**5. Toxicity mechanisms of EDCs**

There are a number of toxicity mechanisms/pathways that are suggested to be caused by estrogenic and antiandrogenic EDCs [17, 18]. These mechanisms are associated with but are not limited to:


The effects of EDCs on cellular metabolism or functions, cellular organelles (peroxisomes, mitochondria, cytoskeleton), DNA damage, chromosomal aberrations, cell cycle checkpoints, translational control, cell death (apoptosis, autophagy, necrosis), immunology/inflammation response, neurological pathways, and development/differentiation are now being studied extensively. However, their effects were also shown to extend beyond these mechanisms/ pathways and may include multiple functions, tissues, and organs such as the liver, kidney, and spleen. Thus, they may have broader impacts—most of them yet to be identified—on disrupting signaling webs and cellular communication [17, 18].

### **6. Phthalates**

The word "plastic" originates from the Greek word "plasticos," which expresses "a material's being capable of molding into different shapes." The first plasticizer was synthesized in 1860. By the progression of technology and increase in the global population growth, plastic materials are now widely used and have very different application fields [19]. Phthalates are dialkyl or alkyl aryl esters of phthalic acid and are abundantly used to make plastic materials more flexible. They are synthesized by reacting phthalic anhydride with alcohol(s) [methanol and ethanol (C1/C2) up to tridecyl alcohol (C13)] either as a straight chain or with some branching. Their main use is for the softening of rigid plastics and polymers. Di(2-ethylhex‐ yl)phthalate (DEHP), the most abundant phthalate derivative, is used 1% to 40% in plastics by weight [20]. Almost 90% of DEHP is used to soften PVC plastics, and in the European Union, 95% of DEHP is used in polymer products as a plasticizer [21].

Phthalates were first synthesized in the 1930s. DEHP was first synthesized in 1933 in Japan and in 1939 in United States in commercial quantities. DEHP was first used in 1949 in United States and has been the most abundantly used phthalate derivative in the Twentieth century. In 1999, the consumption of phthalates were 3.25 million tons and DEHP accounted for 2.1 million tons of the total production. The European Commission reported that 1 million tons of DEHP were used in 2000 [22].

#### **6.1. Occurrence, uses, and exposure to phthalates**

Phthalates migrate out PVC-containing items into food, air, dust, water, and soils and cause human exposure in various ways [23]. Several studies were conducted in different parts of the world, and human blood and urine (mostly spot urine samples) were used as biological fluids to evaluate the exposure to phthalates. The results of these studies revealed that humans are ubiquitously exposed to different phthalates, mostly in industrialized countries [24–27]. On the contrary, workplace inhalation is also of concern as phthalates; particularly, DEHP has low vapor pressure [28].

Diet is the main source of phthalate exposure in the general population. Particularly, fatty food (e.g., fish and oils) can cause high phthalate exposure if contaminated [29, 30]. One other major source is medical exposure by blood storage bags and blood transfusion equipment during receiving blood transfusion [31, 32] or hemodialysis (dialysis bags) [33, 34].

Based on the number of carbon atoms in their alcohol chain, phthalates are divided into two distinct groups, with very different applications, toxicological properties, and classification: high molecular weight (MW) phthalates and low MW phthalates. Their use largely depends on their MW. Higher MW phthalates, such as DEHP, are used in construction materials and in numerous PVC products, including clothing, food and beverage packaging, children products (toys, grip bumpers), and biomedical equipment (e.g., blood transfusion bags, dialysis bags, and umbilical catheterization devices), whereas relatively lower MW phthalates such as dimethyl phthalate (DMP), diethyl phthalate (DEP), and dibutyl phthalate (DBP), are mainly used as odor/color fixatives or as solvents and in cosmetics, textiles, and pharmaceut‐ icals [35].

