168 Biochimica et Biophysica Acta, 116 t (1993) 168 176 @ 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4838/93/$06.00

BBAPRO 34386

Inhibition of intestinal peroxidase activity by nonsteroidal antiinflammatory drugs

Ratna Chatterjee, Uday Bandyopadhyay, Dipak Bhattacharyya and Ranajit K. Banerjee Department of Physiology, Indian Institute of Chemical Biology, Calcutta (India)

(Received 11 August 1992)

Key words: Nonsteroidal antiinflammatory drug; Intestinal peroxidase; Eosinophil peroxidase; Lactoperoxidase; Horseradish peroxidase; Indomethacin; Acetylsalicylic acid

The peroxidase activity of the mitochondrial fraction of rat intestine is inhibited in vitro by non-steroidal antiinflammatory drugs (NSAIDs), such as indomethacin (IMN) and acetylsalicylic acid (ASA), the former being more potent than the latter. The peroxidase was solubilised by cetab-NHaC1 extraction and purified to apparent homogeneity by Sephadex G-150 gel filtration and affinity chromatography on Con-A Sepharose. The purified enzyme activity was 80% inhibited by 150/z M IMN and 50% by 2.67 mM ASA. IMN could also inhibit lactoperoxidase activity to the same extent but not the horseradish peroxidase activity. The inhibition of peroxidase-catalysed iodide oxidation by IMN and ASA was optimal at pH 5.5 and 4.5, respectively. Kinetic studies revealed that the inhibition by IMN was competitive with respect to iodide or guaiacol, while the inhibition by ASA was noncompetitive and reversible in nature. Studies of some structural analogues showed that indole-3-acetic acid was as effective as IMN, while salicylic acid was more potent than ASA. Spectral studies showed a small bathochromic shift of the Soret band of the enzyme by IMN, suggesting its possible interaction at or near the heme moiety. The competitive nature of IMN may be explained as due to its oxidation by the peroxidase to a product absorbing at 412 nm, the formation of which is inhibited by iodide. We suggest that IMN inhibits intestinal peroxidase activity by acting as a competitive substrate for the enzyme. As intestinal peroxidase is mainly contributed by the invading eosinophils, NSAIDs may affect the host defence mechanism by inhibiting the activity of the enzyme.

Introduction

Nonsteroidal antiinflammatory drugs (NSAIDs) are commonly used to alleviate the symptoms of various inflammatory reactions [1,2]. These drugs inhibit prostaglandin biosynthesis by inhibiting prostaglandin synthase [3-6] and other inflammation-associated path- ways [7-9] and enzymes [10,11]. These drugs also affect the defence mechanism exerted by lymphocytes, mono- cytes and neutrophils [12,13]. NSAIDs, especially in- domethacin, inhibit HOC1 production by myeloperoxi- dase [13] by which neutrophils exert bactericidal activ- ity [14]. Eosinophils also take part in the host defence

Correspondence to: R.K. Banerjee, Department of Physiology, In- dian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700032, India. Abbreviations: NSAIDs, non-steroidal antiinflammatory drugs; IMN, indomethacin; ASA, acetylsalicylic acid; IAA, indole-3-acetic acid; cetab, cetyltrimethylammonium bromide; HRP, horseradish peroxi- dase; LPO, lactoperoxidase; MPO, myeloperoxidase; EPO, eosinophil peroxidase; IPO, intestinal peroxidase; BSA, bovine serum albumin; CA, carbonic anhydrase.

mechanism by exerting cytotoxic activity with the help of hypohalous acids produced from H 2 0 2 (generated during the respiratory burst) and a halide [15] accord- ing to the following reaction:

H 2 0 2 + X - q - H+--+ H O X + H 2 0

Where X - = halide ( C 1 - / B r - / I ) and HOX is the corresponding hypohalous acid which acts as the bacte- ricidal agent. Eosinophils exert antiparasitic and an- tibacterial action with the help of eosinophil peroxi- dase, a specific granular protein of molecular weight of 77 kDa consisting of two subunits [16,17] and function- ally distinct from the myeloperoxidase of the neu- trophils [18,19]. Some inhibitors of eosinophil peroxi- dase prevent the killing of antibody and complement opsonised schistosomules by intact eosinophils in serum-containing medium [20], indicating that eosino- phil peroxidase plays an important role in the eosino- phil-mediated killing of parasites.

