- Email: [email protected]
Action of rubratoxin B on mouse liver mitochondria
Toxicology, 6 (1976) 253--261 © Elsevier/North-Holland, Amsterdam -- Printed in The Netherlands
ACTION OF R U B R A T O X I N B ON MOUSE LIVER MITOCHONDRIA
A. WALLACE HAYES
Department of Pharmacology and Toxicology, The University of Mississippi Medical Center, 2500 North State Street, Jackson, Miss. 39216 (U.S.A.) (Received February 17th, 1976) (Revision received March 30th, 1976) (Accepted April 14th, 1976)
SUMMARY
The effect of rubratoxin B on the electron transport system of mouse liver mitochondria was investigated. Oxygen consumption in mitochondria isolated from male mice was measured polarographically in a Gilson Oxygraph Model K-ICC. Oxygen consumption was depressed 73% at a concentration of 1.13 mM rubratoxin. At concentrations as low as 0.08 mM rubratoxin B, 50% inhibition was observed. Rubratoxin B (0.28 mM) depressed oxygen consumption 67% in ADP-coupled mitochondria and 60% in 2,4-dinitrophenol (DNP)-uncoupled mitochondria using either pyridine-nucleotide linked substrates or succinate. By employing the N,N,N',N"-tetramethyl-pphenylenediamine (TMPD) shunt, it was shown that inhibition was n o t between c y t o c h r o m e b and c y t o c h r o m e C1 or C. It appears, based on these studies and comparison with known inhibitors of specific sites along the electron transport system, that the principal site of action of rubratoxin B is between cytochrome C1 or C and the termination of electron flow.
INTRODUCTION
The toxic nature of certain fungal metabolites is well d o c u m e n t e d [2,15, 17]. Unfortunately, the broad implication of the m y c o t o x i n problem was not recognized until the early 1940s when, in Russia, humans eating m o l d y over-wintered grain suffered severe dermal necroses, hemorrhages, leukopenia and bone marrow destruction; mortality was as high as 60% [8]. BiologiAbbreviations: DMSO, dimethylsulfoxide; DNP, 2,4-dinitrophenol; Md, menadione; TMPD, N,N,N',N"-tetramethyl-p-phenylenediamine.
253
cal effects of mycotoxins are varied and, because of diagnostic difficulties only the more acute and dramatic manifestations generally are reported. Many compounds including mycotoxins have been reported to induce effects on mitochondria. Dicoumarol stimulated respiration in rat liver mitochondria [5] and also caused respiratory inhibition by direct inhibition of succinate dehydrogenase and fl-hydroxybutyrate dehydrogenase [10]. Aflatoxin B1 and ochratoxin A, two mycotoxins, inhibited respiration in rat liver mitochondria. Doherty and Campbell [3] reported that 0.25 mM aflatoxin BI depressed oxygen consumption 40% in ADP-coupled mitochondria and 60% in DNP-uncoupled mitochondria. The site of aflatoxin action in rat liver mitochondria in vitro was postulated to be between cytochrome b and cytochrome C1 or C. Moore and Truelove [14] reported complete inhibition of coupled respiration with 0.4 mM ochratoxin. However, Meisner and Chan [13], stated that ochratoxin A acted as a competitive type inhibitor of mitochondrial transport carrier proteins and not on energy-conserving reactions. We previously reported that 0.33 mM rubratoxin B, another mycotoxin, inhibited oxygen uptake of succinate and citrate in mouse liver homogenates [7]. Current data confirm this inhibition by rubratoxin of oxygen consumption of mouse liver mitochondria preparations and indicate that the site of action was probably between c y t o c h r o m e C~ or C and the termination of electron flow (mitochondrial site III respiration). MATERIALS AND METHODS Liver mitochondria from 25--30 g Swiss-Webster male mice (Charles River Mouse Farms) were prepared by the m e t h o d of Johnson and Lardy [9]. Livers were removed, perfused with cold 0.25 M sucrose, weighed, minced and finally homogenized in 10 ml of cold 0.25 M sucrose. All subsequent procedures were at 0--4°C. Homogenates were diluted to 40 ml with cold 0.25 M sucrose, filtered through glass wool and then spun 2 times at 600 g for 10 rain with the supernatant fluid filtered each time. The supernatant fluid was decanted and centrifuged at 15 000 g for 10 min. Care was used to resuspend the buff-colored mitochondrial pellet in cold 0.25 M sucrose while eliminating the bottom-layered erythrocytes and grayish nuclear material. Mitochondria were washed once and resuspended in the oxygen electrode solution described below. This procedure routinely yields 8--10 mg mitochondrial protein per g liver. Respiratory control ratios (oxygen consumption measured in the presence and absence of ADP) between 4.5 and 6 were consistently obtained when succinate was used as substrate. Thus mitochondrial fraction was used immediately after isolation and the respiratory control ratio was obtained before and after each experiment to insure mitochondrial integrity [4]. Respiration rates and respiratory control ratios were measured polarographically in a Gilson Oxygraph Model K-ICC employing a vibrating platinum cathode housed in a 3.0-ml glass reaction vessel which was enclosed in a water jacket maintained at 30°C [4]. The oxygen electrode was calibrated
