The Toxicologist - Society of Toxicology

cyanoacetic acid increased 2-3 fold in plasma and urine. Urinalysis of rats showed a
significant proteinuria. We also studied the role of vehicles in the toxicity of AN.
The effects were variable in various organs. Among the vehicles studied corn oil,
olive oil and safflower oil had a significant effect in increasing or decreasing the levels
of metabolites studied in different organs. [Supported by Grant No.
SO6RR08038 from the Minority Biomedical Research Support Program of the
National Institutes of Health]
1836 DRUGS INTERACTING WITH TRICHLOROETHYLENE
METABOLISM IN RAT AND HUMAN.
M. Cheikh-Rouhou 2 and S. Haddad 1 . 1 Environmental and Occupational Health,
Université de Montréal, Montréal, QC, Canada and 2 Biological Sciences, TOXEN,
Université du Québec à Montréal, Montréal, QC, Canada.
We recently studied the impact of 14 commonly used drugs on the metabolism of
Trichloroethylene (TCE) in rat hepatocytes and identified that 5 altered the kinetics
of formation of trichloroethanol (TCOH) and trichloroacetic acid (TCA). The
objective of this study were i) to determine if the same interactions are observable in
human hepatocytes, and ii) to characterize the stronger interactions using microsomal
assays. First, the interacting drugs have been identified in vitro by measuring
the metabolite formation rates in suspensions of human hepatocytes in the presence
and absence of each of the drugs in closed vials. The concentrations of TCE and its
metabolites TCOH and TCA were measured GC-MS. Similar to observations in
rat hepatocytes, acetyl salicylic acid and naproxen increased significantly TCA and
TCOH levels, while the significant decreased levels in both metabolites were not
observed by the same drugs (rat: valproic acid, acetaminophen and gliclazide; and
human: carbamazepine and erythromycine). Drugs decreasing only TCOH levels
were different in both species: sulfasalazine and valproic acid in humans, and erythromycine
in rat. Decreases in TCA only were observed with gliclazide in humans,
and with cimetidine, diclofenac and amoxicillin in rats. Although metabolites levels
in hepatocytes were shown to increase in the presence of salicylic acid and decrease
with acetaminophen, no effect were were detected using microsomes. Results from
characterization in rats shows that gliclazide (Ki= 566.7μM, TCOH formation)
and valproic acid (Ki= 624.2 μM for TCA formation and Ki= 682 μM for TCOH
formation) altered TCE microsomal oxidation by mixed partial inhibition. And
naproxen partially competitively inhibited TCOH glucuronidation (Ki = 294.8
μM). Combined in vivo rat exposures of TCE with to the identified stronger interacting
drug are underway and in vitro–in vivo extrapolations of interactions will be
validated with PBPK modeling to ultimately enable predictions of interactions in
humans.
1837 HUMAN DIOXIN-INDUCIBLE CYTOCHROME P450,
CYP2S1, METABOLIZES CYCLOOXYGENASE – AND
LIPOXYGENASE – DERIVED EICOSANOIDS.
P. H. Bui 1, 2, 3 , S. Imaizumi 4 , S. Beedanagari 1, 2 , S. T. Reddy 4 and O.
Hankinson 1, 2, 3 . 1 Pathology and Laboratory Medicine, University of California, Los
Angeles, Los Angeles, CA, 2 Molecular Toxicology Interdepartmental Program,
University of California, Los Angeles, Los Angeles, CA, 3 Jonsson Comprehensive
Cancer Center, University of California, Los Angeles, Los Angeles, CA and
4 Department of Molecular and Medical Pharmacology, University of California, Los
Angeles, Los Angeles, CA.
Human CYP2S1 is a recently described dioxin-inducible cytochrome P450 which
can oxidize a number of carcinogens via the peroxide shunt. To further characterize
the biological function of this enzyme, we investigated whether it can metabolize
cyclooxygenase (COX) and lipoxygenase (LOX) derived lipid peroxides in a
NADPH-independent fashion. Human CYP2S1 metabolizes prostaglandin G2
(PGG2) (Km = 0.267±0.072 μM) into several products including 12S-hydroxy-
5Z,8E,10E-heptadecatrienoic acid (12-HHT). It also metabolizes prostaglandin
H2 (PGH2) (Km=11.7 ± 2.8 μM) into malondialdehyde (MDA), 12-HHT, and
thromboxane A2 (TXA2). The turnover to 12-HHT by human CYP2S1
(1.59±0.04 min -1 ) is 40-fold higher than that of TXA2 (0.04 min -1 ). In addition to
PGG2 and PGH2 metabolism, human CYP2S1 efficiently metabolizes the hydroperoxyeicosatetraenoic
acids (HpETE), 5S-, 12S-, 15S- HpETEs and 13S-hydroperoxyoctadecadienoic
acid (13S-HpODE) into 5-oxo-eicosatetraenoic acid (5oxoETE)
(turnover = 16.7 ± 0.3 min -1 ), 12-oxo-eicosatetraenoic acid 1(12-oxoETE
) (11.5 ± 0.9 min -1 ), 15-oxo-eicosatetraenoic acid (15-oxoETE) (16.9 ± 0.8 min -1 ),
and 13-octadecadienoic acid (13-oxoODE) (20.2±0.9 min -1 ), respectively. Other
P450s such as CYP1A1, 1A2, 1B1, and 3A4 carried out similar conversions, but at
slower rates. The fatty acid hydroperoxides were also converted by human CYP2S1
to several epoxy alcohols. Our data indicate that fatty acid endoperoxides and hy-
droperoxides represent endogenous substrates of CYP2S1 and suggest that the enzyme
CYP2S1 may play an important role in the inflammatory process since some
of the products that CYP2S1 produces play important role in inflammation.
