Biomedical subjects

Kristin Schirmer

Publications and source records attributed to Kristin Schirmer.

16 recordsLinked to original sources

PubMed2006

Nonylphenol isomers differ in estrogenic activity.

Nonylphenol belongs to the most investigated xenohormones acting at the estrogen receptor. Technical nonylphenol contains approximately 20 para-substituted isomers. Because of limitations in testing and quantifying all 20 isomers in the mixture, the linear form, 4n-NP, is often used as a reference substance, even though it is not present in the technical mixture. Here, we report on the synthesis and estrogenic potency of six nonylphenol isomers that occur at different proportions in technical nonylphenol mixtures. The relative potency of each isomer was determined by use of the MVLN transcriptional activation cell assay. As well, a subset of isomers was tested in the E-screen assay. One isomer, p353-NP, exhibited the same relative potency as the nonylphenol mixture, whereas the other isomers were found to be less potent. Two isomers, p22-NP and p262 NP, and the linear 4n-NP were found to be weak ER agonists with responses near the detection limit in the MVLN assay. Two isomers, p262-NP and 4n-NP, exhibited measurable activity in the E-screen. Our results demonstrate that defined p-NP isomers are most suitable for reflecting the estrogenic potency of technical NP mixtures. Among other applications, they should be used in the future to explain differences in estrogenic potency due to NPs as detected by various in vitro assays.

Biological Assay↗

PubMed2006

Modeling the impact of a benzene source zone on the transport behavior of PAHs in groundwater.

Aquifers at industrial sites are commonly characterized by a multitude of contaminant source zones. Conceivably, dissolved contaminants originating from an up-gradient residual nonaqueous phase liquid (NAPL) source zone may be transported along the groundwater flow path into another residual NAPL source zone down-gradient. However, if and how contaminants from different zones may affect one another with regard to dissolution and transport has thus far been unknown. To identify and understand such potential interactions, the numerical model BIONAPL3D was applied to simulate the behavior of six dissolved polycyclic aromatic hydrocarbons (PAHs), stemming simultaneously from an up-gradient NAPL source zone, when they encounter a down-gradient NAPL source zone. The down-gradient NAPL source zone was assumed to be a residual benzene phase with a saturation of 10%. When the dissolved PAHs entered the benzene source zone, the aqueous PAH concentrations declined significantly due to their partitioning into the residual benzene phase. As benzene rapidly dissolved intothe aqueous phase,the PAHswere resolubilized with negligible impact due to benzene co-solvency. The degree of resolubilization was much smaller than the initial loss due to partitioning into the benzene phase. Thus, the PAHs formed a new residual NAPL phase that, over time, replaced the original benzene source zone. The new NAPL phase continued to grow even after all of the benzene was dissolved. Our modeling approach is the first theoretical demonstration of a significant interaction of contaminants emanating from multiple source zones. It should be regarded as a starting point to consider source zone interactions at polluted field sites.

Algorithms↗

PubMed2006

Proposal to improve vertebrate cell cultures to establish them as substitutes for the regulatory testing of chemicals and effluents using fish.

Cultures of vertebrate cells are widely applied in mechanistic studies in human toxicology as well as in toxicity identification in ecotoxicology. As in vitro models, they display many advantages over whole animal experimentation, pertaining to such characteristics as availability, reproducibility and costs. As well, they satisfy the societal desire to reduce the number of animals in toxicology. For these reasons vertebrate cell models also appear to be a desirable replacement for animals in regulatory tests. Several vertebrate cell models are now accepted for regulatory purposes in human health sciences, with the test for photocytotoxicity using the 3T3 mouse cell line being one example. However, an in vitro alternative to whole animal tests has not yet been established for regulatory risk assessment in ecotoxicology. This review sets out to outline why such a replacement has not yet been possible and explores avenues to improve vertebrate cell cultures so that a replacement of whole animal tests could more likely be achieved. Inasmuch as fish is the most widely used non-mammalian vertebrate in risk assessment and regulation, focus will be on the replacement, by in vitro vertebrate models, of fish.

