Debasis Bagchi

Oxidative mechanisms in the toxicity of metal ions.

The role of reactive oxygen species, with the subsequent oxidative deterioration of biological macromolecules in the toxicities associated with transition metal ions, is reviewed. Recent studies have shown that metals, including iron, copper, chromium, and vanadium undergo redox cycling, while cadmium, mercury, and nickel, as well as lead, deplete glutathione and protein-bound sulfhydryl groups, resulting in the production of reactive oxygen species as superoxide ion, hydrogen peroxide, and hydroxyl radical. As a consequence, enhanced lipid peroxidation. DNA damage, and altered calcium and sulfhydryl homeostasis occur. Fenton-like reactions may be commonly associated with most membranous fractions including mitochondria, microsomes, and peroxisomes. Phagocytic cells may be another important source of reactive oxygen species in response to metal ions. Furthermore, various studies have suggested that the ability to generate reactive oxygen species by redox cycling quinones and related compounds may require metal ions. Recent studies have suggested that metal ions may enhance the production of tumor necrosis factor alpha (TNF alpha) and activate protein kinase C, as well as induce the production of stress proteins. Thus, some mechanisms associated with the toxicities of metal ions are very similar to the effects produced by many organic xenobiotics. Specific differences in the toxicities of metal ions may be related to differences in solubilities, absorbability, transport, chemical reactivity, and the complexes that are formed within the body. This review summarizes current studies that have been conducted with transition metal ions as well as lead, regarding the production of reactive oxygen species and oxidative tissue damage.

Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinsons disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral keratinocytes. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(2) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection factor in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

In vitro and in vivo generation of reactive oxygen species, DNA damage and lactate dehydrogenase leakage by selected pesticides

Reactive oxygen species may be involved in the toxicity of various pesticides and we have, therefore, examined the in vivo effects of structurally dissimilar polyhalogenated cyclic hydrocarbons (PCH), such as endrin and chlordane, chlorinated acetamide herbicides (CAH), such as alachlor, and organophosphate pesticides (OPS), such as chlorpyrifos and fenthion, on the production of hepatic and brain lipid peroxidation and DNA-single strand breaks (SSB), two indices of oxidative stress and oxidative tissue damage. The selected pesticides were administered p.o. to female Sprague-Dawley rats in two 0.25 LD50 doses at 0 h and 21 h and killed at 24 h. In a parallel set of experiments, we have determined the in vitro effects of these pesticides on the DNA-SSB and enhanced lactate dehydrogenase leakage (LDH) from neuroactive PC-12 cells in culture. In vitro production of reactive oxygen species by these pesticides was also assessed by determining the enhanced chemiluminescence responses of hepatic and brain homogenates. Following treatment of rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, increases of 2.8-, 3.0-, 4.2-, 4.3- and 4.8-fold were observed in hepatic lipid peroxidation, respectively, while at these same doses, increases in lipid peroxidation of 2.4-, 2.1-, 3.6-, 4.6- and 5.3-fold, respectively, were observed in brain homogenates. Increases of 4.4-, 3.9-, 1.6-, 3.0- and 3.5-fold were observed in hepatic DNA-SSB following treatment of the rats with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while at these same doses, increases of 1.9-, 1.7-, 2.2-, 1.4-, 1.4-fold, respectively, were observed in brain nuclear DNA-SSB. Following in vitro incubation of hepatic and brain tissues with 1 nmol/ml of each of the five pesticides, maximum increases in chemiluminescence occurred within 4-7 min of incubation and persisted for over 10 min. Increases of 3.0-, 2.7-, 3.6-, 4.9- and 4.4-fold were observed in chemiluminescence following in vitro incubation of the liver homogenates with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.7-, 1.8-, 2.0-, 3.4- and 3.7-fold, respectively, were observed in the brain homogenates. Increases of 2.2-, 2.3-, 2.9-, 2.9- and 3.4-fold were observed in the chemiluminescence responses in the liver homogenates of the animals treated with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively, while increases of 1.8-, 2.0-, 3.2-, 2.9- and 2.4-fold, respectively, were observed in the brain homogenates. Cultured neuroactive PC-12 cells were incubated with the pesticides and the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular damage and cytotoxicity was examined. Maximal release of LDH from cultured PC-12 cells was observed at 100 nM concentrations of the pesticides. Increases of 2.3-, 2.5-, 2.8-, 3.1 and 3.4-fold were observed in LDH leakage following incubation of the PC-12 cells with endrin, chlordane, alachlor, chlorpyrifos and fenthion, respectively. Following incubation of the cultured PC-12 cells with 100 nM concentrations of these same pesticides, increases in DNA-SSB of 2.5-, 2.2-, 2.1-, 2.4- and 2.5-fold, respectively, were observed. The results clearly demonstrate that these different classes of pesticides induce production of reactive oxygen species and oxidative tissue damage which may contribute to the toxic manifestations of these xenobiotics. Reactive oxygen species may serve as common mediators of programmed cell death (apoptosis) in response to many toxicants and pathological conditions.

