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Micronucleus formation and DNA damage in buccal epithelial cells of Indian street boys addicted to gasp ‘Golden glue’
Mutation Research 721 (2011) 178–183
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Micronucleus formation and DNA damage in buccal epithelial cells of Indian street boys addicted to gasp ‘Golden glue’ Nandan Kumar Mondal a,∗ , Sreenita Ghosh b , Manas Ranjan Ray a a b
Department of Experimental Hematology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, West Bengal 700026, India Department of Zoology, University of Burdwan, Burdwan 713104, India
a r t i c l e
i n f o
Article history: Received 11 July 2010 Received in revised form 1 January 2011 Accepted 22 January 2011 Available online 31 January 2011 Keywords: Micronucleus DNA damage Glue Addiction Street children India
a b s t r a c t Genotoxicity of glue sniffing/huffing and tobacco use has been examined in 302 street boys (median age 13 years) and 50 age-matched control school boys who were neither tobacco nor glue users. All the street boys were tobacco users. In addition, 155 were addicted to gasp an industrial adhesive popularly known as ‘Golden glue’. Micronucleus (MN) frequency was determined as a measure of chromosomal breakage in exfoliated buccal epithelial cells (BECs) and DNA double strand breaks were quantitatively assessed by counting ␥-H2AX foci using immunofluorescence microscopy. Micronucleated cell frequencies (MCFs) in BEC of glue non-addicted (only tobacco) and addicted (tobacco plus glue) street boys were 1.87 ± 1.06‰ and 4.04 ± 2.55‰ respectively, which were significantly higher than that of control (0.32 ± 0.11‰, p < 0.0001). Similarly, the numbers ␥-H2AX foci in nuclei of BEC were 2.3- and 5.2-times more than control in glue non-addicted and addicted street boys respectively (p < 0.0001). Spearman’s rank correlation revealed a strong positive association between years of glue addiction with MCFs and ␥-H2AX foci numbers, and the association between glue addiction and chromosomal and DNA damage remained positive and significant after controlling income, spending on addiction and loss of appetite as potential confounders in multivariate logistic regression analysis. Thus, addiction to tobacco among the street children in India is associated with chromosomal and DNA damage in BECs and the severity of these changes is significantly increased by the habit of sniffing/huffing of industrial glue. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Inhalant dependence and abuse have been reported from various parts of the world [1–4]. Drug usage among minors has created a global generation of addicted and often abandoned children bereft of family support, education and social skills. Chronic exposure to abuse solvents can produce loss of appetite [5], but the underlying mechanism is not well understood. Street children constitute a marginalized population in most of the urban centers throughout the world. In India, street children are abundant in all metros, but their exact number is not well documented. The magnitude of the hardship they experience is also rarely unveiled. The marginalized street children constitute a truly “hidden” population who are not covered by nor find place in the national census, educational or health data, primarily because they are a floating population without any fixed address [6]. It is widely speculated that between 100,000 and 125,000 children live on the streets and railway stations of India’s major cities, and that more than half of them have some form of drug addiction. Street boys living in different rail-
∗ Corresponding author. Tel.: +91 33 2476 5101; fax: +91 33 2475 7606. E-mail address: nandan [email protected] (N.K. Mondal). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.01.011
way stations in West Bengal, a state in eastern India, take care of themselves and each other in an otherwise uncaring environment and they survive through begging, petty theft or hawking goods on the railway platforms. The major part of the paltry sum they earn is spent on tobacco and on tube of ‘Golden glue’ (commercially available as ‘Dendrite’ which is an industrial contact adhesive and rubber cement marketed in the form of glue sticks, tubes and cans in Eastern India, Bangladesh and Bhutan) which is a particular favorite with those children. This golden colored industrial glue containing organic solvents is cheap and readily available and provides faster onset of action and the regular ‘high’ [7,8]. Glue sniffing has been reported also from Mexico among 1% in general population and 22% of street children [9]. It has been estimated that the glue addicts can be exposed to 2000–30,000 ppm of organic solvents within a few minutes [10]. In India, the kids squeeze glue onto a rag and huff its fumes through the mouth. The overall effects of glue inhalation range from an alcohol-like intoxication and intense euphoria to vivid hallucinations, depending on the substance and the dosage. These products are not classified as drugs and young children and adolescents can easily obtain them and abuse them [5]. Besides addiction, street boys in India are usually victims of abuse and harassment, and are extremely vulnerable to trafficking, sex trade and child labor [11]. Glue sniffing can be more
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injurious to health than cigarette smoking as the glue contains a large number of health-damaging chemicals like benzene, xylene, toluene, n-hexane, heptane, styrene, acetone, methyl ethyl ketone, methylene chloride, cyanoacrylates trichloroethane, halogenated aromatic hydrocarbons and mineral spirits [12–16]. A number of these chemicals are potential mutagens or carcinogens. Toluene, a volatile organic compound, is the main toxic component that enters the body through glue sniffing [17]. Its metabolite hippuric acid is nephrotoxic [18] causing kidney failure [12]. Besides, long term glue sniffing can lead to liver disease [19] and damage to the nervous system [20]. Once these solvents or gases are inhaled, the extensive capillary surface of the lungs and the airways readily absorbs them. Since the glue contains human carcinogens including benzene, chronic inhalation of glue may lead to DNA damage particularly in those cells that are at the direct line of exposure such as cells of the nasopharynx, oral cavity, airways and the lung. The literature from India on inhalant abuse is limited to only a few case reports [21,22] and case series [7,23–25]. To our knowledge, no study has ever been carried out in India or elsewhere on the possibility of chromosomal and DNA damage among chronic glue inhalers. DNA double-strand breaks (DSBs) can result in cell death or genetic alterations when cells are subjected to environmental or occupational stresses. A complex DNA-damage-response pathway is activated to repair the damage, and the inability to repair these breaks can lead to carcinogenesis. One of the earliest responses to DSBs is the phosphorylation of the histone H2AX at serine 139 (␥-H2AX), which can be detected by a fluorescent antibody [26]. We examined in this study whether glue sniffing by street children elicits chromosomal and DNA damage in epithelial cells of the oropharynx. To achieve this objective we employed micronucleus (MN) assay for the detection of chromosomal damage and enumeration of ␥-H2AX foci for evaluation of the possibility of DNA DSBs in exfoliated buccal epithelial cells.
2.2. Collection of background data
2. Materials and methods
2.5. Pap staining and MN assay
2.1. Participants
For Papanicolaou (Pap) staining, the fixed slides (two for each individual) were brought to water through graded ethanol. The slides were then stained with Harris’ hematoxylin for 30 s, rinsed in distilled water and placed in Scott’s tap substitute for bluing. Thereafter, the slides were washed in running tap water, dehydrated in 70% and 90% ethanol, stained in Orange-G6 for 30 min and differentiated in 95% alcohol. Then the slides were incubated in EA-50 solution for 30 min in room temperature, dehydrated in ethanol and mounted with distrene plasticizer xylene (DPX) mountant. The slides were coded and examined under light microscope (Leitz Dialux 20, Germany) with 100× oil immersion objective for the presence of micronucleus (MN) and other nuclear anomalies. For MN, 2000 non-overlapping, differentiated, uniformly stained nuclei of BEC of each individual were analyzed by two investigators (NKM and MRR) blindly. Both investigators scored slides from all the subjects independently. The difference in final score between the observers was less than 5%, and the arithmetic mean of two scores was used as individual value. Cells with pyknotic or condensed chromatin or having karyorrhectic nuclei and keratohyalin bodies in the cytoplasm were not considered for MN assay. MN identification was based essentially on the criteria of Tolbert et al. [29] as described in detail by Thomas et al. [30]. In brief, the characteristics of MN are (i) round or oval in shape, (ii) diameter ranges between 1/3rd and 1/16th of the main nucleus, (iii) have the same staining intensity and texture as the main nucleus, (iv) are located within the cytoplasm of the cells, and (v) usually one per cell, but the number could be two or more following genotoxic insults. Besides MN, other nuclear anomalies like ‘broken egg’ (cells with nuclear buds), binucleated cells, pyknosis (shrunken nuclei), karyorrhexis (nuclear disintegration), and karyolysis (dissolution of nucleus) were evaluated following the established criteria [29,30]. ‘Broken egg’ was scored from 2000 cells while 1000 cells were examined for the detection of other nuclear anomalies [30].
