- Email: [email protected]
Intestinal absorption and secretion of ochratoxin A in the rat
rOXICOLOGY
AND
APPLIED
PHARMACOLOGY
Intestinal Absorption
64, 94- 102 (1982)
and Secretion
SUSUMU KUMAGAI'
of Ochratoxin
A in the Rat
AND KAGEAKIAIBARA
Department of Biomedical Research on Food, National Institute of Health, Kamiosaki. Shinagawa-ku. Tokyo, Japan
Received October 22, 1981; accepted February 8. I982 Intestinal Absorption and Secretion of Ochratoxin A in the Rat. KUMAGAI, S., AND AIBARA, K. ( 1982). Toxicol. Appl. Pharmacol. 64,94- 102. The absorption and secretion of ochratoxin A (OA) by the gastrointestinal tract were studied in the rat. When OA was introduced into the lumen at various sites of the gastrointestinal tract, the largest concentration of OA in portal blood was found after the toxin was injected into the lumen at the proximal jejunum. After the injection of OA into a closed loop at the proximal jejunum, the rate of appearance of OA in the mesenteric venous plasma was higher than that in lymph. The rate of appearance in the venous plasma increased with an increase in the luminal concentration of OA while in the lymph the rate remained almost constant with respect to the luminal OA concentration. These results suggest that the site of maximal absorption is the proximal jejunum and that the primary route of absorption is the portal vein although the contribution of the lymphatic route cannot be excluded when low-dose levels of OA are given. When various parts of the gastrointestinal tract were perfused after iv injection of OA, noticeable amounts of the toxin appeared in the intestinal perfusate, suggesting that intestinal secretion may be another route of excretion of OA. Comparison of intestinal secretion and absorption showed asymmetric transfer of OA across the intestinal mucosa, the lumen-to-blood transfer being greater than that in the opposite direction.
exposure of mice to very low levels such as 0.005 pg/kg (Haubeck et al., 1981). Although a mycotoxicosis produced by OA, when it occurs naturally, is caused undoubtedly by ingestion of the toxin, little is known about the gastrointestinal absorption of the toxin. Studies of the fate of OA have shown that po administered toxin is absorbed somewhere in the gastrointestinal tract and appears in the bloodstream within one hour (Nel and Purchase, 1968; Galtier, 1974a; Suzuki et al., 1977; Galtier et al., 1979). Both OA and its metabolites are excreted in urine and feces (Nel and Purchase, 1968; Galtier, 1974b; Chang and Chu, 1977; Suzuki et al., 1977; Nip and Chu, 1979). Fecal excretion has been attributed to biliary excretion (Suzuki et al., 1977). However, the possibility of excretion of OA by intestinal
Ochratoxin A (OA) is a mycotoxin produced by a number of species of Aspergillus and Penicillium. It has been suggested as a possible causative agent of endemic nephropathy in farm animals and humans (Krogh, 1977). Relatively low-dose levels damage the kidney (van Walbeek et al., 1971; Munro et al., 1974; Rutqvist et al., 1978), but highdose levels damage the liver and intestine as well as the kidney (Purchase and Theron, 1968; Munro et al., 1974). The oral LD50 in the rat is 20 to 22 mg/kg (Purchase and Theron, 1968). Teratogenic effects in experimental animals have been studied (Brown et al., 1976; Hood et al., 1976; Szczech and Hood, 1980). Recently immunosuppressive action of the toxin was demonstrated by the ’ To whom correspondence should be addressed. 0041-008X/82/070094-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
94
INTESTINAL
ABSORPTION
secretion cannot be excluded because some drugs have been found to be secreted by the intestinal mucosa (Noach et a/., 1958; Williams et al., 1965; Caldwell et al., 1980). In order to study some of the basic phenomena of gastrointestinal absorption and secretion of OA, we have conducted in vivo studies to determine the site of maximal absorption of OA, absorption of OA into the venous blood and lymph, and secretion of OA by the gastrointestinal tract. METHODS Animals and chemicals. Male Wistar rats 8 to 10 weeks of age were maintained under 12-hr lighting with food and water ad libitum. OA was purchased from Makor Chemical Ltd. (Israel). Unless otherwise stated, OA dissolved in 0. I N NaHC02 ( 1.17 mg/ml) was used for the administration of OA. Determination of OA. Extraction of OA was carried out on the day samples were obtained. After being acidified with 1 N HCl, every sample was extracted three times with 20 vol of chloroform. For the extraction, 100 ~1 of serum, plasma, or lymph, 100 to 200 ~1 of bile, 2 to 3 ml of the stomach or intestinal perfusate, or 1 to 2 ml of tissue homogenate was used. The chloroform extracts were stored in the dark at -20°C until the next step. Within 2 days after the extraction, the chloroform was evaporated to dryness under Nz, redissolved in chloroform, and applied quantitatively to thin-layer plates (DC-Fertigplatten Kieselgel 60, Merck, West Germany) (Suzuki et al., 1977). After the plates were developed with benzene-acetic acid (9:1), they were examined under ultraviolet light (wavelength 365 nm, Nikko Sekiei Works Co., Japan) for the presence of fluorescent spots, and the OA concentration was measured in a spectrophotofluorometer equipped with a TLC scanner (Hitachi Co., Japan) (Suzuki et al., 1977). The activation and emission wavelengths for OA determination were set at 340 and 375 nm, respectively (Chu and Butz, 1970). The recovery of the toxin added to plasma, bile, and lymph was 91.9 r 6.1% (n = 17). 87.6 + 2.0% (n = 5) and 82.7 + 5.1% (n = 6) respectively. ‘The toxin added to heparinized blood was recovered at a quantity of 75.3 + 5.8% (n = 7) from the plasma which was separated after incubation of the blood for I hr at 37’C, indicating that most of the OA in the blood was in the plasma. The site of maximal absorption: Experiment I. OA concentrations in peripheral blood serum were determined after the toxin was injected into the stomach, small intestine, cecum, or colon. Under ether anesthesia, the abdomen was opened by a small incision, and the
OF OCHRATOXIN
A
05
injection site was isolated with ligatures around the intestine. OA solution (OA = 1.4 mg/kg) was injected into the lumen with a tuberculin syringe, after which the abdomen was closed with a clip and the animal was allowed to awaken. Blood specimens were taken by heart puncture 15, 60, 120. and 240 min after the toxin was injected. The site of maximal absorption: Experiment 2. GA was introduced into a tied loop 4 to 8 cm in length in the small intestine or colon, and portal blood was collected. Preceding the experiment, rats were starved overnight but allowed free access to water. After the abdomen was opened in animals under pentobarbital (sodium pentobarbital. 64.8 mg/kg, Pitman-Moore Inc., USA) anesthesia, one loop was selected at various sites of the small intestine or colon and isolated between hgatures. Into this loop, OA solution (OA = I. 17 mg/kg) and 1.0 ml of phosphate buffer (pH 7.0) were introduced. When the same amount of OA solution was mixed with 1.0 ml of phosphate buffer in a test tube. the OA was soluble. After 200 units of heparin sodium solution (Novo Industri, Denmark) was injected into the femoral vein, a polyethylene tube (0.30 mm id., 1.09 mm o.d.) was inserted into the portal vein near its entrance into the liver for blood collection. The size of this cannula allowed only a small amount of venous blood to flow out. Blood was collected at lo-min intervals from 5 min after the introduction of OA. Portal blood was collected in the same manner after the OA sohttion (OA = 1.17 mg/kg) was introduced into the stomach in 1.0 ml of citric acid buffer (pH 3.0). When the same amount of OA solution and I.0 mi ot citric acid buffer were mixed in a test tube, the OA was insoluble. To assess the degradation of OA by the gastrointestinal tissue, 1.0 ng of OA was incubated (37°C. 2 hr) with a homogenate (phosphate buffer, pH 7.4) of stomach or intestinal tissue (0.2 to 0.5 g), and the recover) of the added toxin was determined. The same amount of OA was also incubated with heated tissue homogenates, and its recovery was used as a control. Absorption of OA into blood and lymph. The rate of appearance of OA in mesenteric venous plasma and lymph in the same rat was determined. Rats starved overnight were anesthesized with pentobarbital, and the small intestine was brought to the exterior through an abdominal incision. After heparin was injected into the femoral vein, a polyethylene tube (0.86 mm i.d., 1.27 mm o.d.) was inserted into the mesenteric lymph channel with a ligature when needed. A 2.5 to 4.5-m segment of the small intestine drained bv a single vein was selected. and a polyethylene tube (0.28 mm i.d.. 0.61 mm o.d.) was inserted into this vein and secured with an adhesive agent (Aron Alpha, Toa Gosei Kagaku Co., Japan). After one end of the segment was closed with a ligature, 16 to 96 ~1 of OA solution and 0.3 ml of Krebs-Ringer solution were introduced through a nee-
