IN VlTRO INHIBITION OF RAT INTESTINAL HISTAMINE-METABOLIZING ENZYMES*

S. L. TAYLOR? and E. R. LIEBER

Lertermon Arm11 Insfirrcte of Research. Department of Nutrition Food Hygiene D&ion, Presidio of San Francisco, CA 94129, LISA

(Receiued -77 December IY7N)

Abstract-When used at 10 mM concentrations in assays in virro. seven of 37 chemicals strongly inhibited (> 751,, inhibition) rat jejunal mucosa histamine-N-methyltransferase (HMT), while eight of 37 chemicals strongly inhibited rat jejunal mucosa diamine oxidase (DAO). The HMT inhibitors included tyramine, fi-phenylethylamine, tryptamine. octopamine, agmatine, aminoguanidine and nicotine. The most potent DA0 inhibitors were aminoguanidine, anserine, carnosine, histamine, agmatine, thiamine, cadaverine and tyramine. Since many of these inhibitors occur together with histamine in spoiled tuna, the inhibi- tion of the two major intestinal histaminecatabolizing enzymes, HMT and DAO, may play a vital role in the chemical potentiation of histamine toxicity.

Introduction

Histamine has been implicated as the causative agent in certain food-poisoning episodes, particularly in allergy-like reactions due to consumption of spoiled tuna or mackerel (Merson, Baine, Gangarosa & Swanson, 1974). The involvement of histamine in this type of food poisoning is strongly supported by the similarity of the symptoms to those of iv hista- mine administration, the efficacy of antihistamine therapy, and the consistent association between high histamine levels in implicated foods and the develop- ment of toxic reactions. However, a rather remarkable lack of toxic response occurs after oral dosing of histamine to humans or various laboratory animals (Douglas, 1970; Geiger, 1955; Granerus, 1968). This relative lack of oral histamine toxicity is possibly due to the presence of histamine-catabolizing enzymes in the intestines. Two primary intestinal his- tamine-metabolizing enzymes, histamine-N-methyl- transferase (HMT) and diamine oxidase (DAO), have been identified in several species (Kim, Backus, Harris & Rourke, 1969; Taylor & Lieber, 1978). The paradox between the apparent toxicity of histamine when con- sumed with spoiled tuna and the lack of toxicity of pure histamine remains unexplained. One possible explanation would be the chemical potentiation of histamine toxicity by the coincident existence of inhibitors of the intestinal histamine-catabolizing enzymes in the spoiled tuna. This study was under- taken to assess the potential inhibitory effect on the intestinal histamine-catabolizing enzymes of various chemicals likely to be consumed with tuna.

*The opinions or assertions contained herein are the pri- vate views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.

tPresent address: Food Research Institute, University of Wisconsin, 1925 Willow Drive, Madison, WI, 53706.

Requests for reprints should be sent to Commander, Letterman Army Institute of Research, ATTN: Medical Research Library. Presidio of San Francisco. CA 94129.

Experimental

Enzyme prepararion. Male Sprague-Dawley rats weighing approximately 250-350 g were maintained on a chow diet ad lib. and then killed by cervical dislocation. The jejunal segment of the small intestine was, removed and placed in cold 0.9% NaCl. After expression of the remaining intestinal contents, the jejunal segments were sliced open and rinsed with cold @9% NaCI. The mucosal layer was removed by scraping. All further preparatory procedures were car- ried out at 4°C. The jejunal mucosa was weighed and homogenized on 0.1 M-potassium phosphate buffer (pH 7.4), by four strokes of a glass-Teflon homogen- izer set at 400rpm. The homogenate was filtered through two layers of cheesecloth, and made to a final volume of 10 ml/g mucosa with 0.1 M-potassium phos- phate buffer (pH 7.4). The filtered homogenate was centrifuged at 100,OOOg for 60 min. The resultant supernatant or soluble fraction was used as the enzyme source for all experiments.

Enzyme and protein analysis. HMT was assayed by a modification of the method of Axelrod (1971). The l-ml reaction mixtures contained 0.2 ml of 0.15 mr+ histamine in 0.5 M-potassium phosphate buffer, pH 7.6,0*2 ml of0.5 mM-[ 14C]S-adenosylmethionine with a specific activity of 1.45 x lo3 cpm/nmol, @5 ml of enzyme preparation, and 0.1 ml of water or inhibitor solution. Unless otherwise specified (in Table l), chemicals that were tested as inhibitors were prepared as lOOmu-solutions in water, and the final concen- tration of the inhibitors in the reaction mixture was 1Om~. The inhibitor (or deionized water), enzyme and S-adenosylmethionine were pre-incubated together for 5 min at 37°C before the reaction was initiated by the addition of histamine. Reaction mix- tures were incubated for 20min before stopping by addition of @5 ml of 5 t+NaOH. After stopping the reaction, 6 ml of toluene-isoamyl alcohol (1: 1, v/v) was added and mixed vigorously. The organic layer was removed and filtered through anhydrous sodium sulphate, and a known portion of filtered organic solution was placed in scintillation vials containing

237

238 S. L. TAYLOR and E. R. L&IER

10 ml of ACSTM solution (Amersham Corp., Arlington Heights). The counting efficient was 95%. Radioac-

Y tivity measurements were done n a liquid scintillation counter. Corrections were made for the radioactivity of an enzyme-substrate blank prepared by adding his- tamine after the 5 N-NaOH.

