Toxicological Research

Korean Society of Toxicology & Korea Environmental Mutagen Society (한국독성학회)
권호 표지
DOI QR코드

DOI QR Code

The effects of co-exposure to methyl paraben and dibutyl phthalate on cell line derived from human skin

  • Received : 2021.11.22
  • Accepted : 2022.08.09
  • Published : 2023.01.15
⟨ Previous Next ⟩

Abstract

Data on the cumulative effects of chemical substances are necessary for the proper risk assessment, but their availability is still insufficient. The aim of the study was to evaluate the cytotoxic effect of methyl paraben (MePB) and dibutyl phthalate (DBP) on the cells of the skin line (A431) and to compare the cytotoxic effects of the tested substances after single application to A431 cells with the effects of an equimolar/equitoxic (1:1) binary mixture of these compounds as well as their mixtures in ratio 1:3: and 3:1. On the basis of the obtained results, it was found that there were interactions between the tested compounds in terms of cytotoxic effect on A431, assessed on the basis of metabolic activity of cells (MTT test) and integrity of their cell membranes (NRU test). The obtained values of synergy coefficients (SI) and isobolographic analysis indicate that between the tested chemicals in a two-component equimolar mixture (1:1) there is a synergism of action, which, at a high DBP content in the mixture (>50%) turned into antagonism. Observations using a holotomographic microscope show morphological changes in A431 cells after exposure to both DBP and MePB separately and binary mixtures of these compounds, compared to untreated cells. The observed changes in cell morphology seem to be more pronounced when the cells are exposed to the binary mixtures of DBP and MePB than when exposed to these substances individually, which may confirm the synergy of cytotoxic activity between them (this phenomenon was observed for the higher of the tested concentrations in all tested proportions). It is important to consider such effects when considering the effects of cumulative exposure in the risk assessment in order not to underestimate the risk of adverse effects associated with exposure to chemical mixtures.

Keywords

Acknowledgement

We would like to extend our warmest thanks to Mrs. Lilianna Marciniak for her great help in experimental part of work as well as to Prof. Justyna Wiktorowicz, Ph.D., for her extremely valuable help in statistical analysis.

