Abstract  We improved our previous analytical method to measure phthalate metabolites in urine as biomarkers for phthalate exposure by automating the solid-phase extraction (SPE) procedure and expanding the analytical capability to quantify four additional metabolites: phthalic acid, mono-3-carboxypropyl phthalate, mono-isobutyl phthalate (miBP), and monomethyl isophthalate. The method, which involves automated SPE followed by isotope dilution-high performance liquid chromatography (HPLC)-electrospray ionization (ESI)-tandem mass spectrometry (MS), allows for the quantitative measurement of 15 phthalate metabolites in urine with detection limits in the low ng/ml range. SPE automation allowed for the unattended sequential extraction of up to 100 samples at a time, and resulted in an increased sample throughput, lower solvent use, and better reproducibility than the manual SPE. Furthermore, the modified method permitted for the first time, the separation and quantification of mono-n-butyl phthalate (mBP) and its structural isomer miBP. The method was validated on spiked pooled urine samples and on pooled urine samples from persons with no known exposure to phthalates.

Abstract  Most rodent developmental toxicity studies of dibutylphthalate (DBP) have relied on bolus gavage dosing. This study characterized the developmental toxicity of dietary DBP. Pregnant CD rats were given nominal doses of 0, 100, or 500 mg DBP/kg/day in diet (actual intake 0, 112, and 582 mg/kg/day) from gestational day (GD) 12 through the morning of GD 19. Rats were killed 4 or 24 hr thereafter. DBP dietary exposure resulted in significant dose-dependent reductions in testicular mRNA concentration of scavenger receptor class B, member 1; steroidogenic acute regulatory protein; cytochrome P450, family 11, subfamily a, polypeptide 1; and cytochrome P450 family 17, subfamily a, polypeptide 1. These effects were most pronounced 4 hr after the end of exposure. Testicular testosterone was reduced 24 hr post-exposure in both DBP dose groups and 4 hr after termination of the 500-mg DBP/kg/day exposure. Maternal exposure to 500 mg DBP/kg/day induced a significant reduction in male offspring's anogenital distance indicating in utero disruption of androgen function. Leydig cell aggregates, increased cord diameters, and multinucleated gonocytes were present in DBP-treated rats. Monobutyl phthalate, the developmentally toxic metabolite of DBP, and its glucuronide conjugate were found in maternal and fetal plasma, amniotic fluid, and maternal urine. Our results, when compared to previously conducted gavage studies, indicate that approximately equal doses of oral DBP exposure of pregnant rats, from diet or gavage, result in similar responses in male offspring.

Abstract  Dibutylphthalate (DBP) was administered to male Sprague Dawley rats in oral doses of 0.01, 0.1 and 1.0 mmol/kg for five days. The lower dose was considered relevant to human intake. Additional groups were given a recovery period of four weeks without DBP. DBP significantly increased the liver microsomal concentration of cytochrome P-450 (48%) at the lowest dose and NADPH-cytochrome-c-reductase activity (28-29%), at the two lower doses. The liver microsomal metabolism of n-hexane increased to about the same extent at all dosage levels. The main increase was found in the formation of the preneurotoxic metabolite 2-hexanol. The induction of cytochrome P-450 and NADPH-cytochrome-c-reductase in liver microsomes did not return to normal after the period of recovery, whereas the metabolism of n-hexane normalized during the same period of time, indicating that the majority of the induced forms of cytochrome P-450 were not related to n-hexane metabolism. No major changes were observed in the liver microsomal metabolism of B(a)P. The only effect found in cytochrome P-450 related metabolism in lung microsomes was a decrease of the B(a)P metabolism, especially in the formation of the 9,10- and 4,5-diol metabolites at lower dosage levels. It is suggested that DBP and its hydrolyzed products formed in the intestine after oral administration exert the same effect on some specific forms of cytochrome P-450 in liver and lung. It is concluded that DBP is a moderate to weak inducer of several minor forms of liver microsomal cytochrome P-450 enzymes in doses which may be relevant to human oral intake.

