Indian Journal of Experimental Biology Yol.)8, September 2000, pp. 921-925

Expression of heat shock protein 70 in freshwater prawn Macrobrachium malcolmsonii (H. Milne Edwards) following exposure to Hg and Cu

A Yamuna, V Kabila & P Geraldine *

Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024,lndi a

Received 21 September 1999; revised 29 May 2000

Juveniles of freshwater prawn M. malcolmsonii were exposed to l/6'h concentration of LC50 of Hg and Cu for 48 hr. Sampling was done at I 8, 12, 16, 24, 30, 36, 42 and 48 hr of exposure. Gill and hepatopancreas were di ssected and subjected to one-dimensional electrophoresis. Western blotting was employed to determine the relative concentration of heat shock protein, hsp 70 (stress-70) in each sample. In the gill tissue of the prawn that had been exposed to Hg (0.024 mgHg 1" 1

), stress-70 was detected from the I" hr till the 16'h hr of exposure. But in the gills of Cu exposed prawn, synthesis of stress-70 appeared from the I" hr till the 24'h hr. Synthesis of hsp70 was not recorded after the 24'h hr in the gi lls of exposed prawns. Synthesis of stress-70 was also found to be ti ssue-specific for both metal s in this prawn. When the antibody probe raised against stress-70 was used, synthesis of stress-70 was not observed in hepatopancreas of prawns exposed to Hg or Cu, during the entire period of exposure of 48 hr. The expression of stress-70 in M. malcolmsonii following exposure to Hg and Cu is apparently only transient , and also a differenti al expression of stress-70 between gill and hepatopancreas was observed for both the metal s.

Living organisms respond at the cellular level to unfavourable conditions such as heat-shock, or other stressful situations of many different origins, by the rapid, vigorous, and transient acceleration in the synthesis of a class of proteins known as heat-shock proteins (hsps) or stress-proteins. Originally, they were termed the heat-shock response because of the synthesis of these proteins following hyperthermia. Now, these proteins are known to be induced by diverse classes of physical and chemical agents including metals (Cd, Zn, Hg, Pb and Cu), oxidizing agents and drugs affecting respiration and energy metabolism (arsenite, anoxia, H20 2 and KCN). These hsps are highly conserved and found in all organisms, from archaebacteria to eubacteria, yeasts , plants and vertebrates, including humans 1

'2

. Some hsps are constitutively expressed, that is, present even under normal conditions, and thus are believed to play critical roles in normal cell physiology, growth and development3

.

In many of the model organisms studied, a family of four major heat-shock proteins (hsp) of 90, 70, 60 and 16-12 kDa is the most prominent, and these proteins have been frequently referred to as hsp90,

*Correspondent author (() : 91 431 660 360 Fax: 91 431 660 245 e- mail : gerry@bdu.ernet.in

hsp70, hsp60 and low-molecular weight (LMW) hsps respectively 4

. Owing to its extremely high induction in cells subjected to metabolic stress, considerable attention has been directed towards the characterization of the hsp70 or stress-70. Under adverse environmental conditions, synthesis of these proteins increases and they act to repair and protect cellular proteins from stressor-induced damage and to mtntmtze protein aggregation5

•7

. Stress-70 also facilitates the transport of badly damaged proteins to the lysosomes for breakdown8

.

Although tissue specificity in the heat-shock re­sponse has been observed in a number of aquatic spe­cies9 ' 14

, the physiological significance of these differ­ences is not well understood. At the cellular level , regulation of synthesis of stress-protein is closely linked to protein damage 15

'16

. The extent of cellular damage to a particular tissue is dependent upon : ( I) the distribution of the contaminant among tissues; (2) the ability of each tissue type to detoxify the contami­nant and thus minimize cellular damage; and (3) the molecular mechanisms by which the contaminant ex­erts its toxic effects. The relative concentrations of stress proteins synthesized by tissues may differ sig­nificantly with the stressor used to elicit the stress response as a consequence of the interplay between these physiological processes 14

• Thus, the differences in the synthesis of stress protein may be useful in identifying tissues which are particularly vulnerable

' 922 INDIAN J EXP BIOL, SEPTEMBER 2000

to damage by a specific stressor and in evaluating the extent of that damage.

The present study was undertaken to determine (l) whether synthesis of the highly inductive stress-70 occurs in the freshwater prawn Macrobrachium malcolmsonii on exposure to Hg and Cu; and (2) the differential response of the prawn, if any, to these metals and the possible significance of the response.

Materials and Methods The prawns Macrobrachium malcolmsonii were

collected from the lower Anicut , Tiruchirappalli, which is on the river Coleroon (unpolluted site), and transfened to the laboratory. They were reared in large cement tanks (3 x 3 x 3 ft) and fed ad libitum with chopped boiled goat liver during acclimation and experimental periods. Ju veni le prawns of the intermoult stage ranging from 40 - 50 mm in length and 0.8 - 1.0 g in weight were chosen for the experiments.

