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Electronic cigarettes (ECs) are purported to be tobacco harm-reduction products whose degree of harm has been highly debated. EC use is considered less hazardous than smoking but is not expected to be harmless. Following the banning of e-liquid flavors in countries such as the US, Finland, Ukraine, and Hungary, there are growing concerns regarding the safety profile of e-liquid flavors used in ECs. While these are employed extensively in the food industry and are generally regarded as safe (GRAS) when ingested, GRAS status after inhalation is unclear. The aim of this review was to assess evidence from 38 reports on the adverse effects of flavored e-liquids on the respiratory system in both

Background Low-level exposure to carbon monoxide (CO) is a significant health concern but is difficult to diagnose. This main study aim was to establish the prevalence of low-level CO poisoning in Emergency Department (ED) patients. Methods A prospective cross-sectional study of patients with symptoms of CO exposure was conducted in four UK EDs between December 2018 and March 2020. Data on symptoms, a CO screening tool and carboxyhaemoglobin were collected. An investigation of participants’ homes was undertaken to identify sources of CO exposure. Results Based on an ED assessment of 4175 participants, the prevalence of suspected CO exposure was 0.62% (95% CI; 0.41–0.91%). CO testing in homes confirmed 1 case of CO presence and 21 probable cases. Normal levels of carboxyhaemoglobin were found in 19 cases of probable exposure and in the confirmed case. Conclusion This study provides evidence that ED patients with symptoms suggestive of CO poisoning but no history of CO exposure are at risk from CO poisoning. The findings suggest components of the CO screening tool may be an indicator of CO exposure over and above elevated COHb. Clinicians should have a high index of suspicion for CO exposure so that this important diagnosis is not missed.

A novel analytical workflow for suspect screening of organic acidic contaminants in drinking water is presented, featuring selective extraction by silica-based strong anion-exchange solid-phase extraction, mixed-mode liquid chromatography-high resolution accurate mass spectrometry (LC-HRMS), peak detection, feature reduction and compound identification. The novel use of an ammonium bicarbonate-based elution solvent extended strong anion-exchange solid-phase extraction applicability to LC-HRMS of strong acids. This approach performed with consistently higher recovery and repeatability (88 ± 7 % at 500 ng L-1), improved selectivity and lower matrix interference (mean = 12 %) over a generic mixed-mode weak anion exchange SPE method. In addition, a novel filter for reducing full-scan features from fulvic and humic acids was successfully introduced, reducing workload and potential for false positives. The workflow was then applied to 10 London municipal drinking water samples, revealing the presence of 22 confirmed and 37 tentatively identified substances. Several poorly investigated and potentially harmful compounds were found which included halogenated hydroxy-cyclopentene-diones and dibromomethanesulfonic acid. Some of these compounds have been reported as mutagenic in test systems and thus their presence here requires further investigation. Overall, this approach demonstrated that employing selective extraction improved detection and helped shortlist suspects and potentially toxic chemical contaminants with higher confidence.

Tissue injuries, including burns, are major causes of death and morbidity worldwide. These injuries result in the release of intracellular molecules and subsequent inflammatory reactions, changing the tissues’ chemical milieu and leading to the development of persistent pain through activating pain-sensing primary sensory neurons. However, the majority of pain-inducing agents in injured tissues are unknown. Here, we report that, amongst other important metabolite changes, lysophosphatidylcholines (LPCs) including 18:0 LPC exhibit significant and consistent local burn injury-induced changes in concentration. 18:0 LPC induces immediate pain and the development of hypersensitivities to mechanical and heat stimuli through molecules including the transient receptor potential ion channel, vanilloid sub-family, member 1 and member 2 at least partly via increasing lateral pressure in the membrane. As levels of LPCs including 18:0 LPC increase in other tissue injuries, our data reveal a novel role for these lipids in injury-associated pain. These findings have high potential to improve patient care.

Background Electronic cigarettes (ECs) are considered a less hazardous alternative to tobacco smoking but are not harmless. Growing concerns about the safety profiles of flavors in e-liquids underpin the need for this study. Methods Here, we screened 53 nicotine-free flavored e-liquids (across 15 flavor categories) across a 3-point concentration range (0.25%, 0.5%, and 1% v/v) in a high-throughput fashion in human bronchial epithelial (HBEC-3KT) submerged cell cultures to identify ‘toxic hits’ using in vitro endpoint assays comprising cell count, cell viability, and lactate dehydrogenase (LDH). Results We observed significant, dose-dependent adverse effects only with cinnamon, vanilla tobacco, and hazelnut e-liquids compared to media-only control and PG/VG vehicle controls. Hence, we further analyzed these three flavors for their effects on HBEC-3KT proliferation, mitochondrial health, and oxidative stress. A significant decrease in cell proliferation after 36h was observed for each e-liquid toxic hit compared to media-only and PG/VG controls. Hazelnut (at all concentrations) and vanilla tobacco (1%) increased cytoplasmic reactive oxygen species (ROS) generation compared to media-only and PG/VG controls. Conversely, all three flavors at 0.5% and 1% significantly decreased mitochondrial membrane potential (MP) compared to PG/VG and media-only controls. We hypothesized that the cytotoxic effects of cinnamon flavor in e-liquids might be mediated via the transient protein receptor ankyrin subtype 1 (TRPA1); however, TRPA1 antagonist AP-18 (10 µM) did not mitigate these effects, and cinnamon significantly increased TRPA1 transcript levels. Therefore, pathways that mediate cinnamon’s cytotoxicity warrant further investigations. Conclusion This study could inform public health authorities on the relative health risks assessment following exposure to EC flavor ingredients.

The incidence of lead (Pb) poisoning in children in Georgia has been identified as a major health concern, with a recent national survey identifying that 41% of children aged 2–7 years had blood lead concentrations (BLCs) greater than the blood lead reference value (BLRV) of ≥5 µg dL−1. This study collected samples of blood, spices, paint, soil, dust, flour, tea, toys, milk, and water from 36 households in Georgia where a child had previously been identified as having a BLC > BLRV. The Pb concentrations of these samples were determined and compared to Georgian reference values. Samples from 3 households were analysed for their Pb isotope composition. The Pb isotope composition of the environmental and blood samples were compared to identify the most likely source(s) of Pb exposure. This approach identified that some spice and dust samples were the likely sources of Pb in the blood in these cases. Importantly, some soil, paint, and dust sources with high Pb concentrations could be discounted as contributing to blood Pb based on their distinct isotope composition. The data presented demonstrate the significant contribution that Pb surveillance and Pb isotope ratio analyses can make to managing Pb exposure in regions where high BLCs are identified.