Abstract  A single oral dose of di-n-butyl phthalate (DBP) to male rats caused a sloughing of the germ cells at 6 h, with more severe sloughing at 24 and 48 h. DBP is metabolized to mono-n-butyl phthalate (MBP), which is transported through the blood-tubular barrier into the seminiferous lumen. MBP is incorporated into the lumen at a maximum rate between 1 and 3 h after dosing with DBP. MBP caused decreases in the activities of succinate dehydrogenase in the Sertoli cells and sorbitol dehydrogenase in the germ cells, an increase in the activity of lactate dehydrogenase (LDH) in the germ cells and in the seminiferous lumen and a decrease in testicular iron levels.

Abstract  An extensive body of animal studies has demonstrated that prenatal phthalate exposure, by lowering fetal testicular testosterone, can cause testicular, epididymal, and gubernacular cord agenesis, as well as shortened anogenital distance (AGD). This cluster of abnormalities has been termed the "phthalate syndrome". We present data from the first study to examine AGD and related endpoints in relation to in utero phthalate exposure in humans. Methods: A standardized measure of AGD and some of the genital measurements used to identify the phthalate syndrome in rodents were obtained in boys 2-30 months of age. Alternative methods for adjusting AGD and body weight at examination were compared. Markers of genital development were examined in relation to phthalate metabolite levels in stored prenatal urine samples. These phthalate levels were compared to those measured in a national sample and to those causing phthalate syndrome in rodents. Results: Four phthalate metabolites [monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and mono-isobutyl phthalate (MiBP)] were inversely related to AGD (p-values from 0.012 (MEP) to 0.055 (MBzP). Comparing concentration in the highest to the lowest quartiles, the odds ratio for a shorter-than-predicted AGD ranged from 3.8 to 10.2 (all p-values < 0.05). AGD was more strongly related to a measure of joint exposure to these four phthalate metabolites. Shorter AGD was correlated with a measure of testicular descent and penile volume. The urinary phthalate concentrations associated with shorter AGD were consistent with those that have been measured in one-quarter of the female population of the United States. The median intake estimates associated with reduced AGD are two orders of magnitude below the USEPA reference doses for these chemicals. Discussion: We demonstrated associations between phthalates and genital development that are consistent with the anti-androgenic action of phthalates and the development of the phthalate syndrome previously identified in rodents at higher doses. This study supports the hypothesis that prenatal phthalate exposure can adversely affect reproductive development in male infants and that current regulatory levels may not be adequately protective.

Abstract  A capillary gas chromatographic method with flame ionization detector (GC-FID) for the detection of the six phthalates (dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), di-n-octyl phthalate (DOP)) in the cosmetics was developed. The phthalates were extracted from cosmetics with methanol under ultrasonication and then separated with high-speed centrifugation. The supernatant was dehydrated and filtrated through membrane with 0.5 microm pore diameter. The filtrate was injected into the GC system for analysis. Then the positive results observed in the GC-FID chromatogram were confirmed by gas chromatography-electron impact-mass detection (GC-EI-MS) analysis. Retention times of the peaks could be applied for qualitative analysis. External standard method was used for quantitative analysis. The recoveries of the six phthalates were between 82.90% and 109.50%. The relative standard deviations were between 2.1% and 4.6%. The detection limits of the method were: 0.1 ng for DMP, DEP, DBP and BBP, and 0.5 ng for DEHP and DOP, respectively. The method presented the advantages of high precision, high sensitivity, small sample size, and simple pretreatment. The method can be used to test the six phthalates in the cosmetics.

Abstract  The objective of this study was to determine the adverse effects of monobutyl phthalate (MBuP), a major metabolite of dibutyl phthalate (DBP), on development of the reproductive system in offspring following maternal administration during late pregnancy, and to assess the role of MBuP in the antiandrogenic effects of DBP. Pregnant rats were given MBuP by gastric intubation at 250, 500, or 750 mg/kg on days 15 through 17 of pregnancy. Maternal body weight gain and food consumption during the administration period were significantly decreased at 500 mg/kg and higher and at 750 mg/kg, respectively. A significant increase in the incidence of postimplantation embryonic loss was found at 500 mg/kg and higher. The body weights of male and female fetuses were significantly lower at 750 mg/kg. A significant increase in the incidence of fetuses with undescended testes was found at 250 mg/kg and higher. A significant decrease in the anogenital distance (AGD) of male fetuses was observed at 250 mg/kg and higher. The AGD/body weight ratio and AGD/cube root of body weight ratio in male fetuses was also significantly reduced at 250 mg/kg and higher. The AGD, AGD/body weight ratio and AGD/cube root of body weight ratio in female fetuses in the MBuP-treated groups were comparable to those in the control group. The present study indicates that MBuP on days 15 to 17 of pregnancy produced adverse effects on the development of reproductive system in male offspring and suggest that MBuP may be responsible for the induction of the antiandrogenic effects of DBP.

