Genotoxic effects of heavy metal cadmium on growth, biochemical, cyto-physiological parameters and detection of DNA polymorphism by RAPD in Capsicum annuum L. – An important spice crop of India

Abstract

The present study was designed to investigate the effects of cadmium (Cd) on biochemical, physiological and cytological parameters of Capsicum annuum L. treated with five different concentrations (20, 40, 60, 80 and 100 ppm) of the metal. Shoot–root length, pigment and protein content showed a continuous decrease with increasing Cd concentrations and the maximal decline was observed at the higher concentration. Proline content was found to be increased upto 60 ppm while at higher concentrations it gradually decreased. MDA content and chromosomal aberrations increased as the concentration increased. Additionally Random amplified polymorphic DNA (RAPD) technique was used for the detection of genotoxicity induced by Cd. A total of 184 bands (62 polymorphic and 122 monomorphic) were generated in 5 different concentrations with 10 primers where primer OPA-02 generated the highest percentage of polymorphism (52.63%). Dendrogram showed that control, R1 and R2 showed similar cluster and R4 and R5 grouped with R3 into one cluster, which showed that plants from higher doses showed much difference than the plants selected at mild doses which resemble control at the DNA level. This investigation showed that RAPD marker is a useful tool for evaluation of genetic diversity and relationship among different metal concentrations.

1. Introduction

Heavy metal toxicity includes the binding of heavy metals more strongly to functional sites that are normally occupied by essential functional groups of biologically important molecules such as enzymes, changing the conformation of the biological molecules, proteins and nucleic acids thus disrupting the integrity of entire cells and their membranes, making them inactive, decomposing essential metabolites and changing the osmotic balance around the cells (Jjemba, 2004). Heavy metals in the soil through leachates on the one hand accelerates the vegetation (Pascual et al., 1999), attributed these increases with the increased total organic carbon content coupled with increased enzyme activity, but on the other hand the growth of the leguminous crops was reduced in the fields which receive heavy metal contaminated sludge (Bhogal et al., 2003), similarly the yield of green gram declined in pots when heavy metals (Cd, Pb, Cu, Zn, Cr and Ni) were added individually or combined in various mixtures (Athar and Ahmad, 2002). The contamination of soil and water resources by genotoxic compounds is a worldwide issue (Alam et al., 2009, 2010; Tabrez and Ahmad, 2011). Genotoxicity testing of complex vehicular effluents contaminating various agricultural soils has demonstrated that these environmental mixtures contain many unidentified and, therefore, unregulated toxicants that are potential carcinogens (Magdaleno et al., 2001; Ohe et al., 2004). Among the heavy metals cadmium is a very genotoxic metal. Numerous studies have shown that genotoxicity of Cd is directly related to its effect on structure and function of DNA, which may be determined using a number of laboratory methods (Liu et al., 2009a,b; Cambier et al., 2010). In plants, cadmium is known to inhibit seed germination and root growth (Chakravarty and Srivastava, 1992; Liu et al., 1992) and induces chromosomal aberrations and micronucleus formation (Zhang and Xiao, 1998).

Capsicum annuum L. belongs to the family Solanaceae (2n = 24) and they are variously used as a pungent flavor in food, natural plant color, pharmaceutical ingredient and as sprays for riot control and self-defense. The pungent flavor of chilli is due to a group of closely related alkaloid called capsaicinoids found only in the genus Capsicum (Hoffman et al., 1983). Capsaicin content of peppers is one of the major parameters that determine Capsicum’s commercial quality (Kawabata et al., 2006; Hachiya et al., 2007). It is also a rich source of vitamins C (ascorbic acid), A and E. It has also been reported to show anticancer effect (Morre and Morre, 2003) and to be active against neurogenic inflammation (Szolcsanyi, 2004), protective effects against high cholesterol levels and obesity (Kempaiah et al., 2005), anti-mutagenicity effect (Macho et al., 2003; Morre and Morre, 2003) and a high antioxidant activity (Sim and Sil, 2008).

