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Heat-shock protein (Hsp70) and cytochrome P-450 (CYP1A) in the whitemullet Mugil curema (Pisces:Mugilidae) as biomarkers to assessenvironmental quality in coastal lagoonsJulián Ríos-Sicairos a; Miguel Betancourt-Lozano a; Beatriz Leal-Tarín a; Rubí Hernández-Cornejo a;Gabriela Aguilar-Zárate a; Luz María García-De-La-Parra a; Jesús N. Gutiérrez b; Facundo Márquez-Rocha c; Alejandra García-Gasca a

a Centro de Investigación en Alimentación y Desarrollo, Unidad Mazatlan, Mazatlán, Mexico b Centrode Investigaciones Biológicas del Noroeste, La Paz, Mexico c Centro de Investigación Científica yEducación Superior de Ensenada (CICESE), Ensenada, Mexico

Online publication date: 15 December 2009

To cite this Article Ríos-Sicairos, Julián, Betancourt-Lozano, Miguel, Leal-Tarín, Beatriz, Hernández-Cornejo, Rubí,Aguilar-Zárate, Gabriela, García-De-La-Parra, Luz María, Gutiérrez, Jesús N., Márquez-Rocha, Facundo and García-Gasca, Alejandra(2010) 'Heat-shock protein (Hsp70) and cytochrome P-450 (CYP1A) in the white mullet Mugil curema(Pisces:Mugilidae) as biomarkers to assess environmental quality in coastal lagoons', Journal of Environmental Scienceand Health, Part A, 45: 1, 68 — 74To link to this Article: DOI: 10.1080/10934520903388855URL: http://dx.doi.org/10.1080/10934520903388855

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Journal of Environmental Science and Health Part A (2010) 45, 68–74Copyright C© Taylor & Francis Group, LLCISSN: 1093-4529 (Print); 1532-4117 (Online)DOI: 10.1080/10934520903388855

Heat-shock protein (Hsp70) and cytochrome P-450 (CYP1A)in the white mullet Mugil curema (Pisces:Mugilidae) asbiomarkers to assess environmental quality in coastallagoons

JULIAN RIOS-SICAIROS1, MIGUEL BETANCOURT-LOZANO1, BEATRIZ LEAL-TARIN1, RUBIHERNANDEZ-CORNEJO1, GABRIELA AGUILAR-ZARATE1, LUZ MARIA GARCIA-DE-LA-PARRA1,JESUS N. GUTIERREZ2, FACUNDO MARQUEZ-ROCHA3 and ALEJANDRA GARCIA-GASCA1

1Centro de Investigacion en Alimentacion y Desarrollo, Unidad Mazatlan, Mazatlan, Mexico2Centro de Investigaciones Biologicas del Noroeste, S.C., La Paz, Mexico3Centro de Investigacion Cientıfica y Educacion Superior de Ensenada (CICESE), Ensenada, Mexico

Biomarkers have been useful tools to monitor some effects of pollution in coastal environments. Hepatic expression of heat-shockprotein 70 (Hsp70) and cytochrome P450 1A (CYP1A) were analyzed in white mullet (Mugil curema) by RT-PCR from July, 2005until July, 2006 in three coastal lagoons located in the southern Gulf of California, Mexico. These three coastal systems receivecontaminants derived from local anthropogenic activities. Heat-shock proteins function to maintain protein integrity in the presenceof stressors (such as heat or chemicals) and can be used as biomarkers of homeostatic alterations in polluted environments, whereascytochrome P450 family members participate in steroid hormone synthesis and metabolism, and in xenobiotic transformation as adetoxification mechanism. The expression levels of both genes showed consistency in time and space, and presented a high overallcorrelation (r = 0.731, P < 0.001). Regardless of a high individual variability, both genes presented higher expression levels in theUrias Estuary (P < 0.001 and P < 0.05 for CYP1A and Hsp70, respectively), which was considered the most polluted among thethree systems, especially during the rainy season (summer to fall). Gene expression levels were significantly associated with non-halogenated hydrocarbon concentrations in sediments during the sampling period (r = 0.686, P = 0.019 for CYP1A and r = 0.91,P < 0.001 for Hsp70), suggesting that both genes respond to chemicals in the environment. The results indicate that Mugil curema isa good candidate species to implement biomonitoring programs in tropical coastal environments.

Keywords: Hsp70, CYP1A, Mugil curema, cellular stress, pollution, coastal environments.

