Mono-2-ethylhexyl phthalate, a metabolite of di-(2-ethylhexyl) phthalate, causally linked to testicular atrophy in rats

Acute testicular atrophy results when appropriate dosages of di-(2-ethylhexyl) phthalate (DEHP) or its hydrolysis product mono-2-ethylhexyl phthalate (MEHP) are given to male rats. Events thought to be involved in this pathological effect also occur in cultures of testicular cells in vitro, but require MEHP rather than DEHP. Primary cultures of hepatocytes, Sertoli cells, and Leydig cells were incubated with 14C-labeled MEHP [8 microM] for up to 24 hr. No significant reduction in viability was produced under these conditions. In contrast to the hepatocytes, which extensively metabolized MEHP to a variety of products in 1 hr, the testicular cell cultures were apparently unable to metabolize MEHP (beyond a slight hydrolysis to phthalic acid by Sertoli cells) in 18-24 hr. MEHP was efficiently taken up by hepatocytes, but much less so by testicular cells. These results, combined with related observations from the literature, support the hypothesis that MEHP itself is the metabolite of DEHP responsible for testicular atrophy in rats.     

Assessment of the teratogenicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in mice

Di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were administered PO or IP to pregnant ICR mice at varying doses on days 7, 8, and 9 of gestation. In groups given DEHP orally, resorptions and malformed fetuses increased significantly at 1,000 mg/kg. Fetal weights were also significantly suppressed. Anterior neural tube defects (anencephaly and exencephaly) were the malformations most commonly produced. No teratogenic effects were revealed by IP doses of DEHP and PO or IP doses of MEHP, although high doses were abortifacient and lethal to pregnant females. Thus DEHP is highly embryotoxic and teratogenic in mice when given PO but not IP. The difference in metabolism, disposition, or excretion by the route of administration may be responsible for the difference in DEHP teratogenicity. Although MEHP is a principal metabolite of DEHP and is several times more toxic than DEHP to adult mice, it seems that MEHP and its metabolites are not teratogenic in ICR mice.     

Quantitative evaluation of alternative mechanisms of blood and testes disposition of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in rats.

Di(2-ethylhexyl) phthalate (DEHP), a commercially important plasticizer, induces testicular toxicity in laboratory animals at high doses. After oral exposure, most of the DEHP is rapidly metabolized in the gut to mono(2-ethylhexyl) phthalate (MEHP), which is the active metabolite for induction of testicular toxicity. To quantify the testes dose of MEHP with various routes of exposure and dose levels, we developed a physiologically based pharmacokinetic (PBPK) model for DEHP and MEHP in rats. Tissue:blood partition coefficients for DEHP were estimated from the n-octanol: water partition coefficient, while partition coefficients for MEHP were determined experimentally using a vial equilibration technique. All other parameters were either found in the literature or estimated from blood or tissue levels following oral or intravenous exposure to DEHP or MEHP. A flow-limited model failed to adequately simulate the available data. Alternative plausible mechanisms were explored, including diffusion-limited membrane transport, enterohepatic circulation, and MEHP ionization (pH-trapping model). In the pH-trapping model, only nonionized MEHP is free to become partitioned into the tissues, where it is equilibrated and trapped as ionized MEHP until it is deionized and released. All three alternative models significantly improved predictions of DEHP and MEHP blood concentrations over the flow-limited model predictions. The pH-trapping model gave the best predictions with the largest value of the log likelihood function. Predicted MEHP blood and testes concentrations were compared to measured concentrations in juvenile rats to validate the pH-trapping model. Thus, MEHP ionization may be an important mechanism of MEHP blood and testes disposition in rats.     

Toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in rats

The toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP, was investigated in Sprague-Dawley rats orally treated with a single dose of 14C-DEHP. Urinary excretion of total 14C-DEHP and of its metabolites was followed by liquid scintillation counting (LSC). Concentrations of DEHP and MEHP were determined 6, 24, and 48 h after treatment in rat serum and 6, 12, 24, and 48 h after treatment in urine by high-performance liquid chromatography (HPLC). After 24 h, peak concentrations of MEHP in both urine and serum were observed in animals treated with 40, 200, or 1000 mg DEHP/kg. HPLC showed that general toxicokinetic parameters, such as Tmax (h), Cmax (microg/ml), Ke (1/h), and AUC (microg-h/ml/) were greater for MEHP than DEHP in both urine and serum. In contrast, the half-lives (t1/2 [h]) of DEHP were greater than those of MEHP. The AUC ratios between DEHP and MEHP were relatively smaller in serum than in urine, suggesting the important role of urinary DEHP data for exposure assessment of DEHP. The toxicokinetic relationship between DEHP and MEHP in rats suggests that DEHP exposure assessment should be based on DEHP and MEHP in urine and serum for risk assessment applications.     

No background concentrations of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in blood of rats

We measured the background levels of di(2-ethylhexyl) phthalate (DEHP) and its hydrolytic metabolite mono(2-ethylhexyl) phthalate (MEHP) in blood from naive female Sprague-Dawley rats and in de-ionized charcoal-purified water using an analytical procedure that is based on sample treatment with acetonitrile, n-hexane extraction and analysis by gas chromatography. In blood, blank values of 91.3 +/- 34.7 micrograms DEHP/l (n = 31) and 30.1 +/- 13.1 micrograms MEHP/l (n = 20) were obtained, and in water, values of 91.6 +/- 44.2 micrograms DEHP/l (n = 26) and 26.7 +/- 10.4 micrograms MEHP/l (n = 15) were found. Since there is no difference between the background valves obtained from blood of naive rats and water, we conclude that DEHP and MEHP result from contamination during the analytical procedure.     

Effect of sterilization process on the formation of mono(2-ethylhexyl)phthalate from di(2-ethylhexyl)phthalate

The risk assessment of di(2-ethylhexyl)phthalate (DEHP) migrating from polyvinyl chloride (PVC) medical devices is an important issue. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags is hydrolyzed by esterase into mono(2-ethylhexyl)phthalate (MEHP). However, MEHP is thought to be even more toxic than the parent compound. Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The limits of quantification (LOQs) of DEHP and MEHP were 2.5 and 0.75 ng/ml, respectively. In this study, the effect of sterilization process on the levels of DEHP and MEHP migration was investigated. The level of migration of DEHP from gamma(gamma)-ray sterilized PVC sheet was low compared with that of the unsterilized control. By contrast, the level of MEHP migration from the gamma-ray sterilized PVC sheet was high compared with that of the unsterilized control. In addition, a high content of MEHP was found in the gamma-ray sterilized PVC sheet.     

Disposition of orally administered di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate in the rat

The dispositon of di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) was studied in the rat. Three hours after a single oral dose of DEHP (2.8 g/kg), plasma concentrations of 8.8±1.7 g/ml DEHP and 63.2±8.7 g/ml MEHP were reached. MEHP levels declined with a half-life of 5.2±0.5 h. The ratio of the area under the plasma concentration-time curve of MEHP to that of DEHP was 16.1±6.1. When ¹⁴CDEHP was administered, 19.3±3.3% of the radioactivity was excreted in the urine within 72 h, the rest being excreted in the faeces. The urinary excretion rate of total radioactivity declined with a half-life of 7.9±0.5 h. Single administration of MEHP (0.4 g/kg) resulted in plasma concentrations of 84.1±14.9 g/ml 3 h after dosing; the half-life of MEHP was 5.5±1.1 h. Multiple dosing with DEHP (2.8 g/kg/day) for 7 consecutive days produced no accumulation of DEHP or MEHP in plasma.     

