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Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Effects of the aflatoxins on ATPase activities in mouse and rat liver a
a
a
D. Desaiah , T. D. Phillips , A. W. Hayes & I. K. Ho
a
a
Department of Pharmacology and Toxicology , University of Mississippi Medical Center , Jackson, Mississippi, 39216 Published online: 21 Nov 2008.
To cite this article: D. Desaiah , T. D. Phillips , A. W. Hayes & I. K. Ho (1979) Effects of the aflatoxins on ATPase activities in mouse and rat liver, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 14:3, 265-278, DOI: 10.1080/03601237909372127 To link to this article: http://dx.doi.org/10.1080/03601237909372127
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and
Downloaded by [McGill University Library] at 11:28 10 December 2014
views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
J. ENVIRON. SCI. HEALTH, B14(3), 265-278 (1979)
EFFECTS OF THE AFLATOXINS ON ATPase ACTIVITIES
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IN MOUSE AND RAT LIVER Key Words: Aflatoxins, ATPase, Mouse, Rat, Liver D. Desaiah, T. D. Phillips, A. W. Hayes and I. K. Ho Department of Pharmacology and Toxicology University of Mississippi Medical Center Jackson, Mississippi 39216 ABSTRACT The effects of the aflatoxins on ATPase a c t i v i t i e s in mouse and r a t tissues were investigated in vitro. The hepatic oligomycin-sensitive (O.S.) Mg++ ATPase was inhibited significantly. The order of inhibition was G1 > B1 > G2 > B2. Mouse O.S. Mg++ ATPase was more sensitive than the corresponding r a t enzyme. The oligomycin-insensitive (O.I.) Mg++ ATPase a c t i v i t i e s in r a t and mouse liver were not altered.
Although aflatoxins G1 and
B1 were more potent inhibitors of hepatic O.S. Mg++ ATPase, no concentration-response was observed, whereas aflatoxins G2 and B2 inhibited enzyme activity in a concentration-dependent manner. Spectral analysis of aflatoxin G1 solutions suggested that solubility was not related to the observed effects.
In addi-
tion, the effects of aflatoxin B1 and G1 on mouse brain microsomal Na+-K+ ATPase were examined. Although aflatoxin B1 was 265 Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
266
DESAIAH ET AL.
more potent that G1, both mycotoxins significantly inhibited enzyme activity in a concentration-dependent fashion. INTRODUCTION Although aflatoxin contamination of foodstuffs has long been Downloaded by [McGill University Library] at 11:28 10 December 2014
considered a health hazard in societies where primitive methods of food storage prevail, such contamination i s generally not a human health problem in modern societies.
The aflatoxins are potent
hepatocarcinogens in experimental animals,^"^ and aflatoxin B\ may be an important human carcinogen.^ The l i t e r a t u r e pertaining to toxicity, metabolism and mode of action of the aflatoxins i s well documented.5-8 The liver i s the primary organ for aflatoxin accumulation.9
Consequently, the liver and i t s cellular organ-
ell es, including mitochondria, have been investigated in an effort to understand the mechanism(s) of toxicity.10-12 Mitochondrial ATPase, which i s involved in oxidative phosphorylation,!^ however, has received limited attention.
Aflatoxin Bj increased uncoupler
stimulated mitochondrial ATPase in mouse b r a i n ^ and l i v e r l ^ , but had no effect on renal and cardiac mitochondrial ATPase.^ In contrast, Pai et.£l_. 1 6 reported that aflatoxin B} increased mitochondrial ATPase activity but diminished the uncoupler stimulated ATPase activity in r a t liver. In view of existing discrepancy in the l i t e r a t u r e or a t least species variability regarding the sensitivity of mitochondrial ATPase to the aflatoxins, this study was undertaken t o investigate the in vitro effects of aflatoxins Bj, B2, Gj and G2 on mitochondrial ATPase in r a t and mouse l i v e r , employing two d i s t i n c t
EFFECTS OF THE AFLATOXINS
methods t o determine ATPase activity.
267
The i £ vitro response of
Na+-K+ ATPase, a biochemical manifestation of the Na+ pump, t o the aflatoxins also was examined using a mouse brain microsomal preparation.
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MATERIALS AND METHODS Animals: Male ICR mice weighing 25-30 g and male Sprague-Dawley rats weighing 250-275 g (Charles River Co., Wilmington, MA) were used. Animals were housed in a i r conditioned quarters with free access t o food and water. Chemicals: Purity of aflatoxins Bi, B2, Gi, G2 (Makor Chemical Jerusalem, Israel) was confirmed by melting point, infrared spectrum and thin layer chromatography. The structural formulae of these toxins are shown in Figure 1. All other chemicals were from Sigma Chemical Co. (St. Louis, MO). Tissue Fractionation:
Livers of both species were used for mito-
chondrial preparations but only mouse brain for microsomal preparation. Preparation of hepatic mitochondria: Mice and rats were decapitated and the l i v e r s quickly removed and placed in an ice-cold 0.32 M sucrose solution containing 1 mM EDTA and 10 mM imidazole, pH 7.5. Individual l i v e r s were homogenized separately in 9 vol of the sucrose solution using a ground glass homogenizer. The heavy mito-
chondria were obtained by centrifugation 1 fiand were resuspended in the sucrose solution, quick frozen in liquid nitrogen, and stored at -85° C until used for the ATPase assay.
