The Role of 1, 2-Epoxyindene in the Metabolism of Indene in vivo

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tion and are grateful to Dr.Norrnan Lazarus of the Wellcorne Foundation Ltd., Dartford, Kent,. U.K. for ... We appreciate the interest shown in the work by Dr. W.

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low an efficiency. Our limited project also points to an insensitive relationship between plasma immunoreactive insulin of exogenous origin and biochemical hypoglycaemia. This agrees with Galloway & Root (1972) and so appears to be liposome-independent. Should diabetic subjects show similar response patterns, future investigations will have two principal directions: (a ) human studies of liposomes densely charged with insulin and (6) efforts towards a rational improvement in the efficiency of the uptake and subsequent metabolic response. Economic and therapeutic advantages of the latter approach have already led us to experiments for determining factors that presently limit efficacy of the liposomal hormone. H. M. P. andB. E. R. gratefullyacknowledge financialhelp from the British Diabetic Association and are grateful to Dr.Norrnan Lazarus of the Wellcorne Foundation Ltd., Dartford, Kent, U.K. for the gift of insulin. We appreciate the interest shown in the work by Dr. W. Manderson, Dr. J. Ratcliffe and Dr. Robin Russell. Dapergolas, G . & Gregoriadis, G . (1976) Lancet ii, 824-827 Engel, R. H., Riggi, S. J. & Fahrenbach, M. J. (1968)Nature (London) 219, 856-857 Galloway, J . A. & Root, M. A. (1972) Diabetes 21, Suppl. 2 , 637-648 Inouye, W . Y . & Mars, H. M. (1962) Surg. Forum 13, 316 Laskowski, M., Haessler, H. A . , Miech, R. P., Peanasky, R. J. &Laskowski,O. (1959)Science 127, 1115-1117

Patel, H. M. & Ryman, B. E. (1976) FEBS Lett. 62, 60-63 Patel, H. M. & Ryrnan, B. E. (1977) Biochem. Soc. Trans. 5, 1054-1055 Speth, R. M. & Christian, H. J. (1963) Diabetes 12, 243-245

The Role of 1,2-Epoxyindene in the Metabolism of Indene in vivo F. A. KERDEL, R. J. BICK, R. P. HOPKINS and P. CALLAGHAN Department of Biochemistry, St. Thomas’s Hospital Medical School, London SE1 7EH, U.K.

Epoxides produced during the metabolism of aromatic hydrocarbons are reactive intermediates that may bind to macromolecules (such as DNA, RNA and protein) or may undergo further conversion into phenols, dihydrodiols and GSH (reduced glutathione) conjugates (Jerina & Daly, 1974), the last giving rise to mercapturic acids (Boyland & Chasseaud, 1969). Evidence has been found for the occurrence of 1,2epoxyindene as an intermediate in the conversion of indene into tuuns-indane-l,2-diol (Leibman & Ortiz, 1968, 1970) and into S-(hydroxyindany1)glutathione (Francis et al., 1975) by liver preparations. The first of these compounds has been found during studies of the metabolism of indene in the whole animal (Brooks & Young, 1956), but no evidence for a mercapturic acid was obtained from such experiments. The more recent demonstration of a GSH conjugate, however, would point to the likely existence of a mercapturic acid. The present work provides evidence for the urinary excretion of a mercapturic acid by rats administered indene and a number of its derivatives, including the I ,2-oxide. Mercapturic acids were synthesized as reference compounds from 1-chloroindane by interaction with N-acetyl-Land indene bromohydrin (2-bromo-1-hydroxyindane) cysteine under alkaline conditions, as described by Clapp & Young (1970) for benzylmercapturic acid. 1-Chloroindane yielded N-acetyl-S-(1,2-dihydroinden-l -yl)-L-cysteine, i.e. indan-1-ylmercapturic acid, m.p. 176175°C (Found: C, 60.4; H, 6.2; N, 5.2; S, 11.7%. Cl4HI7NSO3requires C, 60.2; H, 6.3; N, 5.0; S, 11.5%). Indene bromohydrin yielded N-acetyl-S-( 1,2-dihydro-1-hydroxyinden-2-yl)-~-cysteine, i.e. l-hydroxyindan-2-ylmercapturic acid, m.p. 147°C (Found: C, 56.7; H, 5.8; N, 4.7; S, 10.6%; CI4Hl7NSO4requires C, 56.9; H, 5.8; N, 4.7; S, 10.9 %). 1.r. spectra of these compounds

