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Extrahepatic sites of metabolism of carbon tetrachloride in rats
299
Chrmdiol. Interactiona. 46 (19A3) 299-316 Elwvier Scientific PublisherR Ireland Ltd.
EPATIC TETRACHWRIDE
SITES OF IN RATS
LISM OF CA
HANS TJi6LVE and BOBL LC)FBEW
Departmentof Ta~kology. Universityof Uppsah. Box 573, ~7.5123 Uppsaia (Shmh) (ReceivedJanuary 29th. 1983) fR.ovision received April 14th, 1963) (Accepted April 19th. 1983) SUMMARY
Rats were injected i.v. and i.p. with [“Clcarbon tetrachloride and the localization and binding of metabolites in the tissues were studied by autoradiography. Based on the autoradiographic findings, various tissues were tested for their capacity to form “CO, from [“Clcarbon tetrachloride in vitro. Autoradiography in vitro was used to localize the sites of [“Clcarbon tetrachloride metabolism under in vitro conditions. The results showed that several tissues accumulating metabolites in vivo had an ability to form ‘“CO, in vitro, and accumulation of metabolites was observed also under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. The structures identified to have a marked carbon tetrachloride-metabolizing capacity were, besides the liver, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, the oesophageal mucosa. the mucosa of the larynx, the tongue and the cheeks, the lateral nasal gland and the kidney cortex. It is well established that the degradation of carbon tetrachloride involves the cytochrome P-450 system, and the metabolism of the substance in the mentioned tissues is probably correlated to high concentrations of cytochrorne P-450. The nasal olfactory mucosa was found to be the tissue with the highest capacity to form “CO* from the [“Clcarbon tetrachlnride and microautoradiography indicated that in this tissue the cells of the subepithelial glands of the lamina propria mucosae are most actively engaged in the metabolism. It was also shown that cytcmc rome P-450 is present in ths nasal olfactory mucosa. Key words: Carlon Cytochromc P-450 -.
tetract loride - Metabolism
- Autoradiography
-
I_-
INTRODUCTION
The liver Nonetheless,
plays the principal role in the extrahepatic drug metabolism
OOOSJ-2797/33/$03.00
0 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
breakdown may be
of xenobioticn. toxicologically
300
significant. Drugs may be inactivated by locally acting enzymes and the formation of toxic or reactive metabolites within cells of nonhepatic tissues may induce local toxicity. Extrahepatic metabolism may also be of great importance in chemical carcinogenesis. It is thus of interest to trace the extrahepatic tissues which are active iu degrading xenobiotics. Studies at our department have concerned the fate of several N=nitrosamines in experimental animals L41. The carcinogenicity of these compounds usually shows an expressed organ specificity, and our studies aimed at determining whether this could be correlated with a local metabolism of the N-nitrosamines in the target tissues. In these experiments, whole-body autoradiography was used to trace the extrahepatic tissues accumulating radioactivity. Many N-nitrosamines are volatile and autoradiography with dried sections could therefore be used to show the tissue-disposition of the metabolites. By washing the sections with trichloroacetic acid and organic solvents before the autoradiography it was also possible to localize tissuebound metabolites. Based on the autoradiographic observations, experiments were performed in vitro, in which the ability of the tissues to degrade the N-nitrosamines was determined. For non-polar N-nitrosamines, which are able to freely pass the ce!lular membranes, it was also possible to perform autoradiography in vitro with tissue pieces incubated with the labelled N-nitrosamines. In the present study, the extrahepatic metabolism of carbon tetrachloride has been examined in rats using the test battery outlined above. Carbon tetrachloride is a well known hepatotoxin, but noxious effects may also be induced in other tissues, notably the kidney and the lung [5,6]. It is well established that the metabolism of carbon tetrachloride involves the cytochrome P-450 system [7-81 and a mapping of the tissues engaged in carbon tetrachloride degradation will therefore provide information concerning the presence of cytochrome P-450 in the tissues. Bergman t101 used whole-body autoradiography in mice to study the disposition of inhaled carbon tetrachloride. In the present study, the compound was given i.v. or Lp. in order to circumgo the possibility of an excessive accumulation of metabolites in the upper respiratory and digestive system due to the inhalation es:posure. Metabolic experiments were performed in vitro and the formation of “CO2 from the IWlcarbon t.etrachloride was then used as a measure of the metabolism. Carbon dioxide is a major metabolite of carbon tetrachloride both in vivo and in vitro, reflecting the complete oxidative dechlorination of the compound [ 1 l-131. MATERIALS AND METHODS
Animals Female Sprague-Dawley rats, body wt. about 100 g, were used. The animals were fed a standard pellet diet for laboratory rodents and were given tap water ad libitum.
301
]‘“C]Carbon tetrachloride, spec. act., 2.8mCi/mmol, of :; 99% purity by gas=liquid radiochromatography, was obtained from the New England Nuclear, Dreieichenhein, F.R.G. Thin-layer chromatography on glass plates coated with 256 pm of silica gel, performed at -2O”C, using chloroform as solvent system and autoradiography at -80°C to localize radioactive materials, showed only one labelled spot containing >99% of the radioactivity on the plate.
Whole- body autoradiograph y k&s were injected i.v. (in a tail vein) or i.p, with 10 PCi (0.34 ~1) of [l4C]carbon tetrachioride dissolved in 10 ~1 absolute ethanol (the injections were performed with a Hamilton syringe). The animals were sacrificed by Cot2 asphyxiation after 5 min (one injected i.v. and one injected i.p.), 30 min (two injected i.v. and one injected i.p.) and 4 h (one injected i.v. and one injected i.p.). They were immediately embedded in a carboxymethyl cellulose gel and sectioned on tape (200pm thick sections) in a cryomicrotome according to published procedure [14]. From one animal killed 30 min after i.v. injection of [“C]carbon tetrachloride, transversal sections were taken through the head. All other rats were sectioned sagittally through the entire bodies. Fifteen duplicate sections were taken from each animal and were freezedried. To localize tissue-bound metabolites, every other freeze-dried section was washed with trichloroacetic acid and organic solvents, as described previously 121. The sections were then dried and apposed to X-ray film together with the adjacent non-extracted freeze-dried sections.
Micmautoradiography One rat was injected i.v. and one rat was injected i.p. with 1OpCi (0.34 ~1) of [14C]carbon tetrachloride in 10 ~1 absolute ethanol and the animals were killed after 1 h by COz asphyxiation. Pieces of the ethmoturbinates of the nose, the lung, the oesophagus and the liver were removed and fixed in 4% formaldehyde in a phosphate buffer (pH 7.0). After the fixation, the nasal region was decalcified in 44% formic acid/708 ethanol (1: 1) for 5 days. The tissues were then dehydrated in an ethanol series and embedded in paraffin. Five-micrometer thick sections were mounted on glras slides. After deparaffinization of the sections in ethanol, the slides were dipped in NTB-2 (Eastman-Kodak) liquid film emulsion and stored for 12 weeks at 4°C. After exposure, the slides were developed an stained with haematoxylineosin. In autoradiograms made under these conditions, it is assumed that only tissue=bound radioactivity is left in the tissues.
