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.     

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.     

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.     

Modulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in F344 rats by di(2-ethylhexyl)phthalate

The effects of cotreatment with a hyperlipidemic chemical, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and a hypolipidemic agent, di(2-ethylhexyl)-phthalate (DEHP), on lipid metabolism and toxicologic responses were studied in F344 rats. Treatment with TCDD alone (160 micrograms/kg) caused an increase in serum triglycerides and cholesterol while treatment with DEHP alone (2 g/kg/day) caused a decrease in triglycerides and cholesterol versus untreated controls. When administered before or after TCDD, DEHP caused a decrease in TCDD-induced hyperlipidemia. This change was attributed to enhanced hepatic peroxisomal beta-oxidation and decreased hepatic lipid synthesis resulting from treatment with DEHP. TCDD treatment produced a fatty liver, as determined by gravimetric analysis of extracted lipid and microscopic examination of liver sections which revealed extensive cytoplasmic vacuolization that stained positive with Oil Red 0, but did not induce peroxisomal beta-oxidation. Thus, an increase in hepatic or serum lipid levels is not sufficient for induction of peroxisome proliferation. Neither TCDD nor DEHP treatment affected mitochondrial beta-oxidation. Pretreatment of rats with DEHP, followed by daily exposure to this hypolipidemic agent after treatment with TCDD, had a partial protective effect against TCDD-induced fatty liver, body weight loss and mortality. Microscopic examination of liver sections confirmed the suppression of TCDD-induced fatty liver by pretreatment with DEHP. When DEHP treatment was initiated after the TCDD dose, there was less protection against the above parameters of TCDD toxicity. This study demonstrates that TCDD-induced fatty liver, hyperlipidemia and mortality can be antagonized by treatment with a hypolipidemic agent such as DEHP.     

Pregnancy-associated changes in plasma concentration of the endocrine disruptor di(2-ethylhexyl) phthalate in a sheep model

The plasticizer di(2-ethylhexyl)phthalate (DEHP), used for producing polyvinyl chloride (PVC), acts as an endocrine disruptor with toxic effects on reproductive and developmental processes. Exposure to DEHP in humans is mainly by environment and food. Thus, our aim was to determine plasma levels in livestock animals using the ewe (Ovis aries) as a model. In a first trial, 150 samples from ewes of different ages (2 to 7 yr) and reproductive status (pregnant and nonpregnant) were analyzed by high-performance liquid chromatography (HPLC). DEHP was detected in 34.7% of the samples, with a mean level of 0.45 ± 0.01 μg/mL (range, 0.05 to 2.81 μg/mL). The percentage of nonpregnant animals with DEHP traces was higher in animals older than 4 yr (n = 66, 37.9%) than in younger animals (n = 69, 17.4%; P < 0.05), although the mean levels in ewes with residues were similar (0.16 ± 0.01 vs. 0.16 ± 0.02 μg/mL). All the pregnant ewes (n = 15) showed presence of DEHP, with higher plasma levels than that in nonpregnant females (1.42 ± 0.18 vs. 0.16 ± 0.01 μg/mL; P < 0.0001). For confirming the effect of pregnancy on mobilization of DEHP from body fat, 101 ewes of the same age were sampled in a second trial at a different farm. The percentage of animals with DEHP traces was higher in pregnant ewes (n = 32, 71.9%; P < 0.005) than in nonpregnant ewes (n = 37, 35.1%) or in ewes that recently gave birth (n = 32, 21.9%), although mean levels were similar (0.42 ± 0.02, 0.33 ± 0.02, and 0.34 ± 0.05 μg/mL, respectively). In conclusion, current results indicate a high incidence of ewes reared in the field showing accumulation of phthalates; percentage of animals with presence of DEHP increases with age, due to an extended period of exposure, but mainly during pregnancy, due to the mobilization of body reserves.     

Simultaneous analysis of the di(2-ethylhexyl)phthalate metabolites 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine by gas chromatography–mass spectrometry

A gas chromatographic–mass spectrometric method was developed for the quantitative analysis of the three Di(2-ethylhexyl)phthalate (DEHP) metabolites, 2-ethylhexanoic acid, 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in urine. After oximation with O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine hydrochloride and sample clean-up with Chromosorb P filled glass tubes, all three organic acids were converted to their tert.-butyldimethylsilyl derivatives. Quantitation was done with trans-cinnamic acid as internal standard and GC–MS analysis in the selected ion monitoring mode (SIM). Calibration curves for all three acids in the range from 20 to 1000 μg/l showed correlation coefficients from 0.9972 to 0.9986. The relative standard deviation (RSD) values determined in the observed concentration range were between 1.3 and 8.9% for all three acids. Here we report for the first time the identification of 2-ethyl-3-hydroxyhexanoic acid and 2-ethyl-3-oxohexanoic acid in human urine next to the known DEHP metabolite 2-ethylhexanoic acid. In 28 urine samples from healthy persons we found all three acids with mean concentrations of 56.1±13.5 μg/l for 2-ethylhexanoic acid, 104.8± 80.6 μg/l for 2-ethyl-3-hydroxyhexanoic acid and 482.2± 389.5 μg/l for 2-ethyl-3-oxohexanoic acid.     

