Novel Use of a Whole Cell E. coli Bioreporter as a Urinary Exposure

Dec 11, 2008 - Laboratory, Prospect Place, The Hoe, Plymouth, U.K.,. PL1 3DH, and School of Biology, Georgia Institute of. Technology, 311 Ferst Drive...
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Environ. Sci. Technol. 2009, 43, 423–428

Novel Use of a Whole Cell E. coli Bioreporter as a Urinary Exposure Biomarker C E R I L E W I S , * ,† S I H A M B E G G A H , ‡ CHRIS POOK,† CARLOS GUITART,§ CLARE REDSHAW,| JAN ROELOF VAN DER MEER,‡ JAMES W. READMAN,§ AND TAMARA GALLOWAY† School of Biosciences, Hatherly Laboratories, University of Exeter, Prince of Wales Road, Exeter, U.K., EX4 4PS, Department of Fundamental Microbiology, Université de Lausanne, 1015 Lausanne, Switzerland, Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth, U.K., PL1 3DH, and School of Biology, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, Georgia 30332

Received May 15, 2008. Revised manuscript received October 16, 2008. Accepted October 30, 2008.

Bacterial bioreporters have substantial potential for contaminant assessment, but their real world application is currently impaired by a lack of sensitivity. Here, we exploit the bioconcentration of chemicals in the urine of animals to facilitate pollutant detection. The shore crab Carcinus maenas was exposed to the organic contaminant 2-hydroxybiphenyl, and urine was screened using an Escherichia coli-based luciferase gene (luxAB) reporter assay specific to this compound. Bioassay measurements differentiated between the original contaminant and its metabolites, quantifying bioconcentration factors of up to one hundred-fold in crab urine. Our results reveal the substantial potential of using bacterial bioreporter assays inreal-timemonitoringofbiologicalmatricestodetermineexposure histories, with wide ranging potential for the in situ measurement of xenobiotics in risk assessments and epidemiology.

Introduction Exposure measurement is a vital analytical tool in environment and human health risk assessment, forming a basic component of epidemiology. Faced with an increasingly polluted environment and rising incidence of chemicals with the potential to harm environment, wildlife, and human health, there is an increasing need for new, rapid, low cost techniques for large scale exposure studies. One emerging technology with great potential for contaminant assessment involves the use of whole cell living bacterial bioreporters. These are genetically engineered bacteria designed to react to the presence of chemical signals with the rapid production of an easily quantifiable marker protein (1-3). Bacterial bioreporters have been developed for a large range of analytes and study areas, with increasing focus on compounds of environmental concern (1, 4, 5). Their real world application, * Corresponding author phone: 0(44) 1392 263436; fax 0(44) 1392 263700, e-mai: [email protected]. † University of Exeter. ‡ Universite´ de Lausanne. § Plymouth Marine Laboratory. | Georgia Institute of Technology. 10.1021/es801325u CCC: $40.75

