Selenium lethality: Role of glutathione and metallothionein

Toxicology Letters, 66 (1993) 213-219

273

0 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4274/93/$06.00

TOXLET 02883

Selenium lethality: Role of glutathione and metallothionein

Vani K. Nonavinakerea, Ronald0 A. Potmisa, Hamid R. Rasekh”, Rosonald R. Bellb and Johnnie L. Early II” “Florida A&M University, College of Pharmacy and Pharmaceutical Sciences, Tallahassee, FL (USA) bThe Upjohn Company, Kalamazoo, MI (USA)

and

(Received 28 August 1992) (Accepted 10 November 1992) Key words: Selenium lethality; Diethylmaleate; Glutathione; Metallothionein

SUMMARY Male Sprague-Dawley rats (200-300 g) were pretreated (i.p.) with diethylmaleate (DEM; 3.1 mmol/kg) or propylene glycol (PG). After 1 h, three PG and three DEM groups received saline or sodium selenite (Se: 0.8 or 1.6 mg/kg) i.p. Eighty to one hundred percent mortality occurred within 3 h after Se in DEMpretreated groups. Except for one PG and one DEM group, which were sacrificed after 1 h, the remaining groups received saline or Se (1.6 mg/kg) 25 h after pretreatment. No mortality occurred within 3 h after Se. Liver and kidney GSH decreased at 1 h, while liver MT increased at 28 h. The changes are related to Se-induced lethality.

INTRODUCTION

Apart from its importance as an essential trace element, selenium (Se) has been recognized for its toxic effects [l-3]. Biochemically, Se is required as an integral part of the enzyme glutathione peroxidase (GSH-Px) [4]. Other biochemical functions of this element are still being investigated [5]. Selenium interacts similar to the heavy metals with sulphydryl groups [6]. The tissue richest in biological components containing nonprotein and protein sulphydryl groups is the liver [7]. GSH is a nonprotein sulphydryl compound that protects SH-groups on enzymes [8]. GSH can be depleted by reacting with diethylmaleate (DEM) [9].

Correspondence to: Vani K. Nonavinakere, Florida A&M University, College of Pharmacy and Pharmaceutical Sciences, Tallahassee, FL 32307, USA.

274

One-third of the amino acids found in metallothionein (MT), a low molecular weight protein, are cysteines. MT has, thus, an abundance of sulphydryl groups, which are believed to be extremely important in the functioning of this protein [lo]. The interaction of Se with metallothionein-like proteins (MTLPs) of similar weight range as MT has been investigated, and the binding to MTLP was comparable to that of Se binding to GSH-Px [l I]. Mammalian MTs are induced by high doses of heavy metals [12,13] and recent reports indicate that MT be associated with resistance to various forms of oxidative stress [l&16]. Bauman et al. have recently shown that the induction of MT levels in various tissues after DEM administration occurs at the mRNA level [17]. Sodium selenite has been shown to increase lipid peroxidation in vivo [ 181. GSH-Px reduces lipid hydroperoxides to the corresponding alcohol, and hydrogen peroxide to water. In both reactions GSH is utilized. It has also been shown that MT can decrease lipid peroxidation [ 191. The objectives of this study were to investigate the significance of GSH in Se toxicity with respect to DEM-induced changes in MT levels. MATERIALS

AND METHODS

Animuls Male albino Sprague-Dawley rats weighing 200-300 g (Harlan Sprague-Dawley. Indianapolis, IN) were used in this study. Animals were housed in metal cages, at 21°C under a 12-h light/dark cycle (lights on 06:OO h), and had free access to food and water. Rats were acclimated for 7 days prior to experimentation.

Chemicals Diethylmaleate (DEM; maleic acid diethyl ester) was mixed with propylene glycol (PG) to result in a final concentration of 3.1 mmol/ml. The above chemicals and chemicals for GSH and MT determinations except ““Cd, were obtained from Sigma Chemical Co., St Louis, MO. Sodium selenite (Alfa products, Danvers, MA) was dissolved in double distilled deionized water to achieve Se concentrations of 0.8 and “‘Cd, specific activity 2.67 ,Kil mg, was purchased from New 1.6 mg/ml. Cadmium, England Nuclear (Boston, MA).