Distribution and elimination of di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylhexyl phthalate (MEHP) after a single oral administration of DEHP in rats

The distribution and elimination of di-2-ethylhexyl phthalate (DEHP) and mono-2-ethylhexyl phthalate (MEHP) after a single oral administration of DEHP (25 mmol/kg) were studied. A gas-liquid Chromatographic method was used for the simultaneous determination of MEHP and DEHP. The compounds were extracted with methylene chloride and the monoester was alkylated to the hexyl derivative by solid-liquid phase transfer catalysis in methylethyl ketone. The coefficients of variation of this method for determination of DEHP and MEHP were 8.3% and 11.4% respectively. The concentration of DEHP and MEHP in blood and tissues increased to maximum within 6–24 h after dosing, while the highest levels observed in the heart and lungs occurred within 1 h. At 6 h after administration, the highest ratio of MEHP/DEHP (mol%) were recorded in testes (210%) while the other tissues exhibited less than 100%. MEHP disappeared exponentially with t _1/2 values ranging from 23 to 68 h; DEHP t _1/2 ranged from 8 to 156 h and the t _1/2 values of MEHP in several tissues were slightly longer than DEHP. The t _1/2 values in blood were 23.8 h and 18.6 h for MEHP and DEHP, respectively.     

Di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate inhibit growth and reduce estradiol levels of antral follicles in vitro

Any insult that affects survival of ovarian antral follicles can cause abnormal estradiol production and fertility problems. Phthalate esters (PEs) are plasticizers used in a wide range of consumer and industrial products. Exposure to these chemicals has been linked to reduced fertility in humans and animal models. Di-(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) decrease serum estradiol levels and aromatase (Arom) expression, prolong estrous cycles, and cause anovulation in animal and culture models. These observations suggest PEs directly target antral follicles. We therefore tested the hypothesis that DEHP (1-100 μg/ml) and MEHP (0.1-10 μg/ml) directly inhibit antral follicular growth and estradiol production. Antral follicles from adult mice were cultured with DEHP or MEHP, and/or estradiol for 96 h. During culture, follicle size was measured every 24 h as a measurement of follicle growth. After culture, media were collected for measurement of estradiol levels and follicles were subjected to measurement of cylin-D-2 (Ccnd2), cyclin-dependant-kinase-4 (Cdk4), and Arom. We found that DEHP and MEHP inhibited growth of follicles and decreased estradiol production compared to controls at the highest doses. DEHP and MEHP also decreased mRNA expression of Ccnd2, Cdk4, and Arom at the highest dose. Addition of estradiol to the culture medium prevented the follicles from DEHP- and MEHP-induced inhibition of growth, reduction in estradiol levels, and decreased Ccnd2 and Cdk4 expression. Collectively, our results indicate that DEHP and MEHP may directly inhibit antral follicle growth via a mechanism that partially includes reduction in levels of estradiol production and decreased expression of cell cycle regulators.     

Pharmacokinetics and effects on serum cholinesterase activities of organophosphorus pesticides acephate and chlorpyrifos in chimeric mice transplanted with human hepatocytes

Organophosphorus pesticides acephate and chlorpyrifos in foods have potential to impact human health. The aim of the current study was to investigate the pharmacokinetics of acephate and chlorpyrifos orally administered at lowest-observed-adverse-effect-level doses in chimeric mice transplanted with human hepatocytes. Absorbed acephate and its metabolite methamidophos were detected in serum from wild type mice and chimeric mice orally administered 150 mg/kg. Approximately 70% inhibition of cholinesterase was evident in plasma of chimeric mice with humanized liver (which have higher serum cholinesterase activities than wild type mice) 1 day after oral administrations of acephate. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated plasma concentrations of acephate and chlorpyrifos in humans were consistent with reported concentrations. Acephate cleared similarly in humans and chimeric mice but accidental/incidental overdose levels of chlorpyrifos cleared (dependent on liver metabolism) more slowly from plasma in humans than it did in mice. The data presented here illustrate how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of toxicological potential of organophosphorus pesticides.     