2.1.1. Street boys A total of 302 homeless street boys aged between 8 and 16 years were recruited from different railway stations in and around Kolkata (former Calcutta) in eastern India. The inclusion criteria for street boys were (i) apparently healthy, homeless, 8–16 year-old boys surviving through begging or hawking goods on the railway platforms for the past 6 months or more, (ii) having a body mass index (BMI) >15 and <25 kg/m2 , and (iii) users of tobacco or tobacco plus glue. They were all either smokers and/or chewers of tobacco. They usually smoke 3–10 (median 5) beedi (tobacco rolled on dried Tendu leaf [Diospyros melanoxylon Family Ebenaceae]) or cheap brands of cigarettes per day and chew raw tobacco with lime (khaini) or tobacco in combination with betel nut and catechu (guthka) available in local shops in small pouches. Out of the 302 street boys, 155 (median age 12 years) were addicted to ‘Golden glue’, an industrial adhesive marketed with the brand name of ‘Dendrite’. They breathe in the glue directly from its container (called sniffing or snorting) or they place a rag soaked in the glue over their nose and mouth and inhaling it (called huffing). Sniffing or huffing of the glue provides a quick, powerful ‘high’. The remaining 147 street boys (median age 13 years) were not addicted to glue. We recorded spots or sores around the mouth, red eyes, rhinorrhea, chemical odor on the breath, and a dazed appearance as signs of recent inhalant abuse [27]. Dietary habits of the participants were surveyed using a food quality and frequency questionnaire. In general, the street boys purchase/beg their food from small restaurants and eateries at the railway stations. We came across only 12 boys (3.9%) who used to cook their own food in makeshift ovens near the railway track using waste paper, cardboard, packing boxes, etc. as cooking fuel. 2.1.2. Control (school) boys For comparison, we enrolled 50 school boys aged between 8 and 16 years (median age 12 years) from suburban areas of Kolkata. Majority of these boys (34/50) were studying in class IV–VII in local schools. The average distance from home to the schools was 1.0 km and they either walk or bicycles to reach the schools. The boys came from families with a monthly income of US$ 88–275 (median 124). They were all nonsmokers and non-chewers of tobacco and they did not have the habit of glue inhalation. Accordingly they were considered as control.
Members of the research team interviewed each participant separately. In general, they were asked to furnish background information about age, education, food, tobacco smoking and chewing habit. Boys in the control group were asked in addition about their parent’s occupation, family size, housing, type of cooking fuel used at home, and presence of smoker in family. The street boys were asked about their means of living (hawking/begging/ragpicking/other), tobacco smoking (beedi/cigarette, number per day) and chewing habit (khaini/guthka, number of pouches per day), consumption of alcohol (yes/no), addiction to glue (duration, number of sniffing per day) and source of food and drinking water. Large majority of the street boys were illiterate; hence the researchers entered their responses in structured questionnaire forms on their behalf. The Ethics Committee of Chittaranjan National Cancer Institute, Kolkata approved the study protocol. 2.3. Chemicals Hematoxylin (CAS No. 517-28-2), orange-G6 (CAS No. 1936-15-8), eosin Y (CAS No. 17372-87-1), light green SF yellowish (CAS No. 5141-20-8), Bismarck brown (CAS No. 10114-58-6), phosphotungstic acid (CAS No. 12067-99-1), paraformaldehyde (CAS No. 30525-89-4), Triton-X 100 (CAS No. 9002-93-1), and BSA (CAS No. 9048-46-8), were purchased from Sigma–Aldrich Chemicals, Saint Louis, MO, USA. Potash alum (CAS No. 7784-24-9), and GAA (CAS No. 64-19-7) were obtained from Merck, Mumbai, India. Lithium carbonate (CAS No. 554-13-2) was from SISCO Research Laboratories, India. Ethanol (CAS No. 64-17-5) was from Bengal Chemicals & Pharmaceuticals Ltd., Kolkata, India. All the chemicals were of analytical reagent grade. Primary antibody ‘rabbit polyclonal ␥-H2AX [phosphor S-139]’ (Cat No. ab2839) and secondary antibody ‘FITC-conjugated goat anti-rabbit IgG’ (Cat No. sc-2012) were purchased from Abcam, UK and Santacruz, Biotechnology Inc., USA respectively. 2.4. Collection and fixation of exfoliated buccal epithelial cells All the participants were asked to wash their mouth with sterile 0.9% saline (NaCl). Epithelial cells were collected from buccal mucosa scraping the middle part of the inner cheeks with sterile plastic spatula. Four slides were prepared for each participant. Two slides were immediately fixed in 95% ethanol and brought to the laboratory and stained with Papanicolaou (Pap) method following the procedure of Hughes and Dodds [28]. Remaining two slides were fixed in 4% paraformaldehyde at the site of collection for immunocytochemical localization of ␥-H2AX foci.
2.6. Immunofluorescent microscopy and quantification of -H2AX foci Immunofluorescent microscopic study was carried out following the procedure of Zhou et al. [31], with slight modifications. In short, paraformaldehyde fixed slides (two for each individual) were washed with PBS, and permeabilized with 0.2% Triton X-100. After blocking with 3% bovine serum albumin (BSA) for 1.5 h, samples were incubated with rabbit polyclonal ␥-H2AX (phosphor S 139; diluted 1:1000 in 1% BSA) for 2 h, followed with FITC-conjugated goat anti-rabbit IgG secondary antibody (1:500) for 1 h in darkness and examined immediately using a fluorescence microscope (Leica DM 4000B, Germany). To prevent bias in selection of cells that
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display foci, all the cells were counted in the field of vision. Image Pro Plus (Media Cybernetics, Silver Spring, MD) was used to count the ␥-H2AX foci in each cell.
with that of control. Like MN, nuclear anomalies were more frequent among glue addicted street boys than those without glue addiction (p < 0.0001, Table 2).
2.7. Statistical analysis Results are presented as mean ± SD (standard deviation) or median with range. Statistical significance of the differences in different parameters between street boys and control group was determined by Mann–Whitney U test, Chi-square test or Student’s t test, as applicable. Statistical analyses of collected data were done using EPI info 6 and SPSS statistical software (Statistical Package for Social Sciences for windows, release 10.0, SPSS Inc., Chicago, USA) and p < 0.05 was considered significant.