Published on Web 12/11/2008

 2009 American Chemical Society

however, is currently impeded by poor sensitivity to environmentally relevant concentrations and a lack of information regarding matrix interferences. Traditional chemical techniques including mass spectrometry involving gas or liquid phase chromatography can detect pollutants in the nanomolar range, but most bacterial assays except those for heavy metals perform inaccurately at analyte concentrations below 0.1 µM (6). Solvent extraction and concentration of samples could improve sensitivity, but organic solvents generally interfere with the biological function of the bioreporters. In human exposure assessment, epidemiologists often exploit the fact that the large pool of endogenous metabolites in a tissue or body fluid also changes in response to chemical exposure, and that urine is often one of the most informationrich biofluids available (7). This raises the possibility of an alternative application of bacterial bioreporters, which utilizes the observed tendency of organic contaminants to bioaccumulate in the tissues and body fluids of biological receptors, to overcome their lack of sensitivity to environmentally realistic concentrations. Here, we test whether a chemical specific bacterial bioreporter can be applied as a urinary exposure biomarker, and, second, whether the natural bioaccumulation of chemicals in the urine of animals improves the method detection limit for the bioreporter assay. Hydrophobic compounds may be bioaccumulated by many orders of magnitude from surrounding water bodies (8, 9), and biomonitoring of sessile filter feeding species such as marine mussels is widely applied in environmental biomonitoring programs (e.g., the CCMA Musselwatch Program, the longest continuous contaminant monitoring program in U.S. coastal waters [http://ccma.nos.noaa.gov/stressors/pollution/ nsandt/]. The biotransformation and metabolism of all but the most inert organic compounds, to form hydrophilic conjugated metabolites, has deterred the application of biosensors to these media, as in general, direct and specific binding of the parent compound forms the basis of the regulatory protein’s mechanism of recognition (10). Using urine for exposure assessment has the added advantage of identifying the bioavailable fraction of the contaminant in question, since this is, by definition, the fraction producing the deleterious effects. As a test in case, we investigate the application of an Escherichia coli-based gene reporter assay (the bacterial bioreporter) to a biological matrix, using the organic contaminant 2-hydroxybiphenyl (2-HBP) as a test compound and urine collected from the common shore crab, Carcinus maenas, as a test matrix. 2-HBP is an antimicrobial agent with wide commercial use as a disinfectant, fungicide for citrus fruits, and component of household detergents (11). As such, there is wide human exposure to these compounds. In addition, historical release of 2-HBP as a byproduct of the synthesis of phenols and in the microbial desulfurization of dibenzothiophene in fossil fuels has led to continued contamination of ground and surface waters. Evidence suggests that 2-HBP is metabolically activated by the cytochrome P450 enzyme system to form quinones and semiquinones, conjugated to glucuronide (2-HBP-G) and sulfate (2-HBP-S) and excreted in the urine (12-14). Studies of 2-HBP exposure in humans, mice, and rats demonstrate that nearly all of an administered dose of 2-HBP is eliminated in the urine (11, 12). Metabolites of 2-HBP have been associated with DNA single strand breaks (15) and dose-dependent hyperplastic and neoplastic responses in the urinary bladder of rats (16). As such, 2-HBP is classified as an irritant and potential carcinogen. VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Schematic outline of the HbpR-activated reporter gene cassette in Escherichia coli drawn to scale (kb). Constitutive expression of hbpR under its native promoter produces the transcription activator HbpR which binds to a site nearby the σ54-dependent PC promoter. Addition of 2-HBP to the reporter cells will cause a biochemical interaction with HbpR, which then activates the RNA polymerase-σ54 complex stalled at the PC promoter. Transcription from the PC promoter will result in expression of the luciferase genes (luxAB) and production of bioluminescence. In most bacterial bioreporters, the sensory function of a regulatory system in the bacterial cell is exploited to detect analyte presence and drive expression of a specific reporter gene, such as bacterial luciferase or green fluorescent protein, which can then be measured using luminometry or fluorometry, respectively. Certain bacterial strains are able to use hydroxybiphenyls as sole carbon and energy sources (17). In the case of Pseudomonas azelaica, regulation of 2-HBP degradation is mediated by the regulatory gene hbpR which encodes a transcriptional activator protein of the so-called NtrC family, regulatory proteins which activate RNA polymerase bound to the auxiliary sigma factor σ54 (18). The HbpR protein activates transcription from a promoter (PC) in front of the first gene for 2-HBP degradation, hbpC, following interaction of 2-HBP with the protein’s amino terminal A domain (19). This activation cycle was used to reconstruct a quantitative gene reporter assay in E. coli DH5R (Figure 1) in which the hbpR gene controls and activates expression of the luxAB luciferase reporter genes of Vibrio harveyi (20) via the PC promoter in the presence of 2-HBP (19). The common shore crab Carcinus maenas is widely distributed around the shores and estuaries of northern Europe, including areas of high anthropogenic impact and industrial pollution. Crabs are able both to bioaccumulate and biotransform aromatic hydrocarbons from the surrounding medium (21) evidenced by the excretion of conjugated metabolites in the urine, allowing their use as bioindicator species. Urine samples are easily collected on site, and the analysis of bioaccumulated contaminants in urine from crabs inhabiting environments contaminated with petroleum and combustion-derived aromatic hydrocarbons has previously been demonstrated as a viable means of exposure assessment using nonspecific immunometric and fluorescence-based analysis methods (21).