A novel and generalisable analytical workflow for non-target analysis (NTA) and suspect screening (SS) of potentially toxic organic acids in drinking water is presented, featuring selective extraction by silica-based strong anion exchange solid-phase extraction (SAX-SPE), mixed-mode liquid chromatography-high resolution mass spectrometric (LC-HRMS) analysis, non-targeted peak detection, features reduction and SS. To achieve this, a selection of 23 structurally diverse compounds with a broad range of polarities were evaluated. Importantly, the novel introduction of an elution solvent containing ammonium bicarbonate extended the current applicability of SAX-SPE for strong acids and subsequent mass spectrometry analysis. This new method performed with consistently higher recovery (88 ± 7 % at 500 ng L-1), improved selectivity and lower matrix interference (mean = 12%) over a generic mixed-mode weak anion exchange SPE method. In addition, a novel filter for reducing full-scan features from fulvic and humic acids was successfully introduced, reducing workload and potential for false positives. The workflow was then applied to NTA/SS of 10 London municipal drinking water samples, allowing the identification of 22 confirmed substances. Several poorly investigated and potentially harmful compounds were found. In particular, predicted mutagenicity and occurrence frequency indicated the need for further investigation of halogenated hydroxy-cyclopentene-diones (HCDs) and dibromomethanesulfonic acid in drinking water. Overall, this approach demonstrated that employing some selectivity for general molecular properties in NTA overall helped shortlist suspect and potentially toxic chemical contaminants with higher confidence, in this case for organic acids.

Allergic airway disease (AAD) is a collective term for respiratory disorders that can be exacerbated upon exposure to airborne allergens, including fungi and other air pollutants. Fungi are of particular interest as they are present in both outdoor and indoor air and are associated with asthma and allergy. A recent literature review identified key pathways contributing to the allergic responses to fungal allergens. However, due to the heterogeneity of models and methods used, it is not clear whether there are common or specific pathways induced by different fungi/fungal components. Air-liquid interface (ALI) culture was used to induce differentiation of nasal (RPMI 2650), bronchial (HBEC3-KT, BEAS-2B, Calu-3) and small airways (A549) epithelial cells into a columnar stratified epithelium with cilia, goblet cells, and the formation of tight junctions. Cultures were harvested at 0, 7, 14, 21 and 28 days, and the expression of molecular markers of basal, ciliated and mucus producing cells were assessed with reverse-transcription quantitative PCR (RT-qPCR). Different cell lines produce subtly different differentiated models, with downregulation of basal (undifferentiated) cell markers and upregulation of markers of ciliated and mucus producing cells from day 7. While no ciliated cells were observed in any of the differentiated cell lines, mucus was produced in some. The differentiation of primary cell lines at ALI, and expression of genes of interest (including known fungal receptors and genes identified in driving fungal allergy) were also measured to inform selection of appropriate model(s) to better understand the allergenic response to fungi/fungal components.

Currently, any association between smoking and COVID-19 susceptibility is conflicted as there is limited research especially on the impact of vaping. This systematic literature review will identify genes and signalling pathways that are affected by smoking or vaping that may play a role in altering the susceptibility of an individual to COVID-19. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, OVID and Web of Science databases, were searched (1946 to Nov 2021). Abstracts and subsequent full texts were screened for eligibility by RB, with EM and AB screening 5% each. Discrepancies were discussed and agreed between all. Papers were excluded for reasons such as focussing on comorbidities, immune cells or those with no exposure. Data from included papers detailing the exposure (type, duration and dose if applicable), pathway or gene of interest, sample type (species and cell type) and the overall findings, were extracted. Pathway analysis was undertaken to identify those pathways in common between smoking, vaping, nicotine and COVID-19 (g:Profiler and Cytoscape). We identified genes within the respiratory tract involved in both the initial viral response and those affected by smoking, vaping or nicotine exposure. This enabled us to determine signalling pathways of interest and therefore areas for further study. This systematic review will inform further research to identify any altered susceptibility and the risk of severe COVID-19 posed to smokers and vapers. This will inform Public Health Policies, highlighting vulnerable groups and provide guidance of relevance to both COVID-19 and other respiratory viruses.

Air-liquid interface (ALI) cultures are commonly used for 3D modelling of the human airways and to examine the effects of direct exposure to airborne toxicants. We explored the suitability of human epithelial cell lines (RPMI 2560, HBEC-3KT, CALU-3 and A549) and primary cells (nasal, bronchiolar and small airways) grown at ALI as a physiologically relevant model to examine whether environmental exposures can increase the susceptibility of the human airways to SARS-COV-2 infection. Cultures were kept at ALI for up to 28 days to allow differentiation. Trans-epithelial electrical resistance (TEER) was measured weekly. Cells were harvested at 0, 7, 14, 21 and 28 days, and transcriptional markers of basal, ciliated and mucus-producing cells together with SARS-COV-2 viral entry genes (ACE2, TMPRSS2 and Furin) and other genes of interest, were assessed by reverse-transcription quantitative PCR. Each cell line yielded differentiated models, with upregulation of markers of ciliated and mucus producing cells from day 7. In contrast to primary cells, immortalised cell lines did not produce cilia but, mucus was produced. TEER values increased with time, as expected, but values and therefore formation of tight junctions differed between models. Similarly, expression of viral entry genes increased as cells differentiated with marked differences across the different models. This study will be used to select the most appropriate ALI model for investigating the effect of environmental exposures such as smoking and vaping on susceptibility to SARS-COV-2 infection.

Decontamination of skin by washing may increase dermal absorption, a phenomenon known as the wash-in effect. The wash-in effect is frequently discussed in studies investigating casualty decontamination where potentially life-saving interventions may enhance the dermal penetration of toxic chemicals, leading to an increase in incidence of morbidity and rates of mortality. However, the wash-in effect is seldom investigated within the context of mass casualty decontamination and real-life consequences are therefore poorly understood. This paper reviews the existing literature on the wash-in effect to highlight the proposed mechanisms for enhanced absorption and evaluate the wash-in effect within the context of mass casualty chemical decontamination.

Introduction:To date, all human studies of mass-casualty decontamination for chemical incidents have relied on the collection and analysis of external samples, including skin and hair, to determine decontamination efficacy. The removal of a simulant contaminant from the surface of the body with the assumption that this translates to reduced systemic exposure and reduced risk of secondary contamination has been the main outcome measure of these studies. Some studies have investigated systemic exposure through urinary levels of simulant metabolites. The data obtained in these studies were confounded by high background concentrations from dietary sources. The unmetabolized simulants have never been analyzed in urine for the purposes of decontamination efficacy assessment.Study Objective:Urinary simulant analysis could obviate the need to collect skin or hair samples during decontamination trials and provide a better estimate of both decontamination efficacy and systemic exposure. The study objective therefore was to determine whether gross skin contamination as part of a decontamination study would yield urine levels of simulants sufficient to evaluate systemic availability free from dietary confounders.Methods:In this study, a gas chromatography-tandem mass spectrometry method was developed for the analysis of two chemical simulants, methyl salicylate (MeS) and benzyl salicylate (BeS), in urine. An extraction and sample clean-up method was validated, enabling quantitation of these simulants in urine. The method was then applied to urine collected over a 24-hour period following simulant application to the skin of volunteers.Results:Both MeS and BeS were present in all urine samples and were significantly increased in all post-application samples. The MeS levels peaked one hour after skin application. The remaining urinary levels were variable, possibly due to additional MeS exposures such as inhalation. In contrast, the urinary excretion pattern for BeS was more typical for urinary excretion curves, increasing clearly above baseline from four hours post-dose and peaking between 12.5 and 21 hours, a pattern consistent with dermal absorption and rapid excretion.Conclusion:The authors propose BeS is a useful simulant for use in decontamination studies and that its measurement in urine can be used to model systemic exposures following skin application and therefore likely health consequences.

