FUNDAMENTAL AND APPLIED TOXICOLOGY 33 , 9 1 - 9 9 (1996)

ARTICLE NO. 0146

Inducibility of Metallothionein mRNA Expression and CadmiumTolerance in Larvae of a Marine Teleost,

the Turbot (Scophthalmus maximus)

STEPHEN G. GEORGE,* PAUL A. HODGSON,* PETER TYTLER,| AND KEITH TODD*

*NERC Unit of Aquatic Biochemistry, University of Stirling, and t Department of Biologicaland Molecular Sciences, University of Stirling, Stirling FK94LA, Scotland

Received December 19, 1995; accepted April 26, 1996

Inducibility of Metallothionein mRNA Expression and Cad-mium Tolerance in Larvae of a Marine Teleost, the Turbot {Scoph-thalmus maximus). GEORGE, S. G., HODGSON, P. A., TYTLER, P.,AND TODD, K. (1996) Fundam. Appl. Toxicol. 33, 91-99.

An acute (48-hr) lethal toxicity study of turbot at various stagesfrom hatch to juveniles showed that prior to first exogenous feed-ing, larvae of turbot were an order of magnitude more sensitiveto cadmium toxicity than later developmental stages (L.C50 of0.18-0.23 ppm Cd versus 2-5 ppm Cd). To investigate the possiblerole of metallothionein (MT) in Cd tolerance, the concentrationsof MT mRNA were determined in control and Cd-exposed animalsusing a plaice MT cDNA probe. MT mRNA was expressedthroughout embryonic and early larval development at levels ap-proximately twice those in juvenile turbot liver apart from theperiod around hatching, when there was a decreased level of ex-pression. During endogenous feeding, despite larval drinking, MTmRNA levels did not appear to be inducible by exposure to Cd inthe water within 3 days of hatching but were induced three- tofivefold after 48 hr exposure to waterborne Cd at all later stagesof development. The results suggest that Cd tolerance is associatedwith metal induction of MT gene transcription and that the en-hanced sensitivity of very early stage larvae is attributable to adeficiency in MT gene transcription, c 1996 soday of Toxicology

The heavy metal cadmium occurs naturally in associationwith zinc ores and its recent extensive industrial uses inelectroplating and as both a stabilizer and pigment in plasticshave resulted in a marked elevation in cadmium concentra-tions in inshore waters, which are important breeding andnursery areas for many fish species. Fish larvae are moresensitive to pollutant toxicity than juveniles (McKim, 1977)and short-term acute Cd exposure studies carried out at dif-ferent stages of marine fish development have reported LC50values of 12 mg Cd liter"1 for 7-day-old larvae comparedwith 43 mg Cd liter"' for adults of Fundulus heteroclitus(Middaugh and Dean, 1977) and 8 mg Cd liter"' for larvalcompared with 34 mg Cd liter"' for juvenile Mugil cephalus(Hilmy et al., 1985). The reasons for this increased suscepti-bility to heavy metal pollutants are unknown and could be

due to many factors including differential rates of uptake,distribution, or detoxication.

Metallothioneins (MTs) are low-molecular weight cys-teine-rich proteins capable of binding 6-7 g atoms of cad-mium or zinc per mole (reviewed by Hamer, 1986; Kagiand Schaeffer, 1988) and have been identified in most Eucar-yotes, including fish (reviewed by Kille et al., 1992; Georgeand Olsson, 1994). Functionally they have been implicatedin copper and zinc metabolism (Bremner, 1987; Cousins,1985), detoxication of the pollutant metals cadmium andmercury (Kagi and Kojima, 1987), and as free radical scav-engers (Thomas et al., 1986; Lazo et al., 1995). In juvenileand adult fish, synthesis is strongly induced by transcrip-tional activation of MT gene expression by exposure togroup 2B metals such as cadmium and zinc (George et al.,1992, 1996; George and Olsson, 1994; Zafarullah et al.,1989), and the metals are sequestered by the newly synthe-sized protein.

In contrast to the situation in the adult, fetal and neonatallivers of mammals contain relatively high concentrations ofMT, Zn, and Cu (Bremner et al., 1987; Riordan and Rich-ards, 1980; Clough et al, 1986). The finding that ZnMTwas localized primarily in the nucleus led to the suggestionthat the fundamental role of MT is as a Zn donor duringgrowth and development (Panemangalore et al., 1983). Re-cent reports that mice with disrupted MT genes developnormally (Michalska and Choo, 1993; Masters et al., 1994)appear to conflict with this hypothesis; however, the en-hanced sensitivity of these transgenic mouse embryos to Cd(Masters et al., 1994) or cells cultured from them to chemi-cally mediated oxidative stress (Lazo et al., 1995) demon-strates the protective role of MT against such stressors. Ina study of preimplantation development of mice, Peters etal., (1995) suggested that during very early embryonic devel-opment the regulation of MT expression by metals may befunctionally different from that in adults. At later develop-mental stages, weak regulation of mammalian fetal MT lev-els by glucocorticoids (Quaife et al., 1986) as well as induc-tion by metals has been reported, although the placental

91 0272-059(V96 $18.00Copyright O 1996 by the Society of ToxicologyAll rights of reproduction in any form reserved

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92 GEORGE ET AL.

barrier may modulate response considerably (Andrews etal.,1984, 1987; Fleet and McCormick, 1988).

