A complex effect of arsenite on the formation of α-ketoglutarate in rat liver mitochondria

ARCHIVES

Vol.

OF BIOCHEMISTRY

283, No. 2, December,

AND

BIOPHYSICS

pp. 388-396,

1990

A Complex Effect of Arsenite on the Formation of wKetoglutarate in Rat Liver Mitochondria Ewa Lenartowicz Department

Received

June

of Cellular

21,1990,

Biochemistry,

and in revised

Nencki Institute

form

July

of Experimental

Biology, Pasteura

3, 02-093 Warsaw, Poland

25, 1990

This investigation presents disturbances of the mitochondrial metabolism by arsenite, a hydrophilic dithiol reagent known as an inhibitor of mitochondrial a-keto acid dehydrogenases. Arsenite at concentrations of O.l1.0 mM was shown to induce a considerable oxidation of intramitochondrial NADPH, NADH, and glutathione without decreasing the mitochondrial membrane potential. The oxidation of NAD(P)H required the presence of phosphate and was sensitive to ruthenium red, but occurred without the addition of calcium salts. Mitochondrial reactions producing a-ketoglutarate from glutamate and isocitrate were modulated by arsenite through various mechanisms: (i) both glutamate transaminations, with oxaloacetate and with pyruvate, were inhibited by accumulating a-ketoglutarate; however, at low concentrations of cY-ketoglutarate the aspartate aminotransferase reaction was stimulated due to the increase of NAD+ content; (ii) the oxidation of isocitrate was stimulated at its low concentration only, due to the oxidation of NADPH and NADH; this oxidation was prevented by concentrations of citrate or isocitrate greater than 1 mM; (iii) the conversion of isocitrate to citrate was suppressed, presumably as a result of the decrease of Mga+ concentration in mitochondria. Thus the depletion of mitochondrial vicinal thiol groups in hydrophilic domains disturbs the mitochondrial metabolism not only by the inhibition of a-keto acid dehydrogenases but also by the oxidation of NAD(P)H and, possibly, by the change in the ion concentrations. 0 ~ssoA~~~.~~~cP~~,I~c.

Trivalent arsenicals readily react with thiol groups, but they form stable mercaptides with dithiol compounds only (1). Arsenite, which is easily taken up by rat liver mitochondria in competition with phosphate, has often been applied as an inhibitor of mitochondrial pyruvate and (Yketoglutarate dehydrogenases (2). This inhibition is due

to the reaction of arsenite with the vicinal thiol groups of reduced lipoic acid (1,3). The same arsenite-sensitive component is also present in complexes of mitochondrial branched-chain keto acid dehydrogenase (4). In intact mitochondria arsenite reacts with thiol groups of numerous proteins and therefore its action is not limited to the inhibition of a-keto acid dehydrogenases. Arsenite was found to stimulate the respiration of mitochondria utilizing succinate (5) and glutamate plus malate (6). Similar to other thiol reagents, it also increases the permeability of mitochondrial membranes. This was exemplified by the acceleration of the depletion of matrix adenine nucleotides in the presence of phosphate (7) and by the induction of Ca2+ efllux from respiring mitochondria (8). The latter effect is associated with oxidation of intramitochondrial NADPH (9). However, the oxidation of intramitochondrial nicotinamide nucleotides in response to arsenite has until now been observed only in uncoupled mitochondria (10). Arsenite was also shown to suppress oxidation of fatty acids due to the inhibition of thiolase (11). In contrast to hydrophobic arsenicals, as phenylarsine oxide, hydrophilic inorganic arsenite does not uncouple oxidative phosphorylation (1, 12). The present investigation has shown that addition of 0.1-1.0 mM arsenite to energized mitochondria in the presence of phosphate causes the oxidation of intramitochondrial NADPH and NADH without decreasing mitochondrial membrane potential. This oxidation of NAD(P)H was associated with the uptake of Ca2+ and the oxidation of mitochondrial GSH. Various mechanisms of arsenite action in mitochondria were examined in the course of reactions producing a-ketoglutarate from glutamate and isocitrate, since these reactions could be modulated by both the redox state of mitochondrial NAD(P) and the accumulation of cY-ketoglutarate. MATERIAL Chemicals. fluorocitrate

AND METHODS Sodium arsenite was obtained from

388 All

was purchased from Calbiochem. Ruthenium

Copyright 0 1990 rights of reproduction

Merck. Barium red, mersalyl,

0003-9861/90 by Academic in any form

$3.00

Press, Inc. reserved.

EFFECT

OF

ARSENITE

NEM’ and BHT were from Sigma, while BCNU was from Bristol oratories. Enzymes and coenzymes used for enzymatic analyses from Boehringer. All other reagents were of analytical grade.