In uitro autorudiography Rats were decapitated and pieces of the nose, the lung plus the trachea, and the oesophagus were removed. The tissues were then incubated at 37°C for 60 min with 44.6 KM [“Clcarbon tetrachloride (0.25 &i; dissolved in 1~1 absolute ethanol) in 2 ml of Krebs-Ringer phosphate buffer @H 7.0) containing 10 n&I glucose. After the incubations, the tissues were rinsed for 1 min in
302 physiological saline and used for autoradiography as described above, either with tape sections or according to the microautoradiographic technique. Oxygen was always used as atmosphere for the tissues which were employed for m:croautoradiography. For the tissues which were tape-sectioned, either oxygen, nitrogen or carbon monoxide were used as atmospheres. The vials were flushed with each gas for 5min before the addition of the [‘“Clcarbon tetrachloride and then during the whole incl;,bation period. In vitro metabolism Rats were decapitated and selected tissues were excised and sliced. The tissues (30-100 mg) were then incubated In Warburg respiratory vessels at 37°C for 60 min with 44.6 FM [‘4C]carbon tetrachloride (0.25 FCi; dissolved in 1 p 1 absolute ethanol) in 2 ml of Krebs-Ringer phosphate buffer (pH 7.0) containing 10 mM glucose. Oxygen was always used as atmosphere and the vials were flushed for 5min with the gas and then stoppered with a thin was deterrubber cap. The 14C0.,_ formed from the [‘4C]carbon tet,rachloride mined by trapping on filter papers moistened with KOH as described previou,Jy [15]. Incubations with boiled livers served as controls and the obtajned blank values were subtracted before the calculation of the L4C0,form&ion. In the nasal region, the olfactory mucosa of the ethmot,urbinates is distinguishable by its light-brown colour and it. can be scraped off with a scalpel. The median regions of the nasal cavity with the naso- and maxilloturbinates and the septum have a respiratory mucosa and this can also be scraped off with a scalpel. We performed incubations both with the olfactory and the respiratory mucosa of nasal cavity. Determ ina lion of cytochrome P-450 in the nasal olfactory m ucosa As will appear in the results, the nasal olfactory mucosa was found to have a high capacity of form 14C02 from the I14C]carbon tetrachloride and it was therefore decided to determine the contents of cytochrome P-450 in this tissue. Due to the small amount of tissue available, the cytochrome P-450 in the nasal olfactory mucosa was determined in whole tissue homogenates according to the method of McLean and Day 1161. As a comparison, the cytochrome P-450 was also determined in liver homogenates. Protein was measured according to Lowry et al. [17]. RESULTS
Waole-body autoradiography The whole-body autol,adiography showed a localization of radioactivity in several tissues with similar distrib-ution patterns in the i.v. and the i-p. injected animals, and only small variations in the pictures at the different survival intervals. Washings of the sections with trichloroacetic acid and organic solvents betore the autoradiography removed some of the radioactivity from all tissues, but the relative labelling between the various tissues was the same in autoradiograms of freeze-dried and washed sections.
asal mucosa
ucosa of the tongue Nasal mucosa
Kidnev
ronchi
Oesophageal
Lateral nasal gland
mucosa
Liver Bronchi
Liver
ucosa of the tongue
Fig. 1. Whole-body autoradiograms of rats (A) 30min chloride and (B) 4 h after i.p. injwtton of ]“(‘]carbon
&trr i v InJection of ]:‘C]carkon tetr+ ttat~.~~nl~rr~&. ThtA frtwzr-drlt4. s.+_~tt.~l
sections were exposed for 3 months.
The liver showed
a strong labelling at all survival intervals (Fig. 1). .Jit was rather evenly distributed in the parenchyma but at later survival intervals t.hrrtl was an irregular pattern, with thta highest labelling in the central parts of the lobuli. A strong radioactivity was prrsent at all survival intervals in the mucosa of the oesophagus. the IalFns. thr tongue and the cheeks (Figs. 1 and 2). The mucosu of the palate was only weakly labelled. In the nose a strong labelling was prevent at all times in the mucosa uf the ethmoturbinates and in the lateral nasal gland (Steno‘s gland), whicT1 occupies the submucosa anterior, lateral and inferior to the maxillaq 5 min, the radioactivity
UCOSAOF THECHEEKS
MUCOSA OF THE
MUCOSAOF
BULB)
THE TONGUE
BRAlNlOLFACTORY
MAXILLARY SINUS
COSAOF THE CHEEK
MUCOSAOF THE ETHMOTURBINATES
MUCOSA OF THE TONGUE
LATERAL NASAL GLANDS
TONGUE
BULB)
LATERAL NASAL GLANDS ETHM~TuRBINATEs
.” : : : :
; : :
Fig. 2. A, B: details of autoradiograms of a rat 30 mm after an i.v. injection of i’Y~~carLon w.rauhiortdc. (‘. D: haematoxyhn-eosin corresponding to A and H respectwely. (A, C) are from the postrlrior portion of the nose. (B, D) are from the median portion freeze-dried, transversal sections were exposed for 3 months.
CHEEK
t
.za
.’
OLFACTORY
stained sections of the nose. The
sinus (Figs. 1 and 2). A close examination of labelling of the ethmoturbinates showed that the highest radioactivity was present in the lamina propria mucosae, below the surface epithelium. There was also a marked labelling of the mucosa of the median regions of the nasal cavity. The mucosa of the trachea and the mucosa of the bronchial tree were labelled to a moderate extent. The lung parenchyma was only weakly labelled. In the abdomen a marked radioactivity was present in the kidney cortex and in the contents of the intestines and the stomach- the gastric labelling being highest on the inner side of the mucosa. The blolod was labelled to a low extent at all survival intervals and a low radioactivity was also present in the bonemarrow, the salivary glands, brown fat and the adrenal cortex. Microautoradiogrczphy The microautoradiograms of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands (Bowman’s glands) of the lamina propria mucosae (Fig. 3A). The olfactory epithelium was labelled to a lower extent. In the lung a labelling was present in the epithelium of the bronchi and the bronchioles, whereas the radioactivity in the parenchyma was low. In the oesophagus there was a strong labelling of the stratified squamous epithelium (Fig. 3B). In the liver, the strongest labelling was present over the central parts of the lobuli (Fig. 3C). In vitro autoradiography In the autoradiograms obtained with tape sections of lungs incubated with [“C]carbon tetrachloride under an atmosphere of oxygen or nitrogen, a strong labelling was present in the epithelium of the bronchial tree (Figs. 4A and 4B). The lung parenchyma was labelled to a lower extent. There was also a marked radioactivity in tracheal epithelium. In the incubations in an atmosphere of carbon monoxide there was almost no labelling of the lung or the trachea (Fig. 4C). The incubations with the oesophagus showed a strong labelling of the epithelium when oxygen or nitrogen were used as atmospheres (Figs. 5A and 5B). A carbon monoxide atmosphere very markedly depressed this labelling (Fig. 5C). The incubations with the nose resulted in a strong labelling of the mucosa of the ethmoturbinates in oxygen or nitrogen atmospheres, but only a very low labelling in a carbon monoxide atmosphere (data not shown). The in vitro microautoradiography gave similar results as described in vivo, with a labelling of the subepithelial glands of the olfactory mucosa, the epithelium of the bronchial tree of the lung (Fig. 3D) and the squamous epithelium of the oesophagus. In aitrw metabolism The nasal olfactory mucosa was found to have the highest capacity to form n4C02from the [“Clcarbon tetrachloride (Table I). As was expected, the liver was also very active. i4COz-formation in decreasing order was also noted in the oesophagus, the nasal respiratory mucosa, the cheeks, the larynx, the
Fig. 3. Micraautoradiograms showing: the nasal olfactory mucosa Ih after iv. injection of [“C]carbon tetrachioride (A), the oesophagus 1 h after iv. injection of [“Clcarbon tetrachloride (B), the liver Ih after i.p. injection of [“Clcarbon tetrachloride (C) and the lung after in vitro incubation for 1 h in a buffor solution containing [‘Clcarbon tetrachloride (IN The autoradiograms show tissue-bound radioactivity. In (A) there is a labclling of the olfactory subepithelial glands of the lamina propria mucosae. In (B) there is a labelling of the stratified squamous epithelium of the oesophagus. In (C) the strongest labelling is present in the central part of the liver lobule around the central vein. In (D) there iti a marked labelling of the bronchial epithelium, whereas the labelling of the alveoli is low. OL, olfactory epithelium: SC. subepithelial glands (Bowman’s glands): SE. squamous epithelium of the oesophagus; CV. central vein; B. bronchus. x460.
308
4. A-C:
autoradlograms
of
1UII~Sand tracheas Incubated
for 1 h in buffer
solutions
containing
[W]carbon
tetrachlorlde.
l_t
: Li
:
:
0
: :
II-F.
haematoxplin-
rosin stained sections correspondmg to A, I3 and (:. reqprctlvely The atmospheres used during the incubations were for (A. DI oxygen. (B. I?, nitrogen and (CT.I?) carbon monoxide. The tissue srrtions were extracted with trlchloroacetlc acid and organic solvents before the autoradiography In (A) and (I%) there is a strong labelllng of the rplthellum of the bronchial trrr and the trachea and ;I low labrlling of the lung parenchyma. in (Ci nc, Iwhrlling of the tissues can hr. d tected. Thr exposure-time was 2 months.
