Znt. J . Cancer: 19, 538-545 (1977)

INDUCTION BY CIGARETTE SMOKE OF ARYL HYDROCARBON HYDROXYLASE ACTIVITY I N THE RAT KIDNEY AND LUNG1 Jacques VANCANTFORT and Jacques GIELEN Laboratoire de Chimie Mkdicale, Institut de Pathologie, Unite' de Biochimie, B-4000 SART TILMAN par Likge I , Belgium

In both the rat kidney and lung, inhalation of cigarette smoke diluted with air (1115) for a limited period of time (15 min) specifically induces aryl hydrocarbon hydroxylase ( A H H ) ' in, less than 4 h. U p to four successive inhalations administered a t 2-h intervals additively induce both lung and kidney hydroxylase activities. The maximal effect corresponds to about 10 times the control value. Compared to the kidney enzyme, the lung A H H activity is about three or four times more sensitive to small concentrations of cigarette smoke. The biological half-life of the lung A H H activity i s longer than 24 h, while it i s only 3-4 h in the kidney. I n both tissues, the induced enzyme presents the same in vitro thermolability and sensitivity to various inhibitors. For the establishment of the A H H induction, protein synthesis i s continuously required, while R N A synthesis i s only necessary during the first 2 h following the smoke treatment.

Aryl hydrocarbon hydroxylase (AHH) belongs to the group of monooxygenases (Hayaishi, 1969; Mason, 1957) and represents the microsomal enzymatic complex responsible for the biotransformation of the polycyclic hydrocarbons, such as benzo(a)pyrene, benz(a)anthracene, or 3-methylcholanthrene. Present in most mammalian tissues, this enzymatic activity can be enhanced in animals by the administration of a variety of chemicals (Conney, 1967) like the polycyclic hydrocarbons themselves, B-naphthoflavone, 2,3,7,8-tetrachlorodibenzo-p-dioxin (Hook et al., 1975; Poland et al., 1974). The end products of the polycyclic hydrocarbon metabolism, i.e. phenols, trans-dihydrodiols, quinones and thiol conjugates generally result from the further enzymatic or non-enzymatic transformation of reactive arene oxide (or epoxide) intermediates (Jerina et al., 1974; Sims and Grover, 1974) which are themselves the first products of the AHH catalyzed enzymatic reaction. That those epoxides are most likely the ultimate or proximate carcinogens is supported by several experimental facts, such as: (1) their ability to bind covalently to the cellular macromolecules (Baird et al., 1975; Daudel et al., 1975; Miller, 1970); (2) their mutagenic activity in bacteria (Ames, 1972; Felton and Nebert, 1975) and (3) their malignant transformation activity in cell culture (Grover et al., 1971; Heidelberger, 1973).

Important differences have been demonstrated regarding the mutagenic as well as the cytotoxic activities of a number of benzo(a)pyrene epoxides (Wood et al., 1975). On the other hand, it is largely documented that the pattern of metabolites produced from a single polycyclic hydrocarbon varies both quantitatively and qualitatively as a function of biological and biochemical parameters such as the activity of the various enzymes involved in the metabolic pathway (Holder et al., 1974; Yang et al., 1975), the polycyclic hydrocarbon used as a substrate (Felton and Nebert, 1975), the animal species or the tissue analyzed (Selkirk et al., 1975). Thus, it becomes quite obvious that any chemicals likely to modify the activity of the AHH shoulJ be considered as potential hazards. Cigarette smoking is known to induce AHH activity in the lungs of animals (Abramson and Hutton, 1975; Holt and Keast, 1973; Okamoto et al., 1972; Welch et al., 1971, 1972) as well as in human alveolar macrophages (Cantrell et al., 1973) and placenta (Nebert et al., 1969; Welch et al., 1969). More recently, it was reported that cigarette smoke induces lung AHH activity in both the inducible and non-inducible strains of mice (Abramson and Hutton, 1975; Kouri et al., 1973; Van Cantfort and Gielen, 1975). It should also be emphasized that in rats and in responsive mice, AHH activity is selectively induced by the smoke in two tissues, the lung and the kidney (Van Cantfort and Gielen, 1975). The existence of a relationship between the inducibility of AHH activity and susceptibility to the development of skin tumors in mice (Nebert et al., 1974) and possibly lung cancer in man (Kellermann et al., 1973) after prolonged cigarette Received : November 11, 1976. Requests for reprints should be addressed to J. Gielen. This work was presented in part at the meeting of the International Agency for Research in Cancer (Gielen ef al., 1975) (Brussels, June 9-11, 1975) and at the International Meeting of Pharmacology (Van Cantfort et a/., 1975~)(Helsinki, July 20-25, 1975). * Abbreviations used in this paper: AHH, aryl hydrocarbon hydroxylase; MC, 3-methylcholanthrene.

