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Year : 2020  |  Volume : 4  |  Issue : 3  |  Page : 220-224

“Evaluation of salivary levels of heat shock protein 70 using ELISA among nonsmokers, conventional smokers, and reverse smokers”: A prospective study

Department of Oral and Maxillofacial Pathology, Vishnu Dental College, Bhimavaram, Andhra Pradesh, India

Date of Submission27-Mar-2020
Date of Acceptance21-May-2020
Date of Web Publication12-Sep-2020

Correspondence Address:
Dr. Swetha Pasupuleti
Department of Oral and Maxillofacial Pathology, Vishnu Dental College, Bhimavaram - 534 202, Andhra Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/bbrj.bbrj_47_20

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Background: Heat shock response is coordinated by five families of proteins with varying molecular weights known as heat shock proteins (HSP100, 90, 70, 60 and small HSPs). Members of HSP70 family are expressed during tumor cell differentiation, elevated temperatures, necrosis, exposure to nicotine etc., and act as chaperones facilitating newly synthesized proteins through intracellular compartments. Reverse smoking, where intraoral temperature could is very deleterious, well documented to cause malignancies and other pathological change. This present study was conducted to analyze the salivary levels of HSP 70 in conventional, reverse and non-smokers by using ELISA. Materials and Methods: A total of 90 individuals were included in the study comprising of conventional smokers (30), Reverse smokers (30) and non-smokers (30). 2ml of saliva was collected from all the individuals and subjected to centrifuge at 3000 rpm for 20mins. The collected supernatant was subjected to ELISA. Results: Increased salivary levels of HSP70 were observed in controls followed by reverse and conventional smokers (p value 0.000). Post Hoc analysis of HSP70 among conventional and reverse smokers, conventional and non-smokers, reverse and non-smokers was statistically significant between the all groups. Conclusion: Increased salivary HSP70 levels in non-smokers could be because the control group is due to their higher metabolism and possible hormonal imbalance in younger individuals. Reverse smokers had higher levels of salivary HSP70 due to the increased intraoral temperature and oxidative stress caused by the smoke. Hence, Smoking in any form could significantly alter the salivary HSP70 levels.

Keywords: Conventional smokers, ELISA, HSP70, Non-smokers, reverse smokers

How to cite this article:
Bobbili MK, Pasupuleti S, Manyam R. “Evaluation of salivary levels of heat shock protein 70 using ELISA among nonsmokers, conventional smokers, and reverse smokers”: A prospective study. Biomed Biotechnol Res J 2020;4:220-4

How to cite this URL:
Bobbili MK, Pasupuleti S, Manyam R. “Evaluation of salivary levels of heat shock protein 70 using ELISA among nonsmokers, conventional smokers, and reverse smokers”: A prospective study. Biomed Biotechnol Res J [serial online] 2020 [cited 2022 Jan 24];4:220-4. Available from: https://www.bmbtrj.org/text.asp?2020/4/3/220/294857

  Introduction Top

Tobacco, either in smoke or smokeless form, has its adverse effects on the overall well-being of an individual.[1] It is well described about the consequences and course of its by-products on oral mucosa. Among various forms of tobacco intake, smoking has a wide regional variation like conventional and reverse smoking. The habit of reverse smoking, in particularly, observed in low socioeconomic status and fishermen.[2]

Heat shock proteins (HSPs) or stress proteins play a vital role in maintaining and for the survival of the cell and are released during the regular activities, especially in response to physiological stress either as thermal or oxidative stress.[3] It was stated that tobacco smoke exerts its toxicity by oxidative stress leading to the release of reactive nitrogen species (RNS) and reactive oxygen species (ROS), thus leads to the release of HSPs,[4] especially in reverse smoking by providing a tight lip seal.

HSPs (molecular chaperones)[5] are conserved group of protective proteins and play an important role in cellular homeostasis importantly during stress.[6] Activation of HSP is through heat shock factors (HSFs) that includes HSF 1–5. When a cell is under stress either physical or chemical, these proteins get damaged and release HSF from its HSP70/90 and then slow down the functions like transport of ions and proteotoxic stresses. Under these conditions, HSPs are induced to maintain normal cellular functions.[7]

Among HSPs, HSP70 is considered to be important[8] and is present in both intracellularly and sometimes extracellularly. HSP70 in saliva helps in maintaining and repairing the integrity of the mucosal surface of the oral cavity and salivary gland cells.[9] Other functions include facilitation of passage and accompanying of various proteins across intracellular compartments, assist in correct folding of polypeptide chain into a functional protein, and they help in refolding or degradation of denatured proteins.[10] Extracellular and membrane-bound HSPs are involved in binding antigens and presenting them to the immune system where they are characterized as “molecular chaperones” as they assist in the repair of denatured proteins or promote their degradation after stress or injury.[11]

There is a little evidence regarding the status of HSP70 in humans, particularly in nonsmokers, conventional smokers, and reverse smokers; hence, this study aims to estimate the levels of HSP70 in saliva among the nonsmokers and smokers (conventional and reverse smoking) in predicting the disease status of conventional and reverse smokers.

