Metallothionein induction and metal homeostasis in rainbow trout hepatocytes exposed to mercury

To.~-icolog~ Lerrers,

99

5 1 (1990) 999107

Elsevier

TOXLET

02303

and metal homeostasis exposed to mercury

Metallothionein induction rainbow trout hepatocytes

Frangois TOXEN.

Gag&,

DPpartement

Michel Marion de Chimie,

(Received

30 June 1989)

(Accepted

29 October

Universik

and Francine

de Quebec 2 MontrfM

in

Denizeau Montrhzl,

QuPbec (Canada)

1989)

Key tvords: Metallothionein:

Metal homeostasis;

Mercury;

Hepatocytes

SUMMARY Rainbow

trout

hepatocytes

0. 5. 10, 15, 20,25. of Hg. The presence molecular-weight

of Hg led to elevated

components,

gest that when hepatocytes sequestration

were exposed

to a sublethal

30 and 35 h at 15°C. The hepatocytes although

concentration

of mercury

were found to accumulate

(Hg) (100 nM) for

appreciable

Ca, Cu and Zn levels in cells. Hg was bound

it induced the synthesis

of metallothionein

to low-

(MT). The results sug-

are exposed to low levels of Hg. MT does not play a significant

of the metal. The elevated cellular

quantities

mainly

Cu and Zn levels could be associated

role in the

with the induction

of MT.

INTRODUCTION

Mercury (Hg) is a highly toxic heavy metal and it is known to induce the synthesis of mellathionein (MT) [l]. MT is a low-molecular-weight metal-binding protein (6 kDa) which exhibits a high cysteine content (30%) and is devoid of aromatic amino acids and histidine [2,3]. MT binds copper (Cu) and zinc (Zn) under normal physiological conditions but has a high potential for binding several non-essential heavy metals such as Hg and cadmium (Cd) [4]. In addition to its induction by heavy metals, MT is also inducible by glucocorticoid hormones, alkylating agents and vitamin C [5-71. It has been proposed that MT is implicated in the regulation of Cu and Zn homeostasis [8]. Moreover, a detoxification potential against heavy metals has been attributed to MT. However, the MT action mechanism in heavy metal resistance is still unclear, although it appears, as has been suggested for Cd, that sequestration Addressfor

correspondence:

Succ. A, Montreal

0378-4274/90/$3.50

Dr. Francine

(P. Quebec),

Denizeau,

H3C 3P8, Canada.

Universite

du Quebec a Montreal,

Tel. (514) 987-8229.

@ 1990 Elsevier Science Publishers

B.V. (Biomedical

Division)

Casier postal 8888,

100

of the metal by MT produces a less toxic form of the latter [9]. However, this mechanism is still controversial in the case of rainbow trout because when organisms are environmentally exposed to Cd, MT is found not to bind the metal significantly [IO]. As far as Hg is concerned, there is a lack of data on the possible role of MT in the detoxification

of the metal in rainbow

trout.

The use of hepatocytes isolated from fish to study the mechanisms of action of toxic agents is now well recognized [ 111.At the same time, as previously pointed out, there are virtually no studies aimed at clarifying the detailed biochemical mechanisms related to Hg toxicity and MT in fish using cellular models. In order to examine the metabolism of MT and Hg toxicity, we used a cell culture system which consists of exposing freshly isolated rainbow trout (S&no gairdnevi) hepatocytes to Hg in vitro. The kinetics of MT induction were determined under sublethal exposure conditions as defined by LDH release into the extracellular medium. Cellular levels of calcium (Ca), Cu and Zn were also followed in cells exposed to Hg. Relationships between cellular Cu, Zn and Hg, cell survival, and MT levels in trout hepatocytes exposed to Hg are proposed. MATERIALS

AND METHODS

Preparation of trout hepatocytes Primary cultures of trout hepatocytes were prepared according to the method of Klauning et al. [12]. Cell viability was assayed by the trypan blue exclusion test. The cells were suspended in Williams’ Medium E (WME) containing 10% fetal bovine

I

Q +

IOOnMHg Control

time (h)

Fig.

