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Nonylphenol ethoxylate plastic additives inhibit mitochondrial respiratory chain complex I.

To the Editor:

The first and major entry point of electrons into the mitochondrial respiratory chain (MRC) [1] occurs through NADH-coenzyme Q reductase (complex I). Decreased complex I activity is associated with a wide range of conditions, including inherited mitochondrial diseases and neurodegenerative conditions such as Parkinson disease (1-2). More than 60 natural and commercial compounds are reported to inhibit complex I activity, among them certain pesticides, agrochemicals, and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), a neurotoxin that induces parkinsonism in humans; thus, exposure to these compounds may be harmful (3). We report here that nonylphenol ethoxylate (NP) surfactants leach from disposable laboratory plastic-ware and inhibit mitochondrial complex I.

During a routine diagnostic workup designed to diagnose mitochondrial diseases from patients' muscle biopsy samples, we observed a substantial (50%-66%) decrease of mitochondrial enzymatic activities related to complex I compared with historical values determined spectrophotometrically in our laboratory. Furthermore, a 71% decrease in complex I-dependent oxygen consumption was also observed with glutamate + malate as substrates (Oxygraph, Hansatech Instruments) (2). Notably, other MRC activities, as well as citrate synthase, a mitochondrial marker enzyme, were largely unaffected. It was deter

mined that inhibition resulted from a single brief period of contact between assay reagents and blue polypropylene 1-mL pipette tips ("blue tips") manufactured by Scientific Specialties (Table 1).

Methanol washes of blue tips were subjected to positive ion-mode electrospray mass spectrometry (Waters Mariner BioSpectrometry Workstation oa-TOF); these analyses confirmed the presence of NP compounds, marketed as surfactants from the Tergitol NP series. The prevalent ion at m/z 683.5 was consistent with the use of NP-10 as a dye-solubilizing agent in the plastic manufacturing process, whereas m/z peaks indicative of shorter (NP-9) and longer (NP-11) side-chain surfactants are characteristic of mass spectral data for these compounds (4).

Subsequently we evaluated MRC function in the presence of commercial samples of NP-10 and NP-9 and detected inhibition of complex I enzymatic activity, with [IC.sub.50] values of 4 [micro]mol/L and 3.7 [micro]mol/L, respectively. Other respiratory chain complexes were unaffected in the presence of these surfactants at 10 [micro]mol/L; however, a marked decrease in complex I-dependent oxygen consumption was evident (Table 1). Oxygen consumption with complex I-independent substrates was not affected (not shown).

The inhibition of oxygen consumption in whole mitochondria by NP-10 and NP-9 indicates that these compounds penetrate the inner mitochondrial membrane. We also studied the impact of leachate from blue tips and of NP surfactants on growing fibroblasts in culture and confirmed that these substances penetrate whole cell membranes. Growth in MRC-dependent, restrictive medium lacking glucose was partially inhibited by NP-10 or NP-9, whereas growth in MRC independent, permissive glucose-containing medium was minimally affected. The use of blue tips to pipette culture medium was deleterious for cell growth in restrictive medium, but affected growth in permissive medium to a lesser extent (Table 1). Other nonionic detergents--dodecyl maltoside and Tween 20--and clear colorless tips from a different manufacturer had no effects under either set of conditions.

Our data clearly show that blue tips leached NP-10 into assay buffers and interfered with MRC complex I while leaving other complexes unaffected. Previously, McDonald et al. reported that disposable laboratory plasticware (pipette tips, microfuge tubes) leached the processing agents DiHEMDA [di(2-hydroxyethyl)-methyldodecylammonium] and oleamide into buffers, resulting in profound interference with biological assays (5) We did not detect these compounds in blue-tip leachates, neither did they interfere with complex I function (not shown), suggesting that leachates from various disposables might affect a variety of assays in different ways

In addition to being used in plastic manufacturing, NP surfactants are also found in household cleaners, laundry detergents, spermicides, and cosmetics Consequently these compounds are present in sewage waters and soil in micromolar quantities, and their alkylphenol breakdown products are estrogenic and persist in the environment Furthermore, human exposure and absorption have been confirmed through detection of NP surfactants in human urine (4). Our data show that NP-10 and NP-9 penetrate both the human cell membrane and the mitochondrial inner membrane, and other investigators have confirmed these compounds as substrates for the human P-glycoprotein transporter (4). The possibility thus exists that these and related compounds may pose an environmental hazard through inhibition of oxidative phosphorylation via the MRC. Of greater concern to researchers is the addition of these surfactants to the growing list of bioactive compounds present in disposable laboratory plasticware, highlighting the need for individuals to screen plastics as a potential source of interferences in life science experiments.

Author Contributions: All authors confirmed they have contributed to the intellectual content oft his paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.

Authors' Disclosures of Potential Conflicts of Interest: No authors declared any potential conflicts of interest.

Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.

Acknowledgments: The authors thank Sarah Weissman for technical assistance. Professors Orly Elpeleg and Randy Whittal are acknowledged for helpful discussions.

References

(1.) Pagliarini DJ, Calvo SE, Chang B, Sheth SA, Vafai SB, Ong SE, et al. Mitochondrial protein compendium elucidates complex I disease biology. Cell 2008;134:112-23.

(2.) Saada A, Bar-Meir M, Belaiche C, Miller C, Elpeleg O. Evaluation of enzymatic assays and compounds affecting ATP production in mitochondrial respiratory chain complex I deficiency. Anal Biochem 2004;335:66-72.

(3.) Degli Esposti M. Inhibitors of NADH-ubiquinone reductase: an overview. Biochim Biophys Acta 1998;1364:222-35.

(4.) Charuk HM, Grey AA, Reithmeier AR. Identification of the synthetic surfactant nonylphenol ethoxylate: a P-glycoprotein substrate in human urine. Am J Physiol 1998;274:1127-39.

(5.) McDonald GR, Hudson AL, Dunn SM, You H, Baker GB, Whittal RM, et al. Bioactive contaminants leach from disposable laboratory plastic ware. Science (Wash DC) 2008;322:917.

Corinne Belaiche [2] Andrew Holt [3] Ann Saada [2] *

[1] Nonstandard abbreviations: MRC, mitochondrial respiratory chain; NP, nonylphenol ethoxylate.

[2] Metabolic Disease Unit Hadassah-Hebrew UniversityMedical Center Jerusalem, Israel

[3] Department ofPharmacology University ofAlberta Edmonton, Canada

* Address correspondence to this author at: Metabolic Disease Unit Hadassah-Hebrew University Medical Center POB.12000, Jerusalem 91120, Israel E-mails annsr@hadassah.org.il, saada-ann@macam.ac.il

Previously published online at DOI: 10.1373/clinchem.2009.130054
Table 1. MRC function in the presence of blue-tip leachate and NP
surfactants. (a)

 No
Assay additive

Complex I (c) (NADH-CoQ 174
 reductase)
Complex I + III (c) (NADH- 532
 cytochrome c reductase)
Complex II (c) (succinate 46
 dehydrogenase)
Complex II + III (c) (succinate- 240
 cytochrome c reductase)
Complex IV (c) (cytochrome c 1156
 oxidase)
Citrate synthase (c) 1800
Glutamate + malate 66
 oxidation (d)
Viable cell count, restrictive 62 X [10.sup.3]
 medium
Viable cell count, permissive 84 X [10.sup.3]
 medium

Assay Blue tip

Complex I (c) (NADH-CoQ 87 (50%)
 reductase)
Complex I + III (c) (NADH- 181 (34%)
 cytochrome c reductase)
Complex II (c) (succinate 46 (100%)
 dehydrogenase)
Complex II + III (c) (succinate- 240 (100%)
 cytochrome c reductase)
Complex IV (c) (cytochrome c 1156 (100%)
 oxidase)
Citrate synthase (c) 1800 (100%)
Glutamate + malate 19(29%)
 oxidation (d)
Viable cell count, restrictive 7.5 X [10.sup.3] (12%)
 medium
Viable cell count, permissive 42 X [10.sup.3] (50%)
 medium

