XENOBIOTICA,

1975, VOL. 5,

NO.

9, 539-551

Metabolism of Nicotine by the Isolated Perfused Dog Lung D. M. TURNER", A. K. ARMITAGE, R. H. BRIANT and C. T. DOLLERY

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Department of Cardiovascular Pharmacology, Tobacco Research Council Laboratories, Harrogate, Yorkshire, and Department of Clinical Pharmacology, Royal Postgraduate Medical School, Hammersmith Hospital, London W12, U.K.

(Received 29 November 1974)

1. Metabolism of [14C]nicotinehas been studied in the isolated perfused dog lung. [14C]Nicotine, 50 pg every 30 s for 10 min administered via the pulmonary artery, undergoes first pass metabolism to a small extent. [14C]Cotinine was detected in the venous blood. Of the injected activity, 6% was in the lung at the end of experiment ; 60% being present as ['4C]nicotine and 20% as [14C]nicotine1'-oxide. 2. When [14C]nicotine was administered in cigarette smoke a greater degree of metabolism was observed at first pass. Pyrolysis products of [14C]nicotine also were present in the venous blood. Lungs after smoke exposure contained 30% of administered radioactivity, with a substantial proportion of [14C]nicotine-1 '-oxide. 3. Administration of [14C]nicotine-labelled smoke to lung preparations, on closed circuit, gave significant amounts of [14C]cotinine and other metabolites over a 2 h period. Lung tissue contained approx. 40% of injected dose, of which 25 yo only was [14C]nicotine. Large proportions of [14C]cotinine and ['*C]nicotine-1'-oxide were present but 45% of the activity was present as other unidentified pyrolysis products. ['4C]Dernethyl cotinine was detected.

Introduction Tobacco smokers commonly inhale the smoke into their lungs from which some of the smoke components transfer to the blood. Although components such as nicotine transfer from lung tissue to the circulation extremely rapidly, it is important to elucidate whether the lung plays an active part in the fate and subsequent distribution of these components. The non-respiratory functions of lung tissue (Heinemann & Fishman, 1969) and in particular its capacity to metabolize foreign compounds (Kiese & Uehleke, 1961) have recently been extensively studied. The deposition of tobacco smoke components in lung has been investigated (Dalhamn, Edfors & Rylander, 1968 ; Davis, Houseman & Roderick, 1973) but only limited attention has been given to possible metabolism of such compounds by the lung. Tissue distribution studies have been reported (Turner, 1969 ; Nakashirna, 1972) which give some indication of the capacity of the lung to metabolize nicotine in vivo. In addition, the capacity of mouse lung tissue to metabolize nicotine in vitro (Hansson,

* To whom correspondence should be addressed, at: Department of Biochemistry and Drug Metabolism, Hazleton Laboratories Europe Limited, Otley Road, Harrogate HG3 lPY, North Yorkshire, U.K. 2N2

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Hoffmann & Schmiterlow, 1964) and rat lung to metabolize other smoke constituents (Wattenberg & Leong, 1965) have been measured and some idea thus obtained of its detoxicating ability in those animals. Similar studies in rabbit, on the fate of other xenobiotics (Nakazawa & Costa, 1971 ; Oppelt et al., 1970) indicate a significant, though minor role for the lung in drug metabolism compared with the liver. Although the lung has low capacity to metabolize foreign compounds in vitro, cigarette smoke will induce this enzyme activity (Okamoto, Chan & So, 1972 ; Welch, Cavallito & Loh, 1972). Since assays in vitro do not necessarily reflect activity in z’iz‘o, we have studied the metabolism of nicotine in the isolated perfused lung of dog, so that metabolism in other organs was excluded. T h e techniques for preparation of the isolated dog lung are well documented and the physiological state of the organ under the conditions we used is relatively normal (West, Dollery & Naimark, 1964 ; West & Dollery, 1965). T h e lung perfusion method has recently been used to study the metabolism of drugs (Rosenbloom & Bass, 1970 ; Orton et al., 1973).

