Renal changes and apoptosis caused by subacute exposure to Aroclor 1254 in selenium-deficient and selenium-supplemented rats

All chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). A1254 (analytical standard) was obtained from the German, Manheim branch of Sigma-Aldrich. Assay kits for measuring total antioxidant capacity (TAOC), thiobarbituric acid reactive substances (TBARS), protein carbonyls (colorimetric), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase 1 (GPx1) were supplied by Cayman Chemical Company (Ann Arbor, MI, USA).

The study included 36 three-week-old male Sprague-Dawley rats purchased from the Experimental Animals Production Centre of Hacettepe University (Ankara, Turkey). All experimental procedures and animal use were approved by the Animals Ethics Committee of Hacettepe University. The experiments followed the regulations of the Ministry of Food, Agriculture, and Livestock and Hacettepe University Animal Ethics Committee (30, 31).

The animals had free access to food and water during the five-week experiment and were randomly divided in six groups of six. The first, control group was receiving a regular rat chow containing 0.15 mg/kg of Se. The second was receiving rat chow supplemented with 1 mg/kg of Se (SeS group). The third group was receiving Se-deficient rat chow (≤0.05 mg/kg of Se) (SeD group). The fourth group was receiving regular rat chow with 0.15 mg/kg of Se and 10 mg/kg of Aroclor 1254 by gavage for the last 15 days of feeding (A group). The fifth group was receiving Se-supplemented rat chow (1 mg/kg Se) and 10 mg/kg of A1254 by gavage for the last 15 days of feeding (ASeS group). The sixth group was receiving Se-deficient rat chow (≤0.05 mg/kg of Se) and 10 mg/kg of A1254 by gavage for the last 15 days of feeding (ASeD group).

The A1254 dose of 10 mg/kg/day corresponded to 1 % of median lethal oral dose (LD₅₀) of 1010 mg/kg (5) and was diluted in 1 mL of corn oil as vehicle. Corn oil was also given by gavage to the control, SeS, and SeD groups over the last 15 days of feeding.

Control, Se-deficient, and Se-supplemented diets were obtained from the Scientific Animal Food and Engineering (SAFE) laboratories (Augy, France) and contained sodium selenite as a source of Se. Se doses in both supplemented and deficient chow were based on recent studies (32, 33, 34), Sundae (34) in particular, which has showed that 0.05 mg/kg bw of Se ensures 50 % of normal GPx1 activity, while 0.15 mg/kg ensures normal activity. Diet containing 1 mg/kg was evidenced to provide supraphysiological doses of Se (23, 34).

The animals were decapitated under total anaesthesia 24 h after having received the last dose of A1254, and their kidneys removed and weighed. The right kidney was prepared for histopathological analysis and apoptotic cell count, whereas the left kidney was frozen at -80 °C until tissue homogenisation for oxidant/antioxidant parameter analysis.

Samples of one half of the right kidney were fixed in 2.5 % glutaraldehyde solution for 4 h and post-fixed in 1 % osmium-tetroxide solution in 0.1 mol/L phosphate-buffer for 1 h. They were then dehydrated in a graded series of ethanol and embedded in Araldite/Epon812 (Cat. No. 13940, EMS, PA, USA). After heat polymerisation, the samples were cut in 1 μm thick sections, stained with methylene blue-azure II, and examined at 400x magnification under a light microscope (Leica DM6000B, Wetzlar, Germany) with a DC490 digital camera (Leica). Ultrathin (70 nm thick) sections cut with an ultramicrotome (Leica ultracut R) were double-stained with uranyl acetate and lead citrate (Leica EM AC20), examined under a JEOL-JEM 1400 electron microscope, and photographed with a CCD camera (Gatan, Pleasanton, CA, USA).

Samples of the other half of the right kidney were fixed in 10 % formalin solution, dehydrated, and embedded in paraffin. Their sections were then incubated with 0.1 % Triton X-100 in 0.1 % sodium citrate for permeabilisation at 4 °C for 8 min, washed, and incubated with a TUNEL reagent at 37 °C for 1 h and washed again. They were then treated with a converter reagent at 37 °C for 30 min. After washing, the sections were incubated with Fast Red substrate solution for 10 min. Negative control sections were incubated with a reaction mixture without terminal deoxynucleotidyl transferase (TdT). TUNEL-positive apoptotic cells (red-labelled) were counted in ten random fields of cortex and medulla with a light microscope at 400x magnification (one sample per animal). The count is given as the mean number of TUNEL-positive cells per field per group.

