Approaches to chemoprevention of lung cancer based on carcinogens in tobacco smoke.

Chemoprevention may be one way to prevent lung cancer in smokers who are motivated to quit but cannot stop. The approach to chemoprevention of lung cancer described in this article is based on an understanding of the lung carcinogens present in tobacco smoke. The available data indicate that the compounds in cigarette smoke most likely involved in the induction of lung cancer in humans are the complex of polynuclear aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P) and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). A large number of compounds are now available that inhibit lung tumorigenesis by B[a]P or NNK in rodents. Inhibition of NNK-induced lung carcinogenesis by phenethyl isothiocyanate (PEITC) and inhibition of B[a]P-induced lung carcinogenesis by benzyl isothiocyanate (BITC) are discussed as examples. Studies with PEITC in rodents clearly demonstrate that it inhibits NNK-induced lung tumorigenesis by inhibiting the metabolic activation of NNK. Similar changes appear to occur in humans according to data generated in smokers who ate watercress, a source of PEITC. It is likely that mixtures of chemopreventive agents with activity against carcinogens in tobacco smoke, such as NNK and B[a]P, will be useful in chemoprevention of lung cancer in smokers. Furthermore, there is a need to develop suppressing agents for lung cancer that might be applicable in both smokers and ex-smokers.


Introduction
Lung cancer is the leading cause of cancer death in the United States, with over 160,000 deaths expected in 1997 (1). Smoking causes at least 80% of lung cancer (2). Therefore, smoking cessation is dearly the best way to decrease incidence and mortality from the great majority of lung This paper is based on a presentation at the symposium on Mechanisms and Prevention of Environmentally Caused Cancers held 21-25 October 1995 in Santa Fe, New Mexico. Manuscript received cancer; however, smoking cessation has not been uniformly successful. Available data indicate that approximately 26% of the adult population in the United States still smokes, in spite of widespread knowledge of the associated hazards (3). Many of these people are addicted to nicotine and cannot stop smoking even after participation in smoking cessation programs and use of the nicotine patch. Chemoprevention may be a way to prevent lung cancer in those smokers who are motivated to stop yet have failed in smoking cessation Table 1. Smoking and lung cancer: causative agents.a programs. Considering the immense death toll from lung cancer, chemoprevention would make a significant impact even if it were successful in a relatively small percentage ofsmokers.
Our approach to chemoprevention of lung cancer is based on an understanding of the carcinogens in tobacco smoke. Tobacco smoke is a complex mixture of compounds and contains at least 40 known carcinogens. Carcinogens identified in cigarette smoke include polynuclear aromatic hydrocarbons (PAHs), aza-arenes, which are PAHs containing a nitrogen in the ring system; nitrosamines; aromatic amines; aldehydes; miscellaneous organic compounds such as benzene, acrylonitrile, vinyl chloride, 2-nitropropane, and ethyl carbamate; and inorganic compounds such as hydrazine and various metals (4). Among these, the PAHs and nitrosamines have in their families the strongest respiratory carcinogens, while certain aldehydes and metals are also known respiratory carcinogens. In contrast, some of the other carcinogens such as aromatic amines and benzene are associated with other cancers, such as bladder cancer and leukemia. The role of specific carcinogens of tobacco smoke in human cancers can be assessed by considering the amounts of the carcinogens in tobacco products, their target tissues and carcinogenic potency in laboratory animals, and biochemical evidence that humans and laboratory animals respond in similar ways. Likely causative agents for lung cancer are summarized in Table 1. PAHs, typified by benzo[a]pyrene (B[a]P), are formed by the incomplete combustion of tobacco during smoking. They are well-recognized carcinogens that induce tumors of the lung in laboratory animals exposed by inhalation, instillation in the trachea, or implantation in the lung (5-7  (4). This argument is bolstered by biochemical studies that have demonstrated that human lung tissue can metabolize PAHs by pathways that lead to covalent modification of DNA and by the detection of the relevant DNA adducts in lung tissue of smokers.
