Bayse Gladys S, Hammonds-Odie Latanya P, Jackson Kimberly M, Tucker Deidre K, Kirlin Ward G
Department of Chemistry, Spelman College, Atlanta, USA.
Department of Biology, Spelman College, Atlanta, USA.
Adv Biol Chem. 2013 Aug;3(4):389-396. doi: 10.4236/abc.2013.34041.
The benzenearsonate, Roxarsone, has been used since 1944 as an antimicrobial, growth-promoting poultry feed additive. USGS and EPA report that Roxarsone (4-hydroxy-3-nitrobenzenearsonate) and metabolites, including AHBA (3-amino-4-hydroxybenzenearsonate), contaminate waterways at greater than 1100 tons annually. To assess human impact of these organic arsenic water contaminants, it was important to study their potential absorption. The human adenocarcinoma cell line, Caco-2, is a model for intestinal absorption. We found proliferative effects on Caco-2 cells at micromolar levels of these compounds, as monitored by [H]-thymidine incorporation into DNA. Flow cytometry cell cycle analysis confirmed accumulation in S phase from 21% (control) to 36% (24 hour exposure to 10 μM AHBA). Confluent Caco-2 cells grown on collagen-coated Transwell plates were dosed on the apical side. After exposure, media from apical and basolateral sides were collected separately. Following removal of FBS by 30K centrifugal filtration, the benzenearsonates in the collected media were analyzed by HPLC. Analyses were at wavelengths in the ultraviolet/visible range where the absorbance values were linear with respect to concentration. Concentrations were calculated by comparison with analytically-prepared commercial standards. Results from cells dosed at 10 μM for 24 hours with AHBA, Roxarsone, or Acetarsone indicated 6% - 29% permeation occurring from apical to basolateral side, modeling absorption across intestinal epithelium to the circulatory system. Benzenearsonate feed additives are frequently applied in combination with antibiotics, raising additional health concerns. We conclude that micromolar levels of these benzenearsonates are adequate to stimulate Caco-2 cell proliferation.
苯胂酸（洛克沙胂）自1944年起就被用作抗菌、促进生长的家禽饲料添加剂。美国地质调查局和美国环境保护局报告称，洛克沙胂（4-羟基-3-硝基苯胂酸）及其代谢产物，包括AHBA（3-氨基-4-羟基苯胂酸），每年对水道的污染超过1100吨。为了评估这些有机砷类水污染物对人类的影响，研究它们的潜在吸收情况很重要。人腺癌细胞系Caco-2是肠道吸收的模型。我们发现，通过[H]-胸腺嘧啶核苷掺入DNA监测，这些化合物在微摩尔浓度水平对Caco-2细胞有增殖作用。流式细胞术细胞周期分析证实，S期细胞积累从21%（对照）增加到36%（暴露于10μM AHBA 24小时）。在胶原包被的Transwell板上生长的汇合Caco-2细胞从顶端侧给药。暴露后，分别收集顶端侧和基底外侧的培养基。通过30K离心过滤去除胎牛血清后，用高效液相色谱法分析收集的培养基中的苯胂酸盐。分析在紫外/可见光范围内的波长进行，吸光度值与浓度呈线性关系。通过与分析制备的商业标准品比较计算浓度。用AHBA、洛克沙胂或乙酰胂酸以10μM剂量处理细胞24小时的结果表明，从顶端侧到基底外侧的渗透为6% - 29%，模拟了从肠道上皮到循环系统的吸收。苯胂酸饲料添加剂经常与抗生素联合使用，这引发了更多的健康问题。我们得出结论，这些苯胂酸盐的微摩尔浓度足以刺激Caco-2细胞增殖。
