Mol Cell Toxicol (2014) 10:75-82DOI 10.1007/s13273-014-0009-8

Abstract Present study demonstrates the therapeu-tic role of Emblica officinalis (EO) against arsenic-induced DNA and hepatic damage in female rats. Ourearlier study on arsenic-exposed human unveils a linkbetween tissue necrosis and carcinogenesis with im-paired antioxidant system-associated DNA damage.Here we show ingestion of EO extract (500 mg in 0.1mL water) in combination with sodium arsenite (0.4ppm)/100 g b.w. for 24 days to rats offered significantprotection against arsenic-induced oxidative damagesof DNA and hepatic tissue architecture. Arsenic onlyexposure decreased hepatic superoxide dismutase,catalase activities and the level of non protein solublethiol with a concomitant increase in thiobarbituric acidreactive substances and conjugated di-enes which arerestrained by EO with a restoration of antioxidantcomponents. In conclusion, restricted generation offree radicals is correlated to DNA protection resultingin prevention of tissue necrosis and possible carcino-genesis.

Keywords Arsenic, Antioxidant systems, DNA frag-mentation, Hepatic carcinogenesis, Emblica officinalis

Arsenic, a major water pollutant exerts its carcino-genic and genotoxic effects mainly in the form ofhyperkeratosis, gangrene, skin cancer, hepatic, alveo-lar, ovarian and adrenal tumors1,2. Severe diabetic dis-order and reproductive abnormalities have also beenreported3. Our recent finding demonstrate that deple-tion of antioxidant components and high rate of DNAfragmentation in arsenic exposed human populationresult in tissue necrosis and carcinogenesis in skinand other organs4.

Metabolic processing of arsenical compounds isrelated to the production of free radicals and reactiveoxygen species which ultimately induces oxidativestress by a significant depletion of reduced glutathioneand increase in DNA breakage5,6. A few drugs likeBritish Anti Lewsite (BAL), dimercaptosuccinic acid(DMSA) are available in the market as the arsenicchelating agent, but these have some side effects, suchas nausea, etching, abdominal pain, hypertension7.Some previous studies have explored that vitamin C8,vitamin E and selenium9 play important nutritionalrole against arsenic toxicity. Recently, it has beensown that human chorionic gonadotropin (hCG) iseffective in the restoration in arsenic mediated ovarianand uterine steroidogenesis via hypophyseal-gonadaland hypophyseal-adrenal axis10. Herbal remediation ofarsenic-induced hepatocellular toxicity by the extractsof Moringa olifera seed has also been reported11. Sev-eral other components of herbal origin like quercetin,combination of monoisoamyl dimercaptosuccinic acidand Moringa oleifera seed powder, Hippophae rham-noides L., curcumin, resveratrol and some importantphytochemicals have been demonstrated to show pro-tection against arsenic-induced oxidative stress, DNAbreakage, hepatic damage, fibrosis and carcinogene-sis12,13. In the present study, we investigated the thera-peutic role of Emblica officinalis (Indian gooseberry,

ORIGINAL PAPER

Emblica officinalis (amla) ameliorates arsenic-induced liverdamage via DNA protection by antioxidant systems

Smarajit Maiti1, Sandip Chattopadhyay2, Nirmallya Acharyya1, Bimal Deb2 & Amiya Kumar Hati3

Received: 14 January 2012 / Accepted: 16 July 2012�The Korean Society of Toxicogenomics and Toxicoproteomics and Springer 2014

1Post Graduate Department of Biochemistry, Cell and MolecularTherapeutics Laboratory, Oriental Institute of Science andTechnology, Vidyasagar University, Midnapore-721102, West Bengal, India2Department of Bio-Medical Laboratory Science and Management,(UGC Innovative Department), Vidyasagar University, Midnapore-721 102, West Bengal, India3Former Director, School of Tropical Medicine, Calcutta, Present address: Department of Pathology, Gautam Laboratories 9A,K. K. Street, Calcutta 700 007Correspondence and requests for materials should be addressed to S. Maiti ( maitism@rediffmail.com)

locally known as amla) fruit extract on arsenic-inducedoxidative stress, DNA breakage and hepatic damagein rat. Amla, frequently used in traditional medicationpractice is also regarded as a divine fruit in Indianmythology. Reports reveal that amla fruit extract showsantiviral and antimicrobial14, apoptotic and antican-cer15 efficacies. The antidiabatic, anti-inflammatory,anti-dyslipidemic, anti-oxidant and anti-aging role ofthis fruit extract have also been demonstrated16. Theefficiency for cholesterol and triglyceride dispositionand several other activities of amla fruit are also evi-dent. In the present study, protective role of amlaagainst arsenic-induced tissue damage has been inves-tigated.

