Detoxication of xenobiotics by earthworms

Camp. Biochem. Phvsiol. Vol. 78C, No. 2, pp. 249-252. 1984

0306~4492/84 $3.00 + 0.00 fi: 1984 Pergamon Press Ltd

Printed in Great Britain

MINIREVIEW DETOXICATION

OF XENOBIOTICS J~RGEN

Norwegian

Plant

Protection

Institute,

STENERSEN

Box 70, N-1432

AS-NLH,

(Received 21 November

INTRODUCTION

More than a hundred years ago Charles Darwin recognized the importance of the earthworm in the decomposition of organic litter such as leaves and animal manure. However, even today, very little attention has been paid to the possible importance of these animals in the degradation of chemicals such as man-made xenobiotics or natural products. The toxicity of chemicals to earthworms, however, has been studied frequently. Reviews have been written by Edwards and Thompson (1973), Edwards and Lofty (1977) and by Thompson (1973). Terrestrial animals, such as insects and mammals, have evolved complicated enzyme systems in order to cope with xenobiotic substances-that is substances of little or no nutritional value which may be toxic or accumulate if not degraded. Such substances are frequently more soluble in oils and organic solvents than in water, but to be excreted they must be transferred via the water phase.

MICROSOMAL

BY EARTHWORMS

MONOOXYGENASES

Oxidation is a common route that make many xenobiotics more water soluble. Several animals of terrestrial phyla, e.g. arthropods and vertebrates, have developed a complicated system named microsomal monooxygenases or mixed function oxidases (MFO-system). This system is able to catalyse the oxidation of many natural and man-made oil-soluble substances. It is probably one of the more popular subjects for biochemical research. Between 1970 and 1980, Chemical Abstracts refers to about 4000 publications on this topic. The properties and role of the MFOsystem are less well-documented for aquatic animals than for the terrestrial ones, although a comprehensive review on “Pesticide and Xenobiotic Metabolism in Aquatic Organisms” appeared recently (Khan et al., 1979). The MFO-system is present in earthworms. Nelson et al. (1976) found that aldrin was converted to dieldrin by Lumbricus terrestris. The reaction proceeded after injection of aldrin into living worms, as

parathion

Telephone:

(02) 94 94 00

1983)

well as by enzyme preparations. Subcellular distribution of aldrin epoxidase, requirement for the cosubstrates oxygen and NADPH and the sensitivity to inhibition by carbon monoxide indicated that the aldrin epoxidase of earthworms is a typical microsomal monooxygenase involving the hemoprotein cytochrome P-450. The authors were not able to identify cytochrome P-450, however, by the standard difference-spectrophotometric method because a high amount of erythrocruorin in the preparations interfered. The highest epoxidase activity was found in the gut wall and the typhlosole (Fig. 1). By using gel filtration on Sepharose 2B (Pharmacia) Liimatainen and Hgnninen (1982) have recently separated cytochrome P-450 from the interfering blood pigments. Their preparations gave a carbon monoxide difference spectrum characteristic for cytochrome P-450. 7-Ethoxycoumarin was O-dealkylated, whereas benzo(a)pyrene or 7-ethoxyresorufin were not substrates. The oxidative metabolism of a few other substances has also been reported. Nakatsugawa and Nelson (1972) reported that diethylphosphate was produced from parathion (OO-diethyl O-Cnitrophenylphosphorothioate) in L. terrestris. Similar results were found by Stenersen (1979b) using “E. foetida” (These worms are now divided into two species, Oien and Stenersen, 1984). The worm used has the proposed name E. unicolor (Andrt). The product actually formed by the mixed function oxidases is paraoxon (diethyl 4-nitrophenyl phosphate), but the microsomal preparation contains an esterase that converts paraoxon to the diester. Paraoxon formation was therefore demonstrated by the presence of its degradation product, diethylphosphate (Fig. 2).

paraoxon 249

Norway.

diethylphosphate

250

JC~RGEN STENERSEN

We detected only N,N-dimethylformamide

I

VTGuGiCE

P

Organ Fig. 1. Enzymatic conversion of aldrin to dieldrin by different tissues from the earthworm Lumhricus terrestris. I, intestine; V, viscera; T, typhlosole; Gu, gut wall; Gi, gizzard; C, crop; E, eosophagus; P, pharynx. The figure is drawn from the data of Nelson et ul. (1976).

