Restoration of Arsenite Induced Hepato-Toxicity by Crude Tannin Rich Fraction of Theobroma Cacao in Sprague Dawley Rats

Food Research International 50 (2013) 46–54

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Food Research International

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Restoration of arsenite induced hepato-toxicity by crude tannin rich fraction of Theobroma cacao in Sprague Dawley rats

Chandronitha Chandranayagam a, Gayathri Veeraraghavan a, Ananthi Subash a, Hannah Rachel Vasanthi a,b,⁎ a Herbal and Indian Medicine Research Laboratory (HIMRL), Department of Biochemistry, Sri Ramachandra University, Chennai 600 116, India b Department of Biotechnology, School of Life Sciences, Pondicherry University, Pondicherry 605 014, India article info abstract

Article history: The present study was designed to investigate the protective efficacy of tannin rich cocoa (Theobroma cacao L.) Received 25 April 2012 in comparison with its detannified fraction. Arsenic as sodium-m-arsenite at 100 ppm dose was orally adminis- Accepted 14 September 2012 tered via drinking water constantly for 28 days to Sprague Dawley female rats. The hepatic cellular thiol levels Available online 27 September 2012 such as total sulfhydryl content, protein bound sulfhydryl, non-protein bound sulfhydryl content, cellular oxidative stress and damage markers such as reduced glutathione (GSH), glutathione peroxidase (GPx), superoxide Keywords: dismutase (SOD) and the index of nitrite/nitrate (NO ) levels and a biomarker for arsenic toxicity namely Theobroma cacao x Arsenic delta-aminolevulinic acid dehydratase (ALAD) were substantially reduced and elevation of thiobarbituric acid Hepatotoxicity reactive substances (TBARS) was observed in arsenic-exposed groups. The supplementation of tannin rich Cellular thiol levels cocoa fraction (TCF) enhanced the levels of cellular thiols, markers of oxidative stress and levels of arsenic bio- Arsenic biomarker markers and decreased the level of oxidative damage in treatment groups. Histopathologically, the liver of Oxidative stress arsenic-exposed rats showed some degenerative lesions with few cells showing appearance of apoptosis Oxidative damage markers which was effectively antagonized by tannin rich cocoa feeding. Treatment with detannified cocoa (DCF) was not that protective compared to the tannin rich cocoa (TCF). These results demonstrate that tannin rich cocoa can be considered as a functional food which effectively antagonizes many of the adverse effects of arsenic intoxication. © 2012 Elsevier Ltd. All rights reserved.

1. Introduction arsenite exposure leads to oxidative stress and subsequent cellular re- sponses, which are implicated in arsenical toxicity and carcinogenicity Sodium arsenite (As III) is known to be 60 times more toxic than (Shi, Shi, & Liu, 2004). Arsenic is known to induce tumors of the urinary sodium arsenate (As V). The trivalent state of inorganic arsenic (arsenite, bladder, skin, liver and lung (Beyersmann & Hartwig, 2008; Dopp, von As III), as said above, has been reported to be more toxic than the penta- Recklinghausen, Diaz-Bone, Hirner, & Rettenmeier, 2010). Chronic ex- valent form (arsenate, As V) which is capable of binding to macromolec- posure to arsenite causes a wide range of toxic effects and this metal- ular thiols (Tapio & Grosche, 2006). As these macromolecular thiols are loid is classiﬁed as a carcinogen in humans (Gupta & Flora, 2006a). present in several molecules participating in the cellular redox balance, Arsenic has been related epidemiologically to an elevated risk of cancer in the skin, bladder, kidney, colon, lung, liver and other organic dys- functions, such as diabetes mellitus, nephrotoxicity, and cerebral ische- Abbreviations: GSH, reduced glutathione; GPx, glutathione peroxidase; SOD, super- mia (Meliker, Wahl, Cameron, & Nriagu, 2007). The main source of oxide dismutase; NOx, nitrite/nitrate; ALAD, delta-aminolevulinic acid dehydratase; TBARS, thiobarbituric acid reactive substances; TCF, tannin rich cocoa fraction; DCF, human exposure to inorganic arsenic worldwide occurs through con- detanniﬁed cocoa fraction; As III, sodium arsenite; As V, sodium arsenate; CL, chemilu- sumption of drinking water drawn from groundwater sources that minescence; NaAsO2, sodium-m-arsenite; T. cacao, Theobroma cacao; OD, optical densi- contain dissolved inorganic arsenic, which has become a serious prob- ty; CPCSEA, Committee for the Purpose of Control and Supervision on Experimental lem, with considerable impact on public health (Kapaj, Peterson, Liber, Animals; ALA, aminolevulinic acid; TCA, trichloroacetic acid; LPO, lipid peroxidation; PMS-NBT, phenazine methosulfate-nitro blue tetrazolium; HCL, hydrochloric acid; & Bhattacharya, 2006; I.A.R.C. Working Group on the Evaluation of

DTNB, 5′-dithiobis (2-nitro benzoic acid); ANOVA, analysis of variance; H2O2, hydrogen Carcinogenic Risks to Humans, 2004). Besides the natural sources of ar- peroxide; DNA, deoxyribonucleic acid; ROS, reactive oxygen species; NO, nitric oxide; senic contamination in drinking water, the use of arsenic-contaminating •\ • RSNOs, S-nitrosothiols; CAT, catalase; O2 , superoxide anion; OH, hydroxyl radical. herbicides, insecticides, rodenticides and preservatives and by products ⁎ Corresponding author. Present address: Department of Biotechnology, School of of fossil fuels are also potential sources of toxicity (Flora et al., 2004). Life Sciences, Pondicherry University, Pondicherry 605 014, India. Tel.: +91 413 2654745. The toxicological effects of arsenic on health are multidimensional in E-mail address: [email protected] (H.R. Vasanthi). both human beings and animal. The liver and kidneys are mainly