ON

MITOCHONDRIAL

Labwere

Isolation of mitochondriu. Mitochondria from livers of male Wistar rats were obtained according to the procedure of Schneider and Hogeboom (13), in a homogenization medium containing 225 mM mannitol, ‘75 mM sucrose, and 0.1 mM EGTA. All washings were done with the same solution without EGTA. The layer of fat was carefully removed after sedimentation of mitochondria. Protein was determined by the biuret method (14). Incubation of mitochondria. Incubation of mitochondria (3-5 mg protein/ml) was performed at 3O’C. The compositions of media are given in the legends to the figures and tables. Determination of NAD’, NADPC, and metabolites. The reaction was started by the addition of mitochondria and stopped by the addition of HCIOl to a final concentration of 4%. The extracts were neutralized with 0.5 M triethanolamine plus 5 M KOH and, after centrifugation, the nucleotides and metabolites were determined in the supernatants by enzymatic assays. NAD+, NADP+, a-ketoglutarate, aspartate, isocitrate, and citrate were determined according to Williamson and Corkey (15), alanine according to Williamson (16), and ammonia according to Kun and Kaerney (17). GSSG was measured with glutathione reductase and NADPH in a solution containing 50 mM triethanolamine, 10 mM MgC12, and 1 mM EDTA (pH 7.4). Determination of NADH and NADPH. The reaction was started by addition of mitochondria and stopped by mixing the samples with 0.5 vol of 1 M KOH in 90% ethanol. After 5 min at room temperature 0.1 vol of 0.5 M triethanolamine-HCl buffer (pH 8.5) was added and the pH was adjusted to 9.0 with 6% HCIO1. After short centrifugation NADH and NADPH were measured immediately in the supernatants according to Williamson and Corkey (15). Measurement of enzymatic activities in mitochondrial extract. The extraction procedure was as follows: 1 ml of freshly isolated mitochondria was mixed with 0.5 ml of rabbit serum and 0.75 ml of 0.33% deoxycholate. This solution was frozen in a solid carbon dioxide/acetone bath and thawed rapidly using running tap water. This freeze-thaw procedure was repeated two times and the suspension was centrifuged to remove mitochondrial membranes. All enzymatic activities in this extract were measured spectrophotometrically at 340 nm in the medium consisting of 100 mM KHzPOl and 5 mM MgCl, (pH 7.4). The activities of isocitrate dehydrogenases were estimated in the presence of 5 mM isocitrate and either 0.5 mM NAD+ or 0.5 mM NADP* while the activity of aconitase in the presence of 5 mM citrate and 0.5 mM NADP+. For determination of the activity of glutathione reductase incubated mitochondrial samples were treated with 15 ~1 Triton X-100 per milliliter and, after centrifugation, the oxidation of NADPH by supernatant was measured fluorometrically in the presence of GSSG according to Eklow et al. (18).

389

METABOLISM

phosphate caused an extensive oxidation of NADH and NADPH (Fig. 1). In this system the production of NADH occurs mainly in the reaction of malate dehydrogenase supplying oxaloacetate for the transamination with glutamate. It can be noticed that arsenite was unable to induce the oxidation of nicotinamide nucleotides unless phosphate was also present. Arsenite plus phosphate brought about a considerable depletion of both NADH and NADPH, which corresponded to the increment of NADf and NADP+. Only traces of nicotinamide nucleotides were found in supernatants after removal of mitochondria by short centrifugation from samples incubated without or with arsenite. The content of NAD+ and NADP+ in incubated mitochondria initially increased with increasing concentration of added arsenite; however, at concentrations of arsenite higher than 1 mM this increase was only transient (Fig. 2). Since the content of mitochondrial NADPH and NADH in response to arsenite addition was decreasing at all arsenite concentrations tested (not shown), it can be assumed that arsenite at concentrations higher than 1 mM brought about a hydrolysis of oxidized nicotinamide nucleotides in mitochondria similar to that shown for organelles treated with hydroperoxides (20).

I

.f 5 ho .E”

NADPH

NADH

NAD+

z E

Measurement of mitochondrial membrane potential. The distribution of TPP+ across the inner mitochondrial membrane was measured using a TPP+-selective electrode according to Kamo et al. (19). Mitochondria were incubated at 25’C in a medium containing 100 mM KCl, 40 mM Tris-HCl, 5 mM KH2POI, 10 mM glutamate, 10 mM malate, 10 pM TPP+, and various concentrations of NaAsOz (pH 7.25).

