Di(2-ethylhexyl) Phthalate Treatment Research Articles

Effects of DI-(2-Ethylhexyl) Phthalate on Rat Ovarian Function

Effects of DI-(2-Ethylhexyl) Phthalate on Rat Ovarian FunctionThis study aimed to evaluate the effects of di-(2-ethylhexyl)phthalate (DEHP) on estrous cycle, sex hormone levels and ovary histological features. A total of 80 female SD rats were randomly divided into 8 groups (n=10 per group): short-course control group, short-course low-dose group, short-course medium-dose group, and short-course high-dose group, long-course control group, long-course low-dose group, long-course medium-dose group and long-course high-dose group. Intragastrical DEHP was administrated at 1000 mg/kg/d (low dose), 2000 mg/kg/d (medium dose) and 3000 mg/kg/d (high dose) independently for 14 days (short course) or 28 days (long course). Rats in control groups were untreated. Vaginal smearing was used to detect the estrous cycle and rats were weighed at every Monday and Thursday to evaluate the growth status. At the end of study, rats were sacrificed and bilateral ovaries were obtained for histological examination. In addition, ELISA determined levels of serum progesterone, estradiol, testosterone, follicle stimulating hormone and luteinizing hormone. DEHP treatment limited body weight gain (p<0.05), prolonged the estrous cycle (p<0.05), decreased the ovarian mass index (p<0.05) and ovarian weight. No evident degeneration, necrosis or other pathological features were found in the ovaries. The testosterone levels were decreased by DEHP treatment in a dose dependent manner. DEHP treatment could increase serum testosterone level, inhibit ovulation and prolong the estrous cycle of rats, exerting reproductive toxicity in a dose dependent manner. We speculate DEHP can affect the endocrine regulatory function of the ovary and limit the body weight gain, resulting in chronic toxicity.

Effects of endocrine disruptors on imprinted gene expression in the mouse embryo

Environmental endocrine disruptors (EDs) are synthetic chemicals that resemble natural hormones and are known to cause epigenetic perturbations. EDs have profound effects on development and fertility. Imprinted genes had been identified as susceptible loci to environmental insults by EDs because they are functionally haploid, and because the imprints undergo epigenetic resetting between generations. To screen for possible epigenetic perturbations caused by EDs at imprinted loci, we treated pregnant mice daily between 8.5 and 12.5 days post coitum (dpc) with di-(2-ethylhexyl)-phthalate (DEHP), bisphenol A (BPA), vinclozolin (VZ), or control oil vehicle. After isolating RNA from the placenta, yolk sac, amnion, head, body, heart, liver, lung, stomach, and intestines of 13.5 dpc embryos we measured the allele-specific expression of 38 imprinted transcripts using multiplex single nucleotide primer extension (SNuPE) assays. In this representative data set we identified only a small number of transcripts that exhibited a substantial relaxation of imprinted expression with statistical significance: Slc22a18 with 10% relaxation in the embryo after BPA treatment; Rtl1as with 11 and 16% relaxation in the lung and placenta, respectively after BPA treatment; and Rtl1 with 12% relaxation in the yolk sac after DEHP treatment. Additionally, the standard deviation of allele-specificity increased in various organs after ED treatment for several transcripts including Igf2r, Rasgrf1, Usp29, Slc38a4, and Xist. Our data suggest that the maintenance of strongly biased monoallelic expression of imprinted genes is generally insensitive to EDs in the 13.5 dpc embryo and extra-embryonic organs, but is not immune to those effects.

Dose-Response Assessment of Fetal Testosterone Production and Gene Expression Levels in Rat Testes Following InUtero Exposure to Diethylhexyl Phthalate, Diisobutyl Phthalate, Diisoheptyl Phthalate, and Diisononyl Phthalate

