Tissue distribution and binding of radioactivity in mouse after intravenous administration of [14C]3-chloro-p-toluidine

Toxicology, 11 (1978) 153--165 © Elsevier/North-Holland Scientific Publishers Ltd.

TISSUE DISTRIBUTION AND BINDING OF RADIOACTIVITY IN MOUSE AFTER INTRAVENOUS ADMINISTRATION OF [ 14C]3-CHLORO-p-TOLUIDINE

SHRI N. GIRI, DAVID M. SIEGEL and STUART A. PEOPLES

Department of Physiological Sciences, School of Veterinary Medicine, University of California, Davis, Calif. 95616 (U.S.A.) (Received June 12th, 1978) (Accepted July 20th, 1978)

SUMMARY

The avicide ['4C]3-chloro-p-toluidine (CPT) HCL, ring labeled, was injected intravenously to mice. The radioactivity associated with this compound was found to be unevenly distributed in different parts of the body. It leaves the plasma, as well as many tissues, with 2 elimination rate constants, the fast and the slow. The faster component of the [ I a c ] c P T decay curve of the plasma was similar to the faster components of the decay curves of brain, lung, heart, intestine, testicle and kidney. The retention half-life of the radioactivity for the slower component of the decay curve varied a great deal from tissue to tissue, being shortest (14.55 h) in the intestine and longest (326 h) in the adipose tissue. Of the 10 tissues examined, a substantial amount of [14C]CPT radioactivity was found to be cov~ently bound only to liver, kidney, lung and RBC protein. There was no cause and effect relationship between the covalent binding of radioactivity and the tissue pathology, since no remarkable histopathological lesions were found in the liver and kidney of treated mice. The tissue retention of [14C]CPT radioactivity did not parallel the covalent binding of the compound to tissue protein. The covalent binding of [~4C]CPT radioactivity to RBC was suggestive of the conversion of the parent compound into a reactive metabolite responsible for the generation of methemoglobin in mice. The percent distribution of radioactivity in subcellular fractions of liver and kidney correlated with the amount of protein associated with subcellular fractions. The 102 000 g supernatant fraction of the liver contained the highest proportion of radioactivity, both in terms of absolute percent radioactivity as well as specific activity (dpm/mg of protein). This was also Abbreviations : CPT, 3-chloro-p-toluidine ; dimethyl POPOP, 1,4-bis-[ 2-methyl-5-phenyloxazolyl ] -benzene ; PPO, 2,5-diphenyloxazole.

153

true for the 102 000 g supernatant fraction of the kidney. The majority of radioactivity in the 102 000 g supernatant fraction of liver appears to be bound to one or more p o l y p e p t i d e sized proteins with a mol. wt. of approx. 1000--2000.

INTRODUCTION

The avicide, CPT hydrochloride has been studied in this laboratory and elsewhere to determine its safety for non-target species as well as its usefulness in controlling the population of starlings [1,2]. The desirability o f this c o m p o u n d as an effective avicide is based on its high avian toxicity and low mammalian toxicity; for example, the oral LDs0 in most birds is less than 20 mg/kg, in contrast to 650 mg/kg in rats [1,2]. This c o m p o u n d has been cleared for use to control the population of starlings (Sturnus vulgaris) in many areas of the United States and the c o m p o u n d is commercially sold by Ralston Purina under the trade name Starlicide. The biochemical and pathological lesions which presumably contribute to lethality of birds due to a toxic dose o f CPT include metabolic disturbances [ 3 ] , hepato- and nephrotoxicity [ 1,4]. The mechanism w h e r e b y CPT exerts" such a selective toxicity in starlings and chickens has been partly attributed to the presence of N-deacetylase in the kidneys of these birds, b u t not in rodents [ 5 ] . In an earlier s t u d y [ 4 ] , we have reported some o f the basic fundamental pharmacodynamic and pharmacokinetic aspects of ['4C]CPT in starlings, such as half-life, tissue distribution, binding, localization and binding characteristics. None of this information is available for ['4C]CPT in any mammalian species. Therefore, we investigated the pharmacokinetic and pharmacodynamic aspects of ['4C]CPT in mice in the present study. M A T E R I A L S AND METHODS

