Toxicology, 72 (1992) 251-263 251 Elsevier Scientific Publishers Ireland Ltd.

Effect of ethanol on the distribution of cadmium between the cadmium metallothionein- and

non-metallothionein-bound cadmium pools in cadmium-exposed rats

Gee ta Sharma, Rav ind ra N a t h and Ki ran 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 cadmium accumulation, metallothionein (MT) synthesis, Cd-binding capacity and lipid peroxidation, rats were administered either Cd, ethanol or their combina- tion 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 MT 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 tox- icologically more important than total Cd levels.

Key words: Cadmium; Metallothionein; Lipid peroxidation; Rat

Introduction

Cadmium has been established as a serious environmental pollutant which ac- cumulates 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 in- take 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 in- tracellular Cd bound to MT is non-toxic [5]. Nomiyama and Nomiyama [6] put for- ward 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 sug- gested 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 relation- ship 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 in-

tragastrically 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 simultane- ously. The animals of the pairfed groups were given diet restricted in quantity com- pared 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 kill- ed 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 mM HNO3 and Cd was read directly in/zg/ml on an atomic absorp- tion 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 MT was estimated according to the Cd-saturation/haemoglobin method described

by Onosaka and Cherian [14]. For this 10 ppm 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. MT 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°9Cd and 65Zn binding studies The livers from all four groups were homogenized, heat -treated for 1 min and cen-

trifuged 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 gamma 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 (MDA) and measured by the th iobarbi tur ic acid (TBA) method as

described by Wills [15]. Since M D A is a degradat ion product of peroxidized lipids, the development of color with the characteristics (having absorpt ion maxima at

532 nm) of a TBA-malond ia ldehyde chromophore is taken as an index of lipid peroxidat ion.

Statistical analysis Statistical analysis was done by using one way analysis of variance (ANOVA). The

significance was calculated using preplanned or thogonal contrasts compar ing two groups. F values having a P < 0.05 were considered significant.

Since the results of the pairfed groups were not significantly different from control groups, the data on pairfed groups are omitted.

R e s u l t s

Figure l(a,b,c) shows the cadmium content of various organs of rats treated with cadmium, ethanol or their co-exposure. As expected, Cd accumula t ion was observed in all the organs of rats exposed to Cd. The max imum retent ion of Cd was observed in liver after Cd t reatment alone followed by kidney, intestine, spleen, heart and

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Fig. 1. (a,b,c) Total cadmium, cadmium bound to metallothionein (Cd-MT) and cadmium not bound to metallothionein (non-MT-Cd) values in various organs of rats from different groups. I"1 Controls (n = 6) received diet and water ad libitum ; DI, cadmium-treated rats (n = 8) received 10 mg cadmium/kg body wt;~, ethanol-treated group (n = 8) received 5.56 ethanol/kg body wt; I , cadmium + ethanol-treated group (n = 8) received cadmium (10 mg/kg body wt) and ethanol 5.56 g/kg body wt. Values are mean 4- S.D. of 6-8 observations, *Significantly different from control group. *'+Significantly different from control as well as cadmium-treated group.

255

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256

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. In- testine, spleen and lungs showed around 2.2-, 1.5- and 1.9-fold increases in Cd ac- cumulation 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, MT was induced in all organs of rats exposed to Cd (Fig. 2). Maximum MT 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 cir- cumstances this Cd can be present as a non-MT-Cd fraction also. The values of Cd bound to MT and not bound to MT 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

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Fig. 2. Metallothionein content of various organs from different groups of rats. r-I Control (n = 6) receiv- ed diet and water ad libitum; I~1, 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; i , 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 observations. *Significantly different from control group. *'+Significantly different from control

as well as cadmium-treated group.

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 o f 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 treat- ment 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 concentra- tion 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 o f l ° 9Cd 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 °9Cd w a s eluted in the same single peak but the binding of l ° gCd 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).

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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 con- trast 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. *Significantly different from control group. *'+Significantly different from control as well as cadmium-treated group.

Discussion

Cadmium from the environment enters the body through lungs and gastro- intestinal tract. Cd thus absorbed is t ransported 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 cadmium [16]. The resultant high accumulat ion o f Cd in kidneys may be explained by a high MT-inducing ability o f kidney proximal tubule cells. Mobil izat ion o f Cd bound to several proteins f rom destructed liver cells also contr ibute to this accumulation. F rom the kidneys where Cd-MT is catabolized, cadmium is set free and deposited via circulation to various organs o f the body 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 of experimental design Tandon and Tweari [17] also observed a significantly higher accumulat ion 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 induct ion o f metallothionein synthesis in response to Cd exposure. Max imum induction is observed in liver followed by kidney. It is reported that when cadmium in the form of 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 of metallothionein was observed although it was much less as compared to either liver or kidney. Corresponding to increased accumulation of Cd in the Cd + ethanol-exposed group, an enhanced MT synthesis in liver, kidney and in- testines 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 of protein and calcium influence the intracellular distribution of 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 non-MT-Cd in comparison to the only Cd-treated group. Ravi et al. [19] also demonstrated a significant alteration in the distribution of intracellular cadmium between MT and the non-MT 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 MT 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-MT- Cd 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 sug- gesting 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 + ethanol- exposed 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 non- MT-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 co- exposed 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

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metals. Life Sci., 23 (1978) 1. 19 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.