Recently there has been increased interest in the use offish as models for vertebrate development. In comparisonwith higher vertebrates, development of many organ systemsis only rudimentary in the first free-living larval stages offish. For example, at hatch, turbot {Scophthalmus maximus)have an open hemocoel, no kidney glomeruli, no gills, andincompletely differentiated gut and liver (Segner et al., 1994,Tytler et al., 1993, 1996). Thus they may provide usefulinsights into gene regulation and the functional developmentof organ systems.

In view of the apparently greater sensitivity of larval fishto Cd toxicity and the known role of MT in detoxificationof Cd in fully developed animals it is of interest to determinethe ontogeny of metal regulation at various developmentalstages of fish. We have therefore studied the sensitivity ofturbot (S. maximus) to waterborne Cd exposure at differentstages of development and established the profile of em-bryo-larval MT mRNA expression and the regulation oflarval MT gene expression by Cd exposure. Our results indi-cate that the hypersensitivity of early-stage larval fish to Cdtoxicity is correlated with a lack of inducibility of MT geneexpression by Cd during the early stages of development ofthe free-living individual.

MATERIALS AND METHODS

Animals and rearing. Turbot eggs, larvae, and juveniles were obtainedfrom the marine hatchery facility of Golden Sea Produce Ltd., HunterstonAyrshire. Fish were reared at 15 ± 1°C in seawater of 34%c salinity ateither Hunterston or Stirling. At Day 2 after hatching, algae {hochrysisgalbana tahitian) and rotifers (Brachionus plicatilis) were added to theculture tanks to maintain a density of 10 rotifers/ml. Exogenous feedingcommenced around Day 4 after hatching, just prior to complete resorptionof the yolk sac. The larvae were weaned from this diet onto one of algal-enriched brine shrimp (Anemia salina) by introduction of the Anemia tothe mixed culture between Days 10 and 13. Around metamorphosis (22days) they were gradually weaned onto a commercial pelleted trout feed

Cd exposures. All experiments were carried out using the same set oftanks at a temperature of 15 ± I°C in seawater of 34%o salinity To avoidstress, water for cadmium exposures was taken from the rearing tanks andprefiltered through activated charcoal and then through a 0.22-^m glass-fiber filter. CdCl2 was added to the required concentration from freshlyprepared stocks in distilled water. The experimental tanks were preequili-brated with cadmium for 48 hr to saturate cadmium-binding sites on thetank walls; the water was then changed before addition of animals. Fishwere distributed evenly between triplicate sets of tanks containing controlor five different concentrations of added cadmium Actual cadmium concen-trations in the water determined at the end of the experiments by atomicabsorption spectroscopy using standard methods did not differ by more than5% from the initial water concentrations. For larval fish 500-ml dishes,each containing 350 ml water, were used, and experiments at 10, 18, and27 days posthatch were carried out with both fed and starved animals. Forjuveniles 7-liter tanks containing 5 liters water were used.

Microscopic examination of heartbeat for early-stage larvae or tactilestimulus of older larvae and juveniles was used to confirm mortality. Alldead animals were removed at 6 hr, 12 hr, and then every succeeding 12hr until 48 or 96 hr.

