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Effects of microsomal enzyme inducers upon UDP-glucuronic acid concentration and UDP-glucuronosyltransferase activity in the rat intestine and liver
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
115,253-260
(1992)
Effects of Microsomal Enzyme Inducers upon UDP-Glucuronic Acid Concentration and UDP-Glucuronosyltransferase Activity in the Rat Intestine and Liver’ DANIEL GooN’,~
AND CURTIS
D. KLAASSEN~
Environmentai Heaith and Occupational Medicine Center, Deparrtnent qf Phartnac&gy, Tm5cology and Therapeutics. University ~fKan.ras Medical Center, Kansas City, Kansas 66103 Received June 13, 1991; accepted March 16, 1992
Effects of Microsomal Enzyme Inducers upon UDP-Glucuranic Acid Concentration and UDP-Glucuronosyltransferase Activity in the Rat Intestine and Liver. GOON, D. AND KLAASSEN, C. D. (1992). Toxic& Appf. Pharmaeol. 115,253-260. This study was conducted to evaluate UDP-glucuronosyltransferase(UDP-GT) activity, UDP-glucuronic acid (UDP-GA) concentration, and UDP-glucose (UDPG) concentration in the rat intestine and liver following oral administration of butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone-16a-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or trans-stilbeneoxide (TSO). Microsomal UDP-GT activity was assayedin vitro with acetaminophen (AA), harm01(HA), and I-naphthol (NA) as the aglycones. Intestinal HA and AA glucuronidationwereenhancedby BHA, BaP, and TSO, whereas 3MC, PB, PCN, and TCDD augmentedhepatic HA-glucuronide formation and BHA, PB, PCN, TCDD, and TSO significantly increasedhepatic AA glucuronidation. All inducing agentsexcept PB and PCN markedly increasedboth intestinal and hepatic NA glucuronidation. PB, PCN, and TCDD paradoxically decreasedintestinalglucuronidation of AA and HA. A similar effect upon hepatic glucuronidation was not observedwith any of the agentsstudied. Hepatic UDP-GA concentration was increased significantly by all inducers studied except PCN and TCDD, whereashepatic UDPG concentration was increasedonly by BHA. In the intestine, significant increasesin UDP-GA concentration were produced only by BHA and BaP, which also elevated intestinal UDPG. These results demonstrate that microsomalenzyme inducersevoke different effectsupon intestinal and hepatic glucuronidation. These differences are manifested with regard to induced changesin UDP-GT activity aswell as treatment-induced alterations in UDP-GA content. Thus, the
present study further underscores the marked variance of intestinal and hepatic xenobiotic glucuronidation. G) 1992 Academic
compounds (e.g., bilirubin, steroids, and catecholamines) are substrates for glucuronidation. The reaction involves the transfer of glucuronic acid from uridine diphospho-a-Dglucuronic acid (UDP-GA), the endogenous activated reaction cosubstrate, to an electronegative group on the target substrate through the mediation of UDP-glucuronosyltransferase (UDP-GT), a family of inducible microsomal isozymes. The enhanced water solubility of the conjugated product greatly facilitates excretion into bile or urine. The liver is generally recognized to be the major site of glucuronidation in the body. However, numerous extrahepatic organs also possess the ability to conjugate compounds with glucuronic acid: including the kidneys, lungs, and gastrointestinal tract (Aitio, 1974; Aitio and Marniemi, 1980: Dutton, 1980). Since initial reports of glucuronidation in the intestinal mucosa (Hartiala, 1954, 1955; Shirai and Ohkubo, 1954) the intestine has received considerable attention with regard to its glucuronidation capabilities (Hietanen and Lang, 1978; Grafstrom et nl., 1979; Koster and Noordhoek, 1983a; Koster, 1985). Compounds bearing a phenolic moiety are particularly prone to glucuronidation in the intestine (Hartiala, 1973, 1977). For example, intestinal glucuronidation significantly decreases the systemic bioavailability of such variegated agents as ethinyl estradiol (Hirai ef al., 198 1; Schwenk et al., 1982), harm01 (Goon and Klaassen, 1991) morphine (Iwamoto and Klaassen, 1977a), nalorphine (Iwamoto and Klaassen, 1977b), 1-naphthol (Bock and Winne, 1975; Goon and Klaassen. 199 l), and salicylamide (Barr and Riegelman, 1970; Shibasaki et al., 198 1). Early studies conducted with tissue slices and subcellular fractions in vitro reported a steep oral-to-aboral gradient in ’ Presented in part at the 27th Annual Meeting for the Society of Toxicology, Dallas, TX. February 1988. ’ Present address: Molecular Toxicology and Environmental Health Sciences Program. School of Pharmacy, University of Colorado. Boulder. CO 80309. 3 Supported by U.S. Public Health Service Grant ES-07079 and a Procter and Gamble Fellowship. 4 To whom reprint requests should be addressed.
Press. Inc.
Glucuronidation is a major phase II biotransformation reaction of considerable importance in pharmacology and toxicology (for review see Dutton, 1980; Kasper and Henton, 1980; Caldwell, 1985). Both xenobiotics and endogenous 253
0041-008X/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
254
GOON AND KLAASSEN
intestinal UDP-GT activity, with the highest activity in the duodenum (Hartiala et al., 1964; Hanninen et al., 1968). More recent studies conducted with perfused intestinal segments in vitro and in situ have failed to note appreciable differences in the total glucuronidation capacity of various regions of the intestine &asker and Rickert, 1978; Koster and Noordhoek, 1983b). Regardless, it is apparent that UDPGT activity is distributed throughout the entire length of the intestinal tract (Hartiala et al., 1964; Hinninen et al., 1968). Similar to the liver, the activity of UDP-GT in the intestine can be increased by certain chemicals, such as benzo[a]pyrene (Aitio et al., 1972; Turner et al., 1977) 3methylcholanthrene (Aitio et al., 1972; Hietanen and Lang, 1978; Hietanen et al., 1980), and 2,3,7,8-tetrachlorodibenzop-dioxin (Marselos et al., 1978; Schiller and Lucier, 1978). Watkins and Klaassen ( 1983) have shown that many inducers of hepatic microsomal UDP-GT activity also have the propensity to significantly elevate UDP-GA concentration in the liver, including the three agents cited above. In contrast to the liver, however, relatively little information is available regarding the induction of intestinal UDP-GT activity and even less is known about effects upon intestinal UDP-GA concentration. The primary aim of this study was to evaluate the effect of oral administration of known inducers of hepatic UDPGT activity upon both UDP-GT activity and UDP-GA concentration in the rat intestine. The effect upon tissue concentrations of UDP-glucose (UDPG), the immediate precursor of UDP-GA, was also assessed. In addition, hepatic UDP-GT activity, UDPG and UDP-GA concentrations were assessed following oral administration of the inducing agents. Oral administration was selected as intestinal UDPGT activity is increased more effectively by this route rather than with intraperitoneal administration (Hietanen et al., 1980). Specifically, the agents butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone- 16cy-carbonitrile (PCN), trans-stilbene oxide (TSO), and 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) were studied. Microsomal UDP-GT activity was assessedin vitro with acetaminophen (AA), harm01 (HA), and 1-naphthol (NA) as the aglycones. METHODS Materials and animals. AA, aprotinin (from bovine lung), BHA, BaP. ethylenediaminetetraacetic acid (EDTA; tetrasodium salt), HA, leupeptin (hemisulfate salt), magnesium chloride (hexahydrate), 3MC. NA, I-[ I“C]naphthol ([‘“Cl NA), D-saccharic- I ,Clactone, sucrose, Triton X- 100, Trizma base, urethane, UDPG, and UDP-GA were purchased from Sigma Chemical Co. (St. Louis, MO). Heparin was acquired from Elkin-Sinn (Cherry Hill, NJ), dithiothreitol from Calbiochem (San Diego, CA), PB from Merck and Co. (Rahway, NJ), TSO from Aldrich Chemical Co. (Milwaukee, WI), and TCDD (purity >99%) from Cambridge Isotope Laboratories (Woburn. ME). PCN was a gift from The Upjohn Co. (Kalamazoo, MI). Male Sprague-Dawley rats (Sasco, Inc., Omaha, NE) weighing 250-350 g were used in all experiments (four or five animals per group). Animals
were maintained on a 12-hr light/dark cycle at 23-27°C and were allowed free access to food (Purina Laboratory Rodent Chow, Ralston-Purina, St. Louis, MO) and water. Animals were acclimatized a minimum of 5 days prior to treatment with inducing agents. Treatment of animals. BHA was administered in a pulverized diet (Purina Laboratory Rodent Chow) at a concentration of 1% (w/w) for 10 days (Hazelton et al., 1985; Hjelle et al., 1985). TCDD was prepared in corn oil:acetic acid (95:5, w/v) and was administered once at a dosage of 8 fig/ kg (po; 4 ml/kg) 10 days prior to tissue sample collection (Schiller and Lucier, 1978). PB was administered in the drinking water for 4 daysat a concentration of 1 mg/ml (Turner et al., 1977). PCN was suspended in 2% Tween 80 (Fisher Scientific, St. Louis, MO) in saline and administered po for 4 days at a daily dosage of 75 mg/kg (Watkins et al.. 1982). BaP and 3MC were each administered po 24 hr prior to tissue sample collection as a suspension in corn oil (5 ml/kg) at a dosage of 100 mg/kg (Aitio et al., 1972). TSO was administered for 5 days at a daily dosage of 400 mg/kg (Seidegird and DePierre, 1982). The dosage of TSO was split on the first 2 days of treatment and administered 8 hr apart. TSO was prepared in corn oil and administered po (5 ml/kg). Administration protocols for BHA. TCDD, PB, BaP. and 3MC were based upon previous studies investigating intestinal glucuronidation (as cited above). whereas treatment regimens for PCN and TSO were based on reported hepatic effects.Control animals received either 2% Tween 80 in saline for 4 days (PO). corn oil once or for 4 days, or were untreated. Results from the various control treatments demonstrated no differences and were subsequently pooled. Sample collection and preparation. Twenty-four hours after completion of the respective dosing regimens, rats were anesthetized with urethane ( 1.5 mg/kg; ip) and two I .O g samples of both proximal jejunum and liver were collected. One sample set was frozen immediately in liquid nitrogen and processed for determination of tissue UDPG and UDP-GA concentrations by modification of the method of Dills and Klaassen (1985). Under liquid nitrogen, perchloric acid (3.96%; Mallinckrodt, Inc., St. Louis, MO) was added drop-wise (15.5 and 1:6.5 w/v ratio of intestinal and hepatic tissue, respectively. to acid) and ground with the tissue using a mortar and pestle precooled to -70°C. The frozen powder was transferred to a 30-ml Corex tube (precooled and kept in ice water) and homogenized with a Brinkman Polytron (Kinematica GmbH, Lucerne, Switzerland) until fluid. The pH of the homogenate was adjusted to approximately 5.5 with 5 M potassium carbonate (typically about 0.32 and 0.45 ml per gram of intestine and liver. respectively) and separated by centrifugation (20.00013 for 10 min at 4°C). The pH of the resulting supernatant was adjusted to 5.5 with 1 N HCl and the supernatant cleaned by sequential centrifugation (10,OOOgfor 4 min at 4°C) and filtration. The filtration assembly was composed of a 0.45~pm membrane filter contained within a Swinnex-25 disc filter holder (Millipore Corp., Bedford, MA) attached to a ~-CCsyringe. The filtration assembly was cooled to 0°C prior to use. The filtrate was stored in 500~~1 aliquots at -70°C for no more than 72 hr prior to analysis. Intestinal and hepatic microsomes were prepared from the second set of tissue samples following the protocol of Lu and Levin (1972) with some modifications. All procedures were performed at 4°C. Tissue samples were finely minced with scissors and mixed with 2 ml of homogenization buffer. The homogenization buffer for liver samples was 50 mM Tris-HCl (pH 7.0 at 22°C) containing 150 mM KCl. For intestinal samples, the homogenization buffer used for preparation of hepatic microsomes was modified by the addition of 1 mM dithiothreitol, 1 mM EDTA, 10 pM leupeptin, 200 KIU aprotinin/ml (approximately 0.22 TIU/ml), 25 units heparin/ml, and 20% glycerol (v/v). The modifications to the homogenization buffer were incorporated in accordance with recommendations by various investigators (Tredger and Chhabra, 1976; Fang and Strobe], 1978; Jones et al.. 1980: Harada and Omura, 1983). Both liver and intestinal samples were homogenized with a Brinkman Polytron (Kinematica GMBH) and microsomes prepared by ultracentrifugation (100,OOOg for 60 min) of the postmitochondrial supernatant (10,OOOg for 20 min). The microsomes were washed by homogenization in 10 mM EDTA (pH 7.4) containing 150 mM KC1 and
EFFECTS OF INDUCERS re-isolated by ultracentrifugation. The final microsomal pellet was resuspended in 0.25 M sucrose (0.8 ml) and stored at -70°C. Determination of tissue UDPG and UDP-GA concentrations. Hepatic and intestinal concentrations of UDPG and UDP-GA were measured by isocratic, reverse-phase, ion-pair HPLC with uv absorbance detection (254 nm) modified from the method of Aw and Jones (1982). Samples were injected onto an Adsorbosphere HS Cr8-5 pm analytical HPLC-column (Alltech Assoc.. Inc., Deerfield, IL) and eluted with 100 mM potassium acetate buffer (pH 5.5) containing 0.01% n-octylamine (Aldrich Chemical Co.. Inc.) as the ion-pairing agent. A guard column packed with 5 pm Adsorbosphere HS CL8 silica (Alltech) and a saturator column containing 200/425 mesh Adsorbosil silica (Alltech) were included in the analytical system. Retention times of UDPG and UDP-GA were determined by cochromatography of standards purchased from Sigma Chemical Co. Quantitation of UDPG and UDP-GA were based upon integration of absorbance peak areas. The concentrations of UDPG and UDP-GA were determined from standard calibration curves for the respective agents. Determination of UDP-GT activity. Reaction mixtures and incubation conditions for the measurement of UDP-GT activity in vitro were essentially as described previously (Hazelton et al., 1985; Hjelle et al., 1985; Arlotto et al.. 1986). The final incubation volume was 0.5 ml and contained Tris-HC1 (200 mM. pH 7.7, at room temperature), magnesium chloride (10 mM). UDP-GA (4 mM), D-saccharic- I ,4-lactone (I .25 mM), microsomal protein (0.5-4.0 mg/ml), and aglycone (5 mM AA, 0.8 mivt HA, or I mM [“‘GINA, 0.2 $Zi/jd) at the final concentrations indicated. Hepatic microsomal UDPGT activity was assessedseparately for each animal. Due to the smaller yield of microsomal protein per gram of tissue, intestinal microsomes prepared from individual animals were pooled within a treatment group for the determination of UDP-GT activity. All assayswere performed in duplicate with two concentrations of microsomal protein at 37°C with mechanical agitation (90 cycles/min). Reactions were initiated by the addition of UDPGA after preincubation for 10 min and stopped after 20 min by the addition of 0.5 ml ice-cold ethanol. Unreacted [“‘C]NA (>99%) was extracted from the reaction mixture with two 5-ml volumes of chloroform and NA-glucuronide formation determined by liquid scintillation analysis ofthe aqueous phase (Hazelton et al., 1985). Unreacted AA was extracted with cyclohexanone saturated with calcium chloride (Hjelle et al., 1985) and AA-glucuronide formation assessedby HPLC analysis of the aqueous phase (Howie et al., 1977 as modified in Goon and Klaassen, 1990). In assaysinvolving HA as the aglycone, microsomes were solubilized in 0.01% Triton X-100 for I5 min prior to preincubation with the reaction mixture. The precipitate formed upon addition of ethanol was pelleted by centrifugation (10,OOOg for 5 min) and the HA-glucuronide content of the supernatant measured by thin-layer chromatographic separation with fluorometric detection (Mulder and Hagedoorn, 1974, as modified in Goon and Klaassen, 1991). Experiments and assaysinvolving HA were conducted under subdued, red lighting due to the sensitivity of HA to light. For both intestinal and hepatic microsomes, preliminary studies were performed with each aglycone to establish conditions under which glucuronide formation was proportional to protein concentration and incubation time. Microsomal protein concentrations were determined by the method of Lowry et al.. (1951) with bovine serum albumin as standard. Statistics. Results derived from experiments conducted with pooled samples (i.e., intestinal microsomes) represent the mean + standard deviation of duplicate assaysperformed at two protein concentrations. Data obtained from experiments performed with individual samples are expressed as means ? standard error (SE). Comparisons between treated groups and controls were conducted with Dunnett’s multiple comparison test. The acceptable level of statistically significant difference was set at p < 0.05 (Steel and Torrie. 1980). RESULTS
The effects of orally administered microsomal enzyme inducers upon hepatic and intestinal AA glucuronidation in
ON UDP-GT
255
AND UDP-GA
vitro are presented in Figs. 1A and 2A, respectively. Hepatic
microsomal UDP-GT activity directed toward AA was increased significantly by treatment with all inducing agents studied except BaP and 3MC (Fig. 1A). The formation of AA-glucuronide by hepatic microsomes was increased 20 to 40% in rats orally administered BHA, PB, TCDD, or TSO. The greatest increase in hepatic AA-glucuronide formation was observed in microsomes prepared from animals treated with PCN (80%). The various inducing agents elicited different effects upon UDP-GT activity directed toward AA in the intestine. AA-glucuronide formation by intestinal microsomes prepared from rats treated with BHA, BaP, or TSO was 250, 220, and 260% of control levels, respectively (Fig. 2A). Administration of 3MC produced minimal effects upon AA-glucuronide formation by intestinal microsomes. Paradoxically, treatment with PB, PCN, or TCDD in vivo decreased microsomal AA-glucuronidation in the intestine (25, 36, and 48% of control values, respectively). The formation of HA-glucuronide by hepatic and intestinal microsomes prepared from rats orally administered the various inducing agents is depicted in Figs. 1B and 2B, re-
HEPATIC AA:lJDP-GT
ACTNITY
HEPATIC HkUDP-GT
ACTIVIM
(A)
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i
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HEPATIC NkUDP-GT
ACTIVITY
l
03
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2 0 2 8 I
100
50
CON
BHA
BP
3MC
PB
PCN
TCDD
TSO
FIG. 1. Effect of orally administered microsomal enzyme inducers upon the formation of acetaminophen-glucuronide (A), harmol-glucuronide (B), and I-naphthol-glucuronide (C) by hepatic microsomes. Results represent the mean i standard error of four or five rats. Individual assayswere performed for each animal within a treatment group. Assays were conducted in duplicate with two concentrations of microsomal protein. Asterisks indicate statistically significant difference (p < 0.05) from control.
