. In a similar kind o f experimental design T a n d o n and Tweari [17] also observed a significantly higher accumulation o f Cd in organs like the liver, kidney and spleen after co-exposure to Cd and ethanol. The data in Fig. 2 show that there is induction o f metallothionein synthesis in response to Cd exposure. M a x i m u m induction is observed in liver followed by kidney. It is reported that when c a d m i u m in the form o f its salts is administered to experimental animals, the highest percentage o f dose gets distributed to the liver
261
[18], hence maximum induction takes place in liver. In other organs too, a significant induction o f metallothionein was observed although it was much less as compared to either liver or kidney. Corresponding to increased accumulation o f Cd in the Cd + ethanol-exposed group, an enhanced M T synthesis in liver, kidney and intestines too was observed in this group. As discussed earlier Cd accumulation leads to the synthesis o f Cd-thionein which binds to Cd apart from Zn and Cu. It has been demonstrated that various dietary factors such as a deficient intake o f protein and calcium influence the intracellular distribution o f Cd between the metallothionein and non-metallothionein fractions [3]. Taking this as a working hypothesis, we observed in our data that in the Cd + ethanol-exposed group there was a significant increase in the percentage of n o n - M T - C d in comparison to the only Cd-treated group. Ravi et al. [19] also demonstrated a significant alteration in the distribution o f intracellular cadmium between M T and the n o n - M T fraction in protein calorie malnourished and calcium deficient monkeys exposed to cadmium as compared to the only Cd-treated monkeys. A particularly interesting feature was observed in hearts where no further increase in either Cd or M T levels was observed on Cd and ethanol co-exposure.
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262 However, the distribution of Cd amongst the two fractions i.e. Cd-MT and non-MTCd was significantly altered on Cd and ethanol co-exposure. A significant increase in non-MT-Cd levels was observed as compared to the only Cd-treated group suggesting that ethanol is affecting the distribution of Cd in both fractions. To confirm the redistributing effect of ethanol, hepatic supernatants from all four groups were chromatographed on Sepahdex G-75 columns and it was observed that a significantly lesser binding of l°9Cd to the peak fractions of the Cd + ethanolexposed hepatic supernatant was observed when compared to the only Cd-treated rat liver supernatant (Fig. 3a-d). Ethanol consumption can thus act as a nutritional stress condition and can alter the distribution of Cd between the Cd-MT and nonMT-Cd pools. Stacey et al. [11] postulated that intracellular Cd interacts with the cell membranes leading to lipid peroxidation. Since non-MT-Cd has been proposed to be the toxicologically reactive entity, an attempt was made to find a relationship between the non-MT-Cd and lipid peroxidation, if any. The data in Fig. 4 show that maximum lipid peroxidation was observed in the liver followed by kidney after Cd exposure and the same further increased significantly when the animals were coexposed to Cd and ethanol. It was also observed that the maximum non-MT-Cd levels were present in the liver and kidney of the same group Fig. l(a). A positive correlation between the levels of non-MT-Cd and lipid peroxidation was observed after statistical analysis of the coefficient of correlation. Furthermore, in the heart, although no increase in lipid peroxidation was observed in the Cd-treated rats, a significant increase relative to controls was observed in Cd + ethanol-exposed rats. This again relates well with the non-MT-Cd levels in heart, which increased significantly in animals co-exposed to Cd and ethanol when compared to the only Cd-treated group. Figure 5 depicts the relationship between non-MT-Cd and lipid peroxidation. A linear increase in L-px was observed with respect to increasing levels of non-MT-Cd. The data thus imply that the levels of non-MT-Cd can be more crucial for the induction of lipid peroxidation which eventually leads to peroxidative injury to the cell membranes and other subcellular organelles. In conclusion the results of this study demonstrate that ethanol not only increases the uptake and retention of cadmium but also significantly alters the distribution of Cd between MT and non-MT fractions.
Acknowledgement Financial assistance provided by Indian Council of Medical Research, New Delhi is gratefully acknowledged.
References 1
L. Friberg, M. Piscator, G.F. Nordbergand T. Kjellstrom. Cadmium in the Environment, 2nd Edn., CRC Press, Cleveland, 1974, p. 248. 2 R.L. Singhal, Z. Merali and P.D. Hrdina, Aspects of the biochemical toxicologyof cadmium. Fed. Proc., 35 (1976) 75. 3 M. Webb, The metallothioneins: The Chemistry, Biochemistry and Biologyof Cadmium, in M. Webb (Ed.), Elsevier North Holland, Biomedical Press, NY, 1979, pp. 195-266. 4 C.S. Lieber, Alcohol and the liver: Metabolism of ethanol, metaboic effects and pathogenesis of injury. Acta Med. Scand., 703 (1985) 11.
263 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
G.F. Nordberg, Effect of acute and chronic cadmium exposure with special reference to protective effects of metallothionein. Environ. Physiol. Biochem., 1 (1971) 171. K. Nomiyama and H. Nomiyama, Critical concentration of unbound cadmium in rabbit renal cortex. Experientia, 42 (1986) 49. M. Sato and Y. Nagai, Mode of existence of cadmium in rat liver and kidney after prolonged subcutaneous administration. Toxicol. Appl. Pharmacol., 54 (1980) 90-99. Z.A. Shaikh, Metallothionein as a storage protein for cadmium. Its toxicological implications, in E.C. Foulkes (Ed.), Biological Role of Metallothionein, Elsevier, New York, 1982, pp. 69-76. K. Waku, The chemical form of cadmium in subcellular fractions following cadmium exposure. Environ. Health Perspect., 54 (1984) 3744. R.A. Goyer, C.R. Miller, S. Zhu and W. Victery, Non-metallothionein bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat. Toxicol. Appl. Pharmacol., 101 (1989) 232. N.H. Stacey, L.R. Cantilena Jr. and C.D. Klaassen, Cadmium toxicity and lipid peroxidation in isolated rat hepatocytes. Toxicol. Appl. Pharmacol., 53 (1980) 470. G.L. Plaa and H. Witschi, Chemicals, drugs and lipid peroxidation. Annu. Rev, Pharmacol. Toxicol., 16 (1976) 125. M.A. Everson and C.T. Anderson Jr., Ultramicro analysis for copper, cadmium and zinc in human liver tissue by use of atomic absorption spectrophotometry. Clin. Chem., 21 (1975) 537. S. Onosaka and M.G. Cherian, Comparison of metallothionein determination by polarographic and cadmium saturation methods. Toxicol. Appl. Pharmacol., 23 (1982) 270. E.D. Wills, Mechanism of lipid peroxide formation in animal tissues. Biochem. J., 99 (1966) 667. T.J. Caperna and M.L. Failla, Cadmium metabolism by rat liver endothelial and Kupffer cells. Biochem. J., 221 (1984) 631. S.K. Tandon and P.C. Tewari, Effect ofcoexposure to ethanol and cadmium in rats. Bull. Environ. Contam, Toxicol., 39 (1987) 633. M.G. Cherian and R.A. Goyer, Metallothioneins and their role in the metabolism and toxicity of metals. Life Sci., 23 (1978) 1. K. Ravi, V.K. Paliwal and R. Nath, Induction of Cd-Metallothionein in cadmium-exposed monkeys under different nutritional stresses. Toxicol. Lett., 22 (1984) 21.