3.1. Analysis of standard solution with DNA biosensor
The effect of three benzene, two naphthalene and four anthracene derivatives were analysed in this work with DNA biosensor because widely present in the ACNA site. The molecular structures of the compounds analysed are reported in
Figure 1. The voltammetric behaviour of the biosensor with and without 1.0 μM 2-anthramine standard solution is shown in
Figure 2. Different concentrations for each compound were analysed and the inhibition of the guanine oxidation peak increased with analyte concentration (
Table 1 and
Figure 3).
The results demonstrated that the compounds presented different genotoxic effect. Generally, the effect was higher with increased ring number of the molecule, and also the type of the group had a relevance. For example, the amino group was more toxic than a sulfonate one at the same molecular concentration. The results showed the highest toxic effect for molecules with three rings. In fact the naphthalene and anthracene derivatives analysed are DNA intercalating agents [
5,
6,
11], while the benzene derivatives are not double helix intercalant but only a weak DNA interactive (i.e. as reported in [
11], 4-chloroaniline is described as non genotoxic and its cytotoxicity, measured as cell viability assay, is relatively low compared with other chemicals).
This work demonstrated that naphthalen-sulfonate derivatives with two and three aromatic rings and aromatic amines with two and three rings gave a positive result with the biosensor in a concentration range of micro and submicro-molar. The EC50 value was 2.0 μmol/L for 2-anthracenecarboxylic acid and 1,2-diamineanthraquinone and 1.0 μmol/L for 2-anthramine.
3.3. Analysis of real soil samples with DNA biosensor
In ACNA site the environmental risk arises from pollution diffused in the ground, accumulation of waste materials and pollution of the Bormida river. Four zones in a specific ACNA's area (hill n°5) were sampled in March 2003: zone 1 low contamination level; zone 2 pseudo-reference; zone 3 moderate pollution level and zone 4 high ecological risk.
Subsequently, the hill was destroyed and another sampling at the basement was made in January 2004. A polluted soil sample of this last sampling was mixed with a control soil (1:5 and 1:10) and the extraction was performed with milli-Q water (1:3 v/v) by the Department of Environmental and Life Sciences (DISAV), University of Eastern Piedmont, Alessandria, Italy.
ACNA samples were analysed with DNA biosensor and the different zones and dilutions were compared.
The comparison of the zones with the superficial sampling (max 5 cm depth) didn't showed a significant difference between them (
Figure 4). The comparison of the zones with 20 cm sampling depth showed differences between them, showing a trend of pollution as expected (
Figure 5): the biosensor was able to distinguish different soil contamination sites. The comparison between the different depths and extraction pHs of the zone 4 showed a contamination increasing with the sampling depth and small differences between pHs (
Figure 6). The results confirmed the fact that this zone is the most polluted of the hill n°5.
ACNA samples were analysed also with micronuclei and Comet tests performed by ISPESL/DIPIA laboratory (Rome, Italy). These are simple and sensitive techniques for analysis and measure of DNA damage in individual mammalian (and to some extent prokaryotic) cells. Seed of
Vicia faba var minor were put in ACNA soil samples and the top roots were used for the micronuclei and Comet tests in order to analyse a vegetable mutation after 5 days of exposure. The length of the top roots was also calculated in order to establish a phyto-toxicity. After, half of the roots were prepared for the micronuclei test, the other half for the Comet test. The genotoxic effect was based on the frequency of irregular anaphases and micronucleate cells. The results showed an increase in pollution from zone 1 to 4 and within the zone, with the sampling depth for all methods (
Table 5).
Concerning the Comet test, the analyisis showed an increase in DNA damage, corresponding to an increase in pollution, from zone 1 to 4 and with the sampling depth. The results are reported in
Table 5 and in
Figure 7 which shows an extension of the “comet tail” corresponding to an increase of damage, according to a visual scoring classification from 0 to 4 damage classes.
The analysis with the biosensor on ACNA samples from the 2004 sampling (after the destroy of the hill) showed a stronger genotoxic effect corresponding to a stronger pollution level with the depth in comparison with the superficial sampling of 2003 and a lower genotoxic effect mixing the sample with a control soil (
Figure 8). These results were confirmed by the analysis with other bioassays performed by DISAV (
Table 6). For example, phytoxicity was calculated by measuring the roots length of
allium porrum exposed to the polluted soil (the root is the first plant zone influenced by soil pollutants and its length diminish after a stress); plant genotoxicity was calculated measuring the mitotic anomalies of
allium porrum; stress syndrome of earthworms (
Lumbricus rubellus) was evaluated by a biomarker battery after 10 days of exposure: lysosomal membrane stability in coelomocytes, Ca
2+ ATPase activity on intestinal epithelium, metallothionein content in toto organism, mortality rate.
Interestingly, the results obtained analysing the ACNA soil samples with DNA biosensor were confirmed by other bioassays, hence this kind of biosensor can be very useful as a rapid screening method of analysis.