Role of saturated and unsaturated fatty acids on dicarbonyl-albumin derived advanced glycation end products in vitro.

Glycation is a non-enzymatic reaction that occurs between the free amino group of proteins and reducing sugars and/or lipids, leading to the formation of advanced glycation end products (AGEs). The reaction also produces reactive oxygen species that have detrimental effects on cellular and extracellular proteins. Aminoguanidine is a known inhibitor of AGEs, and some fatty acids are known to have a beneficial role in vivo by reducing inflammation and oxidative stress. However, the role of fatty acids on AGE formation has not been thoroughly reported. We investigated the role of a range of fatty acids in the formation of AGEs and their reactive intermediates using an in vitro BSA-dicarbonyl model. The model assessed a time-dependent (0-72 h) and dicarbonyl concentration (0-2 mM) -dependent studies for the optimal formation of AGEs. A 72 h time point was found to be optimal for the reaction of BSA with either methylglyoxal (MGO) or glyoxal (GO) to generate AGE-BSA complexes. When arachidonic, eicosapentaenoic or docosahexaenoic acids were included in the reaction, a significant decrease in protein-bound fluorescent AGEs was seen compared to the respective controls. In contrast, saturated and 18 carbon polyunsaturated fatty acids showed no significant activity. Liquid chromatography-mass spectrometry (LC-MS/MS) analysis showed saturated fatty acids significantly decreased the production of Nε-carboxymethyllysine (CML) and Nε-carboxyethyllysine (CEL) from GO and MGO models, respectively, whilst increasing methylglyoxal-derived hydroimidazolone (MG-H1). In contrast, arachidonic, eicosapentaenoic and docosahexaenoic acids did not significantly change either CEL or MG-H1 compared to no treatment controls whilst significantly reducing CML levels.

Pentafulvene-Maleimide Cycloaddition for Bioorthogonal Ligation.

The applications of bioconjugation chemistry are rapidly expanding, and the addition of new strategies to the bioconjugation and ligation toolbox will further advance progress in this field. Herein, we present a detailed study of the Diels-Alder cycloaddition (DAC) reaction between pentafulvenes and maleimides in aqueous solutions and investigate the reaction as an emerging bioconjugation strategy. The DAC reactions were found to proceed efficiently, quantitatively yielding cycloadducts with reaction rates ranging up to ∼0.7 M-1 s-1 for a series of maleimides, including maleimide-derivatized peptides and proteins. The absence of cross-reactivity of the pentafulvene with a large panel of functional (bio)molecules and biological media further demonstrated the bioorthogonality of this approach. The utility of the DAC reaction for bioorthogonal bioconjugation applications was further demonstrated in the presence of biological media and proteins, as well as through protein derivatization and labeling, which was comparable to the widely employed sulfhydryl-maleimide coupling chemistry.

Genotoxicity of advanced glycation end products in vitro is influenced by their preparation temperature, purification and cell exposure time.

Advanced glycation end products (AGEs) are formed via non-enzymatic reactions between amino groups of proteins and the carbonyl groups of reducing sugars. Previous studies have shown that highly glycated albumin prepared using a glucose-bovine serum albumin (Glu-BSA) model system incubated at 60°C for 6 weeks induces genotoxicity in WIL2-NS cells at 9 days of exposure measured by the cytokinesis-block micronucleus cytome (CBMNcyt) assay. However, this AGE model system is not physiologically relevant as normal body temperature is 37°C and the degree of glycation may exceed the extent of albumin modification in vivo. We hypothesised that the incubation temperature and purification method used in these studies may cause changes to the chemical profile of the glycated albumin and may influence the extent of genotoxicity observed at 3, 6 and 9 days of exposure. We prepared AGEs generated using Glu-BSA model systems incubated at 60°C or 37°C purified using trichloroacetic acid (TCA) precipitation or ultrafiltration (UF) and compared their chemical profile (glycation, oxidation, and aggregation) and genotoxicity in WIL2-NS cells using the CBMNcyt assay after 3, 6 and 9 days of exposure. The number of micronuclei (MNi) was significantly higher for cells treated with Glu-BSA incubated at 60°C and purified via TCA (12 ± 1 MNi/1000 binucleated cells) compared to Glu-BSA incubated at 37°C and purified using UF (6 ± 1 MNi/1000 binucleated cells) after 9 days (P < 0.0001). The increase in genotoxicity observed could be explained by a higher level of protein glycation, oxidation, and aggregation of the Glu-BSA model system incubated at 60°C relative to 37°C. This study highlighted that the incubation temperature, purification method and cell exposure time are important variables to consider when generating AGEs in vitro and will enable future studies to better reflect in vivo situations of albumin glycation.

