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Stress, hormonal changes, alcohol, food constituents and drugs: factors that advance the incidence of tobacco smoke-related cancer?
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Stress, hormonal changes, alcohol, food constituents and drugs: factors that advance the incidence of tobacco smoke-related cancer? Edmund Maser The genotoxicity of the most potent carcinogen in cigarette smoke [4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK)] is dependent on the relationship between its activation by cytochrome P450 enzymes and its detoxification by carbonyl reduction to NNK alcohol (NNAL) followed by glucuronidation. Recently, ‘11b-hydroxysteroid dehydrogenase’ (11b-HSD 1) was identified to be responsible for NNK carbonyl reduction. It is now speculated that differences in tissue expression of 11b-HSD 1, as well as genetic polymorphisms, may have profound influences on the organospecificity and potency of NNK-induced cancerogenesis. Moreover, endogenous and exogenous substrates or inhibitors of 11b-HSD 1 may shift the NNK/NNAL equilibrium and favour NNK toxification in a variety of physiological and therapeutic situations. These issues are discussed here by Edmund Maser, who also describes how recent observations could provide the experimental base for epidemiological or clinical studies, which focus on polymorphisms in 11b-HSD 1 enzyme expression, as well as on implications of exposure to 11b-HSD 1 modulators and concurrent smoking.
E. Maser, Associate Professor, Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-vonFrisch-Strasse 1, D-35033 Marburg/Lahn, Germany.
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In the past few years the health effects of active and passive smoking have become a matter of debate in scientific and popular areas, as several epidemiological studies have suggested that exposure to environmental tobacco smoke could be a possible risk factor for lung cancer. Recent estimates indicate that cigarette smoking causes approximately 80–90% of lung cancer in the United States1 and that smoking during pregnancy2, as well as passive exposure of children to cigarette smoke3, may increase the risk of cancer in children and young adults. Tobacco smoke is a complex mixture of more than 4000 substances4, among which at least 40 have been identified as carcinogens, tumour initiators or promoters
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T in laboratory animals1. Tobacco-specific nitrosamines, which are formed by nitrosation of nicotine during maturation and air-curing of tobacco, represent the most abundant and potent carcinogens in tobacco products and tobacco smoke5, and convincing evidence for their role as causative factors in several adult human cancers has been discussed6–8.
NNK is one of the strongest nitrosamine carcinogens in tobacco products The most potent carcinogenic agent contained in cigarette smoke is the nitrosamine 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK)9, which is thought to be an important etiological factor in tobacco smoke-related human cancer10. In laboratory animals, NNK has a remarkable specificity for the lung 5,11, which is independent of the route of administration, for example, whether it is applied topically to the skin, taken orally, or administered by intraperitoneal, subcutaneous (s.c.) or intravenous (i.v.) injection. NNK concentrations vary between 1–100 mg per cigarette12,13, and the fact that sidestream smoke often contains higher amounts of NNK than mainstream smoke is significant with respect to the question of cancer induction by passive exposure to tobacco smoke5. The presence of NNK metabolites in the urine of nonsmokers who were exposed to sidestream cigarette smoke14 provides evidence for the link between exposure to environmental tobacco smoke and the risk of lung cancer. Another consideration is that of an individual’s capacity to metabolically activate or detoxify these substances. NNK requires metabolic activation in order to exert its carcinogenic effect. This activation involves the a-hydroxylation of either the a-methylene or methyl carbon of NNK, resulting in the formation of electrophiles which can methylate and pyridyloxobutylate, respectively, haemoglobin and DNA (for review see Ref. 7). Reduction of the carbonyl group of NNK to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a very efficient pathway of NNK metabolism in animals and human tissues15–22, and initiates NNK detoxification by providing the butanoyl-hydroxy function necessary for glucuronidation and excretion. Minor detoxification pathways include pyridine-Noxidation of both NNK and NNAL (Refs 7, 23).
Carbonyl reduction to NNAL constitutes the major metabolic route of NNK in smokers It is well established that carbonyl reduction to NNAL constitutes a major metabolic route of NNK in laboratory animals and in many isolated human tissues in vitro15–22, accounting for, under certain conditions, 75% of the sum of all NNK metabolites formed17. Adams and co-workers24 have observed that in hamster, mouse, rat and baboon the in vivo equilibrium between NNK and NNAL in plasma strongly favours the reduced form NNAL. The ratio of NNAL to NNK was greatest in hamster (90:1) 1 h after i.v. administration of NNK.
Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0165 – 6147/97/$17.00 PII: S0165-6147(97)01090-0
V Moreover, the actual rate of NNAL formation may be underestimated, because NNAL has been shown to become conjugated with glucuronic acid to a significant extent in vivo, which is then excreted as NNAL-glucuronide in urine and bile21,25–30. In a study with two patas monkeys approximately 30% of the urinary metabolites resulted from carbonyl reduction of NNK followed by glucuronidation or N-oxidation31. The first quantitation of metabolites of tobaccospecific nitrosamines in human urine was described by Carmella et al.28 Mean levels of NNAL and NNALglucuronide were 0.67 and 3.16 mg 24 h–1, respectively. In four of the smokers, information on the levels of NNK in their cigarettes and the number of cigarettes smoked in the previous 24 h had been available. From these data, it was estimated that 39–101% of the dose was excreted as NNAL and NNAL-glucuronide28. In another study by Carmella et al.32 products of NNK carbonyl reduction were determined in the urine of 61 smokers. Amounts of NNAL ranged from 0.08 to 4.89 pmol mg–1 creatinine, and NNAL-glucuronide levels varied from 0.16 to 19.0 pmol mg–1 creatinine. From these results it appeared that intra-individual differences were generally small, whereas interindividual differences were large. Very high levels of NNAL and NNAL-glucuronide were observed in the urine of people from Sudan who used an oral tobacco product – toombak – known to contain unusually high levels of NNK (Ref. 33). Interestingly, NNAL and NNAL-glucuronide have also been identified in the urine of nonsmokers who were exposed to environmental tobacco smoke14. This indicates that nonsmokers take up and metabolize NNK and provides support for the proposal that environmental tobacco smoke can cause lung cancer. However, there are apparently wide variations among individuals in their ability to metabolize NNK by carbonyl reduction; these differences might be due to genetic polymorphisms or environmental factors. It is these individual differences which are of greatest interest, as they may be linked to individual susceptibility to cancer.
