Development and Application of a Microplate Assay for Toxicity Testing on Aquatic Cyanobacteria.

Regulatory environmental risk assessment, applied to establish a protection limit for all bacterial diversity in surface waters, relies on a growth inhibition test performed on a single species of cyanobacteria and the activated sludge respiration inhibition test. Recently, the ability of this approach to protect adequately for bacteria that provide important ecosystem services has been questioned, and empirical data on additional species to further investigate the effectiveness of the environmental risk assessment are urgently required. We present the development and validation of a cost-effective and time-efficient microplate assay that is comparable to the traditional shake flask test for measurement of cyanobacteria growth rate after chemical exposure. The assay has been optimized to ensure that comparisons of cyanobacteria sensitivity under exponential growth are assessed across equivalent experimental conditions using phycocyanin fluorescence as a surrogate for cell density. The test system is validated using potassium dichromate, and the results are compared with those obtained in an Organisation for Economic Co-operation and Development (2011) test guideline 201 shake flask test system. This assay is suitable for the screening of new and legacy chemicals (including antibiotics) for which ecotoxicology data are lacking across a wide range of cyanobacteria, with the aim of developing more comprehensive environmental risk assessment. Environ Toxicol Chem 2020;39:705-720. © 2019 SETAC.

Vision of a near future: Bridging the human health-environment divide. Toward an integrated strategy to understand mechanisms across species for chemical safety assessment.

There is a growing recognition that application of mechanistic approaches to understand cross-species shared molecular targets and pathway conservation in the context of hazard characterization, provide significant opportunities in risk assessment (RA) for both human health and environmental safety. Specifically, it has been recognized that a more comprehensive and reliable understanding of similarities and differences in biological pathways across a variety of species will better enable cross-species extrapolation of potential adverse toxicological effects. Ultimately, this would also advance the generation and use of mechanistic data for both human health and environmental RA. A workshop brought together representatives from industry, academia and government to discuss how to improve the use of existing data, and to generate new NAMs data to derive better mechanistic understanding between humans and environmentally-relevant species, ultimately resulting in holistic chemical safety decisions. Thanks to a thorough dialogue among all participants, key challenges, current gaps and research needs were identified, and potential solutions proposed. This discussion highlighted the common objective to progress toward more predictive, mechanistically based, data-driven and animal-free chemical safety assessments. Overall, the participants recognized that there is no single approach which would provide all the answers for bridging the gap between mechanism-based human health and environmental RA, but acknowledged we now have the incentive, tools and data availability to address this concept, maximizing the potential for improvements in both human health and environmental RA.

Variability in cyanobacteria sensitivity to antibiotics and implications for environmental risk assessment.

Once released into the environment antibiotics can kill or inhibit the growth of bacteria, and in turn potentially have effects on bacterial community structure and ecosystem function. Environmental risk assessment (ERA) seeks to establish protection limits to minimise chemical impacts on the environment, but recent evidence suggests that the current regulatory approaches for ERA for antibiotics may not be adequate for protecting bacteria that have fundamental roles in ecosystem function. In this study we assess the differences in interspecies sensitivity of eight species of cyanobacteria to seven antibiotics (cefazolin, cefotaxime, ampicillin, sufamethazine, sulfadiazine, azithromycin and erythromycin) with three different modes of action. We found that variability in the sensitivity to these antibiotics between species was dependent on the mode of action and varied by up to 70 times for ß-lactams. Probabilistic analysis using species sensitivity distributions suggest that the current predicted no effect concentration PNEC for the antibiotics may be either over or under protective of cyanobacteria dependent on the species on which it is based and the mode of action of the antibiotic; the PNECs derived for the macrolide antibiotics were over protective but PNECs for ß-lactams were generally under protective. For some geographical locations we identify a significant risk to cyanobacteria populations based upon measured environmental concentrations of selected antibiotics. We conclude that protection limits, as determined according to current regulatory guidance, may not always be protective and might be better derived using SSDs and that including toxicity data for a wider range of (cyano-) bacteria would improve confidence for the ERA of antibiotics.

Pharmacology beyond the patient - The environmental risks of human drugs.