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DESAIAH ET
AL.
KÁ, Figure 1. Structural formulas of aflatoxins.
Preparation of brain microsomes: Mice were decapitated, the brains quickly removed and homogenized in the,sucrose solution as described above. The microsomes were prepared by centrifuging the 13,000 x g supernatant at 105,000 x g for 1 hr and the pellet was resuspended in the sucrose solution, quick frozen in liquid nitrogen and stored at -85° C until used. ATPase assay: The enzyme activity in mitochondrial preparations was determined by using two methods routinely used in our laboratories. 19-20 Continuous (or enzymatic) method: ATPase activity was determined by continuously monitoring the oxidation of NADH at 340 nm on a Beckman Acta III Recording Spectrophotometer, equipped with a constant temperature (37° C) water bath.
EFFECTS OF THE AFLATOXINS
269
A 3-ml reaction medium contained 5 mM ATP, 5 mM Mg++, 135 mM imidazole-HCl buffer (pH 7.5), 0.2 mM NADH, 0.5 mM phosphoenolpyruvate, approximately 9 units of pyruvate kinase, and 12 units of l a c t i c acid dehydrogenase. 21
A 100 yl mitochondrial fraction was
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used with a protein content of 50 yg. Absorbance changes in the reaction mixture were measured over a 10 min period. Specific activity was expressed as micromoles of inorganic phosphate per milligram of protein per hour. Discontinuous (or inorganic) phosphate method: Mitochondrial ATPase and mouse brain microsomal Na+-K+ a c t i v i t i e s were measured using endpoint phosphate a n a l y s i s . 2 0 A 1-ml reaction mixture contained 5 mM ATP, 5 mM Mg++, 100 mM Na+, 20 mM K+, 135 mM imidazole-HCl buffer (pH 7.5) and 50 yg enzyme protein.
The total ATPase activity was measured with Na+,
K+ and Mg++ present in the reaction mixtures. The basal Mg++-ATPase component was measured by omitting both Na+ and K+. Thus, delineation of the Na+-K+ activated component of ATPase was obtained by difference between total ATPase (Na+ + K+ + Mg++) and basal Mg++ ATPase (Mg++ only) activity.
The Na+-K+ ATPase activity was
consistently ouabain sensitive. Treated and control preparations, reagent and enzyme blanks (to correct f o r inherent phosphate present in samples) were preincubated 'simultaneously a t 37° C for 3 min prior t o initiation of the reaction with ATP. Incubation was stopped after 10 min with the addition of TCA (trichloroacetic acid) a t a final concentration of 5% w/v in the reaction mixture. Samples then were assayed
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DESAIAH ET AL.
for inorganic phosphate using the method of Lowry and Lopez2^ as modified by Phillips et^al_. 2 2 In both procedures, the Mg++ATPase activity was delineated into O.S. and O.I. by adding 5 x 10~6 M oligomycin (basedrupon a
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combined molecular weight of 401.20 from 15% oligomycin A and 85% oligomycin B in ethanol) to the reaction mixture. The volume of ethano'i used was 1 ul in 3 ml of reaction mixture. Oligomycin i s a potent inhibitor of mitochondria! Mg++ ATPase activity involved in oxidative phosphorylation. 23 " 2 ^ Protein was determined by the method of Lowry ert al_. 2 ^ using bovine serum albumin as the standard. Aflatoxins Bi, B2> Gj and G2 were dissolved in DMSO. Different toxin concentrations were preincubated with the enzyme in the reaction mixture for 3 min prior to the addition of ATP. Enzyme activities were determined in the presence and absence of toxin and percent effects calculated. Since the differences between the two methods were not s t a t i s t i c a l l y significant (Table 1), the data were combined for the remaining experiments to calculate the percent inhibition. RESULTS The specific activities of mitochondrial Mg++ ATPase in mouse and r a t liver (Table 1) were comparable to those previously reported 2 1 » 2 6 , further, the variability between the two methods was s t a t i s t i c a l l y insignificant. Mitochondrial (O.S.) Mg"1"1" ATPase activity in the r a t liver was determined in the absence and in the presence of aflatoxins Bj,
EFFECTS OF THE AFLATOXINS
271
TABLE 1 Determination of Mg++ ATPase Activity in Rat and House Liver Mitochondria by two Assay Methods
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Specific Activity ± S.E.M.*
Method
Rat Oligomycin Sensitive Insensitive
Mouse Oligomycin Sensitive Insensitive
Coupled Enzymatic
26.8tl.79
4.9±0.17
36.3±0.23
8.0±0.30
Inorganic Phosphate
. 25.6±1.47
5.0±0.23
35.9±0.69
7.7+0.23
*Specific Activity = pmoles of Pi/mg protein/hr. S.E.M. - Standard error of the mean of four separate liver preparations and each preparation was assayed at least four times. No s t a t i s t i c a l l y significant difference was observed between two assay methods as determined by Student's t_ test (P < 0.05).