Cytotoxicity and oxidative mechanisms of different forms of chromium

Chromium exists mostly in two valence states in nature: hexavalent chromium [chromium(VI)] and trivalent chromium [chromium(III)]. Chromium(VI) is commonly used in industrial chrome plating, welding, painting, metal finishes, steel manufacturing, alloy, cast iron and wood treatment, and is a proven toxin, mutagen and carcinogen. The mechanistic cytotoxicity of chromium(VI) is not completely understood, however, a large number of studies demonstrated that chromium(VI) induces oxidative stress, DNA damage, apoptotic cell death and altered gene expression. Conversely, chromium(III) is essential for proper insulin function and is required for normal protein, fat and carbohydrate metabolism, and is acknowledged as a dietary supplement. In this paper, comparative concentration- and time-dependent effects of chromium(VI) and chromium(III) were demonstrated on increased production of reactive oxygen species (ROS) and lipid peroxidation, enhanced excretion of urinary lipid metabolites, DNA fragmentation and apoptotic cell death in both in vitro and in vivo models. Chromium(VI) demonstrated significantly higher toxicity as compared with chromium(III). To evaluate the role of p53 gene, the dose-dependent effects of chromium(VI) were assessed in female C57BL/6Ntac and p53-deficient C57BL/6TSG p53 mice on enhanced production of ROS, lipid peroxidation and DNA fragmentation in hepatic and brain tissues. Chromium(VI) induced more pronounced oxidative damage in multiple target organs in p53 deficient mice. Comparative studies of chromium(III) picolinate and niacin-bound chromium(III), two popular dietary supplements, reveal that chromium(III) picolinate produces significantly more oxidative stress and DNA damage. Studies have implicated the toxicity of chromium picolinate in renal impairment, skin blisters and pustules, anemia, hemolysis, tissue edema, liver dysfunction; neuronal cell injury, impaired cognitive, perceptual and motor activity; enhanced production of hydroxyl radicals, chromosomal aberration, depletion of antioxidant enzymes, and DNA damage. Recently, chromium picolinate has been shown to be mutagenic and picolinic acid moiety appears to be responsible as studies show that picolinic acid alone is clastogenic. Niacin-bound chromium(III) has been demonstrated to be more bioavailable and efficacious and no toxicity has been reported. In summary, these studies demonstrate that a cascade of cellular events including oxidative stress, genomic DNA damage and modulation of apoptotic regulatory gene p53 are involved in chromium(VI)-induced toxicity and carcinogenesis. The safety of chromium(III) is largely dependent on the ligand, and adequate clinical studies are warranted to demonstrate the safety and efficacy of chromium(III) for human consumption.

Protective effects of grape seed proanthocyanidins and selected antioxidants against TPA-induced hepatic and brain lipid peroxidation and DNA fragmentation, and peritoneal macrophage activation in mice.

1. The comparative protective abilities of a grape seed proanthocyanidin extract (GSPE) (25-100 mg/kg), vitamin C (100 mg/kg), vitamin E succinate (VES) (100 mg/kg) and beta-carotene (50 mg/kg) on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced lipid peroxidation and DNA fragmentation in the hepatic and brain tissues, as well as production of reactive oxygen species by peritoneal macrophages, were assessed. 2. Treatment of mice with GSPE (100 mg/kg), vitamin C, VES and beta-carotene decreased TPA-induced production of reactive oxygen species, as evidenced by decreases in the chemiluminescence response in peritoneal macrophages by approximately 70%, 18%, 47% and 16%, respectively, and cytochrome c reduction by approximately 65%, 15%, 37% and 19%, respectively, compared with controls. 3. GSPE, vitamin C, VES and beta-carotene decreased TPA-induced DNA fragmentation by approximately 47%, 10%, 30% and 11%, respectively, in the hepatic tissues, and 50%, 14%, 31% and 11%, respectively, in the brain tissues, at the doses that were used. Similar results were observed with respect to lipid peroxidation in hepatic mitochondria and microsomes and in brain homogenates. 4. GSPE exhibited a dose-dependent inhibition of TPA-induced lipid peroxidation and DNA fragmentation in liver and brain, as well as a dose-dependent inhibition of TPA-induced reactive oxygen species production in peritoneal macrophages. 5. GSPE and other antioxidants provided significant protection against TPA-induced oxidative damage, with GSPE providing better protection than did other antioxidants at the doses that were employed.

