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Effect of sulfamethazine on phenobarbital and benzo[a]pyrene induced hepatic microsomal mixed function oxidase system in rats
Toxicology Letters ELSEVIER
Toxicology Letters 87 (1996) 25-30
Effect of sulfamethazine on phenobarbital and benzo[a]pyrene induced hepatic microsomal mixed function oxidase system in rats KM. Kodam, S.S. Adav, S.P. Govindwar* Deportment of Biochemistry, Shivoji University, Kolhopur 416 004, India
Received 8 November 1995; revised 12 March 1996; accepted 27 March 1996
AbStMCt Administration of sulfamethaxine (300 mgkg, i.p., single dose) to phenobarbital (80 mgkg, i.p., 3 days) pretreated rats showed significant decrease in microsomal protein, electron transport components and drug metabolizing enzyme activities, compared with phenobarbital administration alone. Induction of mixed function oxidase enzymes due to phenobarbital was not affected by the pretreatment of sulfamethazine. Sulfamethaxine administration to benxo[a]pyrene (20 mgkg, ip., 2 days in oil) pretreated rats showed no significant change, but there was a slight decrease in cytochrome P450 and aminopyrine Ndemethylase activity, compared with benxo[a]pyrene administration alone. A significant inhibition wals observed in aminopyrine Ndemethylase activity due to in vitro addition of sulfamethaxine (3.5 mM) to microsomal incubations from untreated, sulfamethaxine, phenobarbital and benxo[a]pyrene-treated rats. The results indicate that the phenobarbital induced cytochrome P450 is more susceptible to sulfamethazine than benxo[a]pyrene induced cytochrome P450.
Keywordr: Sulfamethaxine; Cytochrome P450; Mixed function oxidase
1. Introduction
Sulfamethazine is widely used in human and veterinary medicine for prophylaxis and as an antibacterial in cattle, sheep and chickens. Sulfamethaxine administration (150 mg/kg, i.p., 3 days) to chickens induced hepatic microsomal electron transport components and drug metabolizing enzymes [l]. lSulfamethaxine is metabolized to 6-hydroxymethyl sulfamethazine (6CHzOHSMZ) 5-hydroxy and sulfamethazine (SOHSMZ) by microsomal mixed function oxi* Corresponding author.
dase, and appears to be oxidized by the male specific cytochrome P450 2Cll [2,3]. We have previously observed that sulfamethaxine (300 mg&g) treatment induces a significant decrease in the level of total cytochrome P450 and aminopyrine Ndemethylase activity in rats (Kodam and Govindwar, unpublished data). In vitro addition of sulfamethaxine (3.5 mM) in microsomal incubates showed inhibition of aminopyrine iVdemethylase activity. The present work is designed to study the inhibitory effect of sulfamethazine on different cytochrome P450 enzymes (phenobarbital inducible CYP 2B, 3A and benxo[a]pyrene inducible CYP 1Al) [4,5].
0378-4274196BJ5.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO378-4274(96)03695-8
26
K. M. Koah
et al. / Toxicology Letters 87 (1996) 25-30
2. Materials and methods
890 and 9:00 h. The volume injected into rats of body weight 200 g was 1 ml.
2.1. Animals
2.4. Preparation of microsomes Adult (200-220 g, 3 months) male Wistar rats were obtained from Haflkine Institute, Bombay, India. The animals were housed in plastic cages and were given an appropriate standard laboratory diet (Hindustan Lever Ltd., Bombay) and tap water ad libitum. 2.2. Chemicals Nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), nicotinamide adenine dinucleotide phosphate (NADP), cytochrome c, glucose 6_phosphate, glucose 6-phosphate dehydrogenase, sulfamethazine, aminopyrine, aniline hydrochloride, HEPES and benzo[a]pyrene were obtained from Sigma Chemical Co. (St. Louis, MO). Phenobarbital was obtained from John Baker (Colorado Springs, CO). Sucrose, phenol, trichloroacetic acid, sodium chloride, potassium chloride, calcium chloride and other chemicals were of analytical grade, obtained from Qualigens Chemical (Bombay). 2.3. Treatment Adult male rats were divided into 8 groups of 6 animals each. Group 1 was injected with 0.9% saline and served as control. Group 2 was treated with sulfamethaxine (300 mg/kg in saline, i.p., 1 day). Group 3 was injected with phenobarbital (80 mgikg in saline, i.p., 3 days). Group 4 received phenobarbital (80 mg/kg in saline, i.p., 3 days) prior to sulfamethazine treatment (300 mg/kg in saline, i.p., 1 day). Group 5 received sulfamethazine (300 mg/kg in saline, i.p., 1 day) prior to phenobarbital treatment (80 mg/kg in saline, i.p., 3 days). Group 6 was treated with benxo[a]pyrene (20 mg/kg in oil, i.p., 2 days). Group 7 was injected with sulfamethazine (300 mg/kg in saline, i.p., 1 day) after benxo[a]pyrene pretreatment (20 mg/kg in oil, i.p., 2 days) and group 8 received sulfamethaxine (300 mg/kg in saline, i.p., 1 day) prior to benzo[a]pyrene treatment (20 mg/kg in oil, i.p., 2 days). The animals were injected between
The rats used in this study were killed by cervical dislocation 24 h after the last treatment. Livers were perfused in situ with ice-cold 1.15% KC1 solution containing 0.05 mM EDTA, rapidly excised, blotted dry, weighed, minced and homogenized with 2 volumes of ice-cold 0.25 M sucrose solution in Potter-Elvehjem type homogenizer. The homogenate was centrifuged at 10 000 x g for 30 min in a refrigerated REM1 C-24 centrifuge. Microsomes from the supematant fraction were isolated by the procedure of Cinti et al. [6]. The microsomal pellet was washed with 1.15% KC1 solution containing 0.05 mM EDTA, resuspended in phosphate buffer (0.1 M, pH 7.4) and the suspension was used for microsomal enzyme assays. Microsomal protein was estimated by the biuret method [7] using bovine serum albumin as standard.
2.5. Enzyme assays The levels of microsomal electron transport components, cytochrome P450 and cytochrome b, were determined using Shimadzu W-visible recording spectrophotometer (W- 16OA)by the procedure of Omura and Sato [8]. Cytochrome c reductase activity was determined by the method of Masters et al. 191.Aminopyrine Ndemethylase activity was assayed according to the procedure of Schenkman et al. [lo]. Formaldehyde liberated during N-demethylation was estimated by the procedure of Nash [ 111. Aniline hydroxylase assay was performed using the procedure reported by Govindwar and Dalvi [12]. Sulfamethaxine dissolved in glass distilled water was added to the incubates at 3.5 mM concentration and activities of aminopyrine Ndemethylase and aniline hydroxylase were determined. In another experiment to study the in vitro effect of sulfamethaxine on the spectral and catalytic activity of cytochrome P450, the phenobarbital and benxo[a]pyrene treated rat liver microsomes were divided into 4 equal groups. Group 2 was incubated with sulfamethaxine only, group 3 con-
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tained the NADPH-generating system only, group 4 contained sulfamethazine and the generating system. All the groups were incubated for 10 min at 37OC. Croup 1 was incubated with phosphate buffer (0.1 M, pH 7.4) without addition of sulfamethazine and NADPH-generating system for 10 min at 37°C and served as control. All four incubates were centrifuged at 10 000 x g for 30 min in a refrigerated centrifuge (REMI-C24). The microsomal pellet was resuspended in phosphate buffer (0.1 M, pH 7.4). Microsomal cytochrome P450 level and the aminopyrine N-demethylase activity of in vitro treated microsomes were determined by using standard procedures &IO,1 I].
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Table 2 Alteration in rat liver microsomal drug metabolizing enxymes due to sulfamethaxine (300 mgkg, i.p., 1 day), phenobarbital (80 m&g, Lp., 3 days) and benxo[a]pyrene (20 mg/kg, i.p., 2 days) treatment
2.6. Analysis of data Statistical analysis was done on IBM-PC software, pharmacological calculation system, by Student’s t-test. The level of significance was set at 0.05.
Group
Aminopyrine N-demethylasep
Aniline hydroxylaseb
Control Sulfamethaxine Phenobarbital Phenobarbital + sulfamethaxine Sulfamethaxine + phenobarbital Benxo[a]pyrene Benxo]a]pyrene + sulfamethaxine Sulfamethaxine + benxo[a]pyrene
4.23 * 3.76 f 7.36 * 4.90 *
0.93 f 0.88 f 1.49 l 1.08 f
0.05 0.06 0.06* 0.047
l
o.os*t
0.12 0.08’ 0.25* 0.1s*t
6.80 f 0.45*
I.17
4.81 l 0.1 I* 4.58 f 0.13
1.00 f 0.04 1.03 f 0.08
4.65 f 0.33
1.05 f 0.02
Values are means of three experiments * S.E.M.; six animals in each group. ?nnol formaldehyde IiberatedAnin per mg microsomal protein. bnmol paminophenol formed/mm per mg microsomal protein. lSignificantly different from control value at P < 0.05 (by Student’s t-test). tSignificantly different from phenobarbital value at P < 0.05.
3. Rem&s and discmsion Our earlier studies on inhibition of catalytic and spectral activity of cytochrome P450 due to sulfamethazine metabolites and significant decrease in phenobarbital sleeping time due to prior administration of sulfamethazine in male rats encouraged
to study the effect of sulfamethazine on the activity of various cytochrome P450 isozymes. The objective was to study the effect of sulfamethazine on phenobarbital induced CYP 2B, 3A [4] and us
Table 1 Alteration in rat liver microsomal protein and electron transport components due to sulfamethaxine (300 mgkg, i.p., I day), phenobarbital (80 mgkg, i.p., .3 days) and benzo[a]pyrene (20 mgikg, i.p., 2 days) treatment Group
MicrosomaP protein
Cytochromeb b5
Cytochromeb P450
Cytochrome cc reductase
Control Sulfamethaxine Phenobarbital Phenobarbital + sulfametlhaxine Sulfamethaxine + phenob~arbital Benxo[a]pyrene Benxo[a]pyrene + sulfamethazine Sulfamethaxine + benxo]a]pyrene
9.45 f 14.40 t 12.00 * 9.00 f Il.37 f 10.83 f 13.67 f 10.50 f
0.20 0.18 0.31 0.26 0.30 0.23 0.22 0.21
0.37 0.24 I.12 0.42 I.12 0.47 0.41 0.48
35.85 zk 4.65 34.80 + 0.48 97.70 * 9.48. 65.33 f 2.72.t 72.66 f 5.60* 36.50 zt 0.76 36.66 zt 3.33 30.66 f 2.30
0.46 0.76* 0.74* 0.76t 0.43* 2.12 1.87’ 1.89
* f f f f i
0.01 0.01 0.01* O.Ol* 0.02’ O.Ol* l 0.01 f 0.01
Values are means of three experiments f S.E.M.; six animals in each group. “mg protein/g liver. bmnoUmg microsomal protein. %mol cytochrome c reduced/min per mg microsomal protein. *Significantly different from control value at P < 0.05 (by Student’s t-test). $Signifkantly different from phenobarbital value at P < 0.05.