254
according to methods described by the manufacturer in which the appropriate solution was adequately aerated at the experimental temperature and the calibration for oxygen content was based on standard oxygen solubilities. Oxygen consumption per mg mitochondrial protein for control preparations obtained on different days varied no more than 5%. The final volume of the oxygen electrode solution was 3.0 ml and contained 1.5-ml basal reaction medium (0.25 M sucrose, 0.02 M KC1, 0.003 M potassium phosphate, pH 7.4, 0.05 M Tris buffer, pH 7.4 and 0.03 M MgC12). Mitochondria suspended in 0.25 M sucrose were added in a volume of 0.1 ml containing approximately 1 mg protein. Mitochondrial protein was estimated after each experiment [12]. Rubratoxin B was isolated from Penicillium rubrum [6]. Purity was established by melting point, infrared and mass spectra and thin-layer chromatography. After crystallization, the m y c o t o x i n was stored in the dark. Concentrations of rubratoxin, dissolved in a maximum of 25 #l DMSO which previously had been shown to have no effect, tested ranged from 0.028 to 1.1 mM. Each concentration was run on triplicate samples from mitochondrial preparations obtained from 6 separate animals. The effects of roteone, Md, antimycin A, aflatoxin B,, sodium cyanide, sodium azide, all known inhibitors of mitochondrial respiration, also were compared to polarographic traces of state 3 respiration in the presence of rubratoxin. ADP:O ratios were measured polarographically by the m e t h o d of Estabrook [4]. Rubratoxin was added in a maximum of 25 pl of DMSO which alone had no effect on the ADP :O ratio. Approx. 2 mg of mitochondrial pr Otein was routinely employed in this study. For administration to animals, rubratoxin was dissolved in propylene glycol, and dosed i.p. at the rate of 0.017, 0.167 amd 1.67 mg/kg. Animals were killed by decapitation 3 h after receiving the toxin at which time the liver was removed and weighed. The isolation of mitochondria and respiration measurements were as in the in vitro study. RESULTS Rubratoxin (0.083 mM) caused a 60% reduction in the rate of oxygen consumption by state 3 mitochondria when succinate was the substrate (Fig. la). A dose-related inhibition which rose rapidly and then leveled off was observed (Fig. 2). The data are expressed as percent inhibition in the presence of approx. 1.0 mg of mitochondrial protein during state 3 respiration. Similar inhibition of the rate of oxygen consumption was observed during the metabolism of glutamate (data n o t presented). Md, which is known to overcome inhibition caused by rotenone (a site I inhibitor), failed to overcome this inhibition (Fig. la). Inhibition also occurred in the presence of DNP using glutamate as substrate (Fig. lb). The average inhibition of rate of oxygen uptake with 0.28 mM rubratoxin was 47%. Inhibition of oxygen uptake of ADP~oupled mitochondria averaged 58% in the presence of 0.28 mM rubratoxin B (Fig. lc).
255
"•70C ~ 60C
700 t.
~
Mit
50C
f_M*t
600
£5cc
.~40C
c .~400
Rub
=e ~oo
~ 200
~39
~I00
~r22 \
0
J
2,
I
4
m inu '
Time
60G
i
r,me
m
nu'es
)
~. M i t
c 500 ~ub
40C E300
o200 c ~10£
f
2
Time
3
4
(minutes)
Fig. 1. Rubratoxin inhibition of whole mitochondria preparations with variously mediated electron transport systems. Rubratoxin B added in 25 #l DMSO to a final concentration of 0.083 mM (plate A) or 0.28 mM (plates B and C). Other conditions presented in the text; representative tracings were repeated at least 10 times each. Final concentrations of added compounds were (A) succinate, 5.0 raM, ADP, 1.0 mM; (B) glutamate, 5.0 mM, DNP, 37.5 pm; (C) glutamate, 5.0 mM, ADP, 1.0 mM.
IO0
8O
60
r j .~
zo
i
Rubrat
~
i
J×ln
,
i
b
i
i
,
,
!,u~/cen~ra*lon
,
J
,
(mM)
Fig. 2. I n h i b i t i o n o f state 3 m i t o c h o n d r i a l o x y g e n c o n s u m p t i o n as a f u n c t i o n o f rubrat o x i n B c o n c e n t r a t i o n . S u c c i n a t e ( 3 . 3 raM) w a s u s e d as substrate to m e a s u r e the inhibition o f o x y g e n c o n s u m p t i o n b y r u b r a t o x i n B in the p r e s e n c e o f A D P ( 1 . 0 m M ) and 3 . 0 ml of t h e o x y g e n e l e c t r o d e s o l u t i o n d e s c r i b e d in the t e x t . R u b r a t o x i n B w a s a d d e d in a v o l u m e o f 25 pl of D M S O , w h i c h a l o n e was s h o w n to have n o effect.
256
Similar patterns of inhibition were observed using succinate (data not presented). Because of similarity in degree to which succinate and NAD~lependent substrates were inhibited, inhibition of mitochondrial oxygen consumption by rubratoxin B was postulated to involve the electron transport chain. Results of experiments designed to test this postulation and to ascertain the location of the inhibition follow. Data showing the minimal inhibition of rubratoxin B on the oxidation of succinate and the failure of 0.2 M TMPD to overcome this inhibition are in Fig. 3a. Inhibition in control systems using either antimycin A or aflatoxin B~, known site II inhibitors, was removed by 0.2 M TMPD (Fig. 4). Since this concentration of TMPD is generally effective in overcoming inhibition between cyt b and cyt C~ or C, it was felt that rubratoxin B did not have an effect similar to either of these inhibitors. Since glutamate is metabolized to succinate within the mitochondria and cannot be strictly said to be an NADdependent substrate, 13-hydroxybutyrate, which is metabolized to acetoace-
"~700
700
~ 600
600 500
M~.tRu b
4_M i t
~500
Rub
_o 4 0 0
._o,~oo
~00
~_
~PD
300
o
u 200
o 200
~100
o
I00
0 t Time
2 3 ( mt n,.~te~ )
4
?
T ~e
~
a
r ~*e ~
700 ~630
~500 ~AD °
24J0 DC~ o 200
tOO o I
2
:
4
Time ( rinutes
5
6
7
8
)
Fig. 3. Rubratoxin B inhibition of oxygen consumption by mitochondria preparations modified by various treatments. Rubratoxin B (0.28 raM) added in 25 pl DMSO to 3.0 ml of the oxygen electrode solution described in the text. Other conditions presented in the text; representative tracings were repeated at least 10 times each. Final concentrations of added components were (A) succinate, 5.0 raM, ADP, 1.0 mM, TMPD, 0.2 mM; (B) J3hydroxybutyrate, 10 raM, TMPD, 0.2 raM, and (C) ascorbate, 1.5 raM, TMPD, 1.0 raM.
257
60( o
~E 50(
.
MIT
¢
c 0
40C
30C
"
AB1
°t
E =
¢
20C
c
100
.
TMPD
> x
O
1
2
Time
3
4
( m i n u t e s )
Fig. 4. Reversal by 0.2 mM TMPD of aflatoxin B1 inhibition of the rate o f o x y g e n u p t a k e by m i t o c h o n d r i a l preparations. S u c c i n a t e (5.0 mM) was the substrate and 3.0 ml of the o x y g e n electrode s o l u t i o n was as described in the text. Representative tracing was rep e a t e d 6 times. A f l a t o x i n B1 (4.8 mM) was added in a v o l u m e o f 25 #1 of DMSO, w h i c h alone had no effect.
tate but no further by liver mitochondria, was used as an NAD-dependent model compound to test for the inhibitory effect of rubratoxin between the substrate and cyt b. These results are presented as a minimal effect in Fig. 3b. Rubratoxin (0.28 mM) inhibited the rate of oxygen uptake 75% and again the inhibition was not overcome by TMPD. TMPD is known to be reduced by cytochrome b and oxidized by cytochrome C (or C1), thereby bridging the second crossover point (11). As a result of these data, respiration sites I and II were eliminated as locations for rubratoxin inhibition. Therefore, only site III remained as a possibility. The procedure of Sanadi and Jacobs (16) was employed to test for the effect of rubratoxin B between cyt C, or C and terminal 02. In this system, coupling site III is the only active coupling site and remains operative via electrons supplied by TMPD, which is kept in the reduced state with exogenous ascorbate. Rotenone, an inhibitor of NAD-dependent substrates, was used to suppress the oxidation of endogenous substrates. Site III was inhibited 38% by 0.28 mM rubratoxin B thus indicating site III as a possible location for the inhibition of the electron flow by rubratoxin (Fig. 3c). In addition, the effect of sodium azide and potassium cyanide, known site III inhibitors, gave polarographic tracings similar to rubratoxin B using succinate as the substrate. A reduction in the ADP :O ratio by approximately one also was observed in the presence of rubratoxin further supporting the hypothesis
258
TABLE I EFFECT OR RUBRATOXIN B ON THE ADP : 0 RATIO OF HEPATIC MITOCHONDRIA FROM UNTREATED MALE MICE Rubratoxin B (mM)
Control Solvent b 0.029 0.138 0.55 1.10 RCR d a b c d
M i t o c h o n d r i a l activities a ~-Hydroxybutyrate
Succinate
Ascorbate + TMPD
2.36 2.39 2.31 2.26 1.41 1.22 8.9
1.57 1.51 1.49 1.53 1.58 0.75 5.3
1.03 0.98 0.98 0.42 0.08 0.14 2.1
± 0.12 ± 0.10 ± 0.05 ± 0.08 +- 0 . 0 9 c ± 0.07 c
± 0.10 ± 0.12 ± 0.09 +- 0 . 0 4 ± 0.07 +- 0 . 0 5 c
± ± ± ± ± ±
0.04 0.05 0.06 0.01 c 0.06 c 0.03 c
M e a n ± S E o f six m i c e p e r t r e a t m e n t . D i m e t h y l s u l f o x i d e , 25 pl. p < 0 . 0 1 ; S t u d e n t ' s t-test. Respiratory control rate.
that rubratoxin B inhibits electron transport at the third coupling site (Table I). This reduction of one in the ADP:O ratio was observed regardless of the entry point into the electron transport chain. Results of in vivo studies showed a similar dose-related inhibition of mitochondrial oxygen consumption by rubratoxin B with succinate as the substrate. A 56% reduction was observed in the rate of oxygen consumption by state 3 mitochondria which had been isolated from mice treated, i.p., 3 h earlier with 1.67 mg/kg rubratoxin B. DISCUSSION
At least two recent papers have appeared in the literature stating that rubratoxin :B is capable of inhibiting oxygen consumption. Neither, however, reports a specific site of inhibition although Bernard and Dumas [1] speculated that this m y c o t o x i n affects one of the dehydrogenases or inhibits in the terminal region of the cytochrome chain. The possibility that one of the dehydrogenases such as succinic dehydrogenase is the specific site can n o t y e t be eliminated because, in our experience, TMPD does overcome the inhibition of succinate oxidation. However, the effect of rubratoxin on the oxidation of a number of NAD-dependent substrates of the tricarboxylic acid cycle by mouse liver mitochondria has been examined and no additional sites of inhibition have been observed. Concentrations of rubratoxin B near the m a x i m u m inhibition level induced no inhition at any point in the electron transport chain other than observed in the terminal region of the cytochrome chain. Furthermore, a reduction of aporoximately one in the ADP:O ratio indi-
259
cated that rubratoxin does affect phosphoration at the third site and n o t at the first or second site. This supports the contention that there is more than one ADP:O phosphoration site in the electron transport chain of mouse liver mitochondria because the third site was different from the other two sites since it is the only one affected even at the highest toxin concentration. Until respiration rate studies have been carried out with submitochondrial particles and it is determined whether rubratoxin B has an affect on these particles, it will be impossible to rule out the concept of a competitive type of inhibitory pattern with ~espect to mitochondrial transport carriers located in the membrane. Neither:lhas direct evidence been presented here concerning whether the mitochondrion is the physiological site of action of rubratoxin B. However, these findings suggest that rubratoxin may function outside the cell nucleus and the likelihood of interaction with mitochondria being related to its necrogenic property is a possible consequence. The data of Hayes and Hannah [7] have shown that oxygen consumption of liver homogenates could be inhibited with similar concentrations of rubratoxin B which would suggest that liver cell necrosis, as part of the hepatotoxic effect of rubratoxin, m a y be due in part to inhibition of cell respiration. The mechanism of this inhibition would seem to be localized in the electron transport chain between c y t o c h r o m e C (C1) and the terminal oxygen shown here. Moreover, the concentration of rubratoxin B required to elicit this response is within the concentrations t h a t might be expected from dosages promoting acute hepatotoxicity and liver cell necrosis. ACKNOWLEDGEMENTS
Support was provided by U.S. Public Health Grants ES01351 and ES01352 from NIEHS. REFERENCES 1 2 3 4
5 6 7 8
9
10 11
C. Bernard and P. Dumas, Mycopathologia, 55 (1975) 53. A. Ciegler, Lloydia, 38 (1975) 21. W.P. Doherty and T.C. Campbell, Chem.-Biol. Interact., 7 (1973) 63. R.W. Estabrook, Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios, in R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. 10, Academic Press, New York, 1967, p. 41. A.L. Greenbaum, J.B. Clark and P. Melean, Biochem. J., 96 (1965) 507. A.W. Hayes and B.J. Wilson, Appl. Microbioi., 16 (1968) 1163. A.W. Hayes and C.J. Hannah, Toxicol. Appl. Pharmacol., 25 (1973) 30. A. Joffe, Alimentary toxic aleukia, in S. Kadis, A. Ciegler and S.J. Ajl (Eds.), Microbial Toxins, Vol. 7, Algae and Fungal Toxins, Academic Press, New York, 1971, p. 139. D. Johnson and H. Lardy, Isolation of liver or kidney mitochondria, in R.W. Estabrook and M.E. Pullman (Eds.) Methods in Enzymology, Vol. 10, Academic Press, New York, 1967, p. 94. P. Jurtshuk, L. Schuzu and D.H. Green, J. Biol. Chem., 238 (1963) 3595. C.P. Lee, K. Nordenbrand and L. Ernster, Electron transport and oxidative phosphorylation in cytochrome b-c region of the respiratory chain as studied with the
260
12 13 14 15 16
17
tetramethylphenylenediamine shunt over the antimycin A sensitive site, in Proc. Intern. Symp. on Oxidases and Related Redox Systems, Vol. II (1964), Amherst, Mass., Witey, New York, 1965, p. 960. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. H. Meisner and S. Chan, Biochemistry, 13 (1974) 2795. J.H. Moore and B. Truelove, Science, 168 {1970) 1102. P.M. Newberne, Clin. Toxicol., 7 (1974) 161. D.R. Sanadi and E.E. Jacobs, Assay of oxidative phosphorylation at the cytochrome oxidase region (site III), in R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. 10, Academic Press, New York, 1967, p. 38. B.J. Wilson and A.W. Hayes, Microbial toxins, in F.M. Strong (Ed.), Toxicants Occurring Naturally in Foods, National Academy of Science, Washington, D.C., 1973.
