Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke.

BACKGROUND: Data are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. AIMS: To assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. METHODS: This prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. 'Dipyridamole-high on-treatment platelet reactivity (HTPR)' was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. RESULTS: Dipyridamole-HTPR was identified in 71.4-75% of patients on PFA-100 C-ADP, 83.9-86.8% of patients on VerifyNow P2Y12, and 81.5-83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. DISCUSSION: Additional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.

Non-melanoma skin cancer activity during the COVID-19 pandemic- A single UK tertiary centre experience.

The pandemic caused by SARS-CoV-2 virus, also known as COVID-19, has generated shockwaves in medical and surgical practice. It has necessitated re-deployment of staff and resources to cater for the unpredictable increase in footfall and demand on healthcare systems. This study aimed to investigate how the restructuring of our service altered the triage and management of non-melanoma skin cancer (NMSC) during the pandemic's first wave rise and peak. We retrospectively analysed all patients who underwent a skin excision under local anaesthetic which revealed the presence of a basal cell carcinoma (BCC) or squamous cell carcinoma (SCC) on histopathological analysis between 1st February 2020 - 31st May 2020 compared with the same period in 2019. There was a 158% increase in patients with excision of lesions confirmed on histopathological analysis as a NMSC during the COVID-19 period (168 vs. 65). In 2020, more excisions were performed by consultants (42.9% v 21.5%, p = 0.002) with a lower proportion of excisions with a close margin (27.7% v 17.8%, p = 0.096) and an involved margin (3.1% v 1.8%, p = 0.62). Five of these patients had their further management altered due to service constraints at this time The resource constraints secondary to the pandemic have yielded beneficial service adaptations with the incorporation of a more efficient model for the NMSC service. The sustainability of this model and its impact on training will require further examination when non-urgent and benign elective workload is slowly reinstated and plastic surgery trainees return to their original posts.

Inflammatory cytokines, high-sensitivity C-reactive protein, and risk of one-year vascular events, death, and poor functional outcome after stroke and transient ischemic attack.

BACKGROUND: Inflammation driven by pro-inflammatory cytokines is a new therapeutic target in coronary disease. Few data exist on the association of key upstream cytokines and post-stroke recurrence. In a prospective cohort study, we investigated the association between pivotal cytokines, high-sensitivity C-reactive protein (hsCRP) and one-year outcomes. METHODS: BIO-STROKETIA is a multi-center prospective cohort study of non-severe ischemic stroke (modified Rankin score ≤ 3) and transient ischemic attack. Controls were patients with transient symptoms attending transient ischemic attack clinics with non-ischemic final diagnosis. Exclusion criteria were severe stroke, infection, and other pro-inflammatory disease; hsCRP and cytokines (interleukin (IL) 6, IL-1ß, IL-8, IL-10, IL-12, interferon-γ (IFN-γ), tumor-necrosis factor-α (TNF-α)) were measured. The primary outcome was one-year recurrent stroke/coronary events (fatal and non-fatal). RESULTS: In this study, 680 patients (439 stroke, 241 transient ischemic attack) and 68 controls were included. IL-6, IL-1ß, IL-8, IFN-γ, TNF-α, and hsCRP were higher in stroke/transient ischemic attack cases (p ≤ 0.01 for all). On multivariable Cox regression, IL-6, IL-8, and hsCRP independently predicted one-year recurrent vascular events (adjusted hazard ratios (aHR) per-quartile increase IL-6 1.31, confidence interval (CI) 1.02-1.68, p = 0.03; IL-8 1.47, CI 1.15-1.89, p = 0.002; hsCRP 1.28, CI 1.01-1.62, p = 0.04). IL-6 (aHR 1.98, CI 1.26-3.14, p = 0.003) and hsCRP (aHR 1.81, CI 1.20-2.74, p = 0.005) independently predicted one-year fatality. IL-6 and hsCRP (adjusted odds ratio per-unit increase 1.02, CI 1.01-1.04) predicted poor functional outcome, with a trend for IL-1ß (p = 0.054). CONCLUSION: Baseline inflammatory cytokines independently predicted late recurrence, supporting a rationale for randomized trials of anti-inflammatory agents for prevention after stroke and suggesting that targeted therapy to high-risk patients with high baseline inflammation may be beneficial.

The views of public service managers on the implementation of National Health Insurance in primary care: a case of Johannesburg Health District, Gauteng Province, Republic of South Africa.

