In vitro metabolism of di(2-ethylhexyl) phthalate (DEHP) by various tissues and cytochrome P450s of human and rat

In vitro metabolism of DEHP by subcellular fractions of human brain, intestine, kidney, liver, lung, skin, testis, rat liver and recombinant CYP isoforms of human and rat was investigated using LC–MS/MS. DEHP was rapidly hydrolyzed to mono(2-ethylhexyl) phthalate (MEHP) in 12 microsomal/cytosolic fractions of selected 7 human organs and rat liver but not in microsomal fractions of human brain and human female skin. MEHP was metabolized to CYP-mediated oxidative and dealkylated metabolites in human and rat liver and at a lower rate in human intestine. Measurable amounts of mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-Oxo MEHP), mono(2-ethyl-5-carboxypentyl) phthalate (5-carboxy MEPP), mono(2-carboxymethyl-hexyl) phthalate (2-carboxy MMHP) and phthalic acid (PA) were formed by human liver fractions. Human CYP2C9¹1, CYP2C19 and rat CYP2C6 were the major CYP isoforms producing 5-OH MEHP and 5-Oxo MEHP metabolites; however, only human CYP2C9¹1 and 2C9¹2 produced 5-carboxy MEPP from MEHP. Additionally, human CYP3A4 and rat CYP3A2 were the primary enzymes for PA production via heteroatom dealkylation of MEHP. Percent total normalized rates (%TNR) by CYP2C9¹1 in human liver microsomes (HLM) were 94%, 98% and 100%, respectively, for 5-OH MEHP, 5-Oxo MEHP, 5-carboxy MEPP, and 76% for PA production by CYP3A4.     

New metabolites of di(2-ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP

The metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans was studied after three doses of 0.35 mg (4.7 g/kg), 2.15 mg (28.7 g/kg) and 48.5 mg (650 g/kg) of D4-ring-labelled DEHP were administered orally to a male volunteer. Two new metabolites, mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) were monitored for 44 h in urine and for 8 h in serum for the high-dose case, in addition to the three metabolites previously analysed: mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP). For the medium- and low-dose cases, 24 h urine samples were analysed. Up to 12 h after the dose, 5OH-MEHP was the major urinary metabolite, after 12 h it was 5cx-MEPP, and after 24 h it was 2cx-MMHP. The elimination half-lives of 5cx-MEHP and 2cx-MMHP were between 15 and 24 h. After 24 h 67.0% (range: 65.8–70.5%) of the DEHP dose was excreted in urine, comprising 5OH-MEHP (23.3%), 5cx-MEPP (18.5%), 5oxo-MEHP (15.0%), MEHP (5.9%) and 2cx-MMHP (4.2%). An additional 3.8% of the DEHP dose was excreted on the second day, comprising 2cx-MMHP (1.6%), 5cx-MEPP (1.2%), 5OH-MEHP (0.6%) and 5oxo-MEHP (0.4%). In total about 75% of the administered DEHP dose was excreted in urine after two days. Therefore, in contrast to previous studies, most of the orally administered DEHP is systemically absorbed and excreted in urine. No dose dependency in metabolism and excretion was observed. The secondary metabolites of DEHP are superior biomonitoring markers compared to any other parameters, such as MEHP in urine or blood. 5OH-MEHP and 5oxo-MEHP in urine reflect short-term and 5cx-MEHP and 2cx-MMHP long-term exposure. All secondary metabolites are unsusceptible to contamination. Furthermore, there are strong hints that the secondary oxidised DEHP metabolites—not DEHP or MEHP—are the ultimate developmental toxicants.     

