Early antitumor activity of oral Langerhans cells is compromised by a carcinogen.

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Spermidine Suppresses Oral Carcinogenesis through Autophagy Induction, DNA Damage Repair, and Oxidative Stress Reduction.

Autophagy has been proposed to play a dual role in cancer-as a tumor suppressor in early stages and oncogenic in late stages of tumorigenesis. This study investigated the role of autophagy in oral carcinogenesis using the model of oral squamous cell carcinoma (OSCC) induced by carcinogen 4-nitroquinoline 1-oxide (4NQO), mimicking molecular and histopathologic aspects of human OSCC. The induction of autophagy by spermidine (SPD) treatment reduced the severity of lesions and the incidence of OSCC in mice exposed to 4NQO. On the other hand, autophagy inhibition by chloroquine treatment had no protection. The comet assay indicated that SPD reduced 4NQO-induced DNA damage, likely related to the activation of DNA repair and the decrease of reactive oxygen species. As sphingolipid alterations have been reported in OSCC, sphingolipids in the tongue and plasma of animals were analyzed and plasma C16 ceramide levels were shown to increase proportionally to lesion severity, indicating its potential as a biomarker. Mice exposed to 4NQO plus SPD had lower levels of C16 ceramide than the 4NQO group, which indicated SPD's ability to prevent the 4NQO-induced carcinogenesis. Together, these data indicate that activation of autophagy has a tumor suppressor role during the early stages of oral carcinogenesis. Because of its ability to induce autophagy accompanied by reduced oxidative stress and DNA damage, SPD may have a protective action against chemically induced oral cancer.

BRCA1/Trp53 heterozygosity and replication stress drive esophageal cancer development in a mouse model.

BRCA1 germline mutations are associated with an increased risk of breast and ovarian cancer. Recent findings of others suggest that BRCA1 mutation carriers also bear an increased risk of esophageal and gastric cancer. Here, we employ a Brca1/Trp53 mouse model to show that unresolved replication stress (RS) in BRCA1 heterozygous cells drives esophageal tumorigenesis in a model of the human equivalent. This model employs 4-nitroquinoline-1-oxide (4NQO) as an RS-inducing agent. Upon drinking 4NQO-containing water, Brca1 heterozygous mice formed squamous cell carcinomas of the distal esophagus and forestomach at a much higher frequency and speed (∼90 to 120 d) than did wild-type (WT) mice, which remained largely tumor free. Their esophageal tissue, but not that of WT control mice, revealed evidence of overt RS as reflected by intracellular CHK1 phosphorylation and 53BP1 staining. These Brca1 mutant tumors also revealed higher genome mutation rates than those of control animals; the mutational signature SBS4, which is associated with tobacco-induced tumorigenesis; and a loss of Brca1 heterozygosity (LOH). This uniquely accelerated Brca1 tumor model is also relevant to human esophageal squamous cell carcinoma, an often lethal tumor.

Dietary fat and male sex increase histopathological changes in a mouse model of oral cancer.

OBJECTIVE: To compare the effects of dietary fat and sex on murine oral squamous cell carcinoma pathology. MATERIALS AND METHODS: Male and female C57Bl/6 mice (36/sex) received a low-fat (10 kcal%) or high-fat (60 kcal%) diet. Water (control), vehicle, or 4-nitroquinoline-1-oxide in vehicle (50 µg/ml) was provided for 17 weeks followed by six additional weeks of water. Oral lesion development was recorded weekly. Histopathologic changes in tongues were examined, and T cells (CD3+), macrophages (CD68+), and neutrophils (Ly6+) were quantified. RESULTS: All 4-nitroquinoline-1-oxide-treated mice developed oral tumors. High-fat diet exacerbated pathology, demonstrated by an increased final tumor burden (10.9 ± 4.5 vs. 7.9 ± 2.5, mm/mouse, p < .05; high-fat diet vs. low-fat diet, respectively), and a greater histopathology score. When dietary groups were combined, 4-nitroquinoline-1-oxide-treated males displayed higher histopathology scores than females (4.2 ± 0.3 vs. 3.6 ± 0.2, respectively, p < .05). Lymphoid cell infiltration was greater in the 4-nitroquinoline-1-oxide mouse tongues than controls: T cells (14.0 vs. 0.96 cells/mm2 ), macrophages (3.6 vs. 1.8 cells/mm2 ), and neutrophils (12.0 vs. 0.38 cells/mm2 ). CONCLUSION: High-fat diet and male sex increased the pathology of 4-nitroquinoline-1-oxide-induced oral cancer. Elevated lymphoid cell infiltration contributed to disease pathology.

