Clin. Bioehem. 12 (2) 56-58 (1979)
Amphetamine Metabolism in Mice Exposed Chronically to Phenobarbital and to Phenobarbital with Carbon Tetrachloride Y.W. LEE and L.I. WIEBE Division of Bionucleonics and Radiopharmacy, University of Alberta, Edmonton, Canada T6G 2N8 (Accepted September 12, 1978)
CLBIA, 12, (2) 56-58 (1979)
Clin. Biochem. Lee, Y.W., and Wiebe, L.I.
Division of Bionucleonies and Radiopharmacy, University of Alberta, Edmonton, Alberta T6G 2N8 A M P H E T A M I N E METABOLISM IN MICE EXPOSED CHRONICALLY TO P H E N O B A R B I T A L AND TO P H E N O B A R B I T A L W I T H CARBON TETRACHLORIDE. The metabolism of amphetamine was studied in three groups of mice, including normal mice (N), mice given chronic doses of phenobarbital via their drinking water (PB), and mice exposed to carbon tetrachloride vapors daily during the course of chronic phenobarbital consumption (PB/CC14). Renal and hepatic tissue from animals of each group were examined by electron microscopy. Mice in the PB/CC14 group demonstrated the classical symptoms of carbon tetrachloride-induced hepatic cirrhosis, and structural damage to the kidney. The PB group presented a normal renal pathology, but ultrastructural changes including swollen mitochondria and dilation of the endoplasmic reticulum were evident in the hepatocytes. The N, PB and PB/CC14 mice excreted 84.5, 61.5 and 72.3 percent respectively of a dose of 14C-amphetamine sulfate in the 0-72 hour urine. Seven major u r i n a r y metabolites were detected in the normal group, 4 in PB group, and 3 in the PB/CC14 group. Unchanged amphetamine, p-hydroxyamphetamine and benzoic acid were tentatively identified by combined techniques of gas chromatograhy and thin-layer chromatography with autoradiography.
ALTHOUGH INVESTIGATIONS OF THE MAMMALIAN METABOLISM of a m p h e t a m i n e a n d its d e r i v a t i v e s cont i n u e , little or no i n f o r m a t i o n is available w i t h respect to its m e t a b o l i s m in diseased a n i m a l s ' " . The widespread occurrence of h e p a t i t i s i n c o n j u n c t i o n w i t h d r u g abuse ~ , a n d the p o t e n t i a l of occurrence of This work was supported in part by R.O.D.A. scholarships (to Y.W.L.), Health and Welfare Canada. Correspondence: Leonard I. Wiebe, Ph.D. Associate Professor Division of Bionucleonics and Radiopharmacy University of Alberta Edmonton, Canada T6G 2N8
hepatic c i r r h o s i s as a r e s u l t of exposure to c a r b o n t e t r a c h l o r i d e - c o n t a i n i n g commercial p r e p a r a t i o n s c~ made such a s t u d y of a m p h e t a m i n e m e t a b o l i s m desirable. The carbon t e t r a c h l o r i d e - i n d u c e d c i r r h o t i c model of McLean et al (4) was selected for t h i s work, because of the ease and r e p r o d u c i b i l i t y of i n d u c t i o n of the chronic c i r r h o t i c state. T h i s model, which was regarded as a morphological s i m i l a r i t y to L a e n n e c ' s c i r r h o s i s in m a n (5), r e q u i r e d i n d u c t i o n by combined p h e n o b a r b i t a l and c a r b o n t e t r a c h l o r i d e t r e a t m e n t s . Because of the lack of i n f o r m a t i o n c o n c e r n i n g the effects of chronic p h e n o b a r b i t a l t r e a t m e n t on xenobiotic metabolism, a p h e n o b a r b i t a l t r e a t e d g r o u p was studied in a d d i t i o n to the c i r r h o t i c model. The m e t a b o l i s m of a m p h e t a m i n e in the mouse h a s been shown to involve p r i m a r i l y a r o m a t i c h y d r o x y lation and side c h a i n b r e a k d o w n ; u r i n a r y e l i m i n a t i o n of p - h y d r o x y a m p h e t a m i n e , benzoic acid, and unchanged a m p h e t a m i n e accounted for 15, 32 and 30 p e r c e n t respectively of a single dose '6). We now r e p o r t q u a n t i t a t i v e and q u a l i t a t i v e data from a s t u d y of a m p h e t a m i n e m e t a b o l i s m in c i r r h o t i c mice and in mice fed p h e n o b a r b i t a l chronically. R e l e v a n t aspects of hepatic and r e n a l pathology are discussed. MATERIALS AND METHODS Cirrhosis was induced according to the methods of McLean et al (4). Thirty male white mice (25-30g; strain ALAS, Health Sciences Animal Centre, University of Alberta) were divided into 3 groups, all receiving the same laboratory chow. The f i r s t group of 12 animals served as the control. A group of 6 animals, designated the PB group, had their drinking water replaced by sodium phenobarbital water (0.25 g ml "1 for a total of 37 days. The remaining 12 animals, the cirrhotic ( P B / CCl4) group, received phenobarbital drinking water for 7 days prior to and during the entire carbon tetrachloride intoxication period. A desiccator (10 1 internal volume) was used as an inhalation chamber. The mice were placed inside the chamber in groups of 4, and CC14 (1 ml) was introduced via a pipette onto a filter paper previously placed at the bottom of the chamber. The filter paper was separated from the animals by a porcelain grid. Exposure time was 10 minutes daily for 30 days. Metabolic studies were carried out 4 days after cessation of treatments. Autoradiography was carried out with Agfa-GavaertT M X-ray films. The films were developed in Kodak T M X-ray developer and fixed in Kodak T M X-ray fixer. All procedures and washing were carried out at room temperature. Thin layer chromatography (TLC) was done on Silica-
A M P H E T A M I N E METABOLISM IN E X P E R I M E N T A L CIRRHOSIS
57
TABLE 1. URINARY
pH,
URINARY EXCRETION, FECAL ELIMINATION OF NC-RADIOACTIVITY AFTER I.P.INJECTION OF NC-AMPHETAMINE. SIX ANIMALS W E R E U S E D IN THE P B A N D PB-CCI4 GROUPS, 5 IN THE CONTROL GROUP.