The present study was conducted to estimate the genotoxicity of Cadmium nitrate (CdNO₃)₂ on chilli crop (C. annuum L.) grown in cadmium amended soils near the industrial areas of Aligarh and Kanpur. For genotoxicity tests we used chromosomal aberrations and biochemical parameters and RAPD technique for DNA polymorphism detection.

2. Materials and methods

2.1. Plant material and heavy metal

Certified healthy seeds of C. annuum L. were obtained from Indian Agriculture Institute New Delhi, India. Healthy seeds were presoaked in distilled water at 24 ± 2 °C for 7 h and then treated with five different concentrations of cadmium nitrate (20, 40, 60, 80 and 100 ppm) at 24 ± 2 °C for 24 h with constant intermittent stirring. One set of seeds was soaked in distilled water to act as control. The treated sets of seeds were washed with tap water to remove the residual heavy metal adhering to the seed coat. Each set of seeds was sown in a field with (CRBD design) with a spacing of 12 cms in order to avoid interference of roots to obtain plants.

2.2. Growth parameters

Growth parameters in the form of shoot length (cm), root length (cm), fresh weight (g/plant) and tolerance index (%) were recorded at 90 days of treatment following the method of (Choudhary et al., 2012).

2.3. Chromosomal aberration studies

For chromosomal abnormalities the root tips of germinated seeds from each dosage level were fixed in a Carnoy’s fluid containing absolute alcohol and galacial acetic acid (3:1) for 24 h and stored in 70% alcohol until further use. The fixed roots were hydrolyzed at 60 °C for 5–10 min, in 1 N HCl. After washing the root tips were transferred to 1% Iron alum for 25–30 min. Finally the root tips were transferred to 2% aceto-hematoxylin stain for 2 h and then the stained region of the root tip was squashed in 1% propinocarmine on a slide, mounted and observed under Dsx Olympus microscope and photographs were taken from permanent slides.

2.4. Mitotic index

Mitotic index was determined by the formula No. of cells in division phase/Total number of cells studied × 100 following the method of (Siddiqui et al., 2009).

2.5. Biochemical analysis

2.5.1. Determination of pigment content

The pigment content was determined after extraction of the pigment with 80% acetone following the method of (Arnon, 1949). Fresh mass of leaves (200 mg) was grounded in small volumes of acetone solutions and filter with Whatman No. 1 filter paper. The extract obtained was diluted to a final volume of 10 mL. Absorbance of chlorophyll content at 663 and 645 nm and carotenoid content at 480 and 510 nm was determined using UV–Vis Spectrophotometer (UV-1700 Pharma Spec, Shimadzu, Kyoto, Japan).

2.5.2. Proline estimation

The proline estimation was determined following the method of (Bates et al., 1973). Fresh material (300 g) was homogenized in 10 mL of 30% aqueous sulphosalicylic acid. The homogenate was centrifuged at 9000g for 15 min. A 2 mL aliquot of the supernatant was mixed with an equal volume of acetic acid and ninhydrin (1.25 g) in 30 mL acetic acid and 20 mL of 6 NH₃PO₄ and incubated for 1 h at 100 °C. The reaction was terminated in an ice bath and extracted with 4 mL of toluene. The extract was vortexed for 20 s. The chromatophore containing toluene was then aspirated from the aqueous phase, and absorbance read at 520 nm using UV–Visible Spectrophotometer, Shimadzu, Japan.

2.5.3. Lipid peroxidation

Lipid peroxidation was determined by measuring the MDA equivalents according to (Hodges et al., 1999) and expressed as mg/g FW.

