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Infants and young children (IYC) remain the most vulnerable population group to environmental hazards worldwide, especially in economically developing regions such as sub-Saharan Africa (SSA). As a result, several governmental and non-governmental institutions including health, environmental and food safety networks and researchers have been proactive toward protecting this group. Mycotoxins, toxic secondary fungal metabolites, contribute largely to the health risks of this young population. In SSA, the scenario is worsened by socioeconomic status, poor agricultural and storage practices, and low level of awareness, as well as the non-establishment and lack of enforcement of regulatory limits in the region. Studies have revealed mycotoxin occurrence in breast milk and other weaning foods. Of concern is the early exposure of infants to mycotoxins through transplacental transfer and breast milk as a consequence of maternal exposure, which may result in adverse health effects. The current paper presents an overview of mycotoxin occurrence in foods intended for IYC in SSA. It discusses the imperative evidence of mycotoxin exposure of this population group in SSA, taking into account consumption data and the occurrence of mycotoxins in food, as well as biomonitoring approaches. Additionally, it discusses the health implications associated with IYC exposure to mycotoxins in SSA.
1.2-Dioxetanes, very reactive and high energy molecules. are involved as labile intermediates in dioxygenase- activated aerobic metabolism and in physiological processes. Various toxico1ogica1 tests reveal that dioxetanes are indeed genotoxic. In supercoiled DNA of bacteriophage PM2 they induce endonucleasesensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, fonnamidopyrimidines). Pyrimidinedimersand sites ofbase loss (AP sites) which were probed by UV endonuclease and exonuclease 111 are minor lesions in this system. While the alky1-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S. typhimurium strain TA I 00. DNA adducts formed with an intermediary alkyJating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane. We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter. since benzofuran epoxides are highly mutagenic in the S. typhimurium strain TAIOO and they form DNA adducts. as detected by the 212Ppostlabelling technique. Our results imply that the type of D NA darnage promoted by dioxetanes is dependent on the structural feature of dioxetanes. Furthermore, the direct photochemical DNA darnage by energy transfer. i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes. Instead, photooxidation dominates in isolated DNA. while radical darnage and alkylation prevail in the cellular system.
Spatially restricting cAMP production to discrete subcellular locations permits selective regulation of specific functional responses. But exactly where and how cAMP signaling is confined is not fully understood. Different receptors and adenylyl cyclase isoforms responsible for cAMP production are not uniformly distributed between lipid raft and non-lipid raft domains of the plasma membrane. We sought to determine the role that these membrane domains play in organizing cAMP responses in HEK293 cells. The freely diffusible FRET-based biosensor Epac2-camps was used to measure global cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. Disruption of lipid rafts by cholesterol depletion selectively altered cAMP responses produced by raft-associated receptors. The results indicate that receptors associated with lipid raft as well as non-lipid raft domains can contribute to global cAMP responses. In addition, basal cAMP activity was found to be significantly higher in non-raft domains. This was supported by the fact that pharmacologic inhibition of adenylyl cyclase activity reduced basal cAMP activity detected by Epac2-CAAX but not Epac2-MyrPalm or Epac2-camps. Responses detected by Epac2-CAAX were also more sensitive to direct stimulation of adenylyl cyclase activity, but less sensitive to inhibition of phosphodiesterase activity. Quantitative modeling was used to demonstrate that differences in adenylyl cyclase and phosphodiesterase activities are necessary but not sufficient to explain compartmentation of cAMP associated with different microdomains of the plasma membrane.
