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Small Intestinal Cytochromes P450 Laurence S. Kaminsky & Michael J. Fasco Published online: 03 May 2015.
To cite this article: Laurence S. Kaminsky & Michael J. Fasco (1992) Small Intestinal Cytochromes P450, Critical Reviews in Toxicology, 21:6, 407-422 To link to this article: http://dx.doi.org/10.3109/10408449209089881
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Toxicology
1. INTRODUCTION
Small Intestinal Cytochromes P450
The small intestine provides a major route for exposure to xenobiotics via ingestion of food and liquids, and secondarily by swallowing inhaled xenobiotics after clearance from the tracheobronchial tree. Enzymatic biotransformation in intestinal mucosal cells has the potential to detoxify these generally hydrophobic xenobiotics by directly facilitating their excretion to the intestinal lumen, or by conjugation with subsequent excretion. This first-pass metabolism could substantially prevent many xenobiotics from being distributed throughout the body. However, the biotransformation could also potentially activate some xenobiotics, with toxic consequences for the organism. The predominant biotransfonnation system in the small intestine is the monooxygenase system, with the heme protein, cytochrome P450 (P450), as the terminal oxidase. This P450 system is primarily situated in the endoplasmic reticulum and accepts electrons from NADPH via a flavoprotein, NADPHP450 reductase. P450 exists as a superfamily of genes - 154 had been characterized by October, 1990, with 38 in rats.' To date, 27 gene families and numerous subfamilies have been described. A recently updated revision of the P450 nomenclature has been published and will be used whenever possible in this review. However, in some instances where identification of P450 forms is not clear when one uses this nomenclature, the nomenclature used in the original publication will be retained. Small intestinal P450s have been investigated in their own right, or as part of broader studies to assess extrahepatic P450s. There have been several reviews of aspects of intestinal P450s and intestinal microsomal metaboli~m.~-~ The most recent was published in 1986, prior to a surge in investigations of this system, and the current review emphasizes the more recent data. In this review, the status of small intestinal P450s with respect to regulation and metabolism is described, as well as the toxicologic consequences of the function of these enzymes.
Laurence S. Kaminsky, Ph.D. and Michael J. Fasco, Ph.D.
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ABSTRACT Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally -being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occumng in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics - with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin 0-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly - iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3 .Zfold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.
II. INTESTINAL MORPHOLOGY AND CYTOCHROME P450 LOCATION Knowledge of the morphology of the small intestine is essential for developing methods to evaluate the regulation of intestinal P450s and the toxicologic consequences. The small intestine is approximately 7 m in length in man and 90 cm in the rat.6 It comprises the duodenum, the jejunum, and the ileum, which are coiled to varying extents. In man, the small intestine contains submucosal folds, plicae circulares or folds Laurence S. Kaminsky, Ph.D. (corresponding author) and Michael J. Fasco, Ph.D., Wadsworth Center for Laboratories and Research, New York State Department of Health, P.O. Box 509, Albany, New York 12201-0509.
Key Words: cytochrome P450, small intestine, intestinal metabolism. 1992
407
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Critical Reviews In
I____
of Kerckring, which greatly increase the surface area, most prominently in the distal duodenum and proximal jejunum, but are absent in the distal ileum. The mucosal surface is studded with finger-like projections called villi (Figure 1). In rodents, the small intestine contains no plicae, but the villi are twice as long as that in man. The villi and plicae together greatly enhance the absorptive surface of the small intestine. The morphology of the small intestine can be altered by dietary factors. A high-fiber diet has been reported to broaden the villi.'
OPENING OF A CRYPT
I
ViLLl
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I
stitutive or induced. It has been demonstrated, by using a monoclonal antibody to P4503A3, that P450 content is greatest in human intestinal segments near the pylorus and is lower distally.* This major component of human small intestine was not detected in the large intestine.' A monoclonal antibody to P450 hA7, a P4503A form, has been used to probe the localization of human small intestinal P 4 5 0 ~ The . ~ columnar absorptive epithelial cells of the villus and the mast cells in the small intestine wall exhibited the strongest immunoreactivity, while no immunostaining was detected in the goblet cells or in the epithelial cells of the crypts.' Notwithstanding these reports of specific P4503A forms being present, the actual form or forms making this major contribution have still not been resolved. NADPH-P450 reductase has been localized by Western blotting techniques in human intestinal tissue, with the highest concentrations in the enterocytes, maximally at the tips of the villi. l o
-
d A M l N A PROPRIA
\ CRYPTS
OF LIEBERKUEHN
FIGURE 1, Human small intestine showing the villi and crypts.
At the base of the villi are the crypts of Lieberkuehn (Figure 1 1. The crypt epithelium comprises undifferentiated cells, gob-
let cells, enteroendocrine cells, and Paneth cells, while the villous epithelium also consists of enterocytes. The stem cells at the base of the crypt give rise to the columnar crypt cells, Paneth cells, goblet cells, and enteroendocrine cells.6 From stem cell division to appearance of the cell at the crypt surface takes approximately 4 days in man and 1 day in rodents. The period for these cells to move from the crypt surface to villous tip, while maturing to the various types of cells, is 3 days in man and 2 days in rodents. The rapidity of this migration and maturation complicates studies on the regulation of the P450 components and is probably a basis for the frequent contradictions in published reports.
-
A. Human Cytochromes P450 Location in Small Intestine Several P450 forms have been detected in human small intestine, but it could not be determined whether they are con408
B. Animal Cytochromes P450 Location in Small Intestine P4501A1 has been readily detected by immunoblots in untreated rat duodenum, but it was beneath detectable levels in the jejunum and ileum, while P4502B1 and 3A1/2 were detected in all three sections, also with highest levels in the duodenum." P4503A1/2 mRNA was also detected at substantial levels in the small intestine of untreated adult Holtzmann rats. l 2 In contrast, neither Western blotting nor enzyme activity measurements were reported to detect P4501A1I3 or 2B1 or 2B214 in untreated rat intestine. Intestinal P4501A1 was also reported to be absent from untreated rats," as probed by immunohistochemical techniques, and pretreatment with neither PB nor clofibrate increased its levels to above detection. However, in the case of P4502B1, localization techniques have demonstrated its presence in the enterocytes of untreated rat small intestine,I6 graded in quantity from crypt to maximally at the tips of villi." AHH and P450 concentration were determined in villous tip cells of sequential segments of the small intestine of rats fed commercial rat chow." The results are presented in Figure 2 and indicate that AHH activity is maximal in the segments closest to the pylorus, while P450 concentrations are maximal more distally - suggesting that various P450s may be differentially distributed along the length of the small intestine. P450 concentrations also varied along the villus - with a tenfold higher concentration at the villous tip relative to the crypt.'* Several studies have also been conducted to localize induced P450s in the intestine. In PB-induced rats, total P450 concentration and P4502B1 concentration varied along the length of the small intestine, being highest in the duodenum and lowest in the ileum (Table 1).19 In this study, P4502B1 concentration was 3.4-fold higher in the upper villus than in the lower villus and this P450 was not detected in the crypt." However, im-
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Tox icoIogy was also observed by using a polyclonal antibody to P4501A1 I in rats administered BNF i.p.'O AHH activity measurements o.20
were undertaken on rat intestinal cells isolated in ten fractions originating from the villous tip to the crypt.'' AHH activity was greatest in the midvillous region, slightly lower at the villous tip, and lowest in the crypt. This differential in activity was most noticeable when AHH activity was plotted against the percent total protein in each cell fraction.21These apparent discrepancies in the localization of P450s along the villus may be a consequence of differences in the length of times between the various observations and administration of the inducing agent. In many studies, hepatic and extrahepatic-specific P450s have been sought in the intestine, and in a few instances have, in fact, been detected. Thus, the mRNA for P450 ka-2, but not for P450 ka-1, is expressed constitutively in rabbit intestine.22Rabbit nasal P450s NMa and NMb are also not expressed in rabbit intestine.23Other P450 forms reported to be at levels beneath detection of immunoblotting techniques in untreated rat intestine are P4501A2, 2Cl1, and 2E1."
I0.15
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I
I
I
I
I
I
J
0.00
LENGTH FROM PYLORUS (cm)
FIGURE 2. P450 and AHH activities of rat intestinal, villous tip cells as a function of distance along intestine from pylorus. (a),AHH activity, (A), P450 concentration. (Data adapted from Hoensch, H., Woo, C. H., Raffin, S. B., and Schmid, R., Gastroenterology, 70, 1063, 1976.)
Rats are the most extensively studied species with respect to isolation methods, development, and characterization of intestinal epithelial cells and their microsomal components. The two principal methods employed for enterocyte isolation are scraping and elution of the intestinal mucosa, and extensive variations of these approaches exist in the literature. In the vast majority of experiments, intestinal segments ranging from 15 to 60 cm from the pyloric valve have been used as the cell source due to the relative paucity of P450 in the distal onethird of the small intestine. Protease inhibitors, such as soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, and metal chelators, such as EDTA, are commonly included in the buffer solutions used for cell isolation and homogenization. The proteases apparently responsible for the degradation of P450 have not been identified. In situ perfusion of the small intestine is also advantageous to remove blood contaminants, particularly hemoglobin. Indiscriminate scraping of enterocytes from the intestinal mucosa of the small intestine yields a mixture of villous tip, midvillous, and crypt cells, which combined exhibit a low specific content of P450 for reasons previously discussed. P420, arising by denaturation of cytochrome P450, and/or hemoglobin contamination are also disadvantages of this method.Iz Lindeskog et al.24 found that a substantial portion of the microsomal P450 in scraped enterocytes sedimented with the 10,000 X g pellet. Resuspension of the 10,OOO X g pellet followed by reisolation of the microsomal component by centrifugation increased the overall yield of P450. The specific
Table 1 Concentration of Total P450 and P450261 in Rat Intestinal Cells as a Function of Distance Along Small Intestine P4502B1 Total P450 (prnoUrng mierosomal (pmoUmg mierosornal protein) Distance along intestine protein) Proximal third Middle third Distal third
208.0 f 22.4 210.0 ? 40.2 99.9 2 34.0
116.0 137.0 50.4
f
6.4
?
24.2
111. ISOLATION OF ENTEROCYTES AND PREPARATION OF MICROSOMES
* 14.2
Data adapted from Reference 19.
munohistochemical localization using a monoclonal antibody to P4502B 1/2 revealed a strong immunostain along the length of the villus, from tip to crypt.I6 The reasons for the greater response of proximal than distal enterocytes to P450-inducing agents has not been resolved. Possibly the higher concentrations of pancreatic, biliary, and gastric secretions may play a role - but further study is necessary to resolve this question. A monoclonal antibody to rat P4501A1 was used to immunocytochemically probe small intestine from P-napththoflavone (BNF)-induced male rats. l5 When BNF was administered orally, immunoreactive P450 was evident throughout the length of the villus, but not in the crypts.I5 However, i.p. administration of BNF resulted in positive staining both along the villi and in the crypts.I5 Staining along the whole villus 1992
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Critical Reviews In P450 contents of scraped enterocytes from untreated and BNFinduced rats of 2 and 39 pmolimg microsomal protein, respectively, were determined by immunological methods, which do not discriminate between P450 and denatured P450 (P420) present in the preparations. Severe mucus contamination is also a potential disadvantage of this technique. ‘ 2 * 2 5 Hoensch et a1 , I 8 modified a previously published scraping technique26for the separation of villous tip, lower villous, and crypt cells. The tip cells were obtained by light hand scraping with a metal spatula, lower villus with increasing pressure, and crypt cells by abrasive scraping. At 1 h following 3Hthymidine administration, crypt cells isolated by this method contained the highest levels of radiolabel, followed by the lower villous and finally the villous tip cells. By 48 h after administration. a reverse gradient of radiolabel was obtained -- consistent with results obtained with elution methods and the fact that the crypt cells had differentiated and migrated to the villous tips. 27.2x The specific P450 microsomal contents from the three enterocyte populations were 1 15, 70. and 11 pmol/mg protein in the tip cells, the lower villous cells, and the crypt cells, respectively. Cytochrome P420 (P420) was a contaminant of only the crypt cell microsomes. Both cytochrome b, and NADPH-P450 reductase exhibited a declining concentration gradient from villous tip to crypt cell, but the slopes were not as steep as that of P450. The differential scraping technique has been used in a number of investigation^.^^-^^ Correia and co-workersLy~30 reported villous tip cell P450 concentrations in untreated and BNF-induced rats of approximately 24 and 150 pmol/mg microsomal protein, respectively. Stohs et al. ’I reported 1 1 pmolimg microsomal protein for untreated rats and 75 pmol/mg microsomal protein for 3-MC-induced rats. It is probable that these pronounced differences in the specific content of P450 are at least partially attributable to variations in the application of this scraping approach. In conjunction with hyaluronidase/collagenase treatment, Grafstrom et a1.12were able to obtain villous tip cells by this method that were maintained in culture long enough to permit metabolism studies, including conjugation. The numerous elution methods that have been used to remove various enterocyte populations from the small intestine can be categorized as enzymatic, vibrational, or chelator based. Invariably, the tip cells are removed first, followed by the midvillous and then the crypt cells. A number of enzymes have been used in such studies including tryp~in,’~ hyaluronida~e,~‘ and coifagenase.2xP450 was not characterized in many of these studies. Hartrnann et employed an in sifu collagenasevascular pcrfusion method to loosen the enterocytes from the small intestinal mucosa. The intestinal lumen was flushed at various times during the collagenase perfusion to obtain villous tip {IS rnin), midvillous (25 rnin), and lower villousicrypt cells ( 3 5 min). The isoiated cell populations were determined to be intact and viable by a number of criteria, including ultrastructural appearance. They synthesized heme and bilirubin in cul41 0
Volume 2
ture and were characterized as villous tip, midvillous, and crypt cells based on levels of alkaline phosphate and sucrase activities, glycoprotein formation, and 3H-thymidineincorporation. Each of these activities compared favorably with results obtained by W e i ~ e r , ~who ’ employed a chelation-elution method (see below) to obtain comparable enterocyte populations. The mean P450 concentrations for the villous tip, midvillous, and crypt cells were, respectively, 38, 15, and 10 pmol/mg microsomal protein. This represents one of the best characterized methods for isolation of the various enterocyte populations. It suffers from being tedious and too time-consuming for the preparation of large quantities of cells. Numerous vibrational methods for enterocyte preparation have also been d e ~ c r i b e d . ’ ~ . ~The ~ - ~technique ’ involves eversion of intestinal segments over steel rods which are placed in buffered solution, usually containing EDTA to facilitate cell detachment, and subjected to mechanical stress with a vrbromixer. By varying the degree of vibrational stress, villous and crypt cells can be ~eparated,~’ but the extent of cross-contamination of each fraction with the midvillous cells was not determined. Cells isolated by this method have not been as well characterized as those obtained by the collagenase perfusion and less vigorous elution method^,^^.^' although Shirk et al.37 reported a specific P450 content of approximately 3 1 pmol/mg microsomal protein in their “tip cell” preparations, which compares favorably with values obtained by the other methods cited above. The majority of nonvibrational elution techniques are modifications of the method of Weiser.” Incubation of the small intestinal lumen with phosphate-buffered citrate (solution A) at 37°C for 30 min, followed by several timed fillings and discharges with phosphate-buffered saline containing EDTA and dithiothreitol (solution B) at 37°C produced an elution gradient from villous tip to crypt cell. Each cell population was well resolved as assessed by alkaline phosphatase and sucrase activities, ’H-thymidine incorporation, and glycoprotein formation. A more rapid method of obtaining the villous tip cells was developed by Bonkovsky et al. l9 Incubation with solution A was performed as described.” The tip cells were separated from the villi by filling the intestinal segment with ice-cold solution B, followed by gentle tapping of the intestine with the fingers. The cells were harvested and the procedure was repeated twice more. Washing in a histidine buffer yielded a villous tip cell preparation that was essentially free of mucosal cells. The microsomal P450 was free of P420 contamination, and its specific content, determined by immunological methods, was approximatcly 70 pmolimg microsomal protein. Watkin5 et al.38 used a slightly modified technique with similar results. This approach is the most technically straightforward, inexpensive, and suitable for handling larger numbers of animals. It is not clear, however, whether the modifications of the original procedure have altered the separations of the various enterocyte populations.
