Chem. Res. Toxicol. 1988,1, 69-73
69
Carboxylesterase Isoenzyme Specific Deacylation of Diacetoxyscirpenol (Anguidine) Su-Er Wut and Michael A. Marletta" Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Received September 23, 1987
The deacylation reaction of diacetoxyscirpenol (DAS), a trichothecene mycotoxin, was studied by using five carboxylesterase isoenzymes isolated from mouse liver microsomes. The simultaneous isolation of the five microsomal carboxylesterase isoenzymes was accomplished by chromatofocusing. DAS serves as a unique substrate since both acetyl groups are accessible for hydrolysis, providing an opportunity to investigate the regioselective hydrolysis of DAS with the various isoenzymes. A novel basic carboxylesterase isoenzyme, pI8.4-8.2, was isolated. This isoenzyme exhibits strict regioselectivity toward DAS hydrolysis; only the acetyl group a t (2-4, but not that at C-15, is hydrolyzed. Moreover, this is the major isoenzyme responsible for the deacylation of DAS in mouse hepatic microsomes. The rate constant determined for deacylation of DAS at C-4 for this isoenzyme is 130-1000 times greater than that of the other carboxylesterases. This isoenzyme was also the most efficient for the hydrolysis of 4-monoacetoxyscirpenediol with a rate constant 30-100 times greater than that of the other isoenzymes.
Scheme I
Introduction The trichothecene mycotoxins are a group of fungal metabolites that exhibit a range of significant biological properties including cyto- and phytotoxicity (I). The deacylation reaction catalyzed by microsomal carboxylesterase (CE)' (EC 3.1.1.1) has been shown to be a key step in the metabolic transformation of trichothecene esters2 (2-7). In general, deacylation of trichothecenes renders them much less toxic. In particular, in our laboratory the metabolic fate of diacetoxyscirpenol (anguidine, DAS) was investigated with rat (6, 7) and CD-1 mouse hepatic microsomes.2 The acetyl group at C-4 or C-15 of DAS was OAc
DAS
OH
'v
15-MA
/
\'
\r
OAc
on
DAS
Triol
y@qA ;c HO
'4-MA
rapidly hydrolyzed to form 15-monoacetoxyscirpendiol (15-MA) or 4-monoacetoxyscirpendiol(4-MA)respectively and the remaining acetyl group (at C-15 or C-4) was further hydrolyzed to form scirpentriol (Triol) (Scheme I). Conjugation with glucuronic acid was found to be a concurrent pathway with the hydrolysis reaction (6, 7).2 Glucuronidation was highly regiospecific, with the formation of a p-glycosyl bond only with the hydroxyl group at the C-3 position. While glucuronyl conjugates were found to form with DAS as well as 15-MA and the Triol2 the conjugates derived from the hydrolysis products predominated. Furthermore, the relative amounts of the DAS hydrolysis products varied substantially when rat microsomes were compared to mouse microsomes.2 This was one indication that isoenzyme regiospecific hydrolysis may be occurring.
The microsomal CE are enzymes that catalyze the hydrolysis of esters, amides and thio esters (8, 9). The nonspecific CE are involved in the detoxification of xenobiotics (9,lO) and in the activation of ester and amide type prodrugs (9). The physiological roles of these enzymes are still uncertain, although their involvement in steroid metabolism (11)and hydrolysis of several classes of natural lipid esters (12, 13) is known. After isolation and characterization of five rat microsomal CE isoenzymes (14,151 it became evident that these isoenzymes differ in their isoelectric points and their specificities toward ester and amide substrates (12, 13). The unique feature of DAS as a substrate for esterases is that both acetyl groups are accessible for hydrolysis,
*To whom correspondence should be addressed at: Division of Medicinal Chemistry, College of Pharmacy, The University of Michigan, Ann Arbor, MI 48109-1065. Present Address: Department of Animal Drug Metabolism, Merck, Sharp & Dohme Research Laboratories, Rahway, NJ 07065.
Abbreviations: CE, microsomal carboxylesterase; DAS, diacetoxyscirpenol; 4-MA, 4-monoacetoxyscirpendiol; 15-MA, 15-monoacetoxyscirpendiol; Triol, scirpentriol; Brij 35, polyoxyethylene 23 lauryl ether; PNP, p-nitrophenol;PNPA, p-nitrophenyl acetate. * Wu, S. E., Russo-Rodriguez, S.G . ,Roush, W. R., and Marletta, M. A., unpublished results.