256
GOON AND KLAASSEN 0.60
2 z
60
P k-l
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INTESTINAL AAzUDP-GT
ACTIVITY
INTESTINAL
HkUDP-GT
ACTIVITY
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NkUDP-GT
ACTIWY
(A)
_
(a
w
z 20
CON
BHA
BP
3MC
PB
PCN
TCDD
TSO
FIG. 2. Effect of orally administered microsomal enzyme inducers upon the formation of acetaminophen-glucuronide (A), harmol-glucuronide (B), and 1-naphthol-glucuronide (C) by intestinal microsomes. Results represent the mean f standard deviation of assays conducted in duplicate at two protein concentrations with pooled intestinal microsomes prepared from four or five rats per treatment group.
spectively. Hepatic UDP-GT activity directed toward HA was enhanced significantly following treatment with 3MC. PB, PCN, or TCDD (Fig. 1B). HA glucuronidation by hepatic microsomes was increased 20 to 40% after oral administration of 3MC, PB, or TCDD. Similar to AA, the greatest increase in hepatic HA-glucuronide formation was noted in microsomes prepared from rats treated with PCN (90%). Treatment with BHA, BaP, or TSO produced essentially no change in the glucuronidation of HA by hepatic microsomes. As seen in Fig. 2B, HA-glucuronide formation by intestinal microsomes was increased approximately 90% following administration of BHA, BaP. or TSO in vivo. Additionally, administration of 3MC increased the formation of HA-glucuronide by intestinal microsomes 30%. As was observed with AA, oral administration of PB, PCN, or TCDD decreased intestinal glucuronidation of HA in vitro (56, 77, and 76% of control levels, respectively). The activities of hepatic and intestinal microsomal UDPGT directed toward NA following oral administration of various microsomal enzyme inducers are presented in Fig. 1C and 2C, respectively. The formation of NA-glucuronide by hepatic microsomes was enhanced significantly by all in-
ducing agents studied except PB and PCN (Fig. 1C). Treatment with BHA, BaP, 3MC, and TSO increased hepatic microsomal NA-glucuronide formation 120,20,40, and I lo%, respectively. The greatest induction of hepatic UDP-GT activity was observed in microsomes prepared from rats orally administered TCDD, which exhibited a greater than 250% increase in NA glucuronidation. As noted with hepatic microsomes, intestinal NA glucuronidation in vitro was increased by all agents studied except PB and PCN (Fig. 2C). The greatest effects upon UDP-GT activity in the intestine were observed following oral administration of BHA, BaP, 3MC, or TSO, which enhanced microsomal formation of NA-glucuronide 80 to 120%. TCDD moderately increased intestinal NA glucuronidation in vitro (30%). PCN produced no change and PB slightly decreased NA-glucuronide formation ( I 1%) by intestinal microsomes. The effects of orally administered microsomal enzyme inducers upon hepatic and intestinal UDPG and UDP-GA concentrations are presented in Figs. 3 and 4. In the liver, the concentration of UDPG was increased nearly 50% following treatment with BHA (Fig. 3A). All other inducing agents studied (i.e., BaP, 3MC, PB, PCN, TCDD, and TSO)
600 HEPATIC
UDPG
HEPATIC
UDP-GA
6’)
600
*
0) * T
:
3MC
PB
200
CON
BHA
BaP
PCN TCDD
TSO
FIG. 3. Effect of orally administered microsomal enzyme inducers upon concentrations of UDPG (A) and UDP-GA (B) in the liver. Results represent the mean + standard error of four or five rats per treatment group. Asterisks indicate statistically significant difference (p < 0.05) from control.
EFFECTS OF INDUCERS
INTESTINAL UDPG
250
(4
*
3 z
150
T2 $
100
E 5 g
50
d 5 8
250
E Y
200
d Li 2
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“: 8
100
INTESTINAL UDP-GA
@I
50
0 CON
BHA
BaP
3MC
PB
PCN TCDD
TSO
FIG. 4. Effect of orally administered microsomal enzyme inducers upon concentrations of UDPG (A) and UDP-GA (B) in the intestine. Results represent the mean + standard error of four or five rats per treatment group. Asterisks indicate statistically significant difference (p < 0.05) from control.