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Toxicology Microsomes from enterocytes have been prepared essentially received the greatest attention. A summary of these studies is by methods developed for liver microsomes. The cells are provided in Table 2. disrupted by either homogeni~ation'~ or sonicationJ8in a suitThe influence of circadian variations and age on intestinal able buffer containing sucrose. The 10,000 x g supernatant P450s has been studied. In the rat, intestinal AHH activity is recovered and the microsomes separated by either pelleting achieved significant peaks at approximately 5:00 am and 7:OO at 105,000 x g or CaC1,-induced precipitation. pm, a pattern which differed from that of the liver.45Rabbits Small intestinal P450 from rabbits was first partially purified exhibited similar circadian patterns in the intestine, but with in 1979.39When reconstituted, it catalyzed the hydroxylation more limited peak-to-trough differences.45 The development of fatty acids. Subsequently, a combination of 6-amino-n-hexyl of P450 activities has been investigated in rabbit intestine.46 Sepherose 4B, CM-Sephadex C-50, and hydroxylapatite A variety of activities were undetectable for 2 days after birth chromatography produced three partially purified P450s from and remained low for 20 days. A rapid two- to fivefold increase rabbit intestine -one form metabolized hexadecane and other in microsomal P450 activities then occurred and adult levels fatty acids, particularly myristic acid, and benzo[a]pyrene were achieved at 30 days after birth. Activities decreased there(B[a]P).a*41 after until 50 days, and then rose to adult levels at 75 days. Three intestinal P450s (P450ia, ib, and ic) were more highly NADPH-P450 reductase followed a similar development patpurified from untreated rabbits and were reported to be contern.46Morphological proliferation observed during the period stitutive, although the feed composition was not i n d i ~ a t e d . ~ ~ . ~from ~ approximately 30 to 50 days may be associated with the P450ia had a specific activity of 10.6 nmol/mg protein, was decrease in intestinal activities during this period, but other putative causes such as dietary factors were not reported electrophoretically homogeneous with a molecular weight of 53,000, and catalyzed the metabolism of prostaglandins. P45Oib Intestinal P450 activity has been demonstrated to increase with and ic had molecular weights of 56,000 and 49,000, respecperinatal age in swine, but to a much lesser extent than in the liver. 47 tively, and both demethylated aminopyrine, benzphetamine, and N,N-dimethylaniline on recon~titution.~~ P45Oic exhibited A few studies have investigated animal species differences in intestinal P450 activities. When rats, mice, guinea pigs, and virtually identical properties to rabbit liver P450LM2 (P4502B4). 3-MC-induced rabbits yielded two purified intestinal P450s hamsters were compared, only rabbits and guinea pigs yielded ethylmorphine N-demethylase activity, while rabbits exhibited (P448a and P448b), which on reconstitution equivalently meThe same species were used to tabolized B[a]P, 7-ethoxycoumarin, and 7-etho~yresorufin.~~ the highest AHH investigate species differences in susceptibilities to PB and 3P448a was electrophoretically homogeneous, molecular weight MC induction of intestinal P450.49PB induced AHH activity 58,000,and was in the low spin state - probably P4501A1. in mice intestine only by 5-fold, and induced ECOD activity P44Sb had a molecular weight of 55,500. in mice, rats, and guinea pigs by 4-, 2 . 5 , and l.6-fold, respectively. 3-MC-induced AHH activity in rats, mice, guinea IV. INTESTINAL CYTOCHROME P450 pigs, and rabbits by 16-, 5-, 2.4-, and 0-fold, respectively, and ECOD by 12-fold in rats only.49In contrast to other studies, REGULATION these authors reported no difference in intestinal induction following i.p. or oral administration of agents. A number of factors combine to complicate investigations Several reports have demonstrated the capacity of 3-MC and of small intestinal P450 regulation. These include the relatively other polycyclic aromatics to induce intestinal P450s and asshort life of enterocytes, the marked effect of route of adminsociated catalytic activities. 2,3,7,8-Tetrachlorodibenzo-pistration of inducing agents, quantitative and possibly qualidioxin (TCDD) induced AHH activity in rats and tative variations in P450 along the length of the intestine, P4501A113,14s2 and EROD were induced by 3-MC variations in P450 between the crypt and villous tip, variations by greater than 70- and 4l-fold, respectively. P4501A2 was, in the procedures for preparation of enterocytes and their mihowever, not induced by 3-MC in rat intestine, and immucrosomes, and a significant role for undefined dietary factors noblotting using monoclonal antibodies confirmed the inducin intestinal P450 regulation. Together these factors provide a tion of P4501A1, and the absence of induction of P4501A2, basis for the many inconsistencies in reported experimental in rat52 and mouse54 intestine. In the mouse intestine, AHH findings on intestinal P450 regulation and function. and estradiol 2-hydroxylase activities were also coordinately induced by 3-MC.54 Several agents were compared as to the A. Xenobiotic Induction potency of their inducing capacities, based on intestinal AHH Probes of intestinal P450s have utilized Western and Northand EROD a c t i v i t i e ~The . ~ ~ results were reported as TCDD > ern blotting techniques as well as activity measurements. Rel3-MC > B[a]P > Aroclor 1254, but since the agents were not atively few xenobiotics have been investigated for their inadministered in equimolar doses these relative effectivenesses duction of intestinal P450s. Of these, PB and 3-MC have
1992
41 1
Critical Reviews In Table 2 induction of Intestinal P450s P450 Route of Inducing q e n r
adminitration
B[alP
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Estrone Estradiol Estriol Benz[a]anthracene Lkxamethasone
Species
P450
C57BU6N ntouse Rat Toad fish Female rat Female rat Female rat Mouse Rat
P4501 A 1
PCN N-Benzyl-lmidazole
Oral Gastric lavage
Rat Rat
N-[ 2-naphthylmethyl] imidazole Cimetidine Disulfiram
Gastric lavage
Rat
Oral Oral oral. s.c Oral Oral Oral 1.p. 1.p.
Rat Rat Rat Rat Rat Rat Rabbit Rabbit
1.P
C57BU6N mouse Rat
Cobalt
Ethanol Indole-3-carbinol Clofibrate Di(2-ethylhexyl) phthalate Isosafrole Sodium fluonde
Oral
TCDD BNF PCBs
Oral Oral
Aroclor 1242 Aroclor 1254
1.g.
Rat Rat Rat Rat Rat
3-MC
3-MC
PB
Rat Rat Rat Japanese quail Rat Rat
i.p. 1.p. Oral 1.p. i.p. i.p. Oral i.p. i.p. i.p.
Rat Rat C57BU6N mouse Guinea pig Rabbit
concentration (pmoVmg) AHH
59
AHH, EROD AHH AHH, ECOD AHH, ECOD AHH AHH Erythromycin demethylase
55 24 55 77, 122, 123 71 68 38
None EROD, p-nitroanisole, and erythromycin demethylase EROD, p-nitroanisole, and erythromycin demethylase AHH, ECOD AHH AHH AHH AHH, 2-amino-fluorene EROD, ECOD Prostaglandin A, w-hydroxylase Prostaglandin A, w-hydroxylase
70 67
Estradiol 2-hydroxylase
54 73
530
Aminopyrine N-demethylase, acetanilide hydrox ylase AHH, EROD AHH EROD, ECOD EROD, ECOD
103
AHH, EROD
55 84
58 370 80
248 P4503A
236 43.4
44 41 26 38
133 26 150
P450l A 1 P4502B 1 P4502B 1
589 75
I20 I16 P450l A I 260 340
Mouse Mouse Rat Rat Rat Rat Rabbit Guinea pig
P4502B I P4502B I
Ref.