DEHP has a very low degree of acute toxicity, with oral lethal dose 50 (LD50) values ranging from 26 to >34 g/kg in a variety of species. In a study by Lawrence et al. [36], the lethal effect of this compound appeared to be cumulative, because the LD50 value for intraperitoneal (i.p.) administration to mice five times weekly for 10 weeks was 1.36 g/kg in comparison to a singledose value of 37.8 g/kg. Autian [37] concluded that this was because biotransformation was required before DEHP produces toxic effects. In rats, lethal concentration 50 (LC50) by inhala‐ tion (1 h) was found to be >23.670 mg/m3 (1457 ppm) and inhalation LC50 (6 h) value was >600 mg/m3 (37 ppm) [37]. The oral administration LD50 value for rats (Wistar, male) was suggested to be between 26.000 and 34.000 mg/kg body weight and the LD50 value for i.p. administration in rats was found to be between 30.600 to 49.000 mg/kg body weight [38].

#### **6.1. Biotransformation of phthalates**

Phthalates are not covalently bound to plastic products and therefore may leak out to con‐ taminate blood or food products and can be ingested. When administered orally to humans and rodents, phthalates are rapidly hydrolyzed by esterases in the gut and other tissues to produce the corresponding active monoesters and their further oxidized metabolites. During phase I biotransformation, the relatively polar and low MW phthalates (e.g., DEP) are primarily metabolized to their hydrolytic monoesters by hydrolysis of one of the ester bonds. In contrast, the high MW phthalates are first metabolized to their respective hydrolytic monoesters and then, after enzymatic oxidation of the alkyl chain, to more hydrophilic, oxidative metabolites [39]. For example, DEHP is metabolized to its monoester metabolite, mono-2-ethylhexyl phthalate (MEHP), which is more toxic than the parent compound. MEHP is further metabolized to secondary oxidative metabolites [40]:


Some phthalates are subject to phase II (particularly to glucuronidation and, to a lesser extent, sulfation) metabolic reactions. Glucuronidation not only provided higher urinary excretion of phthalate metabolites but also can reduce their biological activity. Monoesters and the oxidative metabolites of phthalates are excreted in urine (95%) and, to a very lesser extent, eliminated by feces (5%) [39].

#### **6.2. Genotoxic effects of phthalates**

The biological effects of phthalates are of major concern but so far elusive. Phthalates are shown to cause cytogenetic damage to animals and humans. In 1980s, phthalates were evaluated as epigenetic carcinogens because of their peroxisome proliferative effects. However, in the 1990s and in the Twenty first century, several studies confirmed their genotoxic effects [41–43].

Chromosomal aberration test, unscheduled DNA synthesis (UDS), Ames test, micronucleus test, and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutation test were applied to evaluate the genotoxic potentials of phthalates. DEHP was shown to induce single chro‐ matid aberrations and sister chromatid exchange (SCE) in human lymphocytes [44]. Besides, DEHP caused lymphatic mitotic inhibition after 4 h of exposure and caused an increase in the doubling time of human lymphocytes [44]. Later, Stenchever et al. [45] reported that DEHP caused chromosomal breaks in human lymphocytes; mitotic rate decreased and DEHP caused polyploidy and aneuploidy in human fetal lung cells.

Phillips et al. [46] reported that, in Chinese hamster ovary (CHO) cells, MEHP caused chro‐ mosome damage without affecting in the SCE and HPRT mutation test. However, after DEHP treatment in rat hepatocytes, Astill et al. [47] did not find a change in Ames test, mouse lymphoma activation assay, micronucleus test, UDS, and cell transformation tests. A study performed on both CHO cells and RL4 liver cells showed that MEHP caused chromosomal aberrations. However, S-9 mix (a mix of cytosolic and microsomal drug-metabolizing enzymes and cofactors) had no effect on the chromosome damage produced by MEHP in CHO cells [48].

A study using mouse hepatocytes evaluated the genotoxicity of these compounds (i.e., DNA repair or UDS). No changes were observed in DNA repair capacity. However, UDS of the hepatocytes obtained from mice treated with DEHP containing diet showed significantly higher UDS compared to control cells [49]. Lindahl-Kiessling et al. [50] showed that DEHP induced SCE in human lymphocytes, which were co-cultured with rat liver cells. Müller-Tegethoff et al. [51] observed that peroxisome proliferators (nafenopin, Wy-14,643) and DEHP did not induce any changes in micronucleus test in rat hepatocytes. Kim et al. [52] showed that DBP caused both chromatid and chromosomal type chromosomal aberrations (break and exchange) in the lymphocytes of B6C3F1 mice and this aberrations showed marked increases and these alterations show marked elevations dependent on the time of exposure. McKee et al. [53] reported that di(isononyl) phthalate (DINP) was not found to be mutagenic in Ames test, *in vitro* cytogenetic assay, and mouse micronucleus assay. Using Ames test, Lee and Lee [54] observed that the phthalic acid and terephthalic acid did not produce any mutagenic responses in the absence or presence of S9 mix on the *Salmonella typhimurium* strains in Ames test. Besides, phthalic acid and terephthalic acid did not show any significant cytogenetic effect on CHO cells in the chromosomal aberration test and in the mouse micronucleus test [54].

Many studies are performed on the genotoxicity of phthalates using Comet assay in the last 30 years. Anderson et al. [55] showed that both DEHP and its major metabolite MEHP induced DNA damage in human leukocytes as evidenced by increases in tail moment in Comet assay. Kleinsasser et al. [56] compared susceptibilities to DBP and di-iso-butyl-phthalate (DiBP) in nontumor patients to those in patients with squamous cell cancer (SCC) of the oropharynx or larynx using Comet assay and indicated that DBP and DiBP produced significant differences in the Olive tail moment (OTM) between oropharynx (TO), larynx (TL), and TO plus TL groups and the nontumor donors. The same researchers also determined the correlation between the genotoxic sensitivities to DBP and its isomer DiBP in mucosal epithelial cells or lymphocytes using Comet assay, and both phthalates showed significant genotoxicity on both cells and lymphocytes where the genotoxic effect of DiBP was higher than DBP in both cell types [57]. Biscardi et al. [58] reported that DEHP can leach out of polyethylene terephthalate (PET) bottles in time, especially after almost 10 months of storage, and this phenomenon can cause increases in both total tail length and number of cells in human leukocytes *in vitro*.

DEHP, BPA, nonylphenol, and paraquat dichloride were tested for their genotoxicity poten‐ tials on HeLa cells. DEHP showed genotoxicity (>90 µM) with significant increases in tail moment [59]. In a recent study performed on HepG2 cells exposed to various concentrations of DEHP for 24 or 48 h, DNA damage increased significantly in a dose-dependent manner [60]. Throughout our studies on DEHP and MEHP, we determined that both DEHP and MEHP were both cytotoxic and genotoxic in LNCaP cells (human prostate cancer cell line) and Leydig cells (mouse Leydig carcinoma cell line). We also observed that selenium supplementation in either organic form (selenomethionine at 10 µM) or inorganic form (sodium selenite at 30 nM) was highly protective against the cytotoxicity and genotoxicity exerted by these particular phthalate derivatives [42, 43].

The correlation between urinary phthalate levels and sperm DNA damage is being investi‐ gated by several studies. Duty et al. [61] analyzed semen and urine samples of 141 subjects were for five phthalate metabolites using neutral Comet assay and DNA was only correlated with monoethyl phthalate (MEP) levels, although there was no correlation with other phthalate metabolites [monobenzyl phthalate (MBzP), mono-*n*-butyl phthalate (MBP), MEHP, and monomethyl phthalate (MMP)]. Hauser et al. [62] determined the urinary concentrations of phthalate metabolites among men (*n*=379) who were admitted to an infertility clinic. Sperm DNA damage was associated with MEP and MEHP. Recently, Ahbab et al. [63] determined the possible genotoxicity of two different phthalate esters, namely, di-*n*-hexyl phthalate (DHP) and dicyclohexyl phthalate (DCHP), at different concentrations using Comet assay in male rat pups from gestational day (GD) 6 to GD19 at different doses [0 (vehicle), 20, 100, and 500 mg/ kg/day]. Male rats were allowed to grow until different ages (prepubertal, pubertal, and adulthood). The Comet assay was performed on blood lymphocytes and testes samples of adult male rats and the results showed that DHP significantly induced genotoxicity at doses of 100 and 500 mg/kg/day versus control; however, DCHP did not show the same effect [63].

