Non-toxic Level of Acetaminophen Potentiates Carbon Tetrachloride-Induced Hepatotoxicity in Mice.

A wide range of medications are routinely used to maintain and improve human health. Hence, it is essential that we understand and predict adverse effects caused by the combined use of multiple medications. In the present study, we investigated whether the combination of carbon tetrachloride (CCl4) and acetaminophen (APAP) had a detrimental effect on the liver. Mice injected with APAP (100 mg/kg) showed no significant changes in hepatic injury markers (alanine aminotransferase and aspartate aminotransferase), histopathological findings, pro-inflammatory cytokine levels, or hepatic oxidative stress. In contrast, a single injection of CCl4 (15 mg/kg) led to a significant increase in hepatic injury, in addition to an increase in pro-inflammatory cytokine levels and oxidative stress. Co-administration of APAP and CCl4 resulted in exacerbation of these hepatic injuries. Our results suggest that a non-toxic dose of APAP has the potential to increase CCl4-induced liver damage and oxidative stress.

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

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.

Kampo formula "Hochu-ekki-to" suppressed carbon tetrachloride-induced hepatotoxicity in mice.

OBJECTIVE: The aim of this study was to investigate whether pretreatment with the Japanese herbal medicine "Hochu-ekki-to" (TJ-41) has an ameliorative effect on carbon tetrachloride (CCl4)-induced hepatotoxicity through anorexia prevention. METHODS: Twenty-four hours before CCl4 injection, TJ-41 or saline solution was intraperitoneally administered. Furthermore, 24 h after TJ-41 injection, mice were intraperitoneally administered 1.6 g/kg CCl4 or olive oil. Moreover, 24 h after CCl4/olive oil injection, mice from each group were euthanized and bled for plasma analysis. RESULTS: Mice injected with CCl4 exhibited severe anorexia. Moreover, CCl4 increased the plasma levels of hepatic injury markers (i.e., alanine aminotransferase and aspartate aminotransferase) as well as lipid peroxidation and hepatic Ca levels. Pretreatment with TJ-41 recovered the CCl4-induced anorexia and plasma levels of the hepatic injury markers. Moreover, CCl4-induced lipid peroxidation and hepatic Ca levels decreased upon TJ-41 pretreatment. In addition, hepatic metallothionein levels in the TJ-41 + CCl4-treated group were decreased by >50 % compared with the levels in the TJ-41-treated group, implying that metallothionein was consumed by CCl4-induced radicals. CONCLUSION: Our results suggest that TJ-41 attenuates CCl4-induced hepatotoxicity, presumably by the induction of metallothionein, which in turn scavenges radicals induced by CCl4 exposure.

Suppressive Effect of Kampo Formula "Juzen-taiho-to" on Carbon Tetrachloride-Induced Hepatotoxicity in Mice.

The aim of the present study was to investigate whether pretreatment with the Japanese herbal medicine, "Juzen-taiho-to" (JTX), had an ameliorative effect on carbon tetrachloride (CCl4)-induced hepatotoxicity through anorexia prevention. Mice injected with CCl4 exhibited severe anorexia. Moreover, CCl4 increased the plasma levels of hepatic injury markers (i.e., alanine aminotransferase and aspartate aminotransferase), lipid peroxidation, and hepatic Ca(2+) levels. Pretreatment with JTX recovered the CCl4-induced anorexia. In addition, JTX pretreatment decreased CCl4-induced plasma levels of hepatic injury markers. Increased Ca(2+) is a known indicator of the final progression to hepatocyte death, and CCl4-induced hepatotoxicity is mainly caused by oxidative stress. The present study indicated CCl4-induced lipid peroxidation and hepatic Ca(2+) content decreased with JTX pretreatment. Our results suggest that JTX has potential to protect of CCl4-induced anorexia, and the modulation of oxidative stress.

Calcium-deficient diet attenuates carbon tetrachloride-induced hepatotoxicity in mice through suppression of lipid peroxidation and inflammatory response.

