Short-term test for the toxicogenomic assessment of ecotoxic modes of action in Myriophyllum spicatum.

In environmental risk assessment of substances, the 14-day growth inhibition test following OECD test guideline 239 is employed to assess toxicity in the macrophyte Myriophyllum spicatum. Currently, this test evaluates physiological parameters and does not allow the identification of the mode of action (MoA) by which adverse effects are induced. However, for an improved ecotoxicity assessment of substances, knowledge about their ecotoxic MoA in non-target organisms is required. It has previously been suggested that the identification of gene expression changes can contribute to MoA identification. Therefore, we developed a shortened three-day assay for M. spicatum including the transcriptomic assessment of global gene expression changes and applied this assay to two model substances, the herbicide and photosynthesis inhibitor bentazone and the pharmaceutical and HMG-CoA reductase inhibitor atorvastatin. Due to the lack of a reference genome for M. spicatum we performed a de novo transcriptome assembly followed by a functional annotation to use the toxicogenomic results for MoA discrimination. The gene expression changes induced by low effect concentrations of these substances were used to identify differentially expressed genes (DEGs) and impaired biological functions for the respective MoA. We observed both concentration-dependent numbers and differentiated patterns of DEGs for both substances. While bentazone impaired genes involved in the response to reactive oxygen species as well as light response, and also genes involved in developmental processes, atorvastatin exposure led to a differential regulation of genes related to brassinosteroid response as well as potential metabolic shifts between the mevalonate and methyl erythritol 4-phosphate pathway. Based on these responses, we identified biomarker candidates for the assessment of MoA in M. spicatum. Utilizing the shortened assay developed in this study, the investigation of the identified biomarker candidates may contribute to the development of future MoA-specific screening approaches in the ecotoxicological hazard prediction using aquatic non-standard model organisms.

National Assessment of Long-Term Groundwater Response to Pesticide Regulation.

Quantitative assessments of long-term, national-scale responses of groundwater quality to pesticide applications are essential to evaluate the effectiveness of pesticide regulations. Retardation time in the unsaturated zone (Ru) was estimated for selected herbicides (atrazine, simazine, and bentazon) and degradation products (desethylatrazine (DEA), desisopropylatrazine (DIA), desethyldesisopropylatrazine (DEIA), and BAM) using a multidecadal time series of groundwater solute chemistry (∼30 years) and herbicide sales (∼60 years). The sampling year was converted to recharge year using groundwater age. Then, Ru was estimated using a cross-correlation analysis of the sales and the frequencies of detection and exceedance of the drinking water standard (0.1 µg/L) of each selected compound. The results showed no retardation of the highly polar, thus mobile, parent compounds (i.e., bentazon), while Ru of the moderately polar compounds (i.e., simazine) was about a decade, and their degradation products showed even longer Ru. The temporal trends of the degradation products did not mirror those of the sale data, which were attributed to the various sale periods of the parent compounds, sorption of the parent compounds, and complex degradation pathways. The longer Ru in clayey/organic sediments than in sandy sediments further confirmed the role of soil-specific retardation as an important factor to consider in groundwater protection.

Bentazone in water and human urine in Wuhan, central China: exposure assessment.

