Degradation of bis(2-ethylhexyl) phthalate constituents under methanogenic conditions

The degradation of bis(2-ethylhexyl) phthalate (DEHP) and its intermediary hydrolysis products 2-ethylhexanol (2-EH) and mono(2-ethylhexyl) phthalate (MEHP) was investigated in a methanogenic phthalic acid ester-degrading enrichment culture at 37°C. 2-Ethylhexanoic acid (2-EHA), a plausible degradation product of 2-EH, was also studied. The culture readily degraded 2-EH via 2-EHA to methane which was formed in stoichiometric amounts assuming complete degradation of 2-EH to methane and carbon dioxide. MEHP was degraded to stoichiometric amounts of methane with phthalic acid as a transient intermediate. DEHP remained unaffected throughout the experimental period (330 days).Abbreviations 2-EH 2-ethylhexyl alcohol - 2-EHA 2-ethylhexanoic acid - BBP butylbenzyl phthalate - Be-CoA benzoyl Coenzyme A - CoA Coenzyme A - DEHP bis(2-ethylhexyl) phthalate - MEHP mono(2-ethylhexyl) phthalate - MSW municipal solid waste - PA phthalic acid - PAE phthalic acid ester - TMS trimethylsilyl derivative     

Measurement of eight urinary metabolites of di(2-ethylhexyl) phthalate as biomarkers for human exposure assessment.

Human metabolism of di(2-ethylhexyl) phthalate (DEHP) is complex and yields mono(2-ethylhexyl) phthalate (MEHP) and numerous oxidative metabolites. The oxidative metabolites, mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) and mono(2-carboxymethylhexyl) phthalate (MCMHP), have been considered to be better biomarkers for DEHP exposure assessment than MEHP because urinary levels of these metabolites are generally higher than MEHP, and their measurements are not subject to contamination. The urinary levels of the above metabolites, and of three other recently identified DEHP oxidative metabolites, mono(2-ethyl-3-carboxypropyl) phthalate (MECPrP), mono-2-(1-oxoethylhexyl) phthalate (MOEHP), and mono(2-ethyl-4-carboxybutyl) phthalate (MECBP), were measured in 129 adults. MECPP, MCMHP and MEHHP were present in all the samples analysed. MEHP and the other oxidative metabolites were detected less frequently: MEOHP (99%), MECBP (88%), MECPrP (84%), MEHP (83%) and MOEHP (77%). The levels of all DEHP metabolites were highly correlated (p<0.0001) with each other, confirming a common parent. The ? and ?-1 oxidative metabolites (MECPP, MCMHP, MEHHP and MEOHP) comprised 87.1% of all metabolites measured, and thus are most likely the best biomarkers for DEHP exposure assessment. The percentage of the unglucuronidated free form excreted in urine was higher for the ester linkage carboxylated DEHP metabolites compared with alcoholic and ketonic DEHP metabolites. The percentage of the unglucuronidated free form excreted in urine was higher for the DEHP metabolites with a carboxylated ester side-chain compared with alcoholic and ketonic metabolites. Further, differences were found between the DEHP metabolite profile between this adult population and that of six neonates exposed to high doses of DEHP through extensive medical treatment. In the neonates, MEHP represented 0.6% and MECPP 65.5% of the eight DEHP metabolites measured compared to 6.6% (MEHP) and 31.8% (MECPP) in the adults. Whether the observed differences reflect differences in route/duration of the exposure, age and/or health status of the individuals is presently unknown.     

Genotoxicity studies of di(2-ethylhexyl)phthalate and its metabolites in CHO cells

The widely used plasticizer di(2-ethylhexyl)phthalate (DEHP), its hydrolysis products mono(2-ethylhexyl)phthalate (MEHP) and 2-ethylhexanol, and also phthalic acid have been tested for clastogenic activity in cultured Chinese hamster ovary (CHO) cells. Only MEHP was found to cause chromosome damage. MEHP was without effect in the SCE and HGPRT mutation test in CHO cells. The clastogenicity of MEHP suggests a role for this compound in the observed carcinogenicity of DEHP and its positive effect in the dominant lethal assay.     

