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).

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.

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.

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.