Experimental procedures Expt. 1. Groups of five rats were pretreated

with DEM (3.1 mmol/kg) or PG (i.p.). One hour later, these rats received saline or Se: 0.8 or 1.6 mg/kg, i.p. Mortality, was monitored over a 3-h period. Expt. 2. Rats (n = 4-5) were pretreated with PG or DEM. After 1 h, one PG- and one DEM-pretreated group was sacrificed by decapitation. Liver and kidney samples were excised and analyzed for GSH and MT. The remaining PG- or DEM-pretreated groups (n = 4-5) were challenged 25 h later with Se (1.6 mg/kg) or saline, i.p., and were sacrificed 3 h following the Se challenge. Liver and kidney samples were again analyzed for MT and GSH. GSH was measured

215

in order to ascertain that the alterations in GSH that might have occurred 1 h after DEM did not prevail at 25 h. Glututhione assay. Liver and kidney (cortex) samples were weighed, homogenized in ice-cold 0.02 M ethylenediaminetetraacetic acid (EDTA). One ml 50% trichloroacetic acid (TCA) was added to 5 ml of homogenate after diluting with 4 ml of distilled water. The above solutions were vortexed and centrifuged for 15 min at 3000 x g. Two ml of the supernatant was mixed with 4 ml of 0.4 M Tris buffer (pH 8.9) and 100 ~1 of 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB; 99 mg/25 ml methanol). The absorbance was read at 412 mu against reagent blank within 5 min as described by Sedlak and Lindsay [20]. Metallothionein assay. Liver samples were weighed, homogenized (1:3 w/v) in ice-cold 10 mM Tris-HCl buffer (pH 7.4). The liver homogenates were centrifuged at 10 000 x g for 10 min and the resulting supernatant was further centrifuged at 100 000 x g for 1 h. Metallothionein was measured by the Cd/hemoglobin radioassay of Onosaka et al. [21], as modified by Eaton and Toal [22]. Statistical analysis Data on mortality, GSH, and MT were analyzed by analysis of variance (ANOVA) under the General Linear Models of the Statistical Analysis System (SAS). Multiple mean comparisons were done according to Duncan [23]. The probability level was set at P < 0.05. RESULTS

Table I shows Se lethality after DEM as compared to PG pretreated controls. Selenium lethality is indicated as No. of rats dead/No. of rats treated in groups of n = 5. Selenium (0.8 or 1.6 mg/kg, i./p.) injected 1 h after DEM (3.1 mmol/kg, i.p.) resulted in 80 to 100% mortality. No mortality was observed in all groups pretreated with PG, and in groups treated with saline or Se 25 h after DEM. Mortality in groups treated with Se 1 h after DEM was found to be highly significant (P < O.OOOl),when compared to 0% mortality in all remaining groups. Liver and kidney GSH levels WmoYg tissue) after PG or DEM are indicated in Table II. A decrease in liver GSH (P < 0.05) and in kidney GSH (P c 0.0001) was observed 1 h post DEM administration as compared to the respective GSH levels of PG pretreated controls. Liver and kidney GSH at 28 h after DEM were not different from their respective controls (not shown in the table). Liver MT levels are shown in Fig. 1. A significant increase in liver MT (P < 0.0001) at 28 h after DEM was observed when compared to liver MT after PG (1 and 28 h), and to liver MT 1 h after DEM. Metallothionein levels of Se-injected groups were greater than saline-administered controls. Liver MT 1 h after DEM increased, however, this increase was not of the same magnitude as liver MT 28 h after DEM. There was no significant increase in kidney MT levels after DEM pretreatment when compared to PG-pretreated controls.

