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Journal of EnvironmentalScience and Health,Part B: Pesticides, FoodContaminants, andAgricultural WastesPublication details, including instructionsfor authors and subscription information:http://www.tandfonline.com/loi/lesb20
Protective effects ofsodium bicarbonate onmurine ochratoxicosisS. Yong a , M. Albassam a & M. Prior aa Animal Sciences Wing , AlbertaEnvironmental Centre , Bag 4000,Vegreville, Alberta, T0B 4L0, CanadaPublished online: 21 Nov 2008.
To cite this article: S. Yong , M. Albassam & M. Prior (1987) Protectiveeffects of sodium bicarbonate on murine ochratoxicosis, Journal ofEnvironmental Science and Health, Part B: Pesticides, Food Contaminants,and Agricultural Wastes, 22:4, 455-470
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J. ENVIRON. SCI. HEALTH, B22(4), 455-470 (1987)
PROTECTIVE EFFECTS OF SODIUM BICARBONATE
ON MURINE OCHRATOXICOSIS
Keywords: urinary modifiers, rat, ochratoxicosis,
heart lesions
S. YONG*, M. ALBASSAM AND M. PRIOR
Animal Sciences Wing, Alberta Environmental Centre,Bag 4000, Vegreville, Alberta, Canada. T0B 4L0
ABSTRACT
The protective effect of sodium bicarbonate
(NaHCO3), a urine modifier, to alleviate murine
ochratoxicosis was investigated. The study included
two trials. Urinary pH was altered before oral
administration of ochratoxin A (OA) in Trial 1, and
*to whom correspondence should be addressed
455
Copyright © 1987 by Marcel Dekker, Inc.
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456 YONG, ALBASSAM, AND PRIOR
animals were given combined doses of OA and ethyl
biscoumacetate (Eb) in Trial 2. Acute toxicity of
OA as measured by LD50 values was reduced by 23%
and 20% in rats treated with NaHCO3 for Trials 1
and 2 respectively. Bicarbonate-treated rats dosed
with 20 mg/kg OA or with a combination dose of OA at
17 mg/kg and Eb at 50 mg/kg, had a lower frequency
of histological lesions in kidneys, liver, lung,
spleen and heart. Two types of heart lesions found
in the present study are described.
INTRODUCTION
Ochratoxin A (OA), a mycotoxin produced by fungi
of .genera Aspergiiius and Penicillium. has been
reported to induce renal lesions in poultry, pigs,
and man following natural exposure to contaminated
cereal and legume crops (Buckly, 1971; Peckham,
1978; Austwick, 1975). Similar lesions have been
reproduced experimentally in laboratory rats (Munro
et §1., 1974; Chang and Chu, 1977). The biological
effects of ochratoxins in other species were
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PROTECTIVE EFFECTS OF SODIUM BICARBONATE 457
reviewed (Steyn 1984; Harwig et al_., 1983). OA and
its metabolites are primarily excreted in the urine
(Chang and Chu, 1977). Because OA is a weak organic
acid (pKa 7.1), the transfer of OA between blood and
urine will occur mostly in the pH range of 5 and 9.
Calculations using the Henderson-Hasselbach equation
(Baggot, 1977) indicate that the theoretical
equilibrium concentration ratio of unbound
ochratoxin A in the urine, relative to 100 in blood
would be 35 at pH 5.8, 50 at pH 6.8 and 453 at pH
8.2. Ameliorative measures aimed at increasing the
excretion of OA in the urine would be more
successful if the urine were made more basic.
Sodium bicarbonate (NaHCO ) has long been
established as a urinary alkalinizer in animals
(Brander and Pugh, 1971). Ammonium chloride
(NH Cl) has been used in promoting transient
diuresis after urinary acidification (Brander and
Pugh, 1971), and was used as part of the controls in
the present study. Alteration of urinary pH by
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458 YONG, ALBASSAM, AND PRIOR
these urinary modifiers might hasten OA excretion
and therefore, reduce the acute toxicity.
Ethyl biscoumacetate (Eb), an acidic drug, has
been shown to competitively inhibit the binding of
OA to plasma albumin in. vitro and synergistically
increase OA toxicity (Galtier et aj_., 1980). It was
administered with OA to test the effectiveness of
the two urinary modifiers.
MATERIALS AND METHODS
Mature, male Long Evans rats (average body
weight 222.9 ± 5.95 g) were housed individually in
stainless steel mesh-bottomed cages. The animal
rooms were maintained at 22 + 2°C, relative humidity
50 + 20%, and a photoperiod of 12h light and 12h
dark. Five days prior to commencement of each
trial, rats were randomly allocated to three
experimental groups given either reverse osmosis
water (Group A), 1.42% NH Cl solution (Group B),
or 1.42% NaHCO solution (Group C) as the
drinking fluid. Water and a certified rodent chow
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PROTECTIVE EFFECTS OF SODIUM BICARBONATE 459
were provided ad. libitum. Food was withheld
overnight before oral dosing with OA. OA (Sigma)
was dissolved in 0.1M NaHCO for administration.3
For Trial 1, 40 rats were assigned to each of
Groups A, B and C. Ten rats from each Group
received either 0, 10, 20 or 40 mg/kg Body Weight of
OA. For Trial 2, 25 rats were assigned to each of
Groups A, B and C. Five rats from each group
received either 0, 12, 17, 22 or 28 mg/kg Body
Weight of OA combined with 50 mg/kg of Eb.
