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The interaction of aflatoxin B1 with vitamin K, phenylbutazone, and sulfamethoxine in rats
BIOCHEMICAL
MEDICINE
AND METABOLIC
BIOLOGY
39, 158-167 (1988)
The Interaction of Aflatoxin B, with Vitamin K, Phenylbutazone, and Sulfamethoxine in Rats I. U. ASUZU,* S. N. *Department
of Veterinary and fDepartment
SHETTY,*
Physiology and Pharmacology, of Biochemistry, University
AND
0.
University of Nigeria,
OBIDOA~ of Nigeria, Nsukka, Nsukka, Nigeria
Nigeria,
Received March 11, 1987, and in revised form June 8, 1987
Aflatoxins are metabolites of toxigenic fungi of strains of Aspergillus flavus and A. parasiticus. These fungi are common contaminants of agricultural products especially in the tropics where warm temperatures and high humidity are prevalent (1). Bassir and Babunmi (2) showed that aflatoxin B, (AFB,) possesses some anticoagulant activities probably due to its chemical relationship with the coumarins. Aflatoxicosis has also been associated with gastrointestinal hemorrhages (3,4). The anticoagulant effect of aflatoxin is due to the suppressive effect it has on the synthesis of prothrombin and other clotting factors (V, VII, and X) in the liver and not as a result of hepatocellular damage usually observed in aflatoxicosis (2).
Compounds like alfatoxin which contain the coumarin ring are known to bind to serum/plasma albumin and this effect affects their pharmacokinetic properties (5). Interaction of aflatoxin with other serum/plasma protein-binding drugs may lead to displacement of the former from its binding site. Such a situation will obviously modify the pharmacokinetic profile of the toxin in terms of its concentration in the blood, metabolism, excretion, and ultimately the toxin-induced pathology. This theoretical consideration has led to the present study. Phenylbutazone and sulfamethoxine have been selected for this study because they are known protein-binding drugs which are commonly used for treating disease conditions in both human beings and animals. Vitamin K in addition to its protein binding property has been selected because of its antihemorrhagic effect which is antagonized by coumarin anticoagulants (6). Vitamin K is also claimed to offer temporary relief to dogs suffering from aflatoxicosis (7). MATERIALS
AND METHODS
Animals
Albino rats of both sexes (9-11 weeks old) and weighing between 100 and 150 g were used for this study. The rats were housed in stainless metal cages and 158 0885-4505188 $3.00 Copyright All rights
0 1988 by Academic Press. Inc. of reproduction in any form reserved
AFLATOXIN
B, DRUG
INTERACTION
159
were allowed free access to pelleted feed and water throughout the period of study. They were randomly divided into five groups consisting of 18 (male and female) rats each. Drug
Treatment
The first group of rats served as the untreated control group; the rats in this group did not receive any drug/AFB, treatment. The second group of rats served as the positive control group, treated with 25, pg/kg of AFB, (Sigma) for 5 days. The rats in groups 3, 4, and 5 were treated with vitamin K (Synkavit, HoffmanLa Roche, 5 mg/kg), phenylbutazone (Butazolidine, Geigy, 50 mg/kg), and sulfamethoxine (Fanasil, Roche, 50 mg/kg), respectively, for 3 days immediately after 5 days of therapy with AFB, (25 pg/kg). All treatments were by the intraperitoneal route and the total volume of fluid injected into each rat per day did not exceed 1 ml. All drug solutions except AFB, were made in distilled water. AFB, was first dissolved in a minimal amount of dimethyl sulfoxide (DMSO) and then diluted with distilled water to make a 6.25 pg/ml concentration. Analytical
and Clinicopathological
Procedures
Three rats from each group were randomly selected on 1, 4, 7, 14, 21, 28, and 35 days post-treatment. In addition to clinical observations the following parameters were determined for each rat: the live body weight, whole blood clotting time, and relative wet weights of the liver, kidney, and spleen. Whole blood clotting time was determined by the methods of Riley (8) and Bush (9). A nonheparinized capillary tube was inserted into the medial eye canthus and gently rotated thereby opening some blood capillaries in the region. Immediately after the capillary tube was filled with blood a stopwatch was started. The tube was then broken from one end in bits at intervals of 5 set until a strand of coagulated (clotted) blood was observed linking the ends of the broken capillary tube. At that point the stopwatch was stopped and the time was recorded. Three readings were obtained for each rat and the means + standard deviation were calculated. Relative wet weights of the liver, kidneys, and spleen were determined after sacrificing the rats by decapitation. The organs were removed, lightly blotted, and weighed on an analytical balance. The relative wet weights of these organs were calculated using relative wet weight =
weight of organ Xi!!! weight of animal 1 ’
All the visceral organs were examined in situ for gross pathological changes, if any. Representative samples of the organs were preserved in 10% formal-saline solution for histopathology. The organ samples were embedded in paraffin wax and sections were cut at 5 pm and stained with hematoxylin-eosin (H-E). Plasma
Protein
Equilibrium total protein
Binding
dialysis experiments were performed using rat plasma (4 mg/ml content). The heparinized plasma from rat blood was diluted with
