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Intestinal organic anion transport, glutathione transferase and aryl hydrocarbon hydroxylase activity: Effect of dioxin
Life Sciences, Vol . 24, pp . 1373-1380 Printed in the U.S .A .
Pergamon Press
INTESTINAL ORGANIC ANION TRANSPORT, GLUTATHIONE TRANSFERASE AND ARYL HYDROCARBON HYDRORYLASE ACTIVITY : EFFECT OF DIORIN James Mania and Robert Apap Division of Gastroenterology, Department of Medicine State University of New York, Downatate Medical Center Brooklyn, New York 11203 (Received in final form February 26, 1979)
u
Summary
Benzyl penicillin, p-aminohippurate and phlorizia are passively nspotted across the intestinal wall of rat averted gut sacs . mid-intestine takes up more organic anion than the duodenum or terminal ileum and also moves significantly more anion into the serosal fluid than the more proximal or distal segments of intestine. About the same amount (40X) of the anion taken up by the various regions of the intestine remains is the wall of the gut sac after incubation . Thin pattern of organic anion transport down the intestine is different than the aboral gradient of glutathione S-transferase activity found in the intestine, suggesting that the two mehanisms are not directly related is the intestine. Oral and intraperitoneal 2,3,7,8-tetrachlorodibenzo~pdioxin (TCDD) increases iron transport sad aryl hydrocarbon hydroxylase activity in the intestine and aryl hydrocarbon hydroxylase and glutathione S-traasferase activity in the liver. By contrast, intestinal glutathione S-transferase and organic anion transport are not affected by TCDD treatment . Intestinal transport and enzymatic mechanisms apparently respond differently to TCDD than those previously described in the liver and kidney .
Studies of the intestinal absorption of organic anions _in vivo and _in vitro suggest that moat of these substances are absorbed by passive processes (1-3) . Notable exceptions are bile salts which are absorbed by a well defined active transport mechanism in the ileum (1,4-6) . By contrast, liver and kidney possess similar active transport mechanisms for the transport of sulphonic acids, hippurates, sulfonamides and beazyl penicillin (7-10) . Furthermore, it has been suggested that glutathione S-transferase B (ligandin, a soluble protein) functions se a component of the organic anion transport system in liver and kidney (9) . Glutathione S-transferase (GSA-T) activity in the small intestine is greatest in the duodenum and jejunum and 3X of the extractable protein from these tissues cross-react with antibody to GSH-T A,B and E (11) . Other workers measured 2X of rat intestinal extractable protein se immunoreactive glutathione S-tranaferase B, suggesting that the major fraction of total intestinal tranaferase activity is GSH-T B or ligandin (12) . Furthermore, phenobarbital was ineffective as an inducer of intestinal glutathione S-transferase B, measured by radial immunodiffusioa (12), whereas intestinal GSH-T activity, measured eazymatically with 1-chloro-2,4-dinitrobenzene as substrate, was increased by phenobarbital treatment (11) . More significantly, 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) increased renal GSH-T B and urinary excretion of beazyl penicillin, whereas phenobarbital had no effect on either mechanism 0024-3205/79/151373-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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in the kidney (9) suggesting that this inducer was a more effective stimulant for the anion transport mechanism than phenobarbital . TCDD is a potent inducer of several enzyme systems, including aryl hydrocarbon hydroxylase (AHH) (13,14) and is a potent stimulant of the active transport mechanism for iron in the rat intestine (15) . The following experiments further study the organic anion transport mechanism in rat intestine and its relationship to GSH-T and AHH activity in normal and TCDD-treated rats . Methods Male albino Sprague-Dawley rats, 80-120 g (Carworth Farms, New City, N .Y .) were fed Teklad Mouse/Rat Diet (Teklad Diets, Winfield, Iowa) and housed in cages with galvanized wire mesh bottoms . The rate were fasted for 17 hours prior to sacrifice by exsanguination after a blow on the head . Everted gut sacs were prepared as described previously (16) . The sacs were filled with 0 .5 ml of the following medium (M) : NaCl, 0 .145 ; sodium phosphate buffer, 0 .004, pH 7 .4 ; CaC12, 0 .0001; fructose, 0 .04 and appropriate concentrations of stable organic anion and radioisotopic organic anion to give adequate counting rates in a liquid scintillation counter(Packard Model 2425, Packard Instrument Co ., Downers Grove, .Ill .) with Bray's Solution (17) . The sacs were placed in 2 .5 ml of medium in a 25 ml Erlenmeyer flask and incubated in a Dubnoff Incubation Apparatus under 100X oxygen for 2 .5 hours at 37°C . After incubation the sacs were drained into calibrated tubes and the volume measured . Recovery of inside fluid was 86-105X in these experiments . The anions studied included, stable phlorisin (ICN Pharmaceuticals, Inc ., Plainview, N .Y .), benzyl penicillin and p-aminohippurate (Sigma Chemical Co ., St . Louie, Mo .) and radioisotopic (3fi~phlorizin, (14 C)p-aminohippurate (New England Nuclear, Boston, Mass .) and ( 4C) benzyl penicillin (Amersham/Searle, Arlington Heights, I11 .) . Iron transport was studied as described previously (16) with 59FeS04 (New England Nuclear, Boston, Mase .) . Animals treated with TCDD were given the inducer dissolved in 1,4-dioxane . When injected intraperitoneally the concentration of TCDD was adjusted so that the volume of dioxane never exceeded 0 .05 ml . The oral dose of TCDD was given in 0 .1 ml of dioxane by gastric tube . Control rata received identical volumes of dioaane without TCDD . Animals dosed orally were fasted overnight