Correspondence Satomi Onosaka (E-mail onosakanutrkobegakuinacjp)

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

Hiroki Yoshioka12 Haruki Usuda2 Tsunemasa Nonogaki2 and Satomi Onosaka1

1Faculty of Nutrition Kobe Gakuin University Ikawadani-cho Nishi-ku Kobe Hyogo 651-2180 Japan2Department of Pharmacy College of Pharmacy Kinjo Gakuin University

2-1723 Omori Moriyamaku Nagoya Aichi 463-8521 Japan

(Received September 18 2015 Accepted October 27 2015)

ABSTRACT mdash 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 sub-lethal 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 rad-icals on CCl4-induced lethal toxicity of mice To induce MT production in male ddY mice we adminis-tered Zn (as ZnSO4) at 50 mgkg as a once-daily subcutaneous injection for 3 days prior to a single intra-peritoneal administration of 4 gkg 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 injec-tion Our results showed that three times pretreatment with Zn yielded gt 40-fold induction of hepatic MT protein levels compared to control group Zn pretreatment completely abolished the CCl4-induced mortal-ity 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 prophy-laxis with Zn protects mice from CCl4-induced acute hepatic toxicity and mortality presumably by induc-tion of radical-scavenging MT

Key words Carbon-tetrachloride Liver Zinc Metallothionein Mortality

INTRODUCTION

Liver is one of the most important organs in the human body providing multiple functions including detoxifi-cation protein synthesis and production of biochemi-cals necessary for digestion (Ma et al 2014) Addition-ally liver is the most vulnerable to toxic chemical agents (Wang et al 2007 Bhondave et al 2014) Acute liver injury typically causes rapid development of hepatocel-lular dysfunction and has a poor prognosis Liver dam-age frequently results from the induction of drugs virus infection toxins or hepatic ischemic-reperfusion injury (Auzinger and Wendon 2008 Huang et al 2012)

Carbon tetrachloride (CCl4) is a potent hepatotox-ic chemical which has been widely used in experimen-tal animal models of liver injury that mimic human hepatic toxicity (Weber et al 2003 Lee and Lim 2008 Singh et al 2008) CCl4 is metabolized to trichlorome-thyl radicals and trichloromethyl peroxy radical by cyto-

chrome P450 in liver microsomes These free radicals are thought to react with membrane lipids to induce peroxi-dation and subsequent dysfunction of membranes there-by causing injuries to liver kidney heart testis and brain (Ciccoli et al 1978 Ahmad et al 1987 Singh et al 2008 Domitrović et al 2011 Liu et al 2013)

Metallothionein (MT) was first isolated in 1957 from cortex of mouse kidney as a cadmium-binding protein (Margoshes and Vallee 1957) This protein belongs to the class of low-molecular weight cysteine-rich metal-bind-ing proteins MT production is induced in many organs not only by various metals such as Zn Cu and Cd but also by nonmetallic compounds (Suzuki and Yamamura 1980 Onosaka et al 1984 Min et al 1991) Due to its sulfhydryl moieties MT has properties like those of agents that provide detoxification of heavy metal agents antioxidation against reactive oxygen species (ROS) and scavenging of free radicals (Cagen and Klaassen 1979 Thornalley and Vasak 1985 Sato and Bremner 1993

Original Article

The Journal of Toxicological Sciences (J Toxicol Sci)Vol41 No1 55-63 2016

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Sato and Kondoh 2002) Owing to these abilities the protective role of MT against metal toxicity has been extensively investigated For instance pretreatment with Zn or low doses of Cd as a MT inducer results in reduced lethality after administration of high doses of Cd (Webb 1972 Probst et al 1977 Coogan et al 1994) Addition-ally we previously showed that multiple injections with Zn or Cd significantly increased MT levels compared to single injections with Zn or Cd (Onosaka and Cherian 1981 1982)

Several studies have shown some compounds show protective effects against CCl4-induced hepatotoxicity (Singh et al 2008 Huang et al 2012 Ma et al 2014 Zhang et al 2014) These investigations focused prima-rily on blood levels of liver enzymes alanine aminotrans-ferase (ALT) and aspartate aminotransferase (AST) (markers of liver damage) histological evaluation lipid peroxidation and expression levels of cytochromes P450 These papers did not examine CCl4-induced lethal toxici-ty because the dose of CCl4 used in these studies was low-er than the LD50 Therefore prevention of CCl4-induced lethal toxicity has not been reported In the present study we investigated whether multiple pretreatments with Zn were sufficient to attenuate CCl4-induced lethal toxicity

MATERIALS AND METHODS

Animal treatmentMale ddY mice were purchased from Japan SLC

(Shizuoka Japan) and were maintained under standard conditions of controlled temperature (24 plusmn 1degC) humid-ity (55 plusmn 5) and light (1212 hr lightdark cycles) with free access to water and food Experimental treatments were performed using eight-week-old animals All exper-iments were approved by the Institutional Animal Care and Experiment Committee of Kobe Gakuin University (NOA14-22)

Administration of CCl4 and evaluation of mortality

Mice were divided into three groups of five mice each Members of each group were treated at doses of CCl4 (Wako Chemical Tokyo Japan) ranging from 1 to 16 gkg administered as emulsions in olive oil and delivered by intraperitoneal (ip) injection by subcutaneous (sc) injection or by oral gavage (per os po) The volume of administered CCl4 was 10 mLkg body weight (for dos-ing at 16 gkg) or 5 mLkg body weight (for other dose levels) Mortality was tracked for 24 hr following CCl4-exposure Following the experiment any surviving mice were sacrificed using pentobarbital

Induction of MT by Zn or Cd injectionMice were divided into two groups of ten or eleven

mice each At -72 -48 and -24 hr animals of the group-1 were administered at 24-hr intervals by sc injection with 50 mgkg ZnSO4 (Nacalai Tesque Kyoto Japan) Twenty-four hours after the final ZnSO4 injection both group-1 and group-2 (not treated with ZnSO4) were injected ip with 4 gkg (at 5 mLkg) CCl4 At 6 hr after the CCl4 injection three randomly selected mice from each group were euth-anized and bled for plasma The resulting plasma sam-ples were stored at -80degC (pending alanine aminotrans-ferase (ALT) assays) Livers were harvested from each of these animals and separate samples from each liver were stored at -80degC (pending quantitative reverse transcription (RT)-PCR assay) or fixed in 15 neutral buffered forma-lin (pH 72) (pending processing for histological evalua-tion) The remaining seven or eight mice of each group were monitored every 3 hr until 24 hr after CCl4 admin-istration to determine acute mortality the surviving ani-mals were weighed and then terminated Using equiva-lent protocols protection from CCl4 lethal toxicity was assessed using single pre-CCl4 injections with 50 mgkg ZnSO4 or with 3 mgkg CdCl2 25H2O (Wako Chemical) administered sc

Measurement of ALTPlasma ALT activities were measured using the

Transaminase CII Test Wako (Wako Chemical) accord-ing to the manufacturerrsquos instructions Each plasma sam-ple (20 μL) was mixed with substrate mixture (1 mL) incubated at 37degC for 20 min and then quenched by addi-tion of stopping solution (2 mL) The absorbance of blue pigment formed by the reaction was measured at 555 nm For relative quantification calibration curves were pre-pared using a standard solution

Histopathological studiesFor histological analysis a portion of liver left lobe

was fixed using 15 neutral buffered formalin (pH 72) processed using standard methodologies and embedded in paraffin Embedded tissues were sectioned at 4-μm thicknesses mounted on slides and stained with hema-toxylin and eosin (HampE) or periodic acid-Schiff (PAS) using standard methodologies Histopathological features in the slices were observed with a light microscope

Isolation of total RNA and RT-PCR assayTotal RNA was extracted from 01-g liver sections

using ISOGEN II (Nippon Gene Tokyo Japan) Aliquots (1 μg) of total RNA from each specimen were reverse-transcribed using Super Script III Reverse Transcriptase

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reagent (Invitrogen Carlsbad CA USA) with oligo-dT as a primer and a 1-hr incubation at 50degC the reaction mixture containing synthesized cDNA then was diluted three-fold with Tris-EDTA buffer

For the quantitative RT-PCR assay aliquots (2 μL) of diluted reverse-transcription products were amplified using an Applied Biosystems 7300 (Applied Biosystems Foster City CA USA) in a reaction mixture containing SYBR Premix Ex Taq II (Takara Bio Shiga Japan) and each primer at 05 μM After pre-incubation of the reac-tion mixtures at 95degC for 30 sec real-time PCR amplifi-cation was performed with the PCR conditions and prim-er pairs shown in Table 1 Relative expression of each mRNA was determined using the standard curve method The amount of each quantified target mRNA was normal-ized against that of GAPDH-encoding mRNA

