KDM5 Lysine Demethylases in Pathogenesis, from Basic Science Discovery to the Clinic.

The histone lysine demethylase 5 (KDM5) family proteins are Fe2+ and α-ketoglutarate-dependent dioxygenases, with jumonji C (JmjC) domain as their catalytic core and several plant homeodomains (PHDs) to bind different histone methylation marks. These enzymes are capable of demethylating tri-, di- and mono-methylated lysine 4 in histone H3 (H3K4me3/2/1), the key epigenetic marks for active chromatin. Thus, this H3K4 demethylase family plays critical roles in cell fate determination during development as well as malignant transformation. KDM5 demethylases have both oncogenic and tumor suppressive functions in a cancer type-dependent manner. In solid tumors, KDM5A/B are generally oncogenic, whereas KDM5C/D have tumor suppressive roles. Their involvement in de-differentiation, cancer metastasis, drug resistance, and tumor immunoevasion indicated that KDM5 family proteins are promising drug targets for cancer therapy. Significant efforts from both academia and industry have led to the development of potent and selective KDM5 inhibitors for preclinical experiments and phase I clinical trials. However, a better understanding of the roles of KDM5 demethylases in different physiological and pathological conditions is critical for further developing KDM5 modulators for clinical applications.

KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements.

Tumours use various strategies to evade immune surveillance1,2. Immunotherapies targeting tumour immune evasion such as immune checkpoint blockade have shown considerable efficacy on multiple cancers3,4 but are ineffective for most patients due to primary or acquired resistance5-7. Recent studies showed that some epigenetic regulators suppress anti-tumour immunity2,8-12, suggesting that epigenetic therapies could boost anti-tumour immune responses and overcome resistance to current immunotherapies. Here we show that, in mouse melanoma models, depletion of KDM5B-an H3K4 demethylase that is critical for melanoma maintenance and drug resistance13-15-induces robust adaptive immune responses and enhances responses to immune checkpoint blockade. Mechanistically, KDM5B recruits the H3K9 methyltransferase SETDB1 to repress endogenous retroelements such as MMVL30 in a demethylase-independent manner. Derepression of these retroelements activates cytosolic RNA-sensing and DNA-sensing pathways and the subsequent type-I interferon response, leading to tumour rejection and induction of immune memory. Our results demonstrate that KDM5B suppresses anti-tumour immunity by epigenetic silencing of retroelements. We therefore reveal roles of KDM5B in heterochromatin regulation and immune evasion in melanoma, opening new paths for the development of KDM5B-targeting and SETDB1-targeting therapies to enhance tumour immunogenicity and overcome immunotherapy resistance.

Human iPS Cell-derived Tissue Engineered Vascular Graft: Recent Advances and Future Directions.

Tissue engineered vascular grafts (TEVGs) generated from human primary cells represent a promising vascular interventional therapy. However, generation and application of these TEVGs may be significantly hindered by the limited accessibility, finite expandability, donor-donor functional variation and immune-incompatibility of primary seed cells from donors. Alternatively, human induced pluripotent stem cells (hiPSCs) offer an infinite source to obtain functional vascular cells in large quantity and comparable quality for TEVG construction. To date, TEVGs (hiPSC-TEVGs) with significant mechanical strength and implantability have been generated using hiPSC-derived seed cells. Despite being in its incipient stage, this emerging field of hiPSC-TEVG research has achieved significant progress and presented promising future potential. Meanwhile, a series of challenges pertaining hiPSC differentiation, vascular tissue engineering technologies and future production and application await to be addressed. Herein, we have composed this review to introduce progress in TEVG generation using hiPSCs, summarize the current major challenges, and encapsulate the future directions of research on hiPSC-based TEVGs. Graphical abstract.

Efficient Differentiation of Human Induced Pluripotent Stem Cells into Endothelial Cells under Xenogeneic-free Conditions for Vascular Tissue Engineering.

Tissue engineered vascular grafts (TEVGs) represent a promising therapeutic option for emergency vascular intervention. Although the application of small-diameter TEVGs using patient-specific primary endothelial cells (ECs) to prevent thrombosis and occlusion prior to implantation could be hindered by the long time course required for in vitro endothelialization, human induced pluripotent stem cells (hiPSCs) provide a robust source to derive immunocompatible ECs (hiPSC-ECs) for immediate TEVG endothelialization. To achieve clinical application, hiPSC-ECs should be derived under culture conditions without the use of animal-derived reagents (xenogeneic-free conditions), to avoid unwanted host immune responses from xenogeneic reagents. However, a completely xenogeneic-free method of hiPSC-EC generation has not previously been established. Herein, we substituted animal-derived reagents used in a standard method of xenogeneic hiPSC-EC differentiation with functional counterparts of human origin. As a result, we generated xenogeneic-free hiPSC-ECs (XF-hiPSC-ECs) with similar marker expression and function to those of human primary ECs. Furthermore, XF-hiPSC-ECs functionally responded to shear stress with typical cell alignment and gene expression. Finally, we successfully endothelialized decellularized human vessels with XF-hiPSC-ECs in a dynamic bioreactor system. In conclusion, we developed xenogeneic-free conditions for generating functional hiPSC-ECs suitable for vascular tissue engineering, which will further move TEVG therapy toward clinical application.

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.

KDM5B Is Essential for the Hyperactivation of PI3K/AKT Signaling in Prostate Tumorigenesis.

KDM5B (lysine[K]-specific demethylase 5B) is frequently upregulated in various human cancers including prostate cancer. KDM5B controls H3K4me3/2 levels and regulates gene transcription and cell differentiation, yet the contributions of KDM5B to prostate cancer tumorigenesis remain unknown. In this study, we investigated the functional role of KDM5B in epigenetic dysregulation and prostate cancer progression in cultured cells and in mouse models of prostate epithelium-specific mutant Pten/Kdm5b. Kdm5b deficiency resulted in a significant delay in the onset of prostate cancer in Pten-null mice, whereas Kdm5b loss alone caused no morphologic abnormalities in mouse prostates. At 6 months of age, the prostate weight of Pten/Kdm5b mice was reduced by up to 70% compared with that of Pten mice. Pathologic analysis revealed Pten/Kdm5b mice displayed mild morphologic changes with hyperplasia in prostates, whereas age-matched Pten littermates developed high-grade prostatic intraepithelial neoplasia and prostate cancer. Mechanistically, KDM5B governed PI3K/AKT signaling in prostate cancer in vitro and in vivo. KDM5B directly bound the PIK3CA promoter, and KDM5B knockout resulted in a significant reduction of P110α and PIP3 levels and subsequent decrease in proliferation of human prostate cancer cells. Conversely, KDM5B overexpression resulted in increased PI3K/AKT signaling. Loss of Kdm5b abrogated the hyperactivation of AKT signaling by decreasing P110α/P85 levels in Pten/Kdm5b mice. Taken together, our findings reveal that KDM5B acts as a key regulator of PI3K/AKT signaling; they also support the concept that targeting KDM5B is a novel and effective therapeutic strategy against prostate cancer. SIGNIFICANCE: This study demonstrates that levels of histone modification enzyme KDM5B determine hyperactivation of PI3K/AKT signaling in prostate cancer and that targeting KDM5B could be a novel strategy against prostate cancer.

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-ß signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.

Tissue-Engineered Vascular Grafts with Advanced Mechanical Strength from Human iPSCs.

Vascular smooth muscle cells (VSMCs) can be derived in large numbers from human induced pluripotent stem cells (hiPSCs) for producing tissue-engineered vascular grafts (TEVGs). However, hiPSC-derived TEVGs are hampered by low mechanical strength and significant radial dilation after implantation. Here, we report generation of hiPSC-derived TEVGs with mechanical strength comparable to native vessels used in arterial bypass grafts by utilizing biodegradable scaffolds, incremental pulsatile stretching, and optimal culture conditions. Following implantation into a rat aortic model, hiPSC-derived TEVGs show excellent patency without luminal dilation and effectively maintain mechanical and contractile function. This study provides a foundation for future production of non-immunogenic, cellularized hiPSC-derived TEVGs composed of allogenic vascular cells, potentially serving needs to a considerable number of patients whose dysfunctional vascular cells preclude TEVG generation via other methods.

Application of Human Induced Pluripotent Stem Cells in Generating Tissue-Engineered Blood Vessels as Vascular Grafts.

In pace with the advancement of tissue engineering during recent decades, tissue-engineered blood vessels (TEBVs) have been generated using primary seed cells, and their impressive success in clinical trials have demonstrated the great potential of these TEBVs as implantable vascular grafts in human regenerative medicine. However, the production, therapeutic efficacy, and readiness in emergencies of current TEBVs could be hindered by the accessibility, expandability, and donor-donor variation of patient-specific primary seed cells. Alternatively, using human induced pluripotent stem cells (hiPSCs) to derive seed vascular cells for vascular tissue engineering could fundamentally address this current dilemma in TEBV production. As an emerging research field with a promising future, the generation of hiPSC-based TEBVs has been reported recently with significant progress. Simultaneously, to further promote hiPSC-based TEBVs into vascular grafts for clinical use, several challenges related to the safety, readiness, and structural integrity of vascular tissue need to be addressed. Herein, this review will focus on the evolution and role of hiPSCs in vascular tissue engineering technology and summarize the current progress, challenges, and future directions of research on hiPSC-based TEBVs.

Large Animal Models for the Clinical Application of Human Induced Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology offers a practically infinite and ethically acceptable source to obtain a variety of somatic cells. Coupled with the biotechnologies of cell therapy or tissue engineering, iPSC technology will enormously contribute to human regenerative medicine. Before clinical application, such human iPSC (hiPSC)-based therapies should be assessed using large animal models that more closely match biological or biomechanical properties of human patients. Therefore, it is critical to generate large animal iPSCs, obtain their iPSC-derived somatic cells, and preclinically evaluate their therapeutic efficacy and safety in large animals. During the past decade, the establishment of iPSC lines of a series of large animal species has been documented, and the acquisition and preclinical evaluation of iPSC-derived somatic cells has also been reported. Despite this progress, significant obstacles, such as obtaining or preserving the bona fide pluripotency of large animal iPSCs, have been encountered. Simultaneously, studies of large animal iPSCs have been overlooked in comparison with those of mouse and hiPSCs, and this field deserves more attention and support due to its important preclinical relevance. Herein, this review will focus on the large animal models of pigs, dogs, horses, and sheep/goats, and summarize current progress, challenges, and potential future directions of research on large animal iPSCs.

KDM5B Promotes Drug Resistance by Regulating Melanoma-Propagating Cell Subpopulations.

Tumor heterogeneity is a major challenge for cancer treatment, especially due to the presence of various subpopulations with stem cell or progenitor cell properties. In mouse melanomas, both CD34+p75- (CD34+) and CD34-p75- (CD34-) tumor subpopulations were characterized as melanoma-propagating cells (MPC) that exhibit some of those key features. However, these two subpopulations differ from each other in tumorigenic potential, ability to recapitulate heterogeneity, and chemoresistance. In this study, we demonstrate that CD34+ and CD34- subpopulations carrying the BRAFV600E mutation confer differential sensitivity to targeted BRAF inhibition. Through elevated KDM5B expression, melanoma cells shift toward a more drug-tolerant, CD34- state upon exposure to BRAF inhibitor or combined BRAF inhibitor and MEK inhibitor treatment. KDM5B loss or inhibition shifts melanoma cells to the more BRAF inhibitor-sensitive CD34+ state. These results support that KDM5B is a critical epigenetic regulator that governs the transition of key MPC subpopulations with distinct drug sensitivity. This study also emphasizes the importance of continuing to advance our understanding of intratumor heterogeneity and ultimately develop novel therapeutics by altering the heterogeneous characteristics of melanoma.

Effects of chronic alcohol consumption on DNA damage and immune regulation induced by the environmental pollutant dibenzo[a,l]pyrene in oral tissues of mice.

Previously, we showed that oral application of the environmental pollutant dibenzo[a,l]pyrene (DB[a,l]P) induces oral tumors in mice. Thus, in the present investigation we examined the effect of alcohol on DB[a,l]P-induced DNA damage and immune regulation; we showed that alcohol (6.4% v/v in the diet, 35% of Calories) significantly enhanced the levels of (-)-anti-trans-DB[a,l]P-dA while decreased the levels of GSH in the mouse oral tissues. Analysis of RNA expression revealed that DB[a,l]P alone upregulates inflammatory genes while alcohol suppresses several markers of immune surveillance. Collectively, these results suggest that alcohol may enhance oral carcinogenesis induced by DB[a,l]P.

Carcinogenesis of the Oral Cavity: Environmental Causes and Potential Prevention by Black Raspberry.

Worldwide, cancers of the oral cavity and pharynx comprise the sixth most common malignancies. Histologically, more than 90% of oral cancers are squamous cell carcinoma (SCC). Epidemiologic data strongly support the role of exogenous factors such as tobacco, alcohol, and human papilloma virus infection as major causative agents. Avoidance of risk factors has only been partially successful, and survival rates have not improved despite advances in therapeutic approaches. Therefore, new or improved approaches to prevention and/or early detection are critical. Better understanding of the mechanisms of oral carcinogenesis can assist in the development of novel biomarkers for early detection and strategies for disease prevention. Toward this goal, several animal models for carcinogenesis in the oral cavity have been developed. Among these are xenograft, and transgenic animal models, and others employing the synthetic carcinogens such as 7,12-dimethylbenz[a]anthracene in hamster cheek pouch and 4-nitroquinoline-N-oxide in rats and mice. Additional animal models employing environmental carcinogens such as benzo[a]pyrene and N'-nitrosonornicotine have been reported. Each model has certain advantages and disadvantages. Models that (1) utilize environmental carcinogens, (2) reflect tumor heterogeneity, and (3) accurately represent the cellular and molecular changes involved in the initiation and progression of oral cancer in humans could provide a realistic platform. To achieve this goal, we introduced a novel nonsurgical mouse model to study oral carcinogenesis induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and tobacco smoke constituent, and its diol epoxide metabolite (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene [(±)-anti-DB[a,l]PDE]. On the basis of a detailed comparison of oral cancer induced by DB[a,l]P with that induced by the other above-mentioned oral carcinogens with respect to dose, duration, species and strain, cellular and molecular targets, and relative carcinogenic potency, our animal model may offer a more realistic platform to study oral carcinogenesis. In this perspective, we also discuss our preclinical studies to demonstrate the potential of black raspberry extracts on the prevention of OSCC. Specifically, we were the first to demonstrate that black raspberry inhibited DB[a,l]P-DNA binding and of particular importance its capacity to enhance the repair of DB[a,l]P-induced bulky lesions in DNA. We believe that the information presented in this perspective will stimulate further research on the impact of environmental carcinogens in the development of oral cancer and may lead to novel strategies toward the control and prevention of this disease.

An easy and efficient inducible CRISPR/Cas9 platform with improved specificity for multiple gene targeting.

The CRISPR/Cas9 system is a powerful genome editing tool and has been widely used for biomedical research. However, many challenges, such as off-target effects and lack of easy solutions for multiplex targeting, are still limiting its applications. To overcome these challenges, we first developed a highly efficient doxycycline-inducible Cas9-EGFP vector. This vector allowed us to track the cells for uniform temporal control and efficient gene disruption, even in a polyclonal setting. Furthermore, the inducible CRISPR/Cas9 system dramatically decreased off-target effects with a pulse exposure of the genome to the Cas9/sgRNA complex. To target multiple genes simultaneously, we established simple one-step cloning approaches for expression of multiple sgRNAs with improved vectors. By combining our inducible and multiplex genome editing approaches, we were able to simultaneously delete Lysine Demethylase (KDM) 5A, 5B and 5C efficiently in vitro and in vivo This user friendly and highly efficient toolbox provides a solution for easy genome editing with tight temporal control, minimal off-target effects and multiplex targeting.

Simultaneous detection of deoxyadenosine and deoxyguanosine adducts in the tongue and other oral tissues of mice treated with Dibenzo[a,l]pyrene.

We were the first to demonstrate that direct application of the environmental pollutant and tobacco smoke constituent dibenzo[a,l]pyrene (DB[a,l]P) into the oral cavity of mice induced squamous cell carcinoma (SCC) in oral tissues but not in the tongue; however, the mechanisms that can account for the varied carcinogenicity remain to be determined. Furthermore, we also showed that not only dA adducts, but also dG adducts can account for the mutagenic activity of DB[a,l]P in the oral tissues in vivo. In this study, we initially focused on DB[a,l]P-induced genotoxic effects in both oral and tongue tissues. Therefore, to fully assess the contribution of these DNA adducts in the initiation stage of carcinogenesis induced by DB[a,l]P, an LC-MS/MS method to simultaneously detect and quantify DB[a,l]PDE-dG and -dA adducts was developed. Mice were orally administered with DB[a,l]P (24 nmole, 3 times per week for 5 weeks) or its fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE, 12 nmole, single application); animals were sacrificed at 2, 7, 14, and 28 days after the last dose of carcinogen administration. Oral and tongue tissues were obtained and DNA were isolated followed by enzymatic hydrolysis. Following the development of an isotope dilution LC-MS/MS method, we successfully detected (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(2)-dG, as well as (-)-anti-cis- and (-)-anti-trans-DB[a,l]PDE-N(6)-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (-)-anti-trans-DB[a,l]PDE-N(6)-dA were ≥2 folds higher than (-)-anti-cis-DB[a,l]PDE-N(6)-dA adduct and those of dG adducts in the oral tissues and tongue at all time points selected after the cessation of DB[a,l]P treatment. Levels of dG adducts were comparable in both tissues. Collectively, our results support that DB[a,l]P is predominantly metabolized to (-)-anti-DB[a,l]PDE, and the levels and persistence of (-)-anti-trans-DB[a,l]PDE-N(6)-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

Mechanisms of oral carcinogenesis induced by dibenzo[a,l]pyrene: an environmental pollutant and a tobacco smoke constituent.

We previously reported that dibenzo[a,l]pyrene (DB[a,l]P), the most potent known environmental carcinogen among polycyclic aromatic hydrocarbons (PAH) congeners, is carcinogenic in the oral tissues of mice. We have now developed a new mouse model which employs the oral application of the fjord region diol epoxide, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE), a metabolite of the tobacco smoke constituent DB[a,l]P, and we show its specific induction of oral squamous cell carcinoma (OSCC) in both tongue and other oral tissues. Groups of B6C3F1 mice (20/group) received 6 or 3 nmol of (±)-anti-DB[a,l]PDE administered into the oral cavity; 3 times per week for 38 weeks. Additional groups received the vehicle alone or were left untreated. Mice were sacrificed 42 weeks after the first carcinogen administration. The high dose induced 74 and 100% OSCC in the tongue and other oral tissues, respectively; the corresponding values at the lower dose were 45 and 89%. Using immunohistochemistry, we showed that DB[a,l]PDE resulted in overexpression of p53 and COX-2 proteins in malignant tissues when compared to normal oral tissues and tongues. Consistent with the carcinogenicity, we demonstrated powerful mutagenicity in cII gene in B6C3F1 (Big Blue) mouse tongue. The mutational profile in lacI reporter gene is similar to those detected in human head and neck cancer, and p53 mutations were observed in mouse oral tumor tissues. Taken together, we conclude that the formation of diol epoxides plays a major role among the mechanisms by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity.