RESULTS

The addition of 0.5 mM arsenite to mitochondria incubated with glutamate plus malate in the presence of 1 Abbreviations used: BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; BHT, butylhydroxytoluene; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; FCCP, carbonyl cyanide p-trifiuoromethoxyphenylhydrazone; NEM, N-ethylmaleimide; TPP+, tetraphenylphosphonium cation.

0

5

10

Time

(min)

FIG. 1. Effect of arsenite and phosphate on the content of nicotinamide nucleotides in mitochondria. Mitochondria were incubated in medium containing 100 mM KCl, 40 mM Tris-HCl, 10 mM glutamate, 10 mM malate, and the following additions: none (Cl), 5 mM KH*PO, (0), 0.5 mM NaAsOz (A), and 5 mM KHPPOp plus 0.5 mM NaAsOZ (U), pH 7.25. Data of one from three similar experiments are presented.

390

EWA NAD+

NADP

LENARTOWICZ

oxidation was prevented by NEM and mersalyl, which are known to inhibit the uptake of both phosphate and arsenite into mitochondria (2). Both of these compounds react with thiol groups; however, mersalyl does not penetrate into mitochondria (21) while NEM was reported to promote the oxidation of mitochondrial NAD(P)H in the presence of added calcium salts only (22). Arseniteinduced NAD(P)H oxidation is also dependent on Ca2+ uptake by mitochondria since it was sensitive to ruthenium red and EGTA. However, very low concentrations of Cazf are sufficient for arsenite to induce NAD(P)H oxidation since it occurred without the addition of calcium salts. The addition of 0.1 mM CaClz increased the extent of NAD(P)H oxidation without arsenite while the oxidation promoted by arsenite increased only transiently, within two initial minutes of incubation (not shown). Thus, the addition of Ca2+ potentiates the ability of arsenite to promote the hydrolysis of nicotinamide nucleotides, similarly to what was observed for hydroperoxide (20). Another important factor involved in the arsenite-induced intramitochondrial NAD(P)H oxidation seems to be the release of Mp from mitochondria. This oxidation was suppressed by external MgC12 and blocked by citrate and isocitrate known to complex Mg2+ inside mitochon-

l

Time (min)

FIG. 2. The content of NAD+ and NADP+ in mitochondria incubated with various concentrations of arsenite. Mitochondria were incubated in medium containing 100 mM KCl, 40 mM Tris-HCl, 10 mM glutamate, 10 mM malate, 5 mM KHPPOI, either without NaAsO, (0) or with following concentrations of NaAsO,: 0.1 mM (A), 1.0 mM (V), 2.0 mM (O), and 5.0 mM (O), pH 7.25. Data of one from two similar experiments are presented.

Conditions protecting against intramitochondrial dation of NADPH and NADH induced by arsenite phosphate in mitochondria incubated in KCl-Tris dium are presented in Table I. As could be expected,

oxiplus methe

TABLE Effect

of Various

Compounds

on the Arsenite-Induced

I

Increase

of NADP+

and NAD+

Content

Content of NADP+ (nmol/mg protein) Additions bM) None Additional substrates Isocitrate (10) Citrate (10) Citrate (10) + Auorocitrate Inhibitors Mersalyl (0.02) NEM (0.1) Ruthenium red (0.01) BHT (0.1) BCNU (0.25)d FCCP (0.005) Inorganic salts Md% (10) CaCl* (0.1) Other ATP (2.5) EGTA (2.5)

Without

(0.1)

arsenite

in Mitochondria Content (nmol/mg

With

arsenite

Without

arsenite

of NAD+ protein) With

arsenite

0.33 + 0.17

1.86 + 0.44”

1.29 f 0.30

2.37 + 0.26”

0.27 + 0.05 0.20 f 0.08 0.20 f 0.10

0.56 + 0.26’ 0.37 + 0.06’ 0.36 zk 0.14”

1.25 + 0.32 1.28 + 0.10 1.27 + 0.12

1.56 + 0.48’ 1.55 f. 0.10* 1.45 f 0.28’

0.21 0.19 0.10 0.18 0.41 3.56

0.30 0.28 0.18 0.96 1.02 4.79

1.10 1.09 0.82 1.06 1.22 3.00

1.28 1.26 1.08 1.85 1.95 3.88

f + + + + f

0.06 0.06 0.03 0.07 0.15 0.17

+ + + + + +

0.10’ o.loc 0.06’ 0.18’ 0.27* 0.32”

+ f f If: IL f

0.20 0.25 0.06 0.11 0.23 0.06

f f If: + f +

0.25’ 0.26’ 0.06” 0.24” 0.25* 0.15”