produced no significant perturbations in hepatic UDPG concentration. Hepatic UDP-GA concentrations were unaffected after oral administration of PCN or TCDD (Fig. 3B). In contrast, the concentration of UDP-GA in the liver was increased significantly after treatment with BHA, BaP, 3MC, PB, or TSO (40 to 80%). In the intestine, neither UDPG nor UDP-GA concentrations (Figs. 4A and 4B, respectively) were altered appreciably following oral administration of 3MC, PB, PCN, TCDD, or TSO. Conversely, significant increases in both intestinal UDPG and UDP-GA concentrations were noted in rats treated with either BHA (50 and 40%, respectively) or BaP (30 and 60%, respectively). DISCUSSION The results presented regarding the effects of 3MC and BaP upon the capacity of intestinal microsomes to glucuronidate NA concur with previous investigations (Aitio et al., 1972; Hietanen and Lang, 1978; Hietanen et al., 1980), which assessed intestinal glucuronidation of p-nitrophenol in vitro after pretreatment in vivo with 3MC or BaP. Both NA and
ON UDP-CT
AND UDP-GA
257
p-nitrophenol are classified as group 1 substrates for UDPGT, i.e., substrates whose conjugation with glucuronic acid is catalyzed by the 3MC-inducible form of UDP-GT (Bock et al., 1983). Furthermore, the results of the present study regarding NA-glucuronide formation by hepatic microsomes after oral administration of the various inducing agents correspond well with the previous work of Watkins et al. (1982) in which the inducers were administered by intraperitoneal injection. In both studies, NA-glucuronide formation by hepatic microsomes was enhanced significantly upon treatment with BaP. 3MC, TCDD, or TSO in vivo. However, intraperitoneal administration of BHA (500 mg/kg/day for 10 days) produced negligible effects upon hepatic NA glucuronidation (Watkins et al., 1982) whereas oral administration of BHA ( 1% of diet for 10 days) markedly increased NA-glucuronide formation by hepatic microsomes in the present study. Turner el uf. (1977) previously demonstrated increased intestinal NA glucuronidation in situ in rats pretreated with BaP. However, results of the present study demonstrate that, in addition to increasing UDP-GT activity, BaP treatment in vivo also significantly elevates endogenous UDPG and UDP-GA concentrations in the intestine. Thus, enhanced intestinal NA glucuronidation in situ following BaP administration cannot be attributed exclusively to induction of intestinal UDP-GT activity without prior knowledge of kinetic properties of intestinal UDP-GT; specifically, the relationship between endogenous UDP-GA concentration and the K,,, of UDP-GT for UDP-GA in the intestine. Similar to NA. HA is also classified as a group 1 UDPGT substrate (Koster, 1985). As expected, HA-glucuronide formation by hepatic microsomes prepared from 3MCtreated rats was significantly greater than control hepatic microsomes. In contrast, treatment with 3MC only slightly increased the glucuronidation of HA by intestinal microsomes. Moreover, the effects of the various microsomal enzyme inducers upon intestinal HA glucuronidation were extremely disparate from the effects produced upon hepatic HA glucuronidation. None of the inducing agents studied effectively increased HA glucuronidation by both hepatic and intestinal microsomes. A degree of disparity was also observed between intestinal and hepatic microsomal glucuronidation of AA in response to treatment with the various inducing agents. Intestinal AA glucuronidation was selectively enhanced by BaP administration, whereas treatment with PB, PCN, and TCDD selectively increased AA-glucuronide formation by hepatic microsomes. Unlike HA, however, oral administration of BHA or TSO increased AA glucuronidation by both intestinal and liver microsomes. In contrast to HA and AA, UDP-GT activity directed towards NA in hepatic and intestinal microsomes was affected similarly by the various microsomal enzyme inducers. All inducing agents that effectively increased hepatic NA glucuronidation (i.e., BHA, BaP, 3MC. TCDD, and TSO) also augmented formation of
258
GOON AND KLAASSEN
NA-glucuronide by intestinal microsomes, although the extent to which NA glucuronidation was increased differed between the two tissues. Administration of PB, PCN, and TCDD paradoxically decreased the formation of AA-glucuronide and HA-glucuronide by intestinal microsomes. With all three inducing agents, the decrease was more severe with AA as the aglycone. Intestinal microsomes prepared from PB-treated rats also demonstrated slightly decreased activity toward NA. The cause(s) for this observation is not known, however, the effect appears to be restricted to the intestine. Hepatic microsomal glucuronidation of AA and HA was enhanced significantly by treatment with these agents. In addition, formation of NA-glucuronide by liver microsomes was increased profoundly by TCDD treatment and was similar to control levels after treatment with PB or PCN. The significant increases in hepatic UDP-GA concentrations manifested upon oral administration of BHA, BaP. 3MC, PB, and TSO are in close agreement with the earlier work of Watkins and Klaassen ( 1983). Moreover, PCN minimally affected hepatic UDP-GA content in both studies. However, the two investigations are at odds with regard to the effect of TCDD upon the concentration of UDP-GA in the liver. Although similar dosages were studied (8 and 10 pg/kg), TCDD significantly increased the hepatic concentration of UDP-GA (1 .&fold) in the previous study, whereas the effect of TCDD upon hepatic UDP-GA content was negligible in the present study. The discrepancy between the two studies may be due in part to differences in the route of administration employed. In the former study, TCDD was administered intraperitoneally, whereas the oral route of administration was used in the present study. The results of the present study further illustrate the differences between hepatic and intestinal glucuronidation. In contrast to the liver, intestinal UDP-GA concentrations were less prone to alteration upon administration of the various microsomal enzyme inducers. Significant increases in UDPGA content of the intestine were noted only after administration of BHA or BaP, whereas all inducers studied, except PCN and TCDD, substantially increased hepatic UDP-GA concentrations. Thus, UDP-GA concentrations in the liver appear to be susceptible to a greater number and variety of microsomal enzyme inducers than in the intestine. The effect of microsomal enzyme inducers upon tissue UDPG concentration has not been reported previously. In the intestine, UDPG concentrations were increased by the same two agents that elicited significant increases in intestinal UDP-GA levels (i.e., BHA and BaP). In general, UDPG concentrations in the liver were more resilient than hepatic UDPGA concentrations. Specifically, only BHA produced an appreciable change in hepatic UDPG concentration, whereas hepatic UDP-GA levels were significantly increased by BHA. BaP, 3MC, PB, and TSO. These observations imply that the increases noted in hepatic UDP-GA concentrations following
treatment with various microsomal enzyme inducers (except BHA) may result from increased activity, or possibly induction, of UDP-glucose dehydrogenase, the cytosolic enzyme that catalyzes the formation of UDP-GA from UDPG, in the liver. Conversely, the coordinate increase in UDPG and UDP-GA in the intestine after administration of BHA or BaP, and in the liver upon administration of BHA, suggests that enhanced synthesis of UDPG may be causative in augmenting endogenous tissue concentrations of UDP-GA. Orally administered BHA, BaP, and TSO effectively enhanced intestinal microsomal UDP-GT activity directed towards all three aglycones studied (i.e., AA, HA, and NA). In addition, 3MC markedly increased the formation of NAglucuronide by intestinal microsomes, but only marginally affected the glucuronidation of AA and HA in vitro. Treatment of rats with BHA or BaP also altered intestinal cosubstrate levels in vivo. Specifically, intestinal concentrations of both UDP-GA and its immediate precursor, UDPG, were increased significantly following oral administration of BHA or BaP. In contrast, intestinal UDPG and UDP-GA concentrations were unaffected by oral administration of 3MC or TSO. Thus, of the four agents found to substantially augment the glucuronidation capacity of intestinal microsomes, 3MC and TSO were unique in effectively inducing intestinal UDPGT activity without appreciably altering the concentrations of UDP-GA or UDPG in the intestine. A similar effect in the liver was demonstrated following oral administration of PCN or TCDD. Specifically, PCN enhanced hepatic microsomal glucuronidation of AA and HA, whereas TCDD significantly increased hepatic UDP-GT activity directed toward all three substrates studied. Concurrently, hepatic UDPG and UDP-GA concentrations were unaffected by orally administered PCN or TCDD. In summary, the data presented indicate that orally administered microsomal enzyme inducers exert different effects upon the glucuronidation capacity of the intestine relative to the liver. These differences are manifested not only with regard to induced changes in UDP-GT activity directed toward the aglycones acetaminophen, harmol, and 1-naphthol, but also in terms of effects upon the concentration of UDP-GA in the two organs. As demonstrated previously in the liver, the present results show that significant increases in intestinal UDP-GA concentration may be produced after treatment with various microsomal enzyme inducers. However, it appears that hepatic UDP-GA content is susceptible to elevation by a greater variety of microsomal enzyme inducers than intestinal UDP-GA. Moreover, 3MC and TSO are unique agents capable of inducing intestinal UDP-GT activity without appreciably affecting intestinal UDP-GA concentration. Thus, 3MC and TSO are potentially powerful and valuable investigational tools for studying the mechanism(s) of capacity-limited glucuronidation in the rat intestine.
EFFECTS OF INDUCERS
ACKNOWLEDGMENTS The authors express their appreciation to Dr. Andrew Parkinson and Michael P. Arlotto for their guidance in the isolation and handling of hepatic and intestinal microsomes.
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Steei, R. G. D.. and Torrie, J. H. (1980). Principles qf.Statistics: ..1 Biometrical Approach. 2nd ed. McGraw-Hill. New York. Tredger, J. M.. and Chhabra. R. S. ( 1976). Preservation of various microsomal drug metabolizing components in tissue preparations from the livers. lungs. and small intestines of rodents. Drug Metab. Dispos. 4, 4.5 l-459. Turner, J. C.. Shanks. V.. Kelly, W. J.. and Green, R. S. (1977). The effect of sodium phenobarbital and 3.4-benzpyrene on the glucuronidation of I-naphthol in rat small intestinal loops in viva. Gen. Pharmacol. 8, 5 l53. Watkins. J. B.. Gregus, Z., Thompson, T. N., and Klaassen. C. D. (I 982). Induction studies on the functional heterogeneity of rat liver UDP-glucuronosyltransferases. To.xicol. Appl. Pharmacol. 64, 439-446. Watkins, J. B., and Klaassen, C. D. (1983). Chemically-induced alteration of UDP-glucuronic acid concentration in rat liver. Drug Metab. Dispos. 11, 37-40.
