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The effect of sodium phenobarbital and 3,4-benzpyrene on the glucuronidation of 1-naphthol in rat small intestinal loops in vivo
(;~., P/lar,,ic~c.. 1977 IoI 8. Pl'. 51 re, 53 Per~lamon Pres.s. Printed in Great Br:tain
THE EFFECT OF SODIUM PHENOBARBITAL A N D 3,4-BENZPYRENE ON THE G L U C U R O N I D A T I O N OF I-NAPHTHOL IN RAT SMALL INTESTINAL LOOPS IN V I V O J. C. Tt;a.~r.a, V. SrlA~'KS, W. J. Kr~LLY and R. S. GaFE'~ Wallaccville Animal Research Centre, Research Division, Ministry of Agriculture and Fisherics. Private Bag, Upper Hutt, New Zealand (Recei~'ed 26 October 1976)
Abstract I. After 0.1 mM l-naphthol was injected into intestinal loops, 70 90"., of the I-naphthol in the intestinal vcnous blood was present as l-naphthol glucuronide. For 1.0 and 2.0 mM injections the proportion of l-naphthol present as l-naphthol glucuronide was 25-50~'~,,. 2. The proportion of I-naphthol glucuronide in the intestinal venous blood was increased by oral dosing of eithcr sodium phenobarbital or 3,4-benzpyrene but these compounds did not alter the rate of appearance of l-naphthol, free or conjugated. INTRODUCFION
Ltd., Poole. England, and l-naphthol phosphate from Koch-Light Lab. Ltd., Colnbrook England.
There is increasing interest in drug metabolism in cxtrahepatic tissues ( C h h a b r a et al., 1974; Pantuck et al., 1974; T u r n e r et al., 1976). Although the gastrointestinal tract is the first system encountered by many drugs a n d pesticides its role in the absorption a n d dctoxication of xenobiotics is poorly understood. Most of the work has been carried out using in vitro techniques ( C h h a b r a et al., 1974; Del Villar et al., 1974; Pantuck et al., 1974). In the few in t,it'o studies that have been reported it was usual to measure the a m o u n t of the substrate beforc and after a period of incubation in an intestinal loop (Doluisio et al., 1969). G l u c u r o n i d a t i o n of phenols is one of the major conjugations that occurs in the intestinal mucosa (Bock & Winne, 1975; Rance & Shillingford, 1976). The present study was carried out to investigate the effects in rivo of dosing two inducing agents on the ability of the intestine to glucuronidate a simple phenol, l-naphthol. The cannulation technique described below was chosen because it enabled the venous blood draining the intestinal loop to be collected and thus thc I-naphthol metabolites could bc quantitatcd. Any change in the mctabolite pattern due to the inducing agents could also be detected, t4C-ln a p h t h o l was chosen as the substrate since the major metabolite is l-naphthol glucuronide with little formation of l - n a p h t h o l sulphate ( B o c k & Winnc, 1975).
Analytical techniques Blood samples were assayed for l-naphthol and l-naphthol glucuronide by a slight modification of the rapid radioassay de~ribed by Bock & White (1974). Aliquots (20pl) of whole blood were digested in thc counting vial with 0.4ml NCS tissue solubiliscr [AmcrshamSearle Corp., Arlington Heights, 1113. The ~mplcs werc decolorized with 0.2 ml .saturated solution of benzoyl pcroxidc in toluene. Tissue samples were treated as described previously (Turner et al., 19761. Confirmation of the presence of l-naphthol and its mctabolites in intestinal venous blood was made by TLC on silica gel plates in n-butanol-n-propanol 2 M" NH.,OH fl :6:3 v/v) and by paper chromatography on Whatman No. I paper developed with butanol-aqueous NH 3 (0.88) H 2 0 (4:1:5 v/'v) (Binning et al., 1967). The [ollowing standards were co-chromatographed with the whole blood extracts: l-naphthol, I-naphthol glucuronidc. l-naphthol sulphatc and l-naphthol phosphate. Standards were visualized as described by' Binning et al. (1967). Two methods of preparing whole blood cxtracts for chromatography were used. In the tirst method, one w~lume of blood was precipitated with two volumes of acetone, centrifuged for 2 3 min at 15(X).qand thc supernatant was rotary evaporated just to dryncss at 45 C. In the second method, whole blood ~mples wcre extracted twice with equal volumes of ethyl acetate. The combined ethyl acetate extracts were rotary evaporated just to dryness at 45'C. The residues obtained from both methods of extraction were taken up in 8()'~;, ethanol and aliquots chromatographed. Samples scraped from T L C platcs aftcr development were counted as described previously (Tt, rncr et al.. 1976). The paper chromatograms werc cut into 0.5 x 2.0cm wide strips from the origin to the sol~.cnt front and the radioactivity in the strips counted. The positions of the radioactive regions were compared with thc standard compounds. Details of the scintillation counter, phosphors and counting procedure have been describcd previously (Turner et al., 1976).
