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Human testis steroidogenesis is inhibited by phthalates
Abstract
Phthalic acid esters are widely used in the manufacture of plastics. Numerous studies have shown that these phthalates impair testicular testosterone production in the rat. However, the scarce and contradictory data concerning humans have cast doubt over whether these compounds are also anti-androgenic in man. We therefore investigated the direct effects of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on organo-cultured adult human testis and a human cell line.
Adult human testis explants or NCI-H295R adrenocortical human cells were cultured with DEHP or MEHP. The effects of ketoconazole, used as a reference molecule, were also assessed.
In both models, DEHP and MEHP significantly inhibited testosterone production. The effects of both phthalates appeared to be specific for steroidogenesis, as INSL3 production by Leydig cells was not altered. Furthermore, the phthalates of interest had no effect on inhibin B production by Sertoli cells or on germ cell apoptosis. As only a small fraction of the phthalates added was found in the testis explants, and as these compounds were found to be metabolized, we estimate that the anti-androgenic effects observed occurred at concentrations of phthalates that are of the same order of magnitude as exposures reported in the literature for men.
We provide the first evidence that DEHP and MEHP can inhibit testosterone production in the adult human testis. This is consistent with recent epidemiological findings of an inverse correlation between exposure to MEHP and testosterone concentrations.
... Since ECS is involved in reproductive function and data about the effect of cannabis consumption on reproductive health are conflicting, our study aimed to determine whether THC and CBD, alone or in a mixture, directly impact testis steroidogenic function and germ cell lineage. To this end, we tested the effect of these cannabinoids in our well-characterized organotypic culture of human adult testis, which is routinely used to identify anti-androgenic compounds [21][22][23][24]. ...
... Immunohistochemistry was performed on PFA 4%-fixed histological sections of explants to observe Leydig cells and estimate the number of testicular cells undergoing apoptosis and proliferation, as previously described [22][23][24]. Briefly, Leydig cells were labeled with a rabbit primary antibody (1:500; Sigma-Aldrich) against the cytochrome P450, family 11, subfamily A, polypeptide 1 (CYP11A1) followed by biotinylated goat anti-rabbit secondary antibody (1:200; Dako) [23]. ...
... This study is the first to investigate the direct impact of the plant-extract cannabinoids CBD and THC, alone or in a combination, on the human adult testis. Using our validated model of human adult testis in organotypic culture [21][22][23][24]26], we investigated whether exposure to cannabinoids used for recreational and therapeutic purposes may directly impact testis functions. We provide evidence that 48 hours or 9 day exposures of testis explants to CBD and THC alone or in a mixture -which duration spans the 1.3 days halflife of cannabis measured in infrequent users [27] for the range of cannabinoid concentrations used -has no major effect on steroidogenic function and germ cell lineage. ...
Purpose:
While an increased risk of developing germ cell tumors has been established in regular cannabis consumers, there is conflicting evidence about an association between cannabis use and testosterone levels in those regular consumers. In this context, we aimed to determine whether Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), the two major and best-studied cannabinoids present in cannabis, also the most used for therapeutic applications, can directly impact the steroidogenic function and germ cell lineage of the human adult testis.
Materials and methods:
We used our well-characterized organotypic culture of human testis, in which adult testis explants were exposed to CBD, THC, or CBD/THC [ratio 1:1] from 10-9 to 10-5 M for up to either 48 hours or 9 days of culture. The testes were obtained from multi-organ donors (n=13; mean age: 55.15±5.62 y).
Results:
The testosterone production and the spatial distribution of Leydig cells did not change upon CBD and/or THC exposure versus controls after 48 hours or 9 days. The overall tissue morphology of the cannabinoids-exposed testis explants was similar to their control upon 24 hours or 9 days of exposure, a finding confirmed by morphometric analyses on short-term cultures. In line, the number of apoptotic cells was not affected by either 48 hours or 9 days of cannabinoids treatment versus mock. Cannabinoids had no impact on the number of proliferating cells nor on mRNA expression of genes encoding proteins involved in germ cell differentiation, meiosis, or Sertoli and Leydig functions after 24 hours exposure.
Conclusions:
Altogether, these findings show an absence of acute direct effects of exposure to cannabis-derived cannabinoids THC and CBD on testicular testosterone production and germ cells ex vivo. Further studies are warranted to explore an indirect impact of cannabinoids on testis functions through the hypothalamic-pituitary-testis axis, as well as the potential effects of long-term exposures.
... Following dewaxing and rehydration, antigen retrieval was performed for all immunostaining by treating the sections with 10 mM citrate buffer (pH 6.0) at 80 °C for 45 min or Tris-Ethylenediaminetetraacetic acid (EDTA buffer (pH 9.0) at 80 °C for 30 min before cooling them at room temperature (RT). Non-specific sites were then blocked with a 10% BSA or chicken serum treatment for 1 h at RT and sections were incubated overnight at 4 °C with the primary antibody diluted in Dako antibody diluent (Dako, Ref [29]. Signal corresponding to the secondary antibody was amplified by an incubation with an avidin and biotinylated enzyme complex (ABC Vectastain Elite Kit, Vector Laboratories). ...
... Using our original and well characterized ex vivo model of human fetal testis [24,25,28,29], we evaluated the effects of the main components of cannabis, CBD, and THC on 8-12 DW testes. This model has already enabled us to identify the toxic effects of environmental compounds visible at the macroscopic level in short-term culture [25,60]. ...
Background
Cannabis consumption by pregnant women continues to increase worldwide, raising concerns about adverse effects on fetal growth and deleterious impacts on the newborn, in connection with evidence of placental transfer of cannabis compound. Cannabis action is mediated by the endocannabinoid system (ECS), which expression is well established in the brain but unknown in the developing testis. The fetal testis, whose endocrine function orchestrates the masculinization of many distant organs, is particularly sensitive to disruption by xenobiotics. In this context, we aimed to determine whether cannabis exposure has the potential to directly impact the human fetal testis.
Methods
We determined the expression of components of the ECS in the human fetal testis from 6 to 17 developmental weeks and assessed the direct effects of phytocannabinoids Δ9-trans-tetrahydrocannabinol (THC) and cannabidiol (CBD) on the testis morphology and cell functions ex vivo.
Results
We demonstrate the presence in the human fetal testis of two key endocannabinoids, 2-arachidonylglycerol (2-AG) and to a lower level anandamide (AEA), as well as a range of enzymes and receptors for the ECS. Ex vivo exposure of first trimester testes to CBD, THC, or CBD/THC [ratio 1:1] at 10⁻⁷ to 10⁻⁵ M altered testosterone secretion by Leydig cells, AMH secretion by Sertoli cells, and impacted testicular cell proliferation and viability as early as 72 h post-exposure. Transcriptomic analysis on 72 h-exposed fetal testis explants revealed 187 differentially expressed genes (DEGs), including genes involved in steroid synthesis and toxic substance response. Depending on the molecules and testis age, highly deleterious effects of phytocannabinoid exposure were observed on testis tissue after 14 days, including Sertoli and germ cell death.
Conclusions
Our study is the first to evidence the presence of the ECS in the human fetal testis and to highlight the potential adverse effect of cannabis consumption by pregnant women onto the development of the male gonad.
... Of all the TEHTM metabolites tested, only the mixture of 2/1-MEHTM monoester metabolites increased the E 2 concentration and decreased the T concentration in a dose-dependent fashion (from 5 µg/ml). Phthalates have been shown to affect human testis steroidogenesis, inhibiting testosterone production Desdoits-Lethimonier et al. (2012) through an increased production of E 2 , and MEHP is known to affect aromatase activity in cultured rat granulosa cells (Lovekamp and Davis 2001). These data could be due to a change in aromatase activity, an enzyme regulated by FSH mediated pathways and androgens, transforming testosterone into estrogens. ...
... The observed effect could be attributed to the presence of TEHTM monoester metabolites in the culture medium as it has been proven that the metabolic pathways are present in H295R cells. (Desdoits-Lethimonier et al. 2012) showed that DEHP was processed into MEHP in human testis explants and H295R cells, with the latter being further oxidized into 5OH-MEHP. ...
Tri-(2-ethylhexyl) trimellitate (TEHTM) is a plasticizer for polyvinyl chloride (PVC) material used in medical devices. It is an alternative to di-(2-ethylhexyl) phthalate (DEHP), a well-known reprotoxic and endocrine disruptor. As plasticizers are known to easily migrate when in contact with fatty biological fluids, patient exposure to TEHTM is highly probable. However, there is currently no data on the potential endocrine-disrupting effects of its human metabolites. To evaluate the effects of TEHTM metabolites on endocrine activity, they were first synthesized and their effects on estrogen, androgen and thyroid receptors, as well as steroid synthesis, were investigated by combining in vitro and in silico approaches. Among the primary metabolites, only 4-MEHTM (4-mono-(2-ethylhexyl) trimellitate) showed agonist activities on ERs and TRs, while three diesters were TR antagonists at non-cytotoxic concentrations. These results were completed by docking experiments which specified the ER and TR isoforms involved. A mixture of 2/1-MEHTM significantly increased the estradiol level and reduced the testosterone level in H295R cell culture supernatants. The oxidized secondary metabolites of TEHTM had no effect on ER, AR, TR receptors or on steroid hormone synthesis. Among the fourteen metabolites, these data showed that two of them (4-MEHTM and 2/1-MEHTM) induced effect on hormonal activities in vitro. However, by comparing the concentrations of the primary metabolites found in human urine with the active concentrations determined in bioassays, it can be suggested that the metabolites will not be active with regard to estrogen, androgen, thyroid receptors and steroidogenesis-mediated effects.
... Furthermore, the expression of genes involved in T biosynthesis, namely P450scc, 3β-HSD, and P450c17, was downregulated, which probably contributes to T deficiency [154]. It seems that the plasticizer DEHP exerts anti-androgenic effects directly onto LCs, inhibiting T synthesis probably through dysfunction of CYP17 [155]. However, the impact of its metabolite, MEHP, is much more pronounced since it inhibited the expression of all steroidogenic enzymes, as well as all T precursors, in both the ∆4 and ∆5 steroidogenic pathways [155]. ...
... It seems that the plasticizer DEHP exerts anti-androgenic effects directly onto LCs, inhibiting T synthesis probably through dysfunction of CYP17 [155]. However, the impact of its metabolite, MEHP, is much more pronounced since it inhibited the expression of all steroidogenic enzymes, as well as all T precursors, in both the ∆4 and ∆5 steroidogenic pathways [155]. The effects of both phthalates appeared to be specific for steroidogenesis since they did not alter the expression of the insulin-like 3 gene, a specific marker of LCs. ...
The current scenario of male infertility is not yet fully elucidated; however, there is increasing evidence that it is associated with the widespread exposure to endocrine-disrupting chemicals (EDCs), and in particular to obesogens. These compounds interfere with hormones involved in the regulation of metabolism and are associated with weight gain, being also able to change the functioning of the male reproductive axis and, consequently, the testicular physiology and metabolism that are pivotal for spermatogenesis. The disruption of these tightly regulated metabolic pathways leads to adverse reproductive outcomes. The permanent exposure to obesogens has raised serious health concerns. Evidence suggests that obesogens are one of the leading causes of the marked decline of male fertility and key players in shaping the future health outcomes not only for those who are directly exposed but also for upcoming generations. In addition to the changes that lead to inefficient functioning of the male gametes, obesogens induce alterations that are “imprinted” on the genes of the male gametes, establishing a link between generations and contributing to the transmission of defects. Unveiling the molecular mechanisms by which obesogens induce toxicity that may end-up in epigenetic modifications is imperative. This review describes and discusses the suggested molecular targets and potential mechanisms for obesogenic–disrupting chemicals and the subsequent effects on male reproductive health.
... NP exposure resulted in an increase of apoptosis in teleost fish gonad (Kaptaner and Ünal, 2011;Sayed et al., 2012). DEHP increased apoptosis in a rodent fetal testis cells model (Muczynski et al., 2012) and exhibited antiandrogenic effects in cultured human testis lines (Desdoits-Lethimonier et al., 2012). FS has not been studied as extensively, but was determined to incite cytotoxicity (Khanavi et al., 2012;Permeh et al., 2012). ...
... Strong interactions with estrogen and androgen systems were demonstrated for BPA (Hatef et al., 2012;Kinch et al., 2015) and NP (Lee et al., 2003). DEHP interacts with androgenic pathways (Desdoits-Lethimonier et al., 2012), as well as with estrogenic systems at higher concentrations (Uren-Webster et al., 2010). BPA, NP and DEHP have been found to interact with thyroid hormone signaling (Ghisari and Bonefeld-Jorgensen, 2009). ...
Endocrine disrupting chemicals mimic or disrupt action of the natural hormones, adversely impacting hormonal function as well as cardiovascular, reproductive, and metabolic health. Goldfish are seasonal breeders with an annual reproductive cycle regulated by neuroendocrine signaling which involves allocation of metabolic energy to sustain growth and reproduction. We hypothesize that seasonal changes in physiology alter overall vulnerability of goldfish to metabolic perturbation induced by environmental contaminants. In this study, we assess effects of endogenous hormones, individual contaminants and their mixture on metabolism of goldfish at different reproductive stages. Exposure effects were assessed using 1H-NMR metabolomics profiling of male goldfish midbrain, gonad and liver harvested during early recrudescence (October), mid-recrudescence (February) and late recrudescence (June). Compounds assessed include bisphenol A, nonylphenol, bis(2-ethylhexyl) phthalate, fucosterol and a tertiary mixture (DEHP + NP + FS). Metabolome-level responses induced by contaminant exposure across tissues and seasons were benchmarked against responses induced by 17β-estradiol, testosterone and thyroid hormone (T3). We observe a clear seasonal dependence to metabolome-level alteration induced by hormone or contaminant exposures, with February (mid-recrudescence) the stage at which male goldfish are most vulnerable to metabolic perturbation. Responses induced by contaminant exposures differed from those induced by the natural hormones in a season-specific manner. Exposure to the tertiary mixture induced a functional gain at the level of biochemical pathways modeling over responses induced by individual components in select tissues and seasons. We demonstrate the importance of seasonally driven changes in physiology altering overall vulnerability of goldfish to metabolic perturbation induced by environmental contaminants, the relevance of which likely extends to other seasonally-breeding species.
... Exposure to phthalates was shown to inhibit the production of testosterone, as well as induce oxidative damage, and insulin resistance in skeletal muscle (Lethimonier et al. 2012;Chen et al. 2013;Chen et al. 2020;Wei et al. 2020). Androgens such as testosterone play an important role in muscle satellite cell proliferation and differentiation, and low level of free testosterone is related with loss of muscle mass (Chen et al. 2005;Yuki et al. 2013). ...
... An in vitro study using luciferase reporter gene assay found phthalate exposure suppressed dihydrotestosterone-stimulated activity of AR (Engel et al. 2017). In addition, phthalates were reported to inhibit testosterone production via downregulating the transcription of steroidogenic enzymes such as steroidogenic acute regulatory (StAR), cholesterol side-chain cleavage enzyme (P450scc), 17α-hydroxylase (CYP17), 3βhydroxycortisol dehydrogenase (3β-HSD) (Lethimonier et al. 2012;Chen et al. 2013;Motoh et al. 2016). Negative association of phthalate exposure with decreased serum total testosterone levels has also been identified in population studies Watkins et al. 2014). ...
