Document aJND548jdb2qXravmX78V2z6Y

Covance Studies The attached report summarizes the results from the assessment of mitochondrial bioenergetics of hepatic mitochondria isolated from rats treated for 3 weeks with one of 4 doses (plus control) of dietary FC10. What is obvious from the data is the ability to detect dramatic differences among the 13 animals tested. However, attempts to group animals according to degree of effect failed to correlate with the exposure dose. Further inquiry revealed that all animals were euthanized with ca., 50 mg/kg pentobarbital, which we subsequently demonstrated to have profound effects on inhibiting pyruvate oxidation at complex I of the mitochondrial electron transport chain. We attribute our inability to establish a dose-response correlation to this effect of pentobarbital. Subsequent attempts to eliminate this confounding effect from the analyses were unsuccessful. However, we rem ain convinced of the importance of collecting data describing mitochondrial deficits in tissues from exposed animals and in our ability to detect such effects with sufficient sensitivity. Rather than relying on the isolation of high integrity, intact mitochondria for measuring coupled bioenergetic properties, we suggest m easuring glycolytic and tricarboxylic metabolites in frozen tissues in order to derive a profile of intermediary metabolism for each exposed animal. From the relative concentrations of the individual metabolites (e.g., the oxoglutarate-to-pyruvate or succinate-to-fumarate ratios), we can estimate relative degrees of metabolic dysfunction in exposed animals4. Rather than requiring one of us to be present on site at the time of sacrifice, we can perform such analyses on tissues that are collected in liquid nitrogen and kept frozen for shipping to our laboratory. This is considerably more convenient for all parties involved and we are anxious to discuss potential opportunities to continue to contribute to the in vivo monitoring studies. 4 Colson, C. and Klapper, M.H. (1979). Sequential measurements o f glycolytic and tricarboxylic acid cycle metabolites in single sample aliquots. Anal. Biochem. 97, 394399. G041S4 PROBE DESCRIPTIONS The introduction. The rationale of the following experiments was to determine as many "independent" characteristics of energetics of mitochondria as it is possible from a single probe and having time limitations. Due to this, the specific experimental procedure and conditions were designed. The pyruvate -supported respiration and the membrane potential were measured in a simple slightly hypotonic incubation media. Other compounds, such as ADP, C a^, and succinate, were added to mitochondria at saturating concentrations and at different times but at least 2 min after the mitochondria were introduced to the pyruvate -containing medium. No inhibitors of oxidative phosphorylation, the respiratory chain, Ca2+transport, or uncouplers were used. Also, no attempts were taken to reduce the concentration of free fatty acids co-isolated with mitochondria or generated during incubation. The logic of the experimental design was to measure in a single probe the most vulnerable characteristics of mitochondria, such as pyruvate-supported respiration and oxidative phosphorylation, and the most "insensitive", easily reproducible parameters such as succinate-supported respiration and C a^ transport. Under such limitations, there is no possibility to obtain the precise characteristics for any of the parameters. However, the combinatory analysis of all the measured parameters allows to estimate, at first approximation, the energetics of mitochondria, and make some conclusions about the in vivo conditions, in a tissue, where the mitochondria were isolated from. The interpretation of any in vitro data, and especially the backward deduction of in vivo conditions in a tissue from the data obtained with isolated mitochondria, is always a kind of "might be" guessing. It is rath er. tempting to thing in terms like "bad mitochondria", say, if they can not oxidize a substrate fast enough, or "good mitochondria", if they exerted an RCI more then 20. We would prefer another mode of thinking, namely that mitochondria are good when the energetics characteristics they exert fit the cellular demands. Arid the particular values for mitochondrial energetics usually depend on the particular experimental conditions. Due to this, it seems to be necessary to remind shortly the peculiarities of mitochondrial biochemistry underlying the experimental conditions we have chosen. The most vulnerable functions of mitochondria are pyruvate - supported respiration and oxidative phosphorylation The oxidation of pyruvate by liver mitochondria depends on the availability of CoASH in the mitochondrial matrix, on the overall activity o f the TCA cycle, and on the activity of respiratory chain (especially, on the Complex I