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Activities of hepatic and extrahepatic microsomal mixed function oxidase enzymes in diabetic and gonadectomized-diabetic rats
Gen. Pharrauc, Vol. 12. pp. 345 to 350. 1981
0306-3623/81/090345-O9502.00/0 Copyright O 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
ACTIVITIES OF HEPATIC AND EXTRAHEPATIC MICROSOMAL MIXED FUNCTION OXIDASE ENZYMES IN DIABETIC AND GONADECTOMIZED-DIABETIC RATS W A L I D A. A L - T U R K , SIDNEY J. STOHS a n d E D W A R D B. ROCHE
Department of Biomedicinal Chemistry, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68105, U.S.A. IReceired 25 February 1981)
Abstract--1. Streptozotocin (STZ~-induced diabetes results in a decrease in hepatic aryl hydrocarbon hydroxylase (AHH) activity in male rats and an increase in female rats, while hepatic 7-ethoxycoumarin O-deethylase (ECD) activity is increased in both sexes. 2. In castrated male rats, induction of the diabetic state resulted in a further decrease in hepatic AHH activity. In diabetic female rats, ovariectomy did not alter hepatic AHH or ECD activities. 3. STZ-induced diabetes produced an increase in hepatic cytochrome P-450 content. The diabetic state failed to produce an increase in hepatic cytochrome P-450 content of castrated male animals, and only a small increase in castrated female rats. 4. AHH and ECD activities are decreased in lungs of STZ-induced diabetic male and female rats. In diabetic-castrated rats a further decrease in pulmonary ECD activity occurs in both sexes, and in the AHH activity of female rats. 5. The induction of diabetes with STZ increased intestinal AHH and ECD activities in both sexes. In diabetic-castrated rats, intestinal ECD activities were identical to the activities in diabetic male and female animals, while intestinal AHH activities were lower than the activities in diabetic animals but still significantly higher than in control animals of both sexes.
INTRODUCTION Experimentally induced diabetes has been shown to alter the hepatic microsomal metabolism of some xenobiotics. Kato & Gillette (1963, 1965) observed that liver microsomal hexobarbital hydroxylase and aminopyrine N-demethylase were depressed in alloxan-diabetic male rats, whereas the activities were increased in diabetic female rats. Aniline hydroxylase was increased in both male and female alloxan-diabetic rats. The activity of zoxazolamine hydroxylase was not altered in diabetic males, but increased significantly in diabetic female rats (Kato & Gillette, 1963, 1965). The metabolism of hexobarbital and aminopyrine were not depressed in castrated male and female rats by alloxan-induced diabetes, but the metabolism of these substrates was depressed in castrated-diabetic male and female rats receiving methyltestosterone. From these findings Kato & Gillette (1965) suggested that diabetes impaired the action of androgens to increase the sex-dependent activity of drug metabolizing enzymes. We have found that liver microsomal aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase are depressed in streptozotocin (STZ)diabetic male rats, whereas the activities are increased in diabetic female rats (Reinke et al., 1978, 1979; Stohs et al., 1979). Aniline hydroxylase was also increased in both male and female rats with STZinduced diabetes (Reinke et al., 1978). The above metabolic alterations in streptozotocin diabetic rats were reversed by treating with insulin (Reinke et al., 1978, 1979). We have also observed that liver and
intestinal microsomal 7-ethoxycoumarin 0-deethylase (ECD) activities are increased in STZ diabetic male and female rats, whereas the lung activity is depressed in both sexes (Al-Turk et al., 1980a). Hepatic microsomal cytochrome P-450 content has been reported to increase as a result of STZ-induced diabetes in both male and female rats (Reinke et al., 1978, 1979), and is increased in alloxan-induced diabetic female rats but remains unaltered in alloxandiabetic male rats (Ackerman & Leibman, 1977; Kato et al., 1970). The effects of gonadectomy on the mixed function monooxygenase enzymes of experimentally induced diabetic animals has received little attention. In order to determine the influence of other hormones on the alterations of xenobiotic metabolism which occur in diabetic animals, we have examined the effects of gonadectomy on hepatic and extrahepatic microsomai aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin 0-deethylase (ECD) activities in streptozotocin-induced diabetic male and female rats. MATERIALS AND METHODS Animals and Treatment
Male and female rats, age 40-45 days, of a SpragueDawley derived strain (Sasco Inc., Omaha, NE, USA) w e r e individually caged and allowed free access to water and Purina lab chow for at least 3-5 days before experimental use. The animals were maintained at a temperature of 21~C, with lighting from 0600 to 1800 hr daily. Male rats were castrated by scrotal incision, and female rats were ovariectomized by dorsal incision. All control 345
346
WAI,n) A. At.-Tt:RK ¢'t ul.
