Zhang S Z, Lipsky M M, Trump B F, Hsu I C
Department of Pathology, University of Maryland, School of Medicine, Baltimore.
Cell Biol Toxicol. 1990 Apr;6(2):219-34. doi: 10.1007/BF00249595.
Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.
培养基中的中性红（NR）被培养的大鼠和人肝细胞的溶酶体吸收并浓缩。NR摄取量随孵育时间增加，2小时后达到平台期。摄取量与NR溶液浓度和活肝细胞数量成正比。肝细胞的长时间培养增加了溶酶体数量，从而增加了染料积累。NR可从溶酶体中提取，用于定量测定肝细胞活力和异生物素的细胞毒性。通过该测定法，比较了几种无血清培养基（如Waymouth's、MEM、LHC-8等）在体外维持活肝细胞的能力。有趣的是，用于培养人支气管上皮细胞的LHC-8培养基能最好地保存活的大鼠肝细胞。通过NR测定法检测了二甲基亚硝胺（DMN）和黄曲霉毒素B1（AFB1）对大鼠和人肝细胞培养物的细胞毒性作用，发现其依赖于暴露剂量和时间。在大鼠肝细胞中，暴露24小时时，DMN的NR50为20 mM，AFB1为0.072 microM；暴露48小时时，DMN降至12.5 mM，AFB1降至0.053 microM。人肝细胞对这两种化学物质的毒性更具抗性；24小时处理时，DMN和AFB1的NR50值分别为100 mM和1.8 microM。与乳酸脱氢酶（LDH）泄漏试验相比，NR测定法在测定肝细胞原代培养物中异生物素的活力和细胞毒性方面更简单、更灵敏。
