The Effects of Black Raspberry as a Whole Food-Based Approach on Biomarkers of Oxidative Stress in Buccal Cells and Urine of Smokers

Abstract

Cigarette smoke is a rich source of free radicals that can promote oxidative stress and carcinogenesis including head and neck squamous cell carcinoma (HNSCC) development; importantly, 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-iso-prostaglandin F_2α (8-isoprostane) are biomarkers of oxidative stress. Several mechanisms including the antioxidant properties of black raspberry (BRB) account for their chemopreventive effects. In the present clinical trial, we tested the hypothesis that BRB administration reduces biomarkers levels of oxidative stress in buccal cells and urine of smokers. One week after enrolling 21 smokers, baseline buccal cells and urine samples were collected before the administration of BRB lozenges for 8 weeks (5/day, 1 gm BRB/lozenge). Buccal cells and urine samples were collected at the middle and the end of BRB administration. The last samples were collected after the BRB cessation (washout period). We analyzed levels of 8-oxodG and 8-isoprostane (LC-MS/MS), urinary cotinine (ELISA) and creatinine (spectrophotometry). BRB significantly reduced the levels of 8-oxodG by 17.08% (p = 0.00079) in buccal cells and 12.44% (p = 0.034) in urine at the middle of BRB administration as compared to baseline; the corresponding values at the end of BRB administration were 16.46% (p = 0.026) in buccal cells and 25.72% (p = 0.202) in urine. BRB had no significant effect on the levels of urinary 8-isoprostane. BRB’s capacity to inhibit 8-oxodG formation of smokers’ buccal cells and urine is clearly evident and the reduction in 8-oxodG suggests antioxidant abilities are central to BRB’s HNSCC chemopreventive properties.

Introduction

Over 40% of all cancer diagnoses in the USA are tobacco related, and around 30% of cancer deaths are attributed to cigarette smoke (1). Head and neck squamous cell carcinoma (HNSCC) accounted for 878,348 new cases and 444,347 deaths worldwide (2); the corresponding values in the USA in 2023 are 54,540 new cases and 11,580 deaths (3). There are over 7,000 chemicals present in the tobacco smoke with about 70 compounds known to exert toxic and/or carcinogenic effects in animal models (4). Tobacco smoke is also a rich source of reactive oxidants (e.g. free radicals and aldehydes) that can lead to oxidative damage and inflammation which can contribute to the initiation and more so in the post-initiation phases of carcinogenesis (5–8). Oxidative stress occurs when production and accumulation of highly reactive species such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) exceed the ability of the biosystem to detoxify these species. It was previously reported that oral and lung cancer risks are increased in individuals with decreased capacity to protect against oxidative stress (5).

Buccal mucosa cells are directly exposed to numerous toxic and carcinogenic agents in tobacco smoke, making them the most valuable cell type to explore various molecular targets involved in the carcinogenesis process such as oxidative DNA damage (9). Notably, 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) is a key biomarker of endogenous oxidative stress as it is produced in vivo and readily quantifiable following target issue DNA hydrolysis (10) (Figure 1). Furthermore, the easy access and the non-invasive collection make oral mucosa cells a highly suitable cell type to monitor the impact of preventive agents on various oral mucosal damages-induced by tobacco smoking. Similarly, urine analyses also benefit from a non-invasive collection. Also, due to its reduced levels of organic or inorganic metals, urine samples are less likely than blood to reflect artifactually increased oxidative levels (11). The urinary biomarkers 8-oxodG and 8-iso-prostaglandin F_2α (8-isoprostane), a product of lipid peroxidation, are important markers (Figure 1) commonly used to assess the pathologic consequences of reactive oxygen species (ROS) on a systemic level (7,12,13). Cigarette smoking has been found to increase levels of oxidative stress biomarkers including urinary levels of 8-oxodG and 8-isoprostane (14). 8-Isoprostane belongs to the F₂-isoprostane family, and it is the oxidative product of arachidonic acid present in the membrane phospholipids of the body’s cells. Accordingly, 8-isoprostane has also been considered as a reliable biomarker of oxidative stress in vivo (15). Therefore, buccal cells and urinary levels of biomarkers of oxidative stress in human and rodents have been used as a non-invasive biomarker for the determination of reactive oxygen species (ROS) exposure/damage (16).

Figure 1.

Chemical structures of 8-oxodG and 8-isoprostane. Both 8-oxodG and 8-isoprostane are commonly used as biomarkers for oxidative stress and can be detected and accurately quantified in several biological fluids such as buccal cells, urine, blood and tissues obtained from animals and humans. In this study, we examined the effects of BRB on both biomarkers in buccal cells and urine of active smokers.