216

DISCUSSION

Diethylmaleate has been recognized as an oxidative stress agent that decreases GSH levels [17,24]. It has also been observed that DEM predisposes the rats to Se lethality [24,25]. It has been reported that DEM conjugates with GSH, rendering it unavailable for selenite reduction [26]. Selenite itself is known to increase peroxidation which further emphasizes the need for GSH in selenium detoxification [27]. Selenium challenge at 1 h post DEM resulted in 80-100% mortality, whereas no deaths occurred when the Se challenge was delayed for 25 h after DEM (Table I). The differential response to Se in DEM-pretreated rats may be due in part to time-dependent depletion of reduced glutathione (GSH) (Table II). The findings of this study showed no decrease in liver and kidney GSH levels at 28 h after DEM when compared to PG-injected controls unlike the decrease observed at 1 h after DEM indicating that the GSH depletion by DEM is short lived. The repletion rate of GSH in kidney has been reported to be higher than that in liver [28]. The results obtained indicate that GSH levels are resumed after initially being depleted. Adequate tissue TABLE EFFECT

I OF DEM (3.1 mmol/kg

BODY WEIGHT,

i.p.) ON SE LETHALITY

Rats

Lethality

(No.

dead/No.

treated)

(2-3 h) Control

1 h after PG: O/S

(PG injected)

saline

(n = 5)

Se (0.8 mg/kg)

O/5

Se (1.6 m&kg)

015

25 h after PG: Se (0.8 mg/kg)

015 015

Se (1.6 mg/kg)

015

saline

DEM injected (n = 5)

1 h after DEM: saline

015

Se (0.8 mg/kg)

4/5** 5/5**

Se (1.6 mg/kg) 25 h after DEM: saline

o/5

Se (0.8 mg/kg) Se (1.6 mg/kg)

o/5 o/5

Male Sprague-Dawley rats (20&300 g) were treated as indicated. **Significantly different from the remaining groups (F= 99999.99; Mortality occurred between 2-3 h after Se.

df = 24; P < 0.0001)

211

60-

-

PG

!5Sl DEM (3.1

mmol/kg) **

501 40-

30-

20-

oL

10-

1

28 Saline TIME

Se

(HOURS)

Fig. 1. Effect of DEM on liver M,, 1 and 28 h after injection. Male Sprague-Dawley rats (200-300 g) were treated as indicated. Values represent mean f SE (n = 45). **Significant difference as compared to means of PG-injected controls, and mean of 1 h after DEM group (F= 27.67; df= 21; P < 0.0001)

TABLE II EFFECT OF DEM (3.1 mmol/kg BODY WEIGHT, i.p.) ON LIVER AND KIDNEY GSH 1 h AFTER THE INJECTION Rats

Liver GSH @mol/g tissue)

Kidney GSH @mol/g tissue)

Control (PG injected) (n = 45)

3.201 t 0.485

3.008 ? 0.204

DEM injected (n = 4-5)

1.728 f 0.238*

0.356 dz0.064**

Male Sprague-Dawley rats (200-300 g) were treated as indicated. Kidney cortex was used to determine GSH. Data are means f SE. *Significant decrease as compared to PG-injected control. (F = 6.33; df = 7; P < 0.05). **Significant decrease as compared to PG-injected control (F= 19.67; df= 14; P < 0.0001).

