The Toxicologist - Society of Toxicology

studies showed that AF exposure in rural residents of Burkina Faso is high and other
effective and targeted intervention strategy, in addition to education, are urgently
needed.
1689 EMPLOYING A NEW ANALYTICAL METHOD FOR
MEASURING THE CYANIDE METABOLITE, 2-
AMINOTHIAZOLINE-4-CARBOXYLIC ACID, IN
BIOLOGICAL SAMPLES.
J. C. Yu 2 , I. Petrikovics 1 , S. Martin 1, 2 , J. Nasr 1 , D. Thompson 1 and S. Holmes 1 .
1 Chemistry, Sam Houston State University, Huntsville, TX and 2 Forensic Science, Sam
Houston State University, Huntsville, TX.
A new, sensitive method was developed for the identification and quantification of
2-aminothiazoline-4-carboxylic acid (ATCA) in biological samples, such as blood,
urine, liver, and brain samples. The purpose of this study was to determine the optimal
conditions for the quantification of ACTA in various biological fluids and organs
using solid phase extraction (SPE) with a mixed-mode cation exchange cartridge,
and high performance liquid chromatography coupled to tandem mass
spectrometry (LC-MS/MS). Conditions associated with SPE sample loading, washing
and final elution were studied and refined. The effect of ATCA signal fluctuations
arising from electrospray ionization suppression was minimized by using a
structural analogue of ATCA (2-aminothiazole-4-carboxylic acid, ATZA) as an internal
standard. 0.5% trifluoroacetic acid in methanol was found to be a mobile
phase that effectively eluted ATCA and ATZA. Symmetric peak shapes were obtained
that were well suited to quantification. Mice received three different sublethal
doses (6, 8, and 10 mg/kg) of KCN subcutaneously (3 mice / doses) and were
terminated 15 mins after cyanide exposure. Exogenously elevated levels of ATCA
were compared to endogenous levels of ATCA present in the biological fluids and
organs. ATCA concentration in mice blood samples increased from 189 +/- 28
ng/mL to 413 +/- 66 ng/mL after a 10 mg/kg body weight dose of KCN was introduced
subcutaneously. The endogenous ATCA concentrations found were as follows:
1.2 +/- 0.1 μg/g for kidney samples, 1.6 +/- 0.1 μg/g for brain samples, 1.8
+/- 0.2 μg/g for lung samples, 2.9 +/- 0.1 μg/g for heart samples, and 3.6 +/- 0.9
μg/g for liver samples. This method, which uses LC-MS/MS to detect ATCA as a
biomarker for cyanide poisoning will be used in future toxicokinetic studies involving
different biological matrices, and other animal species. The fundamental goal
will be to gain a more complete picture of the toxicokinetics of cyanide poisoning.
1690 MOLECULAR DOSIMETRY AND HALF LIFE OF N 2 -
HYDROXYMETHYL-DG ADDUCT IN RATS EXPOSED
TO FORMALDEHYDE.
K. Lu 1 , B. Moeller 2 , M. Doyle-Eisele 3 , J. McDonald 3 and J. A. Swenberg 1, 2 .
1 Environmental Sciences and Engineering, University of North Carolina, Chapel Hill,
NC, 2 Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC and
3 Lovelace Respiratory Research Institute, Albuquerque, NM.
Formaldehyde is classified as a known human and animal carcinogen, causing nasopharyngeal
cancer. Additionally, limited evidence for leukemia in humans is
available; however, this is inconsistent across studies. Both genotoxicty and cytotoxicity
are key events in formaldehyde nasal carcinogenicity in rats, but no mechanistic
data exist for leukemia. In this study, a highly sensitive nano-LC-MS/MS-SRM
method was developed and [ 13 CD 2 ]-formaldehyde exposures utilized, allowing differentiation
of DNA adducts originating from endogenous and inhalation-derived
formaldehyde exposure. Rats were exposed to 0.7, 2, 6, 10, 15 ppm [ 13 CD 2 ]formaldehyde
for 6h and N 2 -hydroxymethyl-dG adducts in DNA from rat nasal
epithelium were quantified by mass spectrometry. The number of exogenous N 2 -
HO 13 CD 2 -dG were 0.039±0.019, 0.19±0.08, 1.04±0.24, 2.02±0.44, 11.15±3.01
adducts/10 7 dG in rats exposed to 0.7, 2, 6, 10, 15 ppm [ 13 CD 2 ]-formaldehyde, respectively.
Endogenous N 2 -HOCH 2 -dG in the nasal tissue was 4.70±1.80
adducts/10 7 dG. The results clearly show a nonlinear dose response, as demonstrated
by the fact that 21 fold increase in exposure (0.7 to 15 ppm) induced 287
fold higher amounts of exogenous DNA adducts in nasal epithelium. In addition,
we have performed a study to determine the half life of exogenous formaldehyde
DNA adducts by exposing rats to 10 ppm [ 13 CD 2 ]-formaldehyde for 1 day (6h),
followed by sacrificing animals at 6, 12, 24, 48 and 72 h, respectively. Our preliminary
data indicate a rapid loss of nearly half of the adducts during the first 6 hours
post exposure, followed by a constant loss over the rest of the 72 hour experiment
with a half-life about 94 hours. This study provides strong data to support mode of
action for inhaled formaldehyde in the target tissue for carcinogenesis and critical
evidence for the science-based risk assessment of formaldehyde exposure.
