Animals ; Benzo(a)pyrene/toxicity* ; Cytokines ; Dibutyl Phthalate/toxicity* ; Female ; Liver ; Male ; NF-kappa B ; Rats ; Rats, Sprague-Dawley

OBJECTIVES: Maintaining the constant exposure to hydrophobic organic compouds in acute toxicity tests is one of the most difficult issues in the evaluation of their toxicity and corresponding risks. Passive dosing is an emerging tool to keep constant aqueous concentration because of the overwhelming mass loaded in the dosing phase. The primary objectives of this study were to develop the constant exposure condition for an acute mortality test and to compare the performance of the passive dosing method with the conventional spiking with co-solvent. METHODS: A custom cut polydimethylsiloxane (PDMS) tubing loaded with benzyl butyl phthalate (BBP) was placed in each well of a 24-well plate containing assay medium. The rate of the release of BBP from PDMS was evaluated by measuring the change in the concentration of BBP in the assay medium. The efficiency of maintaining constant exposure condition was also evaluated using a simple two-compartment mass transport model employing a film-diffusion theory. An acute mortality test using 10 C. elegans in each well was conducted for the evaluation of the validity of passive dosing and the comparative evaluation of the passive dosing method and the conventional spiking method. RESULTS: Free concentration in the assay medium reached 95% steady state value within 2.2 hours without test organisms, indicating that this passive dosing method is useful for an acute toxicity test in 24 hours. The measured concentration after the mortality test agreed well with the estimated values from partitioning between PDMS and the assay medium. However, the difference between the nominal and the free concentration became larger as the spiked concentration approached water solubility, indicating the instability of the conventional spiking with a co-solvent. CONCLUSIONS: The results in this study support that passive dosing provides a stable exposure condition for an acute toxicity test. Thus, it is likely that more reliable toxicity assessment can be made for hydrophobic chemicals using passive dosing.
Benzophenones ; Biological Availability ; Boronic Acids ; Caenorhabditis ; Caenorhabditis elegans ; Dibutyl Phthalate ; Dimethylpolysiloxanes ; Phthalic Acids ; Solubility ; Toxicity Tests, Acute

OBJECTIVETo detect the differential expression of Notch1 in the genital tubercle (GT) of fetal male rats with hypospadias induced by maternal exposure to Di-n-butyl phthalate (DBP) and that in normal control fetal rats in order to further explore the role of Notch1 in DBP-induced hypospadias.

METHODSTwenty pregnant SD rats were equally and randomly divided into an experimental and a control group, the former given DBP and the latter soybean oil intragastrically at 800 mg/(kg x d) and 2 ml/d respectively from gestation day (GD) 14 to GD 18. On GD 19, the birth weight (BW), anogenital distance (AGD) and hypospadias incidence were recorded, GTs of the fetal male rats collected, and the expression of Notch1 analyzed by Western blot and immunohistochemistry.

RESULTSThe BW of the fetal male rats was (4.40 +/- 0.30) g in the experimental group, significantly lower than (6.11 +/- 0.40) g in the control (P <0.05), and the AGD was (2.17 +/- 0.18) mm in the former, markedly shorter than (3.28 +/- 0.16) mm in the latter (P<0.05). The incidence of hypospadias was 42.9%. The relative expression of Notch1 was remarkably lower in the hypospadiac rats than in the normal controls (0.671 +/- 0.021 vs 1.327 +/- 0.031, P<0.05), and it was mainly located in the epithelial cells of the GT. The staining intensity was obviously weaker in the hypospadias than in the normal control group.

CONCLUSIONDBP has an obvious toxic effect on fetal male rats and can change the expression of Notch1 in the GT. It possibly affects cell proliferation and apoptosis and epithelial-to-mesenchymal transition (EMT), resulting in the occurrence of hypospadias.

Animals ; Dibutyl Phthalate ; toxicity ; Female ; Fetus ; Hypospadias ; chemically induced ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; metabolism

OBJECTIVETo study the reversibility of di-n-butyl phthalate (DBP) effects on F(1) generation rat testes.

METHODSPregnant rats were treated with different dose of DBP (0, 50, 250 and 500 mg per kg per day) by gavage from GD1 to PND21. The adverse effects of DBP on testes of F(1) male rats in different developmental period (PND14, 21 and 70) were observed by anatomy and pathological methods.

RESULTSThere was no difference in rat testis weight and testis/body weight between DBP-treated group and the control. From the results of pathology and sertoli cell counting, comparing with the control, thinner seminiferous epithelium, decreased cell number and vacuole cells were observed in PND14 male DBP-treated rats. In PND21 rats, the number and form of sertoli cells were recovered and few exfoliated spermatogenic cells were found. When maturing to PND70, few rats were found irreversible damages such as seminiferous tubule degeneration, seminiferous epithelium atrophy, etc.

CONCLUSIONThese results suggest that adverse effects of DBP on young rat testes should be reversibility.

Animals ; Dibutyl Phthalate ; toxicity ; Female ; Male ; Rats ; Rats, Sprague-Dawley ; Sertoli Cells ; pathology ; Testis ; drug effects ; pathology

OBJECTIVETo investigate the effects of prepubertal continuous exposure to dibutyl phthalate (DBP) on the testis development in SD rats.

