Mitochondrial Oxygen Consumption by the Foreskin and its Fibroblast-rich Culture.

OBJECTIVES: This study investigated the feasibility of using a phosphorescence oxygen analyser to measure cellular respiration (mitochondrial O2 consumption) in foreskin samples and their fibroblast-rich cultures. METHODS: Foreskin specimens from normal infants were collected immediately after circumcision and processed for measuring cellular respiration and for culture. Cellular mitochondrial O2 consumption was determined as a function of time from the phosphorescence decay of the Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. RESULTS: In sealed vials containing a foreskin specimen and glucose, O2 concentration decreased linearly with time, confirming the zero-order kinetics of O2 consumption by cytochrome oxidase. Cyanide inhibited O2 consumption, confirming that the oxidation occurred mainly in the mitochondrial respiratory chain. The rate of foreskin respiration (mean ± SD) was 0.074 ± 0.02 µM O2 min(-1) mg(-1) (n = 23). The corresponding rate for fibroblast-rich cultures was 9.84 ± 2.43 µM O2 min(-1) per 10(7) cells (n = 15). Fibroblast respiration was significantly lower in a male infant with dihydrolipoamide dehydrogenase gene mutations, but normalised with the addition of thiamine or carnitine. CONCLUSION: The foreskin and its fibroblast-rich culture are suitable for assessment of cellular respiration. However, the clinical utility of foreskin specimens to detect disorders of impaired cellular bioenergetics requires further investigation.

The phosphorescence oxygen analyzer as a screening tool for disorders with impaired lymphocyte bioenergetics.

This study aimed to show the feasibility of using the phosphorescence oxygen analyzer to screen for clinical disorders with impaired cellular bioenergetics. [O(2)] was determined as function of time from the phosphorescence decay of Pd (II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. In sealed vials, O(2) consumption by peripheral blood mononuclear cells was linear with time, confirming its zero-order kinetics. Cyanide inhibited O(2) consumption, confirming the oxidation occurred in the mitochondrial respiratory chain. The rate of respiration (mean±SD, in µM O(2) per min per 10(7) cells, set as the negative of the slope of [O(2)] vs. t) for adults was 2.1±0.8 (n=18), for children 2.0±0.9 (n=20), and for newborns (umbilical cord samples) 0.8±0.4 (n=18), p<0.0001. For an 8-year-old patient with reduced NADH dehydrogenase and pyruvate dehydrogenase activities in the muscle, the rate was 0.7±0.2 (n=3) µM O(2) per min per 10(7) cells. For a 3-month-old patient with hepatocerebral mitochondrial DNA depletion syndrome (MDS) with confirmed mutations in the MPV17 gene, the rate was 0.6µM O(2) per min per 10(7) cells. For an18 month-old patient with MDS and confirmed mutations in the POLG gene, the rate was 0.5 µM O(2) per min per 10(7) cells. For a 6-year-old patient with MDS and confirmed mutations in the POLG gene, the rate was 0.6 µM O(2) per min per 10(7) cells. For 1-week-old patient with congenital lactic acidemia and hypotonia (confirmed mutations in DLD gene), the rate was 1.5 µM O(2) per min per 10(7) cells. For three siblings (9-year-old male, 8-year-old male and 2-month-old female) with congenital progressive myopathy, the rates were 0.9, 0.6 and 1.2 µM O(2) per min per 10(7) cells, respectively. Four patients with congenital lactic acidemia (with inadequate work-up) were also studied; their rates were 0.2, 1.5, 0.3 and 1.7 µM O(2) per min per 10(7) cells. This novel approach permits non-invasive, preliminary assessment of cellular bioenergetics. Potential applications and limitations of this technique are discussed.

Measurement of oxygen consumption by murine tissues in vitro.

