Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues

Comparative Biochemistry and Physiology, Part C 142 (2006) 128 – 135 www.elsevier.com/locate/cbpc

Cadmium toxicity related to cysteine metabolism and glutathione levels in frog Rana ridibunda tissues Piotr Sura a , Natalia Ristic b , Patrycja Bronowicka b , Maria Wróbel b,⁎ a

Department of Human Developmental Biology, Collegium Medicum, Jagiellonian University, Kopernika 7, 31-034 Kraków, Poland b Institute of Medical Biochemistry, Collegium Medicum, Jagiellonian University, Kopernika 7, 31-034 Kraków, Poland Received 19 July 2005; received in revised form 16 November 2005; accepted 17 November 2005 Available online 27 December 2005

Abstract The level of glutathione and sulfane sulfur and sulfurtransferases activity in adult frogs Rana ridibunda were investigated after the exposure to 40 mg or 80 mg CdCl2 L− 1 for 96 h or 240 h. Cd accumulation in the liver, kidneys and testes was confirmed, and the highest Cd level was found in the testes. In the liver, the exposure to Cd resulted in an increase of GSH level and the activity of rhodanese, while the activity of 3mercaptopyruvate sulfurtransferase and cystathionase decreased. The kidneys and brain showed the elevated level of GSH and the activity of all investigated sulfurtransferases, as well as sulfane sulfur especially in brain. In such tissues as the testes, muscles and heart, the level of GSH and the activity of 3-mercaptopyruvate sulfurtransferase were significantly diminished. The increased level of sulfane sulfur was determined in the testes and muscles and the increased activity of rhodanese in the testes and the heart. These findings suggest the possible role of sulfane sulfur and/ or sulfurtransferases in the antioxidation processes, which can be generated in cells by cadmium. © 2005 Elsevier Inc. All rights reserved. Keywords: Cadmium; γ-Cystathionase; Glutathione; 3-Mercaptopyruvate sulfurtransferase; Rana ridibunda tissues; Rhodanese; Sulfane sulfur

1. Introduction There are two amino acids used in animals as a source of sulfur: methionine and cysteine (Scheme 1). Cysteine is one of substrates in the synthesis of glutathione—the most important cellular antioxidant. It may also undergo oxidative metabolism leading to sulfates and taurine (Bagley and Stipanuk, 1995) or non-oxidative desulfuration that produces compounds containing sulfane sulfur atoms (atoms of sulfur bound only to other sulfur atoms and so having an oxidation state 0 or − 1) (Drake et al., 1987; Toohey, 1989; Ogasawara et al., 1994, 1998). There are three sulfur-transferring enzymes on the desulfuration pathway. All of them contain sulfhydryl groups in their active site and these groups bind a transferred atom of sulfur forming persulfides in the case of rhodanese and 3-mercaptopyruvate sulfurtransferase (Nagahara and Nishino, 1996) or polysulfides in the case of γ-cystathionase (Yamanishi and Tuboi, 1981). Rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) and 3-

⁎ Corresponding author. Tel.: +48 12 424 72 29; fax: +48 12 422 32 72. E-mail address: [email protected] (M. Wróbel). 1532-0456/$ - see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2005.11.007

mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) are enzymes found in the majority of living organisms: plants, fungi, bacteria and animals (Westley, 1973). In animal cells, MPST is located in cytosol and mitochondria, while rhodanese is found mainly in mitochondria, but in lower vertebrates, such as amphibians, reptiles and fish, it is also present in cytosol (Dudek et al., 1980; Nagahara et al., 1998). Among tissues, their highest activity is found in the liver and kidney. Rhodanese transfers the sulfur atom from various donors (sulfane sulfur containing compounds) to various thiophilic acceptors (Scheme 1). MPST catalyses the transfer of sulfur atom from 3mercaptopyruvate (the single donor) to various acceptors, which often produce sulfane sulfur-containing compounds (Westley et al., 1983). γ-Cystathionase (cystathionine γ-lyase, CST, EC 4.4.1.1) is an enzyme found in cytosol of eukaryotic cells, with its highest activity in the liver and kidney (Ogasawara et al., 1994). CST plays a role in the pathway of cysteine synthesis from methionine and–what is more important–is involved in sulfane sulfur generation in cells (Scheme 1) (Szczepkowski and Wood, 1967). At least five ways requiring sulfane sulfur have been identified so far. In these processes, the atom of sulfur is transferred

