Protection against paraquat-induced toxicity with sulfite or thiosulfate in mice

Toxicology, 79 (1993) 37-43 Elsevier Scientific Publishers Ireland Ltd.

37

Protection against paraquat-induced toxicity with sulfite or thiosulfate in mice Hiro-aki Yamamoto Department of Environmental Medicine, Institute of Community Mec~cine, The University of Tsukuba, Tsukuba, lbaraki 305 (Japan)

(Received July 20th, 1992; accepted October 15th, 1992)

Summary The toxicity of the herbicide paraquat in mice, measured by the single dose LD50 after 7 days, was significantly decreased by coinjection of thiosulfite (1 g/kg; one time per day for 3 days) or sulfite (0.2 g/kg; one time per day for 3 days). However, the toxicity of paraquat was not changed by coinjection of sulfate (1 g/kg; one time per day for 3 days). The body weight of mice was significantly decreased by paraquat treatment (30 mg/kg, i.p.). However, the decrease of body weight was abolished by coinjection of thiosulfate or sulfite but not by coinjection of sulfate. On the other hand, paraquat significantly decreased reduced glutathione contents in liver. The depletion of the glutathione contents was also abolished by coinjection of thiosulfate or sulfite but not by coinjection of sulfate. These results suggest that the preventive effect against paraquat-induced toxicity with thiosulfate or sulfite may involve the glutathionedependent detoxication in mice. Key words." Paraquat; Thiosulfate; Sulfite; Acute toxicity; Glutathione antagonist

Introduction P a r a q u a t (1,1 ' - d i m e t h y l - 4 , 4 ' - b i p y r i d i u m ion) is widely used as b r o a d s p e c t r u m herbicide a n d has caused d e a t h s due to its toxicity from accidental o r suicidal ingestion. G l u t a t h i o n e , a s c o r b a t e a n d c~-tocopherol are n o t effective as a n t i d o t a l drugs against p a r a q u a t toxicity. E x p o s u r e to high levels o f p a r a q u a t p r o d u c e s lung, kidney a n d liver injury in h u m a n s [1,2] a n d a n i m a l s [3,4]. P a r a q u a t is readily c o n v e r t e d by one e l e c t r o n - r e d u c t i o n to a free radical which reacts very r a p i d l y with m o l e c u l a r oxygen [5]. T h e r e a c t i o n regenerates the native b i p y r i d y l a n d converts oxygen to a high reactive molecule such as super-oxide a n i o n radicals (. 0 2 - ) [6]. P a r a q u a t toxicity is e n h a n c e d b y the presence o f high c o n c e n t r a t i o n s o f oxygen [7]. F u r t h e r m o r e , p a r a q u a t stimulates lipid p e r o x i d a t i o n in vitro, n o t a b l y in liver a n d lung m i c r o s o m e s [8,9] a n d has also been shown to r e d o x cycle in vivo [10], a l t h o u g h some in vivo studCorrespondence to." Hiro-aki Yamamoto, Department of Environmental Medicine, Institute of Community Medicine, The University of Tsukuba, Tsukuba, Ibaraki, 305 Japan.