Abstract  The effects of monobutyl phthalate (MBuP) on reproductive function were determined in pregnant and pseudopregnant rats. Rats were given MBuP by gastric intubation at 250, 500, 750, or 1000 mg/kg on days 0 to 8 of pregnancy and pregnancy outcome was determined on day 20 of pregnancy. The effects of MBuP on the uterine function, as a cause of early embryonic loss, were also determined in pseudopregnant rats, with an induced decidual cell response. The same doses of MBuP were given to pseudopregnant rats on days 0 to 8 of pseudopregnancy and the uterine weight on day 9 served as an index of uterine decidualization. MBuP at 1000 mg/kg caused significant increases in the incidences of preimplantation loss in females successfully mated and of postimplantation loss in females having implantations. Uterine decidualization in pseudopregnant rats was significantly decreased at 1000 mg/kg. These findings suggest that early embryonic loss due to MBuP is mediated, at least in part, via suppression of uterine decidualization, an impairment of uterine function.

Abstract  Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To explore the possible actions of endocrine disruptors on the metabolic fate of free AA into these two pathways, we investigated the effects of nonylphenol (NP), bisphenol A (BPA), di-n-butyl phthalate (DBP), benzyl-n-butyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) on the formation of PG and AA-CoA from 5 microM AA (close to the physiological concentration of the substrate) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 5 microM [(14)C]-AA in 0.1 M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. NP (1-200 microM) strongly enhanced the AA-CoA formation with a coincident decrease in the PG formation. BPA, DBP, BBP and DEHP failed to show any effect on the PG and AA-CoA formation up to 200 microM. Experiments utilizing 60 microM AA as the substrate concentration indicated that, under a low concentration of AA, NP decreases PG formation by inhibiting the COX activity, and reduces the AA flow into the COX pathway through inhibition on the COX activity, increasing availability of the substrate for the ACS and leading to enhanced AA-CoA formation. These results firstly show that NP has the potential to disturb the balance of PG and AA-CoA formations under normal physiological conditions.

Abstract  To date, regulatory agencies have not considered conducting cumulative risk assessments for mixtures of chemicals with diverse mechanisms of toxicity because it is assumed that the chemicals will act independently and the individual chemical doses are not additive. However, this assumption is not supported by new research addressing the joint effects of chemicals that disrupt reproductive tract development in the male rat by disrupting the androgen signalling pathway via diverse mechanisms of toxicity [i.e. androgen receptor (AR) antagonism in the reproductive tract vs. inhibition of androgen synthesis in the foetal testis]. In this study, pregnant rats were exposed to four dilutions of a mixture containing vinclozolin, procymidone, linuron, prochloraz, benzyl butyl phthalate, dibutyl phthalate and diethylhexyl phthalate during the period of sexual differentiation and male offspring were assessed for effects on hormone sensitive endpoints including: anogenital distance, infant areolae retention and reproductive tract tissue weights and malformations. The ratio of the chemicals in the mixture was based upon each chemical's ED(50) for inducing reproductive tract malformations (hypospadias or epididymal agenesis). The observed responses from the mixture were compared with predicted responses generated with a toxic equivalency approach and models of dose addition, response addition or integrated addition. As hypothesized, we found that the mixture of chemicals that alter the androgen signalling pathway via diverse mechanisms disrupted male rat reproductive tract differentiation and induced malformations in a cumulative, dose-additive manner. The toxic equivalency and dose addition models provided the best fit to observed responses even though the chemicals do not act via a common cellular mechanism of action. The current regulatory framework for conducting cumulative risk assessments needs to consider the results, including those presented herein, which indicate that chemicals that disrupt foetal tissues during sexual differentiation act in a cumulative, dose-additive manner irrespective of the specific cellular mechanism of toxicity.

