Methoxychlor Inhibits Brain Mitochondrial Respiration and Increases Hydrogen Peroxide Production and CREB Phosphorylation

Rosemary A. Schuh*,{dagger},{ddagger}, Tibor Kristián*, Rupesh K. Gupta{dagger},{ddagger}, Jodi A. Flaws{dagger},{ddagger} and Gary Fiskum*,{ddagger},1

* Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, Maryland 21201; {dagger} Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201; {ddagger} Program in Toxicology, University of Maryland School of Medicine, Baltimore, Maryland 21201

1 To whom correspondence should be addressed at Department of Anesthesiology, University of Maryland School of Medicine, 685 W. Baltimore Street, MSTF 5–34, Baltimore, MD 21201. Fax: (410) 706-2550. E-mail: gfisk001{at}umaryland.edu.

Received July 28, 2005; accepted September 14, 2005


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The organochlorine insecticide methoxychlor (mxc) is an established reproductive toxicant that affects other systems including the central nervous system (CNS), possibly by mechanisms involving oxidative stress. This study tested the hypothesis that mxc inhibits brain mitochondrial respiration, resulting in increased production of reactive oxygen species (ROS). Oxygen electrode measurements of mitochondrial respiration and Amplex Red measurements of H2O2 production were performed with rat brain mitochondria exposed in vitro to mxc (0–10 µg/ml) and with brain mitochondria from mice chronically exposed in vivo to mxc (0–64 mg/kg/day) for 20 days by intraperitoneal injection. In vitro mxc exposure inhibited ADP-dependent respiration (state 3) using both complex I- and II-supported substrates. Similarly, state 3 respiration was inhibited following in vivo mxc exposure using complex I substrates. H2O2 production was stimulated after in vitro mxc treatment in the presence of complex I substrates, but not in mitochondria isolated from in vivo mxc-treated mice. Because previous studies demonstrated a relationship between oxidative stress and CREB phosphorylation, we also tested the hypothesis that mxc elevates phosphorylated CREB (pCREB) in mitochondria. Enzyme-linked immunosorbent assay (ELISA) measurements demonstrated that pCREB immunoreactivity was elevated by in vitro mxc exposure in the presence or absence of respiratory substrates, indicating that stimulation of H2O2 production is not necessary for this effect. These multiple effects of mxc on mitochondria may play an important role in its toxicity, particularly in the CNS.

Key Words: methoxychlor; mitochondria; CREB; oxidative stress.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Organochlorines are a diverse group of synthetic chemicals including pesticides and industrial products that are persistent environmental pollutants due to their high lipophilicity and subsequent bioaccumulation in the food chain. The organochlorine pesticides dichlorodiphenoxytrichloroethane (DDT) and 1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane (methoxychlor, mxc) have been shown in several studies to possess estrogenic properties resulting in adverse effects on the reproductive system in both animal models (Borgeest et al., 2002Go; Cummings, 1997Go; Gray et al., 1989Go) and cell lines (Chedrese and Feyles, 2001Go; Hodges et al., 2000Go; Okubo et al., 2004Go; Shekhar et al., 1997Go). These studies indicate that exposure to organochlorine pesticides and related compounds are of concern in terms of human health.

Botella et al. (2004)Go determined the levels of several organochlorine pesticide residues in adipose tissue and blood samples from 200 women living in Southern Spain. The highest concentrations found were for 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p'-DDE), the major metabolite of DDT. Methoxychlor residues were also identified in this study, but at lower levels than the other pesticides tested. In addition, a recent study by Rudel et al., (2003)Go determined the levels of 89 target chemicals including pesticides, designated as endocrine disruptors in urine samples, house dust, and indoor air from 120 homes in Cape Cod, MA. The results from the study indicate that DDT and mxc were present in at least 50% of the homes tested at relatively high concentrations.

In addition to their estrogenic properties, certain organochlorines including hexachlorocyclohexane (Sahoo and Chainy, 1998Go) and endosulfan (Kannan and Jain, 2003Go) have been demonstrated to play a role in cellular oxidative stress. Studies by Latchoumycandane and Mathur (2002)Go demonstrated depletion of antioxidant enzymes in mitochondria and microsomes from rat testis following exposure to mxc. Additionally, Chen et al. (1999)Go demonstrated increased superoxide production in rat liver mitochondria following exposure to the synthetic estrogen ethinyl estradiol. Other pesticides, including paraquat and rotenone, inhibit mitochondrial respiration and stimulate mitochondrial production of reactive oxygen species (ROS) in rat brain (Meyer et al., 2004Go; Starkov et al., 2004Go; Tawara et al., 1996Go). The mitochondrial effects of environmental toxicants, e.g., rotenone, are likely responsible for their induction of neurodegeneration (Greenamyre et al., 1999Go). Considering the epidemiological evidence that pesticide exposure may be linked to Parkinson's disease, a more extensive assessment of the effects of these toxicants on mitochondrial functions, including ROS production is needed.

