Chem. Res. Toxicol. 1992,5,355-360 (33) RamaKrishna, N.V. S., Cavalieri, E. L., Rogan, E. G., Dolnikowski, G., Cerny, R. L., Gross, M. L., Jeong, H., Jankowiak, R., and Small, G. J. (1992)Synthesis and structure determination of the adducts of the potent carcinogen 7,12-dimethylbenz[a]anthracene and deoxyribonucleosides formed by electrochemical oxidation: models for metabolic activation by one-electron oxidation. J. Am. Chem. SOC.114,1863-1874. (34) Cremonesi, P., and Cavalieri, E. (1985)Determination of oxidation potentials of aromatic hydrocarbons by cyclic voltammetry. In 1985 Electroanalytical Symposium, pp 67-70, BAS Press, West Lafayette, IN. (35) Nelsen, S. F., Blackstock, S. C., Petillo, P. A., Agmon, I., and Kaftory, M. (1987)One-electron oxidation equilibria for acylated 109,5724-5731. hydrazines. J. Am. Chem. SOC. (36)Amatore, C.,Azzabi, M., Calas, P., Jutand, A., Lefron, C., and Rollin, Y. (1990)Absolute determination of electron consumption in transient or steady state electrochemical techniques. J. Electroanal. Chem. 288,4543. (37) King, P. F., and O'Malley, R. F. (1984)Fluorination of poly-
355
cyclic aromatic hydrocarbons: charge vs. frontier orbital control in substitution reaction of radical cations. J . Org. Chem. 49, 2803-2807. (38) Jeftic, L., and Adams, R. N. (1970)Electrochemical oxidation pathways of benzo[a]pyrene. J. Am. Chem. SOC.92,1332-1337. (39) Podany, V., Benesova, M., and Bahna, L. (1979)Electrochemical properties of polycyclic compounds studied by the polarographic method in anhydrous systems. VII. Polarographic oxidation of carcinogenic hydrocarbons in dimethylformamide. Neoplasma 26,685-689. (40) Cavalieri, E. L.,Rogan, E. G., Cremonesi, P., and Devanesan, P. D. (1988)Radical cations as precursors in the metabolic formation of quinones from benzo[a]pyrene and 6-fluorobenzo[a]pyrene. Fluoro substitution as a probe for one-electron oxidation in aromatic substrates. Biochem. Pharmacol. 37, 2173-2182. (41) Cavalieri, E.,Rogan, E., Cremonesi, P., Higginbotham, S., and Salmasi, S. (1988)Tumorigenicity of 6-halogenated derivatives of benzo[a]pyrenein mouse skin and rat mammary gland. J . Cancer Res. Clin. Oncol. 114,10-15.
Antioxidant Activity of the Pyridoindole Stobadine. Pulse Radiolytic Characterization of One-Electron-Oxidized Stobadine and Quenching of Singlet Molecular Oxygen Steen Steenken,t Alfred R. Sundquist,i Slobodan V. Jovanovic,t Rowena Crockett,t and Helmut Sies*f* Institut fur Physiologische Chemie I, Uniuersitat Diisseldorf, Moorenstrasse 5, W-4000 Diisseldorf, Germany, and Max-Planck-Institut fur Strahlenchemie, Stiftstrasse 34-36, W-4330 Mulheim, Germany Received January 22, 1992 Antioxidant properties of stobadine, a pyridoindole derivative described to exhibit cardioprotective properties, were characterized. The radical scavenging potential of stobadine was evaluated using pulse radiolysis with optical detection, by which it is shown that one-electron oxidation of stobadine with radicals such as C6H50*,CC1302', Br2'-, and HO' (reaction rate constants 4X 108-1010 M-' s-l) leads to the radical cation (absorbance maxima a t 280 and 445 nm) which deprotonates from the indolic nitrogen (pK, = 5.0) to give a nitrogen-centered radical (absorbance maxima a t 275,335, and 410 nm), probably bearing a positive charge a t the pyrido nitrogen. The radical of stobadine reacts with Trolox (i.e., 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) with a rate constant of 1.2 X lo7 M-' s-l at pH 7.0 by one-electron oxidation to yield the phenoxyl-type radical of Trolox. This reaction is reversible (12 = 2 X lo5 M-l s-l). The redox potential of stobadine at pH 7 is 0.58 V/NHE. Stobadine is also a quencher of singlet molecular oxygen (lo2) with an overall quenching rate constant of 1.3 X lo8 M-l s-l, determined with the endoperoxide of 3,3'-( 1,4-naphthylene)dipropionate(NDP02)as lo2source and by monitoring lo2photoemission with a germanium diode.