Animals and radioactive compound Male albino mice (Swiss Webster) weighing 25--30 g were purchased from Simonson Laboratory, Inc., Gilroy, California. The animals were maintained in animal care facilities fully accredited by the American Association for the Accreditation of Laboratory Animal Care. The mice were housed in group of 4 per cage and had access to laboratory c h o w and water ad lib. ['4C] ring labeled 34zhloro-p-toluidine (CPT) HC1 was custom synthesized by New England Nuclear Corp., Boston, Mass. The specific activity was 2.62 mCi/ mmol. Radiochemical purity was checked by thin-layer chromatography on silica gel G using benzene--isopropyl ether (3 : 2) mixture as the developing solvent. Radiochemical purity was greater than 98%.

Administration of radioactivity The [14C]CPT HC1 was dissolved in sterile isotonic saline to give a concen-

154

tration of 2 mg/ml saline. Each mouse was injected with 3 ~Ci of [14C]CPT (5.9 mg/kg) in a vol. of 0.1 ml through the tail vein. The mice were anesthetized by chloroform at different times following the administration of [ '4C] CPT and blood was collected in a heparinized syringe directly from the heart. Blood samples were centrifuged at 2000 rev./min for 15 min in an International Centrifuge. After aspirating the plasma, the erythrocytes were washed twice with isotonic saline and resuspended in saline to the original whole blood volume. Different organs including liver, kidney, heart, testicle, spleen, skeletal muscle, lungs, intestine, epididymal fat pad and brain were dissected out immediately after collecting the blood. All organs were rinsed 3 times in cold isotonic saline to get rid of blood and then cleaned of the extraneous tissue. In the case of the intestine, it was split open, and then the contents were washed out before rinsing in saline. A small piece of tissue was excised from each organ for determination of radioactivity and the rest was quickly frozen on dry ice for other studies.

Determination of radioactivity Aliquots of resuspended erythrocytes (0.05 ml), plasma (0.1 ml) or tissue samples (40--60 rag) were placed in scintillation vials containing 1 ml Protosol tissue solubilizer (New England Nuclear Corp.). The vials were heated a£ 60°C for 12--24 h until the material was thoroughly digested. A 0.2 ml solution of benzoyl peroxide in toluene (20% w/v), as a decolorizing agent, was added to the vials containing erythrocyte digest and heated at 60°C for 30 min. 15 ml of scintillation fluid containing 0.0075% (w/v) 1,4-bis-[2-(methyl-5-phenyloxazolyl)]-benzene (Dimethyl POPOP) and 0.6% (w/v) 2,5-diphenyloxazole (PPO) in toluene was added to each vial. The samples were allowed to equilibrate 24 h before determining the radioactivity in a Packard Tri-Carb liquid scintillation spectrometer 3320. The quench correction for each tissue was carried out by adding 10 ~1 of [~4C]toluene (4.5 × l 0 s dpm/ml, New England Nuclear Corp.) to the vials. The counting efficiency ranged from 70 to 80%. Subcellular distribution of ['4C]CPT in liver and kidney Subcellular distribution of [14C]CPT radioactivity in liver and kidney was carried out in mice sacrificed at 6 h following i.v. administration of 3 pCi of the isotope. Tissues were thawed and a 500 mg sample was homogenized in a glass mortar at 4°C in 2.5 ml of 0.25 M sucrose using a motor-driven Teflon pestle (Tri-R). The homogenate was centrifuged in a refrigerated centrifuge at 4°C in the following sequence: 3000 g for 20 min; 10 000 g for 20 min (Sorvall RC-2B, SS-34 rotor), and 102 000 g for 60 min (IEC International ultracentrifuge Model B-60, A-321 rotor). This procedure yielded the nuclear-heavy mitochondrial pellet (3000 g), light mitochondrial pellet (10 000 g), microsomal pellet (102 000 g), and soluble fraction (102 000 g supernatant) [6]. Pellets were resuspended in 4 ml of distilled deionized water. An aliquot (1 ml) of resuspended pellet was transferred into a scintillation vial and another aliquot was used for protein estimation [ 7 ]. In the