Animals were observed for 14 days after dosing.
Complete necropsies were performed on survivors and
animals found dead during the course of the study.
Tissues were fixed in buffered formalin and stained
with hematoxylin and eosin. The L0 values5O
were calculated by the method of Litchfield and
Wilcoxon (Litchfield and Wilcoxon, 1949). The
number of histological lesions were tested by
analysis of variance (Raktoe and Hubert, 1979).
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460 YONG, ALBASSAM, AND PRIOR
RESULTS AND DISCUSSION
In a preliminary experiment, both NaHCO and
NH Cl were effective in maintaining urine in the
alkaline (pH > 8) or acidic (pH < 6.5) range for at
least a week without any adverse effect on animals.
In Trial 1, alkalinization of urine decreased the
toxicity of OA by 23% as the LD value of Groupso
C increased to 25.25 mg/kg (95% confidence interval
18.1 - 35.0) from 20.5 mg/kg (12.6 - 33.3) of Group
A. Acidification of urine, as expected, had no
beneficial effect (Table 1). Histologically, the
kidneys, liver, lungs, spleen and heart were the
most affected organs. The changes were mainly seen
in animals that died within the first 48 hours after
dosing, and no significant pathological alterations
were observed in the survivors. NaHCO -treated3
rats (Group C) had significantly (p < 0.01) fewer
lesions in all affected organs than NH Cl-
treated and control rats (Table 2). The number of
lesions in the cortical tubules of NaHCO -3
treated rats was markedly reduced (Table 2).
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PROTECTIVE EFFECTS OF SODIUM BICARBONATE 461
TABLE 1Effect of Alteration of Urinary pH on L0so ofRats Treated with Ochratoxin A Alone or Ochratoxin Aand 50 mg/kg of Ethyl Biscoumacetate.
5OUrinary pH Range Calculated 95%
TREATMENT (overnight samples) (confidence interval)
Trial 1 - Ochratoxin A aloneGroupGroupGroup
Trial
GroupGroupGroup
ABC
2
ABC
6.8 - 7<6.5>8.0
- Ochratoxin A +biscoumacetate
6.5 - 7<6.5>8.0
.5
50
.8
20.520.525.2
mg/kg of
19.323.223.2
mg/kgmg/kgmg/kg
Ethyl
mg/kgmg/kgmg/kg
(12.6-33.3)(12.6-33.3)(18.1-35.0)
(17.0-21.8)(19.9-27.0)(19.9-27.0)
Group A: control, RO water (Reverse osmosis water)Group B: 1.42% NH4C1 SolutionGroup C: 1.42% NaHCO3 Solution
In Trial 2, when Eb was administered
concurrently with OA, both acidification and
alkalinization of urine lowered toxicity by 20%.
The LO values of Groups B and C were increased
to 23.2 mg/kg (19.9 - 27.0) from 19.3 mg/kg (17.03 -
21.87) of Group A (Table 1). A reduction in
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462 YONG, ALBASSAM, AND PRIOR
TABLE 2Effects of Urine Modifiers (1.42% NH4C1 and1.42% NaHCO3 Solutions) on the Number of Lesionsin Rats Dosed with 20 mg/kg Ochratoxin A - Trial 1.
Incidence of Lesions'*
AcuteTREATMENT Deatha Kidneys Liver Lungs Spleen Heart
Group A: Control, RO Water7/10 8/10 7/10 9/10 6/10 10/10
Group B: 1.42% NH4C1 Solution7/10 8/10 6/10 10/10 5/10 6/10
Group C: 1.42% NaCH04 Solution3/10 1/10 2/10 6/10 3/10 3/10
a Fractions indicate No. of Animals died/No, ofAnimals in the group.
D Fraction indicate No. of Animals/No, of animalstested.
mortality and decreased frequency of lesions in
affected organs was especially noticeable in rats
receiving the combined dosage of 17 mg/kg OA and 50
mg/kg Eb in either Group B or C (Table 3). However,
this favorable response was not as evident in rats
receiving higher doses of OA. The reason why
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PROTECTIVE EFFECTS OF SODIUM BICARBONATE 463
TABLE 3Effects of Urine Modifiers (1.42% NH4C1 and1.42% NaHCO4 Solutions) on the Severity andNumber of Lesions in Rats given a Combined Oral Doseof Ochratoxin A (17, 22 or 28 mg/kg and 50 mg/kg ofEthyl Biscoumacetate - Trial 2.