Fernale BALB/c mice were administered intragastrically with equimolar amounts of either [2-\(^{14}\)C]2-amino-3,8-dimethyi[ 4,5-J]qulnoxaline (MeiQx) or 2-acetylamino[9-\(^{14}\)C]fluorene (2AAF). DNA was isolated from tissues of mice killed either 6 or 24 h after administration. Analysis of liver DNA nucleotide digests by HPLC analysis revealed that all of the radioactivity was attributable to adduct formation. Tbe specific activities of DNA samples were converted to covalent bindlog indices (CBI, J.LIDOI adduct per mol DNA nucleotides/mmol chemical app6ed per kg animal body weight). CBI values of 25 and 9 were detennined for 2AAF and MeiQx in tbe llvers of mice killed 6 h after dosing. The values were in general agreement with the moderate carcinogenic potency of these compounds. The specific activities of DNA preparations obtained from the lddneys, spleens, stomachs, small intestines and large intestlnes of mice treated witb MeiQx and killed 6 h after doslng were S- to 35-times less tban those obtained witb the llver. DNA isolated from tbe lungs (a target organ for MeiQx tumorigenicity) of MeiQx-treated mice was not radiolabeUed at tbe limit of detection (CBI <0.3). With tbe exception of tbe gastrolntestinal tract, the specific activities of DNA samples isolated from mice killed 6 h after administration were higher than those from mice killed after 24 h.
Cyclic adenosine monophosphate (cAMP), the ubiquitous second messenger produced upon stimulation of GPCRs which couple to the stimulatory GS protein, orchestrates an array of physiological processes including cardiac function, neuronal plasticity, immune responses, cellular proliferation and apoptosis. By interacting with various effector proteins, among others protein kinase A (PKA) and exchange proteins directly activated by cAMP (Epac), it triggers signaling cascades for the cellular response. Although the functional outcomes of GSPCR-activation are very diverse depending on the extracellular stimulus, they are all mediated exclusively by this single second messenger. Thus, the question arises how specificity in such responses may be attained. A hypothesis to explain signaling specificity is that cellular signaling architecture, and thus precise operation of cAMP in space and time would appear to be essential to achieve signaling specificity. Compartments with elevated cAMP levels would allow specific signal relay from receptors to effectors within a micro- or nanometer range, setting the molecular basis for signaling specificity. Although the paradigm of signaling compartmentation gains continuous recognition and is thoroughly being investigated, the molecular composition of such compartments and how they are maintained remains to be elucidated. In addition, such compartments would require very restricted diffusion of cAMP, but all direct measurements have indicated that it can diffuse in cells almost freely.
In this work, we present the identification and characterize of a cAMP signaling compartment at a GSPCR. We created a Förster resonance energy transfer (FRET)-based receptor-sensor conjugate, allowing us to study cAMP dynamics in direct vicinity of the human glucagone-like peptide 1 receptor (hGLP1R). Additional targeting of analogous sensors to the plasma membrane and the cytosol enables assessment of cAMP dynamics in different subcellular regions. We compare both basal and stimulated cAMP levels and study cAMP crosstalk of different receptors. With the design of novel receptor nanorulers up to 60nm in length, which allow mapping cAMP levels in nanometer distance from the hGLP1R, we identify a cAMP nanodomain surrounding it. Further, we show that phosphodiesterases (PDEs), the only enzymes known to degrade cAMP, are decisive in constraining cAMP diffusion into the cytosol thereby maintaining a cAMP gradient. Following the discovery of this nanodomain, we sought to investigate whether downstream effectors such as PKA are present and active within the domain, additionally studying the role of A-kinase anchoring proteins (AKAPs) in targeting PKA to the receptor compartment. We demonstrate that GLP1-produced cAMP signals translate into local nanodomain-restricted PKA phosphorylation and determine that AKAP-tethering is essential for nanodomain PKA.
Taken together, our results provide evidence for the existence of a dynamic, receptor associated cAMP nanodomain and give prospect for which key proteins are likely to be involved in its formation. These conditions would allow cAMP to exert its function in a spatially and temporally restricted manner, setting the basis for a cell to achieve signaling specificity. Understanding the molecular mechanism of cAMP signaling would allow modulation and thus regulation of GPCR signaling, taking advantage of it for pharmacological treatment.