Fig.
RONCnlOLES
TRACI-IEA
MUSCLE MUCOSA
MUCOSA
MUSCLE
3
MUCOSA
MUCOSA
MUSCLE i
Fig. 5. A-C: autoradiograrns of oesophagus incubated for 1 h in buffer solutions containing [‘“C~arbon tetrachloride. D-F: hae stained sections corresponding to A, B and C, respectively. The atmospheres used during the incubations were for (A, D) oxygen, (B, (6, F) carbon monoxide. The tissue sections were extracted with trichloroacetic acid and organic solvents before the autoradiograph> there is a strong labelhng of the oesophageal mucosa. In (C) th? labelling is markedly reduced. The exposure time was 2 months.
MUCOSA
MUCOSA
w c,
311
Y VARIOIB
r papers
erpres res within
TISSUES
IN
37°C under an atmos(0.25pCi) in 2 ml of ‘CO2 formed during the e results show the radioacminute (dpm) per mg brackets denote the number of in-
cubations. Tissue
W!On-formation (dpmlmg of wet wt) -
Nasal olfactory Liver (12)
mucosa (12)
Cheek (3)
Trachea (6) Small intestine Lung (6) Adrenal (3)
(3)
salivary
gland (6)
Glandular stomach (6) Forestomach (6)
idney cortex, y the other ti
75.5 2 4.4 62.1) 4.4 30.6 2 I.7 27.2 -t 5.6 19.9 z 4.0
16.8~6.6 15.523.9 12.822.1 8.6r0.3 3.4*Q.6 3.2% 1.1 3.020.7 2.8-cO.3 2.420.6 1.8~0.7 1.5t0.3 1.220.3 1.220.8 1.0zO.6 0.8-rO.S 0.6-cO.4
lateral nasal gland and the csngue. The 14C 9 tested wa8 low.
was detected in the homogenates of tLe nasal olfactory about one third of t e level in the liver, ased on the Gssue weight, and on the
f?W Qf
the co
tetrac tion of
rnetaboli tetrachlo
312 TABLEII CYTOCH’ROME P4610 CONTENTSOF THE NASAL OLFACTORYMUCOSA AND THE LiVER The cytochrw e 8’460 was determinedin homogenatee of rattisauee aa etated in Materialsand Methods.ResuIt~~ are expresseda~ means2 S.E. (n = 7).
-Neal
Liver
olfactory
muco8a
nmolP-GO/g of wet wt.
nmolPMOlmg of protein
9.9 It0.9 26.1 -c1.2
0.22 r 0.01 0.612 0.06
P-450 and the subsequent reduction of the complex by NADPH in the catalytic presence of cytochrome P-450 reductaee [8,9,181. The free trichloro=
methyl radical (CC&J may initiate peroxidation of polyunsaturated fatty acids or it may react with macromolecules, such as proteins and lipids,:and these events will ultimately induce the noxious effects in the tissues [7,8]. By hydrogen abstraction, chloroform (CHCls) is also formed 17,181. The formation of carbon dioxide from carbon tetrachloride is due to the oxidative dechlorination of chloroform and this process also proceeds via cytochrome P-450 dependent pathways [12,13,19]. Previous distribution studies by low-temperature autoradiography have indicated that the non-metabolized carbon tetrachloride can freely pass the biological membranes and reach all tissues of the body [lo]. The autoradiography in the present study, in which freeze-dried or extracted tissues were used, will reveal only the localization of metabolites. The results showed that several tissues accumulating metabolites in vivo had an ability to form “COZ in vitro, and accumulation of metabolites was also observed under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. Since the metabolism of carbon tetra= chloride proceeds by cytochrome P-450mdependent pathways, it is of interest to examine whether our results can be correlated, to the sites of cytochrome P-450-activity in the tissues. In the lung, the bronchiolar Clara cells are the main sites of the cyto. chrome P-450 system, but a low level is also present in the alveolar type=11cells 120,211. The administration of carbon tetrachloride to rats has been shown to induce an acute toxic effect on the Clara cells [6]. Our autoradiography indicated a marked metabolism of carbon tetrachloride in the epithelial lining of the bronchial tree and a low metabolism in the lung parenchyma, and these results probably reflect the metabolic capacity of the mentioned cells. Our results also indicated a metabolic capacity of the tracheal epithelium. Oxidative enzymes have been shown histochemically in the surface epithelium of the trachea [22]. The results showed that the epithelium of the oesophagus is very active’in
313
metabolizing carbon tetrachloride. Cytochrome P-450 has been demonstrated in the oesophagus [231. Our results in addition showed a high metabolism of carbon tetrachloride in the mucosa of the larynx, the cheeks and the t&ngue, which are covered with an epithelium of a similar type as in the oesophagus. It should be noted that the calculations of the i4COrproduction by the oesophagus, the larynx, the cheeks and the tongue, and also by the lung and the trachea, are based on the weights of heterogeneous tissues. The metabolizing capacity of the muccsal cells in these tissues are therefore probabiy much higher than is shown by the “COz-production, and may be better reflected by the intensity of the autoradiographic labelling. The nasal olfactory epithelium was found to have a very high capacity to form “COp from the [“Clcarbon tetrachloride, and the microautoradiography indicated that in this tissue the cells of the subepithelial glands (Bowman’s glands) are most actively engaged in the metabolism. Histochemical studies have shown high contents of oxidative enzymes in the subepithelial glands 1241 and the cells of these glands are also rich in endoplasmic reticulum [25], which is the major source of cytochrome P-450.dependent enzymes. We have previously shown that N-nitrosamines are metabolized in the nasal mucosa, and as for carbon tetrachloride, microautoradiography showed the highest labelling over the subepithelial glands [26,27]. Our determination of cytochrome P-450 in the nasal olfactory mucosa showed an amount which was about one third of the concentration in the liver. The level recorded was higher than the one reported by Hadley and Dahl in the nasal mucosa If F344 rats 1281.This may partly 1103J!&ed to the fact that those authors ued nasal areas which included both the olfactory and the respiratory mucosa, whereas we only used the olfactory mucosa, and also to the fact that they used a microsomal preparation, whereas we used tissue homogenates. The nasal respiratory mucosa was in the present study found to have a marked capacity to degrade carbon tetrachloride, but the activity was considerably lower than in the olfactory mucosa. In spite of the higher cytochrome P-450 concentration in the liver than in the nasal olfactory mucosa, the latter tissue produced somewhat more *‘CO2 from the [14C]earbon tetrachloride than the liver. The correlation between cytochrome P-450 concentration and metabolism appears to be qualitatively good, but not strictly quantitatively valid. This may be due to the fact that cytochrome P-450 is not a single entity but consists of various forms which may have different substrate specificities [291. Metabolism of carbon tetraihloride was observed in the lateral nasal gland (Steno’s gland), The acinar cells of this gland contain a well developed endop]asmic reticulum [30]. We have previously observed tissue-bound metabolites in the lateral nasal gland in rats injected with two tobaccospecific N=nitrosamines 14,261. Carbon tetrachloride is a nephrotoxic agent, inducing injury mainly of the kidney cortex [5]. Cytochrome P-450 has been shown in the kidney, with higher activity in the cortex than in the medulla 1311. In agreement with these results, our study showed a “COP-formation from [‘“Clcarbon tetra-
314 chloride by the kidney cortex, but not the kidney meldulla, and the autoradiography showed a higher labelling of the cortex than of the medulla. In the liver, the strongest labelling was observed over the central parts of the lobuli. This can be correlated to the centrilobular liver injury which carbon tetrachloride induces 1321. The in vitro autoradiography showed that incubations performed in a carbon monoxide atmosphere induced a strong inhibition of the binding of metabolites in the oesophagus, the lung and the nose as compared with the incubations performed in oxygen or nitrogen atmospheres. Reduction by carbon monoxide of the binding of carbon tetrachloride to liver microsomal pro+eins has been shov:7 previously and is indicative of a cytochrome P-460dependent pathway [7,9]. The initinl step in carbon tetrachloride metabolism, leading to the formation of the reactive trichloromethyl radical is purely reductive and should proceed under anaerobic conditions. This concords with our autoradiographic observation of a strong binding in a nitrogen atmosphere, and similar results l..qve been obtained with liver microsomal preparations [7,9]. The tissue disposition of carbon tetrachloride metabolites in the rats in the present study is similar to the pattern observed in mice by Bergman [lo]. An exception, however, is the gastrointestinal mucosa, which was more strongly labelled in the mice than in the rats. It is possible that this at least partially may be due to the differences in the routes of the carbon tetrachloride exposure: At inhalation, as performed by Bergman [lo], a part of the [“Clcarbon tetrachloride may be mixed with the secretions of the nose and the mouth and, by swallowing, reach the gastrointestinal contents. This, in turn, may give opportunity for a local degradation of the [“C]carbon tetrachloride in the alimentary canal and as a consequence a labelling of the gastrointestinal walls. The present study has disclosed that carbon tetrachloride metabolism is remarkably active in the mucosal linings of tissues which are exposed orally or by inhalation to drugs and other environmental chemicals. The cytochrome P-450 system which appears to be active at these sites, may constitute a protection mechanism defending the body from unrestrained uptake of xenobiotics. In certain instances, however, and this applies to carbon tetrachloride, the metabolism may instead resuPt in a bioactivation locally in the cells. Carbon tetrachloride should thus be potentially toxic towards the extrahepatic metabolizing tissues. As mentioned previously, noxious effects have been shown in tissues such as the kidney and the lung [6,7], but no attempts were made in the present study to establish whether injuries can also be induced in -the other tissues with metabolizing capacity, REFERENCES 1 E.B. Brittebo, B. LiMberg end H. Tjiilvr, Sites of metabolism mice, C&em.-Biol. Interact., 34 (1981) 209.
