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

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.

Keywords: (5R)-5-hydroxytriptolide; RNA polymerase II; androgen receptor; cytochrome P450; functional knockout; testes; γ-H2AX.

Figure 1

Hepatic Cpr knockout aggravated LLDT-8 induced testicular injury. (A) Body weights of WT and KO mice; (B) Testis relative weight (absolute testis weight vs. body weight); (C) Epididymis relative weight (absolute epididymis weight vs. body weight); H&E sections of the left testicle of WT (D) and KO (E) mouse (× 10, × 40); Star: reduction of germinal layers; Asterisk: vacuolar degeneration; Arrow: abnormally developed spermatids. Significant difference was determined by one way ANOVA, mean ±SD, n = 3, ^*p <0.05, ^***p <0.001 vs. Saline group.

Figure 2

TUNEL assay of LLDT-8 induced testis injury. The paraffin-embedded testis sections of WT (A) and KO (B) mice were labeled with TUNEL reaction mixture to distinguish the TUNEL positive cell (× 10, × 40). Arrow: TUNEL positive cell. (C) Quantification of TUNEL positive cells. A significant difference was determined by one way ANOVA, mean ± SD, n = 3, ^**p < 0.01, ^***p < 0.001 vs. Saline group, #p < 0.05 vs. WT mice at the same dosage.

Figure 3

Levels of LLDT-8 in the blood, liver, kidney, testis, and epididymis of KO and WT mice following a single oral dose of LLDT-8. Levels of LLDT-8 in the plasma from 0.5 mg/kg (A) and 1.0 mg/kg (B) group, and the LLDT-8 level in the liver, kidney testis, and epididymis (C) were determined by LC–MS/MS. ND, not detectable; mean ± SD, n = 5 for each time point, ^**p < 0.01, ^***p < 0.001 vs. WT 1.0 mg/kg, t-test. (D) LLDT-8 metabolites (M1 and M2, m/z 391.1) were detected in the microsomal samples from WT mice. Mass spectrometry of metabolite M1 (E) and M2 (F) were characterized by fargments m/z 391.1-373.1. The retention time and peak area of each metabolite were summarized in Table 3. Inhibitory effects of CYP inhibitors on the metabolism of LLDT-8 in rat liver microsomes (G). LLDT-8 (2 μM) was incubated with rat liver microsomes for 0.5 and 1 h, the residual LLDT-8 was analyzed by LC-MS/MS, mean ± SD, all incubations were carried out in three independent experiments in triplicate. ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. 0 h.

Figure 4

LLDT-8 dose-dependently decreased γ-H2AX in testis but not in liver. The localization of γ-H2AX in the testes of WT (A,E) and KO (B,F) mice were shown (A,B ×20, Scale bar: 100 μm; E,F ×63, Scale bar: 20 μm); The protein level of γ-H2AX in the testes (C), liver (D) of WT and KO mice. Yellow arrow heads indicated leptotene or zygotene spermatocytes marked by the nuclei distribution of γ-H2AX, and the yellow arrows pointed the XY bodies in the process of meiosis. The protein level was quantified with ImageQuant software (C,D). Beta-Actin was used as a loading control. Significant difference was determined by one way ANOVA, mean ± SD, n = 3, ^*p < 0.05, ^***p < 0.001 vs. Saline group (0 mg/kg).

Figure 5

LLDT-8 decreased RNA Pol II in vivo and in vitro. The protein levels of Pol II were detected by western blot in the testes (A), liver (B), spermatogonia-like GC-1spg cells (C), sertoli-like cells (D) and spermatocyte-like cells (E). The protein level was quantified with ImageQuant software. Beta-Actin was used as a loading control. Significant difference was determined by one way ANOVA, mean ± SD, n = 3, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 vs. control group.

Figure 6

LLDT-8 did not reduce the expression of AR-related genes in sertoli cells. AR (A), Rhox5 (B), Cldn11(C), Cst12 (D), ABP (E), and FABP (F) were determined by qPCR. Mean ± SD, n = 3, ^*p < 0.05, ^***p < 0.001 vs. control group (0 mg/kg, Saline group).