f 0.01 f 0.02* f 0.10* zt O.Ol*t f 0.11’ * 0.02. l 0.01* l 0.04*
28
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benzo[a]pyrene induced CYP 1Al [5] rat liver microsomal cytochrome P450. As can be seen from the data in Tables 1 and 2, administration of sulfamethazine caused decrease in cytochrome P450 and aminopyrine Ndemethylase activity. Phenobarbital treatment resulted inthe significant induction of microsomal protein, electron transport components and drug metabolizing enzyme activities. Benzo[a]pyrene treatment caused a significant increase in cytochrome b5, cytochrome P450 and aminopyrine N-demethylase activity. Sulfamethazine treatment of phenobarbital-pretreated rats caused a significant decrease in the levels of microsomal protein, cytochrome P450 and in the activities of aminopyrine N-demethylase, aniline hydroxylase and cytochrome c reductase, which indicates the susceptibility of CYP 2B, 3A to sulfamethazine or its metabolites. Post-treatment of sulfamethazine of benzo[a]pyrene-treated rats showed a slight decrease in cytochrome P450 and aminopyrine Ndemethylase activity compared with benzo[a]pyrene treatment alone. Phenobarbital and benzo[a]pyrene treatments of sulfamethazine pretreated rats showed no significant change in electron transport components and drug metabolizing enzymes when compared with phenobarbital and benzo[a]pyrene treatments alone, respectively, indicating no effect of sulfamethazine or sulfamethazine metabolites on the induction mechanism of phenobarbital and benzo[a]pyrene. These results explain why the phenobarbital sleeping time is not prolonged due to prior treatment of sulfamethazine, even though
Letters 87 (19%)
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sulfamethazine has an inhibitory effect on mixed function ox&se, at this dose level. Higher plasma clearance (half life 3.6 f 0.7 h) of sulfamethazine [3] and the involvement of cytochrome P450 2Cll in the oxidation of phenobarbital [ 13,141 might have caused a significant decrease in phenobarbital sleeping time. To extend our observations of inhibition of mixed function oxidases due to sulfamethazine, experiments were performed in vitro. Sulfamethazine (3.5 mM), when incubated with microsomes from control, sulfamethazine, phenobarbital and benzo[a]pyrene-treated rats with aminopyrine, resulted in significant inhibition of 3 l%, 22%, 30% and 33% of activity, respectively. This decrease may be because of the competition of aminopyrine and sulfamethazine towards the active site or by destruction of cytochrome P450 by sulfamethazine metabolites. The significant inhibition in aniline hydroxylase activity was observed in microsomes of control and sulfamethazine-treated rats; however, no significant change was observed in microsomes of phenobarbital and benzo[a]pyrenetreated rats due to in vitro addition of sulfamethazine (Table 3). In order to find out the cause of significant inhibition of electron transport components and drug metabolizing enzymes due to sulfamethazine treatment of phenobarbital-pretreated rats, experiments were performed in vitro. In vitro treatment of sulfamethazine (10 mM) with microsomes from phenobarbital and benzo[a]pyrene-treated rats with or without NADPH-generating system showed no significant change in the spectral and cataly-
Table 3 Effect of in vitro addition of sulfamethaxine (SMZ) (3.5 mM) on aminopyrine N-demethylase and aniline hydroxylase activities Croup
Control Sulfamethaxine Phenobarbital Eenxo[a]pyrene
Aminopyrine N-demethyla&
Aniline hydroxylaseb
Without SMZ
With SMZ
Without SMZ
With SMZ
4.32 f 3.63 f 7.85 f 4.80 f
2.98 f 2.82 * 5.49 l 3.21 +
0.97 l 0.82 t 1.41 t 1.01 f
0.57 f 0.59 f 1.32 f 0.96 f
0.10 0.07 0.18 0.12
0.08+ 0.0.5* 0.10. 0.07’
0.05 0.06 0.08 0.03
O.lo* 0.05* 0.06 0.03
Values are means of three experiments t S.E.M.; six animals in each group. %mol formaldehyde liberated/min per mg microsomal protein. bmnol p-aminophenol formed/mm per mg microsomal protein. *Significantly different from respective control value (activity without stdfamethaxine at P < 0.05 (by Student’s t-test)).