General observations

Food and water consumption was unaltered in allgroups of animal throughout the experimental sche-dule. At the end of the experiment, general somaticgrowth of arsenic-treated and EO co-administeredgroups of rat did not differ significantly from control(Table 1). Here, organo-somatic indices refer to theorgan weight in gram per 100 g of animal body weight.This parameter represents the general health and toxi-city status of the individual33. After 24 days of arsenictreatment, as shown in Table 1, there was no signifi-cant alteration in the hepato-somatic index comparedto the control group. However, there were significantlylower reno-somatic index in arsenic intoxicated rats.EO co-treatment with arsenic in rats was effective to

protect kidney. This suggests that EO possibly couldinhibit the arsenic induced renal tissue degeneration.Further investigation is necessary to characterize theprotective efficacy of EO with more certainty.

Liver and kidney function-markers

In order to assess the hepatocellular status, the activi-ties of ALT, AST and ALP in serum were estimatedin the present investigation after arsenic exposure tofemale rats. These enzymatic activities were signifi-cantly increased except ALP when compared to thecontrol group (P⁄0.001) (Table 2). A noticeable re-storation of these enzymatic activities was observed inEO supplemented arsenic exposed rats. The marker

76 Mol Cell Toxicol (2014) 10:75-82

Table 1. Effect of Embelica officinalis seed extract on somat-ic growth and hepato-reno-somatic indices in arsenic exposedrats (mean++SE, N==6). As compared with control, P⁄0.01(ANOVA followed by multiple comparison two tailed t test).Same superscript did not differ from each other significantly. Cstands for control, As for arsenic treated and As++EO denotesarsenic treated with Embelica officinalis supplementation.

Condition Organo-somatic indices(g %)

Body weight (g)Liver Kidney

Initial Final

C 148±7a 170±7a 3.06±0.31a 0.68±0.02a

As 150±4a 167±6a 3.52±0.15a 0.62±0.02b

As++EO 146±5a 169±5a 3.36±0.19a 0.81±0.01c

Table 2. Effects of Emblica officinalis on arsenic-induced hepatic functional markers, metabolic and antioxidant parameters infemale rats (data in the table represent mean±SE, N==6). The arsenic (As) or arsenic++EO treated groups compared with control,P⁄0.001 (ANOVA followed by multiple comparison two tailed t test). Same superscript did not differ from each other signifi-cantly.

Parameters studied Control Arsenic treated Arsenic and Emblica officinalis(amla) treated

Liver and kidney functionALP (U/L) 268.5±25.64a 265.33±22.9a 270.5±9.43a

AST (U/L) 128.13±11.46a 324.34±19.3b 132.89±15.51a

ALT (U/L) 54.67±4.64a 140.37±20.27b 41.81±5.54a

Total protein (g/dL) 8.39±0.76a 7.45±0.22b 7.63±0.61b

Urea (mg/dL) 32.39±2.45a 45.18±4.56b 34.7±3.36a

Creatinine (mg/dL) 0.82±0.06a 0.92±0.04b 0.83±0.03a

Lipid profile (mg/dL)Cholesterol 59.2±2.63a 77.8±5.56b 45.7±1.25c

Triglyceride 88.3±6.54a 120.1±13.09b 85.1±9.56a

HDL 45.29±1.45a 42.73±2.21b 48.88±1.73a

LDL 11.7±.96a 21.4±2.23b 9.5±.76a

Free radical/Antioxidant profileMDA (nmole/mg tissue) 20.91±2.67a 33.67±3.95b 12.3±1.45c

CD (nmole/mg tissue) 176.42±12.31a 286.34±10.27b 194.23±6.64a

Catalase (unit/mg protein) 47.5±1.18a 27.3±2.61b 48.6±1.14a

SOD (unit/mg protein) 9.13±0.32a 2.30±0.47b 6.63±0.59c

NPSH (μg/g protein) 32.43±1.21a 23.18±2.10b 28.59±1.43a

molecule of kidney function like urea and creatinineincreased significantly in arsenic exposed rat whichwas restored at EO co-administered animals.

