Di(2‐ethylhexyl) phthalate induces apoptosis through mitochondrial pathway in GC‐2spd cells

Di(2‐ethylhexyl) phthalate (DEHP), a plasticizer of synthetic polymers, is a well‐known endocrine disrupting chemical (EDC) and reproductive toxicant. Addressing the unclear mechanism of DEHP‐induced reproductive dysfunction, this study used GC‐2spd cells to investigate the molecular mechanism involved in the DEHP‐induced toxicity in the male reproductive system. The results indicated that the apoptotic cell death was significantly induced by DEHP exposure over 100 μM. Furthermore, DEHP treatment could induce oxidative stress in GC‐2spd cells involving in the decrease of superoxide dismutase (SOD) activity (200 μM) and glutathione peroxidase (GSH‐Px) activity (50 and 100 μM). In addition, DEHP induction also caused the elevated ratios of Bax/Bcl‐2, release of cytochrome c and decomposition of procaspase‐3 and procaspase‐9 in GC‐2spd cells. Taken together, our work provided the evidence that DEHP exposure might induce apoptosis of GC‐2spd cells via mitochondria pathway mediated by oxidative stress. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1055–1064, 2017.     

Calponin gene expression in Chironomus riparius exposed to di(2‐ethylhexyl) phthalate

Di(2‐ethylhexyl) phthalate, which is a ubiquitous environmental contaminant because of its extensive use as a plasticizer, is a potential nongenotoxic carcinogen. To assess the effects of DEHP exposure on the cytoskeleton of Chironomus, we characterized full‐length cDNA sequences of the calponin gene from Chironomus riparius. The expression of the calponin gene was analyzed during different life‐history stages and under various DEHP concentrations for short and long periods. A phylogenetic investigation was then conducted to compare different orders of insects using sequence database analysis. The complete cDNA sequence of the calponin gene was found to be 555 bp in length. The results of phylogenetic analysis revealed that C. riparius calponin is most closely related to that of beetles. The basal level of calponin mRNA was highly expressed during different life‐history stages. In addition, calponin gene expression decreased within 1 h of short‐term exposure to DEHP, regardless of the concentration. We also investigated expression of the calponin gene following long‐term exposure (10 days). Calponin gene expression was found to decrease significantly in C. riparius that were exposed to a low dose of DEHP, and this response was found to occur in a dose‐dependent manner. Taken together, these results suggest that DEHP affects the functions of Ca²⁺ binding muscle proteins such as calponin in Chironomus species. © 2008 Wiley Periodicals, Inc. Environ Toxicol, 2009.     

Di‐2‐ethylhexyl phthalate (DEHP) induces apoptosis and autophagy of mouse GC‐1 spg cells

As a widely used plasticizer in industry, di‐2‐ethylhexylphthalate (DEHP) can cause testicular toxicity, yet little is known about the potential mechanism. In this study, DEHP exposure dramatically inhibited cellviability and induced apoptosis of mouse GC‐1 spg cells. Furthermore, DEHP significantly increased the levels of autophagy proteins LC3‐II, Beclin1 and Atg5, as well as the ratio ofLC3‐II/LC3‐I. Transmission electron microscopy (TEM) further confirmed that DEHP induced autophagy of mouse GC‐1 spg cells. DEHP was also shown to induceoxidative stress; while inhibition of oxidative stress with NAC could increase cell viability and inhibit DEHP‐induced apoptosis and autophagy. These results suggested that DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress. 3‐MA, an inhibitor of autophagy, could rescue DEHP‐induced apoptosis. In summary, DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress, and autophagy might exert a cytotoxic effect on DEHP‐induced apoptosis.     

Synthesis of 18F‐labeled cyclooxygenase‐2 (COX‐2) inhibitor as a potential PET imaging agent

A new PET tracer for COX‐2 imaging, the 6‐ethoxy‐3‐(4‐methanesulfonylphenyl)‐4‐(4‐[¹⁸F]fluorophenyl)pyran‐2‐one ([¹⁸F]EFMP), was synthesized. For F‐18 radiolabeling, a trimethylammonium precursor and a brominated precursor were synthesized from 1,1,2,3‐tetrachlorocycloprop‐2‐ene in 6 steps. The radiolabeling was achieved through nucleophilic substitution using no‐carrier‐added (n.c.a.) fluorine‐18. Solid‐phase extraction and semi‐preparative‐HPLC purification produced [¹⁸F]EFMP in 14.6±3.3% (n =4) decay corrected radiochemical yield with a specific activity of 487±85.1 (n =4) Ci/mmol and greater than 98% radiochemical purity. Copyright © 2006 John Wiley & Sons, Ltd.     

