Chem.-Biol. interactions, 10 (1975) 123431
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECT OF AFLATOXINS ON OXIDATIVE RAT LIVER MITOCHONDRIA
M. RAMACHANDRA
PAI, N. JAYANTHI
BAI
AND
PHOSPHORYLATION
123
BY
T. A. VENICITASUBRAMANIAN
Department of Biochemistry, Vallabhbhai Pate1 Chest Institute, University of Delhi, Delhi-110007 (India) (Received July 22nd, 1974) (Revision received September 4th, 1974)
SUMMARY
The in vitro effect of aflatoxins Ml, B1 and Gl on oxidative phosphorylation by rat liver mitochondria with succinate as substrate has been studied. All these toxins inhibit the electron transport chain at a 1 - 10-a M concentration and the site of inhibition is between cytochrome b and cytochrome c or cl. Aflatoxin Ml (AFMI) uncouples oxidative phosphorylation at a concentration of 1 - 10-S M and reduces the ADP : 0 ratio, whereas aflatoxin B1 (AFB1) at 1 - lo-6 M concentration uncouples oxidative phosphorylation but does not affect the ADP : 0 ratio. At a concentration of 1 10-s M, AFBl also decreases the ADP : 0 ratio along with the uncoupling of oxidative phosphorylation. Aflatoxin G1 (AFGl) acts as an uncoupler at a relatively higher concentration of 1 * 10-d M. Preincubation of mitochondria with these aflatoxins resulted in inhibition of respiration and uncoupling of rat liver mitochondria. l
INTRODUCTION
Aflatoxins, the bifuranocoumarins, produced as secondary metabolites by Aspergillus fravus are highly toxic and hepatocarcinogenic to a number of animals including rat. AFBl is lethal to rat at a single dose of 0.6 mg/kg and higherl. The in vitro and in vivo effects of AFBl on oxidative phosphorylation by rat liver mitochondria have been reported. When added to actively respiring mitochondria, AFBl inhibits the electron transport flows. Following an oral dose of
Abbreviations: AFB1, aflatoxin B1; AFG1, sflatoxin Gl; AFMl, aflatoxin Ml; DMSO, dimethyl sulphoxide; DNP, 2Pdinitrophenol; HQNO, 2-heptyl4hydroxyquinoline-N-oxide; RCR, respiratory control ratios; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride.
124 0.45 mg pure AFBl/kg body weight, both oxygen consumption and phosphorylation were inhibited after 24 h of administering the toxin; respiration returned to normal after 72 h, but not the phosphorylations. Clifford and Rees4 observed no change in respiration or P : 0 ratio even up to 24 h on administration of 7 mg AFBl/kg body weight in spite of using a number of substrates. Inhibition of oxygen uptake by mitochoudrial fraction was observed by Strufaldi et al.5 when succinate was used as substrate. All these studies were confined to AFBr. There are no reports so far on the action of other aflatoxins on oxidative phosphorylation. The present study deals with the effect, in vitro, of AFM;, BI and Gl on oxidation and phosphorylation by rat liver mitochondria.
AFMr, Br and G1 were prepared from Aspergillusjlavus, NRRL 3240 and 3251 on a semisynthetic mediums. The toxins were separated from the chloroform extract of the media and mycelia by a modification of Steyn’s’ method. The purity of the ~zolourless crystalline aflatoxins so obtained was checked by thin-layer chromatography and molar absorption. AFBl and GI moved as single spots in chloroform : acetone (9 : 1, v/v) and chloroform : methanol (98 : 2, v/v). AFMl moved as a single spot in Zpropanol : water : acetone : chloroform (1 : 1.5 : 12 : 88, v/v/v/v)*. ADP, DNP, HQNO, bovine serum albumin and tris-(hydroxymethyl)-aminomethane (Tris) were from Sigma Chemical Company, St. Louis, Missouri. All other reagents were of analar grade from BDH or Merck. Preparation of mitochondria
Mitochondria were isolated from albino rats weighing 80-120 g. Rats were fed Hindustan Lever pellet diet and water ad libitum. They were not starved and were killed at the same time of the day. The rats were stunned by a blow on the head, livers removed quickly and dropped into ice-cold 0.25 M sucrose, pH 7. The weighed livers were minced and washed free of blood. Mitochondria were then isolated by the method of Johnson and Lardye. The mitochondrial pellet was washed twice with 9.25 M sucrose and finally suspended in 0.25 M sucrose such that one ml of the suspension contained about IO mg of mitochondrial proteins. The above operations were carried out at O-4’. Mitochondrial preparations giving RCR of 3-4 and ADP : 0 ratios of 1.9-2 with succinate as substrate were used in the experiments. All experiments with mitochondria were completed within 3-4 h of killing the rats. Measurement of respiration and oxidative phosphorylation
Respiration and phosphorylation were measured polarographically at 30“ using a Clark Oxygen electrode of Yellow Springs Instrument Company, Ohio. Oxygen content of the medium was traced on a graphic recorder of Varian Associates and calibrated from the standard solubility of oxygen. Respiratory control ratio and ADP : 0 ratio were determined according to the method of Chance and Williams as given by Estabrooklo. The capacity of the
