The detection of biomarkers of genotoxin exposure in the European flounder (Platichthys flesus) collected from the River Tyne Estuary

Mutation Research 446 Ž1999. 111–119 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres

The detection of biomarkers of genotoxin exposure in the European flounder žPlatichthys flesus / collected from the River Tyne Estuary B.P. Lyons a

b

a,)

, C. Stewart a , M.F. Kirby

b

The Centre for EnÕironment Fisheries and Aquaculture Sciences (CEFAS), Lowestoft Laboratory, Pakefield Road, Lowestoft, Suffolk NR33 OHT, UK The Centre for EnÕironment Fisheries and Aquaculture Sciences (CEFAS), Rememberance AÕenue, Burnham-on-Crouch, Essex CM0 8HA, UK Received 11 May 1999; received in revised form 4 August 1999; accepted 10 August 1999

Abstract The Tyne Estuary ŽNorth East England. is known to contain elevated levels of polycyclic aromatic hydrocarbons ŽPAH., compared with other less industrialised English waterways. Previous studies suggest that such contamination is responsible for the toxicity detected in invertebrate bioassays conducted on water and sediment samples collected from the Tyne. Here we present data from a biomonitoring study using hepatic DNA adducts Ž32 P-postlabelling assay. and bile metabolites Žsynchronous fluorescence spectrometry. to investigate genotoxic exposure in flounder Ž Platichthys flesus . collected from three sites ŽScotswood, Newcastle and Redheugh. along the Tyne Estuary. Flounder were also collected from a clean reference site, the Alde Estuary. Levels of bile metabolites Žmg kgy1 wet weight 1-OH pyrene equivalents. were elevated in flounder caught from the Tyne ŽScotswoods 22 247 " 3408; Newcastles 14 572 " 1888; Redheughs 21 872 " 2935. compared with those collected from the Alde Ž632 " 56.. The levels of DNA adducts Žadducted nucleotides per 10 8 normal nucleotides. were also elevated in Tyne flounder ŽScotswoods 24.6 " 3.2; Newcastles 34.4 " 3.7; Redheughs 27.6 " 6.3. compared with fish collected from the Alde Ž10.1 " 4.8., suggesting that a proportion of the bioavailable PAH was being converted into genotoxic metabolites. All DNA adduct profiles in flounder collected from the Tyne consisted of diagonal radioactive zones of radiolabelled adducts, which were not present in fish sampled from the Alde. The in vivo dosing of flounder with benzow axpyrene ŽBaP. to produced DNA adducts in similar chromatographic positions to the diagonal radioactive zones in the Tyne caught flounder are also described. q 1999 Elsevier Science B.V. All rights reserved. Keywords: DNA adducts; 32 P-postlabelling; Bile metabolites; Synchronous fluorescence spectrometry ŽSFS.; Flounder Ž Platichthys flesus .; Tyne Estuary

)

Corresponding author. Tel.: q44-1502-524420; fax: q44-1502-513865; e-mail: [email protected]

1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 1 5 4 - 0

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1. Introduction In recent years there has been increasing concern over the possible degradation of our estuarine environments from chemical pollution. This has led to increased efforts attempting to evaluate the causal relationships between contaminant exposure and observable biological effects in aquatic organisms w1x. Previous studies have demonstrated that sediments within the River Tyne Estuary Žsee Fig. 1. contain levels of contaminants capable of inducing toxicity in a number of invertebrate bioassays w2,3x. Furthermore, we have recently shown that the mixed function oxygenase ŽMFO. system of the benthic flatfish flounder Ž Platichthys flesus . is significantly elevated in the Tyne compared with less contaminated estuaries w4x. While the MFO system is essential for the detoxification of a diverse array of xenobiotics, including polycyclic aromatic hydrocarbons ŽPAHs. and polychlorinated biphenyls ŽPCBs., its induction may also produce deleterious side effects. For example while the majority of contaminants are rendered less harmful, others Žcertain 4–5 ringed PAH. may form genotoxic metabolites with potentially mutagenic andror carcinogenic properties. Previous studies have indicated that the levels of genotoxin-DNA adducts in selected aquatic species could be used as biomarkers of environmental contamination w5–8x. DNA adducts are covalently bound addition products formed when electrophilic chemical species attack the nucleophilic sites in DNA. Furthermore, the quantitative analysis of DNA adducts enables the determination of the biologically relevant levels of exposure to genotoxic chemicals, so integrating the physiological factors such as absorption, metabolism and detoxification involved in genotoxin adduct formation. Here we describe the use of the 32 P-postlabelling assay w9,10x to detect the presence of aromatic andror hydrophobic DNA adducts in liver tissue of the benthic flatfish P. flesus collected from three sites situated in the River Tyne Estuary ŽScotswood, Newcastle and Redheugh.. Levels of bile metabolites, as determined by synchronous fluorescence spectrometry ŽSFS., were also assessed to provide an indication of uptake and excretion of PAH in P. flesus. Fish were also collected from the Alde Estuary ŽSouth East England., an area known to receive

