Cadmium-induced genomic instability in Arabidopsis: Molecular toxicological biomarkers for early diagnosis of cadmium stress

Highlights

• MSI occurred in Arabidopsis seedlings only under Cd stress of 8.0 mg L⁻¹ for 15 d.

• Aberrations in DNA methylation were induced by Cd of 0.25–5.0 mg L⁻¹ using MSAP-PCR.

• Sensitivity of biomarkers for Cd followed: DNA methylation loci >> RAPD > MSI.

• MSAP-PCR emerged as a powerful tool for early diagnosis of Cd stress.

Abstract

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0–5.0 mg L⁻¹ cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5–34.5 at CpG and of 14.5–20 at CHG sites under Cd stress of 5.0 mg L⁻¹ by RAPD and of 0.25–5.0 mg L⁻¹ by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L⁻¹, and an additional high dose (8.0 mg L⁻¹) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology.

Introduction

Environmental contamination with heavy metals has increased substantially over the past decades, which has aroused attention from numerous researchers responsible for contamination diagnosis, environmental protection and ecological remediation. Cd, as one of the most highly toxic and accumulative heavy metals, has contaminated soils and water through atmospheric deposition, urban-industrial activities and agricultural practices (Sun et al., 2008, Liu et al., 2011). Thus, the exploration of Cd toxicity, especially its genotoxic effects, has become a focus in ecotoxicology research (Herbette et al., 2006, Azevedo et al., 2007, Greco et al., 2012, Pierron et al., 2014).

Several classic assays have been applied to examine the genotoxic effects of heavy metals on organisms, e.g. the micronucleus assay and the comet assay. However, neither is sensitive enough to determine the Cd contamination level or able to provide information at the DNA level (Cambier et al., 2010). With the rapid development of molecular biology, several PCR-based techniques have been used to evaluate DNA damage in toxicology studies. Random-amplified polymorphic DNA (RAPD) comprises a single “arbitrary” primer in a PCR reaction and results in the amplification of several discrete DNA products (Welsh and McClelland, 1990, Williams et al., 1990). RAPD has been used extensively in eco-toxicology studies on animals and humans (Misra et al., 2001, Singh and Roy, 2001, Singh and Roy, 2004, Garnis et al., 2005). Recently, RAPD has been used in plant eco-toxicology studies, detecting genetic variation and mutation induced by stresses (Miki et al., 2001, Liu et al., 2005, Liu et al., 2007, Chao et al., 2008, 2009; 2012; Gjorgieva et al., 2013, Malar et al., 2014). RAPD analysis is extremely efficient for DNA analysis in complex genomes. Its use in investigating genomic DNA to detect different kinds of DNA damage and mutations suggests that RAPD can potentially form the basis of novel biomarker assays for the detection of DNA damage and mutations (Lopez-Moreno et al., 2010, Korpe and Aras, 2011).

Regarding DNA repair, Cd suppresses both eukaryotic MutS homolog MSH2-MSH6- and MSH2-MSH3-dependent mismatch repair (MMR) activity through a nonspecific mechanism whereby Cd binds to multiple sites in MSH6 (Banerjee and Flores-Rozas, 2005, Wieland et al., 2009, Li et al., 2013). Loss or low activity of MMR proteins results in losses and gains of dinucleotide repeats in microsatellites, also known as simple sequence repeats (SSRs), leading to microsatellite instability (MSI). Previously, MSI was found at two “hotspots” of microsatellite mutation in mice by analysis of a panel of microsatellite markers under Cd stress (Oliveira et al., 2012, Du et al., 2014). Also, SSRs mutations can be promoted by heavy metals, e.g. Lead, Chromium and Cd (Rodriguez et al., 2013a, Rodriguez et al., 2013b, Du et al., 2014). Monteiro et al. (Monteiro et al., 2009). demonstrated that SSR analyses could be used to evaluate plant genomic DNA instability for deciphering DNA damage induced by Cd in a genotoxicity test. Thus, MSI can be considered as a biomarker of high risk for human cancer and for the assessment of different genotoxic effects of pollutants on plants (Kovalchuk et al., 2000, Soreide et al., 2006). However, little information is available about microsatellite mutations in Arabidopsis under Cd stress.

