Biomarkers of Environmental Pollutants

BioMed Research International

Guest Editors: Anilava Kaviraj, Erhan Unlu, Abhik Gupta, and Ahmed El Nemr Biomarkers of Environmental Pollutants BioMed Research International Biomarkers of Environmental Pollutants

This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents

Biomarkers of Environmental Pollutants, Anilava Kaviraj, Erhan Unlu, Abhik Gupta, and Ahmed El Nemr Volume2014,ArticleID806598,2pages The Presence of Biomarker Enzymes of Selected Scleractinian Corals of Palk Bay, Southeast Coast of India,R.Anithajothi,K.Duraikannu,G.Umagowsalya,andC.M.Ramakritinan Volume2014,ArticleID684874,6pages Synergistic Effects of Toxic Elements on Heat Shock Proteins, Khalid Mahmood, Saima Jadoon, Qaisar Mahmood, Muhammad Irshad, and Jamshaid Hussain Volume2014,ArticleID564136,17pages Protective Role of Omega-3 Polyunsaturated Fatty Acid against Lead Acetate-Induced Toxicity in Liver and Kidney of Female Rats, Heba M. Abdou and Mohamed A. Hassan Volume 2014, Article ID 435857, 11 pages Assessment of DNA Damage and Telomerase Activity in Exfoliated Urinary Cells as Sensitive and Noninvasive Biomarkers for Early Diagnosis of Bladder Cancer in Ex-Workers of a Rubber Tyres Industry, Delia Cavallo, Valentina Casadio, Sara Bravaccini, Sergio Iavicoli, Enrico Pira, Canzio Romano, Anna Maria Fresegna, Raffaele Maiello, Aureliano Ciervo, Giuliana Buresti, Wainer Zoli, and Daniele Calistri Volume 2014, Article ID 370907, 8 pages Effects of Freshwater Pollution on the Genetics of Zebra Mussels (Dreissena polymorpha)atthe Molecular and Population Level,EmiliaG.Thomas,MajaSrut,ˇ Anamaria Stambuk,ˇ Goran¨ I. V. Klobucar,ˇ Alfred Seitz, and Eva Maria Griebeler Volume 2014, Article ID 795481, 11 pages Biomarkers of Type II Synthetic Pyrethroid Pesticides in Freshwater Fish, Anilava Kaviraj and Abhik Gupta Volume2014,ArticleID928063,7pages Metallothionein Induction in the Coelomic Fluid of the Earthworm Lumbricus terrestris following Heavy Metal Exposure: A Short Report,A.Calisi,M.G.Lionetto,E.DeLorenzis,A.Leomanni, and T. Schettino Volume 2014, Article ID 109386, 6 pages Reduced Bone and Body Mass in Young Male Rats Exposed to Lead, Fellipe Augusto Tocchini de Figueiredo, Raquel Fernanda Gerlach, MarciaAndreiaMesquitaSilvadaVeiga,´ Flavio Venancio Nakadi, Junia Ramos, Erika Reiko Kawakita, Carolina de Souza Guerra, and Joao˜ Paulo Mardegan Issa Volume 2014, Article ID 571065, 5 pages MercurySpeciationinHairofChildreninThreeCommunitiesoftheAmazon,Brazil, Jamile Salim Marinho, Marcelo Oliveira Lima, Elisabeth Conceic¸ao˜ de Oliveira Santos, IracinaMauradeJesus,MariadaConceic¸ao˜ N. Pinheiro, Claudio´ Nahum Alves, and Regina Celi Sarkis Muller Volume 2014, Article ID 945963, 9 pages Involvement of Oxidative Stress and Inflammation in Liver Injury Caused by Perfluorooctanoic Acid Exposure in Mice, Bei Yang, Weiying Zou, Zhenzhen Hu, Fangming Liu, Ling Zhou, Shulong Yang, Haibin Kuang, Lei Wu, Jie Wei, Jinglei Wang, Ting Zou, and Dalei Zhang Volume2014,ArticleID409837,7pages Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 806598, 2 pages http://dx.doi.org/10.1155/2014/806598

Editorial Biomarkers of Environmental Pollutants

Anilava Kaviraj,1 Erhan Unlu,2 Abhik Gupta,3 and Ahmed El Nemr4

1 Department of Zoology, University of Kalyani, Kalyani, West Bengal 741235, India 2Department of Biology, University of Dicle, 21280 Diyarbakir, Turkey 3School of Environmental Sciences, Assam University, Silchar 788011, India 4Marine Pollution Department, National Institute of Oceanography and Fisheries, Kayet Bey, Elanfoushy, Alexandria 21556, Egypt

Correspondence should be addressed to Anilava Kaviraj; [email protected]

Received 7 September 2014; Accepted 7 September 2014; Published 22 December 2014

Copyright © 2014 Anilava Kaviraj et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Thousands of pollutants enter daily into environment and Afewpapersdealwithasinglesensitivebiomarker,while exert various kinds of stress on organisms and ecosystems. others deal with combinations of selected biomarkers. The Risk assessment of these pollutants to organisms and ecosys- papershavebeencategorizedunderthreemaingroups. tems is challenging because of diversities in chemical nature and mode of toxicity of the pollutants as well as variation in Biomarkers of Susceptibility/Oxidative Biomarkers.Two sensitivities of the organisms exposed to the pollutants. Even papers have been published in this category. One paper deals low, relatively innocuous concentrations of pollutants often with perfluorooctanoic acid (PFOA), which is widely present produce deleterious effects on organisms, which are difficult in the environment. Oxidative stress induced by PFOA on to be predicted, because measurable effects are expressed mice was evaluated by malondialdehyde (MDA) formation only after prolonged exposure. But it may be too late to take and hydrogen peroxide generation, which were considered remedial actions or steps to reduce risk when these incipient as reliable biomarkers of anthropogenic stresses on mice. effects are expressed. Therefore, it is necessary to develop The second paper in this category also deals with MDA. early warning signals or biomarkers that convincingly reflect Elevation in MDA associated with depletion in antioxidant adverse biological responses towards anthropogenic environ- enzymes activities in the tissues was measured in liver and mental toxins even at minute concentrations [1]. A biomarker kidney of female rats exposed to lead acetate. The authors is defined as “a change in biological response, ranging from observed that Omega-3 polyunsaturated fatty acid provided molecular through cellular and physiological responses to a protection against lead acetate toxicity in rats. behavioral changes, which can be related to exposure to or toxic effects of environmental chemicals” [2]. Recent Biomarkers of Exposure. Four papers have been published developments in molecular biology and biotechnology and in this category. In one paper the authors established inventions of sophisticated instruments have led to the that deposition of mercury in hair served as an excellent development of novel, more sensitive validated biomarkers biomarker of exposure of human population to mercury. In of exposure, effect, and susceptibility to the adverse effects another paper differences in whole blood versus tibia lead of terrestrial and aquatic pollutants [3]. Validity of these concentrations over time in growing rats were evaluated biomarkers has been tested in several laboratory and field prenatally and higher amount of lead found in the bone of studies [1]. In addition, systematic use of multiple biomarkers older animals reinforced the importance of using bone lead as has been found as most useful in the assessment of pollutants’ an exposure biomarker. In the third paper it was established effects [4]. that coelomic fluid instead of whole earthworm could be This special issue on biomarkers of environmental pollu- used to identify ecologically relevant end points. Induction of tants contains ten papers, which discussed some recent appli- metallothionein in the coelomic fluid of earthworms served cations of biomarkers in terrestrial and aquatic environment. as sensitive biomarkers of heavy metal pollution in soil. 2 BioMed Research International

The fourth paper discussed application of multiple biomark- [3] T. S. Galloway, “Biomarkers in environmental and human ers to assess risk of corals. Several biomarkers enzymes health risk assessment,” Marine Pollution Bulletin,vol.53,no. involved in melanin synthesis pathway (phenoloxidase (PO) 10–12, pp. 606–613, 2006. and peroxidases (POD)) and free radical scavenging enzymes [4]C.Tsangaris,K.Kormas,E.Strogyloudietal.,“Multiple (super oxide dismutase (SOD), catalase (CAT)) and glu- biomarkers of pollution effects in caged mussels on the Greek tathione peroxidase (Gpx) were determined in selected scle- coastline,” Comparative Biochemistry and Physiology Part C: ractinian corals to evaluate stress induced by coral pathogen Toxicology & Pharmacology,vol.151,no.3,pp.369–378,2010. and to predict health and future existence of corals.

Biomarkers of DNA Damage. Two papers have been published in this category. One deals with effects of contaminants on DNA damages of Zebra Mussels Dreissena polymorpha evaluatedbyCometassayandmicronucleustest(MNT)and another on DNA damage and telomerase activity in exfoliated urinary cells of human workers of rubber tire industry evaluatedbyCometassayandquantitativetelomeraserepeat amplification protocol (TRAP) and fluorescence in situ hybridization (FISH) assay. MNT appeared as better tool than Comet assay in Zebra mussel while TRAP, Comet, and FISH assays worked well as early biomarkers of procarcinogenic effects on rubber factory workers.

