Anguilla anguilla L. Biochemical and Genotoxic Responses to Benzo[ a]pyrene

Ecotoxicology and Environmental Safety 53, 86}92 (2002)

Environmental Research, Section B

doi:10.1006/eesa.2002.2205

Anguilla anguilla L. Biochemical and Genotoxic Responsesto Benzo[a]pyrene

V. L. Maria,*�-�� A. C. Correia,- and M. A. Santos*��*Animal Physiology/Ecotoxicology Sector, Department of Biology, and -Microbiology/Genetic Sector, Department of Biology, University of Aveiro,

3810-193 Aveiro, Portugal

Received June 18, 2001

Eels (Anguilla anguilla L.) were exposed for 2, 4, 6, 8, 16, 24,48, 72, 144, and 216 h to 0 (control), 0.3, 0.9, and 2.7 �Mbenzo[a]pyrene (BaP). The biotransformation induced by BaPwas measured as liver ethoxyresoru5n O-deethylase (EROD)activity and cytochrome P450 content, and compared with thegenotoxic e4ects, such as erythrocytic nuclear abnormalities(ENA), and blood and liver DNA strand breaks. The liverexhibited a highly signi5cant EROD activity increase from 2 upto 216 h exposure to 0.9 and 2.7 �M BaP, whereas 0.3 �M BaPexposure induced a signi5cant liver EROD increase from 2 up to144 h. Liver cytochrome P-450 content was signi5cantly in-creased at 8 h to 2.7 �MBaP exposure. Liver DNA integrity wasdecreased at 16 h, from 8 up to 144 h and 8 up to 72 h exposureto 0.3, 0.9, and 2.7 �M BaP, respectively. A signi5cant decreasein blood DNA integrity was observed at 48, 72, 144 h, from 8 upto 72, and from 6 up to 72 h exposure to 0.3, 0.9, and 2.7 �MBaP, respectively. The A. anguilla L. genotoxic response to BaP,measured as ENA induction, was signi5cantly increased at 144 hexposure to 0.3�M BaP. The intermediate BaP concentrationtested (0.9 �M) induced a signi5cant three fold ENA increase at48 and 72 h exposure compared to their controls. The highest BaPconcentration (2.7 �M) induced a signi5cant increase in ENAfrequency at 72, 144 and 216 h exposure. � 2002 Elsevier Science (USA)

Key Words: Anguilla anguilla L.; liver EROD; cytochromeP-450; genotoxicity; DNA strand-breaks; erythrocytic nuclearabnormalities.

INTRODUCTION

Molecular biomarkers can be early warning tools, pre-venting irreversible damage in whole organisms, communi-ties, and ecosystems (Livingstone, 1993; Lopez-Barea, 1995;Lopez-Barea and Pueyo, 1998). Prior to excretion, apolarpollutants are converted into water-soluble derivatives bya wide variety of inducible biotransformation detoxifyingenzymes. Marine pollution biomarkers such as cytochrome

�To whom correspondence should be addressed. E-mail: [email protected].

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0147-6513/02 $35.00� 2002 Elsevier Science (USA)All rights reserved.

P-450 (P450) content and liver ethoxyresoru"n O-deethylase (EROD) activity are induced in "sh by pollutantsand involved in biotransformation (Phase I) (Pacheco andSantos, 1997, 1998, 1999; Lemaire et al., 1996; Goksoyr andHusoy, 1998; Goksoyr and Forlin, 1992; Jaksic et al., 1998).Biotransformation enzymes are also responsible for theactivation of foreign chemicals to reactive intermediatesthat may ultimately damage DNA and a!ect its integrity(Di Guillio et al., 1989), resulting in genotoxicity and car-cinogenicity (Buhler and Williams, 1988) if conjugation(Phase II) fails.Benzo[a]pyrene (BaP) is a ubiquitous environmental pol-

lutant whose metabolic activationmay be followed by DNAdamage (Gamper et al., 1977). This progenotoxic compoundhas received most attention in "sh species, with a primaryfocus on hepatic metabolism and adduct formation(Varanasi et al., 1989; Hofe and Pu!er, 1986; Lemaire et al.,1990). BaP has been shown experimentally to be carcino-genic to "sh following both waterborne (Hawkins et al.,1988) and oral exposures (Hendricks et al., 1985).Genotoxicity biomarkers such as DNA adducts (Harvey

