American Journal of Respiratory Cell and Molecular Biology

Induction of the carcinogen-metabolizing enzyme cytochrome P4501A1 (CYP1A1) is a key step in the development of tobacco-related cancers. To determine if marijuana smoke activates CYP1A1, a murine hepatoma cell line expressing an inducible CYP1A1 gene (Hepa-1) was exposed in vitro to tar extracts prepared from either tobacco, marijuana, or placebo marijuana cigarettes. Marijuana tar induced higher levels of CYP1A1 messenger RNA (mRNA) than did tobacco tar, yet resulted in much lower CYP1A1 enzyme activity. These differences between marijuana and tobacco were primarily due to Δ9-tetrahydrocannabinol ( Δ9-THC), the psychoactive component of marijuana. Here we show that Δ9-THC acts through the aryl hydrocarbon receptor complex to activate transcription of CYP1A1. A 2- μ g/ml concentration of Δ9-THC produced an average 2.5-fold induction of CYP1A1 mRNA, whereas a 10- μ g/ml concentration of Δ9-THC produced a 4.3-fold induction. No induction was observed in Hepa-1 mutants lacking functional aryl-hydrocarbon receptor or aryl-hydrocarbon receptor nuclear translocator genes. At the same time, Δ9-THC competitively inhibited the CYP1A1 enzyme, reducing its ability to metabolize other substrates. Spiking tobacco tar with Δ9-THC resulted in a dose-dependent decrease in the ability to generate CYP1A1 enzyme activity as measured by the ethoxyresorufin-o-deethylase (EROD) assay. This inhibitory effect was confirmed by Michaelis-Menton kinetic analyses using recombinant human CYP1A1 enzyme expressed in insect microsomes. This complex regulation of CYP1A1 by marijuana smoke and the Δ9-THC that it contains has implications for the role of marijuana as a cancer risk factor.

Marijuana is often perceived as a natural substance that poses little risk when smoked (1). However, airway biopsies obtained from marijuana smokers exhibit precancerous histopathologic and molecular abnormalities similar to those observed in age-matched tobacco smokers (2, 3). Along the same lines, Ammenheuser and coworkers (4), documented a 3-fold higher frequency of somatic mutations in marijuana-smoking mothers and their newborn infants when compared with nonsmoking control subjects. These cellular, molecular, and genetic alterations suggest a definite carcinogenic potential. This hypothesis is supported by a recent case-control cancer study (5). Zhang and associates (5) analyzed 173 patients with head and neck cancer as well as 176 cancer-free control subjects and observed a significant relationship between the presence of cancer and a history of marijuana use (odds ratio, 2.6). Cancer risk, controlling for a variety of other factors including tobacco exposure, age, sex, education, and race, independently correlated with the number of marijuana cigarettes smoked per day and the years of marijuana use.

These findings, in part, prompted us to evaluate the interaction between marijuana tar and cytochrome P4501A1 (CYP1A1). Like tobacco, marijuana smoke contains several known carcinogens and tumor promoters, including vinyl chlorides, phenols, aldehydes, nitrosamines, reactive oxygen species, and a variety of polycyclic aromatic hydrocarbons (PAHs) (6, 7). Benzo[a]pyrene and benz[a]anthracene, two highly procarcinogenic PAHs, have been reported to occur at 25 to 75% higher concentrations in marijuana tar as compared with tobacco tar (6, 8). CYP1A1 is a key enzyme that converts PAHs into active carcinogens (9, 10). PAHs present in tobacco smoke activate transcription of the CYP1A1 gene and increase pulmonary CYP1A1 activity severalfold (11, 12). This induction of CYP1A1 is time- and exposure-dependent and results in a marked increase in the conversion of smoked PAHs into carcinogens, an increase in DNA mutations in lung tissue, and an increased risk for developing lung cancer (13-15). Benzo[a]pyrene, for example, is metabolized by CYP1A1 into a diol-epoxide that preferentially binds to the human p53 tumor suppressor gene at mutational hotspots associated with respiratory tract cancer (16).

In the present study, tar extracts were prepared from marijuana and tobacco cigarettes, analyzed for their composition, and evaluated for their ability to induce CYP1A1 messenger RNA (mRNA) and enzymatic activity. Marijuana tar induced greater levels of CYP1A1 mRNA than similar amounts of tobacco tar. We found that Δ9-tetrahydrocannabinol (Δ9-THC), the psychotropic component of marijuana, was responsible for this extra induction and that Δ9-THC acted as an independent regulator of CYP1A1.