539

AHH INDUCTION BY CIGARETTE SMOKE

smoking, has initiated a great deal of work designed to understand the biochemical reasons for such a linkage. To approach this problem, we have focused our attention on cigarette-smoke-induced AHH activity. In this report, we will describe some of the fundamental characteristics of this phenomenon in the rat lung and kidney. We will also compare a number of in vivo and in vitro properties of the cigarettesmoke- and MC-induced enzyme. MATERIAL AND METHODS

Chemicals The source of most of the chemicals has been indicated elsewhere (Van Cantfort et al., 19756). Actinomycin D and cycloheximide were obtained from Sigma (St. Louis, USA). Treatment of animals The smoke was generated by a Hamburg type I1 inhalation apparatus (Heinz Borgwald, Hamburg 50, Germany) with standard Belgian cigarettes. The smoke was delivered in the form of a 30-1111 puff diluted with a variable volume of air. Under our standard conditions, the diluting air volume was 420 ml, corresponding to a 1/15 cigarette smoke/air dilution. Every 2 seconds, the air/smoke mixture was blown into the closed area in which the rats were located. By other methods, we have verified that the stress induced by the experimental conditions was not responsible for the AHH induction in the lung and the kidney (J. Van Cantfort, unpublished results). Immediately after treatment (usually 15 min of inhalation), the animals were returned to their normal cages until the moment of killing. The concentration of smoke in the cage was varied by alternating the quantity of air diluting the cigarette smoke. Cycloheximide ( 5 mg/kg) and actinomycin D (1 mg/kg) were administered intraperitoneally, the control animals being treated with a saline solution. Preparation of the subcellular fractions Sprague-Dawley rats weighing 200 g (Centre des Oncins, Lyons, France) were killed by a blow on the head and carefully bled. The organs were removed and rapidly cooled in ice-cold KCI isotonic solution. All of the following manipulations were performed at 4" C. The livers were pressed through a metal disc perforated with 1.5-mm holes. The resulting pulp was then diluted with 4 parts of the same solution and homogenized in a PotterElvehjem tube with a teflon pestle. The homogenate was centrifuged for 10 min at 9,000 g in a refrigerated Sorvall apparatus. The supernatant was stored at - 18" C until the enzymatic measurements were performed.

Enzymatic assays The AHH activity was determined by fluorometric measurement of the 3-hydroxybenzo(a)pyrene formed according to a method described elsewhere (Van Cantfort and Rondia, 1973). The measurements of aminopyrine-N-demethylase (Christensen and Wissig, 1972) and progesterone16a-hydroxylase (Van Cantfort et al., 19756) were also described. In every case we have used the method of Lowry (Lowry et al., 1951) to verify that the treatment did not influence the protein concentration of the organ. RESULTS

Selective induction in the rats A 15-min exposure to a 1/15 dilution of cigarette smoke induced AHH activity in the rat lung and kidney (Fig. l), but did not affect the same enzymatic activity in any of the other tissues studied (liver, bowel, testes, adrenal glands, brain, skin). Furthermore, no induction was recorded in the liver for three other cytochrome P-450 linked enzymes, i. e. aminopyrine-N-demethylase, cholesterol-7ahydroxylase, ' progesterone-1 6a-hydroxylase (results not shown). In the lung and kidney, there was a 2-h lag between the smoke inhalation and the enzyme induction onset. Thereafter, the AHH activity increased very rapidly to peak about 2 h later, reaching a maximal activity which corresponded to 3 or 4 times that of normal rats. The hydroxylase activity then decreased, but much more rapidly in the kidney than in the lung. In fact, the lung AHH activity was still induced to a level approximately 2/3 that of the peak activity, while the kidney enzyme was already back to the control level. The lung and kidney AHH activities were affected differently when either the length of the smoking period or the dilution of the cigarette smoke inhaled for 15 minutes was modified (Fig. 2). Actually, the lung enzyme was much more sensitive to the cigarette smoke and was already maximally induced 4 h after a 3-min exposure to the cigarette smoke or after a 15 min exposure to 1/3 of the standard dose. Under these conditions, the kidney enzyme was also induced, but to a much smaller extent. It is also important to emphasize that, under our experimental conditions, a plateau was rapidly reached when inducing the lung enzyme; in the kidney, a direct relationship could be observed between the amount of cigarette smoke inhaled and the enzymatic induction, over a much wider range of smoke concentrations. Finally, a 1-min exposure to the usual 1/15 dilution of cigarette smoke or a 15-min exposure to l/lOth of the standard smoke

VAN CANTFORT AND GIELEN

FIGURE 1

Effect of a 15-min cigarette smoke inhalation on AHH activity in the lungs (m) and kidneys ( 0 ) . Each group of 6 rats was killed at the same time of the day corresponding to different times after the cigarette smoke treatment. Results are given as the mean bSD.

