The formation of nicotinamide by administration of di(2-ethylhexyl)phthalate does not cause growth retardation in young rats in contrast with excess exogenous administration of nicotinamide

We investigated the relationship between protein and tryptophan intake and the adverse-effect-level of di(2-ethylhexyl)phthalate (DEHP). Growth retardation of young rats due to DEHP was strengthened by increasing protein level. The addition of tryptophan to the diet caused extreme increases in the nicotinamide formation, but no growth retardation was observed.     

Heat shock protein synthesis is induced by diethyl phthalate but not by di(2-ethylhexyl) phthalate in radish (Raphanus sativus)

The toxicity and effects on protein synthesis of the phthalate esters diethyl phthalate (DEP) and di(2-ethylhexyl) phthalate (DEHP) was studied in radish seedlings (Raphanus sativus cv. Kööpenhaminan tori). Phthalate esters are a class of commercially important compounds used mainly as plasticizers in high molecular-weight polymers such as many plastics. They can enter soil through various routes and can affect plant growth and development. First the effect of DEP and DEHP on the growth of radish seedlings was determined in an aqueous medium. It was found that DEP, but not DEHP, caused retardation of growth in radish. A further investigation on protein synthesis during DEP-stress was executed by in vivo protein labeling combined with two-dimensional gel electrophoresis (2D-PAGE). For comparisons with known stress-induced proteins a similar experiment was done with heat shock, and the induced heat shock proteins (HSPs) were compared with those of DEP-stress. The results showed that certain HSPs can be used as an indicator of DEP-stress, although the synthesis of most HSPs was not affected by DEP. DEP also elicited the synthesis of numerous proteins found only in DEP-treated roots. The toxic effect of phthalate esters and the roles of the induced proteins are discussed.     

Elucidation of the toxic mechanism of the plasticizers,phthalic acid esters,putative endocrine disrupters: effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in rats

We have previously reported that the administration of a large amount of di(n-butyl)phthalate (DBP) increased the conversion ratio of tryptophan to niacin in rats. In the present experiment, the effect of di(2-ethylhexyl)phthalate (DEHP) on the conversion ratio and how altering the conversion ratio of tryptophan to niacin depended on the concentration of DEHP were investigated to elucidate the toxic mechanism of phthalic acid esters (PhE). Rats were fed with a diet containing 0%, 0.01%, 0.05%, 0.1%, 0.5%, 1.0%, or 3.0% DEHP for 21 days. To assess the conversion ratio of tryptophan to niacin, urine samples were collected at the last day of the experiment and measured for metabolites on the tryptophan-niacin pathway. The conversion ratio increased with increasing dietary concentration of DEHP above 0.05%; the conversion ratio was about 2% in the control group, whereas it was 28% in the 3.0% DEHP group. It is suggested that the inhibition of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) by DEHP or its metabolites caused this increase in the conversion ratio. We conclude that PhE such as DEHP and DBP disturbed the tryptophan-niacin metabolism.     

Comparison of the effects of di(2-ethylhexyl)phthalate, a peroxisome proliferator, on the vitamin metabolism involved in the energy formation in rats fed with a casein or gluten diet

In order to find an alleviation method for the adverse effect of environmental endocrine disrupters, we studied the effects of the putative endocrine disrupter and peroxisome proliferator, di(2-ethylhexyl)phthalate (DEHP), on animal growth and vitamin metabolism. It is known that the effects of chemical compounds such as xenobiotics differ according to the dietary protein source. We compared the effects of dietary DEHP administration on rats fed with a diet containing milk casein or wheat gluten. The increased conversion ratio of tryptophan to nicotinamide by DEHP administration was significantly higher in the casein group than in the gluten group. We also investigated the effects of DEHP on the urinary excretion of other vitamins. DEHP administration resulted in decreased urinary excretion of vitamin B(1), vitamin B(2), and pantothenic acid.     

Identification of toxicological biomarkers of di(2‐ethylhexyl) phthalate in proteins secreted by HepG2 cells using proteomic analysis

The effects of di(2‐ethylhexyl) phthalate (DEHP) on proteins secreted by HepG2 cells were studied using a proteomic approach. HepG2 cells were exposed to various concentrations of DEHP (0, 2.5, 5, 10, 25, 50, 100, and 250 μM) for 24 or 48 h. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) and comet assays were then conducted to determine the cytotoxicity and genotoxicity of DEHP, respectively. The MTT assay showed that 10 μM DEHP was the maximum concentration that did not cause cell death. In addition, the DNA damage in HepG2 cells exposed to DEHP was found to increase in a dose‐ and time‐dependent fashion. Proteomic analysis using two different pI ranges (4–7 and 6–9) and large size 2‐DE revealed the presence of 2776 protein spots. A total of 35 (19 up‐ and 16 down‐regulated) proteins were identified as biomarkers of DEHP by ESI‐MS/MS. Several differentiated protein groups were also found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up‐ or down‐regulated. Among these, the identities of cystatin C, Rho GDP inhibitor, retinol binding protein 4, gelsolin, DEK protein, Raf kinase inhibitory protein, triose phosphate isomerase, cofilin‐1, and haptoglobin‐related protein were confirmed by Western blot assay. Therefore, these proteins could be used as potential biomarkers of DEHP and human disease associated with DEHP.     

Effects of dietary di(2-ethylhexyl)phthalate, a putative endocrine disrupter, on enzyme activities involved in the metabolism of tryptophan to niacin in rats

We have reported that the conversion ratio of tryptophan to niacin increased with increasing dietary concentration of di(2-ethylhexyl)phthalate (DEHP); the conversion ratio was about 2.0% in the control rat, which increased by about 30% in the rat fed with 3.0% DEHP diet. In this study, we investigated whether this abnormal increase in the conversion ratio by DEHP occurred through the alteration of the enzyme activities involved in the metabolism of tryptophan to niacin. Rats were fed with a diet containing 0%, 0.1%, 0.5%, or 1.0% DEHP for 21 days. The nine kinds of enzyme activities involved in the biosynthesis and catabolism in the liver and kidney were measured. Based on previous findings that the formation of quinolinic acid and its' metabolites significantly increased with DEHP administration, we proposed that the activity of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase would be inhibited by DEHP intake. However, we found that the activities in the liver and kidney did not decrease in the rat fed with DEHP-containing diet. We discuss the discrepancy between the metabolite results and the enzyme activities.     

Effects of dietary di(2-ethylhexyl)phthalate on the metabolism of tryptophan to niacin in mice.

We have reported the effect of di(2-ethylhexyl)phthalate (DEHP) on the tryptophan (Trp)-niacin pathway in rats. To clarify the universal effect of DEHP on rodents, we studied whether DEHP also has an effect on Trp metabolism in mice. Mice were fed a niacin-free, 20% casein diet supplemented with DEHP for 21 days. Feeding with DEHP decreased the body weight gain and increased the liver weight in correlation with the dose level of DEHP. The administration of DEHP significantly increased the formation of quinolinic acid and the lower metabolites of the Trp-niacin pathway. The flux of niacin in the lower part of the Trp-niacin pathway in mice was enhanced by feeding with DEHP.     

Interactive effects of benzo(a)pyrene and cadmium and effects of di(2-ethylhexyl) phthalate on antioxidant and peroxisomal enzymes and peroxisomal volume density in the digestive gland of mussel Mytilus galloprovincialis Lmk

Exposure of marine animals to certain organic and metal pollutants is thought to enhance reactive oxygen species (ROS) production with concomitant alterations of antioxidant defence mechanisms. Some of these organic pollutants cause peroxisome proliferation, a process resulting also in possible enhanced production of ROS. The aim of this study was to investigate the effects of two organic xenobiotics, benzo(a)pyrene (B(a)P) and di(2-ethylhexyl)phthalate (DEHP), as well as the effects of cadmium (Cd), on antioxidant and peroxisomal enzymes and on peroxisomal volume density in the digestive gland of mussel, Mytilus galloprovincialis Lmk., experimentally exposed for 21 days. Special attention was paid to the interactive effects of organic and metal compounds by exposing one group of mussels to a mixture of B(a)P and Cd. Exposure of mussels to Cd caused a decrease in superoxide dismutase (SOD) activity, in Mn-SOD protein levels and in volume density of peroxisomes. B(a)P exposure significantly increased catalase and glutathione peroxidase (GPX) and inhibited Mn-SOD after 21 days of exposure. B(a)P also caused a slight increase in acyl-CoA oxidase (AOX) activity and peroxisomal volume density after 21 days of exposure. Cd tended to inhibit changes provoked by B(a)P, indicating that responses to organic xenobiotics can be modulated by concomitant exposure to metal contaminants. Exposure to DEHP increased catalase and AOX and inhibited SOD activity and Mn-SOD protein levels. In conclusion, peroxisome proliferation, measured as an increase of the peroxisomal enzymes catalase and AOX (up to 1.53-fold for AOX), is a specific response to organic contaminants such as B(a)P and DEHP, whereas Cd does not cause peroxisome proliferation. Thus, peroxisome proliferation may be a specific biomarker of organic pollutants in mussels. Both organic and metal pollutants inhibited SOD activity and protein levels (up to 0.21-fold for Mn-SOD protein levels), the latter offering potential as general marker of pollution.     