96
KUMAGAI
die into the lumen at the other end, and the segment was closed with a ligature just inside the point of the needle. A microsyringe (100 ~1) was used for the introduction of the OA solution and a tuberculin syringe for the Krebs-Ringer solution. In a test tube, the pH of the mixture of 16 to 96 ~1 of the OA solution and 0.3 ml of the Krebs-Ringer solution was 7.4 to 7.8; OA was soluble. After the introduction of the toxin, lymph and blood were collected at IO-min intervals for 30 min. At the same intervals, 0.5 to 1.0 ml of 0.9% NaCl solution was injected into the femoral vein to compensate for the loss of blood and lymph. The flow rate of the blood plasma and lymph was determined by measuring the volume of each. The rate of appearance of OA in each circulation system was calculated from the toxin concentration and the flow rate. For comparison, OA was injected into the femoral vein instead of being introduced into the intestinal loop, and the appearance rate of OA in the lymph was determined in the same manner as described above. To determine the amount of OA that permeated directly from the intestinal lumen to the serosal side, a 2.5 to 4.5-cm loop of the jejunum was filled with a mixture of the OA solution and the Krebs-Ringer solution, and the jejunum then was placed in a bath containing 0.5 to 1.0 ml of phosphate buffer (pH 7.0). During the experimental period, rats were anesthesized with pentobarbital and the circulatory systems to the loop were left intact. The buffer solution was changed at 30min intervals. The amount of the toxin permeating during two 30-min periods was assessedby determining the OA content of the buffer solution. Mucosal secretion of OA. To evaluate the secretion of OA by the gastrointestinal mucosa, various sites of the gastrointestinal tract were perfused and the rate of appearance of OA in the perfusion medium was determined after the OA solution was injected iv in rats not fed overnight. While rats were under pentobarbital anesthesia, the bile duct was cannulated with a polyethylene tube (0.28 mm i.d., 0.61 mm o.d.). A perfusion tube connected to a peristaltic pump (Tokyo Rika Kikai Co., Japan) was inserted into the stomach, the upper (proximal end: 10 cm from the pylorus; mean length, 23.3 cm; SE, 2.4; n = 3) or lower (distal end: distal end of the ileum; mean length, 20.6 cm; SE, 4.5; n = 4) small intestine, or the colon (proximal end: proximal end of the colon; mean length, 6.5 cm; SE, 1.5; n = 3) from the proximal side of each. Another tube was inserted into each lumen from the distal side. Each tube was secured with a ligature. After the luminal contents were flushed out with slowly infused 0.9% NaCl solution, the OA solution (OA = 1.4 mg/kg) was injected into the femoral vein. Each lumen was perfused with 0.9% NaCl solution at a speed of 0.28 ml/min. Bile and perfusate were collected at IO-min intervals for 30 to 60 min. A peripheral blood specimen was taken by heart puncture 30 or 60 min after the injection of OA.
AND AIBARA
0 0
.
0
I I I ‘b I*6-PHM I I 0
1
A-4 I
I
2
I(hrs)
Time after
injection
FIG. 1. Peripheral blood levels of OA after injection into the stomach (A, nonfasting rats, A, fasting rats) or the small intestine (0, nonfasting rats). For comparing secretion with absorption by the intestine, a loop made at the proximal jejunum was perfused with 0.9% NaCl containing 10 rg of OA/ml, and the rate of appearance of OA in the mesenteric venous plasma was determined in the same manner as described previously. Statistics. The statistical significance of differences between means was determined by Student’s t test.
RESULTS The Site of the Maximal
Absorption
Experiment 1. The peripheral blood level of the toxin was above 5.0 pg/ml of serum 15 min after injection into the small intestine, whereas the level was below 1.5 @g/ml of serum after the injection into the stomach even in fasting rats (Fig. 1). Two rats each received the injection in the colon in the presence or absence of its contents and two other rats each received it in the cecum, also in the presence or absence of its contents. In all these rats, the peripheral blood levels of
INTESTINAL
ABSORPTION
OF OCHRATOXIN
‘37
A
trations in the portal blood plasma were below 1.4 pg/ml. Degradation of OA by the tissue homogenate was not observed. Recovery rates of OA added to the tissue homogenates were similar to those of OA added to heated tissue homogenates (stomach, 99.9% of control recovery rate, n = 2; intestine, 103.2% of control recovery rate, n = 6). 60
Y 2
N.D. 0 I 0.1 Distance (length small
0.1 1 0.3
cl.3 I 0.7
from the pylorus of distance/length intestine)
0.7 I 1.0 to the loop of the
FIG. 2. OA levels in portal venous plasma after the injection of 1.17 mg of OA/kg into a loop made at various sites of the small intestine. The site of each loop is expressed by the distance from the pylorus to the midpoint of the loop. Samples taken at 5-15 min and 15-25 min are shown in striped columns and black columns. respectively. Mean + SE was calculated from values of detectable levels. Vertical bars show SE. Numbers over the vertical bars and those within the column are the numbers of rats with detectable levels and those with undetectable levels, respectively.
OA remained lower than 2.0 pg/ml of serum for 2 hr. Experiment 2. OA levels of portal blood plasma are expressed as the levels per wet weight of the tissue of the intestinal loop. As shown in Fig. 2, the highest level of OA was noted when OA was injected into the loop at the site l/10 to 3/10 of the entire length of the small intestine from the pylorus. The levels were below 2.0 pg/ml/g when the toxin was injected into the colon. When it was injected into the stomach, OA concen-
OL -
59
Luminal
113 concentration (sg/ml)
206
284 of
DA
FIG. 3. Rate of appearance of OA in mesenteric venous plasma (upper panel) and in lymph (lower panel) as a function of the luminal OA concentration. Blood and lymph were collected at lo-min intervals after the injection of OA into the loop at the proximal jejunum. Mean +_ SE of combined data from the second and third periods are shown. Vertical bars and numbers over them show SE and the numbers of samples, respectively. Significant differences (p < 0.05) were noted in the appearance rate in the venous plasma between luminal concentrations of 59 vs 113, 59 vs 206, and 206 vs 284 i&ml.
98
KUMAGAI
AND AIBARA TABLE
RATE
OF APPEARANCE AFTER
Treatment Injection to the femoral vein 1.O mg/kg Injection into the jejunal loop 113 pg/ml 206 rg/ml
OF
iv
OA
IN THE LYMPH
INJECTION
AND
OR INJECTION
1 ITS CONCENTRATION INTO
THE JEJUNAL
Appearance rate” in lymph G.410 mid
OA concentration“ in lymph (a/ml)
0.50 +- 0.09 (6)
2.69 + 0.39 (6)
0.30 + 0.04 (7) 0.44 -t 0.08 (6)
1.51 _+ 0.24 (7) 2.16 2 0.40 (6)
IN BLOOD
AND
LYMPH
LOOP
OA concentration* in blood &g/ml serum or plasma) Peripheral serum 13.2 zk 0.9 (3) Mesenteric venous plasma 10.3 f 2.2 (7) 22.1 a 3.6 (8)
a Appearance rate and concentration of OA in lymph are the pooled data from the second and third IO-min periods. Data on rats injected via the jejunal loop were used in Fig. 3. b Peripheral blood was taken 30 min after OA injection. Values for the mesenteric venous plasma were used for the calculation of the appearance rates shown in Fig. 3. ’ Mean + SE. Numbers in parentheses are the numbers of samples.