Phthalates have been extensively detected in environmental and biological matrices. Exposure to phthalates is implicated in various human diseases. In this study, we conducted a cross-sectional study to determine whether urinary phthalate metabolite concentrations were correlated with prevalence of sarcopenia in US adult population. We included 3562 participants with detailed information on skeletal muscle mass and urinary phthalate metabolites based on National Health and Nutrition Examination Survey (NHANES) 1999–2006 data. A total of 7 main phthalate metabolites were analyzed in the urine sample of each participant. Appendicular skeletal muscle mass (ASM) was measured using dual-energy X-ray absorptiometry. Multivariable linear regression models were conducted following adjustment for multiple covariates. ASM adjusted by body mass index (ASM/BMI) was calculated, and sarcopenia was defined as the lowest quintile for ASM/BMI value. Compared with participants in quartile 1, those in quartile 2 of urinary mono-n-butyl phthalate (MnBP) and quartile 4 of urinary monobenzyl phthalate (MBzP) had decreased ASM/BMI. Urinary MnBP in quartile 4, as well as urinary MBzP in quartile 2, was shown to be significantly correlated with higher sarcopenia prevalence. In subgroup analysis, negative association of MBzP with ASM/BMI was observed in both males and females, while this negative association was only observed in males for MnBP. Females with higher urinary monoethyl phthalate (MEP) concentrations had higher sarcopenia risk. Taken together, the present study found several urinary phthalate metabolites were positively associated with sarcopenia prevalence in US adult population. These findings indicated phthalate exposure might be an important environmental risk factor contributing to sarcopenia development.
... Some phthalates (dicylohexyl phthalate, benzylbutyl phthalate, dipentyl phthalate, diisohexyl phthalate, dihexyl phthalate, and diisoheptyl phthalate) demonstrated both antiandrogenic and weakly estrogenic properties [47]. Phthalates inhibited testosterone biosynthesis in vitro in human testis [48]. In vivo, phthalates can decrease testosterone levels and alter sexual differentiation [49]. ...
Context
Phthalates are ubiquitous endocrine-disrupting chemicals and suspected obesogens. However, the associations with fat distribution and associated cardiometabolic complications remain unclear.
Objective
We examined the associations between phthalate exposure, body fat (total and distribution patterns), and metabolic syndrome (MetS) among US adolescents and adults.
Methods
We analyzed cross-sectional data from 829 adolescents and 3905 adults in the 2013 to 2018 National Health and Nutrition Examination Survey. Total percentage body fat (%BF), visceral adipose tissue (VAT) mass, and android to gynoid (A/G) ratio were determined using dual-energy x-ray absorptiometry. Associations between molar sums of low molecular weight (∑LMW), high molecular weight (∑HMW), and di(2-ethylhexyl) phthalate (∑DEHP) metabolites, and adiposity indicators and MetS were analyzed with multivariable linear and logistic regressions. Models included sex interaction terms, were stratified by age group, and adjusted for relevant covariates.
Results
∑HMW and ∑DEHP exposures were positively associated with %BF in males, and all phthalate groups were associated with greater VAT mass and A/G ratio in adolescent males. Five-fold increases in ∑HMW and ∑DEHP metabolites were associated with 21.7% (95% CI, 10.5-33.9) and 18.0% (95% CI, 7.72-29.2) greater VAT mass among adolescent males, respectively. Sex modified the relationship between ∑HMW exposure and A/G ratio among adolescents (interaction P value = .0185). Phthalates were not associated with odds of MetS. When assessing individual MetS components, phthalates were associated with hyperglycemia in adult males.
Conclusion
Greater exposure to phthalates was associated with greater %BF in all males, and with fat distribution in adolescent males; however, phthalates were not linked to MetS.
... PAEs find application as solvents (mainly low molecular weight) in various industries, such as industrial and cosmetic products (Koniecki et al., 2011). Concerning toxicity, field studies carried out on humans have shown effects on the reproductive system, such as infertility (Rozati et al., 2002;Tranfo et al., 2012), damage to sperm (Rozati et al., 2002), and neurodevelopment effects (Engel et al., 2010;Miodovnik et al., 2011); while in vitro studies have shown anomalies in the reproductive tract in humans (Desdoits-Lethimonier et al., 2012) and in zebrafish (Santangeli et al., 2017). PAEs have also been found to be endocrine disruptors in fish in laboratory studies (Bhatia et al., 2014), and in marine mammals (in the field study of (Routti et al., 2021)). ...
... Another potential mechanism that could explain the sex differences is the antiandrogenic effects of phthalates. Exposure to DEHP metabolites was found to inhibit human testis steroidogenesis (testosterone production) (Desdoits-Lethimonier et al., 2012), and urinary levels associated with low testosterone levels in adult men (Meeker et al., 2009). Low testosterone levels in men, but higher androgen levels in females are associated with higher BMI or greater risk for overweight/obesity and metabolic syndrome (Wang et al., 2011, Barber et al., 2006. ...
... Review transport and metabolism or genes encoding steroidogenic enzymes. 82 In addition, DEHP is a peroxisome proliferator that can produce a lot of reactive oxygen species (ROS) in vivo. 83 Prenatal exposure to DEHP leads to a decrease in antioxidant capacity by reducing riboflavin 84 and biotin 85 metabolism. ...
Di(2-ethylhexyl) phthalate (DEHP), an environmental endocrine disruptor, has hormone-like activity and endocrine-disrupting effects. However, the types of reproductive hormones associated with DEHP vary across the studies. Thus, we conducted a systematic review and meta-analysis to pool existing epidemiological evidence. We searched three databases up to January 31, 2024, for eligible original studies to ultimately include 37 studies from eight countries with a total of 28 911 participants. DEHP exposure was evaluated with urinary metabolites. Since the main types, production sites, blood concentrations, and functions of reproductive hormones differ between men and women, we reported the combined effect values by gender. Subgroup analyses were conducted by age, subfertility status, and the national sociodemographic index (SDI) level. Furthermore, the effect of maternal exposure during pregnancy on children’s reproductive hormone levels was analyzed separately. Overall, in general, in men, DEHP was positively correlated with sex hormone binding-globulin (SHBG) and adversely correlated with total testosterone (TT), free androgen index (FAI), and follicle-stimulating hormone (FSH). Results indicated that among men of reproductive age, DEHP exposure was associated with more significant hormonal suppression in infertile men compared with fertile men. Notably, age subgroup analysis among women revealed that postmenopausal women were more vulnerable to DEHP, which was related to lower TT and estradiol (E2). However, this study did not observe a significant association between prenatal DEHP metabolites and reproductive hormone levels in children. Our research identifies the most susceptible hormones (androgen suppression) after DEHP exposure and suggests that infertile men and postmenopausal women are in great need of more attention as sensitive populations.
... Prolonged DEHP exposure has been demonstrated to cause various health issues, including endocrine disturbances, renal failure, liver dysfunction, and cardiovascular disease Wen et al., 2022;Zhao et al., 2022). Furthermore, since DEHP functions as an environmental hormone, its reproductive toxicity significantly impacts human reproductive function, potentially contributing to the increasing global infertility problem (Cobellis et al., 2003;Desdoits-Lethimonier et al., 2012;Messerlian et al., 2016;Sun et al., 2020). During mammalian spermatogenesis, histone acetylation plays a crucial role in orchestrating the reorganization of germ cell chromatin (Luense et al., 2019). ...
... Epidemiological studies reported an association between phthalates exposure and altered seminal parameters [86]. It is important to note that exposure of infants to phthalates is due to both maternal exposure and breastfeeding. ...
Many andrological pathologies seen in adults, including infertility, actually arose in younger age, due to the strong susceptibility and vulnerability of male gonads to external insults, starting from gestation age and during all growth phases. Three main phases are particularly susceptible for subsequent normal testis development and function: the intrauterine phase, the neonatal phase comprising the so called “minipuberty” and puberty. However, even during infancy, damaging causes with permanent effects on testicular function can occur. Among risk factors for alterations of sexual and reproductive organs and function, endocrine-disrupting chemicals (EDCs) have gained particular attention in last decades, given their ability to disrupt them at different levels and at different ages, with long-term consequences and possibly also transgenerational effects. Bisphenol, phthalates, perfluoroalkyl substances, heavy metals and dioxins are particularly intriguing, given the strong experimental evidence of effects on hormone nuclear receptors, hypothalamus–pituitary–testis axis and direct action on spermatogenesis and steroidogenesis. Although epidemiological studies in humans have shown controversial and inconsistent results, the overall conclusion points towards a positive association between exposure to EDCs and alteration of the reproductive system.
... BPA was also linked to recurrent miscarriage in women, confirmed by the inhibitory CYP19 activities of BPA at >5 μM in different cell lines, viz., human fetal osteoblastic (SV-HFO), ovarian granulosa-like KGN (Sugiura-Ogasawara et al., 2005;Watanabe et al., 2012). Experiments with NCI-H295R human adrenocortical cells and human testis explants confirmed the inhibitory effect of DEHP on testosterone activity (Desdoits-Lethimonier et al., 2012). Certain phthalates like benzyl butyl phthalate (BBP), DBP and DEHP exhibited properties in vitro, such as binding estrogen receptors, weakening estrogenic activities (Harris et al., 1997), and proliferating MCF-7 cells (Okubo et al., 2003). ...
A steep rise in global plastic production and significant discharge of plastic waste are expected in the near future. Plastics pose a threat to the ecosystem and human health through the generation of particulate plastics that act as carriers for other emerging contaminants, and the release of toxic chemical additives. Since plastic additives are not covalently bound, they can freely leach into the environment. Due to their occurrence in various environmental settings, the additives exert significant ecotoxicity. However, only 25% of plastic additives have been characterized for their potential ecological concern. Despite global market statistics highlighting the substantial environmental burden caused by the unrestricted production and use of plastic additives, information on their ecotoxicity remains incomplete. By focusing on the ecological impacts of plastic additives, the present review aims to provide detailed insights into the following aspects: (i) diversity and occurrence in the environment, (ii) leaching from plastic materials, (iii) trophic transfer, (iv) human exposure, (v) risks to ecosystem and human health, and (vi) legal guidelines and mitigation strategies. These insights are of immense value in restricting the
use of toxic additives, searching for eco-friendly alternatives, and establishing or revising guidelines on plastic additives by global health and environmental agencies.
... Our testis model has been extensively used to determine the impact of various biological and chemical agents on testicular morphology and functions, including alterations of testosterone secretion (36,41,56,58,(67)(68)(69). Nevertheless, this model has several limitations regarding testicular pathogenesis: first, we cannot assess the deleterious effect of systemic inflammation and immune cells infiltrating the testis. ...
SARS-CoV-2, the etiological agent of the ongoing Covid-19 pandemic, is a respiratory virus that infects several organs beyond the lungs. The alterations of semen parameters, testicular morphology, and testosteronemia reported in Covid-19 patients, along with the high expression of SARS-CoV-2 main receptor ACE2 in the testis, raise questions about the underlying mechanisms. Using a previously validated ex vivo model of human testis, we show that SARS-CoV-2 infects ACE2-positive Leydig and Sertoli cells. Slow virus replication kinetics were observed, with a peak of infection at day 6 post-infection and a decline of infectious virions at day 9. The infection had no major impact on testicular morphology or main hormonal functions, but some steroidogenic enzymes were decreased at day 9. While antiviral effectors were upregulated, there was no transcriptional induction of key pro-inflammatory cytokines. Altogether, these data indicate that SARS-CoV-2 replication in the human testis ex vivo is limited and suggest that testicular damages in infected individuals are unlikely to result from direct deleterious effects of SARS-CoV-2 on this organ.
IMPORTANCE
SARS-CoV-2 is a new virus responsible for the Covid-19 pandemic. Although SARS-CoV-2 primarily affects the lungs, other organs are infected. Alterations of testosteronemia and spermatozoa motility in infected men have raised questions about testicular infection, along with high level in the testis of ACE2, the main receptor used by SARS-CoV-2 to enter host cells. Using an organotypic culture of human testis, we found that SARS-CoV-2 replicated with slow kinetics in the testis. The virus first targeted testosterone-producing Leydig cells and then germ-cell nursing Sertoli cells. After a peak followed by the upregulation of antiviral effectors, viral replication in the testis decreased and did not induce any major damage to the tissue. Altogether, our data show that SARS-CoV-2 replicates in the human testis to a limited extent and suggest that testicular damages in infected patients are more likely to result from systemic infection and inflammation than from viral replication in the testis.
... Установлено, что фталаты могут являться фактором нарушения фертильности у мужчин, отмечаемого у 40-50 % супружеских пар с бесплодием [24]. Репротоксические эффекты фталатов могут включать про-Пути метаболизма фталатов (адаптировано из [17]) Phthalate metabolism pathways (adapted from [17]) [25,26]. Как EDC фталаты могут вызывать эпигенетические изменения в сперматогенных клетках, которые, в свою очередь, влияют на эпигеном эмбриона, что может приводить к негативным трансгенерационным эффектам у потомства [27]. ...
The paper overviews the negative impact of phthalates on the male reproductive system, spermatogenesis, semen parameters and male fertility. The analysis of the literature revealed few studies devoted to the study of the effect of phthalates on reproductive health, gametogenesis and fertility in humans and laboratory models (rats, mice). Although epidemiological studies on the effect of phthalates on the male reproductive health are not big, some toxicological studies show that some phthalates are potential reprotoxicants.
... Long-term (>3 days) exposure of human semen samples to the metabolite DEHP reduced sperm motility and induced cytotoxicity (Pant et al., 2011). In vitro grown human testes explants showed that DHEP inhibited testosterone synthesis (Desdoits-Lethimonier et al., 2012). ...
Confluence of environmental, genetic, and lifestyle variables is responsible for deterioration of human fecundity. Endocrine disruptors or endocrine disrupting chemicals (EDCs) may be found in a variety of foods, water, air, beverages, and tobacco smoke. It has been demonstrated in experimental investigations that a wide range of endocrine disrupting chemicals have negative effects on human reproductive function. However, evidence on the reproductive consequences of human exposure to endocrine disrupting chemicals is sparse and/or conflicting in the scientific literature. The combined toxicological assessment is a practical method for assessing the hazards of cocktails of chemicals, co-existing in the environment. The current review provides a comprehensive overview of studies emphasizing the combined toxicity of endocrine disrupting chemicals on human reproduction. Endocrine disrupting chemicals interact with each other to disrupt the different endocrine axes, resulting in severe gonadal dysfunctions. Transgenerational epigenetic effects have also been induced in germ cells, mostly through DNA methylation and epimutations. Similarly, after acute or chronic exposure to endocrine disrupting chemicals combinations, increased oxidative stress (OS), elevated antioxidant enzymatic activity, disrupted reproductive cycle, and reduced steroidogenesis are often reported consequences. The article also discusses the concentration addition (CA) and independent action (IA) prediction models, which reveal the importance of various synergistic actions of endocrine disrupting chemicals mixtures. More crucially, this evidence-based study addresses the research limitations and information gaps, as well as particularly presents the future research views on combined endocrine disrupting chemicals toxicity on human reproduction.
... There is also clear evidence that most tumours developed by children are highly related to the exposure of parents to carcinogens (O'Leary et al., 1991, Vinson et al., 2011. Researchers started discovering the dangers of indoor pollution initially from domestic chemicals of common use (Hannon and Flaws, 2015, Goncharov et al., 2009, Desdoits-Lethimonier et al., 2012, Steenland and Winquist, 2020, Sjödin et al., 2008, Calafat et al., 2008, Seachrist et al., 2016, Durando et al., 2007, Ho et al., 2006, Snedeker, 2007, Wilson et al., 2007. However, exposure to outdoor chemicals, mainly those employed in intensive agriculture, and pollutants, particularly from industry, is nowadays widely considered one of the main risk factors for human health (Gilliom et al., 2006, Straub et al., 2007, Authman et al., 2015, Quirós-Alcalá et al., 2011. ...
Tumours are nowadays the second world‑leading cause of death after cardiovascular diseases. During the last decades of cancer research, lifestyle and random/genetic factors have been blamed for cancer mortality, with obesity, sedentary habits, alcoholism, and smoking contributing as supposed major causes. However, there is an emerging consensus that environmental pollution should be considered one of the main triggers. Unfortunately, all this preliminary scientific evidence has not always been followed by governments and institutions, which still fail to pursue research on cancer's environmental connections. In this unprecedented national-scale detailed study, we analyzed the links between cancer mortality, socio-economic factors, and sources of environmental pollution in Italy, both at wider regional and finer provincial scales, with an artificial intelligence approach. Overall, we found that cancer mortality does not have a random or spatial distribution and exceeds the national average mainly when environmental pollution is also higher, despite healthier lifestyle habits. Our machine learning analysis of 35 environmental sources of pollution showed that air quality ranks first for importance concerning the average cancer mortality rate, followed by sites to be reclaimed, urban areas, and motor vehicle density. Moreover, other environmental sources of pollution proved to be relevant for the mortality of some specific cancer types. Given these alarming results, we call for a rearrangement of the priority of cancer research and care that sees the reduction and prevention of environmental contamination as a priority action to put in place in the tough struggle against cancer.
