Metabolism-dependent mutagenicity of a compound containing a piperazinyl indazole motif: Role of a novel p450-mediated metabolic reaction involving a putative oxaziridine intermediate.

Compound 1a (6-chloro-5-{3-[4-(1H-indazol-3-yl)-piperazin-1-yl]-propyl}-3,3-dimethyl-1,3-dihydro-indol-2-one) was mutagenic to Salmonella typhimurium TA98 in the presence of rat liver S9 subcellular fraction. The metabolism of 1a in rat liver S9 or microsomes demonstrated that it underwent a P450-mediated N-deindazolation (loss of indazole ring) as a predominant metabolic pathway. To investigate a possible link between metabolism and mutagenicity, a structural analogue 1b (6-chloro-5-{3-[4-(1H-indazol-3-yl)-piperidin-1-yl]-propyl}-3,3-dimethyl-1,3-dihydro-indol-2-one), the cleaved product 2a (6-chloro-3,3-dimethyl-5-(3-piperazin-1-yl-propyl)-1,3-dihydro-indol-2-one), and the core motif 3a (3-piperazinyl indazole) were evaluated in the Ames assay. It was found that 1b was not mutagenic to Salmonella typhimurium TA98 in the absence or presence of a metabolic activating system. In contrast to 1a, 1b did not undergo the metabolic cleavage (loss of indazole ring). Marginal mutagenicity of 2a to TA98 was observed with rat liver S9, whereas 3a was shown to be a promutagen. It was further demonstrated that 1a inactivated P450 3A, the principle enzyme catalyzing the N-deindazolation reaction, in an NADPH-, time-, and concentration-dependent manner. The kinetics of inactivation was characterized by a K(I) of 8.1 microM and k(inact) of 0.114 min(-1). The differences in mutagenicity between 1a and 1b suggest that a chemical bond extending from the 3-position of the indazole to a heteroatom (as part of another cyclic ring) is a prerequisite for the toxicity. The metabolic process leading to the elimination of the indazole from the rest of the molecule apparently plays a key role in causing mutagenicity. It is postulated that the N-deindazolation of 1a proceeds via an oxaziridine intermediate, the formation of which is indirectly inferred from the presence of benzoic acid in microsomal incubations. Benzoic acid is thought to be derived from the hydrolysis of 3-indazolone, an unstable product generated from the oxaziridine. Evidence suggests that the electrophilic oxaziridine intermediate may be responsible for the mutagenicity and inactivation of P450 3A.

Photoreceptor cell apoptosis induced by the 2-nitroimidazole radiosensitizer, CI-1010, is mediated by p53-linked activation of caspase-3.

The nitroimidazole radiosensitizer CI-1010 ((R)-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide) causes selective, irreversible, retinal photoreceptor apoptosis in vivo. The mouse 661 W photoreceptor cell line was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in 661 W cells, as determined by ultrastructural analysis, agarose electrophoresis and analysis of TUNEL-positive nuclei. CI-1010 caused a loss of viability in 661 W cells, as measured by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). A clear link was established between the onset of apoptosis and activity of caspase-3 and caspase-8, prior to poly[ADP-ribose]polymerase (PARP) cleavage. Pretreatment with caspase inhibitors, ZVAD.fmk or DEVD-CHO, prevented morphological changes in most CI-1010-treated cells. Evaluation of mitochondrial inner membrane potential (Deltapsi(m)) in live 661 W cells using the fluorescent dye, tetramethylrhodamine methyl ester revealed retention of (Deltapsi(m)) until after caspase activation. Absence of cytochrome c in the cytoplasm in treated cells further supports the hypothesis of a mitochondrial-independent mechanism of cell death. Significant increase in DNA crosslinks observed in 661 W cells correlates with induction and phosphorylation of p53 at multiple serine sites. Cell cycle analysis of 661 W cells reveals a G(2) arrest in response to CI-1010-induced DNA damage and neuronal cell death. Increased protein expression of Bax, Fas, and FasL, concomitant to the loss of Bcl-xL in treated 661 W cells may be modulated by p53. This study evaluates in vitro mechanisms of CI-1010-induced cell death in photoreceptors and provides evidence in support of a p53-linked activation of caspase-3 in response to DNA damage caused by CI-1010.

Apoptosis and nitrative stress associated with phosphodiesterase inhibitor-induced mesenteric vasculitis in rats.

