Marine Environmental Research 28 (1989) 41M6

Fish and Mammalian Liver Cytosolic Glutathione S-Transferases: Substrate Specificities and

Immunological Comparison

Stephen George, Gordon Buchanan

NERC Unit of Aquatic Biochemistry, University of Stirling, FK94LA, UK

Ian N i m m o

Department of Biochemistry, University of Edinburgh, UK

&

John Hayes

Department of Clinical Chemistry, Royal Infirmary, Edinburgh, UK

A BSTRA CT

The substrate specificities of cytosolic glutathione S-tran,~J'erase (GST) activi O, were compared in several common marine .fish, anadromous and J?eshwater salmonids. Polyclonal antisera raised against purffi'ed rat and plaice GST subunits were used to investigate phylogenetic relationships between the mammalian and fish enzymes. Fla{fish contained two mq/or isoJ~rms, one of which is related to the mammalian group l family. The major GST ~[" salmonids and the cod was related to the mammalian group 3 tran,sferase used as a preneoplastic marker.

The glutathione S-transferases (GSTs) are a multi-gene family of proteins which in mammals serve three complementary roles in detoxication: (1) They conjugate many electrophilic compounds with glutathione (GSH) to produce more soluble, excretable adducts; (2) they covalently bind highly reactive electrophiles (such as azo dye carcinogens) in a 'suicide' reaction; and (3) they bind potentially toxic endobiotics (such as bile acids and pigments) and act as intracellular transporters for their metabolism.1 In rat

41 Marine Environ. Res. 0141-1136/90/$03"50 (c" 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain

42 Stephen George et al.

liver at least 13 isoforms are present which are dimers with subunits of M r 25-28 kDa. The subunits are classified into three multi-gene families on the basis of structure and activities: group one Ya (M r 25-5 kDa), Yc (M r 28.5 kDa), Yk (M r 25 kDa); group two Yb (M r 26.3 kDa), Yn (M r 26 kDa) and group three Y f ( M r 24.8 kDa). 2'3 The sensitivity offish to endogenous toxins (e.g. bilirubin), the mycotoxin Aflatoxin B~ and environmental carcinogens could be due to the absence of certain GST functions. In this study we have used enzymic characteristics and immunological techniques to carry out a comparative study of the spectrum of GSTs present in a number of marine and freshwater fish species.

Cytosols were prepared in 0"25M sucrose, 10raM potassium phosphate, lmM 2-mercaptoethanol, pH 7-5. GST activities were measured spectro- photometrically by the procedures described by Habig & Jakoby 4 and GSH-peroxidase activity was determined with cumene hydroperoxide as substrate. 5 Partial purification of fish GSTs was achieved by affinity chromatography of cytosols on GSH-agarose as described by Simons & Vander Jagt. 6 Purification of GSTs, production of antisera, SDS-PAGE and immunoblott ing have been described previously, v- 1o

The substrate specificities of cytosolic GST activities of cod (Gadus morhua), flounder (Platichthys flesus), plaice (Pleuronectes platessa), sole (Solea solea), turbot (Scopthalmus maximus), brown and sea trout (Salmo trutta), brook trout (Salvelinus Jontinalis), rainbow trout (Oncorhynchus mykiss) and atlantic salmon (Salmo salar) are given in Table 1. In common with mammals, transferase activity with 1-chloro-2,4-dinitrobenzene (CDNB) as acceptor substrate was greatest. Activity in the cod was two to seven times greater than in the marine flatfish. The salmonid activities were very similar apart from the two farmed species, rainbow trout and salmon, which had much higher activities. This is probably attributable to induction by ethoxyquin, an anti-oxidant in the artificial diets which induces rodent GST activity 11 and has been found to produce an even greater (7-fold) induction in salmon (George, unpublished experiments). Activities with bromosulphalein (BSP) and 1,2-dichloro-4-nitrobenzene (DCNB), charac- teristic of the mammalian Yb subunits, were low in all species, rainbow trout exhibiting the highest activities. The cod and trout were the most active with nitrobenzylchloride (NBC), whilst conjugation of ethacrynic acid, a characteristic activity of the Yf subunit, was highest in both cod and rainbow trout. This activity was not tested in the salmon, however, it is possible that the high enzyme activities in the rainbow trout are the result of dietary induction. GSH-peroxidase activity was comparable in all species.

