Effect of Human Glutathione S-Transferases on Glutathione-Dependent Inactivation of Cytochrome P450-Dependent Reactive Intermediates of Diclofenac

Abstract

Idiosyncratic adverse drug reactions due to the anti-inflammatory drug diclofenac have been proposed to be caused by the generation of reactive acyl glucuronides and oxidative metabolites. For the oxidative metabolism of diclofenac by cytochromes P450 at least five different reactive intermediates have been proposed previously based on structural identification of their corresponding GSH-conjugates. In the present study, the ability of four human glutathione S-transferases (hGSTs) to catalyze the GSH-conjugation of the different reactive intermediates formed by P450s was investigated. Addition of pooled human liver cytosol and recombinant hGSTA1-1, hGSTM1-1, and hGSTP1-1 to incubations of diclofenac with human liver microsomes or purified CYP102A1M11 L437N as a model system significantly increased total GSH-conjugation. The strongest increase of total GSH-conjugation was observed by adding hGSTP1-1, whereas hGSTM1-1 and hGSTA1-1 showed lower activity. Addition of hGSTT1-1 only showed a minor effect. When considering the effects of hGSTs on GSH-conjugation of the different quinoneimines of diclofenac, it was found that hGSTP1-1 showed the highest activity in GSH-conjugation of the quinoneimine derived from 5-hydroxydiclofenac (5-OH-DF). hGSTM1-1 showed the highest activity in inactivation of the quinoneimine derived from 4'-hydroxydiclofenac (4'-OH-DF). Separate incubations with 5-OH-DF and 4'-OH-DF as substrates confirmed these results. hGSTs also catalyzed GSH-conjugation of the o-iminemethide formed by oxidative decarboxylation of diclofenac as well as the substitution of one of the chlorine atoms of DF by GSH. hGSTP1-1 showed the highest activity for the formation of these minor GSH-conjugates. These results suggest that hGSTs may play an important role in the inactivation of DF quinoneimines and its minor reactive intermediates especially in stress conditions when tissue levels of GSH are decreased.

Differential sensitivity of metabolically competent and non-competent HepaRG cells to apoptosis induced by diclofenac combined or not with TNF-α

“…DCF quinone imine metabolites have been shown to be reduced by NAD(P)H:quinone oxido-reductase1 and conjugated to GSH by GSTs (Dragovic et al, 2013;Vredenburg et al, 2014). Our data confirming that differentiated HepaRG cells contained higher levels of GST A1/2 and M1/2 than their undifferentiated counterparts and HepG2 cells and showing their higher sensitivity to DCF following GST inhibition by ethacrynic acid, brought further support to their lower sensitivity to apoptotic effects of DCF.…”

Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes

“…In recent studies with diclofenac and clozapine, GSTs have been shown to increase GSH-conjugation rates, and also completely GST-dependent conjugation reactions have been observed with cytosolic human GSTs [30,31]. Here, the formation of GSH conjugates was very similar in incubations with liver microsomes and S9 fractions, suggesting that cytosolic GSTs did not have a clear role in formation in the reactions.…”

“…Here, the formation of GSH conjugates was very similar in incubations with liver microsomes and S9 fractions, suggesting that cytosolic GSTs did not have a clear role in formation in the reactions. This may be due to involvement of microsomal GSTs, or due to a high added GSH-concentration (4 mM), which has been earlier shown to decrease the GSTdependency of conjugate formation [30,31]. It was anticipated that the GSH conjugation reactions would have demonstrated a high rate with hepatocyte experiments, yet possibly with different conjugates than those observed with liver microsomes.…”

Idiosyncratic Drug-Induced Liver Injury (IDILI): Potential Mechanisms and Predictive Assays

“…In the case of GST, levels and activity can be quantified via the removal of chlorine from substrate 1-chloro-2,4-dinitrobenzene (CDNB) [138]. While CDNB can be used as a substrate for total GST activity, p -nitrobenzyl chloride (NBC), 1,2-chloro-4-nitrobenzene (DCNB), and trans-4-phenyl-3-buten-2-one (PBO) can be used to detect GSTM activity, and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and dichloromethane (DCM) can be used as substrates for GSTTs [139, 140]. …”

“…Surendradoss et al used HPLC and MS to quantify valproyl 1-O-acyl-glucuronide (VPA-G), the phase II metabolized drug of valproic acid [142, 143]. Additionally, diclofenac conjugated to GSH has been measured via HPLC to assay for GST activity [140]. The drawback to using these technologies is that metabolites can only be quantified in relative terms without giving exact values as to the forms of drug present in a solution.…”