INTRODUCTION: A novel in vitro system was developed to measure O2 consumption by murine tissues over several hours. METHODS: Tissue specimens (7-35 mg) excised from male Balb/c mice were immediately immersed in ice-cold Krebs-Henseleit buffer, saturated with 95% O2:5% CO2. The specimens were incubated at 37 °C in the buffer, continuously gassed with O2:CO2 (95:5). [O2] was determined as a function of time from the phosphorescence decay rates (1/τ) of Pd(II) meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin. The values of 1/τ were linear with [O2]: 1/τ=1/τo + kq [O2]; 1/τo=the decay rate for zero O2, kq=the rate constant in s⁻¹ µM⁻¹. RESULTS: NaCN inhibited O2 consumption, confirming oxidation occurred in the mitochondrial respiratory chain. The rate of respiration in lung specimens incubated in vitro for 3.9≤t≤12.4 h was 0.24±0.03 µM O2 min⁻¹ mg⁻¹ (mean±SD, n=28). The corresponding rate for the liver was 0.27±0.13 (n=11, t≤4.7 h), spleen 0.28± 0.07 (n=10, t≤5h), kidney 0.34±0.12 (n=7, t≤5h) and pancreas 0.35±0.09 (n=10, t≤4h). Normal tissue histology at hour 5 was confirmed by light and electron microscopy. There was negligible number of apoptotic cells by caspase 3 staining. DISCUSSION: This approach allows accurate assessment of tissue bioenergetics in vitro.

Biocompatibility of calcined mesoporous silica particles with cellular bioenergetics in murine tissues.

A novel in vitro system was developed to investigate the effects of two forms of calcined mesoporous silica particles (MCM41-cal and SBA15-cal) on cellular respiration of mouse tissues. O(2) consumption by lung, liver, kidney, spleen, and pancreatic tissues was unaffected by exposure to 200 µg/mL MCM41-cal or SBA15-cal for several hours. Normal tissue histology was confirmed by light microscopy. Intracellular accumulation of the particles in the studied tissues was evident by electron microscopy. The results show reasonable in vitro biocompatibility of the mesoporous silicas with murine tissue bioenergetics.

Cannabinoids inhibit cellular respiration of human oral cancer cells.

BACKGROUND AND PURPOSE: The primary cannabinoids, Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and Delta(8)-tetrahydrocannabinol (Delta(8)-THC) are known to disturb the mitochondrial function and possess antitumor activities. These observations prompted us to investigate their effects on the mitochondrial O(2) consumption in human oral cancer cells (Tu183). This epithelial cell line overexpresses bcl-2 and is highly resistant to anticancer drugs. EXPERIMENTAL APPROACH: A phosphorescence analyzer that measures the time-dependence of O(2) concentration in cellular or mitochondrial suspensions was used for this purpose. KEY RESULTS: A rapid decline in the rate of respiration was observed when Delta(9)-THC or Delta(8)-THC was added to the cells. The inhibition was concentration-dependent, and Delta(9)-THC was the more potent of the two compounds. Anandamide (an endocannabinoid) was ineffective; suggesting the effects of Delta(9)-THC and Delta(8)-THC were not mediated by the cannabinoidreceptors. Inhibition of O(2) consumption by cyanide confirmed the oxidations occurred in the mitochondrial respiratory chain. Delta(9)-THC inhibited the respiration of isolated mitochondria from beef heart. CONCLUSIONS AND IMPLICATIONS: These results show the cannabinoids are potent inhibitors of Tu183 cellular respiration and are toxic to this highly malignant tumor.

Oxygen measurements via phosphorescence.

Accurate measurements of dissolved O(2) as a function of time have numerous chemical and biological applications. The Pd (II) complex of meso-tetra-(4-sulfonatophenyl)-tetrabenzoporphyrin (Pd phosphor) was used for this purpose. Detection is based on the principle that the phosphorescence of this oxygen probe is inversely related to dissolved O(2) (O(2) quenches the phosphorescence). Biologic samples containing the Pd phosphor were flashed (10/s) with a peak output of 625nm; emitted light was detected at 800nm. Amplified pulses of phosphorescence were digitized at 1-2MHz using an analog/digital converter (PCI-DAS 4020/12 I/O Board) with outputs ranging from 1 to 20MHz. Assessment revealed a customized program was necessary. Pulses were captured using a developed software at 0.1-4MHz, depending on the speed of the computer. O(2) concentration was calculated by fitting to an exponential the decay of the phosphorescence. Twelve tasks were identified, which allowed full control and customization of the data acquisition, storage and analysis. The program used Microsoft Visual Basic 6 (VB6), Microsoft Access Database 2007, and a Universal Library component that allowed direct reading from the PCI-DAS 4020/12 I/O Board. It involved a relational database design to store experiments, pulses and pulse metadata, including phosphorescence decay rates. The method permitted reliable measurements of cellular O(2) consumption over several hours.

