Mohammad Abdulkader Akbarsha

Hepatotoxic effect of ochratoxin A and citrinin, alone and in combination, and protective effect of vitamin E: In vitro study in HepG2 cell

Ochratoxin A (OTA) and citrinin (CTN) are the most commonly co-occurring mycotoxins in a wide variety of food and feed commodities. The major target organ of these toxins is kidney but liver could also be a target organ. The combined toxicity of these two toxins in kidney cells has been studied but not in liver cell. In this study HepG2 cells were exposed to OTA and CTN, alone and in combination, with a view to compare the molecular and cellular mechanisms underlying OTA, CTN and OTA + CTN hepatotoxicity. OTA and CTN alone as well as in combination affected the viability of HepG2 cells in a dose-dependent manner. OTA + CTN, at a dose of 20% of IC50 of each, produced effect almost similar to that produced by either of the toxins at its IC50 concentration, indicating that the two toxins in combination act synergistically. The cytotoxicity of OTA + CTN on hepatocytes is mediated by increased level of intracellular ROS followed/accompanied by DNA strand breaks and mitochondria-mediated intrinsic apoptosis. Co-treatment of vitamin E (Vit E) with OTA, CTN and OTA + CTN reduced the levels of ROS and the cytotoxicity. But the genotoxic effect of OTA and OTA + CTN was not completely alleviated by Vit E treatment whereas the DNA damage as caused by CTN when treated alone was obviated, indicating that OTA induces DNA damage directly whereas CTN induces ROS-mediated DNA damage and OTA + CTN combination induces DNA damage not exclusively relying on but influenced by ROS generation. Taken together, these findings indicate that OTA and CTN in combination affect hepatocytes at very low concentrations and, thereby, pose a potential threat to public and animal health.

DNA/RNA binding and anticancer/antimicrobial activities of polymer-copper(II) complexes.

Water soluble polymer-copper(II) complexes with various degrees of coordination in the polymer chain were synthesized and characterized by elemental analysis, IR, UV-visible and EPR spectra. The DNA/RNA binding behavior of these polymer-copper(II) complexes was examined by UV-visible absorption, emission and circular dichroism spectroscopic methods, and cyclic voltammetry techniques. The binding of the polymer-copper(II) complexes with DNA/RNA was mainly through intercalation but some amount of electrostatic interaction was also observed. This binding capacity increased with the degree of coordination of the complexes. The polymer-copper(II) complex having the highest degree of coordination was subjected to analysis of cytotoxic and antimicrobial properties. The cytotoxicity study indicated that the polymer-copper(II) complexes affected the viability of MCF-7 mammary carcinoma cells, and the cells responded to the treatment with mostly through apoptosis although a few cells succumbed to necrosis. The antimicrobial screening showed activity against some human pathogens.

Surfactant–copper(II) Schiff base complexes: synthesis, structural investigation, DNA interaction, docking studies, and cytotoxic activity

A series of surfactant–copper(II) Schiff base complexes (1–6) of the general formula, [Cu(sal-R2)2] and [Cu(5-OMe-sal-R2)2], {where, sal = salicylaldehyde, 5-OMe-sal = 5-methoxy- salicylaldehyde, and R2 = dodecylamine (DA), tetradecylamine (TA), or cetylamine (CA)} have been synthesized and characterized by spectroscopic, ESI-MS, and elemental analysis methods. For a special reason, the structure of one of the complexes (2) was resolved by single crystal X-ray diffraction analysis and it indicates the presence of a distorted square-planar geometry in the complex. Analysis of the binding of these complexes with DNA has been carried out adapting UV-visible-, fluorescence-, as well as circular dichroism spectroscopic methods and viscosity experiments. The results indicate that the complexes bind via minor groove mode involving the hydrophobic surfactant chain. Increase in the length of the aliphatic chain of the ligands facilitates the binding. Further, molecular docking calculations have been performed to understand the nature as well as order of binding of these complexes with DNA. This docking analysis also suggested that the complexes interact with DNA through the alkyl chain present in the Schiff base ligands via the minor groove. In addition, the cytotoxic property of the surfactant–copper(II) Schiff base complexes have been studied against a breast cancer cell line. All six complexes reduced the visibility of the cells but complexes 2, 3, 5, and 6 brought about this effect at fairly low concentrations. Analyzed further, but a small percentage of cells succumbed to necrosis. Of these complexes (6) proved to be the most efficient aptotoxic agent.

