Download - Protective effects of sodium bicarbonate on murine ochratoxicosis

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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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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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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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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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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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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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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



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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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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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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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.

REFERENCES

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AUSTWICK, P.K.C. Pro. Roy. Soc. Med., 68 (1975)219-221.

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HARWIG, J., KUIPER-GOODMAN, T., AND SCOTT, P.M."Microbial food toxicants: ochratoxins in Handbookof foodborne disease of biological origin", ed. M.Rechcigl, Jr., CRC Press, Boca Raton, Florida(1983), 193:238.

KAMISAWA, H., SUZUKI, S., KOZUKA, Y. AND YAMAZAKA,M. Toxicol. Appl. Pharmacol., 42 (1977) 55-64.

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PECKHAH, J.C. "Ochratoxicosis in poultry, inMycotoxic fungi, mycotoxins, mycotoxicosis", Vol.2. Eds. T.D. Wylie and L.G. Morehouse, MarcelDekker Inc., New York, 1978.

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STEYN, P.S. "Ochratoxins and relateddihydroisocoumarins in Mycotoxins". Production,Isolation, Separation and Purification, ed. V.Betina, Elsevier, Amsterdam (1984), 183-216.

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Received: December 8, 1986

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Download - Protective effects of sodium bicarbonate on murine ochratoxicosis

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