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Evaluation of genotoxic effects of lead in pottery-glaze workers using micronucleus assay, alkaline comet assay and DNA diffusion assay

Abstract

Purpose

We investigated genotoxic effects of occupational exposure to lead acetate in pottery-glaze ceramic workers.

Methods

The study was carried out in 30 exposed workers and 30 matched controls, to whom several biochemical parameters—the blood lead (B-Pb; range: exposed, 41.68–404.77; controls, 12–52) and cadmium (B-Cd) level, the activity of delta-aminolevulinic acid dehydratase (ALAD), erythrocyte protoporphyrin (EP), the level of vitamin B12 and folate in serum—were measured. The genotoxic effects were evaluated by the alkaline comet assay, the DNA diffusion assay and micronucleus test in peripheral blood lymphocytes.

Results

Subjects exposed to lead had significantly higher B-Pb level and, consequently, increased values of tail intensity (TI), frequency of apoptotic and necrotic cells, and frequency of micronuclei (MN). In contrast, their activity of ALAD, the level of vitamin B12 and folate in serum were significantly lower compared to controls. Poisson regression analysis showed a significant correlation of profession, duration of exposure, smoking, level of cadmium in blood, ALAD and EP with primary DNA damage. A majority of primary damage repairs in a short period after exposure to a genotoxic agent. In addition, the influence of gender and level of vitamin B12 and folate in serum MN frequency in exposed group was observed.

Conclusions

In this study, DNA diffusion and micronucleus test showed higher influence of tested parameters to DNA damage. The results indicate a need for concomitant use of at least two different biomarkers of exposure when estimating a genetic risk of lead exposure.

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References

  • Ademuyiwa O, Ugbaja RN, Ojo DA, Owoigbe AO, Adeokun SE (2005) Reversal of aminolevulinic acid dehydratase (ALAD) inhibition and reduction of erythrocyte protoporphyrin levels by Vitamin C in occupational lead exposure in Abeokuta. Nigeria Environ Tox Pharmacol 20:404–411

    CAS  Article  Google Scholar 

  • Agency for toxic substances and disease registry (ATSDR) (1999) Toxicological profile for Lead. US Department of Health and Human Services, Atlanta

    Google Scholar 

  • Alessio L, Castoldi MR, Odone P, Franchini I (1981) Behaviour of indicators of exposure and effect after cessation of occupational exposure to lead. Br J Ind Med 38:262–267

    CAS  Google Scholar 

  • Al-Hakkak ZSH, Hamamy HA, Murad AMB, Hussain AF (1986) Chromosome aberrations in workers at a storage battery plant in Iraq. Mutat Res 171:53–60

    CAS  Article  Google Scholar 

  • ARC (Seventh Annual report on carcinogens) (1994) National toxicology program. National Institute of environmental Health Sciences. Research Triangle Park, North Carolina

    Google Scholar 

  • Asomaning K, Miller DP, Liu G, Wain JC, Lynch TJ, Su L, Christiani DC (2008) Second hand smoke, age of exposure and lung cancer risk. Lung Cancer 61:13–20

    Article  Google Scholar 

  • Barale R, Marrazzini A, Bacci E, Disibio A, Tessa A, Cocchi L, Scarcelli V, Lubrano V, Vassalle C, Landi S (1998) Sister chromatid exchange and micronucleus frequency in human lymphocytes of 1650 subjects in an Italian population: I. Contribution of methodological factors. Environ Mol Mutagen 31:218–227

    CAS  Article  Google Scholar 

  • Basaran N, Shubair M, Ündeger Ü, Kars A (2003) Monitoring of DNA damage in foundry and pottery workers exposed to silica by the alkaline comet assay. Am J Ind Med 43:602–610

    CAS  Article  Google Scholar 

  • Beetstra S, Thomas P, Salisbury C, Turner J, Fenech M (2005) Folic acid deficiency increases chromosomal instability, chromosome 21 aneuploidy and sensitivity to radiation-induced micronuclei. Mutat Res 578:317–326