Cannabinoids inhibit the respiration of human sperm.

OBJECTIVE: To investigate the effects of the psychotropic compounds Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and Delta(8)-tetrahydrocannabinol (Delta(8)-THC) on sperm mitochondrial O(2) consumption (respiration). SETTING: State University of New York Upstate Medical University, Syracuse, New York. PATIENT(S): Forty-one men who visited the andrology laboratory for fertility evaluation. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): A phosphorescence analyzer that measures O(2) concentration in sperm suspensions as a function of time was used to measure respiration. RESULT(S): An immediate decline in the rate of respiration was observed when Delta(9)-THC or Delta(8)-THC was added to washed sperm. The inhibition was concentration dependent, and Delta(9)-THC was the more potent of the two compounds. Respiration was much less affected when Delta(9)-THC or Delta(8)-THC was added to neat semen, suggesting the presence of protective factors in seminal plasma. Both compounds inhibited the respiration of isolated mitochondria, illustrating that direct mitochondrial damage is likely the primary mechanism of action. CONCLUSION(S): The two main active cannabinoids of the marijuana plant, Delta(9)- and Delta(8)-THC, are potent inhibitors of mitochondrial O(2) consumption in human sperm. These findings emphasize the adverse effects of these toxins on male fertility. The cytoprotective capacity of seminal plasma deserves further investigation.

Mesoporous silica nanoparticles inhibit cellular respiration.

We studied the effect of two types of mesoporous silica nanoparticles, MCM-41 and SBA-15, on mitochondrial O 2 consumption (respiration) in HL-60 (myeloid) cells, Jurkat (lymphoid) cells, and isolated mitochondria. SBA-15 inhibited cellular respiration at 25-500 microg/mL; the inhibition was concentration-dependent and time-dependent. The cellular ATP profile paralleled that of respiration. MCM-41 had no noticeable effect on respiration rate. In cells depleted of metabolic fuels, 50 microg/mL SBA-15 delayed the onset of glucose-supported respiration by 12 min and 200 microg/mL SBA-15 by 34 min; MCM-41 also delayed the onset of glucose-supported respiration. Neither SBA-15 nor MCM-41 affected cellular glutathione. Both nanoparticles inhibited respiration of isolated mitochondria and submitochondrial particles.

Caspase activation by anticancer drugs: the caspase storm.

This study measures the time-dependence of cellular caspase activation by anticancer drugs and compares it with that of cellular respiration. Intracellular caspase activation and cellular respiration were measured during continuous exposure of Jurkat, HL-60, and HL-60/MX2 (deficient in topoisomerase-II) cells to dactinomycin, doxorubicin, and the platinum (Pt) compounds cisplatin, carboplatin, and oxaliplatin. Caspase activation was measured using the fluorogenic compound N-acetyl-asp-glu-val-asp-7-amino-4-trifluoromethyl coumarin (Ac-DEVD-AFC). We show that this substrate rapidly enters cells where it is efficiently cleaved at the aspartate residue by specific caspases, yielding the fluorescent compound 7-amino-4-trifluoromethyl coumarin (AFC). Following cell disruption, released AFC was separated on HPLC and detected by fluorescence. The appearance of AFC in cells was blocked by the pancaspase inhibitor benzyloxycarbonyl-val-ala-asp-fluoromethylketone, thus establishing that intracellular caspases were responsible for the cleavage. Caspase activity was first noted after about 2 h of incubation with doxorubicin or dactinomycin, the production of AFC being linear with time afterward. Caspase activation by doxorubicin was delayed in HL-60/MX2 cells, reflecting the critical role of topoisomerase-II in doxorubicin cytotoxicity. For both drugs, caspase activity increased rapidly between approximately 2 and approximately 6 h, went through a maximum, and decreased after approximately 8 h ("caspase storm"). Cisplatin treatment induced noticeable caspase activity only after approximately 14 h of incubation, and the fluorescent intensity of AFC became linear with time at approximately 16 h. Exposure of the cells to all of the drugs studied led to impaired cellular respiration and decreased cellular ATP, concomitant with caspase activation. Thus, the mitochondria are rapidly targeted by active caspases.

Dactinomycin impairs cellular respiration and reduces accompanying ATP formation.