Several phthalate esters have been linked to the Phthalate Syndrome, affecting male reproductive development when administered to pregnant rats during in utero sexual differentiation. The goal of the current study was to enhance understanding of this class of compounds in the Sprague Dawley (SD) fetal rat following exposure on gestational days (GDs) 14-18 by determining the relative potency factors for several phthalates on fetal testes endpoints, the effects of a nine phthalate mixture on fetal testosterone (T) production, and differences in SD and Wistar (W) strain responses of fetal T production and testicular gene expression to di(2-ethylhexyl) phthalate (DEHP). We determined that diisobutyl phthalate (DIBP) and diisoheptyl phthalate (DIHP) reduced fetal testicular T production with similar potency to DEHP, whereas diisononyl phthalate (DINP) was 2.3-fold less potent. DINP was also less potent at reducing StAR and Cyp11a gene expression levels, whereas DIBP was slightly more potent than DEHP. We observed that administration of dilutions of a mixture of nine phthalates (DEHP, DIHP, DIBP, dibutyl-, benzyl butyl-, dicyclohexyl-, diheptyl-, dihexyl-, and dipentyl phthalate) reduced fetal T production in a dose-dependent manner best predicted by dose addition. Finally, we found that the differential effects of in utero DEHP treatment on epididymal and gubernacular differentiation in male SD and W rats (0, 100, 300, 500, 625, 750, or 875 mg DEHP/kg/day) are likely due to tissue-specific strain differences in the androgen and insl3 signaling pathways rather than differential effects of DEHP on fetal testis T and insl3 production.

In Utero Exposure to the Antiandrogen Di-(2-Ethylhexyl) Phthalate Decreases Adrenal Aldosterone Production in the Adult Rat1

We previously reported that in utero exposure of the male fetus to the plasticizer di-(2-ethylhexyl) phthalate (DEHP) resulted in decreased circulating levels of testosterone in the adult without affecting Leydig cell numbers, luteinizing hormone levels, or steroidogenic enzyme expression. Fetal exposure to DEHP resulted in reduced mineralocorticoid receptor (MR; NR3C2) expression in adult Leydig cells. In the present studies, treatment of pregnant Sprague-Dawley dams from Gestational Day 14 until birth with 20, 50, 100, 300, or 750 mg kg(-1) day(-1) of DEHP resulted in significant sex-specific decreases in serum aldosterone but not corticosterone levels at Postnatal Day 60 (PND60) but not at PND21. There was no effect on circulating levels of potassium, angiotensin II or adrenocorticotropin hormone (ACTH). However, there was reduced expression of AT receptor Agtr1a, Agtr1b, and Agtr2 mRNAs. The mRNA levels of proteins and enzymes implicated in aldosterone biosynthesis were not affected by in utero DEHP treatment except for Cyp11b2, which was decreased at high (≥ 500 mg kg(-1) day(-1)) doses. The data presented herein, together with our previous observation that aldosterone stimulates testosterone production via an MR-mediated mechanism, suggest that in utero exposure to DEHP causes reduction in both adrenal aldosterone synthesis and MR expression in Leydig cells, leading to reduced testosterone production in the adult. Moreover, these results suggest the existence of a DEHP-sensitive adrenal-testis axis regulating androgen formation.

Functional Role of Phospholipase D (PLD) in Di(2-Ethylhexyl) Phthalate-Induced Hepatotoxicity in Sprague-Dawley Rats

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidyl choline (PC) to generate phosphatidic acid (PA) and choline. PLD is believed to play an important role in cell proliferation, survival signaling, cell transformation, and tumor progression. However, it remains to be determined whether enhanced expression of PLD in liver is sufficient to induce hepatotoxicity. The aim of this study was to investigate the possible role of PLD in di(2-ethylhexyl) phthalate (DEHP)-induced hepatotoxicity in Sprague-Dawley rats. The phthalate, DEHP (500 mg/kg/d), was administered orally, daily to prepubertal rats (4 wk of age, weighing approximately 70-90 g) for 1, 7, or 28 d. In this study, protein expression levels of PLD1/2, peroxisome proliferator-activated receptor (PPAR), and cytochrome P-450 (CYP) were determined by Western blot analysis using specific antibodies. Liver weight was significantly increased in the DEHP treatment groups. Immunohistochemical analysis demonstrated that DEHP produced strong staining of proliferating cell nuclear antigen (PCNA) at 28 d of exposure, suggestive of hepatocyte proliferation. A significant rise in PLD1/2 expression was observed in liver of DEHP-exposed rats after 7 d. Further, PPARα, constitutive androstane receptor (CAR), pregnane X receptor (PXR), and CYP2B1 protein expression levels were markedly elevated in DEHP-treated groups. Our results suggest that DEHP significantly enhanced the expression of PLD, which may be correlated with PPARα-induced hepatotoxicity through a complex interaction with nuclear receptors including CAR and PXR.

Determination of the di-(2-ethylhexyl) phthalate NOAEL for reproductive development in the rat: importance of the retention of extra animals to adulthood.

Deriving No Observed Adverse Effect Level (NOAEL) or benchmark dose is important for risk assessment and can be influenced by study design considerations. In order to define the di-(2-ethylhexyl) phthalate (DEHP) dose-response curve for reproductive malformations, we retained more offspring to adulthood to improve detection of these malformations in the reproductive assessment by continuous breeding study design. Sprague-Dawley rats were given a dietary administration of 1.5 (control), 10, 30, 100, 300, 1000, 7500, and 10,000 ppm DEHP. Male pups were evaluated for gross reproductive tract malformations (RTMs) associated with the "phthalate syndrome." DEHP treatment had minimal effects on P0 males. There was a statistically significant increase in F1 and F2 total RTMs (testis, epididymides, seminal vesicle, and prostate) in the 7500-ppm dose group and F1 10,000-ppm dose group. The 10,000-ppm exposed F1 males did not produce an F2 generation. The NOAEL for F1 and F2 RTM combined data, because in utero exposures were similar, were 100 ppm (4.8 mg/kg/day), which was close to the 5% response benchmark dose lower confidence limit of 142 ppm. The utility of evaluating more pups per litter was examined by generating power curves from a Monte Carlo simulation. These curves indicate a substantial increase in detection rate when three males are evaluated per litter rather than one. A 10% effect across male pups would be detected 5% of the time if one pup per litter was evaluated, but these effects would be detected 66% of the time if three pups per litter were evaluated. Taken together, this study provides a well-defined dose response of DEHP-induced RTMs and demonstrates that retention of more adult F1 and F2 males per litter, animals that were already produced, increases the ability to detect RTMs and presumably other low-incidence phenomena.

Pubertal Administration of DEHP Delays Puberty, Suppresses Testosterone Production, and Inhibits Reproductive Tract Development in Male Sprague-Dawley and Long-Evans Rats

Although is clear that exposure to high dosage levels of some phthalates delays the onset of puberty in the male rat, it has been hypothesized that low levels of di(2-ethylhexyl) phthalate (DEHP) accelerate puberty by enhancing testicular androgen synthesis. The current study was designed to determine if the dose response to DEHP was nonmonotonic, as hypothesized. Pubertal administration of DEHP delayed the onset of puberty and reduced androgen-dependent tissue weights in both Long-Evans (LE) and Sprague-Dawley (SD) male rats 300 and 900 mg DEHP/kg/day. These effects were generally of greater magnitude in LE than SD rats. By contrast, alterations in testis histopathology (300 and 900 mg/kg/day) were more severe in SD than in LE rats. Taken together, these results suggest that DEHP may be acting on the pubertal male rat testis via two modes of action; one via the Leydig cells and the other via the Sertoli cells. Treatment with DEHP generally reduced serum testosterone and increased serum luteinizing hormone (LH) levels, demonstrating that the reduction in testosterone was due to the effect of DEHP on the testis and not via an inhibition of LH from hypothalamic-pituitary axis. Testosterone production ex vivo (with and without human chorionic gonadotropin stimulation) was consistently reduced in males at the time of puberty and shortly thereafter. DEHP treatment did not accelerate the age at puberty or enhance testosterone levels at 10 or 100 mg/kg/day in either LE or SD rats, as some have hypothesized. Taken together, these results do not provide any evidence of a nonmonotonic dose response to DEHP during puberty.