Many studies have identified oxidative stress as an inducer of post-translational protein modification, resulting in transcriptional activation. For example, some studies indicate that oxidative stress during ischemia/reperfusion causes phosphorylation of Ca2+/cAMP response element binding protein (pCREB) (Mabuchi et al., 2001Go; Tanaka, 2001Go). In an earlier study, we determined that exposure of rat primary cortical and hippocampal neurons to the organophosphate insecticide chlorpyrifos increases pCREB immunoreactivity via a noncholinesterase mechanism (Schuh et al., 2002Go). CREB phosphorylation is implicated in the induction of transcriptional activity that stimulates the expression of antioxidant genes (Bedogni et al., 2003Go). In addition to the effects of pCREB on nuclear gene transcription, mitochondrial gene expression may be under the control of mitochondrial-localized pCREB (Ryu et al., 2003Go). pCREB is present in mitochondria from different tissues, including brain, and its phosphorylation state is regulated by Ca2+, an important intracellular modulator of gene expression (Schuh et al., 2005Go).

Although mxc and other endocrine disruptive compounds have been clearly identified as reproductive toxicants, other systems including the central nervous system (CNS) may also be targeted (Cooper et al., 1999Go; Gore, A. C., 2002Go; Lafuente et al., 2003Go). Furthermore, the mechanism(s) of action of these compounds within the CNS have not been fully investigated at the organelle level. Therefore, the primary objective of the present study was to determine the effects of in vitro and in vivo mxc exposure on brain mitochondrial respiration and ROS production. Considering the recent identification of mitochondrial CREB, we also tested the hypothesis that mxc increases mitochondrial CREB phosphorylation, possibly via stimulation of ROS production. The results of this study provide new insight into non-estrogenic effects of mxc that alter mitochondrial bioenergetics, producing oxidative stress. Mitochondrial pCREB is also identified as a new potential target of organochlorines.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and reagents.
1,1,1-trichloro-2,2-bis(p-methoxyphenyl)ethane (methoxychlor, mxc) was purchased from ChemService (West Chester, PA) in a powdered form and was 99% pure. All other reagents were purchased from Sigma (St. Louis, MO) unless otherwise stated.

Animals.
Female CD-1 mice (25 g, 39 days old) were housed five animals per cage, and male Sprague-Dawley rats (300 g, 90 days old) were housed three animals per cage at the University of Maryland School of Medicine Central Animal Facility and provided food and water ad libitum. Animals were subjected to 12-h light:dark cycles. Mice were dosed via intraperitoneal injection with 16, 32, or 64 mg/kg/day mxc, or sesame oil (vehicle) for 20 continuous days. The mice were sacrificed when in estrus to minimize variability due to hormonal fluctuations within 24–72 h after the final mxc treatment. The in vivo mxc doses were selected based on earlier studies showing deleterious effects in the ovaries (Borgeest et al., 2002Go, 2004Go). The University of Maryland School of Medicine Institutional Animal Use and Care Committee approved all procedures involving animal care, euthanasia, and tissue collection.

Mitochondrial isolation.
Male Sprague-Dawley rat brains and female CD-1 mouse brains were rapidly dissected then further processed to isolate non-synaptosomal mitochondria using the Percoll isolation method described by Sims (1990)Go. Briefly, after decapitation, the forebrain was rapidly removed and placed in ice-cold mannitol-sucrose (MS) buffer pH 7.4 (225 mM mannitol, 75 mM sucrose, 5 mM Hepes, 1 mg/ml fatty acid free BSA, 1 mM EGTA). The brain was homogenized then centrifuged twice at 1317 x g for 3 min. After a further 10 min centrifugation at 21,074 x g, the pellet was resuspended in 15% Percoll (Amersham Biosciences, Piscataway, NJ) then layered on a discontinuous Percoll gradient and spun at 29,718 x g for 8 min. The mitochondrial fraction was centrifuged at 16,599 x g for 10 min then spun at 6668 x g for 10 min. The mitochondrial pellet was resuspended in the above buffer but without BSA or EGTA. Protein concentrations were determined by the method described by Lowry et al. (1951)Go.

