DNA ADDUCT DETECTION IN MUSSELS EXPOSED TO BULKY AROMATIC COMPOUNDS IN LABORATORY AND FIELD CONDITIONS

Biomarkers in Marine Organisms: A Practical Approach. Ph. Garrigues, H. Barth, C.H. Walker and J.F. Narbonne, editors. 9 2001 Elsevier Science B.V. All rights reserved.

Chapter 4 DNA ADDUCT DETECTION IN MUSSELS EXPOSED TO BULKY AROMATIC COMPOUNDS IN LABORATORY AND FIELD CONDITIONS P. Venier

Department of Biology, University of Padova, Via Bassi 58 B, 35131 Padova (Italy) Abstract

Carcinogens and mutagens of different chemical structures share the ability to bind covalently to DNA, either directly or after biotransformation to electrophilic intermediates. DNA binding basically depends on its molecular structure and fimctional state (accessibility of nucleophilic target sites) while physiological and biochemical features determine differences in adduct formation among tissues and across species. DNA adducts can be detected by the 32p-postlabelling assay in cells and organisms exposed to model genotoxins or unknown chemical mixtures, thus confirming the biological relevance of exposure and recognizing relationships between initial DNA lesions and ensuing biological effects (e.g. mutations, tumors). Regarding mussels, sentinel species used world-wide in pollution monitoring, DNA adduct detection presents some drawbacks but can also provide interesting insights into benzo[a]pyrene-induced damage. Compared with vertebrates, lower adduct formation is likely to occur in mussels exposed to 'environmental' genotoxin doses; yet, substantial bioaccumulation of miscellaneous pollutants and diversified intracellular reactions, altogether, may explain the appearance of benzo[a]pyrene-related DNA adducts. Irrespective of molecular mechanisms, the 32p-postlabelling assay can be used to ascertain mussel exposure to genotoxins, being limited only by the complexity and cost of the applied technique. Evidence of unresolved DNA adducts in mussels from one industrial area (Venice lagoon, Italy) is consistent with other studies indicating exposure to genotoxic water pollutants. On the other hand, the non-appearance of specific adducts in mussels from sites still contaminated by polycyclic aromatic hydrocarbons (Galician coast, Spain) suggests that other aromatic compounds could have caused earlier DNA adduct formation. Key words : mussel, DNA, adducts, benzo[a]pyrene, genotoxins, Venice lagoon 65

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I. INTRODUCTION 1.1. Biological relevance of DNA adducts The mutagenic and carcinogenic effects of structurally different DNA-reactive compounds (mustard gas, polycyclic aromatic hydrocarbom and other alkylating agents) were demonstrated in pionering studies several years before the identification of DNA as a transforming factor (1944) and the 'double helix' hypothesis of Watson and Crick (1953) (Lawley, 1994; Pitot and Dragan, 1996). Nowadays, we are aware that hundreds of different DNA adducts are produced by some 20 classes of carcinogens and mutagens, either directly or following metabolic activation to DNA-reactive intermediates (Kleihues, 1994). Electrophilic chemicals, or metabolites, react specifically to DNA sites having different accessibility, position, nucleophilicity and sequence context, and produce characteristic adduct spectra. It appears that N3, N7 ring nitrogens of guanine and adenine as well as exocyclic base oxygens are common nucleophilic sites. In addition to the chemical specificity, spontaneous depurination of modified bases and differential DNA repair can further account for differences in adduct spectra (Lawley, 1994; Hemminki et al., 1994). As an example, the binding of benzo[a]pyrene 7,8-dihydrodiol 9,10-oxide (one of the metabolites derived from the parent compound B[a]P) to the exocyclic amino group of guanine and adenine produces rather persistent adducts, substantially impairing DNA replication and possibly disrupting gene expression (Johnson et al., 1997). On the other hand, a large proportion of B[a]P-DNA adducts (-~ 80 %) are easily lost through depurination (Devanesan et al., 1996). Therefore, DNA adducts are structural changes involving covalent attachment of a chemical to DNA and represent a dynamic index of exposure. In fact, adduct levels at any time are determined by processes such as chemical uptake, rate of activating/deactivating reactions, spontaneous or enzyme-mediated adduct loss and fate of DNA-damaged cells. Interestingly, the existence of 'indigenous' adducts has been recognized (I-compounds) whose spectra and levels exhibit marked species differences (Randerath et al., 1993). These modified nucleotides are most likely generated by normal nutrient metabolism without known exposure to genotoxins and appear strongly influenced by diet and animal age. Through DNA replication or erroneous repair the presence of adducts may cause critical mutations and trigger a cascade of subsequent events leading to cell death, uncontrolled cell replication and other cell disfunctiom (Pitot and Dragan, 1996). As suggested by B. Kurelec, biological consequences of the induced mutations other than cancer might play a major role in lower organisms with scarce or no tumor incidence and have even greater ecological relevance (Kurelec, 1993). Overall, we cannot predict the impact of genotoxic pollutants in the ecosystem and we need suitable biomarkers to investigate their presence and their effects on