animals were sham-operatcd. Four groups of animals of each sex were utilized: control, sham-operated control, diabetic and castrated-diabetic animals. Diabetes was induced by a single tail vein injection of streptozotocin (STZ). 60 mg kg. freshly prepared in 0.05 M citrate buffer, pH 4.5, as previously described (Reinke (,t ,I., 1978). After four days, animals excreting at least 0.5?. glucose in the urine. as detcrmined by Tes-Tapc (Eli Lilly Co.. Indianapolis, Inc.), werc judged to be diabetic. The animals were killed 10 days after S T Z administration. The diabetic state was confirmed by a blood glucosc level of at least 200mgl00ml as determined by the glucose oxidase method (Sigma Chemical Co.. St. Louis. MO). Micro,some Prepto'ation
Liver microsomes were prepared in 1.15",. KCI plus 0.05 M Tris buffer, pII 7.4. by diffcrential centrifugation as previously described [Stohs et u/.. 1971). Preparation of intestinal microsomes was accomplished by the method of Stohs ct ul., (1976). ]'he upper villous layer of the mucosa was homogenized in a pit 7.8 "l-ris KCI buffer (0.05 M Tris chloride in 1.15",, KCI) containing trypsin inhibitor 15 m g g wet weight of small intestine), glycerol (20% v v final concentrationl and heparin (3 units:ml) to decrease agglutination and degradation of the monooxygenase system (Stohs et ul.. 1976). The washed microsomal pellet was resuspended to a concentration of approximately 2.0 mg microsomal proteinml in the Tris-KCI buffer, pH 7.8. Lung microsomes were isolated by a modification of the procedure of Capdevila et ul.. (1975). [ h e lungs were perfused in situ with 30ml of ice-cold normal saline, removed, weighed, blotted and minced. Homogenates werc prepared in a pH 7.6 Tris-KCI buffer 10.05 M Tris chloride in 1.15".. KCI) containing glycerol (10".. ,, vl. The v,ashed microsomes were resuspendcd to a tinal concentration of approximately 3.0 mg of microsomal protein ml in the ptt 7.6 Tris-KCI buffer. A s s a y Procedure.s
Incubations for determination of ary[ hydrocarbon hydroxylase [AHH) activities were conducted in 25 ml Edenmeyer flasks containing 1.06/~mole NADP +. 5.37~mole trisodium isocitrate, 0.05 ml of 0.10 M MgCI2, 0.88 units isocitrate dehydrogenase (all purchased from Sigma Chemical Co.. St. Louis, MO, USA) and approximately 0.5 mg of liver or intestinal microsomal protein or 1.0 mg of lung microsomal protein, in a tinal volume of 1.0 ml. After a 2 rain preincubation at 37 C. the incubations were initiated by the addition of 25 td of a 6.0 mM solution of benzoIa)pyrene in acetone. Thc reactions were conducted in air in an Eberbach shaker, at 75 oscillations:rain, exposed to yellow light. After 5 min, the reactions were terminated by the addition of 0.30 ml of triethylamineTriton X-100 (9:1), follov,ed by 2.0ml of aqueous 0.05". EDTA solution. AHH activity was determined by the assay procedure of Dehnen et al. 11973) on an Aminco-Bowman spectrophotofluorometer. One AHH unit is defined as the amount of enzyme which catalyzes the formation of 1.0 nmole of product:min/mg microsomal protein, using 3-hydroxybenzo(a)pyrene as the reference standard. 7-Ethoxycoumarin 0-deethylase IECD) activity was assayed in 25ml Erlenmeyer flasks, containing the NADPH-generating system described above, and approximately 1.0 mg of lung or intestinal microsomal protein or 0.5 mg of liver microsomal protein, in a final volume of 1.0 ml. After a 2 min preincubation at 37 C, the incubations were initiated by the addition of lO01tl of a 0.10 M solution of 7-ethoxycoumarin prepared in the respective buffers used for isolation of the microsomal fractions. The reactions were conducted in air in an Eberbach shaker, at 75 oscillations/min. After 10 min, the reactions were terminated by the addition of 0.125ml of 15°. Iw/v) trichloroacetic acid. The extractions and 7-ethoxycoumarin
0-deethylase assays were performed according to the procedures of Greenlec & Poland (1978) on an Aminco Bowman spectrophotofluorometer. The quantity of 7hydroxycoumarin formed was calculated by comparing the net fluorescence obscr',ed (sample minus blankl in the final alkaline extract with standard solution of 7-hydroxy coumarin. One ECD unit is defined as the amount of enzyme which catalyzes the formation of 1.0 nmole of product min mg microsomal protein. Cytochrome P-450 content was determined by the method of Omura & Sara (1964). Protein analyses were performed on the microsomal suspensions used in each assay (Lowry et ul., 1951). All absorption measurements were conducted ~ith a Beckman Acta M-VI Spectrophotometcr. "Stuti,~tical M e t h o d ~
Each value represents the mean + SD of 4 animals. The data was subjected to one-way analysis of variance (ANOVA) using Scheffe's S method, and mean values with P < 0.05 were considered significantly different.
RESUI.'IS The effects of STZ-induced diabetes on liver, lung and intestinal microsomal metabolism of benzo(a)pyrene in control and gonadectomized male and female rats are presented in Table 1. The sham operations on control and diabetic animals had no effect on AHH activities in microsomes of the three tissues. The diabetic state resulted in a 50", reduction in hepatic AHH activity in male rats, and was further significantly reduced in castrated animals. In female rats. the diabetic state resulted in a significant increase in hepatic AHH activity in both control and ovariectomized animals (Table 1). In the lung. STZ-induced diabetes significantly decreased AHH activity in both control and castrated male and female rats. In the intestine, both male and female diabetic rats had signilicantly higher AHH activities than the control animals (Table 1). In both sexes, the diabetic-castrated rats had increased AHH activities as compared to the controls, but the increase were not as high as the increases produced in the diabetic animals. The effects of STZ-induced diabetes on the hepatic. pulmonary and intestinal metabolism of 7-ethoxycoumarin in gonadectomized male and female rats is presented in Table 2. Sham operations again had no effect on ECD activities of microsomes from the three tissues. 7-Ethoxycoumarin 0-deethylase activity (ECD) in hepatic microsomes from diabetic male and female rats was increased approximately by 4Y'o and 80'!0. respectively, over control values. In the castrated male but not the female animals. STZ-induced diabetes failed to increase hepatic ECD activity. In the lung. ECD activity was significantly decreased as a result of STZ-induced diabetes in both male and female rats. The diabetic state in both castrated male and female rats resulted in a further decrease in pulmonary ECD activity as compared to the control animals. The induction of the diabctic state by STZ in male and female rats produced a significant increase in intestinal ECD activity as compared to control animals, similar to the results found in the liver (Table 2). In castrated animals of both sexes. STZ-induced dia-
5.23 5.44 2.82 2.62 1.41
+ + + + +
0.41 0.61 0.42 ~ 0.52" 0.14 ''h
Male 0.77 0.76 1.14 1.13 0.98
+ + + + +
0.07 0.13 0.08" 0.06" 0.09"
Female
Liver (Unitsj
51.2 54.2 38.3 37.9 42.0
~- 4.4 + 5.6 + 3.1J + 2.7 ~ + 5.3 ~
Male 47.8 48.9 18.4 17.8 28.9
+ + + + +
4.6 4.6 2.1 ~ 2.0 ~ 3.5 ~'h
Female
Lungs IMilliunitsl
51.4 50.7 142.4 139.2 75.1
± + ± + ±
Male 6.2 3.4 22.3' 21.5 ~ 11.5""
32.3 32.1 64.6 64.0 44.4
+ + + + +
3.5 4.6 11.8 ~ 10.9" 4.6 ~'"
Female
Intestine (MilliunitsJ
96.2 93.1 163.6 165.8 91.1
± 5.2 +_ 4.1 _+ 1(I.5' _+ 11.4 ~ + 2.7"
Male
Li',cr
84.3 80.5 175.4 178.4 180.2
+_ 4.6 _+ 3.9 L 12.4' + 13.8" + 10.9'
Female 34.7 33.5 25.6 27.0 17.5
+ 4.2 + 4.3 ± 3.3" + 3.6 J +_ 2.1 ''h
Male
Lung
36.4 36.1 26.2 26.3 10.8
_+ 3.9 + 3.1 ± 3.4 ~ + 3.2 ~ + 2.6""
Female
2.5 2.6 7.5 7.6 6.9
_+ 0.3 _+ 0.4 i 2.(P + 2.2 ~ + 1.9"
Male
Intestine
1.9 1.9 9.2 10.2 10.8
L + + + +
0.6 0.8 2.5 ~ 3.7" 2.6 ~
Female
A n i m a l s received STZ, 60 mg kg. i.v.. or the 0.05 M citrate buffer, pH 4.5. vehicle one day after surgery and 10 days prior to sacritice. Values are the m e a n s of 4 a n i m a l s _+SD. JP < 0.05 with respect to c o r r e s p o n d i n g c o n t r o l groups, bp < 0.05 with respect to d i a b e t i c animals.