minimal anthropogenic input and therefore regarded as a clean reference site. The results of these studies indicate that P. flesus collected from the Tyne had elevated levels of DNA adducts and bile metabolites compared with those specimens captured from the Alde. Additionally, laboratory studies were performed to examine the induction of DNA adducts in P. flesus following exposure to the ubiquitous environmental carcinogen benzow axpyrene ŽBaP..

2. Material and methods 2.1. Laboratory DNA adduct induction study P. flesus used in the laboratory experiments were collected from the Alde River in Suffolk ŽFig. 1.. The Alde was chosen as a clean location with minimal industrial, agricultural or domestic inputs Žsee Section 2.2.. Fish were kept in 200-l tanks for 2 weeks prior to the experiment to acclimatise them to laboratory conditions. Fish were injected intraperitoneally ŽI.P.. with a single dose of the carcinogen BaP, at levels of 1 and 20 mg kgy1 body weight Ždissolved in corn oil.. A group of fish, serving as the carrier controls, was injected with corn oil alone. Ten fish were sacrificed at the initiation of the experiment and then 4–5 fish were sampled from each treatment group 2, 4, 8, 14 and 21 days post exposure. Fish were killed by cervical dislocation, and the livers removed and immediately placed in liquid nitrogen. 2.2. Field sampling and study area Sampling locations are shown in Fig. 1. P. flesus were caught using 2–3 m beam trawls from Environment Agency research vessels during October 1997. Trawls were towed at 3–4 knots for 20–30 min. The River Tyne Estuary is known to receive considerable amounts of anthropogenic inputs, including municipal sewage discharge Ž268 652 m3 dayy1 . and industrial effluent Ž1 162 931 m3 dayy1 . w11x. This is thought to contribute significantly to the high levels of PAH sediment contamination in the Tyne Ž12 548–43 470 mg kgy1 dry weight, Ý15 PAH.,

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Fig. 1. Map showing sample sites along the River Tyne Estuary and River Alde Estuary UK.

including carcinogens such as BaP and benzow axanthracene at sediment concentrations of 952–

3310 and 945–4130 mg kgy1 dry weight, respectively w12x. The estuary of the river Alde on the

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Suffolk coast was chosen as the clean reference site as, unlike the Tyne, it has no recorded industrial inputs and only minimal municipal wastes entering its waters Ž145 m3 dayy1 .. Once on deck the flounder were placed in tanks containing flowing estuarine water. Fish over 15 cm in length were taken and killed by cervical dislocation, and the liver and bile were removed and immediately placed in liquid nitrogen for cryogenic storage. 2.3. DNA extraction and

