Induction of peroxisomal Lon protease in rat liver after di-(2-ethylhexyl)phthalate treatment

Previously, we identified a peroxisome-specific isoform of Lon protease using subcellular proteomics. In the present study, we investigated changes in the level of the Lon protease in peroxisomes during recovery from peroxisomal proliferation induced by di-(2-ethylhexyl)phthalate (DEHP) to elucidate the function of peroxisomal Lon protease (PSLP). Following a 2-week treatment with DEHP, the level of PSLP was monitored for 15 days. The amount of protease was greatly increased after the 2-week treatment, followed by a further increase 3 days after cessation of the treatment. Afterward, it decreased and reached the control level on day 15. On the other hand, level peroxisomal β-oxidation enzymes induced to express by DEHP started to decrease soon after discontinuation of treatment. The results suggest that PSLP functions to degrade β-oxidation enzymes induced by DEHP during recovery from perxisomal proliferation.     

Specific changes in the protein composition of rat liver in response to the peroxisome proliferators ciprofibrate, Wy-14,643 and di-(2-ethylhexyl)phthalate.

The hypolipidaemic agents ciprofibrate and Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid) and the phthalate-ester plasticizer di-(2-ethylhexyl)-phthalate (DEHP), like other peroxisome proliferators, produce a significant hepatomegaly and induce the peroxisomal fatty acid beta-oxidation enzyme system together with profound proliferation of peroxisomes in hepatic parenchymal cells. Changes in the profile of liver proteins in rats following induction of peroxisome proliferation by ciprofibrate, Wy-14,643 and DEHP have been analysed by high-resolution two-dimensional gel electrophoresis. The proteins of whole liver homogenates from normal and peroxisome-proliferator-treated rats were separated by two-dimensional gel electrophoresis using isoelectric focusing for acidic proteins and nonequilibrium pH gradient electrophoresis for basic proteins. In the whole liver homogenates, the quantities of six proteins in acidic gels and six proteins in the basic gels increased following induction of peroxisome proliferation. Peroxisome proliferator administration caused a repression of three acidic proteins in the liver homogenates. By the immunoblot method using polyspecific antiserum against soluble peroxisomal proteins and monospecific antiserum against peroxisome proliferation associated Mr 80000 polypeptide (polypeptide PPA-80), the majority of basic proteins induced by these peroxisome proliferators appeared to be peroxisomal proteins. Polypeptide PPA-80 becomes the most abundant protein in the total liver homogenates of peroxisome-proliferator-treated rats. These results indicate that ciprofibrate, DEHP and Wy-14,643 induce marked changes in the profile of specific hepatic proteins and that some of these changes should serve as a baseline to identify a set of gene products that may assist in defining the specific 'peroxisome proliferator domain'.     

The activities of peroxisomal oxidases in periportal and perivenous zones of the rat liver acinus

Summary Using a new biochemical microassay the activities of three peroxisomal oxidases in single microdissected periportal and perivenous zones of the liver acinus were measured. Whereas urate oxidase is homogeneously distributed through the acinus, the activities of D-aminoacid oxidase and -hydroxyacid oxidase are respectively 1.80-and 2.71-fold higher in the periportal hepatocytes than in the perivenous hepatocytes.     

Co-induction by peroxisome proliferators of microsomal 1-acylglycerophosphocholine acyltransferase with peroxisomal beta-oxidation in rat liver

Administration of clofibric acid, 2,2'-(decamethylenedithio)diethanol, di(2-ethylhexyl)phthalate or perfluorooctanoic acid to male rates increased markedly microsomal 1-acylglycerophosphocholine (a-acyl-GPC) acyltransferase in a dose-dependent manner in liver. Simultaneous administration of actinomycin D or cycloheximide completely abolished the increase in the enzyme activity. The treatment of rats with clofibric acid did not affect the rate of decay of 1-acyl-GPC acyltransferase. Regardless of a great difference in the chemical structures of the peroxisome proliferators, high correlation was observed between the induced activities of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation. Stearoyl-CoA desaturase was induced by peroxisome proliferators in a dose-dependent manner; nevertheless, high correlation was not seen between the induced activities of desaturase and peroxisomal beta-oxidation. Hormonal (adrenalectomy, diabetes, hyperthyroidism and hypothyroidism) and nutritional (starvation, starvation-refeeding, fat-free diet feeding and high-fat diet feeding) alterations hardly affected the activity of 1-acyl-GPC acyltransferase. The present results indicate that microsomal 1-acyl-GPC acyltransferase is a useful parameter responsive to the challenges by peroxisome proliferators and suggest that a similar regulatory mechanism operates for the inductions of microsomal 1-acyl-GPC acyltransferase and peroxisomal beta-oxidation.     