Introduction

In the environmental context, biomarkers are consideredsensitive indicators of early effects of xenobiotics in ex-posed organisms, and have been useful tools to monitorsome effects of pollution in coastal environments.[1] Dur-ing the last few decades, aquatic organisms have been usedas sentinel species to assess the effects of xenobiotics un-der experimental conditions and in polluted environments.Heat-shock proteins function to maintain protein integrityin the presence of stressors (such as heat or chemicals)[2],and can be used as biomarkers of homeostatic and en-docrine alterations in the environment.[3,4] Low-specificity

Address correspondence to Alejandra Garcia-Gasca, Centro deInvestigacion en Alimentacion y Desarrollo, Mazatlan, Mexico.E-mail: [email protected]

biomarkers (such as Hsp70) have been studied togetherwith chemical analyses and other biomarkers at differentlevels of biological organization.

For instance, Triebskorn et al.[5] analyzed sedimentsand wild fish exposed to industrial and urban compounds,finding a relationship between Hsp70 induction and theconcentration of some chemicals in the environment.Furthermore, Wang et al.[6] studied the expression ofseveral heat-shock proteins (Hsp70, Hsp27, Hsp47, andHsp60) in gold fish (Carassius auratus) inhabiting a mod-erately polluted lake (Gaobeidian Lake) in Beijing, China,concluding that Hsp gene expression is season-dependent,and that Hsp30 and Hsp70 were up-regulated duringthe winter. Between the two, Hsp30 showed differentexpression levels between the polluted lake and the cleanerreference site (Huairou Reservoir). In another study, thestriped mullet, Mugil cephalus, was collected from two

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Biomarkers in white mullet in coastal lagoons 69

different estuaries: one polluted (Ennore) and the otherunpolluted (Kovalam), during a 2-year period.[7,8]

The polluted estuary had elevated levels of nitrite andnitrate, and Hsp70 expression was significantly higherin fish from this estuary compared with fish from theunpolluted site. The authors concluded that Hsp70 in-duction may be protecting the fish against nitrativestress. On the other hand, several cytochrome P450 fam-ily members participate in steroid hormone synthesisand metabolism[9,10], and in xenobiotic transformationas a detoxification mechanism.[9] CYP1A mRNA and 7-ethoxyresorufin O-deethylase (EROD) activity inductionare common biomarkers to monitor exposure to toxic sub-stances in fish, such as polycyclic aromatic hydrocarbons(PAHs) and polychlorobiphenyls (PCBs).[11,12]

For example, industrial effluents induce EROD activityin the flat fish, Pleuronectes americanus.[13] Also, van Veldet al.[14] reported the presence of PAHs in sediments andhigh cytochrome P450 activity in fish liver and intestine.

White mullet (Mugil curema) have been captured in Mex-ico since the early 60s, surpassing 10,000 tons per yearduring the 1980s; although since 1999, the average cap-ture has decreased to 9,000 tons per year.[15] The stateof Sinaloa alone (located in the centre-south of the Gulfof California) sustains 48% of the national fishery, whichrepresents an important source of food and employmentfor the local population.[15] M. curema spends most ofits life cycle in shallow waters, such as estuaries, makingit an accessible resource for local fishermen. The repro-ductive season spans from March to August[16]; sexuallymature fish leave the estuary to spawn, and juveniles en-ter the estuaries where they reach adulthood and sexualmaturity. [17−19]

In Mexico, some coastal systems have been reservoirsof large amounts of contaminants for many years, facingsevere environmental problems such as the loss of biodi-versity. The white mullet is one of the species that can befound in these impacted environments, and therefore wasselected as a sentinel species due to its close association toestuarine sediments (detritivorous feeder), use for humanconsumption, tolerance to changing environmental condi-tions, and its permanent residence in coastal lagoons witha wide geographic distribution.[19]

This study is part of a one-year biomonitoring pro-gram of coastal systems in Northwest Mexico based onthe measurement of hepatic expression of Hsp70 andCYP1A in white mullet by RT-PCR from July, 2005 toJuly, 2006 in three coastal lagoons impacted by differ-ent sources of contaminants of anthropogenic origin. Inthe present study, non-halogenated hydrocarbon concen-trations (which include petroleum-derived hydrocarbons)were measured in sediments from the three coastal lagoonsas indicative of the degree of pollution. Correspondencesbetween individual gene expression levels and overall geneexpression levels with hydrocarbon concentrations wereassessed.

Fig. 1. Study Area. The three systems (Ensenada del Pabellon,Urias Estuary, and Teacapan Estuary) are located in the State ofSinaloa, at the south of the Gulf of California.