Chromatographic fractionation and analysis by mass spectrometry of conjugated metabolites of bis(2-ethylhexyl)phthalate in urine

Mono(2-ethylhexyl)phthalate (MEHP), the primary metabolite of the plasticizer bis(2-ethylhexyl)phthalate (DEHP), was given to guinea pigs and mice and the methods for the isolation, separation and analysis of its metabolites in urine were developed. Following solid-phase extraction with octadecylsilane-bonded silica, individual metabolites were purified and separated using a combination of ion-exchange chromatography on lipophilic gels and reversed-phase high performance liquid chromatography. Analysis of intact conjugates, as well as nonconjugated metabolites, was performed by fast atom bombardment mass spectrometry (FAB-MS) and, after derivation, by gas chromatography-mass spectrometry. Enzymatic methods were used for further characterization. The study confirms glucuronidation as the major conjugation pathway for MEHP in the investigated species. Although less important quantitatively, glucosidation is shown to be an alternative conjugation pathway in mice. The methods developed were applied to a sample of urine from a hyperbilirubinemic newborn infant subjected to DEHP-exposure in conjunction with an exchange transfusion. It was demonstrated that metabolites of DEHP were excreted in amounts which could be analyzed by FAB-MS.     

Role of Ca/CaN/NFAT signaling in IL-4 expression by splenic lymphocytes exposed to phthalate (2-ethylhexyl) ester in spleen lymphocytes

The aims of present study were to investigate the effect of phthalate (2-ethylhexyl) ester (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) on Th1/Th2 balance signaling for interleukin 4 (IL-4) expression in splenic lymphocytes, and contribution of MEHP to any hypothesized changes in vitro. Primary splenic lymphocytes were exposed to DEHP/MEHP. ELISA and Western blotting were used to detect proteins. Confocal-microscopy was used to examine nuclear translocation. Nuclear factor of activated T cells (NFAT) DNA binding activity was examined by electrophoretic mobility-shift assay. DEHP significantly increased IL-4 and interferon gamma (IFN-γ) level, and reduced Th1/Th2 ratio (reflected by IFN-γ/IL-4) with 5 μg/L Concanavalin A (ConA) treatment. While MEHP reduced Th1/Th2 ratio (represented by IFN-γ/IL-6). IL-4 mRNA was significantly increased by DEHP but not by MEHP after PMA and Ion treatment. DEHP significantly inhibited NFATp protein in cytosol and nucleus. DEHP augmented nuclear translocation of NFATc in transfected EL4 cells and NFAT DNA-binding activity. DEHP-mediated enhancement of calcium-dependent phosphatase calcineurin (CaN) protein, and NFAT and IL-4 expression were abrogated by calcium antagonist verapamil and CaN inhibitor tarcolimus. Ca²⁺/calmodulin antagonist chlorpromazine significantly suppressed IL-4 and CaN production with no NFAT mRNA change. Our study suggests that DEHP and MEHP impact Th1/Th2 balance by modulating different cytokines. DEHP-affected IL-4 expression through Ca/CaN/NFAT signaling pathway, but no effect was discovered for MEHP.     

The endocrine disruptor mono-(2-ethylhexyl)phthalate promotes adipocyte differentiation and induces obesity in mice

The environmental obesogen hypothesis proposes that exposure to endocrine disruptors during developmental ‘window’ contributes to adipogenesis and the development of obesity. MEHP [mono-(2-ethylhexyl) phthalate], a metabolite of the widespread plasticizer DEHP [di-(2-ethylhexyl) phthalate], has been found in exposed organisms and identified as a selective PPARγ (peroxisome-proliferator-activated receptor γ) modulator. However, implication of MEHP on adipose tissue development has been poorly investigated. In the present study, we show the dose-dependent effects of MEHP on adipocyte differentiation and GPDH (glycerol-3-phosphate dehydrogenase) activity in the murine 3T3-L1 cell model. MEHP induced the expression of PPARγ as well as its target genes required for adipogenesis in vitro. Moreover, MEHP perturbed key regulators of adipogenesis and lipogenic pathway in vivo. In utero exposure to a low dose of MEHP significantly increased b.w. (body weight) and fat pad weight in male offspring at PND (postnatal day) 60. In addition, serum cholesterol, TAG (triacylglycerol) and glucose levels were also significantly elevated. These results suggest that perinatal exposure to MEHP may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders.     

