High-Protein Diet Ameliorates Arsenic-Induced Oxidative Stress and Antagonizes Uterine Apoptosis in Rats

  • Prerona Biswas
  • Aparna Mukhopadhyay
  • Syed Nazrul Kabir
  • Prabir Kumar MukhopadhyayEmail author


Arsenic toxicity purportedly threats a broad spectrum of female reproductive functions. We investigated the remedial role of a casein- and pea protein-enriched high-protein diet (HPD) in combating the arsenic insult. Cyclic female rats maintained on standard diet (n = 6) or an isocaloric HPD (n = 6) were gavaged with As2O3 at 3 mg/kg BW/rat/day (n = 12) for 28 days. Vehicle-fed rats (n = 6) maintained on the standard diet served as the control. We monitored the estrus cycles and performed the histomorphometric analyses of the uterus and ovary. Uterine luminal epithelial (ULE) ultrastructure was appraised by scanning electron microscopy. Uterine oxidative stress was evaluated in the forms of ROS generation and activities of the ROS scavengers. The uterine apoptotic manifestation was blueprinted by Western blot analysis of caspase-3 and Bax expression. Arsenic treatment arrested the follicular maturation and disrupted the estrus cycles with a typical increase in the diestrus index. Shrunken endometrial glands and thinned microvilli density of the ULE reflected loss of cell polarity and mislaid uterine homeostasis. Increased ROS generation and attenuated activities of the ROS scavengers marked a state of uterine oxidative imbalance and loss of redox regulation. Superfluous expression of procaspase-3, cleaved caspase-3, and Bax mirrored an inflated state of uterine apoptosis. HPD supplementation, by and large, counteracted these arsenic impacts and maintained the frameworks close to the control levels. In conclusion, arsenic mediates its reproductive toxicity, at least in part, by upsetting the uterine ROS homeostasis and redox regulation. Pea proteins and casein-supplemented HPD can counteract the arsenic effects and maintain the reproductive functions.


Arsenic Uterine microvilli Pea protein Casein Apoptosis Oxidative stress 



Analysis of variance


5-bromo-4-chloro-3-indolyl phosphate


Body weight


Corpus luteum


Ethylenediaminetetracetic acid


Former follicular cavity


Glutathione SH


High-protein diet


Immunoglobulin G


Least significant difference


Mitochondrial outer membrane permeabilization


Nitro blue tetrazolium


Polyacrylamide gel electrophoresis


Phosphate buffered saline


Protein carbonyl content


Polyvinylidene fluoride


Radioimmunoprecipitation assay


Reactive oxygen species


Sodium dodecyl sulphate


Scanning electron microscopy


Statistical Package for the Social Sciences


Tris buffered saline


World Health Organization


Funding Information

This study is supported from the Faculty Research and Professional Development Fund, Presidency University and Department of Science and Technology, Government of West Bengal [265(Sanc.)/ST/P/S&T/1G-44/2017].

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.


