Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation

April Human Normal Bronchial Epithelial Cells: A Novel In Vitro Cell Model for Toxicity Evaluation Wenqiang Feng 0 1 2 3 Juanjuan Guo 0 1 2 3 Haiyan Huang 0 1 2 3 Bo Xia 0 1 2 3 Hongya Liu 0 1 2 3 Jie Li 0 1 2 3 Shaolin Lin 0 1 2 3 Tiyuan Li 0 1 2 3 Jianjun Liu 0 1 2 3 Hui Li 0 1 2 3 0 Funding: This work was supported by the Science and Technology Program of Shenzhen (No. GJHZ20130417155931486; JCYJ20140419141154244; CXZZ20140902162618036). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 1 Data Availability Statement: All relevant data are within the paper 2 Academic Editor: Xuefeng Liu, Georgetown University , UNITED STATES 3 1 State Key Laboratory of Virology, Institute of Medical Virology, Wuhan University School of Basic Medical Sciences , Wuhan, Hubei , China , 2 Shenzhen R&D center of State Key Laboratory of Virology, Wuhan University Shenzhen Institute , Shenzhen, Guangdong , China , 3 Key Laboratory of Modern Toxicology of Shenzhen, Shenzhen Center for Disease Control and Prevention , Shenzhen, Guangdong , China , 4 Shenzhen People's Hospital , Shenzhen, Guangdong , China Human normal cell-based systems are needed for drug discovery and toxicity evaluation. hTERT or viral genes transduced human cells are currently widely used for these studies, while these cells exhibited abnormal differentiation potential or response to biological and chemical signals. In this study, we established human normal bronchial epithelial cells (HNBEC) using a defined primary epithelial cell culture medium without transduction of exogenous genes. This system may involve decreased IL-1 signaling and enhanced Wnt signaling in cells. Our data demonstrated that HNBEC exhibited a normal diploid karyotype. They formed well-defined spheres in matrigel 3D culture while cancer cells (HeLa) formed disorganized aggregates. HNBEC cells possessed a normal cellular response to DNA damage and did not induce tumor formation in vivo by xenograft assays. Importantly, we assessed the potential of these cells in toxicity evaluation of the common occupational toxicants that may affect human respiratory system. Our results demonstrated that HNBEC cells are more sensitive to exposure of 10~20 nm-sized SiO2, Cr(VI) and B(a)P compared to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). This study provides a novel in vitro human cells-based model for toxicity evaluation, may also be facilitating studies in basic cell biology, cancer biology and drug discovery. - Competing Interests: The authors have declared that no competing interests exist. toxicants. Usually ones use spontaneously immortalized cell lines (e.g., HaCaT) or viral genes/ cellular genes immortalized cell lines for these studies[14]. Nanometer silicon dioxide (nano-SiO2) has recently become one of the most popular nanomaterials in many fields, such as industrial manufacturing, engineering and biomedicine. However, nano-SiO2 is easily evaporated into air. It has been shown that inhalation of nano-SiO2 causes pulmonary and cardiovascular alteration and damages in old rats [5] and lung fibrogenesis in rats [6]. Hexavalent chromium (Cr (VI)) is widely used in the manufacture of various industrial products, such as stainless steel. Occupational exposure to Cr (VI) may cause respiratory cancer. Studies on chromate production workers indicate that exposure to Cr (VI) was responsible for an increased relative risk of developing lung cancer [7]. Benzo[a]pyrene (B [a]P) is the common toxicant which is present in car exhaust, incomplete fossil fuel combustion, tobacco smoke and occupational exposures. International Agency for Research of Cancer sets B[a]P as Class 2 group A human carcinogen. B(a)P is a known carcinogen to lung, stomach and skin [8, 9]. All of these three toxicants cause damages or even cancer in lung. Studies have been done to investigate the underlying mechanisms. Skin and lung tissue are the potential primary routes and targets of occupational nano-SiO2 exposure. It has been shown that nano-SiO2 alters the expression of proteins associated with oxidative stress and apoptosis [1] and epigenetic regulation of a cellular repair gene poly(ADP-ribose) polymerases-1(PARP-1) in HaCaT cells [10]. However, HaCaT cells are spontaneously immortalized keratinocytes with an aneuploid chromosome [11] which has the biological properties far from normal. Other studies used cancer cell lines or viral/oncogene immortalized cell lines to investigate the cytotoxicity/genocytoxicity exposed to nano-SiO2, Cr(VI) and B(a)P [24, 12]. Recent studies have demonstrated that exogenous gene-immortalized cells have an altered genetic background/signaling pathways and exhibit an abnormal biological characters [13, 14]. That is the major reason why studies turn out to be controversial results using cancer cells or gene-immortalized cells as models. Recently, murine fibroblastic 3T3 feeder cells derived from mouse embryo and fetal bovine serum (FBS) are used to promote epithelial cell proliferation in vitro [13, 15]. Ideally, these xenogeneic materials from the culture should be eliminated for human cell based studies, especially for the long term regenerative medicine studies. In this study, we established human normal bronchial epithelial cells (HNBEC) from the discarded adjacent normal tissue of lung cancer after surgery from a Chinese patient in Shenzhen China. The short tandem repeats (STR) analysis showed that HNBEC cells have 15 loci and the Y-specific Amelogenin locus that do not match any other cell lines published or registered before. We also verified its normal biological features by karyotype analysis, matrigel 3D culture, DNA damage response and xenograft assays. Moreover, we evaluated its potential applications to toxicological model by common occupational toxicants exposure to lung tissue. Our results demonstrated that HNBEC cells were more sensitive to exposure of 10~20nmsized SiO2, Cr(VI) and B(a)P compare to 16HBE cells (a SV40-immortalized human bronchial epithelial cells). Since HNBEC cells were established without any genetic background changes, the cellular responses to toxicants are real physiological status. HNBEC cells provide a valuable in vitro human cells-based model for toxicological research. Materials and Methods 1. Cell isolation and propagation Adjacent normal lung tissue from a surgical specimen of lung tumor was obtained with the written informed consent of the patient. Institutional review boards at Wuhan University Shenzhen Institute and Shenzhen Peoples Hospital approved this study. To avoid contamination of tumor tissue for the specimen, the normal tissue should be obtained as far from the tumor lesion as possible. Besides, mirror structure of specimen were separated and sent for histology. Uncontaminated sample was delivered to research lab and tissue was minced and dispersed into single cells by digestion with collagenase ( (...truncated)