METHODSTwenty-one-day-old weanling prepubertal male SD rats were randomly divided into a control (n = 24) and an experiment group (n = 54), gavaged daily with corn oil vehicle or corn oil + DBP at the repeated dose of 0 mg/(kg x d) (control), 50 mg/(kg x d) (low-dose), 200 mg/(kg x d) (medium-dose) and 600 mg/(kg x d) (high-dose) for 14, 21 and 28 days, and then sacrificed by decapitation on PND35, PND42 and PND49. The body weight gain, the testis weight and volume and the weight of accessory sex organs were measured, the serum testosterone level assayed by chemoluminescence technique, the testis tissues stained by H&E and observed under the light microscope for morphological alteration, the mean diameter of the seminiferous tubules determined and testicular biopsy scores obtained.

RESULTSDisordered arrangement of spermatogenic cells was found in some seminiferous tubules on PND35 in the low-dose group, but testis development and spermatogenesis were normal on PND42 and PND49. In the medium-dose group, disordered arrangement and decreased number of spermatogenic cells were observed on PND35 and PND42, but without testicular atrophy, and various grades of spermatogenic cells and sperm were seen on PND49. High-dose DBP slowed down the body weight gain, decreased serum T levels and induced degeneration of seminiferous tubules, arrest of spermatogenic epithelium development and necrosis of spermatogenic cells. The pubertal rats (PND49) showed testicular atrophy, azoospermia and delayed development of accessory sex organs.

CONCLUSIONPrepubertal continuous exposure to DBP induces damages to testicular development and spermatogenesis in a dose-dependent manner, and those induced by high-dose DBP cannot be recuperated in the phase of prepubertal development, while the slight adverse effects on the testis induced by low- and medium-dose DBP could be completely or partly reversible before PND49.

Animals ; Dibutyl Phthalate ; toxicity ; Environmental Exposure ; Growth ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; drug effects ; growth & development

OBJECTIVETo explore the effect of environmental estrogens on the proliferation of breast cancer cell line MCF-7.

METHODSThe tested compounds were n-4-nonyphenol, Bisphenol A and dibutylphthalate. Human estradiol-dependent MCF-7 breast cancer cells were grown in DMEM medium containing 10% bovine serum. Five days before the addition of the test compounds, the cells were washed by phosphate-buffered saline, and the medium was substituted with a phenol red-free DMEM medium containing 5% dextral charcoal-stripped FBS. The respective test compound was added in fresh medium and the control cell received only the vehicle (ethanol). The proliferation of MCF-7 was analyzed by the MTT assay, (3)H-TdR incorporation assay and flow cytometry.

RESULTSCompared with the ethanol control, the proliferation and DNA synthesis of the test cells treated with n-4-nonyphenol (8 x 10(-7) mol/L, 96 h), Bisphenol A (8 x 10(-7) mol/L, 96 h) or dibutylphthalate (32 x 10(-6) mol/L, 96 h) treatment was markedly enhanced in a time-dependent and dose-dependent manner.

CONCLUSIONn-4-Nonyphenol, Bisphenol A and dibutylphthalate enhanced the proliferation of human breast cancer cell in vitro, which may demonstrate an estrogenic activity.

Benzhydryl Compounds ; Breast Neoplasms ; pathology ; Cell Division ; drug effects ; Cell Line, Tumor ; Dibutyl Phthalate ; toxicity ; Environmental Pollutants ; toxicity ; Estrogens, Non-Steroidal ; toxicity ; Female ; Humans ; Phenols ; toxicity

OBJECTIVEThe aim of the present study was to investigate the effects of paternal Di-N-butyl-phthalate (DBP) exposure pre- and postnatally on F1 generation offspring, and prenatally on F2 generation offspring.

METHODSMale mice were exposed to either 500 mg/kg or 2 000 mg/kg of DBP for 8 weeks, and mated with non-exposed females. Three-quarters of the females were sacrificed a day prior to parturition, and examined for the number of living and dead implantations, and incidence of gross malformations. Pups from the remaining females were assessed for developmental markers, growth parameters, as well as sperm quantity and quality.

RESULTSThere were no changes in the fertility of parents and in intrauterine development of the offspring. Pups of DBP-exposed males demonstrated growth-retardation. Following paternal exposure to 500 mg/kg bw of DBP, there were almost twice the number of males than females born in the F1 generation. F1 generation females had a 2.5-day delay in vaginal opening. Paternal exposure to 2 000 mg/kg bw of DBP increased the incidence of sperm head malformations in F1 generation males; however, there were no changes in the fertility and viability of foetuses in the F2 generation.

CONCLUSIONPaternal DBP exposure may disturb the sex ratio of the offspring, delay female sexual maturation, and deteriorate the sperm quality of F1 generation males.

