Following the adverse outcome pathway from micronucleus to cancer using H2B-eGFP transgenic healthy stem cells.

In vitro assessment of genotoxicity as an early warning tool for carcinogenicity mainly relies on recording cytogenetic damages (micronuclei, nucleoplasmic bridges) in tumour-derived mammalian cell lines like V79 or CHO. The forecasting power of the corresponding standardised test is based on epidemiological evidence between micronuclei frequencies and cancer incidence. As an alternative to destructive staining of nuclear structures a fish stem cell line transgenic for a fusion protein of histone 2B (H2B) and enhanced green fluorescent protein (eGFP) was established. The cells are derived from koi carp brain (KCB) and distinguish from mammalian culturable cells by non-tumour-driven self-renewal. This technology enables the analysis of genotoxic- and malign downstream effects in situ in a combined approach. In proof-of concept-experiments, we used known carcinogens (4-Nitroquinoline 1-oxide, colchicine, diethylstilbestrol, ethyl methanesulfonate) and observed a significant increase in micronuclei (MNi) frequencies in a dose-dependent manner. The concentration ranges for MNi induction were comparable to human/mammalian cells (i.e. VH-16, CHL and HepG2). Cannabidiol caused the same specific cytogenetic damage pattern as observed in human cells, in particular nucleoplasmic bridges. Metabolic activation of aflatoxin B1 and cyclophosphamide could be demonstrated by pre-incubation of the test compounds using either conventional rat derived S9 mix as well as an in vitro generated biotechnological alternative product ewoS9R. The presented high throughput live H2B-eGFP imaging technology using non-transformed stem cells opens new perspectives in the field of in vitro toxicology. The technology offers experimental access to investigate the effects of carcinogens on cell cycle control, gene expression pattern and motility in the course of malign transformation. The new technology enables the definition of Adverse Outcome Pathways leading to malign cell transformation and contributes to the replacement of animal testing. Summary: Complementation of genotoxicity testing by addressing initiating events leading to malign transformation is suggested. A vertebrate cell model showing "healthy" stemness is recommended, in contrast to malign transformed cells used in toxicology/oncocology.

Green toxicological investigation for biofuel candidates.

To avoid potential risks of biofuels on the environment and human, ecotoxicity investigation should be integrated into the early design stage for promising biofuel candidates. In the present study, a green toxicology testing strategy combining experimental bioassays with in silico tools was established to investigate the potential ecotoxicity of biofuel candidates. Experimental results obtained from the acute immobilisation test, the fish embryo acute toxicity test and the in vitro micronucleus assay (Chinese hamster lung fibroblast cell line V79) were compared with model prediction results by ECOSAR and OECD QSAR Toolbox. Both our experimental and model prediction results showed that 1-Octanol (1-Oct) and Di-n-butyl ether (DNBE) were the most toxic to Daphnia magna and zebrafish among all the biofuel candidates we investigated, while Methyl ethyl ketone (MEK), Dimethoxymethane (DMM) and Diethoxymethane (DEM) were the least toxic. Moreover, both in vitro micronucleus assay and OECD QSAR Toolbox evaluation suggested that the metabolites present higher genotoxicity than biofuel candidates themselves. Overall, our results proved that this green toxicology testing strategy is a useful tool for assessing ecotoxicity of biofuel candidates.

Is a liver comparable to a liver? A comparison of different rat-derived S9-fractions with a biotechnological animal-free alternative in the Ames fluctuation assay.

Metabolism has to be considered during the toxicological assessment of chemical and environmental samples because it is an important process in the mammalian liver. It can be assessed in vitro via liver homogenates called S9-fractions, an external metabolic activation system. However, the external metabolic activation systems can vary greatly in their composition due to biological variations among individual animals and animal strains that the S9-fraction are derived as well as the differences in the production treatment. To gain more insight into these variances, three different but commonly used rat-derived S9-fractions were compared in the present study for their variance and performance with a reference compound in the Ames fluctuation assay with Salmonella typhimurium strains TA 98 and TA 100 according to ISO 11350. Severe shortcomings of conventional rat-derived S9-fractions were observed in the present study, such that S9-fractions differed significantly within the same rat strain and for different types of induction procedures in regards to the metabolic capability. An intrinsic mutagenic potential of the three rat-derived S9-fractions were identified in the Ames fluctuation assay with varying S9-fraction concentrations. To address some of the shortcomings of the animal-derived S9-fraction, the present study investigated the use and performance of a biotechnological, animal-free alternative, ewoS9R, in comparison to one of the rat-derived S9-fraction as the others showed a mutagenic potential themselves. Specifically, 12 different chemicals were used as a reference to determine if ewoS9R could serve as an adequate and more consistent replacement of traditional rat-derived metabolic activation systems: 8 pro-mutagenic compounds (i.e., require metabolic activation to show a mutagenic potential), one pro-mutagenic compound but not in the tested strains, one mutagenic compound without metabolic activation and two compounds that are equivocal in the literature. EwoS9R was evaluated as a promising approach in the Ames fluctuation assay with 5 compounds observed to have similar results with both rat-derived S9-fraction and ewoS9R (41%), for 3 compounds ewoS9R was a better metabolization system than the rat-derived S9-fraction (16%). Further research is necessary to determine the full potential of ewoS9R in comparison to rat-derived S9-fractions.

