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ANESTHETIC PHARMACOLOGY

The Plasticizer Di(2-ethylhexyl) Phthalate Modulates -Aminobutyric Acid Type A and Glycine Receptor Function

Liya Yang, PhD^*, Pavle S. Milutinovic, MS^*, Robert J. Brosnan, DVM, PhD, Edmond I. Eger, II, MD^*, and James M. Sonner, MD^*

From the *Department of Anesthesia and Perioperative Care, University of California, San Francisco, California; and The Department of Surgical and Radiological Sciences, University of California, School of Veterinary Medicine, Davis, California.

Address correspondence to Sonner, MD, Department of Anesthesia, Room S-455i, Box 0464, 513 Parnassus Ave., University of California, San Francisco, CA 94143-0464. Address e-mail to sonnerj{at}anesthesia.ucsf.edu.

	Abstract

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INTRODUCTION: Intravenous (IV) fluid bags made of polyvinyl chloride (PVC) often contain the plasticizer di(2-ethylhexyl) phthalate (DEHP) to make the PVC flexible. Phthalate esters have been reported to inhibit neuronal nicotinic acetylcholine receptors, which are sensitive to many inhaled anesthetics. This raises the possibility that DEHP might modulate the function of other cys-loop receptors, such as -amino butyric acid type A (GABA_A) and glycine receptors, and that DEHP-plasticized PVC might interfere with electrophysiologic studies of anesthetic mechanisms on those receptors.

METHODS: ₁ß₂ GABA_A and ₁ glycine receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. We then measured the effect of buffers from IV bags containing DEHP-plasticized PVC, and of buffers saturated with DEHP, on agonist-induced currents.

RESULTS: Agonist-induced currents from glycine receptors were enhanced by buffers from IV bags containing DEHP-plasticized PVC by 291.9% ± 84.5% (mean ± se) and from saturated solutions of DEHP by 70.8% ± 16.7%. Agonist-induced currents from ₁ß₂ GABA_A receptors were inhibited by buffers from IV bags containing DEHP-plasticized PVC by 19.3% ± 3.2% and by 31.7% ± 7.0% from buffers saturated with DEHP.

CONCLUSIONS: The plasticizer DEHP modulates the function of both GABA_A and glycine receptors. DEHP contamination can confound the results of electrophysiologic studies of anesthetic mechanisms on these receptors if DEHP-plasticized PVC is present in the experimental apparatus.

	Introduction

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Plasticizers are used to render plastics containing polyvinyl chloride (PVC) flexible. Di(2-ethylhexyl) phthalate (DEHP), the most common of these plasticizers, comprises as much as 30%–40% of the plastics it is used in (1). IV fluid bags, tubing, and catheters are often composed of DEHP-plasticized PVC. Because DEHP is not covalently bound to the PVC polymer, it can leach out of these products. This has raised concerns about toxicity, primarily the estrogen-like effects of DEHP on reproductive health (2,3) [although the relevance of some animal studies to human exposures has been questioned (4)], and the possible carcinogenicity of DEHP (5,6).

Phthalate esters inhibit the cytosolic free calcium concentration induced by neuronal nicotinic acetylcholine receptor (nAChR) agonists (7). This was of interest to us because nAChRs are sensitive to volatile anesthetics (8). IV bags and tubing composed of DEHP are often used to contain buffers and drugs applied in studies of the physiology of anesthetic-sensitive ion channels because these bags and tubing provide a gas-tight containment system that can drain under gravity. The use of such bags and tubing could confound studies of anesthetic mechanisms using nAChR and related ion channels, owing to the presence of phthalates in the perfusate. We accordingly studied the response of two anesthetic-sensitive ion channels [-amino butyric acid type A (GABA_A) and glycine receptors] from the same cys-loop superfamily of receptors as the nAChR to perfusates in IV bags containing DEHP plasticized PVC and to perfusates saturated with DEHP.

	METHODS

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GABA_A and glycine receptor clones were a gift of Professor R. A. Harris (University of Texas, Austin). With permission of the Institutional Animal Care and Use Committee of University of California, San Francisco, oocytes were harvested from female Xenopus laevis frogs. Stage V and VI oocytes were defolliculated by gentle rotation in 0.5 U/mL collagenase type 1 (Worthingtom Biochemical Corporation, Lakewood, NJ) for 1 h at room temperature. GABA_A receptors comprised of human ₁ and rat ß₂ subunits, and homomeric human ₁ glycine receptors were expressed by microinjection of Xenopus laevis oocytes with 0.25–1 ng cDNA subcloned into PCIS2 and PBK-CMV vectors, respectively. Oocytes were maintained at 18°C in modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 20 mM HEPES, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.41 mM CaCl₂, with 5 mM sodium pyruvate, 50 µg/mL gentamycin, 50 U/mL penicillin, and 50 µg/mL streptomycin, filtered and adjusted to pH 7.4).