... FGD1 and PAFAB1H1, when treated with 100 μM DEHP for ten days (Hokanson et al., 2006). Exposure to DEHP (at 100 μM-250 μM concentrations) and its metabolic derivative MEHP (at 80 μM-250 μM concentrations) also induced apoptosis in human TK6 lymphoblast cells by altering mitochondrial membrane potential, enhancing ROS production and activating caspase cascade (Desdoits-Lethimonier et al., 2012;Rosado-Berrios et al., 2011). DBP also upregulates the gene expressions of interleukin-Iβ (IL-Iβ) and IL 8 in human thyroid follicular carcinoma FTC-238 cell line and IL6 in a human corneal endothelial B4G12 cell line (Krüger et al., 2012;Song et al., 2012). ...
Phthalates are a family of reprotoxicant compounds, predominantly used as a plasticizer to improve the flexibility and longevity of consumable plastic goods. After their use these plastic products find their way to the waste disposal sites where they leach out the hazardous phthalates present within them, into the surrounding environment, contaminating soil, groundwater resources, and the nearby water bodies. Subsequently, phthalates move into the living system through the food chain and exhibit the well-known phenomenon of biological magnification. Phthalates as a primary pollutant have been classified as 1B reprotoxicants and teratogens by different government authorities and they have thus imposed restrictions on their use. Nevertheless, the release of these compounds in the environment is unabated. Bioremediation has been suggested as one of the ways of mitigating this menace, but studies regarding the field applications of phthalate utilizing microbes for this purpose are limited. Through this review, we endeavor to make a deeper understanding of the cause and concern of the problem and to find out a possible solution to it. The review critically emphasizes the various aspects of phthalates toxicity, including their chemical nature, human health risks, phytoaccumulation and entry into the food chain, microbial role in phthalate degradation processes, and future challenges.
... In addition, PCBs are endocrine-disruptor chemicals (EDCs), a type of contaminant that can affect the endocrine system of animals and humans, even at very low concentrations (ng/L) (Vilela et al., 2018). Some of the main effects of EDCs are reproductive anomalies, infertility, obesity, cancer, diabetes, and problems in hormonal metabolism (Carlsen et al., 1992;Hugo et al., 2008;Casals-Casas & Desvergne, 2011;Pant et al., 2011;Desdoits-Lethimonier et al., 2012;MacLoughlin et al., 2016;Darbre, 2017;Fénichel & Chevalier, 2017;Quagliariello et al., 2017;Rattan et al., 2017;Rochefort, 2017;Kar et al., 2021). ...
Micropollutants (MPs) include a wide range of biological disruptors that can be toxic to wildlife and humans at very low concentrations (<1 μg/L). These mainly anthropogenic pollutants have been widely detected in different areas of the planet, including the deep sea, and have impacts on marine life. Because of this potential toxicity, the global distribution, quantity, incidence, and potential impacts of deep-sea MPs were investigated in a systematic review of the literature. The results showed that MPs have reached different zones of the ocean and are more frequently reported in the Northern Hemisphere, where higher concentrations are found. MPs are also concentrated in depths up to 3000 m, where they are also more frequently studied, but also extend deeper than 10,000 m. Potentially toxic metals (PTMs), polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDTs), organotins, and polycyclic aromatic hydrocarbons (PAHs) were identified as the most prevalent and widely distributed MPs at ≥200 m depth. PTMs are widely distributed in the deep sea in high concentrations; aluminum is the most prevalent up to 3000 m depth, followed by zinc and copper. PCBs, organotins, hexachlorocyclohexanes (HCHs), PAHs, and phenols were detected accumulated in both organisms and environmental samples above legislated thresholds or known toxicity levels. Our assessment indicated that the deep sea can be considered a sink for MPs.
... Results from a previous epidemiological study indicated there was an association between utilization of MEHP in occupational settings and lesser testosterone concentrations (Meeker et al., 2009). In an in vitro study, treatment of human testis explants of reproductively mature men with DEHP or MEHP led to an impaired testosterone secretion, most likely by inhibiting the production of testosterone precursor steroids (Desdoits-Lethimonier et al., 2012). In rats, phthalates were reported to have antiandrogenic activity, resulting in lesser testosterone concentrations and abnormalities in the reproductive tract (Foster et al., 2001;Johnson et al., 2012;Marcoccia et al., 2017) indicating there was an indirect aberrant effect on steroidogenesis. ...
The decreasing trend in human and domestic animal fertility in recent decades has resulted in the question of whether reduced sperm quality is associated with changes in global climate and the environment. Proposed causes for reduced sperm quality include environmental contaminants, which enter into the body of animals through the food chain and are transported to the reproductive tract, where contaminating agents can have effects on fertilization capacities of gametes. In this review, there is a focus on various environmental contaminants and potential effects on male fertility. Human-derived contaminants, particularly endocrine-disrupting phthalates and the pesticide atrazine, are discussed. Naturally occurring toxins are also addressed, in particular mycotoxins such as aflatoxin which can be components in food consumed by humans and animals. Mechanisms by which environmental contaminants reduce male fertility are not clearly defined; however, are apparently multifactorial (i.e., direct and indirect effects) with there being diverse modes of action. Results from studies with humans, rodents and domestic animals indicate there are deleterious effects of contaminants on male gametes at various stages of spermatogenesis (i.e., in the testis) during passage through the epididymis, and in mature spermatozoa, after ejaculation and during capacitation. Considering there is never detection of a single contaminant, this review addresses synergistic or additive effects of combinations of contaminants. There is new evidence highlighted for the long-lasting effects of environmental contaminants on spermatozoa and developing embryos. Understanding the risk associated with environmental contaminants for animal reproduction may lead to new management strategies, thereby improving reproductive processes.
... In rats, in utero exposure to DEHP causes reduced anogenital distance, cryptorchidism, hypospadias, multinucleated germ cells, increased aggregation of Leydig cells, and decreased testosterone production in male offspring [30][31][32][33][34][35]. In humans, exposure of human fetal testis explants in culture to DEHP results in decreased testosterone production [36] and is associated with reduced Leydig cell function [37,38]. In all studies, DEHP toxicity was found to be conferred mainly by MEHP [20,39,40]. ...
Steroid production in Leydig cells is stimulated mainly by the pituitary luteinizing hormone, which leads to increased expression of genes involved in steroidogenesis, including the gene encoding the steroidogenic acute regulatory (STAR) protein. Mono(2-ethylhexyl)phthalate (MEHP), the active metabolite of the widely used plasticizer DEHP, is known to disrupt Leydig steroidogenesis but its mechanisms of action remain poorly understood. We found that MEHP caused a significant reduction in hormone-induced steroid hormone production in two Leydig cell lines, MA-10 and MLTC-1. Consistent with disrupted cholesterol transport, we found that MEHP represses cAMP-induced Star promoter activity. MEHP responsiveness was mapped to the proximal Star promoter, which contains multiple binding sites for several transcription factors. In addition to STAR, we found that MEHP also reduced the levels of ferredoxin reductase, a protein essential for electron transport during steroidogenesis. Finally, we tested new plasticizers as alternatives to phthalates. Two plasticizers, dioctyl succinate and 1,6-hexanediol dibenzoate, had no significant effect on hormone-induced steroidogenesis. Our current findings reveal that MEHP represses steroidogenesis by affecting cholesterol transport and its conversion into pregnenolone. We also found that two novel molecules with desirable plasticizer properties have no impact on Leydig cell steroidogenesis and could be suitable phthalate replacements.
... Although these bisphenols are commonly utilized to make a range of daily household items, little is known about their capacity to influence and disrupt steroidogenic pathways, as well as the method by which these chemicals might interfere with steroidogenic enzyme function. Various endocrine disruptors including BPB, BPF, and BPS can have mixed effects on the endocrine system of animals and humans, by acting as direct agonists or antagonists of steroid receptors, as well as potential inducers or inhibitors of steroidogenic enzymes [5,44,45]. The influence of various quantities of BPB, BPF, and BPS on the steroidogenesis of the human H295R cell line is demonstrated in these in vitro experiments (specifically the production of progesterone and testosterone). ...
In recent years, bisphenol analogues such as bisphenol B (BPB), bisphenol F (BPF), and bisphenol S (BPS) have come to replace bisphenol A (BPA) in food packaging and food containers, since BPA has been shown to leach into food and water, causing numerous negative health effects. Although much information on the endocrine activity of BPA is available, a proper human hazard assessment of analogues that are believed to have a less harmful toxicity profile is lacking. The aim of our in vitro study was to assess the potential effect of bisphenol B, F, and S on the biosynthesis of steroid hormones in human H295R adrenocortical carcinoma cells, using the enzyme-linked immunosorbent assay. In addition, we evaluated mitochondrial activity using the MTT test and viability using triple assay. Adrenocortical carcinoma cells were cultivated for 24 h in the presence of bisphenol B, F, or S (0.1, 0.5, 1, 10, 25, 50, 75, 100 μM). We demonstrated that BPB, BPF, and BPS could affect progesterone and testosterone secretion, as well as affect cell mitochondrial, lysosomal, and metabolic activity, as well as plasma membrane integrity, but considerably more detailed and systematic research is required for a better understanding of risks associated with the effects of bisphenols on steroidogenesis.
... 31 Other studies have also reported that prenatal maternal exposure to phthalates affects the hormonal environment in infants. [32][33][34][35][36] The evidence for an association between phthalates and the incidence of cryptorchidism is disputed. A 3-year prospective study demonstrated that children with cryptorchidism were more common among mothers who were exposed to phthalates during their occupations. ...
Cryptorchidism, the absence of testes from the scrotum, is the most common genital disorder in boys and a risk factor for reduced fertility and testicular cancer. The mechanism responsible for cryptorchidism involves two discrete stages: a transabdominal and an inguinoscrotal phase. These phases of testicular descent are regulated by the prenatal sex hormone environment, including levels of testosterone, insulin‐like factor 3, and calcitonin gene‐related peptide. Environmental endocrine disruptors, which are unfavorable environmental factors, may also affect testicular descent through prenatal sex hormones. This review examined the effects of environmental factors, particularly environmental endocrine disruptors, such as phthalates, organochlorine pesticides, diethylstilbestrol, bisphenol A, dioxins/dioxin‐like compounds, and perfluoroalkyl substances, and parental lifestyles on the risk of cryptorchidism. Although some studies have shown that environmental endocrine disruptors can affect testicular descent by changing the hormonal environment during the prenatal period, no significant association has been established between exposure to environmental endocrine disruptors and the incidence of cryptorchidism. Therefore, the role played by environmental endocrine disruptor exposure (if any) in the pathogenesis of cryptorchidism remains unknown. Further studies are needed to examine these issues.
... For phthalates, di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) exposition on organo-cultured adult human testis did not affect Leydig cell INSL3 concentrations [101]. ...
Endocrine-disrupting chemicals have received significant concern, since they ubiquitously persist in the environment and are able to induce adverse effects on health, and more particularly on reproductive function. Most of the studies focused on nuclear hormone receptors as mediators of sex steroid hormones signaling. However, there are increasing evidences that peptides hormones of the Hypothalamo-Pituitary-Gonadal axis are targets of endocrine-disrupting chemicals (as Gonadotropin-Releasing Hormone, Follicle-Stimulating Hormone, Luteinizing Hormone…). The majority of these hormones act on G protein-coupled membrane receptors. This review summarizes the effects of endocrine-disrupting chemicals on homeostasis of peptides hormone of Hypothalamo-Pituitary-Gonadal axis and on their G protein-coupled membrane receptors signaling revealed by experimental, clinical, and epidemiological studies in human.
Plastic additives are intensively used in the plastic industry. Namely, plasticizers, flame retardants, stabilizers, and antioxidants have raised significant environmental concerns. These additives, characterized by relatively high toxicity and bioaccumulation rates, pose substantial risks to human health. When plastics break down into smaller fragments (i.e., micro/nanoplastics (MNPs)), these additives can be released into aquatic environments, where they may interact with other pollutants through various mechanisms, and multiple factors can affect such interactions. This can influence the occurrence of MNP-associated pollutants in water environments and further impact the environment and human health. Although MNP additives and their associated pollutants pose significant risks, research on their behavior and impacts remains limited. This review maps out the current understanding of MNP additives and associated pollutants, and identifies critical knowledge gaps, setting a foundation for future research directions that will further unravel the complexities of MNPs in water environments and their broader implications.
The improper disposal and degradation of plastics causes the formation and spread of micro and nano-sized plastic particles in the ecosystem. The widespread presence of these micro and nanoplastics leads to their accumulation in the biotic and abiotic components of the environment, thereby affecting the cellular and metabolic functions of organisms. Despite being classified as xenobiotic agents, information about their sources and exposure related to reproductive health is limited. Micro and nano plastic exposure during early developmental stages can cause abnormal embryonic development. It can trigger neurotoxicity and inflammatory responses as well in the developing embryo. In embryonic development, a comprehensive study of their role in pluripotency, gastrulation, and multi-differentiation potential is scarce. Due to ethical concerns associated with the direct use of human embryos, pluripotent cells and its 3D in vitro models (with cell lines) are an alternative source for effective research. Thus, the 3D Embryoid body (EB) model provides a platform for conducting embryotoxicity and multi-differentiation potential research. Pluripotent stem cells such as embryonic and induced pluripotent stem cells derived embryoid bodies (EBs) serve as a robust 3D in vitro model that mimics characteristics similar to that of human embryos. Thus, the 3D EB model provides a platform for conducting embryotoxicity and multi-differentiation potential research. Accordingly, this review discusses the significance of 3D in vitro models in conducting effective embryotoxicity research. Further, we also evaluated the possible sources/routes of microplastic generation and analyzed their surface chemistry and cytotoxic effects reported till date.
It remains unclear whether phthalates are associated with gallstones and whether the associations of phthalate alternatives with gallstones are different from traditional phthalates. In this study, 1735 participants from the NHANES 2017–2018 were included and their urine was used to detect phthalate metabolites. We used logistic and restricted cubic spline regressions to assess individual associations and dose–response relationships between phthalate metabolites and gallstones, quantile g-computation and Bayesian kernel machine regression to assess mixed associations of phthalate metabolites with gallstones, and subgroup analyses to explore potential effect modifiers. We observed that individual associations of cyclohexane-1,2-dicarboxylic acid-mono(carboxyoctyl) ester phthalate (MCOCHP) (OR: 1.423, 95% CI: 1.098–1.844) and cyclohexane 1,2-dicarboxylic acid monohydroxy isononyl ester (MHNCH) (OR: 1.380, 95% CI: 1.080–1.763) with gallstones were linearly positive, mixed association of phthalate metabolites (OR: 2.453, 95% CI: 1.054–5.708) with gallstones was also positive, and MCOCHP and MHNCH had positive weights. The associations of phthalate metabolites with gallstones were higher in the males and participants with age ≥ 60 years, BMI ≥ 30 kg/m², hypertension, and diabetes. MCOCHP and MHNCH had the highest groupPIP (groupPIP: 0.941), mono(2-ethyl-5-carboxypentyl) terephthalate (MECPTP) had the highest condPIP (condPIP: 0.721), and mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), MCOCHP, and mono-isobutyl phthalate (MiBP) also had high condPIPs (condPIP > 0.5). The trend in mixed associations of phthalate metabolites with gallstones was positive. Our study suggests that traditional phthalates and phthalate alternatives are associated with gallstones, especially in the elderly, men, obese, hypertensive populations, and diabetic populations, and phthalate alternatives are associated with a higher risk of gallstones.
Background
Di(2-ethylhexyl) phthalate (DEHP) is a commonly used plasticizer known for its toxic effects on the male reproductive system. Green tea polyphenols (GTPs), recognized for their antioxidant and anti-inflammatory properties, have demonstrated protective effects on various organs, but the mechanisms by which GTPs mitigate DEHP-induced testicular damage remain unclear.