0963-9969/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodres.2012.09.021 C. Chandranayagam et al. / Food Research International 50 (2013) 46–54 47 considered to be the primary targets of its toxicopathological manifes- (Germany), BDH Chemicals (Mumbai, India) and Sigma Chemicals tations (Nandi, Patra, & Swarup, 2006). (USA). Cocoa (Theobroma cacao L.) is an important crop in several countries such as Ghana, Ivory Coast, Nigeria, Indonesia and Malaysia. Cocoa is 2.2. Plant material consumed worldwide in the form of chocolates and its consumption rate is rising due to the increasing popularity of chocolate confectioner- Beans of T. cacao were collected from Kerala, India. The collected ies globally. Other applications of cocoa can also be found in beverages, material was authenticated and the voucher specimens are stored in cosmetics, pharmaceuticals and toiletry products (Tafuri, Ferracane, the department's herbarium at Sri Ramachandra University, Chennai. & Ritieni, 2004). Studies have been reported that show increasing scien- tific interest in the potential cardiovascular health benefits associated 2.3. Extraction and detannification with regular cocoa consumption (Cooper, Donovan, Waterhouse, & Williamson, 2008). Moreover, extracts prepared from cocoa powder The authenticated, dried, beans of T. cacao were subjected to and cocoa beans were shown to exhibit anti-hyperglycemic effects on ethanolic extraction (70%), by hot percolation technique. The extracts streptozotocin-induced diabetic rats (Amin, Faizul, & Azl, 2004; Ruzaidi, were filtered, concentrated to dryness in vacuo at controlled temper- Amin, Nawalyah, Hamid, & Faizul, 2005). Amin, Koh, and Asmah (2004) atures (40–50 °C) using a rotary flash evaporator apparatus to get a demonstrated that an ethanolic extract prepared from Malaysian mass of crude fraction (14% w/w). Detannified fraction was prepared cocoa liquor exhibited decreased severity of hepatocarcinogenesis as per a previously standardized procedure in our lab using gelatin in rats. (Chandronitha et al., 2010). Crude tannin rich fraction (TCF) and Many in vitro and in vivo studies have shown that cocoa and its detannified fraction (DCF) were subsequently tested for arsenic in- flavonoids possess beneficial effects against oxidative stress related duced hepato-toxicity. diseases by directly scavenging free radicals (Lamuela-Raventós, Romero-Pérez, Andrés-Lacueva, & Tornero, 2005) and by increasing 2.4. Tannin estimation the activities of antioxidant enzymes (Ramiro Puig et al., 2007). It has been recently shown that the protective activity of total phenolic The amount of tannins present in TCF and DCF was estimated by the extract of cocoa on liver cells is the prevention of apoptosis induced Folin–Ciocalteu colorimetric method of McDonald, Prenzler, Autolovich, by celecoxib (Arlorio et al., 2009). It has also been described that and Robards (2001) using gallic acid as a standard. The OD measure- the protective action toward O2 deprivation is through its antioxidant ment was taken at 640 nm for the estimation of tannin, and from the action in a cellular model of modulated ischemia (Arlorio et al., 2005). calibration curve the tannin content was calculated. Tannins are present in a variety of beverage plants such as tea, coffee and cocoa and are utilized as both food and feed due to its po- 2.5. Animal experimental protocol tential biological activity. Although cocoa consists of a number of polyphenols (Sanchez Rabaneda et al., 2003) the role of tannins in Female Sprague Dawley rats (170–200 g) were obtained from the biological actions is less studied. Three groups of polyphenols can be central animal facility of Sri Ramachandra University, Chennai. The distinguished namely catechins or flavan-3-ols (37%), anthocyanins animals were acclimatized for 7 days prior to their use in experi- (4%) and proanthocyanidins (58%). Total phenol content in cocoa varies ments and housed in polypropylene cages in an air-conditioned from 12 to 18% of defatted and dry weight. The main constituents of room maintained at 25±2 °C and 12 h with alternate day and night cocoa are (+)-catechin, (−)-epicatechin and 60% of proanthocyanidin cycles and relative humidity of 45%–55%. Locally procured paddy half of which are dimers (Borchers, Keen, Hannum, & Gershwin, 2000; husk was used after sterilization as bedding in the cages. Rats were Ortega et al., 2008; Sanchez Rabaneda et al., 2003; Wollgast & Anklam, allowed standard pellet diet (Amrut Feeds, New Delhi, metal con- 2000). The inhibitory effect of tannins has been shown to be due to tents, ppm dry weight Zn 45, Cu 10, Mn 55, Fe 70, and Co 5) through- the reduction of enzyme activity, dysfunctioning of cell membrane out the study. Drinking water solution was prepared by dissolving and deprivation of substrate metal ions and minerals (Goel, Puniya, measured quantities of arsenic (sodium arsenite) in water, and its Aguliar, & Singh, 2005). concentrations were checked by estimating representative samples Chelation therapy has been the core treatment against arsenic for total arsenic content using an atomic absorption spectrophotometer toxicity by formation of metal complexes and allowing removal of ex- (AAS), Perkin Elmer, USA. The arsenic-treated water was replaced every cess or toxic metal from the system rendering it immediately nontoxic alternative day. The laboratory animal protocol used for this study was and reducing the late effects. Although a range of metal chelators is approved by the Committee for the Purpose of Control and Supervision now available for arsenic chelation, development of biomolecules hav- on Experimental Animals (CPCSEA) at Sri Ramachandra University, ing less side effects can be applied. Thereby, prescribing antioxidants or Chennai (IAEC XIII/SRU/91/2008). nutraceuticals may be more seriously considered as good recommen- dations of chelation therapy. Hence, this work was undertaken for the 2.6. Experimental design for evaluation of T. cacao on arsenic toxicity purpose of studying the toxicology of sodium-m-arsenite (NaAsO2) and also the beneficial role of T. cacao in relation to tannins in preventing Female rats were randomized into eight groups of six rats each the arsenic induced hepato-toxicity in the reversal of toxic manifesta- and were treated as below for a period of 4 weeks: tions and altered biochemical variables by studying the markers of metal toxicity, markers of oxidative damage and stress and the changes Group I: control/vehicle treated in cellular thiol groups. Group II: arsenic as sodium arsenite, 100 ppm, via drinking H2O Group III: arsenic (as in group II)+vitamin C, 150 mg/kg, oral gavage, once daily 2. Materials and methods Group IV: arsenic (as in group II)+TCF, 30 mg/kg, oral gavage, once daily 2.1. Chemicals Group V: arsenic (as in group II)+TCF, 100 mg/kg, oral gavage, once daily Sodium-m-arsenite, NaAsO2, MW 130, Spectrochem (Mumbai, India), δ-aminolevulinic acid (Sigma Chemicals, USA) and all other Group VI: arsenic (as in group II)+TCF, 300 mg/kg, oral gavage, chemicals were of analytical grade and were purchased from Merck once daily 48 C. Chandranayagam et al. / Food Research International 50 (2013) 46–54