prior to treatment, were kept fasted for 1-2 hours after intubation, then were refed as usual until killed . Appropriate precautions were observed in handling and disposing of TCDD-contaminated material (13) . GSH-T activity was estimated in appropriate extracts with 1-chloro-2,4-diaitrochlorobenzene as substrate (18) . Portions of liver were removed at the time of sacrifice and the mucoea was scraped from the intestinal sacs after incubation and draining . The tissues were homogenized in 10 volumes of 0 .25M sucrose-O.O1M KP04 buffer, pH 7 .4 (0-5oC) for one minute with a Potter-Elvenhjem homogenizer . Aliquots of homogenate were centrifuged for 60 minutes at 100,000 x g and the supernatant used for the transferase assay. Preliminary experiments had shown that GSH-T activity of mucosa from incubated sacs was unchanged from that of mucosa from unincubated intestine. Aryl hydrocarbon hydroxylase (AHH) activity was measured with aliquots of crude liver homogenate with benzo(a)pyrene ae substrate (19) . Protein was measured with crystalline bovine albumin as a reference standard (20) . Results Location and characteristics of the organic anion transport system in the intestine : The results obtained from incubating everted gut sacs prepared from different regions of the intestine in medium containing p-aminohippurate (PAH) are illustrated in Figure 1 . Sacs about 4 cm long were tied from the entire small intestine from each of sin rate and PAH transport was measured as described in Methods and the results averaged . Net uptake of PAH by the intestine (Mucosal uptake) gradually increased 12X in the proximal third of the
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8.0 v v
6 .0
0
aM O v - 4 .0
c
E a 2 .0 a a 0
50 20 30 40 10 DISTANCE from Pylorus (cm )
60
FIG . 1 Mucosal uptake and eeroeal transfer of PAH by evened gut sate from various regions of the small intestine of the rat. PAH concentration in the incubation medium initially was 4x10-5li . The mesas of sin sate are illustrated +SE . intestine then decreased until the terminal ileum took up leas PAH than the duodenum . Movement of PAH to the inside of the sac (Serosal transfer) roughly followed the pattern of the mucoeal uptake except that the proportional increase in the mid-intestine was greater, 25X compared to 12X for mucosal uptake . The eeroeal transfer accounted for about 58X of the PAH that entered the mucoea . The remaining fraction was retained in the intestine but the tissue location cannot be determined from these data . The mucoea took up about 19X of the total PAH added to the initial incubation medium, and the eeroeal transfer and retained intestinal fraction accounted for 11 and 8X of this total respectively . Total isotope recovery in this and subsequent eapariaents ranged from 93±6 to 100+3X (SE) suggesting that losses of isotope due to metabolic conversion to carbon dioxide or water vapor were insignificant is these eaperiments . Final concentration ratios of PAH across the intestine (inside medium PAH concentration/outside medium PAH concentration) never ezceeded 1 .0 in any of the segments . Furthermore, additional experiments wherein Tris buffer replaced phosphate, the concentration of RC1 and CaC12 vas varied and mannose or glucose replaced fructose failed to increase PAH concentration ratios to more than 1 .0 . Since saturation of a transport mechanism could result in inability to establish concentration gradients across the intestine greater than 1 .0, groups of gut sacs were incubated in media with increasing concentrations of stable PAH . Five gut sacs were tied from the middle 30 cm of intestine from each of five rate and one sac from each rat was incubated at the different PAH concentrations illustrated in Figure 2 . Both mucosal uptake and eeroeal transfer of
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0.75 O v a, v N c ~- 0.50 v
â
E
0 .25 0
0.4
0.8
1.2
PAH concentration
1 .6
~X 10 -3 M
FIG . 2 Mucosal uptake and serosal transfer of PAH by everted gut sacs from the mid-intestine of five rate . Data shown are the means +SE. PAH were linear over the 20-fold concentration range used in this experiment . Furthermore, PAH concentration ratios across the sacs were never greater than 1 .0 . 20X of the total initial PAH added to the incubation medium was taken up by the mucosa regardless of the concentration of PAH (range 18-22X for the five different concentrations tented) . Also similar to the data in Figure 1, 53% (range 46-61X) of the PAH was moved into the inside fluid while the remainder was retained in the intestinal tissue without any detectable trend of increased retention as the PAH concentration of the incubation medium was increased . Comparison of PAH, benzyl Penicillin (PCN) and phlorizin transport : Two additional anions were studied and their transport by the intestine compared with that of PAH as listed in Table I . These results are the averages of four experiments in which a sac was prepared from four different regions of the intestine from each rat . PAH, PCN and phlorizin transport were qualitatively similar to that illustrated in Figure 1 for PAH . The decrease in aerosal transfer in the terminal ileum was proportional to the percent decrease in the mucosal uptake . No anion was concentrated sufficiently in the inside medium to establish concentration gradients greater than 1 .0 across the sac wall . PAH and PCN were quantitatively taken up and moved to the inside medium to a comparable degree . Phlorizin, however, was taken up about five times more effectively and moved to the inside of the sac 3-5 times more effectively than PAH or PCN . Effect of TCDD on intestinal transport, GSH-T and AHH activity in the intestine and liver: Earlier experiments reported that iron transport was increased in gut sacs prepared from the intestine of rate treated with TCDD (15) .