References

  1. Yang RSH (1994) Introduction to the toxicology of chemical mixtures. In: Yang RSH (ed) Toxicology of chemical mixtures: case studies, mechanisms and novel approaches. Academic Press, New York, pp 1-10. https://doi.org/10.1016/B978-0-12-768350-8.50007-4
  2. Kortenkamp A (2008) Low dose mixture effects of endocrine disrupters: implications for risk assessment and epidemiology. Int J Androl 31:233-240. https://doi.org/10.1111/j.1365-2605.2007.00862.x
  3. Gao H-T, Xu R, Cao W-X, Di Q-N, Li R-X, Lu L, Xu Q, Shu-Qin Yu S-Q (2018) Combined effects of simultaneous exposure to six phthalates and emulsifier glycerol monosterate on male reproductive system in rats. Toxicol Appl Pharmacol 341:87-97. https://doi.org/10.1016/j.taap.2018.01.013
  4. Huang P-C, Liao K-W, Chang J-W, Chan S-H, Lee C-C (2018) Characterization of phthalates exposure and risk for cosmetics and perfume sales clerks. Environ Pollut 233:577-587. https://doi.org/10.1016/j.envpol.2017.10.079
  5. Zamkowska D, Karwacka A, Jurewicz J, Radwan M (2018) Environmental exposure to non-persistent endocrine disrupting chemicals and semen quality: an overview of the current epidemiological evidence. Int J Occup Med Environ Health 31:377-414. https://doi.org/10.13075/ijomeh.1896.01195
  6. Nowak K, Ratajczak-Wrona W, Gorska M, Jablonska E (2018) Parabens and their effects on the endocrine system. Mol Cell Endocrinol 474:238-251. https://doi.org/10.1016/j.mce.2018.03.014
  7. Shen H-Y, Jiang H-L, Mao H-L, Pan G, Zhou L, Cao Y-F (2007) Simultaneous determination of seven phthalates and four parabens in cosmetic products using HPLC-DAD and GC-MS methods. J Sep Sci 30:48-54. https://doi.org/10.1002/jssc.200600215
  8. van Meeuwen JA, van Son O, Piersma AH, de Jong PC, van den Berg M (2008) Aromatase inhibiting and combined estrogenic effects of parabens and estrogenic effects of other additives in cosmetics. Toxicol Appl Pharmacol 230:372-382. https://doi.org/10.1016/j.taap.2008.03.002
  9. Guo Y, Wang L, Kannan K (2014) Phthalates and parabens in personal care products from China: concentrations and human exposure. Arch Environ Contam Toxicol 66:113-119. https://doi.org/10.1007/s00244-013-9937-x
  10. Kolatorova L, Vitku J, Hampl R, Adamcova K, Skodova T, Simkova M, Parizek A, Starka L, Duskova M (2018) Exposure to bisphenols and parabens during pregnancy and relations to steroid changes. Environ Res 163:115-122. https://doi.org/10.1016/j.envres.2018.01.031
  11. Debowska R (2016) Konserwanty znane i nieznane. Przemysl Farmaceutyczny 2:88-94
  12. Muszynski Z, Ratajczak M (2009) Konserwacja przeciwdrobnoustrojowa lekow. Farm Pol 65:132-137
  13. Larsson K, Ljung Bjorklund K, Palm B et al (2024) Exposure determinants of phthalates, parabens, bisphenol A and triclosan in Swedish mothers and their children. Environ Int 73:323-333. https://doi.org/10.1016/j.envint.2014.08.014
  14. Marzulli FN, Brown DW, Maibach HI (1969) Techniques for studying skin penetration. Toxicol Appl Pharmacol 14:76-83. https://doi.org/10.1016/S0041-008X(69)80012-8
  15. Seo JE, Kim S, Kim BH (2017) In vitro skin absorption tests of three types of parabens using a Franz diffusion cell. J Expo Sci Environ Epidemiol 27:320-325. https://doi.org/10.1038/jes.2016.33
  16. Pazourekova S, Hojerova J, Klimova Z, Lucova M (2013) Dermal absorption and hydrolysis of methylparaben in different vehicles through intact and damaged skin: using a pig-ear model in vitro. Food Chem Toxicol 59:754-765. https://doi.org/10.1016/j.fct.2013.07.025
  17. Pan TL, Wang PW, Aljufali IA, Hung YY, Lin CF, Fang JY (2014) Dermal toxicity elicited by phthalates: evaluation of skin absorption, immunohistology, and functional proteomics. Food Chem Toxicol 65:105-114. https://doi.org/10.1016/j.fct.2013.12.033
