Download Human cytosolic enzymes involved in the

Carcinogenesis vol.24 no.10 pp.1695±1703, 2003
DOI: 10.1093/carcin/bgg119
Human cytosolic enzymes involved in the metabolic activation of carcinogenic
aristolochic acid: evidence for reductive activation by human
NAD(P)H:quinone oxidoreductase
Marie Stiborova1,3, Eva Frei2, Bruno Sopko1, Klara
kova1,
a1, Martina Lan
Sopkova1, Vladimira Markov
Tereza Kumstyrov
a1, Manfred Wiessler2 and Heinz
H.Schmeiser2
X-ray structures. The results demonstrate for the first
time the potential of human NQO1 to activate AAI by
nitroreduction.
1
Department of Biochemistry, Faculty of Science, Charles University,
Albertov 2030, 128 40 Prague 2, The Czech Republic and 2Division of
Molecular Toxicology, German Cancer Research Center, Im Neuenheimer
Feld 280, D-69120 Heidelberg, Germany
Introduction
Abbreviations: AA, aristolochic acid; AAN, aristolochic acid nephropathy;
AAI, 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid;
AAII, 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid; dA±AAI,
7-(deoxyadenosin-N6 -yl)aristolactam I; dA±AAII, 7-(deoxyadenosin-N6 yl)aristolactam II; dG±AAI, 7-(deoxyguanosin-N2 -yl) aristolactam I; dG±
AAII, 7-(deoxyguanosin-N2 -yl) aristolactam II; NQO1, NAD(P)H:quinone
oxidoreductase; polyethylenimine; XO, xanthine oxidase.
The so-called Chinese herbs nephropathy (CHN), a unique
type of rapidly progressive renal fibrosis associated with the
prolonged intake of Chinese herbs during a slimming regimen,
was observed for the first time in Belgium in 1991 (1,2). About
100 CHN cases have been identified so far in Belgium, half of
which needed renal replacement therapy, mostly including
renal transplantation (3±5). The observed nephrotoxicity has
been traced to the ingestion of Aristolochia fangchi containing
carcinogenic and nephrotoxic aristolochic acid (AA) inadvertently included in slimming pills (2). CHN has been described
in patients in other European and in Asian countries and in the
USA (about 170 cases) (6), who were exposed to Aristolochia
species containing AA and had no relationship with the
Belgian slimming clinic. Therefore, it has been proposed to
designate the interstitial nephropathy in which the unequivocal
role of AA has been fully documented as aristolochic acid
nephropathy (AAN) (7,8). Recently, a high prevalence of
urothelial cancer was found in a large cohort of AAN patients
in Belgium (9,10) and a case with urothelial cancer has also
been described in the UK (11). It is also noteworthy that AA
consumption may be a potential cause of the development of a
similar type of fibrosis of the kidneys with malignant transformation of the urothelium, the Balkan endemic nephropathy
(BEN) (12±15), which is widely found in certain areas of
Rumania, Croatia, Bosnia, Serbia and Bulgaria along the
Danube river basin (12,15). This highlights the carcinogenic
potential of AA in human beings. AA is a mixture of structurally related nitrophenanthrene carboxylic acids, with 8-methoxy6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI)
and 6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid
(AAII), being the major components (Figure 1).
Since the demonstration that AA forms covalent DNA
adducts in rodents (16±18) as well as in AAN patients
(6,19±22), AA±DNA adducts have been used as biomarkers
of exposure and to investigate the mutagenic and carcinogenic
potential of AA.
The predominant AA±DNA adduct in vivo, 7-(deoxyadenosinN6 -yl)aristolactam I (dA±AAI), which is the most persistent of
the adducts in target tissue, is a mutagenic lesion leading to
A ! T transversions in vitro (23,24). This transversion mutation is found at high frequency in codon 61 of the H-ras
oncogene in tumors of rodents induced by AAI, suggesting
that dA±AAI might be the critical lesion in the carcinogenic
process in rodents. DNA binding studies confirmed that both
AAs bind to the adenines of codon 61 in the H-ras mouse gene
Carcinogenesis vol.24 no.10 # Oxford University Press; all rights reserved.
1695
3
To whom correspondence should be addressed
Email: [email protected] or [email protected]
Aristolochic acid (AA), a naturally occurring nephrotoxin
and carcinogen, has been associated with the development
of urothelial cancer in humans. Understanding which human
enzymes are involved in AA metabolism is important in the
assessment of an individual's susceptibility to this carcinogen. Using the 32 P-postlabeling assay we examined the
ability of enzymes of cytosolic samples from 10 different
human livers and from one human kidney to activate the
major component of the plant extract AA, 8-methoxy6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid
(AAI), to metabolites forming adducts in DNA. Cytosolic
fractions of both organs generated AAI±DNA adduct patterns reproducing those found in renal tissues from
humans exposed to AA. 7-(Deoxyadenosin-N6 -yl)aristolactam I, 7-(deoxyguanosin-N2 -yl)aristolactam I and 7(deoxyadenosin-N6 -yl)aristolactam II, indicating a possible
demethoxylation reaction of AAI, were identified as AA±
DNA adducts formed from AAI by all human hepatic and
renal cytosols. To define the role of human cytosolic reductases in the activation of AAI, we investigated the modulation of AAI±DNA adduct formation by cofactors or
selective inhibitors of the NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO) and aldehyde oxidase.