Materials and methods

Study area

The three coastal systems are located in the state of Sinaloa,Mexico (Fig. 1) and present different types of effluents fromanthropogenic activities. The Urias Estuary is a small (6.18square miles), highly polluted system, located in the Port ofMazatlan (between 23◦09′–23◦13′ N and 106◦18′–106◦25′W), which receives a variety of urban and industrial efflu-ents, together with residues from fishing vessels, tourism,a thermoelectric plant, agriculture and aquaculture farms.Ensenada del Pabellon (located between 24◦19′–24◦35′ Nand 107◦28′–107◦45′ W) is a larger system (48.65 squaremiles), moderately polluted, which receives millions of cu-bic meters per year of effluents from the cities of Culia-can and Navolato via the Culiacan River. Furthermore,it receives effluents from almost 667,200 acres of inten-sive agriculture and residual effluents from two sugar pro-cessing plants. The Teacapan Estuary is part of a largersystem (579.15 square miles) called Teacapan-Agua Brava-Marismas Nacionales situated between 22◦04′–22◦35′ Nand 105◦20′–150◦50′ W. It is the least polluted of the threesystems, and receives effluents from agriculture, cattle andmining activities through two main rivers (Baluarte andCanas), along with several tributary creeks.

Sample collection

Mullets were collected every 4 months from July, 2005 toJuly, 2006 using a casting net. A total of 25 to 30 specimens


70 Rıos-Sicairos et al.

were sampled per system per month, except for July, 2005in Ensenada del Pabellon, in which animals were notfound. Animals were measured and weighed. The liver waspreserved in RNAlater (Ambion UNIPARTS, Mexico) andthe gonad was preserved in 4% formaldehyde to performmolecular and histological analyses, respectively. Sampleswere transported to the laboratory for further processing.

RNA isolation, cDNA synthesis and PCR amplification

Total RNA was isolated using Trizol reagent (Gibco-Invitrogen, Accesolab Mexico) followed by DNAse I treat-ment. cDNA synthesis was performed at 45◦C with 5 µg oftotal RNA, MMLV reverse transcriptase (Promega, UNI-PARTS Mexico), and random primers (Promega, UNI-PARTS Mexico). PCR amplification for M. curema Hsp70and CYP1A genes was carried out with the followingprimers: Hsp70-forward 5′ AAG AGC ACT GGC AAGGAG AAC AA 3′ and Hsp70-reverse 5′ CTG ATC TTGCCC TTC AGC TTC TC 3′; cyp-forward 5′ ACC ATGATC CTG AGC TGT GGA AA 3′ and cyp-reverse 5′ GAAGTG CAG CTT TTG GAT GAG GA 3′. In both cases,PCR conditions were performed as follows: one cycle at94◦C for 1.5 min, and 30 cycles at 94◦C 1 min, 60◦C 1 min,72◦C 1 min, rendering products of 211 bp and 200 bp forHsp70 and CYP1A, respectively.

Both gene fragments were previously cloned in ourlaboratory; initial PCR products were amplified usingdegenerate primers. They were purified and ligated into apGEM-T cloning vector (Promega, UNIPARTS Mexico).Transformations were performed using DH5α competentcells (Gibco-Invitrogen, Accesolab Mexico). Positive cloneswere sequenced using a LICOR IR2 DNA sequencer. Bothsequences were registered in GenBank (accession numbersDQ310712 and AY827103 for Hsp70 and CYP1A, respec-tively). Primers for M. curema 28S rRNA gene (mc28S-F 5′GCG GTA CAC CTG TCA AAC GT AAC 3′ and mc28S-R GAC ACC TCC TGC TTA AAA CCC AAA 3′) weredesigned to render a product of 178 bp as an endogenouscontrol of RNA integrity. Semi-quantitative expressionanalysis was performed by agarose gel electrophoresisstained with ethidium bromide. All genes (Hsp70, CYP1Aand 28S rRNA) were amplified using the same cDNAsample from each fish, visualized in a 2% agarose gel inTris-Acetate (TAE) buffer, stained with ethidium bromide,digitalized and analyzed in an Epi Chemi II gel documen-tation system from UVP, using the Labworks Imaging andAnalysis software (UVP BioImaging Systems). Expressionlevels are represented as Hsp70 or CYP1A fluorescenceintensity values normalized with 28S rRNA values. Thisanalysis was performed individually in all samples.