Bentazone is a widely used post-emergence herbicide, while no data was available on its concentrations in tap water from China and in urine among the general population. It was determined in the source (Wuhan section of the Yangtze River watershed), treated, and tap water (n = 20, 20, and 170, respectively) in different seasons (2019) in Wuhan, central China. Also, urine samples (n = 38) collected from healthy adults in Wuhan (September 2020) were analyzed to characterize its urinary concentration. Bentazone was detected in all the source and treated water samples. Its concentrations in the source water in July were higher than those in February (median: 17.9 ng/L vs. 2.86 ng/L) (p < 0.05). It cannot be removed efficiently (27.8-27.9%) by conventional drinking water treatment using NaClO, but it can be efficiently removed by using chlorine dioxide or ozone combined with activated carbon. Bentazone was frequently detected (detection frequency: 96.3%) in 160 tap water samples (underwent conventional treatment) (median: 1.95 ng/L, range: <0.02-47.0 ng/L), while it was not detectable in tap water samples that underwent ozone combined with activated carbon. Seasonal variations were found, with the lowest median concentration (ng/L) in April (0.46) and the highest in July (17.6). In addition, bentazone was frequently (92.1%) detected in human urine samples (median: 0.02 ng/mL; range: < 0.01-0.11 ng/mL). The estimated daily intake of bentazone based on its median concentration in tap water (0.04 ng/kg-body weight [bw]/day) accounted for approximately 8% of that based on the median urinary concentration (0.48 ng/kg-bw/day). This is the first time to characterize its occurrence in drinking water from China and its occurrence in the urine of the general population.

Geographical and temporal assessment of the photochemical decontamination potential of river waters from agrochemicals: A first application to the Piedmont region (NW Italy).

This work shows the potential of using photochemical modelling to assess the river-water ability to photodegrade agrochemicals on a geographic and temporal scale. The case of flowing water requires different data treatment compared to more stationary water bodies (e.g., lakes), but it could allow for the identification of particularly vulnerable environments. Five pesticides were considered here, and the photodegradation rate followed the order bentazon > isoproturon > dimethomorph âˆ¼ chlortoluron > atrazine. The modelled photodegradation kinetics was particularly fast in the river Po, which receives significant input of agricultural nitrate from groundwater and features higher steady-state [•OH] than most other rivers in the region. The fact that the Po eventually collects all river waters in Piedmont is positive, from the point of view of comprehensive photodegradation of pesticides. However, this paradoxical situation of agricultural pollution (nitrate) helping fight pollution from the same source (pesticides) has two important limitations: (i) when compared to the parent compounds, some intermediates deriving from •OH reactions are either more harmful (N-formyl derivatives of phenylureas), or about as harmful (desethyl atrazine); (ii) banned atrazine is no longer sprayed over fields during the plant growth season, but it reaches surface waters from legacy groundwater inputs. The latter are operational also during winter, when photochemistry is least active. Therefore, photochemistry might not ensure considerable attenuation of atrazine during wintertime. Overall, bentazon would be the safest among the studied pesticides because of fast degradation by direct photolysis, and of low ecotoxicological impact of its phototransformation intermediates.

The inadequacies of pre-market chemical risk assessment's toxicity studies-the implications.

Industry provides essentially all the data for most (pre-market) chemical risk assessments (RA); academics study a chemical once it is marketed. For two randomly-chosen high production chemicals, despite new European Union mandates to evaluate all data, just 13% of the herbicide bentazon and 15% of the flame-retardant hexabromocyclododecane's published toxicity studies were found in their pre-market RA, and a systematic review on bentazon concludes it has greater hazards than indicated in its RA. More important, for both, academia's toxicity studies were designated as lower quality than industries were, despite showing hazards at lower doses. The accuracy of industry's test methods is analyzed and found to be replicable but insensitive, thus inaccurate. The synthetic pharmaceutical industry originated them, and by 1983 the Organization for Economic Cooperation & Development mandated their test guidelines (TG) methods be accepted for any new study for pre-market RA. For existing studies, industry's "Klimisch" criterion is universally used to evaluate quality, but it only states that TG studies produce the best data. However, no TG can answer the realistic exposure effect hypotheses of academics; therefore, crucially in pre-market RA, tens of thousands of published experimental findings (increasingly at low dose) are ignored to determine the safe dose. Few appreciate this, so scientific debate on the most accurate elements of toxicity tests is urgently indicated. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of the partial renewal of in situ phytoplankton microcosms and application to the impact assessment of bentazon and dimethenamid.

