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Angiogenic potential of 3-nitro-4-hydroxy benzene arsonic acid (roxarsone).

Basu P, Ghosh RN, Grove LE, Klei L, Barchowsky A - Environ. Health Perspect. (2008)

Bottom Line: Roxarsone was found to exhibit a higher angiogenic index than As(III) at lower concentrations.Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression.The results demonstrate that roxarsone and As(III) promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Duquesne University, Pittsburgh, Pennsylvania 15282, USA. basu@duq.edu

ABSTRACT

Background: Roxarsone (3-nitro-4-hydroxy benzene arsonic acid) is an arsenic compound widely used in the poultry industry as a feed additive to prevent coccidiosis, stimulate growth, and to improve tissue pigmentation. Little is known about the potential human health effects from roxarsone released into the environment from chicken waste or from residual compound in chicken products.

Objective: The growth potentiation and enhanced tissue pigmentation suggest that low levels of roxarsone exposure may have an angiogenic potential similar to that of inorganic arsenite (As(III)). The goal of this investigation was to test the hypothesis described above using cultured human aortic and lung microvascular endothelial cells in high-content imaging tube-forming assays and begin developing a molecular level understanding of the process.

Methods: We used a three-dimensional Matrigel assay for probing angiogenesis in cultured human endothelial cells, and a polymerase chain reaction (PCR) array to probe the gene changes as a function of roxarsone or As(III) treatment. In addition, we used Western blot analysis for changes in protein concentration and activation.

Results: Roxarsone was found to exhibit a higher angiogenic index than As(III) at lower concentrations. Increased endothelial nitric oxide synthase (eNOS) activity was observed for roxarsone but not for As(III)-induced angiogenesis. However, As(III) caused more rapid and pronounced phosphorylation of eNOS. Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression.

Conclusions: The results demonstrate that roxarsone and As(III) promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.

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Related in: MedlinePlus

Effects of L-NAME (A ) and D-NAME (B ) on angiogenic index. HAEC were treated as follows: (a) no treatment, (b) a mixture of growth factors as a positive control, (c) roxarsone (0.1 μM), and (d) AsIII (0.1 μM). Open bars represent cells with no NAME; blue bars represent cells treated with different isomers of NAME (50 μM). In each case, 12 wells were investigated, and each well was imaged at five different fields. Angiogenic index ± SD is plotted in each case.*p < 0.05 and ***p < 0.001 determined from two-way ANOVA analysis, followed by the Bonferroni post-test.
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f3-ehp0116-000520: Effects of L-NAME (A ) and D-NAME (B ) on angiogenic index. HAEC were treated as follows: (a) no treatment, (b) a mixture of growth factors as a positive control, (c) roxarsone (0.1 μM), and (d) AsIII (0.1 μM). Open bars represent cells with no NAME; blue bars represent cells treated with different isomers of NAME (50 μM). In each case, 12 wells were investigated, and each well was imaged at five different fields. Angiogenic index ± SD is plotted in each case.*p < 0.05 and ***p < 0.001 determined from two-way ANOVA analysis, followed by the Bonferroni post-test.

Mentions: Nitric oxide (NO) production is required for certain stimuli to promote angiogenesis (Yu et al. 2005), but AsIII is known to promote reactive oxygen that limits available NO release in endothelial cells (Barchowsky et al. 1999a, Bunderson et al. 2006). The data in Figure 3 demonstrate that roxarsone requires NO generation to increase angiogenesis. Its effects were blocked by the eNOS inhibitor l-NG-nitroarginine methylester (L-NAME), but not its inactive enantiomer d -NG-nitroarginine methylester (D-NAME). In contrast, both NAME enantiomers prevented AsIII from increasing the angiogenic index. This would suggest either that AsIII inhibition by NAME was a nonstereospecific chemical effect or that D-NAME interacted with AsIII in a unique manner relative to roxarsone. Thus, roxarsone and inorganic AsIII appeared to have differential effects on signaling for NO-mediated angiogenesis.


Angiogenic potential of 3-nitro-4-hydroxy benzene arsonic acid (roxarsone).