Electronic cigarettes divide opinions. Some consider them key to reducing smoking incidence while others are concerned over potential for detrimental health consequences. It will take many years to identify the health consequences of e-cigarette use if we rely only upon human data. However, there is a growing body of work using rodent models that inform on these potential toxicities. These studies have focused upon the pulmonary, cardiovascular and central nervous systems. Observations include perturbations of pro-inflammatory, pro-fibrotic and oxidative stress markers, sometimes together with DNA damage and downregulation of DNA repair and antioxidant enzymes. However, the markers affected are often different between studies. A more consistent observation has been the increase in airway hyperresponsiveness, a characteristic of asthma, on exposure to electronic cigarettes, across mouse strains, sex and ages. Detrimental effects in this and other susceptible animal models such as the apolipoprotein E knock-out mouse model of atherosclerosis, suggest greater risk where there is an existing predisposition. Other adverse reactions, including weight loss, oxidative stress and angiogenesis, are reported in animal studies with nicotine-containing devices. These effects remain less severe than cigarette smoke, where investigated. Animal studies have identified therefore that e-cigarettes are potentially hazardous, especially in susceptible populations, nicotine is integral to risk of health effects, but overall e-cigarettes are much less hazardous than cigarettes.

In recent years, reports of lead contamination have dramatically increased in Georgia. Given concerns about the exposure of children to lead (Pb), the National Multiple Indicator Cluster Survey (MICS-2018) included a blood sampling component. The results showed that 41% of the children that participated had blood Pb levels (BLL) ≥ 5 µg/dL and that BLL in children living in Western Georgia were higher than those in Eastern regions. In response to these findings, NCDC implemented written and verbal advice to the families of children who participated in the MICS-2018 on how to reduce Pb exposure. From August 2019 onwards, the state program of clinical follow-up was implemented. The design of this study was a longitudinal study. The intervention of interest was the public health advice and medical follow-up, and the outcome was defined as the difference in BLL between the MICS-2018 survey and the state program follow-up. We observed a significant overall reduction in median BLL between MICS-2018 and state program follow-up in both August 2019 and the latest results (until December 2019). However, we did not observe any significant further reduction between August and the most recent BLL results. In the Georgian setting, written and verbal communication targeting individual households, alongside home visits to the most exposed, effectively reduced BLL in children.

One of the more important classes of potentially toxic indoor air chemicals are the Volatile Organic Compounds (VOCs). However, due to a limited understanding of the relationships between indoor concentrations of individual VOCs and health outcomes, there are currently no universal health-based guideline values for VOCs within Europe including the UK. In this study, a systematic search was conducted designed to capture evidence on concentrations, emissions from indoor sources, and health effects for VOCs measured in European residences. We identified 65 individual VOCs, and the most commonly measured were aromatic hydrocarbons (14 chemicals), alkane hydrocarbons (9), aldehydes (8), aliphatic hydrocarbons (5), terpenes (6), chlorinated hydrocarbons (4), glycol and glycol ethers (3) and esters (2). The pathway of interest was inhalation and 8 individual aromatic hydrocarbons, 7 alkanes and 6 aldehydes were associated with respiratory health effects. Members of the chlorinated hydrocarbon family were associated with cardiovascular neurological and carcinogenic health effects and some were irritants as were esters and terpenes. Eight individual aromatic hydrocarbons, 7 alkanes and 6 aldehydes identified in European residences were associated with respiratory health effects. Of the 65 individual VOCs, 52 were from sources associated with building and construction materials (e.g. brick, wood products, adhesives and materials for flooring installation etc.), 41 were linked with consumer products (passive, electric and combustible air fresheners, hair sprays, deodorants) and 9 VOCs were associated with space heating, which may reflect the relatively small number of studies discussing emissions from this category of sources. A clear decrease in concentrations of formaldehyde was observed over the last few years, whilst acetone was found to be one of the most abundant but underreported species. A new approach based on the operational indoor air quality surveillance will both reveal trends in known VOCs and identify new compounds.

Planning for major incidents involving the release of hazardous chemicals has been informed by a multi-disciplinary research agenda which has sought to inform all aspects of emergency response, but with a focus in recent years on mass casualty decontamination. In vitro and human volunteer studies have established the relative effectiveness of different decontamination protocols for a range of chemical agents. In parallel, a programme of research has focused on communicating with and managing large numbers of contaminated casualties at the scene of an incident. We present an accessible overview of the evidence underpinning current casualty decontamination strategies. We highlight where research outcomes can directly inform response planning, including the critical importance of beginning the decontamination process as soon as possible, the benefits of early removal of contaminated clothing, the evidence under-pinning dry and wet decontamination and how effective communication is essential to any decontamination response. We identify a range of priority areas for future research including establishing the significance of the ‘wash-in’ effect and developing effective strategies for the decontamination of hair. We also highlight several areas of future methodological development, such as the need for novel chemical simulants. Whilst considerable progress has been made towards incorporating research outcomes into operational policy and practice, we outline how this developing evidence-base might be used to inform future iterations of mass casualty decontamination guidance.

The decontamination of exposed persons is a priority following the release of toxic chemicals. Efficacious decontamination reduces the risk of harm to those directly affected and prevents the uncontrolled spread of contamination. Human studies examining the effectiveness of emergency decontamination procedures have primarily focused on decontaminating skin, with few examining the decontamination of hair and scalp. We report the outcome of two studies designed to evaluate the efficacy of current United Kingdom (UK) improvised, interim and specialist mass casualty decontamination protocols when conducted in sequence. Decontamination efficacy was evaluated using two chemical simulants, methyl salicylate (MeS) and benzyl salicylate (BeS) applied to and recovered from the hair of volunteers. Twenty-four-hour urinary MeS and BeS were measured as a surrogate for systemic bioavailability. Current UK decontamination methods performed in sequence were partially effective at removing MeS and BeS from hair and underlying scalp. BeS and MeS levels in urine indicated that decontamination had no significant effect on systemic exposure raising important considerations with respect to the speed of decontamination. The decontamination of hair may therefore be challenging for first responders, requiring careful management of exposed persons following decontamination. Further work to extend these studies is required with a broader range of chemical simulants, a larger group of volunteers and at different intervention times.

