Elsevier

Toxicology Letters

Volume 133, Issues 2–3, 21 July 2002, Pages 171-179
Toxicology Letters

Synergistic cytotoxicity of Δ9-tetrahydrocannabinol and butylated hydroxyanisole

https://doi.org/10.1016/S0378-4274(02)00134-0Get rights and content

Abstract

We examined the food additive, butylated hydroxyanisole (BHA), for its capacity to modulate the cytotoxic effects of Δ9-tetrahydrocannabinol (THC). THC was not cytotoxic when added to cultures of A549 lung tumor cells at concentrations<5 μg/ml, but induced cell necrosis at higher levels with an LC50=16–18 μg/ml. BHA alone, at concentrations of 10–200 μM, produced limited cell toxicity but significantly enhanced the necrotic death resulting from concurrent exposure to THC. In the presence of BHA at 200 μM, the LC50 for THC decreased to 10–12 μg/ml. Similar results were obtained with smoke extracts prepared from marijuana cigarettes, but not with extracts from tobacco or placebo marijuana cigarettes (containing no THC). Two different mechanisms for this synergistic cytotoxicity were investigated. Experiments were repeated in the presence of either diphenyleneiodonium or dicumarol as inhibitors of the redox cycling pathway. Neither of these compounds protected cells from the effects of combined THC and BHA, but rather enhanced necrotic cell death. Measurements of cellular ATP revealed that both THC and BHA reduced ATP levels in A549 cells, consistent with toxic effects on mitochondrial electron transport. The combination was synergistic in this respect, reducing ATP levels to <15% of control. Exposure to marijuana smoke in conjunction with BHA, a common food additive, may promote deleterious health effects in the lung.

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.

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