A modification of the procedure of Okuyama & Kobayashi (1961) was used in the assay of DAO. The l-ml reaction mixtures contained 0*4ml of 0.5 M- potassium phosphate buffer (pH 72), &l ml of 28 mr+ putrescine with a specific activity of 87.2 cpm/nmol, O-3 ml of enzyme preparation, 01 ml of water and O-1 ml of water or inhibitor solution. Inhibitor solu- tions and concentrations were identical to those used in the HMT assays. A 5-min pre-incubation period at 37°C for the inhibitor and enzyme was used. The reaction was initiated by addition of putrescine. After 60min incubation at 37”C, the reaction was stopped by addition of 02 ml of 10% HC104. One ml of alka- line NaHCO, (Okuyama & Kobayashi, 1961) was added, mixed and 6*0ml of toluene was added. After mixing, the organic layer was removed and filtered through anhydrous sodium sulphate. A known por- tion of the filtered toluene phase was placed in scintil- lation vials containing 10ml of ACSTM solution and radioactivity ‘measured as before. Corrections were made for the radioactivity of enzyme-substrate blanks prepared by adding the HClO* before putrescine.

The protein concentration of the enzyme prep- aration was measured by the modified Folin phenol procedure (Lowry, Rosebrough, Farr & Randall, 1951; Miller, 1959). The protein concentration of the soluble fraction used as the enzyme source for these studies was 4.32 mg/rnl.

R@3lllts

The effects of 38 chemicals on the in oitro activity of rat jejunal mucosal HMT and DA0 are presented in Table 1. The most potent inhibitors of HMT ac- tivity were tyramine (99% inhibition), /I-phenylethyl- amine (99% inhibition), tryptamine (98% inhibition), octopamine (94% inhibition), agmatine (87% inhibi- tion), aminoguanidine (81% inhibition) and nicotine (78% inhibition), when tested at 10m~. The most potent inhibitors of DA0 activity were aminoguani- dine (100% inhibition), anserine (100% inhibition), carnosine (lOOo/, inhibition), histamine (99% inhibi- tion), agmatine (97% inhibition), thiamine (92% inhi- bition), cadaverine (87% inhibition) and tyramine (77% inhibition), when tested at 10m~. Several chemicals gave intermediate levels of inhibition in- cluding cadaverine, indole, tartrazine, theophylline, thiamine and trimethylamine with HMT and caffeine, hypoxanthine, indole, 1-methylhistidine, 3-methylhis- tidine, nicotine, octopamine, b-phenylethylamine, piperazine, spezmidine, spermine, synephrine, theo- bromine, .theophylline, tryptamine and xanthine with DAO. Many chemicals such as the amino acids in- cluding arginine, glycine, histidine, lysine, omithine and tryptophan, had essentially no effect on HMT and/or DAO. A few chemicals appeared to activate the enzymes. For example, 144% of the original HMT activity was observed after inclusion of lOmh+anser- ine in the reaction mixtures. Ethanol and glycine activated DA0 substantially, whiie anserine, caffeine,

Table 1. Effect of various chemicals on the in vitro activity of rat intestinal histamine-N-methyltransferase (HMT) and

diamine oxidase (DAO)

Final Percentage of inhibitor original activity*

concn Potential inhibitor bM) HMT DA0

Aknoguanidine Anserine Arginine Cadaverine Caffeine Carnosine Ethanol Glucosamine Glycine Histamine Histidine Hypoxanthinet Indole Lysine I-Methylhistidine 3-Methylhistidine Niacinamide Nicotine Octopamine Ornithine I-Phenylethylamine Piperazine Putrescine Spermidine Spermine Synephrinet Tartrazine Theobrominet Theophylline Thiamine Trimethylamine Tryptamine Tryptophan Tyramine Urea Uric acid? Xanthinet

10 10 10 10 10 10 10 10 10 10 10 10 5 10 10 10 10 10 10 10 10 10 10 10 10 10 5 10 5 10 10 10 10 10 10 10 5 5

13 19 144 111 65 123 125 111 112 104

98 140 61 102 111

;i 22 5.9 98 l-0 110 89 120 116 98 68 136 60

::, 2.3 91 @6 96 138 120

2.6 0 0 109 13 51 0 123 107 121 1.0 116 55 78 105 71 75 115 75 59 103 58 73

76 71 56 101 64 74 7.5 104 56

i? 84 85 58

*Average of two assays. tThese chemicals were dissolved in C-05 N-NaOH, while

all other chemicals listed were dissolved in water.

carnosine, hypoxanthine, spermidine, theobromine, uric acid and xanthine activated HMT to at least 120% of its original activity.