125 electrode vessel was 3 ml and it contained 0.25 M sucrose; 10 mM potassium phosphate, pH 7.4; 5 mM MgClz; 20 mM KCl; 20 mM Tris-HCl, pH 7.4 and 3.3 mM sodium succinate, pH 7.4. To each reaction system was added 0.1 ml of mitochondrial suspension containing approximately 1 mg of protein and 0.1 ml of 0.15 M KC1 containing 0.6 pM ADP. In the studies on the effect of aflatoxins, the compounds were dissolved in DMSO so that 30 ~1 of the solution contained the required amount of the toxin. The solution was added with a micropipette just before the addition of ADP. In the experiments where mitochondria preincubated with aflatoxins were used, the appropriate toxin solutions were added to 0.25 M sucrose, followed by mitochondrial suspension. The mixture was incubated in a water bath at 30” for 10 min without shaking. Mitochondrial swelling was studied in sucrose (0.25 M) and NaCl(O.15 M) in presence !,f various concentrations of allatoxins. There was no swelling of Imitochondria ‘!jyaflatoxins over that caused by the solvent. Prtiein was estimatced by the method of Lowry et ~1.11 using bovine serum albumin as the standard. RESULTS
The inhibition of respiration of rat liver mitochondria by AFBi has been shown to be pronounced when succinate is used as the substrate. Strufaldi et ~2.5found that NAD and FAD dependent substrates other than succinate showed no inhibitory effect when AFBl was added to mitochondrial fraction. Doherty and Campbell2 have shown that there is no effect of AFBi on the electron transport chain after cytochrome c or before cytochrome b. Hence succinate was used in all the following studies. The integrity of mitochondria was checked before and after each day’s experiments. Tightly coupled mitochondria as evidenced by RCR 3-4, ADP : 0 ratio 1.9-2 and state 3 respiration 96-100 nmoles oxygen per minute per mg mitochondrial protein obtained on different days of preparation alone were used in the experiments. Results discussed here are typical of a minimum of six different preparations of mitochondria, with the experiments done in duplicate or in triplicate. Of the various solvents tried for aflatoxins, DMSO was chosen for the experiment I since only DMSO did not affect the respiration, phosphorylation and RCR of norm;rl mitochondria. Dimethylformamide inhibited respiration by about 10% and ethar.J or propanediol were not good solvents for these toxins. The maximum concentration of the toxins employed was 3 . IO-* M, as higher concentrations resulted in precipitation when added to the reaction system. Addition of varying concentrations of aflatoxins, 1 * 10-S M to 3 * lo+ M, to rat liver mitochondria resulted in uncoupling of oxidative phosphorylation and inhibition of electron transport. The rates of state 3 and state 4 respirations, ADP : 0 ratio, RCR and the dominant mechanism operating in presence of AFMI are summarised in Table I. At 1 * IO-6 M concentration AFMr lowers the RCR and ADP : 0 ratio. Since state 3 respiration remains the same as normal and state 4 respiration is
126 TABLE 1 rmCr OF AFLATOXIN Ml
ON RESPIRATORY RATES,
RATIO AND RESPIRATORY CONTROL RATIO
ADP : 0
OF RAT LIVER WTOCHONDRIA
AFMI, at concentrations indicated, was added in a volume of 30 ~1 DMSO to 3 ml of the reaction medium describedin the text. kspiratory rates are expressed as nmoles oxygen per minute per mg protein. All values are Mean f S.E.M. of six separateexperiments.
AFMl
Slate 3
State 4
ADP : 0 ratb
RCR
Dominant mechanism
%f 1.2 96 f 0.7 96 f 0.6
30 f 0.6 44 f 0.6
1.93 f 0.02 1.26 A 0.02 1.19 f 0.01 -
3.20 2.18
Uncoupling and inhibition Uncoupling and inhibition Inhibition and uncoupling Inhibition and uncoupling
concentrat&n (Ml
0 (DMSO) 1 ’ IO-@ 1 * IO-” I ’ 10-a
3*10-4
59 f 0.4 44 & 0.5
44 f 0.6 -
2.18
-
elevated, the toxin has uncoupled oxidation even at this low concentration. The decreasein the ADP : 0 ratio is suggestive of inhibition of electron transport and this has been confirmed in studies with higher concentrations of the toxin. The uncoupling
effect counteracts the inhibitory effect on state 3 respiration with the net result that state 3 respiration remains normal. The various possibilities for the lowering of RCR are inhibition of electron transport, uncoupling of oxidative phosphorylation and inhibition of only phosphorylation. When electron transport is inhibited, state 3 and state 4 respirations are inhibited. In the uncoupling of oxidative phosphorylation, state 3 respiration is unaffected or is affected only slightly and state 4 respiration is ittcrea&. When it is inhibition of only phosphorylation and not of oxidation. state
3 respiration is depressed during the inhibition of phosphorylation and state 4 is um&cted*~. The third possibility is ruled out since state 4 respiration is increased at this concentration of AFMr. At a higher concentration