Control C o n t r o l - s h a m operated Diabetic Diabetic-sham operated Diabetic-castrated
Experimental groups
7 - E t h o x } c o u m a r i n 0-deethylase ( E C D ) activity (units)
T a b l e 2. 7-Ethox~,coumarin 0-dccthylasc activity of liver, lung and intestinal m u c o s a from control, d i a b e t i c and d i a b e t i c - c a s t r a t e d male and female rats
Animals received S'IZ, 60 mg. kg, i.v., or the 0.05 M citrate buffer, pH 4.5. vehicle one day after surgery and 10 da}s p r i o r to sacritice. Values are the means of 4 a n i m a l s + SD. ~P < 0.05 with respect to c o r r e s p o n d i n g control groups, r,p < 0.05 with respect .t.o the d i a b e t i c groups.
Control Control-sham operated Diabetic D i a b e t i c - s h a m operated Diabetic-castrated
|:xperimcntal groups
Aryl I i y d r o c a r b o n H y d r o x y l a s e { A H H I Activity
T a b l e 1. Aryl h y d r o c a r b o n h y d r o x y l a s c activity of liver, lung and intestinal m u c o s a from control, diabetic, and d i a b e t i c - c a s t r a t e d male and female rats
g-
G X
3
348
WALIDA. AL-TURKet ul. Table 3. Cytochrome P-450 content of liver microsomes from control, diabetic, and diabetic-castrated male and female rats Cytochrome P-450 Content (nmol/mg microsomal protein) Experimental groups Control Control-sham operated Diabetic Diabetic-sham operated Diabetic-castrated
Male 1.07 + 0.20 1.09 + 0.21 1.60 + 0.09~ 1.66 _+0.04~ 1.08 _+0.17h
Female 0.87 + 0.06 0.90 + 0.05 1.39 _+0.08~ 1.46 __.0.07° 1.07 + 0.06~'b
Animals received STZ, 60 mg/kg, i.v., or the 0.05 M citrate buffer, pH 4.5, vehicle 1 day after surgery and 10 days prior to sacrifice. Values are the means of 4 animals + SD. JP < 0.05 with respect to control animals. "P < 0.05 with respect to diabetic animals.
betes produced an increase in intestinal ECD that was similar to the response observed with diabetes alone. The effects of castration on the hepatic cytochrome P-450 content of STZ-induced diabetic male and female rats are shown in Table 3. Sham operations on control and diabetic animals had no effect on hepatic cytochrome P-450 content. An increase in cytochrome P-450 content occurred as a result of STZ-induced diabetes in rats, agreeing with previously reported observations (Reinke et al., 1978). However, the diabetic state failed to increase the hepatic cytochrome P-450 content in castrated male rats, and produced only a small increase in cytochrome P-450 content in ovariectomized female rats. DISCUSSION Alterations observed in xenobiotic metabolism as the result of an induced diabetic state in experimental animals are dependent upon the substrate, the tissue examined and the sex of the animals (Reinke et al., 1978; 1979; Stohs et al., 1979; AI-Turk et al., 1980a). Some of these metabolic changes may reflect interference of the diabetic state with steroid-hormone dependent metabolic pathways (Kato & Gillette, 1965). We have therefore examined the metabolism of benzo(a)pyrene and 7-ethoxycoumarin in gonadectomized STZ-diabetic male and female rats. The responses of the AHH and ECD enzymes in liver, lung and intestinal microsomes of diabetic male and female rats which have been gonadectomized are markedly different. The results in Tables 1 and 2 agree with the previously reported observations that STZ-induced diabetes results in a decrease in hepatic AHH activity in male rats (Reinke et al., 19791 and an increase in female rats, while hepatic ECD activity is increased in both male and female rats (Al-Turk et al,, 1980a). The diabetic state in the castrated male rats resulted in a further decrease in the activity of AHH in the liver as compared to the hepatic AHH activity of diabetic male rats which had not been gonadectomized (Table 1) while the hepatic ECD activity was not affected by diabetes in castrated male animals (Table 2). Therefore, androgens do exert a regulatory role on hepatic mixed function oxidases in the diabetic male rat. Several recent investigations have shown that hepatic ECD and AHH activities respond differentially to dietary factors tHietanen, 1980; Hie-
tanen et al., 1980), and therefore lack of agreement in the responses of the two enzymes may be expected. In diabetic female rats, ovariectomy did not alter either ECD or AHH activities in liver. Castration of male rats but not female rats decreased hepatic mixed function oxidase activity in these diabetic rats. The hepatic metabolism of both substrates is dependent on the production of androgens by the testes but not on the production of estrogens by the ovaries (Marshall, 1971; AI-Turk, et al., 1980b). In the diabetic male rat, an apparent inconsistency exists in the form of a decreased metabolism of aminopyrine, hexobarbital, codeine, chlorpromazine and phenylbutazone (Kato & Gillette, 1963, 1965; Dixon et al., 1961; Weiner et al., 1970), as well as benzo(a}pyrene in the presence of increased levels of cytochrome P-450 in hepatic microsomes. A decreased binding of aminopyrine and hexobarbital to cytochrome P-450 in hepatic microsomes from diabetic male rats has been reported (Kato et al., 1971; Kato & Takahashi, 1969), while the binding of aniline was slightly increased (Kato & Takahashi, 1969). The binding of certain substrates to cytochrome P-450 is itself an androgen-dependent phenomenon in the rat (Kato et al., 1971), since castration markedly decreased the binding of substrates such as aminopyrine and hexobarbital, but not substrates such as aniline and zoxazolamine. Therefore, the interference in microsomal metabolism produced by the diabetic state may be the result of a decreased ability of cytochrome P-450 to bind to those substrates metabolized by androgen-dependent pathways, and may therefore not be determined by the absolute amount of cytochrome P-450 present. Another possible explanation lies with the known multiplicity of microsomal hemoproteins (Ryan et al., 1979}. The metabolism of aniline and aminopyrine as well as benzo~aJpyrene and 7-ethoxycoumarin are thought to be mediated by separate cytochrome P-450 forms (Kato & Takahashi, 1969; Ryan et al., 1979; Weiner et al., 1972), and the diabetic state may favor the formation of a specific cytochrome form, thus favoring enzymes as AHH and ECD. In male and female rats, hepatic ECD activity is increased by diabetes (AI-Turk et al., 1980a), and decreased only by castration and not by ovariectomy (AI-Turk et al., 1980b). In the castrated-diabetic male rat, the two effects counteract each other (Table 2). The results indicate that the hepatic metabolism of
