Screening for reactive metabolites using electro-optical arrays featuring human liver cytosol and microsomal enzyme sources and DNA

Article abstract of DOI:10.1039/b909372a

We demonstrate for the first time the combination of human liver cytosol and microsomal enzyme sources into an electro-optical array to screen for reactive metabolites produced in multi-enzyme metabolic processes. The Royal Society of Chemistry 2009.

Full text of DOI:10.1039/b909372a

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Screening for reactive metabolites using electro-optical arrays featuring
human liver cytosol and microsomal enzyme sources and DNAw
a
b
ab
Linlin Zhao, John B. Schenkman and James F. Rusling*
Received (in Cambridge, UK) 12th May 2009, Accepted 4th August 2009
First published as an Advance Article on the web 17th August 2009
DOI: 10.1039/b909372a
We demonstrate for the first time the combination of human liver
cytosol and microsomal enzyme sources into an electro-optical
array to screen for reactive metabolites produced in
multi-enzyme metabolic processes.
converted into potentially reactive metabolites that are
trapped by DNA in the spots as nucleobase adducts. DNA
damage in the array is detected by an electro-optical technique
called electrochemiluminescence (ECL), using a ruthenium
metallopolymer in the spot that provides more ECL
light when the DNA is disordered by nucleobase adduct
The generation of reactive metabolites represents a potential
1
stumbling block in drug discovery and development. Reactive
8,9
formation. After the enzyme/DNA reaction step, ECL from
metabolites generated by metabolic bioactivation of drugs or
environmental chemicals in the liver have the potential to
the array is measured in an open-top electrochemical cell in a
8
a
dark box and detected by a CCD camera. Using microsomes
as a convenient source of cyt P450s avoids tedious purification
of multiple enzymes while providing valuable information on
2
damage proteins and DNA, which may result in toxicity.
Thus, it is important to identify reactive metabolites of drug
and environmental chemical candidates in order to predict
toxicities in the early stages of development. Oxidations
catalyzed by liver cytochrome P450s (cyt P450s) are a major
source of reactive metabolites of lipophilic molecules.
However, bioconjugation processes account for B25% of
marketed drug metabolism and can also contribute to bio-
8
c
toxic metabolic pathways via cyt P450 oxidations, as well as
1
0
glucuronyltransferase-mediated detoxification. In addition,
we demonstrated a voltammetric sensor capable of screening
compounds that form reactive metabolites using cyt P450 1A2
1
1
and NAT. Ultimately, toxicity screening arrays must be
capable of screening reactive metabolites generated from a
complete spectrum of metabolic reactions including oxidation
and conjugation reactions.
2
activation alone or sequentially with cyt P450s.
Different in vitro human liver enzyme sources, such as
microsomes, supersomes, cytosol, S9 fraction, various cell
lines and liver slices, have been employed as tools to investigate
drug metabolism and inhibition, and to predict in vivo
Herein we combine for the first time human liver cytosol
and microsomes as enzyme sources in electro-optical arrays to
screen reactive metabolites produced via bioconjugation with
or without oxidation. Array spots containing ECL ruthenium
3
,4
biotransformations. The liver cytosol contains three major
2
+
enzymes: N-acetyl transferase (NAT), glutathione S-transferase
polymer ([Ru(bpy)
2
PVP10
]
; PVP = polyvinylpyridine,
(
GST), and sulfotransferase (SULT). These enzymes are
RuPVP), DNA, human liver cytosol and microsomes were
2
assembled on 1 in pyrolytic graphite (PG) chip using
recognized for their roles in detoxification and clearance of
foreign compounds. However, they can also lead to bio-
activation and subsequent damage to cellular constituents
8
a,c
established protocols.
Amounts of cytosol, microsomes,
DNA and RuPVP in the spots were estimated by a quartz
crystal microbalance (QCM) (ESIw, Fig. S1, Table S1), which
also confirmed reproducible film formation.
2
,5
causing toxicity. In concert with awareness of this fact,
the liver cytosol has attracted increasing attention in drug
4
biotransformation research. Examples include the conversion
Ethylene dibromide was selected as an initial test
compound because of its known toxic pathways mediated
6
by glutathione S-transferase (Scheme 1). To prove that
of the pesticide ethylene dibromide (EDB) to a half-mustard/
episulfonium ion (Scheme 1A, 3) by glutathione transferases,
and the activation of 2-aminofluorene (2-AF) by acetylation
with or without hydroxylation to generate good leaving
metabolic activation of ethylene dibromide is achieved
by cytosolic GST in the array, spots with architectures
6
,7
groups and form reactive nitrenium ions (Scheme 1B, 9).