Group VII: arsenic (as in group II)+DCF, 100 mg/kg, oral gavage, 10 min. To 1 mL aliquot of supernatant, 2 tin balls combined with once daily 1 mL of 6 N HCL were added and shaken. To this, 2 mL of Griess re- Group VIII: arsenic (as in group II)+DCF, 300 mg/kg, oral gavage, agent was added and mixed well. The mixture was allowed to stand for 20 min under dark conditions. The color intensity of the chromogen once daily. was read at 540 nm. The values are expressed as μmolg−1 tissue. The dose for arsenic was selected on the basis of the induction of toxicity study which was previously carried out to determine the dose 2.7.6. Thiol group's estimation range and exposure duration to induce biochemical alterations and clinical signs of arsenic toxicity in Sprague Dawley rats (Chandronitha et al., 2.7.6.1. Total sulfhydryl content. The level of acid soluble sulfhydryl 2010). group was estimated in liver as total sulfhydryl group by the method The crude (TCF) and detannified (DCF) fractions of T. cacao were of Ellman (1959). The total sulfhydryl (\SH) reacts with 5,5′-dithiobis subsequently administered orally to the rats exposed to arsenic toxic- (2-nitro benzoic acid) and Ellman's reagent (DTNB) to give a yellow- ity. The weekly body weight, water intake and feed intake of individ- colored complex. Homogenates were mixed with 200 μL of saline, ual rats were measured during the entire period of the experiment. 250 μL of 0.2 M phosphate buffer (pH 8.0) and 500 μL of DTNB After 28 days, the animals in different groups were fasted and were (0.6 mM in 0.2 M phosphate buffer, pH 8.0) and incubated for sacrificed under light chloroform anesthesia at the end of the experi- 15 min at 37 °C. The precipitate was removed after centrifugation ment. A portion of the liver tissue from each rat was processed im- at 3000 rpm for 10 min. The total \SH was estimated in an aliquot mediately to appraise the organ weight, and biochemical assays and of supernatant. The absorbance was read at 412 nm using a UV/Vis small representative tissue slices were taken for histopathological spectrophotometer. examinations. 2.7.6.2. Non-protein bound sulfhydryl content (GSH). The level of acid 2.7. Biochemical assays soluble sulfhydryl group was estimated in liver as total non-protein sulfhydryl group by the method of Moren et al. (1979). The method 2.7.1. Tissue delta-aminolevulinic acid dehydratase (ALAD) activity is based on the reaction of reduced glutathione with 5,5′-dithiobis Tissue delta-aminolevulinic acid dehydratase (ALAD) activity was (2-nitro benzoic acid) to give a yellow-colored complex that has ab- determined in accordance with the protocol of Berlin and Schaller sorbance at 412 nm. The values are expressed as μmol of GSH content (1974). The assay system was made up of 0.25 mL 10% tissue homog- g−1 tissue. enate and 0.05 mL standard aminolevulinic acid (ALA) which was incubated for 60 min at 37 °C. The reaction was then stopped after 1 h 2.7.6.3. Protein bound sulfhydryl content. The level of acid soluble sulf- by the addition of 1 mL of 10% w/v TCA. An equal volume of Ehrlich's hydryl group was estimated in liver as protein bound sulfhydryl reagent was added to the supernatant and the absorbance was read group by the method of Ellman (1959). The total sulfhydryl level − − at 556 nm after 5 min. The results are expressed as μg min 1 mg 1 minus non protein bound sulfhydryl will give the level of protein protein. bound sulfhydryl. The values are expressed as μmol of GSH content g−1 tissue. 2.7.2. Tissue reduced glutathione (GSH) and lipid peroxidation (LPO) Reduced glutathione (GSH) was determined according to the 2.8. Histology analysis method of Moren, Desplerra, and Mannervick (1979). The method is based on the reaction of reduced glutathione with 5,5′-dithiobis A sample of liver tissue was fixed in 10% formalin for 24 h, (2-nitro benzoic acid), which gives a yellow-colored complex that dehydrated, embedded in paraffin and then sections of 5 μm thickness has absorbance at 412 nm. The values are expressed as μmol of GSH were cut. The sections were stained with hematoxylin and eosin and − content g 1 tissue. Tissue lipid peroxidation (LPO) was measured as examined under a light microscope. TBA-reactive substances according to the method of Ohkawa, Ohishi, and Yagi (1979). The values are expressed in μmol of malondialdehyde 2.9. Statistical analysis g−1 of protein. The experimental results are expressed as the mean±SEM. To 2.7.3. Tissue superoxide dismutase (SOD) and glutathione peroxidase assess significant protection in the treatment groups one-way ANOVA (GPx) was performed followed by Bonferroni test using statistical software. Superoxide dismutase was assayed by the method of Kakkar, Das, The p values were set at pb0.05 and 0.01 levels. and Viswanathan (1984). The activity was calculated as the amount of protein required to give 50% inhibition of PMS-NBT autooxidation. The 3. Results results are expressed as unit min−1 mg−1 protein. Glutathione peroxidase (GPx) was determined according to the method of Rotruck et al. The change in general parameters such as body weight, feed in- − − (1973). The results are expressed as μmol of GSH utilized min 1 mg 1 take, and organ weight (liver) were observed in the arsenic-induced of protein. experimental rats for 28 days and the data are shown in Tables 1, 2 and 3. There were significant (pb0.01) body weight and organ weight 2.7.4. Tissue protein content changes in the arsenic intoxicated experimental animals. The growth Protein content in liver tissue was estimated by the method of was comparatively reduced in arsenic-intoxicated rats when com- Lowry, Rosenberg, Farr, and Randal (1951). A standard graph of pro- pared to the normal, vitamin C and tannin rich cocoa fraction (TCF) tein was plotted, from which the protein content of the tissue was treated group of animals. All experimental animals showed gradual determined. improvement in body weight and organ weight after treatment with tannin rich cocoa fraction (TCF) (Tables 1 and 3). However, there were 2.7.5. Tissue nitrite/nitrate no significant changes noted in the feed intake of the all experimental Estimation of nitrate/nitrite was assayed by the method of Green groups (Table 2). et al. (1982). The assay mixture consisted of 0.2 mL of 10% homoge- A significant (pb0.01) decrease in the activity of the specific bio- nate, 1.8 mL of saline and 0.4 mL of SSA for protein precipitation. marker (ALAD) for metal toxicity was observed in the hepatic tissue

