Redox and essential metal status in the brain of Wistar rats acutely exposed to a cadmium and lead mixture

Lead acetate (Pb(CH₃COOH)₂x3H₂O, Centrohem, Stara Pazova, Serbia) and cadmium chloride (CdCl₂xH₂O, Merck, Darmstadt, Germany) solutions were used for rat treatment. Standard solutions of Cd, Pb, Zn, Cu, Mn, and Fe used for calibration were purchased from Merck. Nitric acid (65 % HNO₃, Merck) and hydrogen peroxide (30 % H₂O₂, Sigma-Aldrich, Steinheim, Germany) were used for wet digestion of the tissues. All chemicals and reagents for biochemical assays were purchased from Sigma-Aldrich.

The experiment was performed on thirty-three 8–12-week-old male albino Wistar rats weighing 250–350 g [body weight (bw)]. They were purchased from the Military Medical Academy, Belgrade, Serbia. After acclimatisation to the new surroundings, the animals were randomised into five groups: one control and four experimental. Each group counted between 6 and 8 animals. The control group received distilled water. Other groups received aqueous solutions of Pb and/or Cd at acute doses of 15 or 30 mg/kg bw of Cd alone (Cd15 and Cd30 group, respectively) or 150 mg/kg bw of Pb alone (Pb150 group), or their combination (150 mg/kg bw of Pb plus 15 mg/kg bw of Cd; Cd15+Pb150 group) by oral gavage. We chose doses higher than those environmental, as we wanted to obtain observable toxic effects, based on previous reports (18, 30, 36).

All experimental groups had free access to standard pellet food (Veterinary Institute Subotica, Subotica, Serbia) and drinking water. All animals were sacrificed with ketamine administered intraperitoneally 24 h after treatment. The brain was removed, washed with ice-cold saline (0.9 % NaCl), and frozen to -80 °C until metal and oxidative stress analysis. All experimental procedures were approved by the Ethics Committee on Animal Experimentation of the University of Belgrade Faculty of Pharmacy (No. 323-07-11822/2018-05). The study was carried out in accordance with the Animal Welfare Act of the Republic of Serbia (37).

Brain samples for metal analysis were digested with cHNO₃ (7 mL) and H₂O₂ (1 mL) in a microwave digestion system (START D, SK-10T, Milestone Srl, Sorisole, Italy) according to manufacturer’s recommendations. Then we added 25 mL of deionised water to the digested samples. For Pb and Cd measurement we used graphite furnace atomic absorption spectroscopy (GFAAS, with graphite tube atomizer GTA 120 and 200 Series AA; Agilent Technologies, Santa Clara, CA, USA) with 0.5 % NH₄H₂PO₄ as matrix modifier, while for Zn, Cu, Mn, and Fe we used flame atomic absorption spectrometry (FAAS, 240FS AA, Agilent Technologies). The accuracy of AAS analyses was validated with standard reference material (SRM 1577c – bovine liver, National Institute of Standards and Technology, Gaithersburg, MD, USA).

Brain samples for redox status analysis were homogenised with 0.1 mol/L phosphate buffer (pH 7.4) in the 1:9 weight-to-volume ratio using a T10 basic Ultra-Turrax homogeniser (IKA, Germany). One part of the homogenate was kept at -80 °C to measure thiobarbituric acid-reactive substances (TBARS), and the rest was first centrifuged in a cooling centrifuge at 800 ×g for 10 min and then at 9500 ×g for 20 min to obtain a postmitochondrial supernatant. Postmitochondrial supernatant was also stored at -80 °C until biochemical analysis.

Superoxide anion (O₂·⁻) was measured as described by Auclair and Voisin (38) by reducing yellow-stained nitroblue tetrazolium (NBT) to blue formazan. The rate of superoxide anion generation was measured as the rate of formation of reduced NBT and expressed as μmol/min/g of protein.

Total oxidative status (TOS) was measured with a spectrophotometric method optimised by Erel (39) and based on oxidant capacity to oxidise ferrous ion–o-dianisidine complex to ferric ion, which forms a coloured complex with xylenol-orange in an acidic medium. Absorbance was measured at 560 nm and reflects the total content of oxidants in the sample, such as H₂O₂ and lipid hydroperoxides. Calibration was performed with hydrogen peroxide, and results were expresed in μmol of H₂O₂ equivalent per g of protein.

