Systematic Evaluation of the In-Sample Stability of Selected Pharmaceuticals, Illicit Drugs, and Their Metabolites in Wastewater.

The in-sample stability of selected pharmaceuticals, illicit drugs, and their metabolites in wastewater was assessed under six different conditions-untreated, addition of hydrochloric acid or sodium metabisulfite solution, combined with or without sterile filtration, and at four representative temperatures, at 35 °C for up to 28 days, 22 °C for 56 days, and 4 °C and -20 °C for 196 days, or freeze/thaw cycles for 24 weeks. Paracetamol, 6-monoacetylmorphine, morphine, and cocaine were poorly stable in untreated wastewater-e.g., with 50% transformation within 1.2-8.1 days at 22 °C, and acidification reduced their in-sample transformations. Acesulfame, carbamazepine, cotinine, methamphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), ketamine, norfentanyl, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and norbuprenorphine were highly or moderately stable over the observed period, even in untreated wastewater. Fitting of pseudo-first-order kinetics and the Arrhenius equation was used to develop a multistage transformation estimation model combined with an interactive tool to evaluate possible transformation scenarios of selected biomarkers for the processes from sampling to preanalysis. However, as the wastewater composition can vary between sites and over time, the variability of in-sample stability requires further exploration.

Toxicity of the herbicides diuron, propazine, tebuthiuron, and haloxyfop to the diatom Chaetoceros muelleri.

Conventional photosystem II (PSII) herbicides applied in agriculture can pose significant environmental risks to aquatic environments. In response to the frequent detection of these herbicides in the Great Barrier Reef (GBR) catchment area, transitions towards 'alternative' herbicides are now widely supported. However, water quality guideline values (WQGVs) for alternative herbicides are lacking and their potential ecological impacts on tropical marine species are generally unknown. To improve our understanding of the risks posed by some of these alternative herbicides on marine species under tropical conditions, we tested the effects of four herbicides on the widely distributed diatom Chaetoceros muelleri. The PSII herbicides diuron, propazine, and tebuthiuron induced substantial reductions in both 24 h effective quantum yields (ΔF/Fm') and 3-day specific growth rates (SGR). The effect concentrations, which reduced ΔF/Fm' by 50% (EC50), ranged from 4.25 µg L-1 diuron to 48.6 µg L-1 propazine, while the EC50s for SGR were on average threefold higher, ranging from 12.4 µg L-1 diuron to 187 µg L-1 tebuthiuron. Our results clearly demonstrated that inhibition of ΔF/Fm' in PSII is directly linked to reduced growth (R2 = 0.95) in this species, further supporting application of ΔF/Fm' inhibition as a valid bioindicator of ecological relevance for PSII herbicides that could contribute to deriving future WQGVs. In contrast, SGR and ΔF/Fm' of C. muelleri were nonresponsive to the non-PSII herbicide haloxyfop at the highest concentration tested (4570 µg L-1), suggesting haloxyfop does not pose a risk to C. muelleri. The toxicity thresholds (e.g. no effect concentrations; NECs) identified in this study will contribute to the derivation of high-reliability marine WQGVs for some alternative herbicides detected in GBR waters and support future assessments of the cumulative risks of complex herbicide mixtures commonly detected in coastal waters.

Effect of the glucocorticoid receptor antagonist RU486 on MK-801 induced behavioural sensitisation.

Stress is known to modulate sensitisation to repeated psychostimulant exposure. However, there is no direct evidence linking glucocorticoids and sensitisation achieved by repeated administration of the NMDA receptor antagonist MK-801. We tested the hypothesis that co-administration of RU486, a glucocorticoid receptor (GR) antagonist, prior to repeated daily MK-801 injections would block the expression of locomotor sensitisation due to its dual effects on corticosterone and dopamine. We employed a repeated MK-801 administration locomotor sensitisation paradigm in male Sprague Dawley rats. RU486 or a dimethyl sulfoxide (DMSO) vehicle was co-administered with MK-801 or saline during the induction phase. Subsequent to withdrawal, rats were challenged with MK-801 alone to test for the expression of sensitisation. In a separate cohort of rats, plasma corticosterone levels were quantified from blood samples taken on the 1st, 4th and 7th day of induction and at expression. One day after challenge, nucleus accumbens tissue levels of dopamine and its metabolites DOPAC and HVA were measured. During the induction phase, RU486 progressively enhanced locomotor sensitisation to MK-801. RU486 and MK-801 both showed stimulatory effects on corticosterone levels and this was further augmented when given in combination. Contrary to our hypothesis, RU486 did not block the expression of locomotor sensitisation to MK-801 and actually increased levels of dopamine, DOPAC and HVA in nucleus accumbens tissue. Our results showed that RU486 has augmentative rather than inhibitory effects on MK-801-induced sensitisation. This study indicates a divergent role for glucocorticoids in sensitisation to MK-801 compared to sensitisation with other psychostimulants.

Short- and long-term effects of risperidone on catalepsy sensitisation and acquisition of conditioned avoidance response: Adolescent vs adult rats.

