Effects of dibutyl phthalate and di (2-ethylhexyl) phthalate on hepatic structure and function of adult male mice.

The objective of the present research was to determine if dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) alone and combined exposure induced pathological alterations in laboratory reared albino mice. Adult male mice were equally divided (n = 10) into Control, corn oil (CO), DBP, DEHP, and DBP+DEHP treated groups. Dibutyl phthalate (250 mg/kg), DEHP (300 mg/kg), and DBP+DEHP (250+300 mg/kg), respectively, were administered by oral gavage mixed in corn oil (0.2 mL) for 28 days. All animals were sacrificed following 28 days of treatment and blood was collected for serum lipid profiles and liver function tests. Liver samples were also collected for observation of histological changes. Microphotographs of hematoxylin and eosin-stained sections were used for computer-based micrometry. CO, DBP, DEHP, and DBP+DEHP treatment resulted in a significant increase in the mean body and liver weights as compared with the Control group. Histological examination of the livers with DBP and/or DEHP treatment showed marked alterations leading to hepatic hypertrophy. In the treated groups, a significant increase in the mean number of mononucleated, binucleated cells, and oval cells per unit area was noticed with disorganized trabecular arrangement as compared with the Control group. Treatment with DBP and/or DEHP resulted in large regeneration zones in the liver and an increased relative nucleo-cytoplasmic index of mononuclear shepatocytes when compared with the Control group. All treatments caused a significant increases in the liver enzymes and proteins as well as altered serum cholesterol, triglycerides, LDL, and VLDL levels. The histopathological and serological findings confirmed the toxic potentials to hepatic tissue of DBP and DEHP either given alone or in combination.

Maternal Exposure to Dibutyl Phthalate (DBP) or Diethylstilbestrol (DES) Leads to Long-Term Changes in Hypothalamic Gene Expression and Sexual Behavior.

Xenobiotic exposure during pregnancy and lactation has been linked to perinatal changes in male reproductive outcomes and other endocrine parameters. This pilot study wished to assess whether brief maternal exposure of rats to xenobiotics dibutyl phthalate (DBP) or diethylstilbestrol (DES) might also cause long-term changes in hypothalamic gene expression or in reproductive behavior of the resulting offspring. Time-mated female Sprague Dawley rats were given either DBP (500 mg/kg body weight, every second day from GD14.5 to PND6), DES (125 µg/kg body weight at GD14.5 and GD16.5 only), or vehicle (n = 8-12 per group) and mild endocrine disruption was confirmed by monitoring postnatal anogenital distance. Hypothalamic RNA from male and female offspring at PND10, PND24 and PND90 was analyzed by qRT-PCR for expression of aromatase, oxytocin, vasopressin, ER-alpha, ER-beta, kisspeptin, and GnRH genes. Reproductive behavior was monitored in male and female offspring from PND60 to PND90. Particularly, DES treatment led to significant changes in hypothalamic gene expression, which for the oxytocin gene was still evident at PND90, as well as in sexual behavior. In conclusion, maternal xenobiotic exposure may not only alter endocrine systems in offspring but, by impacting on brain development at a critical time, can have long-term effects on male or female sexual behavior.

Curcumin decreases dibutyl phthalate-induced renal dysfunction in Kunming mice via inhibiting oxidative stress and apoptosis.

Curcumin (Cur) has been used extensively in dietary supplement with antioxidant and anti-apoptotic properties. Although dibutyl phthalate (DBP) has adverse effects on the kidney, any association between DBP exposure and the role of Cur is unclear. We tested the hypothesis that exposure to DBP has adverse consequences on renal dysfunction in mice and the potential protective role of Cur in decreasing DBP-induced renal dysfunction via inhibiting oxidative stress and apoptosis. Kidney function, oxidative stress biomarkers, and apoptosis factors as well as Bcl-2 and Bax were investigated. The results showed a marked increase of renal dysfunction, oxidative stress and apoptosis level after DBP exposure compared to the control. While administration of Cur to DBP-treated mice may reduce these adverse biochemical changes compared with DBP-alone group. Overall, these results suggest that oxidative stress and apoptosis are involved in DBP-induced renal disorder, whereas Cur plays a protective role in inhibiting these two pathways.

Effects of di-n-butyl phthalate and di-2-ethylhexyl phthalate on pollutant removal and microbial community during wastewater treatment.