Discussion

Many inhibitors of HMT and DA0 have been identified previously. HMT is known to be inhibited by S-adenosyl-L-methionine analogues such as S-adenosyl+homocysteine (Borchardt & Wu, 1974; Borchardt, Huber & Wu, 1974). HMT can also be inhibited by a variety of antihistaminic drugs (Hanna & Borchardt, 1974; Taylor & Snyder, 1972). Addi- tionally, HMT is subject to substrate inhibition by high concentrations of histamine (Taylor & Leiber, 1978; Taylor & Snyder, 1972). DA0 is known to be sensitive to inhibition by numerous substances, par- ticularly bases, such as amidines and guanidines, car-

I~I rirro inhibition of rat intestinal histamine-metabolizing enzymes 239

bony1 reagents, substituted hydrazines. and chelating REFERENCES

agents (Buffoni. 1966; Zeller. 1963). Many of these known DA0 inhibitors belong to a class of drugs Axelrod. J. (1971). Histamine-N-methyltransferase (nia referred to as monoamine oxidase inhibitors (Crabbe liver). In Mrrhocls in En;Jmo/oy.r. Edited by H. Tabor & Bardsley, 1974). DA0 is also subject to substrate and C. W. Tabor. Vol. 17, Dart B. D. 766. Academic inhibition, when certain diamines are used as sub- Press, New York. strates (Beavan & Shaff. 1975; Taylor & Lieber. 1978). Beaven. M. A. & Shaff. R. E. (1975). Study of the relation-

Despite the rather lengthy list of known inhibitors ship of histaminase and diamine oxidase activities in

for both HMT and DAO, very few chemicals com- various rat tissues and plasma by sensitive isotopic assay

monly found in foods were known to be inhibitors procedures. Biochem. Phurmnc. 24, 979.

of the enzymes. Thiamine and certain aliphatic dia- Blackwell. B. & Marley. E. (1966). Interactions of cheese

and its constituents with monoamine oxidase inhibitors. mines, which are listed as DA0 inhibitors (ButToni, Br. .I. Phormuc. 26, 120. 1966). were the only previously identified food-borne Borchardt, R. T.. Huber, J. A. & Wu, Y. S. (1974). Potential inhibitors of either enzyme. However. as shown in inhibitors of S-adenosylmethionine-dependent methyl- Table I, many chemicals often found in foods can transferases. 2. Modification of the base portion of inhibit HMT and/or DA0 in oirro. Most of the S-adenosylhomocysteine. J. me&/ Chem. 17, 868.

chemicals selected for testing in this study were nitro- Borchardt. R. T. & Wu. Y. S. (1974). Potential inhibitors

gen-containing bases, since similar substances had of S-adenosylmethionine-dependent methyltransferases.

been shown to be inhibitors of these enzymes. The I. Modification of the amino acid portion of S-adenosyl-

most potent inhibitors were monoamines, diamines homocysteine. J. mednl Chem. 17, 862.

Buffoni. F. (1966). Histaminase and related amine oxidases. or guanidines. although correlations between chemi- Phormac. Rec. 18. 1163. cal structure and inhibitory activity were difficult to Crabbe. M. J. C. & Bardsley. W. G. (1974). Monoamine define. oxidase inhibitors and other drugs as inhibitors of dia-

Many of the identified inhibitory chemicals might mine oxidase from human placenta and kidney. B&hem.

be found in tuna along with histamine. For example. Pharmuc. 23, 2983.

among the most potent inhibitors of HMT and DA0 Douglas. W. W. (1970). Histamine and antihistamines:

were a number of amines including tyramine, p- 5hydroxytryptamine and antagonists. In The Phormtrco-

phenylethylamine. tryptamine, cadaverine, putrescine loyicrtl Basi.s of Therupeurics. 4th Ed. Edited by L. S.

and agmatine. which are decarboxylation products of Goodman and A. G. Gilman. p. 621. MacMillan. New York.

tyrosine, phenylalanine, tryptophan. lysine, ornithine Foo, L. Y. (1976). Scombroid poisoning. Isolation and and arginine, respectively. Histamine is a decarboxy- lation product of histidine. which is formed in tuna

identification of ‘saurine’. J. Sci. Fd A@. 27, 807. Gale, E. F. (1946). The bacterial amino actd decarboxylases.

