Cancer Letters, 1(1976)189--196 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
189
POLYCYCLIC AROMATIC HYDROCARBONS IN HUMAN BRONCHIAL CARCINOMA
RENE TOMINGAS, FRIEDRICH POTT and WALTER DEHNEN Medizinisches Institut ft~r Lufthygiene und Silikoseforschung an der Universit~t DUsseldorf, Gurlittstrasse 53, D-4000 DUsseldorf (G.F.R.)
(Received 19 August 1975) (Revised version received 12 September 1975)
SUMMARY A study was made to determine whether and to what e x t e n t polycyclic aromatic h y d r o c a r b o n s (PAH) are present in human bronchial carcinoma. T w e n t y - f o u r carcinomas, obtained from surgical operations and autopsies, were examined. The samples were tested for 12 PAH; these were determined by direct fluorescence analysis on thin-layer plates. Only 4 of the 12 PAH were detected in the cancerous tissue: benzo(a)pyrene, fluoranthene, benzo(ghi)perylene and perylene. Benzo(a)pyrene was found in all carcinomas. The reasons for increased concentration of the detected PAH in cancerous tissue are discussed with respect to deposition and elimination of inhaled particles as well as the metabolism of these compounds.
INTRODUCTION Many investigators have examined the lungs of deceased humans for polycyclic aromatic hydrocarbons (PAH), especially b e n z o ( a ) p y r e n e [6--10, 16]. With the exception of the finding of Mallet and Heros-Dekeierel [ 1 0 ] , their results were practically identical in that in most cases no b e n z o ( a ) p y r e n e was found; a few did contain small amounts, a b o u t 1 pg in the total lung. In all these cases the material examined was n o t altered by neoplasm. On the other hand a considerable a m o u n t of b e n z o ( a ) p y r e n e was found in a bronchial carcinoma [15]. The purpose o f this investigation was to study several examples of such cancerous tissue in order to elucidate the nature o f their PAH-content. The analysed tissue samples were obtained from surgical operations and autopsies. MATERIALS AND METHODS T w e n t y - f o u r broncial carcinomas were examined. T w e n t y - o n e were surgical
190
preparations; 3 carcinomas were obtained from autopsies. Some data concerning age, sex, occupation and smoking habits taken from case reports are listed in Table 1. Parts of lungs separated from the carcinoma tissue served as blanks. Surgery of the lungs with the thermocauter was carried o u t in t w o cases; the carcinoma tissue was n o t touched by this instrument. The determination of the PAH was made b y direct fluorescence analysis on thin-layer plates. The method has been described for benzo(a)pyrene [11]. After treating the sample with hot 4N potassium hydroxide and extractions of the solution with cyclohexane, the extracts were combined and concentrated. A first purification of the PAH was achieved b y column chromatography on silica gel. Separation of the PAH was done on Sephadex LH 20 with propanol-2 as eluent. The fractions were concentrated to 0.5 ml, and 50--100 pl were taken for thin-layer chromatography. In a few cases, when the concentration was t o o low, the whole fraction volume was applied. The analysis included 12 PAH: pyrene, fluoranthene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, TABLE 1 A G E , SEX, O C C U P A T I O N A N D S M O K I N G H A B I T O F T H E P A T I E N T S W H O S E L U N G TISSUE WAS A N A L Y Z E D No.