Absorption
of OA into Blood and Lymph
OA was introduced into a loop made at the proximal jejunum (distance between the loop and the pylorus, 15-27 cm) and the rate of appearance of OA in the mesenteric venous plasma and lymph was determined. The flow rate of the venous plasma did not differ among the dose groups nor among the time groups (mean flow rate, 324 pl/lO mitt; SE, 15; n = 43). The lymphatic flow rate tended to decrease during the experimental period, but did not differ among the dose groups when compared at the same time intervals (mean flow rate, 246 ccl/IO min; SE, 15; n = 41). Since OA was not detected in lymph in the first lo-min period in several rats and the appearance rate in each circulation system remained almost constant from the second to the third interval, data of the second and third time periods were combined and are shown in Fig. 3. The appearance rate in the blood increased with an increase in the luminal concentration of OA (r = 0.78) and was higher than that in the lymph over the range of the luminal OA concentrations used. The difference in the rate between the two circulatory systems increased with an increase in the luminal OA concentration,
because of the nearly constant rate in the lymph. When OA was introduced into a loop in the lower half of the small intestine, the appearance rate in each circulatory system was much lower than that observed when the toxin was introduced into a loop at the proximal jejunum. The rates for the venous and lymphatic circulation during the experimental period were less than 7.5 fig/g/l0 min and less than 0.29 pug/g/l0 min (number of rats = 3), respectively, when 206 pg of OA/ ml was introduced. The combined data on the appearance of iv-injected OA in the lymph in the second and third time periods are shown in Table 1. The rate was almost the same as that observed after the introduction of OA into the loop at the proximal jejunum. Peripheral blood levels of OA were raised by iv injection of OA to a level comparable to that in the mesenteric venous plasma after introduction of OA into the jejunal loop. Permeation of the luminal OA across the intestinal wall to the serosal side was determined by placing the jejunal loop containing 206 hug of OA/ml in buffer solution. The amount of OA that permeated was 0.27 f 0.11 rg/g wet weight/30 min (n = 3) in the first period and 0.55 + 0.13 Kg/g wet
INTESTINAL
ABSORPTION
OF OCHRATOXIN
Mucosal
0
Time
85 &L&l 30
after
60(min)
OA inject.
FIG. 4. Intestinal secretion rate, biliary excretion rate, and peripheral blood serum concentrations of OA after iv injection. Intestinal perfusates of the upper and the lower small intestine and bile were collected at lo-min intervals. Vertical bars and numbers over them show SE and the numbers of rats, respectively.
weight/30 min (n = 3) in the second period. The peripheral blood levels in the same animals were 4.87 + 1.34 fig/ml of serum at 30 min and 6.33 f 0.33 pg/ml of serum at 60 min, suggesting that permeation to the serosal side is unimportant as an absorption route. To ascertain whether fluorescent metabolites of OA appear in the mesenteric vein or lymph, a thin-layer plate was examined. No fluorescent spots other than OA were found in either the blood plasma or the lymph.
A
99
Secretion of UA
For evaluating the possibility of secretion of OA by the gastrointestinal mucosa into the lumen, perfusion at the various sites of the gastrointestinal tract was carried out in rats bearing a cannula in their bile duct. The results expressed as the amount per intestinal wet weight, shown in Fig. 4, show that the appearance of OA in the perfusate is similar in the upper and the lower small intestine. If the total wet weight of the small intestine is taken into account (ca. 6 g), the amount of OA secreted by the entire small intestine is comparable to that excreted through bile. When the colon was perfused, OA was also detected in the perfusate and the maximal rate of appearance was 122.8 ng,Jg/ IO min (n = 3). The rates in the stomach perfusate were less than 65.0 ng/stomach/lO min (n = 3). For comparing secretion with absorption under similar conditions, the proximal jejunum was perfused with 0.9% NaCl containing OA, and the blood was collected from the mesenteric vein at 20-min intervals. The concentration of OA in the perfusion medium chosen for the experiment was 10 pg/ml, which is less than the OA concentrations in the peripheral blood serum observed when the toxin secretion was exatnined (Fig. 4). The results are shown in Fig. 5. In the first time period, OA was below the detectable level. In the following periods. the rates of appearance in the venous plasma were 10 times higher than those in the intestinal perfusate in iv-injected rats (Fig. 4). DISCUSSION The present study shows that the primary site of OA absorption is the small intestine and the site of maximal absorption is the proximal jejunum. The ability of various sites to absorb the toxin may depend on the mucosal surface area, because the villus structure of the small intestine extends its
100
KUMAGAI
surface area especially at the jejunum. Since absorption was assessed by the rate of appearance of OA in the circulatory systems and OA has been reported to be hydrolyzed to a fluorescent metabolite, ochratoxin a, by the intestinal tissue (Doster and Sinnhuber, 1972; Galtier, 1974b; Suzuki et al., 1977), it seems possible that the difference in the absorption rate at various sites might reflect the difference in their metabolic activity. However, the results suggest that hydrolyzation of OA by the intestinal tissue, if any, may have only an insignificant influence on OA absorption. Degradation of OA was not observed in the incubated gastrointestinal tissue and furthermore no spots other than OA were visualized on thin-layer plates either in the extracts of mesenteric venous plasma or in those of lymph. The rate of appearance of OA after injection into the intestinal loop was higher in venous plasma than in lymph over the range of the luminal concentrations. Permeation of OA through the intestinal wall was only slight. These results demonstrate that the primary route of the transport of the toxin is the portal vein. A higher rate of appearance of the toxin in plasma than in lymph would be due to the greater flow rate of blood than lymph. Since it is generally recognized that the exchange of small molecules takes place between lymphatic capillaries and blood capillaries (Yoffey and Courtice, 1970), it seems possible that the rate of appearance of OA in the lymph after injection into an intestinal loop might reflect only the escape of the toxin from the blood. However, comparison of the appearance of OA in the lymph after its injection into the intestinal loop with that after iv injection excludes this possibility. The toxin in lymph was derived from only a short (2.5-4.5 cm) segment of the small intestine when given in the intestinal loop, while it was derived from the entire gastrointestinal tract when given iv. As shown in Table 1, the rate of appearance of OA in the lymph of rats injected via the intestinal
AND AIBARA
4
--
0
20
40
60
2'0
4'0
60,
8b
Time (min) FIG. 5. Rate of appearance of OA in the plasma of the mesenteric vein from the jejunum perfused with 10 rg of OA/ml. Blood was collected at 20-min intervals. Mean -+ SE of two rats is shown. SE is shown by vertical bars.
loop was not lower than that in iv-injected rats. Both treatments raised the blood level of OA to a similar extent. Therefore, the toxin appearing in the lymph after injection into the loop would not have been the toxin escaping from blood but that taken up by the lacteal. The appearance rate of OA in the blood increased with an increase in the luminal concentration of the toxin, suggesting that absorption of OA takes place by passive diffusion and that the concentration of the toxin in the intercellular space also increases with an increase in the luminal OA concentration. The flow rate of lymph in the mesenteric lymph channel was not changed by the increased luminal concentration of OA, but impairment of the lymphatic flow at the site of the loop remains possible. Although no macroscopic damage was observed within the experimental period in the present study, the toxin has been reported to induce enteritis and increase the secretion of the fluid into the intestinal lumen 1.5 hr after oral administration (Suzuki et al., 1977). Such abnormal movement of the fluid at the intestinal mucosa could be accompanied by obstruction of the lymphatic flow and consequently reduce the transport of the toxin by the lymph.