of AFMl, 1 10-s M, state 3, state 4 respirations and RCR remain the same as with 1 10-e M, but the ADP : 0 ratio falls further. When the concentration is 1 mlo-4 M or 3 10-d M, state 3 respiration is depressed; evidently electron transport is inhibited. Further, state 3 and state 4 respiration could not bedistinguished from each other. Together with theinhibition of electron transport uncoupling is also in operation as state 3 respiration gradually increases with increasingtime. Hence the dominant mechanism is inhibition of electron transport. The inhibition of electron transport has been further confirmed by locating the inhibition to be at site II of the electron transport chain. The rates of state 3 and state 4 respirations in presence of drfferent concentrations of AFMl studied are shown in Fig. 1. The effect of different concentrations of AFBl on mitochondrial function is shown in Table II. When the concentration of AFBl is 1 . 10-S M, state 3 respiration and ADP : 0 ratio remain normal, but RCR falls. This is suggestive of uncoupling ofoxidation from phosphorylation, where state 3 is unaffected and state 4 is increased. l
l
l
127 TABLE II EFFECT OF AFLATOXIN BI ON RFSPIRATORY RATES, OF RAT LIVER MITOCHONDRIA
ADP : 0
RATIO,
AND RESPIRAlQRY
CONTROL RATIO
Al%, at concentrations indicated, was added in a volume of 30 ~1 DMSO to 3 ml of the reaction medium described in the text. Respiratory rates are expressed as nmoles oxygen per minute per mg protein. All values are Mean i S.E.M. of six separate experiments. AFBx Sfate 3 concentration /Ml 0 (DMSO) 1 * 10" 1 * 10-j 1 - 10-a 3 - 10-d
96 f 96 i 96 f 48 i 24 rt
0.8 1.3 0.5 0.4 0.3
State 4
ADP : 0 rutiu RCR
Dominant mechanism
32 + 0.4 48 f 0.5 48 + 0.5 -
1.93 f 0.02 I.93 * 0.02 1.35 f 0.01 -
Uncoupling Uncoupling and inhibition Inhibition and uncoupling Inhibition and uncoupling
3.0 2.0 2.0 -
At a higher concentration of 1 10-s A# AFBl, ADP : 0 ratio is decreased denoting the inhibition on the electron transport as well. With 1 10-d or 3 - 10-a M concen; tration, state 3 is decreased and here again state 4 could not be distinguished from state 3. The effects seen with AFB1 are similar to those with AFMl. The respiratory rates in presence of AFBl are shown in Fig. 2. l
l
\E RIE
WE
Fig. 1. Effect of varying con~nt~tions of AFMl on respiratory rates of rat liver mit~hondria. Appropriate amounts of AFMl in 30 ~1 of DMSO were added to 3 ml of the solution in the reaction vessel as described in the text. Respiratory rates in presence of AFMl at conwntrations (JW] of: (A) 0 (30 ~1DMSO); (B) 1 *10-6; (C) 1 - lo-“; (D) 1 - lo-” and (E) 3 * lo-“. Figures on traces denote respiratory rates in nmoles oxygen per minute per mg protein. Fig. 2. Effect of various concentrations of AFBI on respiratory rates of rat liver mitochondria. Detaifs of the reaction system are as described in the text. Rates of respiration in presence of AFBl at concentrations (M): (A) 0 (30 yl DMSO); (B) 1 +lo-s;(C) 1 - 10-S; (D) 1 =lo-,‘and (E) 3 * lO+. Figures on traces denote respiratory rates in nanomdes oxygen per minute per mg protein. Fig. 3. Effect of AFGl on respiratory rates of rat liver mitochondria. AFGI in 30 pl DMSO was added to the reaction vessel as de&bed in the text. Rates of respiration in presence of AFG at concentrations (Mj: (A) 0 (30 cd DMSO); (18)1 - 10-S; (C) 1 * 10-h; (D) 1 -IO-'and (El 3 - 10-5.
TABLE 111 EWECT OFAFLATDXIN OFRATUVER
G1 ON RESPIRATORY RATES,
ADP : 0
RATIO AND RESPIRATORY CONTROL RAT0
WTOCMONDRIA
AFCh, at concentrations indicated, was added in a volume of 30 pl DMSO to 3 ml of the reaction medium de&bed in the text. Resgiratory rates are expressed as nmoles oxygen per minute per mg protein. All values are Mean f S.E.M. of six separateexperiments.
AFG
State 3
State 4
ADP:O
RCR
Dominant mechanism
96 f 1.3
30 5 0.5 30 f 0.5 30 i 0.5 -
1.93 _I 0.02 1.93 _t 0.02 1.93 f 0.02 -
3.2 3.2 3.2 -
-
cwfuwtm~n (W 0 (DMSO) 1’ 10-a I - IO-5 I ’ 10-J 3. IO-”
%f 1.3 %f 1.3 46*o.s 48 f 0.5
inhibition and uncoupling Inhibition and uncoupling
Table II1 shows the effect of AFGl at various concentrations on oxidation and phosphorylazion. In contrast to AFMl or AFBl, AFGl does not affect the normal functioning of mitochondria at concentrations of 1 - 10-G M or 1 * 10-S M. However, at 1 10-a M or 3 IO-4 M concentration, AFGl also inhibits electron transport just like the other two toxins. Moreover, state 3 and state 4 respirations are indistinguishable from each other. It is evident therefore that AFGl can inhibit electron transport at the same concentration as AFMl or AFBl, but can uncouple oxidative phosphorylation, only at a relatively higher concentration. The effect of AFGl on the respiratory rates is shown in Fig. 3. l
l
Site of inhibition of aflutoxins AFBr has been shown to inhibit the electron transport chain between cytochrome b and c (cl) (ref. 2). ‘This study was extended to AFMl and AFGl also, to see whether ull these toxins act at the same or different sites of the electron transport chain. The inhibition of ADP stimulated respiration of all these toxins was reversed by TMPD (Fig. 4). It is apparent therefore that AFMl and AFGl also inhibit site II of the
Fii 4. Reversal of inhibition of aflatoxins by TMPD. Aflatoxin concentration: 3 - 1O-4M; ADP, 0.8 mM.
