Hanumanthappa Krishnamurthy

Nitric oxide donors release extracellular traps from human neutrophils by augmenting free radical generation.

High availability of NO, oxidative stress and neutrophil extracellular trap (NETs) contents are often noticed at the site of inflammation/infection. Studies from this lab and others have reported NO mediated free radical generation from neutrophils; role of NO in NETs formation however remains undefined so far. The present study was therefore undertaken to explore the effect of NO donors on NET release from human neutrophils (PMNs), using confocal/scanning microscopy, measuring the extracellular DNA content and NET-bound elastase activity. Addition of NO donors (SNAP and SNP) to adhered PMNs led to a time and concentration dependent NETs release, which was blocked by N-acetyl cysteine, suggesting involvement of free radicals in NETs formation. Free radical formation by NO donors was assessed by using DCF-DA, DMPO-nitrone antibody and by p47 phox migration to the neutrophils membrane. NO mediated formation of free radicals and NETs was significantly reduced by the pretreatment of neutrophils with diphenyleneiodonium (DPI), a NADPH-oxidase inhibitor and 4-aminobenzoic acid hydrazide (ABAH), a myeloperoxidase inhibitor, suggesting role of enzymatic free radical generation by NO donors. We thus demonstrate that NO by augmenting free radical formation in human neutrophils mediates NETs release.

Neutrophil extracellular traps contain mitochondrial as well as nuclear DNA and exhibit inflammatory potential

Neutrophils expel extracellular traps (NETs) to entrap and exterminate the invaded micro‐organisms. Acute/chronic inflammatory disorders are often observed with aberrantly enhanced NETs formation and high nitric oxide (NO) availability. Recent study from this laboratory demonstrated release of NETs from human neutrophils following treatment with SNP or SNAP. This study is an extension of our previous finding to explore the extracellular bacterial killing, source of DNA in the expelled NETs, their ability to induce proinflammatory cytokines release from platelets/THP‐1 cells, and assessment of NO‐mediated free radical formation by using a consistent NO donor, DETA‐NONOate. NO‐mediated NETs exhibited extracellular bacterial killing as determined by colony forming units. NO‐mediated NETs formation was due to the activation of NADPH oxidase and myeloperoxidase. NO‐ or PMA‐mediated NETs were positive for both nuclear and mitochondrial DNA as well as proteolytic enzymes. Incubation of NETs with human platelets enhanced the release of IL‐1β and IL‐8, while with THP‐1 cells, release of IL‐1β, IL‐8, and TNFα was observed. This study demonstrates that NO by augmenting enzymatic free radical generation release NETs to promote extracellular bacterial killing. These NETs were made up of mitochondrial and nuclear DNA and potentiated release of proinflammatory cytokines.

Poor sperm quality and advancing age are associated with increased sperm DNA damage in infertile men

With increasing evidence for faulty paternal contribution to reproduction, there has been a steady increase in studies highlighting an association between sperm DNA damage, failed/delayed fertilisation and aberrant embryo development. Owing to prevailing ambiguity, the aims of the study were to analyse the genetic integrity of the male gamete and then to understand its association with age, standard semen parameters, lifestyle and occupational factors. The study included 504 subjects, attending university infertility clinic for fertility evaluation and treatment. Semen characteristics were analysed by standard criteria; terminal deoxynucelotidyl transferase–mediated nick end‐labelling assay was employed for DNA damage assessment. The average incidence of sperm DNA damage in patients with normozoospermic semen parameters was <10%. Patients with oligozoospermia, severe oligozoospermia, oligoasthenoteratospermia, asthenoteratozoospermia and necrozoospermia had significantly higher level of sperm DNA damage (P < 0.001). Patients above 40 years of age had significantly high levels of DNA damage (P < 0.001) compared with their counterparts. Patients with varicocele and a history of alcohol consumption had higher incidence of spermatozoa with DNA damage (P < 0.01). Poor sperm characteristics in the ejaculate are associated with increased sperm DNA damage. Age‐related increase in sperm DNA damage and association of the same with varicocele and alcohol consumption are also demonstrated.

Semen abnormalities, sperm DNA damage and global hypermethylation in health workers occupationally exposed to ionizing radiation.

Background Cytogenetic studies have demonstrated that low levels of chronic radiation exposure can potentially increase the frequency of chromosomal aberrations and aneuploidy in somatic cells. Epidemiological studies have shown that health workers occupationally exposed to ionizing radiation bear an increased risk of hematological malignancies. Objectives To find the influence of occupational radiation exposure on semen characteristics, including genetic and epigenetic integrity of spermatozoa in a chronically exposed population. Methods This cross sectional study included 134 male volunteers of which 83 were occupationally exposed to ionizing radiation and 51 were non-exposed control subjects. Semen characteristics, sperm DNA fragmentation, aneuploidy and incidence of global hypermethylation in the spermatozoa were determined and compared between the non-exposed and the exposed group. Results Direct comparison of the semen characteristics between the non-exposed and the exposed population revealed significant differences in motility characteristics, viability, and morphological abnormalities (P<0.05–0.0001). Although, the level of sperm DNA fragmentation was significantly higher in the exposed group as compared to the non-exposed group (P<0.05–0.0001), the incidence of sperm aneuploidy was not statistically different between the two groups. However, a significant number of hypermethylated spermatozoa were observed in the exposed group in comparison to non-exposed group (P<0.05). Conclusions We provide the first evidence on the detrimental effects of occupational radiation exposure on functional, genetic and epigenetic integrity of sperm in health workers. However, further studies are required to confirm the potential detrimental effects of ionizing radiation in these subjects.

