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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA–DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

AbstractBackground: Community pharmacists are among the frontline health professionals who manage patients with an opioid-related disorder (ORD). Pharmacists frequently have a negative attitude toward these patients, which could have a negative impact on their management. However, education on ORD may improve the attitude of future healthcare professionals. This cross-sectional study aimed to assess French pharmacy students’ perceptions of ORD. Methods: This online survey was performed by emails sent to French pharmacy schools (between January 14, 2019 and May 31, 2019). The primary outcome was the perception (visual analogic scale) of ORD as a disease, the roles of community pharmacies (delivery of opioid agonist therapy—OAT and harm reduction kits), and the efficacy of OAT. The secondary outcomes assessed professional experience, university experience of and education on ORD, and the individual characteristics of students. Results: Among the 1,994 students included, 76.3% perceived ORD as a disease and felt that it was normal for pharmacists to deliver OAT (78.9%) and harm reduction kits (74.6%). However, only 46.9% perceived OAT as being effective. Multivariable analyses showed that females had a more positive perception in recognizing ORD as a disease. The progression through university years increased the positive perception of ORD as a disease and the delivery of OAT and harm reduction kits by pharmacists. Education on substance-related disorders had no impact on any scores. Students who had already delivered OAT had a negative perception of their efficacy. The students who had already performed pharmacy jobs or traineeships had a negative perception of harm reduction kit delivery. Conclusion: Education on substance-related disorders had no impact on students’ perceptions. It seemed that the maturity acquired through university years had a stronger impact on the students’ perceptions of ORD. Efforts must be made to improve our teaching methods and reinforce the confidence of students in the roles of community pharmacists.

Background Community pharmacists are among the frontline health professionals who manage patients with an opioid-related disorder (ORD). Pharmacists frequently have a negative attitude toward these patients, which could have a negative impact on their management. However, education on ORD may improve the attitude of future healthcare professionals. This cross-sectional study aimed to assess French pharmacy students’ perceptions of ORD.Methods This online survey was performed by emails sent to French pharmacy schools (between January 14, 2019 and May 31, 2019). The primary outcome was the perception (visual analogic scale) of ORD as a disease, the roles of community pharmacies (delivery of opioid agonist therapy—OAT and harm reduction kits), and the efficacy of OAT. The secondary outcomes assessed professional experience, university experience of and education on ORD, and the individual characteristics of students.Results Among the 1,994 students included, 76.3% perceived ORD as a disease and felt that it was normal for pharmacists to deliver OAT (78.9%) and harm reduction kits (74.6%). However, only 46.9% perceived OAT as being effective. Multivariable analyses showed that females had a more positive perception in recognizing ORD as a disease. The progression through university years increased the positive perception of ORD as a disease and the delivery of OAT and harm reduction kits by pharmacists. Education on substance-related disorders had no impact on any scores. Students who had already delivered OAT had a negative perception of their efficacy. The students who had already performed pharmacy jobs or traineeships had a negative perception of harm reduction kit delivery.Conclusion Education on substance-related disorders had no impact on students’ perceptions. It seemed that the maturity acquired through university years had a stronger impact on the students’ perceptions of ORD. Efforts must be made to improve our teaching methods and reinforce the confidence of students in the roles of community pharmacists.

Following exposure to xenobiotics, cellular mechanisms will take place to prevent damages relative to i) DNA, to maintain genome integrity, ii) proteins, to maintain their activities, iii) lipids, to limit peroxidation. This system of cellular defence and resistance is energetically costly. In this commentary review, we discuss the impact of DNA damage and the DNA damage response (DDR) on energetic metabolism. Conversely, we address the question about how energetic metabolism would influence DDR. These points will be highlighted in the context of cancer, in which genome instability and aerobic glycolysis are cancer hallmarks.

Beside their crucial role in xenobiotic biotransformation, AhR and Nrf2 are involved in the regulation of energetic metabolism. Nrf2, as a cytoprotective transcription factor, supply energy for detoxification and antioxidant response. AhR is ligand-activated transcriptional factor implicated in several physiological function. Following pollutant exposure, it acts as a chemosensor to eliminate harmful chemicals. Altogether, AhR and Nrf2 cooperate to contribute to protective responses upon contaminant exposure, including metabolic remodelling. This fine-tuned energetic coupling with detoxification can lead to prominent dysfunction under a chronic exposure to pollutants, and promote a long-term reprogramming that affects multiple cellular activities.