Individual molecular species of phosphatidylcholines and phosphatidylinositols in liver of rats fed bis(2-ethylhexyl)phthalate

Rats were given a diet containing 1% bis(2-ethylhexyl)phthalate (DEHP) for 3 weeks, and their hepatic lipids analyzed. Phosphatidylcholines increased by 20%, while other phospholipid classes and cholesterol remained unchanged and triglycerides fell. The composition of molecular species of phosphatidylcholines was changed. Thus, the hepatic content of the major species, 1-palmitoyl-2-oleoyl-, 1-palmitoyl-2-arachidonoyl- and 1-stearoyl-2-arachidonoylphosphatidylcholines, rose by about 150%, 90% and 70%, respectively. The content of the other major species, 1-palmitoyl-2-linoleoyl- and 1-stearoyl-2-linoleoylphosphatidylcholine fell by about 20% and 30%, respectively. The content of alkyl-acyl analogues of phosphatidylcholines increased by about 70%, but the composition of molecular species remained the same. The composition of molecular species of phosphatidylinositols was also unchanged. Thus, the analyses show that DEHP can induce selective changes in molecular species of certain phospholipids in the liver. This could be important for the functioning of membrane structures in the hepatocyte.     

Developmental exposure to di(2-ethylhexyl) phthalate impairs endocrine pancreas and leads to long-term adverse effects on glucose homeostasis in the rat

-Di(2-ethylhexyl) phthalate (DEHP), a typical endocrine-disrupting chemical (EDC), is widely used as plasticizer. DEHP exposure in humans is virtually ubiquitous, and those undergoing certain medical procedures can be especially high. In this study, we investigated whether developmental DEHP exposure disrupted glucose homeostasis in the rat and whether this was associated with the early impairment in endocrine pancreas. Pregnant Wistar rats were administered DEHP (1.25 and 6.25 mg·kg(-1)·day(-1)) or corn oil throughout gestation and lactation by oral gavage. Body weight, glucose and insulin tolerance, and β-cell morphometry and function were examined in offspring during the growth. In this study, developmental DEHP exposure led to abnormal β-cell ultrastructure, reduced β-cell mass, and pancreatic insulin content as well as alterations in the expression of genes involved in pancreas development and β-cell function in offspring at weaning. At adulthood, female DEHP-exposed offspring exhibited elevated blood glucose, reduced serum insulin, impaired glucose tolerance, and insulin secretion. Male DEHP-exposed offspring had increased serum insulin, although there were no significant differences in blood glucose at fasting and during glucose tolerance test. In addition, both male and female DEHP-exposed offspring had significantly lower birth weight and maintained relatively lower body weight up to 27 wk of age. These results suggest that developmental exposure to DEHP gives rise to β-cell dysfunction and the whole body glucometabolic abnormalities in the rat. DEHP exposure in critical periods of development can be a potential risk factor, at least in part, for developing diabetes.     

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.     

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.     

Antiandrogenic effect of perinatal exposure to the endocrine disruptor di-(2-ethylhexyl) phthalate increases anxiety-like behavior in male rats during sexual maturation

Di-2-ethylhexyl phthalate (DEHP) is the most widely used phthalate to convey flexibility and transparency to plastic products made of polyvinyl chloride. It has been recognized as endocrine disruptor and associated with reproductive toxic effects. We examined the effects of perinatal exposure to DEHP on anxiety-like behavior, using the Elevated Plus Maze (EPM) test, in male and female rats at different stages of sexual development. Anxiety-like behavior was expressed as a) frequency of open arm entries over the total arm entries (% FEO); b) time spent in them compared with total time the animal stayed in the EPM (% TSO) and c) time spent in closed arms (TSC). Because DEHP has anti-androgenic action we also tested control and exposed immature male rats pretreated with testosterone. We found sex differences in behavior induced by DEHP; while male rats of 45 and 60 days of age showed a significant decrease in FEO and TSO percentages, as well as an increase in TSC, no changes were observed in anxiety-like behavior in perinatal DEHP exposed females at these ages of sexual maturation. In 60-day-old male rats, DEHP exposure produced a significant decrease in serum testosterone levels. Testosterone replacement was able to antagonize the adverse effects of DEHP exposure on LH, activating the negative feed-back mechanism of this steroid on reproductive axis, as well as increasing FEO and TSO percentages to similar values observed in the control group. These findings suggest that the anti-androgenic action of this chemical could be one possible mechanism underlie anxiogenic-like behavior produced by perinatal DEHP exposure in 60-day-old male rats.     

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.     