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Table 4 In vitro effect of sulfamethaxine (SMZ) (10 mM) on cytochrome P450 of phenobarbital (PB) and benxo[a]pyrene (BP) induced rat liver microsomes In vitro treatment groups
P450 remaining Spectral activity0 PB
Microsomes Microsomes + SMZ Microsomes + NADPH gen. system Microsomes + SMZ + NADPH gen. system
1.15 l 1.13 * 1.12 * 1.08 f
Catalytic activityb BP
0.10 0.11 0.10 0.11
0.52 0.52 0.51 0.52
PB f * f +
0.02 0.05 0.04 0.03
7.85 zt 7.85 * 7.46 l 7.01 *
BP 0.18 0.10 0.13 0.12
4.71 4.71 4.62 4.56
f f f *
0.10 0.12 0.15 0.17
Values are means of three experiments f S.E.M.; six animals in each group. %mol cytochrome P45Oqmgmicrosomal protein. bnmol formaldehyde liberated/min per mg microsomal protein (aminopyrine N-demethylase activity).
tic activity of cytochrome P450 (Table 4). These results indicate that phenobarbital induced microsomes could not produce enough sulfamethazine metabolites that inhibit cytochrome P450, as has been observed in vivo. Sulfamethazine is primarily hydroxylated by a male specific isozyme which is constitutively expressed and not inducible by phenobarbital. Earlier reports on the excretion of 6CH,OHSMZ (lO%-12% of the dose given) and SOHSMZ (l%-2% of the dose given) in the urine of male rats [2] and a lower rate of formation of 6CHzOHSMZ and no change in the formation of SOHSMZ in phenobarbital treated (0.1% in drinking water for 5 days) rats [3] lead to the assump tion that destruction ‘of cytochrome P450 is possibly caused by the SMZ metabolite 6CHzOHSMZ. Results from this study indicate that the phenobarbital induced cytochrome P450 (CYP 2B, 3A) is susceptible to sulfamethazine metabolites, especially 6CH20HSMZ. Benzo[a]pyrene induced cytochrome P450 QCYP 1Al) is resistant to sulfamethazine metabolites. Prior treatment of sulfamethazine does not affect the induction mechanism of benzo[a]pyrene and phenobarbital. Acknowledgements One of the authors (K.M.K.) thanks UGC, New Delhi for financial assistance.
VI Kodam, K.M. and Govindwar, S.P. (1995) Effect of sulfamethaxine on mixed function oxidase in chickens. Vet. Hum. Toxicol. 37, 340-342. Vl Witkamp, R.F., Yun, HI., Van’t Klooster, G.A.E., Van Mosel, J.F., Van Mosel, M., Ensink, J.M., Noordhoek, J. and Van Miert, A.S.J.P.A.M. (1992) Comparative aspectsand sexual differentiation of sulfamethaxine plasma elimination and metabolite formation. Studies in rats, rabbits, dwarf goats and cattle. Am. J. Vet. Res. 53, 1830-1835. [31 Witkamp, R.F., Nijmeijer, SM., Yun, H., Noordhoek, J. and Van Miert, A.S.J.P.A.M. (1993) Sulfametbaxine as a model compound to assess sex hormone dependent cytochrome P450 activity in rats. Drug Metab. Dispos. 2L441-446. I41 Fujii-Kuriyama, Y., Mixukami, Y., Kawajiri, K., Sagawa, K. and Muramatsu, M. (1982) Primary structure of a cytochrome P450: coding nucleotide sequence of phenobarbital-inducible cytochrome P450 cDNA from rat liver. Proc. Natl. Acad. Sci. USA 79, 2793-2797. Thomas, P.E., Reik, L.M., Ryan, D.E. and Levitt, W. (1983) Induction of two immunochemically related rat liver cytochrome P450 isoaymes, cytcchrome P45Oc and P45Od, by structurally diverse xenobiotics. J. Biol. Chem. 258, 4590-4598. 161 Cinti, D.L., Moldeus, P. and Schenkman, J.B. (1972) Kinetic parameters of drug metabolizing enxymes in Ca2+ sedimented microsomes from rat liver. Biochem. Pharmacol. 21, 3249-3256. r71 Gomall, A.G., Bardawill, C.J. and David, M.M. (1949) Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751-766. 181 Gnmra, T. and Sate, R. (1964) The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370-2378.
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]9] Masters, B.S.S., Williams, C.H. and Kamin, H. (1967) The preparation and properties of microsomal TPNH cytochrome c reductase from pig liver. In: R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. 10, Academic Press, New York, pp. 565-573. [IO] S&et&man, J.B., Remmer, H. and Estabrook, R.W. (1967) Spectral studies of drug interaction with hepatic microsomal cytochrome. Mol. Pharmacol. 3, 113-123. (1l] Nash, T. (1953) The calorimetric estimation of formaldehyde by means of hantxsch reaction. Biochem. J. 55, 416-421.
[12] Govindwar, S.P. and Dalvi, R.R. (1990) Age dependent toxicity of acorn extract in young and old nude rats. Vet. Hum. Toxicol. 32, 23-26. [13] Waxman, D.J., Dannan, G.A. and GuenSerich, F.P. (1985) Regulation of rat hepatic cytochrome P450: agedependent expression, hormonal imprintiq, and xenobiotic inducibility of sex-speciBc isoenxymes. Biochemistry 24, 44094417. [14] Kobliakov, V., Popova, N. and Rossi, L. (1991) Regulation of the expression of the sex-speciBc isofomui of cytochrome P450 in rat liver. Eur. J. Biochem. 195, 585-591.