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TABLE I Transport of Various Organic Anions by Everted Gut Sacs from Rata No . of rate
PAH Mucoeal uptake Seroeal transfer
6 6
Benzyl penicillin (PCN) Mucoeal uptake 8 Seroeal transfer 8 Phlorizin Mucoeal uptake Seroeal transfer
6 6
PAH Net Transport (cpm/gut sac)+ Intestine Segment Number 2 3 4
1
1748_+162 698+34
2057+_110*** 1993+182NS 1119+62* 1157+51*
1428_+123*** 693+96NS
2250+279 636+29
2248+265NS 986+49*
1293+293* 476+61*
13070+830 2667+419
1919±312NS 860+97*
17619+1989**11683±2526NS 4194±757* 3508+213*** 3086+549NS 1017+96*
+All data are adjusted to reflect an initial specific activity of 100 cpm/nmole of unlabeled carrier . Four evened gut sacs were prepared from the following areas of the small intestine : l~duodenum, 220 cm from the pylorus, 340 cm from the pylorus, 4~terminal ileum. PAH and PCN concentration in the incubation medium initially ~ 4x10 -5M, Phlorizin ~ 2x10 -4M. Values are means _+SE . *P<0 .001, **P<0 .02, ***P<0 .05, NS~not significant . TABLE II Effect of Oral TCDD on Iron and PCN Transport ; Effect on GSH-T and AHH Activity in the Intestine and Liver of the Rat No . of rata
Control TCDD
5 5
59Fe Net Transport (cpm x 103/gut sac) Mucoeal Seroeal uptake transfer 21 .1_+0 .2 21 .4+0 .3
2 .1+0 .2 2 .9+0 .4*
GSH-T Activity (nE/min/mg protein) Control TCDD
5 5
PCN Net Transport (cpm x 10 3/gut sac) Mucoeal Seroeal uptake transfer 7.0+0 .2 6 .6+0 .3
Intestine 2 .38+0 .37 2 .38+0 .11
2 .2+_0 .3 1 .9+0 .1
Liver 3 .13+0 .06 5 .31+0 .21**
AHH Activity (pmoles 3-HBP/mg protein) Control TCDD
5 5
-
89_+9 .4 1783+101**
Values are means +SE . Rata were given 0 .1 ml of dioxane with and without 17 ug/kg of TCDD by stomach tube 2 days before sacrifice . Gut sacs were prepared from the duodenum and the mid-intestine for measurement of 59Fe and PCN transport respectively . FaS04 and PCN concentration in the incubation media were 4x10 -5M respectively . AHH activity was measured ae pmolee of 3-hydroaybenzpyrene formed/mg protein . *P<0 .05, **P<0.001 .
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TABLE III Effect of TCDD on Penicillin and GSH-T Activity in the Rat Intestine
No . of rate
PCN Net Transport (cpm x 10 3/gut sac) Mucoeal Serosal uptake transfer
GSH-T Activity (QE/min/mg protein) Intestine Liver
1 day Control TCDD
3 3
3 .5_+0.4 3 .3+0 .9
2 .4_+0 .3 2 .3+0 .3
2 .32+0 .6 2 .62+0 .5
4.18+_0 .6 5.09+1 .3
4 day Control TCDD
3 3
2 .7+1 .0 1 .5+0 .2
2 .0_+0.2 2 .0+0 .2
2 .35_+0 .6 2 .69+0 .2
3.84+_0 .1 5 .65+0 .1**
11 day Control TODD
4 4
6 .2+0 .9 7 .1+0 .3
2 .5_+0.2 2 .9+0 .5
2 .99+0 .5 1 .88+0 .2
3.36+0 .4 6.37+0 .3*
14 day Control TCDD
3 4
3 .9+0 .8 4 .8+1 .0
2 .0_+0.2 2 .6+0 .3
2 .31_+0 .2 3 .96+0 .3
5.14+0 .4 5 .48+0 .9
Values are means +SE . One sac was prepared from the mid-intestine of each rat . PCN concentration in the incubation medium was 4x10 -5M . Rata were injected intraperitoneally with 0.05 ml of dioxane with or without 17 ug/kg of TCDD the number of days before sacrifice noted in the TABLE . *P<0 .01, **P~0 .001 . Accordingly, experiments were done in which 59 Fe and ~4C)PCN transport were studied in gut sacs prepared from the appropriate segments of intestine from the same rats treated with 17 ug/kg of TCDD by stomach tube 2 days before sacrifice . GSH-T and AHH activity were measured in each liver and the transferase activity was measured in the gut sac mucosa after incubation and the results are shown in Table II . Serosal transfer of iron was increased by 38X in the treated rate (P~0 .05) and the 59Fe concentration gradient almost doubled from 6 .8_+1 .8 to 12 .1+2 .5 (P<0 .05), whereas mucosal uptake of iron remained unchanged By contrast, PCN transport and GSH-T activity in the intestinal mucosa was not stimulated by TCDD treatment despite the 70X increase in transferase activity in the liver and the 20-fold increase in AHH activity in the liver . The direct action of TCDD on the intestine was demonstrated by the oral and iatraperitonal treatment of 12 rata with TCDD prior to the assay of AHH activity in mucosal homogenates from the proaimal, middle and distal regions of the intestine . Low but measurable AHH activity was present in the three regions of intestine from control dioxane-treated rate (5 pmol 3-HBP/mg protein) . By contrast TCDD treatment increased AHH activity about 100-fold to 685 pmol 3-HBP/ mg protein . These data and those in Table II demonstrate that TCDD treatment stimulates two separate and distinct biological pathways in the intestine, while leaving two others unaffected, suggesting that these effects are not the result of general metabolic cheagee . PCN traps ort: effect of TCDD at He atic and intestinal GSH-T activit different times after treatment : Groups of rate were injected with 17 ug kg of TCDD sad sacrificed at different times after treatment whereupon PCN transport sad GSH-T activity were studied as described in Methods sad the results are listed in Table III . GSH-T activity was stimulated significantly in the liver at 4 and 11 days sad was close to control levels at 14 days . By contrast, neither intestinal GSH-T activity nor PCN transport was affected by TCDD
