PCR inhibitors and facilitators - Their role in forensic DNA analysis.

Since its inception, DNA typing technology has been practiced as a robust tool in criminal investigations. Experts usually utilize STR profiles to identify and individualize the suspect. However, mtDNA and Y STR analyses are also considered in some sample-limiting conditions. Based on DNA profiles thus generated, forensic scientists often opine the results as Inclusion, exclusion, and inconclusive. Inclusion and exclusion were defined as concordant results; the inconclusive opinions create problems in conferring justice in a trial- since nothing concrete can be interpreted from the profile generated. The presence of inhibitor molecules in the sample is the primary factor behind these indefinite results. Recently, researchers have been emphasizing studying the sources of PCR inhibitors and their mechanism of inhibition. Furthermore, several mitigation strategies- to facilitate the DNA amplification reaction -have now found their place in the routine DNA typing assays with compromised biological samples. The present review paper attempts to provide a comprehensive review of PCR inhibitors, their source, mechanism of inhibition, and ways to mitigate their effect using PCR facilitators.

Detection and separation of proteins using micro/nanofluidics devices.

Microfluidics is the technology or system wherein the behavior of fluids' is studied onto a miniaturized device composed of chambers and tunnels. In biological and biomedical sciences, microfluidic technology/system or device serves as an ultra-high-output approach capable of detecting and separating the biomolecules present even in trace quantities. Given the essential role of protein, the identification and quantification of proteins help understand the various living systems' biological function regulation. Microfluidics has enormous potential to enable biological investigation at the cellular and molecular level and maybe a fair substitution of the sophisticated instruments/equipment used for proteomics, genomics, and metabolomics analysis. The current advancement in microfluidic systems' development is achieving momentum and opening new avenues in developing innovative and hybrid methodologies/technologies. This chapter attempts to expound the micro/nanofluidic systems/devices for their wide-ranging application to detect and separate protein. It covers microfluidic chip electrophoresis, microchip gel electrophoresis, and nanofluidic systems as protein separation systems, while methods such as spectrophotometric, mass spectrometry, electrochemical detection, magneto-resistive sensors and dynamic light scattering (DLS) are discussed as proteins' detection system.

Genotoxic Potential of Nanoparticles: Structural and Functional Modifications in DNA.

The rapid advancement of nanotechnology enhances the production of different nanoparticles that meet the demand of various fields like biomedical sciences, industrial, material sciences and biotechnology, etc. This technological development increases the chances of nanoparticles exposure to human beings, which can threaten their health. It is well known that various cellular processes (transcription, translation, and replication during cell proliferation, cell cycle, cell differentiation) in which genetic materials (DNA and RNA) are involved play a vital role to maintain any structural and functional modification into it. When nanoparticles come into the vicinity of the cellular system, chances of uptake become high due to their small size. This cellular uptake of nanoparticles enhances its interaction with DNA, leading to structural and functional modification (DNA damage/repair, DNA methylation) into the DNA. These modifications exhibit adverse effects on the cellular system, consequently showing its inadvertent effect on human health. Therefore, in the present study, an attempt has been made to elucidate the genotoxic mechanism of nanoparticles in the context of structural and functional modifications of DNA.

TiO2 NPs Induce DNA Damage in Lymphocytes from Healthy Individuals and Patients with Respiratory Diseases-An Ex Vivo/In Vitro Study.

Little is known of the effects of nanoparticles in human systems, let alone in diseased individuals and nanotechnology has preceded nanotoxicology. Therefore, the effects of titanium dioxide (TiO2) nanoparticles in peripheral blood lymphocytes from patients with respiratory diseases [lung cancer, chronic obstructive pulmonary disease (COPD) and asthma] were compared with those in healthy Individuals, to determine differences in sensitivity to nanochemical insult. The Comet assay was performed according to recommended guidelines. The micronucleus assay and ras oncoprotein detection were conducted according to published standard methods. The results showed statistically significant concentration-dependent genotoxic effects of TiO2 NPs in both respiratory patient and control groups in the Comet assay. The TiO2 NPs caused DNA damage in a concentration dependent manner in both groups (respiratory and healthy controls) with the exception of the lowest TiO2 concentration (10 µg/ml) which did not induce significant damage in healthy controls (n.s). When OTM data were used to compare the whole patient group and the control group, the patient group had more DNA damage (p > 0.001) with the exception of 10 µg/ml of TiO2 that caused less significant damage to patient lymphocytes (p < 0.05). Similarly, there was an increase in the pattern of cytogenetic damage measured in the MN assay without statistical significance except when compared to the negative control of healthy individuals. Furthermore, when modulation of ras p21 expression was investigated, regardless of TiO2 treatment, only lung cancer and COPD patients expressed measurable ras p21 levels. Results were achieved in the absence of cytotoxicity.

