Publications
263 results found
Cohen SM, Gooderham N, 2018, GRAS 28 Flavouring Substances, Food Technology, ISSN: 0015-6639
Smith RL, Cohen SM, Fukushima S, et al., 2018, The safety evaluation of food flavouring substances: the role of metabolic studies, Toxicology Research, Vol: 7, Pages: 618-646, ISSN: 2045-452X
The safety assessment of a flavour substance examines several factors, including metabolic and physiological disposition data. The present article provides an overview of the metabolism and disposition of flavour substances by identifying general applicable principles of metabolism to illustrate how information on metabolic fate is taken into account in their safety evaluation. The metabolism of the majority of flavour substances involves both a series of enzymatic and non-enzymatic biotransformation that often result in products that are more hydrophilic and more readily excretable than their precursors. Flavours can undergo metabolic reactions, such as oxidation, reduction, or hydrolysis that alter a functional group relative to the parent compound. The altered functional group may serve as a reaction site for a subsequent metabolic transformation. Metabolic intermediates undergo conjugation with an endogenous agent such as glucuronic acid, sulphate, glutathione, amino acids, or acetate. Such conjugates are typically readily excreted through the kidneys and liver. This paper summarizes the types of metabolic reactions that have been documented for flavour substances that are added to the human food chain, the methodologies available for metabolic studies, and the factors that affect the metabolic fate of a flavour substance.
Alkandari A, Ashrafian H, Sathyapalan T, et al., 2018, Improved physiology and metabolic flux after Roux-en-Y gastric bypass is associated with temporal changes in the circulating microRNAome: a longitudinal study in humans., BMC Obesity, Vol: 5, ISSN: 2052-9538
BackgroundThe global pandemic of obesity and the metabolic syndrome are leading causes of mortality and morbidity. Bariatric surgery leads to sustained weight loss and improves obesity-associated morbidity including remission of type 2 diabetes. MicroRNAs are small, endogenous RNAs that regulate gene expression post-transcriptionally, controlling most of the human transcriptome and contributing to the regulation of systemic metabolism. This preliminary, longitudinal, repeat sampling study, in which subjects acted as their own control, aimed to assess the temporal effect of bariatric surgery on circulating microRNA expression profiles.MethodsWe used Exiqon’s optimized circulating microRNA panel (comprising 179 validated miRNAs) and miRCURY locked nucleic acid plasma/serum Polymerase Chain Reaction (PCR) to assess circulating microRNA expression. The microRNAome was determined for Roux-en-Y gastric bypass (RYGB) patients examined preoperatively and at 1 month, 3 months, 6 months, 9 months and 12 months postoperatively. Data was analysed using multivariate and univariate statistics.ResultsCompared to the preoperative circulating microRNA expression profile, RYGB altered the circulating microRNAome in a time dependent manner and the expression of 48 circulating microRNAs were significantly different. Importantly, these latter microRNAs are associated with pathways involved in regulation and rescue from metabolic dysfunction and correlated with BMI, the percentage of excess weight loss and fasting blood glucose levels.ConclusionsThe results of this pilot study show that RYGB fundamentally alters microRNA expression in circulation with a time-dependent progressive departure in profile from the preoperative baseline and indicate that microRNAs are potentially novel biomarkers for the benefits of bariatric surgery.
Malik D-E-S, David RM, Gooderham NJ, 2018, Ethanol potentiates the genotoxicity of the food-derived mammary carcinogen PhIP in human estrogen receptor-positive mammary cells: mechanistic support for lifestyle factors (cooked red meat and ethanol) associated with mammary cancer, Archives of Toxicology, Vol: 92, Pages: 1639-1655, ISSN: 0340-5761
Consumption of cooked/processed meat and ethanol are lifestyle risk factors in the aetiology of breast cancer. Cooking meat generates heterocyclic amines such as 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Epidemiology, mechanistic and animal studies indicate that PhIP is a mammary carcinogen that could be causally linked to breast cancer incidence; PhIP is DNA damaging, mutagenic and oestrogenic. PhIP toxicity involves cytochrome P450 (CYP1 family)-mediated metabolic activation to DNA-damaging species, and transcriptional responses through Aryl hydrocarbon receptor (AhR) and estrogen-receptor-α (ER-α). Ethanol consumption is a modifiable lifestyle factor strongly associated with breast cancer risk. Ethanol toxicity involves alcohol dehydrogenase metabolism to reactive acetaldehyde, and is also a substrate for CYP2E1, which when uncoupled generates reactive oxygen species (ROS) and DNA damage. Here, using human mammary cells that differ in estrogen-receptor status, we explore genotoxicity of PhIP and ethanol and mechanisms behind this toxicity. Treatment with PhIP (10-7-10-4 M) significantly induced genotoxicity (micronuclei formation) preferentially in ER-α positive human mammary cell lines (MCF-7, ER-α+) compared to MDA-MB-231 (ER-α-) cells. PhIP-induced CYP1A2 in both cell lines but CYP1B1 was selectively induced in ER-α(+) cells. ER-α inhibition in MCF-7 cells attenuated PhIP-mediated micronuclei formation and CYP1B1 induction. PhIP-induced CYP2E1 and ROS via ER-α-STAT-3 pathway, but only in ER-α (+) MCF-7 cells. Importantly, simultaneous treatments of physiological concentrations ethanol (10-3-10-1 M) with PhIP (10-7-10-4 M) increased oxidative stress and genotoxicity in MCF-7 cells, compared to the individual chemicals. Collectively, these data offer a mechanistic basis for the increased risk of breast cancer associated with dietary cooked meat and ethanol lifestyle choices.