B2> G, and Gg (Table 2). The enzyme activity was sensitive to all four toxins; however, a concentration-response was not observed. The response was biphasic for aflatoxins B^ and G^ with maximum inhibition a t 6 x 10"7 M. Of the aflatoxins tested, aflatoxin Gj was the most potent aflatoxin inhibitor of rat liver mitochondrial Mg++ ATPase. Aflatoxins Bj, B2 and Gi also showed a biphasic effect on mouse liver mitochondrial Mg4"1" ATPase activity (Table 3). Maximum inhibition was observed at 6 x 10"^ M with decreased inhibition with increased toxin concentration.
As in the r a t , afla-
toxin Gj was the most potent inhibitor. Hepatic O.I. Mg++ ATPase
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DESAIAH ET AL.
TABLE 2 Percent Inhibition of Rat Liver Mitochondrial (O.S.) Mg++
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ATPase Activity by Aflatoxins
Toxin cone. (M) 6
X
10-9 8
Percent Inhibition ± S.E.M.* Bl
B2
G2
12.0+1.40
19.0±3.50
19.5±4.60
13.0±4.20
6
X
10-
16.0±2.80
19.0±l.00
32.5±1.60
25.5±0.35
6
X
10-7
24.0±0.70
21.010.71
32.5±1.06
25.0±2.30
6
X
10"6
19.5±0.35
19.0±0.71
27.5±?.5O
9.5±1.00
5
16.0±4.20
24.0±3.50
17.5+3.10
38.3±0.88
6
X
10"
lach value represents the mean of four different tissue preparations and each preparation was assayed in duplicate by two different assay methods and the average was used for calculating the S.E.M. The control specific activities in the presence of 5 yl of DMSO were 28.7+1.9; 25.0+0.24; 25.4+0; 25.6±0.18 ytnoles Pi/mg protein/hr for aflatoxins B\, B2J Gl and G2» respectively.
activity was not affected by any of the aflatoxins tested in either species (data not presented). The Na+-K ATPase activity in mouse brain microsomes was determined in the absence and presence of different concentrations of aflatoxins Bi and Gj and the data are presented in Table 4. Na+-K ATPase activity was inhibited in a concentration-dependent fashion by aflatoxins B\ and Gj with maximum inhibition of 66% and 42%, respectively.
EFFECTS OF THE AFLATOXINS
273
TABLE 3 Percent Inhibition of Mouse Liver Mitochondrial (O.S.) Mg++
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ATPase Activity by Aflatoxins Percent Inhibition ± S.E.M." B2
Toxin cone. (M)
Bl
6 X 10-9
19.8+2.2
22.6+10.0
38.0+1.1
10.0+2.8
îo- 8
36.0+0.0
27.5±3.1
45.4±1.7
17.5±3.8
6 X 10-7
29.5±8.1
19.0+2.1
40.0+1.4
21.5±6.0
6 X 10"6
18.0±2.4
18.5±3.8
30.5±1.7
21.5+1.4
6 X 10"5
11.0±7.7
26.5±1.1
24.0±6.3
34.4±3.8
6X
G2
Each value represents the mean of four different tissue preparations and each preparation was assayed in duplicate by 2 different assay methods and the average was used for calculating the S.E.M. The control specific activities in the presence of 5 yl DMSO were 36.7+0; 36.0+0 yg; 36.2+0.32 and 36.0+0.53 ymoles Pi/mg protein/hr.
No change in the molar absorptivity of high concentrations of aflatoxin Gj at 366 nm in either water, protein or buffer was observed (Fig. 2). DISCUSSION Our results that aflatoxins Bj, B2, Gj and G2 inhibited Mg++ dependent O.S. ATPase activity in r a t and mouse liver mitochondria are in contrast to earlier r e p o r t s . 1 4 " 1 6 For example aflatoxin Gi increased the basal Mg++ ATPase and decreased the DNPstimulated ATPase in rat l i v e r , 1 6 whereas aflatoxin Bi had no effect on either ATPase.16 Aflatoxin Bj also has been reported to
274
DESAIAH ET AL.