3. Results 3.1. Demography Demographic and socioeconomic characteristics of the two groups of street boys and controls are summarized in Table 1. The street boys differed significantly from the control group with respect to BMI (lower in street children), education (street children were mostly illiterate) and tobacco smoking and chewing habits (none among controls). Glue-addicted street boys often had red eyes and a dazed appearance. In addition, they complained about transient loss of memory, delusions or hallucinations, slurred speech and reduced sense of smell. However, none of these signs was specific for glue addiction as glue non-addicted street boys also had many of these signs and symptoms. None of the participants were user of alcoholic beverages. Although control and street children had mixed food habit (both vegetarian and non-vegetarian foods), control group was better nourished as their food contains substantial amount of fish, fresh vegetables, fruits and dairy products which were mostly lacking in food of the street boys. Among the street boys, 114 (37.7%) were ragpickers, 84 (27.8%) were beggars, 46 (15.2%) were mobile hawkers on railway platforms and local trains, and 58 (19.2%) were a combination of ragpickers and beggars. Their daily average income was 38 rupees (US$ 0.85). All of them were tobacco users (Table 1). Out of a total of 233 street boys who smoked, 60.0% smoked cigarettes, 3.4% smoked beedi which is cheaper than cigarette, and 36.5% used to smoke both beedi and cigarettes. One hundred and fifty-five street boys (51.3% of total) were addicted to ‘Golden glue’. They used to sniff and/or huff 2–8 times a day and expend about one-third of their daily income on addiction. Glue-addicted and non-addicted boys were comparable with respect to age, BMI, tobacco smoking and/or chewing habit and education. However, they differed significantly (p < 0.05) with regard to income, expense on habits and the prevalence of loss of appetite which were more in glue addicts (p < 0.05 in Chi-square test, Table 1). Seventeen control boys (34%) were exposed to environmental tobacco smoke (ETS) as one or more male members of their families were regular smokers. In contrast, 98% of street boys were exposed to ETS (p < 0.05, Table 1). 3.2. Micronucleus and other nuclear anomalies The street boys of this study were all tobacco users in the form of smoking and/or chewing. They had 9-times higher micronucleated cell frequencies (MCFs) in exfoliated BEC than the control boys (2.98 ± 1.66 [SD] vs. 0.32 ± 0.11‰, p < 0.0001). In addition to tobacco, 155 street boys were addicted to glue sniffing/huffing. They had a MCF of 4.04 ± 2.55‰ which was 2.1-times more than the MCF of glue non-addicted street boys (p < 0.001, Table 2). MCFs of both groups of street boys (tobacco + glue and tobacco only), however, were significantly higher than that of control boys without addiction to tobacco or glue (p < 0.0001). Besides MN, the frequencies of other nuclear anomalies like ‘broken egg’, binucleation, karyorrhexis, karyolysis and pyknosis were significantly higher (p < 0.0001) in BEC of both groups of street boys when compared
3.3. Immunofluorescence microscopy for -H2AX foci count in BEC Compared with control, the mean and median numbers of ␥H2AX foci were significantly higher in BEC of street boys (p < 0.0001, Table 3, Fig. 1). Among the street boys those who were addicted to glue had about 2.5-times more ␥-H2AX foci than those without glue addiction. The difference in the number of ␥-H2AX foci in BEC between glue-addicted and non-addicted groups was highly significant (p < 0.0001) both in Student’s t-test (difference of mean) and Mann–Whitney U-test (difference of median, Table 3). 3.4. MCFs and -H2AX foci in relation to years of addiction The street boys were divided into five categories on the basis of duration of addiction (exposure years) to tobacco or tobacco plus glue: 0.5, 1.0, 1.5, 2.0 and 2.5 years or more. We found progressive rise in MCFs and ␥-H2AX foci in BEC in relation to exposure years. Highest MCFs and ␥-H2AX foci were recorded in those boys who were exposed to tobacco or tobacco plus glue for the past 2.5 years or more (Table 4). In each exposure category, tobacco plus glue users had significantly higher MN and ␥-H2AX foci than the boys who used only tobacco (p < 0.001, Table 4). Even the street boys with lowest duration of exposure to tobacco (1.44 ± 1.03‰) or tobacco plus glue (3.32 ± 1.86‰) had significantly higher MN frequency than the school (control) boys (0.32 ± 0.11‰, p < 0.001). Spearman’s rank correlation analysis revealed strong positive associations between years of glue addiction and MCFs ( = 0.9438, p < 0.0001) and ␥-H2AX foci numbers ( = 0.9826, p < 0.0001). Positive association was also found between years of tobacco use and MCFs ( = 0.752, p < 0.001) and ␥-H2AX foci numbers ( = 0.9643, p < 0.001). The positive association between glue addiction and MCF and ␥-H2AX foci number remained significant after controlling for income, spending on addiction, loss of appetite and tobacco smoking and chewing habit in multivariate logistic regression analysis (OR = 1.76, 95% confidence interval 1.33–2.68 for MCF and OR = 1.88, 95% confidence interval 1.52–4.16 for ␥-H2AX foci number). 4. Discussion The present study was designed to examine whether inhalant abuse in the form of industrial glue among 8–16-year old street boys elicits genotoxic alterations in buccal epithelial cells. We found significantly elevated MCFs and ␥-H2AX foci, considered as biomarkers of chromosomal and DNA damage respectively, among the street boys compared with age-matched controls who were local school boys without the habit of tobacco and glue. Street boys recruited in this study were all users of smoking and/or chewing tobacco. Tobacco increases MCFs in peripheral blood lymphocytes and apoptosis in buccal cells in young smokers [32]. Moreover, expression of ␥-H2AX, an indicator of potentially carcinogenic DNA double strand breaks, has been shown in lung cells of users of conventional and nonconventional tobacco products [33]. Chewing (smokeless) tobacco also induces MN formation [34], and ␥-H2AX foci expression and apoptosis in human oral cells [35]. Besides, smokeless tobacco increases the frequency of binucleated cells in human buccal epithelial cells [34]. Thus, the habit of smoking and chewing tobacco among street boys of this study seems to have contributed significantly to genotoxic changes observed in their BEC. In addition to active smoking, 98% of street children were exposed to ETS in their work and living places, whereas only one-third of control boys were exposed to ETS at home. This difference might
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Table 1 Demographic and socio-economic characteristics of the participants. Control (n = 50)
Age in years, median (range) Body mass index in kg/m2 , median (range) Tobacco smoking and chewing habit (%) Smoking Chewing Smoking plus chewing Consumption of alcoholic beverages (%) Food habit, mixed (%) Years of schooling (% in each category) 0 1–3 4–6 7–10 Personal income per day in US$ Expense on addiction per day in US$ Loss of appetite (%) Exposure to environmental tobacco smoke (%) a b c d e
Street boys (n = 302) Glue non-addicted (n = 147)
Glue addicted (n = 155)
12 (8–16) 20.2 (18.3–22.4)
12 (8–16) 19.2 (17.5–20.4)
13 (8–16) 18.8 (17.3–19.5)a
0 0 0 0 100
29.3 23.1 47.6 0 100
29.0 22.6 48.4 0 100
0 10 38 52 0 0 2.0 34
88.4 11.6 0 0 0.66 ± 0.23 0.22 ± 0.07 5.4 95.9b
89.0 11.0 0 0 1.04 ± 0.25c,d 0.35 ± 0.11c,d 20.6b,e 100b
p < 0.05 compared with control in Mann–Whitney U test. p < 0.05 compared with control in Chi-square test. p < 0.05 compared with control in Student’s t-test. p < 0.05 compared with glue non-addicted group in Student’s t-test. p < 0.05 compared with glue non-addicted group in Chi-square test.
Table 2 Changes in micronucleus frequencies and other nuclear anomalies in exfoliated buccal epithelial cells of control, glue-addicted and non-addicted street boys. Control (n = 50)
Street boys (n = 302) Glue non-addicted (n = 147)
Micronucleus per 1000 cells (‰) ‘Broken egg’ (‰) Binucleation (‰) Karyorrhexis (‰) Karyolysis (‰) Pyknosis (‰)
0.32 2.16 1.34 0.46 0.37 0.78
± ± ± ± ± ±
0.11 1.82 0.96 0.29 0.23 0.52
1.87 5.13 2.88 1.82 0.76 1.43
± ± ± ± ± ±
Glue addicted (n = 155)
a
1.06 3.61a 1.75a 1.31a 0.44a 1.07a
4.04 8.35 6.11 3.11 1.57 2.32
± ± ± ± ± ±
2.55a,b 6.22a,b 4.23a,b 2.43a,b 1.05a,b 1.68a,b
Results are expressed as mean ± standard deviation. For each participant, 2000 cells were screened for micronucleus and broken egg assay while 1000 cells were scored for remaining nuclear changes. a p < 0.0001 compared with control. b p < 0.0001 compared with glue non-addicted street boys in Student’s t-test. Table 3 The number of ␥-H2AX foci in exfoliated buccal epithelial cells of glue non-addicted and addicted street boys with respect to control. Number of ␥-H2AX foci per cell
Mean ± SD Median (range)
Control (n = 50)
4.08 ± 1.40 3.5 (1.3–6.8)
Street boys (n = 302) Glue non-addicted (n = 147)
Glue addicted (n = 155)
9.73 ± 4.35 8.5 (4.3–21.7)c
21.12 ± 4.72a,b 20.5 (10.3–31.7)c,d
a
For each participant, an average of 400 cells were screened for detection of ␥-H2AX foci. a p < 0.0001 compared with control. b p < 0.05 compared with glue non-addicted group in Student’s t-test. c p < 0.0001 compared with control. d p < 0.0001 compared with glue non-addicted boys in Mann–Whitney U-test.
Fig. 1. Immunofluorescence showing ␥-H2AX foci formation in nuclei of exfoliated buccal epithelial cells of the participants. (a) Control, (b) glue non-addicted and (c) glue addicted street boys. Note highest number of ␥-H2AX foci in nuclei of glue users. Magnification: 1000×.
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Table 4 Micronucleus formation and ␥-H2AX foci expression in exfoliated buccal epithelial cells of street boys in relation to duration of exposure to either tobacco or tobacco plus Golden glue. Years of addiction 0 (control)
0.5
Micronucleus per 1000 cells (‰) Only tobacco users 0.32 ± 0.11 (50) 1.44 Tobacco + glue users 3.32 -H2AX foci per nucleus Only tobacco users 4.08 ± 1.40 7.97 Tobacco + glue users 15.89
1.0 ± 1.03 (26) ± 1.86a (29) ± 3.71 ± 3.81b
1.5
1.45 ± 1.12 (34) 3.66 ± 2.05a (38) 8.95 ± 4.02 17.47 ± 3.86b
1.89 ± 1.22 (38) 3.98 ± 2.33a (31) 9.30 ± 4.15 19.88 ± 4.60b
2.0 2.08 ± 1.11 (26) 4.53 ± 3.16a (30) 10.11 ± 4.81 24.95 ± 4.71b
2.5 or more 2.31 ± 1.41 (23) 4.71 ± 2.97a (27) 12.34 ± 5.25 27.39 ± 4.52b
Results are expressed as mean ± SD. Numbers in parentheses are the number of street boys in each exposure category. a p < 0.001. b p < 0.0001 compared with only tobacco users in Student’s t-test.
have contributed to some extent to higher frequency of MN and ␥-H2AX foci among street boys, because chronic exposure to ETS has been shown to cause DNA damage in children [36]. Besides MN, epithelial cells of street boys exhibited an excess of several other nuclear anomalies such as broken egg (cells with nuclear buds), binucleation, pyknosis, karyorrhexis, and karyolysis. Broken egg formation is believed to be related to the elimination of amplified DNA or DNA repair [37], while binucleation may indicate dysregulation of nuclear division such as failure of cytokinesis in the last nuclear division in the basal cell layer [30]. Pyknosis and karyorrhexis indicate both cytotoxicity (necrosis and keratinization) and genotoxicity (apoptosis) while rise in karyolysis suggests cytotoxicity [29] in epithelial cells of the street boys enrolled in this study. A subgroup of the street boys had the habit of sniffing/huffing glue in addition to tobacco use. They had significantly higher MCFs and ␥-H2AX foci in BEC than glue non-addicted street boys, implying significant genotoxicity associated with glue exposure. Since we did not have a subgroup of glue addicted street boys who were not users of tobacco, it is difficult to ascertain whether the genotoxicity of tobacco and glue was additive or synergistic. Increase in DSB type of DNA damage, as reflected by elevated number of ␥-H2AX foci in nuclei, represents one of the most serious forms of genetic damage. Since both strands of the DNA double helix are broken, chromosomal fragmentation, translocation, and deletions can easily occur in these cells making them more vulnerable to genomic instability [38] and tumor development if the lesion occurs in a critical gene, such as tumor suppressor gene [39]. However, buccal epithelial cells have a rapid turnover, so that damaged cells are removed and do not accumulate, and would therefore not increase with time. The mechanism by which glue sniffing/huffing elicits genotoxicity is currently unknown. Oxidative stress could be involved because chronic inhalation of volatile substances in glue and thinner has been shown to alter the levels of antioxidant enzymes leading to lipid peroxidation in children [40]. The glue contains a large number of organic chemicals and it is difficult to pinpoint the agent(s) responsible for mediating the chromosomal and DNA damage. Since the effect is elicited through sniffing and/or huffing, the causative agent could be volatile organic compound(s) such as benzene, toluene and xylene. Since 1982, benzene is classified as a human carcinogen by the International Agency for Research on Cancer (IARC) and the American Conference of Governmental Industrial Hygienists (ACGIH) [41,42]. Toluene or xylene, on the other hand, were not proven to be genotoxic or carcinogenic for humans or laboratory animals [43,44], and they are considered as ‘not classifiable as a human carcinogen’ by IARC and ACGIH. In addition to tobacco and glue, environmental and nutritional factors can play a role in eliciting genotoxicity among the street boys. For example, arsenic contamination in drinking water, a
health problem in eastern India, may cause genetic damage [45]. However, areas from where the boys were recruited for this study were arsenic-free, and both the street boys and school boys used to drink same underground water lifted by tube wells by the local administration or the railway authority. Thus it seems arsenic was not a contributing factor to genotoxicity. The street boys spent most of the time in polluted areas, sleep on top of the cornices, under staircases and in other neglected unhygienic corners of the station. Although we have not measured the air pollution level in railway stations, it seems that they were chronically exposed to high level of air pollution in a busy railway station and this can results in rise in MCFs and ␥-H2AX foci among street boys. Besides being homeless and bereft of parental care, the street boys were malnourished having a lower BMI than the controls, and malnutrition can lead to DNA damage. For example, deficiencies of magnesium or zinc, two metals required for DNA synthesis, may increase spontaneous chromosomal damage, and may strongly influence the genotoxic response to other dietary factors [46]. Similarly, folic acid and vitamin B12 deficiency causes chromosome breaks [47] and micronucleus formation in blood and epithelial cells and damage to both nuclear and mitochondrial DNA [48]. Besides, folic acid and other nutritional deficiencies can make the individual more susceptible to damage caused by other agents. This may be the case with glue sniffing. Most of the volatile components of the glue are not genotoxic or are very weak, but the boys’ nutritional deficiencies would tend to make them more susceptible to the chemicals’ effects. In agreement with this, the average comet tail length of cells from malnourished children was found twice of those well-nourished children [49]. Similarly, lymphocytes from malnourished children are more susceptible to DNA damage by antibiotics [50], and their ability to repair damaged DNA is lower than the well-nourished children [51]. In essence, the study, first of its kind in India or elsewhere, suggests that tobacco smoking and/or chewing habit among street boys is associated with increased frequency of micronucleus and ␥-H2AX foci formation in oral mucosa, implying chromosomal and DNA damage respectively. Additional habit of sniffing/huffing of industrial glue enhances the severity of genotoxic changes in BEC of these children. Earlier we observed marked increase in argyrophilic nuclear organizer region (AgNOR) in BEC of glue-addicted children, suggesting up-regulation of ribosome biogenesis in these cells [52]. Taken together, the studies indicate genetic changes in oral mucosa of street boys in association with tobacco and glue sniffing or huffing habit. Based on the present findings we recommend use of MN and ␥-H2AX foci formation as biomarkers of genetic damage in inhalant abuse intervention programs. Genetic markers could provide a useful means of detecting early mutagenic events for assessing cancer risk associated with inhalant abuse which is quite prevalent in developed countries also. In the United States, for example, nearly 20% of young persons have experimented with
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inhalants at least once by the time they are in eighth grade and the mean age of first-time inhalant abuse is 13 years [53]. More importantly, children who abuse inhalants early in life are more likely later to use other illicit drugs [54,55]. Thus inhalant abuse intervention programs seem important from the community health perspective also.
[27] [28] [29] [30]
Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgement The authors gratefully acknowledge the financial support received from Central Pollution Control Board, Government of India, Delhi in carrying out this study.
[31]
[32]
[33]
[34]
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Micronucleus formation and DNA damage in buccal epithelial cells of Indian street boys addicted to gasp ‘Golden glue’ Nandan Kumar Mondal a,∗ , Sreenita Ghosh b , Manas Ranjan Ray a a b
Department of Experimental Hematology, Chittaranjan National Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, West Bengal 700026, India Department of Zoology, University of Burdwan, Burdwan 713104, India
a r t i c l e
i n f o
Article history: Received 11 July 2010 Received in revised form 1 January 2011 Accepted 22 January 2011 Available online 31 January 2011 Keywords: Micronucleus DNA damage Glue Addiction Street children India
a b s t r a c t Genotoxicity of glue sniffing/huffing and tobacco use has been examined in 302 street boys (median age 13 years) and 50 age-matched control school boys who were neither tobacco nor glue users. All the street boys were tobacco users. In addition, 155 were addicted to gasp an industrial adhesive popularly known as ‘Golden glue’. Micronucleus (MN) frequency was determined as a measure of chromosomal breakage in exfoliated buccal epithelial cells (BECs) and DNA double strand breaks were quantitatively assessed by counting ␥-H2AX foci using immunofluorescence microscopy. Micronucleated cell frequencies (MCFs) in BEC of glue non-addicted (only tobacco) and addicted (tobacco plus glue) street boys were 1.87 ± 1.06‰ and 4.04 ± 2.55‰ respectively, which were significantly higher than that of control (0.32 ± 0.11‰, p < 0.0001). Similarly, the numbers ␥-H2AX foci in nuclei of BEC were 2.3- and 5.2-times more than control in glue non-addicted and addicted street boys respectively (p < 0.0001). Spearman’s rank correlation revealed a strong positive association between years of glue addiction with MCFs and ␥-H2AX foci numbers, and the association between glue addiction and chromosomal and DNA damage remained positive and significant after controlling income, spending on addiction and loss of appetite as potential confounders in multivariate logistic regression analysis. Thus, addiction to tobacco among the street children in India is associated with chromosomal and DNA damage in BECs and the severity of these changes is significantly increased by the habit of sniffing/huffing of industrial glue. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Inhalant dependence and abuse have been reported from various parts of the world [1–4]. Drug usage among minors has created a global generation of addicted and often abandoned children bereft of family support, education and social skills. Chronic exposure to abuse solvents can produce loss of appetite [5], but the underlying mechanism is not well understood. Street children constitute a marginalized population in most of the urban centers throughout the world. In India, street children are abundant in all metros, but their exact number is not well documented. The magnitude of the hardship they experience is also rarely unveiled. The marginalized street children constitute a truly “hidden” population who are not covered by nor find place in the national census, educational or health data, primarily because they are a floating population without any fixed address [6]. It is widely speculated that between 100,000 and 125,000 children live on the streets and railway stations of India’s major cities, and that more than half of them have some form of drug addiction. Street boys living in different rail-
∗ Corresponding author. Tel.: +91 33 2476 5101; fax: +91 33 2475 7606. E-mail address: nandan [email protected] (N.K. Mondal). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.01.011
way stations in West Bengal, a state in eastern India, take care of themselves and each other in an otherwise uncaring environment and they survive through begging, petty theft or hawking goods on the railway platforms. The major part of the paltry sum they earn is spent on tobacco and on tube of ‘Golden glue’ (commercially available as ‘Dendrite’ which is an industrial contact adhesive and rubber cement marketed in the form of glue sticks, tubes and cans in Eastern India, Bangladesh and Bhutan) which is a particular favorite with those children. This golden colored industrial glue containing organic solvents is cheap and readily available and provides faster onset of action and the regular ‘high’ [7,8]. Glue sniffing has been reported also from Mexico among 1% in general population and 22% of street children [9]. It has been estimated that the glue addicts can be exposed to 2000–30,000 ppm of organic solvents within a few minutes [10]. In India, the kids squeeze glue onto a rag and huff its fumes through the mouth. The overall effects of glue inhalation range from an alcohol-like intoxication and intense euphoria to vivid hallucinations, depending on the substance and the dosage. These products are not classified as drugs and young children and adolescents can easily obtain them and abuse them [5]. Besides addiction, street boys in India are usually victims of abuse and harassment, and are extremely vulnerable to trafficking, sex trade and child labor [11]. Glue sniffing can be more
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injurious to health than cigarette smoking as the glue contains a large number of health-damaging chemicals like benzene, xylene, toluene, n-hexane, heptane, styrene, acetone, methyl ethyl ketone, methylene chloride, cyanoacrylates trichloroethane, halogenated aromatic hydrocarbons and mineral spirits [12–16]. A number of these chemicals are potential mutagens or carcinogens. Toluene, a volatile organic compound, is the main toxic component that enters the body through glue sniffing [17]. Its metabolite hippuric acid is nephrotoxic [18] causing kidney failure [12]. Besides, long term glue sniffing can lead to liver disease [19] and damage to the nervous system [20]. Once these solvents or gases are inhaled, the extensive capillary surface of the lungs and the airways readily absorbs them. Since the glue contains human carcinogens including benzene, chronic inhalation of glue may lead to DNA damage particularly in those cells that are at the direct line of exposure such as cells of the nasopharynx, oral cavity, airways and the lung. The literature from India on inhalant abuse is limited to only a few case reports [21,22] and case series [7,23–25]. To our knowledge, no study has ever been carried out in India or elsewhere on the possibility of chromosomal and DNA damage among chronic glue inhalers. DNA double-strand breaks (DSBs) can result in cell death or genetic alterations when cells are subjected to environmental or occupational stresses. A complex DNA-damage-response pathway is activated to repair the damage, and the inability to repair these breaks can lead to carcinogenesis. One of the earliest responses to DSBs is the phosphorylation of the histone H2AX at serine 139 (␥-H2AX), which can be detected by a fluorescent antibody [26]. We examined in this study whether glue sniffing by street children elicits chromosomal and DNA damage in epithelial cells of the oropharynx. To achieve this objective we employed micronucleus (MN) assay for the detection of chromosomal damage and enumeration of ␥-H2AX foci for evaluation of the possibility of DNA DSBs in exfoliated buccal epithelial cells.
2.2. Collection of background data
2. Materials and methods
2.5. Pap staining and MN assay
2.1. Participants
For Papanicolaou (Pap) staining, the fixed slides (two for each individual) were brought to water through graded ethanol. The slides were then stained with Harris’ hematoxylin for 30 s, rinsed in distilled water and placed in Scott’s tap substitute for bluing. Thereafter, the slides were washed in running tap water, dehydrated in 70% and 90% ethanol, stained in Orange-G6 for 30 min and differentiated in 95% alcohol. Then the slides were incubated in EA-50 solution for 30 min in room temperature, dehydrated in ethanol and mounted with distrene plasticizer xylene (DPX) mountant. The slides were coded and examined under light microscope (Leitz Dialux 20, Germany) with 100× oil immersion objective for the presence of micronucleus (MN) and other nuclear anomalies. For MN, 2000 non-overlapping, differentiated, uniformly stained nuclei of BEC of each individual were analyzed by two investigators (NKM and MRR) blindly. Both investigators scored slides from all the subjects independently. The difference in final score between the observers was less than 5%, and the arithmetic mean of two scores was used as individual value. Cells with pyknotic or condensed chromatin or having karyorrhectic nuclei and keratohyalin bodies in the cytoplasm were not considered for MN assay. MN identification was based essentially on the criteria of Tolbert et al. [29] as described in detail by Thomas et al. [30]. In brief, the characteristics of MN are (i) round or oval in shape, (ii) diameter ranges between 1/3rd and 1/16th of the main nucleus, (iii) have the same staining intensity and texture as the main nucleus, (iv) are located within the cytoplasm of the cells, and (v) usually one per cell, but the number could be two or more following genotoxic insults. Besides MN, other nuclear anomalies like ‘broken egg’ (cells with nuclear buds), binucleated cells, pyknosis (shrunken nuclei), karyorrhexis (nuclear disintegration), and karyolysis (dissolution of nucleus) were evaluated following the established criteria [29,30]. ‘Broken egg’ was scored from 2000 cells while 1000 cells were examined for the detection of other nuclear anomalies [30].