I. Extracellular

activity

of LDH in trout

hepatocytes.

The cells were incubated

wth

100 nM Hg or

without Hg. After each incubation time, the cells were centrifuged at 1000 x R for 5 min and the activity of LDH in the supernatant was assayed. Data represent means and standard errors from 3 separate experiments.

101

serum (FBS), seeded in 60 mm Petri dishes (8 x 10’ viable cells/dish) and incubated for 2 h at 5°C in a humidified atmosphere of 95% air and 5% CO*. At the end of this period, the cells were washed twice with a cystine-free Minimum Essential Medium (MEM) containing 1% FBS. The cells were redistributed in Petri dishes (1 x 10’ viable cells/60 mm dish) using the previous medium. [3H]Cysteine (spec. activity: 920 mCi/mmol) was added to the Petri dishes at a final concentration of 1.l &i/ml. *03HgC12(spec. activity: 2 mCi/mg Hg) was mixed with a 10,~M Hg solution and added to the Petri dishes to obtain a concentration of 100 nM Hg (20 ng/ml) and 1.6 &i/ml. Time course studies

The hepatocytes were allowed to incubate for 0, 5, 10, 15, 20, 25, 30 and 35 h at 15°C under the conditions described above. For each determination, the material from 8 samples was pooled. Cytotoxicity evaluation

After each period, the cells were harvested with a siliconized Pasteur pipette. The cell suspension was then centrifuged at 1000 xg and 4°C for 5 min. The activity of LDH in the supernatant was assayed by the method of Mold&s et Hogberg [13]. Tests in our laboratory have shown that at the concentration used, Hg did not affect the activity of the enzyme. The cell pellet was washed twice with Hanks’ balanced salt solution (containing no Ca) by successive centrifugation-resuspension steps at 1000 x g and 4°C for 5 min. Afterwards, the cell pellet was resuspended in a 50 mM Tris-acetate buffer (pH 7.4) containing 250 mM sucrose, 5 mM /3-mercaptoethanol and 0.01% sodium azide. Aliquots of the cell suspension were taken for the analysis of Ca, Cu, Zn, 3H and *03Hg. 203Hg and 3H were evaluated by liquid scintillation 10

1

8-

tlmo(h)

Fig. 2. Accumulation

of Hg by the hepatocytes

Hg were determined.

Data represent

as a function

of exposure

means and standard

time. Total cellular and cytosolic

errors from 3 separate

experiments.

102

counting

(Packard,

Minaxi-8

tri-carb

4000 series). Ca. Cu and Zn were analyzed

by

atomic absorption spectrophotometry with background correction using a standard air-acetylene flame (aajae Instrument Laboratory). Protein concentration was evaluated by the method of Bradford [14]. Put$cation and analysis sf metallothionein The cell suspension was sonicated at 4”C, and the cytosol was obtained by ultracentrifugation at 107000 XR for 90 min (Beckman L8-M ultracentrifuge, rotor SW-41 Ti). The cytosol preparations were stored at -2O’C under a nitrogen atmosphere until they were analyzed as follows. The cytosol (950 ~1) was chromatographed on a Sephadex G-75 column (36 x I .2 cm) under an atmosphere of nitrogen at room temperature. The elution buffer consisted of 10 mM Tris-acetate (pH 7.4) with 5 mM P-mercaptoethanol and 0.01% sodium azide. Fractions of 950 ,~l were collected and analyzed for 3H and 2n3Hg. Cu was measured with a graphite furnace atomic absorption spectrophotometer equipped with background correction (Varian AA-1475. GTA-95). The fractions containing MT (V,/V, = [1.9-2.31) were pooled and analyzed for MT using the silver saturation assay [ 151. Silver was analyzed using a graphite furnace atomic absorption spectrophotometer as mentioned above. The levels of MT were determined on the basis of the relationship (17.4 g-atoms of silver per mole of MT) known for mammalian MT; thus the calculated MT levels are in units of mammalian MT equivalents. Stutistiul unu1j~si.s The experiments were carried out in triplicate. The data were subjected ysis of variance and to a post hoc test (Scheffk F-test).

