Rajiv Gandhi University of Health Sciences, Karnataka, Bangalore s38
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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE
ANNEXURE-2 PROFORMA FOR REGISRATION OF SUBJECTS FOR DISSERTATION
1. Name of the candidate ANNE DSOUZA and address Postgraduate student Department of Anatomy St. John’s Medical College Bangalore-560034
Residential address Sampige Nagar Udyavar Post Udupi District- 574118
2. Name of the institution St. John’s Medical College Bangalore-560034
3. Course of study and M.D. Anatomy Subject
4. Date of admission 18th March 2009
5. Title of the topic
“DNA DAMAGE IN PEOPLE EXPOSED TO FORMALDEHYDE – A CYTOGENETIC STUDY” 6. Brief resume of the intended work
6.1 Need for the study:
Formaldehyde is said to be a carcinogenic agent. Acute exposure is highly irritating to the eyes, nose and throat, severe allergic reactions of the skin, eyes and respiratory tract.
Long term exposure to low levels of formaldehyde cause asthma like respiratory problems and skin irritation, such as dermatitis and itching. People, who work in laboratories exposed to formaldehyde as a liquid or vapor, absorb it through skin as a liquid. Especially healthcare professionals and medical lab technicians, including anatomy dissection laboratory workers, mortuary workers, teachers as well as students are at higher exposure as they handle specimens stored in formaldehyde. Thus, people exposed to formaldehyde at variable concentrations are at risk of having DNA damage, and they need to be assessed biologically.
In the regular experience of cytogenetic culture setup, the peripheral lymphocyte culture from people exposed to formaldehyde used to fail continuously and there was either a poor yield of good metaphase spreads or there was total failure to grow any chromosomes and this brought the idea of biological monitoring. Even though reports from the west exist, the anatomical laboratory setups differ in Indian setup and people working in anatomy laboratories in India are more exposed to formaldehyde. Thus, the present study could bring the information about the DNA changes in Indian setup. 6. 2 Aims and objectives of the study:
1. To evaluate DNA damage in the form of micro nuclear assay (MN) in people exposed to formaldehyde.
2. To analyze the DNA damage in relation to the duration of exposure to formaldehyde.
3. To compare the frequency of micronuclei in buccal smear, nasal mucosal cells and peripheral lymphocytes.
6.3 Review of literature:
Formaldehyde (FA) is an important colorless, strong smelling, industrial compound with numerous applications ranging from production of resins to medicine. It is commonly used as a preservative in medical laboratories and mortuaries. Formaldehyde is commonly termed as formalin which describes it as aqueous solution, particularly those containing 37 to 50 percent formaldehyde and 6 to 15 percent alcohol stabilizer.
Formaldehyde is a sensitizing agent that can cause an immune system response upon initial exposure. Acute exposure is highly irritating to the eyes, nose and throat, and may even produce severe allergic reactions of the skin, eyes and respiratory tract. Ingestion of formaldehyde can be fatal. Long term exposure to low levels of formaldehyde cause asthma like respiratory problems and skin irritation, such as dermatitis and itching. People, who work in laboratories exposed to formaldehyde as a liquid or vapor, absorb it through skin. Especially healthcare professionals and medical lab technicians, including
Anatomy dissection laboratory workers, mortuary workers, teachers as well as students are at higher exposure as they handle specimens stored in formaldehyde. The increased incidence of nasopharyngeal cancer was reported in large of number of studies (Blair et al
19901, Partnen et al 19932, Armstrong et al 20003, Vaghuan et al 20004, Hildsheim et al
20015, Coggan et al 20036, Hauptmann et al 20047). International agency for research and cancer (IARC) (20068) classified formaldehyde as carcinogenic agent to humans.
Epidemiological studies of industrial workers, anatomy and pathology workers, embalmers, showed elevated risk of cancer, at various sites, such as, nasal cavities, lung, brain, pancreas and hemopoietic system. Induction of micronucleus (MN), sister chromatid exchange (SCE), chromosomal aberrations (CA), DNA-protein crosslinks
(DPC) and DNA damage were reported in industrial workers, embalmers and pathology laboratory workers exposed to formaldehyde. It was reported that occupational exposure of formaldehyde occurs in anatomy and pathology laboratories, where it is used exclusively as cytological fixative to preserve the integrity of cell structures for wide variety of purposes including cell, tissue and organ descriptive examination and
Developmental studies. In anatomical and pathological laboratory settings, the formaldehyde absorption is mainly through inhalation. Inhaled formaldehyde primarily affects the upper respiratory tract. According to Code of Federal Regulations (CFR)9, the permissible exposure limits
(PELs) of formaldehyde in the workspace covered by the standard are 0.75ppm. Several studies show that, air borne formaldehyde in anatomy exceeded recommended exposure criteria. (Shaham et al 200210, Akbar-Khanzadeh and Pulido et al, 200311). The results of the earlier studies indicate that there requires a close monitoring of formaldehyde exposures. Currently, wide range of methods is used to determine the early biological effects of DNA damaging agents. Micronuclei (MN) and sister chromatid exchanges
(SCE) are found to be suitable cytogenetic techniques that are useful to assess the DNA damage at chromosomal level in human biomonitoring. Bonnasi et al 200712 report that micronuclei test (MN) is also an effective test for the predictive cancer risks in human populations. Human peripheral lymphocytes, which are most frequently used cells for cytogenetic monitoring, appear to be an appropriate method for monitoring the genotoxic effects on formaldehyde exposures. Buccal epithelial cells provide an alternative source of tissue for monitoring the occupational exposure. This method has an advantage of rapid and easy sampling by brushing the buccal mucosa. Norppa et al 199313 studied and reported increased incidence of micronuclei in buccal cells of workers in plywood plant and fiber glass factory. Titenko-holland et al 199614 and Suruda et al 199315 reported an increased micronuclei frequency, found after monitoring the students of mortuary science exposed to formaldehyde. Ye X et al 200516 have reported an increased frequency of micronuclei in nasal mucosa cells in workers exposed to formaldehyde than found in control group. 7. MATERIAL AND METHODS:
7.1 Source of the data and sample size:
Thirty subjects including both male and female helpers, working in department of
Anatomy in various medical colleges who are exposed to formaldehyde will be selected for the study after taking an informed consent. The number of days/months/years exposed to formaldehyde will be noted. 30 subjects, not exposed to formaldehyde, will be taken as controls. In the groups, the general health conditions, medical history, long term medication, diagnostic tests (X-rays etc), lifestyle factors including habits and addictions, and details of protective measures will be assessed by an appropriate questionnaire. Hundred cells from each subject and the control group will be analyzed.
7.2 Inclusion criteria:
1. People exposed to formaldehyde, for both long and short term – as study subjects.
2. People not exposed to formaldehyde at any period of time - as controls.
7.3 Exclusion criteria:
1. Subjects with history of cancer and treatment.
2. People exposed to frequent X- Rays and other kinds of radiation.
3. People working in paint and pesticide industry (known carcinogens) are excluded
both from study and control groups.
7.4 Study design: Case control study 7.5 Protocol of the procedure:
1. Cell sampling:
Buccal cells will be sampled with a brush from inside of the cheeks and placed in 50ml vials containing 25ml of buffer solution (0.1m EDTA, 0.01m Tris and 0.02m NaCl). The nasal mucosal cells are collected from the middle meatus with an endocervical brush.
The cells will be washed thrice in buffer solution by centrifugation and slides for microscopic analysis will be prepared. Cell suspension will be dropped onto clean slides and cell density will be checked using microscope. The cells are allowed to dry and then fixed in 80% cold (00C) methanol. The cells will be stained in 10% Giemsa solution.
(Poppova et al 2007)17.
2ml of heparinized venous blood samples will be collected from each subject. Aliquots of 0.5ml of heparinized whole blood sample will be used to duplicate the lymphocyte culture for cytokinesis blocked micronuclei test. Cultures will be incubated at 370C in dark for a total of 72 hours; 6µg/ml of colchicine will be added at 70th hour to prevent cytokinesis. Cells will be collected by centrifugation and treated twice with RPMI supplemented with 2% fetal bovine serum (FBS). The cells will be centrifuged again and submitted to a mild hypotonic treatment in a mixture of RPMI and deionized water (1:4 v/v), supplemented with 2% fetal bovine serum. The centrifuged cells will be placed on dry slides, smears are prepared. After air-drying, the slides will be fixed with cold methanol:acetic acid (3:1 v/v). Air dried slides will be stained with 4% Giemsa, in pH
6.8 buffer solution. 2. Micronuclei analysis:
The frequency of buccal epithelial cells and nasal mucosal cells will be evaluated by scoring 100 buccal epithelial cells and 100 lymphocyte cells with well preserved cytoplasm, per subject. The buccal epithelial cells and nasal mucosal cells will be observed under 100X magnification.
3. Statistical applications:
Various statistical tests such as Student’s t-test, Mann-Whitney test and Pearson’s correlation will be used depending on the nature of data and type of analysis used.
7.6 Does the study require any investigations or interventions to be
conducted on patients or other human or animals? If so please
describe briefly.
The study includes collection of buccal cells, nasal mucosal cells and 2ml of venous blood from the subjects.
Has ethical clearance obtained from your institution in case of 7.6?