Treating cancers including HNSCC at late stages even with improved targeted therapies continues to be a major challenge and thus interception/prevention remains a desirable approach to manage and control this disease (5,17). Consumption of fruit and vegetable-based diets has the potential to lower the risk of cancer. This observation may reflect, in part, the high content of agents that inhibit carcinogen initiation and progression in these natural products (5). Several sources of phytochemicals have been proposed, and black raspberry (BRB) is among these candidates that showed great promise in cancer prevention in both preclinical and clinical investigations (17). BRB is very rich in antioxidants such as anthocyanin, which are known to exert various biological activities, including antioxidant, anti-tumor, anti-inflammatory, anti-lipid peroxidation, anti-cardiovascular disease, and modulation of hypoglycemic conditions (18). In addition, as BRB is approximately 90% water, freeze-drying concentrates BRB components by approximately 10-fold (19). To examine the effects of dietary consumption of freeze-dried BRB on the levels of oxidative stress in current smokers, this study focused on determining the levels of 8-oxodG in buccal cells and urine as well as urinary 8-isoprostane levels in smokers enrolled in a clinical trial to assess the potential cancer prevention effects of an oral lozenge delivering BRB.

Materials and Methods

Rigor and Reproducibility.

As a routine practice in our laboratory, the structural identity of biomarkers of oxidative stress is based on spectral analysis (NMR,MS) and the purity (>98%) was confirmed by LC-MS/MS analysis. Commercially available antibodies were authenticated using appropriate positive and negative controls. All data analyses, including details of statistical methodology, significance levels and statistical software are provided below. Participant were adult cigarette smokers, 21–75 years of age and include both men and women. This is a Phase 0 clinical trial; therefore, randomization and blinding were not required. In this study, we seriously considered the potential artifactual formation of 8-oxodG during sample processing. As recommended in previous studies, including our own, we added 8-hydroxyquinoline, an antioxidant, to the DNA extraction buffer solution to prevent the artifact formation of 8-oxodG (20,21).

Test Agent (Black Raspberry [BRB] Lozenges).

A dose of 5g freeze-dried BRB per day was provided to each participant in the form of dissolvable slow-release BRB lozenges (each containing 1g BRB freeze-dried powder, BerriHealth, Corvallis, OR). Each participant consumed 1 lozenge, 5 times/day and was instructed to not eat or drink 30 minutes following lozenge use. Typically, we found this formulation provides 15–30 minutes of target tissue contact time during dissolution in the mouth when not chewed. A comparable dose regiment in the form of troches has been used in previous Phase 0 clinical trials (22). All lozenges were prepared from a single harvest/year lot of black raspberry (Rubus occidentalis of the Munger cultivar) obtained from Corbett and Sandy Oregon. This form (lozenge) of BRB administration was selected over other forms previously used including consumption in diet or application in toothpaste like gels, based on ease of use and the ability to more accurately control dose (17). Inert ingredients and binders in each lozenge include 150 mg organic honey crystal, 150mg organic rice extract and cellulose. Lozenges were packaged in light protected PETE tamper proof bottles with oxygen absorbers and desiccants. The lozenge shelf life is over 3 years when unopened and 3 months after opening at normal room conditions according to manufacturer’s instruction. Lozenges possess a dark reddish-purple color with a total mass of 1.3 g each; the shape is round (7.43 mm x 22.79 mm). Immediately after receiving the lozenges, they were analyzed for compositions of their constituents (vitamins, minerals, anthocyanosides, free anthocyanidins, ellagic acid and quercetin dihydrate) by Eurofins Food Chemistry Testing (Madison, WI). The total anthocyanosides and total free anthocyanidins in our formulation based on the Certificate of Analysis is 41.8 mg/serving size; more details are provided in Supplementary Table S1.

Chemicals and Enzymes.