Animal Testing Alternatives↗

PubMed2006

Toxic potential of microcystin-containing cyanobacterial extracts from three Romanian freshwaters.

The aim of this study was to characterise for the first time the toxin profile and in vivo/in vitro toxicological effects of cyanobacteria obtained from three Romanian freshwater blooms. Two strains of Microcystis aeruginosa were isolated from blooms occurring in different fish ponds. As well, a complex bloom sample obtained from a recreational lake area and dominated by Microcystis aeruginosa and Microcystis viridis was explored. The morphological identity of the cyanobacterial species could be confirmed by sequence analysis of the intergenic spacer region of the phycocyanin operon, PC-IGS. As well, both the strains and the field sample were confirmed to possess mcyA and mcyB genes, which correlated with the formation of several microcystins (MCYSTs). A number of cyanopeptolins and a new aeruginosin were also found. Despite these mixtures of compounds, in vivo as well as in vitro toxicological responses resembled those previously found for MCYST-LR. These include strong hepatotoxicity in mice with parenchymal disintegration and the emergence of haemorrhagic centres, a lack of responsiveness of human and fish cell lines most likely due to a lack of active transport systems, and a significant subcellular damage most prominent on the level of the lysosomes in primary hepatocytes of mice and rainbow trout. We thus conclude that the formation of blooms in the Romanian fish ponds and the recreational area may impact on animal and human health. A broader survey of cyanotoxin occurrence and related toxicity is necessary in Romania in order to assess the extent of this animal and human health hazard.

Animals↗

PubMed2006

Development of a solvent-free, solid-phase in vitro bioassay using vertebrate cells.

Miniaturized bioassays offer many advantages in exploring the toxic potential of chemicals, including small sample volumes and compatibility with high-throughput screening. One problem common to miniaturized systems, however, is the loss of test chemicals because of sorption. The idea of the current study was to use the sorption phenomenon in a positive way. It was found that contaminants sorbed to the growth surface in wells of tissue-culture plates or to the surface of selected sorbent bead materials are available to vertebrate cells growing in direct contact with the contaminant-coated surface. The use of beads provided more flexibility with regard to surface area, materials, and assay format. Biosilon, a bead cell-culture carrier made of polystyrene, was found to be most suitable. It supported cell adherence and allowed the detection of reproducible dose-response curves of an increase in cytochrome CYP1A enzyme activity by sorbed polycyclic aromatic hydrocarbons in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1. The resulting bead assay provides a miniaturized, solvent-free exposure system. Potential future applications include the coupling to environmental sampling, in which the bead material is used as solid receiving phase before serving as a surface for vertebrate cells to attach and respond.

7-Alkoxycoumarin O-Dealkylase↗

PubMed2005

Microcystin-LR induced cellular effects in mammalian and fish primary hepatocyte cultures and cell lines: a comparative study.

The impact of microcystin-LR, one of the most common cyanobacterial toxins, on liver and gut cells originating from mammals and fish was compared. Upon exposure of human and rainbow trout (Oncorhynchus mykiss) cell lines up to 2.5 microM microcystin-LR, no alteration in cell viability was observed as assessed with three fluorescent indicators dyes, CFDA-AM, Alamar Blue and neutral red. The lack of sensitivity of the trout cell lines coincided with an absence of detectable mRNA levels of organic anion transporter polypeptide (OATP), which is implicated in the uptake of microcystin-LR. In contrast to the cell lines, primary rainbow trout and mouse hepatocytes showed damage to subcellular structures, particularly the lysosomes, as indicated by neutral red. This led us to propose a thus far undetected role of lysosomes as targets and mediators of microcystin-LR elicited cellular damage. An inhibitor of OATP, rifampicin, partly protected hepatocytes from this damage. Yet, the sensitivity of rainbow trout hepatocytes rapidly declined in culture, accompanied by decreasing levels of OATP mRNA. The sensitivity of mouse hepatocytes toward microcystin-LR also declined in culture but overall was about 25-fold greater than that of the trout cells. These differences mirror those observed in vivo and suggest the use hepatocytes for deciphering the species differences.

