* Miami Valley Laboratories, The Procter and Gamble Company, 11810 East Miami River Road, Cincinnati, Ohio 45252; University of Wisconsin-Madison, Rennebohm Hall School of Pharmacy, 777 Highland Ave., Madison, Wisconsin 53705; and
Bristol-Myers Squibb Company, P.O. Box 4000, Princeton, New Jersey 08543
Received February 26, 2004; accepted April 27, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: coumarin; hepatotoxicity; epoxide; GSH conjugation; aldehyde dehydrogenase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although hepatotoxic in rats, coumarin is not hepatotoxic in other species, including mice, hamsters, and gerbils (Fentem and Fry, 1993; Lake and Grasso, 1996
). Species differences in coumarin toxicity appear to be metabolism-mediated, and two major Phase I metabolic pathways are thought to be important to hepatotoxic outcome (outlined in Fig. 1). In most species, particularly humans, coumarin is hydroxylated to 7-hydroxycoumarin (7-HC), a nontoxic metabolite. This reaction is catalyzed by CYP2A enzymes (Negishi et al., 1989
; Pearce et al., 1992
; Raunio et al., 1988
). However, in rats, CYP2A enzymes preferentially catalyze 7
-hydroxylation of testosterone rather than coumarin 7-hydroxylation (Pearce et al., 1992
). As a result, the formation of 7-HC is extremely low in rats (Kaighen and Williams, 1961
; Steensma et al., 1994
), and the lack of this reaction is thought to render rats more susceptible to hepatotoxicity (Cohen, 1979
; Lake, 1999
). The formation of 7-HC is a major metabolic pathway in humans (Cholerton et al., 1992
; Rautio et al., 1992
; Shilling et al., 1969
). In this regard, polymorphisms in the human enzyme have been defined (Fernandez-Salguero et al., 1995
; Hadidi et al., 1998
; Oscarson et al., 1998
) and have been implicated as a possible risk factor for coumarin-induced hepatotoxicity in humans (Hadidi et al., 1997
, 1998
).
|
CE does not appear to be a substrate for epoxide hydrolase, as no diol metabolites of this intermediate have been reported (Kaighen and Williams, 1961; Huwer et al., 1991
), but it does rearrange spontaneously to o-HPA (Born et al., 1997
). Accordingly, there are two possible fates for CE: it can be conjugated with GSH or it can rearrange to o-HPA. This aldehyde is hepatotoxic, and is detoxified by further oxidation to o-HPAA or reduction to o-HPE (Born et al., 2000b
; Fentem et al., 1991
; Lake et al., 1992
; Steensma et al., 1994
).
The lack of a direct correlation between coumarin epoxidation and species sensitivity to hepatotoxicity suggests that factors other than metabolic activation are important determinants of hepatotoxic outcome. The purpose of the present work was to test the hypothesis that detoxification pathways, namely GSH conjugation of CE or oxidation of o-HPA to o-HPAA, are important determinants of species differences in susceptibility to coumarin-induced hepatotoxicity. To test this hypothesis, kinetic characteristics of the reactions were evaluated by in vitro experiments. Furthermore, to determine the human relevance of these findings, detoxification reactions were evaluated in human samples.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Other reagents. GSH, NAD+, NADH, and NADP were purchased from the Sigma Chemical Company (St. Louis, MO). GSH was prepared in nitrogen purged water. All other reagents were HPLC grade or the highest grade available.
Animals. Male F344 rats (210220 g) and female B6C3F1 mice (2025 g) were purchased from Charles River Laboratories (Portage, MI). The strain and sex of animals were selected based on the coumarin bioassay data (NTP, 1993). These data indicated rat liver necrosis was more severe in males, and female mice were more susceptible to coumarin-induced lung tumor formation. Animals were housed in humidity and temperature controlled rooms and allowed free access to a standard lab diet (Purina Laboratory Rodent chow, Ralston-Purina, St. Louis, MO) and water.
Preparation of rodent hepatic microsomes and cytosol. Hepatic microsomes were prepared from untreated female B6C3F1 mice (n = 25/pool) and untreated male F344 rats (n = 15/pool) which were 3 months old. Microsomes and cytosol were prepared via differential centrifugation (Guengerich, 1989). Hepatic cytosol from mice and rats was processed as described below for human cytosol to remove GSH and cofactors. Protein was determined by the Bradford assay with bovine serum albumin as the standard (Bradford, 1976
). Microsomes and cytosol were stored at 80°C until time of use.