Activities of hepatic mixed function oxidase enzymes in diabetic rats both A H H and E C D are regulated by pancreatic and androgenic functions, and suggest that the diabetic state does not interfere with the metabolic control exerted by androgens on hepatic mixed function oxidases. The microsomal metabolism of b o t h benzo(a)pyrene (Table l) and 7-ethoxycoumarin (Table 2) are decreased in lungs of STZ-diabetic male and female rats, agreeing with previously reported observations (Stohs et al., 1979: AI-Turk et al., 1980a). Therefore, the mixed function oxidases from the lung tissue are regulated by pancreatic function. G o n a d e c t o m y of male and female rats has no effect on p u l m o n a r y A H H and E C D activities (AI-Turk et al., 1980b), while STZ-induced diabetes results in a decrease in the activities of A H H (Table l) and E C D (Table 2) activities in both sexes. Substrate-dependent differences occurred when animals are castrated and diabetic. The c o m b i n a t i o n of diabetes and castration of both sexes results in pulmonary E C D activities that are lower than the activities of diabetic or control animals (Table 2). However, pulmonary A H H activities (Table l) of diabetic-castrated male and female animals are lower than the activities of control animals, but not lower than the activities of diabetic animals. Castration has no effect on the A H H and E C D activities of intestinal microsomes from male and female rats (AI-Turk et al., 1980b), while in the diabetic state the activities of the two enzymes are increased in both sexes as compared to control animals (Tables 1 and 21. In diabetic-castrated animals, substrate-dependent responses in A H H and E C D activities of intestinal microsomes are observed. The activities of intestinal E C D in castrated-diabetic male and female rats do not differ significantly from the activities in diabetic animals (Table 2L However, in diabetic-gonadectomized male and female rats, intestinal A H H activities are decreased as compared to the activities of diabetic animals, but were still significantly higher than the control values. Therefore, the results suggest that an interrelationship may exist in the regulation of intestinal A H H activity by pancreatic and gonadal hormones. In addition, nutritional status is k n o w n to influence A H H activity of intestinal microsomes (Burke & Orrenius, 1979; Vainio & Hietanen, 1980L and food intake and absorption after the various interventions may contribute to the results which were observed. SUMMARY The results of these investigations support the concept of androgen-dependent mechanisms in hepatic metabolism in the male rat. Substrate dependent differences occur in intestinal and p u l m o n a r y metabolism in diabetic-gonadectomized animals. Shamoperations on control and diabetic rats had no effect on the A H H and E C D activities of liver, lung and intestinal mucosa. Alterations in xenobiotic metabolism as the result of an induced diabetic state in rats are dependent u p o n the substrate, the tissue examined, and the sex of the animals. Acknowledgements--Financial support was provided W. A. T. by a Blanche Widaman Fellowship. We thank Mrs Judy Williams for technical assistance.
349
REFERENCES ACKERMAN M. D. & LEIBMAN K. C. (1977) Effect of experimental diabetes on drug metabolism in the rat. Dru9 Metab. Dispos. 5, 405-410. AL-TURK W. A., SroHs S. J. & ROCHE E. B. (1980a) Altered metabolism of 7-ethoxycoumarin by hepatic, pulmonary and intestinal microsomes from streptozotocin-diabetic rats. Druy Metab. Dispos. 8, 44-45. AL-TURK W. A., STOHS S. J. & ROCHE E. B. (1980b) Effect of gonadectomy on hepatic, lung and intestinal mixed function monooxygenase enzymes in the rat. Horm. Metab. Res. 12, 404-409. BURKE M. D. & ORRr.NIUS S. (19791 Isolation and comparison of endoplasmic reticulum membranes and their mixed function oxidase activities from mammalian extrahepatic tissues. Pharmac. Therap. 7, 549-600. CAPDEVILA J., JAKOBSSONS. W., JERNSTR6M B., HELIA O. & ORRENIUS S. (1975) Characterization of a rat lung microsomal fraction obtained by scpharose 2B ultrafiltration. Cancer Res. 35, 2820 2829. DEHNEN W., TIMINGAS R. & RODS J. (1973) A modified method for the assay of benzo(a)pyrenc hydroxylase. Anal. Biochem. 53, 378-383. DIXON R. L., HART L. G. & FOUTS J. R. {1961) The metabolism of drugs by liver microsomes from alloxan-diabetic rats. J. Pharmac. exp. Thee. 133, 7-11. GREENLEE W. F. d~. POLAND A. (1978) An improved assay of 7-ethoxycoumarin 0-deethylase activity: Induction of hepatic enzyme activity in C57BL/6J and DBA/2J mice by phenobarbital, 3-methylcholanthrene and 2,3,7,8tetrachlorodibenzo-p-dioxin. J. Pharmac. exp. Thee. 205, 596-605. HIE'rANEN E. (1980) Modification of hepatic drug metabolizing enzyme activities and their induction by dietary protein. Gen. Pharmac. I 1,443-450. HIETANEN E., KOIVUSAARI U., LAITINEN M. & NORLING A. (1980) Hepatic drug metabolism during ethanol ingestion in riboflavin deficient rats. Toxicology 16, 103-111. KATO R. & GXLLErTE J. R. (1963) Sex differences in the effects of starvation, alloxan diabetes and morphine on the metabolism of drugs by rat liver microsomes. Pharmacoloyist 5, 240. KATO R. 8z GILLETTE J. R. (1965) Sex differences in the effects of abnormal physiological states on the metabolism of drugs by rat liver microsomes. J. Pharmac. exp. Thee. 150, 285-291. KAro R. & TAKAHASHIA. (1969) Effect of phenobarbital on the activities of drug metabolizing enzymes and electron transport system of liver microsomes in alloxan diabetes and thyroxine treated rats. Jap. J. Pharmac. 