  1. 1.
    Chakraborty T, De M (2009) Clastogenic effects of inorganic arsenic salts on human chromosomes in vitro. Drug Chem Toxicol 32(2):169–173. PubMedCrossRefGoogle Scholar
  2. 2.
    Sarkar A, Paul B (2016) The global menace of arsenic and its conventional remediation - a critical review. Chemosphere 158:37–49. PubMedCrossRefGoogle Scholar
  3. 3.
    Chakraborti D, Singh S, Rahman M, Dutta R, Mukherjee S, Pati S, Kar P (2018) Groundwater arsenic contamination in the Ganga River Basin: a future health danger. Int J Environ Res Public Health 15(2):180. PubMedCentralCrossRefGoogle Scholar
  4. 4.
    Ahmad SA, Sayed MH, Barua S, Khan MH, Faruquee MH, Jalil A, Hadi SA, Talukder HK (2001) Arsenic in drinking water and pregnancy outcomes. Environ Health Perspect 109(6):629–631PubMedPubMedCentralCrossRefGoogle Scholar
  5. 5.
    Sen J, Chaudhuri AB (2008) Arsenic exposure through drinking water and its effect on pregnancy outcome in Bengali women. Arch Ind Hyg Toxicol 59:271–275. CrossRefGoogle Scholar
  6. 6.
    Shen J, Liu J, Xie Y, Diwan BA, Waalkes MP (2007) Fetal onset of aberrant gene expression relevant to pulmonary carcinogenesis in lung adenocarcinoma development induced by in utero arsenic exposure. Toxicol Sci 95(2):313–320. PubMedCrossRefGoogle Scholar
  7. 7.
    Chattopadhyay S, Pal S, Chaki S, Debnath J, Ghosh D (1999) Effect of sodium arsenite on plasma levels of gonadotrophins and ovarian steroidogenesis in mature albino rats: duration-dependent response. J Toxicol Sci 24(5):425–431PubMedCrossRefGoogle Scholar
  8. 8.
    Akram Z, Jalali S, Shami SA, Ahmad L, Batool S, Kalsoom O (2010) Adverse effects of arsenic exposure on uterine function and structure in female rat. Exp Toxicol Pathol 62(4):451–459. PubMedCrossRefGoogle Scholar
  9. 9.
    Chattopadhyay S, Ghosh D (2010) Role of dietary GSH in the amelioration of sodium arsenite-induced ovarian and uterine disorders. Reprod Toxicol 30(3):481–488. PubMedCrossRefGoogle Scholar
  10. 10.
    Chatterjee A, Chatterji U (2011) All-trans retinoic acid protects against arsenic-induced uterine toxicity in female Sprague–Dawley rats. Toxicol Appl Pharmacol 257(2):250–263. PubMedCrossRefGoogle Scholar
  11. 11.
    Mondal S, Mukherjee S, Chaudhuri K, Kabir SN, Mukhopadhyay PK (2013) Prevention of arsenic-mediated reproductive toxicity in adult female rats by high protein diet. Pharm Biol 51(11):1363–1371. PubMedCrossRefGoogle Scholar
  12. 12.
    Chatterjee A, Chatterji U (2010) Arsenic abrogates the estrogen-signaling pathway in the rat uterus. Reprod Biol Endocrinol 8(1):80. PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Chatterjee A, Chatterji U (2017) All-trans retinoic acid ameliorates arsenic-induced oxidative stress and apoptosis in the rat uterus by modulating MAPK signaling proteins. J Cell Biochem 118(11):3796–3809. PubMedCrossRefGoogle Scholar
  14. 14.
    Flora SJS, Mittal M, Mehta A (2008) Heavy metal induced oxidative stress & its possible reversal by chelation therapy. Indian J Med Res 128(4):501–523PubMedGoogle Scholar
  15. 15.
    Pokorný J (2007) Are natural antioxidants better – and safer – than synthetic antioxidants? Eur J Lipid Sci Technol 109:629–642. CrossRefGoogle Scholar
  16. 16.
    Chattopadhyay S, Ghosh S, Debnath J, Ghosh D (2001) Protection of sodium arsenite-induced ovarian toxicity by coadministration of L-ascorbate (vitamin C) in mature wistar strain rat. Arch Environ Contam Toxicol 41(1):83–89. PubMedCrossRefGoogle Scholar
  17. 17.
    Chattopadhyay S, Ghosh SP, Ghosh D, Debnath J (2003) Effect of dietary co-administration of sodium selenite on sodium arsenite-induced ovarian and uterine disorders in mature albino rats. Toxicol Sci 75(2):412–422. PubMedCrossRefGoogle Scholar
  18. 18.
    Maity M, Perveen H, Dash M, Jana S, Khatun S, Dey A, Mandal AK, Chattopadhyay S (2018) Arjunolic acid improves the serum level of vitamin B12 and folate in the process of the attenuation of arsenic induced uterine oxidative stress. Biol Trace Elem Res 182(1):78–90. PubMedCrossRefGoogle Scholar
  19. 19.
    Sharma A, Flora SJS (2018) Nutritional management can assist a significant role in alleviation of arsenicosis. J Trace Elem Med Biol 45:11–20. PubMedCrossRefGoogle Scholar
  20. 20.
    Mukherjee S, Das D, Darbar S, Mitra C (2003) Dietary intervention affects arsenic-generated nitric oxide and reactive oxygen intermediate toxicity in islet cells of rats. Curr Sci 25:786–793Google Scholar
  21. 21.
    Pownall TL, Udenigwe CC, Aluko RE (2010) Amino acid composition and antioxidant properties of pea seed (Pisum sativum L.) enzymatic protein hydrolysate fractions. J Agric Food Chem 58(8):4712–4718. PubMedCrossRefGoogle Scholar
  22. 22.
    Roy P, Saha A (2002) Metabolism and toxicity of arsenic: a human carcinogen. Curr Sci 10:38–45Google Scholar
  23. 23.
    Mukherjee S, Mukhopadhyay P (2009) Studies on arsenic toxicity in male rat gonads and its protection by high dietary protein supplementation. Al Ameen J Med Sci 2(1):73–77Google Scholar
  24. 24.
    Mukhopadhyay PK, Dey A, Mukherjee S, Pradhan NK (2013) The effect of coadministration of α-tocopherol and ascorbic acid on arsenic trioxide-induced testicular toxicity in adult rats. J Basic Clin Physiol Pharmacol 24(4):245–253. PubMedCrossRefGoogle Scholar
  25. 25.
    Marcondes FK, Bianchi FJ, Tanno AP (2002) Determination of the estrous cycle phases of rats: some helpful considerations. Braz J Biol 62(4A):609–614PubMedCrossRefGoogle Scholar
  26. 26.
    Mahapatra D, Chandra AK (2017) Biphasic action of iodine in excess at different doses on ovary in adult rats. J Trace Elem Med Biol 39:210–220. PubMedCrossRefGoogle Scholar
  27. 27.
    Martin JP, Dailey M, Sugarman E (1987) Negative and positive assays of superoxide dismutase based on hematoxylin autoxidation. Arch Biochem Biophys 255(2):329–336PubMedCrossRefGoogle Scholar
  28. 28.
    Beers RF, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195(1):133–140PubMedGoogle Scholar
  29. 29.
    Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358PubMedCrossRefGoogle Scholar
  30. 30.
    Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357PubMedCrossRefGoogle Scholar
  31. 31.
    Lowry OH, Rosebrough NJ, Farr AL, RandalL RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193(1):265–275Google Scholar
  32. 32.
    Rupnow HL, Phernetton TM, Shaw CE, Modrick ML, Bird IM, Magness RR (2001) Endothelial vasodilator production by uterine and systemic arteries. VII. Estrogen and progesterone effects on eNOS. Am J Phys Heart Circ Phys 280(4):H1699–H1705. CrossRefGoogle Scholar
  33. 33.
    Organisation for Economic Co-operation and Development (2008) Test No. 407: repeated dose 28-day oral toxicity study in rodents. OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.
  34. 34.
    Nain S, Smits JEG (2012) Pathological, immunological and biochemical markers of subchronic arsenic toxicity in rats. Environ Toxicol 27(4):244–254PubMedCrossRefGoogle Scholar
  35. 35.
    Chandravanshi LP, Patel DK (2017) Subchronic early life arsenic exposure at low doses impaired the biogenic amine neurotransmitter and nitric oxide levels in different brain regions of rats. J Environ Anal Toxicol 7(477):2161–0525Google Scholar
  36. 36.
    Batra S, Batra N, Nagori BP (2013) Preliminary phytochemical studies and evaluation of antidiabetic activity of stem bark of Acacia senegal (L.) Willd. in alloxan induced diabetic albino rats. Int J Med Res Rev 1(8):611–616Google Scholar
  37. 37.
    Sun X, Li J, Zhao H, Wang Y, Liu J, Shao Y, Xue Y, Xing M (2018) Synergistic effect of copper and arsenic upon oxidative stress, inflammation and autophagy alterations in brain tissues of Gallus gallus. J Inorg Biochem 178(1):54–62PubMedCrossRefGoogle Scholar
  38. 38.
    Matsuda F, Inoue N, Manabe N, Ohkura S (2012) Follicular growth and atresia in mammalian ovaries: regulation by survival and death of granulosa cells. J Reprod Dev 58(1):44–50PubMedCrossRefGoogle Scholar
  39. 39.
    Young SL (2013) Oestrogen and progesterone action on endometrium: a translational approach to understanding endometrial receptivity. Reprod BioMed Online 27(5):497–505. PubMedCrossRefGoogle Scholar
