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.


Introduction
Carbon tetrachloride (CCl 4 ) is widely used in experimental animal models of liver failure that mimic human hepatic toxicity. The mechanism of CCl 4 hepatotoxicity has been thoroughly studied since 1967, including the use of in vivo models of acute and chronic CCl 4 poisoning, ex vivo perfusion of livers, and the use of isolated or cultured hepatocytes [1,2]. CCl 4 -induced toxicity is a multifactorial process involving the generation of CCl 4 -derived free radicals [2][3][4][5]. The first step is metabolic activation of CCl 4 by CYP2E1, whereby CCl 4 is converted to free a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 radicals (trichloromethyl and trichloromethyl peroxy radicals). The second step is binding of these radicals to antioxidant enzymes, including the sulfhydryl (protein thiol) groups of glutathione (GSH). In the third step, these overproduced free radicals increase membrane lipid peroxidation, bind covalently to macromolecules, deplete ATP, and interfere with calcium homeostasis [6][7][8]. Since sulfhydryl groups are essential elements of the molecular arrangements responsible for the Ca 2+ transport across cellular membranes, loss of function of these proteins is expected to impair the capacity of microsomes and mitochondria to regulate cellular calcium levels.
Recently, we found that cadmium (Cd) -induced cell cytotoxicity is attenuated by calciumfree medium in vitro (unpublished data). These data suggest that calcium is directly involved in Cd-induced toxicity. Because Cd-related toxicity is mediated by GSH depletion, lipid peroxidation, and mitochondrial dysfunction [9][10][11] (that is, by processes similar to those of CCl 4induced toxicity), we hypothesized that calcium might also exacerbate CCl 4 toxicity.
It is well known that some drugs (e.g., thiazide diuretics) cause hypercalcemia [12,13]. Treatment with vitamin D commonly has been used to investigate hypercalcemia in animal models [14][15][16]. In calcium homeostasis, vitamin D3 (V.D3) is a potent serum calcium-raising agent that regulates both calcitonin (CT) and parathyroid hormone (PTH) gene expression [17][18][19]. Serum calcium is the major secretagogue for CT, a hormone product whose biosynthesis is the main biological activity of thyroid C-cells. Taking advantage of this regulatory mechanism, vitamin D3-induced hypercalcemia has been extensively used.
Therefore, in the current study, we investigated whether hypercalcemic mice exhibited increased CCl 4 -induced toxicity. To examine the effect of calcium on acute CCl 4 toxicity, we pre-treated animals with V.D3, before determining plasma biochemical markers, hepatic lipid peroxidation, and hepatic calcium levels.

Animal treatment
Male ddY mice were purchased from Japan SLC (Hamamatsu, Japan) and were maintained under standard conditions of controlled temperature (24 ± 1˚C), humidity (55 ± 5%), and light (12:12 h light/dark cycles) with free access to water and food. Experimental treatments were performed using eight-week-old animals. Following the experiment, any surviving mice were sacrificed using pentobarbital. All experiments were approved by the Institutional Animal Care and Experiment Committee of Kinjo Gakuin University (No. 110).

Evaluation of the effect of vitamin D3 on CCl 4 toxicity
Mice were divided into two groups (olive oil + CCl 4 and V.D3 + CCl 4 ) of twelve mice each. On Days -4 to -1 (i.e., each of the four days prior to CCl 4 injection), animals were administered once daily (at 24-h intervals) by oral gavage (per os; p.o.) with cholecalciferol (vitamin D3; V.D3; Tokyo Chemical Industry, Tokyo, Japan; formulated in olive oil (Nacalai Tesque, Kyoto, Japan)) at 5 mg/kg, or with an equivalent volume of olive oil vehicle alone. On the nominal Day 0 (i.e., twenty-four hours after the final gavage), each mouse was injected intraperitoneally (i.p.) with CCl 4 (Wako Chemical, Osaka, Japan) at 2 g/kg (5 mL/kg). Before the CCl 4 injection, we collected pre-dose blood samples from each mouse; these specimens were used to confirm the effects of V.D3 on plasma Ca concentrations. At 24 h after the CCl 4 injection, three randomly selected mice from each group were euthanized; livers were harvested from each of these animals and flash frozen for storage at -80˚C. The remaining mice (nine per group) were maintained on study through Day 7. Once daily following CCl 4 injection, animals were checked for mortality and body weight was recorded. Additionally, on Days 1, 3, and 7, remaining animals were subjected to blood sampling for determination of blood functional markers. Following the Day-7 procedures, any surviving mice were sacrificed using pentobarbital. Experimental procedure is described in Fig 1.