Nitric oxide may play a role in phosphodiesterase (PDE) inhibitor-induced rat mesenteric vasculitis. The present study was conducted to identify cellular sources of iNOS, determine the distribution of nitrotyrosine (NT) residues as a footprint of peroxynitrite (ONOO-) production, and evaluate their association with vascular apoptosis. To dissociate primary events from secondary changes associated with the inflammatory response, rats were given the PDE IV inhibitor CI-1018 orally at 750 mg/kg alone or concurrently with dexamethasone (DEX) intraperitoneally at 1 mg/kg for 4-5 days. Neutrophil (PMN) involvement in apoptosis was investigated in CI-1018 treated rats dosed with rabbit anti-rat PMN serum (APS). iNOS expression, NT residues, and caspase-3 were detected by immuno-histochemistry. Apoptosis was evaluated by TUNEL assay. CI-1018 induced vascular lesions were associated with iNOS expression in endothelial cells and inflammatory infiltrates; NT was evident only in the latter. Caspase-3 and TUNEL-positive staining were prominent only in medial smooth muscle cells (SMC) from CI-1018-treated rats and only when associated with active inflammation. iNOS- and NT-positive inflammatory cells were present in close proximity to SMC with caspase-3 staining. Inflammatory infiltrates were absent in rats given DEX with minimal SMC necrosis and hemorrhage remained. DEX eliminated apoptosis and immunoreactivity associated with caspase-3, iNOS, and NT. APS depletion of PMNs decreased the incidence and severity of vasculitis but failed to abolish completely caspase-3 immunoreactivity. Expression patterns for caspase-3, iNOS, and NT demonstrated that nitrative stress is a prominent feature of PDE inhibitor-induced vasculitis, with a possible role in medial SMC apoptosis. Further, medial SMC apoptosis may not be a primary event, but instead may be secondary to the inflammatory response.

CI-1010 induced opening of the mitochondrial permeability transition pore precedes oxidative stress and apoptosis in SY5Y neuroblastoma cells.

The hetero-bifunctional nitroimidazole radiosensitizer CI-1010, R-alpha-[[(2-bromoethyl)-amino]methyl]-2-nitro-1H-imidazole-1-ethanol monohydrobromide, causes selective irreversible apoptotic loss of retinal photoreceptor cells in vivo. The human neuroblastoma cell line, SH-SY5Y, was used as a neuronotypic model of CI-1010-mediated retinal degeneration. Exposure to CI-1010 for 24 h induced apoptosis in neuroblastoma cells, as determined by histopathological and ultrastructural analysis and by TUNEL technique. CI-1010 causes a dose-dependent decrease in cell viability in SY5Y cells, as measured by the reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Superoxide dismutase reduced loss of cell viability following CI-1010 treatment suggesting an oxidative stress-mediated mechanism of toxicity. The effects of CI-1010 on mitochondrial membrane potential and intracellular levels of reactive oxygen species were assessed in live SY5Y cells by confocal microscopy using the fluorescent dyes, tetramethylrhodamine methyl ester and 5,6-carboxy-2',7'-dihydrodichlorofluorescein diacetate. CI-1010 caused a rapid depolarization of mitochondria in SY5Y cells followed by an increase in ROS. Both CI-1010-induced mitochondrial depolarization and subsequent increases in ROS were prevented by pretreatment with either the permeability transition pore inhibitor, cyclosporin A (CsA), and by the antioxidant, alpha-tocopherol. However, CsA and alpha-tocopherol were unable to prevent apoptosis in CI-1010-treated cells, suggesting the influence of additional mechanism(s) of CI-1010-induced toxicity. This study evaluates intracellular oxidative stress associated with pore opening prior to apoptosis and provides evidence in support of a mitochondrial mechanism of CI-1010-induced neuronal cell death.

Effect of dexamethasone on the metabonomics profile associated with phosphodiesterase inhibitor-induced vascular lesions in rats.

Metabonomics is the evaluation of the multiparametric metabolic response of biological systems to pathophysiological stimuli. High-resolution nuclear magnetic resonance (NMR) spectroscopy of biofluids coupled with pattern recognition-based chemometric analysis is an emerging approach to the study of metabonomics and may be used for the prediction of toxicity in vivo and for identification of surrogate markers of toxicity. Previously, we established that metabonomic analysis of urine samples has significant potential for identification of phosphodiesterase type 4 (PDE-4) inhibitor-induced vascular lesions in rats. It was not clear, however, whether the observed changes in metabonomics profile were related mechanistically to the pathogenesis of these vascular lesions or whether these changes were reflective primarily of the ensuing inflammatory response. In the present study, dexamethasone was used to suppress inflammation associated with vascular lesions induced in rats by the PDE-4 inhibitor CI-1018 and urine samples were evaluated for resultant changes in metabonomic profile. Female Wistar rats were given CI-1018 by gavage at 750 mg/kg with or without concurrent intraperitoneal administration of dexamethasone at 1 mg/kg for 4 days. Dexamethasone induced a characteristic lymphoid depletion and lymphocytolysis but no evidence of vascular lesions. Rats dosed with CI-1018 had mild vascular changes in liver and/or marked vascular lesions in mesentery characterized by medial necrosis, hemorrhage, and/or edema accompanied by perivascular mixed inflammatory cell infiltrates. Inflammatory infiltrates associated with these lesions were eliminated in rats given dexamethasone, yet minimal medial smooth muscle necrosis and degeneration still occurred, suggestive of etiologic changes rather than effects secondary to the inflammatory response. Principle component analysis of urine NMR spectra produced a clear pattern separation within 48 to 72 h between CI-1018-treated rats with vascular lesions and vehicle controls or rats given dexamethasone alone. There was no pattern separation, however, between rats given CI-1018 alone and rats given CI-1018 and dexamethasone concurrently, suggesting that CI-1018-induced urine spectral changes were associated with the vascular lesions, yet were independent of the inflammatory response. These findings provide new insight into the mechanism(s) of PDE inhibitor-induced vasculitis and support the potential use of metabonomics for developing reliable noninvasive methods for detecting vascular changes in rats.