Histology

In order to characterize gonad development and gender,gonad samples were preserved in 4% formalin for 24 hours,

then washed in running water, and dehydrated with in-creasing concentrations of ethanol. Samples were processedby conventional histology according to Lightner[20], andstained with Hematoxylin-Eosin. Observations were per-formed by light microscopy. Gonad developmental stageswere characterized according to Solomon et al.[17]

Non-halogenated hydrocarbon determination

For solid–liquid extraction, 5 g of sediments were extractedtwice with dichloromethane. The organic phase was passedthrough NaSO4 and concentrated to 0.2 mL. Hydrocarbondetermination in sediment extracts was performed accord-ing to the EPA 1815D method by gas chromatography (GC)(Perkin Elmer Autosystem) with a flame ionizing detector(FID) using an Alltech AT-1301 column (30 m × 0.2 µm ×0.25 mm DI). Temperature programming conditions wereas follows: initial temperature of 200◦C for 2 min, thenincreased at 6◦C/min to 280◦C, hold for 10 min.

This method is used to determine non-halogenated,volatile organic compounds, semi-volatile organic com-pounds, and petroleum-derived hydrocarbons, which in-clude gasoline range organics (GRO) corresponding to therange of alkanes from C6 to C10 and covering a boilingpoint range of approximately 60◦C to 170◦C, and dieselrange organics (DRO) corresponding to the range of alka-nes from C10 to C28 and covering a boiling point rangeof approximately 170◦C at 430◦C. The lineal range wasdetermined, as well as the minimum detectable concentra-tion and reproducibility. Quantification was performed byexternal standardization. Since diesel and gasoline are dif-ferent compounds from those in the samples, the extractof the most concentrated sample was used as a standardaccording to the method. From this extract (24,400 ppm),dilutions were prepared to reach the minimum detectableconcentration (0.64 ppm). Standards for n-alkanes C10 toC28 were also used.

Statistical analyses

Gene expression data was square root-transformed and atwo-way ANOVA with a Holm-Sidak multiple compar-isons test was performed to identify differences betweenlocalities and months. Correspondences among variableswere computed by Pearson correlation analysis. The signif-icance level was P < 0.05.

Results and discussion

Total length and weight of collected fish were 27.3 ± 1.4 cmand 187.1 ± 32.7 g, respectively. Most collected fish werefemales; overall sex ratio was 6:1. The reproductive seasonwas observed from March to July, with a peak from Aprilto June, consistent with Collins[19] and Solomon et al.[16]



The expression of hepatic Hsp70 and CYP1A genes wasanalyzed by RT-PCR. The heat-shock protein Hsp70 as-sists other proteins in damage repair as response to cellu-lar stress, playing an important role in cytoprotection.[21]

Hsp70 induction has been identified in different fish speciesand cells exposed to adverse environmental conditions orchemicals associated to homeostatic and macromolecularalterations.[3,22] CYPA1 participates in xenobiotic trans-formation resulting in bioactive metabolites which areconjugated with products derived from the endogenousmetabolism, and then eliminated from the cell.[23] Severalstudies have reported the induction of CYP1A in aquaticanimals exposed to environmental contaminants like PAHsand PCBs.[11,24−26]

A relationship between CYP1A and Hsp70 inductionhas already been observed under experimental conditionsby Weber et al.[27] The authors measured both gene prod-ucts by Western blot in liver and ovary of the rainbowtrout (Oncorhynchus mykiss) exposed for 4 days to beta-naphthoflavone (BNF). Protein levels were determined bydensitometry. The authors found that hepatic CYP1A andHsp70 proteins significantly increased in fish treated withBNF. In another study, quantitative expression of CYP1AmRNA was compared to EROD activity in the Europeaneel (Anguilla anguilla) exposed to BNF in a time-courseexperiment.[28] The authors found that CYP1A mRNA lev-els were higher at day 1, especially with the highest concen-tration of BNF (3000 nM) while EROD activity was higherat day 1 only in animals exposed to lower concentrations ofBNF (187.5 and 750 nM), and at day 3 in animals exposedto the highest concentration of BNF (3000 nM), indicat-ing a shift between CYP1A mRNA expression and ERODactivity.

In the current study, both hepatic CYP1A and Hsp70 ex-pression in wild mullets showed good correspondence withnon-halogenated hydrocarbon concentrations (r = 0.686,P = 0.019 for CYP1A and r = 0.91, P < 0.001 for Hsp70)in the three systems (Figs. 2 and 3). Despite the high individ-ual variability in gene expression, overall expression levelswere significantly higher in the Urias Estuary (P < 0.001and P < 0.05 for CYP1A and Hsp70, respectively) com-pared to the other two sites, whereas Ensenada del Pabellonwas significantly higher than Teacapan (P < 0.001 and P =0.008 for CYP1A and Hsp70, respectively). The Urias Es-tuary is the smallest system and, because of its location, re-ceives discharges from a variety of sources (fish-processingplants, a thermoelectric plant and untreated domestic andindustrial wastes among others). The volume of the efflu-ents may increase during the rainy season (June to October)when higher levels of non-halogenated hydrocarbons wererecorded, corresponding to higher levels of CYP1A andHsp70 expression (P < 0.05; Fig. 2B). Previous studies inthis system revealed the presence of heavy metals[29−32], andorganic compounds such as polycyclic aromatic hydrocar-bons (PAHs) and organochlorine pesticides.[33] Compar-atively, Ensenada del Pabellon and Teacapan are larger

