TOXICOLOGY
AND
APPLIED
Tissue
PHARMACOLOGY
Distribution
32,21-31
(1975)
of[3H]Nicotine Mon keys1
in Dogs and Rhesus
AKIRA TSUJIMOTO,~ TOSHIKATSUNAKASHIMA,~ SHIROTANINO,~ TOSHIHIRO DOHI,~ AND YUTAKA KIJROGOCHI~ Department of Pharmacology, Hiroshima University School of Dentistry, Hiroshima; Department of Pharmacology, Nara Medical School, Kashihara, Nara, Japan Received June 4, 1974; accepted September 20, 1974
Tissue Distribution of [3H]Nicotine in Dogs and RhesusMonkeys. A., NAKASHIMA, T., TANINO, S., DOHI, T. AND KUROGOCHI, Y. (1975). Toxicol. Appl. Pharmacol. 32, 21-31. The distribution of [3H]nicotine amongdifferent tissueswasexaminedin unanesthetizeddogsand monkeys following iv injection of small dose, 100 fig/kg. Nicotine was rapidly distributed throughout tissues.Five minutesafter injection, adrenal medullaand cerebralcortex containedhigh concentrationsof nicotine (961 and 505 rig/g for dogs, 1163and 310 rig/g for monkeys,respectively) and tissue/serumconcentrationratios for respectivetissuewere 13.7and 7.2 for dogs,20.7 and 5.5 for monkeys.Concentrationsin spleen,adrenalcortex, kidney, and pancreaswere relatively high in both species.There were significant regional differencesin the concentration in the CNS of both species.Concentrations in various areas of CNS in monkeys were markedly lower than those in dogs. Skeletal muscleconcentrationsand tissue/serumconcentrationsratios in monkeyswere almosttwice thosein dogs.Lower brain content in monkeysmay be due to the high affinity of skeletalmusclefor nicotine, and may partially explain the lessersensitivity to nicotine of the monkey ascomparedto the dog. Thirty minutesafter injection, kidney, gastric and intestinal mucosa, and salivary glands had relatively high concentrationsin both speciesand almostall tissueconcentrations in monkeys were markedly higher than those in dogs. Lowest levelswerefound in adiposetissue(10-38 rig/g) in both species.Tissueconcentrations of nicotine were roughly proportional to dose.Pentobarbital significantly reducednicotine concentrationsin CNS and adrenalmedulla in dogs. TSUJIMOTO,
Only limited information concerning the fate of nicotine in tissuesof higher mammals such asthe dog is available and there have been no previous reports for the rhesusmonkey (Larson et al., 1961; Larson and Silvette, 1968, 1971). In the autoradiographic studies by Hansson and Schmiterliiw (1962) on the tissue distribution of radioactive nicotine in mice, it has been suggestedthat the accumulation and subsequentreduction 1Thisstudywasaidedby a Grant from theAmericanMedicalAssociationEducationandResearch Foundation. ZDepartmentof Pharmacology, HiroshimaUniversitySchoolof Dentistry. 3Departmentof Pharmacology, Nara MedicalSchool. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
21
22
TSUJIMOTO ETAL.
of radioactivity in target tissues (the brain and adrenal medulla) appeared correlated with the pharmacological activity of nicotine. Thus, data on tissue concentration of nicotine in the dog and the monkey would be useful in the interpretation of experiments concerning the effects of nicotine in these higher species. It has been demonstrated that the rhesus monkey was five to ten times less sensitive than the dog to the effect of nicotine on gastrointestinal contractility (Hug and Bass, 1970), and respiratory and cardiac rate effects (Hug and Carlson, personal communication). The present investigation was undertaken to provide basic information on the tissue distribution of a small dose of nicotine administered intravenously to unanesthetized dogs and monkeys. Such information may be useful in understanding the basis for the quantitative differences in the responses to nicotine of the two species.
METHODS
Radioactive Nicotine
The [3H]nicotine of free base 3H-labeled generally (324-390 mCi/mmol) was obtained from the Radiochemical Center, Amersham. The compound was identical to authentic, nonradioactive, redistilled nicotine in absorbancy spectra (&,,X 259 nm) and in R, value on paper chromatograms developed in the ammonia and formic acid solvent system (McKennis et al., 1959; McKennis et al., 1962). Approximately 15 % of the tritium-label was exchangeable in aqueous solution of [‘HInicotine and gave spuriously low recoveries in the solvent extraction procedure. The labile tritium could be removed by extraction of the rH]nicotine from an alkaline aqueous solution in the n-hexane (as described below). The [3H]nicotine was recovered in aqueous solution by extraction of the hexane withO. N HCl solution. The amount of nicotine was estimated spectrophotometrically at 259 nm. The radioactive nicotine was diluted with nonradioactive, redistilled nicotine to provide a total dose of 5 and 10 &i/kg of body wt. Radiochemical purity was constantly checked by the paper chromatography and the solvent extraction procedure and there was no evidence for the change of the specific activity during 2 mo. Estimation of Radioactive Nicotine
Tissue samples weighing approximately 1 g were homogenized in 10 ml of ice-cold 0.5 N HCl in a Waring Blendor and a Potter-Elvejhem-type glass homogenizer with the exception that the adrenal medulla (dog: about 150 mg, monkey: about 100 mg), adrenal cortex (dog: about 500 mg, monkey: about 200 mg), and hypophysis (about 40 mg in both species) were homogenized in 3 ml. Duplicate samples of 1.Oml of homogenate and serum (0.5 ml of serum in a few cases) were transferred to glass stoppered 50-ml centrifuge tubes containing 0.2 ml of a 0.1 N HCl solution of nonradioactive nicotine base (2.5 mg/ml). [3H]Nicotine in biological materials was estimated essentially according to the method reported by Hug (1970). The pH was adjusted over 9 with three drops of ammonium hydroxide reagent, 1 ml of 40 % K,PO, was added followed by 10 ml of n-hexane. The mixture was shaken for 30 min at 280-300 oscillations/min and then centrifuged at 3000 rpm for 5 min. From each sample, 8 ml of the organic phase was transferred to a 20-ml glass scintillation-counting vial and 8 ml of a toluene phosphor
TISSUE
DISTRIBUTION
OF NICOTINE
23
solution (containing 6 g of 2,5-diphenyloxazole (ppo) and 200 mg of 1,4-bis-2-(5-phenyloxazolyl)benzene (popop) dissolved in 1 liter of analytical grade toluene) was added. Samples were counted in a liquid scintillation spectrometer (Packard Model 3320 or Aloka LSC-601). The recoveries of 50-1000 ng quantities of [3H]nicotine added to homogenate averaged 96 &- 1.4 % (SE, n = 5) and added to serum were 99 -1:1.7 % (SE, n = 6). Readings of counts per minute were converted to disintegration per minute by external standardization. Preliminary experiments were performed to check if the solvent extraction procedure was specific for nicotine. Considering that cotinine was the major metabolite of nicotine (McKennis et al., 1959; Schmiterlow et al., 1967) the recoveries of cotinine and total metabolites (containing cotinine and other metabolites) by this procedure were determined. [3H]Cotinine and total radioactive metabolites were obtained from dog plasma as follows : the rH]nicotine (100 &i/kg) was injected into femoral vein of a dog in a dose of 100 pg/kg. The plasma was obtained by the centrifugation of an arterial blood sample at 30 min after the injection. To 9 ml of the plasma, 1.O ml of 28 ‘A ammonium hydroxide, 3 ml of 40% K3P04, and 100 ml of CHCI, were added. After shaking for 30 min and centrifugation, the aqueous layer was extracted again with 100 ml of nhexane. The chloroform and n-hexane layers were combined and divided into two portions. Each portion was concentrated and chromatographed on silica gel plates along with authentic nicotine and cotinine by development with 85:15 CHCl,-MeOH. The spots of authentic compounds were stained with acidified iodoplatinate spray and the radioactivities were located by means of an Aloka-type JTC-201 radio thin-layer chromatogram scanner. On the plates, the silica gel layers corresponding to cotinine (R, = 0.75) were scraped off carefully and extracted with 0.1 N HCI. After centrifugation at 12,OOOgfor 10 min, the clear supernatant contained t3H]cotinine. On the other plates, silica gel layers corresponding to nicotine (R, = 0.62) were removed and the remaining silica gel layers were extracted with 0.1 N HCI and then 0.3 N NaOH. The acid and alkaline extracts were combined and contained all metabolites. One milliliter solution of [3H]cotinine and total metabolites were extracted with n-hexane by the same procedure as the nicotine extraction. The recoveries of [3H]cotinine and total metabolites were 1.6 + 0.1 and 2.7 + 0.1% (SE, n = 3) respectively. These data showed that small amounts of metabolites were transferred to the organic phase. From these results, it seems that the interference of metabolites was negligible. Animal Experiments Dogs weighing 7-12 kg, and rhesus monkeys weighing 2-3.5 kg, of either sex were housed in air-conditioned quarters. On the day of the experiment, monkeys were placed in restraining chairs. Dogs were trained to lie quietly on a table; IO-500 pg/kg of [3H]nicotine were injected over 10 set through a polyethylene or steel tube inserted into a vein of the dorsal aspect of the forearm of monkeys and forepaw of dogs. Animals were injected iv with 30 mg/kg of pentobarbital sodium 30 set before sacrifice by exsanguination via the carotid artery. In some experiments, to examine the tissue distribution in anesthetized animals, dogs were anesthetized with iv pentobarbital30 min before nicotine administration. Blood samples were collected in cold beakers and kept on ice. Because the liver possessed activity for metabolizing nicotine, it was excised first, followed by removal of other tissues. Tissues of the central nervous system were carefully
24
TSUJIMOTO
ET AL.