Toxicology Letters 87 (1996) 25-30
Effect of sulfamethazine on phenobarbital and benzo[a]pyrene induced hepatic microsomal mixed function oxidase system in rats KM. Kodam, S.S. Adav, S.P. Govindwar* Deportment of Biochemistry, Shivoji University, Kolhopur 416 004, India
Received 8 November 1995; revised 12 March 1996; accepted 27 March 1996
AbStMCt Administration of sulfamethaxine (300 mgkg, i.p., single dose) to phenobarbital (80 mgkg, i.p., 3 days) pretreated rats showed significant decrease in microsomal protein, electron transport components and drug metabolizing enzyme activities, compared with phenobarbital administration alone. Induction of mixed function oxidase enzymes due to phenobarbital was not affected by the pretreatment of sulfamethazine. Sulfamethaxine administration to benxo[a]pyrene (20 mgkg, ip., 2 days in oil) pretreated rats showed no significant change, but there was a slight decrease in cytochrome P450 and aminopyrine Ndemethylase activity, compared with benxo[a]pyrene administration alone. A significant inhibition wals observed in aminopyrine Ndemethylase activity due to in vitro addition of sulfamethaxine (3.5 mM) to microsomal incubations from untreated, sulfamethaxine, phenobarbital and benxo[a]pyrene-treated rats. The results indicate that the phenobarbital induced cytochrome P450 is more susceptible to sulfamethazine than benxo[a]pyrene induced cytochrome P450.
Keywordr: Sulfamethaxine; Cytochrome P450; Mixed function oxidase
1. Introduction
Sulfamethazine is widely used in human and veterinary medicine for prophylaxis and as an antibacterial in cattle, sheep and chickens. Sulfamethaxine administration (150 mg/kg, i.p., 3 days) to chickens induced hepatic microsomal electron transport components and drug metabolizing enzymes [l]. lSulfamethaxine is metabolized to 6-hydroxymethyl sulfamethazine (6CHzOHSMZ) 5-hydroxy and sulfamethazine (SOHSMZ) by microsomal mixed function oxi* Corresponding author.
dase, and appears to be oxidized by the male specific cytochrome P450 2Cll [2,3]. We have previously observed that sulfamethaxine (300 mg&g) treatment induces a significant decrease in the level of total cytochrome P450 and aminopyrine Ndemethylase activity in rats (Kodam and Govindwar, unpublished data). In vitro addition of sulfamethaxine (3.5 mM) in microsomal incubates showed inhibition of aminopyrine iVdemethylase activity. The present work is designed to study the inhibitory effect of sulfamethazine on different cytochrome P450 enzymes (phenobarbital inducible CYP 2B, 3A and benxo[a]pyrene inducible CYP 1Al) [4,5].
0378-4274196BJ5.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO378-4274(96)03695-8
26
K. M. Koah
et al. / Toxicology Letters 87 (1996) 25-30
2. Materials and methods
890 and 9:00 h. The volume injected into rats of body weight 200 g was 1 ml.
2.1. Animals
2.4. Preparation of microsomes Adult (200-220 g, 3 months) male Wistar rats were obtained from Haflkine Institute, Bombay, India. The animals were housed in plastic cages and were given an appropriate standard laboratory diet (Hindustan Lever Ltd., Bombay) and tap water ad libitum. 2.2. Chemicals Nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), nicotinamide adenine dinucleotide phosphate (NADP), cytochrome c, glucose 6_phosphate, glucose 6-phosphate dehydrogenase, sulfamethazine, aminopyrine, aniline hydrochloride, HEPES and benzo[a]pyrene were obtained from Sigma Chemical Co. (St. Louis, MO). Phenobarbital was obtained from John Baker (Colorado Springs, CO). Sucrose, phenol, trichloroacetic acid, sodium chloride, potassium chloride, calcium chloride and other chemicals were of analytical grade, obtained from Qualigens Chemical (Bombay). 2.3. Treatment Adult male rats were divided into 8 groups of 6 animals each. Group 1 was injected with 0.9% saline and served as control. Group 2 was treated with sulfamethaxine (300 mg/kg in saline, i.p., 1 day). Group 3 was injected with phenobarbital (80 mgikg in saline, i.p., 3 days). Group 4 received phenobarbital (80 mg/kg in saline, i.p., 3 days) prior to sulfamethazine treatment (300 mg/kg in saline, i.p., 1 day). Group 5 received sulfamethazine (300 mg/kg in saline, i.p., 1 day) prior to phenobarbital treatment (80 mg/kg in saline, i.p., 3 days). Group 6 was treated with benxo[a]pyrene (20 mg/kg in oil, i.p., 2 days). Group 7 was injected with sulfamethazine (300 mg/kg in saline, i.p., 1 day) after benxo[a]pyrene pretreatment (20 mg/kg in oil, i.p., 2 days) and group 8 received sulfamethaxine (300 mg/kg in saline, i.p., 1 day) prior to benzo[a]pyrene treatment (20 mg/kg in oil, i.p., 2 days). The animals were injected between
The rats used in this study were killed by cervical dislocation 24 h after the last treatment. Livers were perfused in situ with ice-cold 1.15% KC1 solution containing 0.05 mM EDTA, rapidly excised, blotted dry, weighed, minced and homogenized with 2 volumes of ice-cold 0.25 M sucrose solution in Potter-Elvehjem type homogenizer. The homogenate was centrifuged at 10 000 x g for 30 min in a refrigerated REM1 C-24 centrifuge. Microsomes from the supematant fraction were isolated by the procedure of Cinti et al. [6]. The microsomal pellet was washed with 1.15% KC1 solution containing 0.05 mM EDTA, resuspended in phosphate buffer (0.1 M, pH 7.4) and the suspension was used for microsomal enzyme assays. Microsomal protein was estimated by the biuret method [7] using bovine serum albumin as standard.