I ntroductlon Stobadine (Figure l),a novel drug of a pyridoindole structure, has been described to exhibit cardioprotective effects and to decrease functional damage to isolated rat hearts subjected to a period of ischemia followed by reperfusion (1, 2). Experiments to detect peroxidative damage to lipids suggested that these pharmacological effects arise from antioxidant properties of stobadine (3-6). In order to characterize the radical-scavenging properties of stobadine, and the spectroscopic and acidlbase prop-
* To whom correspondence should be addressed at the Institut fur Physiologische Chemie I, Universitiit Diisseldorf, Moorenstrasse 5, D-4000 Dusseldorf, Germany. + Max-Planck-Institut fiir Strahlenchemie. Universitiit Diisseldorf.
*
erties of its one-electron-oxidizedform, experiments with pulse radiolysis and time-resolved optical detection were performed. Further, the activity of stobadine as a quencher of singlet oxygen (lo2)' was assessed using the endo-peroxide of 3,3'-(1,4-naphthylene)dipropionateas a water-soluble '02 source and monitoring the infrared (1270-nm) emission of lo2(7,8). The antioxidant activity of dehydrostobadine (Figure 1) was also examined.
Materials and Methods Reagents. Stobadine, &-(-)-2,3,4,4a,5,9b-hexahydro-2,8-dimethyl-lH-pyrido[4,3-b]indole,and related compounds were synthesized at the Institute of Experimental Pharmacology, Slovak Abbreviations: lo2,singlet molecular oxygen; NDP02, the endoperoxide of 3,3'-(1,4-naphthylene)dipropionate.
0893-228x/92/2705-0355$03.00/00 1992 American Chemical Society
Steenken et al.
356 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
H
-CH3
CH 3
i5
t
N: HH
HI J f ki
4
Sto badine (cis(-))
2
E
C H 3 m - C H 3 N H
'
Dehydrosto badine
-
00
5
10
3
15
[STOBAOINE] ( 1 0 ' M )
50001/;$
4
5
6
PH
7
8
I
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250
H C H 3 m - C H 3
350
450
550
650
750
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Figure 2. Absorption spectra of transients obtained by pulse radiolysis with stobadine. Spectra were obtained 100 c(s after pulse radiolysis of N,O-saturated aqueous solutions containing 0.2 mM stobadine, 10 mM KBr, and 10 mM phosphate a t pH = 7.4 (diamonds) and a t pH = 3.9 (triangles), and 140 ps after pulse radiolysis of an aerated aqueous solution of 1 M 2-propanol, 50 mM acetone, 50 mM carbon tetrachloride, and 0.2 mM stobadine a t pH = 3.8 (filled circles). Inset a shows the plot of kobs vs stobadine concentration for the reaction of stobadine with C13CO; a t pH 7.0. Inset b shows the effect of p H on the optical density a t 450 nm of the transient formed on oxidation of stobadine by Br2'-. The inflection point (=pK) is a t 5.0 f 0.1.
