Kun-Ming Chen

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.

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.

Inhibition of Nuclear Factor-κB DNA Binding by Organoselenocyanates through Covalent Modification of the p50 Subunit

Most known chemopreventive agents including certain selenium compounds suppress the activation of the nuclear factor kappaB (NF-kappaB), but the mechanisms remain largely elusive. Toward this end, we initially showed that the inhibition of NF-kappaB DNA binding by benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) was reversed by the addition of DTT; this suggests the formation of DTT-reducible selenium-sulfur bonds between selenocyanate moieties and cysteine residues in NF-kappaB (p50) protein. Furthermore, the inhibitory effect of selenocyanates on NF-kappaB was not altered in the presence of physiologic level of reduced glutathione (1 mmol/L), suggesting that selenocyanates can also inhibit NF-kappaB in vivo. Using both matrix-assisted laser desorption/ionization-time of flight and tandem mass spectrometry fragmentation, we showed for the first time that the Cys(62) residue in the active site of NF-kappaB (p50) protein was modified by BSC through the formation of a selenium-sulfur bond. In addition, p-XSC-bound NF-kappaB (p50) protein was also detected by a radiotracer method. To provide further support, molecular models of both BSC and p-XSC positioned in the DNA binding pocket of the p50 were constructed through the covalent modification of Cys(62); the models reveal that DNA substrate could be hindered to enter its DNA binding region. This study shows for the first time that BSC and p-XSC may exert their chemopreventive activity, at least in part, by inhibiting NF-kappaB through covalent modification of Cys(62) of the p50 subunit of NF-kappaB.

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-N2-dG, as well as (−)-anti-cis- and (−)-anti-trans-DB[a,l]PDE-N6-dA in oral and tongue tissues of mice treated with DB[a,l]P. Levels of (−)-anti-trans-DB[a,l]PDE-N6-dA were ≥2 folds higher than (−)-anti-cis-DB[a,l]PDE-N6-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-N6-dA may, in part, explain the carcinogenicity of DB[a,l]P in the oral tissues but not in the tongue.

Modulations of benzo[a]pyrene-induced DNA adduct, cyclin D1 and PCNA in oral tissue by 1,4-phenylenebis(methylene)selenocyanate

Tobacco smoking is an important cause of human oral squamous cell carcinoma (SCC). Tobacco smoke contains multiple carcinogens include polycyclic aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P). Surgery is the conventional treatment approach for SCC, but it remains imperfect. However, chemoprevention is a plausible strategy and we had previously demonstrated that 1,4-phenylenebis(methylene)selenocyanate (p-XSC) significantly inhibited tongue tumors-induced by the synthetic 4-nitroquinoline-N-oxide (not present in tobacco smoke). In this study, we demonstrated that p-XSC is capable of inhibiting B[a]P-DNA adduct formation, cell proliferation, cyclin D1 expression in human oral cells in vitro. In addition, we showed that dietary p-XSC inhibits B[a]P-DNA adduct formation, cell proliferation and cyclin D1 protein expression in the mouse tongue in vivo. The results of this study are encouraging to further evaluate the chemopreventive efficacy of p-XSC initially against B[a]P-induced tongue tumors in mice and ultimately in the clinic.

Effects of Black Raspberry Extract and Protocatechuic Acid on Carcinogen-DNA Adducts and Mutagenesis, and Oxidative Stress in Rat and Human Oral Cells

Effects of black raspberry (BRB) extract and protocatechuic acid (PCA) on DNA adduct formation and mutagenesis induced by metabolites of dibenzo[a,l]pyrene (DBP) were investigated in rat oral fibroblasts. The DBP metabolites, (±)-anti-11,12-dihydroxy-11,12,-dihydrodibenzo[a,l]pyrene (DBP-diol) and 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) induced dose-dependent DNA adducts and mutations. DBPDE was considerably more potent, whereas the parent compound had no significant effect. Treatment with BRB extract (BRBE) and PCA resulted in reduced DBP-derived DNA adduct levels and reduced mutagenesis induced by DBP-diol, but only BRBE was similarly effective against (DBPDE). BRBE did not directly inactivate DBPDE, but rather induced a cellular response—enhanced DNA repair. When BRBE was added to cells 1 day after the DBP-diol, the BRBE greatly enhanced removal of DBP-derived DNA adducts. As oxidative stress can contribute to several stages of carcinogenesis, BRBE and PCA were investigated for their abilities to reduce oxidative stress in a human leukoplakia cell line by monitoring the redox indicator, 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF) in cellular and acellular systems. BRBE effectively inhibited the oxidation, but PCA was only minimally effective against H2DCF. These results taken together provide evidence that BRBE and PCA can inhibit initiation of carcinogenesis by polycyclic aromatic hydrocarbons; and in addition, BRBE reduces oxidative stress. Cancer Prev Res; 9(8); 704–12. ©2016 AACR.