0.22 f 0.06 0.86 f 0.29

1.16 + 0.22” 1.79 + 0.35*

1.04 f 0.25 1.86 + 0.33

1.69 -t 0.25’ 2.66 +- 0.27’

0.25 + 0.09 0.18 + 0.06

0.95 + 0.34’ 0.32 + 0.06*

1.40 f 0.10 1.05 -c 0.10

2.22 + 0.15” 1.27 t- 0.15’

Note. Mitochondria were incubated for 5 min in medium containing 100 mM KCl, 40 mM Tris-HCl, 5 mM KH2POI, 10 mM glutamate and 10 malate (pH 7.25). Where indicated 0.5 mM NaAs.0, was added. The significance of change caused by arsenite calculated by Student’s pair test: a P < 0.01, * P < 0.05, ’ not significant. d The activity of glutathione reductase was inhibited by 28 and 39% in the samples incubated in the absence and presence of NaAsO,, respectively. Mean values for three experiments + SD are presented.

mM

EFFECT

OF

ARSENITE

ON

MITOCHONDRIAL TABLE

Effect

of Arsenite

on the Content

II of GSSG

(nmol Without Additions None BHT (0.1 mM) BCNU (0.25 mM) Note. Mitochondria ’ Significant change

Without

arsenite

0.89 * 0.31 0.48 k 0.06 3.17 + 0.40

391

METABOLISM

in Mitochondria

Content of GSSG GSH eq/mg protein)

CaCl,

With With

arsenite

Without

3.99 + 0.95” 0.97 z!z 0.25’ 4.48 f 0.55”

were incubated under conditions described in Table caused by arsenite, P < 0.05 calculated by Student’s

dria and to chelate Cazf (23,24). The effect of citrate was also exerted in the presence of fluorocitrate, an inhibitor of aconitase, indicating that it was not due to isocitrate oxidation. The suppression of arsenite-induced NAD(P)H oxidation by BHT, a free radical scavenger, points to the participation in this effect of free radical reactions. These reactions result in hydroperoxide production, known to stimulate mitochondrial oxidation of glutathione and, in consequence, oxidation of NADPH by glutathione reductase (25). Incubation of mitochondria with 0.25 mM BCNU, an inhibitor of glutathione reductase, suppressed its activity measured in mitochondrial extract by 68 and 90% after 15 and 30 min incubation, respectively. The addition of 0.5 mM arsenite was also observed to inhibit glutathione reductase during mitochondrial incubation, but after a longer time and to a smaller degree than with BCNU, namely by 10 and 48% after 15 and 30 min of incubation, respectively. In the presence of BCNU the extent of the NAD(P)+ increase in mitochondria caused by arsenite was diminished to a degree similar to that in the presence of BHT. A similar protection against arsenite-induced NAD(P)H oxidation was obtained by substituting 225 mM mannitol (another free radical scavenger) for KC1 in the incubation medium. This protective effect exerted by both scavengers, BHT and mannitol, was increased by M?+ and was complete in the presence of 10 mM MgClz. The addition of arsenite to uncoupled mitochondria increased the mitochondrial NAD+ and NADP+ content by about 30%, in agreement with previous observations (10). The values for NAD’ and NADP+ content found in the presence of FCCP plus arsenite could be assumed to represent the total pool of the nicotinamide nucleotides in mitochondria. The mitochondrial membrane potential during 5 min of incubation was not decreased by the addition of arsenite up to 1 mM unless calcium salt, at least 10 pM, was also added (not shown). Concomitant with the arsenite-induced oxidation of NAD(P)H (Fig. 1) was an increase of GSSG content (Ta-

0.1

mM

CaCl,

arsenite

With

4.40 k 0.20 4.33 iz 0.30 5.43 + 0.30

I. Mean values pair test.

for three

experiments

arsenite

5.15 f 0.75 4.67 + 0.65 6.87 + 0.62” + SD are presented.

ble II). The latter process was abolished by BHT and potentiated by BCNU. In the presence of 0.1 mM CaClz the content of GSSG was greatly increased, but the effects of arsenite and BCNU were still visible. The addition of arsenite to mitochondria incubated with 10 mM glutamate and 10 mM malate resulted in an ac-

a-KETOGLUTARATE

NADP+

Oo-

5

10

Time

no-

10

(min)

FIG. 3. Effect of arsenite on the accumulation of a-ketoglutarate and aspartate by mitochondria incubated with glutamate plus malate. Mitochondria were incubated in medium containing 100 mM KCl, 40 mM Tris-HCl, 20 mM KH2POI, 5 mM MgCl,, 0.5 mM EDTA, 10 mM glutamate, and 10 mM malate, pH 7.25. State 3 was produced by addition of 1 mM ADP, 30 mM glucose, and 10 units of hexokinase/ml. Incubations were performed in State 4 (Cl, n ) and in State 3 (0,O) either without further additions (0, 0) or with 0.5 mM NaAsO, (m, 0). Data of one from three similar experiments are presented.

392

EWA

LENARTOWICZ TABLE

Effect

of Arsenite

on the

III

Utilization

of Isocitrate

by Mitochondria

Ftate (nmol/mg State Metabolic

processes

Without

Accumulation of ol-ketoglutarate Utilization of isocitrate Formation of citrate without fluorocitrate Formation of citrate with fluorocitrate Fluorocitrate-sensitive formation of citrate Oxidation of isocitrate*

12.5 203.5 188.5 23.1 165.4 38.1

protein/5

min)

4

State

arsenite

With

arsenits

+ 2.6 + 18.0 + 29.0 + 4.8 + 27.0 f 9.0

33.6 152.0 133.5 16.0 117.5 34.5

f + f + f +

Without

5.1” 17.1” 31.1 2.9 28.2 12.0

3

arsenite

25.0 209.5 120.5 28.0 92.5 117.0

+ 2.4 + 18.0 + 11.1 + 2.0 f 9.0 + 14.0

With

arsenite

105.3 155.0 73.8 22.1 51.7 103.3

+ 7.1° f 32.0 f 15.1’ + 4.1 + 2.5” + 36.0

Note. Mitochondria were incubated for 5 min in medium containing 100 mM KCI, 40 mM Tris-HCl, 20 mM KHpPOl, 5 mM MgC&, 0.5 mM EDTA, 2.5 mM isocitrate, 1.0 mM malate and, where indicated, 0.1 mM fluorocitrate and/or 0.5 mM NaAsO, (pH 7.25). State 3 was produced as described in the legend to Fig. 2. Mean values for three experiments f SD are presented. a Significant change caused by arsenite, P < 0.05 calculated by Student’s pair test. * Oxidation of isocitrate was calculated from the difference between the amount of utilized isocitrate and the amount of citrate formed in the fluorocitrate-sensitive reaction.

TABLE Effect

of Arsenite

on the

Utilization

of Glutamate of Metabolites

IV

and Citrate Plus Isocitrate Involved in a-Ketoglutarate

by Mitochondria Formation Content

State Nicotinamide metabolic

nucleotides processes

and

Without arsenite

of NAD+ of NADP+

4

State With arsenite

1.2 + 0.2 0.2 AZ 0.1

2.3 + 0.7 k

24.8 31.8 17.1 -0.5 48.4 33.1 +19.1 14.0 62.4

f +f f + + + + f

4.1 3.5 3.0 0.2 5.9 5.8 5.7 3.5 7.5

107.3 54.3 10.5 -1.6 63.2 19.5 -28.5 48.0 111.2

3

Without arsenite

With arsenite

1.7 f 0.2 0.3 +_ 0.1

2.4 f 1.2 f

protein)

0.5d 0.2d (nmol/mg

Accumulation of a-ketoglutarate Accumulation of aspartate Accumulation of alanine Change in the content of ammonia Total utilization of glutamate” Formation of citrate (with fluorocitrate) Change in the content of citrate plus isocitrate Oxidation of isocitrate” Formation of a-ketoglutarate’

Concentrations

or rate

(nmol/mg Content Content

at Physiological

protein/5

If: 10.gd f 5.0d + 0.6d 2 0.2d k 3.6d f 5.5d f 5.7d f 6.0d f 5.7d

Note. Mitochondria were incubated for 5 min in medium containing 100 mM KCl, 40 mM Tris-HCl, KHCOs, 5 mM glutamate, 1 mM malate, 0.3 mM pyruvate, 1 mM citrate, 0.1 mM isocitrate and, where 3 was produced by addition of 2.5 mM ADP. To measure the formation of citrate parallel samples fluorocitrate. All values are means for three experiments f SD. The indicated rates were calculated: alanine, and ammonia, * from the difference between the amount of citrate formed during incubation change in the content of citrate plus isocitrate, as described previously (38), and ’ from the sum of amount of oxidized isocitrate. d Significant change caused by arsenite, P < 0.05 calculated by Student’s pair test.