The NNK/NNAL equilibrium is likely to play a role in the organospecificity and cancerogenic potency of NNK Animal carcinogenicity studies have shown that NNK has a high degree of organoselectivity for induction of lung tumours, independent of the route of administration5. It is hypothesized that, depending on the extent of the competing pathways – a-hydroxylation versus carbonyl reduction followed by glucuronidation – the susceptibility of a tissue to NNK-induced tumourigenesis may be influenced. For example, the existence of low Km (10–20 M) cytochrome P450 isozymes for NNK bioactivation in the lung has been suggested to partially account for the higher susceptibility of the lung than the liver to NNK-induced tumourigenesis19. The lung is
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composed of a variety of different cell types, and it is postulated that tumours arise preferentially from Clara cells (nonciliated, nonmucous secretory cells in the surface epithelium of the pulmonary airways)34, which are known to have high levels of a-hydroxylation and low levels of NNK carbonyl reduction35. In the liver itself NNK has been found to be predominantly metabolized via carbonyl reduction and glucuronidation15,19,21, thus possibly explaining the lower tumour incidence of the liver after NNK exposure. Moreover, of all tissues studied the liver expresses the highest levels of NNK carbonyl reduction, and hepatic clearance has been expected to reduce the exposure of pulmonary tissue to NNK. The absence of NNAL formation in the nasal microsomes19,36–38 might also be a causative factor in the organospecific incidence of tumours there, a fact which could arise from a lack of carbonyl reductase in this tissue in contrast to high levels of activating P450 isozymes19,36,37. In addition, the different extent of NNK carbonyl reduction by organs involved in the first-pass metabolism after s.c. (lung) or oral (small intestine, liver and lung) administration provides an explanation for the different tumour spectrum observed after NNK administration by either s.c. injection (lung, liver, nose) or in the drinking water (lung, liver, pancreas)5,9.
11b-Hydroxysteroid dehydrogenase 1 acts as carbonyl reductase for NNK Several studies have shown that cytochrome P450 enzymes are involved in the a-carbon hydroxylation and pyridine N-oxidation of NNK (Refs 7, 39). However, although constituting the major pathway of NNK metabolism in several tissues15–22, the enzyme that catalyses the carbonyl reduction of NNK to NNAL in microsomal fractions has awaited its characterization since 1980 (Ref. 40). Recent investigations finally revealed the microsomal enzyme that catalyses the carbonyl reduction of NNK as being identical to 11b-hydroxysteroid dehydrogenase 1 (11b-HSD 1) (EC 1.1.1.146)41, the physiological function of which is the oxidoreduction of glucocorticoids. Hence, 11b-HSD 1 can be considered to be the central initiating enzyme of the final detoxification of NNK because, by ketone reduction of NNK, it provides the butanoyl-hydroxy function necessary for glucuronidation and excretion. This liver type 11b-HSD is a pluripotent enzyme in that it is capable of catalysing the reduction of several nonsteroidal xenobiotic carbonyl compounds, in addition to its specificity towards its physiological steroid substrates42,43. The involvement of purified mouse liver 11b-HSD 1 in NNK carbonyl reduction was confirmed on the basis of a homogeneously purified enzyme preparation as well as on the inhibition characteristics of polyclonal antibodies specific for the 11b-HSD 1 protein. In addition, the diagnostic 11b-HSD inhibitor glycyrrhetinic acid decreased NNK carbonyl reduction with a Ki value of 1.5 mM. Moreover, endogenous glucocorticoids also
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very efficiently inhibited NNK carbonyl reduction by purified 11b-HSD 1 with Ki values in the range of 16 mM and 30 mM. The relatively high Km value for NNK carbonyl reduction of purified mouse liver 11b-HSD 1 (1.75 mM) may, on the one hand, be due to the dehydrogenase nature of this enzyme (reaction reversibility) and the absence of glucuronyl transferase, i.e. the lack of successive glucuronidation in the homogenous enzyme preparation which results in an accumulation of the reaction product NNAL. On the other hand, the Km value for purified mouse liver 11b-HSD 1 is within the same range as that observed in microsomes of several other animal tissues. In addition, it should be noted that NNK metabolism in humans is generally quantitatively different from that observed in rodents in that levels of carbonyl reduction to NNAL in humans are usually greater whereas levels of a-hydroxylation are lower than in rodents44, a fact which may result from genuine rodent–human differences. For example, in human lung microsomes, more than 90% of the initial NNK undergo carbonyl reduction, even though the reaction exhibits a Km of ‘only’ 0.57 mM (Ref. 39). However, once they have been characterized in a purified state, human isoforms of 11b-HSD are likely to express lower Km values for NNK carbonyl reduction.
Physiological role of 11b-HSD 11b-HSD is a microsomal enzyme responsible for the interconversion of active 11b-hydroxyglucocorticoids to inactive 11-oxo forms45. By this action 11b-HSD protects the nonselective mineralocorticoid receptor from exposure to cortisol or corticosterone, and modulates access of glucocorticoids to glucocorticoid receptors46,47. It is generally accepted that there are at least two isoforms of 11b-HSD in mammals, a ubiquitous low affinity NADP+ (nicotinamide adenine dinucleotide phosphate)dependent enzyme (11b-HSD 1)43,48 with the liver being the major site of expression49–51, and a tissue specific, high affinity NAD+-dependent form (11b-HSD 2) which is mainly expressed in placenta and aldosterone target tissues such as the kidney and colon52,53. Defective 11b-dehydrogenase activity has been documented in syndromes characterized by cortisoldependent mineralocorticoid excess and hypertension, e.g. the congenital syndrome of mineralocorticoid excess54 and the syndrome of ectopic secretion of adrenocorticotrophic hormone (ACTH)55. Enzyme activity is also impaired in some patients with essential hypertension56,57, hyperthyroidism58, chronic renal failure59 and alcohol excess60. Several endo- and xenobiotics have been found to inhibit 11b-HSD in vivo and in vitro; these include bile acids, cholesterol61,62, progestagens63 or hydroxyprogesterones64, glycyrrhetinic acid65, carbenoxolone66, naringenin67,68, dexamethasone69, furosemide67,70, gossypol71,72, metyrapone19,73, ketoconazole74 and ethanol75,76. Pathophysiological states caused by idiopathic impairment of 11b-HSD expression or druginduced deficiency of enzyme activity are generally 272
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characterized by an abnormally high ratio of cortisol to cortisone metabolites in the urine.
Are 11b-HSD 1 polymorphisms related to lung cancer susceptibility? A number of studies have demonstrated a familial component to lung cancer risk, but association studies on polymorphic genes coding for cytochrome P450 enzymes or glutathione transferases that may play a role in the activation and detoxification of carcinogens in tobacco smoke have often yielded conflicting results (see Ref. 77 for review). However, multiple forms of human glucuronyl transferase have been characterized, and numerous variables including cigarette smoking, diet, disease state, drug therapy, ethnicity, age, genetic factors and hormonal conditions can influence drug glucuronidation in humans78. It has been suggested that polymorphisms in 11b-HSD activity are a risk factor for the development of essential hypertension79. It may be of interest to isolate polymorphic markers linked to 11b-HSD, in order to perform correlation analyses among affected lung cancer patients. As mentioned above, two isozymes of 11b-HSD have already been described. The human gene for 11b-HSD 1 is located on chromosome 1 (Ref. 50), and mutations in this gene have not been detected until recently. In contrast, the gene for human 11b-HSD 2 has been localized on chromosome 16, and for this isozyme mutations have been found in eight of nine kindreds affected with the syndrome of apparent mineralocorticoid excess80. Unfortunately, all attempts failed to purify 11b-HSD 2 in a functional state, such that it is not yet possible to ascribe NNK carbonyl reducing activity to this isozyme. However, both 11b-HSD forms should be considered when searching for the existence of genetic polymorphisms in 11b-HSD expression.