BACKGROUND: The presence of pharmaceuticals in the environment is a growing global concern and although environmental risk assessment is required for approval of new drugs in Europe and the USA, the adequacy of the current triggers and the effects-based assessments has been questioned. OBJECTIVE: To provide a comprehensive analysis of all regulatory compliant aquatic ecotoxicity data and evaluate the current triggers and effects-based environmental assessments to facilitate the development of more efficient approaches for pharmaceuticals toxicity testing. METHODS: Publicly-available regulatory compliant ecotoxicity data for drugs targeting human proteins was compiled together with pharmacological information including drug targets, Cmax and lipophilicity. Possible links between these factors and the ecotoxicity data for effects on, growth, mortality and/or reproduction, were evaluated. The environmental risks were then assessed based on a combined analysis of drug toxicity and predicted environmental concentrations based on European patient consumption data. RESULTS: For most (88%) of the of 975 approved small molecule drugs targeting human proteins a complete set of regulatory compliant ecotoxicity data in the public domain was lacking, highlighting the need for both intelligent approaches to prioritize legacy human drugs for a tailored environmental risk assessment and a transparent database that captures environmental data. We show that presence/absence of drug-target orthologues are predictive of susceptible species for the more potent drugs. Drugs that target the endocrine system represent the highest potency and greatest risk. However, for most drugs (>80%) with a full set of ecotoxicity data, risk quotients assuming worst-case exposure assessments were below one in all European countries indicating low environmental risks for the endpoints assessed. CONCLUSION: We believe that the presented analysis can guide improvements to current testing procedures, and provide valuable approaches for prioritising legacy drugs (i.e. those registered before 2006) for further ecotoxicity testing. For drugs where effects of possible concern (e.g. behaviour) are not captured in regulatory tests, additional mechanistic testing may be required to provide the highest confidence for avoiding environmental impacts.

Cardiovascular Effects and Molecular Mechanisms of Bisphenol A and Its Metabolite MBP in Zebrafish.

The plastic monomer bisphenol A (BPA) is one of the highest production volume chemicals in the world and is frequently detected in wildlife and humans, particularly children. BPA has been associated with numerous adverse health outcomes relating to its estrogenic and other hormonal properties, but direct causal links are unclear in humans and animal models. Here we simulated measured (1×) and predicted worst-case (10× ) maximum fetal exposures for BPA, or equivalent concentrations of its metabolite MBP, using fluorescent reporter embryo-larval zebrafish, capable of quantifying Estrogen Response Element (ERE) activation throughout the body. Heart valves were primary sites for ERE activation by BPA and MBP, and transcriptomic analysis of microdissected heart tissues showed that both chemicals targeted several molecular pathways constituting biomarkers for calcific aortic valve disease (CAVD), including extra-cellular matrix (ECM) alteration. ECM collagen deficiency and impact on heart valve structural integrity were confirmed by histopathology for high-level MBP exposure, and structural defects (abnormal curvature) of the atrio-ventricular valves corresponded with impaired cardiovascular function (reduced ventricular beat rate and blood flow). Our results are the first to demonstrate plausible mechanistic links between ERE activation in the heart valves by BPA's reactive metabolite MBP and the development of valvular-cardiovascular disease states.

ECOdrug: a database connecting drugs and conservation of their targets across species.

Pharmaceuticals are designed to interact with specific molecular targets in humans and these targets generally have orthologs in other species. This provides opportunities for the drug discovery community to use alternative model species for drug development. It also means, however, there is potential for mode of action related effects in non-target wildlife species as many pharmaceuticals reach the environment through patient use and manufacturing wastes. Acquiring insight in drug target ortholog predictions across species and taxonomic groups has proven difficult because of the lack of an optimal strategy and because necessary information is spread across multiple and diverse sources and platforms. We introduce a new research platform tool, ECOdrug, that reliably connects drugs to their protein targets across divergent species. It harmonizes ortholog predictions from multiple sources via a simple user interface underpinning critical applications for a wide range of studies in pharmacology, ecotoxicology and comparative evolutionary biology. ECOdrug can be used to identify species with drug targets and identify drugs that interact with those targets. As such, it can be applied to support intelligent targeted drug safety testing by ensuring appropriate and relevant species are selected in ecological risk assessments. ECOdrug is freely accessible and available at: http://www.ecodrug.org.

Integrating human and environmental health in antibiotic risk assessment: A critical analysis of protection goals, species sensitivity and antimicrobial resistance.