Lamivudine (3TC), a drug used in the treatment of HIV infection, needs to cross the plasma membrane to exert its therapeutic action. Human Organic cation transporter 1 (hOCT1), encoded by the SLC22A1 gene, is the transporter responsible for its uptake into target cells. As SLC22A1 is a highly polymorphic gene, the aim of this study was to determine how SNPs in the OCT1-encoding gene affected 3TC internalization and its interaction with other co-administered drugs. HEK293 cells stably transfected with either the wild type form or the polymorphic variants of hOCT1 were used to perform kinetic and drug-drug interaction studies. Protein co-immunoprecipitation was used to assess the impact of selected polymorphic cysteines on the oligomerization of the transporter. Results showed that 3TC transport efficiency was reduced in all polymorphic variants tested (R61C, C88R, S189L, M420del, and G465R). This was not caused by lack of oligomerization in case of variants located at the transporter extracellular loop (R61C and C88R). Drug-drug interaction measurements showed that co-administered drugs [abacavir (ABC), zidovudine (AZT), emtricitabine (FTC), tenofovir diproxil fumarate (TDF), efavirenz (EFV) and raltegravir (RAL)], differently inhibited 3TC uptake depending upon the polymorphic variant analyzed. These data highlight the need for accurate analysis of drug transporter polymorphic variants of clinical relevance, because polymorphisms can impact on substrate (3TC) translocation but even more importantly they can differentially affect drug-drug interactions at the transporter level.
Diabetes mellitus (DM) is one of the major current health problems due to lifestyle changes. Before diagnosis and in the early years of disease, insulin blood levels are elevated. However, insulin generates low levels of reactive oxygen species (ROS) which are integral to the regulation of a variety of intracellular signaling pathways, but excess levels of insulin may also lead to DNA oxidation and DNA damage. Three pharmaceutical compounds, resveratrol, lovastatin and the mTOR-inhibitor RAD-001, were investigated due to their known beneficial effects. They showed protective properties against genotoxic damage and significantly reduced ROS after in vitro treatment of cultured cells with insulin. Therefore, the selected pharmaceuticals may be attractive candidates to be considered for support of DM therapy.
Effect of inhalation exposure regimen on DNA binding potency of 1,2-dichloroethane in the rat
(1991)
1 ,2-Dichloroethane (DCE) was reported to be carcinogenic in rats in a long-tenn bioassay using gavage in com oil (24 and 48 mg/kg/day), but not by inhalation (up to 150-250 ppm, 7 h/day, 5 days/week). The daily dose metabolized was similar in the two experiments. In order to address this discrepancy, the genotoxicity of DCE was investigated in vivo under different exposure conditions. Fernale F-344 rats (183-188 g) were exposed to [1,2-14C]DCE in a closed inhalation chamber to either a low, constant concentration (0.3 mg/l = 80 ppm for 4 h) or to a peak concentration (up to 18 mg/1 = 4400 ppm) for a few minutes. After 12 h in the chamber, the dose metabolized under the two conditions was 34 mg/kg and 140 mg/k:g. DNA was isolated from liver and lung and was purified to constant specific radioactivity. DNA was enzymaticaBy hydrolyzed to the 3' -nucleotides which were separated by reverse phase HPLC. Most radioactivity eluted without detectable or with little optical density' indicating that the major part of the DNA radioactivity was due to covalent binding of the test compound. The Ievel of DNA adducts was expressed in the dose-nonnalized units ofthe Covalent Binding Index, CBI = f.Lmol adduct per mol DNA nucleotide/ mmol DCE per kg body wt. In liver DNA, the different exposure regimens resulted in markedly different CBI values of 1.8 and 69, for "constant-low" and ''peak" DCE exposure Ievels. In the Jung, the respective values were 0.9 and 31. It is concluded that the DNA darnage by DCE depends upon the concentration-time profile and that the carcinogenic potency determined in the gavage study should not be used for low-Ievel inhalation exposure.