Higher plants can rapidly adapt in response to stress, but this can lead to genome instability and changes in DNA methylation patterns throughout the genome and at specific loci of DNA (Boyko et al., 2010, Chatterjee and Vinson, 2012). For example, plants employ important and sophisticated epigenetic regulatory strategies, such as DNA methylation, to maintain genomic plasticity. This allows the simultaneous regulation of many genes involved in plant defense and in facilitating relatively rapid adaptation to new internal and environmental stress conditions without changes in DNA sequence (Steward et al., 2002, Santoyo et al., 2011). The genomic DNA hypermethylation pattern is correlated with water deficit in pea (Pisum sativum L.), pathogen stress in tobacco (Nicotiana tabacum), radioactive contamination in Pinus silvestris, salt, UVC, cold, heat and flood stresses in Arabidopsis, and chromium stress in Brassica napus (Labra et al., 2002, Kovalchuk et al., 2003, Labra et al., 2004, Boyko et al., 2007, Mason et al., 2008). Also, DNA hypomethylation at several genomic loci occurred in response to heavy metal stress in hemp and clover, to pathogen stress in tobacco (Nicotiana tabacum), and to cold treatment in nucleosome core regions of maize (Zea mays) seedlings (Steward et al., 2002, Aina et al., 2004, Boyko et al., 2007). Potentially, global DNA hypomethylation is often viewed as a sign of genomic instability, which manifests as gross chromosomal aberrations, genome rearrangement, MSI, deletions/insertions, and point mutations, thus facilitating gene divergence and evolution of novel gene functions (Boyko et al., 2007, Duan et al., 2013, Harris et al., 2013).

Huang et al. (Huang et al., 2012). reported that tumor cells usually exhibit diverse patterns of DNA methylation, and that the CpG dinucleotide at any given site of genomic DNA might differ in methylation status among subpopulations of tumor cells, reflecting the heterogeneity and decreased epigenetic fidelity of cancer cells. Thus, CpG dinucleotides are also called methylation variable positions (MVPs) in epigenetic studies. Analysis of MVP profiles might reveal stress-specific methylation patterns, and the methylation frequency at any given MVPs could serve as a novel epigenetic biomarker for physiological and pathologic status (Huang et al., 2012, Kit et al., 2012). Moreover, minimal traces/differences of aberrant methylation profiles in blood DNA could serve as early surrogate biomarkers for cancer diagnosis and for risk assessment of PAHs exposure, respectively (Yang et al., 2012, Shin et al., 2013). Similarly, global DNA methylation in earthworms is apparently affected by the overall effect of metals/metalloids undergoing methylation and this parameter might be a candidate biomarker of epigenetic risks related to the presence of the metal elements in terrestrial environments (Santoyo et al., 2011).

Arabidopsis, with its vast resource of mutants, has long been used as a model dicotyledonous higher plant for studies of plant physiology and plant molecular biology. However, little information is available about potential associations between the status of global DNA methylation and genomic instability such as MSI and RAPD DNA polymorphisms in Arabidopsis seedlings under Cd stress. Therefore, the principal aims of this work were to (1) evaluate the extent of genomic instability using a panel of 36 microsatellite markers and 3 RAPD primers; (2) determine the status of global DNA methylation by MSAP-PCR under Cd stress; (3) explore potential associations between the actual status of global DNA methylation and genomic instability such as MSI and RAPD DNA polymorphisms in Arabidopsis under Cd stress. In-so-doing, we were able to evaluate the aforementioned parameters as candidate biomarkers for early diagnosis of genetic and epigenetic risks related to the presence of Cd in the soil and water environment.