Genomic and Hormonal Biomarkers of Phthalate-Induced Male Rat Reproductive Developmental Toxicity Part II: A Targeted RT-qPCR Array Approach That Defines a Unique Adverse Outcome Pathway

Abstract

Previously, we demonstrated that exposure to some diortho-phthalate esters during sexual differentiation disrupts male reproductive development by reducing fetal rat testis testosterone production (T Prod) and gene expression in a dose-related manner. The objectives of the current project were to expand the number of test compounds that might reduce fetal T Prod, including phthalates, phthalate alternatives, pesticides, and drugs, and to compare reductions in T Prod with altered testis mRNA expression. We found that PEs that disrupt T Prod also reduced expression of a unique "cluster" of mRNAs for about 35 genes related to sterol transport, testosterone and insulin-like hormone 3 hormone syntheses, and lipoprotein signaling and cholesterol synthesis. However, phthalates had little or no effect on mRNA expression of genes in peroxisome proliferator-activated receptor (PPAR) pathways in the fetal liver, whereas the 3 PPAR agonists induced the expression of mRNA for multiple fetal liver PPAR pathway genes without reducing testis T Prod. In summary, phthalates that disrupt T Prod act via a novel adverse outcome pathway including down regulation of mRNA for genes involved in fetal endocrine function and cholesterol synthesis and metabolism. This profile was not displayed by PEs that did not reduce T Prod, PPAR agonists or the other chemicals. Reductions in fetal testis gene expression and T Prod in utero can be used to establish relative potency factors that can be used quantitatively to predict the doses of individual PEs and mixtures of phthalates that produce adverse reproductive tract effects in male offspring.

Keywords: in utero phthalate; AOP; PPAR pathway; cholesterol metabolism; fetal testis gene expression; testosterone production.

Figure 1.

Experimental design and logical sequence of experiments in the current study.

Figure 2a.

Fetal testis testosterone production ex vivo and custom RT-qPCR array mRNA expression from blocks in which dams were exposed by oral gavage to a high dose level of phthalates, phthalate alternatives and pesticides and the drug flutamide. Doses are mg/kg/d. Testosterone production T Prod is % of the concurrent control mRNA values are fold versus control =1.0. Shaded (yellow) T Prod mRNA values differ significantly from controls. Chemicals that reduce testosterone are “positives” and those that do not are “negatives”.

Figure 2b.

Fetal testis testosterone production ex vivo and custom RT-qPCR array mRNA expression from blocks in which dams were exposed to phthalates by oral gavage at several dose levels. The strain of rat used is indicated and phthalates that reduce testosterone are “positives” and those that do not are “negatives”.

Figure 2b.

Fetal testis testosterone production ex vivo and custom RT-qPCR array mRNA expression from blocks in which dams were exposed to phthalates by oral gavage at several dose levels. The strain of rat used is indicated and phthalates that reduce testosterone are “positives” and those that do not are “negatives”.

Figure 2c.

Fetal testis testosterone production ex vivo and custom RT-qPCR array mRNA expression from blocks in which dams were exposed to a phthalate, a phthalate alternative, two pesticides, two industrial chemicals or two drugs by oral gavage at several dose levels. The strain of rat used is indicated and phthalates that reduce testosterone are “positives” and those that do not are “negatives”.

Figure 2d.

Fetal testis testosterone production ex vivo and custom RT-qPCR array mRNA expression from blocks in which dams were exposed to PPAR agonists or a phthalate.

Figure 3.

Factor analysis scores for the effects of the four different treatment groups on the fetal testis mRNA levels on the custom RT-qPCR arrays demonstrating that cluster of transcripts affected by phthalates that reduce T Prod differs significantly from all the other groups. This confirms that these phthalates disrupt key events in a novel adverse outcome pathway.

Figure 4a.

Effects of gestational phthalate ester maternal oral treatment on fetal testis mRNA transcript expression on the RT² Profiler™ PCR Array for rat Lipoprotein Signaling & Cholesterol Metabolism (PARN 080).

Figure 4b.

Dose related effects of dibutyl phthalate (DBP) on fetal rat testis mRNA expression on the Lipid signaling and cholesterol metabolism arrays. Variables are ranked by the ED₅₀ values with the lowest value at the top. Shaded values differ significantly from control fold values. mRNA expression levels for 16 genes were altered consistently by the eight phthalates that reduce testis ex vivo T Prod but not DEP, a phthalate that does not reduce T Prod.

Figure 5.

Effect of phthalates that significantly reduce (p< 0.0001) T Prod also affect mRNA expression for genes on the Phase 1 Drug Metabolism Array. mRNA expression levels are ranked in the figure with the transcript with the lowest fold versus control value (most affected) for the eight phthalates that reduce T Prod at the top.

Figure 6.

PPAR agonists do not recapitulate the maternal and fetal effects of phthalates. PPAR agonists increase maternal liver weight when administered from days 14 to 18 of pregnancy whereas the phthalates DiBP and DPeP do not increase liver weight. Furthermore, the phthalates significantly reduce fetal testis ex vivo T Prod, whereas the PPAR agonists do not affect T Prod.

Figure 7.

The PPAR agonists WY 14643, clofibrate and HFPO-DA (GenX) (columns 4, 6 to 17) significantly induce mRNA expression levels of multiple transcripts in the fetal liver to a greater degree than does the phthalate DPeP (column 2). In addition, WY 14643 has little effect on the PPAR pathway in the fetal testis (column 5, 3 transcripts induced) as compared the effects in the fetal liver (column 4, 25 transcripts induced).

Figure 8.

Heat maps showing the effects of the PPAR agonist WY 14643, HFPO-DA (GenX) and clofibrate and the phthalate DPeP on fetal liver PPAR pathway transcript expression. The heat map on the left compares the fold values to control (fold =1) and the heat map on the right contrasts the fold values of the three PPAR agonists to DPeP with the expression of transcripts by DPeP set to fold =1 (22 transcripts are induced to a significantly greater level by the PPAR agonists versus the phthalate DPeP).

Figure 9.

Factor analysis scores for the effects of the three PPAR agonists on factor 1 differ significantly from the DPeP factor 1 score on fetal liver PPAR pathway mRNA transcript expression (all 84 transcripts were included in the analysis).