Induced activity'
25 333
86 76 63 124 125 75 72 72
50, 55
64 29 61
69 AHH, EROD ECOD, phenacetin 0-deethylase AHH, ECOD AHH, EROD, ECOD, biphenyl 2- & 4-hydroxylase
55
56 49 70
7,12-Dimethylbenz(a]anthracene
15
Phenacetin 0-deethylase AHH, Estradiol-2-hydroxylase
14 49, 54
AHH
49 49 79 49
ECOD AHH, ECOD 7, I2-Dimethylbenz[a]anthracene EROD, ECOD ECOD
400
23 I
67
ECOD
15
69 70 49 49 49
BNF, P-naphthoflavone, PCB, pol Akbbreviations BlalP, benzo[a]pyrene, PCN, pregnenolone- 16a-carbonitnk TCDD, 2,3,7,R-tetrachlorodihenzo-p-d10~1n, ychlorinated biphenyl, 3-MC, 3-methylcholanthrene, PB, phenobarbital, AHH, aryl hydrocarbon hydroxylase, EROD, 7 ethoxyresoruftn Odcethyfase ECOD 7-ethoxycoumann 0-deethylase
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Toxicology are questionable. The rates of rat intestinal phenacetin O-deethylation and ECOD activities with microsomes from control and 3-MC-induced rats were m o n ~ p h a s i cin~ contrast ~ to the corresponding hepatic rates, which were biphasic. The K, values for both reactions were significantly lower in intestines of 3-MC-treated rats than in controls - implying that different enzymes were functioning in control and 3-MC-induced intestines.56 The time course of 3-MC induction of rat intestinal P450, as determined by AHH activity, has been in~estigated.~~ By 1 h after i.g. administration of 3-MC to rats, AHH activity was increased and this increase continued for up to 18 h. By 24 h after administration, substantial decreases in activity were ob~erved.~' The dose response for 3-MC induction of rat intestinal P450 is shown in Figure 3.57There were incremental increases in P450 concentration with increases in 3-MC administered up to 40 mg/kg. BNF (0.1 to 100 mg/kg diet, equivalent to approximately 0.01 to 10 mg/kg body weighvday) administered in the feed of rats for 7 days produced dose-dependent increases in intestinal ECOD and EROD activities from controls at all doses - increasing by 80- to 90-fold across the range.58At the doses used in this study, hepatic P450s were virtually unaffected, demonstrating that intestinal P450 is more sensitive to orally administered BNF. A role for the Ah receptor in the polycyclic aromatic hydrocarbon induction of intestinal P450 was confirmed by the induction of intestinal AHH activity in C57BU6N but not DBN2 mice by B[a]P.59 The latter mice do not express a functional Ah receptor. A number of studies have been undertaken to compare hepatic and intestinal P450s and their regulation. Intestinal P450s respond more rapidly than hepatic P450s to inducers, possibly because of more direct a~ailability.~' Comparisons of kinetic parameters (V,,,, K,) for untreated hepatic and intestinal microsomal metabolism of a variety of substrates (Table 3) showed significant and variable differences.60 These results suggested that induced forms of P450 in the two organs were functionally different. In the Japanese quail, orally administered 2,4,2',4'tetrachlorobiphenyl induced intestinal P450 3.3-fold, whereas 3,4,3',4'-tetrachlorobiphenyl was without effect. Both congeners produced marked induction in the liver.61A series of 15 flavonoids have been used to compare rat hepatic and intestinal microsomal P450s using ECOD activity as a probe.62
2oo
9
0 0
10
20
30
40
50
3-METHYLCHOLANTHRENE (mg/kg) FIGURE 3. Dose response for 3-MC induction of rat intestinal P450 concentrations. (O),24 h after administration; (@), 48 h after administration. (Data adapted from Born, P., Frankhuijzen Sierevogel, A,, and Noordhcek, J., Biochem. Pharmucol., 31, 3701, 1982.)
The flavones 5,6-benzoflavone, 7,8-benzoflavone, and flavone, when added to microsomal preparations, all increased hepatic activity but markedly inhibited intestinal activity; the remaining 12 flavonoids all inhibited both intestinal and hepatic microsomal metabolism. Oral or S.C.administration of cobalt to rats increased intestinal P450 and AHH activity and decreased the corresponding hepatic v a l u e ~The . ~ ~differential in 3-MC induction of intestinal P4501A1 and 1A2 described above indicates a regulatory difference between liver and intestine which also occurs with TCDD" and BNF15as inducing agents. Similar regulatory differences occurred with 2-methoxy-4-aminoazobenzene, which induced P4501A1 in the liver but not in the i n t e ~ t i n eand , ~ ~with tryptophan pyrolysate components in the diets of rats and mice, which induced both P4501A1 and 1A2 in liver, but neither in intestine.65P4502B1, but not 2B2, is expressed in the intestine, in contrast to the liver, where both are expressed.66A series of seven N-substituted imidazoles have been used to compare hepatic and extrahepatic P450
1992
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Critical Reviews In Table 3 Kinetic Values for Rat Hepatic and Intestinal Microsomal Metabolism Intestine
Liver Km
Substrate
v,
Km
(10-5 x M)
(nmoYmirdmg)
6.2 1.7
0.123 0.24
7-Ethoxycoumarin Biphenyl Benzphetamine
1.3
v,,,
(lo-' x M)
(nmoYmirdmg)
1.9
0.8 3.3 13
17
Data adapted from Reference 60.
induction Two of the imidazoles produced fivefold increases in rat intestinal P450 concentrations. However, overall the imidazoles differentially affected hepatic and intestinal P ~ ~ O S , ~ ' with clotrimazole being the most effective hepatic inducer and without any effect in the intestine. The ratios of intestinal to hepatic AHH activities after i.p. administration to a variety of mouse strains was very variable - further proof for differing P450 regulatory mechanisms for these two tissues.6R Western blotting and activity measurements have detected intestinal P4502B1 induction in rats treated with PB," but P4502B2 could only be detected by activity measurements. l 4 The expression of P4502B1 and 2B2 in rat intestinal microsomes was investigated following i.g. or i.p. administration of PB and several environmental contaminants including Aroclor 1254, Firemaster BP, y-chlordane. and trans-n~nachlor.~~ Dot blots were performed using 32P-labeledcDNA which recognized both P~SOS,while Northern blots were performed using oligonucleotide probes specific for each of the P450s. The cDNA probe showed marked induction of P4502B112 mRNA predominantly in proximal and also in distal small intestine equivalently after i.g. or i.p. administration of all of these agents. By using the oligonucleotide probes, low levels on P4502B2 mRNA were detected in contro1 intestine, but this was not induced by PB or Aroclor 1254. However. the weak band for P4502B 1, also detected in control intestine, was markedly induced by PB and Aroclor 1254. Thus, regulation of P4502B 1 and 2B2 in the intestine differs from that in the liver.6y However, in studies with polymerase chain reaction amplification only 2BI and not 2B2 sequences were amplified from small intestinal RNA." These results indicated that P4502B 1 was induced in rat intestine by an increase in gene transcription following i.p. administration of PB. Induction was rapid mRNA was increased as early as 1 h after administration of a single i.p. dose of PB and was markedly induced by 6 h. No further increases were noted by 12 h after administration of PB and levels were equivalent to that noted after 4 days of consecutive administration of PB. The amplified segment of thc PR-inducible P450 mRNA in intestine was identical to the same segment of the hepatic mRNA.% Intestinal P4SOs were probed in rats, which were either un-
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Critical Reviews in Toxicology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/itxc20
Small Intestinal Cytochromes P450 Laurence S. Kaminsky & Michael J. Fasco Published online: 03 May 2015.