2.6. RAPD analysis

About 0.5 g of fresh young leaves from each dosage level was taken for DNA isolation. The sample was ground to fine powder using liquid nitrogen with pre chilled pestle and mortar. The powder of each sample was transferred into15 ml polypropylene centrifuge tubes separately, containing 5 ml of pre warmed extraction buffer. DNA was extracted by CTAB method following (Saghai et al., 1984). The solution was vortexed for 30–40 s for gentle mixing. Then 30 ml of Chloroform: iso-amyl alcohol (24:1) was added to emulsify. The solution was centrifuged at 15,000 rpm for 10 min at room temperature. The aqueous phase was removed with a wide pore pipette, transferred to a clean tube and then mixed by quick gentle inversion by adding 2/3 volume of isopropanol. DNA was spooled using a bent pasture pipette and transferred to another tube. If the DNA appeared flocculent, centrifugation was done at 5000 rpm for 2 min by gently pouring off the supernatant. Then DNA pellet was washed using 70% ethanol and dried pellet was dissolved in 500 μl TE buffer. The purification of DNA was done using RNAase by treating the samples with proteinase K. RAPD analysis was done individually with four random decamer primers. Denaturation was done at 95 °C for 4 min, Primer annealing at 54 °C for 1 min and Primer extension at 73 °C for 2 min.

2.6.1. Polymerase chain reaction

PCR amplification was performed in Eppendrof thermal-cycler (Eppendrof, Germany) with a temperature program consisting of the thermal cycling conditions were denaturation at 94 °C for 4 min; annealing at 35 °C for 1 min; extension at 72 °C for 2 min. The amplification product along with 2 μl of loading dye (bromophenol blue) was separated on 1.5% agarose gel using 0.5 × Tris–borate-EDTA (TBE) buffer at pH 8.0 containing ethidium bromide (0.5 μg/ml) of gel. The gel was viewed under ultraviolent (UV) transilluminator and photographed using gel documentation.

2.7. Statistical analysis

A total of 12 replicates for each treatment were conducted. Statistical analysis of data was done with SPSS 17.0 for Windows (SPSS, Chicago, IL, USA). One way ANOVA was performed with DMRT test to determine the least significant difference (LSD) Photomicrographs were taken from both permanent and temporary slides.

3. Results

3.1. Effect of cadmium on growth parameters

Fig. 1 shows that the shoot length, root length and fresh weight of control plant were 46.04 ± 0.39, 16.36 ± 0.48 and 23.08 ± 1.11 while it significantly (p < 0.01) decreased from 42.80 ± 0.70 to 27.19 ± 0.59, 14.58 ± 0.34 to 8.04 ± 0.47 and 21.61 ± 0.54 to 16.22 ± 0.47 in 20 ppm–100 ppm concentrations. 100 ppm shows the higher toxicity in all the growth parameters.

Figure 1.

Effect of Cadmium nitrate on Shoot length (cm), Root length (cm) and Fresh weight (g/plant) of Capsicum annuum L. Data mean of five replicate ± SD (ANOVA using DMRT ^∗∗p < 0.01).

3.2. Effect of Cd on chromosomal aberrations

Cd induced a number of mitotic abnormalities in C. annuum L. (Table 1, Fig. 2). The percentage of abnormalities was higher (16.76 ± 0.76) in 100 ppm concentration of Cd, statistically significant (p < 0.01) differences were found when compared to control (1.65 ± 0.32). As the Cd concentration increased chromosomal abnormalities i.e. micronuclei, stickiness, laggards, bridge and fragments also increased. While the mitotic index which indicates the cell division frequency was gradually significantly (p < 0.01) decreased with the increase of Cd concentration (Table 1).

Table 1.

Mitotic index (MI) and chromosomal aberrations revealing the genotoxic potential of cadmium nitrate on C. annuum L.

Conc.	Chromosomal aberrations								Total Abnormal cells
	No of cells examined	Cells in mitosis	MI(±SD)	Mn.	Stk.	L	Br	Fr
Control	208	90	43.20 ± 1.26	1	2	–	–	1	1.65 ± 0.32
20 ppm	200	84	42.57 ± 1.42⁎⁎	1	3	–	2	2	3.84 ± 0.77⁎⁎
40 ppm	210	80	38.70 ± 1.74⁎⁎	4	3	4	3	2	7.58 ± 1.00⁎⁎
60 ppm	220	72	32.28 ± 1.72⁎⁎	5	4	6	6	5	11.39 ± 1.48⁎⁎
80 ppm	211	61	28.72 ± 1.86⁎⁎	5	6	6	7	6	14.90 ± 0.99⁎⁎
100 ppm	208	56	26.38 ± 2.17⁎⁎	6	8	8	7	7	16.76 ± 0.76⁎⁎

(Mn = Micronuclei, Stk = Stickiness, L = Laggards, Br = Bridges, Fr = Fragments) each datum represents the mean ± SD (n = 5 for each dose) ANOVA using DMRT.