19, 45-52. KATO R., ONODA K. & TAKANAKAA. (1970) Species differences in drug metabolism by liver microsomes in alloxan diabetic or fasted animals (I) The activities of drug metabolizing enzymes and electron transport system. ,lap. J. Pharmac. 20, 546 561. KATO R., TAKANAKAA. & ONODA K. (1971) Effect of adrenalectomy or alloxan diabetes on the substrate interaction with cytochrome P-450 in the oxidation of drugs by liver microsomes. Biochem. Pharmac. 20, 447-458. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. MARSHALL W. J. (1971) Role of steroid hormones in hepatic microsomal enzyme induction. Biochem. Pharmac. 20, 1723-1725. OMURA T. & SATO R. (1964) The carbon monoxide-binding pigment of liver microsomes. 1. Evidence for its hemoprotein nature. J. biol. Chem. 239, 2320-2378. REINKE L. A., STOHS S. J. R, ROSENBERG H. (1978) Altered activity of hepatic mixed function monooxygenase enzymes in streptozotocin-induced diabetic rats. Xenobiotica g, 611-,-619. RE1NKE L. A., STOHS S. J. & ROSENBERGH. (1979) Increased
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0306-3623/81/090345-O9502.00/0 Copyright O 1981 Pergamon Press Ltd
Printed in Great Britain. All rights reserved
ACTIVITIES OF HEPATIC AND EXTRAHEPATIC MICROSOMAL MIXED FUNCTION OXIDASE ENZYMES IN DIABETIC AND GONADECTOMIZED-DIABETIC RATS W A L I D A. A L - T U R K , SIDNEY J. STOHS a n d E D W A R D B. ROCHE
Department of Biomedicinal Chemistry, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68105, U.S.A. IReceired 25 February 1981)
Abstract--1. Streptozotocin (STZ~-induced diabetes results in a decrease in hepatic aryl hydrocarbon hydroxylase (AHH) activity in male rats and an increase in female rats, while hepatic 7-ethoxycoumarin O-deethylase (ECD) activity is increased in both sexes. 2. In castrated male rats, induction of the diabetic state resulted in a further decrease in hepatic AHH activity. In diabetic female rats, ovariectomy did not alter hepatic AHH or ECD activities. 3. STZ-induced diabetes produced an increase in hepatic cytochrome P-450 content. The diabetic state failed to produce an increase in hepatic cytochrome P-450 content of castrated male animals, and only a small increase in castrated female rats. 4. AHH and ECD activities are decreased in lungs of STZ-induced diabetic male and female rats. In diabetic-castrated rats a further decrease in pulmonary ECD activity occurs in both sexes, and in the AHH activity of female rats. 5. The induction of diabetes with STZ increased intestinal AHH and ECD activities in both sexes. In diabetic-castrated rats, intestinal ECD activities were identical to the activities in diabetic male and female animals, while intestinal AHH activities were lower than the activities in diabetic animals but still significantly higher than in control animals of both sexes.
INTRODUCTION Experimentally induced diabetes has been shown to alter the hepatic microsomal metabolism of some xenobiotics. Kato & Gillette (1963, 1965) observed that liver microsomal hexobarbital hydroxylase and aminopyrine N-demethylase were depressed in alloxan-diabetic male rats, whereas the activities were increased in diabetic female rats. Aniline hydroxylase was increased in both male and female alloxan-diabetic rats. The activity of zoxazolamine hydroxylase was not altered in diabetic males, but increased significantly in diabetic female rats (Kato & Gillette, 1963, 1965). The metabolism of hexobarbital and aminopyrine were not depressed in castrated male and female rats by alloxan-induced diabetes, but the metabolism of these substrates was depressed in castrated-diabetic male and female rats receiving methyltestosterone. From these findings Kato & Gillette (1965) suggested that diabetes impaired the action of androgens to increase the sex-dependent activity of drug metabolizing enzymes. We have found that liver microsomal aryl hydrocarbon hydroxylase (AHH) and aminopyrine N-demethylase are depressed in streptozotocin (STZ)diabetic male rats, whereas the activities are increased in diabetic female rats (Reinke et al., 1978, 1979; Stohs et al., 1979). Aniline hydroxylase was also increased in both male and female rats with STZinduced diabetes (Reinke et al., 1978). The above metabolic alterations in streptozotocin diabetic rats were reversed by treating with insulin (Reinke et al., 1978, 1979). We have also observed that liver and
intestinal microsomal 7-ethoxycoumarin 0-deethylase (ECD) activities are increased in STZ diabetic male and female rats, whereas the lung activity is depressed in both sexes (Al-Turk et al., 1980a). Hepatic microsomal cytochrome P-450 content has been reported to increase as a result of STZ-induced diabetes in both male and female rats (Reinke et al., 1978, 1979), and is increased in alloxan-induced diabetic female rats but remains unaltered in alloxandiabetic male rats (Ackerman & Leibman, 1977; Kato et al., 1970). The effects of gonadectomy on the mixed function monooxygenase enzymes of experimentally induced diabetic animals has received little attention. In order to determine the influence of other hormones on the alterations of xenobiotic metabolism which occur in diabetic animals, we have examined the effects of gonadectomy on hepatic and extrahepatic microsomai aryl hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin 0-deethylase (ECD) activities in streptozotocin-induced diabetic male and female rats. MATERIALS AND METHODS Animals and Treatment
Male and female rats, age 40-45 days, of a SpragueDawley derived strain (Sasco Inc., Omaha, NE, USA) w e r e individually caged and allowed free access to water and Purina lab chow for at least 3-5 days before experimental use. The animals were maintained at a temperature of 21~C, with lighting from 0600 to 1800 hr daily. Male rats were castrated by scrotal incision, and female rats were ovariectomized by dorsal incision. All control 345
346
WAI,n) A. At.-Tt:RK ¢'t ul.
