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Exploring the Boundaries of Additivity: Mixtures of NADH:Quinone Oxidoreductase Inhibitors Jonathan Boyd,*,†,‡ Anshu Saksena,† Julia B. Patrone,† Holly N. Williams,‡ Nathan Boggs,† Huong Le,† and Mellisa Theodore† † ‡
The Johns Hopkins Applied Physics Laboratory, 11100 Johns Hopkins Road, Laurel, Maryland 20723, United States Department of Chemistry, West Virginia University, 217 Clark Hall, Prospect Street, Morgantown, West Virginia 26506, United States ABSTRACT:
The activity of mitochondrial complex I of the electron transport chain (ETC) is known to be affected by an extraordinarily large number of diverse xenobiotics, and dysfunction at complex I has been associated with a variety of disparate human diseases, including those with potentially environmentally relevant etiologies. However, the risks associated with mixtures of complex I inhibitors have not been fully explored, and this warrants further examination of potentially greater than additive effects that could lead to toxicity. A potential complication for the prediction of mixture effects arises because mammalian mitochondrial complex I has been shown to exist in two distinct dynamic conformations based upon substrate availability. In this study, we tested the accepted models of additivity as applied to mixtures of rotenone, deguelin, and pyridaben, with and without substrate limitation. These compounds represent both natural and synthetic inhibitors of complex I of the ETC, and experimental evidence to date indicates that these inhibitors share a common binding domain with partially overlapping binding sites. Therefore, we hypothesized that prediction of their mixtures effects would follow dose addition. Using human hepatocytes, we analyzed the effects of these mixtures at doses between 0.001 and 100 μM on overall cellular viability. Analysis of the doseresponse curves resulting from challenge with all possible binary and ternary mixtures revealed that the appropriate model was not clear. All of the mixtures tested were found to be in agreement with response addition, but only rotenone plus deguelin and the ternary mixture followed dose addition. To determine if conformational regulation via substrate limitation could improve model selection and our predictions, we tested the models of additivity for the binary and ternary mixtures of inhibitors when coexposed with 2-deoxy-D-glucose (2-DG), which limits NADH via upstream inhibition of glycolysis. Coexposure of inhibitors with 2-DG did facilitate model selection: Rotenone plus pyridaben and the ternary mixture were in sole agreement with dose addition, while deguelin plus pyridaben was in sole agreement with response addition. The only ambiguous result was the agreement of both models with the mixture of rotenone plus deguelin with 2-DG, which may be explained by deguelin's well-known affinity for protein kinase B (Akt) in addition to complex I. Thus, our findings indicate that predictive models for mixtures of mitochondrial complex I inhibitors appear to be compound specific, and our research highlights the need to control for dynamic conformational changes to improve our mechanistic understanding of additivity with these inhibitors.
’ INTRODUCTION Eukaryotic NADH:ubiquinone oxidoreductase (complex I) consists of 46 different subunits that total a molecular mass of almost 1000 kDa and is the largest and most complicated enzyme in the electron transport chain (ETC). It functions to shuttle electrons from NADH to ubiquinone, an activity that is coupled to the transfer of protons across the inner mitochondrial membrane and thus maintains membrane potential,1 which is key to the production of ATP via oxidative phosphorylation. Many structurally distinct inhibitors of complex I (natural and synthetic) have been shown to interfere with ubiquinone reduction,1 and although multiple binding sites within complex r 2011 American Chemical Society
I have been proposed, their existence has not been definitively shown. Rather, experimental evidence to date indicates that many inhibitors share a common binding domain spanning at least three subunits (PSST, ND1, and ND5) with partially overlapping binding sites.13 This binding domain may have the capacity to interact with multiple inhibitors simultaneously; therefore, their combined toxicity may not follow simple additivity. Furthermore, mammalian mitochondrial complex I has been shown to exist in two conformationally distinct forms, Received: March 3, 2011 Published: July 12, 2011 1242
dx.doi.org/10.1021/tx200098r | Chem. Res. Toxicol. 2011, 24, 1242–1250
Chemical Research in Toxicology
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Figure 1. Rotenone, deguelin, and pyridaben.
