In Vitro Human Liver Model for Toxicity Assessment with Clinical and Preclinical Instrumentation.

The existing in vitro toxicological models lack translational potential, which makes difficult the application of gathered information to clinical usage. To tackle this issue, we built a model with four different types of primary liver cells: hepatic sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells and hepatocytes. We cultured them in different combinations of composition and volumes of cell medium, hepatocyte proportions of total cells and additions of extracellular matrixes. We added rifampicin (RIF), ibuprofen (IBU) and 5-fluorouracil (5-FU) to this model and observed the microanatomy and physiology changes for a week with preclinical and clinical instruments. Among the different model configurations, we selected the feature combination of the in vitro model that had similar biomarker values to those measured in clinical diagnostics. When we exposed the selected model configuration to RIF, IBU and 5-FU, we observed similar glucose, triglyceride and albumin dynamics as in vivo (from clinical data). Therefore, we have built an in vitro liver model that resembles the liver microenvironment, and we have analysed it with clinical instrumentation to facilitate data translation. Furthermore, during these observations, we found that Kupffer and LSEC cells are suitable candidates for the search for clinical diagnostic markers of liver function.

In Vitro Assessment of Ozone-Treated Deoxynivalenol by Measuring Cytotoxicity and Wheat Quality.

Deoxynivalenol (DON), a trichothecene mycotoxin, could lead to cytotoxicity in both animal bodies and plant seed cells. Ozone degradation technology has been applied to DON control. However, the safety and quality of the contaminated grain after DON degradation are largely obscured. In this work, we evaluated the cytotoxicity of ozone-treated DON through seed germination experiments and cytotoxicity tests. Cell experiments showed that the inhibition rate of HepG2 viability gradually increased within the concentrations of 1-10 mg/L of DON, alongside which an IC50 (half maximal inhibitory concentration) of 9.1 mg/L was determined. In contrast, degrading DON had no significant inhibitory effect on cell growth. Moreover, a 1-10 mg/L concentration of DON increased production of a large amount of reactive oxygen radicals in HepG2, with obvious fluorescence color development. However, fluorescence intensity decreased after DON degradation. Further, DON at a concentration of >1 mg/L significantly inhibited the germination of mung bean seeds, whereas no significant inhibition of their germination or growth were observed if DON degraded. Changes in total protein content, fatty acid value, and starch content were insignificant in wheat samples suffering ozone degradation, compared to the untreated group. Lastly, the ozone-treated wheat samples exhibited higher tenacity and whiteness. Together, our study indicated that the toxicity of DON-contaminated wheat was significantly reduced after ozone degradation.

Hybrid radical coupling during MnO2-mediated transformation of a mixture of organic UV filters: Chemistry and toxicity assessment.

Manganese oxide (MnO2) is one of the most abundant metal oxides, and it is renowned for its ability to degrade various phenolic micropollutants. However, under MnO2-mediated transformation, BP-3 transforms into 12 different radical-coupled transformation products (TPs) out of 15 identified TPs. These radical-coupled TPs are reported with adverse environmental impacts. This study explored the effects of MnO2 on organic UV filter mixtures and different water constituents (i.e., bicarbonate ion (HCO3-), humic acid (HA) and halide ions) in terms of degradation efficiency and transformation chemistry. When a mixture of benzophenone-3 (BP-3) and avobenzone (AVO) underwent transformation by MnO2, hybrid radical-coupled TPs derived from both organic UV filters were generated. These hybrid radical-coupled TPs were evaluated by an in silico prediction tool and Vibrio fischeri bioluminescence inhibition assay (VFBIA). Results showed that these TPs were potentially toxic to aquatic organisms, even more so than their parent compounds. The higher the concentration of HCO3-, HA, chloride ion (Cl-) and bromide ion (Br-), the greater the reduction in the efficiencies of degrading BP-3 and AVO. Contrastingly, in the presence of iodide ion (I-), degradation efficiencies of BP-3 and AVO were enhanced; however, iodinated TPs and iodinated radical-coupled TPs were formed, with questionable toxicity. This study has revealed the environmental risks of hybrid radical-coupled TPs, iodinated TPs and iodinated radical-coupled TPs when the organic UV filters BP-3 and AVO are transformed by MnO2.

