Genotoxicity evaluation of water soil leachates by Ames test, comet assay, and preliminary Tradescantia micronucleus assay

Abstract

Combining genotoxicity/mutagenicity tests and physico-chemical methodologies can be useful for determining the potential genotoxic contaminants in soil samples. The aim of our study was to evaluate the genotoxicity of soil by applying an integrated physico-chemical-biological approach. Soil samples were collected at six sampling points in a Slovenian industrial and agricultural region where contamination by heavy metals and sulphur dioxide (SO(2)) are primarily caused by a nearby power plant. The in vitro alkaline version of the comet assay on water soil leachates was performed with Caco-2 and HepG2 cells. A parallel genotoxicity evaluation of the samples was performed by Ames test using Salmonella typhimurium and the Tradescantia micronucleus test. Pedological analyses, heavy metal content determination, and different physico-chemical analyses, were also performed utilizing standard methodology. Water leachates of soil samples were prepared according to standard methods. Since only a battery of biotests with prokaryotic and eukaryotic organisms or cells can accurately estimate the effects of (geno)toxicants in soil samples and water soil leachates, a combination of three bioassays, with cells or organisms belonging to different trophic levels, was used. Genotoxicity of all six water soil leachates was proven by the comet assay on both human cell lines, however no positive results were detected by bacterial assay, Ames test. The Tradescantia micronucleus assay showed increase in micronuclei formation for three samples. According to these results we can assume that the comet assay was the most sensitive assay, followed by the micronucleus test. The Ames test does not appear to be sensitive enough for water soil leachates genotoxicity evaluations where heavy metal contamination is anticipated.

In Vitro Genotoxicity of Settat Town Landfill Leachate, Morocco

“…It appears that the addition of volumes up to 1000 µL is suitable for (geno)toxicity assessment of water. Our fi ndings for dump leachate and ground water are in accordance with several other studies (6,9,12,20,26,27). Sang et al (26) found cytogenetic damage in root tips of Hordeum vulgare exposed to municipal landfi ll leachate.…”

“…The MN test is a well-known and validated endpoint that can predict increased cancer risk in a human population (22). This test has an advantage over tests with bacteria such as the Ames assay, which are not sensitive enough for water and soil leachate genotoxicity evaluations, especially when heavy metal contamination is anticipated (12,23). Nowadays the MN test is sometimes accompanied by counting apoptotic and necrotic cells, nuclear buds, and nucleoplasmic bridges (the cytome assay) (24).…”

“…This is how leachates containing high amounts of pollutants (3,4) can contaminate ground and surface waters and affect biodiversity of aquatic ecosystems, contaminate food chains, and pose a risk to human health. Several in vitro and in vivo studies of the ecotoxicological impact of environmental chemicals have confi rmed that many chemicals identifi ed at city dumps and in leachates have a genotoxic and carcinogenic potential (5)(6)(7)(8)(9)(10)(11)(12). Determination of the chemical composition and the genotoxic potential of wastewaters, surface, and ground waters is considered crucial for environmental protection and public health (13).…”

Bioindicators and Biomarkers in the Assessment of Soil Toxicity

“…This test, also known as Salmonella/microsome, consists, basically, in the employment of strains of the auxotrophic bacteria Salmonella typhimurium, i.e., deficient in the synthesis of the aminoacid histidine; the strains of these cells are unable to grow in minimum medium, where the mutagenic compounds are able to restore the synthesis capacity of this aminoacid, thus, the mutagenic expression corresponds to the growth of the colony in a minimum culture medium and it can be easily detected by counting the colonies (Umbuzeiro & Vargas, 2003). However, due to the low sensitivity of the Ames test for heavy metals, more studies should be directed to the development of bioassays with higher organisms (Gatehouse et al, 1990, as cited in Lah et al, 2008). Meristematic cells of A. cepa and V. faba, for example, constitute an effective cytogenetic material to analyze chromosome aberrations (figure 6) caused by soil pollution.…”

In Vitro Genotoxicity of Wastewaters from the Town of Settat, Morocco

“…as a result of disturbances in the mitotic spindle). Furthermore, it can be more sensitive than the bacterial Ames assay, especially when heavy metal contamination is anticipated (15,16). Recently, an inter-laboratory comparison study has also demonstrated that the in vitro micronucleus test is indeed very suitable for routine wastewater testing (17).…”