The effects of mixtures of chemicals like the ubiquitous phthalates are of concern since humans are exposed to multiple phthalates at one time (Silva 2004
; Wolff et al. 2007
). In order to address this issue, in 2006 the U.S. Environmental Protection Agency (EPA) requested that the National Academy of Sciences (NAS) establish a panel to provide the EPA with recommendations on whether to perform a cumulative risk assessment of the phthalates. This review will present the major conclusions of the NAS panel (Box from p 116 of the NAS report) and discuss the data from our laboratory on the reproductive effects of mixtures.
Our working hypothesis is that chemicals that disrupt a common system or tissue during development contribute to cumulative toxicity and should therefore, be included in cumulative risk assessments. The results of our studies support this hypothesis and are in agreement with the conclusions of the NAS Report (2008) (). This represents a shift from the current cumulative risk assessment process which only includes chemicals which share a narrowly defined mechanism of action.
Main points from National Academy of Science report: “Phthalates and Cumulative Risk Assessment: The Task Ahead”
As a result of growing concerns about mixtures, the field of “mixtures toxicology” is emerging as an area of increasing scientific and regulatory focus. For example, in 1996 the EPA began considering the cumulative risk of chemicals that act via a common mechanism of toxicity as mandated in the Food Quality Protection Act (FQPA). The EPA’s Offices of Water and Research and Development and the EPA Superfund, Solid Waste and Air Programs also have ongoing programs in the area of mixtures toxicology. In this regard, the research from our laboratory, described herein, is intended to contribute to the development of a guidance framework for assessing cumulative risk to reproduction and development from exposures during pregnancy.
Our research has included mixtures of pesticides, phthalates and 2,3,7,8 TCDD. These chemicals disrupt sexual differentiation by acting as androgen receptor (AR) antagonists, inhibitors of fetal testosterone synthesis or as an aryl hydrocarbon receptor (AhR) agonist ( and ).
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 (more ...)
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 (more ...)
We initially conducted a series of binary mixture studies exposing pregnant dams during fetal sexual differentiation with pairs of chemicals (Gray et al. 2001b
; Hotchkiss et al. 2004a
; Howdeshell et al. 2007
; Howdeshell et al. 2008b
; Rider et al. 2009
). In the binary studies, rats were dosed with chemicals singly or in pairs at dosage levels equivalent to about one half of the ED50 for hypospadias or epididymal agenesis (2 × 2 factorial designs). We predicted that by itself each chemical in a pair would cause a minimal rate of malformations, whereas when two chemicals were mixed together they would produce predictable, dose-additive effects on common target tissues. We found that the binary combinations produced cumulative, dose-additive effects on the androgen-dependent tissues that were a common component of each chemical’s phenotype. Several of the studies have been reviewed in detail previously (Gray 2002
; Gray et al. 2001a
; Howdeshell et al. 2008b
; Rider et al. 2008
; Rider et al. 2009
) and will only be briefly reviewed, whereas the phthalate plus dioxin binary study is new and will be presented in more detail.
In addition to the binary studies, we also have conducted three mixture studies with larger numbers of chemicals. One study combined five phthalates together in the mixture to determine if they produced dose-additive effects on fetal testosterone production (Howdeshell et al. 2008b
), fetal testis gene expression and postnatal development of the male and female offspring. These phthalates all shared a common mechanism of toxicity. The remaining two multi-component mixture studies combined chemicals that elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e. AR antagonist or inhibition of androgen synthesis). One study combined seven chemicals together and the second combined 10 chemicals in the mixture.
The paper that follows will 1) briefly describe the mechanisms and modes of toxicity in vitro and in vivo of the individual chemicals that we selected to study in our research program since these have been reviewed in detail previously (Gray et al. 2001b
; Howdeshell et al. 2008b
; Rider et al. 2008
; Rider et al. 2009
), 2) describe the mathematical modeling procedures that we use to describe the effects of the individual chemicals and derive predictions for how the mixtures will behave, 3) present a summary of the results of our mixture studies on chemicals that act via common mechanisms of toxicity (Section A), chemicals that act via disparate mechanisms of toxicity but disrupt the same signaling pathway (Section B), and chemicals that disrupt common developing tissues via alteration of diverse signaling pathways (Section C), and 4) describe a proposed framework for cumulative risk assessment based upon disruption of a common developing system. The review will include new data from two studies, one a multi-component mixture study of ten chemicals that disrupt androgen signaling by diverse mechanisms of toxicity and another study combining chemicals that disrupt different signaling pathways, the aryl hydrocarbon receptor (AhR) agonist 2,3,7,8 TCDD and dibutyl phthalate that reduces fetal testosterone levels.