The national response for reducing lead (Pb) exposure in Georgia is coordinated by the National Centre for Disease Control and Public Health (NCDC&PH) and implemented as a multi-agency (CDC, UNICEF, WHO, University of Emory) response. Given concerns about the extent of Pb exposure, in 2018 Multiple Indicator Cluster Surveys (MICS) of representative samples of children have been conducted to study several demographic and health aspects, including a study of the prevalence of blood Pb levels among 2 to 7 years old children (n = 1578). This survey was conducted in collaboration with the Italian Instituto Superiore di Sanita' (ISS), UNICEF and NCDC. The laboratory analyses were conducted at ISS in Italy. Initial results showed that in 41% of all children, blood Pb concentration was ≥ 5 µg/dl, a challenge which motivated public agencies to establish an initial public health action plan to assess environmental samples (paint, dust, water, soil, selected food items such as spices and imported sweets) in families where Pb concentrations were ≥ 10 µg/dL. A State intervention programme, monitoring Pb blood concentration among MICS children and their family members, provided relevant information on exposed households and led to a reduction of Pb blood concentration across the most exposed households. In collaboration with Public Health England, NCDC has conducted a small Pb isotope ratio study aimed at identifying the most relevant sources of Pb exposure contributing to elevated blood Pb in MICS children. It is expected that these data will support the design of more detailed public health interventions to reduce exposure to key sources of Pb, thus leading to further reduction of Pb-induced health effects in Georgia. In addition, this experience will clarify elements of an ongoing monitoring of environmental factors such as an Environmental Public Health Tracking system, to support national capacity to manage the risks to public health. Key messages Environmental health response requires extensive research and multi-agency approach. If State implements adequate intervention it is possible to reduce blood lead (Pb) level.

Experimental modelling to identify specific inhalation hazards for nanomaterials has in the main focussed on in vivo approaches. However, these models suffer from uncertainties surrounding species specific differences and cellular targets for biological response. In terms of pulmonary exposure, approaches which combine ‘inhalation-like’ nanoparticulate aerosol deposition with relevant human cell and tissue air-liquid interface cultures are considered an important complement to in vivo work. In this study, we utilized such a model system to build on previous results from in vivo exposures, which highlighted the small airway epithelium as a target for silver nanoparticle (AgNP) deposition. RNA-SEQ was used to characterise alterations in mRNA and miRNA within the lung. Organotypic reconstituted 3D human primary small airway epithelial cell cultures (SmallAir) were exposed to the same spark generated AgNP and at the same dose used in vivo, in an aerosol-exposure air-liquid-interface (AE-ALI) system. Adverse effects were characterised using lactate, LDH release and alterations in mRNA and miRNA. Modest toxicological effects were paralleled by significant regulation in gene expression, reflective mainly of specific inflammatory events. Importantly, there was a level of concordance between gene expression changes observed in vitro and in vivo. We also observed a significant correlation between AgNP and mass equivalent silver ion (Ag(+)) induced transcriptional changes in SmallAir cultures. In addition to key mechanistic information relevant for our understanding of the potential health risks associated with AgNP inhalation exposure, this work further highlights the small airway epithelium as an important target for adverse effects.

Introduction: We aim to study the effect of fatty acid amide hydrolase (FAAH) blockade on bladder hyperactivity and on fatty acid amides levels during cystitis. Material and Methods: Cystitis was induced in female Wistar rats using 5 mg/mL lipopolysaccharide (LPS). Control group were intravesical instilled with saline. LPS and control groups received intravenously (caudal vein) during cystometry: URB 937 (URB; FAAH antagonist) in doses of 0.007, 0.07, 0.7 and 7 mg/kg (cumulative, with 10 minutes interval). Using the maximal effective dose of URB (0.7 mg/kg, see below) animals received 10 uM MJ15 (CB1 receptor antagonist) or 0.3 mg SR144528 /kg (SR; CB2 receptor antagonist). At dose of 7 mg/kg, animals receive 1.4 μg SB366791/kg (SB; TRPV1 antagonist). Control and inflamed (without and with 0.7 and 7 mg/kg URB) group were euthanized and the bladder was harvested for the determination of anandamide (AEA) and palmitoylethanolamide (PEA) by mass spectrometry. Results: Frequency of control was not changed by URB treatment at any dose. LPS increase bladder frequency. 0.007 mg and 0.07 mg URB decrease bladder frequency of LPS-inflamed rats. 0.7 URB reversed LPS-induced bladder hyperactivity. At 7 mg, URB was unable to reverse or reduce LPS-induced bladder hyperactivity. The administration of CB1, CB2 and TRPV1 antagonists did not change the frequency of voiding contractions of naïve animals. CB1 antagonist reversed the effect of 0.7 URB while TRPV1 antagonist reduced the effect of 7 URB. AEA levels increase during inflammation. Treating LPS-inflamed animals with 0.7 mg URB brought AEA levels to control levels. Treating LPS-inflamed animals with 7 mg URB did not change AEA levels, compared to LPS-inflamed animals. PEA levels decrease during inflammation. Treating LPS-inflamed animals with 0.7 mg URB brought AEA levels to control levels. Treating LPS-inflamed animals with 7 mg URB, decreased PEA levels to values similar to the ones observed in LPS- -inflamed animals. Conclusion: During cystitis, the FAAH inhibitor raises the levels of PEA and reverses the urinary frequency by a CB1 receptor- mediated mechanism. When used in very high doses, the FAAH antagonist raises the levels of AEA and increases the urinary frequency by a TRPV1-dependent mechanism. Therefore, the choice of FAAH inhibitor dosage to be used in the clinics should consider the putative effects over the endocannabinoid levels in the system.

Chemical simulants have long been used in human trials of mass decontamination to determine the efficacy of decontamination interventions against more toxic agents. Until now, reliance has mostly been on individual chemicals as surrogates to specific agents (e.g. methyl salicylate for sulphur mustard). A literature review was conducted to identify chemicals that had been previously tested on human volunteers and that represent diverse physicochemical characteristics in order to create a repository for chemical simulants. Of the 171 unique chemicals identified 78 were discounted for the risk they could pose to human volunteers, 39 were deemed suitable for use and a further 54 were considered to be possible simulants but would require further research. Suitable simulants included both solid and liquid chemicals spanning a wide range of physicochemical properties including molecular weight, octanol/ water partition coefficient, vapour pressure and solubility. This review has identified an array of potential simulants suitable for use in human volunteer decontamination studies and is of relevance to future studies on systemic absorption and surface decontamination.

Incidents involving the release of chemical agents can pose significant risks to public health. In such an event, emergency decontamination of affected casualties may need to be undertaken to reduce injury and possible loss of life. To ensure these methods are effective, human volunteer trials (HVTs) of decontamination protocols, using simulant contaminants, have been conducted. Simulants must be used to mimic the physicochemical properties of more harmful chemicals, while remaining non‐toxic at the dose applied. This review focuses on studies that employed chemical warfare agent simulants in decontamination contexts, to identify those simulants most suitable for use in HVTs of emergency decontamination. Twenty‐two simulants were identified, of which 17 were determined unsuitable for use in HVTs. The remaining simulants (n = 5) were further scrutinized for potential suitability according to toxicity, physicochemical properties and similarities to their equivalent toxic counterparts. Three suitable simulants, for use in HVTs were identified; methyl salicylate (simulant for sulphur mustard), diethyl malonate (simulant for soman) and malathion (simulant for VX or toxic industrial chemicals). All have been safely used in previous HVTs, and have a range of physicochemical properties that would allow useful inference to more toxic chemicals when employed in future studies of emergency decontamination systems., To conduct human volunteer trials on the decontamination of chemical warfare agents, simulant chemicals that mimic the properties of the live agents must be used. For the first time, this review identifies previously used chemical simulants and assesses their suitability according to toxicity, physicochemical properties and similarity to their equivalent toxic counterparts.

Human studies investigating the efficacy of emergency decontamination protocols for chemical incidents require the use of non-hazardous chemical simulants. Methyl salicylate (MeS) has almost exclusively been used for this purpose. Whilst MeS is a simulant of the chemical warfare agent (CWA) sulphur mustard, it is not an ideal simulant for many other chemical threats with greater persistence and lower volatility. Benzyl salicylate (BeS) has been investigated here as a low toxicity simulant for lower volatility, persistent chemical threat agents and toxic industrial chemicals (TICs). To evaluate the suitability of BeS as a simulant for human decontamination studies a gas chromatography-triple quadrupole mass spectrometry method was designed, optimised and validated, for the analysis of human skin and hair. Quantification was achieved using isotope-dilution, EI and collision-induced dissociation and multiple reaction monitoring for both qualifier and quantifier ion transitions. The mass transitions were m/z 285 → 91 and m/z 210 → 181, respectively for the quantifier and qualifier ions of BeS, and m/z 289 → 91 and m/z 214 → 185 for the quantifier and qualifier ions for the BeS-d4 internal standard, respectively. The method exhibited excellent coefficients of determination (R2 = 0.9992 to 0.9999) with LOD and LOQ values at 0.023 ng/ml and 0.23ng/ml. Across three Quality Controls (QCs), 11.5 ng/ml, 115 ng/ml and 1150 ng/ml) average accuracy (intra-day 95.6% to 100.3%, inter-day 98.5% to 104.91%) and precision (intra-day RSD (%) 2% to 13.7%, inter-day RSD (%) 3.3% to 8.8%) were determined. The validated method was applied in a proof of principle volunteer study for the determination of BeS recovered from skin and hair. The average total BeS recovery after 70 minutes was 37.9% from skin and there was a significant increase between baseline and post-intervention levels for hair. These data demonstrate that BeS is an appropriate simulant for persistent chemicals and that the analytical method employed here is suitable for BeS analysis in human studies.

Fatty acid amide hydrolase inhibition may be used to control bladder function and pain by modulating endocannabinoid levels in cystitis. We studied the effect of the peripherally restricted fatty acid amide hydrolase inhibitor URB937 in bladder reflex activity and bladder pain using the lipopolysaccharide model of cystitis. We also correlated the URB937's effects with tissue levels of the endocannabinoids anandamide and palmitoylethanolamine. URB937 did not change the reflex activity of normal bladders. In inflamed bladders, URB937 had a U-shaped dose-response curve; following an initial cannabinoid receptor type 1-mediated reduction in pain responses and normalisation of bladder reflex activity, URB937 gradually increased both pain responses and bladder reflex activity through the transient receptor potential ion channel subfamily V member 1. Chronic cystitis increased the tissue levels of anandamide and decreased those of palmitoylethanolamine. At the dose that normalised bladder reflex activity and decreased pain responses, URB937 normalised the levels of anandamide and palmitoylethanolamine in the bladder. At high doses that induced excitatory effects, URB937 apparently did not change anandamide and palmitoylethanolamine levels, which therefore were in the range of the inflamed bladder. Fatty acid amide hydrolase inhibition results in complex changes in bladder endocannabinoid levels. The therapeutic effect of fatty acid amide hydrolase inhibitors is not related to increase in anandamide levels but rather a normalisation of the anandamide and palmitoylethanolamine level ratio.

Cerium dioxide nanoparticles (CeO2NPs) have been used as diesel fuel-borne catalysts for improved efficiency and pollutant emissions. Concerns that such material may influence diesel exhaust particle (DEP) effects within the lung upon inhalation, prompted us to examine particle responses in mice in the presence and absence of the common allergen house dust mite (HDM). Repeated intranasal instillation of combined HDM and DEP increased airway mucin, eosinophils, lymphocytes, IL-5, IL-13, IL-17A and plasma IgE, which were further increased with CeO2NPs co-exposure. A single co-exposure of CeO2NPs and DEP after repeated HDM exposure increased macrophage and IL-17A levels above DEP induced levels. CeO2NPs exposure in the absence of HDM also resulted in increased levels of plasma IgE and airway mucin staining, changes not observed with repeated DEP exposure alone. These observations indicate that CeO2NPs can modify exhaust particulate and allergen induced inflammatory events in the lung with the potential to influence conditions such as allergic airway disease.

Methyl salicylate has a long history of use as a chemical warfare agent simulant for volatile lipophilic compounds such as sulphur mustard. An improved isotope dilution GC–MS/MS method was developed, optimised and validated for the analysis of methyl salicylate in human skin and hair samples, for use in emergency decontamination volunteer studies. Following derivatisation, quantification was measured on a triple quadrupole mass spectrometer, set to EI mode and conducting multiple reaction monitoring of target ions. The mass transitions were 209 → 179 and 213 → 161 for quantitation of methyl salicylate and methyl salicylate D4, respectively whereas qualifier ion transitions used to verify identity were 209 → 169 and 213 → 89. The method achieved excellent coefficient of determination (R2 > 0.9968 to 0.9999) over the range of 0.5–5000 ng/ml and the LOD and LOQ were 0.05 ng/ml and 0.5 ng/ml. The method was further validated for accuracy (intra-day and inter-day average 100.28% to 102.03% and 99.48% to 102.33%, respectively) and precision (intra-day RSD 1.43% to 2.35%, inter-day RSD 1.91% to 2.97%) at three concentrations (25, 250 and 2500 ng/ml). The validated method was successfully used to identify methyl salicylate in samples of human skin generated during volunteer studies of emergency decontamination systems and in hair of staff conducting these studies.

Introduction This project aims to monitor human volunteers as they switch from heavy (>10 cigarette/day) tobacco smoking to electronic cigarette (EC) use. Monitoring of markers will comprise expired carbon monoxide, DNA methylation, oxytocin, cortisol, nicotine cotinine and tobacco specific nitrosamine levels in urine and saliva together with psychometric data. In addition electroencephalography will be used to search for electrical changes in the brain correlated with changes in the above markers after transition to EC use. Methods Volunteers were recruited and consented at St George’s University of London. Volunteers were allowed to select their own EC and e-fluid including nicotine concentration. Blood, saliva, urine and buccal cell samples and questionnaire data (nicotine dependence, withdrawal, anxiety, depression, mood, quality of life and sleep quality) were collected at baseline (day-2 and day-1) and throughout the 28-day EC trial (days 3,21,28) and EEG data (eyes open-eyes fixed-eyes closed, each 3 x 30s) were collected at baseline (day-1) and on EC day 21. Nicotine, cotinine, NNK, NNAL and NNN were determined by UHPLC-HESI-HRMS, 5methyl cytosine by HPLC and oxytocin and cortisol by ELISA. Results Urinary and salivary nicotine, cotinine and tobacco specific nitrosamines and expired carbon monoxide decrease significantly following transition to EC. Behavioural data have revealed modest changes in nicotine craving, nicotine withdrawal symptoms, social anxiety and sleep quality following the transition to e-cigarettes. Preliminary comparison of regional spectral power extracted from EEG data collected pre- and post- transition to e-cigarettes use has identified significant changes in specific spectral bands across the brain. Conclusions Switching to EC quickly induces beneficial changes in exposure to toxicants and psychometrics. Changes in regional spectral power observed by EEG vary will be correlated to these other biomarkers. Funding This work is supported by H2020 funding from the European Union and by a PHE-funded PhD studentship.

