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Potential use of DNA adducts to detect mutagenic compounds in soil
Environmental Pollution 157 (2009) 916–921
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Potential use of DNA adducts to detect mutagenic compounds in soil Guoxiong Hua a, *, Brett Lyons b, Ken Killham c, Ian Singleton a, * a
School of Biology, Institute for Research on the Environment and Sustainability, Devonshire Building, Newcastle University, NE1 7RU, UK CEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK c School of Biology, Cruickshank Building, University of Aberdeen, AB24 3UU, UK b
A novel DNA adduct assay may provide a potential technique to detect mutagenic compounds in contaminated soil.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2008 Received in revised form 24 October 2008 Accepted 29 October 2008
In this study, three different soils with contrasting features, spiked with 300 mg benzo[a]pyrene (BaP)/kg dry soil, were incubated at 20 C and 60% water holding capacity for 540 days. At different time points, BaP and DNA were extracted and quantified, and DNA adducts were quantified by 32P-postlabelling. After 540 days incubation, 69.3, 81.6 and 83.2% of initial BaP added remained in Cruden Bay, Boyndie and Insch soils, respectively. Meanwhile, a significantly different amount of DNA–BaP adducts were found in the three soils exposed to BaP over time. The work demonstrates the concept that DNA adducts can be detected on DNA extracted from soil. Results suggest the technique is not able to directly reflect bioavailability of BaP transformation products. However, this new method provides a potential way to detect mutagenic compounds in contaminated soil and to assess the outcomes of soil remediation. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords: Polycyclic aromatic hydrocarbons (PAHs) DNA-PAH adducts 32 P-postlabelling assay Soil pollution
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are pollutants of great environmental concern because of their toxic, mutagenic, and carcinogenic properties (Shabad, 1980; Fang and Smith, 2001; Yan et al., 2004; Toyooka and Ibuki, 2007). There is an urgent need to develop methods to monitor such contamination to assess ecosystem and human health risks and to recommend remediation action. Generally, risk analysis is based on chemical analysis (assessing total level of pollutants present), but this does not necessarily reflect actual or potential toxicity caused by pollutants and chemical analysis alone may not detect the presence of all pollutants. Ecotoxicological assays for mutagenic compounds (such as PAHs) in soil have been developed and are extremely useful. However, assays generally involve matrix extraction and subsequent exposure of extracts/leachates to single species (Rocheleau and Cimpoia, 1999; Alexander and Alexander, 2000; Watanabe and Goto, 2000) and this use of extracts (often solvents) may not truly reflect the actual availability and toxicity of pollutants to different soil dwelling species. Useful solid phase toxicity assays have also been developed but these again generally involve a surrogate for actual organisms and may not actually reflect uptake of pollutants observed in living organisms (Bergknut et al., 2007). To overcome
* Corresponding authors. Tel.: þ44 1334 463829; fax: þ44 1334 463384. E-mail addresses: [email protected] (G. Hua), [email protected] (I. Singleton).
the limitations of chemical analysis and current combined solvent extraction/toxicity assays, an improved toxicity method would be able to detect the presence of a wide variety of mutagenic compounds in soils and determine if the compounds present are actually capable of exerting a toxic effect in soil. Such a method could also prove useful for bioremediation where there is potential for microbial transformation to form a wide variety of products that would be difficult and expensive to characterise chemically. Soil DNA can be assumed to be mainly derived from microorganisms, representing a wide variety of species, and is subject to attack by electrophilic reagents due to the presence of nucleophilic sites on its purine and pyrimidine bases, guanine, adenine, thymine, and cytosine. The most active of these sites comprise nitrogen atoms at position 7 of guanine and position 3 of adenine (La and Swenberg, 1996). Electrophilic compounds can react with one of the DNA bases, acting as a nucleophile, causing a covalent modification of the DNA base that is referred to as a DNA adduct. DNA adducts represent excellent biomarkers for determining the extent of damage to genetic material and have long been of interest for understanding the mechanism of carcinogenesis and in the assessment of cancer risk posed by various chemicals or processes (Lamb et al., 2000; Cerniglia and Sutherland, 2001). However, as far as the authors are aware no-one has previously attempted to use DNA extracted from soil living organisms to determine if mutagenic compounds in soil are either present or capable of causing DNA damage in the form of DNA adducts. BaP and other PAHs are activated enzymatically to reactive forms that attack DNA resulting in formation of adducts that lead
0269-7491/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.10.026
G. Hua et al. / Environmental Pollution 157 (2009) 916–921
initially to human mutations, carcinogens, and/or developmental toxicants (Harvey, 1991; Jeffrey et al., 1977; Jennette et al., 1977). BaP is widely used as a representative PAH because concentrations of individual PAHs in the urban setting are highly intercorrelated (Perera et al., 2003). BaP inhalation activates the ary hydrocarbon receptor (AhR), which forms an active transcription factor heterodimer with AhR nuclear translocator (ARNT), and induces expression of a group of genes called the [Ah] gene battery, which includes the cytochrome P450 enzyme CYP1A1, CYP1A2 and CYP1B1 genes (Nebert et al., 2000). To produce carcinogenic, mutagenic and cytotoxic effects, BaP is thought to be converted to toxic metabolites through an AhR- dependent mechanism (Hankinson et al., 1991). Among the numerous metabolites identified in BaP-treated cells, BaP-7,8-diol-9,10-epoxide (BPDE) most of effectively forms DNA adducts and serves as a putative carcinogen (Miller and Ramos, 2001; Smithgall et al., 1986, 1988; Penning et al., 1996; Flowers et al., 1997). Therefore, two BaP metabolites: benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (), (anti) and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol () are ideal additives for experiments in this study. This work aimed to examine the potential of using DNA adducts present in soil DNA extracts to determine if mutagenic compounds can be detected in soil without the need for analytical chemistry. This ‘soil DNA adduct assay’ should indicate that the presence of BaP in soil has the potential to cause a biological effect and also that potential mutagens had been formed from BaP. It must be noted that BaP itself does not form adducts but that only metabolites or chemical transformation products of BaP form adducts (Poirier and Santella, 2000; McGrath and Sington, 2000). The individual objectives of the work were to: (a) show that DNA adducts could be detected on DNA extracted directly from soil, (b) determine if DNA adducts could be found in different soil types, and (c) examine the potential of the assay to determine the bioavailability of BaP in different soil types.
