The Toxicologist - Society of Toxicology

1820 RAT LIVER MICROSOMES ENANTIOSELECTIVELY
CATALYZE THE FORMATION OF HYDROXYLATED
METABOLITES OF 2, 2’, 3, 3’, 6, 6’-
HEXACHLOROBIPHENYL (PCB136).
I. Kania-Korwel 1 , M. W. Duffel 2 , S. Bandiera 3 and H. Lehmler 1 . 1 Department of
Occupational and Environmental Health, University of Iowa, Iowa City, IA,
2 Division of Medicinal and Natural Products Chemistry, University of Iowa, Iowa
City, IA and 3 Faculty of Pharmaceutical Sciences, University of British Columbia,
Vancouver, BC, Canada.
Chiral PCB congeners such as PCB136 undergo enantiomeric enrichment in vivo.
The biotransformation processes responsible for this enantiomeric enrichment are
still poorly understood. A series of metabolism studies was performed to test the hypothesis
that metabolism of PCB136 is enantioselective and isoform-dependent in
liver microsomes from rats treated with phenobarbital (PB, CYP2B induced), dexamethasone
(DEX, CYP3A induced) or corn oil (CO). Incubations of all three
types of microsomes with racemic PCB136 (50 μM) resulted in the time-dependent
formation of hydroxylated metabolites. Microsomes from PB-treated animals
generated the highest amount of metabolites, followed by microsomes from DEX
and CO- treated rats. The major metabolite in all preparations was 5-OH-PCB 136
and the rank order was 5-OH-PCB 136 > 4-OH-PCB136 > 4,5-diOH-PCB136.
However, the relative contribution of 4-OH-PCB136 depended on the microsomal
preparation and increased in microsomes from DEX and CO-treated rats. Carbon
monoxide inhibited the formation of metabolites, which is consistent with the involvement
of cytochrome P450 isoforms in the metabolism of PCB136.
Incubation of microsomal preparations from both PB- and DEX-treated rats with
(+)-, (-)-, and racemic PCB 136 revealed no clear differences in metabolite profiles.
Although no enantiomeric enrichment of PCB136 was observed, the second eluting
atropisomers of 4-OH-PCB136 and 5-OH-PCB136 were slightly enriched in
incubations with racemic PCB136. Incubations with pure PCB 136 atropisomers
demonstrated that the second eluting atropisomers of both metabolites are formed
from (+)-PCB136. Overall, these studies demonstrate the enantioselective and isoform-dependent
formation of hydroxylated PCB 136 metabolites by cytochrome
P450 enzymes (Supported by NIH ES017425).
1821 MYELOPEROXIDASE-MEDIATED BIOACTIVATION OF
5-HYDROXYTHIABENDAZOLE: A POSSIBLE
MECHANISM BEHIND THE TOXICITIES ASSOCIATED
WITH THIABENDAZOLE.
J. Jamieson, E. Smith, D. Dalvie, G. Stevens and G. Yanochko. Pfizer, Inc., San
Diego, CA.
Thiabendazole (TBZ), an antihelminthic and antifungal agent, is associated with a
host of adverse effects including nephrotoxicity, hepatotoxicity, and teratogenicity.
Bioactivation of the primary metabolite of TBZ, 5-hydroxythiabendazole, has been
proposed to yield a reactive intermediate. Here we show that this reactive intermediate
can be generated by myeloperoxidase, a neutrophil-bourne peroxidase. Using
a cell viability endpoint, we examined the toxicity of TBZ, 5OH-TBZ, and MPOgenerated
metabolites in cell-based models including primary rat proximal tubule
epithelial cells, NRK-52E rat proximal tubule cells, and H9C2 rat myocardial cells.
Timecourse experiments with MPO showed complete turnover of 5OH-TBZ
within 15 minutes and a dramatic leftward shift in dose-response curves after 12
hours. After a 24 hour exposure in vitro, the LC50 of this reactive intermediate was
22 ± 1μM compared to 117 ± 20μM from 5OH-TBZ alone, a greater than 5-fold
decrease. LC50 values were equal in all cell types used. This toxicity can be completely
rescued via incubation with rutin, an inhibitor of MPO. These results suggest
that MPO-mediated biotransformation of 5OH-TBZ yields a reactive intermediate
which may play a role in TBZ-induced toxicity.
1822 METABOLISM OF 2-HYDROXY-4-
METHOXYBENZOPHENONE DEPENDS ON SPECIES
AND SEX.
C. Wegerski 1 , S. S. Auerbach 2 , J. M. Sanders 2 , M. Doyle-Eisele 1 and J. D.
McDonald 1 . 1 Lovelace Respiratory Research Institute, Albuquerque, NM and
2 NTP/NIEHS, Research Triangle Park, NC.
Metabolism and disposition studies of 2-Hydroxy-4-methoxybenzophenone
(HMB) are being conducted to aid in interpretation of toxicology results and interspecies
and sex extrapolation. To assess species and sex differences within rodents, in
vivo studies were conducted by oral dose administration of 14C labeled HMB in
male and female Sprague-Dawley rats and B6C3F1 mice. Urine samples were analyzed
for HMB metabolites. To confirm these results and extend to human metab-
390 SOT 2011 ANNUAL MEETING
olism, in vitro studies were conducted by incubating 14C HMB with male and female
liver hepatocytes from Sprague-Dawley rats, B6C3F1 mice, and humans.
