Nitric oxide donor, V-PROLI/NO, provides protection against arsenical induced toxicity in rat liver cells: Requirement for Cyp1a1

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Abstract

Arsenic is a cancer chemotherapeutic but hepatotoxicity can be a limiting side effect. O2-vinyl 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) is a nitric oxide (NO) donor prodrug and metabolized by liver cytochromes P450 (CYP450) to release NO. The effects of V-PROLI/NO pretreatment on the toxicity of arsenic (as NaAsO2) were studied in a rat liver cell line (TRL 1215). The cells acted upon the prodrug to release NO, as assessed by nitrite levels, in a time-dependent fashion to maximal levels of 8-fold above basal levels. Pretreatment with V-PROLI/NO markedly reduced arsenic cytolethality which was directly related to the level of NO produced by V-PROLI/NO treatment. Cyp1a1 expression was directly related to the level of NO production and to reduced arsenic cytotoxicity. V-PROLI/NO pretreatment markedly reduced arsenic-induced apoptosis and suppressed phosphorylation of JNK1/2. V-PROLI/NO pretreatment facilitated additional increases in arsenic-induced metallothionein, a metal-binding protein important in arsenic tolerance. Thus, V-PROLI/NO protects against arsenic toxicity in rat liver cells, reducing cytolethality, apoptosis and dysregulation of MAPKs, through generation of NO formed after metabolism by liver cell enzymes, possibly including Cyp1a1. CYP450 required for NO production from V-PROLI/NO treatment in the rat and human appears to differ as we have previously studied the ability of V-PROLI/NO to prevent arsenic toxicity in human liver cells where it reduced toxicity apparently through a CYP2E1-mediated metabolic mechanism. None-the-less, it appears that both rat and human liver cells act upon V-PROLI/NO via a CYP450-related mechanism to produce NO and subsequently reduce arsenic toxicity.

Highlights

► V-PROLI/NO protects against the adverse effects of arsenic in rat liver cells. ► NO-releasing prodrug, V-PROLI/NO generates NO possibly via Cyp1a1 in rat liver cells. ► V-PROLI/NO reduces arsenic-induced toxicity and apoptosis. ► CYP450 required for NO production from V-PROLI/NO differs from rat and human.

Introduction

Inorganic arsenic is now a first choice cancer chemotherapeutic against certain leukemias and is thought to have potential against a variety of other cancers, including solid tumors [1], [2]. Specifically, arsenic trioxide is used in the treatment of acute promyelocytic leukemia and greatly improves the clinical outcome even in refractory or multiple relapsed cases [3], [4]. However, toxic side effects of arsenicals are often a major concern, including the potential for fatal hepatotoxicity [5]. The liver is a major target organ for both arsenic metabolism and toxicity [6]. Thus, an agent able to reduce the toxic potential of arsenic in liver cells would clearly be a useful adjuvant for arsenical chemotherapy, regardless of where the primary tumor of concern was located.

Nitric oxide (NO) is a simple chemical signaling mediator molecule produced by various cells [7]. The study of NO biochemistry and physiology has provided promising avenues for the development of numerous pharmaceuticals based on their ability to release NO [8]. Development of site-specific NO prodrugs is an important trend in production of agents with pharmacologic potential [9]. O2-vinyl 1-[2-(carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (V-PROLI/NO) is a NO donating prodrug that appears to have liver specificity, as it is metabolized to release NO by liver cytochrome P450s (CYP450) [10]. Emerging evidence indicates that cytochrome P450 1a1 (Cyp1a1), a hepatic and extrahepatic enzyme, is regulated by metal exposure [11] and may play an important role in the detoxication of environmental carcinogens [12]. Recently, we have demonstrated that V-PROLI/NO protects liver cells from arsenic-induced toxicity in a human liver cell line (HepG2) through generation of NO likely formed after metabolism by liver cell enzyme cytochrome P450 2E1 (CYP2E1) [13]. HepG2 is a human heptocarcinoma cell line commonly used as a liver model and it is of interest to see if the same phenomenon occurs in non-tumor cell lines or liver cell lines from non-human species. Whether V-PROLI/NO is metabolized by CYP450 and can be protective against arsenic toxicity is universal in mammalian cells has not been demonstrated.

Mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that include extracellular signal-regulated kinases (ERKs) and c-Jun NH2-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) [14]. ERKs transduce growth factor signals inducing cell proliferation or differentiation. In contrast, JNKs/SAPKs are strongly activated in responses to various stresses, growth arrest, DNA-damaging agents and apoptosis [15]. The levels of phosphorylated JNK1/2 and JNK kinase activity are markedly decreased in cells chronically exposed to arsenic [16]. Activation of the JNK pathway can be critical to apoptosis, and pretreatment with V-PROLI/NO negatively regulates arsenic-induced JNK activation in human HepG2 liver cells [13], indicating that NO has the capacity to alter the adverse effects of arsenic with regard to apoptotic signaling.

Metallothionein (MT) is a small, cysteine-rich, metal-sequestering protein that detoxicates various inorganics [17]. For instance, arsenic enhances MT expression [18] and binds to MT [19]. MT appears to prevent arsenic toxicity on several levels [20]. A recent study showed that human populations poorly expressing MT may be more sensitive to chronic arsenic intoxication [21]. NO released from NO-donating compounds interacts with MT indirectly by releasing bound metal and inducing the protein [22]. Indeed, MT, heme oxygenase-1 (HO-1) and glutathione S-transferase (GST) are well-known markers of acute arsenic-induced oxidative stress. GST plays a key role in phase II metabolism typically observed with detoxification. Polymorphisms in GST genes may affect the behavior of several enzymes involved with the maintenance of cellular glutathione (GSH) levels [23], which protect against cellular reactive oxygen species (ROS) and are thought to be generated in cells that methylate inorganic arsenic [24].

Thus, the purpose of this study was to determine if V-PROLI/NO acts as a potential hepatoprotectant prodrug by blocking arsenic-induced toxicity and apoptosis at the cellular level in the rat liver cell line TRL 1215. We found that although V-PROLI/NO reduced arsenic toxicity in both rat (present work) and human liver cells [13], the rat cells required Cyp1a1 for NO release while the human cells required CYP2E1, an important species difference reported in this present work.

Section snippets

Chemicals

Sodium arsenite and α-naphthoflavone were purchased from Sigma Chemical Company (St. Louis, MO). V-PROLI/NO was synthesized as previously described [25]. Chemical structure of V-PROLI/NO and its metabolic pathway are shown in Fig. 1. Anti-phospho-JNK, anti-phospho-ERK, and anti-JNK1/2 antibodies were purchased from New England Biolabs, Inc. (Beverly, MA).

Cell culture

The TRL 1215 rat liver cells were cultured as monolayers in William’s E medium containing 10% fetal bovine serum at 37 °C in a humidified 5% CO2

V-PROLI/NO toxicity

Initially, we tested the toxicity of V-PROLI/NO in TRL 1215 cells (Fig. 2). The results showed V-PROLI/NO had a very low toxicity and 200 μM was non-toxic at 24 h (Fig. 2A). This was true for the entire 24 h treatment period (Fig. 2B). Thus, concentrations of 100 and 200 μM and a 24 h treatment period for V-PROLI/NO were used for the remainder of this study.

Nitrite formation after V-PROLI/NO exposure and V-PROLI/NO-induced arsenic tolerance

To show that V-PROLI/NO was acted upon by rat liver cells to release NO, cells were exposed to V-PROLI/NO for 24 h at 200 μM for the time

Discussion

Recently, arsenic has been re-introduced into cancer chemotherapy, showing stunning efficacy in the treatment of certain leukemias [1], [5]. However, inorganic arsenic can have profound toxic effects even following short-term use. For instance, potentially fatal hepatic toxicity has been reported in a subset of patients receiving arsenic chemotherapy [5]. Individual variation in susceptibility to arsenic-induced toxicity clearly exists, possibly related to polymorphisms, arsenic methylation

Conflict of interest statement

None.

Acknowledgments

The authors thank Drs. Erik Tokar and Yang Sun for critical review of this manuscript. This research was supported in part by the National Toxicology Program, National Institute of Environmental Health Sciences (NIEHS) and by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute, Center for Cancer Research. Additional support came from Contract HHSN261200800001E with SAIC Frederick, Inc. This article may be the work product of an employee or a

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