Substrate Report for: DEHP

A novel bacterium assimilating di-2-ethylhexyl phthalate as a sole carbon source was isolated, and identified as a Rhodococcus species and the strain was named EG-5. The strain has a mono-2-ethylhexyl phthalate (MEHP) hydrolase (EG-5 MehpH), which exhibits some different enzymatic features when compared with the previously reported MEHP hydrolase (P8219 MehpH) from Gordonia sp. These differences include different pH optimum activity, maximal reaction temperature and heat stability. The Km and Vmax values of EG-5 MehpH were significantly higher than those of P8219 MehpH. The primary structure of EG-5 MehpH showed the highest sequence identity to that of P8219 MehpH (39%) among hydrolases. The phylogenetic tree suggested that EG-5 MehpH and P8219 MehpH were categorized in different groups of the novel MEHP hydrolase family. Mutation of a conserved R(109) residue of EG-5 MehpH to a hydrophobic residue resulted in a dramatic reduction in the Vmax value towards MEHP without affecting the Km value. These results indicate that this residue may neutralize the negative charge of a carboxylate anion of MEHP, and thus inhibit the catalytic nucleophile from attacking the ester bond. In other words, the R residue blocks inhibition from the carboxylate anion of MEHP. Recently, registered hypothetical proteins exhibiting 98% or 99% identities for EG-5 MehpH or for P8219 MehpH were found from some pathogens belonging to Actinomycetes. The protein may have other activities besides MEHP hydrolysis and function in other physiological reactions in some Actinomycetes.

A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di-n-propyl phthalate (DPrP), di-n-butyl phthalate (DBP), di-n-pentyl phthalate (DPeP), di-n-hexyl phthalate (DHP), DEHP, n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 mumole) except for BBP and DCHP within 15 min; BBP and DCHP were hydrolyzed within 30 min and 6h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.

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.

Di-(2-ethylhexyl) phthalate (DEHP) is extensively used as an additive to produce plastics, but it may damage non-target organisms in soil. In this study, the effects of DEHP on Folsomia candida in terms of survival, reproduction, enzyme activities, and DNA damage were investigated in spiked artificial soil using a multi-biomarker strategy. The 7-day LC(50) (median lethal concentration) and 28-day EC(50) (median effect concentration) values of DEHP were 1256.25 and 19.72 mg a.i. (active ingredient) kg(-1) dry soil, respectively. Biomarkers involved in antioxidant defense including catalase (CAT-catalase), glutathione S-transferases (GST), detoxifying enzymes including acetylcholinesterase (AChE), Cytochrome P450 (CYP450), and peroxidative damage (LPO-lipid peroxide) were also measured (EC(10), EC(20), and EC(50)) after exposure for 2, 4, 7, and 14 days. The Comet assay was also applied to assess the level of genetic damage. The activity of CAT and LPO was drastically enhanced by the highest dose (EC(50)) of DEHP on day two. The activities of GST and AChE in DEHP treatment groups were found to be blocked. In contrast, the activity of CYP450 was significantly enhanced compared to the respective control groups during the first four days of incubation. The Comet assay in F.candida demonstrated that DEHP (EC(50)) could induce DNA damage. The obtained multi-biomarker data were analyzed using an integrated biomarker response (IBR) index, indicating that limited-time exposure triggered higher stress than long-term exposure at low concentrations of DEHP. These results demonstrate that DEHP may cause biochemical and genetic toxicity to F. candida, which illustrated the potential risks of DEHP in the soil environment and might affect soil ecosystem processes. Further studies are necessary to elucidate the toxic mechanisms of DEHP on other non-target organisms in soil.

