Introduction

The FDA estimates that acetaminophen (APAP) is the most widely used drug in the United States, with approximately 28 billion doses purchased per year1. As a consequence, APAP overdose is the leading cause of drug-induced liver injury in the United States2. Upon ingestion, APAP is absorbed in the intestine and travels to the liver via the portal circulation where it is rapidly glucuronidated or sulfonylated allowing for its harmless excretion3. If the liver is exposed to an excess of APAP, however, these detoxification processes are overwhelmed, and APAP is instead metabolized by the cytochrome P450 enzyme CYP2E1 into the highly reactive and toxic metabolite NAPQI4. NAPQI rapidly forms adducts with critical cellular proteins, impairs mitochondrial membrane integrity, and results in a profound production of reactive oxygen species (ROS) within hepatocytes5. If these ROS overwhelm the cellular antioxidant response, cell death and tissue necrosis occurs, resulting in severe hepatotoxicity that can lead to death6.

Despite the clinical importance of APAP overdose, treatment remains limited7. Current therapies include limiting absorption of APAP by administering activated charcoal8,9,10, replenishing glutathione stores by administering the glutathione precursor n-acetylcysteine (NAC)11,12, the JNK/CYP2E1 inhibitor fomepizole (13), and supportive care. A complicating factor in clinical trials has been the significant inter-individual variability in susceptibility to APAP toxicity14. It has been proposed that this variability is due to genetic differences (i.e. altered expression of Cyp2e1 or conjugation enzymes15,16) or environmental exposures (alcohol17, drugs14,18, or microbiome composition19). However, investigation of these factors in preclinical studies is limited due to the time and expense of generating transgenic mice and the variability of the currently available rodent models20.

Herein, we present a novel, highly reproducible, Drosophila-based system for studying acetaminophen toxicity. Our data demonstrate that acetaminophen accumulates in Drosophila, resulting in the generation of ROS in the fat body (an organ analogous to the mammalian liver21), a rapid depletion of systemic glutathione, and subsequent mortality. We utilize this system to investigate the effect of the microbiome and aging on APAP toxicity, two variables that have proven difficult to definitively investigate in human or murine systems. Our data demonstrate that the presence of the microbiome is protective in the context of APAP toxicity and that advanced age may play a significant role in susceptibility to drug induced liver injury. Finally, as both increasing age and germ-free conditions have been associated with a decline Drosophila antioxidant responses22,23, we defined the requirement of the antioxidant response system in our model and find, in agreement with vertebrate studies, that genetic and pharmacologic manipulations of antioxidant response pathways results in altered sensitivity to APAP toxicity24,25.

Results

Acetaminophen accumulates and results in dose-dependent mortality in WT Drosophila

To determine the effect of acetaminophen on adult Drosophila, we assessed the viability of 5-day old male (Fig. 1A) and female (Fig. 1B) wild-type flies exposed to APAP at a concentration of 100 mM, 50 mM, 25 mM, 12.5 mM, or 0 mM (vehicle control). APAP exposure resulted in a dose-dependent mortality in both male and female Drosophila, with female flies displaying increased resistance to APAP relative to males at all doses. Intriguingly, this effect required continuous exposure to APAP, as animals fed APAP overnight (to simulate a brief/singular dose) showed a mild reduction in survival, but the reduction in survival was not as significant as those who experienced continuous exposure (Supplemental Fig. 1).

Figure 1
figure 1

Acetaminophen toxicity and accumulation in adult Drosophila. Mortality observed due to APAP exposure in 5-day old w1118 (A) male and (B) female Drosophila. Log-Rank p = 0.0001, n = 60. CLARITY staining for DAPI and anti-APAP antibodies in Drosophila treated for 12 h with (C) vehicle and (D) APAP.

