Crotyl Alcohol Bioactivation and Protein Carbonylation
Chem. Res. Toxicol., Vol. 15, No. 8, 2002 1057
comparable changes in gene expression in vivo.
(9) Marnett, L. J ., Hurd, H. K., Hollstein, M. C., Levin, D. E.,
Esterbauer, H., and Ames, B. N. (1985) Naturally occurring
carbonyl coumpounds are mutagens in Salmonella tester strain
TA104. Mutat. Res. 148, 25-34.
(10) Cederbaum, A. I., Pietrusko, R., Hempal, J ., Becker, F. F., and
Rubin, E. (1975) Characterization of a nonhepatic alcohol dehy-
drogenase from rat hepatocellular carcinoma and stomach. Arch.
Biochem. Biophys. 171, 348-360.
11) Burcham, P. C., and Fontaine, F. (2001) Extensive protein
carbonylation precedes acrolein-mediated cell death in mouse
hepatocytes. J . Biochem. Mol. Toxicol. 15, 309-316.
12) Summerfield, F. W., and Tappel, A. L. (1984) Cross-linking of
DNA liver and testes of rats fed 1,3-propanediol. Chem.-Biol.
Interact. 50, 87-96.
(13) Neely, M. D., Amarnath, V., Weitlauf, C., and Montine, T. J .
(2002) Synthesis and cellular effects of an intracellularly activated
analogue of 4-hydroxynonenal. Chem. Res. Toxicol. 15, 40-47.
Given that CrOH exhibits pronounced ADH-dependent
toxicity in freshly isolated mouse hepatocytes, it is safe
to predict that this compound will prove hepatotoxic in
intact rodents. However, before CrOH could be used in
future in vivo studies, it is important that the in vivo
hepatotoxic potential of CrOH is first defined in rats and
mice. Such information is currently lacking, although a
single early study did report findings regarding CrOH
biotransformation in rats (38). The major urinary me-
tabolite in CrOH-treated animals was 3-hydroxy-1-
methylpropylmercapturic acid, with 2-carboxy-1-methyl-
ethylmercapturic acid formed in lesser quantities. Im-
portantly, the same metabolites predominated in the
urine of rats treated directly with CA itself, confirming
that CrOH does indeed undergo efficient conversion to
CA in vivo (38).
(
(
(
14) Chen, R. F. (1967) Removal of fatty acid from serum albumin by
charcoal treatment. J . Biol. Chem. 242, 173-181.
(15) Rikans, L. E., and Moore, D. R. (1987) Effect of age and sex on
allyl alcohol hepatotoxicity in rats: role of liver alcohol and
aldehyde dehydrogenase activities. J . Pharmacol. Exp. Ther. 243,
In conclusion, our current work demonstrates that the
toxic lipid peroxidation product CA can be generated in
an ADH-catalyzed reaction from an unsaturated alcohol
precursor, CrOH. In keeping with expectations, CrOH
was highly toxic to isolated mouse hepatocytes, producing
dose- and time-dependent toxicity that was blocked by
prior treatment of cells with an ADH inhibitor, 4-MP. In
addition to blocking cytotoxicity, 4-MP also prevented
GSH depletion and protein carbonylation in CrOH-
treated hepatocytes. These collective findings indicate
that CrOH permits CA formation directly within the
internal environment of cells, and thus is a useful tool
for clarifying the molecular events underlying the toxicity
of this aldehyde.
2
0-26.
(
16) Harman, A. W., McCamish, L. E., and Henry, C. A. (1987)
Isolation of hepatocytes from postnatal mice. J . Pharmacol.
Methods 17, 157-63.
17) Hartley, D. P., and Petersen, D. R. (1997) Co-metabolism of
ethanol, ethanol-derived acetaldehyde, and 4-hydroxynonenal in
isolated rat hepatocytes. Alcohol. Clin. Exp. Res. 21, 298-304.
(
(18) Esterbauer, H., Cheeseman, K. H., Dianzani, M. U., Poli, G., and
Slater, T. F. (1982) Separation and characterization of the
2
+
aldehydic products of lipid peroxidation stimulated by ADP-Fe
in rat liver microsomes. Biochem. J . 208, 129-140.
(
(
(
19) Hissin, P. J ., and Hilf, R. (1976) A fluorometric method for
determination of oxidized and reduced glutathione in tissues.
Anal. Biochem. 74, 214-226.
20) Richards, A. H., Lubinski, R. M., and Vanderlinde, R. E. (1975)
Studies on the kinetic assay of lactate dehydrogenase activity.
Clin. Chem. 21, 1018-1023.
21) Segel, I. H. (1993) Enzyme kinetics: Behavior and analysis of
rapid equilibrium and steady-state enzyme systems, J . Wiley &
Sons, New York.
22) Pietruszko, R. (1973) Mammalian liver alcohol dehydrogenases.
Adv. Exp. Med. Biol. 56, 1-29.
23) Pietruszko, R. (1982) Alcohol dehydrogenase from horse liver,
steroid active SS isozyme. Methods Enzymol. 89, 428-434.