[¹⁵N₅]-Deoxyguanosine ([¹⁵N₅]-dG) was obtained from Spectra Stable Isotopes (Columbia, MD). 8-Isoprostane and [²H₄]-8-isoprostane were purchased from Cayman Chemicals (Ann Arbor, MI). Optima LC-MS grade water, acetonitrile and methanol were purchased from Fisher Scientific (New Jersey, USA). Other Chemicals and enzymes including proteinase K used in the present study were purchased from Sigma-Aldrich (St. Louis, MO). 8-oxodG and its [¹⁵N₅]-labeled 8-oxodG as an internal standard were prepared according to our previously published method (21). In brief, [¹⁵N₅]-dG (2.5 mg) or dG were dissolved in 2.5 mL of distilled H₂O water in ice bath. Subsequently, equal amount of freshly prepared 170 mM ascorbic acid and of 20 mM copper (II) sulfate were added. To the above mixture, a total of125 μL of 30% H₂O₂ was added. The reaction mixture was kept in an ice bath for another 2 hr; then, the resulted 8-oxodG was purified on an Agilent series 1100 HPLC system (Palo Alto, CA) that employed a ODS-AQ reversed-phase 5 μm 6.0 mm × 250 mm (120 Å) column (YMC, Wilmington, NC) and a CH₃CN/H₂O gradient monitored by UV absorbance at 254 nm. The fractions containing 8-oxodG were collected, pooled and further purified with another round of HPLC purification using the same HPLC condition described above. The collected sample was dried by Speed-Vac or under a mild stream of N₂.

Participant Demographics, Inclusion and Exclusion Criteria.

The age of the participants, 21–75, were recruited from a tobacco user research registry maintained by researchers at the Penn State College of Medicine in Hershey, Pennsylvania. These tobacco users were recruited into the registry through Penn State IRB-approved social media sites, hospital on-hold messaging system, flyers, and word of mouth. Potential participants were contacted by our clinical coordinator to be screened for eligibility and those eligible were invited to the study center for the first study visit. During this visit, a signed informed consent was obtained and final eligibility was determined according to the US Common Rule. Eligibility criteria included adult cigarette smokers, current use of at least 5 cigarettes per day for the past 12 months, an exhaled carbon monoxide (CO) reading of greater than 6 ppm, women not currently pregnant or nursing, ability to read and write in English and provide consent, no serious quit attempt or used of smoking cessation medication in the past 30 days, not planning to quit smoking in the next four months or plans to reduce consumption, no unstable or significant medical condition that could affect participant safety or biomarker data in the past 3 months, no abnormalities found from the oral health screening exam, no use of any non-cigarette nicotine delivery product in the past 7 days, no use of marijuana or other illegal drugs weekly or daily in the past 3 months, no allergies to black raspberry, no use of any high dose antioxidant supplements in the past month, no current use of antibiotics; and no heavy drinking (>4 drinks/day, 5 days/week). Participants completed baseline questionnaires on demographics, cigarette consumption, medical history, and concomitant medications. At study enrollment, and at every subsequent visit, all 21 participants’ medication usage (including over the counter) was reviewed and any changes were recorded. Out of the 21 participants whose data are reported, we had four participants with five instances of anti-inflammatory medication usage. One participant takes meloxicam qd while another takes meloxicam prn. One participant takes a daily low dose ASA and another used Advil Cold and Sinus twice. Our study design, in which every participant serves as their own internal control, assists by having an internal control for those who, by virtue of medical conditions, need to take anti-inflammatory medications daily. For those participants, the possible effects of daily meloxicam and low dose ASA are already recorded on their baseline levels. The participants who take daily medications should not be a problem as we are looking at changes to their baseline levels. Any additional reduction in oxidative stress related effects to their baseline status would therefore reflect the impact of the BRB lozenges. Weight, height, blood pressure/pulse, and exhaled CO were taken at each visit (height only at first visit). In this study, 21 smokers were included, as shown in the demographic data summarized in the Supplementary Table S2. The compliance was determined based on the following equation, and the participants deemed compliant based on a published report that if their rates were 80% or more (23): (number of lozenges provided - number of lozenges returned) / (number of lozenges instructed to take per day x number of days until return to the study) = rate of compliance.

Study Design.

The study design is summarized in Figure 2. This study was approved by the Penn State University Institutional Review Board (IRB#13621). This clinical is registered at clinicaltrials.gov (NCT04372914). It is a Phase 0 clinical trial which does not require randomization and each participant serves as their own control. One week after being enrolled in the study urine samples were collected at baseline (week 1), mid-BRB (week 5), end-BRB (week 9) and washout period (week 13). The amount of each of the 4 urine collections was more than sufficient for quantitative analysis of the biomarkers of oxidative stress. However, to ensure that adequate levels of DNA are obtained from buccal cells for the analysis of biomarkers of oxidative stress, two sample collections (one week apart) for each collection period were obtained as follows: baseline (weeks 0, 1), during the middle of BRB administration (weeks 4, 5), at the end of BRB administration (weeks 8, 9), wash out period after BRB cessation (weeks 12, 13). The 1-week waiting period between sampling allows for adequate cell recovery and will enable release of the most differentiated keratinocytes that have been directly exposed to BRB. The participant was instructed to consume 5 BRB lozenges per day (each lozenge containing 1g BRB freeze-dried powder), continuously for 8 weeks. All of the collected samples were aliquoted and kept in a −80 °C freezer.

