Chemical Research in Toxicology
Article
Kong University of Science and Technology (HKUST) Committee
on Research Practice. Male Sprague−Dawley rats (170−200 g)
obtained from the HKUST Animal and Plant Care Facility were used
for the study. After acclimatizing for a week, rats were dosed with a
single oral dose of 10 mg/kg (n = 3) or 30 mg/kg (n = 3) of AAI
dissolved in 1% sodium bicarbonate (w/v) to investigate the
concentration-dependent formation of ALI−protein adducts. Rats
(n = 3) that received the same volume of the dosing vehicle were used
as the control. Twenty-four hours after the AAI administration, rats
were sacrificed by decapitation, and the kidney, liver, and whole blood
were collected for analysis. In another study to investigate the
formation and elimination kinetics of the ALI−protein adducts, rats
(n = 24) that received a single oral dose of 30 mg/kg of AAI were
sacrificed on days 1, 2, 3, 8, 14, 21, 28, and 60 postdosing for analysis.
Protein Sample Preparation. Kidney and liver proteins of AAI-
exposed rats were isolated by ammonium sulfate precipitation after
slices of kidney and liver tissues were homogenized using a high-speed
tissue-tearor homogenizer, as described previously.38,45 The whole
blood was allowed to sit undisturbed at room temperature for 30 min,
followed by centrifugation (4000 g, 10 min, 4 °C) to remove blood
clots. Saturated ammonium sulfate solution was added dropwise to
the rat serum until ∼60% saturation, agitated gently at room
temperature for 30 min, and centrifuged (18 000 g, 10 min) to
precipitate out the globulins.46,47 The supernatant, the albumin
solution, was desalted, washed, and concentrated by centrifugation
(4500 g, 10 min) in a Nanosep 10K centrifugal device. After the
protein content was quantified using UV spectrophotometry at
generates a signal three times the background noise in blank HSA
sample matrix.18
The efficiency of the hydrolysis and extraction steps was
determined using the ALI−GSH adduct, which provides a critical
correction for the loss of ALI during sample processing. The
validation entailed spiking 100 μg of HSA with 2.2, 22, and 220 fmol
of ALI−GSH standard followed by heat and alkaline-catalyzed
hydrolysis of the adduct, extraction of ALI from the hydrolysates
using EA, and LC−MS/MS analysis of extracts, as described above.
The overall efficiency of the method was calculated as the measured
quantities of liberated ALI divided by the quantities of added ALI−
GSH.
RESULTS AND DISCUSSION
■
To the best of our knowledge, the chemical structures of AA−
protein adducts had not been reported until recent
identification of a series of AAI−aminothiol conjugates.40 It
was proposed that AAI is converted into a reactive
aristolactam−nitrenium ion intermediate through enzymatic
reduction of its nitro group, which reacts with the nucleophilic
thiol group of Cys residues to form covalent carbon−sulfur
linkages (Figure 1), similar to that of the AL−DNA adduct
formation.22,41−44
Because AL−DNA adducts are formed by reacting the
aristolactam−nitrenium ion intermediate with the exocyclic
amino group of purine nucleotides in DNA, and previous
studies identified a broad spectrum of Lys adduct formed by
reacting Lys residues with chemical carcinogens such as
aflatoxin and formaldehyde,33,42,45 we further tested the
plausible conjugation reaction between the AAI reactive
intermediate and Lys residues. Incubation of AAI and Lys in
the presence of Zn/H+ yielded no predicted adduct peak in the
chromatogram (Figure S3), indicating that the aristolactam−
nitrenium ion intermediate is not targeting the amino group of
Lys residues. Furthermore, no signal of predicted adduct peaks
was detected when Lys was replaced with other amino acids
that possess a nucleophilic side chain (Arg, His, Asp, Glu, Ser,
Thr, Asn, and Gln).
A
280 nm, the protein samples were further washed by liquid−liquid
extraction of 2 volumes of ethyl acetate (EA) and hydrolyzed to
release ALI for LC−MS/MS analysis.
