In vitro kinetics of the mouse hepatic arginase inhibition by hexachlorophene

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

ln Vitro Kinetics

of the Mouse Hepatic by Hexachlorophene

G.~ENKATESWARA Molecular

Physiology

Division,

28, 308-317 (1987)

Deporrmenf

Arginase

PRASAD, W. RAJENDRA,AND of Zoology.

S. V. University,

Inhibition

K. INDIRA~ Tirupati-517

502, A. P., Indiu

Received August 18, 1986, accepted February 23, 1987 Hexachlorophene (HCP) inhibited mouse hepatic arginase with a half-inhibitory concentration (I,,) of 1.3 x 10e6 M. The enzyme-HCP interaction coefficients denoted alteration of inhibitory sites on the enzyme, depending on the concentration of HCP. The K, of hepatic arginase was 8.3 mM which was not significantly affected in the presence of HCP. whereas the V,,, and Vmax-to-K, ratios were reduced by HCP by the same factor, denoting a pure noncompetitive inhibition which was further confirmed by the finding that the inhibitor constants K, and Ki’ were equal to one another and also to the I,,. The enzyme in the presence of HCP demanded higher than the normal amount of activation energy (E.J. The MnZ+ -activated arginase was less susceptible than was the MI-$+-deficient enzyme to HCP inhibition. These findings, together with recovery studies employing bovine serum albumin. denoted that HCP may induce conformational changes on the enzyme, probably by exerting strong interactions at the noncatalytic sites of the enzyme. c 19x7 Academic

Press. Inc.

INTRODUCTION

ammonia uptake (10) and resulting in hyencephalopathy (1 I), Although hexachlorophene (HCP) is perammonemic tempted us to investigate the effects of used extensively in medical, agricultural, HCP on the hepatic arginase, an enzyme and consumer products (l-3), its efficacy in many of these preparations has been involved in the detoxication of ammonia. questioned recently because of the inherent MATERIALS AND METHODS toxicity caused by this compound. Animals. Adult male mice (Mus booSymptoms of poisoning by hexachlorophene are mainly neurological (4-S). But duga), maintained under laboratory conditions (temperature 30 ? 2”C, relative hueven in lethal cases, the morphological midity 75%, and a light period of 12 hr) changes in the brain are limited to a mild with free access to food and water were spongy degeneration of white matter which used in the present study. This species has are yet to be related to precise biochemical been used successfully earlier in our laboalterations. Recent studies in our laboraratory as an animal model in the toxicologtory demonstrated a pronounced elevation ical investigations (12- 14). of cerebral and blood ammonia concentraPurification of hepatic arginase. Mouse tions of mice intoxicated with HCP, which (M. booduga) hepatic arginase (E.C. coincided with a sharp reduction in hepatic arginase activity levels (9). However, the 3.5.3.1, L-arginine ureohydrolase) was puactual mechanism of the loss of hepatic ar- rified by the method of Schimke (1.5) with ginase activities and manifestation of hy- slight modifications as described previously perammonemia is not clear. The fact that (9). The mouse liver was isolated from freshly decapitated mice and homogenized the impairment of hepatic ammonia detoxifying function allows the diversion of am- (10% w/v) in a solution containing 0.2 M KCl, 0.05 M MnSO,, and 0.01 M Tris chlomonia to the systemic circulation, causing ride, pH 7.2. The homogenate was centrihyperammonemia and increased cerebral fuged at 2000g for 20 min at 4°C and the ’ To whom all correspondence should be addressed. supernatant fluid was collected. The res308 0048-3575187 $3.00 Copyright All rights

C 1987 by Academic Press, Inc. of reproduction in any form reserved.