155

case of whole homogenate and soluble fraction, a 0.2 ml aliquot was transferred into a scintillation vial containing 0.8 ml of distilled deionized water and another aliquot was used for protein determination. 15 ml of Aquasol (New England Nuclear Corp.) was added to each vial and radioactivity was determined in the Packard Tri-Carb liquid spectrometer. Quench correction was carried out using [14C]toluene as an internal standard. The counting efficiency ranged from 75 to 85%.

Covalent binding of [14C]CPT radioactivity to tissue protein Covalent binding of [~4C]CPT radioactivity to various tissue proteins was carried out in mice sacrificed at 12, 18, 24 and 36 h after i.v. administration of [~4C]CPT. Frozen tissues were thawed, a 250 to 500 mg piece, 0.5 ml of plasma or 0.5 ml RBC suspension was used for determination of covalently bound radioactivity to protein according to the procedure described by Busch et al. [8] with the following 2 modifications. First, after extraction with cold TCA, the protein was additionally extracted with hot (90°C) TCA (1.5 N). Second, following an extraction with chloroform--methanol, the protein was successively extracted 5 times with hot methanol at 60°C. This step removes virtually all of the radioactivity non~ovalently bound to protein [9]. The supernatant resulting from the final wash (ethyl ether) was collected in the scintillation vial. The protein pellet and the supernatant were dried in a hood at room temperature. There was no detectable level of radioactivity in the supernatant after the background correction. A 15--25 mg portion of the dried protein was transferred into a scintillation vial and solubilized in 0.15 ml H20 and 1 ml of NCS solubilizer (Amersham/Searle) by heating at 60°C for 4 h. 15 ml of the toluene scintillation mixture was added to each vial. After 24 h of equilibration, the samples were counted in the Packard Tri-Carb liquid scintillation spectrometer. Quench correction for each sample was done by using [ ~4C]toluene.

H istopa thology A 2 mm slice of liver and kidney from each mouse sacrificed at 18 h after the injection of [14C]CPT was fixed in buffered formalin. Paraffin sections 8--10 pm thick were prepared by standard technique [10] and stained with hematoxylin and eosin. The sections were examined microscopically for histopathologic changes associated with [ 14C]CPT or saline treatment.

Gel filtration chromatography Mice were injected i.v. with 3 t~Ci of [14C]CPT and sacrificed 3 h later. The soluble fraction of the liver homogenate (102 000 g supernatant) was obtained according to the procedure described earlier. An aliquot of the soluble fraction was added to and eluted from a column of Sephadex G-25 (Pharmacia) at room temperature with the following specifications: bed dimensions, 0.9 × 28 cm; flow rate, 4.4 ml/h; elutant, phosphate buffer (0.05 M, pH 8.4); the void volume was 8 ml. 1-ml fractions were collected and assayed for radioactivity and protein.

156

RESULTS

A semilogarithmic plot of radioactivity in plasma, liver and kidney up to 36 h following i.v. administration of [14C]CPT in mice is shown in Fig. 1. It appears that there are at least 2 components in the plasma decay curve of [14C]CPT; a component with a faster elimination rate constant and another with a slower elimination rate constant. A somewhat similar curve of [ 14C]CPT radioactivity was found in most of the tissues studied, except for the RBC. The [~4C]CPT radioactivity in RBC tends to remain constant with time, as shown in Fig. 2. The apparent half-lives of the faster and slower components of the [~4C]CPT decay in plasma and various tissues are summarized in Table I. In calculating the half-life of either of the components, the best fit line was drawn by the method of least squares [11]. This allowed us to compute the half-lives of the faster and slower components of the [14C]CPT decay for plasma as well as for various tissues (Table I). The halfJife of [~4C]CPT radioactivity in tissues lacking the fast component was computed after the radioactivity began to decline in a linear fashion. This included spleen, brain and liver.

