Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities

Health Status of Sand Flathead (Platycephalus bassensis), Inhabiting an Industrialised and Urbanised Embayment, Port Phillip Bay, Victoria as Measured by Biomarkers of Exposure and Effects

Port Phillip Bay, Australia, is a large semi-closed bay with over four million people living in its catchment basin. The Bay receives waters from the Yarra River which drains the city of Melbourne, as well as receiving the discharges of sewage treatment plants and petrochemical and agricultural chemicals. A 1999 study demonstrated that fish inhabiting Port Phillip Bay showed signs of effects related to pollutant exposure despite pollution management practices having been implemented for over a decade. To assess the current health status of the fish inhabiting the Bay, a follow up survey was conducted in 2015. A suite of biomarkers of exposure and effects were measured to determine the health status of Port Phillip Bay sand flathead (Platycephalus bassensis), namely ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbons (PAH) biliary metabolites, carboxylesterase activity (CbE) and DNA damage (8-oxo-dG). The reduction in EROD activity in the present study suggests a decline in the presence of EROD activity-inducing chemicals within the Bay since the 1990s. Fish collected in the most industrialised/urbanised sites did not display higher PAH metabolite levels than those in less developed areas of the Bay. Ratios of PAH biliary metabolite types were used to indicate PAH contaminant origin. Ratios indicated fish collected at Corio Bay and Hobsons Bay were subjected to increased low molecular weight hydrocarbons of petrogenic origin, likely attributed to the close proximity of these sites to oil refineries, compared to PAH biliary metabolites in fish from Geelong Arm and Mordialloc.Quantification of DNA damage indicated a localised effect of exposure to pollutants, with a 10-fold higher DNA damage level in fish sampled from the industrial site of Corio Bay relative to the less developed site of Sorrento. Overall, integration of biomarkers by multivariate analysis indicated that the health of fish collected in industrialised areas was compromised, with biologically significant biomarkers of effects (LSI, CF and DNA damage) discriminating between individuals collected in industrialised areas from observations made in fish collected in less developed areas of the Bay

Freshwater shrimp (Palaemonetes australis) as a potential bioindicator of crustacean health

Palaemonetes australis is a euryhaline shrimp found in south-western Australian estuaries. To determine if P. australis is a suitable bioindicator species for monitoring the health of estuarine biota, they were exposed to measured concentrations of the polycyclic aromatic hydrocarbon, benzo[a]pyrene (B[a]P) at 0.01, 0.1 or 1 ppm for 14 days under laboratory conditions. At the end of exposure the shrimp were sacrificed for biomarker [ethoxycoumarin O-deethylase (ECOD), 8-oxo-dG concentration, and sorbitol dehydrogenase (SDH) activity] analyses. Gender did not appear to influence biomarker responses of the shrimp in this study. ECOD activity was induced in the treatment groups in a linear fashion from 3 (0.01 ppm) times to 12 (1 ppm) times the negative controls. 8-oxo-dG concentration was reduced 3 times in treatment groups below the controls suggesting impaired DNA repair pathways. There was no increase in SDH, signifying hepatopancreatic cell damage had not occurred in any treatment group. The response of P australis to B[a]P exposure indicates that this crustacean is suitable bioindicator species for both laboratory studies and field monitoring. A combination of ECOD and SDH activities and 8-oxo-dG concentration represent a suitable suite of biomarkers for environmental monitoring of the sublethal effects of organic pollution to crustaceans from an estuarine environment

Integrating Multiple Biomarkers of Fish Health: A Case Study of Fish Health in Ports

Biomarkers of fish health are recognised as valuable biomonitoring tools that inform on the impact of pollution on biota. The integration of a suite of biomarkers in a statistical analysis that better illustrates the effects of exposure to xenobiotics on living organisms is most informative; however, most published ecotoxicological studies base the interpretation of results on individual biomarkers rather than on the information they carry as a set. To compare the interpretation of results from individual biomarkers with an interpretation based on multivariate analysis, a case study was selected where fish health was examined in two species of fish captured in two ports located in Western Australia. The suite of variables selected included chemical analysis of white muscle, body condition index, liver somatic index (LSI), hepatic ethoxyresorufin-O-deethylase activity, serum sorbitol dehydrogenase activity, biliary polycyclic aromatic hydrocarbon metabolites, oxidative DNA damage as measured by serum 8-oxo-dG, and stress protein HSP70 measured on gill tissue. Statistical analysis of individual biomarkers suggested little consistent evidence of the effects of contaminants on fish health. However, when biomarkers were integrated as a set by principal component analysis, there was evidence that the health status of fish in Fremantle port was compromised mainly due to increased LSI and greater oxidative DNA damage in fish captured within the port area relative to fish captured at a remote site. The conclusions achieved using the integrated set of biomarkers show the importance of viewing biomarkers of fish health as a set of variables rather than as isolated biomarkers of fish health

