Synergistic cytotoxicity of Δ9-tetrahydrocannabinol and butylated hydroxyanisole
Introduction
Marijuana is a widely used recreational and medicinal substance, the major active component of which is Δ9-tetrahydrocannabinol (THC). Contrary to prevalent opinion of an absence of harmful toxic effects of marijuana exposure, numerous studies have characterized varying degrees of tissue and cell injury from such exposure. Visual inspection of lungs of chronic marijuana smokers has revealed distal airway inflammation, goblet cell hyperplasia and squamous cell metaplasia—conditions consistent with a pre-malignant state (Fligiel et al., 1997, Roth et al., 1998, Barsky et al., 1998). Several recent reports indicate that THC can induce apoptotic cell death by activating cell type-specific signaling pathways (Zhu et al., 1998, Sanchez et al., 1998, Galve-Roperh et al., 2000, Downer et al., 2001, Ruiz et al., 1999). Furthermore, we have demonstrated significant oxidative stress in cells exposed to marijuana smoke (Sarafian et al., 1999). However, using the adenocarcinoma cell line, A549, we observed that marijuana smoke and THC suppressed anti-fas-induced apoptosis while promoting necrotic cell death (Sarafian et al., 2001).
To determine if induced oxidative stress contributes to A549 cytotoxicity, the effect of antioxidants on THC-induced cell death was examined. One of these antioxidants, the common food additive butylated hydroxyanisole (BHA) (Verhagen et al., 1991, Ito and Hirose, 1989) was found to exacerbate THC cytotoxicity. Among the known mechanisms of action of BHA, two major pathways were examined in this study. First, as a reducing agent, BHA may act by enhancing redox-cycling generation of superoxide anion (Kahl and Hildebrandt, 1986, Kahl et al., 1989). Secondly, BHA is known to disturb mitochondrial electron transport and ATP generation (Thompson and Moldéus, 1988, Ferreira, 1990, Fusi et al., 1992, Hampson et al., 1998), compromising cellular energetics. Our results suggest that the second of these mechanisms may be involved in THC-mediated cytotoxicity.
Section snippets
Materials
A549 cells were from American Tissue Culture Collection (Bethesda, MD, CCL-185). Cell culture materials were purchased from Irvine Scientific (Irvine, CA). THC was obtained from the National Institute on Drug Abuse (Bethesda, MD). Propidium iodide, digitonin, BHA, butylated hydroxytoluene (BHT), dicumarol, and lyopholyzed firefly extract were from Sigma (St. Louis, MO) and γ-interferon was from Prepro Tech (Rocky Hill, NJ). Anti-fas IgM antibody (clone IPO-4) was from Kamiya Biomedical
Results
BHA was initially added to A549 cells in conjunction with marijuana tar extract or THC as an antioxidant to investigate the role of oxidative stress in marijuana-induced cytotoxicity. However, instead of protecting against THC-induced cytotoxicity, BHA enhanced cell death. To investigate this effect, BHA dose-response studies were conducted using A549 cells concomitantly treated with cigarette smoke extracts or THC. Twenty four hour treatment of A549 cells with BHA alone produced negligible
Discussion
Over 60% of the US population has smoked marijuana and 2–3% smoke it on a daily basis (Adams and Martin, 1996). During the 1990s, use of marijuana increased among teenagers in parallel with the perception that there is no harm or health risk associated with marijuana smoking. Numerous studies have indicated neurologic and immunologic disturbances caused by marijuana smoking (Adams and Martin, 1996, Heyser et al., 1993, Baldwin et al., 1997, Klein et al., 1998, Roth et al., 1998). However, it is
Acknowledgements
The authors would like to thank Matthew Schibler and the UCLA Carol Moss Spivak Imaging Center for assistance with fluorescent microscopic studies. We are also grateful to Dr Genhong Cheng for use of the luminometer, Dr Robert Strieter for use of the spectrophotometer, and Farnaz Khoshaghideh for technical assistance. This work was supported by NIH Grant R37DA030-18.
References (36)
- et al.
Rapid induction of forestomach tumors in partially hepatectomized Wistar rats given butylated hydroxyanisole
Exp. Mol. Pathol.
(1986) - et al.
Augmentation of chrysotile-induced oxidative stress by BHA in mice lungs
Food Chem. Toxicol.
(1995) - et al.
epidermal growth factor (EGF)-induced generation of hydrogen peroxide. Role in EGF receptor-mediated tyrosine phosphorylation
J. Biol. Chem.
(1997) - et al.
Butylated hydroxyanisole-stimulated NADPH oxidase activity in rat liver microsomal fractions
J. Biol. Chem.
(1983) Effect of butylated hydroxyanisole on electron transport in rat liver mitochondria
Biochem. Pharmacol.
(1990)- et al.
Tracheobronchial histopathology in habitual smokers of cocaine, marijuana, and/or tobacco
Chest
(1997) - et al.
Interaction of butylated hydroxyanisole with mitochondrial oxidative phosphorylation
Biochem. Pharmacol.
(1992) - et al.
Antioxidants—carcinogenic and chemopreventive properties
Adv. Cancer Res.
(1989) - et al.
Methodology for studying antioxidant activity and mechanisms of action of antioxidants
Food Chem. Toxicol.
(1986) - et al.
Production of reactive oxygen species due to metabolic activation of butylated hydroxyanisole
Toxicology
(1989)