AND
APPLIED
PHARMACOLOGY
115,253-260
(1992)
Effects of Microsomal Enzyme Inducers upon UDP-Glucuronic Acid Concentration and UDP-Glucuronosyltransferase Activity in the Rat Intestine and Liver’ DANIEL GooN’,~
AND CURTIS
D. KLAASSEN~
Environmentai Heaith and Occupational Medicine Center, Deparrtnent qf Phartnac&gy, Tm5cology and Therapeutics. University ~fKan.ras Medical Center, Kansas City, Kansas 66103 Received June 13, 1991; accepted March 16, 1992
Effects of Microsomal Enzyme Inducers upon UDP-Glucuranic Acid Concentration and UDP-Glucuronosyltransferase Activity in the Rat Intestine and Liver. GOON, D. AND KLAASSEN, C. D. (1992). Toxic& Appf. Pharmaeol. 115,253-260. This study was conducted to evaluate UDP-glucuronosyltransferase(UDP-GT) activity, UDP-glucuronic acid (UDP-GA) concentration, and UDP-glucose (UDPG) concentration in the rat intestine and liver following oral administration of butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone-16a-carbonitrile (PCN), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or trans-stilbeneoxide (TSO). Microsomal UDP-GT activity was assayedin vitro with acetaminophen (AA), harm01(HA), and I-naphthol (NA) as the aglycones. Intestinal HA and AA glucuronidationwereenhancedby BHA, BaP, and TSO, whereas 3MC, PB, PCN, and TCDD augmentedhepatic HA-glucuronide formation and BHA, PB, PCN, TCDD, and TSO significantly increasedhepatic AA glucuronidation. All inducing agentsexcept PB and PCN markedly increasedboth intestinal and hepatic NA glucuronidation. PB, PCN, and TCDD paradoxically decreasedintestinalglucuronidation of AA and HA. A similar effect upon hepatic glucuronidation was not observedwith any of the agentsstudied. Hepatic UDP-GA concentration was increased significantly by all inducers studied except PCN and TCDD, whereashepatic UDPG concentration was increasedonly by BHA. In the intestine, significant increasesin UDP-GA concentration were produced only by BHA and BaP, which also elevated intestinal UDPG. These results demonstrate that microsomalenzyme inducersevoke different effectsupon intestinal and hepatic glucuronidation. These differences are manifested with regard to induced changesin UDP-GT activity aswell as treatment-induced alterations in UDP-GA content. Thus, the
present study further underscores the marked variance of intestinal and hepatic xenobiotic glucuronidation. G) 1992 Academic
compounds (e.g., bilirubin, steroids, and catecholamines) are substrates for glucuronidation. The reaction involves the transfer of glucuronic acid from uridine diphospho-a-Dglucuronic acid (UDP-GA), the endogenous activated reaction cosubstrate, to an electronegative group on the target substrate through the mediation of UDP-glucuronosyltransferase (UDP-GT), a family of inducible microsomal isozymes. The enhanced water solubility of the conjugated product greatly facilitates excretion into bile or urine. The liver is generally recognized to be the major site of glucuronidation in the body. However, numerous extrahepatic organs also possess the ability to conjugate compounds with glucuronic acid: including the kidneys, lungs, and gastrointestinal tract (Aitio, 1974; Aitio and Marniemi, 1980: Dutton, 1980). Since initial reports of glucuronidation in the intestinal mucosa (Hartiala, 1954, 1955; Shirai and Ohkubo, 1954) the intestine has received considerable attention with regard to its glucuronidation capabilities (Hietanen and Lang, 1978; Grafstrom et nl., 1979; Koster and Noordhoek, 1983a; Koster, 1985). Compounds bearing a phenolic moiety are particularly prone to glucuronidation in the intestine (Hartiala, 1973, 1977). For example, intestinal glucuronidation significantly decreases the systemic bioavailability of such variegated agents as ethinyl estradiol (Hirai ef al., 198 1; Schwenk et al., 1982), harm01 (Goon and Klaassen, 1991) morphine (Iwamoto and Klaassen, 1977a), nalorphine (Iwamoto and Klaassen, 1977b), 1-naphthol (Bock and Winne, 1975; Goon and Klaassen. 199 l), and salicylamide (Barr and Riegelman, 1970; Shibasaki et al., 198 1). Early studies conducted with tissue slices and subcellular fractions in vitro reported a steep oral-to-aboral gradient in ’ Presented in part at the 27th Annual Meeting for the Society of Toxicology, Dallas, TX. February 1988. ’ Present address: Molecular Toxicology and Environmental Health Sciences Program. School of Pharmacy, University of Colorado. Boulder. CO 80309. 3 Supported by U.S. Public Health Service Grant ES-07079 and a Procter and Gamble Fellowship. 4 To whom reprint requests should be addressed.
Press. Inc.
Glucuronidation is a major phase II biotransformation reaction of considerable importance in pharmacology and toxicology (for review see Dutton, 1980; Kasper and Henton, 1980; Caldwell, 1985). Both xenobiotics and endogenous 253
0041-008X/92 $5.00 Copyright Q 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
254
GOON AND KLAASSEN
intestinal UDP-GT activity, with the highest activity in the duodenum (Hartiala et al., 1964; Hanninen et al., 1968). More recent studies conducted with perfused intestinal segments in vitro and in situ have failed to note appreciable differences in the total glucuronidation capacity of various regions of the intestine &asker and Rickert, 1978; Koster and Noordhoek, 1983b). Regardless, it is apparent that UDPGT activity is distributed throughout the entire length of the intestinal tract (Hartiala et al., 1964; Hinninen et al., 1968). Similar to the liver, the activity of UDP-GT in the intestine can be increased by certain chemicals, such as benzo[a]pyrene (Aitio et al., 1972; Turner et al., 1977) 3methylcholanthrene (Aitio et al., 1972; Hietanen and Lang, 1978; Hietanen et al., 1980), and 2,3,7,8-tetrachlorodibenzop-dioxin (Marselos et al., 1978; Schiller and Lucier, 1978). Watkins and Klaassen ( 1983) have shown that many inducers of hepatic microsomal UDP-GT activity also have the propensity to significantly elevate UDP-GA concentration in the liver, including the three agents cited above. In contrast to the liver, however, relatively little information is available regarding the induction of intestinal UDP-GT activity and even less is known about effects upon intestinal UDP-GA concentration. The primary aim of this study was to evaluate the effect of oral administration of known inducers of hepatic UDPGT activity upon both UDP-GT activity and UDP-GA concentration in the rat intestine. The effect upon tissue concentrations of UDP-glucose (UDPG), the immediate precursor of UDP-GA, was also assessed. In addition, hepatic UDP-GT activity, UDPG and UDP-GA concentrations were assessed following oral administration of the inducing agents. Oral administration was selected as intestinal UDPGT activity is increased more effectively by this route rather than with intraperitoneal administration (Hietanen et al., 1980). Specifically, the agents butylated hydroxyanisole (BHA), benzo[a]pyrene (BaP), 3-methylcholanthrene (3MC), phenobarbital (PB), pregnenolone- 16cy-carbonitrile (PCN), trans-stilbene oxide (TSO), and 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) were studied. Microsomal UDP-GT activity was assessedin vitro with acetaminophen (AA), harm01 (HA), and 1-naphthol (NA) as the aglycones. METHODS Materials and animals. AA, aprotinin (from bovine lung), BHA, BaP. ethylenediaminetetraacetic acid (EDTA; tetrasodium salt), HA, leupeptin (hemisulfate salt), magnesium chloride (hexahydrate), 3MC. NA, I-[ I“C]naphthol ([‘“Cl NA), D-saccharic- I ,Clactone, sucrose, Triton X- 100, Trizma base, urethane, UDPG, and UDP-GA were purchased from Sigma Chemical Co. (St. Louis, MO). Heparin was acquired from Elkin-Sinn (Cherry Hill, NJ), dithiothreitol from Calbiochem (San Diego, CA), PB from Merck and Co. (Rahway, NJ), TSO from Aldrich Chemical Co. (Milwaukee, WI), and TCDD (purity >99%) from Cambridge Isotope Laboratories (Woburn. ME). PCN was a gift from The Upjohn Co. (Kalamazoo, MI). Male Sprague-Dawley rats (Sasco, Inc., Omaha, NE) weighing 250-350 g were used in all experiments (four or five animals per group). Animals
