A comparative study based on activity, conformation and computational analysis on the inhibition of human salivary aldehyde dehydrogenase by phthalate plasticizers: Implications in assessing the safety of packaged food items.

Phthalate plasticizers are commonly used in various consumer-end products. Human salivary aldehyde dehydrogenase (hsALDH) is a detoxifying enzyme which defends us from the toxic aldehydes. Here, the effect of phthalates [Di-2-ethylhexyl phthalate (DEHP), Diethyl phthalate (DEP) and Dibutyl phthalate (DBP)] on hsALDH has been investigated. These plasticizers inhibited hsALDH, and the IC50 values were 0.48 ± 0.04, 283.20 ± 0.09 and 285.00 ± 0.14 µM for DEHP, DEP and DBP, respectively. DEHP was the most potent inhibitor among the three plasticizers. They exhibited mixed-type linear inhibition with inclination towards competitive-non-competitive inhibition. They induced both tertiary and secondary structural changes in the enzyme. Quenching of intrinsic hsALDH fluorescence in a constant manner was observed with a binding constant (Kb) of 8.91 × 106, 2.80 × 104, and 1.31 × 105 M-1, for DEHP, DEP and DBP, respectively. Computational analysis showed that these plasticizers bind stably in the proximity of hsALDH catalytic site, reciprocating via non-covalent interactions with some of the amino acids which are evolutionary conserved. Therefore, exposure to these plasticizers inhibits hsALDH which increases the risk of aldehyde induced toxicity, adversely affecting oral health. The study has implications in assessing the safety of packaged food items which utilize phthalates.

Dose-related morphological changes in the epididymal region of sexually active adult male Japanese quail treated with di-n-butyl phthalate (DBP) commencing during the pre-pubertal stage.

Di-n-butyl phthalate (DBP) is widely used as a plasticizer in personal care and medical products and is known to induce toxicity in the male reproductive organs in both mammals and birds. In this study, there was investigation of the effects of DBP on the epithelium of the rete testis, proximal, and distal efferent ductules and epididymal duct of adult Japanese quail (Coturnix japonica) following treatment with varying doses during the pre-pubertal and peri-pubertal periods. Pre-pubertal quail (n = 25) 4 weeks post-hatching were dosed orally with 10, 50, 200 and 400 mg DBP/kg/d, for 30 days and control birds were administered corn-oil only (n = 5 per group). Histo-metrically, there was lesser (P <  0.001) epithelial heights of the rete testis and efferent ductules in all quail DBP-treated groups, but not in the epididymal duct epithelium. There were no morphological change effects as a result of DBP treatments in the rete testis epithelium, while there were epithelial cytoplasmic vacuoles detected in the distal efferent ductule and epididymal duct of birds treated with 50, 200 and 400 mg DPB/kg/d. There were several lesions, including degenerative changes, cytoplasmic vacuoles, apoptosis and autophagy in the epithelium of the proximal efferent ductule in quail treated with 200 and 400 mg DBP/kg/d. Overall, the results indicate that treatment with DBP during the pre-pubertal period induced dose-dependent histometric and morphological changes in the epithelium of the epididymal region. It is concluded that the proximal efferent ductule was a highly sensitive component of the epididymal tissues of Japanese quail following treatment with DBP during the pre-pubertal period.

Intrauterine exposure to low-dose DBP in the mice induces obesity in offspring via suppression of UCP1 mediated ER stress.

Dibutyl phthalate (DBP) is recognized as an environmental endocrine disruptor that has been detected in fetal and postnatal samples. Recent evidence found that in utero DBP exposure was associated with an increase of adipose tissue weight and serum lipids in offspring, but the precise mechanism is unknown. Here we aimed to study the effects of in utero DBP exposure on obesity in offspring and examine possible mechanisms. SPF C57BL/6J pregnant mice were gavaged with either DBP (5 mg /kg/day) or corn oil, from gestational day 12 until postnatal day 7. After the offspring were weaned, the mice were fed a standard diet for 21 weeks, and in the last 2 weeks 20 mice were selected for TUDCA treatment. Intrauterine exposure to low-dose DBP promoted obesity in offspring, with evidence of glucose and lipid metabolic disorders and a decreased metabolic rate. Compared to controls, the DBP exposed mice had lower expression of UCP1 and significantly higher expression of Bip and Chop, known markers of endoplasmic reticulum (ER) stress. However, TUDCA treatment of DBP exposed mice returned these parameters nearly to the levels of the controls, with increased expression of UCP1, lower expression of Bip and Chop and ameliorated obesity. Intrauterine exposure of mice to low-dose DBP appears to promote obesity in offspring by inhibiting UCP1 via ER stress, a process that was largely reversed by treatment with TUDCA.

Steady-State Human Pharmacokinetics of Monobutyl Phthalate Predicted by Physiologically Based Pharmacokinetic Modeling Using Single-Dose Data from Humanized-Liver Mice Orally Administered with Dibutyl Phthalate.

Dibutyl phthalate (DBP) was widely used as a plasticizer but it has been recently replaced with other kinds of phthalates such as di(2-ethylhexyl)phthalate and diisononyl phthalate because of its toxicity. To evaluate the human risk of DBP, forward and reverse dosimetry was conducted using in silico simplified physiologically based pharmacokinetic (PBPK) modeling based on in vivo experimental pharmacokinetic data in humanized-liver mice (HL-mice) obtained after an oral dose of 100 mg/kg. Absorbed DBP was converted to monobutyl phthalate (MBP) and its glucuronide extensively in vivo. HL-mice had higher concentrations of MBP glucuronide in plasma than did the control mice. Concentrations of MBP glucuronide in 0-7 h accumulated urine samples from HL-mice were significantly higher than those in control mice. Similarly, in vitro MBP glucuronidation rates mediated by pooled microsomes from rat or mouse livers were lower than those mediated by human liver microsomes. Liver damage by MBP to humanized liver was detected by measuring human albumin mRNA in HL-mouse plasma. By simple PBPK modeling, in silico concentration curves in plasma, liver, or urine following virtual oral administration of DBP were created for rats, control mice, and HL-mice. A human PBPK model for MBP was established based on the HL-mouse PBPK model using allometric scaling without consideration of interspecies factors in terms of liver metabolism. Human PBPK models were used to estimate urinary and plasma concentrations of MBP and its glucuronide throughout 14 days of oral DBP administration (1.2 and 13 µg/kg/day). Reverse dosimetry PBPK modeling found that reported 50th and 95th percentile MBP urine and plasma concentrations of the general population could potentially imply exposures similar to or exceeding tolerable daily intake levels (5-10 µg/kg/day) recommended by the European and Japanese authorities. Further in-depth assessment of DBP is needed to assess the validity of assumptions made based on human biomonitoring data.

Oxidative Damage and Genetic Toxicity Induced by DBP in Earthworms (Eisenia fetida).

Di-n-butyl phthalate (DBP) is one of the most ubiquitous plasticizers used worldwide. However, it has negatives effects on the soil, water, atmosphere, and other environmental media and can cause serious pollution. According to the artificial soil test and previous studies, this study was conducted to evaluate the toxicity of earthworms induced by DBP at different concentrations (0, 0.1, 1.0, 10, and 50 mg kg-1) on the 7th, 14th, 21st, and 28th days of exposure. The variations in the antioxidant activities of enzymes, such as catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and glutathione-S-transferase (GST), in the amounts of malondialdehyde (MDA) and reactive oxygen species (ROS) and in the amount of DNA damage were measured to evaluate the toxic impact of DBP in earthworms. Upon exposure to DBP, the SOD, CAT, POD, and GST activities were significantly increased, with the exception of the 0.1 mg kg-1 treatment dose. High concentrations of DBP (10 and 50 mg kg-1) induced superfluous ROS to be produced and caused the MDA content to increase significantly. Therefore, we proposed that DBP led to DNA damage in earthworm coelomocytes in a dose-dependent manner, which means that DBP is a source of oxidative damage and genetic toxicity in earthworms.

Oral exposure to dibutyl phthalate exacerbates chronic lymphocytic thyroiditis through oxidative stress in female Wistar rats.

