BRCA1 protein dose-dependent risk for embryonic oxidative DNA damage, embryopathies and neurodevelopmental disorders with and without ethanol exposure.

Although widely known as a tumor suppressor, the breast cancer 1 susceptibility protein (BRCA1) is also important in development, where it regulates fetal DNA repair pathways that protect against DNA damage caused by physiological and drug-enhanced levels of reactive oxygen species (ROS). We previously showed that conditional heterozygous (+/-) knockout (cKO) mouse embryos with a minor 28% BRCA1 deficiency developed normally in culture, but when exposed to the ROS-initiating drug, alcohol (ethanol, EtOH), exhibited embryopathies not evident in wild-type (+/+) littermates. Herein, we characterized a directBrca1 +/- knockout (KO) model with a 2-fold greater (58%) reduction in BRCA1 protein vs. the cKO model. We also characterized and compared learning & memory deficits in both the cKO and KO models. Even saline-exposed Brca1 +/- vs. +/+ KO progeny exhibited enhanced oxidative DNA damage and embryopathies in embryo culture and learning & memory deficits in females in vivo, which were not observed in the cKO model, revealing the potential pathogenicity of physiological ROS levels. The embryopathic EtOH concentration for cultured direct KO embryos was half that for cKO embryos, and EtOH affected Brca1 +/+ embryos only in the direct KO model. The spectrum and severity of EtOH embryopathies in culture were greater in both Brca1 +/- vs. +/+ embryos, and direct KO vs. cKO +/- embryos. Motor coordination deficits were evident in both male and female Brca1 +/- KO progeny exposed in utero to EtOH. The results in our direct KO model with a greater BRCA1 deficiency vs. cKO mice provide the first evidence for BRCA1 protein dose-dependent susceptibility to developmental disorders caused by physiological and drug-enhanced oxidative stress.

Identification of Emerging Novel Psychoactive Substances by Retrospective Analysis of Population-Scale Mass Spectrometry Data Sets.

Over the last two decades, hundreds of new psychoactive substances (NPSs), also known as "designer drugs", have emerged on the illicit drug market. The toxic and potentially fatal effects of these compounds oblige laboratories around the world to screen for NPS in seized materials and biological samples, commonly using high-resolution mass spectrometry. However, unambiguous identification of a NPS by mass spectrometry requires comparison to data from analytical reference materials, acquired on the same instrument. The sheer number of NPSs that are available on the illicit market, and the pace at which new compounds are introduced, means that forensic laboratories must make difficult decisions about which reference materials to acquire. Here, we asked whether retrospective suspect screening of population-scale mass spectrometry data could provide a data-driven platform to prioritize emerging NPSs for assay development. We curated a suspect database of precursor and diagnostic fragment ion masses for 83 emerging NPSs and used this database to retrospectively screen mass spectrometry data from 12,727 urine drug screens from one Canadian province. We developed integrative computational strategies to prioritize the most reliable identifications and tracked the frequency of these identifications over a 3 year study period between August 2019 and August 2022. The resulting data were used to guide the acquisition of new reference materials, which were in turn used to validate a subset of the retrospective identifications. Last, we took advantage of matching clinical reports for all 12,727 samples to systematically benchmark the accuracy of our retrospective data analysis approach. Our work opens up new avenues to enable the rapid detection of emerging illicit drugs through large-scale reanalysis of mass spectrometry data.

Desalkylgidazepam blood concentrations in 63 forensic investigation cases.

Desalkylgidazepam, also known as bromonordiazepam, is the latest designer benzodiazepine to appear in postmortem blood samples in British Columbia. Our laboratory was first alerted to the presence of desalkylgidazepam in seized drug samples in May 2022, and the analyte was added to an in-house library shortly thereafter. Previously acquired spectra from routine death investigation cases were reprocessed using the updated library with the first presumptive identification of desalkylgidazepam occurring in a sample received in April 2022. A standard addition method for the quantitation of desalkylgidazepam in blood samples (from femoral, iliac, jugular and subclavian veins) was validated and consequently used to confirm presence and concentrations of the drug in 63 cases, with an average concentration of 42.2 ± 44.0 ng/mL (median concentration: 24.5 ng/mL; range: 3.7-220.6 ng/mL). Similar to detections of other novel benzodiazepines, co-occurrence of desalkylgidazepam with opioids and/or stimulants was common. To our knowledge, this paper is the first to report desalkylgidazepam concentrations in postmortem blood samples.

Bromazolam Blood Concentrations in Postmortem Cases-A British Columbia Perspective.