Dek overexpression in murine epithelia increases overt esophageal squamous cell carcinoma incidence.

Esophageal cancer occurs as either squamous cell carcinoma (ESCC) or adenocarcinoma. ESCCs comprise almost 90% of cases worldwide, and recur with a less than 15% five-year survival rate despite available treatments. The identification of new ESCC drivers and therapeutic targets is critical for improving outcomes. Here we report that expression of the human DEK oncogene is strongly upregulated in esophageal SCC based on data in the cancer genome atlas (TCGA). DEK is a chromatin-associated protein with important roles in several nuclear processes including gene transcription, epigenetics, and DNA repair. Our previous data have utilized a murine knockout model to demonstrate that Dek expression is required for oral and esophageal SCC growth. Also, DEK overexpression in human keratinocytes, the cell of origin for SCC, was sufficient to cause hyperplasia in 3D organotypic raft cultures that mimic human skin, thus linking high DEK expression in keratinocytes to oncogenic phenotypes. However, the role of DEK over-expression in ESCC development remains unknown in human cells or genetic mouse models. To define the consequences of Dek overexpression in vivo, we generated and validated a tetracycline responsive Dek transgenic mouse model referred to as Bi-L-Dek. Dek overexpression was induced in the basal keratinocytes of stratified squamous epithelium by crossing Bi-L-Dek mice to keratin 5 tetracycline transactivator (K5-tTA) mice. Conditional transgene expression was validated in the resulting Bi-L-Dek_K5-tTA mice and was suppressed with doxycycline treatment in the tetracycline-off system. The mice were subjected to an established HNSCC and esophageal carcinogenesis protocol using the chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Dek overexpression stimulated gross esophageal tumor development, when compared to doxycycline treated control mice. Furthermore, high Dek expression caused a trend toward esophageal hyperplasia in 4NQO treated mice. Taken together, these data demonstrate that Dek overexpression in the cell of origin for SCC is sufficient to promote esophageal SCC development in vivo.

Investigation of in vivo unscheduled DNA synthesis in rabbit corneas following instillation of genotoxic agents.

PURPOSE: An unscheduled DNA synthesis (UDS) test is used for in vitro or in vivo genotoxicity evaluation. The UDS test with hepatocytes is well established; however, drug exposure levels at the application site for topically administered drugs (e.g. ophthalmic drugs) often exceed the exposure levels for systemic administration. To establish in vivo genotoxicity on the ocular surface, we performed the UDS test using rabbit corneas from eyes subjected to instillation of genotoxic agents. MATERIALS AND METHODS: Five genotoxic agents - 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat); acridine orange; ethidium bromide; acrylamide; and 4-nitroquinoline 1-oxide (4-NQO) - were instilled once onto both eyes of male Japanese white rabbits. Physiological saline or a general vehicle for ophthalmic solution were instilled as the negative controls. Dimethyl sulfoxide was instilled as the vehicle control. Isolated corneas were incubated with tritium-labelled thymidine and the number of sparsely labelled cells (SLCs, cells undergoing UDS) was counted by autoradiography. RESULTS: Statistically significant increases in the mean appearance rates of SLCs in the corneal epithelium were noted in paraquat-, acridine orange-, ethidium bromide-, and 4-NQO-treated eyes compared with those of the controls. These increases generally appeared in a dose-dependent manner. Acrylamide did not induce an increase in the mean appearance rates of SLCs, presumably because it caused the generation of fewer metabolites in the cornea. CONCLUSIONS: UDS tests revealed DNA damage in the cornea epitheliums treated with well-known genotoxic agents. These results suggest that the UDS test is one of the useful tools for the assessment of in vivo genotoxicity on the ocular surface in the development of ophthalmic drugs.