Human liver microsomes and cytosol. Human hepatic microsomes (ID number H0017) were purchased from XenoTech LLC (Kansas City, KS). This sample was used because it showed the highest rate of CE formation relative to a series of other human microsomal samples evaluated (Born et al., 2000a). Two different lots of human hepatic cytosol (Catalog number H861, lots 1 and 2) were purchased from GENTEST (Woburn, MA). Each lot of human cytosol was prepared from a pool of 10 human livers, and the two lots varied with respect to the liver samples used to generate them. Unless otherwise noted, cytosol from lot 1 was used in all experiments. If both lots were used for an experiment, they are designated as human-1 and human-2, respectively. The cytosol was processed through a PD-10 desalting column (Amersham Pharmacia Biotech AB, Uppsala, Sweden) to remove GSH and cofactors and stored at 80°C until time of use.
Determination of the fate of coumarin 3,4-epoxide: GSH conjugation. Direct conjugation of GSH to CE was evaluated in a 50 µl reaction containing only potassium phosphate buffer (100 mM, pH 7.4) and GSH (5 mM). A 30-s incubation at 25°C was initiated by the addition of CE (1 to 1000 µM final concentration) in DMSO (final concentration 1%).
Conjugation of GSH to CE was evaluated in rat, mouse, and human hepatic microsomal reaction mixtures (250 µg protein). The final reaction contained potassium phosphate buffer (100 mM, pH 7.4), GSH (5 mM), EDTA (1 mM), MgCl2 (3 mM), glucose 6-phosphate (5 mM), glucose 6-phosphate dehydrogenase (1 IU/ml), and coumarin (10 to 2000 µM) in a final volume of 1 ml containing 1% DMSO. Following a 2-min pre-incubation in a 37°C shaking water bath, NADP (1 mM) was added to initiate the reaction and the samples were incubated for 30 min.
In a second experiment, mouse microsomes were used to generate CE, and GSH conjugation catalyzed by enzymes present in rat, mouse, or human liver cytosol was determined. Cytosolic GST activity was expressed in units, where one unit is equivalent to the amount of enzyme that catalyzes the conjugation of GSH to 1 µmol of 1-chloro-2,4-dinitrobenzene (CDNB) in 1 min at room temperature (Habig et al., 1974). For all reactions, 1 unit of GST activity was used. Finally, the ability of recombinant rat or human GST isozymes (1 µg protein/reaction) to catalyze the conjugation of GSH to CE was evaluated. GST isozymes were from Oxford Biomedical Research, Inc. (Oxford, MI).
Determination of the fate of coumarin 3,4-epoxide: Oxidation and reduction of o-HPA. Oxidation of o-HPA to o-HPAA was determined in rat, mouse, and human hepatic cytosol (25 µg protein). The reaction mixture contained potassium phosphate buffer (100 mM, pH 7.4) and NAD+ (1 mM) in a final volume of 1 ml containing 1% DMSO. Following a 2-min pre-incubation in a 25°C shaking water bath, o-HPA (0.75 to 1000 µM) was added to initiate the reaction and the samples were incubated for up to 30 min. Reduction of o-HPA to o-HPE was determined using these same conditions with the exception that 250 µg of cytosolic protein was used and NAD+ was replaced by NADH (0.5 mM). Oxidation of o-HPE to o-HPA was determined using these same conditions in the presence of 250 µg of cytosolic protein and NAD+ (1 mM) instead of NADH.
Contribution of aldehyde dehydrogenase and aldehyde oxidase to o-HPA oxidation in hepatic cytosol. To determine whether cytosolic aldehyde dehydrogenase (ALDH) and/or aldehyde oxidase (AO) catalyzed the oxidation of o-HPA to o-HPAA, formation of o-HPAA was evaluated in rat, mouse, and human hepatic cytosol (250 µg protein) with 1 mM o-HPA substrate in the presence of 1 mM disulfiram. This level of disulfiram was selected to fully inhibit activity of ALDH isozymes (Hiratsuka et al., 2000; Lam et al., 1997
). Since ALDH requires NAD+ for activity but AO does not, removing this cofactor from the reaction mixture enabled an indirect assessment of the contribution of AO to o-HPA oxidation.
Species differences in the overall fate of coumarin 3,4-epoxide. To compare the metabolic fate of CE in rats, mice, and humans, mouse liver microsomes, which show the highest rate of epoxidation, were used to generate CE. Since CE is unstable and spontaneously rearranges to o-HPA, the rate of epoxidation was determined by measuring o-HPA formation in a mouse hepatic microsomal reaction mixture in the absence of GSH and hepatic cytosol.
Species differences in the detoxification of CE were evaluated in the mouse microsomal reaction mixture containing rat, mouse or human liver cytosol equal to 1 unit of GST activity. The final reaction was replete with cofactors required for oxidation, reduction and GSH conjugation, as it contained NAD+ (1 mM), NADH (0.5 mM), and GSH (5 mM).
Sample preparation and RP-HPLC analysis of o-HPAA, o-HPE, o-HPA, and CE-SG. All metabolic reactions were terminated by the addition of ice-cold 15% trichloroacetic acid (TCA). Following centrifugation, supernatants were analyzed for o-HPAA, o-HPE, o-HPA, and CE-SG. Linearity of product formation was demonstrated with time and protein for o-HPAA, o-HPE, o-HPA, and CE-SG, and reaction conditions were adjusted in order to maintain initial rates of reaction (Vassallo et al., 2003). The ring-opened coumarin metabolites (o-HPAA, o-HPE, and o-HPA) were separated by HPLC and quantified by UV detection at 275 nm as described by Vassallo et al. (2003)
. CE-SG was separated using the same HPLC system, column and mobile phases as the ring-opened coumarin metabolites, but was detected and quantified at a UV absorbance of 332 nm (Vassallo et al., 2003
). The limit of quantification for the ring-opened metabolites and CE-SG was 10 pmol and 0.5 pmol on column, respectively (Vassallo et al., 2003
). There was no overlap in detection or retention times between the ring-opened metabolites and the GSH conjugate.
Data analysis. The apparent Km and Vmax for each reaction were initially evaluated using Michaelis-Menten, Lineweaver-Burk, and Eadie-Hofstee plots. These data suggested that formation of CE-SG, and oxidation of o-HPA to o-HPAA or o-HPE to o-HPA were biphasic with a high and low affinity site. In contrast, reduction of o-HPA to o-HPE was best described by a single-site model. Curve-fitting of the data was conducted using the Enzyme Kinetics Module (SigmaPlot, v 1.1, Chicago, IL). The best fit lines of the single-site model were generated using the Michaelis-Menten quation. The best fit lines of the two-site model were generated using the isozyme analysis provided in the Enzyme Kinetics Module. The isozyme analysis fits the data to the equation:
![]() |
All analyses were carried out as at least three independent analyses and the results are presented as mean ± SE of the replicates. Student's t-test (p < 0.05) was used for statistical analyses when conducted.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
The second major pathway for detoxification of CE involves the oxidation of o-HPA, which forms spontaneously from CE, to o-HPAA. The highest oxidation activity was observed in human cytosols, and the rat was nearly 10 times lower than human (Fig. 4). Eadie-Hofstee analysis of the o-HPA oxidation data indicated o-HPAA formation in rats, mice, and humans was biphasic (results not shown). Therefore, the apparent kinetic constants for o-HPA oxidation were calculated using a two site-model (Table 3). The high affinity Km for the rat, mouse, and average human cytosol was 11.8, 1.7, and 0.8 µM, respectively. Furthermore, the Vmax of the high affinity site in human and mouse was similar and nearly four times higher than rat cytosol. The Km of the low affinity site in rats exceeded 2 mM, whereas the low affinity Km in mouse and human was approximately 200 µM. Collectively, the total intrinsic clearance via oxidation to o-HPAA was about 50 and 20 times higher in the human and mouse, respectively, than in rat.
|
|
To generally determine the cytosolic enzymes involved in o-HPA oxidation, o-HPAA formation was determined in the presence of disulfiram to inhibit ALDH or in the absence of NAD+. Disulfiram reduced the formation of o-HPAA by an average of 90% in the rat, mouse, and human. In the absence of NAD+, o-HPAA formation was also reduced by an average of 96% in rats, mice and humans (Fig. 5).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Conjugation of epoxide intermediates with GSH is an important and well-characterized detoxification reaction. Differences in sensitivity to aflatoxin-induced hepatotoxicity between rats and mice are determined by the rates of GSH conjugation with aflatoxin B1-8,9-epoxide (O'Brien et al., 1983), and the mouse liver efficiently detoxifies the reactive intermediate via alpha GST isozyme (Ramsdell and Eaton, 1990
). GSH conjugation of CE has previously been demonstrated from in vivo studies in rats (Huwer et al., 1991
), and depletion of GSH is reported after high dose coumarin treatment in rats (Lake, 1984
). The present in vitro data indicated that, whereas much of the GSH conjugation of CE occurred nonenzymatically, the total rate of hepatic GSH conjugation of the epoxide is not different between rats and mice. Therefore, it is unlikely that GSH conjugation of CE is a major determinant of species differences in sensitivity to hepatotoxicity.
o-HPA, formed spontaneously from CE, is a hepatotoxic aldehyde (Born et al., 2000b). This metabolite has also been identified as a major intermediate detected following incubation of coumarin with microsomes from many species (Fentem et al., 1991
; Lake et al., 1992
). Therefore, oxidation of this intermediate to the nontoxic o-HPAA was the second detoxification reaction evaluated. Major differences in this reaction were observed, as the clearance of o-HPA through this pathway proceeded more than 20 times faster in mice than in rats. This is consistent with the observation that in mice, all of the o-HPA formed was oxidized to o-HPAA, whereas in rat, o-HPA remained as a major component detected in the microsomal reaction mixture. Based on the requirement for NAD+ and its inhibition by disulfiram, this reaction is likely to be catalyzed by ALDH isozymes.
In vivo studies indicate that rats do excrete o-HPAA in urine after administration of coumarin (Born et al., 2003; Huwer et al., 1991
; Kaighen and Williams, 1961
). Thus, it is not that rats do not oxidize any o-HPA to o-HPAA, but rather, this reaction appears to occur more slowly in rats than in mice. Moreover, in rats, the Km for reduction of o-HPA to o-HPE is very similar to that for the oxidation reaction, a pattern not observed in mice. Since o-HPE can also be oxidized back to o-HPA, the tendency for rats to reduce o-HPA to its corresponding alcohol may contribute to a detrimental cycle of reduction and oxidation that contributes to slower hepatic clearance of the toxic aldehyde.
The present results also have important implications regarding human hepatic metabolism of coumarin. In human cytosol, GSH conjugation of CE was a minor metabolite relative to the oxidation of o-HPA to o-HPAA, and clearance of o-HPA through the oxidation pathway proceeded more than 50 times faster in the average pooled human liver cytosols than in the rat. These results indicate that the rate of detoxification of CE in human liver favors oxidation of the aldehyde. The Km for CE formation in human liver microsomes is at least 1000 µM, which is much higher than that reported for mouse or rat (approximately 40 µM; Born et al., 2000a). At the same time, the Km for o-HPAA formation is about 1 µM in human liver cytosol. Therefore, in human liver, conversion to CE is likely to be very low, whereas oxidation of o-HPA to o-HPAA should occur efficiently.
The major metabolic pathway for coumarin metabolism in humans is 7-hydroxylation, and the Km for this reaction, catalyzed by CYP2A6, is approximately 1 µM (Honkakoski et al., 1993; Pelkonen et al., 2000
; Shimada et al., 1996
). It has been suggested that individuals who are polymorphic for CYP2A enzymes may be at increased risk of hepatotoxicity following coumarin exposure (Hadidi et al., 1997
, 1998
). The genetic polymorphism of CYP2A6 involves more than 10 different allelic variants, with the most common being the CYP2A6*2 variant that encodes an inactive enzyme, and the CYP2A6*4A variant that represents a gene deletion (Fernandez-Salguero et al., 1995
; Oscarson, 2001
; Raunio et al., 2001
; Yamano et al., 1990
). Overall, the variant allelic frequencies are reported to occur in less than 2% of the Caucasian population (Chen et al., 1999
; Paschke et al., 2001
), whereas the CYP2A6*4 gene deletion is present in Asian populations at a 1520% frequency (Oscarson et al., 1999
). Hadidi et al. (1997)
described an individual who was homozygous for the CYP2A6*2 allele, and this subject showed no 7-hydroxylation of coumarin. In fact, the subject excreted approximately 50% of a 2 mg dose of coumarin as o-HPAA, a result that is predicted by and consistent with the in vitro metabolism data presented herein.
More recently, Burian et al. (2003) determined the CYP2A6 genotype of patients who were dosed with 90 mg coumarin/day as part of a clinical evaluation of the efficacy and safety of coumarin for the treatment of chronic venous insufficiency. From 231 patients, 16 (7.4%) were found to be defective for the CYP2A6 genotype, and all were heterozygous for the CYP2A6*2 alleles. During the course of the 16-week study, nine patients showed elevations in serum liver enzymes, with only one patient with a CYP2A6*2 variant allele showing evidence of hepatotoxicity. All other affected patients were identified as wild-type homozygotes. This study represents the most extensive analysis of the contribution of the CYP2A6 polymorphism to coumarin-induced hepatotoxicity conducted to date and indicates that genetic polymorphism in CYP2A6 is not a direct cause of liver dysfunction observed with therapeutic dosages of coumarin.
The results of the present study suggest that oxidation of o-HPA is catalyzed by ALDH. As such, the activity of ALDH isozymes may be a factor that contributes to coumarin-induced liver abnormalities in humans. There is a known polymorphism in mitochondrial ALDH2 that contributes to marked sensitivity to acetaldehyde, especially among Oriental populations (Harada et al., 1980, 1981
; Yoshida et al., 1983
). Other isoforms in the ALDH superfamily are not as well characterized, but there are clear differences in subcellular localization and substrate specificities. ALDH1A1 is a major cytosolic enzyme with affinity for retinaldehyde and aliphatic aldehydes, and cytosolic ALDH3A1 and microsomal ALDH3A2 catalyze the oxidation of medium chain and aromatic aldehydes (Sophos and Vasiliou, 2003
; Vasiliou and Pappa, 2000
). Accordingly, isozymes in the ALDH1A or ALDH3A families may be important in the fate of o-HPA. Clinical evaluation of liver function in coumarin-treated patients with known ALDH isozyme activity coupled with analysis of urinary excretion of o-HPAA is needed to determine whether detoxification of the aldehyde intermediate may contribute to a potential risk for hepatotoxic outcome.
The present results implicate the kinetics of o-HPA detoxification following metabolic activation as the major determinant of species differences in coumarin-induced hepatotoxicity. Whereas the rat appears to be the most susceptible to this toxicity, humans are generally resistant. More importantly, at levels of exposure associated with unintentional exposure to coumarin from food stuffs or consumer products (approximately 0.06 mg/kg/day; Lake, 1999), the results suggest that coumarin metabolism in humans is not likely to result in coumarin-induced toxicity.
![]() |
SUPPLEMENTARY DATA |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed. Fax: (513) 627-1760. E-mail: vassallo.jd{at}PG.com.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Born, S. L., Caudill, D., Fliter, K. L., and Purdon, M. P. (2002). Identification of the cytochromes P450 that catalyze coumarin 3,4-epoxidation and 3-hydroxylation. Drug Metab. Dispos. 30(5), 483487.
Born, S. L., Caudill, D., Smith, B. J., and Lehman-McKeeman, L. D. (2000a). In vitro kinetics of coumarin 3,4-epoxidation: Application to species differences in toxicity and carcinogenicity. Toxicol. Sci. 58, 2331.
Born, S. L., Hu, J. K., and Lehman-McKeeman, L. D. (2000b). o-Hydroxyphenylacetaldehyde is a hepatotoxic metabolite of coumarin. Drug Metab. Dispos. 28, 218223.
Born, S. L., Rodriguez, P. A., Eddy, C. L., and Lehman-McKeeman, L. D. (1997). Synthesis and reactivity of coumarin 3,4-epoxide. Drug Metab. Dispos. 25, 13181324.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of the microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248254.[CrossRef][ISI][Medline]
Bruce, J. M., and Creed, D. (1970). Light-induced and related reactions of quinones: Part VI. Reactions of some p-quinones carrying formyl groups. J. Chem. Soc. C, 649653.
Burian, M., Freudenstein, J., Tegtmeier, M., Naser-Hijazi, B., Henneicke-von Zepelin, H. H., and Legrum, W. (2003). Single copy of variant CYP2A6 alleles does not confer susceptibility to liver dysfunction in patients treated with coumarin. Int. J. Clin. Pharmacol. Ther. 41, 141147.[ISI][Medline]
Chen, G. F., Tang, Y. M., Green, B., Lin, D. X., Guengerich, F. P., Daly, A. K., Caporaso, N. E., and Kadlubar, F. F. (1999). Low frequency of CYP2A6 gene polymorphism as revealed by a one-step polymerase chain reaction method. Pharmacogenetics 9, 327332.[ISI][Medline]
Cholerton, S., Idle, M. E., Vas, A., Gonzalesz, F. J., and Idle, J. R. (1992). Comparison of a novel thin-layer chromatographic fluorescence detection method with a spectrophotofluorometric method for the determination of 7-hydroxycoumarin in human urine. J. Chromatogr. 575, 325330.[Medline]
Cohen, A. J. (1979). Critical review of the toxicology of coumarin with special reference to interspecies differences in metabolism and hepatotoxic response and their significance to man. Food Comest. Toxicol. 17, 277289.[CrossRef]
Egan, D., O'Kennedy, R., Moran, E., Cox, D., Prosser, E., and Thornes, R. D. (1990). The pharmacology, metabolism, analysis, and applications of coumarin and coumarin-related compounds. Drug Metab. Rev. 22, 503529.[ISI][Medline]
Fentem, J. H., and Fry, J. R. (1993). Species differences in the metabolism and hepatotoxicity of coumarin. Comp. Biochem. Physiol. 104C, 18.
Fentem, J. H., Fry, J. R., and Whiting, D. A. (1991). o-Hydroxyphenylacetaldehyde: A major novel metabolite of coumarin formed by rat, gerbil and human liver microsomes. Biochem. Biophys. Res. Commun. 179, 197203.[ISI][Medline]
Fernandez-Salguero, P., Hoffman, S. M., Cholerton, S., Mohrenweiser, H., Raunio, H., Rautio, A., Pelkonen, O., Huang, J. D., Evans, W. E., Idle, J. R., and Gonzalez, F. J. (1995). A genetic polymorphism in coumarin 7-hydroxylation: Sequence of the human CYP2A genes and identification of variant CYP2A6 alleles. Am. J. Hum. Genet. 57, 651660.[Medline]
Guengerich, F. P. (1989). Analysis and characterization of enzymes. In Principles and Methods of Toxciology (A. W. Hayes, Ed.), pp. 777814. Raven Press, New York.
Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974). The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 249, 71307139.
Hadidi, H., Irshaid, Y., Vagbo, C. B., Brunsvik, A., Cholerton, S., Zahlsen, K., and Idle, J. R. (1998). Variability of coumarin 7- and 3-hydroxylation in a Jordanian population is suggestive of a functional polymorphism in cytochrome P450 CYP2A6. Eur. J. Clin. Pharmacol. 54, 437441.[CrossRef][ISI][Medline]
Hadidi, H., Zahlsen, K., Idle, J. R., and Cholerton, S. (1997). A single amino acid substitution (Leu160His) in cytochrome P450 CYP2A6 causes switching from 7-hydroxylation to 3-hydroxylation of coumarin. Food Chem. Toxicol. 35, 903907.[CrossRef][ISI][Medline]
Harada, S., Agarwal, D. P., and Goedde, H. W. (1981). Aldehyde dehyrogenase deficiency as cause of facial flushing reaction to alcohol in Japanese. Lancet 2, 982.[ISI][Medline]
Harada, S., Misawa, S., Agarwal, D. P., and Goedde, H. W. (1980). Liver alcohol dehydrogenase and aldehyde dehydrogenase in Japanese-isoenzyme variation and its possible role in alcohol intoxication. Am. J. Hum. Genet. 32, 815.[ISI][Medline]
Hazleton, L. W., Tusing, T. W., Zeitlin, H. R., Thiessen Jr., R., and Murer, H. K. (1956). Toxicity of coumarin. J. Pharmacol. Exp. Ther. 118, 348358.[ISI][Medline]
Hiratsuka, A., Hirose, K., Saito, H., and Watabe, T. (2000). 4-Hydroxy-2(E)-nonenal enantiomers: (S)-selective inactivation of glyceraldehydes-3-phosphate dehydrogenase and detoxification by rat glutathione S-transferase A4-4. Biochem. J. 349, 729735.[CrossRef][Medline]
Honkakoski, P., Maenpaa, J., Leikola, J., Pasanen, M., Juvonen, R., Lang, M. A., Pelkonene, O., and Raunio, H. (1993). Cytochrome P4502A-mediated coumarin 7-hydroxylation and testosterone hydroxylation in mouse and rat lung. Pharmacol. Toxicol. 72(2), 107112.[ISI][Medline]
Huwer, T., Altmann, H. J., Grunow, W., Lehnardt, S., Przybylski, M., and Eisenbrand, G. (1991). Coumarin mercapturic acid isolated from rat urine indicates metabolic formation of coumarin 3, 4-epoxide. Chem. Res. Toxicol. 4, 586590.[ISI][Medline]
Kaighen, M., and Williams, R. T. (1961). The metabolism of [3-14C] coumarin. J. Med. Pharm. Chem. 3, 2543.[ISI]
Lake, B. G. (1999). Coumarin metabolism, toxicity and carcinogenicity: Relevance for human risk assessment. Food Chem. Toxicol. 37, 423453.[CrossRef][ISI][Medline]
Lake, B. G. (1984). Investigations into the mechanism of coumarin-induced hepatotoxicity in the rat. Arch. Toxicol. 7(Suppl.), 1629.
Lake, B. G., Evans, J. G., Lewis, D. F. V., and Price, R. J. (1994). Comparison of the hepatic effects of coumarin, 3,4-dimethylcoumarin, dihydrocoumarin and 6-methylcoumarin in the rat. Food Chem. Toxicol. 32, 743751.[ISI][Medline]
Lake, B. G., and Grasso, P. (1996). Comparison of hepatotoxicity of coumarin in the rat, mouse and Syrian hamster: A dose and time response study. Fund. Appl. Toxicol. 34, 105117.[CrossRef][ISI][Medline]
Lake, B. G., Gray, T. J. B., Evans, J. G., Lewis, D. F. V., Beamand, J. A., and Hue, K. L. (1989). Studies on the mechanism of coumarin-induced toxicity in rat hepatocytes: Comparison with dihydrocoumarin and other coumarin metabolites. Toxicol. Appl. Pharmacol. 97, 311323.[ISI][Medline]
Lake, B. G., Osborne, D. J., Walters, D. G., and Price, R. J. (1992). Identification of o-hydroxyphenylacetaldehyde as a major metabolite of coumarin in rat hepatic microsomes. Food Chem. Toxicol. 30, 99104.[CrossRef][ISI][Medline]
Lam, J. P., Mays, D. C., and Lipsky, J. J. (1997). Inhibition of recombinant human mitochondrial and cytosolic aldehyde dehydrogenases by two candidates for the active metabolites of disulfiram. Biochemistry 36, 1374813754.[CrossRef][ISI][Medline]
Marshall, M. E., Mohler, J. L., Edmonds, K., Williams, B., Butler, K., Ryles, M., Weiss, L., Urban, D., Bueschen, A., Markiewicz, M. and Cloud, G. (1994). An updated review of the clinical development of coumarin (1,2-benzopyrone) and 7-hydroxycoumarin. J. Cancer. Res. Clin. Oncol. 120(Suppl.), S39S42.[ISI][Medline]
National Toxicology Program (1993). Toxicology and carcinogenicity studies of coumarin (CAS No 91-64-5) in F344/N rats and B6C3F1 mice (gavage studies). Technical Report no. TR 422. NIH Publication No 93-3153, US Dept. of Health and Human Services, National Institutes of Health, Research Triangle Park, NC.
Negishi, M., Lindberg, R., Burkhart, B., Ichikawa, T., Honkakoski, P., and Lang, M. (1989). Mouse steroid 15-hydroxylase gene family: Identification of Type II P-450 15
as coumarin 7-hydroxylase. Biochemistry 28, 41694172.[ISI][Medline]
O'Brien, K., Moss, E., Judah, D., and Neal, G. (1983). Metabolic basis of the species difference to aflatoxin B1 induced hepatotoxicity. Biochem. Biophys. Res. Commun. 114(2), 813821.[ISI][Medline]
Oscarson, M. (2001). Genetic polymorphisms in the cytochrome P450 2A6 (CYP2A6) gene: Implications for the interindividual differences in nicotine metabolism. Drug Metab. Dispos. 29, 9195.