Bromazolam is a designer benzodiazepine that was first detected in British Columbia in January 2021. Postmortem cases were analyzed using a comprehensive blood drug screening procedure by liquid chromatography-high-resolution mass spectrometry before being retrospectively analyzed using an in-house novel psychoactive substances data processing method. Bromazolam was detected in 41 postmortem cases in 2021 and quantitatively confirmed by standard addition, using liquid chromatography-tandem mass spectrometry. The mean bromazolam concentration observed was 11.4 ± 53.7 ng/mL (median concentration: 1.6 ng/mL), with a range from 0.5 to 319.3 ng/mL and the majority of cases co-occurring with fentanyl. These low concentrations may be indicative of a presumed enhancement of opioid effects, rather than being used as a stand-alone drug. Bromazolam was always detected with opioids (fentanyl and carfentanil), stimulants (methamphetamine) and/or other benzodiazepines (etizolam and flualprazolam). To our knowledge, this is the first report to provide concentrations of bromazolam in postmortem blood samples in Canada.

Examining fentanyl and its analogues in the unregulated drug supply of British Columbia, Canada using drug checking technologies.

INTRODUCTION: The emergence of fentanyl and its analogues have contributed to a drastic rise in overdose-related mortality in recent years. The objective of this study was to determine the number of drug checking samples containing fentanyl and fentanyl analogues using both point of care and confirmatory drug checking technologies. METHODS: Point-of-care drug checking data, using a combination of fentanyl immunoassay strips and Fourier-transform infrared spectroscopy (FTIR), were collected at harm reduction sites in Vancouver and Surrey, British Columbia. Based on current recommendations from the British Columbia Centre on Substance Use Drug Checking Project, a subset of these samples was sent for confirmatory analysis using quantitative nuclear resonance spectroscopy, gas chromatography-mass spectrometry and/or liquid chromatography-mass spectrometry. RESULTS: A total of 22,916 samples were tested using FTIR and fentanyl immunoassay strips, of which 6125 (29%) were positive for fentanyl and/or fentanyl analogues. FTIR identified a fentanyl analogue in five samples (all carfentanil). Of the 1467 samples sent for confirmatory analysis, fentanyl was identified in 855 (58%) and fentanyl analogues in 85 (6%), including: carfentanil (n = 56), acetyl fentanyl (n = 15), furanyl fentanyl (n = 9) and cyclopropyl fentanyl (n = 5). DISCUSSION AND CONCLUSION: Our research found that FTIR does not consistently distinguish between fentanyl and its analogues at point of care and that highly sensitive confirmatory drug checking technologies are needed to identify fentanyl analogues. These findings underscore the limitations of current drug checking technologies and the importance of using both point of care and confirmatory drug checking initiatives for monitoring changes in the drug supply.

Fatal ingestion of Taxus baccata: English yew.

Poisoning from consumption of foraged alternative medicine products is an uncommon yet recognized occurrence. Here, presented is the case of a 40-year-old woman who was witnessed to collapse with labored breathing and subsequently died despite emergency medical personnel attendance and resuscitation efforts. Autopsy revealed the presence of plant matter that was visually identified as leaves from Taxus baccata - the English Yew. Isolation of alkaloids from the plant material and subsequent identification of the same alkaloids in the decedent's blood by liquid chromatography-tandem mass spectrometry indicated a toxicological cause of death. This case illustrates a collaborative team approach among subject matter experts to unexpectedly discover and then confirm the sudden death of this woman from T. baccata toxicity.

An outbreak of novel psychoactive substance benzodiazepines in the unregulated drug supply: Preliminary results from a community drug checking program using point-of-care and confirmatory methods.