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living species (Wtkgler and Kramer, 1992; Anderson et al., 1994; Evenden and Depledge, 1997; Theodorakis et al., 1997). 1.2. The 32p-postlabelling assay A number of procedures including immunoassays, fluorometric analysis, chromatographic and mass spectral techniques have been used in the detection and identification of DNA adducts (Talaska et al., 1992; Santella, 1997; Hemminki et al., 1997; Apruzzese and Vouros, 1998). The 32p-postlabelling assay appeared in the early 1980s and has been applied with many procedural versions to detect DNA adducts produced by known carcinogens or complex mixtures in vitro and in vivo (Beach and Gupta, 1992). Its importance is testified by monographic issues, procedural papers and inter-laboratory calibration studies (Phillips et al., 1993; Hemminki et al., 1994; Reichert and French, 1994; Phillips, 1997). The great advantage of this technique is that DNA adduct levels as low as one adduct per 109-101~nucleotides can be detected without prior knowledge of their chemical identity in some micrograms of DNA purified from any tissue of interest (La and Swenberg, 1996; Baan et al., 1997). Briefly, the assay involves DNA purification, digestion to normal and adduct-modified 3'-mononucleotides, removal of normal nucleotides (via enzymatic digestion, solvent extraction or chromatographic methods), 32p-labelling at the 5'-hydroxyl position of adducted nucleotides followed by two-dimensional chromatographic separation, detection and quantification (via autoradiography, liquid scintillation spectrometry or phosphor screen imaging). Results are usually expressed as the number of adducts per l0 g nucleotides and hereinafter are indicated as RAL (relative adduct labelling). Although largely applied to bulky aromatic DNA adducts, the technique can be adapted to detect other very different DNA lesions (Phillips et al., 1993; Phillips, 1997). 1.3. DNA adduct detection and environmental monitoring Most published studies have considered human carcinogenic-DNA adducts since accurate estimation and identification of adducts are expected to be predictive of human disease risk and assume particular relevance for occupational, dietary, tobacco and drug exposures (Farmer et al., 1996). Following the 32p-postlabelling assay, complex mixtures of bulky aromatic chemicals (e.g. from tobacco smoke) cause the appearance of the 'diagonal radioactive zone' (DGZ), chromatographic profiles consisting of discrete overlapping adduct spots, only partially resolved. In major target tissues adduct levels are consistent with smoking habits and epidemiological evidence of lung cancer risk (Randerath and Randerath, 1993). As examples, 1133 and 389 amol/~tg DNA (about 34 and 12 RAL) were detected in normal oral tissue of smokers with intraoral squamous cell carcinoma and in