TABLE 4 Percent Inhibition of Mouse Brain Microsomal Na+-K ATPase Activity by Aflatoxins Percent Inhibition ± S.E.M.*
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Toxin cone. (M)
Bj
Gj
6 x 10-8
35.9 + 1.3
17.9 + 2 . 2
6 x 10" 7
24.5 ± 2.0
26.0 ± 1.1
6 x 10" 6
31.0 ± 1.0
28.0 ± 1.0
5
53.1 ± 0.9
35.4 ± 2.2
3 x 10" 4
66.1 ± 1.7
42.0 ± 1.5
6 x 10"
Each value represents the mean of 3 different tissue preparations and each preparation was assayed in duplicate by using the inorganic phosphate method.22 The control specific activity in the presence of 5 yl of DMSO was 43.5 ± 1.5 ymoles Pi/mg protein/hr.
increase DNP-stimulated ATPase in rat brain and liver mitochondria but was without effect on kidney and heart mitochondria.14"15 It was suggested, based on these earlier results, that the aflatoxins acted as uncouplers of oxidative phosphorylation.16 The O.S. Mg"1"1" ATPase, localized in the inner mitochondrial membrane, is involved in oxidative phosphorylation.13 Since the mitochondria were ruptured when quick frozen to expose the inner membrane, the in vitro system used in this study provided a convenient representation of the inside-out model. Enzymes located on the inner membrane now are accessible to chemicals. However, in the present study as well as in earlier reports 1 4 " 1 6 , the inhi-
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EFFECTS OF THE
275
AFLATOXINS
Figure 2. Molar absorptivity of aflatoxin Gj was measured at 366 nm using Beckman Acta III Scanning spectrophotometer in A) water: B) water + 50 yg protein and C) imidazole buffer. Concentrations of the toxin were 1) 30; 2) 15; 3) 6 and 4) 0.6 yM.
bition of this enzyme activity was not
concentration-dependent.
In fact, a greater inhibition was observed at lower concentrations than at higher toxin concentrations. We assumed that the toxins may be dissociated from protein at higher concentrations. Spectral data showed no change in the UV absorbance for aflatoxin Gj at high concentrations either in the presence of HgO, protein or buffer, indicating a lack of solubility effects or interference of the solvent at the toxin concentration tested. This biphasic effect cannot be explained in terms of insolubility of the aflatoxin in the reaction medium. Further evaluation of these effects on the ATPase system is warranted. The fact that all aflatoxins tested showed same inhibition of mitochondrial Mg++ ATPase suggested that these compounds may be
276
DESAIAH ET AL.
interfering at the energy conservation site in mitochondria. However, the present results do not reveal a relationship between the inhibition of the Mg++ ATPase and the potency of the toxin. For example, aflatoxin Bi, the most potent hepatotoxin in the
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series exhibited less effect on the enzyme activity than aflatoxin Gi.
However, the Na+-K+ ATPase activity, which is involved in
active ion transport across cell membranes, was more sensitive to aflatoxin Bj than Gj. A comparative study using Na+-K+ ATPase from different tissues and species with a variety of aflatoxins is needed to establish the potency of the toxin and the sensitivity of the enzyme. ACKNOWLEDGMENT Support was provided by NIH g r a n t s ES01351 and ES01352. REFERENCES 1.
Shank, R. C., C. H. Bourgeois, N. Keschamras and P. Chandavimol, Food Cosmet. Toxicol. 9, 501 (1971)
2.
Lancaster, M. C., F. P. Jenkins and J. McL. P h i l i p , Nature (London) 192, 1095 (1961)
3.
Wogan, G. N., "Methods in Cancer Research" ed. H. Busch, Academic P r e s s , NY, p. 309 (1973)
4.
Campbell, T. C. and L. S t o l o f f , J. Agr. Food Chem. 22, 1006 (1974)
5.
Wogan, G. N., B a c t e r i o l . Rev. 30, 460 (1966)
6.
Wogan, G. N. and P. M. Newberne, Cancer Res. 27, 2370 (1967)
7.
P a t t e r s o n , D. S. P., B. A. Roberts and R. A l l c r o f t , Food Cosmet. Toxicol. 7, 277 (1969)
EFFECTS OF THE AFLATOXINS
8.
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Campbell, T. C. and J. R. Hayes, Toxicol. Appl. Pharmacol. 3 5 , 199 (1976)
9.
Shank, R. C. and G. N. Wogan, Fed. Proc. 24, 627 (1965)
10.
Doherty, W. P. and T. C. Campbell, Chem. Biol. I n t e r a c t . 7,
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63 (1973) 11.
Goodall, C. M. and W. H. B u t l e r , I n t . J. Cancer 4, 422 (1969)
12.
Gurtoo, H. L. and T. C. Campbell, Mol. Pharmacol. 10, 776 (1974)
13.
Boyer, P. D., B. Chance, L. E r n e s t e r , P. M i t c h e l l , E. Racker and E. C. S l a t e r , Ann. Rev. Biochem. 46, 955 (1977)
14.
Bababunmi, E. A., Brain Res. 70, 559 (1974)
15.