The aim of this study is to investigate whether a Ca-deficient diet has an attenuating effect on carbon tetrachloride (CCl4)-induced hepatotoxicity. Four-week-old male ddY mice were fed a Ca-deficient diet for 4 weeks as a part of the experimental protocol. While hypocalcemia was observed, there was no significant change in body weight. The CCl4-exposed hypocalcemic mice exhibited a significant decrease in alanine aminotransferase and aspartate aminotransferase activities at both 6 h and 24 h even though markers of renal function remained unchanged. Moreover, lipid peroxidation was impaired and total antioxidant power was partially recovered in the liver. Studies conducted in parallel with the biochemical analysis revealed that hepatic histopathological damage was attenuated 24 h post CCl4 injection in hypocalcemic mice fed the Ca-deficient diet. Finally, this diet impaired CCl4-induced inflammatory responses. Although upregulation of Ca concentration is a known indicator of terminal progression to cell death in the liver, these results suggest that Ca is also involved in other phases of CCl4-induced hepatotoxicity, via regulation of oxidative stress and inflammatory responses.

Carbon Tetrachloride-Induced Nephrotoxicity in Mice Is Prevented by Pretreatment with Zinc Sulfate.

Carbon tetrachloride (CCl4) is commonly used as a chemical inducer of experimental liver injury. In addition, many studies showed that CCl4 can induce kidney damage. In the current study, we evaluated the protective effect of zinc (Zn) against CCl4-induced nephrotoxicity. We hypothesized that this protective effect would result from the ability of Zn to serve as an inducer of metallothionein (MT), a known endogenous scavenger of free radicals. We administered Zn (as ZnSO4) 50 mg/kg subcutaneously once daily for 3 successive days prior to a single intraperitoneal administration of CCl4 4 g/kg in male ddY mice. Our results showed that Zn pretreatment significantly decreased creatinine and blood urea nitrogen levels and reduced renal histopathological damage at 6 h post-CCl4 injection, observations consistent with enhanced antioxidative activity in the kidney. Moreover, kidney MT levels in the Zn+CCl4-treated group decreased by greater than 70% compared with levels in the Zn-alone group, implying that MT was consumed by CCl4-induced radicals. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute nephrotoxicity, presumably by induction of MT, which in turn scavenges radicals induced by CCl4 exposure.

Bromobenzene-induced lethal toxicity in mouse is prevented by pretreatment with zinc sulfate.

In the current study, we evaluated the protective effect of zinc (Zn) against bromobenzene (BB) -induced lethal toxicity. We used Zn because this element is known to be an inducer of metallothionein (MT), which is in turn known to serve as an endogenous scavenger of free radicals. We administered Zn (as ZnSO4) at 50 mg/kg subcutaneously once-daily for 3 successive days prior to a single intraperitoneal administration of 1.2 g/kg BB in male ddY mice. Our results showed that pretreatment with Zn completely abolished the BB-induced mortality of mice until 48 h. We also found that pretreatment of mice with Zn significantly decreased the functional marker levels and reduced the histological damage both in liver and kidney as assessed at 18 h post-BB. We also showed that pretreatment with Zn enhanced antioxidative activity, resulting in decreased lipid peroxidation in both liver and kidney. Moreover, BB-induced calcium levels were downregulated by pretreatment with Zn. In addition, Zn-induced MT was decreased in Zn + BB-treated animals, implying that MT was consumed by BB-induced radicals. These findings suggest that prophylaxis with Zn protects mice from BB-induced lethal toxicity by decreasing oxidative stress in liver and kidney, presumably by induction of MT, which scavenges radicals induced by BB exposure.

Carbon tetrachloride-induced lethality in mouse is prevented by multiple pretreatment with zinc sulfate.