Animals↗

PubMed2005

Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout.

We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.

Actins↗

PubMed2005

Time-integrated monitoring of polycyclic aromatic hydrocarbons (Pahs) in groundwater using the ceramic dosimeter passive sampling device.

Passive sampling relies on the uptake of contaminants into appropriate sampling devices along a diffusion gradient without using pumps or bailers. Thus, for example, in groundwater sampling, changes to flow due to pumping can be avoided. If the diffusion gradient can be maintained for extended periods, contaminants can be sampled continuously over time without any action, allowing to determine time-weighted average contaminant concentrations. We here show that the Ceramic Dosimeter, a solid receiving phase passive sampler using a ceramic membrane as sorbent container and diffusion barrier, can be used without calibration for the long-term monitoring of polycyclic aromatic hydrocarbons (PAHs) in groundwater.

Diffusion↗

PubMed2005

Toxic potential of five freshwater Phormidium species (Cyanoprokaryota).

Among the Cyanoprokaryota (blue-green algae), the genus Phormidium has thus far rarely been studied with respect to toxin production and potentially resulting human and environmental health effects. We here show that five previously unexplored freshwater species of this genus (Ph. bijugatum, Ph. molle, Ph. papyraceum, Ph. uncinatum, Ph. autumnale) are indeed capable of producing bioactive compounds. Phormidium extracts caused weight loss as well as neuro/hepatotoxic symptoms in mice, and in the case of Ph. bijugatum even death. Very low levels of saxitoxins and microcystins, as confirmed by ELISA, were insufficient to explain this toxicity and the differing toxic potencies of the Phormidium species. Qualitative HPLC analyses confirmed different substance patterns and in the future could aid in the separation of fractions for more detailed substance characterisation. The results in vivo were confirmed in vitro using cells of human, mouse and fish. The fish cells responded least sensitive but proved useful in studying the temperature dependence of the toxicity by the Phormidium samples. Further, the human cells were more sensitive than the mouse cells thus suggesting that the former may be a more appropriate choice for studying the impact of Phormidium to man. Among the human cells, two cancer cell lines were more responsive to one of the samples than a normal cell line, thereby indicating a potential anti-tumour activity. Thus, the five freshwater Phormidium species should be considered in environmental risk assessment but as well, as a source of therapeutic agents.

Animals↗

PubMed2005

Analysis of estrogenic effects by quantification of green fluorescent protein in juvenile fish of a transgenic medaka.

TheChgH-GFP strain of the teleost medaka contains a regulatory region of the estrogen-responsive choriogenin H (chgH) gene fussed to the green fluorescent protein (GFP) gene. The strain was developed for the identification of environmental estrogens by noninvasive analysis of GFP fluorescence. In the present study, a quantification method for GFP by image analysis was establishedand applied to the analysis of time- and concentration-dependent GFP fluorescence in juvenile fish. Concentration-response analyses were performed with fish exposed for 14 d to 17beta-estradiol (0.37-367 pM), genistein (0.37-367 nM), or p-nonylphenol (0.367-1,835 nM). By means of image analysis, it was shown that ChgH-GFP was induced at 183.5 pM or greater 17beta-estradiol. Time-course and recovery experiments indicated a strong accumulation of GFP in the liver. Results of reverse transcriptase-polymerase chain reaction analysis of choriogenin H and vitellogenin demonstrated induction of gene expression for the same rangeof concentrations as that for GFP analysis. Neither expression of these genes nor GFP fluorescence was induced by genistein and p-nonylphenol. Although the ChgH-GFP strain failed to detect these weakly estrogenic compounds, the simplicity of the GFP quantification during early life stages of fish offers promising possibilities for further developments of transgenic strains using different target regulatory sequences.

Animals↗

PubMed2005

Effect-directed analysis of mutagens and ethoxyresorufin-o-deethylase inducers in aquatic sediments.