Basu P, Ghosh RN, Grove LE, Klei L, Barchowsky A - Environ. Health Perspect. (2008)

Effects of L-NAME (A ) and D-NAME (B ) on angiogenic index. HAEC were treated as follows: (a) no treatment, (b) a mixture of growth factors as a positive control, (c) roxarsone (0.1 μM), and (d) AsIII (0.1 μM). Open bars represent cells with no NAME; blue bars represent cells treated with different isomers of NAME (50 μM). In each case, 12 wells were investigated, and each well was imaged at five different fields. Angiogenic index ± SD is plotted in each case.*p < 0.05 and ***p < 0.001 determined from two-way ANOVA analysis, followed by the Bonferroni post-test.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2290980&req=5

f3-ehp0116-000520: Effects of L-NAME (A ) and D-NAME (B ) on angiogenic index. HAEC were treated as follows: (a) no treatment, (b) a mixture of growth factors as a positive control, (c) roxarsone (0.1 μM), and (d) AsIII (0.1 μM). Open bars represent cells with no NAME; blue bars represent cells treated with different isomers of NAME (50 μM). In each case, 12 wells were investigated, and each well was imaged at five different fields. Angiogenic index ± SD is plotted in each case.*p < 0.05 and ***p < 0.001 determined from two-way ANOVA analysis, followed by the Bonferroni post-test.
Mentions: Nitric oxide (NO) production is required for certain stimuli to promote angiogenesis (Yu et al. 2005), but AsIII is known to promote reactive oxygen that limits available NO release in endothelial cells (Barchowsky et al. 1999a, Bunderson et al. 2006). The data in Figure 3 demonstrate that roxarsone requires NO generation to increase angiogenesis. Its effects were blocked by the eNOS inhibitor l-NG-nitroarginine methylester (L-NAME), but not its inactive enantiomer d -NG-nitroarginine methylester (D-NAME). In contrast, both NAME enantiomers prevented AsIII from increasing the angiogenic index. This would suggest either that AsIII inhibition by NAME was a nonstereospecific chemical effect or that D-NAME interacted with AsIII in a unique manner relative to roxarsone. Thus, roxarsone and inorganic AsIII appeared to have differential effects on signaling for NO-mediated angiogenesis.

Bottom Line: Roxarsone was found to exhibit a higher angiogenic index than As(III) at lower concentrations.Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression.The results demonstrate that roxarsone and As(III) promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Duquesne University, Pittsburgh, Pennsylvania 15282, USA. basu@duq.edu

ABSTRACT

Background: Roxarsone (3-nitro-4-hydroxy benzene arsonic acid) is an arsenic compound widely used in the poultry industry as a feed additive to prevent coccidiosis, stimulate growth, and to improve tissue pigmentation. Little is known about the potential human health effects from roxarsone released into the environment from chicken waste or from residual compound in chicken products.

Objective: The growth potentiation and enhanced tissue pigmentation suggest that low levels of roxarsone exposure may have an angiogenic potential similar to that of inorganic arsenite (As(III)). The goal of this investigation was to test the hypothesis described above using cultured human aortic and lung microvascular endothelial cells in high-content imaging tube-forming assays and begin developing a molecular level understanding of the process.

Methods: We used a three-dimensional Matrigel assay for probing angiogenesis in cultured human endothelial cells, and a polymerase chain reaction (PCR) array to probe the gene changes as a function of roxarsone or As(III) treatment. In addition, we used Western blot analysis for changes in protein concentration and activation.

Results: Roxarsone was found to exhibit a higher angiogenic index than As(III) at lower concentrations. Increased endothelial nitric oxide synthase (eNOS) activity was observed for roxarsone but not for As(III)-induced angiogenesis. However, As(III) caused more rapid and pronounced phosphorylation of eNOS. Quantitative PCR array on select genes revealed that the two compounds have different and often opposite effects on angiogenic gene expression.

Conclusions: The results demonstrate that roxarsone and As(III) promote angiogenic phenotype in human endothelial cells through distinctly different signaling mechanisms.

Show MeSH
Related in: MedlinePlus