2.5. Enzyme assays The levels of microsomal electron transport components, cytochrome P450 and cytochrome b, were determined using Shimadzu W-visible recording spectrophotometer (W- 16OA)by the procedure of Omura and Sato [8]. Cytochrome c reductase activity was determined by the method of Masters et al. 191.Aminopyrine Ndemethylase activity was assayed according to the procedure of Schenkman et al. [lo]. Formaldehyde liberated during N-demethylation was estimated by the procedure of Nash [ 111. Aniline hydroxylase assay was performed using the procedure reported by Govindwar and Dalvi [12]. Sulfamethaxine dissolved in glass distilled water was added to the incubates at 3.5 mM concentration and activities of aminopyrine Ndemethylase and aniline hydroxylase were determined. In another experiment to study the in vitro effect of sulfamethaxine on the spectral and catalytic activity of cytochrome P450, the phenobarbital and benxo[a]pyrene treated rat liver microsomes were divided into 4 equal groups. Group 2 was incubated with sulfamethaxine only, group 3 con-
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tained the NADPH-generating system only, group 4 contained sulfamethazine and the generating system. All the groups were incubated for 10 min at 37OC. Croup 1 was incubated with phosphate buffer (0.1 M, pH 7.4) without addition of sulfamethazine and NADPH-generating system for 10 min at 37°C and served as control. All four incubates were centrifuged at 10 000 x g for 30 min in a refrigerated centrifuge (REMI-C24). The microsomal pellet was resuspended in phosphate buffer (0.1 M, pH 7.4). Microsomal cytochrome P450 level and the aminopyrine N-demethylase activity of in vitro treated microsomes were determined by using standard procedures &IO,1 I].
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Table 2 Alteration in rat liver microsomal drug metabolizing enxymes due to sulfamethaxine (300 mgkg, i.p., 1 day), phenobarbital (80 m&g, Lp., 3 days) and benxo[a]pyrene (20 mg/kg, i.p., 2 days) treatment
2.6. Analysis of data Statistical analysis was done on IBM-PC software, pharmacological calculation system, by Student’s t-test. The level of significance was set at 0.05.
Group
Aminopyrine N-demethylasep
Aniline hydroxylaseb
Control Sulfamethaxine Phenobarbital Phenobarbital + sulfamethaxine Sulfamethaxine + phenobarbital Benxo[a]pyrene Benxo]a]pyrene + sulfamethaxine Sulfamethaxine + benxo[a]pyrene
4.23 * 3.76 f 7.36 * 4.90 *
0.93 f 0.88 f 1.49 l 1.08 f
0.05 0.06 0.06* 0.047
l
o.os*t
0.12 0.08’ 0.25* 0.1s*t
6.80 f 0.45*
I.17
4.81 l 0.1 I* 4.58 f 0.13
1.00 f 0.04 1.03 f 0.08
4.65 f 0.33
1.05 f 0.02
Values are means of three experiments * S.E.M.; six animals in each group. ?nnol formaldehyde IiberatedAnin per mg microsomal protein. bnmol paminophenol formed/mm per mg microsomal protein. lSignificantly different from control value at P < 0.05 (by Student’s t-test). tSignificantly different from phenobarbital value at P < 0.05.
