Papers by Sebastian Buchinger
Over the last few years, the physical dimensions of microchip devices have decreased, enhancing t... more Over the last few years, the physical dimensions of microchip devices have decreased, enhancing the interest in the integration of various devices and complex operations onto a compatible "lab on a chip" system with desirable characteristics and capabilities. This work presents a novel μ-fluidics whole cell biosensor for water toxicity analysis. The biosensor is based on bacterial cells genetically "tailored" to generate an electrochemical bio-signal in the presence of toxic materials. The μ-chip was electrochemically characterized, and demonstrated the potential toxicity analysis with a model toxicant. A novel concept of bacterial biosensors deposition by means of electrophoretic force was examined for the first time. Preliminary results demonstrated the ability to detect electrochemical signal generated by the deposited bacterial cells indicating the potential use for patterning of bacterial cells on solid-state surfaces for the use in bio-sensing.
Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assa... more Ever since the introduction of the Salmonella typhi- murium mammalian microsome mutagenicity assay (the ‘Ames test’) over three decades ago, there has been a constant development of additional genotox- icity assays based upon the use of genetically engi- neered microorganisms. Such assays rely either on reversion principles similar to those of the Ames test, or on promoter–reporter fusions that generate a quantifiable dose-dependent signal in the presence of potential DNA damaging compounds and the induc- tion of repair mechanisms; the latter group is the subject of the present review. Some of these assays were only briefly described in the scientific literature, whereas others have been developed all the way to commercial products. Out of these, only one, the umu-test, has been fully validated and ISO- and OECD standardized. Here we review the main directions undertaken in the construction and testing of bacterial-based genotoxicity bioassays, including the attempts to incorporate at least a partial metabolic activation capacity into the molecular design. We list the genetic modifications introduced into the tester strains, compare the performance of the different assays, and briefly describe the first attempts to incorporate such bacterial reporters into actual geno- toxicity testing devices.
This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The bi... more This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosen- sor presented here is based on bacterial cells that are genetically “tailored” to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemi- cal active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 m and 300 m. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different geno- toxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 g/ml for NA using 30 min for induction and 0.31 M for IQ using 120 min for induction, both 3 min after the addition of the substrate material.
Nature protocols, 2015
This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measur... more This protocol describes a quantitative and robust 96-well-plate-reader-based assay for the measurement of ethoxyresorufin-O-deethylase (EROD) activity using the rat hepatoma cell line H4IIE. The assay can be used to determine the cytochrome P450 subfamily 1A (CYP1A)-inducing potential of single substances, as well as of mixtures and extracts of samples. It is based on the aryl hydrocarbon receptor (AhR)-mediated induction of cytochrome P450 enzymes (subfamily 1A) in cells after exposure to dioxins and dioxin-like compounds. One enzymatic reaction catalyzed by CYP1A is the deethylation of the exogenous substrate 7-ethoxyresorufin to the fluorescent product resorufin, which is measured as EROD activity in the assay. The CYP1A-inducing potential of a sample can be reliably quantified by comparing the EROD activity with the concentration-response curve of the standard substance 2,3,7,8-tetrachlorodibenzo-p-dioxin, which can be detected at concentrations down to the picogram per liter ra...
Umweltwissenschaften und Schadstoff-Forschung
Science of The Total Environment, 2016
There is significant concern regarding the contamination of riverine sediments with dioxins and d... more There is significant concern regarding the contamination of riverine sediments with dioxins and dioxin-like compounds (DLCs), including polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and some polycyclic aromatic hydrocarbons (PAHs). The majority of studies investigating the ecotoxicology of DLCs in fish have focused on a few standard model species. However, there is significant uncertainty as to whether these model species are representative of native river fish, particularly in Europe. In this study, the transcriptional responses following exposure to equipotent concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), PCB 156 or the dioxin-like PAH, benzo[k]fluoranthene (BkF), were investigated in juvenile roach (Rutilus rutilus), a fish species that constitutes a large proportion of the fish biomass in freshwater bodies throughout Europe. To this end, RNA sequencing analysis was used to comprehensively characterise the molecular mechanisms and pathways of toxicity of these DLCs. Whole transcriptome analyses using ClueGO software revealed that DLCs have the potential to disrupt a number of important processes, including energy metabolism, oogenesis, the immune system, apoptosis and the response to oxidative stress. However, despite using equipotent concentrations, there was very little conservation of the transcriptional responses observed in fish exposed to different DLCs. TCDD provoked significant specific changes in the levels of transcripts related to immunotoxicity and carbohydrate metabolism, while PCB 156 caused virtually no specific effects. Exposure to BkF affected the most diverse suite of molecular functions and biological processes, including blood coagulation, oxidative stress responses, unspecific responses to organic or inorganic substances/stimuli, cellular redox homeostasis and specific receptor pathways. To our knowledge, this is the first study of the transcriptome-wide effects of different classes of DLCs in fish. These findings represent an important step towards describing complete toxicity pathways of DLCs, which will be important in the context of informing risk assessments of DLC toxicity in native fish species.
Aquatic toxicology (Amsterdam, Netherlands), 2015
Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxi... more Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-s...
