Changes in Activity of Three Sulfurtransferases in Response to Exposure to Cadmium, Lead and Mercury Ions

Marta Kaczor-Kamińska, Piotr Sura, Maria Wróbel

Medical Biochemistry, Jagiellonian University Medical College, Kraków, Poland.

DOI: 10.4236/jep.2013.46A003                

Abstract

Cadmium, lead and mercury are environmentally persistent toxicants that affect tissues and cellular components or exert an effect on generation of reactive oxygen species causing a decreased level of available antioxidant reserves. Sulfurtransferases are enzymes that are widespread in nature. Rhodanese, 3-mercaptopyruvate sulfurtransferase and γ-cystathionase play an important role in the metabolism of L-cysteine. Heavy metal ions can bind to -SH groups of cysteine residues in their active sites and, therefore, decrease the activity of these enzymes and result in changes in the level of sulfane sulfur-containing compounds, products of L-cysteine desulfuration. Changes in the activity of sulfurtransferases were investigated in the kidneys, heart, brain, liver and skeletal muscle of Marsh frogs (Pelophylax ridibundus) after 10 days of exposure to Pb(NO₃)₂ at the concentration of 28 mg/L and CdCl₂ at the concentration of 40 mg or 80 mg/L, and in Xenopus laevies tissues after 7 and 14 days of exposure to HgCl₂ at the concentration of 1.353 mg/L. The investigated heavy metal ions have a tendency to inhibit the activity of sulfurtransferases and decrease the level of glutathione, what can result in oxidative stress and oxidation of cysteine -SH groups to -SOH. This reversible oxidation and reduction of these redox sensitive groups can play a role in defenses against oxidative stress. Based on the presented results, one can surmise that also the expression of the three sulfurtransferases depends on heavy metal ions and/or some parameters of oxidative stress, what can explain the increase of the activity of MPST and CST in the kidney.

Keywords

Sulfurtransferases; Heavy Metal Ions; Oxidative Stress

1. Introduction

Environmental contamination is a growing problem around the world. One of the most important global issues is chronic, low-level exposure to heavy metals. Cadmium, lead and mercury are toxicants that cause neurological, hepatological, reproductive and gastrointestinal pathologies [1-4]. There are three main reasons of heavy metal toxicity: 1) direct interactions with proteins due to their high affinities for thiol-, histydyl-, carboxyl-groups, influencing their structure, catalytic and transport functions in cells; 2) stimulated generation of reactive oxygen species (ROS) that modify the antioxidant defense and increase oxidative stress; 3) displacement of essential cations from specific binding sites, causing major disruption of their function [5]. Lead is chemically very similar to calcium and it competes with or mimics the action of calcium [6]. Lead in picomolar concentrations can compete for binding sites in the cerebellum for phosphokinase C, causing inhibition of cellular respiration and alterations of calcium-based reactions and neuronal signaling [7].

The cellular targets for metal toxicity include such organs as the kidney, liver, heart, testicles, as well as immune and nervous systems [8-12]. Cadmium, lead and mercury demonstrate multi-directional toxicity [13]. It is also known that several transition metals, such as zinc, iron, copper, cobalt and manganese participate in the control of various metabolic and signaling pathways. However, in excess, heavy metal ions can break down mechanisms guarding cellular homeostasis by binding to protein sites other than those tailored for that purpose or by displacement of other metals from their natural binding sites [14]. Cadmium, lead and mercury (sulfhydryl-reactive metals) are particularly insidious and can affect a vast array of biochemical and nutritional processes [6, 15].

Endogenous sulfur-containing compounds play an important role in numerous physiological processes in organisms, such as stabilization of protein structure, regulation of enzymatic activity, and they are engaged in redox reactions (glutathione, thioredoxine) [16]. There are two amino acids used in animals as a source of sulfur: methionine and cysteine. Cysteine is an intermediate for the synthesis of glutathione, taurine and sulfate [17]. Free sulfhydryl group present in cysteine are considered crucial for the biological functions of proteins [17]. Sulfurtransferases are enzymes widespread in nature. Rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1), 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) and γ-cystathionase (CST, cystathionine γ-lyase, EC 4.4.1.1) play an important role in the metabolism of L-cysteine [18]. Rhodanese is an enzyme, which is responsible for transfer of sulfane sulfur atoms (atoms of sulfur bound only to other sulfur atoms and so having an oxidation state 0 or −1) from various donors to acceptors. MPST and CST catalyze formation of sulfane sulfur-containing compounds from cysteine [18]. The catalytic activity of these enzymes depends on cysteine residues in their active sites [16]. Pollutants and xenobiotics can bind to −SH groups and, therefore, decrease the activity of enzymes and change the level of sulfane sulfur, a product of L-cysteine desulfuration. Thiol group of a redox active cysteine in the catalytic site of MPST and rhodanese may locally serve as an antioxidant. The sulfhydryl groups in the active site of the above-mentioned enzymes can bind heavy metal ions. Oxidation of these groups can inhibit the activity of the enzymes with redox-active cysteine in the active site, while reduction with thioredoxine or glutathione can recover the activity of these enzymes [16].

