Identification of aldo-keto reductase (AKR7A1) and glutathione S-transferase pi (GSTP1) as novel renal damage biomarkers following exposure to mercury.

The kidney is one of the main targets for toxicity induced by xenobiotics. Sensitive detection of early impairment is critical to assess chemical-associated renal toxicity. The aim of this study was to identify potential nephrotoxic biomarkers in rat kidney tissues after exposure to mercury (Hg), a representative nephrotoxicant, and to evaluate these new biomarkers employing in vivo and in vitro systems. Mercuric chloride was administered orally to Sprague-Dawley rats for 2 weeks. Proteomic analysis revealed that aldo-keto reductase (AKR7A1) and glutathione S-transferase pi (GSTP1) were significantly elevated in kidney after Hg exposure. While the levels of conventional nephrotoxic clinical markers including blood urea nitrogen and serum creatinine were not elevated, the mRNA and protein levels of AKR7A1 and GSTP1 were increased upon Hg exposure in a dose-dependent manner. The increases in AKR7A1 and GSTP1 were also observed in rat kidneys after an extended exposure for 6 weeks to low-dose Hg. In in vitro rat kidney proximal tubular cells, changes in AKR7A1 and GSTP1 levels correlated well with the extent of cytotoxicity induced by Hg, cadmium, or cisplatin. AKR7A1 and GSTP1 were identified as new candidates for Hg-induced nephrotoxicity, suggesting that these biomarkers have potential for evaluating or predicting nephrotoxicity.

Procoagulant and prothrombotic effects of the herbal medicine, Dipsacus asper and its active ingredient, dipsacus saponin C, on human platelets.

BACKGROUND: In spite of the growing popularity of herbal medicines and natural food supplements, their effects on cardiovascular homeostasis remain largely unknown, especially regarding pro-thrombotic risks. OBJECTIVE: In the present study, 21 herbal tea extracts were screened for the procoagulant activities on platelets, an important promoter of thrombosis to examine if herbal medicines or natural products may have prothrombotic risks. We discovered that Dipsacus asper (DA), known to have analgesic and anti-inflammatory effects, potently induced procoagulant activities in platelets. We tried to identify the active ingredient and elucidate the underlying mechanism. RESULTS: Among 10 major ingredients of DA, dipsacus saponin C (DSC) was identified as a key active ingredient in DA-induced procoagulant activities. DSC-induced procoagulant activities were achieved by the exposure of phosphatidylserine (PS) and PS-bearing microparticle generation that were caused by the alteration in the activities of phospholipid translocases: scramblase and flippase. These events were initiated by increased intracellular calcium and ATP depletion. Notably, DSC induced a series of apoptotic events including the disruption of mitochondrial membrane potential, translocation of Bax and Bak, cytochrome c release and caspase-3 activation. The key roles of apoptotic pathway and caspase activation were demonstrated by the reversal of DSC-induced PS exposure and procoagulant activities with the pretreatment of caspase inhibitors. Interestingly, EGTA reversed DSC-induced procoagulant activities and apoptotic events suggesting that an intracellular calcium increase may play a central role. These results were also confirmed in vivo where platelets of the rats exposed to DSC or DA exhibited PS exposure. Most importantly, DSC or DA administration led to increased thrombus formation. CONCLUSION: These results demonstrate that herbal medicines or natural products such as DA or DSC might have prothrombotic risks through procoagulant activation of platelets.

Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.

BACKGROUND: Doxorubicin (DOX) is a widely used anticancer drug for solid tumors and hematologic malignancy, but its active use is hampered by serious adverse effects, including thrombocytopenia. Although bone marrow toxicity of DOX has been suggested to be the sole mechanism underlying the reduced platelet counts, the direct effects of DOX on platelets have never been examined. OBJECTIVE: Here, we investigated the DOX-induced platelet cytotoxicity and its underlying mechanism in an effort to elucidate the contribution of platelet cytotoxicity to DOX-induced thrombocytopenia. RESULTS: In freshly isolated human platelets, DOX induced platelet cytotoxicity in a time-dependent and concentration-dependent manner. Reactive oxygen species (ROS) generation, decreased glutathione levels and subsequent protein thiol depletion were shown to underlie the DOX-induced platelet cytotoxicity. Conspicuously, DOX-treated platelets displayed apoptotic features such as caspase-3 activation, reduced mitochondrial transmembrane potential, and phosphatidylserine exposure. Decreased glutathiolation of procaspase-3 was shown to be a link between protein thiol depletion and caspase-3 activation. It is of note that DOX-mediated platelet cytotoxicity was significantly enhanced by shear stress, a common complicating factor in cancer patients. These in vitro results were further confirmed by an in vivo animal model, where administration of DOX induced a platelet count decrease, ROS generation, caspase-3 activation, protein thiol depletion, and damaged platelet integrity. CONCLUSION: We demonstrated that DOX can directly induce platelet cytotoxicity through ROS generation, decreased glutathione levels, and protein thiol depletion. We believe that this study provides important evidence for the role of DOX-induced platelet cytotoxicity in the development of thrombocytopenia in DOX-treated patients.