Introduction Electronic cigarettes (EC) represent a safer alternative to tobacco, however, there is limited information on adverse health effects, especially after long term use. Among potential harmful constituents of EC aerosols nicotine is the major addictive chemical and is a precursor for carcinogenic tobacco-specific nitrosamines (TSNAs). Multiple studies have highlighted the ability of nicotine to compromise blood brain barrier (BBB) integrity, altering transport and receptor systems. One knowledge gap is the effect of e-fluids on enzymes responsible for metabolism of toxicants including carcinogens, Therefore, the aim of this study was to examine the effect of nicotine and e-fluid on the expression of cytochrome P450 enzymes (CYPs) in an in vitro model of human BBB, the hCMEC/D3 cell line. Methods The hCMEC/D3 cells were used to investigate time and dose-dependent responses of nicotine or e-fluid and EC aerosol condensate (normalised to nicotine concentration). High quality RNA was extracted and analysed by qRT-PCR for the expression of genes encoding enzymes involved in nicotine metabolism and bioactivation of procarcinogens (CYP1A1, CYP1A2, CYP2A6, CYP2E1, CYP2A13, CYP3A4) and recently described extrahepatic CYP isoforms (CYP2U1 and CYP2S1), which are highly expressed in BBB. Results qRT-PCR results were normalised using UBC reference gene and expressed as a fold change between non treated and treated cells (n=3, p

Aim To monitor how switching to e-cigarettes from cigarette smoking affects psychometric and clinical parameters and relapse rate over a period of 4 weeks. Methods Heavy smokers (≥10cig/day) attended 6 visits where psychometric parameters associated with craving, nicotine withdrawal symptoms, sleep and quality of life were monitored using well validated questionnaires. Heart rate and blood pressure were also measured at each visit. Visit 1 and 2 were baseline visits where smokers continued to smoke and then asked to quit and transition to e-cigarette use. The subsequent visits occurred 3, 7, 21 and 28 days after the quit date. Compliance was monitored via exhaled CO. Completed participants received weekly questionnaires for 12 months and two follow up calls 6 and 12 months after their final visit to monitor for relapse. Results One month relapse rate amongst eligible participants (n=29) was 28%. At 6 months (n=5), 40% used either e-cigarettes or nothing (non-relapsers) and 60% partially relapsed or were dual users. Repeated measures One-Way ANOVA(n=20) showed a significant effect for parameters: ‘irritability’ (p

Introduction Use of electronic cigarettes (ECs) has increased rapidly since they were introduced in Europe in 2006 especially in the UK. Claims have been made for a role in tobacco harm reduction and smoking cessation. Key to these claims is the ability of EC to maintain nicotine levels to limit cravings, withdrawal and nicotine dependence. Therefore, the aim of the current study is to monitor nicotine intake in heavy smokers (>10 cig/day, n=13 completed) transitioning from cigarette smoking to EC-only use whilst collecting psychometric data on nicotine dependence (Fagerstrom), withdrawal (Minnesota), anxiety, depression and sleep quality. Material and Methods Urine and saliva samples as well as psychometric questionnaire data were collected at baseline (visits 1 and 2) and at days 3, 7, 21 and 28 following start of EC use. An UHPLC-HESI-HRMS method was developed, validated and applied to the determination of nicotine and its main metabolite cotinine in urine and saliva. Results The baseline median (interquartile range) urinary nicotine and cotinine levels were determined as 1534 ng/mg creatinine (317-2478) and 2082 ng/mg creatinine (1727-3632), whereas salivary nicotine and cotinine levels were 145 ng/ml (26-265) and 329 ng/ml (198-440), respectively. One-way repeated measures Anova demonstrated a significant decrease in urinary nicotine and cotinine concentration from baseline at 3 post-transition visits. A significant reduction was also observed in salivary nicotine levels on days 3 and 21 after EC transition and in salivary cotinine levels at each post-transition visit compared to baseline. Both urge and strength of urges to smoke were significantly decreased by 28days compared to baseline. Conclusions These findings offer support that EC can be effective in reducing nicotine consumption and cravings for cigarettes, suggesting the potential use of EC as a smoking cessation tool. However, the inability to achieve levels of nicotine comparable to smokers may contribute to relapse. Funding Statement Funded by European Commission Horizon 2020 programme.

Introduction The aim of this study was to quantify levels of carcinogenic tobacco specific nitrosamines (TSNAs) in human volunteers transitioning from tobacco use to electronic cigarettes (EC). ECs have increased in popularity, with consumers using ECs to aid with smoking cessation, reduce cigarette consumption, or to minimise withdrawal symptoms when smoking but not vaping is prohibited. There has been little investigation into the long term health impacts of ECs, but they are generally considered less toxic than conventional tobacco cigarettes. This is due to the absence or decreased levels of harmful chemicals commonly found in tobacco cigarettes. However, some studies have shown the presence of traces of TSNAs in e-liquid, and the endogenous nitrosation of nicotine to TSNAs in nicotine replacement therapies (NRT). These TSNAs include NNK, NNN, and NNAL, which are potent oesophageal and pulmonary carcinogens. Therefore, inhalation of these compounds may provide an added risk to the use of ECs, when compared to other NRT products. Methods Biofluids have been analysed for TSNAs in the urine and saliva of heavy smokers (>10 cigarettes/day) who quit and transition to EC use for 28 days. A solid phase extraction method using TSNA specific molecular imprinted polymers was used to concentrate samples, which were subsequently analysed using UHPLC-ESI-HRMS. Results Analysis of longitudinal urine and saliva samples (n=13) has shown a decrease in TSNA levels over the period of transition from tobacco use to EC only use. Conclusions The findings are consistent with the notion that substituting conventional cigarettes with ECs significantly lowers exposure to carcinogenic TSNAs. Further work is needed to determine whether TSNAs present in urine and saliva represent continued low level exposure to TSNAs as contaminants in EC or markers of nitrosation. Funding This work is funded by Horizon 2020, and Public Health England