Marine sponge-derived Streptomyces sp SBT343 extract inhibits staphylococcal biofilm formation
(2017)
Staphylococcus epidermidis and Staphylococcus aureus are opportunistic pathogens that cause nosocomial and chronic biofilm-associated infections. Indwelling medical devices and contact lenses are ideal ecological niches for formation of staphylococcal biofilms. Bacteria within biofilms are known to display reduced susceptibilities to antimicrobials and are protected from the host immune system. High rates of acquired antibiotic resistances in staphylococci and other biofilm-forming bacteria further hamper treatment options and highlight the need for new anti-biofilm strategies. Here, we aimed to evaluate the potential of marine sponge-derived actinomycetes in inhibiting biofilm formation of several strains of S. epidermidis, S. aureus, and Pseudomonas aeruginosa. Results from in vitro biofilm-formation assays, as well as scanning electron and confocal microscopy, revealed that an organic extract derived from the marine sponge-associated bacterium Streptomyces sp. SBT343 significantly inhibited staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces, without affecting bacterial growth. The extract also displayed similar antagonistic effects towards the biofilm formation of other S. epidermidis and S. aureus strains tested but had no inhibitory effects towards Pseudomonas biofilms. Interestingly the extract, at lower effective concentrations, did not exhibit cytotoxic effects on mouse fibroblast, macrophage and human corneal epithelial cell lines. Chemical analysis by High Resolution Fourier Transform Mass Spectrometry (HRMS) of the Streptomyces sp. SBT343 extract proportion revealed its chemical richness and complexity. Preliminary physico-chemical characterization of the extract highlighted the heat-stable and non-proteinaceous nature of the active component(s). The combined data suggest that the Streptomyces sp. SBT343 extract selectively inhibits staphylococcal biofilm formation without interfering with bacterial cell viability. Due to absence of cell toxicity, the extract might represent a good starting material to develop a future remedy to block staphylococcal biofilm formation on contact lenses and thereby to prevent intractable contact lens-mediated ocular infections.
The number of bariatric surgeries being performed worldwide has markedly risen. While the improvement in obesity-associated comorbidities after bariatric surgery is well-established, very little is known about its impact on cancer risk. The peripheral lymphocyte micronucleus test is a widely used method for the monitoring of chromosomal damage levels in vivo, and micronucleus frequency positively correlates with cancer risk. Therefore, the aim of this study was to compare the micronucleus frequency before and after bariatric surgery in obese subjects. Peripheral blood mononuclear cells were collected from 45 obese subjects before and at two time-points after bariatric surgery (6 and 12 months) to assess spontaneous micronucleus frequency. Consistent with the increased cancer risk previously shown, bariatric surgery-induced weight loss led to a significant reduction in lymphocyte micronucleus frequency after 12 months. Interestingly, comorbidities such as type 2 diabetes mellitus and metabolic syndrome further seemed to have an impact on the lymphocyte micronucleus frequency. Our findings may indicate a successful reduction of cancer risk in patients following weight loss caused by bariatric surgery.
The comet assay is widely used in basic research, genotoxicity testing, and human biomonitoring. However, interpretation of the comet assay data might benefit from a better understanding of the future fate of a cell with DNA damage. DNA damage is in principle repairable, or if extensive, can lead to cell death. Here, we have correlated the maximally induced DNA damage with three test substances in TK6 cells with the survival of the cells. For this, we selected hydrogen peroxide (H\(_{2}\)O\(_{2}\)) as an oxidizing agent, methyl methanesulfonate (MMS) as an alkylating agent and etoposide as a topoisomerase II inhibitor. We measured cell viability, cell proliferation, apoptosis, and micronucleus frequency on the following day, in the same cell culture, which had been analyzed in the comet assay. After treatment, a concentration dependent increase in DNA damage and in the percentage of non-vital and apoptotic cells was found for each substance. Values greater than 20-30% DNA in tail caused the death of more than 50% of the cells, with etoposide causing slightly more cell death than H\(_{2}\)O\(_{2}\) or MMS. Despite that, cells seemed to repair of at least some DNA damage within few hours after substance removal. Overall, the reduction of DNA damage over time is due to both DNA repair and death of heavily damaged cells. We recommend that in experiments with induction of DNA damage of more than 20% DNA in tail, survival data for the cells are provided.
