Toxicology Letters, 3 (1979) 299-302 Q Elsevier/North-Holland Biomedical Press

299

COVALENT BINDING OF DBCP TO PROTEINS IN VITRO

YASUHIRO KATO, OSAMI MATANO and SHINKO GOT0

institute of Environmental Toxicology, Suzukicho 2-772, Kodairu, Tokyo 18’7 (Japan)

(Received January 2&h, 1979) (Accepted February 8th, 1979)

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SUMMARY

[ 14C] 1,2-Dibromo-3-chloropropane (DBCP) was incubated with rat liver 9000 X g supernatant in order to investigate the covalent binding of DBCP to proteins. Radioactivity incorporated into proteins was dependent on the microsomal oxidase system, but was not prevented by sufficient puromycin to inhibit protein synthesis. From these results DBCP was considered to bind to proteins covalently after activation by microsomal oxidase.

INTRODUCTION

DBCP has been used worldwide as an effective nematicide. Experimentally, it has been found to induce necrosis of the liver and kidney, atrophy of the testes, reduction of sperm cell number, development of abnormally formed sperm cells, and carcinoma in rats and some other mammals [2,8,10,12]. In a preceding paper we reported that after dosing rats with [ 14C] DBCP some 14C02 was eliminated in the expired air and that there were radioactive residues in target tissues which were not extractable with organic solvents, suggesting the alkylation of cellular macromolecules 141. During the past decade it has become increasingly evident that reactive metabolites or alkylating agents that combine with tissue macromolecules may cause carcinogenesis 171, mutagenesis 1131, cellular necrosis [3], and dominant lethality [ll] , i.e. most of the adverse effects reported with DBCP. It is important to confirm the alkylating potential of DBCP and we have therefore carried out in vitro experiments.

MATERIALS AND METHODS

Labelled compound [‘*Cl DBCP (New England Nuclear Co., Boston) was used. The radiochemical

purity was 94%.

Abbreviations: BSA, bovine serum albumin; DBCP, 1,2-dibromo-3-chloropropane; TCA, trichloroacetic acid.

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Rat liver 9000 X g supernatant

Livers were collected from male Wistar Imamichi rats (9 weeks of age) after cardiac perfusion of chilled 0.05 M sucrose in physiological saline, homogenized with 3 vol. of 0.25 M sucrose in 0.05 M phosphate buffer (pH 7.4), and centrifuged at 9000 X g for 20 min.

Incubation Reaction vessels (final vol. 2.0 ml of 0.05 M phosphate buffer, pH 7.4)

contained: 9000 X g supernatant (175 mg of fresh liver weight) with or without boiling for 5 min, 100 mM of NADPH, 20 ~1 of [‘“Cl DBCP in ethanol (30 pg, 2.00. lo5 dpm), and test substances (1% BSA, 1.0 and 10 mM sesamex, and 1.0 and 5.0 mM puromycin dihydrochloride). Incuba- tion was carried out aerobically at 37°C for 30 min and stopped with 0.6 ml 25% TCA and 0.5 ml 10% BSA to decrease protein loss in the extraction process.

Extraction The reaction mixture was agitated vigorously in a voltex mixer (3 min),

and extracted 5 successive times with more than 20 vol. of 5% TCA, acetone, and 70% methanol by repeated suspension, agitation and centrifugation. Sig- nificant radioactivity was not detected in the last washing of methanol. Finally the pellet was digested with 2 ml of NCS tissue solubilizer (Searle, Amersham) and radioassayed by liquid scintillation counting.

RESULTS

When [ 14C] DBCP was incubated only with BSA or boiled liver 9000 X g supernatant, no significant radioactivity was detected in the TCA- and organic solvents-insoluble protein pellet. Radioactive incorporation into proteins was observed only when unboiled liver 9000 X g supernatant was added, indicating the requirement of enzymatic reaction for the protein radiolabelling. This protein labelling was stimulated by NADPH and prevented by sesamex, an in- hibitor of microsomal oxidation [6]. Puromycin, an inhibitor of protein synthesis [9] did not prevent the radioactive incorporation of [ 14C] DBCP into proteins (Table I).

DISCUSSION

Our concern was to conform the covalent interaction of DBCP and cellular macromolecules. The results demonstrated that radiocarbon due to [ 14C] DBCP was incorporated into proteins after activation by microsomal oxidase system. Generally the incorporation of radioactivity into cellular macromolecules is caused by the following two mechanisms: (1) the substance is metabolized into endogenous substrates for the synthesis of cellular macromolecules; (2)

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TABLE I

INCORPORATION OF [“CIDBCP INTO RAT LIVER 9000 x g SUPERNATANT PROTEINS IN VITRO

Incubation mixture

BSA BSA + NADPH 9000 x g sup. 9000 x g sup. + BSA Boiled 9000 x g sup. + BSA Boiled 9000 x g sup + BSA + NADPH 9000 x g sup. + BSA + NADPH

(control) Control + sesamex (1 mM)

+ sesamex (10 mM) Control + puromycin (1 mM)

+ puromycin (5 mM)

N = 6, mean 2 S.D., **P < 0.01.

“C in protein precipitate dpm

89 * 11** 84 f 9**

664 * 75** 707 * 46**

69+ 21** 111 * 36**

10192 87

257 * 68** 179 f 69**

1120 f 221 1016 f 133

the substance or its activated metabolite binds covalently to macromolecules. The first mechanism would not cause any significant toxicological reactions, but the second might pose a risk linked to cellular necrosis; (3) dominant lethality [ 111 or, by covalent interaction with proteins, mutagenicity and carcinogenicity [ 131. Since radioactive carbon dioxide is formed from [ 14C] - DBCP in rats [4], the observed radioactive incorporation might be attributed to the first mechanism. If that were so, the incorporation should be dependent upon protein synthesis. However, puromycin sufficient to inhibit protein synthesis (Km for rat liver peptidyl transferase, 8 FM) [9] did not exert any significant effect on the incorporation. It is therefore concluded that DBCP binds covalently to proteins in vitro, or that DBCP is converted to some alkylating agent by microsomal oxidase system in vitro.

We previously reported the formation of radioactive substances unextract- able with organic solvents in rat liver, kidney, and testes [4] reported as target organs of DBCP, showing cellular necrosis and sperm cell defection [2,10,12]. The present results may be related to these adverse effects. Mutagenic potential has been found in a series of DBCP-related lower haloalkanes (e.g., ethylene- dibromide, 1-bromo-2-chloroethane, 1,2-dibromo-2-methyl-propane, and 1,5- dibromopentene), while the radioactive incorporation of [“Cl DBCP into rat liver DNA in vivo [ 51 further supports the suggestion of mutagenic risk.

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