Possible consequences of concurrent exposure to tobacco smoke and 11b-HSD 1 modulators The fact that a hydroxysteroid dehydrogenase that is involved in the metabolism of endogenous steroids is also responsible for the metabolism of a cancerogenic nitrosamine, is of particular interest in view of its role in regulating the actions of its physiological steroid substrates as well as in participating in the elimination of xenobiotics81.
Glucocorticoids, sex hormones and cholic acids It is clear that in response to ACTH secretion from the anterior pituitary gland during stress, elevated levels of endogenous glucocorticoids occupy the 11b-HSD 1 enzyme. This would result in a shift of the NNK/NNAL equilibrium towards NNK, which, upon metabolic activation via a-carbon hydroxylation, exerts its cancerogenic effect. The same consequences would be expected upon administration of exogenous glucocorticoids during glucocorticoid therapy and concurrent exposure to tobacco smoke.
V High levels of oestradiol and progesterone during pregnancy would also affect NNK detoxification, as both sex hormones have been shown to inhibit 11b-HSD 1. In vivo, oestradiol treatment increases renal and placental 11b-HSD 2 activity, whereas 11b-HSD 1 gene expression and activity are nearly abolished upon oestradiol treatment82,83. Early studies by Lax et al.84 already demonstrated that hepatic 11b-HSD levels are almost completely suppressed by oestradiol in both male and female rats. Progesterone has been reported to attenuate placental 11b-HSD 2 (Refs 82, 85). In other studies, progesterones as well as its 11-hydroxy derivatives were shown to be potent inhibitors of both 11b-HSD 1 and 11b-HSD 2 at 1027 and 1026M (Refs 63, 64, 86, 87). The use of oral contraceptives concurrently with smoking may have similar relevance. It is also suggested that elevated levels of cholesterol, lanosterol and particularly bile acids, which were shown to be potent inhibitors of both 11b-HSD isoforms in vitro61,62, affect the activity of 11b-HSD 1, and thus promote tobacco smoke-related cancerogenesis. In cholestatic states, normal bile acid values can increase by a factor of 100 and may reach millimolar ranges, which can clearly impair 11b-HSD activity. Several reports indicate an abnormal metabolic transformation of cortisol in cholestatic states88,89, a situation which, particularly in smokers with alcoholic liver cirrhosis, would exacerbate NNK activation.
Food constituents and alcohol Cholesterol and lanosterol have been shown to inhibit 11b-HSD 1 in vitro62, providing an interesting link to the proposal that tobacco smoke-related lung cancer has a dietary fat determinant (see Ref. 90 for review). However, it seems reasonable to identify the type of dietary fat that might be expected to change the delivery of NNK metabolites to the lung. Several other dietary compounds are known to inhibit 11b-HSD activities in vitro and in vivo, such as glycyrrhetinic acid65,70, the principal constituent of licorice, and naringenin67,68, a flavonoid with high concentrations in grapefruit juice, as well as some other flavonoids known to occur in fruit (kaempferol, apigenin, quercetin, hesperetin)68. In addition to its established role as a potent and competitive inhibitor of 11b-HSD 1 and 2 activities, glycyrrhetinic acid exerts a pretranslational inhibition of 11b-HSD 1 and 2 expression both in vitro and in vivo91. The popularity of licorice flavouring in candy and in other products such as chewing tobacco persists to this day, as do the problems in electrolyte and blood pressure homeostasis that occur due to the hypermineralocorticoid effects of glycyrrhetinic acid caused by 11b-HSD inhibition and subsequent rise in cortisol levels65. A similar inhibition of 11b-HSD, followed by a decrease in the urinary cortisone/cortisol ratio has been described after ingestion of grapefruit juice, which contains high levels of naringenin68. Furthermore, flavonoids are sold as dietary supplements in tablet form in health-food stores
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and drug stores. It may be hypothesized that smokers consuming licorice and/or grapefruit juice, or taking large quantities of flavonoids, may have a higher risk for the development of lung cancer, due to inhibition of 11b-HSD 1 and impaired NNAL formation. Interestingly, the fact that chronic alcohol consumption greatly enhances the carcinogenic response to NNK (Refs 92–94) might now be explained on the basis that ethanol has been shown to be a potent inhibitor of 11b-HSDs (Refs 75, 76).
Drugs and pharmacotherapy Several pharmacologically relevant substances interfere with 11b-HSD activity and might therefore enhance the lung cancer risk of smokers or nonsmokers exposed to environmental tobacco smoke. Glycyrrhetinic acid has long been known to promote the healing of ulcers and is currently used as a medication and healing agent in Asian and Oriental countries. Carbenoxolone is the hemisuccinate of glycyrrhetinic acid, and has been widely used as a successful treatment for peptic ulcer. However, both have long been associated with mineralocorticoid-like effects, now known to be exerted through inhibition of 11b-HSDs (Refs 66, 91). IC50 values of both compounds for 11b-HSD 1 range from ~10–9 to 10–7 M (Refs 64, 87). Furosemide inhibits 11b-HSD 1 and 2 in vitro and in vivo (Ki values ~10–5 M)67,70, and ethacrynic acid showed IC50 values similar to those of glycyrrhetinic acid67, such that a pharmacotherapy with these diuretics may favour NNK activation. Gossypol, an oral contraceptive for men, has been shown to affect liver 11b-HSD 1 activity with an IC50 value of 0.7 3 10–6 M (Ref. 71). Furthermore, it is conceivable that treatment with exogenous cortisol or cortisone during glucocorticoid therapy dramatically interferes with 11b-HSD 1 activity, thus impairing NNK detoxification. Interestingly, chronic administration of dexamethasone does not only inhibit 11b-HSD 1 enzyme activity, but even correlates with a downregulation of 11b-HSD 1 mRNA expression69. Evidence that modulators of 11b-HSD 1 activity could impair NNK carbonyl reduction and shift the NNK/NNAL equilibrium towards the generation of alkylating species has been provided by studies with metyrapone, a well-known inhibitor of adrenal steroid biosynthesis which is clinically used in the treatment of Cushing’s syndrome. On the one hand, metyrapone turned out to be a nonsteroidic substrate of 11b-HSD 1 (Ref. 42); on the other hand, metyrapone was shown to significantly impair NNK carbonyl reduction19,73. From these facts, the proposal remains that pharmacotherapeutic regimens with the aforementioned drugs, including the antifungal ketoconazol74, favour cytochrome P450-mediated NNK toxification by competing for 11b-HSD 1. The concentrations of drug used in the above studies to show a significant inhibition of 11b-HSD 1 were of the same magnitude as those found in humans after pharmacotherapy. However, research is
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smoking excretion TSNA
alkylating species
11β-HSD 1
P450 NNK
stress cholic acids pregnancy
UDPGT NNAL
NNAL glucuronide
glucocorticoids furosemide carbenoxolone contraceptives glycyrrhetinic acid naringenin alcohol
Fig. 1. The metaabolic activation and inactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK carbonyl reduction by 11b-hydroxysteroid dehydrogenase (11b-HSD 1) may be influenced by a variety of endogenous (left-hand column) and exogenous (right-hand column) enzyme modulators. NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; TSNA, tobacco-specific nitrosamines; UDPGT, uridine diphosphate glucuronosyl transferase.
still needed to verify if 11b-HSD 1 inhibition in these cases is sufficient to promote tobacco smoke-related lung cancer, at least in smokers.
