Abstract ID 25830

Poster Board 591

Background: Intravenous acetaminophen (APAP; paracetamol) is used commonly in the intensive care unit or post-surgery for its analgesic effect. Intravenous administration of APAP is well documented to cause severe transient hypotension. The mechanism underlying these hemodynamic changes is still not clear, but we showed previously that the APAP metabolite N-acetyl-p-benzoquinone-imine (NAPQI) can directly and indirectly active voltage-gated Kv7 channels in vascular smooth muscle cells promoting a vasodilation.

APAP metabolism to NAPQI requires cytochrome P450 enzymes, which are also present in the vascular wall. Furthermore, another pathway of NAPQI formation is activated myeloperoxidase (MPO). MPO is a peroxidase enzyme that is primarily used by neutrophils as part of the innate immune system response and the circulating level of MPO is elevated in critically ill patients. MPO could be the missing link of why critically ill patients experience the most severe hypotension and hemodynamic instability caused by intravenous paracetamol.

Method: Human Coronary Artery Endothelial Cells (HCAEC) and HEK293 cells were treated with APAP (0 – 50 mM) or APAP metabolites (0 – 50 μM) to see the effect on viability and thiol concentration levels.

The formation of APAP-adducts by cytochrome P450 and MPO was investigated by immunocytochemistry (ICC), enzyme activity assays based on thiol assays, Western Blot (WB) and Mass Spectrometry (MS).

Furthermore, individual cytochrome P450 expression and location was investigated by overexpression of CYP20A1, immunocytochemistry, Western Blot and Mass Spectrometry.

Results: APAP decreased viability and thiol levels in a concentration dependent manner similar to NAPQI, but not AM404 (another APAP metabolite) in HCAEC cells. The cytochrome P450 inhibitor Ketoconazole partially rescued the drop in viability and thiol levels. Using ICC, we visualized APAP-adducts, which formed when treating HCAEC cells with APAP. One of the last orphan cytochrome p450 enzymes, CYP20A1, is expressed in both HEK293 and HCAEC and are upregulated in response to treatment of APAP. By overexpressing CYP20A1 in HEK293B cells, we identify APAP as the first substrate of CYP20A1.

In addition to investigating how NAPQI is formed by CYP450 enzymes in endothelial cells, we examined the formation of NAPQI by MPO. Using thiol assays, western blot and mass spectrometry, we found that MPO can form high levels of NAPQI.

Summary: Our findings show that NAPQI formation from APAP can occur via two pathways: cytochrome P450 enzymes and MPO. Both of these are potential mechanisms underlying IV APAP-induced hypotension, with the MPO pathway likely driving NAPQI production in the critically ill. CYP20A1 is a potential candidate involved the metabolism of APAP in endothelial cells. By improving our understanding of the metabolism of IV APAP, we might be able to prevent the iatrogenic hypotension.

This work was support by a Department of Biomedical Sciences (University of Copenhagen) Collaboration grant.