The comet assay is a commonly used method to determine DNA damage and repair activity in many types of samples. In recent years, the use of the comet assay in human biomonitoring became highly attractive due to its various modified versions, which may be useful to determine individual susceptibility in blood samples. However, in human biomonitoring studies, working with large sample numbers that are acquired over an extended time period requires some additional considerations. One of the most important issues is the storage of samples and its effect on the outcome of the comet assay. Another important question is the suitability of different blood preparations. In this study, we analysed the effect of cryopreservation on DNA damage and repair activity in human blood samples. In addition, we investigated the suitability of different blood preparations. The alkaline and FPG as well as two different types of repair comet assay and an in vitro hydrogen peroxide challenge were applied. Our results confirmed that cryopreserved blood preparations are suitable for investigating DNA damage in the alkaline and FPG comet assay in whole blood, buffy coat and PBMCs. Ex vivo hydrogen peroxide challenge yielded its optimal effect in isolated PBMCs. The utilised repair comet assay with either UVC or hydrogen peroxide-induced lesions and an aphidicolin block worked well in fresh PBMCs. Cryopreserved PBMCs could not be used immediately after thawing. However, a 16-h recovery with or without mitotic stimulation enabled the application of the repair comet assay, albeit only in a surviving cell fraction.
Role of PTEN in Oxidative Stress and DNA Damage in the Liver of Whole-Body Pten Haplodeficient Mice
(2016)
Type 2 diabetes (T2DM) and obesity are frequently associated with non-alcoholic fatty liver disease (NAFLD) and with an elevated cancer incidence. The molecular mechanisms of carcinogenesis in this context are only partially understood. High blood insulin levels are typical in early T2DM and excessive insulin can cause elevated reactive oxygen species (ROS) production and genomic instability. ROS are important for various cellular functions in signaling and host defense. However, elevated ROS formation is thought to be involved in cancer induction. In the molecular events from insulin receptor binding to genomic damage, some signaling steps have been identified, pointing at the PI3K/AKT pathway. For further elucidation Phosphatase and Tensin homolog (Pten), a tumour suppressor phosphatase that plays a role in insulin signaling by negative regulation of PI3K/AKT and its downstream targets, was investigated here. Dihydroethidium (DHE) staining was used to detect ROS formation in immortalized human hepatocytes. Comet assay and micronucleus test were performed to investigate genomic damage in vitro. In liver samples, DHE staining and western blot detection of HSP70 and HO-1 were performed to evaluate oxidative stress response. DNA double strand breaks (DSBs) were detected by immunohistostaining. Inhibition of PTEN with the pharmacologic inhibitor VO-OHpic resulted in increased ROS production and genomic damage in a liver cell line. Knockdown of Pten in a mouse model yielded increased oxidative stress levels, detected by ROS levels and expression of the two stress-proteins HSP70 and HO-1 and elevated genomic damage in the liver, which was significant in mice fed with a high fat diet. We conclude that PTEN is involved in oxidative stress and genomic damage induction in vitro and that this may also explain the in vivo observations. This further supports the hypothesis that the PI3K/AKT pathway is responsible for damaging effects of high levels of insulin.
The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.