separated into anatomical areas (Table 1). Samples of tissues were blotted, wrapped in aluminium foil, and frozen immediately in dry ice. RESULTS [3H]Nicotine (lOOpg/kg) Injection to Dogs The dog showed grossly observable effects of nicotine within the first minute and the effects dissipated within 10 min or less after the injection. The dose of 100 pg/kg of nicotine caused marked restlessness, hyperpnea, miosis, salivation, and retching. Usually vomiting, defecation, and urination occurred. Tissue concentrations of nicotine in dogs 3, 5, 10, and 30 min after iv injection of [3H]nicotine are shown in Table 1. Nicotine was very rapidly distributed throughout the tissues of animals. Three minutes after the injection, the concentrations of nicotine in all tissues with the exception of the skeletal muscle and adipose tissue were higher than those of respective tissue at 5 min. At 3 and 5 min, the adrenal medulla had the highest concentration of unchanged nicotine, 1229 and 961 rig/g tissue, respectively. The concentrations in the central nervous system (CNS) also were relatively high, particularly the hypophysis and the cerebral cortex (gray matter). The tissue/serum concentration ratios were 13.7 and 7.2 for the adrenal medulla and cerebral cortex, respectively. The adrenal cortex, spleen, kidney, and salivary glands also had the high concentrations. The liver which possessed nicotine-metabolizing-activity contained low concentrations. The levels in subcutaneous fat, great omentum, skeletal muscle, and serum were also low. At 10 min, the adrenal glands and CNS contained smaller concentrations as compared to those at 3 and 5 min, and these tissue concentrations already were slightly lower than those of spleen and kidney. At 30 min, the nicotine concentrations of almost all tissues were one-half to one-tenth of those in the same tissues at 5 min. The salivary glands, adrenal medulla, spleen, gastric mucosa, and kidney still had high concentrations of nicotine relative to other tissues. The nicotine content of salivary glands and gastric mucosa were not lower than those in kidney. In rats (Yamamoto et al., 1968) and mice (Hansson and Schmiterlow, 1962) high radioactivities in salivary glands also have been shown following [‘“Clnicotine administration. There were no significant differences in the tissue concentrations of nicotine between male and female animals. [3H]Nicotine (10 pg/kg) Injection to dogs The tissue distribution of nicotine in dogs injected with 10 ,ng/kg of the drug which is within the smoking range (Larson and Silvette, 1968) were examined. With this dose of nicotine, slight restlessness and hyperpnea were observed and then slight ataxia occurred occasionally. The results are shown in Table 1. At 5 min, highest concentrations were found in the adrenal medulla (63 rig/g). The spleen, kidney, and cerebral cortex (31 rig/g) also had relatively high concentrations. The tissue/serum concentration ratios for the adrenal medulla and cerebral cortex were approximately equivalent to the respective value in dogs given 100 pg/kg. The skeletal muscle, adipose tissue, and serum had low concentrations relative to
-
628 III 72 (8)” 213 f 29(8) 484k 30(7) 541* 51(7) 297k 39(8) 300+ 32(8) 952f 106(6) 487i 61(8) 252+ 32(7) 1229+270(5) 685 + 140 (4) 679+ 134(8) 209f 39(6) 382 + 110(5) 114_+ 20(5) 245+ 29(5) 166_+ 14(5) 429t50(8) 165+ 14(7) 589f 67(6) 270+ 19(7) 36f S(7) 26+ S(8) 7_+ 2(5) 169+ 17(13) 125+ 15(8)
3
46+ 4(7) 146rt 15(7) 105f S(6) 387f35(14) 163 + 14(14) 502*49(15) 260*28(15) 41 f 4(15) 30f 7(14) 12f 2(7) 77 f 9(14) 70& 4(14)
101* 15(8)
505 f 46(15)" 181+ 16(15) 355f 39(15) 388+36(15) 234 + 20(15) 261f 24(15) 689f 86(15) 379* 30(15) 171?15(14) 961 f 82(9) 526?78(9) 322f 57(13) 176+25(14)
5
S(4) 5(4) 66+ 12(4) 57?T 7(3)
47+ 265
189+20(4)
389,48(4) 135k 15(3) 453 k49(4)
670+ 91(4) 265 +21(4) 141f20(4) 3202 50(3) 254+47(3) 145 &21(3) 136+43(4) 115+28(3) 175?74(3) 172*16(4)
191f 28 (4)
285 k26(4) 188_+25(4)
190+ 25 (4) 244_+27 (4)
361 ?28(4)"
Minutes after injection 10 ~___
Concentration of nicotine (rig/g wet tissue) Dose: 100@g/kg
0Valuesrepresentthe mean+ SE. Numberin parentheses indicatesthe numberof animals. * Submandibular gland. CSartorius. dInterscapularregion.
Cerebral cortex white Hypothalamus Thalamus Pons Medulla Hypophysis Cerebellum Spinal cord Adrenal medulla cortex Salivary glandb Lung Gastric mucosa muscle Intestinal mucosa muscle Kidney Heart Spleen Pancreas Skeletal muscle’ Great omentum Subcutaneousfatd Liver Serum
Tissues
1
30 ___~ 70 2? lO(7)" 49 + 120) 51+ 8(6) 54+ 7(6) 4031 4(6) 4ok S(7) 76+ 9(6) 53+ 8(7) 49f 6(6) 176_+20(6) 92+ 11 (6) 133 _+ M(7) 38k 4(7) 102k 27(7) 34+ 2(6) 47+ S(7) 4Ok S(7) 103 Ik 14(7) 27k 3(7) 14of 19(7) 83 rir 8(7) 31zk 3(7) 12+ 3(7) lo+ 2(6) 46k 6(7) 14k l(7)
5 ~-- ~-31 F 2 (7)" 10 * l(7) 21 f. 2(.5) 29 + 6(7) 15 k l(5) 14 + 1 (5) 33 zk 7(7) 24+2(S) 12 + l(5) 63 _+ 9(7) 35 i- 3(7) 23 _+ 5(6) 18+2(5) 12+2(6) 7 iz l(7) 17+2(7) 13+2(7) 41 Ifr S(7) 12f l(5) 47 f 4(5) 3Ok 5(5) 6?1(7) 4 * 0.4(7) 4+ l(7) 10* 1 (7) 5zk l(7)
Dose: 10pg/kg
CONCENTRATIONS OF[3H]N~~~~~~~OFVARIOUSTISSUES IN UNANESTHETIZED Does AFTERIV INJECTION OF 100 AND10 pg/kg OFDRUG
TABLE
26
TSUJIMOTO
ET
other tissues. These results were approximately 100 pg/kg of nicotine.