129
Fig. 5. Effect of preincubation of mitochondria with aflatoxins. Mitochondria preincubated with aflatoxins at concentrations of 3 * 1O-4Mat 30” for 10 min. ADP, 0.2 mM. Figures on traces denote respiratory rates @moles oxygen per minute per mg protein).
electron transport chain just like AFBI. In order to confirm these results, HQNO was used in place of aflatoxins. 40 yg HQNO per mg mitochondrial protein completely inhibited state 3 respiration and the inhibition was reversed by TMPD or DNP. E&ct of preincubation of mitochondria with aflatoxins The aflatoxins studied inhibited state 3 respiration at a concentration of 1 - 10-a M, whether the toxins were added prior to the addition of ADP or during ADP stimulated respiration. In presence of aflatoxins state 3 respiration gradually increased with time and could not be distinguished from state 4. Hence the effect of preincubation of mitochondria with aflatoxins was investigated and the results are presented in Fig. 5. Control experiments were carried out where mitochondria were preincubated without the aflatoxins. Stimulation of respiration by ADP was lower in mitochondria preincubated with aflatoxins. Without ADP the rate of respiration was much higher in aflatoxin-treated mitochondria than in control mitochondria. DISCUSSION
The results of the present study are in agreement with the in vitro inhibition of electron transport by AFBl reported by Doherty and Campbell2 and Strufaldi et a1.5. Doherty and Campbelle reported an inhibition of 25-44%, when 2.5-4.8 lo-4 A4 AFBl was added to actively respiring mitochondria. In our studies, state 3 respiration is inhibited by about 50 y0 and 75 “/oin presence of AFBr at concentrations of 1 - IO-4M and 3 * 10-d M, respectively. The higher rates of inhibition observed in our studies could be due to the lower concentration of ADP in the system (0.2 mM). When 0.8 mM ADP was added to stimulate respira,tion, AFBr at a concentration of 3 * 10e4 M inhibited respiration by 30% only (Fig. 4). It is apparent from these results that the inhibition caused by AFBl is at least partly reversible with ADP. However, there were differences in the degree of inhibition by several preparations of mitochondria, but the nature of inhibition by the toxins remained the same. The present study shows that aflatoxins have two distinct effects on mitochondrial function, as uncouplers at lower concentrations and as inhibitors at higher l
130 concentrations. AFMl and Bl are uncouplers at a concentration of 1 IO-6 M (Tables I and II). At concentration of I - 10-a M or higher AFMl, BI and G1 inhibit the electron transport. The uncoupling nature of AFBl has not been shown earlier, even though its inhibitory effect on respiration has been investigated in detail. AFGl differs from the other two, only in that it uncouples phosphorylation at a relatively higher concentration of 1 lO-%f. However, all these toxins inhibit the electron transport chain at site II, though the degree of inhibition varies. The inhibition decreases in the order BI, Ml and Gl. The effect of uncouplers on mitochondria could be due to either a gross change in the structure of mitcehondrial membrane or a binding at specific sites responsible for oxidative phosphorylation. The latter mechanism seems more probable in the case of aflatoxins (evidence for binding of aflatoxins with mitochondria is presented elsewhere). It is pertinent to add that even though in the typical experiments depicted in Tables I and II the inhibition by AFMl is lower than that by AFBl, in several preparations of mitochondria, these toxins inhibited state 3 respiration to the same extent. But with all the preparations, at a concentration of 1 * 10V6IkZ,AFMl but not Bl, lowered the ADP : 0 ratio. Hence it is reasonable to assume that AFMl (4hydroxy AFBl) is a more efficient inhibitor of electron transport in rat liver mitochondria. This isina ment with the results of our studies (details of which are not presented here) as part of the structure activity relationship of naturally occurring coumarins, where the hydroxycoumarins exert profound effects on mitochondrlal function. l
l
This investigation was supported in part by PL-480 grant No. FG-In-438.
d G. N. Wogan, Toxicity and bmchemical and fine structural effects of synthetic nd 111in rat liver, J. h’nrl. Cancer fnsr., 47 (1971) 585-592. y and T. C. Campbell, Aflatoxin inhibition of rat liver mitochondria, Chem.-Biol.
H. 1. Grady and J. Higginson, Aflatoxin BI injury in rat and monkey liver, Am. J.
J. I. Clifford and K. R. Rees, The action of allatoxin BI on the rat liver, Biochem. J., 102 (1967) 65-75. II. Strufaldi, D. M. Noyucira and F. I. Pedroso, Effect of aflatoxin Br on metabolic activity of t tic mitochondrial fraction from Rattu.9 Norwegicus (rat), Rev. Farm. Bioquim. Univ. Sao Pado, 8 (1970) l-23. 6 N. D. Davis, U. L. Diener and D. W. Eldridge, Production of aflatoxins BI and Gr by Aspergillus fklvus in a semi*ynthetic mecium, Appl. Mi~*ro6iol., I4 (1966) 378-380. n, Rapid method for the isolation of pure afiatoxim, J. Assoc. Offic. And. Chem., 53 19-621, D. Stuhbkfield, G. M. Shannon and 0. L. Shotwqll, Aflatoxins MI and Mz: preparation and aurification, /. Am. Oil Chem. Sot., 47 (1970) 389-590. 9
JohnsOn and H. Lardy, Isolation of liver or kidney mitochondria, in R. W. Estabrook and E. Pulhan U2ds.h Methods in Enzymology, Vol. X, Academic Press, New York, 1967, pp.
101.
131 10 R. W. Estabrook, Mitochondrial respiratory control and the polarographic measurement of ADP : 0 ratios, in R. W. Estabrook and M. E. Pullman (Eds.), Methods in Enzymology, Vol. X, Academic Press, New York, 1967, pp. 41-47. 11 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurcxent with the Folin phenol reagent, J. Biof. Chem., 193 (1951) 265-275. 12 A. L. Lehninger, The Mitochondrion, Renjamin, New York, 1964, pp. 135-188.