Microcosms, each consisting of 2L natural surface seawater maintained in 2.3-L glass bottles, were immersed at a depth of 6m. The renewal of 10% of microcosm volumes was carried out every other day. Phytoplankton-containing seawater was used for renewal (previously filtered through 25-, 50- or 200-µm cut-off). Phytoplankton community pigment analysis (by HPLC) and flow cytometry analysis were performed. After 13 days, data exhibited phytoplankton characteristics in microcosms in the same range as that of the natural surrounding sea water over the same period. Furthermore, in these microcosms, a negative correlation was observed between the filtration cut-off used for renewal water, and the total cell count. Herbicides were tested as commercial mixtures at 1, 10 and 100 µgL(-1) active substance. Both Frontier® (dimethenamid) and Basamais® (bentazon) induced significant modifications of the phytoplankton populations at every concentration tested. Such results suggest a possible disturbance in polluted coastal areas.

Environmentally friendly iron-catalyzed cascade synthesis of 1,2,4-benzothiadiazine 1,1-dioxide and quinazolinone derivatives.

We have developed an efficient iron-catalyzed method for the cascade synthesis of 1,2,4-benzothiadiazine 1,1-dioxide and quinazolinone derivatives. The protocol uses inexpensive and environmentally friendly FeCl(3) as the catalyst, and no ligand or additive was required. This is the first example of construction of nitrogen-containing heterocycles via iron-catalyzed N-arylation in the absence of ligand. Therefore, this method is of practical application for the synthesis of the two different nitrogen-containing heterocycles.

In-situ microcosms, a tool for assessment of pesticide impacts on oyster spat (Crassostrea gigas).

Effects of the herbicide Basamaïs (bentazon) and the fungicide Opus (epoxiconazole) on oyster spat (Crassostrea gigas) were assessed using in-situ microcosms in a field experiment lasting 13 days. Six-week-old hatchery spat (mean size 1.1 mm), previously collected on PVC plates, was immersed in glass bottles filled with 200 mum filtered seawater. Bottles were maintained underwater at 6 m depth and their water content changed every other day. Growth, measured as shell area index increase, was 126 +/- 4% in the control bottles. While no growth differences were observed between control and individual pesticide treatments at 10 microg l(-1), oysters treated with a mix of 10 microg l(-1) Opus and 10 microg l(-1) Basamaïs showed a 50% growth reduction compared with the control (P < 0.0001), suggesting a synergistic effect of these contaminants. Laboratory controls in microcosms maintained in a water bath with filtered natural light, were not significantly different from in-situ microcosm controls in the field, for organic weight content or growth. This in-situ experiment in microcosms allowed us to conclude that: (1) oyster spat can achieve significant growth in bottles immersed in situ without supplementary food; (2) this microcosm system is reliable and easy to use for environmental toxicity tests with C. gigas spat; (3) such microcosm systems can also be run in a laboratory water bath instead of more technically difficult immersed field experiments; (4) the synergistic effect observed here, at a concentration simulating a peak agricultural runoff event, suggests that the impacts of pesticides could be a real threat for oysters in estuarine areas.

Prescribing patterns for antihypertensive drugs after the Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial: report of experience in a health maintenance organization.