Reviews on Biomarkers. Two review papers have been published. One review paper discussed variations in the expression levels of heat shock protein (HSP) under a variety of toxic conditions. The paper discussed current knowledge on expression of HSPs and concluded that HSP was unreliable as biomarker due to synergistic effects of toxicants and other environmental factors. Another review paper deals with biomarkers of type-II synthetic pyrethroid pesticides. Appli- cations of hematological, enzymatic, cytological, genetic, omic, and other types of biomarkers to evaluate toxicity of the major type-II pyrethroid pesticides in freshwater fish species have been documented. The papers discussed the present status of researches on type-II pyrethroids and the possible future directions. It is concluded that till today oxidative biomarkers are the most frequently used category of biomarkers to assess adverse effects of a wide variety of pollutants. In the quest to develop more specific biomarkers researchers are concentrating on emerging areas with metabolomics and proteomics forming the frontier area of biomarker research. These trends are being facilitated by the development of increasingly precise and sensitive instrumentation in recent years.

Anilava Kaviraj Erhan Unlu Abhik Gupta Ahmed El Nemr

References

[1] R. van der, J. Oost, and N. P. E. Vermeulen, “Fish bioaccumulation and biomarkers in environmental risk assessment: a review,” Environmental Toxicology and Pharmacology,vol.13, no.2,pp.57–149,2003. [2]D.W.PeakallandC.H.Walker,“Theroleofbiomarkersin environmental assessment (3),” Ecotoxicology,vol.3,no.3,pp. 173–179, 1994. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 684874, 6 pages http://dx.doi.org/10.1155/2014/684874

Research Article The Presence of Biomarker Enzymes of Selected Scleractinian Corals of Palk Bay, Southeast Coast of India

R. Anithajothi, K. Duraikannu, G. Umagowsalya, and C. M. Ramakritinan

Department of Marine and Coastal Studies, Madurai Kamaraj University, Tamilnadu 623526, India

Correspondence should be addressed to R. Anithajothi; [email protected]

Received 14 February 2014; Revised 6 June 2014; Accepted 28 July 2014; Published 18 August 2014

Academic Editor: Abhik Gupta

Copyright © 2014 R. Anithajothi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The health and existence of coral reefs are in danger by an increasing range of environmental and anthropogenic impacts. The causes of coral reef decline include worldwide climate change, shoreline development, habitat destruction, pollution, sedimentation and overexploitation. These disasters have contributed to an estimated loss of 27% of the reefs. If the current pressure continues unabated, the estimated loss of coral reef will be about 60% by the year 2030. Therefore, the present study was aimed to analyze the enzymes involved in stress induced by coral pathogen and its resistance. We focused on the enzymes involved in melanin synthesis pathway (phenoloxidase (PO) and peroxidases (POD)) and free radical scavenging enzymes (super oxide dismutase (SOD), catalase (CAT)) and glutathione peroxidase (Gpx) in selected scleractinian corals such as Acropora formosa, Echinopora lamellosa,Faviafavus,Faviteshalicora,Poritessp., and Anacropora forbesi. Overall, PO activity of coral was significantly lower than that of zooxanthellae except for Favia favus. Coral colonies with lower PO and POD activities are prone to disease. Maximum antioxidant defensive enzymes were observed in Favia favus followed by Echinopora lamellose. It is concluded that assay of these enzymes can be used as biomarkers for identifying the susceptibility of corals towards coral bleaching induced by pathogen.

1. Introduction microbes trigger the synthesis of melanin through the cleavage of prophenoloxidase (PPO) into phenoloxidase (PO). The coral holobiont is comprised not only of coral animal Various phenoloxidases catalyse monophenol hydroxylation and endosymbiotic dinoflagellates (zooxanthellae) but also of and diphenol oxidation as well as autocatalytic reactions microbial communities such as bacteria, archaea, and fungi which frequently lead to the formation of the pigment, as well as numerous viruses [1, 2]. As a primitive organism, melanin [14–16]. Oxidative burst is resulting in upregulation corals do have an innate immune system similar to that of ofPPOactivitybecausePPOactivityistheprimarysources other invertebrates [3–6]butdonotpossesstheadaptive of oxidative stress during an invertebrate immune response immune system of vertebrates. The defensive mechanism of the immune system plays a vital role in prevention of [17, 18]. This leads to the production of antioxidant enzymes infection and in the maintenance of tissue integrity of corals such as SOD, CAT, POD, and GPx. Our study focused on [7, 8]. The recognition of the coral defensive system involved the biomarker enzymes involved in defensive mechanism of in immune responses like other invertebrates includes Toll- selected corals of Palk Bay situated at southeast of India. like receptors [9] and the synthesis of melanin. The melanin synthesis is activated by phenoloxidase, immune cells and antioxidants, and peroxidase [10–13]. During PO pathways, 2. Materials and Methods the cytotoxic intermediates lead to inflammatory defense 2.1. Tissue Homogenate for Enzyme Assays. Six different coral [5, 14]. specimens were collected from three different locations, that ∘ 󸀠 󸀠󸀠 ∘ 󸀠 Prophenoloxidase is an important enzyme and a key com- is, from villundi theertham (lat.: 9 17 33.81 N, long.: 79 12 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ponent of innate immunity system of corals [15]. Pathogenic 46.69 E), Pamban (lat.: 9 17 1.21 N, long.: 79 12 44.20 E), 2 BioMed Research International

∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 and Olaikuda (lat.: 9 18 30.12 N, long.: 79 20 4.44 E) of the observed by measuring the decrease in H2O2 concentration Palk Bay, southeast coast of India. Specimens were collected at 240 nm. Working solution of 340 𝜇L of 100 mM phosphate during low tide in the early morning in the month of April buffer with pH 7.0, 10 mM H2O2,and660𝜇L of sample extract 2012. These were identified based on the morphological were mixed in a cuvette. The change in absorbance per minute features and keys observed during collection of samples. at 240nm was calculated. Enzyme activity was expressed in −1 The identified corals were (1) Acropora formosa (AC), (2) units of CAT activity and was expressed as U⋅mg protein. Echinopora lamellosa (EL), (3) Favia favus (FF), (4) Favites One unit of CAT activity was defined as the amount of halicora (FH), (5) Porites sp. (POR), and (6) Anacropora 𝜇 ⋅ −1 2 enzyme needed to reduce 1 MH2O2 min [24]. forbesi (AN). Coral fragments of about 2 cm were collected using a hammer, stored in an ice pack, and immediately transported to the laboratory. 2.5. Glutathione Peroxidase Activity. Glutathione peroxidase The coral samples were crushed using sterile sea water (GPx) activity was determined using a slightly modified protocol of Rotruck et al., 1973 [25]. The reaction mixture and the homogenate was obtained by centrifugation at 𝜇 𝜇 15,000 RPM for 15 minutes to separate the supernatant and was prepared by adding 500 L of tissue homogenate, 200 L of phosphate buffer (0.4 M pH-7), 200 𝜇Lof0.45mMEDTA, pellets. The supernatant thus obtained was considered as 𝜇 𝜇 substratefortheanalysisofproteinandenzymeactivitiesof 100 Lof10mMsodiumazide,and200 LofGSHsolution followed by 0.1 mL H2O2.Thecontentswereincubatedfor coral host [19] and the pellet was considered to be a substrate ∘ foranalysisofproteinandenzymeactivitiesofzooxanthellae. 10 min at 37 C. In this mixture, 0.4 mL of 10% TCA was The pellets containing the zooxanthellae were suspended added to stop the reaction and the mixture was centrifuged −1 at 3200 ×g for 20 min. The pellet was discarded and 0.5 mL in 2 mL of 100 mmol phosphate buffer (pH-7.0) and were of DTNB was added to the supernatant. The supernatant dissolved by sonication for 30 minutes in an ice bath. was assayed spectrophotometrically (SPECRTA max M2e) Triton X-100 (0.05%) solution was added to the sonicated at340nm.Oneunitofglutathioneperoxidaseactivitywas suspension and kept for 10 minutes at room temperature and defined as the amount of enzyme that oxidizes 1 𝜇mol of then the suspension was centrifuged at 14,000 rpm for 30 reduced glutathione and GPx activity was expressed as minutes and used as the zooxanthellae extract for protein and ⋅ −1 enzyme assays. The protein was estimated using standardized U mg protein. protocols prescribed by Lowry et al., 1951 [20]. 2.6. Statistical Analysis. Data were analyzed by one-way 2.2. Peroxidase and Phenoloxidase. Peroxidase activity was analysis of variance (ANOVA) with the Graph-Pad Prism 5 assayed spectrophotometrically (SPECRTA max M2e) at software and the least significant differences were compared 470 nm using guaiacol as a phenolic substrate with hydrogen at 𝑃 < 0.05. peroxidase [21]. Five milliliters of the assay mixture for the peroxidase activity comprised 35 𝜇Lofphosphatebuffer −1 3. Results (10 mmol pH 6.0), 10 𝜇L of sample extract (coral tissue and zooxanthellae solution of six different coral samples), 40 𝜇L 3.1. Phenoloxidase Activity. Phenoloxidase activity of both −1 −1 of guaiacol (25 mmol ), and 25 𝜇LofH2O2 (20 mmol ). The zooxanthellae and coral tissues of six healthy coral samples ∙ reaction was stopped by adding 5% H2SO4 POD activity was collected from the Palk Bay was presented in Figure 1.The −1 expressed as U⋅mg protein. Units were calculated using a phenoloxidase activity of zooxanthellae of Porites sp., A. −1 −1 molarabsorptivityof2.66× 104 M cm for tetraguaiacol forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus 󸀠 󸀠 −1 or 3,3 -dimethoxy-4,4 -biphenoquinone [22]. Phenoloxidase was 3.6, 1.8, 7.6, 1.5, 2.0, and 5.7 U⋅mg protein, respectively. activity was calculated using the same procedure followed for Maximum PO activity was noticed in zooxanthellae of E. the analysis of peroxidase but without peroxide. lamellosa and the minimum was noticed in F. halicora zooxanthellae extract. Maximum PO activities of zooxanthellae 2.3. Superoxide Dismutase Activity. Quantification of SOD are exhibited by E. lamellosa. activity was based on the ability of SOD to inhibit the reduc- The phenoloxidase activity of coral tissues of six different tion of NBT by superoxide [23]. Two hundred microlitres corals such as Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus was 2.7, 0.5, 1.5, 0.95, 1.2, and 5.9 of enzyme extract was added to a tube containing 0.2 mL of −1 0.1 M EDTA solution, 0.3 mM sodium cyanide, and 0.1 mL of U⋅mg protein, respectively. Minimum and maximum PO 1.5 mM NBT. Then, 0.05 mL of 0.12 mM riboflavin was added activities were reported in the coral tissues of A. forbesi and F. at zero timed intervals and then all the tubes were incubated favus,respectively(Figure 1). Maximum PO activity of coral in a light box for 12 minutes and absorbance was recorded tissue was exhibited by F. favus.ExceptinF. favus,thePO at560nmusingSPECRTAmaxM2e.OneunitofSODwas activity of zooxanthellae was significantly higher than coral defined as the enzyme causing half the maximum inhibition tissues in all species. The PO activity of zooxanthellae was −1 of NBT reduction and SOD activity was expressed as U⋅mg significantly higher than that of their respective coral tissues protein. except F. favus.