Compartmentation of the plant metabolic enzymes for the persistent plasticizer chemical,bis-2(-ethylhexyl)-phthalate

The environmentally persistent plasticizer chemical, bis-(2-ethylhexyl)-phthalate (DEHP), was not translocated or metabolized over 17 days after application to intact wheat leaves. The chemical was specifically associated with the cuticle fraction. In contrast, metabolism was observed in injured leaves and in leaf homogenates.DEHP esterase was 10-fold purified from the wheat leaf homogenate, and an apparent molecular weight of 38 000 was determined by gel permeation chromatography.Cleavage of DEHP by the partially purified esterase was strongly inhibited by a wheat cell wall and by a triglyceride preparation. Cleavage of the water-soluble substrate, 4-nitrophenylacetate, was not inhibited under the same conditions.Compartmentation of metabolic enzymes is proposed as an explanation for the persistence of DEHP and certain other environmental chemicals in plants.     

Intermittent fasting ameliorates di-(2-ethylhexyl) phthalate-induced precocious puberty in female rats: A study of the hypothalamic–pituitary–gonadal axis

Di-(2-ethylhexyl) phthalate has been reported to interfere with the development and function of animal reproductive systems. However, hardly any studies provide methods to minimize or prevent the adverse effects of DEHP on reproduction. The energy balance state of mammals is closely related to reproductive activities, and the reproductive axis can regulate reproductive activities according to changes in the body's energy balance state. In this study, the effects of every other day fasting (EODF), as a way of intermittent fasting, on preventing the precocious puberty induced by DEHP in female rats was studied. EODF significantly improved the advancement of vaginal opening age (as the markers of puberty onset) and elevated serum levels of luteinizing hormone and estradiol (detected by ELISA) induced by 5 mg kg^?1 DEHP exposure (D5). The mRNA and western blot results showed that the EODF could minimized the increase of gonadotropin-releasing hormone expression induced by DEHP exposure. The administration of DEHP could elevate the levels of kisspeptin protein and the number of kisspeptin-immunoreactive neurons in anteroventral periventricular nucleu, and this increase was diminished considerably by EODF treatment. In contrast, the D5 and D0 groups showed no remarkable difference in the level of Kiss1 expression in arcuate nucleus, whereas the D5 + EODF group had a remarkable decrease in kisspeptin expression as compared with the other two groups. Our results indicated that EODF might inhibit the acceleration of puberty onset induced by DEHP exposure via HPG axis.     

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。     

Effect of propranolol and cycloheximide on the ethanol-induced increase in liver tryptophan oxygenase activity in starved rats

Increased supply of trytophan to the liver, resulting from the lipolytic action of ethanol, is suggested to be responsible for the increased activity of liver tryptophan oxygenase after ingestion of a single large dose of ethanol. This hypothesis was tested using an antilipolytic drug, propranolol, prior to ethanol treatment. It was found that, while propronolol did inhibit the ethanol-induced increase in blood unesterified fatty acids and free tryptophan concentrations it did not prevent the activation of tryptophan oxygenase by ethanol. In another experiment, where cycloheximide was used to block protein synthesis, it was found that increased protein synthesis rather than decreased protein degradation is probably responsible for the accumulation of liver tryptophan oxygenase after ethanol ingestion.     

