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Title: Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes

Abstract

The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenicIII takes place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams Medium E with various concentrations of arsenicIII (sodium m-arsenite). Aliquots of the cell suspension were lysed with 1.0% Triton X-100 and analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMAIII from sodium arsenite (1 ?M) was linear with respect to time for >90 min. DMAIII formation did not become significant until 60 min. MMAV and DMAV were not consistently observed in the incubations. These results suggest that the reduction of MMAV to MMAIII is rapid relative to the methylation rate since MMAV was not observed as a major product of arsenicIII metabolism in mouse hepatocytes. Metabolism of arsenicV was not observed in mouse hepatocytes, consistent with inhibition of arsenicV active cellular uptake by phosphate in the medium. The formation of MMAIII increased with increasing arsenicIII concentrations up to approximately 2 ?Mmore » and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by substrate inhibition of the reaction by arsenicIII. Kinetic analysis of the data suggested an apparent KM of approximately 3.6 ?M arsenicIII, an apparent Vmax of approximately 38.9 ?g MMAIII formed/L/hr/million cells, and an apparent KI of approximately 1.3 ?M arsenicIII. The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMAIII in liver and other tissues.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
887365
Report Number(s):
PNWD-SA-7249
Journal ID: ISSN 0009-2797; CBINA8; TRN: US200618%%13
DOE Contract Number:
AC05-76RL01830
Resource Type:
Journal Article
Resource Relation:
Journal Name: Chemico-Biological Interactions; Journal Volume: 161; Journal Issue: 2
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ARSENIC; BIOCHEMICAL REACTION KINETICS; LIVER CELLS; MALES; METABOLISM; METHYLATION; MICE; SPECTROSCOPY

Citation Formats

Kedderis, Gregory L., Elmore, Amy R., Crecelius, Eric A., Yager, Janice W., and Goldsworthy, Thomas L. Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes. United States: N. p., 2006. Web. doi:10.1016/j.cbi.2006.04.001.
Kedderis, Gregory L., Elmore, Amy R., Crecelius, Eric A., Yager, Janice W., & Goldsworthy, Thomas L. Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes. United States. doi:10.1016/j.cbi.2006.04.001.
Kedderis, Gregory L., Elmore, Amy R., Crecelius, Eric A., Yager, Janice W., and Goldsworthy, Thomas L. Sat . "Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes". United States. doi:10.1016/j.cbi.2006.04.001.
@article{osti_887365,
title = {Kinetics of Arsenic Methylation by Freshly Isolated B6C3F1 Mouse Hepatocytes},
author = {Kedderis, Gregory L. and Elmore, Amy R. and Crecelius, Eric A. and Yager, Janice W. and Goldsworthy, Thomas L.},
abstractNote = {The toxic and carcinogenic effects of arsenic may be mediated by both inorganic and methylated arsenic species. The methylation of arsenicIII takes place via sequential oxidative methylation and reduction steps to form monomethylarsenic (MMA) and dimethylarsenic (DMA) species. The kinetics of arsenic methylation were determined in freshly isolated hepatocytes from male B6C3F1 mice. Hepatocytes (>90% viability) were isolated by collagenase perfusion and suspended in Williams Medium E with various concentrations of arsenicIII (sodium m-arsenite). Aliquots of the cell suspension were lysed with 1.0% Triton X-100 and analyzed for arsenic species by hydride generation-atomic absorption spectrometry. The formation of MMAIII from sodium arsenite (1 ?M) was linear with respect to time for >90 min. DMAIII formation did not become significant until 60 min. MMAV and DMAV were not consistently observed in the incubations. These results suggest that the reduction of MMAV to MMAIII is rapid relative to the methylation rate since MMAV was not observed as a major product of arsenicIII metabolism in mouse hepatocytes. Metabolism of arsenicV was not observed in mouse hepatocytes, consistent with inhibition of arsenicV active cellular uptake by phosphate in the medium. The formation of MMAIII increased with increasing arsenicIII concentrations up to approximately 2 ?M and declined thereafter. The concentration dependence is consistent with a saturable methylation reaction accompanied by substrate inhibition of the reaction by arsenicIII. Kinetic analysis of the data suggested an apparent KM of approximately 3.6 ?M arsenicIII, an apparent Vmax of approximately 38.9 ?g MMAIII formed/L/hr/million cells, and an apparent KI of approximately 1.3 ?M arsenicIII. The results of this study can be used in the physiologically based pharmacokinetic model for arsenic disposition in mice to predict the concentration of MMAIII in liver and other tissues.},
doi = {10.1016/j.cbi.2006.04.001},
journal = {Chemico-Biological Interactions},
number = 2,
volume = 161,
place = {United States},
year = {Sat Jun 10 00:00:00 EDT 2006},
month = {Sat Jun 10 00:00:00 EDT 2006}
}
  • L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increasesmore » in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.« less
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