TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

117,

17%

179 ( 1992)

In Vitro and in Vivo Effect of Methyl lsocyanate on Rat Liver Mitochondrial Respiration K. JEEVARATNAM,*” *Defence

Research

and Development

Establishment,

S. VIDYA,~ AND C. S. VAIDYANATHAN~ Gwalior-474002

India;

and tlndian

Institute

of Science,

Bangalore-

India

Received March 9, 1992; accepted July 14, 1992

and pulmonary irritant effects and extensive alveolar damage leading to pulmonary edema (Nemery et al., 1985; Kimmerle and Eben, 1964; Ferguson et al., 1986; Fowler and Dodd, VAIDYANATHAN, C. S. (1992). Toxicol. Appt. Pharmacol. 117, 1986). However, Kolb et al. (1987) suggested that MIC ex172-179. posure by inhalation leads to a shock-like condition in guinea Previous work has shown that irrespective of the route of ex- pigs before death, resulting in loss of fluid from the circulatory posure methyl isocyanate (MIC) caused acute lactic acidosis in system. Recently, we reported that MIC administered subrats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, cutaneously (SC) causes a severe degree of hypovolemic hy3 14-3 19, 1990) and the hypoxia was of stagnant type due to potension (Jeevarathinam et al., 1988) resulting from fluid tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et loss from vascular compartment due to altered permeability al., Toxicology 51, 223-240, 1988). The present study was de- to cations through inhibition of Na+-Kf ATPase and probsigned to investigate whether MIC could induce histotoxic hyp- ably also water transport of the plasma membrane (Jeevaratoxia through its effects on mitochondrial respiration. Male Wis- nam and Vaidyanathan, 1992a). This leads to tissue hypoperfusion and subsequently hypoxia, known as stagnant tar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled hypoxia. Ferguson et al. ( 1988) demonstrated that there was mitochondria in vitro resulted in stimulation of state 4 respi- a rapid uptake and distribution of 14C to blood and various ration, abolition of respiratory control, decrease in ADP/O ratio, tissues during and following exposure through inhalation to and inhibition of state 3 oxidation. The oxidation of NAD+[‘4C]MIC in mammals, while Bhattacharya et al. (1988) relinked substrates (glutamate + malate) was more sensitive (five- ported that MIC either inhaled or administered ip reached to sixfold) to the inhibitory action of MIC than succinate while the systemic circulation in its native or active form and was cytochrome oxidase remained unaffected. MIC induced twofold similarly distributed to various organs, as evidenced by cardelay in the onset of anerobiosis, and cytochrome b reduction bamylation of tissue proteins. Hematological and biochemin SMP with NADH in vitro confirms inhibition of electron ical changes were also essentially similar except for the diftransport at complex I region. MIC also stimulated the ATPase ferences in magnitude in rats when MIC was administered activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine either by inhalation or by SCroute (Jeevaratnam et al., 1990) and N,N’-dimethylurea failed to elicit any change in vitro; these and they are also similar in rabbits, reflecting the species effects reveal that MIC per se acts as an inhibitor of electron similarity of systemic toxic effects of MIC in mammals (Jeetransport and a weak uncoupler. Administration of MIC SCat varatnam et al., 199 1). lethal dose caused a similar change only with NAD+-linked subThe predominant systemic effect of MIC intoxication in strates, reflecting impairment of mitochondrial respiration at mammals is severe tissue hypoxia leading to clinical lactic complex I region and thereby induction of histotoxic hypoxia acidosis (Jeevarathinam et al., 1988; Jeevaratnam et al., bl ViVO. 0 1992 Academic Press, Inc. 1990). We have shown that in rabbits administered MIC SC, the hypoxia is of the stagnant type resulting from hypovoMethyl isocyanate (MIC) is a sensory and pulmonary ir- lemic hypotension (Jeevarathinam et al., 1988). However, the occurrence of concurrent histotoxic hypoxia due to the ritant causing extensive alveolar damage (Nemery et al., effects of MIC on the mitochondrial respiration has not been 1985). Most of the earlier studies were confined to the effects ruled out. Hence, in the present study, we have investigated of MIC on the respiratory system, which revealed respiratory the in vitro effects of MIC on the isolated rat liver mito’ To whom correspondence should be addressed at Defence R&D Estab- chondrial respiration. In order to find out whether such eflishment, Tansen Road, Gwalior, MP-474002, India. fects are due to MIC per se or its hydrolysis products the In Vitro and in Viva Effect of Methyl Isocyanate on Rat Liver Mitochondrial Respiration. JEEVARATNAM, K., VIDYA, S., and

0041-008X/92 $5.00 Copyright 0 1992by Academic Press,Inc. All rights of reproduction in any form reserved.