Due to the wide use of plastic products and the releasability of plasticizer into surrounding environment, the hazards, residues and effects of phthalic acid esters (PAEs) in ecosystems have been paid more and more attention. Little information is available about the effects of PAEs on the normal wastewater treatment, although the distribution of PAEs in soil and other ecosystems is closely related to the discharge of sewage. In this study, the effects of high concentrations of di-n-butyl phthalate (DBP) and di-2-ethylhexyl phthalate (DEHP) on pollutant removal and the microbial community during landfill leachate treatment was investigated. After domestication, the activated sludge was used in the co-treatment of landfill leachate and simulated domestic wastewater. We verified that this process reduced the toxicity of landfill leachate. However, high concentrations of added DBP and DEHP were removed first, while the removal of these pollutants from raw landfill leachate was limited. The results of high-throughput sequencing revealed that the bacterial diversity was diminished and the microbial community structure was significantly affected by the addition of DBP and DEHP. The DBP and DEHP samples had 79.05% and 82.25% operational taxonomic units (OTU), respectively, in common with the raw activated sludge. Many genera of PAE-degrading bacteria that had no significant evolutionary relationship were found in the raw activated sludge. And the widespread presence of PAE-degrading bacteria could effectively keep the concentrations of PAEs low during the wastewater treatment.

In utero exposure to DBP stimulates release of GnRH by increasing the secretion of PGE2 in the astrocytes of the hypothalamus in the offspring mice.

Di-n-butyl phthalate (DBP), the most commonly used plasticizer and typical endocrine disrupting chemicals (EDCs), has shown its characteristics of causing reproductive and developmental toxicity in males, while the neuroendocrine toxicity induced by DBP exposure in utero and the mechanism beneath still remain unclear. Here, the pregnant mice were treated with corn oil (control) or DBP at three different doses by oral gavage during gestational days (GD) 12.5-21.5. The results showed that in utero exposure to DBP induced a significant increase of gonadotropin releasing hormone (GnRH) content in serum, as well as activation and proliferation of astrocytes in the hypothalamus of offspring male mice on postnatal day (PND) 22. However, in in vitro study, mono-n-butyl phthalate (MBP), the metabolite of DBP, could not increase the release of GnRH after GnRH neurons were exposed to MBP. Further studies identified that MBP-mediated activation and proliferation of astrocytes resulted in increased secretion of prostaglandin E2 (PGE2), which might be responsible for the increased release of GnRH from GnRH neurons. This study highlights the neuroendocrine toxicity of current plasticizer DBP exposure, laying the foundation for identifying potential molecular targets for related diseases.

Dibutyl phthalate aggravated asthma-like symptoms through oxidative stress and increasing calcitonin gene-related peptide release.

Dibutyl phthalate (DBP) is one of the most ubiquitous phthalate esters found in everyday products, and is receiving increased attention as an immunologic adjuvant. However, information regarding DBP-aggravated allergic asthma is still limited. This study used a mouse model sensitized with ovalbumin (OVA) to determine any adverse effects of DBP on allergic asthma. Our results reveal that allergic asthmatic mice exposed to DBP for an extended period had a significant increase in inflammatory cell infiltration; a significant increase in levels of serum immunoglobulin and T helper 2 cell (Th2) and T helper 17 cell (Th17) cytokines in lung tissue; and significant changes in lung histology and AHR, all of which are typical asthmatic symptoms. The levels of oxidative stress and levels of the neuropeptide, calcitonin gene related peptide (CGRP), were also elevated after DBP exposure. Interestingly, blocking oxidative stress by administering melatonin (MT) not only reduced oxidative stress and CGRP levels, but also ameliorated the asthmatic symptoms. Collectively, these results show that DBP exacerbates asthma-like pathologies by increasing the expression of CGRP mediated by oxidative stress.

Dibutyl phthalate-mediated oxidative stress induces splenic injury in mice and the attenuating effects of vitamin E and curcumin.