0.6 0.4d

min) 64.2 128.5 10.3 +4.0 142.8 41.3 -12.7 54.0 196.8

2~ 8.3 k 13.1 + 0.6 * 0.4 + 13.3 iT 3.0 f 3.5 3~ 6.0 +- 13.5

215.1 111.7 6.8 +5.5 124.0 33.6 -62.4 96.0 220.0

f 22.3d f 7.0 5 0.5d + 0.6d + 7.0 f 5.3 f 6.6d f 9.0d f 12.5

5 mM KHxPO*, 0.5 mM MgCl*, 20 mM indicated, 0.5 mM NaAsOp (pH 7.25). State were incubated in the presence of 0.1 mM ’ from the sum of accumulated aspartate, (in the presence of fluorocitrate) and the the total utilization of glutamate and the

EFFECT

A

ASPARTATE

+----T--

B

OF C

ALANINE

z no+ (I - Ketoglutarate

ARSENITE

ON

MITOCHONDRIAL

UTILIZATION OF CITRATE PLUS ISOCITRATE

z no+

I

(mM)

FIG. 4. Effect of external cY-ketoglutarate on the production of aspartate and alanine and on the utilization of citrate plus isocitrate by mitochondria. Mitochondria were incubated for 5 min in medium containing 100 mM KCl, 40 mM Tris-HCl, 5 mM KH2POI, 0.5 mM MgCle, 20 mM KHCOs, 1 mM malate, various concentrations of a-ketoglutarate and either 5 mM glutamate plus 0.3 mM pyruvate (A and B) or 1 mM citrate plus 0.1 mM isocitrate, without and with 0.1 mM fluorocitrate (C), pH 7.25. The formation of aspartate (A), alanine (B), and the utilization of citrate plus isocitrate (C) were estimated in samples incubated with mitochondria in State 4 (0) and in State 3 (0). The utilization of citrate plus isocitrate, equal to the oxidation of isocitrate, was calculated as described in the legend for Table IV. Points represent means for four experiments.

cumulation of c-u-ketoglutarate and aspartate in nearly equimolar amounts (Fig. 3). In the absence of arsenite aketoglutarate was removed by oxidation, but its production could be reflected by that of aspartate. It is shown that arsenite initially stimulated the formation of aspartate, hence also of a-ketoglutarate. This stimulation was presumably due to the increased level of NAD+ which, in turn, increased the availability of oxaloacetate for transamination with glutamate. Only later on aspartate formation was inhibited by accumulating a-ketoglutarate. In mitochondria incubated with unphysiologically high concentrations of isocitrate, 2.5 mM, arsenite did not produce an increase of NAD+ and NADP+ content (not shown) and did not affect the rate of the isocitrate oxidation (Table III). The activities of both isocitrate dehydrogenases, NADP- and NAD-linked, measured in mitochondrial extracts were also unaffected by arsenite (not shown). Nevertheless, the utilization of isocitrate by mitochondria was lower by 25% in the presence of arsenite. This was due only to the suppression of the conversion into citrate, since no difference in isocitrate utilization was observed in the presence of 0.1 mM fluorocitrate (not shown). However, the activity of aconitase measured in mitochondrial extract was not affected by arsenite (not shown). The formation of a-ketoglutarate by mitochondria at low, close to physiological concentrations of substrates and Mg*+ was affected by arsenite through various ways (Table IV). Under these conditions the addition of arsenite caused a considerable increase of the content of

393

METABOLISM

NAD+ and NADP+ in mitochondria and an accumulation of a-ketoglutarate. The effect of added a-ketoglutarate on both glutamate transaminations, namely that with OXaloacetate and that with pyruvate, as well as on the utilization of citrate plus isocitrate is shown in Fig. 4. It can be noticed that a-ketoglutarate strongly inhibited the aspartate aminotransferase reaction, in agreement with the previous observation of Strzelecki et al. (26), and to a smaller degree the alanine aminotransferase reaction, whereas isocitrate oxidation was scarcely affected. The effect of arsenite on the time course of various reactions contributing to a-ketoglutarate production in mitochondria at physiological concentrations of substrates is presented in Fig. 5. Mitochondrial production of aspartate was enhanced by arsenite only in State 4 while in State 3 the reaction was strongly inhibited after a few minutes of incubation. The alanine aminotransferase reaction, as reflected by the formation of alanine, was

STATE

STATE

4

Time

3

(min)

FIG. 5. Effect of arsenite on the rate of various reactions producing a-ketoglutarate in mitochondria at physiological concentrations of substrates. (A) Production of a-ketoglutarate from glutamate by aspartate and alanine aminotransferases. (B) Total production of a-ketoglutarate and its parts derived from glutamate and from isocitrate. The incubation medium and the calculations of the cu-ketoglutarate formation, the glutamate utilization, and the isocitrate oxidation were the same as described in the legend to Table IV. The symbols represent the formation of aspartate (A, A), alanine (0, +), ol-ketoglutarate (V, v), the total utilization of glutamate (0, n ), and the oxidation of isocitrate (0, l ), either without NaAsO, (open symbols) or with 0.5 mM NaAsO, (closed symbols). The results of one from two experiments are presented.