32

P-postlabelling analysis

DNA was isolated from hepatic tissue of P. flesus using a high salt extraction method w13x. All isolates were quantified spectrometrically and deemed to be free from RNA and protein contamination. The DNA samples were then adjusted to a final concentration of 1 mg mly1 and stored at y708C prior to 32 P-postlabelling. The DNA from three individuals was pooled into one sample for each 32 P-postlabelling reaction. DNA adducts were determined using the butanol version of the 32 P-postlabelling assay w14x, as described previously w6,7x. Briefly, 10 mg samples of DNA were digested to deoxyribonucleoside 3Xmonophosphates in a total volume of 30 ml of digestion mix. Twenty microliters of this mix was then subjected to a butanol enhancement, evaporated to dryness and then redissolved in 9.5 ml of water. The remaining 1 ml of the DNA digest was diluted and held for the labelling of the normal undamaged

nucleotides for subsequent quantification w15x. Adducted and normal nucleotides were labelled separately, but simultaneously, for 2 h using 2 ml of labelling buffer Ž100 mM bicine NaOH, pH 7.6, 100 mM MgCl 2 , 100 mM dithiothreitol, 10 mM spermidine., 0.5 ml T4 polynucleotide kinase Ž10 U mly1 ; Boehringer Mannheim. and 8 ml and 2 ml of w g-32 Px ATP Ž) 6000 Ci moly1 , 10 mCi mly1 ; Amersham., respectively. The adducted deoxyribonucleoside-3X-5X-biphosphates present in the sample were then purified and separated from their normal undamaged counterparts using multidimensional anion exchange thin layer chromatography ŽTLC., on polyethyleneimine ŽPEI.-cellulose plates ŽCamlab, Cambridge, UK.. The levels of DNA adduct radioactivity were determined using an AMBIS radioanalytical scanning system ŽLabLogic, 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 10 8 undamaged nucleotides. Appropriate negative and positive DNA controls were analysed throughout the studies as described by Harvey and Parry w16x. Negative controls consisted of calf thymus DNA, while positive controls were generated in vitro by the addition of 1.5 mM BaP diol-epoxide ŽNCI Chemical Carcinogens Repository, Kansas City, MO. to 1 mg of flounder DNA, followed by phenolchloroform-isoamyl alcohol Ž24:24:1. extraction and isopropanol precipitation.

Fig. 2. ŽA–D.: Typical DNA adduct profiles produced following the 32 P-postlabelling of hepatic DNA from, ŽA. P. flesus administered corn oil only via I.P. injection. ŽB. P. flesus administered 1 mg kgy1 BaP in corn oil via I.P. injection. ŽC. P. flesus administered 20 mg kgy1 BaP in corn oil via I.P. injection. ŽD. Positive control consisting of P. flesus hepatic DNA treated in vitro with 1.5 mM BaP diol epoxide.

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2.4. Synchronous fluorescence spectrometry for determining bile metabolites Bile samples were analysed for fluorescent bile metabolites using synchronous fluorescence spectrometry ŽSFS., as described by Ariese et al. w17x. Briefly, bile samples were thawed in an ultrasonic bath and diluted with ethanolrwater Ž50:50 vrv.. SFS spectra were measured in a 1 cm quartz cuvette using a Perkin Elmer LS50 spectrofluorimeter. For quantification, the net peak area from excitation wavelength 323–423 nm was measured and expressed as mg kgy1 wet weight 1-OH pyrene equivalents. 3. Results 3.1. Analysis of positiÕe and negatiÕe controls for the 32 P-postlabelling assay To ensure accurate data interpretation, positive control DNA Ž P. flesus DNA treated in vitro with 1.5 mM BaP diol-epoxide and negative control DNA Žcalf thymus. was assayed by the 32 P-postlabelling assay alongside samples collected from the field. Previous studies have suggested that such steps are essential when undertaking environmental biomonitoring studies which, unlike laboratory toxicology studies, are often devoid of suitable control samples w6x. The routine analysis of the BaP diol-epoxide control ŽFigs. 2D and 3D. ensures against large-scale inter-experimental variation interfering with data in-