Antibiotics are vital in the treatment of bacterial infectious diseases but when released into the environment they may impact non-target organisms that perform vital ecosystem services and enhance antimicrobial resistance development with significant consequences for human health. We evaluate whether the current environmental risk assessment regulatory guidance is protective of antibiotic impacts on the environment, protective of antimicrobial resistance, and propose science-based protection goals for antibiotic manufacturing discharges. A review and meta-analysis was conducted of aquatic ecotoxicity data for antibiotics and for minimum selective concentration data derived from clinically relevant bacteria. Relative species sensitivity was investigated applying general linear models, and predicted no effect concentrations were generated for toxicity to aquatic organisms and compared with predicted no effect concentrations for resistance development. Prokaryotes were most sensitive to antibiotics but the range of sensitivities spanned up to several orders of magnitude. We show reliance on one species of (cyano)bacteria and the 'activated sludge respiration inhibition test' is not sufficient to set protection levels for the environment. Individually, neither traditional aquatic predicted no effect concentrations nor predicted no effect concentrations suggested to safeguard for antimicrobial resistance, protect against environmental or human health effects (via antimicrobial resistance development). Including data from clinically relevant bacteria and also more species of environmentally relevant bacteria in the regulatory framework would help in defining safe discharge concentrations for antibiotics for patient use and manufacturing that would protect environmental and human health. It would also support ending unnecessary testing on metazoan species.

Progesterone-mediated effects on gene expression and oocyte-cumulus complex transport in the mouse fallopian tube.

BACKGROUND: The fallopian tube transports the gametes to the fertilization site and delivers the embryo to the uterus at the optimal time for implantation. Progesterone and the classical progesterone receptor are involved in regulating both tubal ciliary beating and muscular contractions, likely via both genomic and non-genomic actions. METHODS: To provide more details of the underlying mechanisms, we investigated the effect of progesterone on gene expression in mice fallopian tubes in vitro at 20 min, 2 h and 8 h post progesterone treatment using microarray and/or quantitative PCR. In parallel, oocyte cumulus complex transport was investigated in ovulating mice that were injected with one of the progesterone receptor antagonists, Org 31710 or CDB2194. RESULTS: Microarray analyses did not reveal any apparently regulated genes 20 min after progesterone treatment, consistent with the proposed non-genomic action of progesterone controlling ciliary beating. After 2 h, 11 genes were identified as up-regulated. Analyses using quantitative PCR at 2 h and 8 h showed a consistent up-regulation of endothelin1 and a down-regulation of its receptor Endothelin receptor A by progesterone. We also confirmed that treatment with progesterone receptor antagonists before ovulation accelerates the transport of the oocyte cumulus complex. CONCLUSIONS: This is the first study showing that progesterone regulates the expression of endothelin1 and endothelin receptor A in the fallopian tube. Together with previous studies of the effects of endothelin on muscular contractions in the fallopian tube, the results from this study suggest that endothelin is a mediator of the progesterone-controlled effects on muscular contraction and eventually gamete transport in the fallopian tube.

Improving environmental risk assessment of human pharmaceuticals.

This paper presents 10 recommendations for improving the European Medicines Agency's guidance for environmental risk assessment of human pharmaceutical products. The recommendations are based on up-to-date, available science in combination with experiences from other chemical frameworks such as the REACH-legislation for industrial chemicals. The recommendations concern: expanding the scope of the current guideline; requirements to assess the risk for development of antibiotic resistance; jointly performed assessments; refinement of the test proposal; mixture toxicity assessments on active pharmaceutical ingredients with similar modes of action; use of all available ecotoxicity studies; mandatory reviews; increased transparency; inclusion of emission data from production; and a risk management option. We believe that implementation of our recommendations would strengthen the protection of the environment and be beneficial to society. Legislation and guidance documents need to be updated at regular intervals in order to incorporate new knowledge from the scientific community. This is particularly important for regulatory documents concerning pharmaceuticals in the environment since this is a research field that has been growing substantially in the last decades.

Waterborne beclomethasone dipropionate affects the physiology of fish while its metabolite beclomethasone is not taken up.

