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Affinity of [14C]nitrosopiperidine and metabolites for mouse epithelial tissues
TOXICOLOGYANDAPPLIEDPHAFtMACOLOGY
67, 110-116 (1983)
Affinity of [‘4C]Nitrosopiperidine and Metabolites for Mouse Epithelial Tissues’ CAROLYN MARLOWE* AND WILLIAM
J. WADDELL
Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, LouisviNe, Kentucky 40292
Received July 2, 1982; accepted September 23, I982 Affinity of [r4C]Nitrosopiperidine and Metabolites for Mouse Epithelial Tissues. MARLOWE, W. J. (1983). Toxicol. Appl. Pharmacol. 67, 110-I 16. Male C57BL/6J mice were each administered iv 1.2 mg/kg (6.0 to 7.6 &i) of [‘%Y]nitrosopiperidine ([‘4C]NPIP) and frozen by immersion in dry ice/hexane at 0.1, 0.33, 1, 3, 9, and 24 hr after injection. The mice were processed for whole-body autoradiography without thawing or the use of any solvents; sagittal sections of the frozen mice were freeze-dried and placed on X-ray film to reveal areas of localization of radioactivity. The autoradiographs revealed intense localization of radioactivity at 6 min in the epithelium of the nose and bronchi, as well as in the liver, kidney, and salivary glands. There is virtually no alBnity of [‘4C]NPIP for melanin. Most of the same localizations persisted from 6 min through 24 hr. At 24 hr the most intense accumulation was in the epithelium of the bronchi, nose, salivary gland ducts, and esophagus as well as the liver and Harder’s gland. The results are interpreted to suggest that at least one metabolite of NPIP which localizes in the sites where tumors occur may be. similar to a metabolite of NNN. The distribution is consistent with metabolic conversion of [i’C]NPIP in liver and epithelium of nose and bronchi with subsequent localization of the metabolite in epithelium of esophagus and salivary gland ducts. C., AND WADDELL,
An earlier report from this laboratory (Waddell and Marlowe, 1980) demonstrated that unidentified metabolites of the tobacco carcinogen, N’-nitrosonornicotine (NNN),3 accumulate in specific epithelial tissues of the mouse, i.e., nasal, bronchial, esophageal, and salivary duct epithelium; these are the same sites in which tumors appear after administration of this carcinogen. Studies are con’ Presented at the Eighth International Congress of Pharmacology, July 19-24, 1981, Tokyo, Japan. Sup ported by Pharmacon Research Foundation, Inc. and by the University of Kentucky Tobacco and Health Research Institute. ’ To whom requests for reprints should be addressed. ’ Abbreviations used: [‘4C]NPIP, N-(2,6-‘4C]nitrosopiperidine; NNN, iV’-nitrosonomicotine; NPIP, nitrosopiperdine.
0041-008X/83/0101
10-07%03.00/0
Copyright Q 1983 by Academic Press., Inc. All fights of reproduction in any form rmrved.