Phthalate and di-(2-ethylhexyl) adipate (DEHA) intake by German infants based on the results of a duplicate diet study and biomonitoring data (INES 2)

Phthalates as well as di-(2-ethylhexyl) adipate (DEHA) are used as plasticizers in diverse applications and are of toxicological concern.The study was conducted with a study population of 25 German subjects aged between 15 and 21 months. Overall, 16 phthalates and DEHA were measured by gas chromatography–mass spectrometry in a total of 171 duplicate diet samples collected over 7 consecutive days, and 20 phthalate metabolites were analyzed in the urine samples collected over 7 consecutive days using a liquid chromatography–tandem mass spectrometry method.The median “high” daily dietary intake based on 95th percentiles was 4.66 μg/kg b.w. for di-2-ethylhexyl phthalate (DEHP), 1.03 μg/kg b.w. for di-isobutyl phthalate (DiBP), and 0.70 μg/kg b.w. for di-n-butyl phthalate (DnBP), and 1.0 μg/kg b.w. for DEHA. The “high” daily total intake from biomonitoring data was 4.9 μg/kg b.w. for DEHP, 2.2 μg/kg b.w. for DnBP, 3.9 μg/kg b.w. for DiBP, and 2.6 μg/kg b.w. for di-isononyl phthalate.The comparison of the two intake estimates indicates that the dominant intake source of DEHP was food ingestion, whereas other sources considerably contributed to the total intake of other phthalates. Using our “high” intake scenario, none of the analyzed phthalates reached the recommended tolerable daily intake levels.     

Kinetics of orally administered di(2-ethylhexyl) phthalate and its metabolite,mono(2-ethylhexyl) phthalate,in male pigs

Di(2-ethylhexyl) phthalate (DEHP) is used as a plastic softener in the polymer industry and is widespread in medical devices. DEHP has been incriminated as an endocrine-disrupting chemical, and the effects of DEHP in various species have included disturbances in the reproductive system. The effects of the chemical have varied, depending upon exposure routes and species. This study was performed in order to characterise the kinetics of DEHP and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in the young male pig, an omnivore model-species for research in reproductive toxicology. Eight pigs were given 1000 mg DEHP/kg bodyweight by oral gavage. The concentrations of DEHP and MEHP were then measured in the plasma and tissues of the pigs at different time points after administration. There was no consistent rise above contamination levels of concentrations of DEHP in the plasma of the pigs. However, the metabolite MEHP reached the systemic blood circulation. The half-life of MEHP in the systemic blood circulation was calculated to be 6.3 h. Absorption from the intestine was biphasic in six of the eight pigs and the mono-exponential elimination-phase started 16 h after the after the administration of DEHP. To conclude, MEHP consistently reaches the systemic circulation in the pig when DEHP is administered orally. The kinetic pattern of the parent substance on the other hand is more difficult to characterise.     

Blood burden of di(2-ethylhexyl) phthalate and its primary metabolite mono(2-ethylhexyl) phthalate in pregnant and nonpregnant rats and marmosets

A comparison of the dose-dependent blood burden of di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP) in pregnant and nonpregnant rats and marmosets is presented. Sprague-Dawley rats and marmosets were treated orally with 30 or 500 mg DEHP/kg per day, nonpregnant animals on 7 (rats) and 29 (marmosets) consecutive days, pregnant animals on gestation days 14-19 (rats) and 96-124 (marmosets). In addition, rats received a single dose of 1000 mg DEHP/kg. Blood was collected up to 48 h after dosing. Concentrations of DEHP and MEHP in blood were determined by GC/MS. In rats, normalized areas under the concentration-time curves (AUCs) of DEHP were two orders of magnitude smaller than the normalized AUCs of the first metabolite MEHP. Metabolism of MEHP was saturable. Repeated DEHP treatment and pregnancy had only little influence on the normalized AUC of MEHP. In marmosets, most of MEHP concentration-time courses oscillated. Normalized AUCs of DEHP were at least one order of magnitude smaller than those of MEHP. In pregnant marmosets, normalized AUCs of MEHP were similar to those in nonpregnant animals with the exception that at 500 mg DEHP/kg per day, the normalized AUCs determined on gestation days 103, 117, and 124 were distinctly smaller. The maximum concentrations of MEHP in blood of marmosets were up to 7.5 times and the normalized AUCs up to 16 times lower than in rats receiving the same daily oral DEHP dose per kilogram of body weight. From this toxicokinetic comparison, DEHP can be expected to be several times less effective in the offspring of marmosets than in that of rats if the blood burden by MEHP in dams can be regarded as a dose surrogate for the MEHP burden in their fetuses.     

Mono-(2-ethylhexyl) phthalate (MEHP) affects intercellular junctions of Sertoli cell: A potential role of oxidative stress

We analyzed the potential role of oxidative stress induced by mono (2-ethylhexyl) phthalate (MEHP) in adherent cell junction protein expression of prepubertal rat Sertoli cells (SC) in vitro. Five-day SC cultures were treated with MEHP (200 μM) for 24 h and compared to cells in basal conditions. Western blot and immunofluorescent (IF) analyses showed that MEHP induced increase of N-cadherin and catenin expression, modifying its distribution. Concomitantly, Cx-43 expression decreased significantly and delocalization of the IF signal for tight junction proteins (occludin, claudin-11 and ZO-1) occurred. Indicative of oxidative stress, MEHP induced in SC an increase of lipoperoxides, a decrease in glutathione (GSH) levels and a concomitant increase in Glutathione S-Transferases (GST) activity. Antioxidant N-acetyl-cysteine (1 mM) treatment prevented GSH decrease and N-cadherin and α-catenin up-regulation induced by MEHP. Our data suggest that oxidative stress signaling is a mechanism involved in adherent cell junctions disruption induced by MEHP in SC cultures.     

Induction of propranolol metabolism in isolated rats hepatocytes treated by di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP).

Blood lines of polyvinyl chloride (PVC) for hemodialysis usually contain di(2-ethylhexyl) phthalate (DEHP) as a plasticizer. Previous studies show that 1 mg/kg of this plasticizer can leach into the blood during one dialysis session. It is rapidly metabolized in the liver. Mono(2-ehtylhexyl) phthalate (MEHP), its main metabolite can be detected as well. After oral administration to rodents, both compounds caused a variety of adverse biological effects such as testicular atrophy, peroxisome proliferation and hepatic peroxisomal enzyme induction. Male wistar rats were treated intraperitoneally by DEHP and MEHP using twice the dose of that involved in human exposure during a dialysis session. Propranolol metabolism by hepatocytes was investigated after fresh isolation from treated and untreated rats by means of reverse phase HPLC. The choice of propranolol as a substrate was made because of its rather quick liver metabolisation. Phenobarbital was chosen in the study as a reference of enzymatic inducer to evaluate the inducing effect of DEHP and MEHP. Propranolol was metabolized by the hepatocytes of both treated and untreated rats. Hepatocytes isolated from rats treated by phenobarbital, MEHP and DEHP were shown to have a higher speed constant of metabolism indicating a rapid metabolism of propranolol. Under these conditions, in fact, propranolol metabolisation was found to be respectively 6, 2.7, 2 times faster than the propranolol metabolisation of untreated rats. The hypothesis that DEHP and MEHP are enzymatic inducers, particularly cytochrome P450 (CYP) inducers of the xenobiotics metabolism on the intact liver after IP administration has become been found to be valid. The results obtained in this study confirm the value of isolated hepatocytes as an in vivo drug metabolism predictive model.     

Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate

Two studies were designed to examine amniotic fluid and maternal urine concentrations of the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethylhexyl) phthalate (MEHP) and the di-n-butyl phthalate (DBP) metabolite monobutyl phthalate (MBP) after administration of DEHP and DBP during pregnancy. In the first study, pregnant Sprague-Dawley rats were administered 0, 11, 33, 100, or 300 mg DEHP/kg/day by oral gavage starting on gestational day (GD) 7. In the second study, DBP was administered by oral gavage to pregnant Sprague-Dawley rats at doses of 0, 100, or 250 mg/kg/day starting on GD 13. Maternal urine and amniotic fluid were collected and analyzed to determine the free and glucuronidated levels of MEHP and MBP. In urine, MEHP and MBP were mostly glucuronidated. By contrast, free MEHP and free MBP predominated in amniotic fluid. Statistically significant correlations were found between maternal DEHP dose and total maternal urinary MEHP (p=0.0117), and between maternal DEHP dose and total amniotic fluid MEHP levels (p=0.0021). Total maternal urinary MEHP and total amniotic fluid MEHP levels were correlated (Pearson correlation coefficient=0.968). Statistically significant differences were found in amniotic MBP levels between animals within the same DBP dose treatment group (p<0.0001) and between animals in different dose treatment groups (p<0.0001). Amniotic fluid MBP levels increased with increasing DBP doses, and high variability in maternal urinary levels of MBP between rats was observed. Although no firm conclusions could be drawn from the urinary MBP data, the MEHP results suggest that maternal urinary MEHP levels may be useful surrogate markers for fetal exposure to DEHP.     

