European Journal of Cardiovascular Medicine

Asbestos is international occupational health hazard.¹ Amphibole asbestos fibre are thin and straight, when inhaled, penetrate deeply into the lungs.² In alveoli, they activate the lung’s local immune system and activates an inflammatory reaction.³ They are predominantly responsible for interstitial and malignant pulmonary human disease.⁴ Asbestosis also involves the scarring of lung tissue around the terminal bronchioles and alveolar ducts.⁵

The asbestos fibres are embedded in lung tissue and are coated with endogenous iron, iron containing proteins such as ferritin, mucopolysaccharides and other material on bio-persistent fibres, and they form asbestos bodies which is the hallmark of asbestos exposure.⁵ The presence of transition metals in the fibres suggested the toxic and carcinogenic effects of asbestos. They may trigger asbestos related diseases, with potential DNA damage and apoptosis resistance.⁶

 Bronchoalveolar lavage and sputum are cytological sample used for diagnosing lung asbestosis and related morbidity. Sputum analysis for asbestos bodies is a non-invasive investigation. The presence of asbestos bodies in the sputum are correlated with the intensity of asbestos exposure even though it has stopped for many years. Some studies suggested a correlation between Bronchoalveolar cell and asbestos bodies differential count with survival in patients with asbestosis. These method may contribute in identification of subjects at high risk of lung cancer. ⁶

Asbestos toxicity is the pathogenic mechanisms that triggers pulmonary toxicity and neoplastic transformation, however this is not fully understood. Some researchers are considering the mechanisms relevant to asbestos’s pathogenicity. These include generation of Fe derived free radicals, reactive nitrogen species, release of cytokines, induced genotoxicity and alteration of immune responses.⁷ The research studies indicates asbestos toxicity as a consequence of some characteristic physio-chemical properties of the asbestos fibre.⁸

Over the last two decades, asbestos utilization by countries such as India, China, and Indonesia has increased significantly. In these countries, secondary and environmental exposures are very high due to lack of regulation in occupational setups.^9,10 At present, half of the occupational cancers in the world occur because of asbestos, and its continued use in high quantities can elevate this proportion further. There is no restriction in India on the use or on import–export of asbestos.¹¹

The study of p53 gene mutation in sputum samples of workers using asbestos as raw material is not available. Moreover much less molecular level research in asbestos exposed mutations in cancer is done in India. 

The Indian subcontinent scientific data related to asbestos toxicity, immune response and gene mutation of p53 gene is not available.

Asbestos workers were studied in different factories manufacturing car break insulation etc. These companies were mainly in industrial area around Delhi. A detail history in a pre-textured and pretested questionnaire was recorded. A general physical and systemic examination was done. The sputum and blood samples were collected from 100 workers. Smear was made from sputum and blood for pathological examination. A part of blood was clotted and serum was collected for estimation of IgG, IgA, IgM by using immune-diffusion technique kit.

Two workers had blood streak sputum. Their detail history showed a recent upper respiratory tract infection. Examination showed congestion of throat. However, rest of the general physical examination and systemic examination was within normal limit. Rest of the workers did not show any abnormality.

Occupational history

Most of the workers were recruited for a short duration of time ranging from 6 months to 12 years. Ten workers were employed for 5 to 6 years and only one worker was working in the same industry for 14 years. Most of the time the workers were rotated through different sections of the industry.

Asbestos dust from the mixing equipment polluted the working environment. Most of the industry had a separate mixing room. However the room was always dirty due to leakage of asbestos from mixing equipment. Major work was done in grinding room (Fig: 1). The exhaust system was a total failure. There was huge dust in grinding room.

Figure 1. Approximate plan of asbestos industry

Blood Finding

Peripheral smear showed toxic granules (Fig: 2) in 56 out of 100 cases. There were hypersegmented neutrophils (Fig: 3) in 56 cases. 70 cases showed transformed lymphocytes (Fig: 4). Most of lymphocytes looked like Monocytoid. Plasmacytoid lymphocytes were seen in 14 cases.