Evaluation of role of calcium in CCl 4 toxicity
Mice were divided into three groups (Ca + olive oil, saline + CCl 4 , and Ca + CCl 4 ) of six mice each. Animals were administered i.p. with calcium chloride (CaCl 2 : Wako Chemical; formulated in physiological saline) at 150 mg/kg or with an equivalent volume of saline vehicle. Ten minutes later, animals were administered i.p. with CCl 4 at 2 g/kg or with an equivalent volume of olive oil. Whole blood was collected at 10 and 30 min and at 1, 3, 6, 12, and 24 h (the last by terminal bleed) after CaCl 2 injection. At each time point, whole blood specimens were centrifuged (3000× g, 10 min), and the plasma supernatants were frozen and stored at -80˚C pending use for determination of plasma calcium concentrations (all time points) or hepatic injury markers (terminal samples). Following the terminal bleeds (at 24 h after i.p. injections), mice of each group were euthanized and livers were harvested. Liver specimens were flash-frozen and stored at -80˚C pending use for determination of hepatic calcium levels.

Plasma biochemical analysis
Plasma calcium levels were measured using the calcium-E test (Wako Chemical) according to the manufacturer's instructions. Plasma sample (2.5 μL) was mixed with substrate buffer (100 μL) and coloring reagent (50 μL). The absorbance of the reaction mixture was measured at 610 nm.
Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured using the Transaminase CII Test Wako (Wako Chemical) according to the manufacturer's instructions and as previously described [20,21]. Concentrations of plasma creatinine and blood urea nitrogen (BUN) were measured using Creatinine Liquid Reagents Assay (DIAZYME, Poway, CA) and BUN Wako Test (Wako Chemical), respectively, according to the manufacturer's instructions and as previously described [22,23]. For relative quantification, calibration curves were prepared using standard solutions.

Isolation of total RNA and qRT-PCR assay
Total RNA was extracted from 0.1 g liver sections using the ISOGEN II kit (Nippon Gene, Tokyo, Japan). qRT-PCR was performed with One Step SYBR PrimeScript PLUS RT-PCR kit Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice (Perfect Real Time) (Takara Bio, Shiga, Japan) using an Applied Biosystems 7300 system (Applied Biosystems, Foster City, CA). PCR conditions were as previously described [24]. Primer pairs are shown in Table 1. Relative expression of each mRNA was determined using the standard curve method. The amount of each target mRNA quantified was normalized against that of GAPDH-encoding mRNA.

Histopathological findings
For histological analysis, a portion of the left liver lobe from each animal were perfused with 15% phosphate-buffered neutral formalin (pH 7.2), dehydrated, and embedded in paraffin. Embedded tissues were sectioned at 4 μm and stained with hematoxylin and eosin (H&E), Masson trichrome (MT), or von Kossa. MT stain kit and von Kossa stain were purchased from ScyTek Laboratories, Inc. (Logan, UT, USA) and conducted accordance with manufacture's instructions. Histopathological features were observed using a light microscope.

Measurement of malondialdehyde levels in the liver
The total malondialdehyde (MDA) levels and total antioxidant power in the liver were examined by colorimetric microplate assay (Oxford Biochemical Research, Oxford, MI) according to the manufacturer's protocol and as previously described [22,23].

Determination of glutathione (GSH) levels in the liver
Hepatic GSH levels were measured using GSSG/GSH quantification kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer's instructions and as previously described [25].

Measurement of ATP and NADPH levels in the liver
Hepatic ATP and NADPH levels were measured using ATP Colorimetric / Fluorometric Assay kit (BioVision, Inc., Mountain View, CA, USA) and NADH/NADPH Assay kit (BioAssay Systems, Hayward, CA, USA), respectively. These tests were conducted accordance with manufacture's instructions.

Determination of liver calcium concentrations
Individual liver specimens (0.2-0.3 g each) were digested in 0.5 mL of concentrated nitric acid in glass test tubes. The temperature was held at 80˚C for 1 h, then gradually increased (at 10˚C per h) to 130˚C. When the acid-digested specimens became transparent, volumes of the digests Table 1. Oligonucleotide primer sequences and PCR conditions for real-time RT-PCR.

Gene
Primer sequences PCR Product Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice were raised to 5 mL with distilled water, and calcium concentrations were determined by atomic absorption using a Z-2300 (Hitachi, Tokyo, Japan).