394

EWA

LENARTOWICZ

suppressed by arsenite in both States 4 and 3. The total formation of a-ketoglutarate from glutamate (measured as the sum of accumulating aspartate, alanine, and ammonia) changed in response to arsenite similarly to its main reaction, the aspartate aminotransferase. The oxidation of isocitrate was strongly stimulated in the presence of arsenite, more in State 4 than in State 3. As result, arsenite-induced changes in the rates of glutamate utilization and isocitrate oxidation led to the stimulation of n-ketoglutarate production by about 75 and 10% in States 4 and 3, respectively, after 7.5 min of incubation. Simultaneously, the ratio of contribution by glutamate and isocitrate to a-ketoglutarate formation decreased from 2.5 3.5 to 1.1-1.4 in the presence of arsenite. It should be added that the extent of arsenite-induced NAD(P)H oxidation in the latter experiments could be diminished by an increase of citrate and/or MgC& concentrations and by a decrease of phosphate concentration. Opposite changes in the concentrations of these compounds could augment the effect of arsenite. DISCUSSION Arsenite is shown to produce an extensive oxidation of intramitochondrial NAD(P)H. This effect could only partly be due to inhibition of cu-keto acid dehydrogenases, since it was potentiated by phosphate and prevented by EGTA, ruthenium red, and citrate. This suggests the importance of Ca2+ in the arsenite-mediated oxidation of mitochondrial nicotinamide nucleotides. Ca’+-dependent oxidation of intramitochondrial NAD(P)H has been observed in mitochondria treated with various thiol reagents, such as NEM, diamide, mercurials, Pb2+, and alloxan (22, 27-29). However, it occurred in the presence of added calcium salts and in most cases was accompanied by a decline in the mitochondrial membrane potential. In contrast to that, 0.1-1.0 mM arsenite induced oxidation of mitochondrial NAD(P)H without addition of calcium salts while the addition of Ca2’ was indispensable for the decline of the membrane potential. This indicates that arsenite requires much lower concentrations of calcium for the disturbance of the mitochondrial redox state than for the decrease of the membrane potential. Similar relations have recently been observed in benzoquinonetreated mitochondria in which the release of Ca2+, which is known to follow mitochondrial NADPH oxidation, was not initiated by a collapse of the membrane potential (30). It is known that, in the presence of arsenite, similarly to other thiol reagents, Ca2+ taken by mitochondria is immediately released (8). This release of Ca2+ is accompanied by a similar efflux of Mgz+ (31). Citrate was observed to protect mitochondria against the loss of Mg2+ and against Ca2+-induced disturbances of membrane properties (23). The dependence on phosphate has been described for many Ca2+-involved effects in mitochondria,

such as the decline of the mitochondrial membrane potential, oxidation of NAD(P)H, and efflux of divalent cations from mitochondria induced by hydroperoxides, diamide, and acetoacetate in the presence of low concentrations of Ca2+ and by higher concentrations of Ca2+ alone (29, 32, 33). All these effects are assumed to be associated with the modulation of mitochondrial thiol groups (32,34). It is here noteworthy that phosphate potentiates the uptake of Ca2+ by increasing the membrane potential (35). A number of mechanisms can be proposed to contribute to the oxidation of intramitochondrial NAD(P)H in the presence of arsenite (Fig. 6). One of them is certainly the inhibition of cr-keto acid dehydrogenases, which diminishes the reduction of mitochondrial NAD+. Another important mechanism can result from the sensitivity of nicotinamide nucleotide transhydrogenation to trivalent arsenicals (36). The NAD(P) transhydrogenase reaction has been suggested to be responsible for changes in the redox state of NAD(P) under all conditions affecting mitochondrial thiol groups (9,28,30). Thus arsenite can be assumed to supress the mitochondrial formation of NADPH. Shortages of NADPH suppress the reduction of GSSG by glutathione reductase and the subsequent decrease of GSH content promotes the increase of the concentration of hydroperoxides, which derive from free radicals. Moreover, the decrease of vicinal SH groyby diamide was shown to favor lipid peroxidation in ml&chondria (37), which can enrich the production of hydroperoxides. The increase of hydroperoxide concentration stimulates the oxidation of GSH leading to accumulation of GSSG, which is, similarly to trivalent arsenicals, inhibitory to nicotinamide nucleotide transhydrogenase (36). The oxidation of mitochondrial NADPH by glutathione cycling is strongly stimulated by Ca2+ (25). Hence the arsenite-induced oxidation of NAD(P)H is dependent