Carbon tetrachloride (CCl4) is commonly used as a chemical inducer of experimental liver injury. Several compounds have been demonstrated to attenuate the hepatic damage caused by sublethal doses of CCl4. However, rescue from lethal toxicity of CCl4 has not been reported. In the present study, we evaluated the protective effect of metallothionein (MT), an endogenous scavenger of free radicals, on CCl4-induced lethal toxicity of mice. To induce MT production in male ddY mice, we administered Zn (as ZnSO4) at 50 mg/kg as a once-daily subcutaneous injection for 3 days prior to a single intraperitoneal administration of 4 g/kg CCl4. Animals were observed for mortality every 3 hr for 24 hr after CCl4 injection. Liver damage was assessed by determining (in a subset of these mice) blood levels of alanine aminotransferase (ALT; a marker of liver injury) and liver histopathology at 6 hr after CCl4 injection. Our results showed that three times pretreatment with Zn yielded > 40-fold induction of hepatic MT protein levels compared to control group. Zn pretreatment completely abolished the CCl4-induced mortality of mice. We also found that pretreatment of mice with Zn significantly decreased the ALT levels and reduced the histological liver damage as assessed at 6 hr post-CCl4. These findings suggest that prophylaxis with Zn protects mice from CCl4-induced acute hepatic toxicity and mortality, presumably by induction of radical-scavenging MT.

Effect of hemolytic and iron-deficiency anemia on intestinal absorption and tissue accumulation of cadmium.

Abnormal iron (Fe) metabolism induces iron-deficiency anemia (FeDA) and also affects body cadmium (Cd) accumulation. However, whether hemolytic anemia also affects Cd metabolism is not known. We compared the intestinal absorption and tissue accumulation of Cd after oral administration of Cd to mice with hemolytic anemia induced by treatment with phenylhydrazine (PHA mice) to that in mice with FeDA. Although the hematocrit decreased significantly in mice with either type of anemia, the Fe concentration decreased in the livers and kidneys of FeDA mice, but increased in those of PHA mice. After an oral administration with various amounts of Cd, hepatic and renal Cd concentrations significantly increased in both FeDA and PHA mice. An intraduodenal injection of Fe raised the hepatic Fe content in FeDA mice to the control level and raised the hepatic Fe content in PHA mice to 2.4 times that in control mice. Intestinal divalent metal transporter 1 (DMT1) expression increased significantly in mice with both types of anemia. These data indicate that, despite the accumulation of hepatic Fe associated with PHA, PHA also significantly increases hepatic and renal Cd accumulation according to an stimulation of intestinal DMT1 expression, as occurs in FeDA mice. This suggests that anemia may be a risk factor for Cd accumulation.

Metallothionein-enriched hepatocytes are resistant to ferric nitriloacetate toxicity during conditions of glutathione depletion.

Metallothionein (MT) is involved not only in heavy metal homeostasis/detoxification but also in radical scavenging, yet the relevance to other antioxidant systems and physiological significance under oxidative stress has not been clarified. We studied that ability of MT, induced by zinc and cadmium, to protect against oxidative damage induced by ferric nitrilotriacetate (Fe-NTA) in glutathione depleted primary cell cultures. Treatment with Fe-NTA resulted in significant decreases in cell survival and increases in medium LDH activity in control cells following depletion of glutathione. The toxic effects of Fe-NTA were modulated in Zn-MT-enriched cells. In glutathione-depleted cells, but not in non-treated cells, Cd-binding properties of cellular Zn-MT decreased with increasing concentration of Fe-NTA. Both Zn-MT and Cd-MT-enriched cells were resistant to higher doses of Fe-NTA. These results indicate that MT may act a cellular radical scavenger in the absence of GSH. Thus, MT may function as a secondary antioxidant in a cellular protection system.

Induction of hepatic and renal metallothionein synthesis by ferric nitrilotriacetate in mice: the role of MT as an antioxidant.