Age
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 31 32 33
72 66 59 55 66 55 52 50 62 56 51 64 60 61 49 48 60 55 51 53 53 62 60 58
m m m m m m m m m m m m m m m m m m m m m m f m
Occupation
Smoking habit cigarettes/day
salesman employee craftsman w o r k e r in c h e m i c a l p l a n t forestry officer automobile mechanic engine-driver teacher railway a d m i n i s t r a t i o n o f f i c e r crane operator
20 + 0 20 + + 25 10 25 +
employee turner gardener w o r k e r in l a c q u e r p l a n t baker mason salesman mechanic
50 + 20 0 20 20 30 20
+ Smoker, n u m b e r of cigarettes u n k n o w n
191 perylene, dibenz(a,h)anthracene, benzo(ghi)perylene and coronene. The PAH were identified by fluorescence spectra taken from the spots on the thin-layer plates. The detection limits were determined by applying standard solutions in decreasing concentrations on thin-layer plates and were calculated according to the formula given by Doerffel [5]. Strictly, a detection limit refers to one c o m p o u n d only, since different substances in equal concentration and under optimized conditions will give different fluorescence intensity. RESULTS The results are shown in Tables 2 and 3. Three points seem to be noteworthy: (1) From the 12 PAH tested for in the cancerous tissue, only 4 could be detected: benzo(a)pyrene, benzo(b)fluoranthene, fluoranthene and perylene. The content of the remaining 8 PAH was below the detection limit. (2) Benzo(a)pyrene was found in all carcinoma tissue. Fluoranthene and benzo(b)fluoranthene were sometimes present, as well as perylene, although the latter was not detected in autopsy material. (3) The PAH-content in the cancerous tissue was not significantly different with respect to fluoranthene and benzo(b)fluoranthene, but a large difference was found for benzo(a)pyrene as demonstrated by the U-test. Even with the median values this was significantly higher: 3.5 pg benzo(a)pryene/g tissue in the carcinoma and 0.09 pg in the tumour-free tissue, a ratio of 38:1. DISCUSSION The results demonstrated increased concentration of benzo(a)pyrene in areas of the lung, which proved to be bronchial carcinoma by histological diagnosis. In non-cancerous tissue only small amounts of benzo(a)pyrene were found. When discussing the reasons for an enrichment of PAH in bronchial carcinomas, attention should be focused upon all alterations of deposition, elimination and metabolism of PAH. The deposition of inhaled particles in the area of a carcinoma may be increased by an alteration of bronchial air flow; a disturbance of the elimination of particles may not only cause a prolonged retention time of the deposited particles there but also, and more importantly, may result in a damning up of the substances moving from the terminal air passages toward the trachea. In addition to this incommodation of the 'mechanical' lung clearance, a disturbance of the resorption, as well as the metabolism, of foreign substances must be taken into account. Alveolar macrophages and some other lung cells (e.g., epithelial cells of the bronchus) are able to transform PAH by mixed-function-oxidases into products which are then easily removed. It has been known for some time that macrophages of guinea pigs, in vitro, will dissolve and metabolize PAH from soot particles [12,13]. This enzyme activity can be induced by benzo(a)pyrene and by other PAH as well [3,4]. Even human alveolar macrophages have oxidases which can be induced. Thus, macrophages of smokers have been
192
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189
POLYCYCLIC AROMATIC HYDROCARBONS IN HUMAN BRONCHIAL CARCINOMA
RENE TOMINGAS, FRIEDRICH POTT and WALTER DEHNEN Medizinisches Institut ft~r Lufthygiene und Silikoseforschung an der Universit~t DUsseldorf, Gurlittstrasse 53, D-4000 DUsseldorf (G.F.R.)
(Received 19 August 1975) (Revised version received 12 September 1975)
SUMMARY A study was made to determine whether and to what e x t e n t polycyclic aromatic h y d r o c a r b o n s (PAH) are present in human bronchial carcinoma. T w e n t y - f o u r carcinomas, obtained from surgical operations and autopsies, were examined. The samples were tested for 12 PAH; these were determined by direct fluorescence analysis on thin-layer plates. Only 4 of the 12 PAH were detected in the cancerous tissue: benzo(a)pyrene, fluoranthene, benzo(ghi)perylene and perylene. Benzo(a)pyrene was found in all carcinomas. The reasons for increased concentration of the detected PAH in cancerous tissue are discussed with respect to deposition and elimination of inhaled particles as well as the metabolism of these compounds.