Statistical analysis
All data from the control and treatment groups were obtained from the same numbers of replicated experiments. All experiments were performed independently at least two times. Twogroup comparisons were made using Student's t-test or Welch's t-test; multiple comparisons were analyzed using One-Way ANOVA with post-hoc Tukey-Kramer's test. Tests were twotailed. The results of the survival tests were analyzed by means of χ 2 analysis. All statistical analyses were performed using SPSS 19.0J software (Chicago, IL). Values of P < 0.05 were considered statistically significant.

Results
Effect of pre-treatment with V.D3 on CCl 4 acute toxicity, as assessed by body weight and mortality To determine the effects of V.D3 pre-treatment, we performed analysis of plasma biochemical markers. Four-time, once-daily pre-treatment with V.D3 significantly increased plasma Ca concentrations to 13.0 mg/dL compared to the control value of 7.7 mg/dL ( Table 2); these elevated levels would be classified as severe hypercalcemia. In contrast, plasma levels of ALT and AST (markers of hepatic injury; Fig 2) and of creatinine and BUN (markers of kidney injury; Fig 3) were comparable between V.D3-and olive oil-treated groups. These pre-treated animals were administered i.p. with CCl 4 at 2 g/kg. Animals pre-treated with olive oil (instead of V.D3) and then injected with CCl 4 exhibited a transient loss of approximately 10% body weight on the first day and subsequent recovery from Day 2 ( Fig 4A). Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice In contrast, weight loss in the hypercalcemic mice (pre-treated with V.D3) continued in the days following CCl 4 injection, achieving approximately 30% loss of weight by Day 7 (compared to baseline), a change that was significant compared to that in the control group. In addition, mortality was significantly elevated in the V.D3 + CCl 4 treatment group compared to the control animals ( Fig 4B). Notably, none of the mice died following CCl 4 injection, while 55.6% (5 of 9; 4 on Day 2 and 1 on Day 7) of the hypercalcemic mice were found dead in the week following CCl 4 injection.
Changes in hepatic and renal injury markers in CCl 4 -exposed mice pretreated with V.D3 To reveal the target organ of CCl 4 -induced toxicity under hypercalcemic conditions, we next examined hepatic injury markers in the CCl 4 -treated mice. As shown in Fig 2, pre-treatment with V.D3 significantly potentiated the increase in plasma ALT and AST levels seen following CCl 4 injection; these parameters recovered by the 7 th day after CCl 4 injection.
In parallel with the measurement of ALT and AST, we evaluated plasma creatinine and BUN levels, which are markers of renal injury. As shown in Fig 3A, CCl 4 exposure yielded significant increases (in both groups) in creatinine levels at Days 1 and 3 (compared to respective baseline values), but these effects did not differ significantly between groups (i.e., for animals pre-treated with V.D3 rather than olive oil). On the other hand, although CCl 4 exposure yielded an increase (compared to baseline) in Day-1 BUN in animals pre-treated with V.D3, this effect was not significant (at any of the time points) compared to the values obtained with animals pre-treated with olive oil (Fig 3B).  Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice Effect of pre-treatment with V.D3 on CCl 4 acute toxicity, as assessed by hepatic CYP2E1 levels In addition to plasma injury markers, we measured hepatic CYP2E1 mRNA levels since CYP2E1 is a major CYP contribution to CCl 4 activation [26]. As shown in Effect of pre-treatment with V.D3 on CCl 4 acute toxicity, as assessed by MT stain Next, we conducted Masson Trichrome stain since CCl 4 is well known to induce liver fibrosis [27,28]. However, hepatic fibrosis was not observed in all groups (Fig 6), suggests generation of hepatic fibrosis need to inject multiple times.