Induction of ovarian cancer and DNA adducts by Dibenzo[a,l]pyrene in the mouse.

Tobacco smoking is an etiological factor of ovarian cacner; however, the mechanisms remain largely undefined. Therefore, as an initial investigation, we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P, a tobacco smoke constituent and environmental pollutant. Ovarian tumors in B6C3F1 mice were induced by direct application of DB[a,l]P (24, 12, 6, and 3 nmol/mouse, three times a week for 38 weeks) into the oral cavity of mice. At 6 nmol, DB[a,l]P induced the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total ovarian tumor incidence was 75%, and the incidence of malignancy was 65%. In addition to ovarian tumors, at the dose of 24 nmol, DB[a,l]P induced lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues, which were rare at doses lower than 24 nmol. Another bioassay was conducted to detect and quantify DNA adducts induced by DB[a,l]P (24 nmol, three times a week for 5 weeks) in the ovary at 48 h and 1, 2, and 4 weeks after the last administration of DB[a,l]P. DNA was isolated, and the dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE)-DNA adducts were analyzed by a LC-MS/MS method. DB[a,l]P resulted in the formation of (-)-anti-cis-DB[a,l]PDE-dA and (-)-anti-trans-DB[a,l]PDE-dA adducts, which were 0.8 and 1.6 fmol/10(6) dA, respectively, in ovaries of mice within 48 h, and the level of adducts decreased over a week. Our results indicated that DB[a,l]P can be metabolized to form (-)-anti-DB[a,l]PDE; the latter may, in part, account for DB[a,l]P-induced ovarian cancer. This animal model should assist to better understand the mechanisms, account for the induction of ovarian cancer by tobacco carcinogens, and facilitate the development of chemopreventive agents against ovarian cancer.

Mutagenesis and carcinogenesis induced by dibenzo[a,l]pyrene in the mouse oral cavity: a potential new model for oral cancer.

Cancer of the oral cavity is a serious disease, affecting about 30,000 individuals in US annually. There are several animal models of oral cancer, but each has certain disadvantages. As a new model, we investigated whether topical application of the tobacco smoke carcinogen, dibenzo[a,l]pyrene (DB[a,l]P) is mutagenic and carcinogenic in the oral cavity of the B6C3F1 lacI and B6C3F1 mouse, respectively. B6C3F1 lacI mice received DB[a,l]P (0, 3, 6, 12 nmol) 3× per week. B6C3F1 mice received the same doses and also 24 nmol. At 38 weeks mutagenesis was measured in oral tissues in lacI mice. For the high dose group, the mutant fraction (MF) in upper mucosa and tongue increased about twofold relative to that in vehicle-alone. The increases were statistically significant. The mutational profile in the DB[a,l]P-induced mutants was compared with that induced by benzo[a]pyrene (BaP) in oral tissue. BaP is mutagenic in many tissues when administered by gavage. The mutational profile for DB[a,l]P was more similar to that reported for p53 mutations in head and neck cancers than was that of BaP. At 47 weeks, oral squamous cell carcinomas (OSCC) were found in 31% of the high-dose B6C3F1 group. Elevations of p53 and COX-2 protein were observed in tumor and dysplastic tissue. As DB[a,l]P induces mutations and tumors in the oral cavity, and has a mutational profile in oral tissue similar to that found in p53 in human OSCC, the treatment protocol described here may represent a new and relevant model for cancer of the oral cavity.

Identification and quantification of DNA adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a,l]pyrene by HPLC-MS/MS.

Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(5)]-(±)-anti-DB[a,l]PDE-N(6)-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 µg of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/µg DNA) of (-)-anti-trans adducts (4.03 ± 0.27 to 1.77 ± 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 ± 0.42 to 0.72 ± 0.04) at each time point (p < 0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.