Cohen SM, Eisenbrand G, Fukushima S, et al., 2018, Updated procedure for the safety evaluation of natural flavor complexes used as ingredients in food, Food and Chemical Toxicology, Vol: 113, Pages: 171-178, ISSN: 0278-6915
An effective and thorough approach for the safety evaluation of natural flavor complexes (NFCs) was published in 2005 by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). An updated procedure is provided here, which maintains the essential concepts of the use of the congeneric group approach and the reliance on the Threshold of Toxicological Concern (TTC) concept. The updated procedure emphasizes more rigorous considerations of unidentified constituents and the genotoxic potential of constituents. The update of the previously established procedure is the first step in a multi-year project to conduct safety re-evaluations for more than 250 NFCs that have uses that are currently considered Generally Recognized as Safe (GRAS) by the FEMA Expert Panel. In addition, this procedure can be more generally employed in the safety evaluation of NFCs.
Saleh AF, Bachman M, Priestley CC, et al., 2018, 2'-O-(2-methoxyethyl) nucleosides are not phosphorylated or incorporated into the genome of Human Lymphoblastoid TK6 Cells, Toxicological Sciences, Vol: 163, Pages: 70-78, ISSN: 1096-0929
Nucleoside analogues with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogues that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA and RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogues have been investigated. Using LC/MS/MS, we showed that enzymes in the nucleotide salvage pathway including deoxycytidine kinase (dCK) and thymidine kinase (TK1) displayed poor reactivity toward 2'-O-MOE nucleoside analogues. On the other hand, 2'-fluoro (F) nucleosides, regardless of the nucleobase, were efficiently phosphorylated to their monophosphate forms by dCK and TK1. Consistent with their efficient phosphorylation by dCK and TK1, 2'-F nucleosides analogues were incorporated into cellular DNA and RNA while no incorporation was detected with 2'-O-MOE nucleoside analogues. In conclusion, these data suggest that the inability of dCK and TK1 to create the monophosphates of 2'-O-MOE nucleoside analogues reduces the risk of their incorporation into cellular DNA and RNA.
Harling L, Athanasiou T, Ashrafian H, et al., 2017, Pre-operative serum VCAM-1 as a biomarker of atrial fibrillation after coronary artery bypass grafting, Journal of Cardiothoracic Surgery, Vol: 12, ISSN: 1749-8090
ObjectiveSystemic inflammation is a recognised contributory factor in the pathogenesis of de novo post-operative atrial fibrillation after cardiac surgery. This study aims to determine whether serum soluble vascular endothelial cell adhesion molecule (sVCAM-1) may predict the onset of POAF in patients under going coronary artery bypass grafting.Methods34 patients undergoing non-emergent, on-pump CABG were prospectively recruited. Plasma was obtained at 24 h pre-operatively and at 48 and 96 h post-operatively. POAF was defined by continuous Holter recording. Inter-group comparisons were performed using student t-test or ANOVA as appropriate.ResultsThirteen (13/34) patients developed POAF at a mean of 2.5 days post-operatively. Serum sVCAM-1 was significantly increased in the pre-operative serum of POAF when compared to non-POAF patients (p = 0.022). No significant difference was observed between the groups at 48 h (p = 0.073) or 96 h (p = 0.135) post-operatively. sVCAM-1 had a sensitivity of 60.0% and specificity of 77.27%, with an overall diagnostic accuracy of 75.2% in predicting POAF.ConclusionssVCAM-1 concentration in the pre-operative serum of patients undergoing CABG may accurately predict the onset of de novo POAF. As such, serum sVCAM-1 may be used as a predictive biomarker for this common arrhythmia. Further work must now perform prospective, targeted validation of these results in a larger patient cohort.
Kamola PJ, Maratou K, Wilson PA, et al., 2017, Strategies for in vivo screening and mitigation of hepatotoxicity associated with antisense drugs, Molecular Therapy - Nucleic Acids, Vol: 8, Pages: 383-394, ISSN: 2162-2531
Antisense oligonucleotide (ASO) gapmers downregulate gene expression by inducing enzyme-dependent degradation of targeted RNA and represent a promising therapeutic platform for addressing previously undruggable genes. Unfortunately, their therapeutic application, particularly that of the more potent chemistries (e.g., locked-nucleic-acid-containing gapmers), has been hampered by their frequent hepatoxicity, which could be driven by hybridization-mediated interactions. An early de-risking of this liability is a crucial component of developing safe, ASO-based drugs. To rank ASOs based on their effect on the liver, we have developed an acute screen in the mouse that can be applied early in the drug development cycle. A single-dose (3-day) screen with streamlined endpoints (i.e., plasma transaminase levels and liver weights) was observed to be predictive of ASO hepatotoxicity ranking established based on a repeat-dose (15 day) study. Furthermore, to study the underlying mechanisms of liver toxicity, we applied transcriptome profiling and pathway analyses and show that adverse in vivo liver phenotypes correlate with the number of potent, hybridization-mediated off-target effects (OTEs). We propose that a combination of in silico OTE predictions, streamlined in vivo hepatotoxicity screening, and a transcriptome-wide selectivity screen is a valid approach to identifying and progressing safer compounds.