BACKGROUND: The South African government is implementing National Health Insurance (NHI) as a monopsony health care financing mechanism to drive the country towards Universal Health Coverage (UHC). Strategic purchasing, with separation of funder, purchaser and provider, underpins this initiative. The NHI plans Contracting Units for Primary healthcare (PHC) Services (CUPS) to function as either independent sub-district purchasers or public providers and District Health Management Offices (DHMOs) to support and monitor these CUPS. This decentralised operational unit of PHC, the heartbeat of NHI, is critical to the success of NHI. The views of district-level managers, who are responsible for these units, are fundamental to this NHI implementation. This qualitative study aimed to explore district and sub-district managerial views on NHI and their role in its implementation. METHODS: Purposive sampling was used to identify key respondents from a major urban district in Gauteng, South Africa, for participation in in-depth interviews. This study used framework analysis methodology within MaxQDA software. RESULTS: Three main themes were identified: managerial engagement in NHI policy development (with two sub-themes), managerial views on NHI (with three sub-themes) and perceptions of current NHI implementation (with six sub-themes). The managers viewed NHI as a social and moral imperative but lacked clarity and insight into the NHI Bill as well as the associated implementation strategies. The majority of respondents had not had the opportunity to engage in NHI policy formulation. Managers cited several pitfalls in current organisational operations. The respondents felt that national and provincial governments continue to function in a detached and rigid top-down hierarchy. Managers highlighted the need for their inclusion in NHI policy formulation and training and development for them to oversee the implementation strategies. CONCLUSIONS: It appears that strategic purchasing is not being operationalised in PHC. NHI policy implementation appears to function in a rigid top-down hierarchy that excludes key stakeholders in the NHI implementation strategy. The findings of this study suggest an inadequate decentralisation of healthcare governance within the public sector necessary to attain UHC. District managers need to be engaged and capacitated to operationalise the planned decentralised purchasing-provision function of the DHS within the NHI Bill.

Retinal microvascular parameters are not significantly associated with mild cognitive impairment in the Northern Ireland Cohort for the Longitudinal Study of Ageing.

BACKGROUND: The retinal and cerebral microvasculature share similar embryological origins and physiological characteristics. Improved imaging technologies provide opportunistic non-invasive assessment of retinal microvascular parameters (RMPs) against cognitive outcomes. We evaluated baseline measures for associations between RMPs and mild cognitive impairment (MCI) from participants of the Northern Ireland Cohort for the Longitudinal Study of Ageing (NICOLA). METHODS: RMPs (central retinal arteriolar / venular equivalents, arteriole to venular ratio, fractal dimension and tortuosity) were measured from optic disc centred fundus images and analysed using semi-automated software. Associations between RMPs and MCI were assessed by multivariable logistic regression with adjustment for potential confounders including age, sex, alcohol consumption, smoking status, educational attainment, physical activity, cardiovascular disease (CVD), hypertension, mean arterial blood pressure, triglycerides, diabetes, body mass index, and high density lipoprotein levels. P < 0.05 was considered statistically significant. RESULTS: Data were available for 1431 participants, of which 156 (10.9%) were classified with MCI defined by a Montreal Cognitive Assessment (MoCA) score ≤ 26, with subjective cognitive decline, in the absence of depression or problems with activities of daily living. Participants had a mean age of 62.4 ± 8.5 yrs. and 52% were female. As expected, individuals with MCI had a lower MoCA score than those without (23.5 ± 2.6 versus 26.3 ± 2.7, respectively), were more likely to be female, have a lower level of educational attainment, be less physically active, more likely to have CVD, have higher levels of triglycerides and lower levels of high density lipoprotein. No significant associations between RMPs and MCI were detected in unadjusted, minimally adjusted or fully adjusted regression models or subsequent sensitivity analyses. CONCLUSION: Previous studies have reported both increased retinal venular calibre and reduced fractal dimension in association with mild cognitive impairment. Our study failed to detect any associations between RMPs and those individuals at an early stage of cognitive loss in an older community-based cohort.

Change over time in adolescent smoking, cannabis use and their association: findings from the School Health Research Network in Wales.