Sputum Cytology

Numerous asbestos bodies were seen in all the cases. There were dumbel shaped with hemosiderin deposition. Free asbestos fibers were also seen in all the cases. Carbon laden macrophages were also seen in all the cases. Seven workers had acute inflammatory exudates (Fig: 5). However no dysplastic or malignant cells were seen.

Molecular Study

The sputum was collected from the asbestos workers (n=100). The DNA was extracted from the samples by following method. The sputum was mixed with 90.5M) and sodium citrate (0.05M). They were homogenized in vortex shaker and were centrifuged at 3000 rpm. The pallet obtained was washed with PBS and was treated with Tris HCL (50 mM). It was inactivated at 80 ^o C and was treated with chloroform.

Figure 6. Determination of quantity and quality of genomic DNA from oral sputum samples visualized on an ethidium bromide stain 1% agarose gel. 1-12 samples genomic DNA from sputum. M: Hind III DIGESTED Lambda-DNA molecular weight marker.

The extract of the sample (Fig: 6) was taken and amplified by PCR. The PCR amplification of p53 gene of exon-5 and exon-7 was done. The following primers were used.

Exon 5’

F-5’ TAC TCC CCT GCC CTC AAC AA3’

R-5’ CAT CGC TAT CTG AGC AGC 3’

Exon 7’

F-5’ TCT CCT AGG TTG GCT CTG 3’

R-5’ TCC TGA CCA GGA GTC TTC CAG 3’

The following temperature profiles were used for amplification.

Step 1. Initial denaturation at 95 ^o C for 4 min (4 Cycle)

Step 2. 30 cycles of denaturation at 95 ^o C for 30 sec, primer annealing at 58 ^o C   for 20 sec and extension at 72 degree for 20 sec.

Step 3. In the last cycle the extension at 72 was allowed for 5 min.

Hundred samples of sputum were used for analysis of exon 5 and exon 7 of p53 gene. DNA could be extracted from 15 samples. In rest of samples the quantity of sputum was very less. Amplification was done by polymerase chain reaction, using the primers designed for exon 5 (Fig: 7) and exon 7 (Fig: 8) of p53 gene. DNA extracted was studied by single stand conformation polymorphism, one mutation in exon 5 was detected (Fig 9).

Figure 7. PCR amplification on exon5 of p53 tumor suppressor gene showing amplimer of 184 bp

Lane 1: Normal control DNA

Lane 2-12 Sputum DNA

M: Hae-111 digested ꬾX 174 DNA molecular weight marker

Figure 8. PCR amplification of exone 7 of p53 tumor suppressor gene showing amplimer of 125 bp

Lane 1: Normal control DNA

Lane 2-12 Sputum DNA

M: Hae-III digested ꬾX 174 DNA molecular weight marker

Immunoglobulins

Immunoglobulin G was increased with mean of 1878.0 mg/dl and range 1820-1910 mg/dl (normal 850-1500 mg/dl). Rest of the immunoglobulins were within normal limits.

Immunofluorescence

Immunofluorescence: study showed numerous asbestos fibers coated with IgG (Fig: 10).

Asbestos is known to cause numerous diseases ranging from simple inflammation to squamous cell carcinoma and mesothelioma.¹¹ However no such data is available for India. We found some early changes in blood. There were toxic granules and plasma and Monocytoid changes. These changes are seen in variety of condition and are thought to be non-specific. However these changes signify that the WBC’s are reacting to asbestos fibers. Sputum did not show any dysplastic or malignant changes though asbestos bodies were seen in all the cases. Numerous asbestos bodies were seen in all the cases. There were dumbel shaped with hemosiderin deposition. Free asbestos fibers were also seen in all the cases. Carbon laden macrophages were also seen in all the cases. This is probably because of lesser duration of exposure, the average being 6 months.

