Lower expressions of the human bitter taste receptor TAS2R in smokers: reverse transcriptase-polymerase chain reaction analysis

Background Despite the fact that smokers have deficit in detecting taste, particularly bitter taste, no study has investigated its biological correlate. Methods In this context, we compared the expression of the bitter taste receptor gene, taste 2 receptor (TAS2R) in the tongues of smokers and non-smokers. Tissue samples were collected from the lateral portion of the tongues of 22 smokers and 22 age- and gender-matched healthy volunteers (19 males and three females) with no history of smoking. Reverse transcriptase-polymerase chain reaction was used to examine the expression of TAS2R in the two groups, and the effect of aging on TAS2R expression was also assessed. Results TAS2R expression was significantly lower among smokers than non-smokers (t = 6.525, P < .0001, 11.36 ± 6.0 vs. 2.09 ± 2.8, mean ± SD, non-smokers vs. smokers). Further, a positive correlation between age and expression of TAS2R was observed in non-smokers (r = .642, P = .001), but not smokers (r = .124, P = .584). This correlation difference was significant (Z = 1.96, P = .0496). Conclusions Smokers showed a significantly lower expression of the bitter taste receptor gene than non-smokers, which is potentially caused by their inability to acquire such receptors with age because of cigarette smoking, in contrast to non-smokers.


Background
Accumulating evidence has demonstrated that cigarette smoking induces non-critical adverse effects [1][2][3][4][5] in addition to the well-established critical adverse effects such as the development of cancers and coronary artery diseases [6,7]. Among the non-critical adverse effects of smoking, bitter taste impairment is particularly important in the field of public health [8][9][10][11], not only because it affects the quality of life of smokers but also because it may promote the continuation of smoking which will eventually cause critical adverse effects in both smokers and their relatives. However, despite the importance of this as a potential mechanism for the continuation of smoking [12][13][14], to the best of our knowledge no study has investigated the biological mechanism of this bitter taste disability.
Bitter tastes are perceived by taste receptor cells (TRC) which are assembled into taste buds [15]. In humans, TRCs detect bitter stimuli via a bitter taste receptor, the taste 2 receptor (TAS2R) G-protein-coupled receptor (GPCR) [15]. Although their roles have yet to be elucidated, human genome studies have shown that the TRC-expressed GPCR multi-gene family consists of 25 T2R members [16]. They are named by adding numbers at the end, such as TAS2R1 or TAS2R3.
There are some studies that suggested potential genetic effect of smoking cigarette, such as association between smoking cigarettes and SNP expression [17]. We hypothesized that cigarette smoking induces a lower expression of TAS2R in smokers compared with nonsmokers. To test this, we performed reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of TAS2R expression in tongue tissue samples of both groups.

Participants and clinical evaluation
Twenty-two smokers (20 males and two females) with no history of taste disorders were recruited to participate in the study. They are mainly college students and acquaintances of the authors. The following information was obtained from each of these participants: age (years), smoking duration (years), and the number of cigarettes smoked per day; these data were also used to determine the Brinkman index, which is defined as the number of cigarettes smoked daily multiplied by the number of smoking years. Twenty-two age-and gender-matched healthy volunteers (19 males and three females) with no history of smoking were employed as controls. To avoid age-associated loss of expression, we excluded participants aged 65 years or over from both groups.
The ethics committee of Sanyo Gakuen College, Showa women's University and Medical school of Nihon University approved the current study. After a complete explanation of the study to the subjects, written informed consent was obtained from every participant.

Sample collection
We adopted a scraping method to collect tissue samples for this study because it was difficult to perform an excisional biopsy in several cases. This scraping method is so minimally invasive that it is often used to collect tissue samples from the oral cavity. We considered it suitable as a tissuesampling method in the present study for the examination of taste receptor gene expression of the tongue. Thus, by scraping with plastic tube, we obtain the epithelium specimen from tip, dorsum and foliate papilla in the tongue. None of the participants had anything to eat or drink for at least 90 min before scraping the tongue surface. The specimens were vortexed with TRIzol Reagent (Invitrogen) of 0.5ml for 30 seconds. Then, upper aqueous phase was transferred carefully into fresh tube without disturbing the interphase and used as specimens of interest. The lower red phenol-chloroform phase was used for assessment of potential contamination of DNA. Specimens were refrigerated at temperature of -5°C for a night then -35°C for a few days before RT-PCR. Based on the instruction of TRIzol Reagent, all RNAs were purified from the specimen. To make sample to be suitable for RT-PCR, the pellet of total RNA was dissolved in DEPC-treated water of 10 μL. The concentration of total RNA in the sample was measured. The sample was stored at temperature of -80°C.

Reverse transcriptase-polymerase chain reaction
RT-PCR was performed as previously described [18]. Briefly, the primer pairs (Table 1) were designed from 5' sides and 3' sides of TAS2R genes. Using SuperScript III (Invitrogen, Carlsbad, CA, USA) and random primers (Invitrogen, Carlsbad, CA, USA), first strand cDNAs were reverse-transcribed from 0.65μg total RNA. The cDNA of 0.5μL was used as a template for PCR reaction. The PCR was performed with ExTaq (Takara Bio Inc., Shiga, Japan). The PCR amplification was started with a denaturation step at 95°C for 5min followed by 35 cycles consisting of denaturation (95°C for 30 sec), annealing (59°C for 30 sec) and extension (72°C for 60 sec). The oligonucleotides used for TAS2R amplification is shown in Table 1. The sample was incubated for seven minutes at 72°C. After PCR reaction, the specimen was stored at 4°C. The length and amount of PCR product was analyzed using 2100 Agilent Bioanalyzer (Agilent Technology, Tokyo, Japan) with 7500 Agilent DNA Reagent.

Assessment of potential contamination of DNA
After ethanol precipitation of the lower red phenolchloroform phase of the specimen, genomic DNA was extracted. Then, extracted genomic DNA was analyzed by agarose gel electrophoresis.

Statistical analyses
Independent t-tests were performed to compare the differences in age between smokers and non-smokers, and in the expression of the bitter taste receptor gene TAS2R between smokers and non-smokers. Statistical significance was set at P < .05.
Independent t-tests were also performed to compare the difference in the expression of TAS2R subfamily genes between smokers and non-smokers. Because we had no a priori hypothesis for potential expression level differences in the TAS2R subfamily, statistical significance was set at P < .0024 (=.05/21, number of TAS2R subfamily members successfully measured), correcting multiple comparisons using Bonferroni method.

Correlation analysis
To explore the potential effect of age on the expression of bitter taste receptor genes, we performed Pearson's correlation analyses between age and TAS2R expression in non-smokers and smokers separately. Differences in correlations between groups were examined with the Fisher r-to-z transformation [19]. The statistical threshold was set at P < .05.
Further, to examine the effect of potential factors related to bitter taste receptor gene expression among smokers, we conducted Pearson's correlation analyses between TAS2R expression and period of smoking (years), number of cigarettes smoked daily, and the Brinkman index. The statistical threshold was also set at P < .05.

Assessment of potential contamination of DNA
As shown in Figure 1, genomic DNAs were fragmented into less than 200bp.

Comparison of TAS2R expression between smokers and non-smokers
The mean expression of TAS2R in smokers and nonsmokers was 2.09 and 11.36, respectively. This difference was statistically significant, with smokers having a significantly lower TAS2R expression compared with non-smokers (t = 6.525, P < .0001, Table 3).

Correlation analyses
Age was significantly positively correlated with the expression of TAS2R in the non-smoker group (r = .642, P = .001) but not in the smoker group (r = .124, P = .584) (Figure 2a). Fisher's r-to-z transformation revealed a significant difference in the correlations between the smokers and non-smokers (Z = 1.96, P = .0496), indicating that the age-expression of bitter taste receptor relationship in smokers was significantly different from that of non-smokers (Figure 2a). Pearson's correlation analyses found no significant association between TAS2R expression and duration of smoking (r = .217, P = .331) (Figure 2b), number of cigarettes smoked daily (r = -.105, P = .641) (Figure 2c), or the Brinkman index (r = .104, P = .645) (Figure 2c).