Menadione induces endothelial dysfunction mediated by oxidative stress and arylation.

Our previous studies showed that menadione causes endothelial dysfunction which results in decreased relaxation and increased contraction of blood vessels. This investigation examined the role of two possible mechanisms (oxidative stress and arylation) in menadione-induced endothelial dysfunction. Menadione increased superoxide anion generation in aortic rings in a dose-dependent manner. Superoxide dismutase (SOD), reversed the inhibitory effects of menadione on vascular relaxation. The relaxation induced by the NO donor, sodium nitroprusside, was inhibited by menadione pretreatment in a dose-dependent manner. Endothelial nitric oxide synthase activity (eNOS) was suppressed by menadione. Menadione resulted in a dose-dependent reduction of cGMP levels accumulated by acetylcholine. This reduction of cGMP levels was blocked by SOD treatment, suggesting that superoxide anion generated by menadione could play a role in the inhibition of the nitric oxide pathway. Evidence supporting a possible role for arylation in impaired vascular relaxation was suggested by the observation that benzoquinone, which does not induce oxidative stress in aortic rings, inhibited acetylcholine-induced vascular relaxation to the same extent as menadione. Collectively, these results suggest that menadione can cause endothelial dysfunction in blood vessels by the inhibition of the nitric oxide pathway via superoxide anion generation and that arylation activity may also be another important mechanism.

Adverse consequences of erythrocyte exposure to menadione: involvement of reactive oxygen species generation in plasma.

Previous studies demonstrated that menadione, a representative quinone compound, reacts nonenzymatically with thiols in plasma, resulting in the generation of reactive oxygen species and potentiation of menadione-induced platelet damage. Because of the reported association of menadione with hemolytic anemia in vivo, investigations were undertaken to identify the free radicals generated from the interaction of menadione with plasma, and to assess the potential role of plasma-generated free-radical species in menadione-dependent erythrocyte toxicity. In rat plasma, menadione increased the rate of oxygen consumption and both luminol- and lucigenin-amplified chemiluminescence in a concentration-dependent manner. Superoxide dismutase (SOD) inhibited lucigenin-amplified chemiluminescence, suggesting formation of superoxide anion. Menadione also induced significant increases in chemiluminescence when erythrocytes were suspended in plasma, but not when cells were suspended in buffer. Consistent with these findings, menadione-dependent hemolysis of erythrocytes occurred only when the cells were suspended in plasma. Various free-radical inhibitors were tested for their ability to inhibit menadione-induced hemolysis. Catalase and mannitol each produced significant inhibition, including an additive effect when both compounds were present, while SOD had no marked effect. In addition, pretreatment with 3-amino-1,2,4-triazole, an intracellular catalase inhibitor, potentiated menadione-induced cytotoxicity in the presence of plasma. These results suggest that both hydrogen peroxide and hydroxyl radicals are involved in menadione-mediated plasma erythrocyte cytotoxicity; however, superoxide anion does not appear to play a direct role.

Association of quinone-induced platelet anti-aggregation with cytotoxicity.

Various anti-platelet drugs, including quinones, are being investigated as potential treatments for cardiovascular disease because of their ability to prevent excessive platelet aggregation. In the present investigation 3 naphthoquinones (2,3-dimethoxy-1,4-naphthoquinone [DMNQ], menadione, and 1,4-naphthoquinone [4-NQ]) were compared for their abilities to inhibit platelet aggregation, deplete glutathione (GSH) and protein thiols, and cause cytotoxicity. Platelet-rich plasma, isolated from Sprague-Dawley rats, was used for all experiments. The relative potency of the 3 quinones to inhibit platelet aggregation, deplete intracellular GSH and protein thiols, and cause cytotoxicity was 1,4-NQ > menadione >> DMNQ. Experiments using 2 thiol-modifying agents, dithiothreitol (DTT) and 1-chloro-2,4-dintrobenzene (CDNB), confirmed the key roles for GSH in quinone-induced platelet anti-aggregation and for protein thiols in quinone-induced cytotoxicity. Furthermore, the anti-aggregative effects of a group of 12 additional quinone derivatives were positively correlated with their ability to cause platelet cytotoxicity. Quinones that had a weak anti-aggregative effect did not induce cytotoxicity (measured as LDH leakage), whereas quinones that had a potent anti-aggregative effect resulted in significant LDH leakage (84-96%). In one instance, however, p-chloranil demonstrated a potent anti-aggregative effect, but did not induce significant LDH leakage. This can be explained by the inability of p-chloranil to deplete protein thiols, even though intracellular GSH levels decreased rapidly. These results suggest that quinones that deplete GSH in platelets demonstrate a marked anti-aggregative effect. If this anti-aggregative effect is subsequently followed by depletion of protein thiols, cytotoxicity results.