I H c-0
I
CH3 N-Acet y I S t o bad ine Figure 1. Stobadine and derivatives. Academy of Sciences, Bratislava, Czechoslovakia, and used as hydrochlorides. The pK, values for stobadine, 3.2 and 8.7 (reaction l),were determined by spectrophotometric and potentiometric
where [Q] is the drug concentration, So/S is the degree of quenching, with Soand S the germanium photodiode signal immediately before and after addition of the drug, ( k , + k,) is the overall quenching rate constant, comprised of the rate constanb for physical quenching (k,) and chemical reactivity (k,), and T is the '02lifetime. The T value for 'OZin the assay buffer was determined to be 53 ws according to Valduga et al. ( 1 2 ) using time-resolved measurements with laser excitation of methylene blue as lo, source;2 the value is in accord with literature values of 56-68 ps quoted in ref 12.
dO: Me
,
+- HH*+
H'
pK I3.2
Me
Me
H
H
pK = 8.5
titrations ( 9 ) , respectively. NDPOz was prepared as described (8). Trolox was a gift from Prof. Hartmann, Sao Paulo, Brasil. Pulse Radiolysis Experiments. These were performed as previously described (IO) using a 3-MeV van de Graaff accelerator, a LSI 11/73+ computer to control the experiment and (pre)analyze the data, and a Microvax for final data analysis. Extinction coefficients were obtained assuming G(stobadine radical) = G(Br;-) = G(H0') = 6.0 and taking N,O-saturated 10 mM KSCN for dosimetry [G[(SCN),'-] = 6.0, e[(SCN)z'-] = 7600 M-' cm-'I. The stobadine redox potential was determined at 20 f 1 "C against (a) Trolox (5.1 mM stobadine and 0.025-1.33 mM Trolox in 0.1 M KBr, pH 7.0) and (b) 4-methoxyphenol(O.9 M ethylene glycol, pH 13.5). lo2Quenching Assay. Singlet oxygen quenching assays were carried out in 50 mM sodium phosphate in DzO (pD 7.4) at 37 "C using the thermal decomposition of the water-soluble endoperoxide NDPOZ as the source of lo2: 2NDPOz
37 ' C
2NDP
+ '02+ 302
and monitoring '0, photoemission at 1270 nm with a germanium photodiode detector (Model EO-817L, North Coast Scientific Co., Santa Rosa, CA) (8). The overall quenching rate constant was determined from the Stern-Volmer equation:
so/s = ( k , + k , ) ~ [ Q l + 1
Results Pulse Radiolysis Studies with Stobadine. Aqueous solutions containing 10 mM KBr and saturated with N20 (to convert the hydrated electron, e-, , into HO') were irradiated with 100-400-ns pulses of 3-deV electrons with doses such that ==3pM radicals were produced. Under these conditions the HO' generated by water radiolysis are quantitatively converted into Br,' - (absorbance maximum = 370 nm), which typically acts as a one-electron oxidizing agent toward electron-rich organic compounds (13). On addition of 0.03-0.4 mM stobadine to these solutions, the absorption due to Brz*-decreased and absorption bands at other wavelengths appeared, indicating that Br,'- reacts with stobadine to give rise to a stobadine radical. The rates of decrease of Brz'- and of formation of the new transient radical were the same, and both rates increased with increasing concentration of stobadine. From the linear dependence of k,, for these processes on the concentration of stobadine the rate constant for reaction of stobadine with Br2*-at pH 7.5, a pH where the molecule is protonated at the pyrido nitrogen, is obtained as 1.2 X lo9 M-l S-1.
The absorption spectrum of one-electron-oxidized stobadine changed with pH in a systematic way. At pH I 4, the absorption spectrum is characterized by bands at 445 and 280 nm, whereas at pH 7.4 there is a broad band at 410 nm, a narrower band at 335, and a sharp band at 275 nm (Figure 2). The dependence on pH of the optical
* Courtesy of Prof. S. Braslavsky and Dr. D. Martire, Miilheim, FRG.