Three groups of 90 prawns each were used in this study. The first group served as control. The second group was exposed to 0.024 mgHg r 1 (1/6111 of LC50 of Hg; 96 hr LC50 value - 0.145 mgHg r 1

) and the third group to 0.151 mgCu r 1 (1/6111 of LC50 of Cu; 96 hr LC50 value - 0.908 mgCu r 1

). The experiment was canied out for 48 hr. Sampling was done after 1, 8, 12, 16, 24, 30, 36, 42 and 48 hr of exposure. The prawns were observed for mortality at each sampling period, and the mortality was found to be nil. At each sampling hour, 10 prawns were sampled from each group. Two samples were pooled to constitute a single observation, and thus five such observations were made.

The principal tissues; · namely, the gill and hepatopancreas (Hp), were dissected out and were homogenized using Tris buffer (20 mM, pH 7.5), O.lmM dithiothreitol and 0.1 mM phenylmethyl­sulfonyltluoride. They were centrifuged at 7000 rpm for 30 min at 4°C. Total protein concentration in each supernatant was determined by standard method 17

•

The samples were denatured for 1 min in a boiling water bath with sodium dodecyl sulfate (SDS) sample buffer18 and equal quantities of protein ( 100 f.,lg) were loaded on a 12% polyacrylamide gel with 6.5 % stacking gel, using the buffer system as described earlier 18

•

Western blotting was employed to determine the relative concentration of stress-70 in each sample. Samples were subjected to 1-D gel electrophoresis and electroblotted onto a nitrocellulose membrane 19

•

Jembranes were blocked with 5% milk powder in Tris buffer saline (10 mM Tris , pH 7.5, 0.9% NaCI) and Tween-20 (0.005%). A st ress-70 polyclonal antibody raised against a synthetic peptide from the highly conserved carboxyl terminu ~ of hsp70 (1: 250 dilution) was used as a probe. Goal anti-rabbit IgG (1:500 dilution) conjugated with horseradish peroxidase was used to detect the antibody probes, and the substrate hydrogen peroxide, in the presence of the chromophore 4,chloro-l naphthol, was used to detect the bound antibodies. ·

Results Synthesis of stress-70 was recorded in the gill

tissue of M. rnalcolmsonii exposed to Hg and Cu (Figs 1 and 3). Tissue specificity was also apparent.

Immunoblotting with the polyclonal antibody raised against the synthetic peptide of a conserved segment of hsp70 showed specific reactivity with one protein band of approximately 70 kDa in the gill tissue of both Hg - and Cu - exposed prawns (Figs l and 3). In the gill ti ssue of the prawn that had been exposed to Hg (0.024 mgHg r 1>, stress-70 was detected from the 151 hour till the 16111 hour of exposure (Fig. 1). But in the gills of the prawn exposed to Cu, synthesis of stress-70 appeared from the 151 hr till the 24th hr (Fig. 3). Synthesis of hsp70 was not recorded after the 24th hr of exposure in the gills of the prawns exposed to Hg or Cu.

Synthesis of stress-70 was also found to be tissue­specific for both metals in this prawn. When the antibody probe raised against stress-70 was used, synthesis of stress-70 was not observed either in Hp of prawns exposed to Hg or Cu, during the entire period of exposure of 48 hr (Figs 2 and 4).

Discussion

In the present study, Hg and Cu have been shown to induce synthesis of stress-70 in the freshwater prawn M. malcolmsonii. Exposure to trace metals has been found to induce the synthesis of stress-70 in aquatic organisms such as Salmo gairdnerii due to arsenite20

, Oncorhynchus tshawtscha, exposed to zinc and cadmiurn21

, Pimephales promelas due to chromium22 and arsenite 10

, and Mytilus edulis due to copper13

'14

. In the present study, the induction of hsp70 following exposure to both metals was evident only in the gills of prawn M. malcolmsonii and the response was not elicited in Hp. Thus, a tissue­specific difference upon exposure to these metals was observed. In recent studies, tissue-specific differences

YAMUNA et. al.: EXPRESSION OF HEAT SHOCK PROTEIN IN MA CROBRA CHIUM

Hsp 70

Hsp . 70

la

24 16 12 8

2o

24 16 1 2 8

3a

24 12 8

4a

24 12 8 0

lb

2b

24 16

Exposure (hr)

3b

1 0 24 12 8

:xposure (hr)

4b

24 12 8

Exposure (hr)

0

923

Fig. 1-Electrophoretic rrofile of proteins of gill of M. nralcolmsonii (b) and the corresponding immunoblot (a) following exposure to Hg. Fig. 2-Electrophoretic profile of proteins of hepatopancreas of M. malcolmson ii (b) and the corresponding immunoblot (a) following exposure to Hg. Fig. 3---Electrophoretic profile of proteins of gill of M. malcolmsonii (b) and the corresponding im­munoblot (a) following exposure to Cu. Fig. 4--Electrophoretic profile of proteins of hcpatopancreas of M. malcolm.sonii (b) and the corresponding immunoblot (a) followin g exposure to Cu.

924 INDI AN J EXP BIOL, SEPTEMBER 2000

have also been observed when organi sms are exposed to di fferent chemical stressors. In fathead minnows, a tissue-spec ific stress response has been induced with

. h 1' d d d' . . 1011 arsemte, c romate, 1n ane an 1azmm · . · Interes tingly, differences in expression at the tissue level are a refl ec ti on of differences in the mode of ac tion of each chemica l, and induction is hi ghest in the respective ta rget ti ssues. In Mytilus edulis, exposed to a range of Cu concentrations, synthes is of chaperonin 60 (cpn 60) and stress-70 by the gill was greater than that observed in the mantle tissue 14.

In thi s study, the di ffe rential expression of stress-70 in gill and Hp of the prawn on exposure to Hg and Cu, was possibly due to the di ffering modes of ent ry of tox icant into the prawn. Another poss ibility is that the quantum of metal accumulated in the Hp duri ng the exposure period did not cause cellular damage to the extent of ind uci ng the synthesis of stress-70. The extent of cellular damage for a partic ular ti ssue, in turn , is reported to be dependent on the uptake and distri buti on of the contaminant by tissues and on their ability to detox ify the contaminants14. It is also possible that synthes is of other classes of stress protein, such as stress-90 or low molec ular weight proteins, which were not probed in thi s study, could have occurred in the Hp. The latter poss ibility has been reported in fathead minnows 10'11 exposed to ar eni te; stress proteins of 74, 72, 70, 40 and 20kDa were induced in gill s, whereas proteins of 90, 70, 68, 30 and 20kDa were induced in the musc le tissue.

Protein denaturati on is a primary mechani sm of tox icity which arises out of exposure to adverse phys ical and chemical conditions. Thi s results in the weakening of polar bonds and exposure of hydrophobic groups, resulting in mi sfolding and protein aggregation, a condition known as proteotox icity23

. The resultant denatured or misfolded, or large pool of unfolded, proteins act as key components for the recogniti on of stress by the cells24. At the cellular level, Cu has several primary mechani sms of tox icity that can alter protein conformation and biological activi ty: it can impair protein function th rough denaturation and metal . substitution; it catalyses perox idation reacti ons and the generati on of free radi cals which can damage li pids and proteins25. Hg is a sulfhydryl reacti ve compound which causes depletion of intracellular thiols, which in volves the synthes is of stress proteins26

·27

. In the present study also, Hg may have triggered induction of the synthesis of stress-70 in M. malcolmsonii by depleting intracellular thiols while

Cu, by causing alterations in protein confo rmation, may have induced the synthes is of stress-70.

The heat-shock response is believed to be transient under mildly stressful conditions because translational patterns revert to those found in cont rols within hours of continuous exposure to high, but not extreme,

19 28 ?9 temperatures or to trace metals · - . It had been reported that the synthesis of stress protein in bluegi ll sunfi sh was transient; when this organism was ex posed to sediments contaminated with metals, accumulation of stress proteins did not persist throughout the experiment30. It is op ined that a transient heat-shock response would be less likely to have an important role in physiological adaptati on 13

.

Al so, at both the cellular and organi smal level, induction of synthesis of st ress proteins correlates with acquired tolerance, the phenomenon in which exposure to a mild stress increases the ab il ity to survive a subsequent, more severe stress that would otherwise be lethal27'31'32

. In the present study, since the synthesis of stress-70 in the prawn fo llowing exposure to both Hg and Cu is apparently onl y transient, th is response to stress is less li ke ly to have any ro le in the physiologica l adaptation in thi s organ ism and also does not suppo1t the suggesti on that synthes is of stress protein correlates with acquired tolerance. However, thi s rema ins a matter of conjecture, and can only be confirmed by testing with a range of Cu and Hg concentrations.

In summary, the results suggest that the induction of stress-70 occurs in the gill s of the prawn M. malcolmsonii, fo ll owing exposure to Hg and Cu, although the response is apparently only transient. A diffe renti al expression of stress-70 between gill and Hp was observed fo r both the metals. The inducti on of response in the gill ti ssue of the prawn may be due to conformational changes in protein caused by both Hg and Cu.

Acknowledgement A. Y is the recipient of Senior Research Fellowship

of CSill.., New Delhi . The authors gratefully ac knowledge Dr. Brenda M Sanders, Molecular Ecology Institute, Uni versity of Califo rni a, USA for readily supplying the hsp70 antibodies .The authors are al so thankful to Dr. Philip A. Thomas, Professor, Dept. of Mic obiology, Post Graduate Insti tute of Ophthalmology, Tiruchirappalli and Dr. M. Kri shnan, Department of Biotechnology, Bhara thidasan Uni versity, Ti ruchirappalli for their cri tical evaluati on of the manuscript.

YAMUNA et. al. :EXPRESS ION OF HEAT SHOCK PROTEIN IN MACROBRACHIUM 925

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