When purified

parathion (10 pg) was injected into the level of cholinesterase activity decreased gradually to 25% of the original level during a week (Stenersen, 1979b). This is further evidence for an oxidative metabolism of parathion because the cholinesterases are not inhibited by parathion, but rather by paraoxon which is formed through oxidation. The metabolism of the insecticidal carbamate, carbofuran, is very complex in houseflies and rats (Dorough, 1967) involving oxidation, hydrolysis and conjugation. Similar reactions seem to be operative in earthworms (Stenersen et al., 1973; Gilman and Vardanis, 1974). 3-Hydroxy carbofuran is a typical product of the MFO system and was found in both L. terrestris and E. ,fetida. On the other hand, the oxime carbamate, oxamyl, which has a low toxicity to earthworms (Stenersen, 1979a) does not seem to be metabolized by oxidative pathways in earthworms. Eiseniu

unicolor,

1

2

3

4

1234

Substance Fig. 2. Conversion of 14C-1abelled parathion 5 days after injection (0.5 pg per worm) into Eisenia unicolor. (A) The distribution of radioactivity between various metabolites inside the worms and (B) in ihe surrounding medium (sand). 1, Dimethylphosphate; 2. OO-dimethvlahosnhorothioate: O-m&y1 _ 0-4-nitrophenylphosihordthioate; 4, 3, unchanged parathion. The figure is drawn from the data of Stenersen (1979b).

trace amounts of l-cyanoin earthworms treated with oxamyl (Stenersen and Mien, 1980) although this substance is formed through oxidation catalysed by rat liver microsomes (Harvey and Han, 1978). Oxidation and hydrolysis, followed by conjugation are the major metabolic pathways of carbaryl (1-naphthyl methylcarbamate) in many species (Matsumura, 1975). This carbamate pesticide is very toxic to L. terrestris (Asp&k and An der Lan, 1963), but in E. unicolor it produces long-lasting non-lethal symptoms (Stenersen, 1979a). indicating a very slow metabolism. No direct evidence of the recalcitrance of carbaryl towards detoxication enzymes in earthworms has, however, been produced. We must. with the present level of our knowledge, conclude that a role of the microsomal monooxygenases as a general detoxication system in earthworms needs further elucidation. GLUTATHIONE S-TRANSFERASES

The glutathione S-transferases catalyse the reaction of the tripeptide glutathione (GSH) with hydrophobic substances bearing an electrophilic center. The conjugation may proceed through addition of the glutathione ion to a double bond (I), by cleavage of an epoxide bridge (II) or by substitution of halogen or other leaving groups (III): I:

RCH:CHR’

II: RCH-CHR’

+ GSH + RH(SG)CHR’ + GSH + RCH(OH)CH(SG)R’

‘0’ III: R-X

+ GSH -+ R-SG.