Background Nanomaterial inhalation represents a potential hazard for respiratory conditions such as asthma. Cerium dioxide nanoparticles (CeO2NPs) have the ability to modify disease outcome but have not been investigated for their effect on models of asthma and inflammatory lung disease. The aim of this study was to examine the impact of CeO2NPs in a house dust mite (HDM) induced murine model of asthma. Results Repeated intranasal instillation of CeO2NPs in the presence of HDM caused the induction of a type II inflammatory response, characterised by increased bronchoalveolar lavage eosinophils, mast cells, total plasma IgE and goblet cell metaplasia. This was accompanied by increases in IL-4, CCL11 and MCPT1 gene expression together with increases in the mucin and inflammatory regulators CLCA1 and SLC26A4. CLCA1 and SLC26A4 were also induced by CeO2NPs + HDM co-exposure in air liquid interface cultures of human primary bronchial epithelial cells. HDM induced airway hyperresponsiveness and airway remodelling in mice were not altered with CeO2NPs co-exposure. Repeated HMD instillations followed by a single exposure to CeO2NPs failed to produce changes in type II inflammatory endpoints but did result in alterations in the neutrophil marker CD177. Treatment of mice with CeO2NPs in the absence of HDM did not have any significant effects. RNA-SEQ was used to explore early effects 24 h after single treatment exposures. Changes in SAA3 expression paralleled increased neutrophil BAL levels, while no changes in eosinophil or lymphocyte levels were observed. HDM resulted in a strong induction of type I interferon and IRF3 dependent gene expression, which was inhibited with CeO2NPs co-exposure. Changes in the expression of genes including CCL20, CXCL10, NLRC5, IRF7 and CLEC10A suggest regulation of dendritic cells, macrophage functionality and IRF3 modulation as key early events in how CeO2NPs may guide pulmonary responses to HDM towards type II inflammation. Conclusions CeO2NPs were observed to modulate the murine pulmonary response to house dust mite allergen exposure towards a type II inflammatory environment. As this type of response is present within asthmatic endotypes this finding may have implications for how occupational or incidental exposure to CeO2NPs should be considered for those susceptible to disease. Electronic supplementary material The online version of this article (10.1186/s12989-018-0261-5) contains supplementary material, which is available to authorized users.

Acute inhalation studies are conducted in animals as part of chemical hazard identification and for classification and labelling. Current methods employ death as an endpoint (Organisation for Economic Co-operation and Development (OECD) test guideline (TG) 403 and TG436) while the recently approved fixed concentration procedure (FCP) (OECD TG433) uses fewer animals and replaces lethality as an endpoint with evident toxicity. Evident toxicity is the presence of clinical signs that predict that exposure to the next highest concentration will cause severe toxicity or death in most animals. Approval of TG433 was the result of an international initiative, led by the National Centre for the Replacement, Refinement & Reduction of Animals in Research (NC3Rs), which collected data from six laboratories on clinical signs recorded for inhalation studies on 172 substances. This paper summarises previously published data and describes the additional analyses of the dataset that were essential for approval of the TG.

Background Concerns have been expressed that inhaled nanoparticles may behave differently to larger particles in terms of lung clearance and translocation, with potential implications for their toxicity. Studies undertaken to investigate this have typically involved limited post-exposure periods. There is a shortage of information on longer-term clearance and translocation patterns and their dependence on particle size, which this study aimed to address. Methods Rats were exposed (

WOS: 000319871200001, PubMed ID: 23198723, Aims: Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed 'spot' samples showed strong correlations with 24 h excretion (the 'gold' standard) of urinary 8-oxodG (r(p) 0.67-0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended., ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5:"Food Quality and Safety" [FOOD-CT-2005-513943]; ECNIS2, a coordination and support action within the European Union FP7 Cooperation Theme 2 Food, Agriculture, Fisheries and Biotechnologies; CISBO; Ingeborg; Leo Dannin Foundation; National Science Council, TaiwanNational Science Council of Taiwan [NSC 97-2314-B-040-015-MY3, NSC 100-2628-B-040-001-MY4]; US NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [P30ES009089]; Instituto Carlos III division of the Government for Clinical Research [PI-10/00802, RD06/0045/0006]; Generalitat ValencianaGeneralitat Valenciana [ACOM/2012/238]; Swedish Council for Working Life and Social ResearchSwedish Research CouncilSwedish Research Council for Health Working Life & Welfare (Forte); TUBITAK (Technical and Scientific Research Council of Turkey)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108Y049]; Grant Agency of the Czech RepublicGrant Agency of the Czech Republic [P503/11/0084]; Sahlgrenska University Hospital, Gothenburg; UK Medical Research Council via a People Exchange Programme Research Leader Fellowship award [G1001808/98136], Some of the authors of this work were partners in, and this work was partly supported by, ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5:"Food Quality and Safety" (Contract No. FOOD-CT-2005-513943), and also ECNIS2, a coordination and support action within the European Union FP7 Cooperation Theme 2 Food, Agriculture, Fisheries and Biotechnologies.; P Moller and S Loft are supported by CISBO and the Ingeborg and Leo Dannin Foundation.; M-R Chao and C-W Hu acknowledge financial support from the National Science Council, Taiwan (Grants NSC 97-2314-B-040-015-MY3 and NSC 100-2628-B-040-001-MY4).; R Santella acknowledges the contribution of Qiao Wang, and support from US NIH P30ES009089.; G Saez and C Cerda acknowledge financial support from the Instituto Carlos III division of the Government for Clinical Research (Grants PI-10/00802 and RD06/0045/0006) and Grant ACOM/2012/238 from Generalitat Valenciana.; K Broberg, C Lindh, and M Hossain acknowledge financial support from the Swedish Council for Working Life and Social Research; H Orhan and N Senduran acknowledge financial support from TUBITAK (Technical and Scientific Research Council of Turkey), grant number 108Y049.; P Rossner, Jr. and RJ Sram acknowledge support from the Grant Agency of the Czech Republic (P503/11/0084).; L Barregard acknowledges financial support from the Sahlgrenska University Hospital, Gothenburg.; MS Cooke acknowledges support from the UK Medical Research Council via a People Exchange Programme Research Leader Fellowship award (G1001808/98136).

Acute inhalation studies are conducted in animals as part of chemical hazard identification and characterisation, including for classification and labelling purposes. Current accepted methods use death as an endpoint (OECD TG403 and TG436), whereas the fixed concentration procedure (FCP) (draft OECD TG433) uses fewer animals and replaces lethality as an endpoint with ‘evident toxicity.’ Evident toxicity is defined as clear signs of toxicity that predict exposure to the next highest concentration will cause severe toxicity or death in most animals. A global initiative including 20 organisations, led by the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) has shared data on the clinical signs recorded during acute inhalation studies for 172 substances (primarily dusts or mists) with the aim of making evident toxicity more objective and transferable between laboratories. Pairs of studies (5 male or 5 female rats) with at least a two-fold change in concentration were analysed to determine if there are any signs at the lower dose that could have predicted severe toxicity or death at the higher concentration. The results show that signs such as body weight loss (>10% pre-dosing weight), irregular respiration, tremors and hypoactivity, seen at least once in at least one animal after the day of dosing are highly predictive (positive predictive value > 90%) of severe toxicity or death at the next highest concentration. The working group has used these data to propose changes to TG433 that incorporate a clear indication of the clinical signs that define evident toxicity.

