持久性有机污染物对水生动物芳香烃受体通道的毒理机制及其早期监测

周海龙，张林宝，廖春阳，韦双双，郑继平，薛钦昭，*

1．中国科学院烟台海岸带研究所，烟台264003

2．海南大学农学院生物技术系，海口570228

3．中国科学院研究生院，北京100049

Persistent organic pollutants（POPs）are organic compounds of natural or anthropogenic origin that resist photolytic,chemical,and biological degradation. They are low water solubility and high lipid solubility,resulting in bioaccumulation in adipose tissues of living organisms（Mehmetli and Koumanova, 2007）.They are not only toxic,but also prone to long-range transport.Most of them can be classified into three groups:（1）Industrial chemical product such as polychlorinated biphenyls（PCBs）;（2）Combustion and by-products such as polychlorinated dibenzo-pdioxins and dibenzofurans（PCDD/Fs）,and 2,3,7,8-tetrachlorodibenzo-p-dioxin（2,2,7,8-TCDD）;（3）and Pesticides such as dichlorodiphenyl-trichloroethane（DDT）,dihedron,and toxaphene.

POPs can enter aquatic ecosystem in effluent, atmospheric deposition, runoff, and groundwater. They are not only very harmful to the health of aquatic animalsbutalso thatofhumans.Many results show that POPs may impair most systems of human.Forexample,PCBsandOH-PCBsmay damage brain（Kimura-Kuroda et al.,2007）.Polycyclic aromatic hydrocarbons（PAHs）damage the immunity system（Davila et al.,1995）and the reproductive system（Den Besten et al.,1990）,and cause DNA damage（Lemiere et al.,2005）.PCB,PAH and other POPs are the most important risk factor to cause breast cancer（Gammon et al.,2004）,lung cancer（Okona-Mensah et al.,2005）and prostate cancer（Ritchie et al.,2005）.So the widespread occurrence of POPs has attracted considerable attention.In order to avoid or reduce the damagethat caused by POPs,we should comprehensively understand the toxicological mechanism of POPs,as well as related monitoring methods.

1 The regulation mechanism of POPs toxicology

A series of experiments have revealed that aryl hydrocarbon receptor（AhR）pathway plays a pivotal role in the mediation of POPs toxicology in aquatic animals.AhR pathway genes（e.g.AhR,AHRR）have been found in many aquatic animals,and are detectable in many tissues.Their structure and function have been studied in aquatic animals, including killfish,rainbow trout,zebrafish,harbour seal,medaka,red seabream etc..A large body of experiments have revealed that the mechanisms of the AhR-dependent CYP1A1 gene induction（see Fig. 1）.There are four major steps of toxicology regulation,including formation of cytosolic complex, translocation ofAhR,heterodimerization ofAhR, and induction of CYP1A.

1.1 Formation of cytosolic complex

In absence of ligands,AhR is associated with a cytoplasmic protein complex with two molecules of heatshock protein 90（Hsp90）（Perdew,1988）, immunophilin-like protein XAP2（also referred to as AIP or ARA9）（Carver and Bradfield,1997）,and a 23-kDa co-chaperone protein（p23）（Kazlauskas et al.,1999）.Hsp90 is an essential component of the AhR-signaling pathway,and loss of Hsp90 most likely results in an improperly folded or destabilized receptorprotein Hsp90,onesubunitoftheAhR complex, appears to direct proper folding and maintenance of the high affinity ligand binding conformation of the AhR in some species（Soshilov et al.,2006）.

Though the othertwo membersofcytosolic complex are not essential for the AhR signaling,but they play an important role in stabilizing the cytosoliccomplex.Thecapability ofXAP2,also known as AhR interacting protein（Ma and Whitlock Jr,1997）to modulate AhR function has been studied extensively in cell culture systems.XAP2 is capable of stabilizing the AhR, as well as enhancing cytoplasmic localization of the receptor.And it binds to both the AhR and Hsp90 in the receptor complex, and is also capable of binding independently to both Hsp90 and the AhR（Petrulis and Perdew,2002）, but it is not a limiting component in AhR regulation（Hollingshead et al.,2006）.Mechanistically,p23 appears to promote AhR/ARNT/DRE complex formation in an Hsp90-dependent manner by assisting with the heterodimerization of the AhR and ARNT. Further research of Cox and Miller Iii（2004）show that p23 can inhibit Hsp90 ATPase activity,thereby stabilizing ATP-Hsp90-client protein complexes. However,p23 does not interact directly with either the AhR or ARNT（Kazlauskas et al.,1999）.