MOUSE

HEPATIC

ARGINASE

INHIBITION

idue was washed twice with homogenizing medium to ensure complete extraction. The enzyme extract was brought to 0°C and 4 vol acetone was added with constant stirring in a flat-bottomed flask jacketed with freezing mixture. The precipitate was collected after centrifugation at 15,OOOg for 10 min at - 10°C. A l-g sample of the precipitate was extracted with three 15ml portions of 0.01 M Tris chloride buffer, pH 7.2, containing 0.05 M MnSO,, and the sediment was removed by centrifugation and the supernatant dialyzed overnight against the same buffer. The dialyzed extract was passed through a DEAE-cellulose column equilibrated with the extraction solution (0.01 M Tris chloride, pH 7.2, containing 0.05 M MnSO,). The enzyme was not adsorbed onto the column. The DEAE-cellulose column effluent was heated at 55°C for 10 min in a final concentration of 0.05 M MnSO, to activate the enzyme, and the protein that was precipitated was removed after centrifugation. The activated sample was then passed through a CM-cellulose column equilibrated with 0.01 M Tris chloride, pH 6.2. This process led to the adsorption of arginase to the column. The enzyme was eluted from the column with different concentrations of NaCl solutions (0.05 to 0.3 M in 0.01 M Tris acetate, pH 6.2). To the fractions containing arginase, 0.1 M MnSO, was added to a final concentration of 0.05 M. Arginase was precipitated from the active fractions with (NH& SO, between 2.0 and 2.5 M. The precipitate was dissolved in the required volume of 0.01 M Tris chloride, pH 7.5, containing 0.05 M MnSO,. The average purification obtained by the above method was 320-fold over the crude extract. The specific activity in the purified preparation was 1250. Assay of arginase. Arginase was assayed considering the amount of urea produced by employing the diacetyl monoxime reaction as outlined by Paik et al. (16). Purified preparation containing 1.5 units (unless otherwise stated) was incubated at 37°C for

BY HEXACHLOROPHENE

309

10 min with a reaction mixture containing 0.15 M L-arginine (unless otherwise stated) preadjusted to pH 9.5 with 1 N HCl, glycine:NaOH buffer (0.1 M, pH 9.5), and MnSO, (0.05 M) in a final volume of 1 ml. The reaction was stopped by adding 2.5 ml of 10% HClO,. To 0.5 ml of the clear supernatant obtained after the centrifugation of the above mixture, 5 ml of H,SO,-H,PO, ( 1: 3) mixture was added followed by 0.5 ml of 0.1 M FeCI, and 1 ml of O-75% diacetyl monoxime in ethanol. The contents were mixed thoroughly in a vortex-200 cyclomixer and heated in a water bath at 90°C for 15 min. After cooling in an ice bath for 5 min, the samples were read at 480 nm against the reagent blank in a Systronics spectrophotometer. The enzyme unit was defined as the amount of enzyme that produces 1 Frnol of urea x min- l at 37°C. The protein content of the enzyme source was estimated by using crystalline bovine serum albumin as standard (17). Modifications of the basic assay are given wherever applicable. Half-inhibitory corzcentration (ZsO). Hexachlorophene (2,2’-methylenebis-3,4,6trichlorophenol: 99.8% pure), obtained from Sigma Chemical Co. (St. Louis, MO), was recrystallized twice from isopropanolwater to yield a chromatographically pure compound which was used throughout the investigation. The pure HCP was dissolved in 0.05 M NaOH (vehicle), and this preparation (20 ~1) was injected into the reaction mixture with a microsyringe to give a desired final concentration of HCP. Arginase was assayed over different concentrations of HCP. The control assays contained an isomolar quantity of the vehicle containing no HCP. The half-inhibitory concentration (I,,) of HCP against mouse hepatic arginase was determined by the method of Job et al. (18). Enzyme-HCP

interactions. The cooperativity of the binding of HCP to the enzyme was assessed based on the interaction coefficients (n’ values) obtained from the Hill plots (19). Kinetic parameters. Arginase was as-

310

VENKATESWARA

PRASAD,

sayed over a range of L-arginine concentrations (lo-100 mM), and the MichaelisMenten constants (K,) and maximal velocities (V,,) were determined by employing the least squares as the best fit (20). Effect of HCP on substrate kinetics was studied at selected HCP concentrations. The inhibitory constants Ki and Ki for hepatic arginase with HCP were estimated by Dixon plots (21) and Cornish-Bowden plots (22), respectively. Temperature-dependent activity. The effect of temperature on the arginase activity was examined in the presence and absence of HCP from IO-60°C at intervals of lO”C, pH 9.5, and 300 mM L-arginine concentrations. The incubation was for 10 min. Other conditions of the assay remain the same as described earlier. The apparent energy of activation (&) was calculated by fitting the data to the Arrhenius equation (23). Removal of Mn*+ by dialysis. Dilute purified arginase extracts containing 50 mM Mn*+ were dialyzed against 0.01 M TrisHCl buffer, pH 7.5, for 2 days with four changes of the dialysis bath. The removal of Mn*+ was verified by reactivation studies as described earlier (9). The dialysis process led to the removal of 50% of the enzyme-bound Mn*+. The remaining 50% of the Mn*+ could not be removed by continued dialysis, since that fraction was bound very tightly to the enzyme and its removal by other methods caused irreversible denaturation of the enzyme, also a characteristic of other basic types of arginases (24). Statistics. The statistical significance was assessed by Student’s t test. RESULTS