0300-483X/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

38 ies have failed to show increased rates of lipid peroxidation during paraquat toxicity [11,12]. On the other hand, it has been reported that paraquat significantly decreases reduced glutathione concentrations in liver [13]. Hagen, et al. [14] have shown that exogenous glutathione leads to the protection against injury induced by paraquat. Inorganic sulfite and thiosulfate are a naturally occuring sulphur compounds with strong nucleophilicity and appreciable redox activity. A redox activity of sulfite allows it to interact with cysteinyl disulfides in both low molecular weight compounds such as oxidized glutathione and protein disulfides through sulfitolysis [15,16], a process resembling thiol-disulfide exchange. Sulfite is used extensively as an antioxidant and antimicrobial agent in a variety of chemical, pharmaceutical and food manufacturing industries and is produced during the fermentation of wine and beer [17]. It is a potent allergen in human [17] and produces a variety of toxic effects in animals [18,19]. Thiosulfate is used as an antidotal drug against cyanide toxicity and produces few toxic effects in animals and humans. Both sulfite and thiosulfate are produced as a systemic metabolic intermediate of amino acid cysteine [20], one of amino acid sequence in glutathione. Recently, a number of reports have suggested that sulfite is able to diminish the toxicity of allyl alcohol [21], acrolein [22], Nacetyl-p-benzo-quinone imine [22], menadione [23] and diquat [23] in isolated hepatocytes. In the present experiments, we examined in vivo whether sulfite or thiosulfate, sulfur compound possessing nucleophilicity and redox activity and a metabolic intermediate of cysteine, can protect against paraquat-induced toxicity. Materials and methods

Male ddy mice (Charles river, Shizuoka, Japan), weighing 20-28 g, were used throughout the experiments and were maintained on a standard laboratory feed (Oriental kobo) and water ad libitum. The room temperature was kept at 22-24°C and humidity was 40-80%. Artificial light was the only source and the animals were set on a 14-h light/dark cycle with lights on 0600 h. Paraquat (I,1 '-dimethyl-4,4'-dipyridium chloride), was purchased from Tokyo Kasei Ltd. Sodium thiosulfate, sodium sulfite and sodium sulfate were obtained from Nacalai Chemical Co. (Kyoto, Japan). All drugs were dissolved in saline. Paraquat was injected intraperitoneally in 0.9% NaC1 solution containing 0.3-0.5% paraquat at a dose of 30, 35, 40, 45 or 50 mg/kg just before the first administration of thiosulfate, sulfite or sulfate. The thiosulfate, the sulfite or the sulfate was injected subcutaneously in 0.9% solution containing 10% thiosulfate, 2% sulfite or 10% sulfate one time per day for 3 days. The median lethal dose was determined for paraquat either alone or in combination with thiosulfate, sulfite or sulfate. The experimental values were obtained for three or more groups of mice containing 10-20 mice in each group. LDs0 values based on 7-day effect were calculated by the method of Litchfield-Wilcoxon (1949) with confidence limits of 95% probability. The body weight was determined for 7 days after paraquat injection.

Determination of hepatic reduced glutathione contents Mice treated with paraqaut, thiosulfate + paraquat, sulfite + paraquat or saline were killed by decapitation at 24 h after administration and their livers were rapidly

39 TABLE I EFFECT OF THIOSULFATE, SULFITE OR SULFATE ON THE LETHAL EFFECTS OF PARAQUAT IN MICE a Treatment (g/kg)

LDs0 of paraquat (95% confidence) (mg/kg)

Saline Thiosulfate (1.0) Sulfite (0.2) Sulfate (1.0) Thiosulfate (1.0)+ Sulfite (0.2)

33.0 43.0 45.0 30.0 44.0

(29.9-36.4) (38.7-47.8)* (39.9-50.8)* (26.8-33.5) (38.8-50.0)*

aThiosulfate, sulfite or sulfate was injected subcutaneously one time per day for 3 days in mice (see Method). *Significantly different from control (P < 0.05).

removed and immediately frozen. These samples were used for an assay of reduced glutathione. Reduced glutathione content in the liver was determined using the colorimetric method of Ball [25].