Mechanisms and modes of action of the individual chemicals used in mixture studies
Over the last several decades our laboratory has studied the postnatal effects of in utero exposure to a variety of environmental chemicals with diverse endocrine and non-endocrine mechanisms of reproductive toxicity. We are now using the dose response information from these studies to design mixture studies to determine how chemicals from different classes with similar and diverse mechanisms and modes of action interact in the mixture. The objective of our research is to define a framework for conducting cumulative risk assessments of reproductive toxicants.
The chemicals selected for our current mixture studies are shown in . In this figure, the red 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 (dark red to light red) will interact jointly when combined in a mixture, but those with black squares will have no effect when included in the mixture. For example, of the chemicals in this chart, only the phthalates reduce insulin-like peptide hormone-3 (insl3) and cause gubernacular agenesis. Therefore, combining a phthalate with any of the other chemicals in the chart will not enhance the phthalate-induced reduction in insl3 mRNA levels in the fetal testis or increase the incidence of gubernacular agenesis.
Dicarboximide fungicides: Vinclozolin and Procymidone
While some toxicants disrupt sexual differentiation predominantly via one mechanism of toxicity (i.e. AR antagonists or inhibitors of testosterone synthesis), some pesticides including linuron and prochloraz act via dual mechanisms of toxicity. These pesticides display AR antagonist activity and inhibit testosterone synthesis with varying potencies.
Prochloraz is a fungicide that also disrupts reproductive development and function by several modes of action (Noriega et al. 2005
; Vinggaard et al. 2005
; Vinggaard et al. 2006
). Prochloraz inhibits the steroidogenic enzymes 17, 20 lyase and aromatase and it also is an AR antagonist (Blystone et al. 2007
). In a study in which rat dams were dosed from GD 14–18, Wilson et al. (Wilson et al. 2004
) found that prochloraz reduced fetal testis testosterone levels and increased progesterone production tenfold on GD 18 without affecting Leydig cell insl3 mRNA levels.
In utero, some phthalates alter male and female reproductive tract differentiation via unknown mechanisms of action. The mode of action in the male involves altered Leydig cell migration and differentiation and abnormal gonocytes development (Hallmark et al. 2007
; Mahood et al. 2005
; Mahood et al. 2006
; Parks et al. 2000
). The Leydig cell alterations result in reductions in fetal testis testosterone production, and mRNA levels for key proteins in the steroidogenic pathway including StAR and CYP11, as well as insl-3, which is critical for gubernacular development and testis descent (Hughes and Acerini 2008
; Kumagai et al. 2002
2,3,7,8 Tetrachlorodibenzo Dioxin (TCDD)
Several studies have shown that TCDD administration does not
reduce adult (Gray et al. 1995
; Theobald et al. 2000
) or fetal androgen levels in the male rat or mouse (Ko et al. 2004
) (Haavisto et al. 2001
; Haavisto et al. 2006
), even though several of the effects in the male offspring include suppressed development of some androgen-dependent tissues. In mice, it is known that TCDD directly inhibits the androgen-dependent processes by which the urogenital sinus of fetal mice forms prostatic epithelial buds (Lin et al. 2004
) without affecting androgen levels (Akingbemi et al. 2004
). Additional mechanistic studies proposed that TCDD acted directly on AhR, ARNT, and AhR-induced transcripts in the periprostatic mesenchyme (Vezina et al. 2008
; Vezina et al. 2009
). This tissue intimately contacts urogenital sinus epithelium where buds are specified and they proposed that activation of AhR signaling disrupted dorsoventral patterning of the urogenital sinus, reprogramming the areas where prostatic buds are specified, and prostate lobes are formed.
Administration of TCDD on the 15th day of gestation (GD 15) at doses lower than or equal to 1 μg/kg both demasculinizes and feminizes male rat reproductive morphology and behavior. In our first series of studies, Long Evans Hooded and Holtzman rats were dosed by gavage with 1 μg TCDD/kg on GD 8 (a period of major organogenesis), or Syrian hamsters, a species relatively insensitive to the lethal effects of TCDD, were dosed on GD 11 (a period equivalent to GD 15 in the rat), with TCDD at 2 μg/kg. In Long Evans and Holtzman F1 male rats exposed on GD 15 or hamsters exposed on GD 11, puberty (preputial separation) was delayed by about 3 days, ejaculated sperm counts were reduced by at least 58% and epididymal sperm storage was reduced by 30%. Testicular sperm production was less affected. The sex accessory glands were also reduced in size in Long Evans offspring treated on GD 15 in spite of the fact that serum testosterone levels, testosterone production by the testis in vitro
, and AR levels were not reduced. Some reproductive measures, like anogenital distance and male sex behavior, were altered by TCDD-treatment in rat but not hamster offspring (Gray et al. 1995