ToxSci Advance Access originally published online on June 25, 2008
Toxicological Sciences 2008 105(1):1-4; doi:10.1093/toxsci/kfn123
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
"Additional" Effects of Phthalate Mixtures on Fetal Testosterone Production
MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
For correspondence via. Fax: 44-131-242-6231. E-mail: r.sharpe{at}hrsu.mrc.ac.uk.
Received June 17, 2008; accepted June 17, 2008
The difference between becoming a male rather than a female is about as fundamental as you can get, as it will alter that individual's place in society, transform the shape of his body, reshape his inherent abilities, his thought processes and his behaviors. Whilst it is a constant source of debate and amusement as to whether this "transformation" process represents an improvement or not, when compared with the "set-up" program which would have led to a female, it is becomingly increasingly clear that "making a male" is a rather perilous process. Major disorders of sexual differentiation in genetic males, such as intersex and sex reversal, are rare but milder disorders such as cryptorchidism and hypospadias, are amongst the commonest congenital disorders in humans, affecting 2–9% and 0.3–1% of boys at birth, respectively (Sharpe and Skakkebaek, 2008
). It is accepted that hormones, in particular androgens, play key roles in testis descent and penile development, but it remains largely unknown why cryptorchidism and hypospadias are so common—and perhaps becoming more common. It is increasingly accepted that these two disorders may represent the earliest manifestations of what is termed "testicular dysgenesis syndrome" (TDS) which can also comprise adult-onset disorders such as testicular germ cell cancer and some cases of low sperm counts, and arguably more subtle disorders such as low adult testosterone levels (Sharpe and Skakkebaek, 2008). For all of these disorders, fetal deficiency in androgen production/action is considered an important risk factor.
Though it is the SRY gene that triggers the snowball effect that leads down the male pathway, at the business end it is androgens such as testosterone and dihydrotestosterone that actually cause the body-wide transformation. We have known this for a long time, yet as the mystery about the origins/causes of cryptorchidism and hypospadias illustrate, our understanding of how this actually happens is woeful. For example, the molecular events that mediate androgen-driven masculinization are all but unknown. One explanation for this deficiency may be that we have been looking for these events at the wrong time as, just recently, we have learnt that masculinization of the male reproductive tract can only occur during a relatively narrow, and early, fetal time window, a time that precedes actual morphological differentiation of the reproductive organs. Moreover, cryptorchidism and hypospadias can only arise due to deficient androgen action within this "male programming window" (Welsh et al., 2008). Though this is enlightening and emphasizes the central importance of androgens, it takes us no nearer to understanding why cryptorchidism and hypospadias are so common in boys. In an age of concern about health effects from environmental chemicals, in particular endocrine disruptors, it is not surprising that these have been advocated as potential causes for TDS disorders such as cryptorchidism and hypospadias in humans. This possibility gained considerable momentum from numerous studies in rats showing that exposure of pregnant rats to certain phthalate esters, such as di(n-butyl) phthalate (DBP) or diethylhexyl phthalate (DEHP), could causes a spectrum of cellular changes and reproductive disorders remarkably similar to TDS in humans and also termed "phthalate syndrome" in rats (Foster, 2006). Arguably the most important of these effects was the profound, dose-dependent suppression of testosterone production by the fetal testis—the hormone on which masculinization ultimately depends.
Initially it was reckoned that the high doses of DBP/DEHP used to induce TDS-like disorders in rats were inordinately higher than the likely levels of exposure of humans. However, this reckoning has been eroded considerably by large-scale studies of human exposure, via determination of specific phthalate metabolites in urine, followed by comparison of levels of these metabolites in amniotic fluid in fetal humans (Silva et al., 2004) with those in fetal rats after experimental dosing (Calafat et al., 2006). As a consequence, it is evident that a subsection of highly exposed human fetuses have levels of monobutyl phthalate (the primary metabolite of DBP) that are within fivefold of those associated with induction of reproductive abnormalities in male rat offspring (Calafat et al., 2006). However, the residual comfort of that fivefold difference has now been effectively destroyed by a series of recent studies showing the additive effects of mixtures of certain phthalates or of phthalates with other "antiandrogenic" environmental chemicals in rats (Christiansen et al., 2008; Rider et al., 2008). The largest of these in terms of the number of phthalates used (N = 5) to make up the exposure mixture is that published in the present issue of this journal (Howdeshell et al., 2008). This is also the first of these "mixtures studies" to have directly measured the impact on testosterone levels/production by the fetal testis. The study shows that exposure of pregnant rats to a mixture of five phthalate esters has an additive effect in suppressing steroidogenesis by the fetal rat testis. Such mixtures effects can occur with concentrations at which individual phthalates exert no significant effect or only minor effects, a finding endorsed by other studies using mixtures of various antiandrogenic environmental chemicals rather than just phthalates (Christiansen et al., 2008; Rider et al., 2008). As indicated above, and as shown in various studies using fetal exposure of rats to individual phthalate esters, suppression of fetal testosterone levels is likely to lead to male reproductive disorders if it occurs within the male programming window.