Sediment extracts from a creek in the Neckar river basin (Germany), which received the discharge of treated hospital wastewater, were found to exhibit strong aromatic hydrocarbon (Ah) receptor-mediated effects in a rainbow trout liver cell line (RTL-WI) as well as high mutagenicity in the Salmonella/microsome assay after fractionation. The crude extract did not exhibit a clear mutagenic response. Apparently, cleanup or fractionation before mutagenicity testing is necessary to minimize the risk of false-negative results. Effect-directed fractionation and analysis were applied to characterize and identify the toxicants that cause these effects. Major ethoxyresorufin-O-deethylase induction potency and mutagenicity were detected in different polyaromatic fractions, indicating different sets of toxicants that induce metabolic activation and mutagenicity. Dioxin-like halogenated aromatic hydrocarbons, including polychlorinated biphenyls, naphthalenes, dibenzo-p-dioxins and furans, and priority polycyclic aromatic hydrocarbons, contributed to Ah receptor-mediated activity only to a minor extent. Benzo[a]pyrene, benzo[a]fluoranthene, and perylene could be confirmed as important contributors to mutagenicity. The nonpriority pollutants 11H-indeno[2,1,7-cde]pyrene, a methylbenzo[e]pyrene, and a methylperylene were tentatively identified as major components, representing 82% of the peak area of a highly mutagenic fraction of the sediment extract. This suggests that hazard and risk assessment of complex environmental mixtures should make increasing attempts to identify and consider hazardous key pollutants rather than focusing on a priori-selected key pollutants alone.

Cytochrome P-450 CYP1A1↗

PubMed2004

Applying whole water samples to cell bioassays for detecting dioxin-like compounds at contaminated sites.

Methodology was developed in order to rapidly and cost-efficiently screen whole water samples without extraction for the presence of dioxin-like compounds using a cell bioassay approach. Presence of dioxin-like compounds was indicated by the induction in the rainbow trout (Oncorhynchus mykiss) liver cell line, RTL-W1, of cytochrome CYP1A, which was measured as 7-ethoxyresorufin-O-deethylase (EROD) activity. Two simple culture media, L-15/ex and Earle's-G, prepared in tissue culture water and supplemented with 5% serum, proved suitable for supporting RTL-W1 cell viability and induction of EROD activity by the model inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (BaP). Preparation of the same simplified media using whole surface and ground water instead of tissue culture water again allowed EROD induction by spiked TCDD and BaP to be detected but higher concentrations of inducers were necessary. Despite this reduced sensitivity, RTL-W1 cells responded to 4 out of 40 ground water samples from a former oil and lignite processing site with significant EROD induction. In the future, the value of the bioassay is as an inexpensive means of quickly screening ground and surface water samples to identify high contaminant levels particularly at industrial sites, where detailed site-investigations and long-term monitoring programs are required.

Animals↗

PubMed2004

Evaluating the toxicity of Triton X-100 to protozoan, fish, and mammalian cells using fluorescent dyes as indicators of cell viability.

Three viability assays using fluorescent dyes effectively detected a loss of viability in cultures of three mammalian cell lines (H4IIE, Caco2, and HepG-2), two fish cell lines (RTgill-W1 and RTL-W1), and a ciliated protozoan, Tetrahymena thermophila, after exposure to Triton X-100, used as a model toxicant. The dyes were Alamar Blue (AB), neutral red (NR), and propidium iodide, which respectively monitored energy metabolism, lysosomal activity, and membrane integrity. A fourth fluorescent dye, 5-carboxyfluorescein diacetate acetoxymethyl ester, was problematic. For 2-h Triton X-100 exposures, mammalian cell lines were as susceptible as piscine cell lines, whereas T. thermophila was approximately twofold less sensitive as detected with AB and NR. Despite being less sensitive, cytotoxicity tests on T. thermophila could be done in spring water, which means that unlike animal cells they could be directly exposed to most industrial effluents without osmolality adjustments. Therefore, T. thermophila could be a useful complement to animal cells as alternatives to fish in toxicity testing.

Animal Testing Alternatives↗

PubMed2003

The freshwater cyanobacterium Lyngbya aerugineo-coerulea produces compounds toxic to mice and to mammalian and fish cells.