Materials and methods Experiments were performed using the lungs of greyhound or mongrel dogs weighing 23-33 kg, prepared by the technique of West, Dollery & Naimark (1964). Animals were anaesthetized with pentobarbitone sodium (30 mg/kg) followed by intravenous injection of heparin (2000 i.u./kg). T h e animal was exsanguinated and 500 ml blood was immediately used to prime the perfusion circuit. T h e left lung was then removed via a large intercostal incision. Glass cannulae were tied into the pulmonary artery, left main bronchus and excised left atrium. T h e lung was placed in a sealed, humidified, Perspex box in a horizontal position and the cannulated artery and vein connected to the perfusion circuit. T h e box was sealed and an intermittent partial vacuum applied to ventilate the lung twelve times per minute with a 12% 0, : 5% CO, : 83% N, mixture. After establishing that the preparation was functioning satisfactorily the bronchial cannula, for 4 of the experiments, was connected to a smoking device (Armitage et al., 1974) which could be adjusted to take puffs of cigarette smoke of predetermined volume at pre-set times and present a portion of that puff to the lung together with sufficient of the 0,: CO, mixture to make up the tidal volume on the next appropriate inspiration. In a further experiment [14C]nicotine was administered into the arterial cannula as a series of injections of 50 pg, every 30 s, for 10 min. For the smoke administration experiments, filter-tip cigarettes similar to commercially available brands, were impregnated with 42.8 pCi of 1-[2’14C]nicotine di(p-toluyl tartrate) (sp. activity 10 mCi/mmol) using a spiking device described by Houseman and Heneage (1973 a). T h e mean specific radioactivity of the [14C]nicotine in the mainstream smoke from six such cigarettes when smoked under the analytical conditions with the same puff parameters as used in these experiments was 0.82 mCi/mmol. T h e ( - )-[2’-14C]nicotine was synthesized in these laboratories using the method of Decker and Sammeck (1964) and resolved by Dr. T. H. Houseman. For the intra-arterial experiments [l4CC]nicotine was administered as its dihydrogen tartrate (sp. activity 19-3 mCi/mmol). I n 3 out of 5 experiments (lA, OC1 and OC2) blood passed through an open perfusion circuit. T h e blood volume of the circuit was maintained at constant

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level of 500 ml for the duration of each experiment (approximately 20 min) but the venous output from the lung was collected in approximately 10 ml portions continuously over that period. I n the remaining two experiments (CC1 and CC2) the perfusion circuit was closed, the blood volume of the system being constant at 500 ml, and allowed to circulate for 2 h. Blood samples (10 ml) were withdrawn from the inflow of the reservoir at pre-determined intervals and an equal volume of blood replaced. Blood samples were assayed for [14C]nicotineand [14C]cotinine by the method of Turner (1969). The lung tissue was homogenized in dichloromethanemethanol (2 : 1, vlv) and portions of the two liquid phases thus produced were concentrated and subjected to two-dimensional paper chromatography on Whatman 3MM, using the solvent systems described by Papadopoulos and Kintzios (1963). I n one experiment pooled blood samples were also extracted with dichloromethane-methanol in order to identify some of the metabolites. Total radioactivity in all blood samples was measured by combustion of portions of each sample to 14C02using a Packard Model 305 Tricarb sample oxidizer (Packard Instruments, Wembley, Middlesex). Sections of the paper chromatograms which contained nicotine and its metabolites were visualized, using cyanogen bromide (Turner 1969), cut out and combusted also. Non-extractable radioactivity in the pooled blood samples and lung tissue was similarly measured by combustion of portions of extracted solid residue. The 14C02produced was taken up into methanolic ethanolamine which was further diluted with a toluene-based scintillator (Packard Instruction Manual No. 21 18, 1972). Liquid scintillation counting of all samples was performed using a Packard 3375 liquid scintillation spectrometer (Packard Instruments, Wembley, Middlesex) as described by Turner (1969). All doses of [14C]nicotine refer to the base. The metabolism of [14C]nicotine administered via the pulmonary artery to a perfused lung on open circuit (Experiment 1A) A series of injections of [14C]nicotinewas made into the pulmonary artery of a perfused lung preparation with a circulating blood capacity of 500 ml. The blood flow rate was 90 ml/min. [WINicotine (50 pg in 0.3 ml of phosphate buffered saline (pH 7.4)) was injected into the circulating blood every 30 s for 10 min. When the first nicotine injection was given, the perfusion circuit was broken adjacent to the venous outflow and blood samples (11 ml) continuously collected during the period of nicotine administration and for a short period thereafter. The blood level in the reservoir was maintained at 500 ml by frequent addition of heparinized blood collected from the donor dog. The time of collection of each sample was recorded. After continuous collection of 100 samples the experiment was terminated and the contents of the reservoir allowed to empty. This blood was pooled in a suitable container, the volume measured, and a portion retained for analysis. At the end of experiment the lungs were removed, weighed and stored frozen until processed. The metabolism of [Wlnicotine in the perfused lung on open circuit exposed to [14C]nicotine-labelledsmoke (Experiments OC1 and OC2) Two experiments (OC1 and OC2) were performed in which [14C]nicotinelabelled smoke was administered every 30 s using the cigarette-smoking device described earlier. As with the intra-arterial nicotine experiment blood samples