et al., 1997), DNA strand breaks (Everaarts et al., 1993,1998; Everaarts and Sarkar, 1996; Everaarts, 1995),micronuclei (Jaylet et al., 1986; Zoll-Moreaux and Ferrier,1999), and erythrocytic nuclear abnormalities (ENA)(Pacheco and Santos, 1996, 1997, 1998, 1999) assume par-ticular importance in the evaluation of DNA damage. DNAdamage can be observed as di!erent types of lesions, such assingle-strand breaks, double-strand breaks, alkali-labilesites, interstrand cross-links, DNA}protein cross-links, andprotein-associated strand breaks (Kohn, 1986; Russo et al.,1989).Several biochemical techniques, based on di!erent mech-

anisms and performed under di!erent conditions, have beendeveloped for the DNA strand break measurement. Alka-line DNA unwinding by chromatography on hydroxylapa-tite (Ahnstrom, 1988; Ahnstrom and Erixon, 1981) andalkaline elution of DNA are among these techniques(Brunborg et al., 1988; Kohn, and Grimek-Ewig, 1973;

87A. anguilla RESPONSES TO BENZO[a]PYRENE

Kohn et al., 1981). The alkaline DNA unwinding techniqueis based on renatured and denatured DNA discrimination.Previous studies have demonstrated a signi"cant total

EROD induction in juvenile eels exposed for 3 days to 0.9and 2.7 �M BaP, whereas ENA frequency was increasedonly at 0.9 �M BaP (Pacheco and Santos, 1997). Addition-ally, adult eels exposed for 8, 16, and 24 h to 2.7 �M BaPdemonstrated a signi"cant EROD increase at 20 and 253C(Pacheco and Santos, 2001).The above results obviously need more thorough study

concerning the relation between liver P450, EROD, ENA,and blood and liver strand breakage induction in adult eelsexposed to 0.3, 0.9, 2.7 �MBaP for longer time periods suchas 2, 4, 6, 8, 16, 24, 48, 72, 144, 216 h. These types ofexperiments will allow determination of the relationshipbetween Phase I biotransformation enzymes and di!erentlevels of genotoxicity biomarkers such as DNA strandbreaks and ENA.

MATERIALS AND METHODS

Chemicals

All chemicals were of analytical grade, obtained fromSigma Chemical Co. (St. Louis, MO), Boehringer}MannheimGmbH (Germany), and E. Merck}Darmstadt (Germany).Benzo[a]pyrene was obtained from Sigma.

Fish

Anguilla anguilla L. (eel), with an average weight of 50 g,were collected from the Aveiro lagoon. The eels were trans-ported in anoxia and acclimated to laboratory conditions inaerated, "ltered, and dechlorinated tap water in 200-Laquaria for 1 week, at 203C.

Experimental Protocols

The experiment took place in aquaria with 80-L of cleanand dechlorinated tap water. The eels were not fed duringthe experiment. Eels were exposed for 2, 4, 6, 8, 16, 24, 48, 72,144, and 216 h to 0 (control), 0.3, 0.9, and 2.7 �MBaP. Eachexperiment was carried out using test groups of "ve eels(n"5). The BaP was previously dissolved in 1 ml ofdimethyl sulfoxide (DMSO) and added to the water of theexperimental aquaria. The BaP solutions were not replacedduring the entire exposure period. Fish were killed by de-capitation and blood and liver were sampled. Each liver wasdivided into to halves, with one stored at !203C afterbeing immediately frozen in liquid nitrogen and the otherone stored in TNES}urea bu!er with proteinase K solution("nal concentration, was 0.8mg/ml) for DNA isolation(Genomic DNA Puri"cation Kit, Fermentas). The freshblood samples were prepared for DNA isolation and storedat 43C.

Genotoxicity Assays

Genotoxicity was tested using the ENA and the DNAalkaline unwinding assay. ENA test was carried out in eelmature erythrocytes, according to the procedures of Schimd(1976), Carrasco et al. (1990), and Smith (1990) as adaptedby Pacheco and Santos (1996). Each group "nal result waspresented as the mean value (%o) of the sums for the all theindividual lesions observed, and scored in 1000 cells per "shblood smear. The blood and live r DNA integrity measure-ment (%) was performed according to Rao et al. (1996), withminor modi"cations. Data from blood and liver DNA alka-line unwinding technique are expressed as F values (%),which represent the fraction of double-stranded DNApresent following alkaline unwinding.

EROD Assay

Microsomes were obtained according to Lange et al.(1992), and Monod and Vindimian (1991) and adaptedby Pacheco and Santos (1998). Liver EROD activity wasmeasured as described by Burke and Mayer (1974).

Cytochrome P-450 Content

P-450 content was quanti"ed by measuring the 490 to450-nm absorbance spectrum as described by Hermenset al. (1990).