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Indene

2-Bromoindene

1-Chloroindane

H SR

S-(Indan-l-yl)glutathione

1.2-Epoxyindene

I

I

H S-CH @Hz

CH-COzH I-'

NH-CO-CH3

HZ S-( I-Hydroxyindan-2-y1)glutathione

&;-cHz-yH-coz

NH-CO-CHn H

HZ

Indan-l -ylmercapturicacid

R = CH z- CH-CO-NH-CHz-COzH I

NH-CO-CHI-CHZ-CH

I -Hydroxyindan-2-ylmercapturic acid

I

-COzH

NHZ

Scheme 1. Proposedpazhways of formation of indanylmercapturic acids

were consistent with the rnercapturic acid structure (Fuson et al., 1952; George et al., 1966). 1-Hydroxyindan-2-ylmercapturic acid was detected by g.l.c., t.1.c. and paper chromatography in the urine of rats dosed with indene, 1,2-epoxyindene, 1-chloroindane and 2-bromoindene. In addition, 1,2-epoxyindene gave rise to a second, as yet unidentified, mercapturic acid, and 1-chloroindane gave rise to the same mercapturic acid as is made from it by synthesis, i.e. indan-1-ylmercapturic acid. Indene bromohydrin was excreted largely unchanged, and gave negligible amounts of rnercapturic acid when administered to rats. In an attempt to isolate 1-hydroxyindan-2-ylmercapturicacid as a metabolite of indene, 24g of the hydrocarbon was administered by subcutaneous injection into 30 rats. Fractionation of the urine produced a brown gum that was shown by chromato-

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graphic procedures to contain the mercapturic acid. Attempts to crystallize the acid from the gum have proved unsuccessful, and attempts to form the dicyclohexylamine salt produced only a small amount of a crystalline product. This material, however, displayed chromatographic properties similar to those of the dicyclohexylamine salt prepared from synthetic 1-hydroxyindan-2-ylmercapturicacid. In the present study, whichextends the work of Francis et al. (1975), it has been shown that a consequence in uivo of conjugation of 1,2-epoxyindene with GSH is the excretion of a mercapturic acid. Although hydroxyindanes are the principal metabolites of indene in viuo (Brooks & Young, 1956), and are probably formed through the intermediacy of l,Zepoxyindene, it is clear from the present work that significant amounts of 1-hydroxyindan-2-ylmercapturic acid are excreted, despite the fact that the compound could not be crystallized from urine extracts. The formation of 1-hydroxyindan-2-ylmercapturic acid from indene, 1-chloroindane and 2-bromoindene in viuo is consistent with the metabolism of these compounds through 1,2-epoxyindene. The excretion of indan-1-ylmercapturic acid after the administration of 1-chloroindane results from the direct replacement of the halogen by glutathione as occurs with the alkyl halides (Chasseaud, 1976). The fact that indene bromohydrin is excreted largely unchanged suggests that this compound is not metabolized through the epoxide, nor is the bromine readily replaced. These proposed pathways are shown in Scheme 1. Boyland, E. & Chasseaud, L. F. (1969) Adv. Enzymol. Relat. Areas Mol. Biol. 32, 173-219 Brooks, C. J. W. & Young, L. (1956) Biochem. J . 63, 264269 Chasseaud,L. F. (1976) in Glutathione: Metabolism and Function (Arias, I. M. & Jacoby, W. B., eds.), Raven Press, New York Clapp, J. J. & Young, L. (1970) Biochem. J . 118, 765-771 Francis, T. J. R., Bick, R. J., Callaghan, P. & Hopkins, R. P. (1975) Biochem. SOC. Trans. 3 , 1244-1246 Fuson, N., Josien, M. L. & Powell, R. L. (1952) J . Am. Chem. SOC. 74,l-5 George, W. O., Goodman, R. C. W. &Green, J. H. S. (1966) Spectrochim. Acta22,1741-1749 Jerina, D. M. & Daly, J. W. (1974) Science 185, 573-582 Leibman, K. C. & Ortiz, E. 0. (1968) Mol. Pharmacol. 4, 201-207 Leibrnan, K. C. & Ortiz, E. 0. (1970) J. Pharmacol. Exp. Ther. 173, 242-246

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