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Hepatic mixed-function oxidase activity in mice infected with Trypanosoma brucei gambiense or treated with trypanocides
Molecular and Biochemical Parasitology, 3 (1981) 199-204 Elsevier/North-Holland Biomedical Press
199
HEPATIC MIXED-FUNCTION OXIDASE ACTIVITY IN MICE INFECTED WITH TR YPANOSOMA BR UCEI G A M B I E N S E OR TREATED WITH TRYPANOCIDES
HOWARD G. SHERTZER 1, JAMES EDWIN HALL 2 and JOHN R. SEED 2 I Kettering Laboratory, University o f CincinnatiMedical Center, Cincinnati, OH 4526 7, and Department o f Biology, Texas A and M University, College Station, TX 77843, U.S.A. (Received 17 September 1980; accepted 19 December 1980)
Three days after mice were inoculated with Trypanosoma brucei gambiense, total hepatic eytochrome P-450 levels were decreased 14% from control values, while the specific mixed-function oxidase activity (per nmole of cytochrome P-450) was inhibited almost 40% in infected animals. Furthermore, drugs used to treat trypanosomiasis (Suramin and Melarsoprol B) are themselves potent in vitro inia~itors of hepatic mixed-function oxidase activity. These two trypanoeides also produced a decrease in mixed-function oxidations and an increase in pentobaxbital sleeping time when administered intraperitoneally in vivo. These results demonstrate that mice with trypanosomiasis or undergoing trypanosome chemotherapy have a significantly impaked capacity to metabolize foreign compounds. Key words: Trypanosoma brucei gambiense, Mice, Mixed-function oxidase, Cytochrome P-450, Suramin, Melarsoprol, Liver.
INTRODUCTION Lumsden et al. [1] reported various cytopathologic alterations in the rough and smooth endoplasmic reticulum o f hepatocytes from trypanosome-infected guinea pigs. These findings suggested to us that hepatic cytochrome P-450 and associated mixedfunction oxidase activity, localized in the microsomal membrane, m a y be affected during trypanosomiasis. In order to assess the acute hepatic effects o f trypanosome infection on monooxygenase activity, we used a laboratory mammal, the white mouse, that exhibits fulminating parasitemia about 3 days after inoculation. We also used this mouse model to assess the effects o f the commonly used trypanocides, Suramin and Melarsoprol, on drug metabolism. MATERIALS AND METHODS Materials. Male Texas ICR Swiss randomly bred mice were obtained from TIMCO, Houston, TX. Animals were maintained on standard Purina Chow and provided with water
0166-6851/81/0000-0000/$02.50 O Elsevier/North-Holland Biomedical Press
200 ad libitum. Glucose 6-phosphate, NADP÷, glucose-6-phosphate dehydrogenase, bovine serum albumin, aniline, anthracene, p-nitroanisole, p-aminophenol, benzo(a)pyrene and p-nitrophenol were obtained from Sigma Chemical Co., St. Louis, MO. Sodium pentobarbital (65 mg/ml) was obtained from W.A. Butler Co., Columbus, OH. Na Suramin (8 ~'-[carbonylbis[imino-3,1 -phenylenecarbonylimino (4-methyl-3,1-phenylene)carbonylimino]]bis-l,3,5-naphthalenetrisulfonic acid hexasodium salt), USP quality, was from Mobay Chemical Corp., FBA Pharamaceuticals, 425 Park Place, New York, NY. Melarsoprol (2.[4-[(4,6-diamino- 1,3,5-triazin-2-yl)amino]phenyl]-I ,3,2-dithiarsolane-4methanol) was kindly supplied by LTC, VC, Dr. David E. Davidson, Department of Biology, Walter Reed Army Institute of Research. The Melarsoprol was Walter Reed No. WR 216692.