Bababunmi, E. A. and O. B a s s i r , FEBS L e t t e r s 26, 102 (1972)
16.
P a i , M. R., N. J. Bai and T. A. Venkitasubramanian, Bull. Environ. Contam. Toxicol. 13, 638 (1975)
17.
Skou, J. C., "Membrane Transport and Metabolism" eds: A. K l e i n z e l l e r and A. Kotyk, Academic P r e s s , NY, p. 228 (1961)
18.
Green, D. E., R. L. L e s t e r and D. M. Z i e g l e r , Biochim. Biophys. Acta 23, 516 (1957)
19.
F r i t z , P. J. and M. E. Hamrick, Enzymol. Acta Biocat. 30, 57 (1966)
20.
Lowry, O. H. and J. A. Lopez, J . B i o l . Chem. 162, 421 (1946)
21.
Desaiah, D., A. W. Hayes and I. K. Ho, Toxicol. Appl. Pharmacol. 39, 71 (1977)
22.
P h i l l i p s , T. D., A. W. Hayes, I . K. Ho and D. Desaiah, J . Biol. Chem. 253 (1978)
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Lardy, H. A., P. Witonsky and W. C. McMurray, Arch. Biochem. Biophys. 78, 587 (1958)
24.
Desaiah, D., L. K. Cutkomp, R. B. Koch and H. H. Yap, L i f e Sci. 11, 389 (1972)
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25.
Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall, J. Biol. Chem. 193, 265 (1951)
26.
Desaiah, D., I . K. Ho and H. M. Mehendale, Toxicol. Appl. Pharmacol. 39, 219 (1977)
Received: May 13, 1978.
Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/lesb20
Effects of the aflatoxins on ATPase activities in mouse and rat liver a
a
a
D. Desaiah , T. D. Phillips , A. W. Hayes & I. K. Ho
a
a
Department of Pharmacology and Toxicology , University of Mississippi Medical Center , Jackson, Mississippi, 39216 Published online: 21 Nov 2008.
To cite this article: D. Desaiah , T. D. Phillips , A. W. Hayes & I. K. Ho (1979) Effects of the aflatoxins on ATPase activities in mouse and rat liver, Journal of Environmental Science and Health, Part B: Pesticides, Food Contaminants, and Agricultural Wastes, 14:3, 265-278, DOI: 10.1080/03601237909372127 To link to this article: http://dx.doi.org/10.1080/03601237909372127
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and
Downloaded by [McGill University Library] at 11:28 10 December 2014
views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
J. ENVIRON. SCI. HEALTH, B14(3), 265-278 (1979)
EFFECTS OF THE AFLATOXINS ON ATPase ACTIVITIES
Downloaded by [McGill University Library] at 11:28 10 December 2014
IN MOUSE AND RAT LIVER Key Words: Aflatoxins, ATPase, Mouse, Rat, Liver D. Desaiah, T. D. Phillips, A. W. Hayes and I. K. Ho Department of Pharmacology and Toxicology University of Mississippi Medical Center Jackson, Mississippi 39216 ABSTRACT The effects of the aflatoxins on ATPase a c t i v i t i e s in mouse and r a t tissues were investigated in vitro. The hepatic oligomycin-sensitive (O.S.) Mg++ ATPase was inhibited significantly. The order of inhibition was G1 > B1 > G2 > B2. Mouse O.S. Mg++ ATPase was more sensitive than the corresponding r a t enzyme. The oligomycin-insensitive (O.I.) Mg++ ATPase a c t i v i t i e s in r a t and mouse liver were not altered.
Although aflatoxins G1 and
B1 were more potent inhibitors of hepatic O.S. Mg++ ATPase, no concentration-response was observed, whereas aflatoxins G2 and B2 inhibited enzyme activity in a concentration-dependent manner. Spectral analysis of aflatoxin G1 solutions suggested that solubility was not related to the observed effects.
In addi-
tion, the effects of aflatoxin B1 and G1 on mouse brain microsomal Na+-K+ ATPase were examined. Although aflatoxin B1 was 265 Copyright © 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
266
DESAIAH ET AL.
more potent that G1, both mycotoxins significantly inhibited enzyme activity in a concentration-dependent fashion. INTRODUCTION Although aflatoxin contamination of foodstuffs has long been Downloaded by [McGill University Library] at 11:28 10 December 2014
considered a health hazard in societies where primitive methods of food storage prevail, such contamination i s generally not a human health problem in modern societies.
The aflatoxins are potent
hepatocarcinogens in experimental animals,^"^ and aflatoxin B\ may be an important human carcinogen.^ The l i t e r a t u r e pertaining to toxicity, metabolism and mode of action of the aflatoxins i s well documented.5-8 The liver i s the primary organ for aflatoxin accumulation.9
Consequently, the liver and i t s cellular organ-
ell es, including mitochondria, have been investigated in an effort to understand the mechanism(s) of toxicity.10-12 Mitochondrial ATPase, which i s involved in oxidative phosphorylation,!^ however, has received limited attention.
