Metallothionein-I accumulation in the rat lung following a single paraquat administration

Toxicology Letters, 45 (1989) 41-47 Elsevier

41

TXL 02084

Metallothionein-I accumulation in the rat lung following a single paraquat administration

Masao Sato, Atsushi Ohtake, Koichiro Takeda, Hiroshi Mizunuma and Yasushi Nagai Division of Environmental Pollution Research, Research Laboratory, Fukushima Medical College, Fukushima 960-12 (Japan] (Received 20 May 1988) (Revision received 25 July 1988) (Accepted 24 August 1988) Key words: Metallothionein;

Paraquat; Lung

SUMMARY The ability of paraquat (PQ), a free radical inducible chemicat, to increase metallothionein-I (MT-I) content in the tissues was determined using radioimmunoassay. A single dose of PQ into the rat caused a sevenfold increase in pulmonary MT-I concentration on day 1 and its concentration returned to the control level by day 5. Paraquat-induced increase in MT-I was not observed in the kidney, but was seen in the liver. The data show the difference in accumulation of MT-I in liver, kidney and lung after intraperitoneal injection of a high dose of PQ.

INTRODUCTION

Paraquat (PQ), a readily available bipyridilium herbicide, is wideiy used. It is weli-known that PQ has specific effects on the lung, e.g. causing pulmonary edema, hemorrhage, interstitial and alveolar fibrosis and bronchial epithelial proliferation [l]. The mechanism is thought to be due to lipid peroxidation mediated through superoxide radical formation [2]. ~etallothionein (MT) is a low molecular weight protein which is rich in cysteine Address for correspondence: Dr. Masao Sato, Division of Environmental Pollution Research, Research Laboratory, Fukushima Medical College, 1, Hikarigaoka, Fukushima 960-12, Japan. 0378-4274/89/$ 03.50 0 1989 Elsevier Science Publishers B.V. (Biomedjcal Division)

42

and binds

metals

[3]. Although

the biological

function

of MT is still unclear,

MT

is thought to be involved in the regulation of essential metals such as zinc (Zn) and copper (Cu), and in the detoxification of toxic heavy metals such as cadmium (Cd) and mercury (Hg). In recent years particular attention has been paid to a possible role of MT in the scavenging of free radicals [4]. Metallothionien can be induced by many agents and environmental conditions such as partial hepatectomy [5], adrenalectomy [6], endotoxin [7], carrageenan [S], ethanol [9], interleukin-1 [lo] or interferon [ll]. It has been reported that exposure to high-dose X-irradiation [ 121, high-tension oxygen [ 131, and carbon tetrachloride (CCld) [14] produces lipid peroxidation and results in an increase in MT in the liver and kidney. Since PQ produces lipid peroxidation [2], the same as CC14 [15] or Xirradiation [ 161, we examined whether or not PQ could increase MT formation in the lung. The rate of radical formation from PQ in the lung was different from that in the liver and kidney [17]. In the present study the levels of MT in liver, kidney and lung of rats injected with PQ were determined by radioimmunoassay. MATERIALS

AND

METHODS

Reagents Pure PQ was obtained from Wako Pure Chem. Ind. Ltd. (Osaka). Bolton-Hunter reagent was obtained from ICN Biochemicals Inc. (Irvine, CA). Donkey anti-sheep IgG antiserum as the second antibody in radioimmunoassay was obtained from the Scottish Antibody Production Unit (Carluke, Scotland, U.K.).

Animals Japan) weighing approxMale Wistar rats (Funabashi Nojo Ltd., Funabashi, imately 130 g were used. Animals were allowed free access to water and a commerRats were intraperitoneally cial laboratory chow (Oriental Yeast Co., Tokyo). injected with PQ (22.5 mg/kg). Saline was injected as the control (0.5 ml/kg). Five rats were used in each group. Blood was collected by cardiac puncture under light ether anesthesia, and the rats were killed 1, 3, 5, 7, 14, 21 d after injection. Heparinized plasma was stored at 4°C and lung, liver and kidney were stored at - 40°C.

Experimental Two forms of MTs have been identified in the rat and referred to as metallothionein-I (MT-I) and a metallothionein-II [3]. In the present study, MT-I was determined by radioimmunoassay which was developed by Mehra and Bremner [18]. Sheep anti-rat liver MT-I antiserum was kindly provided by Dr. Ian Bremner, Scotland. Samples of lung, liver and kidney were homogenized in 9 vol of 10 mM Tris-acetate buffer, pH 8.0. For metal analysis, a portion of the homogenates was digested with mixed acids (HNO3/HC104/HzS04, 5:2:1 v/v) [19]. After digestion

43

the inorganic residues were dissolved in redistilled water and metal analysis was performed with a Perkin-Elmer 403 atomic absorption spectrometer. The resulting homogenate was centrifuged at 1500 x g for 15 min, and the supernatant was used for estimation of MT-I in the radioimmunoassay. MT-I and metal concentrations were expressed as pg per g of wet tissue. Statistics

Student’s t-test was used for the determination of the statistical significance of difference between means. A value of P < 0.05was accepted as significant. RESULTS

The dose of PQ used in the experiment (22.5 mg/kg) was determined as the dose which did not cause death, but did cause great body weight loss. Death occurred 25% or 100% of the time at a dose of 25 or 30 mg/kg, respectively, while at 20 mglkg death did not occur. As Sharp et al. [ZO] showed, death occurred in 2-5 d, in relation to the extent of body weight loss. When compared with control rats, the MT-I content in the lung increased sevenfold by 24 h following a single PQ administration (Fig. 1). Increased accumulation of MT-I in the lung was observed at 1 and 3 d and its concentration returned to the control level by day 5. The wet weight of lung did not change after PQ injection. An intraperitone~ injection of PQ produced at MT-I response in plasma that was above the controI level at day I (Fig. 2).

