Phloretin cytoprotection and toxicity

Abstract

Phloretin (Phl) is a dihydrochalcone flavonoid with significant cytoprotective properties; e.g., free radical trapping, electrophile scavenging. Based on this, it has been suggested that Phl might be useful in the treatment of pathogenic processes and prevention of drug toxicities. Therefore, we determined the ability of Phl to provide route- and dose-dependent hepatoprotection in a mouse model of acetaminophen (APAP) overdose. Intraperitoneal (i.p.) administration of Phl produced a bimodal effect; i.e., the highest dose (2.40 mmol/kg) did not prevent APAP-induced lethality, whereas lower doses (0.2-0.4 mmol/kg) afforded modest hepatoprotection. When given alone, the highest i.p. Phl dose was lethal within 24 h, whereas the lower doses were not toxic. Oral Phl (0.40-2.40 mmol/kg) did not prevent APAP-induced hepatotoxicity. The highest oral dose given alone (2.4 mmol/kg) produced 64% lethality, whereas lower doses were not lethal. This toxicity profile was reflected in a study using APAP-exposed isolated mouse hepatocytes, which showed that the Phl pharmacophores, 1,3,5-trihydroxyacetophenone (PG) and 2',4',6'-trihydroxyacetophenone (THA) where protective. Corroborative cell free studies showed that polyphenol protectants prevented glutathione loss mediated by the APAP metabolite, N-acetyl-p-benzoquinone imine (NAPQI). Thus, in spite of possessing cytoprotective attributes, Phl was generally toxic in our APAP models. These and earlier findings suggest that Phl is not a candidate for drug design. In contrast, we have found that the enol-forming pharmacophores, THA and PG, are potential platforms for pharmacotherapeutic development.

Keywords: Acetaminophen overdose; Drug-induced toxicity; Enol-based cytoprotectants; Hepatoprotection; Phytopolyphenol.

Figure 1.

(A) Dose-dependent hepatoprotective effects of Phl (0.20 – 2.40 mmol/kg) administered by intraperitoneal injection 20 minutes prior to oral APAP (500 mg/kg) overdose. (B) Dose-dependent toxicity (lethality) of Phl (0.20 – 2.40 mmol/kg) administered alone by intraperitoneal injection. Kaplan-Meier survival curves illustrate the cumulative percent daily lethality in the APAP alone and APAP/Phl groups (n= 15 mice/group). Joining lines indicate statistically significant differences in treatment groups at *p<0.05, *p<0.01 and *** p<0.001 levels of significance.

Figure 2

(A) Dose-dependent hepatoprotective effects of oral Phl (0.40 – 2.40 mmol/kg) administered 20 minutes prior to oral APAP (500 mg/kg) overdose. (B) Dosedependent toxicity (lethality) of oral Phl (0.40 – 2.40 mmol/kg). Kaplan-Meier survival curves illustrate the cumulative percent daily lethality in the APAP alone, APAP/Phl groups (n= 15 mice/group). Joining line indicates statistically significant differences in treatment groups at *** p<0.001 level of significance.

Figure 3

(A) Effects of graded Phl concentrations (0.1 – 1.0 mM) on the viability of APAP (1.0 mM × 4 hrs)-exposed freshly isolated mouse hepatocytes. Data are expressed as mean percent of control ± SEM cell viability (n = 6–8). Exposure of hepatocytes to APAP alone (1.0 mM × 4 hrs) caused a decrease in mean cell viability of 48 ± 8% (see ordinate of Fig. 3A). Exposure of hepatocytes to phloretin concentrations ≤ 0.75 mM did not alter viability, whereas the 1.0 mM concentration caused a 59 ± 11% loss of viability (data not shown). (B) Concentration-dependent effects of phloretin (0.25 and 1.0 mM) on alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) measured in incubation medium. Data are expressed as mean IU/mg protein ± SEM cell viability (n = 4–6). Statistically significant differences in treatment groups at *p<0.05 and **p<0.01 levels of significance.

Figure 4

Effects of 0.25 mM protectant concentrations on APAP (1.0 mM × 4 hrs)induced changes in medium (A) alanine aminotransferase (ALT) and (B) lactate dehydrogenase (LDH) activities. Data are expressed as mean IU/mg protein ± SEM cell viability (n = 3–6). Statistically significant differences in treatment groups at *p<0.05 and **p<0.01 levels of significance. Abbreviations: NAC – N-acetylcysteine; 2-ACP – 2- acetylcyclopentanone; THA - 2’,4’,6’-trihydroxyacetophenone; PHL – phloretin; PG - 1,3,5-trihydroxyacetophenone.

Figure 5

Effects of 1.0 mM protectant concentrations on APAP (1.0 mM × 4 hrs)induced changes in medium (A) alanine aminotransferase (ALT) and (B) lactate dehydrogenase (LDH) activities. Data are expressed as mean IU/mg protein ± SEM cell viability (n = 3–6). Statistically significant differences in treatment groups at *p<0.05 and **p<0.01 levels of significance. Abbreviations: NAC – N-acetylcysteine; 2-ACP – 2- acetylcyclopentanone; THA - 2’,4’,6’-trihydroxyacetophenone; Phl – phloretin; PG - 1,3,5-trihydroxyacetophenone.

Figure 6

Cell free in chemico evaluation of the relative abilities of selected cytoprotectants to inhibit NAPQI-induced loss of sulfhydryl groups on GSH. Graded concentrations of NAPQI were incubated with putative hepatoprotectants. Data are expressed as mean percent control ± SEM (n = 3 to 4 separate experiments) and corresponding IC₅₀ values are provided in parentheses. Abbreviations: NAC – N-acetylcysteine; 2-ACP – 2-acetylcyclopentanone; PG - 1,3,5-trihydroxyacetophenone; THA - 2’,4’,6’-trihydroxyacetophenone; Phl – phloretin and Curc - curcumin.