The Green toxicology approach: Insight towards the eco-toxicologically safe development of benign catalysts.

Green toxicology is a novel approach increasingly applied for the development of materials and chemicals that are more benign to the environment and human health than their conventional counterparts. It includes predictive eco-toxicological assessments of chemicals during the early developmental process to exclude adverse effects. In the present study, two guanidine zinc catalysts for the ring-opening polymerization of lactide were investigated using eco-toxicological tools. Namely, the fish embryo toxicity assay for teratogenic effects, the ER (α) CALUX assay for endocrine activity and the Ames fluctuation assay for mutagenic potential were applied. Both complexes showed no endocrine activity, mutagenicity or acute aquatic toxicity, however a delayed hatch could be observed, therefore suggesting potential effects on a molecular level. This proof-of-concept study aims to assess the toxicity of guanidine zinc catalysts and is a first step towards the incorporation of toxicological assessments into chemical developmental processes to achieve a sustainable and safe production of catalysts.

Using a high-throughput method in the micronucleus assay to compare animal-free with rat-derived S9.

This study presents a high-throughput (HTP) micronucleus assay in multi-well plates with an automated evaluation for risk assessment applications. The evaluation of genotoxicity via the micronucleus assays according to international guidelines ISO 21427-2 with Chinese hamster (Cricetulus griseus) V79 cells was the starting point to develop our methodology. A drawback of this assay is that it is very time consuming and cost intensive. Our HTP micronucleus assay in a 48-well plate format allows for the simultaneous assessment of five different sample-concentrations with additional positive, negative and solvent controls with six technical replicates each within a quarter of the time required for the equivalent evaluation using the traditional slide method. In accordance with the 3R principle, animal compounds should be replaced with animal-free alternatives. However, traditional cell culture-based methods still require animal derived compounds like rat-liver derived S9-fraction, which is used to simulate the mammalian metabolism in in vitro assays that do show intrinsic metabolization capabilities. In the present study, a recently developed animal-free biotechnological alternative (ewoS9R) was investigated in the new high-throughput micronucleus assay. In total, 12 different mutagenic or genotoxic chemicals were investigated to assess the potential use of the animal-free metabolization system (ewoS9R) in comparison to a common rat-derived product. Out of the 12 compounds, one compound did not induce micronuclei in any treatment and 2 substances showed a genotoxic potential without the need for a metabolization system. EwoS9R demonstrated promising potential for future applications as it shows comparable results to the rat-derived S9 for 6 of the 9 pro-genotoxic substances tested. The remaining 3 substances (2-Acetamidofluorene, Benzo[a]pyrene, Cyclophosphamide) were only metabolized by rat-derived S9. A potential explanation is that ewoS9R was investigated with an approx. 10-fold lower enzyme concentration and was only optimized for CYP1A metabolization that may be improved with a modified production procedure. Future applications of ewoS9R go beyond the micronucleus assay, but further research is necessary.

Alternative type of Ames test allows for dynamic mutagenicity detection by online monitoring of respiration activity.

The Ames test is the most commonly used mutagenicity test worldwide. It is based on a microbial system that uses histidine auxotrophic Salmonella typhimurium strains. Due to either spontaneous mutations or mutations induced by a mutagenic compound, the cells can regain their ability to grow without histidine supplementation. The degree of mutagenicity of a sample correlates with the number of cells that are able to grow in media that lack histidine. All test variants published up to now are endpoint determinations providing no information about cell growth and respiration activity during the cultivation time. This study aimed to develop an alternative type of Ames test by characterizing the respiration activity of Salmonella typhimurium over time for dynamic mutagenicity detection. It focuses on elucidating the mechanisms underlying this novel test system, and serves as a general proof of principle. Respiration activity (oxygen transfer and uptake rate) and biomass growth of Salmonella typhimurium TA 100 and TA 98 were mechanistically modeled to understand and predict the behavior of the bacteria during the Ames test. The results simulated by the model were experimentally validated by the online monitoring of respiration activity over cultivation time using a Respiration Activity MOnitoring System (RAMOS). The simulated prediction was observed to fit well to the experimental data. When a mutagenic compound was added, its mutagenicity could be detected online due to the elevated cell number and respiration of histidine prototrophic cells. Laborious manual evaluation of mutagenicity after cultivation is not necessary. Mutagenicity evaluation with the presented alternative Ames RAMOS test fitted well to results from an Ames fluctuation test. In the future, a miniaturized RAMOS device for microtiter plates should allow for a high-throughput Ames RAMOS test.