MATERIALS AND ME'rHODS ('h{,micul~
I-[ l -~'~C]-napht hol (20.8 mCi/mmole) was obtained from the Radiochemical Centre, Amersham, England. The radiochemical purity of the compound was greater than 97~ as determined by TLC on silica gel with benzene-methanol (19:1 v/v). 3,4-benzpyrene. l-naphthol glucuronide and I-naphthol sulphate were obtained from Sigma Chemical Co. {St. Louis. Mo.), I-naphthol from B.D.H. Chemicals
Preparation of intestitlal loops Male Wistar rats (300 350 g) wcrc used. Some animals were dosed per os with 3,4-benzpyrenc (30 mg:kg body
51
52
J C. Tt:R,'qER,V. SHANKS,W. J. KELLYAND R. S. GREEN
weight) in peanut oil 24 hr before the operation. Controls were dosed with peanut oil only. Some animals were given sodium phenobarbital in their drinking water (1 mg/ml) for 4 days. Freshly prepared solutions of sodium phenobarbital were supplied each day. Controls had water only. All animals were starved for 18hr before surgery to ensure that intestinal loops were free of food. Intestinal loops were prepared with the rats under light ether anaesthesia. A jejunal loop, about 10cm from the duodenal flexure and about 5 cm long. was exteriorized and kept moist with isotonic saline. A polythene eannula was inserted into the vein draining the entire loop and secured with tissue glue (Ethieon Buerylat, Ethicon, Hamburg, Germany). The loop was then ligated. The blood flow was 0.8 1.0ml/min. Heparinized blood from donor rats was transfused into the femoral vein to replace the blood collected from the cannula. The prepared loop was injccted with 0.5 ml of 1-naphthol solution (containing 0.35/~Ci t4C-l-naphthol and 0.500 v/v ethanol) in isotonic saline buffered to pH 7.2 with phosphate buffer. Three concentrations of l-naphthol, 0.1, 1.0 and 2.0mM were used. Blood samples (80 100/~1) wcre takcn at intervals for 15 min. The blood collected between these samplcs was deep-frozen and used for the TLC and paper chromatography analyses. At lhe end of the blood collection period the rat was killed and the intestinal loop was removed. The mucosa was .scraped off with a spatula and homogenized and both mucosa and luminal contents were assayed for l-naphthol and l-naphthol glucuronide as dcscribcd for blood.
100 806040-
20-
%
o
,
,
,
,
(hgl)
0 1raM
,
,--"
,
~-nophlho[ i
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40-
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1- n o t p h t h o l
(fig3)20mM
1-naphthol
5
,b
%0 B0604020-
%0 1
2 3 4
7
,~
Time ( m ~ n s l
RESULTS
l-Naphthol glucuronide was the major metabolite of I-naphthol. Thc sulphate conjugate of l-naphthol comprised less than 4% of the dose at all three concentrations of l-naphthol used, both in control and dosed animals. Other metabolites of l-naphthol were present as traces only. Figures 1-3 show the percentage of I-naphthol present as the glucuronide in the intestinal venous blood at various times after the introduction of 0.1, 1.0 or 2.0 mM 1-naphthol, respectively, into rat intestinal loops. The figures also show the effect of dosing with sodium phenobarbital on thc proportion of l-naphthol glucuronide in the venous blood. Figures 2 and 3 show the effect of dosing with peanut oil and benzpyrene on the proportion of l-naphthol glucuronide in the blood. After injecting 0.1 mM I-naphthol, 70 -90,°.,,iof the l-naphthol was found to bc present as I-naphthol glucuronide from I min after dosing until blood collection ceased at 15min. Sodium phenobarbital had no effect on the proportion of I-naphthol glucuronide formed. At the 1.0 and 2.0 mM I-naphthol dose rates the proportion of 1-naphthol glucuronide in the venous blood increased from 25 to 50%. Sodium phenobarbital increased the rate of glucuronidation compared to the controls. Dosing with peanut oil (a control for the benzpyrene-dosing cxperiment) gave a marked increase in the proportion of glucuronide (Figs. 2 and 3} with both 1.0 and 2.0 and 2.0mM l-naphthol; in fact the proportion of glucuronide formed was greater than that found in rats exposed to sodium phenobarbital. After dosing with benzpyrene the percentage of 1-naphthol glucuronidated increased with 1.0mM l-naphthol in the intestinal loop. About 80'J,;~ of the l-naphthol was present as l-naphthol glucuronide. With 2.0 mM l-naphthol, bcnzpyrene had little effect
Figs. 1, 2 and 3. The percentage of l-naphthol present as the glucuronide in the intestinal venous blood after injecting 1-naphthol solution into intestinal loops. (Values are means of 3-6 animals.) O, Control (water); O, phenobarbital; A, peanut oil; A, benzpyrene. on the percentage of glucuronide formed compared to the peanut oil controls. In general, there was a trend towards an increase in the proportion of I-naphthol glucuronide during the course of the 15-min blood collection period (Figs. 1-3). No alteration was observed in the rate of appearance of total 14C-activity in the intestinal venous blood after dosing either benzpyrene or sodium phenobarbital. At the end of the blood collection period the lumen contents contained a high proportion of the glucuronide, ranging from 45 to 80%.
DISCUSSION
The validity of the rapid radio-assay used for the assay of 1-naphthol and l-naphthol glucuronide depends on negligible quantities of other metabolites being present. Because analysis of blood samples showed that the amount of the sulphate conjugate and other metabolites did not increase after dosing either benzpyrene or sodium phenobarbital, the rapid radio-assay was used throughout. l'he increase in the proportion of l-naphthol glucuronide in the intestinal venous blood from rat loops containing 1.0 or 2.0raM I-naphthol after dosing with sodium phenobarbital, peanut oil or benzpyrene suggests that induction of the glueuronidation system had occurred. Induction of this reaction in intestine by 3,4-benzpyrene has been demonstrated using an in vitro assay (Aitio et al., 1972).