1838 CYTOCHROME P450’S VARY IN THEIR ABILITY TO
GENERATE REACTIVE OXYGEN SPECIES.
V. Mishin 1 , D. E. Heck 2 , D. L. Laskin 1 and J. D. Laskin 3 . 1 Pharmacology &
Toxicology, Rutgers University, Piscataway, NJ, 2 Environmental Health Sciences, New
York Medical College, Valhalla, NY and 3 Environmental & Occupational Medicine,
UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ.
In the presence of NAD(P)H, the microsomal enzymes are able to generate hydrogen
peroxide as a result of the non-productive utilization of reducing equivalents, a
reaction known as ‘uncoupling’. The precise origin of hydrogen peroxide in this reaction
is not known and may be generated directly or indirectly via the formation
of superoxide anion. Hydrogen peroxide generated by this process can form highly
toxic hydroxyl radicals in the presence of redox active transition metals; depending
on the cytochrome P450’s present in tissues, this may compromise cellular functioning
and contribute to tissue injury. In present studies we quantified the relative
‘uncoupling’ activity of different human recombinant cytochrome P450’s using a
modified end-point Amplex Red/horse radish peroxidase assay. The complex of
human recombinant NADPH-cytochrome P450 reductase and cytochrome b5 (in
the absence of cytochrome P450 enzymes) generated low, but detectable amounts
of hydrogen peroxide (0.05-0.1 nmoles hydrogen peroxide/min/100 Units of reductase).
Significantly higher activity was found when NADPH cytochrome P450
reductase/b5 reductase was coexpressed with various cytochrome P450’s. Activities
ranged from 6.0 nmoles hydrogen peroxide formed/nmole P450 to 0.4
nmole/nmole P450 with CYP1A1 as the most active, followed CYP2D6, CYP3A4,
CYP4A11, CYP2E1, CYP1A2 and CYP2C. Substrate binding is thought to facilitate
oxygen activation by the cytochrome P450 enzymes. In contrast, we found that
the hydrogen peroxide generating activity of the recombinant CYP’s did not require
the addition of any cytochrome P450 substrates. Since CYP3A4/5 and
CYP2C8/9/18/19 represent up to 80% of total cytochrome P450 in the liver, these
enzymes most likely represent a major source of hydrogen peroxide in human liver
microsomes. Supported by AR055073, NS072097, ES005022, GM034310,
ES004738 and CA132624.
1839 BACKGROUND LEVELS OF THE TRYPTOPHAN
PHOTOPRODUCT 6-FORMYLINDOLO[3, 2b]CARBAZOLE
(FICZ) DETERMINE THE OUTCOME
OF IN VITRO BIOASSAYS FOR AH-RECEPTOR
ACTIVATION.
U. Rannug 1 , E. Wincent 1, 2 , A. Mohammadi Bardbori 2 , T. Alsberg 3 and A.
Rannug 2 . 1 Department of Genetics, Microbiology and Toxicology, Stockholm
University, Stockholm, Sweden, 2 Institute of Environmental Medicine, Karolinska
Institutet, Stockholm, Sweden and 3 Department of Applied Environmental Science,
Stockholm University, Stockholm, Sweden.
6-Formylindolo[3,2-b]carbazole (FICZ) is formed when tryptophan containing
cell culture media are exposed to UV radiation or visible light. This substance binds
to the aryl hydrocarbon receptor (AHR) with very high affinity and is a claimed endogenous
AHR ligand (Rannug et al., J Biol Chem, 262, 1987, 15422-27). FICZ
is a potent inducer of CYP1 enzymes and an excellent substrate for CYP1A1 and
may thus interfere with in vitro bioassays for AHR activation and toxicity of AHR
dependent xenobiotics.
Our earlier analyses employing HPLC/MS demonstrated that Dulbecco’s Modified
Eagle Medium (DMEM) exposed to light contained FICZ at concentrations up to
8 pM (Öberg et al., Tox Sci, 85, 2005, 935-43). Here we describe analyses of lightprotected
commercial media and again we could detect FICZ, but at concentrations
around 0.2 pM. The biological consequences of these low background concentrations
of FICZ were analyzed in HaCaT cells grown in DMEM. The cells
were exposed to H 2 O 2 or 3’-methoxy-4’-nitroflavone (MNF), two substances reported
to have AHR activating properties. Both substances increased CYP1A1
mRNA expression and 7-ethoxyresorufin O-deethylase activity. Interestingly, these
effects were only seen in commercial medium and not in DMEM prepared with purified
tryptophan and hence lacking FICZ. When FICZ was added to this medium
at a concentration of 0.1 pM the CYP1A1 activation by MNF was regained. Both
substances also inhibited the CYP1A1 catalyzed turnover of FICZ, and if present
together with FICZ, they prolonged the otherwise transient AHR-mediated
CYP1A1 induction. These data show that background levels of FICZ in commercial
media are high enough to explain activation of the AHR and that erroneous
conclusions could be drawn from in vitro bioassays in screening for AHR activators.
SOT 2011 ANNUAL MEETING 393