Reproductive Effects of Di(2-ethylhexyl)phthalate in Immature Male Rats and Its Relation to Cholesterol, Testosterone, and Thyroxin Levels

Phthalates are chemicals employed in several industrial products and there is a growing body of evidence demonstrating that they induce numerous adverse effects on the reproductive system. This study was carried out to assess possible alterations induced by the plasticizer di(2-ethylhexyl phthalate (DEHP) on cholesterol, testosterone, and thyroxine (total T4) levels, as well as to discuss the significance of these data in global changes observed in the reproductive tract of pubertal animals. Wistar rats aged 21 days received DEHP orally at 0, 250, 500, and 750 mg/kg/day for 30 days and were examined for different reproductive endpoints. At the end of the treatment, significant decreases in relative weight of testosterone-dependent organs, delayed preputial separation, and low serum testosterone were observed at the highest DEHP dose. The plot of the relationship between DEHP dose and serum cholesterol revealed a biphasic effect. The concentration of cholesterol in serum was significantly reduced at 250 mg/kg/day DEHP but returned to control values at 750 mg/kg/day. Cholesterol levels measured in testicular tissue increased with DEHP treatment. Serum T4 levels were not affected by DEHP at any dose, indicating the absence of a link between total thyroxin concentration and phthalate effects on cholesterol levels. Taken together these results indicate that effects observed in serum and testicular cholesterol levels may reflect distinct effects of DEHP on cholesterol synthesis and usage. These results confirm and extend previously reported findings showing that alterations in cholesterol balance may play a role in the suppression of steroidogenesis induced by DEHP in rats.

Clinically relevant concentrations of di (2-ethylhexyl) phthalate (DEHP) uncouple cardiac syncytium

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer found in a variety of polyvinyl chloride (PVC) medical products. The results of studies in experimental animals suggest that DEHP leached from flexible PVC tubing may cause health problems in some patient populations. While the cancerogenic and reproductive effects of DEHP are well recognized, little is known about the potential adverse impact of phthalates on the heart. This study examined the effects of clinically relevant concentrations of DEHP on neonatal rat cardiomyocytes. It was found that application of DEHP to a confluent, synchronously beating cardiac cell network, leads to a marked, concentration-dependent decrease in conduction velocity and asynchronous cell beating. The mechanism behind these changes was a loss of gap junctional connexin-43, documented using Western blot analysis, dye-transfer assay and immunofluorescence. In addition to its effect on electrical coupling, DEHP treatment also affected the mechanical movement of myocyte layers. The latter was linked to the decreased stiffness of the underlying fibroblasts, as the amount of triton-insoluble vimentin was significantly decreased in DEHP-treated samples. The data indicate that DEHP, in clinically relevant concentrations, can impair the electrical and mechanical behavior of a cardiac cell network. Applicability of these findings to human patients remains to be established.

Di‐(2‐ethylhexyl) Phthalate Upregulates ATF3 Expression and Suppresses Apoptosis in Mouse Genital Tubercle

To investigate the effect of di-(2-ethylhexyl) phthalate (DEHP) on the expression of activating transcription factor 3 (ATF3) and apoptosis of fetal mouse genital tubercle (GT). In this developmental toxicity study, pregnant C57BL/6 mice were exposed to corn oil or DEHP (100 or 500 mg/kg/day) from embryonic day 12 (ED12) to ED16. Apoptosis was characterized by Terminal transferase dUTP nick end labeling (TUNEL) assay. Using RT-PCR and western blot, the expressions of ATF3 and apoptosis-related genes (P53, Bcl-2 and Bax) were investigated. Apoptosis of fetal mouse GT cells notably decreased after DEHP treatment. DEHP activated ATF3 both at the mRNA and protein levels in GT. Furthermore, pro-apoptotic P53 was downregulated and the ratio of anti-apoptotic (Bcl-2)/pro-apoptotic (Bax) was not significantly changed. These results suggest that DEHP may induce external genital defects via a mechanism involving apoptosis, which might correlate with the regulation of ATF3 and P53 expressions.

Effects of Di(2-ethylhexyl) Phthalate on Regulation of Steroidogenesis or Spermatogenesis in Testes of Sprague-Dawley Rats