The effect of dactinomycin on cellular respiration and accompanying ATP formation was investigated in Jurkat and HL-60 cells. Cellular mitochondrial oxygen consumption (measured by a homemade phosphorescence analyzer) and ATP content (measured by the luciferin-luciferase bioluminescence system) were determined as functions of time t during continuous exposure to the drug. The rate of respiration, k, was the negative of the slope of [O2] versus t. Oxygen consumption and ATP content were diminished by cyanide, confirming that both processes involved oxidations in the mitochondrial respiratory chain. In the presence of dactinomycin, k decreased gradually with t, the decrease being more pronounced at higher drug concentrations. Cellular ATP remained constant for 5 h in untreated cells, but in the presence of 20 microM dactinomycin it decreased gradually (to one-tenth the value at 5 h for untreated cells). The drug-induced inhibition of respiration and decrease in ATP were blocked by the pancaspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethyl ketone (zVAD-fmk). A rapid but temporary decrease in cellular ATP observed on the addition of zVAD-fmk was shown to be due to DMSO (added with zVAD-fmk). The effect of dactinomycin on respiration differed from that of doxorubicin. Plots of [O2] versus t were curved for dactinomycin so that k decreased gradually with t. The corresponding plots for doxorubicin were well fit by two straight lines; so k was constant for approximately 150 min, at which time k decreased, remaining constant at a lower level thereafter. The results for cells treated with mixtures of the two drugs indicated that the drugs acted synergistically. These results show the onset and severity of mitochondrial dysfunction in cells undergoing apoptosis induced by dactinomycin.

Inhibition of cellular respiration by doxorubicin.

Doxorubicin executes apoptosis, a process known to produce leakage of cytochrome c and opening of the mitochondrial permeability transition pores. To define the loss of mitochondrial function by apoptosis, we monitored cellular respiration during continuous exposure to doxorubicin. A phosphorescence analyzer capable of stable measurements over at least 5 h was used to measure [O(2)]. In solutions containing glucose and cells, [O(2)] declined linearly with time, showing that the kinetics of oxygen consumption was zero order. Complete inhibition of oxygen consumption by cyanide indicated that oxidations occurred in the respiratory chain. A decline in the rate of respiration was evident in Jurkat and HL-60 cells exposed to doxorubicin. The decline was abrupt, occurring after about 2 h of incubation. The inhibition was concentration-dependent and was completely blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Respiration in resistant HL-60/MX2 cells, characterized by an altered topoisomerase II activity, was not inhibited by doxorubicin. A decline in cellular ATP was measured in Jurkat cells after 2-4 h of incubation with 20 microM doxorubicin, paralleling the decline in respiration rate. Thus, cells incubated with doxorubicin exhibit caspase-mediated inhibition of oxidative phosphorylation.

Enhanced cellular respiration in cells exposed to doxorubicin.

Doxorubicin executes topoisomerase II mediated apoptosis, a process known to result in mitochondrial dysfunction, such as the leakage of cytochrome c and the opening of mitochondrial permeability transition pores (PTP). To further define the effects of doxorubicin on cell metabolism, we measured cellular respiration, cellular ATP, DNA fragmentation, and cytochrome c leakage in Jurkat (supersensitive), human leukemia-60 (HL-60, sensitive), and HL-60/MX2 (resistant) cells following exposure to 1.0 microM doxorubicin for 30 min. The measurements were made after 24 h of exposure to the drug. In Jurkat and HL-60 cells, doxorubicin treatment increased cellular mitochondrial oxygen consumption and ATP content by 2-3-fold. The increment in oxygen consumption was blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone (zVAD-fmk) and by the PTP inhibitor cyclosporin A. In HL-60/MX2 cells, which are resistant because of a reduced topoisomerase II activity, doxorubicin treatment was without effect on either respiration or ATP content, suggesting that topoisomerase II was essential for induction of apoptosis and stimulation of respiration and ATP content. The conclusion that both of the latter processes were products of oxidations in the mitochondrial respiratory chain was supported by the further observation that rotenone and sodium cyanide inhibited oxygen consumption and substantially lowered ATP content in the treated and untreated cells. Thus, oxidative phosphorylation is enhanced in cells briefly incubated with doxorubicin for as long as 24 h post drug exposure despite apoptosis-associated mitochondrial insults caused by the drug.

Pi binding by the F1-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane.