1.2 Translocation of AhR

Upon binding to aligand（TCDD orother POPs）,the AhR complex translocates into the nucleus and the AhR dissociates from Hsp90 complex to form a heterodimerwith itspartner molecule,ARNT（Poland and Knutson,1982）.AhR has been extensively studied as a mediator of toxicity of a diverse group of xenobiotics,including PCDD/Fs,PCBs,and PAHs etc.（Ma,2001）.AhR is a ligand-activated transcription factor and a member of the basic helix-loop-helix/PER-ARNTSIM family of DNA-binding proteins（Chen and Perdew,1994）,and it contains three well-conserved domainsinvolved in substrate binding.The first domain located in theN-terminalregion ofthe molecule,consists of the bHLH domain found in many transcription factors（e.g.MyoD,c-myc,and Max）（Olson and Klein,1994;Murre et al.,1989; Kadesch,1992）.The second domain is very similar to the Drosophila circadian rhythm gene per and the Drosophila single-minded protein sim and,therefore, is referred to as the PAS domain（Hoffman et al., 1991;Takahashi,1992）.The third domain,located at the C-terminal end of the molecule,is glutamine（Q）-rich.The ligand-binding function apparently resides in the PAS region of AhR（Dolwick et al., 1993）.

In addition,a large body ofliterature has implicated AhR in multiple signal transduction pathways.AhR is known to interact with signaling pathways that are mediated by estrogen receptor and other hormone receptors,hypoxia,nuclear factor B, and retinoblastoma protein（Carlson and Perdew, 2002）.Furthermore,AhR complexes may affect cellular signaling through interactionswith various otherregulatory and signaling proteins,including PAS heterodimerization partners, chaperone and immunophilin-like proteins, protein kinases and phosphatases（Carlson and Perdew,2002）.So we can conclude that AhR not only play a crucial role in the POPs toxicology but also has an important physiological function in aquatic animals.

1.3 Heterodimerization of AhR

When AhR binds to ligand,it is translocated to the nucleus and dissociates from the Hsp90 complex to form a heterodimer with ARNT.The AhR/ARNT heterodimer binds to the xenobiotic response elements（XRE）sequence in the promoter regions of target genes encoding drug-metabolizing enzymes,including CYP1A1,quinone reductase,etc.,and alters their expression（Kikuchi et al.,2003）.ARNT belongs to the bHLH-PAS family.Binding with AhR,ARNT also interacts with SIM1（Single Minded 1）,SIM2（Single Minded 2）,HIF1α（hypoxia-inducible factor 1α）,CHF1（Cardiovascular helix-loop-helix factor 1）and EPAS1（Endothelial PAS domain protein 1）to regulate neurogenesis, the hypoxia response, cardiovascular development and pathological angiogenesis（Mimuraand Fujii-Kuriyama,2003; Swanson,2002;Taylor and Zhulin,1999）.Therefore, ARNT may serve as a central player in regulating these diverse signaling pathways.AHRR is an AhR related protein, and represses the transcription activity of AhR by competing with AhR by heterodimerization with ARNT and subsequently binding to XRE sequence（Mimura et al.,1999）. These results indicate that AhR and AHRR form a regulatory feedback loop （Mimura and Fujii-Kuriyama,2003）.Recently,Evansetal.（2008）propose amechanism ofAHRR action involving“transrepression”of AhR signalling through proteinprotein interactions rather than by inhibition of the formation orDNA binding ofthe AhR-ARNT complex.In the future,targeted knock-down of one or both AHRR proteins by application of morpholino oligonucleotides can be used to further characterize these duplicate zebrafish AHRRs and to elucidate theirpotentialroles in developmentand in the developmental toxicity of chemicals such as TCDD.

Now,although it remains to be studied how AhR and AHRR are involving in the other TCDD-induced biological effects such as teratogenesis and immunosuppression than induction of XMEs,it is well known that these adverse biological effects are caused by untimely activation of gene expression by ligand-activated AhR and AHRR in the biological processes.