2.4. Catalase Activity. Catalase activity was estimated by the 3.2. Peroxidase Activity. Peroxidase (POD) activity of both method described by Aebi, 1984 [24]. Catalase activity was zooxanthellae and coral tissues of six different coral species BioMed Research International 3

9 Significant increase in peroxidase activity was observed 8 in zooxanthellae extracts when compared with coral tissue 7 extracts of all coral samples except F. favus (Figure 2). 6 5 3.3. Antioxidant Defensive Factors of Corals. The three major

of protein) of antioxidant enzymes are the superoxide dismutase (SOD) 4 ∙− −1 which produces H2O2 by converting O2 ,then,theenzyme

mg 3 · catalase (CAT) that splits H2O2 to oxygen and water, and

(U 2 Phenoloxidase activity Phenoloxidase the third antioxidant enzyme, that is, glutathione peroxides 1 (GPx), that converts H2O2 and organic peroxides. Increased 0 level of ROS activates these three antioxidant enzymes. POR AN EL FH AC FF Coral species 3.3.1. Superoxide Dismutase Activity. Superoxide dismutase Zooxanthellae activity of both zooxanthellae and coral tissues of six dif- Coral tissue ferent coral species is presented in Figure 3. SOD activity of zooxanthellae extract of Porites sp., A. forbesi, E. lamellosa, Figure 1: Phenoloxidase activity of coral tissue and zooxanthellae 𝑛=3 F. halicora, A. formosa, and F. favus was 19.4, 4.7, 24, 4.7, ( ). POR: Porites sp., AN: A. forbesi,EL:E. lamellosa, FH: F. ⋅ −1 halicora,AC:A. formosa, and FF: F. favus. 14.1, and 51.4 U mg protein, respectively. Maximum SOD activity was noticed in the zooxanthellae extract of F. favus, while A. forbesi and F. halicora exhibited low SOD activity (Figure 3). 12 SODactivityofcoraltissuesofPorites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus was 11.7, 3.3, 10 −1

of protein) of 19.3, 3.5, 2.4, and 9.4 U⋅mg protein, respectively. The coral −1 8 tissues of A. formosa exhibited minimum SOD activity and mg · E. lamellosa tissues exhibited maximum activity (Figure 3). 6 SOD activity of E. lamellosa was 8-fold higher than that of A. formosa and5-foldhigherthanthatofPorites sp. 4 SOD activity of F. favus was found to be 11-fold higher 2 than that of A. forbesi and F. halicora followed by 5- and 4-fold higher activity than E. lamellosa and Porites sp., respectively.

Peroxidase activity (U activity Peroxidase 0 Except A. forbesi and F. halicora, the SOD activity significantly POR AN EL FH AC FF varied between zooxanthellae and coral tissue in all species. Coral species Zooxanthellae 3.3.2. Catalase Activity. Catalase (CAT) activities of zooxan- Coral tissue thellae and coral tissues of six different coral species have Figure 2: Peroxidase activity of coral tissue and zooxanthellae (𝑛= been presented in Figure 4. CAT activity of zooxanthellae of 3). POR: Porites sp., AN: A. forbesi,EL:E. lamellosa, FH: F. halicora, Porites sp.,A.forbesi,E.lamellosa,F.halicora,A.formosa, −1 AC: A. formosa, and FF: F. favus. and F. favus was 5.1, 1.6, 8.7, 3.2, 1.8, and 3.4 U⋅mg protein, respectively (Figure 4). CAT activity of zooxanthellae extract of E. lamellosa exhibited maximum activity while zooxan- is presented in Figure 2.Theperoxidaseactivityofzooxan- thellae of A. forbesi exhibited minimum catalase activity thellae of Porites sp., A. forbesi, E. lamellosa, F. halicora, A. (Figure 4). CAT activity of E. lamellosa was 5.4-fold higher formosa, and F. favus was 5.65, 0.43, 7.1, 0.65, 1.22, and 6.91 than that of A. forbesi and 3.2-fold higher than that of Porites −1 sp. U⋅mg protein, respectively. Maximum POD activity was observed in zooxanthellae of E. lamellosa and the minimum Similarly, CAT activity of coral tissues of Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, and F. favus wasobservedinzooxanthellaeofA. forbesi (Figure 2). POD −1 activity of A. forbesi was 17-fold higher than that of E. was 2.1, 0.6, 2.2, 1.1, 2.1, and 5.0 U⋅mg protein, respectively lamellosa. (Figure 4). The coral tissues of A. forbesi and F. favus exhib- The peroxidase activity of coral tissues of six different ited minimum and maximum CAT activities, respectively coral species such as Porites sp., A. forbesi, E. lamellosa, (Figure 4). CAT activity of tissues of A. forbesi was 8.3-fold F. halicora, A. formosa, and F. favus was 3.21, 0.34, 1.73, higher than that of F. favus and 3.7-fold higher than that of E. −1 1.13, 0.67, and 10.7 U⋅mg protein, respectively. Maximum lamellosa. POD activity of coral tissues was found in F. favus and the Comparing CAT activity between coral zooxanthellae minimum activity was found in A. forbesi (Figure 2). The and coral tissues of the six different species, it was observed POD activity of F. favus was 32-fold higher than that of A. that, except A. formosa and F. favus, CAT activity was higher forbesi (Figure 2). inzooxanthellaethanthatofcoraltissuesinallspecies. 4 BioMed Research International

60 0.3

50 0.25

40 0.2 of protein) of

of protein) of 30 0.15 −1 −1 mg ·

mg 20 0.1 · (U (U 10 0.05 Glutathione peroxidase activity peroxidase Glutathione Superoxide dismutase activity dismutase Superoxide 0 0 POR AN EL FH AC FF POR AN EL FH AC FF Coral species Coral samples Zooxanthellae Zooxanthellae Coral tissue Coral tissue

Figure3:SODactivityofcoraltissueandzooxanthellaeof6coral Figure 5: Glutathione peroxidase activity of coral tissue and species (𝑛=3). POR: Porites sp., AN: A. forbesi,EL:E. lamellosa,FH: zooxanthellae (𝑛=3). POR: Porites sp., AN: A. forbesi,EL:E. F. halicora,AC:A. formosa, and FF: F. favus. lamellosa,FH:F. halicora,AC:A. formosa, and FF: F. favus.