Concluding remarks and future directions From the evidence discussed, it seems that the genotoxicity of NNK is dependent on the relationship between its metabolic activation by cytochrome P450 enzymes and its final detoxification by carbonyl reduction followed by glucuronidation. Therefore, the position of the NNK/NNAL equilibrium in various tissues and species is likely to play a role in the organospecificity and carcinogenic potency of NNK. This equilibrium, on the one hand, is dependent on the levels of expression of 11b-HSD/NNK carbonyl reductase, which may result from environmental or genetic (polymorphism) factors. On the other hand, it may be influenced directly by competition of endogenous or exogenous 11b-HSD/NNK carbonyl reductase substrates or inhibitors. It is proposed that endogenous steroids (glucocorticoids, oestrogen, progesterone, cholesterol, bile acids), exogenous steroids (glucocorticoids, dexamethasone, oral contraceptives), drugs (carbenoxolone, furosemide, ethacrynic acid, gossypol, ketoconazole, metyrapone) and dietary compounds (naringenin, glycyrrhetinic acid, flavonoids) may modulate NNK-induced carcinogenicity by shifting the NNK/NNAL equilibrium towards NNK and favouring cytochrome P450-mediated NNK toxification (Fig. 1). The discovery that a hydroxysteroid dehydrogenase initiates detoxification of the tobacco-specific nitrosamine NNK is of great interest and provides the basis for further research. The influence of 11b-HSD 1 modulators on the metabolism of NNK in animal and human tissues, or cells in culture, needs to be directly investigated. Studies on NNK-related tumour incidence in 274
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rodents and primates being simultaneously exposed to 11b- HSD 1 modulators, stress or pregnancy may elucidate these interactions in vivo. Molecular epidemiology studies, by evaluating relations between tobacco smokerelated human cancers and levels of 11b-HSD 1 expression in respective tumour biopsies, may help to understand the significance in humans. The incidence of tobacco smoke-related tumours in population collectives like pregnant women, patients under specific pharmacological regimens, women taking oral contraceptives, humans addicted to special forms of diet, or people suffering from stress, could be compared to that of respective control collectives. In conclusion, such findings may have potentially important implications for active and passive smokers, who could be expressing low levels of 11b-HSD/NNK carbonyl reductase and/or concurrently being exposed to 11b-HSD 1 modulators. Selected references 1 International Agency for Research on Cancer (1986) IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 38, 127–163 2 Stjernfeldt, M., Berglund, K., Lindsten, J. and Ludvigsson, J. (1986) Lancet 1 1350–1352 3 Sandler, D. P., Everson, R. B., Wilcox, A. J. and Browder, J. P. (1985) Am. J. Public Health 75, 487–492 4 Roberts, D. L. (1988) Rec. Adv. Tobacco Sci. 14, 49–81 5 Hecht, S. S. and Hoffmann, D. (1988) Carcinogenesis 9, 875–884 6 Hoffmann, D. and Hecht, S. S. (1985) Cancer Res. 45, 935–944 7 Hecht, S. S. (1994) Drug Metab. Rev. 26, 373–390 8 Preston-Martin, S. (1991) Crit. Rev. Toxicol. 21, 295–298 9 Rivenson, A., Hoffmann, D., Prokopczyk, B., Amin, S. and Hecht, S. S. (1988) Cancer Res. 48, 6912–6917 10 Hecht, S. S. and Hoffmann, D. (1989) Cancer Surv. 8, 273–294 11 Castonguay, A. et al. (1983) Cancer Res. 43, 1223–1229 12 International Agency for Research on Cancer (1985) IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 37, 37–136 13 Tricker, A. R., Ditrich, C. and Preussmann, R. (1991) Carcinogenesis 12, 257–261 14 Hecht, S. S. et al. (1993) New Engl. J. Med. 329, 1543–1546 15 Liu, L. L., Alaoui-Jamali, M. A., el Alami, N. and Castonguay, A. (1990) Cancer Res. 50, 1810–1816
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60 Stewart, P. M., Burra, P., Shackleton, C. H., Sheppard, M. C. and Elias, E. (1993) J. Clin. Endocrinol. Metab. 76, 748–751 61 Perschel, F. H., Bühler, H. and Hierholzer, K. (1991) Pflügers Arch. 418, 538–543 62 Bühler, H., Perschel, F. H., Fitzner, R. and Hierholzer, K. (1994) Steroids 59, 131–135 63 Latif, S. A., Hartmann, L. R., Souness, G. W. and Morris, D. J. (1994) Steroids 59, 352–356 64 Souness, G. W., Latif, S. A., Laurenzo, J. L. and Morris, D. J. (1995) Endocrinology 136, 1809–1812 65 Stewart, P. M. et al. (1987) Lancet ii, 821–824 66 Stewart, P. M., Wallace, A. M., Atherden, S. M., Shearing, C. H. and Edwards, C. R. (1990) Clin. Sci. 78, 49–54 67 Zhang, Y. D., Lorenzo, B. and Reidenberg, M. M. (1994) J. Steroid Biochem. Mol. Biol. 49, 81–85 68 Lee, Y. S., Lorenzo, B. J., Koufis, T. and Reidenberg, M. M. (1996) Clin. Pharmacol. Ther. 59, 62–71 69 Brem, A. S., Bina, R. B., Klinger, J. R., King, T. and Werner, J. C. (1995) Proc. Soc. Exp. Biol. Med. 209, 146–151 70 Escher, G., Meyer, K. V., Vishwanath, B. S., Frey, B. M. and Frey, F. J. (1995) Endocrinology 136, 1759–1765 71 Song, D., Lorenzo, B. and Reidenberg, M. M. (1992) J. Lab. Clin. Med. 120, 792–797 72 Sang, G. W., Lorenzo, B. and Reidenberg, M. M. (1991) J. Steroid Biochem. Mol. Biol. 39, 169–176 73 Castonguay, A. and Rossignol, G. (1992) Toxicol. Vitro 6, 397–404 74 Bühler, H., Lichtenstein, I., Perschel, F. H., Siebe, H. and Hierholzer, K. (1991) Pflügers Arch. 418, R118 75 Riddle, M. C. and McDaniel, P. A. (1993) Metabolism 42, 1370–1374 76 Valentino, R. et al. (1995) Horm. Res. 43, 176–180 77 Schwartz, A. G., Yang, P. and Swanson, G. M. (1996) Am. J. Epidemiol. 144, 554–562 78 Miners, J. O. and Mackenzie, P. I. (1991) Pharmacol. Ther. 51, 347–369 79 Edwards, C. R., Benediktsson, R., Lindsay, R. S. and Seckl, J. R. (1993) Lancet 341, 355–357 80 Mune, T., Rogerson, F. M., Nikkila, H., Agarwal, A. K. and White, P. C. (1995) Nat. Genet. 10, 394–399 81 Maser, E. (1995) Biochem. Pharmacol. 49, 421–440 82 Baggia, S., Albrecht, E. D. and Pepe, G. J. (1990) Endocrinology 126, 2742–2748 83 Low, S. C. et al. (1993) J. Endocrinol. 139, 27–35 84 Lax, E. R., Ghraf, R. and Schriefers, H. (1978) Acta Endocrinol. 89, 352–358 85 Pepe, G. J., Waddell, B. J., Stahl, S. J. and Albrecht, E. D. (1988) Endocrinology 122, 78–83 86 Latif, S. A., Sheff, M. F., Ribeiro, C. E. and Morris, D. J. (1997) Steroids 62, 230–237 87 Bujalska, I., Shimojo, M., Howie, A. and Stewart, P. M. (1997) Steroids 62, 77–82 88 Zumoff, B., Bradlow, H. L., Gallagher, T. F. and Hellman, L. (1971) J. Clin. Endocrinol. 32, 36–41 89 Gerdes, H., Riemenschneider, H. and Littmann, K. P. (1973) Acta Endocrinol. (Suppl.) 173, 10 90 El–Bayoumy, K. et al. (1996) Nutr. Cancer 26, 1–10 91 Whorwood, C. B., Sheppard, M. C. and Stewart, P. M. (1993) Endocrinology 132, 2287–2292 92 McCoy, G. D. and Wynder, E. L. (1979) Cancer Res. 39, 2844–2850 93 Tuyns, F. J. (1979) Cancer Res. 39, 2840–2843 94 Driver, H. E. and Swann, P. E. (1987) Anticancer Res. 7, 309–320
