Effects of Curcumin Attenuated Hepatitis in Mice with Paracetamol Overdose ABSTRACT

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1 Originl Article 43 Effects of Curcumin Attenuted Heptitis in Mice with Prcetmol Overdose Somnwt K 1 Thong-Ngm D 1 Klikew N 2 ABSTRACT Bckground: N-cetyl-p-minophenol () or prcetmol overdose cuses incresing of toxic metolites, which disrupting heptocyte function, nd liver injury occurs. Curcumin hs een used for tretment of inflmmtory conditions such s heptitis. Therefore, this study ims to determine effects of curcumin ttenute heptitis in mice with overdose. Methods: Mle mice (25-3 grm) were divided into four groups. Group I (control); mice were gvged with distilled wter. Group II (); mice were gvged with single dose of 4 mg/kg of. Group III ( + CUR 2); mice were gvged with single dose of 4 mg/kg of nd 2 mg/kg of curcumin. Group IV ( + CUR 6); mice were gvged with single dose of 4 mg/kg of nd 6 mg/kg of curcumin. The serum ws collected to determine liver enzymes nd liver tissues for heptic MDA, heptic GSH nd histopthology. Results: Serum ALT, AST nd heptic MDA were significntly incresed in when compred with control nd significntly decresed in + CUR 2 nd + CUR 6 when compred with. Heptic GSH ws significntly decresed in when compred with control nd significntly incresed in + CUR 2 nd + CUR 6 when compred with. Histopthology of showed cute centriloulr hemorrhgic heptic necrosis involving ll zones nd the improvement of liver pthology reveled in + CUR 2 nd + CUR 6. Conclusion: These results suggest tht overdose is relted to liver toxicity. Our results show curcumin cn prevent the dmge y induction of heptic GSH, reduction of oxidtive stress, ttenution of liver inflmmtion, nd the improvement of liver pthology. In ddition, curcumin t the dose of 6 mg/kg tends to e more potent thn 2 mg/kg. Key words : prcetmol, curcumin, heptitis, oxidtive stress [Thi J Gstroenterol 212; 13(1): ] 1 Deprtment of Physiology, 2 Deprtment of Pthology, Fculty of Medicine, Chullongkorn University, Bngkok 133, Thilnd. Address for Correspondence: Assoc. Prof. Dungporn Thong-Ngm, M.D., Deprtment of Physiology, Fculty of Medicine, Chullongkorn University, Bngkok 133, Thilnd.

