Stimulation of cytoprotective autophagy and components of mitochondrial biogenesis / proteostasis in response to ionizing radiation as a credible pro-survival strategy.

The present study illustrates mitochondria-mediated impact of ionizing radiation which is paralleled by activation of several pro-adaptive responses in normal human dermal fibroblast cells. Irradiation of cells with X-rays (5 Gy) led to an increase in fragmentation and mitochondrial mass. Distinct temporal changes in cytosolic and mitochondrial reactive oxygen species (ROS) were noted in response to radiation, which was associated with depletion in mitochondrial membrane potential followed by decrease in ATP levels. Long Amplicon-Polymerase Chain Reaction (LA-PCR) analysis showed time-dependent increase in mitochondrial DNA damage that preceded mitochondrial ROS generation. Irradiation of cells led to an initial G2/M arrest at 8 h that persisted till 16 h, with subsequent block at G0/G1 measured at 48 and 72 h time points. Interestingly, cells activated autophagy as a countermeasure against radiation-mediated cellular insults and aided in removal of damaged mitochondria. Blocking autophagy using 3-methyladenine led to cell death via activation of enhanced ROS, PARP-1 and caspase 3 cleavage. Upregulation of mitochondrial biogenesis factors Nrf1/PGC-1α, following irradiation was observed. Irradiated cells exhibited an increase in the phosphorylation of GCN2, PERK and eIF2α that might be responsible for the up-regulation of ATF4 and CHOP thereby regulating autophagy and components of integrated stress response. Apart from this, we measured accumulation of mitochondrial chaperones (HSP60/HSP10) and ATF5 which is a major molecule involved in mitochondrial stress. Taken together, mitochondria are one of the major cytoplasmic targets for ionizing radiation and possibly act as an early indicator of cellular insult. The findings also show that stressed mitochondria might influence endoplasmic reticulum (ER)-related signalling leading to the activation of adaptive mechanisms like cytoprotective autophagy, and molecules responsible for mitochondrial biogenesis and protein quality control in order to replenish mitochondrial pool and maintain cellular homeostasis.

Harmonization of Mangiferin on methylmercury engendered mitochondrial dysfunction.

Mangiferin (MGN), a C-glucosylxanthone abundantly found in mango plants, was studied for its potential to ameliorate methylmercury (MeHg) induced mitochondrial damage in HepG2 (human hepatocarcinoma) cell line. Cell viability experiments performed using 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide (MTT) showed protective property of MGN in annulling MeHg-induced cytotoxicity. Conditioning the cells with optimal dose of MGN (50 µM) lowered MeHg-induced oxidative stress, calcium influx/efflux, depletion of mitochondrial trans-membrane potential and prevented mitochondrial fission as observed by decrease in Mitotracker red fluorescence, expression of pDRP1 (serine 616), and DRP1 levels. MGN pre-treated cells demonstrated elevation in the activities of glutathione (GSH), Glutathione-S-transferase (GST), Glutathione peroxidase (GPx), Glutathione reductase (GR), reduced levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) and mitochondrial electron transport chain (ETC) enzyme complexes. In addition, the anti-apoptotic effect of MGN was clearly indicated by the reduction in MeHg-induced apoptotic cells analyzed by flowcytometric analysis after Annexin V-FITC/propidium iodide staining. In conclusion, the present work demonstrates the ability of a dietary polyphenol, MGN to ameliorate MeHg-mediated mitochondrial dysfunction in human hepatic cells in vitro. This hepatoprotective potential may be attributed predominantly to the free radical scavenging/antioxidant property of MGN, by facilitating the balancing of cellular Ca2+ ions, maintenance of redox homeostasis and intracellular antioxidant activities, ultimately preserving the mitochondrial function and cell viability after MeHg intoxication. As MeHg intoxication occurs over a period of time, continuous consumption of such dietary compounds may prove to be very useful in promoting human health. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 630-644, 2017.

Hydroxytyrosol, a dietary phenolic compound forestalls the toxic effects of methylmercury-induced toxicity in IMR-32 human neuroblastoma cells.

This study demonstrates the protective potential of hydroxytyrosol (HT), an olive oil phenol, against methylmercury (MeHg)-induced neurotoxicity using IMR-32 human neuroblastoma cell line. HT inhibited MeHg-induced cytotoxicity and genotoxicity as confirmed by MTT, micronucleus, and comet assays. Cells preconditioned with HT showed reduction of MeHg-induced cellular oxidative stress along with the maintenance of glutathione, superoxide dismutase, glutathione-S-tranferase, and catalase. Fluorescence microscopy and DNA ladder assays indicated the inhibitory effect of HT against MeHg-induced apoptosis, which was further established by Western blotting. An effective concentration of 5 µM HT caused downregulation of p53, bax, cytochrome c, and caspase 3 and upregulation of prosurvival proteins including nuclear factor erythroid 2-related factor 2 (Nrf2) and metallothionein. This work indicates the cytoprotective potential of HT against MeHg-induced toxicity primarily by the lowering of oxidative stress, which may be endorsed to its antigenotoxic and antiapoptotic potential, in addition to its free radical scavenging ability. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1264-1275, 2016.

Thymol, a monoterpene phenolic derivative of cymene, abrogates mercury-induced oxidative stress resultant cytotoxicity and genotoxicity in hepatocarcinoma cells.

Thymol (TOH) was investigated for its ability to protect against mercuric chloride (HgCl2 )-induced cytotoxicity and genotoxicity using human hepatocarcinoma (HepG2) cell line. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay confirmed the efficacy of TOH pretreatment in attenuating HgCl2 -induced cytotoxicity. Pretreatment with TOH inhibited HgCl2 -induced genotoxicity, depolarization of mitochondrial membrane, oxidative stress, and mitochondrial superoxide levels. Interestingly, TOH (100 µM) alone elevated the intracellular basal glutathione S-transferase (GST) levels and TOH pretreatment abrogated the decrease in glutathione, GST, superoxide dismutase, and catalase levels even after HgCl2 intoxication. Furthermore, TOH was also capable of inhibiting HgCl2 -induced apoptotic as well as necrotic cell death analyzed by flowcytometric analysis of cells dual stained with Annexin-FITC/propidium iodide. The present findings clearly indicate the cytoprotective potential of TOH against HgCl2 -induced toxicity, which may be attributed to its free radical scavenging ability which facilitated in reducing oxidative stress and mitochondrial damage thereby inhibiting cell death.

Objective assessment of endogenous collagen in vivo during tissue repair by laser induced fluorescence.

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm2 He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.

The extent of paternal sperm DNA damage influences early post-natal survival of first generation mouse offspring.

OBJECTIVES: To study the post-natal characteristics and the survival of offspring derived from DNA damaged sperm. STUDY DESIGN: This experimental prospective study was conducted on Swiss Albino mice (Mus musculus). Sperm DNA damage was induced by different doses of γ-irradiation in male mice who were then mated with healthy female mice. The post-natal characteristics including the survival of first generation offspring were studied and then correlated with the amount of paternal sperm DNA damage. RESULTS: A significant reduction of survival in the early post-natal period was observed in the first generation offspring derived from the DNA damaged sperm, and a strong association was observed between the extent of sperm DNA damage and the survival of the offspring. CONCLUSION: The DNA damage load in sperm at the time of fertilization influences early post-natal survival of the mouse offspring.

Spectroscopic and histological evaluation of wound healing progression following Low Level Laser Therapy (LLLT).

The present study focuses on the evaluation of the effect of He-Ne laser on tissue regeneration by monitoring collagen synthesis in wound granulation tissues in Swiss albino mice using analysis of laser induced fluorescence (LIF) and light microscopy techniques. The spectral analyses of the wound granulation tissues have indicated a dose dependent increase in collagen levels during the post-wounding days. The histological examinations on the other hand have also shown a significant increase in collagen deposition along with the reduced edema, leukocytes, increased granulation tissue, and fibroblast number in the optimal laser dose treated group compared to the non-illuminated controls.

Effect of laser dose and treatment schedule on excision wound healing in diabetic mice.

The present study was undertaken to evaluate a He-Ne laser (632.8 nm; 7 mW; 4.02 mW cm(-2); 15 mm spot size) dose and the treatment schedule on diabetic wound healing in a mouse model. Circular wounds of 15 mm diameter were created on streptozotocin induced diabetic Swiss albino mice, and were uniformly illuminated with the single exposure of various He-Ne laser doses of 1, 2, 3, 4 and 5 J cm(-2) respectively. Further, the treatment schedule was also optimized by exposing the wounds with 3 J cm(-2) at 0, 24 h, 48 h postwounding. Contraction kinetics, mean area under the curve and the mean healing time of the wounds were computed along with the collagen and the glucosamine levels in the wound ground tissues at various postwounding treatment schedules. Results of this study indicated that the single exposure of 3 J cm(-2) laser dose applied immediately after the wounding caused a significant reduction in the mean area under the curve and the mean healing time along with the elevated levels of collagen and glucosamine contents in the tissue compared to the controls. In conclusion, He-Ne laser dose of 3 J cm(-2) applied immediately after the wounding has demonstrated optimum wound healing compared to the other doses and treatment schedules.

Development and evaluation of fiber optic probe-based helium-neon low-level laser therapy system for tissue regeneration--an in vivo experimental study.

We report the design and development of an optical fiber probe-based Helium-Neon (He-Ne) low-level laser therapy system for tissue regeneration. Full thickness excision wounds on Swiss albino mice of diameter 15 mm were exposed to various laser doses of 1, 2, 3, 4, 6, 8 and 10 J cm(-2) of the system with appropriate controls, and 2 J cm(-2) showing optimum healing was selected. The treatment schedule for applying the selected laser dose was also standardized by irradiating the wounds at different postwounding times (0, 24 and 48 h). The tissue regeneration potential was evaluated by monitoring the progression of wound contraction and mean wound healing time along with the hydroxyproline and glucosamine estimation on wound ground tissues. The wounds exposed to 2 J cm(-2) immediately after wounding showed considerable contraction on days 5, 9, 12, 14, 16 and 19 of postirradiation compared with the controls and other treatment schedules, showing significant (P < 0.001) decrease in the healing time. A significant increase in hydroxyproline and glucosamine levels was observed for the 2 J cm(-2) irradiation group compared with the controls and other treatment groups. In conclusion, the wounds treated with 2 J cm(-2) immediately after the wounding show better healing compared with the controls.