The precipitate was removed by centrifugation at 4000 rpm for of NaAsO2 intoxicated experimental rats (Fig. 1). Administration of C. Chandranayagam et al. / Food Research International 50 (2013) 46–54 49

Table 1 Effect of crude tannin rich cocoa fraction (TCF) and detanniﬁed cocoa fraction (DCF) of arsenic-induced rats on body weight.

Treatment Body weight (g)

0 week 1st week 2nd week 3rd week 4th week

Vehicle 175.50±4.55 179.50±4.22 182.83±7.25 186.50±5.59 192.00±5.05 PC (As 100) 166.37±5.89^^ 167.17±7.47^^ 162.00±6.67^^ 156.87±7.66^^ 151.67±8.06^^ Vitamin C+As 100 181.33±4.73 186.50±5.45 189.17±5.64 190.83±7.25 194.33±8.60 TCF low+As 100 179.93±5.47 175.67±5.35 179.33±5.47 183.67±5.04 186.67±6.36 TCF mid+As 100 181.60±4.60 177.67±4.81 181.50±6.04 185.17±3.98 188.00±4.96 TCF high+As 100 183.30±1.93 181.67±2.58 185.67±2.87 188.50±3.76 191.50±4.63 DCF low+As 100 178.23±2.80 174.67±5.35 176.17±3.71 178.83±5.34 176.67±5.22 DCF high+As 100 182.87±2.01 177.67±1.58 171.50±2.56 184.70±4.52 187.83±3.68

Body weight changes in experimental animals during 0 week, ﬁrst, second, third, and fourth weeks are depicted. Values are expressed in mean±SEM (n=6). One way variance followed by Dunnet multiple comparisons. The p values ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) treated rats.

tannin rich cocoa (TCF) showed a dose dependent significant (pb0.01) Tannin rich cocoa (TCF) showed a significant (pb0.01) dose dependent protection as evidenced by ALAD activity. TCF at a dose of 300 mg/kg elevation in liver SOD activity whereas, with detannified cocoa (DCF) body weight for 28 days showed better protection of hepatic ALAD treatment the SOD activity did not show better activity as compared activity. Vitamin C treated rats also showed a markedly significant to the TCF treated animals. Treatment with vitamin C showed a signif- (pb0.01) increase in ALAD activity. But, treatment with detannified icant (pb0.01) preventive role against arsenic toxicity. GPx is the most cocoa (DCF) showed only slight protection, which was comparatively important enzyme against the toxic effects of oxygen metabolism. Ar- poor when compared to the tannin rich TCF treated. senic administration produced a significant decline (pb0.01) in liver GSH level in the liver tissue of the experimental animals has been GPx activity (Fig. 5), whereas tannin rich cocoa (TCF) treatment against depicted in Fig. 2. The results show that administration of NaAsO2 arsenic toxicity showed significant (pb0.01) increase in liver GPx ac- caused a significant (pb0.01) reduction in the GSH level compared tivity similar to the vitamin C treated rat. Administration of TCF at a to the normal control. Co-treatment with mid and high doses of TCF dose of 100 mg/kg body weight showed better activity against arsenic to the toxin administered rats showed significant protection against toxicity whereas, with detannified cocoa (DCF) treatment the GPx ac- arsenic intoxication. DCF treated groups did not show better elevation tivity did not show better activity as compared to the TCF treated of GSH levels than TCF treated groups. Administration of vitamin C animals. significantly (pb0.01) increased the GSH levels proving it to be a The measurement of nitrite in extracellular medium is often used very good antioxidant against arsenic intoxication. Exposure to arsenic to measure NO generation. The administration of NaAsO2 caused a produced an appreciable change in hepatic TBARS levels suggesting significant (pb0.01) reduction in the nitrite level compared to the lipid peroxidation, a marker for oxidative damage. Arsenic exposure normal control (Fig. 6). The administration of low and mid doses of produced significant adverse effects on the redox status of liver, which tannin rich cocoa (TCF) in rats caused a significant (pb0.01) increase isevidencedbyasignificant (pb0.01) increase in thiobarbituric acid in nitrate levels. Administration of DCF did not show any statistically reactive substance level as shown in Fig. 3. Treatment with TCF followed significant increase in nitrite levels. by arsenite administration decreased the TBARS level compared to the The levels of the hepatic sulfhydryl groups (total, protein and toxin control. TCF at mid and high dose demonstrated a significant non-protein bound) in different groups are indicated in Table 4.NaAsO2 (pb0.01) protection against arsenic toxicity. Vitamin C treatment also administration reduced the thiol content significantly compared to proved to be a significant (pb0.01) protection. High dose of DCF that of the normal control. Co-treatment with TCF followed by toxin showed slight significant protection (pb0.05) against arsenic toxicity administration could attenuate the toxic effect of NaAsO2. The results but statistical significance proved that TCF treatment showed better showed a significant dose dependent (pb0.01) increase in the sulfhy- protection than DCF treatment. dryl levels. DCF treatment also showed a marked elevation in sulfhy-