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were taken continuously during and after smoke administration. T h e smoking device took a 25 ml puff at the appropriate time and was set to deliver a nominal 15 ml of each puff to the lung on inhalation, exhausting the remaining 10 ml to atmosphere. I n OC1 16 puffs were obtained from the cigarette before reaching standard butt length (49 mm rod smoked) and in OC2 19 puffs were obtained. For each experiment a glass fibre filter disc (Cambridge Filter Corporation) collected the particulate phase (and hence almost all of the radioactivity) from the portions of each puff which were exhausted. A further filter disc collected that radioactivity exhaled by the lung between each puff.

The metabolism of [14C]nicotine in the perfused lung no closed circuit exposed to labelled smoke (Experiments CC1 and CCZ) Two further experiments (CC1 and CC2) were performed in which puff rate was every 60 s for 9 min (i.e. 10 puffs for each experiment) and the circulating blood was maintained in a closed system for 2 h at a flow rate of 402 ml/min. Blood samples were withdrawn via the outflow from the pulmonary vein at predetermined intervals. Filter discs collected the exhausted and exhaled radioactivity in an identical manner to that described above. Though the smoking machine was set to deliver 15 ml of a 25 ml puff to the lung, subsequent analytical studies of the 'split' ratio (volume of puff exhausted/ volume presented to lung) showed that the actual delivery was less. In any particular experiment, however, the volume delivered to the lung in each puff was constant. T h e particulate phase proportion of smoke vented to atmosphere before inhalation of the nominal 15 ml was collected on a glass fibre filter (exhaust) and the smoke from the exhalations after each inhaled puff was collected on another filter (exhale). Since the majority of the 14C in the smoke is transferred with the particulate phase it is possible to make some assessment, for each experiment, by measurement of the activity deposited on the exhale and exhaust Table 1. Recoveries of 14C in the four smoking experiments oc1

( a ) Total exhausted ( b ) [14C]Nicotine exhausted (c) Total exhaled ( d ) Recovered from lung and blood ( e ) Inhaled by lung : ( c + d ) (f) Percentage retained by lung : (100je) ( g ) Volume of smoke inhaled : [25e/(e a)] ( h ) ['TC]Nicotine absorbed (pg) : (162b/0%2n)

+

2.70 2.50 1.18 1.08 2.26 47.794 11.4 ml 197

oc2 14C-activity Wi) 3.32 2.99 1.42 1.37 2.79 49.1 7' 11.4 ml 243

cc1

cc2

2.32 1.94 1.42 0.46 1.88 24.594 11.2 ml

2-08 1.87 1.04 1.07 2-10 50-9'6 12.6 ml

76

189

The smoking device took a 25 ml puff of smoke, exhausted a portion (nominally 10 ml in these experiments) to atmosphere through a filter and held, momentarily, the remaining smoke (nominally 15 ml in these experiments) which was then inhaled by the lung. Smoke exhaled by the lung was passed through a second filter. Radioactivity collected on the filters was then extracted into methanol and portions of each extract counted. T h e amount of radioactivity due to [14C]nicotine on the exhaust filter was measured by the method of Houseman (1973 b).

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filters, of the activity ‘inhaled’ by the lung. On the assumption that overall radioactive recovery in each experiment is quantitative, and using the measured activities of smoke recovered from the exhaust and exhale filters, the estimated volume of inhaled smoke for each experiment can be calculated as is shown in Table 1. T h e activity inhaled, in each experiment, is calculated on the assumption of quantitative recovery of exhaled activity together with that from lung tissue and blood samples i.e. (c + d ) . T h e volume of smoke inhaled was derived from this inhaled radioactivity since the volume of smoke containing activity ( a ) and that containing activity (e) is 25 ml. T h e amounts of [14C]nicotine absorbed are calculated using the mean sp. activity of 0-82 mCi/mmol and on assumption that for a particular experiment the proportion of [l*C]nicotine in the I4C exhausted is the same as in the activity inhaled.