160
ASUZU,
SHE’M’Y,
AND
OBIDOA
0.067 M sodium phosphate buffer (pH 7.4) to obtain the required protein concentration. The dialysis tubing (Visking, size 24A, mol. wt cutoff between 12,000 and 14,000) used for the experiment was cut to suitable lengths and washed several times in deionized water and then soaked in 0.067 M sodium phosphate buffer (pH 7.4) maintained at 4°C for 2 days. One end of the tube was securely tied and the sac was filled with 3 ml plasma plus 1 ml AFB, (3 pg) plus 1 ml of drug (250 pg vitamin K, 2500 pg phenylbutazone, or 2500 pg sulfamethoxine as the case may be). The dialysis tubings with their contents were simultaneously dialyzed at 37°C for 6 hr. The absorbance of the dialysate was determined at 363 nm on a uv spectrophotometer (Sp-500, Eye Unican) using a standard reference alfatoxin solution to quantitate the dialyzed amount of free aflatoxin. Values thus obtained were used to .determine the amount of alfatoxin displaced by each drug. Determination
of Possible AFB,-Drug
Complexing
AFB, (10 pg/ml), vitamin K (250 ,ug/ml), phenylbutazone (2500 pg/ml), and sulfamethoxine (2500 pug/ml) were spotted on a 10 x IO-cm-thin layer chromatoplate (TLC) which was developed in a methanol:choloroform (2:98) solvent system. Each of the drugs was also mixed with AFB,, and the mixture was spotted on TLC alongside the respective drugs. The migration of AFB, and the drugs on the TLC plate was viewed under short and long wavelength uv light. The R, values were calculated for comparison. RESULTS Clinical
Findings
The significant findings were profuse bleeding AFB,-treated rats 1 day following AFB, treatment and vulva in some rats on Day 3 post-treatment. Gross Necropsy
from minor injuries and the enlargement
in most of testis
Findings
The liver of rats in groups 2, 4, and 5 were congested and friable. Echymosis and petechial hemorrhage were also observed in the subcutaneous tissues of these rats. The kidneys and spleen of these animals appeared normal. Control rats (group 1) and rats of group 3 (AFB, plus vitamin K) had apparently normal liver, kidneys, and spleen. These observations were made on Days 1 and 7 posttreatment. By Day 14 post-treatment, rats of groups 2, 3, 4, and 5 showed a swollen, congested, and friable liver, whereas the kidneys and spleen remained normal. Organs of control rats did not show these changes. On Days 21 and 28 post-treatment, lesions observed on Day 14 post-treatment persisted in all treated rats except in those of group 5 in which the lesions were of milder degree. By Day 35 there was obvious recovery in all the treated rats other than those in group 4, in which the repair process was slow. Whole Blood Clotting Time The blood clotting time of group 2 rats was significantly (P < 0.05) higher than that of the control and peak prolongation of clotting time occurred on Day
AFLATOXIN
B , DRUG INTERACTION
Post-treatment
161
Day
FIG. 1. The effect of AFB,, AFB,, + Vitamin K, AFB, + phenylbutazone, famethoxine on the mean clotting time in rats.
and AFB, + sul-
4 post-treatment (Fig. 1). The clotting time in rats of group 3 remained normal throughout the experiment. There were significant (P < 0.05) differences between group 4 rats and those of group 1 and group 2 on Days 1 and 4 post-treatment, with the clotting time of group 4 rats being higher than that of group 1 and lower than those of group 2 rats. On Days 7, 14, and 21 post-treatment, clotting time of group 4 rats was significantly higher than that of group 1 rats. The clotting time of group 5 rats was significantly lower than group 2 rats but was not different from readings obtained from group 1 rats on Day 1 post-treatment. However, by Day 4 the clotting time of group 5 rats was significantly higher than that of group 1 and lower than that of group 2 rats. On Days 7, 14, and 21 post-treatment there was no significant difference between the clotting times of rats in group 5 and group 2. There was no significant difference in clotting time values between all the groups on Days 28 and 35 post-treatment. Relative
Weights of Visceral Organs
There was no significant difference between the relative liver weight of AFB,treated rats and that of control rats except on Days 21 and 28 (Table 1). Similar results were seen in rats treated with AFB, plus vitamin K and AFBr plus sulfamethoxine. However, AFBl plus phenylbutazone-treated rats showed significantly (P < 0.05) higher liver weights compared to AFB,-treated and control groups on the 28th day post-treatment (Table 1). There was no significant difference in relative kidney weights of group 2 and group 1 rats (Table 1). However, the kidneys of group 4 rats were significantly (P < 0.05) smaller than those of group 1 rats on Days 14 and 35 post-treatment. The relative kidney weights of group
Liver Kidney Spleen
Liver Kidney Spleen
Liver Kidney Spleen
Liver Kidney Spleen
14
21
28
35
3.11 + 0.01 1.00 + 0.20 0.51 f 0.07
3.56 f 0.01 1.02 + 0.01 0.41 + 0.01
3.63 + 0.17 1.07 2 0.20 0.40 + 0.04
3.82 + 0.18 1.25 + 0.16 0.69 + 0.14
4.00 + 0.14 0.99 + 0.04 0.51 + 0.04
Group 1 untreated controls
2.96 + 0.11 0.94 f 0.08 0.47 f 0.07
3.93 f 0.12* 0.95 f 0.09 0.41 + 0.07
4.39 + 0.18* 1.02 + 0.07 0.51 AZ 0.15
3.72 IL 0.29 1.07 -c 0.02 0.48 f 0.14
4.12 f 0.14 1.18 k 0.25 0.62 2 0.35
Group 2 AFB,
* Significantly (P < 0.05) different from untreated rats. ** Significantly (P < 0.05) different from AFB, treated rats.
