Fig 1.
Schematic experimental design of pre-treatment with V.D3 and CCl4 injection.
Table 1.
Oligonucleotide primer sequences and PCR conditions for real-time RT-PCR.
Fig 2.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by plasma ALT and AST levels.
Mice were pre-treated with olive oil (vehicle) or with V.D3 (at 5 mg/kg) administered as four once-daily p.o. doses. At 24 h after the final pre-treatment, mice of both groups were injected i.p. with CCl4 (at 2 g/kg). Plasma ALT (A) and AST (B) activities were determined at 0, 1, 3, and 7 days after CCl4 injection. Data are presented as mean ± S.D. of 4–9 mice. # P < 0.05, ## P < 0.01 versus CCl4 group on the respective day.
Fig 3.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by creatinine and BUN levels.
Mice were treated as described in legend for Fig 2. Plasma creatinine (A) and BUN (B) levels were determined at 0, 1, 3, and 7 days after CCl4 injection. Data are presented as mean ± S.D. of 4–9 mice.
Table 2.
Effect of pre-treatment with V.D3 on plasma calcium concentrations.
Fig 4.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by body weight change and mortality.
Mice were treated as described in legend for Fig 2. Body weights (normalized to baseline) (A) and mortality (B) were recorded every 24 h through the 7th day after CCl4 injection. Data are presented as mean ± S.D. of 4–9 mice. ## P < 0.01 versus CCl4 group on the respective day.
Fig 5.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by hepatic CYP2E1 mRNA level.
Mice were treated as described in legend for Fig 2. Hepatic CYP2E1 mRNA levels were determined at 0, 1, 3, and 7 days after CCl4 injection. Data are presented as mean ± S.D. of 4–9 mice.
Fig 6.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by live fibrosis.
Animals were treated as described in legend for Fig 2, and livers were harvested at 24 h, 72 h, or 168 h after CCl4 injection. Liver specimens were fixed and stained with MT. Micrographs provide 10× magnified images of representative MT-stained liver sections obtained from the control (A), V.D3 (B), CCl4 (C), and V.D3 + CCl4 (D) groups at Day 1, control (E), V.D3 (F), CCl4 (G), and V.D3 + CCl4 (H) groups at Day 3, control (I), V.D3 (J), CCl4 (K), and V.D3 + CCl4 (L) groups at Day 7, respectivity.
Fig 7.
Pretreatment with V.D3 becomes worse animals from acute CCl4-induced hepatotoxicity, as assessed by H&E staining.
Mice were treated as described in legend for Fig 2. At 24 h after CCl4 injection, animals were euthanized and livers were harvested at necropsy. Liver specimens were fixed and processed by standard methods, and sections were stained with H&E (A–D). Micrographs provide 10× magnified images of representative H&E-stained liver sections obtained from the control (A), V.D3 (B), CCl4 (C), and V.D3 + CCl4 (D) groups. Black arrows indicate area of necrosis.
Fig 8.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by Day-1 MDA levels, antioxidant power, hepatic ATP levels, and NADPH levels.
Mice were treated as described in legend for Fig 2. At 24 h after CCl4 injection, animals were euthanized and livers were collected for determination of MDA levels (A), total antioxidant power (B), hepatic ATP levels (C), and hepatic NADPH levels (D). Data are presented as mean ± S.D. of 6 mice. * P < 0.05 and ** P < 0.01 versus control, # P < 0.05 and ## P < 0.01 versus CCl4 group.
Fig 9.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by Day-1 hepatic GSH levels, GCLC, and GCLM levels.
Mice were treated as described in legend for Fig 2. At 24 h after CCl4 injection, animals were euthanized and livers were collected for determination of GSH levels (A), GCLC mRNA (B), and GCLM mRNA (C). Data are presented as mean ± S.D. of 6 mice. * P < 0.05 and ** P < 0.01 versus control, # P < 0.05 and ## P < 0.01 versus CCl4 group.
Fig 10.
Effect of pre-treatment with V.D3 on CCl4 toxicity, as assessed by hepatic calcium levels and calcium stain.
Animals were treated as described in legend for Fig 2, and livers were harvested at 24 h after CCl4 injection. (A): Hepatic calcium levels at 24 h were determined by atomic absorption spectrometry. Data are presented as mean ± S.D. of 6 mice. * P < 0.05 and ** P < 0.01 versus control, ## P < 0.01 versus CCl4 group. (B–E): Liver specimens were fixed and stained with von Kossa. Micrographs provide 10× magnified images of representative von Kossa-stained liver sections obtained from the control (B), V.D3 (C), CCl4 (D), and V.D3 + CCl4 (E) groups.
Fig 11.
Effect of intraperitoneal injection with CaCl2 on plasma calcium levels.
Mice were injected i.p. with CaCl2 at 150 mg/kg. Plasma calcium levels were determined after 10 and 30 min and at 1, 3, 6, 12, and 24 h after CaCl2 injection. (A) and (B) show the schematic experimental design of CaCl2 injection and the results, respectively. Data are presented as mean ± S.D. of 6 mice.
Table 3.
Effect of pre-treatment with calcium on various parameters associated with CCl4-induced acute hepatotoxicity.