Results Open circuit, intra-arterial [14C]nicotine (Experiment 1A) T h e radioactivity appearing in the venous blood is shown in Fig. 1. There are fluctuations .uring the rise of radioactivity while smoking and during the

30 2c

25

20

g 3 X

*

0 I

15

-3

10 5

5

0

i!i!i!i!i!ilt I I ,

, , , , , , 1 2 3 4 5 6 7 8 9 1011 1 2 1 3 1 4 1 5 1 6

0

min

Fig. 1. Blood radioactivity in the open circuit experiments. 00 , intra-arterial administration of 50 pg [14C]nicotine every 30 s for 9.5 min, administration of 16 puffs of smoke from a left-hand ordinate (IA). A-A, cigarette labelled with [14C]nicotine, right-hand ordinate (OC1).

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subsequent fall. Analysis of a representative group of the blood samples for [14C]nicotine and [14C]cotinine gave results shown in Fig. 2 ; unidentified radioactivity was calculated as nicotine. It may be seen that [14C]nicotine comprises the major proportion of the blood activity, [14C]cotinine is present in small though significant amounts and similar levels of unidentified activity are present. T e n blood samples which contained the peak radioactivity (and which included, therefore, three individually analysed blood samples) were pooled, extracted as described in the methods section, and portions of the concentrated ' organic ' and ' aqueous ' phase extracts thus obtained were subjected to paper chromatography in two dimensions. T h e recovery of radioactivity by this method was almost quantitative (90.7%) as is shown below : Total 14C activity in pooled blood, 9.24 pCi ; in aqueous phase of extract, 1-21pCi ; organic phase of extract, 7.16 pCi.

900-

800 -

700 -

800-

B

0 500n

-E

\ F400

-

300

-

200 -

P

Fig. 2. Radioactivity due to nicotine, cotinine and unidentified activity during the intraarterial nicotine experiment. The arrows indicate the time and number of injections of [14C]nicotine ; nicotine ( o), cotinine (a)and unidentified activity ( 0 )are shown.

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Table 2 shows the percentage proportions of the identified metabolites of nicotine in the total extracted radioactivity by summation of the aqueous and organic phase activities in their respective proportions. Table 2. The percentage proportions of [14C]nicotine and its metabolites in the extractable activities in lung and blood

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Experiment : Extractable 14C-activity (yoof dose) Metabolites Nicotine Cotinine Demethylcotinine Nicotine N-oxide Unidentified

oc1

oc2

Lung

Lung

1A Blood

1A Lung

90.7

85.0 79.2 73.4 65.0 Proportion of extractable activity (yo) 60.1 56.4 51.5 24.5 7.8 8.1 16.3 19.0 3.9 20.4 16.4 11.2 8.8 11.7 19.1 17.1 47.7

90.8 4.3 1.6 1.2 2.0

CCI Lung

cc2 Lung

62.9 25.3 10.7 18.7 45.3

The percentage proportions of nicotine and cotinine of the extractable 14C activity in pooled blood agrees reasonably well with the mean proportional activity calculated from the three individually analysed blood samples from the same time period, namely 96.7% nicotine and 2.3% cotinine. Incubation of oxygenated blood with [14C]nicotine over a period of 2 h, and subsequent extraction, yielded no evidence of metabolism. The lungs were also homogenized in dichloromethane-methanol and the extractable radioactivity analysed as for the pooled blood samples. Overall recovery of radioactivity was almost quantitative (85%) as shown : Total 14C activity in lungs, 6.7 pCi ; I4C activity in organic extract, 3.9 pCi ; 14C activity in aqueous extract, 1.8 pCi. The percentage proportions of identified metabolites is also shown in Table 2 (1A-lung). The most striking differences between blood and lung 14C composition are the marked decrease in [Wlnicotine content and increase in [14C]nicotine-l’-oxide content of the lung. Unidentified 14C activity also comprised a much greater proportion of the overall extractable radioactivity. Paper chromatography of a portion of eluted [14C]nicotine-l’-oxide using the solvent system described by Booth and Boyland (1970) resulted in a separation into two components which are believed to be the stereoisomers of the compound. The ratio between the amounts of laevo- and dextrorotatory isomers was 7.5. The recovery of radioactivity from all blood and tissue samples was almost quantitative (93.8%) as illustrated : Total 14Cinjected, 118.9 pCi ; 14Cin lung, 6.7 pCi ; 14Cin blood samples, 104.8 pCi. [14C]Nicotine labelled smoke-open circuit (Experiment OC1 and OC2) Fig. 1 also shows the appearance and decline of radioactivity in the blood during and after smoking in experiment OC1. As with the pulsed intra-arterial injections of nicotine the blood radioactivity increased and decreased erratically. I n experiment OC2 the change in blood radioactivity was similar. Selected