The effect of sodium phenobarbital and 3,4-bcnzpyrene The lack of any observable effect of sodium phenobarbital when 0.1 mM I-naphthol was used is probably an indication that little l-naphthol remained since about 85°,/, of the dose was glucuronidated in the control rats. Therefore any quantitative increase in UDP-glucuronyltransferase would have been superfluous at this low dose rate. For this reason the effect of benzpyrene on thc metabolism of 0.1 mM l-naphthol in the loop was not investigated. Thc increase in glucuronide formation after dosing with peanut oil was probably due to peroxides of sterols present in the oil. Peanut oil contains small amounts of sterols and peroxides are known to be formed during storage. Sterol peroxides have been shown to induce liver microsomal mixed-function oxidases (Brown et al., 1954). The similarity in percentagc glucuronidation in the first few minutes between the benzpyrene-treated rats and the peanut oil-dosed rats with 2.0 mM l-naphthol may have been due to the high concentration of l-naphthol in thc mucosal cells. Assuming glucuronidation occurs at the maximum rate, the proportion of l-naphthol glucuronidc would increase as the concentration of l-naphthol in the mucosal cells decreased during the 15 minute blood collection period. Differences in the absolute amount of UDP-glucuronyltransferase between the benzpyrcne-treated animals and the peanut oil-dosed controls would therefore become more apparent as the l-naphthol concentration decreased. This trend is apparent from the 5th minute onwards in Fig. 3. The capacity of the glucuronidation enzyme system appears to be limited since there was a general tendency for the proportion of l-naphthol glucuronide formcd to increase during the course of an experiment. This increase was probably due to the concentration of l-naphthol in the intestinal loop falling during the course of the experiment so that less l-naphthol reached the mucosal cells. Bock & Winne (1975) showed that l-naphthol glucuronide injected into intestinal loops was slowly absorbed. However, the rate at which this occurrcd was very low compared with the rate of l-naphthol glucuronidation in the mucosa; in this case secretion of l-naphthol glucuronide from the mucosa into the lumen and reabsorption would not contribute significantly to the percentage of I-naphthol glucuronidc found in the intestinal venous blood. Dosing with sodium phenobarbital and benzpyrene had no effect on the ratc of appearance of total 14C activity in the intestinal venous blood. This lack of effect on the absorption rate is not unexpected if the normal absorptive process of l-naphthol is by passive diffusion. Passive diffusion is thought to be the mechanism of absorption of many drugs, particularly if they are not structurally similar to normal dietary constituents (Schanker, 1971 ). The rate of passage into the bloodstream is to some extent governed by the rate of blood flow (Parsons, 1968). The presence of 1-naphthol glucuronide in the lumen contents indicates that the glucuronide can be secreted into either venous blood or the intestinal lumen. This confirms the findings of Bock & Winne (1975). The results of this work suggest that with small quantities of substratc the glucuronidating capacity
53
of the intestine is sufficicat to conjugate most of the compound being absorbed. The liver has previously been assumed to be the major site of metabolism of foreign compounds but the present studies and those of others (Powell et al., 1974: Rance & Shillingford, 1976) indicate that the gastrointestinal tract plays a significant role in the conjugation of drugs. The results reported here considered together with those on the effect of benzpyrene on the dealkylation of phenacetin (Pantuck et al., 1974; Pantuck et al., 1975). justify further studies of substances that may alter the intestinal metabolism of drugs thereby affecting their bioavailability.
REFERENCES