Mitochondrial oxygen consumption.
Oxidizable respiratory substrates consisting of either 5 mM L-malate plus 5 mM L-glutamate, 5 mM succinate plus 1 µM rotenone, or 0.02 mM N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) plus 2 mM ascorbate and 1 µM antimycin A in potassium chloride buffer (30°C) containing 125 mM KCl ultrapure (Merck, Whitehouse Station, NJ), 20 mM Hepes, 2 mM K2HPO4, 0.01 mM EGTA, and 1 mM MgCl2 (pH 7.0) were placed in a thermostatically controlled Clarke-type O2 electrode (Hansatech Instruments, Norfolk, England). Isolated non-synaptosomal rat or mouse brain mitochondria (0.25 mg/ml) were added to the chamber and the rates of oxygen consumption were measured. For the in vitro mxc treatment studies, mxc (0–10 µg/ml) in dimethyl sulfoxide (DMSO) was added prior to mitochondrial addition. State 3 respiration was initiated 2 min after the addition of mitochondria by the addition of 0.8 mM ADP. Approximately 2 min later, state 3 respiration was terminated and state 4o respiration (resting) was initiated with addition of 1.25 µg/ml oligomycin, an inhibitor of the mitochondrial ATP synthase. While the state 4o respiration measured in the presence of oligomycin is not equivalent to the classical state 4 rate obtained after a small bolus of ADP is almost completely converted to ATP, the use of oligomycin eliminates the contribution of ATP cycling via hydrolysis by contaminating ATPases and resynthesis by the mitochondrial ATP synthase to state 4 respiration. The oligomycin-induced state 4o rate of respiration is therefore a more specific indicator of the inner membrane proton leakiness. The maximal rate of uncoupled respiration was subsequently measured by titration with 54 nM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP). The mitochondrial suspensions were centrifuged at 18,522 x g for 3 min and the pellet resuspended in lysis buffer (pH 7.4) containing 0.5% NP40 (USB, Cleveland, OH), 1% Triton X-100, 150 mM NaCl, 10 mM Tris, and 1% protease inhibitor cocktail (Calbiochem, San Diego, CA). The aliquots were stored at –70°C.

Mitochondrial membrane potential.
Mitochondrial membrane potential changes in isolated non-synaptosomal brain mitochondria (0.25 mg/ml) were followed qualitatively by monitoring the fluorescence of tetramethyl rhodamine methyl ester (TMRM, Molecular Probes, Eugene, OR), a cationic lipid-soluble probe that accumulates in energized mitochondria. Fluorescence intensity was measured in a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan) using the 549 nm wavelength for excitation and the emission wavelength set at 580 nm. An increase in fluorescence represents dequenching of TMRM when the probe is released into the medium upon mitochondrial depolarization. Briefly, the potassium chloride buffer (30°C) mentioned above was used, with addition of TMRM (100 nM) and oxidizable respiratory substrates consisting of 5 mM L-malate plus 5 mM L-glutamate. Following mitochondrial addition, sequential mxc (total amount 10 µg/ml) and FCCP (54 nM) were added.

Mitochondrial hydrogen peroxide production.
Hydrogen peroxide (H2O2) production from isolated non-synaptosomal mitochondria from rat and mouse brains was measured fluorimetrically utilizing Amplex Red (Molecular Probes, Eugene, OR) as previously described (Starkov and Fiskum, 2003Go). Briefly, the potassium chloride buffer (30°C) mentioned above was used, with addition of 5 U/ml horseradish peroxidase, 40 U/ml Cu, Zn superoxide dismutase, and 1 µM Amplex Red. Measurements were initiated prior to addition of mxc and mitochondria to identify background rates. Methoxychlor (0–10 µg/ml) in DMSO was added to the cuvette prior to mitochondria in studies using rat brain mitochondria. After mitochondrial addition, the oxidizable substrates 5 mM L-malate plus 5 mM L-glutamate were added. Adenosine diphosphate (0.8 mM) was added a minute later, followed by oligomycin (1.25 µg/ml). When malate/glutamate were used as substrates, 1 µM rotenone was added, following oligomycin treatment. For experiments using alternative substrates, 1 µM rotenone was added followed by 5 mM succinate and 1 µM antimycin A addition after the oligomycin. Detection of H2O2 production was measured as an increase in fluorescence of Amplex Red dye at 585 nm with the excitation wavelength set at 550 nm. The dye response was calibrated with addition of a known amount of H2O2 (1 nmol). The concentration of the H2O2 stock was calculated from light absorbance at 240 nm employing E240 = 43.6 M–1 cm–1.

Experiments using DMSO (vehicle control) alone were performed for all the procedures mentioned above and DMSO was determined to have no effect on the parameters measured. The DMSO concentrations in all experiments were kept below 0.5% (data not shown).

pCREB immunoreactivity.
Mitochondrial samples were assessed using an enzyme-linked immunosorbent assay (ELISA kit; BioSource International, Camarillo, CA) that recognized pCREB phosphorylated at serine 133. pCREB levels were determined according to the manufacturer's protocol.