278

GSH levels may be in part the reason for the 100% survival observed in DEMpretreated rats when the Se challenge was delayed for 25 h. Our findings confirm the finding of Bauman et al., that oxidative stress induced by DEM depletion of liver GSH results in induction of MTs [17]. Increased liver MT levels 29.52 + 4.65 and 45.49 & 5.86,uglg tissue, observed 28 h after DEM were significantly greater than the increased MT (13.42 + 0.45 ,ug/g tissue) 1 h after DEM (Fig. 1). The oxidative nature of selenite might be the reason for the greater increase in MT levels observed 28 h after DEM in the Se-treated group (Fig. 1). However, MT levels of saline- and Se-treated groups did not differ significantly in PG-pretreated controls. A role for MT in amino acid transport analogous to GSH has been proposed [29]. Although the function of MT remains unknown, it is possible that the regulation of metallothionein expression is part of the host defense component. Thornalley and Vasak suggested that metal-thiolate clusters on MT serve to scavenge hydroxyl radicals [30]. The observed increase in liver MT is a DEM-induced oxidative stress response, that can be enhanced by selenite administration. Whether the increased MT at 28 h after DEM pretreatment plays a role in resisting Se challenge needs to be further investigated. Binding of Se to MTLP has been observed [ 111. Results from this study indicate that selenium lethality in DEM-pretreated rats is influenced by the time elapsed between administration of Se and DEM. Both, an increase in the thiol-rich metal-binding protein, MT, and resumed GSH levels 28 h after DEM play a significant role in protecting against mortality due to Se. ACKNOWLEDGEMENTS

The authors thank Drs. J.W. Bauman and M. Iszard (Kansas Medical Center) for sharing the information. The authors appreciate Dr. F.K. Stino’s advice on statistical analysis. The technical assistance by Z. Mallory is acknowledged. The authors thank Ms. F. James for typing the manuscript. The research was financially supported by NIH/NIGMS/MBRS grant (SO6 GM 08111) and partially by NIH/NIRR/RCMI grant (RR03020). REFERENCES 1 Diplock,

A.P. (1976) Metabolic

aspects

of selenium

action

and toxicity.

CRC Crit. Rev. Toxicol.

4,

be cured? CRC Crit. Rev. Toxicol.

1,

272-329. 2 Frost, D.V. (1972) The two faces of selenium 4677514. 3 Burk, R.F. (1975) Selenium

Can selenophobia

in man. In: A.S. Prasad

and D. Oberlas

(Eds.), Trace Elements

in Human

Health and Disease, Vol. ii, Academic Press, New York, pp. 1055135. 4 Rotruck, J.T., Pope, A.L., Ganther, H.E., Swanson, A.B., Hafeman, D.G. and Hoekstra, W.G. (1973) Selenium: Biochemical role as a component of glutathione peroxidase. Science 179, 588590. 5 Burk, R.F. (1989) Protection 385.

against

free radical

injury by selenoenzymes.

Pharmacol.

6 Ganther, H.E. (1979) Metabolism of hydrogen selenide and methylated selenides. (Ed.), Advances in Nutritional Research, Plenum Press, New York, pp. 2-107.

Ther. 45, 383In: H.H.

Draper

219

7 Jocelyn,

P.C. (1972) Thiols

Academic 8 Jocelyn,

Press, London,

and disulphides

P.C. (1972) Biochemistry

9 Plummer,

tissues.

In: Biochemistry

of the SH Group,

Academic

Press, London,

J.L., Smith, B.R., Seis, H. and Bend, J.R. (1981) Chemical

Methods

Enzymol.

IO Hamer,

in animal

of the SH Group,

p. 261. pp. xvi-xvii.

depletion

of glutathione

D.H. (1986) Metallothionein.

Annu.

Rev. Biochem.

11 Viljoen, A.J. and Tappel, A.L. (1988) Interactions

55, 913-951.

of selenium and cadmium

with metallothionein-like

and other cytosolic proteins of rat kidney and liver. J. Inorg. Biochem. 34, 2777290. 12 Valle, B.L. (1979) Metallothionein: Historical review and perspectives. In: J.H.R. Nordberg 13 Vasak,

(Eds.), Metallothionein, M. and Kagi, J.H.R.

Metal Ions in Biological 14 Bakka,

A., Johnsen,

content

Birkhauser-Verlag,

B.A., Voss, G.W.,

cadmium

aerosol

17 Bauman,

J.W., Mckim

diethylmaleate. 18 Dougherty,

Shatos,

of metallothionein.

the protective

J. (1990) Cross tolerance

G.J. and Girotti,

and zinc-metallothioneins.

of lipid peroxidation

A.W. (1987) Inhibition

Biochim.

in biochemical

D.B. (1955) Multiple

24 Early, J.L. and Schnell, drug metabolism 25 Nonavinakere, selenium

Biophys.

