Mercury is a global environmental pollutant, accumulating mainly in the kidney and liver inducing hepatorenal toxicity, oxidative stress, and tissue damage. Oxidative stress is caused by an imbalance between free radicals’ production and cellular antioxidant defense systems. In the present study, we investigated the effect of N N′-diphenyl-1, 4-phenylenediamine (DPPD) antioxidant activity against mercury chloride- (HgCl2-) induced renal and hepatic toxicity. Thirty adult female Sprague Dawley rats were divided into three equal groups: the first group was injected with saline only and served as a control, the second group was injected with HgCl2, and the third group received DPPD + HgCl2 rats injected with HgCl2 without treatment showing a significant increase in alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea, creatinine, and uric acids compared to control. Moreover, the second group showed a significant reduction in the activity of the antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH)) in addition to a marked increase in the malondialdehyde (MDA) content, histopathological alterations, collagen deposition, CD8%, CD4%, and TGF-
Mercury is one of the most toxic metals responsible for environmental pollution [
N N′-diphenyl-1, 4-phenylenediamine, a grey or dark grey powder, is used as an antioxidant in rubber and oils, especially for tires in industry due to its colour and stability [
All chemicals and reagents were of the highest purity grade. DPPD (≥99.8%) and HgCl2 (≥99.5%) were obtained from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). In addition to serum ALT, AST, and ALP activities, urea, uric acid, and creatinine levels were determined using colourimetric diagnostic kits (Biodiagnostic, Cairo, Egypt) according to the manufacturer’s instructions. TGF-
Rats were assigned to groups by using the Statistical Package of Social Science (SPSS) program for Windows (Standard version 21). Thirty female Sprague Dawley rats, weighing approximately 170–220 gm, were purchased from the Medical Experimental Research Center (MERC), Faculty of Medicine, Mansoura University, Mansoura, Egypt. The animals were kept in polypropylene cages under standard laboratory conditions of relative humidity (45 ± 5%) and temperature (25 ± 2°C) with 12 h light/dark cycle and provided with food pellets and tap water ad libitum. Principles of laboratory animals caring (NIH publication no. 85–23, that revised 1985) were followed. Ethical protocols for laboratory animal care and use were approved and followed under the supervision of Faculty of Postgraduate Studies for Advanced Sciences (PSAS), Beni-Suef University, Experimental Animals Ethical Committee (No. BSU/EAEC/PSAS/16/112018).
Rats were randomly divided into 3 groups (10 rats/group). The HgCl2 dose was 4 mg/kg, i.p.: Group I (control): rats received saline i.p. for 14 days and served as the control Group II (HgCl2): rats were injected with a single dose of HgCl2 (4 mg/kg, i.p.) at day one of the experiment Group III (HgCl2 + DPPD): rats were injected with a single dose of HgCl2 (4 mg/kg, i.p.) at day one of the experiments and, then, treated with DPPD (0.5 g/kg, i.p.) according to [
All rats were exposed to sevoflurane anesthesia and killed by decapitation (24 h after the last injection), and urine and blood samples were collected from each rat after 14 days of HgCl2 (or saline) injection. The liver and kidneys tissues were dissected and used for biochemical, flow cytometry, and histopathological examinations.
To assess liver functions, serum ALT, AST, and ALP enzyme activities were detected. Also, serum urea, uric acid, and creatinine levels were determined to assess kidney functions using colourimetric diagnostic kits (Biodiagnostic, Cairo, Egypt) according to the manufacturer’s instructions.
The activities of the antioxidant enzymes SOD, CAT, and GSH in addition to the MDA content in liver and kidney tissues were all measured using commercial laboratory diagnostic kits (Biodiagnostic Co., Cairo, Egypt).
Flow cytometry detection of CD8%, CD4%, and TGF-
About 50 mg of kidney or liver tissue specimens were hydrolyzed, and then, chloramine
The formalin-embedded liver and kidney tissues were cut into 4
Data were analyzed using SPSS software version 22 for Windows (IBM, Armonk, NY, USA). Descriptive statistics were calculated in the form of Mean ± Standard deviation (SD). ANOVA and Tukey’s post hoc tests were used for comparison between groups. A level of
The potential effects of HgCl2 and DPPD treatment on renal and liver function parameters are summarized in Tables
Kidney injury parameters. Values are expressed as
Variables | Control group | HgCl2 group | HgCl2 + DPPD group |
---|---|---|---|
Serum: | |||
Creatinine (mg/dl) | 0.46 ± 0.03 | 1.53 ± 0.21 | 0.57 ± 0.10 |
Urea (mg/dl) | 27.83 ± 3.60 | 73.43 ± 5.87 | 32.23 ± 2.30 |
Uric acid (mg/dI) | 2.57 ± 0.25 | 4.53 ± 0.76 | 3.16 ± 0.98 |
Homogenate: | |||
Hydroxyproline (ug/mg tissue) | 22.58 ± 0.63 | 41.23 ± 9.25 | 30.24 ± 5.23 |
MDA (mmol/g tissue) | 59.79 ± 4.99 | 92.25 ± 13.05 | 70.62 ± 9.69 |
SOD (U/mg protein) | 11.66 ± 0.64 | 6.71 ± 1.51 | 9.63 ± 1.48 |
CAT (mol/min/gm) | 0.66 ± 0.09 | 0.35 ± 0.05 | 0.58 ± 0.09 |
Glutathione ( | 32.13 ± 2.07 | 21.71 ± 6.22 | 28.21 ± 3.51 |
TGF- | 33.27 ± 3.43 | 49.20 ± 9.10 | 38.60 ± 9.19 |
CD4 (ng/ml) | 21.42 ± 1.04 | 43.70 ± 5.31 | 34.30 ± 5.01 |
CD8 (ng/ml) | 23.62 ± 3.88 | 44.06 ± 6.77 | 25.66 ± 3.87 |
SD: standard deviation; P : probability;
Liver injury parameters. Values are expressed as
Variables | Control group | HgCl2 group | HgCl2 + DPPD group |
---|---|---|---|
Serum: | |||
ALT (U/L) | 33.30 ± 3.30 | 64.70 ± 14.16 | 41.80 ± 11.14 |
AST (U/L) | 59.40 ± 5.12 | 114.70 ± 15.96 | 74.00 ± 5.43 |
ALP (U/L) | 215.45 ± 5.42 | 333.97 ± 32.48 | 247.10 ± 25.91 |
Homogenate: | |||
Hydroxyproline (ug/mg tissue) | 19.80 ± 1.84 | 39.20 ± 1.77 | 24.36 ± 4.08 |
MDA (mmol/g tissue) | 81.83 ± 4.58 | 112.72 ± 10.48 | 81.93 ± 4.02 |
SOD (U/mg protein) | 20.99 ± 1.75 | 14.87 ± 2.65 | 19.94 ± 3.71 |
CAT (mol/min/gm) | 1.05 ± 0.10 | 0.60 ± 0.28 | 0.93 ± 0.13 |
Glutathione ( | 25.35 ± 1.50 | 18.97 ± 3.10 | 22.24 ± 2.01 |
TGF- | 32.31 ± 2.45 | 50.60 ± 8.83 | 34.60 ± 4.92 |
CD4 (ng/ml) | 24.44 ± 2.64 | 39.66 ± 1.60 | 25.30 ± 3.31 |
CD8 (ng/ml) | 19.73 ± 1.13 | 38.5 ± 2.88 | 21.07 ± 3.84 |
SD: standard deviation; P : probability;
The data of lipid peroxidation, CAT, GSH, and SOD activities in the renal and hepatic tissues are shown in Tables
The hydroxyproline content is a specific marker for collagen deposition. The HgCl2 group showed a significantly (
As shown in Tables
H&E-stained and Masson trichrome-stained kidney sections are shown in Figure
H&E-stained and Masson-stained kidney tissues of rats from different groups (magnification ×200). (a) Section of the control group showing the normal architecture of the kidney. (b) Significant increase in tubular dilatation and degenerative changes observed in HgCl2-injured rats. (c) Treatment with DPPD significantly attenuated the renal histopathological changes. (d) Masson’s trichrome staining indicated no abnormal collagen in the control group. (e) Sections of HgCl2-treated group indicated an increase in fibrosis stained in blue. (f) Kidney section of rats cotreated with HgCl2 + DPPD showed a significant decrease in collagen deposits. (g) Pathological scoring showed a significant increase in the tubular injury score in the HgCl2-treated group when compared with other groups. (h) Comparison between different groups in the Masson% area. Data were mean ± SD.
H&E-stained and Masson-stained liver tissues of rats from different groups (magnification ×200). (a) Hepatic histology of the control group, showing normal hepatic lobular architecture. (b) Hepatic degenerative changes with extensive cell necrosis observed in HgCl2-injured rats. (c) Rats treated with DPPD showed a significant modulation in the hepatic histology towards normal. (d) Control group stained with Masson’s trichrome showed that the natural structure and collagen fibers cannot be seen. (e) Accumulation and progression of collagen fibers in the liver of the HgCl2 group. (f) Significant decrease in collagen fibers observed in rats cotreated with HgCl2 + DPPD. (g) Pathological scoring showed a significant increase in the hepatic injury score in the HgCl2-treated group compared with other groups. (h) Comparison between different groups in the Masson% area. Data were mean ± SD.
The collagen content was assessed by Masson’s trichrome stain, and the control group showed a normal collagen content in the kidney (Figure
HgCl2 generates free radicals and subsequently increases oxidative stress, which leads to nephrotoxicity and accelerates hepatotoxicity [
In the present study, liver and renal functions were detrimentally altered after HgCl2 administration causing hepatorenal dysfunction evidenced by a significant elevation in AST, ALT, and ALP enzyme activities and urea, uric acid, and creatinine levels. Similar results were reported by [
HgCl2 administration initiates the formation of highly reactive substances such as reactive oxygen species in addition to the stimulation of oxidative stress [
In the present study, we found that HgCl2 significantly diminishes the activities of the antioxidant enzymes SOD and CAT in addition to GSH in kidney and liver tissues, whereas the end product of lipid peroxidation (MDA contents) was significantly increased compared with the control group. A variety of experiments have demonstrated parallel results [
Hydroxyproline is used for the estimation of the collagen content, considering that collagen contained 12.7% hydroxyproline by weight [
Our results reported a significant increase in both renal and hepatic TGF-
In the present study, we performed a histopathological examination to further support the biochemical and immunological evidence. We compared the morphological structure among each group using H&E stain. In the control group, there were no injuries or histological changes detected in the kidneys (Figure
Masson’s trichrome stain was used for distinguishing collagen deposition. The control group showed a normal collagen content in the kidney (Figure
Finally, we conclude that the antioxidant DPPD can retard the progression of hepatorenal fibrosis and collagen deposition induced by HgCl2. Further studies are needed to explain the intrinsic and extrinsic pathways of DPPD antifibrotic efficacy.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.