Effect of a hypolipidemic drug, Di (2-ethylhexyl) phthalate, on mRNA-expression associated fatty acid and acetate metabolism in rat tissues

Di (2-ehtylhexyl) phthalate (DEHP) is a peroxisome proliferator and a drug having a hypolipidemic effect. The body-weight change of rats treated with DEHP was lower than that of rats in an untreated control group. Expressions of long-chain acyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase, which are involved in fatty acid oxidation and acetate formation in mitochondria, showed an increase in the liver and testes of rats treated with DEHP. The expression of acetyl-CoA synthetase 1 was significantly decreased in the testes and relatively decreased in the liver, while the expression of acetyl-CoA synthetase 2 was significantly increased in the heart. Furthermore, the expressions of acetyl-CoA carboxylase in heart and testes showed a tendency to decrease. From these results, it is suggested that DEHP-treatment increased fatty acid oxidation and acetate formation in liver and testes, and that acetate utilization was increased in peripheral tissues such as the heart.     

Inhibition of membrane Na(+)-K+ Atpase of the brain, liver and RBC in rats administered di(2-ethyl hexyl) phthalate (DEHP) a plasticizer used in polyvinyl chloride (PVC) blood storage bags

Significant amounts of di(2-ethylhexyl) phthalate (DEHP) leach out into blood stored in DEHP plasticized polyvinyl chloride (PVC) bags resulting in the exposure of recipients of blood transfusion to this compound. The aim of this study was to find out whether DEHP at these low levels has any effect on the activity of membrane Na(+)-K+ ATPase, since a decrease in this enzyme activity has been reported to take place in a number of disorders like neurodegenerative and psychiatric disorders, coronary artery disease and stroke, syndrome-X, tumours etc. DEHP was administered (ip) at a low dose of 750 microg/100 g body weight to rats and the activity of membrane Na(+)-K+ ATPase in liver, brain and RBC was estimated. Histopathology of brain, activity of HMG CoA reductase (a major rate limiting enzyme in the isoprenoid pathway of which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is a product), intracellular concentration of Ca2+ and Mg2+ in RBC (which is altered as a result of inhibition of Na(+)-K+ ATPase) were also studied. (In the light of the observation of increase of intracellular Ca2+ load and intracellular depletion of Mg2+ when Na(+)-K+ ATPase is inhibited). Histopathology of brain revealed areas of degeneration in the rats administered DEHP. There was significant inhibition of membrane Na(+)-K+ ATPase in brain, liver and RBC. Intracellular Ca2+ increased in the RBC while intracellular Mg2+ decreased. However activity of hepatic HMG CoA reductase decreased. Activity of Na(+)-K+ ATPase and HMG CoA reductase, however returned to normal levels within 7 days of stopping administration of DEHP. The inhibition of membrane Na(+)-K+ ATPase activity by DEHP may indicate the possibility of predisposing recipients of transfusion of blood or hemodialysis to the various disorders mentioned above. However since this effect is reversed when DEHP administration is stopped, it may not be a serious problem in the case of a few transfusion; but in patients receiving repeated blood transfusion as in thalassemia patients or patients undergoing hemodialysis, possibility of this risk has to be considered. This inhibition is a direct effect of DEHP or its metabolites, since activity of HMG CoA reductase, (an enzyme which catalyses a major rate limiting step in the isoprenoid pathway by which digoxin, the physiological inhibitor of Na(+)-K+ ATPase is synthesized) showed a decrease.     

Content of polyunsaturated fatty acids (PUFAs) in serum and liver of rats fed restricted diets supplemented with vitamins B2, B6 and folic acid

The aim of study was to investigate an influence of nutritional deficiency and dietary addition of vit. B(2), B(6) and folic acid on PUFAs content in rats' serum and liver. Limitation of consumption full value diet to 50% of its previously determined daily consumption, enriched with m/a vitamins, significant decreased of linoleic (LA) and alpha-linolenic (ALA) acids as well as distinctly increased arachidonic (AA) and docosahexaenoic (DHA) acids content in serum in 30th day. In 60th day lower content of AA and DHA fatty acids was found. Nutrition with such diet, lasting 90 days caused decrease of LA content and increase of AA. Diet limitation to its 30% of daily consumption decreased of eicosapentaenoic acid (EPA) and DHA in the 30th day, while AA and DHA content was increased in the 60th day. Distinct decrease of AA content and increase of EPA content were found in the 90th day of experiment. Use of diets, with limited consumption to 50% caused increase of LA and ALA acids content while AA and DHA acids content were significantly decreased in the liver, in 90th day. Limited consumption supplemented diet to 30% caused in liver significant decrease of LA and increase of EPA acids content.     

Novel benzoylpiperidine-based stearoyl-CoA desaturase-1 inhibitors: Identification of 6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxylic acid (2-hydroxy-2-pyridin-3-ylethyl)amide and its plasma triglyceride-lowering effects in Zucker fatty rats

Starting from a known piperazine-based SCD-1 inhibitor, we obtained more potent benzoylpiperidine analogs. Optimization of the structure of the benzoylpiperidine-based SCD-1 inhibitors resulted in the identification of 6-[4-(2-methylbenzoyl)piperidin-1-yl]pyridazine-3-carboxylic acid (2-hydroxy-2-pyridin-3-yl-ethyl)amide (24) which showed strong inhibitory activity against both human and murine SCD-1. In addition, this compound exhibited good oral bioavailability and demonstrated plasma triglyceride lowering effects in Zucker fatty rats in a dose-dependent manner after a 7-day oral administration (qd).