BACKGROUND: From mid-2018, an increase in novel psychoactive substance (NPS) benzodiazepines was noted on surveillance of the unregulated drug market around Vancouver, British Columbia, Canada. The rise was concordant with an outbreak of atypical overdoses suspicious for benzodiazepine adulteration of unregulated opioids. This study sought to describe the number and type of NPS benzodiazepines in a sample drawn from a community drug checking program during this period, and to explore accuracy of point-of-care drug checking technologies when compared to confirmatory methods in this sample. METHODS: Point-of-care drug checking data using fentanyl and benzodiazepine test strips as well as Fourier transform infrared spectroscopy were gathered at harm reduction sites in the Vancouver area from October 2018 to January 2020. A convenience subsample underwent confirmatory testing with gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or quantitative nuclear magnetic resonance spectroscopy. RESULTS: Of 159 samples with both point-of-care and confirmatory results, 24 (15.1%) contained at least one NPS benzodiazepine, including etizolam (n = 18), flubromazolam (n = 3), flualprazolam (4), and flubromazepam (n = 1). Of 114 confirmatory samples expected by participants on self-report to contain opioids, 18 (15.8%) contained some NPS benzodiazepine, with 16 (14.0%) containing both an NPS benzodiazepine and an opioid, always fentanyl. False positive and negative rates were 15.5% and 37.5% for test strips, and 3.9% and 91.7% for FTIR, respectively. Combined together, false positive and negative rates of point-of-care methods were 17.8% and 29.2%. CONCLUSIONS: NPS benzodiazepine adulteration in an unregulated drug supply sample reveals new risks compounding ongoing harms associated with the synthetic opioid epidemic. Given substantial false positive and false negative rates noted in our sample for point-of-care detection methods, cautious use of combined point-of-care methods, routinely paired with confirmatory drug checking may aid in early detection and monitoring of unregulated drug markets and inform targeted harm reduction strategies and health policy approaches.

Detection of synthetic cannabinoid adulteration in the unregulated drug supply in three Canadian settings.

INTRODUCTION: Novel psychoactive substances (NPS) are increasingly being consumed worldwide, with synthetic cannabinoids and synthetic opioids being the second and third most commonly used NPS, respectively. Certain synthetic cannabinoids can produce significant harms, particularly when used with opioids. The objective of this study was to characterise the presence of synthetic cannabinoids in the unregulated drug supply in three Canadian settings METHODS: In the British Columbia setting, all samples were first analysed at point-of-care using combination Fourier-transform infrared (FTIR) spectroscopy and fentanyl immunoassay strips prior to confirmatory testing using quantitative nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry (GC/MS) and/or liquid chromatography/mass spectrometry (LC/MS). In the Toronto, Ontario setting, the samples were analysed directly by GC/MS, LC/MS liquid chromatography-high resolution/mass spectrometry. RESULTS: Between January 2018 and December 2019, 38 (2.8%) synthetic cannabinoid samples were detected in the unregulated drug supply (25/909 in British Columbia and 13/440 in Ontario). In British Columbia and Ontario, 76% and 85% of samples, respectively, were expected by individuals to be an opioid. Synthetic cannabinoids detected included AMB-FUBINACA, AB-FUBINACA, 5-fluoro-MDMB-PINACA, and 5-fluoro-MDMB-PICA, and largely co-occurred with fentanyl. In the British Columbia context, Fourier-transform infrared spectroscopy failed to detect synthetic cannabinoid compounds in almost half (48%) of the samples at point-of-care. DISCUSSION AND CONCLUSIONS: As point-of-care technologies failed to detect these compounds in many occasions, our findings demonstrate the importance of laboratory confirmatory analysis to identify NPS. Given the high risk of harm associated with the consumption of synthetic cannabinoids, further research should investigate the reasons for adulteration.

Drug checking identifies counterfeit alprazolam tablets.

BACKGROUND: Non-prescribed benzodiazepine use is increasing in North America, especially among youth. Owing to increasing demand, counterfeit benzodiazepine tablets are mass-produced in clandestine, unregulated environments and sold as legitimate pharmaceuticals. This study aimed to examine the contents of counterfeit alprazolam tablets available in the unregulated drug market in British Columbia, Canada. METHODS: Data were collected from an ongoing evaluation of a community drug checking service in British Columbia between October 2017 and March 2020. The service operates point-of-care in harm reduction sites using Fourier-transform infrared (FTIR) spectrometers coupled with fentanyl and benzodiazepine immunoassay strips. A subset of samples were sent for confirmatory analysis at partner laboratories and underwent one or more of gas chromatography/mass spectrometry, liquid chromatography/mass spectrometry, and quantitative nuclear magnetic resonance analysis. RESULTS: During the study period, 10,814 total samples were submitted for drug checking, 139 of which were expected to be Xanax (alprazolam) or generic tablets and met the criteria for inclusion. Using FTIR analysis, 33 (23.7 %) samples were identified to contain alprazolam. Only 122 samples were checked using benzodiazepine immunoassay strips and 88 (72.1 %) tested positive. Qualitative results from the 20 samples submitted for confirmatory analysis included various new psychoactive substances and only 2 contained only alprazolam. CONCLUSIONS: Our findings provide evidence that Xanax tablets obtained from the unregulated drug market are likely to be counterfeit and may not contain alprazolam. Drug checking offers people who use drugs a valuable means to determine the contents of their substances; however, limitations of point-of-care technologies must be considered.

Association of Racial/Ethnic Segregation With Treatment Capacity for Opioid Use Disorder in Counties in the United States.

Importance: Treatment with methadone or buprenorphine is the current standard of care for opioid use disorder. Given the paucity of research identifying which patients will respond best to which medication, both medications should be accessible to all patients so that patients can determine which works best for them. However, given differences in the historical contexts of their initial implementation, access to each of these medications may vary along racial/ethnic lines. Objective: To examine the extent to which capacity to provide methadone and buprenorphine vary with measures of racial/ethnic segregation. Design, Setting, and Participants: This cross-sectional study included all counties and county-equivalent divisions in the US in 2016. Data on racial/ethnic population distribution were derived from the American Community Survey, and data on locations of facilities providing methadone and buprenorphine were obtained from Substance Abuse and Mental Health Services Administration databases. Data were analyzed from August 22, 2018, to September 11, 2019. Exposures: Two county-level measures of racial/ethnic segregation, including dissimilarity (representing the proportion of African American or Hispanic/Latino residents who would need to move census tracts to achieve a uniform spatial distribution of the population by race/ethnicity) and interaction (representing the probability that an African American or Hispanic/Latino resident will interact with a white resident and vice versa, assuming random mixing across census tracts). Main Outcomes and Measures: County-level capacity to provide methadone or buprenorphine, defined as the number of facilities providing a medication per 100 000 population. Results: Among 3142 US counties, there were 1698 facilities providing methadone (0.6 facilities per 100 000 population) and 18 868 facilities providing buprenorphine (5.9 facilities per 100 000 population). Each 1% decrease in probability of interaction of an African American resident with a white resident was associated with 0.6 more facilities providing methadone per 100 000 population. Similarly, each 1% decrease in probability of interaction of a Hispanic/Latino resident with a white resident was associated with 0.3 more facilities providing methadone per 100 000 population. Each 1% decrease in the probability of interaction of a white resident with an African American resident was associated with 8.17 more facilities providing buprenorphine per 100 000 population. Similarly, each 1% decrease in the probability of interaction of a white resident with a Hispanic/Latino resident was associated with 1.61 more facilities providing buprenorphine per 100 000 population. Conclusions and Relevance: These findings suggest that the racial/ethnic composition of a community was associated with which medications residents would likely be able to access when seeking treatment for opioid use disorder. Reforms to existing regulations governing the provisions of these medications are needed to ensure that both medications are equally accessible to all.

A call to maximize impact of the SUPPORT for Patients and Communities Act through standard inclusion of opioid use disorder treatment curricula in medical schools.

Physicians who want to prescribe buprenorphine to treat opioid use disorder require a waiver established by the Drug Addiction Treatment Act (DATA) of 2000, often through completion of an eight-hour training course. This is an issue for a number of reasons, including that opioid overdose deaths continue to rise nationally. However, on October 24, 2018, the SUPPORT (Substance Use-Disorder Prevention that Promotes Opioid Recovery and Treatment) for Patients and Communities Act was signed into law. This bill allows any physician who graduates in good standing from an allopathic or osteopathic medical school in the United States that incorporates necessary material around opioid misuse in their standard curriculum, without need for any additional training, to prescribe buprenorphine. This perspective piece describes why this is an important first step and what more needs to be done within medical education to combat the opioid epidemic.

Measurement of the Oxidative DNA Lesion 8-Oxoguanine (8-oxoG) by ELISA or by High-Performance Liquid Chromatography (HPLC) with Electrochemical Detection.

Reactive oxygen species (ROS) can oxidize cellular macromolecules like DNA, causing DNA damage. The most common form of DNA damage is the 8-oxoguanine (8-oxoG) lesion, typically repaired by the base excision repair (BER) pathway, which is initiated by the enzyme oxoguanine glycosylase 1 (OGG1). ROS are produced endogenously and can be enhanced by environmental factors, such as xenobiotics, radiation, and microbial pathogens. As a commonly used biomarker of oxidative damage, 8-oxoG can be measured in two different ways described herein. Commercially available ELISA kits allow for easy detection of the 8-oxoG lesion, while more difficult HPLC assays with UV and electrochemical detection allow for a more definitive identification and quantification of 8-oxoG.

Breast cancer 1 (BRCA1)-deficient embryos develop normally but are more susceptible to ethanol-initiated DNA damage and embryopathies.