AL.
similar to those in dogs injected with
[3H]Nicotine (lOO,ug/kg) Injection to Pentobarbital Anesthetized Dogs In anesthetized dogs, most effects of nicotine which were grossly observable in unanesthetized animals could not be produced. Only hyperpnea was observed. The tissue concentrations of nicotine in pentobarbital-anesthetized dogs and the ratio of the concentrations in anesthetized dogs to those of respective tissue in unanesthetized dogs 5 min after the administration are shown in Table 2. It was found that the pentobarbital anesthesia significantly reduced nicotine concentrations in the CNS, adrenal glands, heart and serum at 5 min after the injection of nicotine. The cerebral cortex concentrations were about 56 % of those in unanesthetized dogs. On the other hand, concentrations in liver, gastric mucosa, intestinal muscle, pancreas, and fatty tissue were significantly higher relative to those of respective tissue in unanesthetized animals. [jH]Nicotine
(100 pg/kg) Injection to Monkeys
In monkeys, 100 pg/kg of nicotine produced miosis and facial redness, and restlessness, hyperpnea, and coughing were observed occasionally. The tissue concentrations of nicotine in monkeys 5 and 30 min after intravenous administration of [3H]nicotine are shown in Table 3. Five minutes following administration of nicotine, the adrenal glands and kidney had the highest concentrations. The CNS and spleen also were high relative to other tissues such as the lung and pancreas. In the CNS, the highest concentrations of nicotine were found in the hypophysis and cerebral cortex followed by the cerebellum, thalamus, hypothalamus, medulla, pons, spinal cord, and cerebral white matter, in order of decreasing concentration similarly to that observed in dogs. The tissue/serum concentration ratios for the adrenal medulla and cerebral cortex were 20.7 and 5.5, respectively. The concentrations in liver, skeletal muscle, and adipose tissue were low. At 30 min, the adrenal glands followed by kidney, pancreas, spleen, intestinal mucosa, cerebral cortex, and salivary glands had relatively high nicotine concentrations. Concentrations in almost all tissues were reduced to about one-half of those at 5 min following the injection. The concentrations in the salivary glands, skeletal muscle, and intestinal mucosa remained relatively constant throughout the 30 min. The decrement in the tissue content between 5 and 30 min in monkeys was much less marked than that in dogs. C3H]Nicotine (500 pg/kg) Injection to Monkeys In monkeys, the 500qg/kg dose of nicotine produced restlessness, hyperpnea, miosis, and retching. Usually vomiting and urination occurred. The intensity of these effects was roughly similar to that in dogs injected with 100 pg/kg of nicotine. The tissue concentrations of nicotine in monkeys 5 min after iv injection of 500 fig/kg of drug are shown in Table 3. The highest concentrations were in the adrenal medulla followed by hypophysis, kidney, and adrenal cortex. The lung, cerebral cortex, and salivary glands also had relatively high concentrations. The tissue/serum concentration ratios for the adrenal
0.58 0.64 0.44 0.57 0.76 0.61 0.57 0.89 1.02
0.56b 0.77 0.59 0.60 0.05 0.01 0.02 NS 0.01 0.05 NS NS
0.01” 0.05 0.05 0.01 Heart Kidney Spleen Pancreas Skeletal muscle Great omentum Subcutaneous fat Liver Serum
Gastric mucosa muscle Intestinal mucosa muscle
of nicotine (rig/g wet tissue)
AFTER
102++ 42(7) 20(4) 377 500 + 104 (4) 337+ 8(4) 32k 5 (7) 42+ 6(7) 22 + 4 (7) 124+ 10(5) 52 rfI 5 (7)
0.63 0.97 1.OO 1.30 0.78 1.40 1.83 1.61 0.14
2.35b 1.67 1.29 1.41
IV INJECTION
237+ 34(5) 77+ 14(7) 188i 14(7) 148 k 9 (7)
MINUTES
animals.
DOGS 5
a Values represent the mean k SE. Number in parentheses indicates the number of animals. * Ratio of the nicotine concentration in anesthetized dogs to that of respective tissue in unanesthetized c Probability of difference from unanesthetized dogs.
151 rt+ 13 19 (4) 150 303 f 31 (6) 214 * 25 (4) 130-t lO(4) 559 f 82 (7) 297 + 36 (7) 287 + 40 (5) 180*46(4)
Medulla Pons Hypophysis Cerebellum Spinal cord Adrenal medulla cortex Salivary gland Lung
17 (7)” 6(7) 20 (4) 12 (7)
2
IN PENTOBARBITAL ANESTHETIZED OF 100 pg/kg OF DRUG
Concentration
OF [3H]N~~~~~~~
282 + 139k 208 + 233 f:
CONCENTRATIONS
Cerebral cortex white Hypothalamus Thalamus
TISSUE
TABLE
0.05 NS NS 0.01 NS NS 0.05 0.01 0.01
0.05” NS NS 0.01
Z
% z 8 =! 5
3
5 f: 3 2 B
OF
0.61bvc 0.56c 0.51C 0.56” 0.60’ 0.55’ 0.50” 0.58” 0.67’ 1.27 1.06 0.48” 1.39 1.57” 1.30 1.36” 1.31 1.42’ 0.52” 0.60’ 0.95 1.95’ 1.17 3.17’ 0.48’ 0.80’
Minutes 151 + 18 (3) 79 f 11 (3) 89 I!I 9 (3) 97 f 9 (3) 78 + 7(3) 80 + 10 (3) 119kl4(3) 113 f 11(3) 77 + 6 (3) 429 + 21 (3) 209 f 20 (3) 148 + 28 (3) 133 +_ 3 (3) 89 + 23 (3) 42 + 5 (3) 175 + 9(3) 88 f 9(3) 301 f 55 (3) 47 + 6(3) 185 f 15 (3) 187 rt 10 (3) 74_+ 8 (3) 27 f Z(3) 24f 2 (3) 22+_ 3 (3) 23 f 4 (3)
after injection 30
Concentration of nicotine Dose : 100 pgg/kg
TABLE 3 OF VARIOUS TISSUES IN UNANESTHETIZED 100 AND 500 pg/kg OF DRUG
310+ 28 (7) 101* 13 (7) 182& 15(7) 218 f 21 (7) 141+ 14(7) 143 + 14(7) 342 f 69 (7) 221 + 23 (7) 114f 11 (7) 1163 f 137 (4) 556 f 140 (4) 156k 20(7) 245 +_ 48 (7) 159+ 17(4) 60 f 6 (4) 199+ 13(4) 138+_ 23 (4) 550* 59 (7) 84 f 6 (7) 300 + 31 (7) 247+_ 38(7) SO_+ 11 (7) 35 -I 6(7) 38+ S(4) 37 f 4(7) 56f 5 (7)
5
[3H]N~~~~~~~ AFTER
2.16”,= 1.61 1.75” 1.80” 1.95’ 2.00’ 1.57” 2.13’ 1.57” 2.44” 2.27’ 1.11 3.50’ 0.87 1.23 3.72’ 2.20’ 2.92” 1.74” 1.32 2.25’ 2.39” 2.25’ 2.40” 0.48’ 1.64
(rig/g wet tissue)
MONKEYS
u Values represent the mean f SE. Number in parentheses indicates the number of animals. b Ratio of the nicotine concentration in monkeys to that of respective tissue in dogs. c Significant at p values of 0.05 or less.