0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
EFFECT OF AFLATOXINS ON OXIDATIVE RAT LIVER MITOCHONDRIA
M. RAMACHANDRA
PAI, N. JAYANTHI
BAI
AND
PHOSPHORYLATION
123
BY
T. A. VENICITASUBRAMANIAN
Department of Biochemistry, Vallabhbhai Pate1 Chest Institute, University of Delhi, Delhi-110007 (India) (Received July 22nd, 1974) (Revision received September 4th, 1974)
SUMMARY
The in vitro effect of aflatoxins Ml, B1 and Gl on oxidative phosphorylation by rat liver mitochondria with succinate as substrate has been studied. All these toxins inhibit the electron transport chain at a 1 - 10-a M concentration and the site of inhibition is between cytochrome b and cytochrome c or cl. Aflatoxin Ml (AFMI) uncouples oxidative phosphorylation at a concentration of 1 - 10-S M and reduces the ADP : 0 ratio, whereas aflatoxin B1 (AFB1) at 1 - lo-6 M concentration uncouples oxidative phosphorylation but does not affect the ADP : 0 ratio. At a concentration of 1 10-s M, AFBl also decreases the ADP : 0 ratio along with the uncoupling of oxidative phosphorylation. Aflatoxin G1 (AFGl) acts as an uncoupler at a relatively higher concentration of 1 * 10-d M. Preincubation of mitochondria with these aflatoxins resulted in inhibition of respiration and uncoupling of rat liver mitochondria. l
INTRODUCTION
Aflatoxins, the bifuranocoumarins, produced as secondary metabolites by Aspergillus fravus are highly toxic and hepatocarcinogenic to a number of animals including rat. AFBl is lethal to rat at a single dose of 0.6 mg/kg and higherl. The in vitro and in vivo effects of AFBl on oxidative phosphorylation by rat liver mitochondria have been reported. When added to actively respiring mitochondria, AFBl inhibits the electron transport flows. Following an oral dose of
Abbreviations: AFB1, aflatoxin B1; AFG1, sflatoxin Gl; AFMl, aflatoxin Ml; DMSO, dimethyl sulphoxide; DNP, 2Pdinitrophenol; HQNO, 2-heptyl4hydroxyquinoline-N-oxide; RCR, respiratory control ratios; TMPD, N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride.
124 0.45 mg pure AFBl/kg body weight, both oxygen consumption and phosphorylation were inhibited after 24 h of administering the toxin; respiration returned to normal after 72 h, but not the phosphorylations. Clifford and Rees4 observed no change in respiration or P : 0 ratio even up to 24 h on administration of 7 mg AFBl/kg body weight in spite of using a number of substrates. Inhibition of oxygen uptake by mitochoudrial fraction was observed by Strufaldi et al.5 when succinate was used as substrate. All these studies were confined to AFBr. There are no reports so far on the action of other aflatoxins on oxidative phosphorylation. The present study deals with the effect, in vitro, of AFM;, BI and Gl on oxidation and phosphorylation by rat liver mitochondria.
AFMr, Br and G1 were prepared from Aspergillusjlavus, NRRL 3240 and 3251 on a semisynthetic mediums. The toxins were separated from the chloroform extract of the media and mycelia by a modification of Steyn’s’ method. The purity of the ~zolourless crystalline aflatoxins so obtained was checked by thin-layer chromatography and molar absorption. AFBl and GI moved as single spots in chloroform : acetone (9 : 1, v/v) and chloroform : methanol (98 : 2, v/v). AFMl moved as a single spot in Zpropanol : water : acetone : chloroform (1 : 1.5 : 12 : 88, v/v/v/v)*. ADP, DNP, HQNO, bovine serum albumin and tris-(hydroxymethyl)-aminomethane (Tris) were from Sigma Chemical Company, St. Louis, Missouri. All other reagents were of analar grade from BDH or Merck. Preparation of mitochondria
Mitochondria were isolated from albino rats weighing 80-120 g. Rats were fed Hindustan Lever pellet diet and water ad libitum. They were not starved and were killed at the same time of the day. The rats were stunned by a blow on the head, livers removed quickly and dropped into ice-cold 0.25 M sucrose, pH 7. The weighed livers were minced and washed free of blood. Mitochondria were then isolated by the method of Johnson and Lardye. The mitochondrial pellet was washed twice with 9.25 M sucrose and finally suspended in 0.25 M sucrose such that one ml of the suspension contained about IO mg of mitochondrial proteins. The above operations were carried out at O-4’. Mitochondrial preparations giving RCR of 3-4 and ADP : 0 ratios of 1.9-2 with succinate as substrate were used in the experiments. All experiments with mitochondria were completed within 3-4 h of killing the rats. Measurement of respiration and oxidative phosphorylation
Respiration and phosphorylation were measured polarographically at 30“ using a Clark Oxygen electrode of Yellow Springs Instrument Company, Ohio. Oxygen content of the medium was traced on a graphic recorder of Varian Associates and calibrated from the standard solubility of oxygen. Respiratory control ratio and ADP : 0 ratio were determined according to the method of Chance and Williams as given by Estabrooklo. The capacity of the