activity). In addition to these, maximal rates of pyruvate oxidation usually require the presence of catalytic amounts of malate in the incubation medium (to stimulate the complete turnover of TCA cycle). Also, it is under allosteric control by of naturally occurring endogenous compounds, such as free fatty acids, acetyl-CoA, and ATP inhibiting the pyruvate dehydrogenase activity. The respiration rate of pyruvate-supported mitochondria phosphorylating ADP depends on all the mentioned parameters plus the activities of oxidative phosphorylation system (Pi transporter, ADP/ATP-translocase, and ATP-synthase). These latter are also can be negatively regulated by free long chain fatty acids and long-chain acyl-CoA. The oxidation of succinate is not so vulnerable. The sensitivity of succinate dehydrogenase and Complex in to free fatty acids is much less then that of pyruvate dehydrogenase and Complex I. Practically, under any "regular" experimental conditions the rate of succinate-supported respiration is limited only by transmembrane electrochemical profdft gradient The only know' strong endogenous inhibitor of succinate dehydrogenase is oxaloacetic acid C041S5 However, in the absence o f malate and the presence of sufficient amounts of pyruvate in the incubation medium, the accumulation of oxaloacetate could not be (2)expected. The respiration rate that can be observed during Ca2+ uptake is practically insensitive to any endogenous compounds, which may be co-isolated with mitochondria. Providing that phosphate concentration is sufficient, the respiration rate during Ca2+ transport can be considered as the maximal possible rate of oxidation of a respiratory substrate by the respiratory chain. It is limited mainly by the activity of a Ca2+ transporting system, which is kinetically faster, then any other transport system in mitochondria, including the transport of the majority of respiratory substrates, and much fester then ADP transport. However, when a NAD - dependent substrate is used, the respiration may become inhibited due to fast opening of a Ca2+ -induced pore in the inner mitochondrial membrane, and subsequent release and dilution of mitochondrial NAD. For succinate-supported respiration, the same event would provide the fastest respiration rate because the succinate dehydrogenase is a FAD-dependent enzyme, and pore opening completely discharge the transmembrane proton gradient (2) due to kinetic limitation to TCA cycle arising from overreduction of NAD and fast removal of oxaloacetyc acid stimulated by pyruvate-derived acetyl-CoA. 004136 M ATERIALS AND METHODS The isolation of m itochondria. Mitochondria were isolated from liver pieces (~1.5 g wet weight) of adult male and female rats by a conventional differential centrifugation procedure. Liver pieces were cooled in 20 ml of isolation medium (210 mM mannitol, 10 mM sucrose, 5mM HEPES-KOH (pH 7.4), 1 mM EGTA). Cooled tissue was minced with scissors and washed once with 20 ml of isolation medium, then diluted with the same medium and homogenized for -45 sec with a motor-driven Potter homogenizer (Teflon pestle, glass beaker) and placed on ice. The tissue to medium ratio was approximately 1:20 (g:ml). This procedure was applied sequentially to 8 liver specimens. The time interval between homogenizing of the first and the last liver pieces did not exceedf1) 9 min. The homogenates were simultaneously centrifuged for 10 min x 700 g, t - 4 C, and mitochondrial pellets were recovered by centrifugation at 10,000 g x 10 min. The pellets were resuspended in 2 ml of washing medium (210 mM mannitol, 10 mM sucrose, 5mM HEPES-KOH, pH 7.4). The suspensions of mitochondria were diluted to 35 ml with the same medium, and centrifuged at 10,000 g x 10 min. The final mitochondrial pellets were resuspended in washing medium to a protein concentration of 80-120 mg x ml'1and stored on ice. The isolation procedure took exactly 1 hour. This isolation procedure was repeated for the second set of 8 liver specimens. The processing o f the first set (samples 1 - 8 ) was completed at - 10:35 AM and the processing of the second set (samples 9 - 16) was started at -12:35 AM and completed within 1 hour. The measurements were started at -2:00 PM. M easurem ents. Mitochondrial membrane potential (A'F) was estimated from TFP* ions distribution measured with a TPP+- selective electrode. Mitochondrial membrane potential was calculated as described elsewhere. The rate o f oxygen consumption by mitochondria was measured with a hand-made Clark -type oxygen electrode. Both the mitochondrial membrane potential and the respiration rate were recorded simultaneously using a multichannel incubation chamber equipped with a magnetic stirrer. The volume of the chamber was 1.8 ml. All experiments were performed at room temperature (22 C). The TPP+ -sensitive electrode was calibrated by sequential additions of known amounts of TPP^Cl' before the addition of mitochondria (see Fig.l). The respiration rates were calculated assuming the initial oxygen concentration to be equal to 240 pM. The incubation medium containing 210 mM mannitol, 10 mM sucrose, 5mM HEPES-KOH, pH 7.4, 4 mM KH2PO4, and 10 mM pyruvate was used in all experiments. The same volumes of mitochondrial suspension (30 pi) were added, regardless of the protein concentrations. Protein concentrations were determined after the experiment by the Bradford assay. Bovine serum albumin was used as a standard. Additions. Where mentioned, the additions were as following: 5 mM succinate, 200 pM ADP, and 264 pM CaCl2. 