The toxic effect of di(2-ethylhexyl) phthalate (DEHP) on prepubertal testes was examined in this study. We treated 3-week-old male mice with 4.8 mg/kg/day (milligram/kilogram/day) (no observed adverse effect level), 30 mg/kg/day (high exposure dose relative to humans), 100 mg/kg/day (level causing a reproductive system disorder), and 500 mg/kg/day (dose causing a multigenerational reproductive system disorder) of DEHP via gavage. Obvious abnormalities in the testicular organ coefficient, spermatogenic epithelium, and testosterone levels occurred in the 500 mg/kg DEHP group. Ribonucleic acid sequencing (RNA-seq) showed that differentially expressed genes (DEGs) in each group could enrich reproduction and reproductive process terms according to the gene ontology (GO) results, and coenrichment of metabolism pathway was observed by the Reactome pathway analysis. Through the analysis of common genes in the metabolism pathway, we discovered that DEHP exposure at 4.8 to 500 mg/kg or 100 mg/kg caused the same damages to the prepubertal testis. In general, we identified two key transcriptional biomarkers (fatty acid binding protein 3 (Fabp3) and carboxylesterase (Ces) 1d), which provided new insight into the gene regulatory mechanism associated with DEHP exposure and will contribute to the prediction and diagnosis of prepuberty testis injury caused by DEHP.

Phthalate esters (PAEs) are harmful to human health and have been repeatedly identified in Baijiu samples. In our study, the distribution and degradation characteristics of 14 PAEs in Baijiu raw materials (BRMs) and Baijiu during distillation were detected using QuEChERS or vortex-assisted surfactant-enhanced-emulsification liquid-liquid micro-extraction (VSLLME) methods coupled with gas chromatography-mass spectrometry. The same five PAEs were detected in all tested samples, values ranged from 0.003 to 0.292 mg/kg; however, higher concentrations existed in BRMs compared to Baijiu samples. Using multivariate statistical analysis, detailed distinctions between different varieties of Baijiu and BRMs and separation-related PAE markers were revealed. PAEs concentration during Baijiu distillation showed a decreasing trend. The highest concentrations detected in distillate heads, were 1.6-, 2.3-, and 8.1-fold higher than those in heart1, heart2, and tail distillates, respectively. These findings revealed that PAEs may migrate from BRMs; moreover, that PAEs content can be regulated by distillation.

Di(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer, which is considered an endocrine disrupting pollutant. Growth kinetics and esterases activity by biochemical tests and polyacrylamide gel electrophoresis were characterized for Fusarium culmorum grown in DEHP-supplemented (1000mg/L) medium as the only carbon source and in control medium with glucose. Intermediate compounds of biodegraded DEHP were identified by GC-MS. F. culmorum degraded 92% of DEHP within 36h. DEHP was degraded to butanol, hexanal, catechol and acetic acid. It is suggested that the first two compounds would transform into butanediol and the last two would enter into the Krebs cycle and would be mineralized to CO2 and H2O. DEHP induced eight esterase isoforms, which were different to those constitutive isoforms produced in the control medium. It is suggested that five enzymes (25.7, 29.5, 31.8, 97.6 and 144.5kDa) detected during the first 36h be involved in the primary biodegradation of DEHP. The rest of the enzymes (45.9, 66.6 and 202.9kDa) might be involved in the final steps for DEHP metabolism. F. culmorum has a promising practical application in the treatment of DEHP-contaminated environments because it can secrete specific esterase to breakdown high concentrations of DEHP in a short period of time. This research represents the first approach for the study of esterase involved in the DEHP degradation by fungi using this phthalate as the sole source of carbon and energy.

Herein, we report a double enzyme system to degrade 12 phthalate esters (PAEs), particularly bulky PAEs, such as the widely used bis(2-ethylhexyl) phthalate (DEHP), in a one-pot cascade process. A PAE-degrading bacterium, Gordonia sp. strain 5F, was isolated from soil polluted with plastic waste. From this strain, a novel esterase (GoEst15) and a mono(2-ethylhexyl) phthalate hydrolase (GoEstM1) were identified by homology-based cloning. GoEst15 showed broad substrate specificity, hydrolyzing DEHP and 10 other PAEs to monoalkyl phthalates, which were further degraded by GoEstM1 to phthalic acid. GoEst15 and GoEstM1 were heterologously coexpressed in Escherichia coli BL21 (DE3), which could then completely degrade 12 PAEs (5 mM), within 1 and 24 h for small and bulky substrates, respectively. To our knowledge, GoEst15 is the first DEHP hydrolase with a known protein sequence, which will enable protein engineering to enhance its catalytic performance in the future.