Cohen SM, Fukushima S, Gooderham NJ, et al., 2016, Safety evaluation of substituted thiophenes used as flavoring ingredients., Food and Chemical Toxicology, Vol: 99, Pages: 40-59, ISSN: 1873-6351
This publication is the second in a series by the Expert Panel of the Flavor and Extract Manufacturers Association summarizing the conclusions of its third systematic re-evaluation of the safety of flavorings previously considered to be generally recognized as safe (GRAS) under conditions of intended use. Re-evaluation of GRAS status for flavorings is based on updated considerations of exposure, structural analogy, metabolism, pharmacokinetics and toxicology and includes a comprehensive review of the scientific information on the flavorings and structurally related substances. Of the 12 substituted thiophenes reviewed here, 11 were reaffirmed as GRAS based on their rapid absorption, metabolism and excretion in humans and animals; the low estimated dietary exposure from flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels; and the lack of significant genotoxic and mutagenic potential. For one of the substituted thiophenes, 3-acetyl-2,5-dimethylthiophene, it was concluded that more detailed exposure information, comparative metabolism studies and comprehensive toxicity data, including an in-depth evaluation of the mechanism of action for any adverse effects observed, are required for continuation of its FEMA GRAS™ status. In the absence of these data, the compound was removed from the FEMA GRAS list.
Akingbasote JA, Foster AJ, Jones HB, et al., 2016, Improved hepatic physiology in hepatic cytochrome P450 reductase null (HRN™) mice dosed orally with fenclozic acid, Toxicology Research, Vol: 6, Pages: 81-88, ISSN: 2045-4538
Hepatic NADPH-cytochrome P450 oxidoreductase null (HRN™) mice exhibit no functional expression of hepatic cytochrome P450 (P450) when compared to wild type (WT) mice, but have normal hepatic and extrahepatic expression of other biotransformation enzymes. We have assessed the utility of HRN™ mice for investigation of the role of metabolic bioactivation in liver toxicity caused by the nonsteroidal anti-inflammatory drug (NSAID) fenclozic acid. In vitro studies revealed significant NADPH-dependent (i.e. P450-mediated) covalent binding of [14C]-fenclozic acid to liver microsomes from WT mice and HRN™ mice, whereas no in vitro covalent binding was observed in the presence of the UDP-glucuronyltransferase cofactor UDPGA. Oral fenclozic acid administration did not alter the liver histopathology or elevate the plasma liver enzyme activities of WT mice, or affect their hepatic miRNA contents. Livers from HRN™ mice exhibited abnormal liver histopathology (enhanced lipid accumulation, bile duct proliferation, hepatocellular degeneration, necrosis, inflammatory cell infiltration) and plasma clinical chemistry (elevated alanine aminotransferase, glutamate dehydrogenase and alkaline phosphatase activities). Modest apparent improvements in these abnormalities were observed when HRN™ mice dosed orally with fenclozic acid for 7 days at 100 mg/kg/day. Previously we observed more marked effects on liver histopathology and integrity in HRN™ mice dosed orally with the NSAID diclofenac for 7 days at 30 mg/kg/day. We conclude that HRN™ mice are valuable for assessing P450-related hepatic drug biotransformation, but not for drug toxicity studies due to underlying liver dysfunction. Nonetheless, HRN™ mice may provide novel insights into the role of inflammation in liver injury, thereby aiding its treatment.
Cohen SM, Fukushima S, Gooderham NJ, et al., 2016, FEMA expert panel review of p-mentha-1,8-dien-7-al genotoxicity testing results, Food and Chemical Toxicology, Vol: 98, Pages: 201-209, ISSN: 1873-6351
p-Mentha-1,8-dien-7-al is a naturally occurring cyclic alpha,beta-unsaturated aldehyde that is used as a flavoring substance throughout the world. Due to the chemical structure and the potential DNA reactivity of the alpha,beta-unsaturated carbonyl moiety, a battery of genotoxicity assays was requested by the European Food Safety Authority. Previous genotoxicity studies on the substance gave mixed results, but both positive and negative results were hampered by not always being performed to any standard guideline. The new test battery data indicated some evidence of mutagenicity in vitro, but an in vivo comet/micronucleus combination assay performed in rats was concluded by the study directors to not result in any biologically relevant positive responses. However, EFSA concluded that the in vivo assay gave evidence that p-mentha-1,8-dien-7-al was of potential genotoxic concern. The Expert Panel of the Flavor and Extract Manufacturers Association (FEMA) has reviewed the newly available data and considered its interpretation relative to standard guidelines such as that established by the Organization for Economic Cooperation and Development, and has concluded that the results in the comet/micronucleus combination assay are consistent with the interpretation by the study directors; namely, that p-mentha-1,8-dien-7-al does not appear to have any in vivo genotoxic potential.
