Associations between serum phthalates and biomarkers of reproductive function in 589 adult men
Introduction
The hypothalamic–pituitary–gonadal axis and associated hormones are imperative in the regulation of spermatogenesis. Spermatogenesis may consequently be affected by exogenous compounds if they interfere with the endogenous hormone regulation (Gazouli et al., 2002). The ubiquitous phthalates show anti-androgenic action in animal models and in vitro. They therefore represent a class of chemicals that is suspected to harm male fertility in humans (Borch et al., 2006, Christen et al., 2010, Dalsenter et al., 2006). For example, di-2-ethylhexyl phthalate (DEHP) exposure in the pre- and post-pubertal male rat decreases serum testosterone levels and adversely affects Leydig cell proliferation in the adult rat testis (Li et al., 2012, Noriega et al., 2009), but DEHP has also been found to antagonize the androgen receptor in vitro (Christen et al., 2010, Shen et al., 2009).
Phthalates are di-esters of 1,2-benzenedicarboxylic acid (phthalic acid). They have low water solubility, which decreases with increasing length of the side chain and with higher molecular weight (Lyche et al., 2009). The use of phthalates in industrial production mainly depends on their molecular weight. High molecular weight phthalates (ester side-chain lengths with five or more carbons), such as DEHP and diisononyl phthalate (DiNP), are used as plasticizers in numerous polyvinyl chloride products, such as raincoats and footwear, wall coverings and flooring, food packaging, gloves, toys and medical devices (Kavlock et al., 2002a, Kavlock et al., 2002b, Rock et al., 1986). Low weight phthalates, such as dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP), are used as solvents in personal care products, lacquers, insecticides and coatings (Duty et al., 2005a). Humans are mainly exposed to phthalates through ingestion and dermal contact while inhalation is of minor importance outside the occupational setting (Bradley et al., 2013, Wormuth et al., 2006). The compounds or their metabolites have been detected in urine in more than 95% of the investigated men and women in Western parts of the world (Wittassek et al., 2007).
The biological half-life of phthalates is short and therefore they do not accumulate in the body. A study of a healthy man intentionally ingesting DEHP observed 65–70% urinary excretion 44 h after oral intake (Koch et al., 2004). For DiNP, the urinary excretion was 43.6% after 49 h (Wittassek and Angerer, 2008). The di-ester phthalates are rapidly hydrolyzed into mono-esters during absorption. The mono-esters of the less polar phthalates are further metabolized at different positions in the ester chain by oxidation and hydroxylation into secondary metabolites, which are excreted in the urine as such or after conjugation to glucuronic acid (Hans-Detlev Gilsing et al., 2002, Lyche et al., 2009). The primary metabolite of DEHP, the mono-ester mono-2-ethylhexyl phthalate (MEHP), can be metabolized to, among others, 2-ethyl-5-hydroxy-hexyl phthalate (5OH-MEHP) and 2-ethyl-5-oxyhexyl phthalate (5oxo-MEHP) or 5-carboxy-mono-2-ethylpenty phthalate (5cx-MEPP) (Fig. 1). In humans, urinary levels of the DEHP secondary metabolites 5OH-MEHP and 5oxo-MEHP are several folds higher than the levels of MEHP (Koch et al., 2003). Analysis of these metabolites may therefore increase the analytical sensitivity compared to analysis of MEHP (Koch et al., 2003). Furthermore, the chance of contamination during preparation and analysis of urine samples is greater for MEHP than for the secondary metabolites (Frederiksen et al., 2010, Koch et al., 2003). Finally, most reproductive effects are exerted by the primary- or secondary mono-ester metabolites and not by the di-ester itself (Martino-Andrade and Chahoud, 2010). The metabolism of DiNP is similar to that of DEHP (Fig. 1). Metabolic pathways may impact the toxicity of the phthalate, but only a few studies regarding metabolism and reproductive health exist (Joensen et al., 2012, Meeker et al., 2009).