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

ABSTRACT 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.

CK0403, a 9‑aminoacridine, is a potent anti‑cancer agent in human breast cancer cells

3‑({4‑[4‑(Acridin‑9‑ylamino)phenylthio]phenyl}(3‑hydroxypropyl)amino)propan‑1‑ol (CK0403) is a sulfur‑containing 9‑anilinoacridine analogue of amsacrine and was found to be more potent than its analogue 2-({4-[4-(acridin-9-ylamino)phenylthio]phenyl}(2‑hydroxyethyl)amino)ethan‑1‑ol (CK0402) and amsacrine in the inhibition of the topoisomerase II‑catalyzed decatenation reaction. A previous study by our group reported that CK0402 was effective against numerous breast cancer cell lines, and the combination of CK0402 with herceptin enhanced its activity in HER2(+) SKBR‑3 cells. In order to identify novel chemotherapeutic agents with enhanced potency, the present study explored the potential of CK0403 in the treatment of breast cancer. First, the growth inhibitory activity of CK0403 in the breast cancer cell lines MCF‑7, MDA‑MB‑231, BT474 and SKBR‑3, as well as in the non‑cancerous MCF‑10A cell line, was examined using a sulforhodamine B assay. The results showed that CK0403 exerted more potent growth inhibitory activity than CK0402 in all of the breast cancer cell lines except MCF‑7. SKBR‑3 and MDA‑MB‑231 were the most sensitive cell lines tested, and the combination of CK0403 with herceptin in HER2(+) SKBR‑3 cells enhanced the growth inhibitory activity of CK0403. Analysis of cell cycle alterations induced by CK0403 in SKBR‑3 cells revealed that, similarly to CK0402, CK0403 induced G2/M‑phase arrest with a decreased S- and G0/G1-phase ratio. In addition, it was shown that CK0403 induced apoptosis more effectively than CK0402 in SKBR‑3 cells. Further analysis of autophagy protein 5 (Atg5) indicated that CK0403 induced more cleaved Atg5 than CK0402 and other chemotherapeutic agents tested. Of note, although still relatively potent, CK0403 exhibited reduced growth inhibitory activity under hypoxic conditions, which can induce autophagy. Collectively, the present results supported that CK0403 is highly potent and more effective than CK0402 against estrogen receptor-negative and HER2‑overexpressing breast cancer cell lines, suggesting its future application for chemotherapy in breast cancer.

Tissue Distribution, Excretion and Pharmacokinetics of the Environmental Pollutant Dibenzo[def,p]chrysene in Mice

Dibenzo[def,p]chrysene (DBP), a representative example of the class of polycyclic aromatic hydrocarbon (PAH), is known to induce tumors in multiple organ sites including the ovary, lung, mammary glands, and oral cavity in rodents. The goal of this study was to test the hypothesis that the levels of DBP and its metabolites that reach and retain the levels for an extended time in the target organs as well as the capacity of these organs to metabolize this carcinogen to active metabolites that can damage DNA may account for its tissue selective tumorigenicity. Therefore, we used the radiolabeled [(3)H] DBP to accurately assess the tissue distribution, excretion, and pharmacokinetics of this carcinogen. We also compared the levels of DBPDE-DNA adducts in a select target organ (ovary) and nontarget organs (kidney and liver) in mice treated orally with DBP. Our results showed that after 1 week, 91.40 ± 7.23% of the radioactivity was recovered in the feces; the corresponding value excreted in the urine was less than 2% after 1 week. After 24 h, the stomach had the highest radioactivity followed by the intestine and the liver; however, after 1 week, levels of the radioactivity in these organs were the lowest among tissues examined including the ovary and liver; the pharmacokinetic analysis of DBP was conducted using a one compartment open model. The level of (-)-anti-trans-DBPDE-dA in the ovaries (8.91 ± 0.08 adducts/10(7) dA) was significantly higher (p < 0.01) than the levels of adducts in kidneys (0.69 ± 0.09 adducts/10(7) dA) and livers (0.63 ± 0.11 adducts/10(7) dA). Collectively, the results of the tissue distribution and pharmacokinetic analysis may not fully support our hypothesis, but the capacity of the target organs vs nontarget organs to metabolize DBP to active intermediates that can damage DNA may account for its tissue selective tumorigenicity.