Abnormalities, Drug-Induced ; Animals ; Dibutyl Phthalate ; toxicity ; Female ; Male ; Mice ; Paternal Exposure ; adverse effects ; Plasticizers ; toxicity ; Pregnancy ; Sex Ratio ; Sexual Development ; drug effects ; Sperm Head ; drug effects ; pathology

OBJECTIVETo observe the effects of dibutyl phthalate (DBP) and monobutyl phthalate (MBP) on the mRNA and protein expression of insulin-like factor 3 (INSL3) in the Leydig tumor cells (MA-10) of mice and the level of testosterone secreted from MA-10 cells.

METHODSThe MA-10 cells of mice, used as a cellular model, were exposed to DBP and MBP. The content of testosterone in the supernatant medium was measured by enzyme-linked immunosorbent assay; the mRNA and protein expression levels of INSL3 in MA-10 cells were measured by quantitative PCR and Western Blot.

RESULTSCompared with the control group, MA-10 cells showed increased synthesis of testosterone when exposed to low concentrations of DBP and MBP (10(-9) ∼ 10(-6) mol/L) and inhibited synthesis of testosterone when exposed to high concentrations of DBP and MBP (10(-3) mol/L), and the typical two-way effects became more significant as the time went one and the concentrations increased (P < 0.05). Compared with the control group, MA-10 cells showed significantly lower mRNA and protein expression levels of INSL3 when exposed to 10(-6) and 10(-4) mol/L DBP (P < 0.05); MA-10 cells showed increased protein expression of INSL3 when exposed to 10(-7) mol/L MBP, and the mRNA and protein expression levels of INSL3 decreased as the concentration of MBP increased.

CONCLUSIONDBP and MBP can inhibit the secretion of testosterone from MA-10 cells at high concentrations, but stimulate the secretion of testosterone at low concentrations. Both DBP and MBP have inhibitory effects on the mRNA and protein expression of INSL3 in MA-10 cells.

Animals ; Cell Line ; Dibutyl Phthalate ; toxicity ; Insulin ; metabolism ; Leydig Cells ; drug effects ; metabolism ; Male ; Mice ; Phthalic Acids ; toxicity ; Proteins ; metabolism ; Testosterone ; secretion

OBJECTIVETo evaluate the effect of dibutyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) on urine superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in rats.

METHODSAccording to 2×2 factorial analysis, 60 adult male SD rats were randomized into 10 groups (n=6), including a control group (fed with sesame oil), 3 DBP groups (fed with DBP at the doses of 30, 100 and 300 mg/kg), 3 DEHP groups (with DEHP at 50, 150, and 450 mg/kg), and 3 DBP+DEHP groups (with 30 mg/kg DBP+50 mg/kg DEHP, 100 mg/kg DBP+150 mg/kg DEHP, and 300 mg/kg DBP +450 mg/kg DEHP). The agents were administered in a single dose through gavage in a volume of 2 ml. After the treatments, the 24, 48, 72, and 96 h urine samples were collected to determine the SOD activity and MDA content.

RESULTSDBP and DEHP, either alone or in combination, significantly decreased SOD activity and increased MDA content in the urine collected at 24 h but not at the other time points. Such changes were gradually reversed with time.

CONCLUSIONDBP or DEHP treatment alone can result in significant oxidative damage in the kidney of rats, and the toxic effect of the combined exposure is even more obvious.

Animals ; Dibutyl Phthalate ; toxicity ; Diethylhexyl Phthalate ; toxicity ; Environmental Pollutants ; toxicity ; Kidney ; drug effects ; physiopathology ; Male ; Malondialdehyde ; urine ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; urine

OBJECTIVETo evaluate the effects of di-butyl phthalate(DBP) on the sperm motility and oxidative stress in rats.

METHODSHealthy 6-week-old male Sprague Dawlay rats were randomly divided into 4 groups with 8 in each group. DBP dissolved in peanut oil was administered by gavage at dosage of 0, 250, 500, 1,000 mg/(kg.d). After 4-week DBP exposure, the animals were killed and the organs were selected and weighed. The sperm VCL, VSL, VAP, BCF, ALH, LIN, MAD and STR in the cauda epididymis were assessed by computer assisted sperm analysis (CASA), and the activities of superoxide dismutase (SOD) and the contents of malondialdehyde (MDA) in serum and testis homogenate were measured simultaneously. The increase of body weight per day and the organ body weight ratio changes of the liver, testes and epididymides were also observed.

RESULTSThe liver organ body weight ratios of the treated groups were higher than those of the control (P < 0.01), while the testis organ body weight ratios were lower at dosage of 1,000 mg/(kg.d) DBP. Compared with the control group, the parameters of rat sperm VCL and ALH declined significantly at dosage of 1,000 mg/(kg.d) DBP. In addition, DBP showed inhibiting effect on SOD activities in the testis, and it was significant in the highest exposure group compared with the control (P < 0.05). However, there were no differences in serum SOD activities between the treated groups and the control.

CONCLUSIONDBP exposure may affect the sperm motility and the anti-oxidative systems. The testis is a vital target organ influenced by DBP.

Animals ; Dibutyl Phthalate ; toxicity ; Dose-Response Relationship, Drug ; Male ; Oxidative Stress ; drug effects ; Rats ; Rats, Sprague-Dawley ; Sperm Motility ; drug effects