^⁎⁎p < 0.01.

Figure 2.

Chromosomal abnormalities in the root tip cells of Capsicum annuum L. on exposure of different treatments of Cadmium nitrate (A) Multivalents, (B) Chromosome stitching at anaphase I, (C) Bridge at anaphase I and (D) Laggard at anaphase I.

3.3. Effect of cadmium on chlorophyll and carotenoid content

The levels of photosynthetic pigments i.e. total chlorophyll and carotenoids significantly decreased (p < 0.01) as the Cd concentrations increased, compared with control. Control plant shows 3.62 ± 0.05 and 7.05 ± 0.42 chlorophyll and carotenoid contents respectively. The maximum decline in chlorophyll was observed at 100 ppm concentration (0.21 ± 0.13). In addition to this, carotenoid content at p < 0.01 significantly decreased from 5.59 ± 0.24–0.73 ± 0.23 in 20–100 ppm Cd treatment (Fig. 3).

Figure 3.

Effect of Cadmium nitrate on Chlorophyll (mg/g FW) and Carotenoid content (mg/g FW) of Capsicum annuum L. Data mean of five replicate ± SD (ANOVA using DMRT ^∗∗p < 0.01).

3.4. Effect of cadmium on proline content

The results pertaining to the effect of Cd on proline content are presented in (Fig. 4). In this study, proline content was significantly increased (p < 0.01) from 20–60 ppm concentration (7.3 ± 0.27 to 11.18 ± 0.50 μmol/g DM) as compared to control, while gradually significantly decreased at higher concentrations (80 ppm and 100 ppm) from 9.78 ± 0.13 to 6.16 ± 0.48 (p < 0.01) in comparison with the control (Fig. 4).

Figure 4.

Effect of Cadmium nitrate on Proline content (μmol/g FW) of Capsicum annuum L. Data mean of five replicate ± SD (ANOVA using DMRT ^∗∗p < 0.01).

3.5. Effect of Cd on lipid peroxidation

Fig. 5, shows that MDA content was significantly increased (p < 0.01) from lower to higher concentrations (20–100 ppm) of Cd (6.84 ± 0.08 to 13.68 ± 0.21 mg/g FW) over control plants.

Figure 5.

Effect of Cadmium nitrate on MDA (mg/g FW) and Protein content (mg/g FW) of Capsicum annuum L. Data mean of five replicate ± SD (ANOVA using DMRT ^∗∗p < 0.01).

3.6. Effect of Cd on protein content

The result pertaining to the effect of Cd on protein content is presented in (Fig. 5). Control plant shows 2.92 ± 0.08 mg/g DM protein content while it is significantly decreased (p < 0.01) as the Cd concentration increased from 20 to 100 ppm (2.48 ± 0.04 to 0.82 ± 0.10). The higher concentrations of Cd (100 ppm) show a significantly decreased (p < 0.01) protein content to 28.08% compared to that of control.

3.7. RAPD analysis

A total of 184 scorable bands were generated in 5 different concentrations with 10 primers (Fig. 6a, b; Table 2). Out of 184 bands, 62 bands were polymorphic and 122 monomorphic. The highest percentage of polymorphism was generated by primer OPA-02 (52.63%) and the lowest percentage of polymorphism was generated by primer OPA-09 (5.88%). Higher number of co migrating bands monitors the reproducibility of amplification pattern while polymorphic ones provided the key to genotype identification. The genetic distance was computed considering all the genotypes from the pooled data and the dendrogram was constructed (Fig. 7). Dendrogram showed that control, R1 and R2 showed similar cluster and R4 and R5 grouped with R3 into one cluster, which showed that plants from higher doses showed much difference than the plants selected at mild doses which resemble control at the DNA level. The polymorphism percentage showed that cadmium caused some changes in the DNA band level at higher concentrations which is quite determinant by disappearing of old bands and appearing of new bands.