Discussion
In the current study, we detected a significantly lower expression of TAS2R in smokers compared with age-and gender-matched non-smokers. Sub-analyses also demonstrated that bitter taste receptor gene expression was significantly lower in five of the 21 receptors studied, TAS2R7, 9, 30 38, and 45. Although we did not investigate whether the current smokers have impaired taste, our results strongly suggest that the well-documented disturbance of taste seen in smokers derives from the lower-than-normal expression of TAS2R caused by smoking [8][9][10][11]. There are several potential substances that may affect the expression of TAS2R, the most potentially relevant of which is nicotine [20]. Indeed, previous studies have reported that nicotine decreased the expression of several genes [21,22]. Moreover, nicotine is known to induce the development of various cancers by causing DNA damage [23].
We also demonstrated that age-related changes in the expression of bitter taste receptors in smokers were significantly different from those in non-smokers, which is indicative of the potential accumulative effects of smoking. Although the association between age and the expression of bitter taste receptors is controversial and the relationship may not be consistent [24], it was reported to be greatly influenced by various environmental factors [25]. For instance, we previously reported that bitter taste receptor expression was influenced by exposure to a wide variety of foods; in other words the more food types people experience, the greater their expression of bitter taste receptors (presented at 15 th International Symposium on Olfaction and Taste at San Francisco). In the present study, we only included participants under 65 years of age. Thus, although additional factors might affect the relationship between age and bitter taste receptor expression, our results suggests that cigarette smoking impairs the acquisition of bitter taste receptors that occurs with aging and exposure to more tastes. We observed no significant association between bitter taste receptor expression and smoking history, such as its duration and the number of cigarettes smoked daily. A possible explanation for this is that the expression of bitter taste receptors is so sensitive to environmental factors that it is conceivable that the expression would decrease to zero very quickly [25].
The current investigation has a number of methodological limitations. First, although we enlisted an age-and gender-matched non-smoker comparison group, several potential confounding factors were not considered that may affect TAS2R expression, such as genetic variability. Second, although this control group had no history of smoking, the potential effect of secondhand smoke was not investigated. However, unlike smoking, secondhand smoke does not enter the oral cavity, so its effect on TAS2R expression is potentially limited. Moreover, we mainly recruited adult males as the control group, and the Japanese Ministry of Health, Labour and Welfare reported that the prevalence of smoking among females is very low if their husbands do not smoke. Thus, although this should have been confirmed directly by questionnaire, the likelihood of exposure to secondhand smoke in our control group was low. Third, we  Relation between number of expression of TAS2R and age and smoking behaviors. A significant positive correlation was observed between age and bitter taste receptor expression in non-smokers but not in smokers (a). There was no significant association between bitter taste receptor expression and duration of smoking (b), the number of cigarettes smoked daily (c), and the Brinkman index (d).
did not investigate the mechanism by which cigarette smoke affects TAS2R expression, but it is assumed that there is a biochemical pathway that nicotine interferes with that decreases TAS2R expression. Fourth, although we have collected samples from the foliate papillae of the lateral portion of the tongue which is assumed to express bitter taste receptor well, and adopted standard method of the RT-PCR, no controls for presence of taste bud material for the RT-PCR experiments were performed.
As it is state-of-the-art to perform and show control reaction such as samples amplified from not incubated with reverse transcription, further studies that adopted such method to improve the quality and reliability of the data are expected. Fifth, although we have shown that the lower phase of the TRIzol Reagent treated specimen included only genomic DNA fragments whose sizes are less than 200bp, suggesting that genomic DNA was fragmented into small factions by a process of homogenate we have not conducted enzymatic treatment of DNA degrading. Thus, there still remains possibility that the result of the present study is influenced by genomic DNA contamination. Sixth, although experimenter tried to obtain samples from tongue in the same manner in every participant, it may be rational criticism that the tongues of smokers and non-smokers are different in tissue. Concretely, it may be possible that the epithelium may be more keratinized in smokers than non-smokers, provoking the possibility that the difference in TAS2R expression between smokers and non-smokers derives from the difference in degree of keratinization rather than expression of the receptors. Similarly, the relation between smoking history and number of TAS2R expression may possibly reflect the effect of cigarette on lingual epithelium over years, rather than the mechanism we have assumed in the section of discussion. Further, although there was aging effect of number of expression of TAS2R among non-smoker between age of 20 and 65, it should be noted that such kind of aging effect may not continue after age of 65. There remain some unanswered questions. For example, we were unable to examine the temporal relationship between smoking and TAS2R expression. It would be interesting to determine which receptor decreases its expression after the beginning of smoking. Additionally, it is not clear whether TAS2R expression recovers after smoking abstinence. Future research should investigate these areas.

Conclusions
The current study demonstrated a significantly lower expression of the bitter taste receptor gene TAS2R in individuals who smoke cigarettes compared with non-smokers. Further, cigarette smoking may impede the acquisition of bitter taste receptors that occurs with experience.