#### **6.3. Epigenetic effects of phthalates**

The results of many studies strongly point out that that EDC exposure can caused by epigenetic mechanisms, which can lead to cumulative adverse effects on future generations. The epige‐ netic marks can induce up/down alterations in gene expression that may persist throughout a lifetime. These permanent changes will result in adverse health effects, such as neural and immune disorders, infertility, and late-onset complex diseases (cancers and diabetes) [64].

Phthalates are long suggested to be epigenetic carcinogens because of their peroxisome proliferator effects. The *in utero* and neonatal exposure to phthalates [particularly to DEHP, MEHP, benz-butyl phthalate (BBP), DBP, and MBP] may cause methylation changes in DNA at CpG islands near gene promoter regions, different histone modifications (acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP ribosylation), and alterations in the expression of noncoding RNAs, including microRNAs (miRNAs) [65].

The treatment of human breast cancer MCF-7 cells with BBP led to the demethylation of ERα promoter-associated CpG islands, indicating that altered ERα mRNA expression by BBP can induce aberrant DNA methylation in the promoter region of this gene. Maternal exposure to DEHP was shown to induce DNA methylation and different DNA methyltransferase expressions in mouse testis. Fetal testis is suggested to be the main target for DEHP. DEHP can lead to testicular dysgenesis syndrome (TDS) due to a reduction in insulin-like hormone 3 (INSL3) expression and testosterone production [66]. During the period of embryonic sex determination, transient exposure to a plastic mixture (BPA and phthalates) of gestating female rats was shown to promote early-onset puberty transgenerationally (F3 generation) and decrease the pool size of ovarian primordial follicles in female pups. On the contrary, in male pups, spermatogenic cell apoptosis was also affected transgenerationally, and differential DNA methylation of the F3 generation sperm promoter regions was also observed [67].

#### **6.4. Carcinogenicity of phthalates**

Phthalates are well-known peroxisome proliferators that can alter gene and protein expres‐ sions. This capability may result in the promotion of hepatic carcinogenesis in rodents [68]. On the contrary, there are data in the literature that indicate that phthalates increase oxidative stress in the rodent liver even before peroxisomal oxidases are induced. In addition, Kupffer cells have been suggested to be a potential source of oxidants in rodent liver after treatment with DEHP [69, 70]. It appears that molecular events, which may be a consequence of increase oxidative stress, could interact with other pathways activated by peroxisome proliferation in rodent liver [69, 70]. Although several studies including the studies by our group (in the last decade) pointed out that DEHP can induce reactive oxygen species (ROS) production and lead to increased cellular oxidative stress both *in vivo* and *in vitro*, there are no convincing data to prove whether the induction of ROS production is a one of the major pathways or whether ROS elimination is not efficiently achieved after a series of molecular events induced by phthalates, particularly by DEHP [42, 43, 70, 71].

In the 1980s, the hepatocarcinogenic effects of DEHP, due to its peroxisome proliferator effect, was shown by several studies. Back then, some concerns started rising about the safety of this substance. In 2000, the International Agency for Research on Cancer (IARC) classified DEHP as a Group III carcinogen (not classifiable as to its carcinogenicity to humans), as peroxisome proliferation has not been documented in human hepatocyte cultures exposed to DEHP nor in the liver of exposed nonhuman primates [71]. Therefore, the mechanism by which DEHP increases the incidence of hepatocellular tumors in rats and mice is not relevant to humans. However, later in 2013, DEHP was classified as a Group IIB carcinogen due to some concerns [72]. On the contrary, butyl benzyl phthalate (BBP) is classified as a Group III carcinogen in 1999 [73]. Blom et al. [74] showed that exposure to different phthalate esters, particularly to DBP, can lead to high proliferation of human breast cancer cell lines, which was explained in part by the potency of phthalates in terms of a "xenoestrogenic impact," although phthalates are usually classified as "antiandrogens" and not "xenoestrogens" [74, 75]. This effect is suggested to be related to a direct ER binding of some, but not all, phthalates [76, 77].