Figure 2.

The design of the clinical study demonstrating the duration of BRB supplementation and time points for the collection of urine and buccal cells in smokers.

Buccal 8-oxodG analysis.

Buccal cells of the participants were collected following our previous reported procedure (24). In brief, after preliminary rinses, participants used a soft bristle toothbrush to brush the inside cheeks of the mouth following with a 2-minute rinse with 20 ml of saline, and then collected the solution, and the toothbrush was agitated in the solution to loosen cells. DNA isolation from oral buccal cells was conducted using a phenol-chloroform extraction method as previously described by us (24) with the addition of 8-hydroxyquinoline, an antioxidant, in the alkaline lysis buffer (21). Buccal cell DNA was pooled from two collections from each period (baseline, mid-BRB, end-BRB, and washout periods). Similarly, DNA hydrolysis was conducted using our reported method (21). The DNA solution was spiked with 4 pmol of [¹⁵N₅]8-oxodG before the addition of deoxyribonuclease I (1 unit, Thermoscientific, U.S.A.). The resulting solution was then incubated with nuclease P1, and alkaline phosphatase. HPLC was used to quantify 2’-deoxyguanosine (dG) of the DNA hydrolysate followed by purification with solid phase extraction (SPE) using Env+ Isolute cartridges as previously published (21). Briefly, after preconditioning of the SPE column, a 1:1 diluted sample was loaded onto the column and washed twice with 300 μl H₂O. The column was further washed with 1 mL of 2% MeOH; then the fraction contains 8-oxodG was eluted in 2 × 500 μl of 20% (v/v) acetonitrile in methanol, and concentrated under mild N₂ stream to dryness. The 8-oxodG analysis was conducted using a Sciex QTRAP 6500+ mass spectrometry coupled with a Sciex EXion HPLC separation system. We used a 1.7 μm Acquity UPLC BEH C18 analytical column (2.1 × 100 mm, Waters, Ireland) to separate analytes from impurities. A flow rate of 0.3 mL/min with the following gradient elution conditions was used: initial at 5% mobile phase B (0.1 % formic acid in acetonitrile) and 95 % mobile phase A (0.1% formic acid in water); this condition was kept for 2.5 min, followed by a linear gradient to 90 % mobile phase B in 0.5 minute, and kept at 90% mobile phase B for 1.5 additional minutes to flush the column before returning back to initial conditions to equilibrate the column.

The Sciex QTrap 6500+ mass spectrometer was equipped with an electrospray ionization probe operated in positive mode under the following condition: the decluster potential (DP) was 16V; the entrance potential (EP) was 10 V; the collision energy (CE) was 23 V; the collision cell exit potential (CXP) was 10 V. 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 20 L/h, and gas 2 was 15 L/h. We used multiple reaction monitoring mode (MRM) to analyze and quantify 8-oxodG and the internal standard [¹⁵N₅]-8-oxodG, with the transitions of m/z 284 > 168 for 8-oxodG, 289 > 173 for [¹⁵N₅]-8-oxodG. All peaks were integrated and quantified by Sciex OS 1.5 software.

Urinary 8-oxodG analysis.

A volume of 0.15 mL of urine sample containing 4 pmole of [¹⁵N₅]-8-oxodG was partially purified using the Env+ Isolute cartridges as described above. Then, the urinary 8-oxodG was analyzed by the LC-MS/MS method described above. Urine creatinine levels were measured (see below) and used to normalize 8-oxodG levels.

Urinary 8-isoprostane analysis.