Determination of MT levels in the liverThe hepatic MT protein levels were determined by Cd

saturation-hemolysate method (Cd-hem method) Liver tissue was homogenized with 5 vols of 025 M sucrose The homogenates were centrifuged at 18000 g for 20 min at 4degC to separate the postmitochondrial supernatant and suitable aliquots were used for MT assay by the Cd-hem method as described previously (Onosaka and Cherian 1981 1982)

Statistical AnalysisAll data from the control and treatment groups were

obtained from the same numbers of replicated experi-ments All experiments were performed independent-ly at least two times The results of the acute CCl4 tox-icity were analyzed by means of Kruskal-Wallis test ALT value comparisons were made by using post-hoc Tukey-Kramerrsquos test All statistical analyses were per-formed using SPSS 190J software (Chicago IL USA) Values of P lt 005 were considered statistically signifi-cant

RESULTS AND DISCUSSION

Difference in mortality by administration routes of CCl4

First to determine the lethal dose of CCl4 in our model (male ddY mice) we performed an escalating-dose exper-iment with CCl4 administered by either of three different routes Mortality was estimated at 24 hr after administra-tion As shown in Table 2 mouse death was not observed in the sc-administered group even at the highest tested dose-level (16 gkg) In contrast ip administration at 4 and more than 8 gkg yielded 80 and 100 mortality respectively In the po-administered group 40 of mice died with dosing at 16 gkg mouse death was not observed at lower test dose-levels Previous reports indicated LD50 values for CCl4 in mice of 304 gkg 09-47 gkg and 121-144 gkg when administered via sc ip and po routes respectively (Klaassen and Plaa 1966 1967 Gehring 1968) Thus our results were consistent with lit-erature values In addition ip administration more than 8 gkg was observed sudden death within 3 hr after CCl4 injection These doses were difficult to use in the sub-sequent protection assays because toxic levels were too strong to evaluate the defensive role Based on our results

Table 1 Oligonucleotide primer sequences and PCR conditions for real-time RT-PCRGene(Accession No)

Primer sequences PCR condition PCR Product length (bp)Sequence (5rsquo to 3rsquo) Denaturation Elongation Cycle No

MT-I Forward TTC ACC AGA TCT CGG AAT GG 95 degC 60 degC40 99

(NM_013602) Reverse GAG GTG CAC TTG CAG TTC TTG 5 sec 30 secMT-II Forward CCT GCA AAT GCA AAC AAT GC 95 degC 60 degC

40 118(NM_008630) Reverse CAC TTG TCG GAA GCC TCT TTG 5 sec 30 secGAPDH Forward TGG TGA AGG TCG GTG TGA AC 95 degC 60 degC

40 98(NM_008084) Reverse GTC GTT GAT GGC AAC AAT CTC C 5 sec 30 sec

Table 2 Mortality of three different administration routes on acute CCl4 toxicity

CCl4 dose (gkg) ip po sc1 05 05 052 05 05 054 45 05 058 55 05 0516 55 25 05Mortality was tracked for 24 hr following exposure at a given dose level

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we selected a CCl4 dose of 4 gkg ip for use in the next assays

Effect of Zn and Cd against acute CCl4 toxicityTo evaluate whether MT rescues CCl4-induced lethal

toxicity we selected Zn as an inducer of MT because we and other groups demonstrated that Zn is the most potent inducers of MT (Onosaka and Cherian 1982 Onosaka et al 1984 Santon et al 2006) In the present study 50 mgkg of ZnSO4 was pretreated to mice subcutaneous-ly for one or three times (at 24-hr intervals) We assessed induction of MT-I- and MT-II-encoding genes by using quantitative RT-PCR to measure the liver levels of the corresponding transcripts (Fig 1) Single pretreatment with ZnSO4 yielded significant enrichment in liver levels of MT-I and MT-II mRNAs (150- and 58-fold respective-ly compared to control) Moreover animals treated pro-phylactically with 3-times of ZnSO4 (50 mgkg sc qd) exhibited significant elevations of MT-I and MT-II mRNA

levels (224- and 95-fold respectively) compared to con-trol animals (Fig 1A and B) In addition we determined hepatic MT protein levels by Cd-hem method MT pro-tein induction is observed in a dose dependent manner (Fig 1C) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Zn (Onosaka and Cherian 1982)

We next examined the ability of our ZnSO4 prophy-laxis (single or three sc qd doses at 50 mgkg) to coun-teract acute CCl4 toxicity we monitored mortality every 3 hr until 24 hr after CCl4 ip injection As shown in Fig 2 lethal toxicity of CCl4 started to be observed from 6 hr after injection and the percentage of death reached to 714 (57) at 24 hr after injection consistent with our earlier escalating-dose study (Table 1) Single sc pre-treatment with 50 mgkg ZnSO4 reduced mortality to 28 (27) Furthermore 3-times pretreatment with ZnSO4 completely prevented CCl4-induced acute lethal toxicity with all seven test animals survived

Fig 1 Effect of multiple pretreatment with Zn on MT levels in mouse liver Mice were injected sc with 50 mgkg ZnSO4 every 24 hr for three times (A) and (B) Total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then per-formed The amount of quantified target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respectively (C) The liver samples were collected followed by determining the MT protein levels Data are expressed as mean plusmn SD of three or four mice significantly different from compared values (P lt 005 and P lt 001)

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Zn is well known as an essential trace element for a variety of biological activities Zn has catalytic and struc-tural roles in more than 300 metalloenzymes as well as regulatory roles in diverse cellular processes such as sig-naling transduction and gene expression (McCall et al 2000) Many studies have demonstrated that Zn per se functions as antioxidant and also plays an important role in regulation of cellular glutathione (GSH) (Parat et al 1997 Powell 2000 Zhou et al 2005) Although GSH is the most important molecule involved in cellular anti-oxidant defense radical scavenging activity of GSH is 300-times lower than that of MT (Thornalley and Vasak 1985) Moreover Santon et al (2006) reported Zn alone does not completely exert anti-oxidant effect against met-al toxicity using MT knock out cell line which implies the protective effect of Zn against oxidative stress is mainly due to the induction of MT rather than a direct action of Zn

To further examine the protective involvement of MTs against CCl4-induced lethal toxicity we investigated the ability of 3 mgkg Cd pretreatment to prevent CCl4-in-

duced lethal toxicity As shown in Figs 3 and 4 ip administration of CCl4 yielded 86 (67) mortality after 24 hr injection single-dose pretreatment with Cd reduced frequency of death to 25 (28) (Fig 3) Thus single-dose Cd prophylaxis protects from CCl4-induced lethal toxicity as well as single-dose Zn although potency was weaker than that of 3-times Zn prophylaxis (in that some mice still died) As with Zn pretreatment Cd exposure also induced liver MT-I and MT-II mRNA to levels 94- and 30-fold (respectively) higher than those detected in the control and MT protein levels were also increased significantly (Fig 4) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Cd (Onosaka and Cherian 1981)

Cd is classified as non-essential trace element and Cd stimulates the expression of various types of genes The most dramatic regulatory effect of Cd (non-toxic concen-trations) is its capability to induce the expression of the genes for the syntheses of MT and GSH at low concentra-tions (Goering and Klaassen 1983 Hatcher et al 1995) At more elevated concentrations Cd also induces a series of stress proteins like HSP70 (Goering et al 1993) Moreover Goering et al reported that a low dose of Cd renders animals highly tolerant to Cd-induced lethality and that this effect is mediated through induction of MT expression (Goering and Klaassen 1983) Taken together these findings suggest that the main regulatory effect of

Fig 2 Effect of multiple pretreatment with Zn on acute CCl4 toxicity (A) Schematic experimental design of pre-treatment with Zn and CCl4 injection (B) Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr After pretreatment mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortality in each group Data indi-cate seven mice The CCl4 1xZn + CCl4 and 3xZn + CCl4 groups are represented with white triangles white circles and black circles respectively P lt 005 ver-sus 3xZn + CCl4 group

Fig 3 Effect of single pretreatment with Cd on acute CCl4 toxicity Mice were injected sc once with 3 mgkg CdCl2 24 hr before CCl4 injection Twenty-four hr lat-er mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortal-ity in each group Data indicate seven or eight mice The CCl4 and Cd + CCl4 groups are represented with white triangles and black squares respectively

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Cd is to induce MT synthesis and we postulate that the Cd prophylaxis in the present study protected against CCl4-induced lethality through induction of MT expression