172

MIC EFFECTS ON MITOCHONDRIAL

influence of methylamine (MA) and N,N’-dimethylurea (DMU) has also been studied in vitro. Besides, we have investigated whether MIC could induce histotoxic hypoxia as well through its effect on the mitochondrial respiration, in vivo, in rats administered MIC SC.Subcutaneous application was chosen to avoid possible dosing inaccuracies encountered in the inhalation procedure and the effects of pulmonary reflexes. MATERIALS

AND METHODS

MIC (99% purity) was synthesized and characterized in our laboratory as described earlier (Kaushik et al., 1987; Jeevarathinam et al., 1988). MA (40% aqueous solution) was obtained from E. Merck (India) and DMU from Sigma (USA). All chemicals used were of analytical grade procured from either Sigma or E. Merck. For in vitro studies, chilled aqueous solutions of MIC in distilled water were prepared freshly and used within 5 min. MIC was stable up to 5 min in aqueous solution when it was kept chilled in an ice-salt mixture as analyzed by gas chromatograph (data not shown), while Meshram and Rao (1988) reported the persistence of only 70, 50, and 23% of MIC in aqueous solutions at 25°C for 2, 15, and 30 min, respectively. For in vivo studies, as MIC is highly volatile and reactive, it was dissolved in olive oil, where the compound is stable for 24 hr for injection purposes. Fresh solutions of MIC were prepared always and used immediately (Jeevarathinam et al., 1988). Acute LD50 of MIC administered SCin male Wistar rats is 328.6 mg/kg as determined by Finney’s probit analysis (Jeevaratnam et al., 1990). Forty male Wistar rats, bred in the animal house of Indian Institute of Science, Bangalore, weighing 140 + 10 g were used throughout the experiments. The rats were housed four per cage over rice-husk bedding and had accessto a commercial rat feed (Lipton India Ltd., Bangalore, India) and water ad libitum at all times under a 12-hr photoperiod. Sixteen rats were used for in vitro studies to prepare liver mitochondria and submitochondrial particles (SMP). The remaining animals were randomly divided into three groups (each n = 8): (i) Control receiving olive-oil (vehicle) injected SC,(ii) and (iii) MIC administered receiving sc injection of MIC at two doses, 0.5 and 1.O LD50, respectively. Rats were fasted overnight and during the experiment while water was allowed ad libitum. As the resultant biochemical changes were maximum and highly significant 4 hr after MIC administration (Jeevaratnam et al., 1990), rats were killed by cervical dislocation 4 hr after MIC administration SC,either at 0.5 LD50 or 1.0 LD50 or olive oil SCin control group. Liver mitochondria were prepared according to the standard procedure (Johnson and Lardy, 1967) with minor modifications (Kurup et al., 1970) notably the applied centrifugal force in the differential centrifugation. The liver homogenate was initially centrifuged at 700g instead of 600g and later 1OOOOgfor 10 min instead of 15OOOgfor 5 min in the standard procedure. Liver SMP were prepared by sonic disruption and washed by ultracentrifugation (Graven et al., 1967). Protein was determined by the modified biuret method (Gornall er al., 1949). Oxidative phospho~lation. Respiratory control and oxygen uptake were determined at 30°C by polarography in a Gilson Oxygraph Model 5/6H (Gilson Medical Electronics, Inc., Middleton, WI) fitted with a Clark-type oxygen electrode (Rasheed et al., 1980). For polarographic experiments, about 0.8 mg mitochondrial protein was used with glutamate + malate while 0.5 mg protein was used with succinate. Respiratory control index (RCI) was calculated as the ratio of oxidation in “state 3” (ADP present) to that in “state 4” (ADP exhausted) (Chance and Williams, 1955). Assay ofenwymes. The frozen and thawed mitochondria were used for various mitochondrial enzyme assays.About 4 mg protein treated with different concentrations of MIC (35, 70, 105, and 140 pM per mg of protein)

