In the current study, we evaluated the dose-related effects of several PEs on fetal testis gene expression using custom-designed 96-gene real-time (RT) PCR arrays. We compared the sensitivity of the affected genes to T production to identify potential genomic biomarkers of effect and exposure. These data indicate that the PEs which reduce T production act through a similar mode of action in the fetal testis, due to the consistency in reducing expression of a subset of genes involved in steroid transport and synthesis. The order of sensitivity from most to least affected genes was Cyp11b1 > Star = Scarb1> Cyp17a1 =T production > Cyp11a1 = Hsd3b = Insl3 > Cyp11b2. Interestingly, two of these consistently downregulated fetal testis genes, Cyp11b1 and Cyp11b2, code for adrenal enzymes in the adult and are not present in adult LCs. Although several genes involved in androgen synthesis and steroid transport are dramatically downregulated in the fetal testis, genes in the PPARα pathway were not induced by any PE treatment, suggesting that this pathway is not involved in PE-induced fetal testis toxicity.
We determined that the order of potency for reducing expression of the affected genes was generally consistent across most of the genes with the order of potency for reducing T production (DPeP > DHP > DIBP ≥ DHeP > DINP; ). We also demonstrated that DIDP was not an active antiandrogenic phthalate, as defined by the lack of an effect on fetal T production.
The relative potency of each phthalate for reducing gene expression was used to conduct a mixture study with nine phthalates in which we found that dose-addition models adequately predicted the mixture effects on the genes (being slightly and statistically superior to RA predictions for two of seven modeled genes and equal to RA predictions for the other five).
We previously evaluated the potencies of DPeP, DINP, DIBP, DEHP, and DIHP for reducing fetal testicular T production (Hannas et al., 2011a
). DPeP is the most potent phthalate tested to date for reducing T production and inducing postnatal reproductive tract malformations (Hannas et al., 2011a
) and testicular toxicity in the pubertal male model (Foster et al., 1980
). Based on the results of the current study, DHP is the second most potent for reduction of fetal T production. DHP induces malformations of the reproductive tract in male rat offspring, reduces AGD and induces permanent female-like nipples (Saillenfait et al., 2009
) at slightly higher dosage levels than does DPeP. When DHP is administered to pregnant SD rats (GD 12–21) at doses of 0, 50, 125, 250, or 500 mg/kg/day, AGD was reduced and areola/nipples were retained (in infants and adults) at 250 and 500 mg DHP/kg/day. In addition, low incidences of severe reproductive malformations were observed in young adult males at 125 and 250 mg DnHP/kg/day, whereas most males were malformed at 500 mg/kg/day. We previously determined that the ED50 for reduction of male AGD on PND 2 by DPeP was 252.3 mg/kg/day (Hannas et al., 2011a
) with severe malformations occurring at lower doses (Gray, personal communication). Together, these data indicate that the potency of DHP appears to verge upon that of DPeP for inhibiting differentiation of the reproductive tract of the fetal male rat.
DHeP, which was less potent than both DPeP and DHP for reducing T production and testis gene expression, has one and two additional carbons in the ester side chain compared with DHP and DPeP, respectively. Saillenfait et al. (2011)
demonstrated that DHeP only reduced AGD in male fetuses exposed in utero
(GD 6–20) at doses of 500–1000 mg/kg/day administered orally to the dam, whereas testis descent was normal for this stage of development in all dosage groups.
Our observations that DIDP was negative and DINP was the least potent active PE for reducing T production and testis gene expression are consistent with published data on the ability of these PEs to induce the Phthalate Syndrome in male rats. For example, DINP only reduces AGD (Boberg et al., 2011
) and induces reproductive tract malformations (Boberg et al., 2011
; Gray et al., 2000
) at dosage levels of 900 and 750 mg/kg/day, respectively. DIDP, which is a mixture of isomers containing two carbon chains with 9–11 carbons, was inactive in the current fetal screening protocol. This result was expected based on both its structure and the lack of effect on reproductive endpoints examined in two separate two-generation studies (Hushka et al., 2001
). In those studies, the male offspring (F1) of Sprague-Dawley rats (F0) administered DIDP in feed from 10 weeks prior to mating through female lactation, displayed no reproductive abnormalities.
