Smrati Bhadauria

Isoniazid-induced apoptosis in HepG2 cells: Generation of oxidative stress and Bcl-2 down-regulation

Isoniazid (INH) is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. However it has a serious limitation of being hepatotoxic. Delineating the mechanism underlying INH-induced hepatotoxicity may be beneficial in devising ways to counteract its toxic manifestations. Studies in human hepatoma HepG2 cells have indicated that INH exposure causes induction of apoptosis. This study was aimed at identifying the key components/pathways of the INH-induced apoptotic pathway using HepG2 cells. HepG2 cells were exposed to increasing concentrations of INH (6.5, 13, 26, and 52 mM). Hydrogen peroxide (0.3 mM) served as positive control. After incubating for specific time intervals cells were harvested and evidences of cytotoxicity, oxidative stress, and apoptosis were sought. The findings indicated that INH exposure causes increased ROS generation along with alteration in levels of enzymatic antioxidants such as Superoxide dismutase, Catalase, and Glucose-6-Phosphate dehydrogenase. Altered Bcl-2/Bax content, cytochrome-c translocation, caspase activation, and DNA fragmentation emphasized involvement of apoptosis.

Expression of an insecticidal fern protein in cotton protects against whitefly

Whitefly (Bemisia tabaci) damages field crops by sucking sap and transmitting viral diseases. None of the insecticidal proteins used in genetically modified (GM) crop plants to date are effective against whitefly. We report the identification of a protein (Tma12) from an edible fern, Tectaria macrodonta (Fee) C. Chr., that is insecticidal to whitefly (median lethal concentration = 1.49 μg/ml in in vitro feeding assays) and interferes with its life cycle at sublethal doses. Transgenic cotton lines that express Tma12 at ∼0.01% of total soluble leaf protein were resistant to whitefly infestation in contained field trials, with no detectable yield penalty. The transgenic cotton lines were also protected from whitefly-borne cotton leaf curl viral disease. Rats fed Tma12 showed no detectable histological or biochemical changes, and this, together with the predicted absence of allergenic domains in Tma12, indicates that Tma12 might be well suited for deployment in GM crops to control whitefly and the viruses it carries.

Dose-dependent adverse effects of salinomycin on male reproductive organs and fertility in mice.

Salinomycin is used as an antibiotic in animal husbandry. Its implication in cancer therapy has recently been proposed. Present study evaluated the toxic effects of Salinomycin on male reproductive system of mice. Doses of 1, 3 or 5 mg/kg of Salinomycin were administered daily for 28 days. Half of the mice were sacrificed after 24 h of the last treatment and other half were sacrificed 28 days after withdrawal of treatment. Effects of SAL on body and reproductive organ weights were studied. Histoarchitecture of testis and epididymis was evaluated along with ultrastructural changes in Leydig cells. Serum and testicular testosterone and luteinizing hormones were estimated. Superoxide dismutase, reduced glutathione, lipid peroxidation, catalase and lactate dehydrogenase activities were measured. Spermatozoa count, morphology, motility and fertility were evaluated. Expression patterns of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage proteins (CYP11A1) were assessed by Western blotting. Salinomycin treatment was lethal to few mice and retarded body growth in others with decreased weight of testes and seminal vesicles in a dose dependent manner. Seminiferous tubules in testes were disrupted and the epithelium of epididymis showed frequent occurrence of vacuolization and necrosis. Leydig cells showed hypertrophied cytoplasm with shrunken nuclei, condensed mitochondria, proliferated endoplasmic reticulum and increased number of lipid droplets. Salinomycin decreased motility and spermatozoa count with increased number of abnormal spermatozoa leading to infertility. The testosterone and luteinizing hormone levels were decreased in testis but increased in serum at higher doses. Depletion of superoxide dismutase and reduced glutathione with increased lipid peroxidation in both testis and epididymis indicated generation of oxidative stress. Suppressed expression of StAR and CYP11A1 proteins indicates inhibition of steroidogenesis. Spermatogenesis was however observed in testis 28 days after Salinomycin withdrawal. The results indicate reversible dose-dependent adverse effects of Salinomycin on male reproductive system of mice.

Targeting tumor associated macrophages (TAMs) via nanocarriers

&NA; Recruitment of inflammatory cells to tumor has been well documented, with the most frequent inhabitants being macrophages termed as tumor associated macrophages, (TAMs). Their presence was thought to be an evidence of immune system initiating a fight response towards the tumor, i.e. immune surveillance. This is the case too initially, when TAMs majorly exhibit an M1 phenotype, but their continued presence in tumor microenvironment brings about their polarization to M2 phenotype, which not only participate in continued sustenance of existing tumor but also open up deleterious avenues for further progression and metastasis of cancer. Current perspective is built around this very premise and focuses specifically on TAMs and how they are being targeted by researchers working in annals of nanomedicine. To do so, we dwell into tumor microenvironment and focus on nanotechnology based drug delivery aspects which have either been already or can be potentially employed in the future to target tumor associated macrophages for improved immunoadjuvant therapy of cancer. Graphical abstract Figure. No caption available.