INTRODUCTION Many investigators have examined the lungs of deceased humans for polycyclic aromatic hydrocarbons (PAH), especially b e n z o ( a ) p y r e n e [6--10, 16]. With the exception of the finding of Mallet and Heros-Dekeierel [ 1 0 ] , their results were practically identical in that in most cases no b e n z o ( a ) p y r e n e was found; a few did contain small amounts, a b o u t 1 pg in the total lung. In all these cases the material examined was n o t altered by neoplasm. On the other hand a considerable a m o u n t of b e n z o ( a ) p y r e n e was found in a bronchial carcinoma [15]. The purpose o f this investigation was to study several examples of such cancerous tissue in order to elucidate the nature o f their PAH-content. The analysed tissue samples were obtained from surgical operations and autopsies. MATERIALS AND METHODS T w e n t y - f o u r broncial carcinomas were examined. T w e n t y - o n e were surgical
190
preparations; 3 carcinomas were obtained from autopsies. Some data concerning age, sex, occupation and smoking habits taken from case reports are listed in Table 1. Parts of lungs separated from the carcinoma tissue served as blanks. Surgery of the lungs with the thermocauter was carried o u t in t w o cases; the carcinoma tissue was n o t touched by this instrument. The determination of the PAH was made b y direct fluorescence analysis on thin-layer plates. The method has been described for benzo(a)pyrene [11]. After treating the sample with hot 4N potassium hydroxide and extractions of the solution with cyclohexane, the extracts were combined and concentrated. A first purification of the PAH was achieved b y column chromatography on silica gel. Separation of the PAH was done on Sephadex LH 20 with propanol-2 as eluent. The fractions were concentrated to 0.5 ml, and 50--100 pl were taken for thin-layer chromatography. In a few cases, when the concentration was t o o low, the whole fraction volume was applied. The analysis included 12 PAH: pyrene, fluoranthene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, TABLE 1 A G E , SEX, O C C U P A T I O N A N D S M O K I N G H A B I T O F T H E P A T I E N T S W H O S E L U N G TISSUE WAS A N A L Y Z E D No.
Age
Sex
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 31 32 33
72 66 59 55 66 55 52 50 62 56 51 64 60 61 49 48 60 55 51 53 53 62 60 58
m m m m m m m m m m m m m m m m m m m m m m f m
Occupation
Smoking habit cigarettes/day
salesman employee craftsman w o r k e r in c h e m i c a l p l a n t forestry officer automobile mechanic engine-driver teacher railway a d m i n i s t r a t i o n o f f i c e r crane operator
20 + 0 20 + + 25 10 25 +
employee turner gardener w o r k e r in l a c q u e r p l a n t baker mason salesman mechanic
50 + 20 0 20 20 30 20
+ Smoker, n u m b e r of cigarettes u n k n o w n
191 perylene, dibenz(a,h)anthracene, benzo(ghi)perylene and coronene. The PAH were identified by fluorescence spectra taken from the spots on the thin-layer plates. The detection limits were determined by applying standard solutions in decreasing concentrations on thin-layer plates and were calculated according to the formula given by Doerffel [5]. Strictly, a detection limit refers to one c o m p o u n d only, since different substances in equal concentration and under optimized conditions will give different fluorescence intensity. RESULTS The results are shown in Tables 2 and 3. Three points seem to be noteworthy: (1) From the 12 PAH tested for in the cancerous tissue, only 4 could be detected: benzo(a)pyrene, benzo(b)fluoranthene, fluoranthene and perylene. The content of the remaining 8 PAH was below the detection limit. (2) Benzo(a)pyrene was found in all carcinoma tissue. Fluoranthene and benzo(b)fluoranthene were sometimes present, as well as perylene, although the latter was not detected in autopsy material. (3) The PAH-content in the cancerous tissue was not significantly different with respect to fluoranthene and benzo(b)fluoranthene, but a large difference was found for benzo(a)pyrene as demonstrated by the U-test. Even with the median values this was significantly higher: 3.5 pg benzo(a)pryene/g tissue in the carcinoma and 0.09 pg in the tumour-free tissue, a ratio of 38:1. DISCUSSION The results demonstrated increased concentration of benzo(a)pyrene in areas of the lung, which proved to be bronchial carcinoma by histological diagnosis. In non-cancerous tissue only small amounts of benzo(a)pyrene were found. When discussing the reasons for an enrichment of PAH in bronchial carcinomas, attention should be focused upon all alterations of deposition, elimination and metabolism of PAH. The deposition of inhaled particles in the area of a carcinoma may be increased by an alteration of bronchial air flow; a disturbance of the elimination of particles may not only cause a prolonged retention time of the deposited particles there but also, and more importantly, may result in a damning up of the substances moving from the terminal air passages toward the trachea. In addition to this incommodation of the 'mechanical' lung clearance, a disturbance of the resorption, as well as the metabolism, of foreign substances must be taken into account. Alveolar macrophages and some other lung cells (e.g., epithelial cells of the bronchus) are able to transform PAH by mixed-function-oxidases into products which are then easily removed. It has been known for some time that macrophages of guinea pigs, in vitro, will dissolve and metabolize PAH from soot particles [12,13]. This enzyme activity can be induced by benzo(a)pyrene and by other PAH as well [3,4]. Even human alveolar macrophages have oxidases which can be induced. Thus, macrophages of smokers have been
192
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