Hepatoprotective pathway against CCl4 is report-ed through MT-dependent pathway in sub-lethal dose (Klaassen and Cagen 1981 Clarke and Lui 1986) On the other hand another pathway is also observed using MT knock-out (KO) mice (Itoh et al 1997 Liu et al 1998) Although gene KO mice have the advantage of determining the function of that protein a compensato-ry mechanism might work In addition major pathway canrsquot be discussed between gene KO pathway and inde-pendent pathway because the gene itself is lost in KO mice It is still unclear whether major protective pathway against CCl4 toxicity is MT-dependent or not in sub-le-thal dose our results suggest that the MT-dependent path-way may be the primary mechanism of response to expo-sure to CCl4 levels that evoke lethal toxicity Even if

Fig 4 Effect of single pretreatment with Cd on MT levels in mouse liver Mice were injected sc with 3 mgkg CdCl2 (A) and (B) Twenty-four hr later total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then performed The amount of quantifi ed target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respec-tively (C) The liver samples were collected and the MT protein levels were determined Data are expressed as mean plusmn SD of three mice P lt 001 versus control group

Fig 5 Effect of multiple Zn pretreatment on ALT levels Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 The ALT activity in plasma was determined 6 hr after the injection Data indicate mean plusmn SD of three or four mice P lt 001 versus control group

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Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

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MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

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Hepatoprotection by zinc against carbon tetrachloride-induced lethality

Sato and Kondoh 2002) Owing to these abilities the protective role of MT against metal toxicity has been extensively investigated For instance pretreatment with Zn or low doses of Cd as a MT inducer results in reduced lethality after administration of high doses of Cd (Webb 1972 Probst et al 1977 Coogan et al 1994) Addition-ally we previously showed that multiple injections with Zn or Cd significantly increased MT levels compared to single injections with Zn or Cd (Onosaka and Cherian 1981 1982)

Several studies have shown some compounds show protective effects against CCl4-induced hepatotoxicity (Singh et al 2008 Huang et al 2012 Ma et al 2014 Zhang et al 2014) These investigations focused prima-rily on blood levels of liver enzymes alanine aminotrans-ferase (ALT) and aspartate aminotransferase (AST) (markers of liver damage) histological evaluation lipid peroxidation and expression levels of cytochromes P450 These papers did not examine CCl4-induced lethal toxici-ty because the dose of CCl4 used in these studies was low-er than the LD50 Therefore prevention of CCl4-induced lethal toxicity has not been reported In the present study we investigated whether multiple pretreatments with Zn were sufficient to attenuate CCl4-induced lethal toxicity

MATERIALS AND METHODS

Animal treatmentMale ddY mice were purchased from Japan SLC

(Shizuoka Japan) and were maintained under standard conditions of controlled temperature (24 plusmn 1degC) humid-ity (55 plusmn 5) and light (1212 hr lightdark cycles) with free access to water and food Experimental treatments were performed using eight-week-old animals All exper-iments were approved by the Institutional Animal Care and Experiment Committee of Kobe Gakuin University (NOA14-22)

Administration of CCl4 and evaluation of mortality

Mice were divided into three groups of five mice each Members of each group were treated at doses of CCl4 (Wako Chemical Tokyo Japan) ranging from 1 to 16 gkg administered as emulsions in olive oil and delivered by intraperitoneal (ip) injection by subcutaneous (sc) injection or by oral gavage (per os po) The volume of administered CCl4 was 10 mLkg body weight (for dos-ing at 16 gkg) or 5 mLkg body weight (for other dose levels) Mortality was tracked for 24 hr following CCl4-exposure Following the experiment any surviving mice were sacrificed using pentobarbital

Induction of MT by Zn or Cd injectionMice were divided into two groups of ten or eleven

mice each At -72 -48 and -24 hr animals of the group-1 were administered at 24-hr intervals by sc injection with 50 mgkg ZnSO4 (Nacalai Tesque Kyoto Japan) Twenty-four hours after the final ZnSO4 injection both group-1 and group-2 (not treated with ZnSO4) were injected ip with 4 gkg (at 5 mLkg) CCl4 At 6 hr after the CCl4 injection three randomly selected mice from each group were euth-anized and bled for plasma The resulting plasma sam-ples were stored at -80degC (pending alanine aminotrans-ferase (ALT) assays) Livers were harvested from each of these animals and separate samples from each liver were stored at -80degC (pending quantitative reverse transcription (RT)-PCR assay) or fixed in 15 neutral buffered forma-lin (pH 72) (pending processing for histological evalua-tion) The remaining seven or eight mice of each group were monitored every 3 hr until 24 hr after CCl4 admin-istration to determine acute mortality the surviving ani-mals were weighed and then terminated Using equiva-lent protocols protection from CCl4 lethal toxicity was assessed using single pre-CCl4 injections with 50 mgkg ZnSO4 or with 3 mgkg CdCl2 25H2O (Wako Chemical) administered sc

Measurement of ALTPlasma ALT activities were measured using the

Transaminase CII Test Wako (Wako Chemical) accord-ing to the manufacturerrsquos instructions Each plasma sam-ple (20 μL) was mixed with substrate mixture (1 mL) incubated at 37degC for 20 min and then quenched by addi-tion of stopping solution (2 mL) The absorbance of blue pigment formed by the reaction was measured at 555 nm For relative quantification calibration curves were pre-pared using a standard solution

Histopathological studiesFor histological analysis a portion of liver left lobe

was fixed using 15 neutral buffered formalin (pH 72) processed using standard methodologies and embedded in paraffin Embedded tissues were sectioned at 4-μm thicknesses mounted on slides and stained with hema-toxylin and eosin (HampE) or periodic acid-Schiff (PAS) using standard methodologies Histopathological features in the slices were observed with a light microscope

Isolation of total RNA and RT-PCR assayTotal RNA was extracted from 01-g liver sections

using ISOGEN II (Nippon Gene Tokyo Japan) Aliquots (1 μg) of total RNA from each specimen were reverse-transcribed using Super Script III Reverse Transcriptase

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reagent (Invitrogen Carlsbad CA USA) with oligo-dT as a primer and a 1-hr incubation at 50degC the reaction mixture containing synthesized cDNA then was diluted three-fold with Tris-EDTA buffer

For the quantitative RT-PCR assay aliquots (2 μL) of diluted reverse-transcription products were amplified using an Applied Biosystems 7300 (Applied Biosystems Foster City CA USA) in a reaction mixture containing SYBR Premix Ex Taq II (Takara Bio Shiga Japan) and each primer at 05 μM After pre-incubation of the reac-tion mixtures at 95degC for 30 sec real-time PCR amplifi-cation was performed with the PCR conditions and prim-er pairs shown in Table 1 Relative expression of each mRNA was determined using the standard curve method The amount of each quantified target mRNA was normal-ized against that of GAPDH-encoding mRNA

Determination of MT levels in the liverThe hepatic MT protein levels were determined by Cd

saturation-hemolysate method (Cd-hem method) Liver tissue was homogenized with 5 vols of 025 M sucrose The homogenates were centrifuged at 18000 g for 20 min at 4degC to separate the postmitochondrial supernatant and suitable aliquots were used for MT assay by the Cd-hem method as described previously (Onosaka and Cherian 1981 1982)

Statistical AnalysisAll data from the control and treatment groups were

obtained from the same numbers of replicated experi-ments All experiments were performed independent-ly at least two times The results of the acute CCl4 tox-icity were analyzed by means of Kruskal-Wallis test ALT value comparisons were made by using post-hoc Tukey-Kramerrsquos test All statistical analyses were per-formed using SPSS 190J software (Chicago IL USA) Values of P lt 005 were considered statistically signifi-cant

RESULTS AND DISCUSSION

Difference in mortality by administration routes of CCl4

First to determine the lethal dose of CCl4 in our model (male ddY mice) we performed an escalating-dose exper-iment with CCl4 administered by either of three different routes Mortality was estimated at 24 hr after administra-tion As shown in Table 2 mouse death was not observed in the sc-administered group even at the highest tested dose-level (16 gkg) In contrast ip administration at 4 and more than 8 gkg yielded 80 and 100 mortality respectively In the po-administered group 40 of mice died with dosing at 16 gkg mouse death was not observed at lower test dose-levels Previous reports indicated LD50 values for CCl4 in mice of 304 gkg 09-47 gkg and 121-144 gkg when administered via sc ip and po routes respectively (Klaassen and Plaa 1966 1967 Gehring 1968) Thus our results were consistent with lit-erature values In addition ip administration more than 8 gkg was observed sudden death within 3 hr after CCl4 injection These doses were difficult to use in the sub-sequent protection assays because toxic levels were too strong to evaluate the defensive role Based on our results

Table 1 Oligonucleotide primer sequences and PCR conditions for real-time RT-PCRGene(Accession No)

Primer sequences PCR condition PCR Product length (bp)Sequence (5rsquo to 3rsquo) Denaturation Elongation Cycle No