261
ACTION OF R U B R A T O X I N B ON MOUSE LIVER MITOCHONDRIA
A. WALLACE HAYES
Department of Pharmacology and Toxicology, The University of Mississippi Medical Center, 2500 North State Street, Jackson, Miss. 39216 (U.S.A.) (Received February 17th, 1976) (Revision received March 30th, 1976) (Accepted April 14th, 1976)
SUMMARY
The effect of rubratoxin B on the electron transport system of mouse liver mitochondria was investigated. Oxygen consumption in mitochondria isolated from male mice was measured polarographically in a Gilson Oxygraph Model K-ICC. Oxygen consumption was depressed 73% at a concentration of 1.13 mM rubratoxin. At concentrations as low as 0.08 mM rubratoxin B, 50% inhibition was observed. Rubratoxin B (0.28 mM) depressed oxygen consumption 67% in ADP-coupled mitochondria and 60% in 2,4-dinitrophenol (DNP)-uncoupled mitochondria using either pyridine-nucleotide linked substrates or succinate. By employing the N,N,N',N"-tetramethyl-pphenylenediamine (TMPD) shunt, it was shown that inhibition was n o t between c y t o c h r o m e b and c y t o c h r o m e C1 or C. It appears, based on these studies and comparison with known inhibitors of specific sites along the electron transport system, that the principal site of action of rubratoxin B is between cytochrome C1 or C and the termination of electron flow.
INTRODUCTION
The toxic nature of certain fungal metabolites is well d o c u m e n t e d [2,15, 17]. Unfortunately, the broad implication of the m y c o t o x i n problem was not recognized until the early 1940s when, in Russia, humans eating m o l d y over-wintered grain suffered severe dermal necroses, hemorrhages, leukopenia and bone marrow destruction; mortality was as high as 60% [8]. BiologiAbbreviations: DMSO, dimethylsulfoxide; DNP, 2,4-dinitrophenol; Md, menadione; TMPD, N,N,N',N"-tetramethyl-p-phenylenediamine.
253
cal effects of mycotoxins are varied and, because of diagnostic difficulties only the more acute and dramatic manifestations generally are reported. Many compounds including mycotoxins have been reported to induce effects on mitochondria. Dicoumarol stimulated respiration in rat liver mitochondria [5] and also caused respiratory inhibition by direct inhibition of succinate dehydrogenase and fl-hydroxybutyrate dehydrogenase [10]. Aflatoxin B1 and ochratoxin A, two mycotoxins, inhibited respiration in rat liver mitochondria. Doherty and Campbell [3] reported that 0.25 mM aflatoxin BI depressed oxygen consumption 40% in ADP-coupled mitochondria and 60% in DNP-uncoupled mitochondria. The site of aflatoxin action in rat liver mitochondria in vitro was postulated to be between cytochrome b and cytochrome C1 or C. Moore and Truelove [14] reported complete inhibition of coupled respiration with 0.4 mM ochratoxin. However, Meisner and Chan [13], stated that ochratoxin A acted as a competitive type inhibitor of mitochondrial transport carrier proteins and not on energy-conserving reactions. We previously reported that 0.33 mM rubratoxin B, another mycotoxin, inhibited oxygen uptake of succinate and citrate in mouse liver homogenates [7]. Current data confirm this inhibition by rubratoxin of oxygen consumption of mouse liver mitochondria preparations and indicate that the site of action was probably between c y t o c h r o m e C~ or C and the termination of electron flow (mitochondrial site III respiration). MATERIALS AND METHODS Liver mitochondria from 25--30 g Swiss-Webster male mice (Charles River Mouse Farms) were prepared by the m e t h o d of Johnson and Lardy [9]. Livers were removed, perfused with cold 0.25 M sucrose, weighed, minced and finally homogenized in 10 ml of cold 0.25 M sucrose. All subsequent procedures were at 0--4°C. Homogenates were diluted to 40 ml with cold 0.25 M sucrose, filtered through glass wool and then spun 2 times at 600 g for 10 rain with the supernatant fluid filtered each time. The supernatant fluid was decanted and centrifuged at 15 000 g for 10 min. Care was used to resuspend the buff-colored mitochondrial pellet in cold 0.25 M sucrose while eliminating the bottom-layered erythrocytes and grayish nuclear material. Mitochondria were washed once and resuspended in the oxygen electrode solution described below. This procedure routinely yields 8--10 mg mitochondrial protein per g liver. Respiratory control ratios (oxygen consumption measured in the presence and absence of ADP) between 4.5 and 6 were consistently obtained when succinate was used as substrate. Thus mitochondrial fraction was used immediately after isolation and the respiratory control ratio was obtained before and after each experiment to insure mitochondrial integrity [4]. Respiration rates and respiratory control ratios were measured polarographically in a Gilson Oxygraph Model K-ICC employing a vibrating platinum cathode housed in a 3.0-ml glass reaction vessel which was enclosed in a water jacket maintained at 30°C [4]. The oxygen electrode was calibrated
