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
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