Hussain Arif

Cancer Therapy by Catechins Involves Redox Cycling of Copper Ions and Generation of Reactive Oxygen Species

Catechins, the dietary phytochemicals present in green tea and other beverages, are considered to be potent inducers of apoptosis and cytotoxicity to cancer cells. While it is believed that the antioxidant properties of catechins and related dietary agents may contribute to lowering the risk of cancer induction by impeding oxidative injury to DNA, these properties cannot account for apoptosis induction and chemotherapeutic observations. Catechin (C), epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG) are the four major constituents of green tea. In this article, using human peripheral lymphocytes and comet assay, we show that C, EC, EGC and EGCG cause cellular DNA breakage and can alternatively switch to a prooxidant action in the presence of transition metals such as copper. The cellular DNA breakage was found to be significantly enhanced in the presence of copper ions. Catechins were found to be effective in providing protection against oxidative stress induced by tertbutylhydroperoxide, as measured by oxidative DNA breakage in lymphocytes. The prooxidant action of catechins involved production of hydroxyl radicals through redox recycling of copper ions. We also determined that catechins, particularly EGCG, inhibit proliferation of breast cancer cell line MDA-MB-231 leading to a prooxidant cell death. Since it is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies, cancer cells would be more subject to redox cycling between copper ions and catechins to generate reactive oxygen species (ROS) responsible for DNA breakage. Such a copper dependent prooxidant cytotoxic mechanism better explains the anticancer activity and preferential cytotoxicity of dietary phytochemicals against cancer cells.

Mobilization of Copper ions by Flavonoids in Human Peripheral Lymphocytes Leads to Oxidative DNA Breakage: A Structure Activity Study

Epidemiological studies have linked dietary consumption of plant polyphenols with lower incidence of various cancers. In particular, flavonoids (present in onion, tomato and other plant sources) induce apoptosis and cytotoxicity in cancer cells. These can therefore be used as lead compounds for the synthesis of novel anticancer drugs with greater bioavailability. In the present study, we examined the chemical basis of cytotoxicity of flavonoids by studying the structure–activity relationship of myricetin (MN), fisetin (FN), quercetin (QN), kaempferol (KL) and galangin (GN). Using single cell alkaline gel electrophoresis (comet assay), we established the relative efficiency of cellular DNA breakage as MN > FN > QN > KL > GN. Also, we determined that the cellular DNA breakage was the result of mobilization of chromatin-bound copper ions and the generation of reactive oxygen species. The relative DNA binding affinity order was further confirmed using molecular docking and thermodynamic studies through the interaction of flavonoids with calf thymus DNA. Our results suggest that novel anti-cancer molecules should have ortho-dihydroxy groups in B-ring and hydroxyl groups at positions 3 and 5 in the A-ring system. Additional hydroxyl groups at other positions further enhance the cellular cytotoxicity of the flavonoids.

Mobilization of copper ions in human peripheral lymphocytes by catechins leading to oxidative DNA breakage: A structure activity study

Epidemiological studies suggest that dietary consumption of plant polyphenols is related to a lower incidence of various cancers. Among these compounds catechins (present in green tea and other beverages) are considered to be potent inducers of apoptosis and cytotoxicity to cancer cells. Thus these compounds can be used as leads to synthesize novel anticancer drugs with greater bioavailability. In view of this in this paper we have examined the chemical basis of cytotoxicity of catechins by studying the structure-activity relationship between catechin (C), epicatechin (EC), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG). Using single cell alkaline gel electrophoresis (comet assay) we have established the relative efficiency of cellular DNA breakage as EGCG>EGC>EC>C. We also show that cellular DNA breakage is the result of mobilization of copper ions bound to chromatin and the generation of reactive oxygen species. Further the relative DNA binding affinity order was confirmed using molecular docking and thermodynamic studies by studying the interaction of catechins with calf thymus DNA. The results suggest that the synthesis of any novel anti cancer molecule based on the structure of catechins should have as many galloyl moieties as possible resulting in an increased number of hydroxyl groups that may facilitate the binding of the molecule to cellular DNA.

Deciphering the interaction of bovine heart cystatin with ZnO nanoparticles: Spectroscopic and thermodynamic approach

ZnO-NPs have been widely used in biomedical fields such as therapeutics, cellular imaging, and drug delivery. However, the risk of exposure of nanoparticles to the biological system is not well understood. Nanoparticle-protein interaction is pivotal to understand their biological behavior and predict nanoparticle toxicity that is crucial for its safer applications. In the present study zinc oxide nanoparticles (ZnO-NPs) were synthesized and subjected to interact with buffalo heart cystatin (BHC), purified from buffalo heart, to assess the effect(s) of ZnO-NPs on the structure and function of BHC. In vitro toxicity assessments revealed that BHC, upon interaction with ZnO-NPs, led to the altered protein conformation and perturbed function. A decrease in the anti-papain activity of BHC was observed. Spectroscopic studies demonstrated that formation of BHC-ZnO-NPs complex accompanied by structural changes in BHC along with a significant decrease in its α-helical content. ITC determined the thermodynamic parameters of binding between ZnO-NPs and BHC quantitatively. Increased surface hydrophobicity (change in the tertiary structure) was observed by ANS fluorescence that demonstrated the formation of molten globular intermediates that were found to be stable without any signs of aggregation as depicted by ThT fluorescence. TEM images gave the physical evidence of the formation of ZnO-NPs-BHC corona.