Statistical analysis.
Data are expressed as means ± SE, and the comparisons between experimental groups were made with SPSS statistical software (SPSS, Inc., Chicago, IL) using a regression analysis (test for trend). Statistical significance was assumed at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Methoxychlor Alters Rat Brain Mitochondrial Respiration and Membrane Potential
After isolation of non-synaptosomal mitochondria from rat forebrain, we determined whether and how mxc affects respiration. In untreated mitochondria, ADP addition to the mitochondrial suspension in the presence of the oxidizable substrates malate and glutamate initiated state 3 respiration (Fig. 1A). Addition of the mitochondrial ATP synthase inhibitor oligomycin reduced the rate of O2 consumption to that of state 4o respiration, limited by the proton permeability of the inner membrane. In a few experiments, the rate of respiration was measured in the presence of the protonophore uncoupler FCCP, which stimulated respiration due to rapid futile cycling of protons across the inner membrane and collapse of the electrochemical gradient (Fig. 1A). The rate of respiration in the presence of FCCP is limited by the rate of electron transport rather than the ATP synthase or adenine nucleotide translocase activities, which can, under some circumstances, limit the rate of state 3 respiration. The lower trace in Figure 1A details control conditions without mxc addition, whereas the upper trace demonstrates that the presence of mxc (10 µg/ml) resulted in a 48% inhibition of state 3 respiration and a 43% increase in the state 4o rate. Methoxychlor also inhibited FCCP-stimulated respiration by 35%, suggesting a site of action at the electron transport chain rather than the ATP synthase.



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FIG. 1. Oxygen electrode measurements of respiration and fluorescence measurements of membrane potential using isolated rat brain mitochondria. A. Representative traces of mitochondrial oxygen consumption ± in vitro methoxychlor (mxc, 10 µg/ml) treatment in the presence of L-malate (5 mM), L-glutamate (5 mM), ADP (0.8 mM) to initiate state 3 respiration, and oligomycin (1.25 µg/ml) to induce state 4o respiration. Maximal, uncoupled respiration was initiated with the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 54 nM). The traces are representative of three to six separate experiments. B. Representative trace of changes in mitochondrial membrane potential ({Delta}{Psi}) following sequential additions of mxc (total amount 10 µg/ml) in the presence of L-malate (5 mM) plus L-glutamate (5 mM). Maximal {Delta}{Psi} in uncoupled mitochondria was initiated with FCCP (54 nM). The trace is representative of three separate experiments. A.U., arbitrary units.

 
The finding that 10 µg/ml mxc both inhibits state 3 respiration and stimulates state 4o respiration suggests that mitochondrial membrane potential could be impaired. Qualitative, fluorescent TMRM measurements of mitochondrial membrane potential were performed under the same conditions used for the respiratory measurements, except that ADP was absent (Fig. 1B). Using suspensions of isolated mitochondria, depolarization causes an increase in TMRM fluorescence due to dequenching upon release of the fluorophore from mitochondria into the medium. After sequential additions of mxc totaling 10 µg/ml, the TMRM fluorescence increased toward the level obtained in the presence of FCCP; however, no mitochondrial depolarization was apparent at total doses of either 1 or 5 µg/ml.

The dose–response relationships for mxc and mitochondrial respiration are shown in Figure 2. The mxc doses used in these experiments were based on the studies of Miller et al. (2005)Go assessing the effects of mxc on apoptosis in vitro. Exposure to mxc (0–10 µg/ml) significantly reduced state 3 respiration when electron transport chain complex I substrates malate and glutamate were present (p < 0.001 compared to vehicle control) (Fig. 2A). In the presence of the complex II substrate succinate and the complex I inhibitor rotenone, mxc also demonstrated significant inhibition of state 3 respiration (p < 0.001) (Fig. 2A). Complex I-dependent respiration appeared more sensitive to inhibition than that of complex II (5 µg/ml mxc resulted in a 40% inhibition of respiration on malate plus glutamate compared to a 19% inhibition for succinate plus rotenone). Ascorbate plus TMPD were used to donate electrons to cytochrome c and then through complex IV to O2 to probe for any effects of mxc on this distal portion of the electron transport chain. No significant effect on state 3 respiration following addition of mxc was observed under these conditions (p = 0.61, Fig. 2A).



