Molecular pathogenesis of chewable tobacco

In Pakistan, extensive use of several precarious chewable tobacco formulations has made oral cancer the second leading malignancy. Selection of literature was done by a survey of studies published from 1990 to 2017 mainly, from PUBMED and few from other search engines, on naswar, gutka, areca nut and betel quid, which included published reviews, original articles and other data sources on chewable tobacco, its epidemiology, pathological implications, and psychological effects. These studies have revealed that the chemicals in these formulations bind and mutate DNA of oral mucosa through down regulating cellular repair pathways and upregulating genetic networks associated with pathogenesis. Areca nut, having aercoline [the major alkaloid] causes carcinogenicity, mutagenicity, and genotoxicity of oral mucosa through increased production of growth factors and corticotrophin-releasing hormone, and genetic alteration in expression of CASP8, APAF-1, BAX, BAD, and upregulation of caspas-3. Gutka addiction leads to precancerous lesions resulting in characteristic facial abnormalities, following trismus. Naswar, in addition to oral cancer, causes adverse cardiovascular events by reducing glutathione per oxidase [GPx] and super-oxide dismutase [SOD], serum levels of HDL, whereas, increasing the ratio of cholesterol, LDL, triglycerides and LDL-C/HDL-C. Betel quid [Paan], causes psychoactivity affecting central and autonomic nervous systems leading to dependence with decreased cognition, euphoria, sweating, salivation, palpitation, heightened alertness and zest to work. Metabolically, cardio-acceleration, cortical desynchronisation of EEG, elevated plasma noradrenaline and adrenaline were found. This review highlights the corrosive effects of various most popular chewable tobacco formulations; and damage done by their cocktail of carcinogenic substances and added ingredients, leading to oropharangeal cancer

Reviewing a manuscript: disparity amongst peer reviewers' priorities from basic health sciences and clinicians

Objective: to determine the disparities and the priorities of basic health scientists and clinicians while assessing a manuscript

Study Design: cross-sectional study

Place and Duration of Study: the study was conducted at Karachi, from January to April 2014

Methodology: the study was a proforma-based research, employed convenient sampling for data collection, in which 100 reviewers belonging to clinical and basic health science background were selected. The questionnaire used was a selfadministered one with close-ended questions. The results were analysed using SPSS software

Results: out of 100 respondents, 50 were from Basic Health Sciences Reviewers [BRs] and 50 Clinicians Reviewers [CRs]. The results depicted grammatical and improper referencing amongst formatting mistakes as the most common observation by the reviewers. The reviewers also raised concerns regarding validity of statistical analysis, use of outdated references and lack of acknowledgment of the sources of funding utilised by the authors. There were elements of manuscripts that BRs and CRs reviewers agreed upon and prioritised, but there were elements of the manuscript that they approached mostly in methods, results and conclusion components

Conclusion: the priorities of reviewers from Basic Health Sciences and Clinical background differed markedly in certain respects. This impacts how they review the manuscripts

Understanding the fat gene. Obesity common variant FTO RS9939609

Obesity, attributable to the interaction of genetic makeup, lifestyle and environment Factors are a multifactorial disease. However, not all people with similar genetics or same environmental setup become obese. Currently, extensive research is being conducted regarding increases in the prevalence of obesity, apparent in many societies, studying their eating habits and physical activity. Although, people with obesity have the equal distribution of body fat, yet they do not suffer from the same medical ailments. The recent identification of obesity-risk FTO variants rs9939609 associated with body mass index [BMI] and risk of obesity in multiple populations has led to the major success in the field of obesity genetics, making FTO the first locus unequivocally associated with adiposity. The role of FTO gene as a risk factor of obesity has also been identified by the Genome wide association which is entirely a new approach. The function of FTO gene was unknown, but recent investigation showed that the FTO gene regulates ghrelin a key mediator of ingestive behavior, which might explain a predisposition to increased food intake leading to obesity. People with rs9939609 variant of the FTO gene are at increased risk of obesity they have higher circulating levels of the hunger hormone ghrelin, and feel hungry soon after eating a meal. This review focuses on the genetics of FTO gene and the association of each additionally minor allele of [FTO] rs9939609 single nucleotide polymorphism [SNPs] with the risk of obesity