0893-228~/88/2701-0069$01.50/0
0 1988 American Chemical Society
70 Chem. Res. Toxicol., Vol. 1, No. 1, 1988
Wu and Marletta
thus, providing an opportunity to investigate the regioselective hydrolysis of DAS by various CE isoenzymes. In this paper, we report the isolation of five CE isoenzymes from CD-1 mouse hepatic microsomes by chromatofocusing and the kinetics of these isoenzymes with DAS, 15-MA as substrates.
Kinetics of DAS Hydrolysis. The reaction solution (total volume 100 pL) containing 15 mM MgC12, 50 mM phosphate buffer (pH 7.7), various amount of purified isoenzymes, and 247 pM [I4C]DAS (or 106 pM [14C]-15-MAor 106 pM [14C]-4-MA) was incubated at 37 "C. An aliquot was removed at various times and the amount of [14C]DAS, [14C]-15-MA, [14C]-4-MA,and [ 14C]Triolwas quantitated after HPLC separation. HPLC Analysis. HPLC separations were carried out on a Waters Assoc., Inc. solvent delivery system (Model 6000 A) and gradient controller system (Model 680) employing a pBondapak C-18 precolumn and pBondapak C-18 analytical column (3.9 mm X 30 cm). Buffer A consisted of methanol and Buffer B consisted of 10 mM potassium phosphate buffer (pH 7.0). The elution gradient was programmed as follows: 5% buffer A for 5 min, followed by a linear gradient of 5-60% buffer A from 5 to 45 min and then 60% buffer A for another 10 min. The elution times for the Triol, &MA, 4-MA, and DAS were 28-30,32-35,35-37, and 45-48 min, respectively.
Materials and Methods Chemicals. ["CIDAS (sp act. 19 mCi/mmol) was a generous gift from Dr. W. R. Roush, synthesized according to Roush & Russo-Rodriguez (16).p-Nitrophenylacetate (PNPA) was purchased from Aldrich. Bis-Tris, iminodiacetic acid, piperazine, N-methylpiperazine, polyoxyethylene 23 lauryl ether (Brij 3 9 , and sodium iminodiacetate were purchased from Sigma. Mono P (HR 5/20), Polybuffer 74 and 96, and PD-10 Sephadex G-25M prepack columns were purchased from Pharmacia. The reverse-phase pBondapak C-18 (3.9 mm x 30 cm) column was purchased from Waters Assoc., Inc. Male CD-1 mice (40-50 days old) were purchased from Charles River Breeding Laboratories, Inc. (Wilmington, MA). Solubilization of Microsomal CE Isoenzymes. Mouse livers were perfused with 1.15% KCl, minced and homogenized in 3 volumes of 10 mM EDTA, 1.15% KCl, and 50 mM potassium phosphate buffer (pH 7.5). This homogenate was centrifuged for 10 min at 13000g, and the resulting supernatant was centrifuged a t 105000g for 60 min. The pellet was resuspended in 10 mM EDTA and 1.15% KC1 (pH 7.4) and centrifuged a t 105000g for 60 min. The microsomal pellet was solubilized by stirring with the nonionic detergent Brij 35 (1%) in 0.1 M Tris buffer (pH 7.5) at 4 "C for 90 min. After solubilization, the microsomal preparation was centrifuged a t 105000g for 60 min, and the resulting supernatant was chromatographed on a Sephadex G-75-120 (40-120 pm) column, that was preequilibrated and eluted with 25 mM bis-Tris buffer containing 1% Brij 35 (pH was adjusted to 7.1 with 1 N iminodiacetic acid). The CE activities were monitored by the PNPA assay (see below for details) and the protein concentrations were determined by a modified Lowry assay (17). Chromatofocusing. The fractions containing CE activity were pooled and concentrated by pressure filtration using a PM-10 membrane (Amicon, Inc.). All the samples were filtered through a 0.22-pm sterile filter before chromatofocusing on the Mono P columm ( P h m a c i a , Inc.). For any given pH gradient range, the Mono P column was first equilibrated with starting buffer. After sample application, the column was eluted with Polybuffer (74 or 96)) which consists of amphoteric buffering substances with different pK, values. The volumes (mL) of starting buffer were programmed as suggested by the manufacturer; however, the volumes (mL) of total eluent were modified as follows: 60, 36, 32,37, and 40 mL for pH gradients 7-4,8.5-7.5,6.5-5.5,5.5-4.5, and 5-4, respectively. All the buffers were prepared in the presence of 1% Brij 35. The CE activities were monitored by the PNPA assay (see below) and the pH was determined by a flatsurface electrode (Fisher Model 13-639-83). Each CE isoenzyme was applied to PD-10 Sephadex G-25M column to exchange the buffer to bis-Tris buffer (25 mM) containing 1%Brij 35 (pH 7.1) before kinetic studies. P N P A Assay. The reaction solution contained 1.8 mL of 0.5 M Tris buffer (pH 8.0) and 0.2 mL of a fresh PNPA solution prepared by dissolving 18.1 mg of PNPA in 1 mL of acetonitrile and adjusting the final volume to 100 mL with water. After the addition of enzyme, the formation of p-nitrophenol (PNP) was followed spectrophotometrically at 405 nm (e = 1.81 X LO-' cm-' pM-l) for 2-5 min. The nonenzymatic hydrolysis of PNPA was also measured for the same period of time. The amount of PNP formed enzymatically was calculated after correcting for the amount of P N P formed in the control solutions. Preparation of [WI-lS-MA and [%]-4-MA. A solution (0.5 mL) containing 15 mM MgCl,, 50 mM phosphate buffer (pH 7.7), 628 pM [14C]DAS,and CD-1 mouse microsomes (3.9 mg protein) was incubated at 37 OC for 30 min. The resulting mixture was centrifuged to remove microsomal proteins prior to HPLC separation. The pooled peaks of [l4C]-l5-MAand [14C]-4-MAwere lyophilized and redissolved in a 20% methanol solution.
Results and Discussion We have previously found that the glucuronides from DAS and T-2 toxin formed the P-glucuronylbond only at the C-3 position (6, 7)) and while glucuronides can be formed from DAS and T-2 toxin? the quantitatively significant glucuronides are those that result from conjugation of the hydrolysis products of DAS and T-2 toxin (6, 7). The glucuronides are essentially devoid of toxicity as judged by the ability to inhibit protein synthesis in vitro. Therefore, the competing reactions of hydrolysis versus conjugation are important and will directly influence the toxicity of these compounds. The main goal of the studies reported here was to understand the extent that the nonspecific CE isoenzymes participated in the hydrolysis of DAS and if any regiospecificity was preferred. Five rat microsomal CE have been isolated previously (14,15), and their specificities for a variety of ester and amide substrates were investigated (12,13).However, very little was known about CE activity in the mouse. Our preliminary studies on the hydrolysis of DAS showed that rat and mouse hepatic microsomal CE activity had different regioselective preferences.2 In order to understand this regiospecificity, we isolated five CE isoenzymes from CD-1 mouse hepatic microsomes (Table I) and studied the hydrolysis kinetics of these isoenzymes. These particular isoenzymes can be separated based on their isoelectric points. The application of chromatofocusing to separate isoenzymes has become increasingly popular (18, 19). Proteins bind to this weak anionic exchanger at pH's that are higher than their isoelectric points. The elution procedure generates a pH gradient resulting in the sequential elution of proteins. As shown in Figure 1, five CE isoenzymes were separated in the pH gradient range of 7-4, and their CE activities were determined by the hydrolysis of PNPA. In order to avoid any contamination by neighboring isoenzymes, each peak of activity was rechromatographed at a narrower pH gradient range on the same Mono P column, and, as an example, the elution profile of peak 1 is shown in Figure 2. Even though the protein preparation obtained may not be homogeneous, each peak of CE isoenzyme activity contains no other isoenzyme of CE. The p l range of five isoenzymes were as follows: 8.4-8.2, 5.8-5.6, 5.0-4.9, 4.7-4.6, and 4.3-4.2 (Table I). The p l determined by chromatofocusing is always lower than that determined by gel electrophoresis~.~Although it may be difficult to directly compare mouse isoenzymes obtained here to those isolated from the rat (14, 15), the isolation of a basic CE isoenzyme, p l ~
~~
Chromatofocusing with Posbuffer and PBE, Technical Booklet Series (1980),Pharmacia Fine Chemicals, Uppsala, Sweden.