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terpretation. Minimal experimental variation was detected upon the comparisons of the adduct levels of the positive controls assayed in this study Ž259.5 " 37.7 adducts per 10 8 normal nucleotides, n s 5.. The analysis of the positive and negative Žadduct free calf thymus DNA. controls along side environmental samples ensures against the possibility of procedural artefacts, as described by previous authors w16x, interfering with data analysis. 3.2. Detection of DNA adducts in P. flesus following exposure to the ubiquitous carcinogen BaP The results obtained following the exposure of P. flesus to BaP and corn oil alone are displayed in Fig. 2A–C and Table 1. No DNA adducts were detectable in either the samples taken at the start of the experiment or subsequently in corn oil treated fish. Treatment with 20 mg kgy1 BaP resulted in the induction of a single adduct, which was detectable after 4 days and persisted through until day 21 ŽFig. 2C.. Administration of a single I.P. injection of 1 mg kgy1 BaP also induced a qualitatively similar adduct in hepatic tissue of P. flesus. However, the adduct was only detectable 21 days after treatment ŽFig. 2B.. While interpreting the data it was noted that the chromatographic migrations of the same adduct would differ between experiments Žsee Figs. 2D and 3D.. This problem is thought to be due to different batches of chromatography plates used and serves to highlight existing draw-backs of the 32 P-postlabel-

Fig. 3. ŽA–D.: Typical DNA adduct profiles obtained from 32 P-postlabelling. ŽA. Pooled P. flesus hepatic DNA sample displaying a low level of DNA adducts from individuals collected from the Alde Estuary. ŽB. Diagonal radioactive zone ŽDRZ. of radiolabelled P. flesus hepatic DNA adducts from a pooled sample from Scotswood on the Tyne Estuary. ŽC. Representative DNA adduct profile from a pooled sample of P. flesus hepatic DNA from individuals collected from Newcastle Bridge on the Tyne Estuary. ŽD. Positive control consisting of P. flesus hepatic DNA treated in vitro with 1.5 mM BaP diol epoxide.

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Table 1 Levels of hepatic DNA adducts Žadducted nucleotides per 10 8 normal nucleotides. detected in P. flesus following exposure to BaP Ž1 mg kgy1 and 20 mg kgy1 . and corn oil Days post exposure

1 mg kgy1 BaP

20 mg kgy1 BaP

Corn oil

0 2 4 8 14 21

ND a ND ND ND ND 1.9"0.3

ND ND 1.8"0.2 b 2.5"0.2 2.3"0.2 1.6"0.3

ND ND ND ND ND ND

a b

ND: not detectable under current assay conditions. Mean adduct levels"SE Ž ns 2., 4–5 fish pooled per sample.

ling assay in conjunction with TLC chromatography. Previous authors have also discussed the problem of adduct mobility differing between, and even within batches of commercially available plates w18x. Such qualitative differences in adduct migration patterns between experiments potentially hinders the application of the 32 P-postlabelling assay to the biomonitoring of adducts caused by unknown mixtures of carcinogens, and serves to highlight the need to run known standards with each batch of environmental samples. 3.3. Analysis of bile metabolites and hepatic DNA adducts in P. flesus specimens collected from the Tyne and Alde Estuaries Clear differences were observed in flounder captured from the contaminated Tyne Estuary and the reference Alde Estuary for both hepatic DNA adducts

and bile metabolites ŽTable 2.. Results show that levels of bile metabolites were considerably higher in flounder sampled from the Tyne compared with those captured from the reference Alde Estuary. A similar result, though not as striking was observed for the detection of DNA adducts with samples analysed from the Tyne containing elevated adduct levels compared with fish collected from the reference Alde Estuary. Representative autoradiograms of 32 P-postlabelled DNA from pooled hepatic tissues of P. flesus are displayed in Fig. 3A–C. All DNA adduct profiles observed in fish captured in the Tyne consisted of a diagonal radioactive zone ŽDRZ. of 32 P-labelled adducts ŽFig. 3B–C.. In contrast, autoradiograms obtained from P. flesus samples from the Alde Estuary lacked distinct DRZs and generally displayed faint areas of labelled DNA adducts ŽFig. 3A.. Previous studies have demonstrated DRZs to be representative of exposure to complex mixtures of aromatic andror hydrophobic genotoxins, such as those formed by PAH. For example, similar DNA adduct profiles were observed in laboratory studies following exposure of English sole Ž Parophrys Õetulus . to sediment extract contaminated with PAH. Field investigations have also detected DNA adduct profiles consisting of DRZs when fish are sampled from PAH contaminated waterways w5–8,19x. The co-migration of the BaP diol-epoxide standard ŽFigs. 2D and 3D. along with the adduct detected following in vivo exposure of P. flesus to BaP ŽFig. 2B–C. to similar positions on the chromatogram further supports the assumption that PAH-adducts are responsi-