Pi binding by the F(1)-ATPase of beef heart mitochondria and of the Escherichia coli plasma membrane (E. coli F(1)) was examined by two methods: the centrifuge column procedure [Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899] and the Paulus pressure dialysis cell [Paulus, H. (1969) Anal. Biochem. 32, 91-100]. The latter is an equilibrium dialysis-type procedure. Pi binding by beef heart F(1) could be determined by either procedure. However, direct binding of Pi to E. coli F(1) could be determined adequately only in the Paulus cell which indicated more than two binding sites per mol of enzyme with a K(d) in the range of 0.1 mM. It is concluded that previous failure to observe Pi binding to E. coli F(1) with the centrifuge column procedure is due to a rapid rate of dissociation of Pi from the E. coli enzyme which results in loss of Pi during transit of the enzyme-Pi complex through the column.

Effects of cisplatin on mitochondrial function in Jurkat cells.

In this work, we measured the effects of pharmacological concentrations of cisplatin (cis-diaminedichloroplatinum II) on mitochondrial function, cell viability, and DNA fragmentation in Jurkat cells. The exposure of cells to 0-25 microM cisplatin for 3 h had no immediate effect on cellular mitochondrial oxygen consumption, measured using a palladium-porphyrin oxygen sensing phosphor. Similarly, the cell viability as measured by trypan blue staining was unchanged immediately following exposure to the drug, and no small DNA fragments, characteristic of drug-induced apoptosis, appeared. At 24 h after exposure to cisplatin, cellular respiration and viability decreased relative to controls and the amount of small DNA fragments, measured using quantitative agarose gel electrophoresis, was proportional to the concentration of cisplatin present during the drug exposure period. The small DNA fragments showed the banding pattern (with a spacing of approximately 300 bp) characteristic of drug-induced cell death by apoptosis. The changes in respiration and DNA fragmentation correlated linearly with the amount of platinum bound to DNA, determined by atomic absorption spectroscopy immediately following drug exposure. The oxygen consumption by beef heart mitochondria was not affected 0-24 h after exposure to 25 microM cisplatin or to solutions containing the monoaquated form of the drug, suggesting that the drug does not attack the mitochondrial respiratory chain directly. Cells exposed to the peptide benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone, which blocks apoptosis by the caspase pathway, showed a decrease in cisplatin-induced DNA fragmentation but not in the impairment of cellular respiration. Thus, although apoptosis is caspase-dependent, the impairment of cellular respiration is independent of the caspase system. Collectively, these results suggest that alteration in mitochondrial function is a secondary effect of cisplatin cytotoxicity in Jurkat cells.

Immediate effects of anticancer drugs on mitochondrial oxygen consumption.

The evolving role of mitochondria as a target for many anticancer drugs (e.g. platinum-based compounds, alkylating agents and anthracyclines) prompted us to investigate their immediate effects on the mitochondrial respiratory chain. For this purpose, we used a phosphorescence analyzer that measures [O(2)] in solution. The [O(2)] of solutions containing an appropriate substrate and various cell lines, tumors from patients or beef heart submitochondrial particles (SMPs) declined almost linearly (r>0.99) as a function of time, indicating that the kinetics of cellular oxygen consumption were zero order. Rotenone inhibited respiration, confirming that oxygen was consumed by the respiratory chain. Exposure to a clinically relevant concentration of cisplatin (5 microM at 37 degrees for 1-3 hr) had no effect on the respiration in cells or in SMP. Higher cisplatin concentrations (10-99 microM at 37 degrees for 1-3 hr) produced <25% inhibition. Incubations with 4-hydroperoxycyclophosphamide (50-100 microM at 37 degrees for 1 hr) inhibited oxygen consumption in SMP ( approximately 70% inhibition at 50 microM) and in cells ( approximately 30% inhibition at 50 microM). Incubations (37 degrees for 1 hr) of SMP with doxorubicin (25-100 microM) and daunorubicin (25-100 microM) had no inhibitory effect on the respiration. By contrast, incubations (37 degrees for 1 hr) of cells with doxorubicin (5-20 microM) and daunorubicin (2-20 microM) produced significant inhibition. We conclude that cisplatin does not directly damage the energy converting mechanism of mitochondria. On the other hand, comparable exposures to alkylating agents and anthracyclines produce immediate and dose-dependent impairment of cellular respiration.