Nephrin: an emerging biomarker for detecting damage of glomerular filtration barrier

Nephropathy due to damage of filtration barrier is a primary origin of end stage renal disease in Diabetics. It is associated with considerable morbidity and mortality. Mostly clinicians rely on urine analysis of Albumin. In the recent years extensive research has been conducted to find out a novel biomarker which can help in the early diagnosis of nephropathy. A search was conducted regarding publications of new diagnostic biomarkers for nephropathy. Among many renal biomarker researched lately, Nephrin was identified as the one that can become an early predictor of nephropathy and the levels of which can also ascertain the severity of the disease

Relation of serum cotinine with passive smoking

Background: Cotinine is a major metabolite of nicotine and retains for a substantial time in different body fluids. It is considered as a passive smoke exposure marker. Studies on different biochemical markers of tobacco smoke exposure are lacking in Pakistan and serum continine levels in nonsmokers provide a comprehensive measure of passive smoking from all sources in the last two to three days

Objective: To compare the frequency of serum cotinine detection in smokers, passive smokers and never smokers

Methods: Serum cotinine was analyzed on 135 self reported smokers, never smokers and passive smokers by 17-A gas chromatograph with flame ionization detector [FID] equipped with Supelco SPB-5 fused silica capillary column attached with Class GC 10 Software. Association between the qualitative variables [cotinine present/absent] between groups [smoker, never smoker and passive smoker] was taken out using Pearson chi-square test. The mean difference for serum cotinine between the groups [smoker, never smoker and passive smoker] was evaluated by using kruskal wallis test. P-value less than 0.05 was taken as significant

Results: Serum cotinine was detectable in 72.1% of passive smokers which was a significantly higher proportion than smokers and never smokers [p< 0.001]

Conclusion: Cotinine was detectable in the serum of smokers and passive smokers. Passive smoking may contribute to increased serum cotinine levels. Large sample size may be required to establish it as a marker for environmental tobacco smoke exposure marker and for validation of smoking status

[VDR] gene polymorphisms - rapid and noninvasive oral detection method

Background: In recent years, the importance of vitamin D receptor [VDR] gene restriction fragment length polymorphisms for various types of disease and cancers has been investigated by a great number of studies. A non invasive method could be employed for extracting DNA. Oral rinse has been found to be one of the sources for collecting genomic DNA

Objective: To develop a rapid and non-invasive method for the detection of Vitamin D Receptor [VDRFok1] Gene from oral rinse

Methods: Oral rinse samples were collected normal individuals with informed consent. Individuals included were healthy adults between 20-40 years of age. Oral rinse [40ml] was taken after gentle brushing over the lesions. DNA extraction was done according to Lucky MH et al and PCR was performed using beta globin primers GH02/PC04 and VDR-Fok1 primers. A 256bp amplified products was visualized by Gel Doc Hero Lab software [Germany]. The PCR-RFLP results showed the 20 or 40% FF genotype [homozygote of common allele] with one band of 265bp

Results: The mean concentration of 60 DNA samples was 14.484 +/- 10.63ug/ml. The results of VDR-Fok1 gene polymorphism shows that out of 60 subjects 48 were normal [FF 80%], 12 were Heterozygous [Ff 20%] and 0 were mutated [ff 0%]

Conclusion: Oral rinse is a perfect medium for rapid and non invasive diagnostic applications of VDR gene may be optimized for other salivary biomarkers

Cytotoxic activity of honey in hepatoma cells: in vitro evaluation

Background: Honey is used extensively in south East Asia, Middle and Far Eastern countries as a sweetener as well as medication for its anti-bacterial, anti-fungal, and anti-viral properties with no risk of resistance or side effects. Recent studies suggest that honey can modulate tumor growth by reducing cell proliferation and increasing apoptosis susceptibility