ALT Assay

Liver alanine transaminase (ALT) activity was measuredas described by Reitman and Frankel (1957) in the super-natant resulting from liver microsomal isolation as sugges-ted by Chambers and Yarbrough (1979) and Santos et al.(1990).

Protein Measurement

Microsomal protein content and supernatant proteinconcentration were determined according to the Biuretmethod (Gornall et al., 1949), using bovine serum albumin(E. Merck}Darmstadt) as a standard.

Statistical Analysis

The results are expressed as means $ standard errors(SE) and statistical analysis was performed using a two-tailed Student t test (Bailey, 1959).

RESULTS

¸iver EROD and A¸¹ ResponsesThe A. anguilla L. biotransformation response to BaP,

measured as liver EROD activity displayed a signi"cant

FIG. 1. A. anguilla L. liver EROD activity induction (pmol/ min/ mg protein) after 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h exposure to benzo[a]pyrene(BaP), 0, 0.3, 0.9, and 2.7 �M. Values represent mean $ SD. Di!erences from control: (O) P(0.01 (�) P(0.001.

FIG. 2. A. anguilla L. liver ALT (alanine transaminase, U/g) activityafter 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h exposure to benzo[a]pyrene(BaP), 0, 0.3, 0.9, and 2.7 �M. Values represent mean $ SD. Di!erencesfrom control: (*) P(0.05; (O) P(0.01.

88 MARIA, CORREIA, AND SANTOS

increase after 2 h exposure to 0.3 (P(0.001), 0.9(P(0.001), and 2.7 (P(0.001) �M, when compared totheir controls (Fig. 1). Furthermore, 0.9 and 2.7 �M BaPrevealed a signi"cant hepatic EROD activity increase from2 up to 216 h exposure. However, 0.3 �M BaP at 216 hexposure did not induce a signi"cant liver EROD activityincrease. Maximum EROD induction was observed at 16 hexposure to 0.3 (17-fold) and 0.9 �MBaP (26.4-fold), where-as 2.7 �M BaP induced maximum (15-fold) liver ERODactivity at 72 h exposure (Fig. 1). Liver ALT activity signi"-cantly decreased at 24 (P(0.02) and 72 (P(0.05) h expo-sure to 0.3 and 2.7 �M BaP (Fig. 2).

Liver Cytochrome P-450 Content

Eel liver P450 content (Fig. 3) signi"cantly decreased at8 h exposure (P(0.02) to 2.7 �M BaP. However, it in-creased in a signi"cant (P(0.01) manner from 8 up to 72 hexposure to 2.7 �M BaP and remained high from 72 up to216 h exposure. The 0.3 and 0.9 �M BaP exposure did notsigni"cantly induce cytochrome P-450 during the experi-mental time length, when compared to their controls.

Genotoxicity Responses

A signi"cant decrease in blood DNA integrity (Fig. 4A) at48 (P(0.01), 72 (P(0.02), and 144 (P(0.05) h exposure

was observed in A. anguilla L., at 0.3 �M BaP, when com-pared to their controls. Furthermore, a signi"cant bloodDNA integrity decrease from 8 (P(0.001) up to 72(P(0.01) h was observed at 0.9 �M BaP. Moreover,2.7 �M BaP promoted a signi"cant decrease in blood DNA

FIG. 3. A. anguilla L. liver cytochrome P-450 content (nmol/mg pro-tein) after 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216 h exposure to benzo[a]pyrene (BaP), 0, 0.3, 0.9, and 2.7 �M. Values represent mean $ SD.Di!erences from control: (#) P(0.02

FIG. 4. A. anguilla L. (A) blood DNA integrity (%) and (B) ENAfrequency (%o) after 2, 4, 6, 8, 16, 24, 48, 72, 144, and 216h exposure tobenzo[a]pyrene (BaP), 0, 0.3, 0.9, and 2.7 �M. Values represent mean$ SD. Di!erences from control: (*) P(0.05; (#) P(0.02; (O)P(0.01; (�) P(0.001.