Methods
Healthy male C57BL/6 J mice were divided into five groups: Control, DEHP, DEHP + GTP treatment, GTP, and Oil groups. Testicular histopathological changes were assessed using hematoxylin–eosin (H&E), periodic acid-Schiff (PAS), and Masson staining. Ultrastructural alterations were examined through transmission electron microscopy. High-throughput sequencing was performed to analyze the expression of mRNA, miRNA, and lncRNA, and to construct an lncRNA–miRNA–mRNA regulatory network for identifying key regulatory axes.
Results
Mice in the DEHP group exhibited significant testicular damage, including reduced sperm count, mitochondrial deformation, and endoplasmic reticulum dilation. GTP treatment notably improved testicular structural integrity, restored sperm count, and alleviated mitochondrial and endoplasmic reticulum damage. Additionally, DEHP significantly increased activated CD8+ T cells, which were reduced with GTP treatment. High-throughput sequencing revealed that GTP treatment exerted protective effects through the regulation of six key lncRNA–miRNA–mRNA axes.
Conclusion
GTPs significantly protect against DEHP-induced testicular damage, and the lncRNA–miRNA–mRNA regulatory axes play a potential role in this process.
Obesity in children remains a major public health problem, with the current prevalence in youth ages 2–19 years estimated to be 19.7%. Despite progress in identifying risk factors, current models do not accurately predict development of obesity in early childhood. There is also substantial individual variability in response to a given intervention that is not well understood. On April 29–30, 2021, the National Institutes of Health convened a virtual workshop on “Understanding Risk and Causal Mechanisms for Developing Obesity in Infants and Young Children.” The workshop brought together scientists from diverse disciplines to discuss (1) what is known regarding epidemiology and underlying biological and behavioral mechanisms for rapid weight gain and development of obesity and (2) what new approaches can improve risk prediction and gain novel insights into causes of obesity in early life. Participants identified gaps and opportunities for future research to advance understanding of risk and underlying mechanisms for development of obesity in early life. It was emphasized that future studies will require multi‐disciplinary efforts across basic, behavioral, and clinical sciences. An exposome framework is needed to elucidate how behavioral, biological, and environmental risk factors interact. Use of novel statistical methods may provide greater insights into causal mechanisms.
Background
Phthalates are ubiquitous anti-androgenic endocrine disrupting chemicals found in personal care products, medications, and many plastics. Studies have shown a racial disparity in phthalates exposure among U.S. women, which may also impact fetal development.
Methods
We conducted a prospective cohort study of gestational exposure to a phthalates mixture in a racially-diverse population to determine their association with genital development. Mid-gestation (18–22 weeks) urine was collected from 152 women who self-identified as non-Hispanic Black and 158 women who self-identified as non-Hispanic White in Charleston, South Carolina between 2011 and 2014. We measured eight phthalate monoester metabolites in urine using liquid chromatography tandem-mass spectrometry. Mid-gestational penile dimensions were measured using ultrasound and anogenital distances were measured postnatally. We used Bayesian kernel machine regression to estimate the associations among the mixture of phthalate metabolites and mid-gestation penile dimensions and postnatal anogenital distance measures among singleton male (n = 179) and female (n = 131) infants, adjusted for urinary specific gravity, maternal age, body mass index, education level, cigarette smoking, and gestational age at enrollment or birth weight z-score.
Results
We found a stronger association between greater phthalates and decreased anopenile distance among infants born to women who self-identified as Black. Mono (2-ethylhexyl) phthalate (MEHP) was the driving mixture component among Black women, and monobutyl phthalate (MBP) and monoethyl phthalate (MEP) were drivers among White women. We also identified a non-linear association between phthalates and lesser ultrasound penile volume among women who self-identified as Black with monoisobutyl phthalate (MiBP) and MBP being most important. We also found an association between greater phthalates and shorter anoclitoral distance among infants born to women who self-identified as Black, with MEP and monobenzyl phthalate (MBzP) contributing most to this association.
Conclusion
Our results suggest a disparity in the association between gestational exposure to a mixture of phthalates and fetal genital development among women who self-identified as Black compared to White.
Background:
Early onset of puberty could have significant impacts on childhood health, but the extent to which it was affected by phthalate esters (PAEs) and sex hormone disruption was not understood. The aim of this study is to investigate the associations between exposure to PAEs and sex hormone disruption and early onset of puberty in children.
Methods:
A longitudinal cohort study was conducted in China from May 2017 to Oct 2020, involving 740 children during consecutive visits. The onset of puberty was evaluated using Tanner definition, and early puberty was defined as an onset age less than the first 25 %, with cut-offs of 10.33 and 8.97 years for boys and girls, respectively. Serum testosterone (TT), estradiol (E2) and urinary PAE metabolites were measured during three visits. Generalized linear models were used to explore the associations between PAE and sex hormones with the age of puberty onset, while log-binomial regressions were applied to assess the associations of persistent exposure to PAEs and sex hormones with early pubertal onset.
Results:
Approximately 86.0 % of boys and 90.2 % of girls completed puberty onset from pre-puberty, and more than 95 % of participants had PAE concentrations higher than the limit of detection. Boys showed higher exposure to PAE pollutants and higher TT levels. Persistent exposure to PAEs was positively associated with early pubertal onset in girls (ARR = 1.97, 95 %CI = 1.12, 3.46). Moreover, persistent exposure to PAEs and E2 had synergistic associations with early pubertal onset in both boys (ARR = 4.77, 95 %CI = 1.06, 21.54) and girls (ARR = 7.07, 95 %CI = 1.51, 33.10). However, PAEs and TT had antagonistic associations only in boys (ARR = 0.44, 95 %CI = 0.07, 2.58).
Conclusion:
Long-term exposure to PAEs might increase the risk of early pubertal onset, and it appears to work in synergy with E2, while in antagonism with TT in boys' early pubertal onset. Reducing PAEs exposure might promote pubertal health.
Di (2-ethyl hexyl) Phthalate (DEHP) is one of the plasticizers widely used in the manufacturing of plastics to make it flexible and durable. Present study is focussed to observe the deleterious effects of DEHP on male reproductive system of animals. For this, 1000 mg/kg body wt. of DEHP was administered to different groups of male Wistar rat for 2, 4, 6 and 8 weeks. After each interval, rats were sacrificed and histological alterations in testis of rats were observed. On hormonal assay, testosterone level decreased significantly in DEHP exposed groups. The histological structure of the testis was also observed to be disrupted significantly with increasing duration of DEHP exposure. Organisation of seminiferous tubule was found distorted and disoriented showing large gaps between them along with degenerated epithelium. Evident changes in morphology of spermatozoa were seen with gradual loss of head and tail structure. Decrease in the number of Leydig cells and sertoli cells were also found suggesting DEHP as a potent toxicant for male reproductive system.
Many andrological pathologies seen in adults, including infertility, actually arose in younger age, due to the strong susceptibility and vulnerability of male gonad to external insults, starting from gestation age and during all growth phases. Three main phases are particularly susceptible for subsequent normal testis development and function: the intrauterine phase, the neonatal phase comprising the so called minipuberty, and puberty. However, even during infancy, when the testes are apparently “sleeping,” damaging causes with permanent effects on testicular function can occur. Among risk factors for alterations of sexual and reproductive organs and function, endocrine-disrupting chemicals (EDCs) have gained particular attention in last decades, given their ability to disrupt them at different levels and at different ages, with long-term consequences and possibly also transgenerational effects. Bisphenol, phthalates, perfluoroalkyl substances, heavy metals, and dioxins are particularly intriguing, given the strong experimental evidence of effects on hormone nuclear receptors, hypothalamus-pituitary-testis axis, and direct action on spermatogenesis and steroidogenesis. Although epidemiological studies in humans have shown controversial and inconsistent results, the overall conclusion points toward a positive association between exposure to EDCs and alteration of the reproductive system.
Background
The structural similarity between sex hormones and exogenous phthalates (PAEs) enabled them as disrupters in regulating childhood blood pressure (BP). We aim to explore the association of sex hormones homeostasis and PAEs metabolites with childhood high BP (HBP).
Methods
A cohort study was conducted with 1416 children aged 7-13 years at baseline and with 824, 819, and 801 children completing three waves' follow up. Serum testosterone (TT) and estradiol (E2) in children during three consecutive waves of surveys were measured by radioimmunoassay, and then TT/E2 ratio calculated as TT divided by E2 were used to represent sex hormones homeostasis. Seven urinary PAEs metabolites were measured in children of first wave. The BP Z-Scores and HBP across waves were obtained by sex, age, and height specific percentiles. Log-binomial regression models with adjusted risk ratios (aRR) after adjusting for confounders were utilized.
Results
The prevalence of HBP at the baseline survey was 25.5%, and increased from 26.3% in the first wave of survey to 35.0% in the third wave of survey. PAEs were negatively correlated with E2, while positively correlated with TT and TT/E2 ratio. A positive association of the serum TT levels, TT/E2 ratio, and total PAEs was found with HBP prevalence (in wave 1, 2 and 3 with TT (aRR): 1.63, 1.37 and 1.45; with TT/E2: 1.63, 1.42 and 1.20; with PAEs: 1.40, 1.32 and 1.32), persistent HBP (with TT (aRR): 2.19; TT/E2: 2.16; PAEs: 2.57), occasional HBP (with TT (aRR): 1.94; TT/E2: 1.72; PAEs: 1.38), and new HBP incidence (with TT (aRR): 1.44; TT/E2: 1.57; PAEs: 1.67), but E2 had a negative association with HBP phenotypes (HBP prevalence in wave 1, 2 and 3 (aRR): 0.77, 0.93, and 1.10; persistent HBP: 0.47; occasional HBP: 0.96; new HBP incidence: 0.81). The E2 and PAEs had antagonistic effects on HBP risks in children, particularly in girls and those with high BMI group, but the TT levels, TT/E2 ratio and PAEs had synergistic effects on HBP risks in children, particularly in boys and those with high BMI group.
Conclusion
Exogenous PAEs exposure and endogenous sex hormones homeostasis disruption independently increase the risks of HBP. Moreover, the exogenous PAEs exposure could disrupt the endogenous sex hormones homeostasis in children, thereby combinedly increased risks of childhood HBP.
Di-(2-ethylhexyl) phthalate (DEHP) is the world's most widely used polyvinyl chloride (PVC) plasticizer and is used in virtually every category of flexible PVC. In fact, DEHP is extensively used in food cosmetics and medical packaging. It has become a serious problem in recent years. DEHP can be absorbed into the human body through the air, food, water, and skin. The current study involved intraperitoneal injection of DEHP dissolved in corn oil once daily for 21 consecutive days to investigate the effects of DEHP on the thyroid and the reproductive system in female rats. Results show that ovarian hormones (progesterone and estrogen) decreased significantly in the rats treated with DEHP compared to control. This result is supported by the alteration of folliculogenesis, the decrease of the follicles viability, and the apoptosis of the granulosa cells observed on histological sections of ovary and thyroid in female rats exposed to low doses of DEHP. Histopathological study revealed that DEHP could damage thyroid tissue and disrupt these functions. We also observed cellular damage, particularly in the liver cells, and a significant increase in biochemical parameters such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared to the control group.
HIGHLIGHTS
Occurrence of contaminants di-(2-ethylhexyl) phthalate (DEHP) is an emerging concern.;
DHEP can disrupt reproductive function, thyroid, and hepatic synthesis.;
In vivo toxicity of DEHP.;
This study investigated the effects of exposure to DEHP on the thyroid and the reproductive system in female rats.;
Environmental pollution with phthalates.;
This study investigated the efficacy of chewable tablets made from the Pleurotus djamor zinc polysaccharide (PCT‐Zn) against the nonylphenol (NPs) induced male reproductive damage in mice. The trial took the form of constructing a model by infusing NPs into mice, followed by treatment with PCT‐Zn solution infusion one hour later, for a total of eight weeks. It was found that PCT‐Zn showed potential effects on increasing the testicular coefficients, enhancing the antioxidant activities, and inhibiting the apoptotic and inflammatory responses, which indicated that PCT‐Zn had the effect of alleviating the NPs‐induced reproductive damages. The results of the experiment suggested that the Pleurotus djamor zinc polysaccharide could be used as a dietary supplement to develop appropriate products to alleviate reproductive damage caused by environmental pollutants. This article is protected by copyright. All rights reserved
Exposure to phthalates (PAEs), phenols, and parabens has been linked with sex hormone imbalance; however, previous studies were predominantly limited to adults and failed to examine the combined effects of these chemicals mixture among adolescents. Thus, we used the data from the National Health and Nutrition Examination Survey (2013-2016) to explore the associations of urinary PAEs, phenols, and parabens biomarkers with sex hormones among participants aged 12-19 years old (n = 613). Latent class analysis (LCA) and quantile-based g-computation (QGC) were applied to assess the associations of the latent exposure profiles and chemicals mixture with sex hormone indicators, including steroid hormones and sex hormone binding globulin (SHBG), in adolescents and by sex. Using LCA, four latent classes were identified among all participants. Compared with the class characterized by "Low exposure", the class represented by "High PAEs" [mono (2-ethyl-5-carboxypentyl) phthalate (MECPP), mono (2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and monobenzyl phthalate (MBZP)] had lower level of estradiol (E2) [β = -0.249, 95% confidence interval (CI): -0.419, -0.08], free androgen index (FAI) (β = -0.258, 95%CI: -0.512, -0.005) and free testosterone (FT) (β = -0.248, 95%CI: -0.496, -0.001) among male adolescents. These results were echoed by the results in QGC analyses, where PAEs mixture was negatively associated with E2 (β = -0.137, 95% CI: -0.263, -0.011), FAI (β = -0.198, 95%CI: -0.387, -0.008) and FT (β = -0.189, 95%CI: -0.375, -0.002) among male adolescents. By contrast, the associations of the identified latent classes or chemicals mixture with sex hormone indicators were generally nonsignificant among female counterparts, except for a positive association between PAEs mixture and SHBG (β = 0.121, 95%CI: 0.012, 0.23). Our study demonstrated that exposure to PAEs, particularly MECPP, MEHHP, and MBZP, would be a threat to the sex hormone homeostasis of male adolescents.
Phthalate esters (PAEs) as hazardous air pollutants can be easily released during the life cycle of plastic products. In this study, a thermal desorption aerosol comprehensive two-dimensional gas chromatography mass spectrometer coupled with a dual-trap was developed and used to measure the hourly-resolved PAEs characteristics in atmospheric PM2.5 at an urban site. Dimethyl phthalate (DMP), diethyl (DEP), dibutyl (DnBP), benzyl butyl (BBP), di(2-ethylhexyl) (DEHP), and di-n-octyl phthalate (DnOP) in PM2.5 were analyzed. The most abundant compounds were DEHP and DMP, followed by DnBP and DEP. The mass concentrations of the detected PAEs are comparable to those at other urban sites measured using offline methods with a lower time resolution. The concentrations of PAEs showed intense change with the variation in PM2.5 mass concentration. The proportion of DEHP increased while that of DMP decreased with the increase in PM2.5 pollution. Positive correlations between PAEs and PM2.5, organic carbon, and elemental carbon were observed, while PAEs had negative correlation with the ambient temperature. Our observation provides evidences on understanding the volatile and semi-volatile PAEs in the ambient aerosols.
Many andrological pathologies seen in adults, including infertility, actually arose in younger age, due to the strong susceptibility and vulnerability of male gonads to external insults, starting from gestational age and during all growth phases. Three main phases are particularly susceptible for normal testis development and function: the intrauterine phase, the neonatal phase comprising the so-called minipuberty, and puberty. However, even during infancy, when the testes are apparently “sleeping,” damaging causes with permanent effects on testicular function can occur. Among risk factors for alterations of sexual and reproductive organs and function, endocrine-disrupting chemicals (EDCs) have gained particular attention in the last decades, given their ability to disrupt them at different levels and at different ages, with long-term consequences and possibly also transgenerational effects. Bisphenol A, phthalates, and perfluoroalkyl substances are particularly intriguing chemicals, given the strong experimental evidence suggesting effects on hormone nuclear receptors, hypothalamus-pituitary-testis axis, and direct action on spermatogenesis and steroidogenesis. Although epidemiological studies in humans have shown controversial and inconsistent results, the overall conclusion points toward a positive association between exposure to EDCs and alteration of male reproductive system.