Lipid profile

The administration of arsenic in female rats caused asignificant increase (P⁄0.001) of total cholesterolvalue in plasma (Table 2). Elevated plasma triglyce-ride, LDL-C (P⁄0.001) along with low level of HDL-C (P⁄0.001) were observed after sodium arsenitetreatment in female rats. EO co-administration to arse-nic treated rats significantly protected the lipid profileand reduced the toxic effects of arsenic.

Status of oxidative stress markers

In this set of experimentations we examined the effectsof arsenic on the hepatic antioxidant status. The MDAand CD content in liver homogenates significantlyincreased in the sodium arsenite-exposed rats. How-ever, administration with EO extract combined witharsenic prevented MDA and CD elevation when com-pared to the arsenic only treated group (P⁄0.001).There was a dramatic decrease in hepatic SOD andcatalase activities in all arsenic treated rats when com-pared to control group (P⁄0.001). Interestingly, arestoration of SOD and catalase activities were observ-

Mol Cell Toxicol (2014) 10:75-82 77

Figure 1. Hepatic histo-architecture. Magnification×100 offemale rat treated with arsenic. Control rat (a) or treated withsodium arsenite (b) or sodium arsenite++E. officinalis (c).

(a)

(b)

(c)

Figure 2. Hepatic DNA status. DNA fragmentation results inliver of female rat treated with arsenic. Control rat (lanes 1,2) or treated with sodium arsenite (lanes 3, 4) or sodium arsen-ite++E. officinalis (5, 6). The extended ladder portion of lane3 and 4 (arsenic treated group) are shown by the arrow.

1 2 3 4 5 6

ed in the EO combination group when compared toonly arsenic-exposed group (P⁄0.001). A partial re-storation of NPSH was observed in EO co-administer-ed rats; when arsenic exposure depleted total thiol by~28% (32.43 Vs 23.18 μg/g protein) then the deple-tion was minimized to ~11% (32.43 Vs 28.59 μg/gprotein) in EO supplemented arsenic exposed groupwith comparison to control group.

Hepatic tissue architecture and DNA fragmentationresult

Arsenic ingestion with the present dose and durationresulted in hepatocyte disarrangement with lobulardegeneration. But the EO co-administration in arsenic-exposed animals shows partial but significant protec-tion which is evident from the histoarchitecture picture(Figure 1). Results of agarose-gel electrophoresis ofliver DNA from different experimental groups show-ed an enhanced DNA ladder (lane 3 and 4, demarcat-ed by the arrow) in arsenic only treated group, whichwas restrained partially in EO supplemented arsenic-exposed group in lane 5 and 6 (Figure 2).

Discussion

In the present study, arsenic treatment for 24 days viadrinking water to female Wister rats induced hepaticinjury as evident by the alterations in the liver func-tion markers. Liu et al. (1999) observed that ingestionof arsenic contaminated drinking water caused infiltra-tion of inflammatory cells in the periportal area in liverbiopsy samples34. This supports our present finding.It is evident that arsenic-induced liver damages likenecrosis, apoptosis and carcinogenesis could be theresult from oxidative stress which is substantiated bythe liver function test and histological manifestations35.Results of the present study revealed a moderate de-crease in total protein levels in liver in arsenic treatedrat. This could be related to the inhibition of proteinsynthesis by accumulation of free amino acids andalteration of numerous sulfhydryl-containing proteinsin liver of arsenic exposed rats36.