(24) Comporti, M. (1998) Lipid peroxidation and biogenic aldehydes:
from the identification of 4-hydroxynonenal to further achieve-
ments in biopathology. Free Radical Res. Commun. 28, 623-635.
25) Moldeus, P., Hogberg J ., and Orrenius S. (1978) Isolation and use
of liver cells. Methods Enzymol. 52, 60-71.
26) Shacter, E., Williams, J . A., Stadtman, E. R., and Levine, R. L.
Ack n ow led gm en t. This work was partially sup-
ported by the National Health & Medical Research
Council of Australia (Project Grant 104848, P.C.B. and
F.R.F.) and NIH/NIAAA Grant 09300 (D.R.P.). This
publication is based on a poster presentation delivered
at the 40th Annual Meeting of the Society of Toxicology,
San Fransisco, March 2001. The authors are grateful for
the assistance of Simon Pyke and Phil Clements during
the mass spectrometry experiments reported in this
paper.
(
(
(
(
(1996) Determination of carbonyl groups in oxidised proteins. In
Free Radicals, A Practical Approach (Punchard, N. A., and Kelly,
F. J . Eds.) pp 159-170, IRL Press, Oxford.
Refer en ces
(
1) IARC (1995) Crotonaldehyde. In IARC Monographs on the evalu-
ation of carcinogenic risks to humans. Dry cleaning, some chlo-
rinated solvents and other industrial chemicals, World Health
Organisation and International Agency for Research on Cancer,
U.K.
(27) Keller, R. J ., Halmes, N. C., Hinson, J . A., and Pumford, N. R.
(1993) Immunochemical detection of oxidized proteins. Chem. Res.
Toxicol. 6, 430-433.
(28) Serafini-Cessi, F. (1972) Conversion of allyl alcohol into acrolein
by rat liver. Biochem. J . 128, 1103-1107.
(29) Smith, P. F., Fisher, R., Shubat, P. J ., Gandolfi, A. J ., Krumdieck,
C. L., and Brendel, K. (1987) In vitro cytotoxicity of allyl alcohol
and bromobenzene in a novel organ culture system. Toxicol. Appl.
Pharmacol. 87, 509-522.
(30) Hartley, D. P., Kroll, D. J ., and Petersen, D. R. (1997) Prooxidant-
initiated lipid peroxidation in isolated rat hepatocytes: detection
of 4-hydroxynonenal- and malondialdehyde-protein adducts. Chem.
Res. Toxicol. 10, 895-905.
(31) Qiu, Y., Benet, L. Z., and Burlingame, A. L. (1998) Identification
of the hepatic protein targets of reactive metabolites of acet-
aminophen in vivo in mice using two-dimensional gel electro-
phoresis and mass spectrometry. J . Biol. Chem. 273, 17940-
17953.
(32) Cooper, K. O., Witmer, C. M., and Witz G. (1987) Inhibition of
microsomal cytochrome c reductase activity by a series of R,â-
unsaturated aldehydes. Biochem. Pharmacol. 36, 627-631.
(33) Mitchell, D. Y., and Petersen, D. R. (1993) Inhibition of rat liver
mitochondrial and cytosolic aldehyde dehydrogenases by croton-
aldehyde. Drug Metab. Dispos. 21, 396-399.
(
(
(
2) Schuler, B. D., and Eder, E. (2000) Development of a 32P-post-
2
labeling method for the detection of 1,N -propanodeoxyguanosine
adducts of crotonaldehyde in vivo. Arch. Toxicol. 74, 404-414.
3) Eder, E., Schuler, D., and Budiawan (1999) Cancer risk assess-
ment for crotonaldehyde and 2-hexenal: an approach. IARC Sci.
Publ. 150, 219-232.
2
4) Budiawan, and Eder, E. (2000) Detection of 1,N -propanodeoxy-
3
2
guanosine adducts in DNA of Fisher 344 rats by an adapted P-
post labeling technique after per os application of crotonaldehyde.
Carcinogenesis 21, 1191-1196.
(
(
(
(
5) Zlatkis, A., Poole, C. F., Brazeli, R., Bafus, D. A., and Spencer, P.
S. (1980) Volatile metabolites in sera of normal and diabetic
patients. J . Chromatogr. 182, 137-145.
6) Ichihashi, K., Toshihiko, O., Toyokuni, S., and Uchida, K. (2001)
Endogenous formation of protein adducts with carcinogenic
aldehydes. J . Biol. Chem. 276, 23903-23913.
7) Burcham, P. C. (1998) Genotoxic lipid peroxidation products: their
DNA damaging properties and role in formation of endogenous
DNA adducts. Mutagenesis 13, 101-119.
8) Neudecker, T., Eder, E., Deininger, C., and Henschler D. (1989)
Crotonaldehyde is mutagenic in Salmonella typhimurium TA100.
Environ. Mol. Mutagen. 14, 146-148.
(34) Cooper, K. O., Witz, G., and Witmer, C. (1992) The effects of R,â-
unsaturated aldehydes on hepatic thiols and thiol-containing
enzymes. Fundam. Appl. Toxicol. 19, 343-349.