We also determined whether the activities of NQO1 and
XO in different human hepatic cytosolic samples correlated with the levels of AAI±DNA adducts formed by the
same cytosolic samples. Based on these studies, we attribute
most of the activation of AA in human cytosols to NQO1,
although a role of cytosolic XO cannot be ruled out.
With purified NQO1 from rat liver and kidney and
XO from buttermilk, the major role of NQO1 in the
formation of AAI±DNA adducts was confirmed. The orientation of AAI in the active site of human NQO1 was
predicted from molecular modeling based on published
M.Stiborova et al.
Fig. 1. Metabolic activation and DNA adduct formation of AA (Umlagerung ˆ rearrangement).
(23,24) and preferentially to purines in the human p53 gene
(5,6,25).
Interestingly, to date only 5% of the patients treated with the
slimming regimen in Belgium have suffered from nephropathy. Taking into account that AA is toxic, should it not have
affected more of the patients? One possible explanation for the
different responses of patients may be individual differences in
the activities of the enzymes catalyzing the biotransformation (detoxication and/or activation) of AA. Many genes of
enzymes metabolizing carcinogens are known to exist in variant forms or show polymorphisms resulting in differing activities of the gene products. These genetic variations appear to
be important determinants of cancer risk (26). Therefore,
screening AAN patients, as well as healthy persons treated
with the slimming regimen, for genetic variations in the genes
of the enzymes involved in AA metabolism should lead to
possible relationships between genotypes and nephropathy.
Thus, the identification of the enzymes principally involved
in the activation of AA in humans and a detailed knowledge of
their catalytic specificities is of major importance.
The detection of specific DNA adducts (16±22) by 32 Ppostlabeling analyses has allowed us to use AA±DNA binding
1696
as a probe for metabolic activation of AA in in vitro systems.
Recently we found that in vitro, both microsomal and cytosolic
enzymes activate AAI and AAII to form the same DNA
adducts found in vivo in rodents (27±30) and in humans suffering from AAN (6,19±22). However, in these experiments,
only enzymes from animals, and human recombinant enzymes,
were tested for their capabilities to activate AA. Rat hepatic
microsomal and recombinant human cytochromes P450
1A1 and 1A2, rabbit NADPH:cytochrome P450 reductase
(29,30), rat liver cytosolic NAD(P)H:quinone oxidoreductase
(NQO1, DT-diaphorase) (31), buttermilk xanthine oxidase
(XO) (27) and peroxidases such as ovine prostaglandin H
synthase (28) and milk lactoperoxidase (27) were efficient in
reductive activation of AA. However, animal enzymes or
human recombinant systems are usually not the ideal models
of the catalytic properties of enzymes in human organs. Moreover, no data are available on the participation of authentic
human reductases in AA activation, but these are necessary to
evaluate the susceptibility of humans to develop AAN or BEN.
The present study was therefore undertaken to determine the
capability of human organs to activate the major component of
aristolochiacea extract, AAI and to identify whether human
Activation of AA by NAD(P)H:quinone oxidoreductase
hepatic and renal cytosolic enzymes (and which of them) are
involved in adduct formation by AAI.
Materials and methods
Caution
AAs are mutagenic and carcinogenic and should be handled with care. Exposure to 32 P should be avoided, by working in a confined laboratory area, with
protective clothing, plexiglass shielding, Geiger counters and body dosimeters.
Wastes must be discarded according to appropriate safety procedures.
Chemicals
Chemicals were obtained from the following sources: NADH, NADPH, nuclease P1, deoxyadenosine 30 -monophosphate (dAp), deoxyguanosine 30 -monophosphate (dGp), buttermilk XO, hypoxanthine, cytochrome c, sodium
dodecyl sulfate (SDS), dicoumarol, allopurinol and 2-hydroxypyrimidine
from Sigma Chemical Co. (St Louis, MO), bicinchoninic acid from Pierce,
(Rockford, IL), Sephadex G-150 from Amersham-Pharmacia (Uppsala, Sweden),
menadione from Merck (Darmstadt, Germany), Affi-Gel Blue (Cibacron
Blue Agarose, Porcine Blue HB, C.T. 61211 Agarose) from Bio-Rad
(Richmond, CA), Sudan I from The British Drug Houses (UK), N1 -methylnicotinamide from Aldrich Chemical Co. (Milwaukee, WI) and calf thymus
DNA from Roche Diagnostics Mannheim (Germany). The natural mixture
AA consisting of 65% AAI and 34% AAII was a gift from Madaus (Cologne,
Germany). AAI and AAII were isolated from the mixture by preparative
HPLC; their purity was 99.7% as estimated by HPLC (32). All other
chemicals were of analytical purity or better. Enzymes and chemicals for
the 32 P-postlabeling assay were obtained commercially from sources
described previously (19,27).