Molecular mechanisms of cardioprotection by a novel grape seed proanthocyanidin extract

Free radicals and oxidative stress play a crucial role in the pathophysiology of a broad spectrum of cardiovascular diseases including congestive heart failure, valvular heart disease, cardiomyopathy, hypertrophy, atherosclerosis and ischemic heart disease. We have demonstrated that IH636 grape seed proanthocyanidin extract (GSPE) provides superior antioxidant efficacy as compared to Vitamins C, E and beta-carotene. A series of studies were conducted using GSPE to demonstrate its cardioprotective ability in animals and humans. GSPE supplementation improved cardiac functional assessment including post-ischemic left ventricular function, reduced myocardial infarct size, reduced ventricular fibrillation (VF) and tachycardia, decreased the amount of reactive oxygen species (ROS) as detected by ESR spectroscopy and reduced malondialdehyde (MDA) formation in the heart perfusate. Cardiomyocyte apoptosis detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining. In concert, the proapoptotic signals mediated by JNK-l and c-fos proteins were also reduced suggesting that the novel cardioprotective properties of GSPE may be at least partially attributed to its ability to block anti-death signaling mediated through the proapoptotic transcription factors and genes such as JNK-1 and c-JUN. In a separate study, GSPE pretreatment significantly inhibited doxorubicin-induced cardiotoxicity as demonstrated by reduced serum creatine kinase (CK) activity, DNA damage and histopathological changes in the cardiac tissue of mice. Concentration-dependent efficacy of GSPE was also assessed in a hamster atherosclerosis model. Approximately 49 and 63% reduction in foam cells, a biomarker of early stage atherosclerosis, were observed following supplementation of 50 and 100 mg GSPE/kg body weight, respectively. A human clinical trial was conducted on hypercholesterolemic subjects. GSPE supplementation significantly reduced oxidized LDL, a biomarker of cardiovascular diseases. Finally, a cDNA microarray study demonstrated significant inhibition of inducible endothelial CD36 expression, a novel cardioregulatory gene, by GSPE. These results demonstrate that GSPE may serve as a potential therapeutic tool in promoting cardiovascular health via a number of novel mechanisms.

Anti-angiogenic, antioxidant, and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula

Edible berry anthocyanins possess a broad spectrum of therapeutic and anti-carcinogenic properties. Berries are rich in anthocyanins, compounds that provide pigmentation to fruits and serve as natural antioxidants. Anthocyanins repair and protect genomic DNA integrity. Earlier studies have shown that berry anthocyanins are beneficial in reducing age-associated oxidative stress, as well as in improving neuronal and cognitive brain function. Six berry extracts (wild blueberry, bilberry, cranberry, elderberry, raspberry seeds, and strawberry) were studied for antioxidant efficacy, cytotoxic potential, cellular uptake, and anti-angiogenic (the ability to reduce unwanted growth of blood vessels, which can lead to varicose veins and tumor formation) properties. We evaluated various combinations of edible berry extracts and developed a synergistic formula, OptiBerry IH141, which exhibited high ORAC (Oxygen-Radical Absorbing Capacity) value, low cytotoxicity, and superior anti-angiogenic properties compared to the other combinations tested. Anti-angiogenic approaches to treat cancer represent a priority area in vascular tumor biology. OptiBerry significantly inhibited both H2O2- and TNF-α-induced VEGF (Vascular Endothelial Growth Factor) expression by human keratinocytes. VEGF is a key regulator of tumor angiogenesis. Matrigel assay using human microvascular endothelial cells showed that OptiBerry impaired angiogenesis. In anin vivo model of angiogenesis, OptiBerry significantly inhibited basal MCP-1 and inducible NF-κB transcriptions. Endothelioma cells pretreated with OptiBerry showed a diminished ability to form hemangioma and markedly decreased tumor growth by more than 50%. In essence, these studies highlight the novel anti-angiogenic, antioxidant, and anti-carcinogenic potential of a novel anthocyanin-rich berry extract formula, OptiBerry.