Fig. 2. . Relative hepatic mRNA expression (arbitrary units) ofCYP1A and Hsp70 in mullets from: (A) Ensenada del Pabellon,(B) Urias Estuary, (C) Teacapan Estuary. Total hydrocarbon con-centrations (mg g−1) in sediments are indicated as a continuousline. Different letters indicate significant differences in gene ex-pression over time, lowercase letters refer to Hsp70 expression,and capital letters refer to CYP1A expression.

systems, and although they receive effluents from agricul-ture, aquaculture, and cattle farming, the dilution factorand water exchange are higher than in Urias. Significantdifferences in CYP1A expression were detected in Ense-nada del Pabellon and Teacapan during the spring of 2006(P = 0.006) and summer of 2006 (P < 0.001), respectively(Figs. 2A and C). Hsp70 also showed significant differencesin gene expression in Teacapan during the summer of 2006(P < 0.05) (Fig. 2C). Previous reports indicate the pres-ence of agrochemicals (such as DDT, endosulfan, lindane,



Fig. 3. Correlation analysis of gene expression (arbitrary units)and hydrocarbon concentration (mg g−1): (A) CYP1A (r = 0.686,P = 0.019), (B) Hsp70 (r = 0.91, P < 0.001) (n = 11).

and methyl-parathion) in mussel tissues from Ensenadadel Pabellon[34], and organochlorine pesticides (such as lin-dane) in clam and shrimp tissues from Teacapan[33,35], aswell as Hg in fish tissues.[36,37]

Overall gene expression data over time indicate thatCYP1A transcription was significantly higher during thesummer of 2006 (P < 0.001), whereas Hsp70 did not showsignificant differences (Fig. 2). Nevertheless, our resultsshow a significant correlation between individual Hsp70and CYP1A levels in each of the three systems (r = 0.622,P < 0.001 for Ensenada del Pabellon; r = 0.724, P < 0.001for Urias Estuary; r = 0.738, P < 0.001 for Teacapan Es-tuary) (Fig. 4) and overall (r = 0.731, P < 0.001, n = 292)(Fig. 5). Hsp70 is an unspecific biomarker that could belinked to CYP1A since both gene products respond to cel-lular stress and are involved (by different mechanisms) incytoprotection. Based on these results, Hsp70 and CYP1Agene expression in the white mullet correlate with anthro-pogenic contamination in the field, suggesting a response

Fig. 4. Correlation analysis between CYP1A and Hsp70 expres-sion levels (arbitrary units) in: (A) Ensenada del Pabellon (r =0.622, P < 0.001, n = 80), (B) Urias Estuary (r = 0.724, P <

0.001, n = 110), (C) Teacapan Estuary (r = 0.738, P < 0.001,n = 102).

Fig. 5. Overall correlation analysis between CYP1A and Hsp70expression levels (arbitrary units) (r = 0.731, P < 0.001, n = 292).



to complex mixtures of contaminants likely occurring inthe study sites.

Conclusion

Hepatic expression of CYP1A and HSP70 in the white mul-let showed significant correlations with non-halogenatedhydrocarbon concentrations in the three systems. Higherexpression levels and hydrocarbon concentrations were de-tected in the smallest, most polluted system, the Urias Es-tuary, which receives discharges from several sources. Theseresults indicate that it is possible to use the white mullet, M.curema, as a sentinel species, and that both gene productsare good indicators of environmental stress associated withpollution.

Acknowledgments

The authors would like to thank S. Abad, I. Martınez-Rodriguez, and R. Hernandez-Guzman for technical assis-tance, as well as C. Bastidas and D. Herrera for samplingassistance. Thank you to local fishermen Hipolito Holague,Juan Sarmiento, Ines Pardo, Willy, Arturo Jimenez, andDelfino for assistance in the field. This work was fundedby FOSEMARNAT-2004–01-199 grant awarded to AGG,and Fomix Conacyt-Espana J200–142 grant awarded toLMGP.

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