Chronic lymphocytic thyroiditis (CLT) is a common autoimmune disorder. The possible pathogenic role and mechanism of dibutyl phthalate (DBP) in CLT is still controversial. Experiments were conducted after 35-days of oral exposure to the three concentrations of DBP or saline, and three immunizations with thyroglobulin (TG). Healthy female Wistar rats were randomly divided into ten exposure groups (n = 8 each): (A) saline control, (B) 0.5 mg/kg/d DBP, (C) 5 mg/kg/d DBP, (D) 50 mg/kg/d DBP, (E) TG-immunized group, (F) TG- combined with 0.5 mg/kg/d DBP, (G) TG- combined with 5 mg/kg/d DBP, (H) TG- combined with 50 mg/kg/d DBP, (I) TG- combined with 50 mg/kg/d DBP plus 100 mg/kg/d vitamin C; (J) 100 mg/kg/d vitamin C. We showed that oral exposure DBP can aggravate CLT in rats. This deterioration was concomitant with increased thyroid auto antibodies, Th1/Th2 imbalance and Th17 immune response, activated pro-inflammatory and apoptosis pathways, and increased thyroid dysfunction in rats. Our results also suggested that DBP could promote oxidative damage. The study also found that vitamin C reduced the levels of oxidative stress and alleviated CLT. In short, the study showed that DBP exacerbated CLT through oxidative stress.

Di-n-butyl phthalate prompts interruption of spermatogenesis, steroidogenesis, and fertility associated with increased testicular oxidative stress in adult male rats.

Di-n-butyl phthalate (DBP) is extensively used as plasticizer, and it was ubiquitary released into the environment. The present study was aimed to investigate the effect of DBP on reproductive competence in adult male rats. Adult male rats were received corn oil or DBP injection intraperitoneally (ip) at 100 and 500 mg/kg body weight on 90, 97, 104, and 111 days. Following completion of the experimental period, adult male rats were cohabitated with untreated proestrus female rats for determination of fertilization capacity. Then, adult male rats were sacrificed, and other reproductive endpoints were determined by histopathology and biochemical analysis. The results revealed significant reduction of fertilization potential by decrease mating, fertility indices with increase pre-implantation and post-implantation losses, and resorptions in normal female rat cohabitation with DBP-treated adult male rats. The testes, seminal vesicle tissue somatic indices, epididymal sperm count, motility, viability, and hypoosmotic swelling (HOS) sperm were significantly decreased with increased sperm morphological abnormalities in DBP-treated adult male rats. The disorientation of spermatogenic cells decreased the diameter and epithelial thickness of seminiferous tubule in the testicular histopathology of DBP-exposed rats. Significant reduction of testicular 3ß-hydroxysteroid dehydrogenase and 17ß-hydroxysteroid dehydrogenase enzyme levels and serum testosterone with increased follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were observed in DBP-treated groups. Higher testicular oxidative stress marker (lipid peroxidation product) with lower antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase levels in DBP-exposed groups was observed. From these results, it can be concluded that DBP increases oxidative stress; it leads to impairment of spermatogenesis, steroidogenesis, and fertility in adult male rats.

In Utero Exposure to Di( n-butyl)phthalate Induces Morphological and Biochemical Changes in Rats Postpuberty.

Pregnant Sprague-Dawley rats were orally administered di( n-butyl)phthalate (DBP; 100 mg/kg/day) on gestation days (GD) 12 to 21. We investigated the male offspring and probed morphological alterations in Sertoli cells at 7, 9, 14, and 17 weeks of age. Parameters assessed in this study included offspring number, sex ratios, body weights, testis weights, seminiferous tubule (ST) profile numbers and diameters, number of vimentin-labeled Sertoli cells, and both testosterone and follicle-stimulating hormone (FSH) levels. Testicular weight/body weight ratios and the numbers and diameters of ST in maximum transverse testicular sections were statistically similar at weeks 7 and 9; however, at weeks 14 and 17, they were statistically different and displayed higher BrdU-positive Sertoli cells/Sertoli cell ratios in the DBP treatment group. Noteworthily, the serum FSH levels were higher and testicular testosterone levels were lower in the DBP treatment group. To our knowledge, the present study is the first to report that in utero DBP exposure significantly increased Sertoli cell numbers and their cellular proliferation from postpuberty to adulthood, with a significant decrease in testicular testosterone and an increase in FSH.

Prenatal exposure to di-n-butyl phthalate disrupts the development of adult Leydig cells in male rats during puberty.

