Katy Drieu

Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Ginkgo biloba extracts and cancer: a research area in its infancy

Recent studies conducted with various molecular, cellular and whole animal models have revealed that leaf extracts of Ginkgo biloba may have anticancer (chemopreventive) properties that are related to their antioxidant, anti‐angiogenic and gene‐regulatory actions. The antioxidant and associated anti‐lipoperoxidative effects of Ginkgo extracts appear to involve both their flavonoid and terpenoid constituents. The anti‐angiogenic activity of the extracts may involve their antioxidant activity and their ability to inhibit both inducible and endothelial forms of nitric oxide synthase. With regard to gene expression, a Ginkgo extract and one of its terpenoid constituents, ginkgolide B, inhibited the proliferation of a highly aggressive human breast cancer cell line and xenografts of this cell line in nude mice. cDNA microarray analyses have shown that exposure of human breast cancer cells to a Ginkgo extract altered the expression of genes that are involved in the regulation of cell proliferation, cell differentiation or apoptosis, and that exposure of human bladder cancer cells to a Ginkgo extract produced an adaptive transcriptional response that augments antioxidant status and inhibits DNA damage. In humans, Ginkgo extracts inhibit the formation of radiation‐induced (chromosome‐damaging) clastogenic factors and ultraviolet light‐induced oxidative stress – effects that may also be associated with anticancer activity. Flavonoid and terpenoid constituents of Ginkgo extracts may act in a complementary manner to inhibit several carcinogenesis‐related processes, and therefore the total extracts may be required for producing optimal effects.

The Ginkgo biloba extract EGb 761 rescues the PC12 neuronal cells from β-amyloid-induced cell death by inhibiting the formation of β-amyloid-derived diffusible neurotoxic ligands

Abstract β Amyloid (Aβ) treatment induced free radical production and increased glucose uptake, apoptosis and cell death in PC12 nerve cells. Addition of the standardized extract of Ginkgo biloba leaves, EGb 761 together with the Aβ protein prevented, in a dose-dependent manner, the Aβ-induced free radical production, increased glucose uptake, apoptosis and cell death. However, pretreatment of the cells with EGb 761 did not rescue the cells from the Aβ-induced toxicity although it prevented the Aβ-induced reactive oxygen species generation. Moreover, the terpene and flavonoid-free EGb 761 extract, HE 208, although inhibited the Aβ-induced increased glucose uptake, it failed to protect the cells from apoptosis and cytotoxicity induced by Aβ. In conclusion, these results indicate that the terpenoid and flavonoid constituents of EGb 761, acting probably in combination with components present in HE 208, are responsible for rescuing the neuronal cells from Aβ-induced apoptosis and cell death; their mechanism of action being distinct of their antioxidant properties. Because pre- and post-treatment with EGb 761 did not protect the cells from Aβ-induced neurotoxicity, we examined whether EGb 761 interacts directly with Aβ. Indeed, in vitro reconstitution studies demonstrated that EGb 761 inhibits, in a dose-dependent manner, the formation of β-amyloid-derived diffusible neurotoxic soluble ligands (ADDLs), suggested to be involved in the pathogenesis of Alzheimer’s disease.

Protection of hypoxia-induced ATP decrease in endothelial cells by ginkgo biloba extract and bilobalide

Due to their localization at the interface between blood and tissue, endothelial cells are the first target of any change occurring within the blood, and alterations of their functions can seriously impair organs. During hypoxia, which mimics in vivo ischemia, a cascade of events occurs in the endothelial cells, starting with a decrease in ATP content and leading to their activation and release of inflammatory mediators. EGb 761 and one of its constituents, bilobalide, were shown to inhibit the hypoxia-induced decrease in ATP content in endothelial cells in vitro. Under these conditions, glycolysis was activated, as evidenced by increased glucose transport, as well as increased lactate production. Bilobalide was found to increase glucose transport under normoxic but not hypoxic conditions. In addition, EGb and bilobalide prevented the increase in total lactate production observed after 60 min of hypoxia. However, after 120 min of hypoxia, the total lactate production was similar under normoxic and hypoxic conditions, and both compounds increased this production. These results indicate that glycolysis slowed down between the 60th and 120th minute of hypoxia, while EGb and bilobalide delayed the onset of glycolysis activation. In another experimental model, both compounds were shown to increase the respiratory control ratio of mitochondria isolated from liver of rats treated orally. Since ischemia is known to uncouple mitochondria, the protection of ATP content and the delay in glycolysis activation observed during hypoxia in the presence of EGb 761 or bilobalide is best explained by a protection of mitochondrial respiratory activity, at least during the first 60 min of hypoxia incubation. Both products retain the ability to form ATP, thereby reducing the cells need to induce glycolysis, probably by preserving ATP regeneration by mitochondria as long as oxygen is available.

