Elsevier

Toxicology and Applied Pharmacology

Volume 306, 1 September 2016, Pages 86-97
Toxicology and Applied Pharmacology

Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death

https://doi.org/10.1016/j.taap.2016.06.004Get rights and content

Highlights

  • Carbendazim induced transcriptional activity of the aryl hydrocarbon response element.

  • Carbendazim induced nuclear translocation of the aryl hydrocarbon receptor (AhR).

  • Carbendazim induced the expression of cytochrome P450 (CYP) 1A1, an AhR-targeted gene.

  • Carbendazim is an AhR agonist.

  • The activated AhR decreased carbendazim-induced cell toxicity and death.

Abstract

Carbendazim inhibits microtubule assembly, thus blocking mitosis and inhibiting cancer cell proliferation. Accordingly, carbendazim is being explored as an anticancer drug. Data show that carbendazim increased mRNA and protein expressions and promoter activity of CYP1A1. In addition, carbendazim activated transcriptional activity of the aryl hydrocarbon response element, and induced nuclear translocation of the aryl hydrocarbon receptor (AhR), a sign the AhR is activated. Carbendazim-induced CYP1A1 expression was blocked by AhR antagonists, and was abolished in AhR signal-deficient cells. Results demonstrated that carbendazim activated the AhR, thereby stimulating CYP1A1 expression. In order to understand whether AhR-induced metabolic enzymes turn carbendazim into less-toxic metabolites, Hoechst 33342 staining to reveal carbendazim-induced nuclear changes and flow cytometry to reveal the subG0/G1 population were applied to monitor carbendazim-induced cell apoptosis. Carbendazim induced less apoptosis in Hepa-1c1c7 cells than in AhR signal-deficient Hepa-1c1c7 mutant cells. Pretreatment with β-NF, an AhR agonist that highly induces CYP1A1 expression, decreased carbendazim-induced cell death. In addition, the lower the level of AhR was, the lower the vitality present in carbendazim-treated cells, including hepatoma cells and their derivatives with AhR RNA interference, also embryonic kidney cells, bladder carcinoma cells, and AhR signal-deficient Hepa-1c1c7 cells. In summary, carbendazim is an AhR agonist. The toxicity of carbendazim was lower in cells with the AhR signal. This report provides clues indicating that carbendazim is more potent at inducing cell death in tissues without than in those with the AhR signal, an important reference for applying carbendazim in cancer chemotherapy.

Introduction

Carbendazim (Fig. 1) is a broad-spectrum benzimidazole fungicide and a metabolite of benomyl (McCarroll et al., 2002). It is widely used in agriculture to control plant diseases of cereals and fruits, such as strawberries, pineapples, pomes, citrus and bananas (Yenjerla et al., 2009). In addition to acting on fungal tubulin, carbendazim also weakly inhibits polymerization of mammalian tubulin into microtubules; therefore, it is able to inhibit mitosis, arrest the cell cycle at the G2/M phase, and induce apoptosis (Yenjerla et al., 2009). It inhibits cancer cell proliferation, including drug-resistant, multiple drug-resistant and p53-deficient cell lines (Hammond et al., 2001, Yenjerla et al., 2009). Due to its promising preclinical antitumor activity, carbendazim has potential to be applied as an anti-cancer drug. Carbendazim is under further clinical development for cancer treatment, and the phase I clinical trials were performed for chemotherapy treating patients with solid tumors (Yenjerla et al., 2009).

Aryl hydrocarbon receptor (AhR), a cytosolic receptor, is activated by xenobiotics and their endogenous ligands, such as tryptophan metabolites (Murray et al., 2014). The xenobiotics include 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo[a]pyrene (BaP), 1-nitropyrene (1-NP), and many other polycyclic aromatic hydrocarbons (PAHs) (Murray et al., 2014, Su et al., 2008). Upon activation by binding to its agonist, AhR translocates to nucleus, heterodimerizes with the AhR nuclear translocator (ARNT), and, as a transcription factor, binds to aryl hydrocarbon response element (AHRE), also named as xenobiotic response element (XRE), of its target genes for the expressions (Murray et al., 2014, Nakata et al., 2006).

The AhR-induced detoxification process has been divided into three stages: phase I, II, and III, and AhR induces drug-metabolic enzymes in every stage (Nakata et al., 2006). The functions of phase I, II, and III are the introduction of hydroxyl group on the aryl hydrocarbons, the conjugation with glutathione, sulfate, or glucuronide, and the elimination of phase metabolites from cells, respectively. However, in phase I, some reactive intermediates may interact with DNA and other cellular omponents, resulting in toxic effects. Both groups of enzymes, cytochrome p450 (CYP) and aldoketo reductases (AKRs), can be referred to as phase I drug-metabolizing enzymes (Zhang et al., 2012). CYP1A1 is one of the major phase I enzymes, and the most well-known AhR-targeted genes.