AtTIO A., VAINIO H. & HANNINENO. (19721 Enhancement of drug oxidation and conjugation by carcinogens in different rat tissucs. FEBS Lett. 24(3), 237 240. BINNING A., DARBV F. J., HtiENAN M. P. & S~IH J. N. (1967) The conjugation of phenols with phosphate in grass grubs and flies. Biochem. J. 103, 42-48. BOCK K. W. ~ WHITE I. N. H. (1974) UDP-glucuronyltransferasc in perfused rat liver and in microsomes: influence of phenobarbital and 3-methylcholanthrene. Eur. J. Biochem. 46, 451-459. ~ : K K. W. & WINrqE D. (1975) Glucuronidation of I-naphthol in the rat intestinal loop. Biochem. Pharmac. 24. 859-862. BROWN R. R., MILLER J. A. & MILLER E. C. (1954) The metabolism of methylated aminoazo dyes. IV. Dietary factors enhancing demethylation in vitro. J. biol. Chem. 209, 211 222. CtIHABRA R. S., PC)IlL R. J. & F'C.L:'I3J. R. (1974) A comparative study of xenobiotic-metabolising enzymes in liver and intestinc of various animal species. Dr~, Metab. Disp. 2, 443 447. DH, VILLAR E., SANCHEZE. & TI'PHLY T. R. (1974) Morphine metabolism. I!. Studies on morphine glucuronyltransfcrase activity in intestinal microsomcs of rats. Drug Metab. Disp. 2, 370-374. DOLUISIO J. T., BU,Lt:I,S N. F., DIrTERT L. W., SUGITA E. T. & SWINTOSKV J. V. (1969) Drug absorption I: An in situ rat gut technique yielding realistic absorption rates..1. Pharm. Sci. 58, 1196 1200. PAr¢I'UCK E. J., HSlAO K-C.. KAt'LAX S. A., Kt;NTZMAN R. & CONNI-:YA, H. (1974) Effects of enzyme induction on intestinal phenacetin metabolism in the rat. d. Pharmac. exp. Ther. 191, 45-52. PANTUCK E. J., HSIAO K-C., KUNTZMANR. & CONXI!YA. H. (1975) Intestinal metabolism of phcnacctin in the rat: effect of charcoal-broiled beef and rat chow. Science 187, 744 745. PARSONS D. S. (1968) Methods for the investigation of intestinal absorption. In Handbook of Physioloqy, (Edited by HI~tDFL W. & CoDr~ C. F.), Section 6. Vol. II1. pp. 1177-1216. American Physiology Society, Washington, D.C. POWELL G. M., MILLI!R J. J., OI,AVI'kSENA. H. & CURTIS C. G. (1974) Liver as major organ of phenol detoxication? Nature, Lond. 252, 234 235. RAN¢:E M. J. & S81LIJ.~GIrORI)J. S. (1976) The role of the gut in the metabolism of strong analgesics. Biochem. Pharmac. 25. 735 741. SC~tA,~KER L. S. (1971) Absorption of drugs from the gastrointestinal tract. In Handbook of Experimental Pharmacology, (Edited by BRODII-:B. B. & GIt,LI-:rTE J. R.), Vol. XXVIIL Part I, pp. 9 24. Springer-Vcrlag, Berlin. TuRNr~R J. C., GRliEI~ R. S. & KI!I,LY W. J. (1976) In vitro metabolism of aniline by sheep intestine. Gen. Pharmac. 7, 289.
THE EFFECT OF SODIUM PHENOBARBITAL A N D 3,4-BENZPYRENE ON THE G L U C U R O N I D A T I O N OF I-NAPHTHOL IN RAT SMALL INTESTINAL LOOPS IN V I V O J. C. Tt;a.~r.a, V. SrlA~'KS, W. J. Kr~LLY and R. S. GaFE'~ Wallaccville Animal Research Centre, Research Division, Ministry of Agriculture and Fisherics. Private Bag, Upper Hutt, New Zealand (Recei~'ed 26 October 1976)
Abstract I. After 0.1 mM l-naphthol was injected into intestinal loops, 70 90"., of the I-naphthol in the intestinal vcnous blood was present as l-naphthol glucuronide. For 1.0 and 2.0 mM injections the proportion of l-naphthol present as l-naphthol glucuronide was 25-50~'~,,. 2. The proportion of I-naphthol glucuronide in the intestinal venous blood was increased by oral dosing of eithcr sodium phenobarbital or 3,4-benzpyrene but these compounds did not alter the rate of appearance of l-naphthol, free or conjugated. INTRODUCFION
Ltd., Poole. England, and l-naphthol phosphate from Koch-Light Lab. Ltd., Colnbrook England.
There is increasing interest in drug metabolism in cxtrahepatic tissues ( C h h a b r a et al., 1974; Pantuck et al., 1974; T u r n e r et al., 1976). Although the gastrointestinal tract is the first system encountered by many drugs a n d pesticides its role in the absorption a n d dctoxication of xenobiotics is poorly understood. Most of the work has been carried out using in vitro techniques ( C h h a b r a et al., 1974; Del Villar et al., 1974; Pantuck et al., 1974). In the few in t,it'o studies that have been reported it was usual to measure the a m o u n t of the substrate beforc and after a period of incubation in an intestinal loop (Doluisio et al., 1969). G l u c u r o n i d a t i o n of phenols is one of the major conjugations that occurs in the intestinal mucosa (Bock & Winne, 1975; Rance & Shillingford, 1976). The present study was carried out to investigate the effects in rivo of dosing two inducing agents on the ability of the intestine to glucuronidate a simple phenol, l-naphthol. The cannulation technique described below was chosen because it enabled the venous blood draining the intestinal loop to be collected and thus thc I-naphthol metabolites could bc quantitatcd. Any change in the mctabolite pattern due to the inducing agents could also be detected, t4C-ln a p h t h o l was chosen as the substrate since the major metabolite is l-naphthol glucuronide with little formation of l - n a p h t h o l sulphate ( B o c k & Winnc, 1975).