Aflatoxin Bj increased uncoupler
stimulated mitochondrial ATPase in mouse b r a i n ^ and l i v e r l ^ , but had no effect on renal and cardiac mitochondrial ATPase.^ In contrast, Pai et.£l_. 1 6 reported that aflatoxin B} increased mitochondrial ATPase activity but diminished the uncoupler stimulated ATPase activity in r a t liver. In view of existing discrepancy in the l i t e r a t u r e or a t least species variability regarding the sensitivity of mitochondrial ATPase to the aflatoxins, this study was undertaken t o investigate the in vitro effects of aflatoxins Bj, B2, Gj and G2 on mitochondrial ATPase in r a t and mouse l i v e r , employing two d i s t i n c t
EFFECTS OF THE AFLATOXINS
methods t o determine ATPase activity.
267
The i £ vitro response of
Na+-K+ ATPase, a biochemical manifestation of the Na+ pump, t o the aflatoxins also was examined using a mouse brain microsomal preparation.
Downloaded by [McGill University Library] at 11:28 10 December 2014
MATERIALS AND METHODS Animals: Male ICR mice weighing 25-30 g and male Sprague-Dawley rats weighing 250-275 g (Charles River Co., Wilmington, MA) were used. Animals were housed in a i r conditioned quarters with free access t o food and water. Chemicals: Purity of aflatoxins Bi, B2, Gi, G2 (Makor Chemical Jerusalem, Israel) was confirmed by melting point, infrared spectrum and thin layer chromatography. The structural formulae of these toxins are shown in Figure 1. All other chemicals were from Sigma Chemical Co. (St. Louis, MO). Tissue Fractionation:
Livers of both species were used for mito-
chondrial preparations but only mouse brain for microsomal preparation. Preparation of hepatic mitochondria: Mice and rats were decapitated and the l i v e r s quickly removed and placed in an ice-cold 0.32 M sucrose solution containing 1 mM EDTA and 10 mM imidazole, pH 7.5. Individual l i v e r s were homogenized separately in 9 vol of the sucrose solution using a ground glass homogenizer. The heavy mito-
chondria were obtained by centrifugation 1 fiand were resuspended in the sucrose solution, quick frozen in liquid nitrogen, and stored at -85° C until used for the ATPase assay.
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AL.
KÁ, Figure 1. Structural formulas of aflatoxins.
Preparation of brain microsomes: Mice were decapitated, the brains quickly removed and homogenized in the,sucrose solution as described above. The microsomes were prepared by centrifuging the 13,000 x g supernatant at 105,000 x g for 1 hr and the pellet was resuspended in the sucrose solution, quick frozen in liquid nitrogen and stored at -85° C until used. ATPase assay: The enzyme activity in mitochondrial preparations was determined by using two methods routinely used in our laboratories. 19-20 Continuous (or enzymatic) method: ATPase activity was determined by continuously monitoring the oxidation of NADH at 340 nm on a Beckman Acta III Recording Spectrophotometer, equipped with a constant temperature (37° C) water bath.
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269
A 3-ml reaction medium contained 5 mM ATP, 5 mM Mg++, 135 mM imidazole-HCl buffer (pH 7.5), 0.2 mM NADH, 0.5 mM phosphoenolpyruvate, approximately 9 units of pyruvate kinase, and 12 units of l a c t i c acid dehydrogenase. 21
A 100 yl mitochondrial fraction was
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used with a protein content of 50 yg. Absorbance changes in the reaction mixture were measured over a 10 min period. Specific activity was expressed as micromoles of inorganic phosphate per milligram of protein per hour. Discontinuous (or inorganic) phosphate method: Mitochondrial ATPase and mouse brain microsomal Na+-K+ a c t i v i t i e s were measured using endpoint phosphate a n a l y s i s . 2 0 A 1-ml reaction mixture contained 5 mM ATP, 5 mM Mg++, 100 mM Na+, 20 mM K+, 135 mM imidazole-HCl buffer (pH 7.5) and 50 yg enzyme protein.
The total ATPase activity was measured with Na+,
K+ and Mg++ present in the reaction mixtures. The basal Mg++-ATPase component was measured by omitting both Na+ and K+. Thus, delineation of the Na+-K+ activated component of ATPase was obtained by difference between total ATPase (Na+ + K+ + Mg++) and basal Mg++ ATPase (Mg++ only) activity.
