Measurement of the mitochondrial membrane potential and pH gradient from the redox poise of the hemes of the bc1 complex.

The redox potentials of the hemes of the mitochondrial bc(1) complex are dependent on the proton-motive force due to the energy transduction. This allows the membrane potential and pH gradient components to be calculated from the oxidation state of the hemes measured with multi-wavelength cell spectroscopy. Oxidation states were measured in living RAW 264.7 cells under varying electron flux and membrane potential obtained by a combination of oligomycin and titration with a proton ionophore. A stochastic model of bc(1) turnover was used to confirm that the membrane potential and redox potential of the ubiquinone pool could be measured from the redox poise of the b-hemes under physiological conditions assuming the redox couples are in equilibrium. The pH gradient was then calculated from the difference in redox potentials of cytochrome c and ubiquinone pool using the stochastic model to evaluate the ΔG of the bc(1) complex. The technique allows absolute quantification of the membrane potential, pH gradient, and proton-motive force without the need for genetic manipulation or exogenous compounds.

Spectral components of the α-band of cytochrome oxidase.

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high (a(H)) or low (a(L)) midpoint potential (E(m)) by comparing their occupancy during redox titrations carried out when the membrane potential (ΔΨ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a(L) has a maximum at 605nm whereas the spectrum of a(H) has a maximum at 602nm. The ΔΨ-dependent shift in the E(m) of a(H) was too great to be accounted for by electron transfer from cytochrome c to heme a against ΔΨ but was consistent with a model in which a(H) is formed after proton uptake against ΔΨ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E(m) changes during redox titrations. The redox anti-cooperativity between heme a and heme a(3), and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a(3) with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.

Prooxidant-antioxidant shift induced by androgen treatment of human prostate carcinoma cells.

BACKGROUND: Prostate cancer is a disease associated with aging. Also commonly associated with increasing age is a shift in the prooxidant-antioxidant balance of many tissues toward a more oxidative state, i.e., increased oxidative stress. We hypothesize that androgen exposure, which has long been associated with the development of prostate cancer, may be a means by which the prooxidant-antioxidant balance of prostate cells is altered. PURPOSE: Using established prostate carcinoma cell lines, we studied the effect of androgens on various parameters of oxidative state (e.g., generation of hydrogen peroxide and hydroxyl radicals, lipid peroxidation, and oxygen consumption) and antioxidant defense mechanisms (e.g., the glutathione system and catalase). METHODS: The androgen-responsive LNCaP and the androgen-independent DU145 prostate carcinoma cell lines were exposed to 5 alpha-dihydrotestosterone (DHT) and to the synthetic androgen R1881. The cellular proliferation responses were measured by use of a fluorometric assay to quantitate the amount of DNA. The generation of reactive oxygen species was measured by use of 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals. Lipid peroxidation was quantitated by use of a chromogen specific for malonaldehyde and 4-hydroxy-2(E)-nonenal. General mitochondrial activity was determined by assaying 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. A Clark-type electrode was used to assess oxygen consumption per cell. Intracellular glutathione concentrations and the activities of catalase and gamma-glutamyl transpeptidase were measured spectrophotometrically. All P values resulted from two-sided tests. RESULTS: DHT at less than 1 to 100 nM (a concentration range encompassing the physiologic levels of DHT considering all ages) and R1881 at 0.1-1 nM concentrations were effective in inducing in LNCaP cells comparable proliferative responses and changes in oxidative stress. In contrast, neither DHT nor R1881 had any effect on the oxidative stress in DU145 cells. The mitochondrial activity in LNCaP cells, as measured by MTT reduction, was significantly elevated above the levels of the untreated controls by DHT (0.1-1000 nM) and R1881 (0.05-1 nM) (P < .001 in both). Oxygen consumption and catalase activity were increased in LNCaP cells in the presence of 1 nM R1881 by 60% and 40%, respectively, over the values in the untreated control cells (P < .03 and P < .01, respectively). The same concentration of R1881 resulted in a decrease in intracellular glutathione concentrations and an increase in gamma-glutamyl transpeptidase activity in LNCaP cells. Treatment with the oxidizing agents H2O2 and menadione produced an increase in gamma-glutamyl transpeptidase activity in LNCaP cells, whereas treatment with the antioxidant compound ascorbic acid (100 mM) reduced the oxidative stress produced in LNCaP cells by 1 nM R1881 and completely blocked the gamma-glutamyl transpeptidase activity. CONCLUSIONS: Physiologic levels of androgens are capable of increasing oxidative stress in androgen-responsive LNCaP prostate carcinoma cells. The evidence suggests that this result is due in part to increased mitochondrial activity. Androgens also alter intracellular glutathione levels and the activity of certain detoxification enzymes, such as gamma-glutamyl transpeptidase, that are important for maintenance of the cellular prooxidant-antioxidant balance.

Effect of antioxidants on androgen-induced AP-1 and NF-kappaB DNA-binding activity in prostate carcinoma cells.