4-(Methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK), a nitrosamine formed from the major tobacco constituent nicotine during tobacco processing and smoking, is a powerful and organ-selective lung carcinogen in laboratory animals (8). NNK is one of a family of nicotine-derived nitrosamines that are collectively called tobacco-specific nitrosamines. Adenocarcinoma of the lung is the main type of lung cancer induced by NNK. The total amount of NNK required to produce lung cancer in rats is similar to the total amount of this compound to which a smoker would be exposed in a lifetime of smoking (9,10). These data support the role of NNK in the induction of lung cancer, particularly adenocarcinoma. Moreover, human lung tissue metabolically activates NNK, although not as efficiently as rodent lung tissue (11). DNA adducts specific to NNK and the related nitrosamine N'-nitrosonornicotine (NNN) have been detected in smokers' lungs, and metabolites of NNK are present in smokers' urine (10,12). Table 1 lists some other tobacco-smoke constituents that could be involved in lung cancer induction; the evidence suggesting a role for these compounds is weaker than that discussed above for PAHs and NNK. Polonium-210 is present in cigarette mainstream smoke and is a strong pulmonary carcinogen, inducing tumors of the lung upon inhalation in rats or on intratracheal instillation in Syrian golden hamsters (4). The significance of polonium-210 in tobacco-induced lung cancer has been questioned based on comparisons of doses experienced by smokers versus miners. It has been estimated that about 1% of the lung cancer risk associated with cigarette smoking could be ascribed to polonium-210.
Chromium, cadmium, and nickel are all present in cigarette smoke (4). Calcium chromate is carcinogenic in rats, inducing lung tumors after instillation. Cadmium chloride aerosols produce adenocarcinoma and squamous cell carcinoma in rats.
Nickel subsulfide yields lung cancer in rats upon inhalation. Because levels of exposure to chromium, cadmium, and nickel compounds in cigarette smoke may be comparable to those of some PAHs, these metal ions may play some role in lung cancer induction.
Inhalation studies of formaldehyde and acetaldehyde have demonstrated that they are respir.atory carcinogens in the rat, inducing mainly nasal cavity tumors (4). There may be a direct effect of these compounds on the lung upon inhalation in tobacco smoke. Although they are weak respiratory carcinogens, the levels of formaldehyde and acetaldehyde in cigarette smoke are at least 1000 times greater than those of PAHs and nitrosamines.
Cigarette smoke contains some stable free radicals and is known to induce oxidative damage (4,13). Products that result from oxidative damage to both lipids and DNA have been detected in smokers and their levels are higher than in nonsmokers (14,15). Although the direct role of such products in carcinogenesis is unclear, 8-oxoguanine, a DNA adduct detected at elevated levels in smokers, has miscoding properties associated with the cancer induction process.
Collectively, the available evidence favors PAHs and NNK as important compounds responsible for lung cancer induction in smokers. Their role in lung cancer is consistent with results of analyses of mutations in the p53 and ras genes from human lung tumors. These analyses have demonstrated the presence of a large number of G -* T transversions and G -4 A transitions in these genes, which is consistent with mutational spectra expected from PAHs and NNK (16)(17)(18)(19)(20).
With each cigarette, the smoker is exposed to PAHs and NNK. As illustrated in Figure 1, these carcinogens undergo metabolic activation to DNA adducts. If these adducts persist unrepaired during DNA replication, miscoding can occur, leading to permanent mutations in critical genes such as p53 and ras, which are likely to be important in the lung cancer induction process. Blocking any one of these steps would decrease the probability of lung cancer development. Our strategy has been to block the metabolic activation step. This approach will be discussed in this report, using phenethyl isothiocyanate (PEITC), a chemopreventive agent against NNK-induced lung tumorigenesis, as one example, and benzyl isothiocyanate (BITC), an inhibitor of B[a]P-induced lung tumorigenesis, as another. Other known inhibitors of NNK-and B[a]Pinduced lung tumorigenesis will also be reviewed. We emphasize that the approach discussed here is only one example of strategies that can be employed for chemoprevention of lung cancer.