The Na+-K+ ATPase activity was
consistently ouabain sensitive. Treated and control preparations, reagent and enzyme blanks (to correct f o r inherent phosphate present in samples) were preincubated 'simultaneously a t 37° C for 3 min prior t o initiation of the reaction with ATP. Incubation was stopped after 10 min with the addition of TCA (trichloroacetic acid) a t a final concentration of 5% w/v in the reaction mixture. Samples then were assayed
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for inorganic phosphate using the method of Lowry and Lopez2^ as modified by Phillips et^al_. 2 2 In both procedures, the Mg++ATPase activity was delineated into O.S. and O.I. by adding 5 x 10~6 M oligomycin (basedrupon a
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combined molecular weight of 401.20 from 15% oligomycin A and 85% oligomycin B in ethanol) to the reaction mixture. The volume of ethano'i used was 1 ul in 3 ml of reaction mixture. Oligomycin i s a potent inhibitor of mitochondria! Mg++ ATPase activity involved in oxidative phosphorylation. 23 " 2 ^ Protein was determined by the method of Lowry ert al_. 2 ^ using bovine serum albumin as the standard. Aflatoxins Bi, B2> Gj and G2 were dissolved in DMSO. Different toxin concentrations were preincubated with the enzyme in the reaction mixture for 3 min prior to the addition of ATP. Enzyme activities were determined in the presence and absence of toxin and percent effects calculated. Since the differences between the two methods were not s t a t i s t i c a l l y significant (Table 1), the data were combined for the remaining experiments to calculate the percent inhibition. RESULTS The specific activities of mitochondrial Mg++ ATPase in mouse and r a t liver (Table 1) were comparable to those previously reported 2 1 » 2 6 , further, the variability between the two methods was s t a t i s t i c a l l y insignificant. Mitochondrial (O.S.) Mg"1"1" ATPase activity in the r a t liver was determined in the absence and in the presence of aflatoxins Bj,
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TABLE 1 Determination of Mg++ ATPase Activity in Rat and House Liver Mitochondria by two Assay Methods
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Specific Activity ± S.E.M.*
Method
Rat Oligomycin Sensitive Insensitive
Mouse Oligomycin Sensitive Insensitive
Coupled Enzymatic
26.8tl.79
4.9±0.17
36.3±0.23
8.0±0.30
Inorganic Phosphate
. 25.6±1.47
5.0±0.23
35.9±0.69
7.7+0.23
*Specific Activity = pmoles of Pi/mg protein/hr. S.E.M. - Standard error of the mean of four separate liver preparations and each preparation was assayed at least four times. No s t a t i s t i c a l l y significant difference was observed between two assay methods as determined by Student's t_ test (P < 0.05).
B2> G, and Gg (Table 2). The enzyme activity was sensitive to all four toxins; however, a concentration-response was not observed. The response was biphasic for aflatoxins B^ and G^ with maximum inhibition a t 6 x 10"7 M. Of the aflatoxins tested, aflatoxin Gj was the most potent aflatoxin inhibitor of rat liver mitochondrial Mg++ ATPase. Aflatoxins Bj, B2 and Gi also showed a biphasic effect on mouse liver mitochondrial Mg4"1" ATPase activity (Table 3). Maximum inhibition was observed at 6 x 10"^ M with decreased inhibition with increased toxin concentration.
As in the r a t , afla-
toxin Gj was the most potent inhibitor. Hepatic O.I. Mg++ ATPase
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TABLE 2 Percent Inhibition of Rat Liver Mitochondrial (O.S.) Mg++
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ATPase Activity by Aflatoxins
Toxin cone. (M) 6
X
10-9 8
Percent Inhibition ± S.E.M.* Bl
B2
G2
12.0+1.40
19.0±3.50
19.5±4.60
13.0±4.20
6
X
10-
16.0±2.80
19.0±l.00
32.5±1.60
25.5±0.35
6
X
10-7
24.0±0.70
21.010.71
32.5±1.06
25.0±2.30
6
X
10"6
19.5±0.35
19.0±0.71
27.5±?.5O
9.5±1.00
5
16.0±4.20
24.0±3.50
17.5+3.10
38.3±0.88
6
X
10"
lach value represents the mean of four different tissue preparations and each preparation was assayed in duplicate by two different assay methods and the average was used for calculating the S.E.M. The control specific activities in the presence of 5 yl of DMSO were 28.7+1.9; 25.0+0.24; 25.4+0; 25.6±0.18 ytnoles Pi/mg protein/hr for aflatoxins B\, B2J Gl and G2» respectively.
activity was not affected by any of the aflatoxins tested in either species (data not presented). The Na+-K ATPase activity in mouse brain microsomes was determined in the absence and presence of different concentrations of aflatoxins Bi and Gj and the data are presented in Table 4. Na+-K ATPase activity was inhibited in a concentration-dependent fashion by aflatoxins B\ and Gj with maximum inhibition of 66% and 42%, respectively.