of N-nitroaodiethylamine
in
315 2 E.B. Brittebo and H. Tjiilve. Formation of tissue-bound N’-nitrosonomlcotjne metabolih the t-get tieauen of Sprague-Dawley and Fisher rata, Carcinogeneeis. 2 (1981) 959.
by
3 E. Brittobo, B. LBfherg and H. Tjiilve, Extrahepatic sites of metabolism of N.nitmmpyr. rolidine in mice and rata, Xenobiotica. 11 (19131) 619. tonguay, H. Wilvr: and S.S. Hecht, Tiesue distribution of the tobaxo-specific car. cinol?en C(methylnit~eminor.l~(Sl~yyridyl)-l-butnnone. and its metabolites in F.344 rats, Cancer Rec.. 43 (1983) 630. 15 R.B. Jennings and W.M. Keens, Necrotizing nephronis in the rat following administration of carbon tetrachloride. Arch. Pathol., F6 (1963) 348. 6 MR. Boyd, C.N. Statham and N.S Longo, The pulmonary Clara cells as a target for toxic chemicals requiring metabolic activation; Studies with carbon tetrachloride, J. Pbamlacol. Exp. Ther., 212 (19801 109. 7 H. Uehleke. K.H. Hellmer and S. Tabarelli, Binding of “C-carbon tetrachloride to micro. somal proteins in vitro and formation of CHC13 by reduced liver microsome, Xenobiotica. 3 (1973) 1. 8 ML Dial time& J.A. Castro, E.C. de Ferreyra, N. D’Acosta and C.R. de Castro, Irreversible bir.+ng of “C from “CC4 to liver microsomal lipids and proteins from rats pretreated with compounda altering microsomal mixed-function oxygenase activity, Toxicol. Appl. Pharmacol.. 25 (1973) 534. 9 LG. Sipes, G. Krishna and J.R. Gillette, Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane; role of cytoehrome P-450. Life Sci, 20 (1977) 1541. 10 K. Bergman, Whole-body autoradiography and allied tracer techniques in distribution and elimination studies of some organic solvents, Stand. J. Work Environ. Health, 5 (1979) Suppl. 1. 11 B.B. Paul and D. Rubinstein, Metabolism of carbon tetrachloride and chloroform by the rat, J. Pharmacol. Exp. Ther.. 141 (1963) 141. 12 D. Rubinstein and L. Kanics, The conversion of carbon tetrachloride and chloroform to carbon dioxide by rat liver homogenates, Can. J. Biochem., 42 (1964) 1577. 13 D.H. Hathway. Chemical, biochemical and tnxicological differences between carbon tetrachloride and chloroform, Anneim.-Forsch., 24 (1974) 173. 1.4 S. Ullberg, The technique of whole-body autoradiography. Cryosectioning of large specimens. Science Tools, Special Issue (1977). 15 E. Johansson-Brittebo and H. Tjlilve. Studies on the distribution and metabolism of “Cdimethylnitmsamine in foetal and young mice, Acta Pharmacol. Toxicol.. 45 (1979) 73. 16 A.E.M. McLean and P.A. Day, The use of new methods to measure: the effect of diet and inducers of microsomal enzyme synthesis on cytochrome P-450 in liver homogenates. and the metabolism of dimethylnitrosamine. Biochem. Pharmacol., 23 (1974) 1173. 17 O.H. Lowry, N.J. Rosebrough. A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem.. 193 (1951) 265. 18 H.J. Ahr. L.J. King, W. Nastainczyk and V. Ullrich, The mechanism of chloroform and carbon monoxide formation from carbon tetrachloride by microsomal cytochrome P-450. Biochcm. Pharmacol., 29 (1980) 2855. 19 A.E.M. McLean, The effect of protein deficiency and microsomal enzyme induction by DDT and phenobarbitone on the acute toxicity of chloroform and a pyrrolizidine alkaloid. Retrosine, Br. J. Exp. Pathol., 61 (1970) 317. 20 M. Boyd. Evidence for the Clara cell as a site of cytochrome P-450.dependent mixed-function oxidase activity in the lung, Natun,. 269 (1977) 713. 21 T.R. Devereux, C. &rbjit-Singh, S.R. Slaughter, C.R. WOK. R.M. Philpot and J.R. Fo&s. Identification of cytochrome P-460 isozymes in non-ciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated from rabbit lung, Exp. Lung Res., 21 (1981) 221. 22 A. Azzopardi and W.M. Thurlbeck, Oxidative enzyme pattern of the bronchial mucous glands, Am. Rev. Respir. Die., 97 (1968) 1038. 23 G.E. Labuc and M.C. Archer, Esophageal and hepatic microsomal methabolism of Nnitroaomethylbenzylamine and N-nitrosodimethylamine in the rat, Cancer bs., 42 (1982) 3181.
24 E. Mira, Cxidative and Hydrolytic enzymes in Bowman’s glands, Acta Cto-Laryng., 66 (1963) 706. 25 D. Frisch, Ultrastructure of mouse olfactory mucosa, Am. J. Anat., 121 (1967) 87. 26 B. Liifberg, E.B. Brittebo and H. Tjiilve, Localization and binding of IV’-nitrosonomicotine metabolites in the nasal region and in some other tissues of Sprague-Dawley rats, Cancer Res., 42 (1982) 2877. 27 H. Tjiilve and A. Castonguay, The in vivo tissue disposition and in vitro target-tissue metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino).I-(3.pyridyl)-1.butanone in Syrian golden hamsters, Carcinogenesie (1983) in press. 28 W.M. Hadley and A.R. Dahl, Cytochrome P-450 dependent monooxygenase activity in rat nasal epithelia) membranes, Toxicology Lett., 10 (1982) 417. 29 A.Y.H. Lw and S.B. West, Multiplicity of mammalian microsomal cytochromes P-450, Phannacol. Rev., 31 (1980) 277. 39 H. Moe and F. Bojsen-Moller. The fine structure of the lateral nasal gland (Steno’s gland) of the rat, J. Ultrastruct. Res., 36 (1971) 127. 31 Y. Ichikawa and T. Yamano, Electron spin resonance of microsomal cytochromes, Arch. Biochem. Biophys.. 121(1967) 742. 32 E.H. Ledue and J.W. Wilson, Injury to liver cells in carbon tetrachloride poisoning, Arch. Pathol., 65 (1958) 147.
Chrmdiol. Interactiona. 46 (19A3) 299-316 Elwvier Scientific PublisherR Ireland Ltd.