Abstract  Ozark Hellbender (Cryptobranchus alleganiensis bishopi) populations are in decrease throughout their native range with rare recruitment of young. Increased estrogenic chemical levels and alterations of physicochemical properties in their habitat may play a significant role in this phenomenon. We report here the first systematic, comprehensive study of organic chemical concentrations and physical and nutrient parameters in two rivers containing Ozark hellbender populations. Water samples were collected monthly from August 2003 to November 2004. Concentrations of 21 organic chemicals were determined using gas chromatography-mass spectrometry. Nine organic chemicals were detected. Benzyl butyl phthalate, dibutyl phthalate, bisphenol A, and beta-sitosterol were all detected >85% of the time, with median concentrations of 18 to 234 ng/L and maximum concentrations of 198 to 4141 ng/L. Individually, concentrations of nutrients and organic chemicals were much lower than those shown previously in laboratory and field experiments to have reproductive effects on amphibians. Nevertheless, hellbenders are exposed to a variety of chemicals with potential estrogenic effects. Our study establishes the basis to examine the specific effects of the detected concentrations, alone and in combination, on the Ozark hellbenders.

Abstract  Phthalic acid esters (PAEs) are used in many branches of industry and are produced in huge amounts throughout the world. An investigation on particulate- and gas-phase distribution of PAEs has been conducted in Nanjing (China). The 12-h daily sampling program (from 8:00 am to 8:00 pm) for ten consecutive days was conducted in April, July and October 2005, and in January 2006 at about 1.5m above the ground level. For comparative purposes, sampling events were simultaneously conducted at two stations, one at the urban center and the other about 12 km from city center for suburban background monitoring. It was observed that the most abundant members of the PAE group were dimethyl phthalate (DMP) (10.1 ng m(-3), average), diethyl phthalate (DEP) (3.4 ng m(-3)), dibutyl phthalate (DBP) (58.8 ng m(-3)), butylbenzyl phthalate (BBP) (3.2 ng m(-3)), di-2-ethylhexyl phthalate (DEHP) (20.3 ng m(-3)) and di-n-octyl phthalate (DOP) (1.2 ng m(-3)). The average contribution of PAEs in the gas phase to the total PAE concentration (Sigma(6)PAE, sum of six PAE congeners) ranged from 75.0% to 89.2%. Both particulate- and gas-phase Sigma(6)PAE concentrations decreased with increasing temperature. Experimentally determined gas-particle partitioning (K(p)) of PAEs is well-correlated with their vapor pressure. The Sigma(6)PAE levels in the urban area are approximately 3.5 times as high as the levels found at the suburban station. The vertical profiles from 1.5 to 30.0m above the ground display slight height dependence.

Abstract  The objective of this study was to determine the characterization of the developmental toxicity of di-n-butyl phthalate (DBP) in rats. Pregnant rats were given DBP by gastric intubation at a dose of 0.75, 1.0 or 1.5 g/kg on days 7-9, 10-12 or 13-15 of pregnancy. Postimplantation loss was 100% for each period of dosing at 1.5 g/kg. A significant increase in the postimplantation loss was found in dams given DBP at doses of 0.75 and 1.0 g/kg regardless of the days of treatment. No evidence of teratogenicity was detected when DBP was given on days 10-12. Treatment on days 7-9 with DBP at doses of 0.75 and 1.0 g/kg caused a significant increase in the number of skeletal malformations such as deformity of the vertebral column in the cervical and thoracic regions and of the ribs, but neither external nor internal malformations. Treatment with DBP on days 13-15 at doses of 0.75 and 1.0 g/kg resulted in a significant increase in the incidence of fetuses with external and skeletal malformations such as cleft palate and fusion of the sternebrae. The frequency of malformations increased as the dose of DBP was increased. The highest incidence of malformed fetuses occurred after treatment with DBP on days 13-15. It could be concluded that susceptibility to the teratogenicity of DBP varies with the developmental stage at the time of administration.