tinuing in this laboratory with possible metabolites of NNN and with compounds similar in structure to those metabolites. Nitrosopiperidine (NPIP) is present in the condensate of cigarette smoke (McCormick et al., 1973), induces cancer of the nose, esophagus, liver, and lung in rodents (Magee and Barnes, 1967), and is the nitroso derivative of the saturated pyridine half of the nicotine molecule. NPIP is metabolized to 5-hydroxypentanal (Leung et al., 1978) and Nnitrosopyrrolidine to 4-hydroxybutanal (Hecht et al., 1978) suggesting that the nitroso derivatives of either the six- or five-membered rings of NNN may be metabolized by similar routes. Information on the tissue affinities and metabolism of NPIP should provide further clues for the identification of the proximal
110
[‘4C]NITROSOPIPERIDINE
compound involved in carcinogenesis with NNN, NPIP, and closely similar nitrosamines. METHODS Adult male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were allowed standard food (Purina Lab Chow, No. 500 1) and water ad libitum until time of treatment. The mice killed at 3, 9, or 24 hr were housed, following administration of the isotope, in a cage with a wire mesh bottom. N-[2,6-‘4C]nitrosopiperidine (lot 1229-011) was purchased from New England Nuclear (Boston, Mass.). This material has a specific activity of 18.8 mCi/mmol. Immediately prior to the experiment, the radiochemical purity was determined on thin-layer chromatographic plates coated with silica gel. Plates spotted with aliquots of the [i4C]NPIP solution were developed in hexane:ether: methylene chloride (4:3:2). After exposing these developed plates against X-ray film, a single radioactive spot with an R, of 0.46 was detected. Mice weighing 30 to 37 g were injected iv in the tail vein with 0.2 pCi/g body weight of the [‘4C]NPIP dissolved in 0.9% NaCl (w/v); this dose corresponds to 1.2 mg NPIP/kg body weight. Six minutes, 20 minutes, 1, 3, 9, or 24 hours after the injection, each mouse was briefly anesthetized with ether and killed by freezing in a dry ice/hexane bath. The frozen mouse was attached to a microtome stage and whole-body sag&al sections, 20 and 40 pm thick, were taken onto Scotch tape at -20°C. The freeze-dried sections were exposed against Kodak AA Xray film to produce autoradiographs. These whole-body autoradiographic procedures do not allow thawing or contact with any solvents; thus, there is no loss or translocation of radioactivity. Sites of radioactive uptake in the autoradiographs, therefore, represent the in viva disposition of [‘4C]NPIP and/or its metabolites at the time the mouse was killed. Details of these autoradiographic prccedures for studying water-soluble compounds have been published (Waddell and Marlowe, 1977).
IN MOUSE TISSUES
111
Activity was much lower in all the remaining tissues. There was gradually increasing radioactivity in the epithelium of the oral cavity and esophagus as the time intervals increased. By 3 hr after injection (Fig. 2) the highest concentrations were in nasal, bronchial, oral, and esophageal epithelium and Harder’s gland; liver, and sublingual and submaxillary glands had the next highest concentrations. Radioactivity was decreasing in kidney, but the contents of the upper gastrointestinal tract showed occasionally high activity, and there was noticeable activity in the pancreas and preputial gland. By 24 hr after receiving [ “C]NPIP (Figs. 3 and 4), the highest concentrations of radioactivity were in Harder’s gland, liver, and the epithelium of salivary gland ducts, oral cavity, esophagus, nose, trachea, and bronchi. The concentration in the kidney and urine was clearly decreasing but slight activity was now present in the cortex of the thymus, bone marrow, wall of intestine, and preputial gland. The activity in the liver and kidney was not homogeneous; higher activity was seen in the periportal area than around the central vein in the liver. The radioactivity in the kidney was confined almost completely to the distal tubules (Longley, 1969). DISCUSSION
The very rapid accumulation and prolonged retention of the labeled NPIP in nasal and bronchial epithelium and in liver may be explained by one of at least several mechanisms. One possibility for the rapid accuRESULTS mulation is specific binding of unchanged NPIP to proteins or other molecules in these At the earliest time interval, 6 min, after cells. Also, active transport may account for receiving the [‘4C]NPIP, there was already a the accumulation. A third and more likely sharp pattern of localization of radioactivity possibility is that NPIP is interacting with en(Fig. 1). The nasal and bronchial epithelium zymes in these sites where it is metabolized had the highest concentrations; kidney, urine, to other compounds. A salient argument for liver, and salivary glands had the next highest metabolic conversion in these sites is the evconcentrations. Some accumulation was seen idence accumulating for high P-450 activity in Harder’s gland and the conjunctival sac. in these tissues (Harris et al., 1977; Waddell
GLAND
SAC
BLOOD
IN HEART
BRONCHI
LIVER
INTESTINE
KIDNEY
FIG. 1. A print of a whole-body autoradiograph of a 20-pm-thick section from a male C57BL/6J mouse which was frozen 6 min aftex receiving 1.2 mg [‘4C]NPIP/kg iv. White areas correspond to radioactivity. X2.6.