2.1.1. Street boys A total of 302 homeless street boys aged between 8 and 16 years were recruited from different railway stations in and around Kolkata (former Calcutta) in eastern India. The inclusion criteria for street boys were (i) apparently healthy, homeless, 8–16 year-old boys surviving through begging or hawking goods on the railway platforms for the past 6 months or more, (ii) having a body mass index (BMI) >15 and <25 kg/m2 , and (iii) users of tobacco or tobacco plus glue. They were all either smokers and/or chewers of tobacco. They usually smoke 3–10 (median 5) beedi (tobacco rolled on dried Tendu leaf [Diospyros melanoxylon Family Ebenaceae]) or cheap brands of cigarettes per day and chew raw tobacco with lime (khaini) or tobacco in combination with betel nut and catechu (guthka) available in local shops in small pouches. Out of the 302 street boys, 155 (median age 12 years) were addicted to ‘Golden glue’, an industrial adhesive marketed with the brand name of ‘Dendrite’. They breathe in the glue directly from its container (called sniffing or snorting) or they place a rag soaked in the glue over their nose and mouth and inhaling it (called huffing). Sniffing or huffing of the glue provides a quick, powerful ‘high’. The remaining 147 street boys (median age 13 years) were not addicted to glue. We recorded spots or sores around the mouth, red eyes, rhinorrhea, chemical odor on the breath, and a dazed appearance as signs of recent inhalant abuse [27]. Dietary habits of the participants were surveyed using a food quality and frequency questionnaire. In general, the street boys purchase/beg their food from small restaurants and eateries at the railway stations. We came across only 12 boys (3.9%) who used to cook their own food in makeshift ovens near the railway track using waste paper, cardboard, packing boxes, etc. as cooking fuel. 2.1.2. Control (school) boys For comparison, we enrolled 50 school boys aged between 8 and 16 years (median age 12 years) from suburban areas of Kolkata. Majority of these boys (34/50) were studying in class IV–VII in local schools. The average distance from home to the schools was 1.0 km and they either walk or bicycles to reach the schools. The boys came from families with a monthly income of US$ 88–275 (median 124). They were all nonsmokers and non-chewers of tobacco and they did not have the habit of glue inhalation. Accordingly they were considered as control.
Members of the research team interviewed each participant separately. In general, they were asked to furnish background information about age, education, food, tobacco smoking and chewing habit. Boys in the control group were asked in addition about their parent’s occupation, family size, housing, type of cooking fuel used at home, and presence of smoker in family. The street boys were asked about their means of living (hawking/begging/ragpicking/other), tobacco smoking (beedi/cigarette, number per day) and chewing habit (khaini/guthka, number of pouches per day), consumption of alcohol (yes/no), addiction to glue (duration, number of sniffing per day) and source of food and drinking water. Large majority of the street boys were illiterate; hence the researchers entered their responses in structured questionnaire forms on their behalf. The Ethics Committee of Chittaranjan National Cancer Institute, Kolkata approved the study protocol. 2.3. Chemicals Hematoxylin (CAS No. 517-28-2), orange-G6 (CAS No. 1936-15-8), eosin Y (CAS No. 17372-87-1), light green SF yellowish (CAS No. 5141-20-8), Bismarck brown (CAS No. 10114-58-6), phosphotungstic acid (CAS No. 12067-99-1), paraformaldehyde (CAS No. 30525-89-4), Triton-X 100 (CAS No. 9002-93-1), and BSA (CAS No. 9048-46-8), were purchased from Sigma–Aldrich Chemicals, Saint Louis, MO, USA. Potash alum (CAS No. 7784-24-9), and GAA (CAS No. 64-19-7) were obtained from Merck, Mumbai, India. Lithium carbonate (CAS No. 554-13-2) was from SISCO Research Laboratories, India. Ethanol (CAS No. 64-17-5) was from Bengal Chemicals & Pharmaceuticals Ltd., Kolkata, India. All the chemicals were of analytical reagent grade. Primary antibody ‘rabbit polyclonal ␥-H2AX [phosphor S-139]’ (Cat No. ab2839) and secondary antibody ‘FITC-conjugated goat anti-rabbit IgG’ (Cat No. sc-2012) were purchased from Abcam, UK and Santacruz, Biotechnology Inc., USA respectively. 2.4. Collection and fixation of exfoliated buccal epithelial cells All the participants were asked to wash their mouth with sterile 0.9% saline (NaCl). Epithelial cells were collected from buccal mucosa scraping the middle part of the inner cheeks with sterile plastic spatula. Four slides were prepared for each participant. Two slides were immediately fixed in 95% ethanol and brought to the laboratory and stained with Papanicolaou (Pap) method following the procedure of Hughes and Dodds [28]. Remaining two slides were fixed in 4% paraformaldehyde at the site of collection for immunocytochemical localization of ␥-H2AX foci.
2.6. Immunofluorescent microscopy and quantification of -H2AX foci Immunofluorescent microscopic study was carried out following the procedure of Zhou et al. [31], with slight modifications. In short, paraformaldehyde fixed slides (two for each individual) were washed with PBS, and permeabilized with 0.2% Triton X-100. After blocking with 3% bovine serum albumin (BSA) for 1.5 h, samples were incubated with rabbit polyclonal ␥-H2AX (phosphor S 139; diluted 1:1000 in 1% BSA) for 2 h, followed with FITC-conjugated goat anti-rabbit IgG secondary antibody (1:500) for 1 h in darkness and examined immediately using a fluorescence microscope (Leica DM 4000B, Germany). To prevent bias in selection of cells that
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display foci, all the cells were counted in the field of vision. Image Pro Plus (Media Cybernetics, Silver Spring, MD) was used to count the ␥-H2AX foci in each cell.
with that of control. Like MN, nuclear anomalies were more frequent among glue addicted street boys than those without glue addiction (p < 0.0001, Table 2).
2.7. Statistical analysis Results are presented as mean ± SD (standard deviation) or median with range. Statistical significance of the differences in different parameters between street boys and control group was determined by Mann–Whitney U test, Chi-square test or Student’s t test, as applicable. Statistical analyses of collected data were done using EPI info 6 and SPSS statistical software (Statistical Package for Social Sciences for windows, release 10.0, SPSS Inc., Chicago, USA) and p < 0.05 was considered significant.
