Genotoxic Potential of Agricultural Soils of Amritsar

INTRODUCTION

In recent years, soil contamination with heavy metals has been increasing due to use of pesticides, fertilizers, continuous air emissions from industrial sources and vehicular traffic. The factors controlling the total and bioavailable concentrations of heavy metals are of equal importance to human toxicology and agricultural productivity (Alloway, 1995). At present, the problem of preserving environmental purity is becoming very acute, thus necessitating a strict evaluation of the potential genotoxic effects of environmental mutagens. A qualitative/quantitative evaluation of environmental genetic hazard requires the building up of a screening system, which is able to detect different genotoxic/mutagenic events at molecular levels. In order to protect humans against the consequences of genotoxic effects, a variety of methods have been developed which enable the detection of mutagenic chemicals in an environment. Due to the conservative structure of DNA such investigations can be carried out not only with mammalian cells but also with bacteria, plants and insects among others as indicator organisms.

A number of earlier studies all over the world have shown the presence of different mutagenic substances in different soils using Ames test (Smith, 1982; Knize et al., 1987; Kool et al., 1989). However, in some studies, Ames test has yielded negative results (Steinkellner et al., 1998). Since 1970s, higher plant bioassays have been recommended for genotoxic evaluation of complex environmental mixtures by Anonymous (1973), Committee 17 of Environmental Mutagen Society (Drake et al., 1975) the World Health Organization (1985) and National Swedish Environmental Protection Board in 1989 (Cabrera et al., 1999) Considering the potential health hazards posed by heavy metals in soil and non-availability of data on content of heavy metals in agricultural soils of Amritsar, the present study was planned to evaluate the genotoxic potential of extracts of soil samples collected from different agricultural fields of Amritsar (31°37`N and 74°52`E), India, employing Ames and Allium root anaphase aberration assay (Al-RAAA).

MATERIALS AND METHODS

Sample Collection
Four soil samples (Table 1) were collected from different agricultural fields in September, 2003. Two samples were collected from Fatehgarh Churian, one from Chabba and fourth from the Botanical Garden of Guru Nanak Dev University, Amritsar.

The soil samples were collected by digging the soils to 10x10x20 cm³, dried at room temperature for 72 h and then ground to fine powder (Cabrera and Rodriguez, 1999).

Estimation of Heavy Metals
The soil extract was prepared by digesting 1 g of soil sample in 10 mL aqua regia. Final volume was made to 100 mL with distilled water. It was further diluted 10 times before feeding to atomic absorption spectrophotometer and mercury analyzer. The heavy metals like: chromium, cobalt, copper, manganese, nickel and zinc were estimated using atomic absorption spectrophotometer model AA6200 make Shimadzu and mercury was estimated using mercury analyzer model MA5800/E make Electronics Corporation of India, Ltd.

Estimation of Other Physico Chemical Parameters
Other physico chemical parameters like pH, bulk density, water holding capacity, moisture content, nitrates, phosphates and potassium were estimated as per procedures described by Rao (1998). pH was measured by pH meter model μ pH system 361 make Shimadzu. Bulk density was estimated by weight/volume method. Moisture content was estimated by oven drying method and water holding capacity by Brass box method. Nitrates and phosphates were estimated using Spectrophotometer model 2202 make Systronics and potassium by flame photometer model CL 26D make ELICO.

Estimation of Mutagenic Potential Employing Ames Assay
Water extraction of the soil samples was carried out using distilled water (soil:water, 1:2 w/v). The solution was filtered using Whatman No. 1 filter paper after 24 h of shaking. The extract was evaporated to dryness. The resulting dried extracts were further dissolved in distilled water. Different concentrations of extract corresponding to 0.25, 0.5, 1.0, 2.0, 2.5 g equivalent of soil per plate were used with Salmonella typhimurium TA98 and TA100 strains, with and without in vitro metabolic activation (S9) to detect direct and indirect pro mutagenic compounds.

