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Validation of an automated counting procedure for phthalate-induced testicular multinucleated germ cells

Daniel J Spade1, Cathy Yue Bai1, Christy Lambright2

  • 1Department of Pathology and Laboratory Medicine, Brown University, Providence, RI, USA.

Toxicology Letters
|March 25, 2018
PubMed

Insights

In utero exposure to phthalates causes testicular toxicity, marked by multinucleated germ cells (MNGs). An automated ImageJ method accurately quantifies MNGs, aiding phthalate-induced reproductive toxicity research.

Area of Science:

  • Reproductive Toxicology
  • Developmental Biology
  • Environmental Health

Background:

  • In utero exposure to phthalate esters induces testicular toxicity, including multinucleated germ cells (MNGs) and reduced testosterone in rats.
  • Quantifying phthalate-induced MNGs via manual counting is time-consuming, hindering dose-response studies.
  • Anti-androgenic effects of phthalates are better understood than their direct testicular pathology.

Purpose of the Study:

  • To develop and validate an automated ImageJ-based method for quantifying MNGs in rat fetal testis tissue.
  • To compare the automated MNG counting method with traditional manual counting.
  • To assess the correlation between MNG induction and anti-androgenic activity of various phthalates.

Main Methods:

  • An automated ImageJ method was developed to quantify MNGs in digital images of hematoxylin-stained rat fetal testis sections.
  • Timed pregnant Sprague Dawley rats were exposed to eight phthalate compounds or vehicle from gestation day 17 to 21.
  • Both manual and automated methods were used to identify phthalates inducing MNGs, and results were correlated (r=0.923).

Main Results:

  • The automated method successfully identified di-n-butyl phthalate, butyl benzyl phthalate, dipentyl phthalate, and di-(2-ethylhexyl) phthalate as MNG inducers.
  • Dimethyl phthalate, diethyl phthalate, di-(2-ethylhexyl) tetrabromophthalate, and dioctyl terephthalate did not induce MNGs.
  • MNG induction results aligned with anti-androgenic activities confirmed by an ex vivo testosterone production assay.

Conclusions:

  • A reliable automated image analysis method for quantifying MNGs has been developed.
  • This method facilitates dose-response studies for phthalate-induced MNGs.
  • The findings support the utility of MNG analysis in assessing reproductive toxicity of phthalates.
Keywords:
Multinucleated germ cellsPhthalate estersQuantitative pathology

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