Evaluation of the direct toxicity of trioctyltrimellitate (TOTM), di(2-ethylhexyl) phthalate (DEHP) and their hydrolysis products on isolated rat hepatocytes.

Plasticizers are added to polyvinyl chloride (PVC) to confer flexibility to the polymer. Di(2-ethylhexyl) phthalate (DEHP) is the most commonly used of them. However, due to its non covalent bond to the PVC, DEHP tends to vaporize easily. A significant exposure has been recorded in dialyzed patients since medical tubings. Most animal species metabolize DEHP rapidly into monoethylhexyl phthalate (MEHP) and 2-ethylhexanol (2-EH). Because of the suspected toxicity of DEHP, an alternative plasticizer, trioctyltrimellitate (TOTM) has aroused increasing interest. The aim of this study was to determine on isolated rat hepatocytes in vitro, the direct hepatotoxic potential of both DEHP and TOTM and their hydrolytic products. To evaluate the possible toxic liver risk resulting from exposure to DEHP and TOTM, isolated rat hepatocytes were incubated with either DEHP, TOTM, MEHP or their common metabolite (2-EH) for 3 hours. Cell viability was periodically estimated thanks to trypan blue tests (15 - 180 min). The activity of lactate dehydrogenase (LDH) was also monitored (1h, 2h, 3h). The results obtained with trypan blue test and with direct LDH activity measurements, were satisfactorily correlated. Hepatocytes treated with both plasticizers and metabolites on the one hand, and the controls (untreated suspension) on the other hand, showed important differences as for cell viability. The acute toxicity on hepatocytes is mainly due to MEHP. Among DEHP, TOTM, MEHP, 2-EH and after intraperitoneal injection of those compounds, only DEHP and MEHP were able to induce a significant hydrogen peroxide (H2O2) production by the rat hepatocytes. These observations enable us to confirm the hypothesis according to which DEHP and MEHP cause an imbalance between the synthesis and the degradation of H2O2. Our results suggest a short-term in vitro cytotoxicity of MEHP. Even if trypan blue and LDH tests offered good results and were easily branded, further assays as well as MTT-tests should performed in order to confirm the cytotoxicity of the compounds tested.

Induction of propranolol metabolism in isolated rats hepatocytes treated by di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate (MEHP).

Blood lines of polyvinyl chloride (PVC) for hemodialysis usually contain di(2-ethylhexyl) phthalate (DEHP) as a plasticizer. Previous studies show that 1 mg/kg of this plasticizer can leach into the blood during one dialysis session. It is rapidly metabolized in the liver. Mono(2-ehtylhexyl) phthalate (MEHP), its main metabolite can be detected as well. After oral administration to rodents, both compounds caused a variety of adverse biological effects such as testicular atrophy, peroxisome proliferation and hepatic peroxisomal enzyme induction. Male wistar rats were treated intraperitoneally by DEHP and MEHP using twice the dose of that involved in human exposure during a dialysis session. Propranolol metabolism by hepatocytes was investigated after fresh isolation from treated and untreated rats by means of reverse phase HPLC. The choice of propranolol as a substrate was made because of its rather quick liver metabolisation. Phenobarbital was chosen in the study as a reference of enzymatic inducer to evaluate the inducing effect of DEHP and MEHP. Propranolol was metabolized by the hepatocytes of both treated and untreated rats. Hepatocytes isolated from rats treated by phenobarbital, MEHP and DEHP were shown to have a higher speed constant of metabolism indicating a rapid metabolism of propranolol. Under these conditions, in fact, propranolol metabolisation was found to be respectively 6, 2.7, 2 times faster than the propranolol metabolisation of untreated rats. The hypothesis that DEHP and MEHP are enzymatic inducers, particularly cytochrome P450 (CYP) inducers of the xenobiotics metabolism on the intact liver after IP administration has become been found to be valid. The results obtained in this study confirm the value of isolated hepatocytes as an in vivo drug metabolism predictive model.

Evaluation of childhood exposure to di(2-ethylhexyl) phthalate from perfusion kits during long-term parenteral nutrition.

Leachability of the plasticizer di(2-ethylhexyl) phthalate (DEHP) from administration sets into intravenous parenteral emulsions containing fat was investigated. DEHP is added to polyvinyl chloride (PVC) to impart flexibility. However, DEHP is a lipid-soluble suspected carcinogen that is hepatotoxic and teratogenic in rodents, and has been shown to leach from PVC products containing lipophilic mixtures. Consequently, total parenteral nutrition (TPN) mixtures containing fat emulsions should be stored in ethylvinyl acetate (EVA) bags rather than PVC packs. However, while TPN bags are made of EVA, they contain PVC-DEHP residues and the lines used between TPN bags and venous catheters are made of PVC-DEHP. The present study quantified the amount of DEHP leached from bags and tubing that could potentially contaminate patients during home TPN. Four types of emulsions containing fat were studied. Levels of DEHP in the bag and at the outlet tubing were measured by high-performance liquid chromatography (HPLC). This was measured during simulated TPN at different times after starting perfusion, 1 day after reconstitution of solutions in the bags, and 1 week later after storage at 4 degrees C. Detectable and stable amounts of DEHP were found to leach from bags (0.2 +/- 0.008 mg to 0.7 +/- 0.02 mg) and DEHP content increased in the outlet tubing (0.8 +/- 0.09 mg to 2 +/- 0.07 mg) during simulated infusions. The same phenomenon was observed after 1 week of storage at 4 degrees C. DEHP extraction by TPN depends on the lipid content of each TPN preparation and the flow rate. These results suggest that children treated with prolonged TPN are regularly exposed to significant amounts of DEHP.

