And 50 xylene, followed by pure xylene for one and a half hour. Samples were then impregnated with molten 4EGI-1 paraffin wax, then embedded and blocked out. Paraffin sections (4? um) were stained with IQ-1 hematoxylin and eosin, the conventional staining technic [31]. Stained sections were examined for necrosis, apoptosis, inflammation and vascular changes in renal tissue. The hepatic tissue was evaluated for any alterations in the architecture, portal or lobular inflammation, sinusoidal dilatation and congestion along with presence of granulomas, degeneration, necrosis and fatty change. b) Histopathological grading for renal lesions. Renal lesions in [Au(en)Cl2]Cl dosed rats were assessed by light microscopy and graded into five categories by utilizing a scale of 0 to 5 as mentioned and adopted by Zhang et al [32]: 0 = normal histology, 1 = tubular epithelial cell degeneration, without significant necrosis/apoptosis; 2? = ,25 , ,50 , ,75 and .75 of the tubules showing tubular epithelial cell necrosis/apoptosis, respectively, accompanied by other concomitant alterations.c) Histopathological categorization of hepatic lesions. The hepatic lesions were categorized according 
to theMod/ marked100 (5)80 (4)60 (3)Congestion60 (3) 20 (1) ??20 (1)?40 (2)Mild??Inflammation portal/lobularMarked???Mod??????60 (3)20 (1)80 (4)20 (1)-40 (2)Mild40 (2)??20 (1)40 (2)criteria mentioned below by Ramchandran et al [33] (table 1).????Results*Capsular inflammation with peritonitis was discerned in 40 of cases.(fibrinopurulent exudates on the surface). **An occasional microgranuloma was present in 20 of cases. doi:10.1371/journal.pone.0051889.t002 Hepatocellular necrosis/degeneration Necrosis with inflammationThe results of the study are depicted in tables 2, 3, 4 and Figures 2, 3, 4, 5, 6, 7, -8.20 (1)Acute ToxicityRenal Microscopic Findings. The renal lesion in all groups of this batch demonstrated variable extent of renal tubular necrosis/apoptosis (Fig. 2) with one grade showing slight predominance over the other. No single group specific necrosis grade was evident in the entire series. All the 5 rats in group A/I (Dose: 1500 mg/kg) died before sacrificing. The renal microscopy revealed normal histology in three animals and tubular necrosis of grade 2 severity i.e. comprising less than 25 of the total tubular tissue, in the remaining two cases (Fig. 3a and 3b). Scattered occasional tubules with vacuolated cytoplasm were also seen along with one of the case showing cells with strongly eosinophilic cytoplasm. In group B/I (Dose: 750 mg/kg), four out of five animals died before sacrificing. Again, a large range of necrosis was discerned, with 24786787 three animals revealing grade 1 (Fig. 3c and 3d), one grade 4 and the last grade 5 tubular necrosis. In group C/I (Dose: 375 mg/kg), three out of five animals died before sacrificing. All animals showed renal tubular necrosis comprising 75 or more of the total renal tissue examined (grade 5, Fig. 3e and 3f). Group D/I (Dose: 187.5 mg/kg) had two dead animals out of five, before sacrificing. A wide range of renal tubular necrosis comprising around 25 to more than 75 of total tissue (predominantly grade 2) was 
discerned. Group E/I (Dose: 93.75 mg/kg) with all 5 animals alive at necropsy, revealed renal tubular necrosis varying in range from individual cell necrosis/apoptosis to necrosis constituting less than 50 of the total renal tissue examined (predominantly grade 2?).??Table 2. Acute toxicity, salient hepatic micros.And 50 xylene, followed by pure xylene for one and a half hour. Samples were then impregnated with molten paraffin wax, then embedded and blocked out. Paraffin sections (4? um) were stained with hematoxylin and eosin, the conventional staining technic [31]. Stained sections were examined for necrosis, apoptosis, inflammation and vascular changes in renal tissue. The hepatic tissue was evaluated for any alterations in the architecture, portal or lobular inflammation, sinusoidal dilatation and congestion along with presence of granulomas, degeneration, necrosis and fatty change. b) Histopathological grading for renal lesions. Renal lesions in [Au(en)Cl2]Cl dosed rats were assessed by light microscopy and graded into five categories by utilizing a scale of 0 to 5 as mentioned and adopted by Zhang et al [32]: 0 = normal histology, 1 = tubular epithelial cell degeneration, without significant necrosis/apoptosis; 2? = ,25 , ,50 , ,75 and .75 of the tubules showing tubular epithelial cell necrosis/apoptosis, respectively, accompanied by other concomitant alterations.c) Histopathological categorization of hepatic lesions. The hepatic lesions were categorized according to theMod/ marked100 (5)80 (4)60 (3)Congestion60 (3) 20 (1) ??20 (1)?40 (2)Mild??Inflammation portal/lobularMarked???Mod??????60 (3)20 (1)80 (4)20 (1)-40 (2)Mild40 (2)??20 (1)40 (2)criteria mentioned below by Ramchandran et al [33] (table 1).????Results*Capsular inflammation with peritonitis was discerned in 40 of cases.(fibrinopurulent exudates on the surface). **An occasional microgranuloma was present in 20 of cases. doi:10.1371/journal.pone.0051889.t002 Hepatocellular necrosis/degeneration Necrosis with inflammationThe results of the study are depicted in tables 2, 3, 4 and Figures 2, 3, 4, 5, 6, 7, -8.20 (1)Acute ToxicityRenal Microscopic Findings. The renal lesion in all groups of this batch demonstrated variable extent of renal tubular necrosis/apoptosis (Fig. 2) with one grade showing slight predominance over the other. No single group specific necrosis grade was evident in the entire series. All the 5 rats in group A/I (Dose: 1500 mg/kg) died before sacrificing. The renal microscopy revealed normal histology in three animals and tubular necrosis of grade 2 severity i.e. comprising less than 25 of the total tubular tissue, in the remaining two cases (Fig. 3a and 3b). Scattered occasional tubules with vacuolated cytoplasm were also seen along with one of the case showing cells with strongly eosinophilic cytoplasm. In group B/I (Dose: 750 mg/kg), four out of five animals died before sacrificing. Again, a large range of necrosis was discerned, with 24786787 three animals revealing grade 1 (Fig. 3c and 3d), one grade 4 and the last grade 5 tubular necrosis. In group C/I (Dose: 375 mg/kg), three out of five animals died before sacrificing. All animals showed renal tubular necrosis comprising 75 or more of the total renal tissue examined (grade 5, Fig. 3e and 3f). Group D/I (Dose: 187.5 mg/kg) had two dead animals out of five, before sacrificing. A wide range of renal tubular necrosis comprising around 25 to more than 75 of total tissue (predominantly grade 2) was discerned. Group E/I (Dose: 93.75 mg/kg) with all 5 animals alive at necropsy, revealed renal tubular necrosis varying in range from individual cell necrosis/apoptosis to necrosis constituting less than 50 of the total renal tissue examined (predominantly grade 2?).??Table 2. Acute toxicity, salient hepatic micros.