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treatment . Similar treatment of another group of rata and measurement of aryl hydrocarbon hydroaylase activity in the intestine, demonstrated that a similar single dose of TODD increased intestinal AHH activity for up to 10 days . Since the data in Figure 1 and Table I demonstrate that organic anion transport varies significantly in different regions of the intestine, four sacs were prepared as described in Table I from each of 15 rate treated with 17 ug/ kg of TODD IP and 15 controls and PAH transport and GSH-T activity were estimated . No changes in PAH transport or intestinal GSH-T activity were noted despite the almost two-fold increase in hepatic GSH-T activity from 4 .61_+0 .2 to 7 .74_+0 .7 (P<0 .0001) . The GSH-T activity in the intestine decreased aborally in a pattern distinct from that for PAH transport (Table I) (2 .15_+0 .3 and 0 .59+_0 .1 pE/min/mg protein duodenum and terminal ileum, respectively), suggesting a lack of direct correlation between transferase activity and PAH transport . Discussion Earlier studies had shown that several organic anions were probably absorbed passively by the intestine . In 1964, Tidball measured the biliary excretion of bromosulphophthalein (BSP), rose bengal and phenoaulphonphthalein instilled into loops of rat intestine (3) . Lack and Weiner measured hippurate, PAH, phenol red and BSP transport by evened gut sate from guinea pigs (10) . Finally, Lanmaa et al measured the disappearance of hippurate, PAH, phenol red and sulphonilic acid from the entire intestine of rate _in vivo (2) . The present studies are consistent with, but also extend the earlier hypothesis, by demonstrating a decreasing uptake and movement of anion across the gut wall by the mid-intestine, duodenum and ileum, respectively, indicating that regional differences in the passive transport mechanism may exist . The different experimental techniques in the cited studies are probably the reason these regional variations were not apparent previously . The factors involved in the variation of anion transport are not apparent from the data reported here but GSH-T does not appear to be directly related to anion transport is the intestine of the rat . The eacretion of many organic anions by the liver and kidney have been described as congruent by several investigators (7-10,21) and active transport mechanisms for penicillin, PAH and other anions have been characterized in both organs . Moreover, urinary PCN eacretion increased in rata treated with TODD concomitant with as increase in hepatic and renal GSH-T B (ligandin, a soluble protein) suggesting that this tranaferase ie a component of the organic anion transport system in the kidney as well as in the liver (9) . TODD treatment did not affect either organic anion transport or GSH-T activity in the intestine in our studies, therefore suggesting that the intestinal processes for organic anion movement are different than the congruent renal and hepatic mechanisms . Total GSH-T activity was measured in our studies, not GSH-T B (22), therefore, it is possible the latter may be related to intestinal anion transport but our data suggest that other factors probably participate in this mechanism. The anions tested in these studies bind to ligandin to a lesser degree than other substances (9), therefore it is possible that transport of other compounds might show correlations with transferase activity . TODD and phenobarbital are representatives of two different classes of iaducers of microsomal enzyme systems . The phenobarbital group stimulates a wider variety of pathways whereas the polcyclic hydrocarbon group (TODD) stimulates a more limited group of reactions (23) . The present study demonatratea that TODD stimulates two intestinal mechanisms (iron transport and AHH activity) and fails to affect GSH-T and organic anion transport, while phenobarbital is reported to stimulate GSH-T in the intestine (11) . These activities in different organs are consistent with the demonstrated differences between
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the two classes of microsomal enzyme inducers . Abbreviations used : Dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin are synonomous and the abbreviation TCDD is used in the text . PCN~benzyl penicillin AHH~aryl hydrocarbon hydroxylase, PAH~p-aminohippurate, GSA-T~glutathione transferase. Acknowle~ements We thank Dr . I .M . Arias and Z . Gatmaitan for their advice and help in setting up the GSH-T assay . We thank Dr . A. Poland far the gift of TCDD . This work was supported by a grant from the Public Health Service (AM-18247) . References 1 . L. LACK and I .M . WEIHER, Am . J . Physiol . _210 1142-1152 (1966) . 2 . R.C . LANMAN, C .E . STREMATERFER and L .S . SCHANKER, Renobiotica _1 613-619 (1971) . 3 . C .S . TIDBALL, Am . J . Physiol . _206 239-242 (1964) . 4 . P .R . HOLT, Am . J. Physiol . _207 1-7 (1963) . 5 . L . LACK and I .M . WEIHER, Am . J . Physiol . _200 313-317 (1961) . 6 . M.R . PLATOUST and K .J . ISSELBACHER, J . Clin . Invest . _43 467-476 (1964) . 7 . A. DESPOPOULOS, Am . J . Physiol . _210 760-764 (1966) . 8 . A. DESPOPOULOS and H . SONNENBERG, Am . J . Physiol . _212 1117-1122 (1967) . 9 . R. KIRSCH, G. FLEISCHHER, R . KAMISAKA and I .M . ARIAS, J . Clin . Invest . _55 1009-1019 (1975) . 10 . I . SPERBER, Pharmacol . Rev. _11 109-134 (1959) . 11 . L .M . PINKiJS, J .N . RETLEY and W.B . JAROBY, Biochem . Pharmacol . _26 2359-2363 (1977) . 12 . G . FLEISCHHER, J. ROBBINS and I .M . ARIAS, J . Clin . Invest . _51 677-684 (1972) 13 . A. POLAND and E . GLOVER, Mol . Pharmacol . _10 349-359 (1974) . 14 . A. POLAND and E . GLOVER, J . Biol . Chem . _2S1 4936-4946 (1976) . 15 . J . MANIS and G . KIM, Arch . Environ . Health, (In Press) . 16 . J .G . MANIS and D. SCHACHTER, Am . J . Physiol . _203 73080 (1962) . 17 . G.A. BRAY, Anal . Biochem . _1 279-285 (1960) . 18 . W.H . HABIG, M.J . PABST and W .B . JAROBY, J. Biol . Chem . 249 7130-7139 (1974) . 19 . D .W . NEBERT and H .V . GELBOIN, J . Biol . Chem . _243 6242-6249 (1968) . 20 . O .H . LOWRY, N.J . ROSEBROUGH, A.L . FARR and R.L . RANDALL, J . Biol . Chem . 193 265-275 (1951) . 21 . E .H . BÂRÂNY, Acta Physiol. Scand . _88 491-504 (1973) . 22 . W. HABIG, M . PABST, G . FLEISCHHER, Z . GATMAITAN, I .M . ARIAS and W . JAROBY, Proc . Natl . Acad . Sci . _71 3879-3882 (1974) . 23 . A.H . CONVEY, Pharmacol . Rev . 19 317-366 (1967) .
Pergamon Press
INTESTINAL ORGANIC ANION TRANSPORT, GLUTATHIONE TRANSFERASE AND ARYL HYDROCARBON HYDRORYLASE ACTIVITY : EFFECT OF DIORIN James Mania and Robert Apap Division of Gastroenterology, Department of Medicine State University of New York, Downatate Medical Center Brooklyn, New York 11203 (Received in final form February 26, 1979)
u
Summary
Benzyl penicillin, p-aminohippurate and phlorizia are passively nspotted across the intestinal wall of rat averted gut sacs . mid-intestine takes up more organic anion than the duodenum or terminal ileum and also moves significantly more anion into the serosal fluid than the more proximal or distal segments of intestine. About the same amount (40X) of the anion taken up by the various regions of the intestine remains is the wall of the gut sac after incubation . Thin pattern of organic anion transport down the intestine is different than the aboral gradient of glutathione S-transferase activity found in the intestine, suggesting that the two mehanisms are not directly related is the intestine. Oral and intraperitoneal 2,3,7,8-tetrachlorodibenzo~pdioxin (TCDD) increases iron transport sad aryl hydrocarbon hydroxylase activity in the intestine and aryl hydrocarbon hydroxylase and glutathione S-traasferase activity in the liver. By contrast, intestinal glutathione S-transferase and organic anion transport are not affected by TCDD treatment . Intestinal transport and enzymatic mechanisms apparently respond differently to TCDD than those previously described in the liver and kidney .
Studies of the intestinal absorption of organic anions _in vivo and _in vitro suggest that moat of these substances are absorbed by passive processes (1-3) . Notable exceptions are bile salts which are absorbed by a well defined active transport mechanism in the ileum (1,4-6) . By contrast, liver and kidney possess similar active transport mechanisms for the transport of sulphonic acids, hippurates, sulfonamides and beazyl penicillin (7-10) . Furthermore, it has been suggested that glutathione S-transferase B (ligandin, a soluble protein) functions se a component of the organic anion transport system in liver and kidney (9) . Glutathione S-transferase (GSA-T) activity in the small intestine is greatest in the duodenum and jejunum and 3X of the extractable protein from these tissues cross-react with antibody to GSH-T A,B and E (11) . Other workers measured 2X of rat intestinal extractable protein se immunoreactive glutathione S-tranaferase B, suggesting that the major fraction of total intestinal tranaferase activity is GSH-T B or ligandin (12) . Furthermore, phenobarbital was ineffective as an inducer of intestinal glutathione S-transferase B, measured by radial immunodiffusioa (12), whereas intestinal GSH-T activity, measured eazymatically with 1-chloro-2,4-dinitrobenzene as substrate, was increased by phenobarbital treatment (11) . More significantly, 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) increased renal GSH-T B and urinary excretion of beazyl penicillin, whereas phenobarbital had no effect on either mechanism 0024-3205/79/151373-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd
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in the kidney (9) suggesting that this inducer was a more effective stimulant for the anion transport mechanism than phenobarbital . TCDD is a potent inducer of several enzyme systems, including aryl hydrocarbon hydroxylase (AHH) (13,14) and is a potent stimulant of the active transport mechanism for iron in the rat intestine (15) . The following experiments further study the organic anion transport mechanism in rat intestine and its relationship to GSH-T and AHH activity in normal and TCDD-treated rats . Methods Male albino Sprague-Dawley rats, 80-120 g (Carworth Farms, New City, N .Y .) were fed Teklad Mouse/Rat Diet (Teklad Diets, Winfield, Iowa) and housed in cages with galvanized wire mesh bottoms . The rate were fasted for 17 hours prior to sacrifice by exsanguination after a blow on the head . Everted gut sacs were prepared as described previously (16) . The sacs were filled with 0 .5 ml of the following medium (M) : NaCl, 0 .145 ; sodium phosphate buffer, 0 .004, pH 7 .4 ; CaC12, 0 .0001; fructose, 0 .04 and appropriate concentrations of stable organic anion and radioisotopic organic anion to give adequate counting rates in a liquid scintillation counter(Packard Model 2425, Packard Instrument Co ., Downers Grove, .Ill .) with Bray's Solution (17) . The sacs were placed in 2 .5 ml of medium in a 25 ml Erlenmeyer flask and incubated in a Dubnoff Incubation Apparatus under 100X oxygen for 2 .5 hours at 37°C . After incubation the sacs were drained into calibrated tubes and the volume measured . Recovery of inside fluid was 86-105X in these experiments . The anions studied included, stable phlorisin (ICN Pharmaceuticals, Inc ., Plainview, N .Y .), benzyl penicillin and p-aminohippurate (Sigma Chemical Co ., St . Louie, Mo .) and radioisotopic (3fi~phlorizin, (14 C)p-aminohippurate (New England Nuclear, Boston, Mass .) and ( 4C) benzyl penicillin (Amersham/Searle, Arlington Heights, I11 .) . Iron transport was studied as described previously (16) with 59FeS04 (New England Nuclear, Boston, Mase .) . Animals treated with TCDD were given the inducer dissolved in 1,4-dioxane . When injected intraperitoneally the concentration of TCDD was adjusted so that the volume of dioxane never exceeded 0 .05 ml . The oral dose of TCDD was given in 0 .1 ml of dioxane by gastric tube . Control rata received identical volumes of dioaane without TCDD . Animals dosed orally were fasted overnight prior to treatment, were kept fasted for 1-2 hours after intubation, then were refed as usual until killed . Appropriate precautions were observed in handling and disposing of TCDD-contaminated material (13) . GSH-T activity was estimated in appropriate extracts with 1-chloro-2,4-diaitrochlorobenzene as substrate (18) . Portions of liver were removed at the time of sacrifice and the mucoea was scraped from the intestinal sacs after incubation and draining . The tissues were homogenized in 10 volumes of 0 .25M sucrose-O.O1M KP04 buffer, pH 7 .4 (0-5oC) for one minute with a Potter-Elvenhjem homogenizer . Aliquots of homogenate were centrifuged for 60 minutes at 100,000 x g and the supernatant used for the transferase assay. Preliminary experiments had shown that GSH-T activity of mucosa from incubated sacs was unchanged from that of mucosa from unincubated intestine. Aryl hydrocarbon hydroxylase (AHH) activity was measured with aliquots of crude liver homogenate with benzo(a)pyrene ae substrate (19) . Protein was measured with crystalline bovine albumin as a reference standard (20) . Results Location and characteristics of the organic anion transport system in the intestine : The results obtained from incubating everted gut sacs prepared from different regions of the intestine in medium containing p-aminohippurate (PAH) are illustrated in Figure 1 . Sacs about 4 cm long were tied from the entire small intestine from each of sin rate and PAH transport was measured as described in Methods and the results averaged . Net uptake of PAH by the intestine (Mucosal uptake) gradually increased 12X in the proximal third of the
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8.0 v v
6 .0
0
aM O v - 4 .0
c
E a 2 .0 a a 0
50 20 30 40 10 DISTANCE from Pylorus (cm )
60
FIG . 1 Mucosal uptake and eeroeal transfer of PAH by evened gut sate from various regions of the small intestine of the rat. PAH concentration in the incubation medium initially was 4x10-5li . The mesas of sin sate are illustrated +SE . intestine then decreased until the terminal ileum took up leas PAH than the duodenum . Movement of PAH to the inside of the sac (Serosal transfer) roughly followed the pattern of the mucoeal uptake except that the proportional increase in the mid-intestine was greater, 25X compared to 12X for mucosal uptake . The eeroeal transfer accounted for about 58X of the PAH that entered the mucoea . The remaining fraction was retained in the intestine but the tissue location cannot be determined from these data . The mucoea took up about 19X of the total PAH added to the initial incubation medium, and the eeroeal transfer and retained intestinal fraction accounted for 11 and 8X of this total respectively . Total isotope recovery in this and subsequent eapariaents ranged from 93±6 to 100+3X (SE) suggesting that losses of isotope due to metabolic conversion to carbon dioxide or water vapor were insignificant is these eaperiments . Final concentration ratios of PAH across the intestine (inside medium PAH concentration/outside medium PAH concentration) never ezceeded 1 .0 in any of the segments . Furthermore, additional experiments wherein Tris buffer replaced phosphate, the concentration of RC1 and CaC12 vas varied and mannose or glucose replaced fructose failed to increase PAH concentration ratios to more than 1 .0 . Since saturation of a transport mechanism could result in inability to establish concentration gradients across the intestine greater than 1 .0, groups of gut sacs were incubated in media with increasing concentrations of stable PAH . Five gut sacs were tied from the middle 30 cm of intestine from each of five rate and one sac from each rat was incubated at the different PAH concentrations illustrated in Figure 2 . Both mucosal uptake and eeroeal transfer of
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24, No . 15, 1979
0.75 O v a, v N c ~- 0.50 v
â
E
0 .25 0
0.4
0.8
1.2
PAH concentration
1 .6
~X 10 -3 M
FIG . 2 Mucosal uptake and serosal transfer of PAH by everted gut sacs from the mid-intestine of five rate . Data shown are the means +SE. PAH were linear over the 20-fold concentration range used in this experiment . Furthermore, PAH concentration ratios across the sacs were never greater than 1 .0 . 20X of the total initial PAH added to the incubation medium was taken up by the mucosa regardless of the concentration of PAH (range 18-22X for the five different concentrations tented) . Also similar to the data in Figure 1, 53% (range 46-61X) of the PAH was moved into the inside fluid while the remainder was retained in the intestinal tissue without any detectable trend of increased retention as the PAH concentration of the incubation medium was increased . Comparison of PAH, benzyl Penicillin (PCN) and phlorizin transport : Two additional anions were studied and their transport by the intestine compared with that of PAH as listed in Table I . These results are the averages of four experiments in which a sac was prepared from four different regions of the intestine from each rat . PAH, PCN and phlorizin transport were qualitatively similar to that illustrated in Figure 1 for PAH . The decrease in aerosal transfer in the terminal ileum was proportional to the percent decrease in the mucosal uptake . No anion was concentrated sufficiently in the inside medium to establish concentration gradients greater than 1 .0 across the sac wall . PAH and PCN were quantitatively taken up and moved to the inside medium to a comparable degree . Phlorizin, however, was taken up about five times more effectively and moved to the inside of the sac 3-5 times more effectively than PAH or PCN . Effect of TCDD on intestinal transport, GSH-T and AHH activity in the intestine and liver: Earlier experiments reported that iron transport was increased in gut sacs prepared from the intestine of rate treated with TCDD (15) .
