TCDD decreases ATP levels and increases reactive oxygen production through changes in mitochondrial F(0)F(1)-ATP synthase and ubiquinone

Abstract

Mitochondria generate ATP and participate in signal transduction and cellular pathology and/or cell death. TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) decreases hepatic ATP levels and generates mitochondrial oxidative DNA damage, which is exacerbated by increasing mitochondrial glutathione redox state and by inner membrane hyperpolarization. This study identifies mitochondrial targets of TCDD that initiate and sustain reactive oxygen production and decreased ATP levels. One week after treating mice with TCDD, liver ubiquinone (Q) levels were significantly decreased, while rates of succinoxidase and Q-cytochrome c oxidoreductase activities were increased. However, the expected increase in Q reduction state following TCDD treatment did not occur; instead, Q was more oxidized. These results could be explained by an ATP synthase defect, a premise supported by the unusual finding that TCDD lowers ATP/O ratios without concomitant changes in respiratory control ratios. Such results suggest either a futile cycle in ATP synthesis, or hydrolysis of newly synthesized ATP prior to release. The TCDD-mediated decrease in Q, concomitant with an increase in respiration, increases complex 3 redox cycling. This acts in concert with glutathione to increase membrane potential and reactive oxygen production. The proposed defect in ATP synthase explains both the greater respiratory rates and the lower tissue ATP levels.

Fig. 1. UCP2 expression and function

Mitochondria were prepared from mouse liver, 7 days after treatment with corn oil (open circles) or TCDD (closed circles). A Western immunoblot using a UCP2 antibody was performed using mitochondrial protein from 3 corn oil-treated mice (the left 3 blots) and from 3 TCDD-treated mice (the right 3 blots). The influence of effectors of UCP2 on H₂O₂ production was estimated using luminol chemiluminescence (center row, expressed as luminescence units X 10⁻³ min⁻¹mg⁻¹). The fatty acid oleate activates UCP2, while the purine nucleotide GDP inhibits. The influence of effectors of UCP2 on mitochondrial membrane potential was determined using JC-1 fluorescence ratios (lower row). A fluorescence ratio of 4 is approximately the equivalent of –180 mV. Data are presented as the mean value ± S.E. (n = 3). Data were evaluated statistically using a two-way ANOVA, with the factors being TCDD and GDP concentration (left panels), and TCDD and oleate concentration (right panels). A three-way ANOVA was also performed and discussed in the Results section, with factors being oleate concentration, GDP concentration and TCDD treatment. *P<0.05 versus 0 μM oleate (left panels), and versus 0 μM GDP (right panels), using Student-Newman-Keuls test for pairwise comparison.

Fig. 2. Mitochondrial enzyme activities after TCDD treatment

Electron transport activities for different regions of the respiratory chain were determined in liver mitochondria prepared from corn oil-treated (open bars) or TCDD-treated (closed bars) mice. Activities are expressed as nmol min⁻¹ mg protein⁻¹ for SOX (succinoxidase), SCR (succinate cytochrome c reductase), SDH (succinate dehydrogenase), SQR (succinate ubiquinol reductase) and QCR (ubiquinol cytochrome c reductase). Activity is expressed as a first order rate constant [s⁻¹ mg protein⁻¹] for COX (cytochrome oxidase). Data are presented as the mean value ± S.E. (n = 6). A t-test was used to evaluate statistical significance of differences between group sample mean values. *P<0.05 versus vehicle-treated mice.

Fig. 3. Effects of TCDD treatment on mitochondrial ubiquinones Q₉ and Q₁₀

Liver mitochondria were prepared from corn oil-treated (open bars) or TCDD-treated (closed bars) mice. The levels of mitochondrial oxidized and reduced Q₉ and Q₁₀ were determined by HPLC (left panel), and the percent reduced Q (right panel) were calculated. Data are presented as the mean value ± S.E. (n = 6). A t-test was used to evaluate statistical significance of differences between group sample mean values. *P<0.05 versus vehicle-treated mice.

Fig. 4. Effect of TCDD treatment on mitochondrial membrane fluidity

Mitochondria were prepared from mouse liver 7 days after treatment with corn oil (open bars) or TCDD (closed bars). Mitochondrial membrane fluidity was calculated as the fluorescence polarization anisotropy, determined using the fluorescence probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl- 1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), to probe the hydrophobic lipid interior, and the membrane surface, respectively. Data are presented as the mean value ± S.E. (n = 6). A t-test was used to evaluate statistical significance of differences between group sample mean values. *P<0.05 versus vehicle-treated mice.

Fig. 5. Relationship between mitochondrial respiration, membrane potential and H₂O₂ production

Mitochondria were partially uncoupled by exposure to increasing concentrations of free Ca⁺⁺. The concentration of free Ca⁺⁺ was controlled using EGTA buffer in respiratory buffer (KCl-RB), and free Ca⁺⁺ was calculated assuming a Ca⁺⁺-EGTA K_d = 327 nM. Data were evaluated statistically using two-way ANOVAs, with the factors being [Ca⁺⁺] and JC-1 fluorescence, and [Ca⁺⁺] and RCR. *P<0.05 versus either JC-1 fluorescence or RCR at the same [Ca⁺⁺], using Student-Newman-Keuls test for pairwise comparison.

Fig. 6. Models for proposed electron transfer and F₀F₁-ATP synthase pathways

TCDD is proposed to decrease the efficiency of ATP formation catalyzed by the β subunit of F₁ within the F₀F₁-ATP synthase complex. Respiration would still depend on ADP, and RCR would remain unchanged. However, ATP/O ratio would decrease due to uncoupling. Electrons from complex 2 feed into complex 3, primarily utilizing Q₁₀, from which electrons flow to Q₉ and enter the proton-motive Q cycle. An oxidation state crossover point exists within complex 3, somewhere between Q₁₀ and Q₉. Abbreviations: AA, antimycin-A; b_L and b_H, respectively, low energy and high energy forms of cytochrome b; ISPs, iron-sulfur proteins; SDH, succinate dehydrogenase.

Fig. 7. Proposed mechanism for the TCDD-mediated decrease in Q levels

TCDD regulates the expression level for several genes encoding enzymes involved in retinoic acid (RA) metabolism. The resultant increase in trans-RA and decrease in cis-RA activate the trans-retinoic acid receptor (RAR) and inhibit activation of the cis-retinoic acid receptor (RXR), resulting in the inhibition of the rate of synthesis for HMG-CoA, a precursor for Q.