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FIG. 2. Dose-dependent effects of in vitro methoxychlor treatment on rat brain mitochondria respiring on different oxidizable substrates. A. Mean state 3 oxygen consumption rates following exposure of isolated mitochondria to in vitro mxc (0–10 µg/ml) in the presence of either L-malate (5 mM) plus L-glutamate (5 mM); succinate (5 mM) and rotenone (1 µM); or N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD, 0.02 mM), ascorbate (2 mM), and antimycin A (1 µM), measured as shown in Figure 1A. (B) Mean state 4o oxygen consumption rates following addition of 1.25 µg/ml oligomycin were as shown in Figure 1A. Data are expressed as mean oxygen consumption rates (nmol oxygen/min/mg mitochondrial protein) and represent the mean ± SEM of three to six separate experiments. **Significantly different (p < 0.001) from control. C. Mean respiratory control ratios following exposure of isolated mitochondria to in vitro mxc (0–10 µg/ml) in the presence of L-malate (5 mM) plus L-glutamate (5 mM). Data are expressed as the ratio of state 3 rates:state 4o rates and represent the mean ± SEM of three to six separate experiments. **Significantly different (p < 0.001) from control.

 
After addition of oligomycin, in vitro mxc treatment produced a small but significant increase in state 4o respiration in the presence of the complex II-linked substrate succinate (p < 0.001 as compared to vehicle control; Fig. 2B). No significant effects of mxc on state 4o respiration were observed when using either malate plus glutamate or ascorbate/TMPD as oxidizable substrates (p = 0.147 and p = 0.333, respectively; Fig. 2B). Despite the lack of an effect on state 4o respiration in the presence of malate plus glutamate, mxc caused a dose-dependent reduction in the respiratory control ratio (RCR) (p < 0.001 compared to vehicle control; Fig. 2C). For example, in the presence of mxc at 1 and 10 µg/ml, the RCR values were 3.38 ± 1.04 and 2.21 ± 0.54, respectively, compared to 6.41 ± 1.22 in the absence of mxc (Fig. 2C).

In Vitro Methoxychlor Treatment Increases ROS Production by Rat Brain Mitochondria
Because inhibition of mitochondrial respiration can, under some circumstances, result in increased production of ROS, we next determined whether mxc stimulated mitochondrial ROS production. As described previously (Starkov and Fiskum, 2003Go), fluorescent Amplex Red measurements of H2O2 were made with isolated brain mitochondria exposed to subsequent additions of malate plus glutamate, ADP, oligomycin, and rotenone (Fig. 3A and B). The presence of 10 µg/ml mxc resulted in an approximately sevenfold increase in H2O2 production during state 3 respiration (Fig. 3B) as compared with control (Fig. 3A).



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FIG. 3. Fluorescent Amplex Red measurements of H2O2 production in isolated rat brain mitochondria exposed in vitro to methoxychlor. Representative spectrofluorometer measurements of H2O2 production (A) without mxc or (B) plus mxc (10 µg/ml) in the presence of L-malate (5 mM) plus L-glutamate (5 mM), ADP (0.8 mM) to initiate state 3 respiration, and oligomycin (1.25 µg/ml) to induce state 4o respiration. Mean H2O2 production rates after exposure of actively respiring mitochondria to mxc (0–10 µg/ml) in the presence of complex I-linked substrates (C) or complex II-linked substrates (D). Values represent mean H2O2 production rates (pmol/min/mg mitochondrial protein) ± SEM of three to four separate experiments. *Significantly different (p < 0.01); **significantly different (p < 0.001) from control.

 
Since mxc inhibition of respiration was greatest in the presence of complex I substrates, we hypothesized that the effect of mxc on ROS production would be more pronounced with malate plus glutamate than with succinate in the presence of rotenone. The rate of H2O2 production at state 3 respiration with malate plus glutamate as substrates increased significantly with increasing doses of mxc as compared with vehicle control (p < 0.001; Fig. 3C). In the presence of oligomycin (state 4o), mxc had no significant effect on mitochondrial H2O2 generation (p = 0.65; Fig. 3C). In contrast to the stimulatory effect of mxc on ROS production with malate plus glutamate, mxc caused a significant reduction in succinate-supported ROS generation under both state 3 and state 4o respiration (p < 0.001 and p < 0.01, respectively; Fig. 3D).

Effect of Chronic Methoxychlor Exposure in Mice on Brain Mitochondrial Respiration and H2O2 Production
Considering the effects of mxc on mitochondrial respiration and ROS production in vitro, experiments were performed to probe for possible effects of mxc on brain mitochondria in vivo. Female CD-1 mice were treated with mxc (0–64 mg/kg/day) in sesame oil via ip injection for 20 consecutive days prior to the mitochondrial isolation. This dose range was used because it produces follicular atresia (Borgeest et al., 2002Go, 2004Go) and oxidative injury to the testes (Latchoumycandane and Mathur, 2002Go). State 3 respiration with the complex I-linked substrates malate and glutamate was significantly lower for brain mitochondria isolated from the mxc-treated mice compared to those treated with the drug vehicle (p < 0.05; Fig. 4A). There was a similar trend toward inhibition for respiration using the complex II-linked substrate succinate with rotenone (p = 0.08; Fig. 4A). No significant changes in state 4o respiration were observed with either complex I- or complex II-linked substrates (p = 0.19 and p = 0.56, respectively; Fig. 4B).