cortex white Hypothalamus Thalamus Pons Medulla Hypophysis Cerebelhun Spinal cord Adrenal medulla cortex Salivary gland Lung Gastric mucosa muscle Intestinal mucosa muscle Kidney Heart Spleen Pancreas Skeletal muscle Great omentum Subcutaneous fat Liver Serum
Cerebral
Tissues
CONCENTRATIONS
OF
1572 + 394 (3) 609+ 98 (3) 1082 f 204 (3) 1123 + 214 (3) 796 + 86(3) 72Of 24 (3) 3590 +_ 1037 (3) 1055 + 323 (3) 596 + 102 (3) 5824 f 379 (3) 3013 + 193 (3) 1465 + 727 (3) 2116 + 642 (3) 590 + 137 (3) 316+ 49(3) 774 + 217 (3) 469 +_ 116 (3) 3380 + 878 (3) 530 f 32(3) 1130+ 260(3) 1399 f 78 (3) 346_+ 56 (3) 293 IL 86(3) 227 + 23 (3) 133 f 26(3) 329f 32(3)
5
Dose : 500 pug/kg
IV INJECTION
TISSUE
DISTRIBUTION
OF NICOTINE
29
medulla and cerebral cortex were approximately equivalent to the respective values in monkeys given 100 pug/kg. The liver and adipose tissue concentrations were the lowest. DISCUSSION
Nicotine was quickly distributed throughout the tissues of dogs and monkeys after iv injection of 100 pg/kg of drug. It should be noted that levels of nicotine and tissueserum concentration ratios in target tissues such as the adrenal medulla and CNS were very high shortly following the iv injection of nicotine in both species. These findings are in agreement with those reported in mice using an autoradiographic method (Hansson and Schmiterlow, 1962), in cats using a methylene dichloride extraction procedure (Turner, 1969), and in rats using the present procedure (Hug, 1970). The blood supply to the brain is large and extracellular fluid space is small and the capillaries are completely invested with a cellular wrapping of glia cells in the CNS (Bonnycastle, 1965). The vascular density of adrenal glands also is conspicuously high (Nair et al., 1960). At pH 7.4 and a temperature of 37°C nicotine has been calculated to be about 31% nonionized base which can readily penetrate the blood-brain barrier (Domino, 1965). In addition, nicotine has high lipid solubility (Bowman et al., 1964). These physiological and anatomical characteristics, and physicochemical properties of nicotine may account for the faster penetration and egress of nicotine from these tissues. Nicotine concentration in various areas of CNS in dogs and monkeys arranged in order of decreasing concentration was as follows: the hypophysis, cerebral cortex, cerebellum, thalamus, hypothalamus, medulla, pons, spinal cord, and cerebral white matter. These results are in general agreement with those reported for the cat injected with small multiple doses of nicotine (Turner, 1971). In the autoradiographic study of brain in rats given [14C]nicotine, Schmiterlow et al. (1967) suggested that radioactivity was concentrated in the nerve cells. In the study of tissue distribution of morphine (Mule and Woods, 1962) it was suggested that the multiple membranes of the laminated myelin sheaths which are found in white matter may function as a barrier to penetration of the drug. Furthermore, the vascularity of the CNS is not uniform, e.g., the vascular density of cerebral cortex is much greater than that of the spinal cord and white matter (Nair et al., 1960). Therefore, the differences in cell density and vascular density of the CNS may account for the differences in the concentration of nicotine among various anatomical areas. Pentobarbital was found to reduce the concentration of nicotine in brain and adrenal medulla. Most effects of nicotine which were grossly observable in unanesthetized dogs could not be produced in the anesthetized animals. Barbiturates have a protective action against nicotine-induced convulsions (Yamamoto et al., 1966) and death (Larson et al., 1949; Yamamoto et al., 1966). The decrease in nicotine concentration in “target tissue” may result in part in the antagonism of the effects of nicotine by pentobarbital. In the comparison of tissue concentrations of nicotine (Table 3) and tissue/serum concentrations (Table 4) in dogs and monkeys 5 min after nicotine administration, it is noteworthy that concentrations in the CNS in monkeys were lower relative to those in dogs. In addition, the tissue/serum concentration ratios in heart, a tissue rich in catecholamine granules, were also considerably lower in monkeys as compared to dogs. The monkey appear to be five to ten times less sensitive than the dog to the effects of
30
TSUJIMOTO
ET AL.
TABLE 4 TISSUE/SERUM CONCENTRATION RAnos IN SOME TISSUES OF Does AND MONKEYS 5 MIN FOLLOWING IV INJECTION OF 100 pg/kg OF [‘HINICOTINE
Tissues Cerebral cortex Hypothalamus Heart Adrenal medulla Skeletal muscle
Tissue/serum ratios Monkey Dog 7.2 5.1 2.3 13.7 0.6
5.5 3.3 1.5 20.8 1.4
nicotine on gastrointestinal contractility (Hug and Bass, 1970), and respiratory and cardiac rate (Hug and Carlson, personal communication). Many of the peripheral actions of nicotine including the increase of heart rate have been considered as secondary effects, due to catecholamine liberated from the terminal adrenergic nerve fibres by nicotine and the adrenal medulla (Burn and Rand, 1958; Westfall and Watts, 1963). Furthermore, nicotine has been reported to increase not only the respiratory and heart rate but to inhibit the contractile activity of the gastrointestinal tract through stimulation of the CNS (Carlson et al., 1970). Tissue/serum concentration ratios in the adrenal medulla were higher than those in dogs. In addition, all tissue concentrations with the exception of the liver and spleen in monkeys given 500 pg/kg of nicotine were three times or more higher than those of respective tissue in dogs given 100 pg/kg even though the intensity of the observable effects in monkeys was roughly similar to dogs. Therefore, factors causing the species differences in quantitative responses to nicotine are not solely due to the differences in distribution. Recently, it was shown that the effect of nicotine in catecholamine-release from the adrenal glands in monkeys was less potent than that in dogs (Tsujimoto et al., 1974). In the previous report, it was suggested that the rate of nicotine destruction in monkeys might be faster than that in dogs (Tsujimoto et al., 1972); this is in agreement with findings that nicotine metabolizing enzyme activities of monkey liver are higher than those of dog liver (Dohi et al., 1973). The skeletal muscle and tissue/serum ratios in monkeys were almost twice as high as those in dogs. The skeletal muscle occupies a great proportion of the body mass, approximately 40-50 % of body weight in the monkey (Hayashi, 1965). Therefore, it could be considered that the higher affinities of skeletal muscle for nicotine and the faster destruction of nicotine in monkeys compared to that of dogs may account for the lower concentrations in the CNS, some other tissues and serum. ACKNOWLEDGMENTS
The authors express their appreciation to Dr. M. H. Seevers for advice and continued interest in this investigation. We thank Dr. H. McKennis, Jr., and Dr. E. R. Bowman for a generous supply of authentic cotinine. REFERENCES BONNYCASTLE, D. D. (1965). Intimate
study of drug action I: Absorption
and distribution.
In
Drill’s Pharmacologyin Medicine (J. R. Dipalma, Ed.), 3rd ed., pp. 16-25. McGraw-Hill
Book Company, New York.
TISSUE
DISTRIBUTION
OF NICOTINE
31
E. R., HANSSON, E., TURNBULL, L. B., MCKENNIS, H., JR. AND SCHMITERL~W, C. G. (1964). Disposition and fate of (-)-cotinine-H3 in the mouse. J. Phurmacol. Exp. Ther. 143. 301-308. BURN, J. H. AND RAND, M. J. (1958). Noradrenaline in artery walls and its dispersalby reserpine.Brit. J. Pharmacol.1, 903-908. CARLSON, G. M., RUDDON, R. W., HUG, C. C., JR., SCHMIEGE, S. K. AND BASS, P. (1970).Analysis of the site of nicotine action on gastric antral and dudodenalcontractile activity. f. Pharmacol.Exp. Ther. 172, 377-383. DOHI, T., KOJIMA, S. AND TSUJIMOTO, A. (1973). Comparative studiesof hepatic nicotine metabolizingenzymeactivities in monkeysand dogs.JapanJ. Phurmacol.13,748-751. DOMINO, E. F. (1965).Somecomparative pharmacologicalactionsof (-)-nicotine, its optical isomer, and related compounds.In TobaccoAlkaloids and Related Compounds (U.S. von Euler, Ed.), pp. 303-319.PergamonPress,Oxford. HANSSON, E. AND SCHMITERL~W, C. G. (1962).Physiologicaldispositionandfate of C1s-labelled nicotinein miceandrats.J. Pharmacol.Exp. Ther. 137,91-102. HAYASHI, K. (1965).Kaibogakuoyobi SeirigakuKeisu,2nd ed., pp. 65. Bunkyo Shoin, Tokyo. HUG, C. C., JR. (1970).Distribution of nicotine in the rat. Pharmacologist12, 220. HUG, C. C., JR. AND BASS, P. (1970).Effect of nicotine on gastrointestinalcontractile activity. Univ. Mich. Med. Cent. J. 36, 246. LARSON, P. S., FINNEGAN, J. K., BIBB, J. C. AND HAAG, H. B. (1949).Speciesvariation in the protective action of anesthesia againstthe acutely lethal effect of nicotine. Fed.Proc. 8, 313. LARSON, P. S., HAAG, H. B. AND SILVETTE, H. (1961). Tobacco,Experimentaland Clinical Studies,pp. 10-12. The Williams and Wilkins Company, Baltimore. LARSC)N, P. S. AND SILVETTE, H. (1968).Tobacco,ExperimentalandClinicalStudies,Supplement I, pp. 5, 6, 45, 683, 684. The Williams and Wilkins Company, Baltimore. LARSON, P. S. AND SILVETTE, H. (1971).Tobacco,ExperimentalandClinicalStudies,Supplement II, pp. 3-5. The Williams and Wilkins Company, Baltimore. MCKENNIS, H., JR., TURNBULL, L. B., SCHWARZ, S. L., BOWMAN, E. R. AND WADA, E. (1959). Demethylation of cotinine in vivo. J. Amer. Chem.Sot. 81, 3951-3954. MCKENNIS H., JR., TURNBULL, L. B., SCHWARZ, S. L., TAMAKI, E. AND BOWMAN, E. R. (1962). Demethylation in the metabolismof (-)-nicotine. J. Biol. Chem.237, 541-546. MULE, S. J. AND WOODS, L. A. (1962). Distribution of N-C14-methyllabelled morphine: I. In central nervoussystemof nontolerant and tolerant dogs.J. Pharmacol.Exp. Ther. 136, BOWMAN,
232-241.