111--this modification o f the conventional isolatimi procedure can be expected to decrease the quality of mitochondria. According to our experience, 10 min o f storing of homogenized tissue on ice can produce approximately 20 - 30% decrease in RC1, depending on the perfectness of oooling 004137 The results Table 1 shows the relation of sample labeling used in the following text to the labels of liver specimens. Table 1. The sample labels : 12 3 4567 C90753 m C90719 m' C91230 f C90935 m C91024 mf C91350 f v\ C90890 m 8 C91165 f' 11 C91205 . f 12 C91430 f* 13 C90814 m 14 C91135 fl 15 C91169 fv The samples were measured in the following time sequence: 3 - 4 --5 --6 --8 --12 --13 --11 --14 --2 - 1- 7-15 An average time spent for each probe was about 12 min, with approximately 5 min interval between probes. All measurements were completed within 4 hours. Table 2 shows the values of the respiration rate obtained in the presence of various additions. Table 2. The respiration parameters measured. For the incubation medium and other conditions, see Materials & Methods. For the sequence of additions, see Probe Descriptions chapter. Data are expressed in nmol Qj /min/mg of protein. Vpyr, the rate of pyruvate - supported respiration; Vdp, the rate of respiration produced by ADP addition (St. 3); Vra, the rate of respiration during Ca2+uptake; Vuicc, succinate- supported respiration in the presence of pyruvate and in the absence of other additions (unless stated otherwise); Ca/O, the ratio of Ca2* transported (nmol) to the amount of oxygen consumed (nmol); ADP/O, the ratio of ADP phosphoxylated (nmol) to the amount of oxygen consumed (nmol). Vpyr V adp Vc. i, 3 2> 4 5 67 11 12 13 w . 1 5 < 3.7 3.1 2 6.8 5.5 4 5.7 0 4.6 2.1 1.8 2.7 2.7 0 '5 .7 30.5 50.5 '10.2 **5.1 0 ND p8 19 '7 .7 42.7 34.1 44.5 94.3 73.2 ND '6 .4 96 36.8 ND ND 78.9 '14.2 ND 46.9 V SUCC 10.4 14.7 13.6 33.4 *119 28.8 15.9 24 *69 10.6 *87 16.7 12.3 C a/O 7.6 7.3 4.3 ND '7 .2 4.9 5.5 ND ND 4.4 '1 2 ND 4.7 A D P /O -- N.R. N.R N.R. 3.6 N.R. -- ND 3.1 N.R. 3.5 0.7 1 ND - not determined N.R. - not resolved (see Probe Description chapter) P- pyruvate -supported respiration * - the respiration rate was measured in the presence of the excess of Ca2+, which induced the opening of pore, so it should be considered as the maximal rate of succinate oxidation. C041S8 Probe 1. The sequence of additions was ADP, succinate, Ca2+, Ca2+. Mitochondria were able to maintain the membrane potential of 144 mV by pyruvate oxidatioa However^ D P did not stimulate the respirationTylthough induced the AH' decrease to 130 mV. The subsequent addition of succinate stimulated the respiration and increased the AH' to 170 mV. The increase in respiration and in the AH' was biphasic, indicating incomplete phosphorylation of ADP to the moment of the addition of succinate. Only steady-state values for AH' and respiration are shown. RCI for phosphorylation was about 1.2. Subsequent addition of Ca2+ induced ~4 fold transient stimulation of respiration and decrease in AH' to 149 mV. These mitochondria consumed 360 nmol Ca2+/mg of protein, the pore was not observed. Interpretation: the pyruvate transport system or pyruvate oxidation is inhibited; the activity of respiratory chain with succinate during Ca2+ uptake is ~2 fold less then usual (or the Ca2+ transporting system is inhibited); the resistance of these mitochondria to pore opening is in the upper top of normal range (or even slightly higher) for a well - coupled rat liver mitochondria. Probe 2. The sequence of additions was as in Probe l(T heresponses on ADP, succinate, and Ca2+ were as in Probe 1, to o . The AH' value for pyruvate oxidation was 138 mV, the ADP -induced AH' decrease was to 127 mV, the succinate - supported AH' was 166 mV, and Ca2+ induced the decrease in AH' to 127 mV. Interpretations: The same as for Probe 1 for except that apparent Ca2+ capacity (the resistance to pore opening, determined by the characteristic AH' decrease) was less then for Probe 1 (between 200 and 360 nmol Ca2+/mg of protein). Probe 3. The sequence of additions was succinate, ADP, Ca2+. These mitochondria were unable to maintain the AH' with pyruvate. The AH' with succinate was also low, 144 mV. The addition of ADP induced the decrease