Metallothionein (MT) demonstrates strong antioxidant properties, yet the physiological relevance of its antioxidant action is not clear. Injection of mice with ferric nitrilotriacetate (Fe-NTA) caused a dose-dependent increase in hepatic and renal MT. Fe-NTA caused a greater increase in hepatic and renal MT concentration (2.5- and 4-fold) compared with FeCl(3) at the same dose of ferric ion. MT mRNA levels were markedly elevated in both of tissues. Thiobarbituric acid (TBA) values in both tissues reached a maximum after 2-4 h. The MT concentrations were significantly increased after 2-4 h in liver and after 8-16 h in kidneys. Plasma concentrations of cytokines such as IL-6 and TNFalpha were elevated by 4 h; IL-6 levels were 24 times higher after Fe-NTA than that after injection of FeCl(3). Pretreatment of mice with ZnSO(4) attenuated nephrotoxicity induced by Fe-NTA after 2 h, but was not effective 4 h after injection. After a Fe-NTA injection, a loss of Cd-binding properties of preinduced MT was observed only in kidneys of Zn-pretreated mice but not in liver. Treatment with BSO, glutathione (GSH) depletor, intensified a loss of its Cd-binding properties after a Fe-NTA injection. These results indicate that induction of MT synthesis may result from reactive oxygen species (ROS) generated by Fe-NTA, and MT may act in vivo as a complementary antioxidant.

Dietary cadmium inhibits spontaneous hepatocarcinogenesis in C3H/HeN mice and hepatitis in A/J mice, but not in C57BL/6 mice.

Cadmium is known to be a potent carcinogenic and mutagenic metal. However, we demonstrated that dietary supplementation with 50 ppm cadmium inhibits spontaneous carcinogenesis in C3H/HeN and spontaneous hepatitis in A/J mice. We found that the frequencies of spontaneous hepatocarcinogenesis in C3H/HeN mice and of spontaneous hepatitis in A/J mice fed low-dose cadmium for 54 weeks were significantly lower than those in the respective control groups. A cadmium-induced increase in metallothionein production itself and/or metallothionein-associated increases in hepatic zinc concentrations may be involved in the observed preventive effects of cadmium. Our results suggest that low doses of cadmium in the diet or environment may play a beneficial role in the prevention of hepatic disease in humans and animals.

Paradigm shift in zinc: metal pathology.

Zinc (Zn) is an essential, common metal in animal tissues. Zn levels were elevated in only four tissues after Zn administration, the highest increase being in the pancreas. Zn concentration was increased by metallothionein induction. Metallothionein-bound Zn significantly reduced the toxicity of the metals Cd, Cu and Hg. It should be noted that tissue Zn levels are different in experimental animals and humans. Acute pancreatitis was observed following the injection of a large dose of Zn. Different metals have different target organs. Using metal pathology, treatments may be developed to save patients suffering from hepatic and renal diseases because Zn is used to a model animal of hepatic or renal disease.

Glucocorticoids suppress the inflammation-mediated tolerance to acute toxicity of cadmium in mice.

Several compounds have been shown to cause acute toxicity to cadmium (Cd). The mechanism of tolerance to Cd toxicity induced by glucocorticoids or by inflammation involves induction of metallothionein (MT) synthesis via glucocorticoid response elements or by inflammatory cytokines. We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses inflammation-mediated induction of hepatic MT synthesis. Here we investigated the effect of glucocorticoid on tolerance to Cd induced by inflammation in mice. The LD50 of Cd for mice with induced inflammation by injection with turpentine oil (Tur-mice) was higher than the LD50 in control mice. Pretreatment of Tur-mice with dexamethasone to the Tur-mice (Dex+Tur-mice) resulted in a decrease in LD50 after Cd treatment. A significant increase in plasma alanine aminotransferase and aspartate aminotransferase levels in the Dex+Tur-mice was observed at lower doses of Cd than in the Tur-mice and at higher doses of Cd than in control mice. Dexamethasone did not suppress tolerance to cadmium toxicity in the testes of the Tur-mice. Pretreatment of Tur-mice with dexamethasone resulted in suppression of both plasma interleukin (IL)-6 elevation and in suppression of hepatic MT levels when induced by inflammation but not when induced by Cd. These data suggest that suppression of tolerance to Cd toxicity induced by glucocorticoid may involve hepatic MT synthesis mediated by inflammatory cytokines, such as IL-6. We suggest that the inflammatory response can modulate Cd toxicity by induction of MT by inflammatory cytokines.