A Multiparametric Assay Platform for Simultaneous In Vivo Assessment of Pronephric Morphology, Renal Function and Heart Rate in Larval Zebrafish.

Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.

Binary combinations of organophosphorus and synthetic pyrethroids are more potent acetylcholinesterase inhibitors than organophosphorus and carbamate mixtures: An in vitro assessment.

Anticholinesterase insecticides such as organophosphorous (OP) and carbamates pesticides (CB); and synthetic pyrethroids (SP) pesticides commonly co-occur in the environment. This raises the possibility of antagonistic, additive, or synergistic neurotoxicity in exposed organisms. Acetylcholinesterase (AChE) inhibition has been demonstrated to be useful as a biomarker for exposure to OP and CBs in many environments. This study investigated the response of housefly (Musca domestica) head AChE (HF-AChE) exposed to five OPs; chlorpyrifos (CPF), malathion (MLT), triazophos (TRZ), monocrotophos (MCP) and profenofos (PRF) and two CBs; carbaryl (CRB) and carbofuran (CBF) as individual compounds and as binary mixtures of OPs and CBs under in vitro conditions. In addition, the selected OPs and CBs were evaluated for their toxicity in binary combinations with two SPs; deltamethrin (DLT) and cypermethrin (CYP) at fixed concentrations of 0.1 and 10µg/L. The toxicological interaction of five OPs with two CBs pesticides was evaluated under oxidised and un-oxidised conditions using a toxic unit (TU) approach and a concentration addition (CA) model. Pyrethroid combinations were assessed only under oxidised conditions. Since OPs and CBs act by a similar mechanism of inhibition of AChE, a dose additive effect was expected, but not conclusively found. TRZ with either CBF or CRB exhibited synergism under oxidised and un-oxidised conditions but the degree of synergism was stronger under un-oxidised conditions. Additivity was exhibited by CBF+MCP, CRB+MCP, CRB+MLT and CBF+MCP under un-oxidised conditions and CRB+MCP and CRB+CPF under oxidised conditions. Pyrethorids in combination with OPs (TRZ, MLT and CPF) were highly synergistic. In the present study, we used pure housefly head AChE without any interference of monooxygenase and/or esterase enzyme activities. Therefore these other enzymes were not producing the observed deviations from concentration-addition in the binary combinations between OPs, CBs and SPs. The mechanisms of OP, CB and SP interactions in pesticide mixtures requires further investigation.

A case study for assessment of microbial community dynamics in genetically modified Bt cotton crop fields.

Bt cotton was the first genetically modified crop approved for use in India. However, only a few studies have been conducted to assess the feasibility of its commercial application. Bt cotton is genetically modified to express a proteinaceous endotoxin (Cry) encoded by cry gene of Bacillus thuringiensis that has specific insecticidal activity against bollworms. Therefore, the amount of pesticides used for growing Bt cotton is postulated to be considerably low as compared to their non-Bt counterparts. Alternatively, it is also speculated that application of a genetically modified crop may alter the bio-geochemical balance of the agriculture field(s). Microbial community composition and dynamics is an important descriptor for assessment of such alterations. In the present study, we have assessed the culturable and non-culturable microbial diversities in Bt cotton and non-Bt cotton soils to determine the ecological consequences of application of Bt cotton. The analyses of microbial community structures indicated that cropping of Bt cotton did not adversely affect the diversity of the microbial communities.

Bacterial community structure of a pesticide-contaminated site and assessment of changes induced in community structure during bioremediation.

The introduction of culture-independent molecular screening techniques, especially based on 16S rRNA gene sequences, has allowed microbiologists to examine a facet of microbial diversity not necessarily reflected by the results of culturing studies. The bacterial community structure was studied for a pesticide-contaminated site that was subsequently remediated using an efficient degradative strain Arthrobacter protophormiae RKJ100. The efficiency of the bioremediation process was assessed by monitoring the depletion of the pollutant, and the effect of addition of an exogenous strain on the existing soil community structure was determined using molecular techniques. The 16S rRNA gene pool amplified from the soil metagenome was cloned and restriction fragment length polymorphism studies revealed 46 different phylotypes on the basis of similar banding patterns. Sequencing of representative clones of each phylotype showed that the community structure of the pesticide-contaminated soil was mainly constituted by Proteobacteria and Actinomycetes. Terminal restriction fragment length polymorphism analysis showed only nonsignificant changes in community structure during the process of bioremediation. Immobilized cells of strain RKJ100 enhanced pollutant degradation but seemed to have no detectable effects on the existing bacterial community structure.