After spiking 1 mL urine with 4 μL internal standard [²H₄]-8-isoprostane (1.2 ng), the sample was diluted with 400 μL of 5% methanol followed by 400 μL of 1% formic acid to maintain acidic condition. The sample was votexed and centrifuged at 4°C, 4000 rpm for 3 min before loading onto a Phenomenex Strata-X cartridge for SPE analysis. The SPE cartridge was preconditioned by 1 mL methanol, and then was equilibrated by 1 mL 0.1% formic acid. After loading the samples, the SPE cartridge was washed by 0.1% formic acid and hexane. The cartridge was dried before the analytes were eluted out with 1mL of 100% ethyl acetate. The eluent was then dried by Speed Vac and followed by reconstitution with 40 μL 50% methanol containing 0.1% formic acid. The reconstituted sample was centrifuged at 4°C, 4000 rpm for 5 min before injected into HPLC-MS-MS system. The analysis of 8-isoprostane was also conducted by the above mentioned Sciex QTRAP 6500+ and EXion UHPLC-MS/MS system using a 1.7 μm Acquity UPLC BEH C18 analytical column (1.0 × 100 mm, Waters, Ireland) to separate 8-isoprostane and its metabolites, isomers as well as other impurities. The eluting gradient was conducted using a flow rate of 0.15 mL/min with the following conditions: 7.5 minutes in 35% mobile phase B (0.1% acetic acid in acetonitrile:methanol (50:50) and 65% solvent A (0.1 % acetic acid in water), a linear gradient to 100% mobile phase B in 0.5 minute, and keep the 100% mobile phase B for 1.5 minutes to flush the column before back to initial conditions to equilibrate the column.

The Sciex QTrap 6500+ mass spectrometer was equipped with an electrospray ionization probe operated in negative mode. The decluster potential (DP) was −86 V for; the entrance potential (EP) was −10 V, the collision energy (CE) was −34 V and the collision cell exit potential (CXP) was −15 V, while the curtain gas (CUR) was 35 L/h, the collision gas (CAD) was medium. The ionSpray voltage was −4500 V, the temperature was 530 °C, gas 1 was 25 L/h, and gas 2 was 30 L/h. The multiple reaction monitoring mode (MRM) was used to analyze and quantify 8-isoprostane and 8-isoprostane-d4, with the transitions of m/z 353.1 > 193.2 for 8-isoprostane and 357.1 > 197.2 for 8-isoprostane-d4. All peaks were integrated and quantified by Sciex OS 3.0 software.

Creatinine analysis.

Levels of urinary creatinine were determined spectrophotometrically based on the Jaffe’s reaction between creatinine and alkaline picrate (25). Briefly, urine was diluted and 50 μL of each sample were mixed with 200 μL of 0.12% of picric acid in 0.15 N sodium hydroxide in a 96 well plate. The plate was incubated with shaking, at room temperature for 30 minutes. Absorbance at 490 nm was measured using a microplate reader (Biotek Synergy HTX). Creatinine levels were quantified based on constructing a creatinine standard curve.

Cotinine analysis.

Levels of urinary cotinine were determined using a commercially available competitive enzyme-linked immunoassay (ELISA) kit from Calbiotech (El Cajon, CA, USA) following the manufacturer’s protocol. Briefly, standards, controls, and diluted urine samples were added in duplicate to wells coated with a polyclonal antibody to cotinine. Enzyme conjugate was added and the plates were incubated, protected from light, at room temperature for one hour. All wells were thoroughly washed. After the addition of a substrate reagent, each plate was incubated for an additional 20 minutes. Subsequently, stop solution was added and absorbance was measured at 450 nm using the microplate reader. The concentration of cotinine was calculated against the standard curve generated from the standards supplied in the kit.

Sample size calculation

The 21 participants enrolled in this study came from a larger project studying the impact of black raspberry on several biomarkers in the urine and buccal cells of active smokers. In this larger project, 47 participants are required based on the tobacco specific nitrosamine Ń-nitrosonornicotine (NNN)-releasing adduct (4-hydroxy-1-(3-pyridyl)-1-butanone [HPB]) as the primary biomarker and powered to detect a change of proportion of HPB-releasing adduct values after black raspberry administration. However, the biomarkers examined in this paper (8-oxodG and 8-isoprostane) are considered as secondary endpoints. At the time of data analysis, samples obtained from 21 participants were available. Although no power analysis was performed for the secondary biomarkers in this paper, it does not affect the validity of our results (26).

Statistical analysis.

This is a smaller longitudinal design in which each participant is measured at 4 time points: baseline (weeks 0, 1), during the middle of BRB administration (weeks 4, 5), at the end of BRB administration (weeks 8, 9), wash out period after BRB cessation (weeks 12, 13). Our analysis first computes the change from baseline (for time point 2, time point 3 and time point 4) for each participant and then tests if this change differs from 0 on the population level at time point 2, time point 3 and time point 4, respectively. Statistical power is enhanced by treating each participant (at baseline) as own control at later time points. Statistical robustness is increased by including the Wilcoxon rank test. For all endpoints, their distribution at baseline, mid-BRB, end-BRB and washout period were first inspected using boxplots; a log transformation was then applied to each endpoint so that its distribution is more symmetric and closer to a normal distribution. The change of these variables at each period, respectively from their corresponding value at baseline was calculated in the transformed log-scale. The t-test and Wilcoxon rank sum test were used to test if each change has an underlying mean or median of 0. A longitudinal approach is also used to analyze the trajectory of each endpoint over the four periods with similar results. The correlation of 8-oxodG levels between buccal cells and urine was plotted and summarized by Spearman’s rank correlation. For all endpoints, their distribution at baseline, mid-BRB, end-BRB and washout period were first inspected using boxplots; a log transformation was then applied to each endpoint so that its distribution is more symmetric and closer to a normal distribution. The change of these variables at each period, respectively from their corresponding value at baseline was calculated in the transformed log-scale.