Liver Kidney Spleen
Organ
7
Posttreatment day
3.05 + 0.08 0.94 + 0.12 0.31 k 0.05*.**
3.88 2 0.31 1.03 f 0.19 0.47 + 0.08
4.12 2 0.13* 1.01 + 0.10 0.31 r 0.03*
3.24 + 0.77 1.07 + 0.09 0.38 f 0.10*
3.66 k 0.24* 0.80 f 0.09 0.32 + 0.05*
Group 3 AFB, + vitamin K
3.05 c 0.22 0.81 2 0.03* 0.32 + 0.03*.**
4.20? 0.11 1.04 k 0.02 0.38 f 0.05
4.21 f 0.50 1.04 + 0.13 0.41 2 0.06
3.51 + 0.19 0 87 t 0 03*,** 0:31 + 0:10*
3.61 k 0.18* 1.16 f 0.18 0.59 f 0.22
Group 4 AFB, + phenylbutazone
f k + rt f k + + +
0.45 0.07 0.08 0.28* 0.13 O.lO* 0.61 O.lO* 0.09 2.91 2 0.19 0.85 f 0.09* 0.39 2 0.04
4.13 1.13 0.51 4.43 1.05 0.61 3.91 0.85 0.39
3.99 + 0.37 1.01 2 0.20 0.70 f 0.22
Group 5 AFB, + sulfamethoxine
TABLE 1 Relative Weights (Mean + SD, N = 3) of the Organs of Rats Treated with AFB, and AFB, followed by Other Drugs
g 9
zi E g
5
!
8
%i s“C
AFLATOXIN
B, DRUG
INTERACTION
163
4 rats were significantly lower than those of group 2. Likewise, a significant difference existed between kidney weights of group 5 and group 1 rats on Days 28 and 35 post-treatment (Table 1). There was no significant difference between the relative splenic weights of group 2 and group 1 rats. Group 3 rats showed a significant decrease in relative splenic weight when compared to group 1 on the 7th, 14th, 21st, and 35th day post-treatment and compared to group 2 rats on the 35th day post-treatment. Group 5 rats had significantly (P < 0.05) heavier spleens than group 1 rats on Day 21 post-treatment. Histopathological
Change
The liver, kidneys, and spleen of group 1 rats were normal. In group 2, the liver showed bile duct proliferation and periportal cell necrosis with heavy fatty infiltration in the centrilobular areas as well as bile duct hyperplasia on Days 7 to 28 post-treatment. The kidneys of group 2 rats showed mild nephrosis involving mainly the proximal convoluted tubules. The lesions in the kidneys were observed to be undergoing regeneration by the 14th day post-treatment. There was blood pooling in the sinuses with a large number of immature lymphocytes present in the spleen of group 2 rats on Days 7 and 14 post-treatment. By Day 28 following treatment, the blood pooling had disappeared but the immature lymphocytes were still present. Lesions in the liver and kidneys of group 3 rats were similar to those found in group 2 rats. The liver and kidneys of group 4 rats showed lesions similar to those observed in group 2 rats. Blood pooling in the spleen was observed on Day 7 post-treatment but disappeared by Day 14. Immature lymphocytes were still present by Day 28 post-treatment. Among group 5 rats the liver showed lesions similar to those of group 2 rats up to the 28th day posttreatment. Lesions in the kidneys of group 5 rats were similar to those observed in group 2. The renal lesions however started undergoing regeneration by Day 7 post-treatment (Fig. 2), while the spleen did not show any significant histopathological change throughout the experiment (Fig. 3). Plasma
Protein
Binding
Phenylbutazone and sulfamethoxine significantly (P < 0.05) displaced AFB, from its binding sites in plasma (Table 2). The displacement of AFB, by vitamin K was not significant. Determination
of Possible
AFB, Drug
Complexing
The Rf value of AFB, (0.52) did not change even when it was co-spotted with the other drugs. The Rf value of sulfamethoxine (0.45) also did not change even when the drug was co-spotted with AFB,. DISCUSSION
Profuse bleeding from minor injuries was observed in rats 1 day after intraperitoneal treatment with 25 pg/kg of AFB,. This confirmed that the dose of AFB, selected for the study was sufficient to cause aflatoxin-induced coagulopathy in rats. By the 5th day of treatment with AFB,, a steady state of the toxin had been established as indicated by the mean clotting time of 44.33 min on the 4th
164
ASUZU,
SHE-l-l-Y, AND OBIDOA
FIG. 2. Kidney of rat treated with AFB, + sulfamethoxine regeneration.
showing tubular cells undergoing
FIG. 3. Spleen of rat treated with AFB, + sulfamethoxine.
AFLATOXIN
TABLE 2 Displacement of AFB, from Plasma by Vitamin K, Phenylbutazone,
Dw AFB, AFB, + Vitamin K AFB, + Phenylbutazone AFB, Sulfamethoxine
Amount of AFB, in dialysate (PM) 8.116 8.188 8.551 9.130
165
B, DRUG INTERACTION
2 0.126 If: 0.125 2 0.126 2 0.376
and Sulfamethoxine
Amount of AFB, displaced (%)
n
P value”
0.9 5.4 12.5
3 3 3 3
P > 0.05 P < 0.05 P < 0.05
-
y Compared to AFB, alone.
and 5th days of AFB, treatment. Gross pathological change due to AFB, toxicity had been observed by Day 1 post-treatment (i.e., after 5 days of AFB, treatment followed by 3 days of drug treatment). On Day 1 post-treatment, AFB,-treated rats had significantly (P < 0.05) higher clotting time than the control rats and the rats treated with vitamin K, phenylbutazone, or sulfamethoxine. Vitamin K and sulfamethoxine effectively counteracted the initial rise in clotting time caused by AFB, because there was no significant difference between the clotting time in these groups and that of the control rats (Fig. 1). Though phenylbutazone treatment was able to reduce the clotting time compared to that of AFB,-treated rats, the clotting time recorded in the former group was still significantly higher than that of the control rats. Peak increase in clotting time was established on Day 4 post-treatment in rats treated with AFB, alone, AFB, plus phenylbutazone, and AFB, plus sulfamethoxine. Though phenylbutazone and sulfamethoxine significantly (P < 0.05) reduced the clotting time compared with the AFB,-treated group, they were not able to offer complete protection since their clotting-time values were still significantly higher than values of untreated controls. Among the agents used, vitamin K was the only drug which continued to offer complete protection against prolonged clotting time caused by AFB, by the 4th day post-treatment. Vitamin K maintained this protective capability for the duration of the experiment. By Days 7 and 14 the effect of AFBi on clotting time had declined, although the clotting time was still significantly higher than those of control values. There was at this time no significant difference between the AFB,-treated group and the groups treated with phenylbutazone and sulfamethoxine. From the 21st day of the study, clottingtime values in four out of five treatment groups had returned to normal with the clotting time of rats treated with phenylbutazone showing significantly higher values than those of the controls. It appears that the increase in clotting time persists more in phenylbutazone-treated rats than in all other groups. It is not possible to explain the exact mechanism of action of the drugs used in this study in reducing AFB ,-induced coagulopathy. However, the equilibrium dialysis experiment indicated that phenylbutazone and sulfamethoxine signilicantly (P -=c 0.05) displaced AFB, from rat plasma in vitro (Table 2). Therefore the displacement of AFB, from the binding sites in plasma could be one of the
166
ASUZU,
SHETTY,
AND
OBIDOA
mechanisms involved in the observed effect of phenylbutazone and sulfamethoxine on clotting time. The involvement of other mechanisms is likely because displacement of AFB, from its plasma binding sites alone should have intensified the anticoagulant effect (6). This is, however, contrary to what has been observed in this study. Displacement of drugs from bound to free form also can result in increased rate of metabolism and excretion (10). It is known that the alteration in the rate of one parameter, such as metabolism, could alter the biologic activity of AFBi (11). Phenylbutazone is a known inducer of liver microsomal enzymes and this property is likely to enhance the metabolic rate of free AFB,. Though vitamin K is known to bind extensively to plasma proteins (12), the equilibrium dialysis experiment with AFB, plus vitamin K did not show any significant displacement of AFBi by vitamin K. However, vitamin K is necessary for the synthesis in the liver of not only prothrombin (coagulation factor II) itself but also of proconvertin (VII), plasma thromboplastin component (IX), and StuartPrower (X) factors (9). AFB,, on the contrary, suppresses the synthesis of prothrombin, factors V, VII, and X of the clotting mechanism in the liver (2). This may well explain why vitamin K was able to completely suppress the increase in clotting time caused by AFB,. Direct complexing of vitamin K, phenylbutazone, or sulfamethoxine with AFB, does not seem to contribute to the results of this study as shown by the TLC of the mixture of AFB, with each of the other drugs. Results of this study suggest that though vitamin K was able to completely suppress the effect of AFB, on clotting time, it had no protective effect on the organs especially the liver and kidney. It did, however, have protection over the spleen. Sulfamethoxine also had protection over the spleen and the kidneys where the cells were undergoing regenerative changes by Day 7 post-treatment, 1 week earlier than was observed in the AFB,-treated group. The effect of the different treatments (drugs plus AFBJ on organ weight was not consistent and was therefore considered insignificant in this study. In conclusion, the increased blood clotting time in rats caused by AFB, was suppressed to varying degrees by vitamin K, phenylbutazone, and sulfamethoxine with vitamin K offering complete suppression throughout the period of experimentation. However, this suppressive effect appears to be limited to blood clotting time alone since no significant protection was offered to the liver and kidney of the affected rats. Vitamin K in particular may therefore offer temporary relief in acute alIatoxicosis but cannot be used as a sole cure for animals suffering from the condition. Sulfamethoxine was not only capable of reducing the severity of allatoxin-induced coagulopathy but may also provide some beneficial protective effects against tissue damage caused by AFB,. SUMMARY
The interactions of aflatoxin B, (AFB,) with vitamin K, phenylbutazone, and sulfamethoxine were investigated in albino rats. Vitamin K (5 mg/kg) was able to completely suppress the increase in whole blood clotting time caused by AFB, (25 pg/kg). Phenylbutazone (50 mg/kg) and sulfamethoxine (50 mg/kg) also significantly (P < 0.05) lowered the increased clotting time caused by AFB,.
AFLATOXIN
B, DRUG INTERACTION
167
Equilibrium dialysis was performed on rat plasma (4 mg/ml protein content) to investigate the displacement of AFB, (3 pg) from its bound form by vitamin K (250 pug), phenylbutazone (2500 pg), and sulfamethoxine (2500 pg). Phenylbutazone and sulfamethoxine significantly (P < 0.05) displaced AFB, from rat plasma protein. Histopathological examinations performed on the liver, kidneys, and spleen of control and treated rats showed that none of the drugs used appeared to offer any significant organ protection against AFB, except in the spleen. ACKNOWLEDGMENTS The authors are grateful to Dr. E. Onyekweodiri for interpreting the histopathological slides and Mr. P. A. Nnadi for his contribution to this work. This work was partially supported by the University of Nigeria Senate Research Grant 00422/81.