AND

RAJENDRA,

AND

INDIRA

2% (mean 2 SD) inhibition on the enzyme when tested in six replicates. Interaction of HCP with the enzyme was assessed by fitting the data obtained from dose-inhibition studies to the Hill equation. Results showed that small changes in HCP concentration produced large changes in V,,,. The Hill plot exhibited bilinearity showing sharp transition at higher concentrations of HCP (Fig. I). The interaction coefficient (n’) at HCP concentrations below I,, was 1.1, while at higher concentrations (>I,,) a value of 2.5 was recorded. These findings may reflect the alteration of inhibitory sites on the enzyme depending on the concentration of HCP. Since HCP does not irreversibly inhibit or denature the arginase, the Hill coefficients suggest the binding of HCP to three or fewer sites on each enzyme molecule in a cooperative manner. A similar sort of cooperative binding of HCP has been reported with dehydrogenase enzymes (25, 26). The results obtained from the substrate kinetic studies suggest that the enzyme displays hyperbolic kinetics with respect to substrate saturation. The hydrolysis of arginine was dependent on its concentration up to 40 mM. Above this concentration, the velocity became substrate independent. The Michaelis-Menten constant (K,) was 8.3 mM under defined conditions (Table 1).

DISCUSSION

Hexachlorophene in micromolar concentrations strongly inhibited the mouse hepatic arginase. The half-inhibitory concentration (I,,,) for HCP determined by the method of Job et al. (18) was found to be 1.3 x 1O-6 M when 1.36 pg of enzyme protein was used in the assay. The I,, obtained by the above method produced 50 ?

\

I -6.0 LOG

CHCPI

I -5.0

(Ml

FIG. 1. Hill plot of the inhibition of hepatic arginase by HCP, from which interaction coefficient (n’) values were obtained. Each point is the mean of six observations with less than 10% variability.

I

MOUSE HEPATIC

ARGINASE

INHIBITION

BY HEXACHLOROPHENE

311

The HCP inhibition studies show that the K, is not significantly altered in the presence of HCP up to 2.6 x 1O-6 M concentration, whereas the V,,, was depressed drastically at different concentrations of HCP (Table 1 and Fig. 2). The V,, and the ratio of V,,,,,IK, were decreased in the presence of HCP by the same factor (Table 1). Such an effect is characteristic of a pure noncompetitive inhibition, as suggested by a common intercept on the abscissa in the Lineweaver-Burk plots (Fig. 2), Dixon plots (Fig. 5), and Cornish-Bowden plots (Fig. 6). The secondary plots or replots (20, 27) of the slopes and intercepts obtained in the double reciprocal plots of the hepatic arginase inhibition by different concentrations of HCP yielded parabolic curves (Figs. 3 and 4), indicating that the inhibition of the enzyme by HCP is a slope-parabolic, intercept-parabolic, noncompetitive inhibition. The nonlinearity of the secondary plots may indicate a complex type of inhibition involving two or more mechanisms to exert an inhibitory effect (20). A plot of reciprocal velocity versus HCP concentration yielded at three different fixed substrate concentrations a family of lines which intersected at a common point on the abscissa (Fig. 5). This intersecting point denotes - Ki where Ki is the dissociation constant of EI complex (21). A Ki

FIG. 2. Lineweaver-Burk showing the effect of HCP mouse hepatic arginase: (A) IOF M HCP (O), (C) 1.95 (D) 2.6 x IOF M HCP (0). expressed as pm01 urealmin vidual assays with less thnn

double reciprocal plots on substrate kinetics of no HCP (0). (B) 1.3 X x IO-6 M HCP (O), and Enzyme activities (v-l) are means of six indi10% variability.

312

VENKATESWARA

PRASAD,

AND

INDIRA

L

-Ye--+-

[HCPI (Mx 106)

FIG. 3. Replots of slopes reciprocal plots (Fig. 2).

FtAJENDRA.

obtained

from

the double

value of 1.35 x lop6 M (if HCP concentrations of not more than I,, were used) was obtained from such a plot. Similarly a family of lines given by Cornish-Bowden plots (Fig. 6) have shared a common intercept on the abscissa, giving a value of 1.26 x 10e6 M for Ki, the dissociation constant of EIS complex. An advantage of treating the same data according to both Dixon (Fig. 5) and Cornish-Bowden (Fig. 6) was that these two methods yield two different inhibitor constants (Ki and Ki, respectively) which are highly useful in determining or confirming the type of inhibition (21. 22, 28). From the results it is clear that the affinity of the enzyme for the inhibitor is not changed when the enzyme also binds substrate; i.e., within the experimental error the dissociation constants of both EI com-

FIG. 5. Dixon plots showing the inhibition pattern of mouse hepatic arginase at three substrate concentrations: (A) 80 mM, (B) 20 mM, and CC’) 10 mM. Each point represents the mean of six assays with less than 10% variability.

plex (Ki) and EIS complex (Kf) are equal to one another and also to the half-inhibitory concentration, strongly confirming that HCP exerts purely noncompetitive inhibition on hepatic arginase. However, the inhibitor constants mentioned in the foregoing discussion were obtained with HCP concentrations equal to or less than half-inhibitory concentration. At higher inhibitor concentrations, both the plots exhibited diversions from the linearity (Figs. 5 and 6). which may be due to the operation of new inhibitor binding sites of the enzyme as was also indicated by the Hill plots (Fig. 1). The inhibitor constants were also determined at high concentrations of HCP and are presented in Table 1. Low inhibitor constants

tHCP1

I

1.0 [HCPI

FIG. 4. Replots of intercepts reciprocal plots (Fig. 2).

I 2.0

(M x 10~)

obtained

from

doable

I"‘1061

FIG. 6. Plots of (S)/v versus inhibitor (HCP) concentration (Cornish-Bowden plots) showing the effect of HCP on arginase at three different L-arginine concentrations: (A) 80 mM, (B) 20 mM, and(C) IO mM. Each point is the mean of six observations.

MOUSE

HEPATIC

ARGINASE

INHIBITION

at high concentrations of HCP suggest an increased affinity of HCP to the enzyme at HCP concentrations above I,,, which may be due to the operation of multiple inhibitor binding sites which when occupied may increase inhibitory effect on the enzyme. It is also reported that within the experimental error the Ki values observed at HCP > I,, were equal to the Ki values and also to the I,, values derived at similar concentrations of HCP, indicating that the type of inhibition is noncompetitive even at higher concentrations of HCP. The effect of temperature on the inhibition of arginase by HCP is shown in Fig. 7, in the form of Arrhenius plots of log,, V,,, versus the reciprocal of the incubation temperature. In the absence of HCP the enzyme activity was increased with the rise of temperature up to 60°C and a straight line was obtained in the plot. The optimal temperature was 60°C under the defined conditions. From the Arrhenius plots, the apparent activation energy (E,) of the arginase in the absence of HCP ranged from 16,731 to 17,082 cal/mole over the temperature range of lo-60°C. In the presence of 2.3 x lop6 M HCP (near half-inhibitory concentration), arginase showed a break in the Arrhenius function at temperatures

‘ii-j;; Ibo \\ ( 360

10’

x

‘/,

360

(*K-l1

FIG. 7. Arrhenius plots qf log V,,,,, versas IIT (“K-l) from which the activation energy (E,) values were obtained. Each point is the mean of six observations with less than 10% variability. The reaction mixture contained (A) no HCP and (B) 2.3 x IOm6 M HCP. The incubation was for 5 min with 4.5 units of arginase. The L-arginine concentration in the basic nssav medium was increased to 200 mM.

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above about 45°C probably as a result of HCP-induced transconformational changes in the enzyme protein. The E, of arginase in the presence of HCP ranged from 18,016 to 20,617 cal/mole over the temperature range lo-4O”C, whereas at higher temperatures (40-60°C) it was decreased to a range of 7122 to 11,804 Cal/mole (Table 2). The results suggest that at each given temperature range (up to 4o”C), the enzyme demanded a relatively higher amount of the energy of activation in the presence than in the absence of HCP, suggesting that the enzyme is catalytically less efficient in the presence of HCP. Since the bovine serum albumin (BSA) is known to bind HCP strongly (29) and reported to reverse the inhibitory effect of HCP on various enzymes (25) and mitochondrial phosphorylation (30), experiments were carried out to study whether BSA can reverse the inhibitory effect of HCP on arginase. When 1.36 pg (1.7 units) of enzyme protein was used, addition of 40 mg/ml of BSA completely protected the enzyme from inhibition caused by 1.3 x 1O-6 M HCP under defined conditions (Fig. 8). Requirement of such a large concentration of BSA to prevent HCP inhibition suggests a higher degree of affinity of the binding of HCP to the enzyme protein. The addition of similar quantities of BSA to the enzyme preincubated with HCP for 60 set resulted in only a partial reversibility of the HCP inhibition. The prevention and partial reversibility of HCP inhibitory effect by BSA point out the importance of in viva protein concentration as a protective factor against HCP effects. The potent inhibitory effect of HCP may be related to the high degree of chlorination of the drug as suggested by Nakaue et al. (31), who have demonstrated an increased uncoupling activity of the chlorinated bisphenols with increased chlorination of the aromatic rings. The existence of this type of correlation was further confirmed by Wang and Buhler (26), who reported very low half-inhibitory concentrations for HCP

314

VENKATESWARA

PRASAD,

RAJENDRA,

TABLE

AND

INDIRA

2

Effect of HCP (2.3 x 10-e M) on the Activation Energy (E,) Requirements of Mouse Hepatic Arginase Activation Temperature range (“C)

No HCP

10-20 20-30 30-40 40-50 50-60

17,082 16,731 16,839 17,049 17,070

Note. The assay conditions were SD of six determinations. Student’s

+ ” + ? 2

(EJ in Cal/mole

energy 2.3 x 1O-6

4.50 420 640 470 600

18,016 18,270 20,617 11,804 7,122

i i ?I 2 i-

the same as those given for Arrhenius r test: *P < 0.001; **P < 0.01.

as compared with other bisphenols of comparatively low degree of chlorination when tested on torula yeast glucose-6-phosphate dehydrogenase. Since HCP is known to bind tightly to bovine serum albumin (32), erythrocyte membranes, and various dehydrogenases (25) through hydrophobic interactions, a similar interaction of HCP with hepatic arginase might have caused the enzyme inhibition. The data presented in Table 3 show that the Mn2+-activated arginase was less susceptible to HCP inhibition as evinced by higher values of I,, as compared with arginase preparations from which 50% of the Mn2+ was removed by dialysis. Since MS+ is known to act as a co-factor for ar-

M HCP

% Alteration

500”” 460* 590* 380* 220* plots

+ 5.5 + 9.2 +22.4 - 30.8 -58.3 (Fig.

7). & values

are means

2

ginase and to activate the enzyme by stabilizing the active conformation of the enzyme protein (24, 33), it can be predicted from the present findings that HCP may induce conformational changes on the enzyme molecule, probably by exerting strong interactions at the noncatalytic sites of the enzyme molecule as denoted by the unaffected K, in the presence of HCP (Table 1 and Fig. 2). Furthermore, when the concentration of the enzyme protein was increased in the assay medium, the I,, values were increased gradually (Table 4). Thus a likely explanation for the mechanism of the inhibition caused by HCP may be that the hydrophobic properties (34, 35) of the drug may induce conformational changes in the enzyme protein. Although numerous studies (36-39) have

z TABLE

3

Effect of Mn2+ on the Inhibition Arginase by HCP Sample

40 BSA

final

concentration

fmgiml)

FIG. 8. Protective effect of bovine serum albumin (BSA) on HCP-induced inhibition of mouse hepatic arginase. (A) Enzyme preincubated with HCP for 60 set at 3PC was added to the reaction mixture containing BSA. (B) Enzyme-BSA mixture was added to the reaction mixture containing HCP. The data points are means of six observations with less than 10% variability.

Mn*+ removed enzyme preparationa Enzyme activated at a final Mnr+ concentration of 50 mW Enzyme activated at a final MnZ+ concentration of 100 mW

of Hepatic I, HCP CM 0.9 k 0.06 x 1O-6 1.3 + 0.11 x 10-k 1.2 It 0.09 x 10-k

Note. Values are means f SD of six observations. a Dilute purified preparation of the hepatic arginase was dialyzed against 0.01 MTris-HCI buffer. pH 7.5. for 2 days to remove Mn*+ partially. b The reactivation of the enzyme was carried out at 55°C for 10 min. c Significantly different from Mnr+-removed enzyme preparation (P < 0.001).

MOUSE HEPATIC

ARGINASE

INHIBITION

TABLE 4 Relation between the Concentration of the Enzyme (Arginase) Protein and the Degree of Inhibition by HCP Concentration of enzyme protein (kg/ml) 1.4 2.8 5.6 11.2

Half-inhibitory concentration (I,,) HCP (M) 1.3 1.9 2.5 3.8

k ? k k

0.11 0.16 0.23 0.26

x x x x

10-G 1O-6 10m6 10-e

Note. Values are means 4 SD of six individual observations. L-arginine concentration in the basic assay medium was increased to 0.5 M.

dealt with the toxic effects of HCP, the precise biochemical mechanism of HCP action have remained obscure. Despite various proposed mechanisms (30,34,40,41), none could account clearly for the high toxic potentials of HCP, while some considered that HCP has multiple targets of action (42, 43) which may exert a synergistic effect. HCP was reported to inhibit various hepatic and brain enzymes (13, 25, 44) and uncouple the phosphorylating respiration in liver and brain mitochondria (31, 40). The results of the present study suggest that hepatic arginase is inhibited strongly by HCP. Since it is well established that the capture of ammonia in the Krebs-Henseleit cycle constitutes a potential route of ammonia disposal (11, 45), inhibition of arginase may contribute to the accumulation of toxic ammonia. It may be difficult at present to correlate the in vitro effects of HCP with the in viva effects because the tissue concentrations of HCP were not detected in this study. However, it is plausible that when HCP is given orally, the drug is transported first to the liver through the hepatic portal system and may lead to the accumulation of a large concentration of HCP in the liver because of the complex HCP-protein interactions (34). Further, HCP is highly resistant to oxidative metabolism and conjugation (46) and even if HCP is oxidized via a cytochrome P-450 mixed function oxidase system in the liver, it produces highly

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315

reactive metabolites which can bind covalently to protein and exert toxic effects (35). Labeled studies suggest that the halflife of radioactivity from [14C]HCP is quite long in liver, indicating the persistence of HCP or its reactive metabolites in vivo for appreciable duration (35). In agreement with these findings, Chow et al. (47) and Kawashima et al. (48) have reported the highest concentrations of HCP in liver among various organs tested, following an acute oral administration of HCP. In consolidation, the previous reports suggest the possibility of a high degree of HCP accumulation in liver. Persistence of a high in viva concentration of HCP in liver may exert inhibitory effects on hepatic arginase. Recent studies in our laboratory demonstrated that when HCP was administered orally to mice at a dosage of 60 mg/kg/day for 7 consecutive days, the hepatic arginase showed 79% loss in the activity levels with subsequent increases in blood and cerebral ammonia concentrations of 105 and 140%, respectively, as compared with those of controls (9). These findings are compatible with the hypothesis that similar sort of inhibitory effects of HCP on hepatic arginase as observed in the present study may also manifest in viva and contribute to the hyperammonemia during HCP intoxication. It is an established fact that excess ammonia causes severe metabolic disturbances in the brain (49-51) and exerts toxic effects on brain membranes (52, 53). The direct effects of HCP on brain include altered membrane properties and membrane-bound enzymes (13, 41, 54). Hence it can be suggested that the elevated levels of ammonia may act in concert with direct effects of HCP on the brain and thus may deserve consideration in the expression of neurotoxicity. However, further studies involving the effects of HCP on the other enzymes of the ammonia detoxication system are in progress which may offer an elucidative explanation on the mechanism of the manifestation of hyperammonemia during HCP ad-

316

VENKATESWARA

ministration establishment

and its implications of HCP neurotoxicity.

PRASAD,

in the

ACKNOWLEDGMENT We are thankful to the Council of Scientific and Industrial Research, New Delhi, India, for the financial assistance in the form of a Senior Research Fellowship to Dr. G. V. Prasad.

1. 2.

3.

4. 5.

6.

7. 8.

9.

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11. 12.

RAJENDRA,

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30.

31.

32.

33. 34.

35.

36.

37. 38.

39.

40.

ARGINASE

INHIBITION

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