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TABLE I Transport of Various Organic Anions by Everted Gut Sacs from Rata No . of rate
PAH Mucoeal uptake Seroeal transfer
6 6
Benzyl penicillin (PCN) Mucoeal uptake 8 Seroeal transfer 8 Phlorizin Mucoeal uptake Seroeal transfer
6 6
PAH Net Transport (cpm/gut sac)+ Intestine Segment Number 2 3 4
1
1748_+162 698+34
2057+_110*** 1993+182NS 1119+62* 1157+51*
1428_+123*** 693+96NS
2250+279 636+29
2248+265NS 986+49*
1293+293* 476+61*
13070+830 2667+419
1919±312NS 860+97*
17619+1989**11683±2526NS 4194±757* 3508+213*** 3086+549NS 1017+96*
+All data are adjusted to reflect an initial specific activity of 100 cpm/nmole of unlabeled carrier . Four evened gut sacs were prepared from the following areas of the small intestine : l~duodenum, 220 cm from the pylorus, 340 cm from the pylorus, 4~terminal ileum. PAH and PCN concentration in the incubation medium initially ~ 4x10 -5M, Phlorizin ~ 2x10 -4M. Values are means _+SE . *P<0 .001, **P<0 .02, ***P<0 .05, NS~not significant . TABLE II Effect of Oral TCDD on Iron and PCN Transport ; Effect on GSH-T and AHH Activity in the Intestine and Liver of the Rat No . of rata
Control TCDD
5 5
59Fe Net Transport (cpm x 103/gut sac) Mucoeal Seroeal uptake transfer 21 .1_+0 .2 21 .4+0 .3
2 .1+0 .2 2 .9+0 .4*
GSH-T Activity (nE/min/mg protein) Control TCDD
5 5
PCN Net Transport (cpm x 10 3/gut sac) Mucoeal Seroeal uptake transfer 7.0+0 .2 6 .6+0 .3
Intestine 2 .38+0 .37 2 .38+0 .11
2 .2+_0 .3 1 .9+0 .1
Liver 3 .13+0 .06 5 .31+0 .21**
AHH Activity (pmoles 3-HBP/mg protein) Control TCDD
5 5
-
89_+9 .4 1783+101**
Values are means +SE . Rata were given 0 .1 ml of dioxane with and without 17 ug/kg of TCDD by stomach tube 2 days before sacrifice . Gut sacs were prepared from the duodenum and the mid-intestine for measurement of 59Fe and PCN transport respectively . FaS04 and PCN concentration in the incubation media were 4x10 -5M respectively . AHH activity was measured ae pmolee of 3-hydroaybenzpyrene formed/mg protein . *P<0 .05, **P<0.001 .
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TABLE III Effect of TCDD on Penicillin and GSH-T Activity in the Rat Intestine
No . of rate
PCN Net Transport (cpm x 10 3/gut sac) Mucoeal Serosal uptake transfer
GSH-T Activity (QE/min/mg protein) Intestine Liver
1 day Control TCDD
3 3
3 .5_+0.4 3 .3+0 .9
2 .4_+0 .3 2 .3+0 .3
2 .32+0 .6 2 .62+0 .5
4.18+_0 .6 5.09+1 .3
4 day Control TCDD
3 3
2 .7+1 .0 1 .5+0 .2
2 .0_+0.2 2 .0+0 .2
2 .35_+0 .6 2 .69+0 .2
3.84+_0 .1 5 .65+0 .1**
11 day Control TODD
4 4
6 .2+0 .9 7 .1+0 .3
2 .5_+0.2 2 .9+0 .5
2 .99+0 .5 1 .88+0 .2
3.36+0 .4 6.37+0 .3*
14 day Control TCDD
3 4
3 .9+0 .8 4 .8+1 .0
2 .0_+0.2 2 .6+0 .3
2 .31_+0 .2 3 .96+0 .3
5.14+0 .4 5 .48+0 .9
Values are means +SE . One sac was prepared from the mid-intestine of each rat . PCN concentration in the incubation medium was 4x10 -5M . Rata were injected intraperitoneally with 0.05 ml of dioxane with or without 17 ug/kg of TCDD the number of days before sacrifice noted in the TABLE . *P<0 .01, **P~0 .001 . Accordingly, experiments were done in which 59 Fe and ~4C)PCN transport were studied in gut sacs prepared from the appropriate segments of intestine from the same rats treated with 17 ug/kg of TCDD by stomach tube 2 days before sacrifice . GSH-T and AHH activity were measured in each liver and the transferase activity was measured in the gut sac mucosa after incubation and the results are shown in Table II . Serosal transfer of iron was increased by 38X in the treated rate (P~0 .05) and the 59Fe concentration gradient almost doubled from 6 .8_+1 .8 to 12 .1+2 .5 (P<0 .05), whereas mucosal uptake of iron remained unchanged By contrast, PCN transport and GSH-T activity in the intestinal mucosa was not stimulated by TCDD treatment despite the 70X increase in transferase activity in the liver and the 20-fold increase in AHH activity in the liver . The direct action of TCDD on the intestine was demonstrated by the oral and iatraperitonal treatment of 12 rata with TCDD prior to the assay of AHH activity in mucosal homogenates from the proaimal, middle and distal regions of the intestine . Low but measurable AHH activity was present in the three regions of intestine from control dioxane-treated rate (5 pmol 3-HBP/mg protein) . By contrast TCDD treatment increased AHH activity about 100-fold to 685 pmol 3-HBP/ mg protein . These data and those in Table II demonstrate that TCDD treatment stimulates two separate and distinct biological pathways in the intestine, while leaving two others unaffected, suggesting that these effects are not the result of general metabolic cheagee . PCN traps ort: effect of TCDD at He atic and intestinal GSH-T activit different times after treatment : Groups of rate were injected with 17 ug kg of TCDD sad sacrificed at different times after treatment whereupon PCN transport sad GSH-T activity were studied as described in Methods sad the results are listed in Table III . GSH-T activity was stimulated significantly in the liver at 4 and 11 days sad was close to control levels at 14 days . By contrast, neither intestinal GSH-T activity nor PCN transport was affected by TCDD