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase in rat liver. The effects of di-(2-ethylhexyl)phthalate, a peroxisome proliferator

Immunocytochemical localization of delta 3, delta 2-enoyl-CoA isomerase (isomerase) was investigated in rat liver. Livers of di-(2-ethylhexyl)phthalate (DEHP)-treated or untreated rats were perfusion-fixed and embedded in Epon or Lowicryl K4M. By light microscopy, reaction deposits for the enzyme were present in the cytoplasmic granules of hepatocytes and interlobular bile duct epithelium. Weak staining was noted in sinus-lining cells. After administration of DEHP, the granular staining of the hepatocytes was markedly enhanced, whereas the staining reaction of the sinus-lining cells decreased. The isomerase staining pattern was quite similar to that of long-chain acyl-CoA dehydrogenase (a mitochondrial marker), but different from that of catalase (a peroxisomal marker). Under electron microscopy, gold particles for isomerase were seen to be confined mainly to mitochondria of the hepatocytes, the bile duct epithelial cells and sinus-lining cells. Peroxisomes were weakly labeled. After DEHP administration, the peroxisomes were markedly induced, but the mitochondria were not. Quantitative analysis showed that the induction of the peroxisomal isomerase was only 2-fold whereas the mitochondrial isomerase was enhanced about 5-fold, 40 times as high as the peroxisomal enzyme. The results show that the mitochondria are the main intracellular site for isomerase and the peroxisomes a minor site. The mitochondrial isomerase of the rat liver is markedly induced by peroxisome proliferators, DEHP and clofibrate.     

Induction of rat liver mitochondrial fatty acid elongation by the administration of peroxisome proliferator di-(2-ethylhexyl)phthalate: absence of elongation activity in peroxisomes

The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male Sprague-Dawley rats resulted in more than a threefold increase in activity of acetyl CoA-dependent hepatic mitochondrial fatty acid elongation. Peroxisomes obtained either from control or DEHP-treated rats were not capable of elongating any of the fatty acyl CoAs tested. Furthermore, the peroxisomes possessed no trans-2-enoyl CoA reductase activity. Therefore, the elongation activity in the 7500g fraction from both control and DEHP-fed animals can be attributed totally to the mitochondria. Maximal incorporation of acetyl CoA occurred in the presence of both NADH and NADPH, and octanoyl CoA (8:0) and decanoyl CoA (10:0) were found to be optimal primers for fatty acid elongation in both control and DEHP-treated animals. The apparent Km for 8:0 CoA was 17 microM in both animal groups while the Vmax was increased from 4.5 to 12.5 nmol/min/mg following treatment. The apparent Km for 10:0 CoA was 10 microM in both control and DEHP-treated groups while the apparent Vmax increased from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor primer for chain elongation. Although the acetyl CoA-dependent fatty acid elongation was stimulated by DEHP treatment, the mitochondrial trans-2-enoyl CoA reductase activity was unaffected. The mitochondrial total elongation activity following DEHP-treatment using 8:0 CoA as primer was about two times higher than enoyl CoA reductase activity using trans-2-decenoyl CoA (10:1). This was the result of accumulation of intermediates, which were identified as trans-2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and elongated saturated product 10:0 (24%). Elongation by one acetate unit was found in both the control and DEHP-treated animals. The results are discussed in terms of physiological significance.     

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.     

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     

Mutagenicity of the peroxisome proliferators clofibrate, Wyeth 14,643 and di-2-ethylhexyl phthalate in the lacZ plasmid-based transgenic mouse mutation assay

BACKGROUND: Peroxisome proliferators are considered rodent carcinogens that are putative human non-carcinogens based on the presumed absence of direct genetic toxicity in rodent and human cells and the resistance of human cells to the induction of peroxisomes by peroxisome proliferators. The highly sensitive lacZ plasmid-based transgenic mouse mutation assay was employed to investigate the mutagenicity of several peroxisome proliferators based on several lines of evidence suggesting that these agents may in fact exert a genotoxic effect. METHODS: Male and female lacZ-plasmid based transgenic mice were treated at 4 months of age with 6 doses of 2,333 mg di-2-ethylhexyl phthalate (DHEP), 200 mg Wyeth-14,643, or 90 mg clofibrate per kg of bodyweight, respectively, over a two-week period. Control animals were treated with the respective vehicles only (35% propyl glycol for DEHP and Wyeth-14,643 treatment controls and sterile water for clofibrate treatment controls).The mutant frequency in liver, kidney and spleen DNA was determined as the proportion of retrieved mutant and wild-type lacZ plasmids expressed in Escherichia Coli C host cells employing a positive selection system for mutant plasmids. RESULTS: Exposure to DEHP or Wyeth-14,643 significantly increased the mutant frequency in liver, but not in kidney or spleen, of both female and male mice. Treatment with clofibrate did not lead to an increased mutant frequency in any of the organs studied. CONCLUSION: The results indicate that some peroxisome proliferators display an organ-specific mutagenicity in lacZ plasmid-based transgenic mice consistent with historical observations of organ- and compound-specific carcinogenicity.     

