Inhibition of human alcohol and aldehyde dehydrogenases by acetaminophen: assessment
of the effects on first-pass metabolism of ethanol
Yeung-Pin Lia, Jian-Tong Liaob, Ya-Wen Chengb, Ting-Lun Wub, Shou-Lun Leec,
Jong-Kang Liua, Shih-Jiun Yinb,*
a
Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung,
Taiwan
b
c
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan
Department of Biological Science and Technology, China Medical University, Taichung,
Taiwan
*Corresponding author. Department of Biochemistry, National Defense Medical Center,
161 Minchuan East Road Section 6, Taipei 11453, Taiwan. Tel.: +886-2-8792-3100 ext.
18800; fax: +886-2-8792-4818.
E-mail address: yinsj@ndmc.idv.tw (S.-J. Yin).
Abstract
Acetaminophen is one of the most widely used over-the-counter analgesic, antipyretic
medications. Use of acetaminophen and alcohol are commonly associated. Previous
studies showed that acetaminophen may affect bioavailability of ethanol by inhibiting
gastric alcohol dehydrogenase (ADH). However, potential inhibitions by the drug of
human ADH family and relevant aldehyde dehydrogenase (ALDH) isozymes in relation
to first-pass metabolism (FPM) of ethanol remain undefined. ADH and ALDH, both
exhibiting racial distinct allozymes and tissue-specific distribution of isozymes, are
principal enzymes responsible for ethanol metabolism in humans. In this study, we
investigated acetaminophen inhibition of ethanol oxidation with recombinant human
ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and
of acetaldehyde oxidation with recombinant human ALDH1A1 and ALDH2 at near
physiological pH 7.5 and a cytoplasmic coenzyme concentration, 0.5 mM NAD+.
Acetaminophen acted as noncompetitive inhibitor for ADH family with the slope
inhibition constants (Kis) ranging from 0.90 mM (ADH2) to 20 mM (ADH1A), and the
intercept inhibition constants (Kii) ranging from 1.4 mM (ADH1C allozymes) to 19 mM
(ADH1A). Acetaminophen exhibited noncompetitive inhibition for ALDH2 (Kis = 3.0
mM and Kii = 2.2 mM) but competitive inhibition for ALDH1A1 (Kis = 0.96 mM). The
metabolic interactions between acetaminophen and ethanol/acetaldehyde were assessed
by computer simulation using the inhibition equations and the determined kinetic
constants. At therapeutic to subtoxic plasma levels of drug (i.e., 0.2 to 0.5 mM) and
physiologically relevant concentrations of ethanol (10 mM) and acetaldehyde (10 μM) in
target tissues, acetaminophen could inhibit ADH1C allozymes (12–26%) and ADH2
(14–28%) in liver and small intestine, ADH4 (15–31%) in stomach, ALDH1A1 (16–33%)
and ALDH2 (8.3–19%) in the three tissues. The results suggest that inhibition of hepatic
and gastrointestinal FPM of ethanol through ADH and ALDH pathway by
acetaminophen might become significant at higher, subtoxic levels of the drug.
Keywords: Acetaminophen and ethanol; Metabolic interaction; Alcohol dehydrogenase;
Aldehyde dehydrogenase; Inhibition kinetics; First-pass metabolism of ethanol
Introduction
Acetaminophen (paracetamol; N-acetyl-p-aminophenol) is a common analgesic and
antipyretic drug, one of the most widely used over the counter medication in the world. It
has been well documented that chronic excessive alcohol exposure enhance hepatoxicity
of acetaminophen by elevation of ethanol-inducible cytochrome P450 2E1 (CYP2E1),
which converts the drug into reactive toxic intermediates; in contrast, simultaneous
alcohol exposure may exert protective effect due to inhibition of the CYP2E1
biotransformation of acetaminophen by ethanol (Cederbaum, 2012; Lieber, 2004;
Riordan and Williams, 2002). However, interaction of acetaminophen with ethanol
metabolism has received much less attention. Acetaminophen was reported to inhibit
gastric alcohol dehydrogenase (ADH) activity (Palmer et al., 1991; Roine et al., 1991); its
potential inhibition of other ADH family members and aldehyde dehydrogenase (ALDH)
isozymes in relation to ethanol metabolism remains unknown. Use of acetaminophen and
alcohol are commonly associated. Acetaminophen may increase blood alcohol levels in
vivo, particularly at a low alcohol dose, thus having potential clinical consequences as
well as influence on performance of drinking drivers (Jones, 2010; Lieber et al., 1996).
