Companion animals get close to the toxic aspects of antropogenic world: cytotoxicity of phthalates and bisphenol A on dog testicular primary cells

Tekin, Koray; Arslan, Pinar; Cil, Beste; Filazi, Ayhan; Akçay, Ergun; Yurdakok-Dikmen, Begum

doi:10.1007/s10616-020-00401-y

Original Article
Published: 20 May 2020

Companion animals get close to the toxic aspects of antropogenic world: cytotoxicity of phthalates and bisphenol A on dog testicular primary cells

Koray Tekin¹,
Pinar Arslan²,
Beste Cil¹,
Ayhan Filazi³,
Ergun Akçay¹ &
Begum Yurdakok-Dikmen ORCID: orcid.org/0000-0002-0385-3602³

Cytotechnology volume 72, pages 629–638 (2020)Cite this article

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Abstract

Phthalates, which are among the most abundant plasticizers, have detrimental effects on the reproductive system. Similar to human, dogs are prominently exposed to phthalates in daily routines at low concentrations; while toys, training devices and commercial dog foods are considered as the primary sources of exposure. This study aimed to reveal and compare the cytotoxic effects of selected phthalates (Benzyl butyl phthalate (BBP), Diethyl phthalate (DEP), Bis (2-ethylhexyl) phthalate (DEHP), Di-‘isobutyl’ phthalate (DIBP), Di-‘isodecyl’ phthalate (-DIDP) Di-‘isononyl’ phthalate (DINP), Dimethyl phthalate (DMP), Di-n-octyl phthalate (DNOP)), and Bisphenol A (BPA) following 24 h exposure on primary testicular parenchymal cells of dog in vitro at concentrations between 0.001 and 2.5 nM. According to cytotoxicity results, DEHP was found to be the most toxic phthalate with IC₅₀ at 22.53 µM; while DMP was the least (169.17 nM). IC₅₀ of BPA was 161.81 nM, less than the average (61.95 nM) of phthalates. In addition, dog primary testicular cells were found more susceptible to the high molecular weight phthalates (DNOP, DEHP, DINP, DIDP) than low molecular weight phthalates (DMP, DEP, DIBP, BBP). Further studies should focus on morphological, physiological and molecular differences to comprehend the mechanisms involved as well as decreasing the risk for impaired spermatogenesis caused by environmental toxicants in companion animal medicine.