Applied for the ethical clearance and the results are awaited. 8. List of references:
1. Blair A, Saracci R, Stewart PA, Hayes RB, Shy C. Epidemiologic evidence on
the relationship between formaldehyde exposure and cancer. Scand J Work
Environ Heatlh 1990;16:381-93.
2. Partanen T. Formaldehyde exposure and respiratory cancer — a meta-analysis of
the epidemiologic evidence. Scand J Work Environ Health 1993;19: 8–15.
3. Armstrong RW, Imrey PB, Lye MS, Armstrong MJ, Yu MC, Sani S.
Nasopharyngeal carcinoma in Malaysian Chinese: occupational exposures to
particles, formaldehyde and heat. Int J Epidemiol 2000;29: 991–98.
4. Vaughan TL, Stewart PA, Teschke K, Lynch CF, Swanson GM, Swanson G et al.
Occupational exposure to formaldehyde and wood dust and nasopharyngeal
carcinoma. Occup Environ Med 2000;57: 376–84.
5. Hildesheim A, Dosemeci M, Chan CC, Chen CJ, Cheng YJ, Hsu MM et al.
Occupational exposure to wood, formaldehyde, and solvents and risk of
nasopharyngeal carcinoma. Cancer Epidemiol Biomarkers Prev 2001;10:1145–
115. 6. Coggon D, Harris, EC, Poole J, Palmer KT. Extended follow up of a cohort of
British chemical workers exposed to formaldehyde. J Natl Cancer Inst 2003;
95:1608–15.
7. Hauptmann M, Lubin JH, Stewart PA, Hayes RB, Blair, A. Mortality from
solid cancers among workers in formaldehyde industries. Am J Epidemiol
2004;159:1117–30.
8. Cogliano VJ, Grosse Y, Baan RA, Straif K, Secretan MB, Ghissassi FE et al.
Meeting Report: Summary of IARC Monograph on formaldehyde, 2-
butoxyethanol and 1- tert- butoxy- 2- propanol. Environ Health Perspect
2005;113(9):1205-1208
9. Formaldehyde fact sheet, OSHA’s Safety and Health Program Management
Guidelines Federal Register 1989;54:3904-16
10. Shaham J, Gurvich R, Kaufman Z. Sister chromatid exchange in pathology
staff occupationally exposed to formaldehyde. Mutat Res 2002;15:115–123.
11. Akbar-Khanzadeh F, Pulido EV. Using respirators and goggles to control
exposure to air pollutants in an anatomy laborator. Am J Ind Med 2003;
43:326–331.
12. Bonassi S, Znaor A, Ceppi M, Lando C, Chang WP, Holland N et al. An
incresead micronucleus frequency in peripheral blood lymphocytes predicts
the risk of cancer in humans. Carcinogenesis 2007;28:625–631. 13. Norppa H, Luomahaara S, Heikanen H, Roth S, Sorsa M, Renzi L et al.
Micronucleus assay in lymphocytes as a tool to biomonitor human exposure to
aneuploidogens and clastogens. Environ Health Perspect 1993;101:519–525.
14. Titenko-Holland N, Levine AJ, Smith MT, Quintana PJ, Boeniger M et al.
Quantification of epithelial cell micronuclei by fluorescence in situ
hybridization (FISH) in mortuary science students exposed to formaldehyde.
Mutat Res 1996;371:237–248.
15. Suruda A, Schulte P, BoenigerM, Hayes RB, Livingston GK, Steenland K et
al. Cytogenetic effects of formaldehyde exposure in students of mortuary
science. Cancer Epidemiol Biomarkers 1993;2:453–460.
16. Ye X, Yan W, Xie H, Zhao M, Ying C Cytogenetic analysis of nasal mucosa
cells and lymphocytes from high- level long- term formaldehyde exposed
workers and low- level short- term exposed waiters. Mutat Res 2005;588(1):
22-7.
17. Popova L, Kishkilova D, Hadjidekova VB, Hristova RP, Atanasova P,
Hadjidekova VV, et al. Micronuclei test in buccal epithelium cells from
patients subjected to panoramic radiography. Dentomaxillofacial Radiology
2007; 36:168-71. 9. Signature of candidate:
10. Remarks of Guide:
10.0 Name and designation of
10.1 Guide:
Dr. Rema Devi Professor & Head Division of Human Genetics Department of Anatomy St. John’s Medical College Bangalore – 560 034
10.2 Signature. 10.3 Co-guide: -
10.4 Signature: -
10.5 Head of the Department:
Dr. ROOPA RAVINDRANATH Professor, Department of Anatomy, St. John’s Medical College, Bangalore- 560034.
10.6 Signature:
11.1 Remarks of the chairman & Principal;
11.2 Signature: