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. 2022 Jun 24;43(8):746–753. doi: 10.1093/carcin/bgac058

Detection of DNA adducts derived from the tobacco carcinogens, benzo[a]pyrene and dibenzo[def,p]chrysene in human oral buccal cells

Kun-Ming Chen 1, Yuan-Wan Sun 2, Nicolle M Krebs 3, Dongxiao Sun 4, Jacek Krzeminski 5, Lisa Reinhart 6, Krishne Gowda 7, Shantu Amin 8, Susan Mallery 9, John P Richie Jr 10, Karam El-Bayoumy 11,✉
PMCID: PMC9486590  PMID: 35749296

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are recognized as potential etiological agents in the development of oral cancer in smokers. In particular, benzo[a]pyrene (B[a]P) and dibenzo[def,p]chrysene (DB[a,l]P) are detected in cigarette smoke and the environment and can induce DNA damage, mutagenesis and carcinogenesis in the oral cavity of rodents. Consequently, DNA adducts are regarded as the most direct markers of genotoxicity and can be used as biomarkers of cancer risk. Thus, this study used LC-MS/MS analysis with isotope labeled internal standard to detect and quantify DNA adducts derived from B[a]P and DB[a,l]P in buccal cells of cigarette smokers and non-smokers. Participants in this study include 21 smokers and 16 non-smokers. Our data are the first to report that levels (mean ± SD) of BPDE-N2-dG were significantly (P < 0.001) higher in smokers (20.18 ± 8.40 adducts/108 dG) than in non-smokers (0.84 ± 1.02 adducts/108 dG). Likewise, levels of DBPDE-N6-dA in smokers (5.49 ± 3.41 adducts/108 dA) were significantly higher (P = 0.019) than non-smokers (2.76 ± 2.29 adducts/108 dA). Collectively, the results of this clinical study support that PAHs in tobacco smoke can contribute to the development of oral cancer in humans.

We demonstrated for the first time that levels of BPDE-N2-dG and DBPDE-N6-dA were significantly higher in smokers than non-smokers. Our results support that PAHs in tobacco smoke can contribute to the development of oral cancer in humans.

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Introduction

Head and neck squamous cell carcinoma (HNSCC) ranks as the sixth most common malignancy in the world, and oral squamous cell carcinoma (OSCC) is the most predominant type of this disease (1). Epidemiological data provide strong support for exogenous factors such as tobacco use, alcohol drinking, human papillomavirus (HPV) infection and betel quid chewing as major causative agents in the development of HNSCC (1–5). More than 60 carcinogens (animal or human) have been identified in tobacco smoke (6). Although it is not clear which compounds in tobacco smoke contribute to the development of HNSCC in humans, certain classes of chemical carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and tobacco-specific nitrosamines (TSNA) are recognized as potential etiological agents for oral cancer (4–6). In addition, previous studies from our laboratories have confirmed that human oral epithelium possesses every enzyme associated with nitrosamine metabolism (7) and human oral mucosal explants can bioactivate PAHs, consistent with the presence of functional P-4501A1/1B1 (CYP1A1/1B1) (8).

PAHs represent a large class of aromatic compounds that are generated by incomplete combustion or pyrolysis processes such as cigarette smoking, vehicle generated exhausts, industrial processes, grilled and smoked food (9). Benzo[a]pyrene (B[a]P) is a prototype environmental PAH carcinogen that has been extensively used as a model compound to study biological mechanism(s) of action and to assess exposure to PAHs (10). When fed in diet for 2 years, B[a]P induced papillomas and carcinomas of the tongue as well as tumors at distal sites in mice (4,11). In addition, B[a]P also induced both papillomas and carcinomas in the hamster cheek pouch (4). B[a]P can be metabolically activated by cytochrome P-4501A1/1B1 and epoxide hydrolase leading to the formation of B[a]P-dihydrodiol (B[a]P-DHD) intermediate and the ultimate carcinogen 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) which can react with deoxyguanosine (dG) to form BPDE-N2-dG adducts (Figure 1) (4). The detection of the major DNA adduct resulting from exposure to the ultimate tumorigenic B[a]P metabolite, BPDE, in human oral buccal cells using a mass-spectrometry method has been elusive (12). However, Weng et al. using immunohistochemistry detected BPDE-N2-dG adducts in buccal cells but levels were not different between smokers and non-smokers (13). Unlike B[a]P, which was classified by the IARC as Group 1 carcinogen and is listed as a priority PAHs contaminant by United States–Environmental Protection Agency (US-EPA), dibenzo[def,p]chrysene (also known as dibenzo[a,l]pyrene; DB[a,l]P), the most potent known environmental carcinogen among PAHs congeners, is not on the list (9); therefore, the toxic potential of DB[a,l]P has been underinvestigated. Previously, we demonstrated that topical intraoral application of DB[a,l]P, at the dose of 24 nmol a day, 3 times a week for 38 weeks, induced OSCC in approximately one-third (31%) of B6C3F1 mice (14). This treatment also doubled the mutant fraction (MF) in upper mucosa and tongue relative to control animals (14). Similar to B[a]P, DB[a,l]P (Figure 1) can be metabolically activated by CYP1A1/1B1 and epoxide hydrolase through the formation of its dihydrodiol to its ultimate carcinogen, dibenzo[def,p]chrysene-11,12-dihydrodiol-13,14-epoxide (DBPDE) (4,15). We demonstrated that the formation of diol epoxides is a major mechanism by which DB[a,l]P exerts its oral mutagenicity and tumorigenicity; at the dose of 6 nmol, DB[a,l]P diol epoxides induced 74 and 100% OSCC in the tongue and other oral tissues of mice, respectively (15). DBPDE can react with DNA to form both deoxyadenosine and deoxyguanosine adducts and both have been detected in the oral cavity of mice treated with DB[a,l]P with the former adduct being the major (16,17).

Figure 1.

Figure 1.

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Metabolic activation of B[a]P and DB[a,l]P to the corresponding diol epoxides that can damage DNA.

As the formation of DNA adducts derived from PAHs is a prerequisite step in the multi-step carcinogenesis process, adduct formation is a valid biomarker of cancer risk. In the current study we focused on translating our basic findings in mice (16,17) by conducting an analysis of DNA adducts derived from B[a]P and DB[a,l]P (BPDE-N2-dG and DBPDE-N6-dA, respectively) in human oral buccal cells of smokers and non-smokers by a quantitative LC-MS/MS method.

Materials and methods

The experimental design is summarized in Figure 2. All DNA samples from each subject were isolated, hydrolyzed and analyzed side by side for the analysis of each adduct.

Figure 2.

Figure 2.

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Schematic presentation of the HPLC-MS/MS method for analysis of BPDE-N2-dG and DBPDE-N6-dA in human oral buccal cells.

Chemicals and enzymes

Protease K, RNase A, 8-hydroxyquinoline, other chemicals and enzymes used in the present study were obtained from Sigma–Aldrich (St Louis, MO). [15N5]-dG were obtained from Spectra Stable Isotopes (Columbia, MD). (−)-anti-trans- DB[a,l]P DE-N6-dA (DBPDE-N6-dA) adduct standards were prepared in our laboratory as previously reported (16).

BPDE-N2-dG were prepared by us using a direct microwave supported synthesis. In brief, 2 mg of a racemic (±)-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (6.61 × 10−3 mmol), 20 mg of anhydrous dG (74.8 × 10−3 mmol) and 1 ml of DMF were mixed in a standard 10 ml microwave tube (closed vessel) of CEM Discover instrument. Mixture was irradiated at 100°C for 30 min at the power of 300 W. Solvent was removed under high vacuum at room temperature and residue dissolved in 200 µl of dimethyl sulfoxide (DMSO). C-18 sep-PAK cartridge was activated with 10 ml MeOH followed by 20 ml H2O. The cartridge was loaded with the above DMSO solution of the reaction mixture and elution with 1 ml of H2O followed. The sep-PAK was then washed with additional 9 ml of H2O and finally eluted with 14 ml of MeOH. The methanolic eluate was stripped of solvent and reconstituted in 2 ml of MeOH, centrifuged if necessary (to remove unreacted dG) and purified by high-performance liquid chromatography (HPLC) as follows: Preliminary purification was conducted using a C18 Vydac column 20ITP54 4 × 250 mm, Mobile phase: H2O/MeOH, Gradient: 20–70% of MeOH in 35 min then to 100% of MeOH in 5 min, followed by isocratic elution with 100% of MeOH for 5 min. Flow rate was 1 ml/min, and detected with UV at 347 nm. The instrument is a Hewlett-Packard 1100 Series system. Relevant fractions were combined and further purified using a Ultrasphere RP, ODS-5 2.6 × 250 mm column, Mobile phase is MeOH/H2O, Gradient: 46% MeOH isocratic for 34 min, Flow rate, detection and instrument are same as in the above.

Participants

Participants (smokers) were members of a tobacco user research registry who agreed to be contacted for future tobacco-related research at the Penn State College of Medicine in Hershey, Pennsylvania. Due to COVID-19 restrictions, subject recruitment and participation were done remotely. Prospective participants were contacted by phone and screened for study eligibility. If eligible, participants were mailed a packet with supplies for the buccal cell collection (i.e. soft bristle toothbrush, saline solution packet and sample collection tube). Consented participants (Penn State College of Medicine Institutional Review Board IRB#17017) completed the buccal sample collection during a virtual visit (audio and video) that provided specific instructions. Participants were also asked a brief questionnaire on their demographics and social history, with a focus on OSCC-relevant tobacco and alcohol use. All subjects were remunerated for their participation ($25 per participant). The demographic data of both smokers and non-smokers are summarized in the Supplementary Table 1, available at Carcinogenesis Online. Subjects (21 smokers and 16 non-smokers) were included, but not all participants were analyzed for both DNA adducts as shown in the Supplementary Tables 2 and 3, available at Carcinogenesis Online.

Buccal cell collection methods

First, participants rinsed their mouth twice with water. Next, participants used a soft bristle toothbrush to brush the inside cheeks of the mouth by moving the brush up and down on each side of the cheek for 1 min. The mouth was rinsed with approximate 20 ml of saline (SALJET; Winchester Laboratories LLC, St. Charles, IL) for 2 min. All procedures were timed by the research coordinator for precision. The saline rinse was collected into a 50-ml centrifuge tube. The toothbrush was agitated in the solution to loosen cells that adhered to the bristles and then discarded. The participants placed the sample into a refrigerator until it was retrieved by the research coordinator.

DNA isolation

The isolation of DNA from oral buccal cells was conducted using a phenol-chloroform extraction method (18). Briefly, before the addition of 5 µl proteinase K (20 mg/ml), buccal cells were first suspended in 200 µl alkaline lysis buffer. Cells were incubated for 3–4 h under 58°C followed by the addition of 300 µl of phenol/chloroform/isoamyl alcohol mix (25:24:1, pH 6.7). The solution was mixed, and then centrifuged for 5 min at 13 000 rpm. The top aqueous layer was collected, followed by the addition of sodium acetate buffer (to a final concentration of 0.3 M, pH 5.2) and ethanol for DNA to be precipitated. The resulting DNA pellet was washed with 70% ethanol and the resulted DNA pellet was stored at −20°C.

Enzymatic hydrolysis of DNA, deoxyribonucleosides analysis and purification

Prior to enzymatic digestion, 150 pg of [15N5]-labeled DBPDE-N6-dA or 2 ng of [15N5]-labeled BPDE-N2-dG adducts were added to varied amounts of DNA. DNA hydrolysis and solid phase extraction were carried out under similar conditions as previously reported (16,19). DNA was hydrolyzed in the presence of MgCl2 and DNase I at 37°C for 1.5 h, followed by further incubation with nuclease P1, phosphodiesterase and alkaline phosphatase. An aliquot of the DNA hydrolysate was examined for the completion of hydrolysis by analyzing the hydrolyzed deoxyribonucleosides by HPLC. The remaining hydrolysate was partially purified by a solid phase extraction method using an Oasis HLB column (1 ml, 30 mg; Waters Ltd.).

