Effects of In Utero and Lactational Exposure to New Generation Green Plasticizers on Adult Male Rats: A Comparative Study With Di(2-Ethylhexyl) Phthalate

Abstract

Di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer, is a ubiquitous environmental contaminant and may act as an endocrine disruptor. Early life exposures to DEHP may result in anti-androgenic effects, impairing the development of the male reproductive tract. However, data on the long-lasting consequences of such DEHP exposures on adult male reproductive function are still rare and discrepant. Previously, we identified 2 novel plasticizers, 1,4-butanediol dibenzoate (BDB) and dioctyl succinate (DOS), as potential substitutes for DEHP that did not reproduce classically described endocrine disrupting phenotypes in prepubertal male offspring after maternal exposure. Here, we investigated the consequences of in utero and lactational exposure to BDB and DOS on adult male rat reproductive function in a comparative study with DEHP and a commercially available alternative plasticizer, 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH). Timed pregnant Sprague Dawley rats were gavaged with vehicle or a test chemical (30 or 300 mg/kg/day) from gestation day 8 to postnatal day 21. While DEHP exposure (300 mg/kg/day) significantly increased epididymal weight in the adult, exposure to DINCH, BDB, or DOS did not affect reproductive organ weights, steroid levels, or sperm quality. Using a toxicogenomic microarray approach, we found that adult testicular gene expression was affected by exposure to the higher dose of DEHP; transcripts such as Nr5a2, Ltf, or Runx2 were significantly downregulated, suggesting that DEHP was targeting estrogen signaling. Lesser effects were observed after treatment with either DINCH or BDB. DOS exposure did not produce such effects, confirming its potential as a responsible substitute for DEHP.

Figure 1.

Androgen-sensitive organ weights in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Data are standardized to 100 g body weight (bw) except for testes, which are encapsulated organs. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 8–11 animals from independent litters per group; *p < .05.

Figure 2.

Serum testosterone and gonadotropin levels in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Serum testosterone (A), LH (B) and FSH (D) were assessed in 7–11 males from independent litters. The ratio of testosterone to LH (C), which allows detection of potential compensated Leydig cell failure, was calculated as well. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test.

Figure 3.

Sperm head counts in PND 90 male rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. Sperm heads were counted in the testis (A), caput and corpus epididymides (B) or cauda epididymidis (C) and reported as 10⁸ per gram of each. Daily sperm production is reported for each treatment (D). n = 4–7 males from independent litters. No significant difference was detected using one-way ANOVA corrected by Dunnett’s multiple comparison test.

Figure 4.

Testicular gene expression in PND 90 rats following in utero and lactational exposure to DEHP, DINCH, BDB, or DOS. (A) PCA of single-color microarray data; (B) Number of uniquely mapped transcripts significantly up or downregulated by more than 1.5-fold determined by moderated t test and Benjamini–Hochberg FDR correction (p > .05); (C) Venn diagrams representing commonalities in differential gene expression between treatments at 30 and 300 mg/kg/day, respectively. All data were generated using n = 5–6 male rats from independent litters.

Figure 5.

Gene expression quantification by RT-qPCR for selected transcripts following in utero and lactational exposure to DEHP, DINCH, BDB or DOS. Relative levels of gene amplification compared with the control group and housekeeping gene Ppia were calculated following the ΔΔC_t method. Significance was determined by one-way ANOVA corrected by Dunnett’s multiple comparison test; n = 6 biological replicates plated in triplicate; **p < .01; ****p < .0001.