Saleh AF, Fellows MM, Ying L, et al., 2016, The lack of mutagenic potential of a guanine-rich triplex forming oligonucleotide in physiological conditions, Toxicological Sciences, Vol: 155, Pages: 101-111, ISSN: 1096-6080
Triplex forming oligonucleotides (TFOs) bind in the major groove of DNA duplex in a sequence-specific manner imparted by Hoogsteen hydrogen bonds. There have been several reports demonstrating the ability of guanine-rich TFOs to induce targeted mutagenesis on an exogenous plasmid or an endogenous chromosomal locus. In particular, a 30mer guanine-rich triplex forming oligonucleotide, AG30, optimally designed to target the supFG1 reporter gene was reported to be mutagenic in the absence of DNA reactive agents in cultured cells and in vivo Here, we investigated the mutagenic potential of AG30 using the supFG1 shuttle vector forward mutation assay under physiological conditions. We also assessed the triplex binding potential of AG30 alongside cytotoxic and mutagenic assessment. In a cell free condition, AG30 was able to bind its polypurine target site in the supFG1 gene in the absence of potassium chloride and also aligned with a 5-fold increase in the mutant frequency when AG30 was pre-incubated with the supFG1 plasmid in the absence of potassium prior to transfection into COS-7 cells. However, when we analysed triplex formation of AG30 and the supFG1 target duplex at physiological potassium levels, triplex formation was inhibited due to the formation of competing secondary structures. Subsequent assessment of mutant frequency under physiological conditions, by pre-transfecting COS-7 cells with the supFG1 plasmid prior to AG30 treatment led to a very small increase (1.4-fold) in the mutant frequency. Transfection of cells with even higher concentrations of AG30 did result in an elevated mutagenic response but this was also seen with a scrambled sequence, and was therefore considered unlikely to be biologically relevant as an associated increase in cytotoxicity was also apparent. Our findings also provide further assurance on the low potential of triplex-mediated mutation as a consequence of unintentional genomic DNA binding by therapeutic antisense oligonucleotides.
Aigner A, Buesen R, Gant T, et al., 2016, Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop, Regulatory Toxicology and Pharmacology, Vol: 82, Pages: 127-139, ISSN: 1096-0295
The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context.
Harling L, Lambert J, Ashrafian H, et al., 2016, Elevated serum microRNA 483-5p levels may predict patients at risk of post-operative atrial fibrillation, European Journal of Cardio-thoracic Surgery, Vol: 51, Pages: 73-78, ISSN: 1873-734X
Objective: Atrial fibrillation (POAF) is the commonest post-operative cardiac arrhythmia, affecting approximately 1 in 3 patients undergoing coronary artery bypass grafting (CABG). Although its aetiology is complex, atrial substrate changes may pre-dispose to its onset. This study aims to ascertain the atrial microRNA signature of POAF and determine the potential for circulating microRNA as a pre-operative biomarker for this arrhythmia.Methods: 34 patients undergoing non-emergent, on-pump CABG were prospectively recruited. Right atrial biopsies were taken intra-operatively and snap frozen for RNA extraction. Plasma was obtained at 24 hours pre-operatively and at 2 and 4-days post-operatively. POAF was defined by continuous Holter recording. Inter-group comparisons were performed using student t-test or ANOVA as required. Receiver operating characteristics (ROC) analysis was used to determine the diagnostic accuracy of pre-operative serum miRNA as a POAF biomarker. Results: 16 microRNA were differentially expressed in the atrial myocardium of POAF patients when compared to those maintaining sinus rhythm (SR). MiR-208a was the most under-expressed (FC:2.458) and miR-483-5p the most over-expressed (FC:1.804). Mir-483-5p also demonstrated significant overexpression in the pre-operative serum of these patients, with ROC analysis demonstrating an overall predictive accuracy of 78%. Conclusions: This study provides the first description of atrial myocardial and circulating plasma microRNA in POAF patients. Our findings suggest POAF may be associated with pre-existing atrial substrate differences predisposing to arrhythmogenesis. Moreover, this study highlights the potential for miR-483-5p in biomarker development. Further work must now perform prospective, targeted validation of these results in a larger patient cohort.
Osborne M, Haltalli M, Currie R, et al., 2016, Transdifferentiated rat pancreatic progenitor cells (AR42J-B13/H) respond to phenobarbital in a rat hepatocyte-specific manner, Toxicology, Vol: 363-364, Pages: 10-18, ISSN: 1879-3185
Phenobarbital (PB) is known to produce species-specific effects in the rat and mouse, being carcinogenic in certain mouse strains, but only in rats if treated after a DNA damaging event. PB treatment in the rat and mouse also produces disparate effects on cell signalling and miRNA expression profiles. These responses are induced by short term and prolonged PB exposure, respectively, with the latter treatments being difficult to examine mechanistically in primary hepatocytes due to rapid loss of the original hepatic phenotype and limited sustainability in culture. Here we explore the rat hepatocyte-like B13/H cell line as a model for hepatic response to PB exposure in both short-term and longer duration treatments. We demonstrate that PB with Egf treatment in the B13/H cells, resulted in a significant increase in Erk activation, as determined by the ratio of phospho-Erk to total Erk, compared to Egf alone. We also show that an extended treatment with PB in the B13/H cells produces a miRNA response similar to that seen in the rat in vivo, via the time-dependent induction of miR-182/96. Additionally, we confirm that B13/H cells respond to Car activators in a typical rat-specific manner. These data suggest that the B13/H cells produce temporal responses to PB that are comparable to those reported in short-term primary rat hepatocyte cultures and in the longer term are similar to those in the rat in vivo. Finally, we also show that Car-associated miR-122 expression is decreased by PB treatment in B13/H cells, a PB-induced response that is common to the rat, mouse and human. We conclude that the B13/H cell system produces a qualitative response comparable to the rat, which is different to the response in the mouse, and that this model could be a useful tool for exploring the functional consequences of PB-sensitive miRNA changes and resistance to PB-mediated tumours in the rat.
