The Toxicologist - Society of Toxicology

1849 MICROSOMAL METABOLISM OF NAPHTHALENE (NA)
IN TARGET AND NON-TARGET RODENT TISSUES:
COMPARISON WITH NON-HUMAN PRIMATES.
A. Buckpitt, D. Morin, P. Edwards and L. Van Winkle. Veterinary Medicine,
University of California Davis, Davis, CA.
NA is a volatile aromatic hydrocarbon to which humans are exposed. In rodents,
NA causes dose dependent, species and tissue selective toxicity in murine airways
and nasal epithelium, and rat nasal, but not airway, epithelium. Sites which are susceptible
to acute toxicity are also targeted following long term exposure.
Cytochrome P450 metabolism of NA plays a key role in the downstream tissue responses.
The metabolism of NA has been characterized in a number of systems
(whole tissue microsomes, postmitochondrial supernatant and airway explants) but
side by side comparisons using microsomes prepared from only target areas of the
respiratory tract have not been reported. Accordingly, these studies compared rates
of microsomal NA metabolism in target areas of the respiratory tract of the mouse,
rat and monkey. The highest rates of substrate turnover were in the rat nasal olfactory
epithelium (30 nmoles/mg/min). Rates of metabolism in mouse olfactory microsomes
(16.4) were half those in the rat. Microsomes from monkey nasoturbinates
were less than 10% those of the mouse. Microsomes from dissected
murine airways catalyzed NA metabolism at 10.9 nmole/mg/min whereas metabolism
in rat airways occurred at 5% of this rate. The majority of the metabolites were
accounted for as GSH conjugates of the 1,2-epoxide. At longer incubation times
diGSH conjugates of the diepoxide and GSH adducts of the diol epoxide and 1,4naphthoquinone
were observed in all preparations except rat airway. Under the conditions
used, less than 12% of the total metabolites produced were accounted for by
1-naphthol or dihydrodiol. Microsomes from mouse airways, mouse and rat nasal
olfactory epithelium showed a high degree of stereoselectivity in NA epoxidation
(20:1), rat airways and monkey nasal samples did not. Tissue susceptibility to NAinduced
injury correlates with high rates of substrate turnover; metabolism in the
nasal epithelium of monkeys is 10-50 fold lower than in rat olfactory epithelium.
Supported by the Naphthalene Coalition under the API and by NCRR 00169.
1850 CYTOCHROME P450S MEDIATE THE METABOLISM
AND ARE REGULATED BY BERBERINE IN MICE AND
HUMANS.
Y. J. Guo 1 , F. Li 2 , Y. Chen 1 , Z. Tan 1 , C. Pope 3 , X. Chen 2 , X. Ma 2 , C. D.
Klaassen 2 and H. Zhou 1 . 1 Pharmacogenetics Research Institute, Institute of Clinical
Pharmacology, Central South University, Changsha, China, 2 University of Kansas
Medical Center, Kansas, KS and 3 XenoTech, LLC, Kansas City, KS.
Berberine (BBR) is used in traditional medicine for many metabolic diseases.
However, little is known about BBR metabolism and whether it influences the cytochrome
P450 (CYP) enzymes in vivo. The purpose of this study was to characterize
the metabolism of BBR in liver and its role in regulating cytochrome P450s.
Metabolomic analysis identified 11 metabolites of BBR in urine and feces of mice,
including 5 phase-I metabolites, which were predominant in feces, and 6 phase-II
glucuronide and sulfate metabolites, which were predominant in urine. Of the 5
phase-I metabolites observed in mice, only 3 were detected when BBR was incubated
with human and mice liver microsomes or recombinant human CYPs.
CYP2D6 appeared to be the major enzyme producing the phase-I metabolites, followed
by CYP1A2, 3A4, 2E1 and 2C19. BBR (10, 30, 100, 300 mg/kg, p.o.) was
given to 8-week-old C57BL/6 male mice for 2 weeks. In the highest dosed group,
Cyp3a mRNAs decreased about 75%, and the enzyme activity of Cyp3a and 2d decreased
83% and 67%, respectively. A two-phase randomized crossover clinical
study in healthy male subjects showed that after 2-weeks of BBR, 300 mg t.i.d. p.o,
the Cmax and AUC of midazolam (probe for CYP3A4) increased 36.5% and
45.0%, the oral clearance decreased 24.2%, the t1/2 prolonged from 3.03 h to
3.66 h, and the 1 hr ratio of midazolam/1’-hydroxymidazolam increased 49.6%.
BBR also increased the 0-8 h urinary ratio of dextromethorphan/dextrorphan
(CYP2D6 activity marker) by 900% and Losartan/E-3174 (CYP2C9 activity
marker) by 100%. In conclusion, the metabolites of BBR are similar in human and
mice. BBR treatment decreased CYP450 activities in mice and humans. Therefore,
drug-drug interactions should be considered especially when high doses of BBR are
taken (supported by NIH grants ES-09649, ES-09716, ES-07079, DK 081461
and RR-021940).
1851 CYTOCHROME P450 2S1 MEDIATED REGULATION OF
HUMAN LUNG CELL PROLIFERATION.
A. M. Rowland, T. W. Madanayake, T. P. Fidler and L. Montoya. Chemistry and
Biochemistry, New Mexico State University, Las Cruces, NM.