P. Sura et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 128–135

Methionine

GSH

129

Protein

L-Cysteine

Cys-S-S-Cys CYSTATHIONASE

AMINOTRANSFRASE

COOC=O

Pyruvate + NH 4

CH2SH 3-Mercaptopyruvate

+

Sulfane Sulfur

Cys-S-SH Thiocysteine

2-

SO3

Albumin-S-SH MPST

2-

Protein

Cystine

Cys-S-S-S-Cys Thiocystine

S=SO3 Thiosulfate Pyruvate

So

RHODANESE

Protein(Fe-S)n Protein-S-SH

CN SCN

-

-

Scheme 1. L-Cysteine desulfuration pathways.

by sulfurtransferases to CN− to detoxify it (Frankenberg, 1980; Wing et al., 1992; Baskin et al., 1992,1999; Porter et al., 1996) (Scheme 1). Furthermore, sulfurtransferases are enzymes that incorporate atoms of sulfur into clusters (Fe–S)n (Scheme 1) and so they are indispensable in the synthesis or repair of iron– sulfur proteins (Ogasawara et al., 1995; Bonomi et al., 1985; Taniguchi and Kimura, 1974; Tse Sum Bui et al., 2000). Then, it has been demonstrated that sulfur may be transferred to the tRNA nucleotides by MPST and rhodanese. Such a modification is probably important in the translation process (Wong et al., 1975; Palenchar et al., 2000). What is more, MPST and CST transfer sulfane sulfur atoms to –SH groups in the active sites of many enzymes to regulate their activity (Yamanishi et al., 1983). And finally, sulfane sulfur may play a role as an antioxidant. It has been shown that some sulfane sulfur-containing compounds cause the inhibition of cytochrome P-450, crucial in lipid peroxidation, generating reactive oxygen species (ROS) and converting xenobiotics to radical species (Ogasawara et al., 1997, 1998, 1999). Cadmium is an element very similar to Zn and is easily absorbed by animals. Cd is stored in organs in the form of complex with metallothioneins containing numerous –SH groups. Cd has been demonstrated to exert numerous toxic effects: it induces free radical processes and inhibits some enzymes, especially these containing –SH group in the active site. Cd also alters the oogenesis and the morphology of oocytes in Xenopus laevis (Lienesch et al., 2000), but has no effect on the gonadogenesis in Pleurodeles waltl (Flament et al., 2003). In anuran amphibian testes, Cd causes androgen synthesis inhibition (Gosh et al., 1987) and a significant decrease in secondary spermatogonial and spermatocyte stages (Kasinathan et al., 1987). Frogs subjected to Cd, also show a loss of balance, respiratory problems and slowness in motion and increased mucus secretion (Selvi et al., 2003). Environmental contamination with cadmium has significantly increased

because of its extensive use in anthropogenic activities. Low levels of Cd have little effect on animals, but, after a critical concentration level has been reached, cadmium becomes highly toxic due to the tendency to accumulate in tissues. Declines and losses of amphibian populations are a global problem. Environmental toxicants act directly to kill animals, or indirectly by impairing reproduction, retarding growth rates, disrupting normal development or increasing susceptibility to diseases by immunosuppression or inhibition of immune system development (Carey and Bryant, 1995; Carey et al., 1999; Alford and Richards, 1999; Loumbourdis et al., 1999). In some Rana frogs, and presumably also in other amphibians, the metabolism of sulfane sulfur and the level of glutathione may be crucial for survival and reproduction. Sulfurtransferases activity and sulfane sulfur levels in frog tissues are dependent on the season of the year and change adequately to the changing organism requirements (Wróbel et al., 1992; Wróbel and Frendo, 1993). It is supposed that, in frog liver and gonads, sulfane sulfur compounds are involved in the synthesis of proteins and in frog kidney in the protection against cellular oxidative stress, similarly as in the brain (Wróbel et al., 2000b). The enzymes related to the sulfane sulfur metabolism, MPST, CST and rhodanese, have –SH group in their active sites. Cd binds to this group and therefore may influences the activity of these enzymes and simultaneously the entire non-oxidative metabolism of Cys. The aim of this work was to investigate to what degree the exposure to Cd (40 mg or 80 mg CdCl2 L− 1 water in which the animals were kept for 96 or 240 h) influenced the activity of sulfurtransferases and the level of sulfane sulfurcontaining compounds and GSH in frog tissues (liver, kidneys, brain, testes, heart and skeletal muscle). The content of Cd and sulfur in the liver, kidneys and testes were also assayed.