Abstract  In our previous studies, dibutyl phthalate (DBP) was found to be embryolethal and teratogenic in rats. In this study, the effects of DBP on reproductive function were investigated on pregnant and pseudopregnant rats. Rats were given DBP by gastric intubation at 0, 250, 500, 750, 1000, 1250 or 1500 mg/kg on Days 0 to 8 of pregnancy and the pregnancy outcome was determined on Day 20 of pregnancy. The same doses of DBP were given to pseudopregnant rats, with an induced decidual cell response, on Days 0 to 8 of pseudopregnancy, and the uterine weight on Day 9 served as an index of the uterine decidualization. DBP caused significant increases in the incidences of preimplantation loss in females successfully mated at 1250 and 1500 mg/kg and of postimplantation loss in females having implantations at 750 mg/kg and above. The uterine decidualization in pseudopregnant rats was significantly decreased at 750 mg/kg and above. These findings suggest that early embryonic loss due to DBP may be mediated, at least in part, via the suppression of uterine decidualization, an impairment of uterine function.

Abstract  A single oral dose of di-n-butyl phthalate (DBP) to male rats caused histologically a sloughing of the germ cells at 6 h. On Days 1 and 2 more severe sloughing was seen, followed by atrophy and the dissociationof the germ cells from the Sertoli cells and the spermatogonia. Biochemically, there was elevation of y-glutamyl transferase, a decrease in sorbitol levels at 3 h and a decrease in the activity of aldose reductase at 6 h, in the testes of treated rats. This was followed by decreases in fructose levels and increases in the activity of lactate dehydrogenase (LDH) and in lactate levels at 12 h , and decreases in the activities of sorbitol dehydrogenase and succinate dehydrogenase on Day 2. LDH isoenzymes 4 and 6 increased at 6 h prior to the increase in lactate levels. Increases in the levels of inositol and the activities of alkaline phosphatase and lactate dehdryogenase were also observed. Thus, these data suggest that DBP-induced testicular toxicity is caused by a shortage of energy fuels from glucose metabolism or by an anoxia.

Abstract  The rat has been explored in detail for its in utero susceptibility to male reproductive tract malformation following phthalate exposure. Few other species have been studied in detail, and it is important for both mechanistic and risk assessment purposes to understand the species specificity of this response. We investigated the response of the fetal mouse testis to phthalate exposure and compared these results with those previously obtained from the rat. Initial experiments using a variety of phthalate congeners (monobutyl phthalate, di-(n-butyl) phthalate, or mono (2-ethylhexyl) phthalate) and exposure paradigms did not reduce fetal mouse testis testosterone levels. Pharmacokinetic data after a single 500 mg/kg di-(n-butyl)-phthalate (DBP) exposure on mouse gestation day (gd) 18 demonstrated that the concentrations and kinetics of the active metabolite monobutyl phthalate (MBP) in fetal and maternal plasma were similar to the rat. After a single 500 mg/kg or multiple day 250 mg/kg fetal mouse DBP exposure, rapid and dynamic changes in testis gene expression were observed, including induction of immediate early genes. Unlike the rat, expression of genes involved in cholesterol homeostasis and steroidogenesis were not decreased and were increased in a few cases. Similar to the rat, however, a 250- or 500-mg DBP/kg/day mouse exposure from gd 16 through 18 significantly increased seminiferous cord diameter, the number of multinucleated gonocytes per cord, and the number of nuclei per multinucleated gonocyte. Together, these results demonstrate that fetal mouse and rat phthalate exposure both induce immediate early gene expression and disrupt seminiferous cord and gonocyte development. This response in the mouse occurs without a measurable decrease in testicular testosterone, suggesting that altered seminiferous cord formation and gonocyte multinucleation may not be mechanistically linked to lowered testosterone.

Abstract  BACKGROUND: Certain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human. OBJECTIVES: Our aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset. METHODS: Fetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15-19 weeks of gestation) were cultured for 24-48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP. RESULTS: Exposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5-20.5) DBP treatment. In vitro, MBP (10(-3) M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10(-3) M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2-7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy. CONCLUSIONS: These findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro.

