The aim of these studies was to identify circulating miRNAs that can be used as
biomarkers in patients with paracetamol-induced liver injury. Whether the miRNAs
discovered in humans could be back-translated to zebrafish with the aim of
developing a liver toxicity model to replace rodent use was also investigated.
First, the miRNA signature of DILI induced by paracetamol was defined. Plasma
miRNAs were quantified in paracetamol overdose patients. A signature of 16 miRNAs
was discovered that best separated patients with liver injury from those without liver
injury. This signature was tested in a second cohort and resulted in the detection of
paracetamol-induced liver injury with high specificity and sensitivity. At first
presentation to hospital miR-122-5p was the most sensitive single miRNA and
superior to ALT activity in predicting liver injury.
In order to further qualify miR-122-5p, three detailed studies relevant to possible
clinical scenarios were performed. The effect of acute alcohol ingestion (commonly
co-ingested with paracetamol overdose) on circulating concentrations of miR-122-5p
in healthy volunteers was investigated. Alcohol ingestion induced a small, non-clinically
relevant, increase in miR-122-5p. The effect of chronic kidney disease (CKD)
and haemodialysis (HD) on circulating miR-122-5p concentrations was explored
because kidney dysfunction has been associated with a reduction in the
concentration of circulating miRNAs. HD patients had lower concentrations of miR-
122-5p compared to healthy volunteers and CKD patients. To facilitate miRNA
measurement outwith hospitals, miR-122-5p was measured in a blood drop from a
finger prick. miR-122-5p was readily measurable in finger prick samples and
concentrations were significantly higher in the blood drop from DILI patients
compared with healthy volunteers.
To complement miR-122-5p as a marker of toxicity, circulating paracetamol
metabolites were measured in plasma samples from paracetamol overdose patients.
A higher percentage of circulating metabolites formed by cytochrome P450 enzymes
were present in patients with liver injury and these metabolites were superior to both
ALT and paracetamol concentration with regard to early patient stratification.
To reduce need for rodent studies, miRNAs were back-translated into zebrafish. In
order to study circulating miR-122-5p in adult zebrafish, a bloodletting method by
collecting blood retro-orbitally was developed. After studying different dosing
regimens of paracetamol in adult and larvae zebrafish the model was determined to
be too variable with regard to liver injury. A new drug, triptolide, originating from
traditional Chinese medicine and responsible for DILI in China, was tested as an
alternative model for drug-induced liver injury in zebrafish larvae. miRNA-122-5p
decreased in zebrafish larvae after triptolide treatment and triptolide-induced liver
injury could be tracked by fluorescent microscopy. Selective plane illumination
microscopy was able to track the decrease in liver volume during triptolide exposure.
In order to identify the toxic pathways involved in triptolide-induced liver injury, RNA-sequencing
was performed. This identified KEGG pathways including ribosome,
spliceosome and notch signalling as pathways affected by triptolide.
In summary, miRNAs can be used as highly sensitive biomarkers to detect acute liver
injury in patients and zebrafish. Zebrafish may represent an alternative model species
to study DILI, further work is needed