Migration of phthalates on culture plates – an important challenge to consider for in vitro studies

ABSTRACT

Phthalates are endocrine disruptors of the reproductive system and suspected to influence many other organ and hormone systems. They are also semi-volatile organic compounds present in the gas phase in the environment. Their mode of action has been investigated in numerous in vitro studies. Multi-well culture plates are typically used to study phthalates in cell cultures. In a pilot study, we observed evidence of phthalate migration in 24-well culture plates. As this has not previously been described, we investigated the phenomenon in more detail. Primary human thyroid epithelial cell cultures (n = 8 cultures) were exposed to either di-ethyl phthalate (DEP), di-n-butyl phthalate (DnBP), mono-n-butyl phthalate (MnBP) or di-(2-ethylhexyl) phthalate (DEHP). Measurement of phthalate metabolites by mass spectrometry demonstrated that the short-branched DEP was able to migrate to adjacent wells when added to cell culture plates. DnBP also seemed to be able to migrate, unlike the long-branched DEHP or the monoester MnBP which did not seem to have this ability. High background levels of phthalate metabolites were also observed, which might compromise results from low dose phthalate studies. In conclusion, the migration of phthalates which is probably caused by their volatile properties might lead to false interpretation of study results.

Keywords:

• Primary cell cultures
• chromatography
• dibutyl phthalate
• diethyl phthalate
• diethylhexyl phthalate
• plasticizers
• volatilization
• tandem mass spectrometry

Acknowledgements

We greatly appreciate the kind and helpful cooperation from the surgeons and staff at the Department of Otorhinolaryngology, Head & Neck Surgery and Audiology, Rigshospitalet, who provided the thyroid tissue used in this study. Also, the skilled assistance by Ole Nielsen, Department of Growth and Reproduction, Rigshospitalet, in measuring phthalate metabolites is greatly appreciated. Medical laboratory technician Jane Hinrichsen and MSc Jacob Hofman-Bang, Department of Medical Endocrinology, Rigshospitalet, were both involved in the first interpretations of the pilot study results, which is highly appreciated.

JFH received salary from three Danish public funds and one private fund: The Capital Region of Denmark, Rigshospitalets forskningspuljer, the Danish Council for Independent Research (grant number: 11-107874) and Musikforlæggernes Agnes og Knut Mørks Fond. Equipment and materials were also sponsored by the three public funds as well as by the Novo Nordisk Foundation. Ulla Feldt-Rasmussen has received funding for her research salary by Arvid Nilsson’s Fund.

MLHN is an employee of Novo Nordisk A/S Denmark and MMB is an employee of Dako A/S Denmark. MLHN has received salary from Novo Nordisk A/S during all stages of this study. MMB has been an employee of Rigshospitalet and received salary from the same funds as JFH during the study design and interpretation of the data. During preparation of the manuscript, MMB has been an employee of Dako A/S.

None of the funding agencies nor Dako A/S or Novo Nordisk A/S Denmark had any role or influence on this study.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.