Abstract  Toxic effects of ozone, 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK), and/or dibutyl phthalate (DBP) were examined through NF-kappaB, AP-1, Nrf2, and osteopontin (OPN) in lungs and livers of B6C3F1 mice. Electrophoretic mobility shift assay (EMSA) indicated that mice treated with combination of toxicants induced high NF-kappaB activities. Expression levels of p105, p65, and p50 proteins increased in all treated mice, whereas IkB activity was inhibited in NNK-, DBP-, and combination-treated ones. All treated mice except ozone-treated one showed high AP-1 binding activities. Expression levels of c-fos, c-jun, junB, jun D, Nrf2, and OPN proteins increased in all treated mice. Additive interactions were frequently noted from two-toxicant combination mice compared to ozone-treated one. These results indicate treatment of mixture of toxicants increased toxicity through NF-kappaB, AP-1, Nrf2, and OPN. Our data could be applied to the elucidation of mechanism as well as the risk assessment of mixture-induced toxicity.

Abstract  The genotoxicity of phthalates, widely used plasticizers, has been shown previously for di-butyl-phthalate (DBP) and di-iso-butyl-phthalate (DiBP) in human mucosal cells of the upper aerodigestive tract in a previous study using the Comet assay. Furthermore, higher genotoxic sensitivities of patients with squamous cell carcinomas of either the larynx or the oropharynx compared to non-tumor patients were described. Other authors have demonstrated DNA damage by a different phthalate in human hymphocytes. It was the aim of the present study to determine whether there is a correlation between the genotoxic sensitivities to DBP and its isomer DiBP in either mucosal cells or lymphocytes. The single-cell microgel electrophoresis assay (Comet assay) was applied to detect DNA strand breaks in human epithelial cells of the upper aerodigestive tract (n=132 specimens). Human mucosa was harvested from the oropharynx in non-tumor pateints and patients with squamous cell carcinomas of the oropharynx. Laryngeal mucosa of patients with laryngeal squamous cell carcinomas was harvested as well. Peripheral lymphocytes (n=49 speciments) were separated from peripheral blood. Xenobiotics investigated were DBP, DiBP, and N'methyl-N'-nitro-N-nitrosoguanidine (MNNG) as positive control, respectively. For statistical analysis, the SPSS correlation analysis according to Pearson and the Wilcoxon test were performed. Genotoxicity was found for DBP and DiBP in epithelial cells and lymphocytes (P<0.001). MNNG caused severe DNA damage. In analyzing DPB and DiBP results, genotoxic impacts in mucosal cells showed an intermediate correlation (r=0.570) Correlation in lymphocytes was the same (r=0.570). Phthalates have been investigated as a potential health hazard for a variety of reasons, including the possible xenoestrogenic impact, peroxisome proliferation, and membrane destabilization. The present investigation suggests a correlated DNA-damaging impact of DBP and DiBP in human mucosal cells and in lymphocytes, respectively.

Abstract  Some phthalates such as di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) and their metabolites are suspected of producing teratogenic or endocrine-disrupting effects. To predict possible human exposure to phthalates in cosmetics, the levels of DEHP, diethyl phthalate (DEP), DBP and butylbenzyl phthalate (BBP) were determined by high-performance liquid chromatography (HPLC) in 102 branded hair sprays, perfumes, deodorants and nail polishes. DBP was detected in 19 of the 21 nail polishes and in 11 of the 42 perfumes, and DEP was detected in 24 of the 42 perfumes and 2 of the 8 deodorants. Median exposure levels to phthalates in cosmetics by derman absorption were estimated to be 0.0006 micrograms/kg body weight (bw)/d for DEHP, 0.6 micrograms/kg bw/d for DEP, and 0.103 micrograms/kg bw/d for DBP. Furthermore, if phthalates in cosmetics were assumed to absorbed exclusively via 100% inhalation, the median daily exposure levels to phthalates in cosmetics were estimated to be 0.026 micrograms/kg bw/d for DEHP, 81.471 micrograms/kg bw/d for DEP, and 22.917 micrograms/kg bw/d for DBP, which are far lower than the regulation levels set by the Scientific Committee on Toxicity, Ecotoxicity, and the Environment (CSTEE) (37 micrograms/kg bw/d, DEHP), Agency for Toxic Substances and Disease Registry (ATSDR) (7000 micrograms/kg bw/d, DEP), and International Programme on Chemical Safety (IPCS) (66 micrograms/kg bw/d, DBP), respectively. Based on these data, hazard indices (HI, daily exposure level/regulation level) were calculated to be 0.0007 for DEHP, 0.012 for DEP, and 0.347 for DBP. These data suggest that estimated exposure to phthalates in the cosmetics mentioned are relatively small. However, total exposure levels from several sources may be greater and require further investigation.

