The Toxicologist - Society of Toxicology

1975 DIFFERENTIAL MODULATION OF CYP1A1 BY
ARSENITE IN VIVO AND IN VITRO IN C57BL/6 MICE.
A. Anwar-Mohamed and A. O. El-Kadi. Faculty of Pharmacy and Pharmaceutical
Sciences, University of Alberta, Edmonton, AB, Canada.
Arsenite (As(III)), have been implicated in altering the carcinogenicity of aryl hydrocarbon
receptor (AhR) ligands, typified by 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD), via modulating the induction of the cytochrome P450 1a1 (Cyp1a1) enzyme,
but the mechanism(s) remain unresolved. In this study, the effect of As(III)
on Cyp1a1 expression and activity was investigated in C57BL/6 mice livers and isolated
hepatocytes. For this purpose, C57BL/6 mice were injected intraperitoneally
with As(III) (12.5 mg/kg) in the absence and presence of TCDD (15 μg/kg). After
6 or 24 h, livers were excised and the levels of Cyp1a1 mRNA, protein, and catalytic
activity levels were determined using real time-PCR, Western blot, and 7ethoxyresorufin-O-detethylase
(EROD) analyses, respectively. At in vitro level, isolated
hepatocytes from C57BL/6 mice were treated with increasing concentrations
of As(III) (1, 5, and 10 μM) in the absence and presence of TCDD (1 nM) for different
time-points (0 - 24 h). Our results showed that at in vivo level, As(III) decreased
the TCDD-mediated induction of Cyp1a1 mRNA at 6 h while potentiating
the TCDD-mediated induction of Cyp1a1 mRNA, protein, and catalytic
activity levels at 24 h. In isolated mouse hepatocytes, As(III) decreased the TCDDmediated
induction of Cyp1a1 mRNA in a time-dependent manner. Moreover,
As(III) decreased the TCDD-mediated induction of Cyp1a1 protein and catalytic
activity levels at 24 h. Investigating the effect of co-exposure to As(III) and TCDD
at transcriptional levels revealed that As(III) significantly inhibited TCDD-mediated
induction of AhR-dependent luciferase reporter gene expression. In conclusion,
caution should be taken when extrapolating in vitro data to in vivo situation.
Furthermore, our results denote more complex regulation of Cyp1a1 at the in vivo
level that warrants further investigation. Supported by Natural Sciences and
Engineering Research Council of Canada Discovery Grant RGPIN 250139-07.
1976 ROLE OF CONTINUOUS LOW-LEVEL
MONOMETHYLARSONOUS ACID EXPOSURE IN THE
INHIBITION OF PARP AND CONTRIBUTION TO
INCREASED GENOTOXICITY IN THE MALIGNANT
TRANSFORMATION OF UROTSA CELLS.
S. M. Wnek, M. M. Medeiros, J. M. Camarillo, T. J. Jensen, X. Zheng, B. W.
Futscher and A. Gandolfi. Pharmacology and Toxicology, University of Arizona,
Tucson, AZ.
Exposure of human bladder cells (UROtsa) to 50 nM of the arsenic metabolite,
monomethylarsonous acid, (MMAIII) for 12 wk results in malignant transformation
and a time-dependent increase in DNA damage. Acute studies have suggested
an indirect role of arsenicals in eliciting genotoxicity; however, it is unknown
whether continuous low-level MMAIII exposure can increase genotoxic potential
by inhibiting DNA repair processes. Poly(ADP-ribose) polymerase-1 (PARP-1), a
zinc finger protein, is rapidly activated upon DNA damage. However, in MMAIIIexposed
UROtsa cells, PARP activity does not increase despite the increase in DNA
damage through 12 wk of exposure; when UROtsa cells are removed from MMAIII
exposure (2 wk), PARP activity increases and DNA damage levels decrease.
Interestingly, PARP-1 gene and protein levels are elevated in the presence of
MMAIII, possibly as a means to compensate for the decrease in global protein
poly(ADP-ribosyl)ation. PARP plays a critical role in the cellular response to DNA
damage and maintaining genomic stability; overall, these data suggest a potential
role of MMAIII in the inhibition of PARP. The zinc finger domains of PARP contain
vicinal sulfhydryl groups as a result of closely spaced cysteine amino acids,
which may act as a potential site for MMAIII to bind PARP, displace zinc ion, and
render PARP inactive. In the presence of continuous MMAIII exposure, zinc supplementation
(4 wk) was able to increase PARP activity levels and reduce the genotoxicity
associated with MMAIII, suggesting the possible interference of MMAIII
with PARP zinc-finger motifs. Overall, these results present a potential mechanism
in which MMAIII may inhibit PARP through the displacement of zinc; thus inactivating
PARP and increasing the susceptibility of UROtsa cells to genotoxic insult/malignant
transformation as well as explain the increased genotoxicity associated
with methylated arsenicals when compared to inorganic arsenic.
1977 P53 RESPONSE TO MONOMETHYLARSONOUS ACID
EXPOSURE IN HUMAN BLADDER EPITHELIAL CELLS.
M. K. Medeiros and A. Gandolfi. Pharmacology and Toxicology, University of
Arizona, Tucson, AZ.