Materials and Methods Exposures and Urine Collection. Green, adult male intermoult shore crabs, Carcinus maenas (carapace width 50-70 mm) were collected using baited traps on an incoming tide from estuaries in South Devon, UK. Animals were maintained in holding tanks containing well aerated, filtered (5 µm) seawater (FSW) at 15 °C, until use in the experiments. Crabs (total n ) 15 for each treatment over two exposures) were exposed in individual glass aquaria to 2-HBP at 0.12 or 1.2 µM, chosen to span environmentally realistic exposures (22), and the lowest exposure concentration being three times lower than the bioreporter method detection limit, for up to two weeks. Crabs were fed 1 g of irradiated cockle each, every two days throughout the exposure period, and their water (or treatment solution) was changed 24 h after feeding each 424

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time. Urine was collected nondestructively by applying gentle pressure to the base of the antennal gland and collecting urine into a sterile pipet every 48 h. Samples were immediately frozen and stored at -20 °C in siliconised microcentrifuge tubes until analysis. Determination of 2-HBP in Urine Samples by HPLC-F and GC-MS. Chemical analysis of urine samples from exposed and unexposed crabs was performed to quantify uptake and excretion of 2-HBP and determine any metabolic transformation of the parent compound. HPLC fluorescence of diluted (10%) crab urine in a matched matrix was performed using a Kontron 325 gradient pump (0.5 mL min-1 flow rate) and a Kontron 360 autosampler (20 µL injection volume) supplying a Waters reverse-phase C18 µBondapak analytical column (10 µm particle size, 3.9 mm internal diameter × 300 mm length) fitted with a µBondapak guard column (10 µm particle size, 3.9 mm internal diameter × 20 mm length). Detection was via a Varian 9070 fluorescence detector (excitation λ: 248 nm, emission λ: 330 nm). The gradient elution conditions were as follows: 2 min hold at 75% A; 18 min ramp to 60% B; 20 min ramp to 90% B; 10 min ramp to 95% B; 5 min ramp to 25% B; where A ) deionized water and B ) 0.1% formic acid in methanol. Retention times and calibration were obtained using authentic 2-HBP standards prepared in the linear range 10 µg L-1 to 500 µg L-1. For GC-MS analyses, 2-HBP was isolated from urine by solid-phase extraction (SPE). A 50 µL amount of raw urine was diluted to 1 mL with deionized water (DIW) and then passed through an SPE cartridge (1 g of Isolute C18, International Sorbent Technologies) preconditioned with 5 mL of ethylacetate, methanol, and MilliQ, sequentially. Following the sample extraction, the cartridges were dried for 30 min under a vacuum and were then eluted with 8 mL of ethyl acetate with the eluent passing through an in-line NaSO4 cartridge to eliminate residual H2O. The extract volume was reduced under a gentle stream of pure nitrogen and then transferred to vials to provide a final volume of 300 µL. The internal standard (BPA-d16) was added before the volume was adjusted to approximately 500 µL by adding 200 µL of the derivatization reagent MTBSTFA (Sigma-Aldrich, UK). This mixture was then heated at 70 °C for 2 h. The mean recovery obtained from spiked (20 µM) urine (taken from unexposed crabs) was 74 ( 8% (n ) 2). A linear calibration range for the target analyte (2-HBP) was obtained from 1.8 µM to 18 µM, and analytical data was corrected for method blanks. Analyses were performed using an Agilent 6890 gas chromatograph interfaced with an Agilent 5973N mass spectrometer in EI mode (70 eV). A capillary column HP-MS 5 (30 m × 0.25 mm i.d., 0.25 µm film thickness) was used. The injection volume was 2 µL in splitless mode (280 °C, purge time 0.75 min). The helium carrier flow was maintained at 1 mL min-1. The oven temperature program was 1 min at 70 °C, 30 °C min-1 to 150 °C, 6 °C min-1 to 275 °C, 3 °C min-1 to 300 °C, 10 °C min-1 to 310 °C, and 310 °C was held for 6 min. A SIM program was designed to acquire signals for both quantification and confirmatory m/z fragments for the target analytes (211 and 227 for 2-HBP and 178 and 217 for BPA-d16 derivatives, respectively). The ion source and quadrupole analyzer temperatures were fixed at 230 and 150 °C, respectively. LC-MS Analysis of Urinary Metabolites. Previous studies of 2-HBP exposure in humans, mice, and rats have demonstrated that nearly all of an administered dose of 2-HBP is eliminated in the urine as metabolites, conjugated to mainly glucose or sulfate groups (12, 13). LC-MS analysis of urine from exposed crabs was therefore performed to fully elucidate the metabolic pathway of 2-HBP for Carcinus maenas. Low flow (3 µL min-1) infusion via an electrospray interface fitted to a Finnigan MAT LCQ (Thermo Finnigan, San Jose, CA)