The elevated levels of serum transaminases indica-tor of liver functions, were observed in arsenic treatedrat and this may be related to the extensive alterationsin the liver histology indicating liver damage37. Pre-sent result on kidney function indicates that EO alsoprotect kidney from possible degeneration which isshown as the restored urea and creatinine level. Ourresults show the significant alteration of lipid profileand this correlates with some earlier findings on ische-mic heart disease due to sustained hyperlipidaemiastate where oxidative stress may play as a major con-

tributor38. Arsenic exposure also exhibits oxidativestress through significant reduction of GSH in liver,cultured lung epithelial cells and discrete brain areas39.This finding is corroborated with the result of ourpresent investigation, where sodium arsenite leads tothe formation of free radical significantly via the inhi-bition of catalase and SOD.

Amla or EO is reported to be an antagonistic toapoptosis and lipid peroxidation of human hepatocyteinduced by different environmental pollutants40. Therecovery observed in the present study is probablyrelated to the antioxidant and free radical scavengingproperties of EO via its high vitamin-C content41.Here, clinical implication focuses that arsenic affect-ed people might be protected from arsenic-inducedhepatic disorders through nutritional supplement ofEO in the endemic regions. The E. officinalis hasbeen demonstrated to induce the healing of tissuewound by protecting collagen from free radical relat-ed damages42. Histological and DNA fragmentationstudies in the present investigation clearly reveal theprotective role of EO against arsenic exposure. E. offi-cinalis can protect tissue from oxidative damage andprevent lipid peroxidation43 which is decisively point-ed in the present investigation on arsenic exposure.Report reveals that carbon tetra chloride (CCl4) orthioacitamide induced hepatic damage and tissue car-cinogenesis can be reversed by E. officinalis44. Anti-cancer role of E. offiinalis has also been presented.Several earlier investigations on chemical analysis ofthis fruit reveal that EO contain high amount (445 mg/100 g) of ascorbic acid (vitamin C). Several line ofevidences on this analysis show that hydro-alcoholicextract of E. officinalis fruit contain alkaloids, reduc-ing sugars, glycosides, tannins, resins, saponins, ste-rols and fixed oils45. The fruit contains several otherpolyphenols like flavonoids, kaempferol, ellagic andgallic acid. In the present investigation, the differentialprotective response of amla has been observed againstarsenic toxicity. Amla not only restored cholesterollevel from arsenic exposed group but slightly decreas-ed from the control value also. This trend is applica-ble in MDA level also. Cholesterol and triglyceridedepleting role and radical scavenging efficiency ofamla is noted earlier even in control individual. In ourfindings alterations of some of the parameters havebeen restored partially by the supplementation of amla,as for example total protein and SOD activity. Furtherinvestigation is necessary for the elucidation of activecomponent effective against arsenic toxicity. Presentresult clearly establish the preventive role of E. offici-nalis against arsenic induced tissue and DNA damageindicating its therapeutic efficacy not only at cellularor biochemical level but also at molecular level.

78 Mol Cell Toxicol (2014) 10:75-82

Materials & Methods

Preparation of aqueous extract of Emblica officinalis(EO) fruit

One kilogram of green young EO fruit was crushed inan electrical grinder with 500 mL water and extractionwas performed in a soxhlet apparatus for 18 h. Theextract was dried at reduced pressure and finally lyo-philized.

Animal selection and treatment

Female rats of Wistar strain weighing 150±10 g wereacclimatized for 10 days at 12-hour light-dark cycle,32�C±2�C temperature, 50%-70% humidity and bredin the Central Animal Resource facility of VidyasagarUniversity, India. Those were fed with a standard pel-let diet (Hindustan Lever Ltd, Mumbai, India) andwater ad libitum. Studies were carried out in accor-dance with the National Institutes of Health, USAguidelines and institutional ethical concerns weremaintained throughout the investigation.