Asthma is commonly treated with inhalable glucocorticosteroids, including beclomethasone dipropionate (BDP). This is a synthetic prodrug which is metabolized to the more active monopropionate (BMP) and free beclomethasone in humans. To evaluate potential effects of residual drugs on fish, we conducted a 14 day flow-through exposure experiment with BDP and beclomethasone using rainbow trout, and analyzed effects on plasma glucose, hepatic glutathione and catalase activity together with water and body concentrations of the BDP, BMP and beclomethasone. We also analyzed hepatic gene expression in BDP-exposed fish by microarray and quantitative PCR. Beclomethasone (up to 0.65 µg/L) was not taken up in the fish while BDP (0.65 and 0.07 µg/L) resulted in accumulation of both beclomethasone, BMP and BDP in plasma, reaching levels up to those found in humans during therapy. Accordingly, exposure to 0.65 µg/L of BDP significantly increased blood glucose as well as oxidized glutathione levels and catalase activity in the liver. Exposure to beclomethasone or the low concentration of BDP had no effect on these endpoints. Both exposure concentrations of BDP resulted in significantly higher transcript abundance of phosphoenolpyruvate carboxykinase involved in gluconeogenesis, and of genes involved in immune responses. As only the rapidly metabolized prodrug was potent in fish, the environmental risks associated with the use of BDP are probably small. However, the observed physiological effects in fish of BDP at plasma concentrations known to affect human physiology provides valuable input to the development of read-across approaches in the identification of pharmaceuticals of environmental concern.

A novel method for cross-species gene expression analysis.

BACKGROUND: Analysis of gene expression from different species is a powerful way to identify evolutionarily conserved transcriptional responses. However, due to evolutionary events such as gene duplication, there is no one-to-one correspondence between genes from different species which makes comparison of their expression profiles complex. RESULTS: In this paper we describe a new method for cross-species meta-analysis of gene expression. The method takes the homology structure between compared species into account and can therefore compare expression data from genes with any number of orthologs and paralogs. A simulation study shows that the proposed method results in a substantial increase in statistical power compared to previously suggested procedures. As a proof of concept, we analyzed microarray data from heat stress experiments performed in eight species and identified several well-known evolutionarily conserved transcriptional responses. The method was also applied to gene expression profiles from five studies of estrogen exposed fish and both known and potentially novel responses were identified. CONCLUSIONS: The method described in this paper will further increase the potential and reliability of meta-analysis of gene expression profiles from evolutionarily distant species. The method has been implemented in R and is freely available at http://bioinformatics.math.chalmers.se/Xspecies/.

Oral exposure to industrial effluent with exceptionally high levels of drugs does not indicate acute toxic effects in rats.

The Patancheru area near Hyderabad in India is recognized as a key link in the global supply chain for many bulk drugs. A central treatment plant receives wastewater from approximately 90 different manufacturers, and the resulting complex effluent has contaminated surface, ground, and drinking water in the region. Ecotoxicological testing of the effluent has shown adverse effects for several organisms, including aquatic vertebrates, at high dilutions. In addition, a recent study of microbial communities in river sediment indicated that the contamination of antibiotic substances might contribute to the emergence and spread of antibiotic resistance genes. In an attempt to start investigating how exposure to effluent-contaminated water may directly affect humans and other terrestrial vertebrates, rats were tube-fed effluent. Several pharmaceuticals present in the effluent could be detected in rat blood serum at low concentrations. However, results from exploratory microarray and quantitative polymerase chain reaction assays indicated no marked effects on hepatic gene transcription after 5 d of exposure. Clinical analysis of blood serum constituents, used as biomarkers for human disease did not reveal any significant changes, nor was there an effect on weight gain. The authors could not find evidence for any acute toxicity in the rat; however, the authors cannot rule out that [corrected] higher doses of effluent or a longer exposure time may still be associated with risks for terrestrial vertebrates.

Global hepatic gene expression in rainbow trout exposed to sewage effluents: a comparison of different sewage treatment technologies.

