Genotoxic endpoints in a Pb-accumulating pea cultivar: insights into Pb2+ contamination limits

  • Eleazar Rodriguez
  • Márcia Sousa
  • Anicia Gomes
  • Raquel Azevedo
  • Nuno Mariz-Ponte
  • Sara Sario
  • Rafael José MendesEmail author
  • Conceição Santos
Research Article


Lead (Pb) persists among the most hazardous contaminant metals. Pb-induced genotoxic effects remain a matter of debate as they are a major cause of plant growth impairment, but assessing Pb genotoxicity requires the selection of Pb-sensitive genotoxic biomarkers. Seedlings of the ecotoxicological model species Pisum sativum L. were exposed to Pb2+ (≤ 2000 mg L−1). Flow cytometry (FCM) revealed that 28 days after, Pb2+ arrested root cell cycle at G2 but no eu/aneuploidies were found. Comet assay and FCM-clastogenicity assays showed that Pb2+ increased DNA breaks in roots at concentrations as low as 20 mg L−1. Leaves showed no variation in DNA-ploidy or cell cycle progression but had increased DNA breaks at the highest Pb2+ dose. We conclude that both Comet assay and the full-peak coefficient of variation (FPCV) were the most relevant endpoints of Pb-phytogenotoxicity. Also, the Pb-induced DNA breaks may be related with the arrest at the G2-checkpoint. Data will be relevant to better define Pb2+ ecogenotoxicological effects and their measuring tools and may contribute to a regulatory debate of this pollutant limits.