Biological monitoring of the five major metabolites of di-(2-ethylhexyl)phthalate (DEHP) in human urine using column-switching liquid chromatography-tandem mass spectrometry

We present a fast and reliable on-line clean-up HPLC-method for the simultaneous determination of the five major urinary metabolites of di-(2-ethylhexyl)phthalate (DEHP) namely mono-(2-ethyl-5-carboxypentyl)phthalate (5carboxy-MEPP), mono-[2-(carboxymethyl)hexyl]phthalate (2carboxy-MMHP), mono-(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono-(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono-(2-ethylhexyl)phthalate (MEHP). These metabolites represent about 70% of an oral DEHP dose. We for the first time succeeded to reliably quantify 5carboxy-MEPP and to identify 2carboxy-MMHP as major metabolites in native urines of the general population. The analytical procedure consists of an enzymatic hydrolysis, on-line extraction of the analytes from urinary matrix by a restricted access material column (RAM), back-flush transfer onto the analytical column (betasil phenylhexyl), detection by ESI-tandem mass spectrometry and quantification by isotope dilution (limit of detection (LOD) 0.25 microg/l). Median concentrations of a small collective taken from the general population (n=19) were 85.5 microg/l (5carboxy-MEPP), 47.5 microg/l (5OH-MEHP), 39.7 microg/l (5oxo-MEHP), 9.8 microg/l (MEHP) and about 37 microg/l (2carboxy-MMHP). The presented method can provide insights into the actual internal burden of the general population and certain risk groups. It will help to further explore the human metabolism of DEHP-an occupational and environmental toxicant of great concern.     

Effects of mono-(2-ethylhexyl) phthalate on fetal and neonatal rat testis organ cultures

Di-(2-ethylhexyl) phthalate (DEHP) and its active metabolite, mono-(2-ethylhexyl) phthalate (MEHP), have been shown to cause reproductive toxicity in both developing and adult animals. In this study, we used organ cultures of fetal and neonatal rat testes to assess the in vitro effect of MEHP on seminiferous cord formation in Embryonic Day 13 (E13) testes and on the development of E18 and Postnatal Day 3 (P3) testes. Interestingly, MEHP had no effect on cord formation in the organ cultures of E13 testes, indicating that it has no effect on sexual differentiation of the indifferent gonad to testis. Consistently, the expression of a Sertoli cell-specific protein, mullerian inhibiting substance (MIS), or the number of gonocytes did not change in E13 testes after MEHP treatment. In contrast, MEHP decreased the levels of MIS and GATA-4 proteins in Sertoli cells and impaired Sertoli cell proliferation in the organ cultures of E18 and P3 testes. These results suggest that MEHP negatively influences proliferation and differentiation of Sertoli cells in both fetal and neonatal testes. In addition, MEHP treatment did not alter the number of gonocytes in E18 testes, whereas the number of gonocytes in P3 testes decreased in a dose-dependent manner, apparently due to enhanced apoptosis. These results suggest that MEHP adversely affects the gonocytes, which are mitotically active and undergoing migration and differentiation in neonatal testes, but it has no effect on fetal gonocytes that are mitotically quiescent.     

Polar metabolites of di-(2-ethylhexyl)phthalate in the rat

Di-(2-ethylhexyl)phthalate (DEHP) is an important industrial chemical widely used as a plasticizer for vinyl and other plastics. DEHP is extensively metabolized by mammals, different species showing dramatic differences in metabolite distributions. Previous studies of the metabolism in rats led to the suggestion that the enzymatic processes normally associated with omega-, omega-1, alpha-, and beta-oxidation of fatty acids could account for the known metabolites of DEHP found in the urine. Several additional metabolites of DEHP have been identified in the present study. Their formation requires that the initial hydroxylation process be less specific than fatty acid omega- and omega-1 oxidation are thought to be. Furthermore, it is necessary to postulate either that the aliphatic chain of mono-(2-ethylhexyl)phthalate can be oxidized at two sites simultaneously, or that oxidation products can be recycled for a second hydroxylation prior to excretion.     