Table 1:	Description of different agricultural soil sample codes

The protocol used to test the mutagenic activity of the sample was as proposed by Maron and Ames (1983). A brief outline of the protocol is: to 2 mL of top agar containing 0.5 mM histidine/biotin, 0.1 mL of fresh S. typhimurium culture of tester strain (TA98 and TA100), 0.1 mL of soil extract were added and mixed thoroughly before pouring on a minimal glucose agar plate. The plates were tilted and rotated very quickly to ensure speedy and uniform spreading of top agar. The plates were incubated at 37°C for 48 h. Concurrently with the above experiment, a positive control (20 μg/0.1 mL of NPD for TA98, 2.5 μg/0.1 mL of NQNO for TA100 strain) and a negative control (0.1 mL of distilled water) per plate were also set. To detect indirect mutagenic compounds, rat liver homogenate (S9) was used and 2AF was used as positive control.

Estimation of Genotoxic Potential
The soil extracts were prepared by suspending soil in distilled water in ratio of 1:2 (w/v) for 12 h at room temperature (Cabrera et al., 1999). Different concentrations of extracts (10, 25, 50, 75 and 100%) were used for treatment of roots of Allium cepa. The A. cepa bulbs with freshly emerged roots of about 1-2 cm were treated with different concentrations of soil extracts for 3 h. After thorough washing, the root tips were fixed in Farmer`s fluid (glacial acetic acid and ethanol in 1:3 ratio). The root tips were squashed in aceto-orcein and slides were screened for anaphase aberrations. 0.1% of MNNG was used as a positive control, with distilled water as negative control.

In situ Treatment
In situ conditions were simulated by allowing the onion bulbs to root directly in different soil samples contained in small pots after removing their primary roots. The freshly emerged root tips (1-2 cm) were fixed in Farmer`s fluid and the above mentioned procedure was followed to screen for anaphasic aberrations.

Statistical Analysis
All the physico chemical parameters and genotoxic potential were statistically analyzed by calculating mean values, standard error, regression and student`s t-test of significance (Bailey, 1995).

RESULTS

Table 2 shows that one of the samples from Chabba (CB) contained elevated levels of a number of metals like Cu (5.31 mg g^-1), Cr (3.12 mg g^-1), Co (2.71 mg g^-1) and Ni (5.017 mg g^-1). While the sample from Fatehgarh Churian Road (FC-I) contained comparatively high levels of mercury (0.015 mg g^-1). On the other hand, samples FC-II and BG contained all metals in traces.

The mutagenic effects of different concentrations of soil extracts in Ames assay are shown in Fig. 1 and Table 3. All the samples studied were found to be non-mutagenic in TA98 tester strain with and without S9. The mutagenic potential in TA100 strain for different samples was observed as

Table 2:	Heavy metal content in agricultural soils of Amritsar

ND: Not Detectable, Data shown are mean±SE of three experiments, ^@: Sample codes are mentioned in Table 1


Fig. 1:	Effect of different concentrations of soil extracts on TA98 and TA100 strains of S. typhimurium with and without S9

FC-I>FC-II>CB>BG without supplementation of S9 mix and FC-I>CB>FC-II>BG with S9 mix. The maximum number of histidine revertants were found to be at highest concentration of 2.5 g plate^-1 of FC-I as 636 for TA100-S9 and 556 for TA100+S9. Furthermore, no dose dependent effect was observed for both the strains.

Results on genotoxic effects of different soil extracts employing Al-RAA assay are given in Table 4. A dose dependent increase in genotoxic potential was noticed for almost all the soil samples tested (Fig. 2). Apart from treatment of root tips with soil extracts, in situ conditions were simulated by allowing the onions to root directly in the soil samples collected from different agricultural fields

Table 3:	Effects of extracts of different agricultural soil samples of Amritsar, India on histidine reversion frequency in TA98 and TA100 strains of S. typhimurium

Data are presented as Mean±SE of three independent experiments with triplicate plates/dose/experiment. NPD: 4-Nitro-o-phenylenediamine; NQNO: 4-Nitroquinoline-N-oxide; 2AF: 2-Aminofluorene, ^@: Sample codes are mentioned in Table 1