LC-MS/MS analysis

The analysis of BPDE-N2-dG and DBPDE-N6-dA was conducted using a Sciex QTRAP 6500+ mass spectrometry which is coupled with a Sciex EXion HPLC separation system. A 1.7 µm Acquity UPLC BEH C18 analytical column (2.1 × 50 mm, Waters, Ireland) was used to separate analytes. The gradient elution was conducted using a flow rate of 0.3 ml/min with the following conditions: initial at 40% mobile phase B (0.1% formic acid in methanol) and 60% mobile phase A (0.1% formic acid in water), followed by a linear gradient to 100% mobile phase B in 2 min and kept at 100% mobile phase B for 2 additional minutes to flush the column before going back to initial conditions to equilibrate the column.

The Sciex QTrap 6500+ mass spectrometer was equipped with an electrospray ionization probe, and a positive mode was used. The decluster potential (DP) was 70 V for BPDE-N2-dG and 15N-BPDE-N2-dG, 56 V for DBPDE-N6-dA and 15N-DBPDE-N6-dA; the entrance potential (EP) was 10 V for both BPDE-N2-dG and 15N-BPDE-N2-dG, 7.5 V for DBPDE-N6-dA and 15N-DBPDE-N6-dA; the collision energy (CE) was 17 V for BPDE-N2-dG and 15N-BPDE-N2-dG, 33 V for DBPDE-N6-dA and 15N-DBPDE-N6-dA; the collision cell exit potential (CXP) was 24 V for BPDE-N2-dG and 15N-BPDE-N2-dG, 8 V for DBPDE-N6-dA and 15N-DBPDE-N6-dA. The curtain gas (CUR) was 35 l/h, the collision gas (CAD) was medium. The ionSpray voltage was 5500 V, the temperature was 400°C, gas 1 was 30 l/h and gas 2 was 15 l/h. The analysis was conducted using multiple reaction monitoring mode (MRM) to quantify BPDE-N2-dG and DBPDE-N6-dA as well as their internal standards 15N-BPDE-N2-dG and 15N-DBPDE-N6-dA, with the transitions of m/z 570 → 454 for BPDE-N2-dG, 575 → 459 for 15N-BPDE-N2-dG and 604 → 335 for DBPDE-N6-dA, 609 → 335 for 15N-DBPDE-N6-dA. All peaks were integrated and quantified by Sciex OS 1.5 software.

Results

Calibration curves for both BPDE-N2-dG and DBPDE-N6-dA adducts were constructed to include detection of relatively low levels of adducts. These constructed calibration curves appear to be linear for BPDE-N2-dG and DBPDE-N6-dA (r2 = 0.998 each as illustrated in Figure 3).

Figure 3.

Figure 3.

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Calibration curves for quantification of BPDE-N2-dG (A) and DBPDE-N6-dA (B) by HPLC-MS/MS.

We were routinely able to collect over 30 µg DNA from participants in this study using the above-mentioned buccal cells collection protocol and phenol-chloroform extraction method for DNA isolation. The amount of DNA we obtained is within the range of published data (10–287 µg) using similar methods (20–25). For the detection of BPDE-N2-dG in buccal cells from smokers (n = 13) and non-smokers (n = 15), 5 µg DNA from each participant was used. In Figure 4A, a representative HPLC-MS/MS chromatogram of BPDE-N2-dG analysis of DNA hydrolysate obtained from a participant’s oral buccal cells is shown. The area of the peak which co-eluted with 15N-BPDE-N2-dG (retention time = 2.53) was used to quantitate adduct levels. The levels of BPDE-N2-dG were significantly greater among smokers, which ranged from 5.90 to 33.05 adducts/108 dG with a mean of 20.18 ± 8.40 adducts/108 dG, than among non-smokers, which ranged from 0.15 to 3.84 adducts/108 dG with a mean of 0.84 ± 1.02 adducts/108 dG (P < 0.001) as shown in Figure 5A. The individualized data on BPDE-N2-dG levels in the oral buccal cells of smokers and non-smokers are shown in the supporting information (Supplementary Table 2, available at Carcinogenesis Online).

Figure 4.

Figure 4.

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Representative chromatograms of BPDE-N2-dG (A) and DBPDE-N6-dA (B) obtained from stable isotope dilution HPLC-MS/MS analysis of DNA isolated from human oral buccal cells.

Figure 5.

Figure 5.

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Levels of BPDE-N2-dG (A) and DBPDE-N6-dA (B) in oral buccal cells of smokers and non-smokers.