Zaman J, Harling L, Ashrafian H, et al., 2016, Post-operative atrial fibrillation is associated with a pre-existing structural and electrical substrate in human right atrial myocardium, International Journal of Cardiology, Vol: 220, Pages: 580-588, ISSN: 1874-1754
BackgroundPost-operative atrial fibrillation (POAF) is a major health economic burden. However, the precise mechanisms in POAF remain unclear. In other forms of AF, sites of high dominant frequency (DF) in sinus rhythm (SR) may harbour ‘AF nests’. We studied AF inducibility in relation to substrate changes using epicardial electrograms and cardiomyocyte calcium handling in the atria of AF naïve patients.MethodBipolar electrograms were recorded from the lateral right atrial (RA) wall in 34 patients undergoing coronary surgery using a high-density array in sinus rhythm (NSR). RA burst pacing at 200/500/1000 ms cycle lengths (CL) was performed, recording episodes of AF > 30 s. Co-localised RA tissue was snap frozen for RNA and protein extraction.ResultsElectrograms prolonged during AF (76.64 ± 29.35 ms) vs. NSR/pacing (p < 0.001). Compared to NSR, electrogram amplitude was reduced during AF and during pacing at 200 ms CL (p < 0.001). Electrogram DF was significantly lower in AF (75.87 ± 23.63 Hz) vs. NSR (89.33 ± 25.99 Hz) (p < 0.05), and NSR DF higher in AF inducible patients at the site of AF initiation (p < 0.05). Structurally, POAF atrial myocardium demonstrated reduced sarcolipin gene (p = 0.0080) and protein (p = 0.0242) expression vs. NSR. Phospholamban gene and protein expression was unchanged. SERCA2a protein expression remained unchanged, but MYH6 (p = 0.0297) and SERCA2A (p = 0.0343) gene expression was reduced in POAF.ConclusionsHuman atrial electrograms prolong and reduce in amplitude in induced peri-operative AF vs. NSR or pacing. In those sustaining AF, high DF sites in NSR may indicate ‘AF nests’. This electrical remodelling is accompanied by structural remodelling with altered expression of cardiomyocyte calcium handling detectable before POAF. These novel upstream substrate changes offer a novel mechanism and manifestation of human POAF.
Gooderham N, Ross P, Keltie S, 2016, Toxicology Research: Publishing research that is excellent and innovative, that drives toxicology and has international impact, Toxicology Research, Vol: 5, Pages: 371-372, ISSN: 2045-452X
David R, Ebbels T, Gooderham N, 2016, Synergistic and Antagonistic Mutation Responses of Human MCL-5 Cells to Mixtures of Benzo[a]pyrene and 2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine: Dose-Related Variation in the Joint Effects of Common Dietary Carcinogens., Environmental Health Perspectives, Vol: 124, Pages: 88-96, ISSN: 1552-9924
BACKGROUND: Chemical carcinogens such as benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) may contribute to the etiology of human diet-associated cancer. Individually, these are genotoxic, but the consequences of exposure to mixtures of these chemicals have not been systematically examined. OBJECTIVES: To determine the mutagenic response to mixtures of BaP and PhIP at concentrations relevant to human exposure (mM to sub-nM). METHODS: Human MCL-5 cells (metabolically competent) were exposed to BaP or PhIP individually or in mixtures. Mutagenicity was assessed at the thymidine kinase (TK) locus, CYP1A activity and message determined by Ethoxyresorufin-O-deethylase (EROD) activity and Q-PCR respectively, and cell cycle measured by flow cytometry. RESULTS: Mixtures gave modified dose-responses compared to the individual chemicals; a remarkable increased mutant frequency (MF) at low concentration combinations (not mutagenic individually), and decreased MF at higher concentration combinations, compared to the calculated predicted additive MF of the individual chemicals. EROD activity and CYP1A1 mRNA levels correlated with TK MF supporting involvement of the CYP1A family in mutation. Moreover, a cell cycle G2/M phase block was observed at high dose combinations, consistent with DNA damage sensing and repair. CONCLUSIONS: Mixtures of these genotoxic chemicals produced mutation responses that differed from expectations for additive effects of the individual chemicals. The increase in MF for some combinations of chemicals at low concentrations that were not genotoxic for the individual chemicals, and the non-monotonic dose response, may be important for understanding the mutagenic potential of food and the etiology of diet-associated cancers.