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.

Ginkgo biloba extract (EGb 761) pretreatment limits free radical-induced oxidative stress in patients undergoing coronary bypass surgery.

A growing body of evidence supports the trigger role of free radicals in the delayed functional and metabolic myocardial recovery following cardiopulmonary bypass (CPB) in humans, thus opening the field to specific therapies. This clinical study was designed to evaluate, in 15 patients undergoing aortic valve replacement, whether the extent of CPB- and reperfusion-induced lipid peroxidation, ascorbate depletion, tissue necrosis, and cardiac dysfunction is reduced by orally administered EGb 761, a Ginkgo biloba extract withpotent in vitro antiradical properties. Patients received either EGb 761 (Tanakan, 320 mg/day, n = 8) or a matching placebo (n = 7) for 5 days before surgical intervention. Plasma samples were obtained from the peripheral circulation and the coronary sinus at crucial stages of the operation (i.e., before incision, during ischemia, and within the first 30 minutes post-unclamping), and up to 8 days postoperatively. Upon aortic unclamping, EGb 761 inhibited the transcardiac release of thiobarbituric acid species (p ` 0.05), as assessed by high-performance liquid chromatography, and attenuated the early (5–10 minute) decrease in dimethylsulfoxide/ascorbyl free radical levels, an electron spin resonance index of the plasma ascorbate pool (p ` 0.05). EGb 761 also significantly reduced the more delayed leakage of myoglobin (p = 0.007) and had an almost significant effect on ventricular myosin leakage (p = 0.053, 6 days postoperatively). The clinical outcome of recovery of treated patients was improved, but not significantly, compared with untreated patients. Our results demonstrate the usefulness of adjuvant EGb 761 therapy in limiting oxidative stress in cardiovascular surgery and suggest the possible role of highly bioavailable terpene constituents of the drug.

Complementary cardioprotective effects of flavonoid metabolites and terpenoid constituents of Ginkgo biloba extract (EGb 761) during ischemia and reperfusion.

Abstract Hemodynamic and electron spin resonance (ESR) analyses were performed on isolated ischemic and reperfused rat hearts to assess the cardioprotective and antioxidant effects of therapeutically relevant concentrations of Gingko biloba extract (EGb 761; 5, 50 or 200 μg/ml), its terpenoid constituents (ginkgolide A; 0.05 μg/ml and ginkgolide B; 0.05, 0.25 or 0.50 μg/ml), and a terpene-free fraction of EGb 761 (CP 205; 5 or 50 μg/ml). Hearts underwent 10 min of low-flow ischemia, 30 min of no-flow global ischemia, and 60 min of reperfusion. Test substances were added to the perfusion fluid during the last 10 min of control perfusion, low-flow ischemia and the first 10 min of reperfusion. A separate group of rats were treated with CP 205 (60 mg/kg/day; p.o.) for 15 days, after which the hearts were perfused with plain buffer. In ESR experiments, the spin-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added to the perfusate to determine the effects of treatment on post-ischemic myocardial free radical generation. Results showed that in vitro exposure of hearts to EGb 761 (5 or 50 μg/ml) or to ginkgolides A and B (both at 0.05 μg/ml), or in vivo pretreatment of the rats with CP 205 delayed the onset of contracture during ischemia. The strong reperfusion-induced elevation of left ventricular end-diastolic pressure observed in untreated hearts was significantly reduced by in vitro exposure to the lowest concentrations of EGb 761, by ginkgolide A, and to a lesser extent by ginkgolide B, or by prior oral treatment with CP 205. Post-ischemic functional recovery was significantly improved by in vivo administration of CP 205, by perfusion with 5 μg/ml of EGb 761 or with both terpenoids as compared to untreated group but in vitro CP 205 was not effective. ESR analyses revealed that DMPO-OH (the DMPO / hydroxyl radical spin-adduct) concentrations in coronary effluents were markedly decreased by all treatments, except for the lowest concentration of gingkolide B. Perfusing 5 μg/ml EGb 761 resulted in a better inhibition of baseline DMPO-OH concentration than 5 μg/ml CP 205 (−70% and −48% vs. control, respectively), indicating that both terpenoid and flavonoid constituents of EGb 761 are required to produce this effect. CP 205 was significantly more efficient in reducing DMPO-OH concentration when administered in vivo than when applied in vitro, indicating that the antioxidant effect of flavonoid metabolites (formed in vivo) is superior to that of intact flavonol glycosides (present in vitro). Collectively, these findings provide the first evidence that part of the cardioprotection afforded by EGb 761 is due to a specific action of its terpenoid constituents and that this effect involves a mechanism independent of direct free radical-scavenging. Thus, the terpenoid constituents of EGb 761 and the flavonoid metabolites that are formed after in vivo administration of the extract act in a complementary manner to protect against myocardial ischemia-reperfusion injury.