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2.3. Soil contamination and incubation Soils were spiked with BaP (300 mg of BaP/kg dry soil). The method used was modified from Pinto (Pinto and Moreno (2002)). Briefly, spiking and incubations were carried out in 250-ml brown glass jars containing the equivalent of 50 g dw soil. The soils were treated with the stock solution of BaP. The required amount of BaP stock (acetone solution) was added to a 20% fraction (10 g) of the soil sample and the jars were closed for 10 min to let the solvent disperse. Thereafter, the solvent was evaporated in a fume hood for 48 h at room temperature, and the sub-sample was mixed with the remaining 80% (40 g) of the soil sample. All mixings were performed thoroughly in each separate jar for 2 min with a metal spatula. Finally, distilled water was added to each jar to give soil samples with moisture contents of 60% water holding capacity (WHC). The treated soil was transferred into jars, and the jars were loosely covered with aluminium foil to maintain moisture content and aeration. All soil samples including the soils spiked with BaP and blank soils were incubated at 20 C in a constant temperature room for 540 days. Soil moisture was maintained by adding deionised water when needed. Soils were not sterilised in any part of this study. All experiments were carried out in triplicate (a separate jar being used for each replicate). 2.4. BaP extraction from soil Soil (0.50 g, fresh weight) was mixed with CH3CN (1.5 ml) in a clean microcentrifuge tube and firstly shaken at 5.5 m/ s in a FastPrepÔ instrument for 30 s. After this, the sample was shaken for another 1 min on a Vortex stirrer. The resulting slurry was centrifuged at 10,000 g for 30 min and passed through a 0. 45-mm Whatman filter. The filtrate was made ready for synchronous fluorescence spectroscopy (SFS) measurement by a 125-fold or 250-fold or 5000-fold dilution with aqueous CTAB micellar medium (7.8 mM), respectively. 2.5. BaP analysis by SFS Quantification of BaP was carried out by using a recently published SFS technique (Hua et al., 2006, 2007). All fluorescence spectra were determined using the synchronous mode on a Cary Eclipse luminescence spectrophotometer (PerkinElmer., Rockville, MD). Each sample was analysed by using the excitation and emission monochrometers at a wavelength difference of 20 nm (Dl ¼ 20 nm). Scan speed was 240 nm/s and PMT voltage was kept at 700 V. The corrected synchronous spectra were recorded in an excitation scale in the range 200–500 nm. High-grade UV quartz cuvettes (10-mm path length) were used for all spectroscopic analyses. 2.6. Isolation of soil DNA
2. Materials and methods
Four different DNA extraction procedures were compared in initial experiments in order to obtain the highest level of DNA (adducts) for all soil samples.
2.1. Chemicals All chemicals were purchased from the Sigma-Aldrich chemical company unless otherwise stated. Two BaP metabolism isomers, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (), (anti) and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol () were purchased from the NCI Chemical Carcinogen Repositories (MRI, Kansas City, MO, USA). PicoGreen reagent was obtained from Molecular Probes (Eugene, OR). Water was purified using a Milli-Q system (Millipore, Bedford, MA). All solvents and chemicals were the highest purity available and used without further purification.
2.2. Soils used Three well characterised soils were used in this work with a wide range of texture and organic matter concentration: Cruden Bay (low sand, high clay and high organic matter), Boyndie (high sand, low clay and low organic matter) and Insch (moderate sand, moderate clay and low organic matter). Their key physical and chemical properties are listed in Table 1. All soils were screened through a 2mm sieve after air-drying/hand-grinding and stored at room temperature before use.
Table 1 Selected physical and chemical characteristics of the tested soil samples. Soil Series pH Sand (%) Silt (%) Clay (%) Texture Organic matter (%)
Cruden Bay Tipperty 6.03 43.44 32.31 24.25 Clay loam 7.38
Boyndie Boyndie 3.61 79.10 14.30 6.53 Loamy sand 3.57
Insch Insch 6.97 57.70 30.79 11.51 Sandy loam 3.75
2.6.1. Method (i) DNA was isolated from all soil samples using a commercial Bio 101 kit according to manufacturer’s instructions (BIO101 Inc., Carlsbad, CA, USA). This is a commercial kit made for direct extraction of bacterial DNA from soil. 2.6.2. Method (ii) The second DNA extraction procedure was modified from previously described protocol (Burgmann et al., 2001). In brief, to a mixture of fresh soil (0.50 g) and glass beads (0.75 g, 425–600 mm diameter) in a microcentrifuge tube, the buffer (1.2 ml, 0.2% hexadecyltrimethylammonium bromide (CTAB), 1 mM dithiotreitol (DTT) and sodium phosphate buffer (0.2 M, pH 8.0), 0.1 M NaCl, 50 mM EDTA) was added. After processing the mixture was centrifuged at 13,000 g for 15 min. The supernatant (700 ml) was transferred to a clean microcentrifuge tube for a final extraction with phenol–chloroform using phenol (350 ml) and CIA (350 ml, chloroform/isoamyl alcohol ¼ 24:1). An extra CIA (700 ml) was added to the upper supernatant to remove phenol and the mixture centrifuged at 13,000 g for 10 min at room temperature. Harvest of DNA was performed by incubation with precipitation solution (750 ml, 20% polyethyleneglycol 6000 and 2.5 M NaCl) at 37 C and centrifugation at 13,000 g. 2.6.3. Method (iii) This DNA extraction procedure was modified from Krsek and Wellington (1999): fresh soil (0.50 g) and glass beads (0.50 g, 0.25 g of 150–212 mm and 0.25 g of 425– 600 mm , acid-washed, SIGMA) were added to TE buffer (1.0 ml, 10 mM Tris–HCl and 1 mM EDTA, pH 8.0). This mixture was then incubated with 5 mg/ml lysozyme (650 ml) at 37 C for 1 h and the supernatant (after centrifugation at 13,000 g) was incubated with 10% SDS (132 ml) for 30 min at 70 C. Harvest of DNA was carried out by incubating the supernatant with 50% PEG-6000 (200 ml) and 5 M NaCl (50 ml) overnight at 4 C. 2.6.4. Method (iv) This procedure followed exactly that described by Sandaa and Enger (1998). This is a direct method aimed at extracting bacterial DNA from soil after treatment with lysozyme, proteinase K and lauryl sulphate (SDS).
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The resultant DNA from all different methods was re-suspended in highly pure water (50 ml) for further study. The first DNA extraction procedure was used in the experiment to study the formation of DNA-BaP adducts from all three soils. 2.7. Quantitative determination of total DNA DNA in the soil extracts was quantified by using PicoGreen reagent and fluorescence (Sandaa and Enger, 1998). Briefly, DNA extract (2.0 ml), PicoGreen (2.0 ml) and 1X TE (396.0 ml) were mixed in a polypropylene tube, vortexed, and quantified by fluorescence in a Fluoroskan Accent FL (Thermo Labsystems). DNA standards were prepared from bacteriophage l DNA stocks. Fluorescence was measured at 485 nm excitation and 535 nm emission. Both slits were set to 2.5 nm. 1X TE buffer was used as blank sample. DNA yield was calculated as micrograms of total DNA in solution per gram dry weight of extracted soil.
DNA level (µg DNA/g dry soil)
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25 20 15 10 5 0 Method 1
2.8. DNA purification
2.9.