Effects of mono(2-ethylhexyl)phthalate on cytoplasmic maturation of oocytes – The bovine model

Phthalates are known reproductive toxicants, but their intracellular disruptive effects on oocyte maturation competence are less known. We studied the potential risk associated with acute exposure of oocytes to mono(2-ethylhexyl)phthalate (MEHP). First, bovine oocytes were matured in vitro with or without 50 μM MEHP and examined for mitochondrial features associated with DNA fragmentation. MEHP increased reactive oxygen species levels and reduced the proportion of highly polarized mitochondria along with alterations in genes associated with mitochondrial oxidative phosphorylation (CYC1, MT-CO1 and ATP5B). In a second set of experiments, we associated the effects of MEHP on meiotic progression with those on cytoplasmic maturation. MEHP impaired reorganization of cytoplasmic organelles in matured oocytes reflected by reductions in category I mitochondria, type III cortical granules and class I endoplasmic reticulum. These alterations are associated with the previously reported reduced developmental competence of MEHP-treated bovine oocytes, and reveal the risk associated with acute exposure.     

Non-linearities in the pharmacokinetics of di-(2-ethylhexyl) phthalate and metabolites in male rats

The disposition of the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and four of its major metabolites was studied in male rats given single infusions of a DEHP emulsion in doses of 5, 50 or 500 mg DEHP/kg body weight. Plasma concentrations of DEHP and metabolites were followed for 24 h after the start of the infusion. The kinetics of the primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) was studied separately.The concentrations of DEHP in plasma were at all times considerably higher than those of MEHP, and the concentrations of MEHP were much higher than those of the other investigated metabolites. In animals given 500 mg DEHP/kg, the areas under the plasma concentration-time curves (AUCs) of the other investigated metabolites were at most 15% of that of MEHP. Parallel decreases in the plasma concentrations of DEHP, MEHP and the and (-1) oxidized metabolites indicated that the elimination of DEHP was the rate-limiting step in the disposition of the metabolites. This was partly supported by the observation that the clearance of MEHP was higher than that of DEHP. Nonlinear increases in the AUCs of DEHP and MEHP indicated saturation in the formation as well as the elimination of the potentially toxic metabolite MEHP.     

Di-(2-ethylhexyl) phthalate induces apoptosis of GC-2spd cells via TR4/Bcl-2 pathway

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used environmental endocrine disruptor. Many studies have reported that DEHP exposure causes reproductive toxicity and cells apoptosis. However, the mechanism by which DEHP exposure causes male reproductive toxicity remains unknown. This study investigated the role of the testicular orphan nuclear receptor4 (TR4)/Bcl-2 pathway in apoptosis induced by DEHP, which resulted in reproductive damage. To elucidate the mechanism underpinning the male reproductive toxicity of DEHP, we sought to investigate apoptotic effects, expression levels of TR4/Bcl-2 pathway in GC-2spd cells, including TR4, Bcl-2 and caspase-3. GC-2spd cells were exposed to various concentrations of DEHP (0, 50, 100, or 200 μM). The results indicated that, with the increase of the concentrations of DEHP, the survival rate of cell decreased gradually. DEHP exposure at over 100 μM significantly induced apoptotic cell death. DEHP decreased SOD and GSH-Px activity in 200 μM group. Compared to the control group, the mRNA levels of caspase-3 increased significantly, however, Bcl-2 mRNA decreased (P < 0.05). In addition, there was a significant reduction in TR4, Bcl-2 and procaspase-3 protein levels. Taken together, these results lead us to speculate that in vitro exposure to DEHP might induce apoptosis in GC-2spd cells through the TR4/Bcl-2 pathway.