in AH' to 137 mV and irreversible stimulated the respiration (Table 2). The addition of Ca2+ further stimulated the respiration, but in this case the stimulation was transient (normal response). The pore was opened by 164 nmol Ca2+/mg of protein. Interpretations: These mitochondria a)uncoupled, b)the pyruvate oxidation is inhibited at the level of transport, pyruvate dehydrogenase, or Complex I. The rest of respiratory chain and/or succinate dehydrogenase activity are in the normal range. The sensitivity to Ca2+ is too high. It may be due to accumulation of a pore inducing compound like free long-chain fatty acids. The decrease in Ca/O (Table 1) is probably due to uncoupling and/or incomplete phosphorylation of ADP to the moment of Ca2+ addition. Probe 4. The sequence of additions was as in Probe 3. These mitochondria were not able to maintain the AH' by pyruvate oxidation. The ADP irreversibly slightly stimulated the succinate -supported respiration, the addition of Ca2+ was without further effect. The resistance to pore opening was higher then 135 nmol Ca2+/mg of protein. Interpretations: Mitochondria were uncoupled and inhibited with both substrates. However, pyruvate oxidation was less inhibited then in the probes described above (compare the Vpyr values in Table 1). The amount of Ca2+ required for pore opening bear no information in this case (the protein concentration, which was measured after the experiment, was too high, 1.97 mg/ml, so the added amount of Ca2+ was insufficient). Probe 5. G041S9 The sequence of additions was ADP. Ca2+, Ca2+, and succinate. These mitochondria were able to maintain AT of 143 mV by pyruvate oxidation. The addition of ADP transiently stimulated the respiration and decreased the A'F to 115 mV, the RCI was 2.7. The measurement of the A'F revealed an unusual pattern, after the completion of ADP phosphorylation the AT increased to the higher level, than that before the ADP addition (a hyperpolarisation). The response of respiration on the Ca2+ addition was unusually slower then the response on ADP (see Table 2). The pore was opened by 243 nmol Ca2+/ mg of protein. Succinate-supported respiration was recorded after the pore opening, so it reflects the maximal activity of succinate dehydrogenase + the rest of respiratory chain, and it is in the normal range. Interpretations: These mitochondria were coupled, the respiration with both substrates was not significantly inhibited. However, the response on Ca2+ was unusual, it might indicate some alteration in the Ca2+ transporting system. Actually, the "good" pyruvate oxidation may also reflect the availability of free CoASH in the mitochondrial matrix, that can results from the inhibition of fatty acids (3-oxidation. Probe 6. The sequence of additions was ADP, succinate, Ca2+. These mitochondria maintained the AT of 122 mV by pyruvate oxidation. ADP slightly stimulated the respiration. The response on the succinate, Ca2+ additions were like these in Probe 3, for except that 155 nmol Ca2+/mg o f protein did not open the pore (normal response). Interpretations: These mitochondria were uncoupled, and the pyruvate oxidation inhibited. The data provide no information on the sensitivity to pore by the same reasons as in Probe 4 (insufficient Ca2+ loading). Probe 7. The sequence of additions was ADP, succinate, Ca2+, Ca2+, Ca2+. The responses were essentially as in Probes 1, 2. The AT for pyruvate oxidation was 143 mV, and that for succinate-supported respiration was 167 mV. Ca2+ opened the pore in the range 306 -465 nmol/ mg of protein (see Probe 1). Interpretations: See Probe 1, Probe 2. Probe 8. The only succinate was added because these mitochondria appeared to be unable to maintain the A T both with pyruvate and with succinate. Succinate-supported respiration was partially inhibited. It should be noted also, that the yield of mitochondria from this liver specimen was the lowest, the initial concentration of mitochondria in suspension was 75 mg/ml and that in probe was 1.25 mg/ml Interpretations: for a regular rat liver mitochondria, 1.25 mg/ml concentration in probe is more then sufficient to observe all the mitochondrial fimctjon^TThese mitochondria were inhibited and completely uncoupled?^ Probe 11. The sequence o f additions was ADP, Ca2+, and succinate. These mitochondria were able to maintain the AT o f 136 mV by pyruvate oxidatioa The addition of ADP slightly but reversible stimulated the respiration, but that of Ca2+ was without effect. However, the addition of Ca2+ induced complete deenergization, and the subsequent succinate addition did not increase the AT whereas stimulated the respiration. Interpretations: The absence of the stimulation of pyruvate oxidation by the Ca2+ addition is probably due to the opening of pore (see Introduction).The sensitivity to Ca2+ is unusually high, less then 150 nmol Ca2+/ mg of protein, as in Probe 3. 004190