Consistent with our hypothesis, we were able to identify changes in the transcriptome of peripheral blood cells in subjects treated with a single dose of APAP that did not produce liver injury as detected by currently available liver chemistries. Furthermore, these observations are consistent with whole blood transcriptome changes observed in rats and human exposed to overtly hepatotoxic doses of APAP.
Our observations indicate a distinct putative peripheral blood transcriptomic signature for a sub-toxic dose in humans. Specifically, we observed down-regulation of multiple nuclear DNA encoded and 4 mitochondrial DNA encoded genes for proteins located in mitochondria, particularly those associated with oxidative phosphorylation. Although this phenomenon was seen most clearly when using the power of pooling the 6 clinical replicates, we did see this response in individual subjects. Moreover, directed analysis of data from our rat and human overdose subjects revealed a similar effect on oxidative phosphorylation genes. In rats, we found a dose-dependent down regulation of oxidative phosphorylation genes at toxic doses of APAP at 12 and 24 hours, when liver injury had occurred. In addition, a subgroup of animals treated with toxic doses showed strong down-regulation at 6 hours, when there was no indication of liver injury. Of the 5 human overdose subjects, only two had their blood collected 48 hours after APAP ingestion and each showed clear evidence of down regulation of 6 oxidative phosphorylation genes. Two of the remaining subjects had down-regulation in a total of 3 of the genes that were also down-regulated in the 48 hour subjects. Clearly, more data is needed, but the limited amount at our disposal is consistent with our observations in the supratherapeutic subjects.
Because we measured thousands of mRNAs in only 6 treated subjects of differing ethnicity, false discovery is a concern. However, several lines of evidence support that the changes observed were real. First, the significance of the canonical pathway changes using stringent false discovery rate parameters was even stronger after making appropriate adjustments to the data for ethnicity. Second, these changes were not observed in any of our three placebo patients. Third, down regulation of oxidative phosphorylation genes was temporally associated with a rise in serum lactate when the pooled data from APAP treated and placebos was compared, as would be expected during functional impairment of oxidative phosphorylation. This is therefore an example of the power of “metabolomic anchoring” of transcriptomic data. Fourth, there was a positive correlation among the individual treated subjects between the extent of down regulation of genes associated with mitochondrial function and the production of APAP mercapturate and cysteine conjugates in the urine, an accepted quantitative measure of conversion of acetaminophen to its toxic metabolite, NAPQI. Finally, as discussed below, there are plausible biological mechanisms that could account for the observed changes. Also worthy of note is the absence of changes in complete blood counts in any of the patients during the course of the study. This is important since any such changes could contribute strongly to differential gene expression changes. In aggregate, these observations solidify our conclusion that a non-overtly toxic dose of APAP can produce down regulation of oxidative phosphorylation genes in peripheral blood.
It may be important that, though all complexes of the oxidative phosphorylation pathway were affected, genes of complex I of the oxidative phosphorylation pathway were most consistently down-regulated. Among the complexes of the oxidative phosphorylation chain, complex I dysfunction has been especially linked with lactic acidosis while complex III has been implicated as a sensor of hypoxia and activator of hypoxia inducible factors (10
). Impairment in complex 1 function may therefore account in part for the observed increase in serum lactate. We cannot rule out other tissues as the source of the increased serum lactate.
It is tempting to speculate that the down-regulation of oxidative phosphorylation genes we observed reflects APAP toxic effects on lymphocytes. Mitochondria are known to be a primary target for APAP toxicity in hepatocytes through production of NAPQI, which is chiefly produced in the liver by cytochrome P4502E1 (CYP2E1). NAPQI causes depletion of mitochondrial glutathione (GSH) and resulting oxidative stress (14
), and covalently binds to mitochondrial proteins (15
). Because lymphocytes contain detectable amounts of CYP2E1 mRNA and protein (16
), NAPQI could be produced within lymphocytes and target the lymphocyte mitochondria. Further support for possible mitochondrial toxicity in lymphocytes is our RT-PCR results that demonstrate down-regulation of two mitochondrial DNA encoded genes (MT-RNR1, MTRNR2) that are not involved in oxidative phosphorylation. However, it is also possible that APAP is metabolized to NAPQI in the liver and then released into the serum, resulting in damage to circulating peripheral blood leukocytes. On the other hand, mitochondrial toxicity alone is unlikely to explain our findings because some of the down-regulated mRNAs involved in oxidative phosphorylation are products of nuclear and not mitochondrial gene transcription. It may therefore be relevant that APAP has been shown to induce caspase-dependent apoptosis in cultured primary lymphocytes with no evidence of formation of NAPQI bound proteins (18
). However, in this study we did not detect significant changes in apoptotic pathways across all patients.
Another possible explanation for down-regulation of both mitochondrial and nuclear genes could involve an adaptive metabolic strategy by the leukocytes. Activation of granulocytes, monocytes, and T lymphocytes, as would be expected to occur during overt liver injury, results in a metabolic shift from reliance upon oxidative phosphorylation for energy production to aerobic glycolysis. (20
). Though our observation of down-regulated oxidative phosphorylation genes would be entirely consistent with this hypothesis, we did not see consistent up-regulation of genes involved in glycolysis.
It should be noted that a link between the transcriptome changes and APAP toxicity is suggested by the timing of the changes relative to dose administration. Down-regulation of oxidative phosphorylation pathway and sustained increase in serum lactate were both observed 48 hours post dosing. Though we cannot specifically attribute the increase in lactate to any particular organ or cell type, this timing is consistent with the onset of overt liver injury in clinical overdose cases where abnormal liver chemistries typically do not appear until 24 to 48 hours after ingestion (23
). These observations are consistent with the peripheral blood transcriptome changes being at least associated with some mild liver stress, but presumably they would represent an early, harmless transitory stage in the process.
As a final note, it is unclear whether the tightly controlled clinical environment and dietary intake incorporated in this study were important in detecting these changes. However, environmental factors such as diet and exercise have been shown to significantly influence peripheral blood gene expression (24
In summary, we have demonstrated down-regulation of mitochondrial genes, most prominently in the oxidative phosphorylation pathway, in peripheral blood cells after a single supratherapeutic but not overtly toxic APAP dose. The gene expression changes are supported by our metabolomic finding of a concurrent increase in serum lactate. The basis for these changes are unclear, but they are consistent with known mechanisms underlying APAP liver injury and support our earlier rat work suggesting that certain blood transcripts might provide earlier detection of potential DILI. Further studies will be needed to determine if there are blood transcriptome “signatures” that could be used to both diagnose DILI and potentially identify specific culprit drugs.