The reactive metabolites formed in both cases react with DNA
3 3
(DNA/RuPVP) /(DNA/RuPVP/cytosol) were exposed to
80 mM EDB and 0.5 mM glutathione (GSH) in 50 mM
MES buffer (pH 6.0). Fig. 1A shows spots that increase in
6
,7
and can cause genotoxicity.
We recently developed predictive in vitro arrays that screen
reactive metabolite formation using a DNA damage end
8
point. This technology employs thin film spots containing
DNA and enzymes, either pure or as microsomes containing
multiple or individual cyt P450s. Test compounds are first
a
Department of Chemistry, University of Connecticut, 55 N. Eagleville Rd,
Storrs, CT, USA. E-mail: james.rusling@uconn.edu
Department of Cell Biology, University of Connecticut, Farmington,
CT, USA
b
Scheme 1 Major biotransformations of EDB and 2-AF leading to
w Electronic supplementary information (ESI) available: Experimental
details, Fig. S1, S2, Table S1. See DOI: 10.1039/b909372a
6,7
DNA adducts formation.
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386 | Chem. Commun., 2009, 5386–5388
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Fig. 1 Reconstructed ECL array data for (A) spots of RuPVP/DNA/
human liver cytosol exposed to 80 mM EDB, 0.5 mM glutathione
in 50 mM MES buffer (pH 6.0) for denoted times; (B) spots of
RuPVP/DNA/human liver cytosol/human liver microsomes exposed
to 100 mM 2-AF, 0.5 mM acetyl coenzyme A, 1.6 mM DTT, 0.5 mM
EDTA and NADPH regenerating system in 50 mM MES buffer
Fig. 2 Relative ECL increases for arrays utilizing data in Fig. 1 for
(A) EDB reaction (J) and control with EDB only (n); (B) 2-AF
reaction mediated by cytosolic NAT and microsomal enzymes (K),
2-AF reaction mediated by cytosolic NAT (’), and control reaction
with only 2-AF (m). Error bars represent ÆSD from 8–12 spots.
(pH 6.0); (C) spots of RuPVP/DNA/human liver cytosol exposed to
the same enzymatic reaction solution as in (B) without the NADPH
regenerating system. Control spots in each panel are exposed to the
same concentration of substrates alone.
controls with 2-AF only (Fig. 1B, control). It is likely that
microsomal cyt P450s mediating oxidation and cytosolic NAT
mediating conjugation act synergistically in converting
2-AF into reactive nitrenium ion(s) (Scheme 1, 9) that bind
covalently to DNA. This was further supported by inhibiting
ECL intensity when exposed to EDB for various times.
II
Ru PVP polymer catalyst in the films is oxidized electro-
III
chemically at B1.1 V in the detection step to Ru PVP, which
subsequently oxidizes guanines on DNA to yield electronically
1
3
NAT activation using luteolin, which eliminated the ECL
increase (ESIw, Fig. S2). This result supports the critical role of
NAT mediated conjugations in causing significant DNA
damage by 2-AF.
II
9
excited Ru *PVP that emits visible ECL light at 610 nm.
Reactive metabolites of EDB formed within the film
Scheme 1A) are trapped as DNA base adducts that partially
(
disrupt the double helix and enhance the access of the catalytic
III
Ru -centers to guanines leading to increased oxidation rates
To investigate the role of NAT in activating 2-AF in the
absence of cyt P450s, microsomes were omitted from the array
spots. A smaller ECL increase was found, as illustrated in
Fig. 1C. This suggests that much smaller amounts of DNA
adducts were formed when only the cytosolic NAT mediated
conjugation pathway was active, probably via 2-acetyl-
aminofluorene (6) followed by reactive nitrenium ion (9)
and increased ECL light emission for damaged DNA, as
8
documented extensively for other DNA damage reactions.