Inhibition of NNK-induced Lung Tumorigenesis by PEITC
A naturally occurring isothiocyanate, PEITC (PhCH2CH2N=C=S), is found as its glucosinolate conjugate gluconasturtiin in several vegetables including watercress. PEITC is released from watercress upon chewing by the action of myrosinase, a thioglucosidase present in the plant (21,22). Consumption of approximately 50 g of watercress releases 10 to 15 mg of PEITC (22). When PEITC was added to NIH-07, an open formula rodent diet, at a concentration of 498 ppm (3 pmol/g diet) before and during treatment of male F344 rats with NNK, it caused a significant and selective 50% reduction in the incidence of adenocarcinoma of the lung (23) ( Table 2). There were no toxic effects of PEITC at this dose. A single dose of 5 pmol of PEITC administered to A/J mice 2 hr prior to treatment with 10 pmol of NNK resulted in a significant 62% reduction in lung tumor multiplicity (24). Other studies using multiple doses of PEITC have shown similar results in A/J mice (25,26). Thus PEITC has been firmly established as an effective inhibitor of lung tumorigenesis induced by NNK in both rats and mice.
An overview of the major metabolic activation and detoxification pathways of NNK is illustrated in Figure 2 (27). In laboratory animals and humans, NNK is rapidly converted to 4-(methylnitrosamino)-l -   by carbonyl reductase enzymes. Also a potent pulmonary carcinogen, NNAL is partially converted to its diastereomeric glucuronides, [4-(methylnitrosamino)-1-(3pyridyl)but-1 -yl] -3-O-D-glucosiduronic acid (NNAL-gluc). These glucuronides are likely detoxification' products of NNK. Pyridine N-oxidation of NNK and NNAL gives the corresponding N-oxides, which are detoxification products. Metabolic activation of NNK proceeds by ax-hydroxylation of the methylene and methyl carbons producing unstable intermediates 1 and 2. These spontaneously decompose with formation of aldehydes and the electrophilic diazohydroxides 4 and 5. Diazohydroxide 4 methylates DNA of NNK target tissues, producing permanent mutations, mainly of the G -A type. Diazohydroxide 5 alkylates DNA producing both G -> A and G -* T mutations. It also reacts with hemoglobin to form ester adducts. Hydrolysis of DNA or hemoglobin obtained from animals treated with NNK or from smokers produces 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) (6), which is a biomarker of the metabolic activation of NNK (28). Smokers' urine contains quantifiable amounts of NNAL and NNAL-gluc as biomarkers.
The mechanism of NNK carcinogenesis inhibition by PEITC has been examined. Initial studies demonstrated that PEITC inhibited the metabolic activation of NNK to electrophiles, which methylate and pyridyloxobutylate pulmonary DNA in rats (23). Subsequently, detailed investigations of the effects of PEITC on NNK metabolism in mouse and rat liver and lung, as well as studies of other enzyme activities, have clearly demonstrated that the inhibitory effect of PEITC on NNK carcinogenesis is due mainly to inhibition of NNK metabolic activation to methylating and pyridyloxobutylating electrophiles (29,30). In rats treated with PEITC by gavage or by addition to the diet, a persistent inhibition of metabolic activation of NNK is observed in lung microsomes, which results from inhibition of cytochrome P450 enzymes. In contrast, a persistent inhibition in liver microsomes is not observed. Experiments in vitro have shown that PEITC is a competitive inhibitor of NNK metabolic activation in rat lung microsomes, with the concentration that inhibits 50% ranging from 150 to 210 nM, and in explants of rat lung (30,31).