  Materials and Methodology Top

This is a single-centered study done at the Department of Oral Pathology, Vishnu Dental College. A total of 90 patients with an age range of 21–65 years of age were considered in the study after giving informed consent. Patients were grouped as nonsmokers, conventional smokers, and reverse smokers (chutta smoking) of thirty patients in each group. All the patients had a history of smoking for more than 5 years with not <10 cigars/day. Non-smokers are indeed healthy controls who had never consumed tobacco in any form in their life time. A brief relevant case history was taken and the unstimulated saliva samples were collected from every patient and later transferred into Eppendorf tubes. The study was approved from the institutional ethical committee before the commencement.

Inclusion criteria

  • Patients with a habit of conventional chutta and reverse chutta smoking for the past 5 years
  • Patients who never smoked or had a habit of tobacco, beedi in any form in their lifetime.

Exclusion criteria

  • Patients with any systemic diseases and those under any medication for any diseases
  • Patients with a history of radiotherapy and oral malignancies.

All the participated patients were asked to rinse his/her mouth with water and 2 ml of saliva was collected in a sterile container and was immediately transported to the laboratory. The samples were centrifuged at 3000 rpm for 20 min and the supernatants were carefully collected. All the collected supernatants were then subjected to ELISA (YH Biosearch laboratory).


Salivary HSP70 ELISA kit is based on the simultaneous binding of human HSP70 to two antibodies, one monoclonal immobilized on microwell plates and the other polyclonal conjugated with horseradish peroxidase. After incubation, simple solid-phase washing is performed, in which free unbound antibodies are washed away. Then, the enzyme in the bound fraction reacts with the substrate and develops a blue color that changes into yellow when the stop solution is added. The color intensity is proportional to the HSP70 concentration in the sample.


  • Five different standard concentrations were prepared approximately with the following 20, 40, 80, 160, and 320 μg/ml as (S0, S1, S2, S3, and S4)
  • Content of each vial with × 30 concentration wash solution was diluted with distilled water to attain a final volume of 900 ml.

Before the procedure, all the reagents and saliva samples were allowed to attain a room temperature. The microwells were arranged in the tray in a sequence in accordance with the markings calibrated on the tray. Forty microliter of the test sample was added in each well, followed by the addition of 10 μl HSP70 antibodies and 50 μl streptavidin HRP. Then, the wells were covered with a seal plate membrane and incubated at 370°C for 1 h. Afterward, the seal plate membrane was carefully removed, the liquid was drained by shake off, and then, the plate was dyed such that the bottom of the well showed a clean surface under sunlight. Then, the wells were washed with a washing solution for five times and then blotted. Then, 50 μl chromogen reagent A followed by 50 μl chromogen reagent B was added to each well and then shaken gently to the mixture. Incubation was done for 10 min at 370°C away from light for color development. Finally, 50 μl stop solution was added to each well to stop the reaction. Blank well was taken as zero, and the absorbance was measured at 450 nm wavelength within 10 min. According to the absorbance value of samples, the concentration of the salivary levels of HSP70 was calculated in micrograms per milliliter of saliva (μg/ml). Salivary levels of HSP70 among the three groups were compared by ANOVA test. Chi-square analysis was used for comparison of age, gender, and duration of the smoking habits. Post hoc analysis was done for comparison of salivary levels of HSP 70among conventional and reverse smokers, conventional and non-smokers and reverse and non-smokers. P < 0.05 was considered as statistically significant.

  Results Top

All the sample collections were collected in noninvasive manner and are in compliance with the Helsinki Declaration.

All the obtained samples were subjected to statistical analysis using SPSS 21.0 version software (IBM, USA). Tests used were Chi-square test, post hoc analysis, and ANOVA test. P < 0.05 is considered as statistically significant for all the analysis.