Animal experiments
To induce NQO1, 10 male Wistar rats were injected i.p. with Sudan I in maize
oil (20 mg Sudan I/kg body wt) once a day for 3 consecutive days. Rats were
placed in cages in temperature- and humidity-controlled rooms. Standardized
diet and water were provided ad libitum. Animals were killed 24 h after the last
treatment by cervical dislocation (33). Liver and kidney of animals were
excised immediately after death, quickly frozen in liquid nitrogen and stored
at ÿ80 C until the isolation of cytosolic and microsomal fractions.
Preparation of cytosolic fractions
Liver and renal fractions (cytosol and microsomes) from 10 rats pre-treated
with Sudan I (see above) and renal fractions from one human kidney (specimen
from European transplantation Centre was a gift from Dr J.Nortier, Brussels,
Belgium) were prepared by differential centrifugation as described previously
(31,33). The age, drug and alcohol history of the donor of the kidney used in
the experiment is not known. The 105 000 g supernatant was taken as cytosol
and used for studies presented in the paper. All tissue fractions were stored at
ÿ80 C. Ten human hepatic cytosolic fractions were from Gentest (Woburn,
MA) (numbers of samples are shown in Table I) and stored at ÿ80 C. The
donors ranged in age from 2 to 71 years and included two men and eight
women. A drug and/or alcohol abuse history of the samples is described in
Gentest protocols. Each cytosolic preparation was analyzed for specific NQO1
and XO activities. The assays used were as follows.
NQO1, XO and aldehyde oxidase assays
NQO1 activity was measured essentially as described by Ernster (34). The
standard assay system contained 25 mM Tris±HCl (pH 7.4), 0.2% Tween 20,
0.07% bovine serum albumin, 400 mM NADH (or NADPH) and 100 mM
menadione (2-methyl-1,4-naphthoquinone) dissolved in methanol. The
enzyme activity was determined by following the oxidation of NADH
(NADPH) spectrophotometrically at 340 nm on a Hewlett-Packard 8453
diode array spectrophotometer. One unit of activity is defined as the amount
of enzyme catalyzing the oxidation of 1 mmol of NAD(P)H in 1 min. The
activities of XO in cytosolic fractions were measured as described by Ichikawa
et al. (35) using hypoxanthine as substrate. The aldehyde oxidase activity was
assayed as described by Felsted et al. (36) using N1 -methylnicotinamide as
substrate. Protein concentration was assessed using the bicinchoninic acid
protein assay with serum albumin as a standard (37).
Isolation of NQO1
NQO1 was isolated as described earlier (33). Hepatic and renal cytosolic
fractions from Sudan I-treated rats were used. Briefly, proteins in 20 ml cytosol
(24 and 17.4 mg/ml for hepatic and renal cytosolic fractions, respectively)
were fractionated with ammonium sulfate and the fraction between 30 and
90% saturation containing most of the NQO1 activity was dialyzed against
2000 ml of 150 mM KCl in 50 mM Tris (pH 7.4). The dialyzed enzyme
preparation was chromatographed on a Sephadex G-150 column and NQO1
was eluted with the same buffer. Pooled fractions containing the NQO1
activity were applied onto a column of Affi-Gel Blue and non-NQO1 proteins
were eluted with the same buffer and subsequently with a gradient of NaCl
(0±3.5 M) in this buffer. NQO1 was eluted from Affi-Gel Blue with 20 mM
Tris buffer (pH 10.0) containing 1 mM NADH. In order to remove residual
protein impurities, the NQO1 sample was applied onto a Sephadex G-150
column and re-chromatographed. The eluate was concentrated by ultrafiltration and stored at ÿ80 C. All steps of the isolation procedure were performed
at 4 C.
Table I. XO-, NQO1-dependent catalytic activities and DNA adducts formed by AAI in human hepatic and renal cytosolic samples
Human hepatic (H) and renal (R)
cytosolic samples
XOa
H803
H806
H823
H830
H856
H866
H870
H889
H893
H8112
40.0
20.0
22.8
21.9
20.9
30.4
26.2
38.0
39.0
43.8
Average valuef
R1
30.3 (8.70)
4.3
NQO1a
AAI±DNA adductsb
Without
cofactors
With
2-hydroxypyrimidinec
With
hypoxanthined
With
NADHe
With
NADPHe
70.0
1.0
12.0
4.0
4.0
19.0
5.0
3.0
3.0
10.0
2.7
7.2
1.0
3.2
5.2
5.4
1.4
5.2
5.4
1.4
1.2
4.4
2.9
3.3
5.0
0.3
0.7
0.8
5.9
4.7
3.5
10.8
1.7
3.8
9.7
2.1
5.5
3.5
7.7
3.8
13.2
12.0
4.0
12.2
10.1
10.1
11.9
14.3
8.9
10.5
23.0
14.6
16.2
15.0
14.8
18.2
15.3
15.2
15.4
16.0
13.1 (19.6)
0.2
3.8 (2.0)
0.8 0.04
0.10
0.35
0.005
0.15
0.25
0.25
0.05
0.25
0.25
0.05
0.05
0.20
0.10
0.15
0.25
0.02
0.03
0.04
0.30
0.25
2.9 (1.9)
n.m.