Taken together, these results demonstrate that our fetal T production and gene expression findings are predictive of postnatal androgen- and INSL3-dependent tissue malformations. Numerous studies have demonstrated the ability of PE’s to reduce testicular T levels (Howdeshell et al., 2008
; Lehmann et al., 2004
; Shultz et al., 2001
), which occurs through a nonandrogen receptor (AR)–mediated mechanism (Parks et al., 2000
). Additionally, expression of Insl3
is downregulated by antiandrogenic PEs (Howdeshell et al., 2008
; Wilson et al., 2004
). In the current study, the tested PE’s that were classified as positive for reducing fetal testicular T production were all consistent in downregulating gene expression of steroidogenic enzymes, steroid regulatory, and transport proteins () and Insl3
. Some of these effects on T production and gene expression were expected based on previous work demonstrating that DBP downregulated many of these genes including Star
, and Cyp17a1
(Barlow et al., 2003
; Howdeshell et al., 2008
; Johnson et al., 2007
; Lahousse et al., 2006
; Lehmann et al., 2004
; Plummer et al., 2007
; Shultz et al., 2001
; Wilson et al., 2004
). Nevertheless, only a few of these studies have included PEs other than DBP (Howdeshell et al., 2008
) or included a sufficient range of doses to be able to determine ED50s or relative potency values for these genomic endpoints. Results of the current study also clearly indicate that all PEs that induce the phthalate syndrome alter fetal LC function via a common endocrine and genomic mode of action and that the overall potency in inducing fetal testis alterations is predictive of their potency to induce reproductive tract malformations.
FIG. 7. Steroid biosynthesis pathways of the testes (pathway outside dashed circle) and adrenals (pathway inside dashed circle). Steroidogenesis-related enzymes and transport proteins (noted by parentheses) affected by in utero phthalate exposure are circled. (more ...)
We derived two important comparisons from the dose-response data generated using the PCR gene array: (1) comparison of the individual phthalate potency for reducing expression of a particular gene and (2) comparison of the sensitivity of different genes to phthalate exposure. In comparing phthalate potency, we previously demonstrated a link between the reduction of fetal T production and early postnatal male reproductive tract malformations following dosing during the sexual differentiation period by determining that the potency of DPeP for the fetal endocrine endpoints was predictive of the postnatal endocrine endpoints (Hannas et al., 2011a
). Here, we further demonstrate that phthalate effects on expression of androgen-related genes are linked to reduced T production, based not only on the biological relevancy but also on the congruency in phthalate potency between the endpoints. We thereby further assume that these gene endpoints are also predictive of the postnatal malformations.
The second comparison we made as mentioned above was between the sensitivity of the different affected endpoints to each phthalate. In general, the order of most sensitive to least was consistent for all phthalates. A few of the genomic endpoints ranked more sensitive to PE disruption than T production, including Cyp11b1, Scarb1, and Star, although the difference among Scarb1, Star, and T production was not great. Cyp17a1 was as sensitive to PE disruption as was T production.
Currently, T production is being considered as a critical endpoint for some phthalate risk assessments. Nevertheless, the findings of this study could potentially support the use of genomic endpoints as the critical effect in future risk assessments, with a few caveats. When considering gene expression as the most sensitive endpoint of phthalate exposure, it is critically important to consider the biological role of the products of the genes. This point is illustrated when taking into account the role of the most sensitive genes detected in our assessment. The “most” sensitive gene detected in the fetal testis, Cyp11b1
, does not appear to be biologically linked to the postnatal outcomes of concern. CYP11B1, also known as 11β-hydroxylase, is an enzyme responsible for conversion of 11-deoxycortisol to cortisol in the adrenal cortex. It is not expressed in adult testes. On the contrary, recent studies, which examined mouse fetal testes, have demonstrated that a subpopulation of steroidogenic cells that express Cyp11b1
occur in the fetal LC (Hu et al., 2007
; Val et al., 2006
). Hu et al. (2007)
determined that although Cyp11b1
gene expression was detected, the protein product was not detected by immunohistochemistry, and enzyme activity was also not detected. The authors of this study suggest that translation of the enzyme product is suppressed to prevent high levels of corticosteroid production in the fetal testes. Although our results demonstrate that message of this gene in the fetal testes is highly susceptible to phthalate exposure, this vulnerability is not likely to translate into the postnatal phthalate effects, and therefore, Cyp11b1
gene expression would not be a suitable critical effect endpoint in risk assessment. Likewise, Cyp11b2
, another adrenal enzyme gene detected in the fetal testes, is not likely linked to the postnatal reproductive tract toxicity. In contrast, SR-B1, StAR, and Cyp17A1 are all critical for normal testosterone synthesis. SR-B1 protein facilitates cholesterol uptake into steroidogenic cells, whereas StAR acts as the transport protein for cholesterol across mitochondrial membranes. Cyp17A1 is the steroidogenic enzyme that converts progesterone to the androgen androstenedione, which is then converted to testosterone by another enzyme (). It is reasonable to assume that the vulnerability of these genes is linked to the postnatal reproductive malformations we detect following in utero
phthalate exposure because the products of these genes feed directly into production of T during the critical period for androgen-dependent tissue development. Furthermore, the ED50 values for the effects of PEs on Scarb1
, and Cyp17a1
gene expression do not differ greatly from the ED50 value for T production, suggesting that these genomic biomarkers could be considered additional sensitive critical or supportive endpoints for PE risk assessments.