The cell differential counts and the number of asbestos bodies in broncho-alveolar lavage fluid were obtained from patients with asbestosis, and results were correlated with their survival.¹² Smoking and a high number of ABs (>2 AB/mL) were associated with high total cell counts and high macrophage and low lymphocyte differential counts. Shortened survival was associated with high numbers of ABs (78 vs 165 months; p=0.042) and low lymphocyte (77 vs 179 months; p=0.005), high neutrophil (102 vs 180 months; p=0.016) and high eosinophil (104 vs170 months; p=0.007) differential counts.¹³

The most important finding was involvement of IgG, immunological reaction was induced by the inhaled asbestos fibers. The immunoglobulins were not only high but, were also coating the fibers.¹⁴ This shows, an asbestos fibres is able to create conditions that favour malignant transformation and suppression of the activation of immune surveillance mechanisms responsible for destroying transformed cells. We also examined p53 gene mutation in workers exposed to asbestos fibres and observed 3.33% gene mutation in exon-5 of p53 gene.

We feel this may be an early and initiating etiological factors in asbestos induced changes. It is concluded, thus that a simple blood and sputum examination can help to detect asbestos exposure. Thus all the changes recorded were early, which was basic aim of this study.

Research studies have proven asbestos as a carcinogen. Many countries have banned asbestos while others have restricted its usage. However ill effect of asbestos exposures remains unrestrained in India. It is time for Indian government to appraise its policies associated with asbestos consumption earnestly.

1. International Agency for Research on Cancer. Asbestos. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Humans. Vol. 14. Lyon, France: International Agency for Research on Cancer; 1977. p. 106.
2. F. Dodson and S. P. Hammar, Asbestos: Risk Assessment, Epidemiology, and Health Effects, CRC Press, Boca Ratton, Fla, USA, 2006.
3. L. Roggli, T. D. Oury, and T. A. Sporn, Springer Science and Business Media, Springer Science and Business Media, New York, NY, USA, 2nd edition, 2004.
4. O’Byrne and V. Rusch, Malignant Pleural Mesothelioma, Oxford University Press, New York, NY, USA, 2006.
5. Baldi, Nova Scientific, Nova Scientific Publishers, New York, NY, USA, 2008.
6. Hasselbacher. Binding of immunoglobulin and activation of complement by    asbestos fibers. J Allergy Clin Immunol.1979;64(4):294-8.
7. B Fubini, F Barcelo, CO Areán Ferritin adsorption on amosite fibers: possible implications in the formation and toxicity of asbestos bodies. J. Toxicol. EHealth. 1997;52:343-352.
8. Lorella Pascolo1 , Alessandra Gianoncelli2 , Giulia Schneider3 , Murielle Salome´4 , Manuela Schneider5 , Carla Calligaro6 , Maya Kiskinova2 , Mauro Melato1 & Clara Rizzardi. The interaction of asbestos and iron in lung tissue revealed by synchrotron based scanning X-ray microscopy. Toxicology Letters. 2016; 241: 111–120.
9. In: Indian Minerals Yearbook (Part-III: Mineral Reviews). 56th ed. Government of India, Ministry of Mines, Indian Bureau of Mines; 2017.
10. Frank AL, Joshi TK. The global spread of asbestos. Ann Glob Health. 2014;80:257-62.
11. GBD 2017 Risk Factor Collaborators. Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: A systematic analysis for the global burden of disease study 2017. Lancet 2018;392:1923-94.
12. Paris, F. Galateau-Salle , C. Creveuil , R. Morello, C. Raffaelliz, J.C. Gillon , M.A. Billon-Gallandƒ , J.C. Pairon, L. Chevreau, M. Letourneux. Asbestos bodies in the sputum of asbestos workers: correlation with occupational exposure. Eur Respir J 2002; 20: 1167–1173.
13. Keskitalo E, Varis L, Bloigu R, Kaarteenaho R. Bronchoalveolar cell differential count and the number of asbestos bodies correlate with survival in patients with asbestos.J Occupational and Environmental Medicine.2019;76(10) 765-771.
14. M. Klockars, M. Hedenborg, J. Koistinen ,T. Isobe.  IgG and IgA enhance the chrysotile-induced production of reactive oxygen metabolites by human polymorphonuclear leucocytes. Clin. exp. Immunol. 1989; 78: 372-377.