BACKGROUND: While tobacco smoking has declined among UK youth in recent decades, cannabis use has begun to show some growth. Given their interrelationship, growth in cannabis use may act as a barrier to continued reduction in youth smoking. This paper assesses recent tobacco and cannabis use trends in Wales, and their association, to explore whether change in cannabis use might have impacted youth tobacco smoking prevalence. METHODS: Repeat cross-sectional data on tobacco and cannabis use were obtained from biennial Welsh Student Health and Wellbeing surveys between 2013 and 2019. Data were pooled and analysed using logistic regression with adjustment for school-level clustering. RESULTS: No change in regular youth tobacco smoking was observed between 2013 and 2019. In contrast, current cannabis use increased during this time, and cannabis users had significantly greater odds of regular tobacco smoking. After adjusting for change in cannabis use, a significant decline in youth tobacco smoking was observed (OR 0.95; 95% confidence intervals: 0.92, 0.97). CONCLUSION: Recent growth in cannabis use among young people in Wales may have offset prospective declines in regular tobacco smoking. Further reductions in youth smoking may require more integrated policy approaches to address the co-use of tobacco and cannabis among adolescents.

Mast cell tumours in dogs less than 12 months of age: a multi-institutional retrospective study.

OBJECTIVES: To describe the clinicopathological and genetic characteristics of mast cell tumours in dogs less than 12 months old. MATERIALS AND METHODS: Retrospective review of dogs aged less than 12 months when diagnosed with mast cell tumours at three referral hospitals in the UK. RESULTS: Sixteen pure-bred dogs were included, of which 11 were female. The median age at first presentation and diagnosis were 7.6 and 9 months, respectively. In 13 dogs the mast cell tumours were cutaneous and in three they were subcutaneous. Four cutaneous mast cell tumours were described as high-grade (Patnaik or Kiupel) and nine were Patnaik grade II; three had mitotic index of >5 in 10 high-power fields. Of the three subcutaneous tumours, two had an infiltrative growth pattern and one had mitotic index of 10 per 10 high-power fields. Of 10 tested dogs, seven had c-kit mutations in exon 11 and Ki-67 score was above the cut-off value in nine. Four of 12 cases showed evidence of metastasis in the regional lymph nodes. After varying treatment protocols, all patients were alive and disease free at a median of 1115 days after diagnosis. CLINICAL SIGNIFICANCE: The prognosis of mast cell tumours in dogs less than a year old appears better than the adult counterparts, even without extensive treatment.

Patient perceptions and understanding of obesity related endometrial cancer.

Obesity is the greatest risk factor for endometrial cancer. There is often a lack of recognition amongst patients about this risk. Evidence for weight-loss in the management of endometrial cancer is emerging. This was questionnaire-based study, that examined opinions and attitudes of patients with endometrial cancer and obesity towards obesity as a risk factor for cancer as well as examining their willingness to engage in weight loss interventions as an alternative treatment to endometrial cancer. This survey was conducted in a gynaeoncology out-patient department in Ireland. A total of 45/50 (90%) of questionnaires were completed. The majority of the patients questioned (86.7%; 39/45) agreed that obesity is a disease. Just over half of the cohort (53.3%; 24/45) believed that obesity can cause cancer. Over one-third, 39.9% (18/45) either disagreed or strongly disagreed that obesity is a risk factor for endometrial cancer while 35.5% (16/45) agreed or strongly agreed. Two-thirds (66.6%; 30/45) knew that the greatest amount of weight could be lost through metabolic surgery. Over three-quarters (82.1%; 37/45) of patients surveyed would be willing to engage in a combination of treatments in order to achieve weight-loss should it be proven to have a role in the management of endometrial cancer. This study demonstrates a need for patient education regarding the strong relationship between obesity and endometrial cancer risk. Patients are willing to consider weight loss interventions if they were proven to be as safe and effective as pelvic surgery in the management of endometrial cancer.

Comparison of the utility of clinical breast examination and MRI in the surveillance of women with a high risk of breast cancer.

AIM: To review surveillance magnetic resonance imaging (MRI) and clinical breast examinations (CBE) performed for women at high risk of breast cancer in order to determine recall and cancer-detection rates. MATERIALS AND METHODS: Data were collected on all surveillance MRI examinations performed at St James's Hospital in 2016 for women at high risk of developing breast cancer. Data collected included age, indication for MRI, MRI score, ultrasound indications and scores, and histology findings. Ultrasound scores were recorded from CBEs that received a score of ≥3. RESULTS: A total of 385 breast surveillance MRI examinations and CBEs were performed for women at high risk of breast cancer. A recall rate of 11.2% was documented for breast MRI examinations, whereas a recall rate of 6.2% was identified for CBEs. The biopsy rate was 6.2% for MRI and 0.2% for CBE. The cancer detection rate was 1.6% or 16 per 1,000 for MRI screening and 0% for CBE. CONCLUSION: The high cancer detection rate in the present study supports the unparalleled sensitivity of breast MRI surveillance. Furthermore, the present study did not identify any breast cancers through CBE, suggesting it is not a critical component of the surveillance programme of high-risk women. The current UK guidelines recommending a target recall rate of 7% were not met in the present study or by other studies in the literature, collectively suggesting the guidelines may not be reflective of what is attainable in clinical practice.