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2. Materials and methods Eighteen mature frogs (Rana ridibunda) of both sexes were collected in October in the vicinity of Kraków (southern Poland) and were placed for 1 week in plastic aquaria with dechlorinated tap water. The animals were kept in room temperature with a natural day/night rhythm. After acclimatization, frogs were divided into three groups: control group (four animals, both sexes), experimental group I—water containing 40 mg CdCl2 L− 1 for 96 h (IA—three animals of both sexes) or 240 h (IB—four animals of both sexes) and experimental group II—water containing 80 mg CdCl2 L− 1 for 96 h (IIA—three animals of both sexes) or 240 h (IIB—four animals of both sexes). Water was changed every 24 h to keep a stable concentration of Cd. At the end of the experiment, all the animals were in good physical condition. After 96 h or 240 h, they were decapitated and the spinal cord was pitched. For biochemical determinations, excised livers, kidneys, brain, testes, heart and striated muscle from the thigh were washed with cold saline and homogenized in four volumes of 0.1 M phosphate buffer pH 7.5. The MPST activity was assayed according to the method of Valentine and Frankenfeld (1974). The assays were carried out according to the procedure described by Wróbel et al. (2004). The enzyme units were defined as nanomoles of pyruvate formed during 1 min incubation at 37 °C/1 mg of protein. Rhodanese was assayed according to Sörbo (1955). The assays were carried out according to the procedure described by Wróbel et al. (2004). The enzyme units were defined as nanomoles of SCN− formed during 1 min incubation at 20 °C/1 mg of protein. The γ-cystathionase activity was determined according to Matsuo and Greenberg (1958) with the modification described by Czubak et al. (2002), using cystathionine as a substrate. The activity of cystathionase was expressed as nanomoles of 2-ketobutyrate formed during 1 min incubation at 37 °C/1 mg of protein. Sulfane sulfur was determined by the method of Wood (1987), based on cold cyanolysis and colorimetric detection of ferric thiocyanate complex ion. Determinations of GSH were performed according to Tietze (1969). Protein was determined by the method of Lowry et al. (1951) using crystalline bovine serum albumin as a standard. Cd and sulfur content were determined in 30 μm thick cryostat sections, lyophilized in the Edwards apparatus following mounting and subsequent covering with a carbon powder layer. The content of the element was calculated on the basis of the EDS spectrum (energy dispersion spectrum) obtained by a JED JSH 5410 scanning microscope at the 20 kV voltage and using an EDS detector Voyager 3100 manufactured by Noran. EDS spectrums presented the number of counts for elements versus energy. The results were expressed as millimoles per kilogram of dry mass, the average value ± S.D. (standard deviation) for 15 cryostat sections from each tissue. Sodium 3-mercaptopyruvate, sodium sulfite, N-ethylmaleimide, dithiothreitol, NADH, NADPH, lactate dehydrogenase (EC 1.1.1.27) from pig heart, glutathione reductase, sodium thiosulfate, cystathionine, 5,5′-dithiobis-(2-nitrobenzoic acid) and pyridoxal phosphate were obtained from Sigma Chemical

