Abstract
Di(2-ethylhexyl)phthalate (DEHP)-administration was found to be toxic in mammalian systems and also known to affect the male reproduction in rats. The aim of this study was to investigate the effects of DEHP on the male reproductive system in a rat model. Three groups of six rats each, i.e. first group control (corn oil administered), second group DEHP administered at the rate of 850 mg/kg BW/day, whereas third group DEHP administered at the rate of 950 mg/kg BW/day for a period of 30 days, consecutively. Initially, final testes weights and sperm quality parameters of testes were evaluated. Oxidative stress markers, lysosomal enzyme activities and inflammatory cytokine levels in testicular tissue were analysed. Sex hormones and haematological parameters were also analysed in control and experimental groups of rats. The results obtained for Di(2-ethylhexyl)phthalate (DEHP) administered rats were compared with its respective controls with relevance to sperm count, sperm motility and daily sperm production rates, which were significantly decreased due to DHEP administration, whereas oxidative stress indicators were found to be significantly increased. Lipid peroxidation (LPO) and decreased activity of the antioxidant enzymes as super oxide dismutase (SOD), reduced levels of catalase (CAT) and glutathione content (GSH) recorded under DEHP administration. While testicular enzymes like γ-glutamyl transpeptidase (GGT) and lactate dehydrogenase (LDH) activities were found to be significantly elevated during DEHP administrated rats. Oxidative marker enzyme succinate dehydrogenase (SDH) activity was found to be significantly decreased. β-Glucuronidase activity was significantly increased and acid phosphatase activity was significantly decreased, in response to DEHP administration into rats. Pro-inflammatory cytokinines such as tumour necrosis factor-1 α (TNF-α) and interleukin-1β (IL-1β) levels were significantly increased. Serum testosterone levels decreased, serum follicle-stimulating hormone (FSH) and leutinizing hormone (LH) levels increased. Due to DEHP administration leucocytes, monocytes, neutrophils/granulocytes, erythrocytes and haemoglobin contents were significantly decreased, whereas lymphocytes, mean corpuscular volume (MCV), haematocrit, mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) levels were found to be increased. Results obtained in the present study suggest that DEHP affecting the process of spermatogenesis through changing the activities of the enzymes responsible for the maturation of the sperm. The rate of production of sperm numbers from testes may be responsible for the antifertility effects of DHEP. The results obtained clearly suggests that disruption of cellular energetics in the testes may be responsible for the reported infertility on male rats.
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The data presented in this study are available on request from the corresponding author.
References
Abdul Ghani S, Abdul Ghani R, Qazzaz M, Adbeen Z (2014) The effect of di-butyl phthalates (DBP) and prenatal di (2-ethylhexyl) phthalates (DEHP) on female rats fertility. Int J Pharmceu Sci and Res 5(5):1671–1681
Adaramoye OA, Adedara IA, Farombi EO (2012) Possible ameliorative effects of kolaviron against reproductive toxicity in sub-lethally whole body gamma-irradiated rats. Exp Toxicol Pathol 64:379–385
Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126
Agarwal DK, Eustis S, Lamb IVJC (1986) Effects of di(2- ethylhexyl) phthalate on the gonadal pathophysiology sperm morphology and reproductive performance of male rats. Environ Health Perspect 65:343–350
Araki A, Mitsui T, Goudarzi H, Nakajima T, Miyashita C, Itoh S (2017) Prenatal di (2-ethylhexyl) phthalate exposure and disruption of adrenal androgens and glucocorticoids levels in cord blood: The Hokkaido Study. Sci Total Environ 581:297–304