V.. PALM, D. AND ROTH, L. J. (1960).Relative vascularity of certain anatomicalareasof the brain and other organsof the rat. Nature (London) 188, 497498. SCHMITERL~W, C. G., HANSSON, E., ANDERSON, G., APPELGREN, L. E. AND HOFFMANN, P. C. (1967). Distribution of nicotine in the central nervous system.Ann. N. Y. Acud. Sci. 142, 2-14. TSUJIMOTO, A., KOJIMA, S., IKEDA, M. AND DOHI, T. (1972).Excretion of nicotine and its metabolites in dog and monkey saliva. Toxicol. Appl. Pharmacol.22, 365-374. TSUJIMOTO, A., NISHIKAWA, T., DOHI, T. AND KOJIMA, S. (1974).Comparisonof pharmacological responses to nicotine and releaseof catecholaminesfrom the adrenalglandsin dogs and monkeys.Eur. J. Pharmacol.26, 236-242. TURNER. D. M. (1969).The metabolismof [14C]nicotine in the cat. Biochem.J. 115, 889-896. TURNER. D. M. (1971).Metabolism of smallmultiple dosesof [‘*Cl nicotine in the cat. Brit. J. Pharmacol.41, 521-529. WESTFALL, T. C. AND WATTS, D. T. (1963).Effect of cigarette smokeon epinephrinesecretion in the dog. Proc. Sot. Exp. Biol. Med. 112, 843-847. YAMAMOTO, I., INOKI, R. AND IWATSUBO, K. (1968).Penetration of nicotine-14Cinto several rat tissuein vivo and in vitro. Toxicol. Appl. Pharmacol.12, 560-567. YAMAMOTO, I., OTORI, K. AND INOKI, R. (1966). Pharmacologicalstudieson antagonists againstnicotine-inducedconvulsionsand death. Japan.J. Pharmacol.16, 402-415. NAIR.
AND
APPLIED
Tissue
PHARMACOLOGY
Distribution
32,21-31
(1975)
of[3H]Nicotine Mon keys1
in Dogs and Rhesus
AKIRA TSUJIMOTO,~ TOSHIKATSUNAKASHIMA,~ SHIROTANINO,~ TOSHIHIRO DOHI,~ AND YUTAKA KIJROGOCHI~ Department of Pharmacology, Hiroshima University School of Dentistry, Hiroshima; Department of Pharmacology, Nara Medical School, Kashihara, Nara, Japan Received June 4, 1974; accepted September 20, 1974
Tissue Distribution of [3H]Nicotine in Dogs and RhesusMonkeys. A., NAKASHIMA, T., TANINO, S., DOHI, T. AND KUROGOCHI, Y. (1975). Toxicol. Appl. Pharmacol. 32, 21-31. The distribution of [3H]nicotine amongdifferent tissueswasexaminedin unanesthetizeddogsand monkeys following iv injection of small dose, 100 fig/kg. Nicotine was rapidly distributed throughout tissues.Five minutesafter injection, adrenal medullaand cerebralcortex containedhigh concentrationsof nicotine (961 and 505 rig/g for dogs, 1163and 310 rig/g for monkeys,respectively) and tissue/serumconcentrationratios for respectivetissuewere 13.7and 7.2 for dogs,20.7 and 5.5 for monkeys.Concentrationsin spleen,adrenalcortex, kidney, and pancreaswere relatively high in both species.There were significant regional differencesin the concentration in the CNS of both species.Concentrations in various areas of CNS in monkeys were markedly lower than those in dogs. Skeletal muscleconcentrationsand tissue/serumconcentrationsratios in monkeyswere almosttwice thosein dogs.Lower brain content in monkeysmay be due to the high affinity of skeletalmusclefor nicotine, and may partially explain the lessersensitivity to nicotine of the monkey ascomparedto the dog. Thirty minutesafter injection, kidney, gastric and intestinal mucosa, and salivary glands had relatively high concentrationsin both speciesand almostall tissueconcentrations in monkeys were markedly higher than those in dogs. Lowest levelswerefound in adiposetissue(10-38 rig/g) in both species.Tissueconcentrations of nicotine were roughly proportional to dose.Pentobarbital significantly reducednicotine concentrationsin CNS and adrenalmedulla in dogs. TSUJIMOTO,
Only limited information concerning the fate of nicotine in tissuesof higher mammals such asthe dog is available and there have been no previous reports for the rhesusmonkey (Larson et al., 1961; Larson and Silvette, 1968, 1971). In the autoradiographic studies by Hansson and Schmiterliiw (1962) on the tissue distribution of radioactive nicotine in mice, it has been suggestedthat the accumulation and subsequentreduction 1Thisstudywasaidedby a Grant from theAmericanMedicalAssociationEducationandResearch Foundation. ZDepartmentof Pharmacology, HiroshimaUniversitySchoolof Dentistry. 3Departmentof Pharmacology, Nara MedicalSchool. Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
21
22
TSUJIMOTO ETAL.
of radioactivity in target tissues (the brain and adrenal medulla) appeared correlated with the pharmacological activity of nicotine. Thus, data on tissue concentration of nicotine in the dog and the monkey would be useful in the interpretation of experiments concerning the effects of nicotine in these higher species. It has been demonstrated that the rhesus monkey was five to ten times less sensitive than the dog to the effect of nicotine on gastrointestinal contractility (Hug and Bass, 1970), and respiratory and cardiac rate effects (Hug and Carlson, personal communication). The present investigation was undertaken to provide basic information on the tissue distribution of a small dose of nicotine administered intravenously to unanesthetized dogs and monkeys. Such information may be useful in understanding the basis for the quantitative differences in the responses to nicotine of the two species.