Methods. Mice were housed on Beta Chip hardwood bedding (Northeastern Product Corp., Warrensburg, NY). Animals weighing about 27 g were inoculated intraperitoneaUy with 5 × 104 trypanosomes. A cloned strain of the Wellcome TS strain of Trypanosoma brucei gambiense was used. After 72 h the animals were sacrificed by cervical dislocation, trypanosomes in the blood were quantified by hemocytometer counts, and the livers were excised and placed in 0.9% (w/v) NaC1 at 0°C. The tissue was then washed, trimmed, blotted at 0°C, weighed, and then homogenized (15%, w/v) using a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenization buffer consisted of 250 mM sucrose, 1 mM EDTA, 154 mM KC1, and 100 mM HEPES/KOH (pH 7.6). The whole homogenate was used to determine all of the hepatic parameters described in this paper. Protein was assayed according to Lowry et al. [2], cytochrome P-450 according to Johannesen and DePierre [3] and DNA by the method of Burton [4]. Lipids were quantiffed gravimetrically after Soxhlet extraction in chloroform/methanol (2:1). Mixedfunction oxidase activities were assayed essentially as described previously [5, 6], with a modified assay medium for the metabolism of aniline, p-nitroanisole, anthracene or benzo(a)pyrene. The modified medium contained 1 mM NADP+, 10 mM glucose 6phosphate, 2 units/ml glucose-6-phosphate dehydrogenase, 10 mM MgC12, 10/aM MnC12, 10 mM glucose, about 0.5 nmol cytochrome P-450/ml, and 100 mM HEPES/KOH buffer, pH 7.6. The reactions were started by the addition of substrate: 10 mM aniline, 1 mM p-nitroanisole, or 100 /aM anthracene or benzo(a)pyrene. All of the enzymatic activities assayed in this paper were linear with respect to protein and time under the assay conditions specified. Pentobarbital sleeping times were analyzed as follows. Mice were injected i.p. at 10-11 a.na. with 40 mg/kg body weight of a sterile solution (65 mg/ml) of sodium pentobarbital in 30% propylene glycol and 2% benzyl alcohol. Sleep time commenced when the animal lost its righting reflex (3-5 min) and ceased when the animal righted itself twice within 10 s. The length of sleep is expressed in Table IV. Due to extreme animal variability in sleeping times, values were initially determined on a large group of mice. Only those mice with sleeping times between 80 and 100 rain were used for these experiments. Injections with trypanocides were begun 2 weeks after this screening procedure.
201 RESULTS AND DISCUSSION Table I shows that parasitemia in three day trypanosome infected mice was very high. Although total hepatic protein increased slightly, total tissue DNA decreased 25% and DNA per g liver wet weight decreased over 40% in infected mice. These data suggest that a loss in cellularity with a concomitant influx o f non-cellular protein occurred during the acute infection. A loss in total cell numbers in the liver would also explain the 30% decrease in cytochrome P-450 levels per g liver wet weight. However, when hepatic cytochrome P-450 values are expressed per mg DNA, there is a significant 18% increase produced by the trypanosome infection. This increase in the amount of cytochrome P-450 per cell may be the result o f induction o f cytochrome P-450 synthesis. Potential inducing agents for cytochrome P-450 synthesis are certain indoles that are secreted b y trypanosomes during the course o f infection [7]. Indoles have been shown previously to interact with rabbit hepatic cytochrome P-450 [8] and to be potent inducers ofmixed-func. tion oxidase activity in rat liver [9, 10]. Although total hepatic cytochrome P-450 was only slightly less in infected animals, total mixed-function oxidase activity was inhibited almost 50% (Table II). This was
TABLE I Hepatic alterations during acute trypanosomiasis in mice. Control a
Trypanosomes X 10 -s ml whole blood g body weight g liver wet weight kg body weight mg dry liver weight g wet liver weight mg total liver lipids g wet liver weight mg protein g liver wet weight mg DNA g liver wet weight nmol cytochrome P-450 g liver wet weight nmol cytochrome P-450 mgDNA Total liver nmol P-450
Trypanosome infected a
0
Tryp/Control
5.2 -+ 0.9 (12)
-
29.6 -+ 1.2 (12)
27.9 + 0.6 (12)
0.94
52.7 +3.3
69.0 + 2.3 (12) b
1.31
