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Drug metabolism in liver and in some non-hepatic tissues of partially hepatectomized rats
Pharmacological Research Communications, Vol. 2, No. 4, 1970
277
DRUG METABOLISM IN LIVER AND IN SOME NON-HEPATIC TISSUES OF PARTIALLY HEPATECTOMIZED RATS. L. ~anzo, M. De Bernardi, A. Ferrara and F. Bert@ Department of Pharmacology, University of Pavia, Italy.
Received
8 O c t o b e r 1970
Rapidly growing liver may be deficient in some microsomal enzyme systems which metabolize drugs and several physiological compounds. Lacking or scanty drug metabolism has been observed in different conditions of hepatocellular growth such as fetal or neonatal liver (Fours and Adamson,
1955), various hepatomas (Adamson and Fouts,
1961)
and regenerating liver of different animal species (Von Der Decken and Hultin, 1960; Murphy and Du Bois, 1958). Partial hepatectomy can produce a marked impairment of drug metabolism (Fours, Dixon and Shultice,
1961; Ben~mark, Ekholm and
01sson, 1967). Many investigations, however, suggest that the microsomal enzyme activity in the regenerating liver is not equally affected after partial liver removal (Fours, Dixon and Shultice, and Smuckler,
1970; Ferra~i,
1961; Barker, Arcasoy
1959).
In the present investigation the metabolism of two centrally active compounds was studied in the liver and in various non-hepatic tissues of hepatectomized rats. The metabolic pathways investigated were: a) the biosynthesis of oxazepam glucuronide; b) the oxidative N-demethylation of aminopyrine to 4-aminoantipyrine 4-~cetylaminoantipyrine
(4-AP); c) the acetylation of 4-AP to
(4-ACAP).
In previous studies carried out "in vitro" in the rat, rabbit and guinea-pig (Bert~ et al. 1967, 1968 a,b) and "in vivo" in the dog and
Pharmaco~g~alResearchCommun~ations, VoL 2, No. 4 , 1 9 7 0
278
monkey (Benzi et al., 1967, 1968), we pointed out that the drugs mentioned above are appreciably metabolized not only in hepatic preparations, but also in the lung, brain and kidney.
METHODS
The studies were carried out on 40 female rats of the Sprague-
Dawley strain weighing about 190 g. The rats, maintained under constant environmental conditions (22~
Rh 60 ~ 5%; lighting cycle 14 hrs light
and 10 hrs darkness), were fed on a normal laboratory diet with tap water ad libitum. Hepatectomy was performed according to Higgins and Anderson (1931), under ether anaesthesia and under sterile conditions. Sham-operated animals were anaesthetized, the peritoneum was opened, the liver exteriorized and manipulated (5-7 min.), and the wound closed as in hepatectomized animals. The duration of laparatomy was the same in both groups.
In all
experiments, groups of 8 animals were used. The metabolism of oxazepam and aminopyrine was tested "in vitro" in sham-operated rats and in hepatectomized animals at various times (48, 96, 192 hrs after hepatectomy). The rats were killed, and the liver, kidney, brain and lung were immediately removed and placed at + 4~ Specimens obtained during hepatectomy were also collected and analyzed as "normal manipulated liver". All organs were carefully homogenized in a Potter-Helvehjem type homogenizer with teflon pestle in crushed ice for 5-7 min. Homogenates were prepared by suspending the different tissues in 3.3 vol. of cold isotonic potassium chloride solution. The drug metabolism was studied in the supernatant fraction, obtained by centrifugation of homogenates at 9000 x g for 20 min.
The supernatant corresponding to I. 5 g of fresh tissue was
always used. The experimental conditions and co-factors used have been previously described (Manzo, et al. 1969; Bert& and Benzi, 1967). The aminopyrine metabolism was studied by measuring the amounts of metabolites 4-AP and 4-ACAP formed in enzyme preparations according to
Pharmacological Research Communications, Vol. 2, No. 4, 1970
279
Brodie and Axelrod ( 1950). Oxazepam metabolism was studied by evaluating the amounts of oxazepam glucuronide formed in incubation mixtures from different tissues, as described by Walkenstein
e• al. (1964). The metabolite is almost entirely
hydrolyzed by rat preputial gland ~-glucuronidase. Furthermore the enzymic hydrolisis is inhibited by glucarolactone (Dutton, 1966 ). Preliminary experiments showed that only traces of conjugated metabolite can be detected in the reaction mixture if the supernatant is not strengthened with UDPsodium glucuronate. Protein of the 9000 x g supernatant fraction from the tissue homogenates was evaluated using the method of Lowry et al. (1951), with crystalline bovine serum albumin as standard. Statistical observations Were performed according to "p" calculated by applying Student's test.