32
P-postlabelling assay of DNA adducts
DNA adducts were determined using the standardised nuclease P1 version of the P-postlabelling assay, as described previously (Phillips and Castegnaro, 1999). Briefly, samples of DNA were digested to deoxyribonucleoside 30 -monophosphates in a total volume of 9.5 ml of digestion mix (6 mU/ml calf spleen phosphadiesterase (Calbiochem, UK), 36 mU/ml micrococcal nuclease, 100 mM sodium succinate, 50 mM CaCl2). The DNA digest (2.0 ml) was diluted and held for the labelling of the normal undamaged nucleotides for subsequent quantification. To the remaining 7.5 ml of digested DNA sodium acetate buffer (1.0 ml, final concentration 40 mM), ZnCl2 (1.0 ml, final concentration 0.2 mM) and nuclease P1 solution (1.0 ml, final concentration 0.31 mg/ml) were added to remove normal nucleotides. The reaction mixture was incubated at 37 C for 30 min, and the reaction then stopped by the addition of Tris solution (1.0 ml). Adducted and normal nucleotides were then labelled separately, but simultaneously, for 30 min with labelling buffer (200 mM bicine NaOH, pH 9, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM spermidine), six units T4 polynucleotide kinase (30 mU/ml; Amersham) and 50 mCi of [g-32P] ATP (>7000 Ci/mmol, ICN). The adducted deoxyribonucleoside-30 -50 -biphosphates present in the sample were then purified and separated from their normal undamaged counterparts using multidimensional anion exchange thin layer chromatography (TLC), on 10 10-cm polyethyleneimine (PEI)-cellulose plates (Camlab, Cambridge, UK). The levels of DNA adduct radioactivity were determined using an AMBIS radioanalytical scanning system (LabLogic, Sheffield, UK). Upon the quantification of both the adducted nucleotides and the normal nucleotides, the relative adduct labelling values were calculated and converted to the number of adducted nucleotides per 108 undamaged nucleotides (Phillips and Castegnaro, 1999). Appropriate negative and positive DNA controls were analysed throughout the studies as described by Harvey and Parry (1998). 32
2.10. Control experiment: potential impact of the DNA isolation method on DNA-BaP adduct analysis This control experiment was carried out using DNA isolation method (i). It was considered that during DNA extraction, BaP metabolites could be released from soil particles into soil solution and interact with DNA released from cells. This could potentially increase the amount of BaP metabolites bound to isolated DNA and result in an ‘over-estimation’ of DNA adduct formation. Essentially this means that the DNA adduct assay may not accurately predict the bioavailability of mutagens but would be able to reveal the presence of potential mutagens in soil. To test the above hypothesis, i.e. to determine the potential interaction of metabolites with naked DNA during extraction, the following experiment was performed: Control experiment (i): two mutagenic microbial benzo[a]pyrene metabolites, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (), (anti) and benzo[a]pyrener-7,t-8,c-9,t-10-tetrahydrotetrol () (5 mg/ g dry soil for each), were added to all soils (uncontaminated) immediately in triplicate prior to DNA extraction.
Method 2
Method 3
Method 4
Fig. 1. Efficacy of DNA extraction procedures evaluated on the basis of DNA yield from three different soil types: Cruden Bay, Boyndie and Insch (the values are averages standard errors based on three independent extractions).
2.11. Data analyses Errors are indicated as standard deviation (SD) of the mean of triplicate measurements. The 32P-postlabelling data followed a normal distribution.
3. Results and discussion 3.1. Selection of suitable DNA extraction method The successful application of molecular techniques to a soil relies on the effective recovery of nucleic acid that is of suitable quantity, quality and purity for all subsequent analyses. There have been a number of published protocols for the extraction of total microbial community DNA from soils (Holben, 1994; Picard et al., 1992; Krsek and Wellington, 1999). The direct cell lysis method is the most routinely used with nucleic acids extracted directly from the soil matrix. Several researchers have compared available nucleic acid extraction techniques in order to assess the quantity and quality of nucleic acids and the overall reliability of published techniques (Zhou et al., 1996; Courtois et al., 2001; Torsvik, 2001; Robe et al., 2003). In general, the results indicate that cell lysis by bead-mill homogenisation in the presence of an organic solvent such as phenol and chloroform yield the greatest quantity of DNA. Considering the above discussion, an efficient and reproducible DNA extraction method was essential for this study to maximise yield and purity of DNA (adducts) from all three soil samples. Attention was also paid to the ease and rapidity of the method. Large differences in amounts of DNA isolated from the different
BaP concentration (µg/g dry soil)
Four treatments were investigated: (i) overnight precipitation with two volumes of 30% polyethylene glycol 6000 (PEG) (wt/wt)/1.6 M NaCl at 4 C; (ii) phenol/chloroform purification (Sambrook et al., 1989); (iii) 30-min incubation with 1/2 volume of 8 M potassium acetate on ice; (iv) 30-min incubation with 1/2 volume of 5% cetyltrimethylammonium bromide (CATB) buffer (wt/wt). DNA pellet after each treatment was collected by centrifugation at 13,000 g for 30 min at 4 C, washed with ice-cold 80% ethanol and centrifuged at 13,000 g for 10 min. Ethanol was completely removed and the pellet was allowed to dry in a vacuum (DNA 120 Speed VacÒ, Thermo Savant) to get a dry DNA adduct sample. DNA adducts were stored at 20 C until 32P-postlabelling analysis for DNA adducts. Of the four treatments tested, PEG precipitation produced DNA of sufficient quality for DNA adduct analysis (results not shown), presumably due to efficient removal of humic materials from extraction solutions and this technique was used in all further work.
Cruden Bay Boyndie Insch
280 Cruden Bay Boyndie Insch
250 220 190 160 130 100 0
74
158
284
365
550
Incubation time (day) Fig. 2. BaP concentrations during incubation in three BaP contaminated soils: Cruden Bay, Boyndie and Insch (the values are averages standard errors based on triplicate samples).
G. Hua et al. / Environmental Pollution 157 (2009) 916–921
soils were clearly observed using different DNA extraction methods (Fig. 1). Method (i) (BIO101 kit) gave the highest yield of DNA from all three soils and was considered to be the most rapid and easy method to carry out. Therefore this method was selected to extract DNA for the study of DNA adduct formation for all soil samples in all further studies. 3.2. BaP extraction and analysis Efficient extraction and analysis in studies of PAHs in soils is essential (Eschenbach et al., 1994; Hechler et al., 1995; Haeseler et al., 1999; Song et al., 2002). The classic Soxhlet extraction of PAHs from soils and sediments has been replaced by faster, less solventconsuming and often-automated techniques, which include one or more extraction cycles. Representative examples of these new extraction techniques are ultrasonic extraction (Sun et al., 1998; Femandez et al., 1999), supercritical fluid extraction (Deuster et al., 1997; Sun et al., 1998), pressurised liquid extraction (Hawthowe et al., 2000; Lundstedt et al., 2000), and microwave-assisted extraction (Pensado et al., 2000; Shu et al., 2000). In this study, a shaking extraction procedure, which provides a simple and low cost effective alternative, was used. Though this technique does not provide the highest extraction efficiency of PAHs in environmental samples, it is, however, suitable for most research purposes examining the relative change in PAH levels such as biodegradation experiments in soils/sediments (Hua et al., 2006). We found BaP recovery when acetonitrile was used as extraction reagent ranged from 80 to 100% dependent, to a great extent, on soil type (data not shown). This shaking method was considered satisfactory for the purposes of the work described here as it was well suited to indicating a decrease in the extractability of BaP over time in the soils used.