Di(2-ethylhexyl) phthalate (DEHP), a plasticizer of synthetic polymers, is a well-known endocrine disrupting chemical (EDC) and reproductive toxicant. Addressing the unclear mechanism of DEHP-induced reproductive dysfunction, this study used GC-2spd cells to investigate the molecular mechanism involved in the DEHP-induced toxicity in the male reproductive system. The results indicated that the apoptotic cell death was significantly induced by DEHP exposure over 100 muM. Furthermore, DEHP treatment could induce oxidative stress in GC-2spd cells involving in the decrease of superoxide dismutase (SOD) activity (200 muM) and glutathione peroxidase (GSH-Px) activity (50 and 100 muM). In addition, DEHP induction also caused the elevated ratios of Bax/Bcl-2, release of cytochrome c and decomposition of procaspase-3 and procaspase-9 in GC-2spd cells. Taken together, our work provided the evidence that DEHP exposure might induce apoptosis of GC-2spd cells via mitochondria pathway mediated by oxidative stress.

A novel bacterium assimilating di-2-ethylhexyl phthalate as a sole carbon source was isolated, and identified as a Rhodococcus species and the strain was named EG-5. The strain has a mono-2-ethylhexyl phthalate (MEHP) hydrolase (EG-5 MehpH), which exhibits some different enzymatic features when compared with the previously reported MEHP hydrolase (P8219 MehpH) from Gordonia sp. These differences include different pH optimum activity, maximal reaction temperature and heat stability. The Km and Vmax values of EG-5 MehpH were significantly higher than those of P8219 MehpH. The primary structure of EG-5 MehpH showed the highest sequence identity to that of P8219 MehpH (39%) among hydrolases. The phylogenetic tree suggested that EG-5 MehpH and P8219 MehpH were categorized in different groups of the novel MEHP hydrolase family. Mutation of a conserved R(109) residue of EG-5 MehpH to a hydrophobic residue resulted in a dramatic reduction in the Vmax value towards MEHP without affecting the Km value. These results indicate that this residue may neutralize the negative charge of a carboxylate anion of MEHP, and thus inhibit the catalytic nucleophile from attacking the ester bond. In other words, the R residue blocks inhibition from the carboxylate anion of MEHP. Recently, registered hypothetical proteins exhibiting 98% or 99% identities for EG-5 MehpH or for P8219 MehpH were found from some pathogens belonging to Actinomycetes. The protein may have other activities besides MEHP hydrolysis and function in other physiological reactions in some Actinomycetes.

The di(2-ethylhexyl) phthalate (DEHP) is an ubiquitous environmental chemical with detrimental health effects. The present work was designed to asses some potential mechanisms by which DEHP causes, among others, a reduced body fat retention. Since this effect could be related to an alteration of adipocyte triacylglycerol (TG) metabolism, we evaluated the effects of dietary DEHP in adipose tissues upon (1) the number and size of fat cells; (2) the basal and stimulated lipolysis and (3) the lipoprotein lipase (LPL) activity. Groups of male Wistar rats were fed for 21 days a control diet alone (control group) or the same control diet supplemented with 2% (w/w) of DEHP (DEHP group). The LPL activity of DEHP-fed rats was increased in lumbar and epididymal adipose tissues. These rats had significantly reduced weight in epididymal and lumbar tissues, together with reduced size of epididymal adipocytes. These alterations do not seem to be associated with higher lipid mobility because neither basal lipolysis nor 'in vitro' stimulated lipolysis by noradrenaline (NA) showed to be modified by DEHP. Based on these results, we concluded that the adipose tissue size reduction induced by DEHP intake is not due to changes in lipolysis nor to a decreased LPL activity. More research is needed to achieve a comprehensive understanding of the potential mechanisms by which DEHP causes, among others, a reduced body fat retention.