Figure 6.

DNA Band Profile of five different concentrations of Cadmium in Capsicum annuum L. using RAPD with primer OPA-02, with primer OPB-04. ∗(M-marker, C-control, R1-20 ppm, R2-40 ppm, R3-60 ppm, R4-80 ppm, R5-100 ppm.)

Table 2.

Total number of amplified fragments and number of polymorphic bands generated by RAPD-PCR using ten random decamer primers.

S. no.	Name of primer	Sequence (5′–3′)	Polymorphic bands	Monomorphic bands	Percentage polymorphism	G + C content
1	OPA-02	TGCCGAGCTG	10	9	52.63	70
2	OPA-05	AGGGGTCTTG	8	12	40.00	60
3	OPA-07	GAAACGGGTG	5	13	27.77	60
4	OPA-09	GGGTAACGCC	1	16	5.88	70
5	OPB-03	CATCCCCFTG	9	15	37.5	60
6	OPB-04	GGACTGGAGT	8	14	36.36	60
7	OPC-05	GATGACCGCC	5	13	27.77	70
8	OPD-02	GGACCCAACC	6	14	30.00	70
9	OPK-10	GTGCAACGTG	4	11	26.66	60
10	OPL-14	GTGACAGGCT	6	12	33.33	60

Figure 7.

UPGMA Dendrogram of Capsicum annuum L. treated with five different concentrations of Cadmium based on RAPD markers. (C-control, R1-20 ppm, R2-40 ppm, R3-60 ppm, R4-80 ppm, R5-100 ppm.)

4. Discussion

According to Mohan and Hosetti (1997) and Patra et al. (2004), Cd is a non-essential element that inhibits some vital plant processes such as photosynthesis, mitosis, and water absorption with adverse effects on leaves, wilting of older leaves, stunted foliage, and brown short roots. A greater impact of heavy metal exposure was observed on root growth compared to shoot leading to a larger reduction in length and fresh weight (Elloumi et al., 2007). The reduction in the growth of Capsicum might be also due to suppression of the elongation growth rate of cells, because of an irreversible inhibition exerted by Cd on the proton pump responsible for the process (Aidid and Okamoto, 1993). Retarded shoot growth due to the presence of the root environment with excess of Pb was also found by Seyyedi et al., 1999. The fall in chlorophyll content in plants exposed to Cd²⁺ and Pb²⁺ stress is believed to be due to (a) inhibition of key enzymes such as δ-aminolevulinic acid dehydratase (ALA-dehydratase) (Padmaja et al., 1990) and protochlorophyllide reductase (Van Assche and Clijsters, 1990) associated with chlorophyll biosynthesis; (b) impairment in the supply of Mg²⁺, Fe²⁺, Zn²⁺ and Mg²⁺ (Van Assche and Clijsters, 1990; Kupper et al., 1996). A similar decrease in chlorophyll content under heavy metal stress was reported earlier in cyanobacteria, unicellular chlorophytes (Chlorella), gymnosperms, such as Picea abies and angiosperms, such as Zea mays, Quercus palustris and Acer rubrum (Siedlecka and Krupa, 1996). The decrease in chlorophyll content was also reported in sunflower (Zengin and Munzuroglu, 2006), almonds (Elloumi et al., 2007) and Trigonella (Choudhary et al., 2012).