Fig. 2:	Effects of soil extracts on percent aberrant anaphases

Table 4:	Genotoxic effects of different soil extracts in Al-RAA assay

: Values were found to be significantly higher than negative control at p<=0.05, Data shown are mean±SE of three experiments, each experiment consisted of 100 root tip cells of Allium cepa* at anaphase. MNNG: N-Methyl-N`-Nitro-N-Nitrosoguanidine, ^@: Sample codes are mentioned in Table 1

Table 5:	Physico-chemical parameters of agricultural soils of Amritsar (Mean±SE)

Data shown are mean±SE of three experiments, ^@: Sample codes are mentioned in Table 1

to study their genotoxic effects. Of the four samples studied, the highest genotoxic potential (7.50%) was found in FC-I, which also contained highest content of mercury (0.015 mg g^-1). The soil for Chabba was second in induction of genotoxic effects and contained second highest levels of Hg while concentrations of Cu, Co, Cr and Ni were much higher as compared to sample for FC-I. The genotoxic potential of other soil samples was also found to correlate with the content of Hg with 100% concentration of soil extracts. Results of two different modes of treatments i.e., in situ and in vitro with 100% soil extract were found to be comparable.

FC-I and CB soils were found to be acidic in nature. All the samples contained optimum amounts of nitrates, phosphates and potassium. The water holding capacity and moisture content were found to be very low for all the samples (Table 5).

DISCUSSION

In the present study, strong differences in heavy metal contamination of different soils were found. TA98 tester strain of Salmonella yielded negative results both in presence and absence of S9 mix, while contrary results were obtained for TA100 strain with significant mutagenicity at the highest concentrations of 2.5 g equivalent of soil per plate. Though genotoxic effects of different soil samples in Allium cepa root anaphase aberration assay were found to correlate with the content of Hg, no direct correlation was found with other heavy metals studied. This is in line with the studies of Majer et al. (2002) who investigated genotoxic effects of 20 soil samples from nine locations of Austria, Germany and Slovenia and found no correlation between metal content and genotoxicity of soils. This study indicated that the genotoxicity of soils depended on their physico-chemical properties in addition to heavy metal content. The mobility of heavy metals in soils is known to be influenced by pH value, particle size distribution and carbon content of soil (Alloway and Ayres, 1993). The soils having low pH are expected to be more genotoxic. This was observed in the present study where the soils with higher genotoxicity (FC-I and Chabba soils) were found to have pH value less than 7 (i.e., towards acidity). Environment and human health are directly or indirectly affected by heavy metal contamination of agricultural soils. Currently, most environmental risk assessment of contaminated soils is based on chemical analysis, which reveals the presence of many mutagenic and carcinogenic compounds in the soil (e.g., Heavy metals, polyaromatic hydrocarbons (PAHs) and pesticides etc.). A major limitation of this approach is that most of the compounds present in soils are still unknown and most soil ecotoxicity data relates to relatively less known compounds (Wang et al., 1997). This necessitates the inclusion of different bioassays for genotoxic evaluation of soils as biomonitors in environmental risk assessment of contaminated soils. This led to the study of the mutagenicity of different soil samples employing Ames test (Brooks et al., 1998; Hughes et al., 1998; Monarca et al., 2002), Tradescantia micronucleus test (Knasmuller et al., 1998; Cabrera et al., 1999; Majer et al., 2002), Tradescantia stamen hair mutation (Trad-SHM) (Cabrera et al., 1999; Gichner, 1999) and Vicia root micronucleus assay (Wang, 1999; Cotelle et al., 1999). The present study suggests that Al-RAA is a suitable method to monitor the genotoxic effects of metal contaminated soils in situ and may be a valuable supplement to analytical methods of soil contamination and serve as first alert indication of DNA damage and pollution.

ACKNOWLEDGMENTS

Thanks are due to Prof. A.K. Thukral for his guidance in statistical analysis. J.K. Katnoria is recipient of UGC fellowship.