Arsenite

1 Loss of vicinal Inhibition

of nicotinamide

nuclwtide

transhydrogenase

Sti groups

4 Inhibition

d

acid

of a-k&o

dehydrogenases J

Sup~ressian

of NADPH

formation

Shortage

Accumulation

of NADPH I % Suppression of GSSG reduction

formation

-

Oxidation + )

Shortage GSH

of GSH

I Accumulation

of -

I

of

HZ02 Free radicals

FIG. 6. Proposed state of mitochondrial

mechanism of the action NAD and NADP.

of arsenite

on the redox

EFFECT

OF

ARSENITE

ON

MITOCHONDRIAL

on Ca’+, is decreased by BHT (scavenging free radicals) and BCNU (suppressing the activity of glutathione reductase; Table I), and is associated with a considerable increase of mitochondrial GSSG (Table II). Oxidation of intramitochondrial NAD(P)H is another factor, besides the inhibition of a-keto acid dehydrogenases, modulating mitochondrial metabolism in the presence of arsenite. This is manifested by the effect of arsenite on the utilization of glutamate and isocitrate by mitochondria. In mitochondria glutamate is converted to cu-ketoglutarate mainly in the reaction of aspartate aminotransferase. The addition of arsenite initially stimulated this reaction, which can be explained by the increase in the oxaloacetate/malate ratio resulting from the oxidation of mitochondrial NADH. Oxygen consumption by mitochondria incubated under these conditions in State 3 is also significantly increased by arsenite (6). Only upon prolonged incubation the formation of aspartate was inhibited by accumulating cr-ketoglutarate (Fig. 3), as it was also observed by Strzelecki et al. (26). The alanine aminotransferase reaction is suppressed by arsenite due to both the increase of cY-ketoglutarate concentration and the oxidation of NADH, since the reaction is favored by NAD reduction (38). The oxidation of isocitrate, present at an unphysiologically high concentration, 2.5 mM, was not affected by arsenite, since no NAD(P)H oxidation occurred in mitochondria, and also a-ketoglutarate was found not to be inhibitory in this process (Fig. 5). In contrast to p-benzoquinone, an arylating thiol group reagent (30), arsenite did not affect the activity of either NADP- or NAD-linked isocitrate dehydrogenases in mitochondrial extract. Under these conditions the oxidation of mitochondrial NAD(P)H in the presence of arsenite was prevented by citrate, since the same protection was produced by citrate plus fluorocitrate, similar to that in diamide-treated mitochondria (23). This protection against NAD(P)H oxidation by citrate may be related to the chelation of Mg2+ and Ca2+ within mitochondria. In contrast, a similar protection by isocitrate against mitochondrial NAD(P)H oxidation in mitochondria treated with benzoquinone was due to a direct reduction of NAD(P)+ (29). _ At physiological concentrations of glutamate, malate, pyruvate, isocitrate, citrate, and M$+ arsenite induced a considerable oxidation of intramitochondrial NADPH and NADH resulting in a strong stimulation of isocitrate oxidation. This could be ascribed to stimulation of NADPlinked isocitrate dehydrogenase, much more potent than the NAD-linked enzyme. NAD+-linked isocitrate oxidation, a process very susceptible to inhibition by NADH, was also certainly stimulated in the presence of arsenite (39). Therefore, arsenite does not completely inhibit the production of a-ketoglutarate in mitochondria, in spite of the accumulation of a-ketoglutarate. Under these con-

395

METABOLISM

ditions only its formation from glutamate can be suppressed. It seems interesting that arsenite suppressed by 3035% the conversion of isocitrate to citrate in mitochondria without inhibiting aconitase. In this situation the only explanation of this effect could be a shift in the equilibrium ratio of citratelisocitrate, known to be correlated with the concentration of Mgzf (40). Also, the differences in Mg2+ concentrations between States 4 and 3 (higher Mg2+ in State 4 than in State 3, Ref. (41)) can be a reason for a faster formation of citrate from isocitrate in State 4 than in State 3 (Table III). The effect of arsenite on intramitochondrial concentration of Mg2+ requires further investigation. However, it can be suggested that arsenite can additionally disturb mitochondrial metabolism by changing intramitochondrial concentrations of various ions. ACKNOWLEDGMENT The author thanks Professor Lech Wojtczak for critical reading and correcting of the manuscript. The technical assistance of K. Kropiwnicka is gratefully acknowledged. This work was supported by the Polish Academy of Sciences within CPBP 04.01.

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