2 44 THAI J GASTROENTEROL 212 Effects of Curcumin Attenuted Heptitis in Mice with Prcetmol Overdose Drug-induced liver injury (DILI) is mjor helth prolem tht chllenges not only helth cre professionls ut lso the phrmceuticl industry nd drug regultory gencies (1). According to the United Sttes Acute Liver Filure Study Group, DILI ccounts for more thn 5% of cute liver filure, including heptotoxicity cused y overdose of N-cetyl-pminophenol () or prcetmol (39%) nd idiosyncrtic liver injury triggered y other drugs (13%) (2). Although the exct mechnism of DILI remins lrgely unknown, it ppers to involve 2 pthwys: direct heptotoxicity nd dverse immune rections. In most instnces, DILI is initited y the ioctivtion of drugs to chemiclly rective metolites, which hve the ility to interct with cellulr mcromolecules such s proteins, lipids, nd nucleic cids, leding to protein dysfunction, lipid peroxidtion, DNA dmge, nd oxidtive stress. Additionlly, these rective metolites my induce disruption of ionic grdients nd intrcellulr clcium stores, resulting in mitochondril dysfunction nd loss of energy production. This impirment of cellulr function cn culminte in cell deth nd possile liver filure (3). Curcumin is the min yellow ioctive component of turmeric (Curcum long Linn.). It hs een shown to possess wide spectrum of iologicl ctions. These include nti-inflmmtory, ntioxidnt, nticrcinogenic, ntimutgenic, nticogulnt, nd ntidietic ctivities (4-6). The heptoprotection of curcumin hve een widely cknowledged nd used in trditionl medicine for tretment of inflmmtory conditions such s heptitis (7). Therefore, this study ims to determine effects of curcumin ttenute heptitis in mice with overdose. MATERIALS AND METHODS Chemicls Curcumin in powder form (Cymn Chemicl Compny, USA) ws dissolved in corn oil tht ws freshly prepred for the experiment. A single dose of 2 nd 6 mg/kg of curcumin were dministered to mice y orl gvge. A single dose of 4 mg/kg of lso known s Tylenol which is freshly prepred y dissolving in distilled wter (dh 2 O) nd ws dministered to mice y orl gvge. Animls Mle mice (4-5 weeks of ge), weighing 25-3 grm (g), were purchsed from the Ntionl Lortory Animl Center, Sly Cmpus, Mhidol University, Thilnd nd use s experimentl nimls. The mice were cclimtized t lest 1 week in climtecontrolled room on 12-hour (h) light-drk cycle nd were fed d liitum. The study ws performed in dherence to the Ntionl Institutes of Helth guidelines for the experimentl use of nimls nd followed protocol pproved y the Animl Cre nd Use Committee, Fculty of Medicine, Chullongkorn University, Thilnd. The mice were then fsted for 16 h efore experiments to sensitize mice to toxicity. Mice were divided into four groups Group I (control); mice were gvged with dh 2 O. Group II (); mice were gvged with single dose of 4 mg/kg of. Group III ( + CUR 2); mice were gvged with single dose of 4 mg/kg of with single dose of 2 mg/kg of curcumin. Group IV ( + CUR 6); mice were gvged with single dose of 4 mg/kg of with single dose of 6 mg/kg of curcumin. Study design Twenty-four hours fter dministrtion, ll mice ws nesthetized with intrperitonel (i.p.) injection of thiopentl sodium. The domen ws opened medilly nd the whole liver ws rpidly removed nd wshed with cold norml sline (4-8 C). The tissues were chopped into smll pieces with scissors, frozen in liquid nitrogen, nd stored t -8 C for heptic mlondildehyde (MDA) nd heptic glutthione (GSH). The remining liver ws fixed in 1% formlin solution for histopthology. Susequently, the whole lood of mice ws collected from the hert. The lood ws llowed to cogulte t room temperture for 2 h nd then centrifuged for 2 minutes t 3 revolution per minute (r.p.m.) to otin serum. The serum ws collected to determine liver enzymes including AST nd ALT. Heptic MDA ssy Lipid peroxidtion of mice liver using thiorituric cid (TBA) ws mesured y modified method of Ohkw et l (8). One grm of mice liver tissue ws homogenized in 3 ml of 5 mm potssium phosphte uffer (ph 7.). To.3 ml of liver homogented in test tue, 1.5 ml of 1% trichlorocetic cid (TCA) solution nd 1.5 ml of.8% TBA

3 45 solution were dded. The mixture ws oiled in wterth t 95 C for 6 min nd then cooling with tp wter t room temperture. After centrifugtion t 3 r.p.m. for 15 min, the sornce of smple ws mesured t 532 nm. 1, 1, 3, 3 tetrmethoxy propne (TMP) ws used s stndrd of MDA. The MDA content ws clculted in comprison with stndrd MDA curve nd ws expressed s nmol/mg protein. Heptic GSH ssy Cymn GSH Assy utilizes crefully optimized enzymtic recycling method, using GSH reductse, for the quntifiction of GSH (9). The sulfhydryl group of GSH rects with 5, 5'-dithio-is-(2- nitroenzoic cid) (DTNB), or Ellmn s regent nd produces yellow colored 5-thio-2-nitroenzoic cid (TNB). The mixed disulfide, GSTNB (etween GSH nd TNB) tht is concomitntly produced, is reduced y GSH reductse to recycle the GSH nd produce more TNB. The rte of TNB production is directly proportionl to this recycling rection which is in turn directly proportionl to the concentrtion of GSH in the smple. The opticl density (O.D.) of TNB is then mesured t nm using microplte reder, which provides n ccurte estimtion of GSH in the smple. Histopthology After the liver smples hve een fixed in 1% formlin solution t room temperture, they were processed using stndrd method. Briefly, tissues were emedded in prffin, sectioned t 5 μm, nd stined with Hemtoxylin & Eosin (H & E), nd then picked up on glss slides. The histologicl slides were evluted under light microscope (LM) y n experienced pthologist who is linded to the experiment. All fields in ech section were exmined for grding of heptic necroinflmmtion ccording to the criteri descried y Brunt EM et l (1) from to 3 s follow; score = no heptocyte injury/inflmmtion, score 1= sprse or mild focl zone 3 heptocyte injury/inflmmtion, score 2 = noticele zone 3 heptocyte injury/inflmmtion, nd score 3 = severe zone 3 heptocyte injury/ inflmmtion. Sttisticl nlysis All dt were presented s men ± stndrd devition (SD). For comprison mong ll groups of nimls, one wy nlysis of vrince (one-wy ANOVA) nd Tukey PostHoc comprisons were employed. Differences were considered sttisticlly significnt t p <.5. The dt were nlyzed using the SPSS softwre version 17. for Windows. RESULTS The serum AST nd ALT enzymes were significntly incresed in group when compred with control group (AST; ± vs ± 6.9 U/L nd ALT; 186. ± vs ± 6.95 U/L, p <.5) nd significntly decresed in + CUR 2 (AST; ± vs ± U/L nd ALT; ± 3.11 vs ± U/L, p <.5) nd + CUR 6 groups (AST; ± 8.33 vs ± U/L nd ALT; 47.5 ± 4.72 vs ± U/L, p <.5) when compred with group (Tle 1, Figure 1 nd 2). Heptic GSH ws significntly decresed in group when compred with control group (2.75 ±.16 vs ±.11 nmol/mg protein, p <.5). These were significntly incresed in + CUR 2 (9.16 ±.49 vs ±.16 nmol/mg protein, p <.5) nd + CUR 6 groups (9.72 ±.22 vs ±.16 nmol/mg protein, p <.5) when compred with Tle 1. Summry of prmeters in ll groups (n = 8 ech) Prmeters Group AST ALT GSH MDA (U/L) (U/L) (nmol/mg protein) (nmol/mg protein) ± ± ± ± ± ± ± ±.5 + CUR ± 14.39, ± ±.49, 1.47 ±.1 + CUR ± 8.33,c 47.5 ± ±.22,,c 1.46 ±.1 The results re mens ± SD. p <.5 compre with control group, p <.5 compre with group, nd c p <.5, compre with + CUR 2 group.