To cite this article: Laurence S. Kaminsky & Michael J. Fasco (1992) Small Intestinal Cytochromes P450, Critical Reviews in Toxicology, 21:6, 407-422 To link to this article: http://dx.doi.org/10.3109/10408449209089881
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Toxicology
1. INTRODUCTION
Small Intestinal Cytochromes P450
The small intestine provides a major route for exposure to xenobiotics via ingestion of food and liquids, and secondarily by swallowing inhaled xenobiotics after clearance from the tracheobronchial tree. Enzymatic biotransformation in intestinal mucosal cells has the potential to detoxify these generally hydrophobic xenobiotics by directly facilitating their excretion to the intestinal lumen, or by conjugation with subsequent excretion. This first-pass metabolism could substantially prevent many xenobiotics from being distributed throughout the body. However, the biotransformation could also potentially activate some xenobiotics, with toxic consequences for the organism. The predominant biotransfonnation system in the small intestine is the monooxygenase system, with the heme protein, cytochrome P450 (P450), as the terminal oxidase. This P450 system is primarily situated in the endoplasmic reticulum and accepts electrons from NADPH via a flavoprotein, NADPHP450 reductase. P450 exists as a superfamily of genes - 154 had been characterized by October, 1990, with 38 in rats.' To date, 27 gene families and numerous subfamilies have been described. A recently updated revision of the P450 nomenclature has been published and will be used whenever possible in this review. However, in some instances where identification of P450 forms is not clear when one uses this nomenclature, the nomenclature used in the original publication will be retained. Small intestinal P450s have been investigated in their own right, or as part of broader studies to assess extrahepatic P450s. There have been several reviews of aspects of intestinal P450s and intestinal microsomal metaboli~m.~-~ The most recent was published in 1986, prior to a surge in investigations of this system, and the current review emphasizes the more recent data. In this review, the status of small intestinal P450s with respect to regulation and metabolism is described, as well as the toxicologic consequences of the function of these enzymes.
Laurence S. Kaminsky, Ph.D. and Michael J. Fasco, Ph.D.
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ABSTRACT Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally -being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occumng in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics - with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin 0-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly - iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3 .Zfold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.
II. INTESTINAL MORPHOLOGY AND CYTOCHROME P450 LOCATION Knowledge of the morphology of the small intestine is essential for developing methods to evaluate the regulation of intestinal P450s and the toxicologic consequences. The small intestine is approximately 7 m in length in man and 90 cm in the rat.6 It comprises the duodenum, the jejunum, and the ileum, which are coiled to varying extents. In man, the small intestine contains submucosal folds, plicae circulares or folds Laurence S. Kaminsky, Ph.D. (corresponding author) and Michael J. Fasco, Ph.D., Wadsworth Center for Laboratories and Research, New York State Department of Health, P.O. Box 509, Albany, New York 12201-0509.
Key Words: cytochrome P450, small intestine, intestinal metabolism. 1992
407
-
Critical Reviews In
I____
of Kerckring, which greatly increase the surface area, most prominently in the distal duodenum and proximal jejunum, but are absent in the distal ileum. The mucosal surface is studded with finger-like projections called villi (Figure 1). In rodents, the small intestine contains no plicae, but the villi are twice as long as that in man. The villi and plicae together greatly enhance the absorptive surface of the small intestine. The morphology of the small intestine can be altered by dietary factors. A high-fiber diet has been reported to broaden the villi.'
OPENING OF A CRYPT
I
ViLLl
I
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I
stitutive or induced. It has been demonstrated, by using a monoclonal antibody to P4503A3, that P450 content is greatest in human intestinal segments near the pylorus and is lower distally.* This major component of human small intestine was not detected in the large intestine.' A monoclonal antibody to P450 hA7, a P4503A form, has been used to probe the localization of human small intestinal P 4 5 0 ~ The . ~ columnar absorptive epithelial cells of the villus and the mast cells in the small intestine wall exhibited the strongest immunoreactivity, while no immunostaining was detected in the goblet cells or in the epithelial cells of the crypts.' Notwithstanding these reports of specific P4503A forms being present, the actual form or forms making this major contribution have still not been resolved. NADPH-P450 reductase has been localized by Western blotting techniques in human intestinal tissue, with the highest concentrations in the enterocytes, maximally at the tips of the villi. l o
-
d A M l N A PROPRIA
\ CRYPTS
OF LIEBERKUEHN
FIGURE 1, Human small intestine showing the villi and crypts.
At the base of the villi are the crypts of Lieberkuehn (Figure 1 1. The crypt epithelium comprises undifferentiated cells, gob-
let cells, enteroendocrine cells, and Paneth cells, while the villous epithelium also consists of enterocytes. The stem cells at the base of the crypt give rise to the columnar crypt cells, Paneth cells, goblet cells, and enteroendocrine cells.6 From stem cell division to appearance of the cell at the crypt surface takes approximately 4 days in man and 1 day in rodents. The period for these cells to move from the crypt surface to villous tip, while maturing to the various types of cells, is 3 days in man and 2 days in rodents. The rapidity of this migration and maturation complicates studies on the regulation of the P450 components and is probably a basis for the frequent contradictions in published reports.
-
A. Human Cytochromes P450 Location in Small Intestine Several P450 forms have been detected in human small intestine, but it could not be determined whether they are con408
B. Animal Cytochromes P450 Location in Small Intestine P4501A1 has been readily detected by immunoblots in untreated rat duodenum, but it was beneath detectable levels in the jejunum and ileum, while P4502B1 and 3A1/2 were detected in all three sections, also with highest levels in the duodenum." P4503A1/2 mRNA was also detected at substantial levels in the small intestine of untreated adult Holtzmann rats. l 2 In contrast, neither Western blotting nor enzyme activity measurements were reported to detect P4501A1I3 or 2B1 or 2B214 in untreated rat intestine. Intestinal P4501A1 was also reported to be absent from untreated rats," as probed by immunohistochemical techniques, and pretreatment with neither PB nor clofibrate increased its levels to above detection. However, in the case of P4502B1, localization techniques have demonstrated its presence in the enterocytes of untreated rat small intestine,I6 graded in quantity from crypt to maximally at the tips of villi." AHH and P450 concentration were determined in villous tip cells of sequential segments of the small intestine of rats fed commercial rat chow." The results are presented in Figure 2 and indicate that AHH activity is maximal in the segments closest to the pylorus, while P450 concentrations are maximal more distally - suggesting that various P450s may be differentially distributed along the length of the small intestine. P450 concentrations also varied along the villus - with a tenfold higher concentration at the villous tip relative to the crypt.'* Several studies have also been conducted to localize induced P450s in the intestine. In PB-induced rats, total P450 concentration and P4502B1 concentration varied along the length of the small intestine, being highest in the duodenum and lowest in the ileum (Table 1).19 In this study, P4502B1 concentration was 3.4-fold higher in the upper villus than in the lower villus and this P450 was not detected in the crypt." However, im-
Volume 21, Issue 6
Tox icoIogy was also observed by using a polyclonal antibody to P4501A1 I in rats administered BNF i.p.'O AHH activity measurements o.20
were undertaken on rat intestinal cells isolated in ten fractions originating from the villous tip to the crypt.'' AHH activity was greatest in the midvillous region, slightly lower at the villous tip, and lowest in the crypt. This differential in activity was most noticeable when AHH activity was plotted against the percent total protein in each cell fraction.21These apparent discrepancies in the localization of P450s along the villus may be a consequence of differences in the length of times between the various observations and administration of the inducing agent. In many studies, hepatic and extrahepatic-specific P450s have been sought in the intestine, and in a few instances have, in fact, been detected. Thus, the mRNA for P450 ka-2, but not for P450 ka-1, is expressed constitutively in rabbit intestine.22Rabbit nasal P450s NMa and NMb are also not expressed in rabbit intestine.23Other P450 forms reported to be at levels beneath detection of immunoblotting techniques in untreated rat intestine are P4501A2, 2Cl1, and 2E1."