10 GPx activity and F. favus tissues exhibited maximum activity 9 8 (Figure 5). 7 The GPx activities of both zooxanthellae and the coral 6 tissues of Porites sp.weresimilar.ButtheGPxactivityofcoral

of protein) of 5 tissues of A. forbesi and E. lamellosa was less than that of

−1 4 respective coral zooxanthellae activity. However, the activity mg · 3 was more in coral tissues of F. halicora, A. formosa, and F. Catalase activity Catalase

(U 2 favus than the respective coral zooxanthellae. 1 0 POR AN EL FH AC FF 4. Discussion Coral species As phenoloxidase plays a vital role in defensive mechanism of Zooxanthellae invertebrates, the presence of PO activity in all 6 coral species Coral tissue indicates the presence of baseline level of antimicrobial Figure 4: Catalase activity of coral tissue and zooxanthellae (𝑛=3). defense [13]. Generally, invertebrate animals with decreased POR: Porites sp., AN: A. forbesi,EL:E. lamellosa,FH:F. halicora,AC: phenoloxidase (PO) activity are more susceptible to disease A. formosa, and FF: F. favus. [26–28]. In the present study, A. forbesi, F. halicora, and A. formosa showed lowest phenoloxidase activity. It reveals that these coralsare susceptible to bleaching, thermal stress, and diseases. At the same time, F. favus, E. lamellose, and Porites 3.3.3. Glutathione Peroxidase. Like SOD and CAT activities, sp. indicated higher disease resistance towards pathogens glutathione peroxidase (GPx) activities of both zooxanthellae [13]. The low level of PO activity in A. formosa and A. and coral tissues of six different corals were also estimated forbesi showed that branching corals are more susceptible and presented in Figure 5. GPx activity of zooxanthellae of to diseases. Similar results were observed in the study of Porites sp., A. forbesi, E. lamellosa, F. halicora, A. formosa, susceptible ranking of 15 scleractinian corals by Palmer et al. −1 and F. favus was 0.07, 0.05, 0.21, 0.04, 0.06, and 0.08 U⋅mg [13]. protein, respectively. Peroxidase activity induction includes oxidation of sub- The zooxanthellae of E. lamellosa and F. halicora exhib- strates and cytotoxic molecules [14, 17] which act as antimi- ited maximum and minimum GPx activities, respectively crobial agents [29]. In most cases, peroxidase provides resis- (Figure 5). Comparatively, GPx activity of E. lamellose was tance to the corals against fungal pathogens as in case of 5.3-fold higher than that of F. halicora zooxanthellae. There Aspergillosis [30]. In our findings, the maximum peroxidase was no significant difference between GPx activity of zoox- activity was observed in the tissue of Favia favus and zooxanthellae of Porites sp. and F. favus while the remaining four anthellae of E. lamellosa and the lower activity was observed species showed significant variations in activity. in A. forbesi. Pathogen recognition results in apoplastic −1 Similar to GPx activity of zooxanthellae, the GPx activity generation of superoxide (O ), hydrogen peroxide (H2O2), of coral tissues has been presented in Figure 5 and the activity and hydroxyl radicals [31], thereby initiating the upregulation −1 was 0.07, 0.01, 0.1, 0.07, 0.08, and 0.23 U⋅mg protein, of antioxidant enzymes. ROS production is provided with ∙− respectively. The tissues of A. forbesi exhibited minimum an array of antioxidant enzymes, which either convert O2 BioMed Research International 5

to H2O2 (SOD), convert H2O2 to water and oxygen (CAT), [2] R. Anithajothi, N. Nagarani, G. Umagowsalya, K. Duraikannu, or use H2O2 to oxidize substrates (glutathione peroxidase) and C. M. Ramakritinan, “Screening, isolation and character- [31]. The principal findings reveal that corals such as F. favus ization of protease producing moderately halophilic microor- and E. lamellosa have strong resistance towards bleaching and ganism Halomonas meridiana associated with coral mucus,” disease and, at the same time, Anacropora forbesi are more Toxicological & Environmental Chemistry,2014. susceptible to bleaching and diseases [13]. The SOD activities [3] S. R. Dunn, “Immunorecognition and immunoreceptors in the of zooxanthellae were too high when compared with the SOD Cnidaria,” Invertebrate Survival Journal,vol.6,pp.7–14,2009. activities of coral tissue of all coral samples analyzed in this [4]L.D.MydlarzandR.S.Jacobs,“Aninduciblereleaseofreactive study. The findings suggested the production of SOD by coral oxygen radicals in four species of gorgonian corals,” Marine and host in response to O2 production during photosynthesis of Freshwater Behaviour and Physiology,vol.39,no.2,pp.143–152, its particular host. 2006. The maximum catalase activity was observed in zoox- [5]L.D.Mydlarz,E.S.McGinty,andC.D.Harvell,“Whatarethe anthellae compared to coral tissue. This may be due to the physiological and immunological responses of coral to climate conversion of superoxide ion produced during photosynthe- warming and disease?” JournalofExperimentalBiology,vol.213, sis into hydroxyl ion which was denoted by the increase in pp. 934–945, 2010. GPxandSODactivity,whichinturnresultedinhydrogen [6]K.C.Reed,E.M.Muller,andR.vanWoesik,“Coralimmunol- peroxide free radical in the zooxanthellae. Hence, the increase ogy and resistance to disease,” Diseases of Aquatic Organisms, vol. 90, no. 2, pp. 85–92, 2010. in CAT activities was observed as a result of increased H2O2 concentrations in the zooxanthellae [32]. [7] E. L. Cooper, “Comparative immunology,” Current Pharmaceu- Glutathione peroxidase (GPx) plays a vital role in cleaving tical Design,vol.9,no.2,pp.119–131,2003. H2O2 as a compensatory mechanism under severe oxidative [8]T.L.Stedman,Stedman’s Medical Dictionary, Lippincott, challengewherethecatalaseactivitymaybeinhibited[33, 34]. Williams and Wilkins, Baltimore, Md, USA, 2000. The antioxidant enzymes such as SOD, CAT, and GPx are [9] D. J. Miller, G. Hemmrich, E. E. Ball et al., “The innate immune reported to be increased during thermal and salinity stress repertoire in Cnidaria—ancestral complexity and stochastic in response to ROS generated during these stresses [35]. gene loss,” Genome Biology,vol.8,no.4,articleR59,2007. [10] E. 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While the corals [13]C.V.Palmer,J.C.Bythell,andB.L.Willis,“Levelsofimmunity of A. formosa, F. halicora, and A. forbesi exhibited minimum parameters underpin bleaching and disease susceptibility of reef activities of the defensive enzymes indicating susceptibility to corals,” The FASEB Journal,vol.24,no.6,pp.1935–1946,2010. coral pathogens, these susceptible corals are prone to disease [14] A. J. Nappi and B. M. Christensen, “Melanogenesis and andshouldbeprioritizedtopreventtransmittanceofdisease. associated cytotoxic reactions: applications to insect innate immunity,” Insect Biochemistry and Molecular Biology,vol.35, Conflict of Interests no.5,pp.443–459,2005. [15] L. Cerenius, B. L. Lee, and K. Soderh¨ all,¨ “The proPO-system: The authors declare that there is no conflict of interests pros and cons for its role in invertebrate immunity,” Trends in regarding the publication of this paper. Immunology,vol.29,no.6,pp.263–271,2008. [16] L. Cerenius, S. Kawabata, B. L. Lee, M. 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Review Article Synergistic Effects of Toxic Elements on Heat Shock Proteins

Khalid Mahmood,1 Saima Jadoon,2 Qaisar Mahmood,3 Muhammad Irshad,3 and Jamshaid Hussain3

1 Department of Biology, Government Post-Graduate College Asghar Mall, Rawalpindi, Pakistan 2 Department of Natural Resource Engineering and Management, University of Kurdistan-Hawler Erbil, Kurdistan Region, Iraq 3 Department of Environmental Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan

Correspondence should be addressed to Qaisar Mahmood; [email protected]

Received 23 November 2013; Revised 24 May 2014; Accepted 17 June 2014; Published 20 July 2014

Academic Editor: Abhik Gupta

Copyright © 2014 Khalid Mahmood et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Heat shock proteins show remarkable variations in their expression levels under a variety of toxic conditions. A research span expanded over five decades has revealed their molecular characterization, gene regulation, expression patterns, vast similarity in diverse groups, and broad range of functional capabilities. Their functions include protection and tolerance against cytotoxic conditions through their molecular chaperoning activity, maintaining cytoskeleton stability, and assisting in cell signaling. However, their role as biomarkers for monitoring the environmental risk assessment is controversial due to a number of conflicting, validating, and nonvalidating reports. The current knowledge regarding the interpretation of HSPs expression levels has been discussed in the present review. The candidature of heat shock proteins as biomarkers of toxicity is thus far unreliable due to synergistic effectsof toxicants and other environmental factors. The adoption of heat shock proteins as “suit of biomarkers in a set of organisms” requires further investigation.