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157

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It was interesting to note that the half-life of the fast c o m p o n e n t of the plasma decay curve of [14C]CPT was quite similar to the half-lives of the f a s t c o m p o n e n t for most of the tissues, except for the muscle. For most tissue, this ranged from 1.52 to 3.96 h, while for muscle it was 6.85 h. The half-life TABLE I APPARENT H A L F - L I F E OF R A D I O A C T I V I T Y F O R F A S T AND SLOW COMPONENTS OF THE DECAY C U R V E F O R PLASMA AND V A RI O U S TISSUES OF MICE A F T E R i.v. A D M I N I S T R A T I O N OF 3 uCi ['4C]CPT Tissue

Fat Muscle Heart Spleen Lung Brain Kidney Plasma Liver Intestine Testicle

Apparent half-life a in hours Fast c o m p o n e n t

Slow c o m p o n e n t

1.58 6.85 2.39 b 3.96 b 1.85 2.55 b 1.52 3.75

326.00 97.15 93.98 81.29 41.29 36.23 25.05 17.63 15.91 14.55 c

a Determined by the method of least squares when the radioactivity levels were decreasing in a linear manner and extrapolated to zero time. See the Materials and Methods Section for details. b Fast c o m p o n e n t could n o t be distinguished. c Radioactivity level too low to analyze.

158

of the slow component of the decay curve varied a great deal from one tissue to another (Table I). This fell into 4 distinct groups: First, a very long half-life (326 h) in the adipose tissue; second, a long half-life (81.29--97.15 h) in the spleen, heart and muscle; third, a medium long half-life (25.05-41.29 h) in the kidney, brain and lung; and fourth, a short half-life (15.91-17.63 h) in the intestine, liver and plasma. As CPT has been demonstrated to produce hepatic and kidney lesions in several avian species, an attempt was made to study the subcellular distribution of ['4C]CPT in these 2 organs of mice sacrificed 6 h after drug administration. The results of these studies for liver are shown in Table II, and those for kidney are shown in Table III. The protein content of the subcellular fractions and the radioactivity associated with these fractions of the liver appear to be quantitatively different from those of the kidney. For example, 1 g wet weight of liver contained 62% less protein in nuclear-heavy mitocchondrial fractions than that present in the corresponding fractions of 1 g of the kidney tissue. There were, however, no significant differences in the protein content of any other similar subceUular fractions between liver and kidney (Tables II and III). The percent distribution of radioactivity in subcellular fractions closely parallels the amount of protein present in the fractions. For liver, the percent distribution of radioactivity in different subcellular fractions, as arranged in descending order, were soluble, light mitochondria, microsomes and nuclear-heavy mitochondria (Table II). A similar pattern of radioactivity distribution was also found for the subcellular fractions of the kidney with a noticeable exception that the nuclear-heavy mitochondrial fraction of the kidney contained more protein and consequently more ['4C]CPT radioactivity than the corresponding fraction of the liver (Table III). A direct relationship between the covalent binding of drugs or their meMbolites to protein in vivo and the production of tissue necrosis has been well established for a number of drugs in mammalian species [9,12,13]. In view of the experimental evidence that mice are more resistant to the toxic effects of CPT than several of the avian species, an attempt was made to study the covalent binding of [~4C]CPT radioactivity to different tissues of mice sacrificed at 12, 18, 24 and 36 h after the injection of the compound. The results of this study are summarized in Fig. 3. Of the 10 tissues examined, only liver, kidney, lungs and RBC were found to covalently bind [~4C]CPT radioactivity significantly to acid precipitable protein. In this regard, the kidney manifested a greater ability to bind the radioactivity than any other tissues examined in this study. The binding of [~4C]CPT radioactivity to kidney protein was maximal at 18 h and thereafter it began to decrease gradually with time. A somewhat similar trend in the binding of [ t4C]CPT radioactivity to liver and lung protein was also noticed;the difference being that the maximal binding of radioactivity in the case of the lung and liver occurred between 12 and 18 h after the drug administration. The binding of [~4C]CPT radioactivity to RBC was different from other tissues in