BACKGROUND: Previous studies have suggested that male hormones (androgens) and certain forms of oxygen (reactive oxygen species) are linked to the development of prostate cancer. We hypothesized that androgens contribute to prostate carcinogenesis by increasing oxidative stress. We further hypothesized that antioxidants reduce prostate cancer risk by modulating androgen effects on cellular processes. METHODS: To test these hypotheses, we looked for 1) a change in the level of reactive oxygen species in the presence of androgens, 2) androgen-induced binding activity of transcriptional activators AP-1 and NF-kappaB, whose activities are known to be altered during cell proliferation, and 3) the effect of antioxidants on androgen-induced transcription factor binding. RESULTS: Physiologic concentrations (1 nM) of 5alpha-dihydrotestosterone or 1-10 nM R1881, a synthetic androgen, produced sustained elevation of AP-1 and NF-kappaB DNA-binding activity in LNCaP cells, an androgen-responsive human prostate carcinoma cell line. Androgen-independent DU145 cells (another human prostate carcinoma cell line) were unaffected by R1881 treatment. AP-1-binding activity increased 5 hours after 1 nM R1881 treatment; NF-kappaB DNA-binding activity increased after 36 hours. Both activities remained elevated for at least 120 hours. Nuclear AP-1 and NF-kappaB protein levels were not elevated. Antioxidant vitamins C plus E blocked both androgen-induced DNA-binding activity and production of reactive oxygen species. CONCLUSION: Physiologic concentrations of androgens induce production of reactive oxygen species and cause prolonged AP-1 and NF-kappaB DNA-binding activities, which are diminished by vitamins C and E.

Alteration of glyceraldehyde-3-phosphate dehydrogenase activity and messenger RNA content by androgen in human prostate carcinoma cells.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) activity and mRNA content is altered in the androgen-responsive human prostate carcinoma cell line LNCaP after exposure to the synthetic androgen R1881. Elevation in GAPDH activity is noted as early as 24 h after treatment with 1 nM R1881 and lasts at least 96 h. R1881 has no effect on the activity of GAPDH in androgen-independent DU145 cells. LNCaP GAPDH mRNA content is lowered by treatment with 1 nM R1881; the magnitude of reduction appears to depend on the length of exposure. The results present at least one means by which androgen-responsive tissues may develop alterations in GAPDH mRNA or activity, as is found in certain tumor tissues.

Alteration in gamma-glutamyl transpeptidase activity and messenger RNA of human prostate carcinoma cells by androgen.

Concentrations of the synthetic androgen R1881 that correspond to physiologically relevant concentrations of 5alpha-dihydrotestosterone are capable of altering the activity of gamma-glutamyl transpeptidase (GGT) in human prostate carcinoma cells. GGT activity of the androgen-responsive prostate cancer cell line LNCaP increases >50% above that of the control after a 72-h exposure to 1 nM R1881. This elevation in GGT activity occurs as early as 48 h after treatment and is maintained for at least 96 h. Loss of glutathione (GSH) from media and accumulation of intracellular GSH of cells pretreated with 1 nM R1881 occur at a higher rate than in control cells, suggesting that a greater rate of GSH salvage is associated with the increased GGT activity. Immunohistochemical staining detects an increase in GGT-positive staining in cells treated with 1 nM R1881 for 72 h. Steady-state mRNA levels for GGT are elevated above those of the control 24-72 h after treatment. R1881 has no effect on the GGT activity of the androgen-independent prostate cell line DU145. Growth of LNCaP but not DU145 cells is inhibited by 1 nM R1881 compared to that of the control. Inhibitors of GGT activity, acivicin and serine-borate, are capable of dampening or blocking the effect of R1881 on growth. Growth of LNCaP cells treated with 1 nM R1881 plus 100 mM glycylglycine, a stimulator of GGT activity, is inhibited to a greater extent than the growth of LNCaP cells treated with R1881 alone. These data demonstrate that androgens can elevate GGT activity and increase GGT mRNA and protein levels in human prostate carcinoma cells. In addition, compounds able to alter GGT activity are capable of altering androgen-related growth effects.

Androgen-induced oxidative stress in human LNCaP prostate cancer cells is associated with multiple mitochondrial modifications.

We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.

Measurement of the oxidation state of mitochondrial cytochrome c from the neocortex of the mammalian brain.

Diffuse optical remission spectra from the mammalian neocortex at visible wavelengths contain spectral features originating from the mitochondria. A new algorithm is presented, based on analytically relating the first differential of the attenuation spectrum to the first differential of the chromophore spectra, that can separate and calculate the oxidation state of cytochrome c as well as the absolute concentration and saturation of hemoglobin. The algorithm is validated in phantoms and then tested on the neocortex of the rat during an anoxic challenge. Implementation of the algorithm will provide detailed information of mitochondrial oxygenation and mitochondrial function in physiological studies of the mammalian brain.

Adult-onset energy restriction of rhesus monkeys attenuates oxidative stress-induced cytokine expression by peripheral blood mononuclear cells.

We previously reported that energy restriction (ER) of mice attenuated age-associated increases in serum levels of interleukin-6 (IL-6). Here, we studied peripheral blood mononuclear cells (PBMC) from male rhesus monkeys to investigate the following: 1) the production of IL-6 and other cytokines become dysregulated with aging; 2) ER influences cytokine production and mRNA expression; and, 3) oxidative stress, as induced in vitro by xanthine and xanthine oxidase (X/XOD), influences cytokine mRNA and protein levels. Two types of comparisons were made as follows: 1) between normally fed young (6-9 y) and old monkeys (22-33 y); and 2) between middle-aged monkeys (15-21 y) fed either a normal energy intake or subjected to ER (for 5.5 y at 30% less than base-line intake). IL-6 protein levels and X/XOD-induced IL-6 mRNA levels in PBMC from old monkeys were significantly greater than those in PBMC from young animals. In contrast, interleukin-1beta (IL-1beta) and interleukin-8 mRNA levels were not strongly influenced by advancing age. X/XOD, which increased levels of protein carbonyls (indicative of oxidative damage) in PBMC, induced the expression of all three cytokines. ER reduced IL-6 protein and mRNA levels induced by X/XOD and the unstimulated mRNA levels of IL-1beta. These results indicate that, in a nonhuman primate model, oxidative stress may contribute to age-associated increases in the levels of certain cytokines and that adult-onset ER partially ameliorates this alteration.