Tumor-derived exosomes promote carcinogenesis of murine oral squamous cell carcinoma.

Circulating tumor-derived exosomes (TEX) interact with a variety of cells in cancer-bearing hosts, leading to cellular reprogramming which promotes disease progression. To study TEX effects on the development of solid tumors, immunosuppressive exosomes carrying PD-L1 and FasL were isolated from supernatants of murine or human HNSCC cell lines. TEX were delivered (IV) to immunocompetent C57BL/6 mice bearing premalignant oral/esophageal lesions induced by the carcinogen, 4-nitroquinoline 1-oxide (4NQO). Progression of the premalignant oropharyngeal lesions to malignant tumors was monitored. A single TEX injection increased the number of developing tumors (6.2 versus 3.2 in control mice injected with phosphate-buffered saline; P < 0.0002) and overall tumor burden per mouse (P < 0.037). The numbers of CD4+ and CD8+ T lymphocytes infiltrating the developing tumors were coordinately reduced (P < 0.01) in mice injected with SCCVII-derived TEX relative to controls. Notably, TEX isolated from mouse or human tumors had similar effects on tumor development and immune cells. A single IV injection of TEX was sufficient to condition mice harboring premalignant OSCC lesions for accelerated tumor progression in concert with reduced immune cell migration to the tumor.

Mutations in long-lived epithelial stem cells and their clonal progeny in pre-malignant lesions and in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are the most common cancers of the oral cavity, but the molecular mechanisms driving OSCC carcinogenesis remain unclear. Our group previously established a murine OSCC model based on a 10-week carcinogen [4-nitroquinoline 1-oxide (4-NQO)] treatment. Here we used K14CreERTAM;Rosa26LacZ mice to perform lineage tracing to delineate the mutational profiles in clonal cell populations resulting from single, long-lived epithelial stem cells, here called LacZ+ stem cell clones (LSCCs). Using laser-capture microdissection, we examined mutational changes in LSCCs immediately after the 10-week 4-NQO treatment and >17 weeks after 4-NQO treatment. We found a 1.8-fold ±0.4 (P = 0.009) increase in single-nucleotide variants and insertions/deletions (indels) in tumor compared with pre-neoplastic LSCCs. The percentages of indels and of loss of heterozygosity events were 1.3-fold±0.3 (P = 0.02) and 2.2-fold±0.7 (P = 0.08) higher in pre-neoplastic compared with tumor LSCCs. Mutations in cell adhesion- and development-associated genes occurred in 83% of the tumor LSCCs. Frequently mutated genes in tumor LSCCs were involved in planar cell polarity (Celsr1, Fat4) or development (Notch1). Chromosomal amplifications in 50% of the tumor LSCCs occurred in epidermal growth factor receptor, phosphoinositide 3-kinase and cell adhesion pathways. All pre-neoplastic and tumor LSCCs were characterized by key smoking-associated changes also observed in human OSCC, C>A and G>T. DeconstructSigs analysis identified smoking and head and neck cancer as the most frequent mutational signatures in pre-neoplastic and tumor LSCCs. Thus, this model recapitulates a smoking-associated mutational profile also observed in humans and illustrates the role of LSCCs in early carcinogenesis and OSCCs.

Characterization of CD103+ CD8+ tissue-resident T cells in esophageal squamous cell carcinoma: may be tumor reactive and resurrected by anti-PD-1 blockade.

Though therapy that promotes anti-tumor response about CD8+ tumor-infiltrating lymphocytes (TILs) has shown great potential, clinical responses to CD8+ TILs immunotherapy vary considerably, largely because of different subpopulation of CD8+ TILs exhibiting different biological characters. To define the relationship between subpopulation of CD8+ TILs and the outcome of antitumor reaction, the phenotype and function of CD103+ CD8+ TILs in esophageal squamous cell carcinoma (ESCC) were investigated. CD103+ CD8+ TILs were presented in ESCC, which displayed phenotype of tissue-resident memory T cells and exhibited high expression of immune checkpoints (PD-1, TIM-3). CD103+ CD8+ TILs were positively associated with the overall survivals of ESCC patients. This population of cells elicited potent proliferation and cytotoxic cytokine secretion potential. In addition, CD103+ CD8+ TILs were elicited potent anti-tumor immunity after anti-PD-1 blockade and were not affected by chemotherapy. This study emphasized the feature of CD103+ CD8+ TILs in immune response and identified potentially new targets in ESCC patients.