There are several wide-reaching implications of the present, and other, mixtures studies—these extend to risk assessment for man, regulation of phthalates and other "antiandrogenic" chemicals, and the implications for toxicity testing. Some of these issues have been addressed elsewhere in some detail with regard to both estrogenic and antiandrogenic compounds (Kortenkamp et al., 2007).
Risk assessment is largely undertaken on a chemical by chemical basis with little or no account taken of other concurrent exposures, a practice that clearly has to change if account is to be taken of the combined effect of mixtures of similarly acting endocrine disruptors such as phthalates (Kortenkamp et al., 2007). This will require considerable diligence and ingenuity. Daunting though this task may be, the encouragement is that knowledge of the dose-response relationship of an individual chemical, for example of DEHP and its effects on fetal steroidogenesis, clearly predicts its "additive effect." However, this will only be true for compounds that have a similar endpoint action (it does not necessarily matter if the mechanisms of action is different), such as compounds that affect either androgen production (e.g., certain phthalates) or its actions by blocking the androgen receptor (e.g., vinclozolin). Nevertheless, the biggest challenge is that risk assessment for such mixtures requires detailed and accurate information on human exposure for each of the components of the mixture and a reasonable understanding of just how many similarly acting compounds contribute to overall human exposure. Human exposure data is notoriously limited for most environmental chemicals, as illustrated by our lack of knowledge about phthalate exposure up until about 5 years ago, so this will prove an important hurdle to overcome and will require considerable political will in view of the high monetary cost of obtaining such data. Plans to comprehensively evaluate production/environmental chemicals for their endocrine-disrupting activities are already part of the regulatory agenda in the United States and Europe, so there are some grounds for optimism about being able to count the number of chemicals to be taken into account in a "mixtures risk assessment"—although the stumbling block may again be lack of appropriate human exposure data. Furthermore, we should not underestimate the amount (and cost) of work involved in generating detailed dose-response activity profiles for individual compounds, as illustrated in Howdeshell et al. (2008). To be effective, such analyses will probably have to be undertaken in vivo, but if appropriate in vitro systems are available, and can be relied upon (always an issue because of factors such as pharmacokinetics and metabolism), then they would be inordinately quicker and cheaper (Kortenkamp et al., 2007).
One suggestion from the study by Howdeshell et al. (2008) is that testing for masculinization disruption as part of toxicity evaluation or for regulatory purposes could save time and money by simply measuring the effect of compounds on fetal testis testosterone rather than evaluating downstream endpoint disorders such as hypospadias and cryptorchidism. Though this is an attractive idea, it needs to take account of the fact that "not all reductions in fetal testosterone levels are equal." This has only just become apparent from the demonstration of the "male programming window" outlined above, as this has shown that it is only androgen action within this window (which in the rat is within the period e15.5–e18.5) that is critically important for reproductive tract masculinization (Welsh et al., 2008). Androgen action in the final few days of gestation (e19.5–e21.5) appears to be largely immaterial with regard to masculinization of the reproductive tract and yet this is the period within which phthalates such as DBP exert their greatest % suppression of testosterone levels (Shultz et al., 2001; Thompson et al., 2004) whilst suppression within the male programming window is more modest (Scott et al., 2008). This explains why DBP exposure causes only a relatively modest reduction in anogenital distance (AGD) in male rats, as AGD is also determined by androgen action only within the male programming window (Welsh et al., 2008). This suggests that AGD, measured at birth, would be a more informative parameter to measure than suppression of fetal testosterone levels, if the endpoint is to assess disruption of masculinization; however, the sensitivity and discriminatory ability of AGD as an endpoint needs to be evaluated.