The metabolism of di(2-ethylhexyl) phthalate (DEHP) and mono-(2-ethylhexyl) phthalate (MEHP) in rats: in vivo and in vitro dose and time dependency of metabolism

This study investigated the in vivo metabolism of di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP) in rats after multiple dosing, the metabolism of MEHP in primary rat hepatocyte cultures for periods of up to 3 days, and the biotransformation of some major metabolites of MEHP. Rats were orally administered [14C]DEHP or [14C]MEHP at doses of 50 and 500 mg/kg body wt for three consecutive days. Urine was collected at 24-hr intervals, and metabolite profiles were determined. After a single dose of either compound, urinary metabolite profiles were similar to those previously reported. However, after multiple administration of both DEHP and MEHP at 500 mg/kg, increases in omega-/beta-oxidation products [metabolites I and V, mono(3-carboxy-2-ethylpropyl) phthalate and mono(5-carboxy-2-ethylpentyl) phthalate, respectively] and decreases in omega - 1-oxidation products [metabolites VI and IX, mono(2-ethyl-5-oxohexyl) phthalate and mono(2-ethyl-5-hydroxyhexyl) phthalate, respectively] were seen. At the low dose of 50 mg/kg little or no alteration in urinary metabolite profiles was observed. At 500 mg/kg of MEHP a 4-fold stimulation of CN- -insensitive palmitoyl-CoA oxidation (a peroxisomal beta-oxidation marker) was seen after three consecutive daily doses. At the low dose of 50 mg/kg only a 1.8-fold increase was noted. Similar observations were made with rat hepatocyte cultures. MEHP at concentrations of 50 and 500 microM was extensively metabolized in the rat hepatocyte cultures. Similar metabolic profiles to those seen after in vivo administration of MEHP were observed. At the high (500 microM) concentration of MEHP, changes in the relative proportions of omega- and omega- 1-oxidized metabolites were seen. Over the 3-day experimental period, omega-/beta-oxidation products increased in a time-dependent manner at the expense of omega - 1-oxidation products. At a concentration of 500 microM MEHP, a 12-fold increase of CN- -insensitive palmitoyl CoA oxidation (a peroxisomal beta-oxidation marker) was observed. At the low concentration of MEHP (50 microM) only a 3-fold increase in CN- -insensitive palmitoyl-CoA oxidation was noted and little alteration in the metabolite profile of MEHP was observed with time. Biotransformation studies of the metabolites of MEHP confirmed the postulated metabolic pathways. Metabolites I and VI appeared to be endpoints of metabolism, while metabolite V was converted to metabolite I, and metabolite IX to metabolite VI. It was also possible to reduce the transformation of metabolite X [mono(2-ethyl-6-hydroxyhexyl) phthalate] to metabolite V.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of phthalate ester derivatives including oxidized metabolites on coactivator recruiting by PPARalpha and PPARgamma

Phthalate esters (PEs), a group of environmental chemicals, affect biological systems via endocrine and lipid metabolism modulations. These effects are believed to be mediated in part by peroxisome proliferator-activated receptors (PPARs). Evaluations of PE activities as ligands toward PPARs have been investigated in many studies on their primary metabolites, monoesters. However, the activities of various other metabolites, including oxidized derivatives, remain to be determined. Here, we have evaluated the PPAR ligand activities of these PE derivatives by in vitro coactivator recruiting assay. Mono(2-ethyl-5-hydroxyhexyl)phthalate, the most abundant metabolite of di-(2-ethylhexyl)phthalate (DEHP), was less active than mono(2-ethylhexyl)phthalate (MEHP) as a PPAR ligand. Other derivatives oxidized at the alkyl group and benzene ring of DEHP, MEHP, dibutyl phthalate and its monoester were also investigated and some affected PPAR activities. Unexpectedly, MEHP as well as its further oxidized metabolite did not show clear activity for PPARalpha, although MEHP is believed to interact with PPARalpha. This might imply indirect PPAR-mediated mechanisms that lead to observed biological effects such as peroxisome proliferation.     

Di(2-ethylhexyl)phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuterium-labelled DEHP