159

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DISTRIBUTION

8068 1289 1447 1299 4225

± 1480 ± 134 ± 190 ± 204 ± 857

I00 m g tissue

Total d p m

100.0 16.0 17.9 16.1 52.4

Percent dpm

O F [14C]CPT R A D I O A C T I V I T Y

IN M O U S E

176.72 32.76 38.98 30.40 61.07

± ± ± ± ±

4.94 1.50 2.15 1.52 5.67

Total protein(mg/g) tissue

AND PROTEIN

100.0 18.5 22.1 17.2 34.6

Percent protein

LIVER a

463 395 373 427 766

± 97 ± 44 ± 49 ± 62 ± 190

d p m / m g protein

11989 3464 1894 1095 6000

± 2021 ± 889 ± 335 ± 112 ± 1325

1 0 0 m g tissue

Total dpm

100.0 28.9 15.8 9.1 50.0

Percent dpm

205.30 86.19 42.68 28.93 57.03

± 11.15 ± 8.97 ± 3.18 ± 4.30 ± 4.21

Total p r o t e i n (rng/g) tissue

100.0 42.0 20.8 14.1 27.8

Percent protein

669 392 440 388 1223

± 103 ± 66 ± 57 ± 35 ± 199

dpm/mg Protein

a S u b c e l l u l a r d i s t r i b u t i o n was carried o u t as d e s c r i b e d u n d e r Materials a n d M e t h o d s s e c t i o n , 6 h a f t e r i.v. a d m i n i s t r a t i o n o f 3 uCi [14C]C P T / m o u s e . E a c h value is t h e m e a n o f 4 d e t e r m i n a t i o n s ± S.E. P e r c e n t d i s i n t e g r a t i o n / m i n ( d p m ) a n d p r o t e i n for e a c h f r a c t i o n were c a l c u l a t e d as p e r c e n t o f t h e t o t a l d p m o r p r o t e i n in e a c h h o m o g e n a t e . T o t a l r e c o v e r y o f r a d i o a c t i v i t y was 1 0 3 . 8 % a n d p r o t e i n 104.6%.

Homogenate Nuclear-heavy mitochondrial Light mitochondrial Microsomal Soluble

Subcellular fraction

S U B C E L L U L A R D I S T R I B U T I O N O F [ ' 4 C ] C P T R A D I O A C T I V I T Y A N D P R O T E I N IN M O U S E K I D N E Y a

T A B L E III

a S u b c e l l u l a r d i s t r i b u t i o n was carried o u t as d e s c r i b e d u n d e r Materials a n d M e t h o d s s e c t i o n 6 h a f t e r i.v. a d m i n i s t r a t i o n o f 3 uCi o f [~4C]C P T / m o u s e . E a c h v a l u e is t h e m e a n o f 4 d e t e r m i n a t i o n s ± S.E. P e r c e n t d i s i n t e g r a t i o n / r a i n ( d p m ) a n d p r o t e i n for e a c h f r a c t i o n were c a l c u l a t e d as p e r c e n t of t h e t o t a l d p m o r p r o t e i n in e a c h h o m o g e n a t e . T o t a l r e c o v e r y o f r a d i o a c t i v i t y was 1 0 2 . 4 % a n d p r o t e i n 92.4%.