RNA extraction, electrophoresis, and Hybridization. For measurementof MT mRNA concentrations, total RNA was extracted from animals thathad been rinsed in sterile saJine on a 200-fim nylon-mesh filter by anacid guanidinium thiocyanate-phenol-chloroform procedure as describedpreviously (George et ai, 1992). RNA was extracted from dechorionatedembryos, from whole larvae up to Day 3 posthatch after removal of theyolk sac, from the anterior halves of later-stage larvae, or from liver ofjuvenile fish so that MT mRNA concentrations were determined in RNAderived predominately from visceral tissues. Integrity of the RNA andspecificity of the plaice MT cDNA probe were assessed by Northern blotanalysis as described previously for turbot (George et al. 1992, 1996).Briefly, IO-/Jg samples of glyoxalated total RNA were separated by electro-phoresis on 1.6% agarose gels in the presence of ethidium bromide, trans-ferred by capillary blotting to nylon membranes (Amersham, Hybond N),and probed with a 32P-labeled MT cDNA probe obtained from the entirecoding region of plaice MT cDNA (pPPMTl, EMBL Accession No.X56743). Washing was performed down to a stringency of 0.1 M sodiumcitrate-1 M sodium chloride (0. l x SSC), 0.1% sodium dodecyl sulfate for15 min at 65°C. Slot-blot analysis was used to quantify MT mRNA levels;2- and 4-/xg samples of total RNA (standardized from £260 values anddensitometry of UV transparency of ethidium bromide-stained 23 S RNAbands on agarose gel electrophoresis) denatured in 15% (w/v) formaldehydewere blotted to nylon using a vacuum blotting manifold (BRL, Bethesda,MD) and baked at 80°C before probing as above. The membranes wereautoradiographed using Kodak X-O-MAT film with intensifying screens(Dupont, Cronex) at -80°C.

Calculation and analysis of results. Data from replicate exposure tanksand experiments were pooled and LC50 values were determined by probitanalysis (Finney, 1971). Statistical analysis was performed using Statviewsoftware (Abacus). Relative MT mRNA levels were calculated from thehybridization intensity of the bands on the autoradiographs by densitometricscanning (Shimadzu model CS9000 densitometer) and are expressed asdensitometer units per microgram of total RNA.

RESULTS

Morphological Development

Compared with larvae of many other temperate-water fishspecies, for example, salmonids and clupeids, the embryonicstage of turbot is short (ca. 75 degree days) and the larvaeare only ca. 2-2.5 mm long at hatch. At hatch many organsystems are undeveloped or poorly developed, there is anopen hemocoel, the liver and kidneys are incompletelyformed, and osmoregulation is performed by water absorp-tion from an undifferentiated gut and ion excretion by cuta-neous chloride cells. Subsequent organogenesis is rapid; atfirst feeding some 3 to 4 days after hatch (larvae are ca. 3 -3.5 mm long), the circulatory system, liver, and kidneys areformed and functional, the stomach is formed, the gut haslengthened, and there is a rectal sphincter. Digestion is estab-lished about 24 hr after first feeding commences when theyolk has become fully resorbed and the skin has becomethickened and water impermeable. The gills do not becomefully formed until 12-14 days after hatch when the larvaeare about 5 mm long (this study and Al-Maghazachi andGibson, 1984; Brown and Tytler, 1993; Segner et al., 1994;Tytler and Ireland, 1994; Tytler et al., 1993, 1996).

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FISH METALLOTHIONEIN GENE EXPRESSION AND Cd TOXICITY 93

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FIG. 1. Effects of tank containment and starvation on mortality of artificially reared turbot larvae. Larvae at different stages of development werehoused in 500-ml tanks containing 350 ml filtered seawater, dead animals were removed and mortalities recorded at intervals as described under Materialsand Methods. Open bars indicate dietary regimes; solid bars and lines indicate the period of experimental Cd exposure; dashed lines show extendedperiod of study of control animals. Values are means for at least two replicate experiments, each carried out with three tanks of fish

Mortality Due to Tank Confinement of Larvae

Turbot larvae are pelagic and large tanks of ca. 200 literswere used for rearing; therefore, mortality due to confine-ment in the small vessels used for experimental exposureswas assessed. Use of static conditions with no artificial aera-tion was found to reduce the severe mortality caused byphysical abrasion with vessel walls. Under these conditionsmortality was less than 20% after 48 hr at all stages ofdevelopment other than larvae immediately before metamor-phosis (Fig. 1). Fish at this developmental stage are ex-tremely fragile and do not withstand handling. They are alsoprone to bacterial infections. Consequently there is normallya very high mortality under conditions of artificial rearing(A. Barber, GSP Ltd., personal communication). The normalfeeding regimes for larval rearing are also indicated in Fig.1, and the effect of food deprivation on mortality was alsodetermined to ensure that effects of uptake of waterborneCd rather than uptake of Cd adsorbed to food material wasbeing assessed and that mortality in the tanks containing Cdwas due to the metal rather than an inhibition of feedingleading to starvation. In the first 36-48 hr of food depriva-tion there were no significant differences in mortality be-tween fed and starved groups of larvae between first feedingand metamorphosis (p > 0.05, ANOVA). When larvae werestarved for longer periods (48-96 hr) an increase in mortal-ity was observed only in the experiment performed from 10-to 14 days posthatch at the stage of weaning from an algal/rotifer diet to one of Anemia. Thus to obviate effects of tank

confinement and starvation, Cd exposures were limited to48 hr for mortality assessment and MT mRNA analysis.