#### **6.5. Reproductive toxicity of phthalates**

Recent *in vivo* and *in vitro* studies on phthalates are mainly focusing on their reproductive toxicity potential. Phthalates were suggested to target mainly male reproductive system. The "TDS hypothesis" proposes that a proportion of the male reproductive disorders—cryptorch‐ idism, hypospadias, decline infertility (or loss of fertility), and testicular cancer—may be symptoms of TDS, which is most likely a result of disturbed gonadal development in the embryo. There is a decline in male fertility and increase in the number of cases with TDS in the last decades, and phthalates are suggested to be the major underlying factors. Several studies have shown that fetal exposures to DEHP or DBP induce TDS-like effects and reduce anogenital distance (AGD) in rodents [78].

In testis, Leydig and Sertoli cells are the main targets of phthalates. Many researchers observed that DEHP caused disruption in the function of both cell types. The administration of MEHP to Wistar rats at a single oral dose (400 mg/kg body weight) was toxic to Sertoli cells and caused detachment of germ cells [79]. In fact, Richburg and Boekelheide [80] demonstrated histopa‐ thological disturbances and alterations of cytoplasmic distribution of vimentin in Sertoli cells in testis of 28-day-old Fisher rats after a single oral dose of MEHP (2000 mg/kg). Tay et al. [81] also observed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. We also determined that DEHP exposure caused disruption and collapse of vimentin filaments and significantly induced apoptotic death of germ cells [82].

Exposure to phthalates, particularly to DEHP, resulted in decreased testicular testosterone production in rodents, and most of the reprotoxic effects are suggested to be related to their antiandrogenic potential [83, 84]. In our studies, we determined that DEHP caused abnormal sperm production, decreases in sperm count, and motility when administered to 10-week-old rats at 1000 ppm dose for 10 days. Moreover, we also observed that DEHP caused decreases in testosterone, luteinizing hormone (LH), and follicle stimulating hormone (FSH) levels [84]. Moreover, DEHP induced oxidative stress in rat testis, as evidenced by the significant decrease in GSH/GSSG redox ratio, marked increase in lipid peroxidation, and a significant decrease in GPx4 activity [85].

#### **6.6. Hepatotoxic effects of phthalates**

DEHP and other phthalates, such as di-(2-ethylhexyl) adipate (DEHA) and DINP, are shown to be hepatocarcinogenic in both sexes in mice and rats. These substances were shown to cause both hepatocellular carcinomas and adenomas [86–88]. There are a number of molecular events that underlie the hepatocarcinogenic potential of these substances: Their genotoxicity, peroxisome proliferative property, and epigenetic effects are the most studied mechanisms. Collectively, it appears that, in rodent liver, oxidative stress-related molecular events could interact with other pathways that can be activated by peroxisome proliferation. Previously, we have also shown that DEHP caused peroxisome proliferation, alterations in antioxidant enzyme activities (decreases in glutathione peroxidase 1, glutathione peroxidase 4, superoxide dismutase, and glutathione *S*-transferase activities; increase in thioredoxin reductase activity), and liver enzymes when administered to 10-week-old rats at 1000 ppm dose for 10 days. Besides, DEHP caused cellular disorganization, increases in catalase activity/immunoreactiv‐ ity, and lipid peroxidation [89].

#### **6.7. Effects of phthalates on other organs/systems**

Other than testis and liver, phthalates were suggested to be toxic to kidneys and thyroid [90, 91]. Moreover, phthalates were also shown to affect the neuroendocrine system and the hypothalamus-pituitary-ovarian/testicular axis in rats [92–94].