Two to 3 days after injection, two-electrode voltage clamping was performed on oocytes at room temperature (GeneClamp 500B; Molecular Devices, Axon Instruments, Foster City, CA). Two-electrode voltage clamp experiments were performed using frog Ringer's (FR) solution as perfusate (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂,10 mM HEPES, filtered and adjusted to pH 7.4). An automated perfusion system was used to deliver solutions (Automate ValveBank perfusion system, San Francisco, CA). Teflon tubing connected the reservoirs containing buffer with the automated perfusion system, and the automated perfusion system to the oocyte recording chamber. Recordings were obtained in a 250-µL recording chamber at flow rates of 2–3 mL/min. Signals were filtered using a four-pole low-pass Bessel filter set at a 50–100 Hz cutoff before sampling at 100–1000 Hz.

Oocytes were voltage clamped at –80 mV. Stable inward currents in response to agonist were verified by application of agonist for 20 s before and after test compounds were studied. Two experiments were performed on each receptor. In the first, the effect of compounds leaching from IV bags containing DEHP-plasticized PVC bags was tested by placing FR plus agonist (either 30 µM GABA or 75 µM glycine) in an empty 250-mL capacity IntraVia bag, which contains DEHP-plasticized PVC (Baxter Healthcare Corporation, Deerfield, IL). This perfusate was applied to oocytes for 20 s.

In the second study, oocytes were perfused with a saturated solution made of 0.4 µL/L DEHP (Sigma-Aldrich, St. Louis, MO) in FR. A liter of this solution has 0.8 micromoles of DEHP, a concentration which exceeds the solubility of DEHP in water of 2.9 µg/L, or 0.0074 µM (9). This saturated DEHP solution and agonist in FR were coapplied to oocytes for 20 s.

The effect of perfusates from DEHP plasticized PVC bags, or from perfusates made up with DEHP, was compared with solutions made in FR contained in glass syringes and plastic bottles, which did not contain DEHP plasticizer. Perfusates were used as rapidly as they were made up, that is, we did not allow perfusates placed in DEHP plasticized PVC bags to equilibrate for prolonged times.

Statistics: Data are reported as means ± se. Groups were compared using a Student's t-test. P < 0.05 was considered significant.

	RESULTS

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FR solutions from IntraVia IV bags inhibited GABA-induced currents from ₁ß₂ GABA_A receptors by 19.3% ± 3.2% (P < 0.001) (n = 8 oocytes). Perfusate from these bags enhanced glycine-induced currents in homomeric human ₁ glycine receptors by 291.9% ± 84.5% (P = 0.014) (n = 4 oocytes). See Figure 1 for a summary of these effects, and Figure 2 for representative current tracings.

View larger version (11K): [in this window] [in a new window]   Figure 1. (A) Perfusate from an IV bag containing di(2-ethylhexyl) phthalate (DEHP)-plasticized polyvinyl chloride ("bag") and a saturated solution of DEHP ("DEHP") both inhibit 1ß2 -aminobutyric acid type A receptor function. (B) Perfusate from a bag containing DEHP-plasticized polyvinylchloride markedly potentiates 1 glycine receptor function, with a smaller but significant potentiation by a saturated solution of DEHP.

View larger version (15K): [in this window] [in a new window]   Figure 2. Current tracings. (A) Di(2-ethylhexyl) phthalate (DEHP) coapplied with glycine enhances currents through the glycine receptors. (B) Glycine applied from a bag made from DEHP-plasticizer polyvinyl chloride enhances currents through the glycine receptor compared to glycine applied from a glass syringe. (C) (DEHP) coapplied with -aminobutyric acid (GABA) reduces currents through 1ß2 GABA type A (GABAA) receptors. (D) GABA applied from a bag made from DEHP-plasticizer polyvinyl chloride reduces currents through the GABAA receptor compared to GABA applied from a glass syringe.

Perfusate saturated with DEHP inhibited GABA-induced currents from ₁ß₂ GABA_A receptors by 31.7% ± 7.0% (P = 0.004) (n = 4 oocytes). Perfusate saturated with DEHP enhanced glycine-induced currents in human glycine ₁ receptors by 70.8% ± 16.7% (P = 0.003) (n = 5 oocytes). See Figure 1 for a summary of these effects, and Figure 2 for representative current tracings.

Perfusate from the bag and the DEHP solution did not differ in potentiation of ₁ß₂ GABA_A receptors (P = 0.09). However, perfusate from the bag and the DEHP solution significantly differed in potentiation of ₁ glycine receptors (P = 0.023).

