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.

Initial hazard screening for genotoxicity of photo-transformation products of ciprofloxacin by applying a combination of experimental and in-silico testing.

Ciprofloxacin (CIP) is a broad-spectrum antibiotic found within µg/L concentration range in the aquatic environment. It is a known contributor of umuC induction in hospital wastewater samples. CIP can undergo photolysis to result in many transformation products (TPs) of mostly unknown toxicity. The aims of this study were to determine the genotoxicity of the UV mixtures and to understand the possible genotoxic role of the stable TPs. As such, CIP and its UV-irradiated mixtures were investigated in a battery of genotoxicity and cytotoxicity in vitro assays. The combination index (CI) analysis of residual CIP in the irradiated mixtures was performed for the umu assay. Further, Quantitative Structure-Activity Relationships (QSARs) predicted selected genotoxicity endpoints of the identified TPs. CIP achieved primary elimination after 128 min of irradiation but was not completely mineralized. Nine photo-TPs were identified. The irradiated mixtures were neither mutagenic in the Ames test nor genotoxic in the in vitro micronucleus (MN) test. Like CIP, the irradiated mixtures were umuC inducing. The CI analysis revealed that the irradiated mixtures and the corresponding CIP concentration in the mixtures shared similar umuC potentials. QSAR predictions suggested that the TPs may be capable of inducing chromosome aberration, MN in vivo, bacterial mutation and mammalian mutation. However, the experimental testing for a few genotoxic endpoints did not show significant genotoxic activity for the TPs present as a component of the whole mixture analysis and therefore, further genotoxic endpoints may need to be investigated to fully confirm this.

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.

Modification of the umu-assay (ISO 13829) accounting for cytotoxicity in genotoxicity assessment: a preliminary study.

In this study, the umu-assay (ISO 13829) was modified by addition of the resazurin-reduction assay to assess the cytotoxic potential of toxins. The aim was to develop a test system that was capable of examining both genotoxicity and cytotoxicity on the basis of the metabolic health of the cells so as to provide a better assessment of the negative influence of toxic effects on the evaluation of genotoxicity. The test was established and validated with mitomycin C (MMC), 1,3-dinitropyrene (1,3-DNP), 4-nitroquinoline-1-oxide (4-NQO) and chloramphenicol (CHL) as toxins with known responses in the umu-assay. The results indicate that the modified umu-assay was able to reveal genotoxic responses towards MMC, 1,3-DNP and 4-NQO up to 1µg/ml. Further, the assay was able to determine the cytotoxicity of CHL, MMC and 4-NQO. Hence, the modified umu-assay was proven to be a sensitive test for determining the cytotoxicity and genotoxicity of toxins, with results that were comparable with literature data. Moreover, the modified umu-assay showed that cytotoxic concentrations that cause >50% inhibition of dehydrogenase activity (DHA) can result in unreliable computation of genotoxicity.