Our study reveals numerous miRNAs, notably 574-5p, 135a*, 466g, 1196, 466f-3p, and 877, are upregulated in the setting of lethal compared to sublethal APAP-associated hepatotoxicity, whereas miRNAs 342-3p, 195, 375, 29c, 148a and 652 are markedly downregulated. We demonstrate elevated ALT levels as well as histologic evidence supporting worsened hepatotoxicity in the setting of lethally dosed mice compared to non-lethally dosed mice (Figures and ). With 90% lethality in the lethally dosed mice in relation to no lethality in sublethally dosed mice (P = 0.0011), this supports the premise that unique plasma miRNA profiles may correlate with non- vs life-threatening APAP dosing (Figure ). The fold-change of a variety of miRNAs in the setting of lethally dosed mice compared to sublethal doses is of interest. Of note, more than 40 were both up- and downregulated, with the greatest fold miRNA changes reported (Table ).
A literature search to investigate possible functions of the miRNAs both up- and downregulated was undertaken. Intriguingly, many of those most up- and downregulated in the lethally compared to sublethally dosed mice, namely 574-5p, 466g, 466f-3p, 375, 29c, and 148a, have also been implicated in the development of asthma[22,23
]. For instance, 574-5p may be involved with asthma pathogenesis, with decreased miRNA 574-5p in chronic compared to acute asthma in a mouse model sensitized with ovalbumin[23
]. In another study, a potential relationship between histocompatibility antigen-G, chronic asthma, and miRNA 29c was determined[22
]. This consequently suggests a pathophysiologic relationship between APAP toxicity and asthma[22,23
Interestingly, prior research has shown an association between APAP use and asthma although the exact association is still unclear[24,25
]. For instance, an adult case control study described a relationship between acetaminophen use and asthma[26
]. Additional literature revealed an increased risk of wheeze in children whose mothers used prenatal APAP[27
]. The etiology still remains unclear. However, one theory is that decreased glutathione (due to depletion secondary to APAP toxicity) provides the opportunity for unchecked reactive oxygen species to promote asthma development[24
]. Additional theories include increased prostaglandin E2 production secondary to elevated cyclooxygenase-2 activity in the presence of APAP promoting a T2 allergic response[25
]. A third cause could be direct lung damage from NAPQI, a byproduct of APAP metabolism[28
]. Clearly, more information is needed to further elucidate the relationship between APAP and risk of asthma.
The previous literature describing the function of the other miRNAs up- and downregulated in our study is more varied. For example, some literature reveals a miRNA 135a association with HIF 1-α[15
]. Interestingly, previous literature has demonstrated HIF 1-α induction prior to APAP toxicity in the setting of lethal APAP dosing, with toxicity prevented by the presence of cyclosporine A, a HIF 1-α inhibitor which prevents mitochondrial permeability transition and oxidative stress[29
]. Additional studies have also shown elevation of HIF 1-α in the setting of APAP toxicity, with increased HIF 1-α causing increased glucose transporter-1 expression[30
]. Of note, miRNAs 195 and 342-3p have been shown as involved with hepato cytopathology in the setting of tumor suppression in hepatocellular carcinoma models[21
] and hepatitis B virus hepatocarcinoma diagnosis[10
However, how these miRNAs in total affect hepatotoxicity in the setting of APAP poisoning still needs to be elucidated. Prior studies reveal upregulation of plasma miR-122 in the setting of APAP-associated liver toxicity, while our data suggest downregulation of plasma miR-122 at the 12 h time point. This discrepancy could be potentially explained by examination of upregulation of miR-122 at 1 h, 3 h and 24 h time points, not at a 12 h time point in previous literature. In addition, different APAP dosing levels were used in each study (300 mg/kg compared to 500 mg/kg), again demonstrating the dynamic nature of miRNA regulation across both time and clinical setting[11
]. Of note, we found upregulation of plasma miR-298 and miR-370, whereas other researchers found downregulation of these miRNAs in the setting of APAP-associated hepatotoxicity[31
]. Again, this may be due to evaluation at different time points (6 h compared to 12 h) and differing APAP dosing parameters (1000 mg/kg compared to 500 mg/kg)[31
]. Together, these results demonstrate the need for further identification of additional plasma microRNA profiles at various time points and dosing levels.
Our ultimate goal would be to eventually have a miRNA APAP nomogram to be used for human patient care, similar to the previous effective Rumack-Matthew nomogram[32,33
]. The problem with this current nomogram, however, is that it relies on knowing when a patient initially ingested APAP. This is often difficult, if patients are poor historians or have ingested mind-altering substances such as alcohol at the time of evaluation. In addition, patients may have been taking APAP chronically, not acutely, making use of the Rumack-Matthew nomogram pointless. The importance of our work is to establish a novel miRNA nomogram that would be used for patients who have taken APAP at an unknown time or with chronic ingestions. In turn, this would avoid investigation of patients who have taken it chronically, thus preventing unnecessary treatment and iatrogenic ingestions. Additional studies in this field, with additional time points and dosing levels, however, are clearly still necessary.
The specificity of this profile also requires further improvement. For instance, in a recent report miRNA-122 was shown to be increased in the setting of APAP toxicity, although this increase was under the detection cut-off of our study[11
]. Of note, miRNA-122 is also upregulated in hepatitis C settings which, by itself, is evidently not specific enough to uniquely identify APAP toxicity[14
]. However, with additional future studies and data analysis, this may possible. This approach may also be used as a model to develop profiles for additional disease processes, namely non-alcoholic fatty liver disease and hepatocellular carcinoma, since microRNA profiles are already being used as early biomarkers for numerous pathologic states[9,10
]. Together, this may improve the diagnostic accuracy of hepatopathology, namely early APAP-induced hepatotoxicity. In turn, this may allow clinicians to better and more rapidly distinguish which patients who have ingested APAP will actually mandate therapy. Subsequently, this may result in decreasing the number of patients who receive unnecessary, expensive empiric treatment.
In conclusion, lethal dosing of APAP in a murine model is consistent with hepatotoxicity and up- and downregulation of a unique pattern of circulating plasma miRNAs, which is different from the plasma miRNA profile associated with sublethal APAP dosing. These differences may be useful in the future to distinguish lethal and sublethal APAP toxicity in humans.