animals were sham-operatcd. Four groups of animals of each sex were utilized: control, sham-operated control, diabetic and castrated-diabetic animals. Diabetes was induced by a single tail vein injection of streptozotocin (STZ). 60 mg kg. freshly prepared in 0.05 M citrate buffer, pH 4.5, as previously described (Reinke (,t ,I., 1978). After four days, animals excreting at least 0.5?. glucose in the urine. as detcrmined by Tes-Tapc (Eli Lilly Co.. Indianapolis, Inc.), werc judged to be diabetic. The animals were killed 10 days after S T Z administration. The diabetic state was confirmed by a blood glucosc level of at least 200mgl00ml as determined by the glucose oxidase method (Sigma Chemical Co.. St. Louis. MO). Micro,some Prepto'ation
Liver microsomes were prepared in 1.15",. KCI plus 0.05 M Tris buffer, pII 7.4. by diffcrential centrifugation as previously described [Stohs et u/.. 1971). Preparation of intestinal microsomes was accomplished by the method of Stohs ct ul., (1976). ]'he upper villous layer of the mucosa was homogenized in a pit 7.8 "l-ris KCI buffer (0.05 M Tris chloride in 1.15",, KCI) containing trypsin inhibitor 15 m g g wet weight of small intestine), glycerol (20% v v final concentrationl and heparin (3 units:ml) to decrease agglutination and degradation of the monooxygenase system (Stohs et ul.. 1976). The washed microsomal pellet was resuspended to a concentration of approximately 2.0 mg microsomal proteinml in the Tris-KCI buffer, pH 7.8. Lung microsomes were isolated by a modification of the procedure of Capdevila et ul.. (1975). [ h e lungs were perfused in situ with 30ml of ice-cold normal saline, removed, weighed, blotted and minced. Homogenates werc prepared in a pH 7.6 Tris-KCI buffer 10.05 M Tris chloride in 1.15".. KCI) containing glycerol (10".. ,, vl. The v,ashed microsomes were resuspendcd to a tinal concentration of approximately 3.0 mg of microsomal protein ml in the ptt 7.6 Tris-KCI buffer. A s s a y Procedure.s
Incubations for determination of ary[ hydrocarbon hydroxylase [AHH) activities were conducted in 25 ml Edenmeyer flasks containing 1.06/~mole NADP +. 5.37~mole trisodium isocitrate, 0.05 ml of 0.10 M MgCI2, 0.88 units isocitrate dehydrogenase (all purchased from Sigma Chemical Co.. St. Louis, MO, USA) and approximately 0.5 mg of liver or intestinal microsomal protein or 1.0 mg of lung microsomal protein, in a tinal volume of 1.0 ml. After a 2 rain preincubation at 37 C. the incubations were initiated by the addition of 25 td of a 6.0 mM solution of benzoIa)pyrene in acetone. Thc reactions were conducted in air in an Eberbach shaker, at 75 oscillations:rain, exposed to yellow light. After 5 min, the reactions were terminated by the addition of 0.30 ml of triethylamineTriton X-100 (9:1), follov,ed by 2.0ml of aqueous 0.05". EDTA solution. AHH activity was determined by the assay procedure of Dehnen et al. 11973) on an Aminco-Bowman spectrophotofluorometer. One AHH unit is defined as the amount of enzyme which catalyzes the formation of 1.0 nmole of product:min/mg microsomal protein, using 3-hydroxybenzo(a)pyrene as the reference standard. 7-Ethoxycoumarin 0-deethylase IECD) activity was assayed in 25ml Erlenmeyer flasks, containing the NADPH-generating system described above, and approximately 1.0 mg of lung or intestinal microsomal protein or 0.5 mg of liver microsomal protein, in a final volume of 1.0 ml. After a 2 min preincubation at 37 C, the incubations were initiated by the addition of lO01tl of a 0.10 M solution of 7-ethoxycoumarin prepared in the respective buffers used for isolation of the microsomal fractions. The reactions were conducted in air in an Eberbach shaker, at 75 oscillations/min. After 10 min, the reactions were terminated by the addition of 0.125ml of 15°. Iw/v) trichloroacetic acid. The extractions and 7-ethoxycoumarin
0-deethylase assays were performed according to the procedures of Greenlec & Poland (1978) on an Aminco Bowman spectrophotofluorometer. The quantity of 7hydroxycoumarin formed was calculated by comparing the net fluorescence obscr',ed (sample minus blankl in the final alkaline extract with standard solution of 7-hydroxy coumarin. One ECD unit is defined as the amount of enzyme which catalyzes the formation of 1.0 nmole of product min mg microsomal protein. Cytochrome P-450 content was determined by the method of Omura & Sara (1964). Protein analyses were performed on the microsomal suspensions used in each assay (Lowry et ul., 1951). All absorption measurements were conducted ~ith a Beckman Acta M-VI Spectrophotometcr. "Stuti,~tical M e t h o d ~
Each value represents the mean + SD of 4 animals. The data was subjected to one-way analysis of variance (ANOVA) using Scheffe's S method, and mean values with P < 0.05 were considered significantly different.
