Activation of aryl hydrocarbon receptor reduces carbendazim-induced cell death
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
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
References (28)
- et al.
Cyprodinil as an activator of aryl hydrocarbon receptor
Toxicology
(2013) - et al.
The emerging roles of AhR in physiology and immunity
Biochem. Pharmacol.
(2013) You AhR what you eat: linking diet and immunity
Cell
(2011)- et al.
Screening and detection of apoptosis
J. Surg. Res.
(2007) - et al.
A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim
Mutat. Res.
(2002) - et al.
Potential protective mechanisms of aryl hydrocarbon receptor (AHR) signaling in benign prostatic hyperplasia
Differentiation
(2011) - et al.
Nuclear receptor-mediated transcriptional regulation in Phase I, II, and III xenobiotic metabolizing systems
Drug Metab. Pharmacokinet.
(2006) - et al.
Development of a multiplex quantitative PCR assay for the analysis of human cytokine gene expression in influenza A virus-infected cells
J. Immunol. Methods
(2016) Xenobiotic-metabolizing enzymes involved in activation and detoxification of carcinogenic polycyclic aromatic hydrocarbons
Drug Metab. Pharmacokinet.
(2006)- et al.
Aldo-keto reductase 1C2 is essential for 1-nitropyrene's but not for benzo[a]pyrene's induction of p53 phosphorylation and apoptosis
Toxicology
(2008)
Crosstalk between activated forms of the aryl hydrocarbon receptor and glucocorticoid receptor
Toxicology
The role of endogenous aryl hydrocarbon receptor signaling in cardiovascular physiology
J. Cardiovasc. Dis. Res.
The role of human aldo-keto reductases in the metabolic activation and detoxication of polycyclic aromatic hydrocarbons: interconversion of PAH Catechols and PAH o-Quinones
Front. Pharmacol.
Role of the modulation of CYP1A1 expression and activity in chemoprevention
J. Appl. Toxicol.
Cited by (13)
Etoricoxib enhances aryl hydrocarbon receptor activity
2023, ToxicologySorafenib is an antagonist of the aryl hydrocarbon receptor
2022, ToxicologyCitation Excerpt :CYP1A1 is a well-known AhR-sensitive target, and the expression level of CYP1A1 is often used as an indicator for AhR activation. Beta-naphthoflavone (β-NF) is a synthetic, potent agonist of AhR that greatly increases AhR activity as well as CYP1A1 expression (Bock et al., 1990; Wei et al., 2016b). AhR is involved in other physiological processes as well.
Sublethal effects of carbendazim in Jenynsia multidentata detected by a battery of molecular, biochemical and genetic biomarkers
2020, Ecotoxicology and Environmental SafetyCitation Excerpt :Taking into account that a knockdown of TNF-α in zebrafish caused reductions of liver size (Qi et al., 2010), future studies of fishes chronically exposed to CBM are suggested in order to explore potential consequences of changes in TNF- α expression in J. multidentata. Although in a general basis in humans, rats and rabbit, data show that CBM increased mRNA and protein expressions and promotes activity of CYP1A1 (Rey-Grobellet et al., 1996; Backlund et al., 1999; Rudzok et al., 2009; Wei et al., 2016) other studies showed that human hepatic CYP2D6 activity (an enzyme implicated in the metabolism of several pharmaceutical compounds) was completely inhibited by CBM (Abass et al., 2009). In fishes, a lack of information about CBM detoxification through P450 enzymes is noteworthy, and at the best of our knowledge this is the first report on the evaluation of CYP1A1 expression in this taxa.
Measurement and correlation of solubility of carbendazim in lower alcohols
2018, Thermochimica ActaCitation Excerpt :Carbendazim (Methyl benzimidazol-2-yl carbamate, CAS Registry No.10605-21-7, presented in Fig. 1) is one of the benzimidazole fungicides, which is a widely-used broad-spectrum fungicide [1,2].
The tissue distribution, metabolism and hepatotoxicity of benzoylurea pesticides in male Eremias argus after a single oral administration
2017, ChemosphereCitation Excerpt :The flufenoxuron treatment group showed significant up-regulation of the Cyp1a and Ahr genes in the lizard liver. A previous study has shown that carbendazim with an aromatic hydrocarbon ring in the structure, is also an activator of the Cyp1a and Ahr genes (Wei et al., 2016). Combined with the decrease of hepatocytes in the liver treated with flufenoxuron, the stimulation of the Ahr genes has a negative effect on the cell proliferation.