Human metabolism of di(2-ethylhexyl)phthalate (DEHP) was studied after a single oral dose of 48.1 mg to a male volunteer. To avoid interference by background exposure the D4-ring-labelled DEHP analogue was dosed. Excretion of three metabolites, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP), was monitored for 44 h in urine and for 8 h in serum. Peak concentrations of all metabolites were found in serum after 2 h and in urine after 2 h (MEHP) and after 4 h (5OH-MEHP and 5oxo-MEHP). While the major metabolite in serum was MEHP, the major metabolite in urine was 5OH-MEHP, followed by 5oxo-MEHP and MEHP. Excretion in urine followed a multi-phase elimination model. After an absorption and distribution phase of 4 to 8 h, half-life times of excretion in the first elimination phase were approximately 2 h with slightly higher half-life times for 5OH- and 5oxo-MEHP. Half-life times in the second phase—beginning 14 to 18 h post dose—were 5 h for MEHP and 10 h for 5OH-MEHP and 5oxo-MEHP. In the time window 36 to 44 h, no decrease in excreted concentrations of 5OH- and 5oxo-MEHP was observed. In the first elimination phase (8 to 14 h post dose), mean excretion ratios of MEHP to 5oxo-MEHP and MEHP to 5OH-MEHP were 1 to 1.8 and 1 to 3.1. In the second elimination phase up to 24 h post dose mean excretion ratios of MEHP to 5oxo-MEHP to 5OH-MEHP were 1 to 5.0 to 9.3. The excretion ratio of 5OH-MEHP to 5oxo-MEHP remained constant through time at 1.7 in the mean. After 44 h, 47% of the DEHP dose was excreted in urine, comprising MEHP (7.3%), 5OH-MEHP (24.7%) and 5oxo-MEHP (14.9%).     

Identification of the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate

A primary rat hepatocyte culture system was utilized to determine the proximate peroxisome proliferator(s) derived from di(2-ethylhexyl) phthalate (DEHP). DEHP was administered to rats and the urinary metabolites were identified and isolated. The major metabolites were those resulting from initial omega- or omega - 1-carbon oxidation of the mono(2-ethylhexyl) phthalate (MEHP) moiety. These metabolites, together with MEHP and 2-ethylhexanol, were added to primary rat hepatocyte cultures and the effect on peroxisomal enzyme activity was determined. The omega-carbon oxidation products [mono(3-carboxy-2-ethylpropyl) phthalate (I) and mono(5-carboxy-2-ethylpentyl) phthalate (V)] and 2-ethylhexanol produced little or no effect on CN- -insensitive palmitoyl-CoA oxidation (a peroxisomal marker). MEHP and the omega - 1-carbon oxidation products [mono-(2-ethyl-5-oxohexyl) phthalate (VI) and mono(2-ethyl-5-hydroxyhexyl) phthalate (IX)] produced a large (7- to 11-fold) induction of peroxisomal enzyme activity. Similar structure-activity relationships were observed for the induction of cytochrome P-450-mediated lauric acid hydroxylase and increase in cellular coenzyme A content. This identification of the proximate proliferators will aid in the elucidation of the mechanism by which DEHP causes proliferation of peroxisomes in the rodent liver. Oral administration of MEHP (150 or 250 mg/kg) to male guinea pigs did not produce hepatic peroxisome proliferation. Addition of MEHP (0 to 0.5 mM) or one of the "active" proliferators in the rat (metabolite IX, 0 to 0.5 mM) to primary guinea pig hepatocyte cultures also failed to produce an induction of peroxisomal beta-oxidation. Possible reasons for this species difference are discussed.     

Metabolite profiling and identification in human urine after single oral administration of DEHP

Di(2-ethylhexyl)phthalate (DEHP) is known to be a reproductive toxicant in male rodents, and its primary or secondary metabolites seem to be causative agents. To identify and quantify urinary metabolites of DEHP in humans, urine samples collected from healthy male and female volunteers following oral administration of deuterium labeled DEHP (d(4)-DEHP) at 3mg/person were analyzed by a high performance liquid chromatograph/mass spectrometer (LC-MS), and the excretion behavior of orally taken DEHP was evaluated. Mono(2-ethylhexyl)phthalate (MEHP), OH-MEHP, oxo-MEHP, COOH-MEHP, and their glucuronides were identified as metabolites in the male and female urine. The ratios of the conjugate forms in the total urinary metabolites were remarkably high from immediately after administration (0 to 4-hr post-dose), which were approximately 69% to 86% (male) and 80% to 89% (female) until 36-hr post-dose. It was suggested that DEHP taken orally was promptly converted to MEHP and its oxidative metabolites such as OH-MEHP, oxo-MEHP, and COOH-MEHP, and most of these metabolites received the conjugation reaction and were excreted as glucuronides. Remarkable differences from rodents, in which most of these metabolites were excreted as free forms, were demonstrated.     