1 HX Such electrophilic substances are frequently very toxic. Sometimes they act as carcinogens and mutagens and their elimination is therefore important. Many epoxides formed by the mixed function oxidases are such noxious substances. Our investigations (Stenersen et al., 1979; Stenersen and 0ien, 1981) have shown that earthworms have a high activity of these enzymes when measured with a “good substrate”. In rat liver, these enzymes may constitute approx. lOo/o of the total cytosolic protein (Jakoby et al., 1976). Seven different types have been isolated from rat liver. They have different, but overlapping substrate specificities (Jakoby, 1978). Our studies indicate that the earthworms have a GSH S-transferase system as complicated as the rat. Using isoelectric focusing (Stenersen et cd., 1979) or anion exchange chromatography (Stenersen and Mien, 1981), several forms of GSH S-transferases were demonstrated from all species tested. Using the different substrates quintozene (pentachloronitrobenzene), ethacrynic acid, 1,2-dichloro-4-nitrobenzene and I-chloro-2,4_dinitrobenzene it was shown that the different types present in Eiseniu unicolor had different substrate specificity. The activity towards 1-chloro-2,4_dinitrobenzene and ethacrynic acid seem to be high in all species. We found in nine species that the specific activity varied between 35.9 nkatalimg soluble protein (Allo-

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Detoxication of xenobiotics by earthworms lobophora rosea) and 3.2 (Dendrobaena hortensis), and that the activity towards ethacrynic acid was between 5.8 to 25% of this activity. By the same methods the activities in rat liver were determined to be 24.1 nkatal/mg protein and 2.4% respectively (Stenersen and Mien, 1981). The GSH S-transferases of earthworms were unable or less able to detoxicate many substances which are easily conjugated by one or more forms of the rat liver or housefly enzyme. The pesticides atrazine and gamma-HCH were not, whereas quintozene was, conjugated. Other typical transferase substrates like p-nitrophenoxy 2,3_propyleneoxide and bromosulphophtalein were not conjugated. The classical transferase substrate 1,2-dichloro-4-nitrobenzene was conjugated at a reasonable rate by some species only. Iodomethane or parathion-methyl were not substrates in any species tested. The earthworms therefore seem to lack methyl, glutathione S-transferase activity. Based upon the reaction rate with l-chloro-2,4dinitrobenzene, the earthworms contain a considerable amount of GSH S-transferase in most tissues. We found the highest activity in the nephridia, followed by the crop and the ventral nerve cord. The chloragoneous tissue were low in activity. This tissue is therefore not completely analogous to the vertebrate liver which has high activity compared to other organs (Stenersen and Mien, 1981). We are now investigating purification methods and have made considerable progress using affinity chromatography, essentially after the method of Simons and Jagt (1977). The physiological role of these enzymes (as they appear in earthworms) is obscure. The narrow substrate specificity indicates that their intrinsic function may be to support excretion of endogenous catabolic products, rather than to support detoxication of xenobiotics.

CONCLUDING

REMARKS

Studies of the detoxication enzymes of earthworms and other soil inhabitants could be an interesting contribution to ecotoxicology from the biochemist. More knowledge about this will help us to rationalize the increasing accumulation of data about the toxicity of various substances. There are also some other more theoretical biological aspects which could be elucidated through such studies. The annelids have never conquered the dry land as successfully as mammals and insects. Many biologists have proposed the hypothesis that good detoxication mechanisms are more important for terrestrial than for aquatic animals-because they cannot dilute and excrete noxious substances in a great amount of water (e.g. Brodie and Maickel, 1962). Although being true terrestrial animals, the earthworms need very high humidity to thrive. A better knowledge about detoxication enzymes in such semiterrestrial animals, as well as comparative data between related terrestrial and aquatic groups will elucidate the role of these enzymes in evolution as well as their role in the general toxicology.

REFERENCES

Asp&k H. and An der Lan H. (1963) okologische wirkungen und physiologische Besonderheiten