Bahrami N, Goudarzi M, Hosseinzadeh A, Sabbagh S, Reiter RJ, Mehrzadi S (2018a) Evaluating the protective effects of melatonin on di(2-ethylhexyl) phthalate-induced testicular injury in adult mice. Biomed Pharmacother 108:515–523
Bahrami N, Mehrzadi S, Goudarzi M, Mansouri E, Fatemi I (2018b) Lycopene abrogates di-(2-ethylhexyl) phthalate induced testicular injury by modulating oxidative endocrine and inflammatory changes in mice. Life Sci 207:265–271
Ben Rhouma K, Tebourbi O, Krichah R, Sakly M (2001) Reproductive toxicity of DDT in adult male Rats. Hum Exp Toxicol 20:393–397
Bishop DW (1968) Sorbitol dehydrogenase in relation to spermatogenesis and fertility. J Reprod Fertil 17:410
Blackshaw AW, Hamilton D (1970) The effect of heat on hydrolytic enzymes and spermatogenesis in rat testes. J Reprod Fertil 22:569
Blazak WF, Treinen KA, Juniewicz PE (1993) Application of testicular spermhead counts in the assessment of male reproductive toxicity. Meth Toxicol 3A:86–94
Chapin RE, Norton RM, Popp JA, Bus JS (1982) The effects of 25-hexanedione on reproductive hormones and testicular enzyme activities in the F-344 rat. Toxicol, Appl, Pharmacol 62:262
Crissman JW, Goodman DG, Hildebrandt PK, Maronpot RR, Prater DA, Riley JH (2004) Best practice guideline: toxicologic histopathology. Toxicol Pathol 32:126–131
David RM (2006) Proposed mode of action for in utero effects of some phthalate esters on the developing male reproductive tract. Toxicol Pathol 34(3):209–219
Durairajanayagam D (2018) Lifestyle causes of male infertility. Arab J Urol 16:10–20
Earls A, Axford IP, Braybrook JH (2003) Gas chromatography-mass spectrometry determination of the migration of phthalate plasticisers from polyvinyl chloride toys and childcare articles. J Chromatogr A 983(1–2):237–246
El-Desoky GE, Bashandy SA, Alhazza IM (2013) Improvement of mercuric chloride-induced testes injuries and sperm quality deteriorations by Spirulina platensis in rats. PLoS ONE 8:e59177
Erkekoglu P, Rachidi W, De Rosa V (2010) Protective effect of selenium supplementation on the genotoxicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl) phthalate treatment in LNCaP cells. Free Radic Biol Med 49:559–566
Erkekoglu P, Zeybek ND, Giray B (2011) Reproductive toxicity of di(2-ethylhexyl) phthalate in selenium-supplemented and selenium-deficient rats. Drug Chem Toxicol 34:379–389
Faccio L, Da Silva AS, Tonin AA, França RT, Gressler LT, Copetti MM, Oliveira CB, Sangoi MB, Moresco RN, Bottari NB, Duarte MM, Monteiro SG (2013) Serum levels of LH FSH estradiol and progesterone in female rats experimentally infected by Trypanosoma evansi. Exp Parasitol 135:110–115
Free MJ (1970) Carbohydrate metabolism in the testes in A,D, Johnson W,R, Gomes and N,L, Vandimark (Eds,) The testes. Vol, II Academic Press New York 125
Fu G, Dai J, Li Z, Chen F, Liu L, Yi L (2020) The role of STAT3/p53 and PI3KAkt- mTOR signaling pathway on DEHP-induced reproductive toxicity in pubertal male rat. Toxicol Appl Pharmacol 404:115–151
Gangolli SD (1982) Testicular effects of phthalate esters. Environ, Health Perspect 45:77
Gazouli M, Yao ZX, Boujrad N (2002) Effect of peroxisome proliferators on Leydig cellperipheral-type benzodiazepine receptor gene expression hormonestimulated cholesterol transport and steroidogenesis: role of the peroxisome proliferator-activator receptor alpha. Endocrinology 143:2571–2583
Gozde Karabuluta and Nurhayat Barlas (2018) Genotoxic histologic immunohistochemical morphometric and hormonal effects of di-(2-ethylhexyl)-phthalate (DEHP) on reproductive systems in pre-pubertal male rats. Toxicol Res 7:859
Grance SR, Teixeira MA, Leite RS, Guimaraes EB, De Siqueira JM, De Oliveira Filiu WF (2008) Baccharistrimera: effect on hematological and biochemical parameters and hepatorenal evaluation in pregnant rats. J Ethnopharmacol 117:28–33
Guthrie HD, Welch GR (2012) Effects of reactive oxygen species on sperm function. Theriogenology 78:1700–1708