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.

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.

Association between Sperm DNA Integrity and Seminal Plasma Antioxidant levels in Health workers Occupationally Exposed to Ionizing Radiation

There is a paucity of data regarding the association between occupational radiation exposure and risk to human fertility. Recently, we provided the first evidence on altered sperm functional characteristics, DNA damage and hypermethylation in radiation health workers. However, there is no report elucidating the association between seminal plasma antioxidants and sperm chromatin integrity in occupationally exposed subjects. Here, we assessed the seminal plasma antioxidants and lipid peroxidation level in 83 men who were occupationally exposed to ionizing radiation and then correlated with the sperm chromatin integrity. Flow cytometry based sperm chromatin integrity assay revealed a significant decline in αt value in the exposed group in comparison to the non-exposed group (P<0.0001). Similarly, both total and reduced glutathione levels and total antioxidant capacity in the seminal plasma were significantly higher in exposed group than the non-exposed group (P<0.01, 0.001 and 0.0001, respectively). However, superoxide dismutase level and malondialdehyde level, which is an indicator of lipid peroxidation in the seminal plasma, did not differ significantly between two groups. The total antioxidant capacity (TAC) and GSH level exhibited a positive correlation with sperm DNA integrity in exposed subjects. To conclude, this study distinctly shows that altered sperm chromatin integrity in radiation health workers is associated with increase in seminal plasma antioxidant level. Further, the increased seminal plasma GSH and TAC could be an adaptive measure to tackle the oxidative stress to protect genetic and functional sperm deformities in radiation health workers.

Ethanolic extract of Moringa oleifera Lam. leaves protect the pre-pubertal spermatogonial cells from cyclophosphamide-induced damage

ETHNOPHARMACOLOGICAL RELEVANCE Moringa oleifera Lam. is widely cultivated in Asian and African countries for its medicinal and dietary significance. The leaves are highly nutritious and are known to possess various biological activities. MATERIALS AND METHODS Pre-pubertal Swiss albino male mice were injected with single dose of cyclophosphamide (CP, 200mg/kg body weight) or ethanolic extract of Moringa oleifera leaves (MOE, 100mg/kg body weight) intraperitoneally. In combination group, MOE was administered 24h prior to CP injection. RESULTS CP induced a significant decrease in testicular weight (p<0.01) and depletion of germ cells (p<0.001) and higher level of DNA damage (p<0.001) compared to control. The expression of P53, Bax, Cytochrome C (Cyt C) was increased while there was a decrease in the expression of Bcl2, c-Kit and Oct4. Administration of MOE 24h prior to CP treatment ameliorated the depletion (p<0.001), DNA damage (p<0.001) and apoptosis (p<0.01) of germ cells induced by CP. The mitigating effect of MOE appears to be mediated by up-regulating the expression of c-Kit and Oct4 transcripts in P53-independent manner. CONCLUSION MOE protects the spermatogonial cells from CP-induced damage by modulating the apoptotic response elicited by CP and therefore can be considered as an efficient method of male fertility preservation.

Cytotoxic Property of Surfactant–Cobalt(III) Complexes on a Human Breast Cancer Cell Line

The cancer chemotherapeutic potential of surfactant–cobalt(III) complexes, cis‐[Co(bpy)2(C14H29NH2)Cl](ClO4)2 · 3 H2O (1) and cis‐[Co(phen)2(C14H29NH2)Cl](ClO4)2 · 3 H2O (2) (bpy = 2,2′‐bipyridine, phen = 1,10‐phenanthroline) on MCF‐7 breast cancer cell was determined adopting MTT assay and specific staining techniques. The complexes affected the viability of the cells significantly and the cells succumbed to apoptosis as seen in the changes in the nuclear morphology and cytoplasmic features. Since the complex 2 appeared to be more potent, further assays were carried out on the complex 2. Single‐cell electrophoresis indicated DNA damage. The translocation of phosphatidyl serine and loss of mitochondrial potential was revealed by annexin V‐Cy3 staining and JC‐1 staining respectively. Western blot analysis revealed up‐regulation of pro‐apoptotic p53 and down‐regulation of anti‐apoptotic Bcl‐2 protein. Taken together, the surfactant–cobalt(III) complex 2 would be a potential candidate for further investigation for application as a chemotherapeutic for cancers in general and estrogen receptor‐positive breast cancer in particular.