Antioxidant Activity of Stobadine
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 357
Table I. Rate Constants for Oxidation of Stobadine and Trolox by Radicals4 oxidizing radical HO' Brz' (SCN)2'* C13COz' CH3OZnd C6H6Ooe 4-CH30C6H40' stobadine' dehydrostobadine
kf k1 substrate pH (M-l 8-l) (M-l s-l) stobadine 7.5 7.0 x 109 stobadine 7.5 1.2 x 109 stobadine 7.4 3.6 X lo8 stobadine 7.4 6.6 X lo8 stobadine 7.0 1V (W)] and the amino acids tryptophan [E7.0= 1.03 V (26)] and tyrosine [E,, = 0.94 V (14)],which indicates that stobadine can repair oxidized bases and amino acids by electron donation. The redox potential of stobadine is less positive than the formal reduction potential of O2'-/H2Oz [E7,0 = 0.89 V (231, which suggests that 02'- can be inactivated by stobadine. On the other hand, the redox potential of stobadine is more positive than those of vitamin E [E7,0 = 0.48 V (19)] and vitamin
Antioxidant Activity of Stobadine
C [E,.o = 0.30 V (19)], and thus, at pH 7, stobadine radical formed as a consequence of such antioxidant activity may consume vitamin E and/or C. However, under acidic conditions the reaction of stobadine radical with vitamin E would be less significant, since the difference in redox potential of the two compounds is near 0 at pH < 3. The trialkylammonium group has a pronounced effect on the physicochemical properties of the stobadine radical, as is evident from the different dissociation constants of the stobadine radical (pK, = 5.0) and the N-methyl-ptoluidine radical (pK, = 8.6). The magnitide of this effect is, however, surprising, considering that it is transmitted through two saturated carbons. Such a long-range inductive effect on the acid-base equilibria and redox potential of free radicals has not been previously reported. The physicochemical properties of dehydrostobadine (Figure 1) and its one-electron-oxidized derivative are different from those of stobadine. The pK, of the indolic NH group of dehydrostobadine and of the corresponding radical cation are >13 and 3.7, re~pectively.~ The low pK of the radical compared to its parent is analogous to that observed with stobadine and is a consequence of the one-electron oxidation. The lower pK of the dehydrostobadine radical compared to that of the stobadine radical is to be expected, since indoles are more electron-poor than indolines. This is also reflected in the redox potential of = 1.11 V),3 which is considerably dehydrostobadine more positive than that of stobadine. In fact, it is more positive than the redox potential of tyrosine (see above), which means that the dehydrostobadine radical is capable of oxidizing tyrosine and, consequently, that dehydrostobadine can be expected to be a relatively poor biological antioxidant. Singlet Oxygen Quenching. Stobadine and the analogues of stobadine that demonstrated antioxidant activity against lipid peroxidation4 are also effective quenchers of lo2.The overall quenching constant determined for stobadine (1.3 X lo8 M-' 9-l) is comparable to those for the different tocopherol homologues [=lo* M-l (28)] and is intermediate between the ranges of values determined for carotenoids [lo9 M-' s-l (29)] and thiols [lo6 M-' s-l (30)].Therefore, stobadine, together with ita stereoisomers and dehydrostobadine which were as effective as stobadine at quenching lo2,represent some of the most efficient water-soluble lo2quenchers reported to date. The Nacetyl derivative of stobadine is >lOO-fold less effective than stobadine, which can be explained in terms of (a) the reduction in electron density of the aromatic by the acetyl group and/or (b) the absence of the NH proton. Acknowledgment. This study was supported by the National Foundation for Cancer Research, Bethesda, MD. A.R.S. is a Stipendiat of the Alexander von HumboldtStiftung (Bonn).