  18. Koo HJ, Lee BM (2004) Estimated exposure to phthalates in cosmetics and risk assessment. J Toxicol Environ Health Part A 67:1901-1914. https://doi.org/10.1080/15287390490513300
  19. Sugino M, Hatanaka T, Todo H, Mashimo Y, Suzuki T, Kobayashi M et al (2017) Safety evaluation of dermal exposure to phthalates: Metabolism-dependent percutaneous absorption. Toxicol Appl Pharmacol 328:10-17. https://doi.org/10.1016/j.taap.2017.05.009
  20. Prasad B, Prasad KS, Dave H, Das A, Asodariya G, Talati N et al (2022) Cumulative human exposure and environmental occurrence of phthalate esters: a global perspective. Environ Res 210:112987. https://doi.org/10.1016/j.envres.2022.112987
  21. Zhao A, Wang L, Pang X, Liu F (2022) Phthalates in skin wipes: distribution, sources, and exposure via dermal absorption. Environ Res 204:112041. https://doi.org/10.1016/j.envres.2021.112041
  22. Tennant JR (1964) Evaluation of the trypan blue technique for determination of cell viability. Transplantation 2:685-694. https://doi.org/10.1097/00007890-196411000-00001
  23. Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55-63. https://doi.org/10.1016/0022-1759(83)90303-4
  24. Suzuki T, Ide K, Ishida M (2001) Response of MCF-7 human breast cancer cells to some binary mixtures of oestrogenic compounds in-vitro. J Pharm Pharmacol 53:1549-1554. https://doi.org/10.1211/0022357011777927
  25. Munshi A, Hobbs M, Meyn RE (2005) Clonogenic cell survival assay. Methods Mol Med 110:21-28. https://doi.org/10.1385/1-59259-869-2:021
  26. Puck TT, Markus PI (1956) Action of X-rays on mammalian cells. J Exp Med 103:653-666. https://doi.org/10.1084/jem.103.5.653
  27. Franken NAP, Rodermond HM, Stap J, Haveman J, van Bree C (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315-2319. https://doi.org/10.1038/nprot.2006.339
  28. Kruszewski M, Gradzka I, Bartlomierczyk T, Chwastowska J, Sommer S et al (2013) Oxidative DNA damage corresponds to the long term survival of human cells treated with silver nanoparticles. Toxicol Lett 219:151-159. https://doi.org/10.1016/j.toxlet.2013.03.006
  29. Bedner E, Smolewski P, Amstad P, Darzynkiewicz Z (2000) Activation of caspases measured in situ by binding of fluorochrome-labeled inhibitors of caspases (FLICA): correlation with DNA fragmentation. Exp Cell Res 259:308-313. https://doi.org/10.1006/excr.2000.4955
  30. Li Z, Zhang H, Gibson M, Liu P (2012) An evaluation of the combined effects of phenolic endocrine disruptors on vitellogenin induction in goldfish Carassius auratus. Ecotoxicology 21:1919-1927. https://doi.org/10.1007/s10646-012-0925-0
  31. Couleau N, Falla J, Beillerot A, Battaglia E, D'Innocenzo M, Plancon S et al (2015) Effects of endocrine disruptor compounds, alone or in combination, on human macrophage-like THP-1 cell response. PLoS ONE 10:e0131428. https://doi.org/10.1371/journal.pone.0131428
  32. Bujnakova Mlynarcikova A, Scsukova S (2018) Simultaneous effects of endocrine disruptor bisphenol A and flavonoid fisetin on progesterone production by granulosa cells. Environ Toxicol Pharmacol 59:66-73. https://doi.org/10.1016/j.etap.2018.03.001
  33. Riad MA, Abd-Rabo MM, Abd El Aziz SA, El Behairy AM, Badawy MM (2018) Reproductive toxic impact of subchronic treatment with combined butylparaben and triclosan in weanling male rats. J Biochem Mol Toxicol. https://doi.org/10.1002/jbt.22037
  34. Vingskes AK, Spann N (2018) The toxicity of a mixture of two antiseptics, triclosan and triclocarban, on reproduction and growth of the nematode Caenorhabditis elegans. Ecotoxicology 27:420-429. https://doi.org/10.1007/s10646-018-1905-9
  35. Christen V, Crettaz P, Oberli-Schrammli A, Fent K (2012) Antiandrogenic activity of phthalate mixtures: validity of concentration addition. Toxicol Appl Pharmacol 259:169-176. https://doi.org/10.1016/j.taap.2011.12.021