Di(2-ethylhexyl) phthalate (DEHP) used as a common plasticizer additive in the manufacture of plastics, such as polyvinyl chloride (PVC). This study examined the effect of DEHP on steroidogenesis or spermatogenesis in the testes of Sprague-Dawley male rats treated orally with 250, 500, 750 mg/kg over a 30-day period. The expression levels of the steroidogenic- or spermatogenic-related genes were analyzed in the testis using a reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis. High doses of DEHP (500 and 750 mg/kg) significantly decreased the testicular sper mc ounts and daily sperm production (DSP). In addition, serum testosterone levels were significantly lower in the DEHP treatment groups than in the control. The mRNA levels of SR-B1, StAR, PBR and CYP17 increased in a dose-dependent manner. These increases were significant at 500 and 750 mg/kg. In the other hand, the mRNA levels of CYP19 decreased significantly in testes of rats exposed to DEHP 500 and 750 mg/kg. Dose-dependent decreases in Spag4 and LDHA mRNA in the testis were observed after DEHP exposure, while there was a significant decrease in thyroid hormone receptor (TR)α 1p rotein levels. High doses of DEHP significantly increased the expression of peroxisome proliferator-activated receptor (PPAR)-r and retinoid X receptor (RXR)-α protein but markedly decreased the expression of RXR-r. These results suggest that DEHP exposure can alter the expression of the spermatogenic- or steroidogenic-related genes resulting in a decrease in sperm production in the testis. This study is expected to be helpful in research examining the mechanisms for how DEHP reduces the expression pattern of the genes involved steroid hormone synthesis after chronic exposure to DEHP.

Adjuvant effect of di-(2-ethylhexyl) phthalate on asthma-like pathological changes in ovalbumin-immunised rats

Pollution of phthalates, including di-(2-ethylhexyl)phthalate (DEHP), is widespread because of their use in plastics and other daily consumer products. Epidemiological studies have shown that the largest source of general population exposure to DEHP is on dietary. Studies have suggested an association between exposure to phthalate plasticisers, and increased prevalence of asthma. To investigate the adjuvant effect of DEHP on asthmatic pathological changes in ovalbumin-immunised rat model. Wistar rats were randomly divided into five groups (eight rats of each group): (1) saline; (2) ovalbumin (OVA); (3) OVA+DEHP 0.7mg·kg−1·d−1; (4) OVA+DEHP 70 mg·kg−1·d−1; and (5) DEHP 70 mg·kg−1·d−1. DEHP treatment groups which were administered to 0.7 mg and 70 mg DEHP/kg/d by gastric gavage for 30d before and during the process of OVA immunisation or saline treatment. The in vivo pulmonary function analysis, bronchoalveolar lavage (BAL) fluid collection and section histological observation were conducted. DEHP exposure could significantly increase airway hyperresponsiveness, airway remolding, and eosinophil infiltration in the OVA-immunised rats. But the DEHP exposure alone group does not show significant airway structural change and inflammatory cell infiltration, compared with control group. DEHP administered by gastric gavage play an adjuvant effect on respiratory systems in ovalbumin-immunised rat model.

Di(2-Ethylhexyl) Phthalate (DEHP) Increases Transforming Growth Factor-β1 Expression in Fetal Mouse Genital Tubercles

Phthalates are known to elicit marked effects on the developing male reproductive tract as evidenced by hypospadias. Recently, transforming growth factor-β1 (TGF-β1) was postulated to play an essential role in the development of genital tubercles (GT), and TGF- β1 was found to act as a phthalates-responsive gene. In this study, the effects of di(2-ethylhexyl) phthalate (DEHP) were examined on the expression of TGF-β1 in fetal mice, as GT development is dependent upon this factor. Pregnant C57BL/6 mice were exposed to corn oil or DEHP (100, 200, or 500 mg/kg/d orally) from embryonic day 12 (ED12) to ED17. Data showed a significant inhibition of male fetal GT development following DEHP treatment. Hypospadic-like urethral orifice and abnormal urethra were evaluated macroscopically and by histology at ED19. By using reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, the expression of TGF-β1 was upregulated in DEHP-treated mice. These results suggest that hypospadias may be induced by DEHP exposure involving modification of TGF-β1 levels.