#### **6.8. Epidemiological studies concerning the effects of phthalates on reproductive/endocrine systems**

In the last five decades, a gradual decline in global semen quality has been reported [95]. The effects of phthalate exposure on male fertility has been attracting the attention of researchers for a long period of time, although phthalates exert serious health effects on different organs/ systems. Semen quality defines the sperm count, motility, and morphology. An early study described sperm concentration and DBP measured in the seminal fluid of American students (*N*=21) recruited and these students were classified as low metabolizers (*n*=12) and high metabolizers (*n*= 9). Seminal DBP levels were associated with decreased sperm concentration, and an overall analysis indicated a positive association between DBP and sperm concentration [95]. Total semen phthalates were higher among 21 men with an infertility diagnosis compared to 32 men without (*p*<0.05) in a cross-sectional study of infertility clinic patients in India. Among the infertile men, seminal phthalates were associated with increased sperm abnor‐ mality but not with sperm count or motility [96]. Later, in a cross-sectional study of 234 Swedish military conscripts aged 18 to 21 years, Jönsson et al. [97] observed that urinary MEP concen‐ trations were associated with decreased sperm motility. No associations were determined between semen quality and MBP, MBzP, or MEHP. A positive association between sperm motility and phthalic acid was found, and this finding suggested that an increased ability to metabolize phthalates may be protective. Besides, the researchers reported a cross-sectional association between urinary MEP concentrations and decreased LH levels when comparing the quartile of highest exposure to the lowest [97].

Among 52 Chinese men attending a reproduction clinic in Shanghai, no associations were detected for sperm count or morphology and semen concentrations of DEP, DEHP, and DBP [98]. The majority of studies of phthalate exposure and semen quality have been conducted among infertile populations. In 2006, an American study was conducted on 443 men under‐ going infertility therapy. Higher urinary MBP levels were associated with decreased sperm concentration and motility after adjusting for age, abstinence time, and smoking status. No associations with semen quality were suggested for MEP, MMP, MEHP, MEOHP, or MEHHP [99].

Pant et al. [100] measured the phthalate diesters levels in the semen of 300 Indian men, and correlations were detected between DBP, DEHP, and decreased motility in unadjusted analyses. Unadjusted associations were also reported between DBP, DEHP, DEP, and de‐ creased sperm concentration and for DEHP and increased abnormal morphology. No associ‐ ations were reported for semen DMP or di-*n*-octyl phthalate (DnOP) levels and sperm parameters [100]. Wirth et al. [101] determined the urinary phthalate levels in 45 men who were admitted to an infertility clinic. Urinary MEP levels were associated with decreased sperm concentration (after adjusting for race and for urine specific gravity) and abnormal morphology (after adjusting for urine specific gravity). Urinary mono-3-carboxypropyl phthalate (MCPP) levels were also associated with an increased proportion of morphologically abnormal sperm, and an association was suggested for urinary MEHP with low sperm concentration. However, no associations were indicated for MMP, MBP, MiBP, MBzP, MEHP, MEOHP, and MEHHP [101].

Another study on 349 men who were recruited from a German andrology clinic reported no associations between urinary DEHP metabolites (MEHP, MEHHP, MEOHP, and 5cx-MEPP) and any semen parameters after adjusting for age, smoking, abstinence period, and urine creatinine [102]. In another cross-sectional study, the median semen DBP concentration was higher in oligoasthenospermic men (*n*=65) compared to fertile men (*n*=50), and median DEHP concentration was also higher in oligoasthenospermic men versus in fertile men. In addition, sperm motility was inversely associated with DBP and DEHP in oligoasthenospermic (*n*=65) and asthenospermic (*n*=65) men, respectively. However, the associations were not adjusted for potential confounding variables [103]. Another cross-sectional study recruited 97 men undergoing infertility treatment in China and found the top tertile of urinary MBP to be strongly associated with decreased sperm concentration after adjusting for age, abstinence time, body mass index (BMI), smoking, alcohol consumption, and education. There were no associations reported for sperm parameters and MMP, MEP, MBzP, MEHP, or MEOHP [104]. A more recent work suggested that urinary MBP levels were inversely associated with sperm concentrations in a general population sample of 232 men residing in a heavily industrialized urban area in China. However, no such associations were found for MEP (median=3.10 ng/mL) or MEHP (1.10 ng/mL) or for motility and morphology [105]. A Polish study on men (n=269) under 45 years of age and attending a infertility clinic measured urinary MEHP, MEHHP, monoisononyl phthalate (MiNP), MBzP, MBP, and MEP levels. Inverse associations were detected between sperm motility and log-transformed MEHP, MEHHP, and MiNP levels after adjusting for age, smoking, abstinence period, past diseases, and creatinine as confound‐ ing covariates. No adjusted associations were detected between sperm concentration and morphology and urinary MBzP and MEP levels [106].