active and deactive, which are interconvertible based upon the availability of substrates (NADH and oxidized quinone).4 Both forms differ in their affinity for complex I inhibitors,5 and this dynamic conformational stability could further complicate mixtures additivity. The involvement of complex I in a variety of human degenerative diseases, including those with environmentally relevant etiologies (e.g., Parkinson's disease),68 coupled with the large number of diverse inhibitors9,10 warrants further examination for potentially greater than additive mixtures effects. To date, the most comprehensive guidance for quantitative health risk assessment of a chemical mixture has come from the U.S. Environmental Protection Agency (EPA), which recommends three different approaches, depending upon the type of available data. In the first approach, if toxicity data on the mixture of concern are available, then the risk assessment is performed directly with these preferred data. In the second approach, when toxicity data are not available for the mixture of concern, the EPA guidelines recommend using toxicity data on a sufficiently similar mixture if deemed appropriate. Finally, the third approach is to evaluate the mixture through an analysis of its components, for example, using dose addition for similarly acting chemicals and response addition for independently acting chemicals. These procedures include a general assumption that interaction effects at low dose levels either do not occur at all or are small enough to be insignificant to the risk estimate;11 for a review, see McCarty.12 The dose addition model is based on the theory that noninteracting chemicals behave as dilutions of one another within a mixture and has been applied to chemicals with the same primary mode of action.13,14 Response addition states that the toxic response from the combination of chemicals in a mixture is equal to the conditional sum of component responses as defined for the sum of independent event probabilities.11 The present study examines the toxicity of three mitochondrial complex I inhibitors, rotenone, deguelin, and pyridaben, both alone and as mixtures in whole cells. Rotenone and deguelin are natural isoflavenoids produced by plants of the family Leguminosae,15,16 and pyridaben is a synthetic pesticide modeled after rotenone17 (Figure 1). Beyond their central role in cellular respiration, mitochondria are key players in apoptosis,1820 generation of reactive nitrogen and oxygen species,2124 signal transduction,25,26 and calcium homeostasis.2729 Thus, it is no surprise that while all complex I inhibitors are known to interrupt proton translocation at the inner mitochondrial membrane, they have also been shown to lead to alterations in general cellular processes. Therefore, we chose to utilize a standard 3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay to examine the overall level of cellular viability as a measure of inhibitor-induced toxicity allowing downstream detection of decreased cellular health due to both primary and potential secondary effects,30 rather than focusing on mitochondrionspecific effects.
Figure 2. Glucose and 2-DG.
Another benefit of using the whole cell response is that it allows us to examine the influence of glycolysis inhibition on additivity of complex I inhibitors by coexposing cells to mixtures of inhibitors plus 2-deoxy-D-glucose (2-DG), dosed at the no observable effect level of 2-DG. The inclusion of 2-DG serves two purposes, enhancement of cytotoxic response and regulation of complex I conformational stability. 2-DG is a glucose analogue that limits substrate delivery (namely, NADH) to mitochondrial complex I by inhibiting hexokinase, glucose phosphoisomerase, and protein glycosylation.31,32 The structures of both 2-DG and glucose are shown in Figure 2. Furthermore, 2-DG has been shown to potentially alter complex I conformation via NADH restriction and subsequently affect rotenone binding.4,5,3335 Therefore, 2-DG has the potential to increase the effects of the mitochondrial inhibitors as measured by whole cell response (i.e., potentiation, as defined in Hodgson36) by promoting oxidative phosphorylation and restricting the glycolytic pathway but could also simplify the joint action of the inhibitors by changing the conformation of the binding pocket. Examining the influence of an additional upstream inhibitor on downstream additivity has not yet been explored and could illustrate the need for additional experimental regulation that may be necessary for accurate prediction of mixtures response, even when applying additivity models to chemicals with known modes of action. To fully understand the additivity of complex I inhibitors, human hepatocellular-carcinoma derived cells (HepG2) were exposed to all binary and ternary combinations of rotenone, deguelin, and pyridaben, with and without coexposure to 2-DG for 24 h. Mixture dosing employed a fixed-ray design,37 based upon ratios of the EC50 values of individual inhibitors, with and without 2-DG. In this study, we found that in the absence of 2-DG, the appropriate predictive model was not clear, but when inhibitors were coexposed with 2-DG, model discrimination improved. Thus, our findings demonstrate that when examining mixtures associated with mammalian mitochondrial complex I inhibitors, measures to control for potential dynamic conformational changes may be necessary to enhance our mechanistic understanding of additivity.