MT-I Forward TTC ACC AGA TCT CGG AAT GG 95 degC 60 degC40 99

(NM_013602) Reverse GAG GTG CAC TTG CAG TTC TTG 5 sec 30 secMT-II Forward CCT GCA AAT GCA AAC AAT GC 95 degC 60 degC

40 118(NM_008630) Reverse CAC TTG TCG GAA GCC TCT TTG 5 sec 30 secGAPDH Forward TGG TGA AGG TCG GTG TGA AC 95 degC 60 degC

40 98(NM_008084) Reverse GTC GTT GAT GGC AAC AAT CTC C 5 sec 30 sec

Table 2 Mortality of three different administration routes on acute CCl4 toxicity

CCl4 dose (gkg) ip po sc1 05 05 052 05 05 054 45 05 058 55 05 0516 55 25 05Mortality was tracked for 24 hr following exposure at a given dose level

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we selected a CCl4 dose of 4 gkg ip for use in the next assays

Effect of Zn and Cd against acute CCl4 toxicityTo evaluate whether MT rescues CCl4-induced lethal

toxicity we selected Zn as an inducer of MT because we and other groups demonstrated that Zn is the most potent inducers of MT (Onosaka and Cherian 1982 Onosaka et al 1984 Santon et al 2006) In the present study 50 mgkg of ZnSO4 was pretreated to mice subcutaneous-ly for one or three times (at 24-hr intervals) We assessed induction of MT-I- and MT-II-encoding genes by using quantitative RT-PCR to measure the liver levels of the corresponding transcripts (Fig 1) Single pretreatment with ZnSO4 yielded significant enrichment in liver levels of MT-I and MT-II mRNAs (150- and 58-fold respective-ly compared to control) Moreover animals treated pro-phylactically with 3-times of ZnSO4 (50 mgkg sc qd) exhibited significant elevations of MT-I and MT-II mRNA

levels (224- and 95-fold respectively) compared to con-trol animals (Fig 1A and B) In addition we determined hepatic MT protein levels by Cd-hem method MT pro-tein induction is observed in a dose dependent manner (Fig 1C) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Zn (Onosaka and Cherian 1982)

We next examined the ability of our ZnSO4 prophy-laxis (single or three sc qd doses at 50 mgkg) to coun-teract acute CCl4 toxicity we monitored mortality every 3 hr until 24 hr after CCl4 ip injection As shown in Fig 2 lethal toxicity of CCl4 started to be observed from 6 hr after injection and the percentage of death reached to 714 (57) at 24 hr after injection consistent with our earlier escalating-dose study (Table 1) Single sc pre-treatment with 50 mgkg ZnSO4 reduced mortality to 28 (27) Furthermore 3-times pretreatment with ZnSO4 completely prevented CCl4-induced acute lethal toxicity with all seven test animals survived

Fig 1 Effect of multiple pretreatment with Zn on MT levels in mouse liver Mice were injected sc with 50 mgkg ZnSO4 every 24 hr for three times (A) and (B) Total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then per-formed The amount of quantified target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respectively (C) The liver samples were collected followed by determining the MT protein levels Data are expressed as mean plusmn SD of three or four mice significantly different from compared values (P lt 005 and P lt 001)

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Zn is well known as an essential trace element for a variety of biological activities Zn has catalytic and struc-tural roles in more than 300 metalloenzymes as well as regulatory roles in diverse cellular processes such as sig-naling transduction and gene expression (McCall et al 2000) Many studies have demonstrated that Zn per se functions as antioxidant and also plays an important role in regulation of cellular glutathione (GSH) (Parat et al 1997 Powell 2000 Zhou et al 2005) Although GSH is the most important molecule involved in cellular anti-oxidant defense radical scavenging activity of GSH is 300-times lower than that of MT (Thornalley and Vasak 1985) Moreover Santon et al (2006) reported Zn alone does not completely exert anti-oxidant effect against met-al toxicity using MT knock out cell line which implies the protective effect of Zn against oxidative stress is mainly due to the induction of MT rather than a direct action of Zn

To further examine the protective involvement of MTs against CCl4-induced lethal toxicity we investigated the ability of 3 mgkg Cd pretreatment to prevent CCl4-in-

duced lethal toxicity As shown in Figs 3 and 4 ip administration of CCl4 yielded 86 (67) mortality after 24 hr injection single-dose pretreatment with Cd reduced frequency of death to 25 (28) (Fig 3) Thus single-dose Cd prophylaxis protects from CCl4-induced lethal toxicity as well as single-dose Zn although potency was weaker than that of 3-times Zn prophylaxis (in that some mice still died) As with Zn pretreatment Cd exposure also induced liver MT-I and MT-II mRNA to levels 94- and 30-fold (respectively) higher than those detected in the control and MT protein levels were also increased significantly (Fig 4) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Cd (Onosaka and Cherian 1981)

Cd is classified as non-essential trace element and Cd stimulates the expression of various types of genes The most dramatic regulatory effect of Cd (non-toxic concen-trations) is its capability to induce the expression of the genes for the syntheses of MT and GSH at low concentra-tions (Goering and Klaassen 1983 Hatcher et al 1995) At more elevated concentrations Cd also induces a series of stress proteins like HSP70 (Goering et al 1993) Moreover Goering et al reported that a low dose of Cd renders animals highly tolerant to Cd-induced lethality and that this effect is mediated through induction of MT expression (Goering and Klaassen 1983) Taken together these findings suggest that the main regulatory effect of

Fig 2 Effect of multiple pretreatment with Zn on acute CCl4 toxicity (A) Schematic experimental design of pre-treatment with Zn and CCl4 injection (B) Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr After pretreatment mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortality in each group Data indi-cate seven mice The CCl4 1xZn + CCl4 and 3xZn + CCl4 groups are represented with white triangles white circles and black circles respectively P lt 005 ver-sus 3xZn + CCl4 group

Fig 3 Effect of single pretreatment with Cd on acute CCl4 toxicity Mice were injected sc once with 3 mgkg CdCl2 24 hr before CCl4 injection Twenty-four hr lat-er mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortal-ity in each group Data indicate seven or eight mice The CCl4 and Cd + CCl4 groups are represented with white triangles and black squares respectively

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Cd is to induce MT synthesis and we postulate that the Cd prophylaxis in the present study protected against CCl4-induced lethality through induction of MT expression

Hepatoprotective pathway against CCl4 is report-ed through MT-dependent pathway in sub-lethal dose (Klaassen and Cagen 1981 Clarke and Lui 1986) On the other hand another pathway is also observed using MT knock-out (KO) mice (Itoh et al 1997 Liu et al 1998) Although gene KO mice have the advantage of determining the function of that protein a compensato-ry mechanism might work In addition major pathway canrsquot be discussed between gene KO pathway and inde-pendent pathway because the gene itself is lost in KO mice It is still unclear whether major protective pathway against CCl4 toxicity is MT-dependent or not in sub-le-thal dose our results suggest that the MT-dependent path-way may be the primary mechanism of response to expo-sure to CCl4 levels that evoke lethal toxicity Even if

Fig 4 Effect of single pretreatment with Cd on MT levels in mouse liver Mice were injected sc with 3 mgkg CdCl2 (A) and (B) Twenty-four hr later total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then performed The amount of quantifi ed target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respec-tively (C) The liver samples were collected and the MT protein levels were determined Data are expressed as mean plusmn SD of three mice P lt 001 versus control group

Fig 5 Effect of multiple Zn pretreatment on ALT levels Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 The ALT activity in plasma was determined 6 hr after the injection Data indicate mean plusmn SD of three or four mice P lt 001 versus control group

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Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

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MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

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Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

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reagent (Invitrogen Carlsbad CA USA) with oligo-dT as a primer and a 1-hr incubation at 50degC the reaction mixture containing synthesized cDNA then was diluted three-fold with Tris-EDTA buffer

For the quantitative RT-PCR assay aliquots (2 μL) of diluted reverse-transcription products were amplified using an Applied Biosystems 7300 (Applied Biosystems Foster City CA USA) in a reaction mixture containing SYBR Premix Ex Taq II (Takara Bio Shiga Japan) and each primer at 05 μM After pre-incubation of the reac-tion mixtures at 95degC for 30 sec real-time PCR amplifi-cation was performed with the PCR conditions and prim-er pairs shown in Table 1 Relative expression of each mRNA was determined using the standard curve method The amount of each quantified target mRNA was normal-ized against that of GAPDH-encoding mRNA

Determination of MT levels in the liverThe hepatic MT protein levels were determined by Cd

saturation-hemolysate method (Cd-hem method) Liver tissue was homogenized with 5 vols of 025 M sucrose The homogenates were centrifuged at 18000 g for 20 min at 4degC to separate the postmitochondrial supernatant and suitable aliquots were used for MT assay by the Cd-hem method as described previously (Onosaka and Cherian 1981 1982)

