Circulating Cell‐Free DNAs as a Biomarker and Therapeutic Target for Acetaminophen‐Induced Liver Injury

Abstract Acetaminophen (APAP) overdose is a leading cause of drug‐induced liver injury and acute liver failure, while the detection, prognosis prediction, and therapy for APAP‐induced liver injury (AILI) remain improved. Here, it is determined that the temporal pattern of circulating cell‐free DNA (cfDNA) is strongly associated with damage and inflammation parameters in AILI. CfDNA is comparable to alanine aminotransferase (ALT) in predicting mortality and outperformed ALT when combined with ALT in AILI. The depletion of cfDNA or neutrophils alleviates liver damage, while the addition of cfDNA or adoptive transfer of neutrophils exacerbates the damage. The combination of DNase I and N‐acetylcysteine attenuates AILI significantly. This study establishes that cfDNA is a mechanistic biomarker to predict mortality in AILI mice. The combination of scavenging cfDNA and reducing oxidative damage provides a promising treatment for AILI.


Isolation of peripheral blood leukocytes
For the isolation of peripheral blood leukocytes, mice were anesthetized to obtain the whole blood and the erythrocytes were lysed to isolate leukocytes.

Isolation of non-parenchymal liver cells
For non-parenchymal liver cells (NPC) isolation, the portal vein was cannulated and the liver was perfused with 0.5 mM EGTA (5 mL/min, 37 ℃ preheating) to flush the blood. Then, the liver was perfused with 0.075 % collagenase type IV (Perfusate, 3 mL/min, 37 ℃ preheating, Sigma-Aldrich) for 4 min, after which it was excised, placed in 87.5 % DMEM (Gibco) + 12.5 % Perfusate and brushed into homogenates.
The homogenates were digested for 20 min in a shaking incubator Bluepard) at 37 ℃ at 120 rpm and filtered through 100-mesh nylon screen to obtain single-cell suspensions. After centrifugation (50 g, 5 min) twice, the supernatant was centrifuged for 10 min at 500 g, and then the precipitate was resuspended in 25 % isotonic Percoll and layered over 70% isotonic Percoll (Biosharp). After which the NPCs were separated from the hepatocytes by differential centrifugation (800 g for 15 min) at room temperature.

CpG-induced liver injury model and treatment
After 1 h of sensitization with D-GalN (800 mg/kg, i.p., Adamas), mice were administered with CpG ODN 1826 (0.75 mg/kg, i.p.) to induce acute liver injury. The depletion of neutrophils or macrophages were achieved by anti-mouse Ly6G/Ly6C antibody or Clodronate liposomes as previously described. DNase Ⅰ was administered (80 U/dose, i.v.) at 3 and 10 h after CpG-challenge. After 24 h the mice were sacrificed to detect the levels of cfDNA and ALT.

The isolation and purification of mouse bone marrow-derived neutrophils
Mice were euthanized then doused in 75% ethanol. The tibias and femurs were removed from both legs entirely and bone marrow was flushed from the bones. Then the bone marrow fluid was filtered through 100-mesh nylon screen to obtain singlecell suspensions. After centrifugation (230 g, 6 min), the precipitate was resuspended in DMEM. 2.5 mL 72% Percoll, 2 mL 62% Percoll and 2 mL 52% Percoll were layered in a 15-ml conical tube. Overlay the bone marrow cell suspension on the top and the neutrophils were separated by differential centrifugation (1545 g for 30 min) at room temperature. Count the neutrophils and determine their purity by flow cytometry.
For combination therapy of NAC and DNase Ⅰ, NAC (150 mg/kg, i.p.) was administered at 3 h and different doses of DNase Ⅰ (40, 80, 120 and 160 U/dose) was administered at 3 h and 10 h after APAP challenge. After 24 h, the mice were sacrificed and serum was collected to detect the levels of ALT and AST.

Liver homogenate and redox factors
Liver tissue was homogenized in normal saline to prepare 10% liver homogenate. The supernatant was collected after centrifugation (4 ℃, 3000 rpm, 10 min). Total protein was measured by BCA protein assay kit (Thermo Fisher). The liver homogenate was diluted to different concentrations for following detection. The activity GSH-Px, and the levels of MDA were determined with assay kits, respectively (Nanjing Jiancheng).

Histopathological assessment and immunohistochemistry
Pathological sections of liver were stained with hematoxylin and eosin (H&E).
TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was performed using One Step TUNEL Apoptosis Assay Kit (Beyotime) according to the manufacturer's instructions. The nucleus is labeled with DAPI. TUNEL staining was quantified by the percentage of positive cells.

In vivo biosafety profile
Mice were administrated with NAC and DNase Ⅰ as mentioned previously and the body weight was recorded once a week. After 28 days, the serum was collected to perform analyses. The heart, liver, spleen, lung and kidney were collected for histopathological examination.

Quantitative real-time PCR analysis
Total RNA was isolated from mouse liver samples with TRIzol reagent (Invitrogen) and reverse transcription of 1 μg RNA was performed using the PrimeScript TM RT reagent Kit with gDNA Eraser (Takara) following the manufacturer's protocol. The resulting cDNAs were amplified by PCR with TB Green ® Premix Ex Taq TM Ⅱ Kit (Takara) in a real-time PCR system (LightCycler ® 96 instrument, Roche). The amplified transcripts relative to the GAPDH endogenous control were quantified using the comparative 2 -∆∆Ct method.
The following primer sequences synthesized by Sangon Biotech were used: