Acetaminophen dosing of humans results in blood transcriptome and metabolome changes consistent with impaired oxidative phosphorylation

Abstract

The diagnosis and management of drug-induced liver injury (DILI) is hindered by the limited utility of traditional clinical chemistries. It has recently been shown that hepatotoxicants can produce compound-specific changes in the peripheral blood (PB) transcriptome in rodents, suggesting that the blood transcriptome might provide new biomarkers of DILI. To investigate in humans, we used DNA microarrays as well as serum metabolomic methods to characterize changes in the transcriptome and metabolome in serial PB samples obtained from six healthy adults treated with a 4-g bolus dose of acetaminophen (APAP) and from three receiving placebo. Treatment did not cause liver injury as assessed by traditional liver chemistries. However, 48 hours after exposure, treated subjects showed marked down-regulation of genes involved in oxidative phosphorylation/mitochondrial function that was not observed in the placebos (P < 1.66E-19). The magnitude of down-regulation was positively correlated with the percent of APAP converted to the reactive metabolite N-acetyl-p-benzoquinone-imide (NAPQI) (r= 0.739;P= 0.058). In addition, unbiased analysis of the serum metabolome revealed an increase in serum lactate from 24 to 72 hours postdosing in the treated subjects alone (P< 0.005). Similar PB transcriptome changes were observed in human overdose patients and rats receiving toxic doses.

Conclusion: The single 4-g APAP dose produced a transcriptome signature in PB cells characterized by down-regulation of oxidative phosphorylation genes accompanied by increased serum lactate. Similar gene expression changes were observed in rats and several patients after consuming hepatotoxic doses of APAP. The timing of the changes and the correlation with NAPQI production are consistent with mechanisms known to underlie APAP hepatoxicity. These studies support the further exploration of the blood transcriptome for biomarkers of DILI.

Figures

Figure 1. Serum ALT Levels in APAP Treated Subjects

Serum ALT levels of six subjects receiving APAP at the beginning of clinical day 4. Bloods were drawn for serum chemistries at approximately 6 a.m. each day. Red arrow indicates time of administration of the single 4 g APAP bolus following the blood draw on the 4th day. M= male; F=female.

Figure 2. Serum Acetaminophen Concentrations

Serum APAP concentrations (Log10 µg/ml) for treated subjects through 12 hours post dosing.

Figure 3. Hierarchical clusters of differentially expressed oxidative phosphorylation genes in blood samples

Hierarchical clusters of differentially expressed oxidative phosphorylation genes in blood samples. Log2-calibrated ratios were clustered using commercial software (Partek/St Louis, MS) using an agglomerative method and average linkage clustering. Shade of color in the heat map represents the extent of differential expression: red, up-regulation; blue, down-regulation. Areas of lightest grey indicate no statistical significance at the given condition of time and dose. (a) Clinical subjects – 48 hrs after single 4 gram APAP dose; (b) Human overdose subjects; (c) APAP treated rats. Horizontal experiment dendogram is labeled with dose (H = 2500 mg/kg; I = 1500 mg/kg; L = 150 mg/kg) and colored by time (red = 6 hrs; blue = 12 hrs; green = 24 hrs).

Figure 4. Regression Analysis: Toxic APAP conjugates versus the Ratio of Down Regulated Mitochondrial Function Genes

Regression analysis of a scatter plot of mercapturate/cysteine APAP conjugates (as a percent of total urinary excreted metabolites) and the ratio of genes in the mitochondrial function pathway down regulated in each treated subject. Each data point represents one of the six treated subjects.

Figure 5. Serum Metabolome Time Course Profiles

Targeted Metabolite profiles: Time course of 21 metabolites measured in placebo (a) and APAP treated (b) subjects’ serum. Lactate increases (APAP vs placebo) are seen consistently after 24 hours. All other metabolites showed no significant changes over time.