Metabolism as a Critical Aspect of the Actions of Arsenic as a Toxin and Carcinogen

1
Metabolism as a Critical Aspect of the Actions of
Arsenic as a Toxin and Carcinogen D. J. Thomas1,
M.F. Hughes1, B. Adair1, E.M. Kenyon1, J.T.
Creed2, M. Styblo3, S.B. Waters3, Z. Drobna3, V.
Devesa3 1U.S. Environmental Protection Agency,
Office of Research and Development, NHEERL 2U.S.
EPA, ORD, NERL, 3University of North Carolina,
Chapel Hill, NC
References
Results/Conclusions
Impact and Outcomes
3 Purification of an enzyme from rat liver that
catalyses the methylation of arsenic allows the
isolation and cloning of a gene that encodes this
enzyme. The gene and its product are now
designated Arsenic (3 Oxidation State)
Methyltransferase (AS3MT) trivalent arsenic.
Studies show that AS3MT catalyses both the
transfer of methyl groups to arsenicals and the
reduction of pentavalent arsenic to trivalency.
Thus, the enzyme functions both as a
methyltransferase and as an arsenate reductase.
Li. J., Waters, S.B., Drobna, Z., Devesa,
V., Styblo, M., and Thomas, D.J. Arsenic (3
oxidation state) methyltransferase and the
inorganic arsenic methylation phenotype.
Accepted. Toxicology and Applied Pharmacology.
Drobná, Z., Waters, S.B., Devesa, V., Harmon,
A.W., Thomas, D.J., Stýblo, M. Metabolism and
toxicity of arsenic in human urothelial cells
expressing rat arsenic (3 oxidation
state)-methyltransferase. Accepted. Toxicology
and Applied Pharmacology Waters S.B, Devesa,
V., Fricke, M., Creed, J., Styblo, M., and,
Thomas, D.J. Glutathione modulates recombinant
rat arsenic (3 oxidation state)
methyltransferase-catalyzed formation of
trimethylarsine oxide and trimethylarsine. Chem.
Res. Toxicol., web release date 11/11/04.
Devesa, V., Del Razo, L.M., Adair B. Drobna, Z.,
Waters S.B., Hughes, M.F, Styblo, M., and,
Thomas, D.J. Comprehensive analysis of arsenic
metabolites by pH-specific hydride generation
atomic absorption spectrometry. J. Anal. At.
Spectrom. 19, 1460 - 1467, 2004. Drobna, Z.,
Waters, S.B., Walton, F.S., LeCluyse, E.L.,
Thomas, D.J., and Styblo, M. Interindividual
variation in the metabolism of arsenic in
cultured primary human hepatocytes. Toxicol.
Appl. Pharmacol. 201166-177, 2004. Thomas,
D.J., Waters, S.B., and Styblo, M. Elucidating
the pathway for arsenic methylation. Toxicol.
Appl. Pharmacol. 198319-326, 2004. Waters,
S.B., Styblo, M., and Thomas, D.J., Endogenous
reductants support the catalytic function of
recombinant rat cyt19, an arsenic
methyltransferase. Chem. Res. Toxicol. 17
404-409, 2004. Drobna, Z., Jaspers, I.,
Thomas, D.J. and Styblo, M., Differential
activation of AP-1 in human bladder epithelial
cells by inorganic and methylated arsenicals.
FASEB Journal 17 67-69, 2003. Lin, S., Shi,
Q., Nix, F.B., Styblo, M., Beck, M.A.,
Herbin-Davis, K.M., Hall, L.L., Simeonsson, J.B.,
and Thomas, D.J. A novel S-adenosyl-l-methionine
arsenic(III) methyltransferase from rat liver
cytosol. J. Biol. Chem. 277 10795-10803, 2002.
Simeonsson, J.B., Elwood, S.A., Ezer, M.,
Pacquette, H.L., Swart, D.J., Beach, H.D., and
Thomas, D.J. Development of ultratrace
analytical techniques for arsenic measurements.
Talanta.58 189-199, 2002. Thomas, D.J.,
Styblo, M., and Lin, S. The cellular metabolism
and systemic toxicity of arsenic. Toxicol. Appl.
Pharmacol. 176 127-144, 2001.
The ORD research effort is linked with efforts at
other federal and non-governmental institutions.
ORD scientists actively collaborate with
colleagues to provide insights from their
research to aid in the design and conduct of
research on the metabolism of arsenic and its
roles in the toxicity and carcinogenicity of
arsenic. ORD scientists serve as a resource
working with various regulatory offices in EPA to
provide expertise on issues related to the risk
assessment for inorganic arsenic and for
methylated arsenical pesticides. To date, this
work has shown the feasibility of identifying and
quantifying of a wide range of arsenic-containing
metabolites in biological samples, including
tissue and urine. These data on the occurrence
of arsenicals in tissues improve the internal
dosimetry and contribute to better understanding
of dose-response relationships. Identification
of new metabolites of inorganic arsenic also
contributes to the design and conduct of studies
of the modes of action of arsenicals which may
underline its actions as a toxin and carcinogen.
Science Question
Although epidemiological evidence associates
chronic exposure to inorganic arsenic with
increased risk of cancer and degenerative
diseases, the modes of action of arsenic as a
toxin or carcinogen are not well understood. A
growing body of evidence suggests that many of
the adverse health effects associated with
chronic exposure to inorganic arsenic are in fact
caused by methylated metabolites which are formed
from inorganic arsenic. Two general questions
can be framed for these metabolites. First, how
is inorganic arsenic converted to methylated
products? Second, can the formarion of these
metabolites be linked to specific adverse
effects?
Research Goal
This research effort is divided into two
complementary projects. The first project has
elucidated the enzymatic basis for conversion of
inorganic arsenic into methylated products. The
second project has focused on development and
refinement of analytical techniques for
quantitation of arsenicals with a particular goal
of developing methods to identify the oxidation
state of arsenic in various metabolites.
1 Scheme for the effects of methylated
arsenicals that contain trivalent arsenic. The
generation of arsenicals containing trivalent
arsenic is an activation step producing reactive
species that disrupt a variety of cellular
processes related to redox status of cells and to
cell death or proliferation.
Future Directions
4 - AS3MT originally cloned from rat liver has
been found to be a prototype for arsenic
methyltransferase found in many mammalian
species. Notably, orthologous genes in the mouse
and human genomes encode very similar proteins
which have been cloned and expressed and shown to
be arsenic methyltransferases.
Arsines generated
Methods/Approach
Arsines detected quantified
Cuvette
Integrating processes to understand fate of
arsenicals in cells Use our understanding of
metabolism to identify critical protein targets
involved in mediating the effects of arsenicals..
Arsines separated
Hydride generation atomic absorption
spectrometry combined with pH selective
generation (pH 1 and 6) allows differentiation of
AsIII- and AsV-containing species. Refinement of
conditions for assay allows detection and
quantitation of inorganic arsenicals, methyl
arsenicals, dimethylated arsenicals and
trimethylated arsenicals in biological samples.
Complementary lines of research are undertaken to
answer the two science questions posed above.
The development of improved analytical techniques
able to differentiate arsenicals by the oxidation
state of arsenic and by methylation status makes
it possible to elucidate all the steps in the
methylation process.
5 Understanding the function of AS3MT and
developing better analytical tools has permitted
identification of each metabolite in the scheme
that leads from inorganic arsenic to
trimethylarsine, a volatile arsine.
Identify new metabolites in known pathways (e.g.,
sulfides formed from methylated arsenicals) and
elucidate other pathways to methylated arsenicals
(e.g., from the degradation of arsenosugars).