Co., St. Louis, MO, U.S.A. Potassium cyanide originated from Merck and 2-mercaptoethanol, EDTA-Na2·2H2O from Fluka AG. The statistical significance of differences between groups I and II and the controls were determined using the Student's ttest. The differences were regarded as significant at p b 0.05. 3. Results Table 1 shows Cd accumulation in the liver, kidneys and testes of frogs exposed to 80 mg CdCl2 L−1 in water. It should be noted that it accumulated especially in the testes; after 96 h (IIA), the detected Cd content was 16 times higher in comparison to the control group, while after 240 h (IIB) it increased 45 times. At the same time, the total sulfur content (Fig. 1) in the testes was also significantly higher in both groups, while in the liver it did not change significantly and in the kidneys the increase was observed only in the IIA group. As it can be seen in Table 2, Cd induces an increase of rhodanese activity in the liver. After 96 h of exposure to Cd, the activity of this enzyme shows the value of 160% of the level observed in the controls. The activity of MPST does not change in group I, while in group II a quite marked decrease of activity is observed (down to 80% of the control value). The CST activity is diminished in both groups; the effect is more remarkable in group II, in which the activity of this enzyme drops even by 40% in comparison to the control value. The level of GSH increases in both groups by 60% (group IA and B) or even by 80% (group IIA and B) of the control value. The level of sulfane sulfur-containing compounds does not change for the lower dose of Cd, while in group II it initially decreases to get back to the control value after 240 h. As it follows from Table 3, in the kidneys, the activity of rhodanese in both experimental groups is higher by about 30% as compared to the control value. Similarly, Cd augments the activity of MPST. CST activity also increases in result of exposure to Cd in both experimental groups, but only after 240 h—in group IB, this increase is almost doubled in comparison to the control value. For the concentration of Cd equal 40 mg L− 1, the level of GSH increases almost three times after 96 h, but after 240 h it is only two times higher than in the control group (Table 3). When the concentration of Cd is 80 mg L− 1, the level of GSH increases in a time-dependent manner— after 96 h, it is about 2.5 times higher than in the controls and, after 240 h, it increases more than three times in comparison to Table 1 Cd content in the liver, kidney and testes of frog exposed to 80 mg L− 1 of CdCl2 in water after 96 h (IIA) and 240 h (IIB) exposure Group

Liver

Kidneys

Testes

mg of Cd kg− 1 dry mass Control IIA IIB

120 (24) 180 (24) 200 (32)

200 (24) 220 (16) 290 (24)

10 (8) 160 (8) 450 (8)

Each value is the average of the number of analyses shown in brackets. S.D. was not presented because observed large differences between values, which was dependent on the analyzed region of a section.

% of control

P. Sura et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 128–135 96h

200 180 160 140 120 100 80 60 40 20 0

240h

Table 3 Cd effect on sulfurtransferases activity and the level of GSH and sulfane sulfur in Rana ridibunda kidneys Group

r

ne

id

K

Control IA IB IIA IIB

s

y

ve

Li

131

ste

Te

Fig. 1. Sulfur content in the liver, kidneys and testes of frog exposed to 80 mg/L of CdCl2 in water after 96 h (IIA) and 240 h (IIB). Control values are: 218 ± 29 mmol kg− 1 dry mass in liver, 219 ± 23 in kidneys and 117 ± 12 in testes.

nmol product mg− 1 protein min− 1

nmol mg− 1 protein

Rodanese

MPST

CST

Glutathione

Sulfane sulfur

2860 ± 40 3760 ± 240⁎⁎ 3520 ± 230⁎⁎ 3710 ± 80⁎⁎ 3730 ± 40⁎⁎

799 ± 18 1035 ± 19⁎⁎ 1020 ± 37⁎⁎ 1088 ± 62⁎⁎ 1078 ± 50⁎⁎

1.7 ± 0.5 2.3 ± 0.5 3.7 ± 0.4⁎⁎ 2.0 ± 0.2 2.8 ± 0.1⁎⁎

0.29 ± 0.01 1.04 ± 0.02⁎⁎ 0.69 ± 0.03⁎⁎ 0.75 ± 0.05⁎⁎ 0.92 ± 0.02⁎⁎

185 ± 27 216 ± 13⁎ 172 ± 17 172 ± 21 212 ± 20

⁎ p b 0.05. ⁎⁎ p b 0.001.

the control value. Sulfane sulfur levels do not change significantly—an increase is detected only in the IA group. Table 4 illustrates changes caused by Cd in frog brain. The activity of rhodanese increases by about 20% or even 40% after the exposure to Cd in the concentration of 80 mg L− 1 for 240 h in comparison to the control value. The MPST activity increases by about 20% and 30% in group I and group II, respectively. The level of GSH in group I reaches 130% of the control value after 240 h and, in group II, it amounts to 120% of the control level as early as after 96 h. The levels of sulfane sulfur increase in both groups—this effect is more clear when the concentration of Cd is higher (up to 130% of the control value). Changes resulting from Cd influence in frog testes are demonstrated in Table 5. The activity of rhodanese for lower concentration of Cd in water firstly increases (by 20% in comparison to the control value) and then decreases below the control level (by 20%). For the higher Cd concentration, the rhodanese activity increases in a time-dependent manner to reach almost the double value of the controls after 240 h. The MPST activity increases in both groups by about 20% after 96 h, to undergo a decrease after 240 h—in group II even by 25% below the control value. The level of GSH is diminished by about 10% in both groups after 96 h, but in group I it grows above the control value after 240 h. The level of sulfane sulfur increases after 96 h (in a dosedependent manner) and, after 240 h, it returns to the control value.