INTESTINAL
ABSORPTION
The results show that the lower the luminal OA concentration, the larger the contribution of the lymphatic route. Recently very low levels of OA have been found to induce immunosuppression in mice (Haubeck et al., 1981). If impairment of the function of the intestinal and mesenteric lymphoid tissues is involved in such toxicity of OA, the lymphatic route would play an important role by exposing these tissues to high concentrations of the toxin. In addition to renal and biliary excretion of OA (Suzuki et al., 1977), the results suggest that the mucosal secretion by the gastrointestinal tract is also a route of OA excretion. The amount of OA which would be secreted by the entire gastrointestinal tract was estimated to be comparable to that excreted through bile. Since such experimental conditions as perfusion speed and perfusion medium appear favorable for mucosal secretion, the estimated value may not be exactly the same as the amount of the toxin which would be secreted in the presence of the luminal contents. However, at the site where the luminal OA concentration is far below the concentration of OA in blood, a large amount of the toxin may be secreted from the mucosa. Since the maximal absorption was seen in the proximal jejunum and similar secretion rates were observed in the upper and the lower small intestine, the lower half of the intestine may be the predominant site of OA secretion when the toxin is ingested. Comparison of secretion and absorption showed that transfer of OA takes place more efficiently from the intestinal lumen into the blood stream than in the opposite direction. Such asymmetric transfer of OA between blood and the intestinal lumen is apparently requisite for the persistence of the toxin in the animal body. Morphological and functional characteristics of the intestine which result in the asymmetry of flux of nutrients possibly play a role in the mechanism. However, a more important factor may be the limited diffusion of OA from blood plasma
OF OCHRATOXIN
101
A
into the extracellular space presumably because of binding of OA to serum albumin (Chu, 1971, 1974; Chang and Chu, 1977). This limited diffusion was seen in the much lower OA concentrations in the lymph than in the blood serum after iv injection of the toxin (Table 1). Whether the toxin is secreted directly from the mucosal cells or through the tight junction between them remains uncertain; the limited diffusion from plasma into the extracellular space would result in a lower concentration of the toxin in the intercellular space and the mucosal cells. In the present study, the absorption and secretion of OA were examined under conditions in which no macroscopic damage was observed. Further study will be needed to ascertain whether or not the toxic effects of the toxin on the gastrointestinal tract change the absorption and secretion of OA. REFERENCES BROWN, M. H., SZCZECH, G. M., AND PURMALIS, B. P. (1976). Teratologic and toxic effects of ochratoxin A in rats. Toxicoi. Appi. Pharmacol. 37, .?3 I 338.
CALDWELL, J. H.. CALDWELL, P. B., MURPHY, J. W.. AND BEACHLER, C. W. (1980). Intestinal secretion of digoxin in the rat. Augmentation by feeding activated charcoal. Nauyn-Schmiedeberg’s Arch. Pharmakol.
Exp.
Pathol.
312, 211-215.
CHANG, F. C., AND CHU, F. S. (1977). The fate of ochratoxin A in rats Food Cosmet. Toxicol. 15. 199204.
CHU, F. S. (1971). Interaction of ochratoxin A with bovine serum albumin Arch. Biochem. Biophvs 147, 359-366.
CHU, F. S. (1974). A comparative study of the interaction of ochratoxin A with bovine serum albumin. Biochem. Pharmacol. 23, I 105- 1113. CHU, F. S.. AND BUTZ, M. E. (1970). Spectrophotofluorodensitometric measurement of ochratoxin A in cereal products, J. Assoc. Ojic. Anaf. Chem. 53, 1253-1257. DOSTER, R. C., AND SINNHUBER, R. 0. (1972). Comparative rates of hydrolysis of ochratoxin A and B in vitro.
Food
Cosmet.
Toxicol.
10, 389-394.
GALTIER, P. (1974a). Devenir de I’ochratoxine A dans I’organisme animal. 1. Transport sanguin de la toxine chez le rat. Ann. Rech. Vet. 5, 311-318.
102
KUMAGAI
GALTIER, P. (1974b). Devenir de l’ochratoxin A dans I’organisme animal. II. Distribution tissulaire et Mimination chez le rat. Ann. Rech. Vet. 5, 319-328. GALTIER, P., CHARPENTEAU, J. L., ALVINERIE, M., AND LABOUCHE, C. ( 1979). The pharmacokinetic profile of ochratoxin A in the rat after oral and intravenous administration. Drug Metab. Dispos. 7, 429-434.
HAUBECK, H. D., LORKOWSKI, G., K~LSCH, E., AND R~SCHENTHALER, R. ( 198 1). Immunosuppression by ochratoxin A and its prevention by phenylalanine. Appl. Environ. Microbial. 41, 1040- 1042. HOOD, R., NAUGHTON, M. J., AND HAYES, A. W. (1976). Prenatal effects of ochratoxin A in hamsters. Teratology
13, 1 I- 14.
KROGH, P. (1977). Ochratoxins. In Mycoroxins in Human and Animal Health (J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, eds.), pp. 489-498. Pathotox Pub., Illinois. MUNRO, I. C., MOODIE, C. A., KUIPER-GOODMAN, T., SCOTT, P. M., AND GRICE, H. C. (1974). Toxicologic changes in rats fed graded dietary levels of ochratoxin A. Toxicol. Appl. Pharmacol. 28, 180- 188. NEL, W., AND PURCHASE, I. F. H. (1968). The fate of ochratoxin A in rats. J. South Afr. Chem. Inst. 21, 87-88.
W. K., AND A in goats. J. NOACH, E. L., L. S. (1958). NIP,
CHU, F. S. (1979). Fate of ochratoxin Environ.
Sci. Health
B14,
319-333.
WOODBURY, D. M., AND GOODMAN, Studies on the absorption, distribution,
AND
AIBARA
fate and excretion of 4-C’4-labelled diphenylhydantoin. J. Pharmacol. Exp. Ther. 122, 301-314. PURCHASE, I. F. H., AND THERON, J. J. (1968). The acute toxicity of ochratoxin A in rats. Food Cosmel. Toxicol.
6, 479-483.
RUTQVIST, L., BJ~RKLUND, N.-E., HULT, K., H~KBY, E., AND CARLSSON, B. (1978). Ochratoxin A as the cause of spontaneous nephropathy in fattening pigs. Appl.
Environ.
Microbial.
36, 920-925.
SUZUKI, S., SATOH, T., AND YAMAZAKI, M. ( 1977). The pharmacokinetics of ochratoxin A in rats. Japan. J. Pharmacol.
27, 735-744.
SZCZECH, G. M., AND HOOD, R. D. (1980). Brain necrosis in mouse fetuses transplacentally exposed to the mycotoxin ochratoxin A. Toxieol. Appl. Pharmaeol. 57, 127-137.
VAN WALBEEK, W., MOODIE, C. A., SCOTT, P. M., HARWIG, J., AND GRICE, H. C. ( 197 1). Toxicity and excretion of ochratoxin A in rats intubated with pure ochratoxin A or fed cultures of Penicillium viridicum. Toxicol.
Appl.
Pharmacol.
20, 439-44
1.
WILLIAMS, R. T., MILLBURN, P., AND SMITH, R. L. (1965). The influence of enterohepatic circulation on toxicity of drugs. Ann. N. Y. Acad. Sci. 123, 11 O124.
YOFFEY, J. M., AND COURTICE, F. C. (1970). Exchange of materials within the extracellular fluid phase. In Lymphatics, plex (J. M.
Lymph
and the Lymphomyeloid
Com-
Yoffey and F. C. Courtice, eds.), pp. 72135. Academic Press, London.