Rats were randomly distributed in 3 groups having6 in each. Animals of Group-II and Group-III werefed with 0.5 mL drinking water containing sodiumarsenite at a concentration of 0.4 ppm/100 g b. w./dayfor 24 days. Initially, several dose response studies ofarsenic were conducted on rat model. The present doseusually does not cause animal mortality but exposurefor a moderate time period (more than twenty days)increase liver and kidney toxicity marker and otherclinical marker suggesting cellular toxicity11. Thegroup I as designated control was supplied with sameamount of drinking water for stipulated duration.Group-III animals were supplemented with lyophiliz-ed extract of EO by gavages at a concentration of 500mg dissolved in 0.1 mL distilled water/100 g bodyweight/day at 6.00 a.m. for 24 days17. At the sametime, all the animals of Group-I and Group-II weresupplemented with only 0.1 mL distilled water. Feed-ing habits of all the animals were observed carefullythrough out the experimental schedule. On 25th day ofexperiment, final body weights of all the animalswere noted. Blood was collected from dorsal aorta ofrats at light anesthesia condition (by ether) using aheparinized syringe (21-gauge needle) and plasmasamples were separated. Organs required for biochem-ical and histological experiments were dissected outand stored at -40�C until use.

Biochemical assays of transaminase, phosphataseand total protein

In the present investigation, alanine aminotransferase(ALT) and aspartate aminotransferase (AST) were

assayed each with 0.1 mL of plasma and utilizing L-alanine and α-ketoglutarate as substrate for ALT and,L-aspertate and α-ketoglutarate as substrate for AST.Enzyme activities were measured at 340 nm18,19.

To measure the activity of alkaline phosphatase(ALP), 0.1 mL of plasma was incubated at 37�C inthe presence of a mixture of Tris-HCl (pH 8.0) and p-nitrophenyl phosphate. The activity was measured at405 nm using a visible spectrophotometer20.

Total protein was measured following Biuret methodusing standard kit from Ranbaxy Diagnostic IndiaLimited, Mumbai, India21.

Detection of urea and creatinine level

The urea was determined by modified method where10 μL of plasma was treated with urease at 37�C andfinally chromogen and hypochlorite induced greencolor was measured at 570 nm22.

Following the modified Jaffe’s kinetic method, cre-atinine was measured using alkaline picrate with 0.1mL plasma at 37�C and reading was taken at a wave-length of 520 nm23.

Estimation of lipid profile

The lipid components such as total Cholesterol (TC)24,HDL-C, and triglyceride25 were estimated in plasmaby using standard kits supplied by Ranbaxy Diagnos-tic Limited, Mumbai, India. The level of LDL-C wascalculated from the value of triglyceride, TC andHDL-C by Friedwald and Fredickson’s formula.

Estimation of malondialdehyde (MDA) andconjugated di-ene (CD) levels

Hepatic tissue was homogenized (10% w/v) in ice-cold phosphate buffer (0.1 mol/L, pH 7.4) and thehomogenate was centrifuged at 15,000 g in 4�C for 20min. The supernatant was used for the estimation ofMDA and CD. The MDA assay was conducted follow-ing the method of Buege and Aust, 197826 with aslight modification. To chelate iron and reduce itsinterference in peroxidation reaction of unsaturatedfatty acid, 1 mM EDTA was used in the reaction mix-ture. To reduce the interference caused by a yellow-orange color produced by some carbohydrates, thereaction mixture was heated at 80�C instead of 100�C.Finally the MDA was measured and calculated utiliz-ing the molar extinction coefficient of MDA (1.56×105 cm2/mmol)27. Conjugated dienes were determinedby a standard method28. In brief, lipids were extractedwith chloroform-methanol (2 : 1), followed by centri-fugation at 1,000 g for 5 min and lipid residues weredissolved in 1.5 mL of cyclohexane and the absorbance

Mol Cell Toxicol (2014) 10:75-82 79

was measured at 233 nm to determine the amount ofhydroperoxide formed.

Assay of super oxide dismutase and catalaseactivities

Hepatic tissue was homogenized in chilled 100 mmol/L Tris HCl buffer containing 0.16 mol/L KCl (pH 7.4)to give a tissue concentration of 10% (w/v) and centri-fuged at 10,000 g for 20 min at 4�C. The SOD activityin the supernatant was measured according to a stan-dard protocol29. The reaction mixture was prepared bymixing 800μL of TDB (Merck), 40μL of 7.5 mmol/LNADPH (Sigma), 25 μL of EDTA-MnCl2 and 100 μLof the tissue supernatant. The activity of SOD in thismixture was monitored at 340 nm from the rate of oxi-dation of NADPH.