REFERENCES 1. Heathcote, J. G., and Hibbert, J. R.. “Mycotoxins: Chemical and Biological Aspects,” pp. 1629. Elsevier Scientific, Amsterdam, 1978. 2. Bassir, O., and Babunmi, E. A. W. Afr. J. Biol. Appl. Chem. 12, 28 (1969). 3. Butter, W. H., hit. J. Cancer 18, 756 (1964). 4. Newberne, P. M., Russo, R., and Wogan, G. N., Path&@ Vet. 3, 331 (1966). 5. Dayton, P. G., Israili, Z. H., and Perel, J. M., Ann. N. Y. Acad. Sci. 226, 172 (1973). 6. Wilson, A., Schild, H. O., and Modell, W., “Applied Pharmacology,” 11th ed., pp. 240-245. The English Language Book Society and Churchill Livingstone, Edinburgh, 1975. 7. Nwakanma, E., Police dog section, Nigerian Police Force, Enugu, 1985. 8. Riley, R., Biol. Med. 104, 751 (1960). 9. Bush, B. M., In “Veterinary Laboratory Manual,” 1st ed. Heinemann, London, 1960. 10. Meyers, I. H., Jawetz, E., and Goldfien, A., “Review of Medical Pharmacology,” 4th ed., pp. 165-176. Lange Med. Pub., CA, 1970. Il. Gamer, R. C., World Rev. Nutr. Diet. 29, 178 (1978). 12. Goldstein, A., Aronow, L., and Kalman, S. M., “Principles of Drug Action: The Basis of Pharmacology,” 2nd ed., pp. 819-827. Wiley, New York, 1974.
MEDICINE
AND METABOLIC
BIOLOGY
39, 158-167 (1988)
The Interaction of Aflatoxin B, with Vitamin K, Phenylbutazone, and Sulfamethoxine in Rats I. U. ASUZU,* S. N. *Department
of Veterinary and fDepartment
SHETTY,*
Physiology and Pharmacology, of Biochemistry, University
AND
0.
University of Nigeria,
OBIDOA~ of Nigeria, Nsukka, Nsukka, Nigeria
Nigeria,
Received March 11, 1987, and in revised form June 8, 1987
Aflatoxins are metabolites of toxigenic fungi of strains of Aspergillus flavus and A. parasiticus. These fungi are common contaminants of agricultural products especially in the tropics where warm temperatures and high humidity are prevalent (1). Bassir and Babunmi (2) showed that aflatoxin B, (AFB,) possesses some anticoagulant activities probably due to its chemical relationship with the coumarins. Aflatoxicosis has also been associated with gastrointestinal hemorrhages (3,4). The anticoagulant effect of aflatoxin is due to the suppressive effect it has on the synthesis of prothrombin and other clotting factors (V, VII, and X) in the liver and not as a result of hepatocellular damage usually observed in aflatoxicosis (2).
Compounds like alfatoxin which contain the coumarin ring are known to bind to serum/plasma albumin and this effect affects their pharmacokinetic properties (5). Interaction of aflatoxin with other serum/plasma protein-binding drugs may lead to displacement of the former from its binding site. Such a situation will obviously modify the pharmacokinetic profile of the toxin in terms of its concentration in the blood, metabolism, excretion, and ultimately the toxin-induced pathology. This theoretical consideration has led to the present study. Phenylbutazone and sulfamethoxine have been selected for this study because they are known protein-binding drugs which are commonly used for treating disease conditions in both human beings and animals. Vitamin K in addition to its protein binding property has been selected because of its antihemorrhagic effect which is antagonized by coumarin anticoagulants (6). Vitamin K is also claimed to offer temporary relief to dogs suffering from aflatoxicosis (7). MATERIALS
AND METHODS
Animals
Albino rats of both sexes (9-11 weeks old) and weighing between 100 and 150 g were used for this study. The rats were housed in stainless metal cages and 158 0885-4505188 $3.00 Copyright All rights
0 1988 by Academic Press. Inc. of reproduction in any form reserved
AFLATOXIN
B, DRUG
INTERACTION
159
were allowed free access to pelleted feed and water throughout the period of study. They were randomly divided into five groups consisting of 18 (male and female) rats each. Drug
Treatment
The first group of rats served as the untreated control group; the rats in this group did not receive any drug/AFB, treatment. The second group of rats served as the positive control group, treated with 25, pg/kg of AFB, (Sigma) for 5 days. The rats in groups 3, 4, and 5 were treated with vitamin K (Synkavit, HoffmanLa Roche, 5 mg/kg), phenylbutazone (Butazolidine, Geigy, 50 mg/kg), and sulfamethoxine (Fanasil, Roche, 50 mg/kg), respectively, for 3 days immediately after 5 days of therapy with AFB, (25 pg/kg). All treatments were by the intraperitoneal route and the total volume of fluid injected into each rat per day did not exceed 1 ml. All drug solutions except AFB, were made in distilled water. AFB, was first dissolved in a minimal amount of dimethyl sulfoxide (DMSO) and then diluted with distilled water to make a 6.25 pg/ml concentration. Analytical
and Clinicopathological
Procedures
Three rats from each group were randomly selected on 1, 4, 7, 14, 21, 28, and 35 days post-treatment. In addition to clinical observations the following parameters were determined for each rat: the live body weight, whole blood clotting time, and relative wet weights of the liver, kidney, and spleen. Whole blood clotting time was determined by the methods of Riley (8) and Bush (9). A nonheparinized capillary tube was inserted into the medial eye canthus and gently rotated thereby opening some blood capillaries in the region. Immediately after the capillary tube was filled with blood a stopwatch was started. The tube was then broken from one end in bits at intervals of 5 set until a strand of coagulated (clotted) blood was observed linking the ends of the broken capillary tube. At that point the stopwatch was stopped and the time was recorded. Three readings were obtained for each rat and the means + standard deviation were calculated. Relative wet weights of the liver, kidneys, and spleen were determined after sacrificing the rats by decapitation. The organs were removed, lightly blotted, and weighed on an analytical balance. The relative wet weights of these organs were calculated using relative wet weight =
weight of organ Xi!!! weight of animal 1 ’
All the visceral organs were examined in situ for gross pathological changes, if any. Representative samples of the organs were preserved in 10% formal-saline solution for histopathology. The organ samples were embedded in paraffin wax and sections were cut at 5 pm and stained with hematoxylin-eosin (H-E). Plasma
Protein
Equilibrium total protein
Binding
dialysis experiments were performed using rat plasma (4 mg/ml content). The heparinized plasma from rat blood was diluted with
160
ASUZU,
SHE’M’Y,
AND
OBIDOA
0.067 M sodium phosphate buffer (pH 7.4) to obtain the required protein concentration. The dialysis tubing (Visking, size 24A, mol. wt cutoff between 12,000 and 14,000) used for the experiment was cut to suitable lengths and washed several times in deionized water and then soaked in 0.067 M sodium phosphate buffer (pH 7.4) maintained at 4°C for 2 days. One end of the tube was securely tied and the sac was filled with 3 ml plasma plus 1 ml AFB, (3 pg) plus 1 ml of drug (250 pg vitamin K, 2500 pg phenylbutazone, or 2500 pg sulfamethoxine as the case may be). The dialysis tubings with their contents were simultaneously dialyzed at 37°C for 6 hr. The absorbance of the dialysate was determined at 363 nm on a uv spectrophotometer (Sp-500, Eye Unican) using a standard reference alfatoxin solution to quantitate the dialyzed amount of free aflatoxin. Values thus obtained were used to .determine the amount of alfatoxin displaced by each drug. Determination
of Possible AFB,-Drug
Complexing
AFB, (10 pg/ml), vitamin K (250 ,ug/ml), phenylbutazone (2500 pg/ml), and sulfamethoxine (2500 pug/ml) were spotted on a 10 x IO-cm-thin layer chromatoplate (TLC) which was developed in a methanol:choloroform (2:98) solvent system. Each of the drugs was also mixed with AFB,, and the mixture was spotted on TLC alongside the respective drugs. The migration of AFB, and the drugs on the TLC plate was viewed under short and long wavelength uv light. The R, values were calculated for comparison. RESULTS Clinical
Findings
The significant findings were profuse bleeding AFB,-treated rats 1 day following AFB, treatment and vulva in some rats on Day 3 post-treatment. Gross Necropsy
from minor injuries and the enlargement
in most of testis
Findings
The liver of rats in groups 2, 4, and 5 were congested and friable. Echymosis and petechial hemorrhage were also observed in the subcutaneous tissues of these rats. The kidneys and spleen of these animals appeared normal. Control rats (group 1) and rats of group 3 (AFB, plus vitamin K) had apparently normal liver, kidneys, and spleen. These observations were made on Days 1 and 7 posttreatment. By Day 14 post-treatment, rats of groups 2, 3, 4, and 5 showed a swollen, congested, and friable liver, whereas the kidneys and spleen remained normal. Organs of control rats did not show these changes. On Days 21 and 28 post-treatment, lesions observed on Day 14 post-treatment persisted in all treated rats except in those of group 5 in which the lesions were of milder degree. By Day 35 there was obvious recovery in all the treated rats other than those in group 4, in which the repair process was slow. Whole Blood Clotting Time The blood clotting time of group 2 rats was significantly (P < 0.05) higher than that of the control and peak prolongation of clotting time occurred on Day
AFLATOXIN
B , DRUG INTERACTION
Post-treatment
161
Day
FIG. 1. The effect of AFB,, AFB,, + Vitamin K, AFB, + phenylbutazone, famethoxine on the mean clotting time in rats.
and AFB, + sul-
4 post-treatment (Fig. 1). The clotting time in rats of group 3 remained normal throughout the experiment. There were significant (P < 0.05) differences between group 4 rats and those of group 1 and group 2 on Days 1 and 4 post-treatment, with the clotting time of group 4 rats being higher than that of group 1 and lower than those of group 2 rats. On Days 7, 14, and 21 post-treatment, clotting time of group 4 rats was significantly higher than that of group 1 rats. The clotting time of group 5 rats was significantly lower than group 2 rats but was not different from readings obtained from group 1 rats on Day 1 post-treatment. However, by Day 4 the clotting time of group 5 rats was significantly higher than that of group 1 and lower than that of group 2 rats. On Days 7, 14, and 21 post-treatment there was no significant difference between the clotting times of rats in group 5 and group 2. There was no significant difference in clotting time values between all the groups on Days 28 and 35 post-treatment. Relative
Weights of Visceral Organs
There was no significant difference between the relative liver weight of AFB,treated rats and that of control rats except on Days 21 and 28 (Table 1). Similar results were seen in rats treated with AFB, plus vitamin K and AFBr plus sulfamethoxine. However, AFBl plus phenylbutazone-treated rats showed significantly (P < 0.05) higher liver weights compared to AFB,-treated and control groups on the 28th day post-treatment (Table 1). There was no significant difference in relative kidney weights of group 2 and group 1 rats (Table 1). However, the kidneys of group 4 rats were significantly (P < 0.05) smaller than those of group 1 rats on Days 14 and 35 post-treatment. The relative kidney weights of group
Liver Kidney Spleen
Liver Kidney Spleen
Liver Kidney Spleen
Liver Kidney Spleen
14
21
28
35
3.11 + 0.01 1.00 + 0.20 0.51 f 0.07
3.56 f 0.01 1.02 + 0.01 0.41 + 0.01
3.63 + 0.17 1.07 2 0.20 0.40 + 0.04
3.82 + 0.18 1.25 + 0.16 0.69 + 0.14
4.00 + 0.14 0.99 + 0.04 0.51 + 0.04
Group 1 untreated controls
2.96 + 0.11 0.94 f 0.08 0.47 f 0.07
3.93 f 0.12* 0.95 f 0.09 0.41 + 0.07
4.39 + 0.18* 1.02 + 0.07 0.51 AZ 0.15
3.72 IL 0.29 1.07 -c 0.02 0.48 f 0.14
4.12 f 0.14 1.18 k 0.25 0.62 2 0.35
Group 2 AFB,
* Significantly (P < 0.05) different from untreated rats. ** Significantly (P < 0.05) different from AFB, treated rats.