Homogenate Nuclear-heavy mitochondrial Light mitochondrial Microsomal Soluble

Subcellular Fraction

SUBCELLULAR

TABLE

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TIME (hr) Fig. 3. Covalent binding of [14C]CPT radioactivity to proteins of various tissues of mice. The mice were injected i.v. with [14C]CPT (3 /~Ci/mouse). They were sacrificed at different times, and covalent binding of [14C]CPT radioactivity to acid precipitable protein was determined as described in the Materials and Methods Section. Each point represents the mean of 4 animals. Vertical bars indicate S.E.

the sense that binding increased gradually with time. A considerable reduction in the binding of the radioactivity to RBC occurred only at 36 h after the drug administration. A negligible a m o u n t of covalent binding was found to occur with the plasma protein (Fig. 3). The remaining tissues such as testicle, muscle, intestine, spleen and heart exhibited very little capacity for covalent binding of [ 14C] CPT radioactivity (data n o t shown). The soluble fraction of the liver and kidney appeared to contain 50% of [14C]CPT radioactivity. In addition, the a m o u n t of radioactivity bound to 1 mg of the soluble protein of the liver or kidney was 2--3 times higher than the a m o u n t b o u n d by the proteins of other subcellular fractions (Tables II and HI). Therefore, an a t t e m p t was made to study the nature of the soluble fraction of the protein respons~le for binding [ 14C]CPT radioactivity. This was accomplished by subjecting the 102 000 g supernatant fraction of the liver to molecular sieve chromatography. The elution profile of [14C]CPT radioactivity bound to the soluble proteins from a Sephadex G-25 column indicates that radioactivity elutes in 2 peaks (Fig. 4}. The first is associated with the void volume and is, therefore, presumably bound to a macromolecule whose molecular weight is in excess of 10 000. This peak contains only

161

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30% of the radioactivity. The second peak elutes at a volume which suggests that ['4C]CPT radioactivity is bound to a macromolecule whose molecular weight ranges between 1 0 0 0 and 2000, and this peak contains the majority of the radioactivity {70%). The molecular weight can only be approximated since the standards used (carbohydrate and vitamin) may have s o m e w h a t different elution characteristics from proteins of comparable weight. It was interesting to find that there was no evidence for the presence of free [~4C]CPT in the elution profile of the soluble fraction of the liver protein (Fig. 4). Histopathological studies were done in mice sacrificed at 18 h after [14C]CPT injection. There were no remarkable histopathological lesions in the livers and kidneys of these mice.

162

DISCUSSION

The experimental data collected in the present study reveal certain very basic pharmacokinetic and pharmacodynamic aspects of CPT disposition in mice. The compound seems to be unevenly distributed in different compartments of the body. The radioactivity associated with [14C]CPT appears to leave the plasma as well as many tissues with 2 elimination rate constants, a fast component and a slow component. The faster component of the [~4C]CPT decay curve of the plasma appears to be similar to the faster component of the decay curve of fat, brain, lung, heart, intestine, testicle and kidney. This suggests that [~4C]CPT radioactivity in these tissues is in equilibrium with the plasma during the rapid phase of drug elimination. The retention half-life of the slower component of the decay curve, with a slower elimination rate constant, varies a great deal from one tissue to another. The adipose tissue (epididymal fat pad) has the longest retention half-life (326 h). This suggests that the radioactivity associated with the parent compound and/or its metabolite is extremely lipophylic and is presumably being sequestered by the lipid portion of the cell. It was interesting to find that the retention half-lives of [14C]CPT radioactivity in the heart, skeletal muscle and spleen of mice were approx. 15, 11 and 16 times longer than the corresponding tissues of starlings, respectively, injected with approximately the same amount of [~4C]CPT by i.v. route [4]. This then indicates that these tissues in mice might serve as a reservoir or sink for the compound and thereby help to avoid the drug toxicity in the absence of the availability of an excess amount of drug at the target sites (liver and kidney). If this is true, this will then explain, in part, the reason for the extreme susceptibility of the starling to the toxic effect of CPT over that of the mouse [1,2]. An attempt was made to elucidate the type of binding responsible for retaining the [~4C]CPT radioactivity in different tissues. Of the 10 tissues examined, a considerable amount of radioactivity was found to be tightly associated with the proteins of liver, kidney, lung and RBC. The persistence of this binding to proteins of these tissues, even after extensive solvent extraction, strongly suggests, but does not prove, that the binding is of a covalent nature. The maximal covalent binding occurred at 18 h to kidney protein and at 12--18 h to liver and lung protein. A significant reduction in the covalent binding to proteins of these tissues was found at 36 h after drug administration. The role of covalent binding of [~4C]CPT radioactivity to kidney and liver protein in producing necrotic lesions is questionable. No histopathological changes were observed in these tissues at 18 h after the drug administration at the time when there was a maximal covalent binding of [14C]CPT radioactivity to these tissues. A significant amount of [14C]CPT radioactivity was found to be covalently bound to the lung protein of mice but not to the lung protein of starlings [4]. The significance of this species dependent variation in the binding of [14C]CPT radioactivity to lung proteins is not known.