BACKGROUND: The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) reported that primary cardiovascular outcomes were the same in hypertensive patients treated with thiazide-type diuretics as in those treated with calcium channel blockers or angiotensin-converting enzyme inhibitors. The aim of this study was to assess prescribing patterns of antihypertensive agents in a health maintenance organization (HMO) before and after the publications of ALLHAT results. METHODS: Our analysis used computer-stored information from the pharmacy system of the HMO for the period between January 1, 1996, and December 31, 2003. The study assessed prescribing patterns for antihypertensive drugs in six classes: thiazide-type diuretics, other-type antihypertensive diuretics, angiotensin-converting enzyme inhibitors/angiotensin receptor blockers, calcium channel blockers, beta-blockers, and alpha-blockers. The monthly total number and relative percentage of dispensed prescriptions for each class of antihypertensive agent, including new prescriptions and refills, were calculated. RESULTS: The use of alpha-blockers for all prescriptions began to decrease after the closure of the alpha-blocker (doxazosin) arm of ALLHAT in January 2000. Prescriptions for the thiazide-type diuretics immediately increased after the ALLHAT publication in December 2002. During the first 6 months of 2003, the percentages of thiazide-type diuretics were statistically significantly higher compared with the predicted values. This pattern held for all as well as for new antihypertensive prescriptions. CONCLUSIONS: The ALLHAT publications had large effects on the antihypertensive prescribing patterns in our population. Prescription of thiazide-type diuretics significantly increased after the ALLHAT publication. Our findings establish that the response of physicians to new clinical evidence can be very rapid.

ALLHAT: a critical assessment.

The ALLHAT study has attracted considerable attention in the media and in the research community, partly due to the study's unexpected and controversial conclusions. However, the study has several serious shortcomings. The primary end-points in ALLHAT were negative and the conclusions are based entirely on secondary end-points and subgroup analyses. Moreover, there is good reason for skepticism concerning the findings on heart failure in ALLHAT, because of ambiguity in the diagnosis, lack of information on blood pressure and absence of a "washout" period. The study design was severely flawed and does not reflect clinical reality. Also, blood pressure differences between groups severely complicate interpretation. From a patient perspective in ALLHAT, there are drug safety concerns with the thiazides, as there was evidence of excess diabetes development. The ALLHAT results are difficult to generalize and have limited relevance in European settings. Thus, the ALLHAT study suffers from several major shortcomings and there is a huge body of evidence that contradicts the ALLHAT interpretations.

Incidence of out-of-hospital cardiac arrest.

Estimates of the incidence of out-of-hospital primary cardiac arrest (CA) have typically relied solely upon emergency medical service or death certificate records and have not investigated incidence in clinical subgroups. Overall and temporal patterns of CA incidence were investigated in clinically defined groups using systematic methods to ascertain CA. Estimates of incidence were derived from a population-based case-control study in a large health plan from 1986 to 1994. Subjects were enrollees aged 50 to 79 years who had had CA (n = 1,275). A stratified random sample of enrollees who had not had CA was used to estimate the population at risk with various clinical characteristics (n = 2,323). Poisson's regression was used to estimate incidence overall and for 3-year time periods (1986 to 1988, 1989 to 1991, and 1992 to 1994). The overall CA incidence was 1.89/1,000 subject-years and varied up to 30-fold across clinical subgroups. For example, incidence was 5.98/1,000 subject-years in subjects with any clinically recognized heart disease compared with 0.82/1,000 subject-years in subjects without heart disease. In subgroups with heart disease, incidence was 13.69/1,000 subject-years in subjects with prior myocardial infarction and 21.87/1,000 subject-years in subjects with heart failure. Risk decreased by 20% from the initial to the final time period, with a greater decrease observed in those with (25%) compared with those without (12%) clinical heart disease. Thus, CA incidence varied considerably across clinical groups. The results provide insights regarding absolute and population-attributable risk in clinically defined subgroups, information that may aid strategies aimed at reducing mortality from CA.

Properties of quantal transmission at CA1 synapses.