SciRAPnano: a pragmatic and harmonized approach for quality evaluation of in vitro toxicity data to support risk assessment of nanomaterials.

Large amounts of nanotoxicity data from alternative non-animal (in vitro) test methods have been generated, but there is a lack of harmonized quality evaluation approaches for these types of data. Tools for scientifically sound and structured evaluation of the reliability and relevance of in vitro toxicity data to effectively inform regulatory hazard assessment of nanomaterials (NMs), are needed. Here, we present the development of a pragmatic approach to facilitate such evaluation. The tool was developed based on the Science in Risk Assessment and Policy (SciRAP) tool currently applicable to quality evaluation of chemical toxicity studies. The approach taken to develop the tool, referred to as SciRAPnano, included refinement of the original SciRAP in vitro tool through implementation of identified NM-relevant criteria, and further refined based on a set of case studies involving evaluation of 11 studies investigating in vitro toxicity of nano-sized titanium dioxide. Parameters considered cover key physicochemical properties as well as assay-specific aspects that impact NM toxicity, including NM interference with test methods and NM transformation. The final SciRAPnano tool contains 38 criteria for reporting quality, 19 criteria for methodological quality, and 4 guidance items to evaluate relevance. The approach covers essential parameters for pragmatic and harmonized evaluation of NM in vitro toxicity studies and allows for structured use of in vitro data in regulatory hazard assessment of NMs, including transparency on data quality.

Physicochemical surrogates for in vitro toxicity assessment of liposomal amphotericin B.

Pharmaceutical toxicity evaluations often use in vitro systems involving primary cells, cell lines or red blood cells (RBCs). Cell-based analyses ('bioassays') can be cumbersome and typically rely on hard-to-standardize biological materials. Amphotericin B (AmB) toxicity evaluations are primarily based on potassium release from RBCs and share these limitations. This study evaluates the potential substitution of two physicochemical AmB toxicity approaches for the bioassay: Ultraviolet-visible spectroscopy (UV-vis) and in vitro drug release kinetics. UV-vis spectral analyses indicated that liposomal AmB's (L-AmB) main peak position (λmax) and peak ratio (OD346/OD322) are potential toxicity surrogates. Similarly, two first-order release parameters derived from USP-4 in vitro drug release analyses also provided linear relationships with toxicity. These were the initial, overall drug release rate and the ratio of loose to tight AmB pools. Positive slopes and high correlation coefficients (R2 > 0.9) characterized all interrelations between physicochemical parameters and toxicity. These tests converted the manufacturing variables' nonlinear (i.e., curvilinear) relationships with in vitro toxicity to linear responses. Three different toxicity attenuation approaches (2 manufacturing, 1 formulation), covering formulation composition and process aspects, support this approach's universality. These data suggest that one or more spectral and kinetic physicochemical tests can be surrogates for L-AmB in vitro toxicity testing.

A High-Throughput Toxicity Screen of 42 Per- and Polyfluoroalkyl Substances (PFAS) and Functional Assessment of Migration and Gene Expression in Human Placental Trophoblast Cells.

Per- and polyfluoroalkyl substances (PFAS) have become ubiquitous environmental contaminants that have been associated with adverse pregnancy outcomes in women and experimental research models. Adverse developmental and reproductive outcomes have been investigated for relatively few PFAS, and such studies are not scalable to address the thousands of unique chemical structures. As the placenta has been reported as a PFAS target tissue, the human placental trophoblast JEG-3 cell line was employed in a high-throughput toxicity screen (HTTS) to evaluate the effects of 42 unique PFAS on viability, proliferation, and mitochondrial membrane potential (MMP). HTTS concentration-response curve fitting determined EC50 values for 79% of tested compounds for at least one of the three endpoints. Trophoblast migratory potential was evaluated for a subset of six prioritized PFAS using a scratch wound assay. Migration, measured as the percent of wound closure after 72 h, was most severely inhibited by exposure to 100 µM perfluorooctanoic acid (PFOA; 72% closure), perfluorooctanesulfonic acid (PFOS; 57% closure), or ammonium perfluoro-2-methyl-3-oxahexanoate (GenX; 79% closure). PFOA and GenX were subsequently evaluated for disrupted expression of 46 genes reported to be vital to trophoblast health. Disrupted regulation of oxidative stress was suggested by altered expression of GPEX1 (300 µM GenX and 3 µM GenX), GPER1 (300 µM GenX), and SOD1 and altered cellular response to xenobiotic stress was indicated by upregulation of the placental efflux transporter, ABCG2 (300 µM GenX, 3 µM GenX, and 100 µM PFOA). These findings suggest the placenta is potentially a direct target of PFAS exposure and indicate that trophoblast cell gene expression and function are disrupted at PFAS levels well below the calculated cytotoxicity threshold (EC50). Future work is needed to determine the mechanism(s) of action of PFAS towards placental trophoblasts.