254
according to methods described by the manufacturer in which the appropriate solution was adequately aerated at the experimental temperature and the calibration for oxygen content was based on standard oxygen solubilities. Oxygen consumption per mg mitochondrial protein for control preparations obtained on different days varied no more than 5%. The final volume of the oxygen electrode solution was 3.0 ml and contained 1.5-ml basal reaction medium (0.25 M sucrose, 0.02 M KC1, 0.003 M potassium phosphate, pH 7.4, 0.05 M Tris buffer, pH 7.4 and 0.03 M MgC12). Mitochondria suspended in 0.25 M sucrose were added in a volume of 0.1 ml containing approximately 1 mg protein. Mitochondrial protein was estimated after each experiment [12]. Rubratoxin B was isolated from Penicillium rubrum [6]. Purity was established by melting point, infrared and mass spectra and thin-layer chromatography. After crystallization, the m y c o t o x i n was stored in the dark. Concentrations of rubratoxin, dissolved in a maximum of 25 #l DMSO which previously had been shown to have no effect, tested ranged from 0.028 to 1.1 mM. Each concentration was run on triplicate samples from mitochondrial preparations obtained from 6 separate animals. The effects of roteone, Md, antimycin A, aflatoxin B,, sodium cyanide, sodium azide, all known inhibitors of mitochondrial respiration, also were compared to polarographic traces of state 3 respiration in the presence of rubratoxin. ADP:O ratios were measured polarographically by the m e t h o d of Estabrook [4]. Rubratoxin was added in a maximum of 25 pl of DMSO which alone had no effect on the ADP :O ratio. Approx. 2 mg of mitochondrial pr Otein was routinely employed in this study. For administration to animals, rubratoxin was dissolved in propylene glycol, and dosed i.p. at the rate of 0.017, 0.167 amd 1.67 mg/kg. Animals were killed by decapitation 3 h after receiving the toxin at which time the liver was removed and weighed. The isolation of mitochondria and respiration measurements were as in the in vitro study. RESULTS Rubratoxin (0.083 mM) caused a 60% reduction in the rate of oxygen consumption by state 3 mitochondria when succinate was the substrate (Fig. la). A dose-related inhibition which rose rapidly and then leveled off was observed (Fig. 2). The data are expressed as percent inhibition in the presence of approx. 1.0 mg of mitochondrial protein during state 3 respiration. Similar inhibition of the rate of oxygen consumption was observed during the metabolism of glutamate (data n o t presented). Md, which is known to overcome inhibition caused by rotenone (a site I inhibitor), failed to overcome this inhibition (Fig. la). Inhibition also occurred in the presence of DNP using glutamate as substrate (Fig. lb). The average inhibition of rate of oxygen uptake with 0.28 mM rubratoxin was 47%. Inhibition of oxygen uptake of ADP~oupled mitochondria averaged 58% in the presence of 0.28 mM rubratoxin B (Fig. lc).
255
"•70C ~ 60C
700 t.
~
Mit
50C
f_M*t
600
£5cc
.~40C
c .~400
Rub
=e ~oo
~ 200
~39
~I00
~r22 \
0
J
2,
I
4
m inu '
Time
60G
i
r,me
m
nu'es
)
~. M i t
c 500 ~ub
40C E300
o200 c ~10£
f
2
Time
3
4
(minutes)
Fig. 1. Rubratoxin inhibition of whole mitochondria preparations with variously mediated electron transport systems. Rubratoxin B added in 25 #l DMSO to a final concentration of 0.083 mM (plate A) or 0.28 mM (plates B and C). Other conditions presented in the text; representative tracings were repeated at least 10 times each. Final concentrations of added compounds were (A) succinate, 5.0 raM, ADP, 1.0 mM; (B) glutamate, 5.0 mM, DNP, 37.5 pm; (C) glutamate, 5.0 mM, ADP, 1.0 mM.
IO0
8O
60
r j .~
zo
i
Rubrat
~
i
J×ln
,
i
b
i
i
,
,
!,u~/cen~ra*lon
,
J
,
(mM)
Fig. 2. I n h i b i t i o n o f state 3 m i t o c h o n d r i a l o x y g e n c o n s u m p t i o n as a f u n c t i o n o f rubrat o x i n B c o n c e n t r a t i o n . S u c c i n a t e ( 3 . 3 raM) w a s u s e d as substrate to m e a s u r e the inhibition o f o x y g e n c o n s u m p t i o n b y r u b r a t o x i n B in the p r e s e n c e o f A D P ( 1 . 0 m M ) and 3 . 0 ml of t h e o x y g e n e l e c t r o d e s o l u t i o n d e s c r i b e d in the t e x t . R u b r a t o x i n B w a s a d d e d in a v o l u m e o f 25 pl of D M S O , w h i c h a l o n e was s h o w n to have n o effect.