Preparation of Tar Extracts

A smoking device equipped with an in-line Cambridge filter (Fisher, Orlando, FL) was used to collect the tar phase of mainstream smoke from either commercial tobacco cigarettes with filter tips (average weight, 850 mg, Marlboro Red Hard Pack; Philip Morris Inc., Richmond, VA), nonfiltered research-grade marijuana cigarettes made from leaves containing 3.95% Δ9-THC (average weight, 734 mg; National Institute on Drug Abuse [NIDA], Rockville, MD), or nonfiltered placebo marijuana cigarettes made from ethanol-extracted leaves containing 0% Δ9-THC (average weight, 833 mg; NIDA). Cigarettes were inserted into the smoking device and lit, and 40 ml puffs of smoke were drawn through the filter every 30 s until the entire cigarette was consumed. Tar was extracted from Cambridge filters using dimethyl sulfoxide (DMSO) for biologic studies, methanol for Δ9-THC analyses by high performance liquid chromatography (HPLC), or dichloromethane for gas chromatography–mass spectroscopy (reagents from Sigma Chemical Corp., St. Louis, MO). The mass of recovered tar was determined by comparing the vacuum-desiccated filter weights before and after solvent extraction.

Induction of CYP1A1

A murine hepatoma cell line containing an inducible CYP1A1 gene (Hepa-1) or mutant derivatives lacking either the aryl hydrocarbon receptor (c35 cells) (17) or the aryl hydrocarbon receptor nuclear translocator (c4 cells) (18) was maintained in continuous culture at 37°C in RPMI-1640 supplemented with glutamine (Bio-Whittaker, Walkersville, MD), 0.01 M N-2-hydroxyethylpiperazine-N′-ethane sulfonic acid buffer (GIBCO Laboratories, Grand Island, NY), antibiotic/antimycotic mixture (GIBCO), and 10% heat-inactivated and filtered fetal calf serum (Omega Scientific, Tarzana, CA). To compare the effects of different inducing agents, Hepa-1 cells were cultured for 24 h in the presence of either 10−8 M 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); purified Δ9-THC (1 to 10 μg/ml; NIDA); extracts of tobacco, marijuana, or placebo marijuana tar prepared in DMSO (0.1 to 30 μg/ ml, final DMSO concentration ⩽ 0.2%); or comparable amounts of DMSO as a solvent control. Induction of CYP1A1 was determined in a quantitative manner by Northern blot analysis as previously described (19).

CYP1A1 Enzyme

CYP1A1 activity was assayed using an ethoxyresorufin-o-deethylase (EROD) assay modified for microwell analysis (20, 21). Cell sonicates prepared from control or induced Hepa-1 cells (1.7 × 104 cells) were added to wells of a flat-bottomed 96-well microtiter plate in a total volume of 200 μl of 50 mM Tris buffer (pH 7.5) containing 1% bovine serum albumin (BSA), 5 μM ethoxyresorufin (substrate), 200 mM dicumarol, and 1.67 mM nicotenamide adenine dinucleotide phosphate (all reagents from Sigma). The enzymatic conversion of ethoxyresorufin to resorufin was measured at 37°C using a multiwell plate cytofluorimeter (Cytofluor 2300; PerSeptive Biosystems, Framingham, MA). All assays were performed in duplicate, and six concentrations of resorufin (4 to 40 pmol/well) were included as standards. EROD activity was expressed as picomoles of resorufin formed per minute, per well. In studies evaluating the direct effects of Δ9-THC on recombinant human P4501A1, microsomes prepared from insect cells transduced with a baculovirus vector containing the human CYP1A1 gene were obtained commercially (Human CYP1A1 Supersomes; GENTEST Corporation, Woburn, MA) and used as the source of human P4501A1. Human CYP1A1 Supersomes were diluted in 50 mM Tris buffer–1% BSA at a concentration of 0.2 pM (specific activity, 35.4 pmol product/[min × pmol P4501A1]).