1. Introduction the linking of stress genes, such as HSP70 and metallothionein, with a reporter gene for farmed vegetation that could Human population explosion has led to an era of rapid be employed to enable the use of satellite images to gauge and heavy industrialization which threatens life in almost crop status for environmental health. Furthermore, these bio- all possible habitats. The environment is being increasingly markers may be utilized as tools to gain better understanding polluted with the addition of a large number of heavy metals, of the links existing among the environmental quality, food chemicals, and radiation, which are dangerous not only for chains, and human health [1]. humanbutalsoforotherlivingbeings.Inthesecircumstances Until present, various biomarkers proposed for environ- biologists are playing a pivotal role in creating awareness mental risk assessment have been presented in Table 1. regarding the effects of hazardous materials along other The biomarkers presented in Table 1 have been considered issues towards the restoration and conservation of a healthy as important tools for the identification and quantification environment. Therefore, one key area of focus is the use of of exposure, effects, or susceptibility in individuals of a biomarkers as indicators of biochemical change, providing population under adverse conditions. Therefore, biomarker an early warning of environmental risk and its assessment. selection should be primarily based on the ability of a As such, biochemical markers often parallel changes in the biomarker being sufficiently sensitive to provide an accurate physiochemical characteristics of the environment; the use measurement in a sample. of biomarkers as a detective measure can enable adopting Heat shock proteins (HSPs) are specifically produced some timely preventive measures to avoid certain hazards. To when cells are exposed for a while to temperatures higher achieve these objectives, certain investigations have suggested than their normal growth requirement. The synthesis of HSPs 2 BioMed Research International

Table 1: Biomarkers proposed for environmental risk assessment. studies seems to be the use of varying concentrations of toxicity exposure; some authors have realistically used low Name of biomarker Reference concentrations,somehaveusedhighconcentrations,butonly Esterases [130] very few tested real-world contamination exposures models. Polytene chromosomes of chironomids (Diptera) [131] Furthermore, the existing validated bioassays, mostly based HSP60, HSP70, alpha B-crystallin homologue, lipid on lethality or reproduction, have somewhat limited appli- peroxide, total glutathione level, ubiquitin, [93, 122, cability due to their crude sensitivity, long exposure, or the mitochondrial manganese superoxide dismutase, 123] overall expenses of the test. In contrast, changes at bio- metallothionein, and cytochrome P450 2E homologue chemical level are usually the first detectable response to Numbers of macrophages in liver tissue, changes in environmental disturbance. Therefore, analysis of toxicity- [105] various blood parameters induced changes in gene expression (i.e., alterations in pat- Histological and ultrastructural markers, lysosomal terns of protein synthesis) and resultant cell injury may be [51, 132] membrane stability of coelomocytes, histidine quite handy factors to be considered as biomarkers of toxicity Catalase activity [78] exposure. As these changes underline all effects at higher Metabonomics analysis using NMR techniques [133] organizational level, therefore these have been regarded as highly sensitive indicators of toxicity. P-glycoprotein, major vault protein, topoisomerase-II [103] Acetylcholinesterase inhibition and imposex [134] Digestive enzymes, glycolytic enzymes, and cellular 2. Favoring Evidences: HSPs as a Biomarker [124] energy allocation 2.1. Aquatic Environment. The first favorable report on the Apoptosis in marine sponges and spiders [135, 136] use of HSPs as a toxicity biomarker came from the study of Cochrane et al. [40]. It was reported that exposure of rotifer Brachionus plicatilis to sublethal doses of CuSO4 resulted in is a universal phenomenon in all studied plant and animal a 4-5 fold increase in HSP58, with the maximum increase species including humans. Because HSPs can also be induced occurring at approximately 5% of the LC50 for the species. A by oxidants, toxins, heavy metals, free radicals, viruses, and similar response was seen with tributyltin. Kinetics of induc- other stressors, they are sometimes called the “stress proteins” tion was sigmoid with induction occurring in the range of 20– [2]. Heat shock proteins were initially discovered in 1962 in 30 𝜇g/L. However, no response was observed when exposed Drosophila melanogaster larvae in response to heat shock [3], to Al, Hg, Zn, sodium arsenite, sodium azide, sodium dodecyl and the term “heat shock protein” was coined by Tissieres et sulfate, or pentachlorophenol. It was suggested that HSP58 al. [4]. Heat shock proteins show characteristically marked abundance might act as a biomarker of toxicity exposure. variations in their expression in most of organisms under a Anumberofstudiesconductedonmollusks,mostly range of temperatures [5, 6], heavy metals [7–10], chemicals on mussels, for example, Sanders and Martin [41], reported [11–14], radiations [15–19], metabolites and hormones [20– elevated levels of HSP60 and HSP70 in mussels and fish 24], clinical situations [16, 25–30] or pathogens that may be tissue collected from polluted areas. The collection of data above or beyond their optimal limits [31–34], and drugs [35– on sediment and water chemistry from the sampling sites 37]. and on contaminant body burdens indicated their exposure Heat shock proteins are cosmopolitan in all living organ- to contaminants was long-term. This study suggested that isms and are usually classified as different families according HSP accumulation might provide a method of quantifying to their molecular size—HSP27, HSP47, HSP60, HSP70, adverse biological impacts of exposure to toxicants when HSP90, and HSP110. These families of heat shock proteins examining wild populations from contaminated sites. Simi- play crucial roles in physiological processes such as protein larly, another study conducted by Porte et al. [42]illustrated chaperoning activity, protection against apoptosis, steroido- statistically insignificant differences in the total levels of genesis, and stress tolerance. In addition, heat shock proteins, cytochrome P450 and benzo(a) pyrene hydroxylase activity especially HSP70 and HSP60, have also been proposed as and significantly induced HSP70 which correlated with the biomarkers of exposure levels and toxicity. Their candidature quantities of the PAHs accumulated in mussels Mytilus asbiomarkersisbasedonmanyobservationsshowingthat galloprovincialis (collected from sites polluted with aliphatic the HSP60 induction in certain organisms (e.g., mussel and polycyclic aromatic hydrocarbons—PAH). Again, it was Mytilus edulis and nematode Plectus acuminatus) results in suggested that HSP70 could be used as a biomarker in aseveralfoldsensitiveresponsethantheuseofothercom- Mytilus galloprovincialis against PAH toxicity. Another study parable parameters such as quantifying adverse effects on in mollusks considered HSP70 as biomarkers (in gill, mantle, biomass or reproduction [38, 39]. Such an observation in and digestive gland) and the reason of survival of Macoma mussels (M. edulis) led to their recommendation as one of nasuta (clam) in response to heavy metals (Ni, Cr, and Cu) the most suitable organisms for biomonitoring of aquatic eco- and trace organic pollutants (like PAH and organochlorine systems. pesticides—aldrin and DDT and its metabolites DDD and The role of HSP70/HSP60 as a biomarker is highly topical. DDE) exposure. Pearson and Spearman correlation analysis Certain studies have produced contradictory results; some revealed that mortality and HSP70 in gills were significantly indicate a high HSP70 sensitivity to pollutants, while others correlated with tissue concentrations of DDT and/or its suggest otherwise. The confounding issue in many such metabolites [43]. BioMed Research International 3