The general strategy for quantitation of the ALI−protein adduct
involves successive washing of proteins, release of the protein-bound
ALI in proteins, extraction of ALI, addition of internal standards, and
quantitation by LC−MS/MS. To release the covalently bound ALI
from the adduct, 100 μg of protein sample (dissolved in 100 μL of
water) was first washed twice by liquid−liquid extraction of 2 volumes
of EA and was then incubated at 100 °C in 2.5 M NaOH for 2 h. After
cooling to ambient temperature, the hydrolysates were extracted
thrice with 600 μL of EA. The organic layers were combined and
dried under a nitrogen stream, and the residue was dissolved in 100
μL of 70% aqueous methanol containing 0.16 nM N-methyl-ALI
internal standard for LC−MS/MS analysis.
LC−MS/MS Quantitation of ALI in Tissue-Isolated Proteins
or Serum Albumin. The analysis was performed with 10 μL of the
70% aqueous methanol of each of the reconstituted samples. Samples
were injected onto an Acquity UPLC BEH C18 column (2.1 × 100
mm i.d., 1.7 μm; Waters Corporation, Milford, MA) in a Waters
Acquity UPLC system eluted at a flow rate of 0.4 mL/min and 40 °C
with the following gradient of acetonitrile in 0.2% acetic acid: 0−5
min, 5−100%; 5−8 min, 100%, followed by 3 min of re-equilibration
of the column. The UPLC column was coupled to a Waters Xevo TQ-
XS triple quadrupole MS with an ESI source operated in positive
mode with the following parameters for voltages and source gas: cone
gas, 250 L/h; desolvation gas, 850 L/h; collision gas (argon), 0.15
mL/min; nebulizer gas, 7.0 bar; desolvation temperature, 600 °C;
capillary voltage, 1.5 kV; and cone voltage, 35 V. The MS was
operated in multiple-reaction-monitoring (MRM) mode with the
following MRM transitions: ALI: m/z 294 → 279 (quantitative;
collision energy, 22 eV), m/z 294 → 251 (qualitative; collision
energy, 30 eV); N-methyl-ALI internal standard: m/z 308 → 293
Calibration and Method Validation. A matrix-matched
calibration curve was used for the quantitative analysis. Working
standard solutions were prepared by spiking to blank HSA sample
matrix with different concentrations of ALI (0.01−0.68 nM) and a
fixed concentration of N-methyl-ALI (0.16 nM) and analyzed using
the LC−MS/MS method stated above. The calibration curve was
established by plotting the peak area ratios of ALI to N-methyl-ALI
versus the concentration of ALI in calibration standards (Figure S5).
The limit of detection was estimated as the concentration of ALI that
Based on this foundation and the previous knowledge that
serum albumin contains a free Cys residue at position 34
(Cys34) that is reactive toward many oxidants and electro-
philes,33,48 we developed and validated a method, using ALI−
GSH tripeptide adduct and ALI−HSA, to quantitate ALI−
protein adducts in HSA exposed to AAI, tissues, and sera
collected from AAI-exposed rats.
Choice of Hydrolysis Method and Elimination
Product. Prior to the optimization of the hydrolysis
conditions for LC−MS/MS analysis, we first compared the
performance of enzymatic and chemical hydrolysis using ALI−
GSH adduct. ALI−GSH standard was prepared by HPLC
purification of the reaction product of a reaction of AAI with
GSH, as described in Experimental Section. By spiking 2.2
pmol of ALI−GSH standard into 100 μg of HSA followed by
protease digestion as reported previously,45 it was discovered
that the level of ALI−GSH remained stable, and no
elimination products were observed by HPLC−FLD analysis
Attempts were then made to hydrolyze ALI−GSH at 100 °C
in 1.0 M HCl/NaOH for 1 h, which are the conditions
previously stated by Krumbiegel et al. for hydrolyzing ALI-
conjugated metabolites.49 Results showed heat/H+ treatment
hydrolyzed ALI−GSH to a complex mixture, including ALI−
Cys, two unknown but highly fluorescent products, and ALI as
146
Chem. Res. Toxicol. 2021, 34, 144−153