Circulating liver-specific microRNAs in cynomolgus monkeys

Abstract

Circulating microRNAs (miRNAs) can potentially be used as sensitive and specific biomarkers for tissue injury. However, the usefulness of circulating miRNAs as safety biomarkers in nonclinical toxicological studies using nonhuman primates is debatable owing to the limited information on organ-specific miRNAs. Therefore, a systematic investigation was performed to address this point. We identified organ-specific miRNAs from cynomolgus monkeys by next-generation sequencing analysis, which revealed that miR-122 was only abundant in the liver, whereas miR-192 was abundant in the liver, stomach, intestines, and kidney. The sequences of these miRNAs were identical to their human counterparts. Next, the absolute miR-122 and miR-192 levels were qualified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to determine the circulating levels of the miRNAs. No significant differences in the levels of circulating miRNAs between sexes were noted, and there was greater interindividual variation in miR-122 (20-fold variation) than in miR-192 (8-fold variation), based on their dynamic ranges. Finally, we evaluated the fluctuation in circulating liver-specific miRNAs in a monkey model of acetaminophen-induced hepatotoxicity. Acetaminophen with L-buthionine-(S,R)-sulfoximine induced hepatotoxicity in all the animals, which was characterized histopathologically by centrilobular necrosis and vacuolation of hepatocytes. Circulating miR-122 and miR-192 levels increased more than ALT levels after 24 h, indicating that circulating miR-122 and miR-192 may serve as sensitive biomarkers for the detection of hepatotoxicity in cynomolgus monkeys. This review describes the fundamental profiles of circulating liver-specific miRNAs in cynomolgus monkeys and focusses on their organ specificity, circulating levels, and fluctuations in drug-induced hepatotoxicity.

Keywords: cynomolgus monkeys; drug-induced liver injury; microRNA; next-generation sequencing.

Fig. 1.

Expression profiles of selected microRNAs (miRNAs) in cynomolgus monkeys identified by next-generation sequencing (NGS). The expression levels in each tissue or organ were normalized from the raw count data to reads per million (rpm). The x-axis expresses reads per million.

Fig. 2.

Circulating levels of liver-specific microRNAs (miRNAs) in male and female monkeys. The expression levels of the miRNAs are represented by dot plots. Differences were analyzed by the Mann–Whitney U-test. The ranges of the means ± 2 SD from the male or female data are represented by gray boxes.

Fig. 3.

Intraindividual variations in the expression of each circulating microRNA (miRNA). The plasma levels of the miRNAs on different sampling days (Day 1, Day 8, and Day 56) in the same animal are shown. The ranges of the means ± 2 SD calculated from 50 cynomolgus monkeys are represented by gray boxes.

Fig. 4.

Centrilobular necrosis in the liver of cynomolgus monkeys. A: Control animal. B: Acetaminophen (2,000 mg/kg) with L-buthionine-(S,R)-sulfoximine (BSO; 300 mg/kg; Animal No. 04F01). Marked necrosis and slight vacuolation of hepatocytes were seen in the centrilobular area (C) and periportal area (P), respectively. Hematoxylin and eosin (H&E) stain ×100.

Fig. 5.

Time course of plasma acetaminophen (APAP) and N-acetyl-p-benzoquinone imine (NAPQI) concentrations. The animals were given acetaminophen at 300, 1,000, or 2,000 mg/kg with L-buthionine-(S,R)-sulfoximine (BSO; 300 mg/kg). The data represent means + SDs.

Fig. 6.

Fluctuations in the levels of plasma ALT, miR-122, and miR-192. Acetaminophen (APAP; 2,000 mg/kg) was orally administered to animals 1 h after pretreatment with an intravenous injection of L-buthionine-(S,R)-sulfoximine (BSO; 300 mg/kg). Plasma ALT, miR-122, and miR-192 levels were measured at 0 (pre-dosing), 4, 7, 24, 48, 72, 96, 120, 144, and 168 h after APAP administration. Three animals were euthanized at 48 h for histopathological examination, and the remaining animals were used for time-course analysis. The dotted line indicates the mean of the pre-dose values in each parameter.