I0.15
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\
I
I
I
I
I
I
J
0.00
LENGTH FROM PYLORUS (cm)
FIGURE 2. P450 and AHH activities of rat intestinal, villous tip cells as a function of distance along intestine from pylorus. (a),AHH activity, (A), P450 concentration. (Data adapted from Hoensch, H., Woo, C. H., Raffin, S. B., and Schmid, R., Gastroenterology, 70, 1063, 1976.)
Rats are the most extensively studied species with respect to isolation methods, development, and characterization of intestinal epithelial cells and their microsomal components. The two principal methods employed for enterocyte isolation are scraping and elution of the intestinal mucosa, and extensive variations of these approaches exist in the literature. In the vast majority of experiments, intestinal segments ranging from 15 to 60 cm from the pyloric valve have been used as the cell source due to the relative paucity of P450 in the distal onethird of the small intestine. Protease inhibitors, such as soybean trypsin inhibitor and phenylmethylsulfonyl fluoride, and metal chelators, such as EDTA, are commonly included in the buffer solutions used for cell isolation and homogenization. The proteases apparently responsible for the degradation of P450 have not been identified. In situ perfusion of the small intestine is also advantageous to remove blood contaminants, particularly hemoglobin. Indiscriminate scraping of enterocytes from the intestinal mucosa of the small intestine yields a mixture of villous tip, midvillous, and crypt cells, which combined exhibit a low specific content of P450 for reasons previously discussed. P420, arising by denaturation of cytochrome P450, and/or hemoglobin contamination are also disadvantages of this method.Iz Lindeskog et al.24 found that a substantial portion of the microsomal P450 in scraped enterocytes sedimented with the 10,000 X g pellet. Resuspension of the 10,OOO X g pellet followed by reisolation of the microsomal component by centrifugation increased the overall yield of P450. The specific
Table 1 Concentration of Total P450 and P450261 in Rat Intestinal Cells as a Function of Distance Along Small Intestine P4502B1 Total P450 (prnoUrng mierosomal (pmoUmg mierosornal protein) Distance along intestine protein) Proximal third Middle third Distal third
208.0 f 22.4 210.0 ? 40.2 99.9 2 34.0
116.0 137.0 50.4
f
6.4
?
24.2
111. ISOLATION OF ENTEROCYTES AND PREPARATION OF MICROSOMES
* 14.2
Data adapted from Reference 19.
munohistochemical localization using a monoclonal antibody to P4502B 1/2 revealed a strong immunostain along the length of the villus, from tip to crypt.I6 The reasons for the greater response of proximal than distal enterocytes to P450-inducing agents has not been resolved. Possibly the higher concentrations of pancreatic, biliary, and gastric secretions may play a role - but further study is necessary to resolve this question. A monoclonal antibody to rat P4501A1 was used to immunocytochemically probe small intestine from P-napththoflavone (BNF)-induced male rats. l5 When BNF was administered orally, immunoreactive P450 was evident throughout the length of the villus, but not in the crypts.I5 However, i.p. administration of BNF resulted in positive staining both along the villi and in the crypts.I5 Staining along the whole villus 1992
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Critical Reviews In P450 contents of scraped enterocytes from untreated and BNFinduced rats of 2 and 39 pmolimg microsomal protein, respectively, were determined by immunological methods, which do not discriminate between P450 and denatured P450 (P420) present in the preparations. Severe mucus contamination is also a potential disadvantage of this technique. ‘ 2 * 2 5 Hoensch et a1 , I 8 modified a previously published scraping technique26for the separation of villous tip, lower villous, and crypt cells. The tip cells were obtained by light hand scraping with a metal spatula, lower villus with increasing pressure, and crypt cells by abrasive scraping. At 1 h following 3Hthymidine administration, crypt cells isolated by this method contained the highest levels of radiolabel, followed by the lower villous and finally the villous tip cells. By 48 h after administration. a reverse gradient of radiolabel was obtained -- consistent with results obtained with elution methods and the fact that the crypt cells had differentiated and migrated to the villous tips. 27.2x The specific P450 microsomal contents from the three enterocyte populations were 1 15, 70. and 11 pmol/mg protein in the tip cells, the lower villous cells, and the crypt cells, respectively. Cytochrome P420 (P420) was a contaminant of only the crypt cell microsomes. Both cytochrome b, and NADPH-P450 reductase exhibited a declining concentration gradient from villous tip to crypt cell, but the slopes were not as steep as that of P450. The differential scraping technique has been used in a number of investigation^.^^-^^ Correia and co-workersLy~30 reported villous tip cell P450 concentrations in untreated and BNF-induced rats of approximately 24 and 150 pmol/mg microsomal protein, respectively. Stohs et al. ’I reported 1 1 pmolimg microsomal protein for untreated rats and 75 pmol/mg microsomal protein for 3-MC-induced rats. It is probable that these pronounced differences in the specific content of P450 are at least partially attributable to variations in the application of this scraping approach. In conjunction with hyaluronidase/collagenase treatment, Grafstrom et a1.12were able to obtain villous tip cells by this method that were maintained in culture long enough to permit metabolism studies, including conjugation. The numerous elution methods that have been used to remove various enterocyte populations from the small intestine can be categorized as enzymatic, vibrational, or chelator based. Invariably, the tip cells are removed first, followed by the midvillous and then the crypt cells. A number of enzymes have been used in such studies including tryp~in,’~ hyaluronida~e,~‘ and coifagenase.2xP450 was not characterized in many of these studies. Hartrnann et employed an in sifu collagenasevascular pcrfusion method to loosen the enterocytes from the small intestinal mucosa. The intestinal lumen was flushed at various times during the collagenase perfusion to obtain villous tip {IS rnin), midvillous (25 rnin), and lower villousicrypt cells ( 3 5 min). The isoiated cell populations were determined to be intact and viable by a number of criteria, including ultrastructural appearance. They synthesized heme and bilirubin in cul41 0
Volume 2
ture and were characterized as villous tip, midvillous, and crypt cells based on levels of alkaline phosphate and sucrase activities, glycoprotein formation, and 3H-thymidineincorporation. Each of these activities compared favorably with results obtained by W e i ~ e r , ~who ’ employed a chelation-elution method (see below) to obtain comparable enterocyte populations. The mean P450 concentrations for the villous tip, midvillous, and crypt cells were, respectively, 38, 15, and 10 pmol/mg microsomal protein. This represents one of the best characterized methods for isolation of the various enterocyte populations. It suffers from being tedious and too time-consuming for the preparation of large quantities of cells. Numerous vibrational methods for enterocyte preparation have also been d e ~ c r i b e d . ’ ~ . ~The ~ - ~technique ’ involves eversion of intestinal segments over steel rods which are placed in buffered solution, usually containing EDTA to facilitate cell detachment, and subjected to mechanical stress with a vrbromixer. By varying the degree of vibrational stress, villous and crypt cells can be ~eparated,~’ but the extent of cross-contamination of each fraction with the midvillous cells was not determined. Cells isolated by this method have not been as well characterized as those obtained by the collagenase perfusion and less vigorous elution method^,^^.^' although Shirk et al.37 reported a specific P450 content of approximately 3 1 pmol/mg microsomal protein in their “tip cell” preparations, which compares favorably with values obtained by the other methods cited above. The majority of nonvibrational elution techniques are modifications of the method of Weiser.” Incubation of the small intestinal lumen with phosphate-buffered citrate (solution A) at 37°C for 30 min, followed by several timed fillings and discharges with phosphate-buffered saline containing EDTA and dithiothreitol (solution B) at 37°C produced an elution gradient from villous tip to crypt cell. Each cell population was well resolved as assessed by alkaline phosphatase and sucrase activities, ’H-thymidine incorporation, and glycoprotein formation. A more rapid method of obtaining the villous tip cells was developed by Bonkovsky et al. l9 Incubation with solution A was performed as described.” The tip cells were separated from the villi by filling the intestinal segment with ice-cold solution B, followed by gentle tapping of the intestine with the fingers. The cells were harvested and the procedure was repeated twice more. Washing in a histidine buffer yielded a villous tip cell preparation that was essentially free of mucosal cells. The microsomal P450 was free of P420 contamination, and its specific content, determined by immunological methods, was approximatcly 70 pmolimg microsomal protein. Watkin5 et al.38 used a slightly modified technique with similar results. This approach is the most technically straightforward, inexpensive, and suitable for handling larger numbers of animals. It is not clear, however, whether the modifications of the original procedure have altered the separations of the various enterocyte populations.
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Toxicology Microsomes from enterocytes have been prepared essentially received the greatest attention. A summary of these studies is by methods developed for liver microsomes. The cells are provided in Table 2. disrupted by either homogeni~ation'~ or sonicationJ8in a suitThe influence of circadian variations and age on intestinal able buffer containing sucrose. The 10,000 x g supernatant P450s has been studied. In the rat, intestinal AHH activity is recovered and the microsomes separated by either pelleting achieved significant peaks at approximately 5:00 am and 7:OO at 105,000 x g or CaC1,-induced precipitation. pm, a pattern which differed from that of the liver.45Rabbits Small intestinal P450 from rabbits was first partially purified exhibited similar circadian patterns in the intestine, but with in 1979.39When reconstituted, it catalyzed the hydroxylation more limited peak-to-trough differences.45 The development of fatty acids. Subsequently, a combination of 6-amino-n-hexyl of P450 activities has been investigated in rabbit intestine.46 Sepherose 4B, CM-Sephadex C-50, and hydroxylapatite A variety of activities were undetectable for 2 days after birth chromatography produced three partially purified P450s from and remained low for 20 days. A rapid two- to fivefold increase rabbit intestine -one form metabolized hexadecane and other in microsomal P450 activities then occurred and adult levels fatty acids, particularly myristic acid, and benzo[a]pyrene were achieved at 30 days after birth. Activities decreased there(B[a]P).a*41 after until 50 days, and then rose to adult levels at 75 days. Three intestinal P450s (P450ia, ib, and ic) were more highly NADPH-P450 reductase followed a similar development patpurified from untreated rabbits and were reported to be contern.46Morphological proliferation observed during the period stitutive, although the feed composition was not i n d i ~ a t e d . ~ ~ . ~from ~ approximately 30 to 50 days may be associated with the P450ia had a specific activity of 10.6 nmol/mg protein, was decrease in intestinal activities during this period, but other putative causes such as dietary factors were not reported electrophoretically homogeneous with a molecular weight of 53,000, and catalyzed the metabolism of prostaglandins. P45Oib Intestinal P450 activity has been demonstrated to increase with and ic had molecular weights of 56,000 and 49,000, respecperinatal age in swine, but to a much lesser extent than in the liver. 47 tively, and both demethylated aminopyrine, benzphetamine, and N,N-dimethylaniline on recon~titution.~~ P45Oic exhibited A few studies have investigated animal species differences in intestinal P450 activities. When rats, mice, guinea pigs, and virtually identical properties to rabbit liver P450LM2 (P4502B4). 3-MC-induced rabbits yielded two purified intestinal P450s hamsters were compared, only rabbits and guinea pigs yielded ethylmorphine N-demethylase activity, while rabbits exhibited (P448a and P448b), which on reconstitution equivalently meThe same species were used to tabolized B[a]P, 7-ethoxycoumarin, and 7-etho~yresorufin.~~ the highest AHH investigate species differences in susceptibilities to PB and 3P448a was electrophoretically homogeneous, molecular weight MC induction of intestinal P450.49PB induced AHH activity 58,000,and was in the low spin state - probably P4501A1. in mice intestine only by 5-fold, and induced ECOD activity P44Sb had a molecular weight of 55,500. in mice, rats, and guinea pigs by 4-, 2 . 5 , and l.6-fold, respectively. 3-MC-induced AHH activity in rats, mice, guinea IV. INTESTINAL CYTOCHROME P450 pigs, and rabbits by 16-, 5-, 2.4-, and 0-fold, respectively, and ECOD by 12-fold in rats only.49In contrast to other studies, REGULATION these authors reported no difference in intestinal induction following i.p. or oral administration of agents. A number of factors combine to complicate investigations Several reports have demonstrated the capacity of 3-MC and of small intestinal P450 regulation. These include the relatively other polycyclic aromatics to induce intestinal P450s and asshort life of enterocytes, the marked effect of route of adminsociated catalytic activities. 2,3,7,8-Tetrachlorodibenzo-pistration of inducing agents, quantitative and possibly qualidioxin (TCDD) induced AHH activity in rats and tative variations in P450 along the length of the intestine, P4501A113,14s2 and EROD were induced by 3-MC variations in P450 between the crypt and villous tip, variations by greater than 70- and 4l-fold, respectively. P4501A2 was, in the procedures for preparation of enterocytes and their mihowever, not induced by 3-MC in rat intestine, and immucrosomes, and a significant role for undefined dietary factors noblotting using monoclonal antibodies confirmed the inducin intestinal P450 regulation. Together these factors provide a tion of P4501A1, and the absence of induction of P4501A2, basis for the many inconsistencies in reported experimental in rat52 and mouse54 intestine. In the mouse intestine, AHH findings on intestinal P450 regulation and function. and estradiol 2-hydroxylase activities were also coordinately induced by 3-MC.54 Several agents were compared as to the A. Xenobiotic Induction potency of their inducing capacities, based on intestinal AHH Probes of intestinal P450s have utilized Western and Northand EROD a c t i v i t i e ~The . ~ ~ results were reported as TCDD > ern blotting techniques as well as activity measurements. Rel3-MC > B[a]P > Aroclor 1254, but since the agents were not atively few xenobiotics have been investigated for their inadministered in equimolar doses these relative effectivenesses duction of intestinal P450s. Of these, PB and 3-MC have
1992
41 1
Critical Reviews In Table 2 induction of Intestinal P450s P450 Route of Inducing q e n r
adminitration
B[alP
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Estrone Estradiol Estriol Benz[a]anthracene Lkxamethasone
Species
P450
C57BU6N ntouse Rat Toad fish Female rat Female rat Female rat Mouse Rat
P4501 A 1
PCN N-Benzyl-lmidazole
Oral Gastric lavage
Rat Rat
N-[ 2-naphthylmethyl] imidazole Cimetidine Disulfiram
Gastric lavage
Rat
Oral Oral oral. s.c Oral Oral Oral 1.p. 1.p.
Rat Rat Rat Rat Rat Rat Rabbit Rabbit
1.P
C57BU6N mouse Rat
Cobalt
Ethanol Indole-3-carbinol Clofibrate Di(2-ethylhexyl) phthalate Isosafrole Sodium fluonde
Oral
TCDD BNF PCBs
Oral Oral
Aroclor 1242 Aroclor 1254
1.g.
Rat Rat Rat Rat Rat
3-MC
3-MC
PB
Rat Rat Rat Japanese quail Rat Rat
i.p. 1.p. Oral 1.p. i.p. i.p. Oral i.p. i.p. i.p.
Rat Rat C57BU6N mouse Guinea pig Rabbit
concentration (pmoVmg) AHH
59
AHH, EROD AHH AHH, ECOD AHH, ECOD AHH AHH Erythromycin demethylase
55 24 55 77, 122, 123 71 68 38
None EROD, p-nitroanisole, and erythromycin demethylase EROD, p-nitroanisole, and erythromycin demethylase AHH, ECOD AHH AHH AHH AHH, 2-amino-fluorene EROD, ECOD Prostaglandin A, w-hydroxylase Prostaglandin A, w-hydroxylase
70 67
Estradiol 2-hydroxylase
54 73
530
Aminopyrine N-demethylase, acetanilide hydrox ylase AHH, EROD AHH EROD, ECOD EROD, ECOD
103
AHH, EROD
55 84
58 370 80
248 P4503A
236 43.4
44 41 26 38
133 26 150
P450l A 1 P4502B 1 P4502B 1
589 75
I20 I16 P450l A I 260 340
Mouse Mouse Rat Rat Rat Rat Rabbit Guinea pig
P4502B I P4502B I
Ref.