RESUI.'IS The effects of STZ-induced diabetes on liver, lung and intestinal microsomal metabolism of benzo(a)pyrene in control and gonadectomized male and female rats are presented in Table 1. The sham operations on control and diabetic animals had no effect on AHH activities in microsomes of the three tissues. The diabetic state resulted in a 50", reduction in hepatic AHH activity in male rats, and was further significantly reduced in castrated animals. In female rats. the diabetic state resulted in a significant increase in hepatic AHH activity in both control and ovariectomized animals (Table 1). In the lung. STZ-induced diabetes significantly decreased AHH activity in both control and castrated male and female rats. In the intestine, both male and female diabetic rats had signilicantly higher AHH activities than the control animals (Table 1). In both sexes, the diabetic-castrated rats had increased AHH activities as compared to the controls, but the increase were not as high as the increases produced in the diabetic animals. The effects of STZ-induced diabetes on the hepatic. pulmonary and intestinal metabolism of 7-ethoxycoumarin in gonadectomized male and female rats is presented in Table 2. Sham operations again had no effect on ECD activities of microsomes from the three tissues. 7-Ethoxycoumarin 0-deethylase activity (ECD) in hepatic microsomes from diabetic male and female rats was increased approximately by 4Y'o and 80'!0. respectively, over control values. In the castrated male but not the female animals. STZ-induced diabetes failed to increase hepatic ECD activity. In the lung. ECD activity was significantly decreased as a result of STZ-induced diabetes in both male and female rats. The diabetic state in both castrated male and female rats resulted in a further decrease in pulmonary ECD activity as compared to the control animals. The induction of the diabctic state by STZ in male and female rats produced a significant increase in intestinal ECD activity as compared to control animals, similar to the results found in the liver (Table 2). In castrated animals of both sexes. STZ-induced dia-
5.23 5.44 2.82 2.62 1.41
+ + + + +
0.41 0.61 0.42 ~ 0.52" 0.14 ''h
Male 0.77 0.76 1.14 1.13 0.98
+ + + + +
0.07 0.13 0.08" 0.06" 0.09"
Female
Liver (Unitsj
51.2 54.2 38.3 37.9 42.0
~- 4.4 + 5.6 + 3.1J + 2.7 ~ + 5.3 ~
Male 47.8 48.9 18.4 17.8 28.9
+ + + + +
4.6 4.6 2.1 ~ 2.0 ~ 3.5 ~'h
Female
Lungs IMilliunitsl
51.4 50.7 142.4 139.2 75.1
± + ± + ±
Male 6.2 3.4 22.3' 21.5 ~ 11.5""
32.3 32.1 64.6 64.0 44.4
+ + + + +
3.5 4.6 11.8 ~ 10.9" 4.6 ~'"
Female
Intestine (MilliunitsJ
96.2 93.1 163.6 165.8 91.1
± 5.2 +_ 4.1 _+ 1(I.5' _+ 11.4 ~ + 2.7"
Male
Li',cr
84.3 80.5 175.4 178.4 180.2
+_ 4.6 _+ 3.9 L 12.4' + 13.8" + 10.9'
Female 34.7 33.5 25.6 27.0 17.5
+ 4.2 + 4.3 ± 3.3" + 3.6 J +_ 2.1 ''h
Male
Lung
36.4 36.1 26.2 26.3 10.8
_+ 3.9 + 3.1 ± 3.4 ~ + 3.2 ~ + 2.6""
Female
2.5 2.6 7.5 7.6 6.9
_+ 0.3 _+ 0.4 i 2.(P + 2.2 ~ + 1.9"
Male
Intestine
1.9 1.9 9.2 10.2 10.8
L + + + +
0.6 0.8 2.5 ~ 3.7" 2.6 ~
Female
A n i m a l s received STZ, 60 mg kg. i.v.. or the 0.05 M citrate buffer, pH 4.5. vehicle one day after surgery and 10 days prior to sacritice. Values are the m e a n s of 4 a n i m a l s _+SD. JP < 0.05 with respect to c o r r e s p o n d i n g c o n t r o l groups, bp < 0.05 with respect to d i a b e t i c animals.
Control C o n t r o l - s h a m operated Diabetic Diabetic-sham operated Diabetic-castrated
Experimental groups
7 - E t h o x } c o u m a r i n 0-deethylase ( E C D ) activity (units)
T a b l e 2. 7-Ethox~,coumarin 0-dccthylasc activity of liver, lung and intestinal m u c o s a from control, d i a b e t i c and d i a b e t i c - c a s t r a t e d male and female rats
Animals received S'IZ, 60 mg. kg, i.v., or the 0.05 M citrate buffer, pH 4.5. vehicle one day after surgery and 10 da}s p r i o r to sacritice. Values are the means of 4 a n i m a l s + SD. ~P < 0.05 with respect to c o r r e s p o n d i n g control groups, r,p < 0.05 with respect .t.o the d i a b e t i c groups.
Control Control-sham operated Diabetic D i a b e t i c - s h a m operated Diabetic-castrated
|:xperimcntal groups
Aryl I i y d r o c a r b o n H y d r o x y l a s e { A H H I Activity
T a b l e 1. Aryl h y d r o c a r b o n h y d r o x y l a s c activity of liver, lung and intestinal m u c o s a from control, diabetic, and d i a b e t i c - c a s t r a t e d male and female rats
g-
G X
3
348
WALIDA. AL-TURKet ul. Table 3. Cytochrome P-450 content of liver microsomes from control, diabetic, and diabetic-castrated male and female rats Cytochrome P-450 Content (nmol/mg microsomal protein) Experimental groups Control Control-sham operated Diabetic Diabetic-sham operated Diabetic-castrated
Male 1.07 + 0.20 1.09 + 0.21 1.60 + 0.09~ 1.66 _+0.04~ 1.08 _+0.17h
Female 0.87 + 0.06 0.90 + 0.05 1.39 _+0.08~ 1.46 __.0.07° 1.07 + 0.06~'b
Animals received STZ, 60 mg/kg, i.v., or the 0.05 M citrate buffer, pH 4.5, vehicle 1 day after surgery and 10 days prior to sacrifice. Values are the means of 4 animals + SD. JP < 0.05 with respect to control animals. "P < 0.05 with respect to diabetic animals.