Assay NP-10

Complex I (c) (NADH-CoQ 30 (17%)
 reductase)
Complex I + III (c) (NADH- 8 (1.5%)
 cytochrome c reductase)
Complex II (c) (succinate 45 (98%)
 dehydrogenase)
Complex II + III (c) (succinate- 245 (102%)
 cytochrome c reductase)
Complex IV (c) (cytochrome c 1098 (95%)
 oxidase)
Citrate synthase (c) 1800 (100%)
Glutamate + malate 31 (47%)
 oxidation (d)
Viable cell count, restrictive 35 X [10.sup.3] (56%)
 medium
Viable cell count, permissive 78 X [10.sup.3] (92%)
 medium

Assay N-P9

Complex I (c) (NADH-CoQ 31 (18%)
 reductase)
Complex I + III (c) (NADH- 32 (6%)
 cytochrome c reductase)
Complex II (c) (succinate 42 (92%)
 dehydrogenase)
Complex II + III (c) (succinate- 245 (102%)
 cytochrome c reductase)
Complex IV (c) (cytochrome c 1087 (94%)
 oxidase)
Citrate synthase (c) 1818 (101%)
Glutamate + malate 28 (42%)
 oxidation (d)
Viable cell count, restrictive 28 X [10.sup.3] (45%)
 medium
Viable cell count, permissive 84 X [10.sup.3] (100%)
 medium

Assay DDMb

Complex I (c) (NADH-CoQ 113(65%)
 reductase)
Complex I + III (c) (NADH- 489 (92%)
 cytochrome c reductase)
Complex II (c) (succinate 39 (84%)
 dehydrogenase)
Complex II + III (c) (succinate- 238 (99%)
 cytochrome c reductase)
Complex IV (c) (cytochrome c 1017 (88%)
 oxidase)
Citrate synthase (c) 1764 (98%)
Glutamate + malate 60 (89%)
 oxidation (d)
Viable cell count, restrictive 61 X [10.sup.3] (98%)
 medium
Viable cell count, permissive 78 X [10.sup.3] (92%)
 medium

Assay TW-20

Complex I (c) (NADH-CoQ 131 (75%)
 reductase)
Complex I + III (c) (NADH- 420 (79%)
 cytochrome c reductase)
Complex II (c) (succinate 46 (100%)
 dehydrogenase)
Complex II + III (c) (succinate- 242 (101%)
 cytochrome c reductase)
Complex IV (c) (cytochrome c 1306 (113%)
 oxidase)
Citrate synthase (c) 1854 (103%)
Glutamate + malate 60 (89%)
 oxidation (d)
Viable cell count, restrictive 66 X [10.sup.3] (106%)
 medium
Viable cell count, permissive 81 X [10.sup.3] (96%)
 medium

(a) Enzymatic assays of MRC complexes (I-IV) and oxygen consumption
with glutamate and malate were performed on isolated human muscle
mitochondria in the absence or presence of 10 [micro]mol/L surfactants
or blue tip leachate. Equal amounts of human fibroblasts were seeded
on 24-well plates and grown in restrictive medium lacking glucose or
permissive, glucose-containing medium in the absence or presence of
2.5 [micro] mol/L surfactants for 72 h. Viable cell count was
determined by trypan blue exclusion. Numbers in parenthesis indicate
percentages relative to no additive controls. All values are means of
at least 2 separate determinations.

(b) DDM, dodecyl maltoside; TW, Tween.

(c) Nanomoles per minute per milligram protein.

(d) Nanomoles oxygen per milligram protein in the presence of ADP.
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Title Annotation:Letters to the Editor
Author:Belaiche, Corinne; Holt, Andrew; Saada, Ann
Publication:Clinical Chemistry
Article Type:Letter to the editor
Date:Oct 1, 2009
Words:1670
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