AND
APPLIED
PHARMACOLOGY
Intestinal Absorption
64, 94- 102 (1982)
and Secretion
SUSUMU KUMAGAI'
of Ochratoxin
A in the Rat
AND KAGEAKIAIBARA
Department of Biomedical Research on Food, National Institute of Health, Kamiosaki. Shinagawa-ku. Tokyo, Japan
Received October 22, 1981; accepted February 8. I982 Intestinal Absorption and Secretion of Ochratoxin A in the Rat. KUMAGAI, S., AND AIBARA, K. ( 1982). Toxicol. Appl. Pharmacol. 64,94- 102. The absorption and secretion of ochratoxin A (OA) by the gastrointestinal tract were studied in the rat. When OA was introduced into the lumen at various sites of the gastrointestinal tract, the largest concentration of OA in portal blood was found after the toxin was injected into the lumen at the proximal jejunum. After the injection of OA into a closed loop at the proximal jejunum, the rate of appearance of OA in the mesenteric venous plasma was higher than that in lymph. The rate of appearance in the venous plasma increased with an increase in the luminal concentration of OA while in the lymph the rate remained almost constant with respect to the luminal OA concentration. These results suggest that the site of maximal absorption is the proximal jejunum and that the primary route of absorption is the portal vein although the contribution of the lymphatic route cannot be excluded when low-dose levels of OA are given. When various parts of the gastrointestinal tract were perfused after iv injection of OA, noticeable amounts of the toxin appeared in the intestinal perfusate, suggesting that intestinal secretion may be another route of excretion of OA. Comparison of intestinal secretion and absorption showed asymmetric transfer of OA across the intestinal mucosa, the lumen-to-blood transfer being greater than that in the opposite direction.
exposure of mice to very low levels such as 0.005 pg/kg (Haubeck et al., 1981). Although a mycotoxicosis produced by OA, when it occurs naturally, is caused undoubtedly by ingestion of the toxin, little is known about the gastrointestinal absorption of the toxin. Studies of the fate of OA have shown that po administered toxin is absorbed somewhere in the gastrointestinal tract and appears in the bloodstream within one hour (Nel and Purchase, 1968; Galtier, 1974a; Suzuki et al., 1977; Galtier et al., 1979). Both OA and its metabolites are excreted in urine and feces (Nel and Purchase, 1968; Galtier, 1974b; Chang and Chu, 1977; Suzuki et al., 1977; Nip and Chu, 1979). Fecal excretion has been attributed to biliary excretion (Suzuki et al., 1977). However, the possibility of excretion of OA by intestinal
Ochratoxin A (OA) is a mycotoxin produced by a number of species of Aspergillus and Penicillium. It has been suggested as a possible causative agent of endemic nephropathy in farm animals and humans (Krogh, 1977). Relatively low-dose levels damage the kidney (van Walbeek et al., 1971; Munro et al., 1974; Rutqvist et al., 1978), but highdose levels damage the liver and intestine as well as the kidney (Purchase and Theron, 1968; Munro et al., 1974). The oral LD50 in the rat is 20 to 22 mg/kg (Purchase and Theron, 1968). Teratogenic effects in experimental animals have been studied (Brown et al., 1976; Hood et al., 1976; Szczech and Hood, 1980). Recently immunosuppressive action of the toxin was demonstrated by the ’ To whom correspondence should be addressed. 0041-008X/82/070094-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
94
INTESTINAL
ABSORPTION
secretion cannot be excluded because some drugs have been found to be secreted by the intestinal mucosa (Noach et a/., 1958; Williams et al., 1965; Caldwell et al., 1980). In order to study some of the basic phenomena of gastrointestinal absorption and secretion of OA, we have conducted in vivo studies to determine the site of maximal absorption of OA, absorption of OA into the venous blood and lymph, and secretion of OA by the gastrointestinal tract. METHODS Animals and chemicals. Male Wistar rats 8 to 10 weeks of age were maintained under 12-hr lighting with food and water ad libitum. OA was purchased from Makor Chemical Ltd. (Israel). Unless otherwise stated, OA dissolved in 0. I N NaHC02 ( 1.17 mg/ml) was used for the administration of OA. Determination of OA. Extraction of OA was carried out on the day samples were obtained. After being acidified with 1 N HCl, every sample was extracted three times with 20 vol of chloroform. For the extraction, 100 ~1 of serum, plasma, or lymph, 100 to 200 ~1 of bile, 2 to 3 ml of the stomach or intestinal perfusate, or 1 to 2 ml of tissue homogenate was used. The chloroform extracts were stored in the dark at -20°C until the next step. Within 2 days after the extraction, the chloroform was evaporated to dryness under Nz, redissolved in chloroform, and applied quantitatively to thin-layer plates (DC-Fertigplatten Kieselgel 60, Merck, West Germany) (Suzuki et al., 1977). After the plates were developed with benzene-acetic acid (9:1), they were examined under ultraviolet light (wavelength 365 nm, Nikko Sekiei Works Co., Japan) for the presence of fluorescent spots, and the OA concentration was measured in a spectrophotofluorometer equipped with a TLC scanner (Hitachi Co., Japan) (Suzuki et al., 1977). The activation and emission wavelengths for OA determination were set at 340 and 375 nm, respectively (Chu and Butz, 1970). The recovery of the toxin added to plasma, bile, and lymph was 91.9 r 6.1% (n = 17). 87.6 + 2.0% (n = 5) and 82.7 + 5.1% (n = 6) respectively. ‘The toxin added to heparinized blood was recovered at a quantity of 75.3 + 5.8% (n = 7) from the plasma which was separated after incubation of the blood for I hr at 37’C, indicating that most of the OA in the blood was in the plasma. The site of maximal absorption: Experiment I. OA concentrations in peripheral blood serum were determined after the toxin was injected into the stomach, small intestine, cecum, or colon. Under ether anesthesia, the abdomen was opened by a small incision, and the
OF OCHRATOXIN
A
05
injection site was isolated with ligatures around the intestine. OA solution (OA = 1.4 mg/kg) was injected into the lumen with a tuberculin syringe, after which the abdomen was closed with a clip and the animal was allowed to awaken. Blood specimens were taken by heart puncture 15, 60, 120. and 240 min after the toxin was injected. The site of maximal absorption: Experiment 2. GA was introduced into a tied loop 4 to 8 cm in length in the small intestine or colon, and portal blood was collected. Preceding the experiment, rats were starved overnight but allowed free access to water. After the abdomen was opened in animals under pentobarbital (sodium pentobarbital. 64.8 mg/kg, Pitman-Moore Inc., USA) anesthesia, one loop was selected at various sites of the small intestine or colon and isolated between hgatures. Into this loop, OA solution (OA = I. 17 mg/kg) and 1.0 ml of phosphate buffer (pH 7.0) were introduced. When the same amount of OA solution was mixed with 1.0 ml of phosphate buffer in a test tube. the OA was soluble. After 200 units of heparin sodium solution (Novo Industri, Denmark) was injected into the femoral vein, a polyethylene tube (0.30 mm id., 1.09 mm o.d.) was inserted into the portal vein near its entrance into the liver for blood collection. The size of this cannula allowed only a small amount of venous blood to flow out. Blood was collected at lo-min intervals from 5 min after the introduction of OA. Portal blood was collected in the same manner after the OA sohttion (OA = 1.17 mg/kg) was introduced into the stomach in 1.0 ml of citric acid buffer (pH 3.0). When the same amount of OA solution and I.0 mi ot citric acid buffer were mixed in a test tube, the OA was insoluble. To assess the degradation of OA by the gastrointestinal tissue, 1.0 ng of OA was incubated (37°C. 2 hr) with a homogenate (phosphate buffer, pH 7.4) of stomach or intestinal tissue (0.2 to 0.5 g), and the recover) of the added toxin was determined. The same amount of OA was also incubated with heated tissue homogenates, and its recovery was used as a control. Absorption of OA into blood and lymph. The rate of appearance of OA in mesenteric venous plasma and lymph in the same rat was determined. Rats starved overnight were anesthesized with pentobarbital, and the small intestine was brought to the exterior through an abdominal incision. After heparin was injected into the femoral vein, a polyethylene tube (0.86 mm i.d., 1.27 mm o.d.) was inserted into the mesenteric lymph channel with a ligature when needed. A 2.5 to 4.5-m segment of the small intestine drained bv a single vein was selected. and a polyethylene tube (0.28 mm i.d.. 0.61 mm o.d.) was inserted into this vein and secured with an adhesive agent (Aron Alpha, Toa Gosei Kagaku Co., Japan). After one end of the segment was closed with a ligature, 16 to 96 ~1 of OA solution and 0.3 ml of Krebs-Ringer solution were introduced through a nee-