SALIVARY
CONJUNCTIVAL
BRONCHI
AND SUBMAXILLARY
ORAL CAVITY
FIG. 2. A print of a whole-body autoradiograph of a 20-pm-thick section from a male White areas correspond to radioactivity. ~2.2.
SUBLINGUAL,
EPITHELIUM
HARDER’S,
NASAL
CS’BL/bJ
mouse
GLANDS
LIVER
which
3 hr after receiving 1.2 mg [ 14C]NpIp/kg iv,
Gl CONTENTS PREPUTIAL GLAND BLOOD was frozen
PANCREAS
KIDNEY
SALIu4RY
OLAhtDs
CAVITY
THYMUS
BRONCHI
BLOOD
LIVER
INTESTINAL
KIDNEY
MARROW
CONTENTS
BONE
URINE
FIG. 3. A print of a whole-body autoradiograph of a 20-pm-thick section from a male C57BL./6.J mouse which was frozen 24 hr after receiving 1.3 mg [‘4C]NPIP/kg IV. White areas correspond to radioactivity. X2.3.
ANti
H&b&
ORAL
SAC
CONJUNCTIVAL
GLAND
OUCTS
THYMUS
TRACHEA
ESOPHAGUS
BLOOO
LIVER
BILE
BRONCHI
FIG. 4. A print of an enlargement of an autoradiograph of a 20-pm-thick section from a male C57BL/6J mouse which was frozen 24 hr after receiving 1.3 mg [14C]NPIP/ kg iv. White areas correspond to radioactivity. X7.3.
SALIVARY
BONE MARROW
116
MARLOWE
AND WADDELL
and Marlowe, 1976, 1978). The prolonged retention suggests that a metabolite of NPIP may be alkylating constituents of these cells. The delay in accumulation of radioactivity in oral, esophageal, and salivary duct epithelium and in Harder’s gland suggests that only the metabolites of NPIP interact with constituents of these cells. It is most unlikely that the metabolite is 14C02 because the pattern of distribution of this fragment is different from that seen with NPIP (Waddell et al., 1969). The retention in the distal tubules of the kidney may only reflect the final stages of renal elimination of NPIP and/or metabolites; the extensive renal elimination of radioactivity at early intervals after administration indicates that this radioactivity probably represents unchanged NPIP. Radioactivity in the upper intestine at 3 hr may represent biliary excretion of metabolites of NPIP. The transient appearance of radioactivity in the pancreas at 3 hr with no retention at later time intervals may represent pancreatic secretion of a metabolite of NPIP. Although the retention of radioactivity in the cortex of the thymus and in bone marrow is only minimal at 24 hr, the possibility should not be overlooked that one or more metabelites of NPIP may be affecting the immune system of the mouse. The authors are at a loss to offer tentative explanations for the residual radioactivity in the wall of the intestine and in the preputial gland. The impressive specificity of retention of radioactivity from [‘4C]NPIP in the tissues where tumors have been reported in rodents from administration of NPIP encourages further experiments with other compounds of similar structure. Studies with smaller fragments of NNN and with potential blockers of metabolism and binding of these carcinogenic nitrosamines are continuing in this laboratory.
ACKNOWLEDGMENTS The authors thank Drs. James B. Longley, Frederick K. Hilton, and Richard D. Rink, Department of Anatomy, University of Louisville, for their expert assistance in the identification of the anatomical structures in the whole-body sections and Rhonda Elam for her expert technical assistance.
