Protective effect of Phyllanthus fraternus against mitochondrial dysfunction induced by co-administration of cisplatin and cyclophosphamide.

The evolving role of mitochondria, in mediating chemotherapy-induced apoptosis motivated us for the studies described here. The combination of cisplatin and cyclophosphamide is widely used in treating various types of cancers. The purpose of our study was to understand the mechanism of the toxicity induced by the co-administration of cisplatin and cyclophosphamide, on mitochondrial bioenergetics, and to study the protective effect of prior administration of the medicinal plant extract Phyllanthus fraternus. Our results reveal that co-administration of cisplatin (12 mg/kg, i.p) and cyclophosphamide (150 mg/kg, oral) to wistar rats (100 g) significantly alters mitochondrial structure and hence function. The rate of mitochondrial respiration was decreased significantly with both NAD + and FAD-linked substrates. The respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylating efficiency also were decreased significantly accompanied by elevation in the lipid peroxide levels in liver, kidney homogenate and liver mitochondria respectively. Also, the phospholipid content of the mitochondrial membrane, showed a significant decrease, indicating mitochondrial membrane changes. Prior administration of an aqueous extract of P. fraternus (100 mg/kg) to rats, showed protection on all parameters investigated. Administration of P. fraternus alone did not show any significant changes on mitochondrial membrane bioenergetics. Thus, we propose, that the toxic side effects of cisplatin (+) cyclophosphamide, are due to a chain of interconnected events, within the mitochondrial inner membrane, ultimately leading to hepatotoxicity and nephrotoxicity. Further, our work also suggests that administration of aqueous extract of P. fraternus can enhance the therapeutic potential of anticancer drugs by reducing drug related toxicity.

Beneficial effect of the administration of Hemidesmus indicus against bromobenzene induced oxidative stress in rat liver mitochondria.

AIM OF THE STUDY: To study the beneficial effect of the prior administration of an aqueous extract of Hemidesmus indicus against bromobenzene induced oxidative damage in rat liver mitochondria. MATERIALS AND METHODS: Oxidative stress was induced in rats with bromobenzene (10 mmol/kg body wt.). The rate of respiration, P/O ratios, lipid peroxides, protein carbonyls and sulphydryls were studied. RESULTS: When the rats were administered with bromobenzene, the rate of respiration was decreased significantly and the P/O ratio was completely abolished. There was a significant increase on the levels of lipid peroxide and protein carbonyl and a significant decrease on total sulphydryl groups when compared with control. Administration of rats with an aqueous extract (100mg/kg) prior to bromobenzene administration showed significant beneficial effects like, stimulation in respiration, prevented the rise in lipid peroxides and protein carbonyls, increased the level of sulphydryl groups back to control level. Administration of vitamin E could not reverse as effectively as Hemidesmus indicus. CONCLUSIONS: This study demonstrates a good protective effect of Hemidesmus indicus against the bromobenzene induced oxidative stress.

Protective effect of Phyllanthus fraternus against allyl alcohol-induced oxidative stress in liver mitochondria.

The effect of administration of allyl alcohol on the oxidative stress and the protective effect due to administration of an aqueous extract of Phyllanthus fraternus against allyl alcohol-induced damage in liver mitochondria were studied. When rats were treated with allyl alcohol, the rate of mitochondrial respiration was decreased significantly with both NAD(+)- and FAD-linked substrates. The respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylation efficiency also decreased significantly. There was a significant increase in the lipid peroxide level and the protein carbonyl content. A significant decrease was observed in the total sulphydryl groups and a significant increase in the generation of superoxide radicals. Administration of rats with an aqueous extract of Phyllanthus fraternus (100 mg/kg) prior to allyl alcohol administration showed protection of 72, 40 and 80% using glutamate+malate (NADH oxidation) and 77, 54 and 20% using succinate as substrate on state 3, RCR and P/O ratio, respectively. The protection on lipid peroxide level was 88 and 91% in homogenate and mitochondria, respectively. In case of protein carbonyls, total sulphydryl groups and on the generation of superoxide radicals the protection was 99, 59 and 53%, respectively.