The effects of PEITC on NNK metabolism have also been examined in vivo. In these experiments, the goal was to determine whether the observed inhibition of tumorigenesis was due to specific inhibition of metabolic activation of NNK, or  whether treatment with PEITC might have caused a change in distribution of NNK resulting in diminished amounts of the carcinogen reaching extrahepatic tissues. In experiments carried out using a protocol essentially identical to that employed in the carcinogenicity study described above, it was shown that the levels of NNK and its primary metabolite NNAL were not markedly different in tissues of PEITC treated and control rats. However, the data dearly indicated a decrease in the levels of NNK metabolic activation in the PEITC treated rats in almost all tissues examined (32). The effects of chronic PEITC treatment on hemoglobin adducts and urinary metabolites of NNK have been examined in rats. Results of the urinary metabolite analyses are summarized in Table 3. Chronic PEITC treatment caused significant 4to 6-fold increases in the levels of NNAL and NNAL-gluc in urine; this most likely results from a decrease in metabolic activation of NNK since hemoglobin adducts of NNK also decreased (data not shown). The ratio of NNAL-gluc to NNAL, a potential biomarker of NNK detoxification, increased upon PEITC treatment. Collectively, the results of these studies clearly show that PEITC exerts a specific inhibitory effect on the metabolic activation of NNK without causing any apparent toxic effects in rats.  Table 5. Although this table includes only defined compounds, certain mixtures such as green tea, black tea, snuff extract, orange oil, and NIH-07 diet also inhibit NNK-induced tumorigenesis; however, the responsible compounds have not been identified (43,49,(52)(53)(54).

Inhibition of B[a]P-induced
More than 25 inhibitors of NNK-induced lung tumorigenesis are known (Table 5). Isothiocyanates appear to be the strongest inhibitors according to presently available data. Other inhibitors include natural products such as sinigrin, indole-3carbinol, D-limonene, diallyl sulfide, epigallocatechin-3-gallate, and ellagic acid; antioxidants such as butylated hydroxyanisole; and drugs such as sulindac, ibuprofen, and piroxicam. Other compounds, such as the ipomeanol analogue 7-hydroxy-1-phenyl-1 -octanone, the P450 suicide inhibitor 4-phenyl-1-butyne, and the organoselenium compound 1,4phenylenebis (methylene) selenocyanate, have been developed based on mechanistic considerations and by analogy to other chemopreventive agents. All compounds tested to date have shown activity when administered before or concurrently with NNK. There are no reported suppressors of NNK-induced lung tumorigenesis, e.g., agents that are effective when administered only after NNK treatment. Compounds tested as inhibitors of B[a]P-induced lung tumorigenesis are listed in Table 6. At least 20 inhibitors have been identified. Some of the inhibitory compounds are the same as those that inhibit NNK-induced lung tumorigenesis; these include butylated hydroxyanisole, ellagic acid, and diallyl sulfide. As in the case of NNK, the inhibitors include natural products, drugs, and antioxidants. Only three compoundsmyo-inositol, dexamethasone, and butylated hydroxyanisole-have been shown to inhibit B[a]P-induced lung tumorigenesis when administered after B[a]P.