Statistical AnalysisAll data from the control and treatment groups were

obtained from the same numbers of replicated experi-ments All experiments were performed independent-ly at least two times The results of the acute CCl4 tox-icity were analyzed by means of Kruskal-Wallis test ALT value comparisons were made by using post-hoc Tukey-Kramerrsquos test All statistical analyses were per-formed using SPSS 190J software (Chicago IL USA) Values of P lt 005 were considered statistically signifi-cant

RESULTS AND DISCUSSION

Difference in mortality by administration routes of CCl4

First to determine the lethal dose of CCl4 in our model (male ddY mice) we performed an escalating-dose exper-iment with CCl4 administered by either of three different routes Mortality was estimated at 24 hr after administra-tion As shown in Table 2 mouse death was not observed in the sc-administered group even at the highest tested dose-level (16 gkg) In contrast ip administration at 4 and more than 8 gkg yielded 80 and 100 mortality respectively In the po-administered group 40 of mice died with dosing at 16 gkg mouse death was not observed at lower test dose-levels Previous reports indicated LD50 values for CCl4 in mice of 304 gkg 09-47 gkg and 121-144 gkg when administered via sc ip and po routes respectively (Klaassen and Plaa 1966 1967 Gehring 1968) Thus our results were consistent with lit-erature values In addition ip administration more than 8 gkg was observed sudden death within 3 hr after CCl4 injection These doses were difficult to use in the sub-sequent protection assays because toxic levels were too strong to evaluate the defensive role Based on our results

Table 1 Oligonucleotide primer sequences and PCR conditions for real-time RT-PCRGene(Accession No)

Primer sequences PCR condition PCR Product length (bp)Sequence (5rsquo to 3rsquo) Denaturation Elongation Cycle No

MT-I Forward TTC ACC AGA TCT CGG AAT GG 95 degC 60 degC40 99

(NM_013602) Reverse GAG GTG CAC TTG CAG TTC TTG 5 sec 30 secMT-II Forward CCT GCA AAT GCA AAC AAT GC 95 degC 60 degC

40 118(NM_008630) Reverse CAC TTG TCG GAA GCC TCT TTG 5 sec 30 secGAPDH Forward TGG TGA AGG TCG GTG TGA AC 95 degC 60 degC

40 98(NM_008084) Reverse GTC GTT GAT GGC AAC AAT CTC C 5 sec 30 sec

Table 2 Mortality of three different administration routes on acute CCl4 toxicity

CCl4 dose (gkg) ip po sc1 05 05 052 05 05 054 45 05 058 55 05 0516 55 25 05Mortality was tracked for 24 hr following exposure at a given dose level

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Hepatoprotection by zinc against carbon tetrachloride-induced lethality

we selected a CCl4 dose of 4 gkg ip for use in the next assays

Effect of Zn and Cd against acute CCl4 toxicityTo evaluate whether MT rescues CCl4-induced lethal

toxicity we selected Zn as an inducer of MT because we and other groups demonstrated that Zn is the most potent inducers of MT (Onosaka and Cherian 1982 Onosaka et al 1984 Santon et al 2006) In the present study 50 mgkg of ZnSO4 was pretreated to mice subcutaneous-ly for one or three times (at 24-hr intervals) We assessed induction of MT-I- and MT-II-encoding genes by using quantitative RT-PCR to measure the liver levels of the corresponding transcripts (Fig 1) Single pretreatment with ZnSO4 yielded significant enrichment in liver levels of MT-I and MT-II mRNAs (150- and 58-fold respective-ly compared to control) Moreover animals treated pro-phylactically with 3-times of ZnSO4 (50 mgkg sc qd) exhibited significant elevations of MT-I and MT-II mRNA

levels (224- and 95-fold respectively) compared to con-trol animals (Fig 1A and B) In addition we determined hepatic MT protein levels by Cd-hem method MT pro-tein induction is observed in a dose dependent manner (Fig 1C) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Zn (Onosaka and Cherian 1982)

We next examined the ability of our ZnSO4 prophy-laxis (single or three sc qd doses at 50 mgkg) to coun-teract acute CCl4 toxicity we monitored mortality every 3 hr until 24 hr after CCl4 ip injection As shown in Fig 2 lethal toxicity of CCl4 started to be observed from 6 hr after injection and the percentage of death reached to 714 (57) at 24 hr after injection consistent with our earlier escalating-dose study (Table 1) Single sc pre-treatment with 50 mgkg ZnSO4 reduced mortality to 28 (27) Furthermore 3-times pretreatment with ZnSO4 completely prevented CCl4-induced acute lethal toxicity with all seven test animals survived

Fig 1 Effect of multiple pretreatment with Zn on MT levels in mouse liver Mice were injected sc with 50 mgkg ZnSO4 every 24 hr for three times (A) and (B) Total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then per-formed The amount of quantified target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respectively (C) The liver samples were collected followed by determining the MT protein levels Data are expressed as mean plusmn SD of three or four mice significantly different from compared values (P lt 005 and P lt 001)

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Zn is well known as an essential trace element for a variety of biological activities Zn has catalytic and struc-tural roles in more than 300 metalloenzymes as well as regulatory roles in diverse cellular processes such as sig-naling transduction and gene expression (McCall et al 2000) Many studies have demonstrated that Zn per se functions as antioxidant and also plays an important role in regulation of cellular glutathione (GSH) (Parat et al 1997 Powell 2000 Zhou et al 2005) Although GSH is the most important molecule involved in cellular anti-oxidant defense radical scavenging activity of GSH is 300-times lower than that of MT (Thornalley and Vasak 1985) Moreover Santon et al (2006) reported Zn alone does not completely exert anti-oxidant effect against met-al toxicity using MT knock out cell line which implies the protective effect of Zn against oxidative stress is mainly due to the induction of MT rather than a direct action of Zn

To further examine the protective involvement of MTs against CCl4-induced lethal toxicity we investigated the ability of 3 mgkg Cd pretreatment to prevent CCl4-in-

duced lethal toxicity As shown in Figs 3 and 4 ip administration of CCl4 yielded 86 (67) mortality after 24 hr injection single-dose pretreatment with Cd reduced frequency of death to 25 (28) (Fig 3) Thus single-dose Cd prophylaxis protects from CCl4-induced lethal toxicity as well as single-dose Zn although potency was weaker than that of 3-times Zn prophylaxis (in that some mice still died) As with Zn pretreatment Cd exposure also induced liver MT-I and MT-II mRNA to levels 94- and 30-fold (respectively) higher than those detected in the control and MT protein levels were also increased significantly (Fig 4) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Cd (Onosaka and Cherian 1981)

Cd is classified as non-essential trace element and Cd stimulates the expression of various types of genes The most dramatic regulatory effect of Cd (non-toxic concen-trations) is its capability to induce the expression of the genes for the syntheses of MT and GSH at low concentra-tions (Goering and Klaassen 1983 Hatcher et al 1995) At more elevated concentrations Cd also induces a series of stress proteins like HSP70 (Goering et al 1993) Moreover Goering et al reported that a low dose of Cd renders animals highly tolerant to Cd-induced lethality and that this effect is mediated through induction of MT expression (Goering and Klaassen 1983) Taken together these findings suggest that the main regulatory effect of

Fig 2 Effect of multiple pretreatment with Zn on acute CCl4 toxicity (A) Schematic experimental design of pre-treatment with Zn and CCl4 injection (B) Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr After pretreatment mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortality in each group Data indi-cate seven mice The CCl4 1xZn + CCl4 and 3xZn + CCl4 groups are represented with white triangles white circles and black circles respectively P lt 005 ver-sus 3xZn + CCl4 group

Fig 3 Effect of single pretreatment with Cd on acute CCl4 toxicity Mice were injected sc once with 3 mgkg CdCl2 24 hr before CCl4 injection Twenty-four hr lat-er mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortal-ity in each group Data indicate seven or eight mice The CCl4 and Cd + CCl4 groups are represented with white triangles and black squares respectively

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Cd is to induce MT synthesis and we postulate that the Cd prophylaxis in the present study protected against CCl4-induced lethality through induction of MT expression

Hepatoprotective pathway against CCl4 is report-ed through MT-dependent pathway in sub-lethal dose (Klaassen and Cagen 1981 Clarke and Lui 1986) On the other hand another pathway is also observed using MT knock-out (KO) mice (Itoh et al 1997 Liu et al 1998) Although gene KO mice have the advantage of determining the function of that protein a compensato-ry mechanism might work In addition major pathway canrsquot be discussed between gene KO pathway and inde-pendent pathway because the gene itself is lost in KO mice It is still unclear whether major protective pathway against CCl4 toxicity is MT-dependent or not in sub-le-thal dose our results suggest that the MT-dependent path-way may be the primary mechanism of response to expo-sure to CCl4 levels that evoke lethal toxicity Even if