Table 2 Levels of hepatic DNA adducts Žadducted nucleotides per 10 8 normal nucleotides. and bile metabolites Žmg kgy1 wet weight 1-OH pyrene equivalents. in P. flesus sampled from a reference estuary ŽRiver Alde, South East England. and an estuary known to contain elevated levels of contamination ŽRiver Tyne, North East England. Sample location

Levels of DNA adducts per 10 8 nucleotides

Bile metabolites Žmg kgy1 wet weight 1-OH pyrene equivalents.

Scotswood ŽTyne Estuary. Newcastle ŽTyne Estuary. Redheugh ŽTyne Estuary. Alde Estuary

24.6 " 3.2 a Ž5. b 34.4 " 3.7 Ž5. 27.6 " 6.3 Ž5. 10.1 " 4.8 Ž7.

22 247 " 3408 Ž16. c 14 572 " 1887 Ž13. 21 872 " 2935 Ž27. 632 " 56 Ž14.

a

Mean " SE. Numbers in parentheses represent number of pooled samples analysed for DNA adducts Žfive fish per pooled sampled.. c Numbers in parentheses represent number of individuals analysed for bile metabolites. b

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ble for a high proportion of the adducted DNA in the DRZ.

4. Discussion 4.1. Detection of DNA adducts following exposure to BaP The studies presented here demonstrate the potential of the European flounder to activate BaP to DNA reactive metabolites. The definitive structural assignment of the BaP adduct detected would require further analysis, but previous studies Žas reviewed by Ref. w20x. have suggested that the predominant adduct produced in fish following exposure to BaP is Žq. anti-BaP-7,8-diol-9,10 epoxide-deoxyguanosine. Reverse phase HPLC analysis of w3 HxBaP modified deoxyribonucleotides produced in vitro following studies with English sole Ž Par. Õetulus . and starry flounder Ž Pseudopleuronectes americanus. hepatic microsomes produced DNA adducts in salmon sperm with the identical retention time as a Žq. antiBaPDE-deoxyguanosine standard w21,22x. Similarly, the in vivo exposure of English sole to a single dose of 15 mg gy1 BaP resulted in a single major adduct detectable by 32 P-postlabelling with chromatographic similarities to an anti-BaPDE DNA standard w23x. Time course observations of the adducts produced revealed no significant decline in binding levels up to 60 days post-exposure. The detection of DNA adducts at 21 days post exposure, as presented here would confirm previous studies suggesting that DNA adducts are relatively stable modifications to the DNA following genotoxin exposure and therefore suitable biomarkers of genotoxic exposure in environmental biomonitoring studies w6,18,24x. Following exposure to 1 mg kgy1 BaP, detectable levels of DNA adducts were only seen after 21 days, and this delay is thought to reflect a combination of the biotransformation, activation, detoxification and repair processes resulting in the slow accumulation of DNA adducts to levels detectable by the 32 P-postlabelling assay. Previous studies have indicated that the rate of DNA adduct accumulation in teleosts following exposure to environmentally realistic levels of contamination may be relatively slow. For example, Shugart et al. w25x exposed bluegill sunfish