Hatefi Y, Galante YM (1980) Isolation of cytochrome b560 from complex II (succinate ubiquinone oxidoreductase) and its reconstitution with succinate dehydrogenase. J Biol Chem 255:5530–5537
Hirai S, Naito M, Kuramasu M, Ogawa Y, Terayama H, Qu N (2015) Low-dose exposure to di-(2-ethylhexyl) phthalate (DEHP) increases susceptibility to testicular autoimmunity in mice. Reprod Biol 15:163–171
Holstein A-F, Schulze W, Davidoff M (2003) Understanding spermatogenesis is a prerequisite for treatment. Reprod Biol Endocrinol 1:107
Huang PC, Tien CJ, Sun YM, Hsieh CY, Lee CC (2008) Occurrence of phthalates in sediment and biota: relationship to aquatic factors and the biota-sediment accumulation factor. Chemosphere 73(4):539–544
Jung D, Park Sultan SM, Habeebu CD, Klaassen (2002) Testicular toxicity of di-(2-ethylhexyl)phthalate in young Sprague-Dawley rats. Toxicology 171:105–115
Kasahara E, Sato EF, Miyoshi M, Konaka R, Hiramoto K, Sasaki J (2002) Role of oxidative stress in germ cell apoptosis induced by di (2-ethylhexyl) phthalate. Biochem J 365:849–856
Kawai Y, Anno K (1971) Mucopolysaccharides degrading enzymes from the liver of the squid Ommastrephes solani paciicus. I, Hyaluronidase, Biochim Biophys Acta 242:428–436
King J (1965) Lactate dehydrogenase n practical clinical enzymology. Van Nostrand London 83–93
Li X, Fang EF, Scheibye-Knudsen M, Cui H, Qiu L, Li J (2014) Di-(2-ethylhexyl) phthalate inhibits DNA replication leading to hyperPARylation SIRT1 attenuation and mitochondrial dysfunction in the Testes. Sci Rep 4:6434
Lin H, Ge RS, Chen GR, Hu GX, Dong L, Lian QQ, Hardy DO, Sottas CM, Li XK, Hardy MP (2008) Involvement of testicular growth factors in fetal Leydig cell aggregation after exposure to phthalate in utero. Proc Natl Acad Sci USA 105:7218–7222
Ma H, Li J, Yang JJ (2012) Di-2-ethylhexyl phthalate and its metabolite single-ethylhexyl phthalate affect TGF-beta 1 expression and telomerase activity in the testes of young male rats. Zhonghua Nan Ke Xue 18:783–788
Males JW, Turkington RW (1971) Hormonal control of lysosomal enzymes during spermatogenesis in the rat. Endocrinology 88:579
Mandava V, Rao BG (2008) Antioxidative potential of melatonin against mercury induced intoxication in spermatozoa in vitro. Toxicol in Vitro 22:935–942
Martinez-Arguelles DB, Culty M, Zirkin BR, Papadopoulos V (2009) In utero exposure to di-(2-ethylhexyl) phthalate decreases mineralocorticoid receptor expression in the adult testes. Endocrinology 150(12):5575–5585
Misra HP, Fridovich I (1972) The role of superoxide anion in the autooxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175
Nachlas MM, Margulies SI, Goldberg JD, Seligman AM (1960) The determination of lactic dehydrogenase with a tetrazolium salt. Anal Biochem 1:317–326
Naito M, Terayama H, Hirai S, Qu N, Lustig L, Itoh M (2012) Experimental autoimmune orchitis as a model of immunological male infertility. Med Molec Morphol 45:185–189
Nishioka J, Iwahara C, Kawasaki M, Yoshizaki F, Nakayama H, Takamori K (2012) Di-(2-ethylhexyl) phthalate induces production of inflammatory molecules in human macrophages. Inflamm Res 61:69–78
Nohynek GJ, Borgert CJ, Dietrich D, Rozman KK (2013) Endocrine disruption: fact or urban legend? Toxicol Lett 223(3):295–305
Noriega NC, Howdeshell KL, Furr J, Lambright CR, Wilson VS, JrLE G (2009) Pubertal administration of DEHP delays puberty suppresses testosterone production and inhibits reproductive tract development in male Sprague-Dawley and long-evans rats. Toxicol Sci 111(1):163–178
Okhawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95(2):351–358
Oyagbemi AA, Adedara IA, Saba AB (2010) Role of oxidative stress in reproductive toxicity induced by co-administration of chloramphenicol and multivitamin-haematinics complex in rats. Basic Clin Pharmacol Toxicol 107:703–708
Palani V, Senthikumaran RK, Govindawamy S (1999) Biochemical evaluation of antitumor effect of Muthu marunthu (a herbal formulation) on experimental fibrosarcoma in rats. J Ethnopharmacol 65:257–265