First-pass, or presystemic, metabolism (FPM) of ethanol affects peripheral availability
and intoxicating consequences in the body. The sites of FPM include stomach, small
intestine, and liver but their relative contributions for ethanol metabolism remain
controversial (Badger et al., 2003; Gentry et al., 1994; Levitt, 1994; Yin et al., 2007).
Several factors may affect the extent of the FPM of ethanol, such as food consumption,
concentration of alcoholic beverages, genetic polymorphism of alcohol metabolizing
enzymes, medications that interfere with activity of the metabolizing enzymes or with
absorption of ethanol (Cederbaum, 2012; Jones, 2010; Kalant, 1996; Lee et al., 2006a).
ADH and ALDH catalyze oxidation of various aliphatic/aromatic endogenous and
exogenous alcohols to the corresponding aldehydes, and then to the corresponding
carboxylic acids, respectively (Edenberg and Bosron, 2010; Hoog et al., 2003; Sophos
and Vasiliou, 2003; Wang et al., 2009). Both ADH and ALDH, the principal enzymes
responsible for metabolism of ethanol in humans (Cederbaum, 2012; Yin and Agarwal,
2001), exhibit functional polymorphisms among racial populations and tissue-specific
distributions. Human ADH family members have been categorized into five classes on
the basis of protein sequence and gene organization, electrophoretic, kinetic and
immunochemical features (Duester et al., 1999; Hoog and Ostberg, 2011; Lee et al.,
2006b). The class I ADH contains multiple forms, that is, ADH1A (previously denoted
αα), ADH1B (ββ) and ADH1C (γγ). The classes II−IV ADHs contain a single form each,
that is, ADH2 (ππ), ADH3 (χχ), and ADH4 (μμ or σσ), respectively. ADH1B*1 (encoding
the β1 polypeptide subunit) and ADH1B*2 (encoding β2 subunit) are predominant among
Caucasians and East Asians, respectively; ADH1B*3 (encoding β3 subunit) is found
exclusively in Africans and some tribes of American Indians. ADH1C*1 (encoding γ1
subunit) and ADH1C*2 (encoding γ2 subunit) are approximately equally distributed
among Caucasians and American Indians, but the former is highly prevalent among East
Asian and African populations. Currently, class V ADH is the only family member with
no available data for catalytic function due to its extremely labile activity (Hoog et al.,
2003; Ostberg et al., 2013). All three class I isozymes, ADH2 and ADH3 are expressed in
human liver (Edenberg and Bosron, 2010; Yin and Agarwal, 2001), while ADH4 and
ADH1C are detected in the stomach (Yin et al., 1997), and ADH2 and ADH1C in small
intestine (Chiang et al., 2012b).
In the human ALDH superfamily (Anonymous, 1989; Sladek, 2003; Weiner and Ho,
2007), class I ALDH1A1 and class II ALDH2 are predominantly expressed in human
liver (Yao et al., 1997), and both isozymes are detected in the gastrointestinal tract
(Chiang et al., 2012a,b; Yin et al., 1997), whereas class III ALDH3A1 is a major form
found in the stomach (Yin et al., 1997). Mitochondrial ALDH2 is the major isozyme for
oxidation of acetaldehyde in vivo due to its submicromolar Km and high catalytic
efficiency, whereas cytosolic ALDH1A1 with its high micromolar Km , may be also
contribute, particularly for individuals who lack active ALDH2 (Peng and Yin, 2009; Yin
and Peng, 2005). About 40% of East Asians are deficient in ALDH2 activity due to the
dominant negative variant allele of ALDH2*2 (Crabb et al., 2004; Lai et al., 2013b). This
deficiency has been attributed to protection against development of alcoholism (Chen et
al., 2009a,b), but it is a risk factor for alcohol-related diseases such as esophageal cancer
(Brooks et al., 2009; Yin and Agarwal, 2001).
To investigate potential metabolic interactions between ethanol and acetaminophen, we
report herein that, from an enzymological and pharmacogenetic perspective, the
inhibition of human ADH isozymes/allozymes and relevant ALDH isozymes at a
physiological concentration of coenzyme NAD+ as well as the simulation of the effects at
physiological levels of ethanol and acetaldehyde, respectively.