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References

Andriana BB, Tay TW, Maki I et al (2004) An ultrastructural study on cytotoxic effects of mono(2-ethylhexyl) phthalate (MEHP) on testes in Shiba goat in vitro. J Vet Sci 5:235. https://doi.org/10.4142/jvs.2004.5.3.235
Article PubMed Google Scholar
Axelsson J, Rylander L, Rignell-Hydbom A et al (2015) Phthalate exposure and reproductive parameters in young men from the general Swedish population. Environ Int 85:54–60. https://doi.org/10.1016/j.envint.2015.07.005
CAS Article PubMed Google Scholar
Bingham E, Cohrssen B, Powell CH, John Wiley & Sons I (2001) Patty’s toxicology, 5th edition in 9 volumes. Chem Heal Saf 8:45–46. https://doi.org/10.1016/s1074-9098(01)00241-6
Article Google Scholar
Boekelheide K, Johnson KJ, Richburg JH (2005) Sertoli cell toxicants. In: Skinner MK, Girsworld MD (eds) Sertoli cell biology. Elsevier Science, NewYork, pp 345–382
Chapter Google Scholar
Chauvigné F, Menuet A, Lesné L et al (2009) Time- and dose-related effects of di-(2-ethylhexyl) phthalate and its main metabolites on the function of the rat fetal testis in vitro. Environ Health Perspect 117:515–521. https://doi.org/10.1289/ehp.11870
CAS Article PubMed Google Scholar
ChemsafetyPro (2019) Bisphenol A (BPA). In: ChemsafetyPro. https://www.chemsafetypro.com/Topics/Restriction/Bisphenol_A_(BPA).html. Accessed 2 Dec 2019
Consumer Product Safety Commission (2017) Prohibition of Children’s Toys and Child Care Articles Containing Specified Phthalates. Final rule. Fed Regist 82:49938–49982
Google Scholar
Directive (EU) 2015/863 (2015) Commission Delegated Directive (EU) 2015/863—of 31 March 2015—amending Annex II to Directive 2011/65/EU of the European Parliament and of the Council as regards the list of restricted substances
Dostal LA, Weaver RP, Schwetz BA (1987) Transfer of di(2-ethylhexyl) phthalate through rat milk and effects on milk composition and the mammary gland. Toxicol Appl Pharmacol 91:315–325. https://doi.org/10.1016/0041-008x(87)90054-8
CAS Article PubMed Google Scholar
Fennell TR, Krol WL, Sumner SCJ, Snyder RW (2004) Pharmacokinetics of dibutylphthalate in pregnant rats. Toxicol Sci 82:407–418. https://doi.org/10.1093/toxsci/kfh294
CAS Article PubMed Google Scholar
Filazi A, Yurdakök-Dikmen B (2018) Endokrin Bozucu Kimyasal Maddelerin Belirlenmesinde Kullanılan İn Vitro Yöntemler. Turkiye Klin Vet Sci Toxicol Top 4:15–21
Google Scholar
Fisher JS, Macpherson S, Marchetti N, Sharpe RM (2003) Human “testicular dysgenesis syndrome”: a possible model using in utero exposure of the rat to dibutyl phthalate. Hum Reprod 18:1383–1394. https://doi.org/10.1093/humrep/deg273
CAS Article PubMed Google Scholar
Foster PMD, Cattley RC, Mylchreest E (2000) Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment. Food Chem Toxicol 38:S97–S99. https://doi.org/10.1016/s0278-6915(99)00128-3
CAS Article PubMed Google Scholar
Fredricsson B, Möller L, Pousette Å, Westerholm R (1993) Human sperm motility is affected by plasticizers and diesel particle extracts. Pharmacol Toxicol 72:128–133. https://doi.org/10.1111/j.1600-0773.1993.tb00303.x
CAS Article PubMed Google Scholar
Gray TJB, Beamand JA (1984) Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells. Food Chem Toxicol 22:123–131. https://doi.org/10.1016/0278-6915(84)90092-9
CAS Article PubMed Google Scholar
Grieco V, Riccardi E, Greppi GF et al (2008a) Canine testicular tumours: a study on 232 dogs. J Comp Pathol 138:86–89. https://doi.org/10.1016/j.jcpa.2007.11.002
CAS Article PubMed Google Scholar
Grieco V, Riccardi E, Veronesi MC et al (2008b) Evidence of testicular dysgenesis syndrome in the dog. Theriogenology 70:53–60. https://doi.org/10.1016/j.theriogenology.2008.02.009
CAS Article PubMed Google Scholar
Habert R, Muczynski V, Grisin T et al (2014) Concerns about the widespread use of rodent models for human risk assessments of endocrine disruptors. Reproduction 147:R119–R129. https://doi.org/10.1530/rep-13-0497
CAS Article PubMed PubMed Central Google Scholar
Heindel RJ, Chapin RE (1989) Inhibition of FSH-stimulated cAMP accumulation by mono(2-ethylhexyl) phthalate in primary rat sertoli cell cultures. Toxicol Appl Pharmacol 97:377–385. https://doi.org/10.1016/0041-008x(89)90342-6
CAS Article PubMed Google Scholar
Helmestam M, Davey E, Stavreus-Evers A, Olovsson M (2014) Bisphenol A affects human endometrial endothelial cell angiogenic activity in vitro. Reprod Toxicol 46:69–76. https://doi.org/10.1016/j.reprotox.2014.03.002
CAS Article PubMed Google Scholar
Iida H, Maehara K, Doiguchi M et al (2003) Bisphenol A-induced apoptosis of cultured rat Sertoli cells. Reprod Toxicol 17:457–464. https://doi.org/10.1016/s0890-6238(03)00034-0
CAS Article PubMed Google Scholar
Jones HB, Garside DA, Liu R, Roberts JC (1993) The influence of phthalate esters on leydig cell structure and function in vitro and in vivo. Exp Mol Pathol 58:179–193. https://doi.org/10.1006/exmp.1993.1016
CAS Article PubMed Google Scholar
La Rocca C, Tait S, Guerranti C et al (2015) Exposure to endocrine disruptors and nuclear receptors gene expression in infertile and fertile men from Italian areas with different environmental features. Int J Environ Res Public Health 12:12426–12445. https://doi.org/10.3390/ijerph121012426
CAS Article PubMed PubMed Central Google Scholar
Lara NLM, Costa GMJ, Avelar GF, et al (2018) Testis physiology—overview and histology. Encycl Reprod 105–116
Lea RG, Byers AS, Sumner RN et al (2016) Environmental chemicals impact dog semen quality in vitro and may be associated with a temporal decline in sperm motility and increased cryptorchidism. Sci Rep. https://doi.org/10.1038/srep31281
Article PubMed PubMed Central Google Scholar
Li L-H, Jester WF, Orth JM (1998) Effects of relatively low levels of mono-(2-ethylhexyl) phthalate on cocultured sertoli cells and gonocytes from neonatal rats. Toxicol Appl Pharmacol 153:258–265. https://doi.org/10.1006/taap.1998.8550
CAS Article PubMed Google Scholar
Lukacova J, Jambor T, Knazicka Z et al (2015) Dose- and time-dependent effects of bisphenol A on bovine spermatozoain vitro. J Environ Sci Heal Part A 50:669–676. https://doi.org/10.1080/10934529.2015.1011963
CAS Article Google Scholar
MacLeod DJ, Sharpe RM, Welsh M et al (2010) Androgen action in the masculinization programming window and development of male reproductive organs. Int J Androl 33:279–287. https://doi.org/10.1111/j.1365-2605.2009.01005.x
CAS Article PubMed Google Scholar
Main KM, Mortensen GK, Kaleva MM et al (2006) Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ Health Perspect 114:270–276. https://doi.org/10.1289/ehp.8075