Eosinophils are migrating phagocytic cells which accumulate in tissues with an epithelial lining like the gastrointestinal tract and mediate the extracellular de- struction of large invading metazoan pathogens, such

as the helminthic parasites [21]. On the basis of histo- chemical studies, Ryt6maa and Teir [22] suggested that very high peroxidase activity in some tissues of rat such as intestine [23] is due to the presence of a large number of invading eosinophils rich in peroxidase [24]. The intestinal peroxidase purified from various sources is similar to the eosinphil peroxidase as regards the molecular, spectral, kinetic and immunological proper- ties [25-27]. We have further observed that only 3% of the total intestinal peroxidase may be attributed to be endogenous located in the epithelial cells of the intesti- nal villi while the major part is localized in the lamina propia, the core of the intestinal villi [28] which is known to contain eosinophils [29]. The remarkable similarity in the physicochemical properties of intesti- nal peroxidase with those of eosinophils [28] led us to confirm the earlier view that intestinal peroxidase is contributed by the invading eosinophils [23-27]. As the small number of eosinophils in blood make their isola- tion and subsequent purification of its peroxidase in good yield difficult, the intestine may serve as an alternative good source of eosinophil peroxidase which we have purified to apparent homogeneity with a view to studying the effect of NSAIDs on this enzyme. Shacter et al. have recently demonstrated that NSAIDs strongly inhibit MPO activity of the neutrophils and were competitive with respect to chloride but uncom- petitive to H 2 0 2 [13]. As eosinophils are found in high quantities at the sites of inflammation [30], as well as in the intestine, it was thought interesting to investigate the effect of these drugs on the peroxidase activity of the intestine/eosinophils. The present study indicates that NSAIDs, especially indomethacin, are strong com- petitive inhibitors of the intestinal eosinophil peroxi- dase with respect to the oxidation of its electron donor. Evidence has been presented to show that it inhibits the peroxidase activity by acting as a competitive sub- strate for the enzyme.

Materials and Methods

Chemicals. Indomethacin, acetylsalicylic acid, bovine milk lactoperoxidase (A412/A28o=0.88), horseradish p e r o x i d a s e (A403/A280 = 3), sodium deoxycholate, cetab (cetyltrimethylammonium bromide), a-methyl- mannoside and guaiacol were purchased from Sigma (St. Louis, MO, USA). Sephadex G-150 and Con-A Sepharose were obtained from Pharmacia, Sweden. All other chemicals used were of reagent grade. In- domethacin and acetylsalicylic acid were prepared in 60% and 25% ethanol, respectively.

Purification of intestinal peroxidase. The crude mito- chondrial fraction (12 000 X g pellet) obtained from the cell-free homogenate of the intestine (60 g) of 10-15 Sprague-Dawley rats was used as the starting material. The enzyme was solubilized and purified as described

169

earlier [28] with some modifications. The enzyme was enriched in the membrane fraction by initial extraction with 0.5% sodium deoxycholate and then homogeniza- tion of the pellet in 100 mM Tris-HCl buffer (pH 10) followed by centrifugation at 105 000 X g for 1 h. The pellet was treated with 0.2% cetab, frozen for 40 h, thawed and then 3.6 M NH4CI was added to a final concentration of 1.2 M. After stirring for 4 h, it was centrifuged at 140000 x g for 1 h to get the soluble enzyme. It was concentrated to 4 ml by Amicon ultra- filtration using UM-20 filter and applied to Sephadex G-150 column (1.6 x 75 cm) previously equilibrated with a buffer containing 50 mM sodium phosphate (pH 7.3) and 1.2 M NH4CI. Elution was carried out with the same buffer. The enzyme eluted around V J V o = 1.6, was finally applied to a Con-A-Sepharose column (volume 5 ml), equilibrated with the above buffer. After washing with the above buffer until A280 reached near zero, the enzyme was eluted with the same buffer containing 0.5 M a-methylmannoside (Fig. 1A). The active fractions (32-36) were pooled to get the pure enzyme. The purity was checked by SDS-PAGE [31] and its Rz value was determined from the ratio of absorbance at 412 nm to 280 nm.