Effects of Diethylhexyl Phthalate (DEHP) on INSL3 mRNA Expression by Leydig Cells Derived from Mouse Embryos and in Newborn Mice

Insulin-like factor 3 (INSL3) regulates testicular descent during fetal life, and Insl3 gene inactivation results in cryptorchidism. Little is known, however, about whether the plasticizer diethylhexyl phthalate (DEHP), a contaminant found widely in the environment, influences INSL3 expression. In this study, primary cultures of Leydig cells from mouse embryos were treated in vitro with DEHP. We also treated pregnant mice with DEHP from gestation day 12 to postnatal day 3 in order to study the effect of DEHP in vivo. INSL3 mRNA expression levels in primary Leydig cell cultures and in the testes of newborn mice were significantly lower following DEHP treatment. DEHP also caused detrimental morphological changes in both primary cultures of Leydig cells and the testes of newborn mice. These results suggest that the downregulation of INSL3 mRNA by DEHP might cause abnormalities of gubernacular development, which might be one of the mechanisms for development of cryptorchidism.

Preventive effect of d-psicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury in rat

To investigate the preventive effects of d-psicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury, prepubertal male Sprague–Dawley rats were exposed to DEHP via their diet or orally, while under treatment with d-psicose. The rats given a diet-containing 1% DEHP alone for 7–14 days showed severe testicular atrophy accompanied by aspermatogenesis. On the other hand, those given the diet plus 2% but not 1% d-psicose-supplemented water for 14 days did not develop testicular atrophy, and exhibited an almost complete spermatogenesis. There was no significant difference in plasma mono-(2-ethylhexyl) phthalate (MEHP) levels between the d-psicose-free and d-psicose-treated groups. The testicular malondialdehyde (MDA) level after a single oral administration of 2 g/kg of DEHP showed a similar pattern of increase to the plasma MEHP level and peaked in 24 h suggesting a close and dose-dependent relation between plasma MEHP and testicular reactive oxygen species (ROS) levels. Pretreatment with d-psicose at a concentration of 2% and 4% resulted in an almost complete but not absolute suppression of testicular MDA production among rats administered 2 g/kg of DEHP. The microarray analysis showed the induction of oxidative stress related genes including the thioredoxin, glutathione peroxidase 1 and 2, glutaredoixn 1 after 24 h of the DEHP treatment in the testis. These results show that d-psicose prevents DEHP-induced testicular injury by suppressing the generation of ROS in the rat testis. This effect may be due to the direct scavenging by d-psicose of ROS generated in the testis.

Di(2-ethylhexyl) Phthalate Induces Apoptosis Through Peroxisome Proliferators-Activated Receptor-Gamma and ERK 1/2 Activation in Testis of Sprague-Dawley Rats

Di(2-ethylhexyl) phthalate (DEHP) is a well-known hepatic and reproductive toxicant whose toxicity may be mediated by peroxisome proliferators-activated receptor (PPAR). This study examined the effects of DEHP on the expression of PPAR-regulated genes involved in testicular cells apoptosis. Sprague-Dawley male rats were treated orally with 250, 500, or 750 mg/kg/d DEHP for 28 d, while control rats were given corn oil. The levels of cell cycle regulators (pRb, cyclins, CDKs, and p21) and apoptosis-related proteins were analyzed by Western blot analysis. The role of PPAR-gamma (PPAR-γ), class B scavenger receptor type 1 (SR-B1), and ERK1/2 was further studied to examine the signaling pathway for DEHP-induced apoptosis. Results showed that the levels of pRB, cyclin D, CDK2, cyclin E, and CDK4 were significantly lower in rats given 500 and 750 mg/kg/d DEHP, while levels of p21 were significantly higher in rat testes. Dose-dependent increases in PPAR-γ and RXRα proteins were observed in testes after DEHP exposure, while there was a significant decrease in RXRγ protein levels. In addition to PPAR-γ, DEHP also significantly increased SR-B1 mRNA and phosphorylated ERK1/2 protein levels. Furthermore, DEHP treatment induced pro-caspase-3 and cleavage of its substrate protein, poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner. Data suggest that DEHP exposure may induce the expression of apoptosis-related genes in testes through induction of PPAR-γ and activation of the ERK1/2 pathway.