Urinary excretion data % of dose ± S.D. (pH) Group (dose in ~ Ci)
0-24 hr
Fecal elimination data % of dose ± S.D.
24-48 hr
48-72 hr
0-24 hr
24-48 hr
Control . . . . . . . (1.69) . . . . . . . . .
82.7 (6.7
± 10.4 ± 0.3)
1.5 ± 1.3 (6.5 ± 0.3)
0.3 ± 0.I (6.1 ± 0.3)
0.9 ± 1.0.
0.2 ± 0.I.
PB . . . . . . . . . . . (1.63) . . . . . . . . .
60.3 (6.4
± 7.6 ± 0.3)
0.7 ± 0.5 (6.4 ± 0.4)
0.5 ± 0.1 (6.5 ± 0.3)
1.9 ± 3.0
0.2 4- 0.1
PB-CC1 . . . . . . (1.42) . . . . . . . .
71.2 ± 3.2 (8.41 ± 1.5)
0.5 ± 0.2 (6.3 ± 0.4)
0.6 ± 0.2 (6.2)± 0.6)
2.4 ± 1.7
Amphetamine Metabolism in Mice Exposed Chronically to Phenobarbital and to Phenobarbital with Carbon Tetrachloride Y.W. LEE and L.I. WIEBE Division of Bionucleonics and Radiopharmacy, University of Alberta, Edmonton, Canada T6G 2N8 (Accepted September 12, 1978)
CLBIA, 12, (2) 56-58 (1979)
Clin. Biochem. Lee, Y.W., and Wiebe, L.I.
Division of Bionucleonies and Radiopharmacy, University of Alberta, Edmonton, Alberta T6G 2N8 A M P H E T A M I N E METABOLISM IN MICE EXPOSED CHRONICALLY TO P H E N O B A R B I T A L AND TO P H E N O B A R B I T A L W I T H CARBON TETRACHLORIDE. The metabolism of amphetamine was studied in three groups of mice, including normal mice (N), mice given chronic doses of phenobarbital via their drinking water (PB), and mice exposed to carbon tetrachloride vapors daily during the course of chronic phenobarbital consumption (PB/CC14). Renal and hepatic tissue from animals of each group were examined by electron microscopy. Mice in the PB/CC14 group demonstrated the classical symptoms of carbon tetrachloride-induced hepatic cirrhosis, and structural damage to the kidney. The PB group presented a normal renal pathology, but ultrastructural changes including swollen mitochondria and dilation of the endoplasmic reticulum were evident in the hepatocytes. The N, PB and PB/CC14 mice excreted 84.5, 61.5 and 72.3 percent respectively of a dose of 14C-amphetamine sulfate in the 0-72 hour urine. Seven major u r i n a r y metabolites were detected in the normal group, 4 in PB group, and 3 in the PB/CC14 group. Unchanged amphetamine, p-hydroxyamphetamine and benzoic acid were tentatively identified by combined techniques of gas chromatograhy and thin-layer chromatography with autoradiography.
