* Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, Maryland 21201; Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201;
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 534, Baltimore, MD 21201. Fax: (410) 706-2550. E-mail: gfisk001{at}umaryland.edu.
Received July 28, 2005; accepted September 14, 2005
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: methoxychlor; mitochondria; CREB; oxidative stress.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Botella et al. (2004) 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)
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, 1998) and endosulfan (Kannan and Jain, 2003
) have been demonstrated to play a role in cellular oxidative stress. Studies by Latchoumycandane and Mathur (2002)
demonstrated depletion of antioxidant enzymes in mitochondria and microsomes from rat testis following exposure to mxc. Additionally, Chen et al. (1999)
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., 2004
; Starkov et al., 2004
; Tawara et al., 1996
). The mitochondrial effects of environmental toxicants, e.g., rotenone, are likely responsible for their induction of neurodegeneration (Greenamyre et al., 1999
). 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., 2001; Tanaka, 2001
). 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., 2002
). CREB phosphorylation is implicated in the induction of transcriptional activity that stimulates the expression of antioxidant genes (Bedogni et al., 2003
). 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., 2003
). 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., 2005
).
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., 1999; Gore, A. C., 2002
; Lafuente et al., 2003
). 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2472 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., 2002, 2004
). 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). 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)
.
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 (010 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, 2003). 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 (010 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 M1 cm1.
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The doseresponse 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) assessing the effects of mxc on apoptosis in vitro. Exposure to mxc (010 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).
|
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, 2003), 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).
|
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 (064 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., 2002, 2004
) and oxidative injury to the testes (Latchoumycandane and Mathur, 2002
). 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).
|
|
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 1978; Tawara et al., 1996
). 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 3specific respiratory inhibition observed with mxc. Maximal ROS production in the presence of complex Idependent 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, 2003
). 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)
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, 1983), 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., 1999; Schuh et al., 2005
; Ryu et al., 2003
), and we recently demonstrated how the mitochondrial CREB phosphorylation state is regulated by both physiological and pathological levels of Ca2+ (Schuh et al., 2005
). 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, 2003
). 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, 1998; Zhang and Teng, 2002
), 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., 1989). If the tissue concentrations generated at these doses are only 1% of these levels, the total concentration is in the range of 0.22.0 µg/ml, i.e., comparable to the range of 0.510.0 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., 1994) and Parkinson's disease (Greenamyre et al., (1999)
. 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 |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Borgeest, C., Symonds, D., Mayer, L. P., Hoyer, P. B., and Flaws, J. A. (2002). Methoxychlor may cause ovarian follicular atresia and proliferation of the ovarian epithelium in the mouse. Toxicol. Sci. 68, 473478.
Borgeest, C., Miller, K. P., Gupta, R., Greenfeld, C., Hruska, K. S., Hoyer, P., and Flaws, J. A. (2004). Methoxychlor-induced atresia in the mouse involves Bcl-2 family members, but not gonadotropins or estradiol. Biol. Reprod. 70, 18281835.