Changes in morphology, MDA, total antioxidant levels, ATP, and NADPH levels in CCl 4 -exposed mice pre-treated with V.D3 To further investigate V.D3-induced exacerbation of liver damage, we randomly selected mice from each group, harvested livers from these animals at 24 h after CCl 4 treatment, and conducted histopathological studies. H&E-stained liver sections from the control and V.D3 groups showed a normal cell morphology and well-preserved cytoplasm, in addition to a clear, plump nucleus ( Fig  7A and 7B). In contrast, we observed necrosis in the mice treated with CCl 4 (Fig 7C). In addition, Pretreatment with V.D3 become exacerbated some, but not all, liver cell necrosis (Fig 7D).
In parallel with histopathological studies, we measured liver MDA levels as a marker of lipid peroxidation. CCl 4 treatment significantly increased hepatic MDA levels, both in animals pre-treated with olive oil and in those pre-treated with V.D3 (Fig 8A). Pre-treatment with V. D3 further potentiated the CCl 4 -induced increase in MDA levels (CCl 4 vs. V.D3 + CCl 4 ). Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice Many studies have suggested that total antioxidant power, ATP, and NADPH can be used as an indicator of oxidative stress. As shown in Fig 8B, CCl 4 -treatment markedly decreased the total antioxidant power, and pre-treatment with V.D3 potentiated the CCl 4 -induced decrease in antioxidant power. Notably, for both hepatic MDA and total oxidant power, values did not differ significantly between animals pre-treated with vehicle and with V.D3. This observation demonstrated that hypercalcemia itself does not induce either of these parameters. In addition, hepatic ATP and NAPDH levels were consistent with total antioxidant power ( Fig  8C and 8D).
Moreover, we determined hepatic GSH levels, that is well known to deplete on CCl 4 administration [29][30][31][32]. As shown in Fig 9A, CCl 4 treatment significantly decreased hepatic GSH levels, both in animals pre-treated with olive oil and in those pre-treated with V.D3. Pre-treatment with V.D3 further potentiated the CCl 4 -induced decrease in GSH levels (CCl 4 vs. V.D3 + CCl 4 ). Moreover, we determined glutamate cysteine ligase catalytic subunit (GCLC) and glutamate cysteine ligase modifier subunit (GCLM) by qRT-PCR assay (Fig 9B  and 9C). Although GCLC was same tendency compared with GSH, GCLM was no significant change in all groups in the present study.  Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice Influence of V.D3 on CCl 4 acute toxicity as assessed by hepatic calcium levels and calcium stain As we showed above, pre-treatment with V.D3 yielded increased plasma Ca levels. We next examined whether V.D3 pre-treatment, with or without CCl 4 exposure, also altered hepatic calcium levels at 24 h post CCl 4 injection, which we assessed by atomic absorption spectrometry (Fig 10A). In animals pre-treated with olive oil, CCl 4 injection yielded a significant, 60-fold  Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice increase in liver Ca levels. Injection of CCl 4 in mice pre-treated with V.D3 yielded a further >3-fold elevation in hepatic Ca levels. Notably, pre-treatment with V.D3 yielded a small (1.8-fold) and non-significant increase in hepatic Ca levels compared to pre-treatment with olive oil (in the absence of CCl 4 injection). This observation demonstrated that V.D3 alone does not induce appreciable hypercalcemia of the liver. In further to investigate Ca involvement, we stained hepatic Ca by von Kossa method. In control and V.D3 groups, Ca deposition was not observed (Fig 10B and 10C). In contrast, Injection of CCl 4 in mice was slightly confirmed von Kossa positive staining in the area necrosis is not observed (Fig 10D). Moreover, maximum von Kossa staining was confirmed in V.D3 + CCl 4 group (Fig 10E).

