Susanne Rudzok

Measuring and modeling of binary mixture effects of pharmaceuticals and nickel on cell viability/cytotoxicity in the human hepatoma derived cell line HepG2.

The interaction of drugs and non-therapeutic xenobiotics constitutes a central role in human health risk assessment. Still, available data are rare. Two different models have been established to predict mixture toxicity from single dose data, namely, the concentration addition (CA) and independent action (IA) model. However, chemicals can also act synergistic or antagonistic or in dose level deviation, or in a dose ratio dependent deviation. In the present study we used the MIXTOX model (EU project ENV4-CT97-0507), which incorporates these algorithms, to assess effects of the binary mixtures in the human hepatoma cell line HepG2. These cells possess a liver-like enzyme pattern and a variety of xenobiotic-metabolizing enzymes (phases I and II). We tested binary mixtures of the metal nickel, the anti-inflammatory drug diclofenac, and the antibiotic agent irgasan and compared the experimental data to the mathematical models. Cell viability was determined by three different methods the MTT-, AlamarBlue(R) and NRU assay. The compounds were tested separately and in combinations. We could show that the metal nickel is the dominant component in the mixture, affecting an antagonism at low-dose levels and a synergism at high-dose levels in combination with diclofenac or irgasan, when using the NRU and the AlamarBlue assay. The dose-response surface of irgasan and diclofenac indicated a concentration addition. The experimental data could be described by the algorithms with a regression of up to 90%, revealing the HepG2 cell line and the MIXTOX model as valuable tool for risk assessment of binary mixtures for cytotoxic endpoints. However the model failed to predict a specific mode of action, the CYP1A1 enzyme activity.

Inhibition of endotoxin‐induced perinatal asthma protection by pollutants in an experimental mouse model

One of the most promising strategies to face the increasing asthma prevalence and to prevent disease development might be an early contact with microbial compounds. However, little is known about an interaction between an early‐life contact to microbial compounds leading to asthma protection in the offspring and a co‐exposure to allergy‐promoting pollutants.

Transcriptional signatures of regulatory and toxic responses to benzo-[a]-pyrene exposure

BackgroundSmall molecule ligands often have multiple effects on the transcriptional program of a cell: they trigger a receptor specific response and additional, indirect responses (side effects). Distinguishing those responses is important for understanding side effects of drugs and for elucidating molecular mechanisms of toxic chemicals.ResultsWe explored this problem by exposing cells to the environmental contaminant benzo-[a]-pyrene (B[a]P). B[a]P exposure activates the aryl hydrocarbon receptor (Ahr) and causes toxic stress resulting in transcriptional changes that are not regulated through Ahr. We sought to distinguish these two types of responses based on a time course of expression changes measured after B[a]P exposure. Using Random Forest machine learning we classified 81 primary Ahr responders and 1,308 genes regulated as side effects. Subsequent weighted clustering gave further insight into the connection between expression pattern, mode of regulation, and biological function. Finally, the accuracy of the predictions was supported through extensive experimental validation.ConclusionUsing a combination of machine learning followed by extensive experimental validation, we have further expanded the known catalog of genes regulated by the environmentally sensitive transcription factor Ahr. More broadly, this study presents a strategy for distinguishing receptor-dependent responses and side effects based on expression time courses.

The inducibility of human cytochrome P450 1A by environmental-relevant xenobiotics in the human hepatoma derived cell line HepG2

Overexpression of the CYP1 family, independent of gender, is focal to the evaluation of the risk of human cancer. We have analysed the ability of 17 anthropogenic environmental xenobiotics widely used in Europe within households and agriculture to induce the human cytochrome P450 1A (CYP1A) in the human hepatoma derived cell line HepG2. The xenobiotics were potent to concomitantly induce both CYP1A mRNA and CYP1A activity in a dose-response relationship. Exceptions were shown by the organophosphate insecticide chlorpyrifos and the imidazole fungicide prochloraz in high concentrations which were capable of both inhibiting the basal or abolishing the initially induced CYP1A activity, respectively. A CYP1A induction has been shown for the first time by the aromatic xenobiotics irgasan, permethrin and azoxystrobin, the nonaromatic tributyltinoxide and for humans by the piperonylbutoxide. The xenobiotics additionally differed by their induced CYP1A isoenzyme pattern. A pronounced CYP1A1 and CYP1A2 mRNA induction was given by the phenyl urea herbicide diuron and benzodiazole insecticide piperonylbutoxide, respectively. In conclusion, out of the environmental xenobiotics, we described new members of human CYP1A inducers which extend chemical structures of biotransformation activators.

