Dear, James W

McCrae, Jame C

Sharkey, Noel

Vliegenthart, A D Bastiaan

Webb, David J

Jame C. McCrae

Noel Sharkey

David J. Webb

A. D. Bastiaan Vliegenthart

James W. Dear

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Clinical Toxicology

Ethanol consumption produces a small increase in circulating miR-122 in healthy individuals

INTRODUCTION: MicroRNA 122 (miR-122) is a new circulating biomarker for liver injury, which increases earlier than conventional markers in patients with acetaminophen hepatotoxicity. However, as co-ingestion of ethanol is common with drug overdose, a confounding effect of acute ethanol consumption on serum miR-122 must be examined.METHODS: Blood was collected from healthy volunteers before and after recreational consumption of ethanol. Routine biochemistry and haematology measurements were performed, and serum miR-122 was measured by qPCR. The primary outcome was the difference in serum miR-122 with ethanol consumption.RESULTS: We recruited 18 participants (72% male). Their mean serum ethanol concentration was 113 mg/dl (95% confidence interval [CI] 91-135 mg/dl) after consuming ethanol. Serum miR-122 increased from a mean of 71.3 million (95% CI 29.3-113.2 million) to 139.1 million (95% CI 62.6-215.7 million) copies/ml (2.2-fold increase). There was no significant difference in serum alanine aminotransferase activity before and after ethanol consumption.CONCLUSION: miR-122 increased with moderate ethanol consumption, but the fold change was modest. As increases with acetaminophen toxicity are 100- to 10 000-fold, moderate ethanol intoxication is unlikely to confound the use of this biomarker of hepatotoxicity.</p