were maintained on a 12-hr light/dark cycle at 23-27°C and were allowed free access to food (Purina Laboratory Rodent Chow, Ralston-Purina, St. Louis, MO) and water. Animals were acclimatized a minimum of 5 days prior to treatment with inducing agents. Treatment of animals. BHA was administered in a pulverized diet (Purina Laboratory Rodent Chow) at a concentration of 1% (w/w) for 10 days (Hazelton et al., 1985; Hjelle et al., 1985). TCDD was prepared in corn oil:acetic acid (95:5, w/v) and was administered once at a dosage of 8 fig/ kg (po; 4 ml/kg) 10 days prior to tissue sample collection (Schiller and Lucier, 1978). PB was administered in the drinking water for 4 daysat a concentration of 1 mg/ml (Turner et al., 1977). PCN was suspended in 2% Tween 80 (Fisher Scientific, St. Louis, MO) in saline and administered po for 4 days at a daily dosage of 75 mg/kg (Watkins et al.. 1982). BaP and 3MC were each administered po 24 hr prior to tissue sample collection as a suspension in corn oil (5 ml/kg) at a dosage of 100 mg/kg (Aitio et al., 1972). TSO was administered for 5 days at a daily dosage of 400 mg/kg (Seidegird and DePierre, 1982). The dosage of TSO was split on the first 2 days of treatment and administered 8 hr apart. TSO was prepared in corn oil and administered po (5 ml/kg). Administration protocols for BHA. TCDD, PB, BaP. and 3MC were based upon previous studies investigating intestinal glucuronidation (as cited above). whereas treatment regimens for PCN and TSO were based on reported hepatic effects.Control animals received either 2% Tween 80 in saline for 4 days (PO). corn oil once or for 4 days, or were untreated. Results from the various control treatments demonstrated no differences and were subsequently pooled. Sample collection and preparation. Twenty-four hours after completion of the respective dosing regimens, rats were anesthetized with urethane ( 1.5 mg/kg; ip) and two I .O g samples of both proximal jejunum and liver were collected. One sample set was frozen immediately in liquid nitrogen and processed for determination of tissue UDPG and UDP-GA concentrations by modification of the method of Dills and Klaassen (1985). Under liquid nitrogen, perchloric acid (3.96%; Mallinckrodt, Inc., St. Louis, MO) was added drop-wise (15.5 and 1:6.5 w/v ratio of intestinal and hepatic tissue, respectively. to acid) and ground with the tissue using a mortar and pestle precooled to -70°C. The frozen powder was transferred to a 30-ml Corex tube (precooled and kept in ice water) and homogenized with a Brinkman Polytron (Kinematica GmbH, Lucerne, Switzerland) until fluid. The pH of the homogenate was adjusted to approximately 5.5 with 5 M potassium carbonate (typically about 0.32 and 0.45 ml per gram of intestine and liver. respectively) and separated by centrifugation (20.00013 for 10 min at 4°C). The pH of the resulting supernatant was adjusted to 5.5 with 1 N HCl and the supernatant cleaned by sequential centrifugation (10,OOOgfor 4 min at 4°C) and filtration. The filtration assembly was composed of a 0.45~pm membrane filter contained within a Swinnex-25 disc filter holder (Millipore Corp., Bedford, MA) attached to a ~-CCsyringe. The filtration assembly was cooled to 0°C prior to use. The filtrate was stored in 500~~1 aliquots at -70°C for no more than 72 hr prior to analysis. Intestinal and hepatic microsomes were prepared from the second set of tissue samples following the protocol of Lu and Levin (1972) with some modifications. All procedures were performed at 4°C. Tissue samples were finely minced with scissors and mixed with 2 ml of homogenization buffer. The homogenization buffer for liver samples was 50 mM Tris-HCl (pH 7.0 at 22°C) containing 150 mM KCl. For intestinal samples, the homogenization buffer used for preparation of hepatic microsomes was modified by the addition of 1 mM dithiothreitol, 1 mM EDTA, 10 pM leupeptin, 200 KIU aprotinin/ml (approximately 0.22 TIU/ml), 25 units heparin/ml, and 20% glycerol (v/v). The modifications to the homogenization buffer were incorporated in accordance with recommendations by various investigators (Tredger and Chhabra, 1976; Fang and Strobe], 1978; Jones et al.. 1980: Harada and Omura, 1983). Both liver and intestinal samples were homogenized with a Brinkman Polytron (Kinematica GMBH) and microsomes prepared by ultracentrifugation (100,OOOg for 60 min) of the postmitochondrial supernatant (10,OOOg for 20 min). The microsomes were washed by homogenization in 10 mM EDTA (pH 7.4) containing 150 mM KC1 and
EFFECTS OF INDUCERS re-isolated by ultracentrifugation. The final microsomal pellet was resuspended in 0.25 M sucrose (0.8 ml) and stored at -70°C. Determination of tissue UDPG and UDP-GA concentrations. Hepatic and intestinal concentrations of UDPG and UDP-GA were measured by isocratic, reverse-phase, ion-pair HPLC with uv absorbance detection (254 nm) modified from the method of Aw and Jones (1982). Samples were injected onto an Adsorbosphere HS Cr8-5 pm analytical HPLC-column (Alltech Assoc.. Inc., Deerfield, IL) and eluted with 100 mM potassium acetate buffer (pH 5.5) containing 0.01% n-octylamine (Aldrich Chemical Co.. Inc.) as the ion-pairing agent. A guard column packed with 5 pm Adsorbosphere HS CL8 silica (Alltech) and a saturator column containing 200/425 mesh Adsorbosil silica (Alltech) were included in the analytical system. Retention times of UDPG and UDP-GA were determined by cochromatography of standards purchased from Sigma Chemical Co. Quantitation of UDPG and UDP-GA were based upon integration of absorbance peak areas. The concentrations of UDPG and UDP-GA were determined from standard calibration curves for the respective agents. Determination of UDP-GT activity. Reaction mixtures and incubation conditions for the measurement of UDP-GT activity in vitro were essentially as described previously (Hazelton et al., 1985; Hjelle et al., 1985; Arlotto et al.. 1986). The final incubation volume was 0.5 ml and contained Tris-HC1 (200 mM. pH 7.7, at room temperature), magnesium chloride (10 mM). UDP-GA (4 mM), D-saccharic- I ,4-lactone (I .25 mM), microsomal protein (0.5-4.0 mg/ml), and aglycone (5 mM AA, 0.8 mivt HA, or I mM [“‘GINA, 0.2 $Zi/jd) at the final concentrations indicated. Hepatic microsomal UDPGT activity was assessedseparately for each animal. Due to the smaller yield of microsomal protein per gram of tissue, intestinal microsomes prepared from individual animals were pooled within a treatment group for the determination of UDP-GT activity. All assayswere performed in duplicate with two concentrations of microsomal protein at 37°C with mechanical agitation (90 cycles/min). Reactions were initiated by the addition of UDPGA after preincubation for 10 min and stopped after 20 min by the addition of 0.5 ml ice-cold ethanol. Unreacted [“‘C]NA (>99%) was extracted from the reaction mixture with two 5-ml volumes of chloroform and NA-glucuronide formation determined by liquid scintillation analysis ofthe aqueous phase (Hazelton et al., 1985). Unreacted AA was extracted with cyclohexanone saturated with calcium chloride (Hjelle et al., 1985) and AA-glucuronide formation assessedby HPLC analysis of the aqueous phase (Howie et al., 1977 as modified in Goon and Klaassen, 1990). In assaysinvolving HA as the aglycone, microsomes were solubilized in 0.01% Triton X-100 for I5 min prior to preincubation with the reaction mixture. The precipitate formed upon addition of ethanol was pelleted by centrifugation (10,OOOg for 5 min) and the HA-glucuronide content of the supernatant measured by thin-layer chromatographic separation with fluorometric detection (Mulder and Hagedoorn, 1974, as modified in Goon and Klaassen, 1991). Experiments and assaysinvolving HA were conducted under subdued, red lighting due to the sensitivity of HA to light. For both intestinal and hepatic microsomes, preliminary studies were performed with each aglycone to establish conditions under which glucuronide formation was proportional to protein concentration and incubation time. Microsomal protein concentrations were determined by the method of Lowry et al.. (1951) with bovine serum albumin as standard. Statistics. Results derived from experiments conducted with pooled samples (i.e., intestinal microsomes) represent the mean + standard deviation of duplicate assaysperformed at two protein concentrations. Data obtained from experiments performed with individual samples are expressed as means ? standard error (SE). Comparisons between treated groups and controls were conducted with Dunnett’s multiple comparison test. The acceptable level of statistically significant difference was set at p < 0.05 (Steel and Torrie. 1980). RESULTS
The effects of orally administered microsomal enzyme inducers upon hepatic and intestinal AA glucuronidation in
ON UDP-GT
255
AND UDP-GA
vitro are presented in Figs. 1A and 2A, respectively. Hepatic
microsomal UDP-GT activity directed toward AA was increased significantly by treatment with all inducing agents studied except BaP and 3MC (Fig. 1A). The formation of AA-glucuronide by hepatic microsomes was increased 20 to 40% in rats orally administered BHA, PB, TCDD, or TSO. The greatest increase in hepatic AA-glucuronide formation was observed in microsomes prepared from animals treated with PCN (80%). The various inducing agents elicited different effects upon UDP-GT activity directed toward AA in the intestine. AA-glucuronide formation by intestinal microsomes prepared from rats treated with BHA, BaP, or TSO was 250, 220, and 260% of control levels, respectively (Fig. 2A). Administration of 3MC produced minimal effects upon AA-glucuronide formation by intestinal microsomes. Paradoxically, treatment with PB, PCN, or TCDD in vivo decreased microsomal AA-glucuronidation in the intestine (25, 36, and 48% of control values, respectively). The formation of HA-glucuronide by hepatic and intestinal microsomes prepared from rats orally administered the various inducing agents is depicted in Figs. 1B and 2B, re-
HEPATIC AA:lJDP-GT
ACTNITY
HEPATIC HkUDP-GT
ACTIVIM
(A)
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i
15
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10
z e
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HEPATIC NkUDP-GT
ACTIVITY
l
03
150
2 0 2 8 I
100
50
CON
BHA
BP
3MC
PB
PCN
TCDD
TSO
FIG. 1. Effect of orally administered microsomal enzyme inducers upon the formation of acetaminophen-glucuronide (A), harmol-glucuronide (B), and I-naphthol-glucuronide (C) by hepatic microsomes. Results represent the mean i standard error of four or five rats. Individual assayswere performed for each animal within a treatment group. Assays were conducted in duplicate with two concentrations of microsomal protein. Asterisks indicate statistically significant difference (p < 0.05) from control.
256
GOON AND KLAASSEN 0.60
2 z
60
P k-l
40
INTESTINAL AAzUDP-GT
ACTIVITY
INTESTINAL
HkUDP-GT
ACTIVITY
INTESTINAL
NkUDP-GT
ACTIWY
(A)
_
(a
w
z 20
CON
BHA
BP
3MC
PB
PCN
TCDD
TSO
FIG. 2. Effect of orally administered microsomal enzyme inducers upon the formation of acetaminophen-glucuronide (A), harmol-glucuronide (B), and 1-naphthol-glucuronide (C) by intestinal microsomes. Results represent the mean f standard deviation of assays conducted in duplicate at two protein concentrations with pooled intestinal microsomes prepared from four or five rats per treatment group.
spectively. Hepatic UDP-GT activity directed toward HA was enhanced significantly following treatment with 3MC. PB, PCN, or TCDD (Fig. 1B). HA glucuronidation by hepatic microsomes was increased 20 to 40% after oral administration of 3MC, PB, or TCDD. Similar to AA, the greatest increase in hepatic HA-glucuronide formation was noted in microsomes prepared from rats treated with PCN (90%). Treatment with BHA, BaP, or TSO produced essentially no change in the glucuronidation of HA by hepatic microsomes. As seen in Fig. 2B, HA-glucuronide formation by intestinal microsomes was increased approximately 90% following administration of BHA, BaP. or TSO in vivo. Additionally, administration of 3MC increased the formation of HA-glucuronide by intestinal microsomes 30%. As was observed with AA, oral administration of PB, PCN, or TCDD decreased intestinal glucuronidation of HA in vitro (56, 77, and 76% of control levels, respectively). The activities of hepatic and intestinal microsomal UDPGT directed toward NA following oral administration of various microsomal enzyme inducers are presented in Fig. 1C and 2C, respectively. The formation of NA-glucuronide by hepatic microsomes was enhanced significantly by all in-
ducing agents studied except PB and PCN (Fig. 1C). Treatment with BHA, BaP, 3MC, and TSO increased hepatic microsomal NA-glucuronide formation 120,20,40, and I lo%, respectively. The greatest induction of hepatic UDP-GT activity was observed in microsomes prepared from rats orally administered TCDD, which exhibited a greater than 250% increase in NA glucuronidation. As noted with hepatic microsomes, intestinal NA glucuronidation in vitro was increased by all agents studied except PB and PCN (Fig. 2C). The greatest effects upon UDP-GT activity in the intestine were observed following oral administration of BHA, BaP, 3MC, or TSO, which enhanced microsomal formation of NA-glucuronide 80 to 120%. TCDD moderately increased intestinal NA glucuronidation in vitro (30%). PCN produced no change and PB slightly decreased NA-glucuronide formation ( I 1%) by intestinal microsomes. The effects of orally administered microsomal enzyme inducers upon hepatic and intestinal UDPG and UDP-GA concentrations are presented in Figs. 3 and 4. In the liver, the concentration of UDPG was increased nearly 50% following treatment with BHA (Fig. 3A). All other inducing agents studied (i.e., BaP, 3MC, PB, PCN, TCDD, and TSO)
600 HEPATIC
UDPG
HEPATIC
UDP-GA
6’)
600
*
0) * T
:
3MC
PB
200
CON
BHA
BaP
PCN TCDD
TSO
FIG. 3. Effect of orally administered microsomal enzyme inducers upon concentrations of UDPG (A) and UDP-GA (B) in the liver. Results represent the mean + standard error of four or five rats per treatment group. Asterisks indicate statistically significant difference (p < 0.05) from control.
EFFECTS OF INDUCERS
INTESTINAL UDPG
250
(4
*
3 z
150
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100
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100
INTESTINAL UDP-GA
@I
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0 CON
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BaP
3MC
PB
PCN TCDD
TSO
FIG. 4. Effect of orally administered microsomal enzyme inducers upon concentrations of UDPG (A) and UDP-GA (B) in the intestine. Results represent the mean + standard error of four or five rats per treatment group. Asterisks indicate statistically significant difference (p < 0.05) from control.
