Cumulative effects of in utero administration of mixtures of reproductive toxicants that disrupt common target tissues via diverse mechanisms of toxicity

Abstract

Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act required the US Environmental Protection Agency to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures of chemicals to elucidate mechanisms of joint action at the systemic level with the goal of providing a framework for assessing the cumulative effects of reproductive toxicants. Previous mixture studies conducted with antiandrogenic chemicals are reviewed briefly and two new studies are described. In all binary mixture studies, rats were dosed during pregnancy with chemicals, singly or in pairs, at dosage levels equivalent to approximately one-half of the ED50 for hypospadias or epididymal agenesis. The binary mixtures included androgen receptor (AR) antagonists (vinclozolin plus procymidone), phthalate esters [di(n-butyl) phthalate (DBP) plus benzyl n-butyl phthalate (BBP) and diethyl hexyl phthalate (DEHP) plus DBP], a phthalate ester plus an AR antagonist (DBP plus procymidone), a mixed mechanism androgen signalling disruptor (linuron) plus BBP, and two chemicals which disrupt epididymal differentiation through entirely different toxicity pathways: DBP (AR pathway) plus 2,3,7,8 TCDD (AhR pathway). We also conducted multi-component mixture studies combining several 'antiandrogens'. In the first study, seven chemicals (four pesticides and three phthalates) that elicit antiandrogenic effects at two different sites in the androgen signalling pathway (i.e. AR antagonist or inhibition of androgen synthesis) were combined. In the second study, three additional phthalates were added to make a 10 chemical mixture. In both the binary mixture studies and the multi-component mixture studies, chemicals that targeted male reproductive tract development displayed cumulative effects that exceeded predictions based on a response-addition model and most often were in accordance with predictions based on dose-addition models. In summary, our results indicate that compounds that act by disparate mechanisms of toxicity to disrupt the dynamic interactions among the interconnected signalling pathways in differentiating tissues produce cumulative dose-additive effects, regardless of the mechanism or mode of action of the individual mixture component.

Figure 1

A diagram showing how different mechanisms of toxicity and disruption of diverse toxicity pathways (Androgen- versus Ah receptor signaling pathways) converge as an integrated network of pathways to disrupt the development of the reproductive tract during a critical developmental period.

Figure 2

“Heat map” displaying the intensity of effects of each chemical in vitro, in short-term in vivo screens and on F1 male and female offspring after exposure in utero during sexual differentiation. The red shading indicates stronger effects whereas the black areas indicate the absence of effects. Reviewing the columns associated with each chemical describes the 1) known mechanisms of toxicity as determined from in vitro and short-term in vivo screening studies and 2) the overall phenotype in the male offspring after exposure during fetal life. Comparing the different chemicals by rows (the endpoints) allows one to compare the relative potencies displayed by the toxicants to one another. For each endpoint, we predict that all the chemicals with shading (light red to dark red) will interact jointly when combined in a mixture, but those with black squares will have no effect when included in the mixture.

Figure 3

Effects of the individual chemicals on AGD at 3 days of age (Fig 3A). Phthalates refers to the group of phthalates (DBP, DiBP, DiHP, BBP, and DEHP) which have similar potency and are represented by the dose-response data from DBP and DEHP. DPP is not shown on this graph, but was three times as potent as the other phthalates and was assumed to have a dose-response curve with a similar shape as that of the other phthalates. Observed (OBS) effects of the mixture on AGD at 3 days of age (Fig 3B, male and female) and observed and predicted effects in our new multi-component mixture study with ten chemicals (four pesticides and six phthalates) on AGD in male rats at 3 days of age (Fig 3C). Since AGD is linear in the low dose range, all models (dose (DA), integrated (IA) and response addition (RA) provided similarly accurate predictions for the observed reductions in AGD. The ED50s as % of the top dose (100%) are shown for the observed and predicted effects of the mixture on F1 male rat AGD at three days of age. Predicted ED50 values with shading fall outside of the 95% confidence limits surrounding the ED50 calculated from the observed data. RA provides less accurate predictions than either DA or IA models. Values on the figures are means and standard errors of observed responses and predicted responses.

Figure 4

Summary of the observed (Fig 4A) and predicted effects in our new multi-component mixture study with ten chemicals (four pesticides and six phthalates) on reproductive tract malformations detected in adult F1 males. Individual chemical data that went into models can be found in Rider et al. 2008. Dose addition (DA) models more accurately predicted the effects of the mixture of ten chemicals on the incidences of hypospadias (Fig 4B) and epididymal agenesis (Fig 4C). The integrated addition (IA) model under-predicts effects and the response addition (RA) model predicts no malformations at even the highest dose. The ED50s as % of the top dose (100%) are shown for the observed and predicted effects of the mixture on F1 male rat malformations (hypospadias, epididymal agenesis and undescended testes (Fig 4D)). Predicted ED50 values with shading fall outside of the 95% confidence limits surrounding the ED50 calculated from the observed data. Only the DA model accurately predicted the observed incidences of all three of these malformations. Values on the figures are means and standard errors of observed responses and predicted responses.

Figure 5

Summary of the observed and predicted effects in our new multi-component mixture study with ten chemicals (four pesticides and six phthalates) on reproductive tract organ weights measured in adult F1 males. Individual chemical data that went into models can be found in Rider et al. 2008. The ED50s as % of the top dose (100%) are shown for the observed and predicted effects of the mixture on F1 male rat seminal vesicle (Fig 5A), epididymal (Fig 5B), ventral prostate (Fig 5C) and LABC (Fig 5D) weights. Predicted ED50 values with shading fall outside of the 95% confidence limits surrounding the ED50 calculated from the observed data. The reductions in seminal vesicle, ventral prostate, epididymal and LABC weights were all more accurately predicted by dose addition (DA) than by either integrated addition (IA) or response addition (RA) models. The DA model accurately predicted all the reductions except for the reduction in ventral prostate weight, where it slightly under-predicted the magnitude of the effect. However, it was still clearly the “best” model as compared to IA or RA predictions. Values on the figures are means and standard errors of observed responses and predicted responses.

Figure 6

A summary of the binary mixture study with DBP and 2,3,7,8 TCDD, a mixture of chemicals that disrupts diverse receptor signaling pathways (AR and AhR) is presented here. The incidences of malformed organs in the 100% (TCDD at 2 μg/kg on GD 14 and DBP at 500 mg/kg/d GD 14-18) and 65% mixture group exceeded response addition (RA) for the epididymal and testicular (Fig 6A), vas deferens (Fig 6C), external genitalia (Fig 6F) and liver (Fig 6G) malformations and epididymal (Fig 6B) and testis weights (Fig 6E) and epididymal sperm numbers (Fig 6D). The increases in gross liver pathology and malformations of the external genitalia were unanticipated since TCDD is not known to induce hypospadias and the gross lesions in the liver have not been seen previously with either chemical alone. Values on the figures are means and standard errors of observed responses. The line identified as RA is the response level predicted by the RA model for the effect of the high dose mixture of TCDD and DBP. The effect of the high dose mixture was significantly greater (p<0.03 by t-test of litter mean values) than the level predicted by the response addition model.