Data Availability.

Data generated in this study are available within the article and its supplementary data files.

Results

The results of this study on the effects of BRB on urinary levels of cotinine, 8-oxodG and 8-isoprospane, and buccal cells’ levels of 8-oxodG in smokers are provided in the Supplementary Table S3. As described above, the compliance of all participants exceeded 80% based on a previously reported approach (23).

Analysis of buccal cell levels of 8-oxodG.

Representative chromatograms of the analyses of 8-oxodG and [¹⁵N₅]-8-oxodG by LC-MS/MS are shown in Figure 3A. The area of the peak which co-eluted with [¹⁵N₅]-8-oxodG (retention time = 1.87 min) was used to quantitate the level of 8-oxodG. The baseline level of 8-oxodG (mean ± SD) in the buccal cells of participants before the consumption of BRB is determined to be 3.22 ± 1.21 8-oxodG/ 10⁴ dG. We found that during BRB administration and at the end of BRB administration, the level of 8-oxodG in buccal cells (2.67 ± 1.05 and 2.69 ± 1.10 8-oxodG/ 10⁴ dG, respectively) are lower as compared to baseline (p = 0.00079 and 0.026, respectively from t-test). The levels of buccal cell 8-oxodG (2.95 ± 1.08 8-oxodG/ 10⁴ dG) at the washout period are not significantly different from baseline level. In Figure 3B, the levels of buccal cell 8-oxodG were plotted after log transformation. The effects of BRB on 8-oxodG in buccal cells of the individual participants (spaghetti plot graph) throughout the duration of the clinical trial is shown in Figure 3C; the results showed that BRB decreased, increased, or had no effect on the levels of 8-oxodG in buccal cells.

Figure 3.

HPLC-MS/MS analysis of 8-oxodG in buccal cells of smokers. (A) Representative chromatograms of 8-oxodG and [¹⁵N₅]-8-oxodG obtained from stable isotope dilution HPLC-MS/MS analysis of smokers’ buccal cells; (B) Analysis of levels of 8-oxodG in buccal cells after log transformation; (C) The effects of BRB on 8-oxodG in buccal cells of the individual participants.

Analysis of urinary levels of 8-oxodG.

Before the consumption of BRB, the average level (mean ± SD) of urinary 8-oxodG normalized with urinary creatinine is 13.26 ± 22.25 pmol / mg creatinine; this level is considered as the baseline level to examine the effects of BRB consumption on urinary level of 8-oxodG. We found that at the mid-BRB collection of urine, the levels of urinary 8-oxodG (11.61 ± 22.85 pmol / mg creatinine ) are lower as compared to baseline (p = 0.034). The levels of urinary 8-oxodG at the end-BRB collection (9.85 ± 13.97 pmol / mg creatinine) were reduced but not significantly different from baseline level. However, these urinary levels were significantly (7.77 ± 6.65 pmol / mg creatinine) lower than the baseline (p = 0.017) at the washout period. In Figure 4A, the levels of urinary 8-oxodG was plotted after log transformation. Our results demonstrated that there is a correlation between levels of buccal cells and urinary levels of 8-oxodG as shown in Figure 4B with a correlation coefficient of 0.44 (p = 3.57e-05). The effects of BRB on 8-oxodG in the urine of individual participants (spaghetti plot graph) throughout the duration of the clinical trial is shown in Figure 4C.

Figure 4.

The effect of BRB administration on 8-oxodG in urine samples. (A) Analysis of urinary 8-oxodG levels normalized per their individual creatinine levels during the study after log transformation; (B) Correlation of levels of 8-oxodG in buccal cells and levels of 8-oxodG in urine of participants; (C) The effect of BRB on urinary levels of 8-oxodG of the individual participants.

Analysis of urinary levels of 8-isoprostane.