3. Rem&s and discmsion Our earlier studies on inhibition of catalytic and spectral activity of cytochrome P450 due to sulfamethazine metabolites and significant decrease in phenobarbital sleeping time due to prior administration of sulfamethazine in male rats encouraged
to study the effect of sulfamethazine on the activity of various cytochrome P450 isozymes. The objective was to study the effect of sulfamethazine on phenobarbital induced CYP 2B, 3A [4] and us
Table 1 Alteration in rat liver microsomal protein and electron transport components due to sulfamethaxine (300 mgkg, i.p., I day), phenobarbital (80 mgkg, i.p., .3 days) and benzo[a]pyrene (20 mgikg, i.p., 2 days) treatment Group
MicrosomaP protein
Cytochromeb b5
Cytochromeb P450
Cytochrome cc reductase
Control Sulfamethaxine Phenobarbital Phenobarbital + sulfametlhaxine Sulfamethaxine + phenob~arbital Benxo[a]pyrene Benxo[a]pyrene + sulfamethazine Sulfamethaxine + benxo]a]pyrene
9.45 f 14.40 t 12.00 * 9.00 f Il.37 f 10.83 f 13.67 f 10.50 f
0.20 0.18 0.31 0.26 0.30 0.23 0.22 0.21
0.37 0.24 I.12 0.42 I.12 0.47 0.41 0.48
35.85 zk 4.65 34.80 + 0.48 97.70 * 9.48. 65.33 f 2.72.t 72.66 f 5.60* 36.50 zt 0.76 36.66 zt 3.33 30.66 f 2.30
0.46 0.76* 0.74* 0.76t 0.43* 2.12 1.87’ 1.89
* f f f f i
0.01 0.01 0.01* O.Ol* 0.02’ O.Ol* l 0.01 f 0.01
Values are means of three experiments f S.E.M.; six animals in each group. “mg protein/g liver. bmnoUmg microsomal protein. %mol cytochrome c reduced/min per mg microsomal protein. *Significantly different from control value at P < 0.05 (by Student’s t-test). $Signifkantly different from phenobarbital value at P < 0.05.
f 0.01 f 0.02* f 0.10* zt O.Ol*t f 0.11’ * 0.02. l 0.01* l 0.04*
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et al. / Toxicology
benzo[a]pyrene induced CYP 1Al [5] rat liver microsomal cytochrome P450. As can be seen from the data in Tables 1 and 2, administration of sulfamethazine caused decrease in cytochrome P450 and aminopyrine Ndemethylase activity. Phenobarbital treatment resulted inthe significant induction of microsomal protein, electron transport components and drug metabolizing enzyme activities. Benzo[a]pyrene treatment caused a significant increase in cytochrome b5, cytochrome P450 and aminopyrine N-demethylase activity. Sulfamethazine treatment of phenobarbital-pretreated rats caused a significant decrease in the levels of microsomal protein, cytochrome P450 and in the activities of aminopyrine N-demethylase, aniline hydroxylase and cytochrome c reductase, which indicates the susceptibility of CYP 2B, 3A to sulfamethazine or its metabolites. Post-treatment of sulfamethazine of benzo[a]pyrene-treated rats showed a slight decrease in cytochrome P450 and aminopyrine Ndemethylase activity compared with benzo[a]pyrene treatment alone. Phenobarbital and benzo[a]pyrene treatments of sulfamethazine pretreated rats showed no significant change in electron transport components and drug metabolizing enzymes when compared with phenobarbital and benzo[a]pyrene treatments alone, respectively, indicating no effect of sulfamethazine or sulfamethazine metabolites on the induction mechanism of phenobarbital and benzo[a]pyrene. These results explain why the phenobarbital sleeping time is not prolonged due to prior treatment of sulfamethazine, even though
Letters 87 (19%)
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sulfamethazine has an inhibitory effect on mixed function ox&se, at this dose level. Higher plasma clearance (half life 3.6 f 0.7 h) of sulfamethazine [3] and the involvement of cytochrome P450 2Cll in the oxidation of phenobarbital [ 13,141 might have caused a significant decrease in phenobarbital sleeping time. To extend our observations of inhibition of mixed function oxidases due to sulfamethazine, experiments were performed in vitro. Sulfamethazine (3.5 mM), when incubated with microsomes from control, sulfamethazine, phenobarbital and benzo[a]pyrene-treated rats with aminopyrine, resulted in significant inhibition of 3 l%, 22%, 30% and 33% of activity, respectively. This decrease may be because of the competition of aminopyrine and sulfamethazine towards the active site or by destruction of cytochrome P450 by sulfamethazine metabolites. The significant inhibition in aniline hydroxylase activity was observed in microsomes of control and sulfamethazine-treated rats; however, no significant change was observed in microsomes of phenobarbital and benzo[a]pyrenetreated rats due to in vitro addition of sulfamethazine (Table 3). In order to find out the cause of significant inhibition of electron transport components and drug metabolizing enzymes due to sulfamethazine treatment of phenobarbital-pretreated rats, experiments were performed in vitro. In vitro treatment of sulfamethazine (10 mM) with microsomes from phenobarbital and benzo[a]pyrene-treated rats with or without NADPH-generating system showed no significant change in the spectral and cataly-
Table 3 Effect of in vitro addition of sulfamethaxine (SMZ) (3.5 mM) on aminopyrine N-demethylase and aniline hydroxylase activities Croup
Control Sulfamethaxine Phenobarbital Eenxo[a]pyrene
Aminopyrine N-demethyla&
Aniline hydroxylaseb
Without SMZ
With SMZ
Without SMZ
With SMZ
4.32 f 3.63 f 7.85 f 4.80 f
2.98 f 2.82 * 5.49 l 3.21 +
0.97 l 0.82 t 1.41 t 1.01 f
0.57 f 0.59 f 1.32 f 0.96 f
0.10 0.07 0.18 0.12
0.08+ 0.0.5* 0.10. 0.07’
0.05 0.06 0.08 0.03
O.lo* 0.05* 0.06 0.03
Values are means of three experiments t S.E.M.; six animals in each group. %mol formaldehyde liberated/min per mg microsomal protein. bmnol p-aminophenol formed/mm per mg microsomal protein. *Significantly different from respective control value (activity without stdfamethaxine at P < 0.05 (by Student’s t-test)).