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peripheral blood mononuelear cells of coal miners, respectively (Jones et al., 1993; Qu et al., 1997). More recently, the 32p-postlabelling has been applied to DNA adduet detection in non-mammal species, extending to invertebrates, plants and also bacterial cells (Dunn et al., 1987; Vamnasi et al., 1989b; Pfohl-Leszkowiez et al., 1993; van Shooten et al., 1995; Quillardet et al., 1996). Specifically, DNA adducts have been studied in aquatic organisms, both freshwater and marine species, since water bodies receive pollutants from different environmental compartments and have a well-recognized importance for living organisms and human activities. Bulky aromatic DNA adduets have been found in fish from polluted sites often in association .with hepatic cancers or lesions (Varanasi et al., 1989b; Dunn et al., 1987; Stein et al., 1994). Significant DNA adduet formation characterized fish from polluted v s . unpolluted waters, (Maccubbin et al., 1990; Liu et al., 1991; Ericson et al., 1994; van Shooten et al., 1995; el Adlouni et al., 1995; Lyons et al., 1997). Indicative adduet levels in fish liver were 24 v s . <1 RAL in tilapia (Liu et al., 1991) and 16.6-34.3 v s . 4.7 RAL in eel (van Shooten et al., 1995). In the latter study, DNA adduets (1.5 RAL) were detected also in earthworms exposed to polycyelic aromatic hydrocarbon-contaminated soil. Nevertheless, butanol-enriched DNA samples from five fish species produced conflicting results since 227 and 57 amol/~tg of adduets (about 6.8 and 1.7 RAL) characterized fish from unpolluted and polluted waters, respectively (Kurelee et al., 1989). Influence of natural factors as well as species specificity and seasondependence in adduet formation have been reported (Kurelee et al., 1990; Dunn, 1991; Kurelec and Gupta, 1993). 2. FORMATION AND DETECTION OF DNA ADDUCTS IN MUSSELS One of the main drawback regarding shellfish and other invertebrates is their limited or insignificant competence for the biotransformation of certain chemical pollutants to DNA-reaetive intermediates (Livingstone, 1991). Consequently, the absence of DNA adduets can not be predictive of the absence of genotoxic pollutants and if any adduet appears, its indigenous or pollution-related nature has to be discriminated. The negligible xenobiotie metabolism is just one of the reasons to choose mussels instead of fish, in monitoring marine pollution because piscine metabolism subtracts a substantial fraction of chemical from its analytical determination. Following acute exposure, a few hundred lag and some ~tg per g of w. w. tissue are representative B[a]P levels measured in mussels and rainbow trout, respectively (unpublished results). Indeed, mussels possess a selective flavine-containing monoxygenase that efficiently converts aromatic amines to DNA-reactive and mutagenic intermediates (Kurelec and Gupta, 1993). Hydroxylation of aromatic hydrocarbons (AH) and the

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existence of distinct cytochrome P450 genes have also been recognized (Livingstone and Farrar, 1984; Wootton et al., 1994; Porte et al., 1995). In mussels, mixed function oxygenases are influenced by seasonal factors, environmental exposure or inducing treatments (So16 et al., 1995, 1996; Wootton et al., 1996; Livingstone et al., 1997). In any case, the activity of such an enzymatic mussel system appears to be considerably lower (one-two order of magnitude) than in vertebrate organisms (Ade et al., 1982; Varanasi et al., 1989a). Many other phase I- and phase II-enzymes involved in the xenobiotic metabolism (e.g. epoxide hydrolase and glutathione S-transferase) are present in bivalve and mollusk species (Livingstone and Pipe, 1992). 2.1. DNA adducts in mussels following laboratory exposure to genotoxins Although other end-points of genetic damage have been investigated in mussels intentionally exposed to genotoxins (e.g. DNA breaks, sister chromatid exchanges, chromosomal aberrations, micronuclei) only limited knowledge is available about adduct formation (De Flora et al., 1991). Ten years ago, the appearance was reported of 2-aminofluorene-induced adducts in digestive gland DNA of Mytilus galloprovincialis (Kurelec et al., 1988). Lately, certain compounds and less defined chemical mixtures (2-aminofluorene, 2-acetyl aminofluorene, 2-aminoanthracene, 4-nitroquinoline 1-oxide, and chemically contaminated sediments) induced DNA adducts in vivo, in digestive gland or gills (Kurelec et al., 1990; Marsh et al., 1992; Harvey and Parry, 1997; Harvey et al., 1997). In the former in vitro study B[a]P did produce one very weak and lateral adduct spot, thus suggesting insubstantial benzo[a]pyrene metabolism and according to the exiguous DNA binding detected after injection of mussels with 3H-B[a]P (Kurelec et al., 1988; Marsh et al., 1992). Although debated, B[a]Prelated DNA adduct formation has been ascertained in separate experimental exposures to waterborne B[a]P (Venier et al., 1996; Harvey and Parry, 1997; Canova et al., 1998). Gill and digestive gland DNA from mussels exposed to B[a]P (0.5-1000 ~tg/liter or ppb, nominal doses) showed similar induction of low adduct levels (ordinarily < 1 RAL, although higher values were exceptionally obtained) dose- and timerelated. Significant adduct formation was evident after 2 and 3 days of exposure to 50 and 5 ppb of B[a]P, respectively. Following nuclease P 1-enrichment and cochromatography, the spot typically found in mussels did not show the same chromatographic mobility of the main spot detectable in mammalian DNA (B[a]PN2dG adduct). In addition, spot intensity did not increase by pre-treating mussels with the mixed function monoxygenase inducing agent, Aroclor 1254 (Venier and Canova, 1996; Canova et al., 1998). Altogether, these findings suggest that B[a]P-related DNA adducts in mussels are the result of biochemical pathways distinct from the 'inducible' B[a]P