A weak hydrolyzing activity against bis (2-ethylhexyl) phthalate (DEHP) was discovered in a commercial crude lipase (EC 3.1.1.3) preparation from porcine pancreas. DEHP was hydrolyzed to mono (2-ethylhexyl) phthalate (MEHP) not by a pancreatic lipase but by a cholesterol esterase (CEase, EC 3.1.1.13), a trace contaminant in the crude lipase preparation. Enzymatic hydrolysis of phthalic acid esters (PAEs), suspected to be endocrine-disrupting chemicals, was investigated using CEases from two species of mammals and a microorganism. Eight structurally diverse PAEs, namely diethyl phthalate (DEP), di-n-propyl phthalate (DPrP), di-n-butyl phthalate (DBP), di-n-pentyl phthalate (DPeP), di-n-hexyl phthalate (DHP), DEHP, n-butyl benzyl phthalate (BBP), and dicyclohexyl phthalate (DCHP), were hydrolyzed to their corresponding monoesters by both porcine and bovine pancreatic CEases, while a microbial CEase from Pseudomonas sp. had no hydrolyzing activity against these PAEs. The hydrolysis experiments with bovine pancreatic CEase (50 U) indicated complete hydrolysis of every PAE (5 mumole) except for BBP and DCHP within 15 min; BBP and DCHP were hydrolyzed within 30 min and 6h, respectively. The rates of PAE hydrolysis could be affected by the bulkiness of alkyl side chains in the PAEs. This study provides important evidence that mammalian pancreatic CEases, such as those from porcine and bovine sources, are potential enzymes for nonspecific degradation of structurally diverse PAEs.

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.

To clarify species differences in the metabolism of di(2-ethylhexyl) phthalate (DEHP) we measured the activity of four DEHP-metabolizing enzymes (lipase, UDP-glucuronyltransferase (UGT), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH)) in several organs (the liver, lungs, kidneys, and small intestine) of mice (CD-1), rats (Sprague-Dawley), and marmosets (Callithrix jacchus). Lipase activity, measured by the rate of formation of mono(2-ethylhexyl) phthalate (MEHP) from DEHP, differed by 27- to 357-fold among species; the activity was highest in the small intestines of mice and lowest in the lungs of marmosets. This might be because of the significant differences between Vmax/Km values of lipase for DEHP among the species. UGT activity for MEHP in the liver microsomes was highest in mice, followed by rats and marmosets. These differences, however, were only marginal compared with those for lipase activity. ADH and ALDH activity also differed among species; the activity of the former in the livers of marmosets was 1.6-3.9 times greater than in those of rats or mice; the activity of the latter was higher in rats and marmosets (2-14 times) than in mice. These results were quite different from those for lipase or UGT activity. Because MEHP is considered to be the more potent ligand to peroxisome proliferator-activated receptor alpha involved in different toxic processes, a possibly major difference in MEHP-formation capacity could be also considered on extrapolation from rodents to humans.

The efficiency of two lypolytic enzymes (fungal cutinase, yeast esterase) in the degradation of di-(2-ethylhexyl)-phthalate (DEHP) was investigated. The DEHP-degradation rate of fungal cutinase was surprisingly high, i.e. almost 70% of the initial DEHP (500 mg/l) was decomposed within 2.5 h and nearly 50% of the degraded DEHP disappeared within the initial 15 min. With the yeast esterase, despite the same concentration, more than 85% of the DEHP remained even after 3 days of treatment. During the enzymatic degradation of DEHP, several DEHP-derived compounds were detected and time-course changes in composition were also monitored. During degradation with fungal cutinase, most DEHP was converted into 1,3-isobenzofurandione (IBF) by diester hydrolysis. In the degradation by yeast esterase, two organic chemicals were produced from DEHP: IBF and an unidentified compound (X). The final chemical composition after 3 days was significantly dependent on the enzyme used. Fungal cutinase produced IBF as a major degradation compound. However, in the DEHP degradation by yeast esterase, compound X was produced in abundance in addition to IBF. The toxic effects of the final degradation products were investigated, using various recombinant bioluminescent bacteria and, as a result, the degradation products from yeast esterase were shown to contain a toxic hazard, causing oxidative stress and damage to protein synthesis.