Cadmium Toxicity, LC50s

Replicate tanks containing turbot larvae of different ageswere exposed to a range of concentrations of Cd and themortality monitored for up to 96 hr. Mortality for the differ-ent Cd concentrations in the tank water after 48 hr is shownin Fig. 2, and the 48-hr LC50 values calculated by probitanalysis are summarized in Table 1. The newly hatchedlarvae (yolk sac stage) were very sensitive to Cd toxicity,with a 48-hr LC50 value of 0.18-0.23 mg Cd liter"'. Sensi-

TABLE 1Toxicity of Waterborne Cd at Different Stages of Development

of Turbot (Scophthalmus maximus)

Stage

IA. HatchIIA. First feedingIIB, CIV. PremetamorphosisPos metamorphosis

Age atcommencement

of exposure(days posthatch)

04

101826

LC50

24 h

0

13149

(mg Cd liter"1)

48 h

0.18-0.2325

Estimated as 7-105

Note. LC50 values calculated by probit analysis (Finney, 1971).

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94 GEORGE ET AL.

tivity to Cd decreased with age; in stage IIA larvae (Day 4posthatch, first feeding) it was an order of magnitude less,with a 48-hr LC50 of 2 mg Cd liter1), and for 10-dayposthatch larvae a 48-hr LC50 of 5 mg Cd liter"1 was found.Feeding status had no significant effect on short-term Cdtoxicity at any developmental stage. With Day 18 larvae,immediately prior to metamorphosis, mortality was ex-tremely high in both control and Cd-exposed tanks (see Fig.2). Therefore, LC50 values could not be calculated directly;however, if the data are corrected for the excessive mortalityin the control tanks, values between 7 and 10 mg Cd liter"1

are predicted. Just after metamorphosis (26 days posthatch)the 48-hr LC50 was about 5 mg Cd liter"1. Juveniles (40days posthatch) appeared to be much more robust and onlylow mortality was observed after 48 hr Cd exposure; thus,exposure was monitored for 96 hr to obtain an LC50 value(10 mg Cd liter"1).

Expression of Metallothionein mRNA during Embryo-Larval Development

Because of the extremely small amounts of tissue avail-able and the insensitivity of available assay methods, MTprotein levels could not be determined in early develop-mental stages. Nucleotide probes for MT gene expressionwere sufficiently sensitive and therefore this study focuseson the expression of the turbot MT gene rather than itsprotein product during development and changes in responseto Cd challenge. MT is localized primarily in the liver ofjuvenile turbot, and the two other major MT-containing or-gans, gills and kidneys, contribute the remaining 10% of thetotal body MT (Norey et ai, 1990; George et ai, 1996).After first feeding, the musculature contributes the majorbody mass. Therefore, to avoid dilution by muscle RNAsthe tail of larval animals was removed by dissection beforeRNA extraction so that analyses were predominately of RNAfrom visceral tissues, especially the liver, gut, and kidneys.After metamorphosis only hepatic tissue was used for analy-sis. Ethidium bromide staining of electrophoretically sepa-rated RNA and Northern blot analysis using a plaice MTcDNA probe showed that there was minimal RNA degrada-tion and that the plaice probe hybridized to a single transcriptof about 500 bp. The concentrations of MT mRNA weredetermined by slot-blot analysis of total RNA samples boundto nylon using the same hybridization stringency conditions.Standardization of RNA loading was based on UV ab-sorbance and intensity of ethidium bromide staining of 23S RNA rather than using a probe for a housekeeping genesuch as actin or tubulin, as we have found expression of thelatter to be variable during early embryogenesis of fish andtherefore unreliable for standardization (unpublished results,in preparation for publication). Early-stage embryos, 24 and72 hr after fertilization, contained high levels of MT mRNAand the level dropped to approximately 50% of this level at

hatch between 4.5 and 5 days postfertilization (Fig. 3). MTmRNA levels in RNA from larvae at hatch were the sameas those observed in the livers of fish at metamorphosis andin juveniles. During the period from first exogenous feeding(Day 3 or 4) until metamorphosis (day 22), while there wassome variation in MT mRNA levels between differentbatches of larvae, when levels within batches were comparedwith those at hatch and in juvenile livers, they showed aconsistent slight elevation of some 1.5-fold (Fig. 3). Thusthe data indicate an overall trend of a slowly declining levelof MT mRNA expression from those in embryos 24 hr post-fertilisation to those in livers at metamorphosis, with a tran-sient decrease in expression immediately prior to and duringhatching.