Genome-wide analysis of canine oral malignant melanoma metastasis-associated gene expression.

Oral malignant melanoma (OMM) is the most common canine melanocytic neoplasm. Overlap between the somatic mutation profiles of canine OMM and human mucosal melanomas suggest a shared UV-independent molecular aetiology. In common with human mucosal melanomas, most canine OMM metastasise. There is no reliable means of predicting canine OMM metastasis, and systemic therapies for metastatic disease are largely palliative. Herein, we employed exon microarrays for comparative expression profiling of FFPE biopsies of 18 primary canine OMM that metastasised and 10 primary OMM that did not metastasise. Genes displaying metastasis-associated expression may be targets for anti-metastasis treatments, and biomarkers of OMM metastasis. Reduced expression of CXCL12 in the metastasising OMMs implies that the CXCR4/CXCL12 axis may be involved in OMM metastasis. Increased expression of APOBEC3A in the metastasising OMMs may indicate APOBEC3A-induced double-strand DNA breaks and pro-metastatic hypermutation. DNA double strand breakage triggers the DNA damage response network and two Fanconi anaemia DNA repair pathway members showed elevated expression in the metastasising OMMs. Cross-validation was employed to test a Linear Discriminant Analysis classifier based upon the RT-qPCR-measured expression levels of CXCL12, APOBEC3A and RPL29. Classification accuracies of 94% (metastasising OMMs) and 86% (non-metastasising OMMs) were estimated.

Owners' perception of their dogs' quality of life during and after radiotherapy for cancer.

OBJECTIVES: To determine the owners' perception of dogs' quality of life before, immediately after and 6 weeks after radiotherapy treatments for a variety of neoplasms and assess owner satisfaction over their decision to treat. MATERIALS AND METHODS: Questionnaires were given to owners whose dogs completed a radiotherapy treatment at a referral radiation oncology centre. Questionnaires were given at three time points: before treatment, on the last day of treatment and more than 6 weeks after the treatment was finished. Owners were asked questions regarding their perception of radiotherapy and the quality of life of their pets before, during and after treatment with radiation therapy. Quality of life was scored from 1 (could not be worse) to 10 (could not be better). RESULTS: Seventy-one owners met the inclusion and exclusion criteria. Results showed that 6 weeks or more after treatment, most owners were happy that they had chosen to treat their dogs (92%) and would treat another pet again, if indicated (88%). Across the three time points, median quality of life perception score was 9. CLINICAL SIGNIFICANCE: Radiotherapy was well tolerated by owners and dogs in this study. The great majority of clients were happy to have pursued radiotherapy, would choose to do it again (if indicated) and would recommend it to a friend.

Elevated metabolites of acetaminophen in cord blood of children with obesity.

BACKGROUND: High-throughput metabolomics has been used cross-sectionally to evaluate differential metabolic profiles associated with human obesity. OBJECTIVES: This study longitudinally assessed the cord blood metabolome to explore if metabolic signatures of obesity at age 3-5 are apparent at birth. METHODS: In a nested case-control design, metabolomics analysis was performed on umbilical cord blood of 25 children who developed obesity by age 3-5 years, compared with 25 sex-matched non-obese children enrolled as part of an ongoing birth cohort. Logistic regression models were used to identify significant metabolites, adjusting for maternal pre-pregnancy obesity. RESULTS: Children who had obesity by age 3-5 years had elevated levels of medium and long chain fatty acids including stearate, oleate and palmitate at birth. Children with obesity were also more likely to have elevated levels of acetaminophen metabolites at birth, specifically: 3-(N-acetyl-L-cystein-S-yl) acetaminophen, 2-hydroxyacetaminophen sulfate, 2-methoxyacetaminophen glucuronide and p-acetamidophenyl glucuronide. CONCLUSION: Although the observed increases in lipids are consistent with previous metabolomic studies of obesity, this study is the first to report associations between acetaminophen metabolites and obesity in children; however, we lack mechanistic insights for this link. Larger human studies with longer follow-up and laboratory-controlled animal experiments are needed to clarify associations.