Other methods The data of glutathione contents or animal body weight were statistically analyzed by analysis of variance. Where a significant difference was indicated, the means were determined by Student's t-test [26] using a probability level of 0.05. Results

Effect of thiosulfate, sulfite or sulfate on lethal effect of paraquat The effect of thiosulfate, sulfite or sulfate on the lethal effect of paraquat is shown in Table I. The LDs0 value of paraquat alone (33.0 mg/kg) was similar to a

TABLE II PREVENTIVE POTENCY RATIOS AGAINST LETHAL EFFECT OF PARAQUAT WITH THIOSULFATE OR SULFITE Treatments (g/kg)

Slope function a

Potency ratio b

Saline Thiosulfate (1.0) Sulfite (0.2) Sulfate (1.0) Thiosulfate (1.0)+ sulfite (0.2)

1.22 (1.04-1.42) 1.33 (1.07-1.66) 1.34 (1.03-1.75) 1.29 (1.05-1.59) 1.33 (1.03-1.72)

1 1.30 (1.15-1.47)* 1.36 (I.13-1.63)* 0.91 (0.78-1.06) 1.33 (I.11-1.59)*

aNone of the slope were significantly different from one another. bpotency ratio = LDs0 of paraquat with or without antagonist(s)/LDs0 of paraquat without antagonist. *Significantly increased from saline (P < 0.05).

40

previously reported value [13]. Cotreatment of thiosulfate or sulfite (one time per day for 3 days) in mice significantly increased the paraquat LDs0 to 43.0 or 45.0 mg/kg. However, coadministration of sulfate (1.0 g/kg; one time per day for 3 days) did not change in the paraquat LDs0 (Table I). The slopes of the log dose-mortality effect regression lines were analyzed statistically in all experiments and were not significantly different from each other (Table II); therefore, calculation of the potency ratios were valid. The significant protection against lethal effect of paraquat was provided by thiosulfate (potency ratio = 1.30), sulfite (potency ratio = 1.36) or thiosulfate plus sulfite (potency ratio = 1.33). However, sulfate did not provide a significant protection (potency ratio = 0.91).

,..--,.

2

o~

-1

-2

I

2

3

4

5

6

7 Cday)

Fig. 1. Effect ofparaquat, thiosulfate, sulfite or sulfate on the increase of body weight. Mice (body weight 20-21 g; 4 weeks old) were used in these experiments. Paraquat (30 mg/kg) was intraperitoneally injected at a single dose. Thiosulfate (1 g/kg (.)), sulfite (0.2 g/kg (Q)), sulfate (1 g/kg (0)) or saline (&) in mice just before paraquat injection. Control receiced saline (O) Values represent as means of increased gram body weight 4- S.D. (n = 10). *Siginificantly different from control (P < 0.01). **Significantly different from paraquat plus saline treated mice (P < 0.01). Administration of paraquat plus saline killed by 50% of the mice three days after treatment. Therefore, we determined the body weight during three days in mice treated with paraquat plus saline.

41 T A B L E III E F F E C T O F P A R A Q U A T ON H E P A T I C G L U T A T H I O N E C O N T E N T S IN MICE a Treatments (g/kg)

Glutathione contents (tzmol/g wet liver)

Saline Paraquat Paraquat + thiosulfate Paraquat + sulfite Paraquat + sulfate

7.62 + 0.352 5.50 ± 0.216" 6.56 4- 0.321"* 6.80 4- 0.374** 5.21 ~ 0.248*

aparaquat (30 mg/kg) was injected intraperitoneally in mice treated with or without thiosulfate (1 g/kg, s.c.), sulfite (0.2 g/kg, s.c.), sulfate (1 g/kg, s.c.) or saline. Control received saline. Reduced glutathione contents in liver were determied as described under Methods. Data are represented the mean + S.E. (n = 6). *Significantly different from control (P < 0.001). **Significantly different from paraquat alone-treated (P < 0.05).