Catalase activity was assayed by a colorimetricmethod30. Dichromate in acetic acid was converted toperchromic acid and then to chromic acetate whenheated in the presence of H2O2. The chromic acetateformed was measured at 620 nm. The catalase prepa-ration was allowed to split H2O2 for different periodsof time. The reaction was stopped at different timeintervals by the addition of a dichromate-acetic acidmixture and the remaining H2O2 was determined aschromic acetate. One unit of activity was expressedas a mole of H2O2 consumed/min/mg protein.

Estimation of non protein soluble thiol (NPSH)

The NPSH in 10,000×g supernatant from liver tissuehomogenate (prepared in 0.1 M phosphate buffer, pH7.4) was determined by standard DTNB (5,5′-dithio-bis-2-nitrobenzoic acid) method with a slight modifi-cation31. In brief, the protein was precipitated by sul-fosalisialic acid and clear cytosol was added to 0.1 Msodium phosphate buffer containing 5 μM DTNB.The level of NPSH was determined against a GSHstandard curve.

Histology and DNA fragmentation analysis

Liver tissue was embedded in paraffin, serially sec-tioned at 5 μM, stained with eosin and hematoxylinand observed under a microscope (400×).

Liver tissue was used for DNA preparation andtreated with 500 μL of lysis buffer (50 mM Tris pH8.0, 20 mM EDTA, 10 mM NaCl, 1% SDS, 0.5 mg/mLproteinase K) for 20 min on ice (4�C) and centrifugedin cold at 12,000 g for 30 minutes. The supernatantwas extracted with 1 : 1 mixture of phenol: chloroformwith gentle agitation for 5 min followed by centrifu-gation and precipitated in two equivalence of coldethanol and one-tenth equivalence of sodium acetate.After spinning down and decanting, the precipitate

was re-suspended in 30 μL of deionized water-RNasesolution [0.4 mL water++5 μL of RNase] and 5 μL ofloading buffer for 30 minutes at 37�C. The 0.8% aga-rose gel with ethidium bromide was run at 5 V for 5min before increasing to 100 V and documented in geldocumentation system32.

Statistical analysis

Data from supplemented group was compared withcontrol by utilizing ANOVA followed by multiplecomparison two tailed ‘t’ test.

References

1. Roy, P. & Saha, A. Metabolism and toxicity of arsenic:A human carcinogen. Curr Sci 82:38-45 (2002).

2. Rahman, M. M., Nag, J. C. & Naidu, R. Chronic ex-posure of arsenic via drinking water and its adversehealth impacts on humans. Environ Geochem Health31 Suppl 1:189-200 (2009).

3. Pott, W. A., Benjamin, S. A. & Yang, R. S. Pharma-cokinetics, metabolism, and carcinogenicity of arsenic.Rev Environ Contam Toxicol 169: 165-214 (2001).

4. Maiti, S. et al. Antioxidant and metabolic impairmentresult in DNA damage in arsenic-exposed individualswith severe dermatological manifestations in EasternIndia. Environ Toxicol (2010) [Epub ahead of print]PubMed PMID: 20925122

5. Maiti, S. & Chatterjee, A. K. Effects on levels of glu-tathione and some related enzymes in tissues after anacute arsenic exposure in rats and their relationship todietary protein deficiency. Arch Toxicol 75:531-537(2001).

6. Yamanaka, K. et al. Induction of DNA damage bydimethylarsine, a metabolite of inorganic arsenics, isfor the major part likely due to its peroxyl radical. Bio-chem Res Commun 168:58-64 (1990).

7. Inns, R. H., Rice, P., Bright, J. E. & Marrs, T. C. Eva-luation of the efficacy of dimercapto chelating agentsfor the treatment of systemic organic arsenic poison-ing in rabbits. Hum Exp Toxicol 9:215-220 (1990).

8. Chattopadhyay, S., Ghosh, S., Debnath, J. & Ghosh,D. Protection of sodium arsenite-induced ovarian toxi-city by coadministration of L-ascorbate (vitamin C) inmature wistar strain rat. Arch Environ Contam Toxi-col 41:83-89 (2001).