173

RESPIRATION

for 2 min at 30°C were pelleted in microfuge and then reconstituted with fresh 0.25 M sucrose solution for in vitro studies. Succinate dehydrogenase activity was assayedusing phenazine methosulfate together with 2,6-dichlorophenol indophenol (Nair and Kurup, 1986). The activity of NADH dehydrogenase was measured using potassium ferricyanide as an electron acceptor (King and Howard, 1967). Standard spectrophotometric procedures were used for the assay of NADH oxidase (Mackler, 1967) NADH-cytochrome c reductase (Hatefi and Rieske, 1967), and cytochrome oxidase (Yonetani. 1967) using Shimadzu uv/Vis Spectrophotometer Model 160A (Shimadzu Corporation, Tokyo, Japan). Further, NADH oxidase activity of liver SMP exposed to different concentrations of MIC in vitro was measured polarographically in Gilson Oxygraph (King, 1967) while the activities of NADH oxidase, NADH-cytochrome c reductase, and NADH dehydrogenase of liver SMP of rats administered MIC SCwere also measured spectrophotometrically as stated above. From linear regression equations correlating activity with MIC concentration, the amount required for 50% inhibition (EC50) in vitro was determined. The assay of ATPase was carried out using freshly isolated mitochondria as described by Veldsema-Currie and Slater ( 1968) by estimating the inorganic phosphate, Pi, liberated during 2 min incubation with ATP at 30°C by the spectrophotometric method (Marsh, 1959). For the in vitro study, about 500 rg of protein was treated with either MIC or methylamine (70 and 140 PM per mg protein), or 2,4-dinitrophenol, a known uncoupler (100 pM per mg protein) for 1 min at 30°C prior to ATPase assay. The rate of reduction of cytochrome b in liver SMP was also determined with NADH as electron donor using Hitachi 557 Dual wavelength Spectrophotometer (Hitachi Ltd., Tokyo, Japan) (Chance and Hagihara, 1963). Lipid peroxidation in the frozen thawed mitochondria treated with different concentrations of MIC in vitro for 2 min at 30°C was determined by the malondialdehyde (MDA) formed as assayed by thiobarbituric acid method (Wilber et al.. 1949) after washing once with phosphate buffer to remove sucrose in the samples. The statistical analysis on the data involving various concentrations (Table 1 and Table 5) was done by one-way analysis of variance (Snedcor and Cocharan, 1967) to determine least significant difference for the comparison of means, while Student’s t test was used for the remaining data. The level of significance was set at p < 0.05.

RESULTS Addition of MIC to tightly coupled rat liver mitochondria resulted in the stimulation of “state 4” respiration (ADP exhausted), abolition of respiratory control, decrease in ADP/ 0 ratio, and inhibition of “state 3” respiration (ADP present) (Table 1). These effects reveal its dual mode of action: (i) an inhibitor of state 3 respiration and (ii) an uncoupler. Some typical polarographic tracings are shown in Fig. 1. However, the hydrolysis products of MIC, either MA or DMU up to a concentration of 300 PM per mg of protein has not altered the rat liver mitochondrial respiration (data not shown). The values of EC50 provide a measure of relative inhibitor potency for different substrates. The data in Table 2 show that the oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive to the inhibitory action of MIC than succinate was. The specificity for interaction at complex I region of electron transport has been observed as the inhibition is halved when the concentration of mitochondrial protein is doubled (Fig. 2). Among the enzymes at complex I region, the NADH oxidase was found to be highly suscep-

174

JEEVARATNAM,

VIDYA,

AND VAIDYANATHAN

TABLE 1 In Vitro Effect of MIC on the Oxidative Phosphorylation

of Rat Liver Mitochondria

Oxidative phosphotylation Succinate

Glutamate + malate Group

State 3”

State 4”

RCI

ADP/O

State 3”

State 4”

RCI

ADP/O

Control MIC 35 PM 70 ELM 105 PM 140 PM 350 /IM LSD

128 f 10.6a

14.6 + 1.75a

8.8 + 0.29a

2.7 + 0.03a

216 f 6.la

38.3 f 2.73a

5.6 + 0.21a

1.5 * 0.05a

17.8 f 1.33b 21.6 + 1.36cd 23.5 + 3.36d 19.9 + 1.81bc 2.27

4.6 + 0.13b 3.1 f 0.16~ 2.2 + 0.16d I .8 2 0.09e 0.38

2.5 rt. 0.06b 2.2 + 0.07c x X 0.10

193 + 9.3b 174 + 4.oc 141 + 4.8d 7.8

52.8 f 1.60b 57.5 f 2.26~ 66.0 k 2.83d 3.8

3.6 + 0.17b 3.0 f O.lOc 2.1 + 0.07d 0.21

1.3 + 0.02b I .2 + 0.03c 1.2 i 0.04c 0.06

81 68 51 36

f 4.5b +_ 4.6~ f 2.2d + 4.0e 6.8

Note. The reaction conditions are the same as those described in the legend for Fig. I. The values are means F SD of six independent determinations. ’ State 3 and state 4 respiration is rate of oxygen uptake (ng atom O/min/mg of protein). Means within a column followed by the same letter(s) are not significantly different (p < 0.05, Student’s range test). X values are not given as an apparent increase in ADP/O ratio (paradoxical) to be observed due to very high inhibition of state 3 respiration.

tible to MIC inhibition (Table 3). The cytochrome oxidase activity was not inhibited by MIC up to 300 I.LM per mg of protein while in case of succinate dehydrogenase about 2030% inhibition was observed without any linearity with increased concentration of MIC (data not shown).

Typical polarographic tracings showing the concentrationdependent inhibition of NADH oxidation in liver SMP are given in Fig. 3. The concentration of MIC required to inhibit liver SMP NADH oxidase activity by 50% (EC50) was 95 + 5.2 (means t SD) as calculated by regression analysis. To further confirm our results, we monitored the rate of reduction of cytochrome b in SMP (about 1 mg of protein) treated with 70 PM MIC. There was nearly a twofold delay in the onset of anerobiosis when NADH was used as substrate (Fig. 4) while there was not any appreciable effect with succinate as substrate (data not shown). Table 4 provides the ATPase activity measured in tightly coupled mitochondria treated with either MIC or MA or 2,Cdinitrophenol. A significant increase in ATPase activity was observed in MIC-treated samples but less than that inTABLE 2 In Vitro Effect of MIC on Oxidation Rate and Respiratory Control in Rat Liver Mitochondria

FIG. 1. In vitro effect of MIC on rat liver mitochondrial respiration. [Freshly isolated rat liver mitochondria were used in a reaction volume of 1.4 ml for the determination of respiratory control by polarography. The values in the parentheses refer to the rates of oxygen uptake (ng atom O/ min/mg protein) on stirring in 360 FM ADP for Glu + mal and 260 pM ADP for succ (state 3) and depletion of added ADP (state 4). Glu, glutamate; Mal, malate; succ, succinate.]

Substrate

Function

Glutamate + malate Succinate

Respiratory control “State 3” oxidation Respiratory control “State 3” oxidation

EC50 ( /.LM) 49 84 248 537

+ 3.1 rf: 3.7” + 8.2 + 18.0”.b

Note. The reaction conditions are the same as those described in the legend for Fig. I. The values of MIC concentrations for 50% effect (EC50) calculated from the regression equations are means + SD of six independent determinations. The values of correlation coefficient ranged from 0.928 to 0.987. Statistical analysis by Students t test. ’ Significant oxidation versus respiratory control. b Significant oxidation of succinate versus glutamate + malate.

MIC EFFECTS ON MITOCHONDRIAL

RESPIRATION

175

L

eS Sf

1 MIN

FIG. 2. Effect of mitochondrial

protein concentration on the inhibitory action of MIC in vitro. For details see legend to Fig. 1.

duced by the known uncoupler, 2,4-dinitrophenol, while MA had not affected the ATPase activity. MIC acts as a weak uncoupler. The MDA content of frozen thawed mitochondria treated with MIC in vitro up to 140 PM per mg of protein for 2 min was found to be unaltered (Table 4). Mitochondrial function measured as respiratory activity of mitochondria isolated from liver homogenate of rats receiving subcutaneous injection of MIC showed a significant effect only in the 1.0 LD50 group while no appreciable change was observed in the 0.5 LD50 group. In the 1.O LD50 group, when glutamate and malate were used as substrate there was 20% decrease in state 3 oxidation, and 50% increase in state 4 respiration with significant decrease in RCI and lowering of ADP/O ratio (Table 5). However, there was no change in these mitochondria when succinate was used as substrate. A significant increase in ATPase activity was observed in the mitochondria isolated from 1.0 LD50 MIC treated rats. The ATPase activities (means +- SE, n = 8) in nmol of Pi liberated/min/mg protein were 18 + 0.8 (control) and 30 f 0.8 (1 .O LD50 MIC treated).

To confirm our results further, liver SMP were prepared for the enzyme assays. The activities of NADH oxidase and NADH-cytochrome c reductase were significantly decreased in hepatic SMP of 1.0 LD50 MIC-treated rats as compared to controls while NADH dehydrogenase, succinate oxidase, and cytochrome oxidase activities remained unaltered (Table 6). In agreement with the in vitro observation, there was a significant delay in the onset of anerobiosis, cytochrome b reduction in MIC-treated liver SMP with NADH as substrate while no appreciable change was noticed with succinate as substrate (Table 7). DISCUSSION The present results demonstrate the ability of MIC to impair cellular respiration through inhibition of mitochondrial electron transport and energy transduction, especially at the complex I region of electron transport chain and help to infer the induction of histotoxic hypoxia in vivo by inhibiting the oxidation of NADH and NAD+-linked substrates in rats

TABLE 3 Zn Vitro Inhibitor Potency of MIC for Various Mitochondrial Reactions Reaction NADH oxidase NADH-cytochrome c reductase NADH dehydrogenase (fenicyanide reductase)

EC50 (PM) 102 * 9.0 238 + 7.4 320 f 12.0

Note. Frozen and thawed mitochondria (about 4 mg protein) treated with MIC (35-140 PM per mg of protein) for 2 min at 30°C were used for the various mitochondrial enzyme assays.The values of MIC concentrations for 50% effect (EC50) calculated from the regression equations are means + SD of six independent determinations.

FIG. 3. In vitro effect of MIC on NADH oxidation in rat liver submitochondrial particle (SMP). [Freshly prepared SMP (0.4 mg protein) were used in a reaction volume of 1.4 ml for determination of NADH oxidase activity by polarography. The values in the parentheses refer to the rates of oxygen uptake (ng atom O/min/mg protein) on stirring in 0.18 mM NADH. The value of MIC concentration for 50% effect (EC50) calculated from regression equation is 95 f 5.2 PM (means -+ SD, n = 6).]

176

JEEVARATNAM,

VIDYA,

FIG. 4. In vitro effect of MIC on reduction of cytochrome b in liver submitochondrial particles (SMP). [Substrate, NADH, was added as indicated and the reduction of cytochrome b in the reaction mixture containing I mM KCN was recorded at 562-577 nm. MIC (70 pM) was added to 1 mg of SMP (sample 2) 1 min before addition of NADH. The values (means k SD, n = 4) of the time of onset of anerobiosis are 57 f 2.4 set (control) and 114 + 4.8 set (MIC treated).]

receiving a lethal dose of MIC SC.The ability of MIC to cause respiratory distress (e.g., gasping, dyspnea) in laboratory animals was hitherto attributed to its potent sensory and pulmonary irritant effects leading to respiratory depression (Nemery et al., 1985; Ferguson et al., 1986) and the possible impaired hemoglobin function because of N-terminal carbamylation (Lee, 1976; Ramachandran et al., 1988). However, MIC interaction with hemoglobin was shown to play little role in induction of tissue hypoxia (Maginniss et al., 1987; Jeevaratnam and Vaidyanathan, 1992b). Irrespective of the route of exposure, MIC induced acute lactic acidosis (Jeevaratnam et al., 1990) which cannot be attributed to the respiratory irritant effects of MIC. Subcutaneous administration of MIC in rabbits, wherein the sensory and pulmonary reflexes were eliminated, revealed the cardiovascular effects of MIC causing hypovolemic hypotension leading to reduced perfusion and stagnant hypoxia (Jeevarathinam et al., 1988). The present study provides evidence for the occurrence of concurrent histotoxic hypoxia. The inhibition of state 3 respiration, stimulation of state 4 respiration, abolition of the respiratory control, and decreased ADP/O ratio by MIC in tightly coupled mitochondria in vitro (Fig. 1 and Table 1) reveal its dual mode of action, an inhibitor of state 3 respiration and an uncoupler, similar to that of an azodye, 2-methyl-4-dimethylaminoazobenzene (Saikumar and Kurup, 1984a,b). The uncoupling action of MIC becomes weak at a very high concentration of MIC possibly because of its potent inhibitory action at complex I region (Table 1). Such effects were also observed in the isolated hepatic mitochondria from rats administered 1.0 LD50 MIC sc (Table 5). Based on liquid phase disappearance study, it is presumed that direct toxic effects from MIC would be confined to the tissues of contact such as eye and lungs and systemic toxicity would be due to hydrolysis products

AND VAIDYANATHAN

such as MA and DMU (Andersson et al., 1985). This led us to examine the effects of hydrolysis products of MIC, MA, and DMU on the isolated liver mitochondria in vitro. As there were no appreciable effects by the hydrolysis products up to 300 PM per mg protein (data not shown) it may be concluded that the observed effects are due to MIC per se. Moreover, in spite of its high reactivity, Ferguson et al. (1988) demonstrated a rapid uptake and distribution of 14Cto blood and various tissues during and following exposure through inhalation to [ 14C]MIC in mammals, while Bhattacharya et al. ( 1988) reported that MIC either inhaled or administered ip reached various organs in its active form as evidenced by carbamylation of tissue proteins. Recently, glutathione has been proposed as a possible carrier for transport of MIC in vivo (Pearson et al., 1990; Coghlan, 1991). The vast difference in EC50 values (concentration of MIC required for 50% effect) for the inhibition of oxygen uptake between the substrates, glutamate + malate and succinate, (Table 2) depicts greater susceptibility of NAD+-linked electron transport for inhibition by MIC as nearly five- to sixfold concentration of MIC needed to inhibit succinate oxidation by 50%, while cytochrome oxidase activity remained unaffected (data not shown). To rule out the possibility that the inhibitory action of MIC is caused by interference with the translocation of NAD+-linked substrates (inhibition of adenine nucleotide translocation may be ruled out by the lower potency in the inhibition of succinate oxidation), the effect of the compound on substrate oxidation by liver SMP was tested. The observed concentration-dependent inhibition of NADH oxidation in liver SMP (Fig. 3) with low EC50 value showed the direct effects of MIC on the electron transport system of complex I region of the respiratory chain. MIC was also found to impair mitochondrial respiration in vivo only at complex I region in rats administered 1.O LD50 MIC SC(Table 5). A differential inhibition of the enzymes at comTABLE 4 In Vitro Effect of MIC on the ATPase Activity and Lipid Peroxidation of Rat Liver Mitochondria

Treatment Control MIC, 70 PM MIC, 140 PM MA, 70 PM MA, 140 PM 2,4-DNP, 100 /.tM

ATPase activity (nmol of Pi formed/min/mg protein) 19 + 25 f 36 + 20 f 21 f 96 f

0.8 0.5” 0.7n*b 0.4 0.7 5.1”

Lipid peroxidation (nmol MDA/mg protein) 0.41 f 0.027 0.41 f 0.031 0.39 zt 0.037 ND ND ND

Note. Values are means rfr SD, n = 4; statistical analysis by Student’s t test. ND, not determined. ’ Statistically significant compared to control. b Statistically significant compared to the lower concentration.

MIC EFFECTS ON MITOCHONDRIAL

Effect of MIC Intoxication

177

RESPIRATION

TABLE 5 on Oxidative Phosphorylation

of Rat Liver Mitochondria

Oxidative phosphorylation Glutamate + malate Group Control 0.5 LD50 ( 164.3 1.O LD50 (328.6 LSD

MIC mg/kg) MIC mg/kg)

Succinate

State 3’

State 4’

RCI

ADP/O

State 3”

State 4”

RCI

ADP/O

111 f 6.4a

12 * 2.3a

9.3 _+ 1.38a

2.8 -C 0.12a

211 + 9.6a

35 * 3.5a

5.4 f 0.39a

1.6 r 0.05a

113 f 8.2a

12 f 0.9a

9.4 f 0.7la

2.8 + 0.18a

215 + 16.6a

33 f 4.8a

5.5 + 0.40a

1.4 f 0.05a

86 + 1.4b 9.13

19 f 1.8b 2.35

4.4 f 0.43b 1.17

2.3 f 0.09b 0.17

225 f 5.5a 14.4

40 f 3.3b 4.9

5.6 k 0.44a 0.63

1.5 f 0.1 la 0.11

Note. Values are means + SD; n = 8. a Rate of oxygen uptake (ng atom O/min/mg protein); Means within a column followed by the same letter(s) are not significantly different (p < 0.05, Student’s range test).

plex I region by MIC was observed both in vitro (Table 3) and in vivo (Table 6); NADH-cytochrome c reductase was inhibited to a lesser extent by MIC compared to NADH oxidase while NADH dehydrogenase was not affected in vivo. A similar differential inhibition of enzymes at complex I region was noticed with sodium amytal and p-chloromercuriphenyl sulfonate (Hatefi and Rieske, 1967). As MIC is a very highly reactive molecule we studied the specificity of its interaction at complex I region of the electron transport chain by examining the effect of protein concentration on the inhibitory action of MIC in the polarographic experiments. The observation that doubling the mitochondrial protein concentration halved the extent of inhibition (Fig. 2) has demonstrated the specificity for MIC interaction

TABLE 6 Effect of MIC Intoxication on Various Enzymes of Submitochondrial Particles (SMP) of Rats Enzyme activity (per min per mg of protein) NADH Oxidase (n moles of NADH oxidized) NADH-cytochrome c reductase (n moles of ferricytochrome reduced) NADH dehydrogenase (n moles of K Fe(CN) reduced) Succinate oxidase (ng atom 0) Cytochrome oxidase (n moles of ferrocytochrome oxidized)

MIC treated (328.6 mg/kg)

Control 475+

15

322+

23”

479+

16

432 +

6”

4918 Z!Z136 380 -t 36 960 + 68

5143 * 170 390 rt 28

at complex I region of electron transport chain. The complex I region has a higher concentration of lipids vulnerable to attack by free radicals and nonpolar compounds. Following in vitro exposure to a radical generating system, state 3 was found to be markedly inhibited (Hillered and Ernster, 1983). As the degree of inhibition depended on the ratio of concentration of MIC to that of mitochondrial or SMP protein in the reaction system, we looked into the possibility of some of the observed effects being mediated by augmented lipid peroxidation. This has been ruled out as the MDA content of coupled mitochondria treated with MIC at 70 and 140 PM per mg protein for 2 min has not been altered (Table 4). The in vitro observation of a twofold delay in the onset of anerobiosis, cytochrome b reduction in liver SMP with NADH (Fig. 4) and unaltered with succinate (data not shown), as well as the significant delay in the onset of anerobiosis in liver SMP isolated from rats administered 1.0 LD50 MIC SC, in vivo, only with NADH and not with succinate as substrate (Table 7) confirms that MIC affects the electron transport system of complex I region in the respiratory chain.

TABLE 7 Effect of MIC Intoxication on Reduction of Cytochrome b in Liver Submitochondrial Particle (SMP) of Rats Sample

Substrate

Control (SW

MIC treated bed

SMP

NADH Succinate

56 rt 1.2 162 f 3.3

89 f 3.4“ 165 f 2.5

980 + 85

Note. Values are means * SD, n = 8. u Statistically significant compared to control (Student’s t test).

Note. Values are means + SD, n = 8. For additional details see legend to Fig. 4. a Statistically significant compared to control (Student’s t test).

178

JEEVARATNAM,

VIDYA,

The ability shown by MIC to stimulate state 4 respiration and abolish respiratory control (Fig. 1, Tables 1 and 5) and its influence on ATPase activity (Table 4) reveal its action as an uncoupler but less potent compared to the known uncoupler, 2,4-dinitrophenol. However, MA has not influenced ATPase activity to any extent at the concentrations studied (Table 4) even though it is known to uncouple plant mitochondrial oxidative phosphorylation at millimolar concentrations (Ort, 1978). From the results presented in this report, one can conclude that MIC per se acts as a potent inhibitor of electron transport along with an unrelated weak uncoupling effect and has the ability to impair cellular respiration through inhibition of mitochondrial electron transport and energy transduction, especially at complex I region of respiratory chain and to induce histotoxic hypoxia in viva by inhibiting the oxidation of NADH and NAD+-linked substrates in rats receiving a lethal dose of MIC SC. ACKNOWLEDGMENTS The authors are grateful to Dr. R. V. Swamy, Director, Defence R&D Establishment, Gwalior, for his keen interest in this work. The authors also thank Prof. C. K. R. Kurup and Prof. T. Ramasanna of Indian Institute of Science, Bangalore, for their critical and useful discussions.

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