The levels of DBPDE-N6-dA were expected to be lower than BPDE-N2-dG based on their relative levels in cigarette smoke (4). Therefore, for the detection of DBPDE-N6-dA in smokers (n = 13) and non-smokers (n = 16), 30 µg DNA from buccal cells were used per participant. In Figure 4B, a representative HPLC-MS/MS chromatogram of DBPDE-N6-dA analysis of DNA hydrolysate obtained from a participant’s oral buccal cells is shown. The area of the peak which co-eluted with 15N-DBPDE-N6-dA (retention time = 2.69) was used to quantitate adduct levels. The levels of DBPDE-N6-dA were significantly greater among smokers, which ranged from 0.48 to 14.01 adducts/108 dA with a mean of 5.49 ± 3.41 adducts/108 dA, than among non-smokers, which ranged from 0 to 5.28 adducts/108 dA with a mean of 2.76 ± 2.29 adducts/108 dA (P = 0.019) as shown in Figure 5B. The individualized data on DBPDE-N6-dA levels in buccal cells of smokers and non-smokers are shown in the supporting information (Supplementary Table 3, available at Carcinogenesis Online).

The detection of DNA adducts resulting from exposure to DB[a,l]P in human tissues has not been previously reported. To further confirm the detection of DBPDE-N6-dA in human oral buccal cells, we pooled 300 µg of buccal cell DNA from eight smokers without the addition of the internal standard. The pooled DNA sample was hydrolyzed and purified by SPE using the same procedure described above for other samples prior to HPLC-MS/MS analysis. As shown in Figure 6, the peak eluting at 2.74 min (A) is consistent with the retention time of 15N-DBPDE-N6-dA (B), which was injected into HPLC-MS/MS in a separate run but immediately after the injection of the pooled DNA hydrolysate.

Figure 6.

Figure 6.

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(A) Detection of DBPDE-N6-dA in pooled DNA from oral buccal cells of smokers. without the addition of the internal standard; (B) The internal standard 15N-DBPDE-N6-dA.

Discussion

The human oral cavity is both a carcinogen metabolically active site (7,8) and the first site of exposure to cigarette smoke in smokers. In addition, oral epithelial cells can be easily collected and assessed for DNA damage which is a reliable marker for exposure and cancer risk (26). Among the analytical methods developed for the detection of PAHs derived DNA adducts, including BPDE-N2-dG and DBPDE-N6-dA, LC-MS/MS-based methods have been considered the best (10,27). Advantages include the ability to provide structural confirmation, sensitivity and utilization of stable isotope containing internal standards for unequivocal analyte identification and quantification. The majority of PAHs adducts, with the exception of DBPDE-N6-dA, express a common neutral loss of 2ʹ-deoxyribose fragment (116 µ) after a positive electrospray ionization (ESI)-MS/MS collision-induced dissociation observed in product ion spectra data (10). Therefore, we used the resulting adducted base (m/z 454 for BPDE-N2-dG and m/z 459 for 15N-BPDE-N2-dG) to quantitate BPDE-N2-dG. On the other hand, the major product ion derived from the PAHs moiety of DBPDE-N6-dA (m/z 335) was used to quantitate the level of DBPDE-N6-dA as we had previously reported (16). The fragment 335 is a daughter ion derived from DBPDE-dA after the loss of a deoxyribosyl adenosine fragment and a molecule of water.

Humans are exposed to both B[a]P and DB[a,l]P from cigarette smoke as well as from indoor and outdoor environmental exposures (4,5). The detection of DB[a,l]P in cigarette smoke was reported by Snooke in 1977, but the level was not quantified by this group (28). It is generally accepted that the level of DB[a,l]P present in tobacco smoke is much lower than the level of B[a]P (4). By the year 2000, B[a]P was found at levels of 20–40 ng/cigarette in smoke of non-filter cigarettes; on the other hand, the level of DB[a,l]P was reported in the amounts of 1.7–3.2 ng/cigarette in smoke of non-filter cigarettes (29). Furthermore, it was reported that by indoor air sampling of emissions from unvented coal combustion, the level of DB[a,l]P is 2.3 µg/m3, and the level of B[a]P is 10.3 µg/m3 (30). In soil and sediment samples, the levels of DB[a,l]P were in the range of tens to hundreds ppb for samples with different levels of pollution (31,32). These levels are roughly more than two orders of magnitude lower than those of B[a]P and the gap is reduced to a 40-fold difference in the case of strong contamination.