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Title: Ethanol consumption produces a small increase in circulating miR-122 in healthy individuals.Keywords: Biomarkers; Ethanol; Toxicity; microRNAThis paper has been written in UK English. Authors: McCrae, Jame C, Room 3.08, Ashworth Laboratories, King’s Buildings, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT, Scotland, UKSharkey, Noel, Chancellor’s Building, University of Edinburgh, 49 Little France Crescent, Edinburgh, EH16 4SB, UKWebb, David J, University/BHF Centre for Cardiovascular Science, Queen's Medical Research Institute, University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, Scotland, UKVliegenthart, AD Bastiaan, Pharmacology, Toxicology & Therapeutics, University/BHF Centre for Cardiovascular Science, University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, EdinburghDear, James W. Pharmacology, Toxicology & Therapeutics, University/BHF Centre for Cardiovascular Science, University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, EdinburghCorresponding Author: McCrae, JCEmail: jcmccrae@gmail.com Tel: +44 7824 99 58 37Abstract Introduction MicroRNA 122 (miR-122) is a new circulating biomarker for liver injury, which increases earlier than conventional markers in patients with acetaminophen hepatotoxicity. However, as co-ingestion of ethanol is common with drug overdose, a confounding effect of acute ethanol consumption on serum miR-122 must be examined. Methods Blood was collected from healthy volunteers before and after recreational consumption of ethanol. Routine biochemistry and haematology measurements were performed, and serum miR-122 by was measured by qPCR. The primary outcome was the difference in serum miR-122 with ethanol consumption. Results We recruited eighteen participants (72% male). Their mean serum ethanol concentration was 113 mg/dl (95% CI 91 – 135 mg/dl) after consuming ethanol. Serum miR-122 increased from a mean of 71.3 million (95% CI 29.3– 113.2 million) to 139.1 million (95% CI 62.6 – 215.7 million) copies/ml (2.2x-fold increase). There was no significant difference in serum alanine aminotransferase activity before and after ethanol consumption. Conclusion miR-122 increased with moderate ethanol consumption, but the fold change was modest. As increases with acetaminophen toxicity are 100-10,000 fold, moderate ethanol intoxication is unlikely to confound the use of this biomarker of hepatotoxicity. Word count: 181IntroductionMicroRNAs are short (~22 nucleotides) RNA species which do not code for proteins, but affect gene expression via binding to messenger RNA (mRNA) and blocking translation to protein[1,2]. Some are tissue-specific and stable in the circulation which has led to microRNAs being developed as organ injury biomarkers[3–5].miR-122 is abundant and liver specific, representing over 70% of all hepatocyte microRNA[2]. It has been implicated in liver pathologies such as viral hepatitis, hepatocellular carcinoma, drug-induced liver injury and alcoholic liver disease[2–4]. Recently, we reported that miR-122 is a biomarker of liver injury in paracetamol (acetaminophen – APAP) poisoning. In patients requiring acetylcysteine (NAC) treatment for APAP overdose, circulating miR-122 rose more quickly than alanine aminotransferase (ALT), and could accurately predict subsequent liver injury at first presentation to hospital[6]. If miR-122 is going to have clinical utility as a marker for acute liver injury in APAP poisoning, the effect of acute ethanol ingestion needs to be defined, as co-ingestion of ethanol is common in overdose patients[7]. This study is an examination of circulating miR-122 before and after ethanol ingestion in healthy volunteers. Methods We prospectively recruited healthy volunteers aged 18-65 years, with pre-existing plans to consume ethanol recreationally. Ethical approval was obtained from the South East Scotland research ethics committee and informed consent obtained from all participants. Exclusion criteria were: prior liver disease or hepatobiliary surgery; previous viral hepatitis; recent viral infection; recent strenuous exercise; consumption of cytochrome P450-inducing medication; past medical history of epilepsy, cancer, diabetes mellitus or alcoholism; psychiatric disease; pregnancy or the possibility of pregnancy; and direct connection to clinical staff involved in the study. We collected blood up to 48 hours before, and again 3-5 hours after, the participants had consumed ethanol. The amount of ethanol to be consumed was not specified in advance. In serum the following parameters were measured: full blood count, urea and electrolytes, bilirubin, ALT, alkaline phosphatase, gamma-glutamyl transferase, coagulation profile, ethanol and miR-122. Samples were refrigerated immediately, and prepared for analysis within 24 hours. miR-122 was measured by PCR as previously described[8]. Absolute quantification of miR-122 was achieved by generating a standard curve using synthetic target. All other analyses were performed by laboratory services at the Royal Infirmary of Edinburgh.  	The primary outcome was difference in serum miR-122 before and after ethanol consumption. We calculated the sample size as follows: alpha-level 0.05; beta-level 0.9; standard deviation 0.203 (calculated from the first 5 participants’ pre-consumption serum miR-122); we estimated that to determine a minimal difference in miR-122 delta-Ct of 0.165 (corresponding to a 2x fold increase in copy number/ml) we would require 18 participants. Statistical analysis was performed using Graphpad Prism (GraphPad Software, La Jolla California, USA).Results Eighteen healthy subjects were recruited to the study as per the power calculation, of whom 13 (72%) were male. Table 1 summarises the clinical parameters at baseline and after ethanol consumption. Notably, there was no significant difference between ALT measurements, which were all in the normal range. Several blood parameters were significantly different after alcohol consumption. There were