SOD catalyzes the conversion of superoxide anion into H2O2. dryl levels. Arsenite intoxication caused signiﬁcant depletion (pb0.01) in liver superoxide dismutase (SOD) activity with respect to control (Fig. 4).

Table 3 Effect of crude tannin rich cocoa fraction (TCF) and detannified Table 2 cocoa fraction (DCF) of arsenic-induced rats on organ weight. Effect of crude tannin rich cocoa fraction (TCF) and detannified cocoa fraction (DCF) of arsenic-induced rats on feed intake. Treatment Organ weight (liver) (g) Treatment Feed intake (g) Vehicle 6.64±0.33 1st week 2nd week 3rd week 4th week PC (As 100) 3.15±1.04^^ Vehicle 56.80±1.49 62.55±1.16 65.61±1.43 66.67±1.61 Vitamin C+As 100 5.43±0.33 PC (As 100) 54.17±1.51 70.17±2.11 68.33±2.49 68.33±2.30 TCF low+As 100 4.74±1.11 Vitamin C+As 100 56.20±1.19 66.31±1.13 69.73±1.13 71.55±1.09 TCF mid+As 100 5.25±0.37 TCF low+As 100 67.90±1.75 65.67±1.31 73.37±1.24 72.53±1.57 TCF high+As 100 5.48±0.38 TCF mid+As 100 59.87±1.86 61.22±1.73 65.90±1.85 67.51±1.65 DCF low+As 100 3.97±0.87 TCF high+As 100 56.20±0.99 60.73±2.07 68.50±0.93 63.33±1.74 DCF high+As 100 4.15±1.25 DCF low+As 100 59.07±1.36 63.20±1.33 62.23±1.05 61.73±2.15 Effect of arsenic induced rats on organ (liver) weight is depicted. DCF high+As 100 58.42±1.29 65.73±1.03 67.73±1.13 69.35±1.44 Values are expressed in mean±SEM (n=6). One way variance Effect of arsenic induced rats on feed intake of experimental animals during, first, followed by Dunnet multiple comparisons. The p values ^(b0.05) second, third, and fourth weeks was depicted. Values are expressed in mean±SEM and ^^(b0.01) — normal control compared with arsenic (100 ppm) (n=6). One way variance followed by Dunnet multiple comparisons. treated rats. 50 C. Chandranayagam et al. / Food Research International 50 (2013) 46–54

Fig. 3. Effect of arsenic induced rats on hepatic LPO activity. The results are expressed Fig. 1. Effect of arsenic induced rats on hepatic arsenic bio-marker enzyme (ALAD) ac- as μmol (micromoles) per gram tissue. Values are expressed as mean±SEM (n=6); μ tivity. The results are expressed as g per minute per milligram protein. Values are statistical analysis was done using one way ANOVA, Bonferroni. The p values expressed as mean±SEM (n=6); statistical analysis was done using one way ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) treated ^ b ^^ b — ANOVA, Bonferroni. The p values ( 0.05) and ( 0.01) normal control compared rats, †(b0.05) and ††(b0.01) — arsenic (100 ppm) treated compared with vitamin C, † b †† b — with arsenic (100 ppm) treated rats, ( 0.05) and ( 0.01) arsenic (100 ppm) TCF and DCF treated rats, c,d,e(b0.05) and cc,dd,ee(b0.01) — TCF compared with DCF c,d,e b cc,dd, treated compared with vitamin C, TCF and DCF treated rats, ( 0.05) and treated rats. ee(b0.01) — TCF compared with DCF treated rats.

Histopathological assessments of different hepatic segments are examination of the vitamin C treated group also supported its preven- presented in Fig. 7. In arsenic-treated animals the hepatic sections tive role against NaAsO2-induced hepatic oxidative impairment. showed hepatocellular degenerative lesions along with inflammatory cells and irregular hepatic cells with few cells which showed an ap- 4. Discussion pearance of apoptosis. Treatment with TCF showed prominent recovery in the form of maintained hepatic histoarchitecture. The Dietary antioxidants in the form of nutrients appear to be of great portal triad region showed only few inflammatory cell infiltrations importance in controlling damage by free radicals. However, each nu- with many intact hepatocytes and no severe sinusoidal conjection trient is unique in terms of its structure and its particular antioxidant after TCF treatment was seen whereas, DCF treated animals showed function. Phytochemical substances in plants and plant derived foods severe inflammatory cell infiltrations and few intact hepatocytes in such as cocoa and chocolate have gained growing attention. They are the hepatic artery region with sinusoidal conjection. Histological also being widely investigated for their antioxidant activity and other

Fig. 2. Effect of arsenic induced rats on hepatic GSH activity. The results are expressed Fig. 4. Effect of arsenic induced rats on hepatic SOD activity. The results are expressed as μmol (micromoles) per gram tissue. Values are expressed as mean±SEM (n=6); as units per minute per mg of protein. Values are expressed as mean±SEM (n=6); statistical analysis was done using one way ANOVA, Bonferroni. The p values statistical analysis was done using one way ANOVA, Bonferroni. The p values ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) treated ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) treated rats, †(b0.05) and ††(b0.01) — arsenic (100 ppm) treated compared with vitamin C, rats, †(b0.05) and ††(b0.01) — arsenic (100 ppm) treated compared with vitamin C, TCF and CDT treated rats, c,d,e(b0.05) and cc,dd,ee(b0.01) — TCF compared with DCF TCF and DCF treated rats, c,d,e(b0.05) and cc,dd,ee(b0.01) — TCF compared with DCF treated rats. treated rats. C. Chandranayagam et al. / Food Research International 50 (2013) 46–54 51

Table 4 Effect of crude tannin rich cocoa fraction (TCF) and detanniﬁed cocoa fraction (DCF) of arsenic-induced rats on hepatic thiol activity.