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study.

Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.

In Vitro Assessment Reveals the Effects of Environmentally Persistent Free Radicals on the Toxicity of Photoaged Tire Wear Particles.

Tire wear particles (TWP) have been identified as one of the major sources of microplastics (MPs), and few studies have focused on their environmental behaviors and impacts. However, a thorough characteristic and toxicity assessment associated with environmentally persistent free radicals (EPFRs) on the photoaged TWP is missing. In this study, we investigated EPFRs in the process of TWP photoaging and evaluated their toxicity using in vitro bioassays. Our results showed that a total of around 1.0 × 1017 spins/g EPFRs (g-factors ranging 2.00308-2.00318) was formed on TWP with 60 days of light irradiation, which contained more than 29% of reactive EPFRs (r-EPFRs). Using macrophages as model cells for bioassays, TWP-associated EPFRs trigged endpoints, including the decrease of cell viability (27 to 45%) and the increase of oxidative stress response (46-93%) and inflammatory factor secretion. The enhancement of TWP toxicity with photoaging was confirmed to be attributed to the generated EPFRs combined with other TWP's chemical compositions (e.g., various metals and organics). Most importantly, the toxicity of photoaged TWP was closely correlated with the generated r-EPFRs, which induced reactive oxidant species (ROS) generation. This study provides direct evidence of toxicity on the photoaged TWP particles, revealing the potential contributions of EPFRs to the adverse effect on human health and highlighting the need for an improved understanding of the impacts of EPFRs on the risk assessment of TWP released into the environment.

Combining short-term bioassays using fish and crustacean model organisms with ToxCast in vitro data and broad-spectrum chemical analysis for environmental risk assessment of the river water (Sava, Croatia).

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

The in vitro assessment of the toxicity of volatile, oxidisable, redox-cycling compounds: phenols as an example.

Phenols are regarded as highly toxic chemicals. Their effects are difficult to study in in vitro systems because of their ambiguous fate (degradation, auto-oxidation and volatility). In the course of in vitro studies of a series of redox-cycling phenols, we found evidences of cross-contamination in several in vitro high-throughput test systems, in particular by trimethylbenzene-1, 4-diol/trimethylhydroquinone (TMHQ) and 2,6-di-tertbutyl-4-ethylphenol (DTBEP), and investigated in detail the physicochemical basis for such phenomenon and how to prevent it. TMHQ has fast degradation kinetics followed by significant diffusion rates of the resulting quinone to adjacent wells, other degradation products being able to air-diffuse as well. DTBEP showed lower degradation kinetics, but a higher diffusion rate. In both cases the in vitro toxicity was underestimated because of a decrease in concentration, in addition to cross-contamination to neighbouring wells. We identified four degradation products for TMHQ and five for DTBEP indicating that the current effects measured on cells are not only attributable to the parent phenolic compound. To overcome these drawbacks, we investigated in detail the physicochemical changes occurring in the course of the incubation and made use of gas-permeable and non-permeable plastic seals to prevent it. Diffusion was greatly prevented by the use of both plastic seals, as revealed by GC-MS analysis. Gas non-permeable plastic seals, reduced to a minimum compounds diffusion as well oxidation and did not affect the biological performance of cultured cells. Hence, no toxicological cross-contamination was observed in neighbouring wells, thus allowing a more reliable in vitro assessment of phenol-induced toxicity.

Non-Animal Strategies for Toxicity Assessment of Nanoscale Materials: Role of Adverse Outcome Pathways in the Selection of Endpoints.