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diolepoxide production. In fact, DNA-reactive intermediates (both epoxides and radicals of B[a]P) and reactive oxygen species (ROS) originate during B[a]P metabolism (Morrero and Marnett, 1993; Penning, 1993). In contrast with higher organisms (crustaceans, echinoderms and vertebrates), mussels metabolize B[a]P to predominantly B[a]P quinones (diones) whose potential for ROS production is high (Sj/51in and Livingstone, 1997). Radical formation might be enhanced not only by redox cycling of B[a]P quinones but also by specific reactions occurring atter lysosome destabilization (enzyme release) and in the presence of metals with a catalytic role in ROS production. There is no doubt that mussel filter-feeding activity causes bioaccumulation of miscellaneous pollutants, metals included (Livingstone and Pipe, 1992). Notably, free radical formation is not restricted to B[a]P but is rather common for mutagenic, carcinogenic and teratogenic chemicals (Pitot III and Dragan, 1996; Wells et al., 1997). In the presence of free radicals, lipid peroxidation, DNA oxidations and strand breaks can occur (Dix and Marnett, 1983; Morgenstem et al, 1981; Sbrana et al. 1995, Hanelt et al., 1997). As expected, DNA damage observed via alkaline elution, cytogenetie analysis, 8OHdG electrochemical detection and Comet assay has been found in mussels exposed to B[a]P (Bihari et al., 1990; Venier et al., 1997a; Canova et al., 1998; Mitchelmore et al., 1998). Overall the experimental data above lead to a paradoxical conclusion. For monitoring purposes, the inadequate production of B[a]P diolepoxides is irrelevant since a small fraction of the great excess of bio-aceumulated B[a]P is always available for generation of DNA reactive-intermediates and, concurrent processes of lipid peroxidation and free radical formation can amplify DNA damage detectable with current procedures. The persistence for up to 58 days of fluctuating levels of B[a]P-related DNA adducts in mussels treated for two days with 100 ppb of waterborne B[a]P is consistent with the above assertions and might be explained by new adduct formation (based on the evidence of partial B[a]P depuration) rather than, or in addition to, authentic adduct persistence (Venier et al., 1997b and unpublished data). The latter findings suggest that DNA adducts could be representative of long-term exposure; however, the remarkable and irregular temporal changes observed in adduct levels underline the qualitative value of DNA adduct detection in B[a]P-exposed mussels. The effects of specific enzyme inhibitors, DNA repair capacity and cell turnover still need to be investigated in B[a]P-exposed mussels. 2.2. DNA adducts in mussels following field exposure

A number of critical points should be taken into account when the 32p. postlabelling assay is applied to mussels in field studies. Indigenous adducts, that is 'natural' DNA modifications, can make the evaluation of pollution-related DNA adducts difficult (Dunn 1991; Kurelec and

