Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice

Abstract

The aim of this study was to determine whether calcium potentiates acute carbon tetrachloride (CCl4) -induced toxicity. Elevated calcium levels were induced in mice by pre-treatment with cholecalciferol (vitamin D3; V.D3), a compound that has previously been shown to induce hypercalcemia in human and animal models. As seen previously, mice injected with CCl4 exhibited increased plasma levels of alanine aminotransferase, aspartate aminotransferase, and creatinine; transient body weight loss; and increased lipid peroxidation along with decreased total antioxidant power, glutathione, ATP, and NADPH. Pre-treatment of these animals with V.D3 caused further elevation of the values of these liver functional markers without altering kidney functional markers; continued weight loss; a lower lethal threshold dose of CCl4; and enhanced effects on lipid peroxidation and total antioxidant power. In contrast, exposure to V.D3 alone had no effect on plasma markers of liver or kidney damage or on total antioxidant power or lipid peroxidation. The potentiating effect of V.D3 was positively correlated with elevation of hepatic calcium levels. Furthermore, direct injection of CaCl2 also enhanced CCl4-induced hepatic injury. Since CaCl2 induced hypercalcemia transiently (within 3 h of injection), our results suggest that calcium enhances the CCl4-induced hepatotoxicity at an early stage via potentiation of oxidative stress.

Fig 1. Schematic experimental design of pre-treatment with V.D3 and CCl₄ injection.

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.

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 7^th 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.