In order to verify that our custom PCR arrays responded appropriately to activation of PPARα, we determined that the potent PPARα agonist, Wy-14,643, was effective in increasing liver weights of the exposed dams and upregulating genes in the PPARα pathway including: Rxra, Rxrb, Rxrg, Acox1, Cyp4a1, and Fabp1. In contrast, in the same experiment, DIBP had no effect on these genes. Furthermore, we confirmed that Wy-14,643 had no effect on fetal testicular T production, whereas DIBP significantly reduced T production. From this experiment, we conclude that insensitive methodology is not the cause for the lack of phthalate-induced activation of PPARα target genes in the fetal testis.
We did not detect any Pparg
expression in the fetal testis, also eliminating this as a likely PE pathway for PE-induced reproductive toxicity. Furthermore, in utero
administration of rosiglitazone, a potent PPARγ agonist, did not reduce fetal AGD, T levels, and Insl3
or steroidogenic gene expression or induce histopathological changes in the fetal testis, whereas DIBP administration induced all of these affects (Boberg et al., 2008
). We did not specifically test the effects of a PPARβ agonist on fetal T production; however, there is currently no evidence to suggest that PPARβ is involved in testicular toxicity. The existing data do not support the hypothesis that activation of PPARα or PPARγ pathways is involved in the effects of PEs on sexual differentiation of the male rat.
Despite lack of effect on the PPARα pathway, the current study provides evidence supporting the hypothesis that phthalate exposure reduces T production by interfering with cholesterol regulation. This mode of action can be inferred based on the consistency of the antiandrogenic phthalates in reducing Star
gene expression as well as the reduction of Dhcr7
at relatively high doses of DPeP, DHP, and DINP. DHCR7 or 7-dehydrocholesterol is the enzyme, which mediates the final step in cholesterol production. As previously mentioned, SR-B1 and StAR are involved in transport of cholesterol into the cell and mitochondria, respectively, as the precursor to testosterone. Johnson et al. (2007)
detected reduced Star
expression following 3 h of in utero
exposure (GD 19) to 10 or greater and 100 or greater mg DBP/kg, respectively. Plummer et al. (2007)
also demonstrated downregulation of Scarb1
expression by DBP in a time-dependent fashion between GD 15.5 and 19.5. In light of these collective results, upstream genes in this pathway and earlier time points and/or short phthalate exposure durations during the critical period warrant further investigation.
We previously determined that our 9-PE mixture reduces fetal testicular T production in a dose-additive manner (Hannas et al., 2011b
). The impetus behind that study was to provide data to support the recommendation provided to the U.S. EPA by the National Academy of Science National Research Council committee that a cumulative assessment be conducted for antiandrogenic phthalates (National Academy of Sciences, 2008
). In the current study, we further assessed fetal testicular samples for gene expression changes to determine if the effects could similarly be modeled using a DA mixture model. The mixture was designed so that each of the nine phthalates would contribute equally in terms of potency for reducing fetal T production if they acted in a dose-additive manner. The top dose was expected to dramatically reduce T production and gene expression if the effects of the nine PEs were dose additive. Based on the data from this mixture study, we can conclude that the DA model adequately predicted the observed values for all seven genes that showed consistent dose-related PE-induced downregulation. The DA model was slightly superior to the RA model for two of seven genes, whereas the models were roughly equivalent in their ability to predict the ED50 of the other five genes.
In conclusion, we used a targeted RT-PCR array approach of toxicity assessment in an attempt to address several of the challenges faced in the human health risk assessment process related to phthalate exposure. Based on the results, we confirmed that the antiandrogenic phthalates we assessed act through a similar mode of toxicity, despite not yet fully understanding the proximate molecular target. We additionally demonstrated that the rank of potency of the individual phthalates largely translates from reduction of T production to the downregulation of gene expression, suggesting that most of the consistently downregulated genes from our array plate are directly linked to the postnatal reproductive tract malformations. Finally, we demonstrated that the targeted genomic response of the fetal testis to a mixture of nine antiandrogenic phthalates was predicted using a DA mathematical model, supporting the notion that a cumulative risk assessment of the phthalates would be most protective of human health as compared with assessments of individual phthalates. Using this targeted gene array approach, we can continue to investigate the behavior of phthalates in male fetuses during a critical period of development by focusing on timing within the sexual differentiation period and additional target genes/pathways in future assessments.