Schroder¨ et al. [44]validatedhepaticHSP70asapoten- induction of HSP60 related to increased concentrations of tial biomarker of cellular stress responses in fish Limanda Cu and Cd (4–400 𝜇g/L and 7–700 𝜇g/L, resp.). For copper, limanda at spawning stage 2. Varying levels of HSP70 (con- the induction of HSP60 was three orders of magnitude more sisting of two forms 75 and 73 kDa) among individuals were sensitivethanwastheEC20 for reproduction. For cadmium, observed at different locations of North Sea (Germany), but HSP60 induction was one order of magnitude more sensitive. each was correlated with intensity of DNA damage (single- Their results pointed out that HSP60 induction occurred at strandbreaksandalkalinelabilesites).ItwassuggestedthatL. concentrations that were realistic for the field situation (2 limanda might serve as a useful bioindicator and heat-shock to 4.8 𝜇g of Cu for loamy sand and clayey soil). Therefore, proteins as a useful biomarker for the monitoring of environ- HSP60 was suggested to be suitable as a potential biomarker mental pollution. The fish epidermis is highly susceptible, as to toxicant stress in P. ac uminatu s . About two years later, they it is the interface between the fish and its surrounding aquatic presented a review on the potential and limitations of inver- environment. Heresztyn and Nicholson [45]investigatedthe tebrate biomarkers (including HSPs, metallothioneins and use of HSP70 as a measure of sublethal ecotoxicity in cultured metal-binding proteins, esterases, histological and ultrastruc- skin epidermal cells of rainbow trout Oncorhynchus mykiss tural markers, lysosomal integrity, and the novel biomarker exposed to chemical stress (2,4-dichloroaniline). A positive histidine) for soil risk assessment purposes. They concluded toxicant concentration-dependent increase was noted in the that the HSP response in soil invertebrates was especially concentrations of HSP70 (quantified by immunocytochem- suitable to indicate the effects of exposure to comparatively istry). In addition to skin epidermal cells, the erythrocytes of low concentrations for a range of toxicants and could be certain fishes (e.g., silver sea bream Sparus sarba)havethe regarded as a biomarker of general stress [51]. However, in ability to synthesize HSPs due to presence of a nucleus and another study they suggested that HSP60 response in the therefore are considered as an interesting cellular model for in nematode alone was not a suitable biomarker for heavily vitro toxicological studies. In one such study, their exposure contaminated soils. It had indicative value related to the to sublethal concentrations of Cd, Pb, or Cr-VI as low as HSP70responseintheisopods(Oniscus asellus and Porcellio 0.1 𝜇M (a value which represented threshold concentration in scaber) and could be a suitable biomarker for moderately vitro) for 1 to 2 hours has shown significant overexpression contaminated soils. Furthermore, HSP70 concentrations in of HSP70 [46]. Guizani et al. [47]alsoprovidedfavoring nontolerant individuals of both these isopods species were evidence in support of HSP70 to act as biomarkers of considered to be suitable for use as potential biomarkers for environmental stress. monitoring environmental pollution [52]. In addition to rotifers, mollusks, and fishes, some authors Nadeau et al. [53] reported that HSP70i analysis by explored the potential of HSP70 as a biomarker of stress western blot in intestinal tissues of L. terrestris was a suitable in algae and other plants. For example, Bierkens et al. [48] and sensitive bioassay for the assessment of adverse effects suggested that HSP70 in alga R. subcapitata is a sensitive in earthworms when exposed to chemicals and heavy metals biomarker, as it displayed a dose-dependent increase in (chloroacetamide, pentachlorophenol, Pb, Cd, Cu, and Hg response to a wide range of pollutants (ZnCl2,SeO2,lindane, for1–16days).Theirdataalsoshowedagoodlevelof carbaryl, and SDS—but not pentachlorophenol) at concen- reproducibility despite some individual variations. In addi- trations below the range of classical cytotoxicity (i.e., growth tion, they suggested that the use of animals from pristine inhibition, lethality). However, ZnCl2 and SeO2 were found habitats transposed into contaminated environments is of to be the strongest inducers of HSP70. In another study, high ecological relevance. Induction of HSP70 in earthworms Ireland et al. [49] reported elevated concentrations of HSP70 should represent not only a good wide-spectrum biomarker in toothed wrack Fucus serratus and common duckweed of exposure but also a biomarker of toxicity effects since Lemna minor exposed for 24 hours to osmotic and cadmium known toxicants altered gene expression in these animals. stresses. In both stresses, the production of HSP70 increased Data gathered, therefore, is not simply a measure of accu- to the maximum and subsequently decreased as the stressor mulation of HSP. The detection of HSP70 in earthworms levels increased. They suggested that HSP70, tested by an could represent an early-warning system for the presence indirect competitive enzyme-linked immunosorbent assay, of potentially deleterious agents in soils, particularly in L. could potentially be applied for the stress detection in these terrestris and earthworms in general acting as potential aquatic species. In addition to HSP70 the biomarker response sentinel animal species. In other studies, HSP70 has also of MitosHSP to heat, ChlsHSP to H2O2, and antioxidant been suggested as a sensitive biomarker in coelomocytes of enzymes (Mn-SOD and Fe-SOD) and HSP60 to heat, H2O2, the earthworm Eisenia fetida exposed to metals like Zn, Cu, 2 and Pb in the dinoflagellate Karenia brevis has also been Pb, and Cd (at concentration of 1.32 𝜇g/cm of filter paper) reported by Miller-Morey and Van Dolah [50]. [54] and in McCoy cells exposed to low Hg, Cd, and CuCl2 concentrations (0.7, 1, and 3 𝜇M, resp.) [55]. 2.2. Terrestrial Environment. Various studies have also supported the use of HSPs as biomarkers in monitoring of soil 2.3. HSP in Transgenic Systems. Some authors have empha- pollution. In this context, most attention has been paid to sizedtheuseofHSPsintransgeniccellsandorganismsfor soil invertebrates, especially Plectus acuminatus (nematodes), environmental risk assessment. Examples include transfected Lumbricus terrestris (annelids), and gastropods (mollusks) HeLa cells with firefly luciferase having the HSP22 promoter with the emphasis on HSP70 and/or HSP60 as a biomarker sequence of Drosophila melanogaster for CdCl2,Cd(NO3)2, of toxicity. For example, Kammenga et al. [39]reportedthe NaAsO2, alachlor, fentin acetate, thiram, and maneb in 4 BioMed Research International the concentration range of 0.05–50 𝜇M[56]. Other exam- HSP72 after short-term exposure (2–6 hr) of pulmonary cell plesincludetheuseoftransgenicDrosophila melanogaster line (A549) to acute Cd concentrations (higher than 50 𝜇M) (HSP70-lacZ) for the phthalimide group of chemicals, captan, was considered an early biomarker for occupational exposure captafol, and folpet [57], for cypermethrin (0.002, 0.2, 0.5, to Cd but long-term (1 month) chronic exposure in vivo made and 50.0 ppm), and for effluents of chrome plating industry it doubtful because the expression of HSP72 decreased due containingCr,Zn,Fe,Ni,Mn,Pb,andCu[58, 59]. Embryos of to cellular adaptation to chronic Cd exposure [73]. Similarly a stable transgenic zebrafish with HSP70/eGFP reporter gene in juvenile rainbow trout exposed to Cd (1.5 𝜇g/L) and Zn system, in which HSP70 expression was activated in a tissue- (150 𝜇g/L) for 21 days, an adaptive response, to a lesser specific manner following exposure to a number of different extent, in the liver was shown by an increase in antioxidant toxins including Cd (data not given by author), served as a defenses (total glutathione, superoxide dismutase, and Trolox reliable and extremely quick indicator of cell-specific toxicity equivalent antioxidant capacity) without any impairment [60]. In transgenic zebrafish, reporter gene under human of GSH redox status or induction of HSP70 and HSP60 HSP70 promoter showed sensitivity to detect CuSO4 at doses [74]. as 1.2 m𝜇M[61]. Efremova et al. [75]reportedthatPbandZncaused Thus, HSP70 has often been highlighted as a particularly a strong induction of HSP; organochlorines and pen- sensitive biomarker of exposure to different pollutants in tachlorophenol also caused induction but did not enhance earthworms, mussels, clams, Drosophila, certain fishes, algae, consistently. Wastewater from the Pulp and Paper Plant and some aquatic plants. Quite a few studies have suggested caused a concentration-dependent increase in HSP70 expres- it as a biomarker of other adverse effects. Widely accepted sion in freshwater sponges endemic in Lake Baikal. However, models, such as Drosophila made transgenic for different there was no difference in the basal concentrations of HSP70 stress genes, namely, HSP70, HSP83, and HSP26, tagged with between sponges collected from polluted (with wastewater of reporter genes like 𝛽-galactosidase or GFP have been used to Pulp and Paper Plant) and unpolluted sites. Another study on detect cellular stress caused by environmental chemicals or the sponge Crambe crambe reported that the accumulation their mixtures [62]. The designed assays allowed the quan- and response of HSP54 were higher and quicker than HSP72 tification of stress gene expression following chemical pollu- for Cu exposure. However, HSP72 was significantly induced tion exposure, suggesting the magnitude of cellular toxicity only in the individuals transplanted to the contaminated site. inflicted by the chemicals63 [ –68]. Nisamedtinov et al. [69] Under experimental conditions, both heat shock proteins studied the response of the yeast Saccharomyces cerevisiae to were induced by copper at 30 𝜇g/L and inhibited at 100 𝜇g/L. different stress conditions employing transgenic technology. ThehighestmeanvaluesofHSP54andHSP72corresponded The group used HSP12p-Gfp2p fusion protein construct and to the sponges that showed the lowest mean values of toxicity. demonstrated that the abundance of HSP12p under different Thus, toxicity and production of HSP displayed antagonistic environmental conditions depended on the specific stress trends [76]. In ascidian Pseudodistoma crucigaster, HSPs factor. A rapid shift in stress factors gave higher rates of were induced only where Cu concentrations were under HSP12p synthesis compared to gradually changing stress half of the actual concentration in their polluted harbor. conditions. Similarly, for developmental toxicity studies, TheauthorsuggestedthatHSPwasusefulonlyasanearly zebrafish transgenic for HSP70 or HSP27 tagged with a GFP warning system for sublethal Cu pollution in the ascidian, reporter were exposed to heavy metals to examine the effect as the response was inhibited above a threshold value of of the xenobiotics on different stages of development [70]. the stressing agent, which was variable among species [77]. Furthermore, the effect of Cu (50, 100, and 960 𝜇g/L for 2– ∘ 3. Contradictory Evidences 24 h at 19 C) on the levels of HSP60 analyzed by western blotting analysis of the planarians Dugesia schubarti revealed A number of researchers have criticized the use of HSP70 no changes in HSP60 expression. However, catalase activity or HSP60 as biomarkers. Wieganta et al. [71], for example, was significantly affected. Therefore, it was concluded that reported that different stressors (arsenite, cadmium, dinitro- HSP60 should not be used as biomarker for Cu pollution in phenol, and ethanol) recognized as well-known HSP- planarians [78]. inducers, failed to stimulate specific HSPs in rat hepatoma Insect Tetrix tenuicornis was collected from polluted and cells to a degree that is comparable to the induction of these unpolluted sites for studying heavy metals accumulation and HSPs by thermal shock. Therefore, validation of stressor-spe- itsimpactonstressproteinslevel.Insectscollectedfroma cific risk assessment was considered through further research polluted area had higher concentrations (ranging from 1.5- to with larger groups of proteins. Mirkes et al. [72]reported 42-fold) of Cu, Zn, Pb, and Cd than control insects. Moreover, that the heat shock response, characterized by the synthesis heavy metals accumulations caused only minor variations in and accumulation of HSP72, was not a general biomarker the accumulation of Hsc70 and HSP70i [9]. Furthermore, an in rat embryos for chemical teratogens such as N-acetoxy- inhibition of HSP70 synthesis has also been observed in the 2-acetylaminofluorene, CdCl2, cyclophosphamide, sodium earthworm Lumbricus terrestris in response to a variety of arsenite (AS), and sodium salicylate (SAL). Last two chem- metals such as Pb, Cd, and Cu [53]. While evaluating the icals induced the synthesis and accumulation of HSP72, and power of HSP70 as an environmental biomarker of fish health both have different accumulation kinetics; otherwise, these during field conditions, Webb and Gagnon79 [ ]reportedthat chemicals caused embryotoxicity characterized by abnormal HSP70 measurement alone was insufficient to evaluate fish development and growth retardation. Overexpression of health conditions. BioMed Research International 5