Fig 4 Effect of single pretreatment with Cd on MT levels in mouse liver Mice were injected sc with 3 mgkg CdCl2 (A) and (B) Twenty-four hr later total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then performed The amount of quantifi ed target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respec-tively (C) The liver samples were collected and the MT protein levels were determined Data are expressed as mean plusmn SD of three mice P lt 001 versus control group

Fig 5 Effect of multiple Zn pretreatment on ALT levels Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 The ALT activity in plasma was determined 6 hr after the injection Data indicate mean plusmn SD of three or four mice P lt 001 versus control group

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Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

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MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

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Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

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we selected a CCl4 dose of 4 gkg ip for use in the next assays

Effect of Zn and Cd against acute CCl4 toxicityTo evaluate whether MT rescues CCl4-induced lethal

toxicity we selected Zn as an inducer of MT because we and other groups demonstrated that Zn is the most potent inducers of MT (Onosaka and Cherian 1982 Onosaka et al 1984 Santon et al 2006) In the present study 50 mgkg of ZnSO4 was pretreated to mice subcutaneous-ly for one or three times (at 24-hr intervals) We assessed induction of MT-I- and MT-II-encoding genes by using quantitative RT-PCR to measure the liver levels of the corresponding transcripts (Fig 1) Single pretreatment with ZnSO4 yielded significant enrichment in liver levels of MT-I and MT-II mRNAs (150- and 58-fold respective-ly compared to control) Moreover animals treated pro-phylactically with 3-times of ZnSO4 (50 mgkg sc qd) exhibited significant elevations of MT-I and MT-II mRNA

levels (224- and 95-fold respectively) compared to con-trol animals (Fig 1A and B) In addition we determined hepatic MT protein levels by Cd-hem method MT pro-tein induction is observed in a dose dependent manner (Fig 1C) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Zn (Onosaka and Cherian 1982)

We next examined the ability of our ZnSO4 prophy-laxis (single or three sc qd doses at 50 mgkg) to coun-teract acute CCl4 toxicity we monitored mortality every 3 hr until 24 hr after CCl4 ip injection As shown in Fig 2 lethal toxicity of CCl4 started to be observed from 6 hr after injection and the percentage of death reached to 714 (57) at 24 hr after injection consistent with our earlier escalating-dose study (Table 1) Single sc pre-treatment with 50 mgkg ZnSO4 reduced mortality to 28 (27) Furthermore 3-times pretreatment with ZnSO4 completely prevented CCl4-induced acute lethal toxicity with all seven test animals survived

Fig 1 Effect of multiple pretreatment with Zn on MT levels in mouse liver Mice were injected sc with 50 mgkg ZnSO4 every 24 hr for three times (A) and (B) Total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then per-formed The amount of quantified target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respectively (C) The liver samples were collected followed by determining the MT protein levels Data are expressed as mean plusmn SD of three or four mice significantly different from compared values (P lt 005 and P lt 001)

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H Yoshioka et al

Zn is well known as an essential trace element for a variety of biological activities Zn has catalytic and struc-tural roles in more than 300 metalloenzymes as well as regulatory roles in diverse cellular processes such as sig-naling transduction and gene expression (McCall et al 2000) Many studies have demonstrated that Zn per se functions as antioxidant and also plays an important role in regulation of cellular glutathione (GSH) (Parat et al 1997 Powell 2000 Zhou et al 2005) Although GSH is the most important molecule involved in cellular anti-oxidant defense radical scavenging activity of GSH is 300-times lower than that of MT (Thornalley and Vasak 1985) Moreover Santon et al (2006) reported Zn alone does not completely exert anti-oxidant effect against met-al toxicity using MT knock out cell line which implies the protective effect of Zn against oxidative stress is mainly due to the induction of MT rather than a direct action of Zn

To further examine the protective involvement of MTs against CCl4-induced lethal toxicity we investigated the ability of 3 mgkg Cd pretreatment to prevent CCl4-in-

duced lethal toxicity As shown in Figs 3 and 4 ip administration of CCl4 yielded 86 (67) mortality after 24 hr injection single-dose pretreatment with Cd reduced frequency of death to 25 (28) (Fig 3) Thus single-dose Cd prophylaxis protects from CCl4-induced lethal toxicity as well as single-dose Zn although potency was weaker than that of 3-times Zn prophylaxis (in that some mice still died) As with Zn pretreatment Cd exposure also induced liver MT-I and MT-II mRNA to levels 94- and 30-fold (respectively) higher than those detected in the control and MT protein levels were also increased significantly (Fig 4) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Cd (Onosaka and Cherian 1981)

Cd is classified as non-essential trace element and Cd stimulates the expression of various types of genes The most dramatic regulatory effect of Cd (non-toxic concen-trations) is its capability to induce the expression of the genes for the syntheses of MT and GSH at low concentra-tions (Goering and Klaassen 1983 Hatcher et al 1995) At more elevated concentrations Cd also induces a series of stress proteins like HSP70 (Goering et al 1993) Moreover Goering et al reported that a low dose of Cd renders animals highly tolerant to Cd-induced lethality and that this effect is mediated through induction of MT expression (Goering and Klaassen 1983) Taken together these findings suggest that the main regulatory effect of

Fig 2 Effect of multiple pretreatment with Zn on acute CCl4 toxicity (A) Schematic experimental design of pre-treatment with Zn and CCl4 injection (B) Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr After pretreatment mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortality in each group Data indi-cate seven mice The CCl4 1xZn + CCl4 and 3xZn + CCl4 groups are represented with white triangles white circles and black circles respectively P lt 005 ver-sus 3xZn + CCl4 group

Fig 3 Effect of single pretreatment with Cd on acute CCl4 toxicity Mice were injected sc once with 3 mgkg CdCl2 24 hr before CCl4 injection Twenty-four hr lat-er mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortal-ity in each group Data indicate seven or eight mice The CCl4 and Cd + CCl4 groups are represented with white triangles and black squares respectively

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Cd is to induce MT synthesis and we postulate that the Cd prophylaxis in the present study protected against CCl4-induced lethality through induction of MT expression

Hepatoprotective pathway against CCl4 is report-ed through MT-dependent pathway in sub-lethal dose (Klaassen and Cagen 1981 Clarke and Lui 1986) On the other hand another pathway is also observed using MT knock-out (KO) mice (Itoh et al 1997 Liu et al 1998) Although gene KO mice have the advantage of determining the function of that protein a compensato-ry mechanism might work In addition major pathway canrsquot be discussed between gene KO pathway and inde-pendent pathway because the gene itself is lost in KO mice It is still unclear whether major protective pathway against CCl4 toxicity is MT-dependent or not in sub-le-thal dose our results suggest that the MT-dependent path-way may be the primary mechanism of response to expo-sure to CCl4 levels that evoke lethal toxicity Even if

Fig 4 Effect of single pretreatment with Cd on MT levels in mouse liver Mice were injected sc with 3 mgkg CdCl2 (A) and (B) Twenty-four hr later total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then performed The amount of quantifi ed target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respec-tively (C) The liver samples were collected and the MT protein levels were determined Data are expressed as mean plusmn SD of three mice P lt 001 versus control group

Fig 5 Effect of multiple Zn pretreatment on ALT levels Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 The ALT activity in plasma was determined 6 hr after the injection Data indicate mean plusmn SD of three or four mice P lt 001 versus control group

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Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

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Hepatoprotection by zinc against carbon tetrachloride-induced lethality

MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

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62

H Yoshioka et al

Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

Vol 41 No 1

63

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

Zn is well known as an essential trace element for a variety of biological activities Zn has catalytic and struc-tural roles in more than 300 metalloenzymes as well as regulatory roles in diverse cellular processes such as sig-naling transduction and gene expression (McCall et al 2000) Many studies have demonstrated that Zn per se functions as antioxidant and also plays an important role in regulation of cellular glutathione (GSH) (Parat et al 1997 Powell 2000 Zhou et al 2005) Although GSH is the most important molecule involved in cellular anti-oxidant defense radical scavenging activity of GSH is 300-times lower than that of MT (Thornalley and Vasak 1985) Moreover Santon et al (2006) reported Zn alone does not completely exert anti-oxidant effect against met-al toxicity using MT knock out cell line which implies the protective effect of Zn against oxidative stress is mainly due to the induction of MT rather than a direct action of Zn