Silibinin, a flavonolignan from milk thistle, has intestinal cancer chemopreventive efficacy in rodents. It is a strong antioxidant and modulates the insulin-like growth factor (IGF) system by increasing circulating levels of IGF-binding protein 3 (IGFBP-3) and decreasing levels of IGF-I. Here, the hypothesis was tested that administration of oral silibinin generates agent levels in human blood and colorectal and hepatic tissues consistent with pharmacologic activity. Patients with confirmed colorectal adenocarcinoma received silibinin formulated with phosphatidylcholine (silipide) at dosages of 360, 720, or 1,440 mg silibinin daily for 7 days. Blood and biopsy samples of normal and malignant colorectum or liver were obtained before dosing, and blood and colorectal or hepatic tissues were collected at resection surgery after the final silipide dose. Levels of silibinin were quantified by high-pressure liquid chromatography-UV, and plasma metabolites were identified by liquid chromatography-mass spectrometry. Blood levels of IGFBP-3, IGF-I, and the oxidative DNA damage pyrimidopurinone adduct of deoxyguanosine (M1dG) were determined. Repeated administration of silipide was safe and achieved levels of silibinin of 0.3 to 4 μmol/L in the plasma, 0.3 to 2.5 nmol/g tissue in the liver, and 20 to 141 nmol/g tissue in colorectal tissue. Silibinin monoglucuronide, silibinin diglucuronide, silibinin monosulfate, and silibinin glucuronide sulfate were identified in the plasma. Intervention with silipide did not affect circulating levels of IGFBP-3, IGF-I, or M1dG. The high silibinin levels achieved in the human colorectal mucosa after consumption of safe silibinin doses support its further exploration as a potential human colorectal cancer chemopreventive agent.

Consumption of fruits and berries has been associated with decreased risk of developing cancer. The most abundant flavonoid constituents of fruits and berries are anthocyans ( i.e. anthocyanins, glycosides, and their aglycons, anthocyanidins) that cause intense colouration. In this review, we describe epidemiological evidence hinting at the cancer preventive activity of anthocyan-containing foods in humans, results of chemoprevention studies in rodent models with anthocyans or anthocyan-containing fruit/vegetable extracts, and pharmacological properties of anthocyans. Anthocyanidins have been shown to inhibit malignant cell survival and confound many oncogenic signalling events in the 10 −6 –10 −4 M concentration range. Studies of the pharmacokinetics of anthocyanins after their consumption as single agents, anthocyanin mixtures or berry extracts suggest that anthocyanins reach levels of 10 −8 –10 −7 M in human blood. It is unclear whether such concentrations are sufficient to explain anticarcinogenic effects, and whether anthocyanins exert chemopreventive efficacy themselves, or if they need to undergo hydrolysis to their aglyconic counterparts. The currently available literature provides tantalising hints of the potential usefulness of anthocyans or anthocyan mixtures as cancer chemopreventive interventions. Nevertheless further studies are necessary to help adjudge the propitiousness of their clinical development.

Experimental evidence is presented for the presence in the liver of rats and mice of an Aroclor 1254-inducible, NADH-dependent enzyme system that can catalyse the bioactivation of aromatic and heterocyclic amines to genotoxic metabolites. It differs from the established microsomal cytochrome P450 and flavin monooxygenase systems in its response to treatment with cytochrome P450 inducing agents, optimum protein concentration and in vitro modulation by DMSO. The mutagenic metabolites generated by the NADH-supported system appear to be similar to those generated by the NADPH-mediated systems. Mutagenicity of the aminocompounds in the presence of either cosubstrate was less pronounced in an O-acetyltransferase-deficient bacterial strain, implying the presence of hydroxylamines. Moreover, glutathione potentiated the mutagenic response of both the NADH- and NADPH-generated metabolites. Cytochrome c suppressed markedly the NADPH-dependent mutagenicity of aromatic amines but had no such effect in the presence of NADH. Similarly, antibodies to cytochrome P450 reductase markedly inhibited the NADPH-, but not the NADH-dependent bioactivation of the aromatic amine 2-aminoanthracene. The cytochrome P450 suicide inhibitor, 1-aminobenzotriazole, decreased the mutagenicity of both, the NADH- and NADPH-mediated bioactivation of the aminocompounds. The above findings raise the possibility that a cytochrome P450-like protein, that can receive electrons from NADH, possibly through cytochrome b5 reductase, is present in the hepatic microsomes of rats and mice, and is capable of catalysing the bioactivation of aromatic amines through N-hydroxylation. Such a hypothesis is supported by the findings that NADH could support the O-dealkylation of 7-methoxy- and 7-ethoxy-resorufin, in the absence of NADPH. Finally the NADH-dependent bioactivation of aromatic amines was induced markedly by Aroclor 1254 and benzo(a)pyrene in Ah responsive, but not Ah nonresponsive, mice indicating that it is associated with the Ah locus.

Scope: The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. Methods and results: The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 μg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 μg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 μg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 μg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 μg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 μg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. Conclusion: These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.

Naturally occurring anthocyanins possess colorectal cancer chemopreventive properties in rodent models. We investigated whether mirtocyan, an anthocyanin-rich standardized bilberry extract, causes pharmacodynamic changes consistent with chemopreventive efficacy and generates measurable levels of anthocyanins in blood, urine, and target tissue. Twenty-five colorectal cancer patients scheduled to undergo resection of primary tumor or liver metastases received mirtocyan 1.4, 2.8, or 5.6 grams (containing 0.5-2.0 grams anthocyanins) daily for 7 days before surgery. Bilberry anthocyanins were analyzed by high performance liquid chromatography (HPLC) with visible or mass spectrometric detection. Proliferation was determined by immunohistochemistry of Ki-67 in colorectal tumor. Concentrations of insulin-like growth factor (IGF)-I were measured in plasma. Mirtocyan anthocyanins and methyl and glucuronide metabolites were identified in plasma, colorectal tissue, and urine, but not in liver. Anthocyanin concentrations in plasma and urine were roughly dose-dependent, reaching ∼179 ng/gram in tumor tissue at the highest dose. In tumor tissue from all patients on mirtocyan, proliferation was decreased by 7% compared with preintervention values. The low dose caused a small but nonsignificant reduction in circulating IGF-I concentrations. In conclusion, repeated administration of bilberry anthocyanins exerts pharmacodynamic effects and generates concentrations of anthocyanins in humans resembling those seen in ApcMin mice, a model of FAP adenomas sensitive to the chemopreventive properties of anthocyanins. Studies of doses containing

Purpurin, an anthraquinone constituent from madder root, has previously been reported as antimutagenic in the Ames Salmonella bacterial mutagenicity assay and as antigenotoxic in Drosophila melanogaster, against a range of environmental carcinogens. Short-term dietary supplementation with purpurin inhibits the formation of hepatic DNA adducts in male C57bl6 mice after a single dose of the heterocyclic amine dietary carcinogen Trp-P-2 (30 mg/kg). Inhibition of adduct formation was dose-dependent. No DNA adducts were observed in animals treated only with purpurin. The decrease in adduct formation was accompanied by significant, dose-dependent inductions of hepatic cytochrome P450-dependent dealkylations of methoxy- (CYP1A2), ethoxy- (CYP1A1), and pentoxy- (CYP2B) resorufins, total cytochrome P450, and NADPH cytochrome P450 reductase. It is hypothesized that purpurin exhibits chemopreventive potential by inhibiting the cytochrome P450-dependent metabolism of heterocyclic amines to their genotoxic N-hydroxylamines.