Analytical techniques Blood samples were assayed for l-naphthol and l-naphthol glucuronide by a slight modification of the rapid radioassay de~ribed by Bock & White (1974). Aliquots (20pl) of whole blood were digested in thc counting vial with 0.4ml NCS tissue solubiliscr [AmcrshamSearle Corp., Arlington Heights, 1113. The ~mplcs werc decolorized with 0.2 ml .saturated solution of benzoyl pcroxidc in toluene. Tissue samples were treated as described previously (Turner et al., 19761. Confirmation of the presence of l-naphthol and its mctabolites in intestinal venous blood was made by TLC on silica gel plates in n-butanol-n-propanol 2 M" NH.,OH fl :6:3 v/v) and by paper chromatography on Whatman No. I paper developed with butanol-aqueous NH 3 (0.88) H 2 0 (4:1:5 v/'v) (Binning et al., 1967). The [ollowing standards were co-chromatographed with the whole blood extracts: l-naphthol, I-naphthol glucuronidc. l-naphthol sulphatc and l-naphthol phosphate. Standards were visualized as described by' Binning et al. (1967). Two methods of preparing whole blood cxtracts for chromatography were used. In the tirst method, one w~lume of blood was precipitated with two volumes of acetone, centrifuged for 2 3 min at 15(X).qand thc supernatant was rotary evaporated just to dryncss at 45 C. In the second method, whole blood ~mples wcre extracted twice with equal volumes of ethyl acetate. The combined ethyl acetate extracts were rotary evaporated just to dryness at 45'C. The residues obtained from both methods of extraction were taken up in 8()'~;, ethanol and aliquots chromatographed. Samples scraped from T L C platcs aftcr development were counted as described previously (Tt, rncr et al.. 1976). The paper chromatograms werc cut into 0.5 x 2.0cm wide strips from the origin to the sol~.cnt front and the radioactivity in the strips counted. The positions of the radioactive regions were compared with thc standard compounds. Details of the scintillation counter, phosphors and counting procedure have been describcd previously (Turner et al., 1976).
MATERIALS AND ME'rHODS ('h{,micul~
I-[ l -~'~C]-napht hol (20.8 mCi/mmole) was obtained from the Radiochemical Centre, Amersham, England. The radiochemical purity of the compound was greater than 97~ as determined by TLC on silica gel with benzene-methanol (19:1 v/v). 3,4-benzpyrene. l-naphthol glucuronide and I-naphthol sulphate were obtained from Sigma Chemical Co. {St. Louis. Mo.), I-naphthol from B.D.H. Chemicals
Preparation of intestitlal loops Male Wistar rats (300 350 g) wcrc used. Some animals were dosed per os with 3,4-benzpyrenc (30 mg:kg body
51
52
J C. Tt:R,'qER,V. SHANKS,W. J. KELLYAND R. S. GREEN
weight) in peanut oil 24 hr before the operation. Controls were dosed with peanut oil only. Some animals were given sodium phenobarbital in their drinking water (1 mg/ml) for 4 days. Freshly prepared solutions of sodium phenobarbital were supplied each day. Controls had water only. All animals were starved for 18hr before surgery to ensure that intestinal loops were free of food. Intestinal loops were prepared with the rats under light ether anaesthesia. A jejunal loop, about 10cm from the duodenal flexure and about 5 cm long. was exteriorized and kept moist with isotonic saline. A polythene eannula was inserted into the vein draining the entire loop and secured with tissue glue (Ethieon Buerylat, Ethicon, Hamburg, Germany). The loop was then ligated. The blood flow was 0.8 1.0ml/min. Heparinized blood from donor rats was transfused into the femoral vein to replace the blood collected from the cannula. The prepared loop was injccted with 0.5 ml of 1-naphthol solution (containing 0.35/~Ci t4C-l-naphthol and 0.500 v/v ethanol) in isotonic saline buffered to pH 7.2 with phosphate buffer. Three concentrations of l-naphthol, 0.1, 1.0 and 2.0mM were used. Blood samples (80 100/~1) wcre takcn at intervals for 15 min. The blood collected between these samplcs was deep-frozen and used for the TLC and paper chromatography analyses. At lhe end of the blood collection period the rat was killed and the intestinal loop was removed. The mucosa was .scraped off with a spatula and homogenized and both mucosa and luminal contents were assayed for l-naphthol and l-naphthol glucuronide as dcscribcd for blood.