9. Mahata, J. et al. Effect of selenium and vitamin esupplementation on plasma protein carbonyl levels inpatients with arsenic-related skin lesions. Nutr Can-cer 60:55-60 (2008).

10. Chattopadhyay, S. & Ghosh, D. The involvement ofhypophyseal-gonadal and hypophyseal-adrenal axesin arsenic-mediated ovarian and uterine toxicity: mod-ulation by hCG. J Biochem Mol Toxicol 24:29-41(2010).

11. Chattopadhyay, S. et al. Protective role of Moringa

80 Mol Cell Toxicol (2014) 10:75-82

oleifera (Sajina) seed on arsenic-induced hepatocellu-lar degeneration in female albino rats. Biol Trace ElemRes 142:200-212 (2011).

12. Ghosh, D. et al. Quercetin in vesicular delivery sys-tems: evaluation in combating arsenic-induced acuteliver toxicity associated gene expression in rat model.Chem Biol Interact 186:61-71 (2010).

13. Mishra, D. et al. Co-administration of monoisoamyldimercaptosuccinic acid and Moringa oleifera seedpowder protects arsenic-induced oxidative stress andmetal distribution in mice. Toxicol Mech Methods 19:169-182 (2009).

14. Talwar, G. P. et al. A novel polyherbal microbicidewith inhibitory effect on bacterial, fungal and viralgenital pathogens. Int J Antimicrob Agents 32:180-185 (2008).

15. Ngamkitidechakul, C., Jaijoy, K. & Hansakul, P. Anti-tumour effects of Phyllanthus emblica L.: induction ofcancer cell apoptosis and inhibition of in vivo tumourpromotion and in vitro invasion of human cancer cells.Phytother Res 24:1405-1413 (2010).

16. Yokozawa, T. et al. Amla (Emblica officinalis Gaertn.)prevents dyslipidaemia and oxidative stress in the age-ing process. Br J Nutr 97:1187-1195 (2007).

17. Sharma, A., Sharma, M. K. & Kumar, M. Modulatoryrole of Emblica officinalis fruit extract against arsenicinduced oxidative stress in Swiss albino mice. ChemBiol Interact 180:20-30 (2009).

18. Bergmeyer, H. U., Hørder, M. & Rej, R. InternationalFederation of Clinical Chemistry (IFCC) ScientificCommittee, Analytical Section: approved recommen-dation (1985) on IFCC methods for the measurementof catalytic concentration of enzymes. Part 2. IFCCmethod for aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1). J ClinChem Clin Biochem 24:497-510 (1986).

19. Schumann, G. et al. International Federation of Clini-cal Chemistry and Laboratory Medicine. IFCC pri-mary reference procedures for the measurement ofcatalytic activity concentrations of enzymes at 37degrees C. International Federation of Clinical Chem-istry and Laboratory Medicine. Part 4. Reference pro-cedure for the measurement of catalytic concentrationof alanine aminotransferase. Clin Chem Lab Med 40:718-724 (2002).

20. Copeland, W. H., Nealon, D. A. & Rej, R. Effects oftemperature on measurement of alkaline phosphataseactivity. Clin Chem 31:185-190 (1985).

21. Gornall, A. G. et al. Determination of serum proteinsby means of the biuret reaction. J Biol Chem 177:751-766 (1949).

22. Fawcett, J. K. & Scott, J. E. A rapid and precise me-thod for the determination of urea. J Clin Pathol 13:156-159 (1960).

23. Bowers, L. D. Kinetic serum creatinine assays I. Therole of various factors in determining specificity. ClinChem 26:551-554 (1980)

24. Allain, C. C., Poon, L. S., Chan, C. S., Richmond, W.

& Fu, P. C. Enzymatic determination of total serumcholesterol. Clin Chem 20:470-475 (1974).

25. Werner, M., Gabrielson, D. G. & Eastman, J. Ultra-micro determination of serum triglycerides by bio-luminescent assay. Clin Chem 27:268-271 (1981).