3. Results 3.1. Demography Demographic and socioeconomic characteristics of the two groups of street boys and controls are summarized in Table 1. The street boys differed significantly from the control group with respect to BMI (lower in street children), education (street children were mostly illiterate) and tobacco smoking and chewing habits (none among controls). Glue-addicted street boys often had red eyes and a dazed appearance. In addition, they complained about transient loss of memory, delusions or hallucinations, slurred speech and reduced sense of smell. However, none of these signs was specific for glue addiction as glue non-addicted street boys also had many of these signs and symptoms. None of the participants were user of alcoholic beverages. Although control and street children had mixed food habit (both vegetarian and non-vegetarian foods), control group was better nourished as their food contains substantial amount of fish, fresh vegetables, fruits and dairy products which were mostly lacking in food of the street boys. Among the street boys, 114 (37.7%) were ragpickers, 84 (27.8%) were beggars, 46 (15.2%) were mobile hawkers on railway platforms and local trains, and 58 (19.2%) were a combination of ragpickers and beggars. Their daily average income was 38 rupees (US$ 0.85). All of them were tobacco users (Table 1). Out of a total of 233 street boys who smoked, 60.0% smoked cigarettes, 3.4% smoked beedi which is cheaper than cigarette, and 36.5% used to smoke both beedi and cigarettes. One hundred and fifty-five street boys (51.3% of total) were addicted to ‘Golden glue’. They used to sniff and/or huff 2–8 times a day and expend about one-third of their daily income on addiction. Glue-addicted and non-addicted boys were comparable with respect to age, BMI, tobacco smoking and/or chewing habit and education. However, they differed significantly (p < 0.05) with regard to income, expense on habits and the prevalence of loss of appetite which were more in glue addicts (p < 0.05 in Chi-square test, Table 1). Seventeen control boys (34%) were exposed to environmental tobacco smoke (ETS) as one or more male members of their families were regular smokers. In contrast, 98% of street boys were exposed to ETS (p < 0.05, Table 1). 3.2. Micronucleus and other nuclear anomalies The street boys of this study were all tobacco users in the form of smoking and/or chewing. They had 9-times higher micronucleated cell frequencies (MCFs) in exfoliated BEC than the control boys (2.98 ± 1.66 [SD] vs. 0.32 ± 0.11‰, p < 0.0001). In addition to tobacco, 155 street boys were addicted to glue sniffing/huffing. They had a MCF of 4.04 ± 2.55‰ which was 2.1-times more than the MCF of glue non-addicted street boys (p < 0.001, Table 2). MCFs of both groups of street boys (tobacco + glue and tobacco only), however, were significantly higher than that of control boys without addiction to tobacco or glue (p < 0.0001). Besides MN, the frequencies of other nuclear anomalies like ‘broken egg’, binucleation, karyorrhexis, karyolysis and pyknosis were significantly higher (p < 0.0001) in BEC of both groups of street boys when compared
3.3. Immunofluorescence microscopy for -H2AX foci count in BEC Compared with control, the mean and median numbers of ␥H2AX foci were significantly higher in BEC of street boys (p < 0.0001, Table 3, Fig. 1). Among the street boys those who were addicted to glue had about 2.5-times more ␥-H2AX foci than those without glue addiction. The difference in the number of ␥-H2AX foci in BEC between glue-addicted and non-addicted groups was highly significant (p < 0.0001) both in Student’s t-test (difference of mean) and Mann–Whitney U-test (difference of median, Table 3). 3.4. MCFs and -H2AX foci in relation to years of addiction The street boys were divided into five categories on the basis of duration of addiction (exposure years) to tobacco or tobacco plus glue: 0.5, 1.0, 1.5, 2.0 and 2.5 years or more. We found progressive rise in MCFs and ␥-H2AX foci in BEC in relation to exposure years. Highest MCFs and ␥-H2AX foci were recorded in those boys who were exposed to tobacco or tobacco plus glue for the past 2.5 years or more (Table 4). In each exposure category, tobacco plus glue users had significantly higher MN and ␥-H2AX foci than the boys who used only tobacco (p < 0.001, Table 4). Even the street boys with lowest duration of exposure to tobacco (1.44 ± 1.03‰) or tobacco plus glue (3.32 ± 1.86‰) had significantly higher MN frequency than the school (control) boys (0.32 ± 0.11‰, p < 0.001). Spearman’s rank correlation analysis revealed strong positive associations between years of glue addiction and MCFs ( = 0.9438, p < 0.0001) and ␥-H2AX foci numbers ( = 0.9826, p < 0.0001). Positive association was also found between years of tobacco use and MCFs ( = 0.752, p < 0.001) and ␥-H2AX foci numbers ( = 0.9643, p < 0.001). The positive association between glue addiction and MCF and ␥-H2AX foci number remained significant after controlling for income, spending on addiction, loss of appetite and tobacco smoking and chewing habit in multivariate logistic regression analysis (OR = 1.76, 95% confidence interval 1.33–2.68 for MCF and OR = 1.88, 95% confidence interval 1.52–4.16 for ␥-H2AX foci number). 4. Discussion The present study was designed to examine whether inhalant abuse in the form of industrial glue among 8–16-year old street boys elicits genotoxic alterations in buccal epithelial cells. We found significantly elevated MCFs and ␥-H2AX foci, considered as biomarkers of chromosomal and DNA damage respectively, among the street boys compared with age-matched controls who were local school boys without the habit of tobacco and glue. Street boys recruited in this study were all users of smoking and/or chewing tobacco. Tobacco increases MCFs in peripheral blood lymphocytes and apoptosis in buccal cells in young smokers [32]. Moreover, expression of ␥-H2AX, an indicator of potentially carcinogenic DNA double strand breaks, has been shown in lung cells of users of conventional and nonconventional tobacco products [33]. Chewing (smokeless) tobacco also induces MN formation [34], and ␥-H2AX foci expression and apoptosis in human oral cells [35]. Besides, smokeless tobacco increases the frequency of binucleated cells in human buccal epithelial cells [34]. Thus, the habit of smoking and chewing tobacco among street boys of this study seems to have contributed significantly to genotoxic changes observed in their BEC. In addition to active smoking, 98% of street children were exposed to ETS in their work and living places, whereas only one-third of control boys were exposed to ETS at home. This difference might
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Table 1 Demographic and socio-economic characteristics of the participants. Control (n = 50)
Age in years, median (range) Body mass index in kg/m2 , median (range) Tobacco smoking and chewing habit (%) Smoking Chewing Smoking plus chewing Consumption of alcoholic beverages (%) Food habit, mixed (%) Years of schooling (% in each category) 0 1–3 4–6 7–10 Personal income per day in US$ Expense on addiction per day in US$ Loss of appetite (%) Exposure to environmental tobacco smoke (%) a b c d e
Street boys (n = 302) Glue non-addicted (n = 147)
Glue addicted (n = 155)
12 (8–16) 20.2 (18.3–22.4)
12 (8–16) 19.2 (17.5–20.4)
13 (8–16) 18.8 (17.3–19.5)a
0 0 0 0 100
29.3 23.1 47.6 0 100
29.0 22.6 48.4 0 100
0 10 38 52 0 0 2.0 34
88.4 11.6 0 0 0.66 ± 0.23 0.22 ± 0.07 5.4 95.9b
89.0 11.0 0 0 1.04 ± 0.25c,d 0.35 ± 0.11c,d 20.6b,e 100b
p < 0.05 compared with control in Mann–Whitney U test. p < 0.05 compared with control in Chi-square test. p < 0.05 compared with control in Student’s t-test. p < 0.05 compared with glue non-addicted group in Student’s t-test. p < 0.05 compared with glue non-addicted group in Chi-square test.
Table 2 Changes in micronucleus frequencies and other nuclear anomalies in exfoliated buccal epithelial cells of control, glue-addicted and non-addicted street boys. Control (n = 50)
Street boys (n = 302) Glue non-addicted (n = 147)
Micronucleus per 1000 cells (‰) ‘Broken egg’ (‰) Binucleation (‰) Karyorrhexis (‰) Karyolysis (‰) Pyknosis (‰)
0.32 2.16 1.34 0.46 0.37 0.78
± ± ± ± ± ±
0.11 1.82 0.96 0.29 0.23 0.52
1.87 5.13 2.88 1.82 0.76 1.43
± ± ± ± ± ±
Glue addicted (n = 155)
a
1.06 3.61a 1.75a 1.31a 0.44a 1.07a
4.04 8.35 6.11 3.11 1.57 2.32
± ± ± ± ± ±
2.55a,b 6.22a,b 4.23a,b 2.43a,b 1.05a,b 1.68a,b
Results are expressed as mean ± standard deviation. For each participant, 2000 cells were screened for micronucleus and broken egg assay while 1000 cells were scored for remaining nuclear changes. a p < 0.0001 compared with control. b p < 0.0001 compared with glue non-addicted street boys in Student’s t-test. Table 3 The number of ␥-H2AX foci in exfoliated buccal epithelial cells of glue non-addicted and addicted street boys with respect to control. Number of ␥-H2AX foci per cell
Mean ± SD Median (range)
Control (n = 50)
4.08 ± 1.40 3.5 (1.3–6.8)
Street boys (n = 302) Glue non-addicted (n = 147)
Glue addicted (n = 155)
9.73 ± 4.35 8.5 (4.3–21.7)c
21.12 ± 4.72a,b 20.5 (10.3–31.7)c,d
a
For each participant, an average of 400 cells were screened for detection of ␥-H2AX foci. a p < 0.0001 compared with control. b p < 0.05 compared with glue non-addicted group in Student’s t-test. c p < 0.0001 compared with control. d p < 0.0001 compared with glue non-addicted boys in Mann–Whitney U-test.