Cytochrome P450 2S1 (CYP2S1) is a drug metabolism enzyme that is expressed in
hyperproliferative disease and metabolizes endogenous and exogenous chemicals
linked to lung carcinogenesis and treatment. CYP2S1 is preferentially expressed in
396 SOT 2011 ANNUAL MEETING
extrahepatic tissues and is elevated in hyperproliferative diseases including psoriasis
and epithelial cancers. Recent studies demonstrate that CYP2S1 metabolizes
Benzo[a]pyrene (B[a]P) to the ultimate carcinogen, B[a]P-diolepoxide. Under hypoxic
conditions, CYP2S1 demonstrates a robust reductive metabolism of the prodrug
AQ4N [1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione]
to the active topoisomerase II inhibitor, AQ4
[1,4-bis{[2-(dimethylamino)ethyl]amino}-5,8-dihydroxy-anthracene-9,10-dione].
Conflicting metabolic results have been demonstrated for a potential endogenous
substrate, all-trans retinoic acid (atRA). Using independent heterologous systems,
CYP2S1 either metabolized or failed to metabolize atRA to the 4-hydroxy metabolite.
To determine whether CYP2S1 plays a functional role in pulmonary cells, we
examined the impact of changes in CYP2S1 expression on lung cell proliferation.
We performed stable shRNA knock down of CYP2S1 expression in both human
lung alveolar (A549) and bronchiolar epithelial cells (BEAS-2B). CYP2S1 mRNA
levels were significantly reduced by approximately 70% in both A549 and BEAS-
2B cells. CYP2S1 protein was reduced by 50% and 80% in A549 and BEAS-2B,
respectively. Bronchial cells with reduced CYP2S1 expression exhibit elevated proliferation
rates, as demonstrated through both in vitro proliferation assays and flow
cytometry. These studies suggest an important physiological role for CYP2S1-mediated
metabolism of unknown endogenous morphogen or mophogens.
1852 NEW ROLES OF CYTOCHROME P4501A1 (CYP1A1)
AND THE ENDOGEONUS ARYL HYDROCARBON
RECEPTOR (AHR) LIGAND 6-FORMYLINDOLO[3, 2b]CARBAZOLE
(FICZ) IN REGULATION OF CELL
GROWTH.
A. Rannug 1 , H. Mei 1 , A. Mohammmadi-Bardbori 1 , Y. Wei 2 and U. Rannug 3 .
1 Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden,
2 Department of Community Medicine, Mercer University School of Medicine, Macon,
GA and 3 Department of Genetics, Microbiology and Toxicology, Stockholm University,
Stockholm, Sweden.
CYP1A1 is associated with the metabolism of xenobiotics. Our attention, however,
has focused on the role of this enzyme in cell proliferation. CYP1A1 causes rapid
turnover of FICZ, the candidate endogenous AHR ligand. Since the AHR has been
implicated in the regulation of cell growth, we hypothesize that FICZ can stimulate
cell cycle progression and that this function is controlled by CYP1A1. A pronounced
increase in proliferative ability in cells grown at relatively high density in
medium containing 1 μM FICZ was demonstrated, by using different experimental
methods to document cell proliferation, with both human HaCaT cells and
mouse Hepa-1 cells. Grown under such differentiated conditions, the cells express
high CYP1A1 activity. To study the effects of FICZ under conditions of low
CYP1A1 enzyme activity, HaCaT cells were grown undifferentiated at a low cell
density. Under these conditions, cell growth was stimulated by FICZ, and DNA
synthesis was enhanced as measured by FACS analysis, at concentrations as low as 1
pM. Interestingly, in Hepa-1 cells, we observed that CYP1A1-deficient c37 cells
grow more rapidly than wild type Hepa-1 cells and that transfection with a plasmid
containing a functional CYP1A1 cDNA inhibits the more rapid growth of c37
cells. In medium containing purified L-tryptophan (recrystallized to avoid background
contamination with FICZ) the growth rate of c37 cells was not higher compared
to wild type cells but addition of FICZ could stimulate proliferation of c37
cells at a concentration of 100 fM. Taken together, we propose that FICZ has vitamin/hormone-like
physiological properties and that the AHR and CYP1A1 participate
in an autoregulatory feedback that maintains the steady-state concentrations
of FICZ at low levels in order to maintain homeostasis.
1853 TRANSCRIPTOMIC EVALUATION OF CANINE
SUSPENSION-SHIPPED AND PRE-PLATED
HEPATOCYTES: COMPARISON TO LIVER.
A. Ditewig, M. Liguori, E. Blomme and Y. Yang. Cellular, Molecular and
Exploratory Toxicology, Abbott Laboratories, Abbott Park, IL.
The dog is frequently used for preclinical toxicity testing in the pharmaceutical industry.
In vitro tests using rat and human hepatocytes are commonly conducted;
however these methods are far less established for dog hepatocytes. In particular, little
is known about the effects of plating and culturing on hepatocellular functions.
Our objective was to evaluate transcriptomic changes between hepatocytes and liver
using gene expression profiling. Canine liver samples and hepatocytes isolated from
these livers were shipped either in suspension or pre-plated (P). Upon arrival, an
aliquot of each cell preparation was harvested in Qiazol and the remaining suspension-shipped
(S) cells were plated at a density comparable to the P cells.
Hepatocytes were cultured for 120 hours or were treated with 1 mM Phenobarbital
(PB) and 20 μM Rifampin for 48 hours; RNA was isolated for RT-PCR and mi-