Oscarson, M., Gullsten, H., Ratio, A., Bernal, M. L., Sinues, B., Dahl, M.-L., Stengard, J. H., Pelkonen, O., Raunio, H., and Ingelman-Sundberg, M. (1998). Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase. FEBS Lett. 438, 201205.[CrossRef][ISI][Medline]
Oscarson, M., McLellan, R. A., Gullsten, H., Yue, Q.Y. Lang, M. A., Bernal, M. L., Sinues, B., Hirvonen, A., Raunio, H., Pelkonen, O., and Ingleman-Sundberg, M. (1999). Characterization and PCR-based detection of a CYP2A6 gene deletion found at a high frequency in a Chinese population. FEBS Lett. 448, 105110.[CrossRef][ISI][Medline]
Paschke, T., Riefler, M., Schuler-Metz, A., Wolz, L., Scherer, G., McBride, C. M., and Bepler, G. (2001). Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP genotyping method. Toxicology 168, 259268.[CrossRef][ISI][Medline]
Pearce, R., Greenway, D., and Parkinson, A. (1992). Species differences and interindividual varation in liver microsomal cytochrome P450 2A enzymes: Effects on coumarin, dicoumarol and testosterone oxidation. Arch. Biochem. 298, 211225.[ISI][Medline]
Pelkonen, O., Raution, A., Raunio, H., and Pasanen, M. (2000). CYP2A6: A human coumarin 7-hydroxylase. Toxicology 144, 139147.[CrossRef][ISI][Medline]
Ramsdell, H., and Eaton, D., (1990). Mouse liver glutathione s-transferase isoenzyme activity toward aflatoxin B1-8,9-epoxide and benzo[a] pyrene-7,8-dihydrodiol-9,10-epoxide1. Toxicol. Appl. Pharmacol. 105, 216225.[ISI][Medline]
Raunio, H., Rautio, A., Gullsten, H., and Pelkonen, O. (2001). Polymorphisms of CYP2A6 and its practical consequences. Br. J. Clin. Pharmacol. 52, 357363.[CrossRef][ISI][Medline]
Raunio, H., Syngelma, T., Pasanen, M., Juvonen, R., Honkakoski, P., Kairaluoma, M. A., Sotaniemi, E., Lang, M. A., and Pelkonen, O. (1988). Immunochemical and catalytical studies on hepatic coumarin 7-hydroxylase in man, rat and mouse. Biochem. Pharmacol. 37, 38893895.[CrossRef][ISI][Medline]
Rautio, A., Kraul, H., Koho, A., Salmela, E., and Pelkonen, O. (1992). Interindividual variability of coumarin 7-hydroxylation in healthy volunteers. Pharmacogenetics 2, 227233.[ISI][Medline]
Shilling, W. H., Crampton, R. F. and Longland, R. C. (1969). Metabolism of coumarin in man. Nature 221, 664665.[ISI][Medline]
Shimada, T., Yamazaki, H., and Guengerich, F. P. (1996). Ethnic-related differences in coumarin 7-hydroxylation activities catalyzed by cytochrome P4502A6 in liver microsomes of Japanese and Caucasian populations. Xenobiotica 26, 395403.[ISI][Medline]
Sophos, N. A., and Vasiliou, V. (2003). Aldehyde dehydrogenase gene superfamily: The 2002 update. Chem. Biol. Interact. 143144, 522.[ISI]
Steensma, A., Beamand, J. A., Walters, D. G., Price, R. J., and Lake, B. G. (1994). Metabolism of coumarin and 7-ethoxycoumarin by rat, mouse, guinea pig, Cynomolgus monkey and human precision-cut liver slices. Xenobiotica 24, 893907.[ISI][Medline]
Vasiliou, V., and Pappa, A. (2000). Polymorphisms of human aldehyde dehydrogenases. Consequences for drug metabolism and disease. Pharmacology 61, 192198.[CrossRef][Medline]
Vassallo, J. D., Morall, S. W., Fliter, K. L., Curry, S., M., Daston, G. P., and Lehman-McKeeman, L. D. (2003). Liquid chromatographic determination of the glutathione conjugate and ring opened metabolites formed from coumarin epoxidation. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 794, 257271.[ISI][Medline]
Yamano, S., Tatsuno, J., and Conzalez, F. J. (1990). The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 29, 13221329.[ISI][Medline]
Yoshida, A., Wang, G., and Dave, V. (1983). Determination of genotypes of human aldehyde dehydrogenase ALDH2 Locus. Am. J. Hum. Genet. 35, 11071116.[ISI][Medline]