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Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19

Influence of certain metals on the level of metallothionein‐like proteins in the liver and kidneys of rats a

J. K. Piotrowski & Jadwiga A. Szymańska

b

a

Department of Toxicology Chemistry, Medical Academy , Institute of Environmental Research and Bioanalysis , Narutowicza 120 a, Lódz, 90–145, Poland b

Department of Toxicology Chemistry, Medical Academy , Institute of Environmental Research and Bioanalysis , Lódz, Poland Published online: 19 Oct 2009.

To cite this article: J. K. Piotrowski & Jadwiga A. Szymańska (1976) Influence of certain metals on the level of metallothionein‐like proteins in the liver and kidneys of rats, Journal of Toxicology and Environmental Health: Current Issues, 1:6, 991-1002, DOI: 10.1080/15287397609529402 To link to this article: http://dx.doi.org/10.1080/15287397609529402

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INFLUENCE OF CERTAIN METALS ON THE LEVEL OF METALLOTHIONEIN-LIKE PROTEINS IN THE LIVER AND KIDNEYS OF RATS J. K. Piotrowski, Jadwiga A. Szymańska

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Department of Toxicology Chemistry, Institute of Environmental Research and Bioanalysis, Medical Academy, Lódź, Poland

Rats were given certain metal salts once every other day for six to eight doses and thereafter the levels of metollothionein-like proteins (MTP) were determined in their liver and kidneys. The normal level of those proteins ranged from 0.1 to 0.4 mg/g in the liver and 0.2 to 0.6 mg/g in the kidney. Beryllium, magnesium, barium, strontium, tin, arsenic, selenium, chromium, and nickel administration did not influence the tissue levels of MTP. There was a tendency toward increased MTP levels in the liver after the application of high doses of iron. A significant increase in MTP levels in the liver resulted from cobalt administration and in the kidneys of bismuth-treated rats. Applying molecular filtration it was shown that both metals were partially bound in vivo to protein fractions, the molecular weights of which are close to that of metallothionein.

INTRODUCTION The growing interest in metallothionein has resulted from the fact that this protein is responsible for the long-term cumulation of cadmium and inorganic mercury in the kidneys (Kagi and Vallee, 1960; Piotrowski et al., 1974a). These metals appear to increase the metallothionein levels (Piotrowski et al., 1974a, b; Piscator, 1964), presumably through induced de novo synthesis of this compound (Webb, 1972). Apart from cadmium and mercury, the stimulating influence on metallothionein synthesis has been found for zinc (Trojanowska and Piotrowski, 1974) and copper (Mogilnicka et al., 1975; Premakumar et al., 1974), although the identity of metallothionein and related proteins induced by the latter metal seems controversial (Winge et al., 1974). Not forejudging whether the proteins induced by various metals are identical with metallothionein, which they resemble in such features as molecular weight and high content of SH groups, the term "metallothionein-like proteins" (MTP) will be further used. Of the metals so far investigated, vanadium, manganese, and lead The work was supported by Polish-American research agreement 05-009-2 with National Institute for Occupational Safety and Health, Cincinnati, Ohio. Mrs. Maria Biala and Mrs. Barbara Orlowska participated in the screening experiments in the course of their diploma projects. Skillful technical assistance was given by Mrs. Wieslawa Mlodzianko. Requests for reprints should be sent to J. K. Piotrowski, Department of Toxicology Chemistry, Medical Academy, Narutowicza 120 a, 90-145 Lódź, Poland. 991 Journal of Toxicology and Environmental Health, 1:991-1002, 1976 Copyright © 1976 by Hemisphere Publishing Corporation

992

). K. P10TR0WSKI AND J.SZYMANSKA

did not show stimulating properties (Mogilnicka et al., 1975), although lead can be partially bound in the kidney by MTP, particularly in rats pretreated with cadmium (Bolanowska and Sapota, 1975). The purpose of this work was to determine whether other metals—such as Ni, Mg, As, Se, Bi, Co, Cr, Fe, Ba, Sr, and Sn—will also stimulate the biosynthesis of MTP. METHODS

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Animals Female Wistar white rats were used, aged 3-5 months and weighing 200-250 g. The animals were obtained from various suppliers and in the course of their breeding the intake of heavy metals from their food or their environment had not been controlled. Preceding and during the experiment the animals were housed in standard iron cages and fed standard LSM diet. 1 The series of screening experiments given below was performed during a 3-month period. Preliminary and supplementary experiments were conducted at various seasons of the year. Over 200 rats were used for the experiment. Exposure The animals were exposed to six to eight doses of the metal salts under investigation, injected in saline subcutaneously [Co(NO 3 ) 2 , NiSO 4 , FeCI 3 , Na 2 Se0 3 , NaAsO 3 , Be(NO 3 ) 2 ) MgSO 4 , BaCI 2 , BiCI 3 ] or intraperitoneally (CrCI 3 , SnCI 2 , SrCI 2 ) every 2 days. The doses were intended to correspond approximately to one-third to one-fifth LD S 0 . For some metals where literature data were insufficient, the doses were settled empirically in a preliminary experiment. Twenty-four hours following the last injection the animals were killed, their kidneys and liver removed, and the level of metallothionein determined. Six rats were used for each metal, and they were exposed in two separate series of three rats each. Simultaneously, control rats were saline injected by the respective route. Determination of Metallothionein A radiochemical method was applied for determination (Piotrowski et al., 1973). The procedure is based on tracing in vitro metallothionein contained in the homogenate using 203 HgCI 2 (700 jug Hg/g tissue), deproteinization by 10% trichloroacetic acid, and counting the activity of the supernatant. The technique of internal standards was used for calculation of results. 203 HgCI 2 (obtained from the Nuclear Research Institute, Swierk) was applied with specific activity in various series ranging from 1

Suppliers: Wytwórnia Pasz, Lowicz and Borowo, Poland.

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METALS AND METALL0THI0NEIN-L1KE PROTEINS

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300 to 100 mCi/g. Analytic solutions with the activity of about 2 X 10 5 counts/100 sec per sample were used. The standard metallothionein protein was obtained in this laboratory from the equine renal cortex using a technique close to that described by Pulido et al. (1966). The standard preparation contained 2.20 /Limol SH/mg protein determined by the amperometric titration method (Benesch et al., 1955); the molecular weight, determined by means of gel filtration on Sephadex G-75, was 10,000. Cadmium and zinc, determined colorimetrically following separation by means of thin-layer chromatography (Dutkiewicz et al., 1973) was 2.4% for Cd and 0.8% for Zn. The protein showed a typical uv absorption spectrum. Under conditions of the determination 1 mg standard protein bound 210 jug Hg. As pointed out by Sapota et al. (1974) for some animal tissues, the method appeared unspecific. Therefore, to make certain that the 2 0 3 H g tracer counted in the supernatant was bound to the fraction of metallothionein-like proteins, a spot control was performed using chromatography of the analytic supernatants on Sephadex G-75. Chromatographic Studies of Homogenates The chromatography of the supernatants from tissue homogenates performed on Sephadex G-75 was used to check if the metal inducing metallothionein in tissue can be bound to this protein in vivo. The supernatant (5-6 cm 3 ) was applied on the column (65 X 2 cm), eluted with ammonium formate buffer, pH = 8.0; the flow rate was 20 cm 3 /hr, and 5-cm 3 fractions were collected. In the eluates £ 2 8 o was measured, and SH groups were determined colorimetrically with HEDD, 2 according to Bitny-Szlachto (1972). Metals were determined in fractions pooled in intervals corresponding to the assumed molecular weights; solutions were mineralized with H 2 SO 4 and H N O 3 . Iron was determined colorimetrically with o-phenantroline (Kodama, 1963). The measurements were accomplished at 510 nm in 1-cm cuvettes. Under applied conditions the precision of the method amounted to ±6%. Cobalt was determined colorimetrically with the nitroso R-salt (Furman, 1963). Measurements were accomplished at 520 nm in 3-cm cuvettes; the precision of the method was ±4%. Bismuth was determined colorimetrically with zinc dibenzyldithiocarbamate (Jamane et al., 1972). Measurements were accomplished at 370 nm in 4-cm cuvettes. Copper, however, present in the same eluate fractions, interferes with this method for determining bismuth, the compound gives maximum absorption at 440 nm. Due to this fact, both the metals were determined simultaneously, by differentiating the wavelength and calculating the contents of both components from the system of two algebraic equations. The results of determination should be regarded as approximate. All the spectrophotometric measurements were made on the Zeiss VSU-2 apparatus. The radiochemical measurements were performed on the USB-2 scintillating counter. 2

β-Hydroxyethyl-2,4-dinitrophenyl disulfide.