Enzyme activity assay and spectral studies. Peroxi- dase activity was assayed in a Pye-Unicum SP 8-100 spectrophotometer at room temperature using iodide (I 3 assay at 353 nm) or guaiacol (assay at 470 nm) as electron donor [32]. The enzyme activity was expressed as nmol 13 or btmol of tetraguaiacol formed per min, calculated from the molar and millimolar extinction coefficient of 13 and tetraguaiacol as 26340 [33] and 6.48 [34], respectively. The spectral studies were car- ried out in a Shimadzu UV-2201 spectrophotometer.

TABLE I

Purification of peroxidase from rat intestine

Steps Total Total Specific Yield activity protein activity (%) (Units) (mg) (U/mg)

Mitochondrial suspension 33 460 546 61.28 100 Sodium deoxy- cholate pellet 29 304 122.4 239.4 88 Tris-HC1 (pH 10) pellet 28 400 65 436.92 85 Cetab-NHaCl extract 69 000 40.8 1690.2 206 (100) * Amicon concentrate 58860 15 3924 176 (85) Sephadex G-150 eluate 37000 2.034 18190.8 111 (54) Con-A-Sepharose 25 328 0.628 40 330 76 (37)

* The values in parentheses indicate % yield with respect to the cetab-NH 4C1 extract.

170

1 2

B S A ~ I P O ,

H R P ,

C A ,

o 1 ooo

o ~-- D y e f r 0 I0 20 :30 40

FrocHon no. g

Fig. l. (A), elution pattern of intestinal peroxidase from Con-A-Sepharose. A280 (o) and enzyme activity (e) using iodide as electron donor were determined. The assay system contained 50 mM sodium acetate buffer (pH 5.5), 1.7 mM KI and the enzyme preparation in a final volume of 3 ml. Reaction was started by addition of 0.27 mM H20 2. The arrow indicated start of elution of peroxidase by 0.5 M a-methylmannoside. (B),

SDS-PAGE (12%) of the enzyme (2/xg) followed by silver staining. Lane 1 shows the standards and 2 the pure enzyme preparation.

Results

NSAIDs can inhibit the intestinal peroxidase activity of the crude mitochondrial fraction in a concentration dependent manner (data not shown). Of the two drugs tested, indomethacin (IMN) was more effective, caus- ing 80% inhibition at 150/xM, while acetylsalicylic acid (ASA) caused 70% inhibition at 2.67 mM. Further studies were carried out on the enzyme purified from the mitochondrial fraction as shown in Table I. The enzyme was purified 660-fold with a yield of 37% with respect to the cetab-NH4Cl (which activate the en- zyme) extract. The purified enzyme had a R z (A412/A28o) value of 0.9 and was apparently homoge-

r-

E

[ASA ] mM 0 I 2 .3 4

15 II i J ~ i

O I I I I

0 4 0 8 0 120 160 [ IMN]~IM

Fig. 2. Effect of varying concentrations of IMN and ASA on the purified peroxidase activity. Peroxidase activity in presence or ab- sence of varying concentrations of IMN and ASA was determined with iodide as electron donor; (o), IMN; ([]), ASA. Results are the

mean of three sets of experiments.

nous with a molecular mass of 50 kDa as judged by SDS-PAGE (Fig. 1B).