    CAS  Article  Google Scholar 

  • Berlin A, Schaller H (1974) European method for determination of deltaamino-levulinic acid dehydratase activity in blood. Z Clin Chem Clin Biochim 2:389–390

    Google Scholar 

  • Bermúdez E, Stone K, Carter KM, Pryor WA (1994) Environmental tobacco smoke is just as damaging to DNA as mainstream smoke. Environ Health Perspect 102:870–874

    Google Scholar 

  • Betti C, Davini T, Giannessi L, Loprieno N, Barale R (1995) Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects. Mutat Res 343:201–207

    CAS  Article  Google Scholar 

  • Bolognesi C, Lando C, Forni, Landini E, Scarpato R, Migliore L, Bonassi S (1999) Chromosomal damage and aging: effect on micronuclei frequency in peripheral blood lymphocytes. Age Aging 28:393–397

    CAS  Article  Google Scholar 

  • Bonassi S, Ceppi M, Fontana V, Merlo F (1997) Multiple regression analysis of cytogenetic human data. Mutat Res 313:69–80

    Google Scholar 

  • Bonassi S, Neri M, Lando C, Ceppi M, Lin Y, Chang WP, Holland N, Kirsch-Volders M, Wushou PC, Zeiger E, Fenech M (2003) The HUMN collaborative group, effect of smoking habit on the frequency of micronuclei in human lymphocytes: results from the human MicroNucleus project. Mutat Res 543:155–166

    CAS  Article  Google Scholar 

  • Calderón-Ezquerro C, Guerrero-Guerra C, Sansores-Martínez R, Calderón-Segura ME, Villalobos-Pietrini R, Amador-Muñoz O, Gómez-Arroyo S (2010) Genotoxicity in lymphocytes of smokers living in México City. Rev Int Contam Ambient 26:47–63

    Google Scholar 

  • Chen Z, Jianlin L, Shijie C, Wei Z, Wei W, Lifen J, Hongping D, Jiliang H (2006) Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test. Toxicology 223:219–226

    CAS  Article  Google Scholar 

  • Chislom JJ, Brown DH (1975) Micro-scale photofluorimetric determination of “free erythrocyte protoporphyrin” (protoporphyrin IX). Clin Chem 21:1669

    Google Scholar 

  • Collins AR, Dusinska M, Franklin M, Somorovska M, Petrovska H, Duthie S, Fillion F, Panayiotidis M, Raslova K, Vaughan N (1997) Comet assay in human biomonitoring studies: reliability, validation, and applications. Environ Mol Mutagen 30:139–146

    CAS  Article  Google Scholar 

  • Dandevi K, Rozati R, Banu BS, Rao PH, Grover P (2003) DNA damage in workers exposed to lead using comet assay. Toxicology 187:183–193

    Article  Google Scholar 

  • De Restrepo HG, Sicard D, Torres MM (2000) DNA damage and repair in cells of lead exposed people. Am J Ind Med 38:330–334

    CAS  Article  Google Scholar 

  • Eastmond DA, Tucker JD (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocyttes and anti-kinetochore antibody. Environ Mol Mutagen 13:34–43

    CAS  Article  Google Scholar 

  • Ergurhan-Ilhan I, Cadir B, Koyuncu-Arslan M, Arslan C, Gultepe FM, Ozkan G (2008) Level of oxidative stress and damage in erythrocytes in apprentices indirectly exposed to lead. Pediatr Int 50:45–50

    CAS  Article  Google Scholar 

  • Fenech M (2000) The in vitro micronucleus technique. Mutat Res 455:81–95

    CAS  Article  Google Scholar 

  • Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Protoc 2:1084–1104

    CAS  Article  Google Scholar 

  • Fenech M, Bonassi S (2011) The effect of age, gender, diet and life style on DNA damage measured using micronucleus frequency in human peripheral blood lymphocytes. Mutagenesis 26(1):43–49