CAS Article PubMed Google Scholar
National Research Council (US) Committee on the Health Risks of Phthalates (2008) National Research Council (US) Committee on the Health Risks of Phthalates. Phthalates and Cumulative Risk Assessment: The Tasks Ahead. National Academies Press (US), Washington (DC)
Oishi S, Hiraga K (1980) Testicular atrophy induced by phthalic acid esters: effect on testosterone and zinc concentrations. Toxicol Appl Pharmacol 53:35–41. https://doi.org/10.1016/0041-008x(80)90378-6
CAS Article PubMed Google Scholar
Oskam IC, Lyche JL, Krogenæs A et al (2005) Effects of long-term maternal exposure to low doses of PCB126 and PCB153 on the reproductive system and related hormones of young male goats. Reproduction 130:731–742. https://doi.org/10.1530/rep.1.00690
CAS Article PubMed Google Scholar
Pant N, Pant A, Shukla M et al (2010) Environmental and experimental exposure of phthalate esters: the toxicological consequence on human sperm. Hum Exp Toxicol 30:507–514. https://doi.org/10.1177/0960327110374205
CAS Article PubMed Google Scholar
Parmar D, Srivastava S, Seth P (1988) Effect of di(2-ethylhexyl)phthalate (DEHP) on hepatic mixed function oxidases in different animal species. Toxicol Lett 40:209–217. https://doi.org/10.1016/0378-4274(88)90043-4
CAS Article PubMed Google Scholar
Pocar P, Fiandanese N, Secchi C et al (2012) Exposure to Di(2-ethyl-hexyl) phthalate (DEHP) in uteroand during lactation causes long-term pituitary-gonadal axis disruption in male and female mouse offspring. Endocrinology 153:937–948. https://doi.org/10.1210/en.2011-1450
CAS Article PubMed Google Scholar
Rozati R, Reddy PP, Reddanna P, Mujtaba R (2002) Role of environmental estrogens in the deterioration of male factor fertility. Fertil Steril 78:1187–1194. https://doi.org/10.1016/s0015-0282(02)04389-3
Article PubMed Google Scholar
Saillenfait AM, Payan JP, Fabry JP et al (1998) Assessment of the developmental toxicity, metabolism, and placental transfer of Di-n-butyl phthalate administered to pregnant rats. Toxicol Sci 45:212–224. https://doi.org/10.1093/toxsci/45.2.212
CAS Article PubMed Google Scholar
Sathyanarayana S, Karr CJ, Lozano P et al (2008) Baby care products: possible sources of infant phthalate exposure. Pediatrics 121:e260–e268. https://doi.org/10.1542/peds.2006-3766
Article PubMed Google Scholar
Sharpe RM, Auharek S, Scott HM, de Franca LR, Drake AJ, van den Driesche S (2011) Origin of testicular dysgenesis syndrome disorders in the Masculinization programming Window: relevance to final testis size (=Sperm Production). In: Bourguignon JP, Jégou B, Kerdelhué B, Toppari J, Christen Y (eds) Multi-system endocrine disruption. Research and perspectives in endocrine interactions. Springer, Berlin, Heidelberg
Google Scholar
Silva MJ, Reidy JA, Herbert AR et al (2004) Detection of phthalate metabolites in human amniotic fluid. Bull Environ Contam Toxicol. https://doi.org/10.1007/s00128-004-0374-4
Article PubMed Google Scholar
Singleton DW, Feng Y, Chen Y et al (2004) Bisphenol-A and estradiol exert novel gene regulation in human MCF-7 derived breast cancer cells. Mol Cell Endocrinol 221:47–55. https://doi.org/10.1016/j.mce.2004.04.010
CAS Article PubMed Google Scholar
Skakkebæk NE, Rajpert-De Meyts E, Main KM (2001) Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects: opinion. Hum Reprod 16:972–978. https://doi.org/10.1093/humrep/16.5.972
Article PubMed Google Scholar
Skakkebaek NE, Rajpert-De Meyts E, Buck Louis GM et al (2016) Male reproductive disorders and fertility trends: influences of environment and genetic susceptibility. Physiol Rev 96:55–97. https://doi.org/10.1152/physrev.00017.2015
CAS Article PubMed Google Scholar
The Danish Environmental Protection Agency (2013) Phthalate strategy. The Danish Environmental Protection Agency, Copenhagen
Google Scholar
Vitku J, Heracek J, Sosvorova L et al (2016) Associations of bisphenol A and polychlorinated biphenyls with spermatogenesis and steroidogenesis in two biological fluids from men attending an infertility clinic. Environ Int 89–90:166–173. https://doi.org/10.1016/j.envint.2016.01.021
CAS Article PubMed Google Scholar
Wan HT, Mruk DD, Wong CKC, Cheng CY (2013) Targeting testis-specific proteins to inhibit spermatogenesis: lesson from endocrine disrupting chemicals. Expert Opin Ther Targets 17:839–855. https://doi.org/10.1517/14728222.2013.791679
CAS Article PubMed PubMed Central Google Scholar
Wang S-W, Wang SS-W, Wu D-C et al (2013) Androgen receptor-mediated apoptosis in bovine testicular induced pluripotent stem cells in response to phthalate esters. Cell Death Dis 4:e907. https://doi.org/10.1038/cddis.2013.420
CAS Article PubMed PubMed Central Google Scholar
Wang Y-X, You L, Zeng Q et al (2015) Phthalate exposure and human semen quality: results from an infertility clinic in China. Environ Res 142:1–9. https://doi.org/10.1016/J.ENVRES.2015.06.010
CAS Article PubMed Google Scholar
Welsh M, Saunders PTK, Fisken M et al (2008) Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J Clin Invest 118:1479–1490. https://doi.org/10.1172/jci34241
CAS Article PubMed PubMed Central Google Scholar
Wooten KJ, Smith PN (2013) Canine toys and training devices as sources of exposure to phthalates and bisphenol A: quantitation of chemicals in leachate and in vitro screening for endocrine activity. Chemosphere 93:2245–2253. https://doi.org/10.1016/j.chemosphere.2013.07.075
CAS Article PubMed Google Scholar
Wu W, Zhou F, Wang Y et al (2017) Phthalate levels and related factors in children aged 6–12 years. Environ Pollut 220:990–996. https://doi.org/10.1016/J.ENVPOL.2016.11.049
CAS Article PubMed Google Scholar
Ye L, Zhao B, Hu G et al (2011) Inhibition of human and rat testicular steroidogenic enzyme activities by bisphenol A. Toxicol Lett 207:137–142. https://doi.org/10.1016/j.toxlet.2011.09.001
CAS Article PubMed Google Scholar
Yurdakok B, Tekin K, Daskin A, Filazi A (2015) Effects of polychlorinated biphenyls 28, 30 and 118 on bovine spermatozoa in vitro. Reprod Domest Anim 50:41–47. https://doi.org/10.1111/rda.12447
CAS Article PubMed Google Scholar
Yurdakok-Dikmen B, Stelletta C, Tekin K, et al (2017) Effects of phthalates on bovine primary testicular and epididimal cells in vitro. In: 56th Annual Meeting and ToxExpo. Baltimore, Maryland, USA. Abstract Book p 47
Yurdakok-Dikmen B, Stelletta C, Tekin K et al (2019) Effects of phthalates on bovine primary testicular culture and spermatozoa. Cytotechnology 71:935–947. https://doi.org/10.1007/s10616-019-00336-z
CAS Article PubMed PubMed Central Google Scholar