  36. Liang B, Zhong Y, Huang Y, Lin X, Liu J, Lin L et al (2021) Underestimated health risks: polystyrene micro-and nanoplastics jointly induce intestinal barrier dysfunction by ROS-mediated epithelial cell apoptosis. Part Fibre Toxicol 18:1-19. https://doi.org/10.1186/s12989-021-00414-1
  37. Nguyen T-H-Y, Bertin M, Bodin J, Fouquet N, Bonvallot N, Roquelaure Y (2018) Multiple exposures and coexposures to occupational hazards among agricultural workers: a systematic review of observational studies. Saf Health Work 9:239-248. https://doi.org/10.1016/j.shaw.2018.04.002
  38. Zhong WZ, Yong L, Jia XD, LiN FYX (2013) Combined subchronic toxicity of bisphenol A and dibutyl phthalate on male rats. Biomed Environ Sci 26:63-69. https://doi.org/10.3967/0895-3988.2013.01.008
  39. Wojtowicz AK, Szychowski KA, Wnuk A, Kajta M (2017) Dibutyl phthalate (DBP)-induced apoptosis and neurotoxicity are mediated via the aryl hydrocarbon receptor (AhR) but not by estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), or peroxisome proliferator-activated receptor gamma (PPARγ) in mouse cortical neurons. Neurotox Res 31:77-89. https://doi.org/10.1007/s12640-016-9665-x
  40. Kwapiszewska M (2011) „Rozmowa receptorow" ER - AhR i epigenetyczna pamiec. Mechanizm adaptacyjny czy wzrost toksycznosci srodowiskowych zanieczyszczen?" [ER - AhR "receptor cross-talk" and epigenetic memory. Adaptive mechanism vs. the increase in toxicity of environmental pollutants]. In: Srodowisko a gospodarka hormonalna u kobiet Cz.1 i 2. (red.) Maria Kapiszewska. Krakow: Ofcyna Wydawnicza AFM, 2011, s. 227-244. ISBN 978-83-7571-195-0227-244
  41. Engeli RT, Rohrer SR, Vuorinen A, Herdlinger S, Kaserer T, Leugger S, Schuster D, Odermatt A (2017) Interference of paraben compounds with estrogen metabolism by inhibition of 17β-hydroxysteroid dehydrogenases. Int J Mol Sci 18:2007. https://doi.org/10.3390/ijms18092007
  42. Harris CA, Henttu P, Parker MG, Sumpter JP (1997) The estrogenic activity of phthalate esters in vitro. Environ Health Perspect 105:802-811. https://doi.org/10.1289/ehp.97105802
  43. Evans RM, Scholze M, Kortenkamp A (2012) Additive mixture effects of estrogenic chemicals in human cell-based assays can be influenced by inclusion of chemicals with differing effect profiles. PLoS ONE 7:e43606. https://doi.org/10.1371/journal.pone.0043606
  44. Li XH, Yin PH, Zhao L (2017) Effects of individual and combined toxicity of bisphenol A, dibutyl phthalate and cadmium on oxidative stress and genotoxicity in HepG 2 cells. Food Chem Toxicol 105:73-78. https://doi.org/10.1016/j.fct.2017.03.054
  45. Yu H, Caldwell DJ, Suri RP (2018) In vitro estrogenic activity of representative endocrine disrupting chemicals mixtures at environmentally relevant concentrations. Chemosphere 215:396-403. https://doi.org/10.1016/j.chemosphere.2018.10.067
  46. Yang P, Wang J, Hong A-B, Huang L-L, Xie Q-T, Wang Y-X, Xiong C-L, Meng T-Q, Pan A, Chen D (2022) Exposure profiles and predictors of a cocktail of environmental chemicals in Chinese men of reproductive age. Chemosphere 299:134337. https://doi.org/10.1016/j.chemosphere.2022.134337
  47. D'Almeida M, Sire O, Lardjane S, Duval H (2020) Development of a new approach using mathematical modeling to predict cocktail effects of micropollutants of diverse origins. Environ Res 188:109897. https://doi.org/10.1016/j.envres.2020.109897
  48. Stewart P, Stenzel M (2000) Exposure assessment in the occupational setting. Appl Occup Environ Hyg 15:435-444. https://doi. org/10.1080/104732200301395
  49. Kjaerstad MB, Taxvig C, Andersen HR, Nellemann C (2010) Mixture effects of endocrine disrupting compounds in vitro. Int J Androl 33:425-433. https://doi.org/10.1111/j.1365-2605.2009.01034.x