Induction of peroxisome proliferator-activated receptor alpha (PPARα)-related enzymes by di(2-ethylhexyl) phthalate (DEHP) treatment in mice and rats, but not marmosets

To clarify species differences in the induction of peroxisome proliferator-activated receptor alpha (PPARalpha)-related enzymes by di(2-ethylhexyl)phthalate (DEHP) exposure, we investigated the inductions of PPARalpha and its target genes (mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD) and peroxisomal keto-acyl-CoA thiolase (PT) in liver from mice (CD-1), rats (Sprague-Dawley), and marmosets (Callithrix jacchus) exposed to DEHP. Male mice and rats were treated with 0, 1.25 and 2.5 mmol/kg DEHP for 2 weeks, and marmosets with 0, 0.25, 1.25 and 6.25 mmol/kg DEHP for 15 months by gavage. Hepatic mono(2-ethylhexyl)phthalate (MEHP) levels were significantly higher in mice and rats than in marmosets. The constitutive expression of hepatic PPARalpha was 5-7 times greater in rats and mice than in marmosets, but DEHP treatment did not induce PPARalpha-mRNA in all animals. The treatment-induced PT expression detected either by anti-PT antibody or PT-mRNA levels in the liver only from mice and rats, and the induction of the mRNA was greater in the latter than in the former. Thus, DEHP used in this experiment influenced the peroxisomal enzymes in mice and rats, but did not affect the mitochondrial enzymes in any animals or the peroxisomal enzymes in marmosets. These results suggest that there are species differences in the induction of PPARalpha-related enzymes, especially in peroxisomal enzymes by DEHP treatment, and their underlying mechanism may in part reside in the different constitutive levels of PPARalpha and different forming levels of MEHP.

A dose response study following in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP): Reproductive effects on adult male offspring rats

The reproductive effects of in utero and lactational exposure to di-(2-ethylhexyl) phthalate (DEHP) in adult male offspring rats were investigated. The selected endpoints included reproductive organ weights, testicular function, hormonal status, sexual behaviour and fertility. Two wide ranges of doses, low and high, were tested. Female Wistar rats were treated daily with DEHP and peanut oil (vehicle control) by gavage from gestation day 6 to lactation day 21. The low-doses were 0.015, 0.045, 0.135, 0.405 and 1.215 mg DEHP/kg body weight (bw)/day, and the high-doses were 5, 15, 45, 135 and 405 mg DEHP/kg bw/day. A reduction in daily sperm production of 19–25% in relation to control was observed in animals exposed to 15, 45, 135 and 405 mg/kg/day. Quantitation of specific cell types shows that the observed effects in daily sperm production are not related to changes in the number of Sertoli cells or their capability to support early stages spermatocytes. A low incidence of cryptorchidism was observed in DEHP exposed groups with a lowest observed adverse effect level of 5 mg/kg/day. Serum testosterone concentration was similar to control at most doses but was significantly increased at 0.045, 0.405 and 405 mg DEHP/kg/day. In spite of this effect, the weight of seminal vesicle with coagulating glands was significantly reduced at 405 mg/kg/day. Testis, epididymis and prostate weights were similar among groups. Fertility and sexual behaviour were not affected by DEHP treatment at any dose. Overall, our results show that in utero and lactational DEHP exposure reduces daily sperm production and has the potential to induce reproductive tract abnormalities (of which cryptorchidism seems to be the most sensitive in our rat strain) in male offspring rats. The lowest observed adverse effect levels (LOAELs) for these effects were 15 and 5 mg/kg/day, respectively. Therefore, the no observed adverse effect level (NOAEL) for this study can be set at 1.215 mg/kg/day.

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 microM) 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.

Excess Peroxisomes Are Degraded by Autophagic Machinery in Mammals

Peroxisomes are degraded by autophagic machinery termed "pexophagy" in yeast; however, whether this is essential for peroxisome degradation in mammals remains unknown. Here we have shown that Atg7, an essential gene for autophagy, plays a pivotal role in the degradation of excess peroxisomes in mammals. Following induction of peroxisomes by a 2-week treatment with phthalate esters in control and Atg7-deficient livers, peroxisomal degradation was monitored within 1 week after discontinuation of phthalate esters. Although most of the excess peroxisomes in the control liver were selectively degraded within 1 week, this rapid removal was exclusively impaired in the mutant liver. Furthermore, morphological analysis revealed that surplus peroxisomes, but not mutant hepatocytes, were surrounded by autophagosomes in the control. Our results indicated that the autophagic machinery is essential for the selective clearance of excess peroxisomes in mammals. This is the first direct evidence for the contribution of autophagic machinery in peroxisomal degradation in mammals.