Treatment Liver (μmolg−1)

Total bound Protein bound Non protein bound sulfhydryl sulfhydryl sulfhydryl

Vehicle 33.49±1.81 32.35±1.79 1.14±0.03 PC (As 100) 18.18±0.91^^ 17.64±0.91^^ 0.54±0.01^^ Vitamin C+As 100 28.89±1.26†† 28.04±1.27†† 0.85±0.01†† TCF low+As 100 30.10±1.46†† 29.44±1.46†† 0.65±0.02 TCF mid+As 100 32.52±0.42†† 31.05±0.42†† 1.48±0.01†† TCF high+As 100 35.26±0.78†† 34.13±0.82†† 1.12±0.10†† DCF low+As 100 31.52±2.18†† 30.45±2.19†† 1.07±0.01††,cc,dd DCF high+As 100 31.28±0.82†† 29.54±0.83†† 1.74±0.01††,cc,dd,ee

Effect of arsenic induced rats on hepatic thiol activity. The results are expressed as μmol (micromoles) per gram tissue. Values are expressed as mean±SEM (n=6); Statistical analysis was done using one way ANOVA, Bonferroni. The p values ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) treated rats, †(b0.05) and ††(b0.01) — arsenic (100 ppm) treated compared with vitamin C, TCF and DCF treated rats, c,d,e(b0.05) and cc,dd,ee(b0.01) — TCF compared with DCF treated rats.

Fig. 5. Effect of arsenic induced rats on hepatic GPx activity. The results are expressed as μmol of GSH consumed per minute per mg of protein. Values are expressed as mean± SEM (n=6); Statistical analysis was done using one way ANOVA, Bonferroni. The p values indicated that it inhibits lipid peroxidation, scavenges free radicals ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) treated rats, and chelates transition metal ions (Sarmadi, Ismail, & Hamid, 2011). † †† (b0.05) and (b0.01) — arsenic (100 ppm) treated compared with vitamin C, TCF and The present investigation revealed that arsenic intoxication caused c,d,e b cc,dd,ee b — DCF treated rats, ( 0.05) and ( 0.01) TCF compared with DCF treated rats. asignificant increase in lipid peroxidation along with a significant decrease in body weight, organ weight, thiol levels, delta-aminolevulinic acid dehydratase (ALAD), reduced glutathione (GSH), glutathione per- possible health-promoting potentials. ‘T. cacao’ polyphenols have oxidase (GPx), superoxide dismutase (SOD) and the index of nitrite/ been extensively reported to have health benefits. The supplementa- nitrate (NOx) activities in liver. The main cause of the arsenic induced tion of cocoa extracts to obese-diabetic rats decreased plasma oxida- liver injury is the formation of free radicals and its metabolites. Ascorbic tive stress biomarker and increased superoxide dismutase activity, acid is a well-known antioxidant and has been included in the present attributable to the extracts' polyphenols and methylxanthines (Abbe, study as a standard. Co-administration with vitamin C followed by arse- Amin, Chong, Muhajir, & Hasbullah, 2008). Cocoa, cocoa extracts, and nite treatment normalized the activities of all the antioxidant enzymes purified cocoa flavanols and procyanidins had been proven to exert a as well as the status of the cellular metabolites such as thiol groups strong antioxidant effect in vitro (Keen, Holt, Oteiza, Fraga, & Schmitz, compared to that of the NaAsO2 treated group. Histological examination 2005). The phenolic compounds of cocoa (T. cacao L.) belong to of the vitamin C-treated group also supported its preventive role against many classes of molecules namely catechins, epicatechins, anthocya- NaAsO2-induced hepatic oxidative impairment. nins, pro-anthocyanidins, phenolic acids, condensed tannins, other ALAD is a sulfhydryl-containing enzyme involved in the heme flavonoids and some minor compounds (Arlorio et al., 2005). The synthesis pathway, and its inhibition can be attributed to the binding exact mechanism underlying the antioxidant activity of cocoa has of arsenic with sulfhydryl groups (Thomas, Styblo, & Lin, 2001). Arse- nic exerts its toxicity through reaction with sulfhydryls that exist in the cell (Kitchin, 2001). As described in our study, inhibition of ALAD was observed in the liver. Arsenic has a high affinity for the SH group and it binds with reduced glutathione (GSH). ALAD inacti- vation may also lead to the accumulation of δ-aminolevulinic acid that can cause an overproduction of ROS, which in part could explain arsenic-induced oxidative stress (Bhadauria & Flora, 2003). Few recent studies also suggested a significant inhibition of blood delta-aminolevulinic acid dehydratase (ALAD) after sub chronic and chronic arsenic exposure (Flora, Bhadauria, Kannan, & Singh, 2007). In our study, treatment with crude tannin rich cocoa fraction (TCF) caused marked elevation in the level of ALAD activity. GSH has been reported to play an important role in the process of metal toxicity. The result of our present work shows a decrease of GSH in the liver of arsenic exposed rats indicating oxidative stress. GSH is a non-enzymic antioxidant and its regulation on interaction of metal could be through the neutralization of oxidative radicals (Swaran Flora, Smrati Bhadauria, Satish, & Ram, 2005). Mishra, Mehta, and Flora (2008) reported that glutathione and its linked enzymes may play a vital role in detoxification of arsenic from hepatic cells and Fig. 6. Effect of arsenic induced rats on hepatic nitrate/nitrite activity. The results are prevent apoptosis, which strongly supports our current observation. expressed as μmol (micromoles) per gram tissue. Values are expressed as mean± In addition, inhibition of antioxidant enzymes such as superoxide SEM (n=6); statistical analysis was done using one way ANOVA, Bonferroni. The p dismutase (SOD) and glutathione peroxidase (GPx) in the hepatic tis- values ^(b0.05) and ^^(b0.01) — normal control compared with arsenic (100 ppm) † †† sue of arsenic exposed animals suggests disturbed antioxidant ratio, treated rats, (b0.05) and (b0.01) — arsenic (100 ppm) treated compared with vitamin C, TCF and DCF treated rats, c,d,e(b0.05) and cc,dd,ee(b0.01) — TCF compared with resulting in oxidative stress. Superoxide dismutase (SOD) and glutathi- DCF treated rats. one peroxidase (GPx) are the most important defense enzymes against 52 C. Chandranayagam et al. / Food Research International 50 (2013) 46–54