Context
Accelerating evidence of endocrine-related morbidity has raised alarm about the ubiquitous use of phthalates in the human environment, but studies have not directly evaluated mortality in relation to these exposures.
Objectives
To evaluate associations of phthalate exposure with mortality, and quantify attributable mortality and lost economic productivity in 2013–4 among 55–64 year olds.
Design
This nationally representative cohort study included 5303 adults aged 20 years or older who participated in the US National Health and Nutrition Examination Survey 2001–2010 and provided urine samples for phthalate metabolite measurements. Participants were linked to mortality data from survey date through December 31, 2015. Data analyses were conducted in July 2020.
Main Outcome Measures
Mortality from all causes, cardiovascular disease, and cancer.
Results
Multivariable models identified increased mortality in relation to high-molecular weight (HMW) phthalate metabolites, especially those of di-2-ethylhexylphthalate (DEHP). Hazard ratios (HR) for continuous HMW and DEHP metabolites were 1.14 (95% CI 1.06–1.23) and 1.10 (95% CI 1.03–1.19), respectively, with consistently higher mortality in the third tertile (1.48, 95% CI 1.19–1.86; and 1.42, 95% CI 1.13–1.78). Cardiovascular mortality was significantly increased in relation to a prominent DEHP metabolite, mono-(2-ethyl-5-oxohexyl)phthalate. Extrapolating to the population of 55–64 year old Americans, we identified 90,761–107,283 attributable deaths and 39 billion/year or more. While further studies are needed to corroborate observations and identify mechanisms, regulatory action is urgently needed.
The male reproductive system is exposed to a great number of chemical substances which can interfere with the normal hormonal milieu and reproductive function; these are called endocrine disruptors (EDs). Despite a growing number of studies evaluating the negative effects of EDs, their production is continuously growing although some of which have been prohibited. The prevalence of poor semen quality, hypospadias, cryptorchidism, and testicular cancer have increased in the last decades, and recently, it has been postulated that these could all be part of a unique syndrome called testicular dysgenesis syndrome. This syndrome could be related to exposure to a number of EDs which cause imbalances in the hormonal milieu and oestrogenic over-exposure during the foetal stage. The same EDs can also impair spermatogenesis in offspring and have epigenetic effects. Although studies on animal and in vitro models have raised concerns, data are conflicting. However, these studies must be considered as the basis for future research to promote male reproductive health.
While definitions vary, endocrine-disrupting chemicals (EDCs) have two fundamental features: their disruption of hormone function and their contribution to disease and disability. The unique vulnerability of children to low-level EDC exposures has eroded the notion that only the dose makes the thing a poison, requiring a paradigm shift in scientific and policy practice. In this review, we discuss the unique vulnerability of children as early as fetal life and provide an overview of epidemiological studies on programming effects of EDCs on neuronal, metabolic, and immune pathways as well as on endocrine, reproductive, and renal systems. Building on this accumulating evidence, we dispel and address existing myths about the health effects of EDCs with examples from child health research. Finally, we provide a list of effective actions to reduce exposure, and subsequent harm that are applicable to individuals, communities, and policy-makers.
Expected final online publication date for the Annual Review of Pharmacology and Toxicology, Volume 62 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
Besides our current health concerns due to COVID-19, cancer is a longer-lasting and even more dramatic pandemic that affects almost a third of the human population worldwide. Most of the emphasis on its causes have been posed on genetic predisposition, chance, and wrong lifestyles (mainly, obesity and smoking). Moreover, our medical weapons against cancers have not improved too much during the last century, although research is in progress. Once diagnosed with a malignant tumour, we still rely on surgery, radiotherapy, and chemotherapy. The main problem is that we have focused on fighting a difficult battle instead of preventing it by controlling its triggers. Quite the opposite, our knowledge of the links between environmental pollution and cancer has surged from the 1980s. Carcinogens in water, air, and soil have continued to accumulate disproportionally and grow in number and dose, bringing us to today’s carnage. Here, a synthesis and critical review of the state of the knowledge of the links between cancer and environmental pollution in the three environmental compartments is provided, research gaps are briefly discussed, and some future directions are indicated. New evidence suggests that it is relevant to take into account not only the dose but also the time when we are exposed to carcinogens. The review ends by stressing that more dedication should be put into studying the environmental causes of cancers to prevent and avoid curing them, that the precautionary approach towards environmental pollutants must be much more reactionary, and that there is an urgent need to leave behind the outdated petrochemical-based industry and goods production.
Objectives
Di-2-ethylhexyl phthalate (DEHP) is ubiquitous, known as an endocrine disruptor. DEHP is a widespread prevalence in general and occupational populations which raised great public concerns due to its potentially harmful health effects on the male reproductive system. We aimed to assess occupational levels of DEHP on gonadotropin and gonadal hormones including luteinizing hormone (LH), follicle-stimulating hormone (FSH), total testosterone (TT), and sex hormone binding globulin (SHBG) and evaluate its potential effects on Asp327Asn polymorphisms SHBG gene.
Methods
We measured the levels of DEHP of 90 male workers in one of polyvinyl chloride (PVC) industry plant using enzyme-linked immunosorbent assay. Sex hormones were examined and Asp327Asn polymorphisms SHBG gene were detected by PCR-RFLP in all participants.
Results
The workers were divided into low- and high- DEHP exposed groups based on the geometric mean (GM) levels (183.86 U/L) in serum. TT and TT: LH ratio were negatively correlated to DEHP levels (r=−0.213, p=0.038), (r=−0.225, p=0.027), respectively. The linear regression analysis revealed that a 10-fold increase of serum DEHP was found to be associated with 2.07 fold decreased in TT and a 2.26 fold decreased in TT/LH ratio .
Conclusions
Serum testosterone is negatively associated with DEHP exposure in occupational workers.
Phthalates belong to the endocrine-disrupting chemicals, altering the hormonal balance in humans during pregnancy with further effects on the reproductive system. This study aimed to investigate the associations between maternal hormone levels during early pregnancy (≤15th week of pregnancy) and reproductive markers in infant boys (n = 37; 61.67%; average age 3.51 ± 0.73 months) and girls (n = 23; 38.33%; average age 3.30 ± 0.33 months) concerning prenatal exposure to phthalates. We used high-performance liquid chromatography, tandem mass spectrometry (HPLC-MS/MS), and electro-chemiluminescence immunoassay to quantify urinary concentrations of phthalates and serum concentrations of hormones, respectively. In Mother-Infant Study Cohort (PRENATAL), we observed positive and negative correlations between infants' reproductive markers and phthalate metabolites (p ≤ 0.05). Next, we noticed associations between the penile length and maternal testosterone (β = 0.464) and estradiol levels (β= -0.365) with increasing significance after adjustment to maternal mono-n-butyl phthalate (MnBP) and monobenzyl phthalate (MBzP) (p ≤ 0.05). We observed a positive association (β = 0.337) between penile width and maternal testosterone with increasing significance after adjustment to maternal mono-iso-butyl phthalate (MiBP) (p ≤ 0.05). In a group of girls, we reported a negative association between ACD/AFD ratio and maternal follicle-stimulating hormone (FSH) and estradiol levels with increasing significance after adjustment to maternal monoethyl phthalate (MEP), MnBP, and mono(hydroxy-iso-butyl) phthalate (OH-MiBP). Our results highlight that prenatal phthalate exposure may modulate the effects of maternal hormone levels during early pregnancy on infants' reproductive markers.
Internal plasticization of poly(vinyl chloride) (PVC) was achieved in one‐step using copper‐mediated atom transfer radical polymerization to graft different ratios of random n‐butyl acrylate and 2–2‐(2‐ethoxyethoxy)ethyl acrylate copolymers from defect sites on the PVC chain. Five graft polymers were made with different ratios of poly(butyl acrylate) (PBA) and poly(2–2‐(2‐ethoxyethoxy)ethyl acrylate) (P2EEA); the glass transition temperatures (Tg) of functionalized PVC polymers range from − 25 to − 50°C. Single Tg values were observed for all polymers, indicating good compatibility between PVC and grafted chains, with no evidence of microphase separation. Plasticization efficiency is higher for polyether P2EEA moieties compared with PBA components. The resultant PVC graft copolymers are thermally more stable compared to unmodified PVC. Increasing the reaction scale from 2 to 14 g produces consistent and reproducible results, suggesting this method could be applicable on an industrial scale.
Exposure to phthalates in utero alters fetal rat testis gene expression and testosterone production, but much remains to be done to understand the mechanisms underlying the direct action of phthalate within the fetal testis.
We aimed to investigate the direct mechanisms of action of mono-(2-ethylhexyl) phthalate (MEHP) on the rat fetal testis, focusing on Leydig cell steroidogenesis in particular.
We used an in vitro system based on the culture for three days, with or without MEHP, of rat fetal testes obtained at 14.5 days post-coitum.
Exposure to MEHP led to a dose-dependent decrease in testosterone production. Moreover, the production of 5 alpha-dihydrotestosterone (5α-DHT) (−68%) and androstenedione (−54%) was also inhibited by 10 µM MEHP, whereas 17 alpha-hydroxyprogesterone (17α-OHP) production was found to increase (+41%). Testosterone synthesis was rescued by the addition of androstenedione but not by any of the other precursors used. Thus, the hormone data suggested that steroidogenesis was blocked at the level of the 17,20 lyase activity of the P450c17 enzyme (CYP17), converting 17α-OHP to androstenedione. The subsequent gene expression and protein levels supported this hypothesis. In addition to Cyp17a1, microarray analysis showed that several other genes important for testes development were affected by MEHP. These genes included those encoding insulin-like factor 3 (INSL3), which is involved in controlling testicular descent, and Inha, which encodes the alpha subunit of inhibin B.
These findings indicate that under in vitro conditions known to support normal differentiation of the fetal rat testis, the exposure to MEHP directly inhibits several important Leydig cell factors involved in testis function and that the Cyp17a1 gene is a specific target to MEHP explaining the MEHP-induced suppression of steroidogenesis observed.
Sex steroids are crucial regulators of sexual differentiation and the proper development of secondary sex characteristics and patterns of sexual behavior. Since Leydig cells are the primary major producers of these steroid hormones, maintenance of the normal functions of these cells determines the reproductive capacity and fertility of males. The present minireview discusses recent findings concerning endocrine and paracrine regulation of the proliferation, differentiation and involution of human Leydig cells. The physiology and function of the two distinct fetal and adult populations of human Leydig cells are described, with particular focus on the paracrine environment that triggers their differentiation and functional maturation. The roles of established and more recently discovered paracrine regulators of this maturation, including insulin-like factor 3, platelet-derived growth factor-alpha, desert hedgehog, ghrelin and leptin are considered. A brief description of the origin, ontogenesis and functional markers of human fetal and adult Leydig cells is presented.
Endocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose-response character, and cellular target(s) within the fetal testis are not known.
In this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP), and several of their metabolites on the development of organo-cultured testes from rat fetus.
We removed testes from 14.5-day-old rat fetuses and cultured them for 1-3 days with or without DEHP, MEHP, and the metabolites.
DEHP (10(-5) M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro.
We hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects.
BACKGROUND
Insulin-like factor 3 (INSL3) is a neohormone that has evolved to address specific mammalian traits, in particular, the first phase of testicular descent towards the scrotum during mid-gestation.
METHODS
A thorough literature search was made in PubMed using the terms INSL3, as well as the older synonyms RLF and Ley-IL.
RESULTS
INSL3 is a major secretory product of the testicular Leydig cells in the fetus and in adult men, and in rodent models, reduction in fetal INSL3 expression is an early marker of the testicular dysgenesis syndrome. In women, it is produced in lower amounts by ovarian theca and luteal cells, and circulating levels are increased in women with polycystic ovarian syndrome. During pregnancy, there is evidence for an interaction regulating the feto-placental unit. The presence of INSL3 in amniocentesis samples taken at 12–14 weeks gestation is absolutely specific for male gender, and levels are predictive of subsequent pre-eclampsia and/or birthweight. INSL3 is also involved in adult traits, such as spermatogenesis and bone metabolism. In adult men, INSL3 is constitutively expressed and secreted into the bloodstream at a constant level, reflecting the number and/or functional capacity of the Leydig cells. In complete contrast, testosterone is highly variable within individuals, is acutely responsive to fluctuations in the hypothalamic–pituitary–gonadal axis and appears to have marginal diagnostic value. INSL3 declines consistently with age in adult men.
CONCLUSIONS
INSL3 promises to become an important new diagnostic tool to characterize those men with late-onset hypogonadism and to add clinical diagnostic value at amniocentesis.
Several studies have described an increasing frequency of male reproductive disorders, which may have a common origin in fetal life and which are hypothesized to be caused by endocrine disruptors. Phthalate esters represent a class of environmental endocrine-active chemicals known to disrupt development of the male reproductive tract by decreasing testosterone production in the fetal rat.
Using the organ culture system we developed previously, we investigated the effects on the development of human fetal testis of one phthalate--mono-2-ethylhexyl phthalate (MEHP)--an industrial chemical found in many products, which has been incriminated as a disruptor of male reproductive function.
Human fetal testes were recovered during the first trimester (7-12 weeks) of gestation, a critical period for testicular differentiation, and cultured for 3 days with or without MEHP in basal conditions or stimulated with luteinizing hormone (LH).
Whatever the dose, MEHP treatment had no effect on basal or LH-stimulated testosterone produced by the human fetal testis in vitro, although testosterone production can be modulated in our culture system. MEHP (10(-4) M) did not affect proliferation or apoptosis of Sertoli cells, but it reduced the mRNA expression of anti-Müllerian hormone. MEHP (10(-4) M) reduced the number of germ cells by increasing their apoptosis, measured by the detection of caspase-3-positive germ cells, without modification of their proliferation.
This is the first experimental demonstration that phthalates alter the development of the germ cell lineage in humans. However, in contrast to results observed in the rat, phthalates did not affect steroidogenesis.
A large number of industrial chemicals and environmental pollutants, including trichloroethylene (TCE), di(2-ethylhexyl)phthalate (DEHP), perfluorooctanoic acid (PFOA), and various phenoxyacetic acid herbicides, are nongenotoxic rodent hepatocarcinogens whose human health risk is uncertain. Rodent model studies have identified the receptor involved in the hepatotoxic and hepatocarcinogenic actions of these chemicals as peroxisome proliferator-activated receptor alpha (PPARalpha), a nuclear receptor that is highly expressed in liver. Humans exhibit a weak response to these peroxisome proliferator chemicals, which in part results from the relatively low level of PPARalpha expression in human liver. Cell transfection studies were carried out to investigate the interactions of peroxisome proliferator chemicals with PPARalpha, cloned from human and mouse, and with PPARgamma, a PPAR isoform that is highly expressed in multiple human tissues and is an important regulator of physiological processes such as adipogenesis and hematopoiesis. With three environmental chemicals, TCE, perchloroethylene, and DEHP, PPARalpha was found to be activated by metabolites, but not by the parent chemical. A decreased sensitivity of human PPARalpha compared to mouse PPARalpha to trans-activation was observed with some (Wy-14, 643, PFOA), but not other, peroxisome proliferators (TCE metabolites, trichloroacetate and dichloroacetate; and DEHP metabolites, mono[2-ethylhexyl]phthalate and 2-ethylhexanoic acid). Investigation of human and mouse PPARgamma revealed the transcriptional activity of this receptor to be stimulated by mono(2-ethylhexyl)phthalate, a DEHP metabolite that induces developmental and reproductive organ toxicities in rodents. This finding suggests that PPARgamma, which is highly expressed in human adipose tissue, where many lipophilic foreign chemicals tend to accumulate, as well as in colon, heart, liver, testis, spleen, and hematopoietic cells, may be a heretofore unrecognized target in human cells for a subset of industrial and environmental chemicals of the peroxisome proliferator class.