Acute oral dose of sodium nitrite induces redox imbalance, DNA damage, metabolic and histological changes in rat intestine

Industrialization and unchecked use of nitrate/nitrite salts for various purposes has increased human exposure to high levels of sodium nitrite (NaNO2) which can act as a pro-oxidant and pro-carcinogen. Oral exposure makes the gastrointestinal tract particularly susceptible to nitrite toxicity. In this work, the effect of administration of a single acute oral dose of NaNO2 on rat intestine was studied. Animals were randomly divided into four groups and given single doses of 20, 40, 60 and 75 mg NaNO2/kg body weight. Untreated animals served as the control group. An NaNO2 dose-dependent decline in the activities of brush border membrane enzymes, increase in lipid peroxidation, protein oxidation, hydrogen peroxide levels and decreased thiol content was observed in all treated groups. The activities of various metabolic and antioxidant defense enzymes were also altered. NaNO2 induced a dose-dependent increase in DNA damage and DNA-protein crosslinking. Histopathological studies showed marked morphological damage in intestinal cells. The intestinal damage might be due to nitrite-induced oxidative stress, direct action of nitrite anion or chemical modification by reaction intermediates.

Copper-mediated DNA damage by the neurotransmitter dopamine and L-DOPA: A pro-oxidant mechanism

Oxidative DNA damage has been implicated in the pathogenesis of neurological disorders, cancer and ageing. Owing to the established link between labile copper concentrations and neurological diseases, it is critical to explore the interactions of neurotransmitters and drug supplements with copper. Herein, we investigate the pro-oxidant DNA damage induced by the interaction of L-DOPA and dopamine (DA) with copper. The DNA binding affinity order of the compounds has been determined by in silico molecular docking. Agarose gel electrophoresis reveals that L-DOPA and DA are able to induce strand scission in plasmid pcDNA3.1 (+/-) in a copper dependent reaction. These metabolites also cause cellular DNA breakage in human lymphocytes by mobilizing endogenous copper, as assessed by comet assay. Further, L-DOPA and DA-mediated DNA breaks were detected by the appearance of post-DNA damage sensitive marker γH2AX in cancer cell lines accumulating high copper. Immunofluorescence demonstrated the co-localization of downstream repair factor 53BP1 at the damaged induced γH2AX foci in cancer cells. The present study corroborates and provides a mechanism to the hypothesis that suggests metal-mediated oxidation of catecholamines contributes to the pathogenesis of neurodegenerative diseases.

Acute renal toxicity of sodium chlorate: Redox imbalance, enhanced DNA damage, metabolic alterations and inhibition of brush border membrane enzymes in rats

Sodium chlorate (NaClO3) is widely used in paper and pulp industries and as a non‐selective herbicide. Humans can be exposed to NaClO3 through contaminated drinking water due to its improper and unchecked usage in industries and as herbicide. NaClO3 is also present as a major stable by‐product in drinking water that has been disinfected with chlorine dioxide. In this study, we have investigated the effect of a single acute oral dose of NaClO3 on rat kidney. Adult male Wistar rats were divided into one control and four NaClO3 treated groups that were orally given different doses of NaClO3 and euthanized 24 hr after the treatment. Oral administration of NaClO3 resulted in increased hydrogen peroxide levels, lipid, and protein oxidation while thiol and glutathione content and activities of brush border membrane enzymes were decreased in kidney in a NaClO3 dose‐dependent manner. Significant alterations in the activities of enzymes involved in carbohydrate metabolism and antioxidant defense were also observed. Administration of NaClO3 induced DNA fragmentation and increased DNA–protein cross‐linking. Histological studies showed marked damage in kidney from NaClO3 treated animals. These results strongly suggest that NaClO3 induces nephrotoxicity via redox imbalance that results in DNA and membrane damage, metabolic alterations and brush border membrane enzyme dysfunction.

Glycation induced conformational transitions in cystatin proceed to form biotoxic aggregates: A multidimensional analysis

Hyperglycaemic conditions facilitate the glycation of serum proteins which may have predisposition to aggregation and thus lead to complications. The current study investigates the glycation induced structural and functional modifications of chickpea cystatin (CPC) as well as biological toxicity of the modified protein forms, using CPC-glucose as a model system. Several structural intermediates were formed during the incubation of CPC with glucose (day 4, 8, 12, & 16) as revealed by circular dichroism (CD), altered intrinsic fluorescence, and high ANS binding. Further incubation of CPC with glucose (day 21) formed abundant β structures as revealed by Fourier transform infrared spectroscopy and CD analysis which may be due to the aggregation of protein. High thioflavin T fluorescence intensity and increased Congo red absorbance together with enhanced turbidity and Rayleigh scattering by this modified form confirmed the aggregation. Electron microscopy finally provided the valid physical authentication about the presence of aggregate structures. Functional inactivation of glucose incubated CPC was also observed with time. Single cell electrophoresis of lymphocytes and plasmid nicking assays in the presence of modified CPC showed the DNA damage which confirmed its biological toxicity. Hence, our study suggests that glycation of CPC not only leads to structural and functional alterations in proteins but also to biotoxic AGEs and aggregates.