163

The CPT has been demonstrated to generate a significant level of methemoglobin in mice and rats, but not in chickens [ 14]. We have reported earlier that ['4C]CPT is not covalently bound to RBC of starlings [4]. In view of the data obtained in the present study that the covalent binding of ['4C] CPT radioactivity to the RBC of mice slightly increases with time for 24 h after drug administration, it is tempting to postulate a relationship between the covalent binding of the parent compound or its metabolite to RBC and methemoglobin formation in mice. This is not surprising since rats and mice are capable of converting aromatic amino compounds into reactive metabolites which are responsible for methemoglobin formation and erythrocyte destruction [15]. It is not known if the covalent binding of [14C]CPT radioactivity to RBC of mice is the result of the formation of active metabolites from the parent compound followed by covalent binding and methemoglobin formation. It appears that in several other tissues, ['4C] CPT and/or its metabolites are either non-covalently bound to macromolecules or physically trapped in the tissues and therefore, easily come off following the solvent extraction. It is interesting to note that there was a definite lack of correlation between the retention half-life of radioactivity and the covalent binding of [14C]CPT to many tissues. For example, adipose tissue, skeletal muscle, heart and spleen each had a longer retention half-life than that of kidney, liver and lung, but failed to covalently bind [14C]CPT radioactivity as opposed to tissues in the latter group which covalently bound a substantial amount of [14C]CPT. Therefore, it seems that the retention of any compound in any tissue might be the function of a series of complicated factors. This may include tissue perfusion, chemical composition of the tissue, chemical nature of the compound (lipophylic or lypophobic), the metabolic capacity of the tissue (in case the metabolite is being retained) and redistribution of the compound. Other investigators have also suggested that CPT undergoes an extensive redistribution in the body [16]. Unless these factors are individually studied, it is very difficult to explain the differential distribution of this compound among various tissues in the same species or the same tissue in different species. On the basis of subcellular distribution of p4C]CPT, it appears that the radioactivity is distributed in all subcellular organelles of the liver and kidney cells. The absolute percent distribution parallels the amount of protein present in the organelle. The proteins in the soluble fractions showed a greater specificity to retain ['4C] CPT radioactivity than the proteins in other subcellular fractions. In the case of liver, the binding of ['4C]CPT radioactivity by 1 mg of the protein of the soluble fraction was roughly 2 times as much as that by the proteins of other subcellular fractions. This was also true regarding the binding of radioactivity by the proteins of the soluble fraction of kidney. The nature of the soluble proteins of the liver responsible for binding this compound or its metabolites was studied by molecular sieve chromatography. It appears that ['4C]CPT radioactivity is associated with 2 major protein groups. One has a very high molecular weight, elutes in the

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void v o lu me and contains 30% o f t he radioactivity. T h e o t h e r is o f t h e p o l y p e p t i d e size, appears to have a tool. wt. b e t w e e n 1000 and 2 0 0 0 and contains a majo r p o r t i o n o f the radioactivity (70%). The question o f w h e t h e r t h e p a r e n t molecule or a m e t a b o l i t e is the h o u n d radioactive f o r m has n o t y e t been resolved. ACKNOWLEDGEMENT

This s t u d y was s u p p o r t e d by funds f r o m the Agriculture E x p e r i m e n t S tatio n Starling Control Research Project. We t h a n k Ms. Mary Schiedt and Mr. J o h n S eab ur y f o r their technical help. REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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