89A. anguilla RESPONSES TO BENZO[a]PYRENE

integrity from 6 (P(0.05) up to 72 (P(0.001) h exposurecompared to control (Fig. 4A).Liver DNA integrity (Fig. 5) was decreased at 16 h

(P(0.001), from 8 (P(0.05) up to 144 (P(0.05) h, and8 (P(0.001) up to 72 (P(0.001) h exposure, respectively,to 0.3, 0.9, and 2.7 �M BaP, when compared to theircontrols.The A. anguilla L. genotoxic response to BaP, measured

as ENA induction (Fig. 4B), was signi"cantly increased at144 h exposure to 0.3 �M BaP (P(0.05). The ENAmaximum induction (4.2-fold compared to control) wasobserved at 0.3 �M BaP for 216 h exposure (P(0.0.01).The intermediate BaP concentration tested (0.9 �M) in-duced a signi"cant 3-fold ENA increase at 48 (P(0.001)and 72 (P(0.001) h exposure compared to their controls(Fig. 4B). The highest BaP concentration (2.7 �M) induceda signi"cant increase in ENA frequency at 72 (P(0.01), 144(P(0.001), and 216 (P(0.001) h exposure, when com-pared to their controls. Therefore, until 24 h exposure noneof the tested concentration induced a signi"cant ENAincrease (Fig. 4B).An overall analysis of ENA response (Fig. 4B) as a

function of time demonstrates that the lowest concentration(0.3 �M BaP) induced a signi"cant increase from 144(P(0.05) up to 216 (P(0.001) h exposure. However,0.9 �M BaP demonstrated a signi"cant ENA increase from24 up 48 h (P(0.02), which remained constant at 72 hexposure. An abrupt signi"cant ENA frequency decrease(P(0.001) at 144 h 0.9 �M BaP exposure was observed,and remained low. Furthermore, the ENA frequency signi"-cantly increased 2.6-fold at 2.7 �MBaP until 72 h (P(0.05)and remained high up to 216 h exposure.

DISCUSSION

Several studies have reported that BaP (Wolkers et al.,1996; Peters et al., 1997; Pacheco and Santos, 1997, 2001) isa liver EROD inducer. BaP is a progenotoxin, i.e., a chem-ical substance that must be metabolically activated tobecome a DNA-damaging agent (Johnson, 1992). DNAlesions are the "rst event of a carcinogenetic process in-duced by a genotoxic agent, and the formation of micronuc-lei gives information about the promutagenic character ofthese lesions (Robbiano et al., 1999).Our experimental results demonstrated that A. anguilla L.

liver EROD signi"cantly increased from 2 up to 144 hexposure to 0.3 �M BaP compared to their controls. How-ever, the most abrupt liver EROD increase was found be-tween 8 and 16 h, with a signi"cant decrease at 24 h

FIG. 5. A. anguilla L. liver DNA integrity (%) after 2, 4, 6, 8, 16, 24, 48,72, 144, and 216 h exposure to benzo[a]pyrene (BaP), 0, 0.3, 0.9, and2.7 �M. Values represent mean $ SD. Di!erences from control: (*)P(0.05; (#) P(0.02; (O) P(0.01; (�) P(0.001.

90 MARIA, CORREIA, AND SANTOS

followed by an increase from 24 up to 48 and 72 h witha new decrease between 72 and 144 until 216 h exposure to0.3 �M BaP.In relation to 0.9 �M BaP exposure, liver EROD activity

signi"cantly increased from 2 up to 216 h, compared to theircontrols. However, liver EROD suddenly increased between8 and 16h, followed by a signi"cant decrease at 24 h anda second non signi"cant increase from 48 to 72 h, and thena signi"cant decrease from 72 up to 144 and 216h exposureto 0.9�M BaP. Finally, 2.7 �M BaP exposure signi"cantlyincreased liver EROD activity from 2 up to 216 h exposure,compared to their controls. Liver EROD activity also in-creased over time from 6 up to 24, 48, and 72 h and signi"-cantly decreased from 72 up to 144 and 216 h. According toPacheco and Santos (1997), dose}response studies carriedout with glass eels demonstrate a signi"cant liver ERODactivity increase after 72 h exposure to 0.9 and 2.7 �M BaP.Despite the nonsigni"cant oscillations of liver P450 overtime in general, the results follow the liver EROD patternpro"le.The above results obtained from A. anguilla L. BaP expo-

sure demonstrate that low concentrations (0.3 �M) inducea slight increase in liver EROD activity, whereas highconcentrations (2.7 �M) have inhibitory e!ects, and "nallyintermediate concentrations, such as 0.9 �M, are stronginducers. However, liver P450 results seem inconsistent.Previous studies have demonstrated that several inducers

of CYP1A synthesis could also inhibit mixed-functionoxidase (MFO) catalytic activity (EROD or aryl hydrocar-bon hydroxylase (AHH)) (Stegeman and Hahn, 1994). Isalso known that high polycyclic aromatic hydrocarbon of(PAH) doses �-naphtho#avone (PAH-like substance) and