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3.3. Level of BaP in soils over time BaP concentrations in the three different soils were determined at day 0, 50, 110, 220, 365 and 540 and results are shown in Fig. 2. A significant decrease in BaP level over time was observed in all soils. After 540 days incubation, 69.3, 81.6 and 83.2% of the original amount of BaP extracted (day 0) was found for Cruden Bay, Boyndie and Insch soils, respectively. This reduction in BaP extracted from all soils could have been due to a combination of biological and chemical transformation of BaP thereby reducing total BaP levels, or due to physico-chemical binding of BaP to soil constituents (e.g. organic matter and clays). This is a common issue with all soil biodegradation studies, i.e. is the reduction in pollutant level observed with time due to biological or chemical transformation or due to physical processes that reduce solvent extraction efficiency? It was thought that the DNA adduct assay used in this work could provide at least a partial answer to this issue. Therefore, if DNA adducts were detected on isolated soil DNA this would mean that BaP has been transformed (chemically or biologically) as BaP itself does not form adducts (Poirier and Santella, 2000). This would mean that any reduction in BaP levels extracted from soil could be in part due to BaP transformation.
3.4. DNA adduct formation in soils over time 32
P-postlabelling is a very sensitive method for DNA adduct detection and it was applied successfully in our study to assay DNA adducts formed on soil DNA derived from BaP transformation products. Representative autoradiographs from 32P-postlabelled DNA are shown in Fig. 3. The images clearly show that DNA adducts were formed in BaP contaminated soils.
Fig. 3. Representative DNA adduct images (autoradiograms of TLC plates) produced by the 32P-postlabelling assay. (A) Insch BaP contaminated soil lacking any distinct DNA adducts after 220 days incubation; (B) Cruden Bay BaP contaminated soil (220 days incubation) showing a number of 32P-labelled DNA adducts (highlighted by arrows); (C) Boyndie BaP contaminated soil after 220 days incubation showing a main BaP adduct close to the bottom left hand corner (red arrow), note similarity to positive control (D); and (D) positive control consisting of labelled DNA (115 nucleotides per 108 undamaged nucleotides) run with each batch.
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G. Hua et al. / Environmental Pollution 157 (2009) 916–921
3.5. DNA adduct formation with BaP transformation Fig. 4 shows the quantity of DNA adducts formed over time in the three contaminated soils. High levels of DNA adducts were found in Cruden Bay soil at days 220 and 365, while relatively few DNA adducts were found in Boyndie and Insch soils during this time. No DNA adducts were detected after 550 days incubation in all three soils. The results show that the formation of DNA adducts varied in different soils and also varied over time. Importantly, no DNA adducts were found in any soils at time 0 providing evidence that BaP itself is not able to form DNA adducts. Interestingly, the highest amounts of DNA adducts were found in Cruden Bay soil which has a high organic matter and clay concentration. Given that BaP is hydrophobic it could be expected that the chemical would interact with hydrophobic soil organic matter and thus be made unavailable for biological and/or chemical transformation. However, chemical analysis suggests that BaP was removed to a higher extent in Boyndie soil and this is consistent with the higher level of DNA adduct formation found in this soil. It would appear that BaP is available in this soil and this could perhaps be due to the indigenous microbial population present; such a population should be adapted to transformation of high molecular weight material (associated with soil humic matter) and so may be able to access and transform hydrophobic high molecular weight compounds such as BaP. 3.6. Control experiment: potential impact of the DNA isolation technique on the level of BaP related DNA adducts formed
DNA adducts (per 10ex8 undamaged normal uncleotides)
The DNA extraction technique used may have caused the release of previously unavailable BaP metabolites into solution. This could potentially increase the amount of BaP metabolites bound to the isolated DNA causing an increase in DNA adducts formed. Therefore, the DNA adduct technique would result in an over-estimation of potential in situ biological effects. We initially considered that the presence of large amounts of other soil materials such as colloidal clay, cell material and dissolved and particular organic matter would complex the BaP metabolites released during DNA extraction and therefore prevent them binding to naked DNA. However, the addition of two mutagenic microbial benzo[a]pyrene metabolites, to uncontaminated soils immediately prior to DNA extraction resulted in clear DNA adduct formation on isolated soil DNA (Fig. 5A). This clearly demonstrates that BaP metabolites present in soil are able to complex to naked soil DNA and that this effect must be taken into consideration when interpreting results regarding the potential in situ biological effects. Interestingly, three different DNA-PAH adducts were formed even though only two BaP
1000 900 800
Cruden Bay Boyndie Insch
700 600 500 400 300 200
Fig. 5. Control experiment. Production of DNA adducts on isolated soil DNA caused by addition of BaP metabolites to uncontaminated soil immediately prior to DNA extraction.
metabolites were added. This could be due to the chemical instability of the BaP metabolites used causing some other spontaneous products to be formed that were capable of forming DNA adducts or to chemical impurities in the metabolite formulations used. 4. Conclusions This work shows that DNA adducts can be detected on isolated soil DNA and that this only appears to occur when BaP is transformed in soil. Given that such transformation could be biologically or chemically based the detection of DNA adducts does not fully imply that the BaP was bioavailable to soil organisms and hence biologically transformed. However, the detection of DNA adducts on isolated soil DNA would suggest that the biological or chemical transformation products of BaP were able to enter cells of soil organisms and therefore are bioavailable. To test this hypothesis the control experiment performed (addition of BaP metabolites to soil and immediate DNA extraction) showed that transformation products of BaP can bind to ‘naked’ DNA in soil solution during the DNA extraction process and therefore the presence of DNA adducts alone on soil DNA cannot be used to say that BaP metabolites are bioavailable. It is interesting that the BaP metabolites added to soil did interact with naked DNA despite the presence of many other soil components, e.g. dissolved organic carbon, with potential to complex DNA. This finding does limit the potential of the technique to estimate bioavailability but it can be confidently stated that the soil DNA adduct method proposed has excellent potential for detecting the presence of mutagenic compounds in contaminated soil (many different mutagens cause DNA adduct formation). The method could also be used to assess the success of soil remediation strategies, e.g. does bioremediation increase or decrease levels of mutagenic compounds in soil? Further work to establish and optimise the technique with different soil types and other types of mutagenic compounds e.g. nitro-arenes, is required. It would also be of benefit to compare the DNA adduct method to other existing solid phase assays, e.g. passive sampling devices.
100 0 0
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Incubation time (day) Fig. 4. DNA adduct levels during incubation in three BaP contaminated soils: Cruden Bay, Boyndie and Insch ( the values are averages standard errors based on three independent samples).
Acknowledgements We gratefully acknowledge financial support from the BBSRC (No. 13/D18214, UK). Special thanks to Professor David Phillips (Institute of Cancer Research, Sutton, UK) for kind provision of the positive DNA adduct control samples.