Cellular Protection with Proanthocyanidins Derived from Grape Seeds

Abstract: Grape seed proanthocyanidins have been reported to possess a broad spectrum of pharmacological and medicinal properties against oxidative stress. We have demonstrated that IH636 proanthocyanidin extract (GSPE) provides excellent protection against free radicals in both in vitro and in vivo models. GSPE had significantly better free radical scavenging ability than vitamins C, E and β‐carotene and demonstrated significant cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the growth and viability of normal cells. GSPE protected against tobacco‐induced apoptotic cell death in human oral keratinocytes and provided protection against cancer chemotherapeutic drug‐induced cytotoxicity in human liver cells by modulating cell cycle/apoptosis regulatory genes such as bcl2, p53 and c‐myc. Recently, the bioavailability and mechanistic pathways of cytoprotection by GSPE were examined on acetaminophen‐induced hepatotoxicity and nephrotoxicity, amiodarone‐induced pulmonary toxicity, doxorubicin‐induced cardiotoxicity, DMN‐induced immunotoxicity and MOCAP‐induced neurotoxicity in mice. Serum chemistry changes, integrity of genomic DNA and histopathology were assessed. GSPE pre‐exposure provided near complete protection in terms of serum chemistry changes and DNA damage, as well as abolished apoptotic and necrotic cell death in all tissues. Histopathological examination reconfirmed these findings. GSPE demonstrated concentration‐/dose‐dependent inhibitory effects on the drug metabolizing enzyme cytochrome P450 2E1, and this may be a major pathway for the anti‐toxic potential exerted by GSPE. Furthermore, GSPE treatment significantly decreased TNFα‐induced adherence of T‐cells to HUVEC by inhibiting VCAM‐1 expression. These results demonstrate that GSPE is highly bioavailable and may serve as a potential therapeutic tool in protecting multiple target organs from structurally diverse drug‐ and chemical‐induced toxicity.

Oxygen, Oxidants, and Antioxidants in Wound Healing

Abstract: Disrupted vasculature and high energy‐demand by regenerating tissue results in wound hypoxia. Wound repair may be facilitated by oxygen therapy. Evidence supporting the mode of action of hyperbaric oxygen in promoting wound healing is sketchy, however. Topical oxygen therapy involves local administration of pure oxygen. The advantages of topical oxygen therapy include low cost, the lack of systemic oxygen toxicity, and possibility of home treatment. While this modality of wound care is of outstanding interest, it clearly lacks the support of mechanism‐oriented studies. The search for mechanisms by which oxygen supports wound healing has now taken another step. Respiratory burst‐derived oxidants support healing. Oxidants serve as cellular messengers to promote healing. Although this information is of outstanding significance to the practice of oxygen therapy, it remains largely unexplored. The search for “natural remedies” has drawn attention to herbals. Proanthocyanidins or condensed tannins are a group of biologically active polyphenolic bioflavonoids that are synthesized by many plants. Proanthocyanidins and other tannins facilitate wound healing. A combination of grape seed proanthocyanidin extract and resveratrol facilitates inducible VEGF expression, a key element supporting wound angiogenesis. Strategies to manipulate the redox environment in the wound are likely to be of outstanding significance in wound healing.

The cytotoxic effects of a novel IH636 grape seed proanthocyanidin extract on cultured human cancer cells

Grape seed proanthocyanidins are natural antioxidants which possess a broad spectrum of chemoprotective properties against free radicals and oxidative stress. In this study, we have assessed the cytotoxicity of a novel IH636 grape seed proanthocyanidin extract (GSPE) against MCF-7 human breast cancer cells, A-427 human lung cancer cells, CRL-1739 human gastric adenocarcinoma cells and K562 chronic myelogenous leukemic cells at 25 and 50 mg/lit concentrations for 0-72 h using cytomorphology and MTT cytotoxicity assay. In addition, we compared the effects on normal human gastric mucosal cells and normal J774A.1 murine macrophage cells with the effects on the cancer cell lines. Concentration- and time-dependent cytotoxic effects of GSPE were observed on the MCF-7 breast cancer, A-427 lung cancer and gastric adenocarcinoma cells. Following incubation of the MCF-7 cells with 25 mg/lit of the GSPE approximately 6.5, 30 and 43% inhibitions in cell growth were observed at 24, 48 and 72 h of incubation, respectively, while incubation of the MCF-7 cells with 50 mg/lit of the GSPE resulted in 11, 35 and 47% inhibition in cell growth at these same points, respectively. Similar results were observed in the A-427 and gastric adenocarcinoma cells. GSPE exhibited no cytotoxicity toward the neoplastic K562 myelogenous leukemic cells. However, GSPE enhanced the growth and viability of the normal human gastric mucosal cells and J774A.1 murine macrophage cells. These data demonstrate that GSPE exhibited cytotoxicity towards some cancer cells, while enhancing the growth and viability of the normal cells which were examined.