Cracking the Nut

Kim Boekelheide1

Received June 10, 2004; accepted June 10, 2004

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)Go first described "tubular atrophy and degeneration ... resembling senile changes" (p. 131) in the testis following exposure to di-(2-ethylhexyl) 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., 1995Go), 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., 2000Go; Mylchreest et al., 1998Go; Sharpe et al., 1995Go).

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,p'-DDE, and linuron. Unlike these other antiandrogens, however, phthalate esters produce some unique alterations, such as the generation of multinucleated gonocytes (Mylchreest et al., 2002Go) and do not interact with the androgen receptor.

These observations in animal models have contributed to the conceptualization by Skakkebaek et al. (2001)Go 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., 2003Go; Shultz et al., 2001Go; Thompson et al., 2004Go). 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 anti-androgens without interacting with the androgen receptor. Somewhat surprisingly, there is a coordinate reduction in the expression of these genes and their products occurring at phthalate doses below those that cause physiologic effects on the male reproductive tract, with a lowest observed effect level (LOEL) for decreased fetal testosterone for di-n-butylphthalate in rats of 50 mg/kg.

Although exposure to phthalate esters is universal, critically ill neonates are a particularly vulnerable population who may be infused with 10–20 mg of phthalate diesters per day leached from PVC-based medical devices (Loff et al., 2000Go). Because of these exposure concerns, phthalate esters were the first compounds evaluated several years ago by the newly formed Center for the Evaluation of Risks to Human Reproduction (see http://cerhr.niehs.nih.gov/ for detailed reports evaluating seven phthalate diesters). This group expressed serious concern that exposures to di-(2-ethylhexyl)phthalate in critically ill infants may adversely affect male reproductive tract development.

The work by Lehmann et al. contributes significantly to phthalate ester risk evaluation, and in the process raises important questions for future studies. The di-n-butylphthalate LOEL of 50 mg/kg is easily within an order of magnitude of anticipated phthalate diester exposures in vulnerable human populations. How concerned should we be? Is the observed decrease in rat fetal testosterone an adverse effect or an effect without a physiologic consequence? The exposure model involves repeated daily dosing of the dam for over a week, but how much exposure is needed to produce an effect? This is an important point, and preliminary unpublished observations (Gaido, personal communication) indicate that single high dose exposures of dams on GD 19 to di-n-butylphthalate are sufficient to produce changes within 1 h in the pups' gene expression and testosterone levels. What is the dose response of these acute changes? And will we see them in humans? While these questions do not make the risk assessor's life any easier, they do (or should) help focus research priorities.

These new findings dramatically alter our view of the mechanism of action for phthalate esters. While the Sertoli cell was the consensus target cell for toxicity in young adults, the Leydig cell is where the action is during development. Parsimony forces us to consider that phthalate esters could target the same cell type (Sertoli cell? Leydig cell?) in these two exposure scenarios. If this is the case, then the presumptive unifying molecular target within this cell type must be coupled to an incredibly active and important paracrine signaling system! Any suggestions?

In both young adult and developing animals, phthalate esters act rapidly and specifically to produce their effects. For mechanistic toxicologists, these are great models. However, the possible simplicity of the phthalate mechanism of action has been obscured by the complex biology of the testis itself. This complexity is reinforced by the fact that the young adult and developing animal models of exposure have distinct patterns of toxicity. Truth be told, reproductive biologists and toxicologists would have it no other way, since it is the complex biology of the testis that is most intriguing. Indeed, when the molecular mechanism(s?) of phthalate-induced testicular toxicity is finally revealed, we will know that a nut that has been so hard to crack (pun definitely intended) is finally opening up!

NOTES

1 For correspondence via fax: (401) 863-9008. E-mail: Kim_Boekelheide{at}brown.edu.

REFERENCES

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Gray, L. E., Jr., Ostby, J., Furr, J., Price, M., Veeramachaneni, D. N., and Parks, L. (2000). Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat. Toxicol. Sci. 58, 350–365.[Abstract/Free Full Text]

Jobling, S., Reynolds, T., White, R., Parker, M. G., and Sumpter, J. P. (1995). A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ. Health Perspect. 103, 582–587.[ISI][Medline]

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Shaffer, C. B., Carpenter, C. P., and Smyth, J. H. F. (1945). Acute and subacute toxicity of di(2-ethylhexyl) phthalate with note upon its metabolism. J. Indust. Hyg. Toxicol. 27, 130–135.[ISI]

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Thompson, C. J., Ross, S. M., and Gaido, K. W. (2004). Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanism. Endocrinology 145, 1227–1237.[Abstract/Free Full Text]





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