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Gupta, 1993; Harvey et al., 1997). Parallel testing of mussels from relatively clean sites, similar in size and collected at the same time, or in situ transplantation of homogenous mussel groups can be helpful not only for DNA adduct detection but also for monitoring other pollution-related parameters. Procedural details of the 32p-postlabelling assay (e.g. DNA purification and adduct enrichment) are important in monitoring exposure to miscellaneous pollutants inducing only weak DNA adduct formation (Gupta, 1993; Venier and Canova, 1996; Harvey and Parry 1998). During DNA purification attention must be paid to avoid DNA oxidation, loss of adducted nucleotides, RNA contamination and inaccurate DNA quantification. On the other hand, both nuclease P1 digestion and butanol extraction steps of adduct enrichment should be applied in field monitoring, unless chemical pollutants involved and specific DNA adduct recovery with both enrichment methods are known. Knowledge of mussel physiology, biochemistry and genetics is certainly less than adequate in comparison with information available for human and laboratory species (rodents, yeast, bacteria, Drosophila melanogaster and Caenorhabditis elegans). In the light of this, a multidisciplinary approach and comparative species evaluation should be used for a comprehensive and correct interpretation of field data as well as laboratory studies. According to personal experience and current knowledge on mussel xenobiotic metabolism, adduct levels (sum of individual adduct spots) expected under real field exposures can not be as high as those detected in fish (vertebrate organisms). In addition, lipid content-dependent uptake of lipophilic pollutants and cyclic spawning can explain, at least partially, the variability in adduct levels detected in mussels (Livingstone, 1992; Venier and Canova, 1996; Harvey and Parry, 1997). Despite these critical aspects, pollution-related adducts have been found in mussels, for example in mussels collected in the "mixing zone" of oil refinery wastewaters (Kurelec et al., 1990). Exceptional adduct levels, as high as 308.8, 1565 and 3727.9 attmol/lag DNA (roughly equivalent to 9, 46 and 112 RAL) in nuclease Pl-enriched samples, and values even higher for butanol-enriched samples, have been reported for mussels collected in different sites of the U.K. coast (Harvey and Parry, 1998). In a previous study, the same authors reported mean levels of 6.1 and 44.9 RAL, for nuclease P1- and butanol-enriched DNA samples respectively, from both somatic and reproductive tissues of Mytilus edulis collected along the U.K. coast (Harvey et al., 1997). As suggested by the authors procedural factors may explain the appearance of artefactual adduct spots; on the other hand, the efficiency of chromatographic washing steps and quantification of single spots or large TLC zones can determine differences between adduet levels reported in different studies.

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Although never exceeding 1 RAL, significant adduct increases have been found in gill DNA of mussels from an industrial district compared with less polluted sites of the Venice lagoon (semi-enclosed water-body surrounding Venice, North-East Italy). This finding was reproducible in separate samplings and it was confirmed by parallel detection of DRZ-adducts in gill DNA of grass gobies (Zosterisessor ophiocephalus, Pallas) living in the same industrial area (Venier et al., 1996). Figure 1 shows typical DNA adducts and figure 2 illustrates adduct levels measured in mussels (Mytilus galloprovincialis) of the Venice lagoon over twoyears. Pooled tissue samples (gills or digestive glands) from at least 5 mussels were tested in the 32p-postlabelling assay, as described previously (Venier and Canova, 1996). More recently, improvements related to DNA purification (500xg spinning of tissue homogenates to discard tissue debris, DNA purification from crude nuclei and more efficient digestion mixture with RNAse A, RNAse T1 and a-amylase before incubation with proteinase K) in agreement with recent published literature have been routinely introduced. Panel A

73 Panels B-C-D-E

Figure 1 9 Representative images of mussel DNA adducts (chromatographic origin in bottom left comer, autoradiographic development for 90-96 h at -76~ if not indicated differently). A, positive reference DNA from lymphocytes treated with (+) anti B[a]P diolepoxide (5 h a t 76~ B, digestive gland DNA from unexposed mussels. C, B[a]P-related DNA adduct in exposed-mussels (digestive gland). D and E, gill DNA of mussels from the industrial area of Marghera (Venice, Italy)