Cytostaticity Comet assay DNA breaks Lead Phytogenotoxicity Pisum sativum 


Funding information

This work was funded by FEDER/COMPETE [POCI/01/0145/FEDER/007265; FCT/MEC PT2020 UID/QUI/5006/2019]; Fundação para a Ciência e Tecnologia funded Nuno Mariz-Ponte, SFRH/BD/138187/2018; Sara Sario, SFRH/BD/138186/2018; and Rafael J. Mendes, SFRH/BD/133519/2017.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Azevedo H, Gomes C, Pinto G, Santos C (2005) Cadmium effects in sunflower: nutrient imbalances in leaves and calluses. J Pant Nutr 28(12):2233–2241. CrossRefGoogle Scholar
  2. Azevedo R, Rodriguez E, Mendes RJ, Mariz-Ponte N, Sario S, Lopes JC, Ferreira de Oliveira JMP, Santos C (2018) Inorganic Hg toxicity in plants: a comparison of different genotoxic parameters. Plant Physiol Biochem 125:247–254. CrossRefGoogle Scholar
  3. Cao X, Wang H, Zhuang D, Zhu H, Du Y, Cheng Z, Cui W, Rogers HJ, Zhang Q, Jia C, Yang Y, Tai P, Xie F, Liu W (2018) Roles of MSH2 and MSH6 in cadmium-induced G2/M checkpoint arrest in Arabidopsis roots. Chemosphere 201:586–594. CrossRefGoogle Scholar
  4. Cenkci S, Cigerci IH, Yildiz M, Ozay C, Bozdag A, Terzi H (2010) Lead contamination reduces chlorophyll biosynthesis and genomic template stability in Brassica rapa L. Environ Exp Bot 67:467–473. CrossRefGoogle Scholar
  5. Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23:143–151. CrossRefGoogle Scholar
  6. Dias MC, Mariz-Ponte N, Santos C (2019) Lead induces oxidative stress in Pisum sativum plants and changes the levels of phytohormones with antioxidant role. Plant Physiol Biochem 37:121–129. CrossRefGoogle Scholar
  7. Edelstein M, Ben-Hur M (2018) Heavy metals and metalloids: sources, risks and strategies to reduce their accumulation in horticultural crops. Sci Hortic 234:431–444. CrossRefGoogle Scholar
  8. Ghani A, Khan I, Ahmed I, Mustafa I, Abd-Ur-Rehman MN (2015) Amelioration of lead toxicity in Pisum sativum (L.) by foliar application of salicylic acid. J\ Environ Anal Toxicol 5(292):2161–0525. CrossRefGoogle Scholar
  9. Gichner T, Patkova Z, Szakova J, Znidar I, Mukherjee A (2008a) DNA damage in potato plants induced by cadmium, ethyl methanesulphonate and gamma-rays. Environ Exp Bot 62:113–119. CrossRefGoogle Scholar
  10. Gichner T, Znidar I, Szakova J (2008b) Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants. Mutat Res Genet Toxicol Environ 652:186–190. CrossRefGoogle Scholar
  11. Hardison D Jr, Ma LQ, Luongo T, Harris WG (2004) Lead contamination in shooting range soils from abrasion of lead bullets and subsequent weathering. Sci Total Environ 328:175–183. CrossRefGoogle Scholar
  12. Hattab S, Chouba L, Kheder M, Mahouachi T, Boussetta H (2009) Cadmium- and copper-induced DNA damage in Pisum sativum roots and leaves as determined by the Comet assay. Plant Biosyst 143:S6–S11. CrossRefGoogle Scholar
  13. Jeong H, Kim H, Jang T (2016) Irrigation water quality standards for indirect wastewater reuse in agriculture: a contribution toward sustainable wastewater reuse in South Korea. Water 8(4):169. CrossRefGoogle Scholar
  14. Kumar A, Prasad M, Achary M, Panda B (2013) Elucidation of lead-induced oxidative stress in Talinum triangulare roots by analysis of antioxidant responses and DNA damage at cellular level. Environ Sci Pollut Res 20:4551–4561. CrossRefGoogle Scholar
  15. Liu D, Li T, Jin X, Yang X, Islam E, Mahmood Q (2008) Lead induced changes in the growth and antioxidant metabolism of the lead accumulating and non-accumulating ecotypes of Sedum alfredii. J Integr Plant Biol 50:129–140. CrossRefGoogle Scholar
  16. López-Orenes A, Dias MC, Ferrer MÁ, Calderón A, Moutinho-Pereira J, Correia C, Santos C (2018) Different mechanisms of the metalliferous Zygophyllum fabago shoots and roots to cope with Pb toxicity. Environ Sci Pollut Res 25(2):1319–1330. CrossRefGoogle Scholar
  17. Loureiro J, Capelo A, Brito G, Rodriguez E, Silva S, Santos C (2007) Micropropagation of Juniperus phoenicea L adult plants and analyses of ploidy stability and DNA content in micropragation plants. Biol Plant 51:7–14. CrossRefGoogle Scholar
  18. Malar S, Manikandan R, Favas P, Vikram Sahi S, Venkatachalam P (2014) Effect of lead on phytotoxicity, growth, biochemical alterations and its role on genomic template stability in Sesbania grandiflora: a potential plant for phytoremediation. Ecotoxicol Environ Saf 108:249–257. CrossRefGoogle Scholar
  19. MAOTDR, Ministério do Ambiente, do Ordenamento do Território e do Desenvolvimento Regional (2009) DL 277/2009, 192-2-10-2009, 7154-7165Google Scholar
  20. Monteiro MS, Rodriguez E, Loureiro J, Mann R, Soares A, Santos C (2010) Flow cytometric assessment of Cd genotoxicity in three plants with different metal accumulation and detoxification capacities. Ecotoxicol Environ Saf 73:1231–1237. CrossRefGoogle Scholar
  21. Piechalak A, Tomaszewska B, Baralkiewicz D (2003) Enhancing phytoremediative ability of Pisum sativum by EDTA application. Phytochemistry 64:1239–1251. CrossRefGoogle Scholar
  22. Rodriguez E, Azevedo R, Moreira H, Souto L, Santos C (2013) Pb2+ exposure induced microsatellite instability in Pisum sativum in a locus related with glutamine metabolism. Plant Physiol Biochem 62:19–22. CrossRefGoogle Scholar
  23. Santos C, Pourrut B, Oliveira J (2015) The use of comet assay in plant toxicology: recent advances. Front Genet 30(6):216. CrossRefGoogle Scholar
  24. Seth C, Misra V, Chauhan L (2012) Accumulation, detoxification, and genotoxicity of heavy metals in Indian mustard (Brassica Juncea L). Int J Phytoremed 14:1):1–1)13. CrossRefGoogle Scholar
  25. Shahid M, Dumat C, Pourrut B, Pinelli E (2014) Assessing the effect of metal speciation on lead toxicity to Vicia faba pigment contents. J Geochem Explor 144:290–297. CrossRefGoogle Scholar
  26. Silva S, Santos C, Matos M, Pinto-Carnide O (2012) Al toxicity mechanism in tolerant and sensitive rye genotypes. Environ Exp Bot 75:89–97. CrossRefGoogle Scholar
  27. Silva S, Pinto G, Santos C (2017) Low doses of Pb affected Lactuca sativa photosynthetic performance. Photosynthetica 55(1):50–57. CrossRefGoogle Scholar
  28. Tariq SR, Ashraf A (2016) Comparative evaluation of phytoremediation of metal contaminated soil of firing range by four different plant species. Arab J Chem 9(6):806–814. CrossRefGoogle Scholar
  29. Tawinteung N, Parkpian P, DeLaune R, Jugsujinda A (2005) Evaluation of extraction procedures for removing lead from contaminated soil. J Environ Sci Health A 40(2):385–407. CrossRefGoogle Scholar
  30. Tóth G, Hermann T, Silva M, Montanarella L (2016) Heavy metals in agricultural soils of the European Union with implications for food safety. Environ Int 88:299–309. CrossRefGoogle Scholar
  31. Zeng P, Guo Z, Xiao X, Peng C, Feng W, Xin L, Xu Z (2018) Phytoextraction potential of Pteris vittata L. co-planted with woody species for As, Cd, Pb and Zn in contaminated soil. Sci Total Environ 650:594–603. CrossRefGoogle Scholar
  32. Zhang X, Li M, Yang H, Li X, Cui Z (2018) Physiological responses of Suaeda glauca and Arabidopsis thaliana in phytoremediation of heavy metals. J Environ Manag 223:132–139. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.LBCUniversity of AveiroAveiroPortugal
  2. 2.Department of Biology and LAQV/REQUIMTEFaculty of Sciences of University of PortoPortoPortugal
  3. 3.CITAB—Centre for the Research and Technology of Agro-Environmental and Biological SciencesUniversity of Trás-os-Montes e Alto DouroVila RealPortugal

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