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FIG. 4. Effects of in vivo methoxychlor treatment on mouse brain mitochondria respiring on different oxidizable substrates. A. Mean state 3 oxygen consumption rates in the presence of either L-malate (5 mM) plus L-glutamate (5 mM) or succinate (5 mM) and rotenone (1 µM) following in vivo mxc treatment (0–64 mg/kg/day) prior to mitochondrial isolation. B. Mean state 4o oxygen consumption rates following addition of 1.25 µg/ml oligomycin. Data are expressed as mean oxygen consumption rates (nmol oxygen/min/mg mitochondrial protein) and represent the mean ± SEM of four separate experiments per treatment group. *Significantly different (p < 0.05) from control.

 
Because in vivo mxc treatment resulted in the inhibition of brain mitochondrial respiration, we then determined whether in vivo treatment also results in stimulation of H2O2 production by isolated mitochondria. No significant differences in H2O2 production were observed across treatment groups with either complex I-linked (Fig. 5A), or complex II-linked substrates (Fig. 5B).



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FIG. 5. Fluorescent Amplex Red measurements of H2O2 production in isolated mouse brain mitochondria treated in vivo with methoxychlor. Mean H2O2 production rates following in vivo mxc treatment (0–64 mg/kg/day) prior to mitochondrial isolation in the presence of actively respiring mitochondria utilizing complex I-linked substrates (A) or complex II-linked substrates (B). Values represent mean H2O2 production rates (pmol/min/mg mitochondrial protein) ± SEM for four separate experiments per treatment group. No significant differences were observed between treatment groups and vehicle controls.

 
In Vitro Methoxychlor Treatment Increases the Phosphorylation State of Brain Mitochondrial Ca2+/cAMP Response Element Binding Protein (CREB)
Since it is well established in the literature that CREB can be phosphorylated and therefore activated following oxidative stress, we tested the hypothesis that CREB is a downstream target following mxc inhibition of mitochondrial respiration and stimulation of ROS production. At the end of the experiments measuring the effects of mxc on respiration, the mitochondrial suspensions were centrifuged and the mitochondrial pellet retrieved for ELISA measurements that are specific for the phosphorylated form of CREB (pCREB). As anticipated based on the stimulation of ROS production by mxc in the presence of malate plus glutamate, mxc also caused a significant increase in mitochondrial pCREB immunoreactivity (p < 0.05; Fig. 6A). Methoxychlor also caused a significant elevation of pCREB immunoreactivity in the presence of succinate plus rotenone, even though it did not stimulate ROS production under these conditions (p < 0.001; Fig. 6A).



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FIG. 6. Effects of methoxychlor on mitochondrial pCREB. A. pCREB levels (ng/mg mitochondrial protein) as assessed by ELISA following exposure of respiring rat brain mitochondria to in vitro mxc (0–10 µg/ml) treatment in experiments such as those shown in Figure 2A and B. Mitochondrial pCREB levels after incubation of rat brain mitochondria with mxc (0–10 µg/ml) in the absence of respiratory substrates and in the absence or presence of ATP (3 mM). C. pCREB levels (ng/mg mitochondrial protein) as assessed by ELISA after in vivo mxc (0–64 mg/kg/day) treatment in respiration experiments shown in Figure 4A. Values represent the means ± SEM for three to six separate experiments. *Significantly different (p < 0.05); **significantly different (p < 0.001) from control.

 
To further assess the effect of mxc on CREB phosphorylation, mitochondria were incubated in the absence and presence of mxc in the absence of exogenous respiratory substrates, a condition that virtually eliminates mitochondrial ROS formation (Fig. 3A and B). Additionally, ATP (3 mM) was present to act as a phosphate source for CREB phosphorylation. In the presence of ATP and the absence of respiratory substrates, pCREB immunoreactivity was significantly elevated by mxc (p < 0.001, Fig. 6B), whereas mxc had no effect on pCREB in the absence of ATP. These results indicate that while mxc increases mitochondrial pCREB immunoreactivity, the stimulation of mitochondrial ROS production by mxc is not required to trigger this response.