Objectives: To find out the apoptotic effect of honey on hepatoma cancer cell lines

Methods: The growth of hepatoma cell lines [Huh-7] was checked by adding Honey to the cells before culturing in a 24 well plate. Wells were selected and labeled for each of the variables [controls, honey]. After 2 days, cells were studied under an inverted phase contrast microscope and fields were recorded. Approximately four fields per slide of higher intensity were selected randomly to determine the dead cell density, and the procedure was repeated 10 or more times. Frequency and percentages were calculated for dead or alive cells in controls, honey and their mixture. Odds Ratio was used to compare the qualitative variables

Results: Honey was found to induce cell death in hepatoma cell lines [Huh-7]. At a magnification of 40x, the dead cells were 1% in controls whereas, 70.8% in honey. At magnification of 20x and 10x the dead cells density was 52.3% and 82.4 % respectively in honey treated cells compared to 4% in controls

Conclusion: Honey induced cell death in Huh-7 cells. Huh-7 exposed to honey have high probability of apoptosis/necrosis. Honey should be considered as co-adjuvant treatment against cancer

Perception of HPV in children

Human papillomavirus [HPV], with its more than 100 genotypes, is a cause of different diseases, ranging from malignant epithelial tumors such as squamous cell carcinoma in cervix and mouth to benign skin warts. HPV transmission generally occurs through direct person to person contact and exposure to infected material. Since the involvement of HPV in oral carcinoma in adults has been established, high risk strains are being explored in oral cavity of children. The presence of HPV in children presents a serious problem especially when the modes of viral transmission in child always remain a controversial issue. In children presence of HPV shows a strong indication of sexual abuse, though infection from direct contact cannot be ruled out, since the virus has been proven to thrive successfully at a range of sites and infect healthy people. This review aims to discuss HPV mode of transmission in children and its associated epidemiology

Relationship of human papilloma virus with trismus in chewable tobacco users

Background: Human Papilloma Virus [HPV] is found in the oral tobacco chewers with precancerous lesions such as submucous fibrosis [SMF], leukoplakia and erythroplakia. Trismus is a common symptom seen in patients with SMF and oral cancers. In Pakistan the risk of oral cancers has been raised up to 8.5 to 10 times due to increase in tobacco chewers in the recent years

Objective: To determine the frequency of oral HPV infection in chewable tobacco users with trismus living in the lowincome areas of Karachi

Methods: Camps were set up in 5 different areas of Karachi. In each camp, 100 people were examined for oral lesions by experienced volunteers. Oral rinse [40ml] was taken after gentle brushing over the lesions. DNA was extracted and PCR was performed using Gp5+/Gp6+ consensus primers. The results were analyzed on SPSS version 17.0

Results: Out of 187 subjects with trismus, 157 [84%] were males and 30 [15.51%] were females with average age of 28.7 years [12 -70 years]. Gutka, Naswar, Chalia and Paan users were 45.45%, 37.56%, 12.83% and 6.95% respectively. Mild trismus was found in 113 [60.43%] subjects followed by moderate trismus in 52 [27.81%] and severe trismus in 22[11.76%]. Out of total 36 [19%] HPV positive subjects, 27[14.44%], 7[3.74%] and 2[1.07%] had mild, moderate and severe trismus respectively. 150 controls tested negative for HPV

Conclusion: The frequency of HPV in oral cavity was high in subjects with trismus suggesting risk of oral squamous cell carcinoma. HPV in subjects with moderate to severe trismus should be tested in blood because it is difficult to get suitable oral sample

Trends leading to oral cancer

Oral health disparities have exacerbated since the popularity of chewable tobacco among the general population in Pakistan. Karachi South ranks second in the incidence of oral cancer and this frequency is steadily rising. Several molecular studies have shown a link between the use of chewable tobacco products and oral submucosal fibrosis. Oropharyngeal cancer, is being driven by HPV is now well known around the world. Studies in Pakistan on squamous cell carcinoma and precancerous oral lesions, found a high prevalence of Human Papillomavirus [HPV] in patients with male sex showing significant correlation with HPV infection. This paper highlights the causes and risks of the oral epidemic that is threatening our society