REFERENCES HARRIS, C. C., AUTRUP, H., STONER, G. D., McDowELL, E. M., TRUMP, B. F., AND SCHAFER, A. (1977). Metabolism of acyclic and cyclic N-nitrosamines in cultured human bronchi. J. Nut. Cancer Inst. 59, 140 l1406. HECHT, S. S., CHEN, C. B., AND HOFFMANN, D. (1978). Evidence for metabolic a-hydroxylation of N-nitrosopyrrolidine. Cancer Rex 38, 2 15-2 18. LEUNG, K. H., PARK, K. K., AND ARCHER, M. C. (1978). Alpha-hydroxylation in the metabolism of N-nitrosopiperidine by rat liver microsomes: Formation of 5hydroxypentanal. Res. Commun. Chem. Pathol. Pharmacol. 19, 20 l-2 I 1. LONGLEY, J. B. (1969). Histochemistry of the kidney. In The Kidney (C. Rouiller and A. F. Muller, eds.), Vol. 1, pp. 157-260. Academic Press, New York. MCCORMICK, A., NICHOLSON, M. J., BAYLIS, M. A., AND UNDERWOOD, J. G. (1973). Nitrosamines in cigarette smoke condensate. Nature (London) 244,237-238. MAGEE, P. N., AND BARNES, J. M. (1967). Carcinogenic nitroso compounds. Advan. Cancer Res. 10, 163-246. WADDELL, W. J., AND MARLOWE, C. (1976). Localization of nicotine-T, cotinine-14C, and nicotine-l’-Noxide-‘% in tissues of the mouse. Drug Mefub. Dispos. 4, 530-539. WADDELL, W. J., AND MARLOWE, C. (1977). Autoradiography. In Drug Fate and Metabolism: Methods and Techniques (E. R. Garrett and J. L. Hirtz, eds.), Vol. 1, pp. l-25. Marcel Dekker, New York. WADDELL, W. J., AND MARLOWE, C. (1978). Inhibition by metyrapone of the accumulation of nicotine-14C in bronchial epithelium of mice. Arch. Int. Pharmacodyn. Ther. 234, 294-307.
WADDELL, W. J., AND MARLOWE, C. (1980). Localization of [‘%C]nitrosonomicotine in tissues of the mouse. Cancer
Res. 40, 35 18-3523.
WADDELL, W. J., ULLBERG, S., AND MARLOWE, C. ( 1969). Localization of the bicarbonate and carbonate pools by whole-body autoradiography. Arch. Int. Physiol. Biochem.
71, 1-9.
67, 110-116 (1983)
Affinity of [‘4C]Nitrosopiperidine and Metabolites for Mouse Epithelial Tissues’ CAROLYN MARLOWE* AND WILLIAM
J. WADDELL
Department of Pharmacology and Toxicology, School of Medicine, University of Louisville, LouisviNe, Kentucky 40292
Received July 2, 1982; accepted September 23, I982 Affinity of [r4C]Nitrosopiperidine and Metabolites for Mouse Epithelial Tissues. MARLOWE, W. J. (1983). Toxicol. Appl. Pharmacol. 67, 110-I 16. Male C57BL/6J mice were each administered iv 1.2 mg/kg (6.0 to 7.6 &i) of [‘%Y]nitrosopiperidine ([‘4C]NPIP) and frozen by immersion in dry ice/hexane at 0.1, 0.33, 1, 3, 9, and 24 hr after injection. The mice were processed for whole-body autoradiography without thawing or the use of any solvents; sagittal sections of the frozen mice were freeze-dried and placed on X-ray film to reveal areas of localization of radioactivity. The autoradiographs revealed intense localization of radioactivity at 6 min in the epithelium of the nose and bronchi, as well as in the liver, kidney, and salivary glands. There is virtually no alBnity of [‘4C]NPIP for melanin. Most of the same localizations persisted from 6 min through 24 hr. At 24 hr the most intense accumulation was in the epithelium of the bronchi, nose, salivary gland ducts, and esophagus as well as the liver and Harder’s gland. The results are interpreted to suggest that at least one metabolite of NPIP which localizes in the sites where tumors occur may be. similar to a metabolite of NNN. The distribution is consistent with metabolic conversion of [i’C]NPIP in liver and epithelium of nose and bronchi with subsequent localization of the metabolite in epithelium of esophagus and salivary gland ducts. C., AND WADDELL,
An earlier report from this laboratory (Waddell and Marlowe, 1980) demonstrated that unidentified metabolites of the tobacco carcinogen, N’-nitrosonornicotine (NNN),3 accumulate in specific epithelial tissues of the mouse, i.e., nasal, bronchial, esophageal, and salivary duct epithelium; these are the same sites in which tumors appear after administration of this carcinogen. Studies are con’ Presented at the Eighth International Congress of Pharmacology, July 19-24, 1981, Tokyo, Japan. Sup ported by Pharmacon Research Foundation, Inc. and by the University of Kentucky Tobacco and Health Research Institute. ’ To whom requests for reprints should be addressed. ’ Abbreviations used: [‘4C]NPIP, N-(2,6-‘4C]nitrosopiperidine; NNN, iV’-nitrosonomicotine; NPIP, nitrosopiperdine.