In the muscles, as it is shown in Table 6, the activity of rhodanese decreases significantly after the exposure to Cd in the concentration of 40 mg L− 1 already after 96 h and, when the Cd concentration is higher, a decrease of rhodanese activity is observed only after 240 h. In both groups, the activity of rhodanese after 240 h is about 70% of the control value. The MPST activity does not change significantly if the concentration of Cd is 40 mg L− 1, but, with the higher concentration value, the activity of this enzyme decreases after 96 h by about 40% in comparison to the controls, although it is elevated again after a longer time. The level of GSH decreases in a time and dose-dependent manner (down to the 50% of the control in the case of the higher Cd concentration and a longer time of exposure). Sulfane sulfur level initially increases (by 30% in group IA) and then it decreases, with the level still maintained above the control value. In the heart, as it follows from Table 7, the activity of rhodanese does not change after 96 h in group I and increases by about 30% in group II. After 240 h, the rhodanese activity in both groups is slightly higher than the control value. The MPST activity decreases in both groups—in group I, it is lower by 15% as compared to the controls as early as after 96 h and, in group II, the effect is even more significant, although observed only after 240 h. The levels of GSH initially grow slightly in both groups, but after 240 h they decrease below the control level—for the higher concentration of Cd by 30%. Sulfane sulfur levels drop after Cd exposure—in group I, as soon as after 96 h (by about 30%), while in group II after 240 h, with the effect being less remarkable (a decrease by about 20% in comparison to the control value).

Table 2 Cd effect on sulfurtransferases activity and the level of GSH and sulfane sulfur in the liver of Rana ridibunda

Table 4 Cd effect on sulfurtransferases activity and the level of GSH and sulfane sulfur in Rana ridibunda brain

Group

Control IA IB IIA IIB

nmol product mg− 1 protein min− 1

nmol mg− 1 protein

Rhodanese

MPST

CST

Glutathione

Sulfane sulfur

1580 ± 150 2480 ± 280⁎⁎ 1840 ± 150⁎⁎ 2430 ± 60⁎⁎ 2190 ± 500⁎⁎

903 ± 202 859 ± 103 832 ± 92 699 ± 106⁎⁎ 752 ± 186⁎

1.4 ± 0.3 1.1 ± 0.2⁎ 1.0 ± 0.2⁎ 0.9 ± 0.2⁎ 0.9 ± 0.1⁎⁎

3.7 ± 0.8 6.0 ± 2.0⁎ 6.1 ± 0.9⁎⁎ 6.7 ± 1.3⁎⁎ 6.7 ± 1.6⁎⁎

308 ± 72 303 ± 40 340 ± 84 269 ± 31⁎ 313 ± 55

⁎ p b 0.05. ⁎⁎ p b 0.001.

Group

nmol product mg− 1 protein min− 1 Rhodanese

MPST

Glutathione

Sulfane sulfur

Control IA IB IIA IIB

580 ± 20 690 ± 40⁎⁎ 680 ± 30⁎⁎ 710 ± 10⁎⁎ 820 ± 20⁎⁎

300 ± 13 353 ± 12⁎⁎ 368 ± 10⁎⁎ 390 ± 20⁎⁎ 390 ± 9⁎⁎

2.19 ± 0.18 2.50 ± 0.34 2.87 ± 0.18⁎⁎ 2.76 ± 0.14⁎⁎ 2.56 ± 0.33⁎

191 ± 11 237 ± 8⁎⁎ 234 ± 7⁎⁎ 253 ± 19⁎⁎ 245 ± 18⁎⁎

⁎ p b 0.05. ⁎⁎ p b 0.001.

nmol mg− 1 protein

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Table 5 Cd effect on sulfurtransferases activity and the level of GSH and sulfane sulfur in Rana ridibunda testes Group