I~ESULTS
Table l~o.I shows the oxazepa~ metabolism in the liver of normal,
shsm-operated and hepatectomized rats. The livers of unoperated animals and "normal manipulated liver" were referred to as control, since no differences in their drug metabolizing activity were observed. In the livers of 48 hr
hepatectomized rats, the oxazepam glucuronidatio~
was significantly impaired (P < 0.01). The level of the hepatic glucuronide formation rebounded to control values after 4 days of liver regeneration. No changes in hepatic metabolism of aminopyrine were observed following partial liver removal (Table No.II). The sham-operation causes a slight decrease of the activity of some drug metabolizing enzymes; however no statistical differences between shamoperated and control groups resulted in our experiments. As shown in Tables No.I and No.II partial liver removal produces no alteration of aminopyrine and oxazepam metabolism in kidney, brain and lung preparations.
Pharmacological Research Communications, VoL 2, No. 4, 1970
280
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Pharmacologica/ Research Communications, VoL 2, No. 4, 1370
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Pharmacological Research Communications, Vol. 2, No. 4, 1970
The amounts of protein in 9000 x g supernatant fraction do not differ significantly between controls, sham-operated and hepatectomized animals.
DISCUSSION
Partial hepatectomy in the rat can alter the activity of
some drug metabolizing enzymes in liver microsomes. As shown by Gram et al. (1968) and by Beffgmark, Ekholm and Olsson (1967), the microsomal protein content in regenerating liver is reduced for at least 14 days. Recently Barker, Arcasoy and Smuckler (1970) reported that hepatectomy resulted in reduced levels of both cytocromes b 5 and P450 closely allied to microsomal NADPH linked electron transport systems. In our experiments the hepatic oxazepam glucuronidation was significantly impaired 48 hrs after hepatectomy. The glucuronyl-transferase
activity
reached normal values after approximately 96 hrs, at the end of the first phase of rapid hepatocellular growth (Higgins and Anderson,
1931). On the
contrary, the oxidative N-demethylation of aminopyrine to 4-AP and consequent N-acetylation of the metabolite to 4-ACAP were unaffected by hepatectomy. These results confirmed the great variability of effects produced by partial liver removal on the activity of drug metabolizir.g enzymes (Fouts, Dixon and Shultice,
1961; Ba~ker, Arcasoy and Smuckler,
1970; Ferrari,
1959).
Our data are also consistent with the finding that some glucuronyl-transferases can be distributed differently from the NADPH-requiring microsomal enzymes in the sub-fractions of liver microsomes. The aminopyrine N-demethylase as measured by 4-AP formation~ was concentrated in smooth-surfaced microsomes, whereas various enzyme systems catalyzing glucuronide conjugation were found in the rough surfaces or evenly distributed between rough- and smoothsurfaced microsomes (Fouts and Gram, 1969). The formaldehyde formation caused by the N-demethylation of demethylaminoaniline was decreased in the regenerating liver, whereas the N-oxidation of this compound was enhanced. Therefore the intermediate demethylanilineN-oxide may accumulate in the system
( Barker, Arcasoy
and
Smuckler, 1970).
Pharmacologica/ Research Communications, VoL 2, No. 4, 1970
283
This leads to the conclusion that the metabolism of drugs possessing different metabolic steps, can be influenced in different ways by hepatectomy. Evidence has been obtained that drug metabolism in non-hepatic tissues is unaffected by partial liver removal, both when hepatectomy induces an impairment of the hepatic metabolizing activity and when this is without effect. The absence of a compensating mechanism can increase the risk of drug toxicity in conditions of liver damage.
REFE~NCES Adamson R.H. and Fours J.R.: (1961), Cancer Res. 21, 667. Barker E.A., Arcascy M. and Smuckler E.A. : (1970), Agents and Action 1, 27. Ben@mark A., Ekhclm R. and Olsson R. : (1947), Acta Hepato-Splencl. 14, 80. Benzi G., Bert~ F., Crema A. and Arrigoni E.: (]968), J. Pharm. Sci. 57, 1301. Benzi G., Bert~ F., Crema A. and Frigo G.M.: (1967), J. Pharm. Sci. 56, 1349. Bert~ F. and Benzi G. : (1967), J. Pharm. Pharmaool. I_~, 608. Bert~ F., Benzi G., ~anzo L. and Hokari S.: XIV Congresso Nazionale della Societ~ Itaiiana di Farmacologia - Trieste, June 1967 , c. 68. Bert~ F., Benzi G., Manzo L. and Hokari S. : (1968b), Arch. Intern. Pharmacodyn. 173, 377. Bert~ F., Manzo L. and De Bernandi M. : VI Congr. Intern. Soc. Farm. Mediter. Lat., Granada, May 1968 - (1968a), Ars Farmaceutica ~, 4. Brodie B.B. and Axelrod J.: (1950), J. Pharm. Exptl. Therap. 99, 171 9 Dutton G.J.: (1966), Glucuronic acid, Academic Press, p. 281. Ferrari V. : (]959), Acta vitaminologica 13, 145. Fours J.R. and Adamson R.H.: (1955), Science 129, 897. Fouts J.R., Dixon R.L. and Shultice R.W.: (1961), Biochem. Pharmacol. 7, 265. Fouts J.R. and Gram T.E.: (1969), Microsomes and drugs oxidation, Academic Press, p. 81. Gram T.E., Guarino A.M., Greene F.E., Gigon P.L. and Gillette J.R." (1968), Biochem. Pharmacol. I_~, 1769. Higg• G.M. and Anderson R.M.: (1911), Arch. Path. 12, 186. Lowry O.H., Rosenbrough N.J., Farr A.L. and Randall R.J.: (1951), J. Biol. Chem. 1~, 265. Manzo L., Bert~ F. and De Bernardi M.: (1969), Boll. Chim. Farm. 108, 19. Murphy S.D. and Du Bois K.P.: (1958), J. Pharm. Exptl. Therap. 124, 194. Von Der Decken A. and Hultin T.: (1960), Exp. Cell. Res. 19, 591. Walkenstein S.S., Wiser R., Gudmunsen C.H., Kimmel H.B. and Corradino R.A. : (1964), J. Pharm. Sci. 53, 1181.
U
277
DRUG METABOLISM IN LIVER AND IN SOME NON-HEPATIC TISSUES OF PARTIALLY HEPATECTOMIZED RATS. L. ~anzo, M. De Bernardi, A. Ferrara and F. Bert@ Department of Pharmacology, University of Pavia, Italy.
Received
8 O c t o b e r 1970
Rapidly growing liver may be deficient in some microsomal enzyme systems which metabolize drugs and several physiological compounds. Lacking or scanty drug metabolism has been observed in different conditions of hepatocellular growth such as fetal or neonatal liver (Fours and Adamson,
1955), various hepatomas (Adamson and Fouts,
1961)
and regenerating liver of different animal species (Von Der Decken and Hultin, 1960; Murphy and Du Bois, 1958). Partial hepatectomy can produce a marked impairment of drug metabolism (Fours, Dixon and Shultice,
1961; Ben~mark, Ekholm and
01sson, 1967). Many investigations, however, suggest that the microsomal enzyme activity in the regenerating liver is not equally affected after partial liver removal (Fours, Dixon and Shultice, and Smuckler,
1970; Ferra~i,
1961; Barker, Arcasoy
1959).
In the present investigation the metabolism of two centrally active compounds was studied in the liver and in various non-hepatic tissues of hepatectomized rats. The metabolic pathways investigated were: a) the biosynthesis of oxazepam glucuronide; b) the oxidative N-demethylation of aminopyrine to 4-aminoantipyrine 4-~cetylaminoantipyrine
(4-AP); c) the acetylation of 4-AP to
(4-ACAP).
In previous studies carried out "in vitro" in the rat, rabbit and guinea-pig (Bert~ et al. 1967, 1968 a,b) and "in vivo" in the dog and
Pharmaco~g~alResearchCommun~ations, VoL 2, No. 4 , 1 9 7 0
278
monkey (Benzi et al., 1967, 1968), we pointed out that the drugs mentioned above are appreciably metabolized not only in hepatic preparations, but also in the lung, brain and kidney.