Reduced camptothecin sensitivity of estrogen receptor‐positive human breast cancer cells following exposure to di(2‐ethylhexyl)phthalate (DEHP) is associated with DNA methylation changes

Di(2‐ethylhexyl)phthalate (DEHP) has been considered as an estrogen receptor alpha (ERα) agonist due to its ability to interact with ERα and promote the cell proliferation of ERα‐positive breast cancer cells. The impact of DEHP on the chemical therapy in breast cancer is little known. Two breast cancer cell lines, MCF‐7 (ERα‐dependent) and MDA‐MB‐231 (ERα‐independent) were examined. We found that DEHP impaired the effectiveness of camptothecin (CPT) and alleviated the CPT‐induced formation of reactive oxygen species in ERα‐positive MCF‐7 cells, but not in ERα‐negative MDA‐MB‐231 cells. DEHP also significantly protected MCF‐7 cells against the genotoxicity of CPT. Genome‐wide DNA methylation profiling revealed that after 48 hours of exposure to 100 μM DEHP, MCF‐7 cells exhibited a significant change in their DNA methylation pattern, including hypermethylation of 700 genes and hypomethylation of 221 genes. The impaired therapeutic response to CPT in DEHP‐exposed MCF‐7 cells is probably mediated by epigenetic changes, especially through Wnt/β‐catenin signaling. A zebrafish xenograft model confirmed the disruptive effect of DEHP on CPT‐induced anti‐growth of MCF‐7 cells. In summary, DEHP exposure induces acquired CPT‐resistance in breast cancer cells and epigenetic changes associated with Wnt/β‐catenin signaling activation are probably depending on an ER‐positive status.     

The effects of di 2-ethyl hexyl phthalate (DEHP) on cellular lipid accumulation in HepG2 cells and its potential mechanisms in the molecular level

Diethylhexyl phthalate (DEHP) is suspected to be an inevitable factor related to metabolic disease. Our previous study demonstrated that excess DEHP could exacerbate non-alcoholic fatty liver disease (NAFLD) in SD rats. Addressing the terra incognita in DEHP-induced metabolic dysfunction, this study used HepG2 cells to investigate the potential mechanisms involved in DEHP-induced toxicity in vitro. The cells were established lipid overload model with oleic acid and BSA, then exposed to different concentrations (5, 10, 25, 50, 100?μmol/l DEHP) of DEHP for further analysis. The Oil Red O staining results showed that DEHP could promote lipid accumulation in cells. The level of superoxide dismutase (SOD) and malondialdehyde (MDA) changed suggested the balance of oxidative stress was disrupted. Additionally, western blot analysis showed that DEHP could promote the expression of peroxisome proliferator-activated receptor α (PPARα) and sterol regulatory element-binding protein 1c (SREBP-1c). By quantifying the expressions of the two proteins, it is of interest to determine that DEHP could promote lipid accumulation in hepatocytes via activating the SREBP-1c and PPARα-signaling pathway.     

Nonylphenol regulates cyclooxygenase‐2 expression via Ros‐activated NF‐κB pathway in sertoli TM4 cells

The aim of this study was to investigate the signaling pathways involved in the cyclooxygenase (COX)‐2 regulation induced by nonylphenol (NP) in mouse testis Sertoli TM4 cells. Our results showed that treatment of TM4 cells with NP increased COX‐2 protein expression and interleukin‐6 (IL)‐6 and prostaglandin E2 (PGE2) secretion in a dose‐dependent manner. Pretreatment with reactive oxygen species (ROS) scavenger, N‐acetylcysteine (NAC), attenuated NP‐induced ROS production, COX‐2 expression, and IL‐6 and PGE2 release in TM4 cells. Exposure to NP stimulated activation of NF‐κB, whereas the NF‐κB inhibitor, pyrrolidine dithiocarbamate, attenuated NP‐enhanced COX‐2 expression and IL‐6 and PGE2 release in TM4 cells in a dose‐dependent manner. Furthermore, NAC blocked NP‐induced activation of NF‐κB. In addition, inhibition of COX‐2 mitigated NP‐induced IL‐6 release. In conclusion, NP induced ROS generation, activation of NF‐κB pathway, COX‐2 upregulation, and IL‐6 and PGE2 secretion in TM4 cells. NP may regulate COX‐2 expression via ROS‐activated NF‐κB pathway in Sertoli TM4 cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1144–1152, 2015.     

Effect of natsudaidain isolated from Citrus plants on TNF‐α and cyclooxygenase‐2 expression in RBL‐2H3 cells

Objectives Flavonoids inhibit the activity of chemical mediators released from mast cells. Our aim was to investigate the effects of natsudaidain, a polymethoxyflavone isolated from Citrus plants, on mast cells. Methods We investigated the inhibitory effects of natsudaidain, which is a polymethoxy‐flavone isolated from Citrus plants, on histamine release, tumour necrosis factor‐α production and cyclooxygenase‐2 expression in Ca ionophore‐stimulated rat basophilic leukemia cells (A23187‐stimulated RBL‐2H3 cells) by spectrofluorometric, ELISA and immunoblotting methods. Key findings The percent of histamine release from A23187‐stimulated RBL‐2H3 cells pretreated with natsudaidain at 5, 25 and 50 μM was not changed as compared with non‐treated A23187‐stimulated cells. At 100 and 200 μM, natsudaidain pretreatment resulted in slightly reduced histamine release (% histamine release, 89.8 ± 3.5% and 71.5 ± 5.6% at 100 and 200 μM). Thus, natsudaidain hardly affects histamine release from RBL‐2H3 cells, except at high concentrations. On the other hand, natsudaidain dose‐dependently inhibited tumour necrosis factor‐α protein and mRNA levels in A23187‐stimulated RBL‐2H3 cells; a concentration of 6.8 μM was required for a 50% reduction. In addition, all concentrations of this compound that we tested also inhibited cyclooxygenase‐2 protein expression. The mRNA levels of cyclooxygenase‐2 in A23187‐stimulated RBL‐2H3 cells treated with natsudaidain were also markedly decreased. The phosphorylated‐p38 MAPK protein levels in A23187‐stimulated RBL‐2H3 cells treated with natsudaidain were lower than in the non‐treated cells. Conclusions These findings suggest that natsudaidain inhibits tumour necrosis factor‐α and cyclooxygenase‐2 production by suppressing p38 MAPK phosphorylation but not p65 NFKB phosphorylation, and that natsudaidain might alleviate inflammatory diseases.     

Long‐term exposure to bis(2‐ethylhexyl)phthalate (DEHP) inhibits growth of guppy fish (Poecilia reticulata)

Bis(2‐ethylhexyl)phthalate (DEHP) is a widely used plasticizer that is a commonly found contaminant of aquatic environments. However, little is known about the long‐term effects of DEHP on fish development, as previous studies yielded conflicting results and mostly investigated the effects of concentrations higher than those found in natural habitats. We thus aimed to investigate the effects of DHEP (i) at concentrations present in the environment, and (ii) under conditions that might accentuate any deleterious consequences (larvae rather than adult fish, use of higher temperature). Different concentrations of DEHP (0.1–10 µg l^?1rpar; applied continuously for 91 days were tested on guppy fish that were less than one week old at the beginning of the treatment. As early as 14 days after the start of exposure, guppies treated with 10 µg l^?1 DEHP showed significantly reduced body length as compared with control fish. The inhibitory effect of DEHP was concentration‐dependent and increased with time, leading to a maximal reduction in body length of 15 and 40% at 1 and 10 µg l^?1 DEHP, respectively. The effect was even more pronounced for body weight, which was diminished by up to 40 and 70% at 1 and 10 µg l^?1 DEHP, respectively. The reduction in growth was still significant at 91 days of DEHP treatment, whereas the Fulton's condition factor was unaffected. While DEHP significantly blocked growth in both male and female guppies, no shift in the sexual development was observed. These data show that DEHP, at concentrations present in aquatic environments, can profoundly affect development in fish. Copyright © 2009 John Wiley & Sons, Ltd.     

Chronic toxicity of di(2-ethylhexyl)phthalate in mice.

