Biomarkers of Flutamide-Bioactivation and Oxidative Stress In Vitro and In Vivo

Abstract

The nonsteroidal androgen-receptor antagonist flutamide is associated with hepatic injury. Oxidative stress and reactive metabolite formation are considered contributing factors to liver toxicity. Here we have used flutamide as a model drug to study the generation of reactive drug metabolites that undergo redox cycling to induce oxidative stress (OS) in vitro and in vivo. Lipid peroxidation (LPO) markers, as well as genes regulated by the redox-sensitive Nrf2 pathway, have been identified as surrogates for the characterization of OS. These markers and metabolism biomarkers for drug bioactivation have been investigated to characterize drug-induced hepatic damage. Rat hepatocytes and in vivo studies showed that several LPO markers, namely the isoprostanes 15R-PD 2 , dihydro keto PE 2 , and iPF 2a -VI, as well as hydroxynonenal mercapturic acid metabolites, had increased significantly by 24 hours after flutamide treatment from 4.9 to 15.3-fold in hepatocytes and from 2.6 to 31.0-fold in rat plasma. Induction of mRNA expression levels for Nrf2-regulated genes was evident as well, with heme oxygenase 1, glutathione-S-transferase p1 and NAD(P)H dehydrogenase showing a 3.6-, 4.1-, and 1.9-fold increase in hepatocytes and 5.6-, 7.5-, and 94.1-fold in rat liver. All effects were observed at drug concentrations that did not show overt liver toxicity. Addition of an in situ hydrogen peroxide-generating system to in vitro experiments demonstrated the formation of a reactive di-imine intermediate as the responsible metabolic pathway for the generation of OS. The dataset suggests that hepatic oxidative stress conditions can be mediated via metabolic activation and can be monitored with suitable biomarkers preceding the terminal damage.

Exploring the protective effect of silymarin against flutamide-induced hepatotoxicity in male rats

“…Increased MDA level indicated destruction of lipid macromolecules included in the biological membranes which led to structural and functional alterations of hepatic cells, that ultimately caused hepatotoxicity (Xie et al, 2014). Present finding is consistent with published in vitro and in vivo studies which demonstrated increase in LPO products in hepatocytes and plasma of rats after treatment with FLU (Al Maruf and O'Brien, 2014;Teppner et al, 2016). Also, treatment of rat primary hepatocytes with FLU resulted in a high reactive oxygen species (ROS) production and complete GSH depletion (Leclerc et al, 2014).…”

“…Published data reported that FLU bioactivation using CYP1A2 generated a nitro anion free radical (Vazquez et al, 2004). These radicals can bind covalently to proteins and lipids, or remove hydrogen atoms from polyunsaturated fatty acids in lipids, leading to induction of LPO and subsequently hepatocyte injury (Williams and Burke, 2000;Teppner et al, 2016).…”

“…The drug also caused disturbance in the liver synthetic function as evidenced by decreased serum albumin and elevated lipid profile levels (Tavakkoli et al, 2011;Lateef et al, 2012). However, oxidative tissue damage was characterized by increased production of toxic product and decreased activity of antioxidant defence system was largely implicated in the mechanisms of FLU-induced hepatotoxicity (Leclerc et al, 2014;Teppner et al, 2016).…”

Bioengineered NRF2-siRNA Is Effective to Interfere with NRF2 Pathways and Improve Chemosensitivity of Human Cancer Cells

“…Our finding on the reduction of HO-1 and NQO1 levels in both 143B and MG63 cells demonstrated the consequent effects of knocking down NRF2 on NRF2-regulated gene expression by bioengineered NRF2-siRNA. Furthermore, a higher accumulation of intracellular ROS highlighted the impact of interfering NRF2/HO-1 axis on redox homeostasis (DeNicola et al, 2011;Teppner et al, 2016). In addition, the change of cellular ROS levels following the perturbation of NRF2/HO-1 axis should provide at least partial explanation for the antiproliferative activity of bioengineered NRF2-siRNA against OS cells, as well as the improved cell sensitivity to doxorubicin, cisplatin, and sorafenib.…”

“…The HO-1 enzyme is one of the main targets of NRF2 and a major effector of NRF2-dependent cell response to oxidative stress and xenobiotics (Teppner et al, 2016). A concurrent upregulation of NRF2 and HO-1 is obvious in many types of tumors and correlates well with tumor progression, resistance to therapy, and clinical outcomes (Furfaro et al, 2016).…”