4. Discussion: Critical Analysis be avoided and results should be interpreted on the basis of strength of toxicant. Furthermore, the potency of various A general conclusion regarding the validity of HSP70 and/or toxicants significantly differs in inducing HSP70 promoter. HSP60 as biomarkers of toxicity exposure or effect is difficult For example metals like Cd, Zn, and Hg and organic com- to reach, in part due to the conflicting nature of many of pounds like chlorophenol derivatives, 3, 4-dichloroaniline, the above research reports. However, certain research reports ethyl parathion, benzo(a)pyrene, 2, 4-dichlorophenoxyacetic enable us to draw conclusions regarding the strength of spe- acid, endosulfan, diuron, and 4-nonylphenol are effective at cific biomarker candidates. In analyzing the research to date, noncytotoxic doses, while tetrachlorohydroquinone and 1- we have encountered 9 basic concerns regarding the limi- chloro-2,4-dinitrobenzene induce the promoter at cytotoxic tations of the use of HSP as biomarkers. These are explained doses [13]. Occasionally, one metal enhances the accumu- in detail in the following discussion. The following concerns lation of others. This is the case with a combination of Cu should be addressed by the researchers in order to reach some and Zn which resulted in higher accumulation of Zn in concrete conclusions. HepG2 cells [80]. Occasions were also noted where HSP70 (1) Can these HSPs as biomarkers reveal both the con- concentrations did not correlate with the degree of metal centration and nature of a specific toxicant in an exposure (as in crabs Carcinus maenas for Cu and Zn) [81]. environment? Furthermore, a deficiency of certain metals, for example, Cu, reduced the expression of HSP70 in certain tissues (cardiac) (2) Are uptake, threshold concentration, and inducing and their mitochondria [82], and the exact effect of such ability of all metals/toxicants the same? dietary deficiency on other organs/cases is still in question. (3) Do variability of HSP70 concentrations among various organisms and the acquisition of tolerance signif- 4.3. Interindividual Variability of HSP70 Concentrations and icantly affect the results? Acquisition of Tolerance. Individual differences exist even (4) Do age and gender based differences have no effect on at molecular levels, a factor, for instance, giving rise to expression of HSP70? individual variability in HSP70 expression in intestinal tissues of L. terrestris [82]. Some authors have reported a weak cor- (5) Are physical properties of soil and variable detox- relation between toxicant and HSP70 as biomarkers due to ification mechanisms among species not acting as interindividual variability of HSP70 concentrations. This was confounding factors? thecaseinthebaymusselMytilus trossulus, where the inter- (6)Doesasynergisticeffectofdifferenttoxicantsalong individual variability tended to mask inductions of HSP70 environmental factors modify the expression of heat at low concentrations of As(III), thus making it a less effi- shock proteins? cient biomarker of toxicity. To avoid such problems pre- or (7) Do different techniques for HSP detection produce poststress specimens are required to be analyzed to allow for similar results for a particular toxicant in the same greater HSP70 sensitivity and reliability. Other markers, such organism? astheuseofactin,arerequiredtobeusedascontrols;theuse of HSP70-reporter gene constructs is suggested and detection (8) Are the studies validating HSP as biomarkers broad with HSP60, heme oxygenase-1, metallothionein, CYP450, enough? MXR, or GPx has been recommended by La Porte [83]. (9) Is application of biomarker sets a useful strategy for Another difficulty in validating HSPs as biomarker for assessment of toxicity? ERA is the enhancement of stress response (in form of increased synthesis of HSP70/HSP60) in self-tolerance as 4.1. Concentration and Nature of a Specific Toxicant. These well as in cross-tolerance by nonlethal doses of toxicant in HSPs did not reveal the concentration or the nature of a sensitized cells/tissues (the cells or tissues already exposed to specificstressor,thatis,typeofheavymetalorchemical,as such toxicants in self-tolerance or to other toxicants in cross- these showed a marked variation in their concentrations in tolerance). An example is amphetamine and Cd in hepatic response to a variety of stressed conditions. Moreover, these cells [84, 85]; Hg [86]anduranium[87–89]inthekidney concentrations did not give a true picture of different toxi- are able to induce self-tolerance, while Zn against Cd in rat cants in an environment, that is, how many toxicants were proximal tubule cells [90], Pb against Cd/C2H2 in astroglial present in a specific locality or habitat under investigation. cells [91],As,Cd,Hg,Pb,Cu,menadione,anddiethyldi- thiocarbamate against lethal temperature in Reuber H35 4.2. Uptake, Threshold Concentration, and Induction. Thresh- hepatoma cells [92] were also able to induce cross-tolerance. old metal concentrations for the HSP70 induction vary “How many times?” and “To what extent has an organism among different metals. For example, in zebra musselsDreis- ( been exposed?” are questions to be addressed under the natu- sena polymorpha) metal uptake analysis for Pt, Pd, Rh, Cd, ral environments. Therefore, acquired resistance against cyto- andPbindicatedthehighestuptakeforCdfollowedbyPt,Pb, toxicity as well as cross-tolerance in a large number of org- Pd, and Rh. The highest HSP70 values were observed in the anisms also adds uncertainty in the role of HSP as biomarker caseofexposuretoPdfollowedbyPt,Rh,Pb,andCd.There- in ERA. Likewise, biomarker responses vary in relation to the fore, Pd seems to be a particularly potent inducer of HSP70, duration and level of exposure under laboratory conditions despite its relatively low threshold concentration [8]. There- and are also dependent on the population [93, 94]. Acquisi- fore, simple conclusions on the basis of body burdens should tion of tolerance by certain organisms not only masks the true 6 BioMed Research International picture but also seems as a dubious factor when applying the a battery of heat shock genes. Synergistic activation has issue to the establishment of water quality criteria [95]. also demonstrated the metal-responsive promoters by heavy metals (Zn or Cd) and heat shock in combination. Heat also 4.4. Age and Gender Based Differences. Certain studies have stimulates the intracellular accumulation of Zn and Cd when revealed that not merely the functional ability of HSP70 provided exogenously during a heat shock, (in HEK293- against stress conditions that decreases with aging [96]but mammalian cells) and thus results in a hyperactivation alsoagingisassociatedwithanactualdecreasedconcentra- of the metal response pathway. Interestingly, relatively low tion of HSP70 and subsequently a decreased ability to res- concentrations of these heavy metals alone hardly induced pond to stressed conditions [97]. Certain studies have also transcription at all and served as sufficient trigger for such revealed gender based differences regarding expression of synergistic activation of mammalian HSP70 promoter (Fig- HSPs. For example, HSP70 gene assay in the reproductive ures 2(a) and 2(b))[101]. Similarly, water-soluble fractions of organs of adult flies showed its expression restricted to male different sludge containing varying concentrations of heavy flies [59]. Considering these studies, such gender and age metals(Cd,Cr,Cu,Ni,Pb,andZn),whengivenseparately based differences are also expected in other organisms with to human cultured cells (HT29 cell line from gut mucosa), respect to HSP70 in response to toxicants. The age and sex failed to trigger significant expression of HSP72. When given of an organism should also be considered, especially when in combination, they exerted a strong synergistic effect by studyingtheroleofHSPsasariskassessmentofenviron- causing significant overexpression of HSP72Figure ( 3)[102]. mental pollution’s effects in sex organs. Increasing concentrations of HSP70 have also been observed in HepG2 cells under the synergistic effects of Cu and Zn 4.5. Confounding Factors. In case of soil pollution, Filzek as compared with each metal (Figure 4)[80]. Another study 󸀠 󸀠 et al. [98] emphasized the consideration of the underlying conducted by A¨ıt-A¨ıssa et al. [74] reported that 3,3 ,4,4 - geology,thenatureofthesoil,andthelanduseasessential tetrachlorobiphenyl (1 mg/kg) strongly induced HSP70, while prerequisites to understand the significance of any observed its coexposure with metals did not modulate significantly biological effects. The authors also provided extensive discus- its effects. However, 17-beta-oestradiol in combination with sion on how the availability and mobility of various heavy Cd/Zn had shown a synergistic effect. metals at the selected field sites were influenced by a wide Apart from the synergistic effects of toxicants, environ- range of factors such as pH, organic matter, and clay content. mental factors such as temperature (Figure 5)[103], salinity The significant differences in toxic responses measured in [104], and oxygen supply [105] also influence the expression the laboratory exposed versus field exposed nematodes by of HSPs and may also have a synergistic effect in combination Arts et al. [52] were explained due to confounding factors with toxicants, thus making their consideration as biomark- such as food availability and differences in contaminant ers doubtful. For example, Cd exposure increased the HSP70 uptake routes under the different exposure regimes. It was concentrations in marine clams, whereas salinity markedly also suggested that differences existed between native ani- lowered the same level in that species. A laboratory study mals transplanted to the field and field collected animals, regarding the effects of salinity on HSP70 concentrations partlyattributabletotheincreasedandprobablyinherited indicated that exposure to 0.1 ppt salinity markedly lowered tolerance of the field population. Furthermore, physiological HSP70 concentrations in clams Potamocorbula amurensis differences in the way an individual species handles the compared with those exposed to higher salinities (Figure 6 uptake, detoxification, assimilation, and eventual excretion and Table 2)[106]. Increasing the salinity from 5 to 25 per of accumulated metal also influenced the HSP70 response thousand resulted in lowering the toxicity and concentrations in isopods. Such physiological differences exist not only in of the free metal ions (Figure 7). This effect has been regarded terrestrial organisms but also in aquatic [99]. as the strongest for Cd and Pb, while such smaller effects were observed for Ni, Cu, and Zn [104]. The rate of uranium 4.6. Synergistic Effects among Toxicants as well as between accumulation in the gill tissues of clam Corbicula fluminea Toxicant and Environmental Factors. No data exist regarding was higher under hypoxia than normoxia. At the cellular the synergistic effects of different toxicants and/or with the level uranium instead of hypoxia induced the expression of other aspects of environmental stresses (temperature, pH, multixenobiotic resistance protein. On the contrary, HSP60 salinity,etc.).Forinstance,thetoxiceffectissignificantly was induced by hypoxia instead of uranium [107]. altered under the additive effects of many heavy metals as Some authors have also regarded HSP70 as a biomarker compared to cases of isolated single metal toxicity where two in the algae Raphidocelis subcapitata in response to changes or more metals are found in a combination. Individual dose in pH, temperature, humic acids, nitrates, and phosphates. of 20 𝜇M of As, Cd, and Hg induced only a modest HSP70 Algae responded to these changes by a transient increase increase, whereas their combination at the lowest levels of in HSP70 concentration. Temperature and pH were found toxicity still induced a greater accumulation of these proteins to induce acquired tolerance; that is, algae grown at a pH (Figure 1)[100]. Furthermore, a number of stress genes which or at a temperature different from control conditions were respond to heavy metals (such as HSPs and MT) contain showntohaveacquiredresistancetoasubsequentchallenge 5 metal-response elements (MREs) in their promoter/enhancer with Zn (10 M). These results qualify HSP70 as a biomonitor region, which is activated by a metal-responsive transcription for environmental pollution provided that essential environ- factor-1 (MTF-1). The response to heat shock is mediated mental parameters such as pH and temperature are kept by heat shock transcription factor-1 (HSF-1), which activates constant [108]. Apart from temperature and pH, much similar BioMed Research International 7