234 16.0 155
(12)
+5
(5)
+4.0
(5)
-+
9
3.19 + 0.12
(12) (5)
205
+ 6
II.I + 2.7 138
+10
1.89 + 0.06
(12)
0.89
0.70
14.4 -+ 0.5 52.4
0.69
I.I (12) b
12.2 +-0.4
+- 2.2 (12)
(5)
0.59
27.2 +
60.7
0.88
(5)b
38.9 +-2.2 (12) (5)
(5)b
(5)b
1.18
+ 2.4 (12) b
0.86
a Values shown are means + S.E., with the numbers of experiments in parentheses. b Significant difference from control value at P
202 TABLE II Mixed-function oxidase activities in livers from control and trypanosome infected mice. a Substrate
Aniline PNA Anthracene
nmol/min/nmol P-450
nmollmin/liver
Control
Trypanosomeinfected
Control
1.81 ± 0.17 (15) b 2.00 ± 0.30 (15) 3.34 ± 0.32 (15)
1.10 ± 0.18 (17) c 1.23 ± 0.17 (16) c 2.13 ± 0.24 (16) c
105 ± 8 116 ± 9 194 ± 11
Trypanosomeinfected (15) (15) (15)
55 ± 5 (17) c 61 +- 4 (16) c 106 ± 12 (16) c
a Activities of aniline hydroxylation, p-nitroanisole (PNA) demethylation and anthracene oxidation are expressed as specific activities (per nmole cytochrome P-450, left) and total tissue activities (right). Values determined for benzo(a)pyrene oxidation (data not shown) were the same as those shown for anthracene oxidation. b Values shown are means ± S.E., with the number of experiments in parentheses. c Means are significantly different from control values at P<0.001 level using Student's t-test.
due primarily to an almost 40% decrease in the per nmole cytochrome P-450 metabolism o f each substrate tested. The substrates that were used included type I substrates (p-nitroanisole, anthracene, benzo(a)pyrene) and a type II substrate (aniline). The decrease in enzyme activity per nmole cytochrome P - 4 5 0 could not be attributed to a change in NADPH cytochrome c reductase activity. This value remained constant at 80 -+ 9 nmol cytochrome c reduced per min per mg protein. The consistent decrease in specific activity (activity per nmole cytochrome P-450) with each o f the substrates tested suggests that we are not observing a shift in the relative proportions of different forms o f cytochrome P-450. It is possible that we m a y be observing the effects of a nonspecific inhibitor o f hepatic cytochrome P-450 present in the microsomal fraction. However, if such an inhibitor exists, it must remain tightly b o u n d to the microsomal fraction because o f two lines o f evidence. First, the inhibition persisted in washed hepatic microsomes isolated from trypanosome infected animals. Second, the 105 000 X g supernatant o f liver whole homogenate from trypanosome infected mice had no effect on the mixed-function oxidase activity obtained using control animal hepatic microsomes (data not shown). In light o f this evidence, the mechanism responsible for the decrease in mixed-function oxidase activity per nmole cytochrome P-450 in trypanosome infected mice remains unclear. African sleeping sickness in humans is usually treated with Suramin during early infection before central nervous system involvement. The arsenical Melarsoprol (Mel B) is normally used for late infection after central nervous system involvement. The in vitro inhibition o f drug metabolism b y Suramin and Melarsoprol shown in Table III indicates that each o f these trypanocides are potential in vivo inhibitors o f hepatic mixed-function oxidations. In order to test the possibility that trypanocides themselves might inhibit endogenous mixed-function oxidase activity, we examined their effect on pentobarbital
203 TABLE III In vitro inhibition of mixed-function oxidase activities by trypanocides, a Enzyme activity
ug/ml to inhibit 50%
PNA demethylase Anilinehydroxylase BaP hydroxylase
Suramin
Melarsoprol
52 ± 4 (4) b 111 ± 7 (4) 43 ± 5 (4)
47 ± 6 (4) 99 ± 13 (4) 45 ± 6 (4)
a Suramin or Melarsoprol were added to the reaction mixture 1 min prior to adding substrate. The values shown are the mean concentrations of trypanocide necessary to produce a 5(~o inhibition of mixed-function oxidase activity for p-nitroanisole (PNA) demethylase, aniline hydroxylase or benzo(a)pyrene (BaP) hydroxylase. b The confidence limits shown are means ± S.E. with the number of experiments in parentheses. sleeping time in mice. Table IV indicates t h a t b i w e e k l y injections o f Suramin or Melarsorprol p r o d u c e d significant increases in sleeping times. A l t h o u g h it m a y be argued that t h e trypanocides p r o d u c e d a change in p e n t o b a r b i t a l c o m p a r t m e n t a t i o n w h e n injected, t h e m o r e likely e x p l a n a t i o n is t h a t t h e longer sleeping times were a result o f l o w e r rates o f p e n t o b a r b i t a l m e t a b o l i s m especially by the liver. Melarsoprol t r e a t m e n t clearly lowered hepatic c y t o c h r o m e P - 4 5 0 and aniline h y d r o x y l a s e activities ( d e t e r m i n e d in vitro), perhaps due to t h e arsenical nature o f this c o m p o u n d . The net result o f an almost 50% decrease in total hepatic m i x e d f u n c t i o n oxidase capacity and the p o t e n t i a l inhibition b y trypanocides should be considered in the doseTABLE IV Effects of trypanocide injection on parameters of mixed-function oxidations, a
Control(buffer) Suramin Control(sesame oil) Melarsoprol
PB sleep time (rain)
nmolP-450 per g liver wet weight
Aniline hydroxylase b
92 122 89 167
34.7 32.9 36.0 26.8
1.44 1.31 1.52 1.06
-+ 11 ± 14 ± 13 -+ 24
(14) c (15) d (15) (15) d
± 2.1 ± 2.7 ± 3.0 -+ 2.9
(12) (13) (11) (13) d
± 0.I ± 0.I ± 0.2 -+ 0.1
(12) (13) d (11) (13) d
a Mice were injected i.p. (10-11 a.m.) at 40 mg per kg body weight with either Suramin (12 mg/ml in 0.9% NaC1/25 mM sodium phosphate, pH 7.4) or Melarsoprol (12 mg/ml suspended by grinding in sesame oil). Controls consisted of injections with equal volumes of each vehicle. Injections were Friday and Tuesday of each week for 5 weeks. Animals were used the day after the last injection to determine the various parameters. All of the groups had body weight and liver wet weight/body weight that were the same as control values (34.1 -+ 1.3 and 48.1 ± 2.9 g/kg, respectively). b Expressed as nmoles p-aminophenol formed per min per nmole cytoehrome P-450. c Values shown are means -+S.E., with the number of experiments in parentheses. d Means are significantly different from control values a t P < 0.05 level using Student's t-test.
204 response effects of drugs used to treat patients or experimental animals infected with trypanosomes. This is because elevated levels of certain drugs that are normally metabolized by the mixed-function oxidase enzyme system may produce nonspecific or toxic effects in trypanosome infected animals and man. ACKNOWLEDGEMENTS Supported by NIH grants GM-25368 and GM-27928, and the Trypanosome Component of the UNDG/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. REFERENCES 1 2 3
4
5 6 7 8
9
10
Lumsden, R.D., Marciacq, Y. and Seed, J.R. (1972) Trypanosoma gambiense: Cytopathologic changes in guinea pig hepatocytes. Exp. Parasitol. 32,369-389. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265- 275. Johannesen, K.A.M. and DePierre, J.W. (1978) Measurement of cytochrome P-450 in the presence of large amounts of contaminating hemoglobin and myoglobin. Anal. Biochem. 86,725732. Burton, K. (1968) Determination of DNA concentration with diphenylamine. In: Methods in Enzymology, Vol. XIIB, (Grossman, L. and Moldave, K., eds.), pp. 168-166. Academic Press, New York. Duthu, G.S. and Shertzer, H.G. (1979) Effect of nitrite on rabbit liver mixed-function oxidase activity. Drug Metab. Dispos. 7, 263-269. Shertzer, H.G. and Duthu, G.S. (1979) Nitrite binding to rabbit liver microsomes and effects on aminopyrine demethylation. Biochem. Pharmacol. 28, 873-879. Tizard, I., Nielsen, K.H., Seed, J.R. and Hall, J.E. (1978) Biologically active products from African trypanosomes. Microbiol. Rev. 42,661-681. Shertzer, H.G. (1980) Effects of indole derivatives on mono-oxygenase activity in rabbit liver microsomes. In: Microsomes, Drug Oxidations and Chemical Carcinogenesis, Vol. 2, (Coon, M.J., Conney, A.H., Estabrook, R.W., Gelboin, H.V., Gillette, J.R. and O'Brien, P.J., eds.), pp. 889-892, Academic Press, New York. Loub, W.D., Wattenberg, L.W. and Davis, D.W. (1975) Aryl hydrocarbon hydroxylase induction in rat tissues by naturally occurring indoles of cruciferous plants. J. Natl. Cancer Inst. 54,985988. Babish, J.G. and Stoewsand, G.S. (1978) Effect of dietary indole-3-carbinol on the induction of the mixed-function oxidases of rat tissue. Food Cosmet. Toxicol. 16, 151-155.