IMN (150/xM) inhibited the activity of the purified enzyme by 80% with apparent K i of 30 /xM (Fig. 2). ASA, unlike the mitochondrial fraction, is less effective and inhibited the pure enzyme by 50% at 2.67 mM. Both IMN and ASA inhibit mammalian peroxidases, including lactoperoxidase (LPO), but not the plant enzyme horseradish peroxidase (HRP, Table II). Higher concentrations of IMN (150 /xM), however, inhibited HRP activity by 32% only. ASA, which was less effec- tive in inhibiting LPO activity (31% at 2.67 mM) com- pared to the intestinal peroxidase (50% at 2.67 mM), did not inhibit HRP activity at all.

The inhibition of intestinal peroxidase activity by NSAIDs is dependent on the pH of the assay system

TABLE II

Effect of indomethacin and acetylsalicylic acid on carious peroxidases

Intestinal peroxidase (0.1 tzg), horseradish peroxidase (1 /zg) or lactoperoxidase (0.1 /xg) was assayed in presence or absence of IMN and ASA using iodide as electron donor.

Activity (nmo113/min)

Intestinal Lactoper- Horseradish peroxidase oxidase peroxidase

Control 12 50.9 43.0

Indomethacin 20 kLM 6.9 35.9 42.9

150 ~M 3.6 8.9 29.2

Acetylsalicyclic acid

1.33 mM 8.7 44.0 42.5 2.67 mM 6.02 35.0 42.2

I 0 0 -

8 0

E .o 60 4--

t "

E

~ 4o

2O

I 5.0

ok I M N

\ ASA

I I 6.0 ZO

pH Fig. 3. Effect of pH on the inhibition of peroxidase activity by IMN and ASA. Peroxidase activity was assayed at varying pH using iodide as electron donor. The buffer used was 50 mM acetate (pH 4.0 to 6.5). (o); 150 ~M IMN and iodide; (D), 2.67 mM ASA and I- . Ionic strength of controls was maintained with appropriate concentrations

of NaCl.

(Fig. 3). IMN and ASA were most effective at pH 5.5 and 4.5, respectively, when iodide was used as an electron donor. These drugs also required an acidic environment (pH 5.0 to 5.5) for maximum inhibition when guaiacol was used instead of iodide (data not shown). However, at physiological pH (7.2), IMN did inhibit the activity of the peroxidase by 70% at low concentrations (approx. 1 mM) of guaiacol (results not shown) and the inhibition could be reversed with in° creasing concentration of the electron donor, indicat- ing that inhibition is competitive in nature. Unlike

171

IMN, ASA had no effect on the enzyme activity at this pH. The nature of the inhibition by IMN and ASA was further investigated by studying the effect of increasing concentration of iodide. The double-reciprocal plots (Fig. 4A) for iodide oxidation in presence of varying concentrations of IMN showed that the inhibition by IMN was competitive with respect to iodide and the secondary plot of the slopes (Fig. 4B) revealed Ki -- 32 /xM. The inhibition of peroxidase activity by ASA could not be reversed by higher iodide concentrations as shown in Fig. 5A, indicating that the inhibition was non-competitive with respect to iodide. The secondary plot of the y-intercepts at varying ASA concentrations gave a straight line with K i = 2.7 mM (Fig. 5B). Similar kinetics of inhibition were also obtained with IMN and ASA when guaiacol was used instead of iodide (data not shown). The activity of the enzyme in the presence of these drugs could be fully restored on dilution, suggesting that the inhibition is reversible. Preincuba- tion of the enzyme with only IMN or ASA up to 2 h also had no effect on the enzyme activity (results not shown).

A preliminary investigation was carried out using some structural analogues of these drugs to identify the structural features responsible for the inhibition of peroxidase activity (Fig. 6). Indole-3-acetic acid (IAA), an analogue of IMN, was equally effective having a K i

value of 20/zM, comparable to that (30 #M) for IMN. The inhibition could also be reversed by higher con- centrations of iodide similar to IMN. Salicylic acid, an analogue of ASA, was more effective than ASA having a K i value of 0.5 mM which is more than five times lower than that of ASA. However, similar to ASA, it is noncompetitive with respect to iodide. Benzoic acid, another analogue of ASA, is not effective at all.

T._. 2 0 r - A • B

/ / J I 1 1. l I

- 0 . 5 0 0.5 J.o - 8 0 0 80 160 [ I - f I mM I [IMN:] IJM

Fig. 4. (A), Lineweaver-Burk plot for the inhibition of peroxidase activity by IMN in presence of varying concentration of iodide. Purified peroxidase (0.1 /zg) was assayed in the presence of varying concentrations of iodide (1 to 5 mM) in presence or absence of IMN, keeping the H 202 concentration fixed at 0.27 mM. (o), none; (zx), 40/~ M; ([]), 80/~M; (o), 150/~ M IMN. (B), slope-plot of the data shown in A. The slopes

are plotted for each concentration of IMN from panel A.

172

TE IOFA • 3

0 c~ & E 2 0 >

- 0 . 5 0 0.5 1.0 - 2 0 2 4

[ I ' ]ZImM -I [ A S A ] m M Fig. 5. (A), Lineweaver-Burk Plot for the inhibition of peroxidase by ASA in presence of varying concentration of iodide. The enzyme was assayed as in Fig. 4, except that the concentration of ASA was varied instead of IMN. (©), none; ([]), 0.67 mM; (zx), 1.33 raM; (e), 2.67 mM

ASA. (B), Dixon plot of the data shown in panel A. The 1/Vma x values are plotted for each concentration of ASA.

From the competi t ive effect of IMN on the oxida-

t ion of iodide or guaiacol as e lectron donor , it is more likely that IMN may itself act as a competi t ive sub-

S t r u c t u r e Perox tdose act iv i ty with 1.7mM Iodide

(nmolee 13/rain )

Contro l - 11.40 15.9 5

strate for the enzyme. Fig. 7 and its inset show the

kinetics of the oxidation of IMN by the peroxidase.

The extinct ion of I MN at 280 nm decreases with t ime

Peroxidose activity with inhibi t ion 5.1raM Iodide Kiopp

(nmolee Is / ra in )

H3C O , ~ , . _ ~ C H 2 C O O -

v "N "CH 3 ~- Indomethac in I 2 . 2 0 7 . 5 0 Competit ive 30pM

C=O (150pM)

Cl

CH2COO- - I - I n d o l e - 3 - o c e t i c r,~ I T ~ 0 . 8 7 6 .54 Competitive 20pM

acid (150pM) ~ - ~ " N " H

r>,,~r.o-cocH3 + A c e t y l s o l i c y l i c " 5 . 8 5 acid ( 2 . 6 7 m M ) ~L"~.,,~cooe

Non -

8.77 competi t ive 2.67mM

OH +Sa l icy l ic acid ~ Non-

( 2 . 6 7 m M ) ~ C O 0 - 1 .90 4 . 4 7 competitive 0.50raM

A + B e n z o i c acid [ / l] 1 0 . 4 4 -- - -

( 2 . 6 7 raM) ~ C O 0 -

Fig. 6. Studies on the effect of some structural analogues of IMN and ASA on peroxidase activity.

0.8

173

U

0 J3 b..

0

J3

0.6

0.4

0.2

0

i

4 5

0.8 0 ~ ~ _ ~ ~

0.701..- : ~ o - . - ~ 0 . 6 5 1 -

,~'~ 0.40'

0.20

- 0.08

- 0.06

- 0.04

-~ - 0 . 0 2

_L___ 0 4 5

~t

O ~ 15 3 0 Time (rain)

I I

300 40O 5O0 600 n m

Fig. 7. Kinetics of the oxidation of IMN by the purified peroxidase. The assay system contained 50 mM sodium acetate buffer (pH 5.5), purified peroxidase (0.04/~M) and IMN in a final volume of 1 ml. Reaction was started by addition of H20 2 (0.2 mM). Trace 1, after 3 min; 2, 6 rain; 3, 15 rain; 4, 30 min; 5, 45 min. Inset (o), decrease in A280 at 75 #M IMN and (o), corresponding increase in A412 at above IMN concentration; (,x), decrease in A2s 0 and ( • ) , corresponding increase in A412 in the presence of 20/~M iodide and 75/~M IMN; ( I ) , reversal of iodide effect

on product formation at A412 with higher (150/~M) concentration of IMN.

with concomitant appearance of a new product at 412 nm. The decreases in absorbance of IMN at 280 nm with increase in the formation of the product at 412 nm depend on the enzyme and H 2 0 2 and is inhibited by 1 mM azide (data not shown), suggesting that IMN is peroxidised by the enzyme. The inset of Fig. 7 shows that the concentration of product at 412 nm is reduced

in the presence of 20 ~M iodide and the inhibition is reversed when the concentration of IMN is raised from 75 /~M to 150 /~M, suggesting that I - is competing with IMN for oxidation by the peroxidase. As IMN acts as a substrate for the enzyme, it is expected to bind near the heme crevice. Fig. 8, shows that the optical spectra of the peroxidase were altered by IMN which

174

0 .05

O

o

0 , 0 0 3 0 0 4 0 0 5 0 0

n m

Fig. 8. Spectral studies of the purified peroxidase in presence or absence of IMN. Trace 1, purified peroxidase (0.3/~M); 2, + 15/~M

IMN; 3, 30/~M IMN; 4, 60/.~M IMN; 5, 120/~M IMN.

causes a bathochromic shift of the Soret band at 412 nm. ASA, however, had no effect on the Soret spec- trum of the enzyme (not shown).

Discussion

Intestinal peroxidase was first purified from pig intestine and was shown to be similar to eosinophil peroxidase [25]. Later, peroxidase from rat intestine was purified and characterised by Kimura and Jellinck [26] and was proposed to be contributed by the invad- ing eosinophils [22,27]. As the yield of the enzyme purified by Kimura and Jellinck [26] was small (4%), we modified the procedure earlier [28] and showed that the enzyme is mainly contributed by the invading eosinophil although a small amount (3%) may be of endogenous origin [28]. In the present study, we fur- ther modified the isolation procedure to get a homoge- neous enzyme preparation of 50 kDa with high yield (37%) and R z value of 0.9 which is higher than that (R z = 0.783) reported earlier [26]. This enzyme can be used as a substitute for the eosinophil peroxidase of rat blood, as the latter is not a good source for isolation.

Kinetic studies on the effect of IMN and ASA on the peroxidase revealed that IMN is competitive with respect to both iodide and guaiacol as electron donors while the inhibition by ASA is noncompetitive but reversible in nature. This is in contrast to the irre- versible inhibition of prostaglandin synthase caused by acetylation of a serine residue by ASA [35]. H 2 0 2 could not reverse the inhibition by IMN and ASA, suggesting that the drugs do not compete with H 2 0 2 for binding at the sixth co-ordination position of the heme iron where H 2 0 2 reacts to form the active

enzyme. The small bathochromic shift of the Soret band of the peroxidase by IMN also seems to indicate the interaction of the drug with the heme periphery and not directly with the heme iron centre. The pH-de- pendence of the inhibition of the enzyme activity by IMN and ASA suggests the probable interaction of the drugs, in part, with some residues on the enzyme which presumably ionise around pH 6.5. At pH 5.5 for IMN and 4.5 for ASA, the drugs (pK a around 4.5) will be negatively charged and may hydrogen-bond with the positively charged amino acids, resulting in an increase in the inhibition below pH 6.5 . At pH values lower than these, the probability of the ionization of the drugs is reduced and, hence, the inhibition is de- creased due to decreased interaction. As these drugs inhibit the enzyme at moderately acid pH (4.5-5.5), the phagocytic leucocytes will also be susceptible to the interaction of these drugs with the peroxidase as their intracellular pH is known to decrease to 5 during phagocytosis [36].

Analogue studies have furnished valuable informa- tion on the structural requirement of these drugs for the inhibition of enzyme activity. It appears that in- dole-acetic acid group of the primary structure of IMN (Fig. 6) may be responsible for the inhibition. Similari- ties in the inhibition pattern of IMN and IAA and the competitive effect of IMN in the oxidation of iodide and guaiacol suggest that the drug may itself act as a substrate similar to IAA which is oxidised by turnip peroxidase [37] and HRP [38]. This has been substanti- ated by our observation that intestinal peroxidase does oxidise IMN as evidenced by the spectrophotometric decay of IMN at 280 nm with the appearance of a product at 412 nm, which can be competitively inhib- ited by low concentrations of iodide. Although further studies are required to identify the nature of this oxidation product, this provides convincing evidence that IMN acts as a competitive substrate for this en- zyme. ASA inhibits the enzyme less effectively than IMN. When its phenolic -OH is free as in salicylic acid, it acts as a more effective inhibitor than ASA. Benzoic acid, having no phenolic -OH in its structure, is inef- fective indicating, that the -OH group plays an impor- tant role in the inhibition of the enzyme. The greater potency of salicylic acid than ASA lends support to the idea that ASA may act as a prodrug releasing salicylic acid to the site of action [2].

NSAIDs inhibit both intestinal peroxidase and LPO nearly to the same extent, while HRP is resistant to inhibition. This is probably due to a difference in the protein structural motif between plant and mammalian peroxidases. The similarities in the behaviour of in- testinal peroxidase and LPO may be due to same 'heme-linked ionizable group' as suggested by Kimura and Yamazaki [39]. The two peroxidases may have the same type of heme also [26,40]. IMN also causes a

bathochromic shift of LPO at 412 nm [41] similar to that of the intestinal peroxidase.

Shacter et al. [13] have recently shown that the NSAID IMN inhibits HOCI production by neutrophil peroxidase (myeloperoxidase) with a Ki value of 37 /zM. This is comparable to the value (30/~M) obtained with the intestinal peroxidase. IMN is competitive with respect to chloride in case of myeloperoxidase [13] and this is also comparable to the intestinal peroxidase where IMN is competitive to iodide. It is known that intestinal peroxidase is mainly contributed by the eosinophils [22-28]. The eosinophils participate in a variety of inflammatory states with the help of the eosinophil peroxidase (EPO) and hypohalous acid which is bactericidal [42,43]. The nature of halide sub- strate utilized by EPO is uncertain. The enzyme was shown to preferentially utilize B r - to produce HOBr [18,19], However, the E P O - H 2 0 2 - B r - system is toxic to the host tissuue too [44]. Slungaard and Mahoney [45] have recently shown that S C N - may be the physio- logical substrate for EPO as hypothiocyanous acid (HOSCN) produced from EPO-H2Oa-SCN- system is bacteriostatic rather than bactericidal and, hence, less toxic to the mammalian host cells. Whatever be the nature of the substrate, the present study shows that NSAIDs, especially IMN, are likely to affect the EPO-H202-hal ide / pseudohalide system [15,18,43-45] and may inhibit the host defence mechanism either by decreasing the bactericidal (bacteriostatic) activity of the circulating eosinophils or by preventing the de- struction of the helminthic parasites by the intestinal eosinophils [20,45].

NSAIDs inhibit the iodination reaction catalysed by the thyroid peroxidase [46]. They also inhibit the gas- tric peroxidase activity [41] which may be involved in gastric acid secretion [47]. These drugs also affect the myeloperoxidase activity of the neutrophil [13]. The present study further indicates that these drugs, espe- cially IMN, inhibit the intest inal /eosinophi l peroxi- dase by acting as a competitive substrate for the en- zyme. It appears that inhibition of various animal per- oxidases by IMN may be a generalised phenomenon whereby the enzyme may metabolize this drug and while doing so, the normal site-specific physiological function of this enzyme is likely to be deranged.

Acknowledgement

The technical assistance of Tapan K. Chakraborty and Chayan K. Ganguly is gratefully acknowledged.

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