0.15
0.55
0.10
0.10
0.45
0.10
0.25
0.15
0.35
0.20
5.2 (3.0)
0.4 0.04
0.55
0.60
0.20
0.80
0.50
0.50
0.60
0.70
0.45
0.50
10.7 (2.7)
1.4 0.07
1.00
0.55
0.65
0.75
0.65
0.85
0.75
0.70
0.65
0.80
15.9 (2.5)
3.2 0.4
Data for XO and NQO1 activities are presented as means of duplicate experiments; assays for their activities are described in Materials and methods. n.m., not
measured.
a
XO and NQO1 activities in nmol/min/mg protein.
b
Relative adduct labeling (RAL), adducts/108 nucleotides/mg cytosolic protein. The values are arithmetic means SEM (n ˆ 4) of duplicate incubations, each
DNA sample was determined by two postlabeled analyses.
Cofactor of: c aldehyde oxidase, d XO and e NQO1.
f
Arithmetic means for 10 hepatic cytosolic samples (H803±H8112), values in parentheses are standard deviations, representing the inter-individual variability.
1697
M.Stiborova et al.
Incubations
The de-aerated and argon-purged incubation mixtures contained in a final
volume of 0.75 ml: 50 mM Tris±HCl buffer (pH 7.4) containing 0.2%
Tween 20, 1 mM cofactors for cytosolic enzymes (NADH or NADPH or
hypoxanthine or 2-hydroxypyrimidine), 1 mg cytosolic protein, 0.5 mM AAI
as sodium salt dissolved in water and 1 mg calf thymus DNA (4 mM dNp). The
reaction was initiated by adding cofactor. Control incubations were carried out
either without activating system (cytosol) or with activating system and AAI,
but without DNA or with activating system and DNA but without AAI.
Incubations with purified NQO1 enzymes contained in a final volume of
0.75 ml: 50 mM Tris±HCl buffer (pH 7.4) containing 0.2% Tween 20, 1 mM
NADH or NADPH, 0.5 mM AAI as sodium salts dissolved in water, 1 mg calf
thymus DNA (4 mM) and 0.05±1.0 U of hepatic or renal NQO1 instead of
cytosolic fractions. Incubations with buttermilk XO contained in a final
volume of 0.75 ml: 50 mM sodium phosphate (pH 7.4) containing 1 mM
hypoxanthine, 0.5 mM AAI as sodium salts dissolved in water, 1 mg calf
thymus DNA (4 mM) and 0.05±1.0 U XO instead of cytosolic fractions. All
reaction mixtures were incubated at 37 C, 10±60 min, then extracted twice
with ethyl acetate (2 2 ml). DNA was isolated from the water phase by the
phenol±chloroform extraction as described earlier (19,27±31). The content of
DNA was determined spectrophotometrically (38,39).
Inhibition studies
The following chemicals were used to inhibit the activation of AAI in human
cytosolic fractions: dicoumarol and allopurinol to inhibit NQO1 and XO,
respectively (40±42). Inhibitors dissolved in 7.5 ml of methanol, to yield final
concentrations of 10 mM, were added to the incubation mixtures. An equal
volume of methanol alone was added to the control incubations and proceeded
as described above.
32
P-Postlabeling analysis
The nuclease P1 enrichment version (43) of the assay, known to be the most
suitable version of the 32 P-postlabeling technique for AA±DNA adduct detection and quantification was used and performed exactly as described (6,16,
19±22,27±31,44). Enzymatic synthesis of reference compounds, dAp-AAI,
dGp-AAI and dAp-AAII and their 32 P-postlabeling were carried out as
described earlier (18).
Co-chromatography on polyethylenimine-cellulose
Adduct spots detected by the 32 P-postlabeling assay were excised from the
thin-layer plates, extracted and co-chromatographed with reference 30 ,50
bisphosphate adducts as reported previously (18).
HPLC analysis of 32 P-labeled 30 ,50 -deoxyribonucleoside bisphosphate
adducts
HPLC analysis was performed essentially as described previously (18,27,45).
Individual spots detected by the 32 P-post-labeling assay were excised from thin
layer plates and extracted (46). The dried extracts were redissolved in 100 ml of
methanol±phosphate buffer (pH 3.5) 1:1 (v/v). Aliquots (50 ml) were analysed
on a phenyl-modified reversed-phase column (250 4.6 mm, 5 mm Zorbax
Phenyl; Saulentechnik Dr Knauer, Berlin, Germany) with a linear gradient of
methanol (from 40 to 80% in 45 min) in aqueous 0.5 M sodium phosphate and
0.5 M phosphoric acid (pH 3.5) at a flow rate of 0.9 ml/min. Radioactivity
eluting from the column was measured by monitoring Cerenkov radiation with
a Berthold LB 506 C-1 flow through radioactivity monitor (500 ml cell, dwell
time 6 s).