Di(2-ethylhexyl) phthalate metabolites as markers for blood transfusion in doping control: intra-individual variability of urinary concentrations

To indicate homologous or autologous blood transfusion in sports drug testing, quantification of increased urinary concentrations of di(2-ethylhexyl) phthalate (DEHP) metabolites presents a promising approach; however, the possible intra-individual variation of the metabolite concentrations over time has not been well characterized. The aim of this study was to explore the intra-individual variability of urinary DEHP metabolites among seven volunteers without special occupational exposure to DEHP during one week (n = 253) in order to investigate the possibility of increased urinary concentrations of the metabolites caused by, for example, residential, dietary, or environmental exposure. Quantification of three DEHP metabolites--mono(2-ethylhexyl) phthalate, mono(2-ethyl-5-oxohexyl) phthalate, and mono(2-ethyl-5-hydroxyhexyl) phthalate--was accomplished after enzymatic hydrolysis of urinary glucuronide conjugates and direct injection using isotope-dilution liquid chromatography-tandem mass spectrometry. Although urinary concentrations of DEHP metabolites showed considerable intra-individual variation, no increased values were observed comparable to the concentrations measured in urine specimens collected after blood transfusion.     

Effects of di-(2-ethylhexyl) phthalate and five of its metabolites on rat testis in vivo and in in vitro

In an attempt to establish which compound or compounds are responsible for the testicular damage observed after administration of di-(2-ethylhexyl) phthalate (DEHP) in rats, the effects of the parent compound and five of its major metabolites (mono-(2-ethylhexyl) phthalate (MEHP), 2-ethylhexanol (2-EH), mono-(5-carboxy-2-ethylpentyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate and mono-(2-ethyl-5-hydroxyhexyl) phthalate) were investigated in vivo and in vitro. The concentrations of MEHP and the three MEHP-derived metabolites in plasma were determined after single and multiple oral doses of DEHP. The plasma concentrations and areas under the plasma concentration-time curves (AUC's) of each of the MEHP-derived metabolites were considerably lower than those of MEHP both after single and after repeated administration of 2.7 mmol of DEHP/kg body weight. The mean elimination half-life of MEHP was significantly shorter in animals given repetitive doses than in those given a single dose, but there was no statistically significant difference between the mean AUC values. No testicular damage was observed in young rats given oral doses of 2.7 mmol of DEHP or 2-EH/kg body weight daily for five days. In animals which received corresponding doses of MEHP the number of degenerated spermatocytes and spermatids was increased, whereas no such effects were found in animals given the MEHP-derived metabolites. MEHP was also the only compound that enhanced germ cell detachment from mixed primary cultures of Sertoli and germ cells.     

Human biofluid concentrations of mono(2-ethylhexyl)phthalate extrapolated from pharmacokinetics in chimeric mice with humanized liver administered with di(2-ethylhexyl)phthalate and physiologically based pharmacokinetic modeling

Di(2-ethylhexyl)phthalate (DEHP) is a reproductive toxicant in male rodents. The aim of the current study was to extrapolate the pharmacokinetics and toxicokinetics of mono(2-ethylhexyl)phthalate (MEHP, a primary metabolite of DEHP) in humans by using data from oral administration of DEHP to chimeric mice transplanted with human hepatocytes. MEHP and its glucuronide were detected in plasma from control mice and chimeric mice after single oral doses of 250 mg DEHP/kg body weight. Biphasic plasma concentration–time curves of MEHP and its glucuronide were seen only in control mice. MEHP and its glucuronide were extensively excreted in urine within 24 h in mice with humanized liver. In contrast, fecal excretion levels of MEHP glucuronide were high in control mice compared with those with humanized liver. Adjusted animal biomonitoring equivalents from chimeric mice studies were scaled to human biomonitoring equivalents using known species allometric scaling factors and in vitro metabolic clearance data with a simple physiologically based pharmacokinetic (PBPK) model. Estimated urine MEHP concentrations in humans were consistent with reported concentrations. This research illustrates how chimeric mice transplanted with human hepatocytes in combination with a simple PBPK model can assist evaluations of pharmacokinetics or toxicokinetics of the primary or secondary metabolites of DEHP.     