Fetal exposure to di-n-butyl phthalate (DBP) causes the adult disease such as lower testosterone production and infertility. However, the mechanism is still unknown. The objective of the present study is to determine how DBP affects the involution of fetal Leydig cells during the neonatal period and how this event causes the delayed development of the adult Leydig cells during puberty. The pregnant Sprague Dawley dams were randomly divided into 3 groups and were gavaged with 0 (corn oil, the vehicle control), 100 or 500mg/kg DBP from gestational day 12 (G12) to G21. The blood and testes were collected from male pups on postnatal day 4 (P4), P7, P14, P21, P28, and P56. Serum testosterone concentrations were assessed and the mRNA levels of Leydig cell- or gonadotroph cell-specific genes were measured. Prenatal exposure to DBP caused the aggregation of fetal Leydig cells, which slowly disappeared when compared to the control. This effect was associated with the reduction of testicular testosterone secretion and down-regulation of the mRNA levels of Leydig cell biomarkers including Scarb1, Star, Cyp11a1, Hsd3b1, Hsd11b1, and Hsd17b3 as well as the gonadotroph biomarkers including Lhb and Gnrhr. In conclusion, we demonstrated that the increased aggregation of fetal Leydig cells by DBP delayed fetal Leydig cell involution, thus leading to the disrupted development of the adult Leydig cells.

A study on the in vitro percutaneous absorption of silver nanoparticles in combination with aluminum chloride, methyl paraben or di-n-butyl phthalate.

Some reports indicate that the silver released from dermally applied products containing silver nanoparticles (AgNP) (e.g. wound dressings or cosmetics) can penetrate the skin, particularly if damaged. AgNP were also shown to have cytotoxic and genotoxic activity. In the present study percutaneous absorption of AgNP of two different nominal sizes (Ag15nm or Ag45nm by STEM) and surface modification, i.e. citrate or PEG stabilized nanoparticles, in combination with cosmetic ingredients, i.e. aluminum chloride (AlCl3), methyl paraben (MPB), or di-n-butyl phthalate (DBPH) was assessed using in vitro model based on dermatomed pig skin. The inductively coupled plasma mass spectrometry (ICP-MS) measurements after 24h in receptor fluid indicated low, but detectable silver absorption and no statistically significant differences in the penetration between the 4 types of AgNP studied at 47, 470 or 750µg/ml. Similarly, no significant differences were observed for silver penetration when the AgNP were used in combinations with AlCl3 (500µM), MPB (1250µM) or DBPH (35µM). The measured highest amount of Ag that penetrated was 0.45ng/cm2 (0.365-0.974ng/cm2) for PEG stabilized Ag15nm+MPB.

In utero exposure of high-dose di- n-butyl phthalate resulted in opposite effects on testicular cell apoptosis in late embryonic and pubertal male rat offspring.

OBJECTIVE: To investigate the effects of in utero exposure to high-dose di- n-butyl phthalate (DBP) on testicular cell apoptosis in late embryonic and pubertal male rat offspring. METHODS: Twenty pregnant Sprague-Dawley (SD) rats were divided into two groups. During gestation day (GD) 12 to GD 19, control group was given 1 ml day-1 of olive oil and experimental group was given DBP 500 mg kg-1 day-1 by gavage. On GD 19.5 and postnatal day (PND) 45, the testes were removed. Morphological analysis of the testes was observed by transmission electron microscopy and hematoxylin and eosin (H&E) staining. Testicular cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). The expression of Bcl-2, Bax, and p53 was presented by immunohistochemistry (IHC) and western blot. Data of the two groups was compared using independent samples t-test and Mann-Whitney test by SPSS 20.0. RESULTS: H&E staining showed that spermatogenetic cells were significantly decreased in DBP exposed pubertal rat testis. The apoptosis index of testes in DBP-treated group was significantly lower on GD 19.5 but higher on PND 45 than that of the controls ( p < 0.01). IHC and western blot revealed significantly increased expression of Bcl-2 in GD 19.5 rat testis and Bax and p53 in PND 45 rat testis after DBP exposure, compared with the control ( p < 0.05). CONCLUSION: In utero exposure of high-dose DBP resulted in opposite effects on testicular cell apoptosis in late embryonic and pubertal rat offspring. The overexpression of Bcl-2, Bax, and p53 might be related to the occurrence of abnormal apoptosis and finally produce male infertility.

A crossover-crossback prospective study of dibutyl-phthalate exposure from mesalamine medications and semen quality in men with inflammatory bowel disease.