EPATIC TETRACHWRIDE
SITES OF IN RATS
LISM OF CA
HANS TJi6LVE and BOBL LC)FBEW
Departmentof Ta~kology. Universityof Uppsah. Box 573, ~7.5123 Uppsaia (Shmh) (ReceivedJanuary 29th. 1983) fR.ovision received April 14th, 1963) (Accepted April 19th. 1983) SUMMARY
Rats were injected i.v. and i.p. with [“Clcarbon tetrachloride and the localization and binding of metabolites in the tissues were studied by autoradiography. Based on the autoradiographic findings, various tissues were tested for their capacity to form “CO, from [“Clcarbon tetrachloride in vitro. Autoradiography in vitro was used to localize the sites of [“Clcarbon tetrachloride metabolism under in vitro conditions. The results showed that several tissues accumulating metabolites in vivo had an ability to form ‘“CO, in vitro, and accumulation of metabolites was observed also under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. The structures identified to have a marked carbon tetrachloride-metabolizing capacity were, besides the liver, the mucosa of the bronchial tree, the tracheal mucosa, the olfactory and respiratory nasal mucosa, the oesophageal mucosa. the mucosa of the larynx, the tongue and the cheeks, the lateral nasal gland and the kidney cortex. It is well established that the degradation of carbon tetrachloride involves the cytochrome P-450 system, and the metabolism of the substance in the mentioned tissues is probably correlated to high concentrations of cytochrorne P-450. The nasal olfactory mucosa was found to be the tissue with the highest capacity to form “CO* from the [“Clcarbon tetrachlnride and microautoradiography indicated that in this tissue the cells of the subepithelial glands of the lamina propria mucosae are most actively engaged in the metabolism. It was also shown that cytcmc rome P-450 is present in ths nasal olfactory mucosa. Key words: Carlon Cytochromc P-450 -.
tetract loride - Metabolism
- Autoradiography
-
I_-
INTRODUCTION
The liver Nonetheless,
plays the principal role in the extrahepatic drug metabolism
OOOSJ-2797/33/$03.00
0 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
breakdown may be
of xenobioticn. toxicologically
300
significant. Drugs may be inactivated by locally acting enzymes and the formation of toxic or reactive metabolites within cells of nonhepatic tissues may induce local toxicity. Extrahepatic metabolism may also be of great importance in chemical carcinogenesis. It is thus of interest to trace the extrahepatic tissues which are active iu degrading xenobiotics. Studies at our department have concerned the fate of several N=nitrosamines in experimental animals L41. The carcinogenicity of these compounds usually shows an expressed organ specificity, and our studies aimed at determining whether this could be correlated with a local metabolism of the N-nitrosamines in the target tissues. In these experiments, whole-body autoradiography was used to trace the extrahepatic tissues accumulating radioactivity. Many N-nitrosamines are volatile and autoradiography with dried sections could therefore be used to show the tissue-disposition of the metabolites. By washing the sections with trichloroacetic acid and organic solvents before the autoradiography it was also possible to localize tissuebound metabolites. Based on the autoradiographic observations, experiments were performed in vitro, in which the ability of the tissues to degrade the N-nitrosamines was determined. For non-polar N-nitrosamines, which are able to freely pass the ce!lular membranes, it was also possible to perform autoradiography in vitro with tissue pieces incubated with the labelled N-nitrosamines. In the present study, the extrahepatic metabolism of carbon tetrachloride has been examined in rats using the test battery outlined above. Carbon tetrachloride is a well known hepatotoxin, but noxious effects may also be induced in other tissues, notably the kidney and the lung [5,6]. It is well established that the metabolism of carbon tetrachloride involves the cytochrome P-450 system [7-81 and a mapping of the tissues engaged in carbon tetrachloride degradation will therefore provide information concerning the presence of cytochrome P-450 in the tissues. Bergman t101 used whole-body autoradiography in mice to study the disposition of inhaled carbon tetrachloride. In the present study, the compound was given i.v. or Lp. in order to circumgo the possibility of an excessive accumulation of metabolites in the upper respiratory and digestive system due to the inhalation es:posure. Metabolic experiments were performed in vitro and the formation of “CO2 from the IWlcarbon t.etrachloride was then used as a measure of the metabolism. Carbon dioxide is a major metabolite of carbon tetrachloride both in vivo and in vitro, reflecting the complete oxidative dechlorination of the compound [ 1 l-131. MATERIALS AND METHODS
Animals Female Sprague-Dawley rats, body wt. about 100 g, were used. The animals were fed a standard pellet diet for laboratory rodents and were given tap water ad libitum.
301
]‘“C]Carbon tetrachloride, spec. act., 2.8mCi/mmol, of :; 99% purity by gas=liquid radiochromatography, was obtained from the New England Nuclear, Dreieichenhein, F.R.G. Thin-layer chromatography on glass plates coated with 256 pm of silica gel, performed at -2O”C, using chloroform as solvent system and autoradiography at -80°C to localize radioactive materials, showed only one labelled spot containing >99% of the radioactivity on the plate.
Whole- body autoradiograph y k&s were injected i.v. (in a tail vein) or i.p, with 10 PCi (0.34 ~1) of [l4C]carbon tetrachioride dissolved in 10 ~1 absolute ethanol (the injections were performed with a Hamilton syringe). The animals were sacrificed by Cot2 asphyxiation after 5 min (one injected i.v. and one injected i.p.), 30 min (two injected i.v. and one injected i.p.) and 4 h (one injected i.v. and one injected i.p.). They were immediately embedded in a carboxymethyl cellulose gel and sectioned on tape (200pm thick sections) in a cryomicrotome according to published procedure [14]. From one animal killed 30 min after i.v. injection of [“C]carbon tetrachloride, transversal sections were taken through the head. All other rats were sectioned sagittally through the entire bodies. Fifteen duplicate sections were taken from each animal and were freezedried. To localize tissue-bound metabolites, every other freeze-dried section was washed with trichloroacetic acid and organic solvents, as described previously 121. The sections were then dried and apposed to X-ray film together with the adjacent non-extracted freeze-dried sections.
Micmautoradiography One rat was injected i.v. and one rat was injected i.p. with 1OpCi (0.34 ~1) of [14C]carbon tetrachloride in 10 ~1 absolute ethanol and the animals were killed after 1 h by COz asphyxiation. Pieces of the ethmoturbinates of the nose, the lung, the oesophagus and the liver were removed and fixed in 4% formaldehyde in a phosphate buffer (pH 7.0). After the fixation, the nasal region was decalcified in 44% formic acid/708 ethanol (1: 1) for 5 days. The tissues were then dehydrated in an ethanol series and embedded in paraffin. Five-micrometer thick sections were mounted on glras slides. After deparaffinization of the sections in ethanol, the slides were dipped in NTB-2 (Eastman-Kodak) liquid film emulsion and stored for 12 weeks at 4°C. After exposure, the slides were developed an stained with haematoxylineosin. In autoradiograms made under these conditions, it is assumed that only tissue=bound radioactivity is left in the tissues.