References (1) Stolc, S.,Bauer, V., Benes, L., and Tichy, M. (1985)Swiss Patent 651754. (2) Benes, L., and Stolc, S. (1989)Stobadine. Drugs of the Future 14, 135-137. (3) Stolc, S., and Horakova, L. (1988)Effect of stobadine on postischemic lipid peroxidation in the rat brain. In New Trends in Clinical Neuropharmacology (Bartko, D., Turcani, P., and Stern, G., Eds.) pp 59-63,John Libbey, London. (4) Horakova, L., Lukovic, L., and Stolc, S. (1990)Effect of stobadine and vitamin E on the ischemic reperfused brain tissue. Pharmazie 45,233-224. (5) Ondrias, K., Misik, V., Gergel, D., and Stasko, A. (1989)Lipid L. Horakova, K. Briviba, and H. Sies, unpublished results.
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 359 peroxidation of phosphatidylcholine liposomes depressed by the calcium channel blockers nifedipine and verapamil and by the antiarrhythmic-antihypoxic drug stobadine. Biochim. Biophys. Acta 1003,238-245. (6) Stasko, A., Ondrias, K., Misik, V., Szocsova, H., and Gergel, D. (1990)Stobadine-a novel scavenger of free radicals. Chem. Pap. 44,493-500. (7)Di Mascio, P.,Sundquist, A. R., Devasagayam,T. P. A., and Sies, H. (1992)Assay of lycopene and other carotenoids as singlet oxygen quenchers. Methods Enzymol. (in press). (8) Di Mascio, P., and Sies, H. (1989)Quantification of singlet oxygen generated by thermolysis of 3,3'-(1,4-naphthylidene)dipropionate. Monomol and dimol photoemission and the effects of 1,4-diazabicyclo[2.2.2]octane. J. Am. Chem. SOC. 111, 2909-2914. (9) Stefek, M., Benes, L., and Zelnik, V. (1989)N-oxygenation of stobadine, a gamma-carboline antiarrhythmic and cardioprotective agent: the role of flavin-containing monooxygenase. Xenobiotica 19, 143-150. (10) Jagannadham, V., and Steenken, S. (1984)One-electron reduction of nitrobenzenes by a-hydroxyalkyl radicals via addition/ elimination. An example of an organic inner-sphere electrontransfer reaction. J. Am. Chem. SOC.106,6542-6651. (11) Valduga, G., Nonell, S., Reddi, E., Jori, G., and Braslavsky, S. E. (1988)The production of singlet molecular oxygen by zinc(I1) phthalocyanine in ethanol and in unilamellar vesicles. Chemical quenching and phosphorescence studies. Photochem. Photobiol. 48, 1-5. (12) Rougee, M., Bensasson, R. V., Land, E. J., and Pariente, R. (1988)Deactivation of singlet molecular oxygen by thiols and related compounds, possible protectors against skin photosensitivity. Photochem. Photobiol. 47,485-489. (13) Neta, P.(1976)Application of radiation techniques to the study of organic radicals. Adu. Phys. Org. Chem. 12,224-297. (14)Jovanovic, S.V., Steenken, S., and Simic, M. G. (1991)Kinetics and energetics of one-electron-transfer reactions involving tryptophan neutral and cationic radicals. J. Phys. Chem. 95,684-687. (15) Steenken, S. (1989)Purine bases, nucleosides and nucleotides: aqueous solution redox chemistry and transformation reactions of their radical cations and e- and OH adducts. Chem. Rev. 89, 503-520. (16) Quin, L., Tripathi, G. N. R., and Schuler, R. H. (1985)Radiation chemical studies of the oxidation of aniline in aqueous solution. 