199
HEPATIC MIXED-FUNCTION OXIDASE ACTIVITY IN MICE INFECTED WITH TR YPANOSOMA BR UCEI G A M B I E N S E OR TREATED WITH TRYPANOCIDES
HOWARD G. SHERTZER 1, JAMES EDWIN HALL 2 and JOHN R. SEED 2 I Kettering Laboratory, University o f CincinnatiMedical Center, Cincinnati, OH 4526 7, and Department o f Biology, Texas A and M University, College Station, TX 77843, U.S.A. (Received 17 September 1980; accepted 19 December 1980)
Three days after mice were inoculated with Trypanosoma brucei gambiense, total hepatic eytochrome P-450 levels were decreased 14% from control values, while the specific mixed-function oxidase activity (per nmole of cytochrome P-450) was inhibited almost 40% in infected animals. Furthermore, drugs used to treat trypanosomiasis (Suramin and Melarsoprol B) are themselves potent in vitro inia~itors of hepatic mixed-function oxidase activity. These two trypanoeides also produced a decrease in mixed-function oxidations and an increase in pentobaxbital sleeping time when administered intraperitoneally in vivo. These results demonstrate that mice with trypanosomiasis or undergoing trypanosome chemotherapy have a significantly impaked capacity to metabolize foreign compounds. Key words: Trypanosoma brucei gambiense, Mice, Mixed-function oxidase, Cytochrome P-450, Suramin, Melarsoprol, Liver.
INTRODUCTION Lumsden et al. [1] reported various cytopathologic alterations in the rough and smooth endoplasmic reticulum o f hepatocytes from trypanosome-infected guinea pigs. These findings suggested to us that hepatic cytochrome P-450 and associated mixedfunction oxidase activity, localized in the microsomal membrane, m a y be affected during trypanosomiasis. In order to assess the acute hepatic effects o f trypanosome infection on monooxygenase activity, we used a laboratory mammal, the white mouse, that exhibits fulminating parasitemia about 3 days after inoculation. We also used this mouse model to assess the effects o f the commonly used trypanocides, Suramin and Melarsoprol, on drug metabolism. MATERIALS AND METHODS Materials. Male Texas ICR Swiss randomly bred mice were obtained from TIMCO, Houston, TX. Animals were maintained on standard Purina Chow and provided with water
0166-6851/81/0000-0000/$02.50 O Elsevier/North-Holland Biomedical Press
200 ad libitum. Glucose 6-phosphate, NADP÷, glucose-6-phosphate dehydrogenase, bovine serum albumin, aniline, anthracene, p-nitroanisole, p-aminophenol, benzo(a)pyrene and p-nitrophenol were obtained from Sigma Chemical Co., St. Louis, MO. Sodium pentobarbital (65 mg/ml) was obtained from W.A. Butler Co., Columbus, OH. Na Suramin (8 ~'-[carbonylbis[imino-3,1 -phenylenecarbonylimino (4-methyl-3,1-phenylene)carbonylimino]]bis-l,3,5-naphthalenetrisulfonic acid hexasodium salt), USP quality, was from Mobay Chemical Corp., FBA Pharamaceuticals, 425 Park Place, New York, NY. Melarsoprol (2.[4-[(4,6-diamino- 1,3,5-triazin-2-yl)amino]phenyl]-I ,3,2-dithiarsolane-4methanol) was kindly supplied by LTC, VC, Dr. David E. Davidson, Department of Biology, Walter Reed Army Institute of Research. The Melarsoprol was Walter Reed No. WR 216692.