Abstract  Tests were performed with the freshwater invertebrates Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus to determine the acute toxicity of six phthalate esters, including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), di-n-hexyl phthalate (DHP), and di-2-ethylhexyl phthalate (DEHP). It was possible to derive 10-d LC50 (lethal concentration for 50% of the population) values only for the four lower molecular weight esters (DMP, DEP, DBP, and BBP), for which toxicity increased with increasing octanol-water partition coefficient (Kow) and decreasing water solubility. The LC50 values for DMP, DEP, DBP, and BBP were 28.1, 4.21, 0.63, and 0.46 mg/L for H. azteca; 68.2, 31.0, 2.64, and > 1.76 mg/L for C. tentans; and 246, 102, 2.48, and 1.23 mg/L for L. variegatus, respectively. No significant survival reductions were observed when the three species were exposed to either DHP or DEHP at concentrations approximating their water solubilities

Abstract  Influences of major nutrients (N, P) on the biodegradation and bioconcentration of dibutyl phthalate (DBP) and di-2-ethylexyl phthalate (DEHP) by Chlorella vulgaris in lake water were investigated in this work. Our study demonstrated that nutrient addition obviously influenced biodegradation rate constants and apparent bioconcentration factors (BCFs) of DBP and DEHP in Chlorella vulgaris. The effects of P addition on biodegradation were less pronounced than the effects of N addition as a result of N-limitation status of phytoplankton in the lake water, while addition of both N and P more greatly affected biodegradation than addition of N or P. BCFs of DBP and DEHP decreased with increasing algal exudate as measured by dissolved organic carbon (DOC) and a strong correlation between BCFs and DOC was obtained. The results indicate that DOC plays an important role in the bioconcentration of DBP and DEHP

Abstract  Aquatic organisms are extensively exposed to phthalate esters. We have investigated in trout the effects of diethylhexylphthalate (DEHP) and dibutylphthalate (DBP) on xenobiotic metabolizing enzymes which have been suggested as possible environmental biomarkers. Rainbow trout (Oncorhynchus mykiss) were waterborne exposed to DEHP (1 mg/l) or DBP (0.1 or 1 mg/l) for 72 h. Another group of rainbow trout received daily for 3 days an intraperitoneal injection of 50 mg/kg of DEHP or DBP. Laurate hydroxylation, ethoxyresorufin-o-deethylation, UDP-glucuronyltransferase activity and glutathione-S-transferase activity were measured in liver and extrahepatic tissues. The phthalate esters have been found not to induce these enzymes; in particular, the results do not support the previously described induction of lauric acid hydroxylase in sea bass treated with DEHP [Comp. Biochem. Physiol. B122 (1999) 253.]

Abstract  Two laboratory-based linear horizontal agitation methods for determining a range of phthalate esters from soft polyvinyl chloride (PVC) toys are presented in compliance with EU legislation. Both of these methods were validated through interlaboratory trials using a PVC reference disc and four soft PVC toy/childcare articles intended or likely to be mouthed. Two of these commercial samples contained diisononyl phthalate (DINP), one diisodecyl phthalate (DIDP) and one bis(2-ethylhexyl) phthalate (DEHP). Acceptable repeatability (r, within-laboratory) and reproducibility (R, between-laboratory) data were demonstrated for both the analytical detection technique (GC-MS) (r = 9.8% and R = 8.1%) and agitation/extraction procedure (r=21.9% and R = 35.3% at 37 degrees C; r = 22.7% and R = 31.1% at 65 degrees C) for DINP. This was achieved through the participation of six laboratories. The remaining three phthalates from the EU Scientific Committee for Toxicity, Ecotoxicity and the Environment (CSTEE) list--dibutyl phthalate (DBP), benzyl butyl phthalate (BBP) and di-n-octyl phthalate (DnOP)--were not tested due to the unavailability of suitable materials.