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Potential use of DNA adducts to detect mutagenic compounds in soil Guoxiong Hua a, *, Brett Lyons b, Ken Killham c, Ian Singleton a, * a
School of Biology, Institute for Research on the Environment and Sustainability, Devonshire Building, Newcastle University, NE1 7RU, UK CEFAS Weymouth Laboratory, Barrack Road, The Nothe, Weymouth, Dorset, DT4 8UB, UK c School of Biology, Cruickshank Building, University of Aberdeen, AB24 3UU, UK b
A novel DNA adduct assay may provide a potential technique to detect mutagenic compounds in contaminated soil.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 16 June 2008 Received in revised form 24 October 2008 Accepted 29 October 2008
In this study, three different soils with contrasting features, spiked with 300 mg benzo[a]pyrene (BaP)/kg dry soil, were incubated at 20 C and 60% water holding capacity for 540 days. At different time points, BaP and DNA were extracted and quantified, and DNA adducts were quantified by 32P-postlabelling. After 540 days incubation, 69.3, 81.6 and 83.2% of initial BaP added remained in Cruden Bay, Boyndie and Insch soils, respectively. Meanwhile, a significantly different amount of DNA–BaP adducts were found in the three soils exposed to BaP over time. The work demonstrates the concept that DNA adducts can be detected on DNA extracted from soil. Results suggest the technique is not able to directly reflect bioavailability of BaP transformation products. However, this new method provides a potential way to detect mutagenic compounds in contaminated soil and to assess the outcomes of soil remediation. Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved.
Keywords: Polycyclic aromatic hydrocarbons (PAHs) DNA-PAH adducts 32 P-postlabelling assay Soil pollution
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are pollutants of great environmental concern because of their toxic, mutagenic, and carcinogenic properties (Shabad, 1980; Fang and Smith, 2001; Yan et al., 2004; Toyooka and Ibuki, 2007). There is an urgent need to develop methods to monitor such contamination to assess ecosystem and human health risks and to recommend remediation action. Generally, risk analysis is based on chemical analysis (assessing total level of pollutants present), but this does not necessarily reflect actual or potential toxicity caused by pollutants and chemical analysis alone may not detect the presence of all pollutants. Ecotoxicological assays for mutagenic compounds (such as PAHs) in soil have been developed and are extremely useful. However, assays generally involve matrix extraction and subsequent exposure of extracts/leachates to single species (Rocheleau and Cimpoia, 1999; Alexander and Alexander, 2000; Watanabe and Goto, 2000) and this use of extracts (often solvents) may not truly reflect the actual availability and toxicity of pollutants to different soil dwelling species. Useful solid phase toxicity assays have also been developed but these again generally involve a surrogate for actual organisms and may not actually reflect uptake of pollutants observed in living organisms (Bergknut et al., 2007). To overcome
* Corresponding authors. Tel.: þ44 1334 463829; fax: þ44 1334 463384. E-mail addresses: [email protected] (G. Hua), [email protected] (I. Singleton).
the limitations of chemical analysis and current combined solvent extraction/toxicity assays, an improved toxicity method would be able to detect the presence of a wide variety of mutagenic compounds in soils and determine if the compounds present are actually capable of exerting a toxic effect in soil. Such a method could also prove useful for bioremediation where there is potential for microbial transformation to form a wide variety of products that would be difficult and expensive to characterise chemically. Soil DNA can be assumed to be mainly derived from microorganisms, representing a wide variety of species, and is subject to attack by electrophilic reagents due to the presence of nucleophilic sites on its purine and pyrimidine bases, guanine, adenine, thymine, and cytosine. The most active of these sites comprise nitrogen atoms at position 7 of guanine and position 3 of adenine (La and Swenberg, 1996). Electrophilic compounds can react with one of the DNA bases, acting as a nucleophile, causing a covalent modification of the DNA base that is referred to as a DNA adduct. DNA adducts represent excellent biomarkers for determining the extent of damage to genetic material and have long been of interest for understanding the mechanism of carcinogenesis and in the assessment of cancer risk posed by various chemicals or processes (Lamb et al., 2000; Cerniglia and Sutherland, 2001). However, as far as the authors are aware no-one has previously attempted to use DNA extracted from soil living organisms to determine if mutagenic compounds in soil are either present or capable of causing DNA damage in the form of DNA adducts. BaP and other PAHs are activated enzymatically to reactive forms that attack DNA resulting in formation of adducts that lead
0269-7491/$ – see front matter Crown Copyright Ó 2008 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2008.10.026
G. Hua et al. / Environmental Pollution 157 (2009) 916–921
initially to human mutations, carcinogens, and/or developmental toxicants (Harvey, 1991; Jeffrey et al., 1977; Jennette et al., 1977). BaP is widely used as a representative PAH because concentrations of individual PAHs in the urban setting are highly intercorrelated (Perera et al., 2003). BaP inhalation activates the ary hydrocarbon receptor (AhR), which forms an active transcription factor heterodimer with AhR nuclear translocator (ARNT), and induces expression of a group of genes called the [Ah] gene battery, which includes the cytochrome P450 enzyme CYP1A1, CYP1A2 and CYP1B1 genes (Nebert et al., 2000). To produce carcinogenic, mutagenic and cytotoxic effects, BaP is thought to be converted to toxic metabolites through an AhR- dependent mechanism (Hankinson et al., 1991). Among the numerous metabolites identified in BaP-treated cells, BaP-7,8-diol-9,10-epoxide (BPDE) most of effectively forms DNA adducts and serves as a putative carcinogen (Miller and Ramos, 2001; Smithgall et al., 1986, 1988; Penning et al., 1996; Flowers et al., 1997). Therefore, two BaP metabolites: benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (), (anti) and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol () are ideal additives for experiments in this study. This work aimed to examine the potential of using DNA adducts present in soil DNA extracts to determine if mutagenic compounds can be detected in soil without the need for analytical chemistry. This ‘soil DNA adduct assay’ should indicate that the presence of BaP in soil has the potential to cause a biological effect and also that potential mutagens had been formed from BaP. It must be noted that BaP itself does not form adducts but that only metabolites or chemical transformation products of BaP form adducts (Poirier and Santella, 2000; McGrath and Sington, 2000). The individual objectives of the work were to: (a) show that DNA adducts could be detected on DNA extracted directly from soil, (b) determine if DNA adducts could be found in different soil types, and (c) examine the potential of the assay to determine the bioavailability of BaP in different soil types.