In uitro autorudiography Rats were decapitated and pieces of the nose, the lung plus the trachea, and the oesophagus were removed. The tissues were then incubated at 37°C for 60 min with 44.6 KM [“Clcarbon tetrachloride (0.25 &i; dissolved in 1~1 absolute ethanol) in 2 ml of Krebs-Ringer phosphate buffer @H 7.0) containing 10 n&I glucose. After the incubations, the tissues were rinsed for 1 min in
302 physiological saline and used for autoradiography as described above, either with tape sections or according to the microautoradiographic technique. Oxygen was always used as atmosphere for the tissues which were employed for m:croautoradiography. For the tissues which were tape-sectioned, either oxygen, nitrogen or carbon monoxide were used as atmospheres. The vials were flushed with each gas for 5min before the addition of the [‘“Clcarbon tetrachloride and then during the whole incl;,bation period. In vitro metabolism Rats were decapitated and selected tissues were excised and sliced. The tissues (30-100 mg) were then incubated In Warburg respiratory vessels at 37°C for 60 min with 44.6 FM [‘4C]carbon tetrachloride (0.25 FCi; dissolved in 1 p 1 absolute ethanol) in 2 ml of Krebs-Ringer phosphate buffer (pH 7.0) containing 10 mM glucose. Oxygen was always used as atmosphere and the vials were flushed for 5min with the gas and then stoppered with a thin was deterrubber cap. The 14C0.,_ formed from the [‘4C]carbon tet,rachloride mined by trapping on filter papers moistened with KOH as described previou,Jy [15]. Incubations with boiled livers served as controls and the obtajned blank values were subtracted before the calculation of the L4C0,form&ion. In the nasal region, the olfactory mucosa of the ethmot,urbinates is distinguishable by its light-brown colour and it. can be scraped off with a scalpel. The median regions of the nasal cavity with the naso- and maxilloturbinates and the septum have a respiratory mucosa and this can also be scraped off with a scalpel. We performed incubations both with the olfactory and the respiratory mucosa of nasal cavity. Determ ina lion of cytochrome P-450 in the nasal olfactory m ucosa As will appear in the results, the nasal olfactory mucosa was found to have a high capacity of form 14C02 from the I14C]carbon tetrachloride and it was therefore decided to determine the contents of cytochrome P-450 in this tissue. Due to the small amount of tissue available, the cytochrome P-450 in the nasal olfactory mucosa was determined in whole tissue homogenates according to the method of McLean and Day 1161. As a comparison, the cytochrome P-450 was also determined in liver homogenates. Protein was measured according to Lowry et al. [17]. RESULTS
Waole-body autoradiography The whole-body autol,adiography showed a localization of radioactivity in several tissues with similar distrib-ution patterns in the i.v. and the i-p. injected animals, and only small variations in the pictures at the different survival intervals. Washings of the sections with trichloroacetic acid and organic solvents betore the autoradiography removed some of the radioactivity from all tissues, but the relative labelling between the various tissues was the same in autoradiograms of freeze-dried and washed sections.
asal mucosa
ucosa of the tongue Nasal mucosa
Kidnev
ronchi
Oesophageal
Lateral nasal gland
mucosa
Liver Bronchi
Liver
ucosa of the tongue
Fig. 1. Whole-body autoradiograms of rats (A) 30min chloride and (B) 4 h after i.p. injwtton of ]“(‘]carbon
&trr i v InJection of ]:‘C]carkon tetr+ ttat~.~~nl~rr~&. ThtA frtwzr-drlt4. s.+_~tt.~l
sections were exposed for 3 months.
The liver showed
a strong labelling at all survival intervals (Fig. 1). .Jit was rather evenly distributed in the parenchyma but at later survival intervals t.hrrtl was an irregular pattern, with thta highest labelling in the central parts of the lobuli. A strong radioactivity was prrsent at all survival intervals in the mucosa of the oesophagus. the IalFns. thr tongue and the cheeks (Figs. 1 and 2). The mucosu of the palate was only weakly labelled. In the nose a strong labelling was prevent at all times in the mucosa uf the ethmoturbinates and in the lateral nasal gland (Steno‘s gland), whicT1 occupies the submucosa anterior, lateral and inferior to the maxillaq 5 min, the radioactivity
UCOSAOF THECHEEKS
MUCOSA OF THE
MUCOSAOF
BULB)
THE TONGUE
BRAlNlOLFACTORY
MAXILLARY SINUS
COSAOF THE CHEEK
MUCOSAOF THE ETHMOTURBINATES
MUCOSA OF THE TONGUE
LATERAL NASAL GLANDS
TONGUE
BULB)
LATERAL NASAL GLANDS ETHM~TuRBINATEs
.” : : : :
; : :
Fig. 2. A, B: details of autoradiograms of a rat 30 mm after an i.v. injection of i’Y~~carLon w.rauhiortdc. (‘. D: haematoxyhn-eosin corresponding to A and H respectwely. (A, C) are from the postrlrior portion of the nose. (B, D) are from the median portion freeze-dried, transversal sections were exposed for 3 months.
CHEEK
t
.za
.’
OLFACTORY
stained sections of the nose. The
sinus (Figs. 1 and 2). A close examination of labelling of the ethmoturbinates showed that the highest radioactivity was present in the lamina propria mucosae, below the surface epithelium. There was also a marked labelling of the mucosa of the median regions of the nasal cavity. The mucosa of the trachea and the mucosa of the bronchial tree were labelled to a moderate extent. The lung parenchyma was only weakly labelled. In the abdomen a marked radioactivity was present in the kidney cortex and in the contents of the intestines and the stomach- the gastric labelling being highest on the inner side of the mucosa. The blolod was labelled to a low extent at all survival intervals and a low radioactivity was also present in the bonemarrow, the salivary glands, brown fat and the adrenal cortex. Microautoradiogrczphy The microautoradiograms of the nasal olfactory mucosa showed the highest labelling over the subepithelial glands (Bowman’s glands) of the lamina propria mucosae (Fig. 3A). The olfactory epithelium was labelled to a lower extent. In the lung a labelling was present in the epithelium of the bronchi and the bronchioles, whereas the radioactivity in the parenchyma was low. In the oesophagus there was a strong labelling of the stratified squamous epithelium (Fig. 3B). In the liver, the strongest labelling was present over the central parts of the lobuli (Fig. 3C). In vitro autoradiography In the autoradiograms obtained with tape sections of lungs incubated with [“C]carbon tetrachloride under an atmosphere of oxygen or nitrogen, a strong labelling was present in the epithelium of the bronchial tree (Figs. 4A and 4B). The lung parenchyma was labelled to a lower extent. There was also a marked radioactivity in tracheal epithelium. In the incubations in an atmosphere of carbon monoxide there was almost no labelling of the lung or the trachea (Fig. 4C). The incubations with the oesophagus showed a strong labelling of the epithelium when oxygen or nitrogen were used as atmospheres (Figs. 5A and 5B). A carbon monoxide atmosphere very markedly depressed this labelling (Fig. 5C). The incubations with the nose resulted in a strong labelling of the mucosa of the ethmoturbinates in oxygen or nitrogen atmospheres, but only a very low labelling in a carbon monoxide atmosphere (data not shown). The in vitro microautoradiography gave similar results as described in vivo, with a labelling of the subepithelial glands of the olfactory mucosa, the epithelium of the bronchial tree of the lung (Fig. 3D) and the squamous epithelium of the oesophagus. In aitrw metabolism The nasal olfactory mucosa was found to have the highest capacity to form n4C02from the [“Clcarbon tetrachloride (Table I). As was expected, the liver was also very active. i4COz-formation in decreasing order was also noted in the oesophagus, the nasal respiratory mucosa, the cheeks, the larynx, the
Fig. 3. Micraautoradiograms showing: the nasal olfactory mucosa Ih after iv. injection of [“C]carbon tetrachioride (A), the oesophagus 1 h after iv. injection of [“Clcarbon tetrachloride (B), the liver Ih after i.p. injection of [“Clcarbon tetrachloride (C) and the lung after in vitro incubation for 1 h in a buffor solution containing [‘Clcarbon tetrachloride (IN The autoradiograms show tissue-bound radioactivity. In (A) there is a labclling of the olfactory subepithelial glands of the lamina propria mucosae. In (B) there is a labelling of the stratified squamous epithelium of the oesophagus. In (C) the strongest labelling is present in the central part of the liver lobule around the central vein. In (D) there iti a marked labelling of the bronchial epithelium, whereas the labelling of the alveoli is low. OL, olfactory epithelium: SC. subepithelial glands (Bowman’s glands): SE. squamous epithelium of the oesophagus; CV. central vein; B. bronchus. x460.
308
4. A-C:
autoradlograms
of
1UII~Sand tracheas Incubated
for 1 h in buffer
solutions
containing
[W]carbon
tetrachlorlde.
l_t
: Li
:
:
0
: :
II-F.
haematoxplin-
rosin stained sections correspondmg to A, I3 and (:. reqprctlvely The atmospheres used during the incubations were for (A. DI oxygen. (B. I?, nitrogen and (CT.I?) carbon monoxide. The tissue srrtions were extracted with trlchloroacetlc acid and organic solvents before the autoradiography In (A) and (I%) there is a strong labelllng of the rplthellum of the bronchial trrr and the trachea and ;I low labrlling of the lung parenchyma. in (Ci nc, Iwhrlling of the tissues can hr. d tected. Thr exposure-time was 2 months.