Molecular modeling
Crystalographic coordinates for human NQO1 with bound FAD were obtained
from the Protein Data Bank (47). For modeling purposes, the physiological
dimer form was used (MacroMolecular file 1qrd_1.mmol). The coordinates
were used without further refinement. The modeling of the binding of AAI to
the active site was performed with the program Autodock 3.0.3. (48) and Sybyl
6.6.5 (Tripos GmbH, Germany) by the procedure as described (31,49).
Statistical analyses
Correlation coefficients between the catalytic activities of NQO1 and XO in
human hepatic cytosolic samples and the levels of individual AAI±DNA
adducts formed by the same cytosolic samples were determined by linear
regression using Statistical Analysis System software version 6.12. Correlation
coefficients were based on a sample size of 10. P is two-tailed and considered
significant at the 0.05 level.
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Results
DNA adduct formation by AAI in human hepatic and renal
cytosol
We determined the formation of DNA adducts by AAI in calf
thymus DNA in the presence of cytosolic samples from
10 different human livers and from one human kidney. Both
human hepatic and renal cytosolic fractions were capable of
activating AAI to form DNA adducts. AAI activated by cytosol from liver or kidney generated the same major DNA
adducts as those obtained in vivo in rats and humans reported
previously (6,10,11,14±22). A cluster of three major adducts
(spots 1, 2 and 3) was formed by AAI (Figure 2). These
adduct spots formed by AAI showed the same migration on
polyethylenimine-cellulose TLC plates and on reversed-phase
HPLC as those of synthetic standards (18) (see Supplementary
material, Figure 1). Thus, spot 1 was assigned to 30 ,50 -bisphospho-7-(deoxyguanosin-N2 -yl)-aristolactam I (dG±AAI), spot 2
to 30 ,50 -bisphospho-7-(deoxyadenosin-N6 -yl)-aristolactam I
(dA±AAI) and spot 3 to 30 ,50 -bisphospho-7-(deoxyadenosin-N6 -yl)-aristolactam II (dA±AAII). A minor dA±AAII
adduct was formed from AAI also in previous in vitro studies
utilizing other activating enzymatic systems (27±31) or in
forestomach DNA of rats treated with AAI (18). An explanation may be the initial formation of dA±AAI in doublestranded DNA, with subsequent demethoxylation to
dA±AAII (18). Control incubations carried out in parallel
either without cytosol, without DNA or without AAI were
free of adduct spots in the region of interest even after
prolonged exposure times of the autoradiograms.
The adducts formed by AAI activated with human cytosolic
samples are known to be generated from AAI by nitro reduction (6,18,50,51). Therefore, similar to rat hepatic and renal
cytosols (31), the human cytosolic fractions tested in this study
contain enzymatic systems capable of catalyzing the reductive
activation of AAI. XO, NQO1 and aldehyde oxidase are major
candidates for the reductive activation of AAI in cytosol. To
investigate these possibilities, the influences on AAI±DNA
adduct formation by various structurally diverse compounds,
which are electron donors for these enzymes, were examined.
As shown in Table I the formation of AAI±DNA adducts was
stimulated by the cofactors of NQO1, NADPH and NADH
(52). Adduct levels were significantly higher, 4.2- and 2.8-fold
on average, when NADPH or NADH were added into the
Fig. 2. Autoradiographic profiles of AAI±DNA adducts obtained from calf
thymus DNA after activation by human hepatic cytosol in the presence of
NADPH (sample H803) (A), purified rat renal NQO1 in the presence of
NADPH (B) and by buttermilk XO in the presence of hypoxanthin (C). The
nuclease P1-enrichment procedure was used for analysis. Origins, in the
bottom left-hand corner were cut off before exposure. Screen enhanced
autoradiography was at ÿ80 C for 1 h. Chromatographic conditions: D1, 1 M
sodium phosphate, pH 6.8; D3, 3.5 M lithium formate, 8.5 M urea, pH 4.0; D4,
0.8 M LiCl, 0.5 M Tris±HCl, 8.5 M urea, pH 9.1; D5, 1.7 M NaH2 PO4 ,
pH 6.0. Spot 1, dG±AAI; spot 2, dA±AAI; spot 3, dA±AAII.
Activation of AA by NAD(P)H:quinone oxidoreductase
incubation mixture. Hypoxanthine, a cofactor of XO (53), also
stimulated AAI±DNA adduct formation, but only to a much
lesser extent (Table I). 2-Hydroxypyrimidine, an electron
donor of the cytosolic aldehyde oxidase (54), had practically
no effect. These results suggest a minor, but detectable, role of
XO in AAI activation, NQO1 seems more important for this
activation. If the organs are compared, cytosolic samples from
liver exhibited much higher efficiencies to activate AAI than
the single kidney sample (Table I). The lower efficacy to
activate AAI corresponds to lower activities of reductases in
cytosol of this tissue (Table I).
