Fenton process on single and mixture components of phenothiazine pharmaceuticals: Assessment of intermediaries, fate, and preliminary ecotoxicity.

Pharmaceuticals do not occur isolated in the environment but in multi-component mixtures and may exhibit antagonist, synergistic or additive behavior. Knowledge on this is still scarce. The situation is even more complicated if effluents or potable water is treated by oxidative processes or such transformations occur in the environment. Thus, determining the fate and effects of parent compounds, metabolites and transformation products (TPs) formed by transformation and degradation processes in the environment is needed. This study investigated the fate and preliminary ecotoxicity of the phenothiazine pharmaceuticals, Promazine (PRO), Promethazine (PRM), Chlorpromazine (CPR), and Thioridazine (THI) as single and as components of the resulting mixtures obtained from their treatment by Fenton process. The Fenton process was carried out at pH7 and by using 0.5-2mgL-1 of [Fe2+]0 and 1-12.5mgL-1 of [H2O2]0 at the fixed ratio [Fe2+]0:[H2O2]0 of 1:10 (w:w). No complete mineralization was achieved. Constitutional isomers and some metabolite-like TPs formed were suggested based on their UHPLC-HRMSn data. A degradation pathway was proposed considering interconnected mechanisms such as sulfoxidation, hydroxylation, N-dealkylation, and dechlorination steps. Aerobic biodegradation tests (OECD 301 D and OECD 301 F) were applied to the parent compounds separately, to the mixture of parent compounds, and for the cocktail of TPs present after the treatment by Fenton process. The samples were not readily biodegradable. However, LC-MS analysis revealed that abiotic transformations, such hydrolysis, and autocatalytic transformations occurred. The initial ecotoxicity tested towards Vibrio fischeri as individual compounds featured a reduction in toxicity of PRM and CPR by the treatment process, whereas PRO showed an increase in acute luminescence inhibition and THI a stable luminescence inhibition. Concerning effects of the mixture components, reduction in toxicity by the Fenton process was predicted by concentration addition and independent action models.

Hazard screening of photo-transformation products from pharmaceuticals: Application to selective ß1-blockers atenolol and metoprolol.

The identification of toxic components in cocktail mixtures of pollutants, their metabolites and transformation products (TPs) generated from environmental and treatment processes remains an arduous task. This study expanded in this area by applying a combination of chemical analytics, a battery of in vitro bioassays and an in silico "testing battery" to UV photolysis mixtures of active pharmaceutical ingredients. The objectives were to understand the toxic nature of the mixtures and to prioritize photo-TPs for risk analysis. The selective ß1-blockers Atenolol (ATL) and Metoprolol (MTL) that are ubiquitous in the aquatic environment were used as an example. The photolysis mixtures were cytotoxic to Vibrio fischeri and mammalian cells but not mutagenic in the Ames test or genotoxic in the in vitro micronucleus and umu tests. Potentially hazardous TPs were proposed by relating the observed effects to the kinetics of TP occurrence and applying in silico toxicity predictions for individual photo-TPs. This model study was done to identify principal mechanisms rather than accurately simulating environmental transformation processes. Several photo-TPs were proposed to present a greater hazard than the selected ß-blockers and therefore fate and toxicity assessments may be required to determine their environmental relevance.

Transformation products in the water cycle and the unsolved problem of their proactive assessment: A combined in vitro/in silico approach.

Transformation products (TPs) emerging from incomplete degradation of micropollutants in aquatic systems can retain the biological activity of the parent compound, or may even possess new unexpected toxic properties. The chemical identities of these substances remain largely unknown, and consequently, the risks caused by their presence in the water cycle cannot be assessed thoroughly. In this study, a combined approach for the proactive identification of hazardous elements in the chemical structures of TPs, comprising analytical, bioanalytical and computational methods, was assessed by the example of the pharmaceutically active micropollutant propranolol (PPL). PPL was photo-transformed using ultraviolet (UV) irradiation and 115 newly formed TPs were monitored in the reaction mixtures by LC-MS analysis. The reaction mixtures were screened for emerging effects using a battery of in vitro bioassays and the occurrence of cytotoxic and mutagenic activities in bacteria was found to be significantly correlated with the occurrence of specific TPs during the treatment process. The follow-up analysis of structure-activity-relationships further illustrated that only small chemical transformations, such as the hydroxylation or the oxidative opening of an aromatic ring system, could substantially alter the biological effects of micropollutants in aquatic systems. In conclusion, more efforts should be made to prevent the occurrence and transformation of micropollutants in the water cycle and to identify the principal degradation pathways leading to their toxicological activation. With regard to the latter, the judicious combination of bioanalytical and computational tools represents an appealing approach that should be developed further.

Maternal transfer of emerging brominated and chlorinated flame retardants in European eels.