Materials and methods
Expression and purification of human ADH and ALDH
The expression of recombinant enzymes in Escherichia coli and purification to apparent
homogeneity for human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2,
ADH2, ADH3, ADH4, and for human ALDH1A1, ALDH2, ALDH3A1 were carried out
as described previously (Chiang et al., 2009; Lee et al., 2006a,b). All of the isolated
recombinant enzyme forms exhibited a single coomassie blue-staining protein band with
molecular masses of 40 kDa, 55 kDa, and 54 kDa for ADHs, ALDH1A1/2, and
ALDH3A1, respectively, on sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Protein concentration was determined by the method of Lowry et al. (1951) using bovine
serum albumin as the standard.
Kinetic analysis
Kinetic studies for ADH and ALDH were performed in 0.1 M sodium phosphate at pH
7.5 and 25°C, containing 0.5 mM NAD+, 1 mM ethylenediaminetetraacetate (EDTA;
only for ALDH assay), and varied concentrations of substrate and inhibitor. The cytosolic
NAD+ concentration in rat hepatocytes is reported to be ca. 0.5 mM (Bucher et al., 1972).
The enzyme activity was determined by monitoring the production of NADH at 340 nm
using an absorption coefficient of 6.22 mM-1cm-1 for ADH and ALDH assay (unless
otherwise indicated) or at 460 nm for emission of the fluorescence for the assay of ADH3,
ALDH1A1, and ALDH2. Preparation of formaldehyde and calculation of the
concentration of S-hydroxymethylglutathione from the equilibrium constants were
performed as described previously (Lee et al., 2003). Acetaldehyde and benzaldehyde
were redistilled before use. The reaction was initiated with addition of the enzyme.
Enzyme activity units (U) are expressed as micromoles of NADH formed per minute.
Steady-state kinetic data were analyzed by nonlinear least-squares regression using the
Cleland programs of HYPER, COMP, NONCOMP, and UNCOMP (Cleland, 1979).
Initial velocity data were fitted with HYPER program to the Michaelis–Menten equation.
v = (Vmax × S)/(Km + S)
(1)
The data from dead-end inhibition studies were fitted with the following linear inhibition
equations, that is, the COMP program for competitive inhibition, the NONCOMP for
noncompetitive inhibition, and the UNCOMP for uncompetitive inhibition, respectively.
v = (Vmax × S)/[Km (1 + I/Kis) + S]
(2)
v = (Vmax × S)/[Km (1 + I/Kis) + S (1 + I/Kii)]
(3)
v = (Vmax × S)/[Km + S (1 + I/Kii)]
(4)
where Vmax is the maximum velocity, S is the substrate concentration, Km is the Michaelis
constant, I is the inhibitor concentration, and Kis and Kii are the slope and intercept
inhibition constants, respectively. The type of inhibition was determined by evaluating
the standard errors of the kinetic constants and the residual variance for the equation that
best fit the data (Cleland, 1979). In cases where the intercepts and slopes did not vary
greatly with inhibitor concentration, Student’s t-tests were applied to determine if they
were significantly different. The kinetic experiments were performed in duplicate with
five substrate concentrations usually ranging from 0.5 to 5 Km and five (including one for
control, I = 0) inhibitor concentrations ranging from 0.2 up to 2 Ki when applicable.
Values represent means ± standard error of the mean (SEM). Standard errors of the fits to
the appropriate computer programs were less than 8.4% of the values for Km and Vmax and
less than 16% of those for the inhibition constants, indicating good precision.
Molecular docking
Acetaminophen was docked into the active sites of the X-ray structures of human
ADH1A [PDB ID:1HSO], ADH1B1 [PDB ID:1DEH], ADH1C2 [PDB ID:1HT0], ADH2
[PDB ID:3COS], ADH4 [PDB ID:1D1S], and ALDH2 [PDB ID:1O01], all complexes
with NAD+, using AutoDock4 (Morris et al., 2009). The illustrations were generated
using PyMOL v1.5 for Linux/Ubuntu (Schrodinger, LLC, New York).
Results
Inhibition type
Inhibition patterns and the corresponding kinetic constants of acetaminophen against
ethanol oxidation with human ADH family are shown in Table 1. The inhibition against
oxidation of S-hydroxymethylglutathione, instead of ethanol, for class III ADH3 was
studied because ADH3 is nearly unsaturable with ethanol, S0.5 = 3.4 M (Lee et al., 2003).
All ADH family members exhibited noncompetitive inhibition with slope inhibition
constants ranging from 0.90 mM (class II ADH2) to 29 mM (class III ADH3), and the
intercept inhibition constants ranging from 1.4 mM (class I ADH1C1 and ADH1C2) to
19 mM (class I ADH1A and class III ADH3).
Table 2 shows that acetaminophen was a competitive inhibitor with respect to
acetaldehyde for class I ALDH1A1 (Kis = 0.96 mM) but a noncompetitive inhibitor for
class II ALDH2 (Kis = 3.0 mM and Kii = 2.2 mM). In contrast, the inhibition of class III
ALDH3A1 was not detectable up to 20 mM acetaminophen. Benzaldehyde was used as
substrate for ALDH3A1, instead of acetaldehyde, which has a much higher Km (75 mM)
(Yin et al., 1995). Thus ALDH3A1 contributes negligibly to metabolism of the
ethanol-derived acetaldehyde in vivo (Yin and Agarwal, 2001; Yin et al., 1995).
Metabolic interaction
The interactions between acetaminophen, up to 2 mM, and the oxidation of ethanol, up to
50 mM for ADH1C1, ADH2, ADH4, and the oxidation of acetaldehyde, up to 0.5 mM
for ALDH1A1, at a cytosolic concentration of 0.5 mM NAD+ are shown in Fig. 1. The
ADH and ALDH forms exhibiting slope inhibition constants ≤ 2 mM were chosen for
illustration of the drug inhibition in a three-dimensional way. In the absence of inhibitor
(I = 0) enzyme activities increase with increasing substrate concentration, that is, the
substrate saturation curves reflect the Michaelis constants of the enzymes. The enzyme
activities in the presence of inhibitor reflect both Km and Ki as the substrate concentration
progressively increases. In the presence of high substrate concentrations inhibition of the
noncompetitive type may not be completely overcome. This is because the drug binds to
enzyme species other than the one combining with the competing substrate. This is
illustrated most clearly with ADH1C1 exhibiting a low Km (0.25 mM) and a lower Kii (1.4
mM) than that for the Kis (2.2 mM), where saturating substrate (50 mM ethanol) did not
overcome inhibition by 2 mM acetaminophen. It is important to note that in all cases,
activity increases with increasing concentration of substrate, even as the percentage of
inhibition increases.
Quantitative assessment
The therapeutic and toxic blood plasma levels of acetaminophen in humans are estimated
to be 0.017–0.17 and 1.0 mM, respectively (Schulz and Schmoldt, 1994). The percentage
inhibition of enzyme activity by 0.2, 0.5 and 1.0 mM acetaminophen were assessed at 2
and 10 mM ethanol for classes I, II and IV ADHs (Table 3) and at 10, 50 and 200 μM
acetaldehyde for ALDH1A1 and ALDH2 (Table 4). It has been reported that following
ingestion of a low dose of ethanol (0.2 g/kg body weight, roughly equivalent to a bottle of
beer for a 65-kg man), the peak blood alcohol reaches 2 mM (Peng et al., 1999); 20 mM
blood ethanol in several countries is the legal limit for driving. Hepatic steady-state
concentrations of acetaldehyde reach 10–20 μM in rat livers perfused with ethanol (Yao
et al., 2010), and thus it can be inferred that 200 μM acetaldehyde or even higher levels
may occur in the liver of East Asians with heterozygous ALDH2*1/*2 genotype, whose
blood acetaldehyde concentrations reached 24–76 μM, after intake of low to moderate
alcohol (Peng et al., 1999, 2007).
At 0.2 mM acetaminophen, a therapeutic blood concentration, and 2 mM ethanol, the
inhibition of ADH activities appears to be minimal (< 3.3%) for ADH1A, ADH1B1,
ADH1B2, ADH1B3 whereas appreciable inhibitions (12–17%) are detected for ADH1C1,
ADH1C2, ADH2 and ADH4. At a higher but still subtoxic concentration of
acetaminophen, that is, 0.5 mM, the inhibitions of ADH1C allozymes and class II and IV
ADHs are considerably increased (26–34%) whereas that for ADH1A and ADH1B
allozymes remain low (< 7.9%). Since ethanol concentration in stomach fluid in a social
drinking setting may reach 200 mM or higher (Haber et al., 1996; Yin et al., 1997), the
inhibitions of gastric ADH4 activity by 0.2 and 0.5 mM acetaminophen were assessed to
be 7.4% and 17%, respectively, at 200 mM ethanol. At 10 μM acetaldehyde, 0.2 mM
acetaminophen inhibits activities of ALDH1A1 (16%) and ALDH2 (8.3%); the inhibition
increases to 33% and 19%, respectively, by 0.5 mM drug.
Model docking of acetaminophen binding
The activities of human ADH allozymes/isozymes are inhibited by acetaminophen with
varied potencies. This suggests that the different amino acid residues in the barrel-shaped
hydrophobic substrate pockets of ADH interact with the drug somewhat differently. We
found that acetaminophen could fit into the binding sites of human ADH1A, ADH1B1,
ADH1C2, ADH2, and ADH4 in the presence of NAD+. Model docking of ADH2, which
exhibits the highest affinity with acetaminophen, was chosen to illustrate the binding
mode. In our model, the substrate pocket of ADH2 accommodates well the drug molecule
(Fig. 2a.). The catalytic zinc atom is ligated to oxygen of the hydroxyl group of
acetaminophen and the nicotinamide ring of NAD+ forms stacking contact with phenyl
ring of the drug; the bulky Tyr-94 and Phe-146 also contribute to position a correct
binding of the drug. The weak binding of acetaminophen to ADH1A, which exhibits
more than 20-fold higher Kis than that of ADH2, can be largely attributed to the
substitution of a much smaller amino acid at the equivalent position, that is Ala-93 (cf.
Tyr-94 in ADH2), resulting in poor binding. It is worth noting that the
theoretically-calculated binding energies with acetaminophen molecule varied only up to
14% for the ADH isozymes/allozymes and the magnitudes of the calculated dissociation
constants (data not shown) do not match to those of the observed kinetic inhibition
constants. Thus, the modeling only supports the idea that acetaminophen can bind. To
elucidate the exact molecular structures of binding, future studies require x-ray
crystallographic determination of the ADH coenzyme-drug ternary complexes.
Acetaminophen also can be modeled to fit into the binding pocket of human ALDH2 in
the presence of NAD+ (Fig. 2b). Interestingly, acetamide terminal of drug, instead of the
phenolic end, is oriented toward bottom of substrate pocket with its carbonyl oxygen
binding to γ-amide hydrogen of Asn-169. Phe-170, Phe-459 and Phe-465 constitute part
of a hydrophobic tunnel that well accommodates the aryl moiety of the drug.
Discussion
Metabolic interactions between acetaminophen and ethanol
This is the first comprehensive report on acetaminophen inhibition of human ADHs and
ALDHs at a near physiological pH and cytoplasmic NAD+ concentration, indicating that
the drug may interact with metabolic pathway of ethanol at both the ADH and ALDH
steps. The inhibition patterns and kinetic constants in the ADH family provide an
enzymological basis for quantitative evaluation of the potential interactions of ethanol
metabolism with acetaminophen from a pharmacokinetics perspective.
Acetaminophen acts as noncompetitive inhibitor against ethanol oxidation at 0.5 mM
NAD+, a saturating coenzyme concentration for ADH family except ADH1B2, ADH1B3
and ADH4. The noncompetitive inhibition by drug can be explained by the formation of
a dead-end E-NAD+-inhibitor complex during catalysis, which gives rise to slope
inhibition effect (Kis), and by the formation of an E-NADH-inhibitor complex giving rise
to the intercept inhibition effect (Kii). It has been well documented that ADH conforms to
an ordered sequential bi mechanism with binding of NAD+ first and NADH released last,
rate-limited by the release of coenzyme (Edenberg and Borson, 2010). In agreement with
the proposed mechanism, acetaminophen could fit into the substrate binding pockets of
ADH family in the presence of coenzyme as revealed by molecular model dockings. For
comparison, previous studies using the same assay buffer and 0.5 mM NAD+ (Lee et al.,
2011; Lai et al., 2013a) described that 4-methylpyrazole and cimetidine exhibited slope
inhibitions against ethanol oxidation with human ADH family, and the former also
showed a weaker intercept inhibition for ADH1B3, ADH2 and ADH4 and the latter,
varied intercept inhibitions for ADH1B2, ADH1B3 and ADH2. Thus, acetaminophen
appears to be unique in having both slope and intercept effects with varied inhibition
strengths for human ADH family. It is noted that at subsaturating 0.5 mM NAD+, binding
of drug to free E and E-NAD+ could also result in noncompetitive inhibition, which
cannot be completely ruled out for isozymes with high-Km for NAD+, including ADH1B2,
ADH1B3 ADH4. Clarification is needed in future studies.
With respect to oxidation of acetaldehyde, acetaminophen acts as competitive inhibitor
with human ALDH1A1 but as noncompetitive inhibitor with human ALDH2. In contrast,
the opposite was observed with cimetidine inhibitions (Lai et al., 2013a). It may involve
differential binding modes of the two drugs to substrate pockets of the corresponding
isozymes. Interestingly, ALDH2 has been identified as a major acetaminophen-binding
protein in liver mitochondria of mice administered with the drug (Landin et al., 1996).
Acetaminophen inhibition of the first-pass metabolism
Unlike the vast majority of drugs and xenobiotics, the elimination of ethanol is not
proportional to its concentration in body fluids, that is, first-order kinetics, but exhibits
pseudolinear, near zero-order kinetics at concentrations of ethanol above 2 mM (Kalant,
1996; Yin et al., 2007). This Michaelis–Menten-type pharmacokinetics is of special
interest in light of FPM, which is defined as the presystemic elimination of newly
absorbed ethanol through stomach, small intestine and liver, before reaching peripheral
blood. Variations reported for FPM using measurement of the blood alcohol
concentrations (po vs. iv) are largely due to varying saturation of the liver alcohol
metabolism (Badger et al., 2003; Lee et al., 2006a; Levitt and Levitt, 2000; Yin et al.,
2007). The extent of FPM would depend on the Km and Vmax for ethanol and the Ki for
inhibitors with the responsible ADHs, the amounts of expressed isozymes, and the
concentrations of ethanol and the drug in target tissues.
At therapeutic levels of blood plasma acetaminophen (0.2 mM), the decrease of activities
for class I ADH1C1 and ADH1C2, class II ADH2 and class IV ADH4 at 2–10 mM
ethanol and that of class I ALDH1A1 and class II ALDH2 at 10–50 μM acetaldehyde
would be predicted to be modest, that is, 12–17% for the ADHs and 8.3–16% for the
ALDHs (Tables 3 and 4). However, at a raised but still subtoxic concentration (0.5 mM),
the inhibition of activities with the corresponding ADH and ALDH forms would increase
two times, up to 26–34% and 19–33%, respectively, by computer simulations. The
former prediction appears to be consistent with previous reports that therapeutic doses of
acetaminophen only raised peak blood ethanol concentrations by 7–13% and the areas
under blood alcohol curves by 4–19% (Melander et al., 1995; Roine et al., 1991). ADH2
and ADH1C allozymes are among the major isozymes expressed in liver (Yao et al.,
1997) and they are also detected in small intestine (Chiang et al., 2012b); ADH4 and
ADH1C allozymes are predominantly expressed in stomach (Yin et al., 1997).
ALDH1A1 and ALDH2 are the major isozymes found in liver (Lai et al., 2013b; Yao et
al., 1997); both isozymes are also detected in gastrointestinal tract (Chiang et al., 2012a,b;
Yin et al.1997). Thus, our findings suggest that inhibition of hepatic and gastrointestinal
FPM and hence a potential increase of ethanol bioavailability by acetaminophen may
become significant at higher, subtoxic drug concentrations instead of the therapeutic
levels. Further studies with higher acetaminophen are needed to validate this inference.
To achieve a comprehensive analysis of the overall effects of acetaminophen on ethanol
metabolism, it will require that the actual amounts of all of the isozymes in the target
tissues be determined so that the contributions of each isozyme are accounted for, and
such studies are in progress.
Since ALDHs are involved in the metabolism of a great variety of carbonyl compounds,
the effects of acetaminophen on the metabolism of such compounds should also be
considered. For instance, acetaminophen may potentially reduce efficacy of nitroglycerin,
a common antianginal drug, due to its inhibition of mitochondrial ALDH2 and cytosolic
ALDH1A1 (Table 2), both isozymes are responsible for bioactivation of the drug (Beretta
et al., 2008). Clarification of this possibility is required in future studies. It has been,
indeed, observed that individuals carrying the variant ALDH2*2 lacked an efficacious
clinical response to nitroglycerin (Li et al., 2006).
