In utero exposure to an AR antagonist plus an inhibitor of fetal testosterone synthesis induces cumulative effects on F1 male rats

Abstract

Risk assessments are typically conducted on a chemical-by-chemical basis; however, many regulatory bodies are developing frameworks for assessing the cumulative risk of chemical mixtures of chemicals. The current investigation examined how chemicals that disrupt rat sex differentiation via two diverse mechanisms disrupt F1 male rat reproductive development, when administered together orally on days 14-18 of gestation. Experiment 1 used a mixture of 50 mg/kg-d procymidone and 500 mg/kg-d dibutyl phthalate (DBP), whereas experiment 2 used 150 mg/kg-d procymidone and 1125 mg/kg-d DBP (top dose), or 0, 4.17, 8.33, 16.7, 33.3, 50, 66.7, and 83.3% of the top dose. When we compared the dose and response addition predictions to the observed effects we found that dose addition models were more accurate than response addition models, indicating that compounds that act by different mechanisms of toxicity produce cumulative dose-additive effects.

Fig. 1

Individual chemical dose–response data for procymidone and di-n-butyl phthalate (DBP) were analyzed using a four parameter logistic regression model with GraphPad Prism 5.0 software. The ED50 and Hillslope values from the logistic regression analyses for anogenital distance in neonatal male rats (AGD), retained areolas/nipples in infant male rats and malformations (hypospadias, epididymal agenesis) and organ weights (epididymal, levator ani plus bulbocavernosus (LABC) muscles, seminal vesicles and ventral prostate) in adult male F1 rats. The X axes are in log₁₀ scale.

Fig. 2

Logistic regression analyses of the malformation (A) and organ weight (B) data from the second mixture experiment using nine dilutions of a mixture of procymidone and di-n-butyl phthalate (DBP). The list of endpoints at the right of each panel displays the ED50 values for each endpoint at the right. Abbreviations: GP—glans penis, VP—ventral prostate, SV—seminal vesicle, LABC—levator ani bulbocavernosus muscles, COWS—Cowper’s glands, TWT—testis weight, EPIS—epididymis, Body—body weight. The X axes are in log₁₀ scale.

Fig. 3

Observed (OBS) and dose (DA) and response (RA) addition predictions on the effects of the mixture of procymidone and DBP from experiment two on anogenital distance (AGD) at 3 days of age (A), and retained areolae/nipples in infant (B) and adult male (C) rat offspring. The panels also include the ED50 values from the logistic regression of the observed data and the DA and RA predicted effects. Yellow shaded values differ significantly from the observed ED50s because the values fell outside the 95% confidence limits of the observed ED50s. For AGD, and retained nipples in infants and adult males, DA provided a more accurate prediction than did the RA model. However, for AGD the difference between the ED50 for DA and the ED50 for RA was not great. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 4

Logistic regression plots of the observed (OBS) and dose- (DA) and response- (RA) addition predictions on the effects of the mixture of procymidone and DBP from experiment two on the induction of hypospadias (A), and reduced ventral prostate (B), levator ani plus bulbocavernosus muscles (C) and seminal vesicle weights (D). The panels also include the ED50 values from the logistic regression of the observed data and the DA and RA predicted effects. Yellow shaded values differ significantly from the observed ED50s. These values fell outside the 95% confidence limits of the observed ED50s. The DA but not RA model provided an accurate prediction of the effects of the mixture on hypospadias and seminal vesicle weight and the DA model was more accurate than the RA model in predicting ventral prostate weight reductions. The X axes are in log₁₀ scale. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

Observed (OBS) and dose (DA) and response (RA) addition predictions on the effects of the mixture of procymidone and DBP from experiment two on the induction of epididymal agenesis (A) and reduced epididymal weight (B). The panels also include the ED50 values from the logistic regression of the observed data and the DA and RA predicted effects. Yellow shaded values differ significantly from the observed ED50s. These values fell outside the 95% confidence limits of the observed ED50s. The DA and RA models provided predictions of equivalent accuracy for the effects of the mixture on the epididymis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 6

Observed effects of the individual chemicals PRO (procymidone) and di-n-butyl phthalate (DBP) (A) and the mixture of PRO and DBP from experiment two (B) on the induction of histopathological lesions of the testis. Abbreviations: ST degen.—seminiferous tubule degeneration in the testis, IC—interstitial cell (Leydig cell). The panels also include the ED50 values from the logistic regression of the observed data.