Our previous results demonstrated that CYP1A1 decreased 1-NP-induced cell death and mutagenesis (Su et al., 2011), implicating that CYP1A1 converts 1-NP into less toxic metabolites. In contrast, the monooxygenase activity of CYP1A1 introduces an epoxide to BaP, and in turn, epoxide hydrolase hydrolyzes the epoxide to produce diol derivatives, e.g. B[a]P-7,8-diol (Badal and Delgoda, 2014, Shimada, 2006). B[a]P-7,8-diol may be further converted to B[a]P diol epoxide (BPDE). BDBP has been shown to bind to DNA and is regarded as a carcinogenic derivative (Badal and Delgoda, 2014, Zhang et al., 2012). BDBP may also interact with other cellular components, resulting in cell death or damage.

The development of Ahr knockout (Ahr-/-) mice has demonstrated that AhR is not just involved in the detoxification of xenobiotics. Ahr-/- mice show decreased fertility, decreased liver size, vascular abnormalities in the cardiovascular system, decreased levels of mature follicles in reproductive tract, and formation of uric acid stones in the urinary bladder (Safe et al., 2013). In addition, AhR is essential for the postnatal maintenance of intestinal intraepithelial lymphocytes and skin-resident dendritic epidermal gamma delta T cells (Hao and Whitelaw, 2013). AhR also expresses in tumors of pancreas, prostate, urinary tract, lung, esophagus, pituitary and gliomas (Safe et al., 2013). Mice with overexpression of the active AhR exhibit enhanced stomach and liver cancers, and knockdown of the AhR results in decreased proliferation and/or invasion and migration of cancer cell lines (Safe et al., 2013). Therefore, it is an implication that AhR plays a pro-oncogenic role. Conversely, AhR-deficient transgenic mice spontaneously develop colonic tumors and exhibit enhanced carcinogenesis in carcinogen-induced liver tumors (Safe et al., 2013). AhR activation may associate with a decreased risk of symptomatic benign prostatic hyperplasia in humans (Mehta and Vezina, 2011).

In this report, we demonstrated that carbendazim activated AhR, and thereby CYP1A1 expression was stimulated. Activation of AhR and expression of CYP1A1 may influence the metabolism and toxicity of carbendazim. To utilize carbendazim as a chemotherapeutic drug, it is important to know its potency in inducing cell death. We further examined the influence of the activated AhR signal in the carbendazim-induced cell death. Results indicated that activation of AhR by carbendazim may decrease the potency of carbendazim in inducing cell death.

Section snippets

Reagents and antibodies

Carbendazim, Hoechst 33342, and salicylamide were obtained from Sigma (St. Louis, MO). BaP was obtained from ChemService (West Chester, PA). CH-223191 was obtained from Calbiochem (La Jolla, CA). Minimum essential medium alpha (MEMα), Dulbecco's modified Eagle medium/nutrient mixture F12 (DMEM/F12), and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, NY). RPMI 1640 medium was from GE Healthcare Life Sciences HyClone Laboratories (Logan, Utah). Antibody (Ab) against β-actin was

Carbendazim stimulates transcription of CYP1A1

Treatment with carbendazim greatly increased CYP1A1 mRNA with time course- and dose-dependence in HepG2 and Hepa-1c1c7 cells. The induction of CYP1A1 mRNA expression was detectable after 3 h of treatment with carbendazim (10 μM) in both cells (Fig. 2A and B). It reached a maximum level at 3 and 6 h of treatment of Hepa-1c1c7 and HepG2 cells, respectively, and decreased thereafter. Treatments with carbendazim (5, 10, and 20 μM) for 6 h dose-dependently increased CYP1A1 mRNA expression by 5.8-, 10.9-,

Discussion

Due to the inhibition of polymerization of mammalian tubulin into microtubules, carbendazim is being studied for its potentiality in chemotherapy for solid tumors (Hammond et al., 2001, Yenjerla et al., 2009). Phase I trial to study the effectiveness of carbendazim in treating patients who have advanced solid tumors was sponsored by Jonsson Comprehensive Cancer Center and the University of Texas Health Science Center at San Antonio (//clinicaltrials.gov/ct2/show/NCT00023816?term=Carbendazim&rank=1

Conclusions

In summary, although carbendazim contains only one aromatic hydrocarbon ring, and its structure distinctly differs from that of the prototype of AhR ligands, such as BaP and TCDD, our results indicate that carbendazim activated AhR and also induced expression of the AhR-targeted gene, CYP1A1. This information provides a clue for the studies of AhR ligand structures. In addition to be an agriculture chemical, carbendazim is being studied for its potentiality being a chemotherapeutic drug. Our

Conflict of interest

The authors declare that they have no conflicts of interest.

Transparency document

Transparency document.

Acknowledgements

This work was supported by grants (CMRPG6A0401 and CMRPG6A0402) from Chang Gung Memorial Hospital, Chiayi Branch, and grants (99AS-9.2.3-BQ-B3 and 103AS-10.2.3-BQ-B1) from the Bureau of Animal and Plant Health Inspection and Quarantine, Council of Agriculture, Executive Yuan, Taiwan, ROC. We thank Dr. Alvaro Puga (University of Cincinnati Medical Center, Cincinnati, OH) for the generous gift of the pAhRDtkLuc3 and p-1646P1Luc3 plasmids. We thank Dr. Abraham Amsterdam (The Weizmann Institute of

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