Liver Kidney Spleen
Organ
7
Posttreatment day
3.05 + 0.08 0.94 + 0.12 0.31 k 0.05*.**
3.88 2 0.31 1.03 f 0.19 0.47 + 0.08
4.12 2 0.13* 1.01 + 0.10 0.31 r 0.03*
3.24 + 0.77 1.07 + 0.09 0.38 f 0.10*
3.66 k 0.24* 0.80 f 0.09 0.32 + 0.05*
Group 3 AFB, + vitamin K
3.05 c 0.22 0.81 2 0.03* 0.32 + 0.03*.**
4.20? 0.11 1.04 k 0.02 0.38 f 0.05
4.21 f 0.50 1.04 + 0.13 0.41 2 0.06
3.51 + 0.19 0 87 t 0 03*,** 0:31 + 0:10*
3.61 k 0.18* 1.16 f 0.18 0.59 f 0.22
Group 4 AFB, + phenylbutazone
f k + rt f k + + +
0.45 0.07 0.08 0.28* 0.13 O.lO* 0.61 O.lO* 0.09 2.91 2 0.19 0.85 f 0.09* 0.39 2 0.04
4.13 1.13 0.51 4.43 1.05 0.61 3.91 0.85 0.39
3.99 + 0.37 1.01 2 0.20 0.70 f 0.22
Group 5 AFB, + sulfamethoxine
TABLE 1 Relative Weights (Mean + SD, N = 3) of the Organs of Rats Treated with AFB, and AFB, followed by Other Drugs
g 9
zi E g
5
!
8
%i s“C
AFLATOXIN
B, DRUG
INTERACTION
163
4 rats were significantly lower than those of group 2. Likewise, a significant difference existed between kidney weights of group 5 and group 1 rats on Days 28 and 35 post-treatment (Table 1). There was no significant difference between the relative splenic weights of group 2 and group 1 rats. Group 3 rats showed a significant decrease in relative splenic weight when compared to group 1 on the 7th, 14th, 21st, and 35th day post-treatment and compared to group 2 rats on the 35th day post-treatment. Group 5 rats had significantly (P < 0.05) heavier spleens than group 1 rats on Day 21 post-treatment. Histopathological
Change
The liver, kidneys, and spleen of group 1 rats were normal. In group 2, the liver showed bile duct proliferation and periportal cell necrosis with heavy fatty infiltration in the centrilobular areas as well as bile duct hyperplasia on Days 7 to 28 post-treatment. The kidneys of group 2 rats showed mild nephrosis involving mainly the proximal convoluted tubules. The lesions in the kidneys were observed to be undergoing regeneration by the 14th day post-treatment. There was blood pooling in the sinuses with a large number of immature lymphocytes present in the spleen of group 2 rats on Days 7 and 14 post-treatment. By Day 28 following treatment, the blood pooling had disappeared but the immature lymphocytes were still present. Lesions in the liver and kidneys of group 3 rats were similar to those found in group 2 rats. The liver and kidneys of group 4 rats showed lesions similar to those observed in group 2 rats. Blood pooling in the spleen was observed on Day 7 post-treatment but disappeared by Day 14. Immature lymphocytes were still present by Day 28 post-treatment. Among group 5 rats the liver showed lesions similar to those of group 2 rats up to the 28th day posttreatment. Lesions in the kidneys of group 5 rats were similar to those observed in group 2. The renal lesions however started undergoing regeneration by Day 7 post-treatment (Fig. 2), while the spleen did not show any significant histopathological change throughout the experiment (Fig. 3). Plasma
Protein
Binding
Phenylbutazone and sulfamethoxine significantly (P < 0.05) displaced AFB, from its binding sites in plasma (Table 2). The displacement of AFB, by vitamin K was not significant. Determination
of Possible
AFB, Drug
Complexing
The Rf value of AFB, (0.52) did not change even when it was co-spotted with the other drugs. The Rf value of sulfamethoxine (0.45) also did not change even when the drug was co-spotted with AFB,. DISCUSSION
Profuse bleeding from minor injuries was observed in rats 1 day after intraperitoneal treatment with 25 pg/kg of AFB,. This confirmed that the dose of AFB, selected for the study was sufficient to cause aflatoxin-induced coagulopathy in rats. By the 5th day of treatment with AFB,, a steady state of the toxin had been established as indicated by the mean clotting time of 44.33 min on the 4th
164
ASUZU,
SHE-l-l-Y, AND OBIDOA
FIG. 2. Kidney of rat treated with AFB, + sulfamethoxine regeneration.
showing tubular cells undergoing
FIG. 3. Spleen of rat treated with AFB, + sulfamethoxine.
AFLATOXIN
TABLE 2 Displacement of AFB, from Plasma by Vitamin K, Phenylbutazone,
Dw AFB, AFB, + Vitamin K AFB, + Phenylbutazone AFB, Sulfamethoxine
Amount of AFB, in dialysate (PM) 8.116 8.188 8.551 9.130
165
B, DRUG INTERACTION
2 0.126 If: 0.125 2 0.126 2 0.376
and Sulfamethoxine
Amount of AFB, displaced (%)
n
P value”
0.9 5.4 12.5
3 3 3 3
P > 0.05 P < 0.05 P < 0.05
-
y Compared to AFB, alone.