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blood samples were analysed for [14C]nicotine and [14C]cotinine as before and the results are illustrated in Fig. 3 for both experiments. I n both experiments the peak blood level of [Wlnicotine was approximately 200 ng/ml though the rate of increase in [14C]nicotine concentration was slower in OC2 than in OC1. 225

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200

175

150

U

8 125

a

E

2

100

75

50

25

0

Radioactivity due to nicotine, cotinine and unidentified activity in open circuit experiments Kith [14C]nicotinesmoke. The broad arrows indicate the puff frequency for experiment OC1 (-) and the thin arrows the frequency for experiment OC2 (. . .) ; nicotine is shown as ( C ,0) ;

Fig. 3 .

cotinine as

(a, [I) and unidentified activity as ( 0 , w).

It can be seen that relatively large amounts of unidentified radioactivity are present in the blood from both experiments though the major proportion of the activity is [14C]nicotine. T h e proportions of [14C]cotinine produced in both experiments are higher than was the case with the intra-arterial [14C]nicotine experiment (see Table 1). Fractionation of the radioactivity in each lung was

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performed and the results are shown in Table 2. As with experiment lA, relatively large proportions of [14C]cotinine and [14C]nicotine-l’-oxide were found. T h e proportions of the metabolites found in the lung of the experiment OC1 are similar to those observed in experiment OC2. I n both OC1 and OC2, however, the proportions of cotinine were high which is consistent with the relatively large amounts detected in the blood. Recovery of activity by extraction from each lung was similar in both experiments but both recoveries were less than that obtained in experiment 1A.

[‘*C]Nicotine labelled smoke-closed circuit (Experiments C C l and CC2) Fig. 4 shows the blood levels of [l*C]nicotine and [14C]cotinine obtained in the two experiments (CC1 and CC2). I n both experiments the nicotine levels reached a peak at the end of the smoking period. Cotinine levels climbed slowly over the whole time course of each experiment reaching, in CCl, a level of 28 ng/ml. The unidentified activity in this experiment reached a similar terminal level. Analysis of the activity in the lungs at end of experiment shows the presence of other nicotine metabolites and the percentage proportions are shown in Table 2. T h e recovery of activity from each lung by extraction was lower in both cases than in the open circuit experiments. T h e chief differences in the distribution of metabolites between C C l and CC2 occur in relation to [14C]cotinine and [14C]nicotine-l’-oxide but CC2 was terminated after 70 min due to lung haemorrhage.

rnin

Fig. 4. Radioactivity due to nicotine, cotinine and unidentified activity in closed circuit experiments with [14C]nicotinesmoke. The arrows indicate the time and number of puffs. Nicotine ( 0, 0)cotinine (a, [I) and unidentified activity ( 0 , B) are shown for experiments CC1 (-) and CC2 (. .).

.

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Calculation of overall recoveries of activity is based on the same assumptions as those described for experiments OC1 and OC2 and the results are shown in Table 1. T h e proportion of radioactivity inhaled which was retained in CC2 was similar to that in both OC2 and OC1. I n CC1 however the radioactivity retained was much lower. T h e unidentified radioactivity in all four smoke experiments was significant (approximately 17% of the total activity even in the initial blood samples). Over the course of the experiments however this activity attained proportions of 30q.d. Table 1 shows the analytical data relating to all the smoke experiments from which estimates were made of the radioactivity inhaled by each lung, the volume of smoke inhaled and the amounts of [14C]nicotine absorbed. T h e calculated volumes of smoke inhaled are very similar and in three of the four experiments the percentage activity retained by each lung is almost identical. T h e amounts of [14C]nicotine absorbed in all the experiments are consistent with the total nicotine yields obtained from similar unlabelled cigarettes using the same puff parameters.