BACKGROUND: Phthalates are widely used chemicals with ubiquitous exposure. Dibutyl-phthalate (DBP), a male reproductive toxicant in animals, is understudied in humans. Some mesalamine medications used to treat inflammatory bowel disease (IBD) have DBP in their coating, whereas other mesalamine formulations do not. OBJECTIVES: Taking advantage of differences in mesalamine formulations, we investigated whether high-DBP exposure from mesalamine medications was associated with decreased semen parameters. METHODS: 73 men with IBD taking mesalamine participated in a crossover-crossback prospective study. Men taking non-DBP containing mesalamine at baseline i.e., background exposure, crossed-over for four months to high-DBP mesalamine and then crossed-back for four months to their non-DBP mesalamine (B1HB2-arm;Background1-High-Background2) and vice versa for men taking high-DBP mesalamine at baseline (H1BH2-arm;High1-Background-High2). Men provided up to six semen samples (2: baseline, 2: crossover and 2: crossback). RESULTS: We estimated crossover, crossback and carryover effects using linear mixed models adjusted for abstinence time, age, season and duration on high-DBP mesalamine at baseline. Semen parameters in B1HB2-arm (26 men, 133 samples) decreased after high-DBP mesalamine exposure (crossover versus baseline), especially motility parameters, and continued to decrease further even after crossback to non-DBP mesalamine (crossback versus crossover). The cumulative carryover effect of high-DBP (crossback versus baseline) was a decrease of % total sperm motility by 7.61(CI:-13.1, -2.15), % progressive sperm motility by 4.23(CI:-8.05, -0.4) and motile sperm count by 26.0% (CI:-46.2%, 1.7%). However, H1BH2-arm (47 men, 199 samples) had no significant change during crossover or crossback. CONCLUSIONS: Men newly exposed to high-DBP mesalamine for four months had a cumulative reduction in several semen parameters, primarily sperm motility, that was more pronounced and statistically significant even after exposure ended for four months.

Sublethal concentrations of di-n-butyl phthalate promote biochemical changes and DNA damage in juvenile Nile tilapia (Oreochromis niloticus).

Increase in consumption of consumer items such as plasticizers have resulted in a sharp rise in the presence of xenobiotics like phthalic acid esters (PEs) in freshwater and marine environments due to contaminated runoff and improper release of effluents. The sublethal toxicity of Di-n-butyl phthalate (DBP) was investigated in juvenile Nile tilapia, Oreochromis niloticus, in an attempt to determine the biological effect of exposure to 1/2 and 1/3 median lethal concentration (96-h LC50) which, in our study was experimentally determined to be 11.8 mg/l. Following four days of exposure, indices of the oxidative potential [Malondialdehyde content (MDA)], antioxidant parameters [superoxide dismutase activity (SOD) and reduced glutathione level (GSH)] and DNA damage were evaluated by single-cell gel electrophoresis (Comet assay). Hepato-renal markers [alanine aminotransferase activity (ALT), creatinine and urea level] and cortisol levels were also quantified in serum. Additionally, histopathological investigations of liver, kidney and gill tissues were conducted. Comparative results between the 1/2 96-h LC50 group and the 1/3 96-h LC50 group clearly showed that there was a significant elevation in MDA levels and a marked increase in DNA damage in addition to inhibition of antioxidant barriers as represented by attenuation of SOD activity and GSH level in the group that was exposed to higher concentration of DBP (1/2 96-h LC50). The hepatorenal markers and cortisol levels were also observed to be elevated. Histopathological examination of the liver, kidney and gills showed pathological alterations that could be correlated with changes in the biochemical profile of the exposed fish. Additionally, anomalous clinical signs were noted. Based on these findings, we conclude from our study that exposure of juvenile O. niloticus to DBP has the potential to induce biochemical as well as tissue morphological alterations associated with oxidative injury and DNA damage.

In utero-exposed di(n-butyl) phthalate induce dose dependent, age-related changes of morphology and testosterone-biosynthesis enzymes/associated proteins of Leydig cell mitochondria in rats.