4 46 THAI J GASTROENTEROL 212 Effects of Curcumin Attenuted Heptitis in Mice with Prcetmol Overdose AST (U/L) AST, + CUR 2 + CUR 6, c Figure 1. Heptic enzyme (AST) in serum ws determined. p <.5 compre with control group, p <.5 compre with group, nd c p <.5, compre with + CUR 2 group. GSH (nmol/mg prot.) GSH,,, c + CUR 2 + CUR 6 Figure 3. Heptic GSH ws determined. p <.5 compre with control group, p <.5 compre with group, nd c p <.5, compre with + CUR 2 group. ALT (U/L) ALT + CUR 2 + CUR 6 MDA (nmol/mg prot.) MDA + CUR 2 + CUR 6 Figure 2. Heptic enzyme (ALT) in serum ws determined. p <.5 compre with control group, p <.5 compre with group. Figure 4. Heptic MDA in liver homogente ws determined. p <.5 compre with control group, p <.5 compre with group. Tle 2. Summry of heptic necroinflmmtion score in ll groups. Dt re expressed s the numer of mice exhiiting the grde of heptic necroinflmmtion indicted. Group Numer Heptic necroinflmmtion* = none 1 = mild 2 = moderte 3 = severe CUR CUR *All fields in ech section were exmined for grding of heptic necroinflmmtion ccording to the criteri descried y Brunt EM et l (1). Score = no heptocyte injury/inflmmtion Score 1 = sprse or mild focl zone 3 heptocyte injury/inflmmtion Score 2 = noticele zone 3 heptocyte injury/inflmmtion Score 3 = severe zone 3 heptocyte injury/inflmmtion

5 47 A B C D Figure 5. Liver histopthology of H & E stining (A); group showed norml heptic rchitecture, (B); group showed cute centriloulr hemorrhgic heptic necrosis involving ll zones. (C); + CUR 2 group showed mild focl necrosis with mild ftty chnges nd the heptic rchitecture ws preserved, nd (D); +CUR 6 group showed the mjority of heptic loules preserved the norml rchitecture with limited heptic chnge. group (Tle 1 nd Figure 3). Heptic MDA, mrker of oxidtive stress, ws significntly incresed in group when compred with control group (3.55 ±.5 vs ±.1 nmol/ mg protein, p <.5). These were significntly decresed in + CUR 2 (1.47 ±.1 vs ±.5 nmol/mg protein, p <.5) nd + CUR 6 groups (1.46 ±.1 vs ±.5 nmol/mg protein, p <.5) when compred with group (Tle 1 nd Figure 4). Liver histology in control group showed norml. In group, the heptic necroinflmmtion ws moderte to severe injury. In + CUR 2 nd + CUR 6 groups improved the severity, the heptic necroinflmmtion showed mild injury (Tle 2 nd Figure 5). DISCUSSION In therpeutic dose, is minly metolized vi glucuronidtion nd sulftion nd in conjugted forms is excreted from the ody. Besides, prtly is metolized y cytochrome P45 (CYP 2E1), to produce metolites, minly NAPQI, which re drmti-