Induced activity'
25 333
86 76 63 124 125 75 72 72
50, 55
64 29 61
69 AHH, EROD ECOD, phenacetin 0-deethylase AHH, ECOD AHH, EROD, ECOD, biphenyl 2- & 4-hydroxylase
55
56 49 70
7,12-Dimethylbenz(a]anthracene
15
Phenacetin 0-deethylase AHH, Estradiol-2-hydroxylase
14 49, 54
AHH
49 49 79 49
ECOD AHH, ECOD 7, I2-Dimethylbenz[a]anthracene EROD, ECOD ECOD
400
23 I
67
ECOD
15
69 70 49 49 49
BNF, P-naphthoflavone, PCB, pol Akbbreviations BlalP, benzo[a]pyrene, PCN, pregnenolone- 16a-carbonitnk TCDD, 2,3,7,R-tetrachlorodihenzo-p-d10~1n, ychlorinated biphenyl, 3-MC, 3-methylcholanthrene, PB, phenobarbital, AHH, aryl hydrocarbon hydroxylase, EROD, 7 ethoxyresoruftn Odcethyfase ECOD 7-ethoxycoumann 0-deethylase
41 2
Volume 21, Issue 6
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Toxicology are questionable. The rates of rat intestinal phenacetin O-deethylation and ECOD activities with microsomes from control and 3-MC-induced rats were m o n ~ p h a s i cin~ contrast ~ to the corresponding hepatic rates, which were biphasic. The K, values for both reactions were significantly lower in intestines of 3-MC-treated rats than in controls - implying that different enzymes were functioning in control and 3-MC-induced intestines.56 The time course of 3-MC induction of rat intestinal P450, as determined by AHH activity, has been in~estigated.~~ By 1 h after i.g. administration of 3-MC to rats, AHH activity was increased and this increase continued for up to 18 h. By 24 h after administration, substantial decreases in activity were ob~erved.~' The dose response for 3-MC induction of rat intestinal P450 is shown in Figure 3.57There were incremental increases in P450 concentration with increases in 3-MC administered up to 40 mg/kg. BNF (0.1 to 100 mg/kg diet, equivalent to approximately 0.01 to 10 mg/kg body weighvday) administered in the feed of rats for 7 days produced dose-dependent increases in intestinal ECOD and EROD activities from controls at all doses - increasing by 80- to 90-fold across the range.58At the doses used in this study, hepatic P450s were virtually unaffected, demonstrating that intestinal P450 is more sensitive to orally administered BNF. A role for the Ah receptor in the polycyclic aromatic hydrocarbon induction of intestinal P450 was confirmed by the induction of intestinal AHH activity in C57BU6N but not DBN2 mice by B[a]P.59 The latter mice do not express a functional Ah receptor. A number of studies have been undertaken to compare hepatic and intestinal P450s and their regulation. Intestinal P450s respond more rapidly than hepatic P450s to inducers, possibly because of more direct a~ailability.~' Comparisons of kinetic parameters (V,,,, K,) for untreated hepatic and intestinal microsomal metabolism of a variety of substrates (Table 3) showed significant and variable differences.60 These results suggested that induced forms of P450 in the two organs were functionally different. In the Japanese quail, orally administered 2,4,2',4'tetrachlorobiphenyl induced intestinal P450 3.3-fold, whereas 3,4,3',4'-tetrachlorobiphenyl was without effect. Both congeners produced marked induction in the liver.61A series of 15 flavonoids have been used to compare rat hepatic and intestinal microsomal P450s using ECOD activity as a probe.62
2oo
9
0 0
10
20
30
40
50
3-METHYLCHOLANTHRENE (mg/kg) FIGURE 3. Dose response for 3-MC induction of rat intestinal P450 concentrations. (O),24 h after administration; (@), 48 h after administration. (Data adapted from Born, P., Frankhuijzen Sierevogel, A,, and Noordhcek, J., Biochem. Pharmucol., 31, 3701, 1982.)
The flavones 5,6-benzoflavone, 7,8-benzoflavone, and flavone, when added to microsomal preparations, all increased hepatic activity but markedly inhibited intestinal activity; the remaining 12 flavonoids all inhibited both intestinal and hepatic microsomal metabolism. Oral or S.C.administration of cobalt to rats increased intestinal P450 and AHH activity and decreased the corresponding hepatic v a l u e ~The . ~ ~differential in 3-MC induction of intestinal P4501A1 and 1A2 described above indicates a regulatory difference between liver and intestine which also occurs with TCDD" and BNF15as inducing agents. Similar regulatory differences occurred with 2-methoxy-4-aminoazobenzene, which induced P4501A1 in the liver but not in the i n t e ~ t i n eand , ~ ~with tryptophan pyrolysate components in the diets of rats and mice, which induced both P4501A1 and 1A2 in liver, but neither in intestine.65P4502B1, but not 2B2, is expressed in the intestine, in contrast to the liver, where both are expressed.66A series of seven N-substituted imidazoles have been used to compare hepatic and extrahepatic P450
1992
413
Critical Reviews In Table 3 Kinetic Values for Rat Hepatic and Intestinal Microsomal Metabolism Intestine
Liver Km
Substrate
v,
Km
(10-5 x M)
(nmoYmirdmg)
6.2 1.7
0.123 0.24
7-Ethoxycoumarin Biphenyl Benzphetamine
1.3
v,,,
(lo-' x M)
(nmoYmirdmg)
1.9
0.8 3.3 13
17
Data adapted from Reference 60.
induction Two of the imidazoles produced fivefold increases in rat intestinal P450 concentrations. However, overall the imidazoles differentially affected hepatic and intestinal P ~ ~ O S , ~ ' with clotrimazole being the most effective hepatic inducer and without any effect in the intestine. The ratios of intestinal to hepatic AHH activities after i.p. administration to a variety of mouse strains was very variable - further proof for differing P450 regulatory mechanisms for these two tissues.6R Western blotting and activity measurements have detected intestinal P4502B1 induction in rats treated with PB," but P4502B2 could only be detected by activity measurements. l 4 The expression of P4502B1 and 2B2 in rat intestinal microsomes was investigated following i.g. or i.p. administration of PB and several environmental contaminants including Aroclor 1254, Firemaster BP, y-chlordane. and trans-n~nachlor.~~ Dot blots were performed using 32P-labeledcDNA which recognized both P~SOS,while Northern blots were performed using oligonucleotide probes specific for each of the P450s. The cDNA probe showed marked induction of P4502B112 mRNA predominantly in proximal and also in distal small intestine equivalently after i.g. or i.p. administration of all of these agents. By using the oligonucleotide probes, low levels on P4502B2 mRNA were detected in contro1 intestine, but this was not induced by PB or Aroclor 1254. However. the weak band for P4502B 1, also detected in control intestine, was markedly induced by PB and Aroclor 1254. Thus, regulation of P4502B 1 and 2B2 in the intestine differs from that in the liver.6y However, in studies with polymerase chain reaction amplification only 2BI and not 2B2 sequences were amplified from small intestinal RNA." These results indicated that P4502B 1 was induced in rat intestine by an increase in gene transcription following i.p. administration of PB. Induction was rapid mRNA was increased as early as 1 h after administration of a single i.p. dose of PB and was markedly induced by 6 h. No further increases were noted by 12 h after administration of PB and levels were equivalent to that noted after 4 days of consecutive administration of PB. The amplified segment of thc PR-inducible P450 mRNA in intestine was identical to the same segment of the hepatic mRNA.% Intestinal P4SOs were probed in rats, which were either un-
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