Subregion-specific transcriptomic profiling of rat brain reveals sex-distinct gene expression impacted by adolescent stress.

Stress during adolescence clearly impacts brain development and function. Sex differences in adolescent stress-induced or exacerbated emotional and metabolic vulnerabilities could be due to sex-distinct gene expression in hypothalamic, limbic, and prefrontal brain regions. However, adolescent stress-induced whole-genome expression changes in key subregions of these brain regions were unclear. In this study, female and male adolescent Sprague Dawley rats received one-hour restraint stress daily from postnatal day (PD) 32 to PD44. Corticosterone levels, body weights, food intake, body composition, and circulating adiposity and sex hormones were measured. On PD44, brain and blood samples were collected. Using RNA-sequencing, sex-specific differences in stress-induced differentially expressed (DE) genes were identified in subregions of the hypothalamus, limbic system, and prefrontal cortex. Canonical pathways reflected well-known sex-distinct maladies and diseases, substantiating the therapeutic potential of the DE genes found in the current study. Thus, we proposed specific sex distinct, adolescent stress-induced transcriptional changes found in the current study as examples of the molecular bases for sex differences witnessed in stress induced or exacerbated emotional and metabolic disorders. Future behavioral studies and single-cell studies are warranted to test the implications of the DE genes identified in this study in sex-distinct stress-induced susceptibilities.

Building a Conceptual Model for the Environmental Fate of the Fungicide Benzovindiflupyr.

Degradation of the fungicide benzovindiflupyr was slow in standard regulatory laboratory studies in soil and aquatic systems, suggesting it is a persistent molecule. However, the conditions in these studies differed significantly from actual environmental conditions, particularly the exclusion of light, which prevents potential contributions from the phototrophic microorganisms that are ubiquitous in both aquatic and terrestrial environments. Higher tier laboratory studies that include a more comprehensive range of degradation processes can more accurately describe environmental fate under field conditions. Indirect aqueous photolysis studies with benzovindiflupyr showed that the photolytic half-life in natural surface water can be as short as 10 days, compared with 94 days in pure buffered water. Inclusion of a light-dark cycle in higher tier aquatic metabolism studies, to include the contribution of phototrophic organisms, reduced the total system half-life from >1 year in dark test systems to as little as 23 days. The relevance of these additional processes was confirmed in an outdoor aquatic microcosm study in which the half-life of benzovindiflupyr was 13-58 days. In laboratory soil degradation studies, the degradation rate of benzovindiflupyr was significantly faster in cores with an undisturbed surface microbiotic crust, incubated in a light-dark cycle (half-life of 35 days), than in regulatory studies with sieved soil in the dark (half-life >1 year). A radiolabeled field study validated these observations, showing residue decline with a half-life of approximately 25 days over the initial 4 weeks. Conceptual models of environmental fate based on standard regulatory studies may be incomplete, and additional higher tier laboratory studies can be valuable in elucidating degradation processes and improving the prediction of persistence under actual use conditions. Environ Toxicol Chem 2023;42:995-1009. © 2023 SETAC.

The Impact of Disturbed Soil Structure on the Degradation of 2 Fungicides Under Constant and Variable Moisture.

Degradation of agrochemicals in soil is frequently faster under field conditions than in laboratory studies. Field studies are carried out on relatively undisturbed soil, whereas laboratory studies typically use sieved soil, which can have a significant impact on the physical and microbial nature of the soil and may contribute to differences in degradation between laboratory and field studies. A laboratory study was therefore conducted to determine the importance of soil structure and variable soil moisture on the degradation of 2 fungicides (azoxystrobin and paclobutrazol) that show significant differences between laboratory and field degradation rates in regulatory studies. Degradation rates were measured in undisturbed cores of a sandy clay loam soil (under constant or variable moisture contents) and in sieved soil. For azoxystrobin, degradation rates under all conditions were similar (median degradation time [DegT50] 34-37 d). However, for paclobutrazol, degradation was significantly faster in undisturbed cores (DegT50 255 d in sieved soil and 63 d in undisturbed cores). Varying the moisture content did not further enhance degradation of either fungicide. Further examination into the impact of soil structure on paclobutrazol degradation, comparing undisturbed and sieved/repacked cores, revealed that the impact of sieving could not be mitigated by repacking the soil to a realistic bulk density. Examination of fungal and bacterial community structure using automated ribosomal spacer analysis showed significant initial differences between sieved/repacked and intact soil cores, although such differences were reduced at the end of the study (70 d). The present study demonstrates that disruption of soil structure significantly impacts microbial community structure, and for some compounds this may explain the differences between laboratory and field degradation rates. Environ Toxicol Chem 2021;40:2715-2725. © 2021 SETAC.

High-resolution accurate mass spectrometry as a technique for characterization of complex lysimeter leachate samples.

Lysimeter studies can be used to identify and quantify soil degradates of agrochemicals (metabolites) that have the potential to leach to groundwater. However, the apparent metabolic profile of such lysimeter leachate samples will often be significantly more complex than would be expected in true groundwater samples. This is particularly true for S-metolachlor, which has an extremely complex metabolic pathway. Consequently, it was not practically possible to apply a conventional analytical approach to identify all metabolites in an S-metolachlor lysimeter study, because there was insufficient mass to enable the use of techniques such as nuclear magnetic resonance. Recent advances in high-resolution accurate mass spectrometry, however, allow innovative screening approaches to characterize leachate samples to a greater extent than previously possible. Leachate from the S-metolachlor study was screened for accurate masses (±5 ppm of the nominal mass) corresponding to more than 400 hypothetical metabolite structures. A refined list of plausible metabolites was constructed from these data to provide a comprehensive description of the most likely metabolites present. The properties of these metabolites were then evaluated using a principal component analysis model, based on molecular descriptors, to visualize the entire chemical space and to cluster the metabolites into a number of subclasses. This characterization and principal component analysis evaluation enabled the selection of suitable representative metabolites that were subsequently used as exemplars to assess the toxicological relevance of the leachate as a whole. Environ Toxicol Chem 2016;35:1401-1412. © 2015 SETAC.

Bioinspired Synthesis of a Sedaxane Metabolite Using Catalytic Vanadyl Acetylacetonate and Molecular Oxygen.

A bioinspired synthesis of the sedaxane metabolite 2 from intermediate 3 using catalytic VO(acac)2 and O2 is described. Intermediate 3 was synthesized starting from 2-bromostyrene in four steps. The inner cyclopropyl ring of 3 was assembled with trans geometry using a highly diastereoselective Nishiyama cyclopropanation, and the outer hydroxycyclopropyl ring was installed using the Kulinkovich cyclopropanation. Additionally, conversion of 3 into 2 was demonstrated in in vitro microbial culture experiments consisting of bacteria and fungi.

Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues.

Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10(-7) M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k(cat) as that of wild-type enzyme but with altered K(m) values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of K(m)(GTN) to K(m)(NAD(P)H), with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.