We have used Monte Carlo simulations to understand the generation of quantal responses at the single active zones of CA1 synapses. We constructed a model of AMPA channel activation that accounts for the responses to controlled glutamate application and a model of glutamate diffusion in the synaptic cleft. With no further adjustments to these models, we simulated the response to the release of glutamate from a single vesicle. The predicted response closely matches the rise time of observed responses, which recent measurements show is much faster (<100 micros) than previously thought. The simulations show that initial channel opening is driven by a brief (<100 micros) glutamate spike near the site of vesicle fusion, producing a hotspot of channel activation (diameter: approximately 250 nm) smaller than many synapses. Quantal size therefore depends more strongly on the density of channels than their number, a finding that has important implications for measuring synaptic strength. Recent measurements allow estimation of AMPA receptor density at CA1 synapses. Using this value, our simulations correctly predicts a quantal amplitude of approximately 10 pA. We have also analyzed the properties of excitatory postsynaptic currents (EPSCs) generated by the multivesicular release that can occur during evoked responses. We find that summation is nearly linear and that the existence of multiple narrow peaks in amplitude histograms can be accounted for. It has been unclear how to reconcile the existence of these narrow peaks, which indicate that the variation of quantal amplitude is small (CV < 0.2) with the highly variable amplitude of miniature EPSCs (mEPSCs; CV approximately 0.6). According to one theory, mEPSC variability arises from variation in vesicle glutamate content. However, both our modeling results and recent experimental results indicate that this view cannot account for the observed rise time/amplitude correlation of mEPSCs. In contrast, this correlation and the high mEPSC variability can be accounted for if some mEPSCs are generated by two or more vesicles released with small temporal jitter. We conclude that a broad range of results can be accounted for by simple principles: quantal amplitude (approximately 10 pA) is stereotyped, some mEPSCs are multivesicular at moderate and large synapses, and evoked responses are generated by quasi-linear summation of multiple quanta.

Guidelines for antihypertensive treatment: an update after the ALLHAT study.

The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT) Study, the largest double-blind, randomized trial in hypertensive patients, confirmed and strengthened the clinical relevance of thiazide diuretics in the treatment of hypertension but did not prove the superiority of these drugs. Its claim of the superiority of chlorthalidone was based on some secondary outcomes, principally represented by (1) an increased incidence of stroke in the doxazosin and lisinopril arms, an effect that might be explained by differences in systolic BP; (2) greater morbidity but not mortality for congestive heart failure (CHF) in the doxazosin, amlodipine, and lisinopril arms, a finding that might reflect poor accuracy in the diagnosis and/or especially the switching from diuretic treatment in 90% of patients. Moreover, the ALLHAT study has other limitations, and its conclusions are in contrast with data from overall controlled clinical trials indicating that given the same BP reduction, the benefit of different drug classes is similar. As to whether the ALLHAT study will influence ongoing guidelines concerning the choice of antihypertensive drugs, the answer is "yes" if interpretation of its data in favor of diuretics and cost of drugs become the preponderant considerations, as it was in recent JNC VII guidelines. However, the more liberal approach based on the choice of all available drug classes seems still to be valid, as is in the ESH-ESC guidelines, if the preponderant consideration is that the real benefit of antihypertensive therapy is due to efficient BP control and not to a particular benefit of a single drug class.

The potential savings of using thiazides as the first choice antihypertensive drug: cost-minimisation analysis.

BACKGROUND: All clinical practice guidelines recommend thiazides as a first-choice drug for the management of uncomplicated hypertension. Thiazides are also the lowest priced antihypertensive drugs. Despite this, the use of thiazides is much lower than that of other drug-classes. We wanted to estimate the potential for savings if thiazides were used as the first choice drug for the management of uncomplicated hypertension. METHODS: For six countries (Canada, France, Germany, Norway, the UK and the US) we estimated the number of people that are being treated for hypertension, and the proportion of them that are suitable candidates for thiazide-therapy. By comparing this estimate with thiazide prescribing, we calculated the number of people that could switch from more expensive medication to thiazides. This enabled us to estimate the potential drug-cost savings. The analysis was based on findings from epidemiological studies and drug trials, and data on sales and prescribing provided by IMS for the year 2000. RESULTS: For Canada, France, Germany, Norway, the UK and the US the estimated potential annual savings were US13.8 million dollars, US37.4 million dollars, US72.2 million dollars, US10.7 million dollars, US119.7 million dollars and US433.6 million dollars, respectively.