Enolate-Forming Phloretin Pharmacophores: Hepatoprotection in an Experimental Model of Drug-Induced Toxicity

Abstract

Drug-induced toxicity is often mediated by electrophilic metabolites, such as bioactivation of acetaminophen (APAP) to N-acetylp-benzoquinone imine (NAPQI). We have shown that APAP hepatotoxicity can be prevented by 2-acetylcyclopentanone (2-ACP). This 1,3-dicarbonyl compound ionizes to form an enolate nucleophile that scavenges NAPQI and other electrophilic intermediates. In this study, we expanded our investigation of enolate-forming compounds to include analyses of the phloretin pharmacophores, 29,49,69-trihydroxyacetophenone (THA) and phloroglucinol (PG). Studies in a mouse model of APAP overdose showed that THA provided hepatoprotection when given either by intraperitoneal injection or oral administration, whereas PG was hepatoprotective only when given intraperitoneally. Corroborative research characterized the molecular pharmacology (efficacy, potency) of 2-ACP, THA, and PG in APAP-exposed isolated mouse hepatocytes. For comparative purposes, N-acetylcysteine (NAC) cytoprotection was also evaluated. Measurements of multiple cell parameters (e.g., cell viability, mitochondrial membrane depolarization) indicated that THA and, to a lesser extent, PG provided concentration-dependent protection against APAP toxicity, which exceeded that of 2-ACP or NAC. The enolateforming compounds and NAC truncated ongoing APAP exposure and thereby returned intoxicated hepatocytes toward normal viability. The superior ability of THA to protect is related to multifaceted modes of action that include metal ion chelation, free radical trapping, and scavenging of NAPQI and other soft electrophiles involved in oxidative stress. The rank order of potency for the tested cytoprotectants was consistent with that determined in a parallel mouse model. These data suggest that THA or a derivative might be useful in treating drug-induced toxicities and other conditions that involve electrophile-mediated pathogenesis.

Phloretin cytoprotection and toxicity

“…Kaplan-Meier survival curves were used to illustrate the cumulative percent daily lethality of mice in different experimental groups and were generated in Prism 6.0 (Graphpad software). In previous studies, we (e.g., see Geohagen et al, 2016; Zhang et al, 2013) fully characterized the molecular pathogenesis of APAP-induced hepatotoxicity and enol-based hepatoprotection using temporal measurements of histopathology, liver-specific enzymes, GSH/GSSG ratio, mitochondrial function and unsaturated aldehyde production.…”

“…Hepatocytes were prepared from anesthetized mice (isoflurane inhalation) using the collagenase perfusion method of Geohagen et al (2016). Briefly, to separate dead hepatocytes, isolated cells were centrifuged (140g × 8 mins) in a Percoll gradient and then washed in media (140g × 3 mins) to remove Percoll.…”

“…In this regard, we have previously shown that Phl prevented electrophile (e.g., acrolein)-induced nerve terminal toxicity (LoPachin et al, 2011). We also found that the Phl pharmacophores, 2’,4’,6’-trihydroxyacetophenone (THA) and 1,3,5-trihydroxyacetophenone (phloroglucinol), could prevent hepatotoxicity as indicated by prevention of cell lethality and mitochondrial dysfunction in an isolated mouse hepatocyte model of acetaminophen (APAP) overdose (Geohagen et al, 2016). Results indicated that these derivatives were hepatoprotective and that the corresponding levels of protection were significantly greater than that provided by either 2-acetylcyclopentanone (2-ACP), a 1,3-dicarbonyl enol derivative of curcumin, or N-acetylcysteine (NAC; Mucomyst™), the thiol-based acetaminophen (APAP) antidote (Geohagen et al, 2016; Zhang et al, 2013).…”

“…In addition, Phl has several enolizable sites (e.g., C-3 enol on the A ring) that can ionize to form enolates with modest nucleophilicity (LoPachin et al, 2011). Therefore, Phl cytoprotection could also involve scavenging electrophiles such N -acetyl- p- benzoquinone imine (NAPQI) and unsaturated carbonyl species (e.g., acrolein) that mediate APAP hepatotoxicity (Geohagen et al, 2016; LoPachin et al, 2011; Zhu et al, 2009, 2012; reviewed in LoPachin et al, 2016; Zhu et al, 2011). These chemical properties suggest that Phl could treat pathogenic conditions and drug toxicities that involve electrophiles.…”

“…To evaluate this bimodal effect, corroborative studies were conducted in freshly isolated mouse hepatocytes. Results were compared to findings from previous pharmacological studies involving enol- and thiol-based cytoprotectants (Geohagen et al, 2016; LoPachin et al, 2011; Zhang et al, 2013). Research has provided evidence that electrophile-based GSH depletion was a significant index of cytotoxicity (reviewed in LoPachin et al, 2016).…”