256
Similar patterns of inhibition were observed using succinate (data not presented). Because of similarity in degree to which succinate and NAD~lependent substrates were inhibited, inhibition of mitochondrial oxygen consumption by rubratoxin B was postulated to involve the electron transport chain. Results of experiments designed to test this postulation and to ascertain the location of the inhibition follow. Data showing the minimal inhibition of rubratoxin B on the oxidation of succinate and the failure of 0.2 M TMPD to overcome this inhibition are in Fig. 3a. Inhibition in control systems using either antimycin A or aflatoxin B~, known site II inhibitors, was removed by 0.2 M TMPD (Fig. 4). Since this concentration of TMPD is generally effective in overcoming inhibition between cyt b and cyt C~ or C, it was felt that rubratoxin B did not have an effect similar to either of these inhibitors. Since glutamate is metabolized to succinate within the mitochondria and cannot be strictly said to be an NADdependent substrate, 13-hydroxybutyrate, which is metabolized to acetoace-
"~700
700
~ 600
600 500
M~.tRu b
4_M i t
~500
Rub
_o 4 0 0
._o,~oo
~00
~_
~PD
300
o
u 200
o 200
~100
o
I00
0 t Time
2 3 ( mt n,.~te~ )
4
?
T ~e
~
a
r ~*e ~
700 ~630
~500 ~AD °
24J0 DC~ o 200
tOO o I
2
:
4
Time ( rinutes
5
6
7
8
)
Fig. 3. Rubratoxin B inhibition of oxygen consumption by mitochondria preparations modified by various treatments. Rubratoxin B (0.28 raM) added in 25 pl DMSO to 3.0 ml of the oxygen electrode solution described in the text. Other conditions presented in the text; representative tracings were repeated at least 10 times each. Final concentrations of added components were (A) succinate, 5.0 raM, ADP, 1.0 mM, TMPD, 0.2 mM; (B) J3hydroxybutyrate, 10 raM, TMPD, 0.2 raM, and (C) ascorbate, 1.5 raM, TMPD, 1.0 raM.
257
60( o
~E 50(
.
MIT
¢
c 0
40C
30C
"
AB1
°t
E =
¢
20C
c
100
.
TMPD
> x
O
1
2
Time
3
4
( m i n u t e s )
Fig. 4. Reversal by 0.2 mM TMPD of aflatoxin B1 inhibition of the rate o f o x y g e n u p t a k e by m i t o c h o n d r i a l preparations. S u c c i n a t e (5.0 mM) was the substrate and 3.0 ml of the o x y g e n electrode s o l u t i o n was as described in the text. Representative tracing was rep e a t e d 6 times. A f l a t o x i n B1 (4.8 mM) was added in a v o l u m e o f 25 #1 of DMSO, w h i c h alone had no effect.
tate but no further by liver mitochondria, was used as an NAD-dependent model compound to test for the inhibitory effect of rubratoxin between the substrate and cyt b. These results are presented as a minimal effect in Fig. 3b. Rubratoxin (0.28 mM) inhibited the rate of oxygen uptake 75% and again the inhibition was not overcome by TMPD. TMPD is known to be reduced by cytochrome b and oxidized by cytochrome C (or C1), thereby bridging the second crossover point (11). As a result of these data, respiration sites I and II were eliminated as locations for rubratoxin inhibition. Therefore, only site III remained as a possibility. The procedure of Sanadi and Jacobs (16) was employed to test for the effect of rubratoxin B between cyt C, or C and terminal 02. In this system, coupling site III is the only active coupling site and remains operative via electrons supplied by TMPD, which is kept in the reduced state with exogenous ascorbate. Rotenone, an inhibitor of NAD-dependent substrates, was used to suppress the oxidation of endogenous substrates. Site III was inhibited 38% by 0.28 mM rubratoxin B thus indicating site III as a possible location for the inhibition of the electron flow by rubratoxin (Fig. 3c). In addition, the effect of sodium azide and potassium cyanide, known site III inhibitors, gave polarographic tracings similar to rubratoxin B using succinate as the substrate. A reduction in the ADP :O ratio by approximately one also was observed in the presence of rubratoxin further supporting the hypothesis
258
TABLE I EFFECT OR RUBRATOXIN B ON THE ADP : 0 RATIO OF HEPATIC MITOCHONDRIA FROM UNTREATED MALE MICE Rubratoxin B (mM)
Control Solvent b 0.029 0.138 0.55 1.10 RCR d a b c d
M i t o c h o n d r i a l activities a ~-Hydroxybutyrate
Succinate
Ascorbate + TMPD
2.36 2.39 2.31 2.26 1.41 1.22 8.9
1.57 1.51 1.49 1.53 1.58 0.75 5.3
1.03 0.98 0.98 0.42 0.08 0.14 2.1
± 0.12 ± 0.10 ± 0.05 ± 0.08 +- 0 . 0 9 c ± 0.07 c
± 0.10 ± 0.12 ± 0.09 +- 0 . 0 4 ± 0.07 +- 0 . 0 5 c
± ± ± ± ± ±
0.04 0.05 0.06 0.01 c 0.06 c 0.03 c
M e a n ± S E o f six m i c e p e r t r e a t m e n t . D i m e t h y l s u l f o x i d e , 25 pl. p < 0 . 0 1 ; S t u d e n t ' s t-test. Respiratory control rate.