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Acknowledgement

We thank Ankara University Faculty of Veterinary Medicine for providing the infrastructure and chemicals for the analysis. All datasets used in this study are available upon request from the corresponding author.

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Affiliations

Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Ankara University, Diskapi, 06110, Ankara, Turkey
Koray Tekin, Beste Cil & Ergun Akçay
Department of Biology, Faculty of Science, Ankara University, Tandogan, 06100, Ankara, Turkey
Pinar Arslan
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ankara University, Diskapi, 06110, Ankara, Turkey
Ayhan Filazi & Begum Yurdakok-Dikmen

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Koray Tekin
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Pinar Arslan
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Beste Cil
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Ayhan Filazi
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Ergun Akçay
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Begum Yurdakok-Dikmen
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All the listed authors have made substantial contributions to the research design, analysis and interpretation of data and to drafting the paper and revising it critically.

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Correspondence to Begum Yurdakok-Dikmen.

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Tekin, K., Arslan, P., Cil, B. et al. Companion animals get close to the toxic aspects of antropogenic world: cytotoxicity of phthalates and bisphenol A on dog testicular primary cells. Cytotechnology 72, 629–638 (2020). https://doi.org/10.1007/s10616-020-00401-y

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Received: 04 December 2019
Accepted: 11 May 2020
Published: 20 May 2020
Issue Date: October 2020
DOI: https://doi.org/10.1007/s10616-020-00401-y

Keywords

Bisphenol A
Phthalates
Canine testicular cells
In vitro toxicity