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.     

The influence of prolonged di(2-ethylhexyl)phthalate treatment on the dolichol and dolichyl-P content of rat liver

Dolichols and glycosyl transferase activities were studied in rat liver fractions after treatment with the plasticizer di(2-ethylhexyl)phthalate, an inducer of peroxisomes and mitochondria. After a few weeks of treatment with 2% plasticizer in the diet, the amount of dolichol is more than doubled in the lysosomes but not in the microsomes while dolichyl-P decreased by 50% in the microsomes but not in the lysosomes. The isoprenoid pattern for dolichol and dolichyl-P, respectively, is modified to longer polyprenols in the two fractions as seen in the percent distribution of the individual isoprenes. Dolichyl-P and protein glycosylation by N-acetylglucosamine and mannose decreased considerably. Incubation with mixtures containing exogenous dolichyl-P did not increase protein glycosylation. Phthalate ester treatment for 2 years increased dolichol content above the control values even when the dose was decreased a hundred times, to 0.02%. The results demonstrate a compartmentalization of dolichol and dolichyl-P distribution, and the induction studies suggest that hepatocytes possess separate regulating mechanisms for these two compounds.     

Zonal distribution of peroxisomal 3-oxoacyl-CoA thiolase mRNA in liver from rats treated with di-(2-ethylhexyl)phthalate

Treatment of rats with di-(2-ethylhexyl)phthalate leads to a dramatic increase in peroxisomal 3-oxoacyl-CoA thiolase RNA, the concentration being higher in the pericentral than in periportal hepatocytes. These findings indicate that the production of peroxisomal thiolase and the zonal distribution of the enzyme are regulated at a pretranslational level.     

Effect of the peroxisomal proliferator di(2-ethylhexyl)phthalate on component reactions of the rat hepatic microsomal fatty acid chain elongation system and on other hepatic lipogenic enzymes

The feeding of 2% di(2-ethylhexyl)phthalate (DEHP) to rats increased the hepatic microsomal elongation of palmitoyl-CoA by about twofold, while those of palmitoleoyl-CoA and gamma-linolenoyl-CoA decreased to 83 and 63%, respectively, of the control values. When component reactions of the elongation pathway were measured, it was observed that only the activity of condensing enzyme was increased by twofold, while those of beta-ketostearoyl-CoA reductase, beta-hydroxypalmitoyl-CoA dehydrase, and trans-2-hexadecenoyl-CoA reductases were not affected. Furthermore, the time course for induction of both condensation and elongation of palmitoyl-CoA was similar. In vitro addition of DEHP had no effect on either condensation or elongation. Thus, these results indicate that the peroxisomal proliferator induces only the condensing enzyme which is the regulatory and rate-limiting step of elongation sequence. The DEHP treatment also markedly enhanced the cytosolic NADPH-generating activities of glucose-6-PO4 dehydrogenase (2.2-fold) and malic enzyme (7.3-fold). Unexpectedly, the activities of fatty acid synthetase and citrate cleavage enzyme were unaffected. These results are discussed in light of the fact that these lipogenic enzymes are coordinately induced by diet or hormones.     

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.     

Biochemical alterations in rat fetal liver following in utero exposure to di(2-ethylhexyl)phthalate (DEHP)

Oral administration of DEHP, 1000 mg/kg body weight, to rats daily from 6 to 15 day of gestation resulted in retardation of fetal growth and increase in fetal liver weight which contained significant quantities of DEHP. The activities of mitochondrial succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and adenosine triphosphatase were decreased in fetal liver. The data indicate that exposure of mothers to DEHP during pregnancy could adversely affect the fetal livers by interfering with bioenergetics of the cell.     