Technical Tips Online http://www.elsevier.com/locate/tto ...an essential resource for molecular biologists
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Acknowledgements The work in the author’s laboratory was supported by grants from the A. and U. Kulemann-Stiftung (Marburg) and the P. E. Kempkes-Stiftung (Marburg).
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Stress, hormonal changes, alcohol, food constituents and drugs: factors that advance the incidence of tobacco smoke-related cancer? Edmund Maser The genotoxicity of the most potent carcinogen in cigarette smoke [4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK)] is dependent on the relationship between its activation by cytochrome P450 enzymes and its detoxification by carbonyl reduction to NNK alcohol (NNAL) followed by glucuronidation. Recently, ‘11b-hydroxysteroid dehydrogenase’ (11b-HSD 1) was identified to be responsible for NNK carbonyl reduction. It is now speculated that differences in tissue expression of 11b-HSD 1, as well as genetic polymorphisms, may have profound influences on the organospecificity and potency of NNK-induced cancerogenesis. Moreover, endogenous and exogenous substrates or inhibitors of 11b-HSD 1 may shift the NNK/NNAL equilibrium and favour NNK toxification in a variety of physiological and therapeutic situations. These issues are discussed here by Edmund Maser, who also describes how recent observations could provide the experimental base for epidemiological or clinical studies, which focus on polymorphisms in 11b-HSD 1 enzyme expression, as well as on implications of exposure to 11b-HSD 1 modulators and concurrent smoking.
E. Maser, Associate Professor, Department of Pharmacology and Toxicology, School of Medicine, Philipps-University of Marburg, Karl-vonFrisch-Strasse 1, D-35033 Marburg/Lahn, Germany.
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In the past few years the health effects of active and passive smoking have become a matter of debate in scientific and popular areas, as several epidemiological studies have suggested that exposure to environmental tobacco smoke could be a possible risk factor for lung cancer. Recent estimates indicate that cigarette smoking causes approximately 80–90% of lung cancer in the United States1 and that smoking during pregnancy2, as well as passive exposure of children to cigarette smoke3, may increase the risk of cancer in children and young adults. Tobacco smoke is a complex mixture of more than 4000 substances4, among which at least 40 have been identified as carcinogens, tumour initiators or promoters
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T in laboratory animals1. Tobacco-specific nitrosamines, which are formed by nitrosation of nicotine during maturation and air-curing of tobacco, represent the most abundant and potent carcinogens in tobacco products and tobacco smoke5, and convincing evidence for their role as causative factors in several adult human cancers has been discussed6–8.
NNK is one of the strongest nitrosamine carcinogens in tobacco products The most potent carcinogenic agent contained in cigarette smoke is the nitrosamine 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK)9, which is thought to be an important etiological factor in tobacco smoke-related human cancer10. In laboratory animals, NNK has a remarkable specificity for the lung 5,11, which is independent of the route of administration, for example, whether it is applied topically to the skin, taken orally, or administered by intraperitoneal, subcutaneous (s.c.) or intravenous (i.v.) injection. NNK concentrations vary between 1–100 mg per cigarette12,13, and the fact that sidestream smoke often contains higher amounts of NNK than mainstream smoke is significant with respect to the question of cancer induction by passive exposure to tobacco smoke5. The presence of NNK metabolites in the urine of nonsmokers who were exposed to sidestream cigarette smoke14 provides evidence for the link between exposure to environmental tobacco smoke and the risk of lung cancer. Another consideration is that of an individual’s capacity to metabolically activate or detoxify these substances. NNK requires metabolic activation in order to exert its carcinogenic effect. This activation involves the a-hydroxylation of either the a-methylene or methyl carbon of NNK, resulting in the formation of electrophiles which can methylate and pyridyloxobutylate, respectively, haemoglobin and DNA (for review see Ref. 7). Reduction of the carbonyl group of NNK to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a very efficient pathway of NNK metabolism in animals and human tissues15–22, and initiates NNK detoxification by providing the butanoyl-hydroxy function necessary for glucuronidation and excretion. Minor detoxification pathways include pyridine-Noxidation of both NNK and NNAL (Refs 7, 23).
Carbonyl reduction to NNAL constitutes the major metabolic route of NNK in smokers It is well established that carbonyl reduction to NNAL constitutes a major metabolic route of NNK in laboratory animals and in many isolated human tissues in vitro15–22, accounting for, under certain conditions, 75% of the sum of all NNK metabolites formed17. Adams and co-workers24 have observed that in hamster, mouse, rat and baboon the in vivo equilibrium between NNK and NNAL in plasma strongly favours the reduced form NNAL. The ratio of NNAL to NNK was greatest in hamster (90:1) 1 h after i.v. administration of NNK.