Full size image

To confirm that ingested acetaminophen accumulated in Drosophila tissues, we performed immunofluorescence on whole-mounted treated and untreated Drosophila adults using an anti-acetaminophen antibody, which recognizes native acetaminophen and acetaminophen-adducts in tissues 26,27. Vehicle-fed Drosophila show very little background APAP immunoreactivity at 12 h (Fig. 1C), whereas APAP fed adults show increased APAP immunoreactivity, particularly in the abdomen (Fig. 1D).

APAP administration increases ROS in the drosophila fat body and depletes systemic glutathione

In the mammalian liver, APAP is bio-converted into the toxic metabolite NAPQI by the Cytochrome P450 enzyme CYP2E14. NAPQI is highly reactive and rapidly conjugates with critical cellular enzymes, interfering with normal cellular function and destabilizing the mitochondrial membrane5. Due to its impact on mitochondrial function, a hallmark of APAP hepatotoxicity is the robust production of reactive oxygen species (ROS)26. Cleared Drosophila tissues were rinsed 3 × in PBS with gentle shaking for 12 h at RT, then immersed in blocking solution and processed as outlined above.

HydroCy3 Analysis

Early third-instar Drosophila larvae were fed for 4 h on either PBS (vehicle control) or 100 mM APAP along with HydroCy3 (ROSstar 550, Li-Cor). Fat bodies were dissected, whole-mounted on glass microscope slides and imaged using Nikon eclipse 80i microscope fitted with a R1 Retiga Q Imaging camera. Quantification of fluorescence intensity performed using FIJI software (NIH).

Glutathione measurements

Total glutathione levels and redox potential were determined using HPLC to determine glutathione metabolites following derivatization with dansyl chloride. Briefly, cohorts of 30–50 adult flies (approximately 50 mg of fresh tissue) were collected in Eppendorf tubes containing and transferred to 500 ul ice-cold 50 g/l perchloric acid solution containing 0.2 M boric acid and 10 uM gamma-Glu-Glu on ice. Flies were homogenized for 15 s using a Teflon micropestle and the homogenate centrifuged at 14,000 g for 2 min. Aliquots of 300 ul of the supernatant were transferred to fresh tubes for further analysis, while the remaining supernatant fluid was discarded and the protein pellet resuspended in 200 ul of 1 N NaOH and analyzed for protein quantification using the BioRad DC Assay with BSA as a standard. Samples were stored at − 80 °C until they were derivatized with 60 ul of 7.4 mg/ml sodium iodoacetic acid. The pH was adjusted to 8.8–9.2 with 1 M KOH saturated K3B4O7 and 300 ul of 20 mg/ml dansyl chloride, followed by incubation in the dark at room temperature 24 h. 500 ul of chloroform is then added to each of the samples to extract acetone and “free” dansyl chloride. Analysis by HPLC with fluorescence detection was performed as previously described43,44. Concentrations of thiols and disulfides were determined by integration relative to an internal standard45. Redox potential (Eh) was calculated from the cellular GSH and GSSG concentrations using the Nernst equation as described46. A less negative value indicates a more oxidized redox state.

Generation of germ-free flies

Adult flies were placed in vials with fresh food and left overnight (8–14 h). The vials were emptied and ~ 5 mL dH2O was added to each vial. A paintbrush was used to suspend the remaining eggs in the dH2O, then the dH2O and eggs were poured into 90um cell strainers. In an aseptic environment, the cell strainers were placed in 50% bleach for 10 min, then transferred into sterile dH2O for 1 min three times consecutively. Using a sterile scalpel and forceps, the bottoms of the cell strainers were removed and placed in new, autoclaved vials with germ-free fly food. To verify the absence of bacteria, flies from two different germ-free vials were crushed in ~ 200uL of sterile dH2O in an aseptic environment, then plated on blood agar. The blood agar plate was left overnight at 37 degrees Celsius. No bacteria were observed growing on the plate.

Antibiotic treatment

5-day old Drosophila were transferred to media containing 5% sucrose, 1% agar, 100 ug/mL ampicillin, and 50 ug/mL streptomycin (Sigma-Aldrich). 3 days later, Drosophila were transferred to fresh vials containing acetaminophen without antibiotics and mortality assessed.