2.Naturforsch. A . 40A,1026-1039. (17) Neta, P., Huie, R. E., Mosseri, S., Shastri, L. V., Mittal, J. P., Maruthamuthu, P., and Steenken, S. (1989)Rate constants for reduction of substituted methylperoxyl radicals by ascorbate ions and N,N,N,N-tetramethyl-p-phenylenediamine.J. Phys. Chem. 93,4099-4104. (18) Neta, P., Huie, R. E., Maruthamuthu, P., and Steenken, S. (1989)Solvent effects in the reactions of peroxyl radicals with organic reductants. Evidence for proton-transfer mediated electron transfer. J. Phys. Chem. 93,7654-7659. (19) Neta, P.,and Steenken, S. (1982)One electron redox potentials of phenols, hydroxy- and aminophenols and related compounds of biological interest. J. Phys. Chem. 86,3661-3667. (20) Packer, J. E.,Wilson, R. L., Bahnemann, D., and Asmus, K. D. (1980)Electron transfer reactions of halogenated aliphatic peroxyl radicals: measurement of absolute rate constants by pulse radiolysis. J. Chem. SOC.,Perkin. Trans. 2,296-299. (21)Lind, J., Shen, X., Eriksen, T. E., and Merenyi, G. J. (1990)The one-electron reduction potential of 4-substituted phenoxy1 radicals in water J. Am. Chem. SOC.112,479-482. (22) Jovanovic, S. V., Steenken, S., and Simic, M. G. (1990)Oneelectron reduction potentials of 5-indoxyl radicals. A pulse radiolysis and laser photolysis study. J. Phys. Chem. 94,3583-3588. (23) Hansch, C., and Leo, A. (1979)Substituent constants for correlation analysis in chemistry and biology, Wiley, New York. (24) Holcman, J., and Sehested, K. (1977)Dissociation of the OH adduct of N,N-dimethylaniline in aqueous solution. J. Phys. Chem. 81,1963-1966. (25) Jovanovic, S. V., and Simic, M. G. (1989)The DNA guanyl radical: kinetics and mechanisms of generation and repair. Biochim. Biophys. Acta 1008,39-44. (26) DeFelippis, M. R., Murthy, C. P., Faraggi, M., and Klapper, M. H. (1989)Pulse radiolytic measurement of redox potentials. The tyrosine and tryptophan radicals. Biochemistry 28,4847-4853. (27) Sawyer, D. T. (1988)The redox thermodynamics for dioxygen species (02, 02'-, HOO', HOOH, and HOO-) and monooxygen 'OH, and -OH) in water and aprotic solvents. In species (0,Om-,
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Oxygen radicals in biology and medicine (Simic, M. G., Taylor, K. A., Ward, J. F., and von Sonntag, C., Eds.) pp 11-20, Plenum Press, New York. (28) Kaiser, S., Di Mascio, P., Murphy, M. E., and Sies, H. (1990) Physical and chemical scavenging of singlet molecular oxygen by tocopherols. Arch. Biochem. Biophys. 277, 101-108.
(29) Di Mascio, P., Kaiser, S., and Sies, H. (1989) Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Arch. Biochem. Biophys. 274, 532-538. (30) Devasagayam,T. P. A., Sundquist, A. R., Di Mascio, P., Kaiser, S., and Sies, H. (1991) Activity of thiols as singlet molecular oxygen quenchers. J. Photochem. Photobiol. B: Biol. 9, 105-116.