Neither 5-carotene nor vitamin A has shown reproducible inhibitory effects on Environmental Health Perspectives * Vol 105, Supplement 4 * June 1997

Effects of Watercress Consumption on NNK Metabolism in Smokers
The studies described above demonstrate that PEITC inhibits NNK-induced lung tumorigenesis in rats and mice by inhibiting its metabolic activation. We wanted to determine whether similar effects would occur in smokers. The source of PEITC used in this study was watercress (Nasturtium officinale), which contains substantial amounts of gluconasturtiin, the glucosinolate precursor of PEITC (22). Eleven smokers maintained constant smoking habits and avoided cruciferous vegetables and other sources of isothiocyanates throughout the study (82). They donated 24-hr urine samples on 3 consecutive days (baseline period). After 1 to 3 days, they began the watercress consumption period, 3 days during which they consumed 2 oz (56.8 g) of watercress at each meal and donated 24-hr urine samples on each day. One and two weeks later they again donated 24-hr urine samples on 2 to 3 consecutive days (follow-up periods). The samples were analyzed for two metabolites of NNK; NNAL and NNAL-gluc, as well as N-acetyl-S-(N-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), a metabolite of PEITC. Minimum exposure to PEITC during the watercress consumption period averaged 19 to 38 mg per day. Seven of the eleven subjects had increased levels of urinary NNAL plus NNAL-gluc on days 2 and 3 of the watercress consumption period, compared to the baseline period. Overall, the increase in urinary NNAL plus NNAL-gluc in this period was significant [mean ± SD, 0.924 ± 1.12 nmol/24 hr (33.5%), p< 0.0l]. Urinary levels of NNAL plus NNAL-gluc returned to near baseline levels in the follow-up periods. The percent increase in urinary NNAL plus NNAL-gluc during days 2 and 3 of the watercress consumption period correlated with PEITC intake during this period as measured by total urinary PEITC-NAC (r= 0.62, p= 0.04). The results of this study support our hypothesis that PEITC inhibits the oxidative metabolism of NNK in humans, as seen in rodents, and support further development of PEITC and other compounds as chemopreventive agents against lung cancer.

Summary
The research described in this paper conclusively demonstrates that a large number of compounds can inhibit the lung tumorigenicity of the important tobaccosmoke pulmonary carcinogens NNK and B[a]P in rodent models. Among these, isothiocyanates have been investigated extensively with respect to efficacy and mechanisms of inhibition. One of the most thoroughly studied isothiocyanates, PEITC, inhibits lung tumor induction by NNK in rodents, and apparently in smokers, by inhibiting the metabolic activation of NNK. Considering the large number of chemopreventive agents that are effective against lung tumorigenesis by either NNK or B[a]P, it seems likely that appropriately designed mixtures of agents should be effective against both NNK   Most of the inhibitors discussed in this paper must be present at the time of carcinogen administration to be effective, since in many cases they are inhibitors of metabolic activation or enhancers of carcinogen detoxification. This raises questions about their potential efficacy, depending on how tobacco carcinogenesis is viewed. Two models of tobacco carcinogenesis are outlined in Figure 3.
In the classical sequential model, initiation by compounds such as NNK or B[a]P is followed by promotion and progression. Tobacco smoke contains tumor promoters, and the partial reversibility of lung cancer risk associated with smoking cessation is consistent with the reversibility of promotion. However, this model may be somewhat unrealistic. Smokers are simultaneously exposed to carcinogens, promoters, cocarcinogens, and toxic compounds with every cigarette. Therefore, the chronic exposure model may be more realistic and is consistent with the concept that multiple genetic changes are involved in the carcinogenic process. These changes will be decreased by favorable alteration of several steps involved in carcinogenrelated gene changes ( Figure 1). Protocols should be developed to identify agents that would be effective against all known steps in the lung-cancer induction process; typical animal data are summarized in Tables 5 and 6, and by Moon et al. (84). There is a clear need for identification of suppressing agents (compounds active after carcinogen exposure) in future studies, in part because there are a large number of ex-smokers who would benefit from chemoprevention. Such compounds would inhibit nongenotoxic aspects of lung carcinogenesis. Only a few suppressing agents are known at present that are active against lung tumors induced by tobacco smoke carcinogens. The further development of such agents should be a major research priority. NOTE ADDED IN PROOF: Further studies by Wattenberg and Estensen demonstrated the efficacy of myo-inositol and dexamethasone as suppressors of pulmonary tumorigenesis (85). The inhibitory effect of PEITC against rat lung tumorigenesis induced by NNK was demonstrated in two further studies: Chung et al. (86) and Hecht et al. (87).