Protective effect of Phyllanthus fraternus against carbon tetrachloride-induced mitochondrial dysfunction.

The effect of carbon tetrachloride administration on liver mitochondrial function and the protective effect of an aqueous extract of Phyllanthus fraternus were studied in rats. The following changes were observed in mitochondria due to the administration of carbon tetrachloride. 1) A decrease in the rate of respiration, respiratory control ratio and P/O ratio using glutamate and malate or succinate as substrates. 2) A decrease in the activities of NADH dehydrogenase (35%), succinate dehydrogenase (76%) and cytochrome c oxidase (51%). The rate of electron transfer through site I, site II and site III was studied independently and found to be significantly decreased. 3) A decrease in the content of cytochrome aa3 (34%). 4) A significant decrease in the levels of phospholipids particularly cardiolipin and a significant increase in the lipid peroxide level was observed. The carbon tetrachloride induced toxicity may be partly due to the lipid peroxidation and partly due to the effect on protein synthesis. Administration of rats with an aqueous extract of P. fraternus prior to carbon tetrachloride administration showed significant protection on the carbon tetrachloride induced mitochondrial dysfunction on all the parameters studied.

Protective effect of P. fraternus against ethanol-induced mitochondrial dysfunction.

Chronic ethanol consumption (10 g per kg body weight) significantly decreased the rate of respiration, P/O ratio, and respiratory control ratio (RCR). The activities of NADH dehydrogenase and cytochrome oxidase were significantly decreased in submitochondrial particles by ethanol administration compared to control. No significant difference was observed in membrane potential of submitochondrial particles. Cytochrome b, c and aa3 content of mitochondria were significantly decreased by ethanol feeding. Ethanol-induced inhibition on rate of respiration, P/O ratio, and RCR was relieved to a great extent by the administration of the aqueous extract of Phyllanthus fraternus (100 mg dry powder of the plant per kg body weight) along with ethanol. The decrease in the content of cytochromes due to ethanol administration was revived partially by aqueous extract of P. fraternus.

Studies on cytochrome oxidase in carbon tetrachloride treated rats.

Cytochrome c oxidase was purified from control and CCl4 treated rats and its kinetic properties were studied. The activity of the enzyme was inhibited by 51% in CCl4 (4 g per kg body weight for 24 hr) treated rats. Studies on the kinetic properties showed that the K(m) of the enzyme increased by 60% while Vmax decreased by 44% in CCl4 treated rats compared to controls. The content of cytochrome aa3 was decreased by 34% while cytochrome b and c were not affected by CCl4 treatment. Phosphatidylcholine, phosphatidylethanolamine and cardiolipin were decreased significantly by 40%, 49% and 60% respectively in CCl4 treated rats. A decrease in the cytochrome aa3 content and a change in the lipid environment of the membrane are probably responsible for a decreased rate of electron transfer from cytochrome c to oxygen.

Effect of psychosine on mitochondrial function.

The effect of psychosine on the rate of respiration at different segments of the electron transport chain, respiratory control ratio and the efficiency of phosphorylation was studied. The transfer of electrons through site I, site II and site III was studied independently. The transfer through site I and site III was inhibited by psychosine, whereas the transfer through site II was not inhibited. Cardiolipin, which is essential for the electron transfer through site I and III, was implicated to be responsible for the inhibition of electron transfer by psychosine. Electron carriers of site II are not sensitive to cardiolipin, so psychosine could not inhibit the electron transfer through this site. The ADP/O ratio and respiratory control ratio were inhibited by psychosine showing that it has an uncoupler like effect. Mitochondria isolated from rat liver, kidney and brain behaved essentially the same way in their response to psychosine. Cytochrome c oxidase was significantly inhibited by psychosine and the degree of inhibition was almost same in mitochondria and sub mitochondrial particles. The preence of outer membrane in mitochondria did not make any difference with respect to the action of psychosine on electron transport chain. Psychosine interacts at site I and site III and a change in the lipid environment of the membrane is responsible for the mitochondrial dysfunctions induced by psychosine. This represents a possible mechanism for the destruction of cells in Gaucher's and Krabbe's disease.