Science of The Total Environment, 2015
The European eel (Anguilla anguilla) is a facultatively catadromous fish species with a complex l... more The European eel (Anguilla anguilla) is a facultatively catadromous fish species with a complex life cycle. Its current population status is alarming: recruitment has decreased drastically since the 1980s and its stock is still considered to be outside safe biological limits. Although there is no consensus on the reasons for this situation, it is currently thought to have resulted from a combination of different stressors, including anthropogenic contaminants. To deepen our understanding of the processes leading to the accumulation of lipophilic organic contaminants in yellow eels (i.e. the feeding, continental growth stage), we developed a physiologically based toxicokinetic model using our own data and values from the literature. Such models can predict the uptake and distribution of water-borne organic chemicals in the whole fish and in different tissues at any time during exposure. The predictive power of the model was tested against experimental data for six chemicals with n-octanol-water partitioning coefficient (log Kow) values ranging from 2.13-4.29. Model performance was excellent, with a root mean squared error of 0.28 log units. This model has the potential to help identify suitable habitats for restocking under eel management plans.
Environmental Science and Pollution Research, 2015
Despite the key role of higher plants in aquatic ecosystems as functional and structural elements... more Despite the key role of higher plants in aquatic ecosystems as functional and structural elements, sediment-contact tests with macrophytes are still scarce. Moreover, due to large differences in exposure routes for pollutants as well as in life cycles between the diverse taxa of macrophytes, sensitivities to pollutants vary between taxa. Therefore, the development of new test systems with aquatic macrophytes, in general, is favorable. This study proposes a protocol for a sediment-contact test with Oryza sativa and addresses the main question whether the rice plant is a suitable test organism for sediment toxicity testing with higher plants. As a first evaluation step, the variability and sensitivity of the test was investigated using spiked artificial sediments. Thus, according to the protocol, rice was exposed to arsenic-, cadmium-, chromium-, and nickel-spiked sediments. Additionally, it was investigated which classical endpoints for plant bioassays, such as root and shoot elongation, are suitable for this bioassay. As a second evaluation step, the test system was used for assessment of natural sediments. Thereupon, a sensitivity profile of the presented test protocol was analyzed in comparison to other plant-based test systems. Inhibition of root and shoot elongation turned out to be the most sensitive endpoints for single-substance testing in spiked artificial sediments. However, regarding testing of natural sediments, rice shoots responded more sensitive than rice roots. In conclusion, the rice plant clearly showed pollutant-induced effects on growth in sediments, and thus, it is likely a promising test organism to complement sediment-contact tests with higher plants.
Given the complex interactions of re-suspension processes and bioavailability of sediment-bound p... more Given the complex interactions of re-suspension processes and bioavailability of sediment-bound pollutants, such as dioxin-like chemicals (DLCs), there is need for a better integrative understanding of the cause-effect-relationship of these pollutants. Currently, most studies investigating the role of DLCs focus either on (a) chemical analyses or (b) on the characterization of sediment extracts via in vitro bioassays. These bioassays are rapid and inexpensive and the resulting biological 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCCD) equivalents (Bio-TEQs) are a simple and integrative measure of contamination with DLCs. However, since a range of different compounds with varying physical and chemical properties is described with just one value, it is not possible to forecast the distribution of TEQs between environmental compartments, e.g. in bioaccumulation studies. The fact that, e.g., bioavailability, uptake and elimination rates in vivo are not considered, limits the use of bioassays...
Aquatic toxicology (Amsterdam, Netherlands), 2014
As a consequence of global climate change, we will be likely facing an increasing frequency and i... more As a consequence of global climate change, we will be likely facing an increasing frequency and intensity of flood events. Thus, the ecotoxicological relevance of sediment re-suspension is of growing concern. It is vital to understand contaminant uptake from suspended sediments and relate it to effects in aquatic biota. Here we report on a computational study that utilizes a physiologically based toxicokinetic model to predict uptake, metabolism and excretion of sediment-borne pyrene in rainbow trout (Oncorhynchus mykiss). To this end, data from two experimental studies were compared with the model predictions: (a) batch re-suspension experiments with constant concentration of suspended particulate matter at two different temperatures (12 and 24°C), and (b) simulated flood events in an annular flume. The model predicted both the final concentrations and the kinetics of 1-hydroxypyrene secretion into the gall bladder of exposed rainbow trout well. We were able to show that exhaustive...
Whole Cell Sensing Systems Ii, 2010
Genotoxicity test systems that are based on bacteria display an important role in the detection a... more Genotoxicity test systems that are based on bacteria display an important role in the detection and assessment of DNA damaging chemicals. They belong to the basic line of test systems due to their easy realization, rapidness, broad applicability, high sensitivity and good reproducibility. Since the development of the Salmonella microsomal mutagenicity assay by Ames and coworkers in the early 1970s, significant development in bacterial genotoxicity assays was achieved and is still a subject matter of research. The basic principle of the mutagenicity assay is a reversion of a growth inhibited bacterial strain, e.g., due to auxotrophy, back to a fast growing phenotype (regain of prototrophy). Deeper knowledge of the mutation events allows a mechanistic understanding of the induced DNA-damage by the utilization of base specific tester strains. Collections of such specific tester strains were extended by genetic engineering. Beside the reversion assays, test systems utilizing the bacterial SOS-response were invented. These methods are based on the fusion of various SOS-responsive promoters with a broad variety of reporter genes facilitating numerous methods of signal detection.
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Papers by Sebastian Buchinger