These results, together with our previous data on tumor development in mice, suggest that DB[a,l]P represents a relevant oral carcinogen in tobacco smoke. As we observed previously, mice exposed to DB[a,l]P with a cumulated dose of 2.74 µmol (calculated using a dose of 24 nmol a day, 3 times a week for 38 weeks) induced OSCC in 31% of mice and DB[a,l]P-associated DNA damage and mutations in their oral tissues (14).

In a previous study, despite the detection of BPDE-N2-dG in buccal cells using immunohistochemistry, the levels were not significantly different between smokers and non-smokers (13). In contrast to the immunohistochemistry method, upon using a quantitative LC-MS/MS method in the present report, we showed for the first time, the detection of two PAHs adducts derived from B[a]P and DB[a,l]P in human oral buccal cells, and these levels are significantly higher in smokers than in non-smokers. Despite the failure of detecting BPDE-N2-dG in human prostate tissue, salivary and oral cell DNA (12,33,34), using a sensitive method (LC-NSI-HRMS/MS) Villata et al. detected this adduct in 20 out of 29 DNA samples of lung cancer patients from smokers and non-smokers at levels of 3.1 and 1.3 adducts per 1011 nucleotides, respectively (35) and such levels were significantly lower than those measured by immunohistochemistry methods (36). However, in the present study the levels of BPDE-N2-dG in buccal cells of smokers are exceedingly higher than those found in lung cancer patients (35) which can be explained by the fact that the oral cavity is the first organ site exposed to carcinogens in cigarette smoke. Previous studies detected and quantified the levels of DNA damage derived from TSNA N-nitrosonornicotine (NNN) in buccal cells of smokers and such damage was reported to be an independent risk factor for HNSCC (37). Recent studies reported the detection of acrolein-DNA adducts and apurinic/apyrimidinic sites in the oral cells of smokers and non-smokers (38,39). The mutagenic DNA adducts derived from PAHs and TSNA in oral cavity, if left unrepaired, could provide genotoxic stimulus to act together with other weak mutagenic adducts and inflammatory/oxidative damage processes to lead to oral cancer as observed in smokers (39). Collectively, these biomarkers can provide novel targets not just to evaluate cancer risk following exposure to cigarette smoke and environmental pollutants, but also to monitor potential chemopreventive agents such as black raspberries, which were shown to enhance repair of bulky DNA lesions such as DBPDE-N6-dA (19,40). Finally, our results provide further support for the importance of PAHs in tobacco smoke as an important contributing factor in oral carcinogenesis.

Supplementary Material

bgac058_suppl_Supplementary_Table_S1
Click here for additional data file. (105.8KB, jpeg)
bgac058_suppl_Supplementary_Table_S2
Click here for additional data file. (191.1KB, jpeg)
bgac058_suppl_Supplementary_Table_S3
Click here for additional data file. (161.9KB, jpeg)

Acknowledgements

Carcinogens and their active diol epoxides were synthesized by the Penn State Cancer Institute Organic Synthesis Shared Resource. We thank The Penn State College of Medicine Mass Spectrometry Core Facility for conducting the HPLC-MS/MS analysis.

Glossary

Abbreviations

B[a]P

benzo[a]pyrene

DB[a,l]P

dibenzo[a,l]pyrene

OSCC

oral squamous cell carcinoma

PAHs

polycyclic aromatic hydrocarbons

TSNA

tobacco-specific nitrosamines

Contributor Information

Kun-Ming Chen, Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Yuan-Wan Sun, Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Nicolle M Krebs, Department of Public Health Sciences, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Dongxiao Sun, Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Jacek Krzeminski, Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Lisa Reinhart, Department of Public Health Sciences, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Krishne Gowda, Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Shantu Amin, Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Susan Mallery, College of Dentistry, Division of Oral Maxillofacial Pathology, Ohio State University, Columbus, OH, USA.

John P Richie, Jr, Department of Public Health Sciences, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Karam El-Bayoumy, Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA.

Funding

This work was supported by National Cancer Institute (CA173465).

Conflict of Interest Statement

None declared.

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