quadrupole ion trap mass spectrometer was used to identify metabolites present within the four largest peaks, collected as fractions, from the eluent flow of the HPLC fluorescence system described above. Sequential product ion fragmentation and condition optimization of each fraction was performed, in both positive and negative ionization mode. The most abundant product ion at each stage was selected for sequential fragmentation, until no further MSn transitions could be obtained. This allowed the generation of ESI fragmentation pathways, alongside development of optimized MSn conditions for each compound. Full scan range of 50-2000 m/z was used. LCQ tune software was used for data acquisition and processing. Recording of spectral data for 1 min time periods was started once stable spectra were obtained. An HPLC gradient pump (Dionex P580 quaternary pump) and autosampler (5 µL injection volume; Dionex ASI-100 automated sample injector) connected to the ESI-MSn detector was used for high flow full MS analysis and Data Dependent Mass Spectrometry (DDMS) which includes MS2 fragmentation, using the optimized MSn conditions. High flow analysis was conducted on whole crab urine extracted into methanol and matrix-matched using appropriate volumes of eluents prior to analysis. Detection of Urinary 2-HBP Metabolites Using Bioreporter Assay. The bacteria strain used for sensing and reporting of 2-HBP was Escherichia coli DH5R, carrying the plasmid pHBP109 - strain number 868 of the DMF-UNIL collection (19). This plasmid contains the gene hbpR (Genbank accession number U73900) of Pseudomonas azelaica under control of its native promoter, and, divergently oriented, the luxAB genes for bacterial luciferase under control of the HbpR activatable PC promoter. In the presence of 2-HBP, HbpR will thus activate the synthesis of bacterial luciferase, which can be measured by luminometry. Luciferase activity is proportional to the 2-HBP concentration within the range of 0.5 to 8 µM, above which signal saturation occurs. Strain 868 cells were prepared by inoculating an isolated colony from an agar plate in 5 mL of Luria Broth (LB) medium containing 100 µg of ampicillin per milliliter and culturing overnight at 37 °C with shaking. The next morning 0.1 mL of this culture was transferred to 5 mL of fresh LB in a sterile glass vial, which was incubated with shaking at 37 °C. At a culture turbidity of 0.4 at 600 nm wavelength, cells were harvested by centrifugation for 10 min at 3000 rpm and 25 °C and resuspended in 5 mL of MOPS medium (0.5 g NaCl, 1 g NH4Cl, 9.8 g of 3-(N-morpholino)propanesulfonic acid, 59 mg Na2HPO4 · 2H2O, 45 mg KH2PO4, 2 mM MgCl2, 0.1 mM CaCl2 and 2 g of glucose per liter, pH 7.0). Immediately before preparation of the assay, cells were further diluted 20-fold in MOPS medium to achieve a cell culture turbidity of 0.02 at 600 nm. Assay mixtures (150 µL of diluted cell suspension and 50 µL of sample or standard) were prepared in 96-well plates and incubated at 30 °C and 500 rpm on an orbital microplate shaker. After 2 to 3 h of incubation time, 20 µL of 20 mM n-decanal was added to the assays (to give a final concentration of 2 mM), mixed, and then incubated at room temperature for 3 min. The bacterial luciferase activity was then measured as previously described (23) using a Microlumat LB960 luminometer as the integrated value over a 1 s period. Samples and standard solutions were all assayed in triplicates. In the bioreporter assays, actively growing strain 868 bioreporter bacteria were incubated with crab urine (from control or exposed animals potentially containing 2-HBP) for a period of up to 2 h. Light emission from the bioassay after the induction time was compared to a set of calibration assays with known 2-HBP concentrations carried out simultaneously. The following variations on this assay were