To further resolve which cytosolic enzymes are responsible
for activation of AAI, different experimental approaches were
employed: (i) analysis of the correlation of XO and NQO1
activities with AAI±DNA adduct formation by human cytosols, (ii) selective enzyme inhibition, (iii) utilization of purified XO and NQO1 and (iv) computer modeling of the
NQO1±AAI complex.
Correlation of NQO1- and XO-linked enzyme activities in
human hepatic cytosolic samples with DNA adduct formation
by AAI
Catalytic activities of NQO1 and XO with menadione (55) and
hypoxanthine (35) as substrates, respectively, were analyzed in
human hepatic cytosolic preparations from 10 different
donors. Large inter-individual variations in both catalytic
activities were found (Table I). The activity of another reductase, aldehyde oxidase, measured with N1 -methylnicotinamide
as a substrate (36), was not detectable in any human cytosolic
samples.
Quantitative 32 P-postlabeling analyses, as shown in Table I,
also showed wide individual variations in DNA binding of
AAI in the human cytosolic incubations. Correlations between
the NQO1 or XO activities and AAI±DNA adduct formation
were analyzed to examine the role of human cytosolic reductases in AAI activation. Highly significant correlations were
found between the NQO1 activities measured with NADPH as
cofactor and the levels of total AAI±DNA adducts determined
in the presence of the same cofactor (NADPH) (0.985, P 5
0.001). Likewise, highly significant correlations were found
between the activities of this enzyme and the levels of individual AAI±DNA adducts (0.941, P 5 0.001 for dG±AAI,
0.889, P 5 0.001 for dA±AAI and 0.712, P 5 0.05 for dA±
AAII). The correlations were calculated with data taken from
Supplementary material Table Ie. Catalytic activities of XO
did not exhibit significant correlations with the levels of AAI±
DNA adducts determined in the presence of hypoxanthine as
cofactor (ÿ0.349, P ˆ 0.322). These results suggest that
human cytosolic NQO1 is the predominant enzyme participating in AAI activation in human hepatic cytosol.
Effect of inhibitors of NQO1 and XO on activation of AAI by
human cytosolic samples
Inhibition experiments further supported the role of NQO1 in
the activation of AAI in human cytosolic samples. Two hepatic
cytosolic samples with high NQO1 or XO activities were
selected, and incubations were carried out in the absence and
presence of dicoumarol and allopurinol, known inhibitors of
NQO1 and XO, respectively. The hepatic cytosolic AAI activation with dicoumarol resulted in an 85% decrease in the
levels of AAI±DNA adducts, while no effect of allopurinol
was observed (Table II). Likewise, dicoumarol effectively
inhibited AAI±DNA adduct formation by renal cytosol, by
75% (Table II). These results emphasize the major role of
NQO1 in AAI activation by cytosolic samples of both human
tissues.
Activation of AAI by purified XO and NQO1 enzymes
Even though XO has only a minor contribution to the AAI±
DNA adduct formation in human hepatic cytosols, we tested in
more detail its participation in AAI activation in vitro and
compared its efficacy with that of NQO1. For such studies
we utilized buttermilk XO and NQO1 enzymes isolated from
rat liver and kidney.
Incubations of AAI with DNA in the presence of either
purified NQO1 and its cofactor, NADPH, or XO and its cofactor, hypoxanthine, under the standardized conditions (0.5 mM
AAI, 1 mM cofactors, 0.05±1.0 U of the enzymes, pH 7.4,
37 C, 60 min) resulted in the formation of AAI±DNA adducts
(Table III). The same DNA adducts (dG±AAI, dA±AAI and
dA±AAII, Figure 1) as those determined in either cytosol
(present paper) or in vivo (6,10,11,14±22) were generated by
both enzymes. The identity of adducts was confirmed by
co-chromatography on TLC and HPLC. The NQO1- and
XO-catalyzed binding of AAI was shown to be dependent on
the time of incubation, being linear up to 60 min (not shown).
The levels of individual AAI-adducts formed by XO differ
from those produced by cytosol (present paper) or in vivo
(6,10,11,14±22). In contrast to the adduct formation catalyzed
by NQO1, which resembles that in vivo, where the levels of
Table II. The effect of NQO1 and XO inhibitors on the AAI±DNA adduct formation in human cytosol
Human cytosolic sample
H803 ‡ NADPH
H803 ‡ NADPH ‡ dicoumarol
H893 ‡ hypoxanthine
H893 ‡ hypoxanthine ‡ allopurinol
R1 ‡ NADPH
R1 ‡ NADPH ‡ dicoumarol
R1 ‡ hypoxanthine
R1 ‡ hypoxanthine ‡ allopurinol
RALa (mean SEM/108 nucleotides)
dG±AAI
dA±AAI
dA±AAII
Total
10.2
1.5
3.4
3.4
1.3
0.26
0.15
0.15
13.7
2.1
4.0
4.7
1.5
0.41
0.21
0.21
1.5
0.1
0.3
0.3
0.4
0.14
0.04
0.05
23.0
3.7
7.7
8.4
3.2
0.81
0.40
0.41
0.50
0.05
0.30
0.25
0.05
0.04
0.005
0.005
0.60
0.10
0.35
0.35
0.05
0.03
0.035
0.030
0.05
0.005
0.02
0.05
0.05
0.005
0.005
0.005
1.10
0.20
0.40
0.45
0.20
0.70
0.035
0.040
Numbers are averages SEM (n ˆ 4) of duplicate in vitro incubations, each DNA sample was determined by two post-labeled analyses. Experimental
conditions used for incubations with inhibitors are described in Materials and methods; concentrations of dicoumarol and allopurinol were 10 mM.