METHODS
The studies were carried out on 40 female rats of the Sprague-
Dawley strain weighing about 190 g. The rats, maintained under constant environmental conditions (22~
Rh 60 ~ 5%; lighting cycle 14 hrs light
and 10 hrs darkness), were fed on a normal laboratory diet with tap water ad libitum. Hepatectomy was performed according to Higgins and Anderson (1931), under ether anaesthesia and under sterile conditions. Sham-operated animals were anaesthetized, the peritoneum was opened, the liver exteriorized and manipulated (5-7 min.), and the wound closed as in hepatectomized animals. The duration of laparatomy was the same in both groups.
In all
experiments, groups of 8 animals were used. The metabolism of oxazepam and aminopyrine was tested "in vitro" in sham-operated rats and in hepatectomized animals at various times (48, 96, 192 hrs after hepatectomy). The rats were killed, and the liver, kidney, brain and lung were immediately removed and placed at + 4~ Specimens obtained during hepatectomy were also collected and analyzed as "normal manipulated liver". All organs were carefully homogenized in a Potter-Helvehjem type homogenizer with teflon pestle in crushed ice for 5-7 min. Homogenates were prepared by suspending the different tissues in 3.3 vol. of cold isotonic potassium chloride solution. The drug metabolism was studied in the supernatant fraction, obtained by centrifugation of homogenates at 9000 x g for 20 min.
The supernatant corresponding to I. 5 g of fresh tissue was
always used. The experimental conditions and co-factors used have been previously described (Manzo, et al. 1969; Bert& and Benzi, 1967). The aminopyrine metabolism was studied by measuring the amounts of metabolites 4-AP and 4-ACAP formed in enzyme preparations according to
Pharmacological Research Communications, Vol. 2, No. 4, 1970
279
Brodie and Axelrod ( 1950). Oxazepam metabolism was studied by evaluating the amounts of oxazepam glucuronide formed in incubation mixtures from different tissues, as described by Walkenstein
e• al. (1964). The metabolite is almost entirely
hydrolyzed by rat preputial gland ~-glucuronidase. Furthermore the enzymic hydrolisis is inhibited by glucarolactone (Dutton, 1966 ). Preliminary experiments showed that only traces of conjugated metabolite can be detected in the reaction mixture if the supernatant is not strengthened with UDPsodium glucuronate. Protein of the 9000 x g supernatant fraction from the tissue homogenates was evaluated using the method of Lowry et al. (1951), with crystalline bovine serum albumin as standard. Statistical observations Were performed according to "p" calculated by applying Student's test.
I~ESULTS
Table l~o.I shows the oxazepa~ metabolism in the liver of normal,
shsm-operated and hepatectomized rats. The livers of unoperated animals and "normal manipulated liver" were referred to as control, since no differences in their drug metabolizing activity were observed. In the livers of 48 hr
hepatectomized rats, the oxazepam glucuronidatio~
was significantly impaired (P < 0.01). The level of the hepatic glucuronide formation rebounded to control values after 4 days of liver regeneration. No changes in hepatic metabolism of aminopyrine were observed following partial liver removal (Table No.II). The sham-operation causes a slight decrease of the activity of some drug metabolizing enzymes; however no statistical differences between shamoperated and control groups resulted in our experiments. As shown in Tables No.I and No.II partial liver removal produces no alteration of aminopyrine and oxazepam metabolism in kidney, brain and lung preparations.
Pharmacological Research Communications, VoL 2, No. 4, 1970
280
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Pharmacologica/ Research Communications, VoL 2, No. 4, 1370
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282
Pharmacological Research Communications, Vol. 2, No. 4, 1970
The amounts of protein in 9000 x g supernatant fraction do not differ significantly between controls, sham-operated and hepatectomized animals.
DISCUSSION
Partial hepatectomy in the rat can alter the activity of
some drug metabolizing enzymes in liver microsomes. As shown by Gram et al. (1968) and by Beffgmark, Ekholm and Olsson (1967), the microsomal protein content in regenerating liver is reduced for at least 14 days. Recently Barker, Arcasoy and Smuckler (1970) reported that hepatectomy resulted in reduced levels of both cytocromes b 5 and P450 closely allied to microsomal NADPH linked electron transport systems. In our experiments the hepatic oxazepam glucuronidation was significantly impaired 48 hrs after hepatectomy. The glucuronyl-transferase
activity
reached normal values after approximately 96 hrs, at the end of the first phase of rapid hepatocellular growth (Higgins and Anderson,
1931). On the
contrary, the oxidative N-demethylation of aminopyrine to 4-AP and consequent N-acetylation of the metabolite to 4-ACAP were unaffected by hepatectomy. These results confirmed the great variability of effects produced by partial liver removal on the activity of drug metabolizir.g enzymes (Fouts, Dixon and Shultice,
1961; Ba~ker, Arcasoy and Smuckler,
1970; Ferrari,
1959).
Our data are also consistent with the finding that some glucuronyl-transferases can be distributed differently from the NADPH-requiring microsomal enzymes in the sub-fractions of liver microsomes. The aminopyrine N-demethylase as measured by 4-AP formation~ was concentrated in smooth-surfaced microsomes, whereas various enzyme systems catalyzing glucuronide conjugation were found in the rough surfaces or evenly distributed between rough- and smoothsurfaced microsomes (Fouts and Gram, 1969). The formaldehyde formation caused by the N-demethylation of demethylaminoaniline was decreased in the regenerating liver, whereas the N-oxidation of this compound was enhanced. Therefore the intermediate demethylanilineN-oxide may accumulate in the system
( Barker, Arcasoy
and
Smuckler, 1970).
Pharmacologica/ Research Communications, VoL 2, No. 4, 1970
283
This leads to the conclusion that the metabolism of drugs possessing different metabolic steps, can be influenced in different ways by hepatectomy. Evidence has been obtained that drug metabolism in non-hepatic tissues is unaffected by partial liver removal, both when hepatectomy induces an impairment of the hepatic metabolizing activity and when this is without effect. The absence of a compensating mechanism can increase the risk of drug toxicity in conditions of liver damage.
REFE~NCES Adamson R.H. and Fours J.R.: (1961), Cancer Res. 21, 667. Barker E.A., Arcascy M. and Smuckler E.A. : (1970), Agents and Action 1, 27. Ben@mark A., Ekhclm R. and Olsson R. : (1947), Acta Hepato-Splencl. 14, 80. Benzi G., Bert~ F., Crema A. and Arrigoni E.: (]968), J. Pharm. Sci. 57, 1301. Benzi G., Bert~ F., Crema A. and Frigo G.M.: (1967), J. Pharm. Sci. 56, 1349. Bert~ F. and Benzi G. : (1967), J. Pharm. Pharmaool. I_~, 608. Bert~ F., Benzi G., ~anzo L. and Hokari S.: XIV Congresso Nazionale della Societ~ Itaiiana di Farmacologia - Trieste, June 1967 , c. 68. Bert~ F., Benzi G., Manzo L. and Hokari S. : (1968b), Arch. Intern. Pharmacodyn. 173, 377. Bert~ F., Manzo L. and De Bernandi M. : VI Congr. Intern. Soc. Farm. Mediter. Lat., Granada, May 1968 - (1968a), Ars Farmaceutica ~, 4. Brodie B.B. and Axelrod J.: (1950), J. Pharm. Exptl. Therap. 99, 171 9 Dutton G.J.: (1966), Glucuronic acid, Academic Press, p. 281. Ferrari V. : (]959), Acta vitaminologica 13, 145. Fours J.R. and Adamson R.H.: (1955), Science 129, 897. Fouts J.R., Dixon R.L. and Shultice R.W.: (1961), Biochem. Pharmacol. 7, 265. Fouts J.R. and Gram T.E.: (1969), Microsomes and drugs oxidation, Academic Press, p. 81. Gram T.E., Guarino A.M., Greene F.E., Gigon P.L. and Gillette J.R." (1968), Biochem. Pharmacol. I_~, 1769. Higg• G.M. and Anderson R.M.: (1911), Arch. Path. 12, 186. Lowry O.H., Rosenbrough N.J., Farr A.L. and Randall R.J.: (1951), J. Biol. Chem. 1~, 265. Manzo L., Bert~ F. and De Bernardi M.: (1969), Boll. Chim. Farm. 108, 19. Murphy S.D. and Du Bois K.P.: (1958), J. Pharm. Exptl. Therap. 124, 194. Von Der Decken A. and Hultin T.: (1960), Exp. Cell. Res. 19, 591. Walkenstein S.S., Wiser R., Gudmunsen C.H., Kimmel H.B. and Corradino R.A. : (1964), J. Pharm. Sci. 53, 1181.
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