A biochemical, theoretical and immunohistochemical study comparing the therapeutic efficacy of curcumin and taurine on T-2 toxin induced hepatotoxicity in rats

Abstract

Introduction: Foodborne trichothecene T-2 Toxin, is a highly toxic metabolite produced by Fusarium species contaminating animal and human food, causing multiple organ failure and health hazards. T-2 toxins induce hepatotoxicity via oxidative stress causing hepatocytes cytotoxicity and genotoxicity. In this study, curcumin and taurine were investigated and compared as antioxidants against T-2-provoked hepatotoxicity. Methods: Wistar rats were administrated T-2 toxin sublethal oral dose (0.1 mg/kg) for 2 months, followed by curcumin (80 mg/kg) and taurine (50 mg/kg) for 3 weeks. Biochemical assessment of liver enzymes, lipid profiles, thiobarbituric acid reactive substances (TBARs), AFU, TNF-α, total glutathione, molecular docking, histological and immunohistochemical markers for anti-transforming growth factor-β1 (TGFβ1), double-strand DNA damage (H2AX), regeneration (KI67) and apoptosis (Active caspase3) were done. Results and Discussion: Compared to T-2 toxin, curcumin and taurine treatment significantly ameliorated hepatoxicity as; hemoglobin, hematocrit and glutathione, hepatic glycogen, and KI-67 immune-reactive hepatocytes were significantly increased. Although, liver enzymes, inflammation, fibrosis, TGFβ1 immunoexpressing and H2AX and active caspase 3 positive hepatocytes were significantly decreased. Noteworthy, curcumin's therapeutic effect was superior to taurine by histomorphometry parameters. Furthermore, molecular docking of the structural influence of curcumin and taurine on the DNA sequence showed curcumin's higher binding affinity than taurine. Conclusion: Both curcumin and taurine ameliorated T-2 induced hepatotoxicity as strong antioxidative agents with more effectiveness for curcumin.

Keywords: DNA damage; T-2 toxin; apoptosis; curcumin; fibrosis; hepatotoxicity; molecular docking; taurine.

FIGURE 1

Photomicrographs of H&E-stained liver sections of different groups showing: Control group showing normal liver tissue with classic hepatic lobule formed of central vein (CV) surrounded by portal areas (P) at the periphery. Hepatocytes appear eosinophilic with vesicular nuclei, radiating from the central vein as plates separated by blood sinusoids (A,E,I). T-2 toxin group showing loss of normal hepatic structures, dilated congested CV and portal area (P) (red aster) blood sinusoids (red arrow) with inflammatory cellular infiltration (black arrowhead). Many hepatocytes appear vacuolated and degenerated (blue arrowhead) or dense pyknotic apoptotic nuclei with more eosinophilic cytoplasm (black arrow). (B,F,J). Toxin treated with curcumin groups showing almost normal morphology restoration except for very few cellular infiltration (black arrowhead). Few hepatocytes appear vacuolated (blue arrowhead) or dense pyknotic apoptotic (black arrow) (C,G,K). Toxin treated with taurine group showing restoration of liver tissue but still some congestion in the central vein and the blood sinusoids (red aster). Some hepatocytes still appear vacuolated (blue arrowhead) or with dense pyknotic apoptotic (black arrow) (D,H,L) (H & E section at magnification ×100 (A–D) with scale bar; 100 µm and ×200 (E–L) with scale bar; 50 µm).

FIGURE 2

(A) Collected photomicrographs of liver sections stained by different histochemical stains, Sirius red, Masson trichrome, and PAS of different groups. Sirius red and Masson trichrome stained control group showing few collagen fibers deposited around the central vein and portal area (A,E). T-2 toxin group showing massive collagen fibrosis mainly around the portal area (B,F). Toxin treated with curcumin group showing group showed a marked decrease in collagen deposits around the central vein and portal area (C,G). Toxin treated with taurine group showing a moderate decrease in collagen fibers around the central vein and portal area (D,H). Collagen fibrosis was demonstrated as red and green color by Sirius red and Masson trichrome, respectively, in all groups. The magenta red of the PAS reaction appears to be a normal concentration around the central vein (CV) in control (I). However, it is depleted in the T-2 toxin group (J). PAS magenta red color reappeared stronger in toxin treated with curcumin group (K) than in toxin treated with taurine group (L). (Sirius red at x 100 (A–D) with scale bar; 100 µm. Masson trichrome and PAS magnification at ×200 (E–L) with scale bar; 50 µm). (B) Collected Graphs for histochemical morphometry statistical analysis of Area percentage of Masson trichrome (A), Sirius red (B) for collagen fibrosis and (PAS) for glycogen storage (C). Data are expressed as mean ± SD. ***P = significant difference less than 0.05, analysis of different liver tissue sections from all other groups.

FIGURE 3

(A) Photomicrographs of liver tissue sections immunohistochemically stained for (anti-TGF-β1, and KI 67) from all groups. TGF-β1 showing minimal cytoplasmic immunoreaction (black arrow) in hepatocytes mainly at portal area of the control group (A), which is markedly upregulated in the T-2 toxin group (B). However, marked reduction TGF-β1immunoreactive hepatocytes (black arrow) in toxic group treated with curcumin treatment (C) and to less extent in toxic group treated with taurine (D) compared to toxic group. KI-67 positive immunoreactive cells (black arrow) showing marked decrease in T-2 toxin group with increased sinusoidal Kupffer cells immunoreactive (red arrow inside insert) (F) compared to control group (E). Positive immune reactive cells markedly increased in toxic group treated with curcumin (G) and moderately increased in toxic group treated with taurine (H) compared to toxic group. (Magnifications: Anti- TGF-β1, and KI 67 at 400, scale bar; 20 μm). (B) Photomicrographs of liver tissue sections immunohistochemically stained for (anti- H2AX, and active caspase 3) from all groups. Liver tissue sections immunohistochemically stained for Anti-phospho-Histone (H2AX) showing few nuclear positive hepatocytes (black arrow) around the central vein (CV) in control group (A,E). The number nuclear-positive hepatocytes markedly increased in the T-2 toxin group (B,F). However, the positive nuclear hepatocytes are markedly decreased in toxic group treated with curcumin (C,G) and moderately reduced in toxic group treated with taurine (D,H) compared to toxic group. Active caspase immunostaining showing marked increase in the number of immunoreactive hepatocytes (black arrow) in T-2 toxin (J) compared to control (I). The number of positive immune reactive hepatocytes markedly decreased in toxic group treated with curcumin (K) and moderately decreased in toxic group treated with taurine (L). (Magnifications: Anti- H2AX at 200 (A–D) and 400(E–H) with scale bar; 50 and 20 μm respectively and active caspase3 at 400 (I–L) with scale bar; 20 μm).

FIGURE 4

Collected Graphs for immunohistochemically morphometry statistical analysis of Area percentage of anti- TGF-β1 (A) and the number of positive KI 67 (B) H2AX (C) and Active caspase 3 hepatocytes (D). Data are expressed as mean ± SD. ***P = significant difference less than 0.05, analysis of different liver tissue sections from all other groups.

FIGURE 5

3D optimized structures of curcumin and taurine with their HOMO and LUMO molecular orbitals, calculated at B3LYP/6311g (d, p) level.

FIGURE 6

3D models of (A) superposed docking taurine (light green) and curcumin (orange), binding orientation with H-bonds in yellow of (B) taurine and (C) curcumin in the active pocket of DNA.