205 results found
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 Chem Toxicol, Vol: 113, Pages: 171-178
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.
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., Arch Toxicol
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 (10-7-10-4M) 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 (10-3-10-1M) with PhIP (10-7-10-4M) 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.
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., Toxicol Sci
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.
Akingbasote JA, Foster AJ, Jones HB, et al., 2017, Improved hepatic physiology in hepatic cytochrome P450 reductase null (HRN™) mice dosed orally with fenclozic acid, Toxicology Research, Vol: 6, Pages: 81-88, ISSN: 2045-452X
© The Royal Society of Chemistry. 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 [ 14 C]-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 were dosed orally with fenclozic acid for 7 days at 100 mg kg −1 day −1 . 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 −1 day −1 . 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., 2017, Safety evaluation of substituted thiophenes used as flavoring ingredients., Food Chem Toxicol, Vol: 99, Pages: 40-59
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.
Harling L, Lambert J, Ashrafian H, et al., 2017, 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: 1010-7940
Harling L, Lambert J, 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
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.
Saleh AF, Fellows MD, Ying L, et al., 2017, 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
Aigner A, Buesen R, Gant T, et al., 2016, Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop., Regul Toxicol Pharmacol, Vol: 82, Pages: 127-139
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.
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 Chem Toxicol, Vol: 98, Pages: 201-209
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.
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
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
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 JAB, 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: 0167-5273
Cohen SM, Flikushima S, Gooderham NJ, et al., 2015, GRAS 27 Flavoring Substances, FOOD TECHNOLOGY, Vol: 69, Pages: 41-59, ISSN: 0015-6639
Cohen SM, Fukushima S, Gooderham NJ, et al., 2015, GRAS 27 flavoring substances, Food Technology, Vol: 69, ISSN: 0015-6639
David R, Ebbels T, Gooderham N, 2015, 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.
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
© The Royal Society of Chemistry 2016. 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
Gooderham NJ, 2015, Toxicology research new talents themed issue, Toxicology Research, Vol: 4, ISSN: 2045-452X
Kadir NHA, 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, Pages: 59-71, ISSN: 0300-483X
Kamola PJ, Kitson JDA, 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: 0305-1048
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.
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.
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
Saleh AF, Priestley CC, Gooderham NJ, et al., 2015, Re-evaluation of the Mutagenic Response to Phosphorothioate Nucleotides in Human Lymphoblastoid TK6 Cells., Toxicological Sciences, Vol: 145, Pages: 169-176, ISSN: 1096-6080
The degradation of phosphorothioate oligonucleotides (PS-ONDs) and the release of potentially genotoxic modified mononucleotides raise a safety concern for OND-based therapeutics. Deoxyadenosine monophosphorothioate (dAMPαS), a PS nucleotide analog, has been reported to be a potent in vitro mutagen at the thymidine kinase (TK) locus in human TK6 lymphoblastoid cells. This led us to explore the mechanism behind the apparent positive response induced by dAMPαS in the TK gene-mutation assay in TK6 cells. In this work, treatment of TK6 cells with dAMPαS produced a dose-dependent increase in cytotoxicity and mutant frequency at the TK locus. Surprisingly, when the colonies from dAMPαS were re-challenged with the selective agent trifluorothymidine (TFT), the TFT-resistant phenotype was lost. Moreover, dAMPαS-induced colonies displayed distinct growth kinetics and required longer incubation time than 4-nitroquinoline-1-oxide-induced colonies to start growing. Treatment of TK6 cells with dAMPαS induced cell cycle arrest at the G1 phase, enabling cells to grow, and form a colony after the efficacy of TFT in the culture medium was lost. Our findings suggest that a fraction of parental "nonmutant" TK6 cells escaped the toxicity of TFT, possibly via G1 arrest, and resumed growth after the degradation of TFT. We conclude that dAMPαS did not induce real TFT-resistant mutants and caution should be taken with interpretation of mutation data from TK gene-mutation assay in TK6 cells when assessing modified nucleotides.
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., Sci Rep, Vol: 5
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.
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, ISSN: 1932-6203
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
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.
Wu Q, Li JV, Seyfried F, et al., 2015, Metabolic phenotype-microRNA data fusion analysis of the systemic consequences of Roux-en-Y gastric bypass surgery., International Journal of Obesity, Vol: 2015, Pages: 1126-1134, ISSN: 1476-5497
Background/Objectives: Bariatric surgery offers sustained dramatic weight loss and often remission of type 2 diabetes, yet the mechanisms of establishment of these health benefits are not clear.Subjects/MethodsWe mapped the co-ordinated systemic responses of gut hormones, the circulating miRNAome and the metabolome in a rat model of Roux-en-Y gastric bypass (RYGB) surgery. Results: The response of circulating miRNAs to RYGB was striking and selective. Analysis of 14 significantly altered circulating miRNAs within a pathway context was suggestive of modulation of signalling pathways including G protein signalling, neurodegeneration, inflammation, and growth and apoptosis responses. Concomitant alterations in the metabolome indicated increased glucose transport, accelerated glycolysis and inhibited gluconeogenesis in the liver. Of particular significance, we show significantly decreased circulating miRNA-122 levels and a more modest decline in hepatic levels, following surgery. In mechanistic studies, manipulation of miRNA-122 levels in a cell model induced changes in the activity of key enzymes involved in hepatic energy metabolism, glucose transport, glycolysis, TCA cycle, pentose phosphate shunt, fatty acid oxidation and gluconeogenesis, consistent with the findings of the in vivo surgery-mediated responses, indicating the powerful homeostatic activity of the miRNAs. Conclusions: The close association between energy metabolism, neuronal signalling and gut microbial metabolites derived from the circulating miRNA, plasma, urine and liver metabolite and gut hormone correlations further supports an enhanced gut-brain signaling, which we suggest is hormonally mediated by both traditional gut hormones and miRNAs. This transomic approach to map the crosstalk between the circulating miRNAome and metabolome offers opportunities to understand complex systems biology within a disease and interventional treatment setting.International Journal of Obesity accepted article previe
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