Dibutyl phthalate (DBP) is a ubiquitous environmental contaminant that at certain levels can be harmful to human health. Although DBP has been widely linked to immunotoxicity, any association between DBP exposure and splenic injury remains unknown. The purpose of this study was to investigate whether DBP exposure can induce splenic injury and the antagonistic effects of two antioxidants, vitamin E (VitE) and curcumin (Cur), on DBP-induced splenic injury. The levels of ROS, GSH, T-AOC, IL-1ß, TNF-α, cytochrome C, caspase-8, caspase-9 and caspase-3 in the spleen homogenate of mice were measured. Any histopathological changes in the spleen were observed using H&E and toluidine blue staining. And the morphology of mitochondria was observed using Janus Green B staining. The results indicate that exposure to 50 mg/kg DBP could cause histopathological changes of the spleen and result in inflammation and apoptosis associated with oxidative stress, which may lead to splenic injury in mice. Moreover, both VitE and Cur could antagonize the oxidative stress induced by DBP to reduce splenic injury. These findings help to expand our understanding of DBP-mediated immunotoxicity, and to show that VitE and Cur can alleviate DBP-induced splenic injury and the possible DBP-associated decline in immune function.

Choline, not folate, can attenuate the teratogenic effects ofdibutyl phthalate (DBP) during early chick embryo development.

Dibutyl phthalate (DBP), a persistent environmental pollutant, can induce neural tube abnormal development in animals. The possible effects of DBP exposure on human neural tube defects (NTDs) remain elusive. In this study, the distribution of DBP in the body fluid of human NTDs was detected by GC-MS. Then, chick embryos were used to investigate the effects of DBP on early embryonic development. Oxidative stress indicators in chick embryos and the body fluid of human NTDs were detected by ELISA. The cell apoptosis and total reactive oxygen species (ROS) level in chick embryos were detected by whole-mount TUNEL and oxidized DCFDA, respectively. The study found that the detection ratio of positive DBP and its metabolites in maternal urine was higher in the NTD population than that in normal controls. 8-hydroxy-2 deoxyguanosine (8-OHDG) and malondialdehyde (MDA) were evidently upregulated and superoxide dismutase (SOD) was observably downregulated in amniotic fluid and urine. Animal experiments indicated that DBP treatment induced developmental toxicity in chick embryos by enhancing the levels of oxidative stress and cell apoptosis. MDA was increased and SOD was decreased in DBP-treated embryos. Interestingly, the supplement of high-dose choline (100 µg/µL), not folic acid, could partially restore the teratogenic effects of DBP. Our data collectively suggest that the incidence of NTDs is closely associated with DBP exposure. This study may provide new insight for NTD prevention.

Assessing effects of germline exposure to environmental toxicants by high-throughput screening in C. elegans.

Chemicals that are highly prevalent in our environment, such as phthalates and pesticides, have been linked to problems associated with reproductive health. However, rapid assessment of their impact on reproductive health and understanding how they cause such deleterious effects, remain challenging due to their fast-growing numbers and the limitations of various current toxicity assessment model systems. Here, we performed a high-throughput screen in C. elegans to identify chemicals inducing aneuploidy as a result of impaired germline function. We screened 46 chemicals that are widely present in our environment, but for which effects in the germline remain poorly understood. These included pesticides, phthalates, and chemicals used in hydraulic fracturing and crude oil processing. Of the 46 chemicals tested, 41% exhibited levels of aneuploidy higher than those detected for bisphenol A (BPA), an endocrine disruptor shown to affect meiosis, at concentrations correlating well with mammalian reproductive endpoints. We further examined three candidates eliciting aneuploidy: dibutyl phthalate (DBP), a likely endocrine disruptor and frequently used plasticizer, and the pesticides 2-(thiocyanomethylthio) benzothiazole (TCMTB) and permethrin. Exposure to these chemicals resulted in increased embryonic lethality, elevated DNA double-strand break (DSB) formation, activation of p53/CEP-1-dependent germ cell apoptosis, chromosomal abnormalities in oocytes at diakinesis, impaired chromosome segregation during early embryogenesis, and germline-specific alterations in gene expression. This study indicates that this high-throughput screening system is highly reliable for the identification of environmental chemicals inducing aneuploidy, and provides new insights into the impact of exposure to three widely used chemicals on meiosis and germline function.

Nano-sized selenium attenuates the developmental testicular toxicity induced by di-n-butyl phthalate in pre-pubertal male rats.