There are several studies that were conducted to understand the relationship between sex or reproductive hormones and different phthalate derivatives. One cross-sectional study in China quantified exposure using urinary metabolites in occupationally exposed workers (*n*=74) and unexposed referent workers (*n*=63) matched by age and smoking. Decreases in free testoster‐ one, but not FSH, were associated with MBP exposure in all the men after adjusting for age and alcohol consumption [107]. An earlier work performed on 295 men reported an association between an interquartile range (IQR) increase in urinary MBzP concentrations and decreased FSH levels and a nonsignificant association between an IQR increase in MEHP and decreased testosterone after adjusting for age, BMI, and time of specimen collection [108]. Furthermore, MBP was nonsignificantly associated with increased inhibin B after adjustment for covariates. The results of this study are in contradiction with the results obtained from a study that recruited 118 men seeking infertility treatment in China. Sex steroid hormones were assessed in association with urinary MEP, MBP, MBzP, MEHP, MEHHP, and MEOHP levels among 425 men. The molar sum of DEHP metabolites and percentage MEHP were assessed, and an IQR increase for MEHP was associated with decreased serum estradiol and testosterone levels after adjusting for age, BMI, smoking, and time of specimen collection. A significant association between percentage MEHP and an increase in the testosterone/estradiol ratio was also determined. Adjusted decreases in the free androgen index [FAI; describes the ratio of testosterone to sex hormone binding globulin (SHBG)] were also reported for MEHHP, MEOHP, and the sum of DEHP metabolites. No associations were reported between MBP or

MBzP and serum prolactin, FSH, or LH. However, no associations between concentrations of the other phthalate metabolites and concentrations of the other hormones, including testos‐ terone, estradiol, FSH, inhibin B, and SHBG, were found [109]. Another study performed on 425 men recruited through a U.S. infertility clinic found limited inverse association between MEHP and FAI by the proportion of DEHP metabolites in the urine measured as MEHP (MEHP%), a phenotypic marker of less efficient metabolism of DEHP to its oxidized metabo‐ lites. Finally, the ratio of testosterone to estradiol was positively associated with MEHP and MEHP%, suggesting potential relationships with aromatase suppression [110]. In a crosssectional study of 363 fertile men participating in a multicenter U.S. study, inverse associations were described for urine DEHP metabolites (MEHP, MEHHP, MEOHP, and MECPP) and FAI after adjusting for age, BMI, smoking, ethnicity, urine creatinine, and time of sample collection. A positive correlation was also found between MEHP and SHBG, and the FAI/LH ratio after adjustment for covariates. However, no associations were reported for FSH, estradiol, or LH [111]. A recent study compiled data from two investigations of phthalate exposure and reproductive hormones, combining men from the Massachusetts General Hospital and multicenter U.S. studies. In a combined total of 783 men, there were no associations with urinary concentrations of MEP, MBP, or MBzP and any reproductive hormone measured. However, metabolites of DEHP (MEHP, MEHHP, and MEOHP) were associated with decreased free testosterone and increased SHBG levels [112].

A study performed by our group recruited 40 newly diagnosed pubertal gynecomastia cases and 20 controls. Plasma DEHP and MEHP levels were found to be statistically significantly higher in the pubertal gynecomastia group compared to the control group. There was a statistically significant correlation between plasma DEHP and MEHP levels (*r*=0.58; *P*<0.001). In the pubertal gynecomastia group, no correlation was determined between plasma DEHP and MEHP levels and any of sex hormone levels [24]. However, as a part of the Copenhagen Puberty Study, Mieritz et al. [113] did not find any difference between the urinary phthalate levels of Danish boys (n=555) versus control.