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V Moreover, the actual rate of NNAL formation may be underestimated, because NNAL has been shown to become conjugated with glucuronic acid to a significant extent in vivo, which is then excreted as NNAL-glucuronide in urine and bile21,25–30. In a study with two patas monkeys approximately 30% of the urinary metabolites resulted from carbonyl reduction of NNK followed by glucuronidation or N-oxidation31. The first quantitation of metabolites of tobaccospecific nitrosamines in human urine was described by Carmella et al.28 Mean levels of NNAL and NNALglucuronide were 0.67 and 3.16 mg 24 h–1, respectively. In four of the smokers, information on the levels of NNK in their cigarettes and the number of cigarettes smoked in the previous 24 h had been available. From these data, it was estimated that 39–101% of the dose was excreted as NNAL and NNAL-glucuronide28. In another study by Carmella et al.32 products of NNK carbonyl reduction were determined in the urine of 61 smokers. Amounts of NNAL ranged from 0.08 to 4.89 pmol mg–1 creatinine, and NNAL-glucuronide levels varied from 0.16 to 19.0 pmol mg–1 creatinine. From these results it appeared that intra-individual differences were generally small, whereas interindividual differences were large. Very high levels of NNAL and NNAL-glucuronide were observed in the urine of people from Sudan who used an oral tobacco product – toombak – known to contain unusually high levels of NNK (Ref. 33). Interestingly, NNAL and NNAL-glucuronide have also been identified in the urine of nonsmokers who were exposed to environmental tobacco smoke14. This indicates that nonsmokers take up and metabolize NNK and provides support for the proposal that environmental tobacco smoke can cause lung cancer. However, there are apparently wide variations among individuals in their ability to metabolize NNK by carbonyl reduction; these differences might be due to genetic polymorphisms or environmental factors. It is these individual differences which are of greatest interest, as they may be linked to individual susceptibility to cancer.
The NNK/NNAL equilibrium is likely to play a role in the organospecificity and cancerogenic potency of NNK Animal carcinogenicity studies have shown that NNK has a high degree of organoselectivity for induction of lung tumours, independent of the route of administration5. It is hypothesized that, depending on the extent of the competing pathways – a-hydroxylation versus carbonyl reduction followed by glucuronidation – the susceptibility of a tissue to NNK-induced tumourigenesis may be influenced. For example, the existence of low Km (10–20 M) cytochrome P450 isozymes for NNK bioactivation in the lung has been suggested to partially account for the higher susceptibility of the lung than the liver to NNK-induced tumourigenesis19. The lung is
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composed of a variety of different cell types, and it is postulated that tumours arise preferentially from Clara cells (nonciliated, nonmucous secretory cells in the surface epithelium of the pulmonary airways)34, which are known to have high levels of a-hydroxylation and low levels of NNK carbonyl reduction35. In the liver itself NNK has been found to be predominantly metabolized via carbonyl reduction and glucuronidation15,19,21, thus possibly explaining the lower tumour incidence of the liver after NNK exposure. Moreover, of all tissues studied the liver expresses the highest levels of NNK carbonyl reduction, and hepatic clearance has been expected to reduce the exposure of pulmonary tissue to NNK. The absence of NNAL formation in the nasal microsomes19,36–38 might also be a causative factor in the organospecific incidence of tumours there, a fact which could arise from a lack of carbonyl reductase in this tissue in contrast to high levels of activating P450 isozymes19,36,37. In addition, the different extent of NNK carbonyl reduction by organs involved in the first-pass metabolism after s.c. (lung) or oral (small intestine, liver and lung) administration provides an explanation for the different tumour spectrum observed after NNK administration by either s.c. injection (lung, liver, nose) or in the drinking water (lung, liver, pancreas)5,9.
11b-Hydroxysteroid dehydrogenase 1 acts as carbonyl reductase for NNK Several studies have shown that cytochrome P450 enzymes are involved in the a-carbon hydroxylation and pyridine N-oxidation of NNK (Refs 7, 39). However, although constituting the major pathway of NNK metabolism in several tissues15–22, the enzyme that catalyses the carbonyl reduction of NNK to NNAL in microsomal fractions has awaited its characterization since 1980 (Ref. 40). Recent investigations finally revealed the microsomal enzyme that catalyses the carbonyl reduction of NNK as being identical to 11b-hydroxysteroid dehydrogenase 1 (11b-HSD 1) (EC 1.1.1.146)41, the physiological function of which is the oxidoreduction of glucocorticoids. Hence, 11b-HSD 1 can be considered to be the central initiating enzyme of the final detoxification of NNK because, by ketone reduction of NNK, it provides the butanoyl-hydroxy function necessary for glucuronidation and excretion. This liver type 11b-HSD is a pluripotent enzyme in that it is capable of catalysing the reduction of several nonsteroidal xenobiotic carbonyl compounds, in addition to its specificity towards its physiological steroid substrates42,43. The involvement of purified mouse liver 11b-HSD 1 in NNK carbonyl reduction was confirmed on the basis of a homogeneously purified enzyme preparation as well as on the inhibition characteristics of polyclonal antibodies specific for the 11b-HSD 1 protein. In addition, the diagnostic 11b-HSD inhibitor glycyrrhetinic acid decreased NNK carbonyl reduction with a Ki value of 1.5 mM. Moreover, endogenous glucocorticoids also
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very efficiently inhibited NNK carbonyl reduction by purified 11b-HSD 1 with Ki values in the range of 16 mM and 30 mM. The relatively high Km value for NNK carbonyl reduction of purified mouse liver 11b-HSD 1 (1.75 mM) may, on the one hand, be due to the dehydrogenase nature of this enzyme (reaction reversibility) and the absence of glucuronyl transferase, i.e. the lack of successive glucuronidation in the homogenous enzyme preparation which results in an accumulation of the reaction product NNAL. On the other hand, the Km value for purified mouse liver 11b-HSD 1 is within the same range as that observed in microsomes of several other animal tissues. In addition, it should be noted that NNK metabolism in humans is generally quantitatively different from that observed in rodents in that levels of carbonyl reduction to NNAL in humans are usually greater whereas levels of a-hydroxylation are lower than in rodents44, a fact which may result from genuine rodent–human differences. For example, in human lung microsomes, more than 90% of the initial NNK undergo carbonyl reduction, even though the reaction exhibits a Km of ‘only’ 0.57 mM (Ref. 39). However, once they have been characterized in a purified state, human isoforms of 11b-HSD are likely to express lower Km values for NNK carbonyl reduction.