The current study was conducted to test the possible ameliorative role of selenium nanoparticles (Se-NPs) against oxidative damage of Leyding cells induced by di-n-butyl phthalate (DBP) in pre-pubertal male rat offspring. Forty-two pregnant female rats treated from gestation day (GD) 12 to postnatal day (PND) 14 day with two doses of Se-NPs (0.2 and 0.5 mg/kg/d) against developmental testicular toxicity induced by DBP (500 mg/kg/d). At PND 25 serum and testes of offspring were collected. Serum LH, the Leydig cells performance [total serum testosterone, LH and testosterone (LH/T) ratio, relative gene expression of insulin-like growth factor-3 (INSL3) and mineralocorticoid receptor (MR)], oxidative stress biomarker malondialdehyde (MDA) and antioxidant machinery [reduced glutathione (GSH), and the relative gene expression of antioxidant enzymes: superoxide dismutase (SOD), glutathione peroxidase (GPx)] were estimated in all groups. The obtained results revealed that maternal exposure to DBP significantly reduced total serum testosterone level, relative mRNA expression of INSL3 and MR genes with observed testicular damage revealed by increasing MDA and depressed levels of GSH and antioxidant enzymes. The histopathological changes include necrosis and desquamation of spermatogoneal cells. Co-administration of Se-NPs high dose along with DBP significantly increased serum testosterone, improved LH/T ratio and the relative mRNA expression of INSL3 and MR genes, decreased the level of MDA, and also improved all the antioxidant enzymes expression levels. In conclusion, Se-NPs could be a potent maternal prophylactic agent against the reduced total serum testosterone level and oxidative damage of Leydig cells induced by DBP via reducing the lipid peroxidation (LPO) and enhancing the antioxidant state in pre-pubertal male rat offspring.

Maternal supplementation with corn oil associated or not with di-n-butyl phthalate increases circulating estradiol levels of gerbil offspring and impairs sperm reserve.

This study evaluated the consequences of gestational exposure to di-n-butyl phthalate (DBP) for testicular steroidogenesis and sperm parameters of the adult gerbil and the interference of corn oil (co), a vehicle widely used for administration of liposoluble agents, on DBP effects. Pregnant gerbils received no treatment or were treated from gestational day 8 to 23 via gavage with 0.1 mL/day of co only or containing DBP (100 mg/kg/day). Maternal co intake enhanced serum estradiol levels and testicular content of ERα, and reduced sperm reserve of adult offspring. Gestational DBP exposure caused dyslipidemia, increased serum and intratesticular estradiol levels and reduced sperm reserve and motility. Thus, maternal co supplementation alters circulating estradiol and impairs sperm quantity and quality of offspring. Gestational DBP exposure alters lipid metabolism and testicular steroidogenesis and worsens the negative effects of co on the sperm reserve and motility of gerbil. Therefore, co interferes with the reproductive response to DBP.

[Hypospadias induced by maternal exposure to di-n-butyl phthalate and its mechanisms in male rat offspring , , , , ,].

OBJECTIVE: To induce hypospadias in male rat offspring by maternal exposure to di-n-butyl phthalate (DBP) during late pregnancy and further investigate its mechanisms. METHODS: We randomly divided 20 pregnant rats into a DBP exposure and a control group, the former treated intragastrically with DBP while the latter with soybean oil at 750 mg per kilogram of the body weight per day from gestation days (GD) 14 to 18. On postnatal day (PND) 1, we recorded the incidence rate of hypospadias and observed the histopathological changes in the genital tubercle of the hypospadiac rats. We also measured the level of serum testosterone (T) by radioimmunoassay and determined the mRNA and protein expressions of the androgen receptor (AR), sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4) and fibroblast growth factor 8 (Fgf8) in the genital tubercle by real-time PCR and Western blot. RESULTS: No hypospadiac male rats were found in the control group. The incidence rate of hypospadias in male offspring was 43.6% in the DBP-treatment group. Histological analysis confirmed hypospadiac malformation. The serum testosterone concentration was decreased in the hypospadiac male rats as compared with the controls (ï¼»0.49 ± 0.05ï¼½ vs ï¼»1.12 ± 0.05ï¼½ ng/ml, P <0.05). The mRNA expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle were significantly lower in the hypospadiac male rats than in the controls (AR: 0.50 ± 0.05 vs 1.00 ± 0.12, P <0.05; Shh: 0.65 ± 0.07 vs 1.00 ± 0.15, P <0.05; Bmp4: 0.42 ± 0.05 vs 1.00 ± 0.13, P <0.05; Fgf8: 0.46 ± 0.04 vs 1.00 ± 0.12, P <0.05), and so were their protein expressions (AR: 0.34 ± 0.05 vs 1.00 ± 0.09, P <0.05; Shh: 0.51 ± 0.07 vs 1.00 ± 0.12, P <0.05; Bmp4: 0.43 ± 0.05 vs 1.00 ± 0.11, P <0.05; Fgf8: 0.57 ± 0.04 vs 1.00 ± 0.13, P <0.05). CONCLUSIONS: Maternal exposure to DBP during late pregnancy can induce hypospadias in the male rat offspring. DBP affects the development of the genital tubercle by reducing the serum T concentration and expressions of AR, Shh, Bmp4 and Fgf8 in the genital tubercle, which might underlie the mechanism of DBP inducing hypospadias.