0

2

4

6

8

10

12

fW 20

HOURS AFTER INHALATION

dose was sufficient to very significantly induce the lung enzyme, but did not affect the kidney hydroxylase activity. If successive inhalations were administered to the rats at 2-h intervals, the AHH activity assayed 4 h after the last inhalation was induced in a more pronounced way than after a single smoking period (Fig. 3). Up to four successive treatments induced the lung and the kidney enzymatic activities in a n additive manner, the maximal induced activity obtained corresponding to an eight- to twelve-fold induction. The kidney enzyme was usually induced to a higher value than the lung enzyme. When more

than four successive inhalations were administered, the inducing effect of the cigarette smoke began to diminish. Successive inhalations did not modify the activity of the following liver microsomal enzymes: AHH, aminopyrine-N-demethylase,cholesterol-7~hydroxylase, progesterone-1 6a-hydroxylase (Table I). A slight and transient increase of cholesterol-7~hydroxylase activity was consistently recorded; it reflects the stress of the animals during the treatment, this enzyme being the only one of the measured hydroxylases to be inducible by glucocorticoids and stressing conditions (Gielen et al., 1975; Van Cantfort, 1973).

FIGURE 2

Lung and kidney A activity as a function of the tion of cigarette smoke and the length of the treatment. Groups of 6 rats were exposed three times at 2-h intervals to the indicated treatments, the various cigarette smoke/ air dilutions being administered for 15 min. Results are given as the mean ~ S D . I I -X

I

.

'

"150 'b5 [SMOKE /AIR]

"I 5 01

4

15

LENGTH OF INHALATION(min)

541

AHH INDUCTION BY CIGARETTE SMOKE

FIGURE 3 Effect of successive 15-min ciga-

rette smoke inhalations on lung (white columns) and kidney (shaded columns) AHH activity. The smoke inhalations were given at 2-hourly intervals apd all the rats were killed at the same time of the day, 4 h after the last inhalation. Results are given as the mean 1 s ~ .

0

1

2

4

6

8

NUMBER OF INHALATIONS

R N A and protein synthesis requirements The induction of both kidney and lung AHH activity was completely prevented when either cycloheximide or actinomycin D was injected to the animals 15 min prior to the smoke treatment, confirming the previous work of Welch and coworkers (Welch et al., 1971, 1972). Nevertheless, if actinomycin D was administered 2 h after the smoke inhalation, i.e. just before the onset of the enzyme induction, the AHH activity was still induced to almost the same extent as in the control group 4 h later. However, the simultaneous administration of cycloheximide again completely prevented the induction of the enzymatic activity (Table 11). This experiment allowed us to conclude that protein synthesis is continuously required to induce the AHH activity, but that RNA synthesis is only

necessary in the initial period of the phenomenon. In this respect, the molecular mechanism involved in cigarette smoke AHH induction is similar to that described for polycyclic hydrocarbons and phenobarbital AHH induction in tissue culture (Nebert and Gelboin, 1970; Nebert and Gielen, 1971). In vivo biological half-life of cigarette-smoke- and methylcholanthrene-iduced AHH

To further characterize the lung and kidney AHH activity, the half-life of the cigarette-smoke-induced AHH activity was determined after cycloheximide administration to the animals and compared to the MC-induced enzymatic activity measured under the same conditions. The induced lung AHH activity appeared remarkably stable; no significant decay was detected during the 6 h following the adminis-

TABLE I EFEECT OF SUCCESSIVE I S M I N CIGARETTE SMOKE INHALATIONS O N VARIOUS RAT LIVER ENZYMATIC ACTIVITIES ' Number of inhalations

AHH

Aminopyrine demethylase

None

706k98 777 & 109 758+83 774 & 72 723 f86 771 1 8 1

13200 & 2400 12700 & 2600 12800k800 1190011000 12800f 1400 13400+1700

1 2 4 6 8

Cholesterol la-hydroxylase

32.1 1 3 . 6 45.5h4.6 * 49.5+6.8 * 41.934.0 * 47.216.7 * 43.3k5.8 *

Progesterone 16a-hydroxylase

469 & 68 440351 531181 474k39 494f56 515149

Successive 15-min smoke inhalations were administered at 2-h intervals to groups of six rats. The animals were always killed 4 h after the last smoke inhalation. The results, in nmol of product formed per hour and per g of tissue, are given as the mean *so. * p

Induction by cigarette smoke of aryl hydrocarbon hydroxylase activity in the rat kidney and lung.

Znt. J . Cancer: 19, 538-545 (1977) INDUCTION BY CIGARETTE SMOKE OF ARYL HYDROCARBON HYDROXYLASE ACTIVITY I N THE RAT KIDNEY AND LUNG1 Jacques VANCAN...
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