Botella, B., Crespo, J., Rivas, A., Cerrillo, I., Olea-Serrano, M. F., and Olea, N. (2004). Exposure of women to organochlorine pesticides in Southern Spain. Environ. Res. 96, 3440.[CrossRef][ISI][Medline]
Byczkowski, J. Z., and Tluczkiewicz, J. (1978). Comparative study of respiratory chain inhibition by DDT and DDE in mammalian and plant mitochondria. Bull. Environ. Contam. Toxicol. 20, 505512.[CrossRef][ISI][Medline]
Cammarota, M., Paratcha, G., Bevilaqua, L., Levi de Stein, M., Lopez, M., Pellegrino de Iraldi, A., Izquierdo, I., and Medina, J. H. (1999). Cyclic AMP-responsive element binding protein in brain mitochondria. J. Neurochem. 72, 22722277.[CrossRef][ISI][Medline]
Chedrese, P. J., and Feyles, F. (2001). The diverse mechanism of action of dichlorodiphenyldichloroethylene (DDE) and methoxychlor in ovarian cells in vitro. Reprod. Toxicol. 15, 693698.[CrossRef][ISI][Medline]
Chen, J., Li, Y., Lavigne, J. A., Trush, M. A., and Yager, J. D. (1999). Increased mitochondrial superoxide production in rat liver mitochondria, rat hepatocytes and HepG2 cells following ethinyl estradiol treatment. Toxicol. Sci. 51, 224235.[Abstract]
Chuang, L. F., and Chuang, R. Y. (1998). Heptachlor and the mitogen-activated protein kinase module in human lymphocytes. Toxicology 128, 1723.[CrossRef][ISI][Medline]
Cooper, R. L., Goldman, J. M., and Stoker, T. E. (1999). Neuroendocrine and reproductive effects of contemporary-use pesticides. Toxicol. Ind. Health 15, 2636.[CrossRef][ISI][Medline]
Cummings, A. M. (1997). Methoxychlor as a model for environmental estrogens. Crit. Rev. Toxicol. 27, 367379.[ISI][Medline]
Gore, A. C. (2002). Organochlorine pesticides directly regulate gonadotropin-releasing hormone gene expression and biosynthesis in the GT1-7 hypothalamic cell line. Mol. Cell. Endocrinol. 192, 157170.[CrossRef][ISI][Medline]
Gray, L. E., Jr., Ostby, J., Ferrell, J., Rehnberg, G., Linder, R., Cooper, R., Goldman, J., Slott, V., and Laskey, J. (1989). A doseresponse analysis of methoxychlor-induced alterations of reproductive development and function in the rat. Fundam. Appl. Toxicol. 12, 92108.[CrossRef][ISI][Medline]
Greenamyre, J. T., MacKenzie, G., Peng, T. I., and Stephans, S. E. (1999). Mitochondrial dysfunction in Parkinson's disease. Biochem. Soc. Symp. 66, 8597.[Medline]
Hillered, L., and Ernster, L. (1983). Respiratory activity of isolated rat brain mitochondria following in vitro exposure to oxygen radicals. J. Cereb. Blood Flow Metabol. 3, 207214.[ISI][Medline]
Hodges, L. C., Bergerson, J. S., Hunter, D. S., and Walker, C. L. (2000). Estrogenic effects of organochlorine pesticides on uterine leiomyoma cells in vitro. Toxicol. Sci. 54, 355364.
Kannan, K., and Jain, S. K. (2003). Oxygen radical generation and endosulfan toxicity in Jurkat T-cells. Mol. Cell. Biochem. 247, 17.[CrossRef][ISI][Medline]
Lafuente, A., González-Carracedo, A., Romero, A., and Esquifino, A. I. (2003). Methoxychlor modifies the ultradian excretory pattern of prolactin and affects its TRH response. Med. Sci. Monit. 9, P155P160.
Latchoumycandane, C., and Mathur, P. P. (2002). Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis. Toxicology 176, 6775.[CrossRef][ISI][Medline]
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265275.
Mabuchi, T., Kitagawa, K., Kuwabara, K., Takasawa, K., Ohtsuki, T., Xia, Z., Storm, D., Yanagihara, T., Hori, M., and Matsumoto, M. (2001). Phosphorylation of cAMP response element-binding protein in hippocampal neurons as a protective response after exposure to glutamate in vitro and ischemia in vivo. J. Neurosci. 21, 92049213.
Meyer, M. J., Mosely, D. E., Amarnath, V., and Picklo, M. J. (2004). Metabolism of 4-hydroxy-trans-2-nonenal by central nervous system mitochondria is dependent on age and NAD+ availability. Chem. Res. Toxicol. 17, 12721279.[CrossRef][ISI][Medline]
Miller, K. P., Gupta, R. K., Greenfeld, C. R., Babus, J. K., and Flaws, J. A. (2005). Methoxychlor directly affects ovarian antral follicle growth and atresia through Bcl-2- and Bax-mediated pathways. Toxicol. Sci. Epub Aug. 4, 2005.