OA DOSEGROUP
ANDAcutea
TREATMENT Death
17 mq/kqGroup AGroup BGroup C
22 mq/kqGroup AGroup BGroup C
28 mq/kqGroup AGroup BGroup C
Group A:Group B:Group C:
4/51/51/5
4/53/53/5
5/54/54/5
Control,1.42% NH4
Incidence of
Kidneys I
3/51/51/5
3/53/53/5
3/5*4/54/5
RO water
.iver
4/51/51/5
4/53/53/5
5/54/54/5
Lungs
4/51/51/5
4/53/52/5
5/54/53/5
Lesionsb
Spleen
4/51/51/5
4/53/53/5
5/54/54/5
(Reverse Osmosis ICl Solution
1.42% NaHCO, Soituiona Fractions indicate No
animals in the groupb Fractions indicate No.
of animals tested* Observation on
. of
of
three rats
Heart
4/51/51/5
4/51/53/5
4/54/52/5
rfater)
animals died/No, of
animals affected/No.
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464 YONG, ALBASSAM, AND PRIOR
NH Cl treated rats in this trial fared better4
than the controls was not apparent.
Histopathological changes in ochratoxicosis in
rats have been studied extensively (Kamisawa et aJL,
1977). The lesion found in heart muscle in both
trials of the present study is of particular
interest since it was described only once (Purchase
and Theron, 1968) in an acute study, but no details
were given. Two types of heart lesions were seen in
our study. The first was characterized by mild
individual fibre necrosis, hyalinization and
interstitial edema. The second type was localized
in the papillary muscle and characterized by severe
hemorrhage, necrosis of muscle fibres and leukocytic
infiltration. Many of the leukocytes were at
different stages of nuclear pyknosis, karyorrhexis
and karyolysis (Figures 1 and 2). Further studies
are needed to evaluate these lesions and investigate
their pathogenesis. Other severe histopathological
lesions seen included large necrotic areas
(100-150u) of hepatocytes in the liver; diffuse
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PROTECTIVE EFFECTS OF SODIUM BICARBONATE 465
Figure 1. Photomicrograph of a heart showing markedhemorrhage (arrow) in the papillary muscle of a ratgiven a single oral dose of 20 mg/kg Ochratoxin A(x23).
congestion or hemorrhage with edema fluid in the
alveolar spaces and distended alveolar walls with
necrotic leukocytes in the lung; lymphoid necrosis,
and extensive cellular necrosis with hemorrhage and
fibrin deposition in the red pulp of the spleen; as '
well as extensive swelling of epithelial cells,
together with large numbers of eosinophilic hyaline
droplets in the proximal convoluted tubules in the
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466 YONG, ALBASSAM, AND -PRIOR
Figure 2. High magnification of the papillarymuscle showing necrosis and disruption of themyocardial fiber (arrows) with hemorrhage (H) andmoderate . leukocytic infiltration (I). Manyleukocytes are at different stages of nuclearpyknosis, karyorrhexis and karyolysis (x 462).
kidney. Detailed histopathologic and electron
microscopic studies on the acute toxicity of
Ochratoxin A in rats from this laboratory are
described elsewhere (Albassam et aj..).
Like many coumarin analogues, OA is highly
bound to plasma albumin (Galtier, 1974). Galtier et
aj.. (1980) reported that Eb competes with OA for
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PROTECTIVE EFFECTS OF SODIUM BICARBONATE 467
binding sites on plasma proteins, due to the
similarity of the molecular structure.
Administration of Eb with OA increases free
(non-bound) OA in blood. Increased availability of
OA, together with increased urinary pH, would
increase excretion of free OA and possibly reduce
its toxicity. However, it is possible that the
increased free OA in blood might have caused severe
cellular damage and triggered disseminated
intravascular coagulation (Galtier et §J.., 1979) and
induced a secondary heart lesion.
Like other weak acids, OA is present in solution
as both non-ionized and ionized fractions (Baggot,
1977), and is readily absorbed from the intestine
(Kumagai and Aibara, 1982). Bicarbonate already
abundantly present in the intestinal lumen may have
neutralized the H+ ion from OA, and hydrolysed the
rest of the molecule to a less toxic analogue
(ochratoxin a, Ueno, 1985). Furthermore,
increasing urinary pH will increase ionization or
dissociation of OA. This may be a significant
factor in reducing the severity of the lesions found
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4 6 8 YONG, ALBASSAM, / ^Tr>R
in the kidneys, because highly ionized compounds are
usually excreted rapidly, thus decreasing
reabsorption from tubular fluid and total body
burden.
As a urinary modifier, NaHCO treatment was
effective in both trials in reducing mortality, and
frequency of lesions. It can be conveniently
introduced into the drinking water as a protective
agent against acute ochratoxicosis. Its usefulness
in chronic situations remains to be examined.
ACKNOWLEDGEMENTS
We thank Drs. A. Lopez, R. Coppock and L. Li 11ie
for their advice and criticism and Dr. A. Sharma for
the statistical analysis and Mrs. G. Flato for
typing the manuscript.
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