Ausdes

Pflanzenschutzmittels Sevin (1-Naphthyl-N-methylcarbamate). Z. angew. Zool. SO, 343-380. Brodie B. B. and Maickel R. P. (1962) Comparative biochemistry of drug metabolism. Int. Pharmacol. Meeting1st. 6 (1961), 299-324. Dorough H. W. (1969) Metabolism of Furadan (NIA 10242) in rats and houseflies. J. Agr. Foods Chem. 16, 319-325. Edwards C. A. and Lofty J. R. (1977) The Biology of Earthworms (2nd Edn), 333 pp. Chapman & Hall, London. Edwards C. A. and Thompson A. R. (1973) Pesticides and the soil fauna. Residue Rev. 45, l-80. Gilman A. P. and Vardanis A. (1974) Carbofuran. Comparative toxicity and metabolism in the worms Lumbricus terrestris L. and Eisenia foetida S. J. Agr. Food Chem. 22, 625-628. Harvey J. and Han J. C.-Y. (1978) Metabolism of oxamyl and selected metabolites in the rat. J. Agr. Food Chem. 26, 902-9 10. Jakoby W. B. (1978) The glutathione S-transferases: A group of multi-functional detoxication proteins. Adv. Enzymol. 49, 383-414. Jakoby W. B., Ketley J. N. and Habig W. H. (1976) In Glutathione: Metabolism and Function (Edited by Arias 1. M. and Jakoby W. B.), pp. 213-223. Raven Press, New York. Khan M. A. Q., Lech J. J. and Menn J. J. (1979) Pesticide and xenobiotic metabolism in aquatic organisms. American Chemical Society Symposium series 99. Washington DC. Liimatainen A. and HInninen 0. (1982) Occurrence of cytochrome P-450 in the earthworms Lumbricus ferrestris. In C~tochrome P-450. Biochemisfry, Biophysics and Environmental Implication (Edited by Hietanen E., Lartinen M. and MHnninen), pp. 255-258. Elsevier, Amsterdam. Matsumura F. (1975) Toxicolow of Insecticides, p. 235. Plenum Press,‘New York. I. _ Nakatsugawa T. and Nelson P. A. (1972) Studies of the insecticide detoxication in invertebrates, an enzymological approach to the problem of biological magnification. In Environmental~Toxicology of Pesticides (Edited by Matsumura F., Boush G. M. and Misato T.), pp. 501-524. Academic Press, New York. Nelson P. A., Stewart R. R., Morelli M. A. and Nakatsugawa T. (1976) Aldrin expoxidation in earthworm Lumbricus rerrestris L. Pestic. Biochem. Physiol. 6, 243-253. Oien N. and Stenersen J. (1984) Esterases of earthwormIII. Electrophoresis reveals that Eiseniafetida (Savigny) is two species. Comp. Biochem. Physiol. 78C, 277-282. Simons C. P. and Jagt D. L. V. (1977) Purification of glutathione S-transferases from human liver by glutathione-affinity chromatography. Analyt. Biochem. 82, 334-34 I. Stenersen J. (1979a) Action of pesticides on earthworms. Part I: The toxicity of cholinesterase-inhibiting insecticides to earthworms as evaluated by laboratory tests. Pestic. Sci. 10, 6674. Stenersen J. (1979b) Action of pesticides on earthworms. Part II: Elimination of parathion by the earthworm Eisenia foetida (Savigny). Pestic. Sci. 10, 104-l 12. Stenersen J., Gilman A. and Vardanis A. (1973) Carbofuran: Its toxicity to and metabolism by earthworm (Lumbricus terrestris). J. Agr. Food Chem. 21, 166171. Stenersen J., Guthenberg C. and Mannervik B. (1979) Glutathione S-transferase in earthworms (Lumbricidae). Biochem. J. 181, 47-50. Stenersen J. and 0ien N. (1980) Action of pesticides on

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earthworms. Part IV: Uptake and elimination of oxamyl compared with carbofuran. Pestic. Sci. 11, 396400. Stenersen J. and 0ien N. (198 I) Glutathione S-transferases in earthworms (Lumbricidae). Substrate specificity, tissue and species distribution and molecular weight. Camp. Biochem. Physiol. 69C, 243-252.

Thompson A. R. (1973) Pesticide residues and soil invertebrates. In Enoironmental Pollution by Pesticides (Edited by Edwards C. A.), pp. 87-133. Plenum Press. New York.