Effluents from sewage treatment plants contain a mixture of micropollutants with the potential of harming aquatic organisms. Thus, addition of advanced treatment techniques to complement existing conventional methods has been proposed. Some of the advanced techniques could, however, potentially produce additional compounds affecting exposed organisms by unknown modes of action. In the present study the aim was to improve our understanding of how exposure to different sewage effluents affects fish. This was achieved by explorative microarray and quantitative PCR analyses of hepatic gene expression, as well as relative organ sizes of rainbow trout exposed to different sewage effluents (conventionally treated, granular activated carbon, ozonation (5 or 15 mg/L), 5 mg/L ozone plus a moving bed biofilm reactor, or UV-light treatment in combination with hydrogen peroxide). Exposure to the conventionally treated effluent caused a significant increase in liver and heart somatic indexes, an effect removed by all other treatments. Genes connected to xenobiotic metabolism, including cytochrome p450 1A, were differentially expressed in the fish exposed to the conventionally treated effluents, though only effluent treatment with granular activated carbon or ozone at 15 mg/L completely removed this response. The mRNA expression of heat shock protein 70 kDa was induced in all three groups exposed to ozone-treated effluents, suggesting some form of added stress in these fish. The induction of estrogen-responsive genes in the fish exposed to the conventionally treated effluent was effectively reduced by all investigated advanced treatment technologies, although the moving bed biofilm reactor was least efficient. Taken together, granular activated carbon showed the highest potential of reducing responses in fish induced by exposure to sewage effluents.

Octopamine receptors from the barnacle balanus improvisus are activated by the alpha2-adrenoceptor agonist medetomidine.

G protein-coupled octopamine receptors of insects and other invertebrates represent counterparts of adrenoceptors in vertebrate animals. The alpha(2)-adrenoceptor agonist medetomidine, which is in clinical use as a veterinary sedative agent, was discovered to inhibit the settling process of barnacles, an important step in the ontogeny of this crustacean species. Settling of barnacles onto ship hulls leads to biofouling that has many harmful practical consequences, and medetomidine is currently under development as a novel type of antifouling agent. We now report that medetomidine induces hyperactivity in the barnacle larvae to disrupt the settling process. To identify the molecular targets of medetomidine, we cloned five octopamine receptors from the barnacle Balanus improvisus. We show by phylogenetic analyses that one receptor (BiOctalpha) belongs to the alpha-adrenoceptor-like subfamily, and the other four (BiOctbeta-R1, BiOctbeta-R2, BiOctbeta-R3, and BiOctbeta-R4) belong to the beta-adrenoceptor-like octopamine receptor subfamily. Phylogenetic analyses also indicated that B. improvisus has a different repertoire of beta-adrenoceptor-like octopamine receptors than insects. When expressed in CHO cells, the cloned receptors were activated by both octopamine and medetomidine, resulting in increased intracellular cAMP or calcium levels. Tyramine activated the receptors but with much lesser potency than octopamine. A hypothesis for receptor discrimination between tyramine and octopamine was generated from a homology three-dimensional model. The characterization of B. improvisus octopamine receptors is important for a better functional understanding of these receptors in crustaceans as well as for practical applications in development of environmentally sustainable antifouling agents.

Pharmaceutical industry effluent diluted 1:500 affects global gene expression, cytochrome P450 1A activity, and plasma phosphate in fish.

Patancheru, near Hyderabad, India, is a major production site for the global bulk drug market. Approximately 90 manufacturers send their wastewater to a common treatment plant in Patancheru. Extraordinary high levels of a wide range of pharmaceuticals have recently been demonstrated in the treated effluent. As little as 0.2% of this effluent can strongly reduce the growth rate of tadpoles, but the underlying mechanisms of toxicity are not known. To begin addressing how the effluent affects aquatic vertebrates, rainbow trout (Oncorhynchus mykiss) were exposed to 0.2% effluent for 5 d. Several physiological endpoints, together with effects on global hepatic gene expression patterns, were analyzed. The exposed fish showed both an induction of hepatic cytochrome P450 1A (CYP1A) gene expression, as well as enzyme activity. Clinical blood chemistry analyses revealed an increase in plasma phosphate levels, which in humans indicates impaired kidney function. Several oxidative stress-related genes were induced in the livers; however, no significant changes in antioxidant enzyme activities or in the hepatic glutathione levels were found. Furthermore, estrogen-regulated genes were slightly up-regulated following exposure, and moderate levels of estriol were detected in the effluent. The present study identifies changes in gene expression triggered by exposure to a high dilution of the effluent, supporting the hypothesis that these fish are responding to chemical exposure. The pattern of regulated genes may contribute to the identification of mechanisms of sublethal toxicity, as well as illuminate possible causative agents.

Urinary detection times and metabolite/parent compound ratios after a single dose of buprenorphine.

The objective was to estimate the detection times and metabolite/parent compound ratios in urine after a single dose of buprenorphine. Eighteen healthy volunteers received a single dose of 0.4 mg buprenorphine sublingually. Urine samples were collected prior to dosing and at 2, 4, 6, 8 12, 24, 48, 72, and 96 h post-dose. The samples were screened using cloned enzyme donor immunoassay (CEDIA) reagent and quantitation was performed with liquid chromatography-tandem mass spectrometry (LC-MS-MS) with a cut-off of 0.5 ng/mL for buprenorphine and norbuprenorphine. The mean time of continuous positive results was 9 h (range 4 to 24 h) with CEDIA, whereas for an LC-MS-MS method it was 76 h (range 23-96 h) for buprenorphine, and for norbuprenorphine all samples were positive at 96 h. Some subjects had positive CEDIA results after a negative sample, owing to differences in creatinine concentration. The time when the ratio norbuprenorphine/buprenorphine exceeded 1 was estimated at 7 h. The metabolite/parent ratio may be used to estimate the time of intake even though the individual ratios showed an increased variation the more distant the collection time. We believe that using this ratio, rather than the actual concentrations, it is possible to compensate for urine dilution and different doses, and to improve interpretation.

Evolutionary conservation of human drug targets in organisms used for environmental risk assessments.

Pharmaceuticals are typically found in very low concentrations in the aquatic environment. Accordingly, environmental effects clearly assigned to residual drugs are consistent with high affinity interactions with conserved targets in affected wildlife species rather than with a general toxic effect. Thus, evolutionarily well-conserved targets in a given species are associated with an increased risk. In this study orthologs for 1318 human drug targets were predicted in 16 species of which several are relevant for ecotoxicity testing. The conservation of different functional categories of targets was also analyzed. Zebrafish had orthologs to 86% of the drug targets while only 61% were conserved in Daphnia and 35% in green alga. The predicted presence and absence of orthologs agrees well with published experimental data on the potential for specific drug target interaction in various species. Based on the conservation of targets we propose that aquatic environmental risk assessments for human drugs should always include comprehensive studies on aquatic vertebrates. Furthermore, individual targets, especially enzymes, are well conserved suggesting that tests on evolutionarily distant organisms would be highly relevant for certain drugs. We propose that the results can guide environmental risk assessments by improving the possibilities to identify species sensitive to certain types of pharmaceuticals or to other contaminants that act through well defined mechanisms of action. Moreover, we suggest that the results can be used to interpret the relevance of existing ecotoxicity data.

Sensitive and robust gene expression changes in fish exposed to estrogen--a microarray approach.

BACKGROUND: Vitellogenin is a well established biomarker for estrogenic exposure in fish. However, effects on gonadal differentiation at concentrations of estrogen not sufficient to give rise to a measurable vitellogenin response suggest that more sensitive biomarkers would be useful. Induction of zona pellucida genes may be more sensitive but their specificities are not as clear. The objective of this study was to find additional sensitive and robust candidate biomarkers of estrogenic exposure. RESULTS: Hepatic mRNA expression profiles were characterized in juvenile rainbow trout exposed to a measured concentration of 0.87 and 10 ng ethinylestradiol/L using a salmonid cDNA microarray. The higher concentration was used to guide the subsequent identification of generally more subtle responses at the low concentration not sufficient to induce vitellogenin. A meta-analysis was performed with data from the present study and three similar microarray studies using different fish species and platforms. Within the generated list of presumably robust responses, several well-known estrogen-regulated genes were identified. Two genes, confirmed by quantitative RT-PCR (qPCR), fulfilled both the criteria of high sensitivity and robustness; the induction of the genes encoding zona pellucida protein 3 and a nucleoside diphosphate kinase (nm23). CONCLUSION: The cross-species, cross-platform meta-analysis correctly identified several robust responses. This adds confidence to our approach used for identifying candidate biomarkers. Specifically, we propose that analyses of an nm23 gene together with zona pellucida genes may increase the possibilities to detect an exposure to low levels of estrogenic compounds in fish.