The HIV-1 Transactivator Factor (Tat) Induces Enterocyte Apoptosis through a Redox-Mediated Mechanism

The intestinal mucosa is an important target of human immunodeficiency virus (HIV) infection. HIV virus induces CD4+ T cell loss and epithelial damage which results in increased intestinal permeability. The mechanisms involved in nutrient malabsorption and alterations of intestinal mucosal architecture are unknown. We previously demonstrated that HIV-1 transactivator factor (Tat) induces an enterotoxic effect on intestinal epithelial cells that could be responsible for HIV-associated diarrhea. Since oxidative stress is implicated in the pathogenesis and morbidity of HIV infection, we evaluated whether Tat induces apoptosis of human enterocytes through oxidative stress, and whether the antioxidant N-acetylcysteine (NAC) could prevent it. Caco-2 and HT29 cells or human intestinal mucosa specimens were exposed to Tat alone or combined with NAC. In an in-vitro cell model, Tat increased the generation of reactive oxygen species and decreased antioxidant defenses as judged by a reduction in catalase activity and a reduced (GSH)/oxidized (GSSG) glutathione ratio. Tat also induced cytochrome c release from mitochondria to cytosol, and caspase-3 activation. Rectal dialysis samples from HIV-infected patients were positive for the oxidative stress marker 8-hydroxy-2′-deoxyguanosine. GSH/GSSG imbalance and apoptosis occurred in jejunal specimens from HIV-positive patients at baseline and from HIV-negative specimens exposed to Tat. Experiments with neutralizing anti-Tat antibodies showed that these effects were direct and specific. Pre-treatment with NAC prevented Tat-induced apoptosis and restored the glutathione balance in both the in-vitro and the ex-vivo model. These findings indicate that oxidative stress is one of the mechanism involved in HIV-intestinal disease

Association of Polymorphisms in Oxidative Stress Genes with Clinical Outcomes for Bladder Cancer Treated with Bacillus Calmette-Guérin

Genetic polymorphisms in oxidative stress pathway genes may contribute to carcinogenesis, disease recurrence, treatment response, and clinical outcomes. We applied a pathway-based approach to determine the effects of multiple single nucleotide polymorphisms (SNPs) within this pathway on clinical outcomes in non-muscle-invasive bladder cancer (NMIBC) patients treated with Bacillus Calmette-Guérin (BCG). We genotyped 276 SNPs in 38 genes and evaluated their associations with clinical outcomes in 421 NMIBC patients. Twenty-eight SNPs were associated with recurrence in the BCG-treated group (P<0.05). Six SNPs, including five in NEIL2 gene from the overall and BCG group remained significantly associated with recurrence after multiple comparison adjustments (q<0.1). Cumulative unfavorable genotype analysis showed that the risk of recurrence increased with increasing number of unfavorable genotypes. In the analysis of risk factors associated with progression to disease, rs3890995 in UNG, remained significant after adjustment for multiple comparison (q<0.1). These results support the hypothesis that genetic variations in host oxidative stress genes in NMIBC patients may affect response to therapy with BCG

Traffic-Related Air Pollution and DNA Damage: A Longitudinal Study in Taiwanese Traffic Conductors

BACKGROUND: There is accumulating epidemiologic evidence that exposure to traffic-related air pollutants, including particulate matter (PM) and polyaromatic hydro carbons (PAHs), plays a role in etiology and prognosis of a large scale of illnesses, although the role of specific causal agents and underlying mechanisms for different health outcomes remains unknown. OBJECTIVE: Our general objective was to assess the relations between personal exposure to traffic exhausts, in particular ambient PM(2.5) and PAHs, and the occurrence of DNA strand breaks by applying personal monitoring of PM and biomarkers of exposure (urinary 1-hydroxypyrene-glucuronide, 1-OHPG) and effect (urinary 8-hydroxydeoxyguanosine, 8-OHdG and DNA strand breaks). METHODS: We recruited 91 traffic conductors and 53 indoor office workers between May 2009 and June 2011 in Taipei City, Taiwan. We used PM(2.5) personal samplers to collect breathing-zone particulate PAHs samples. Spot urine and blood samples after work shift of 2 consecutive days were analyzed for 1-OHPG, 8-OHdG and DNA strand breaks, respectively. Statistical methods included linear regression and mixed models. RESULTS: Urinary 8-OHdG levels and the occurrence of DNA strand breaks in traffic conductors significantly exceeded those in indoor office workers in mixed models. Particulate PAHs levels showed a positive association with urinary 1-OHPG in the regression model (β = 0.056, p = 0.01). Urinary 1-OHPG levels were significantly associated with urinary 8-OHdG levels in the mixed model (β = 0.101, p = 0.023). Our results provide evidence that exposure to fine particulates causes DNA damage. Further, particulate PAHs could be biologically active constituents of PM(2.5) with reference to the induction of oxidative DNA damages