1.4 Induction of CYP1A

Theligand-AhR-ARNT heterodimerinteracts with AhR response elements（AhREs;also known as XREs or DREs）to activate or repress gene expression from target genes（Hahn et al.,2005;Hahn et al., 2006）.The best characterized targets of the AhR pathway are Cytochrome P4501A（CYP1A）genes, which are strongly induced by TCDD and PAHs（Whitlock,1999）.They have a broad affinity for polycyclic, aromatic hydrocarbons, as well as aromatic amines,and some endogenoussubstrates（Gonzalez and Kimura,2003;Teraoka et al.,2003）. And they play a central role in biotransformation, detoxication and elimination of various structurally diverse xenobiotics（Monostory and Pascussi,2008）. The induction of CYP1A family member expression is regulated by a heterodimer composed of the AhR and ARNT（Fujii-Kuriyama and Mimura,2005）.In contrast,the expression of CYP2,3,and 4 family members is regulated by the nuclear receptors CAR（Constitutive androstane receptor）,PXR（Pregnenolone X receptor）,and PPAR（Peroxisome proliferator activated receptor）,respectively（Waxman,1999）.

The induction of CYP 1A is an important step in the response to POPs.Some researcheshave identified several consensus response elements,there are eight potential xenobiotic response elements（XREs）in the promoterregion ofthe european flounder CYP 1A gene,but not all of these sequences are necessarily for activation,just only four out of eight different XREs are functional in the regulation of CYP 1A.The activity of these response elements enhances the evidence for considerable diversity in vertebrate CYP1A regulation（Lewis et al.,2004）.In a word,we can conclude that AhR pathway plays a pivotal role in the regulation of POPs toxicology in aquatic animals.

2 Theregulation mechanism oftoxicology in zebrafish development

Zebrafish is a very perfect model aquatic animal.At present,the AhR pathway of zebrafish has been extensively studied.Thus,itis very important for us to comprehend the AhR pathway toxicological mechanism of POPs in zebrafish.

Proper regulation of AhR is needed for normal vertebrate cardiovascular development.In zebrafish, therearetwoAhR genes,zfAhR1andzfAhR2. ZfAhR2 binds TCDD with high affinity and transcriptional active which plays a major role in mediating the developmentaltoxicity of TCDD, whereas zfAhR1 lacks the ability to bind TCDD and activate transcription, and it’s function is not known.A new zebrafish AhR designated AhR1B, which shares 34%amino acid sequence identity with AhR1（AHMA）.TheAhR1B generesideson chromosome22,adjacentto AhR2,whereasthe AhR1A gene is located on chromosome 16.AhR1B is expressed in embryos as early as 24 hours postfertilization and increases through the next 2 days, but expression is not inducible by TCDD.So we can conclude that AhR1B may play a physiological role in embryonic development, whereas AhR2 mediates the response to xenobiotics（Karchner et al., 2005）.

When AhR was hyperactivated by TCDD during the processofzebrafish embryo development,it altered heart morphology and function,culminating in death.Within 1 to 2 hours of exposure,TCDD shows rapidly induced expression,in 42 genes which have function in xenobiotic metabolism,proliferation, heartcontractility and pathwaysthatcan regulate heart development.Furthermore,these expression changespreceded signsofcardiovasculartoxicity, including decreased stroke volume,peripheral blood flow,and a halt in heart growth,these which are good candidates for AhR target genes（Carney et al., 2006）,and so these genes can be used to monitor the pollution condition of early aquatic ecosystem.

Itis wellknown thatTCDD is a potent developmental toxicant in most vertebrates, but several frog species are insensitive to TCDD, especially during early life stages.Some experiments with frog suggestthatTCDD insensitivity results largely from rapid elimination.Butrecentstudy confirms that rapid elimination of TCDD is unlikely to contribute to TCDD insensitivity during development of the cardiovascular system（Philips et al.,2006）. Another research of AhRs from X.laevis demonstrates that these proteins bind TCDD with 25~50-fold lower affinity than AhRs from more sensitive species（Lavine et al.,2005）,a property likely that the structural and functional diversity of AhR proteins may confer species-and strain-specific differences in the sensitivity to toxic AhR ligands（Hahn et al., 2005）.

3 Biomontoring of POPs in aquatic animals

Toxicological effects of POPs in aquatic ecosystem lead to the deterioration of water quality and adversely impact on aquatic animal and human health.The highly lipophilic nature of these pollutants may enter fish through the diet or by water-borne exposure（Wong etal.,2001）.In orderto preventand reduce the harm of POPs for the health of aquatic animals and people,it is very important to explore some idealism biomarkers for early biomonitoring.At present,many investigations have been done,briefly including the following two aspects:

3.1 Biochemical biomarker

Now,many biochemical biomarkers have been studied,such as ethoxyresorufin-O-deethylase（EROD）which hasthe catalytic function ofthe CYP1A, microsomal oxygenase subfamily which is a popular biomarker for exposure to xenobiotics,polyhalogenated aromatic hydrocarbons（PHAHs）in particular.It has been widely used to assess the pollution condition of aquatic environment both in vivo and in vitro（Kuiper etal.,2004）.EROD isalso can be used to determine the quantitiesofPOPsin fish muscle（Havelkova etal.,2007）.A modelto assess pollutant exposure via food supply has been developed for the sentinel organism.Mussels were fed for 4 weeks with Benzo［a］pyrene（B［a］P）-contaminated feed,and the result suggests that B［a］P hydroxylase（BPH）, acetylthiocholine esterase（AChE）,DT-diaphorase（DTD）and catalase（CAT）activity as suitable biomarkers of PAH exposure for these sentinel species（Akcha et al.,2000）.The carboxylesterase activity, metallothionein, total haemolymph protein（Hamm et al.,2003）and vitellogenin（Zhan et al.,2007）were the most useful biochemistry biomarkers.

3.2 Molecular biomarker

Previous researches indicate that AhR plays an important role in the regulation pathway of POPs toxicology.Thus the induction of the CYP1A1 gene of fish has been used as a sensitive,early warning method in the monitoring of contamination in aquatic ecosystems（Wong etal.,2001）.Expression of CYP1A1 gene and its related enzyme activity have long been used as a biomarker for AhR activation and a warning of dioxin-like toxicity（Hu et al., 2007）.At thesametime,weshould pay more attention to other factors in real station. For example, on certain conditions, seasonal factors might affect the biomarker（e.g.,antioxidant and peroxisomal enzymes）responses,to a greater extent than pollution variations（Orbea et al.,2002）.

In addition,micronucleus（MN）frequency and DNA single strand break were used as biomarkers of genotoxicity.Mussels were also analyzed for PAH and heavy metals（Hg and Cd）（Bolognesi et al., 2004）.MN response can be used as a sensitive indicatorofexposure to relatively low levelsof genotoxicants and that MN response in ussel gill cells can be a stable biomarker of genotoxicity（Siu etal.,2004）.MoreoverDNA adductisalso a molecular biomarker of exposure to PAHs,and is now well established in ecotoxicology.DNA adduct level in aquatic organisms has been found to produce a better correlation with PAH exposure than PAH concentration in organisms（Shaw and Connell, 2001）,because relatively high levels of DNA adducts can be produced by some non-carcinogenic PAHs,while other non-carcinogenic compounds do not produce detectable adducts.In addition,it has been shown that all carcinogenic PAHs investigated produce DNA adducts and that a relationship exists between relative adduct formation and carcinogenic potency.

We can conclude that the molecular biomarkers are more effective than biochemical biomarkers,and we should pay more attentions on exploring more useful molecular biomarkers to monitor the status of POPs contamination in the future.

4 Summary

POPs have become ubiquitous and aroused the attentions in the world（Swain,1988）.Over the last decades,numerous studies have been investigated on the formation,distribution,accumulation,bioamplification through the food chain, toxicity, toxicology, genotoxicity,biomonitoring and embryo toxicity of POPs.At present,we can find that most of the aquatic animals all conformed to the same toxicology mechanism,namely theAhR pathway.Itmainly consists of four major steps,including formation of cytosolic complex, translocation of AhR, heterodimerization of AhR,and induction of Cyp1a. In addition, there are some other species are insensitive to the TCDD toxicity,especially at the early life stage,for example,African clawed frog, which factor causes the difference between different animal species？It is still an unresolved issue.

Furthermore,the embryo of aquatic animal is the mostcriticalstage in the totalprocess of development and growth of aquatic animal,and it can be used as one kind of biosensors to monitor the pollutant status of aquatic ecosystem.On the other hand,the research of molecular mechanism of POPs embryo toxicology can elucidate the mechanism ofsome cancer,tumours and teratogenesis.But previous researches just focus on one or few genes in the AhR pathway.Actually,itis universally acknowledged that there are a series of related genes involving in the regulation of POPs toxicology. Then,you may ask that which and how many genes are involved in the regulation,and what’s the relationship between them？With the development of modern molecularbiology and biotechnology,we believe that these issues will be resolved gradually in the future.Forexample,we can use differential display technique and microarray technology to explore it,and it can provide a more quickly and effectively method to monitor the POPs pollution in aquatic ecosystem as early as possible.

Acknowledgements：Specialthanks to Louis van Wijk BPM who kindly supplied a grammar correction.He is a Health, Safety & Environmental Manager from Netherlands.Many thanks to Drs.Dongyan Liu and Jingli Liu for their advice and assistance which have greatly improved the quality of this manuscript.

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