125 electrode vessel was 3 ml and it contained 0.25 M sucrose; 10 mM potassium phosphate, pH 7.4; 5 mM MgClz; 20 mM KCl; 20 mM Tris-HCl, pH 7.4 and 3.3 mM sodium succinate, pH 7.4. To each reaction system was added 0.1 ml of mitochondrial suspension containing approximately 1 mg of protein and 0.1 ml of 0.15 M KC1 containing 0.6 pM ADP. In the studies on the effect of aflatoxins, the compounds were dissolved in DMSO so that 30 ~1 of the solution contained the required amount of the toxin. The solution was added with a micropipette just before the addition of ADP. In the experiments where mitochondria preincubated with aflatoxins were used, the appropriate toxin solutions were added to 0.25 M sucrose, followed by mitochondrial suspension. The mixture was incubated in a water bath at 30” for 10 min without shaking. Mitochondrial swelling was studied in sucrose (0.25 M) and NaCl(O.15 M) in presence !,f various concentrations of allatoxins. There was no swelling of Imitochondria ‘!jyaflatoxins over that caused by the solvent. Prtiein was estimatced by the method of Lowry et ~1.11 using bovine serum albumin as the standard. RESULTS
The inhibition of respiration of rat liver mitochondria by AFBi has been shown to be pronounced when succinate is used as the substrate. Strufaldi et ~2.5found that NAD and FAD dependent substrates other than succinate showed no inhibitory effect when AFBl was added to mitochondrial fraction. Doherty and Campbell2 have shown that there is no effect of AFBi on the electron transport chain after cytochrome c or before cytochrome b. Hence succinate was used in all the following studies. The integrity of mitochondria was checked before and after each day’s experiments. Tightly coupled mitochondria as evidenced by RCR 3-4, ADP : 0 ratio 1.9-2 and state 3 respiration 96-100 nmoles oxygen per minute per mg mitochondrial protein obtained on different days of preparation alone were used in the experiments. Results discussed here are typical of a minimum of six different preparations of mitochondria, with the experiments done in duplicate or in triplicate. Of the various solvents tried for aflatoxins, DMSO was chosen for the experiment I since only DMSO did not affect the respiration, phosphorylation and RCR of norm;rl mitochondria. Dimethylformamide inhibited respiration by about 10% and ethar.J or propanediol were not good solvents for these toxins. The maximum concentration of the toxins employed was 3 . IO-* M, as higher concentrations resulted in precipitation when added to the reaction system. Addition of varying concentrations of aflatoxins, 1 * 10-S M to 3 * lo+ M, to rat liver mitochondria resulted in uncoupling of oxidative phosphorylation and inhibition of electron transport. The rates of state 3 and state 4 respirations, ADP : 0 ratio, RCR and the dominant mechanism operating in presence of AFMI are summarised in Table I. At 1 * IO-6 M concentration AFMr lowers the RCR and ADP : 0 ratio. Since state 3 respiration remains the same as normal and state 4 respiration is
126 TABLE 1 rmCr OF AFLATOXIN Ml
ON RESPIRATORY RATES,
RATIO AND RESPIRATORY CONTROL RATIO
ADP : 0
OF RAT LIVER WTOCHONDRIA
AFMI, at concentrations indicated, was added in a volume of 30 ~1 DMSO to 3 ml of the reaction medium describedin the text. kspiratory rates are expressed as nmoles oxygen per minute per mg protein. All values are Mean f S.E.M. of six separateexperiments.
AFMl
Slate 3
State 4
ADP : 0 ratb
RCR
Dominant mechanism
%f 1.2 96 f 0.7 96 f 0.6
30 f 0.6 44 f 0.6
1.93 f 0.02 1.26 A 0.02 1.19 f 0.01 -
3.20 2.18
Uncoupling and inhibition Uncoupling and inhibition Inhibition and uncoupling Inhibition and uncoupling
concentrat&n (Ml
0 (DMSO) 1 ’ IO-@ 1 * IO-” I ’ 10-a
3*10-4
59 f 0.4 44 & 0.5
44 f 0.6 -
2.18
-
elevated, the toxin has uncoupled oxidation even at this low concentration. The decreasein the ADP : 0 ratio is suggestive of inhibition of electron transport and this has been confirmed in studies with higher concentrations of the toxin. The uncoupling
effect counteracts the inhibitory effect on state 3 respiration with the net result that state 3 respiration remains normal. The various possibilities for the lowering of RCR are inhibition of electron transport, uncoupling of oxidative phosphorylation and inhibition of only phosphorylation. When electron transport is inhibited, state 3 and state 4 respirations are inhibited. In the uncoupling of oxidative phosphorylation, state 3 respiration is unaffected or is affected only slightly and state 4 respiration is ittcrea&. When it is inhibition of only phosphorylation and not of oxidation. state
3 respiration is depressed during the inhibition of phosphorylation and state 4 is um&cted*~. The third possibility is ruled out since state 4 respiration is increased at this concentration of AFMr. At a higher concentration