Methods. Mice were housed on Beta Chip hardwood bedding (Northeastern Product Corp., Warrensburg, NY). Animals weighing about 27 g were inoculated intraperitoneaUy with 5 × 104 trypanosomes. A cloned strain of the Wellcome TS strain of Trypanosoma brucei gambiense was used. After 72 h the animals were sacrificed by cervical dislocation, trypanosomes in the blood were quantified by hemocytometer counts, and the livers were excised and placed in 0.9% (w/v) NaC1 at 0°C. The tissue was then washed, trimmed, blotted at 0°C, weighed, and then homogenized (15%, w/v) using a Potter-Elvejhem homogenizer with a Teflon pestle. The homogenization buffer consisted of 250 mM sucrose, 1 mM EDTA, 154 mM KC1, and 100 mM HEPES/KOH (pH 7.6). The whole homogenate was used to determine all of the hepatic parameters described in this paper. Protein was assayed according to Lowry et al. [2], cytochrome P-450 according to Johannesen and DePierre [3] and DNA by the method of Burton [4]. Lipids were quantiffed gravimetrically after Soxhlet extraction in chloroform/methanol (2:1). Mixedfunction oxidase activities were assayed essentially as described previously [5, 6], with a modified assay medium for the metabolism of aniline, p-nitroanisole, anthracene or benzo(a)pyrene. The modified medium contained 1 mM NADP+, 10 mM glucose 6phosphate, 2 units/ml glucose-6-phosphate dehydrogenase, 10 mM MgC12, 10/aM MnC12, 10 mM glucose, about 0.5 nmol cytochrome P-450/ml, and 100 mM HEPES/KOH buffer, pH 7.6. The reactions were started by the addition of substrate: 10 mM aniline, 1 mM p-nitroanisole, or 100 /aM anthracene or benzo(a)pyrene. All of the enzymatic activities assayed in this paper were linear with respect to protein and time under the assay conditions specified. Pentobarbital sleeping times were analyzed as follows. Mice were injected i.p. at 10-11 a.na. with 40 mg/kg body weight of a sterile solution (65 mg/ml) of sodium pentobarbital in 30% propylene glycol and 2% benzyl alcohol. Sleep time commenced when the animal lost its righting reflex (3-5 min) and ceased when the animal righted itself twice within 10 s. The length of sleep is expressed in Table IV. Due to extreme animal variability in sleeping times, values were initially determined on a large group of mice. Only those mice with sleeping times between 80 and 100 rain were used for these experiments. Injections with trypanocides were begun 2 weeks after this screening procedure.