Pesticide exposure assessment is a key methodological issue for epidemiological studies. The history of pesticide has proven difficult to obtain from individuals’ report because of the wide range of active ingredients (AIs). We developed a crop-exposure matrix, which intends to reconstitute parameters of pesticide exposure in France since 1950. PESTIMAT is composed of tables crossing crops and AIs by year and providing the following metrics: (1) probability (proportion of farmers having used the AIs); (2) frequency (number of treatment days); and (3) intensity (application rate of the AIs in kg/ha). Metrics were obtained by the combination of six sources: (i) registration information from the Agriculture Ministry; (ii) information from agricultural bodies on products marketed; (iii) agricultural recommendations by the Plant Health Protection body; (iv) treatment calendars provided by farmers; (v) data from associations of farmers; and (vi) data from the industry. To date, 529 AIs usable between 1950 and 2010 are included in PESTIMAT: 160 fungicides; 160 herbicides; and 209 insecticides. When combined with duration and determinants of intensity, the metrics in PESTIMAT will make it possible to calculate exposure scores and to search for dose–effect relationships, an important criterion for causality judgment in epidemiology.

Neuroleptic drugs have been suggested to act as inverse agonists at the dopamine D2receptor, but no link between therapeutic efficacy and ligand’s intrinsic activity could be determined. Since the resolving capacity to monitor inverse agonism at dopamine D2receptors is limited, we speculated that receptor constitutive activation could be enhanced by constructing chimeric D2/α1Breceptors. Marked inverse agonist responses with a series of dopamine antagonists were obtained by: 1) exchange of the D2shortreceptor’s 3ICL by that of the α1B-adrenoceptor, 2) incorporation of an activating mutation (Ala279Glu) in the distal portion of its 3ICL, and 3) coexpression with a Gα11protein. This chimeric D2/α1Breceptor construct displayed a ligand binding profile comparable to that of the wild-type (wt) D2shortreceptor and an effector activation profile close to that of the wt α1B-adrenoceptor. Most of the dopamine antagonists attenuated by −54 to −59% basal inositol phosphates (IP) formation, thus clearly acting as inverse agonists. Ziprasidone behaved as a silent antagonist (+5% versus basal IP level) and antagonized both dopamine-mediated (p KB, 7.61) and tropapride-mediated (p KB, 8.52) IP responses. Clozapine, olanzapine, and raclopride displayed partial inverse agonist properties (−31, −67, and −71% versus tropapride, respectively), whereas bromerguride (+63%) and cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino tetralin) [(+)-UH 232] (+88%) demonstrated positive agonism. In conclusion, analyses with the chimeric D2/α1BAla279Glu 3ICL receptor construct suggest that neuroleptic drugs can be differentiated on the basis of their intrinsic activity, as they can either activate, inhibit, or be silent at this receptor construct.

Neuroleptic drugs have been suggested to act as inverse agonists at the dopamine D2 receptor, but no link between therapeutic efficacy and ligand's intrinsic activity could be determined. Since the resolving capacity to monitor inverse agonism at dopamine D2 receptors is limited, we speculated that receptor constitutive activation could be enhanced by constructing chimeric D2/alpha 1B receptors. Marked inverse agonist responses with a series of dopamine antagonists were obtained by: 1) exchange of the D 2short receptor's 3ICL by that of the alpha 1B-adrenoceptor, 2) incorporation of an activating mutation (Ala 279 Glu) in the distal portion of its 3ICL, and 3) coexpression with a G alpha11 protein. This chimeric D2/alpha 1B receptor construct displayed a ligand binding profile comparable to that of the wild-type (wt) D 2short receptor and an effector activation profile close to that of the wt alpha 1B-adrenoceptor. Most of the dopamine antagonists attenuated by -54 to -59% basal inositol phosphates (IP) formation, thus clearly acting as inverse agonists. Ziprasidone behaved as a silent antagonist (+5% versus basal IP level) and antagonized both dopamine-mediated (pK B, 7.61) and tropapride-mediated (pK B, 8.52) IP responses. Clozapine, olanzapine, and raclopride displayed partial inverse agonist properties (-31, -67, and -71% versus tropapride, respectively), whereas bromerguride (+63%) and cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino tetralin) [(+)-UH 232] (+88%) demonstrated positive agonism. In conclusion, analyses with the chimeric D2/alpha 1B Ala 279 Glu 3ICL receptor construct suggest that neuroleptic drugs can be differentiated on the basis of their intrinsic activity, as they can either activate, inhibit, or be silent at this receptor construct.

Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonellaprotects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.

We recently characterized the association between DNA damage and immunoresponse in vivoin colonic mucosa of mice infected with a SalmonellaTyphimurium strain expressing a genotoxin, known as typhoid toxin. In this protocol, we describe the specific steps for assessing DNA damage by alkaline comet assay of colonic mucosal samples. The description of the comet assay protocol follows the international guidelines (Minimum Information for Reporting on the Comet Assay, MIRCA (Moller et al., 2020)).