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Ž Lepomis macrocirus. to BaP at 1 mg ly1 continuously for 40 days. In the subsequent time-course study detectable amounts of DNA adducts were not seen until 10 days after the cessation of exposure. Such a lag in the detection of DNA adducts following low dose exposures may present problems Žas highlighted by Ref. w26x. when using a migratory species in aquatic biomonitoring programmes. 4.2. The detection of DNA adducts and bile metabolites in P. flesus from field sites The high concentration of bile metabolites, detected by SFS in this study demonstrate that PAH contaminants present in the Tyne sediment were bioavailable to P. flesus, and that the enhanced EROD activity w4x was in part due to the uptake and subsequent detoxification of these xenobiotics. Furthermore, the detection of elevated levels of DNA adducts, characteristic of PAH exposure, signifies that a proportion of the bioavailable PAH Žmost probably the carcinogenic 4–5 ringed PAH, such as BaP. were being metabolised by the MFO system to metabolites capable of reacting with the DNA in exposed fish. As mentioned previously, DNA adduct formation is one of the initial stages in the process of chemical carcinogenesis and their detection provides us with an estimation of carcinogenic exposure for P. flesus inhabiting the Tyne. Previous studies have associated the presence of DNA adducts, indicative of exposure to complex PAH mixtures, with increases in the incidence of cancerous lesions in marine flatfish Žfor review see Refs. w27,28x.. To date, no studies have been conducted to establish frequencies of pre-neoplastic and neoplastic lesions in P. flesus inhabiting the Tyne, or other equally contaminated English estuaries. However, data from other European waterways suggest levels of contamination similar to that found in the Tyne may lead to the onset of cancerous diseases w29x. The detection in this study of bile metabolites and DNA adducts along with the observations of w4x for EROD activity demonstrate that the PAH contamination of the Tyne is having a biological effect in the resident flounder, which may lead ultimately to the induction of neoplastic disease. Prolonged chronic exposure to PAH contaminated sediments may also lead to reproductive impair-

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ments, as PAH also have been shown to exhibit an endocrine disrupting ability. Previously, we have shown that flounder inhabiting the Tyne display signs endocrine disruption, which were attributed to the presence of oestrogen mimicking compounds such as 17b-oestradiol w11x. However, it is possible that PAH exposure, as detected in the present study, may have been responsible for some of the signs of endocrine disruption observed. Certain PAH, including acenaphthene, benzow axanthracene, BaP and dibenzw a,h xanthracene, are known to exert antioestrogenic effects in vitro w30,31x. Studies have also shown that a 5-month pre-spawning exposure of maturing male American plaice Ž Hippoglossoides platessoides. to ÝPAH sediment concentrations of 39 800 mg kgy1 dry weight affects sperm viability, and leads to a 48% reduction in the hatching success of larvae from eggs of unexposed females w32x. The European flounder as used in this study, confines itself to its home estuary for up to 8 months of the year, only migrating offshore to spawn. The levels of ÝPAH detected in sediments from the Tyne Ž12 548–43 470 mg kgy1 dry weight. indicate that similar reproductive impairments may be occurring in fish inhabiting both this and other, equally polluted, English estuaries. In summary, the studies presented here, along with those previously described w2–4,11,12x, demonstrate that the use of a suite of biochemical indices enhances our ability to detect exposure, and provides not only an estimate of the bioavailability of a group of compounds, but also any subsequent sub-lethal genotoxic effects. The results presented here suggest that populations of P. flesus in the Tyne Žand potentially other industrialised estuaries. maybe facing a significant threat to their long-term health and reproductive capabilities. More research is required if we are to establish the significance of these findings, and determine in full the potential ecosystem effects of chronic contaminant exposure at present levels.

Acknowledgements This work was funded by the Ministry of Agriculture, Fisheries and Food ŽMAFF. and the Department of Environment Trade and Regions ŽDETR.. The Authors wish to thank Paula Neall and Tina

Tylor for their skilled technical assistance, Irene Gooch for the production of Fig. 1 and the Environment Agency ŽEA. for the use of their Research Vessels.