The study group included a total sample size of 90 nonsmokers, conventional smokers, and reverse smokers (chutta smoking) comprising 30 in each group, comprising 31 males and 59 females showing a female predilection in the study [Table 1].
Table 1: Gender distribution among the three groups

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In this study, conventional smoking was predominantly found in male population and reverse smoking showed female preponderance [Table 2]. Comparison between age and smoking habit showed that there was almost an equal prevalence between conventional and reverse smokers within the age range 41–80 years. However, reverse smoking was predominant in 61–80 years age group, conventional in 41–60 years age group, and nonsmokers in 1–20 years age group.
Table 2: Distribution of age among the three groups

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The mean values of salivary HSP70 in conventional smokers, reverse smokers, and control groups were 112.45 μg/ml, 180.72 μg/ml, and 265.18 μg/ml, respectively. Increased salivary HSP levels were observed in nonsmokers followed by reverse smokers and conventional smokers, and the difference was statistically significant with a test value of 15.881 and P = 0.000 [Table 3].
Table 3: Comparison of HSP70 levels among the three groups using ANOVA

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Intergroup comparison was done using Tukey's HSD test for comparison of salivary levels of HSP70 among conventional and reverse smokers, conventional smokers and nonsmokers, and reverse smokers and nonsmokers which were found to be statistically significant (P = 0.036, 0.000 and 0.007) [Graph 1].

  Discussion Top

Smoking and chewing of tobacco have become common social habits in developing countries like India. Tobacco was introduced in India by Portuguese 400 years ago which has become the third largest producer and second largest consumer of tobacco.[12] Around 60% of Indian men and 2%–10% of women use tobacco in conventional and reverse smoking form.[13],[14] The habit of reverse smoking has been reported from America, Asia (South India), and Europe.[15] On an average, each adult may smoke 3–4 chuttas/day, a type of homemade cigar in reverse direction, in local language known as “Adda Poga” lasting for about 15–30 min. This habit is mainly practised by placing the lit end of cigarette inside the mouth, while the other end is held between teeth and lips, thus providing the seal allowing slow inhalation of smoke. Air is supplied to the zone of combustion through the nonheated extreme of chutta and the smoke is expelled from the mouth and ashes are thrown out or swallowed. The lips keep chutta wet which increases its time of consumption from 2 to 18 min.[16] During combustion of tobacco, the intraoral temperature rises up to 120°C and releases RNS and ROS resulting in increased oxidative stress further damages the cellular proteins rendering them nonfunctional. This triggers the activation of HSPs, mainly HSP70, which helps in the repair of damaged proteins and prevention of further damage. Studies indicate that HSP70 reaches human saliva through passive transport from the salivary gland cells.[9]

HSPs were identified as a set of highly conserved proteins, making up to 5%–10% of total protein content in healthy individuals, but their intracellular concentration can be increased by 2–3 times due to any stress stimuli[17] and also by elevated temperatures. After this discovery, it became clear that the synthesis of HSPs is initiated not only by heat but also by many physical-, pathological-, and chemical-induced stress. The total amount of protein in the whole saliva ranges between 0.5 and 3 mg/ml.[18]

HSPs play a major role in the prebiotic evolution of modern enzymes and are vital throughout the whole lifetime of human cells; however, they are needed even more after environmental stress, which induces protein damage.[7],[19] The expression of HSP messenger RNAs is mediated by a family of transcription factors called as HSF.

Among HSF 1–5, HSF1 plays a major role, and in resting cells, they are complexed with various HSPs, such as HSP70. After stress, damaged proteins become abundant and liberate the HSF from HSP70 complexes. This process sets the stage for the trimerization, nuclear translocation, and phosphorylation of HSF1, which are all prerequisite for its binding to the special nucleotide segments, called heat shock elements in the promoter region of HSP genes.[7] Thus, the activated HSP70 blocks the apoptotic pathway by reducing caspase activation, suppression of mitochondrial damage, and nuclear fragmentation. HSP70 also inhibits cathepsins and lysosomal proteases involved in apoptotic mechanism.[20] Therefore, oxidative stress caused by the ROS and RNS and/or increased temperature causes apoptosis. Studies by K Anbarasi showed that the expression of HSP70 is upregulated in the rat brain exposed to cigarette smoke due to oxidative stress. Studies by Newkirk identified anti-HSP70 autoantibodies in chronic smoke-exposed mice but not in ambient air-exposed controls, which suggests that common environmental insult such as chronic smoke plays a role in a number of diseases.[21]

Various methods have been used to detect the HSPs such as Western blot test, immunohistochemistry, high-performance liquid chromatography, polymerase chain reaction, and ELISA. ELISA is the most popular immunoassay procedure used for the detection of antigen or antibody. ELISA seems to be more reliable, sensitive, and less invasive method. Estimation of salivary levels of HSP70 may help in predicting the status and prognosis of diseases such as smoker's palate, leukoplakia, smoker's melanosis, and other diseases associated with smoking tobacco. Thereby, HSP70 likely provides early detection of the disease status in conventional and reverse smokers. In the present study, salivary levels of HSP70 were qualitatively and quantitatively assessed by direct ELISA procedure.