    Google Scholar 

  • Fenech M, Morley AA (1985) Measurement of micronuclei in lymphocytes. Mutat Res 147:29–36

    CAS  Article  Google Scholar 

  • Fenech M, Morley AA (1986) Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation. Mutat Res 161:193–198

    CAS  Article  Google Scholar 

  • Fenech M, Rinaldi J (1994) The relationship between micronuclei in human lymphocytes and plasma level of vitamin C, vitamin E, vitamin B12 and folic acid. Carcinogenesis 15:1405–1411

    CAS  Article  Google Scholar 

  • Fenech M, Neville S, Rinaldi J (1994) Sex is important variable affecting spontaneous micronucleus frequency in cytokinesis-blocked lymphocytes. Mutat Res 313:203–207

    CAS  Article  Google Scholar 

  • Fenech MF, Dreosti LE, Rinaldi JR (1997) Folate, vitamin B12, homocysteine status and chromosome damage rate in lymphocytes of older men. Carcinogenesis 18:1329–1336

    CAS  Article  Google Scholar 

  • Forni A, Camiaghi G, Sechi GC (1976) Initial occupational exposure to lead: chromosome and biochemical findings. Arch Environ Health 31:73–78

    CAS  Google Scholar 

  • Fracasso ME, Perbellini L, Solda S, Talamini G, Franceschetti P (2002) Lead induced DNA strand breaks in lymphocytes of exposed workers: role of reactive oxygen species and protein kinase C. Mutat Res 515:159–169

    CAS  Article  Google Scholar 

  • Frome EL, Du Frain RJ (1986) Maximum likelihood estimation for cytogenetic dose-response curves. Biometrics 42:73–84

    CAS  Article  Google Scholar 

  • Ganguly BB (1993) Cell division, chromosomal damage and micronucleus formation in peripheral lymphocytes of healthy donors: related to donors age. Mutat Res 295:135–148

    CAS  Article  Google Scholar 

  • Garcia-Leston J, Laffon B, Roma-Torres J, Teixeira JP, Costa C, Coelho P, Monteiro S, Mayan O, Valdiglesias V, Pasaro E, Mendez J (2009) Evaluation of genotoxic effects of occupational exposure to lead bay means of the comet assay. Abstract book of ICEM 2009, August 20–25, Firenze, Italy, pp 287–288

  • Garcia-Leston J, Mendez J, Pasaro E, Laffon B (2010) Genotoxic effects of lead: an updated review. Environ Int 36:623–636

    CAS  Article  Google Scholar 

  • Grover PK, Danadevi K, Mahboob M, Rozati R, Saleha B, Rahman MF (2003) Evaluation of genetic damage in workers employed in pesticide production utilizing comet assay. Mutagenesis 18:201–205

    CAS  Article  Google Scholar 

  • Hamurcu Z, Donmez H, Saraymen R, Demirtas H (2001) Micronucleus frequencies in workers exposed to Lead, Zinc, and Cadmium. Biol Trace Elem Res 83:97–102

    CAS  Article  Google Scholar 

  • Hartmann A, Fender H, Speit G (1998) A comparative biomonitoring study of workers of a waste disposal site using cytogenetic tests and the comet (single cell gel) assay. Environ Mol Mutagen 32:17–24

    CAS  Article  Google Scholar 

  • Hartwig A (1994) Role of DNA repair inhibition in lead- and cadmium-induced genotoxicity: a review. Environ Health Perspect 102:45–50

    CAS  Google Scholar 

  • Hartwig A, Schlepergrell R, Beyersmann D (1990) Indirect mechanism of lead induced genotoxicity in cultured mammalian cells. Mutat Res 241:75–82