	DISCUSSION

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We found that buffer from an IV bag containing DEHP-plasticized PVC inhibited ₁ß₂ GABA_A receptors as much as a saturated solution of DEHP. By contrast, buffer from IV bags containing DEHP had an enhancing effect on ₁ glycine receptors, an effect that was greater than for a saturated solution of DEHP, suggesting that another water soluble compound besides DEHP is present in these bags.

These results imply that DEHP contamination can confound the results of studies of anesthetic mechanisms on a variety of ion channels. Inhaled anesthetics generally enhance the function of both GABA_A and glycine receptors (10). The presence of DEHP would exaggerate the effect of any inhaled anesthetic given concurrently during studies of glycine receptors and decrease the effect during studies of GABA_A receptors. Unexpectedly large or small currents should raise the suspicion of a contaminant such as DEHP. The identification of contamination by plasticizers can be accomplished by comparing these currents with those obtained from perfusates contained in reservoirs that do not contain DEHP (or other phthalate plasticizers, presumably) such as Teflon or glass.

How does DEHP modulate channel function? Possibly, DEHP is modulating channel function in a manner similar to volatile anesthetic compounds. Although anesthetics and alcohols would be expected to enhance, rather than reduce, currents through GABA_A receptors, volatile anesthetics as large as DEHP have not been studied on this receptor. Possibly, they show an inhibition of receptor function, much as anesthetic alcohols show inhibition or enhancement of nACh receptors as a function of the size of the alcohol (11). If DEHP is acting via a mechanism similar to volatile anesthetics, then its octanol solubility should predict its anesthetic potency [the Meyer-Overton relation (10,12)]. Is DEHP sufficiently soluble in octanol to produce anesthetic-like effects? DEHP's water solubility C_water is 7.4 x 10^–9 M and its octanol water partition (K_ow) coefficient is 4.3 x 10⁷ (9). Thus, a saturated solution of DEHP would produce an octanol concentration (C_octanol) of C_waterK_ow = 320 mM. This is indeed sufficient to produce anesthetic effects, since typically, volatile anesthetics require only 25 to 50 mM concentrations in a bulk octanol to produce anesthesia. For example, isoflurane (with a minimum alveolar concentration (MAC) of approximately 1.15% and octanol/gas partition coefficient of 88.2) has a bulk octanol concentration at MAC of 45 mM. Desflurane (with a MAC of approximately 6% and octanol/gas partition coefficient of 17.9) has a bulk octanol concentration at MAC of 46 mM.

However, even if DEHP modulates ion channels in a manner similar to volatile anesthetics in experiments on expressed receptors, DEHP's effects on ion channels are probably not important to the conduct of clinical anesthesia. To see why, we will calculate how much perfusate is required to achieve an anesthetic concentration of DEHP in the membranes of a single oocyte under study, and then of cell membranes in humans. How much perfusate, saturated with DEHP, is required to achieve a bulk octanol concentration in the membrane of an oocyte of 50 mM, the concentration predicted from the Meyer-Overton relation at MAC? Assuming a radius of 1 mm for an oocyte and a thickness of 30 Å for the bilayer gives a volume for the bilayer of 3.77 x 10^–11 L. Further, assuming DEHP is as soluble in cell membranes as it is in octanol, to achieve a bulk octanol concentration of 50 mM would require only 0.2 mL of perfusate saturated with DEHP.

By contrast, achieving these bulk octanol concentrations in the membranes of cells in a human would require the infusion of unrealistically large volumes of DEHP-containing solutions. A Xenopus oocyte that is 1 mm in radius has a volume of 4.2 x 10^–3 cm³. Assuming human tissue has an average density of approximately 1 gm/cm³, a 70 kg human has a volume equal to 1.7 x 10⁷ oocytes. If a human had the same amount of membrane as this many oocytes (an assumption that greatly under-estimates the total DEHP uptake possible since human cells are smaller than Xenopus oocytes), and in addition had no other lipids to absorb DEHP (also an under-estimate, since it assumes no adipose tissue) then to achieve a concentration of 50 mM DEHP in membranes would require 2700 L of aqueous solution saturated with DEHP. Even direct exposure to plastics rather than to IV solutions would not seem to produce exposures large enough to produce neurologic effects in animals, since delivery would be via aqueous extracellular fluids in contact with plastic, in which DEHP would be poorly soluble, even though biochemically measurable effects on processes in neurons have been reported (13).

	Footnotes

 
Accepted for publication April 2, 2007.

Supported in part by NIGMS R01 GM069379 (to JS) and T32 GM08440 (to RB).

Dr. Eger is a paid consultant to Baxter Healthcare Corp.

Reprints will not be available from the author.

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