Optimization of the Ames RAMOS test allows for a reproducible high-throughput mutagenicity test.

The Ames test is one of the most widely used mutagenicity tests. It employs histidine auxotrophic bacteria, which can mutate back to histidine prototrophy and, thus, grow on a histidine deficient medium. These mutants develop predominantly after adding a mutagenic compound during an initial growth phase on 1 mg/L histidine. In the established test systems, an endpoint determination is performed to determine the relative number of mutants. An alternative Ames test, the Ames RAMOS test, has been developed, which enables the online detection of mutagenicity by monitoring respiration activity. The reproducibility of the newly developed test system was investigated. A strong dependence of the test results on the inoculum volume transferred from the preculture was found. The more inoculum was needed to reach the required initial OD, the more mutagenic a positive control was evaluated. This effect was attributed to the histidine transfer from the preculture to the original Ames RAMOS test. The same problem is evident in the Ames fluctuation test. High reproducibility of the Ames RAMOS test could be achieved by performing the preculture on minimal medium with a defined histidine concentration and termination after histidine depletion. By using 5 mg/L initial histidine within the minimal medium, a higher separation efficiency between negative control and mutagenic samples could be achieved. This separation efficiency could be further increased by lowering the cultivation temperature from 37 to 30 °C, i.e. lowering the maximum growth rate. The optimized Ames RAMOS test was then transferred into a 48-well microtiter plate format (µRAMOS) for obtaining a high throughput test. The online detection of mutagenicity leads to a reduction of working time in the laboratory. Due to the optimization of reproducibility and the increase in separation efficiency, a sound mutagenicity evaluation, even of weak mutagenic compounds, can be achieved.

Genotoxicity of three biofuel candidates compared to reference fuels.

Global demand for alternative energy sources increases due to concerns regarding energy security and greenhouse gas emissions. However, little is known regarding the impacts of biofuels to the environment and human health even though the identification of such impacts is important to avoid biofuels leading to undesired effects. In this study mutagenicity and genotoxicity of the three biofuel candidates ethyl levulinate (EL), 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF) were investigated in comparison to two petroleum-derived fuels and a biodiesel. None of the samples induced mutagenicity in the Ames fluctuation test. However, the Micronucleus assay revealed significant effects in Chinese hamster (Cricetulus griseus) V79 cells caused by the potential biofuels. 2-MF revealed the highest toxic potential with significant induction of micronuclei below 20.0 mg/L. EL and 2-MTHF induced micronuclei only at very high concentrations (>1000.0 mg/L). In regard to the genotoxic potential of 2-MF, its usage as biofuel should be critically discussed.

Microscale In Vitro Assays for the Investigation of Neutral Red Retention and Ethoxyresorufin-O-Deethylase of Biofuels and Fossil Fuels.

Only few information on the potential toxic effectiveness of biofuels are available. Due to increasing worldwide demand for energy and fuels during the past decades, biofuels are considered as a promising alternative for fossil fuels in the transport sector. Hence, more information on their hazard potentials are required to understand the toxicological impact of biofuels on the environment. In the German Cluster of Excellence "Tailor-made Fuels from Biomass" design processes for economical, sustainable and environmentally friendly biofuels are investigated. In an unique and interdisciplinary approach, ecotoxicological methods are applied to gain information on potential adverse environmental effects of biofuels at an early phase of their development. In the present study, three potential biofuels, ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran were tested. Furthermore, we investigated a fossil gasoline fuel, a fossil diesel fuel and an established biodiesel. Two in vitro bioassays, one for assessing cytotoxicity and one for aryl hydrocarbon receptor agonism, so called dioxin-like activity, as measured by Ethoxyresorufin-O-Deethylase, were applied using the permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss). The special properties of these fuel samples required modifications of the test design. Points that had to be addressed were high substance volatility, material compatibility and low solubility. For testing of gasoline, diesel and biodiesel, water accommodated fractions and a passive dosing approach were tested to address the high hydrophobicity and low solubility of these complex mixtures. Further work has to focus on an improvement of the chemical analyses of the fuel samples to allow a better comparison of any effects of fossil fuels and biofuels.