produced no significant perturbations in hepatic UDPG concentration. Hepatic UDP-GA concentrations were unaffected after oral administration of PCN or TCDD (Fig. 3B). In contrast, the concentration of UDP-GA in the liver was increased significantly after treatment with BHA, BaP, 3MC, PB, or TSO (40 to 80%). In the intestine, neither UDPG nor UDP-GA concentrations (Figs. 4A and 4B, respectively) were altered appreciably following oral administration of 3MC, PB, PCN, TCDD, or TSO. Conversely, significant increases in both intestinal UDPG and UDP-GA concentrations were noted in rats treated with either BHA (50 and 40%, respectively) or BaP (30 and 60%, respectively). DISCUSSION The results presented regarding the effects of 3MC and BaP upon the capacity of intestinal microsomes to glucuronidate NA concur with previous investigations (Aitio et al., 1972; Hietanen and Lang, 1978; Hietanen et al., 1980), which assessed intestinal glucuronidation of p-nitrophenol in vitro after pretreatment in vivo with 3MC or BaP. Both NA and
ON UDP-CT
AND UDP-GA
257
p-nitrophenol are classified as group 1 substrates for UDPGT, i.e., substrates whose conjugation with glucuronic acid is catalyzed by the 3MC-inducible form of UDP-GT (Bock et al., 1983). Furthermore, the results of the present study regarding NA-glucuronide formation by hepatic microsomes after oral administration of the various inducing agents correspond well with the previous work of Watkins et al. (1982) in which the inducers were administered by intraperitoneal injection. In both studies, NA-glucuronide formation by hepatic microsomes was enhanced significantly upon treatment with BaP. 3MC, TCDD, or TSO in vivo. However, intraperitoneal administration of BHA (500 mg/kg/day for 10 days) produced negligible effects upon hepatic NA glucuronidation (Watkins et al., 1982) whereas oral administration of BHA ( 1% of diet for 10 days) markedly increased NA-glucuronide formation by hepatic microsomes in the present study. Turner el uf. (1977) previously demonstrated increased intestinal NA glucuronidation in situ in rats pretreated with BaP. However, results of the present study demonstrate that, in addition to increasing UDP-GT activity, BaP treatment in vivo also significantly elevates endogenous UDPG and UDP-GA concentrations in the intestine. Thus, enhanced intestinal NA glucuronidation in situ following BaP administration cannot be attributed exclusively to induction of intestinal UDP-GT activity without prior knowledge of kinetic properties of intestinal UDP-GT; specifically, the relationship between endogenous UDP-GA concentration and the K,,, of UDP-GT for UDP-GA in the intestine. Similar to NA. HA is also classified as a group 1 UDPGT substrate (Koster, 1985). As expected, HA-glucuronide formation by hepatic microsomes prepared from 3MCtreated rats was significantly greater than control hepatic microsomes. In contrast, treatment with 3MC only slightly increased the glucuronidation of HA by intestinal microsomes. Moreover, the effects of the various microsomal enzyme inducers upon intestinal HA glucuronidation were extremely disparate from the effects produced upon hepatic HA glucuronidation. None of the inducing agents studied effectively increased HA glucuronidation by both hepatic and intestinal microsomes. A degree of disparity was also observed between intestinal and hepatic microsomal glucuronidation of AA in response to treatment with the various inducing agents. Intestinal AA glucuronidation was selectively enhanced by BaP administration, whereas treatment with PB, PCN, and TCDD selectively increased AA-glucuronide formation by hepatic microsomes. Unlike HA, however, oral administration of BHA or TSO increased AA glucuronidation by both intestinal and liver microsomes. In contrast to HA and AA, UDP-GT activity directed towards NA in hepatic and intestinal microsomes was affected similarly by the various microsomal enzyme inducers. All inducing agents that effectively increased hepatic NA glucuronidation (i.e., BHA, BaP, 3MC. TCDD, and TSO) also augmented formation of
258
GOON AND KLAASSEN
NA-glucuronide by intestinal microsomes, although the extent to which NA glucuronidation was increased differed between the two tissues. Administration of PB, PCN, and TCDD paradoxically decreased the formation of AA-glucuronide and HA-glucuronide by intestinal microsomes. With all three inducing agents, the decrease was more severe with AA as the aglycone. Intestinal microsomes prepared from PB-treated rats also demonstrated slightly decreased activity toward NA. The cause(s) for this observation is not known, however, the effect appears to be restricted to the intestine. Hepatic microsomal glucuronidation of AA and HA was enhanced significantly by treatment with these agents. In addition, formation of NA-glucuronide by liver microsomes was increased profoundly by TCDD treatment and was similar to control levels after treatment with PB or PCN. The significant increases in hepatic UDP-GA concentrations manifested upon oral administration of BHA, BaP. 3MC, PB, and TSO are in close agreement with the earlier work of Watkins and Klaassen ( 1983). Moreover, PCN minimally affected hepatic UDP-GA content in both studies. However, the two investigations are at odds with regard to the effect of TCDD upon the concentration of UDP-GA in the liver. Although similar dosages were studied (8 and 10 pg/kg), TCDD significantly increased the hepatic concentration of UDP-GA (1 .&fold) in the previous study, whereas the effect of TCDD upon hepatic UDP-GA content was negligible in the present study. The discrepancy between the two studies may be due in part to differences in the route of administration employed. In the former study, TCDD was administered intraperitoneally, whereas the oral route of administration was used in the present study. The results of the present study further illustrate the differences between hepatic and intestinal glucuronidation. In contrast to the liver, intestinal UDP-GA concentrations were less prone to alteration upon administration of the various microsomal enzyme inducers. Significant increases in UDPGA content of the intestine were noted only after administration of BHA or BaP, whereas all inducers studied, except PCN and TCDD, substantially increased hepatic UDP-GA concentrations. Thus, UDP-GA concentrations in the liver appear to be susceptible to a greater number and variety of microsomal enzyme inducers than in the intestine. The effect of microsomal enzyme inducers upon tissue UDPG concentration has not been reported previously. In the intestine, UDPG concentrations were increased by the same two agents that elicited significant increases in intestinal UDP-GA levels (i.e., BHA and BaP). In general, UDPG concentrations in the liver were more resilient than hepatic UDPGA concentrations. Specifically, only BHA produced an appreciable change in hepatic UDPG concentration, whereas hepatic UDP-GA levels were significantly increased by BHA. BaP, 3MC, PB, and TSO. These observations imply that the increases noted in hepatic UDP-GA concentrations following
treatment with various microsomal enzyme inducers (except BHA) may result from increased activity, or possibly induction, of UDP-glucose dehydrogenase, the cytosolic enzyme that catalyzes the formation of UDP-GA from UDPG, in the liver. Conversely, the coordinate increase in UDPG and UDP-GA in the intestine after administration of BHA or BaP, and in the liver upon administration of BHA, suggests that enhanced synthesis of UDPG may be causative in augmenting endogenous tissue concentrations of UDP-GA. Orally administered BHA, BaP, and TSO effectively enhanced intestinal microsomal UDP-GT activity directed towards all three aglycones studied (i.e., AA, HA, and NA). In addition, 3MC markedly increased the formation of NAglucuronide by intestinal microsomes, but only marginally affected the glucuronidation of AA and HA in vitro. Treatment of rats with BHA or BaP also altered intestinal cosubstrate levels in vivo. Specifically, intestinal concentrations of both UDP-GA and its immediate precursor, UDPG, were increased significantly following oral administration of BHA or BaP. In contrast, intestinal UDPG and UDP-GA concentrations were unaffected by oral administration of 3MC or TSO. Thus, of the four agents found to substantially augment the glucuronidation capacity of intestinal microsomes, 3MC and TSO were unique in effectively inducing intestinal UDPGT activity without appreciably altering the concentrations of UDP-GA or UDPG in the intestine. A similar effect in the liver was demonstrated following oral administration of PCN or TCDD. Specifically, PCN enhanced hepatic microsomal glucuronidation of AA and HA, whereas TCDD significantly increased hepatic UDP-GT activity directed toward all three substrates studied. Concurrently, hepatic UDPG and UDP-GA concentrations were unaffected by orally administered PCN or TCDD. In summary, the data presented indicate that orally administered microsomal enzyme inducers exert different effects upon the glucuronidation capacity of the intestine relative to the liver. These differences are manifested not only with regard to induced changes in UDP-GT activity directed toward the aglycones acetaminophen, harmol, and 1-naphthol, but also in terms of effects upon the concentration of UDP-GA in the two organs. As demonstrated previously in the liver, the present results show that significant increases in intestinal UDP-GA concentration may be produced after treatment with various microsomal enzyme inducers. However, it appears that hepatic UDP-GA content is susceptible to elevation by a greater variety of microsomal enzyme inducers than intestinal UDP-GA. Moreover, 3MC and TSO are unique agents capable of inducing intestinal UDP-GT activity without appreciably affecting intestinal UDP-GA concentration. Thus, 3MC and TSO are potentially powerful and valuable investigational tools for studying the mechanism(s) of capacity-limited glucuronidation in the rat intestine.
EFFECTS OF INDUCERS
ACKNOWLEDGMENTS The authors express their appreciation to Dr. Andrew Parkinson and Michael P. Arlotto for their guidance in the isolation and handling of hepatic and intestinal microsomes.
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ON UDP-GT
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