    CAS  Article  Google Scholar 

  • Hoffmann M, Hagberg S, Karlsson A, Nilsson R, Ranstam J, Hogstedt B (1984) Inorganic lead exposure does not effect lymphocyte micronuclei in car radiator repair workers. Hereditas 101:223–226

    CAS  Article  Google Scholar 

  • Hogstedt C, Hane M, Agrell A, Bodin L (1983) Neuropsychological test results and symptoms among workers with well-defined long-term exposure to lead. Br J Ind Med 40:99–105

    CAS  Google Scholar 

  • Huang P, Huang B, Weng H, Nakayama K, Morimoto K (2009) Effects of lifestyle on micronuclei frequency in human lymphocytes in Japanese hard-metal workers. Prev Med 48:383–388

    Article  Google Scholar 

  • Huber R, Streng S, Bauchinger M (1983) The suitability of the human lymphocyte micronucleus assay system for biological dosimetry. Mutat Res 111:185–193

    CAS  Article  Google Scholar 

  • Johansson L, Pelliccari CE (1988) Lead induced changes in the stabilization of the mouse sperm chromatin. Toxicology 51:11–24

    CAS  Article  Google Scholar 

  • Johnson FM (1998) The genetic effects of environmental lead. Mutat Res 410:123–140

    CAS  Article  Google Scholar 

  • Jurasović J, Telišman S (1993) Determination of lead and cadmium in human seminal fluid by electrothermal atomic absorption spectrometry. J Anal At Spectrom 8:419–425

    Article  Google Scholar 

  • Kašuba V, Rozgaj R, Milić M, Želježić D, Kopjar N, Pizent A, Kljaković-Gašpić Z (2009) Evaluation of lead exposure in battery-manufacturing workers with focus on different biomarkers. J Appl Tox 30:321–328

    Google Scholar 

  • Kažimirova A, Barančokova M, Krajčovičova-Kudlačkova M, Volkovova K, Staruchova M, Valachovičova M, Paukova V, Blažiček P, Wsolova L, Dušinska M (2006) The relationship between micronuclei in human lymphocytes and selected micronutrients in vegetarians and non-vegetarians. Mutat Res 611:64–70

    Article  Google Scholar 

  • Kelada SN, Shelton E, Kaufmann RB, Khoury MJ (2001) Δ-Aminolevulinic acid dehydratase genotype and lead toxicity: a HuGE. Am J Epidemiol 154:1–13

    CAS  Article  Google Scholar 

  • Manikantan P, Balachander V, Sasikala K (2010) DNA damage in workers occupationally exposed to lead, using comet assay. Int J Biol 2:103–110

    CAS  Google Scholar 

  • Mantere P, Hanninen H, Hernberg S, Luukkonen R (1984) A prospective follow-up study on psychological effects in workers exposed to low levels of lead. Scand J Work Environ Health 10:43–50

    CAS  Article  Google Scholar 

  • McKevith B (2004) Folate and folic acid. British Nutrition Foundation, www.nutrition.org.uk

  • Migliore L, Parrini M, Sbrana I, Biagini C, Battaglia A, Loprieno N (1991) Micronucleated lymphocytes in people occupationally exposed to potential environmental contaminants: the age effect. Mutat Res 256:13–20

    CAS  Article  Google Scholar 

  • Milić M, Kašuba V, Oreščanin V, Želježić D, Kopjar N, Rozgaj R (2008) Chromosome damage in workers in cigarette manufacturing industry. J Appl Tox 28:399–404

    Article  Google Scholar 

  • Minozzo R, Deimling LJ, Petrucci Gigante L, Santos-Mello R (2004) Micronuclei in peripheral blood lymphocytes of workers exposed to lead. Mutat Res 565:53–60

    CAS  Article  Google Scholar 

  • MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: Results of the Medical Researc Council Vitamin Study. Lancet 338:131–137

    Article  Google Scholar 

  • Murata K, Iwata T, Dakeishi M, Karita K (2009) Lead toxicity: does the critical level of lead resulting in adverse effects differ between adults and children? J Occup Health 51:1–12

    CAS  Article  Google Scholar 

  • OSHA (2002) Employee Standard Summary—1910.1025 App B.U.S. Department of Labor, Occupational Health and Safety Administration. Available at: http://www.osha.gov/pls/oshaweb/ovaolisp.show_document?_table=STANDARDS&p_id=10032

  • Palus J, Dziubaltowska E, Rydzynski K (1999) DNA damage detected by the comet assay in the white blood cells of workers in a wooden furniture plant. Mutat Res 444:61–74

    CAS  Article  Google Scholar 

  • Palus J, Rydzynski K, Dziubaltowska E, Wyszynska K, Natarajan AK, Nilsson R (2003) Genotoxic effects of occupational exposure to lead and cadmium. Mutat Res 540:19–28

    CAS  Article  Google Scholar 

  • Pinto D, Ceballos JM, García G, Guzmán P, Del Razo LM, Vera E, Gómez H, García A, Gonsebatt ME (2000) Increased cytogenetic damage in outdoor painters. Mutat Res 467:105–111

    CAS  Article  Google Scholar 

  • Popović M, McNeill FE, Chettle DR, Webber CE, Lee CV, Kaye WE (2005) Impact of occupational exposure on lead levels in women. Environ Med 113:478–484

    Google Scholar 

  • Rozgaj R, Kašuba V, Jazbec A (2001) Preliminary study of cytogenetic damage in personnel exposed to anesthetic gases. Mutagenesis 16:139–143

    CAS  Article  Google Scholar 

  • Sarto F, Stella M, Acqua A (1978) Cytogenetic studies in twenty workers occupationally exposed to lead (in Italian). Med Lav 69:120–180

    Google Scholar 

  • SAS Institute Inc (1999) SAS/STAT® user s guide, Version 8. SAS Institute Inc, Cary

    Google Scholar 

  • Schwartz J, Landrigan PJ, Baker EL Jr, Orenstein WA, von Lindern IH (1990) Lead-induced anaemia: dose-response relationships and evidence for a threshold. Am J Pub Health 80:165–168

    CAS  Article  Google Scholar 

  • Seppäläinen AM, Hernberg S, Vesanto R, Kock B (1983) Early neurotoxic effects of occupational lead exposure: a prospective study. Neurotoxicology 4:181–192

    Google Scholar 

  • Shaik AP, Jamil K (2009) Individual susceptibility and genotoxicity in workers exposed to hazardous materials like lead. J Hazard Mater 168:918–924

    CAS  Article  Google Scholar 

  • Silbergeld EK, Waalkes M, Rice JM (2000) Lead as carcinogen: experimental evidence and mechanisms of action. Am J Ind Med 38:316–323

    CAS  Article  Google Scholar 

  • Singh NP (2000) A simple method for accurate estimation of apoptotic cells. Exp Cell Res 256:328–337

    CAS  Article  Google Scholar 

  • Singh NP (2005) Apoptosis assessment by the DNA diffusion assay. Chemosensitivity Vol.2: In vivo models. Imaging Mole Regulat 111:55–67

    CAS  Google Scholar 

  • Singh N, McCoy M, Tice R, Schneider E (1988) A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191

    CAS  Article  Google Scholar 

  • Stoleski S, Karadžinska-Bislimovska J, Stikova E, Risteska-Kuc S, Mijakovski D, Minov J (2008) Adverse effects in workers exposed to inorganic lead. Arch Ind Hyg Toxicol 59:19–29

    CAS  Article  Google Scholar 

  • Swanson DA, Liu MJ, Baker PJ, Garrett L, Stitzel M, Wu J, Harris M, Banerjee R, Shane B, Brody LC (2001) Targeted disruption of the methionine synthase gene in mice. Mol Cell Biol 21:1058–1065