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2.3. Soil contamination and incubation Soils were spiked with BaP (300 mg of BaP/kg dry soil). The method used was modified from Pinto (Pinto and Moreno (2002)). Briefly, spiking and incubations were carried out in 250-ml brown glass jars containing the equivalent of 50 g dw soil. The soils were treated with the stock solution of BaP. The required amount of BaP stock (acetone solution) was added to a 20% fraction (10 g) of the soil sample and the jars were closed for 10 min to let the solvent disperse. Thereafter, the solvent was evaporated in a fume hood for 48 h at room temperature, and the sub-sample was mixed with the remaining 80% (40 g) of the soil sample. All mixings were performed thoroughly in each separate jar for 2 min with a metal spatula. Finally, distilled water was added to each jar to give soil samples with moisture contents of 60% water holding capacity (WHC). The treated soil was transferred into jars, and the jars were loosely covered with aluminium foil to maintain moisture content and aeration. All soil samples including the soils spiked with BaP and blank soils were incubated at 20 C in a constant temperature room for 540 days. Soil moisture was maintained by adding deionised water when needed. Soils were not sterilised in any part of this study. All experiments were carried out in triplicate (a separate jar being used for each replicate). 2.4. BaP extraction from soil Soil (0.50 g, fresh weight) was mixed with CH3CN (1.5 ml) in a clean microcentrifuge tube and firstly shaken at 5.5 m/ s in a FastPrepÔ instrument for 30 s. After this, the sample was shaken for another 1 min on a Vortex stirrer. The resulting slurry was centrifuged at 10,000 g for 30 min and passed through a 0. 45-mm Whatman filter. The filtrate was made ready for synchronous fluorescence spectroscopy (SFS) measurement by a 125-fold or 250-fold or 5000-fold dilution with aqueous CTAB micellar medium (7.8 mM), respectively. 2.5. BaP analysis by SFS Quantification of BaP was carried out by using a recently published SFS technique (Hua et al., 2006, 2007). All fluorescence spectra were determined using the synchronous mode on a Cary Eclipse luminescence spectrophotometer (PerkinElmer., Rockville, MD). Each sample was analysed by using the excitation and emission monochrometers at a wavelength difference of 20 nm (Dl ¼ 20 nm). Scan speed was 240 nm/s and PMT voltage was kept at 700 V. The corrected synchronous spectra were recorded in an excitation scale in the range 200–500 nm. High-grade UV quartz cuvettes (10-mm path length) were used for all spectroscopic analyses. 2.6. Isolation of soil DNA
2. Materials and methods
Four different DNA extraction procedures were compared in initial experiments in order to obtain the highest level of DNA (adducts) for all soil samples.
2.1. Chemicals All chemicals were purchased from the Sigma-Aldrich chemical company unless otherwise stated. Two BaP metabolism isomers, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (), (anti) and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol () were purchased from the NCI Chemical Carcinogen Repositories (MRI, Kansas City, MO, USA). PicoGreen reagent was obtained from Molecular Probes (Eugene, OR). Water was purified using a Milli-Q system (Millipore, Bedford, MA). All solvents and chemicals were the highest purity available and used without further purification.
2.2. Soils used Three well characterised soils were used in this work with a wide range of texture and organic matter concentration: Cruden Bay (low sand, high clay and high organic matter), Boyndie (high sand, low clay and low organic matter) and Insch (moderate sand, moderate clay and low organic matter). Their key physical and chemical properties are listed in Table 1. All soils were screened through a 2mm sieve after air-drying/hand-grinding and stored at room temperature before use.
Table 1 Selected physical and chemical characteristics of the tested soil samples. Soil Series pH Sand (%) Silt (%) Clay (%) Texture Organic matter (%)
Cruden Bay Tipperty 6.03 43.44 32.31 24.25 Clay loam 7.38
Boyndie Boyndie 3.61 79.10 14.30 6.53 Loamy sand 3.57
Insch Insch 6.97 57.70 30.79 11.51 Sandy loam 3.75
2.6.1. Method (i) DNA was isolated from all soil samples using a commercial Bio 101 kit according to manufacturer’s instructions (BIO101 Inc., Carlsbad, CA, USA). This is a commercial kit made for direct extraction of bacterial DNA from soil. 2.6.2. Method (ii) The second DNA extraction procedure was modified from previously described protocol (Burgmann et al., 2001). In brief, to a mixture of fresh soil (0.50 g) and glass beads (0.75 g, 425–600 mm diameter) in a microcentrifuge tube, the buffer (1.2 ml, 0.2% hexadecyltrimethylammonium bromide (CTAB), 1 mM dithiotreitol (DTT) and sodium phosphate buffer (0.2 M, pH 8.0), 0.1 M NaCl, 50 mM EDTA) was added. After processing the mixture was centrifuged at 13,000 g for 15 min. The supernatant (700 ml) was transferred to a clean microcentrifuge tube for a final extraction with phenol–chloroform using phenol (350 ml) and CIA (350 ml, chloroform/isoamyl alcohol ¼ 24:1). An extra CIA (700 ml) was added to the upper supernatant to remove phenol and the mixture centrifuged at 13,000 g for 10 min at room temperature. Harvest of DNA was performed by incubation with precipitation solution (750 ml, 20% polyethyleneglycol 6000 and 2.5 M NaCl) at 37 C and centrifugation at 13,000 g. 2.6.3. Method (iii) This DNA extraction procedure was modified from Krsek and Wellington (1999): fresh soil (0.50 g) and glass beads (0.50 g, 0.25 g of 150–212 mm and 0.25 g of 425– 600 mm , acid-washed, SIGMA) were added to TE buffer (1.0 ml, 10 mM Tris–HCl and 1 mM EDTA, pH 8.0). This mixture was then incubated with 5 mg/ml lysozyme (650 ml) at 37 C for 1 h and the supernatant (after centrifugation at 13,000 g) was incubated with 10% SDS (132 ml) for 30 min at 70 C. Harvest of DNA was carried out by incubating the supernatant with 50% PEG-6000 (200 ml) and 5 M NaCl (50 ml) overnight at 4 C. 2.6.4. Method (iv) This procedure followed exactly that described by Sandaa and Enger (1998). This is a direct method aimed at extracting bacterial DNA from soil after treatment with lysozyme, proteinase K and lauryl sulphate (SDS).
G. Hua et al. / Environmental Pollution 157 (2009) 916–921
The resultant DNA from all different methods was re-suspended in highly pure water (50 ml) for further study. The first DNA extraction procedure was used in the experiment to study the formation of DNA-BaP adducts from all three soils. 2.7. Quantitative determination of total DNA DNA in the soil extracts was quantified by using PicoGreen reagent and fluorescence (Sandaa and Enger, 1998). Briefly, DNA extract (2.0 ml), PicoGreen (2.0 ml) and 1X TE (396.0 ml) were mixed in a polypropylene tube, vortexed, and quantified by fluorescence in a Fluoroskan Accent FL (Thermo Labsystems). DNA standards were prepared from bacteriophage l DNA stocks. Fluorescence was measured at 485 nm excitation and 535 nm emission. Both slits were set to 2.5 nm. 1X TE buffer was used as blank sample. DNA yield was calculated as micrograms of total DNA in solution per gram dry weight of extracted soil.
DNA level (µg DNA/g dry soil)
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25 20 15 10 5 0 Method 1
2.8. DNA purification
2.9.
32
P-postlabelling assay of DNA adducts
DNA adducts were determined using the standardised nuclease P1 version of the P-postlabelling assay, as described previously (Phillips and Castegnaro, 1999). Briefly, samples of DNA were digested to deoxyribonucleoside 30 -monophosphates in a total volume of 9.5 ml of digestion mix (6 mU/ml calf spleen phosphadiesterase (Calbiochem, UK), 36 mU/ml micrococcal nuclease, 100 mM sodium succinate, 50 mM CaCl2). The DNA digest (2.0 ml) was diluted and held for the labelling of the normal undamaged nucleotides for subsequent quantification. To the remaining 7.5 ml of digested DNA sodium acetate buffer (1.0 ml, final concentration 40 mM), ZnCl2 (1.0 ml, final concentration 0.2 mM) and nuclease P1 solution (1.0 ml, final concentration 0.31 mg/ml) were added to remove normal nucleotides. The reaction mixture was incubated at 37 C for 30 min, and the reaction then stopped by the addition of Tris solution (1.0 ml). Adducted and normal nucleotides were then labelled separately, but simultaneously, for 30 min with labelling buffer (200 mM bicine NaOH, pH 9, 100 mM MgCl2, 100 mM dithiothreitol, 10 mM spermidine), six units T4 polynucleotide kinase (30 mU/ml; Amersham) and 50 mCi of [g-32P] ATP (>7000 Ci/mmol, ICN). The adducted deoxyribonucleoside-30 -50 -biphosphates present in the sample were then purified and separated from their normal undamaged counterparts using multidimensional anion exchange thin layer chromatography (TLC), on 10 10-cm polyethyleneimine (PEI)-cellulose plates (Camlab, Cambridge, UK). The levels of DNA adduct radioactivity were determined using an AMBIS radioanalytical scanning system (LabLogic, Sheffield, UK). Upon the quantification of both the adducted nucleotides and the normal nucleotides, the relative adduct labelling values were calculated and converted to the number of adducted nucleotides per 108 undamaged nucleotides (Phillips and Castegnaro, 1999). Appropriate negative and positive DNA controls were analysed throughout the studies as described by Harvey and Parry (1998). 32
2.10. Control experiment: potential impact of the DNA isolation method on DNA-BaP adduct analysis This control experiment was carried out using DNA isolation method (i). It was considered that during DNA extraction, BaP metabolites could be released from soil particles into soil solution and interact with DNA released from cells. This could potentially increase the amount of BaP metabolites bound to isolated DNA and result in an ‘over-estimation’ of DNA adduct formation. Essentially this means that the DNA adduct assay may not accurately predict the bioavailability of mutagens but would be able to reveal the presence of potential mutagens in soil. To test the above hypothesis, i.e. to determine the potential interaction of metabolites with naked DNA during extraction, the following experiment was performed: Control experiment (i): two mutagenic microbial benzo[a]pyrene metabolites, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (), (anti) and benzo[a]pyrener-7,t-8,c-9,t-10-tetrahydrotetrol () (5 mg/ g dry soil for each), were added to all soils (uncontaminated) immediately in triplicate prior to DNA extraction.
Method 2
Method 3
Method 4
Fig. 1. Efficacy of DNA extraction procedures evaluated on the basis of DNA yield from three different soil types: Cruden Bay, Boyndie and Insch (the values are averages standard errors based on three independent extractions).
2.11. Data analyses Errors are indicated as standard deviation (SD) of the mean of triplicate measurements. The 32P-postlabelling data followed a normal distribution.
3. Results and discussion 3.1. Selection of suitable DNA extraction method The successful application of molecular techniques to a soil relies on the effective recovery of nucleic acid that is of suitable quantity, quality and purity for all subsequent analyses. There have been a number of published protocols for the extraction of total microbial community DNA from soils (Holben, 1994; Picard et al., 1992; Krsek and Wellington, 1999). The direct cell lysis method is the most routinely used with nucleic acids extracted directly from the soil matrix. Several researchers have compared available nucleic acid extraction techniques in order to assess the quantity and quality of nucleic acids and the overall reliability of published techniques (Zhou et al., 1996; Courtois et al., 2001; Torsvik, 2001; Robe et al., 2003). In general, the results indicate that cell lysis by bead-mill homogenisation in the presence of an organic solvent such as phenol and chloroform yield the greatest quantity of DNA. Considering the above discussion, an efficient and reproducible DNA extraction method was essential for this study to maximise yield and purity of DNA (adducts) from all three soil samples. Attention was also paid to the ease and rapidity of the method. Large differences in amounts of DNA isolated from the different
BaP concentration (µg/g dry soil)
Four treatments were investigated: (i) overnight precipitation with two volumes of 30% polyethylene glycol 6000 (PEG) (wt/wt)/1.6 M NaCl at 4 C; (ii) phenol/chloroform purification (Sambrook et al., 1989); (iii) 30-min incubation with 1/2 volume of 8 M potassium acetate on ice; (iv) 30-min incubation with 1/2 volume of 5% cetyltrimethylammonium bromide (CATB) buffer (wt/wt). DNA pellet after each treatment was collected by centrifugation at 13,000 g for 30 min at 4 C, washed with ice-cold 80% ethanol and centrifuged at 13,000 g for 10 min. Ethanol was completely removed and the pellet was allowed to dry in a vacuum (DNA 120 Speed VacÒ, Thermo Savant) to get a dry DNA adduct sample. DNA adducts were stored at 20 C until 32P-postlabelling analysis for DNA adducts. Of the four treatments tested, PEG precipitation produced DNA of sufficient quality for DNA adduct analysis (results not shown), presumably due to efficient removal of humic materials from extraction solutions and this technique was used in all further work.
Cruden Bay Boyndie Insch
280 Cruden Bay Boyndie Insch
250 220 190 160 130 100 0
74
158
284
365
550
Incubation time (day) Fig. 2. BaP concentrations during incubation in three BaP contaminated soils: Cruden Bay, Boyndie and Insch (the values are averages standard errors based on triplicate samples).
G. Hua et al. / Environmental Pollution 157 (2009) 916–921
soils were clearly observed using different DNA extraction methods (Fig. 1). Method (i) (BIO101 kit) gave the highest yield of DNA from all three soils and was considered to be the most rapid and easy method to carry out. Therefore this method was selected to extract DNA for the study of DNA adduct formation for all soil samples in all further studies. 3.2. BaP extraction and analysis Efficient extraction and analysis in studies of PAHs in soils is essential (Eschenbach et al., 1994; Hechler et al., 1995; Haeseler et al., 1999; Song et al., 2002). The classic Soxhlet extraction of PAHs from soils and sediments has been replaced by faster, less solventconsuming and often-automated techniques, which include one or more extraction cycles. Representative examples of these new extraction techniques are ultrasonic extraction (Sun et al., 1998; Femandez et al., 1999), supercritical fluid extraction (Deuster et al., 1997; Sun et al., 1998), pressurised liquid extraction (Hawthowe et al., 2000; Lundstedt et al., 2000), and microwave-assisted extraction (Pensado et al., 2000; Shu et al., 2000). In this study, a shaking extraction procedure, which provides a simple and low cost effective alternative, was used. Though this technique does not provide the highest extraction efficiency of PAHs in environmental samples, it is, however, suitable for most research purposes examining the relative change in PAH levels such as biodegradation experiments in soils/sediments (Hua et al., 2006). We found BaP recovery when acetonitrile was used as extraction reagent ranged from 80 to 100% dependent, to a great extent, on soil type (data not shown). This shaking method was considered satisfactory for the purposes of the work described here as it was well suited to indicating a decrease in the extractability of BaP over time in the soils used.
