JOURNAL OF APPLIED TOXICOLOGY, VOL. 14(3), 181-183 (1994)

Evaluation of Three Antidotes on Arsenic Toxicity in the Common Earthworm (Lumbricus terrestris)

~~~~

Wei Li, Paul K. Chien and Arthur Furstt Department of Biology, University of San Francisco, San Francisco, CA 94117-1080, USA

Key words: earthworms; arsenic toxicity; arsenic antidotes.

The common earthworm (Lumbricus terrestris) is being evaluated in our laboratories as a substitute for mice in metal toxicity studies. These two disparate species have enzymes in common, such as catalase, superoxide dismutase and glutathione-Stransferase. Also, worms respond similarly to these rodents for selenium and nickel toxicity. Worms are less sensitive, however, to metal toxicity. In this study earthworms were challenged with three different arsenic compounds: arsenite, arsenate and the vesicant phenyldichloroarsine (PDA). The median lethal dose for each arsenic compound was determined. The order of toxicity of the arsenic compounds to the worms was PDA > arsenite > arsenate (24 h LD~,, values were 189.5, 191.0 and 519.4 pmol kg-', respectively). Individual mammalian dithiol antidotes, namely the sodium salt of 2,fdimercapto- l-propanesul- fonic acid (DMPS), mesedimercaptosuccinic acid (DMSA) or 2,3-dimercapto-l-propanol (BAL), were injected into the worms 5 min after various doses of the arsenic compound were administered. The decreases in acute toxicity values were recorded. All three antidotes protected the worms against arsenic toxicity with varying degrees of effectiveness. The protective action for the inorganic arsenic compounds was in the order DMPS > DMSA > BAL. For the organic arsenical, PDA, the most effective antidote was BAL.

INTRODUCTION

In a continuing program to evaluate the common earthworm (Lumbritus terristris) as a potential substi- tute for mice (Swiss albino) in metal toxicity, studies were undertaken on the effect of three dithiol antidotes on three arsenic compounds. This earthworm belongs to Oligochaeta, which usually lives in soil and decaying oraganic matter.' The body of the earthworm is segmentally arranged by sepia. Coelomic fluid fills every compartment and supports the gut located in the center of the coelom. By contracting muscles, the common earthworm can push the coelomic fluid to pass through the septa and circulate in the coelom. Earthworms are readily available, inexpensive and easy to handle.

Information from our laboratories show that the two disparate species, mice and earthworms, have many common reactions to metal compounds. In acute toxicity studies, selenite is more toxic than selenate in mice by a factor of almost 3:l . This same ratio was found in the earthworm.* The ratios of the median lethal doses of nickel acetate to the chloride or sulfate are nearly identical in both specie^.^ As a rule, however, the worms are more resistant to the toxicity of metals than the rodents. When relative toxicity values of compounds of the same element are compared both species seem to have parallel responses.

Enzymes like superoxide d i ~ m u t a s e , ~ catalase5 and glutathione-S-transferae (GSH-S-t)6 are active in both species. For GSH-S-t, like in rodents, the substrate 4- nitrodichlorobenzene is superior to 4-nitroquinoline- N-oxide.' Another similarity is that both species induce

two isoforms of metallothionein following the injection of cadmium compounds.*

Arsenic toxicity in the past has been investigated extensively in both dogs and rats9 Because of the binding of arsenic to erythrocytes in the rat, the clearance time is measured in months not in hours as in humans, thus Vahter'O cautions the interpretation of arsenic toxicity when the data are obtained from rats. However, rats and mice are used to evaluate potential arsenic antidotes. l1

This study first determined the median lethal toxic dose of three arsenic compounds, the sodium salts of the water-soluble arsenite and arsenate and the oil- soluble vesicant phenyldichloroarsine (PDA). Follow- ing this study, the efficacies of three dithiol antidotes were evaluated: the sodium salt of 2,3-dimercapto-1- sulfonic acid (DMPS) and meso-dimercaptosuccinic acid (DMSA), both of which are water-soluble, and the oil-soluble 2,3-dimercapto-l-propanol (BAL). The antidotal studies were conducted by injecting worms first with increasing concentrations of arsenic com- pounds followed 5 min later with the same concen- tration of the antidote. The data of lethality vs dose were plotted on probit paper. The decrease in the L D ~ ~ value for each arsenic compound was recorded.

MATERIALS AND METHODS

Worms

The common earthworms (Lurnbricus terrestris) were purchased from a local bait shop in San Francisco. They were kept at room temperature in potting soil

CCC 0260-437X/94/030181-03 0 1994 by John Wiley & Sons, Ltd. Received 30 March 1993

Accepted (revised) 7 June 1993

182 W. LI ET AL.

100

80

- 2

60 h 4J .rl rl

c, m

' 40 s

20

0

Table 1. Acute toxicity of three arsenic compounds as modi- fied by three antidotes

- 1. ARSENATE LD50=519.4

2. WITH DMPS LD50=841.0

- 3. WITH DMSA LD50=607.6

4. WITH BAL LD50=531.2

-

-

-

d h I I

Arsenite Arsenate Phenyldichloroarsine (Fig. 1) (Fig. 2) (Fig. 3)

Antidote Control 191 .O 519.4 189.5 DMPS 254.6 841 .O 287.7 DMSA 222.3 607.6 225.0 BAL 21 1.9 531.2 317.4

fortified with corn meal for no more than 1 day before being placed on test.