The effect of thiosulfate, sulfite or sulfate on paraquat-induced reduction in body weight Paraquat (30 mg/kg, i.p.) significantly reduced the body weight of mice. The reduction in body weight induced by paraquat was in part abolished by cotreatment of thiosulfate (1.0 g/kg) or sulfite (0.2 g/kg) though sulfate (1.0 g/kg) did not abolish the reduction in body weight induced by paraquat (Fig. 1). The effect of thiosulfate, sulfite or sulfate on paraquat induced depletion of glutathione in liver. Paraquat (30 mg/kg) administration significantly decreased reduced glutathione contents in liver (Table III). Cotreatment of thiosulfate (1.0 g/kg) or sulfite (0.2 g/kg) prevented against paraquat-induced glutathione depletion in liver. However, cotreatment with sulfate (1.0 g/kg) did not prevent against the paraquat-induced depletion of glutathione in liver. Discussion

Our present data clearly demonstrated that inorganic sulfite and thiosulfate, sulphur compounds possessing strong nucleophilicity and appreciable redox activity, protected against paraquat-induced toxicity in mice. However, inorganic sulfate, another sulphur compound possessing strong nucleophilicity and no redox activity, did not protect against paraquat-induced toxicity. These results suggest that the protection against paraquat-induced toxicity with sulfite or thiosulfate may be due to its redox activity but not due to its nucleophilicity. Recently, Sun et al. [23] have reported that sulfite reduced diquat-induced toxicity in isolated hepatocytes when cells were incubated at 37°C with sulfite and diquat. They have also suggested that depletion of glutathione levels in isolated hepatocytes induced by paraquat is abolished by treatment of sulfite. These findings suggest that sulfite abolishes the

42 diquat-dependent depletion in glutathione contents of hepatic cells and leads to the protective effect against its toxicity. On the other hand, it is known that paraquat induces acute hepatic toxicity in association with stimulated redox cycling with molecular oxygen [27,28]. This redox cycling rapidly increases the production of reactive oxygen metabolites such as • O2-, H202and •O H - and leads to a stimulation of oxidative stress. Recently, it has also been reported that paraquat causes acute hepatic toxicity in association with oxidative depletion of reduced glutathione, indicating redox cycling in the mechanisms of toxicity of paraquat [28]. We have found that paraquat decreased reduced glutathione contents in liver and its effects of paraquat was abolished by thiosulfate or sulfite but not by sulfate (Table III). These observations led to the following two hypotheses. The first is that thiosulfate or sulfite may compete with reduced glutathione for reaction with • O2-, H202 and •OH-. The second is that sulfite may react directly with oxidized glutathione by sulfitosis to liberate reduced glutathione from disulfide [16,21]. Since it is known that thiosulfate can release sulfite in vivo, thiosulfate may be also able to react indirectly with oxidized glutathione by sulfitosis. An increase in reduced glutathione levels in cells can lead to further glutathione-dependent detoxication by enzyme such as glutathione peroxidases. In support of this, it has been reported that in mice deficient in selenium, which is required for glutathione peroxidase activity [29], paraquat toxicity is significantly increased [30]. On the other hand, it is well known that paraquat toxicity in man is characterized by the lung and liver damage [1,2]. It is also believed that lung damage may be the major cause of death. Recently, Hagen et al. have reported that exogenous reduced glutathione protects against the pulmonary injury induced by paraquat [14]. Sulfite or thiosulfate possessing redox activity may have a similar effect with exogenous reduced glutathione in lung and this may be the cause of the inhibitory effect to lethality induced by paraquat in mice. On the other hand, maximal preventive action against paraquat toxicity was observed when thiosulfate and sulfite was injected in mice with 1.0 and 0.2 g/kg, respectively (data not shown). The maximal preventive value induced by thiosulfate was similar to that induced by sulfite (Table I). Furthermore, the combined administration of thiosulfate and sulfite also elicited a similar preventive action and degree. These results suggest that thiosulfate and sulfite may act at the same operation sites as an antagonist against paraquat toxicity. In conclusion, we have presented evidence that protective action against paraquatinduced toxicity with thiosulfate or sulfite may be associated with direct or indirect blockade of toxic active oxygen by its redox activity. Thiosulfate and sulfite may find use as an antidotal drug for paraquat poisoning although further pharmacological and toxicological experiments with several animal species will be necessary before clinically trials should be considered. References

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