Molecular epidemiology of hepatitis B virus in Pakistan: link with Southern Asia

Viruses and their genotypes reflect the relationship of their hosts. How these strains evolve, is one question and how they cross the borders is another. Since the HBV genotypes have been described by Okamoto and colleagues in 1988, Hepatitis B virus is expressed by a classification of different HBV strains into genetic genotypes and sub genotypes. A degree of 8% divergence between the groups has since then become the definition for HBV genotype. Now HBV is known by its geographic genotype and sub-genotype distribution. They also exhibit combination of both separately or in the form of a hybrid and hybrid of HBV genotypes is a common event in countries where movement of people is frequent across the borders, thus, providing a mixture of variation within individuals and in the population in general. It is seen that if travelling is by road then the different genotypes are prevalent around the borders, but if travelling is by air then they exist in general population as a less prevalent genotype. This review highlights the genotypic link between Pakistan and its South Asian neighbors

Isolation of DNA from oral rinse in HPV positive patients

To extract human genomic DNA from oral rinse in HPV positive patients. Experimental study. Research Laboratory, Ziauddin Medical University, from February to July 2011. Two hundred and fifty oral rinse samples from human papilloma virus [HPV] positive subjects were collected in 50 ml corning tubes. DNA was extracted from 10 ml of oral rinse by using Lysis buffer [sodium dodecyl sulfate, sodium chloride, sodium citrate and EDTA], isopropanol and 3 M sodium acetate and final washing with ethanol. The DNA was quantified by using Qubit Registered dsDNA BR Assay [Qubit Registered 2.0 invitrogen life technologies USA] and the quality was checked by running an aliquot on 0.6% agarose gel stained with ethidium bromide. PCR were done to amplify beta-globin gene on chromosome 11 by using primers GH20/PCO4 of 260 bp fragment. The mean concentration of all 250 samples DNA was 15.648 +/- 10.50 microg/ml determined by using Qubit Registered 2.0. A single intense band without smearing was seen in almost all cases which confirmed the integrity of DNA. The PCR amplification of human beta-globins primers was successfully done. The oral rinse method was found a simple and highly appropriate means for non-invasive sample collection with easy storage, DNA recovery and subsequent PCR amplification in HPV positive patients

Human papilloma virus and oral lesions in Gutka eating subjects in Karachi

To determine the frequency of HPV in eaters of Gutka [betel, areca, lime and tobacco concoction], presenting with oral lesions. A descriptive study. Ziauddin University Research Laboratory, from February to July 2010. Subjects munching Gutka fulfilled the inclusion criteria. Samples were collected in the form of 20 - 40 ml oral rinse from 262 subjects who were habitual eaters of Gutka after an informed consent. Gentle brushings from the lesion were taken from subjects with the help of a brush at the other end of dental floss and the oral rinse was stored at 4[degree sign] C until DNA extraction. DNA was extracted and PCR was performed using HPV consensus primers Gp5+/Gp6+. Oral cavity was examined for the presence of ulcer, trismus, sub-mucosal fibrosis, leukoplakia and/or warts. Out of 262 subjects, 42 were females and 220 males with an average age of 27 +/- 10 years. HPV was positive in 47 subjects [17.9%]. HPV frequency was 2.7% greater in chewers with more than 10 years of habit compared to less than 10 years. Examination of oral cavity showed 78% presenting with more than one complaint including oral ulcers [25%], rough mucosa [62%], sub-mucosal fibrosis [24%], leukoplakia [20%] and erythroplakia [10.6%]. Highest frequency of HPV was observed in erythroplakia [25%]. Association between presence of symptoms and HPV shows an ODDS RATIO: ad/bc= 4982/430=11.6. Oral lesions caused by constant exposure to Gutka are associated with high frequency of HPV infection, which may be a risk factor for squamous cell carcinoma of the oral cavity. People should be educated about the consequences of Gutka abuse