and 5th days of AFB, treatment. Gross pathological change due to AFB, toxicity had been observed by Day 1 post-treatment (i.e., after 5 days of AFB, treatment followed by 3 days of drug treatment). On Day 1 post-treatment, AFB,-treated rats had significantly (P < 0.05) higher clotting time than the control rats and the rats treated with vitamin K, phenylbutazone, or sulfamethoxine. Vitamin K and sulfamethoxine effectively counteracted the initial rise in clotting time caused by AFB, because there was no significant difference between the clotting time in these groups and that of the control rats (Fig. 1). Though phenylbutazone treatment was able to reduce the clotting time compared to that of AFB,-treated rats, the clotting time recorded in the former group was still significantly higher than that of the control rats. Peak increase in clotting time was established on Day 4 post-treatment in rats treated with AFB, alone, AFB, plus phenylbutazone, and AFB, plus sulfamethoxine. Though phenylbutazone and sulfamethoxine significantly (P < 0.05) reduced the clotting time compared with the AFB,-treated group, they were not able to offer complete protection since their clotting-time values were still significantly higher than values of untreated controls. Among the agents used, vitamin K was the only drug which continued to offer complete protection against prolonged clotting time caused by AFB, by the 4th day post-treatment. Vitamin K maintained this protective capability for the duration of the experiment. By Days 7 and 14 the effect of AFBi on clotting time had declined, although the clotting time was still significantly higher than those of control values. There was at this time no significant difference between the AFB,-treated group and the groups treated with phenylbutazone and sulfamethoxine. From the 21st day of the study, clottingtime values in four out of five treatment groups had returned to normal with the clotting time of rats treated with phenylbutazone showing significantly higher values than those of the controls. It appears that the increase in clotting time persists more in phenylbutazone-treated rats than in all other groups. It is not possible to explain the exact mechanism of action of the drugs used in this study in reducing AFB ,-induced coagulopathy. However, the equilibrium dialysis experiment indicated that phenylbutazone and sulfamethoxine signilicantly (P -=c 0.05) displaced AFB, from rat plasma in vitro (Table 2). Therefore the displacement of AFB, from the binding sites in plasma could be one of the
166
ASUZU,
SHETTY,
AND
OBIDOA
mechanisms involved in the observed effect of phenylbutazone and sulfamethoxine on clotting time. The involvement of other mechanisms is likely because displacement of AFB, from its plasma binding sites alone should have intensified the anticoagulant effect (6). This is, however, contrary to what has been observed in this study. Displacement of drugs from bound to free form also can result in increased rate of metabolism and excretion (10). It is known that the alteration in the rate of one parameter, such as metabolism, could alter the biologic activity of AFBi (11). Phenylbutazone is a known inducer of liver microsomal enzymes and this property is likely to enhance the metabolic rate of free AFB,. Though vitamin K is known to bind extensively to plasma proteins (12), the equilibrium dialysis experiment with AFB, plus vitamin K did not show any significant displacement of AFBi by vitamin K. However, vitamin K is necessary for the synthesis in the liver of not only prothrombin (coagulation factor II) itself but also of proconvertin (VII), plasma thromboplastin component (IX), and StuartPrower (X) factors (9). AFB,, on the contrary, suppresses the synthesis of prothrombin, factors V, VII, and X of the clotting mechanism in the liver (2). This may well explain why vitamin K was able to completely suppress the increase in clotting time caused by AFB,. Direct complexing of vitamin K, phenylbutazone, or sulfamethoxine with AFB, does not seem to contribute to the results of this study as shown by the TLC of the mixture of AFB, with each of the other drugs. Results of this study suggest that though vitamin K was able to completely suppress the effect of AFB, on clotting time, it had no protective effect on the organs especially the liver and kidney. It did, however, have protection over the spleen. Sulfamethoxine also had protection over the spleen and the kidneys where the cells were undergoing regenerative changes by Day 7 post-treatment, 1 week earlier than was observed in the AFB,-treated group. The effect of the different treatments (drugs plus AFBJ on organ weight was not consistent and was therefore considered insignificant in this study. In conclusion, the increased blood clotting time in rats caused by AFB, was suppressed to varying degrees by vitamin K, phenylbutazone, and sulfamethoxine with vitamin K offering complete suppression throughout the period of experimentation. However, this suppressive effect appears to be limited to blood clotting time alone since no significant protection was offered to the liver and kidney of the affected rats. Vitamin K in particular may therefore offer temporary relief in acute alIatoxicosis but cannot be used as a sole cure for animals suffering from the condition. Sulfamethoxine was not only capable of reducing the severity of allatoxin-induced coagulopathy but may also provide some beneficial protective effects against tissue damage caused by AFB,. SUMMARY
The interactions of aflatoxin B, (AFB,) with vitamin K, phenylbutazone, and sulfamethoxine were investigated in albino rats. Vitamin K (5 mg/kg) was able to completely suppress the increase in whole blood clotting time caused by AFB, (25 pg/kg). Phenylbutazone (50 mg/kg) and sulfamethoxine (50 mg/kg) also significantly (P < 0.05) lowered the increased clotting time caused by AFB,.
AFLATOXIN
B, DRUG INTERACTION
167
Equilibrium dialysis was performed on rat plasma (4 mg/ml protein content) to investigate the displacement of AFB, (3 pg) from its bound form by vitamin K (250 pug), phenylbutazone (2500 pg), and sulfamethoxine (2500 pg). Phenylbutazone and sulfamethoxine significantly (P < 0.05) displaced AFB, from rat plasma protein. Histopathological examinations performed on the liver, kidneys, and spleen of control and treated rats showed that none of the drugs used appeared to offer any significant organ protection against AFB, except in the spleen. ACKNOWLEDGMENTS The authors are grateful to Dr. E. Onyekweodiri for interpreting the histopathological slides and Mr. P. A. Nnadi for his contribution to this work. This work was partially supported by the University of Nigeria Senate Research Grant 00422/81.
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