Female pregnant Sprague-Dawley rats were intragastrically (ig) administered di(n-butyl) phthalate (DBP) at four doses (0, 10, 50 and 100 mg/kg) during gestation days (GD) 12-21 (n = 5 per group). The age-related morphological changes of Leydig cell mitochondrion (LC-Mt) and testosterone biosynthesis enzymes/associated genes/proteins expression levels were investigated. As compared to the control (no DBP), the 10 mg, and 50 mg DBP dose groups, the 100 mg DBP dose group at weeks 5 and 7 showed a significant amount of small LC-Mt. Thereafter, from weeks 9 to 17, the LC-Mt size and quantity in the 100 mg DBP dose group increased and became statistically similar to the other dose groups; hence, dose and time-dependent LC-Mt changes were observed. Throughout the study, the 100 mg DBP dose group had significantly lower testosterone levels. In addition, the 100 mg DBP dose group displayed lower StAR (StAR, steroidogenic acute regulatory protein) and P450scc (CYP11a1, cholesterol side-chain cleavage enzyme) levels at weeks 5 and 7, but they became statistically similar to all other dose groups at weeks 9 to 17; in contrast, the SR-B1 (Sarb1, scavenger receptor class B member 1) levels were similar for all DBP dose groups. The rats in utero 100 mg DBP /kg/day (GD 12-21) exposure results from this study indicate a dose-dependent, age-related morphological change in LC-Mt which are linked to reductions in testosterone biosynthesis genes / proteins expression, specifically StAR and P450scc.

Dibutyl phthalate induces oxidative stress and impairs spermatogenesis in adult rats.

Phthalates are abundantly produced plasticizers, and dibutyl phthalate (DBP) is the most widely used derivative in various consumer products and medical devices. This study was conducted to further explore the potential testicular toxicity of DBP in adult rats and to elucidate the underlying mechanisms. Adult male albino rats were treated orally with DBP at doses of 0, 200, 400, or 600 mg/kg/day for 15 consecutive days. Testicular weight, sperm count, and motility were significantly decreased. Treatment with DBP decreased serum follicle-stimulating hormone and testosterone levels and testicular lactate dehydrogenase activity. DBP treatment also decreased serum total antioxidant capacity and the activities of the testicular antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione reductase. Further, DBP treatment provoked degeneration with absence of spermatogenesis and sperms and necrosis in some of seminiferous tubules. These results indicated that oxidative stress and subsequent decrease in testosterone secretion were the potential underlying mechanism of DBP-induced testicular toxicity.

Long-term exposures to di-n-butyl phthalate inhibit body growth and impair gonad development in juvenile Murray rainbowfish (Melanotaenia fluviatilis).

The aim of the present study was to evaluate whether long-term exposures to environmentally relevant concentrations of di-n-butyl phthalate (DnBP) disrupt the reproduction-based endpoints in juvenile Murray rainbowfish (Melanotaenia fluviatilis). Fish were exposed to 5, 15 or 50 µg l(-1) DnBP for 30, 60 and 90 days each, and the effects on survival, body growth, whole-body concentrations of sex steroid hormones and gonadal development were investigated. The lowest observed effective concentration to affect the condition factor after 90 days was 5 µg l(-1). Complete feminization of the gonad was noted in fish exposed to 5 µg l(-1) for 90 days and to 15 and 50 µg l(-1) of DnBP for 30 or 60 days. After 90 days of exposure to DnBP, the ovaries were regressed and immature as opposed to the control fish which were in early-vitellogenic stage. Testes, present only in fish exposed to 5 µg l(-1) of DnBP for 30 or 60 days, were immature in comparison to the control fish that contained testes in the mid-spermatogenic phase. The E2/11-KT ratio was significantly higher only after exposures to 5 µg l(-1) DnBP for 90 days and 50 µg l(-1) DnBP for 30 days. Our data suggest that exposures to 5 µg l(-1) DnBP for 30 days did not have profound effects on body growth and gonadal differentiation of fish. However, 30 days of exposure to 15 µg l(-1) could interfere with the gonad development and to 50 µg l(-1) could compromise the hormonal profile of juvenile fish.

Assessment of estrogenic potential of di-n-butyl phthalate and butyl benzyl phthalate in vivo.