The European eel (Anguilla anguilla) is regarded as a critically endangered species. Scientists are in agreement that the "quality of spawners" is a vital factor for the survival of the species. This quality can be impaired by parasites, disease and pollution. Especially endocrine disrupting organic chemicals pose a potential threat to reproduction and development of offspring. To our knowledge, the findings in this publication for the first time describe maternal transfer of contaminants in eels. We analysed the concentrations of in total 53 polybrominated diphenyl ethers (PBDEs) and their halogenated substitutes in muscle, gonads and eggs of artificially matured European eels and in muscle and gonads of untreated European eels that were used for comparison. We found evidence that persistent organic pollutants such as PBDEs, as well as their brominated and chlorinated substitutes are redistributed from muscle tissue to gonads and eggs. Concentrations ranged from 0.001 ng g(-1)ww for sum Dechlorane metabolites (DPMA, aCL10DP, aCl11DP) to 2.1 ng g(-1)ww for TBA in eggs, 0.001 ng g(-1)ww for Dechlorane metabolites to 9.4 ng g(-1)ww for TBA in gonads and 0.002 ng g(-1)ww for Dechlorane metabolites to 54 ng g(-1)ww for TBA in muscle tissue. Average egg muscle ratios (EMRs) for compounds detectable in artificially matured eels from both Schlei Fjord and Ems River ranged from 0.01 for Dechlorane 602 (DDC-DBF) to 10.4 for PBEB. Strong correlations were found between flame retardant concentrations and lipid content in the analysed tissue types, as well as transfer rates and octanol-water partitioning coefficient, indicating that these parameters were the driving factors for the observed maternal transfer. Furthermore, indications were found, that TBP-DBPE, TBP-AE, BATE and TBA have a significant uptake from the surrounding water, rather than just food and might additionally be formed by metabolism or biotransformation processes. Dechloranes seem to be of increasing relevance as contaminants in eels and are transferred to eggs. A change of the isomer pattern in comparison to the technical product of Dechlorane Plus (DP) was observed indicating a redistribution of DP from muscle tissue to gonads during silvering with a preference of the syn-isomer. The highly bioaccumulative DDC-DBF was the most abundant Dechlorane in all fish of the comparison group even though it is not produced or imported in the EU. The aldrin related "experimental flame retardant" dibromoaldrin (DBALD) was detected for the first time in the environment in similar or higher concentrations than DP.

Environmental risk assessment of anti-cancer drugs and their transformation products: A focus on their genotoxicity characterization-state of knowledge and short comings.

Anti-cancer drugs are chemotherapeutic agents that are designed to kill or reduce proliferating cells. Often times, they interfere directly or indirectly with the cell's deoxyribonucleic acid (DNA). Some of these drugs can be detected in the ng/L concentration range in the aquatic environment and have the potential to be very persistent. Environmental risk assessment is available for only a few anti-cancer drugs, derived mainly from predicted data and excluding information on their metabolites and transformation products (TPs). Notably, there is no defined strategy for genotoxicity risk assessment of anti-cancer drugs, their metabolites and TPs in the environment. In fact, the presence of anti-cancer drugs in hospital and municipal wastewaters has not been clearly related to the genotoxic nature of these wastewaters. The few available studies that have sought to investigate the genotoxicity of mixtures derived from treating anti-cancer drugs prior to disposal seem to share the commonality of coupling analytical methods to measure concentration and genotoxic bioassays, namely the Ames test to monitor inactivation. Such limited studies on the environmental fate and effects of these drugs presents an area for further research work. Most importantly, there is a need to characterize the genotoxic effects of anti-cancer drugs towards aquatic organisms. Given current environmental risk assessment strategies, genotoxicity risk assessment of these drugs and their TPs would have to include a combination of appropriate analytical methods, genotoxicity bioassays, (bio) degradability and computer based prediction methods such as QSAR studies.

Identification of phototransformation products of thalidomide and mixture toxicity assessment: an experimental and quantitative structural activity relationships (QSAR) approach.

The fate of thalidomide (TD) was investigated after irradiation with a medium-pressure Hg-lamp. The primary elimination of TD was monitored and structures of phototransformation products (PTPs) were assessed by LC-UV-FL-MS/MS. Environmentally relevant properties of TD and its PTPs as well as hydrolysis products (HTPs) were predicted using in silico QSAR models. Mutagenicity of TD and its PTPs was investigated in the Ames microplate format (MPF) aqua assay (Xenometrix, AG). Furthermore, a modified luminescent bacteria test (kinetic luminescent bacteria test (kinetic LBT)), using the luminescent bacteria species Vibrio fischeri, was applied for the initial screening of environmental toxicity. Additionally, toxicity of phthalimide, one of the identified PTPs, was investigated separately in the kinetic LBT. The UV irradiation eliminated TD itself without complete mineralization and led to the formation of several PTPs. TD and its PTPs did not exhibit mutagenic response in the Salmonella typhimurium strains TA 98, and TA 100 with and without metabolic activation. In contrast, QSAR analysis of PTPs and HTPs provided evidence for mutagenicity, genotoxicity and carcinogenicity using additional endpoints in silico software. QSAR analysis of different ecotoxicological endpoints, such as acute toxicity towards V. fischeri, provided positive alerts for several identified PTPs and HTPs. This was partially confirmed by the results of the kinetic LBT, in which a steady increase of acute and chronic toxicity during the UV-treatment procedure was observed for the photolytic mixtures at the highest tested concentration. Moreover, the number of PTPs within the reaction mixture that might be responsible for the toxification of TD during UV-treatment was successfully narrowed down by correlating the formation kinetics of PTPs with QSAR predictions and experimental toxicity data. Beyond that, further analysis of the commercially available PTP phthalimide indicated that transformation of TD into phthalimide was not the cause for the toxification of TD during UV-treatment. These results provide a path for toxicological assessment of complex chemical mixtures and in detail show the toxic potential of TD and its PTPs as well as its HTPs. This deserves further attention as UV irradiation might not always be a green technology, because it might pose a toxicological risk for the environment in general and specifically for water compartments.