small but statistically significant changes in the leucocyte count, creatinine, urea, potassium and bilirubin. At baseline no participants had a detectable serum ethanol. After consuming ethanol, their mean serum ethanol concentration was 113 mg/dl (range 20–175). There was a 2.2-fold increase of miR-122 from a mean value of 71.3 (95% CI 29.3– 113.2 million) to 139.1 million (95% CI 62.6 – 215.7 million) copies/ml. (see Figure 1; p=0.006). Discussion ALT is a serum marker used to detect hepatocellular injury that has been in common practice for several decades[9]. Despite this, there are several issues with its use: low-level insults may not affect ALT, and there is a considerable delay between the insult to the liver and a subsequent rise in serum. miR-122 is one of a panel of new biomarkers which appear to have increased sensitivity and specificity compared to  ALT with regard to detecting liver injury[5,6]. Co-ingestion of ethanol is common in overdose[7,10], and so its effects on new biomarkers must be determined. We have identified a small but statistically significant increase in miR-122 after consumption of recreational levels of ethanol by healthy young adults. The mean increase in serum ethanol in this study (113mg/dl) represents a moderate level of inebriation, but well above the drink-drive limit in many countries (e.g. 80mg/dl in USA, England, Wales; 50mg/dl in Scotland). In previous studies miR-122 has increased 100-10,000-fold in response to hepatocellular injury[5,8]. It is doubtful, therefore, in mixed overdoses of APAP and ethanol that elevations of miR-122 above around 2-fold could be due to ethanol ingestion alone. These data indicate that ethanol coingestion is unlikely to be a significant contributor to the massive increases in miR-122 seen after APAP overdose. This is essential information for the further development of miR-122 as a biomarker of APAP-mediated hepatocellular injury. Our study has a few limitations: firstly, though the data set is sufficient to address our primary outcome, it does not have sufficient power to determine any relationship between level of ethanol ingestion and miR-122 level. Furthermore, patients taking mixed ethanol/APAP overdoses can present with serum ethanol levels greater than the range covered by our study, and we cannot fully exclude the possibility that these might increase mir-122 more substantially. This will need further research in the clinical setting. The participant groups had some differences in their blood parameters before and after ethanol consumption. Whilst statistically significant, the authors feel that these changes would not be considered clinically important as they were all modest and within their respective normal ranges. Lastly, our participants were healthy individuals with no history of liver disease; it is unknown how applicable our findings are to patients with pre-existing hepatic illness, and further exploration in this patient group is required. Conclusion Acute ethanol ingestion leads to a significant rise in miR-122. This increase is small compared to the biomarker signal with APAP-mediated liver injury[6]. This study contributes to the ongoing clinical development of miR-122 as a liver injury biomarker for use in clinical toxicology.Word count: 984AcknowledgementsThe authors wish to thank the Myre Sim Fund and the Dowager Countess Eleanor Peel Trust for their support. Declarations: The authors report no declarations of interestTable/figure legends:Table 1. Clinical parameters before and after ethanol consumption. Statistically significantly different parameters are highlighted in bold. ALT, alanine aminotransferase; ALP, alkaline phosphatase; GGT, gamma glutamyl transpeptidase; INR, International Normalized Ratio; APTT, Activated Partial Thromboplastin Time; SD, standard deviation.Figure 1. Serum miR-122 before and after ethanol consumption. References:  1. 	McDaniel K, Herrera L, Zhou T, Francis H, Han Y, Levine P, et al. The functional role of microRNAs in alcoholic liver injury. J Cell Mol Med 2014;18:197–207. 2. 	Su T-H, Liu C-H, Liu C-J, Chen C-L, Ting T-T, Tseng T-C, et al. Serum microRNA-122 level correlates with virologic responses to pegylated interferon therapy in chronic hepatitis C. Proc Natl Acad Sci 2013;110:7844–9. 3. 	Zhang Y, Jia Y, Zheng R, Guo Y, Wang Y, Guo H, et al. Plasma microRNA-122 as a biomarker for viral-, alcohol-, and chemical-related hepatic diseases. Clin Chem 2010;56:1830–8. 4. 	Wang K, Zhang S, Marzolf B, Troisch P, Brightman A, Hu Z, et al. Circulating microRNAs, potential biomarkers for drug-induced liver injury. Proc Natl Acad Sci 2009;106:4402–7. 5. 	Vliegenthart ADB, Antoine DJ, Dear JW. Target biomarker profile for the clinical management of paracetamol overdose: Biomarkers for paracetamol-induced liver injury. Br J Clin Pharmacol 2015; ePub ahead of print; accessed 19 August 2015. 6. 	Antoine DJ, Dear JW, Lewis PS, Platt V, Coyle J, Masson M, et al. Mechanistic biomarkers provide early and sensitive detection of acetaminophen-induced acute liver injury at first presentation to hospital. Hepatology 2013;58:777–87. 7. 	Waring WS, Stephen AF, Malkowska AM, Robinson ODG. Acute Ethanol Coingestion Confers a Lower Risk of Hepatotoxicity after Deliberate Acetaminophen Overdose. Acad Emerg Med 2008;15:54–8. 8. 	Starkey Lewis PJ, Dear J, Platt V, Simpson KJ, Craig DGN, Antoine DJ, et al. Circulating microRNAs as potential markers of human drug-induced liver injury. Hepatol Baltim Md 2011;54:1767–76. 9. 	Senior JR. Alanine aminotransferase: a clinical and regulatory tool for detecting liver injury-past, present, and future. Clin Pharmacol Ther 2012;92:332–9. 10. 	Teo AIC, Cooper JG. The epidemiology and management of adult poisonings admitted to the short-stay ward of a large Scottish emergency department. Scott Med J 2013;58:149–53. 1

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Ethanol consumption produces a small increase in circulating miR-122 in healthy individuals

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