Real-time Biosensor for the Assessment of Nanotoxicity and Cancer Electrotherapy

Knowledge of cell electronics has led to their integration to medicine either by physically interfacing electronic devices with biological systems or by using electronics for both detection and characterization of biological materials. In this dissertation, an electrical impedance sensor (EIS) was used to measure the electrode surface impedance changes from cell samples of human and environmental toxicity of nanoscale materials in 2D and 3D cell culture models. The impedimetric response of human lung fibroblasts and rainbow trout gill epithelial cells when exposed to various nanomaterials was tested to determine their kinetic effects towards the cells and to demonstrate the biosensor’s ability to monitor nanotoxicity in real-time. Further, the EIS allowed rapid, real-time and multi-sample analysis creating a versatile, noninvasive tool that is able to provide quantitative information with respect to alteration in cellular function. We then extended the application of the unique capabilities of the EIS to do real-time analysis of cancer cell response to externally applied alternating electric fields at different intermediate frequencies and low-intensity. Decreases in the growth profiles of the ovarian and breast cancer cells were observed with the application of 200 and 100 kHz, respectively, indicating specific inhibitory effects on dividing cells in culture in contrast to the non-cancerous HUVECs and mammary epithelial cells. We then sought to enhance the effects of the electric field by altering the cancer cell’s electronegative membrane properties with HER2 antibody functionalized nanoparticles. An Annexin V/EthD-III assay and zeta potential were performed to determine the cell death mechanism indicating apoptosis and a decrease in zeta potential with the incorporation of the nanoparticles. With more negatively charged HER2-AuNPs attached to the cancer cell membrane, the decrease in membrane potential would thus leave the cells more vulnerable to the detrimental effects of the applied electric field due to the decrease in surface charge. Therefore, by altering the cell membrane potential, one could possibly control the fate of the cell. This whole cell-based biosensor will enhance our understanding of the responsiveness of cancer cells to electric field therapy and demonstrate potential therapeutic opportunities for electric field therapy in the treatment of cancer.

Effect of some environmental factors on the acute toxicity of deltamethrin to common carp: a laboratory study under aerobic condition

The toxicity of xenobiotic in aquatic ecosystems is influenced by many factors such as ambient temperature, water hardness, pond soil type, etc. In the present study, it was observed that air temperature, water hardness and soil sediment have profound influence on the toxicity of deltamethrin to common carp fry (ay. length 3.5 ± 0.5 cm, ay. weight 0.58 ± 0.25 g); 96h LC(sub)50 values for common carp at 38.07 ± 2.20°C maximum and 27.86 ± 1.22°C minimum air temperature in soft and very hard water were 0.102 and 0.495 µg lˉ¹, respectively. This value had increased significantly to 2.37 and 3.02 µg at 30.55 ± 1.21°C maximum and 26.04 ± 0.61°C minimum air temperature, respectively. When sediment was included, 96h LC(sub)50 at 38.07°C maximum temperature in very hard water was 1.808 µg 1ˉ¹ and this had increased to 8.073 µg 1ˉ¹ when tested at 30.55°C maximum temperature. Due to the 7.5°C increase in maximum and 1.7°C in minimum temperature, toxicity increased significantly. Lower toxicity in very hard water in comparison to soft water may be due to the lower solubility of deltarnethrin and high level of calcium. Adsorption reaction of deltamethrin with clay, humus, FeOOH, MnOOH and particulate organic carbon, and complexation reaction with dissolved organic carbon were responsible for the lowered toxicity in the experiment with sediment. Exposure time had no significant effect on acute toxicity of deltamethrin.

Biosensor based toxicity dissection of tannery and associated environmental samples

Kenyan tannery and associated environmental samples were selected for ecotoxicological assessment. A tool-kit of techniques was developed, including whole-cell biosensor and chemical assays. A luminescence based bacterial biosensor (Escherichia coli HB101 pUCD607) (via a multi-copy plasmid) was used for toxicity assessment. Samples were manipulated prior to biosensor interrogation to identify the nature of the toxic contaminants. Untreated samples (before any manipulations) showed a strong toxic effect at the discharge point in comparison to other sampling points. Sparging was used to identify toxicity associated with volatile organics. The toxicity of contaminants, removed by treatment with activated charcoal was identified for all the sampling points except for those upstream of effluent discharges. Filtration identified toxicity associated with suspended solids. Changes in availability of toxic contaminants due to pH adjustment of most samples from the tannery effluent treatment pits were also associated with the extreme pH values (4.0 and 8.0). The approach used has highlighted the complexicity of toxic pollutants in effluent from the tanning industry and the dissection of toxicity points to possible remediation strategies for effluents from the tanning industry.