Mutisya, E. M., Bowling, A. C., and Beal, M. F. (1994). Cortical cytochrome oxidase activity is reduced in Alzheimer's disease. J. Neurochem. 63, 21792184.[ISI][Medline]
Okubo, T., Yokoyama, Y., Kano, K., Soya, Y., and Kano, I. (2004). Estimation of estrogenic and antiestrogenic activities of selected pesticides by MCF-7 cell proliferation assay. Arch. Environ. Contam. Toxicol. 46, 445453.[ISI][Medline]
Rudel, R. A., Camann, D. E., Spengler, J. D., Korn, L. R., and Brody, J. G. (2003). Phthalates, alkylphenols, pesticides, polybrominated diphenyl ethers, and other endocrine-disrupting compounds in indoor air and dust. Environ. Sci. Technol. 37, 45434553.[CrossRef][ISI][Medline]
Ryu, H., Lee, J., Simon, D. K., Aminova, A., Andreyev, A., Murphy, A., Ginty, D., Ferrante, R. J., and Ratan, R. R. (2003). Mitochondrial CREB regulates mitochondrial gene expression and neuronal survival. Soc. Neurosci. 207.3 (abstract).
Sahoo, A., and Chainy, G. B. (1998). Acute hexachlorocyclohexane-induced oxidative stress in rat cerebral hemisphere. Neurochem. Res. 23, 10791084.[CrossRef][ISI][Medline]
Schuh, R. A., Kristían, T., and Fiskum, G. (2005). Calcium-dependent dephosphorylation of brain mitochondrial calcium/cAMP response element binding protein (CREB). J. Neurochem. 92, 388394.[CrossRef][ISI][Medline]
Schuh, R. A., Lein, P. J., Beckles, R. A., and Jett, D. A. (2002). Noncholinesterase mechanisms of chlorpyrifos neurotoxicity: Altered phosphorylation of Ca2+/cAMP response element binding protein in cultured neurons. Toxicol. Appl. Pharmacol. 182, 176185.[CrossRef][ISI][Medline]
Shekhar, P. V., Werdell, J., and Basrur, V. S. (1997). Environmental estrogen stimulation of growth and estrogen receptor function in preneoplastic and cancerous human breast cell lines. J. Natl. Cancer Inst. 89, 17741782.[Abstract]
Sims, N. R. (1990). Rapid isolation of metabolically active mitochondria from rat brain and subregions using percoll density gradient centrifugation. J. Neurochem. 55, 698707.[ISI][Medline]
Starkov, A. A., and Fiskum, G. (2003). Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state. J. Neurochem. 86, 11011107.[CrossRef][ISI][Medline]
Starkov, A. A., Fiskum, G., Chinopoulos, C., Lorenzo, B. J., Browne, S. E., Patel, M. S., and Beal, M. F. (2004). Mitochondrial -ketoglutarate dehydrogenase complex generates reactive oxygen species. J. Neurosci. 24, 77797788.
Tanaka, K. (2001) Alteration of second messengers during acute cerebral ischemiaAdenylate cyclase, cyclic AMP-dependent protein kinase, and cyclic AMP response element binding protein. Prog. Neurobiol. 65, 173207.[CrossRef][ISI][Medline]
Tawara, T., Fukushima, T., Hojo, N., Isobe, A., Shiwaku, K., Setogawa, T., and Yamane, Y. (1996). Effects of paraquat on mitochondrial electron transport system and catecholamine contents in rat brain. Arch. Toxicol. 70, 585589.[CrossRef][ISI][Medline]
Zhang, Z., and Teng, C. T. (2002). Methoxychlor stimulates the mouse lactoferrin gene promoter through a GC-rich element. Biochem. Cell. Biol. 80, 2326.[CrossRef][ISI][Medline]
|