邻苯二甲酸酯在不同品种通菜-土壤系统中的累积效应研究

在邻苯二甲酸(2-乙基己基)酯(DEHP)不同污染程度的水稻土上盆栽不同品种通菜,应用GC／MS联机检测技术研究了通菜-土壤系统中DEHP的环境行为与归宿．结果表明,通菜中DEHP的含量为0．335～12．710mg·kg^-1,与土壤的污染程度呈正相关．不同品种通菜之间对DEHP的吸收累积效应存在显著差异,与其叶子大小存在一定的正相关关系．种植不同品种通菜后土壤中DEHP的残留量(1．424～19．834mg·kg^-1)存在显著差异．通菜对土壤中DEHP的BCFs都小于1．0,与土壤的污染程度呈负相关．不同通菜品种的BCFs之间存在显著差异,中等叶子通菜品种的BCFs相对较大。     

A mono-2-ethylhexyl phthalate hydrolase from a Gordonia sp. that is able to dissimilate di-2-ethylhexyl phthalate

Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The K(m) and V(max) values for mono-2-ethylhexyl phthalate were 26.9 +/- 4.3 microM and 18.1 +/- 0.9 micromol/min . mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.     

On-line clean-up by multidimensional liquid chromatography-electrospray ionization tandem mass spectrometry for high throughput quantification of primary and secondary phthalate metabolites in human urine

We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher((R)) ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5-2.0 microgram/l urine). First results on a small non-exposed group (n=8) ranged for 5OH-MEHP from 0.59 to 124 microgram/l, for 5oxo-MEHP from 相似文献   

Non-mutagenicity of 4 metabolites of di(2-ethylhexyl)phthalate (DEHP) and 3 structurally related derivatives of di(2-ethylhexyl)adipate (DEHA) in the Salmonella mutagenicity assay

Four metabolites of the rat liver carcinogen di(2-ethylhexyl)phthalate (DEHP) (mono-(2-ethylhexyl)phthalate, mono-(2-ethyl-5-hydroxyhexyl)phthalate, mono-(2-ethyl-5-oxohexyl)phthalate, and mono-(5-carboxy-2-ethylpentyl)phthalate) and 3 structurally related derivatives of di(2-ethylhexyl)adipate (DEHA) (mono-(2-ethylhexyl)adipate, mono-(2-ethyl-5-hydroxyhexyl)adipate, and mono-(2-ethyl-5-oxohexyl)adipate) were tested for mutagenicity in the Ames assay using Salmonella typhimurium strains TA97, TA98, TA100, and TA102, with and without a metabolic activation preparation. Aroclor 1254-induced rat liver S9 and DEHP-induced rat liver S9 were used. Concentrations of these compounds up to 1000 micrograms/plate were negative with all tester strains in the presence or absence of metabolic activation.     

Association between Maternal Exposure to di(2-ethylhexyl) Phthalate and Reproductive Hormone Levels in Fetal Blood: The Hokkaido Study on Environment and Children's Health

Prenatal di(2-ethylhexyl) phthalate (DEHP) exposure can produce reproductive toxicity in animal models. Only limited data exist from human studies on maternal DEHP exposure and its effects on infants. We aimed to examine the associations between DEHP exposure in utero and reproductive hormone levels in cord blood. Between 2002 and 2005, 514 pregnant women agreed to participate in the Hokkaido Study Sapporo Cohort. Maternal blood samples were taken from 23–35 weeks of gestation and the concentration of the primary metabolite of DEHP, mono(2-ethylhexyl) phthalate (MEHP), was measured. Concentrations of infant reproductive hormones including estradiol (E2), total testosterone (T), and progesterone (P4), inhibin B, insulin-like factor 3 (INSL3), steroid hormone binding globulin, follicle-stimulating hormone, and luteinizing hormone were measured from cord blood. Two hundred and two samples with both MEHP and hormones'' data were included in statistical analysis. The participants completed a self-administered questionnaire regarding information on maternal characteristics. Gestational age, birth weight and infant sex were obtained from birth records. In an adjusted linear regression analysis fit to all study participants, maternal MEHP levels were found to be associated with reduced levels of T/E2, P4, and inhibin B. For the stratified analyses for sex, inverse associations between maternal MEHP levels T/E2, P4, inhibin B, and INSL3 were statistically significant for males only. In addition, the MEHP quartile model showed a significant p-value trend for P4, inhibin B, and INSL3 decrease in males. Since inhibin B and INSL3 are major secretory products of Sertoli and Leydig cell, respectively, the results of this study suggest that DEHP exposure in utero may have adverse effects on both Sertoli and Leydig cell development in males, which agrees with the results obtained from animal studies. Comprehensive studies investigating phthalates'' exposure in humans, as well as their long-term effects on reproductive development are needed.     

