Protective effects of sodium bicarbonate on murine ochratoxicosis

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  • This article was downloaded by: [University of Auckland Library]On: 24 November 2014, At: 13:45Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number:1072954 Registered office: Mortimer House, 37-41 Mortimer Street,London W1T 3JH, UK

    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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    http://www.tandfonline.com/page/terms-and-conditionshttp://www.tandfonline.com/page/terms-and-conditions

  • 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 + 2C, 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 - 78.0

    - Ochratoxin A +biscoumacetate

    6.5 - 78.0

    .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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