Protection of mitochondrial respiration activity by bilobalide.

Mitochondria alteration is an early event in ischemia-induced damage, and its prevention improves tissue survival upon reperfusion. Adenine translocase and complex I activities are rapidly affected by ischemia. Ginkgo biloba extract demonstrates anti-ischemic properties attributable to the terpenoid fraction, mainly due to the presence of bilobalide. The mechanism of the protection afforded by bilobalide is not yet known. In this work, the effects of bilobalide on mitochondrial respiration were investigated. Mitochondria isolated from rats treated with bilobalide (2 to 8 mg/kg) showed a dose-dependent increase in the respiratory control ratio, due to a lower oxygen consumption during state 4. Bilobalide also decreased the sensitivity of oxygen consumption to inhibition of complex I by Amytal or to inhibition of complex III by antimycin A or myxothiazol. There was no protection of complexes IV and V. It also increased the activity of complex I but not of adenine translocase. Similar effects were also obtained in vitro when control mitochondria were preincubated for 1 hr with 0.8 microg/mL bilobalide. Treatment of the rats with 8 mg/kg bilobalide also prevented the ischemia-induced decrease in state 3 of the mitochondrial respiration and thus the decrease in RCR. The protective effect of bilobalide on cellular ATP content observed under ischemic conditions can be correlated with the above observations. By protecting complex I and III activities, bilobalide allows mitochondria to maintain their respiratory activity under ischemic conditions as long as some oxygen is present, thus delaying the onset of ischemia-induced damage. This mechanism provides a possible explanation for the anti-ischemic properties of bilobalide and of Ginkgo biloba extract in therapeutic interventions.

Ginkgo biloba Extracts EGb 761 and Bilobalide Increase NADH Dehydrogenase mRNA Level and Mitochondrial Respiratory Control Ratio in PC12 Cells

In the present study, we investigated the effect of Ginkgo biloba extract, EGb 761, and one of its components, bilobalide, on gene expression of subunit 1 of mitochondrial NADH dehydrogenase (ND1) in PC12 cells. By Northern blot analysis we found a ∼2-fold significant increase in ND1 mRNA level, after 48 and 72 h exposure to 100 μg/ml EGb 761 and to 10 μg/ml bilobalide. We also evaluated, by oxygraphy measurements, mitochondrial respiration during state 3 and state 4. In cells treated with EGb 761 and bilobalide for 48 and 72 h, state 4 respiration was significantly decreased, and the respiratory control ratio was increased. These results provide evidence that EGb 761 and bilobalide exert their protective effects by up-regulating mitochondrial ND1 gene expression and by decreasing state 4 respiration, whose increase is thought to be responsible for oxidative damage.

Free radicals and lipid peroxidation do not mediate β-amyloid-induced neuronal cell death

Abstract “β Amyloid (Aβ)-induced free radical-mediated neurotoxicity” is a leading hypothesis as a cause of Alzheimers disease (AD). Aβ increased free radical production and lipid peroxidation in PC12 nerve cells, leading to increased 4-hydroxy-2-nonenal (HNE) production and modification of specific mitochondrial target proteins, apoptosis and cell death. Pretreatment of the cells with isolated ginkgolides, the anti-oxidant component of Ginkgo biloba leaves, or vitamin E, prevented the Aβ-induced increase of reactive oxygen species (ROS). Ginkgolides, but not vitamin E, inhibited the Aβ-induced HNE modification of mitochondrial proteins. However, treatment with these anti-oxidants did not rescue the cells from Aβ-induced apoptosis and cell death. These results indicate that free radicals and lipid peroxidation may not mediate Aβ-induced neurotoxicity.