Polyethyleneimine anchored copper(II) complexes: Synthesis, characterization, in vitro DNA binding studies and cytotoxicity studies

The water soluble polyethyleneimine-copper(II) complexes, [Cu(phen)(L-tyr)BPEI]ClO4 (where phen=1,10-phenanthroline, L-tyr=L-tyrosine and BPEI=branched polyethyleneimine) with various degree of copper(II) complex units in the polymer chain were synthesized and characterized by elemental analysis and electronic, FT-IR, EPR spectroscopic techniques. The binding of these complexes with CT-DNA was studied using UV-visible absorption titration, thermal denaturation, emission, circular dichroism spectroscopy and cyclic voltammetric methods. The changes observed in the physicochemcial properties indicated that the binding between the polymer-copper complexes and DNA was mostly through electrostatic mode of binding. Among these complexes, the polymer-copper(II) complex with the highest degrees of copper(II) complex units (higher degrees of coordination) showed higher binding constant than those with lower copper(II) complex units (lower degrees of coordination) complexes. The complex with the highest number of metal centre bound strongly due to the cooperative binding effect. Therefore, anticancer study was carried out using this complex. The cytotoxic activity for this complex on MCF-7 breast cancer cell line was determined adopting MTT assay, acridine orange/ethidium bromide (AO/EB) staining and comet assay techniques, which revealed that the cells were committed to specific mode of cell death either apoptosis or necrosis.

Antiproliferative and apoptosis-induction studies of a metallosurfactant in human breast cancer cell MCF-7

The cytotoxic potential of the surfactant–cobalt(III) complex (metallosurfactant) cis-[Co(trien)(C14H29NH2)Cl](ClO4)2 was tested on the MCF-7 breast cancer cell. The viability of the treated cell was evaluated by adopting MTT assay. The mode of cell death was assessed by adopting different morphological, cellular and molecular methods such as comet assay for DNA damage and apoptosis assays (Hoechst staining, acridine orange & ethidium bromide (AO & EB) staining and Annexin V-Cy3 assay). Mitochondria-mediated apoptosis was tested using JC-1 dye. Cell cycle analysis was made by adopting flow cytometry, and the expression of some key pro- and anti-apoptotic proteins was analyzed by adopting Western blotting. The surfactant–cobalt(III) complex induced cell death in a dose- and time-dependent manner. The mode of cell death was essentially apoptosis though necrosis was also noticed. Flow cytometric analysis indicated that the treatment caused cell cycle arrest, as indicated in the accumulation of cells in the sub-G0 + G1 compartment. Western blot analysis revealed the up-regulation of pro-apoptotic p53 and Bax proteins and down-regulation of anti-apoptotic Bcl-2 protein. The study revealed the antiproliferative and apoptosis-induction properties of the surfactant–cobalt(III) complex in an MCF-7 breast cancer cell, primarily by inducing DNA damage and possibly through elevation of ROS levels.

Verrucarin A induces apoptosis through ROS-mediated EGFR/MAPK/Akt signaling pathways in MDA-MB-231 breast cancer cells.

The present study was carried out to elucidate the mechanisms underlying Verrucarin A (VA)‐induced cytotoxicity in human breast cancer cell line MDA‐MB‐231. VA inhibited the growth of MDA‐MB‐231 cells by induction of reactive oxygen species (ROS)‐dependent mitochondrial apoptosis. Elevation of ROS production, associated with changes in Bax/Bcl‐2 ratio, led to loss of mitochondrial membrane potential (Δψm) and cytochrome c release in VA‐treated cells. Release of cytochrome c from mitochondria to cytosol triggered activation of caspase‐3, PARP cleavage, DNA fragmentation, and finally apoptotic cell death. Furthermore, VA‐induced apoptosis was accompanied by the activation of p38MAPK and inhibition of phosphorylation of EGFR as well as of Akt and ERK1/2. However, pre‐treatment with n‐acetyl cysteine, an ROS scavenger, and SB202190, a p38MAPK inhibitor, significantly inhibited VA‐induced ROS generation, EGFR inhibition, p38MAPK activation and apoptosis. Moreover, pharmacological inhibition of EGFR and ERK1/2 significantly accelerated the VA‐induced apoptosis in MDA‐MB‐231 cells. Collectively, these results indicate that VA‐induces ROS elevation in cancer cells, which results in the activation of p38MAPK and inhibition of EGFR/Akt/ERK signaling cascade and, ultimately, cancer cell death. J. Cell. Biochem. 115: 2022–2032, 2014.