The effects of antipsychotic drugs (APDs) on the adolescent brain are poorly understood despite a dramatic increase in prescription of these drugs in adolescents over the past twenty years. Neuronal systems continue to be remodeled during adolescence. Therefore, when given in adolescence, antipsychotic drugs (APDs) have the potential to affect this remodeling. In this study we investigated the effects of chronic 22-day risperidone treatment (1.3mg/kg/day) in both adolescent and adult rats. We examined short- and long-term changes in behaviour (catalepsy, locomotion and conditioned avoidance response (CAR)), and dopaminergic and serotonergic neurochemistry in the striatum and the nucleus accumbens. Here, we report that, both during chronic treatment and after a lengthy drug-free interval, risperidone induced a sensitised cataleptic response regardless of the age of exposure. Selectively in adolescents, risperidone-induced catalepsy was inversely correlated with striatal dopamine turnover immediately after chronic treatment. After a drug-free interval, a significant proportion of rats with prior adolescent risperidone treatment also failed to acquire CAR to a defined criterion. Our data provide evidence that the same chronic risperidone treatment regimen can induce contrasting short- and long-term neural outcomes in the adolescent and adult brains.

Deep venomics of the Pseudonaja genus reveals inter- and intra-specific variation.

Australian elapid venom remains an under-investigated resource of novel bioactive peptides. In this study, the venom gland transcriptomes and proteomes of the Australian western brown snakes, Pseudonaja aspidorhyncha and Pseudonaja nuchalis, were compared to Pseudonaja textilis. A deep venomics strategy incorporating high throughput 454 pyrosequencing gave a total of 200,911 raw reads for the three venoms. Subsequent annotation identified 5716 transcripts from 20 different toxin families with inter-specific variation between species observed in eight of the less abundant families. Integration of each venom proteome with the corresponding annotated reads identified 65 isoforms from six toxin families; high sequence coverage highlighted subtle differences between sequences and intra and inter-specific variation between species. High quality MS/MS data identified unusual glycoforms with natriuretic peptides from P. aspidorhyncha and P. nuchaliscontaining O-linked trisaccharides with high homology to the glycosylated region of TNPc. Molecular evolutionary assessments indicated the accelerated evolution of all toxin families with the exception of both natriuretic peptides and P. aspidorhyncha PLA2s that were found to be evolutionarily constrained under purifying selection pressures. This study has revealed a wide range of novel peptide sequences from six bioactive peptide families and highlights the subtle differences between toxins in these closely related species. BIOLOGICAL SIGNIFICANCE: Mining Australia's vastly untapped source of toxins from its venomous creatures has been significantly advanced by employing deep venomics methodology. Technological advances in transcriptome analysis using next generation sequencing platforms and proteome analysis by highly sensitive tandem mass spectrometry allowed a more comprehensive interrogation of three underinvestigated brown snake (Pseudonaja) venoms uncovering many novel peptide sequences that are unique to these closely related species. This generic strategy will provide invaluable information when applied to other venomous snakes for a deeper understanding of venom composition, envenomation, venom evolution, as well as identifying research tools and drug leads.

A defined α-helix in the bifunctional O-glycosylated natriuretic peptide TcNPa from the venom of Tropidechis carinatus.

Natriuretic peptides (NP) play important roles in human cardiac physiology through their guanylyl cyclase receptors NPR-A and NPR-B. Described herein is a bifunctional O-glycosylated natriuretic peptide, TcNPa, from Tropidechis carinatus venom and it unusually targets both NPR-A and NPR-B. Characterization using specific glycosidases and ETD-MS identified the glycan as galactosyl-ß(1-3)-N-acetylgalactosamine (Gal-GalNAc) and was α-linked to the C-terminal threonine residue. TcNPa contains the characteristic NP 17-membered disulfide ring with conserved phenylalanine and arginine residues. Both glycosylated and nonglycosylated forms were synthesized by Fmoc solid-phase peptide synthesis and NMR analysis identified an α-helix within the disulfide ring containing the putative pharmacophore for NPR-A. Surprisingly, both forms activated NPR-A and NPR-B and were relatively resistant towards proteolytic degradation in plasma. This work will underpin the future development of bifunctional NP peptide mimetics.

Isolation and structural and pharmacological characterization of α-elapitoxin-Dpp2d, an amidated three finger toxin from black mamba venom.

We isolated a novel, atypical long-chain three-finger toxin (TFT), α-elapitoxin-Dpp2d (α-EPTX-Dpp2d), from black mamba (Dendroaspis polylepis polylepis) venom. Proteolytic digestion with trypsin and V8 protease, together with MS/MS de novo sequencing, indicated that the mature toxin has an amidated C-terminal arginine, a posttranslational modification rarely observed for snake TFTs. α-EPTX-Dpp2d was found to potently inhibit α7 neuronal nicotinic acetylcholine receptors (nAChR; IC50, 58 ± 24 nM) and muscle-type nAChR (IC50, 114 ± 37 nM) but did not affect α3ß2 and α3ß4 nAChR isoforms at 1 µM concentrations. Competitive radioligand binding assays demonstrated that α-EPTX-Dpp2d competes with epibatidine binding to the Lymnea stagnalis acetylcholine-binding protein (Ls-AChBP; IC50, 4.9 ± 2.3 nM). The activity profile and binding data are reminiscent of classical long-chain TFTs with a free carboxyl termini, suggesting that amidation does not significantly affect toxin selectivity. The crystal structure of α-EPTX-Dpp2d was determined at 1.7 Å resolution and displayed a dimeric toxin assembly with each monomer positioned in an antiparallel orientation. The dimeric structure is stabilized by extensive intermolecular hydrogen bonds and electrostatic interactions, which raised the possibility that the toxin may exist as a noncovalent homodimer in solution. However, chemical cross-linking and size-exclusion chromatography coupled with multiangle laser light scattering (MALLS) data indicated that the toxin is predominantly monomeric under physiological conditions. Because of its high potency and selectivity, we expect this toxin to be a valuable pharmacological tool for studying the structure and function of nAChRs.