An Orc1/Cdc6 ortholog functions as a key regulator in the DNA damage response in Archaea.

While bacteria and eukaryotes show distinct mechanisms of DNA damage response (DDR) regulation, investigation of ultraviolet (UV)-responsive expression in a few archaea did not yield any conclusive evidence for an archaeal DDR regulatory network. Nevertheless, expression of Orc1-2, an ortholog of the archaeal origin recognition complex 1/cell division control protein 6 (Orc1/Cdc6) superfamily proteins was strongly activated in Sulfolobus solfataricus and Sulfolobus acidocaldarius upon UV irradiation. Here, a series of experiments were conducted to investigate the possible functions of Orc1-2 in DNA damage repair in Sulfolobus islandicus. Study of DDR in Δorc1-2 revealed that Orc1-2 deficiency abolishes DNA damage-induced differential expression of a large number of genes and the mutant showed hypersensitivity to DNA damage treatment. Reporter gene and DNase I footprinting assays demonstrated that Orc1-2 interacts with a conserved hexanucleotide motif present in several DDR gene promoters and regulates their expression. Manipulation of orc1-2 expression by promoter substitution in this archaeon revealed that a high level of orc1-2 expression is essential but not sufficient to trigger DDR. Together, these results have placed Orc1-2 in the heart of the archaeal DDR regulation, and the resulting Orc1-2-centered regulatory circuit represents the first DDR network identified in Archaea, the third domain of life.

Presence of antigenotoxic and anticytotoxic effects of the chalcone 1E,4E-1-(4-chlorophenyl)-5-(2,6,6-trimethylcyclohexen-1-yl)penta-1,4-dien-3-one using in vitro and in vivo assays.

Chalcones are chemically defined as α,ß-unsaturated ketones with a 1,3-diphenyl-2-propen-1-one nucleus. These compounds occur naturally in plants and are considered precursors of flavonoids. Given that evaluating genetic toxicology tests is essential in investigating the safe use and chemopreventive potential of different natural and synthetic compounds, this study aimed to assess the genotoxic, cytotoxic, antigenotoxic, and anticytotoxic activity of the chalcone 1E,4E-1-(4-chlorophenyl)-5-(2,6,6-trimethylcyclohexen-1-yl)penta-1,4-dien-3-one (CAB7ß). The CAB7ß was synthesized via Claisen-Schmidt reaction. The Ames test was applied using the co-treatment model as well as a micronucleus assay of mouse bone marrow with co-, pre- and post-treatment models. Our results indicate no genotoxic effect for CAB7ß in any of the tests applied. At all the concentrations used, CAB7ß showed a significant DNA protective effect against the mutagenic action of 4-nitroquinoline-1-oxide and sodium azide according to the Ames test, and against doxorubicin in the co-, pre- and post-treatment models of the micronucleus assay. CAB7ß alone displayed cytotoxic activity in the micronucleus test. At concentrations of 12,5 and 50 µg/plate, CAB7ß showed a moderate cytotoxic profile only in Salmonella typhimurium strain TA98. However, an anticytotoxic effect was observed against S. typhimurium strain TA100 for all the concentrations tested and during co-, pre- and post-treatment in the micronucleus assay. It was concluded that CAB7ß exhibited a slightly cytotoxic effect in S. typhimurium strain TA98 and significant antigenotoxic and anticytotoxic effects in cells of mouse, making it a promising candidate in chemoprevention and possibly in the development of new cancer treatments.

Single-Cell Analysis of Different Stages of Oral Cancer Carcinogenesis in a Mouse Model.