[Effects of Yishen Shengjing Capsules on semen quality and gonadal hormone levels in rats with dibutyl phthalate-induced reproductive function injury].

OBJECTIVE: To study the possible pathogenesis of infertility caused by dibutyl phthalate (DBP) and investigate the effects of Yishen Shengjing Capsules (YSC, kidney-tonifying and essence-producing capsules) on DBP-induced reproductive function injury and its possible action mechanisms in male Wistar rats. METHODS: Models of DBP-induced reproductive function injury were made in 80 male Wistar rats and another 20 were used as blank controls. After modeling, the model rats were randomly divided into a model control, a high-dose YSC, a medium-dose YSC, and a low-dose YSC group. Four weeks after intervention, all the animals were sacrified for observation of the histomorphologic changes in the testis under the light microscope, measurement of sperm concentration, motility and abnormality, and determination of the levels of serum testosterone (T), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) by radioimmunoassay. RESULTS: Compared with the blank controls, the model rats showed obvious pathological changes in testicular histomorphology, significantly decreased sperm concentration and motility, increased sperm abnormality, reduced contents of serum T and LH, and elevated the level of serum FSH (P<0.01). After 4 weeks of medication, the animals of the high-, medium-, and low-dose YSC groups, in comparison with the model controls, exhibited different degrees of recovery from testicular histomorphological damage, remarkably increased sperm concentration and motility, decreased sperm abnormality, elevated levels of serum T and LH, and reduced content of serum FSH (P<0.01). There were statistically significant differences in all the parameters above between the high-dose YSC and medium- and low-dose YSC groups (P<0.01). CONCLUSIONS: DBP reduces sperm motility and concentration, increases sperm abnormality, causes damage to the morphological structure of the rat testis, decreases the contents of serum T and LH, and elevates the level of the serum FSH. Yishen Shengjing Capsules can improve DBP-induced productive function injury, increase sperm motility and concentration, decrease sperm abnormality, elevate the level of serum T and LH, reduce the content of serum FSH, improve the morphological structure of the testis, and thus promote the reproductive function of the male rat.

Toxicity study of dibutyl phthalate of Rubia cordifolia fruits: in vivo and in silico analysis.

Natural toxins from plant sources with wide ranges of biological activities reflect the upswing of drug design in the pharmaceutical industry. Rubia cordifolia L. is one of the most important red dye yielding plants. Most of the former researches have focused on the bioactive compounds from the roots of R. cordifolia, while no attention was paid towards the fruits. For the first time, here we report the presence of dibutyl phthalate in the fruits of R. cordifolia. Structural characterization was carried out using Ultraviolet-Visible spectrophotometer (UV-Vis), Fourier transform infrared (FTIR), gas chromatography-mass spectrophotometer (GC-MS), Nuclear magnetic resonance (NMR). Acute toxicity of the crude ethanolic extracts of the R. cordifolia fruits was examined in Swiss albino mice. No mortality was observed in all treated mice with 100, 500, 1000 mg/kg body weight of crude extract of R. cordifolia fruit and it indicates that the LD50 value is higher than 1000 mg/kg body weight. This study exhibited a significant change in the body weight. Alanine transaminase (ALT), total protein, triglycerides, glucose, and also the histopathological analysis of liver for all treated mice showed difference from the control group. The dibutyl phthalate was further evaluated for the toxicity study through in silico analysis. Together, the results highlighted that the toxic potential of R. cordifolia fruits extracts and also the toxicity profile of the fruit should be essential for the future studies dealing with the long term effect in animals. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1059-1067, 2016.

The effect of di-n-butyl phthalate on testis and the potential protective effects of resveratrol.

This study aimed to observe the possible protective effects of resveratrol (RSV) against the damage of di-n-butyl phthalate (DBP) on the testis. The study was conducted in 6 groups of rats with 6 animals in each group aged 20 days. The groups include group 1: control group; group 2: solvent (carboxymethylcellulose (CMC), 10 ml/kg); group 3: 500 mg/kg/day DBP; group 4: 500 mg/kg/day DBP + 20 mg/kg/day RSV; group 5: 1000 mg/kg/day DBP; and group 6: 1000 mg/kg/day DBP + 20 mg/kg/day RSV. Groups were treated by gavage for 30 days. Indirect immunohistochemical staining was performed with c-kit, AT1, and ER-α antibodies. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling (TUNEL) method was used for apoptosis. It was found in the DBP-applied groups the C-kit immunostaining, which is parallel to increasing dose, decreased in comparison with the control. C-kit reactivity was similar to that of the control group in the group applied with 500 mg/kg/day + RSV; however, the reactivity was not same in the 1000 mg/kg/day DBP-applied group. It was observed that the reactivity of AT1 increased in the DBP-applied groups. RSV reversed these changes with its protective effects. While there was not much difference between the groups in terms of estrogen receptor reactivity, it was observed that the high dose of DBP reduced the level of estrogen receptor and the resveratrol was not at enough levels in all doses. In TUNEL analysis, high doses of DBP increased the apoptosis in all types of cells; nevertheless, the resveratrol application decreased the apoptosis in the low-level DBP dose. In the statistical analysis, while the length of epithelium and the diameter of seminiferous tubules decreased for all the other groups, it reverted to its original state in the RSV-applied groups. In conclusion, DBP (with increasing dose) administration caused cycle and hormonal changes in testis, resveratrol were recovered the cyclic changes but in hormonal changes, RSV is efficient too but inadequate.

Influence of oily vehicles on fetal testis and lipid profile of rats exposed to di-butyl phthalate.

It has been hypothesized that oils containing high levels of omega-3 polyunsaturated fatty acids, such as canola and fish oil, could counteract some of the adverse effects induced by phthalates. In the present study, the influence of different oily vehicles on di-butyl phthalate (DBP)-induced testicular toxicity and lipid profile was investigated. Pregnant Wistar rats were treated by oral gavage from gestation days 13 to 20 with DBP (500 mg/kg/day) diluted in three different vehicles: corn, canola or fish oil. Male fetuses were analyzed on gestation day 20. DBP exposure lowered intratesticular testosterone levels and anogenital distance, regardless of the vehicle used. The percentage of seminiferous cords containing multinucleated gonocytes and cord diameter was increased in DBP-exposed groups, compared with vehicle controls, with no difference between the three DBP-exposed groups. Clustering of Leydig cells was seen in all DBP groups. Lipid profile indicated that administration of canola and fish oil can increase the content of omega-3 fatty acids in rat testis. However, content of omega-3 was diminished in DBP-treated groups. Overall, our results indicate that different oily vehicles did not alter fetal rat testicular toxicity induced by a high DBP dose.

Short-term neonatal/prepubertal exposure of dibutyl phthalate (DBP) advanced pubertal timing and affected hypothalamic kisspeptin/GPR54 expression differently in female rats.

Dibutyl phthalate (DBP) had been widely used and its exposure in children has been thought to be one of the reasons causing a trend of advanced pubertal timing in girls. Puberty starts from hypothalamic gonadotropin-releasing hormone release which is controlled by many factors including neurotransmitter kisspeptin and its receptor GPR54. These neural organization or reorganization happens in hypothalamus during neonatal or prepubertal period which may be two target windows of DBP exposure. The present study was designed to determine: (1) the difference between the effects of neonatal and prepubertal DBP exposure on female pubertal timing; (2) whether kisspeptin/GPR54 expression in hypothalamus would respond to neonatal and prepubertal DBP exposure differently. Female Sprague-Dawley rats were exposed by subcutaneous injection of 0.5, 5 and 50mg/kg DBP during Postnatal day (P)1-5 (neonatal) or P26-30 (prepubertal). Physiological data demonstrated that both neonatal and prepubertal DBP exposure could advance pubertal timing significantly accompanied by irregular estrous cycles but only a little gonadal impairment. Exposure-period-related difference was found significant with prepubertal exposure groups having longer estrous cycle duration, heavier at vaginal opening and having higher serum estradiol level compared with neonatal exposure groups. Molecular data showed an up-regulated trend in kisspeptin mRNA and immunoreactivity levels of hypothalamic area arcuate but a down-regulation in GPR54 mRNA expression after P1-5 DBP treatment. In P26-30 groups, kisspeptin mRNA and immunoreactivity levels tended to be lower after DBP treatment. These results demonstrated small dose of DBP could induce earlier pubertal timing in females and both neonatal and prepubertal periods were critical windows for DBP exposure.