Control IA IB IIA IIB

nmol product mg− 1 protein min− 1

nmol mg− 1 protein

Rhodanese

MPST

Glutathione

Sulfane sulfur

100 ± 1 120 ± 1⁎⁎ 80 ± 1⁎⁎ 130 ± 1⁎⁎ 190 ± 1⁎⁎

357 ± 21 443 ± 14⁎⁎ 353 ± 24 415 ± 11⁎⁎ 268 ± 10⁎⁎

8.16 ± 0.15 6.88 ± 0.55⁎⁎ 9.07 ± 0.56* 7.54 ± 0.18⁎⁎ 7.48 ± 0.24⁎⁎

244 ± 9 269 ± 16⁎ 237 ± 20 300 ± 17⁎⁎ 265 ± 14⁎

Table 7 Cd effect on sulfurtransferases activity and the level of GSH and sulfane sulfur in Rana ridibunda heart Group

Control IA IB IIA IIB

nmol product mg− 1 protein min− 1

nmol mg− 1 protein

Rhodanese

MPST

Glutathione

Sulfane sulfur

280 ± 10 300 ± 30 300 ± 10⁎ 360 ± 30⁎⁎ 300 ± 10⁎

636 ± 42 549 ± 29⁎ 544 ± 14⁎ 666 ± 38 490 ± 27⁎⁎

2.54 ± 0.02 2.69 ± 0.08⁎ 2.47 ± 0.07⁎ 2.71 ± 0.32 1.81 ± 0.05⁎⁎

148 ± 5 104 ± 7⁎⁎ 98 ± 6⁎⁎ 141 ± 21 122 ± 7⁎⁎

⁎ p b 0.05. ⁎⁎ p b 0.001.

⁎ p b 0.05. ⁎⁎ p b 0.001.

On the basis of the investigations carried out in the control group of R. ridibunda, there may be determined a pattern of tissue distribution of the examined parameters (Tables 2–7). It can be seen that the kidneys display the highest activity of rhodanese; in the liver it is almost two times lower and in other tissues it is 6% to 37% of the activity assayed for the liver, with the lowest value in the testes. The MPST activity is the highest in the liver. In the kidneys and heart, the activity of this enzyme is, respectively, 88% and 70% of the value assayed for the liver, while in the testes, brain and skeletal muscles, it is only 39% to 21% of this value, respectively. The activity of CST has been assayed in all the tissues, but only in the liver and kidneys, where it was quite similar, has this activity been detected. The highest level of sulfane sulfur-containing compounds has been assayed in the liver and, what is interesting, in the testes (80% of the value for the liver). In the brain, kidneys and heart, the content of these compounds is, respectively, 62%, 60% and 48% of the value assayed for the liver and, in the muscle, only 21%. The level of GSH is the highest in the testes (Table 5). In the liver, heart and brain, it is significantly lower, respectively, 46%, 31% and 27% of the value determined for the testes. The minimal level of GSH is observed in the muscle (13%) and, above all, in the kidneys, only 3% in comparison to the highest value assayed in the testes.

kidneys; this fact is quite obvious if we consider that sulfane sulfur metabolism is important in detoxification processes carried out just in these organs. The influence of Cd on desulfuration of L-cysteine in frog tissues has been examined for the first time. Earlier investigations suggest that this metabolic pathway may be crucial for reproducing and survival of these animals, as they are related to antioxidative and detoxication processes, as well as to the synthesis of some proteins, such as iron–sulfur proteins and other, necessary to produce gametes (Wróbel et al., 2000a; Wróbel, 2001; Wróbel and Papla, 2000). Cadmium may highly influence these processes as it has been demonstrated in other experiments (Yamano et al., 1998). In R. ridibunda, Cd accumulation in the liver and kidneys is very high and appears to be time-dependent (Vogiatzis and Loumbourdis, 1998). These authors found that at the fourth day of exposing the frogs to 200 ppm of CdCl2 dissolved in water, the experiment resulted in a 180-fold increase in Cd concentration in the liver and a slower, 10-fold increase in the kidneys compared to control values. At the 30th day of the exposure, the rate of accumulation in the kidneys was higher than the rate of accumulation in the liver compared to the values obtained after 10 days of exposure. The method employed in our work (EDS) confirms that also this element accumulated in these organs and, to an even much greater degree, in the testes (Table 1). The liver is the main site where the synthesis of GSH from cysteine occurs and where from this compound is transported with blood to other tissues (Kaplowitz et al., 1985). As it follows from Table 2, in the liver, Cd causes an increase of GSH level and a decrease of MPST and CST activity. It suggests that Cys is utilized for GSH synthesis and, for that reason, it lacks sulfurtransferases activity. Cysteine may also be utilized to synthesize metallothionein (Mt). There are two possible reasons for an increased GSH synthesis in Cd presence. Firstly, complexing Cd by GSH is one of the first defensive reactions of organism before the synthesis of Mt can be properly enhanced (Bruggeman et al., 1992). Secondly, Cd induces oxidative processes in cells and GSH is the most efficacious antioxidant (Wang et al., 2004). As the liver quite easily gets rid of Cd, an enhanced GSH synthesis in this organ is presumably related to its production to utilize in other tissues attacked by Cd. Also the rhodanese activity is increased in the liver after Cd exposure. It is quite hard to tell which function of this enzyme is used in the defense against this element, but we