Representative chromatograms of the analysis of 8-isoprostane and its internal standard by LC-MS/MS is shown in Figure 5A. The area of the peak which co-eluted with [²H₄]-8-isoprostane (retention time = 6.60 min) was used to quantitate the level of 8-isoprostane. The levels of urinary 8-isoprostane (mean ± SD) normalized with urinary creatinine before the consumption of BRB were found to be 0.48 ± 0.42 pmol / mg creatinine, and they are considered as the baseline levels of 8-isoprostane which was used for comparison with the detected levels of 8-isoprostane after the consumption of BRB. The levels of 8-isoprostane during BRB administration, at the end of BRB administration and washout collections were 0.47 ± 0.39, 0.48 ± 0.40 and 0.46 ± 0.28 pmol / mg creatinine, respectively. We found that the urinary levels of 8-isoprostane were not significantly changed with the consumption of BRB in log transformation scale (Figure 5B).

Figure 5.

The effect of BRB administration on 8-isoprostane in urine of smokers. (A) Representative chromatograms of 8-isoprostane and [²H₄]-8-isoprostane obtained from stable isotope dilution HPLC-MS/MS analysis of smokers’ urine; (B) Analysis of urinary 8-isoprostane levels normalized per their individual creatinine levels after log transformation in smokers during the study.

Analysis of urinary levels of cotinine and creatinine.

Urinary levels of cotinine and creatinine were analyzed. We found that the levels of cotinine ranged from 550 – 21,000 ng cotinine / mg creatinine in the urine collected from our participants throughout the duration of this study. The levels of cotinine were normalized by dividing the level of cotinine with the corresponding level of creatinine as shown in Supplementary Figure S1. We found that the consumption of BRB did not affect the urinary cotinine level of our participants. Furthermore, the results demonstrate that cotinine levels in each participant differ across the various time points suggesting a modulation of tobacco consumption (Supplementary Figure S2, spaghetti plot graph).

Discussion

Tobacco smoking is causatively associated with several diseases including HNSCC, and literature data suggest that some of these diseases are associated with oxidative stress (7). Previous mechanistic studies of tobacco smoke-induced carcinogenesis demonstrated tumor promotion as well as co-carcinogenesis are mediated by oxidative damage and the ensuing inflammation of smoking (6–8). It is worth noting that we found that the levels of 8-oxodG and 8-isoprostane in smokers analyzed by LC-MS/MS are consistent with data published previously by others (14,27). Our results showed that consumption of BRB at the dose (5 g/day) used in this study for 8 weeks resulted in a significant reduction of the levels of 8-oxodG in the buccal cells during and at the end of BRB administration and urine (at mid-BRB administration) of smokers, but had no effects on the urinary level of 8-isoprostane throughout the study. The observed “rebound” effect after BRB cessation (washout period) whereby 8-oxodG levels in buccal cells returned to levels comparable to those measured at baseline is consistent with an apparent need for continuous antioxidant supplementation. A similar dose of BRB has been used in a previous clinical trial (22) and even a much higher dose was used by others (17). Identification and quantification of 8-oxodG in the oral buccal cells may define smokers at risk for HNSCC development and the inhibition of this biomarker by BRB suggesting the potential protective effects of BRB as a whole food-based approach for interception and prevention of this disease in future clinical chemoprevention trials. In previous clinical trials, both oral and topical application of BRB generally did not produce toxicity, but some patients experienced mild disturbances of the gastrointestinal tract (diarrhea and constipation) which resolved in a few days (28). Despite the relatively low toxicity of BRB (29), the toxicity of its individual constituents remains to be determined. In the event that no individual toxic constituents are identified, sustained use of the BRB lozenges should not be problematic.

Preclinical studies also reported that BRB inhibited the formation of 8-oxodG induced by toxic/carcinogenic agents. Harris et al showed that BRB inhibited azoxymethane induced 8-oxodG in rat urine (30). Another group reported dietary consumption of 10% BRB for 4 weeks was found to reverse the arsenic (both 1 and 10 ppm) induced increase of urinary 8-oxodG in mice measured by LC-MS/MS-ESI (12). Clearly, the results of both preclinical (12,30) and clinical studies including the present study support that consumption of BRB can reduce 8-oxodG, a marker of DNA oxidative damage (13). The dominant bioactive phytochemicals in BRB are polyphenolic compounds such as the anthocyanins which can elicit redox scavenging of ROS that can lead to oxidative stress (31). In fact, BRB itself contains several antioxidant enzymes (catalase, super oxide dismutase, glutathione peroxidase) that can facilitate the catabolism of ROS (31,32). Furthermore, we and others have demonstrated that BRB promotes the biosynthesis of glutathione and several antioxidant enzymes capable of removing free radicals thereby enhancing its antioxidant capacity (33–38).