In view of the high prevalence of TDS disorders in humans and the relatively high exposure of some individuals in utero to various phthalates, the findings by Howdeshell et al. (2008) are of obvious concern. However, the discussion above as applied to phthalates rests on the assumption that these chemicals (or their metabolites) will exert similar effects on testosterone production by the fetal human testis as by the fetal rat testis. Is this a reasonable assumption? Although the process of steroidogenesis is essentially the same across most species, its detailed regulation and preferred pathways (there is more than one way to make testosterone) can show species differences. This is dramatically illustrated by the finding that DBP does not suppress testosterone production by the fetal mouse testis in vivo as occurs in the rat, even though it will induce other characteristic "phthalate" effects on the testis, such as on germ cells (Gaido et al., 2007). So is the human fetal testis like the mouse or the rat? Two studies using fetal human testis explants have failed to find any effect of monobutyl phthalate (MBP) on testosterone production (Hallmark et al., 2007; Lambrot et al., 2008). However, there must always be uncertainties about placing complete reliance on in vitro studies, especially as several pieces of indirect evidence are consistent with phthalates such as DBP/MBP being able to suppress testosterone production/action in the human or other primates. For example, MBP can suppress testosterone levels acutely in the neonatal marmoset, although at this age this suppression appears to be rapidly compensated for, presumably via elevation of luteinizing hormone (LH) levels (Hallmark et al., 2007). One study (Swan et al., 2005) has shown a negative relationship between human pregnancy exposure to various phthalates and AGD in the resulting male offspring, and it is clear that AGD faithfully reflects androgen action specifically within the male programming window (Welsh et al., 2008). Another study found a similar relationship between phthalate metabolite levels in human breast milk and the LH-testosterone axis in 3-month-old infants, in particular a negative association between MBP levels and free testosterone (Main et al., 2006). However, as MBP also correlated positively with levels of sex hormone binding globulin in this study, it is possible that this explains the negative relationship with free testosterone levels rather than any suppressive effect on testosterone production per se. Alternatively, identification of a positive relationship between MBP exposure and the LH:free testosterone ratio in these infants could be indicative of "compensated" suppression of steroidogenesis as was found experimentally in marmosets (Hallmark et al., 2007).
Any balanced review of the foregoing evidence cannot reach a firm conclusion as to whether or not certain phthalates, alone or in mixtures will, or will not, affect fetal testosterone production in the human. This is a pivotal issue and clearly we must await further data before reaching any conclusion. In particular, follow-up of the study by Swan et al. (2005) will prove vital, as more concrete evidence of a negative relationship between phthalate exposure in pregnancy and reduced AGD in newborn boys, or the absence of any such relationship, appears to be our best hope of proving whether or not phthalate exposure of the human fetus affects the masculinization process and potentially leads to TDS disorders. If the answer is Yes, then it should trigger a comprehensive review of the risk posed to humans by phthalates, a risk assessment that this time will have to take into account not only exposure to multiple phthalates but also coexposure to other environmental chemicals that are "antiandrogenic" via one or more activities. In addition, as the study by Howdeshell et al. (2008) shows via effects on fetal mortality, it also appears that phthalates that affect fetal steroidogenesis may also affect maternal/placental steroidogenesis (leading to pregnancy disruption). This being the case, then there may also be wider concerns about phthalate effects in adult as well as fetal humans. Nevertheless, if phthalates do not affect steroidogenesis in humans as they do in the rat, such concerns may be redundant.
Advocates of the precautionary principle will not be happy to wait for more informative data as to whether or not phthalates affect the human fetus. But at the end of the day what is important is to identify what factors cause cryptorchidism, hypospadias, and other TDS disorders in humans, irrespective of whether these are genetic, lifestyle or environmental chemical exposures. For certain, it will not be just one factor or just one chemical or class of chemical that ultimately proves responsible, as history has shown us that disease etiology is usually complex and interactive. Therefore, at the very best (or worst, depending on how you see it), phthalates, even in mixtures, can only be one of several factors that might contribute causally to TDS disorders in humans, but if they are not involved we need to know so that we look elsewhere for causes.