a
Relative adduct labeling.
1699
M.Stiborova et al.
Table III. AAI±DNA adduct formation by buttermilk XO and rat hepatic
and renal NQO1
Enzyme
XOb
Hepatic NQO1
Renal NQO1
RALa (mean SEM/108 nucleotides)
dG±AAI
dA±AAI
dA±AAII
Total
25.3 1.30
10.2 0.50
11.7 0.55
18.3 0.9
31.2 1.5
24.8 1.25
6.4 0.3
4.4 0.2
2.8 0.15
50.0 5.1
45.8 4.6
39.3 1.9
Numbers are averages SEM (n ˆ 4) of duplicate in vitro incubations,
each DNA sample was determined by two post-labeled analyses.
Relative adduct labeling.
b
The DNA binding was assayed as described in Materials and methods
with 0.5 mM AAI, 1 mM hypoxanthine with 0.4 U of XO or 1 mM NADPH
with 0.4 U of rat hepatic or renal NQO1.
a
dA±AAI adduct are always higher than those of dG±AAI, the
dG±AAI adduct is prevalent in the XO-dependent system.
Moreover, different dependencies of AAI±DNA adduct levels
on the amount of NQO1 and XO were observed (Figure 3).
Amounts of XO 40.4 U (400 nmol/min/mg) per incubation
much more effectively activated AAI than the same amount of
NQO1 while at lower more physiological enzyme concentrations NQO1 is more effective (Figure 3). These results might
explain a lower contribution of XO to activate AAI in human
cytosolic samples and confirm the major role of NQO1 in this
human subcellular fraction; XO activities in all cytosolic samples were less than the critical XO activity needed for the
pronounced AAI activation (compare Table I).
Fig. 3. Dependence of DNA adduct formation from 0.5 mM AAI on the
concentration of hepatic and renal NQO1 with NADPH as cofactors, and
buttermilk XO in the presence of hypoxanthine. Experimental details
are described in Materials and methods. aRelative adduct labeling.
Molecular modeling
In order to examine the molecular basis of the reductive
activation of AAI by human NQO1, computer modeling was
used in the present study. Molecular modeling clearly explains
the efficiency of human NQO1 in activating AAI. The calculated model structure for the human NQO1±AAI complex is
shown in Figure 4. It is evident from this figure that AAI fits
well into the active site of human NQO1, being bound near the
isoalloxazine ring of the flavin prosthetic group of the enzyme.
This allows for an electron transfer during the reductive
activation of AAI. The value of the apparent dissociation
constant for the human NQO1±AAI complex is calculated to
be 1.48 mM.
Discussion
Recently, specific AA±DNA adducts were found to be associated with a unique nephropathy, AAN, and urothelial cancer
in a cohort of women who had followed a weight reducing
treatment consisting of Chinese herbs containing AA in
Belgium. This disease was observed also in other European
and Asian countries and in the USA (5,6). Specific AA±DNA
adducts were also detected in renal specimens of two patients
suffering from malignant transformation of urothelium and
living in BEN areas (14).
Since the detection and quantification of specific DNA
adducts by the 32 P-postlabeling procedure has proven a useful
tool to monitor exposure to the plant carcinogen AA in vivo
(6,10,11,14±22) and in vitro (27±31), they were utilized in the
present study to identify human enzymes involved in AA
activation.
In mammalian tissues, both cytosol and microsomes contain
enzymes catalyzing the reduction of nitro aromatic compounds
1700
Fig. 4. AAI is shown docked to the active site of human NQO1 where several
key amino acid residues position the AAI substrate parallel to a flavin
prosthetic group.
(40±42,56,57). We have already identified rat hepatic cytosolic NQO1 to be capable of AA activation, generating AA±
DNA adduct profiles identical to those found in renal tissue in
humans (31). The present paper reports on the identification of
human cytosolic enzymes, which participate in the reductive
bioactivation of the major component of AA, AAI.
Here, we clearly demonstrate that human cytosolic samples
from livers of 10 different human donors and kidney of one
donor are capable of activating AAI leading to the same DNA
Activation of AA by NAD(P)H:quinone oxidoreductase
adduct pattern as that formed in humans exposed to AA
(6,10,11,19±22).