References w1x S. De Flora, M. Bagnasco, P. Zanacchi, Genotoxic, carcinogenic and teratogenic hazards in the marine environment, with special reference to the Mediterranean Sea, Mutat. Res. 258 Ž1991. 285–320. w2x P. Matthiessen, J.E. Thain, R.J. Law, T.W. Fileman, Attempts to assess the environmental hazard posed by complex mixtures of organic chemicals in UK estuaries, Mar. Pollut. Bull. 26 Ž1993. 90–95. w3x P. Matthiessen, S. Bifield, F. Jarrett, M.F. Kirby, R.J. Law, W.R. McMinn, D.A. Sheahan, J.E. Thain, G.F. Whale, An assessment of sediment toxicity in the River Tyne estuary, UK by means of bioassays, Mar. Environ. Res. 45 Ž1998. 1–15. w4x M.F. Kirby, P. Matthiessen, P. Neall, T. Tylor, C.R. Allchin, C.A. Kelly, D.L. Maxwell, J.E. Thain, Hepatic EROD activity in flounder Ž Platichthys flesus . as an indicator of contaminant exposure in English estuaries, Mar. Pollut. Bull. 38 Ž1999. 676–686. w5x U. Varanasi, J.E. Stein, M. Nishimoto, W.L. Riechert, T.K. Collier, 32 P-postlabelling analysis of DNA adducts in liver of wild English sole Ž Parophrys Õetulus . and winter flounder Ž Pseudopleuronectes americanus., Cancer Res. 49 Ž1989. 171–1171. w6x J.S. Harvey, B.P. Lyons, M. Waldock, J.M. Parry, The application of the 32 P-postlabelling assay to the aquatic environment, Mutat. Res. 378 Ž1997. 77–88. w7x B.P. Lyons, J.S. Harvey, J.M. Parry, An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis, Mutat. Res. 390 Ž1997. 263–268. w8x G. Ericson, E. Lindesjoo, L. Balk, DNA adducts and histopathological lesions in perch Ž Perca fluÕiatilis. and northern pike Ž Esox lucius . along a polyclclic aromatic hydrocarbon gradient on the Swedish coastline of the Baltic Sea, Can. J. Fish. Aquat. Sci. 55 Ž1998. 815–824. w9x K. Randerath, M.V. Reddy, R.C. Gupta, 32 P-postlabelling test for DNA damage, Proc. Natl. Acad. Sci. U.S.A. 78 Ž1981. 6126–6129. w10x R.C. Gupta, M.V. Reddy, K. Randerath, 32 P-postlabelling analysis of non-radioactive aromatic carcinogen DNA adducts, Carcinogenesis 3 Ž1982. 1081–1092. w11x P. Matthiessen, Y.T. Allen, C.R. Allchin, S.W. Feist, M.F. Kirby, R.J. Law, A.P. Scott, J.E. Thain, K.V. Thomas, Oestrogenic endocrine disruption in flounder Ž Platichthys flesus . from UK estuarine and marine waters, Centre for Environment, Fisheries and Aquaculture Science Lowestoft, UK, Science Series Technical Report 107, 1998, p.48. w12x R.J. Woodhead, R.J. Law, P. Matthiessen, Polycyclic Aro-