201 RESULTS AND DISCUSSION Table I shows that parasitemia in three day trypanosome infected mice was very high. Although total hepatic protein increased slightly, total tissue DNA decreased 25% and DNA per g liver wet weight decreased over 40% in infected mice. These data suggest that a loss in cellularity with a concomitant influx o f non-cellular protein occurred during the acute infection. A loss in total cell numbers in the liver would also explain the 30% decrease in cytochrome P-450 levels per g liver wet weight. However, when hepatic cytochrome P-450 values are expressed per mg DNA, there is a significant 18% increase produced by the trypanosome infection. This increase in the amount of cytochrome P-450 per cell may be the result o f induction o f cytochrome P-450 synthesis. Potential inducing agents for cytochrome P-450 synthesis are certain indoles that are secreted b y trypanosomes during the course o f infection [7]. Indoles have been shown previously to interact with rabbit hepatic cytochrome P-450 [8] and to be potent inducers ofmixed-func. tion oxidase activity in rat liver [9, 10]. Although total hepatic cytochrome P-450 was only slightly less in infected animals, total mixed-function oxidase activity was inhibited almost 50% (Table II). This was
TABLE I Hepatic alterations during acute trypanosomiasis in mice. Control a
Trypanosomes X 10 -s ml whole blood g body weight g liver wet weight kg body weight mg dry liver weight g wet liver weight mg total liver lipids g wet liver weight mg protein g liver wet weight mg DNA g liver wet weight nmol cytochrome P-450 g liver wet weight nmol cytochrome P-450 mgDNA Total liver nmol P-450
Trypanosome infected a
0
Tryp/Control
5.2 -+ 0.9 (12)
-
29.6 -+ 1.2 (12)
27.9 + 0.6 (12)
0.94
52.7 +3.3
69.0 + 2.3 (12) b
1.31
234 16.0 155
(12)
+5
(5)
+4.0
(5)
-+
9
3.19 + 0.12
(12) (5)
205
+ 6
II.I + 2.7 138
+10
1.89 + 0.06
(12)
0.89
0.70
14.4 -+ 0.5 52.4
0.69
I.I (12) b
12.2 +-0.4
+- 2.2 (12)
(5)
0.59
27.2 +
60.7
0.88
(5)b
38.9 +-2.2 (12) (5)
(5)b
(5)b
1.18
+ 2.4 (12) b
0.86
a Values shown are means + S.E., with the numbers of experiments in parentheses. b Significant difference from control value at P
202 TABLE II Mixed-function oxidase activities in livers from control and trypanosome infected mice. a Substrate
Aniline PNA Anthracene
nmol/min/nmol P-450
nmollmin/liver
Control
Trypanosomeinfected
Control
1.81 ± 0.17 (15) b 2.00 ± 0.30 (15) 3.34 ± 0.32 (15)
1.10 ± 0.18 (17) c 1.23 ± 0.17 (16) c 2.13 ± 0.24 (16) c
105 ± 8 116 ± 9 194 ± 11
Trypanosomeinfected (15) (15) (15)
55 ± 5 (17) c 61 +- 4 (16) c 106 ± 12 (16) c
a Activities of aniline hydroxylation, p-nitroanisole (PNA) demethylation and anthracene oxidation are expressed as specific activities (per nmole cytochrome P-450, left) and total tissue activities (right). Values determined for benzo(a)pyrene oxidation (data not shown) were the same as those shown for anthracene oxidation. b Values shown are means ± S.E., with the number of experiments in parentheses. c Means are significantly different from control values at P<0.001 level using Student's t-test.
due primarily to an almost 40% decrease in the per nmole cytochrome P-450 metabolism o f each substrate tested. The substrates that were used included type I substrates (p-nitroanisole, anthracene, benzo(a)pyrene) and a type II substrate (aniline). The decrease in enzyme activity per nmole cytochrome P - 4 5 0 could not be attributed to a change in NADPH cytochrome c reductase activity. This value remained constant at 80 -+ 9 nmol cytochrome c reduced per min per mg protein. The consistent decrease in specific activity (activity per nmole cytochrome P-450) with each o f the substrates tested suggests that we are not observing a shift in the relative proportions of different forms o f cytochrome P-450. It is possible that we m a y be observing the effects of a nonspecific inhibitor o f hepatic cytochrome P-450 present in the microsomal fraction. However, if such an inhibitor exists, it must remain tightly b o u n d to the microsomal fraction because o f two lines o f evidence. First, the inhibition persisted in washed hepatic microsomes isolated from trypanosome infected animals. Second, the 105 000 X g supernatant o f liver whole homogenate from trypanosome infected mice had no effect