B6C3F1 mice were treated with 0, 100, 500, 1500, or 6000 ppm di(2-ethylhexyl)phthalate (DEHP) in the diet for up to 104 weeks. Blood and urine were analyzed at Weeks 26, 52, 78, and 104 from 10 animals per sex per group. Body weights and food consumption were measured weekly for the first 16 weeks, then monthly thereafter. Survival was reduced for mice receiving 6000 ppm DEHP. Overall weight gains were significantly lower for the 6000-ppm male group, but there was no difference among female groups. Food consumption was not affected by exposure. No biologically significant changes in clinical chemistry, hematology, or urinalysis were observed. After 104 weeks of exposure, kidney weights for the 500- and 1500-ppm male, and 6000-ppm male/female groups were significantly lower than for the controls. Significantly higher liver weight was seen for the 500-, 1500-, and 6000-ppm male groups and the 6000-ppm female group of mice. Testis weights for the 500-, 1500-, and 6000-ppm males were significantly lower than for the controls. Uterine weights for the 6000-ppm group were significantly lower than for the controls. All organs were examined for histopathology. The incidence of hepatocellular lesions has been reported separately (R. M. David et al., 1999. Toxicol. Sci. 50, 195-205). Tumors were observed at > or = 500-ppm dosages, where peroxisome proliferation was significantly increased. A NOEL for both tumors and peroxisome proliferation was 100 ppm. In the study presented here, bilateral hypospermia in the testes of male mice, hepatocyte pigmentation and cytoplasmic eosinophilia in the liver, and chronic progressive nephropathy of male and female mice were observed at 6000 ppm. Hypospermia and chronic progressive nephropathy were also observed at 1500 ppm, where peroxisome proliferation was 2.7-6.8-fold higher than controls. Many lesions observed in rats were not seen in mice. A dose level of 500 ppm (98.5-116.8 mg/kg/day) was identified as a no-observed-adverse-effect level (NOAEL) for noncarcinogenic effects.     

Chronic toxicity of di(2-ethylhexyl)phthalate in rats.

Fischer 344 rats were treated with 0, 100, 500, 2500, or 12,500 ppm di(2-ethylhexyl)phthalate (DEHP) in the diet for up to 104 weeks. Blood and urine were analyzed at weeks 26, 52, 78, and 104 from 10 animals per sex per group. Survival was slightly but not statistically reduced for rats receiving 12,500 ppm DEHP. Body weights and food consumption were significantly reduced for rats receiving the highest dose level of DEHP and occasionally for the male 2500-ppm group. BUN and albumin were significantly higher and globulin lower at nearly every sampling interval for the 12,500-ppm group compared with the controls. There was an increase in the mean activities of AST and ALT at 104 weeks, but no statistically significant differences were seen. Erythrocyte count, hemoglobin, and hematocrit values for the 12,500-ppm group were significantly lower than controls at nearly every sampling interval. No other differences in hematology were seen. No toxicologically significant changes were observed in urinalysis. At termination, relative lung weights for the 2500- and 12,500-ppm male groups of rats were significantly higher than for the controls. Absolute and relative liver and kidney weights for the 2500- and 12,500-ppm male rats, and liver weights for 12,500-ppm female rats were higher compared with the controls. Absolute and relative testes weights for the 12, 500-ppm male rats were lower compared with the controls. All organs were examined for histopathology. The incidence of hepatocellular lesions has been reported separately and correlated with the induction of peroxisomal enzyme activity (David et al., 1999). A dose level of 500 ppm was the NOEL for peroxisome proliferation. Bilateral aspermatogenesis in the testes, castration cells in the pituitary gland, spongiosis hepatis, and pancreatic acinar cell adenoma were observed for 12,500-ppm male rats. Aspermatogenesis and spongiosis hepatis were observed for 2500-ppm male rats, and aspermatogenesis was seen at 500 ppm. DEHP exposure exacerbated age-, species- or strain-related lesions such as mineralization of the renal papilla and chronic progressive nephropathy in male rats. Kupffer cell pigmentation and renal tubule pigmentation were seen in male and female 12,500-ppm rats. The increased incidence of spongiosis hepatis correlated with increased palmitoyl CoA oxidase activity, but the incidence of pancreatic acinar cell adenoma was increased only at the highest dose level of 12,500 ppm. These lesions, although typical of those seen with other peroxisome proliferators, may respond differently depending on the potency of the peroxisome proliferator. A dose level of 500 ppm (28.9-36.1 mg/kg/day) was considered to be the NOAEL.     