Similar measurements were performed with the mitochondrial samples obtained from the in vivo mxc-treated mouse experiments. No significant effects of in vivo mxc treatment on pCREB immunoreactivity were present following incubation of mitochondria in the presence of complex I- or complex II-linked substrates (p = 0.124 and p = 0.897, respectively; Fig. 6C).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study is, to our knowledge, the first to demonstrate the effects of methoxychlor on brain mitochondrial respiration and production of ROS. Our results comparing the effects of mxc on mitochondrial O2 consumption using oxidizable substrates that deliver electrons to different locations within the electron transport chain further define the regions of this pathway that are most affected by mxc. In vitro, mxc treatment significantly inhibits ADP-stimulated (state 3) respiration in the presence of the complex I-linked substrates malate plus glutamate and the complex II-linked substrate succinate (Fig. 2A), but it has no effect on O2 consumption when measured in the presence of ascorbate plus TMPD, which deliver electrons to complex IV via cytochrome c (Fig. 2A). In addition, non-synaptosomal mitochondria isolated from mxc-treated mice exhibit significantly inhibited state 3 respiration in the presence of complex I-linked substrates but not in the presence of succinate plus rotenone (Fig. 4A). Thus, electron flow through complex I and, to a lesser extent complex II or III, is sensitive to inhibition by mxc treatment in vitro whereas only flow through complex I is affected by mxc treatment in vivo.

In addition to the respiratory inhibition most evident using complex I-dependent substrates, in vitro mxc treatment significantly increases state 4o respiration in the presence of succinate plus rotenone (Fig. 2B). However, no significant effect on state 4o respiration was observed across treatment groups in mitochondria isolated from in vivo mxc-treated mice (Fig. 4B). Thus, because state 4o mitochondrial O2 consumption in the presence of the ATP synthase inhibitor oligomycin is limited by the rate of H+ influx across the inner membrane, stimulation of state 4o respiration by mxc is likely due to a nonspecific increase in the ion permeability of the inner membrane caused by this lipophilic compound. This uncoupling effect is not as apparent in the presence of malate plus glutamate, because it is counteracted by more extensive respiratory inhibition. This interpretation is supported by the finding that mxc significantly lowers the respiratory control ratio measured in the presence of these substrates. The effects of mxc on respiratory coupling are also reflected by the partial loss of mitochondrial membrane potential observed at 10 µg/ml mxc (Fig. 1B). In summary, mxc is a respiratory inhibitor, particularly in the presence of complex I-linked substrates, and it is also a relatively mild respiratory uncoupler. The dual adverse actions of mxc on mitochondrial respiration indicate that it has the potential to induce cell death through metabolic failure or through adverse effects on respiration-linked activities, e.g., superoxide formation.

Other toxicants, e.g., DDT and paraquat, which inhibit the normal flow of electrons through the mitochondrial electron transport chain, also have the potential for increasing mitochondrial ROS production (Byczkowski and Tluczkiewicz, 1978Go; Tawara et al., 1996Go). In our experiments, the rate of mitochondrial H2O2 production in the presence of malate plus glutamate and ADP was increased by more than 300% in the presence of 10 µg/ml mxc (Fig. 3C). The stimulation observed under state 3, but not state 4o conditions is consistent with the state 3–specific respiratory inhibition observed with mxc. Maximal ROS production in the presence of complex I–dependent substrates is observed in the presence of a saturating concentration of the pesticide rotenone (Fig. 3A). The observation that mxc slightly inhibits the ROS production in the presence of rotenone (Fig. 3B) indicates that the sites of action of these toxicants within complex I are different and mxc acts at a redox site proximal to the site targeted by rotenone. Methoxychlor inhibits rather than stimulates H2O2 production when using the complex II substrate succinate (Fig. 3D). As mitochondrial ROS generation is regulated by the mitochondrial membrane potential through its influence of the redox state of mitochondrial electron transport chain components, inhibition of succinate-supported H2O2 production is explained by the mild membrane depolarization known to inhibit ROS formation (Starkov and Fiskum, 2003Go). In summary, our direct measurements of the effect of mxc on H2O2 production by isolated brain mitochondria support the hypothesis that the toxicity of mxc is due in part to oxidative stress caused by its inhibition of electron flow through complex I of the electron transport chain. Moreover, our results extend the findings of Latchoumycandane and Mather (2002)Go by demonstrating that mxc-induced oxidative stress is not limited to the male reproductive system.