Fig. 1. Immunofluorescence showing ␥-H2AX foci formation in nuclei of exfoliated buccal epithelial cells of the participants. (a) Control, (b) glue non-addicted and (c) glue addicted street boys. Note highest number of ␥-H2AX foci in nuclei of glue users. Magnification: 1000×.
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Table 4 Micronucleus formation and ␥-H2AX foci expression in exfoliated buccal epithelial cells of street boys in relation to duration of exposure to either tobacco or tobacco plus Golden glue. Years of addiction 0 (control)
0.5
Micronucleus per 1000 cells (‰) Only tobacco users 0.32 ± 0.11 (50) 1.44 Tobacco + glue users 3.32 -H2AX foci per nucleus Only tobacco users 4.08 ± 1.40 7.97 Tobacco + glue users 15.89
1.0 ± 1.03 (26) ± 1.86a (29) ± 3.71 ± 3.81b
1.5
1.45 ± 1.12 (34) 3.66 ± 2.05a (38) 8.95 ± 4.02 17.47 ± 3.86b
1.89 ± 1.22 (38) 3.98 ± 2.33a (31) 9.30 ± 4.15 19.88 ± 4.60b
2.0 2.08 ± 1.11 (26) 4.53 ± 3.16a (30) 10.11 ± 4.81 24.95 ± 4.71b
2.5 or more 2.31 ± 1.41 (23) 4.71 ± 2.97a (27) 12.34 ± 5.25 27.39 ± 4.52b
Results are expressed as mean ± SD. Numbers in parentheses are the number of street boys in each exposure category. a p < 0.001. b p < 0.0001 compared with only tobacco users in Student’s t-test.
have contributed to some extent to higher frequency of MN and ␥-H2AX foci among street boys, because chronic exposure to ETS has been shown to cause DNA damage in children [36]. Besides MN, epithelial cells of street boys exhibited an excess of several other nuclear anomalies such as broken egg (cells with nuclear buds), binucleation, pyknosis, karyorrhexis, and karyolysis. Broken egg formation is believed to be related to the elimination of amplified DNA or DNA repair [37], while binucleation may indicate dysregulation of nuclear division such as failure of cytokinesis in the last nuclear division in the basal cell layer [30]. Pyknosis and karyorrhexis indicate both cytotoxicity (necrosis and keratinization) and genotoxicity (apoptosis) while rise in karyolysis suggests cytotoxicity [29] in epithelial cells of the street boys enrolled in this study. A subgroup of the street boys had the habit of sniffing/huffing glue in addition to tobacco use. They had significantly higher MCFs and ␥-H2AX foci in BEC than glue non-addicted street boys, implying significant genotoxicity associated with glue exposure. Since we did not have a subgroup of glue addicted street boys who were not users of tobacco, it is difficult to ascertain whether the genotoxicity of tobacco and glue was additive or synergistic. Increase in DSB type of DNA damage, as reflected by elevated number of ␥-H2AX foci in nuclei, represents one of the most serious forms of genetic damage. Since both strands of the DNA double helix are broken, chromosomal fragmentation, translocation, and deletions can easily occur in these cells making them more vulnerable to genomic instability [38] and tumor development if the lesion occurs in a critical gene, such as tumor suppressor gene [39]. However, buccal epithelial cells have a rapid turnover, so that damaged cells are removed and do not accumulate, and would therefore not increase with time. The mechanism by which glue sniffing/huffing elicits genotoxicity is currently unknown. Oxidative stress could be involved because chronic inhalation of volatile substances in glue and thinner has been shown to alter the levels of antioxidant enzymes leading to lipid peroxidation in children [40]. The glue contains a large number of organic chemicals and it is difficult to pinpoint the agent(s) responsible for mediating the chromosomal and DNA damage. Since the effect is elicited through sniffing and/or huffing, the causative agent could be volatile organic compound(s) such as benzene, toluene and xylene. Since 1982, benzene is classified as a human carcinogen by the International Agency for Research on Cancer (IARC) and the American Conference of Governmental Industrial Hygienists (ACGIH) [41,42]. Toluene or xylene, on the other hand, were not proven to be genotoxic or carcinogenic for humans or laboratory animals [43,44], and they are considered as ‘not classifiable as a human carcinogen’ by IARC and ACGIH. In addition to tobacco and glue, environmental and nutritional factors can play a role in eliciting genotoxicity among the street boys. For example, arsenic contamination in drinking water, a
health problem in eastern India, may cause genetic damage [45]. However, areas from where the boys were recruited for this study were arsenic-free, and both the street boys and school boys used to drink same underground water lifted by tube wells by the local administration or the railway authority. Thus it seems arsenic was not a contributing factor to genotoxicity. The street boys spent most of the time in polluted areas, sleep on top of the cornices, under staircases and in other neglected unhygienic corners of the station. Although we have not measured the air pollution level in railway stations, it seems that they were chronically exposed to high level of air pollution in a busy railway station and this can results in rise in MCFs and ␥-H2AX foci among street boys. Besides being homeless and bereft of parental care, the street boys were malnourished having a lower BMI than the controls, and malnutrition can lead to DNA damage. For example, deficiencies of magnesium or zinc, two metals required for DNA synthesis, may increase spontaneous chromosomal damage, and may strongly influence the genotoxic response to other dietary factors [46]. Similarly, folic acid and vitamin B12 deficiency causes chromosome breaks [47] and micronucleus formation in blood and epithelial cells and damage to both nuclear and mitochondrial DNA [48]. Besides, folic acid and other nutritional deficiencies can make the individual more susceptible to damage caused by other agents. This may be the case with glue sniffing. Most of the volatile components of the glue are not genotoxic or are very weak, but the boys’ nutritional deficiencies would tend to make them more susceptible to the chemicals’ effects. In agreement with this, the average comet tail length of cells from malnourished children was found twice of those well-nourished children [49]. Similarly, lymphocytes from malnourished children are more susceptible to DNA damage by antibiotics [50], and their ability to repair damaged DNA is lower than the well-nourished children [51]. In essence, the study, first of its kind in India or elsewhere, suggests that tobacco smoking and/or chewing habit among street boys is associated with increased frequency of micronucleus and ␥-H2AX foci formation in oral mucosa, implying chromosomal and DNA damage respectively. Additional habit of sniffing/huffing of industrial glue enhances the severity of genotoxic changes in BEC of these children. Earlier we observed marked increase in argyrophilic nuclear organizer region (AgNOR) in BEC of glue-addicted children, suggesting up-regulation of ribosome biogenesis in these cells [52]. Taken together, the studies indicate genetic changes in oral mucosa of street boys in association with tobacco and glue sniffing or huffing habit. Based on the present findings we recommend use of MN and ␥-H2AX foci formation as biomarkers of genetic damage in inhalant abuse intervention programs. Genetic markers could provide a useful means of detecting early mutagenic events for assessing cancer risk associated with inhalant abuse which is quite prevalent in developed countries also. In the United States, for example, nearly 20% of young persons have experimented with
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inhalants at least once by the time they are in eighth grade and the mean age of first-time inhalant abuse is 13 years [53]. More importantly, children who abuse inhalants early in life are more likely later to use other illicit drugs [54,55]. Thus inhalant abuse intervention programs seem important from the community health perspective also.
[27] [28] [29] [30]
Conflicts of interest statement The authors declare that there are no conflicts of interest. Acknowledgement The authors gratefully acknowledge the financial support received from Central Pollution Control Board, Government of India, Delhi in carrying out this study.
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