by the action of microorganisms possessing the requi- At/r. Enyno/. 6, I. site enzyme. histidine decarboxylase. Many micro- Geiger. E. (1955). Role of histamine in poisoning with organisms also possess the necessary enzymes for the spoiled fish. Science. Iv.Y. 121, 865.

decarboxylation of tyrosine. phenylalanine, trypto- Granerus. G. (1968). Effects of oral histamine. histidine and

phan. lysine, ornithine and arginine (Gale, 1946). Con- diet on urinary excretion of histamine. methylhistamine

scquently. tuna exposed to microbial spoilage might and I-methyl-4-imidazoleacetic acid in man. Scctnd. J. c/in. Ltrh. lncesl. 22. 49.

well contain histamine and a variety of other amines. Hanna. P. E. & Borchardt. R. T. (1974). Histamine Spoiled tuna has rather high levels of cadaverine and N-methyltransferase. Inhibition and potentiation by putrescine (Mietz & Karmas, 1977). Also, spoiled rr~rts- and cis-1.5-diphenyl-3-dimethylaminopyrrolidine. tuna extracts contain numerous ninhydrin-reactive J. mednl Chem. 17. 471.

components (Lieber & Taylor. ‘1978). Additionally, Kawabata. T., Ishizaka. K. & Miura. T. (1955). Studies anserine and carnosine. which were inhibitors of his- on the allergy-like food poisoning associated with putre-

tamine catabolism (Table I). are present in tuna meat faction of marine products. II. Separation of causatrvc

(Lukton & Olcott. 1958). substance and some of its chemical characteristics. Jcrp. J. med. Sci. Biol. 8. 503.

The ability of these amines to inhibit intestinal his- Kim. K. S.. Backus. B., Harris. M. & Rourke, P. (1969). tamine catabolism could magnify the oral toxicity of Distribution of diamine oxidase and imidazole-N- histamine and provide an explanation for the appar- methyltransferase along the gastro-intestinal tract. Camp. ently greater toxicity of histamine consumed with Biochem. Physiol. 31, 137. spoiled tuna. The idea of chemical potentiation of his- Lieber. E. R. & Taylor. S. L. (1978). Thin layer chromato- tamine toxicity is not new. Kawabata, lshizaka 8~ graphic screening methods for histamine in tuna fish.

Miura (1955). discussed the existence of an unidenti- J. Chromctr. 153. 143.

fied potentiator. termed ‘saurine’. Foo (1976) recently Lukton. A. & Olcott. H. S. (1958). Content of free imid:+

determined that ‘saurine’ was actually histamine zole compounds in the muscle tissue of aquatic animals.

phosphate. an artifact of extraction. The critical in- Fd Rex 23, 6 I 1.

Lowry. 0. H.. Rosebrough. N. J.. Farr. A. L. & Randall. volvement of enzyme inhibition in amine toxicity has R. J. (1951). Protein measurement with the Folin phenol been demonstrated by the synergism of monoamine reagent. J. hiol. Chem. 193. 265.

oxidase inhibitors in precipitating toxic reactions to Mersen. M. H.. Baine, W. B., Gangarosa. E. J. & Swanson. tyramine in foods (Blackwell & Marley. 1966). While R. C. (1974). Scombroid fish noisonine. Outbreak traced the hypothesis of chemical potentiation of histamine to commercially canned tuna’ fish. J. >m. n~er/. Ass. 228.

poisoning seems viable and is supported by these pre- 1268.

liminary experiments, further work is needed. aimed Mietz. J. L. & Karmas, E. (1977). Chemical quality index

at the isolation of inhibitors from spoiled tuna and of canned tuna as determined by high-pressure liqutd

demonstration of in riro inhibition of intestinal hista- chromatography. J. Fd Sci. 42, 155.

mine catabolism. Miller. G. C. (1959). Protein determination for large

numbers of samples. Analyr. Chem. 31, 964.

240 S. L. TAYLOR and E. R. LIEBER

Okuyama. T. & Kobayashi, Y. (1961). Determination of Taylor, K. M. & Snyder, S. H. (1972). Histamine methyl- diamine oxidase activity by liquid scintillation counting. transferase: inhibition and potentiation by antihista- Archs Biochem. Biophys. 95, 242. mines. MO/W. Pharmacol. 8, 300.

Taylor, S. L. & Lieber. E. R. (1978). Subcellular distribu- Zkller, E. A. (1963). Diamine oxidases. In The En- tion and properties of intestinal histamine-metabolizing zymes. 2nd Ed. Edited by P. D. Boyer, H. Lardy and enzymes from rats, guinea pigs, and rhesus monkeys. K. Myrback. Vol. 8, p. 313. Academic Press, New Comp. Biochem. Physiol. In press. York.