I

10 Fig. 3. Cellular determined

content

by atomic

of Ca in trout

1

I

20 time(h) hepatocytes

30 exposed

to an anal-

1 40 to Hg. Ca levels in trout

hepatocytes

absorption spectrophotometry. Data represent means and standard separate experiments. *P
errors

were from ?

103

RESULTS

AND DISCUSSION

The cytotoxicity marker used in this study (LDH release) indicates to the cell membrane which is considered irreversible and is associated

severe damage with cell death

[16]. A series of experiments was carried out at first to evaluate the optimum range of Hg concentrations within which cell membrane permeability would not be significantly affected, as judged from the leakage of LDH into the extracellular medium.

0 0

5

10

0

5

10

15 20 time (h)

15

20

25

30

35

25

30

35

time (h)

Fig. 4. Cellular

metal levels in trout hepatocytes

(B) in Hg-exposed represent

and unexposed

means and standard

as a function

cells were determined

errors from 3 separate

of exposure

time. Cellular

Cu (A) and Zn

by atomic absorption spectrophotometry. experiments. *Pi 0.05 as compared to control

Data levels.

Below I ,LIM Hg. no LDH release was detected

for an exposure

time of 40 h. This

is shown for the concentration of Hg used (100 nM). which is well below this threshold. and which did not induce the loss of LDH by the cells (Fig. 1). Metal levels were analyzed

in exposed

hepatocytes.

The cells accumulated

detect-

able amounts of Hg (Fig. 2), and Hg in the cytosol increased with time, as did the total cellular Hg. Although Hg was not cytotoxic, as judged from LDH data, its presence led to significantly increased Ca levels at 20 h of incubation time (Fig. 3). It has been proposed that higher levels of intracellular Ca could be a common pathway leading to the loss of cell viability and this could constitute a critical and early stage in the development of toxicity in hepatocytes [17]. The increase in Ca in this case is a reversible phenomenon. suggesting that the cells were able to protect themselves against severe metabolic perturbations [ 181 and further developments of cytotoxicity. The presence of Hg also led to elevated levels of Cu at 25 h. as compared to the controls (Fig. 4A). The levels of Cu in unexposed cells remained constant during the time course study. In addition, there was no significant (P < 0.05) change in cellular Zn levels in untreated cells (Fig. 4B). In Hg-exposed cells, Zn levels were higher at 5. 10, 25 and 30 h of incubation time. The distribution of Hg and of Cu in the cytosol was analyzed through G-75 chromatography. Figure 5 shows a representative elution profile where MT is eluted in the MMW fractions. The presence of Hg and of Cu in the high molecular weight (HMW. V,/V, = [&I .5]). middle molecular weight (MMW, V,/V, = [1.6-2.51) and low molecular weight (LMW, V,jV,=[2.63.5]) fractions was investigated (Figs. 6A and 6B). 6

40000 MMW

E >

z

30000

p m

20000

pii&Eq 10000

0 ”

1

Fig. 5. Representative G-75 chromatography. high molecular and V,/V,=

Sephadex

weight (HMW).

[2.6-3.51

G-75 elution profile of the cytosol.

The profile is separated corresponds

into 3 distinct

VJV, = (I .6 - 2.51 corresponds to the low molecular

The cytosol was resolved

regions:

to the middle molecular

weight (LMW) components.

30 h. The arrow shows the expected elution peak for MT. [‘Hlcystine

by Sephadex

VJV, = [0- I .5] corresponds

to the

weight (MMW),

Incubation

was added to the incubation

time was medium.