Samples were analyzed by LC-RAD and/or LC-MS/MS. There were sex differences
in the in vivo metabolism of HMB detected in urine between the male and female
rat. The major metabolite in the female rat was not detected in the male rat.
Similarly, the major metabolite in the male rat was a minor metabolite in the female
rat. There were also species differences between the rat and mouse, with the major
metabolite in the male mouse urine present only in trace quantities in the male rat
urine. Both HMB and 2,4-dihydroxy-benzophenone, which were present in the
male mouse and rat urine, were absent or in trace quantities in the female rat urine.
Species and sex differences in HMB metabolism were also observed in isolated rat,
mouse and human hepatocytes. Female rodent hepatocytes produced all HMB-derived
metabolites present in males, as well as a few minor metabolites not evident in
males. Male human hepatocytes produced 10 HMB-derived metabolites compared
to 8 detected in female human hepatocytes. The metabolites detected in these studies
were primarily glucuronide and sulfate conjugates, but some Phase I oxidative
intermediate metabolites were also observed. The in vitro data indicate that the
mouse is a closer metabolic match to humans than the rat. (Work conducted under
NIEHS contract N01-ES-75562).
1823 THE IN VITRO METABOLISM OF METHYL AND
PROPYL PARABENS IN HUMAN AND RAT.
K. Choi, H. Joo, J. Campbell, M. Andersen and H. Clewell. The Hamner
Institutes for Health Sciences, Research Triangle Park, NC.
Parabens are antimicrobial preservatives widely used in cosmetic products and
pharmaceuticals as well as in food and beverage processing. Methyl paraben (MP)
and propyl paraben (PP) are the most predominantly used parabens. In mammals
parabens are extensively hydrolyzed to p-hydroxy benzoic acid (pHBA) and further
undergo phase II biotransformation via glucuronide, glycine and sulfate conjugation.
However, their metabolism in humans has not been well characterized. We investigated
the metabolism of MP and PP using subcellular fractions of various organs
in human and rat. In human, pHBA was mainly produced in the liver,
followed by the lung, skin, intestine and testis (for MP) or the intestine, kidney,
lung, skin and testis (for PP). For MP in human, the velocities for pHBA production
were 94.72±5.35 and 46.23±0.07 in liver microsomes and cytosol and
38.17±3.64 and 13.11±0.45 (nmol mg protein-1 min-1) in lung microsomes and
cytosol, respectively. For PP, the velocities for the production of pHBA were
114.71±0.85 and 45.90±1.48 in liver microsomes and cytosol, 8.79±0.05 and
32.38±5.50 (nmol/mg protein/ min) in intestine microsomes and cytosol, respectively.
In rat liver microsomes, the velocities for the production of pHBA with treatment
of MP and PP were 106.14±2.55 and 104.15±4.88 and in rat liver cytosol
36.53±6.0 and 111.76±3.05 (nmol/mg protein/min), respectively. These metabolism
data provide key parameters for PBPK models that support cross-species extrapolation
of parabens exposures.
1824 PHASE II BIOTRANSFORMATION OF DI(2-
EHTYLHEXYL) PHTHALATE IN HUMAN AND RAT.
H. Joo 1 , K. Choi 1 , J. Grimes 1, 2 , T. O’Connell 1, 2 , R. Clewell 1 , J. Campbell 1
and H. Clewell 1 . 1 The Hamner Institutes for Health Sciences, Research Triangle Park,
NC and 2 The UNC, Chapel Hill, NC.
In this study we demonstrated that three metabolites of di(2-ethylhexyl) phthalate
(DEHP), mono(2-ethylhexyl) phthalate (MEHP), mono(2-ethyl-5-OH-hexyl) phthalate
(5-OH MEHP) and mono(2-ethyl-5-oxo-hexyl) phthalate (5-oxo MEHP),
were metabolized via conjugation with glucuronic acid in pooled human liver
(HLM) and rat liver microsomes (RLM). Production of MEHP-, 5-OH MEHPand
5-oxo MEHP glucuronide was measured by LC-ESI (-)/MS/MS. The metabolic
rates in HLM and RLM were 37,460 ± 5,565 and 4,472 ± 206 for MEHP
glucuronide, 102,229 ± 1,160 and 868 ± 3 for 5-OH MEHP glucuronide and
5,549 ± 350 and 920 ± 28 (AUC mg protein-1 min-1) for 5-oxo MEHP glucuronide,
respectively. We also determined metabolic activities of 12 human uridine
5-diphosphoglucuronosyl transferase (UGT) isoforms (1A1, 1A3, 1A4, 1A6,
1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15 and 2B17) toward MEHP, 5-OH MEHP
and 5-oxo MEHP. Among the 12 UGTs 1A3, 1A8, 1A10, 2B7 and 2B17 were
major isoforms for MEHP glucuronide production, while 1A3, 1A7, 1A8, 1A9,
1A10 and 2B7 were mainly responsible for the production of 5-OH MEHP- and
5-oxo MEHP glucuronide. The percent total normalized rates (%TNR) were 84%
for MEHP glucuronide by UGT2B7 and 80% and 85% for 5-OH MEHP- and 5oxo
MEHP glucuronide by UGT1A9, respectively. Additionally, we developed a
method to biosynthesize milligram quantities of MEHP glucuronide for use as a
reference standard. These metabolism data are being used to support PBPK model
evaluation of cross-species dosimetry and human interindividual variability for
DEHP exposures.