Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways.

AimsLiver‐enriched microRNA‐122 (miR‐122) is a novel circulating biomarker for drug‐induced liver injury (DILI). To date, miR‐122 has been measured in serum or plasma venous samples. If miR‐122 could be measured in capillary blood obtained from a finger prick it would facilitate point‐of‐care testing, such as in resource‐limited settings that have a high burden of DILI.MethodsIn this study, in healthy subjects, miR‐122 was measured by polymerase chain reaction in three capillary blood drops taken from different fingers and in venous blood and plasma (n = 20). miR‐122 was also measured in capillary blood obtained from patients with DILI (n = 8).ResultsCirculating miR‐122 could be readily measured in a capillary blood drop in healthy volunteers with a median (interquartile range) cycle threshold (Ct) of 32.6 (31.1–34.2). The coefficient of variation for intraindividual variability across replicate blood drops was 49.9%. Capillary miR‐122 faithfully reflected the concentration in venous blood and plasma (Pearson R = 0.89, P < 0.0001; 0.88, P < 0.0001, respectively). miR‐122 was 86‐fold higher in DILI patients [median value 1.0 × 108 (interquartile range 1.89 × 107–3.04 × 109) copies/blood drop] compared to healthy subjects [1.85 × 106 (4.92 × 105–5.88 × 106) copies/blood drop]. Receiver operator characteristic analysis demonstrated that capillary miR‐122 sensitively and specifically reported DILI (area under the curve: 0.96, P = 0.0002).ConclusionThis work supports the potential use of miR‐122 as biomarker of human DILI when measured in a capillary blood drop. With development across DILI aetiologies, this could be used by novel point‐of‐care technologies to produce a minimally invasive, near‐patient, diagnostic test.

Aims microRNA‐122 (miR‐122) is a hepatotoxicity biomarker with utility in the management of paracetamol overdose and in drug development. Renal dysfunction and haemodialysis have been associated with a reduction in circulating microRNA. The objective of this study was to determine their effect on miR‐122. Methods Blood samples were collected from 17 patients with end‐stage renal disease (ESRD) on haemodialysis, 22 healthy controls, 30 patients with chronic kidney disease (CKD) and 15 patients post‐kidney transplantation. All had normal standard liver function tests. Samples from ESRD patients were collected immediately pre‐ and post‐haemodialysis. Serum alanine transaminase activity (ALT), miR‐122 and miR‐885 (liver enriched) were compared. Results Circulating miR‐122 was substantially reduced in ESRD patients pre‐haemodialysis compared with the other groups (19.0‐fold lower than healthy controls; 21.7‐fold lower than CKD). Haemodialysis increased miR‐122 from a median value of 6.7 × 103 (2.3 × 103–1.4 × 104) to 1.6 × 104 (5.4 × 103–3.2 × 104) copies ml−1. The increase in miR‐122 did not correlate with dialysis adequacy. miR‐122 was reduced in the argonaute 2 bound fraction pre‐haemodialysis; this fraction was increased post‐dialysis. There was no change in miR‐122 associated with extracellular vesicles. miR‐885 was also reduced in ESRD patients (4‐fold compared to healthy subjects) and increased by haemodialysis. Conclusion miR‐122 is substantially lower in ESRD compared to healthy controls, patients with CKD and transplanted patients. Haemodialysis increases the concentration of miR‐122. These data need to be considered when interpreting liver injury using miR‐122 in patients with ESRD on dialysis, and specific reference ranges that define normal in this setting may need to be developed.