In the heart the β\(_1\)-adrenergic receptor (AR) and the β\(_2\)-AR, two prototypical G protein-coupled receptors (GPCRs), are both activated by the same hormones, namely adrenaline and noradrenaline. Both receptors couple to stimulatory G\(_s\) proteins, mediate an increase in cyclic adenosine monophosphate (cAMP) and influence the contractility and frequency of the heart upon stimulation. However, activation of the β\(_1\)-AR, not the β\(_2\)-AR, lead to other additional effects, such as changes in gene transcription resulting in cardiac hypertrophy, leading to speculations on how distinct effects can arise from receptors coupled to the same downstream signaling pathway.
In this thesis the question of whether this distinct behavior may originate from a differential localization of these two receptors in adult cardiomyocytes is addressed. Therefore, fluorescence spectroscopy tools are developed and implemented in order to elucidate the presence and dynamics of these endogenous receptors at the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. This allows the visualization of confined localization and diffusion of the β\(_2\)-AR to the T-tubular network at endogenous expression. In contrast, the β\(_1\)-AR is found diffusing at both the outer plasma membrane and the T-tubules. Upon overexpression of the β\(_2\)-AR in adult transgenic cardiomyocytes, the receptors experience a loss of this compartmentalization and are also found at the cell surface. These data suggest that distinct signaling and functional effects can be controlled by specific cell surface targeting of the receptor subtypes.
The tools at the basis of this thesis work are a fluorescent adrenergic antagonist in combination of fluorescence fluctuation spectroscopy to monitor the localization and dynamics of the lowly expressed adrenergic receptors. Along the way to optimizing these approaches, I worked on combining widefield and confocal imaging in one setup, as well as implementing a stable autofocus mechanism using electrically tunable lenses.
Prerequisite to any biological laboratory assay employing living animals is consideration about its necessity, feasibility, ethics and the potential harm caused during an experiment. The imperative of these thoughts has led to the formulation of the 3R-principle, which today is a pivotal scientific standard of animal experimentation worldwide. The rising amount of laboratory investigations utilizing living animals throughout the last decades, either for regulatory concerns or for basic science, demands the development of alternative methods in accordance with 3R to help reduce experiments in mammals. This demand has resulted in investigation of additional vertebrate species displaying favourable biological properties. One prominent species among these is the zebrafish (Danio rerio), as these small laboratory ray-finned fish are well established in science today and feature outstanding biological characteristics. In this review, we highlight the advantages and general prerequisites of zebrafish embryos and larvae before free-feeding stages for toxicological testing, with a particular focus on cardio-, neuro, hepato- and nephrotoxicity. Furthermore, we discuss toxicokinetics, current advances in utilizing zebrafish for organ toxicity testing and highlight how advanced laboratory methods (such as automation, advanced imaging and genetic techniques) can refine future toxicological studies in this species.
The biotransformation of 1,1,1,3,3-pentafluoropropane was investigated in rats and in in vitro systems. First, the metabolites were identified in vivo using GC/MS and 19F NMR analysis. The main metabolite was identified as trifluoroacetic acid, the minor metabolite as 3,3,3-trifluoropropionic acid and as a cleavage product, inorganic fluoride was found. As the in vitro system, liver microsomes from rat and human samples and rat liver homogenates were used. Trifluoroacetic acid and 3,3,3 trifluoropropionic acid were confirmed in vitro as metabolic intermediates, following biotransformation of 1,1,1,3,3-pentafluoropropane by the cytochrome P-450-system. Studies, designed for clarifying the cardiotoxicity of 1,1,1,3,3-pentafluoropropane were driven by the hypothesis that 3,3,3-trifluoropropionic acid is the toxic agent. This was based on the lethal toxicity, which was observed in previous in vivo experiments. In addition, the point of its structural similarity to toxic agents as for example monofluoroacetic acid or of possible metabolic intermediates like difluoroacrylic acid with known toxicity were considered to support this assumption. However, trifluoroacetic acid was neglected as the