FIGURE 2. HPLC-F chromatograms of (A) a 3 µM 2-HBP standard and (B) urine from a crab exposed to 1.2 µM1 2-HBP. performed: (1) To test the proportionality of the light emission reaction as a function of 2-HBP concentration in the bioreporter assay, we incubated E. coli strain 868 with increasing 2-HBP concentrations, either in the presence or in the absence of crab urine in mineral-buffered medium with glucose (MOPS medium). (2) To test whether the bioreporter assay could detect exposure to 2-HBP in the urine of Carcinus maenas, we incubated E. coli strain 868 with urine from the crabs exposed for 1 or 2 weeks to either low (0.12 µM) or high (1.2 µM) dose 2-HBP treatments. (3) Since 2-HBP is metabolized by Carcinus maenas during detoxification and excretion (see Results), an enzyme pretreatment was used on the urine samples. Enzymatic deconjugation of sulfate moieties was achieved using a sulfatase enzyme preparation from Helix pomatia (Sigma UK). Briefly, 20 µL of urine was added to 20 µL of 0.1 M citrate buffer pH 5.0, 10 µL of MOPS medium pH 7.0, and 2.7 µL of enzyme preparation equating to 5 units of sulfatase activity. The mixture at pH 6.2 was incubated at 37 °C for 18 h after which the urine-digested sample (50 µL) was directly added to the bioassay. Enzyme digested urine was also prepared for HPLC and analyzed as described previously. To test for possible bioreporter inhibition, assays were spiked with 2 µM 2-HBP (final concentration), and the observed increase of the light signal was compared to the expected increase from the calibration curve with 2-HBP only.

Results and Discussion Chemical Analysis of Urinary Metabolites. HPLC-fluorescence analyses of urine samples from the 2-HBP-exposed crabs revealed a series of peaks (Figure 2) that were not present in the urine of unexposed control crabs. None of these peaks, however, corresponded to the retention time of the 2-HBP standard compound, suggesting that the crabs were metabolizing all of the 2-HBP. GC-MS analyses of derivatized urine extracts from crabs exposed to 1.2 µM 2-HBP revealed a concentration of parent 2-HBP in the urine of less than 0.3 µM. This result was comparable to the average bioreporter assay measurements of 0.5 µM (range from 0 to 0.7 µM) and represents 25% of the exposure dose but was much lower than reference measurements taken using direct fluorescence of the urine at the approximate wavelength for 2-HBP (a less specific analysis, see ref 21 for details). Further analysis using HPLC-ESI(-)-MS revealed two peaks (Figure 3) with m/z of 265 (compound A) and 249 (compound VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Standard calibration curves for the E. coli 2-HBP bioreporter showing luciferase expression, measured as relative light emission units, as a function of increasing 2-HBP concentration in buffer solution (- urine) and buffer with crab urine added (+ urine). The inset shows that at 2-HBP concentrations below 1 µM the proportionality of light emission is exponential. FIGURE 3. LC/MSn analysis of urine extract from crabs exposed to 200 µg L-1 (1.2 µM) of 2-HBP. (A) Total ion chromatogram showing the peaks of metabolite A at 24.7 min and B at 29.3 min and their respective structures. (B1 and B2) MS2 scans from the two metabolite peaks showing the fragmentation pathways of each molecular ion. Metabolite A loses a deprotonated sulfite moiety of m/z 80 to leave a primary product ion of m/z 185. The example shown here is 2,4′-dihydroxybiphenyl; however, the precise isomeric identity of this compound cannot be determined. Metabolite B also loses a deprotonated sulfite moiety to leave the deprotonated parent compound, 2-hydroxybiphenyl, of m/z 169. B), respectively. MS/MS (including low flow ESI-MSn of isolated fractions) of the smaller first peak produced a primary product ion with an m/z of 185 and a secondary product ion with an m/z of 80. Vertebrate studies have shown that biotransformation of 2-HBP by mono-oxygenase enzymes can yield several isomers of dihydroxybiphenyl or phenoxyhydroquinone as metabolites of 2-HBP (12), all of which have molecular masses of 186 Da and undergo deprotonation to produce the primary product ion found here. Similarly, a sulfite group has a molecular weight of 81 Da, corresponding to the secondary product ion. This indicates that compound A is either a dihydroxybiphenyl sulfate (DHB-S) or a phenoxyhydroquinone sulfate (PHQ-S). Equivalent analyses of the larger second peak (with an m/z of 249) yielded a primary product ion m/z of 169 and a secondary product ion m/z of 80, corresponding to hydroxybiphenyl and sulfite, respectively, confirming the identity of compound B, the major metabolite, as hydroxybiphenyl sulfate (2-HBP-S). Quantification of the metabolites was performed by HPLC using enzymatic deconjugation in the absence of authentic metabolite standards. Bioreporter Analysis of Crab Urine. We initially tested the performance of the E. coli 2-HBP biosensor in the presence of crab urine, collected from healthy individuals, to check for matrix effects by spiking the urine directly with 2-HBP. The biosensors provided a linear light emission response in the concentration range of 0-8 µM 2-HBP, either in the presence (R2 ) 0.993) or absence (R2 ) 0.996) of crab 426

