Phthalate-induced Leydig cell hyperplasia is associated with multiple endocrine disturbances

Abstract

The possibility that exposures to environmental agents are associated with reproductive disorders in human populations has generated much public interest recently. Phthalate esters are used most commonly as plasticizers in the food and construction industry, and di-(2-ethylhexyl) phthalate (DEHP) is the most abundant phthalate in the environment. Daily human exposure to DEHP in the U.S. is significant, and occupational and clinical exposures from DEHP-plasticized medical devices, e.g., blood bags, hemodialysis tubing, and nasogastric feeding tubes, increase body burden levels. We investigated the effects of chronic exposures to low environmentally relevant DEHP levels on testicular function. Our data show that prolonged exposures to this agent induced high levels of the gonadotropin luteinizing hormone and increased the serum concentrations of sex hormones [testosterone and 17beta-estradiol (E2)] by >50%. Increased proliferative activity in Leydig cells was evidenced by enhanced expression of cell cycle proteins, as determined by RT-PCR. The numbers of Leydig cells in the testis of DEHP-treated rats were 40-60% higher than in control rats, indicating induction of Leydig cell hyperplasia. DEHP-induced elevations in serum testosterone and E2 levels suggest the possibility of multiple crosstalks between androgen, estrogen, and steroid hormone receptors, whereas the presence of estrogen receptors in nonreproductive tissues, e.g., cardiovascular system and bones, implies that the increases in serum E2 levels have implications beyond reproduction, including systemic physiology. Analysis of the effects of phthalate exposures on gonadotropin and steroid hormone levels should form part of overall risk assessment in human populations.

Fig. 1.

Effect of DEHP treatment (A–D, PND 21–90; E–H, PND 21–120) on serum LH and T levels and Leydig cell T production. Serum LH (A and E)andT(B and F) levels were higher in DEHP-treated rats than control, but basal and LH-stimulated T production per Leydig cell was decreased (C, D, and G, H). Thus, elevated serum T levels in the presence of reduced steroidogenic capacity is presumably due to Leydig cell hyperplasia. Leydig cell T production, normalized to nanograms per 10⁶ cells, was measured by RIA in aliquots of the spent media after incubation of Leydig cells for 3 h. n = 10; *, P < 0.01 compared to control. Data are presented as mean ± SEM.

Fig. 2.

Effect of DEHP treatment on expression of cell cycle proteins. DEHP treatment from 21 to 90 days of age increased expression of several cell cycle proteins [PCNA and cyclins D₃ (A) and the tumor suppressor protein p53 and cyclin G₁ (B)]. Increased proliferative activity in Leydig cells is therefore due to induction of cell cycle proteins by DEHP treatment. Total RNA was obtained from two separate experiments, and rat ribosomal protein S16 (S16) was used as internal control for PCR amplification. p53, tumor suppressor protein; *, P < 0.05 compared to control. Data are presented as mean ± SEM.

Fig. 3.

Effect of DEHP treatment on Leydig cell numbers. After DEHP treatment of rats from 21 to 90 days of age (n = 10), the number of Leydig cells recovered from the testis was increased (A). DEHP treatment from PND 21 to 120 increased thymidine incorporation by Leydig cells (B), and the numbers of Leydig cells, counted in both testes from four animals in each group, were increased by as much as 40–60% in DEHP-treated rats compared to control (C). These observations confirm that chronic DEHP exposures induced Leydig cell hyperplasia. *, P < 0.01 compared to control. Data are presented as mean ± SEM.

Fig. 4.

Effect of DEHP treatment on estrogen biosynthesis. Serum E2 levels (A) were higher due to increased Leydig cell E2 production (B and C) and increased aromatase gene expression (D) after DEHP treatment of rats from 21 to 48 days of age (n = 10). E2 production was normalized to nanograms per 10⁶ cells and measured by RIA in aliquots of the spent media after incubation of Leydig cells for 3 h. S16, rat ribosomal protein S16; *, P < 0.01 compared to control. Data are presented as mean ± SEM.

Fig. 5.

Serum MEHP levels in DEHP-treated rats (n = 10). HPLC chromatogram of MEHP, DNHP, and DEHP is shown in A, indicating retention times of 5.1, 15.1, and 19.9 min, respectively. Serum MEHP levels measured on PND 48 after a 28-day DEHP treatment period (B) were much higher than was measured on PND 90 after a 70-day treatment period (C). DNHP was used as internal standard. *, P < 0.01 compared to control. Data are presented as mean ± SEM.

Fig. 6.

A schema summarizing DEHP effects on Leydig cell function. Chronic DEHP exposures increased the serum levels of the gonadotropin LH and the sex steroids T and estradiol. These effects presumably induce cell cycle proteins and cause Leydig cell proliferation and hyperplasia. Increased serum androgen levels in DEHP-treated rats may result in precocious puberty and induce hyperplasia of the prostatic epithelium as well as promote development of testicular seminomas. Furthermore, induction of the tumor suppressor protein p53 is suggestive of DNA repair activity and represents a potential for neoplasia.