’ EXPERIMENTAL PROCEDURES Materials. Rotenone, deguelin, pyridaben, and 2-DG were obtained from Sigma Aldrich (St. Louis, MO). RPMI-1640, sodium pyruvate, 1243
dx.doi.org/10.1021/tx200098r |Chem. Res. Toxicol. 2011, 24, 1242–1250
Chemical Research in Toxicology HEPES, L-glutamine, fetal bovine serum, and penicillin-streptomycin were obtained from Invitrogen (Carlsbad, CA). Cell lines and MTT assay kits were obtained from American Type Culture Collection (Manassas, VA). Cell Culture. Human hepatocelluar-carcinoma derived HepG2 cells were cultured in RPMI-1640, supplemented with 1 mM sodium pyruvate, 5 mM HEPES, 2 mM L-glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a humidified atmosphere at 37 °C and 5% CO2 and passaged at 80% confluence. Dosing. Cells were seeded into clear-bottom, black-sided 96-well plates at a density of 4 104 cells per well and allowed to grow for 24 h. Media were then aspirated from wells, and cells were challenged with varying concentrations of single and mixed compounds in fresh media. Compounds were prepared in either DMSO or water to concentrations of 0.001100 μM. Higher doses were not possible due to solubility limitations of the compounds. When 2-DG was added to the inhibitor mixtures, it was added simultaneously, to give a final concentration of 5 mM. The effect of 2-DG alone on cellular health was assessed by dosing to a final concentration of 0.0115 mM for 24 h and performing an MTT assay. MTT Assay. After 24 h of exposure to single compounds or mixtures of compounds, cell viability was determined using the MTT assay, according to the manufacturer's protocol. The assay is based on the reduction of tetrazolium MTT to formazan by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. Briefly, MTT reagent was added to the wells of the microplate, and after 2 h of incubation at 37 °C, intracellular formazan crystals were solubilized with the provided detergent solution. Absorbance values were obtained using the Safire2 microplate reader (Tecan US, Raleigh, NC) with a measurement wavelength of 570 nm and a reference wavelength of 700 nm, read from the bottom. From this, a percent viability was determined by subtracting the absorbance of blank wells (consisting of cell culture media, the highest dose of the inhibitor being tested, all MTT reagents, but no cells) from all treatments and controls; we next determined the ratio of the blank-corrected absorbance values for wells containing treated cells relative to the wells containing controls (consisting of everything in the treated wells, except the inhibitor); finally, we multiplied this ratio by 100 to yield the percent viability. Mixtures dosing included all binary and ternary combinations of rotenone, deguelin, and pyridaben, with and without coexposure to 2-DG. All mixtures dosing employed a fixed-ray design37 based upon ratios of the EC50 values of individual inhibitors from a preliminary set of n = 6 replicates. EC50 values are defined as the dose resulting in a 50% decrease in response when compared to controls. Using the fixed-ray design, the EC50 mixture fraction of each individual component was multiplied by the total dose to yield the concentration of the component at that dose. An example is the EC50 mixture fraction of rotenone and deguelin, whereby each total dose is composed of 10.13% rotenone and 89.87% deguelin (Table 1). This experimental design is particularly well suited for analyzing multiple mixtures by employing the accepted dose and response addition models.38 From the preliminary data set, the EC50 values for the individual inhibitors were determined to be 67.19, 596.1, and 179.4 μM for rotenone, deguelin, and pyridaben, respectively. For coexposures with 2-DG, the EC50 values for rotenone + 2-DG, deguelin + 2-DG, and pyridaben + 2-DG were calculated to be 1.550, 6.331, and 5.321 μM, respectively. It is important to note that these values were taken from a preliminary data set and used only to define the dosing ray. Actual tests of additivity were computed from positive controls (consisting of individual inhibitors) run in parallel with the mixtures doses (consisting of equimolar dosing rays corresponding to EC50 values, with and without 2-DG), over the entire effect range. By simultaneously rerunning the individual
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exposures, we are able to account for any potential drift in doseresponse curves (that may be associated with an immortalized cell line) in our determination of additivity. DoseResponse Analysis. The predictions of two additivity models were tested against observed mixture responses. Both additivity models are generalizations of the Gompertz function.39 Fðxi Þ ¼ R þ γ expfexp½ðβ0 þ βi xi Þg
ð1Þ
where F(xi) is viability in response to the inhibitor i at dose xi, R is the minimum response, R + γ is the maximum response, β0 is the intercept on the complementary loglog scale, and βi is the slope parameter for the inhibitor. Residuals of fits to the Gompertz function suggested that a logarithmic transformation of dose would produce better fits. The data were again fit to the Gompertz function modified as follows: Flog ðxi Þ ¼ R þ γ expfexp½ðβ0 þ βi logðxi ÞÞg
ð2Þ
Fits of the data to Flog produced lower sum squared errors and, therefore, higher R2 values than fits to F (data not shown). Thus, all analyses were performed using this function as a basis. Dose Addition Modeling. According to the U.S. EPA11 and ATSDR,40 dose addition may be used for chemicals with similar modes of action. Because rotenone, deguelin, and pyridaben exert toxicity via the same binding pocket, and thus have the same expected mode of action, dose addition should be the accepted no-interaction model for predicting toxicity. The standard Gompertz expression in eq 1 is extrapolated to multiple inhibitors to model these kinds of interactions as follows:39 Fðxmix Þ ¼ R þ γ expfexp½ðβ0 þ β1 x1 þ β2 x2 þ β3 x3 Þg ð3Þ Here, β1, β2, and β3 are the parameters associated with the slope, and x1, x2, and x3 represent the doses of rotenone, deguelin, and pyridaben, respectively. The extrapolation is at the level of the dose in accordance with dose addition and is in agreement with Berenbaum's combination index.39,41 In the absence of a standard extrapolation of the modified Gompertz function of log dose given in eq 2, we chose an expression that adds doses each modified by its own slope parameter in such a way that it reduces to eq 2 when there is only one inhibitor. This is accomplished by first using the fact that the slope parameters are always negative for inhibitors and rewriting eq 2 as follows: 8 2 0 !139 < = 1 ð4Þ Flog ðxi Þ ¼ R þ γ exp exp4@β0 þ log β A5 : ; xi i where the βi exponent of xi is a positive number. This manipulation is performed so that the extrapolation can be done at the level of the dose. Now, the dose addition expression based on log dose follows: Flog ðxmix Þ
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