of AFMl, 1 10-s M, state 3, state 4 respirations and RCR remain the same as with 1 10-e M, but the ADP : 0 ratio falls further. When the concentration is 1 mlo-4 M or 3 10-d M, state 3 respiration is depressed; evidently electron transport is inhibited. Further, state 3 and state 4 respiration could not bedistinguished from each other. Together with theinhibition of electron transport uncoupling is also in operation as state 3 respiration gradually increases with increasingtime. Hence the dominant mechanism is inhibition of electron transport. The inhibition of electron transport has been further confirmed by locating the inhibition to be at site II of the electron transport chain. The rates of state 3 and state 4 respirations in presence of drfferent concentrations of AFMl studied are shown in Fig. 1. The effect of different concentrations of AFBl on mitochondrial function is shown in Table II. When the concentration of AFBl is 1 . 10-S M, state 3 respiration and ADP : 0 ratio remain normal, but RCR falls. This is suggestive of uncoupling ofoxidation from phosphorylation, where state 3 is unaffected and state 4 is increased. l
l
l
127 TABLE II EFFECT OF AFLATOXIN BI ON RFSPIRATORY RATES, OF RAT LIVER MITOCHONDRIA
ADP : 0
RATIO,
AND RESPIRAlQRY
CONTROL RATIO
Al%, at concentrations indicated, was added in a volume of 30 ~1 DMSO to 3 ml of the reaction medium described in the text. Respiratory rates are expressed as nmoles oxygen per minute per mg protein. All values are Mean i S.E.M. of six separate experiments. AFBx Sfate 3 concentration /Ml 0 (DMSO) 1 * 10" 1 * 10-j 1 - 10-a 3 - 10-d
96 f 96 i 96 f 48 i 24 rt
0.8 1.3 0.5 0.4 0.3
State 4
ADP : 0 rutiu RCR
Dominant mechanism
32 + 0.4 48 f 0.5 48 + 0.5 -
1.93 f 0.02 I.93 * 0.02 1.35 f 0.01 -
Uncoupling Uncoupling and inhibition Inhibition and uncoupling Inhibition and uncoupling
3.0 2.0 2.0 -
At a higher concentration of 1 10-s A# AFBl, ADP : 0 ratio is decreased denoting the inhibition on the electron transport as well. With 1 10-d or 3 - 10-a M concen; tration, state 3 is decreased and here again state 4 could not be distinguished from state 3. The effects seen with AFB1 are similar to those with AFMl. The respiratory rates in presence of AFBl are shown in Fig. 2. l
l
\E RIE
WE
Fig. 1. Effect of varying con~nt~tions of AFMl on respiratory rates of rat liver mit~hondria. Appropriate amounts of AFMl in 30 ~1 of DMSO were added to 3 ml of the solution in the reaction vessel as described in the text. Respiratory rates in presence of AFMl at conwntrations (JW] of: (A) 0 (30 ~1DMSO); (B) 1 *10-6; (C) 1 - lo-“; (D) 1 - lo-” and (E) 3 * lo-“. Figures on traces denote respiratory rates in nmoles oxygen per minute per mg protein. Fig. 2. Effect of various concentrations of AFBI on respiratory rates of rat liver mitochondria. Detaifs of the reaction system are as described in the text. Rates of respiration in presence of AFBl at concentrations (M): (A) 0 (30 yl DMSO); (B) 1 +lo-s;(C) 1 - 10-S; (D) 1 =lo-,‘and (E) 3 * lO+. Figures on traces denote respiratory rates in nanomdes oxygen per minute per mg protein. Fig. 3. Effect of AFGl on respiratory rates of rat liver mitochondria. AFGI in 30 pl DMSO was added to the reaction vessel as de&bed in the text. Rates of respiration in presence of AFG at concentrations (Mj: (A) 0 (30 cd DMSO); (18)1 - 10-S; (C) 1 * 10-h; (D) 1 -IO-'and (El 3 - 10-5.
TABLE 111 EWECT OFAFLATDXIN OFRATUVER
G1 ON RESPIRATORY RATES,
ADP : 0
RATIO AND RESPIRATORY CONTROL RAT0
WTOCMONDRIA
AFCh, at concentrations indicated, was added in a volume of 30 pl DMSO to 3 ml of the reaction medium de&bed in the text. Resgiratory rates are expressed as nmoles oxygen per minute per mg protein. All values are Mean f S.E.M. of six separateexperiments.
AFG
State 3
State 4
ADP:O
RCR
Dominant mechanism
96 f 1.3
30 5 0.5 30 f 0.5 30 i 0.5 -
1.93 _I 0.02 1.93 _t 0.02 1.93 f 0.02 -
3.2 3.2 3.2 -
-
cwfuwtm~n (W 0 (DMSO) 1’ 10-a I - IO-5 I ’ 10-J 3. IO-”
%f 1.3 %f 1.3 46*o.s 48 f 0.5
inhibition and uncoupling Inhibition and uncoupling
Table II1 shows the effect of AFGl at various concentrations on oxidation and phosphorylazion. In contrast to AFMl or AFBl, AFGl does not affect the normal functioning of mitochondria at concentrations of 1 - 10-G M or 1 * 10-S M. However, at 1 10-a M or 3 IO-4 M concentration, AFGl also inhibits electron transport just like the other two toxins. Moreover, state 3 and state 4 respirations are indistinguishable from each other. It is evident therefore that AFGl can inhibit electron transport at the same concentration as AFMl or AFBl, but can uncouple oxidative phosphorylation, only at a relatively higher concentration. The effect of AFGl on the respiratory rates is shown in Fig. 3. l
l
Site of inhibition of aflutoxins AFBr has been shown to inhibit the electron transport chain between cytochrome b and c (cl) (ref. 2). ‘This study was extended to AFMl and AFGl also, to see whether ull these toxins act at the same or different sites of the electron transport chain. The inhibition of ADP stimulated respiration of all these toxins was reversed by TMPD (Fig. 4). It is apparent therefore that AFMl and AFGl also inhibit site II of the
Fii 4. Reversal of inhibition of aflatoxins by TMPD. Aflatoxin concentration: 3 - 1O-4M; ADP, 0.8 mM.