B.P. Lyons et al.r Mutation Research 446 (1999) 111–119

w13x

w14x

w15x

w16x

w17x

w18x

w19x

w20x

w21x

w22x

matic Hydrocarbons ŽPAH. in surface sediments around England and Wales, and their possible biological significance. Mar. Poll. Bull., ŽIn Press.. S.A. Miller, D.D. Dykes, H.F. Polesky, A simple salting out procedure for extracting DNA from nucleated cells, Nucleic Acids Res. 16 Ž1988. 1215. R.C. Gupta, Enhanced sensitivity of the 32 P-postlabelling analysis of aromatic carcinogen DNA adducts, Cancer Res. 45 Ž1985. 5656–5662. N.J. Jones, M.A. Kadhim, P.L. Hoskins, J.M. Parry, R. Waters, 32 P-postlabelling analysis and micronuclei induction in primary lung cells exposed to tobacco particulate matter, Carcinogenesis 12 Ž1991. 1507–1514. J.S. Harvey, J.M. Parry, Application of the 32 P-postlabelling assay for the detection of DNA adducts: false positives and artefacts and their implications for environmental biomonitoring, Aquatic Toxicol. 40 Ž1998. 293–308. F. Ariese, J.K. Kok, M. Verkaik, C. Gooijer, N.H. Velthorst, J.W. Hofstraat, Synchronous fluorescence spectrometry of fish bile: a rapid screening method for the biomonitoring of PAH exposure, Aquatic Toxicol. 26 Ž1993. 273–286. A.C. Beach, R.C. Gupta, Human biomonitoring and the 32 P-postlabelling assay, Carcinogenesis 13 Ž1992. 1053– 1074. A.E. Maccubin, J.J. Black, B.P. Dunn, 32 P-postlabelling detection of DNA adducts in fish from chemically contaminated waterways, Sci. Total Environ. 94 Ž1990. 89–104. J.E. Stein, W.L. Reichert, M. Nishimoto, U. Varanasi, Overveiw of studies on liver carcinogenesis in English sole from puget sound; evidence for a xenobiotic chemical etiology: II. Biochemical studies, Sci. Total Environ. 94 Ž1990. 51–69. M. Nisimoto, U. Varanasi, Benzow axpyrene metabolism and DNA adduct formation mediated by English sole liver enzymes, Biochem. Pharmacol. 34 Ž1985. 263–268. M. Nisimoto, U. Varanasi, Metabolism and DNA adduct formation of benzow axpyrene and the 7,8-dihydrodiol of benzow axpyrene by fish liver enzymes, in: M.C. Cooke, A.J. Dennis ŽEds.., Polynuclear Aromatic Hydrocarbons: Fate and Effects, Battelle Press, Columbus, OH, 1986, pp. 685–699.

119

w23x U. Varanasi, W.L. Reichart, B.-T.L. Eberhart, J.E. Stein, Formation and persistence of benzow axpyrene-diolepoxideDNA adducts in liver of English sole Ž Parophrys Õetulus ., Chem. Biol. Interact. 69 Ž1989. 203–216. w24x D.H. Phillips, Detection of DNA modifications by the 32 Ppostlabelling assay, Mutat. Res. 378 Ž1997. 1–12. w25x L.R. Shugart, B.D. Jimenez, J.F. McCarthy, DNA damage as a biological marker in aquatic organisms exposed to benzow axpyrene, in: M. Cook, K. Loening, J. Merritt ŽEds.., Polynuclear Aromatic Hydrocarbons, Measurements, Means and Metabolism, Battelle Press, USA, 1992, pp. 839–846. w26x J.S. Harvey, B.P. Lyons, J.M. Parry, C. Stewart, An assessment of the genotoxic impact of the Sea Empress Oil spill by the measurement of DNA adduct levels in selected vertebrate and invertebrate species, Mutat. Res. 441 Ž1999. 103–114. w27x P.C. Baumann, Epizootics of cancer in fish associated with genotoxins in sediment and water, Mutat. Res. 411 Ž1998. 227–233. w28x M.S. Myers, J.T. Landahl, M.N. Krahn, L.L. Johnson, B.B. McCain, Overview of studies on liver carcinogenesis in English sole from Puget Sound; evidence for a xenobiotic chemical etiology. Pathology and epizootiology, Sci. Total Environ. 94 Ž1990. 33–50. w29x A. Koehler, Identification of contaminant-induced cellular and subcellular lesions in the liver of flounder Ž Platichthys flesus L.. caught at differently polluted estuaries, Aquatic Toxicol. 16 Ž1990. 271–294. w30x D.Q. Tran, C.F. Ide, J.A. McLachlan, S.F. Arnold, The anti-oestrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human estrogen receptor, Biochem. Biophys. Res. Commun. 229 Ž1996. 102–108. w31x K. Chaloupka, V. Krishnan, S. Safe, Polynuclear aromatic hydrocarbon carcinogens as antioestrogens in MCF7 human breast cancer cells: role of the Ah receptor, Carcinogenesis 13 Ž1992. 2233–2239. w32x J.J. Naglar, D.G. Cyr, Exposure of male American plaice Ž Hippoglossoides platessoides. to contaminated marine sediments decreases the hatching success of their progeny, Environ. Toxicol. Chem. 16 Ž1997. 1733–1738.