METHODS
Radioactive Nicotine
The [3H]nicotine of free base 3H-labeled generally (324-390 mCi/mmol) was obtained from the Radiochemical Center, Amersham. The compound was identical to authentic, nonradioactive, redistilled nicotine in absorbancy spectra (&,,X 259 nm) and in R, value on paper chromatograms developed in the ammonia and formic acid solvent system (McKennis et al., 1959; McKennis et al., 1962). Approximately 15 % of the tritium-label was exchangeable in aqueous solution of [‘HInicotine and gave spuriously low recoveries in the solvent extraction procedure. The labile tritium could be removed by extraction of the rH]nicotine from an alkaline aqueous solution in the n-hexane (as described below). The [3H]nicotine was recovered in aqueous solution by extraction of the hexane withO. N HCl solution. The amount of nicotine was estimated spectrophotometrically at 259 nm. The radioactive nicotine was diluted with nonradioactive, redistilled nicotine to provide a total dose of 5 and 10 &i/kg of body wt. Radiochemical purity was constantly checked by the paper chromatography and the solvent extraction procedure and there was no evidence for the change of the specific activity during 2 mo. Estimation of Radioactive Nicotine
Tissue samples weighing approximately 1 g were homogenized in 10 ml of ice-cold 0.5 N HCl in a Waring Blendor and a Potter-Elvejhem-type glass homogenizer with the exception that the adrenal medulla (dog: about 150 mg, monkey: about 100 mg), adrenal cortex (dog: about 500 mg, monkey: about 200 mg), and hypophysis (about 40 mg in both species) were homogenized in 3 ml. Duplicate samples of 1.Oml of homogenate and serum (0.5 ml of serum in a few cases) were transferred to glass stoppered 50-ml centrifuge tubes containing 0.2 ml of a 0.1 N HCl solution of nonradioactive nicotine base (2.5 mg/ml). [3H]Nicotine in biological materials was estimated essentially according to the method reported by Hug (1970). The pH was adjusted over 9 with three drops of ammonium hydroxide reagent, 1 ml of 40 % K,PO, was added followed by 10 ml of n-hexane. The mixture was shaken for 30 min at 280-300 oscillations/min and then centrifuged at 3000 rpm for 5 min. From each sample, 8 ml of the organic phase was transferred to a 20-ml glass scintillation-counting vial and 8 ml of a toluene phosphor
TISSUE
DISTRIBUTION
OF NICOTINE
23
solution (containing 6 g of 2,5-diphenyloxazole (ppo) and 200 mg of 1,4-bis-2-(5-phenyloxazolyl)benzene (popop) dissolved in 1 liter of analytical grade toluene) was added. Samples were counted in a liquid scintillation spectrometer (Packard Model 3320 or Aloka LSC-601). The recoveries of 50-1000 ng quantities of [3H]nicotine added to homogenate averaged 96 &- 1.4 % (SE, n = 5) and added to serum were 99 -1:1.7 % (SE, n = 6). Readings of counts per minute were converted to disintegration per minute by external standardization. Preliminary experiments were performed to check if the solvent extraction procedure was specific for nicotine. Considering that cotinine was the major metabolite of nicotine (McKennis et al., 1959; Schmiterlow et al., 1967) the recoveries of cotinine and total metabolites (containing cotinine and other metabolites) by this procedure were determined. [3H]Cotinine and total radioactive metabolites were obtained from dog plasma as follows : the rH]nicotine (100 &i/kg) was injected into femoral vein of a dog in a dose of 100 pg/kg. The plasma was obtained by the centrifugation of an arterial blood sample at 30 min after the injection. To 9 ml of the plasma, 1.O ml of 28 ‘A ammonium hydroxide, 3 ml of 40% K3P04, and 100 ml of CHCI, were added. After shaking for 30 min and centrifugation, the aqueous layer was extracted again with 100 ml of nhexane. The chloroform and n-hexane layers were combined and divided into two portions. Each portion was concentrated and chromatographed on silica gel plates along with authentic nicotine and cotinine by development with 85:15 CHCl,-MeOH. The spots of authentic compounds were stained with acidified iodoplatinate spray and the radioactivities were located by means of an Aloka-type JTC-201 radio thin-layer chromatogram scanner. On the plates, the silica gel layers corresponding to cotinine (R, = 0.75) were scraped off carefully and extracted with 0.1 N HCI. After centrifugation at 12,OOOgfor 10 min, the clear supernatant contained t3H]cotinine. On the other plates, silica gel layers corresponding to nicotine (R, = 0.62) were removed and the remaining silica gel layers were extracted with 0.1 N HCI and then 0.3 N NaOH. The acid and alkaline extracts were combined and contained all metabolites. One milliliter solution of [3H]cotinine and total metabolites were extracted with n-hexane by the same procedure as the nicotine extraction. The recoveries of [3H]cotinine and total metabolites were 1.6 + 0.1 and 2.7 + 0.1% (SE, n = 3) respectively. These data showed that small amounts of metabolites were transferred to the organic phase. From these results, it seems that the interference of metabolites was negligible. Animal Experiments Dogs weighing 7-12 kg, and rhesus monkeys weighing 2-3.5 kg, of either sex were housed in air-conditioned quarters. On the day of the experiment, monkeys were placed in restraining chairs. Dogs were trained to lie quietly on a table; IO-500 pg/kg of [3H]nicotine were injected over 10 set through a polyethylene or steel tube inserted into a vein of the dorsal aspect of the forearm of monkeys and forepaw of dogs. Animals were injected iv with 30 mg/kg of pentobarbital sodium 30 set before sacrifice by exsanguination via the carotid artery. In some experiments, to examine the tissue distribution in anesthetized animals, dogs were anesthetized with iv pentobarbital30 min before nicotine administration. Blood samples were collected in cold beakers and kept on ice. Because the liver possessed activity for metabolizing nicotine, it was excised first, followed by removal of other tissues. Tissues of the central nervous system were carefully
24
TSUJIMOTO
ET AL.
separated into anatomical areas (Table 1). Samples of tissues were blotted, wrapped in aluminium foil, and frozen immediately in dry ice. RESULTS [3H]Nicotine (lOOpg/kg) Injection to Dogs The dog showed grossly observable effects of nicotine within the first minute and the effects dissipated within 10 min or less after the injection. The dose of 100 pg/kg of nicotine caused marked restlessness, hyperpnea, miosis, salivation, and retching. Usually vomiting, defecation, and urination occurred. Tissue concentrations of nicotine in dogs 3, 5, 10, and 30 min after iv injection of [3H]nicotine are shown in Table 1. Nicotine was very rapidly distributed throughout the tissues of animals. Three minutes after the injection, the concentrations of nicotine in all tissues with the exception of the skeletal muscle and adipose tissue were higher than those of respective tissue at 5 min. At 3 and 5 min, the adrenal medulla had the highest concentration of unchanged nicotine, 1229 and 961 rig/g tissue, respectively. The concentrations in the central nervous system (CNS) also were relatively high, particularly the hypophysis and the cerebral cortex (gray matter). The tissue/serum concentration ratios were 13.7 and 7.2 for the adrenal medulla and cerebral cortex, respectively. The adrenal cortex, spleen, kidney, and salivary glands also had the high concentrations. The liver which possessed nicotine-metabolizing-activity contained low concentrations. The levels in subcutaneous fat, great omentum, skeletal muscle, and serum were also low. At 10 min, the adrenal glands and CNS contained smaller concentrations as compared to those at 3 and 5 min, and these tissue concentrations already were slightly lower than those of spleen and kidney. At 30 min, the nicotine concentrations of almost all tissues were one-half to one-tenth of those in the same tissues at 5 min. The salivary glands, adrenal medulla, spleen, gastric mucosa, and kidney still had high concentrations of nicotine relative to other tissues. The nicotine content of salivary glands and gastric mucosa were not lower than those in kidney. In rats (Yamamoto et al., 1968) and mice (Hansson and Schmiterlow, 1962) high radioactivities in salivary glands also have been shown following [‘“Clnicotine administration. There were no significant differences in the tissue concentrations of nicotine between male and female animals. [3H]Nicotine (10 pg/kg) Injection to dogs The tissue distribution of nicotine in dogs injected with 10 ,ng/kg of the drug which is within the smoking range (Larson and Silvette, 1968) were examined. With this dose of nicotine, slight restlessness and hyperpnea were observed and then slight ataxia occurred occasionally. The results are shown in Table 1. At 5 min, highest concentrations were found in the adrenal medulla (63 rig/g). The spleen, kidney, and cerebral cortex (31 rig/g) also had relatively high concentrations. The tissue/serum concentration ratios for the adrenal medulla and cerebral cortex were approximately equivalent to the respective value in dogs given 100 pg/kg. The skeletal muscle, adipose tissue, and serum had low concentrations relative to
-
628 III 72 (8)” 213 f 29(8) 484k 30(7) 541* 51(7) 297k 39(8) 300+ 32(8) 952f 106(6) 487i 61(8) 252+ 32(7) 1229+270(5) 685 + 140 (4) 679+ 134(8) 209f 39(6) 382 + 110(5) 114_+ 20(5) 245+ 29(5) 166_+ 14(5) 429t50(8) 165+ 14(7) 589f 67(6) 270+ 19(7) 36f S(7) 26+ S(8) 7_+ 2(5) 169+ 17(13) 125+ 15(8)
3
46+ 4(7) 146rt 15(7) 105f S(6) 387f35(14) 163 + 14(14) 502*49(15) 260*28(15) 41 f 4(15) 30f 7(14) 12f 2(7) 77 f 9(14) 70& 4(14)
101* 15(8)
505 f 46(15)" 181+ 16(15) 355f 39(15) 388+36(15) 234 + 20(15) 261f 24(15) 689f 86(15) 379* 30(15) 171?15(14) 961 f 82(9) 526?78(9) 322f 57(13) 176+25(14)
5
S(4) 5(4) 66+ 12(4) 57?T 7(3)
47+ 265
189+20(4)
389,48(4) 135k 15(3) 453 k49(4)
670+ 91(4) 265 +21(4) 141f20(4) 3202 50(3) 254+47(3) 145 &21(3) 136+43(4) 115+28(3) 175?74(3) 172*16(4)
191f 28 (4)
285 k26(4) 188_+25(4)
190+ 25 (4) 244_+27 (4)
361 ?28(4)"
Minutes after injection 10 ~___
Concentration of nicotine (rig/g wet tissue) Dose: 100@g/kg
0Valuesrepresentthe mean+ SE. Numberin parentheses indicatesthe numberof animals. * Submandibular gland. CSartorius. dInterscapularregion.