The JAK inhibitor ruxolitinib reduces inflammation in an ILC3-independent model of innate immune colitis.

Innate immunity contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms of IBD mediated by innate immunity are incompletely understood and there are limited models of spontaneous innate immune colitis to address this question. Here we describe a new robust model of colitis occurring in the absence of adaptive immunity. RAG1-deficient mice expressing TNFAIP3 in intestinal epithelial cells (TRAG mice) spontaneously developed 100% penetrant, early-onset colitis that was limited to the colon and dependent on intestinal microbes but was not transmissible to co-housed littermates. TRAG colitis was associated with increased mucosal numbers of innate lymphoid cells (ILCs) and depletion of ILC prevented colitis in TRAG mice. ILC depletion also therapeutically reversed established colitis in TRAG mice. The colitis in TRAG mice was not prevented by interbreeding to mice lacking group 3 ILC nor by depletion of TNF. Treatment with the JAK inhibitor ruxolitinib ameliorated colitis in TRAG mice. This new model of colitis, with its predictable onset and colon-specific inflammation, will have direct utility in developing a more complete understanding of innate immune mechanisms that can contribute to colitis and in pre-clinical studies for effects of therapeutic agents on innate immune-mediated IBD.

Comparison of minichromosome maintenance protein 7, Ki67 and mitotic index in the prognosis of intermediate Patnaik grade cutaneous mast cell tumours in dogs.

A previous study found that minichromosome maintenance protein 7 (MCM7) score was associated with prognosis in dogs with cutaneous mast cell tumours (MCTs) independent of histological grade. The primary aim of this study was to validate this score in a different cohort of dogs focusing exclusively on patients with Patnaik intermediate grade MCTs treated with surgery alone and followed for a minimum of 1 year. A secondary aim was to evaluate the prognostic performance of MCM7 in relation to Kiupel histological grade, mitotic index (MI) and Ki67 index in the same cohort of dogs. Ninety dogs were identified, 82 were low Kiupel grade and 8 were high Kiupel grade. Seventy-two dogs were alive after a median follow-up of 1136 days and 18 dogs died of MCT-related causes after a median of 116 days. A MI threshold of 5 was associated with a sensitivity of 0.39 and a specificity of 0.99 in predicting MCT-related death; for Ki67 a threshold of 0.018 was associated with a sensitivity of 0.78 and a specificity of 0.83; and for MCM7 a threshold of 0.18 gave a sensitivity of 0.83 and a specificity of 0.86. Combining MI, Ki67 and MCM7 showed an improved accuracy of predicting death compared with each individual variable. Therefore, performing Ki67 and MCM7 in dogs with GII MCT, low Kiupel grade and low MI might be a consideration.

Preoperative CT in patients with surgically resectable pancreatic adenocarcinoma: does the time interval between CT and surgery affect survival?

PURPOSE: The preoperative imaging-to-surgery time interval (ISI) influences the risk of unexpected progression (UP) found at surgery for pancreatic adenocarcinoma. We aimed to assess whether ISI influences disease recurrence and/or survival. METHODS AND MATERIALS: A single-institution, ethics board-approved retrospective analysis of all patients who underwent attempted resection of pancreatic (PDAC) or periampullary adenocarcinoma (AmpAC) between 1st January 2010 and 31st December 2015 was performed. All patients underwent preoperative abdominal computed tomography (CT). Exclusion criteria were borderline resectable disease and neoadjuvant chemo/radiotherapy. Patients were followed up until 30th June 2016. The population was divided into ISI ≥/<25 days. Kaplan-Meier and Cox regression survival analyses were performed. RESULTS: 239 patients underwent surgical exploration. UP was found in 29 (12.1%) and these patients had longer ISI (median 46 vs. 29 days, p < 0.05). When intention-to-treat analysis was performed, there was no difference in overall survival (OS) between patients with ISI ≥/<25. In those who underwent resection, ISI did not influence disease-free survival (DFS) or OS for PDAC (n = 174). For AmpAC (n = 36), ISI ≥ 25 days was associated with longer OS (p < 0.05) but did not influence DFS. Longer ISI was independently associated with improved OS on regression analysis for AmpAC. CONCLUSION: Performing surgery for resectable pancreatic adenocarcinoma within 25 days of abdominal CT reduces the chance of UP but does not confer a survival benefit. For those who undergo resection of AmpAC, a longer ISI was associated with longer OS. This probably represents a more biologically indolent disease in this cohort.