Epidemiological data has established a connection between the development of
bladder cancer and chronic arsenic exposure. Acute effects of arsenical exposure
have been well characterized with immortalized human urothelial cells derived
from the lining of the ureter. In addition, chronic exposure of these immortalized
cells to arsenicals has led to their malignant transformation. In this study, primary
human bladder epithelial (HBE) cells were acquired commercially and maintained
in a proprietary culture media designed to delay differentiation and senescence. A
similar sensitivity to acute exposure of sodium arsenite and monomethylarsenous
acid [MMA(III)], a highly toxic metabolite of arsenite, was observed when compared
to immortalized urothelial cells (UROtsa). Chronic exposure of HBE to
MMA(III) over a period of one year did not lead to a malignant transformation.
HBE do express functional p53. With acute (3 weeks) exposure to 50 nM
MMA(III), p53 and p21 expression is induced in response to DNA damage measured
by the comet assay. To emulate the effects of the oncogenic alterations observed
in UROtsa, HBE cells were transfected with a HPV E6 oncogene. The
HBE(E6) cells demonstrated attenuation of p53 responses to etoposide and these
modified cells have not shown anchorage-independent growth. This model will be
subjected to chronic arsenic exposure and will hopefully serve to illustrate the important
role of a dysfunctional p53 in arsenic carcinogenesis.
1978 IN VIVO CONSEQUENCES OF ARSENIC
METHYLATION IN A DROSOPHILA MODEL.
I. Cartwright 1 , J. Muñiz Ortiz 2 and J. Shang 1 . 1 Molecular Genetics, University of
Cincinnati College of Medicine, Cincinnati, OH and 2 NHEERL, EPA, Research
Triangle Park, NC.
Metabolism of inorganic arsenic (As) into methylated derivatives by vertebrates was
originally presumed to be a detoxification that facilitates export of As from the cell.
However, recent analyses revealed that methylation may be a critical step underlying
the toxicity of low level, chronic inorganic As exposure, e.g., in the development
of various organ cancers. Drosophila, unlike vertebrates, does not possess
As(III) methyltransferase (AS3MT), the enzyme mainly responsible for vertebrate
As methylation. We introduced the human AS3MT gene into flies under conditions
where we can obtain inducible As methylation in vivo. When transgenic flies
induced to express AS3MT were exposed to inorganic arsenite-containing food at
low concentrations (levels that do not produce overt toxicity), chromosomal instability
was significantly enhanced, as determined by a loss of heterozygosity assay,
compared to both wild-type and uninduced transgenic flies similarly exposed.
Interestingly, when such transgenic flies were fed arsenite at significantly higher
concentrations, enough to produce overt, quantifiable toxic effects on viability, we
found that induction of AS3MT led to a significantly increased lifespan as compared
to uninduced or non-transgenic wild type flies similarly exposed. Together,
these experiments shed interesting light on the benefits, or otherwise, to an organism
of As methylation. During acute exposure the capacity to methylate As is beneficial,
presumably because it facilitates export, thereby lowering the cellular arsenic
burden and promoting viability, at least in the short term. By comparison, chronic
low-level As exposure does not precipitate immediate life-threatening effects, but
methylation of As clearly promotes chromosome instability, which may, over time,
predispose to cancer. Teasing out the molecular pathways intersected by As in these
situations should be highly facilitated by pursuing the type of genetic analysis for
which Drosophila is so renowned.
1979 ASSOCIATION OF ARSENIC EXPOSURE AND GENETIC
POLYMORPHISMS WITH ARSENIC METABOLISM IN A
VIETNAMESE POPULATION.
T. Agusa 1, 2 , T. Kunito 3 , J. Fujihara 1 , H. Takeshita 1 , T. B. Minh 4 , P. K. Trang 4 ,
P. H. Viet 4 , S. Tanabe 2 and H. Iwata 2 . 1 Shimane University Faculty of Medicine,
Izumo, Japan, 2 Ehime University, Matsuyama, Japan, 3 Shinshu University,
Matsumoto, Japan and 4 Vietnam National University, Hanoi, Viet Nam.
The present study investigated the arsenic (As) exposure and association of genetic
polymorphisms in glutathione S-transferase (GST) family (GSTO1, O2, M1, P1,
and T1) and arsenic (+3 oxidation state) methyltransferase (AS3MT) with As metabolism
in 190 subjects from the As-contaminated groundwater areas in the Red
River Delta, Vietnam. Concentrations of arsenite (As[III]), monomethylarsonic
acid (MMA), and dimethylarsinic acid (DMA) in human urine were positively correlated
with total As levels in the groundwater, suggesting that people living in these
areas may be exposed to As through the groundwater. The concentration ratio of
urinary DMA/MMA, which is an indicator of second methylation capacity, increased
with the urinary As level. The wild type of GSTM1, hetero types of
GSTO1 Glu155del and GSTP1 Ile105Val, and variant homo type of GSTO2
Asn142Asp had high MMA/inorganic As (IA; As[III] + arsenate (As[V])) ratios.
Higher DMA/MMA was found in the wild types of AS3MT Met287Thr and
GSTM1. The concentration ratio of As[III]/As[V] in the urine of the hetero type of
GSTP1 Ile105Val and the wild type of GSTM1 was significantly higher than that
of other genotypes in each gene. Interestingly, a positive correlation between
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