sought-after toxic agent because of its different toxic effects, known from literature. Investigations on the biotransformation of 3,3,3-trifluoropropionic acid were performed and resulted in no metabolic activity and in poor elimination of 3,3,3-trifluoropropionic acid in vivo. The histopathological effects on the heart, which were observed in the 90-day oral toxicity study of 1,1,1,3,3-pentafluoropropane in rats, namely mononuclear inflammatory cell infiltrations and degenerated myocardial fibers, were not observed after a 28 day repeated exposure of up to 10 mg/kg b.w. of 3,3,3-trifluoropropionic acid. However, a single high dose of 3,3,3-trifluoropropionic acid lead to severe toxicological effects. The difference in the observed toxic effects after a single and repeated administration may be due to adaptive mechanisms in rats. The toxicological effects included clinical signs like ataxia, coma and cramps. The conditions of the rats suggested possible inhibition of the energy supply to the organism. Furthermore, the interference of 3,3,3-trifluoropropionic acid in the functionality of the organism was investigated. Experiments were performed in vitro in rat liver and heart mitochondria to investigate effects on the mitochondrial ß-oxidation. However, the transformation of the substrate [U14C] palmitic acid in the ß oxidation pathway was not inhibited by 3,3,3-trifluoropropionic acid. In addition, no cytotoxicity of 3,3,3 trifluoropropionic acid was observed in the cell culture systems. The main effect after a single dose of 3,3,3-trifluoropropionic acid was seen in clinical pathology and metabonomic analysis. The decrease in blood glucose is considered to have the most far-reaching consequences for the toxicity of 3,3,3-trifluoropropionic acid. If considering this change as the primary effect after a single dose, secondary effects, for example, the above-mentioned clinical signs could be explained. In addition, the observed high level of ketone bodies might have been responsible for life-threatening possible ketoacidosis. In general, ketoacidosis occurs after an imbalance between glycolysis, lipolysis, TCA cycle activity and respiratory function. Based on the results, ß-oxidation of fatty acids was not affected, and due to the decrease in glucose levels and the high levels of acetyl CoA, glycolysis was considered not to be impaired. Increased amounts of acetyl CoA might be a result of insufficient activity of the TCA cycle. However, the inhibition of the TCA cycle can be based on the impairment of specific enzymes and/or on the involvement of messenger substrates like insulin. Supporting the first mentioned aspect are decreased levels of TCA cycle intermediates, like α-ketoglutarate or citrate, as seen in 1H-NMR spectra of urine. However, the second aspect would explain the drop in blood glucose with the impairment of glucose transporters or the impairment of the insulin balance. If a single dose of 3,3,3-trifluoropropionic acid had stimulated the insulin release, glycolysis would be activated, and high amounts of acetyl CoA would be produced. In case of impaired use by the TCA cycle, levels of ketone bodies would be increased. Experiments were designed to characterize the direct effect of 3,3,3-trifluoropropionic acid on rat insulinoma-derived INS-1 cells as possible increase in insulin release. Further investigations are necessary to answer in which step of the metabolic pathway 3,3,3-trifluoropropionic acid interferes or finally which specific enzyme is inhibited or activated by 3,3,3-trifluoropropionic acid, leading to the drop in blood glucose and finally in lethal toxicity.
Kinetic assessment by in vitro approaches - A contribution to reduce animals in toxicity testing
(2015)
The adoption of directives and regulations by the EU requires the development of alternative testing strategies as opposed to animal testing for risk assessment of xenobiotics. Additionally, high attrition rates of drugs late in the discovery phase demand improvement of current test batteries applied in the preclinical phase within the pharmaceutical area. These issues were taken up by the EU founded 7th Framework Program “Predict-IV”; with the overall goal to improve the predictability of safety of an investigational product, after repeated exposure, by integration of “omics” technologies applied on well established in vitro approaches. Three major target organs for drug-induced toxicity were in focus: liver, kidney and central nervous system. To relate obtained dynamic data with the in vivo situation, kinetics of the test compounds have to be evaluated and extrapolated by physiologically based pharmacokinetic modeling.