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In figure 1, panel A illustrates the main spot (putatively corresponding to the B[a]P-N2dG and other adducts) and additional adduct spots formed by the (+) anti B[a]P diolepoxide. Number and intensity of spots depend on the degree of covalent binding and length of autoradiographic development (a few hours up to 72-96 hours as suggested by TLC radioactivity) during the 32p-postlabelling assay. Panels B and C show digestive gland DNA from mussels unexposed and exposed to 100 ppb of waterborne B[a]P for three days, respectively. Variable intensity of the B[a]P-related adduct spot was observed in distinct exposure experiments. Panels D and E illustrate the typical pattern of partially resolved adduct spots in two gill DNA samples (mussels from Marghera, Venice).

DRZ-adducts (gills) =5

.....

0.30.2

:0.0 1 2 3 4 5 3 5 2

53535

S ite (1-5) Figure 2 : Adduct levels (gill DNA) of mussels collected in the Venice lagoon. (1, 2, 3: sites at the lagoon entrances Porto Chioggia, Porto Malamocco; Porto Lido; 4: Southern lagoon site, close to Chioggia; 5: industrial district of Marghera, inner central lagoon)

Figure 2 illustrates DRZ-adduct levels detected in gill DNA of mussels collected in the Venice lagoon. The first five values refer to a preliminary campaign whereas the others are distinct coupled comparisons between one lagoon entrance (2 or 3) and the industrial area of Marghera (5). In spite of the low RAL values, reproducible and significant differences between Marghera and the other lagoon sites emerged, in agreement with the described autoradiographic images (figure 1, panels D and E). However, only occasionally did pooled digestive gland samples from the same mussels show similar significant differences (in the coupled

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comparison between sites 2 and 5 RAL values of 0.079+0.020 and 0.310-~0.078 respectively were found). More efficient B[a]P detoxification and DNA repair, possibly occurring in digestive gland compared with gills, might explain the absence of notable levels of DRZ adducts. Native and transplanted mussels exposed to unknown contaminants within the industrial area of Marghera are currently under study. It is worth remembering the variety of chemical contaminants present in the industrial area of Marghera. In addition to heavy metals, halogenated organic compounds (dioxins, polychlorinated biphenyl congeners, pesticides), aliphatic and aromatic hydrocarbons (Pavoni et al., 1992; Livingstone et al., 1995; Marcomini et al., 1996), the presence of radioactive wastes has been reported in local newspapers in 1998. Most striking are some biological effects specifically detected at Marghera. Shell malformations (Garbisa, 1972), higher endoparasitic infection (personal observation), decreased scope for growth (Widdows et al., 1996), immunemodulation as measured in haemocytes (Pipe et al., 1995), increased expression of CYP1AI-like mRNA and CYP1Al-immunopositive proteins (Livingstone et al., 1995) have been found in mussels. In the latter study, elevated 7-ethoxyresorufin O-deethylase activity, CYP1Al-protein levels and changes in antioxidant enzyme activities have been reported in autochthonous grass gobies. In addition, barnacle populations from the same area showed heterozygote deficiency and significant departures from Hardy-Weinberg expectations (Montero et al., 1994). Indirect evidence of the presence of mutagenic components in sediments of Marghera was obtained by means of the Salmonella/microsome assay (La Rocca et al., 1996). Thus, potential mutagenic, carcinogenic and teratogenic agents are likely to affect organisms living in such a polluted environment: the biological evidence described above suggest priority in the environmental recovery and correct management of human activities. Finally, figures 3 and 4 describe recent 32P-postlabelling results on mussels from the Galician coast (La Corufia, North Spain) where the "Aegean Sea" oil spill occurred in 1992 (So16 et al., 1996). Six months after the accident, chemical analysis and DNA adduct detection were performed on mussels collected within the contaminated area (Mera, Lorb6, Puentedeume, Carifio) and in one clean reference site (Meir~is). Compared with Meir~s, the contaminated sites showed higher concentrations of 13 polycyclic AH (sum) and unresolved chemicals (0.13-0.31 vs. 0.03 ~tg/g d.w. and 47-128 vs. 35 lag/g d.w., respectively) in whole mussel tissues. At the same time, a marked presence of DRZ-DNA adducts (particularly at Puentedeume and Carifio) was revealed in digestive glands of mussels from the contaminated sites following butanol-enrichment and phosphor-imager detection of adducts (So16 et al., 1996). As a matter of fact, 128 and 69.9 mean RAL values respectively were detected in