In most in vitro and rodent studies of phthalate toxicity, the dose-levels are orders of magnitude above environmental levels. Interestingly, low-level dose animal studies have found the same anti-androgenic effects on male rat development (Christiansen et al., 2010). It has been questioned whether it is possible to extrapolate animal data to humans. A recent study of xenografted human or rat fetal testis tissue into castrate male mice treated with DBP or monobutyl phthaltate (a metabolite of DBP) found that contrary to the rat, steroidogenesis of the human fetal testis was not suppressed by DBP (Mitchell et al., 2012). In contrast, an in vitro study based on cultured adult human testis tissue showed that testosterone production was inhibited by DEHP and MEHP (Dsdoits-Lethimonier et al., 2012). Validation studies as well as studies of other phthalates are needed, but these findings indicate that research into the effect of DEHP on testicular function is needed.
The number of epidemiologic studies of male reproductive function and environmental and occupational phthalate exposure has increased during the last few years. Several studies indicate that urinary DEHP metabolites may reduce total and free testosterone levels (Joensen et al., 2012, Meeker et al., 2009, Mendiola et al., 2012, Pan et al., 2006), also decreased estradiol and follicle stimulation hormone (FSH) have been reported (Joensen et al., 2012, Meeker et al., 2009). Studies on semen quality have, however, yielded conflicting results. One study investigating DEHP in semen found significantly negative associations with sperm concentration, normal morphology and motility (Pant et al., 2008), but these results have not been replicated in studies investigating DEHP metabolites in other media (Han et al., 2013, Hauser et al., 2006, Herr et al., 2009, Liu et al., 2012, Toshima et al., 2012). Altogether, these results point towards adverse, although inconsistently so, effects of DEHP and DiNP on reproductive function.
Hypothesizing that DEHP and DiNP metabolites exert anti-androgenic effects in humans, the aim of this study was to assess the associations of semen quality and male reproductive hormones relative to serum concentrations of the phthalates. Second, the aim was to investigate whether the metabolic pathways of DEHP and DiNP influence the effects on semen quality and reproductive hormones.
Section snippets
Study population
The study population and collection of semen and blood samples have previously been described in detail (Specht et al., 2012, Toft et al., 2005). Briefly, between May 2002 and February 2004 a total of 3833 pregnant women and their male partners were invited consecutively at the first routine antenatal care visit at local hospitals in 19 cities and settlements throughout Greenland, a large central hospital in Warsaw, Poland, and from three hospitals and eight antenatal clinics in Kharkiv,
Results
The demographic, seminal and hormonal characteristics of the 589 men from Greenland, Poland and Ukraine with complete data on serum concentrations of the six phthalate metabolites are summarized in Table 1. The men were approximately 30 years of age in Greenland and Poland and 26 years in Ukraine. In Greenland, Poland and Ukraine, 73%, 27% and 67% of the men, respectively, were smokers.
Discussion
In this cross-sectional study of 589 male partners of pregnant women from Greenland, Poland and Ukraine, we summed serum levels of DEHP and DiNP secondary metabolites on a molar basis to obtain a proxy for internal exposure to the mother compounds (Hans-Detlev Gilsing et al., 2002, Koch et al., 2003). We observed inverse associations between serum levels of Proxy-MEHP and Proxy-MiNP metabolites and the serum concentrations of testosterone and SHBG, and semen volume. The inverse associations
Conclusions
Overall, we found that increasing exposure to Proxy-MEHP and some metabolites of DEHP were associated with decreased testosterone levels, semen volume and total sperm count. These findings are indicative of an anti-androgenic action of DEHP. Proxy-MiNP and some DiNP metabolites were found to decrease serum testosterone and SHBG, indicating an androgenic effect of DiNP. Metabolic pathways differed significantly between the three study sites, but without major effect on semen quality or
Conflict of interest statement
The authors have no financial interest or other conflicts of interest in the publication of these results.
The following is the supplementary data related to this article.
Acknowledgments
The study was part of the CLEAR Project: Climate, Environmental Contaminants and Reproductive Health (www.inuendo.dk/clear) supported by the European Commission 7th Framework Programme FP7-ENV-2008-1 Environment (including Climate Change) Grant no.: 226217; the Danish Research Council (10-082745) and the ReproSund and ReproHigh projects.Åsa Amilon, Margareta Maxe and Agneta Kristensen are acknowledged for invaluable help with the phthalate metabolite analyses.
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