6 48 THAI J GASTROENTEROL 212 Effects of Curcumin Attenuted Heptitis in Mice with Prcetmol Overdose clly incresed in high concentrtions; these metolites of re detoxified y GSH nd removed from the ody. Then, in overdose cuses incresing of toxic metolites. These metolites interct with rnge of cellulr proteins vi covlent inding, which disrupting heptocyte functions cusing necrosis nd liver injury occurs (11,12). The present study demonstrtes tht in vivo tretment of mle mice with overdose results in heptic GSH depletion. This result corresponds to previous oservtions studied in model showing tht -induced heptic GSH depletion (13-15). GSH serves nucleophilic co-sustrte to glutthione trnsferse in the detoxifiction of xenoiotics nd is n essentil electron donor to glutthione peroxidses in the reduction of hydroperoxides (16,17). GSH is lso involved in mino cid trnsport nd mintennce of protein sulfhydryl reduction sttus (18,19). These findings suggest tht induction of cytochrome P45 (CYP2E1) in the liver should enhnce heptic GSH depletion. GSH is essentil for conjugtion or detoxifiction of toxic metolites, minly NAPQI, which disrupting heptocyte in -treted mice. GSH (reduced form) is esily oxidized to the disulfide dimer GSSG (oxidized form). GSSH is produced during the reduction of hydroperoxides y glutthione peroxidse enzyme. GSSG is reduced to GSH y glutthione reductse enzyme nd it is the reduced form tht exists minly in conjugtion or detoxifiction of toxic metolites. These findings suggest tht curcumin my possily inducer vi glutthione reductse enzyme to produce GSH or inhiitor vi glutthione peroxidse enzyme. In ddition, curcumin ws lso found to e wek inhiitor of cytochrome P45 (CYP 2E1) (2). The present study corresponds to previous oservtions studied in model showing tht induced heptic MDA (13-15). MDA is the most widely used test for mesuring the extent of lipid peroxidtion. Lipid peroxidtion is chin rection process tht involves the prticiption nd production of free rdicl species. Free rdicls cn cuse cellulr injury when produced in sufficient mounts to overcome the normlly efficient protective mechnism. Lipid peroxidtion is free rdicl medited chin rection which is enhnced s consequence of oxidtive stress ( generl term used to descrie stle of dmge cused y rective oxygen species (ROS), nd it results in n oxidtive deteriortion of memrne polyunsturted ftty cids). It is continuous physiologicl process occurring in cell memrnes (21), which defined s the oxidtive deteriortion of polyunsturted fts. The cell memrnes contins lrge mount of polyunsturted ftty cids which re especilly susceptile to lipid peroxidtion. Lipid peroxidtion of cell memrnes results in decresed memrne fluidity, inility to mintin ionic grdients, cellulr swelling, nd tissue inflmmtion. These findings suggest tht induction of cytochrome P45 (CYP2E1) in the liver should enhnce heptic MDA production. The induction of heptic MDA is essentil for lipid peroxidtion (oxidtive stress), which disrupting heptocyte in -treted mice. The cell memrnes contins lrge mount of polyunsturted ftty cids which re especilly susceptile to lipid peroxidtion. Lipid peroxidtion of cell memrnes results in cellulr swelling nd tissue inflmmtion (21). These findings suggest tht curcumin could e decresed heptic MDA medited vi covlently ound to cellulr mcromolecules inhiition. A previous study demonstrted tht curcumin t the dose of 2 mg/kg nd 6 mg/kg hd n ntioxidnt nd nti-inflmmtory property (7). In this study, 2 mg/kg of curcumin ws sufficient dose for ttenute heptitis. This finding corresponded to previous oservtions studied in model showing tht -induced heptitis ws ttenuted y curcumin (13-15). CONCLUSIONS In conclusion, these results suggest tht toxicity to liver is relted to depletion of heptic GSH concomitnt with the induction of oxidtive stress, liver inflmmtion, nd the dmge of liver pthology. Our results show curcumin cn prevent most of the dmge cused y overdose in mice y induction of heptic GSH, reduction of oxidtive stress, ttenution of liver inflmmtion, nd the improvement of liver pthology. In ddition, curcumin t the dose of 6 mg/kg tends to e more potent thn 2 mg/kg in preventing the effects of toxicity. Hence, curcumin might e new nturl therpeutic gent ginst heptitis induced y. ACKNOWLEDGEMENT The 9 th Anniversry of Chullongkorn University Fund (Rtchdphiseksomphot Endowment Fund)

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