Phthalate compounds are widely used industrial chemicals; when incorporated into polyvinyl chloride, they are not covalently bound and released into the surrounding media. Some of them have estrogenic potential in vitro but data on in vivo studies are scanty. For the 3-day uterotrophic assay, di-n-butyl phthalate (DBP;10 and 100 mg/kg), butyl benzyl phthalate (BBP; 20 and 200 mg/kg), and diethylstilbestrol (DES, 40 µg/kg, positive control) were administered orally to immature female rats for three consecutive days from postnatal day (PND) 21. For the 20-day pubertal onset assay, DBP (10 and 20 mg/kg), BBP (20 and 200 mg/kg), and DES (6 µg/kg) were administered orally from PND 21 daily for 20 days. In the uterotrophic assay, in groups treated with higher dose of DBP and BBP, the uterine wet weight significantly decreased in the higher dose, and there were minor variations in the ovary wet weight, while the wet weight of these organs increased significantly in DES-treated group. In the 20-day pubertal assay, the weight of uterus and ovary declined significantly and changes in vaginal weight were nonsignificant in DBP- and BBP-treated groups. However, in DES-treated group nonsignificant elevation in vagina weight was observed. All the DES-treated animals showed the vaginal opening (VO) on day 26.17 ± 0.16. However, VO was not observed in any of the animals in control, vehicle control, BBP-, and DBP-treated groups up to PND 42, except in one animal each in vehicle control and DBP (100 mg/kg)-treated groups. The data indicated that both DBP and BBP were unable to induce elevation in the uterine and ovarian weight. While DES treatment can accelerate the growth of uterus and ovary and alter the onset of puberty and estrous cyclicity in prepubertal rats. These suggest that these compounds may not have estrogenic potential in vivo.

The effects on steroidogenesis and histopathology of adult male Japanese quails (Coturnix coturnix japonica) testis following pre-pubertal exposure to di(n-butyl) phthalate (DBP).

In the present study, we have investigated the effects of 30-day dietary (pre-pubertal) exposure to different doses (0 (control), 1, 10, 50, 200 and 400 mg/kg bodyweight/day) of di(n-butyl) phthalate (DBP) on Leydig cells of adult male Japanese quails by quantifying the transcript levels for P450 side-chain cleavage (p450scc), P450c17 (CYP17), and 3ß- and 17ß-hydroxysteroid dehydrogenase (hsd) using quantitative (real-time) polymerase chain reaction (qRT-PCR). In addition, the plasma testosterone levels were analysed using radioimmunoassay (RIA) and testis was examined for evidence of gross pathology and histopathology. Our data showed that pre-pubertal exposure to DBP produced alterations in testicular architecture as evident by poorly developed or mis-shaped testis, and altered spermatogenesis due to tubular degeneration and atrophy of seminiferous tubules especially in the high DBP dose (200 and 400 mg/kg) treated groups. In addition, DBP altered several key enzymes involved in testicular steroidogenesis pathways in an apparent dose-dependent manner. For example, biphasic effects of DBP were observed for P450scc and 3ß-hsd mRNA, that were generally increasing at low dose 10 mg/kg, and thereafter, an apparent dose-dependent decrease between 50 and 400mg/kg. The steroidogenic acute regulatory (StAR) protein was at the lowest detectable limits and therefore not quantifiable. These effects did not parallel the non-significant changes observed for plasma testosterone levels. The present data is consistent with previous reports showing that DBP modulates Leydig cell steroidogenesis in several species, with a potential negative effect on reproduction in those avian species that are vulnerable to endocrine disrupting chemicals.

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.

Estimated daily intake and hazard quotients and indices of phthtalate diesters for young danish men.

Because of wide exposure to phthalates, we investigated whether simultaneous exposure to several phthalates reached levels that might cause adverse antiandrogenic effects. Thirty three healthy young Danish men each delivered three 24-h urine samples during a three months period. The daily intakes of the sum of di-n-butyl and di-iso-butyl phthalate, di(2-ethylhexyl) phthalate, di-iso-nonyl phthalate, and butylbenzyl phthalate were estimated based on urinary excretion of the metabolites. Based on a hazard quotient (HQ) of the individual phthalate (i.e., the ratio between the daily intake and an acceptable level of exposure), a hazard index (HI) for each man was calculated as the sum of HQs for the individual phthalates. All men were exposed to all phthalates during the urine collection periods. Median HIs were all below 1 (i.e., below an acceptable cumulative threshold) ranging from 0.11 to 0.17 over the three different sample collections. Of the 33 men, 2 men had HIs above 1 in one of their three samples, indicating that occasionally the combined exposure to the investigated phthalates reached a level that may not be considered safe. Besides the phthalates investigated here, humans are exposed to numerous other chemicals that also may contribute to a cumulative antiandrogenic exposure.