Analysis of immunological relationships and molecular weights of the fish GST subunits displayed clear species relationships (Table 2, Fig. 1). All fish except turbot contained a 25 kDa subunit. In the plaice this subunit

Fish and mammalian glutathione S-transferases 43

( t ransferase BL) d isp lays i m m u n o l o g i c a l c ross - reac t iv i ty with the ra t g r o u p one (Ya, Yc and Yk) family. 12 The low m o l e c u l a r weight t u r b o t subun i t

showed s imilar c ross - reac t iv i ty whilst tha t o f the f lounder was present but very weak . T h e 25 k D a subun i t s o f the cod and s a l m o n i d s were

i m m u n o l o g i c a l l y re la ted to the ra t Y f prote in , a f inding s u p p o r t e d by the higher e thac ryn ic acid c o n j u g a t i o n in the cod and r a i n b o w trout . The

a p p a r e n t lack o f E A con juga t i on in the b r o w n and b r o o k t rou t ana lysed is not readi ly expl icable unless act ivi ty was lost on s torage. The mar ine fish all con t a ined a re la ted 27 k D a subunit , a n t i b o d y to this plaice p ro te in

( t ransferase A H ) does no t react with any ra t p ro te in tested (Ya, Yb, Yc, Yf, Yk, Yn ~2) but reac ted ex t remely weak ly with a t rou t p ro te in o f a b o u t 26 kDa . This ant i-(plaice A H ) se rum s t rongly recognised a 32 k D a pro te in in

the cy tosol o f all the sa lmonids but not in purif ied s a lmon id GSTs , indica t ing tha t this p ro te in does not b ind to the GSH-a f f in i t y matr ix . The

TABLE 1 Glutathione S-Transferase and Peroxidase Activities (nmol/min/mg cytosol protein) in

Hepatic Cytosols of Different Fish

CDNB BSP DCNB EA ENPP NBC COOH ( lmM) (3OHM) ( lmM) (200nM) (2m~) ~ lmM) (5m~t)

Cod 1430+95 (3) <0"1 3"6 9"4 - 16'9 22 Flounder 440 _+ 65 (14) <0'1 7-5 1-1 1.4 22 Plaice 750 _+ 90 (40) 0"l 6-9 0.7 <0.1 0"9 29 Sole 290 _+ 50 (6) <0"1 8-5 3.4 10.5 1.7 20 Turbot 170+65 (6) I'1 6-2 1"0 11"8 0"8 15

Brook trout 290 _+ 130 (3) - - <0"1 <0"1 - - 3'4 Brown trout 340 +40 (4) - - <0-1 <0'1 - - 5"4 Rainbow' trout 2010-+ 500 (10) 1-2 27"1 18-7 14.7 13"9 65 Sea trout 300 (1) . . . . . . Salmon (farmed) 1 220-+ 800 (14) . . . . . Salmon (wild) 160 -+ 40 (7) -

GST activities were determined at 25°C with lmM GSH as substrate as described by Habig & Jakoby; 4 for CDNB, activities are means _+ SEM (n) and for other substrates the cytosols from 3-6 fish were pooled for assay. GSH-peroxidase activities were determined with cumene hydroperoxide as substrate by the method ofWendel. 5 Sole were farmed fish maintained on a natural diet, turbot were fed artificial diets containing no anti-oxidants, rainbow trout and salmon (farmed) were maintained on commercial artificial diets containing anti-oxidants. All other fish were wild.

, not determined. BSP, bromosulphalein~ CDNB, 1-chloro-2,4-dinitrobenzene; COOH, cumene hydroper- oxide; DCNB, 1,2-dichloro-4-nitrobenzene; EA, ethacrynic acid; ENPP, 1,2-epoxy-3- (p-nitrophenoxy)propane: NBC, nitrobenzylchloride.