In biological systems, heavy metal ions mediate reactions in which ROS are produced (e.g. Fenton reaction) and in this way they are a direct cause of increased lipid peroxidation, modification of protein structure and protein functions and DNA damage [11,14]. The level of reactive oxygen species increases in the presence of heavy metal ions in tissues. The effect of heavy metal ions depends on the time of exposure and type of tissue [8,9, 11,12]. The aim of the paper is to present the effect of cadmium, lead and mercury on the activity of three sulfurtransferases containing −SH groups in their active sites, together with changes in sulfane sulfur, cysteine and glutathione levels in frog tissues.

2. Materials and Methods

2.1. Animals

Thirty nine mature frogs Pelophylax ridibundus of both sexes were collected in the vicinity of Krakow (southern Poland) and were placed for 1 week in plastic aquaria with dechlorinated tap water. The animals were kept at room temperature with a natural day/night rhythm. After acclimatization, they were used in two experiments. The frogs were divided into the control group—not exposed to heavy metal ions, and the experimental groups kept in water containing 40 mg or 80 mg of cadmium chloride per one liter of water for 96 h or 240 h, or in water containing lead nitrate Pb(NO₃)₂ at the concentration of 28 mg/L for 10 days. Water was changed every 24 h in order to keep a stable level of heavy metal ions. The frogs absorbed heavy metal ions from the contaminated water through their highly permeable skin.

Twenty-three mature frogs Xenopus laevis of both sexes obtained from private breeding were divided into three groups: the control group—kept in clean dechlorinated water for 7 or 14 days, the experimental groups— kept in water containing 1.353 mg mercury chloride per one liter of water for 7 days or for 14 days.

The licenses were obtained from the Local Ethics Commission (43/OP/2005) and the Polish Ministry of Environment to perform studies on a protected species (ref. No: DOPogiz-4200/II-06/5453/05/aj).

2.2. Tissue Collection

After a determined time of exposure, the frogs were decapitated and the spinal cord was pitched. For biochemical determinations, the brain, liver, heart, kidney and muscle from the thigh were excised. The tissues were washed out in cold saline, immediately frozen in liquid nitrogen and kept at −80˚C for further use. For analysis, the tissues were homogenized in four volumes of 0.1 M phosphate buffer (pH = 7.5) and centrifuged at 1600 g for 5 min. The supernatant was used for the determination of enzyme activities and sulfane sulfur level.

2.3. Methods

The MPST activity was assayed according to the method of Valentine and Frankenfeld (1974) [19] with some modifications described by Wróbel et al. (2004) [20]. The enzyme units were defined as nmols of pyruvate formed during 1 min incubation at 37˚C per 1 mg of protein. The rhodanese activity was assayed by the Sörbo method (1955) [21]. The assays were carried out according to the procedure described by Wróbel et al. (2004) [20]. The enzyme units were defined as μmoles of SCN^-, which formed during 1 min incubation at 20˚C per 1 mg of protein. The γ-cystathionase activity was determined according to Matsuo and Greenberg (1958) [22] with the modification described by Czubak et al. (2002) [23]. The activity of cystathionine was expressed as nmole of 2-ketobutyrate formed during 1 min incubation at 37˚C per 1 mg protein. Sulfane sulfur was determined by the method of Wood (1987) [24], based on cold cyanosis and colorimetric detection of ferric thiocyanate complex ion. The level of sulfane sulfur was expressed as nmole per 1 mg protein. Protein was determined by the method of Lowry et al. (1955) [25] using crystalline bovine serum albumin as a standard. The RP-HPLC method of Dominick et al. (2001) [26] with the modification described by Wróbel et al. (2009) [27] was used to determine the level of reduced (GSH) and oxidized form (GSSG) of glutathione, cysteine and cystine. Standard curves were generated in the supernatant obtained from tissue homogenates in the range from 13 to 75 nmol of each compound per ml.

Cadmium content was 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 based on 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 spectrum presented the number of counts for elements versus energy. The results were expressed as millimoles per kilogram of dry mass, the average value ± SD (standard deviation) for cryostat sections from each tissue. The mercury and lead content in a tissue sample was determined using XRF—X-ray fluorescence spectroscopy [28]. Events of characteristic energy for lead and mercury were counted. The results were compared to a standard curve for lead and mercury, respectively, and were expressed as milligram per kilogram of dry mass of tissue.

The statistical significance of differences between the experimental group and the controls were determined using the Student’s t-test. The differences were regarded as significant at p < 0.05.

3. Results and Discussion

3.1. Heavy Metal Accumulation in Frog Tissues

Figure 1 shows accumulation of heavy metal ions in the liver, kidney, brain and testicles of the frogs exposed to 80 mg CdCl₂/l (10 days), 28 mg Pb(NO₃)₂/l (10 days) and 1.353 mg HgCl₂/l (14 days) in water. It can be observed that cadmium had a tendency to accumulate mainly in the testicle and mercury in the kidney. In the experimental group (exposure to cadmium), the concentration of that toxicant in the testicle was 45 times higher as compared to the control group. In the kidney, after 14 days of exposure to mercury, the concentration of mercury had a value 98 times higher in comparison with the value obtained for the control group. Concentrations of lead in the liver, kidney, brain and testicle were 9.3; 9.1; 1.5 and 4.7 times higher in the experimental group in comparison with the controls. EDS method and XRF analysis confirmed heavy metal accumulation in the tissues of the investigated animals.

Conflicts of Interest

The authors declare no conflicts of interest.

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