Effects of environmental factors on the toxicity of pesticides to zebrafish embryos

During the last century mean global temperatures have been increasing. According to the predictions, the temperature change is expected to exceed 1.5ºC in this century and the warming is likely to continue. Freshwater ecosystems are among the most sensitive mainly due to changes in the hydrologic cycle and consequently changes in several physico-chemical parameters (e.g. pH, dissolved oxygen). Alterations in environmental parameters of freshwater systems are likely to affect distribution, morphology, physiology and richness of a wide range of species leading to important changes in ecosystem biodiversity and function. Moreover, they can also work as co-stressors in environments where organisms have already to cope with chemical contamination (such as pesticides), increasing the environmental risk due to potential interactions. Therefore, the objective of this work was to evaluate the effects of climate change related environmental parameters on the toxicity of pesticides to zebrafish embryos. The following environmental factors were studied: pH (3.0-12.0), dissolved oxygen level (0-8 mg/L) and UV radiation (0-500 mW/m2). The pesticides studied were the carbamate insecticide carbaryl and the benzimidazole fungicide carbendazim. Stressors were firstly tested separately in order to derive concentration- or intensity-response curves to further study the effects of binary combinations (environmental factors x pesticides) by applying mixture models. Characterization of zebrafish embryos response to environmental stress revealed that pH effects were fully established after 24 h of exposure and survival was only affected at pH values below 5 and above 10. Low oxygen levels also affected embryos development at concentrations below 4 mg/L (delay, heart rate decrease and edema), and at concentrations below 0.5 mg/L the survival was drastically reduced. Continuous exposure to UV radiation showed a strong time-dependent impact on embryos survival leading to 100% of mortality after 72 hours of exposure. The toxicity of pesticides carbaryl and carbendazim was characterized at several levels of biological organization including developmental, biochemical and behavioural allowing a mechanistic understanding of the effects and highlighting the usefulness of behavioural responses (locomotion) as a sensitive endpoint in ecotoxicology. Once the individual concentration response relationship of each stressor was established, a combined toxicity study was conducted to evaluate the effects of pH on the toxicity of carbaryl. We have shown that pH can modify the toxicity of the pesticide carbaryl. The conceptual model concentration addition allowed a precise prediction of the toxicity of the jointeffects of acid pH and carbaryl. Nevertheless, for alkaline condition both concepts failed in predicting the effects. Deviations to the model were however easy to explain as high pH values favour the hydrolysis of carbaryl with the consequent formation of the more toxic degradation product 1- naphtol. Although in the present study such explanatory process was easy to establish, for many other combinations the “interactive” nature is not so evident. In the context of the climate change few scenarios predict such increase in the pH of aquatic systems, however this was a first approach focused in the lethal effects only. In a second tier assessment effects at sublethal level would be sought and it is expectable that more subtle pH changes (more realistic in terms of climate changes scenarios) may have an effect at physiological and biochemical levels with possible long term consequences for the population fitness.

The gut microbiota: a major player in the toxicity of environmental pollutants?