Earlier, few investigators have conducted studies to assess HSP70 levels in smokers, but, to the best of our knowledge, this study is unique as there are no studies available in the English literature assessing the salivary levels of HSP70 in smokers. Hence, the main aim of the present study was to assess the effect of tobacco smoke on salivary levels of HSP70 in conventional smokers, reverse smokers, and nonsmokers.

In the present study, though the males outnumber the females in conventional smokers, females have taken a lead in reverse smokers. The predominant female population among reverse smokers has been consistently reported in Americans, the Caribbean area, Columbia, Panama, Venezuela, and in Southern India.[15] This predominance among females has been attributed to the following reasons: initially, there are cultural taboos and beliefs that this habit reduces halitosis and prevents pain of dental origin; second, to prevent the lit end of the chutta from the strong and gusty winds and splashing of water during fishing episodes as well as during weaving of fishing nets to prevent the damage upon the threads from hot ash of burnt chuttas. In addition to it, they wanted to keep their smoking habit a secret from their husband and parents.[22] The habit of reverse smoking is widespread in South India mainly in certain parts of Andhra Pradesh with female: male ratio of 1.7:172 and was 6.23 times higher in females than in males.[23]

In the present study, 86.7% of the reverse smokers were within the age range of 40–80 years and nearly 83.3% within the age of 40–80 years were conventional smokers. Correlation of habit history among the groups revealed that the majority of conventional and reverse smokers have the habit for a duration of 20–30 years and in accordance with the study by Ramesh et al., Patil et al., and others.[22],[24]

In the current study, there is an increase in the salivary levels of HSP70 in reverse smokers (180.72 μg/ml) when compared to conventional smokers (112.45 μg/ml) with statistical significance. Increased levels could be due to increased intraoral temperature and also tobacco smoke resulting in the release of RNS and ROS causing oxidative stress.[16] However, salivary levels of HSP70 were found to be more in nonsmokers (265.18 μg/ml) when compared to the conventional (112.45 μg/ml) and reverse smokers (180.72 μg/ml) which could be due to age-related factors such as higher metabolic rate, hormonal imbalance, lifestyles, and other stresses as the younger individuals are more in nonsmokers. Whereas middle and older age individuals are more in reverse and conventional smokers, there seems to be a gradual decrease in self-defensive mechanisms and exponential accumulation of damage at cellular, molecular, and organism levels.[25]

Jin et al. analyzed the serum HSP70 levels using Western dot blot in 327 healthy male donors aged between 15 and 50 years and showed that serum HSP70 levels were positively correlated with age in patients between 15 and 30 years of age but negatively correlated with age in patients aged between 30 and 50 years. Serum HSP70 levels were highest in individuals aged between 25 and 30 years among all age groups. They stated that more variable levels of physical activities and basal metabolism were found in youngest age group compared with the oldest age group, and also, aging is accompanied by decrease in self-defensive mechanisms.[25]

In our study, comparison of salivary levels of HSP70 among conventional (112.45 μg/ml) and reverse smokers (180.72 μg/ml), conventional smokers (112.45 μg/ml) and controls (265.18 μg/ml), and reverse smokers (180.72 μg/ml) and controls (265.18 μg/ml) using Tukey's HSD analysis showed that there was a statistical significance between the all the three groups with P = 0.036, 0.000, and 0.007, respectively.


Further studies involving a larger sample size are required to confirm the influence of tobacco smoke on salivary HSP70.

  Conclusion Top

Smoking in either reverse or conventional form significantly altered the salivary HSP70 levels, and there was a significant comparison between conventional and reverse smokers and reverse smokers and nonsmokers. Increased levels of salivary HSP70 levels in nonsmokers observed could be due to higher metabolic rate, hormonal imbalance, lifestyles, and other stresses in the younger age group. Reverse smokers had higher levels of salivary HSP70 when compared to conventional smokers due to the increased intraoral temperature and oxidative stress caused by the smoke. This study confirms the deleterious nature of reverse smoking and suggests the need for abstinence from tobacco-related products. Health professionals need to take the initiative to drive local and state governments to enlighten the common people by providing general awareness about harmful effects of tobacco smoking.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

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  [Table 1], [Table 2], [Table 3]


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