Induction of Metallothionein mRNA Expression by CdExposure at Different Stages of Development

RNA was extracted from the viscera of control and Cd-exposed larvae withdrawn from tanks exhibiting a mortality<10% after 48 hr; these were fish exposed to 0.1 mg Cdliter"' from hatch to 2 days, from Days 1 - 3 , and from Days2-4; or at a concentration of 0.5 mg Cd liter"' at all otherlarval stages, including fish after metamorphosis on Days 27and 40. MT mRNA levels were again determined by slot-blot analysis of this total RNA and compared with those innon-Cd-exposed control animals.

No induction of MT mRNA was observed in yolk sacstage larvae exposed to 0.1 mg Cd liter"' for 48 hr betweenhatch and Day 2 or from Days 1 to 3 (Fig. 4). When exposureof animals to the same concentration of Cd in the watercommenced a day later (i.e., between Days 2 and 4 posthatchduring the transition from endogenous to exogenous feed-ing), this resulted in a threefold elevation of MT mRNAlevels. There was also a three- to fivefold induction of MTmRNA levels in visceral tissue of fish exposed to 0.5 mgCd liter"' for 48 hr at all later stages of development, inlarvae before metamorphosis (exposed from Days 6-8 , 8 -10, 13-15, or 18-20), in newly metamorphosed fish at 27 -29 days posthatch, and in livers of juvenile fish 40-42 daysposthatch. Comparison of steady-state MT mRNA levels in10-day-old turbot larvae that had been starved or maintainedin green culture with algae/rotifers from 4 days posthatchshowed that starvation for 48 hr did not induce MT mRNA(results not shown). Thus our findings are attributable to Cdexposure and not food deprivation.

DISCUSSION

This study of turbot (5. maximus) is the first detailedinvestigation of the developmental toxicity of a fish to aheavy metal. There was a dramatic change in sensitivity toCd toxicity during the earliest stages of free-living larvaldevelopment between hatching and the onset of exogenous

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96 GEORGE ET AL.

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FIG. 3. Expression of MT mRNA during development of turbot. The 2- and 4-^ig samples of total RNA extracted from whole embryos, anteriorportions of larvae (i.e., minus tail musculature), or livers (Days 20 and 29) were analyzed by slot-blot hybridization using a 32P-labeled plaice MT cDNAprobe under conditions where nonspecific binding was minimal as described under Materials and Methods. RNA concentrations were normalized fromabsorption at 260 nm and intensity of ethidium bromide staining of the 23 S RNA on agarose gels; MT mRNA concentrations are expressed asdensitometer units//jg total RNA applied. Open circles represent average values, and each data point represents the mean of a triplicate estimation carriedout on different batches of 150-300 larvae.

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FIG. 4. Effect of 48-hr Cd exposures on MT levels in turbot at different stages of development. MT mRNA levels (means ± SD) in total RNA fromtissues of Cd-treated larvae (as in Fig. 3) were determined as described under Materials and Methods and are expressed relative to those in non-Cd-exposed control animals sampled on the day of euthanasia. The age of animals is expressed as time posthatch for the 48-hr exposures, and the concentrationsof Cd in the tank water were as noted. 'Significantly different from controls, p < 0.05. ANOVA, Student's I test

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FISH METALLOTHIONEIN GENE EXPRESSION AND Cd TOXICITY 97

feeding. During later stages of development there was agradual reduction in sensitivity to Cd toxicity as reportedpreviously for other fish species, Fundulus (Middaugh andDean, 1977) and Mugil (Hilmy et al, 1985).

Because of the vast excess of chloride ions in seawater,Cd is normally present as chlorocomplexes (Turner et al.,1981) and previous studies with a variety of marine organ-isms have shown that epithelia are freely permeable to Cd2+

ions and uptake displays characteristics of a facilitated diffu-sion process (George and Carpene, 1981; Jenkins and Sand-ers, 1986). While many fish larvae do not possess gills,they have a large surface-to-volume ratio and they have apermeable skin which enables respiratory and ionic transport(Tytler and Bell, 1989). Additionally, even directly on hatch-ing, marine fish larvae drink to maintain their osmotic bal-ance (Brown and Tytler, 1993; Tytler and Blaxter, 1988;Tytler and Ireland, 1994). Therefore at hatch there is littlebarrier to uptake of soluble metals. During early develop-ment the skin differentiates and becomes multilayered and itspermeability to both gases and solutes is reduced; abundantmucus, which sequesters divalent metal ions (Coombs et al.,1972), is also produced. Therefore, uptake of dissolved Cdby the cutaneous route will decrease as permeability barriersdevelop, and since this is a gradual process it may be pre-dicted that during larval development there might be a grad-ual decrease in the apparent toxicity of metals such as Cd inshort-term exposures through this decrease in permeability.While confirmation is required by direct experimental stud-ies, we propose that this could explain the gradual decreasein acute Cd toxicity observed from about 7 days after hatch-ing through metamorphosis both in the present study and inthose reported previously (Middaugh and Dean, 1977; Hilmyet al., 1985).