Incorporation of radioactivity from labeled Di-(2-ethylhexyl)phthalate into DNA of rat liver in vivo

Di-(2-ethylhexyl)phthalate (DEHP), when fed at high levels in the diet for two years, is reportedly an hepatocarcinogen to rats and mice. Radioactivity from ethylhexyl-labeled, but not from phthalate-labeled, [14C]-DEHP is associated with highly purified DNA from the livers of treated rats and this radioactivity is not accounted for by assumptions of adsorption, intercalation, attachment to RNA or histones, an impurity in the labeled DEHP, or artifactual binding during sample workup. Spontaneous binding of radioactivity to DNA from either ethylhexyl-labeled DEHP or its total urinary metabolites could not be detected. Although rat liver slices generated all of the known metabolites of DEHP in vitro, no binding to DNA occurred. Administration of dual 3H/14C-labeled DEHP to rats yielded liver DNA whose 3H/14C ratio was inconsistent with the attachment of any reasonable multi-carbon fragment from the ethylhexyl portion to the DNA. The observation that roughly 100 times as high a percentage of the 14C administered was found in urea as in total DNA suggests that the 14C entered DNA through carbamyl phosphate, a precursor of both urea and pyrimidine bases. If this is the case, the association of C-1 from the ethylhexyl portion of DEHP with DNA may not involve alteration of the DNA or genetic damage.     

Stereoselective effects of chiral clofibric acid analogs on rat peroxisome proliferator-activated receptor α (rPPARα) activation and peroxisomal fatty acid β-oxidation

Enantiomers of a series of substituted analogs of 2-(4-chloronhenoxy)-acetic acid (CPAA) were synthesized and used to examine the influence of steric and structural parameters on peroxisome proliferation. The effects of these compounds were studied on the activation of the peroxisome proliferator-activated receptor α (PPARα) in CV-1 cells using an in vitro co-transfection assay. Selected sets of isomers were tested for their ability to increase peroxisomal fatty acyl-CoA oxidase (ACO) activity in H4IIEC3 (rat Reuber hepatoma) cells. Of the series of 2-substituted analogs studied, the isomers of the n-propyl and phenyl derivatives of CPAA showed a high degree of stereoselectivity [(S)-isomer ≫ (R)-isomer]. In general, the potency of the compound to activate the receptor increased with the size of the 2-alkyl substituent. Among the 4-chlorobenzyloxy- and 4-(4′-chlorophenyl)benzyloxy- analogs studied, 2-[4-(4′-chlorophenyl)-benzyloxy]-propanoic acid exhibited a high degree of stereoselectivity in both the biological systems studied [(R) ≫ (S)]. The congeners of 2-methyl substituted CPAA showed a reverse stereoselectivity [(R) > (S)] as compared to the other 2-substituted analogs [(S) > (R)]. Our results indicate that (1) both structural and steric characteristics of CPAA analogs play an important role in the activation of rPPARα and on stimulation of peroxisomal ACO activities, and (2) clofibric acid and analogs exert their peroxisome proliferative effects by interaction with a specific site on a protein. The enantiomers of the 2-n-propyl and the 2-phenyl CPAA analogs may be useful as mechanistic probes in elucidating the nature of this binding site. Chirality 9:37–47, 1997. © 1997 Wiley-Liss, Inc.     

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.     

Determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in human serum using liquid chromatography-tandem mass spectrometry

Concentrations of mono(2-ethylhexyl)phthalate (MEHP), and di(2-ethylhexyl)phthalate (DEHP), in serum of healthy volunteers were determined by high performance liquid chromatography (HPLC) with tandem mass spectrometry (LC/MS/MS). The serum was extracted with acetone, followed by hexane extraction under acidic conditions, and then applied to the LC/MS/MS. Recoveries of 20 ng/ml of MEHP and DEHP were 101+/-5.7 (n=6) and 102+/-6.5% (n=6), respectively. The limits of quantification (LOQ) of MEHP and DEHP in the method were 5.0 and 14.0 ng/ml, respectively. The concentration of MEHP in the serum was at or less than the LOQ. The concentration of DEHP in the serum was less than the LOQ. Contaminations of MEHP and DEHP from experimental reagents, apparatus and air during the procedure were less than the LOQ and were estimated to be <1.0 and 2.2+/-0.6 ng/ml, respectively. After subtraction of the contamination, the net concentrations of MEHP and DEHP in the serum were estimated at or <5 and <2 ng/ml, respectively. To decrease contamination by DEHP, the cleanup steps and the apparatus and solvent usage were minimized in the sample preparation procedures. The high selectivity of LC/MS/MS is the key for obtaining reliable experimental data from in the matrix-rich analytical samples and for maintaining a low level contamination of MEHP and DEHP in this experimental system. This method would be a useful tool for the detection of MEHP and DEHP in serum.