Cerebral cortex white Hypothalamus Thalamus Pons Medulla Hypophysis Cerebellum Spinal cord Adrenal medulla cortex Salivary glandb Lung Gastric mucosa muscle Intestinal mucosa muscle Kidney Heart Spleen Pancreas Skeletal muscle’ Great omentum Subcutaneousfatd Liver Serum
Tissues
1
30 ___~ 70 2? lO(7)" 49 + 120) 51+ 8(6) 54+ 7(6) 4031 4(6) 4ok S(7) 76+ 9(6) 53+ 8(7) 49f 6(6) 176_+20(6) 92+ 11 (6) 133 _+ M(7) 38k 4(7) 102k 27(7) 34+ 2(6) 47+ S(7) 4Ok S(7) 103 Ik 14(7) 27k 3(7) 14of 19(7) 83 rir 8(7) 31zk 3(7) 12+ 3(7) lo+ 2(6) 46k 6(7) 14k l(7)
5 ~-- ~-31 F 2 (7)" 10 * l(7) 21 f. 2(.5) 29 + 6(7) 15 k l(5) 14 + 1 (5) 33 zk 7(7) 24+2(S) 12 + l(5) 63 _+ 9(7) 35 i- 3(7) 23 _+ 5(6) 18+2(5) 12+2(6) 7 iz l(7) 17+2(7) 13+2(7) 41 Ifr S(7) 12f l(5) 47 f 4(5) 3Ok 5(5) 6?1(7) 4 * 0.4(7) 4+ l(7) 10* 1 (7) 5zk l(7)
Dose: 10pg/kg
CONCENTRATIONS OF[3H]N~~~~~~~OFVARIOUSTISSUES IN UNANESTHETIZED Does AFTERIV INJECTION OF 100 AND10 pg/kg OFDRUG
TABLE
26
TSUJIMOTO
ET
other tissues. These results were approximately 100 pg/kg of nicotine.
AL.
similar to those in dogs injected with
[3H]Nicotine (lOO,ug/kg) Injection to Pentobarbital Anesthetized Dogs In anesthetized dogs, most effects of nicotine which were grossly observable in unanesthetized animals could not be produced. Only hyperpnea was observed. The tissue concentrations of nicotine in pentobarbital-anesthetized dogs and the ratio of the concentrations in anesthetized dogs to those of respective tissue in unanesthetized dogs 5 min after the administration are shown in Table 2. It was found that the pentobarbital anesthesia significantly reduced nicotine concentrations in the CNS, adrenal glands, heart and serum at 5 min after the injection of nicotine. The cerebral cortex concentrations were about 56 % of those in unanesthetized dogs. On the other hand, concentrations in liver, gastric mucosa, intestinal muscle, pancreas, and fatty tissue were significantly higher relative to those of respective tissue in unanesthetized animals. [jH]Nicotine
(100 pg/kg) Injection to Monkeys
In monkeys, 100 pg/kg of nicotine produced miosis and facial redness, and restlessness, hyperpnea, and coughing were observed occasionally. The tissue concentrations of nicotine in monkeys 5 and 30 min after intravenous administration of [3H]nicotine are shown in Table 3. Five minutes following administration of nicotine, the adrenal glands and kidney had the highest concentrations. The CNS and spleen also were high relative to other tissues such as the lung and pancreas. In the CNS, the highest concentrations of nicotine were found in the hypophysis and cerebral cortex followed by the cerebellum, thalamus, hypothalamus, medulla, pons, spinal cord, and cerebral white matter, in order of decreasing concentration similarly to that observed in dogs. The tissue/serum concentration ratios for the adrenal medulla and cerebral cortex were 20.7 and 5.5, respectively. The concentrations in liver, skeletal muscle, and adipose tissue were low. At 30 min, the adrenal glands followed by kidney, pancreas, spleen, intestinal mucosa, cerebral cortex, and salivary glands had relatively high nicotine concentrations. Concentrations in almost all tissues were reduced to about one-half of those at 5 min following the injection. The concentrations in the salivary glands, skeletal muscle, and intestinal mucosa remained relatively constant throughout the 30 min. The decrement in the tissue content between 5 and 30 min in monkeys was much less marked than that in dogs. C3H]Nicotine (500 pg/kg) Injection to Monkeys In monkeys, the 500qg/kg dose of nicotine produced restlessness, hyperpnea, miosis, and retching. Usually vomiting and urination occurred. The intensity of these effects was roughly similar to that in dogs injected with 100 pg/kg of nicotine. The tissue concentrations of nicotine in monkeys 5 min after iv injection of 500 fig/kg of drug are shown in Table 3. The highest concentrations were in the adrenal medulla followed by hypophysis, kidney, and adrenal cortex. The lung, cerebral cortex, and salivary glands also had relatively high concentrations. The tissue/serum concentration ratios for the adrenal
0.58 0.64 0.44 0.57 0.76 0.61 0.57 0.89 1.02
0.56b 0.77 0.59 0.60 0.05 0.01 0.02 NS 0.01 0.05 NS NS
0.01” 0.05 0.05 0.01 Heart Kidney Spleen Pancreas Skeletal muscle Great omentum Subcutaneous fat Liver Serum
Gastric mucosa muscle Intestinal mucosa muscle
of nicotine (rig/g wet tissue)
AFTER
102++ 42(7) 20(4) 377 500 + 104 (4) 337+ 8(4) 32k 5 (7) 42+ 6(7) 22 + 4 (7) 124+ 10(5) 52 rfI 5 (7)
0.63 0.97 1.OO 1.30 0.78 1.40 1.83 1.61 0.14
2.35b 1.67 1.29 1.41
IV INJECTION
237+ 34(5) 77+ 14(7) 188i 14(7) 148 k 9 (7)
MINUTES
animals.
DOGS 5
a Values represent the mean k SE. Number in parentheses indicates the number of animals. * Ratio of the nicotine concentration in anesthetized dogs to that of respective tissue in unanesthetized c Probability of difference from unanesthetized dogs.
151 rt+ 13 19 (4) 150 303 f 31 (6) 214 * 25 (4) 130-t lO(4) 559 f 82 (7) 297 + 36 (7) 287 + 40 (5) 180*46(4)
Medulla Pons Hypophysis Cerebellum Spinal cord Adrenal medulla cortex Salivary gland Lung
17 (7)” 6(7) 20 (4) 12 (7)
2
IN PENTOBARBITAL ANESTHETIZED OF 100 pg/kg OF DRUG
Concentration
OF [3H]N~~~~~~~
282 + 139k 208 + 233 f:
CONCENTRATIONS
Cerebral cortex white Hypothalamus Thalamus
TISSUE
TABLE
0.05 NS NS 0.01 NS NS 0.05 0.01 0.01
0.05” NS NS 0.01
Z
% z 8 =! 5
3
5 f: 3 2 B
OF
0.61bvc 0.56c 0.51C 0.56” 0.60’ 0.55’ 0.50” 0.58” 0.67’ 1.27 1.06 0.48” 1.39 1.57” 1.30 1.36” 1.31 1.42’ 0.52” 0.60’ 0.95 1.95’ 1.17 3.17’ 0.48’ 0.80’
Minutes 151 + 18 (3) 79 f 11 (3) 89 I!I 9 (3) 97 f 9 (3) 78 + 7(3) 80 + 10 (3) 119kl4(3) 113 f 11(3) 77 + 6 (3) 429 + 21 (3) 209 f 20 (3) 148 + 28 (3) 133 +_ 3 (3) 89 + 23 (3) 42 + 5 (3) 175 + 9(3) 88 f 9(3) 301 f 55 (3) 47 + 6(3) 185 f 15 (3) 187 rt 10 (3) 74_+ 8 (3) 27 f Z(3) 24f 2 (3) 22+_ 3 (3) 23 f 4 (3)
after injection 30
Concentration of nicotine Dose : 100 pgg/kg
TABLE 3 OF VARIOUS TISSUES IN UNANESTHETIZED 100 AND 500 pg/kg OF DRUG
310+ 28 (7) 101* 13 (7) 182& 15(7) 218 f 21 (7) 141+ 14(7) 143 + 14(7) 342 f 69 (7) 221 + 23 (7) 114f 11 (7) 1163 f 137 (4) 556 f 140 (4) 156k 20(7) 245 +_ 48 (7) 159+ 17(4) 60 f 6 (4) 199+ 13(4) 138+_ 23 (4) 550* 59 (7) 84 f 6 (7) 300 + 31 (7) 247+_ 38(7) SO_+ 11 (7) 35 -I 6(7) 38+ S(4) 37 f 4(7) 56f 5 (7)
5
[3H]N~~~~~~~ AFTER
2.16”,= 1.61 1.75” 1.80” 1.95’ 2.00’ 1.57” 2.13’ 1.57” 2.44” 2.27’ 1.11 3.50’ 0.87 1.23 3.72’ 2.20’ 2.92” 1.74” 1.32 2.25’ 2.39” 2.25’ 2.40” 0.48’ 1.64
(rig/g wet tissue)
MONKEYS
u Values represent the mean f SE. Number in parentheses indicates the number of animals. b Ratio of the nicotine concentration in monkeys to that of respective tissue in dogs. c Significant at p values of 0.05 or less.
cortex white Hypothalamus Thalamus Pons Medulla Hypophysis Cerebelhun Spinal cord Adrenal medulla cortex Salivary gland Lung Gastric mucosa muscle Intestinal mucosa muscle Kidney Heart Spleen Pancreas Skeletal muscle Great omentum Subcutaneous fat Liver Serum
Cerebral
Tissues
CONCENTRATIONS
OF
1572 + 394 (3) 609+ 98 (3) 1082 f 204 (3) 1123 + 214 (3) 796 + 86(3) 72Of 24 (3) 3590 +_ 1037 (3) 1055 + 323 (3) 596 + 102 (3) 5824 f 379 (3) 3013 + 193 (3) 1465 + 727 (3) 2116 + 642 (3) 590 + 137 (3) 316+ 49(3) 774 + 217 (3) 469 +_ 116 (3) 3380 + 878 (3) 530 f 32(3) 1130+ 260(3) 1399 f 78 (3) 346_+ 56 (3) 293 IL 86(3) 227 + 23 (3) 133 f 26(3) 329f 32(3)
5
Dose : 500 pug/kg
IV INJECTION
TISSUE
DISTRIBUTION
OF NICOTINE
29
medulla and cerebral cortex were approximately equivalent to the respective values in monkeys given 100 pug/kg. The liver and adipose tissue concentrations were the lowest. DISCUSSION
Nicotine was quickly distributed throughout the tissues of dogs and monkeys after iv injection of 100 pg/kg of drug. It should be noted that levels of nicotine and tissueserum concentration ratios in target tissues such as the adrenal medulla and CNS were very high shortly following the iv injection of nicotine in both species. These findings are in agreement with those reported in mice using an autoradiographic method (Hansson and Schmiterlow, 1962), in cats using a methylene dichloride extraction procedure (Turner, 1969), and in rats using the present procedure (Hug, 1970). The blood supply to the brain is large and extracellular fluid space is small and the capillaries are completely invested with a cellular wrapping of glia cells in the CNS (Bonnycastle, 1965). The vascular density of adrenal glands also is conspicuously high (Nair et al., 1960). At pH 7.4 and a temperature of 37°C nicotine has been calculated to be about 31% nonionized base which can readily penetrate the blood-brain barrier (Domino, 1965). In addition, nicotine has high lipid solubility (Bowman et al., 1964). These physiological and anatomical characteristics, and physicochemical properties of nicotine may account for the faster penetration and egress of nicotine from these tissues. Nicotine concentration in various areas of CNS in dogs and monkeys arranged in order of decreasing concentration was as follows: the hypophysis, cerebral cortex, cerebellum, thalamus, hypothalamus, medulla, pons, spinal cord, and cerebral white matter. These results are in general agreement with those reported for the cat injected with small multiple doses of nicotine (Turner, 1971). In the autoradiographic study of brain in rats given [14C]nicotine, Schmiterlow et al. (1967) suggested that radioactivity was concentrated in the nerve cells. In the study of tissue distribution of morphine (Mule and Woods, 1962) it was suggested that the multiple membranes of the laminated myelin sheaths which are found in white matter may function as a barrier to penetration of the drug. Furthermore, the vascularity of the CNS is not uniform, e.g., the vascular density of cerebral cortex is much greater than that of the spinal cord and white matter (Nair et al., 1960). Therefore, the differences in cell density and vascular density of the CNS may account for the differences in the concentration of nicotine among various anatomical areas. Pentobarbital was found to reduce the concentration of nicotine in brain and adrenal medulla. Most effects of nicotine which were grossly observable in unanesthetized dogs could not be produced in the anesthetized animals. Barbiturates have a protective action against nicotine-induced convulsions (Yamamoto et al., 1966) and death (Larson et al., 1949; Yamamoto et al., 1966). The decrease in nicotine concentration in “target tissue” may result in part in the antagonism of the effects of nicotine by pentobarbital. In the comparison of tissue concentrations of nicotine (Table 3) and tissue/serum concentrations (Table 4) in dogs and monkeys 5 min after nicotine administration, it is noteworthy that concentrations in the CNS in monkeys were lower relative to those in dogs. In addition, the tissue/serum concentration ratios in heart, a tissue rich in catecholamine granules, were also considerably lower in monkeys as compared to dogs. The monkey appear to be five to ten times less sensitive than the dog to the effects of
30
TSUJIMOTO
ET AL.
TABLE 4 TISSUE/SERUM CONCENTRATION RAnos IN SOME TISSUES OF Does AND MONKEYS 5 MIN FOLLOWING IV INJECTION OF 100 pg/kg OF [‘HINICOTINE
Tissues Cerebral cortex Hypothalamus Heart Adrenal medulla Skeletal muscle
Tissue/serum ratios Monkey Dog 7.2 5.1 2.3 13.7 0.6
5.5 3.3 1.5 20.8 1.4
nicotine on gastrointestinal contractility (Hug and Bass, 1970), and respiratory and cardiac rate (Hug and Carlson, personal communication). Many of the peripheral actions of nicotine including the increase of heart rate have been considered as secondary effects, due to catecholamine liberated from the terminal adrenergic nerve fibres by nicotine and the adrenal medulla (Burn and Rand, 1958; Westfall and Watts, 1963). Furthermore, nicotine has been reported to increase not only the respiratory and heart rate but to inhibit the contractile activity of the gastrointestinal tract through stimulation of the CNS (Carlson et al., 1970). Tissue/serum concentration ratios in the adrenal medulla were higher than those in dogs. In addition, all tissue concentrations with the exception of the liver and spleen in monkeys given 500 pg/kg of nicotine were three times or more higher than those of respective tissue in dogs given 100 pg/kg even though the intensity of the observable effects in monkeys was roughly similar to dogs. Therefore, factors causing the species differences in quantitative responses to nicotine are not solely due to the differences in distribution. Recently, it was shown that the effect of nicotine in catecholamine-release from the adrenal glands in monkeys was less potent than that in dogs (Tsujimoto et al., 1974). In the previous report, it was suggested that the rate of nicotine destruction in monkeys might be faster than that in dogs (Tsujimoto et al., 1972); this is in agreement with findings that nicotine metabolizing enzyme activities of monkey liver are higher than those of dog liver (Dohi et al., 1973). The skeletal muscle and tissue/serum ratios in monkeys were almost twice as high as those in dogs. The skeletal muscle occupies a great proportion of the body mass, approximately 40-50 % of body weight in the monkey (Hayashi, 1965). Therefore, it could be considered that the higher affinities of skeletal muscle for nicotine and the faster destruction of nicotine in monkeys compared to that of dogs may account for the lower concentrations in the CNS, some other tissues and serum. ACKNOWLEDGMENTS
The authors express their appreciation to Dr. M. H. Seevers for advice and continued interest in this investigation. We thank Dr. H. McKennis, Jr., and Dr. E. R. Bowman for a generous supply of authentic cotinine. REFERENCES BONNYCASTLE, D. D. (1965). Intimate
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OF NICOTINE
31
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