Human Liver Epithelial Cells

A Decrease in Cell Viability by Exposure to MIT

Methylisothiazolinone (MIT) is widely used in various fields due to its low toxicity and ability to inhibit microbial proliferation. However, increasing evidence suggests that MIT may lead to lung diseases and has been banned from use in humidifier disinfectants, with its toxicity mechanisms remaining unclear. Given that MIT is often used with methylchloroisothiazolinone (MCIT) under the trade name of Kathons, and that intravenously injected Kathons first distributes in the blood and then accumulates in the liver, Park et al. sought to explore the toxicity mechanisms of MIT in human liver epithelial cells. They found that MIT might induce multiple pathways of cell death and inflammatory responses through DNA damage caused by nuclear membrane rupture.

To set cell number and dose-range, Park et al. compared the toxicity of MIT in BEAS-2B (bronchial epithelium), HACAT (keratinocytes) and Chang (liver epithelium) cells which are considered as main target organs of toxicants. They found that the cytotoxic levels depend on the cell number (Fig. 1) and rapidly decrease between 15.625 and 31.25 μg/mL. Based on these results, they chose the highest concentration of 20 μg/mL and the cell number of 2 × 10⁴ cells/mL for viability testing of Chang cells. At exposure of 24 h, the viability of cells exposed with 2.5, 5, 10, and 20 μg/mL of dose was 94.8± 10.7%, 85.2± 16.8%, 84.3±9.2%, and 52.7± 5.5% of control (100.0 ± 8.7%), respectively (Fig. 2).

Fig. 1. Comparison of cell viability among cell types by exposure to MIT (Park, E. J., Kim, S., et al., 2018).

Fig. 2. A decrease of cell viability by exposure to MIT (Park, E. J., Kim, S., et al., 2018).

Quantification of Liver Epithelial Cell IFN-λ Production in Response to Lipoplex Treatment

The immune system has always been one of the most challenging barriers to overcome in nanoparticle-mediated delivery processes. Type III interferons (IFN-λ1: IL-29, IFN-λ2/3: IL-28A/B) are a family of antiviral cytokines responsible for regulating the functions of epithelial and endothelial cell barriers, supplementing type I interferon responses. Some studies have found that the immune response of IFN-λ is relatively unique compared to existing responses and may not adversely affect the pharmacokinetics and efficacy of nanomedicines. However, there are no studies on the interaction between IFN-λ and nanomedicine delivery. Therefore, this study describes the impact of type III interferons (IFN-λ) on the response of lipid nanoparticles complexed with nucleic acids (lipoplexes), suggesting that IFN-λ pretreatment can enhance the efficacy of chemotherapeutic nanomedicines.

The authors first tested whether human microvascular endothelial cells and/or hepatic epithelial cells respond to IFN-λ by the expression of interferon lambda receptor 1 (IFNLR1). The results showed that the expression levels of IFNLR1 in human hepatic epithelial cells significantly increased after IFN-λ1 or IFN-λ2/3 treatment. This indicates that human hepatic epithelial cells respond to IFN-λ. Meanwhile, the authors also tested whether these cells could produce IFN-λ under liposome treatment. Accordingly, human primary liver epithelial cells were treated with varying amounts of lipoplexes for 24 h and cell media was then collected for determination of IFN-A levels by ELISA. Media from liver epithelial cells treated with 1, 0.5, or 0.25 μg/mL lipoplexes showed an average IFN-λ level of 49.57, 33.52, and 22.42 pg/mL 24 h after treatment, respectively (Fig. 3). However, this latter value is below the lowest concentration that can be reliably quantified by the ELISA (LLoQ = 31.30 pg/mL).

Fig. 3. IFN-λ produced by liver epithelial cells in response to lipoplex exposure (Tilden, S. G., Ricco, M. H., et al., 2024).