Faster, cheaper, sensitive, and mechanisms-based animal alternatives are needed to address the safety assessment needs of the growing number of nanomaterials (NM) and their sophisticated property variants. Specifically, strategies that help identify and prioritize alternative schemes involving individual test models, toxicity endpoints, and assays for the assessment of adverse outcomes, as well as strategies that enable validation and refinement of these schemes for the regulatory acceptance are needed. In this review, two strategies 1) the current nanotoxicology literature review and 2) the adverse outcome pathways (AOPs) framework, a systematic process that allows the assembly of available mechanistic information concerning a toxicological response in a simple modular format, are presented. The review highlights 1) the most frequently assessed and reported ad hoc in vivo and in vitro toxicity measurements in the literature, 2) various AOPs of relevance to inhalation toxicity of NM that are presently under development, and 3) their applicability in identifying key events of toxicity for targeted in vitro assay development. Finally, using an existing AOP for lung fibrosis, the specific combinations of cell types, exposure and test systems, and assays that are experimentally supported and thus, can be used for assessing NM-induced lung fibrosis, are proposed.

Toxicity assessment of metal oxide nanomaterials using in vitro screening and murine acute inhalation studies.

Characterizations and in vitro toxicity screening were performed on metal oxide engineered nanomaterials (ENMs) independently comprising ZnO, CuO, CeO2, Fe2O3, WO3, V2O5, TiO2, Al2O3 and MgO. Nanomaterials that exhibited the highest toxicity responses in the in vitro screening assays (ZnO, CuO, and V2O5) and the lesser explored material WO3 were tested for acute pulmonary toxicity in vivo. Female and male mice (C57Bl/6J) were exposed to aerosolized metal oxide ENMs in a nose-only exposure system and toxicity outcomes (biomarkers of cytotoxicity, immunotoxicity, inflammation, and lung histopathology) at 4 and 24 h after the start of exposure were assessed. The studies were performed as part of the NIEHS Nanomaterials Health Implications Research consortium with the purpose of investigating the effects of ENMs on various biological systems. ENMs were supplied by the Engineered Nanomaterials Resource and Coordination Core. Among the ENMs studied, the highest toxicity was observed for CuO and ZnO NPs in both in vitro and in vivo acute models. Compared to sham-exposed controls, there was a significant increase in bronchoalveolar lavage neutrophils and proinflammatory cytokines and a loss of macrophage viability at both 4 h and 24 h for ZnO and CuO but not seen for V2O5 or WO3. These effects were observed in both female and male mice. The cell viability performed after in vitro exposure to ENMs and assessment of lung inflammation after acute inhalation exposure in vivo were shown to be sensitive endpoints to predict ENM acute toxicity.

A comparative in vitro toxicity assessment of electronic vaping product e-liquids and aerosols with tobacco cigarette smoke.

The use of electronic vaping products (EVPs) continues to increase worldwide among adult smokers in parallel with accumulating information on their potential toxicity and relative safety compared to tobacco smoke. At this time, in vitro assessments of many widely available EVPs are limited. In this study, an in vitro battery of established assays was used to examine the cytotoxic (Neutral red uptake), genotoxic (In vitro micronucleus) and mutagenic (Bacterial reverse mutation) responses of two commercial EVPs (blu GO™ disposable and blu PLUS+™ rechargeable) when compared to smoke from a reference cigarette (3R4F). In total, 12 commercial products were tested as e-liquids and as aerosols. In addition, two experimental base liquids containing 1.2% and 2.4% nicotine were also assessed to determine the effect of flavour and nicotine on all three assays. In the bacterial reverse mutation (Ames) and in vitro micronucleus (IVM) assays, exposures to e-liquids and EVP aerosols, with and without nicotine and in a range of flavourings, showed no mutagenic or genotoxic effects compared to tobacco smoke. The neutral red uptake (NRU) assay showed significantly reduced cytotoxicity (P < .05) for whole undiluted EVP aerosols compared to tobacco smoke, which by contrast was markedly cytotoxic even when diluted. The reduced in vitro toxicological responses of the EVPs add to the increasing body of scientific weight-of-evidence supporting the role of high-quality EVPs as a harm reduction tool for adult smokers.

Relative potency factor approach enables the use of in vitro information for estimation of human effect factors for nanoparticle toxicity in life-cycle impact assessment.