BaP can inhibit liver EROD catalytic activity (Goosch et al.,1989; Haash et al., 1993). According to these investigators,the P450 enzymes act either by competitive inhibitionor by a mechanism-based inactivation, in which the inhibi-tor is metabolized by the P450 into a product that covalent-ly modi"es the active site, and thereby inactivates theenzymeThe biotransformation process of most chemical com-

pounds is generally understood to be a detoxifying mecha-nism. Nevertheless, PAH metabolization frequentlyconverts these xenobiotics into highly toxic reactive inter-mediates that are mutagenic or carcinogenic to "sh (Steinet al., 1990; Van der Oost et al., 1994). Previous investiga-tions concerning BaP identi"ed it as strong promutagen in"sh (Hawkins et al., 1990; Pacheco and Santos, 1997).Current results demonstrated signi"cant decreases in

liver DNA integrity from 8 up to 16 h with a fast recoverybetween 16 and 24 h that remained stable up to 216 h for0.3 �M BaP. Blood DNA integrity decreased at 48, 72, and144h, and then signi"cantly increased from 144 up to 216 hcompared to their controls, whereas ENA results demon-strated a signi"cant increase at 144 and 216 h exposure to0.3 �M BaP. Liver DNA integrity decreased at 8, 16, 24, 48,72, and 144 h and recovered at 216 h 0.9 �MBaP exposure,compared to their controls. The liver DNA integrity in-crease at 216 h is signi"cant, when compared to 144 h expo-sure to 0.9 �M BaP. Blood DNA integrity decreased at 8,16, 24, 48, 72 h and then increased at 144 and 216 h, com-pared to their controls. Blood DNA integrity signi"cantlyincreased between 72 and 216 h exposure to 0.9 �M BaP.ENA signi"cantly increased at 48 and 72 hours, decreasingat 144 and 216 h compared to their controls, with a signi"-cant increase between 24 and 48, 72 and abruptly decreasingat 144 h exposure to 0.9 �M BaP.Regarding 2.7 �M BaP exposure, liver DNA integrity

decreased from 8 up to 72 h and increased at 144 and 216hcompared to their controls. Liver DNA integrity also in-creased from 72 up to 216 h. BloodDNA integrity decreasedat 6, 8, 16, 24, 48, 72, and 144 h and then increased at 144and 216 h compared to their controls. Blood DNA integrityincreased between 48 and 216 h exposure to 2.7 �M BaP.ENA frequency signi"cantly increased at 72 and 144 h expo-sure to 2.7 �M BaP compared to their controls. ENA in-creased from 2 up to 16, 24, and 48 h with a sudden increaseat 72, 144, and 216 h exposure to 2.7 �M BaP.Global analysis of A. anguilla L. liver DNA integrity

demonstrates that 0.3 �M BaP has a slight genotoxic e!ectcompared to 0.9 and 2.7 �M BaP. Additionally, recoverybetween 144 and 216 h demonstrates that 0.9 �MBaP is lessgenotoxic than 2.7 �M BaP. Blood DNA integrity resultsdemonstrate a high genotoxic e!ect of 2.7 followed by 0.9and 0.3 �M BaP, since a strong recovery was observed forthe previous two concentrations between 72 and 216hexposure.

91A. anguilla RESPONSES TO BENZO[a]PYRENE

ENA frequency results in A. anguilla L. exposed to 0.3,0.9, and 2.7 �M BaP demonstrate that low concentrations(0.3 �M) induce slight e!ects, with a maximum e!ect at216 h, compared to high concentrations (2.7 �M), whereearly increased ENA frequencies decrease slightly between72 and 216 h. An intermediate concentration such as 0.9 �MBaP is a strong ENA inducer between 48 and 72 h, withrecovery occurring between 72 and 216 h exposure.

CONCLUSIONS

The following results indicate that adult eels can be asensitive biological model for BaP genotoxicity and bio-transformation assessment:1. The genotoxic potential of BaP was demonstrated by

blood and liver DNA integrity decrease and ENA frequencyincrease in eel;2. A decrease in blood and liver DNA integrity was

observed before ENA frequency increase was detected afterBaP exposure;3. High BaP genotoxicity seems to be associated with

liver EROD induction for concentrations which are notcytotoxic;4. High BaP concentrations and long exposure periods

induce important cytotoxic e!ects without cell membranedisruption.

ACKNOWLEDGMENTS

The research work for this article was supported by PRAXISXXI/CAN/C/BIA/175/96. V. L. M. was supported by Grant PRAXISXXI/BD/18254/98. The authors thank the CZCM and CBC for support.

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