Marine Environmental Research 35 (1993) 25-28

Control of Metailothionein Expression by Hormones and Stressors in Cultured Fish Cell Lines

David Burgess, Nick Frerichs & Stephen George

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

A B S T R A CT

In-vitro studies with established cell lines from a marine flatfish (turbot) and an anadromous fish (rainbow trout) were used to study hormonal effects on metallothionein synthesis. Only the turbot cell line responded to gluco- corticoid and progesterone treatments, with a 1.5-2-fold induction of M T levels. Neither cell line displayed a positive response to oestradiol, phorbol ester or bacterial endotoxin. A practical implication of these findings in use offish- metallothionein levels for heavy-metal pollution monitoring is that sexually mature animals must not be used and levels less than 3-fold higher than reference sites, which may be due to hormonal effects, must be disregarded.

The regulation of metallothionein (MT) biosynthesis in fish by heavy metals such as Cd, Cu, and Zn is well characterised both in vitro and in vivo, and the presence of elevated MT levels in fish is now being utilised as a sublethal indication of heavy-metal pollution. 1'2 Whereas there do not appear to be seasonal effects on piscine MT levels, 3 there is an elevation in mature female fish 3'4 that has been attributed to Zn mobilisation rather than a direct induction by oestradiol. 3'5 By utilisation of cell-culture and molecular- biological techniques, it has been shown in mammalian systems that, in addition to heavy-metal induction, MT biosynthesis is also transcriptionally regulated by a large number of environmental and hormonal cues, including glucocorticoids, progesterones, and protein kinase C activators, which result in a modest increase in cellular MT levels. 6 It is therefore necessary to determine such effects on piscine MT biosynthesis for effective use of MT levels for monitoring of the environmental impact of heavy metals. To date, direct studies in piscine species using hormone administration in vivo have

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

26 David Burgess, Nick Frerichs, Stephen George

failed to produce clear results,* and we have therefore investigated effects of hormones and other stressors on MT synthesis in two immortalised cell lines from turbot (TF) and rainbow trout (RTG-2). Cultures (25cm 2) at 95% confluency of RTG-2 cells (passage 95) in 10 ml Eagles MEM/10% FCS or TF cells (passage 34) in 10ml Liebowitz L-15 medium/0.35% NaCl/10% FCS, grown at 22°C in an atmosphere of 5% CO 2, were exposed to hormones or water, ethanol, or DMSO vehicles as controls, for 4 days. Harvesting, homogenisation, and protein determinations were as described previously. 7 MT in TF cells was assayed by a l°9Cd-saturation method utilised in previous studies, 1-3'7 and an ELISA procedure 8 was used for measurement of MT in RTG cells and for confirmation of selected saturation assays in the TF cells.

CONTROL Hydrocortisone, 10 -5 M

H, 10 -4 M Dexamethasone, 10 -6 M

D, 10 -5 M Progesterone, 10 -5 M

P, 10 -4 M Testosterone, 10 -5 M

T, 10 -4 M Oestradiol, 10 -5 M

O, 10 -4 M

0 50 100 150 200 . . . . I • • • = I • = , . i • • • i

////////////////////////////~ ~ / ~ , , RTG Cells ~///////////////////////A=4 j

y/////////////////////////~J//////////~-4

r/////////////////~ 1

~///////////////////~. !

////////////////////////A==I

CONTROL Corticosterone, 10 -5

C, 10 -4 HydrocorUsone, 10 -5

H, 10 -4 Dexamethasone, 10 -6

D, 10 -5 Progesterone, 10 -5

P, 10 -4 Testosterone, 10 -5

Oestradiol, 10 -5 O, 10 -4

M M M M M M M M M M M

0 50 100 150 Metallothionein concentration relative

to control = 100

200

Fig. 1. Hormonal induction of metallothionein in cultured rainbow-trout (RTG-2) and turbot (TF) cell lines.

Growth, exposure, and assays were carried out as in the text. Values mean + SD for 6-9 replicates per treatment group. Statistical significance established by two-tailed Student's t

test at *p<0.01 and **p <0'05.

Control of metallothionein expression in fish cell lines 27

The results, plotted in Fig. 1, clearly demonstrate that MT synthesis in the turbot cell line can be induced up to 2-fold by both natural and synthetic glucocorticoids as well as progesterone, whereas the sex-steroid hormones, oestradiol and testosterone, had no significant effect on MT levels. The degree and specificity of the inductive response in this cell line are closely similar to those obtained with mammalian cell lines. 6 In some of the latter cell lines, the paucity of hormone receptors necessitated the use of higher hormone concentrations than those observed in vivo, and, in this fish cell line, the hormone levels required for induction are also from one to two orders of magnitude higher than those that have been observed in fish plasma. Nevertheless, our results demonstrate that fish cells are capable of responding to these hormones.

In contrast, no significant hormonal induction of MT levels was observed in the established rainbow-trout cell line, which would imply that these cells do not contain the necessary receptors, since a positive inducing effect of glucortocoids has been reported in isolated trout hepatocytes in primary culture. Whereas a lack of oestradiol receptors may explain the lack of induction by oestradiol in these cell lines, our results agree with findings in mammalian systems in which oestradiol does not induce MT synthesis and support the contention that the observed elevation in hepatic MT levels in mature female fish is due to tissue Zn mobilisation 3'8 and not direct hormonal regulation of MT expression.

Treatment of the fish cell lines with up to 5 pg/ml phorbol dibutyrate, a tumour promoter that induces MT m R N A expression in a number of mammalian cell lines via protein kinase C induction, did not increase MT levels, which indicated that this pathway was non-functional in these particular cell lines. Bacterial endotoxin is also an effective inducer of MT in mice, which is believed to act via interleukin-I stimulation of protein kinase C 1°, but endotoxin from S. typhosa (up to 10 ng/ml) had no effect om MT levels in the fish cell lines.

The present results are the first to demonstrate that MT levels in a fish cell can be induced by glucocorticoids and progesterone and also show that oestradiol is not an effective inducer in vitro. By these criteria, measurements of piscine MT levels as an indicator of heavy-metal pollution should be valid only if sexually immature fish are utilised and levels greater than three-times reference values are regarded as diagnostic of metal induction.

REFERENCES

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(1991). 3. Olsson, P.-E., Haux, C. & Forlin, L. Fish Physiol. Biochem., 3, 39-47 (1987).

28 David Burgess, Nick Freriehs, Stephen George

4. Overnell, J., Mclntosh, R. & Fletcher, T. C. J. Fish. Biol., 30, 539-46 (1987). 5. Olsson, P.-E., Zafarullah, M. & Gedamu, L. Biochem. J., 257 555-9 (1989). 6. Karin, M., Imagawa, M., Imbra, R. J., Chiu, R., Heguy, A., Haslinger, A., Cooke,

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