Comparative study of the leachability of di(2-ethylhexyl) phthalate and tri(2-ethylhexyl) trimellitate from haemodialysis tubing.

The leachability of both Di(2-ethylhexyl) phthalate (DEHP) and Tri(2-ethylhexyl) trimellitate (TEHTM) or Trioctyl trimellitate (TOTM) from haemodialysis tubing was investigated in 20 patients with chronic renal failure undergoing maintenance haemodialysis. The blood tubing made of common polyvinyl chloride (PVC) plasticized with DEHP (group 1 patients) were replaced with tubing plasticized with TOTM-DEHP (group 2 patients). The patient blood obtained from the inlet and the outlet of the dialyzer was analyzed during a 4 h-dialysis session. Thus, the circulating concentrations of both DEHP and TOTM resulting from the release from dialyzer tubes were estimated using High-performance Liquid chromatograph (HPLC). With the common PVC-DEHP blood tubing, a DEHP quantity of 122.95+/-33.94 mg was extracted from tubing during a single dialysis session (ranging from 55 to 166.21 mg). During the same period, the total amounts of DEHP retained by the patients were 27.30+/-9.22 mg (ranging from 12.50 to 42.72 mg). As for blood tubing plasticized with TOTM-DEHP, 41.80+/-4.47 mg of DEHP and 75.11+/-25.72 mg of TOTM were extracted. During the same period, the amounts of DEHP and TOTM retained by the patients were 3.42+/-1.37 mg and 4.87+/-2.60 mg, respectively. The extraction rate both plasticizers was correlated with serum lipid content (cholesterol+triglyceride) (r(2)=0.75 for DEHP and r(2)=0.64 for TOTM). In the present investigation, less TOTM and DEHP were apparently released from haemodialysis tubing plasticized with TOTM-DEHP than DEHP released from haemodialysis tubing plasticized with DEHP only. TOTM seems to be a superior alternative to DEHP for use in medical devices because of its potential lower leachability. To recommend it as an alternative plasticizer, its possible toxicity towards human body should be investigated before it can be used routinely. However, patients undergoing haemodialysis using tubing plasticized with DEHP only are regularly exposed to non negligible amounts of DEHP. In view of several biological effects previously reported, it is time to reconsider the use of DEHP only as a plasticizer.

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 microl 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.

High-performance liquid chromatographic determination of naltrexone in plasma of hemodialysis patients.

A simple, sensitive, selective and reliable reversed-phase high-performance liquid chromatographic (HPLC) method with UV detection is described for the determination of naltrexone in plasma samples. Naltrexone and the internal standard, naloxone, were isolated from plasma either with a liquid-liquid extraction method using ethyl acetate or with a solid-phase extraction method using Sep-Pack C18 cartridge before chromatography. The extracts were dried under a stream of nitrogen and the samples were reconstituted in the mobile phase, then 20 microL were injected on a Waters Symmetry C18 column (5 microm particle size, 4.6 x 150 mm). The mobile phase consisted of 0.06% triethylamine (pH 2.8)-acetonitrile (92:8, v/v) pumped at 1 mL/min. The peak-area ratio versus plasma concentration was linear over the range of 10-500 ng/mL and the detection limit was less than 8 ng/mL. Quantification was by ultra-violet detection at 204 nm. The present method was applied to the determination of the plasma concentration of naltrexone in dialyzed patients. Patients (n = 8) with severe generalized pruritus received 50 mg of naltrexone orally per day for 2 weeks. The variability in the therapeutic response in treated patients required plasma concentration investigations of this opioid antagonist.

Exposure of hemodialysis patients to di-2-ethylhexyl phthalate.

The migration of di-2-ethylhexyl phthalate (DEHP) from dialyzers was studied in 21 patients with chronic renal failure undergoing maintenance hemodialysis. The circulating concentrations of DEHP were measured by high performance liquid chromatography in blood of patients obtained from the inlet and the outlet of the dialyzer during a 4-h dialysis session. During treatment of renal failure using plasticized tubing, the plasma level of DEHP increased. On average, an estimated 75.2 mg of DEHP was extracted from the dialyzer during a single dialysis session, with a range of 44.3-197. 1 mg. On the other hand, the total amount of DEHP retained by the patient during the dialysis session was evaluated by the difference between the AUCout and the AUCin and ranged from 3.6 to 59.6 mg. The rate of extraction of DEHP from the dialyzer was correlated (r=0.705, P<0.05) with serum lipid content (cholesterol and triglyceride).So, we confirmed that patients on hemodialysis are always regularly exposed to considerable amounts of DEHP. However, several metabolic effects have been reported in various animal species following treatment with DEHP, such as changes in lipid metabolism and in hepatic microsomal drug-metabolizing enzyme activities. DEHP is now a well-known hepatic peroxisomal proliferator in rodents and an inducer of many peroxisomal and non-peroxisomal enzymes. So, lipid metabolism modifications and hepatic changes observed in hemodialysis patients could be explained from chronic exposition to DEHP. In the coming years, it seems necessary to reconsider the use of DEHP as a plasticizer in medical devices. Highly unacceptable amounts of DEHP leached during the dialysis session could be easily avoided by careful selection of hemodialysis tubing.