Left kidney homogenates were prepared in ice-cold buffer containing Tris (10 mmol/L, adjusted to pH 7.4) and protease inhibitors to obtain 10 % (w/v) whole homogenate. After different stages of centrifugation (1500 x g) at 4 °C, the supernatant was used to measure TAOC, malondialdehyde (MDA) concentration, and protein oxidation.

For all spectrophotometric measurements we used SpectraMax M2 (Molecular Devices, CA, USA). Renal GPx activity was measured indirectly through coupled reaction with glutathione reductase (GR). Absorbance was measured at 340 nm at one-minute intervals, and GPx1 activity expressed as nmol/mg of protein per min.

CAT activity was measured with a commercial spectrophotometric kit, which utilises the peroxidative function of CAT for determination of enzyme activity. The absorbance values were measured at 540 nm, and CAT activity was expressed as nmol/mg of protein per min.

SOD activity was measured with a commercial colorimetric kit with a radical detector [i.e. 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-1), monosodium salt] that produces a water-soluble formazan dye upon reduction with a superoxide anion. One unit of SOD is defined as the amount of enzyme needed to exhibit 50 % dismutation of the superoxide radical. SOD activity was expressed as U/mg of protein.

This assay uses the ability of intracellular antioxidants to inhibit the oxidation of 2,2’-azino-di-(3-ethylbenzthiazoline sulphonate) (ABTS) to ABTS⁺ by metmyoglobin at 405 nm. The capacity of these antioxidants to inhibit ABTS+ oxidation was compared to the same ability of Trolox. TAOC levels were expressed as mmol/L of Trolox equivalents per mg protein.

Renal lipid peroxidation was quantified with a TBARS assay kit, which measures the concentration of MDA as an important indicator of lipid peroxidation. MDA forms a complex with thiobarbituric acid (TBA) at 90–100 °C under acidic conditions. The colour intensity of the MDA-TBA complex was measured at 540 nm spectrophotometrically, and MDA concentration calculated based on MDA standards (0, 0.5, 5, 10, 20, 30, and 50 μmol/L) and expressed as μmol/L.

The level of carbonyl groups as an indicator of protein oxidation was determined by derivatising them with 2,4 dinitrophenylhydrazine (DNPH) to form stable dinitrophenyl (DNP) hydrazone adducts, whose levels are proportional to the carbonyls present and are measured at detected at 360 nm. Carbonyl levels were expressed as nmol/mg protein.

Kidney GSH levels were measured with a commercial kit based on the reaction of its sulphhydryl group with 5,5’-dithio-bis-(2-nitrobenzoic acid) (DTNB) to produce a yellow-coloured compound of GSH and 5-thio-2-nitrobenzoic acid (TNB). The absorbance values of the samples were measured at 414 nm. The results were expressed as nmol/mg of protein.

Protein content of the kidney samples was determined with a protein assay kit bicinchoninic acid as described elsewhere (35).

The obtained data were analysed with one-way analysis of variance (ANOVA), followed by the Mann-Whitney U test. We used the Statistical Package for Social Sciences Program (SPSS, Chicago, IL, USA) version 17.0. Statistical significance was set at p<0.05. The results were given as mean ± standard error of the mean (SEM).

In all A1254-exposed groups, kidney weights decreased significantly compared to control (Figure 1), whereas relative kidney weights were significantly lower only in the ASeD group (12.65 %).

Figure 1

Control and the SeS samples showed normal structure of the renal cortex and the medulla. SeD samples showed desquamation of epithelial cells in several renal tubules. A and ASeD samples, however, showed narrowing of the Bowman’s space, renal corpuscular atrophy, vascular congestion, and oedema. Epithelial cells of the tubules were swollen, and desquamated in the cortex and medulla. In addition, ASeD samples showed necrosis and hyaline cast in the tubules. ASeS samples showed peritubular vascular congestion both in the cortex and medulla, but without corpuscular atrophy (Figures 2 and 3).