364 SOT 2011 ANNUAL MEETING
1691 FORMALDEHDYE-INDUCED HYDROXYMETHYL DNA
ADDUCTS IN RATS EXPOSED TO ISOTOPE LABELED
METHANOL.
H. Gul, K. Lu, P. Upton and J. A. Swenberg. Environmental Sciences and
Engineering, University of North Carolina, Chapel Hill, NC.
Formaldehdye is formed as an intermediate metabolite of methanol. As such, it
could induce DNA damage following methanol exposure. However, it is difficult to
measure the number of DNA adducts caused by methanol exposure since it is confounded
by the presence of a substantial natural background formaldehyde adducts.
In order to distinguish DNA adducts originating from endogenous formaldehdye
and methanol-derived formaldehdye, we used highly sensitive mass spectrometry
coupled with exposure to [ 13 CD 4 ]-methanol. Rats were exposed to 500 mg/kg/day
and 2000 mg/kg/day by gavage for 5 days. Both N 2 -hydroxymethyl-dG and N 6 -hydroxymethyl-dA
adducts were quantified by capilary LC-ESI-MS/MS.
Endogenous N 6 -hydroxymethyl-dA addcuts were present at 1~3 adducts/10 7 dA
across different tissues, while endogenous N 2 -hydroxymethyl-dG addduct were
shown to be 3~6 adducts/10 7 dG. The number of [ 13 CD 4 ]-methanol derived exogenous
dG adducts in the 500 mg/kg/day group was 0.08±0.08, 0.13±0.04,
0.12±0.04, 0.19±0.12, 0.37±0.08, 0.16±0.06, 0.09±0.03 adducts/10 7 dG for liver,
lung, kidney, spleen, bone marrow, thymus and white blood cells, respectively. In
the 2000 mg/kg/day dose group, the exogenous dG number was 0.41±0.14,
0.22±0.06, 0.39±0.09, 0.90±0.26, 1.42±0.29, 0.42±0.03 and 0.19±0.02
adducts/10 7 dG, respectively. In addition, [ 13 CD 4 ]-methanol administration caused
a 1.3-2.3 fold increase in endogenous dG adducts. Interestingly, exogenous dA
adducts were only detected in kidney (0.09±0.09 adduct/10 7 dA for 2000
mg/kg/day group) and bone marrow (0.10±0.08 and 0.29±0.09 adduct/10 7 dA for
500 mg/kg/day and 2000mg/kg/day group, respectively). This study provides the
first unambiguous evidence that exogenous methaol-derived formaldehdye induced
DNA damage is formed in multiple tissues. Our data also demonstrates that exogneous
formaldehyde-dA adducts were formed in lower amounts in kidney and bone
marrow. The reason for this tissue difference is under investigation.
1692 HUMAN BIOMARKERS FOR TRICRESYL PHOSPHATE
EXPOSURE.
J. Marsillach Lopez 1, 2 , R. J. Richter 1, 2 , T. B. Cole 1, 3 , J. H. Kim 4 , R. C.
Stevens 1, 2 , M. J. MacCoss 2 , D. Tomazela 2 , E. J. Hsieh 2 , S. M. Suzuki 1, 2 , L. M.
Schopfer 5 , O. Lockridge 5 and C. E. Furlong 1, 2 . 1 Medicine, Division of Medical
Genetics, University of Washington, Seattle, WA, 2 Genome Sciences, University of
Washington, Seattle, WA, 3 Environmental and Occupational Health Sciences,
University of Washington, Seattle, WA, 4 Anesthesiology, University of Washington,
Seattle, WA and 5 University of Nebraska Medical Center, Omaha, NE.
Tricresyl phosphate (TCP) caused thousands of cases of paralysis during
Prohibition. TCPs are still used in jet-engine lubricants and hydraulic fluids. Air
crew report tremors, memory loss and cognitive problems following cabin oil fume
events. There is controversy whether these symptoms result from TCP exposure.
ToCP isomers are metabolized by the liver into cresyl saligenin cyclic phosphate
(CBDP), a neurotoxic metabolite that inhibits many serine active-site enzymes.
Our aim was to identify biomarkers of exposure in blood samples from exposed individuals.
We focused on plasma butyrylcholinesterase (BChE) and red cell
acylpeptide hydrolase (APH). We developed single-step immunomagnetic beadbased
protocols for purifying BChE and APH. CBDP-inhibited samples were used
to identify and characterize the adducted active-site peptides. Samples were analyzed
by high resolution mass spectrometry (MS). A peptide database search of the
acquired data identified active site serine modifications, corresponding to +80 and
+170. Using Selected Reaction Monitoring (SRM) we detected an increase in the
abundance of the modified active site peptides in exposed samples compared to
controls. We are using a chromatography alignment and feature detection software,
CRAWDAD, to compare and detect differences across the samples. We expect that
either the modification in the active site peptide in subjects is of very low abundance
or the in vivo modifications are different from those observed in vitro.
Supported by U.S. Army Medical Research & Materiel Command (W81XWH-07-
2-0034), NIH (U01 NS058056, P30CA36727, R01ES09883, and P42ES04696),
funding from pilot and flight attendant unions, Royal Australian Air Force,
Norwegian Union of Energy Workers (SAFE), and NYCO SA.