Oxidation of Diclofenac to Reactive Intermediates by Neutrophils, Myeloperoxidase, and Hypochlorous Acid

Abstract

Diclofenac is associated with a low, but significant, incidence of hepatotoxicity and bone marrow toxicity. It has been suggested that this could be due to a reactive acyl glucuronide. An alternative hypothesis is that an oxidative reactive metabolite could be responsible for such reactions and such metabolites formed by the enzymes present in neutrophils could be responsible for bone marrow toxicity. Others had reported the formation of 2,2'-dihydroxyazobenzene during the oxidation of diclofenac by myeloperoxidase/hydrogen peroxide. In contrast, in similar experiments we did not find evidence for the formation of 2,2'-dihydroxyazobenzene, but we did find several products, including a reactive iminoquinone. The same iminoquinone was formed by the oxidation of 5-hydroxydiclofenac. This iminoquinone was also formed by oxidation of diclofenac by HOCl or by activated neutrophils. It reacted with glutathione to form a conjugate. 5-Hydroxydiclofenac is also a major hepatic metabolite of diclofenac, and we found that rat hepatic microsomes oxidized 5-hydroxydiclofenac to the iminoquinone which was trapped with glutathione. This reactive metabolite represents another possible cause of the idiosyncratic reactions associated with the use of diclofenac.

Permanganate oxidation of diclofenac: The pH-dependent reaction kinetics and a ring-opening mechanism

“…6. This quinone-like intermediate product was first reported by Miyamoto et al (1997) during the oxidation of DCF with HOCl, myeloperoxidase (MPO), and activated neutrophils. The oxidation of DCF by biogenic manganese oxides was investigated, concluding the formation of 5-hydroxy-diclofenac and diclofenac-2,5-iminoquinone as main degradation products (Forrez et al, 2010).…”

“…We formulated these degradation products from P1 to P7 respectively, and proposed possible structures of products P1-P5 on the basis of: (1) the masses of molecular ion peaks and the major fragments of MS/MS spectra; (2) the most probable elemental composition of products; (3) the isotopic distribution characteristics of chlorine atoms and the existence of nitrogen atom in the structure; (4) the information on degradation products from former DCF oxidation processes, including chlorination, UV/H 2 O 2 , ozonization, manganese oxides oxidation, and photo-Fenton oxidation (Miyamoto et al, 1997;Vogna et al, 2004;Pérez-Estrada et al, 2005;Hofmann et al, 2007;Sein et al, 2008;Forrez et al, 2010;Madhavan et al, 2010;Soufan et al, 2012).…”

Inflammatory Stress and Idiosyncratic Hepatotoxicity: Hints from Animal Models

“…It is interesting in this regard that the addition of noncytotoxic concentrations of peroxidase/H 2 O 2 to hepatocyte cultures markedly increased DCLF cytotoxicity (Tafazoli et al, 2005). In fact, several novel reactive metabolites of DCLF appeared when the drug was incubated with PMN-derived myeloperoxidase (MPO) (Zuurbier et al, 1990;Miyamoto et al, 1997). Thus, PMNs that accumulate in liver after LPS exposure might render DCLF more cytotoxic by forming MPO-derived metabolites.…”

In vitro gene expression analysis of hepatotoxic drugs in rat primary hepatocytes

“…It appeared reasonable that the number of affected genes was very limited because DF is an idiosyncratic hepatotoxicant. Adduct formation of protein with reactive metabolites (Miyamoto et al, 1997;Pumford et al, 1993) and hypersensitivity (Romano et al, 1994;Salama et al, 1991;Kretz-Rommel and Boelsterli, 1993) have been proposed as mechanisms for DF-induced idiosyncratic hepatotoxicity which is not well understood. Although transcriptional effects of DF on hepatocytes are not well characterized, a previous study showed that DF induced Hmox1 mRNA in rat liver and in rat primary hepatocytes at intensive doses: 300 μM for 3 h in vitro (Cantoni et al, 2003).…”

“…DF, a nonsteroidal antiinflammatory drug (NSAID), is an idiosyncratic hepatotoxicant. DF-induced idiosyncratic hepatotoxicity is not well understood, but adduct formation of protein with reactive metabolites (Miyamoto et al, 1997;Pumford et al, 1993) and hypersensitivity (Romano et al, 1994;Salama et al, 1991;Kretz-Rommel and Boelsterli, 1993) have been proposed. Both DF and DSF have been reported to be idiosyncratic hepatotoxicants (Boelsterli, 2003;Forns et al, 1994).…”