Piscator (1964) first proposed that MT served a protectiverole against Cd toxicity. Later studies have supported thisproposal and it has been shown that (1) MT forms high-affinity thiolate clusters, with Cd thus preventing its reactionwith other macromolecules (Kagi and Schaeffer, 1988); (2)MT gene transcription is induced by Cd (Karin, 1985); (3)cultured cells selected for resistance to Cd have elevatedlevels of MT (Hamer, 1986); (4) overexpression of MT aftergene transfection of a variety of cells reduces their sensitivity(Basu and Lazo, 1990) and most compellingly; (5) targeteddisruption of mouse MTI and MTU genes (gene knockout)enhances their sensitivity to Cd (Michalska and Choo, 1993;Masters et al., 1994).

In a study of trout, Olsson et al. (1990) showed that MTwas expressed throughout development and that at hatchthere was a slight elevation in expression of the mRNA forone of its MT isoforms, MT-A, which they suggested mightbe due to a corticosteroid effect. In the present study wehave also shown that the metallothionein gene of turbot isexpressed throughout development; however, the pattern of

mRNA expression was markedly different from that reportedin trout since we found a general trend of a decreasing levelof MT mRNA expression in turbot embryos from 24 hrafter egg fertilization until premetamorphosis, with a notabledepression immediately prior to and at hatching. The reasonsfor this apparent dissimilarity are unknown; however, it maybe a feature of the very much more advanced stage of organ-ogenesis in the trout at hatch. The gene knockout experi-ments with mice have shown that MT is not essential fornormal development (Michalska and Choo, 1993; Masterset al., 1994), although recent evidence suggests that it mayaffect preimplantation embryo development (Peters et al.,1995) and may protect against oxidative stress in embryoniccells (Lazo et al., 1995). While there is no current evidencefor a role of MT as a protectant from oxidative stressorsduring embryo-larval development of turbot, it is worthnoting that in contrast to the environment in the mammalianwomb, turbot are pelagic in the early developmental stagesand could therefore be exposed to high levels of ozone andUV radiation in upper layers of the sea. Thus if MT proteinlevels were also elevated at these stages then it may confera protective effect against oxidative damage.

More importantly, the present study shows that there is anapparent correlation between the inducibility of MT mRNAexpression by exposure of free-living larvae to Cd and itstoxicity. Newly hatched larvae feeding on endogenous yolkreserves were very sensitive to Cd exposure and MT mRNAlevels were not induced by exposure to 0.1 ppm Cd for 48hr. At all later stages of development the sensitivity to Cdwas an order of magnitude less and 48 hr exposure to 0.1or 0.5 ppm Cd resulted in a three- to fivefold induction ofMT mRNA levels. This dramatic change in sensitivity to Cdin the first 48 hr posthatch cannot be explained by any majorphysiological changes in reliance on endogenous yolk re-serve, drinking, or alterations in permeability that would beexpected to alter Cd availability to the larva. Therefore wepropose that our results indicate that Cd tolerance is corre-lated with the inducibility of MT gene transcription.

The mechanism for the lack of apparent inducibility ofMT expression before 4 days posthatch is unknown, neitheris anything presently known of the distribution of MT inembryonic fish tissues. This question can only be resolvedby histochemical studies. In juvenile trout and turbot, MTis expressed in a tissue-specific manner: more than 90% ispresent in the liver and the remainder is almost exclusivelyfound in the gills and kidneys (Norey et al., 1990; Georgeet al., 1996). In turbot, neither the liver nor the kidneysare fully differentiated at hatch and differentiation occursbetween hatch and first feeding (Tytler et al., 1996; andunpublished observations). Thus the simplest explanation forthe lack of inducibility is that there may be a lag periodbefore the requisite transcription factors required for induc-tion of MT gene expression by Cd are present and it is merely

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98 GEORGE ET AL.

a reflection of the stage of liver differentiation. Experimentalstudies are therefore planned to test this hypothesis.

While the recent elegant gene manipulation studies ofMichalska and Choo (1993) and Masters et al. (1994) haveprovided direct evidence that MT protects against Cd toxicityin a mammal, we believe that the present results, whichshow a correlation between the changes in inducibility ofMT mRNA synthesis and Cd toxicity during larval develop-ment of turbot, also provide evidence for a role of MT inprotection from Cd toxicity in fish and provide a mechanisticexplanation for the increased sensitivity of larval fish toheavy metal toxicity.

REFERENCES

Al-Maghazachi, S. J., and Gibson, R. (1984) The developmental stages oflarval turbot, Scophihalmus maximus L. J Exp. Mar. Binl. Ecol. 82, 35 -

51.

Andrews, G. K , Adamson, E D., and Gedamu, L (1984) The ontogeny ofexpression of murine metallothionein: Comparison with the a-fetoprotein

gene. Dev. Btol. 103, 294-303

Andrews, G. K , Gallant, K. R., and Cherian, G. (1987). Regulation of theontogeny of rat liver metallothionein by zinc Eur. J. Biochem 166, 527-531.

Basil, A , and Lazo, J. S (1990). A hypothesis regarding the protective roleof metallothioneins against the toxicity of DNA interactive anticancerdrugs Toxicol. Lett. 50, 123-134.

Bremner, I. (1987). Involvement of metallothionein in the hepatic metabo-lism of copper. J. Nutr. 117, 19-29.

Bremner, I., Williams, R. B., and Young, B. W. (1987). Distribution ofcopper and zinc in the liver of developing sheep fetus Br. J. Nutr. 38,87-92.

Brown. J. A . and Tytler, P. (1993). Hypoosmoregulation of larvae of theturbot, Scophthalmus maximus: Drinking and gut function in relation toenvironmental salinity. Fish Physivl. Biochem. 10, 475-484

Clough, S. R, Mitra, R S , and Kilkami, A. P. (1986). Qualitative andquantitative aspects of human fetal liver melallothioneins. Biol. Neonate48, 241-254.

Coombs, T L.. Fletcher. T., and White, A. (1972). Interaction of metalions with mucus from the plaice, L. Biochem J 128, 128-129.

Cousins. R. J (1985). Absorption, transport and hepatic metabolism ofcopper and zinc Special reference to metallothionein in biological tissues.Physiol. Rev. 65, 238-309

Finney, D. J. (1971). Probit Analysis. 3rd ed.. p 333 Cambridge Univ.

Press, Cambridge. UK.

Fleet. J. C and McCormick. C C. (1988). The ontogeny and induction byzinc of hepatic chick-embryo metallothionein. Proc. Soc. Exp Biol. Med188, 52-60.

George. S.. Burgess. D.. Leaver. M.. and Frenchs, N. (1992). Metallothio-nein induction in cultured fibroblasts and liver of a marine flatfish, theturbot. Scophihalmus maximus. Fish Physiol. Biochem. 10, 43-54

George. S G.. and Carpene. E. (1981) Absorption of cadmium by gills of

Myiilus edulis (L.). Mot. Physiol. 1, 23-34.

George, S G.. and Olsson, P-E. (1994). Metallothioneins as indicators oftrace metal pollution. In Biological Monitoring of Coastal Waters andEstuaries (K. J. M. Kramer. Ed.), pp. 151-178. CRC Press. Boca RatonFL

George. S . Todd, K . and Wnght. J. (1996). Regulation of metallothionein

in teleosts. Induction of MTmRNA and protein by cadmium in hepaticand extrahepatic tissues of a marine flatfish, the turbot (Scophihalmusmaximus). Camp. Biochem. Physiol., 113C, 109—115.

Hamer, D. (1986) Metallothionein. Annu. Rev. Biochem. 55, 913-952

Hilmy, A. M , Shabana, M. B., and Daabees, A. Y. (1985). Bioaccumulationof cadmium: Toxicity in Mugil Cephalus Comp. Biochem. Physiol. C81, 139-143.

Jenkins, K. D., and Sanders, B. M. (1986) Relationships between freecadmium ion activity in sea water, cadmium accumulation and subcellulardistribution, and growth in Polychaetes. Environ. Health Perspect. 65,205-210.

Kagi, J. H. R., and Kojima, Y. (1987) Metallolhionein II. Experienlia.Suppl. 52, Birkhauser Verlag, Basel.

Kagi, J. H. R., and Schaeffer, A (1988). Biochemistry of metallothionein.Biochemistry 27, 8509-8515.

Kann, M. (1985). Metallothioneins. Proteins in search of a function. Cell41,9-10.

Kille, P., Kay, J., Leaver, M. J., and George, S. (1992). Induction of piscinemetallothioneins as a primary response to heavy metal pollutants. Appli-cability of new sensitive molecular probes Aauat. Toxicol. 22, 279-286.

Lazo, J. S., Kondo, Y., Dellapiazza, D., Michalska, A. E., Choo, K. H. A ,and Pitt, B R. (1995). Enhanced sensitivity to oxidative stress in culturedembryonic cells from transgenic mice deficient in metallothionein I andII genes. J. Biol. Chem. 270, 5506-5510.

Masters, B. A., Kelly, E. J., Quaife, C. J., Brinster, R. L., and Palmiter,R. D. (1994). Targeted disruption of metallothionein I and II genes in-creases sensitivity to cadmium Proc. Nail. Acad. Sci. USA 91, 584-588.