Figure 2

Figure 3

The glomerular ultrastructure of control, SeS, and SeD samples was normal, showing open urinary spaces and regular pedicels (Figures 4A, 4B, 4C), except that SeD samples had a few endothelial, mesangial, and visceral epithelial apoptotic cells with condensed nuclei (Figure 4C). In contrast, A1254 treatment caused degeneration of the glomerulus and tubules. Urinary space in A samples was filled with cytoplasmic extensions of podocytes, whose cytoplasm, in turn, showed lipid droplets and lysosomes. Some podocytes showed typical signs of apoptosis, such as chromatin condensation. In some areas, pedicels were effaced. A samples also showed capillaries filled with electron lucent material and apoptotic endothelial cells (Figure 4D). In ASeS samples pedicels were regular and the distance between them was equal, but some were effaced (Figure 4E). Lipid droplets and lysosomes were prominent in podocyte cytoplasm of ASeD samples, and cytoplasmic extensions were filling the urinary space. These samples also showed apoptotic mesangial and endothelial cells (Figure 4F).

Figure 4

Figure 5 compares tubular ultrastructures between the groups. As expected, proximal and distal tubules showed normal ultrastructure in control and SeS samples (Figures 5A and 5B). SeD samples showed numerous lysosomes with electron-dense areas and karyolitic nuclei in the epithelium of proximal tubules (Figure 5C). A samples showed electron lucent material in spaces between the basal and lateral folds. The nuclei were karyolitic, and tubular epithelial cells showed lysosomes in the cytoplasm. The crista of mitochondria could not be distinguished. Apical microvilli in the proximal tubules were short, flat, and also indistinct in some areas (Figure 5D). ASeS samples showed microvilli in the apical part of proximal tubule epithelial cells (Figure 5E). ASeD samples showed some tubular necrosis. Epithelial cells lost integrity and were sloughed to the lumen in some areas. Collagen fibres were abundant in the interstitium, which also showed oedema (Figure 5F).

Figure 5

Figures 6 and 7 show the apoptotic (TUNEL-positive) cells and their counts in the cortex and medulla. Kidney cortex cell counts did not differ between the control, SeS, and SeD groups, but Se deficiency significantly contributed to medullar apoptosis compared to control. A1254 caused significant increases in apoptotic cell counts in both the cortex (almost 2-fold) and the medulla (3.5-fold) compared to control. Judging by significantly lower apoptotic cell counts in either the cortex and medulla of the ASeS group compared to the A group, Se treatment was highly beneficial against A1254 induced renal apoptotic cell death in rats.

Figure 6

Figure 7

The activities of SOD, CAT, and GPx1 are given in Figure 8. SOD activities did not significantly change in any of the groups, although they were 37.49 % higher in the A group than control. CAT activities significantly dropped in all A1254-exposed groups (52.96 % in the A, 48.98 % in the ASeS, and 52.05 % in the ASeD group; p<0.05). GPx1 activity was significantly higher in the SeS group than control group (3.38-fold increase), but markedly lower in the SeD group (70.41 %). It also significantly dropped in the A and ASeD groups (34.92 % and 86.46 %, respectively; p<0.05) compared to control.

Figure 8

We did not observe any significant changes in renal TAOC and carbonyl levels in the A1254-exposed groups, but lipid peroxidation was significantly higher in the A and ASeD groups vs control (30.45 % and 20.44 %, respectively; p<0.05). In the SeS group, MDA and carbonyl levels were significantly lower vs control (43.59 % and 65.64 %, respectively) and all other study groups (p<0.05) (Figure 9).

Figure 9

The SeS group also had significantly higher total GSH vs control (39.80 %), but in all other study groups it was significantly lower (25.62 % in the SeD, 50 % in the A, 18.15 % in the ASeS, and 70.39 % in the ASeD group). However, Se supplementation provided significant increases (63.68 %) in total GSH levels compared to the A group. Se deficiency in A1254-exposed rats, in contrast, led to significantly lower GSH levels compared to A1254-exposed animals with normal selenium intake (A group) (36.84 %, p<0.05).