Di‐(2‐ethylhexyl) phthalate (DEHP)‐induced testicular toxicity through Nrf2‐mediated Notch1 signaling pathway in Sprague–Dawley rats

Di‐(2‐ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor widely used in China that is harmful to the male reproductive system. Many studies have shown that DEHP causes testicular toxicity through oxidative stress, but the specific mechanism is unknown. Because the Notch pathway is a key mechanism for regulating cell growth and proliferation, we investigated whether Notch is involved in DEHP‐induced testicular toxicity and whether vitamins E and C could rescue testicular impairment in Sprague–Dawley (SD) rats. Compared with the control group, we found that DEHP exposure induced testicular toxicity through oxidative stress injury, and it decreased the testosterone level (P < .01) and upregulated nuclear factor‐erythroid 2 related factor (Nrf2) expression (P < .01). Therefore, because oxidative stress might be the initiating factor of DEHP‐induced testicular toxicity, treatment with the antioxidant vitamins E and C activated the Notch1 signaling pathway in the testis and in Leydig cells. Treatment with vitamins E and C normalized the oxidative stress state after DEHP exposure and restored testicular development to be similar to the control group. In summary, antioxidant vitamins E and C may be used to treat DEHP‐induced testicular toxicity.     

Synthesis of 2‐(methylsulfonyl)‐5‐(4‐(methylsulfonyl) phenyl)‐4‐phenyl‐1H‐[5‐14C]imidazole,a selective COX‐2 inhibitor,via asymmetrical benzoins

4,5‐Diarylimidazoles labeled with carbon‐14 in the 5‐position of the imidazole ring were prepared as a part of three‐step sequence from 2‐hydroxy‐1‐(4‐(methylthio)phenyl)‐2‐phenyl[1‐¹⁴C]ethanone as a key synthetic intermediate which has been synthesized from potassium [¹⁴C]cyanide.     

Design and synthesis of pyrazole–pyrazoline hybrids as cancer‐associated selective COX‐2 inhibitors

In continuation of our previous work on cancer and inflammation, 15 novel pyrazole–pyrazoline hybrids ( WSPP1 – 15 ) were synthesized and fully characterized. The formation of the pyrazoline ring was confirmed by the appearance of three doublets of doublets in ¹H nuclear magnetic resonance spectra exhibiting an AMX pattern for three protons (H_A, H_M, and H_X) of the pyrazoline ring. All the synthesized compounds were screened for their in vitro anticancer activity against five cell lines, that is, MCF‐7, A549, SiHa, COLO205, and HepG2 cells, using the MTT growth inhibition assay. 5‐Fluorouracil was taken as the positive control in the study. It was observed that, among them, WSPP11 was found to be active against A549, SiHa, COLO205, and HepG2 cells, with IC₅₀ values of 4.94, 4.54, 4.86, and 2.09 µM. All the derivatives were also evaluated for their cytotoxicity against HaCaT cells. WSPP11 was also found to be nontoxic against normal cells (cell line HaCaT), with an IC₅₀ value of more than 50 µM. The derivatives were also evaluated for their in vitro anti‐inflammatory activity by the protein (egg albumin) denaturation assay and the red blood cell membrane stabilizing assay, using diclofenac sodium and celecoxib as standard. Compounds that showed significant anticancer and anti‐inflammatory activities were further studied for COX‐2 inhibition. The manifestation of a higher COX‐2 selectivity index of WSPP11 as compared with other derivatives and an in vitro anticancer activity against four cell lines further established that compounds that were more selective toward COX‐2 also exhibited a better spectrum of activity against various cancer cell lines.     

miR‐375‐3p/YWHAZ/β‐catenin axis regulates migration,invasion, EMT in gastric cancer cells

