Critical Role of Hepatic Cyp450s in the Testis-Specific Toxicity of (5R)-5-Hydroxytriptolide in C57BL/6 Mice.

Cunzhi Yu,Lihan Sun,Liying Mo,Xinming Qi,Chenggang Li,Hong Yan,Jin Ren,Chunyong Wu,Chunzhu Li,Mingxia Liu,Man Gao,Yu Li

doi:10.3389/fphar.2017.00832

Abstract

Low solubility, tissue accumulation, and toxicity are chief obstacles to developing triptolide derivatives, so a better understanding of the pharmacokinetics and toxicity of triptolide derivatives will help with these limitations. To address this, we studied pharmacokinetics and toxicity of (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide derivative immunosuppressant in a conditional knockout (KO) mouse model with liver-specific deletion of CYP450 reductase. Compared to wild type (WT) mice, after LLDT-8 treatment, KO mice suffered severe testicular toxicity (decreased testicular weight, spermatocytes apoptosis) unlike WT mice. Moreover, KO mice had greater LLDT-8 exposure as confirmed with elevated AUC and Cmax, increased drug half-life, and greater tissue distribution. γ-H2AX, a marker of meiosis process, its localization and protein level in testis showed a distinct meiosis block induced by LLDT-8. RNA polymerase II (Pol II), an essential factor for RNA storage and synapsis in spermatogenesis, decreased in testes of KO mice after LLDT-8 treatment. Germ-cell line based assays confirmed that LLDT-8 selectively inhibited Pol II in spermatocyte-like cells. Importantly, the analysis of androgen receptor (AR) related genes showed that LLDT-8 did not change AR-related signaling in testes. Thus, hepatic CYP450s were responsible for in vivo metabolism and clearance of LLDT-8 and aggravated testicular injury may be due to increased LLDT-8 exposure in testis and subsequent Pol II reduction.

Highlights

The testicular injury is the main adverse effect of LLDT-8 in rodents, and recently we reported that spermatocytes are the primary target for LLDT-8 in the testes
The epididymis weight was not changed by LLDT-8 treatment LLDT-8 treatment (Figure 1C)
TUNEL assay found the number of TUNEL positive foci in KO mice testis were much higher than that in wild type (WT) mice (Figures 2A,B) after LLDT8 weight after LLDT-8 treatment in WT mice, consistent with another experiments using C57BL/6 mice (Supplementary Figure S1A)

Summary

Introduction

Triptolide is a structurally unique diterpenoid from Tripterygium wilfordii Hook F, and has excellent efficiency against cancers, polycystic kidney disease and rheumatic disease (Leuenroth and Crews, 2008; Zheng et al, 2008; Leuenroth et al, 2010; Mujumdar et al, 2010; Pan, 2010; Liu, 2011; Liu et al, 2011, 2013a,b; Manzo et al, 2012; Kim et al, 2014; Lu et al, 2014; Sangwan et al, 2015; Fan et al, 2016). By inhibiting XPB via covalent binding, a DNA helicase and a component of the TFIIH transcription complex, triptolide induced transcription repression and Pol II degradation in cancer cells (Titov et al, 2011; Chen et al, 2015). Other mechanisms such as Hsp inhibition, JNK and NF-kB signal pathways may play a role (Villicana et al, 2013; Sangwan et al, 2015; Zhang et al, 2016). Clarifying the connection between toxicity and pharmacokinetics of triptolide and its derivatives may help us overcome some limitations of triptolide and allow the clinical use of triptolide and its derivatives (Zhou et al, 2012)

Methods

Results

Conclusion