105

The proportion elevated

of cytosolic

Hg in the LMW fraction

for the rest of the study.

increased

at 10 h and remained

At the same time (10 h), the Hg proportion

de-

creased in the HMW and MMW fractions. In the case of Cu, its proportion was lowest in the MMW fractions at 5 h, while it showed a maximum in the HMW fractions. Afterwards, it diminished in the HMW components, but it increased in the MMW fractions. Moreover, the silver-saturation assay applied to the MMW fractions revealed that MT levels were significantly higher at 20 and 30 h of incubation time (as

A 100

60

60 I” 3 %HMW %MMW %LMW

40

20

0 0

5

10

15

20

25

15 20 time(h)

25

30

35

time (h)

B

60 ; s 40

20

0 0

Fig. 6. Time course data are expressed

5

10

of Hg and Cu distribution as the percentage

35

of hepatocytes

exposed

to 100 nM Hg. The

of the metal in each of the HMW, MMW and LMW fractions

to the total cytosolic

metal recovered.

means

errors

and standard

in the cytosol

30

(A) Percentage

from 3 separate

of Hg. (B) Percentage of Cu. The data *P
ort=OhforCu.

relative represent h for Hg

106

compared to t =O); these remained 35 h (Fig. 7).

elevated

until they returned

to control

values at

Relationships between the various metabolic changes can be proposed. First, Hg appears to induce the synthesis of MT in trout hepatocytes. It can be hypothesized that the elevation

of cellular

Cu and Zn levels is associated

with the induction

of MT.

Indeed, higher Cu and Zn levels are detected at the same time as increased cellular MT. Moreover, there was an initial perturbation of Cu distribution in the cytosol: at 5 h, the proportion of Cu in the HMW was more important than that in the MMW fractions. and also in this particular instance, showed a maximum for the period studied. This phenomenon returned to the profile seen at t = 0 (Fig. 6B). at a time (10 h) when MT induction was detected. In addition, it is interesting to observe that Hg is mainly associated with the LMW components, suggesting that the sequestration of Hg by MT is not the principal mechanism involved in its intracellular distribution. Hg has a high affinity for thiols and could bind with small thiol polypeptides in the LMW region, such as glutathione which contains 30% cysteine. It is possible, therefore, that it is the binding of Hg to the LMW components which protected the cells against cytotoxicity and criticial increases in intracellular Ca. Finally, MT fractions contained mostly Cu and Zn (not shown) and Hg to a lesser extent, suggesting that MT would largely remain involved with the regulation of Cu and Zn homeostasis in cells exposed to low levels of Hg. In summary. the presence of Hg at a low concentration did not cause the liberation of LDH into the extracellular medium. However, its presence led to elevated cellular levels of Ca, Cu and Zn. Hg was mainly associated with LMW components in the cytosol. suggesting that MT was not mainly responsible for the sequestration and 6 l

5

0

5

10

15

20

25

30

35

time(h)

Fig. 7. Cellular

MT levels in relation

The data are expressed

to exposure

in units of mammalian

time. MT was determined MT equivalents

by the silver saturation

(nmol MT/mg

protein).

Data

assay.

represent

means and standard errors from 3 separate experiments. Cellular MT levels in unexposed cells did not change during the time studied: they remained in the range of I .6i 0.7 nmol MT/mg protein. *I’< 0.05 for MT levels in treated cells as compared

to control

levels.

107

detoxification

of the metal.

when the hepatocytes

MT would

remain

are exposed to sublethal

involved

in Cu and Zn homeostasis

levels of Hg.

ACKNOWLEDGEMENTS

This study was funded by World Wildlife Fund Canada and NSERC Canada. The authors wish to thank Dr. Christian Blaise of Environment Canada for providing rainbow trout. REFERENCES I Eaton,

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