To further examine the protective involvement of MTs against CCl4-induced lethal toxicity we investigated the ability of 3 mgkg Cd pretreatment to prevent CCl4-in-

duced lethal toxicity As shown in Figs 3 and 4 ip administration of CCl4 yielded 86 (67) mortality after 24 hr injection single-dose pretreatment with Cd reduced frequency of death to 25 (28) (Fig 3) Thus single-dose Cd prophylaxis protects from CCl4-induced lethal toxicity as well as single-dose Zn although potency was weaker than that of 3-times Zn prophylaxis (in that some mice still died) As with Zn pretreatment Cd exposure also induced liver MT-I and MT-II mRNA to levels 94- and 30-fold (respectively) higher than those detected in the control and MT protein levels were also increased significantly (Fig 4) These results were consistent with our previous demonstration of MT protein accumulation in the liver of mice exposed to Cd (Onosaka and Cherian 1981)

Cd is classified as non-essential trace element and Cd stimulates the expression of various types of genes The most dramatic regulatory effect of Cd (non-toxic concen-trations) is its capability to induce the expression of the genes for the syntheses of MT and GSH at low concentra-tions (Goering and Klaassen 1983 Hatcher et al 1995) At more elevated concentrations Cd also induces a series of stress proteins like HSP70 (Goering et al 1993) Moreover Goering et al reported that a low dose of Cd renders animals highly tolerant to Cd-induced lethality and that this effect is mediated through induction of MT expression (Goering and Klaassen 1983) Taken together these findings suggest that the main regulatory effect of

Fig 2 Effect of multiple pretreatment with Zn on acute CCl4 toxicity (A) Schematic experimental design of pre-treatment with Zn and CCl4 injection (B) Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr After pretreatment mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortality in each group Data indi-cate seven mice The CCl4 1xZn + CCl4 and 3xZn + CCl4 groups are represented with white triangles white circles and black circles respectively P lt 005 ver-sus 3xZn + CCl4 group

Fig 3 Effect of single pretreatment with Cd on acute CCl4 toxicity Mice were injected sc once with 3 mgkg CdCl2 24 hr before CCl4 injection Twenty-four hr lat-er mice were injected ip with 4 gkg CCl4 The mice were observed every 3 hr for 24 hr to calculate mortal-ity in each group Data indicate seven or eight mice The CCl4 and Cd + CCl4 groups are represented with white triangles and black squares respectively

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Hepatoprotection by zinc against carbon tetrachloride-induced lethality

Cd is to induce MT synthesis and we postulate that the Cd prophylaxis in the present study protected against CCl4-induced lethality through induction of MT expression

Hepatoprotective pathway against CCl4 is report-ed through MT-dependent pathway in sub-lethal dose (Klaassen and Cagen 1981 Clarke and Lui 1986) On the other hand another pathway is also observed using MT knock-out (KO) mice (Itoh et al 1997 Liu et al 1998) Although gene KO mice have the advantage of determining the function of that protein a compensato-ry mechanism might work In addition major pathway canrsquot be discussed between gene KO pathway and inde-pendent pathway because the gene itself is lost in KO mice It is still unclear whether major protective pathway against CCl4 toxicity is MT-dependent or not in sub-le-thal dose our results suggest that the MT-dependent path-way may be the primary mechanism of response to expo-sure to CCl4 levels that evoke lethal toxicity Even if

Fig 4 Effect of single pretreatment with Cd on MT levels in mouse liver Mice were injected sc with 3 mgkg CdCl2 (A) and (B) Twenty-four hr later total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then performed The amount of quantifi ed target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respec-tively (C) The liver samples were collected and the MT protein levels were determined Data are expressed as mean plusmn SD of three mice P lt 001 versus control group

Fig 5 Effect of multiple Zn pretreatment on ALT levels Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 The ALT activity in plasma was determined 6 hr after the injection Data indicate mean plusmn SD of three or four mice P lt 001 versus control group

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H Yoshioka et al

Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Vol 41 No 1

61

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

Vol 41 No 1

62

H Yoshioka et al

Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

Vol 41 No 1

63

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

Cd is to induce MT synthesis and we postulate that the Cd prophylaxis in the present study protected against CCl4-induced lethality through induction of MT expression

Hepatoprotective pathway against CCl4 is report-ed through MT-dependent pathway in sub-lethal dose (Klaassen and Cagen 1981 Clarke and Lui 1986) On the other hand another pathway is also observed using MT knock-out (KO) mice (Itoh et al 1997 Liu et al 1998) Although gene KO mice have the advantage of determining the function of that protein a compensato-ry mechanism might work In addition major pathway canrsquot be discussed between gene KO pathway and inde-pendent pathway because the gene itself is lost in KO mice It is still unclear whether major protective pathway against CCl4 toxicity is MT-dependent or not in sub-le-thal dose our results suggest that the MT-dependent path-way may be the primary mechanism of response to expo-sure to CCl4 levels that evoke lethal toxicity Even if

Fig 4 Effect of single pretreatment with Cd on MT levels in mouse liver Mice were injected sc with 3 mgkg CdCl2 (A) and (B) Twenty-four hr later total RNA was isolated from mouse livers Quantitative RT-PCR analysis was then performed The amount of quantifi ed target mRNA was normalized by GAPDH mRNA (A) and (B) indicate MT-I and MT-II respec-tively (C) The liver samples were collected and the MT protein levels were determined Data are expressed as mean plusmn SD of three mice P lt 001 versus control group

Fig 5 Effect of multiple Zn pretreatment on ALT levels Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 The ALT activity in plasma was determined 6 hr after the injection Data indicate mean plusmn SD of three or four mice P lt 001 versus control group

Vol 41 No 1

60

H Yoshioka et al

Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Vol 41 No 1

61

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

Vol 41 No 1

62

H Yoshioka et al

Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

Vol 41 No 1

63

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

Fig 6 Hepatoprotective effect of pretreatment with Zn by HampE staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 HampE staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Fig 7 Glycogen storage effect of pretreatment with Zn by PAS staining on acute CCl4 toxicity Mice were injected sc with 50 mgkg ZnSO4 three times per 24 hr Twenty-four hr after fi nal pretreatment mice were injected ip with 4 gkg CCl4 PAS staining of livers were taken 6 hr after the injection (A) (B) (C) and (D) indicate the control 3xZn CCl4 and 3xZn + CCl4 groups respectively

Vol 41 No 1

61

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

Vol 41 No 1

62

H Yoshioka et al

Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

Vol 41 No 1

63

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

MT-independent pathway is majority in sub-lethal dose the balance between the two pathways presumably chang-es depending on the CCl4 dose to which the animal is exposed

Hepatic protective effect on Zn against acute CCl4 toxicity

The primary target organ for CCl4 toxicity is the liv-er Therefore to assess the protective effect of Zn pre-treatment for CCl4 acute liver injury ALT was measured (Fig 5) Plasma samples were collected at 6 hr after ip injection with CCl4 or saline Three times injection with Zn significantly attenuated CCl4-induced acute increases of blood ALT levels (P lt 001) suggesting that pretreat-ment with Zn attenuated CCl4-induced cell damage in liv-er

In parallel with the measurement of ALT we conduct-ed two kinds of histopathological studies (Figs 6 and 7) Liver sections stained with HampE showed normal cell mor-phology well-preserved cytoplasm and a clear plump nucleus in the control and Zn-treated groups However in CCl4-injected mice marked anomalies of liver cells were observed including nuclear chromatin condensation nuclear loss and acidophilic features The liver sections from animals pretreated with Zn revealed that Zn proph-ylaxis reduced or even prevented CCl4-associated dam-age to liver cells Staining of liver sections with PAS stain showed that CCl4 resulted in almost complete depletion of hepatic glycogen (Fig 7) Livers of animals pretreat-ed with Zn recovered some but not all hepatic glycogen content compared to the CCl4-intoxicated group These results suggest that Zn suppresses CCl4-induced lethal toxicity via reducing liver cell damage

The results of the present study indicate that multi-ple pretreatment with Zn reduced the increased ALT and guarded against acute pathological changes induced by CCl4 in the liver The mechanism of CCl4-induced toxic-ity is well documented The highly reactive trichlorome-thyl radicals are generated from CCl4 by the activity of CYP2E1 These radicals initiate lipid peroxidation and necrosis of the liver Trichloromethyl radicals also impair the cellular antioxidant capacity by deactivating GSH and other antioxidant enzymes (Sahreen et al 2011 Khan et al 2012 Huang et al 2013) In the present investigation we did not determine a mechanism for how Zn suppress-es lethal toxicity induced by CCl4 but postulate that this protection is mediated at least in part by MT induction Investigations detailing the protective mechanism against CCl4-induced lethal toxicity are currently in progress

In summary we have provided evidence that three-times pretreatment with Zn completely rescues CCl4-in-

duced lethal toxicity Further investigations will be need-ed to clarify how Zn suppresses lethal toxicity induced by CCl4 and to determine whether and how other target organs contribute to mortality andor protection The ZnCCl4 protection model is expected to contribute to both basic and applied toxicology studies

ACKNOWLEDGMENTS

The authors thank Dr Nobuyuki Fukuishi (Kinjo Gakuin University Japan) for his kind suggestions