Chemicals

Three dithiol antidotes (DMSA, DMSP, BAL) and the sodium salts of arsenite and arsenate were obtained from Sigma Chemical Co. The PDA was purchased from Research OrganidInorganic Chemical Corp.

Experimental

Each worm was rinsed with tap-water, blotted dry with paper towels and then weighed. For each test group five worms weighing in the range 3-8 g were selected. Dosages in pmol kgg' of the arsenicals (arsenite, arsenate and PDA) and the antidotes (DMPS, DMSA and BAL) were calculated according to the weight of the individual worm. The arsenic compound was injected into the coelom 0.5 cm behind the clitellum in mature worms on the ventral side. Increasing doses were administered and the lethality data at 24 h were used for estimating the median lethal dose of each arsenic compound. A second series of groups was also injected with increasing doses of the arsenic compound and 5 min later the worms were injected 0.5 cm below the first injection sites with the same concentration of the potential antidote.

All injected worms were kept at room temperature. After 24 h, the number of survivals was recorded. The data were plotted on probit paper (probability of death vs log of dose). The various L D ~ ~ values were estimated from the graphs.

RESULTS

The relative acute toxicities ( L D ~ ~ ) of the three arsenic compounds (arsenite, arsenate and PDA) with and without the three dithiol antidotes (DMPS, DMSA and BAL) are given in Table 1. The three graphs give a more complete picture of the antidotal action. As this study was to determine comparative toxicity of the arsenic compounds with and without the antidotes, no detailed statistical analysis was conducted. However, all data were plotted on probit paper, and all graphs were generated using the computers. As to be expected, arsenite is more toxic than arsenate by a ratio of 2.72. Arsenite and PDA are about equally toxic to the earthworm. For both arsenite and arsenate the order

100 -

00 -

A

dP - 60 - h c, -4 rl

CI m k 40 - 2

20 -

1. ARSENITE LD50=191.0

2. WITH DMPS LD50=254.6

3. WITH DMSA LD50=222.3

4. WITH BAL LD50=211.9

6 0 ' 70 80 90100 200 300

Arsenite conc. (pmol kg-')

Figure 1. Acute toxicity of arsenite alone and after treatment with the three thiol antidotes.

of protection of the three antidotes is DMPS > DMSA > BAL. For the oil-soluble PDA, the protection order is BAL > DMPS > DMSA.

DISCUSSION

The toxicity of arsenicals is attributed to their reaction with SH moieties in the molecules rather than other entities.12 Of the three arsenicals tested in this study, the organic compound PDA is a bifunctional arsenical that can act with two available thiol groups per molecule. The product of this reaction is a cyclic species that is thermodynamically more stable than

ANTIDOTES TO ARSENIC TOXICITY IN EARTH WORMS 183

l o o r 1. PDA LD50=189.5

2. WITH DMPS LD50=287.7

3. WITH DMSA LD50=225.0

4. WITH BAL LD50=317.4

PDA conc. (pmol kg-')

Figure 3. Acute toxicity of Phenyldichlororsine (PDA) alone and after treatment with the three thiols.

the reaction product derived from monothiols. This results in greater toxicity of PDA to the earthworms.

In comparison with the data available from mice, earthworms show a threefold greater resistance to arsenite. For PDA the ~ ~ 5 0 value for mice and earthworms are almost the same.

Traditionally, BAL and D-penicillamine have been used for the treatment of arsenic poisoning. This is being replaced by newer water-soluble dithiols; an excellent review is by Aposhian and A p 0 ~ h i a n . l ~ The dithiol antidotes have varying degree of activity against arsenic toxicity in the experimental earthworms. For

both water-soluble sodium salts of arsenite and arsen- ate, the water-soluble antidotes DMPS and DMSA were more efficient than the oil-soluble BAL. The reverse was true for PDA, which is easily dissolved in lipids. For the rodent, DMSA is much more effective than is DMPS for the toxicity of the two inorganic arsenic compounds; BAL was not reported in that study." Both DMPS and DMSA can mobilize tissue arsenic; BAL can increase the concentration of arsenic in the animal brain. l4

2,3-Dimercapto-l-propanol is being replaced by the water-soluble dithiol compounds as antidotes for humans exposed to toxic levels of arsenic. One disad- vantage of BAL is that it is insoluble in water and is susceptible to oxidation; this action has the potential of inducing some toxicity. The two water-soluble dithiols are stable crystalline compounds, and are less toxic in themselves.

This study shows that the common earthworm is sensitive to various arsenicals, such as arsenite, arsenate and PDA, and also that the various antidotes DMPS, DMSA and BAL are selectively effective. If the intracoelomic route is employed, reproducible results can be obtained. The common earthworm may be a good model for studies on acute toxicity of arsenicals and their potential antidotes.

Worms are being considered as the species of choice for preliminary screening of various potential toxic substances. The European Economic Community (EEC) now recommends that the earthworm Eiseniu fetidu, kept in an artificial soil, be used for testing their toxicity of organic compound^.'^ HeimbachI6 compared the toxic response of Eisenia fetidu with Lumbricus terristris for a number of toxic agents and found the correlation to be 0.81; no inorganic substances were tested in this study.

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