that rubratoxin B inhibits electron transport at the third coupling site (Table I). This reduction of one in the ADP:O ratio was observed regardless of the entry point into the electron transport chain. Results of in vivo studies showed a similar dose-related inhibition of mitochondrial oxygen consumption by rubratoxin B with succinate as the substrate. A 56% reduction was observed in the rate of oxygen consumption by state 3 mitochondria which had been isolated from mice treated, i.p., 3 h earlier with 1.67 mg/kg rubratoxin B. DISCUSSION
At least two recent papers have appeared in the literature stating that rubratoxin :B is capable of inhibiting oxygen consumption. Neither, however, reports a specific site of inhibition although Bernard and Dumas [1] speculated that this m y c o t o x i n affects one of the dehydrogenases or inhibits in the terminal region of the cytochrome chain. The possibility that one of the dehydrogenases such as succinic dehydrogenase is the specific site can n o t y e t be eliminated because, in our experience, TMPD does overcome the inhibition of succinate oxidation. However, the effect of rubratoxin on the oxidation of a number of NAD-dependent substrates of the tricarboxylic acid cycle by mouse liver mitochondria has been examined and no additional sites of inhibition have been observed. Concentrations of rubratoxin B near the m a x i m u m inhibition level induced no inhition at any point in the electron transport chain other than observed in the terminal region of the cytochrome chain. Furthermore, a reduction of aporoximately one in the ADP:O ratio indi-
259
cated that rubratoxin does affect phosphoration at the third site and n o t at the first or second site. This supports the contention that there is more than one ADP:O phosphoration site in the electron transport chain of mouse liver mitochondria because the third site was different from the other two sites since it is the only one affected even at the highest toxin concentration. Until respiration rate studies have been carried out with submitochondrial particles and it is determined whether rubratoxin B has an affect on these particles, it will be impossible to rule out the concept of a competitive type of inhibitory pattern with ~espect to mitochondrial transport carriers located in the membrane. Neither:lhas direct evidence been presented here concerning whether the mitochondrion is the physiological site of action of rubratoxin B. However, these findings suggest that rubratoxin may function outside the cell nucleus and the likelihood of interaction with mitochondria being related to its necrogenic property is a possible consequence. The data of Hayes and Hannah [7] have shown that oxygen consumption of liver homogenates could be inhibited with similar concentrations of rubratoxin B which would suggest that liver cell necrosis, as part of the hepatotoxic effect of rubratoxin, m a y be due in part to inhibition of cell respiration. The mechanism of this inhibition would seem to be localized in the electron transport chain between c y t o c h r o m e C (C1) and the terminal oxygen shown here. Moreover, the concentration of rubratoxin B required to elicit this response is within the concentrations t h a t might be expected from dosages promoting acute hepatotoxicity and liver cell necrosis. ACKNOWLEDGEMENTS
Support was provided by U.S. Public Health Grants ES01351 and ES01352 from NIEHS. REFERENCES 1 2 3 4
5 6 7 8
9
10 11
C. Bernard and P. Dumas, Mycopathologia, 55 (1975) 53. A. Ciegler, Lloydia, 38 (1975) 21. W.P. Doherty and T.C. Campbell, Chem.-Biol. Interact., 7 (1973) 63. R.W. Estabrook, Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios, in R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. 10, Academic Press, New York, 1967, p. 41. A.L. Greenbaum, J.B. Clark and P. Melean, Biochem. J., 96 (1965) 507. A.W. Hayes and B.J. Wilson, Appl. Microbioi., 16 (1968) 1163. A.W. Hayes and C.J. Hannah, Toxicol. Appl. Pharmacol., 25 (1973) 30. A. Joffe, Alimentary toxic aleukia, in S. Kadis, A. Ciegler and S.J. Ajl (Eds.), Microbial Toxins, Vol. 7, Algae and Fungal Toxins, Academic Press, New York, 1971, p. 139. D. Johnson and H. Lardy, Isolation of liver or kidney mitochondria, in R.W. Estabrook and M.E. Pullman (Eds.) Methods in Enzymology, Vol. 10, Academic Press, New York, 1967, p. 94. P. Jurtshuk, L. Schuzu and D.H. Green, J. Biol. Chem., 238 (1963) 3595. C.P. Lee, K. Nordenbrand and L. Ernster, Electron transport and oxidative phosphorylation in cytochrome b-c region of the respiratory chain as studied with the
260
12 13 14 15 16
17
tetramethylphenylenediamine shunt over the antimycin A sensitive site, in Proc. Intern. Symp. on Oxidases and Related Redox Systems, Vol. II (1964), Amherst, Mass., Witey, New York, 1965, p. 960. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. H. Meisner and S. Chan, Biochemistry, 13 (1974) 2795. J.H. Moore and B. Truelove, Science, 168 {1970) 1102. P.M. Newberne, Clin. Toxicol., 7 (1974) 161. D.R. Sanadi and E.E. Jacobs, Assay of oxidative phosphorylation at the cytochrome oxidase region (site III), in R.W. Estabrook and M.E. Pullman (Eds.), Methods in Enzymology, Vol. 10, Academic Press, New York, 1967, p. 38. B.J. Wilson and A.W. Hayes, Microbial toxins, in F.M. Strong (Ed.), Toxicants Occurring Naturally in Foods, National Academy of Science, Washington, D.C., 1973.
261