100 806040-
20-
%
o
,
,
,
,
(hgl)
0 1raM
,
,--"
,
~-nophlho[ i
BO60-
40-
20(fig2) 1 0 mM
1- n o t p h t h o l
(fig3)20mM
1-naphthol
5
,b
%0 B0604020-
%0 1
2 3 4
7
,~
Time ( m ~ n s l
RESULTS
l-Naphthol glucuronide was the major metabolite of I-naphthol. Thc sulphate conjugate of l-naphthol comprised less than 4% of the dose at all three concentrations of l-naphthol used, both in control and dosed animals. Other metabolites of l-naphthol were present as traces only. Figures 1-3 show the percentage of I-naphthol present as the glucuronide in the intestinal venous blood at various times after the introduction of 0.1, 1.0 or 2.0 mM 1-naphthol, respectively, into rat intestinal loops. The figures also show the effect of dosing with sodium phenobarbital on thc proportion of l-naphthol glucuronide in the venous blood. Figures 2 and 3 show the effect of dosing with peanut oil and benzpyrene on the proportion of l-naphthol glucuronide in the blood. After injecting 0.1 mM I-naphthol, 70 -90,°.,,iof the l-naphthol was found to bc present as I-naphthol glucuronide from I min after dosing until blood collection ceased at 15min. Sodium phenobarbital had no effect on the proportion of I-naphthol glucuronide formed. At the 1.0 and 2.0 mM I-naphthol dose rates the proportion of 1-naphthol glucuronide in the venous blood increased from 25 to 50%. Sodium phenobarbital increased the rate of glucuronidation compared to the controls. Dosing with peanut oil (a control for the benzpyrene-dosing cxperiment) gave a marked increase in the proportion of glucuronide (Figs. 2 and 3} with both 1.0 and 2.0 and 2.0mM l-naphthol; in fact the proportion of glucuronide formed was greater than that found in rats exposed to sodium phenobarbital. After dosing with benzpyrene the percentage of 1-naphthol glucuronidated increased with 1.0mM l-naphthol in the intestinal loop. About 80'J,;~ of the l-naphthol was present as l-naphthol glucuronide. With 2.0 mM l-naphthol, bcnzpyrene had little effect
Figs. 1, 2 and 3. The percentage of l-naphthol present as the glucuronide in the intestinal venous blood after injecting 1-naphthol solution into intestinal loops. (Values are means of 3-6 animals.) O, Control (water); O, phenobarbital; A, peanut oil; A, benzpyrene. on the percentage of glucuronide formed compared to the peanut oil controls. In general, there was a trend towards an increase in the proportion of I-naphthol glucuronide during the course of the 15-min blood collection period (Figs. 1-3). No alteration was observed in the rate of appearance of total 14C-activity in the intestinal venous blood after dosing either benzpyrene or sodium phenobarbital. At the end of the blood collection period the lumen contents contained a high proportion of the glucuronide, ranging from 45 to 80%.