Direct assessment of Ca effect on CCl 4 acute toxicity
In order to confirm the involvement of calcium in CCl 4 toxicity, we induced hypercalcemia by direct injection of CaCl 2 and monitored plasma calcium levels for the subsequent 24 h, both with and without concomitant CCl 4 exposure. As shown in Fig 11B, i.p. injection of CaCl 2 induced transient (within 3 h) hypercalcemia. When mice with this evanescent hypercalcemia were injected with CCl 4 (Ca + CCl 4 ), the animals exhibited significantly elevated plasma ALT and AST levels and hepatic calcium levels compared to normal-calcemic mice (CCl 4 ) ( Table 3).

Discussion
The present study demonstrated that pre-treatment with V.D3 potentiated CCl 4 -induced hepatotoxicity and enhanced mouse mortality, without increasing renal toxicity and generation of liver fibrosis. Our previous investigation demonstrated that single i.p. injection of mice with a fatal dose of CCl 4 (4 g/kg) induced severe hepatotoxicity and moderate renal toxicity [20,22,24]; however, the critical target organ that led to mouse death following CCl 4 injection was not defined. In the current study, V.D3 potentiation of toxicity was observed only in the liver, as indicated by plasma levels of ALT and AST, biochemical markers of hepatic damage. Although pre-treatment with V.D3 significantly increased renal calcium levels compared to Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice those in animals pre-treated with olive oil, renal calcium content did not differ significantly between mice treated with olive oil + CCl 4 and those treated with V.D3 + CCl 4 (data not shown). Together, these data suggest that the liver is the primary target organ of acute CCl 4 toxicity. CCl 4 is metabolized and activated by multiple CYPs, including CYP2E1, CYP2B1, and CYP2B2 [2]. In particular, CYP2E1 is a major CYP contribution to CCl 4 activation [26]. Several literatures reported pre-treatment with phenobarbital, acarbose, or natural products (such as Salvia officinalis) have been shown to potentiate the CYP2E1-mediated hepatotoxicity of CCl 4 [33][34][35][36]. Although vitamin D is known to induce the expression of CYP3A and CYP2B6 via activation of the vitamin D receptor (VDR), the pregnane X receptor (PXR), and/or the  Vitamin D3-induced hypercalcemia increases carbon tetrachloride-induced hepatotoxicity through elevated oxidative stress in mice constitutive androstane receptor (CAR) [37][38][39], we are not aware of any reports of V. D3-induced expression of CYP2E1, 2B1, or 2B2. In fact, hepatic CYP2E1 expression level was not changed by pretreatment with V.D3. Taken together, these observations indicate that CYPs are not primary mediators of the V.D3 potentiation of CCl 4 toxicity. Several studies suggest that a possible molecular mechanism involved in CCl 4 hepatotoxicity is the disruption of the delicate oxidant/antioxidant balance, which can lead to liver injury via oxidative damage [2,40]. Our results suggest that V.D3 (or a V.D3-induced factor) triggers an enhancement of CCl 4 -induced toxicity. Since V.D3 has no ability to change every parameters such as antioxidant power, MDA levels, ATP levels, NADPH levels, GSH levels, and GCL subunit levels, V.D3 itself is not an oxidant. We hypothesize that calcium is likely the aggravating factor, given that pre-treatment with CaCl 2 yielded potentiation of CCl 4 toxicity similar to that seen with pre-treatment with V.D3, a compound known to induce hypercalcemia. In addition, the extracellular plasma calcium concentration is tightly controlled by a complex homeostatic mechanism involving fluxes of calcium between the extracellular fluid and the kidneys, bones, and hormones. It has been reported that CCl 4 disrupts hepatic calcium homeostasis [41,42]. In the current study, CCl 4 -induced hepatic calcium levels were increased by pre-treatment with V.D3, indicating that calcium is a candidate aggravating factor of CCl 4 toxicity. Moreover, multiple researchers have reported that CCl 4 significantly decreases the total content of reduced GSH, and that CCl 4 -derived radicals can react with sulfhydryl groups of GSH and other protein thiols [29][30][31][32]. Our data also supports these reports since GSH was depleted by CCl 4 and these depletion levels got worse by pretreatment with V.D3. In addition, GSH is sequentially synthesize catalytic subunit d from glutamate, cysteine, and glycine, which is mainly controlled by GCL. GCL is composed of two subunits, the GCLC and the modifier subunit GCLM. Our study indicated that GCLC was same tendency compared with GSH. In contrast, GCLM was no significant change in all groups in the present study. These data suggests that single injection of CCl 4 might attack GCLC rather than GCLM since multiple injection of CCl 4 reduces both parameters [43].
Since some protein thiols are essential components of the molecular rearrangements that are required for Ca 2+ transport across cell membranes, loss of such thiols may affect the calcium sequestration activity of subcellular compartments; mitochondria and microsomes employ this sequestration to regulate cytosolic calcium levels. Hence, pre-treatment with V.D3 might induce the collapse of these cellular functions by disrupting calcium homeostasis in the cell.
We demonstrated that both V.D3-induced hypercalcemia and direct injection of calcium itself potentiate CCl 4 -induced toxicity; these results suggest that calcium potentiates hepatotoxicity. In addition, we speculate that calcium augments the CCl 4 -induced toxicity within several hours after CCl 4 -injection, given that transient hypercalcemia was observed at the earliest time points following CaCl 2 injection [44]. It has been reported that CCl 4 -induced hepatotoxicity occurs within 3 h of exposure [45], consistent with our speculation.
In conclusion, we demonstrated that V.D3-induced hypercalcemia or pre-treatment with CaCl 2 enhances CCl 4 -induced hepatotoxicity, presumably via disruption of calcium homeostasis. To our knowledge, this is the first evidence that calcium enhances CCl 4 -induced hepatotoxicity in the early stage in mice. These findings may have relevance to the mechanism of toxicity of other hepatotoxic compounds.