A preliminary investigation of the effect of sample collection technique on the cell and RNA content of fine-needle aspirates of five canine tumours.

OBJECTIVES: To evaluate the effect of syringe size, needle size, number of needle passes and operator experience on cell yield from tumour fine-needle aspirates, and the quantity and quality of extractable RNA. MATERIALS AND METHODS: Fine-needle aspirates were collected from canine lymphoma, cutaneous mast cell tumour, anal gland adenocarcinoma, fibrosarcoma and oral malignant melanoma using nine different techniques. RESULTS: There was a significant difference in cell yield between fine-needle aspirate techniques for melanoma, lymphoma and anal gland adenocarcinoma. The application of suction yielded the largest number of cells. Cell numbers in lymphoma and fibrosarcoma aspirates collected by different veterinary surgeons were not significantly different. Use of a smaller gauge needle and suction increased the quantity of RNA isolated from fibrosarcoma and anal gland adenocarcinoma aspirates, but did not influence RNA integrity. CLINICAL SIGNIFICANCE: Suction during fine-needle aspiration increases cell numbers obtained from five common canine tumours. Suction increases the quantity of RNA isolated from anal gland adenocarcinoma and fibrosarcoma aspirates without affecting RNA quality. Junior veterinary surgeons gain comparable cell numbers to senior staff.

The role of ethyl acrylate induced GSH depletion in the rodent forestomach and its impact on MTD and in vivo genotoxicity in developing an adverse outcome pathway (AOP).

Adverse outcome pathways (AOP) and mode of action (MOA) frameworks help evaluate the toxicity findings of animal studies and their relevance to humans. To effectively use these tools to improve hazard identification and risk assessments for ethyl acrylate (EA), knowledge gaps in metabolism and genotoxicity were identified and addressed. For EA, hypothesized early key events relate to its irritation potential: concentration dependent irritation and cytotoxicity, progressing to regenerative proliferation and forestomach carcinogenicity after repeated oral bolus application in rodents. The current research quantitated glutathione (GSH) depletion to assess a kinetically-derived maximum tolerated dose (MTD) in the target tissue and used this information to conduct an in vivo genotoxicity study using current methods. In the mouse forestomach, gavage doses of EA caused GSH depletion to 47% of control at 20 mg/kg and 28% at 100 mg/kg. Cellular redox changes and histopathology support saturation of metabolism and an MTD of ∼50 mg/kg. No increases in point mutations or deletions occurred in the stomach or liver following a 28 day treatment of gpt delta transgenic mice at gavage doses up to 50 mg/kg/day. These results provide valuable information for evaluating AOP molecular initiating events or MOA key events for EA and other GSH depleting materials.

Nicotine dependence is associated with functional variation in FMO3, an enzyme that metabolizes nicotine in the brain.

A common haplotype of the flavin-containing monooxygenase gene FMO3 is associated with aberrant mRNA splicing, a twofold reduction in in vivo nicotine N-oxidation and reduced nicotine dependence. Tobacco remains the largest cause of preventable mortality worldwide. CYP2A6, the primary hepatic nicotine metabolism gene, is robustly associated with cigarette consumption but other enzymes contribute to nicotine metabolism. We determined the effects of common variants in FMO3 on plasma levels of nicotine-N-oxide in 170 European Americans administered deuterated nicotine. The polymorphism rs2266780 (E308G) was associated with N-oxidation of both orally administered and ad libitum smoked nicotine (P⩽3.3 × 10-5 controlling for CYP2A6 genotype). In vitro, the FMO3 G308 variant was not associated with reduced activity, but rs2266780 was strongly associated with aberrant FMO3 mRNA splicing in both liver and brain (P⩽6.5 × 10-9). Surprisingly, in treatment-seeking European American smokers (n=1558) this allele was associated with reduced nicotine dependence, specifically with a longer time to first cigarette (P=9.0 × 10-4), but not with reduced cigarette consumption. As N-oxidation accounts for only a small percentage of hepatic nicotine metabolism we hypothesized that FMO3 genotype affects nicotine metabolism in the brain (unlike CYP2A6, FMO3 is expressed in human brain) or that nicotine-N-oxide itself has pharmacological activity. We demonstrate for the first time nicotine N-oxidation in human brain, mediated by FMO3 and FMO1, and show that nicotine-N-oxide modulates human α4ß2 nicotinic receptor activity in vitro. These results indicate possible mechanisms for associations between FMO3 genotype and smoking behaviors, and suggest nicotine N-oxidation as a novel target to enhance smoking cessation.