The major theme of the NRC report "Toxicity Testing in the Twenty-first Century" is to replace animal testing by using alternative in vitro methods. Therefore, it can be expected that in the future in vivo data will be replaced with in vitro data. Hence, there is a need for new strategies to make use of the increasing amount of in vitro data when developing human toxicological effect factors (HEF) to characterize the impact category of human toxicity in life cycle assessment (LCA). Here, we present a new approach for deriving HEF for manufactured nanomaterials (MNMs) based on the combined use of in vitro toxicity data and a relative potency factor (RPF) approach. In vitro toxicity tests with nano-CuO, nano-Ag and nano-ZnO and their corresponding ions were performed on THP-1, CaCo-2 and Hep-G2 cell lines. The ratio of the here calculated EC50 of the ionic form and the nanoform corresponds to the Relative Potency Factor (RPF). Using this approach, HEFs (case/kgintake) for the aforementioned nanoparticles were obtained. Non-carcinogenic HEFs (case/kgintake) for exposure via ingestion of 5.9E-01, 7.5E-03 and 2.5 E-02 were calculated for nano-Ag, nano-CuO and nano-ZnO, respectively. The HEF values here proposed were compared with HEF values extrapolated from in vivo toxicity data reported in the literature. The here presented procedure is the most appropriate approximation currently available for using in vitro toxicity data on MNM for application in the field of LCIA.

The Implementation of the Three Rs in Regulatory Toxicity and Biosafety Assessment: The Indian Perspective.

Animal models have long served as a basis for scientific experimentation, biomedical research, drug development and testing, disease modelling and toxicity studies, as they are widely thought to provide meaningful, human-relevant predictions. However, many of these systems are resource intensive and time-consuming, have low predictive value and are associated with great social and ethical dilemmas. Often drugs appear to be effective and safe in these classical animal models, but later prove to be ineffective and/or unsafe in clinical trials. These issues have paved the way for a paradigm shift from the use of in vivo approaches, toward the 'science of alternatives'. This has fuelled several research and regulatory initiatives, including the ban on the testing of cosmetics on animals. The new paradigm has been shifted toward increasing the relevance of the models for human predictivity and translational efficacy, and this has resulted in the recent development of many new methodologies, from 3-D bio-organoids to bioengineered 'human-on-a-chip' models. These improvements have the potential to significantly advance medical research globally. This paper offers a stance on the existing strategies and practices that utilise alternatives to animals, and outlines progress on the incorporation of these models into basic and applied research and education, specifically in India. It also seeks to provide a strategic roadmap to streamline the future directions for the country's policy changes and investments. This strategic roadmap could be a useful resource to guide research institutions, industries, regulatory agencies, contract research organisations and other stakeholders in transitioning toward modern approaches to safety and risk assessment that could replace or reduce the use of animals without compromising the safety of humans or the environment.

More data on in vitro assessment of comparative and combined toxicity of metal oxide nanoparticles.

Isolated and combined damaging effects of PbO and CuO nanoparticles were estimated on an established line of human fibroblasts by a decrease in: (a) the cellular dehydrogenase activity (MTT Assay), (b) the ATP content (Luminescent Cell Viability Assay), (c) the cellular proliferation, viability, spreading, and attachment to substrate evaluated integrally by continuous impedance-based measurement of the Normalized Cell Index. Using all these indices, we demonstrate an explicit dependence of cell damage on the concentrations of both metal oxide nanoparticle (MeO-NP) species. This dependence is adequately approximated with a hyperbolic function. At equal exposure levels, PbO-NP and CuO-NP demonstrate quantitatively similar cytotoxicities. The same was observed previously for some non-specific in vivo toxicity measures. The combined in vitro cytotoxicity has also been described mathematically using the Response Surface Methodology and found to be represented by various types, thus corroborating, in this respect also, the findings of a previous animal experiment with the same MeO-NPs.

In vitro and In vivo toxicity assessment of phytofabricated ZnO nanoparticles showing bacteriostatic effect and larvicidal efficacy against Culex quinquefasciatus.