4. Discussion This work confirms the tissue distribution of sulfurtransferases and sulfane sulfur-containing compounds in frog tissues (Wróbel et al., 2000a,b) (Tables 2–7: control group). A high activity of sulfurtransferases is observed mainly in the liver and Table 6 Cd effect on sulfurtransferases activity and the level of GSH and sulfane sulfur in Rana ridibunda skeletal muscles Group

nmol product mg− 1 protein min− 1 Rhodanese

MPST

Glutathione

Sulfane sulfur

Control IA IB IIA IIB

360 ± 10 230 ± 10⁎⁎ 260 ± 10⁎⁎ 350 ± 10 260 ± 10⁎⁎

191 ± 16 205 ± 10 183 ± 14 112 ± 18⁎⁎ 165 ± 28⁎

1.07 ± 0.03 0.93 ± 0.13⁎ 0.59 ± 0.02⁎⁎ 0.56 ± 0.01⁎⁎ 0.49 ± 0.02⁎⁎

66 ± 6 86 ± 7⁎⁎ 78 ± 4⁎⁎ 79 ± 7⁎ 73 ± 7⁎

⁎ p b 0.05. ⁎⁎ p b 0.001.

nmol mg− 1 protein

P. Sura et al. / Comparative Biochemistry and Physiology, Part C 142 (2006) 128–135

can suppose that rhodanese may catalyze reactions reconstructing –SH groups (they bind Cd and are reductive), following the reaction: RSS− + SO32−→S2O32− + RSH (Westley, 1980). In the kidneys, after the exposure to Cd, an increase of sulfurtransferases activity is observed, as well as a high (even three-fold) increase of GSH level, while the sulfane sulfur level does not change significantly (Table 3). It is known that the kidney exhibits a high activity of γ-glutamyltranspeptidase (GGTP), the enzyme hydrolyzing GSH with the release of CysGly, and cysteinylglycine dipeptydase, the enzyme hydrolyzing this dipeptide to amino acids. What is more, kidney cells can transport GSH from the serum via the mechanism coupled with Na+ transport (Kaplowitz et al., 1985; Dringen et al., 2000). With such systems, the kidneys have practically an unlimited access to GSH and Cys in the serum. The fact that Cd causes such an increase in GSH level in the kidney is probably related to defense against oxidative processes induced by Cd. The uptaken GSH may also serve as a source of Cys for Mt synthesis or bind Cd itself. On the other hand, Cys is the direct precursor of substrates for rhodanese, MPST and CST and the activity of these enzymes has increased (Table 3). The brain, like the kidneys, exhibits a high activity of GGTP and cysteinylglycine dipeptydase, so it may utilize a needed amount of GSH and Cys from the serum (Dringen et al., 2000; Schulz et al., 2000). This possibility is obvious as the brain,

being an organ with intensive oxygen metabolism, must have a proper defence system against ROS. Cd causes in the brain an increase of GSH and sulfane sulfur levels, and also an increase of rhodanese and MPST activity (Table 4). The increase of GSH level may be a reaction to the enhanced–under Cd influence– production of free radicals or peroxides. Also the elevated activity of sulfurtransferases is supposed to be, like in the kidneys, related to the processes protecting brain cells from oxidative stress. In the situation of enhanced radical forms generation, limiting of their production would be desired and here sulfane sulfur may play a role, as it has been already shown that these compounds have antioxidative properties (Ogasawara et al., 1998). Antioxidative processes are a crucial problem in the brain because this organ, with its high content of lipids with unsaturated fatty acids (Dringen et al., 2000), is very sensitive to oxidative processes and the oxidative stress is the most probable cause of neurodegenerative processes (Rassin, 1996; Schulz et al., 2000). The GSH level decreased in the testes after 96 h when Cd concentration was 40 mg L− 1, but after 240 h it was higher than the control level (Table 5). Presumably, at first, GSH stored in the testes is utilized to bind Cd, and then the accumulating Cd stimulates the GSH synthesis causing its level to increase. When Cd concentration is 80 mg L− 1, the situation is quite different—the level of GSH is diminished already after 96 h and after 240 h, as well. Initially, the MPST activity and the level of