In mammals, exposure to antiandrogenic chemicals during sexual differentiation can produce malformations of the reproductive tract. Perinatal administration of AR antagonists like vinclozolin and procymidone or chemicals like di(2-ethylhexyl) phthalate (DEHP) that inhibit fetal testicular testosterone production demasculinize the males such that they display reduced anogenital distance (AGD), retained nipples, cleft phallus with hypospadias, undescended testes, a vaginal pouch, epididymal agenesis, and small to absent sex accessory glands as adults. In addition to DEHP, di-n-butyl (DBP) also has been shown to display antiandrogenic activity and induce malformations in male rats. In the current investigation, we examined several phthalate esters to determine if they altered sexual differentiation in an antiandrogenic manner. We hypothesized that the phthalate esters that altered testis function in the pubertal male rat would also alter testis function in the fetal male and produce malformations of androgen-dependent tissues. In this regard, we expected that benzyl butyl (BBP) and diethylhexyl (DEHP) phthalate would alter sexual differentiation, while dioctyl tere- (DOTP or DEHT), diethyl (DEP), and dimethyl (DMP) phthalate would not. We expected that the phthalate mixture diisononyl phthalate (DINP) would be weakly active due to the presence of some phthalates with a 6-7 ester group. DEHP, BBP, DINP, DEP, DMP, or DOTP were administered orally to the dam at 0.75 g/kg from gestational day (GD) 14 to postnatal day (PND) 3. None of the treatments induced overt maternal toxicity or reduced litter sizes. While only DEHP treatment reduced maternal weight gain during the entire dosing period by about 15 g, both DEHP and DINP reduced pregnancy weight gain to GD 21 by 24 g and 14 g, respectively. DEHP and BBP treatments reduced pup weight at birth (15%). Male (but not female) pups from the DEHP and BBP groups displayed shortened AGDs (about 30%) and reduced testis weights (about 35%). As infants, males in the DEHP, BBP, and DINP groups displayed femalelike areolas/nipples (87, 70, and 22% (p < 0.01), respectively, versus 0% in other groups). All three of the phthalate treatments that induced areolas also induced a significant incidence of reproductive malformations. The percentages of males with malformations were 82% (p < 0.0001) for DEHP, 84% (p < 0.0001) for BBP, and 7.7% (p < 0.04) in the DINP group. In summary, DEHP, BBP, and DINP all altered sexual differentiation, whereas DOTP, DEP, and DMP were ineffective at this dose. Whereas DEHP and BBP were of equivalent potency, DINP was about an order of magnitude less active.
Phthalate esters (PE) such as DEHP are high production volume plasticizers used in vinyl floors, food wraps, cosmetics, medical products, and toys. In spite of their widespread and long-term use, most PE have not been adequately tested for transgenerational reproductive toxicity. This is cause for concern, because several recent investigations have shown that DEHP, BBP, DBP, and DINP disrupt reproductive tract development of the male rat in an antiandrogenic manner. The present study explored whether the antiandrogenic action of DEHP occurs by (1) inhibiting testosterone (T) production, or by (2) inhibiting androgen action by binding to the androgen receptor (AR). Maternal DEHP treatment at 750 mg/kg/day from gestational day (GD) 14 to postnatal day (PND) 3 caused a reduction in T production, and reduced testicular and whole-body T levels in fetal and neonatal male rats from GD 17 to PND 2. As a consequence, anogenital distance (AGD) on PND 2 was reduced by 36% in exposed male, but not female, offspring. By GD 20, DEHP treatment also reduced testis weight. Histopathological evaluations revealed that testes in the DEHP treatment group displayed enhanced 3ss-HSD staining and increased numbers of multifocal areas of Leydig cell hyperplasia as well as multinucleated gonocytes as compared to controls at GD 20 and PND 3. In contrast to the effects of DEHP on T levels in vivo, neither DEHP nor its metabolite MEHP displayed affinity for the human androgen receptor at concentrations up to 10 microM in vitro. These data indicate that DEHP disrupts male rat sexual differentiation by reducing T to female levels in the fetal male rat during a critical stage of reproductive tract differentiation.
Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.
Male reproductive tract abnormalities associated with testicular dysgenesis in humans also occur in male rats exposed gestationally to some phthalate esters. We examined global gene expression in the fetal testis of the rat following in utero exposure to a panel of phthalate esters. Pregnant Sprague-Dawley rats were treated by gavage daily from Gestational Days 12 through 19 with corn oil vehicle (1 ml/kg) or diethyl phthalate (DEP), dimethyl phthalate (DMP), dioctyl tere-phthalate (DOTP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), or benzyl butyl phthalate (BBP) at 500 mg/kg per day. Testes were isolated on Gestational Day 19, and global changes in gene expression were determined. Of the approximately 30 000 genes queried, expression of 391 genes was significantly altered following exposure to the developmentally toxic phthalates (DBP, BBP, DPP, and DEHP) relative to the control. The developmentally toxic phthalates were indistinguishable in their effects on global gene expression. No significant changes in gene expression were detected in the nondevelopmentally toxic phthalate group (DMP, DEP, and DOTP). Gene pathways disrupted include those previously identified as targets for DBP, including cholesterol transport and steroidogenesis, as well as newly identified pathways involved in intracellular lipid and cholesterol homeostasis, insulin signaling, transcriptional regulation, and oxidative stress. Additional gene targets include alpha inhibin, which is essential for normal Sertoli cell development, and genes involved with communication between Sertoli cells and gonocytes. The common targeting of these genes by a select group of phthalates indicates a role for their associated molecular pathways in testicular development and offers new insight into the molecular mechanisms of testicular dysgenesis.
Cryptorchidism is a common reproductive abnormality, possibly resulting from abnormal hormone production/action by the fetal testis. Insulin-like factor 3 (Insl3) is thought to be involved in gubernaculum development and transabdominal testicular descent, but its importance is unclear, due partly to lack of suitable Insl3 antibodies. We generated (by genetic immunization) and validated a novel antirat Insl3 antibody, which we used to characterize immunoexpression of Insl3 in rat Leydig cells (LCs) from fetal life until adulthood and its relationship to cryptorchidism. Immunoexpression was strong on embryonic day (E) 17.5 and E19.5 and from 35 d of age onward but weak from E21.5 until puberty. Because in utero exposure to di (n-butyl) phthalate (DBP) induces cryptorchidism and suppresses Insl3 gene expression, we investigated Insl3 protein expression in fetal and adult rats exposed to 500 mg/kg.d DBP from E13.5 to E21.5. Expression on E17.5 and E19.5 decreased dramatically after DBP exposure, but there was no consistent correlation between this suppression and abnormal testis position. We also compared expression of Insl3 and P450 side-chain cleavage enzyme in fetal testes from rats exposed in utero to DBP or flutamide (50 mg/kg.d). DBP treatment suppressed expression of both P450 side-chain cleavage enzyme and Insl3 at E19.5, but flutamide exposure had no effect on either protein, demonstrating that Insl3 expression in fetal rat LCs is not androgen regulated. In adult rats, Insl3 expression was suppressed in 80% of cryptorchid and 50% of scrotal testes from rats exposed to DBP, suggesting that prenatal DBP exposure also leads to maldevelopment/malfunction of the adult LC population in some animals.
Prenatal phthalate exposure impairs testicular function and shortens anogenital distance (AGD) in male rodents. We present data from the first study to examine AGD and other genital measurements in relation to prenatal phthalate exposure in humans. A standardized measure of AGD was obtained in 134 boys 2-36 months of age. AGD was significantly correlated with penile volume (R = 0.27, p = 0.001) and the proportion of boys with incomplete testicular descent (R = 0.20, p = 0.02). We defined the anogenital index (AGI) as AGD divided by weight at examination [AGI = AGD/weight (mm/kg)] and calculated the age-adjusted AGI by regression analysis. We examined nine phthalate monoester metabolites, measured in prenatal urine samples, as predictors of age-adjusted AGI in regression and categorical analyses that included all participants with prenatal urine samples (n = 85). Urinary concentrations of four phthalate metabolites [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and monoisobutyl phthalate (MiBP)] were inversely related to AGI. After adjusting for age at examination, p-values for regression coefficients ranged from 0.007 to 0.097. Comparing boys with prenatal MBP concentration in the highest quartile with those in the lowest quartile, the odds ratio for a shorter than expected AGI was 10.2 (95% confidence interval, 2.5 to 42.2). The corresponding odds ratios for MEP, MBzP, and MiBP were 4.7, 3.8, and 9.1, respectively (all p-values < 0.05). We defined a summary phthalate score to quantify joint exposure to these four phthalate metabolites. The age-adjusted AGI decreased significantly with increasing phthalate score (p-value for slope = 0.009). The associations between male genital development and phthalate exposure seen here are consistent with the phthalate-related syndrome of incomplete virilization that has been reported in prenatally exposed rodents. The median concentrations of phthalate metabolites that are associated with short AGI and incomplete testicular descent are below those found in one-quarter of the female population of the United States, based on a nationwide sample. These data support the hypothesis that prenatal phthalate exposure at environmental levels can adversely affect male reproductive development in humans.
Phthalates adversely affect the male reproductive system in animals. We investigated whether phthalate monoester contamination of human breast milk had any influence on the postnatal surge of reproductive hormones in newborn boys as a sign of testicular dysgenesis.
We obtained biologic samples from a prospective Danish-Finnish cohort study on cryptorchidism from 1997 to 2001. We analyzed individual breast milk samples collected as additive aliquots 1-3 months postnatally (n = 130; 62 cryptorchid/68 healthy boys) for phthalate monoesters [mono-methyl phthalate (mMP), mono-ethyl phthalate (mEP), mono-n-butyl phthalate (mBP), mono-benzyl phthalate (mBzP), mono-2-ethylhexyl phthalate (mEHP), mono-isononyl phthalate (miNP)]. We analyzed serum samples (obtained in 74% of all boys) for gonadotropins, sex-hormone binding globulin (SHBG), testosterone, and inhibin B.
All phthalate monoesters were found in breast milk with large variations [medians (minimum-maximum)]: mMP 0.10 (< 0.01-5.53 microg/L), mEP 0.95 (0.07-41.4 microg/L), mBP 9.6 (0.6-10,900 microg/L), mBzP 1.2 (0.2-26 microg/L), mEHP 11 (1.5-1,410 microg/L), miNP 95 (27-469 microg/L). Finnish breast milk had higher concentrations of mBP, mBzP, mEHP, and Danish breast milk had higher values for miNP (p = 0.0001-0.056). No association was found between phthalate monoester levels and cryptorchidism. However, mEP and mBP showed positive correlations with SHBG (r = 0.323, p = 0.002 and r = 0.272, p = 0.01, respectively); mMP, mEP, and mBP with LH:free testosterone ratio (r = 0.21-0.323, p = 0.002-0.044) and miNP with luteinizing hormone (r = 0.243, p = 0.019). mBP was negatively correlated with free testosterone (r = -0.22, p = 0.033). Other phthalate monoesters showed similar but nonsignificant tendencies.
Our data on reproductive hormone profiles and phthalate exposures in newborn boys are in accordance with rodent data and suggest that human Leydig cell development and function may also be vulnerable to perinatal exposure to some phthalates. Our findings are also in line with other recent human data showing incomplete virilization in infant boys exposed to phthalates prenatally.
Over recent decades, recurring efforts have been devoted to developing testicular cell or tissue cultures for basic and clinical research. However, there remains much confusion, particularly concerning the fate of human germ cells in culture.
To reassess the status of human testicular cell types as well as the ability of germ cells to divide and differentiate in organotypic culture.
Human testicular fragments were maintained for 2 weeks in culture. The viability and functionality of testicular cells were assessed using light and electronic microscopy, apoptotic cell labelling, 5-bromo-2'-deoxyuridine (BrdU) incorporation, immunohistochemistry and quantitative PCR against specific cell markers.
A gradual loss of meiotic and post-meiotic germ cells occurred throughout the culture period, irrespective of the presence of gonadotrophins. However, all germ cell types remained traceable for up to 16 days, some still dividing and differentiating at a rate compatible with the in vivo situation. Good maintenance of the general architecture of the explants associated with clearly quantifiable levels of several somatic cell markers was observed.
Although this culture model is clearly unsuitable for preparing germ cells for therapeutic purposes, it does represent a most valuable tool for testing the effects of biological and chemical agents on testicular tissue.
Di(2-ethylhexyl) phthalate (DEHP) produced seminiferous tubular atrophy and reductions in seminal vesicle and prostate weight in 4-week-old, but not in 15 -week-old rats. Di-n-pentyl phthalate (DPP) did produce atrophy in the older rats but this developed more slowly than in young animals. Coadministration of testosterone or gonadotrophins did not protect against phthalate-induced testicular toxicity but did partly reverse the depression of seminal vesicle and prostate weight. Secretion of seminiferous tubule fluid and androgen binding protein by the Sertoli cells was markedly suppressed within 1 hr of a dose of DPP or mono-2-ethylhexyl phthalate (MEHP) in immature rats. This occurred less rapidly in mature rats. (/sup 14/C)mono-n-pentyl phthalate and (/sup 14/C)MEHP penetrated the blood testis barrier only to a very limited extent. These findings and the early morphological changes in the Sertoli cells produced by DPP suggest that phthalate esters may act initially to cause Sertoli cell injury, the subsequent loss of germ cells occurring as a consequence of this.
Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.
Di(n-butyl) phthalate (DBP) has antiandrogenic-like effects on the developing reproductive tract in the male rat and produces regions of interstitial cell hyperplasia and gonocyte degeneration in the developing fetal testes at maternal doses of 100-500 mg/ kg/day. Neither DBP nor its primary metabolites interact with the androgen receptor in vitro. The present study was performed to examine gene expression in the fetal rat testes following in utero DBP exposure. Pregnant Sprague-Dawley rats received corn oil, DBP (500 mg/kg/day), or flutamide (reference antiandrogen, 50 mg/kg/day) by gavage daily from gestation day (GD) 12 to 21. Dose levels were selected to maximize fetal response with minimal maternal toxicity. Testes were isolated on GD 16, 19, and 21. Global changes in gene expression were determined by microarray analysis. Selected genes were further examined by quantitative RT-PCR. DBP, but not flutamide, reduced expression of the steroidogenic enzymes cytochrome P450 side chain cleavage, cytochrome P450c17, and steroidogenic acute regulatory protein. Testicular testosterone and androstenedione were decreased on GD 19 and 21, while progesterone was increased on GD 19 in DBP-exposed testes. Testosterone-repressed prostate message-2 (TRPM-2) was upregulated, while c-kit (stem cell factor receptor) mRNA was downregulated following DBP exposure. TRPM-2 and bcl-2 protein staining was elevated in GD 21 DBP-exposed Leydig and Sertoli cells. Results of this study have led to the identification of several possible mechanisms by which DBP can induce its antiandrogenic effects on the developing male reproductive tract without direct interaction with the androgen receptor. Our results suggest that the antiandrogenic effects of DBP are due to decreased testosterone synthesis. In addition, enhanced expression of cell survival proteins such as TRPM-2 and bcl-2 may be involved in DBP-induced Leydig cell hyperplasia, whereas, downregulation of c-kit may play a role in gonocyte degeneration. Future studies will explore the link between these identified gene expression alterations and ultimate adverse responses.
• Ketoconazole, a new oral drug used to treat systemic and superficial mycoses, inhibits sterol synthesis in fungi. The development of gynecomastia in two patients prompted us to investigate the effect of the drug on testosterone production. After a 200-, 400-, or 600-mg dose, volunteer male testosterone serum concentrations fell markedly, but returned toward baseline eight to 24 hours later as ketoconazole serum concentrations waned. A marked but transient drop in testosterone levels occurred in patients receiving long-term therapy, and continuous testosterone depression was noted in one. A block of synthesis was demonstrated in vitro. Ketoconazole at concentrations achievable in serum with currently used doses blocked basal and gonadotropin-stimulated testosterone production by rat Leydig cells. The diminution of testosterone synthesis could be significant as further therapeutic trials may use larger doses or more than once-daily administration. The paucity of reports of endocrinologic toxicity may relate to the "escape" from the block demonstrated in vivo.