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J. K. PIOTROWSKI AND J. SZYMANSKA

Sephadex gels were products of Pharmacia, Uppsala. RESULTS Repeated subcutaneous administration of metal salts resulted in progressive local necrotic changes of the skin; these changes were most pronounced with ferric chloride. Such changes were not observed in animals given metal salts intraperitoneally. Other toxic symptoms were not noted, and there was not any evident change in the appearance of internal organs.

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Screening Investigation As shown in Table 1, the exposure of animals to the majority of tested metals (Se, As, Be, Mg, Cr, Ba, Ni, Sr, Sn) did not seem to influence the level of MTP in either liver or kidney. Physiologic levels of those proteins differed for individual rats and ranged from 0.11 to 0.31 mg/g in the liver and from 0.20 to 0.58 mg/g in the kidney. The values obtained for the exposed rats were slightly higher and for individual TABLE 1. Level of Metallothionein-like Proteins in the Tissues of Rats Exposed to Metals Metallothionein (mg/g) Kidney

Liver Metal

(mg/kg)

Control"

Exposed

Control"

Exposed

0.14C (0.11-0.16)

0.16 (0.11-0.21) 0.14 (0.11-0.18) 0.30 (0.12-0.57) 0.28 (0.25-0.44) 0.23 (0.18-0.31) 0.22 (0.13-0.30) 0.25 (0.15-0.39) 0.31 (0.15-0.52) 0.29 (0.21-0.35)

0.23c (0.17-0.29)

0.27 (0.15-0.31) 0.27 (0.20-0.32) 0.31 (0.21-0.46) 0.38 (0.27-0.58) 0.23 (0.12-0.31) 0.38 (0.28-0.66) 0.57 (0.14-0.86) 0.48 (0.28-0.62) 0.55 (0.17-1.07)

As

2.5

0.12,0.12

Be

75.0

0.13,0.15

Mg

450.0

0.12,0.23

Cr

50.0

0.19,0.22

Ba

11.25

0.11,0.20

Ni

12.5

0.13,0.28

Sr

5.0

0.18,0.31

Sn

2.0

0.24,0.25

0.32,0.18 0.20, 0.32 0.35, 0.43 0.22,0.16 0.36, 0.48 0.20, 0.50 0.38, 0.58 0.48, 0.56

Individual results, random values in parentheses. Average values for six rats, random values in parentheses. c Mean from four rats.

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TABLE 2. Level of Metallothionein-like Proteins and of Metals in the Tissues of Rats Exposed to Iron, Cobalt, and Bismuth Metallothionein (mg/gj" Exposure

Liver

Kidney

0.23 0.40 0.38 0.53

±0.10 ±0.10 ±0.07 ±0.14

0.48 ±0.10 0.47 + 0.06 0.52 ±0.18 0.36 + 0.03

Control Co, 12.5 mg/kg Co, 25.0 mg/kg Co, 37.5 mg/kg

8 5 6 6 10 6 6 7

0.19 0.45 0.46 0.60

±0.08 ± 0.09* + 0.12b ± 0.226

0.44 0.43 0.27 0.38

Control Bi, 0.5 mg/kg Bi, 1.0 mg/kg Bi, 1.5 mg/kg Bi, 3.0 mg/kg

14 6 6 6 9

0.39 + 0.18

Control Fe, 100 mg/kg Fe, 150 mg/kg Fe, 200 mg/kg

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No. of animals

±0.09 ± 0.20 ± 0.06 ± 0.09

Metal level in tissue (ug/g)c 120, 154, 225, 434,

145 100 160 330

L L L L

Influence of certain metals on the level of metallothionein-like proteins in the liver and kidneys of rats.

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