To cope with DNA damage, living cells have also developed elaborate DNA repair machinery and in mammalian systems such DNA damage is repaired by two overlapping pathways namely nucleotide excision repair (NER) and base excision repair (BER). We recently showed that BRB extract (BRBE) enhanced both NER and BER activity via assessment of an oxidized 8-oxodG product, guanidinohydantoin in HeLa cells (39). BRB’s direct impact, however, on 8-oxodG remains to be determined (39). Interestingly, it appears that BER is not involved in the reduction of urinary level of 8-oxodG by BRB as BER resulted in the release of 8-oxo-guanine, but not 8-oxodG (27,40,41). The enzyme MTH1 (human MutT homologue 1) can hydrolyze oxidized nucleotides, such as 8-oxodGTP in the dNTP pool to 8-oxoGMP to avoid the incorporation of the oxidized nucleobase into DNA to reduce DNA damage (27,41–43). Therefore, the mechanisms by which BRB reduce the urinary levels of 8-oxodG in smokers may involve reduction of ROS by phase II conjugation reaction or modulation of nucleotide metabolic enzymes such as MTH1. In fact, as described above, we previously demonstrated that BRBE can enhance phase II conjugation reaction through increasing GSH synthesis (44).

It is possible that the inhibition of markers different from 8-oxodG may require different levels of BRB, such as the formation of 8-isoprostane. In a previous study the analysis of urinary levels of 8-isoprostane in smokers was compared at 4 and 20 weeks, and the results demonstrated longitudinal stability of this biomarker (45). Considering the stability of this urinary biomarker, it is possible not only a higher dose but also a longer duration of BRB supplementation may be required to reduce the urinary levels of 8-isoprostane. In fact, the results from a longer duration showed that a higher BRB daily dose (26 weeks, 32–45 gm qd) in persons (n = 10) with Barrett esophagitis reduced lipid peroxidation, substantiates this premise; however, the level of 8-oxodG measured by ELISA was not significantly changed (13). Therefore, in the present study we used LC-MS/MS analysis for accurate quantification of biomarkers of oxidative stress. Urine is also an ideal biological fluid for clinical studies on oxidative biomarkers, because it can be obtained noninvasively and the artifactual oxidation of dG or arachidonic acid are less likely to occur because of their low abundance that are present in the urine (27). In this study, all of the buccal cell and urine samples exhibited high inter-sample variations as indicated by the SD at every collection period. These variations appear consistent with the outbred human population and the extensive inter-individual heterogeneity in antioxidant defenses (46).

Cotinine is the most widely used biomarker of tobacco smoke exposure; more than 70% of nicotine is metabolically converted into cotinine which is further converted into trans-3’-hydroxycotinine (47). The normalization of urinary cotinine levels by its ratio to urinary creatinine was previously suggested to be important not only to urinary levels of cotinine, but also to other urinary biomarkers (48). It was suggested that a cut-off level of cotinine/creatinine ratio of over 60 ng/mg can be used to investigate environmental tobacco smoke exposure in association with lower respiratory tract infection (49). A very wide range of cotinine/creatinine ratio (30–550 ng cotinine / mg creatinine) had been determined to differentiate smokers from non-smokers. Clearly, based on the cotinine/creatinine ratio, the urinary cotinine levels detected in our participants are consistent with the reported levels of smokers (7,49,50). In addition, we found that the consumption of BRB did not affect the urinary cotinine level of our participants. Therefore, the consumption of BRB has no effect on the amount of nicotine uptake by our participants.

We acknowledge that there are some limitations in the present study such as the use of only one dose of BRB. Another limitation of the present study is that our results cannot be extrapolated to predict the effects of BRB on oxidative stress markers among smokers of different genders and racial backgrounds. These limitations can be considered in the design of future studies since the incidence and mortality of HNSCC are higher in men than women and African American’s have a higher incidence of advanced disease and poor survival compared to whites (3). Despite such limitations, our results show that oral lozenge delivery of BRB can reduce 8-oxodG, a marker of oxidative DNA damage in both buccal cells and urine of smokers, and suggest BRB’s potential benefits as a chemopreventive agent against the development of HNSCC.

Prevention Relevance Statement.

Cigarette smoke contains highly active components namely free radicals that can promote oxidative stress and oral cancer. We found that black raspberry (BRB) inhibited the formation of oxidative stress markers in the oral cavity and urine of smokers suggesting the antioxidant abilities of BRB in preventing oral cancer.