This thesis assessed in vitro kinetics of the selected test compounds (cyclosporine A, adefovir dipivoxil and cisplatinum) regarding their reliability and relevance to respective in vivo pharmacokinetics. Cells were exposed daily or every other day to the test compounds at two concentration levels (toxic and non-toxic) for up to 14 days. Concentrations of the test compounds or their major biotransformation products were determined by LC-MS/MS or ICP-MS in vehicle, media, cells and plastic adsorption samples generated at five different time-points on the first and the last treatment day.
Cyclosporine A bioaccumulation was evident in primary rat hepatocytes (PRH) at the high concentration, while efficient biotransformation mediated by CYP3A4 and CYP3A5 was determined in primary human hepatocytes (PHH) and HepaRG cells. The lower biotransformation in PRH is in accordance with observation made in vivo with the rat being a poor model for CYP3A biotransformation. Further, inter-assay variability was noticed in PHH caused by biological variability in CYP3A4 and CYP3A5 activity in human donors. The inter-assay variability observed for PRH and HepaRG cells was a result of differences between vehicles regarding their cyclosporine A content. Cyclosporine A biotransformation was more prominent in HepaRG cells due to stable and high CYP3A4 and CYP3A5 activity. In addition, in vitro clearances were calculated and scaled to in vivo. All scaled in vitro clearances were overestimated (PRH: 10-fold, PHH: 2-fold, HepaRG cells: 2-fold). These results should be proven by physiologically-based pharmacokinetic modeling and additional experiments, in order to verify that these overestimations are constant for each system and subsequently can be diminished by implementation of further scaling factors.
Brain cell cultures, primary neuronal culture of mouse cortex cells and primary aggregating rat brain cells, revealed fast achieved steady state levels of cyclosporine A. This indicates a chemical distribution of cyclosporine A between the aqueous and organic phases and only minor involvement of biological processes such as active transport and biotransformation. Hence, cyclosporine A uptake into cells is presumably transport mediated, supported by findings of transporter experiments performed on a parallel artificial membrane and Caco-2 cells. Plastic adsorption of cyclosporine A was significant, but different for each model, and should be considered by physiologically based pharmacokinetic modeling.
Kinetics of adefovir dipivoxil highlights the limits of in vitro approaches. Active transporters are required for adefovir uptake, but were not functional in RPTECT/TERT1. Therefore, adefovir uptake was limited to passive diffusion of adefovir dipivoxil, which itself degrades time-dependently under culture conditions.
Cisplatinum kinetics, studied in RPTEC/TERT1 cells, indicated intracellular enrichment of platinum, while significant bioaccumulation was not noted. This could be due to cisplatinum not reaching steady state levels within 14 days repeated exposure. As shown in vivo, active transport occurred from the basolateral to apical side, but with lower velocity. Hence, obtained data need to be modeled to estimate cellular processes, which can be scaled and compared to in vivo.
Repeated daily exposure to two different drug concentrations makes it possible to account for bioaccumulation at toxic concentrations or biotransformation/extrusion at non-toxic concentrations. Potential errors leading to misinterpretation of data were reduced by analyses of the vehicles as the applied drug concentrations do not necessarily correspond to the nominal concentrations. Finally, analyses of separate compartments (medium, cells, plastic) give insights into a compound’s distribution, reduce misprediction of cellular processes, e.g. biotransformation, and help to interpret kinetic data. On the other hand, the limits of in vitro approaches have also been pointed out. For correct extrapolation to in vivo, it is essential that the studied in vitro system exhibits the functionality of proteins, which play a key role in the specific drug induced toxicity. Considering the benefits and limitations, it is worth to validate this long-term treatment experimental set-up and expand it on co-culture systems and on organs-on-chips with regard to alternative toxicity testing strategies for repeated dose toxicity studies.