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(a) (b) RL Yf P Ybl Yb2 Yc Yk Yn P RL C F T RL P S ST RT B

45

(c) RL B RT ST S F C P

(d) P RL C F T RL P S ST RT B

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Fig. I. Immunoblot analysis of structural relationships between fish and rat glutathione S-transferases. Affinity-purified GSTs were electrophoresed on 12"5% acrylamide. (a) Acrylamide gel stained with Coomassie blue; (b) nitrocellulose transfer reacted with anti- (plaice GST-AH) antiserum; (c) nitrocellulose transfer reacted with anti-(rat GST-Yf) antiserum: and (d) nitrocellulose transfer reacted with anti-(plaice GST-BL) antiserum. B, brown trout: C, cod; F, flounder; P, plaice; RL, rat liver; RT, rainbow trout; S, salmon: ST, sea

trout; Yb l, Yb 2, Yc, Yf, Yk, Yn = rat subunits.

nature of this salmonid protein warrants further investigation. There were no definitive cross-reactions between the anti-(rat Yc) serum and the fish proteins whilst only the cod and sea trout displayed any reaction with anti-(rat Yb) serum. Reaction with the anti-(rat Ya) and anti-(rat Yk) sera in the plaice, sea trout and salmon probably indicates the presence of a structurally but not functionally related protein since the catalytic and binding properties of these rat transferase subunits are not present in these fish (Table 1 and Ref. 12).

These results give rise to a number of interesting speculations regarding the consequences of absence of certain GST activities in different fish. Salmonids are extremely sensitive to hepatic neoplasia elicited by Aflatoxin B x epoxide 13 and produce the glucuronide- rather than the GSH- conjugate formed in rats, thus must have GSTs of different specificities. The presence of a Yf-type protein in the salmonids could also be related to the sensitivity to this mycotoxin-induced neoplasia, since expression of the Yf protein in rat liver is normally a characteristic of preneoplastic nodules.14

46 Stephen George et al.

Several flatfish show an extremely high incidence of PAH-induced hepatocarc inomas 15 which may be due to increased sensitivity to cytochrome P-448 inducers or impaired detoxication abilities of the phase 2 enzyme systems. The characteristic covalent binding of highly reactive electrophiles (ligandin activity) is attributed to the Ya subunits in rats and a common feature of the fish tested appears to be the absence or poor expression of a related immunoreactive protein. It is an interesting speculation that the susceptibility offish to hepatic neoplasia may be related to the spectrum of GSTs present in the fish.

R E F E R E N C E S

1. Mannervik, B., Adv. Enzymol. Relat. Areas Mol. Biol., 5"/(1985) 357417. 2. Jakoby, W. B., Ketterer, B. & Mannervik, B., Biochem. Pharmacol., 33 (1984)

2539-40. 3. Hayes, J. D. & Mantle, T. J., Biochem. J., 233 (1986) 779-88. 4. Habig, W. H. & Jakoby, W. B., Meth. Enzymol., 77 (1981) 398405. 5. Wendel, A., Meth. Enzymol., 77 (1981) 325 33. 6. Simons, P. C. & Vander Jagt, D. E., Anal. Biochem., 82 (1977) 334 41. 7. George, S. G. & Young, P., Mar. Environ. Res., 24 (1986) 93-6. 8. Ramage, P. I. N. & Nimmo, I. A., Comp. Biochem. Physiol., 78B (1984) 189 94. 9. Ramage, P. I. N., Rae, G. H. & Nimmo, I. A., Comp. Biochem. Physiol., 83B

(1986) 23-9. 10. Hayes, J. D., Biochem. J., 224 (1984) 839-52. 11. Wattenberg, L. W., Cancer Res., 43 (1983) 2448s 53s. 12. George, S. G. & Buchanan, G., Fish Physiol. Biochem. (submitted). 13. Loveland, P. M., Nixon, J. E. & Bailey, G. S., Comp. Biochem. Physiol., 78C

(1984) 13-19. 14. Kitahara, A., Satoh, K., Nishimura, K., Ishikawa, T., Ruike, K., Sato, K., Tsuda,

H. & Ito, N., Cancer Res., 44 (1984) 2698-703. 15. Varanasi, U., Stein, J. E., Nishimoto, M., Reichert, W. L. & Collier, T., Environ.

Health Perspect., 71 (1987) 155 70.