Despite a growing awareness of the presence of cyanobacterial toxins, knowledge about the ability of specific species to produce toxic compounds is still rather limited. It was the overall goal of the current work to investigate if probes derived from the freshwater species Lyngbya aerugineo-coerulea (Kutz.) Gomont, a cyanobacterium frequently found in southern Europe and not previously investigated for the presence of bioactive compounds, were capable of eliciting in vivo and in vitro toxicity. The cyanobacterial extract revealed signs of neuro- as well as hepatotoxicity in mice, although these signs could not be explained by the well-known respective cyanobacterial neuro- and hepatotoxins saxitoxin and microcystin. Cytotoxicity was elicited by the cyanobacterial extract in all mammalian cell lines tested. As well, the rainbow trout liver cell line, RTL-W1, was found to be susceptible to the cytotoxic effects of the extract, although the cytotoxicity was dependent on temperature. In contrast, the cyanobacterial growth medium elicited cytotoxicity independent of temperature, leading to morphological changes indicative of alterations to the cytoskeleton. Overall, the results suggest that Lyngbya aerugineo-coerulea is an important cyanobacterium to be considered for its potential to cause health risks on environmental exposure of it to mammals and fish. Applying a combination of mammalian and piscine cell line bioassays is a unique approach that, combined with chemical analysis, could be used in the future to identify the structure and cellular mechanisms of the as-yet-unknown toxic Lyngbya aerugineo-coerulea metabolites in particular and to screen cyanobacterial extracts for their toxicity in general.

Animals↗

PubMed2002

Applying whole-water samples directly to fish cell cultures in order to evaluate the toxicity of industrial effluent.

Methodology was developed for presenting to fish cells in culture whole-water samples without extraction and used to evaluate the toxicity to a rainbow trout gill cell line, RTgill-W1, of more than 30 whole-water samples collected from a paper mill over approximately a year of operation. Presentation to cells was achieved by adding to water samples the amounts of salts, galactose and sodium pyruvate, as solids, that were necessary to give concentrations and osmolality of the basal growth medium, Leibovitz's L-15. Cell viability was measured with three fluorescent indicator dyes: alamar Blue for metabolism, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) for membrane function, and neutral red for lysosomal activity. Eighteen samples were tested with the Daphnia lethality bioassay and 11 of these were toxic. None of these were judged cytotoxic to RTgill-W1. Sixteen samples were tested with the rainbow trout lethality bioassay and only one was toxic. This sample was also the only sample that was cytotoxic to RTgill-W1. Therefore, these methods for presenting water samples and measuring their cytotoxicity to RTgill-W1 are a promising substitute for toxicity tests of industrial effluent with rainbow trout but not with Daphnia.

Animals↗

PubMed2002

Effect-directed fractionation and identification of cytochrome p4501A-inducing halogenated aromatic hydrocarbons in a contaminated sediment.

On the basis of a new fractionation method combined with in vitro ethoxyresorufin-O-deethylase (EROD) induction in a rainbow trout liver cell line (RTL-W1) and chemical analysis, halogenated aromatic hydrocarbons with dioxin-like activity were identified in a sediment extract from Bitterfeld, Germany. The fractionation method allowed a separation of different nonplanar and coplanar polychlorinated biphenyls (PCBs), polychlorinated naphthalenes (PCNs), dibenzo-p-dioxins (PCDDs), and dibenzofurans (PCDFs) with different degrees of chlorination. The dioxin-like activity at the investigated site could be quantitatively assigned to PCDD/Fs. Both PCBs and PCNs could be excluded as the cause of the measured effects on the basis of the fractionation procedure and bioanalytical results. Thus, the method allowed the chemical analysis to focus on PCDD/Fs, with significant reduction of the analytical expense. The EROD-induction potency of sediment-extract fractions was quantified, and toxicants were confirmed by the application of induction equivalent quantities on the basis of fixed-effect-level concentrations that exhibit 15% of the maximum induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin. This approach was designed to minimize methodological limitations due to superimposing inhibitory effects.

Animals↗