Fig.
RONCnlOLES
TRACI-IEA
MUSCLE MUCOSA
MUCOSA
MUSCLE
3
MUCOSA
MUCOSA
MUSCLE i
Fig. 5. A-C: autoradiograrns of oesophagus incubated for 1 h in buffer solutions containing [‘“C~arbon tetrachloride. D-F: hae stained sections corresponding to A, B and C, respectively. The atmospheres used during the incubations were for (A, D) oxygen, (B, (6, F) carbon monoxide. The tissue sections were extracted with trichloroacetic acid and organic solvents before the autoradiograph> there is a strong labelhng of the oesophageal mucosa. In (C) th? labelling is markedly reduced. The exposure time was 2 months.
MUCOSA
MUCOSA
w c,
311
Y VARIOIB
r papers
erpres res within
TISSUES
IN
37°C under an atmos(0.25pCi) in 2 ml of ‘CO2 formed during the e results show the radioacminute (dpm) per mg brackets denote the number of in-
cubations. Tissue
W!On-formation (dpmlmg of wet wt) -
Nasal olfactory Liver (12)
mucosa (12)
Cheek (3)
Trachea (6) Small intestine Lung (6) Adrenal (3)
(3)
salivary
gland (6)
Glandular stomach (6) Forestomach (6)
idney cortex, y the other ti
75.5 2 4.4 62.1) 4.4 30.6 2 I.7 27.2 -t 5.6 19.9 z 4.0
16.8~6.6 15.523.9 12.822.1 8.6r0.3 3.4*Q.6 3.2% 1.1 3.020.7 2.8-cO.3 2.420.6 1.8~0.7 1.5t0.3 1.220.3 1.220.8 1.0zO.6 0.8-rO.S 0.6-cO.4
lateral nasal gland and the csngue. The 14C 9 tested wa8 low.
was detected in the homogenates of tLe nasal olfactory about one third of t e level in the liver, ased on the Gssue weight, and on the
f?W Qf
the co
tetrac tion of
rnetaboli tetrachlo
312 TABLEII CYTOCH’ROME P4610 CONTENTSOF THE NASAL OLFACTORYMUCOSA AND THE LiVER The cytochrw e 8’460 was determinedin homogenatee of rattisauee aa etated in Materialsand Methods.ResuIt~~ are expresseda~ means2 S.E. (n = 7).
-Neal
Liver
olfactory
muco8a
nmolP-GO/g of wet wt.
nmolPMOlmg of protein
9.9 It0.9 26.1 -c1.2
0.22 r 0.01 0.612 0.06
P-450 and the subsequent reduction of the complex by NADPH in the catalytic presence of cytochrome P-450 reductaee [8,9,181. The free trichloro=
methyl radical (CC&J may initiate peroxidation of polyunsaturated fatty acids or it may react with macromolecules, such as proteins and lipids,:and these events will ultimately induce the noxious effects in the tissues [7,8]. By hydrogen abstraction, chloroform (CHCls) is also formed 17,181. The formation of carbon dioxide from carbon tetrachloride is due to the oxidative dechlorination of chloroform and this process also proceeds via cytochrome P-450 dependent pathways [12,13,19]. Previous distribution studies by low-temperature autoradiography have indicated that the non-metabolized carbon tetrachloride can freely pass the biological membranes and reach all tissues of the body [lo]. The autoradiography in the present study, in which freeze-dried or extracted tissues were used, will reveal only the localization of metabolites. The results showed that several tissues accumulating metabolites in vivo had an ability to form “COZ in vitro, and accumulation of metabolites was also observed under the in vitro conditions. These results indicate that carbon tetrachloride is metabolized in many extrahepatic tissues in vivo. Since the metabolism of carbon tetra= chloride proceeds by cytochrome P-450mdependent pathways, it is of interest to examine whether our results can be correlated, to the sites of cytochrome P-450-activity in the tissues. In the lung, the bronchiolar Clara cells are the main sites of the cyto. chrome P-450 system, but a low level is also present in the alveolar type=11cells 120,211. The administration of carbon tetrachloride to rats has been shown to induce an acute toxic effect on the Clara cells [6]. Our autoradiography indicated a marked metabolism of carbon tetrachloride in the epithelial lining of the bronchial tree and a low metabolism in the lung parenchyma, and these results probably reflect the metabolic capacity of the mentioned cells. Our results also indicated a metabolic capacity of the tracheal epithelium. Oxidative enzymes have been shown histochemically in the surface epithelium of the trachea [22]. The results showed that the epithelium of the oesophagus is very active’in
313
metabolizing carbon tetrachloride. Cytochrome P-450 has been demonstrated in the oesophagus [231. Our results in addition showed a high metabolism of carbon tetrachloride in the mucosa of the larynx, the cheeks and the t&ngue, which are covered with an epithelium of a similar type as in the oesophagus. It should be noted that the calculations of the i4COrproduction by the oesophagus, the larynx, the cheeks and the tongue, and also by the lung and the trachea, are based on the weights of heterogeneous tissues. The metabolizing capacity of the muccsal cells in these tissues are therefore probabiy much higher than is shown by the “COz-production, and may be better reflected by the intensity of the autoradiographic labelling. The nasal olfactory epithelium was found to have a very high capacity to form “COp from the [“Clcarbon tetrachloride, and the microautoradiography indicated that in this tissue the cells of the subepithelial glands (Bowman’s glands) are most actively engaged in the metabolism. Histochemical studies have shown high contents of oxidative enzymes in the subepithelial glands 1241 and the cells of these glands are also rich in endoplasmic reticulum [25], which is the major source of cytochrome P-450.dependent enzymes. We have previously shown that N-nitrosamines are metabolized in the nasal mucosa, and as for carbon tetrachloride, microautoradiography showed the highest labelling over the subepithelial glands [26,27]. Our determination of cytochrome P-450 in the nasal olfactory mucosa showed an amount which was about one third of the concentration in the liver. The level recorded was higher than the one reported by Hadley and Dahl in the nasal mucosa If F344 rats 1281.This may partly 1103J!&ed to the fact that those authors ued nasal areas which included both the olfactory and the respiratory mucosa, whereas we only used the olfactory mucosa, and also to the fact that they used a microsomal preparation, whereas we used tissue homogenates. The nasal respiratory mucosa was in the present study found to have a marked capacity to degrade carbon tetrachloride, but the activity was considerably lower than in the olfactory mucosa. In spite of the higher cytochrome P-450 concentration in the liver than in the nasal olfactory mucosa, the latter tissue produced somewhat more *‘CO2 from the [14C]earbon tetrachloride than the liver. The correlation between cytochrome P-450 concentration and metabolism appears to be qualitatively good, but not strictly quantitatively valid. This may be due to the fact that cytochrome P-450 is not a single entity but consists of various forms which may have different substrate specificities [291. Metabolism of carbon tetraihloride was observed in the lateral nasal gland (Steno’s gland), The acinar cells of this gland contain a well developed endop]asmic reticulum [30]. We have previously observed tissue-bound metabolites in the lateral nasal gland in rats injected with two tobaccospecific N=nitrosamines 14,261. Carbon tetrachloride is a nephrotoxic agent, inducing injury mainly of the kidney cortex [5]. Cytochrome P-450 has been shown in the kidney, with higher activity in the cortex than in the medulla 1311. In agreement with these results, our study showed a “COP-formation from [‘“Clcarbon tetra-
314 chloride by the kidney cortex, but not the kidney meldulla, and the autoradiography showed a higher labelling of the cortex than of the medulla. In the liver, the strongest labelling was observed over the central parts of the lobuli. This can be correlated to the centrilobular liver injury which carbon tetrachloride induces 1321. The in vitro autoradiography showed that incubations performed in a carbon monoxide atmosphere induced a strong inhibition of the binding of metabolites in the oesophagus, the lung and the nose as compared with the incubations performed in oxygen or nitrogen atmospheres. Reduction by carbon monoxide of the binding of carbon tetrachloride to liver microsomal pro+eins has been shov:7 previously and is indicative of a cytochrome P-460dependent pathway [7,9]. The initinl step in carbon tetrachloride metabolism, leading to the formation of the reactive trichloromethyl radical is purely reductive and should proceed under anaerobic conditions. This concords with our autoradiographic observation of a strong binding in a nitrogen atmosphere, and similar results l..qve been obtained with liver microsomal preparations [7,9]. The tissue disposition of carbon tetrachloride metabolites in the rats in the present study is similar to the pattern observed in mice by Bergman [lo]. An exception, however, is the gastrointestinal mucosa, which was more strongly labelled in the mice than in the rats. It is possible that this at least partially may be due to the differences in the routes of the carbon tetrachloride exposure: At inhalation, as performed by Bergman [lo], a part of the [“Clcarbon tetrachloride may be mixed with the secretions of the nose and the mouth and, by swallowing, reach the gastrointestinal contents. This, in turn, may give opportunity for a local degradation of the [“C]carbon tetrachloride in the alimentary canal and as a consequence a labelling of the gastrointestinal walls. The present study has disclosed that carbon tetrachloride metabolism is remarkably active in the mucosal linings of tissues which are exposed orally or by inhalation to drugs and other environmental chemicals. The cytochrome P-450 system which appears to be active at these sites, may constitute a protection mechanism defending the body from unrestrained uptake of xenobiotics. In certain instances, however, and this applies to carbon tetrachloride, the metabolism may instead resuPt in a bioactivation locally in the cells. Carbon tetrachloride should thus be potentially toxic towards the extrahepatic metabolizing tissues. As mentioned previously, noxious effects have been shown in tissues such as the kidney and the lung [6,7], but no attempts were made in the present study to establish whether injuries can also be induced in -the other tissues with metabolizing capacity, REFERENCES 1 E.B. Brittebo, B. LiMberg end H. Tjiilvr, Sites of metabolism mice, C&em.-Biol. Interact., 34 (1981) 209.