Conflict of interest---- The authors declare that there is no conflict of interest

REFERENCES

Ahmad FF Cowan DL and Sun AY (1987) Detection of free radical formation in various tissues after acute carbon tetrachlo-ride administration in gerbil Life Sci 41 2469-2475

Auzinger G and Wendon J (2008) Intensive care management of acute liver failure Curr Opin Crit Care 14 179-188

Bhondave PD Devarshi PP Mahadik KR and Harsulkar AM (2014) Ashvagandharishta prepared using yeast con-sortium from Woodfordia fruticosa flowers exhibit hepatopro-tective effect on CCl4 induced liver damage in Wistar rats J Ethnopharmacol 151 183-190

Cagen SZ and Klaassen CD (1979) Protection of carbon tetra-chloride-induced hepatotoxicity by zinc role of metallothionein Toxicol Appl Pharmacol 51 107-116

Ciccoli L Casini AF and Benedetti A (1978) Free radical dam-age produced by carbon tetrachloride in the lipids of various rat tissues Agents Actions 8 303-310

Clarke IS and Lui EM (1986) Interaction of metallothionein and carbon tetrachloride on the protective effect of zinc on hepa-totoxicity Can J Physiol Pharmacol 64 1104-1110

Coogan TP Bare RM Bjornson EJ and Waalkes MP (1994) Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxicity in cultured rat liver cells J Toxicol Environ Health 41 233-245

Domitrović R Jakovac H and Blagojević G (2011) Hepatopro-tective activity of berberine is mediated by inhibition of TNF-α COX-2 and iNOS expression in CCl(4)-intoxicated mice Toxicology 280 33-43

Gehring PJ (1968) Hepatotoxic potency of various chlorinated hydrocarbon vapours relative to their narcotic and lethal poten-cies in mice Toxicol Appl Pharmacol 13 287-298

Goering PL Fisher BR and Kish CL (1993) Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxici-ty Toxicol Appl Pharmacol 122 139-148

Goering PL and Klaassen CD (1983) Altered subcellular distri-bution of cadmium following cadmium pretreatment possible mechanism of tolerance to cadmium-induced lethality Toxicol Appl Pharmacol 70 195-203

Hatcher EL Chen Y and Kang YJ (1995) Cadmium resistance in A549 cells correlates with elevated glutathione content but not antioxidant enzymatic activities Free Radical Biol Med 19 805-812

Vol 41 No 1

62

H Yoshioka et al

Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

Sato M and Bremner I (1993) Oxygen free radicals and metal-lothionein Free Radic Biol Med 14 325-337

Sato M and Kondoh M (2002) Recent studies on metallothionein protection against toxicity of heavy metals and oxygen free radi-cals Tohoku J Exp Med 196 9-22

Singh N Kamath V Narasimhamurthy K and Rajini PS (2008) Protective effect of potato peel extract against carbon tetrachlo-ride-induced liver injury in rats Environ Toxicol Pharmacol 26 241-246

Suzuki KT and Yamamura M (1980) Induction and degradation of copper-induced metallothioneins in rat liver as studied at iso-metallothionein levels Toxicol Lett 6 301-307

Thornalley PJ and Vasaacutek M (1985) Possible role for metal-lothionein in protection against radiation-induced oxidative stress Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals Biochim Biophys Acta 827 36-44

Wang T Shankar K Ronis MJ and Mehendale HM (2007) Mechanisms and outcomes of drug- and toxicant-induced liver toxicity in diabetes Crit Rev Toxicol 37 413-459

Webb M (1972) Protection by zinc against cadmium toxicity Biochem Pharmacol 21 2767-2771

Weber LW Boll M and Stampfl A (2003) Hepatotoxicity and mechanism of action of haloalkanes carbon tetrachloride as a toxicological model Crit Rev Toxicol 33 105-136

Zhang F Wang X Qiu X Wang J Fang H Wang Z Sun Y and Xia Z (2014) The protective effect of esculentoside a on experimental acute liver injury in mice PLoS One 9 e113107

Zhou Z Wang L Song Z Saari JT McClain CJ and Kang YJ (2005) Zinc supplementation prevents alcoholic liver injury in mice through attenuation of oxidative stress Am J Pathol 166 1681-1690

Vol 41 No 1

63

Hepatoprotection by zinc against carbon tetrachloride-induced lethality

Huang GJ Deng JS Chiu CS Liao JC Hsieh WT Sheu MJ and Wu CH (2012) Hispolon protects against acute liv-er damage in the rat by inhibiting lipid peroxidation proin-flammatory cytokine and oxidative stress and downregulat-ing the expressions of iNOS COX-2 and MMP-9 Evid Based Complement Alternat Med 2012 480714

Huang GJ Deng JS Huang SS Lee CY Hou WC Wang SY Sung PJ and Kuo YH (2013) Hepatoprotective effects of eburicoic acid and dehydroeburicoic acid from Antrodia cam-phorata in a mouse model of acute hepatic injury Food Chem 141 3020-3027

Itoh N Kimura T Nakanishi H Muto N Kobayashi M Kitagawa I and Tanaka K (1997) Metallothionein-independ-ent hepatoprotection by zinc and sakuraso-saponin Toxicol Lett 93 135-140

Khan RA Khan MR and Sahreen S (2012) CCl4-induced hepatotoxicity protective effect of rutin on p53 CYP2E1 and the antioxidative status in rat BMC Complement Altern Med 12 178

Klaassen CD and Cagen SZ (1981) Metallothionein as a trap for reactive organic intermediates Adv Exp Med Biol 136 Pt A 633-646

Klaassen CD and Plaa GL (1966) Relative effects of various chlorinated hydrocarbons on liver and kidney function in mice Toxicol Appl Pharmacol 9 139-151

Klaassen CD and Plaa GL (1967) Susceptibility of male and female mice to the nephrotoxic and hepatotoxic properties of chlorinated hydrocarbons Proc Soc Exp Biol Med 124 1163-1166

Lee SJ and Lim KT (2008) Glycoprotein of Zanthoxylum pip-eritum DC has a hepatoprotective effect via anti-oxidative char-acter in vivo and in vitro Toxicol In Vitro 22 376-385

Liu CM Zheng GH Ming QL Chao C and Sun JM (2013) Sesamin protects mouse liver against nickel-induced oxidative DNA damage and apoptosis by the PI3K-Akt pathway J Agric Food Chem 61 1146-1154

Liu Y Hartley DP and Liu J (1998) Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein Toxicol Lett 95 77-85

Ma JQ Ding J Zhang L and Liu CM (2014) Hepatoprotec-tive properties of sesamin against CCl4 induced oxidative stress-mediated apoptosis in mice via JNK pathway Food Chem Toxicol 64 41-48

Margoshes M and Vallee BL (1957) A cadmium protein from equine imaging hyperintensity in Alzheimerrsquos disease correla-tion with kidney cortex J Am Chem Soc 79 4813-4814

McCall KA Huang C and Fierke CA (2000) Function and mechanism of zinc metalloenzymes J Nutr 130 1437S-1446S

Min KS Terano Y Onosaka S and Tanaka K (1991) Induc-tion of hepatic metallothionein by nonmetallic compounds asso-ciated with acute-phase response in inflammation Toxicol Appl Pharmacol 111 152-162

Onosaka S and Cherian MG (1981) The induced synthesis of metallothionein in various tissues of rat in response to metals I Effect of repeated injection of cadmium salts Toxicology 22

91-101Onosaka S and Cherian MG (1982) The induced synthesis of

metallothionein in various tissues of rats in response to metals II Influence of zinc status and specific effect on pancreatic met-allothionein Toxicology 23 11-20

Onosaka S Tanaka K and Cherian MG (1984) Effects of cad-mium and zinc on tissue levels of metallothionein Environ Health Perspect 54 67-72

Parat MO Richard MJ Beacuteani JC and Favier A (1997) Involvement of zinc in intracellular oxidantantioxidant balance Biol Trace Elem Res 60 187-204

Powell SR (2000) The antioxidant properties of zinc J Nutr 130 1447S-1454S

Probst GS Bousquet WF and Miya TS (1977) Correlation of hepatic metallothionein concentrations with acute cadmium tox-icity in the mouse Toxicol Appl Pharmacol 39 61-69

Sahreen S Khan MR and Khan RA (2011) Hepatoprotective effects of methanol extract of Carissa opaca leaves on CCl4-in-duced damage in rat BMC Complement Altern Med 11 48

Santon A Formigari A Albergoni V and Irato P (2006) Effect of Zn treatment on wild type and MT-null cell lines in relation to apoptotic andor necrotic processes and on MT isoform gene expression Biochim Biophys Acta 1763 305-312

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Hepatoprotection by zinc against carbon tetrachloride-induced lethality