,9
No increase in light was observed on the array in the absence
of the cofactor glutathione (GSH) in the enzyme reaction step
(
Fig. 1A, control). When array spots were preincubated with
1
2
14
bilirubin, an inhibitor of GST, no increase in light was
observed (ESIw, Fig. S2), indicating the necessity for metabolic
activation of EDB for DNA damage.
formation in the weakly acidic buffer.
Fig. 2B shows a 6-fold larger slope of ECL increase versus
reaction time when both cyt P450s and cytosolic NAT
enzymes are present compared to spots with cytosolic NAT
alone. This result is consistent with our previous result using
pure NAT in a non-array voltammetric sensor (B20% relative
We previously showed that the slope of the relative ECL
increase vs. reaction time correlates with the rate of nucleobase
adduct formation measured directly by LC-MS/MS, as well as
8
,10
14
with rodent liver toxicity metrics.
The relative ECL increase
current increase in 2 min), under slightly different reaction
for the GST catalyzed EDB reaction was nearly 30% in 120 s
compared to a negligible change in controls (Fig. 2A). The
conditions. Overall, results are consistent with the synergistic
roles of oxidations via cyt P450s and conjugations via NAT in
bioactivation of 2-AF (Scheme 1B).
4
normalized turnover rate was 9 Â 10 (per min per mg cytosol)
for GST catalyzed EDB reactions, obtained from the
slope of ECL increase versus reaction time divided by the
QCM-estimated amount of cytosol in the films.
To confirm formation of DNA adducts in the spots and
elucidate their structures, enzyme reactions were performed
using 500 nm diameter silica nanoparticles coated with
1
5
Next, 2-aminofluorene (2-AF) was tested as an example
whose bioactivations involve both cyt P450 mediated oxidations
7
and NAT conjugations (Scheme 1B). Arrays with spots
analogous enzyme/DNA films as in the arrays. After the
enzyme reactions, enzymatic hydrolysis of DNA on the
particles release adducted deoxynucleosides for LC-MS/MS
analysis. For the EDB reaction, silica nanoparticles with
PDDA/DNA/cytosol films were used to catalyze enzyme
containing cytosol, microsomes, and DNA (ESIw, Table S1)
were used to provide enzymes for the oxidation and conjugation
reactions. First, the enzyme reactions were done using 100 mM
7
reactions. S[2-(N -guanyl)ethyl]glutathione with a m/z 601
2
-AF together with 0.5 mM acetyl coenzyme A (AcCoA),
was identified as a major adduct with similar fragmentation
1
6
1
.6 mM DTT, 0.5 mM EDTA and an NADPH regenerating
pattern as previously proposed.
Fig. 3A shows mass
+
system. An increase in ECL versus reaction time was observed,
transition m/z 601 - 213 ([guanine + thioethyl + 2H] )
using tandem MS in multiple reaction monitoring (MRM)
as illustrated in Fig. 1B. Negligible ECL increase was found in
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routes can be activated selectively or collectively by providing
different co-factors of enzymes as desired. Structures and
formation rates of specific metabolite adducts with DNA bases
can be determined using similar films on nanoparticles with
LC-MS/MS product analysis. Future efforts will focus on
testing a wider range of chemicals with known and unknown
metabolism and toxicity using these platforms, to provide
more extensive validation.
The authors gratefully acknowledge financial support from
the National Institute of Environmental Health Sciences
(
NIEHS), NIH (grant No. ES03154).
Fig. 3 LC-MS chromatograms in multiple reaction monitoring
MRM) mode for detection of deoxyguanosine adducts with
(
fragmentation chemistry shown in insets. (A) Formation of
7
S[2-(N -guanyl)ethyl]glutathione with mass transition m/z 601 - 213,
Notes and references
after 5 min reaction using nanoparticles with DNA/human liver
cytosol films and 80 mM EDB, 0.5 mM glutathione in 50 mM MES
buffer (pH 6.0); (B) Formation of N-(deoxyguanosin-8-yl)-2-amino-
fluorene with mass transition m/z 447 - 330 after 5 min reactions
using nanoparticles with DNA/human liver cytosol films (blue) and
DNA/human liver cytosol/microsome films (red) with 100 mM 2-AF,
1
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2
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2
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8
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3
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(
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activated, consistent with the ECL array observations.
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388 | Chem. Commun., 2009, 5386–5388
This journal is ꢀc The Royal Society of Chemistry 2009