REFERENCES
Calafat AM, Brock JW, Silva MJ, Gray LE Jr, Reidy JA, Barr DB, Needham LL. Urinary and amniotic fluid levels of phthalate monoesters in rats after oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate. Toxicology (2006) 217:22–30.[CrossRef][Web of Science][Medline]
Christiansen S, Scholze M, Axelsatd M, Boberg J, Kortenkamp A, Hass U. Combined exposure to anti-androgens causes markedly increase frequencies of hypospadias in the rat. Int. J. Androl. (2008) 31:241–248.[CrossRef][Web of Science][Medline]
Foster PMD. Disruption of reproductive development in male rat offspring following in utero exposure to phthalate esters. Int. J. Androl. (2006) 29:140–147.[CrossRef][Web of Science][Medline]
Gaido K, Hensley JB, Liu D, Wallace DG, Borghoff S, Johnson KJ, Hall SJ, Boekelheide K. Fetal mouse phthalate exposure shows that gonocyte multinucleation is not associated with decreased testicular testosterone. Toxicol. Sci. (2007) 97:491–503.
Hallmark N, Walker M, McKinnell C, Mahood IK, Scott HM, Bayne R, Coutts S, Anderson RA, Grieg I, Morris K, et al. Effects of monobutyl and Di(n-butyl) phthalate in vitro on steroidogenesis and Leydig cell aggregation in fetal testis explants form the rat: Comparison with effects in vivo in the fetal rat and neonatal marmoset and in vitro in the human. Environ. Health Perspect. (2007) 115:390–396.[Web of Science][Medline]
Howdeshell KL, Wilson VS, Furr J, Lambright CR, Rider CV, Blystone CR, Hotchkiss AK, Gray LE Jr. A mixture of five phthalate esters inhibits fetal testicular testosterone production in the Sprague Dawley rat in a cumulative, dose additive manner. Toxicol. Sci (2008) April 14, 2008, 10.1093/toxsci/kfn077.
Kortenkamp A, Faust M, Scholze M, Bachaus T. Low-level exposure to multiple chemicals: Reason for human health concerns. Environ. Health Perspect. (2007) 115(Suppl. 1):106–114.[Medline]
Lambrot R, Muczynski V, Coffigny H, Lecureuil C, Pairault C, Angenard G, Frydman R, Habert R, Rouiller-Fabre V. In vitro effects of MEHP on the human fetal testis. Miniposter Book of 15th European Testis Workshop (2008) University of Turku, Naantail, Finland, miniposter S.VII.OP.
Main KM, Mortensen GK, Kaleva MM, Boisen KA, Damgaard IN, Chellakooty M, Schmidt IM, Suomi A-M, Virtanen HE, Petersen JH, et al. Human breast milk contamination with phthalates and alterations of endogenous reproductive hormones in infants three months of age. Environ. Health Perspect. (2006) 114:270–276.[Web of Science][Medline]
Rider CV, Furr J, Wilson VS, Gray LE Jr. A mixture of seven antiandrogens induces reproductive malformations in rats. Int. J. Androl. (2008) 31:249–262.[CrossRef][Web of Science][Medline]
Scott HM, Hutchison GR, Jobling MS, McKinnell C, Drake AJ, Sharpe RM. Relationship between androgen action in the "male programming window", fetal Sertoli cell number and adult testis size in the rat. Endocrinology (2008) June 19, 2008, 10.1210/en.2008-0413.
Sharpe RM, Skakkebaek NE. Testicular dysgenesis syndrome: Mechanistic insights and potential new downstream effects. Fertil. Steril. (2008) 89(Suppl. 1):e33–e38.[CrossRef][Medline]
Shultz VD, Phillips S, Sar M, Foster PMD, Gaido KW. Altered gene profiles in fetal rat testes after in utero exposure to di(n-butyl) phthalate. Toxicol. Sci. (2001) 64:233–242.
Silva MJ, Reidy JA, Herbert AR, Preau JL Jr, Needham LL, Calafat AM. Detection of phthalate metabolites in human amniotic fluid. Bull. Environ. Contam. Toxicol. (2004) 72:1226–1231.[Web of Science][Medline]
Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS, Redmon JB, Ternand CL, Sullivan S, et al. Decrease in anogenital distance among infants with prenatal phthalate exposure. Environ. Health Perspect. (2005) 113:1056–1061.[Web of Science][Medline]
Thompson CJ, Ross SM, Gaido K. Di(n-butyl) phthalate impairs cholesterol transport and steroidogenesis in the fetal rat testis through a rapid and reversible mechanisms. Endocrinology (2004) 145:1227–1237.
Welsh M, Saunders PTK, Fisken M, Scott HM, Hutchison GR, Smith LB, Sharpe RM. Identification in rats of a programming window for reproductive tract masculinization, disruption of which leads to hypospadias and cryptorchidism. J. Clin. Invest. (2008) 118:1479–1490.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||