Murraya koenigii berry extract based zinc oxide nanoparticles (Mk-ZnO NPs) were synthesized by simple co-precipitation method and examined for bacteriostatic and larvicidal efficiency. Synthesized Mk-ZnO NPs were characterized by UV-Vis spectroscopy at 336 nm. X-Ray diffraction (XRD) showed crystalline nature as hexagonal. Fourier transform infrared spectroscopy (FTIR) spectrum exhibited strong peak at 3442.80 cm-1. Field emission scanning electron microscopy (FE-SEM) showed hexagonal shape of the particle. Transmission electron microscopy (TEM) measured 10-15 nm sized Mk-ZnO NPs. EDX peaks confirm 71.99% of zinc and 11.42% of oxide in Mk-ZnO NPs. Minimum inhibitory concentration (MIC) analysis reveals Mk-ZnO NPs inhibit growth of Gram positive (Staphylococcus aureus, Lysinibacillus fusiformis) and Gram negative (Proteus vulgaris, Providencia vermicola) bacteria at 40 and 50 µg mL-1 respectively. Live & dead assay confirms that Mk-ZnO NPs inhibits bacterial growth at 50 µg mL-1. Bacterial biofilm thickness significantly reduced by Mk-ZnO NPs at 50 µg mL-1. In vitro toxicity of Mk-ZnO NPs on RAW 264.7 macrophages determines 90-50% cell viability at concentrations of 10-100 µg mL-1. In vivo toxicity assay results indicate the lethal concentration of Artemia nauplii were LC50-78.73 µg mL-1 and LC90-130.03 µg mL-1. Larvicidal activity of Mk-ZnO NPs towards mosquito larvae of Culex quinquefasciatus were observed at LC50-2.1 µg mL-1 and LC90-12.1 µg mL-1. Finally the study discloses, potential bacteriostatic effect and mosquito larvae controlling capacity of Mk-ZnO NPs.

Assessment of hepatotoxicity and dermal toxicity of butyl paraben and methyl paraben using HepG2 and HDFn in vitro models.

Parabens, esters of parahydroxybenzoic acid, are widely used in cosmetic, food and pharmaceutical industries mainly for their antibacterial and fungicidal properties. Methyl paraben has shown very low toxicity in a wide range of in vitro and animal tests. However, butyl paraben and derivatives, such as isobutyl parabens, are classified as allergens and have been shown to induce toxic effects. In the present study the effects of exposure to methyl or butyl paraben (5-1000 µM) on cytotoxicity, oxidative stress, mitochondrial dysfunction and genotoxicity were measured in a hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn). Butyl paraben caused a concentration dependent decrease (above 400 µM) in cell viability for both cell lines. Toxicity of butyl paraben observed appeared to be mediated via ATP depletion as seen from luminescence assays. Depletion of glutathione was also observed for higher concentrations of butyl paraben, which may indicate the involvement of oxidative stress. Methyl paraben, however, did not show any significant decrease in cell viability, reduction in ATP or glutathione levels in HepG2 and HDFn cell lines at the concentrations tested. In vitro studies based on human cell lines can provide information in the early stages of multitier paraben toxicity studies and can be combined with in vivo and ex vivo studies to build more comprehensive, scientifically sound strategies for paraben safety testing. The results obtained in this study could supplement existing in vivo toxicity data for defining more robust limits for human exposure.

In vitro assessment of the toxicity of bushfire emissions: A review.

Bushfires produce many toxic pollutants and the smoke has been shown to have negative effects on human health, especially to the respiratory system. Bushfires are predicted to increase in size and frequency, leading to a greater incidence of smoke and impacts. While there are many epidemiological studies of the potential impact on populations, there are few studies using in vitro methods to investigate the biological effects of bushfire emissions to better understand its toxicity and significance. This review focused on the literature pertaining to in vitro toxicity testing to determine the state of knowledge on current methods and findings on the impacts of bushfire smoke. There was a considerable variation in the experimental conditions, outcomes and test concentrations used by researchers using in vitro methods. Of the studies reviewed, most reported adverse impacts of particulate matter (PM) on cytotoxic and genotoxic responses. Studies on whole smoke were rare. Finer primary particulates from bushfire smoke were generally found to be more toxic than the coarse particulates and the toxicological endpoints of bushfire PM different to ambient PM. However the variation in study designs and experimental conditions made comparisons difficult. This review highlights the need for standard protocols to enable appropriate comparisons between studies to be undertaken including the assessment of physiologically relevant outcomes. Further work is essential to establish the effect of burning different vegetation types and combustion conditions on the toxicity of bushfire emissions to better inform both health and response agencies on the significance of smoke from bushfires.