on the mixed-function oxidase activity obtained using control animal hepatic microsomes (data not shown). In light o f this evidence, the mechanism responsible for the decrease in mixed-function oxidase activity per nmole cytochrome P-450 in trypanosome infected mice remains unclear. African sleeping sickness in humans is usually treated with Suramin during early infection before central nervous system involvement. The arsenical Melarsoprol (Mel B) is normally used for late infection after central nervous system involvement. The in vitro inhibition o f drug metabolism b y Suramin and Melarsoprol shown in Table III indicates that each o f these trypanocides are potential in vivo inhibitors o f hepatic mixed-function oxidations. In order to test the possibility that trypanocides themselves might inhibit endogenous mixed-function oxidase activity, we examined their effect on pentobarbital
203 TABLE III In vitro inhibition of mixed-function oxidase activities by trypanocides, a Enzyme activity
ug/ml to inhibit 50%
PNA demethylase Anilinehydroxylase BaP hydroxylase
Suramin
Melarsoprol
52 ± 4 (4) b 111 ± 7 (4) 43 ± 5 (4)
47 ± 6 (4) 99 ± 13 (4) 45 ± 6 (4)
a Suramin or Melarsoprol were added to the reaction mixture 1 min prior to adding substrate. The values shown are the mean concentrations of trypanocide necessary to produce a 5(~o inhibition of mixed-function oxidase activity for p-nitroanisole (PNA) demethylase, aniline hydroxylase or benzo(a)pyrene (BaP) hydroxylase. b The confidence limits shown are means ± S.E. with the number of experiments in parentheses. sleeping time in mice. Table IV indicates t h a t b i w e e k l y injections o f Suramin or Melarsorprol p r o d u c e d significant increases in sleeping times. A l t h o u g h it m a y be argued that t h e trypanocides p r o d u c e d a change in p e n t o b a r b i t a l c o m p a r t m e n t a t i o n w h e n injected, t h e m o r e likely e x p l a n a t i o n is t h a t t h e longer sleeping times were a result o f l o w e r rates o f p e n t o b a r b i t a l m e t a b o l i s m especially by the liver. Melarsoprol t r e a t m e n t clearly lowered hepatic c y t o c h r o m e P - 4 5 0 and aniline h y d r o x y l a s e activities ( d e t e r m i n e d in vitro), perhaps due to t h e arsenical nature o f this c o m p o u n d . The net result o f an almost 50% decrease in total hepatic m i x e d f u n c t i o n oxidase capacity and the p o t e n t i a l inhibition b y trypanocides should be considered in the doseTABLE IV Effects of trypanocide injection on parameters of mixed-function oxidations, a
Control(buffer) Suramin Control(sesame oil) Melarsoprol
PB sleep time (rain)
nmolP-450 per g liver wet weight
Aniline hydroxylase b
92 122 89 167
34.7 32.9 36.0 26.8
1.44 1.31 1.52 1.06
-+ 11 ± 14 ± 13 -+ 24
(14) c (15) d (15) (15) d
± 2.1 ± 2.7 ± 3.0 -+ 2.9
(12) (13) (11) (13) d
± 0.I ± 0.I ± 0.2 -+ 0.1
(12) (13) d (11) (13) d
a Mice were injected i.p. (10-11 a.m.) at 40 mg per kg body weight with either Suramin (12 mg/ml in 0.9% NaC1/25 mM sodium phosphate, pH 7.4) or Melarsoprol (12 mg/ml suspended by grinding in sesame oil). Controls consisted of injections with equal volumes of each vehicle. Injections were Friday and Tuesday of each week for 5 weeks. Animals were used the day after the last injection to determine the various parameters. All of the groups had body weight and liver wet weight/body weight that were the same as control values (34.1 -+ 1.3 and 48.1 ± 2.9 g/kg, respectively). b Expressed as nmoles p-aminophenol formed per min per nmole cytoehrome P-450. c Values shown are means -+S.E., with the number of experiments in parentheses. d Means are significantly different from control values a t P < 0.05 level using Student's t-test.
204 response effects of drugs used to treat patients or experimental animals infected with trypanosomes. This is because elevated levels of certain drugs that are normally metabolized by the mixed-function oxidase enzyme system may produce nonspecific or toxic effects in trypanosome infected animals and man. ACKNOWLEDGEMENTS Supported by NIH grants GM-25368 and GM-27928, and the Trypanosome Component of the UNDG/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. REFERENCES 1 2 3
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