Oral carcinogenesis involves the progression of the normal mucosa into potentially malignant disorders and finally into cancer. Tumors are heterogeneous, with different clusters of cells expressing different genes and exhibiting different behaviors. 4-nitroquinoline 1-oxide (4-NQO) and arecoline were used to induce oral cancer in mice, and the main factors for gene expression influencing carcinogenesis were identified through single-cell RNA sequencing analysis. Male C57BL/6J mice were divided into two groups: a control group (receiving normal drinking water) and treatment group (receiving drinking water containing 4-NQO (200 mg/L) and arecoline (500 mg/L)) to induce the malignant development of oral cancer. Mice were sacrificed at 8, 16, 20, and 29 weeks. Except for mice sacrificed at 8 weeks, all mice were treated for 16 weeks and then either sacrificed or given normal drinking water for the remaining weeks. Tongue lesions were excised, and all cells obtained from mice in the 29- and 16-week treatment groups were clustered into 17 groups by using the Louvain algorithm. Cells in subtypes 7 (stem cells) and 9 (keratinocytes) were analyzed through gene set enrichment analysis. Results indicated that their genes were associated with the MYC_targets_v1 pathway, and this finding was confirmed by the presence of cisplatin-resistant nasopharyngeal carcinoma cell lines. These cell subtype biomarkers can be applied for the detection of patients with precancerous lesions, the identification of high-risk populations, and as a treatment target.

Impact of combination immunochemotherapies on progression of 4NQO-induced murine oral squamous cell carcinoma.

Advanced oral squamous cell carcinomas (OSCC) have limited therapeutic options. Although immune therapies are emerging as a potentially effective alternative or adjunct to chemotherapies, the therapeutic efficacy of combination immune chemotherapies has yet to be determined. Using a 4-nitroquinolone-N-oxide (4NQO) orthotopic model of OSCC in immunocompetent mice, we evaluated the therapeutic efficacy of single- and combined-agent treatment with a poly-epitope tumor peptide vaccine, cisplatin and/or an A2AR inhibitor, ZM241385. The monotherapies or their combinations resulted in a partial inhibition of tumor growth and, in some cases, a significant but transient upregulation of systemic anti-tumor CD8+ T cell responses. These responses eroded in the face of expanding immunoregulatory cell populations at later stages of tumor progression. Our findings support the need for the further development of combinatorial therapeutic approaches that could more effectively silence dominant immune inhibitory pathways operating in OSCC and provide novel, more beneficial treatment options for this tumor.

No difference in 4-nitroquinoline induced tumorigenesis between germ-free and colonized mice.

Variations in oral bacterial communities have been linked to oral cancer suggesting that the oral microbiome is an etiological factor that can influence oral cancer development. The 4-nitroquinoline 1-oxide (4-NQO)-induced murine oral and esophageal cancer model is frequently used to assess the effects of preventive and/or therapeutic agents. We used this model to assess the impact of the microbiome on tumorigenesis using axenic (germ-free) and conventionally housed mice. Increased toxicity was observed in germ-free mice, however, no difference in tumor incidence, multiplicity, and size was observed. Transcriptional profiling of liver tissue from germ-free and conventionally housed mice identified 254 differentially expressed genes including ten cytochrome p450 enzymes, the largest family of phase-1 drug metabolizing enzymes in the liver. Gene ontology revealed that differentially expressed genes were enriched for liver steatosis, inflammation, and oxidative stress in livers of germ-free mice. Our observations emphasize the importance of the microbiome in mediating chemical toxicity at least in part by altering host gene expression. Studies on the role of the microbiome in chemical-induced cancer using germ-free animal models should consider the potential difference in dose due to the microbiome-mediated changes in host metabolizing capacity, which might influence the ability to draw conclusions especially for tumor induction models that are dose dependent.

Establishing of mouse oral carcinoma cell lines derived from transgenic mice and their use as syngeneic tumorigenesis models.