Red cell concentrates (RCC) are stored for 35 to 42 days in plastic containers manufactured with the liquid plasticizer di(2-ethylhexyl)phthalate (DEHP). DEHP leaches from the polyvinylchloride (PVC) plastic bag, then binds to and stabilizes the RC membrane. This study was undertaken to determine the deformability of the RC membrane using an osmotic gradient ektacytometer and to relate these measurements to the concentration of DEHP in the stored RCC. Pooled RCC was aliquoted into PL146 (PVC), PL732 (polyolefin), and PL732 (with added DEHP) bags with samples removed weekly for analysis of osmotic fragility, deformability, and DEHP concentration. The adenosine triphosphate (ATP) content was also measured. The increase in osmotic fragility during storage was greater when RCC was stored without DEHP. In addition, there was a decrease in the maximum elongation index (El max) when there was decreased DEHP in the storage bag. The osmolarity (Omax) at which El max occurred, as well as the Omin, the osmolarity at which minimum elongation (El min) occurred was higher in the PL732 container than in the PL146 or in the PL732 to which DEHP had been added. These changes could be reversed by addition of DEHP at the beginning of the storage period, showing a direct correlation between DEHP concentration during storage and RC membrane flexibility. By a better understanding of the mechanism of DEHP protection, it might be possible to substitute a less toxic stabilizing compound.

The development of flexible plastic blood bags has permitted effective blood component production and therapy. However, the plasticizer di(2-ethylhexyl)phthalate (DEHP), whose toxicity in humans is still undefined, is known to leach from the plastic into stored blood. Despite the availability of bags made of plastics not using DEHP, the collection and storage of red cells is still done in DEHP plasticized packs, and in fact the storage life for red cells has recently been increased up to 49 days using new anticoagulant-preservative solutions. We examined the relationship between DEHP and stored red cells. We found that 28 percent of available 14C-DEHP binds immediately to sites in both the membrane and cytosol fractions of the red cells, and that the total amount and distribution of 14C-DEHP does not change significantly over 7 days. When red cell concentrates were stored with or without DEHP, using either plastic (polyolefin) bags not containing DEHP or glass, definite reduction in the osmotic stability of the red cells was found in the absence of DEHP. Plasma-free hemoglobin levels were 90.3 mg per dl after 35 days of storage in plastic packs containing DEHP and 181.7 mg per dl in the polyolefin bags. The advantages of improved in vitro stability of red cells stored in plastics containing DEHP must be weighed against the potential hazards of patient exposure to DEHP.

Trout liver homogenates metabolized di-2-ethylhexyl phthalate (DEHP) to monoethylhexyl phthalate (MEHP) without added NADPH and to MEHP and more polar metabolites with added NADPH. Both hydrolysis and oxidative metabolism of DEHP were inhibited by piperonyl butoxide. The 10,000g pellet, 100,000g pellet and 100,000g supernatant fraction from liver homogenates all catalyzed the hydrolysis of DEHP and all but the 100,000g supernatant fraction showed the shift to more polar metabolites with added NADPH; serum also catalyzed the hydrolysis of DEHP. Measurement of the microsomal marker, glucose 6-phosphatase, and the mitochondrial marker, succinic dehydrogenase, revealed that DEHP-hydrolytic activity was associated with microsomes and the 100,000g supernatant fraction, whereas DEHP oxidation was associated only with microsomes.