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urine (Figure 4). At concentrations below 1 µM, the light emission was no longer linearly proportional to 2-HBP concentration but was better represented by an exponential relationship. The light emission produced by the biosensor cells in the presence of 50% (v/v) crab urine in the assay was between 10 and 20% different from the calibration assay without urine, but the effect was not consistently lower or higher (Figure 4), indicating that crab urine can be used in the assay without significantly affecting the biosensor response. Precision, taken as the average standard deviation over all calibration points, was 3%, while the detection limit, taken as the interpolated concentration at the relative light emission in the assay of the blank plus three times the standard deviation of the blank measurement, was 0.4 µM 2-HBP. Urine samples from both control and crabs exposed to the lowest 2-HBP concentration were found to produce no significant light emission in the bacterial assay (Figure 5), whereas those exposed to the highest concentration produced luciferase activity equivalent to ≈0.5 µM 2-HBP (Table 1). This indicates that either no uptake had occurred, or that metabolic transformation of the 2-HBP to conjugated metabolites rendered the bioreporter unresponsive. Since the full chemical analysis of the crab urine confirmed the extent of uptake, the complete metabolism of 2-HBP by the crabs, and the presence of these metabolites in their urine samples, it can be concluded that the bioreporter does not respond to metabolites of 2-HBP. As preliminary tests of urine from crabs exposed to 2-HBP indicated a lack of response of the bioreporter to the metabolites, an enzyme incubation step was included to determine whether the conjugate groups could be removed from the 2-HBP using sulfatase and β-glucuronidase enzyme pretreatment. Initial tests confirmed that the sulfatase/βglucuronidase mixture at the applied concentrations itself did not alter the 2-HBP induced light emission of the bioreporter bacteria by more than 5%. Urine samples from crabs exposed to 1.2 µM of 2-HBP in seawater and incubated for 18 h with 5 U sulfatase and 67 U of β-glucuronidase at 37 °C showed a very significant response in the bacterial assay (Figure 5B), demonstrating that the metabolites had

FIGURE 5. (A) Pathway for 2-HBP metabolism in Carcinus maenas and deconjugation by the enzyme glucuronidase. (B) Urinary 2-HBP content from Carcinus maenas individuals exposed to 0.12 µM and 1.2 µM 2-HBP, measured using the pHBP109 bioreporter assay with and without the β-glucuronidase/sulfatase enzymatic pre-treatment.