YWHAZ (14‐3‐3ζ) plays crucial roles in regulating proliferation, apoptosis, migration, and invasion of gastric cancer (GC) cells. However, its extensive roles and potential mechanisms in GC cells remain unknown, and need to be researched deeply. In this study, we focus on the role of miR‐375/YWHAZ axis in migration, invasion and epithelial‐to‐mesenchymal transition (EMT) of GC cells. YWHAZ level was assessed by western blot and qPCR assays in GC cells. Scratch and transwell assays were used to determine the migration and invasion of GC cells. The protein levels of correlative molecules were detected by western blot. The regulation of miR‐375 on the expression of its target gene YWHAZ was verified by dual‐luciferase report system. According to the results, knockdown of YWHAZ inhibited the migration, invasion and EMT of GC cells. Moreover, silencing of YWHAZ restrained the activation of wnt/β‐catenin signalling pathway. YWHAZ was confirmed to be a target gene of miR‐375, and its expression was regulated by miR‐375 in GC cells. Transfection of miR‐375 inhibitor promoted the migration, invasion, EMT and activation of wnt/β‐catenin pathway in GC cells, which was suppressed by inhibition of YWHAZ. Taken together, this study suggests that miR‐375/YWHAZ axis may be served as a novel therapeutic target for GC patients.     

Perinatal exposure to di‐(2‐ethylhexyl)‐phthalate leads to cognitive dysfunction and phospho‐tau level increase in aged rats

Di‐(2‐ethylhexyl)‐Phthalate (DEHP) can affect glucose and insulin homeostasis in periphery and lead to insulin resistance, especially exposure of DEHP during critical developmental period. Given the potential relationship between insulin resistance and pathogenesis of Alzheimer's disease (AD) in elderly life, we investigated the relationship between perinatal DEHP exposure and AD pathogenesis. Our results suggested that perinatal exposure to DEHP can affect the expression of insulin and insulin‐Akt‐ GSK‐3β signal pathway in hippocampus. Furthermore, impaired cognitive ability and increased level of phospho‐Tau was observed in DEHP‐exposed rat offspring (1.25 ± 0.11 vs. 0.47 ± 0.07, P < 0.05). The present study demonstrates that perinatal exposure to DEHP may be a potential risk factor for AD pathogenesis associated with insulin resistance and insulin metabolism disorder in the hippocampus. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 596–603, 2014.     

Synthesis,molecular docking,and pharmacological evaluation of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives as selective COX‐2 inhibitors and anti‐inflammatory agents

A series of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives ( 3am ) was synthesized and evaluated for their in vitro inhibitory activity against COX‐1 and COX‐2. The compounds with considerable in vitro activity (IC₅₀ < 1 μM) were evaluated in vivo for their anti‐inflammatory potential by the carrageenan‐induced rat paw edema method. Out of 13 newly synthesized compounds, 3a , 3b , 3d , 3g , 3j , and 3k were found to be the most potent COX‐2 inhibitors in the in vitro enzymatic assay, with IC₅₀ values in the range of 0.06–0.71 μM. The in vivo anti‐inflammatory activity of these six compounds ( 3a , 3b , 3d , 3g , 3j , and 3k ) was assessed by the carrageenan‐induced rat paw edema method. Compounds 3d (84.09%), 3g (79.54%), and 3a (70.45%) demonstrated significant anti‐inflammatory activity compared to the standard drug ibuprofen (65.90%) and were also found to be safer than ibuprofen, by ulcerogenic studies. A docking study was done using the crystal structure of human COX‐2, to understand the binding mechanism of these inhibitors to the active site of COX‐2.

     

Di-(2-ethylhexyl) phthalate suppresses tamoxifen-induced apoptosis in GH3 pituitary cells

Tamoxifen, an estrogen receptor antagonist, has been clinically used as an antitumor drug and induces apoptosis in GH3 pituitary cells. Although di-(2-ethylhexyl) phthalate (DEHP) is a well-known environmental estrogen and the exposure to this chemical is well expected, reports are limited regarding effects of DEHP on tamoxifen-induced apoptosis in pituitary cells. In the cytotoxicity assay, the reduced cell viability in tamoxifen-treated GH3 cells was reversed by DEHP (250 μM) treatment for 4 days. To characterize cell death, cells were stained using Hoechst 33258. Apoptotic morphological change such as chromatin condensation induced by tamoxifen was suppressed by treatment with DEHP. Flow cytometric analysis revealed that the number of apoptotic cells induced by tamoxifen was significantly decreased by DEHP treatment. Enhanced poly (ADP-ribose) polymerase (PARP) cleavage by tamoxifen treatment was also inhibited by DEHP. These results suggest that DEHP suppresses tamoxifen-induced apoptosis in association with its estrogenic effect in GH3 cells and might counteract the therapeutic effect of tamoxifen.