Exposure to environmental chemicals has been linked to various health disorders, including obesity, type 2 diabetes, cancer and dysregulation of the immune and reproductive systems, whereas the gastrointestinal microbiota critically contributes to a variety of host metabolic and immune functions. We aimed to evaluate the bidirectional relationship between gut bacteria and environmental pollutants and to assess the toxicological relevance of the bacteria–xenobiotic interplay for the host. We examined studies using isolated bacteria, faecal or caecal suspensions—germ-free or antibiotic-treated animals—as well as animals reassociated with a microbiota exposed to environmental chemicals. The literature indicates that gut microbes have an extensive capacity to metabolise environmental chemicals that can be classified in five core enzymatic families (azoreductases, nitroreductases, β-glucuronidases, sulfatases and β-lyases) unequivocally involved in the metabolism of >30 environmental contaminants. There is clear evidence that bacteria-dependent metabolism of pollutants modulates the toxicity for the host. Conversely, environmental contaminants from various chemical families have been shown to alter the composition and/or the metabolic activity of the gastrointestinal bacteria, which may be an important factor contributing to shape an individual’s microbiotype. The physiological consequences of these alterations have not been studied in details but pollutant-induced alterations of the gut bacteria are likely to contribute to their toxicity. In conclusion, there is a body of evidence suggesting that gut microbiota are a major, yet underestimated element that must be considered to fully evaluate the toxicity of environmental contaminants.

ACUTE TOXICITY and ENVIRONMENTAL RISK of OXYTETRACYLINE and FLORFENICOL ANTIBIOTICS TO PACU (Piaractus mesopotamicus)

The oxytetracyline (OTC) and florfenicol (FLO) are the most used antibiotics for bacterial disease control in fish. The aims are: estimating the lethal concentration 50% initial (LC(I) 50;48h) of OTC (Terramicina (R)) and FLO (Aquaflor (R)) for pacu (Piaractus mesopotamicus); classifying OTC and FLO according to the acute toxicity and the risk of environmental intoxication; and assessing OTC and FLO dilution effect in water quality variables. Fish were acclimatized for 10 days in a bioassay room at 27 +/- 2 degrees C. Then nine animals (three each concentration) were exposed to 7.0; 7.5; 8.0 or 8.5 mg L(-1) of OTC or to 600.0; 700.0; 800.0; 900.0 and 1000.0 mg L(-1) of FLO and a control treatment. The water quality variables recorded daily were: pH, temperature, electric conductivity, and dissolved oxygen. OTC and FLO were classified according to acute toxicity and environmental risk. The estimated OTC LC(I) 50;48h for pacu is 7.6 mg L(-1) and the estimated FLO is > 1000 mg L(-1). OTC reduced the dissolved oxygen concentration, and it was considered moderately toxic, causing risk of environmental intoxication to pacu. FLO is not toxic; it does not cause risk of environmental intoxication to pacu and does not change water quality variables.

SUPEROXIDE RADICAL AND TOXICITY OF ENVIRONMENTAL NICKEL EXPOSURE

Three nickel compounds were tested for pancreatic, hepatic and osteogenic damage in rats by a single i.m. injection Ni++ (7 mg kg(-1)). The nickel induced biochemical alterations included significantly increased levels of serum alkaline phosphatase in rats with NiS (75%) and NiO (50%). Amylase and aspartate transaminase were also increased, and lipoperoxide was increased in rats with NiO (5.6-fold) and NiS (3.4-fold). No serum changes were observed with NiCl2. Daily injection of Cu-Zn superoxide dismutase (SOD) conjugated with polyethylene glycol prevented the serum level changes, indicating that superoxide radical is an important intermediate in toxicity of nickel insoluble compounds.

Organotins: A review of their reproductive toxicity, biochemistry, and environmental fate

The review purposes are to (1) evaluate the experimental evidence for adverse effects on reproduction and metabolism and (2) identify the current knowledge of analytical procedures, biochemistry and environmental aspects relating to organotins. Organotins are pollutants that are used as biocides in antifouling paints. They produce endocrine-disrupting effects in mollusks, such as imposex. In rodents, organotin exposure induces developmental and reproductive toxicity as well as alteration of metabolic homeostasis through its action as an obesogen. The adverse effects that appear in rodents have raised concerns about organotins' potential health risk to humans in relation to organotin exposure. At present, triorganotin, such as tributyltin, have been demonstrated to produce imposex, and mammalian reproductive and metabolic toxicity. For most mammals, triorganotin exposure predominantly occurs through the ingestion, and this compound can cross the placenta. With these risks in mind, it is important to improve our knowledge of organotins' effects on environmental health. © 2012 Elsevier Inc.