In conclusion, the results indicate acetaminophen can inhibit activities of human ADH
family to widely varied degrees and also that of ALDH1A1 and ALDH2. In order to
quantitatively assess the overall effects of inhibition of the component isozymes by
acetaminophen in relation to FPM of ethanol, studies to determine protein contents of the
ADH and ALDH isozymes in target tissues are warranted.
Acknowledgments
This work was supported by the Grants from the National Science Council
90-2320-B016-057, 96-2320-B016-018-MY3 and 99-2320-B016-003-MY2, Republic of
China.
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Figure Legends
Fig. 1. Interactions between acetaminophen and ethanol oxidation with human (a)
ADH1C1, (b) ADH2, (c) ADH4, and between acetaminophen and acetaldehyde oxidation
with human (d) ALDH1A1. Enzyme activity was simulated at 0.5 mM NAD+ and varied
concentrations of ethanol, 0–50 mM, or acetaldehyde, 0–0.5 mM, and inhibitor
acetaminophen, 0–2 mM, using Eq. (2) for competitive inhibition and Eq. (3) for
noncompetitive inhibition. In the absence of inhibitor, both Eq. (2) and Eq. (3) are
virtually reduced to Eq. (1). For inhibition pattern and the kinetic constants of ADHs and
ALDH used for the simulation, see Tables 1 and 2, respectively.
Fig. 2. Modeled acetaminophen binding to the binary complexes of NAD+ with (a) human
ADH2 [PDB 3COS] and (b) human ALDH2 [PDB 1O01]. The side chains of amino acid
residues lining the active site that are in close vicinity, ≦5 Å , to the drug, are shown. In
panel (a), catalytic Zn2+ is ligated to oxygen of the hydroxyl group of acetaminophen,
nicotinamide ring of NAD+ forms stacking contact with the aryl ring of drug. In panel (b),
γ-amide hydrogen of Asn-169 bonds to carbonyl oxygen of the acetamide moiety of
acetaminophen. Phe-170, Phe-459 and Phe-465 form part of a hydrophobic tunnel
accommodating the aryl ring of drug.
Table 1. Kinetic constants for inhibition of ethanol oxidation by acetaminophen with
human ADHs
Enzyme activity was determined in 0.1 M sodium phosphate at pH 7.5 and 25⁰C,
containing 0.5 mM NAD+ and varied substrate concentrations at various fixed
concentrations of inhibitor. Km and Vmax are kinetic constants for substrate ethanol except
ADH3. The Km, Vmax, and Ki for ADH1B2, ADH1B3, and ADH4 are apparent values due
to the subsaturating concentration of 0.5 mM NAD+ used. Values represent means ±
SEM.
a
Substrate, S-hydroxymethylglutathione.
Table 2. Kinetic constants for inhibition of acetaldehyde oxidation by acetaminophen
with human ALDHs
Enzyme activity was determined in 0.1 M sodium phosphate at pH 7.5 and 25⁰C,
containing 0.5 mM NAD+ and 1 mM EDTA, and varied substrate concentrations at
various fixed concentrations of inhibitor. Km and Vmax are kinetic constants for substrate
acetaldehyde except ALDH3A1. Values represent means ± SEM.
a
Substrate, benzaldehyde.
b
No detectable inhibition up to 20 mM acetaminophen at 100 μM benzaldehyde.
Table 3. Quantitative assessment of inhibition of ethanol oxidation by acetaminophen
with human ADHs
Enzyme activity at indicated concentrations of substrate and inhibitor was calculated
using the noncompetitive equation (Eq. (3)) based on the determined inhibition pattern
and the corresponding kinetic constants for ADH isozymes/allozymes shown in Table 1.
The enzyme activity in the absence of inhibitor (Eq. (1)) was used as control for
calculation of the corresponding drug inhibitions.
Table 4. Quantitative assessment of inhibition of acetaldehyde oxidation by
acetaminophen with human ALDHs
Enzyme activity at indicated concentrations of substrate and inhibitor was calculated
using the competitive (Eq. (2)) or noncompetitive (Eq. (3)) equations based on the
determined inhibition pattern and the corresponding kinetic constants for ALDH
isozymes shown in Table 2. The enzyme activity in the absence of inhibitor (Eq. (1)) was
used as control for calculation of the corresponding drug inhibitions.