KM Koaizm et al. / Toxicology Lutters 87 (19%) 25-30
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Table 4 In vitro effect of sulfamethaxine (SMZ) (10 mM) on cytochrome P450 of phenobarbital (PB) and benxo[a]pyrene (BP) induced rat liver microsomes In vitro treatment groups
P450 remaining Spectral activity0 PB
Microsomes Microsomes + SMZ Microsomes + NADPH gen. system Microsomes + SMZ + NADPH gen. system
1.15 l 1.13 * 1.12 * 1.08 f
Catalytic activityb BP
0.10 0.11 0.10 0.11
0.52 0.52 0.51 0.52
PB f * f +
0.02 0.05 0.04 0.03
7.85 zt 7.85 * 7.46 l 7.01 *
BP 0.18 0.10 0.13 0.12
4.71 4.71 4.62 4.56
f f f *
0.10 0.12 0.15 0.17
Values are means of three experiments f S.E.M.; six animals in each group. %mol cytochrome P45Oqmgmicrosomal protein. bnmol formaldehyde liberated/min per mg microsomal protein (aminopyrine N-demethylase activity).
tic activity of cytochrome P450 (Table 4). These results indicate that phenobarbital induced microsomes could not produce enough sulfamethazine metabolites that inhibit cytochrome P450, as has been observed in vivo. Sulfamethazine is primarily hydroxylated by a male specific isozyme which is constitutively expressed and not inducible by phenobarbital. Earlier reports on the excretion of 6CH,OHSMZ (lO%-12% of the dose given) and SOHSMZ (l%-2% of the dose given) in the urine of male rats [2] and a lower rate of formation of 6CHzOHSMZ and no change in the formation of SOHSMZ in phenobarbital treated (0.1% in drinking water for 5 days) rats [3] lead to the assump tion that destruction ‘of cytochrome P450 is possibly caused by the SMZ metabolite 6CHzOHSMZ. Results from this study indicate that the phenobarbital induced cytochrome P450 (CYP 2B, 3A) is susceptible to sulfamethazine metabolites, especially 6CH20HSMZ. Benzo[a]pyrene induced cytochrome P450 QCYP 1Al) is resistant to sulfamethazine metabolites. Prior treatment of sulfamethazine does not affect the induction mechanism of benzo[a]pyrene and phenobarbital. Acknowledgements One of the authors (K.M.K.) thanks UGC, New Delhi for financial assistance.
VI Kodam, K.M. and Govindwar, S.P. (1995) Effect of sulfamethaxine on mixed function oxidase in chickens. Vet. Hum. Toxicol. 37, 340-342. Vl Witkamp, R.F., Yun, HI., Van’t Klooster, G.A.E., Van Mosel, J.F., Van Mosel, M., Ensink, J.M., Noordhoek, J. and Van Miert, A.S.J.P.A.M. (1992) Comparative aspectsand sexual differentiation of sulfamethaxine plasma elimination and metabolite formation. Studies in rats, rabbits, dwarf goats and cattle. Am. J. Vet. Res. 53, 1830-1835. [31 Witkamp, R.F., Nijmeijer, SM., Yun, H., Noordhoek, J. and Van Miert, A.S.J.P.A.M. (1993) Sulfametbaxine as a model compound to assess sex hormone dependent cytochrome P450 activity in rats. Drug Metab. Dispos. 2L441-446. I41 Fujii-Kuriyama, Y., Mixukami, Y., Kawajiri, K., Sagawa, K. and Muramatsu, M. (1982) Primary structure of a cytochrome P450: coding nucleotide sequence of phenobarbital-inducible cytochrome P450 cDNA from rat liver. Proc. Natl. Acad. Sci. USA 79, 2793-2797. Thomas, P.E., Reik, L.M., Ryan, D.E. and Levitt, W. (1983) Induction of two immunochemically related rat liver cytochrome P450 isoaymes, cytcchrome P45Oc and P45Od, by structurally diverse xenobiotics. J. Biol. Chem. 258, 4590-4598. 161 Cinti, D.L., Moldeus, P. and Schenkman, J.B. (1972) Kinetic parameters of drug metabolizing enxymes in Ca2+ sedimented microsomes from rat liver. Biochem. Pharmacol. 21, 3249-3256. r71 Gomall, A.G., Bardawill, C.J. and David, M.M. (1949) Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751-766. 181 Gnmra, T. and Sate, R. (1964) The carbon monoxide binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370-2378.
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[12] Govindwar, S.P. and Dalvi, R.R. (1990) Age dependent toxicity of acorn extract in young and old nude rats. Vet. Hum. Toxicol. 32, 23-26. [13] Waxman, D.J., Dannan, G.A. and GuenSerich, F.P. (1985) Regulation of rat hepatic cytochrome P450: agedependent expression, hormonal imprintiq, and xenobiotic inducibility of sex-speciBc isoenxymes. Biochemistry 24, 44094417. [14] Kobliakov, V., Popova, N. and Rossi, L. (1991) Regulation of the expression of the sex-speciBc isofomui of cytochrome P450 in rat liver. Eur. J. Biochem. 195, 585-591.