Formation of AAI±DNA adducts was strongly correlated
with NQO1 activities in different human hepatic cytosolic
samples. Inhibition of DNA adduct formation by dicoumarol,
the inhibitor of NQO1, provided additional evidence for the
role of this enzyme in AAI activation in humans. Molecular
modeling where the AAI molecule was docked to the active
site of human NQO1 suggests that AAI binds where the other
NQO1 substrates (i.e. duroquinone) (58) are found in the X-ray
structure, with the planar aromatic AAI rings parallel to the
flavin ring. This allows for an efficient electron transfer during
the reductive activation. Recently, we showed that rat cytosolic NQO1 was also efficient to activate AAI (31). This indicates similarities of NQO1 enzymes from both species.
Nevertheless, some differences were observable in catalytic
activities of human and rat NQO1s. NQO1 in human hepatic
cytosolic samples more efficiently activates AAI in the presence of NADPH than NADH (see Table I), while no difference was seen with rat hepatic NQO1 (31). This also
corresponds to a ~2-fold lower activity of human NQO1 to
reduce menadione in the presence of NADH than NADPH
(data not shown). But, even though the NQO1 activities with
menadione as substrate in human hepatic cytosols are lower
than those in hepatic cytosol of rats, similar efficacies of
subcellular systems of both species to activate AAI were
observed (31 and the present paper). The higher binding affinity of AAI for human NQO1 than for the rat enzyme can
explain the observed differences. AAI fits better into the active
centre of human NQO1 than into that of the rat enzyme
(compare Figure 4 in the present paper and figure 8 in 31).
Likewise, the value of the calculated dissociation constant for
the human NQO1±AAI complex (1.48 mM) is lower than for
the rat enzyme (16.4 mM) (31).
In comparison with NQO1, XO had only a minor impact on
the capacity to activate AAI to form DNA adducts in human
samples assayed. In contrast to this finding we observed that
the isolated buttermilk XO was an effective activator of this
compound (27 and the present paper), but the enzyme levels
needed are not physiological. Another reason for the observed
discrepancies might be the different substrate specificities of
human cytosolic and buttermilk XO.
The involvement of human NQO1 in the reductive activation
of nitroaromatics like AAI is consistent with previous reports
demonstrating that the enzyme functions efficiently as a nitroreductase of substrates like dinitropyrenes, nitrophenylaziridines and nitrobenzamides (59 and references cited herein).
NQO1 activity is ubiquitously present in all tissue types (59±
62). Levels of expression and activities of NQO1 in humans
differ considerably among individuals, because the enzyme is
influenced by several factors like smoking, drugs, environmental chemicals and genetic polymorphisms (59,63,64).
Biochemical studies have already demonstrated that NQO1
activity is induced by a wide range of chemicals (59,65±67).
Two distinct regulatory elements in the 50 flanking region of
the NQO1 gene, the antioxidant response element (ARE) and
the xenobiotic response element (XRE), involving the
liganded aromatic hydrocarbon (Ah) receptor, have been
shown to regulate NQO1 expression in many cellular systems.
Moreover, the anti-estrogens tamoxifen and hydroxytamoxifen
stimulate the expression of NQO1 by activating the estrogen
receptor, which is different from the Ah locus (68,69). AREmediated NQO1 gene expression is increased by a variety of
phenolic antioxidants, tumor promoters and H2 O2 (59,65,67).
The XRE of NQO1 shares significant homology with the XRE
of CYP1A1 (70), the gene of the enzyme, which also activates
AAs (6,29). Both NQO1 and CYP1A1 genes can be induced by
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons, Sudan I and b-naphthoflavone (59).
Because NQO1 activity is increased in rats treated with AA
(33), it might also be induced in the Belgian AAN patients.
So far two polymorphisms in the human NQO1 gene have
been found in the general population. One of them the 609 C to
T variant, designated NQO1 2, has profound phenotypic consequences and has been associated with an increased risk of
urothelial tumors (71), therapy-related acute myeloid leukemia
(72), cutaneous basal cell carcinomas (73) and pediatric leukemia (74). The frequency of homozygous NQO1 2 mutation
varies across ethnic groups and was reported to be ~5% in
Caucasians (59).
Collectively, the data suggest that variations of this enzyme
and regulatory proteins controlling its expression (Ah receptor,
or its associated transcription factor, the Ah receptor nuclear
translocator or Arnt protein) (26), might play a role in the risk
of cancer by AAs. To confirm this hypothesis, the study
screening Belgian AAN patients and other participants in the
slimming regimen for polymorphisms of the genes encoding
NQO1, cytochrome P450 1A1 and the Ah receptor is underway in our laboratories.
Supplementary material
Supplementary material can be found at http://www.carcin.
oupjournals.org
Acknowledgments
Supported by Grant Agency of the Czech Republic (grant 203/04/0923) and the
Ministry of Education of the Czech Republic (grant MSM 1131 00001). We are
grateful to A.Breuer and K.Klokow (German Cancer Research Center,
Heidelberg) for excellent technical assistance.
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Received May 2, 2003; revised June 25, 2003; accepted June 30, 2003
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