Lipid peroxidation is a biochemical marker for free radical mediated injury. The present study also showed a rise in the level of lipid peroxidation with increasing concentrations of Cd, indicating that these induce oxidative stress in Capsicum. Our results are in conformity with the observations of Malecka et al., 2001 and Unyayar et al., 2006. MDA formation is used as a general indicator of the extent of lipid peroxidation resulting from oxidative stress. The present study showed that MDA content was greatly affected by the highest concentration of Cd in C. annuum L. This shows that Capsicum seedlings have capability to adapt at lower concentrations of the metal and may be related with the low degree of lipid peroxidation. MDA content increases after Cd exposure in Capsicum. This suggested that heavy metal leads to excessive generation of superoxide radicals by deficient antioxidant defences resulting in increased lipid peroxidation and oxidative stress in Capsicum. The above results confirm the observation of Unyayar et al., 2006. According to Saradhi and Alia Vani (1993) proline an imino acid accumulates in wide variety of organisms ranging from bacteria to higher plants on exposure to abiotic stress. Plants were found to accumulate proline under environmental stress (Ahmad et al., 2006, 2008). Evidence suggested that the proline accumulation might contribute to osmotic adjustment at the cellular level and enzyme protection stabilizing the structure of macromolecules and organelles. An increase in proline content may be either due to de novo synthesis or decreased degradation or both (Kasai et al., 1998). Proline accumulation in shoots of Brassica juncea, Triticum aestivum and Vigna radiata in response to Cd²⁺ toxicity was demonstrated by Dhir et al. (2004) but proline accumulation decreased on exposure to Cd²⁺ in hydrophytes (Ceratophyllum, Wolffia and Hydrilla). Similarly increasing proline content due to Cd²⁺ was also reported in sunflower by Zengin and Munzuroglu (2006).

Cd-mediated toxicity in the form of various chromosomal abnormalities observed in our study is postulated to be a consequence of defective functioning of one or two types of specific non-histone proteins involved in chromosome organization that are needed for chromatid separation and segregation (Gaulden, 1987) or due to disturbances in cyto-chemically balanced reactions (Jayabalan and Rao, 1987). However, it seems most probable that interaction of Cadmium with chromatin proteins leads to incorrect coding of some non-histone proteins involved in chromosome organization, ultimately resulting in cytogenetic abnormalities. The reduction in root length is strongly correlated to the mitotic index of the root tips of C. annuum. Similar effect on mitotic index was observed with other heavy metal like cadmium (Siddiqui et al., 2009). The reductions in the number of mitotic cells in root tips of seeds exposed to Cd could be due to its mechanism of action on cell cycle progression. Pb can inhibit the DNA synthesis (Sudhakar et al., 2001) or may even block the cells in the G2_phase of cell cycle preventing the cells from entering mitosis.

To support the above hypothesis, Cd treated seeds exhibited several chromosomal abnormalities in mitotic cells of root tips such as sticky chromosome, bridge and fragment. Among these abnormalities sticky chromosome (Sc) was the most frequently observed chromosomal aberration in the C. annuum. An increase in the aberrant metaphase plates having sticky chromosomes might be due to the denaturing activity of Pb on nuclear proteins such as DNA topoisomerase II, which might also interfere with chromosome segregation (Panda and Panda, 2002). Similar result was also observed in Pisum sativum (Fusconi et al., 2006, 2007; Siddiqui et al., 2009). The bridges noticed in the root tip cells are probably formed by breakage and fusion of chromosome bridges which increased with Cadmium treatment. According to Gomurgen, 2000; Siddiqui, 2012 chromosome bridges may have occurred due to the chromosomal stickiness and subsequent failure of free anaphase separation or may be attributed to an unequal translocation or inversion of chromosome segment. In the present investigation, fragments noticed in the root tips are probably formed by acentric chromosome and also as a result of inversion. Fragmentation might have arisen due to stickiness of chromosomes and consequent failure of separation of chromatids to poles (Fusconi et al., 2006, 2007). Great variations in the form of DNA bands were seen in Capsicum treated with different concentrations of Cadmium, which is also determined by forming a UPGMA dendrogram in which plants from higher concentrations combined in one cluster and plants from mild (lower) concentrations were combined in another clusters. In our finding, molecular screening of DNA in Capsicum under cadmium stress condition, new DNA bands of different molecular weight have been identified on 100 ppm concentration; this may be due to transition of DNA. Thus differences in the nucleotide sequence of alleles result in the production of slightly different types of amino acids or variant forms of proteins. These proteins code for the development of varied morphological, anatomical and physiological characteristics in the organism (Bhat et al., 2012).

5. Conclusion

Therefore, it is concluded that cadmium nitrate may pose genotoxic effect and induce DNA damages and mutations. These can be successfully detected in conjugation with growth, biochemical, cyto-physiological parameters and DNA polymorphism through RAPD method.