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3.3. Level of BaP in soils over time BaP concentrations in the three different soils were determined at day 0, 50, 110, 220, 365 and 540 and results are shown in Fig. 2. A significant decrease in BaP level over time was observed in all soils. After 540 days incubation, 69.3, 81.6 and 83.2% of the original amount of BaP extracted (day 0) was found for Cruden Bay, Boyndie and Insch soils, respectively. This reduction in BaP extracted from all soils could have been due to a combination of biological and chemical transformation of BaP thereby reducing total BaP levels, or due to physico-chemical binding of BaP to soil constituents (e.g. organic matter and clays). This is a common issue with all soil biodegradation studies, i.e. is the reduction in pollutant level observed with time due to biological or chemical transformation or due to physical processes that reduce solvent extraction efficiency? It was thought that the DNA adduct assay used in this work could provide at least a partial answer to this issue. Therefore, if DNA adducts were detected on isolated soil DNA this would mean that BaP has been transformed (chemically or biologically) as BaP itself does not form adducts (Poirier and Santella, 2000). This would mean that any reduction in BaP levels extracted from soil could be in part due to BaP transformation.
3.4. DNA adduct formation in soils over time 32
P-postlabelling is a very sensitive method for DNA adduct detection and it was applied successfully in our study to assay DNA adducts formed on soil DNA derived from BaP transformation products. Representative autoradiographs from 32P-postlabelled DNA are shown in Fig. 3. The images clearly show that DNA adducts were formed in BaP contaminated soils.
Fig. 3. Representative DNA adduct images (autoradiograms of TLC plates) produced by the 32P-postlabelling assay. (A) Insch BaP contaminated soil lacking any distinct DNA adducts after 220 days incubation; (B) Cruden Bay BaP contaminated soil (220 days incubation) showing a number of 32P-labelled DNA adducts (highlighted by arrows); (C) Boyndie BaP contaminated soil after 220 days incubation showing a main BaP adduct close to the bottom left hand corner (red arrow), note similarity to positive control (D); and (D) positive control consisting of labelled DNA (115 nucleotides per 108 undamaged nucleotides) run with each batch.
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3.5. DNA adduct formation with BaP transformation Fig. 4 shows the quantity of DNA adducts formed over time in the three contaminated soils. High levels of DNA adducts were found in Cruden Bay soil at days 220 and 365, while relatively few DNA adducts were found in Boyndie and Insch soils during this time. No DNA adducts were detected after 550 days incubation in all three soils. The results show that the formation of DNA adducts varied in different soils and also varied over time. Importantly, no DNA adducts were found in any soils at time 0 providing evidence that BaP itself is not able to form DNA adducts. Interestingly, the highest amounts of DNA adducts were found in Cruden Bay soil which has a high organic matter and clay concentration. Given that BaP is hydrophobic it could be expected that the chemical would interact with hydrophobic soil organic matter and thus be made unavailable for biological and/or chemical transformation. However, chemical analysis suggests that BaP was removed to a higher extent in Boyndie soil and this is consistent with the higher level of DNA adduct formation found in this soil. It would appear that BaP is available in this soil and this could perhaps be due to the indigenous microbial population present; such a population should be adapted to transformation of high molecular weight material (associated with soil humic matter) and so may be able to access and transform hydrophobic high molecular weight compounds such as BaP. 3.6. Control experiment: potential impact of the DNA isolation technique on the level of BaP related DNA adducts formed
DNA adducts (per 10ex8 undamaged normal uncleotides)
The DNA extraction technique used may have caused the release of previously unavailable BaP metabolites into solution. This could potentially increase the amount of BaP metabolites bound to the isolated DNA causing an increase in DNA adducts formed. Therefore, the DNA adduct technique would result in an over-estimation of potential in situ biological effects. We initially considered that the presence of large amounts of other soil materials such as colloidal clay, cell material and dissolved and particular organic matter would complex the BaP metabolites released during DNA extraction and therefore prevent them binding to naked DNA. However, the addition of two mutagenic microbial benzo[a]pyrene metabolites, to uncontaminated soils immediately prior to DNA extraction resulted in clear DNA adduct formation on isolated soil DNA (Fig. 5A). This clearly demonstrates that BaP metabolites present in soil are able to complex to naked soil DNA and that this effect must be taken into consideration when interpreting results regarding the potential in situ biological effects. Interestingly, three different DNA-PAH adducts were formed even though only two BaP
1000 900 800
Cruden Bay Boyndie Insch
700 600 500 400 300 200
Fig. 5. Control experiment. Production of DNA adducts on isolated soil DNA caused by addition of BaP metabolites to uncontaminated soil immediately prior to DNA extraction.
metabolites were added. This could be due to the chemical instability of the BaP metabolites used causing some other spontaneous products to be formed that were capable of forming DNA adducts or to chemical impurities in the metabolite formulations used. 4. Conclusions This work shows that DNA adducts can be detected on isolated soil DNA and that this only appears to occur when BaP is transformed in soil. Given that such transformation could be biologically or chemically based the detection of DNA adducts does not fully imply that the BaP was bioavailable to soil organisms and hence biologically transformed. However, the detection of DNA adducts on isolated soil DNA would suggest that the biological or chemical transformation products of BaP were able to enter cells of soil organisms and therefore are bioavailable. To test this hypothesis the control experiment performed (addition of BaP metabolites to soil and immediate DNA extraction) showed that transformation products of BaP can bind to ‘naked’ DNA in soil solution during the DNA extraction process and therefore the presence of DNA adducts alone on soil DNA cannot be used to say that BaP metabolites are bioavailable. It is interesting that the BaP metabolites added to soil did interact with naked DNA despite the presence of many other soil components, e.g. dissolved organic carbon, with potential to complex DNA. This finding does limit the potential of the technique to estimate bioavailability but it can be confidently stated that the soil DNA adduct method proposed has excellent potential for detecting the presence of mutagenic compounds in contaminated soil (many different mutagens cause DNA adduct formation). The method could also be used to assess the success of soil remediation strategies, e.g. does bioremediation increase or decrease levels of mutagenic compounds in soil? Further work to establish and optimise the technique with different soil types and other types of mutagenic compounds e.g. nitro-arenes, is required. It would also be of benefit to compare the DNA adduct method to other existing solid phase assays, e.g. passive sampling devices.
100 0 0
42
110
220
365
550
Incubation time (day) Fig. 4. DNA adduct levels during incubation in three BaP contaminated soils: Cruden Bay, Boyndie and Insch ( the values are averages standard errors based on three independent samples).
Acknowledgements We gratefully acknowledge financial support from the BBSRC (No. 13/D18214, UK). Special thanks to Professor David Phillips (Institute of Cancer Research, Sutton, UK) for kind provision of the positive DNA adduct control samples.
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