0041-008X/83/0101
10-07%03.00/0
Copyright Q 1983 by Academic Press., Inc. All fights of reproduction in any form rmrved.
tinuing in this laboratory with possible metabolites of NNN and with compounds similar in structure to those metabolites. Nitrosopiperidine (NPIP) is present in the condensate of cigarette smoke (McCormick et al., 1973), induces cancer of the nose, esophagus, liver, and lung in rodents (Magee and Barnes, 1967), and is the nitroso derivative of the saturated pyridine half of the nicotine molecule. NPIP is metabolized to 5-hydroxypentanal (Leung et al., 1978) and Nnitrosopyrrolidine to 4-hydroxybutanal (Hecht et al., 1978) suggesting that the nitroso derivatives of either the six- or five-membered rings of NNN may be metabolized by similar routes. Information on the tissue affinities and metabolism of NPIP should provide further clues for the identification of the proximal
110
[‘4C]NITROSOPIPERIDINE
compound involved in carcinogenesis with NNN, NPIP, and closely similar nitrosamines. METHODS Adult male C57BL/6J mice (Jackson Laboratory, Bar Harbor, Maine) were allowed standard food (Purina Lab Chow, No. 500 1) and water ad libitum until time of treatment. The mice killed at 3, 9, or 24 hr were housed, following administration of the isotope, in a cage with a wire mesh bottom. N-[2,6-‘4C]nitrosopiperidine (lot 1229-011) was purchased from New England Nuclear (Boston, Mass.). This material has a specific activity of 18.8 mCi/mmol. Immediately prior to the experiment, the radiochemical purity was determined on thin-layer chromatographic plates coated with silica gel. Plates spotted with aliquots of the [i4C]NPIP solution were developed in hexane:ether: methylene chloride (4:3:2). After exposing these developed plates against X-ray film, a single radioactive spot with an R, of 0.46 was detected. Mice weighing 30 to 37 g were injected iv in the tail vein with 0.2 pCi/g body weight of the [‘4C]NPIP dissolved in 0.9% NaCl (w/v); this dose corresponds to 1.2 mg NPIP/kg body weight. Six minutes, 20 minutes, 1, 3, 9, or 24 hours after the injection, each mouse was briefly anesthetized with ether and killed by freezing in a dry ice/hexane bath. The frozen mouse was attached to a microtome stage and whole-body sag&al sections, 20 and 40 pm thick, were taken onto Scotch tape at -20°C. The freeze-dried sections were exposed against Kodak AA Xray film to produce autoradiographs. These whole-body autoradiographic procedures do not allow thawing or contact with any solvents; thus, there is no loss or translocation of radioactivity. Sites of radioactive uptake in the autoradiographs, therefore, represent the in viva disposition of [‘4C]NPIP and/or its metabolites at the time the mouse was killed. Details of these autoradiographic prccedures for studying water-soluble compounds have been published (Waddell and Marlowe, 1977).