Determination of estrogenic mycotoxins in environmental water samples by low-toxicity dispersive liquid-liquid microextraction and liquid chromatography-tandem mass spectrometry

A novel, simple, rapid and eco-friendly method based on dispersive liquid-liquid microextraction using a bromosolvent was developed to determine six estrogenic mycotoxins (zearalenone, zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol) in water samples by liquid chromatography-electrospray ionization tandem mass spectrometry in the negative mode (LC-ESI-MS/MS). The optimal conditions for this method include the use of 100 mu L bromocyclohexane as an extraction solvent (using a non-dispersion solvent), 10 mL of aqueous sample (adjusted to pH 4), a vortex extraction time of 2 min, centrifugation for 10 min at 3500 rpm and no ionic strength adjustment. The calibration function was linear and was verified by applying the Mandel fitting test with a 95% confidence level. No matrix effect was observed. According to the relative standard deviations (RSDs), the precision was better than 13% for the repeatability and intermediate precision. The average recoveries of the spiked compounds ranged from 81 to 118%. The method limits of detection (LOD) and quantification (LOQ) considering a 125-fold pre-concentration step were 4-20 and 8-40 ng L-1, respectively. Next, the method was applied to the analysis of the environmental aqueous samples, demonstrating the presence of beta-zearalanol and zearalanone in the river water samples. (C) 2015 Elsevier B.V. All rights reserved.

Particle emissions, volatility, and toxicity from an ethanol fumigated compression ignition engine

Particle emissions, volatility, and the concentration of reactive oxygen species (ROS) were investigated for a pre-Euro I compression ignition engine to study the potential health impacts of employing ethanol fumigation technology. Engine testing was performed in two separate experimental campaigns with most testing performed at intermediate speed with four different load settings and various ethanol substitutions. A scanning mobility particle sizer (SMPS) was used to determine particle size distributions, a volatilization tandem differential mobility analyzer (V-TDMA) was used to explore particle volatility, and a new profluorescent nitroxide probe, BPEAnit, was used to investigate the potential toxicity of particles. The greatest particulate mass reduction was achieved with ethanol fumigation at full load, which contributed to the formation of a nucleation mode. Ethanol fumigation increased the volatility of particles by coating the particles with organic material or by making extra organic material available as an external mixture. In addition, the particle-related ROS concentrations increased with ethanol fumigation and were associated with the formation of a nucleation mode. The smaller particles, the increased volatility, and the increase in potential particle toxicity with ethanol fumigation may provide a substantial barrier for the uptake of fumigation technology using ethanol as a supplementary fuel.

Environmental health risk assessment of dioxin exposure through foods in a Dioxin hot spot—Bien Hoa City, Vietnam

This study used the Australian Environmental Health Risk Assessment Framework to assess the human health risk of dioxin exposure through foods for local residents in two wards of Bien Hoa City, Vietnam. These wards are known hot-spots for dioxin and a range of stakeholders from central government to local levels were involved in this process. Publications on dioxin characteristics and toxicity were reviewed and dioxin concentrations in local soil, mud, foods, milk and blood samples were used as data for this risk assessment. A food frequency survey of 400 randomly selected households in these wards was conducted to provide data for exposure assessment. Results showed that local residents who had consumed locally cultivated foods, especially fresh water fish and bottom-feeding fish, free-ranging chicken, duck, and beef were at a very high risk, with their daily dioxin intake far exceeding the tolerable daily intake recommended by the WHO. Based on the results of this assessment, a multifaceted risk management program was developed and has been recognized as the first public health program ever to have been implemented in Vietnam to reduce the risks of dioxin exposure at dioxin hot-spots.

Elemental composition and potential toxicity of airborne particles at some urban schools

Although both the size and chemical composition of ambient particles are important parameters in determining their toxicities, their relative contributions are unclear (Heal et al., 2012). Children are particularly at risk to the detrimental health effects that have been linked to long term exposure to airborne particles (See e.g. Ruckerl et al., 2011). However, there is currently limited understanding of the health effects in children due to long term exposure to airborne particles. Schools are locations within an urban environment where children experience significant exposure to vehicle emissions, and to date there is limited information assessing children’s exposure at school. This study is a part of a large project aimed at gaining a holistic picture of the exposure of children to traffic related pollutants. In the current paper, results from the investigation of the elemental composition of airborne particle at urban schools are presented.