(Arch Intern Med 1982;142:2137-2140)
BACKGROUND: Observations of adverse developmental and reproductive effects in laboratory animals and wildlife have fueled increasing public concern regarding the potential for various chemicals to impair human fertility. OBJECTIVE: Our objective in this study was to assess the effect of occupational exposure to high levels of phthallate esters on the balance of gonadotropin and gonadal hormones including luteinizing hormone, follicle-stimulating hormone, free testosterone (fT), and estradiol. METHODS: We examined urine and blood samples of 74 male workers at a factory producing unfoamed polyvinyl chloride flooring exposed to di-n-butyl phthalate (DBP) and di-2-ethythexyl phthalate (DEHP) and compared them with samples from 63 male workers from a construction company, group matched for age and smoking status. RESULTS: Compared to the unexposed workers, the exposed workers had substantially and significantly elevated concentrations of mono-n-butyl phthalate (MBP; 644.3 vs. 129.6 mu g/g creatinine, p < 0.001) and mono-2-ethylhexyl phthalate (MEHP; 565.7 vs. 5.7 mu g/g creatinine, p < 0.001). fT was significantly lower (8.4 vs. 9.7 mu g/g creatinine, p = 0.019) in exposed workers than in unexposed workers. fT was negatively correlated to MBP (r = -0.25, p = 0.03) and MEHP (r = -0.19, p = 0.095) in the exposed worker group. Regression analyses revealed that fT decreases significantly with increasing total phthalate ester score (the sum of quartiles of MBP and MEHP; r = -0.26, p = 0.002). CONCLUSION: We observed a modest and significant reduction of serum fT in workers with higher levels of urinary MBP and MEHP compared with unexposed workers.
Phthalates are developmental and reproductive toxicants for the fetus in pregnant rodents, and the ability of phthalates to penetrate the placenta have been reported. The aims of this study were to evaluate the association between maternal urine excretion, the exposure of fetus to phthalates in amniotic fluid, and the health of newborns. Amniotic fluid and urine samples from pregnant women were collected to measure five phthalate monoesters using liquid chromatography/tandem mass spectrometry (LC/MS-MS) and the newborns' birth weight, gestational age, and anogenital distance (AGD) were collected. The median levels of three phthalate monoesters in urine and amniotic fluid were 78.4 and 85.2 ng/mL monobutyl phthalate (MBP); 24.9 and 22.8 ng/mL mono-(2-ethylhexyl) phthalate (MEHP); 19.8 and Not Detected monoethyl phthalate (MEP). We found a significant positive correlation only between creatinine adjusted urinary MBP and amniotic fluid MBP (R(2)=0.156, p<0.05) in all infants and, only in female infants, a significantly negative correlation between amniotic fluid MBP, AGD (R=-0.31, p<0.06), and the anogenital index adjusted by birth weight (AGI-W) (R=-0.32, p<0.05). Although the influence of prenatal di-n-butyl phthalate (DBP) exposure on the endocrinology and physiology of the fetus is still a puzzle, our data clearly show that in utero exposure to phthalates in general has anti-androgenic effects on the fetus.
Widely used man-made chemicals, including phthalates, can induce hormonal alterations through a variety of cellular and molecular mechanisms. A number of rodent and observational studies have consistently demonstrated the anti-androgenic effect of several phthalates. However, there are only limited data on the relationship between exposure to these chemicals and reproductive hormone levels in men. All men (n=425) were partners of pregnant women who participated in the Study for Future Families in five US cities and provided urine and serum samples on the same day. Eleven phthalate metabolites were measured in urine and serum samples were analysed for reproductive hormones, including follicle-stimulating hormone, luteinizing hormone, testosterone, inhibin B and oestradiol and sex hormone-binding globulin (SHBG). Pearson correlations and parametric tests were used for unadjusted analyses, and multiple linear regression analysis was performed controlling for appropriate covariates. We observed weak or no associations with urinary phthalates other than di(2-ethylhexyl) phthalate (DEHP). All measures of testosterone [total, calculated free testosterone and the free androgen index (FAI)] were inversely correlated with the urinary concentrations of four DEHP metabolites. After adjustment by appropriate covariates, there was no longer an association between urinary DEHP metabolite concentrations and total testosterone levels; however, FAI was significantly associated with the urinary concentrations of several DEHP metabolites. SHBG was positively related to the urinary concentrations of mono(2-ethylhexyl) phthalate, but not with other DEHP metabolites, an association that was attenuated after adjustment. Our results suggest that DEHP exposure of fertile men is associated with minor alterations of markers of free testosterone.
Knockout of the gene encoding insulin-like factor 3 (INSL3) results in cryptorchidism in mice due to disruption of the transabdominal phase of testicular descent. This finding was essential for understanding the complete course of testis descensus, and wound up years of speculations regarding the endocrine regulation of this process. INSL3 is, along with testosterone, a major secretory product of testicular Leydig cells. In addition to its crucial function in testicular descent, INSL3 is suggested to play a paracrine role in germ cell survival and an endocrine role in bone metabolism. INSL3 is produced in human prenatal and neonatal, and in adult Leydig cells to various extents, and is in a developmental context regulated like testosterone, with production during second trimester, an early postnatal peak and increasing secretion during puberty, resulting in high adult serum levels. INSL3 production is entirely dependent on the state of Leydig cell differentiation, and is stimulated by the long-term trophic effects mediated by luteinizing hormone (LH). Once differentiated, Leydig cells apparently express INSL3 in a constitutive manner, and the hormone is thereby insensitive to the acute, steroidogenic effects of LH, which for example is an important factor in the regulation of testosterone. Clinically, serum INSL3 levels can turn out to be a usable tool to monitor basal Leydig cell function in patients with various disorders affecting Leydig cell function. According to animal studies, foetal INSL3 production is, directly or indirectly, sensitive to oestrogenic or anti-androgenic compounds. This provides important insight into the mechanism by which maternal exposure to endocrine disrupters can result in cryptorchidism in the next generation. Conclusively, INSL3 is an interesting testicular hormone with potential clinical value as a marker for Leydig cell function. It should be considered on a par with testosterone in the evaluation of testicular function and the consequences of Leydig cell dysfunction.
Levels of the phthalates such as di(2-ethylhexyl) phthalate (DEHP), mono(2-ethylhexyl) phthalate (MEHP, a major metabolite of DEHP), di-n-butyl phthalate (DBP), mono-n-butyl phthalate (MBP, a major metabolite of DBP), and phthalic acid (P, (a common metabolite of phthalates, including DEHP and DBP) were determined in the semen samples of 99 healthy volunteers without known prior medicosurgical history. Samples were obtained from young men (age 20-25 yr) who visited a clinic, and the semen concentrations of phthalates were measured using ultra-performance liquid chromatography (UPLC) and tandem mass spectrometry (MS/MS). UPLC/MS/MS showed that mean concentrations in semen samples were 1.07 microg/ml for MEHP, 0.61 microg/ml for DEHP, 0.39 microg/ml for PA, 0.06 microg/ml for MBP, and 0.003 microg/ml for DBP. The concentration of MEHP (the metabolite of DEHP) was highest, and the concentrations of the metabolites including MEHP, MBP, and PA were higher than actual concentrations of parent DEHP and DBP. These findings suggest the detection of phthalates in healthy human semen might require further investigation for effects on human fertility.
Experimental animal studies have demonstrated that exposure to some phthalates may be associated with altered endocrine function and adverse effects on male reproductive development and function, but human studies are limited. In the present study, urine and serum samples were collected from 425 men recruited through a US infertility clinic. Urinary concentrations of mono(2-ethylhexyl) phthalate (MEHP), the hydrolytic metabolite of di(2-ethylhexyl) phthalate (DEHP), and other phthalate monoester metabolites were measured, along with serum levels of testosterone, estradiol, sex hormone-binding globulin (SHBG), follicle-stimulating hormone, luteinizing hormone, inhibin B, and prolactin. Two oxidized urinary metabolites of DEHP were also measured in urine from 221 of the men. In multiple regression models adjusted for potential confounders, MEHP was inversely associated with testosterone, estradiol, and free androgen index (FAI). An interquartile range increase in MEHP was associated with 3.7% (95% confidence interval [CI], -6.8% to -0.5%) and 6.8% (95% CI, -11.2% to -2.4%) declines in testosterone and estradiol, respectively, relative to the population median hormone levels. There was limited evidence for effect modification of the inverse association between MEHP and FAI by the proportion of DEHP metabolites in the urine measured as MEHP (MEHP%), which is considered a phenotypic marker of less efficient metabolism of DEHP to its oxidized metabolites. Finally, the ratio of testosterone to estradiol was positively associated with MEHP (P = .07) and MEHP% (P = .007), suggesting potential relationships with aromatase suppression. In conclusion, these results suggest that urinary metabolites of DEHP are inversely associated with circulating steroid hormone levels in adult men. However, additional research is needed to confirm these findings.
After briefly discussing human exposure to phthalates--diesters of 1,2-benzenedicarboxylic acid (phthalic acid)--this article first presents recent findings from the Study for Future Families, a multi-center pregnancy study in which the human analogue of the phthalate syndrome was first identified. This is one of an increasing number of studies that have investigated human endpoints in relation to environmental exposure to these ubiquitous compounds. This literature, which includes a range of human health endpoints following prenatal, neonatal, childhood, and adult exposures, is then summarized. At least one significant association has been reported for urinary metabolites of di-n-butyl phthalate (DBP), butylbenzyl phthalate (BzBP), diethyl phthlate (DEP), and di-isononyl phthalate (DINP) and for three of the urinary metabolites of di(2-ethylhexyl) phthalate (DEHP). Many of the findings reported in humans--most of which have been in males--are consistent with the anti-androgenic action that has been demonstrated for several phthalates. Replication of the results described here and further mechanistic studies are needed to strengthen links between phthalates and adverse health outcomes.
Inhibin, a glycoprotein that preferentially suppresses follicle-stimulating hormone (FSH) secretion, has been isolated from follicular fluid as a heterodimer of two dissimilar subunits linked by disulphide bonds. The larger subunit is termed alpha and the smaller is designated beta. Two forms of inhibin termed A and B have been isolated, the differences being due to variations in the amino acid sequence of the beta-subunit; Inhibin A consists of alpha-beta and Inhibin B of alpha-beta B. Dimers of the beta-subunit, termed activins, have also been found in follicular fluid; these stimulate pituitary FSH secretion. Inhibin is produced in the female by the granulosa cell and corpus luteum under the control of FSH and luteinizing hormone (LH), respectively. The levels in serum rise to peak at mid-cycle and in the mid-luteal phase of the human menstrual cycle, and decline prior to menstruation. In pregnancy, the late-luteal phase decline in inhibin does not occur and the levels increase slowly. Studies suggest that the levels in pregnancy arise from an embryonic source, particularly the placenta. In the male, inhibin is produced by the Sertoli cells under the control of FSH by mechanisms involving cyclic adenosine 3', 5'-monophosphate. Testosterone exerts a minor inhibitory control at supraphysiological levels (10(-5) M), but human chorionic gonadotropin stimulation results paradoxically in a rise in serum inhibin levels. Disruption of spermatogenesis in the rat by cryptorchidism, heat treatment, or efferent duct ligation results in a decline in inhibin levels and a rise in FSH levels, findings consistent with the negative feedback action of inhibin on FSH secretion. As well as their roles in the reproductive system, inhibin and activin have more widespread actions in the haemopoietic, immune and nervous systems as evidenced by the finding of mRNA for its subunits in a range of tissues. Other studies have shown actions on erythroid differentiation and on mitotic activity in thymocytes. These actions suggest that inhibin and activin may function as growth factors as well as regulators of FSH.
Ethylene dimethane sulphonate (DS) administered to adult male rats in a single dose of 75 mg/kg body weight results in a rapid destruction of Leydig cells which, in turn, is associated with a marked decline in levels of serum testosterone. For 24-72 h after treatment with EDS (post-EDS) the Leydig cells undergo degenerative changes consisting of chromatin condensation and cytoplasmic vacuolation, and testicular macrophages progressively remove Leydig cells from the intertubular tissue by phagocytosis. This results in the total absence of Leydig cells on Days 7-14 and the absence of any detectable specific 125I-hCG binding to testis homogenates. Associated with the low levels of serum testosterone, levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in serum rise, LH to levels found in castrate rats. Morphometric and 125I-hCG binding studies indicate that a new generation of Leydig cells develop from Day 21 and reach control levels by Day 49. Morphologic observations suggest that the Leydig cells arise by differentiation from a pool of connective tissue cells that includes fibroblasts, lymphatic endothelial cells and pericytes. The new Leydig cells, which appear around Day 21 post-EDS, have the features of fetal Leydig cells. The latter appear to transform into Leydig cells typical of normal adult rats between 35-49 days post-EDS. The differentiation of new Leydig cells is associated with a reestablishment of normal levels of testosterone 21 days post-EDS. Serum LH and FSH return to normal at 28 days and 49 days respectively.
The in vitro inhibition of Leydig cell microsomal steroidogenesis by ketoconazole, a potent P-450 dependent enzyme blocker, was evaluated in the human, stallion and pig. Purified Leydig cells were isolated by mechanical dispersion of teased, decapsulated whole testes and sieving through a 0.25 mm stainless steel mesh. The activity of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD), 17-hydroxylase (17-OHase), 17,20-desmolase (17,20D), 17-ketosteroid reductase (17-KSR) and aromatase were measured using a constant amount (50 microM) of 14C-labelled substrates in the presence of varying concentrations of pure ketoconazole. Products were isolated by thin layer chromatography and verified by derivative formation. 17-OHase and 17,20D activities were significantly inhibited (p less than .001) by ketoconazole at concentrations as low as 5 microM. 3 beta-HSD, 17-KSR and aromatase activities were only significantly inhibited by ketoconazole at concentrations of 500 and 5000 microM. These data describe the specific loci of inhibition of ketoconazole on testicular steroidogenesis and confirm the observations that ketoconazole is an effective inhibitor of androgen biosynthesis in several species.
A convenient way to estimate the number of viable cells growing in microtitre tray wells is to use a colorimetric assay and an automatic microplate scanning spectrophotometer. One such assay, developed by Mosmann, depends on the reduction by living cells of tetrazolium salt, MTT, to form a blue formazan product. However the original technique has several technical limitations, namely a less than optimal sensitivity, a variable background due to protein precipitation on adding an organic solvent to dissolve the blue formazan product, and a low solubility of the product. These problems have been overcome by the following modifications: avoidance of serum in the incubation medium, thus overcoming precipitation problems in the organic solvent; avoidance of phenol red in the incubation medium, thus avoiding the use of acid in the final solvent which altered the spectral properties of the formazan; elimination of the medium containing MTT after the reaction and subsequent use of pure propanol or ethanol to rapidly solubilize the formazan; use of a higher concentration of MTT; use of half-area microtitre trays to increase the spectrophotometer readings from a given amount of formazan; use of a more judicious reference wavelength in a dual wavelength spectrophotometer. With these modifications the reliability and sensitivity of the test have been increased to the point where it can in many cases replace the [3H]thymidine uptake assay to measure cell proliferation or survival in growth factor or cytotoxicity assays. Examples of its use in IL-2 assays are given.