Antiproliferative property of n-hexane and chloroform extracts of Anisomeles malabarica (L). R. Br. in HPV16-positive human cervical cancer cells.

Objectives: To find the efficacy of serial extracts of Anisomeles malabarica in inhibiting proliferation of and inducing apoptosis in human cervical cancer cells, SiHa and ME 180, that are HPV 16-positive. Materials and Methods: The whole plant was extracted in n-hexane, chloroform, ethyl acetate, n-butanol, methanol, and water. The cells were treated with the extracts at increasing concentrations to find the IC50, adopting MTT ([3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay. Acridine orange (AO) and ethidium bromide (EB) and Hoechst 33258 staining were adopted to assess the mode of cell death, Annexin V-Cy3 staining to evaluate one of the early apoptotic features, JC-1 staining to assess the mitochondrial membrane depolarization, comet assay for DNA fragmentation, and cell cycle analysis for the distribution of cells after treatment. Results: n-Hexane and chloroform extracts were cytotoxic to the cervical cancer cells in dose- and duration-dependent manner. The cells that responded to the treatments revealed typical apoptotic features. Early features of apoptosis, phosphatidyl serine translocation and loss of mitochondrial trans-membrane potential, were observed in the treated cells, and comet assay revealed DNA damage. In the FACS analysis, the cells accumulated in the sub-G0/G1 phase of the cell cycle, except in n-hexane- and chloroform extract–treated SiHa cells at 24 h, which showed arrest in S- and G2/M phases. Conclusions: n-Hexane and chloroform extracts of A. malabarica inhibit proliferation of and induce death in HPV16-positive cervical cancer cells, mostly by apoptosis and to some extent by necrosis.

Occurrence of ampulla in the ductus deferens of the Indian garden lizard Calotes versicolor daudin

During the breeding season, the terminal end of the ductus deferens of Calotes versicolor appears swollen and is comparable to the ampulla of the mammalian ductus deferens. Its anatomy was studied from paraffin sections. It differentiates along its length into five zones. The first has thick smooth muscle and pesudostratified epithelium; the second has luminal trabeculae with an epithelium showing evidence of secretory activity; the third has the epithelial mucosa abutting against the smooth muscle in the form of pocketlike indentations; the fourth has crypts between epithelial folds; and the fifth zone is a sphincter. The anatomy of this ampullary region is indicative of secretory as well as spermatophagous roles. It undergoes seasonal change and appears to be androgen‐dependent.

Curative property of Withania somnifera Dunal root in the context of carbendazim-induced histopathological changes in the liver and kidney of rat.

The liver and kidney of rat underwent severe histopathological lesions when treated with a single bolus dose of carbendazim, a fungicide, particularly affecting the hepatocytes and the renal corpuscles, respectively. The effects appear to be manifestations of the microtubule-disrupting activity of carbendazim. Treatment of carbendazim-treated rats with the powder of tuberous root of Withania somnifera (Ashwagandha) for 48 days resulted in complete cure of these organs. The results indicate that Withania somnifera would be an effective curative for carbendazim-induced histopathological changes in the liver and kidney.

Buffalo Cervico-Vaginal Fluid Proteomics with Special Reference to Estrous Cycle: Heat Shock Protein (Hsp)-70 Appears to Be an Estrus Indicator

ABSTRACT Cervico-vaginal fluid (CVF) plays significant roles in coitus, sperm transport, and implantation. It is believed to be a good noninvasive biomarker for various diagnostic purposes. In this study, a comprehensive proteomic analysis of buffalo CVF was performed during the estrous cycle in order to document the protein expressions, utilizing SDS-PAGE, mass spectrometry, and immunoblot. The main objective was to screen the CVF of buffalo for one or more estrus-specific proteins. A total of 416 proteins were identified in the CVF of both estrus and diestrus phases. Out of these proteins, 68 estrus-specific proteins have been extensively reviewed in the protein database. The major physiological functions of estrus CVF proteins appeared to be stress response, immune response, and metabolic. Eventually, the expression level of heat shock protein-70 in the CVF during the estrus phase, as revealed in SDS-PAGE analysis, was higher than during diestrus. The identity of the protein was confirmed by immunoblot analysis as heat shock protein-70. The findings provide a potential lead for the evaluation of these proteins for estrus detection in buffalo because CVF biomarker detection is a noninvasive technique. The mass spectrometric data of identified proteins have been deposited at the ProteomeXchange with the identifier PXD000620.