Michaelis-Menton Kinetics

Human CYP1A1 supersomes were adjusted to a final concentration of 200 pmol/well, and the EROD assay was performed in the presence of four different concentrations of ethoxyresorufin (12.5, 25, 50, or 100 pmol/well) and three different concentrations of Δ9-THC (0, 4, or 8 μg/ml). Lineweaver-Burke plots were prepared by plotting 1/v (where v = EROD activity in pmol/well/ min) versus 1/s (where s = substrate concentration) for each of the inhibitor concentrations (0, 4, or 8 mg/ml Δ9-THC).

Data Analysis

Data from duplicate measurements of a single assay condition are expressed as the mean value, and data representing the average results of multiple experiments are expressed as the mean ± 1 standard deviation. The coefficients of variation for duplicate wells in the EROD assay were routinely < 5%. Differences between experimental conditions were compared using a Student's t test. A P value of ⩽ 0.05 was considered statistically significant.

Tar Yield and Composition

Tar extracts from tobacco, marijuana, and placebo marijuana cigarettes were examined for dry weight, PAH composition, and Δ9-THC content. Marijuana cigarettes generated more tar than did filtered tobacco cigarettes (47.0 ± 15.5 versus 29.3 ± 3.3 mg; P < 0.01) and contained more benz[a]anthracene (56 versus 46 ng) and benzo[a]pyrene (22 versus 15 ng) as determined by gas chromatography– mass spectroscopy. HPLC analysis of the marijuana tar demonstrated an average Δ9-THC content of 19.7%, 5-fold higher than the 3.95% present in the plant material used to make the cigarette.

Marijuana Tar Induces EROD Activity, but the Effects Are Inhibited by Δ9-THC

Tar extracts were evaluated for their ability to induce CYP1A1 enzyme activity in Hepa-1 cells. After a 24-h exposure, treatment with tobacco tar increased EROD activity in a concentration- and time-dependent manner. A total of 3 μg/ml of tobacco tar produced an average 20-fold induction of enzyme activity (range, 11- to 29-fold; Figure 1). By comparison, 24 h of exposure to the same concentration of marijuana tar increased EROD activity only 9-fold (44 ± 5% of that observed with tobacco tar, n = 4 experiments; P < 0.01). Neither cell death (as measured by trypan blue dye) nor metabolic viability (as measured by alamar blue dye) appeared to be the cause of this difference. The role of Δ9-THC was examined by comparing the effect of marijuana tar with that of placebo marijuana tar, which contained no Δ9-THC. Placebo marijuana induced EROD activity in a pattern almost identical to that of tobacco (average, 98 ± 14% of the induction observed with tobacco tar, n = 4). To confirm this effect of Δ9-THC, tobacco tar was spiked with increasing concentrations of exogenous Δ9-THC before its use as an inducing agent (Figure 2). Addition of Δ9-THC at 1.0 μg/ml decreased resulting EROD activity by 56% and higher levels resulted in further reductions in EROD activity. Thus, whereas marijuana tar induced CYP1A1, the expression of EROD activity appeared to be limited by the presence and concentration of Δ9-THC.

Δ9-THC Induces CYP1A1 mRNA

To determine if the effects of Δ9-THC were mediated at the level of transcription, tar extracts from marijuana and tobacco cigarettes or different concentrations of Δ9-THC were compared with 10−8 M TCDD (a prototypical CYP1A1 inducer) for their ability to induce CYP1A1 mRNA as determined by Northern blot analysis (Figure 3). In contrast to the EROD assay, a 3-μg/ml concentration of marijuana tar always induced greater levels of CYP1A1 mRNA than did the same concentration of tobacco tar. Adding Δ9-THC to tobacco tar also resulted in a concentration-dependent increase, not a decrease, in CYP1A1 mRNA. The capacity for Δ9-THC to increase steady-state levels of CYP1A1 mRNA was further confirmed by incubating Hepa-1 cells directly with purified Δ9-THC in the absence of other inducing agents (Figure 3B). A total of 2 μg/ml of Δ9-THC produced an average 2.5-fold induction of CYP1A1 mRNA, 10 μg/ml produced an average 4.3-fold induction, and 10−8 M TCDD produced a 16.7-fold induction.