of N-nitroaodiethylamine
in
315 2 E.B. Brittebo and H. Tjiilve. Formation of tissue-bound N’-nitrosonomlcotjne metabolih the t-get tieauen of Sprague-Dawley and Fisher rata, Carcinogeneeis. 2 (1981) 959.
by
3 E. Brittobo, B. LBfherg and H. Tjiilve, Extrahepatic sites of metabolism of N.nitmmpyr. rolidine in mice and rata, Xenobiotica. 11 (19131) 619. tonguay, H. Wilvr: and S.S. Hecht, Tiesue distribution of the tobaxo-specific car. cinol?en C(methylnit~eminor.l~(Sl~yyridyl)-l-butnnone. and its metabolites in F.344 rats, Cancer Rec.. 43 (1983) 630. 15 R.B. Jennings and W.M. Keens, Necrotizing nephronis in the rat following administration of carbon tetrachloride. Arch. Pathol., F6 (1963) 348. 6 MR. Boyd, C.N. Statham and N.S Longo, The pulmonary Clara cells as a target for toxic chemicals requiring metabolic activation; Studies with carbon tetrachloride, J. Pbamlacol. Exp. Ther., 212 (19801 109. 7 H. Uehleke. K.H. Hellmer and S. Tabarelli, Binding of “C-carbon tetrachloride to micro. somal proteins in vitro and formation of CHC13 by reduced liver microsome, Xenobiotica. 3 (1973) 1. 8 ML Dial time& J.A. Castro, E.C. de Ferreyra, N. D’Acosta and C.R. de Castro, Irreversible bir.+ng of “C from “CC4 to liver microsomal lipids and proteins from rats pretreated with compounda altering microsomal mixed-function oxygenase activity, Toxicol. Appl. Pharmacol.. 25 (1973) 534. 9 LG. Sipes, G. Krishna and J.R. Gillette, Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane; role of cytoehrome P-450. Life Sci, 20 (1977) 1541. 10 K. Bergman, Whole-body autoradiography and allied tracer techniques in distribution and elimination studies of some organic solvents, Stand. J. Work Environ. Health, 5 (1979) Suppl. 1. 11 B.B. Paul and D. Rubinstein, Metabolism of carbon tetrachloride and chloroform by the rat, J. Pharmacol. Exp. Ther.. 141 (1963) 141. 12 D. Rubinstein and L. Kanics, The conversion of carbon tetrachloride and chloroform to carbon dioxide by rat liver homogenates, Can. J. Biochem., 42 (1964) 1577. 13 D.H. Hathway. Chemical, biochemical and tnxicological differences between carbon tetrachloride and chloroform, Anneim.-Forsch., 24 (1974) 173. 1.4 S. Ullberg, The technique of whole-body autoradiography. Cryosectioning of large specimens. Science Tools, Special Issue (1977). 15 E. Johansson-Brittebo and H. Tjlilve. Studies on the distribution and metabolism of “Cdimethylnitmsamine in foetal and young mice, Acta Pharmacol. Toxicol.. 45 (1979) 73. 16 A.E.M. McLean and P.A. Day, The use of new methods to measure: the effect of diet and inducers of microsomal enzyme synthesis on cytochrome P-450 in liver homogenates. and the metabolism of dimethylnitrosamine. Biochem. Pharmacol., 23 (1974) 1173. 17 O.H. Lowry, N.J. Rosebrough. A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem.. 193 (1951) 265. 18 H.J. Ahr. L.J. King, W. Nastainczyk and V. Ullrich, The mechanism of chloroform and carbon monoxide formation from carbon tetrachloride by microsomal cytochrome P-450. Biochcm. Pharmacol., 29 (1980) 2855. 19 A.E.M. McLean, The effect of protein deficiency and microsomal enzyme induction by DDT and phenobarbitone on the acute toxicity of chloroform and a pyrrolizidine alkaloid. Retrosine, Br. J. Exp. Pathol., 61 (1970) 317. 20 M. Boyd. Evidence for the Clara cell as a site of cytochrome P-450.dependent mixed-function oxidase activity in the lung, Natun,. 269 (1977) 713. 21 T.R. Devereux, C. &rbjit-Singh, S.R. Slaughter, C.R. WOK. R.M. Philpot and J.R. Fo&s. Identification of cytochrome P-460 isozymes in non-ciliated bronchiolar epithelial (Clara) and alveolar type II cells isolated from rabbit lung, Exp. Lung Res., 21 (1981) 221. 22 A. Azzopardi and W.M. Thurlbeck, Oxidative enzyme pattern of the bronchial mucous glands, Am. Rev. Respir. Die., 97 (1968) 1038. 23 G.E. Labuc and M.C. Archer, Esophageal and hepatic microsomal methabolism of Nnitroaomethylbenzylamine and N-nitrosodimethylamine in the rat, Cancer bs., 42 (1982) 3181.
24 E. Mira, Cxidative and Hydrolytic enzymes in Bowman’s glands, Acta Cto-Laryng., 66 (1963) 706. 25 D. Frisch, Ultrastructure of mouse olfactory mucosa, Am. J. Anat., 121 (1967) 87. 26 B. Liifberg, E.B. Brittebo and H. Tjiilve, Localization and binding of IV’-nitrosonomicotine metabolites in the nasal region and in some other tissues of Sprague-Dawley rats, Cancer Res., 42 (1982) 2877. 27 H. Tjiilve and A. Castonguay, The in vivo tissue disposition and in vitro target-tissue metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino).I-(3.pyridyl)-1.butanone in Syrian golden hamsters, Carcinogenesie (1983) in press. 28 W.M. Hadley and A.R. Dahl, Cytochrome P-450 dependent monooxygenase activity in rat nasal epithelia) membranes, Toxicology Lett., 10 (1982) 417. 29 A.Y.H. Lw and S.B. West, Multiplicity of mammalian microsomal cytochromes P-450, Phannacol. Rev., 31 (1980) 277. 39 H. Moe and F. Bojsen-Moller. The fine structure of the lateral nasal gland (Steno’s gland) of the rat, J. Ultrastruct. Res., 36 (1971) 127. 31 Y. Ichikawa and T. Yamano, Electron spin resonance of microsomal cytochromes, Arch. Biochem. Biophys.. 121(1967) 742. 32 E.H. Ledue and J.W. Wilson, Injury to liver cells in carbon tetrachloride poisoning, Arch. Pathol., 65 (1958) 147.