PURPOSE:To develop a metabolically competent, human immortalized corneal epithelial cell line for use in toxicity and inflammation studies.METHODS:Primary corneal epithelial cells (P-CEPI) were immortalized by a recombinant simian virus (SV)40 T antigen retroviral vector defective for viral replication. The cells were grown in serum-free medium with the addition of bovine pituitary extract, cloned at passage 15 and one of the best-growing clones, CEPI-17-CL4, was extensively characterized for differentiation and metabolic characteristics of the human corneal epithelium. Methods used were immunostaining, reverse transcription-polymerase chain reaction (RT-PCR), northern blot analysis, and enzyme assays.RESULTS:The CEPI-17-CL4 cells showed a typical cobblestone morphology, grew to more than 200 passages and expressed the SV40 T antigen in the nucleus of every cell. Immunofluorescence staining for CEPI-17-CL4 cells was strongly positive for keratins (K)8, K18, and K19 and vimentin; weakly positive for K3, K13, and K17; and negative for K4, K7, and K14. Expression of cytokines (interleukin [IL]-1alpha, IL-1beta, IL-6, IL-8, tumor necrosis factor-alpha, and IL-ra), growth factors (transforming growth factor [TGF]-alpha, epidermal growth factors [EGF], EGF receptor [EGFR], TGF-beta1, TGF-beta2, and platelet-derived growth factor-beta) and cytochrome P450 enzymes (1A1, 2C, 2E1, and 3A5) was similar in CEPI-17-CL4 cells and human corneal epithelial samples obtained in biopsy. The CEPI-17-CL4 cells were metabolically competent for enzymes glutathione S-transferase, quinone reductase, aflatoxin aldehyde reductase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase.CONCLUSIONS:The CEPI-17-CL4 cells are truly immortal and express an extensive array of cytokines, growth factors, and metabolic enzymes that resemble the original tissue. These characteristics, which remain stable up to high passage, will allow reproducible, mechanistic studies on toxicity, inflammation, and wound healing.

The production of DNA strand breaks by N-nitrosodimethylamine (NDMA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been observed in T5-2E1 (expressing human CYP2E1) and T5-1A2 (expressing human CYP1A2) human liver cells respectively, using the Comet assay. Responses were statistically significant (P < 0.05) and concentration dependent (0.01–1 μg ml−1 NDMA and 0.1–10 μg ml−1 IQ) and were not observed in T5-neo cells devoid of cytochrome P450 activity. Sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) (0.1–10 μM) gave a marked inhibition of DNA strand breakage by these carcinogens (P < 0.05 linear regression). This was seen in the absence of cytotoxicity and in the absence of an inhibition of H2O2-induced DNA strand breakage. The ability of sulforaphane to inhibit both CYP2E1 and CYP1A2-mediated genotoxicity therefore is relevant to human isoforms of these enzymes and may contribute to a chemopreventative activity.

Summary— This report characterized one of the major cytochrome P450 isozyme involved in thiabendazole metabolism. This study was undertaken by using both cultured rabbit hepatocytes treated or not with drugs known to specifically induce various cytochromes P450 isoenzymes (ie, P450 1A1/2 by β‐naphthoflavone, P450 2B4 by phenobarbital, P450 3A6 by rifampicine and P450 4A by clofibrate) and human liver (THLE‐5) and bronchial (BEAS‐2B) epithelial cells expressing or not the major constitutive human cytochromes P450 (ie, CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 or 3A4). Only hepatocytes exposed to β‐naphthoflavone and clofibrate significantly metabolized thiabendazole to 5‐hydroxythiabendazole. Extensive biotransformation of this anthelmintic only occurred in human cells expressing CYP1A2. Moreover, experiments performed on rabbit preparations showed good correlations between thiabendazole 5‐hydroxylase activity and both ethoxyresorufin and methoxyresorufin O‐dealkylase activities. Thus, CYP1A2 is a major isoenzyme involved in thiabendazole 5‐hydroxylation.

The human respiratory epithelium is in direct contact with chemical carcinogens and toxins in inhaled air. Therefore, the activities of xenobiotic-metabolising enzymes in this epithelium could modulate respiratory toxicity and carcinogenesis. We determined the expression of several xenobiotic-metabolising enzymes, including phase I and phase II enzymes, in human bronchial mucosa and peripheral lung tissues. Reverse transcription–polymerase chain reaction (RT–PCR) analysis of phase I enzymes showed CYP1A1 and CYP2C (CYP2C8 and CYP2C18) mRNA expression in all of the 14 bronchial mucosa specimens. CYP2A6 and CYP2B6 mRNAs were found in 85% of the samples, whereas 50 and 90% of the tissues displayed CYP2E1 and CYP3A5 expression, respectively. However, CYP1A2, CYP2D6 and CYP3A4 mRNAs were not detected in all samples analysed. Normal human bronchial epithelial cells (NHBE cells) cultured in serum-free conditions showed reduced P450 expression in comparison with the bronchial mucosal samples. Similar to the bronchial mucosa, the peripheral lung tissues expressed CYP1A1, CYP2A6, CYP2B6, CYP2C (CYP2C8 and CYP2C18), CYP2E1 and CYP3A5 mRNAs, but did not show detectable levels of CYP2D6. Additional P450s, such as CYP1A2 and CYP3A4, were detected. The expression of CYP1A1, CYP1A2, CYP2B6, CYP2E1 and CYP3A4/5 in peripheral lung tissues was confirmed at the protein level, whereas CYP2A6 protein was undetectable. The use of specific primers for the detection of the phase II isoenzymes belonging to the glutathione S-transferase mu (GSTμ) and N-acetyl transferase (NAT) families showed that GSTM1 was expressed in 40% of the bronchial mucosa and 25% of the peripheral lung tissues, whereas GSTM3 and NAT1 mRNAs were found in all bronchial and lung samples. Finally, NAT2 expression was detected in all peripheral lung tissues, but was not detected in the bronchus. In conclusion, these results describing the diversity of the xenobiotic-metabolising enzymes expressed in the bronchus and lung tissues indicate that the human respiratory system could significantly and specifically contribute to the activation and metabolism of several environmental procarcinogens.

Many natural dietary phytochemicals found compounds found in fruits, vegetables, spices and tea have been shown in recent years to be protective against cancer in various animal models. In the light of the potential impact of these compounds on human health it is important to elucidate the mechanisms involved. We therefore developed and characterized relevant in vitro models using immortalized human epithelial cell lines derived from target tissues in carcinogenesis, such as lung, liver and colon. Assays were established, allowing the evaluation of the cytotoxic and genotoxic effects of various procarcinogens, including nitrosamines, mycotoxins and heterocyclic amines on these metabolically-competent human epithelial cell lines. These cellular models appeared to be a useful tool to study the capacity of certain food components to block the initiation stage of carcinogenesis. The ability of carnosol and carnosic acid from rosemary as well as the synthetic dithiolethione, oltipraz, to block the formation of DNA adducts, and their effects on the expression of phase I and phase II enzymes was investigated. We have observed that both rosemary extracts and oltipraz inhibited benzo(a)pyrene- or aflatoxin B,-induced DNA adduct formation by strongly inhibiting CYP45° activities and inducing the expression of glutathione S-transferase. These results in human cell models give some insight into the different mechanisms involved in the chemopreventive action of both natural and synthetic compounds in relation to phase I and phase II enzymes.

A panel of human bronchial epithelial (BEAS-2B) cell lines stably expressing one single CYP450 cDNA (CYP2A6, 2B6, 2D6, 2E1, 3A4, and 3A5) was developed by liposome-mediated transfection. The different cell lines showed high levels of CYP450 expression as determined by Western blot analysis. These genetically engineered cell lines were applied to evaluate the specificity of fluorescent substrates, such as 7-alkoxycoumarins, coumarins, and 7-alkroxyresorufins, in the analysis of human CYP450 catalytic activities. Additionally, the activation of procarcinogens (i.e., dimethylnitrosamine and aflatoxin B-1) to cytotoxic and genotoxic compounds was taken as reference to illustrate the potential of this system for pharmacotoxicologic applications.

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first and second position of the codon 250. No significant AFB1-induced p53 mutations could be detected in the T5-2A6 cells. Therefore, the differential expression of specific CYP450 genes in human hepatocytes can modulate the cytotoxicity, DNA adduct levels and frequency of p53 mutations produced by AFB1.

Human liver tissue slices, homogenates, organ cultures, subcellular fractions and primary hepatocyte cultures represent valuable tools for analysis of metabolism, toxicity and efficacy of xenobiotics. However, the limited access to human tissue and the high test variability with different tissue isolates underline the significance of having standardized and defined human liver cell models. This report describes the establishment of replicative cultures of human liver epithelial cells and their immortalization in serum-free medium. Highly efficient immortalization is achieved by the infection of primary liver cells with a retroviral recombinant carrying the simian virus 40 (SV40) large T antigen gene. These cultures have an indefinite life-span, are non-tumorigenic and express differentiation markers and metabolic functions of normal primary hepatocytes. Therefore, these cells are useful for various pharmaco-toxicological applications.

Normal human bronchial epithelial (NHBE) cells are the putative progenitor cells of all types of lung cancer. NHBE cells immortalized by SV40 T‐antigen retain many characteristics of the primary cells and are a useful model for investigating the role of oncogenes, tumor suppressor genes, and certain chemical carcinogens in the molecular pathogenesis of lung cancer. In this study, SV40 T‐antigen‐positive cells (BEAS‐2B) were characterized for their metabolic functions and were shown to continue to express epoxide hydrolase, glutathione S ‐transferrase π, glutathione peroxidase, and catalase. To increase their metabolic activity towards human procarcinogens, human cytochrome P450 1A2 (CYP1A2) was stably expressed by introducing CYP1A2 cDNA into BEAS‐2B cells either by infection with a high‐titer recombinant retrovirus (pXT‐1A2) or by transfection with a CYP1A2 expression vector (pCMV1A2), which produced the cell lines B‐1A2 and B‐CMV1A2, and B‐CMV1A2, respectively. Cell lines established with either expression system expressed enzymatically active CYP1A2 protein and were 50‐ to 400‐fold more sensitive to the cytotoxic effect of the carcinogen aflatoxin B 1 ) (AFB 1 ) than the corresponding control cell lines. The cytotoxic effects of AFB 1 were paralleled by increased metabolism of AFB 1 and enhanced formation of the AFB 1 ‐N 7 guanine adduct in B‐CMV1A2 cells. Cytotoxicity and adduct formation correlated with a significantly higher protein expression of CYP1A2 by the cytomegalovirus promoter‐driven plasmid. Since this human epithelial cell line is the precursor cell type of lung cancer, has normal phase II enzymes, and exhibits highly reproducible expression of phase l enzymes, this in vitro model should aid in the evaluation of putative human carcinogens and anticarcinogens. ©1994 Wiley‐Liss, Inc.