BACKGROUND: The survival of OSCC patient needs to be further improved. miR-211 is oncogenic in OSCC and its upregulation is associated with tumor progression and poor patient survival. K14-EGFP-miR-211 transgenic mice also exhibit augmented potential for OSCC induction. METHODS: Four murine OSCC cell lines, designated MOC-L1 to MOC-L4, are established from tongue tumors induced by 4-nitroquinoline 1-oxide using the K14-EGFP-miR-211 transgenic mouse model. The genetic disruption, in vitro oncogenicity, and the eligibilities of tumorigenesis and metastasis of the cell lines are analyzed. RESULTS: All cell lines show green fluorescence and express a range of epithelial markers. The MOC-L1, MOC-L2 and MOC-L3 cells carry missense mutations in the DNA binding domain of the p53 gene. MOC-L1 exhibits a high level of epithelial-mesenchymal transition and has the aggressive characteristics associated with this. MOC-L1 and MOC-L2 are clonogenic in vitro as well as being tumorigenic when implanted into the dermis or tongue of syngeneic recipients. Nonetheless, only MOC-L1 exhibits immense potential for local regional and distal metastasis. Since the expression of miR-196b in MOC-L1 xenografts is drastically decreased on cisplatin treatment, it would seem that targeting of miR-196b might facilitate tumor abrogation. CONCLUSIONS: As cell lines established in this study originated from the C57BL/6 mouse, the strain most suitable for transgenic engineering, exploring the interplay of these OSCC cells with other genetically modified cells in immune-competent mice would provide important insights into OSCC pathogenesis.

Loss of Notch1 predisposes oro-esophageal epithelium to tumorigenesis.

Notch signaling functions in diverse developmental and homeostatic processes, including stem cell self-renewal and cell fate determination. Notch1-inactivating mutations are frequently detected in skin and oro-esophageal cancers, suggesting a role for Notch1 as a tumor suppressor. Here, we clarify the contribution of Notch1 deficiency to oro-esophageal tumorigenesis using a physiological experimental model. Tongue and esophageal tumors induced in mice by 4-nitroquinoline-1-oxide (4-NQO) showed pathophysiological similarities to human tumors, including decreased Notch1 expression in the basal cells. We created mutant mice (N1cKO), in which the Notch1 gene was disrupted specifically in the squamous epithelium. The epithelium formed normally in N1cKO mice, and although multiple skin tumors were detected at 65 weeks, no tumors developed in the tongue and esophagus. However, 4-NQO-induced tumorigenesis assays revealed that tumor onset occurred earlier in N1cKO mice than in wild-type littermates, and the tumors arose preferentially from the Notch1-negative epithelium, indicating the tumor susceptibility of Notch1-deficient epithelium. Notch1 regulates the expression of TERT, and age-related telomere erosion was more rapid in Notch1-deficient basal cells. Our results indicated that although Notch1 deficiency had little effect on squamous epithelium formation, it predisposed the affected epithelium to tumor development, at least in part through accelerated telomere erosion.

Taiwanin C elicits apoptosis in arecoline and 4-nitroquinoline-1-oxide-induced oral squamous cell carcinoma cells and hinders proliferation via epidermal growth factor receptor/PI3K suppression.

Oral Squamous Cell Carcinoma (OSSC) is a major life-threatening disease with high incidence in the Southeast Asian countries. Chronic exposure to arecoline causes genetic changes in the epithelial cells of the oral mucosa, induces proliferation through activation of the EGF receptor and promotes downstream COX-2 expression. Taiwanin C, a podophyllotoxin derived from Taiwania cryptomerioides Hayata is known to inhibit COX activity and to hinder PGE2 production in macrophages. In this study a tumor cell line T28 and a non-tumor cell line N28 derived from mice OSCC models were used to study the effect of Taiwanin C on PGE2 associated COX-2 expression and cell cycle regulators. Taiwanin C activated p21 protein expression, down-regulated cell cycle regulatory proteins, elevated apoptosis and down-regulated p-PI3K/p-Akt survival mechanism in T28 oral cancer cells. Our results therefore emphasize the therapeutic potential of Taiwanin C against arecoline-induced oral cancer.

Investigation of comet assays under conditions mimicking ocular instillation administration in a three-dimensional reconstructed human corneal epithelial model.

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.