Ramonda R, Foresta C, Ortolan A, Bertoldo A, Oliviero F, Lorenzin M (2014) Influence of tumor necrosis factor α inhibitors on testicular function and semen in spondyloarthritis patients. Fertil Steril 101(2):359–365
Rezvanfar M, Sadrkhanlou R, Ahmadi A (2008) Protection of cyclophosphamide-induced toxicity in reproductive tract histology sperm characteristics and DNA damage by an herbal source: evidence for role of free-radical toxic stress. Hum Exp Toxicol 27:901–910
Saxena DK, Srivastava SP, Chandra SV, Seth PK (1985) Testicular effects of DI-(2-ethylhexyl) phthalate (DEHP): histochemical and histopathological alterations. Ind Health 23:191–198
Seth PK (1982) Hepatic effects of phthalate esters. Environ Health Perspect 45:27
Shelby MD (2006) NTP-CERHR monograph on the potential human reproductive and developmental effects of di (2- ethylhexyl) phthalate (DEHP). NTP-CERHR monograph, 18
Somasundaram D, Manokaran K, Selvanesan B, Bhaskaran R (2017) Impact of di-(2 ethylhexyl) phthalate on the uterus of adult Wistar rats. Hum Exp Toxicol 36:565–572
Steinberger A, Steinberger E (1977) The Sertoli cells in A,D, Johnson and W,R, Gomes (Eds,) The testes. Vol, IV Academic Press New York 371–399
Stenz L, Escoffier J, Rahban R, Nef S, Paoloni-Giacobino A (2017) Testicular dysgenesis syndrome and long-lasting epigenetic silencing of mouse sperm genes involved in the reproductive system after prenatal exposure to DEHP. PLoS ONE 12:e0170441
Takahashi O, Oishi S (2001) Testicular toxicity of dietary 22-bis(4-hydroxyphenyl)propane (bisphenol A) in F344 rat. Arch Toxicol 75:42–51
Tamura K, Hayashi N, George J, Toshikuni N, Arisawa T, Hiratake J, Tsuchishima M, Tsutsumi M (2016) GGsTop a novel and specific gamma-glutamyl transpeptidase inhibitor protects hepatic ischemia-reperfusion injury in rat. Am J Physiol Gastrointest Liver Physiol 311:G305–G312
Tang ZR, Xu XL, Deng SL, Lian ZX, Yu K (2020) Oestrogenic endocrine disruptors in the placenta and the fetus. Int J Mol Sci 21:1519
Theodorus PM, Akerboom Sies H (1981) Assay of glutathione glutathione disulfide and glutathione mixed disulfide in biological samples. Meth in Enzmol 77:373–382
Uzunhisarcikli M (2007) Acute subacute and subchronic administration of methyl parathion induced testicular damage in male rats and protective role of vitamins C and E. Pestic Biochem Phys 87:115–122
Wang J, Zhao T, Chen J, Kang L, Wei Y, Wu Y (2021) Multiple transcriptomic profiling: p53 signaling pathway is involved in DEHP-induced prepubertal testicular injury via promoting cell apoptosis and inhibiting cell proliferation of Leydig cells. J Hazard Mater 406:124316
Wang XJ, Zhang YM, Cui JS (2004) Study of effects of lead acetate on reproductive function in male mice. Chin J Ind Med 17:237–239
Wilson C, Zerban H, Bannasch P (2004) Lifelong exposure to di-(2-ethylhexyl)-phthalate induces tumors in liver and testes of Sprague-Dawley rats. Toxicol 206:359–371
Yokoi K, Uthus EO, Nielsen FH (2003) Nickel deficiency diminishes sperm quantity and movement in rats. Biol Trace Elem Res 93:141–153
Acknowledgements
This work was supported by the Department of Zoology, Sri Venkateswara University, for allowing us to use their laboratories for our research work.
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Conceptualization, P.V.R.; methodology, formal analysis, investigation, resources, data curation, writing-original draft preparation, writing—review and editing, visualization, supervision, M.S.R.; all authors have read and agreed to the published version of the manuscript.
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Poli, V., Motireddy, S.R. Toxicity study of di(2-ethylhexyl)phthalate (DEHP) administration on reproductive parameters in male albino rats. Comp Clin Pathol 33, 75–84 (2024). https://doi.org/10.1007/s00580-023-03524-8
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DOI: https://doi.org/10.1007/s00580-023-03524-8