IN MOUSE TISSUES
111
Activity was much lower in all the remaining tissues. There was gradually increasing radioactivity in the epithelium of the oral cavity and esophagus as the time intervals increased. By 3 hr after injection (Fig. 2) the highest concentrations were in nasal, bronchial, oral, and esophageal epithelium and Harder’s gland; liver, and sublingual and submaxillary glands had the next highest concentrations. Radioactivity was decreasing in kidney, but the contents of the upper gastrointestinal tract showed occasionally high activity, and there was noticeable activity in the pancreas and preputial gland. By 24 hr after receiving [ “C]NPIP (Figs. 3 and 4), the highest concentrations of radioactivity were in Harder’s gland, liver, and the epithelium of salivary gland ducts, oral cavity, esophagus, nose, trachea, and bronchi. The concentration in the kidney and urine was clearly decreasing but slight activity was now present in the cortex of the thymus, bone marrow, wall of intestine, and preputial gland. The activity in the liver and kidney was not homogeneous; higher activity was seen in the periportal area than around the central vein in the liver. The radioactivity in the kidney was confined almost completely to the distal tubules (Longley, 1969). DISCUSSION
The very rapid accumulation and prolonged retention of the labeled NPIP in nasal and bronchial epithelium and in liver may be explained by one of at least several mechanisms. One possibility for the rapid accuRESULTS mulation is specific binding of unchanged NPIP to proteins or other molecules in these At the earliest time interval, 6 min, after cells. Also, active transport may account for receiving the [‘4C]NPIP, there was already a the accumulation. A third and more likely sharp pattern of localization of radioactivity possibility is that NPIP is interacting with en(Fig. 1). The nasal and bronchial epithelium zymes in these sites where it is metabolized had the highest concentrations; kidney, urine, to other compounds. A salient argument for liver, and salivary glands had the next highest metabolic conversion in these sites is the evconcentrations. Some accumulation was seen idence accumulating for high P-450 activity in Harder’s gland and the conjunctival sac. in these tissues (Harris et al., 1977; Waddell
GLAND
SAC
BLOOD
IN HEART
BRONCHI
LIVER
INTESTINE
KIDNEY
FIG. 1. A print of a whole-body autoradiograph of a 20-pm-thick section from a male C57BL/6J mouse which was frozen 6 min aftex receiving 1.2 mg [‘4C]NPIP/kg iv. White areas correspond to radioactivity. X2.6.
SALIVARY
CONJUNCTIVAL
BRONCHI
AND SUBMAXILLARY
ORAL CAVITY
FIG. 2. A print of a whole-body autoradiograph of a 20-pm-thick section from a male White areas correspond to radioactivity. ~2.2.
SUBLINGUAL,
EPITHELIUM
HARDER’S,
NASAL
CS’BL/bJ
mouse
GLANDS
LIVER
which
3 hr after receiving 1.2 mg [ 14C]NpIp/kg iv,
Gl CONTENTS PREPUTIAL GLAND BLOOD was frozen
PANCREAS
KIDNEY
SALIu4RY
OLAhtDs
CAVITY
THYMUS
BRONCHI
BLOOD
LIVER
INTESTINAL
KIDNEY
MARROW
CONTENTS
BONE
URINE
FIG. 3. A print of a whole-body autoradiograph of a 20-pm-thick section from a male C57BL./6.J mouse which was frozen 24 hr after receiving 1.3 mg [‘4C]NPIP/kg IV. White areas correspond to radioactivity. X2.3.
ANti
H&b&
ORAL
SAC
CONJUNCTIVAL
GLAND
OUCTS
THYMUS
TRACHEA
ESOPHAGUS
BLOOO
LIVER
BILE
BRONCHI
FIG. 4. A print of an enlargement of an autoradiograph of a 20-pm-thick section from a male C57BL/6J mouse which was frozen 24 hr after receiving 1.3 mg [14C]NPIP/ kg iv. White areas correspond to radioactivity. X7.3.