Liver GSH synthesis

GGTP

Kidney Brain

Testes - muscle - heart low Cys levels GSH

high Cys levels MPST

and

GSH

muscle R

R

R

MSPT

CST(kidney)

SS

Testes R

SS(brain)

SS

Heart

SS

133

Anti-oxidative mechanisms are stimulated in kidney and brain

Toxic Cd activity involves ROS generation CST -γ-cystathionase GGTP -γ-glutamyltranspeptidase GSH – glutathione MPST – 3-mercaptopyruvate sulfurtransferase R – rhodanese ROS – reactive oxygen species SS – sulfane sulfur Scheme 2. Cadmium effect on anaerobic conversions of cysteine in R. ridibunda tissues.

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sulfane sulfur increase, but, after 240 h, these parameters diminish. All these observations suggest that Cys taken by the testes is firstly utilized in the synthesis of sulfane sulfur, and at the same time, GSH is used—everything to eradicate the results of oxidative stress induced by Cd. After a longer time, most likely the maintenance of proper GSH level (to enable gametes production) appears to be more important and Cys is used to synthesize it—then the activity of MPST decreases. A higher Cd concentration induces rhodanese activity, which may be, like in other tissues, involved in antioxidative processes. There is scant information about GSH and sulfane sulfur metabolism in the heart and skeletal muscles; so far it has been shown that the heart has a small quantity of GSH and that possibilities of reproducing it from its oxidized form (GSSG) are low (Kaplowitz et al., 1985). After Cd exposure, in both muscles, the level of GSH has decreased—this effect is dose and time-dependent (Tables 6 and 7). In the heart, it is clear only after a large dose and longer time of exposure to Cd—it suggests that quite an effective antioxidant mechanisms exists in this organ, which is obvious in view of the fact that the heart has a completely oxygen-dependent metabolism. In the heart, the activity of MPST has decreased and, in consequence, also the level of sulfane sulfur has dropped. In the skeletal muscle, the MPST activity decreases only at first, to return to the control level, and sulfane sulfur level increases. These differences suggest that the skeletal muscle utilizes uptaken Cys to synthesize sulfane sulfur, while the heart uses it to produce GSH, the loss of which in the heart is slower. Producing GSH is probably a better way to deal with oxidative stress, but if the skeletal muscle has a poor possibility of synthesizing it, then the synthesis of sulfane sulfur (possibly also antioxidative) seems to be reasonable. In this work, we have assayed for the first time the sulfurtransferases activity and sulfane sulfur in the skeletal muscle of the frog (Table 6). It appears that, in the muscle, the activity of MPST is low (the lowest of all the examined tissues) and the activity of CST equals practically zero. Both enzymes participate in sulfane sulfur generation, so its amount is in consequence also very low in this tissue. It seems, then, that sulfane sulfur metabolism in the skeletal muscle is minimal. It is probably related to the fact that this tissue contains Cd below the detection limits (Vogiatzis and Loumbourdis, 1997) and is much less involved in detoxification processes than other examined tissues and also it is not absolutely dependent on oxygen metabolism. The same reasons may be a cause of quite low level of GSH in the muscle (only 30% of the value assayed for the liver). Scheme 2 summarizes the results of this work. In the liver, Cd induces mainly the synthesis of glutathione, which can be transported by blood to other tissues. The kidneys and brain are able to enhance GSH and sulfane sulfur levels, as well as the activity of sulfurtransferases, defending themselves against Cd. The other organs, after the exposure to Cd have to decrease the GSH level and MPST activity. What is increased is the level of sulfane sulfur (testes, skeletal muscle) or the activity of rhodanese (testes, heart). On the basis of all the findings resulting from the present work, we can conclude that the main mecha-

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