Sathyapalan T, David R, Gooderham NJ, et al., 2015, Increased expression of circulating miRNA-93 in women with polycystic ovary syndrome may represent a novel, non-invasive biomarker for diagnosis., Scientific Reports, Vol: 5, ISSN: 2045-2322
MicroRNAs (miRNA) are a novel class of small noncoding single-stranded RNA molecules that regulate gene expression. There is increasing evidence of their importance in polycystic ovary syndrome (PCOS). The objective was to determine if miRNA-93 and miRNA-223 are differentially expressed in the circulation of women with PCOS compared to age matched women. A case-control study comparing women with PCOS (n = 25) to age and weight matched controls (n = 24) without PCOS was performed. MiRNA-93 and miRNA-223 were determined by total RNA reverse transcription. Both miRNA-93 and miRNA-223 were significantly increased relative to the control group (p < 0.01, p = 0.029 respectively). In both groups there was no correlation of either miRNA-93 or miRNA-223 with insulin, HOMA-IR, HOMA-β or testosterone levels. The area under the receiver operator characteristic curve for miR-223 and miR-93 was 0.66 and 0.72 respectively, suggesting miR-93 is a more efficient biomarker than miR-223 for diagnosis of PCOS. The combination of the two miRNAs together, tested using multiple logistic regression analysis, did not improve the diagnostic potential. In conclusion, circulating miRNA-93 and miRNA-223 were higher in women with PCOS compared to age and weight matched controls independent of insulin resistance and testosterone levels, and miR-93 may represent a novel diagnostic biomarker for PCOS.
David RM, Gooderham NJ, 2015, Using 3D MCF-7 mammary spheroids to assess the genotoxicity of mixtures of the food-derived carcinogens benzo[a]pyrene and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, Toxicology Research, Vol: 5, Pages: 312-317, ISSN: 2045-452X
Genotoxic carcinogens are present in the human diet, and two important examples are benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). BaP is a polycyclic aromatic hydrocarbon generated by incomplete combustion of organic substances, thus contaminating numerous foodstuffs, and PhIP is a heterocyclic amine formed when meat is cooked. Genotoxicity testing of chemical carcinogens has focussed largely on individual chemicals, particularly in relation to diet, despite mixtures representing a more realistic exposure scenario. We have previously shown that exposure of MCL-5 cells to BaP-PhIP mixtures produces a TK mutation dose response that differs from the predicted additive response, using traditional regulatory-like two-dimensional (2D) cell culture. There is a large gap between 2D cell culture and the whole animal, and three-dimensional (3D) cell culture, shown to better represent in vivo tissue structure, may bridge the gap. The aim of the current study was to use 3D spheroids to characterise the DNA damage response following exposure to mixtures of the mammary carcinogens BaP and PhIP. Mammary MCF-7 cells were grown in 3D spheroids, exposed (24 h) to BaP (10-10 to 10-5 M) or PhIP (10-9 to 10-4 M) individually or in mixtures and DNA damage assessed by micronucleus (MN) formation. A dose-dependent increase in MN was observed for the individual chemicals in 3D cell culture. In line with our previous 2D TK mutation data, 3D mixture exposures gave a modified DNA damage profile compared to the individual chemicals, with a potent response at low dose combinations and a decrease in MN with higher concentrations of BaP in the mixture. Ethoxyresorufin-O-deethylase (CYP1A) activity increased with increasing concentration of BaP in the mixture, and for combinations with 10 μM BaP, CYP1A1 mRNA induction was sustained up to 48 h. These data suggest mixtures of genotoxic chemicals give DNA damage responses that differ considerably from those produc
Kamola PJ, Kitson JD, Turner G, et al., 2015, In silico and in vitro evaluation of exonic and intronic off-target effects form a critical element of therapeutic ASO gapmer optimization., Nucleic Acids Research, Vol: 43, Pages: 8638-8650, ISSN: 1362-4962
With many safety and technical limitations partly mitigated through chemical modifications, antisense oligonucleotides (ASOs) are gaining recognition as therapeutic entities. The increase in potency realized by 'third generation chemistries' may, however, simultaneously increase affinity to unintended targets with partial sequence complementarity. However, putative hybridization-dependent off-target effects (OTEs), a risk historically regarded as low, are not being adequately investigated. Here we show an unexpectedly high OTEs confirmation rate during screening of fully phosphorothioated (PS)-LNA gapmer ASOs designed against the BACH1 transcript. We demonstrate in vitro mRNA and protein knockdown of off-targets with a wide range of mismatch (MM) and gap patterns. Furthermore, with RNase H1 activity residing within the nucleus, hybridization predicted against intronic regions of pre-mRNAs was tested and confirmed. This dramatically increased ASO-binding landscape together with relatively high potency of such interactions translates into a considerable safety concern. We show here that with base pairing-driven target recognition it is possible to predict the putative off-targets and address the liability during lead design and optimization phases. Moreover, in silico analysis performed against both primary as well as spliced transcripts will be invaluable in elucidating the mechanism behind the hepatoxicity observed with some LNA-modified gapmers.
Cohen SM, Fukushima S, Gooderham NJ, et al., 2015, GRAS 27 flavoring substances, Food Technology, Vol: 69, ISSN: 0015-6639
Patel SA, Gooderham NJ, 2015, IL6 mediates immune and colorectal cancer cell crosstalk via miR-21 and miR-29b., Molecular Cancer Research, Vol: 13, ISSN: 1557-3125
Tumors are surrounded and infiltrated by a variety of stromal cell types including fibroblasts, immune cells and vascular endothelial cells, which interact with malignant cells to generate the tumor microenvironment (TME). This complex environment is thought to be regulated by the tumor in order to promote its survival and progression, and thus constitutes a potential target for cancer therapy. However, intercellular communication within the microenvironment is not yet well understood. The current study investigates the mechanism by which cancer and immune cells communicate using an in vitro co-culture model. It is demonstrated that interleukin-6 (IL6), a proinflammatory cytokine, secreted by immune cells promotes colorectal cancer cell invasiveness. Additionally, in the presence of IL6, the cancer cells were able to secrete circulating microRNAs (miRs) miR-21 and miR-29b to further induce immune cell IL6 production. Activated immune cells were also found to release miR-21 into the tumor microenvironment. Taken together, these mechanistic findings provide a better understanding of intercellular communication between immune and cancer cells in the TME and offer insight into some of the key players that mediate this crosstalk. IMPLICATIONS: This study demonstrates that co-cultured cancer and immune cells communicate via IL6 and circulating miRs to sustain chronic inflammation and promote pro-metastatic cancer cell behavior. In addition, critical players are identified that mediate intercellular communication in the TME and suggest possible therapeutic approaches that target the microenvironment.