Pathway and time-resolved benzo[a]pyrene toxicity on Hepa1c1c7 cells at toxic and subtoxic exposure

Benzo[a]pyrene (B[a]P) is an environmental contaminant mainly studied for its toxic/carcinogenic effects. For a comprehensive and pathway orientated mechanistic understanding of the effects directly triggered by a toxic (5 μM) or a subtoxic (50 nM) concentration of B[a]P or indirectly by its metabolites, we conducted time series experiments for up to 24 h to study the effects in murine hepatocytes. These cells rapidly take up and actively metabolize B[a]P, which was followed by quantitative analysis of the concentration of intracellular B[a]P and seven representative degradation products. Exposure with 5 μM B[a]P led to a maximal intracellular concentration of 1604 pmol/5 × 10(4) cells, leveling at 55 pmol/5 × 10(4) cells by the end of the time course. Changes in the global proteome (>1000 protein profiles) and metabolome (163 metabolites) were assessed in combination with B[a]P degradation. Abundance profiles of 236 (both concentrations), 190 (only 5 μM), and 150 (only 50 nM) proteins were found to be regulated in response to B[a]P in a time-dependent manner. At the endogenous metabolite level amino acids, acylcarnitines and glycerophospholipids were particularly affected by B[a]P. The comprehensive chemical, proteome and metabolomic data enabled the identification of effects on the pathway level in a time-resolved manner. So in addition to known alterations, also protein synthesis, lipid metabolism, and membrane dysfunction were identified as B[a]P specific effects.

Toxicity profiles of four metals and 17 xenobiotics in the human hepatoma cell line HepG2 and the protozoa Tetrahymena pyriformis—A comparison

We performed an interspecies comparison for the human hepatoma cell line HepG2 and the eukaryotic single cell organism Tetrahymena pyriformis (T. pyriformis) for 17 xenobiotics with diverse structures and four metals. The cytotoxicity was assessed by four different cell viability assays (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide reduction (MTT), neutral red uptake (NRU), resazurin dye (AlamarBlue), 5‐carboxyfluorescein diacetate acetoxymethyl ester (CFDA‐AM)) for the HepG2 and by cell count and MTT for T. pyriformis. For HepG2 cells, the results revealed interassay variations depending on the compound. The highest assay conformity was found for the metal Hg2+ and the fungicide prochloraz. The AlamarBlue assay was the most sensitive assay according to low‐effect concentrations. By contrast, the NRU assay was comprised of more frequent whole concentration response relationships and was more susceptible to EC50. For T. pyriformis the EC50 values of the two applied assays displayed a high conformity (R2 = 0.97). Comparing the EC50 values obtained by the MTT assay for the two cell models, a direct correlation was absent for the xenobiotics and only present for the metals (Cd2+, Cu2+, and Ni2+). Moreover, the protozoa T. pyriformis displayed a 20 times higher sensitivity than the cell line. The highest interspecies difference of three log degrees was obtained for the polycyclic aromatic hydrocarbon fluoranthene. In addition, a correlation of the EC50 values and octanol‐water partition coefficient (log KOW) of the xenobiotics was performed. No correlation was found for HepG2, and a weak one for T. pyriformis. Interestingly, the interspecies difference of logarithmized EC50 correlated positive with the log KOW (R2 = 0.65). In conclusion, to obtain reliable evidence for human cytotoxicity, more than one viability/cytotoxicity assay had to be applied for cell lines. Second, the human hepatoma cell line was less affected by the organic compounds than the eukaryotic single‐cell organism and was also less dependent on the log KOW of the xenobiotic.