Induction of CYP1A1 by PAHs requires interaction between the inducing agent, the aryl hydrocarbon receptor, and the aryl hydrocarbon receptor nuclear translocator protein (18). The capacity for marijuana tar and Δ9-THC to increase expression of CYP1A1 mRNA was therefore examined in mutant derivatives of the Hepa-1 cell line lacking either the aryl hydrocarbon receptor (c35 cells) or the aryl hydrocarbon receptor nuclear translocator (c4 cells). No induction was observed in these Hepa-1 mutants (data not shown), suggesting that both marijuana and Δ9-THC require a functional aryl hydrocarbon receptor complex in order to induce CYP1A1.

Direct Effects of Δ9-THC on Recombinant Human CYP1A1

To explain the opposite effects of Δ9-THC in the EROD assay and Northern blot analysis, we examined the effect of Δ9-THC directly on the function of recombinant CYP1A1 using microsomes prepared from insect cells transduced with the human CYP1A1 gene (CYP1A1 Supersomes). This approach allowed us to monitor the effect of Δ9-THC on enzyme activity in the absence of an induction step. Δ9-THC produced a concentration-dependent inhibition of EROD activity (Figure 4). This inhibitory effect was further evaluated by Michaelis-Menton kinetics and determined to be competitive in nature (Figure 5).

Carcinogenesis is a highly complex process in which DNA injury, mutation, and altered gene regulation are considered key events. The presence of these abnormalities in the lungs and blood of marijuana smokers raises serious concern about the carcinogenic potential of this drug (2-4, 22). This concern is reinforced by a number of case reports and a recent case-control study suggesting a correlation between marijuana smoking and airway cancer (5, 23-26). The goal of the present study was to examine the particulate phase of marijuana smoke for its interaction with CYP1A1, an inducible enzyme linked to the carcinogenic effects of polycyclic aromatic hydrocarbons. We observed several striking outcomes. First, marijuana tar was more potent than tobacco tar in its ability to increase expression of CYP1A1 mRNA. Second, enhanced expression of CYP1A1 was primarily due to the presence of Δ9-THC. Finally, in contrast to its effect on transcription, Δ9-THC acted as a competitive inhibitor of CYP1A1 at the level of enzyme function.

Several reasons explain why marijuana smoking might induce higher levels of CYP1A1 than tobacco smoking. Marijuana smoke is a complex substance produced by pyrolysis of the marijuana plant Cannabis sativa. As others have reported, marijuana cigarettes produce a high yield of tar and a comparatively high yield of procarcinogenic PAHs such as benzo[a]pyrene and benz[a]anthracene (6– 8). The reason for these findings is due partly to the loose manner in which marijuana is packed into a cigarette, allowing higher temperatures and more effective pyrolysis, as well as the lack of a filter tip, permitting a greater percentage of pyrolysis products to be delivered into the smoker's mouth (27). In real life, the pulmonary deposition of marijuana tar is further magnified by the manner in which marijuana is generally smoked, employing a large puff volume, a deep inhalation, and a long breathholding time. This breathing pattern has been shown to enhance tar deposition into the lung by severalfold (27).

In addition to the PAHs that marijuana and tobacco share in common, marijuana contains another class of cyclic aromatic hydrocarbons, the cannabinoids. Cannabinoids are a structurally related group of cyclic aromatic C21 hydrocarbons unique to the marijuana plant. Although 61 different cannabinoids have been described, Δ9-THC is the predominant form in marijuana and is primarily responsible for its effects on the central nervous system (28). Our work suggests that Δ9-THC becomes highly concentrated in the tar phase of marijuana smoke, reaching levels five times higher than that present in the original plant material. As a result, a single marijuana cigarette yields milligram quantities of Δ9-THC (compared with nanogram to microgram quantities of tobacco-related PAHs). Like PAHs, cannabinoids appear capable of interacting directly with cytochrome P450s. In 1972, Witschi and Saint-Francois (29) administered large oral doses of Δ9-THC to rats (40 to 280 mg/kg) and observed a 3-fold induction of arylhydrocarbon hydroxylase (AHH) activity in lung homogenates. The P450 subfamilies responsible for this AHH activity were never identified. Studies with resected human liver have also demonstrated that cytochrome P450s metabolize Δ9-THC (30). In this case, P4502C and P4503A were identified as the primary subtypes responsible for this metabolism. However, resting human liver expresses minimal levels of P4501A1, and these studies were not designed to examine the interaction between Δ9-THC and CYP1A1.