Bioactivation Mechanism of S-(3-Oxopropyl)-N-acetyl-~-cysteine, the Mercapturic Acid of Acrolein Mazzaz Hashmi,t Spyridon Vamvakas,l and M. W. Anders*it Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642, and Institut fur Toxikologie, Universitat Wurzburg, 0-8700 Wurzburg, Versbacher Strasse 9, Federal Republic of Germany Received January 13, 1992
S-(3-Oxopropyl)glutathione,the glutathione conjugate of acrolein, has been reported to be nephrotoxic. The objective of the present studies was to investigate the bioactivation mechanism of the analogues S-(3-oxopropy1)-N-acetyl- cysteine (1) and S-(3-oxopropyl)-N-acetyl-~-cysteine S-oxide (2) and to test the hypothesis that the cytotoxicity of 1 is associated with its latent potential to release acrolein in kidney cells. Mechanistic considerations indicated that sulfoxidation of sulfide 1 to form S-oxide 2 and a subsequent general-base-catalyzed P-elimination reaction would release the cytotoxin acrolein. Hence the release of acrolein from 1 and 2 was studied in chemical systems, and their cytotoxicity was investigated in cultured LLC-PK1 cells and in isolated rat renal proximal tubular cells. Acrolein formation from S-oxide 2, but not from sulfide 1, was observed under basic conditions and with phosphate as the base. Kinetic analysis indicated that a general-base-catalyzed reaction was involved. Both S-conjugates 1 and 2 were cytotoxic in LLC-PK1 cells and in isolated rat renal proximal tubular cells, and the cytotoxicity of sulfide 1, but not of S-oxide 2, in isolated renal proximal tubular cells was reduced in presence of methimazole, an inhibitor of the flavin-containing monooxygenase. These findings indicate that the cytotoxicity of S-conjugate 1 is associated with a novel bioactivation mechanism that involves sulfoxidation followed by a general-base-catalyzed elimination of acrolein from S-oxide 2. Introductlon The elucidation of mechanisms by which chemicals produce cell damage and death remains a challenging problem in toxicology. It is clear, however, that the organ-selective toxicity of most chemicals is associated with target-organ bioactivation of xenobiotics to reactive intermediates (1-4). Similarly, the nephrotoxicity and, perhaps, nephrocarcinogenicity of xenobiotics is also associated with renal bioactivation (4-6). Recent studies demonstrate that glutathione-dependent bioactivation is an important mechanism for the production of kidney damage (7-9). Glutathione conjugates of vicinal dihaloethanes are half-sulfur mustards and yield reactive episulfonium ions that account for the observed mutagenicity and nephrotoxicity of vicinal dihaloethanes (10-12). Glutathione conjugates of haloalkenes are nephrotoxic and, after hydrolysis to the cysteine S-conjugates and transport to the kidney, undergo activation by renal cysteine conjugate @-lyase(7-9). Acrolein (prop-2-enal), along with many other a,@-unsaturated aldehydes (see ref 13 for review), is toxic. Acrolein is not a potent nephrotoxin, but kidney damage has been reported after intermittent or continuous inhalational exposure to acrolein (14). The acrolein precursor University of Rochester.
* Universitiit Wurzburg.
allyl alcohol is cytotoxic to isolated renal cells (15). The glutathione S-conjugate of acrolein S-(3-0XOpropy1)glutathione has been reported to be nephrotoxic in rats, and its toxicity was blocked by the y-glutamyltransferase inhibitor acivicin (16). This finding prompted the present studies to elaborate the bioactivation mechanism of S-(3-oxopropyl)glutathione.S-(3-Oxopropyl)-Nacetyl-L-cysteine (I), the mercapturic acid of S-(~-OXOpropyl)glutathione, was selected as a model compound for these studies, which were designed to test the hypothesis that the observed cytotoxicity of 1 is associated with its latent potential to release acrolein in kidney cells. Indeed, Esterbauer and co-workers observed that cysteine adducts of a,@-unsaturatedaldehydes slowly release the precursor aldehydes and may serve as prodrugs to deliver cytotoxic a,@-unsaturatedaldehydes to tumor tissue (17-19). Chemical considerations indicated that S-oxidation of sulfide 1 would afford 5'-(3-oxopropy1)-N-acetyl- cysteine S-oxide (21, which may undergo &elimination reaction to release acrolein (Figure 1). Hence the release of acrolein from S-conjugates 1 and 2 was studied in chemical systems, and the cytotoxicity of 1 and 2 was investigated in the pig kidney-derived LLC-PK1 cell line and in isolated rat renal proximal tubular cells. The general-base-catalyzedrelease of acrolein from S-oxide 2, but not from sulfide 1, was observed. Both S-conjugates 1 and 2 were cytotoxic, and the cytotoxicity of sulfide 1 in isolated rat renal proximal tubular cells was reduced in the presence of the flavin-
0893-228~/92/2705-036Q~Q3.~Q/0 @ 1992 American Chemical Society
