doi:10.1016/j.envpol.2005.03.012 
Copyright © 2005 Elsevier Ltd All rights reserved.
A biomarker model of sublethal genotoxicity (DNA single-strand breaks and adducts) using the sentinel organism Aporrectodea longa in spiked soil
Francis L. Martina, 
, 
, Trevor G. Piearcea, Alan Hewerb, David H. Phillipsb and Kirk T. Semplec
aDepartment of Biological Sciences, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK
bInstitute of Cancer Research, Brookes Lawley Building, Cotswold Road, Sutton, Surrey SM2 5NG, UK
cDepartment of Environmental Science, Institute of Environmental and Natural Sciences, Lancaster University, Lancaster LA1 4YQ, UK
Received 11 November 2004;
accepted 17 March 2005.
Available online 13 June 2005.
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Abstract
There is a need to develop risk biomarkers during the remediation of contaminated land. We employed the earthworm, Aporrectodea longa (Ude), to determine whether genotoxicity measures could be applied to this organism's intestinal tissues. Earthworms were added, for 24 h or 7 days, to soil samples spiked with benzo[a]pyrene (B[a]P) and/or lindane. After exposure, intestinal tissues (crop/gizzard or intestine) were removed prior to the measurement in disaggregated cells of DNA single-strand breaks (SSBs) by the alkaline comet assay. Damage was quantified by comet tail length (CTL, μm). B[a]P 24-h exposure induced dose-related increases (P<0.0001) in SSBs. Earthworm intestine was significantly (P<0.0001) more susceptible than crop/gizzard to B[a]P and/or lindane. However, both tissues appeared to acquire resistance following 7-day exposure. B[a]P-DNA adducts, measured by 32P-postlabelling, showed a two-adduct-spot pattern. This preliminary investigation suggests that earthworm tissues may be incorporated into genotoxicity assays to facilitate hazard identification within terrestrial ecosystems.
Sublethal genotoxicity in the sentinel organism A. longa can be used to monitor the effects of contaminants in soil.
Sublethal genotoxicity in the sentinel organism A. longa can be used to monitor the effects of contaminants in soil.
Keywords: Adducts; Benzo[a]pyrene; Contaminated soil; Comet assay; DNA single-strand breaks; Earthworms
Fig. 1. Comet-forming activity of 24-h treatment with B[a]P in A. longa intestine. Following disaggregation of tissues in cold PBS, cell suspensions were incorporated into the comet assay, as described in Materials and methods. Each panel represents the level of DNA SSBs following the indicated treatment of an individual earthworm. These were as follows: (A) solvent control; (B) 24-h exposure to 0.1 ppm B[a]P-spiked soil; (C) 24-h exposure to 1.0 ppm B[a]P-spiked soil; and (D) 24-h exposure to 10.0 ppm B[a]P-spiked soil. P, as compared with solvent control.
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Fig. 2. Comet-forming activity of 24-h treatment with 10 ppm B[a]P in A. longa crop/gizzard or intestine. Following disaggregation of tissues in cold PBS, cell suspensions were incorporated into the comet assay, as described in Materials and methods. Each panel represents the level of DNA SSBs following the indicated treatment of an individual earthworm. These were as follows: (A) solvent crop/gizzard control; (B) crop/gizzard following 24-h exposure to 10.0 ppm B[a]P-spiked soil; (C) crop/gizzard following 24-h exposure to 10.0 ppm lindane-spiked soil; (D) crop/gizzard following 24-h exposure to 10.0 ppm B[a]P plus 10 ppm lindane-spiked soil; (E) solvent intestine control; (F) intestine following 24-h exposure to 10.0 ppm B[a]P-spiked soil; (G) intestine following 24-h exposure to 10.0 ppm lindane-spiked soil; and (H) intestine following 24-h exposure to 10.0 ppm B[a]P plus 10 ppm lindane-spiked soil. P, as compared with corresponding solvent control group; P*, as compared with corresponding crop/gizzard group.
 |
Fig. 3. Comet-forming activity of 7-day treatment with 10 ppm B[a]P in A. longa crop/gizzard or intestine. Following disaggregation of tissues in cold PBS, cell suspensions were incorporated into the comet assay, as described in Materials and methods. Each panel represents the level of DNA SSBs following the indicated treatment of an individual earthworm. These were as follows: (A) solvent crop/gizzard control; (B) crop/gizzard following 7-day exposure to 10.0 ppm B[a]P-spiked soil; (C) crop/gizzard following 7-day exposure to 10.0 ppm lindane-spiked soil; (D) crop/gizzard following 7-day exposure to 10.0 ppm B[a]P plus 10 ppm lindane-spiked soil; (E) solvent intestine control; (F) intestine following 7-day exposure to 10.0 ppm B[a]P-spiked soil; (G) intestine following 7-day exposure to 10.0 ppm lindane-spiked soil; and (H) intestine following 7-day exposure to 10.0 ppm B[a]P plus 10 ppm lindane-spiked soil. P, as compared with corresponding solvent control group; P*, as compared with corresponding crop/gizzard group.
Fig. 4. Autoradiograms of B[a]P-DNA adduct patterns from A. longa exposed to spiked soil samples. Following tissue resection, isolated DNA was subjected to 32P-postlabelling analysis as described in Materials and methods. Examples shown are as follows: (A) solvent control (EW2, 3.17 B[a]P-DNA adducts per 108 nucleotides); (B) 24-h exposure to 10.0 ppm B[a]P-spiked soil (EW4, 11.49 B[a]P-DNA adducts per 108 nucleotides); (C) 24-h exposure to 1.0 ppm B[a]P-spiked soil (EW6, 7.91 B[a]P-DNA adducts per 108 nucleotides); (D) 24-h exposure to 0.1 ppm B[a]P-spiked soil (EW8, 5.08 B[a]P-DNA adducts per 108 nucleotides); and (E) positive control (hepatic-derived DNA isolated from a B[a]P-treated rat). Arrows point to areas of chromatograms where readings of radioactivity were taken, either to calculate levels of adducts or as a background reading.
Table 1.
B[a]P-DNA adduct formation in intestine of A. longa
Individual earthworms were exposed for 24 h to soil samples spiked with solvent, 0.1 ppm, 1.0 or 10.0 ppm B[a]P. Following resection of tissue samples, isolated DNA was subjected to 32P-postlabelling analysis (4 μg per sample) using the nuclease P1 digestion method of sensitivity enhancement (Reddy and Randerath, 1986). Relative levels of DNA modification were calculated from the levels of radioactivity in the DNA adduct spots detected on the postlabelling chromatograms and from the specific activity of the [γ-32P]ATP used in the labelling procedure. For each tissue sample, duplicate analyses were performed.
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