~tarine Environmental Research 3411992) 233-236

Regulation of Hepatic Glutathione S-Transferase Expression in Flounder

Karen Scott, Michael J. Leaver & Stephen G. George

NERC Unit of Aquatic Biochemistry, School of Natural Sciences. University of Stirling. Stirling, UK. FK9 4LA

A B S T R A C T

The major glutathione S-transferase isoform ~/'flounder liver, an antigeni- calh" related structural homologue of plaice GST-A, also displays mRNA homology. A 901hp cRNA probe .[or plaice GST-A cross-hybridised to a 1 lOOhpflounder mRNA on northern blot attalysis. The plaice antibody and cRNA probes were used to study effects of imhtcer treatment ~t GST-A e.wressum in flounder lit'er. Six &lvs after PA H treatment ( 3-methylcholan- threne) total hepatic GSTactivity was hah'ed, h'vels of GST-A were 80% and GST-A mRNA levels were 25% of controls..4 commercial PCB mixture (Aroclor 1254 TM) had little effect on total GST or GST-A let'els ~lespite hahring GST-A mRNA h'vels. An epoxi~le, trans-stilbene oxide imh¢ced total GST aetivit), 1.4-fold and GST-A protein h'vels 1.8-fohl atul its mRNA levels 3-fi~ld. This reduced expression of the major flounder hepatic GST by agents which huhwe evtochrome P4501AI may too&date cvtoxicitv of environ- mental pollutants b7 this species.

The glutathione S-transferases (GSTs; EC 2.5.1.18) are a multigene family of enzymes catalysing the conjugation of numerous electrophiles with reduced glutathione by formation ofa thioether bond. For the majority of substrates the GSH conjugate is less toxic than the parent compound and thus the cell is afforded protection against chemical insult. Some GSTs have peroxidase activity towards organic peroxides, thus protecting against lipid peroxid- ation, while certain enzymes can reduce peroxidised DNA and may therefore be involved in DNA repair. GSTs have also been implicated in intrinsic and acquired resistance to triazine and thiocarbamate herbicides

233

Marine Environ, Res. 0141-1136/92/$05.00 ~ 1992 Elsevier Science Publishers Ltd, England. Printed in Great Britain

234 Karen Scott, Michael J. Leaver, Stephen G. George

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Regulation of Hepatic" GST expression in flounder 235

imaize), the insecticides DDT, Dichloros TM and lindane (houseflies), as well as in drug therapy in mammals.l Expression of GSTs is transcription- ally regulated. 2 Since the level of expression of particular isoforms may be extremely important in determining resistance to pollutant xenobiotics we have studied the expression of one major isoform, GST-A, in liver of the benthic-feeding estuarine flatfish, the flounder, Platichthvsflesus.

Flounders, obtained in April from a non-polluted area by commercial fishing, were transferred to the laboratory and maintained in a recirculating sea water aquarium at 9;C. They were injected intraperitoneally with olive oil (vehicle cc qtrols) or one of three prototypical inducers--a polyaromatic hydrocarbon, 3-methycholanthrene (3MC, 20mg/kg), a polychlorinated biphenyl mixture. Aroclor 1254 TM (ARO, 100 mg/kg) or an epoxide, trans- stilbene oxide (tSOx, 100mg/kg). After 6 days animals were sacrificed, a portion of the liver was frozen in liquid nitrogen and stored at -80 ' :C for RNA analysis and the remainder used for preparation of cytosols as described previously. 3 Total GST activities were measured by 1-chloro 2,4- dinitrobenzene (CDNB) conjugation, GST-A by immunoblotting with rabbit anti-plaice GST-A IgG as described by George and Buchanan "~ and GST-A mRNA by northern blotting using a 901bp plaice pPLAH GST-A cRNA probe as described elsewhere. 5

The major GST isoform of flounder liver is antigenically related to plaice GST-A 6 and cross-reaction of a 25kDa polypeptide is evident on immunoblot analysis (Fig. 1). Extensive homology was also evident at the mRNA level; when total RNA from flounder was probed with a plaice GST- A cRNA there was hybridisation after high stringency washing (0-1 x SSC, 0'1% SDS, 70-~C) to a 1100bp RNA species (Fig. 1). Results of treatment of flounders with prototypical inducers on hepatic GST are summarised in Table 1 and Fig. 1. Inducer treatment did not alter the liver somatic index LSI) or cytosol protein concentrations. Plaice GST activity is inducible b v

3MC: 3'a however, shortly after treatment activity is reduced, a In flounders 6 days alter treatment, 3MC also reduced total GST activity by about 50%, while the level of GST-A mRNA estimated by hybridisation analysis was only about 25% of those in the control animals, although levels of GST-A protein estimated by western blotting were only very slightly reduced (not statistically significant). After treatment with the PCB mixture, total GST activity appeared to show a slight elevation of about 1.3-fold, GST-A protein levels were unchanged and GST mRNA was two-thirds that of the controls. The magnitude of the response to trans-stilbene oxide appeared to be quite variable between animals; there was strong 3-fold induction of GST-A mRNA levels and somewhat less induction of both GST-A protein [l-8-fold) and total CDNB conjugation activity (l.4-fold).

While the total GST activity of flounders and other flatfish is inducible by

236 Karen Scott, Michael J. Leaver, Stephen G. George

TABLE 1 Effect of Inducer Treatments on Glutathione S-Transferase Expression in Flounder Liver"

Control 3MC A RO tSOx

Body weight (g) 49.1 -4-9.6 Liver weight (g) 0.51 +-0.16 LSI h 1"2 -+ 0-3 Cytosol protein (rag/g) 100-1- 15 GST (tlmol CDNB conj. min/mg) 0.76 -+ 0.1 GST-A (units/rag) 58 -+ 4 GST-A mRNA (units 'mg) 8 _+ 1

54.4+21-9 47-3__+ 15"3 42.6_+4-7 0-59+0"16 0-5-+0'23 0"55-+0"1

1.4-+0"4 1'3+0-4 1'3-+0"1 92-+7 92-+7 96-+9

0-37+0-17" 0-81 +0-23 1"13-+0'24" 44+I7 52+13 83+__9*

3-+I" 6+1 33+__6*

" Five fish/treatment group were injected i.p, with olive oil (control), 3-methycholanthrene (3MC, 20mg/kg), Aroclor 1254 TM {ARO, 100mgkg) or trans-stilbene oxide (tSOx, 100mg/kg), held at 9 C and sacrificed alter 6 days. GST-A and GST-A mRNA were estimated by densitometric scanning of immunoblots and northern blots, respectixely (arbitrary units). Values, mean + SD. Significance by Student's-test, ", p < 0-05. h LSI, liver somatic index, liver wt as percent of body ~t.

po lyaromat ic hydrocarbons and polychlor inated biphenyls (George & Buchanan "~ and unpublished resu[ts), the present results demonst ra te that these compounds repress expression of the major GST isoform of f lounder liver. The exact function of this enzyme, like the major i ty o f GSTs, is unclear and we are current ly a t tempt ing to define its role by molecular biological techniques. Reduct ion of expression of this or thologue may well have p rofound effects on the toxicity of polyaromat ic hydrocarbons to the t lounder if it is involved in detoxicat ion of its metabolites. Nevertheless, these results do show that synthesis o f GST-A is induced by an epoxide which may confer increased protect ion against cytotoxic damage by this type of compound .

R E F E R E N C E S

I. Hayes, J. D., Pickett, C. B. & Mantle, T. J, (eds) Glutathione S-Transferases and Drug Resistance. Taylor & Francis, London, 1990, 459pp.

2. Pickett, C. B. Essays 01 Biochemistry, Vo[. 23. Biochemica[ Society, London, 1987, pp. 116-143.

3. George, S. G. & Young, P. Cornp. Biochem. Physiol., 83C, 33-44 (1986). 4. George, S. G. & Buchanan, G, Fish Physiol. Biochem., 8, 437-49 (1990). 5. Leaver, M. J., Scott, K. & George, S. G., 34 237-41 (1992). 6. George, S., Buchanan, G., Nimmol, I. & Hayes, J. Mar. Environ. Res., 28, 41-6

(1989).