Cannabidiol induces expression of human cytochrome P450 1A1 that is possibly mediated through aryl hydrocarbon receptor signaling in HepG2 cells
Introduction
Marijuana is the most widely used illicit drug in the world. Its use is a growing public health concern due to potential adverse effects such as dependence, association with polysubstance use, increased risk of motor vehicle crashes, impaired respiratory function, cardiovascular disease, and various health consequences [1]. Marijuana leaves contain at least 70 cannabinoids [2], with Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), and cannabinol (CBN) being the three main constituents (Fig. 1). Δ9-THC is the principal psychoactive component of marijuana and has various pharmacological effects such as catalepsy, hypothermia, antiinflammation, and antinociception [3]. CBD is not psychoactive, but has several pharmacological effects such as antiepileptic, anxiolytic, and antiemetic actions [4]. CBN is believed to exert minimal pharmacological effects on the central nervous system.
Marijuana is commonly consumed by smoking. Previous studies reported that habitual smokers of marijuana exhibited molecular and histopathological changes that were similar to precancerous lesions observed in the bronchial epithelium of tobacco smokers [5], [6]. Furthermore, an epidemiological study revealed a correlation between marijuana use and head and neck cancer [7]. These findings suggest that a history of marijuana use may increase the risk of developing cancer. Marijuana smoke includes various procarcinogenic polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) and benz[a]anthracene [8], [9], [10]. These PAHs are metabolically activated by cytochrome P450s (CYPs) to exert genotoxicity and carcinogenicity [11]. For example, B[a]P is metabolized by CYP1A1 and epoxide hydrolase to a diol-epoxide, the ultimate carcinogen, the formation of DNA adduct by which plays a critical role in tumor initiation [12]. Some PAHs are also known to potently induce the expression of CYP1A1 [11]. These findings indicated that the potency of the catalytic activity of CYP1A1 and its expression levels are important risk factors for determining cancer induced by marijuana use. Witschi and Saint-François [13] demonstrated that B[a]P hydroxylase activity, an index of CYP1 activity, was increased in the lung homogenates of rats administered Δ9-THC. Furthermore, Δ9-THC has been shown to induce the expression of CYP1A1 in mouse hepatoma Hepa-1 cells, primary human airway epithelial cells, and human breast cancer MDA-MB-231 cells [14], [15], [16]. Phytocannabinoids are present in marijuana smoke at markedly higher concentrations than PAHs [8], [9], [14]. A previous study estimated that the content of Δ9-THC was approximately 9.3 mg per marijuana cigarette whereas the contents of B[a]P and benz[a]anthracene were 22 and 56 ng per marijuana cigarette, respectively [14]. Thus, phytocannabinoids may also contribute to the induction of CYP1A1 by marijuana components. However, it currently remains unclear whether major phytocannabinoids other than Δ9-THC, i.e. CBD and CBN, induce the expression of CYP1A1.
In the present study, we examined the inducibility of human CYP1A1 by the three major phytocannabinoids (Δ9-THC, CBD, and CBN). We showed that CBD was the most potent inducer of the expression of CYP1A1 in human hepatoma HepG2 cells. Furthermore, the results of our study suggest that the induction of CYP1A1 by CBD was mediated through aryl hydrocarbon receptor (AhR) signaling via the activation of protein tyrosine kinases (PTKs).
Section snippets
Materials
Δ9-THC, CBD, and CBN were isolated from cannabis leaves using a previously reported method [17]. CBD-2′-monomethyl ether (CBDM) and CBD-2′,6′-dimethyl ether (CBDD) were prepared as described previously [18]. The purities of these cannabinoids were determined to be above 97% by gas chromatography, except for CBDD, the purity of which was 93% [19]. Other chemicals and materials were obtained from the following sources: olivetol, d-limonene, and an anti-actin (20–33) antibody produced in rabbits
Inducibility of human CYP1A1 expression by Δ9-THC, CBD, and CBN in HepG2 cells
To characterize the inducibility of human CYP1A1 by phytocannabinoids, the effects of Δ9-THC, CBD, and CBN on CYP1A1 expression levels were evaluated with HepG2 cells. Δ9-THC and CBD increased the expression of CYP1A1 mRNA in a concentration-dependent manner; CYP1A1 mRNA levels at 50 μM Δ9-THC and CBD were 1.7- and 4.8-fold higher, respectively, than control levels (Fig. 2A and B). In contrast, CBN had a less marked effect on CYP1A1 expression (Fig. 2C). CYP1A1 mRNA expression levels reached a
Discussion
Previous studies on the inducibility of CYP expression by marijuana components have been conducted using experimental animals and cultured cells. Cannabis resin, marijuana tar, and Δ9-THC have been shown to induce the expression of CYP1A enzymes [13], [14], [15], [16]. We herein demonstrated that CBD induced the expression of CYP1A1 in HepG2 cells; inducibility by CBD was more potent than that by Δ9-THC. To the best of our knowledge, this is the first study to show the induction of CYP1A
Conclusion
We demonstrated that CBD as well as Δ9-THC induced human CYP1A1 expression. Our results suggest that the induction of CYP1A1 by CBD is mediated through the activation of PTK-dependent AhR signaling. Furthermore, two phenolic hydroxyl groups in the resorcinol moiety of CBD may play pivotal roles in CYP1A1 induction, whereas the whole structure of CBD is essential for overall induction. This study has provided useful information to understand the mechanism underlying CBD-mediated CYP1A1
Conflicts of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgments
This work was supported in part by the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Grant-in-Aid for Young Scientists (B) (grant number 21790135) and Grant-in-Aid for Scientific Research (C) (grant number 20590217)] and by the ‘Academic Frontier’ Project for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (2005–2009) (grant number 05F016). We thank the RIKEN cell bank (Tsukuba, Japan) for providing the human
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Present address: Laboratory of Xenobiotic Metabolism and Environmental Toxicology, Faculty of Pharmaceutical Sciences, Hiroshima International University (HIU), 5-1-1 Hiro-koshingai, Kure, Hiroshima 737-0112, Japan.