129
Fig. 5. Effect of preincubation of mitochondria with aflatoxins. Mitochondria preincubated with aflatoxins at concentrations of 3 * 1O-4Mat 30” for 10 min. ADP, 0.2 mM. Figures on traces denote respiratory rates @moles oxygen per minute per mg protein).
electron transport chain just like AFBI. In order to confirm these results, HQNO was used in place of aflatoxins. 40 yg HQNO per mg mitochondrial protein completely inhibited state 3 respiration and the inhibition was reversed by TMPD or DNP. E&ct of preincubation of mitochondria with aflatoxins The aflatoxins studied inhibited state 3 respiration at a concentration of 1 - 10-a M, whether the toxins were added prior to the addition of ADP or during ADP stimulated respiration. In presence of aflatoxins state 3 respiration gradually increased with time and could not be distinguished from state 4. Hence the effect of preincubation of mitochondria with aflatoxins was investigated and the results are presented in Fig. 5. Control experiments were carried out where mitochondria were preincubated without the aflatoxins. Stimulation of respiration by ADP was lower in mitochondria preincubated with aflatoxins. Without ADP the rate of respiration was much higher in aflatoxin-treated mitochondria than in control mitochondria. DISCUSSION
The results of the present study are in agreement with the in vitro inhibition of electron transport by AFBl reported by Doherty and Campbell2 and Strufaldi et a1.5. Doherty and Campbelle reported an inhibition of 25-44%, when 2.5-4.8 lo-4 A4 AFBl was added to actively respiring mitochondria. In our studies, state 3 respiration is inhibited by about 50 y0 and 75 “/oin presence of AFBr at concentrations of 1 - IO-4M and 3 * 10-d M, respectively. The higher rates of inhibition observed in our studies could be due to the lower concentration of ADP in the system (0.2 mM). When 0.8 mM ADP was added to stimulate respira,tion, AFBr at a concentration of 3 * 10e4 M inhibited respiration by 30% only (Fig. 4). It is apparent from these results that the inhibition caused by AFBl is at least partly reversible with ADP. However, there were differences in the degree of inhibition by several preparations of mitochondria, but the nature of inhibition by the toxins remained the same. The present study shows that aflatoxins have two distinct effects on mitochondrial function, as uncouplers at lower concentrations and as inhibitors at higher l
130 concentrations. AFMl and Bl are uncouplers at a concentration of 1 IO-6 M (Tables I and II). At concentration of I - 10-a M or higher AFMl, BI and G1 inhibit the electron transport. The uncoupling nature of AFBl has not been shown earlier, even though its inhibitory effect on respiration has been investigated in detail. AFGl differs from the other two, only in that it uncouples phosphorylation at a relatively higher concentration of 1 lO-%f. However, all these toxins inhibit the electron transport chain at site II, though the degree of inhibition varies. The inhibition decreases in the order BI, Ml and Gl. The effect of uncouplers on mitochondria could be due to either a gross change in the structure of mitcehondrial membrane or a binding at specific sites responsible for oxidative phosphorylation. The latter mechanism seems more probable in the case of aflatoxins (evidence for binding of aflatoxins with mitochondria is presented elsewhere). It is pertinent to add that even though in the typical experiments depicted in Tables I and II the inhibition by AFMl is lower than that by AFBl, in several preparations of mitochondria, these toxins inhibited state 3 respiration to the same extent. But with all the preparations, at a concentration of 1 * 10V6IkZ,AFMl but not Bl, lowered the ADP : 0 ratio. Hence it is reasonable to assume that AFMl (4hydroxy AFBl) is a more efficient inhibitor of electron transport in rat liver mitochondria. This isina ment with the results of our studies (details of which are not presented here) as part of the structure activity relationship of naturally occurring coumarins, where the hydroxycoumarins exert profound effects on mitochondrlal function. l
l
This investigation was supported in part by PL-480 grant No. FG-In-438.
d G. N. Wogan, Toxicity and bmchemical and fine structural effects of synthetic nd 111in rat liver, J. h’nrl. Cancer fnsr., 47 (1971) 585-592. y and T. C. Campbell, Aflatoxin inhibition of rat liver mitochondria, Chem.-Biol.
H. 1. Grady and J. Higginson, Aflatoxin BI injury in rat and monkey liver, Am. J.
J. I. Clifford and K. R. Rees, The action of allatoxin BI on the rat liver, Biochem. J., 102 (1967) 65-75. II. Strufaldi, D. M. Noyucira and F. I. Pedroso, Effect of aflatoxin Br on metabolic activity of t tic mitochondrial fraction from Rattu.9 Norwegicus (rat), Rev. Farm. Bioquim. Univ. Sao Pado, 8 (1970) l-23. 6 N. D. Davis, U. L. Diener and D. W. Eldridge, Production of aflatoxins BI and Gr by Aspergillus fklvus in a semi*ynthetic mecium, Appl. Mi~*ro6iol., I4 (1966) 378-380. n, Rapid method for the isolation of pure afiatoxim, J. Assoc. Offic. And. Chem., 53 19-621, D. Stuhbkfield, G. M. Shannon and 0. L. Shotwqll, Aflatoxins MI and Mz: preparation and aurification, /. Am. Oil Chem. Sot., 47 (1970) 389-590. 9
JohnsOn and H. Lardy, Isolation of liver or kidney mitochondria, in R. W. Estabrook and E. Pulhan U2ds.h Methods in Enzymology, Vol. X, Academic Press, New York, 1967, pp.
101.
131 10 R. W. Estabrook, Mitochondrial respiratory control and the polarographic measurement of ADP : 0 ratios, in R. W. Estabrook and M. E. Pullman (Eds.), Methods in Enzymology, Vol. X, Academic Press, New York, 1967, pp. 41-47. 11 0. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, Protein measurcxent with the Folin phenol reagent, J. Biof. Chem., 193 (1951) 265-275. 12 A. L. Lehninger, The Mitochondrion, Renjamin, New York, 1964, pp. 135-188.