Days after

injection

Fig. 1. Concentration of pulmonary MT-I at various times following paraquat administration. Values represent the mean f SE of 4-5 rats. *Denotes significant difference from the control (P < 0.05). 0, control; e, paraquat.

44

Days Fig. 2. Plasma

concentration

& SE of 3-5 rats.

*Denotes

after

of MT-I following significant

4

2

0

6

injection

a single injection

difference

from control

of paraquat.

Values represent

(P < 0.05).

0,

control;

0,

the mean paraquat.

Increase in MT content in the liver has been observed under conditions such as food restriction and starvation [7]. Since the rats exposed to PQ did not eat, another control group of rats deprived of food for 24-h period was used. MT-I concentration in the lung was 390 k 12 ng MT-I/g lung (n= 3) in the 24-h starved rats, whereas 230 f 12 ng MT-I/g lung (n = 3) was found in the control rats. Thus, the starvation caused the increase in pulmonary MT-I concentration, but the degree of the increase in MT-I content in the lung was smaller in starved rats than in PQtreated rats (Fig. 1). Therefore, the pulmonary increase in MT-I by starvation cannot account for the great accumulation of MT-I in the lung of PQ-treated rat.

40 I t I

2 .-,”

-

4

.* 0

(51

y

20-

0!” Liver

Fig. 3. Concentration 24 h after the injection cant

difference

from

of zinc and copper of paraquat control

Kidney

in the liver and kidney of paraquat-treated

(22.5 mg/kg).

(P < 0.05).

0,

Values are the mean Zn control;

paraquat.

0,

rats. Rats were killed

? SE of 5 rats. *Denotes

Zn paraquat;

A , Cu control;

signifiA , Cu

45

* 1

20

t

0 +

oLiver Fig. 4. Concentration the injection.

of hepatic

Kidney

and renal MT-I in the paraquat-treated

Values are the mean

k SE of 5 rats. 0.05).

0,

control;

*Denotes 0,

significant

rats. Rats were killed 24 h after difference

from control

(P <

paraquat.

To compare the ability of MT synthesis and to investigate the status of metals in PQ-treated rats, concentrations of Zn, Cu and MT-I were determined in the liver and kidney. The abilities of Zn-MT [21] and Cu-MT [22] to modify lipid peroxidation mediated through radical formation have been studied. Administration of PQ resulted in an increase in hepatic Zn concentration, but did not cause any increase in hepatic and renal Cu concentrations (Fig. 3). MT-I concentration significantly increased in the liver of PQ-treated rats, but PQ exposure did not cause the increase in renal MT-I concentration (Fig. 4). The wet weight of liver was 5.68 f 0.17 and 4.38 + 0.18 g (P < 0.01) in the control and PQ-treated rats, respectively. The hepatic content of MT-I was significantly increased in the PQ-treated rats; that is 23.8 + 7.3 in the control and 90.9 + 14.4 pg MT-I/whole liver (P < 0.01) in the PQ-treated rats. The renal wet weight did not change after PQ injection. DISCUSSION

Metallothionein can be synthesized by heavy metals, hormones, external physical and internal chemical stress in many tissues [23]. In the lung, Sampson et al. [24] have reported that repeated exposure to Cd causes an increase in MT. Hart and Garvey [25] have also reported that exposure to Cd aerosols caused synthesis of pulmonary MT which localized with alveolar macrophages, fibroblasts and lymphocytes. The present study showed a significant increase in the pulmonary concentration of MT-I by the administration of PQ (Fig. l), and in MT synthesis in the

46

lung

produced

by a compound

other

than

heavy

metals.

Iijima

et al. [26] have

reported that induction of MT by lipopolysaccharides is mediated by a factor released from macrophages. This factor is different from the known primary inducers of MT, such as heavy metals, glucocorticoid hormones, interleukin-I and interferon. The mechanism of the increase in MT formation induced by PQ and the biological role of the increased MT are under investigation in our laboratory. In the liver of PQ-treated rats, MT-I concentration was significantly increased when compared to the controls (Fig. 4). Since Zn concentration also increased (Fig. 3), the increased MT may be Zn-bound MT. On the other hand, the increase in MT-I concentration was not observed in the kidney at day 1 after injection of PQ (Fig. 4). Although we did not measure PQ in any organ, Sharp et al. [20] reported that PQ was distributed in the greatest amount to lung, somewhat less to the kidney and least to the liver after a single injection of PQ. The difference in the distribution of PQ to liver, kidney and lung may not contribute to the difference in MT-I content among the three tissues in the PQ-treated rats. Further studies are required to elucidate the mechanism of increased MT accumulation in lung and liver after injection of PQ. ACKNOWLEDGEMENTS

The authors are grateful to Ms. D.K. Spencer for revising manuscript and to Ms. I. Kanno for manuscript typing.

the English

of this

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