Discussion In all experiments the dog lung preparations have metabolized [14C]nicotine. In experiment 1A small but significant blood levels of cotinine were measured as was a small amount of unidentified radioactivity. However the greater proportion of the blood activity at all times during the experiment was due to [14C]nicotine. I n this experiment a considerable proportion of the [14C]nicotine appeared to pass almost directly from arterial to venous blood and only 6% of the injected activity was found in the lungs at the end of experiment. However, significantly greater metabolism of [14C]nicotine occurs in the lung than would be suggested by the blood data alone as is shown in Table 2. One notable metabolite which is produced is [14C]nicotine-l'-oxide which is present to the extent of 20%. Since the relative proportions of [14C]cotinine to [14C]-N-oxide are different in the blood it would appear that this latter metabolite is bound to lung tissue. In earlier in vivo experiments in the cat (Turner, 1969) it was suggested that nicotine metabolites may be selectively bound in lung on the basis of distribution studies. Orton et aE. (1973) have studied the metabolism of a series of basic drugs in perfused rabbit lung. They found that, with the exception of methadone, no significant oxidative metabolism occurred. Uehleke (1969) however reported that the perfused cat and rabbit lungs showed remarkably active N-hydroxylation ability. In contrast to the 1A experiment the results of the two open circuit experiments in which ['*C]nicotine-labelled smoke was administered indicated that a much greater proportion of the radioactivity was present in the lungs at the end of experiment when compared to the total activity recovered from the bloodapproximately 30%. This large difference indicates the importance of studying the lung metabolism of foreign compounds, which are normally inhaled, used inhalation techniques as opposed to intra-arterial administration. T h e irregular nature of the increase in blood radioactivity shown in Fig. 1 is expected because

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of the pulsed administration of [14C]nicotine or smoke. However, the decline in radioactivity during the latter part of each experiment is also not smooth. This may be due to the mechanical effects of inflation and deflation of the lungs which would tend to produce a surge. These effects would, of course, be present during the whole period of experiment and would contribute to the effects of puffs of smoke. It has been shown (Isaac & Rand, 1969) that the arterial blood levels of nicotine fluctuate between puffs of cigarette smoke inhaled every minute. Perhaps in the intact animal any ‘ mechanical ’ fluctuations are damped by virtue of a closed circulation. I n our closed circuit experiments the relative smooth increase in blood [14C]nicotine levels during smoke inhalation is not surprising since blood samples were taken at the same time relative to each puff of smoke. T h e total dose of [14C]nicotine was similar in both open circuit experiments as were the maximum blood levels attained. Blood levels of [14C]cotinine comprised a larger proportion of the overall activity than in experiment 1A, consistent with the [f4C]nicotine having been more accessible to the sites of metabolism as a result of inhalation of the compound. T h e unidentified activity which is present in the blood even at the beginning of experiment to the extent of approx. 17% is almost certainly due to the presence of pyrolysis products of [14C]nicotine in the smoke. It has been shown (Armitage, Houseman & Turner, 1974) that some 20% pyrolysis of nicotine can occur during smoking. Approximately half this activity is transferred to mainstream smoke in the particulate phase of smoke and can therefore be trapped on the filters together with [14C]nicotine. T h e vapour phase activity will most certainly include [14C]carbon monoxide. T h e nature of the other pyrolysis products is as yet unknown. Significant metabolism to compounds other than [14C]cotinine occurred, however, since the proportion of unidentified activity in both experiments increased to a maximum of 307;. Lung tissue from experiments OC1 and OC2 contained a higher proportion of unidentified radioactivity than was the case with the 1A experiment and is consistent with the retention of a proportion of [14C]nicotine pyrolysis products in the organ. Significant still though were the proportions of [14C]cotinine and [14C]nicotine-l’-oxide in lung tissue of both smoke experiments. I n the closed circuit experiments much more extensive metabolism occurred ; the levels of [14C]cotinine, in one experiment (CC1) eventually reaching 28 ng/ml. T h e peak [14C]nicotine level in the blood of experiment CC1 was much less than that observed in CC2 during to the retention of much less [14C]nicotine in the lung during the smoking period. There was evidence again of [14C]nicotine pyrolysis and the levels of unidentified radioactivity were large with the proportion exceeding 20% during the latter half of each experiment indicating some metabolism to compounds other than [14C]nicotine. Indeed the extractable radioactivity recoveries from the lungs in these two experiments were smaller than in the two open circuit experiments, consistent with the longer time available for metabolism. Further, the percentage proportion of unidentified radioactivity in each lung was large and the [14C]nicotine correspondingly reduced. T h e proportion of radio-activity remaining in the lungs at the end of these two experiments was approximately 40%. I n experiment CC1 the amount of radioactivity retained by the lung was only half that retained in all other smoke experiments. This could be due to a significant difference in the depth of inhalation perhaps caused by a difference in the elasticity of the lung. I n 3 of the