Di(2-ethylhexyl) phthalate (DEHP) produced seminiferous tubular atrophy and reductions in seminal vesicle and prostate weight in 4-week-old, but not in 15-week-old rats. Di-n-pentyl phthalate (DPP) did produce atrophy in the older rats but this developed more slowly than in young animals. Coadministration of testosterone or gonadotrophins did not protect against phthalate-induced testicular toxicity but did partly reverse the depression of seminal vesicle and prostate weight. Secretion of seminiferous tubule fluid and androgen binding protein by the Sertoli cells was markedly suppressed within 1 hr of a dose of DPP or mono-2-ethylhexyl phthalate (MEHP) in immature rats. This occurred less rapidly in mature rats. [14C]Mono-n-pentyl phthalate and [14C]MEHP penetrated the blood testis barrier only to a very limited extent. These findings and the early morphological changes in the Sertoli cells produced by DPP suggest that phthalate esters may act initially to cause Sertoli cell injury, the subsequent loss of germ cells occurring as a consequence of this. Some features of the testicular lesion could be reproduced in primary cocultures of rat Sertoli and germ cells. Structure activity studies with a range of phthalate monoesters showed good agreement between the induction of germ cell detachment in culture and testicular toxicity in vivo. Three metabolites of MEHP (metabolites V, VI, and IX) were much less toxic in culture than MEHP itself, suggesting that the latter may be the active testicular toxin from DEHP.
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FIGURE 2.
To determine the antisteroidogenic effect of ketoconazole (KTZ) in the human testis, we measured the plasma delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, dehydroepiandrosterone (DHEA), progesterone, 17 alpha-hydroxyprogesterone, androstenedione (A), and testosterone (T) concentrations in three men with previously untreated metastatic prostate cancer at various time intervals for 24 h before and 48 h after the administration of 200 mg oral KTZ every 8 h. The adrenal glands of these three patients were suppressed (as measured by the plasma cortisol levels) by the administration of 1.0 mg dexamethasone daily for 7 days before and during the study. After six doses of KTZ, bilateral orchiectomy was performed, and the intratesticular concentration of the aforementioned seven steroids and the intratesticular activities of the 17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase enzymes in the delta 4-steroidogenic pathway were determined. These seven intratesticular steroids and three intratesticular enzyme activities were compared to those in five men with previously untreated prostate cancer who underwent orchiectomy as primary treatment for their disease. Plasma A, DHEA, and T all significantly decreased during KTZ therapy. There was no significant change in the other four steroids in the plasma. In the testis, delta 5-pregnenolone, delta 5-17 alpha-hydroxypregnenolone, and delta 4-17 alpha-hydroxyprogesterone were all significantly elevated, whereas intratesticular DHEA, A, and T were significantly decreased in the three KTZ-treated patients compared to levels in the five non-KTZ-treated patients. Measurement of the enzyme activities demonstrated a significant reduction in both 17 alpha-hydroxylase and 17,20-desmolase, but no change in 17 beta-hydroxysteroid dehydrogenase, in the KTZ-treated patients compared to the levels in the non-KTZ-treated patients. We conclude that oral KTZ decreases testicular T production by inhibiting the 17,20-desmolase and also the 17 alpha-hydroxylase steps in both the delta 4- and delta 5-T biosynthetic pathways.
Ketoconazole, a new oral drug used to treat systemic and superficial mycoses, inhibits sterol synthesis in fungi. The development of gynecomastia in two patients prompted us to investigate the effect of the drug on testosterone production. After a 200-, 400-, or 600-mg dose, volunteer male testosterone serum concentrations fell markedly, but returned toward baseline eight to 24 hours later as ketoconazole serum concentrations waned. A marked but transient drop in testosterone levels occurred in patients receiving long-term therapy, and continuous testosterone depression was noted in one. A block of synthesis was demonstrated in vitro. Ketoconazole at concentrations achievable in serum with currently used doses blocked basal and gonadotropin-stimulated testosterone production by rat Leydig cells. The diminution of testosterone synthesis could be significant as further therapeutic trials may use larger doses or more than once-daily administration. The paucity of reports of endocrinologic toxicity may relate to the "escape" from the block demonstrated in vivo.
Phathalate esters, which are commonly used as plasticizers for polyvinyl chloride, are also well known to disturb Sertoli cells. This study aims to show the effect of prenatally administered phthalate on testicular descent in pre- and postnatal rats. Pregnant rats were exposed to mono-n-butyl phthalate (MBP) by gavage from the 15th to the 18th gestational days. Rats administered with solvent only were used as controls. In 20-day-old fetuses (n = 15), the degree of transabdominal testicular ascent in relation to the bladder neck was thus found to be significantly higher in MBP-treated rats than that of the controls (n = 19). In addition, in MBP-treated male offspring (n = 26), 22 rats showed either bilateral or unilateral cryptorchidism at the age of 30 to 40 days old, and the occurrence of cryptorchidism was 84.6%. By contrast, the occurrence of cryptorchidism was 0% in the control rats (n = 15, P < .001). It is therefore suggested that prenatal exposure to MBP may disturb the Sertoli cells and elevate the fetal testes relative to the bladder neck while also inducing cryptorchidism postnatally. Sertoli cells may thus play an important role in the transabdominal descent of the testis by secreting Müllerian-inhibiting substance (MIS), which is known to act as a putative mediator of the transabdominal phase.
There is increasing concern that certain chemicals in the environment can cause endocrine disruption in exposed humans and wildlife. Investigations of potential effects on endocrine function have been limited mainly to interactions with hormone receptors. A need exists for the development of alternate in vitro methods to evaluate chemicals for their potential to disturb various endocrine functions via other mechanisms. Our laboratory is using the human H295R adrenocortical carcinoma cell line to examine chemicals for their potential to interfere with the activity and/or expression of several key cytochrome P450 (CYP) enzymes involved in the biosynthesis of steroid hormones. In this report we demonstrated that the commonly used 2-chloro-s-triazine herbicides atrazine, simazine, and propazine dose-dependently (0-30 microM) induced aromatase (CYP19) activity to an apparent maximum of about 2.5-fold in H295R cells. Basal- and triazine-induced aromatase activity was completely inhibited by the irreversible aromatase inhibitor 4-hydroxyandrostenedione (100 microM). The triazines increased levels of CYP19 messenger ribonucleic acid (mRNA) between 1.5- and 2-fold. The time-response profile of the induction of aromatase activity and CYP19 mRNA by the triazines was similar to that by 8-bromo-cyclic adenosine monophosphate, a known stimulant of the protein kinase-A pathway that mediates the induction of aromatase in these cells. The observed induction of aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, may be an underlying explanation for some of the reported hormonal disrupting and tumor promoting properties of these herbicides in vivo.
Targeted inactivation of the insulin-like hormone 3 (insl3) gene in male mice results in altered gubernacular development, disrupted testis decent, and cryptorchidism. Cryptorchidism is a fairly common human malformation, being displayed in about three males per 100 at birth, but only a small percentage can be linked directly to genetic defects. The phthalate esters (PEs) are high production volume, ubiquitous environmental chemicals, some of which when administered during sexual differentiation, induce male rat reproductive tract malformations including gubernacular agenesis. We hypothesized that phthalate-induced gubernacular lesions likely result from an inhibition of insl3 gene expression. Three phthalates, di-n-ethylhexyl phthalate (DEHP), dibutyl phthalate (DBP) and benzyl butyl phthalate (BBP) were administered orally to the dam on gestation day 14 through 18 (GD14-18) and the fetal testes examined on GD18 for effects on steroid hormone production and insl3 gene expression. Compared to chemicals like vinclozolin, linuron, and prochloraz that act as AR antagonists and/or inhibit fetal Leydig cell testosterone production, only the three phthalates significantly reduced both ex vivo testosterone production and insl3 gene expression when quantified by real-time rtPCR. These results provide the first demonstration of PE-induced alteration of insl3 mRNA in the fetal male rat testis.
In utero exposure of male rats to the antiandrogen di(n-butyl) phthalate (DBP) leads to decreased anogenital distance (AGD) on postnatal day (PND) 1, increased areolae retention on PND 13, malformations in the male reproductive tract, and histologic testicular lesions including marked seminiferous epithelial degeneration and a low incidence of Leydig cell (LC) adenomas on PND 90. One objective of this study was to determine the incidence and persistence of decreased AGD, increased areolae retention, and LC adenomas in adult rats following in utero DBP exposure. A second objective was to determine whether AGD and areolae retention during the early postnatal period are associated with lesions in the male reproductive tract. Pregnant Crl:CD(SD)BR rats were gavaged with corn oil or DBP at 100 or 500 mg/kg/day, 10 dams per group. Three replicates of rats (n = 30 rats per replicate) were exposed from gestation day 12 to 21 and the male offspring allowed to mature to 6, 12, or 18 months of age. Gross malformations in the male reproductive tract and histologic lesions in the testes were similar to those previously described. However, testicular dysgenesis, a lesion of proliferating LCs and aberrant tubules that has not been previously described in DBP-exposed testes, was diagnosed. The incidence of this lesion was approximately 20% unilateral and 7-18% bilateral in the high-dose group and was similar among all ages examined, implicating a developmental alteration rather than an age-related change. AGD and areolae retention were found to be permanent changes following in utero exposure to 500 mg/kg/day of DBP. Decreased AGD was a sensitive predictor of lesions in the male reproductive tract, relatively small changes in AGD were associated with a significant incidence of male reproductive malformations. In utero DBP exposure induced proliferative developmental lesions, some of which would have been diagnosed as LC adenomas by the morphological criteria set forth by the Society of Toxicologic Pathology. However, these lesions were dissimilar to traditional LC adenomas as the LCs were poorly differentiated and the lesions contained aberrant seminiferous tubules. While the morphology and incidence of this DBP-induced testicular developmental lesion has been fully characterized by this study, the detailed pathogenesis warrants further investigation.
The plasticizer di(2-ethylhexyl)phthalate (DEHP) exhibits antiandrogenic effects in perinatally exposed male rats. Di(2-ethylhexyl) adipate (DEHA) and diisononyl phthalate (DINP) are currently being evaluated as potential substitutes for DEHP, but similarities in structure and metabolism of DEHP with DEHA and DINP have led to the hypothesis that similarities in action may also exist. Pregnant Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg bodyweight per day), DINP (750 mg/kg bodyweight per day), DEHP (750 mg/kg bodyweight per day) in combination with DEHA (400 mg/kg bodyweight per day), or DEHP (300 mg/kg bodyweight per day) in combination with DINP (750 mg/kg bodyweight per day). DINP and DEHP were both shown to reduce testicular testosterone production ex vivo and testosterone levels in testes and plasma of male fetuses at gestation day 21, indicating a similar mechanism of action for DINP and DEHP. Additionally, plasma LH levels in male fetuses were elevated. Neonatal anogenital distance was reduced and the number of nipples at postnatal day 13 increased in DEHP-exposed male offspring. Serum inhibin B levels were significantly reduced in DEHP-exposed prepubertal male offspring, and in a few adult males. No modulating effects of DEHA on the endocrine effects of DEHP were detected, but a tendency towards an accumulating effect of DEHP and DINP in combination on suppression of testosterone synthesis was seen.
The metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans was studied after three doses of 0.35 mg (4.7 microg/kg), 2.15 mg (28.7 microg/kg) and 48.5 mg (650 microg/kg) of D4-ring-labelled DEHP were administered orally to a male volunteer. Two new metabolites, mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) were monitored for 44 h in urine and for 8 h in serum for the high-dose case, in addition to the three metabolites previously analysed: mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP). For the medium- and low-dose cases, 24 h urine samples were analysed. Up to 12 h after the dose, 5OH-MEHP was the major urinary metabolite, after 12 h it was 5cx-MEPP, and after 24 h it was 2cx-MMHP. The elimination half-lives of 5cx-MEHP and 2cx-MMHP were between 15 and 24 h. After 24 h 67.0% (range: 65.8-70.5%) of the DEHP dose was excreted in urine, comprising 5OH-MEHP (23.3%), 5cx-MEPP (18.5%), 5oxo-MEHP (15.0%), MEHP (5.9%) and 2cx-MMHP (4.2%). An additional 3.8% of the DEHP dose was excreted on the second day, comprising 2cx-MMHP (1.6%), 5cx-MEPP (1.2%), 5OH-MEHP (0.6%) and 5oxo-MEHP (0.4%). In total about 75% of the administered DEHP dose was excreted in urine after two days. Therefore, in contrast to previous studies, most of the orally administered DEHP is systemically absorbed and excreted in urine. No dose dependency in metabolism and excretion was observed. The secondary metabolites of DEHP are superior biomonitoring markers compared to any other parameters, such as MEHP in urine or blood. 5OH-MEHP and 5oxo-MEHP in urine reflect short-term and 5cx-MEHP and 2cx-MMHP long-term exposure. All secondary metabolites are unsusceptible to contamination. Furthermore, there are strong hints that the secondary oxidised DEHP metabolites-not DEHP or MEHP-are the ultimate developmental toxicants.
Di(2-ethylhexyl)phthalate (DEHP) is a plasticizer that can leach from medical devices including storage bags for plateletpheresis concentrates (PCs). In this study, the DEHP exposure to patients receiving PCs was determined and several variables were evaluated to reduce DEHP load to PC recipients.
In 12 patients, serum DEHP was assessed before and after PC transfusion. For in vitro investigations, PCs were produced either with donor plasma or with 65 percent additive solution (AS; T-Sol) and stored for 5 days. Washing of PCs was performed according to AABB standards. DEHP levels were determined by gas chromatography-mass spectrometry.
Transfusion of PCs led to a significant increase in serum DEHP. DEHP levels in the PCs continuously increased during storage, although the accumulation of DEHP was less in PCs stored in the AS, T-Sol, than when stored in plasma. Storage-related accumulation of DEHP contributed to a major part of the total DEHP load in PCs stored for 5 days. Washing of PCs led to a reduction of DEHP load.
Recipients of PCs are exposed to DEHP, although the total amount represents only a small percentage of the defined tolerable intake. Reduction of storage time, the storage of PC in T-Sol, or the exchange of the storage medium before transfusion are practicable means to reduce the DEHP load in PC.
High exposure to phthalates, which are ubiquitous contaminants, has been shown in animal studies to produce detrimental effects on male reproductive functions. A recent study in humans reported dose-response relations between low phthalate levels in urine and human semen parameters, which raises the question whether humans are more sensitive to phthalate exposure than animals.
Urine, serum, and semen samples were collected from 234 young Swedish men at the time of their medical conscript examination. Semen volume, sperm concentration, and motility were measured, together with sperm chromatin integrity (sperm chromatin structure assay) and biochemical markers of epididymal and prostatic function. We analyzed reproductive hormones in serum, and mono ethyl phthalate (MEP), mono ethylhexyl phthaltale (MEHP), mono benzyl phthalate (MBzP), mono butyl phthalate (MBP), and phthalic acid in urine.
For MBP, MBzP, and MEHP, no clear pattern of associations were observed with any of the reproductive biomarkers. Subjects within the highest quartile for MEP had fewer motile sperm (mean difference = 8.8%; 95% confidence interval = 0.8-17), more immotile sperms (8.9%; 0.3-18), and lower luteinizing hormone values (0.7 IU/L; 0.1-1.2), but there was no suggestion of harmful effects for most other endpoints. Phthalic acid actually was associated with improved function, as measured by several markers.
The observed weak associations between 1 phthalate biomarker and impairment of a few aspects of reproductive function biomarkers were not consistent with results from a recent U.S. study. It is not yet possible to conclude whether phthalate exposure may reflect a hazard for human male reproduction.
Phthalate esters have been shown to cause undescended testes (UT) in rats. It has been proposed that Leydig insulin-like factor 3 (INSL3) may play an important role in testicular descent. The aim of this study was to investigate the effect of mono-n-butyl phthalate (MBP) on both the INSL3 gene expression and the transabdominal testicular descent in rats.
On the 19th gestational day, the male fetuses that had been exposed to MBP on the 15th to 18th gestational days were dissected and the degree of testicular ascent was examined. Next, quantitative reverse transcriptase polymerase chain reaction was performed to analyze the testicular INSL3 gene expression. At 60 days of age, the testicular position was recorded in the rest of MBP-treated male offspring.
On the 19th gestational day, INSL3 messenger RNA expression was significantly decreased in the MBP-treated testes, and the associated degree of testicular ascent was significantly higher than in the controls. At 60 days of age, 12 (54.5%) of 22 rats had either unilateral or bilateral UT.
Prenatal MBP may inhibit the INSL3 gene expression associated with the transabdominal testicular ascent in fetal rats, thereby causing UT in postnatal offspring.