Physiological role of 11b-HSD 11b-HSD is a microsomal enzyme responsible for the interconversion of active 11b-hydroxyglucocorticoids to inactive 11-oxo forms45. By this action 11b-HSD protects the nonselective mineralocorticoid receptor from exposure to cortisol or corticosterone, and modulates access of glucocorticoids to glucocorticoid receptors46,47. It is generally accepted that there are at least two isoforms of 11b-HSD in mammals, a ubiquitous low affinity NADP+ (nicotinamide adenine dinucleotide phosphate)dependent enzyme (11b-HSD 1)43,48 with the liver being the major site of expression49–51, and a tissue specific, high affinity NAD+-dependent form (11b-HSD 2) which is mainly expressed in placenta and aldosterone target tissues such as the kidney and colon52,53. Defective 11b-dehydrogenase activity has been documented in syndromes characterized by cortisoldependent mineralocorticoid excess and hypertension, e.g. the congenital syndrome of mineralocorticoid excess54 and the syndrome of ectopic secretion of adrenocorticotrophic hormone (ACTH)55. Enzyme activity is also impaired in some patients with essential hypertension56,57, hyperthyroidism58, chronic renal failure59 and alcohol excess60. Several endo- and xenobiotics have been found to inhibit 11b-HSD in vivo and in vitro; these include bile acids, cholesterol61,62, progestagens63 or hydroxyprogesterones64, glycyrrhetinic acid65, carbenoxolone66, naringenin67,68, dexamethasone69, furosemide67,70, gossypol71,72, metyrapone19,73, ketoconazole74 and ethanol75,76. Pathophysiological states caused by idiopathic impairment of 11b-HSD expression or druginduced deficiency of enzyme activity are generally 272
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characterized by an abnormally high ratio of cortisol to cortisone metabolites in the urine.
Are 11b-HSD 1 polymorphisms related to lung cancer susceptibility? A number of studies have demonstrated a familial component to lung cancer risk, but association studies on polymorphic genes coding for cytochrome P450 enzymes or glutathione transferases that may play a role in the activation and detoxification of carcinogens in tobacco smoke have often yielded conflicting results (see Ref. 77 for review). However, multiple forms of human glucuronyl transferase have been characterized, and numerous variables including cigarette smoking, diet, disease state, drug therapy, ethnicity, age, genetic factors and hormonal conditions can influence drug glucuronidation in humans78. It has been suggested that polymorphisms in 11b-HSD activity are a risk factor for the development of essential hypertension79. It may be of interest to isolate polymorphic markers linked to 11b-HSD, in order to perform correlation analyses among affected lung cancer patients. As mentioned above, two isozymes of 11b-HSD have already been described. The human gene for 11b-HSD 1 is located on chromosome 1 (Ref. 50), and mutations in this gene have not been detected until recently. In contrast, the gene for human 11b-HSD 2 has been localized on chromosome 16, and for this isozyme mutations have been found in eight of nine kindreds affected with the syndrome of apparent mineralocorticoid excess80. Unfortunately, all attempts failed to purify 11b-HSD 2 in a functional state, such that it is not yet possible to ascribe NNK carbonyl reducing activity to this isozyme. However, both 11b-HSD forms should be considered when searching for the existence of genetic polymorphisms in 11b-HSD expression.
Possible consequences of concurrent exposure to tobacco smoke and 11b-HSD 1 modulators The fact that a hydroxysteroid dehydrogenase that is involved in the metabolism of endogenous steroids is also responsible for the metabolism of a cancerogenic nitrosamine, is of particular interest in view of its role in regulating the actions of its physiological steroid substrates as well as in participating in the elimination of xenobiotics81.
Glucocorticoids, sex hormones and cholic acids It is clear that in response to ACTH secretion from the anterior pituitary gland during stress, elevated levels of endogenous glucocorticoids occupy the 11b-HSD 1 enzyme. This would result in a shift of the NNK/NNAL equilibrium towards NNK, which, upon metabolic activation via a-carbon hydroxylation, exerts its cancerogenic effect. The same consequences would be expected upon administration of exogenous glucocorticoids during glucocorticoid therapy and concurrent exposure to tobacco smoke.
V High levels of oestradiol and progesterone during pregnancy would also affect NNK detoxification, as both sex hormones have been shown to inhibit 11b-HSD 1. In vivo, oestradiol treatment increases renal and placental 11b-HSD 2 activity, whereas 11b-HSD 1 gene expression and activity are nearly abolished upon oestradiol treatment82,83. Early studies by Lax et al.84 already demonstrated that hepatic 11b-HSD levels are almost completely suppressed by oestradiol in both male and female rats. Progesterone has been reported to attenuate placental 11b-HSD 2 (Refs 82, 85). In other studies, progesterones as well as its 11-hydroxy derivatives were shown to be potent inhibitors of both 11b-HSD 1 and 11b-HSD 2 at 1027 and 1026M (Refs 63, 64, 86, 87). The use of oral contraceptives concurrently with smoking may have similar relevance. It is also suggested that elevated levels of cholesterol, lanosterol and particularly bile acids, which were shown to be potent inhibitors of both 11b-HSD isoforms in vitro61,62, affect the activity of 11b-HSD 1, and thus promote tobacco smoke-related cancerogenesis. In cholestatic states, normal bile acid values can increase by a factor of 100 and may reach millimolar ranges, which can clearly impair 11b-HSD activity. Several reports indicate an abnormal metabolic transformation of cortisol in cholestatic states88,89, a situation which, particularly in smokers with alcoholic liver cirrhosis, would exacerbate NNK activation.
Food constituents and alcohol Cholesterol and lanosterol have been shown to inhibit 11b-HSD 1 in vitro62, providing an interesting link to the proposal that tobacco smoke-related lung cancer has a dietary fat determinant (see Ref. 90 for review). However, it seems reasonable to identify the type of dietary fat that might be expected to change the delivery of NNK metabolites to the lung. Several other dietary compounds are known to inhibit 11b-HSD activities in vitro and in vivo, such as glycyrrhetinic acid65,70, the principal constituent of licorice, and naringenin67,68, a flavonoid with high concentrations in grapefruit juice, as well as some other flavonoids known to occur in fruit (kaempferol, apigenin, quercetin, hesperetin)68. In addition to its established role as a potent and competitive inhibitor of 11b-HSD 1 and 2 activities, glycyrrhetinic acid exerts a pretranslational inhibition of 11b-HSD 1 and 2 expression both in vitro and in vivo91. The popularity of licorice flavouring in candy and in other products such as chewing tobacco persists to this day, as do the problems in electrolyte and blood pressure homeostasis that occur due to the hypermineralocorticoid effects of glycyrrhetinic acid caused by 11b-HSD inhibition and subsequent rise in cortisol levels65. A similar inhibition of 11b-HSD, followed by a decrease in the urinary cortisone/cortisol ratio has been described after ingestion of grapefruit juice, which contains high levels of naringenin68. Furthermore, flavonoids are sold as dietary supplements in tablet form in health-food stores
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and drug stores. It may be hypothesized that smokers consuming licorice and/or grapefruit juice, or taking large quantities of flavonoids, may have a higher risk for the development of lung cancer, due to inhibition of 11b-HSD 1 and impaired NNAL formation. Interestingly, the fact that chronic alcohol consumption greatly enhances the carcinogenic response to NNK (Refs 92–94) might now be explained on the basis that ethanol has been shown to be a potent inhibitor of 11b-HSDs (Refs 75, 76).