96
KUMAGAI
die into the lumen at the other end, and the segment was closed with a ligature just inside the point of the needle. A microsyringe (100 ~1) was used for the introduction of the OA solution and a tuberculin syringe for the Krebs-Ringer solution. In a test tube, the pH of the mixture of 16 to 96 ~1 of the OA solution and 0.3 ml of the Krebs-Ringer solution was 7.4 to 7.8; OA was soluble. After the introduction of the toxin, lymph and blood were collected at IO-min intervals for 30 min. At the same intervals, 0.5 to 1.0 ml of 0.9% NaCl solution was injected into the femoral vein to compensate for the loss of blood and lymph. The flow rate of the blood plasma and lymph was determined by measuring the volume of each. The rate of appearance of OA in each circulation system was calculated from the toxin concentration and the flow rate. For comparison, OA was injected into the femoral vein instead of being introduced into the intestinal loop, and the appearance rate of OA in the lymph was determined in the same manner as described above. To determine the amount of OA that permeated directly from the intestinal lumen to the serosal side, a 2.5 to 4.5-cm loop of the jejunum was filled with a mixture of the OA solution and the Krebs-Ringer solution, and the jejunum then was placed in a bath containing 0.5 to 1.0 ml of phosphate buffer (pH 7.0). During the experimental period, rats were anesthesized with pentobarbital and the circulatory systems to the loop were left intact. The buffer solution was changed at 30min intervals. The amount of the toxin permeating during two 30-min periods was assessedby determining the OA content of the buffer solution. Mucosal secretion of OA. To evaluate the secretion of OA by the gastrointestinal mucosa, various sites of the gastrointestinal tract were perfused and the rate of appearance of OA in the perfusion medium was determined after the OA solution was injected iv in rats not fed overnight. While rats were under pentobarbital anesthesia, the bile duct was cannulated with a polyethylene tube (0.28 mm i.d., 0.61 mm o.d.). A perfusion tube connected to a peristaltic pump (Tokyo Rika Kikai Co., Japan) was inserted into the stomach, the upper (proximal end: 10 cm from the pylorus; mean length, 23.3 cm; SE, 2.4; n = 3) or lower (distal end: distal end of the ileum; mean length, 20.6 cm; SE, 4.5; n = 4) small intestine, or the colon (proximal end: proximal end of the colon; mean length, 6.5 cm; SE, 1.5; n = 3) from the proximal side of each. Another tube was inserted into each lumen from the distal side. Each tube was secured with a ligature. After the luminal contents were flushed out with slowly infused 0.9% NaCl solution, the OA solution (OA = 1.4 mg/kg) was injected into the femoral vein. Each lumen was perfused with 0.9% NaCl solution at a speed of 0.28 ml/min. Bile and perfusate were collected at IO-min intervals for 30 to 60 min. A peripheral blood specimen was taken by heart puncture 30 or 60 min after the injection of OA.
AND AIBARA
0 0
.
0
I I I ‘b I*6-PHM I I 0
1
A-4 I
I
2
I(hrs)
Time after
injection
FIG. 1. Peripheral blood levels of OA after injection into the stomach (A, nonfasting rats, A, fasting rats) or the small intestine (0, nonfasting rats). For comparing secretion with absorption by the intestine, a loop made at the proximal jejunum was perfused with 0.9% NaCl containing 10 rg of OA/ml, and the rate of appearance of OA in the mesenteric venous plasma was determined in the same manner as described previously. Statistics. The statistical significance of differences between means was determined by Student’s t test.
RESULTS The Site of the Maximal
Absorption
Experiment 1. The peripheral blood level of the toxin was above 5.0 pg/ml of serum 15 min after injection into the small intestine, whereas the level was below 1.5 @g/ml of serum after the injection into the stomach even in fasting rats (Fig. 1). Two rats each received the injection in the colon in the presence or absence of its contents and two other rats each received it in the cecum, also in the presence or absence of its contents. In all these rats, the peripheral blood levels of
INTESTINAL
ABSORPTION
OF OCHRATOXIN
‘37
A
trations in the portal blood plasma were below 1.4 pg/ml. Degradation of OA by the tissue homogenate was not observed. Recovery rates of OA added to the tissue homogenates were similar to those of OA added to heated tissue homogenates (stomach, 99.9% of control recovery rate, n = 2; intestine, 103.2% of control recovery rate, n = 6). 60
Y 2
N.D. 0 I 0.1 Distance (length small
0.1 1 0.3
cl.3 I 0.7
from the pylorus of distance/length intestine)
0.7 I 1.0 to the loop of the
FIG. 2. OA levels in portal venous plasma after the injection of 1.17 mg of OA/kg into a loop made at various sites of the small intestine. The site of each loop is expressed by the distance from the pylorus to the midpoint of the loop. Samples taken at 5-15 min and 15-25 min are shown in striped columns and black columns. respectively. Mean + SE was calculated from values of detectable levels. Vertical bars show SE. Numbers over the vertical bars and those within the column are the numbers of rats with detectable levels and those with undetectable levels, respectively.
OA remained lower than 2.0 pg/ml of serum for 2 hr. Experiment 2. OA levels of portal blood plasma are expressed as the levels per wet weight of the tissue of the intestinal loop. As shown in Fig. 2, the highest level of OA was noted when OA was injected into the loop at the site l/10 to 3/10 of the entire length of the small intestine from the pylorus. The levels were below 2.0 pg/ml/g when the toxin was injected into the colon. When it was injected into the stomach, OA concen-
OL -
59
Luminal
113 concentration (sg/ml)
206
284 of
DA
FIG. 3. Rate of appearance of OA in mesenteric venous plasma (upper panel) and in lymph (lower panel) as a function of the luminal OA concentration. Blood and lymph were collected at lo-min intervals after the injection of OA into the loop at the proximal jejunum. Mean +_ SE of combined data from the second and third periods are shown. Vertical bars and numbers over them show SE and the numbers of samples, respectively. Significant differences (p < 0.05) were noted in the appearance rate in the venous plasma between luminal concentrations of 59 vs 113, 59 vs 206, and 206 vs 284 i&ml.
98
KUMAGAI
AND AIBARA TABLE
RATE
OF APPEARANCE AFTER
Treatment Injection to the femoral vein 1.O mg/kg Injection into the jejunal loop 113 pg/ml 206 rg/ml
OF
iv
OA
IN THE LYMPH
INJECTION
AND
OR INJECTION
1 ITS CONCENTRATION INTO
THE JEJUNAL
Appearance rate” in lymph G.410 mid
OA concentration“ in lymph (a/ml)
0.50 +- 0.09 (6)
2.69 + 0.39 (6)
0.30 + 0.04 (7) 0.44 -t 0.08 (6)
1.51 _+ 0.24 (7) 2.16 2 0.40 (6)
IN BLOOD
AND
LYMPH
LOOP
OA concentration* in blood &g/ml serum or plasma) Peripheral serum 13.2 zk 0.9 (3) Mesenteric venous plasma 10.3 f 2.2 (7) 22.1 a 3.6 (8)
a Appearance rate and concentration of OA in lymph are the pooled data from the second and third IO-min periods. Data on rats injected via the jejunal loop were used in Fig. 3. b Peripheral blood was taken 30 min after OA injection. Values for the mesenteric venous plasma were used for the calculation of the appearance rates shown in Fig. 3. ’ Mean + SE. Numbers in parentheses are the numbers of samples.
Absorption
of OA into Blood and Lymph
OA was introduced into a loop made at the proximal jejunum (distance between the loop and the pylorus, 15-27 cm) and the rate of appearance of OA in the mesenteric venous plasma and lymph was determined. The flow rate of the venous plasma did not differ among the dose groups nor among the time groups (mean flow rate, 324 pl/lO mitt; SE, 15; n = 43). The lymphatic flow rate tended to decrease during the experimental period, but did not differ among the dose groups when compared at the same time intervals (mean flow rate, 246 ccl/IO min; SE, 15; n = 41). Since OA was not detected in lymph in the first lo-min period in several rats and the appearance rate in each circulation system remained almost constant from the second to the third interval, data of the second and third time periods were combined and are shown in Fig. 3. The appearance rate in the blood increased with an increase in the luminal concentration of OA (r = 0.78) and was higher than that in the lymph over the range of the luminal OA concentrations used. The difference in the rate between the two circulatory systems increased with an increase in the luminal OA concentration,
because of the nearly constant rate in the lymph. When OA was introduced into a loop in the lower half of the small intestine, the appearance rate in each circulatory system was much lower than that observed when the toxin was introduced into a loop at the proximal jejunum. The rates for the venous and lymphatic circulation during the experimental period were less than 7.5 fig/g/l0 min and less than 0.29 pug/g/l0 min (number of rats = 3), respectively, when 206 pg of OA/ ml was introduced. The combined data on the appearance of iv-injected OA in the lymph in the second and third time periods are shown in Table 1. The rate was almost the same as that observed after the introduction of OA into the loop at the proximal jejunum. Peripheral blood levels of OA were raised by iv injection of OA to a level comparable to that in the mesenteric venous plasma after introduction of OA into the jejunal loop. Permeation of the luminal OA across the intestinal wall to the serosal side was determined by placing the jejunal loop containing 206 hug of OA/ml in buffer solution. The amount of OA that permeated was 0.27 f 0.11 rg/g wet weight/30 min (n = 3) in the first period and 0.55 + 0.13 Kg/g wet
INTESTINAL
ABSORPTION
OF OCHRATOXIN
Mucosal
0
Time
85 &L&l 30
after
60(min)
OA inject.
FIG. 4. Intestinal secretion rate, biliary excretion rate, and peripheral blood serum concentrations of OA after iv injection. Intestinal perfusates of the upper and the lower small intestine and bile were collected at lo-min intervals. Vertical bars and numbers over them show SE and the numbers of rats, respectively.
weight/30 min (n = 3) in the second period. The peripheral blood levels in the same animals were 4.87 + 1.34 fig/ml of serum at 30 min and 6.33 f 0.33 pg/ml of serum at 60 min, suggesting that permeation to the serosal side is unimportant as an absorption route. To ascertain whether fluorescent metabolites of OA appear in the mesenteric vein or lymph, a thin-layer plate was examined. No fluorescent spots other than OA were found in either the blood plasma or the lymph.
A
99
Secretion of UA
For evaluating the possibility of secretion of OA by the gastrointestinal mucosa into the lumen, perfusion at the various sites of the gastrointestinal tract was carried out in rats bearing a cannula in their bile duct. The results expressed as the amount per intestinal wet weight, shown in Fig. 4, show that the appearance of OA in the perfusate is similar in the upper and the lower small intestine. If the total wet weight of the small intestine is taken into account (ca. 6 g), the amount of OA secreted by the entire small intestine is comparable to that excreted through bile. When the colon was perfused, OA was also detected in the perfusate and the maximal rate of appearance was 122.8 ng,Jg/ IO min (n = 3). The rates in the stomach perfusate were less than 65.0 ng/stomach/lO min (n = 3). For comparing secretion with absorption under similar conditions, the proximal jejunum was perfused with 0.9% NaCl containing OA, and the blood was collected from the mesenteric vein at 20-min intervals. The concentration of OA in the perfusion medium chosen for the experiment was 10 pg/ml, which is less than the OA concentrations in the peripheral blood serum observed when the toxin secretion was exatnined (Fig. 4). The results are shown in Fig. 5. In the first time period, OA was below the detectable level. In the following periods. the rates of appearance in the venous plasma were 10 times higher than those in the intestinal perfusate in iv-injected rats (Fig. 4). DISCUSSION The present study shows that the primary site of OA absorption is the small intestine and the site of maximal absorption is the proximal jejunum. The ability of various sites to absorb the toxin may depend on the mucosal surface area, because the villus structure of the small intestine extends its
100
KUMAGAI
surface area especially at the jejunum. Since absorption was assessed by the rate of appearance of OA in the circulatory systems and OA has been reported to be hydrolyzed to a fluorescent metabolite, ochratoxin a, by the intestinal tissue (Doster and Sinnhuber, 1972; Galtier, 1974b; Suzuki et al., 1977), it seems possible that the difference in the absorption rate at various sites might reflect the difference in their metabolic activity. However, the results suggest that hydrolyzation of OA by the intestinal tissue, if any, may have only an insignificant influence on OA absorption. Degradation of OA was not observed in the incubated gastrointestinal tissue and furthermore no spots other than OA were visualized on thin-layer plates either in the extracts of mesenteric venous plasma or in those of lymph. The rate of appearance of OA after injection into the intestinal loop was higher in venous plasma than in lymph over the range of the luminal concentrations. Permeation of OA through the intestinal wall was only slight. These results demonstrate that the primary route of the transport of the toxin is the portal vein. A higher rate of appearance of the toxin in plasma than in lymph would be due to the greater flow rate of blood than lymph. Since it is generally recognized that the exchange of small molecules takes place between lymphatic capillaries and blood capillaries (Yoffey and Courtice, 1970), it seems possible that the rate of appearance of OA in the lymph after injection into an intestinal loop might reflect only the escape of the toxin from the blood. However, comparison of the appearance of OA in the lymph after its injection into the intestinal loop with that after iv injection excludes this possibility. The toxin in lymph was derived from only a short (2.5-4.5 cm) segment of the small intestine when given in the intestinal loop, while it was derived from the entire gastrointestinal tract when given iv. As shown in Table 1, the rate of appearance of OA in the lymph of rats injected via the intestinal
AND AIBARA
4
--
0
20
40
60
2'0
4'0
60,
8b
Time (min) FIG. 5. Rate of appearance of OA in the plasma of the mesenteric vein from the jejunum perfused with 10 rg of OA/ml. Blood was collected at 20-min intervals. Mean -+ SE of two rats is shown. SE is shown by vertical bars.
loop was not lower than that in iv-injected rats. Both treatments raised the blood level of OA to a similar extent. Therefore, the toxin appearing in the lymph after injection into the loop would not have been the toxin escaping from blood but that taken up by the lacteal. The appearance rate of OA in the blood increased with an increase in the luminal concentration of the toxin, suggesting that absorption of OA takes place by passive diffusion and that the concentration of the toxin in the intercellular space also increases with an increase in the luminal OA concentration. The flow rate of lymph in the mesenteric lymph channel was not changed by the increased luminal concentration of OA, but impairment of the lymphatic flow at the site of the loop remains possible. Although no macroscopic damage was observed within the experimental period in the present study, the toxin has been reported to induce enteritis and increase the secretion of the fluid into the intestinal lumen 1.5 hr after oral administration (Suzuki et al., 1977). Such abnormal movement of the fluid at the intestinal mucosa could be accompanied by obstruction of the lymphatic flow and consequently reduce the transport of the toxin by the lymph.
INTESTINAL
ABSORPTION
The results show that the lower the luminal OA concentration, the larger the contribution of the lymphatic route. Recently very low levels of OA have been found to induce immunosuppression in mice (Haubeck et al., 1981). If impairment of the function of the intestinal and mesenteric lymphoid tissues is involved in such toxicity of OA, the lymphatic route would play an important role by exposing these tissues to high concentrations of the toxin. In addition to renal and biliary excretion of OA (Suzuki et al., 1977), the results suggest that the mucosal secretion by the gastrointestinal tract is also a route of OA excretion. The amount of OA which would be secreted by the entire gastrointestinal tract was estimated to be comparable to that excreted through bile. Since such experimental conditions as perfusion speed and perfusion medium appear favorable for mucosal secretion, the estimated value may not be exactly the same as the amount of the toxin which would be secreted in the presence of the luminal contents. However, at the site where the luminal OA concentration is far below the concentration of OA in blood, a large amount of the toxin may be secreted from the mucosa. Since the maximal absorption was seen in the proximal jejunum and similar secretion rates were observed in the upper and the lower small intestine, the lower half of the intestine may be the predominant site of OA secretion when the toxin is ingested. Comparison of secretion and absorption showed that transfer of OA takes place more efficiently from the intestinal lumen into the blood stream than in the opposite direction. Such asymmetric transfer of OA between blood and the intestinal lumen is apparently requisite for the persistence of the toxin in the animal body. Morphological and functional characteristics of the intestine which result in the asymmetry of flux of nutrients possibly play a role in the mechanism. However, a more important factor may be the limited diffusion of OA from blood plasma
OF OCHRATOXIN
101
A
into the extracellular space presumably because of binding of OA to serum albumin (Chu, 1971, 1974; Chang and Chu, 1977). This limited diffusion was seen in the much lower OA concentrations in the lymph than in the blood serum after iv injection of the toxin (Table 1). Whether the toxin is secreted directly from the mucosal cells or through the tight junction between them remains uncertain; the limited diffusion from plasma into the extracellular space would result in a lower concentration of the toxin in the intercellular space and the mucosal cells. In the present study, the absorption and secretion of OA were examined under conditions in which no macroscopic damage was observed. Further study will be needed to ascertain whether or not the toxic effects of the toxin on the gastrointestinal tract change the absorption and secretion of OA. REFERENCES BROWN, M. H., SZCZECH, G. M., AND PURMALIS, B. P. (1976). Teratologic and toxic effects of ochratoxin A in rats. Toxicoi. Appi. Pharmacol. 37, .?3 I 338.
CALDWELL, J. H.. CALDWELL, P. B., MURPHY, J. W.. AND BEACHLER, C. W. (1980). Intestinal secretion of digoxin in the rat. Augmentation by feeding activated charcoal. Nauyn-Schmiedeberg’s Arch. Pharmakol.
Exp.
Pathol.
312, 211-215.
CHANG, F. C., AND CHU, F. S. (1977). The fate of ochratoxin A in rats Food Cosmet. Toxicol. 15. 199204.
CHU, F. S. (1971). Interaction of ochratoxin A with bovine serum albumin Arch. Biochem. Biophvs 147, 359-366.
CHU, F. S. (1974). A comparative study of the interaction of ochratoxin A with bovine serum albumin. Biochem. Pharmacol. 23, I 105- 1113. CHU, F. S.. AND BUTZ, M. E. (1970). Spectrophotofluorodensitometric measurement of ochratoxin A in cereal products, J. Assoc. Ojic. Anaf. Chem. 53, 1253-1257. DOSTER, R. C., AND SINNHUBER, R. 0. (1972). Comparative rates of hydrolysis of ochratoxin A and B in vitro.
Food
Cosmet.
Toxicol.
10, 389-394.
GALTIER, P. (1974a). Devenir de I’ochratoxine A dans I’organisme animal. 1. Transport sanguin de la toxine chez le rat. Ann. Rech. Vet. 5, 311-318.
102
KUMAGAI
GALTIER, P. (1974b). Devenir de l’ochratoxin A dans I’organisme animal. II. Distribution tissulaire et Mimination chez le rat. Ann. Rech. Vet. 5, 319-328. GALTIER, P., CHARPENTEAU, J. L., ALVINERIE, M., AND LABOUCHE, C. ( 1979). The pharmacokinetic profile of ochratoxin A in the rat after oral and intravenous administration. Drug Metab. Dispos. 7, 429-434.
HAUBECK, H. D., LORKOWSKI, G., K~LSCH, E., AND R~SCHENTHALER, R. ( 198 1). Immunosuppression by ochratoxin A and its prevention by phenylalanine. Appl. Environ. Microbial. 41, 1040- 1042. HOOD, R., NAUGHTON, M. J., AND HAYES, A. W. (1976). Prenatal effects of ochratoxin A in hamsters. Teratology
13, 1 I- 14.
KROGH, P. (1977). Ochratoxins. In Mycoroxins in Human and Animal Health (J. V. Rodricks, C. W. Hesseltine, and M. A. Mehlman, eds.), pp. 489-498. Pathotox Pub., Illinois. MUNRO, I. C., MOODIE, C. A., KUIPER-GOODMAN, T., SCOTT, P. M., AND GRICE, H. C. (1974). Toxicologic changes in rats fed graded dietary levels of ochratoxin A. Toxicol. Appl. Pharmacol. 28, 180- 188. NEL, W., AND PURCHASE, I. F. H. (1968). The fate of ochratoxin A in rats. J. South Afr. Chem. Inst. 21, 87-88.
W. K., AND A in goats. J. NOACH, E. L., L. S. (1958). NIP,
CHU, F. S. (1979). Fate of ochratoxin Environ.
Sci. Health
B14,
319-333.
WOODBURY, D. M., AND GOODMAN, Studies on the absorption, distribution,
AND
AIBARA
fate and excretion of 4-C’4-labelled diphenylhydantoin. J. Pharmacol. Exp. Ther. 122, 301-314. PURCHASE, I. F. H., AND THERON, J. J. (1968). The acute toxicity of ochratoxin A in rats. Food Cosmel. Toxicol.
6, 479-483.
RUTQVIST, L., BJ~RKLUND, N.-E., HULT, K., H~KBY, E., AND CARLSSON, B. (1978). Ochratoxin A as the cause of spontaneous nephropathy in fattening pigs. Appl.
Environ.
Microbial.
36, 920-925.
SUZUKI, S., SATOH, T., AND YAMAZAKI, M. ( 1977). The pharmacokinetics of ochratoxin A in rats. Japan. J. Pharmacol.
27, 735-744.
SZCZECH, G. M., AND HOOD, R. D. (1980). Brain necrosis in mouse fetuses transplacentally exposed to the mycotoxin ochratoxin A. Toxieol. Appl. Pharmaeol. 57, 127-137.
VAN WALBEEK, W., MOODIE, C. A., SCOTT, P. M., HARWIG, J., AND GRICE, H. C. ( 197 1). Toxicity and excretion of ochratoxin A in rats intubated with pure ochratoxin A or fed cultures of Penicillium viridicum. Toxicol.
Appl.
Pharmacol.
20, 439-44
1.
WILLIAMS, R. T., MILLBURN, P., AND SMITH, R. L. (1965). The influence of enterohepatic circulation on toxicity of drugs. Ann. N. Y. Acad. Sci. 123, 11 O124.
YOFFEY, J. M., AND COURTICE, F. C. (1970). Exchange of materials within the extracellular fluid phase. In Lymphatics, plex (J. M.
Lymph
and the Lymphomyeloid
Com-
Yoffey and F. C. Courtice, eds.), pp. 72135. Academic Press, London.