Genotoxicity and oxidative stress in chromium-exposed tannery workers in North India

Trivalent chromium (Cr) is an environmental contaminant, which is extensively used in tanning industries throughout the world and causes various forms of health hazards in tannery workers. Therefore, a cross-sectional study design was used to evaluate the DNA damage and oxidative stress condition in tannery workers exposed to Cr in North India. The study population comprised 100 male tanners in the exposed group and 100 healthy males (no history of Cr exposure) in the comparable control group. Baseline characteristics including age, smoking, alcohol consumption habits and duration of exposure were recorded via interviewing the subjects. Blood Cr level (measured by atomic absorption spectrophotometry), DNA damage (measured by comet assay) and oxidative stress parameters (malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD)) were estimated in both the groups. As a result of statistical analysis, exposed group showed significantly higher level of Cr (p < 0.0001), DNA damage (p < 0.0001), MDA (p < 0.0001), SOD (p < 0.05) and lower level of GSH (p < 0.001) when compared with controls. Smoking, alcohol consumption habits and age had no significant effect (p > 0.05) on DNA damage and oxidative stress parameters in both the groups. In simple and multiple correlation analysis, DNA damage and oxidative stress parameters showed significant correlation with Cr level and duration of exposure in exposed group. The findings of the present study revealed that chronic occupational exposure to trivalent Cr may cause DNA damage and oxidative stress in tannery workers.

Estrogen receptor potentiates mTORC2 signaling in breast cancer cells by upregulating superoxide anions.

The estrogen receptor (ER) plays a cardinal role in estrogen-responsive breast carcinogenesis. It is, however, unclear as to how estrogen-ER interaction potentiates breast cancer progression. Compelling evidence supports estrogen-induced redox alterations, such as augmented reactive oxygen species (ROS) levels, as having a crucial role in breast carcinogenesis. Despite ER being a biological mediator of the majority of estrogen-induced cellular responses; its role in estrogen-induced tissue-specific ROS generation remains largely debatable. We examined a panel of human breast cancer specimens and found that ER-positive breast cancer specimens exhibited a higher incidence of augmented O(2)(•-) levels compared to matched normal tissue. ROS are known to function as signal transducers and ROS-mediated signaling remains a key complementary mechanism that drives carcinogenesis by activating redox-sensitive oncogenic pathways. Additional studies revealed that augmented O(2)(•-) levels in breast cancer specimens coincided with mammalian target of rapamycin complex 2 (mTORC2) hyperactivation. Detailed investigations using in vitro experiments established that 17β-estradiol (E2)-stimulated breast cancer cells exhibited transiently upregulated O(2)(•-) levels, with the presence of ER being a crucial determinant for the phenomenon to take place. Gene expression, ER transactivation, and confocal studies revealed that the E2-induced transient O(2)(•-) upregulation was effected by ER through a nongenomic pathway possibly involving mitochondria. Furthermore, E2 treatment activated mTORC2 in breast cancer cells in a characteristically ER-dependent manner. Interestingly, altering O(2)(•-) anion levels through chemical/genetic methods caused significant modulation of the mTORC2 signaling cascade. Taken together, our findings unravel a novel nongenomic pathway unique to estrogen-responsive breast cancer cells wherein, upon stimulation by E2, ER may regulate mTORC2 activity in a redox-dependent manner by transiently modulating O(2)(•-) levels particularly within mitochondria. The findings suggest that therapies aimed at counteracting these redox alterations and/or resultant signaling cascades may complement conventional treatments for estrogen-responsive breast cancer.

Glycine soya diet synergistically enhances the suppressive effect of tamoxifen and inhibits tamoxifen-promoted hepatocarcinogenesis in 7,12-dimethylbenz[α]anthracene-induced rat mammary tumor model

There is increasing interest in phytoestrogens as potential alternatives to synthetic selective estrogen receptor modulators (SERMs) in the prevention and therapy of breast cancer. The present study is aimed at determining whether dietary glycine soya (Glycine max seeds; GS), which is rich in phytoestrogens, can enhance the anti breast cancer efficacy of the SERM tamoxifen (TAM) and the effect of TAM and GS, either alone or in combination, on DMBA-initiated hepatocarcinogenesis in rat. For determination of enhancing effect, rats bearing palpable 7, 12-dimethylbenz[α] anthracene (DMBA)-induced mammary tumors were treated with TAM (10 mg kg(-1)/day) while being fed AIN-93G diet with or without added GS (3×10(4) mg kg(-1)), and the tumor growth was monitored up to 5 weeks of treatment. For determining the effect on hepatocarcinogenesis, DMBA-initiated rats were exposed to TAM and dietary GS as above for 6 weeks during promotion stage in a medium-term bioassay, and the development of placental form of glutathione-S-transferase (GST-P)-expressing preneoplastic liver lesions was quantified. Exposure to both TAM and dietary GS enhanced the anti tumor efficacy of TAM via a combination of tumor cell apoptosis (determined by TUNEL) and inhibition of tumor cell proliferation (determined by PCNA immunostaining) and suppressed the growth of GST-P-positive liver lesions. The findings show that dietary GS enhances the therapeutic efficacy of TAM against mammary tumors and minimizes TAMs hepatocarcinogenesis promotion potential.

Theophylline, a methylxanthine drug induces osteopenia and alters calciotropic hormones, and prophylactic vitamin D treatment protects against these changes in rats

The drug, theophylline is frequently used as an additive to medications for people suffering from chronic obstructive pulmonary diseases (COPD). We studied the effect of theophylline in bone cells, skeleton and parameters related to systemic calcium homeostasis. Theophylline induced osteoblast apoptosis by increasing reactive oxygen species production that was caused by increased cAMP production. Bone marrow levels of theophylline were higher than its serum levels, indicating skeletal accumulation of this drug. When adult Sprague-Dawley rats were treated with theophylline, bone regeneration at fracture site was diminished compared with control. Theophylline treatment resulted in a time-dependent (at 4- and 8 weeks) bone loss. At 8 weeks, a significant loss of bone mass and deterioration of microarchitecture occurred and the severity was comparable to methylprednisone. Theophylline caused formation of hypomineralized osteoid and increased osteoclast number and surface. Serum bone resorption and formation marker were respectively higher and lower in the theophylline group compared with control. Bone strength was reduced by theophylline treatment. After 8 weeks, serum 25-D3 and liver 25-hydroxylases were decreased in theophylline group than control. Further, theophylline treatment reduced serum 1, 25-(OH)2 vitamin D3 (1,25-D3), and increased parathyroid hormone and fibroblast growth factor-23. Theophylline treated rats had normal serum calcium and phosphate but displayed calciuria and phosphaturia. Co-administration of 25-D3 with theophylline completely abrogated theophylline-induced osteopenia and alterations in calcium homeostasis. In addition, 1,25-D3 protected osteoblasts from theophylline-induced apoptosis and the attendant oxidative stress. We conclude that theophylline has detrimental effects in bone and prophylactic vitamin D supplementation to subjects taking theophylline could be osteoprotective.

Therapeutic effect of centchroman alone and in combination with glycine soya on 7,12-dimethylbenz[α]anthracene-induced breast tumor in rat.

Centchroman is a non-steroidal oral contraceptive and has been found to be a candidate drug for breast cancer exhibiting partial to complete remission of lesions in 40.5% of breast cancer patients. The therapeutic efficacy of centchroman was monitored alone and together with glycine soya on growth of 7,12-dimethylbenz[alpha]anthracene-induced breast tumor in rat. The tumor regression was monitored at different doses of centchroman alone ranging from 0 to 10 mg kg(-1) and with glycine soya from 1x10(4) to 5x10(4) mg kg(-1) per day until 5weeks treatment. An optimum tumor treatment opus was established with varying treatment parameters including doses of therapeutic agents and treatment period. The tumors were found to be static with a strong anti-estrogenic effect. Overall our study shows that both centchroman and glycine soya alone and jointly combat with breast cancer.

Dynamics of physical interaction between HIV-1 Nef and ASK1: identifying the interacting motif(s).

FasL mediated preferential apoptosis of bystander CTLs while protection of infected CD4+T cells remains one of the hallmarks of immune evasion during HIV infection. The property of infected host cells to evade cell-autonomous apoptosis emanates from ability of HIV-1Nef -protein to physically interact with ASK-1 and thereby inhibit its enzymatic activity. The specific domains of HIV-1Nef through which it may interact with ASK1 and thereby impair the ASK1 activity remain unidentified so far and represent a major challenge towards developing clear understanding about the dynamics of this interaction. Using mammalian two hybrid screen in association with site directed mutagenesis and competitive inhibitor peptides, we identified constituent minimal essential domain (152 DEVGEANN 159) through which HIV-1Nef interacts with ASK1 and inhibits its function. Furthermore our study also unravels a novel alternate mechanism underlying HIV-1 Nef mediated ASK1 functional modulation, wherein by potentiating the inhibitory ser967 phosphorylation of ASK1, HIV-1Nef negatively modulated ASK1function.