V.K.,

26 Chasseaud,

pigments.

28 Rodriguez,

and nonprotein procedure

F tests. Biometrics depletion

sulfhydryl

for determination

of

affinity assay for the rapid deter-

Appl. Pharmacol.

Lunan,

S. and Mallory,

Fed. Proc. 2, No. 5, Abstr. with glutathione Metabolism

66, 134142.

11, l-62. on selenium

lethality

and hepatic

Z.Y. (1988) Effect of diethylmaleate

on

6646.

and mercapturic

and Function.

B.Z. (1986) Effect of dietary

J. Am. COB. Toxicol.

J.V., Torres,

lipid peroxidation

Lett. 11, 2533257.

(Eds.), Glutathione:

A.S. and Menken,

lipofuscin

J.L.,

L.F. (1976) Conjugation

and W.B. Jacoby 27 Csallany,

Early,

by

in vivo by injected selenite

ofcell membrane

of the Cd/hemoglobin tissues. Toxicol.

R.C. (1982) Effect of glutathione

hyperglycemia.

of metallothionein

Acta, 884,448461.

K. (1978) A simplified 24, 128-133.

range and multiple

in male rats. Toxicol.

induced

following

Proc. Sot. Exp. Biol. Med. 169, 209-215.

D.L. and Toal, B.F. (1982) Evaluation

23 Duncan,

to hyperoxia

103, 2555270. C.D. (1992) Induction

W.G. (1982) Stimulation

of metallothionein

against

114, 188-196.

21 Onosaka, S., Tanaka, K., Doi, M. and Okahara, metallothionein in animal tissues. Eisei Kagaku mination

in cells with high

role of metallothioneins

20 Sedlak, J. and Lindsay, R.H. (1968) Estimation of total, protein-bound, groups in tissue with Ellman’s reagent. Anal. Biochem. 25, 192-205.

22 Eaton,

In: H. Sigel (Eds.).

Lett. 50, 1233135.

Appl. Pharmacol.

Lui, J. and Klaassen,

by selenate.

J.P., Bachowski,

by cadmium-

regarding

M.A. and Doherty,

Appl. Pharmacol.

J.J. and Hoekstra,

M.

New York, pp. 213-273.

H.E. (1982) Radioresistance

drugs. Toxicol.

Toxicol.

Jr. J.M.,

Toxicol.

and lack of stimulation 19 Thomas,

anticancer

pretreatment.

and

38, 381-383.

15 Basu, A. and Lazo, J.S. (1990) A hypothesis 16 Hart,

Dekker,

L. and Rugstad.

Experientia

of DNA interactive

properties

Vol. 15, Marcel

A.S., Endresen,

Kagi

Basel, pp. 1940.

(1983) Spectroscopic

Systems,

of metallothionein.

the toxicity

in vivo.

77,50-59.

acid excretion.

In: I.M. Arias,

Raven Press, New York, pp. 77-l 14.

selenite on hepatic

organic

solvent-soluble

5, 79985.

A.M. and Elias, M.M. (1987) Renal and hepatic

glutathione

pool modifica-

tions in response to depletion treatments. Can. J. Physiol. Pharmacol. 65, 8486. 29 Kagi, J.H.R., Mimmelhoch, S.R., Whanger, P.D., Bethune, J.L. and Vallee, B.L. (1974) Equine hepatic and renal metallothioneins. Purification, J. Biol. Chem. 249, 353773542. 30 Thornalley, induced Biochim.

P.J. and Vasak,

oxidative Biophys.

molecular

weight, amino acid composition,

M. (1985) Possible role for metallothionein

stress. Kinetics

and mechanism

Acta 827, 3644.

of its reaction

in protection

with superoxide

and metal content. against

radiation

and hydroxyl

radicals.