The ability of marijuana and Δ9-THC to increase expression of CYP1A1 was clearly demonstrated in the present study using Hepa-1 cells as an in vitro model. Induction of CYP1A1 mRNA by marijuana tar reached 80% of that produced by an optimal concentration of TCDD. Adding Δ9-THC to tobacco tar produced a concentration-dependent increase in CYP1A1 mRNA and incubating Hepa-1 cells directly with Δ9-THC, in the absence of other inducing agents, produced a similar effect. A total of 2 μg/ml of Δ9-THC produced an average 2.5-fold induction of CYP1A1 mRNA, whereas 10 μg/ml produced an average 4.3-fold induction. Activation of CYP1A1 requires interaction between the inducing agent, the aryl hydrocarbon receptor, and the aryl hydrocarbon receptor nuclear translocator protein (18). Marijuana tar and purified Δ9-THC appear to operate via the same pathway as neither agent induced CYP1A1 mRNA in Hepa-1 mutants lacking these proteins. In contrast to many of the other biologic effects of Δ9-THC that are mediated by specific cannabinoid receptors (31), the effects of Δ9-THC on CYP1A1 appear to be a consequence of its interaction with the aryl hydrocarbon receptor.

The other intriguing result from this study was the relative inefficiency of marijuana in stimulating CYP1A1 enzyme function as measured by the EROD assay. Despite its superior ability to induce CYP1A1 mRNA, tar from 3.95% marijuana cigarettes stimulated only 40 to 50% as much EROD activity as did tar from either tobacco or placebo marijuana. This difference suggested that Δ9-THC might directly inhibit the CYP1A1 enzyme, an effect confirmed by evaluating Δ9-THC in kinetic studies using recombinant human CYP1A1. A variety of other molecules, including curcumin (32), diosmetin (33), and bilirubin (34), have recently been shown to interact with CYP1A1 in a similar manner. Curcumin, a polyphenolic compound found in the spice turmeric, produced up to an 8-fold induction of CYP1A1 mRNA. A competitive binding assay with radiolabeled TCDD confirmed that curcumin binds to the aryl hydrocarbon receptor and, similar to Δ9-THC, curcumin competitively inhibited CYP1A1 function in the EROD assay. These compounds, like many conventional PAHs such as benzo[a]pyrene, appear to share a common affinity for the aryl hydrocarbon receptor complex, resulting in induction, and an affinity for the active site of CYP1A1, resulting in their own metabolism and competitive inhibition of other substrates.

In summary, we found that tar from marijuana cigarettes was more potent than tobacco tar at inducing expression of CYP1A1 in Hepa-1 cells. This enhanced effect was due to Δ9-THC, which, like conventional PAHs, acted through the aryl hydrocarbon receptor complex to increase CYP1A1 mRNA. The presence of CYP1A1 and the aryl hydrocarbon receptor complex, as well as the capacity of PAHs to induce CYP1A1, are well documented in lung epithelium and in lung cancer cells (11, 12, 35, 36). Our studies therefore raise important questions about the role of marijuana smoking as a lung cancer risk factor. Induction of CYP1A1 by Δ9-THC could result in greater activation of smoke-related procarcinogens and contribute to the high rate of DNA damage and mucosal abnormalities observed in marijuana smokers (2-5). However, the capacity of Δ9-THC to competitively inhibit the CYP1A1 enzyme could moderate these consequences, decreasing the production of carcinogens. The in vivo balance between enzyme induction and competitive antagonism likely depends on many factors that were not addressed in this study. In addition, results with Hepa-1 cells may not exactly predict the regulatory effects of Δ9-THC on lung tissue. Our results support a role for Δ9-THC in promoting carcinogenesis but suggest that further testing is required in order to determine its clinical impact on lung tissue in vivo.

The authors thank I. M. Vankatesan and E. C. Ruth for their analysis of PAHs in tar extracts, T. Sarafian for his assistance with the microplate fluorimeter, and E. Minehart for technical assistance. This study was supported by grant DA03018-16 (D. P. T. and M. D. R.) from the National Institute on Drug Abuse/National Institutes of Health and grant CA28868 (O. H.) from the National Cancer Institute/National Institutes of Health, and performed with resources provided by the Jonsson Comprehensive Cancer Center/UCLA.

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Address correspondence to: Michael D. Roth, M.D., Div. of Pulmonary and Critical Care, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-1690. E-mail:

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