betes produced an increase in intestinal ECD that was similar to the response observed with diabetes alone. The effects of castration on the hepatic cytochrome P-450 content of STZ-induced diabetic male and female rats are shown in Table 3. Sham operations on control and diabetic animals had no effect on hepatic cytochrome P-450 content. An increase in cytochrome P-450 content occurred as a result of STZ-induced diabetes in rats, agreeing with previously reported observations (Reinke et al., 1978). However, the diabetic state failed to increase the hepatic cytochrome P-450 content in castrated male rats, and produced only a small increase in cytochrome P-450 content in ovariectomized female rats. DISCUSSION Alterations observed in xenobiotic metabolism as the result of an induced diabetic state in experimental animals are dependent upon the substrate, the tissue examined and the sex of the animals (Reinke et al., 1978; 1979; Stohs et al., 1979; AI-Turk et al., 1980a). Some of these metabolic changes may reflect interference of the diabetic state with steroid-hormone dependent metabolic pathways (Kato & Gillette, 1965). We have therefore examined the metabolism of benzo(a)pyrene and 7-ethoxycoumarin in gonadectomized STZ-diabetic male and female rats. The responses of the AHH and ECD enzymes in liver, lung and intestinal microsomes of diabetic male and female rats which have been gonadectomized are markedly different. The results in Tables 1 and 2 agree with the previously reported observations that STZ-induced diabetes results in a decrease in hepatic AHH activity in male rats (Reinke et al., 19791 and an increase in female rats, while hepatic ECD activity is increased in both male and female rats (Al-Turk et al,, 1980a). The diabetic state in the castrated male rats resulted in a further decrease in the activity of AHH in the liver as compared to the hepatic AHH activity of diabetic male rats which had not been gonadectomized (Table 1) while the hepatic ECD activity was not affected by diabetes in castrated male animals (Table 2). Therefore, androgens do exert a regulatory role on hepatic mixed function oxidases in the diabetic male rat. Several recent investigations have shown that hepatic ECD and AHH activities respond differentially to dietary factors tHietanen, 1980; Hie-
tanen et al., 1980), and therefore lack of agreement in the responses of the two enzymes may be expected. In diabetic female rats, ovariectomy did not alter either ECD or AHH activities in liver. Castration of male rats but not female rats decreased hepatic mixed function oxidase activity in these diabetic rats. The hepatic metabolism of both substrates is dependent on the production of androgens by the testes but not on the production of estrogens by the ovaries (Marshall, 1971; AI-Turk, et al., 1980b). In the diabetic male rat, an apparent inconsistency exists in the form of a decreased metabolism of aminopyrine, hexobarbital, codeine, chlorpromazine and phenylbutazone (Kato & Gillette, 1963, 1965; Dixon et al., 1961; Weiner et al., 1970), as well as benzo(a}pyrene in the presence of increased levels of cytochrome P-450 in hepatic microsomes. A decreased binding of aminopyrine and hexobarbital to cytochrome P-450 in hepatic microsomes from diabetic male rats has been reported (Kato et al., 1971; Kato & Takahashi, 1969), while the binding of aniline was slightly increased (Kato & Takahashi, 1969). The binding of certain substrates to cytochrome P-450 is itself an androgen-dependent phenomenon in the rat (Kato et al., 1971), since castration markedly decreased the binding of substrates such as aminopyrine and hexobarbital, but not substrates such as aniline and zoxazolamine. Therefore, the interference in microsomal metabolism produced by the diabetic state may be the result of a decreased ability of cytochrome P-450 to bind to those substrates metabolized by androgen-dependent pathways, and may therefore not be determined by the absolute amount of cytochrome P-450 present. Another possible explanation lies with the known multiplicity of microsomal hemoproteins (Ryan et al., 1979}. The metabolism of aniline and aminopyrine as well as benzo~aJpyrene and 7-ethoxycoumarin are thought to be mediated by separate cytochrome P-450 forms (Kato & Takahashi, 1969; Ryan et al., 1979; Weiner et al., 1972), and the diabetic state may favor the formation of a specific cytochrome form, thus favoring enzymes as AHH and ECD. In male and female rats, hepatic ECD activity is increased by diabetes (AI-Turk et al., 1980a), and decreased only by castration and not by ovariectomy (AI-Turk et al., 1980b). In the castrated-diabetic male rat, the two effects counteract each other (Table 2). The results indicate that the hepatic metabolism of
Activities of hepatic mixed function oxidase enzymes in diabetic rats both A H H and E C D are regulated by pancreatic and androgenic functions, and suggest that the diabetic state does not interfere with the metabolic control exerted by androgens on hepatic mixed function oxidases. The microsomal metabolism of b o t h benzo(a)pyrene (Table l) and 7-ethoxycoumarin (Table 2) are decreased in lungs of STZ-diabetic male and female rats, agreeing with previously reported observations (Stohs et al., 1979: AI-Turk et al., 1980a). Therefore, the mixed function oxidases from the lung tissue are regulated by pancreatic function. G o n a d e c t o m y of male and female rats has no effect on p u l m o n a r y A H H and E C D activities (AI-Turk et al., 1980b), while STZ-induced diabetes results in a decrease in the activities of A H H (Table l) and E C D (Table 2) activities in both sexes. Substrate-dependent differences occurred when animals are castrated and diabetic. The c o m b i n a t i o n of diabetes and castration of both sexes results in pulmonary E C D activities that are lower than the activities of diabetic or control animals (Table 2). However, pulmonary A H H activities (Table l) of diabetic-castrated male and female animals are lower than the activities of control animals, but not lower than the activities of diabetic animals. Castration has no effect on the A H H and E C D activities of intestinal microsomes from male and female rats (AI-Turk et al., 1980b), while in the diabetic state the activities of the two enzymes are increased in both sexes as compared to control animals (Tables 1 and 21. In diabetic-castrated animals, substrate-dependent responses in A H H and E C D activities of intestinal microsomes are observed. The activities of intestinal E C D in castrated-diabetic male and female rats do not differ significantly from the activities in diabetic animals (Table 2L However, in diabetic-gonadectomized male and female rats, intestinal A H H activities are decreased as compared to the activities of diabetic animals, but were still significantly higher than the control values. Therefore, the results suggest that an interrelationship may exist in the regulation of intestinal A H H activity by pancreatic and gonadal hormones. In addition, nutritional status is k n o w n to influence A H H activity of intestinal microsomes (Burke & Orrenius, 1979; Vainio & Hietanen, 1980L and food intake and absorption after the various interventions may contribute to the results which were observed. SUMMARY The results of these investigations support the concept of androgen-dependent mechanisms in hepatic metabolism in the male rat. Substrate dependent differences occur in intestinal and p u l m o n a r y metabolism in diabetic-gonadectomized animals. Shamoperations on control and diabetic rats had no effect on the A H H and E C D activities of liver, lung and intestinal mucosa. Alterations in xenobiotic metabolism as the result of an induced diabetic state in rats are dependent u p o n the substrate, the tissue examined, and the sex of the animals. Acknowledgements--Financial support was provided W. A. T. by a Blanche Widaman Fellowship. We thank Mrs Judy Williams for technical assistance.
349
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