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mussels from the two cited locations while the mean RAL value in vehicle dosed rats was 18.1 (the latter figure, rather high, may depend on specific details used for adduct quantification). Recently, tissue samples from mussels collected three years after the accident (immediately frozen and stored at-76~ were subjected to the 32p-postlabelling assay since analytical determination showed polycyclic AH levels as high as those detected soon after the oil spill (0.18-0.54 vs. 0.06 ~tg/g d.w.) (Biosca, 1997). Four tissue pools of gills or digestive glands, representative of 20 mussels per station, were analyzed. Only nuclease P1 digestion was applied became polyCyclic AHderived DNA adducts are well recovered by both nuclease P1 and butanol extraction steps (Beach and Gupta, 1992). As indicated in figure 3, only faint spots were detected in such samples: spot 1 was present in all five sites, spot 2 only in mussels from Lorb~ and spot 3 only in one gill sample of mussels from Carifio.

"r

~,,

1

j,. s.

9

--~

D3

Figure 3 9 Scheme of adduct spots (1,2,3) detected in mussels from the Galician coast (La Corta~, North Spain).

Corresponding RAL values, calculated for each adduct spot, are reported in figure 4.

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Individual adducts

(gills and digestive glands) =o

~"

0.5 0.4 0.3

'

~

, 7<

"-

0.2

0.1 0.0-

',

'

'

'

'

'

'

'

I'DI'EI'AI'BI'CI'DI'E1 C2'C2'

i

"t (A-E) and a d d u c t n u m b e r (1-3) .........

Figure 4 9Adduct levels in gill and digestive gland DNA of mussels from the Galician coast (La Corufia, North Spain). A, Meir/Ls, reference site; B, Mera; C, Lorb6; D, Puentedeume; E, Carifio; 1,2,3: adduct spots; empty and dotted bars: gill and digestive gland samples, respectively.

The presence of adduet spot 2, detected in all pooled samples from the aquaculture area of Lorb6 is difficult to explain; on the other hand, no correspondence exists between these weak adduct spots and the intense DRZadducts previously detected (So16 et al., 1996). Consequently, one may infer that other aromatic chemicals, distinct from polycyclic aromatic hydrocarbons caused adduct formation in mussels analyzed soon aider the "Aegean Sea" oil spill. 3. CONCLUSION Because of their widespread presence and biological features, mussels are organisms of choice in monitoring coastal water pollutants. Apparently, some of these features make the dosimetry of DNA adducts, genetic lesions possibly induced by genotoxic pollutants, more difficult than in vertebrate organisms. The limited formation of DNA-reactive intermediate via mixed function oxygenase reactiom might be counterbalanced by efficient filter-feeding activity (considerably increasing tissue concentration of different pollutants) and high ROS production in mussels. Following acute laboratory exposure, reproducible and doses-effect related induction of DNA adducts can be obtained with model genotoxic compounds, such as B[a]P, and evidence of DNA adduct formation also

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resulted from some field studies on Mytilus galloprovincialis and Mytilus edulis. However, additional studies are still necessary in order to evaluate and improve the application of the 32p-postlabelling assay to field investigations. ACKNOWLEDGEMENTS

I wish to thank Dr. Claudia Zampieron who gave me support during work organization and experimental activity, Prof. P. Garrigues (CNRS, Bordeaux I, France) for the confidence shown towards my work and Dr. C. Porte for providing mussel samples from the Galician coast. Grant by ENV4-CT96-0300 (BIOMAR II project) and Italian MURST. REFERENCES

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