SALIVARY
BONE MARROW
116
MARLOWE
AND WADDELL
and Marlowe, 1976, 1978). The prolonged retention suggests that a metabolite of NPIP may be alkylating constituents of these cells. The delay in accumulation of radioactivity in oral, esophageal, and salivary duct epithelium and in Harder’s gland suggests that only the metabolites of NPIP interact with constituents of these cells. It is most unlikely that the metabolite is 14C02 because the pattern of distribution of this fragment is different from that seen with NPIP (Waddell et al., 1969). The retention in the distal tubules of the kidney may only reflect the final stages of renal elimination of NPIP and/or metabolites; the extensive renal elimination of radioactivity at early intervals after administration indicates that this radioactivity probably represents unchanged NPIP. Radioactivity in the upper intestine at 3 hr may represent biliary excretion of metabolites of NPIP. The transient appearance of radioactivity in the pancreas at 3 hr with no retention at later time intervals may represent pancreatic secretion of a metabolite of NPIP. Although the retention of radioactivity in the cortex of the thymus and in bone marrow is only minimal at 24 hr, the possibility should not be overlooked that one or more metabelites of NPIP may be affecting the immune system of the mouse. The authors are at a loss to offer tentative explanations for the residual radioactivity in the wall of the intestine and in the preputial gland. The impressive specificity of retention of radioactivity from [‘4C]NPIP in the tissues where tumors have been reported in rodents from administration of NPIP encourages further experiments with other compounds of similar structure. Studies with smaller fragments of NNN and with potential blockers of metabolism and binding of these carcinogenic nitrosamines are continuing in this laboratory.
ACKNOWLEDGMENTS The authors thank Drs. James B. Longley, Frederick K. Hilton, and Richard D. Rink, Department of Anatomy, University of Louisville, for their expert assistance in the identification of the anatomical structures in the whole-body sections and Rhonda Elam for her expert technical assistance.
REFERENCES HARRIS, C. C., AUTRUP, H., STONER, G. D., McDowELL, E. M., TRUMP, B. F., AND SCHAFER, A. (1977). Metabolism of acyclic and cyclic N-nitrosamines in cultured human bronchi. J. Nut. Cancer Inst. 59, 140 l1406. HECHT, S. S., CHEN, C. B., AND HOFFMANN, D. (1978). Evidence for metabolic a-hydroxylation of N-nitrosopyrrolidine. Cancer Rex 38, 2 15-2 18. LEUNG, K. H., PARK, K. K., AND ARCHER, M. C. (1978). Alpha-hydroxylation in the metabolism of N-nitrosopiperidine by rat liver microsomes: Formation of 5hydroxypentanal. Res. Commun. Chem. Pathol. Pharmacol. 19, 20 l-2 I 1. LONGLEY, J. B. (1969). Histochemistry of the kidney. In The Kidney (C. Rouiller and A. F. Muller, eds.), Vol. 1, pp. 157-260. Academic Press, New York. MCCORMICK, A., NICHOLSON, M. J., BAYLIS, M. A., AND UNDERWOOD, J. G. (1973). Nitrosamines in cigarette smoke condensate. Nature (London) 244,237-238. MAGEE, P. N., AND BARNES, J. M. (1967). Carcinogenic nitroso compounds. Advan. Cancer Res. 10, 163-246. WADDELL, W. J., AND MARLOWE, C. (1976). Localization of nicotine-T, cotinine-14C, and nicotine-l’-Noxide-‘% in tissues of the mouse. Drug Mefub. Dispos. 4, 530-539. WADDELL, W. J., AND MARLOWE, C. (1977). Autoradiography. In Drug Fate and Metabolism: Methods and Techniques (E. R. Garrett and J. L. Hirtz, eds.), Vol. 1, pp. l-25. Marcel Dekker, New York. WADDELL, W. J., AND MARLOWE, C. (1978). Inhibition by metyrapone of the accumulation of nicotine-14C in bronchial epithelium of mice. Arch. Int. Pharmacodyn. Ther. 234, 294-307.
WADDELL, W. J., AND MARLOWE, C. (1980). Localization of [‘%C]nitrosonomicotine in tissues of the mouse. Cancer
Res. 40, 35 18-3523.
WADDELL, W. J., ULLBERG, S., AND MARLOWE, C. ( 1969). Localization of the bicarbonate and carbonate pools by whole-body autoradiography. Arch. Int. Physiol. Biochem.
71, 1-9.