Chem. Res. Toxicol., Val. 1, No. I , 1988 71
Carboxylesterase Isoenzyme Specificity
Table I. The Purification Chart of CE Isoenzymes column sp act.: condimol/*mg/ enzyme fractions tions total protein, mg min homogenization & centrifugation solubilization with 1% Brij 35 gel filtration Sep G75-120
microsomes soluble soluble
244.6 74.3 56.5
pH 7.1
% act. recvry
100 73.3 65.2
purifictn fold 1 2.4 2.8
act. recvry
purifictn fold
3.4 8.2 9.6
~~
Mono P column pH gradient
peak
elution pH
sp act.: mol/mg/min
total protein, mg
?&
Chromatofocusing I 7-4
1 2 3 4 5
>7.1 5.8-5.3 5.2-4.9 4.8-4.5 4.4-4.1
8.5-7.5 6.5-5.5 5.5-4.5 5.5-4.5 5-4
1 2 3 4 5
8.4-8.2 5.8-5.6 5.0-4.9 4.7-4.6 4.3-4.2
8.7 14.4 3.0 3.0 4.3
5.0 17.3 10.0 3.6 7.7
5.2 30.0 3.6 1.3 4.0
1.5 5.1 3.0 1.1 2.3
20.1 24.5 28.5 7.1 25.4
5.1 13.8 2.4 0.5 2.4
5.6 7.2 8.4 2.1 7.5
Chromatofocusing I1 2.1 4.7 0.7 0.6 0.8
a Specific activities of enzyme fractions were measured with PNPA as substrate and followed the formation of PNP spectrophotometrically (see Materials and Methods).
I
2.51
t
'
I
I
2.0-
0)
'
5:
C
e 0
o n
a
8.4-8.2, is novel. The kinetic studies that follow revealed that this basic isoenzyme is the major isoenzyme responsible for the deacylation of DAS. Scheme I summarizes the two possible pathways for the deacylation reaction of DAS. The acetyl group at C-4 of DAS was hydrolyzed to form 15-MA with rate constant k1 and the remaining acetyl group of 15-MA was further removed to form Triol with rate constant k,. Likewise rate constants k3 and k4 were assigned. The hydrolysis reaction was assumed to be irreversible. The following K,,, values for DAS were determined: G-75 column eluent, 2.5 mM; isoenzyme 1,2.2 mM; isoenzyme 2,2.5 mM; isoenzyme 3, 1.7 mM; isoenzyme 4, 2.7 mM; isoenzyme 5, 4 mM. Since the substrate concentration is much less that the K,, then all of the rate constants represent V / K , values with units of reciprocal time. It would be desirable to divide these kinetic parameters by the appropriate enzyme concentrations to give kat/Km with second-order units. However, even though each peak of CE activity contains no other CE isoenzyme, the protein preparation obtained is not homogeneous; therefore the rate constants were normalized
-
Fraction Number
F r a c t i o n Number
Figure 1. Chromatofocusingon CE isoenzymes. The Mono P (HR 5/20)column was equilibrated with 25 mM bis-Tris buffer (pH 7.1) containing 1%Brij 35 and eluted with 10% Polybuffer 74 (pH 4.0) containing 1%Brij 35. The pH gradient was 7-4 (e-.). The CE activities (0-0)were followed by the PNPA assay (Aa6). The protein profile ( 0 - 0 ) was recorded as AzW
-
: I
Figure 2. Second chromatofocusingof CE isoenzyme. Each CE isoenzyme peak (from Figure 1) was pooled, concentrated and exchanged to the appropriate starting buffer with a prepack PD-10 Sephadex G-25M column (Pharmacia)before chromatofocusing on second Mono P column. The results with peak 1as an example are shown here. The Mono P column was equilibrated with 25 mM Tris buffer (pH 8.8) containing 1% Brij 35 and eluted with a solution (total volume, 100 mL) of 1 % Brij 35, 0.11 mL of Pharmalyte 8-10.5, and 9.5 mL of Polybuffer 96 (pH 7.5). The pH gradient (e-*) was 8.5-7.5. CE activities (0-0)was monitored by the PNPA assay and protein profile (0-0) was recorded as Am
according to the amount of protein used. The kinetic laws for the system are given by the eq 1-4. [DAS], = [DAS]oe-(kl+k3)t (1)
[Triol], = [DAS], - [15-MAIt - [4-MAlt (4) The variables of eq 1-4 could be determined experimentally by subjecting a sample of reaction mixture, a t time t , to HPLC analysis. The sum of rate constants kl and k3 can be easily determined according to eq 1. It is not possible to determine rate constants k , and k 2 from eq 2 alone. Under conditions where the concentration of
72 Chem. Res. Toxicol., Vol. 1, No. 1, 1988
Wu and Marletta
Table 11. Rate Constants of CE Isoenzymes rate constants: min-' isoenzyme k , + k3b 1 (6.0 f 0.2) x 10-1 2 (6.7 + 0.5) x 10-4 3 (5.1 f 0.3) x 4 (1.99 0.03) x 10-3 5 (1.25 f 0.03) x 10-3
hb kl'
