Cracking the Nut

Phthalate esters impart flexibility to hard plastics, are used as inert ingredients in cosmetics and personal hygiene products, and are ubiquitous environmental contaminants. Finding the underlying molecular mechanism of phthalate-induced testicular injury has been a vision quest of male reproductive toxicologists for many decades. Since 1945, when Shaffer et al. (1945) first described ‘‘tubular atrophy and degeneration . . . resembling senile changes’’ (p. 131) in the testis following exposure to di-(2ethylhexyl) phthalate, phthalates have become the most studied and most thoroughly characterized class of testicular toxicants. What is especially exciting about the phthalates for mechanistic toxicologists is the rapid onset of testis-specific abnormalities; ‘‘rapid’’ and ‘‘specific’’ should mean simple and straightforward, right? Wrong! A number of mechanistic hypothesis have been proposed to explain phthalate-induced testicular injury, including alterations in (1) zinc-dependent enzymatic activities, (2) hormonal status, (3) metabolic function, (4) FSH receptor-dependent pathways, (5) peroxisome proliferator-activated receptor-dependent pathways, and (6) Sertoli-germ cell adhesion. These hypotheses have been generated from studies using single high doses of phthalate esters in adult and peripubertal animals and from in vitro studies using the active metabolites. In these models, the Sertoli cell is viewed as the target cell for testicular injury, based upon the early and specific onset of histopathological changes in this cell type following exposure, and on the lack of changes in the usual hormonally responsive endpoints. A decade ago, everything was in place for a methodical examination of the mechanism of phthalate-induced testicular injury—a well-described acute exposure model, an established cellular target (the Sertoli cell), and a rapid and specific pattern of response. For male reproductive toxicologists, a tectonic shift in perspective has occurred with the appreciation that phthalate esters are endocrine disruptors. Following the demonstration that butylbenzylphthalate and di-n-butylphthalate are weakly estrogenic in in vitro assays (Jobling et al., 1995), several major laboratory groups quickly showed that in utero/perinatal exposures to phthalate esters produce a spectrum of developmental anomalies in animal models (Gray et al., 2000; Mylchreest et al., 1998; Sharpe et al., 1995). The observed alterations include an early suppression of Leydig cell function associated with a host of subsequent abnormalities that are both transient and long-lasting, such as reduced anogenital distance, retained nipples, cleft phallus, epididymal dysgenesis and agenesis, Leydig cell hyperplasia, cryptorchidism, hypospadias, the generation of multinucleated gonocytes, small testes, and reduced sperm counts. Phthalate esters alter the developing male reproductive tract similarly to antiandrogens such as flutamide, p,p0-DDE, and linuron. Unlike these other antiandrogens, however, phthalate esters produce some unique alterations, such as the generation of multinucleated gonocytes (Mylchreest et al., 2002) and do not interact with the androgen receptor. These observations in animal models have contributed to the conceptualization by Skakkebaek et al. (2001) of a ‘‘testicular dysgenesis syndrome’’ to explain the temporal changes in various human male reproductive tract abnormalities, including falling sperm counts, and an increasing incidence of hypospadias, cryptorchidism, and testis germ cell cancer. According to Skakkebaek’s hypothesis, an altered in utero and perinatal hormonal environment—possibly due to exposures to endocrine disrupting chemicals—predisposes the human male reproductive tract to dysgenesis, because of the hormonally sensitive nature of the underlying developmental events. The report by Lehmann et al. in this issue exploits the animal model of in utero phthalate exposure as well as a global expression analysis database created with the capabilities of modern ‘‘-omics’’ science (Barlow et al., 2003; Shultz et al., 2001; Thompson et al., 2004). Lehmann et al. report the dose response of changes in mRNA and protein expression of a group of genes involved in cholesterol transport and steroidogenesis. The suppression of these genes and their protein products is tightly coupled to the observed phthalate-induced decrease in fetal testosterone. This explains why the active phthalates behave like 1 For correspondence via fax: (401) 863-9008. E-mail: Kim_Boekelheide@ brown.edu.