Biochemical effects of di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is a widely used plasticizer in the plastics industry. Biochemical changes in rat blood and liver were studied at 21 days after 3 intraperitoneal injections of DEHP on days 1, 5 and 10. In the liver, a decrease in the activity of succinic dehyrogenase (SDH) and adenosine triphosphate and an increase in alkaline phosphatase activity was observed. In serum the activity of acid and alkaline phosphatases and glutamic oxaloacetic and glutamic pyruvic transaminases remained unaltered. Under in vitro conditions, only the activity of SDH was inhibited when homogenates or mitochondrial preparations from normal rat liver were incubated with DEHP and the degree of inhibition was related to the concentration of the plasticizer.     

Biosorption of di(2-ethylhexyl)phthalate by seaweed biomass

Samples of various Sargassum species were collected in the Hong Kong marine environment and used for studies on biosorption of di(2-ethylhexyl)phthalate (DEHP). Batch adsorption experiments were carried out to determine the removal capacity and removal efficiency of the biosorbents. The DEHP removal ability was similar among beached seaweed and three freshly collected Sargassum species. Different physico-chemical factors were evaluated in order to enhance the performance of the biosorbents. Under optimized conditions (25 mg biomass, initial pH 4, 25 °C, 40 mg L^–1 DEHP), the mean removal capacity of beached seaweed and Sargassum siliquastrum was 5.68 and 6.54 mg g^–1, respectively. Examination of the Langmuir and Freundlich adsorption isotherms showed that the biosorption phenomenon by these biosorbents could well be described by these models. Desorption of DEHP was also assessed with methanol, which showed the most satisfactory desorbing ability. Further study in multiple adsorption–desorption of DEHP by the biosorbents demonstrated the reusability of both beached seaweed and S. siliquastrum for biosorption of DEHP.     

Integrated biosorption and photocatalytic oxidation treatment of di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP), a toxic phthalate ester, is a ubiquitous contaminant due to its extensive use and persistence. No available treatment method can cost-effectively remove it from industrial wastewater. In a previous study, DEHP was effectively removed from aqueous solution by adsorption onto the biomass of selected seaweed, i.e., beached seaweed and Sargassum siliquastrum. Since biosorption cannot detoxify DEHP, the degradation (and detoxification) of desorbed DEHP from seaweed biomass by photocatalytic oxidation (PCO) was attempted. The first part of this study was to optimize the conditions for the degradation of desorbed DEHP in aqueous solution by PCO. Under optimized conditions, a total degradation of 20 mg/L of DEHP was achieved within 45 min. The degradation intermediates/products such as phthalic anhydride and 2-ethyhexanol were identified by GC-MS analysis. Total organic carbon analysis was also used to ensure the complete mineralization of DEHP. The Microtox^® test was used to assess the toxicities of the parent and degraded compounds. In the second part of this study, DEHP was first removed and concentrated by adsorption onto seaweed biomass under the conditions optimized in the previous study. It was then desorbed from seaweed biomass and degraded by PCO. Results indicate that the treatment for DEHP by integrating biosorption and PCO is feasible.     

Effect of in utero exposure to di(2-ethylhexyl)phthalate on rat testes.

In utero exposure to di(2-ethylhexyl)phthalate (DEHP; 1000 mg/kg body weight) significantly decreased activities of testicular sorbitol dehydrogenase and acid phosphatase and increased gamma-glutamyl transpeptidase, lactate dehydrogenase and beta-glucuronidase activities at early ages. A decrease in the sperm count of the epididymal spermatozoa was also observed in the sexually matured animals of DEHP exposed group. The data suggest that in utero exposure to DEHP may affect the normal development of testes.