ALTHOUGH INVESTIGATIONS OF THE MAMMALIAN METABOLISM of a m p h e t a m i n e a n d its d e r i v a t i v e s cont i n u e , little or no i n f o r m a t i o n is available w i t h respect to its m e t a b o l i s m in diseased a n i m a l s ' " . The widespread occurrence of h e p a t i t i s i n c o n j u n c t i o n w i t h d r u g abuse ~ , a n d the p o t e n t i a l of occurrence of This work was supported in part by R.O.D.A. scholarships (to Y.W.L.), Health and Welfare Canada. Correspondence: Leonard I. Wiebe, Ph.D. Associate Professor Division of Bionucleonics and Radiopharmacy University of Alberta Edmonton, Canada T6G 2N8
hepatic c i r r h o s i s as a r e s u l t of exposure to c a r b o n t e t r a c h l o r i d e - c o n t a i n i n g commercial p r e p a r a t i o n s c~ made such a s t u d y of a m p h e t a m i n e m e t a b o l i s m desirable. The carbon t e t r a c h l o r i d e - i n d u c e d c i r r h o t i c model of McLean et al (4) was selected for t h i s work, because of the ease and r e p r o d u c i b i l i t y of i n d u c t i o n of the chronic c i r r h o t i c state. T h i s model, which was regarded as a morphological s i m i l a r i t y to L a e n n e c ' s c i r r h o s i s in m a n (5), r e q u i r e d i n d u c t i o n by combined p h e n o b a r b i t a l and c a r b o n t e t r a c h l o r i d e t r e a t m e n t s . Because of the lack of i n f o r m a t i o n c o n c e r n i n g the effects of chronic p h e n o b a r b i t a l t r e a t m e n t on xenobiotic metabolism, a p h e n o b a r b i t a l t r e a t e d g r o u p was studied in a d d i t i o n to the c i r r h o t i c model. The m e t a b o l i s m of a m p h e t a m i n e in the mouse h a s been shown to involve p r i m a r i l y a r o m a t i c h y d r o x y lation and side c h a i n b r e a k d o w n ; u r i n a r y e l i m i n a t i o n of p - h y d r o x y a m p h e t a m i n e , benzoic acid, and unchanged a m p h e t a m i n e accounted for 15, 32 and 30 p e r c e n t respectively of a single dose '6). We now r e p o r t q u a n t i t a t i v e and q u a l i t a t i v e data from a s t u d y of a m p h e t a m i n e m e t a b o l i s m in c i r r h o t i c mice and in mice fed p h e n o b a r b i t a l chronically. R e l e v a n t aspects of hepatic and r e n a l pathology are discussed. MATERIALS AND METHODS Cirrhosis was induced according to the methods of McLean et al (4). Thirty male white mice (25-30g; strain ALAS, Health Sciences Animal Centre, University of Alberta) were divided into 3 groups, all receiving the same laboratory chow. The f i r s t group of 12 animals served as the control. A group of 6 animals, designated the PB group, had their drinking water replaced by sodium phenobarbital water (0.25 g ml "1 for a total of 37 days. The remaining 12 animals, the cirrhotic ( P B / CCl4) group, received phenobarbital drinking water for 7 days prior to and during the entire carbon tetrachloride intoxication period. A desiccator (10 1 internal volume) was used as an inhalation chamber. The mice were placed inside the chamber in groups of 4, and CC14 (1 ml) was introduced via a pipette onto a filter paper previously placed at the bottom of the chamber. The filter paper was separated from the animals by a porcelain grid. Exposure time was 10 minutes daily for 30 days. Metabolic studies were carried out 4 days after cessation of treatments. Autoradiography was carried out with Agfa-GavaertT M X-ray films. The films were developed in Kodak T M X-ray developer and fixed in Kodak T M X-ray fixer. All procedures and washing were carried out at room temperature. Thin layer chromatography (TLC) was done on Silica-
A M P H E T A M I N E METABOLISM IN E X P E R I M E N T A L CIRRHOSIS
57
TABLE 1. URINARY
pH,
URINARY EXCRETION, FECAL ELIMINATION OF NC-RADIOACTIVITY AFTER I.P.INJECTION OF NC-AMPHETAMINE. SIX ANIMALS W E R E U S E D IN THE P B A N D PB-CCI4 GROUPS, 5 IN THE CONTROL GROUP.
Urinary excretion data % of dose ± S.D. (pH) Group (dose in ~ Ci)
0-24 hr
Fecal elimination data % of dose ± S.D.
24-48 hr
48-72 hr
0-24 hr
24-48 hr
Control . . . . . . . (1.69) . . . . . . . . .
82.7 (6.7
± 10.4 ± 0.3)
1.5 ± 1.3 (6.5 ± 0.3)
0.3 ± 0.I (6.1 ± 0.3)
0.9 ± 1.0.
0.2 ± 0.I.
PB . . . . . . . . . . . (1.63) . . . . . . . . .
60.3 (6.4
± 7.6 ± 0.3)
0.7 ± 0.5 (6.4 ± 0.4)
0.5 ± 0.1 (6.5 ± 0.3)
1.9 ± 3.0
0.2 4- 0.1
PB-CC1 . . . . . . (1.42) . . . . . . . .
71.2 ± 3.2 (8.41 ± 1.5)
0.5 ± 0.2 (6.3 ± 0.4)
0.6 ± 0.2 (6.2)± 0.6)
2.4 ± 1.7