26. Buege, J. A. & Aust, S. D. Microsomal lipid peroxi-dation. Methods Enzymol 52:302-310 (1978).

27. Maiti, S. & Chatterjee, A. K. Differential response ofcellular antioxidant mechanism of liver and kidney toarsenic exposure and its relation to dietary proteindeficiency. Environ Toxicol Pharmacol 8:227-235(2000).

28. Slater, T. F. Overview of methods used for detectinglipid peroxidation. Methods Enzymol 105:283-293(1984).

29. Paoletti, F. & Mocali, A. Determination of superoxidedismutase activity by purely chemical system basedon NAD(P)H oxidation. Methods Enzymol 186:209-220 (1990).

30. Sinha, A. K. Colorimetric assay of catalase. Anal Bio-chem 47:389-394 (1972).

31. Forman, H. J. Critical methods in Free Radical Bio-logy & Medicine. Free Radic Biol Med 47 Suppl 2:S207 (2009).

32. Garcia-Martinez, V. et al. Internucleosomal DNA frag-mentation and programmed cell death (apoptosis) inthe interdigital tissue of the embryonic chick leg bud.J Cell Sci 106:201-208 (1993).

33. Schmitt, C. J. & Dethloff, G. M. Biomonitoring ofEnvironmental Status and Trends (BEST) Program:Selected Methods for Monitoring Chemical Contami-nants and their Effects in Aquatic Ecosystems. Infor-mation and Technology Report USGS/BRD/ITR (2000-2005).

34. Liu, K. T. et al. Adverse effects of combined arsenicand fluoride on liver and kidney in rats. Fluoride 32:243-247 (1999).

35. Karimov, Kh.Ia., Inoiatova, F.Kh. & Inoiatov, F.Sh.Toxic effects of various water pollutants on structuraland functional parameters of hepatocytes. Vopr MedKhim 48:174-179 (2002).

36. Palaniappan, P. R. & Vijayasundaram, V. FTIR studyof arsenic induced biochemical changes on the livertissues of fresh water fingerlings Labeo rohita. Rom JBiophys 18:135-144 (2008).

37. World Health Organization. Environmental HealthCriteria 18: Arsenic. Geneva: WHO InternationalProgramme on Chemical Safety; 1981.

38. Chen, C. J., Chiou, H. Y., Chiang, M. H., Lin, L. J. &Tai, T. Y. Dose-response relationship between ische-mic heart disease mortality and long-term arsenicexposure. Arterioscler. Thromb Vasc Biol 16:504-510(1996).

39. Li, M., Cai, J. F. & Chiu, J. F. Arsenic induces oxida-tive stress and activates stress gene expressions incultured lung epithelial cells. J Cell Biochem 87:29-38 (2002).

40. Dhir, H., Agarwal, K., Sharma, A. & Talukder, G.

Mol Cell Toxicol (2014) 10:75-82 81

Modifying role of Phyllanthus emblica and ascorbicacid against nickel clastogenicity in mice. CancerLett 59:9-18 (1991).

41. Poltanov, E. A. et al. Chemical and antioxidant evalu-ation of Indian gooseberry (Emblica officinalis Gaertn.,syn. Phyllanthus emblica L.) supplements. PhytotherRes 23:1309-1315 (2009).

42. Sumitra, M., Manikandan, P., Gayathri, V. S., Mahen-dran, P. & Suguna, L. Emblica officinalis exerts woundhealing action through up-regulation of collagen andextracellular signal-regulated kinases (ERK1/2).

Wound Repair Regen 17:99-107 (2009).43. Naik, G. H. et al. In vitro antioxidant studies and free

radical reactions of triphala, an ayurvedic formulationand its constituents. Phytother Res 19:582-586 (2005).

44. Sultana, S., Ahmad, S., Khan, N. & Jahangir, T. Effectof Emblica officinalis (Gaertn) on CCl4 induced hepat-ic toxicity and DNA synthesis in Wistar rats. Indian JExp Biol 43:430-436 (2005).

45. Pandey, G. & Pandey, S. P. Phytochemical and toxi-city study of E. Officinalis (amla). Int Res J Pharm2:270-272 (2011).

82 Mol Cell Toxicol (2014) 10:75-82