Multisite Calibration of a Microporous Polyethylene Tube Passive Sampler for Quantifying Drugs in Wastewater.

Passive sampling approaches to monitor licit and illicit drugs of concern in wastewater shows promise as a supplementary sampling technique to grab sampling when conventional composite autosampling may not be possible. Recent studies have assessed the applicability of passive sampling at a single wastewater treatment plant (WWTP). However, it remains unknown whether a single-site calibration can be applied to other WWTPs. This study evaluated the in situ calibration of microporous polyethylene tube passive samplers (MPTs) against simultaneously collected composite samples for 22 different WWTPs. Samples were analyzed for methylamphetamine, amphetamine, hydroxycotinine, cotinine, benzoylecgonine, 3,4-methylenedioxymethamphetamine, and noroxycodone. Estimated rates of chemical uptake (sampling rates) were calculated using the mass accumulated in the samplers, the concentration measured in composite samples, and the duration of deployment. The estimated sampling rates were consistent between WWTPs (>90% within a factor of two) and ranged from 5 mL day-1 (amphetamine) to 9 mL day-1 (noroxycodone). The samplers were effective at estimating analyte concentrations, with 77% of results having a normalized difference to 24 h composite samples of below 30%. Our study suggests that MPT passive samplers provide a tool for the spatiotemporal monitoring of drug use where automated integrative sampling techniques may not be feasible.

A wastewater-based evaluation of the effectiveness of codeine control measures in Australia.

BACKGROUND AND AIM: From 1 February 2018, codeine was rescheduled from an over-the-counter (OTC) to a prescription-only medicine in Australia. We used wastewater-based epidemiology to measure changes in population codeine consumption before and after rescheduling. METHODS: We analysed 3703 wastewater samples from 48 wastewater treatment plants, taken between August 2016 and August 2019. Our samples represented 10.6 million people, 45% of the Australian population in state capitals and regional areas in each state or territory. Codeine concentrations were determined by liquid chromatography-tandem mass spectrometry and converted to per-capita consumption estimates using the site daily wastewater volume, catchment populations and codeine excretion kinetics. RESULTS: Average per-capita consumption of codeine decreased by 37% nationally immediately after the rescheduling in February 2018 [95% confidence interval (CI) = 35.3-39.4%] and substantially in all states between 24 and 51% (95% CI = 22.4-27.0% and 41.8-59.4%). The decrease was sustained at the lower level to August 2019. Locations with least pharmacy access decreased by 51% (95% CI = 41.7-61.7%), a greater decrease than 37% observed for those with greater pharmacy access (95% CI = 35.1-39.4%). Regional areas decreased by a smaller margin to cities (32 versus 38%, 95% CI = 30.2-34.1% versus 34.9-40.4%, respectively) from a base per-capita usage approximately 40% higher than cities. CONCLUSION: Wastewater analysis shows that codeine consumption in Australia decreased by approximately 37% following its rescheduling as a prescription-only medicine in 2018. Wastewater-based epidemiology can be used to evaluate changes in population pharmaceutical consumption in responses to changes in drug scheduling.

Methcathinone in wastewater: Drug of choice, or artefact?

Methcathinone is a prevalent Novel Psychoactive Substance (NPS) used illicitly in some countries. Routine analysis of wastewater sampled from catchments in South Australia has shown a consistent low-level presence of the compound, inconsistent with NPS use. This raised the question was the occurrence due to regular use as a drug of choice or was it an artefact being produced from other sources in the sewer system? NPS consumption is generally sporadic and would therefore point to the origin of methcathinone in wastewater being due to in-sewer oxidation of its legal precursor, pseudoephedrine. The present study tested this hypothesis by comparing the levels of pseudoephedrine and methcathinone in wastewater samples collected bimonthly from 8 catchment sites in South Australia. Laboratory experiments exposing pseudoephedrine to common household oxidizing agents (hypochlorite and percarbonate) were also performed and the production of methcathinone was demonstrated and monitored. The results of this study showed that the level of pseudoephedrine and methcathinone measured in wastewater followed a similar pattern. However, there were periods when the levels of each compound diverged. Laboratory experiments showed that when exposed to various oxidizing agents, pseudoephedrine is oxidised to non-stoichiometric quantities of methcathinone. Although the use of methcathinone as a drug of choice remains possible, the results of this study indicate that the low and persistent level of methcathinone found in wastewater may arise in part from the oxidation of pseudoephedrine in the sewer system.

Pholedrine is a marker of direct disposal of methamphetamine.

Consumption of methamphetamine has primarily been estimated in wastewater-based epidemiology by measuring the parent compound. However, this could lead to overestimation when methamphetamine is directly disposed into the sewer system. In this respect, it would be advantageous to measure a specific metabolite of methamphetamine instead. We identified 4-hydroxymethamphetamine (pholedrine) as a potential marker. Stability experiments were performed in both filtered and unfiltered wastewater. Correlations with relative loads in wastewater were used to establish its potential as a marker of direct disposal of methamphetamine, or even as a wastewater-based epidemiology biomarker of methamphetamine consumption. This study then investigated the use of pholedrine in combination with methamphetamine to better detect direct disposal events and its potential as a marker of methamphetamine consumption. Examples from both South Australia and New Zealand exemplify the use of pholedrine to identify potential instances of direct disposal of methamphetamine.

Changes in alcohol consumption associated with social distancing and self-isolation policies triggered by COVID-19 in South Australia: a wastewater analysis study.

AIM: To assess the effects of social distancing and social isolation policies triggered by COVID-19 on alcohol consumption using wastewater analysis in Adelaide, South Australia. DESIGN: Longitudinal quantitative analysis of influent wastewater data for alcohol concentration. SETTING: Adelaide, South Australia. PARTICIPANTS: Wastewater catchment area representative of 1.1 million inhabitants. MEASUREMENTS: Twenty-four hour composite influent wastewater samples were collected from four wastewater treatment plants in Adelaide, South Australia for 7 consecutive days (Wednesday-Tuesday) every 2 months from April 2016-April 2020. The alcohol metabolite ethyl sulfate was measured in samples using chromatography-tandem mass spectrometry. Data were population-weighted adjusted with consumption expressed as standard drinks/day/1000 people. Weekly consumption and weekend to mid-week consumption ratios were analysed to identify changes in weekday alcohol use pattern. FINDINGS: Estimated weekend alcohol consumption was significantly lower (698 standard drinks/day/1000 people) after self-isolation measures were enforced in April 2020 compared with the preceding sampling period in February 2020 (1047 standard drinks/day/1000 people), P < 0.05. Weekend to midweek consumption ratio was 12% lower than the average ratio compared with all previous sampling periods. April 2020 recorded the lowest alcohol consumption relative to April in previous years, dating back to 2016. CONCLUSIONS: Wastewater analysis suggests that introduction of social distancing and isolation policies triggered by COVID-19 in Adelaide, South Australia, was associated with a decrease in population-level weekend alcohol consumption.

Impact of COVID-19 Controls on the Use of Illicit Drugs and Alcohol in Australia.

Methamphetamine, MDMA, cocaine, cannabis, and alcohol in samples from 20 wastewater treatment plants servicing the eight state or territory capitals of Australia were analyzed, with equivalent coverage of >45% of the national population. Trends in drug consumption were calculated and assessed from samples collected from 2016 to 2020, with a focus on pre-COVID-19 (August 2016 to December 2019), versus February to June 2020, when Australia observed a nationwide lockdown. Results showed delayed but significant decreases in methamphetamine, >50% in Western Australia. In contrast, significant increases in cannabis in most jurisdictions were observed. This suggests changes in consumption may be somewhat linked to reduced supply of imported substances, with increased use of locally produced drugs. Initial decreases in cocaine and MDMA consumption were evident in many parts of the country, but pre-COVID trends were re-established after April 2020. Interestingly, weekend-weekday differences were narrowed for cocaine, MDMA, and alcohol during lockdown, which might be expected due to bars being closed and social gathering not allowed. With this study providing insight into the first four months of COVID-19 restrictions in Australia, it remains to be seen what the longer-term effect of the pandemic will be.

Determination of prescribed and designer benzodiazepines and metabolites in influent wastewater.

Benzodiazepines are important prescription pharmaceuticals used to help in the treatment of anxiety and sleep disorders. However, they also have a strong potential for abuse. In this respect, illicit benzodiazepines, i.e. not prescribed in Australia and designer benzodiazepines, which are new compounds that are not legally prescribed in any jurisdiction, have emerged in the illicit Australian market in recent years. Designer benzodiazepines are a new class of new psychoactive substances (NPS) and are particularly dangerous due to limited toxicity information and propensity to be mistaken for conventional benzodiazepines, leading to severe side effects and potentially death. It is therefore important to assess the prevalence of the use of these compounds in the community. The current work presents a validated liquid chromatography-mass spectrometry method for 20 prescribed and designer benzodiazepines and metabolites: 7-amino nimetazepam, alpha-hydroxy alprazolam, alprazolam, clonazepam, delorazepam, deschloroetizolam, diazepam, diclazepam, etizolam, flubromazepam, flunitrazepam, lorazepam, lormetazepam, meclonazepam, midazolam, nimetazepam, nitrazepam, oxazepam, pyrazolam and temazepam. Quetiapine, a prescription sedative drug that has been diverted for non-medical use, was also validated. Limits of quantification were predominantly below 10 ng L-1, except for the ubiquitous oxazepam, quetiapine and temazepam, which were between 75-300 ng L-1. Stability, recovery and matrix effects were also examined. Finally, this method was applied to influent wastewater from South Australia, which showed the presence of many benzodiazepines including the NPS etizolam.

Anabasine-based measurement of cigarette consumption using wastewater analysis.

Community tobacco use can be monitored over time using wastewater-based epidemiological approaches by estimating the mass loads of nicotine and its metabolites, cotinine, or hydroxycotinine, in wastewater. However, due to the use of nicotine in smoking cessation products, other sources of nicotine contribute to cotinine and hydroxycotinine loads. The use of nicotine replacement therapies could vary in space and time and mask the true rates of tobacco consumption. Therefore, this work evaluated the content of tobacco specific markers, anatabine and anabasine, in cigarettes, in urine of smokers, and in wastewater. The results indicated that the anabasine content in both licit and illicit cigarettes in Australia is less variable than anatabine and is therefore considered a better measure of tobacco consumption. A study determining the excretion of tobacco-specific alkaloids of smoking and non-smoking volunteers gave an average urinary mass load of anabasine of 4.38 µg/L/person and a daily mass load of 1.13 µg/day/person. Finally, this was compared with the mass loads of anabasine from wastewater-based epidemiology data of 3 µg/day/person to estimate cigarette rates in a South Australian city: equivalent to 2.6 cigarettes/person/day. The rate of decline of cigarette use was greater when using anabasine as a measure of consumption compared with cotinine. This is the first study to estimate the rate of anabasine excretion, which can be used to estimate tobacco use independent of therapeutically prescribed nicotine.

Simultaneous determination of 24 opioids, stimulants and new psychoactive substances in wastewater.

Wastewater-based epidemiology has become a reputable means to estimate drug consumption within a community. However, these methods typically focus solely on illicit drugs or a single chemical family, with multi-class methods out of favour due to the increased analytical challenges. •A sensitive liquid chromatography - mass spectrometry method was developed for the simultaneous determination of 24 opioids, stimulants and new psychoactive substances in influent wastewater.•Filtered wastewater samples, preserved with sodium metabisulfite, were pretreated and 1000 times concentrated using off-line solid phase extraction.•The method was optimised and fully validated for all compounds, with limits of quantification between 0.2 and 300 ng/L.