Beta-carotene and lung cancer*

Does beta-carotene increase, rather than decrease, human lung cancer rates? A large body of observational epidemiologic study has demonstrated that individuals who eat more fruits and vegetables rich in carotenoids and/or who have higher levels of serum betacarotene have a lower risk of cancer, particularly lung cancer. This inverse relationship has been particularly strong in lung cancer patients with a history of heavy smoking. However, there is contradictory evidence from recent human intervention studies using beta-carotene supplements (20–30 mg per day). An increase in risk of lung cancer among smokers who took beta-carotene supplements was reported in the Alpha Tocopherol, Beta-carotene Cancer Prevention (ATBC) Trial and among smokers and asbestos-exposed workers in the BetaCarotene and Retinol Efficiency Trial (CARET), but not among male physicians in the United States in the Physicians Health Study (only 11 % of whom were current smokers). Whether there is a true hazard associated with beta-carotene has been evaluated in control studies using the ferret. This animal mimics the human tissue metabolism of beta-carotene, and has been used for studies of tobacco smoking and inhalation toxicology. In the first study, ferrets were given a high-dose beta-carotene supplement equivalent to 30 mg per day in humans, and exposed cigarette smoke or both for six months. A strong proliferative response in lung tissue and squamous metaplasia were observed in all beta-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared to the control group, beta-carotene-supplemented animals (with or without smoke exposure) had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited reductions in RAR-beta gene expression (a tumor suppressor gene). Further, ferrets given a high-dose beta-carotene supplement and exposed to tobacco smoke had fourfold elevated expressions of c-jun and c-fos genes. In a second study, ferrets were given either physiologicalor pharmacologic-dose beta-carotene supplementations, which were equivalent to 6 mg vs. 30 mg per day in humans, respectively. The animals were exposed to cigarette smoke for six months. The retinoic acid concentration and RAR beta-gene expression were reduced in the lung tissues, whereas the expression of AP1, cyclin D1, and proliferative cell nuclear antigen were greater in the high-dose, beta-carotene-supplemented animals with or without smoke, as well as the smoke-exposed, low-dose, beta-carotene-supplemented animals—but not in the low-dose, beta-carotene-supplemented animals alone, as compared with the control group. Squamous metaplasia was only observed in the lung tissues of high-dose, beta-carotene exposed groups with or without smoke (but not the low-dose beta-carotene plus smoke group, the low-dose beta-carotene-supplemented group, or the control group). These data show that in contrast with the pharmacologic dose of beta-carotene, a physiologic dose of beta-carotene in smoke-exposed ferrets has no detrimental effect—and, in fact, may afford weak protection against lung damage induced by cigarette smoke. *Lecture presented at the 13th International Symposium on Carotenoids, Honolulu, Hawaii, USA, 6–11 January 2002. Other presentations are presented in this issue, pp. 1369–1477. Further studies from our laboratory have revealed an instability of the beta-carotene molecule in the lungs of cigarette smoke-exposed ferrets. Oxidized beta-carotene metabolites may play a role in lung carcinogenesis: by inducing carcinogen-bioactivating enzymes, facilitating the binding of metabolites of benz[a]pyrene to DNA, enhancing retinoic acid metabolism by P450 enzyme induction with subsequent down-regulation of RAR-beta, and acting as pro-oxidants, causing damage to DNA. Ferret studies under highly controlled experimental conditions using highand low-dose beta-carotene in the presence of alpha tocopherol and ascorbic acid (thereby stabilizing the beta-carotene molecule) showed protective effects against smoke-induced lung squamous metaplasia in ferrets. BETA-CAROTENE AND LUNG CANCER Up until the mid-1990s, there was great hope that beta-carotene would be an effective agent for the prevention of lung cancer, particularly among heavy smokers. A large body of observational epidemiologic studies had consistently demonstrated that individuals eating more fruits and vegetables, and individuals having higher serum beta-carotene levels had a lower risk of developing lung cancer, especially among smokers. But in 1994, two studies were published that showed that supplementation with betacarotene resulted in more—rather than fewer—lung cancers in high risk, smokers [1]. In the ATBC trial conducted in Finland, almost 30 000 male smokers smoking at least one pack of cigarettes per day were exposed to either a 20-mg beta-carotene supplement, a 50-mg supplement of vitamin E, the combination of both, or a placebo, and were followed for a mean of 6.1 years. Vitamin E had no effect on lung cancer incidence over this period of time, whereas high-dose beta-carotene intervention increased the incidence of lung cancers, as compared to the groups not exposed to beta-carotene. A second trial from the United States used daily 30 mg of beta-carotene combined with 25 000 units of retinol in smokers or asbestos-exposed workers who were at high risk for the development of lung cancer. Once again, it was found that beta-carotene supplementation gave rise to an increased number of lung cancers with a 20 % increase in the incidence over a four-year period [2]. A third study conducted among physicians in the United States (only 11 % of whom were smokers) showed neither a detrimental nor beneficial effect of beta-carotene supplementation at a dose of 50 mg given every other day [3]. It is interesting to note in these studies that because of different beta-carotene preparations that were used, average serum levels of beta-carotene reached 200 to 300 μg/dl in the ATBC and CARET studies, whereas in the Physicians Health Study, the average serum beta-carotene level was only 120 μg/dl [1–3]. These levels can be contrasted with those representative of the American population as found in the National Health and Nutrition Examination Survey (NHANES) study, ranging between 5 and 50 μg/dl [4]. Several questions arose due to these intervention studies. First of all, how does one explain the paradox of an apparent beneficial effect of beta-carotene against lung cancer in the observational epidemiologic studies, vs. either a detrimental or no effect in the high-dose intervention studies; and secondly, is there a true hazard associated with high doses of beta-carotene? The ferret model has been used to answer these questions, since this animal mimics human beta-carotene absorption and metabolism in tissues [5]. Moreover, this animal has been used for studies of tobacco smoking and inhalation toxicology [6,7]. Lung beta-carotene and retinol levels are similar in humans and ferrets not exposed to smoke or supplemental beta-carotene, as well as in humans and ferrets exposed to smoke and betacarotene together. These observations set the stage for an intervention study to be conducted in ferrets. In the first intervention study, ferrets were either exposed to supplemental beta-carotene, or not exposed to beta-carotene, at levels that were calculated to be equivalent to 30 mg per day in humans [8]. In each group of animals, beta-caroteneand nonbeta-carotene-supplemented, the ferrets were further divided into smoking and nonsmoking groups. Ferrets were exposed to cigarette smoke twice in the morning and twice in the afternoon, 10 cigarettes over a 30-min period each time, in a chamber connected to a smoking device. Animals that were not exposed to smoke underwent the same procedures R. M. RUSSELL © 2002 IUPAC, Pure and Applied Chemistry 74, 1461–1467 1462