Effect of administration of galactosamine hydrochloride on rat liver mitochondria.

The effect of galactosamine on liver mitochondrial functions was studied in vivo in rats at 12hr, 24hr and 36hr after the administration of the drug. State 3 respiration decreased significantly with both NAD+ linked and FAD linked substrates. Respiratory control ratio, an index of membrane integrity and P/O ratio which is a measure of phosphorylation efficiency decreased significantly. There was a significant decrease in the activities of NADH dehydrogenase, succinate dehydrogenase and cytochrome oxidase. A significant decrease was also seen on membrane potential, cytochrome aa3, cytochrome b, cytochrome c and on phospholipids of mitochondria. The observed mitochondrial dysfunctions were related to increased lipid peroxidation, which could cause loss of membrane integrity and a decreased rate of phosphorylation. It is proposed that increased lipid peroxidation was responsible for the inhibition on both oxidation and phosphorylation in mitochondria in galactosamine treated rats.

Protective effect of vitamin E against ethionine toxicity.

The effect of administration of ethionine on rat liver mitochondrial functions and the protective effect of vitamin E on ethionine induced damage was studied. Ethionine treatment decreased the rate of respiration, respiratory control ratio and P/O ratio. There was a significant decrease in the activities of NADH dehydrogenase, succinate cytochrome C reductase and cytochrome oxidase. A significant decrease was seen on membrane potential and on the levels of ATP. Among the mitochondrial phospholipids only cardiolipin decreased significantly. The lipid peroxide level increased significantly in ethionine treated rats. Administration of vitamin E prior to ethionine treatment relieved the effects (induced by ethionine) on all the parameters studied. This study shows that vitamin E protects against ethionine toxicity.

Direct measurement of the initial and early ratios of proton extrusion to oxygen uptake accompanying cytochrome c oxidation by rat liver mitoplasts.

We have recently described new methods that enable the sharp initiation of a respiratory pulse by photolysis of the CO complex of cytochrome oxidase in a stirred suspension of mitochondria, succinate, O2, and CO (Setty, O. H., R. I. Shrager, B. Bunow, B. Reynafarje, A. L. Lehninger, and R. W. Hendler. 1986. Biophys. J. 50:391-404). Data are collected directly into a microcomputer at 10-ms intervals from fast responding O2 and pH electrodes. These procedures eliminate delays and uncertainties due to mixing times, recorder response, and recovery of the O2 electrode from responding to the injection of O2. Correction procedures were also described for the inherent electrode delays. These procedures revealed an initial burst in medium acidification and a lag in O2 uptake that led to H+/O rates of 20-30 during the first 50 ms and relaxed to "normal" levels by 300 ms. Subsequent changes in [H+] and [O2] followed time courses that appeared to be, but were not strictly, first order. We describe here similar studies in which cytochrome c served as electron donor to site III of rat liver mitoplasts. A qualitatively similar but quantitatively smaller burst in medium acidification and H+/O ratio was seen in these studies. Implications of the previous (Setty et al., 1986) and current studies on defining "mechanistic" H+/O ratios are discussed.

Direct measurement of the initial proton extrusion to oxygen uptake ratio accompanying succinate oxidation by rat liver mitochondria.

The problem of obtaining very early ratios for the H+/O stoichiometry accompanying succinate oxidation by rat liver mitochondria was attacked using new techniques for direct measurement rather than extrapolations based on data obtained after mixing and the recovery of the electrode from initial injection of O2. Respiration was quickly initiated in a thoroughly mixed O2-containing suspension of mitochondria under a CO atmosphere by photolysis of the CO-cytochrome c oxidase complex-. Fast responding O2 and pH electrodes were used to collect data every 10 ms. The response time for each electrode was experimentally measured in each experiment and suitable corrections for electrode relaxations were made. With uncorrected data obtained after 0.8 s, the extrapolation back to zero time on the basis of single-exponential curve fitting confirmed values close to 8.0 as previously reported (Costa et al., 1984). The data directly obtained, however, indicate an initial burst in H+/O ratio that peaked to values of approximately 20 to 30 prior to 50 ms and which was no longer evident after 0.3 s. Newer information and considerations that place all extrapolation methods in question are discussed.

Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli.

An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios. We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment. The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi. (b) An increase in [K+] from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity. (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM [K+], causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV. From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM. (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution. (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors. (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.

Interference of electron donors in the measurement of the proton gradient and membrane potential by flow dialysis.

The three most commonly used electron donors for flow dialysis measurements of membrane potential lead to the development of an apparent but artifactual membrane potential with the interior negative in the presence or absence of membrane vesicles. The same three electron donors used in flow dialysis determinations of delta pH in the presence or absence of membrane vesicles lead to the development of an apparent but artifactual delta pH with the interior acidic. These artifacts have been evaluated using two probes for membrane potential, namely, TPP+ and rubidium in the presence of valinomycin and for two probes of delta pH, namely, acetate and DMO. Measurements were made over a range of ionic strengths.

Effect of heparin on vitamin A induced hyperlipidemia in rats.

Effect of intravenous administration of heparin (5 I.U.) has been studied on vitamin A induced changes in plasma, liver, kidney, heart and adipose tissue of rats. Heparin reversed vitamin A induced hypertriglyceridemia. It reduced plasma TG and PC levels and increased NEFA and LPC levels in control and retinol fed rats. Similar pattern was noted in the palmitate-1-14C radioactivity of these plasma lipids. Heparin administration did not influence the levels of lipids and phospholipids and their palmitate-1-14C radioactivity in liver and kidney of retinol fed rats. In heart heparin improved the uptake of plasma TG in retinol fed rats and increased its FFA levels. Similar pattern was observed in the palmitate-1-14C radioactivity of the above heart lipids. In adipose tissue heparin administration improved the uptake of plasma lipids of vitamin A fed rats.

Molecular species of phosphatidyl choline and phosphatidyl ethanolamine in heart of rats given excess vitamin A.

The effect of 33 mg of vitamin A for two days on heart phospholipids and on the molecular species of phosphatidyl-choline and phosphatidyl-ethanolamine of rats has been studied. Vitamin A reduced the amounts of heart total phospholipids, phosphatidyl-choline and its tetraenoic species and of the hexaenoic species of phosphatidyl-ethanolamine. In rats administered vitamin A the incorporation of NaH232PO4 and choline 1,2-14C chloride into total phospholipids and into the phosphatidyl-choline fraction was reduced. The results of the incorporation of NaH232PO4 into the various molecular species of phosphatidyl-choline and phosphatidyl-ethanolamine showed that excess A impaired the synthesis of these phospholipids via the CDP-choline-ethanolamine pathway.

Interaction of vitamin A and cortisol on liver and plasma lipids of rats.

Effect of vitamin A and cortisol administration either alone or together has been studied on liver and plasma lipids of rats. The effects of cortisol administration on liver and plasma lipids of rats and incorporation of palmitate-1-14C into liver and plasma lipids were similar to that of vitamin A. When both were given together, the effects were additive.

Retinol feeding and low and high density lipoproteins of plasma of rats.

Effect of feeding 33 mg retinol daily for two days has been studied on lipids of plasma low and high density lipoproteins of rats. Triglycerides, nonesterified fatty acids, phosphatidyl choline and its monoenoic, dienoic and tetraenoic species were increased in plasma low density lipoproteins of retinol fed rats. In high density lipoprotein fraction triglycerides, phosphatidyl choline and its tetraenoic species were decreased and that of nonesterified fatty acids levels increased in retinol fed rats. The incorporation of NaH(2)(32)PO4 (counts/min/ml) into phosphatidyl choline and its all molecular species was increased in low density lipoproteins but it was decreased in high density lipoprotein of plasma of rats fed retinol.

Upake of plasma lipids by extrahepatic tissues of vitamin A fed rats.

The uptake of newly synthesized plasma lipids from intraportally injected palmitate 1(-14)C by heart, kidney and adipose tissues of rats fed 33 mg retinol for 2 days have been studied. The uptake of plasma lipids by adipose tissues and heart was decreased and that by kidney increased in rats fed retinol as compared to the controls.