Segal CV, Koufaris C, Powell C, et al., 2015, Effects of treatment with androgen receptor ligands on microRNA expression of prostate cancer cells, Toxicology, Vol: 333, Pages: 45-52, ISSN: 0300-483X
Post-transcriptional regulation by microRNA (miRNA) is an important aspect of androgen receptor (AR) signalling in prostate cancer cells. However, the global profiling of miRNA expression in prostate cancer cells following treatment with AR ligands has not been reported so far. In this study we examined the effect of treatment with two AR agonists (mibolerone (MIB) and dihydrotestosterone (DHT)) and an AR antagonist (bicalutamide (BIC)) on miRNA expression in the human androgen-dependent LNCaP prostate cancer cell line using microarray technology and verification of selected miRNA using quantitative real-time PCR (qRT-PCR). No miRNA was identified as differentially expressed following treatment with the AR antagonist BIC. In contrast, a number of common and compound-specific alterations in miRNA expression were observed following treatment with AR agonists. Unexpectedly it was found that treatment with the AR agonists resulted in the repression of miR-221, a miRNA previously established to be involved with prostate cancer development. This observation indicates that this miRNA may have a more complex role in prostate cancer development than considered previously. Treatment with MIB led to an induction of miR-210 expression, a hypoxia-related miRNA. This miRNA is reported to be involved in cell adaptation to hypoxia and thus induction in conditions of normoxia may be important in driving metabolic changes observed in prostate cancer. Thus examining the effect of AR agonists and antagonists on miRNA expression can provide novel insights into the response of cells to AR ligands and subsequent downstream events.
Saidin NA, Holmes E, Takayama H, et al., 2015, The cellular toxicology of mitragynine, the dominant alkaloid of the narcotic-like herb, Mitragyna speciosa Korth, Toxicology Research, Vol: 4, Pages: 1173-1183, ISSN: 2045-4538
Mitragyna speciosa Korth (Kratom), a herb of the Rubiaceae family is indigenous to southeast Asia. The plant and its dominant alkaloid mitragynine (MIT) are narcotic/analgesic and illicit consumption is widespread in Asia; the toxicological consequences of consumption are poorly documented. We determined cytotoxicity of MIT on human cell lines and report dose and time-dependent stimulation and inhibition of proliferation. Since MIT has powerful opiate-like activity, we focussed on human neuronal SH-SY5Y cell line and found the colony forming ability of cells treated with MIT showed a dose-dependent trend for reduced survival. Studies using metabolically competent MCL-5 cells and chemical inhibitors indicated that CYP 2E1 and 2A6 were involved in the cytotoxicity. Cytotoxicity was preceded by cell cycle arrest mainly at G1 and S phase. To assess whether arrest was due to DNA damage or mutation, we examined genotoxic potential using the L5178 TK+/− mouse lymphoma assay and found that MIT was not genotoxic at the TK locus, even at doses that were highly cytotoxic. To investigate mechanisms of MIT cytotoxicity, we used flow cytometry and annexin V with 7-amino-actinomycin D staining and show apoptosis and necrotic activity. Apoptosis was further supported as MIT rapidly induced the activity of executioner caspases 3/7. However, cytotoxicity of MIT was partially reduced by inclusion of the opioid receptor antagonist naloxone, a μ and δ opioid receptor antagonist, suggesting that cytotoxicity depends in part on opioid signalling, consistent with the known toxicity of other opiates. Based on consumption of 20 leaves per day of Mitragyna speciosa, we estimated daily human exposure to MIT to be about 17 mg MIT for regular consumers, potentially giving plasma concentrations in of 10−9 to 10−7 M. Importantly, fatalities after kratom consumption have been reported to occur in individuals with blood mitragynine concentrations of between 0.45–1.0
Kadir NH, David R, Rossiter JT, et al., 2015, The selective cytotoxicity of the alkenyl glucosinolate hydrolysis products and their presence in Brassica vegetables., Toxicology, Vol: 334, ISSN: 1879-3185
Cruciferous vegetable consumption correlates with reduced risk of cancer. This chemopreventative activity may involve glucosinolates and their hydrolysis products. Glucosinolate-derived isothiocyanates have been studied for their toxicity and chemopreventative properties, but other hydrolysis products (epithionitriles and nitriles) have not been thoroughly examined. We report that these hydrolysis products differ in their cytotoxicity to human cells, with toxicity most strongly associated with isothiocyanates rather than epithionitriles and nitriles. We explored mechanisms of this differential cytotoxicity by examining the role of oxidative metabolism, oxidative stress, mitochondrial permeability, reduced glutathione levels, cell cycle arrest and apoptosis. 2-Propenylisothiocyanate and 3-butenylisothiocyanate both inhibited cytochome P450 1A (CYP1A) enzyme activity in CYP expressing MCL-5 cells at high cytotoxic doses. Incubation of MCL-5 cells with non-cytotoxic doses of 2-propenylisothiocyanate for 24h resulted in a dose-dependent inhibition of ethoxyresorufin O-deethylase, yet failed to affect CYP1A1 mRNA expression indicating interference with enzyme activity rather than inhibition of transcription. Increased reactive oxygen species (ROS) production was observed only for 2-propenylisothiocyanate treatment. 2-Propenylisothiocyanate treatment lowered reduced glutathione levels whereas no changes were noted with 3,4-epithiobutylnitrile. Cell cycle analysis showed that 2-propenylisothiocyanate induced a G2/M block whereas other hydrolysis products showed only marginal effects. We found that 2-propenylisothiocyanate and 3-butenylisothiocyanate induced cell death predominantly via necrosis whereas, 3,4-epithiobutylnitrile promoted both necrosis and apoptosis. Thus the activity of glucosinolate hydrolysis products includes cytotoxicity that is compound-class specific and may contribute to their putative chemoprotection properties.