  40. 40.
    Murphy CR (2000) Understanding the apical surface markers of uterine receptivity: pinopods—or uterodomes? Hum Reprod 15(12):2451–2454PubMedCrossRefGoogle Scholar
  41. 41.
    Murphy CR (2004) Uterine receptivity and the plasma membrane transformation. Cell Res 14(4):259–267. PubMedCrossRefGoogle Scholar
  42. 42.
    Guo H, Hao G, Xia Y, Guo W, Li C, Guo Z (2011) Effects on serum estadiol and progesterone of female mice exposed to arsenic chronically. Wei Sheng Yan Jiu 40(1):120–121PubMedGoogle Scholar
  43. 43.
    Kabir-Salmani M, Murphy CR, Hosseini A, Valojerdi MR (2008) Ultrastructural modifications of human endometrium during the window of implantation. Int J Fertil Steril 2(2):44–59Google Scholar
  44. 44.
    Aplin JD, Ruane PT (2017) Embryo–epithelium interactions during implantation at a glance. J Cell Sci 130(1):15–22. PubMedCrossRefGoogle Scholar
  45. 45.
    Royer C, Lu X (2011) Epithelial cell polarity: a major gatekeeper against cancer? Cell Death Differ 18(9):1470–1477. PubMedPubMedCentralCrossRefGoogle Scholar
  46. 46.
    Gaweł S, Wardas M, Niedworok E, Wardas P (2004) Malondialdehyde (MDA) as a lipid peroxidation marker. Wiad Lek 57(9–10):453–455PubMedGoogle Scholar
  47. 47.
    Birben E, Sahiner UM, Sackesen C, Erzurum S, Kalayci O (2012) Oxidative stress and antioxidant defense. World Allergy Organ J 5(1):9PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Chevion M, Berenshtein E, Stadtman ER (2000) Human studies related to protein oxidation: protein carbonyl content as a marker of damage. Free Radic Res 33:S99–S108PubMedGoogle Scholar
  49. 49.
    Banerjee S, Banerjee S, Saraswat G, Bandyopadhyay SA, Kabir SN (2014) Female reproductive aging is master-planned at the level of ovary. PLoS One 9:e96210. PubMedPubMedCentralCrossRefGoogle Scholar
  50. 50.
    Ruder EH, Hartman TJ, Blumberg J, Goldman MB (2008) Oxidative stress and antioxidants: exposure and impact on female fertility. Hum Reprod Update 14(4):345–357PubMedPubMedCentralCrossRefGoogle Scholar
  51. 51.
    Khan AA, Rahmani AH, Aldebasi YH, Aly SM (2014) Biochemical and pathological studies on peroxidases—an updated review. Global J Health Sci 6(5):87CrossRefGoogle Scholar
  52. 52.
    Baiza-Gutman LA, Flores-Sánchez MM, Dı́az-Flores M, Hicks JJ (2000) Presence of uterine peroxidase activity in the rat early pregnancy. Int J Biochem Cell Biol 32(2):255–262. PubMedCrossRefGoogle Scholar
  53. 53.
    Slot KA, Voorendt M, de Boer-Brouwer M, van Vugt HH, Teerds KJ (2006) Estrous cycle dependent changes in expression and distribution of Fas, Fas ligand, Bcl-2, Bax, and pro-and active caspase-3 in the rat ovary. J Endocrinol 188(2):179–192PubMedCrossRefGoogle Scholar
  54. 54.
    Danial NN, Korsmeyer SJ (2004) Cell death: critical control points. Cell 116(2):205–219PubMedCrossRefGoogle Scholar
  55. 55.
    Walsh JG, Cullen SP, Sheridan C, Lüthi AU, Gerner C, Martin SJ (2008) Executioner caspase-3 and caspase-7 are functionally distinct proteases. Proc Natl Acad Sci 105(35):12815–12819PubMedCrossRefGoogle Scholar
  56. 56.
    Wong PYY, Kitts DD (2003) Chemistry of buttermilk solid antioxidant activity. J Dairy Sci 86(5):1541–1547. PubMedCrossRefGoogle Scholar
  57. 57.
    Rival SG, Boeriu CG, Wichers HJ (2001) Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition. J Agric Food Chem 49(1):295–302PubMedCrossRefGoogle Scholar
  58. 58.
    Maiti S, Chatterjee AK (2001) Effects on levels of glutathione and some related enzymes in tissues after an acute arsenic exposure in rats and their relationship to dietary protein deficiency. Arch Toxicol 75(9):531–537PubMedCrossRefGoogle Scholar
  59. 59.
    Davies-Hoes LD, Scanlon MG, Girgih AT, Aluko RE (2017) Effect of pea flours with different particle sizes on antioxidant activity in pan breads. Cereal Chem 94(5):866–872. CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Life SciencesPresidency UniversityKolkataIndia
  2. 2.CSIR-Indian Institute of Chemical BiologyKolkataIndia

Personalised recommendations