Fig. 7. Histopathological findings of drug treated female rats for 28 days in liver tissues were shown. a represents the normal liver tissue, b represents arsenic induced rats, c represents vitamin C treated, d represents TCF treated and e represents DCF treated. Each white arrow shows the variation of damage. toxic effects of oxygen metabolism. SOD accelerates the dismutation of nitrite can be reduced back to NO under appropriate physiological con- superoxide to H2O2. A decrease in the activity of SOD can be due to ditions and nitrite itself can directly nitrosate thiols to form RSNOs enhanced superoxide production during arsenic metabolism (Gupta (Bryan et al., 2005), have caused intense interest in this molecule & Flora, 2006b). Glutathione peroxidase (GPx) is a Se-dependent en- (Gladwin et al., 2005). NO can react with metals, reduced thiols, molec- zyme and effect of arsenic on synthesis and activity of selenoenzymes ular oxygen and superoxide leading to adverse reactions. Thereby, which may directly interact with Se and form insoluble and inactive co-administration of tannin rich cocoa (TCF) and arsenic led to a signif- As–Se complex (Jain, Yadav, Bozhkov, Padalko, & Flora, 2011) resulting icant increase in the hepatic nitrite levels revealing that NO production in the inhibition of GPx activity. The chronic consumption of diets is reduced in arsenic intoxication. containing 2% cocoa powder, providing 1.57 mg/g diet of flavanols and One of the possible mechanisms for higher toxicity of trivalent procyanidins, was linked with reduced DNA and glutathione oxidation arsenicals compared to the corresponding pentavalent forms is that in rats (Orozco, Wang, & Keen, 2003). In the present study, administra- trivalent species have higher affinity for thiol compounds and gener- tion of tannin rich cocoa (TCF) significantly protected the levels of SOD ate reactive oxygen species (Shiobara, Ogra, & Suzuki, 2001). Arsenic and GPx activities by directly scavenging ROS as well as by inhibiting as potent sulfhydryl-reactive compound has been shown to affect lipid peroxidation suggesting antioxidant properties of cocoa. numerous intracellular signal transduction pathways causing many Lipid peroxidation is known to be one of the major causes of arse- alterations in cellular functions (Rana et al., 2011). The thiol levels nite induced liver toxicity. The estimation of a byproduct of LPO, play an important role in the detoxification process via facilitating re- malondialdehyde (MDA), is a relevant method to evaluate the degree moval of arsenic from the cellular sites and stimulating excretion of of peroxidation damage to cell membrane. Increased production of methylated arsenic (Vahter, 2002), which is responsible for arsenic free radicals and inhibition of antioxidant enzymes have been possible induced cytotoxicity. In the present study, administration of tannin mechanisms to explain arsenic-induced oxidative damage. Studies have rich cocoa (TCF) significantly restores the depleted thiol levels in con- reported that an increased LPO level has a positive correlation with the trast to DCF treatment. dose of arsenic. Membrane LPO is a common mechanism of cell death The liver is a major site for the biotransformation, accumulation that directly causes cell membrane destruction or may change mem- and excretion of exogenous chemicals. Prolonged arsenic ingestion brane fluidity and the membrane potential (Bera et al., 2010). In rats, di- leads to hepatic fibrosis and non-cirrhotic portal hypertension as abetes mellitus-induced cataracts and ex vivo lipid peroxide formation liver is one of the target organs (Rana et al., 2010). Routine histological were decreased when cocoa liquor was given (Osakabe, Yamagishi, studies on liver documented arsenic-induced changes characterized by Natsume, Yasuda, & Osawa, 2004). The preventive properties of tannin dilated sinusoids, formation of intracellular edema, megalocytosis, vac- rich cocoa (TCF) can been related to the inhibition of lipid peroxide uolation and appearance of hepatic cells with distorted nuclei (Datta et formation as evident from the decreased TBARS level. al., 2007). The present investigation suggests that arsenite intoxication Nitrite and nitrate are oxidative products of the metabolism and produces various pathological alterations in the liver of Sprague Dawley have gained much attention. Nitrates and nitrites also play a positive rats such as hepatocellular degenerative lesions along with inflam- role in the human body, protecting against hypertension and supporting matory cells and irregular hepatic cells with few cells which showed the circulatory system (Hord, Tang, & Bryan, 2009). Nitrate serves as a an appearance of apoptosis. Treatment of tannin rich fraction (TCF) source, via successive reduction, for the production of nitrite and nitric prevented any such arsenite induced alterations and preserved the oxide as well as other metabolic products. Earlier studies revealed that hepatic cells histologically almost to normal. nitrite may be reduced to NO in vivo and in vitro representing a huge The toxic effect of arsenic is mediated through the generation of bioactive NO storage pool (Cosby et al., 2003). Nitric oxide (NO), a reactive oxygen species (ROS) and ROS-driven oxidative stress. The short-lived molecule acts as a trans-cellular messenger in numerous anti-oxidant systems such as dietary phytochemicals would play a physiological processes, including vasodilation, neurotransmission major role in treatment-related