DISCUSSION
The validity of the rapid radio-assay used for the assay of 1-naphthol and l-naphthol glucuronide depends on negligible quantities of other metabolites being present. Because analysis of blood samples showed that the amount of the sulphate conjugate and other metabolites did not increase after dosing either benzpyrene or sodium phenobarbital, the rapid radio-assay was used throughout. l'he increase in the proportion of l-naphthol glucuronide in the intestinal venous blood from rat loops containing 1.0 or 2.0raM I-naphthol after dosing with sodium phenobarbital, peanut oil or benzpyrene suggests that induction of the glueuronidation system had occurred. Induction of this reaction in intestine by 3,4-benzpyrene has been demonstrated using an in vitro assay (Aitio et al., 1972).
The effect of sodium phenobarbital and 3,4-bcnzpyrene The lack of any observable effect of sodium phenobarbital when 0.1 mM I-naphthol was used is probably an indication that little l-naphthol remained since about 85°,/, of the dose was glucuronidated in the control rats. Therefore any quantitative increase in UDP-glucuronyltransferase would have been superfluous at this low dose rate. For this reason the effect of benzpyrene on thc metabolism of 0.1 mM l-naphthol in the loop was not investigated. Thc increase in glucuronide formation after dosing with peanut oil was probably due to peroxides of sterols present in the oil. Peanut oil contains small amounts of sterols and peroxides are known to be formed during storage. Sterol peroxides have been shown to induce liver microsomal mixed-function oxidases (Brown et al., 1954). The similarity in percentagc glucuronidation in the first few minutes between the benzpyrene-treated rats and the peanut oil-dosed rats with 2.0 mM l-naphthol may have been due to the high concentration of l-naphthol in thc mucosal cells. Assuming glucuronidation occurs at the maximum rate, the proportion of l-naphthol glucuronidc would increase as the concentration of l-naphthol in the mucosal cells decreased during the 15 minute blood collection period. Differences in the absolute amount of UDP-glucuronyltransferase between the benzpyrcne-treated animals and the peanut oil-dosed controls would therefore become more apparent as the l-naphthol concentration decreased. This trend is apparent from the 5th minute onwards in Fig. 3. The capacity of the glucuronidation enzyme system appears to be limited since there was a general tendency for the proportion of l-naphthol glucuronide formcd to increase during the course of an experiment. This increase was probably due to the concentration of l-naphthol in the intestinal loop falling during the course of the experiment so that less l-naphthol reached the mucosal cells. Bock & Winne (1975) showed that l-naphthol glucuronide injected into intestinal loops was slowly absorbed. However, the rate at which this occurrcd was very low compared with the rate of l-naphthol glucuronidation in the mucosa; in this case secretion of l-naphthol glucuronide from the mucosa into the lumen and reabsorption would not contribute significantly to the percentage of I-naphthol glucuronidc found in the intestinal venous blood. Dosing with sodium phenobarbital and benzpyrene had no effect on the ratc of appearance of total 14C activity in the intestinal venous blood. This lack of effect on the absorption rate is not unexpected if the normal absorptive process of l-naphthol is by passive diffusion. Passive diffusion is thought to be the mechanism of absorption of many drugs, particularly if they are not structurally similar to normal dietary constituents (Schanker, 1971 ). The rate of passage into the bloodstream is to some extent governed by the rate of blood flow (Parsons, 1968). The presence of 1-naphthol glucuronide in the lumen contents indicates that the glucuronide can be secreted into either venous blood or the intestinal lumen. This confirms the findings of Bock & Winne (1975). The results of this work suggest that with small quantities of substratc the glucuronidating capacity
53
of the intestine is sufficicat to conjugate most of the compound being absorbed. The liver has previously been assumed to be the major site of metabolism of foreign compounds but the present studies and those of others (Powell et al., 1974: Rance & Shillingford, 1976) indicate that the gastrointestinal tract plays a significant role in the conjugation of drugs. The results reported here considered together with those on the effect of benzpyrene on the dealkylation of phenacetin (Pantuck et al., 1974; Pantuck et al., 1975). justify further studies of substances that may alter the intestinal metabolism of drugs thereby affecting their bioavailability.
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