邻苯二甲酸二乙基己酯对翡翠贻贝(Perna viridis)生化指标的影响

实验条件下,研究邻苯二甲酸二乙基己酯(DEHP)5个浓度组(0、0.38、1.92、9.60和48.00mg.L-1)长时间胁迫下翡翠贻贝(Perna viridis)内脏团和外套膜中抗氧化酶(SOD和CAT)活性和丙二醛(MDA)含量的变化,以及胁迫解除后这些指标的恢复情况。结果表明:在胁迫过程中,翡翠贻贝内脏团SOD活性表现为先显著升高,随后受抑制而逐渐降低(P<0.05),CAT活性则表现为先被抑制后受诱导,15d后恢复到对照组水平,MDA含量呈显著增加的趋势(P<0.05);外套膜中的SOD活性在胁迫初期在低浓度组被抑制,而在高浓度组则被诱导(P<0.05),4d后SOD活性逐渐恢复到对照组水平,各浓度组MDA含量均出现明显的增加(P<0.05);净化阶段,低浓度组(0.38mg.L-1)内脏团SOD活性和CAT活性逐渐恢复到对照组水平,但MDA含量升高;净化7d后,除高浓度组(48.00mg.L-1)外,其余浓度组外套膜中SOD活性均已经恢复到对照组水平,MDA含量也没有出现明显升高的现象。研究表明,DEHP对翡翠贻贝内脏团和外套膜抗氧化防御系统酶具有明显的影响,DEHP诱导引起2种组织内脂质过氧化损伤,并且短期内这种损伤无法消除。     

The effects of di(2-ethylhexyl) phthalate and/or selenium on trace element levels in different organs of rats

Di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer for synthetic polymers, is known to have endocrine disruptive potential, reproductive toxicity, and induces hepatic carcinogenesis in rodents. Selenium (Se) is a component of several selenoenzymes which are essential for cellular antioxidant defense and for the functions of mammalian reproductive system. The present study was designed to investigate the effects of DEHP exposure on trace element distribution in liver, testis, and kidney tissues and plasma of Se-deficient and Se-supplemented rats. Se deficiency was produced by feeding 3-week old Sprague-Dawley rats with ≤0.05 mg Se/kg diet for 5 weeks, and supplementation group were on 1 mg Se/kg diet. DEHP treated groups received 1000 mg/kg dose by gavage during the last 10 days of feeding period. Se, zinc (Zn), copper (Cu), iron (Fe) and manganese (Mn) levels were measured by inductively coupled plasma mass spectrometry (ICP-MS). Se supplementation caused significant increases in hepatic, renal, and testicular Se levels. With DEHP exposure, plasma Se and Zn, kidney Se, Cu and Mn levels were significantly decreased. Besides, liver Fe decreased markedly in all the DEHP-treated groups. Liver and kidney Mn levels decreased significantly in DEHP/SeD group compared to both DEHP and SeD groups. These results showed the potential of DEHP exposure and/or different Se status to modify the distribution pattern of essential trace elements in various tissues, the importance of which needs to be further evaluated.     

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.     

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-(2-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal β-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal β-oxidation was 5–7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the β-oxidation system and of catalase decreased to the control levels with a half-life of 2–3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.     

High-performance liquid chromatographic method for the determination of di(2-ethylhexyl) phthalate in total parenteral nutrition and in plasma

A simple, rapid and sensitive reversed-phase high-performance liquid chromatographic method with UV detection was developed for the quantification of di(2-ethylhexyl) phthalate (DEHP) in parenteral nutrition admixtures containing fat emulsion and in plasma samples of children daily treated by total parenteral nutrition. The analyte and the internal standard, di-n-heptyl phthalate, were extracted twice using hexane and the organic layer separated and dried under nitrogen. The residues were reconstituted with acetonitrile and 20 μl was injected into a Waters Spherisorb C₁₈ column, the UV detector was set at 202 nm. The mobile phase was acetonitrile–aqueous buffer (triethylamine 0.08% adjusted to pH 2.8 with 1 M phosphoric acid) mixture (88:12, v/v) and it was pumped at 1 ml/min. Average recoveries were 97% or greater. This method was successfully used to investigate the amounts of DEHP which can leach from bags and tubing into fat emulsion and which could contaminate children under long-term parenteral nutrition. On the other hand, the circulating DEHP concentrations were estimated in four children under regular long-term parenteral nutrition.