The breast cancer 1 (brca1) gene is associated with breast and ovarian cancers, and heterozygous (+/-) brca1 knockout progeny develop normally, suggesting a negligible developmental impact. However, our results show BRCA1 plays a broader biological role in protecting the embryo from oxidative stress. Sox2-promoted Cre-expressing hemizygous males were mated with floxed brca1 females, and gestational day 8 +/- brca1 conditional knockout embryos with a 28% reduction in protein expression were exposed in culture to the reactive oxygen species (ROS)-initiating drug ethanol (EtOH). Untreated +/- brca1-deficient embryos developed normally, but when exposed to EtOH exhibited increased levels of oxidatively damaged DNA, measured as 8-oxo-2'-deoxyguanosine, γH2AX, which is a marker of DNA double strand breaks that can result from 8-oxo-2'-deoxyguanosine, formation, and embryopathies at EtOH concentrations that did not affect their brca1-normal littermates. These results reveal that even modest BRCA1 deficiencies render the embryo more susceptible to drug-enhanced ROS formation, and corroborate a role for DNA oxidation in the mechanism of EtOH teratogenesis.

A role for glutathione, independent of oxidative stress, in the developmental toxicity of methanol.

Oxidative stress and reactive oxygen species (ROS) have been implicated in the teratogenicity of methanol (MeOH) in rodents, both in vivo and in embryo culture. We explored the ROS hypothesis further in vivo in pregnant C57BL/6J mice. Following maternal treatment with a teratogenic dose of MeOH, 4 g/kg via intraperitoneal (ip) injection on gestational day (GD) 12, there was no increase 6h later in embryonic ROS formation, measured by 2',7'-dichlorodihydrofluorescin diacetate (DCFH-DA) fluorescence, despite an increase observed with the positive control ethanol (EtOH), nor was there an increase in embryonic oxidatively damaged DNA, quantified as 8-oxo-2'-deoxyguanosine (8-oxodG) formation. MeOH teratogenicity (primarily ophthalmic anomalies, cleft palate) also was not altered by pre- and post-treatment with varying doses of the free radical spin trapping agent alpha-phenyl-N-tert-butylnitrone (PBN). In contrast, pretreatment with L-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, depleted maternal hepatic and embryonic GSH, and enhanced some new anomalies (micrognathia, agnathia, short snout, fused digits, cleft lip, low set ears), but not the most common teratogenic effects of MeOH (ophthalmic anomalies, cleft palate) in this strain. These results suggest that ROS did not contribute to the teratogenic effects of MeOH in this in vivo mouse model, in contrast to results in embryo culture from our laboratory, and that the protective effect of GSH in this model may arise from its role as a cofactor for formaldehyde dehydrogenase in the detoxification of formaldehyde.

Embryonic catalase protects against ethanol-initiated DNA oxidation and teratogenesis in acatalasemic and transgenic human catalase-expressing mice.

Reactive oxygen species (ROS) are implicated in fetal alcohol spectrum disorders (FASD) caused by alcohol (ethanol, EtOH). Although catalase detoxifies hydrogen peroxide, embryonic catalase activity is only about 5% of maternal levels. To determine the roles of ROS and embryonic catalase in FASD, pregnant mice with enhanced (expressing human catalase, hCat) or deficient (acatalasemic, aCat) catalase activity, or their respective wild-type (WT) controls, were treated ip on gestational day 9 with 4 or 6g/kg EtOH or its saline vehicle, and embryos and fetuses were, respectively, evaluated for oxidatively damaged DNA and structural anomalies. Untreated hCat and aCat dams had, respectively, more and less offspring than their WT controls. hCat progenies were protected from all EtOH fetal anomalies at the low dose (p < .01) and from reduced head diameter and resorptions at the high dose (p < .001). Conversely, aCat progenies were more sensitive to dose-dependent EtOH fetal anomalies (p < .001) and exhibited a 50% increase in maternal lethality (p < .05) at the high dose. Maternal pretreatment of aCat mice with polyethylene glycol-conjugated catalase (PEG-Cat) reduced EtOH fetal anomalies (p < .001). EtOH-initiated embryonic DNA oxidation was reduced in hCat and WT mice pretreated with PEG-Cat and enhanced in aCat mice. Plasma concentrations of EtOH in catalase-altered mice were similar to controls, precluding a pharmacokinetic basis for altered EtOH teratogenesis. Endogenous embryonic catalase, despite its low level, is an important embryoprotective enzyme for EtOH teratogenesis and a likely determinant of individual risk.