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4 smoke experiments the proportion of smoke retained in the lung was approximately .SOY/,which is very similar to the retention observed in the anaesthetized cat when given smoke via the same machine (Armitage, Houseman & Turner, 1974). There is a difference in the relative proportions of [14C]cotinine to [14C]nicotine 1’-oxide produced by the two lungs of CC1 and CC2, which may be due to strain difference. T h e donor animal for experiment CC1 was a mongrel type whereas in all the other experiments the donor animals were greyhound. T h e [14C]-IV-oxide isolated from the lungs of experiment lA, yielded a ratio of 7.5 in favour of the laevorotatory isomer. Booth and Boyland (1970) observed that both lung and liver of the guinea-pig synthesize, in vitro, almost exclusively the luevo isomer. Jenner, Gorrod and Beckett (1973) reported that liver supernatants from rats, rabbits, mice and hamsters also formed predominantly the laevo isomer. T h e dextrorotatory isomer, however, was predominant in human smokers‘ urine but the urinary excretion data may be misleading if preferential metabolism of the luevo isomer occurs within the organism. T h e dog lung preparation is capable also of demethylation as evidenced by the presence of [14C]demethyl cotinine. Our results demonstrate the usefulness of the isolated lung perfusion technique as a tool for the study of the metabolism of smoke constituents and illustrate the importance of inhalation administration when assessing the capability of lung tissue to metabolize drugs whose normal route of entry into the organism is via the respiratory tract. Acknowledgments T h e authors express their thanks to Dr. T. H. Houseman for radiochemical analysis of the labelled cigarettes and the Cambridge filters; also to C. M. Sellers, P. S. B. Minty, Miss M. Budden and Miss J. Atkinson for their skilled technical assistance. Thanks are also due to B. Emmett for preparation of the figures. References ARMITAGE, A. K., HOUSEMAN, T. H., TURNER, D. M. & WILSON,D. A. (1974). Q.Jl. exp. Physiol., 59, 43. ARMITAGE,A. K., HOUSEMAN, ‘r.H. & TURNER, D. M. (1974). Q . JZ. exp. Physiol., 59, 55. BOOTH,J. & BOYLAND, E. (1970). Biochem. Pharmac., 19, 733. DALHAMN, T., EDFORS, M. & RYLANDER, R. (1968). Archs envir. Hlth, 17, 746. DAVIS,B. R., HOUSEMAN, T. H. & RODERICK, H. R. (1973). Beitruge zur Tabak, 7, 148. DECKER, K. & SAMMECK, R. (1964). Biochem. Z., 340,326. P. C. & SCHMITERLOW, C. G. (1964). Acta physiol. scund., 61, HANSSON, E., HOFFMANN, 380. A. P. (1969). Physiol. Revs., 49, 1. HEINEMANN, H. 0. & FISHMAN, HOUSEMAN, T. H. & HENEAGE, E. (1973 a). Beitruge zur Tabak, 7 , 138. HOUSEMAN, T. H. (1973 b). Beitruge zur Tabak, 7, 142. ISAAC,P. F. & RAND,M. J. (1969). Eur. J . Pharmac., 8, 269. JENNER, P., GORROD, J. W. & BECKETT, A. H. (1973). Xenobiotica, 3, 573. H . (1961). Naunyn Schmiedeberg’s Arch. exp. Path. Pharmak., 242, KIESE,M. & UEHLEKE, 117. NAKASHIMA, T. (1972). Foliu pharmac. Jap., 68, 29. K. & COSTA,E. (1971). Nature, 234, 48. NAKAZAWA,

Nicotine Metabolism by Dog Lung

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Metabolism of nicotine by the isolated perfused dog lung.

1. Metabolism of [14C]nicotine has been studied in the isolated perfused dog lung. [14C]Nicotine, 50 mug every 30 s for 10 min administered via the pu...
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