McKim, J. M. (1977). Evaluation of tests with early life stages of fish forpredicting long-term toxicity. J Fish Res Bd. Can. 34, I 148-1154.

Michalska, A. E., and Choo, K H. A. (1993). Targeting and germ-linetransmission of a null mutation al the metallothionein I and II loci inmouse Proc Natl Acad Sci. USA 90, 8088-8092.

Middaugh, D. P., and Dean, J. M. (1977). Comparative sensitivity of eggs,larvae and adults of the estuarine teleosts Fundulus heteroclitus andMenidia menedia to cadmium Bull. Environ. Contam. Toxicol. 17, 645-652

Norey C. G., Cryer A . and Kay, J (1990). Induction of metallothioneingene expression by cadmium and retention of the metal in the tissues ofrainbow trout (Salmo gairdneri) Comp. Biochem. Physiol. C 97, 215-220.

Olsson, P-E., Zafarullah, M., Foster, R., Hamor, T., and Gedamu, L. (1990).Developmental regulation of melallothionein mRNA. zinc and copperlevels in rainbow trout, Salmo gairdneri. Eur. J Biochem. 193, 229-235.

Panemangalore, M., Banerjee, D., Onosaka, S.. and Cherian. M. G. (1983).Changes in the intracellular accumulation and distribution of metallothio-nein in rat liver and kidney during postnatal development Dev Biol. 97,95-102.

Peters, J. M., Duncan, J R.. Wiley, L. M.. Rucker, R. B , and Keen, C. L.(1995). Effect of a metallothionein antisense oligonucleotide on embryodevelopment. Reprod. Toxicol 9, 123-130

Piscator. M. (1964). On cadmium in normal human kidney together with areport on the isolation of metallothionein from livers of cadmium exposedrabbits. Nord. Hyg. Tidskr. 45, 76-82.

Quaife, C . Hammer, R. E., Mottet, N. K.. and Palmiter, R. D. (1986).Glucorticoid regulation of metallothionein during murine developmentDev Biol 118, 549-555.

Riordan. J. R.. and Richards. V. (1980). Human fetal liver contains bothzinc and copper-nch forms of metallolhionein J Biol Chem. 255, 5380-5383.

by guest on September 1, 2016

http://toxsci.oxfordjournals.org/D

ownloaded from

FISH METALLOTHIONEIN GENE EXPRESSION AND Cd TOXICITY 99

Segner, H., Storch, V., Reinecke, M.. Kloas. W., and Hanke, W. (1994)The development of functional digestive and metabolic organs in turbot.Scophthalmus maximus. Mar. Biol. 119, 471-486.

Thomas, J. P., Bachowski, G. J , and Girotti. A. W. (1986). Inhibitionof cell-membrane lipid-peroxidation by cadmium-metallothionein andzinc-metallothionein. Biochim Biophys. Acta 884, 448-461.

Turner, D. R., Whitfield, M., and Dickson. A. G. (1981). The equilibriumspeciation of dissolved components in freshwater and seawater at 25°Cand 1 aim pressure. Geochim Cosmochim Ada 45, 855-881.

Tytler. P.. and Bell, M. V. (1989) A study of diffusional permeability ofwater, sodium and chloride in yolk-sac larvae of cod (Gadus morhuaL.). J. Exp. Biol. 147, 125-132.

Tytler, P , Bell, M. V., and Robinson. J. (1993) The ontogeny of osmoregu-lation in marine fish: Effects of changes in salinity and temperature. In

Physiological and Biochemical Aspects of Fish Development (B. T. Wal-ther and H. J. Flynn. Eds.), pp. 249-258. University of Bergen, Bergen.

Tytler, P.. and Blaster. J. H. S. (1988). Drinking in yolk-sac halibut. Hippog-lossus hippoglossus. J. Fish Biol. 32, 493-494.

Tytler. P , and Ireland, J. (1994). Drinking and water absorption by thelarvae of herring (Clupea harengus) and the turbot (Scophthalmus max-imus) J. Fish Biol 44, 103-1 16.

Tytler, P , Ireland, J., and Fitches. E. (1996). A study of the structure andformation of the pronephros in the larvae of the turbot (Scophalmusmaximus) and the hemng (Clupea harengus). Mar. Freshwater BehavPhysioi, in press.

Zafarullah. M.. Olsson, P-E., and Gedamu, L. (1989). Endogenous andheavy metal-ion-induced metallothionein gene expression in salmonidtissues and cell lines. Gene 83, 85—93.

by guest on September 1, 2016

http://toxsci.oxfordjournals.org/D

ownloaded from