    CAS  Article  Google Scholar 

  • Telišman S, Keršanc A, Prpić-Majić D (1982) The relevance of arguments for excluding ALAD from the recommended biological limit values in occupational exposure to inorganic lead WHO 1980. Int Arch Occup Environ Health 50:397–412

    Article  Google Scholar 

  • Telišman S, Prpić-Majić D, Keršanc A (1990) Relationships between blood lead and indicators of effect in cows environmentally exposed to lead. Toxicol Lett 52:347–356

    Article  Google Scholar 

  • Thierens H, Vral A, De Ridder L (1996) A cytogenetic study of radiological workers: effect of age, smoking and radiation burden on the micronucleus frequency. Mutat Res 360:75–82

    CAS  Article  Google Scholar 

  • Vaglenov A, Creus A, Laltchev S, Petkova V, Pavlova S, Marcos R (2001) Occupational exposure to lead and induction of genetic damage. Environ Health Perspect 109:295–298

    CAS  Article  Google Scholar 

  • Wagner C (1995) Biochemical role of folate in cellular metabolism. In: Bailey LB (ed) Folate in health and disease. Marcel Dekker, New York, pp 23–42

    Google Scholar 

  • Wetmur JG (1994) Influence of the common human delta-aminolevulinate dehydratase polymorphism on lead body burden. Environ Health Perspect 102(suppl. 3):215–219

    CAS  Google Scholar 

  • Woźniak K, Blasiak J (2003) In vitro genotoxicity of lead-acetate; induction of single and double DNA strand breaks and DNA-protein cross-links. Mutat Res 535:127–139

    Article  Google Scholar 

  • Zalata A, Yahia S, l El-Bakary A, Elsheikha HM (2007) Increased DNA damage in children caused by passive smoking as assessed by comet assay and oxidative stress. Mutat Res 629:140–147

    CAS  Article  Google Scholar 

  • Zhijian C, Jianlin L, Shije C, Wei Z, Wei W, Lifen J, Hongping D, Jiliang H (2006) Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TRC gene mutation test. Toxicology 223:219–226

    Article  Google Scholar 

  • Zhou W, Heist RS, Liu G, Asomaning K, Miller DP, Neuberg DS, Wain JC, Lynch TJ, Christiani DC (2006) Second hand smoke exposure and survival in early-stage non-small-cell lung cancer patients. Clin Cancer Res 12:7187–7193

    CAS  Article  Google Scholar 

  • Zijno A, Andreoli C, Leopardi P, Marcon F, Rossi S, Caiola S, Verdina A, Galati R, Cafolla A, Crebelli R (2003) Folate status, metabolic genotype, and biomarkers of genotoxicity in healthy subjects. Carcinogenesis 24:1097–1103

    CAS  Article  Google Scholar 

  • Zing JM, Jones PA (1997) Genetic and epigenetic aspects of DNA methylation on genome expression, evolution, mutation and carcinogenesis. Carcinogenesis 18:869–882

    Article  Google Scholar 

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Acknowledgments

This investigation was supported by the Croatian Ministry of Science, Education and Sports as a part of the Projects No: 022-0222148-2137 and No: 022-0222411-2408. We thank Krešimir Nekić from Unit for analytical toxicology and mineral metabolism for performing ALAD and EP measurement.

Conflict of interest

None of authors had any personal or financial conflict of interest.

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Kašuba, V., Rozgaj, R., Milić, M. et al. Evaluation of genotoxic effects of lead in pottery-glaze workers using micronucleus assay, alkaline comet assay and DNA diffusion assay. Int Arch Occup Environ Health 85, 807–818 (2012). https://doi.org/10.1007/s00420-011-0726-4

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  • DOI: https://doi.org/10.1007/s00420-011-0726-4

Keywords

  • Ceramic workers
  • Lead exposure
  • Biological markers
  • Alkaline comet assay
  • Micronucleus assay