Cellular internalization and antioxidant activity of cerium oxide nanoparticles in human monocytic leukemia cells.

Overproduction of free radicals contributes to oxidative stress and inflammation leading to various disease conditions. Cerium oxide nanoparticles (nanoceria) have been shown to scavenge free radicals and have the potential for being used as a therapeutic agent in disease conditions. Therefore, in the present study, human monocytic leukemia cells (THP-1) were used as a model to evaluate the uptake and free radical scavenging activity of nanoceria. Our data showed a significant (P<0.05) increase in the internalization of nanoceria in a concentration-dependent (10-100 µg/mL) manner in THP-1 cells. Although no cytotoxicity was observed at these concentrations, nanoceria significantly (P<0.05) reduced the amount of reactive oxygen species. This was evident by a significant (P<0.05) decrease in the 2,7-dichlorofluorescein diacetate fluorescence observed in flow cytometry and fluorescence microscopy. The present study shows that nanoceria have therapeutic potential in diseases such as cancer.

Synthesis of biocompatible iron oxide nanoparticles as a drug delivery vehicle.

Over the last decade, there has been growing interest in developing novel nanoparticles (NPs) for biomedical applications. A safe-by-design approach was used in this study to synthesize biocompatible iron oxide NPs. The size of the particles obtained was ~100 nm. Although these NPs were significantly (P<0.05) internalized in MCF-7 (human breast adenocarcinoma cell line) cells, no adverse effect was observed in the cells as assessed by cytotoxicity assays (neutral red uptake and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and cell cycle analysis. Our data demonstrate the potential of iron oxide NPs as a biocompatible carrier for targeted drug delivery.

TiO2 nanoparticles induce DNA double strand breaks and cell cycle arrest in human alveolar cells.

TiO2 nanoparticles (NPs) have the second highest global annual production (∼3000 tons) among the metal-containing NPs. These NPs are used as photocatalysts for bacterial disinfection, and in various other consumer products including sunscreen, food packaging, therapeutics, biosensors, surface cleaning agents, and others. Humans are exposed to these NPs during synthesis (laboratory), manufacture (industry), and use (consumer products, devices, medicines, etc.), as well as through environmental exposures (disposal). Hence, there is great concern regarding the health effects caused by exposure to NPs and, in particular, to TiO2 NPs. In the present study, the genotoxic potential of TiO2 NPs in A549 cells was examined, focusing on their potential to induce ROS, different types of DNA damage, and cell cycle arrest. We show that TiO2 NPs can induce DNA damage and a corresponding increase in micronucleus frequency, as evident from the comet and cytokinesis-block micronucleus assays. We demonstrate that DNA damage may be attributed to increased oxidative stress and ROS generation. Furthermore, genomic and proteomic analyses showed increased expression of ATM, P53, and CdC-2 and decreased expression of ATR, H2AX, and Cyclin B1 in A549 cells, suggesting induction of DNA double strand breaks. The occurrence of double strand breaks was correlated with cell cycle arrest in G2/M phase. Overall, the results indicate the potential for genotoxicity following exposure to these TiO2 NPs, suggesting that use should be carefully monitored.

Titanium dioxide nanoparticle-induced oxidative stress triggers DNA damage and hepatic injury in mice.

BACKGROUND: The use of metal oxide nanoparticles (titanium dioxide) in consumer and industrial products improves their quality but also underscores the possible adverse effects to human and environmental health. MATERIALS & METHODS: Mice were exposed orally for 14 consecutive days and analyzed for alteration in different hepatic enzymes, histopathological changes, oxidative stress, DNA damage, tumor suppressor and proapoptotic protein expression in liver cells. RESULTS: We observed a significant alteration in the level of hepatic enzymes and liver histopathology at a dose of 100 mg/kg body weight. Significant oxidative DNA damage was observed in liver cells, which could be attributed to oxidative stress. In addition, the increased expression of p53, BAX, caspase-3 and -9 proteins and decreased expression of antiapoptotic protein Bcl-2, suggest activation of the intrinsic pathway of apoptosis. CONCLUSION: High accumulation of titanium dioxide nanoparticles in the liver tissue would cause DNA damage and apoptosis through the intrinsic pathway.

2.45 GHz microwave irradiation-induced oxidative stress affects implantation or pregnancy in mice, Mus musculus.

The present experiment was designed to study the 2.45 GHz low-level microwave (MW) irradiation-induced stress response and its effect on implantation or pregnancy in female mice. Twelve-week-old mice were exposed to MW radiation (continuous wave for 2 h/day for 45 days, frequency 2.45 GHz, power density=0.033549 mW/cm(2), and specific absorption rate=0.023023 W/kg). At the end of a total of 45 days of exposure, mice were sacrificed, implantation sites were monitored, blood was processed to study stress parameters (hemoglobin, RBC and WBC count, and neutrophil/lymphocyte (N/L) ratio), the brain was processed for comet assay, and plasma was used for nitric oxide (NO), progesterone and estradiol estimation. Reactive oxygen species (ROS) and the activities of ROS-scavenging enzymes- superoxide dismutase, catalase, and glutathione peroxidase-were determined in the liver, kidney and ovary. We observed that implantation sites were affected significantly in MW-irradiated mice as compared to control. Further, in addition to a significant increase in ROS, hemoglobin (p<0.001), RBC and WBC counts (p<0.001), N/L ratio (p<0.01), DNA damage (p<0.001) in brain cells, and plasma estradiol concentration (p<0.05), a significant decrease was observed in NO level (p<0.05) and antioxidant enzyme activities of MW-exposed mice. Our findings led us to conclude that a low level of MW irradiation-induced oxidative stress not only suppresses implantation, but it may also lead to deformity of the embryo in case pregnancy continues. We also suggest that MW radiation-induced oxidative stress by increasing ROS production in the body may lead to DNA strand breakage in the brain cells and implantation failure/resorption or abnormal pregnancy in mice.

TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells.

Titanium dioxide nanoparticles (TiO(2) NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO(2) NPs induce significant (p < 0.05) oxidative DNA damage by the Fpg-Comet assay even at 1 µg/ml concentration. A corresponding increase in the micronucleus frequency was also observed. This could be attributed to the reduced glutathione levels with concomitant increase in lipid peroxidation and reactive oxygen species generation. Furthermore, immunoblot analysis revealed an increased expression of p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 and decreased the level of Bcl-2 thereby indicating that apoptosis induced by TiO(2) NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO(2) NPs induce DNA damage and cause apoptosis in HepG2 cells even at very low concentrations. Hence the use of such nanoparticles should be carefully monitored.

Titanium dioxide nanoparticles induce oxidative stress-mediated apoptosis in human keratinocyte cells.

Titanium dioxide nanoparticles (TiO2 NPs) are the most commonly used metal oxide NPs in various industrial and commercial products. The present study has demonstrated a significant cellular uptake of TiO2 NPs in the human keratinocyte cells (HaCaT) using transmission electron microscopy and flow cytometry. The data exhibited a significant (p < 0.05) concentration dependent decrease in cell viability and glutathione with concomitant increase in lipid peroxidation and reactive oxygen species. The increased oxidative stress further leads to apoptosis after 48 h of exposure. Our study demonstrates oxidative stress mediated apoptosis in human keratinocyte cells exposed to TiO2 NPs.

Stable metal oxide nanoparticle formulation for toxicity studies.