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treatment . Similar treatment of another group of rata and measurement of aryl hydrocarbon hydroaylase activity in the intestine, demonstrated that a similar single dose of TODD increased intestinal AHH activity for up to 10 days . Since the data in Figure 1 and Table I demonstrate that organic anion transport varies significantly in different regions of the intestine, four sacs were prepared as described in Table I from each of 15 rate treated with 17 ug/ kg of TODD IP and 15 controls and PAH transport and GSH-T activity were estimated . No changes in PAH transport or intestinal GSH-T activity were noted despite the almost two-fold increase in hepatic GSH-T activity from 4 .61_+0 .2 to 7 .74_+0 .7 (P<0 .0001) . The GSH-T activity in the intestine decreased aborally in a pattern distinct from that for PAH transport (Table I) (2 .15_+0 .3 and 0 .59+_0 .1 pE/min/mg protein duodenum and terminal ileum, respectively), suggesting a lack of direct correlation between transferase activity and PAH transport . Discussion Earlier studies had shown that several organic anions were probably absorbed passively by the intestine . In 1964, Tidball measured the biliary excretion of bromosulphophthalein (BSP), rose bengal and phenoaulphonphthalein instilled into loops of rat intestine (3) . Lack and Weiner measured hippurate, PAH, phenol red and BSP transport by evened gut sate from guinea pigs (10) . Finally, Lanmaa et al measured the disappearance of hippurate, PAH, phenol red and sulphonilic acid from the entire intestine of rate _in vivo (2) . The present studies are consistent with, but also extend the earlier hypothesis, by demonstrating a decreasing uptake and movement of anion across the gut wall by the mid-intestine, duodenum and ileum, respectively, indicating that regional differences in the passive transport mechanism may exist . The different experimental techniques in the cited studies are probably the reason these regional variations were not apparent previously . The factors involved in the variation of anion transport are not apparent from the data reported here but GSH-T does not appear to be directly related to anion transport is the intestine of the rat . The eacretion of many organic anions by the liver and kidney have been described as congruent by several investigators (7-10,21) and active transport mechanisms for penicillin, PAH and other anions have been characterized in both organs . Moreover, urinary PCN eacretion increased in rata treated with TODD concomitant with as increase in hepatic and renal GSH-T B (ligandin, a soluble protein) suggesting that this tranaferase ie a component of the organic anion transport system in the kidney as well as in the liver (9) . TODD treatment did not affect either organic anion transport or GSH-T activity in the intestine in our studies, therefore suggesting that the intestinal processes for organic anion movement are different than the congruent renal and hepatic mechanisms . Total GSH-T activity was measured in our studies, not GSH-T B (22), therefore, it is possible the latter may be related to intestinal anion transport but our data suggest that other factors probably participate in this mechanism. The anions tested in these studies bind to ligandin to a lesser degree than other substances (9), therefore it is possible that transport of other compounds might show correlations with transferase activity . TODD and phenobarbital are representatives of two different classes of iaducers of microsomal enzyme systems . The phenobarbital group stimulates a wider variety of pathways whereas the polcyclic hydrocarbon group (TODD) stimulates a more limited group of reactions (23) . The present study demonatratea that TODD stimulates two intestinal mechanisms (iron transport and AHH activity) and fails to affect GSH-T and organic anion transport, while phenobarbital is reported to stimulate GSH-T in the intestine (11) . These activities in different organs are consistent with the demonstrated differences between
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the two classes of microsomal enzyme inducers . Abbreviations used : Dioxin and 2,3,7,8-tetrachlorodibenzo-p-dioxin are synonomous and the abbreviation TCDD is used in the text . PCN~benzyl penicillin AHH~aryl hydrocarbon hydroxylase, PAH~p-aminohippurate, GSA-T~glutathione transferase. Acknowle~ements We thank Dr . I .M . Arias and Z . Gatmaitan for their advice and help in setting up the GSH-T assay . We thank Dr . A. Poland far the gift of TCDD . This work was supported by a grant from the Public Health Service (AM-18247) . References 1 . L. LACK and I .M . WEIHER, Am . J . Physiol . _210 1142-1152 (1966) . 2 . R.C . LANMAN, C .E . STREMATERFER and L .S . SCHANKER, Renobiotica _1 613-619 (1971) . 3 . C .S . TIDBALL, Am . J . Physiol . _206 239-242 (1964) . 4 . P .R . HOLT, Am . J. Physiol . _207 1-7 (1963) . 5 . L . LACK and I .M . WEIHER, Am . J . Physiol . _200 313-317 (1961) . 6 . M.R . PLATOUST and K .J . ISSELBACHER, J . Clin . Invest . _43 467-476 (1964) . 7 . A. DESPOPOULOS, Am . J . Physiol . _210 760-764 (1966) . 8 . A. DESPOPOULOS and H . SONNENBERG, Am . J . Physiol . _212 1117-1122 (1967) . 9 . R. KIRSCH, G. FLEISCHHER, R . KAMISAKA and I .M . ARIAS, J . Clin . Invest . _55 1009-1019 (1975) . 10 . I . SPERBER, Pharmacol . Rev. _11 109-134 (1959) . 11 . L .M . PINKiJS, J .N . RETLEY and W.B . JAROBY, Biochem . Pharmacol . _26 2359-2363 (1977) . 12 . G . FLEISCHHER, J. ROBBINS and I .M . ARIAS, J . Clin . Invest . _51 677-684 (1972) 13 . A. POLAND and E . GLOVER, Mol . Pharmacol . _10 349-359 (1974) . 14 . A. POLAND and E . GLOVER, J . Biol . Chem . _2S1 4936-4946 (1976) . 15 . J . MANIS and G . KIM, Arch . Environ . Health, (In Press) . 16 . J .G . MANIS and D. SCHACHTER, Am . J . Physiol . _203 73080 (1962) . 17 . G.A. BRAY, Anal . Biochem . _1 279-285 (1960) . 18 . W.H . HABIG, M.J . PABST and W .B . JAROBY, J. Biol . Chem . 249 7130-7139 (1974) . 19 . D .W . NEBERT and H .V . GELBOIN, J . Biol . Chem . _243 6242-6249 (1968) . 20 . O .H . LOWRY, N.J . ROSEBROUGH, A.L . FARR and R.L . RANDALL, J . Biol . Chem . 193 265-275 (1951) . 21 . E .H . BÂRÂNY, Acta Physiol. Scand . _88 491-504 (1973) . 22 . W. HABIG, M . PABST, G . FLEISCHHER, Z . GATMAITAN, I .M . ARIAS and W . JAROBY, Proc . Natl . Acad . Sci . _71 3879-3882 (1974) . 23 . A.H . CONVEY, Pharmacol . Rev . 19 317-366 (1967) .