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TABLE 3 Percent Inhibition of Mouse Liver Mitochondrial (O.S.) Mg++
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ATPase Activity by Aflatoxins Percent Inhibition ± S.E.M." B2
Toxin cone. (M)
Bl
6 X 10-9
19.8+2.2
22.6+10.0
38.0+1.1
10.0+2.8
îo- 8
36.0+0.0
27.5±3.1
45.4±1.7
17.5±3.8
6 X 10-7
29.5±8.1
19.0+2.1
40.0+1.4
21.5±6.0
6 X 10"6
18.0±2.4
18.5±3.8
30.5±1.7
21.5+1.4
6 X 10"5
11.0±7.7
26.5±1.1
24.0±6.3
34.4±3.8
6X
G2
Each value represents the mean of four different tissue preparations and each preparation was assayed in duplicate by 2 different assay methods and the average was used for calculating the S.E.M. The control specific activities in the presence of 5 yl DMSO were 36.7+0; 36.0+0 yg; 36.2+0.32 and 36.0+0.53 ymoles Pi/mg protein/hr.
No change in the molar absorptivity of high concentrations of aflatoxin Gj at 366 nm in either water, protein or buffer was observed (Fig. 2). DISCUSSION Our results that aflatoxins Bj, B2, Gj and G2 inhibited Mg++ dependent O.S. ATPase activity in r a t and mouse liver mitochondria are in contrast to earlier r e p o r t s . 1 4 " 1 6 For example aflatoxin Gi increased the basal Mg++ ATPase and decreased the DNPstimulated ATPase in rat l i v e r , 1 6 whereas aflatoxin Bi had no effect on either ATPase.16 Aflatoxin Bj also has been reported to
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TABLE 4 Percent Inhibition of Mouse Brain Microsomal Na+-K ATPase Activity by Aflatoxins Percent Inhibition ± S.E.M.*
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Toxin cone. (M)
Bj
Gj
6 x 10-8
35.9 + 1.3
17.9 + 2 . 2
6 x 10" 7
24.5 ± 2.0
26.0 ± 1.1
6 x 10" 6
31.0 ± 1.0
28.0 ± 1.0
5
53.1 ± 0.9
35.4 ± 2.2
3 x 10" 4
66.1 ± 1.7
42.0 ± 1.5
6 x 10"
Each value represents the mean of 3 different tissue preparations and each preparation was assayed in duplicate by using the inorganic phosphate method.22 The control specific activity in the presence of 5 yl of DMSO was 43.5 ± 1.5 ymoles Pi/mg protein/hr.
increase DNP-stimulated ATPase in rat brain and liver mitochondria but was without effect on kidney and heart mitochondria.14"15 It was suggested, based on these earlier results, that the aflatoxins acted as uncouplers of oxidative phosphorylation.16 The O.S. Mg"1"1" ATPase, localized in the inner mitochondrial membrane, is involved in oxidative phosphorylation.13 Since the mitochondria were ruptured when quick frozen to expose the inner membrane, the in vitro system used in this study provided a convenient representation of the inside-out model. Enzymes located on the inner membrane now are accessible to chemicals. However, in the present study as well as in earlier reports 1 4 " 1 6 , the inhi-
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EFFECTS OF THE
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AFLATOXINS
Figure 2. Molar absorptivity of aflatoxin Gj was measured at 366 nm using Beckman Acta III Scanning spectrophotometer in A) water: B) water + 50 yg protein and C) imidazole buffer. Concentrations of the toxin were 1) 30; 2) 15; 3) 6 and 4) 0.6 yM.
bition of this enzyme activity was not
concentration-dependent.
In fact, a greater inhibition was observed at lower concentrations than at higher toxin concentrations. We assumed that the toxins may be dissociated from protein at higher concentrations. Spectral data showed no change in the UV absorbance for aflatoxin Gj at high concentrations either in the presence of HgO, protein or buffer, indicating a lack of solubility effects or interference of the solvent at the toxin concentration tested. This biphasic effect cannot be explained in terms of insolubility of the aflatoxin in the reaction medium. Further evaluation of these effects on the ATPase system is warranted. The fact that all aflatoxins tested showed same inhibition of mitochondrial Mg++ ATPase suggested that these compounds may be
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interfering at the energy conservation site in mitochondria. However, the present results do not reveal a relationship between the inhibition of the Mg++ ATPase and the potency of the toxin. For example, aflatoxin Bi, the most potent hepatotoxin in the
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series exhibited less effect on the enzyme activity than aflatoxin Gi.
However, the Na+-K+ ATPase activity, which is involved in
active ion transport across cell membranes, was more sensitive to aflatoxin Bj than Gj. A comparative study using Na+-K+ ATPase from different tissues and species with a variety of aflatoxins is needed to establish the potency of the toxin and the sensitivity of the enzyme. ACKNOWLEDGMENT Support was provided by NIH g r a n t s ES01351 and ES01352. REFERENCES 1.
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2.
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3.
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4.
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P a t t e r s o n , D. S. P., B. A. Roberts and R. A l l c r o f t , Food Cosmet. Toxicol. 7, 277 (1969)
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Received: May 13, 1978.