Kinetics of di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in blood and of DEHP metabolites in urine of male volunteers after single ingestion of ring-deuterated DEHP

The plasticizer di(2-ethylhexyl) phthalate (DEHP) is suspected to induce antiandrogenic effects in men via its metabolite mono(2-ethylhexyl) phthalate (MEHP). However, there is only little information on the kinetic behavior of DEHP and its metabolites in humans. The toxikokinetics of DEHP was investigated in four male volunteers (28-61 y) who ingested a single dose (645 ± 20 μg/kg body weight) of ring-deuterated DEHP (DEHP-D₄). Concentrations of DEHP-D₄, of free ring-deuterated MEHP (MEHP-D₄), and the sum of free and glucuronidated MEHP-D₄ were measured in blood for up to 24 h; amounts of the monoesters MEHP-D₄, ring-deuterated mono(2-ethyl-5-hydroxyhexyl) phthalate and ring-deuterated mono(2-ethyl-5-oxohexyl) phthalate were determined in urine for up to 46 h after ingestion. The bioavailability of DEHP-D₄ was surprisingly high with an area under the concentration-time curve until 24 h (AUC) amounting to 50% of that of free MEHP-D₄. The AUC of free MEHP-D₄ normalized to DEHP-D₄ dose and body weight (AUC/D) was 2.1 and 8.1 times, that of DEHP-D₄ even 50 and 100 times higher than the corresponding AUC/D values obtained earlier in rat and marmoset, respectively. Time courses of the compounds in blood and urine of the volunteers oscillated widely. Terminal elimination half-lives were short (4.3-6.6 h). Total amounts of metabolites in 22-h urine are correlated linearly with the AUC of free MEHP-D₄ in blood, the parameter regarded as relevant for risk assessment.     

Toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in rats

The toxicokinetic relationship between di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP, was investigated in Sprague-Dawley rats orally treated with a single dose of 14C-DEHP. Urinary excretion of total 14C-DEHP and of its metabolites was followed by liquid scintillation counting (LSC). Concentrations of DEHP and MEHP were determined 6, 24, and 48 h after treatment in rat serum and 6, 12, 24, and 48 h after treatment in urine by high-performance liquid chromatography (HPLC). After 24 h, peak concentrations of MEHP in both urine and serum were observed in animals treated with 40, 200, or 1000 mg DEHP/kg. HPLC showed that general toxicokinetic parameters, such as Tmax (h), Cmax (microg/ml), Ke (1/h), and AUC (microg-h/ml/) were greater for MEHP than DEHP in both urine and serum. In contrast, the half-lives (t1/2 [h]) of DEHP were greater than those of MEHP. The AUC ratios between DEHP and MEHP were relatively smaller in serum than in urine, suggesting the important role of urinary DEHP data for exposure assessment of DEHP. The toxicokinetic relationship between DEHP and MEHP in rats suggests that DEHP exposure assessment should be based on DEHP and MEHP in urine and serum for risk assessment applications.     

Phthalate esters enhance quinolinate production by inhibiting alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD), a key enzyme of the tryptophan pathway.

Tryptophan is metabolized to alpha-amino-beta-carboxymuconate-epsilon-semialdehyde (ACMS) via 3-hydroxyanthranilate (3-HA). ACMS decarboxylase (ACMSD) directs ACMS to acetyl CoA; otherwise ACMS is non-enzymatically converted to quinolinate (QA), leading to the formation of NAD and its degradation products. Thus, ACMSD is a critical enzyme for tryptophan metabolism. Phthalate esters have been suspected of being environmental endocrine disrupters. Because of the structural similarity of phthalate esters with tryptophan metabolites, we examined the effects of phthalate esters on tryptophan metabolism. Phthalate esters containing diets were orally given to rats and the urinary excreted tryptophan metabolites were quantified. Of the phthalate esters with different side chains tested, di(2-ethylhexyl)phthalate (DEHP) and its metabolite, mono(2-ethylhexyl)phthalate (MEHP), most strongly enhanced the production of QA and degradation products of nicotinamide, while 3-HA was unchanged. This pattern of metabolic change led us to assume that these esters lowered ACMSD protein or its activity. Although DEHP could not be tested because of its low solubility, MEHP reversibly inhibited ACMSD from rat liver and mouse kidney, and also the recombinant human enzyme. Correlation between inhibition of ACMSD by phthalate esters with different side chains and urinary excretion of QA supports the notion that phthalate esters perturb tryptophan metabolism by inhibiting ACMSD. Quinolinate is a potential endogenous toxin and has been implicated in the pathogenesis of various disorders. Although toxicity of phthalate esters through accumulation of QA remains to be investigated, they may be detrimental by acting as metabolic disrupters when intake of a tryptophan-rich diet and exposure to phthalate esters occur coincidentally.     