Physicochemical properties and biodegradability of organically functionalized colloidal silica particles in aqueous environment.

Engineered sub-micron particles are being used in many technical applications, leading to an increasing introduction into the aquatic environment. Only a few studies have dealt with the biodegradability of non-functionalized organic particles. In fact the knowledge of organically surface functionalized colloids is nearly non-existent. We have investigated the biodegradability of organically surface functionalized silica (SiO2) particles bearing technically relevant groups such as amino-, carboxyl-, benzyl-, sulfonate-, chloro-, and phosphatoethyl-derivatized alkyls. Essential physicochemical properties including zeta potential, isoelectric point, morphology, surface area, porosity, surface density, and elemental composition of the particles were investigated, followed by biodegradability testing using the Closed Bottle Test (OECD 301D). None of the particles met the biodegradability threshold value of 60%. Only a slight biodegradation was revealed for SiO2-Benzyl (13.7±6.7%) and for SiO2-3-Chlorpropane (10.8±1.5%). For the other particles biodegradability was below the normal background fluctuation of 5%. The results were different of those obtained from structurally similar chemicals not being functionalized on the particle surface and from general rules of structure-biodegradation prediction of organic molecules. Therefore, our results suggest that the attachment of the organic groups heavily reduces their biodegradability, increases their residence time and possibility for adverse effects to environmental species.

Modeling hairy root tissue growth in in vitro environments using an agent-based, structured growth model.

An agent-based model for simulating the in vitro growth of Beta vulgaris hairy root cultures is described. The model fitting is based on experimental results and can be used as a virtual experimentator for root networks. It is implemented in the JAVA language and is designed to be easily modified to describe the growth of diverse biological root networks. The basic principles of the model are outlined, with descriptions of all of the relevant algorithms using the ODD protocol, and a case study is presented in which it is used to simulate the development of hairy root cultures of beetroot (Beta vulgaris) in a Petri dish. The model can predict various properties of the developing network, including the total root length, branching point distribution, segment distribution and secondary metabolite accumulation. It thus provides valuable information that can be used when optimizing cultivation parameters (e.g., medium composition) and the cultivation environment (e.g., the cultivation temperature) as well as how constructional parameters change the morphology of the root network. An image recognition solution was used to acquire experimental data that were used when fitting the model and to evaluate the agreement between the simulated results and practical experiments. Overall, the case study simulation closely reproduced experimental results for the cultures grown under equivalent conditions to those assumed in the simulation. A 3D-visualization solution was created to display the simulated results relating to the state of the root network and its environment (e.g., oxygen and nutrient levels).

Detection of up to 65% of Precancerous Lesions of the Human Colon and Rectum by Mutation Analysis of APC, K-Ras, B-Raf and CTNNB1.

In the present study a recently conceived 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signaling pathways was used to analyze 20 colorectal serrated lesions and 41 colorectal adenoma samples and to determine the percentage of each of the above-mentioned potentially precancerous lesions carrying at least one of the four above-mentioned genes in a mutated form. CTNNB1 and B-Raf were screened by PCR-single-strand conformation polymorphism analysis, K-Ras by restriction fragment length polymorphism analysis and the APC gene mutation cluster region (codons 1243-1567) by direct DNA sequencing. APC mutations were only detected in 10% of the serrated lesions but in 34% of the adenomas. Twenty percent of the serrated lesions and 14% of the adenomas carried a mutated K-Ras. B-Raf was found to be mutated in 50% of the serrated lesions and in 22% of the adenomas. CTNNB1 was altered in 12% of the adenomas, but not in serrated lesions. By using the above gene marker panel it could be shown that 65% of the serrated lesions and 61% of the adenomas carried at least one of the four genes in a mutated form. Based on its excellent performance in detecting mutations in sporadic preneoplastic (in this study) and neoplastic lesions (in a previous study) of the human colon and rectum, this primer combination might also be suited to efficiently and non-invasively detect genetic alterations in stool DNA of patients with early colorectal cancer.

A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in 80% of early (UICC I) colon cancer stages in humans.

BACKGROUND: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. METHODS: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243-1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. RESULTS: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. CONCLUSIONS: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80-90% of human sporadic CRC samples analyzed.