TABLE 1. Summary of 2-Hydroxybiphenyl or Metabolite Equivalent Concentrations in Crab Urine As Determined by the Bacterial Bioreporter Compared with Actual Concentrations Measured for the Same Individuals (all data as mean ± standard deviation)

sample

biosensor measurement of 2-HBP equivalents, µM actual concentration no enzyme enzyme added 2-HBP µM

unexposed urine 0.0 ( 0.0 1.76 ( 2.14 0.12 µM 2-HBP 0.44 ( 0.17 16.06 ( 9.23 1.2 µM 2-HBP 0.17 ( 0.32 68.02 ( 19.64

2.53 ( 1.88 24.21 ( 12.90 98.00 ( 23.40

been deconjugated to re-form the parent compound, 2-HBP. Individual crab urine samples were then incubated with the enzyme mixture, added to the bioreporter assay without further treatment, and the light responses were used to calculate a 2-HBP ‘equivalent’ concentration by interpolation from a calibration curve with pure 2-HBP. Crabs exposed to 0.12 µM 2-HBP produced 2-HBP equivalent concentration in the bioassay of 16.06 µM ( 9.23 (SD), representing a bioaccumulation factor (Bf) of around 100, while those exposed to 1.2 µM 2-HBP had 68.02 ( 19.64 (SD), equivalent to a Bf of ≈50-fold (Figure 5 and Table 1). This bioconcentration factor showed a large interindividual variation between crabs (Figure 5B), as confirmed by analytical chemistry. Data was not normalized for crab size which may reduce this variation. HPLC confirmed that the enzyme incubation step had completely deconjugated the 2-HBP metabolites, and that the biosensor readings fell within the actual concentrations of 2-HBP present within the individual urine samples for both treatments (Table 1). Potential Applications. The findings presented here demonstrate that the E. coli bioreporter assay not only allowed quantitative determination of the pollutant metabolites in the crab urine but differentiated clearly between nonmodified parent pollutant and conjugated metabolites. This, therefore,

allows extrapolation to an exposure history of the organism in that environment. Such exposure history determinations may have an enormous potential for in situ environmental monitoring purposes. In particular, our work illustrated the ease and potential of using marine invertebrates as ‘bioconcentrators’ in combination with simple bacterial gene reporter assays, since they accumulate environmental pollutants within their body fluids and tissues to concentrations that are within the detection limits of bioreporters. The addition of a simple enzyme deconjugation step enabled the biosensor to measure exposure to the parent compound with high accuracy. Enzymatic deconjugation did prolong the total time needed for the bioassay. Whereas the biosensor assay itself takes 2-3 h incubation time before bioluminescence is measured, enzymatic pretreatment of the urine sample increases this by 18 h. Urine is a widely used matrix in human epidemiology and vertebrate wildlife exposure assessment (24, 25) and could, therefore, likewise be assayed in combination with the cheap bacterial bioreporter assays specific to a wide variety of contaminants (e.g., arsenic (26)). Bacterial bioreporters now exist with target specificities for around 20% of priority pollutants (5). Portable assays have been designed for on-site monitoring using freeze-dried bioreporter strains (27), and bioreporter cell arrays have been developed for high throughput analyses (28, 29). There are a range of initiatives and legislative requirements currently in place that force governments worldwide to monitor inputs and temporal trends within the marine environment and investigate the subsequent fate and environmental impact of such inputs (30). Linking environmental pollution with human health impacts is also currently a major research focus among many governmental and research funding bodies. The development of bacterial whole cell bioreporter platforms that can be used for continuous, real-time determination of compounds of environmental concern in biological matrices could therefore be of major benefit in realizing the aims of the many national and international VOL. 43, NO. 2, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Acknowledgments This work was funded by the European Commission through the FP6 FACEiT programme (FP6-2004-Global-3.3.111.3.1), contract 018391. Carlos Guitart was supported by an EIF Fellowship (CT2005-023699) through the European Marie Curie Programme. Thanks to Prof. Steve Rowland and Edwin Sonnenveld for their comments on the manuscript and Trevor Worsey for technical support.

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