Metal oxide nanoparticles such as TiO2 (< 100 nm) are used in lotions and sunscreens. Toxicity studies so far have reported the use of TiO2 NPs with size > 100 nm as determined by dynamic light scattering (DLS) technique. We used non-reactive chemical agents such as propylene glycol (PG), glycerol (G), ethylene glycol (EG) to prevent aggregation and maintain NPs in monodispersed state closer to the reported TEM size. The lowest concentration of PG (0.1%) along with complete culture media (RPMI 1640) showed mean hydrodynamic diameter of TiO2 NPs as 33.71 nm measured by DLS. The present study demonstrates a method to prepare stable TiO2 NPs in culture media for toxicity assessment. This is a significant contribution as the biological properties of NPs vary considerably with decrease in size.

Toxicity of graphene in normal human lung cells (BEAS-2B).

Graphite nanomaterials such as thermally exfoliated graphite oxide (GO) are versatile in many applications. However, little is known about its effects on biological systems. In this study we characrerized the GO using dynamic light scattering (DLS) along with the toxicological aspects related to cytotoxicity and apoptosis in normal human lung cells (BEAS-2B). A significant concentration and time dependent decrease in cell viability was observed at different concentrations (10-100 microg/ml) by the MTT assay after 24 and 48 h of exposure and significant increase of early and late apoptotic cells was observed as compared to control cells. Our study demonstrates that GO induces cytotoxicity and apoptosis in human lung cells.

ROS-mediated genotoxicity induced by titanium dioxide nanoparticles in human epidermal cells.

Titanium dioxide nanoparticles (TiO(2) NPs) are among the top five NPs used in consumer products, paints and pharmaceutical preparations. Since, exposure to such nanoparticles is mainly through the skin and inhalation, the present study was conducted in the human epidermal cells (A431). A mild cytotoxic response of TiO(2) NPs was observed as evident by the MTT and NR uptake assays after 48 h of exposure. However, a statistically significant (p<0.05) induction in the DNA damage was observed by the Fpg-modified Comet assay in cells exposed to 0.8 µg/ml TiO(2) NPs (2.20±0.26 vs. control 1.24±0.04) and higher concentrations for 6 h. A significant (p<0.05) induction in micronucleus formation was also observed at the above concentration (14.67±1.20 vs. control 9.33±1.00). TiO(2) NPs elicited a significant (p<0.05) reduction in glutathione (15.76%) with a concomitant increase in lipid hydroperoxide (60.51%; p<0.05) and reactive oxygen species (ROS) generation (49.2%; p<0.05) after 6h exposure. Our data demonstrate that TiO(2) NPs have a mild cytotoxic potential. However, they induce ROS and oxidative stress leading to oxidative DNA damage and micronucleus formation, a probable mechanism of genotoxicity. This is perhaps the first study on human skin cells demonstrating the cytotoxic and genotoxic potential of TiO(2) NPs.

DNA damaging potential of zinc oxide nanoparticles in human epidermal cells.

At present, more than 20 countries worldwide are manufacturing and marketing different varieties of nanotech-based consumer products of which cosmetics form the largest category. Due to the extremely small size of the nanoparticles (NPs) being used, there is a concern that they may interact directly with macromolecules such as DNA. The present study was aimed to assess the genotoxicity of zinc oxide (ZnO) NPs, one of the widely used ingredients of cosmetics, and other dermatological preparations in human epidermal cell line (A431). A reduction in cell viability as a function of both NP concentration as well as exposure time was observed. ZnO NPs demonstrated a DNA damaging potential as evident from an increased Olive tail moment (OTM) of 2.13 +/- 0.12 (0.8 g/ml) compared to control 1.37 +/- 0.12 in the Comet assay after an exposure of 6 h. ZnO NPs were also found to induce oxidative stress in cells indicated by depletion of glutathione (59% and 51%); catalase (64% and 55%) and superoxide dismutase (72% and 75%) at 0.8 and 0.08 g/ml respectively. Our data demonstrates that ZnO NPs even at low concentrations possess a genotoxic potential in human epidermal cells which may be mediated through lipid peroxidation and oxidative stress. Hence, caution should be taken in their use in dermatological preparations as well as while handling.