Although mxc stimulates mitochondrial ROS production in vitro, the rate of H2O2 production by mitochondria isolated from mice following in vivo mxc treatment was unchanged compared to vehicle-treated controls (Fig. 5). The presence of BSA in the mitochondrial isolation medium likely depletes the mitochondria of any residual mxc present in situ at the time the brain is removed. Thus, any mitochondrial alterations observed following isolation are the sequelae from the effects of mxc in vivo. Because complex I is sensitive to inhibition by ROS (Hillered and Ernster, 1983Go), stimulation of mitochondrial ROS formation by mxc present in the brains of chronically treated mice may result in oxidative modifications to complex I that are manifested as inhibition of state 3 respiration in the presence of malate plus glutamate. The absence of elevated H2O2 production by mitochondria isolated from mxc-treated animals indicates that the degree or nature of respiratory inhibition observed after mitochondrial isolation is insufficient to cause a detectable stimulation of ROS production. It is also possible that the metabolites of mxc generated in vivo have direct or indirect effects on brain mitochondria that are different from what we characterized for mxc in vitro. Further studies are therefore necessary to determine the mechanisms of action of mxc on brain mitochondria in vivo.

A unique finding of this study is that in vitro mxc treatment increases the immunoreactivity of phosphorylated CREB within mitochondria at mxc concentrations similar to those that both inhibit respiration and stimulate mitochondrial ROS production. Several labs have documented the presence of CREB and pCREB in mitochondria (Cammarota et al., 1999Go; Schuh et al., 2005Go; Ryu et al., 2003Go), and we recently demonstrated how the mitochondrial CREB phosphorylation state is regulated by both physiological and pathological levels of Ca2+ (Schuh et al., 2005Go). As oxidative stress can stimulate nuclear gene expression via an increase in cellular CREB phosphorylation, we hypothesized that the mitochondrial oxidative stress caused by mxc increases mitochondrial pCREB levels in vitro. After measurements of respiration, pCREB present within isolated brain mitochondria was measured. pCREB levels were significantly elevated in the presence of in vitro mxc-treated mitochondria respiring on either malate and glutamate or succinate (Fig. 6A). Because mxc inhibits ROS production with succinate as electron donor, we measured the effects of mxc on pCREB levels in the absence of oxidizable substrates where mitochondrial H2O2 production is negligible (Starkov and Fiskum, 2003Go). Methoxychlor increases mitochondrial pCREB immunoreactivity in the absence of respiration and ROS production, and the degree to which pCREB is elevated is greater than that observed in the presence of oxidizable substrates (Fig. 6B).

Because the results do not support the hypothesis that mxc-induced oxidative stress is responsible for increased mitochondrial CREB phosphorylation in vitro, the effect of mxc is likely due either to inhibition of a phosphatase or to activation of a kinase. We did not observe an effect of in vivo mxc treatment on mitochondrial pCREB immunoreactivity; however, the conditions used during the mitochondrial isolation procedure are not sufficient to clamp CREB phosphorylation at the state in which it exists in vivo. As studies implicate the cellular CREB pathway in the response of tissues other than brain to mxc and other organochlorines (Chuang and Chuang, 1998Go; Zhang and Teng, 2002Go), future experiments will determine if the phosphorylation state of mitochondrial and non-mitochondrial CREB in the brain are affected by mxc at the in vivo doses we found influence brain mitochondrial respiration.

The range of mxc concentrations that produce direct effects on mitochondrial respiration, ROS production, and CREB phosphorylation state are within the range of those that could feasibly exist in vivo at doses that elicit oxidative stress and cell death. The doses used in vivo range from approximately 20 to 200 mg/kg/day (Gray et al., 1989Go). If the tissue concentrations generated at these doses are only 1% of these levels, the total concentration is in the range of 0.2–2.0 µg/ml, i.e., comparable to the range of 0.5–10.0 µg/ml used in the in vitro mitochondrial experiments. It is impossible at this juncture to relate these levels to those that exist in humans, as virtually no data on mxc levels in human tissue are available.

It is well established that compromised mitochondrial respiration plays a role in initiation of apoptotic cascades. Additionally, several studies have suggested a relationship between defective energy metabolism and neurodegenerative diseases including Alzheimer's disease (Mutisya et al., 1994Go) and Parkinson's disease (Greenamyre et al., (1999)Go. Thus, the findings that mxc inhibits brain mitochondrial respiration, stimulates ROS production, and increases mitochondrial CREB phosphorylation warrant further investigation into the role that endocrine-disruptive compounds may play utilizing non-estrogenic mechanisms of action, including those present within mitochondria.


    ACKNOWLEDGMENTS
 
This work was supported by National Institute of Environmental Health Sciences (NIEHS) grant ES07263 to R.A.S., NIEHS grant ES13061–01 to J.A.F., National Institutes of Health (NIH) grant R21NS050653 to T.K., and NIH grants NS34152, HD016596, and U.S. Army Medical Research and Material Command Neurotoxin Research Program grant DAMD 17–99–1–9483 to G.F.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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