Speciation of arsenic metabolites in chicken meat and in human cells

• Author / Creator

  Liu, Qingqing

• A phenylarsenical, 3-nitro-4-hydroxyphenylarsonic acid (Roxarsone®, Rox), has been used extensively as a poultry feed additive for over 60 years. However, little is known about the concentrations of different arsenic species present in chicken meat as a consequence of feeding Rox to chickens. The first objective of my thesis research is to study the speciation of Rox in chicken meat, providing relevant information on human exposure to arsenic through the ingestion of chicken meat. The second objective is to study accumulation, transport, and metabolism of Rox and the related arsenic species using human cells, improving a mechanistic understanding. To quantify arsenic species in chicken meat, I first developed an enzyme-assisted extraction method which enhanced the extraction efficiency of arsenic from 28% using water/methanol extraction to 55% using papain digestion. The efficiency was further enhanced to 88% by using ultrasonication combined with the papain digestion. Arsenic species were separated using high performance liquid chromatography (HPLC). Inductively coupled plasma mass spectrometry (ICPMS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used in combination to quantify and identify arsenic species. The method of papain-assisted extraction and HPLC-ICPMS determination achieved detection limits of 1.0-1.8 µg arsenic per kg chicken breast meat for arsenobetaine (AsB), inorganic arsenite (AsIII), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), inorganic arsenate (AsV), Rox, and N-acetyl-4-hydroxy-m-arsanilic acid (NAHAA). To determine whether the feeding of Rox to chicken leads to the increased concentrations of other arsenic species in chicken breast meat, I studied the arsenic species in the breast meat from chickens raised in a 35-day feeding study. Eight hundred chickens were fed a diet supplemented with Rox and another 800 chickens were fed a control diet. The results from the analyses of 229 chicken samples showed that Rox, AsB, AsIII, MMA, DMA and an unidentified arsenic metabolite (Un) were detectable in breast meat from the Rox-fed chickens. The concentrations of arsenic species, except AsB, were significantly higher in the Rox-fed than the control chickens. The concentrations of AsIII, DMA, MMA, and Un decreased after Rox feeding was stopped. Seven days after termination of Rox feeding, the concentrations of AsIII (3.1 μg/kg), Rox (0.4 μg/kg), and Un (0.8 μg/kg) in the Rox-fed chickens were still significantly higher than the control. To examine whether the bioavailability of Rox is lower than AsIII and AsB, I studied the accumulation and transepithelial transport of arsenic species using the Caco-2 cells. After Caco-2 cells were exposed for 24 h to Rox, AsB, or AsIII, the accumulated Rox in Caco-2 cells was 6-20 times less than AsB and AsIII. The permeability of Rox from the apical to basolateral side of the Caco-2 monolayers was much less than AsIII and AsB. To study the metabolism of Rox in human cells, I treated HepG2 cells and human primary hepatocytes with Rox for 24 h and measured the arsenic metabolites. The results showed that both HepG2 and human hepatocytes could metabolize Rox to 3-amino-4-hydroxylphenlarsonic acid (3AHPAA) and AsV. Human primary hepatocytes also had the ability to metabolize Rox to MMA and NAHAA. A new and previously unknown metabolite was identified as a thiolated Rox. The characterization of this new metabolite was achieved using a newly developed HPLC-ESI-Qudrupole Time-of-Flight (QToF) high-resolution mass spectrometry method. To understand how a detected metabolite NAHAA was formed, I assessed whether N-acetyltransferases were responsible for the acetylation of a precursor, 3AHPAA, to NAHAA. Preliminary results showed that the pigeon liver N-acetyltransferases and chicken liver cytosol could acetyl 3AHPAA to NAHAA. The use of human liver cytosol and human N-acetyltransferase 1 did not produce detectable NAHAA. This dissertation shows speciation of arsenic in chicken meat and in human liver and colon cells, with an emphasis on Rox and its related arsenic species. Analytical methods enabled the sensitive detection of arsenic species and the identification of new arsenic metabolites. Feeding of Rox to chickens increased the concentrations of five arsenic species in breast meat. The quantitative determination of arsenic species in chicken meat provided useful information for assessing human exposure to arsenic from consumption of chicken meat. Studies using human colon and liver cells improved understanding of arsenic transport and metabolism.

• Subjects / Keywords

  • Roxarsone
  • Bioavailability
  • Speciation
  • Mass-spectrometry
  • Chicken
  • Arsenic

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R32B8VS9Z

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.