Curcumin and kolaviron ameliorate di-n-butylphthalate-induced testicular damage in rats.

The present study was carried out to evaluate the ameliorative effects of kolaviron (a biflavonoid from the seeds of Garcinia kola) and curcumin (from the rhizome, Curcuma longa L.) on the di-n-butylphthalate (DBP)-induced testicular damage in rats. Administration of DBP to rats at a dose of 2 g/kg for 9 days significantly decreased the relative testicular weights compared to the controls, while the weights of other organs remained unaffected. Curcumin or kolaviron did not affect all the organ weights of the animals. While only DBP treatment significantly increased the testicular malondialdehyde level and gamma-glutamyl transferase activity (gamma-GT), it markedly decreased glutathione level, the testicular catalase, glucose-6-phosphate dehydrogenase, superoxide dismutase, sperm gamma-GT activities and serum testosterone level compared to the control group. Data on cauda epididymal sperm count and live/dead ratio were not significantly affected in the DBP-treated rats. Alone, DBP treatment resulted in a 66% decrease in spermatozoa motility and a 77% increase in abnormal spermatozoa in comparison to control. DBP-treated rats showed marked degeneration of the seminiferous tubules with necrosis and defoliation of spermatocytes. The DBP-induced injuries in biochemical, spermatological parameters and histological structure of testis were recovered by treatment with kolaviron or curcumin. The pattern in the behaviour of these compounds might be correlated with their structural variations. Our results indicate that kolaviron and curcumin protect against testicular oxidative damage induced by DBP. The chemoprotective effects of these compounds may be due to their intrinsic antioxidant properties and as such may prove useful in combating phthalate-induced reproductive toxicity.

Comparison of embryotoxicity of ESBO and phthalate esters using an in vitro battery system.

Epoxidized soy bean oil (ESBO) and phthalate esters have been used as a plasticizer in polyvinyl chloride products. In this study, the embryotoxicity of ESBO and phthalate esters, namely, diethyl hexyl phthalate (DEHP), butylbenzyl phthalate (BBP) and dibutyl phthalate (DBP) was evaluated using short-term in vitro battery system, such as the whole embryo, midbrain and limb bud culture systems. Whole embryos at gestation day 9.5 were cultured for 48 h and the morphological scoring was measured. The cytotoxic effect and differentiation for mid-brain (MB) and limb bud (LB) cell were assessed by 50% inhibition concentration (IC(50)) with neutral red uptake and hematoxylin-stained foci (MB) or Alcian Blue staining (LB), respectively. In the whole embryo culture assay, ESBO (83, 250 and 750 microg/ml) exerted no toxic effect on growth and development of the embryo, whereas phthalate esters (1, 10, 100 microg/ml for DEHP, 10, 100, 1,000 microg/ml for BBP and DBP) inhibited growth and development dose dependently. In mid-brain and limb bud culture, the IC(50) of differentiation and cytotoxicity in BBP was 412.24 and 231. 76 microg/ml for mid-brain, and 40.13 and 182.38 microg/ml for limb bud, respectively. The IC(50) of differentiation and cytotoxicity in DBP was 27.47 and 44.53 microg/ml for mid-brain, and 21.21 and 25.54 microg/ml for limb bud cells, respectively. The lower IC(50) in both cells was obtained from DBP when compared to BBP. From these results, limb bud cells responded more sensitively to BBP and DBP than mid-brain cells. The IC(50) of limb bud cell differentiation and cytotoxicity in DBP is 1.9 and 7.1 less than that of BBP. However, any alteration in cytotoxicity and differentiation was observed with ESBO treatment. These studies suggested that ESBO is not embryotoxic; however, DEHP, BBP and DBP exhibit embryotoxic potential at high concentration.