toxicities. Superoxide dismutase and macrophage-mediated immunity. The recent discoveries that (SOD), glutathione peroxidase (GPx) and catalase (CAT), together C. Chandranayagam et al. / Food Research International 50 (2013) 46–54 53 with glutathione (GSH), form the first-line of defense against ROS. Arlorio, M., Bottini, C., Travaglia, F., Locatelli, M., Bordiga, M., Coisson, J. D., et al. (2009). •\ Protective activity of Theobroma cacao L. phenolic extract on AML12 and MLP29 SOD converts superoxide anion (O2 )toH2O2, which is then de- liver cells by preventing apoptosis and inducing autophagy. Journal of Agricultural toxified to water by GPx and CAT. If not removed, H2O2 itself causes and Food Chemistry, 57, 10612–10618. oxidative damage to biomolecules, and can be converted to a more Arlorio, M., Coïsson, J. D., Travaglia, F., Varsaldi, F., Miglio, G., Lombardi, G., et al. (2005). Antioxidant and biological activity of phenolic pigments from Theobroma cacao damaging hydroxyl radical (•OH) species (Eun-Mi Park et al., 2007). hulls extracted with supercritical CO2. Food Research International, 38, 1009–1014. On the other hand, under stress conditions, an excess of superoxide Bera, A. K., Rana, T., Das, S., Bandyopadhyay, S., Bhattacharya, D., Pan, D., et al. (2010). releases free iron from iron-containing molecules. Phenolic phyto- L-Ascorbate protects rat hepatocytes against sodium arsenite-induced cytotoxicity – chemicals are generally recognized as major antioxidants, but they and oxidative damage. Human and Experimental Toxicology, 29(2), 103 111. Berlin, A., & Schaller, K. H. (1974). European standardized method for the determina- can also exhibit prooxidant activities. In fact, most free-radical scav- tion of delta aminolevulinic acid dehydratase activity in blood. Zeitschrift für engers act in oxidation–reduction reactions that are reversible, and Klinische Chemie und Klinische Biochemie, 12, 389–390. some (such as phenolic phytochemicals) can act both as antioxidants Beyersmann, D., & Hartwig, A. (2008). Carcinogenic metal compounds: Recent insight into molecular and cellular mechanisms. Archives of Toxicology, 82, 493–512. and prooxidants depending on their structure and the reaction condi- Bhadauria, S., & Flora, S. J. S. (2003). Arsenic induced inhibition of D-aminolevulinic acid tions. Amid human focus, the flavonoid-rich chocolate consumption dehydratase activity in rat blood and its response to meso 2,3-dimercapto-succinic increased the plasma antioxidant capacity and reduced the amounts acid and monoisoamyl DMSA. Biomedical and Environmental Sciences, 17, 305–313. Borchers, A. T., Keen, C. L., Hannum, S. M., & Gershwin, M. E. (2000). Cocoa and choco- of plasma 2-thiobarbituric acid reactive substances (Wang et al., late: Composition, bioavailability, and health implications. Journal of Medicinal 2000). While the defined mechanisms underlying the antioxidant Food, 3,77–105. effects of flavonoids have yet to be identified, the research reported Bryan, N. S., Fernandez, B. O., Bauer, S. M., Garcia-Saura, M. F., Milsom, A. B., Rassaf, T., fl et al. (2005). Nitrite is a signaling molecule and regulator of gene expression in that consumption of avanols and procyanidins in the diet can signif- mammalian tissues. Nature Chemical Biology, 1(5), 290–297. icantly supplement the oxidative defense system. Thus, the effect of Chandronitha, C., Ananthi, S., Ramakrishnan, G., Lakshmisundaram, R., Gayathri, V., & dietary phenolic phytochemicals varies with the structures of the in- Vasanthi, H. R. (2010). 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(TCF) plays a possible more protective role against NaAsO2-induced fi Sublethal concentration of arsenic interferes with the proliferation of hepatocytes liver damage than detanni ed cocoa fraction (DCF) due to the pres- and induces in vivo apoptosis in Clarias batrachus L. Comparative Biochemistry and ence of high polyphenolic compounds such as tannins, phenols, and Physiology Part C: Toxicology & Pharmacology, 145, 339–349. flavonoids. On the other hand, the deficiency of tannins in DCF led Dopp, E., von Recklinghausen, U., Diaz-Bone, R., Hirner, A. V., & Rettenmeier, A. W. to the possible declined antioxidant activity. The mechanistic path- (2010). Cellular uptake, subcellular distribution and toxicity of arsenic compounds in methylating and nonmethylating cells. Environmental Research, 110(5), 435–442. ways that the tannin rich TCF used to exhibit its protective action Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry, 82,70–77. against arsenic induced toxicity are not fully clear except for its anti- Flora, S. J. S., Bhadauria, S., Kannan, G. M., & Singh, N. (2007). Arsenic induced oxidative oxidant property. Like vitamin C, tannin rich fraction (TCF) also pos- stress and the role of antioxidant supplementation during chelation: A review. Journal of Environmental Biology, 28(2), 333–347. sesses similar free radical scavenging activity and shows a Flora, S. J. S., Mehta, A., Rao, P. V. L., Kannan, G. M., Bhaokar, A., Dube, S. N., et al. (2004). preventive role against arsenic induced toxicity. So further investiga- Therapeutic potential of monoisoamyl and monomethyl esters of meso 2,3- tions are necessary to find out the exact mechanistic pathways and dimercaptosuccimic acid in gallium arsenide intoxicated rats. Toxicology, 195,127–146. Gladwin, M. T., Schechter, A. N., Kim-Shapiro, D. B., Patel, R. P., Hogg, N., Shiva, S., et al. this work is currently in progress. 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