Sathyapalan T, Thatcher NJ, Hammersley R, et al., 2015, Aspartame Sensitivity? A Double Blind Randomised Crossover Study (vol 10, e0116212, 2015), PLoS One, Vol: 10, Pages: 1-1, ISSN: 1932-6203
[Correction]
Gooderham NJ, 2015, Toxicology research new talents themed issue, Toxicology Research, Vol: 4, Pages: 540-540, ISSN: 2045-452X
Patel SAA, Gooderham NJ, 2015, Interleukin-6 promotes dietary carcinogen-induced DNA damage in colorectal cancer cells, Toxicology Research, Vol: 4, Pages: 858-866, ISSN: 2045-452X
Colorectal cancer (CRC) is the third most common cancer worldwide with 80% of cases being sporadic, arising following a series of environment-induced gene mutations. DNA damaging pro-carcinogens such as benzo[a]pyrene (BaP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) contained in red or processed meats are a potential risk factor for disease. These dietary pro-carcinogens require metabolic activation to their genotoxic agents by cytochrome P450 (CYP) family 1 enzymes. We have previously demonstrated that the pro-inflammatory cytokine interleukin-6 (IL6) promotes CYP1B1 expression in CRC cells grown as 2D monolayers and that these two proteins are overexpressed in malignant tissue resected from CRC patients, indicating that inflammation influences metabolic competency in CRC cells. To determine whether IL6 can influence BaP and PhIP activation, we investigated IL6 effect on BaP- and PhIP-induced DNA damage in CRC cell lines grown as 2D monolayers and as 3D spheroids using the in vitro micronucleus (MN) assay. We also investigated the involvement of p53 and CYPs in the observed effects. MN formation was increased dose-dependently following treatment with BaP and PhIP while pre-treatment with IL6 further enhanced DNA damage. We confirmed that IL6-mediated effects were not caused by p53 expression changes but rather by CYP1B1 expression induction through miR27b downregulation. Taken together, these data demonstrate that inflammatory cytokines can promote dietary pro-carcinogen activation and DNA damage in CRC cells.
Sathyapalan T, Thatcher NJ, Hammersley R, et al., 2015, Aspartame Sensitivity? A Double Blind Randomised Crossover Study, PLOS One, Vol: 10, ISSN: 1932-6203
BackgroundAspartame is a commonly used intense artificial sweetener, being approximately 200 timessweeter than sucrose. There have been concerns over aspartame since approval in the1980s including a large anecdotal database reporting severe symptoms. The objective ofthis study was to compare the acute symptom effects of aspartame to a control preparation.MethodsThis was a double-blind randomized cross over study conducted in a clinical research unitin United Kingdom. Forty-eight individual who has self reported sensitivity to aspartamewere compared to 48 age and gender matched aspartame non-sensitive individuals. Theywere given aspartame (100mg)-containing or control snack bars randomly at least 7 daysapart. The main outcome measures were acute effects of aspartame measured using repeatedratings of 14 symptoms, biochemistry and metabonomics.ResultsAspartame sensitive and non-sensitive participants differed psychologically at baseline inhandling feelings and perceived stress. Sensitive participants had higher triglycerides (2.05± 1.44 vs. 1.26 ± 0.84mmol/L; p value 0.008) and lower HDL-C (1.16 ± 0.34 vs. 1.35 ± 0.54mmol/L; p value 0.04), reflected in 1H NMR serum analysis that showed differences in thebaseline lipid content between the two groups. Urine metabonomic studies showed no significantdifferences. None of the rated symptoms differed between aspartame and controlbars, or between sensitive and control participants. However, aspartame sensitive participantsrated more symptoms particularly in the first test session, whether this was placebo or control. Aspartame and control bars affected GLP-1, GIP, tyrosine and phenylalanine levelsequally in both aspartame sensitive and non-sensitive subjects.ConclusionUsing a comprehensive battery of psychological tests, biochemistry and state of the artmetabonomics there was no evidence of any acute adverse responses to aspartame. Thisindependent study gives reassurance to both regulatory bodies an
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