Drugs and pharmacotherapy Several pharmacologically relevant substances interfere with 11b-HSD activity and might therefore enhance the lung cancer risk of smokers or nonsmokers exposed to environmental tobacco smoke. Glycyrrhetinic acid has long been known to promote the healing of ulcers and is currently used as a medication and healing agent in Asian and Oriental countries. Carbenoxolone is the hemisuccinate of glycyrrhetinic acid, and has been widely used as a successful treatment for peptic ulcer. However, both have long been associated with mineralocorticoid-like effects, now known to be exerted through inhibition of 11b-HSDs (Refs 66, 91). IC50 values of both compounds for 11b-HSD 1 range from ~10–9 to 10–7 M (Refs 64, 87). Furosemide inhibits 11b-HSD 1 and 2 in vitro and in vivo (Ki values ~10–5 M)67,70, and ethacrynic acid showed IC50 values similar to those of glycyrrhetinic acid67, such that a pharmacotherapy with these diuretics may favour NNK activation. Gossypol, an oral contraceptive for men, has been shown to affect liver 11b-HSD 1 activity with an IC50 value of 0.7 3 10–6 M (Ref. 71). Furthermore, it is conceivable that treatment with exogenous cortisol or cortisone during glucocorticoid therapy dramatically interferes with 11b-HSD 1 activity, thus impairing NNK detoxification. Interestingly, chronic administration of dexamethasone does not only inhibit 11b-HSD 1 enzyme activity, but even correlates with a downregulation of 11b-HSD 1 mRNA expression69. Evidence that modulators of 11b-HSD 1 activity could impair NNK carbonyl reduction and shift the NNK/NNAL equilibrium towards the generation of alkylating species has been provided by studies with metyrapone, a well-known inhibitor of adrenal steroid biosynthesis which is clinically used in the treatment of Cushing’s syndrome. On the one hand, metyrapone turned out to be a nonsteroidic substrate of 11b-HSD 1 (Ref. 42); on the other hand, metyrapone was shown to significantly impair NNK carbonyl reduction19,73. From these facts, the proposal remains that pharmacotherapeutic regimens with the aforementioned drugs, including the antifungal ketoconazol74, favour cytochrome P450-mediated NNK toxification by competing for 11b-HSD 1. The concentrations of drug used in the above studies to show a significant inhibition of 11b-HSD 1 were of the same magnitude as those found in humans after pharmacotherapy. However, research is
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smoking excretion TSNA
alkylating species
11β-HSD 1
P450 NNK
stress cholic acids pregnancy
UDPGT NNAL
NNAL glucuronide
glucocorticoids furosemide carbenoxolone contraceptives glycyrrhetinic acid naringenin alcohol
Fig. 1. The metaabolic activation and inactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). NNK carbonyl reduction by 11b-hydroxysteroid dehydrogenase (11b-HSD 1) may be influenced by a variety of endogenous (left-hand column) and exogenous (right-hand column) enzyme modulators. NNAL, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol; TSNA, tobacco-specific nitrosamines; UDPGT, uridine diphosphate glucuronosyl transferase.
still needed to verify if 11b-HSD 1 inhibition in these cases is sufficient to promote tobacco smoke-related lung cancer, at least in smokers.
Concluding remarks and future directions From the evidence discussed, it seems that the genotoxicity of NNK is dependent on the relationship between its metabolic activation by cytochrome P450 enzymes and its final detoxification by carbonyl reduction followed by glucuronidation. Therefore, the position of the NNK/NNAL equilibrium in various tissues and species is likely to play a role in the organospecificity and carcinogenic potency of NNK. This equilibrium, on the one hand, is dependent on the levels of expression of 11b-HSD/NNK carbonyl reductase, which may result from environmental or genetic (polymorphism) factors. On the other hand, it may be influenced directly by competition of endogenous or exogenous 11b-HSD/NNK carbonyl reductase substrates or inhibitors. It is proposed that endogenous steroids (glucocorticoids, oestrogen, progesterone, cholesterol, bile acids), exogenous steroids (glucocorticoids, dexamethasone, oral contraceptives), drugs (carbenoxolone, furosemide, ethacrynic acid, gossypol, ketoconazole, metyrapone) and dietary compounds (naringenin, glycyrrhetinic acid, flavonoids) may modulate NNK-induced carcinogenicity by shifting the NNK/NNAL equilibrium towards NNK and favouring cytochrome P450-mediated NNK toxification (Fig. 1). The discovery that a hydroxysteroid dehydrogenase initiates detoxification of the tobacco-specific nitrosamine NNK is of great interest and provides the basis for further research. The influence of 11b-HSD 1 modulators on the metabolism of NNK in animal and human tissues, or cells in culture, needs to be directly investigated. Studies on NNK-related tumour incidence in 274
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rodents and primates being simultaneously exposed to 11b- HSD 1 modulators, stress or pregnancy may elucidate these interactions in vivo. Molecular epidemiology studies, by evaluating relations between tobacco smokerelated human cancers and levels of 11b-HSD 1 expression in respective tumour biopsies, may help to understand the significance in humans. The incidence of tobacco smoke-related tumours in population collectives like pregnant women, patients under specific pharmacological regimens, women taking oral contraceptives, humans addicted to special forms of diet, or people suffering from stress, could be compared to that of respective control collectives. In conclusion, such findings may have potentially important implications for active and passive smokers, who could be expressing low levels of 11b-HSD/NNK carbonyl reductase and/or concurrently being exposed to 11b-HSD 1 modulators. Selected references 1 International Agency for Research on Cancer (1986) IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 38, 127–163 2 Stjernfeldt, M., Berglund, K., Lindsten, J. and Ludvigsson, J. (1986) Lancet 1 1350–1352 3 Sandler, D. P., Everson, R. B., Wilcox, A. J. and Browder, J. P. (1985) Am. J. Public Health 75, 487–492 4 Roberts, D. L. (1988) Rec. Adv. Tobacco Sci. 14, 49–81 5 Hecht, S. S. and Hoffmann, D. (1988) Carcinogenesis 9, 875–884 6 Hoffmann, D. and Hecht, S. S. (1985) Cancer Res. 45, 935–944 7 Hecht, S. S. (1994) Drug Metab. Rev. 26, 373–390 8 Preston-Martin, S. (1991) Crit. Rev. Toxicol. 21, 295–298 9 Rivenson, A., Hoffmann, D., Prokopczyk, B., Amin, S. and Hecht, S. S. (1988) Cancer Res. 48, 6912–6917 10 Hecht, S. S. and Hoffmann, D. (1989) Cancer Surv. 8, 273–294 11 Castonguay, A. et al. (1983) Cancer Res. 43, 1223–1229 12 International Agency for Research on Cancer (1985) IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 37, 37–136 13 Tricker, A. R., Ditrich, C. and Preussmann, R. (1991) Carcinogenesis 12, 257–261 14 Hecht, S. S. et al. (1993) New Engl. J. Med. 329, 1543–1546 15 Liu, L. L., Alaoui-Jamali, M. A., el Alami, N. and Castonguay, A. (1990) Cancer Res. 50, 1810–1816
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TiPS – August 1997 (Vol. 18)
Acknowledgements The work in the author’s laboratory was supported by grants from the A. and U. Kulemann-Stiftung (Marburg) and the P. E. Kempkes-Stiftung (Marburg).
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