Assessment of the teratogenicity of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in mice

Di(2-ethylhexyl)phthalate (DEHP) and mono(2-ethylhexyl)phthalate (MEHP) were administered PO or IP to pregnant ICR mice at varying doses on days 7, 8, and 9 of gestation. In groups given DEHP orally, resorptions and malformed fetuses increased significantly at 1,000 mg/kg. Fetal weights were also significantly suppressed. Anterior neural tube defects (anencephaly and exencephaly) were the malformations most commonly produced. No teratogenic effects were revealed by IP doses of DEHP and PO or IP doses of MEHP, although high doses were abortifacient and lethal to pregnant females. Thus DEHP is highly embryotoxic and teratogenic in mice when given PO but not IP. The difference in metabolism, disposition, or excretion by the route of administration may be responsible for the difference in DEHP teratogenicity. Although MEHP is a principal metabolite of DEHP and is several times more toxic than DEHP to adult mice, it seems that MEHP and its metabolites are not teratogenic in ICR mice.     

Urinary and amniotic fluid levels of phthalate monoesters in rats after the oral administration of di(2-ethylhexyl) phthalate and di-n-butyl phthalate

Two studies were designed to examine amniotic fluid and maternal urine concentrations of the di(2-ethylhexyl) phthalate (DEHP) metabolite mono(2-ethylhexyl) phthalate (MEHP) and the di-n-butyl phthalate (DBP) metabolite monobutyl phthalate (MBP) after administration of DEHP and DBP during pregnancy. In the first study, pregnant Sprague-Dawley rats were administered 0, 11, 33, 100, or 300 mg DEHP/kg/day by oral gavage starting on gestational day (GD) 7. In the second study, DBP was administered by oral gavage to pregnant Sprague-Dawley rats at doses of 0, 100, or 250 mg/kg/day starting on GD 13. Maternal urine and amniotic fluid were collected and analyzed to determine the free and glucuronidated levels of MEHP and MBP. In urine, MEHP and MBP were mostly glucuronidated. By contrast, free MEHP and free MBP predominated in amniotic fluid. Statistically significant correlations were found between maternal DEHP dose and total maternal urinary MEHP (p=0.0117), and between maternal DEHP dose and total amniotic fluid MEHP levels (p=0.0021). Total maternal urinary MEHP and total amniotic fluid MEHP levels were correlated (Pearson correlation coefficient=0.968). Statistically significant differences were found in amniotic MBP levels between animals within the same DBP dose treatment group (p<0.0001) and between animals in different dose treatment groups (p<0.0001). Amniotic fluid MBP levels increased with increasing DBP doses, and high variability in maternal urinary levels of MBP between rats was observed. Although no firm conclusions could be drawn from the urinary MBP data, the MEHP results suggest that maternal urinary MEHP levels may be useful surrogate markers for fetal exposure to DEHP.     

High-throughput determination of mono- and di(2-ethylhexyl)phthalate migration from PVC tubing to drugs using liquid chromatography-tandem mass spectrometry

The risk assessment of di(2-ethylhexyl) phthalate (DEHP) that migrated from polyvinyl chloride (PVC) medical devices is an important issue for hospitalized patients. Many studies have been conducted to determine the level of DEHP migration. A recent report has indicated that DEHP in blood bags was hydrolyzed by esterase to mono(2-ethylhexyl) phthalate (MEHP). Therefore, a method for the simultaneous determination of DEHP and MEHP was developed. The migration of DEHP and MEHP from PVC tubing to drugs was examined. Although we detected MEHP in the drugs, we found no enzymatic activity involved in the migration process. Some reports have indicated that hydrolysis may have occurred during sterilization by autoclaving. However, we did not perform any heat treatment. It is speculated that the MEHP migrated directly from the PVC tubing. The simultaneous determination of DEHP and MEHP is required for risk assessment, as MEHP may be even more toxic than the parent compound.     

Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium

Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates’ testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 μM selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.     

Non-linearities in the pharmacokinetics of di-(2-ethylhexyl) phthalate and metabolites in male rats

The disposition of the plasticizer di-(2-ethylhexyl) phthalate (DEHP) and four of its major metabolites was studied in male rats given single infusions of a DEHP emulsion in doses of 5, 50 or 500 mg DEHP/kg body weight. Plasma concentrations of DEHP and metabolites were followed for 24 h after the start of the infusion. The kinetics of the primary metabolite mono-(2-ethylhexyl) phthalate (MEHP) was studied separately.The concentrations of DEHP in plasma were at all times considerably higher than those of MEHP, and the concentrations of MEHP were much higher than those of the other investigated metabolites. In animals given 500 mg DEHP/kg, the areas under the plasma concentration-time curves (AUCs) of the other investigated metabolites were at most 15% of that of MEHP. Parallel decreases in the plasma concentrations of DEHP, MEHP and the and (-1) oxidized metabolites indicated that the elimination of DEHP was the rate-limiting step in the disposition of the metabolites. This was partly supported by the observation that the clearance of MEHP was higher than that of DEHP. Nonlinear increases in the AUCs of DEHP and MEHP indicated saturation in the formation as well as the elimination of the potentially toxic metabolite MEHP.     

No background concentrations of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in blood of rats

We measured the background levels of di(2-ethylhexyl) phthalate (DEHP) and its hydrolytic metabolite mono(2-ethylhexyl) phthalate (MEHP) in blood from naive female Sprague-Dawley rats and in de-ionized charcoal-purified water using an analytical procedure that is based on sample treatment with acetonitrile, n-hexane extraction and analysis by gas chromatography. In blood, blank values of 91.3 +/- 34.7 micrograms DEHP/l (n = 31) and 30.1 +/- 13.1 micrograms MEHP/l (n = 20) were obtained, and in water, values of 91.6 +/- 44.2 micrograms DEHP/l (n = 26) and 26.7 +/- 10.4 micrograms MEHP/l (n = 15) were found. Since there is no difference between the background valves obtained from blood of naive rats and water, we conclude that DEHP and MEHP result from contamination during the analytical procedure.     

Urinary biomarkers of di-isononyl phthalate in rats

Commercial di-isononyl phthalate (DiNP) is a mixture of various branched-chain dialkyl phthalates mainly containing nine-carbon alkyl isomers. At high doses in rodents, DiNP is a carcinogen, and a developmental toxicant. After exposure, the diester isomers are de-esterified to form hydrolytic monoesters, monoisononyl phthalates (MiNP), which subsequently metabolize to form oxidative metabolites. These metabolites can be excreted in urine or feces. The urinary excretion of DiNP metabolites was monitored in adult female Sprague-Dawley rats after oral administration of a single dose (300 mg/kg) of commercial DiNP. The metabolites were extracted from urine, resolved with high performance liquid chromatography, analyzed by mass spectrometry, and tentatively identified based on their chromatographic separation and mass spectrometric fragmentation pattern. Because DiNP is an isomeric mixture, its metabolites were also isomeric mixtures that eluted from the HPLC column with close retention times. Mono(carboxy-isooctyl)phthalate (MCiOP) was identified as the major metabolite of DiNP; in addition, mono(hydroxy-isononyl)phthalate (MHiNP) and mono(oxo-isononyl)phthalate (MOiNP) were present. Furthermore, metabolites of di-isooctyl phthalate (DiOP) and di-isodecyl phthalate (DiDP) were also detected. Excretion toxicokinetics of the DiNP metabolites in urine followed a biphasic pattern with initial rapid decay in concentration. Despite potential differences in the metabolism of DiNP among species, MCiOP, MHiNP and MOiNP were detected in humans with no known exposure to DiNP at levels significantly higher than MiNP suggesting that these oxidative metabolites may be better urinary biomarkers of human exposure to DiNP than is MiNP.