The spectrophotometric method we used for measuring total antioxidative status (TAS) has also been described by Erel (40). The method is based on oxidation of 2,2′-azinobis(3-ethylbenzo-thiazoline-6-sulfonate) (ABTS) to colour ABTS⁺ radical cation. Discoloration caused by sample antioxidants was measured at 660 nm. The results were expressed in μmol of Trolox equivalent per g protein.

Superoxide-dismutase (SOD) activity was performed according to the modified method of Misra and Fridovich (41), based on the ability of the enzyme to inhibit auto-oxidation of epinephrine in alkaline medium (pH 10.2). The activity of this enzyme is expressed in relative units, which are obtained by measuring the absorbance of the resulting red oxidation product at 480 nm and 25 °C. SOD activity in the sample is calculated as the percentage of inhibition of epinephrine auto-oxidation (U/g).

Levels of total SH-groups were measured as described by Ellman (42), using 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) as reagent in alkaline medium (pH 9.0). Absorbance of yellow reaction product (dianion of 5-thio-2-nitrobenzoic acid) was measured at 412 nm.

MDA was determined as TBARS, according to the method described by Girotti (43). It is based on the absorption maximum of MDA and other TBARS with thiobarbituric acid at 535 nm. Absorbance was measured after heating samples and reagents at 100 °C for 5 min, then ice-cooling them, and then centrifuging them at 10,000 ×g and + 4 °C for 10 min. Calibration was performed with MDA solutions.

Protein concentration in brain tissue was determined with the method described by Bradford (44), in which bovine serum albumin is used as the standard. This is a spectrophotometric method based on the binding of Coomassie Brilliant Blue G-250 to a protein molecule in a sample, which causes the absorption maximum to shift from 465 to 595 nm.

Advanced oxidation protein products (AOPP) were determined with a spectrophotometric method described by Witko et al. (45), using glacial acetic acid, 1.16 mol/L potassium-iodide solution, and phosphate buffer (pH 7.4). The absorbance was measured at 340 nm. The reaction was calibrated with chloramine-T, and results expressed as μmol of chloramine-T equivalent per g of protein.

Oxidative stress index (OSI) as an indicator of the degree of oxidative stress was calculated as a ratio between TOS and TAS and presented in arbitrary units (40). All spectrophotometric methods run on ILAB 300 Plus analyser (Instrumentation Laboratory, Milan, Italy), with the exception of TBARS determination, run on a Cary60 UV– VIS spectrophotometer (Agilent Technologies).

For all statistical analyses we used the IBM SPSS Statistics version 18 for Windows (IBM, Armonk, NY, USA). After normality and homogeneity of variance check, differences between the groups were analysed with one-way ANOVA or Kruskal-Wallis test, followed by LSD or the Mann-Whitney U test, respectively, with P<0.05 set as the level of significance. All values are expressed as mean ± standard deviation or median and range. Graphs were made using the GraphPad Prism 5 software (GraphPad software, Inc., La Jolla, CA, USA).

The levels of Pb (Figure 1) and Cd (Figure 2) in rat brains are expressed as ng of metal per g of wet tissue. Lead brain levels were significantly higher in the Pb150 and Cd15+Pb150 groups than controls (P<0.001), but the two groups did not differ significantly. Cd levels were significantly higher in the Cd15 and Cd30 groups compared to control (P<0.01) and in the Cd30 group compared to Cd15. The Cd15+Pb150 group, however, had significantly lower Cd brain levels than the Cd15 group.

Figure 1

Figure 2

There were no significant differences in Zn, Cu, Mn, and Fe brain levels between the groups (Table 1).

Table 2 shows the effects of Pb and/or Cd treatment on oxidative stress parameters in rat brain. MDA and TBARS levels were significantly higher in the Cd30 group compared to control (P<0.01) and the Cd15 group (P<0.05). Co-treatment significantly increased MDA and TBARS compared to control (P<0.001) and either metal administered alone (Cd15 P<0.001, Pb150 P<0.05). However, we observed no significant changes in AOPP, O₂^·−, SOD, and SH. Exposure to toxic metals resulted in significant increase in TAS in all treated groups compared to control, but TOS did not change significantly. Consequently, OSI was significantly lower in the Cd15 (P<0.05), Cd30 (P<0.01), and Cd15+Pb150 group (P<0.05) compared to control.