1.0 1.0 g protein) g protein) 𝜇 𝜇 0.5 0.5 i(ng/ i(ng/ 70 70 ∗ Hsp

Hsp As

As ∗∗

0.0 0.0 Cd Hg As Cd + Hg Cd + As Hg + As Cd + Hg + As

10 𝜇m 10 𝜇m 20 𝜇m 20 𝜇m 40 𝜇m 40 𝜇m (a) (b)

Figure 1: Toxic metals induce HSP70i accumulation in podocytes in a dose-dependent manner. Results of a quantitative western blot analysis of HSP70i accumulation in podocytes treated with various concentrations of individual toxic metals (a) or combinations of two and three toxicmetalstotaling10,20,or40mM(b)for3days.ValuesareexpressedasngofHSP70ipermgtotalprotein.BasalHSP70ilevelswere below the limits of detection [100].

500 8

400 7

6 300 5 200 g/g protein

𝜇 4

100 3 Fold activation Fold 2 0 1 h) h) Zn Cd 1h) , ,1 ,1 + +

C 0 M M

42 1 2 3 4 5 6 7 8 9 10 11 12 Heat Heat 60 𝜇 Nontreated 100 𝜇 Zinc (100 𝜇M) − ++−− −−++−− − Heat ( Cd ( Cd 60 𝜇 ) −−++++ −− −− −−

Zn ( Zn Cadmium ( M Zn Heat shock (43 C, 1 h) − −−+ + + −−−+ ++ Cd mHSF-1 (2𝜇g) − −−−−−+ + + + + + (a) (b)

Figure 2: (a) Cellular accumulation of Zn and Cd is boosted by heat shock. After addition of Zn and Cd to final concentration of 100 ∘ and60uM,respectively,withorwithoutheatshock(42C for 1 h). HEK293 cells were harvested and analyzed by ICP-MS. The data from three independent determinations has been shown [101].(b)ExpressionofHSP70promoterbyCdandheatinpresenceorabsenceof HSF-1. HEK293 cells were transfected with HSP70-Luc promoter-reporter construct, the CMV-LacZ reference construct, and mouse HSF-1 ∘ expression vector. 36 h after transfection, cells were treated with 100 𝜇M ZnCl2 or 60 𝜇M CdCl2 with or without heat shock at 43 Cfor1h. The cells were collected and reporter gene activities were determined by luciferase assay. The basal level was taken as 1 to calculate the fold activation [101]. 8 BioMed Research International

∗ Table 2: HSP70 levels in clams during adaptation to various salinities .

HSP70 (relative density ×103) Salinity (ppt) After 24 h After 24 h After 24h 0.1 17.91 17.41 14.59 3 17.72 23.92 38.08 627.3439.2535.82 10 (ambient) 31.05 26.20 17.80 14 19.56 36.10 32.6 27 37.53 31.08 35.56 ∗ Clams collected from Martinez marina (salinity: 10 ppt). Values represent relative density arbitrary units of bands detected by western blotting of pooled samples of five clams Potamocorbula amurensis, with no significant differences in temperature but salinity increasing in gradient manner in the sampling sites (Werner and Hinton, 2000 [106]).

150 S1 125

C 0.6 1.2 0.6 1.2 0.6 1.2 C 0.6 1.2 0.6 1.2 0.6 1.2 0.6 1.2 0.6 1.2 C SE SE + HM HM SE SE + HM HM SE SE + HM 100 S2 75

CC2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 2.5 5 SE SE + HM HM SE SE + HM HM SE SE + HM HM (%) Control 50 S3 25

C 0.7 1.5 0.7 1.5 0.7 1.5 C 0.7 1.5 0.7 1.5 0.7 1.5 0.7 1.5 0.7 1.5 0.7 1.5 0 SE SE + HM HM SE SE + HM HM SE SE + HM HM 2 2+1 2+24 4 4+1 4+24 Time (hr) Figure 3: Immunoblots showing HSP72 levels in HT29 cells after a 24 h exposure to the different sludge extracts at different Cu concentrations. Cells were subjected to soluble sludge extracts alone Zn (SE), to soluble sludge extracts + heavy metal solution (SE + HM), or Cu + Zn to heavy metal solution alone (HM) at the indicated concentrations Figure 4: Scanning densitometry analysis of HSP70 in Cu expressed as grams per kilogram of dry material. Untreated cells are 𝜇 𝜇 control (C) S1, S2, S3, different sludge [102]. (30 g/mL) and Zn (50 g/mL) alone or in combination treated HepG2 cells expressed as percent of control at the time described [80]. emphasis has also been placed on the analysis of nutrients, electrolytes, and dissolved oxygen content [105]. Another environmental factor influencing the HSP C NA 27 CNA+27C expression is seasonal variation, which acts partially according to the corresponding temperature regimes. Seasonal variations in HSP70 as noted in the mussel (Mytilus galloprovincialis) at two sites of Mediterranean Sea Carteau (native site) and La Fourcade (transplantation site) in a two-year study may be more likely a result of combined environmental ∘ Figure 5: Effect of 2 h heat shock (27 C) and sodium arsenate factors (temperature, salinity, and turbidity) and chemical 𝜇 ∘ contamination levels [109]. Bodin et al. [109]conducteda (10 m) in A6 cells of Xenopus laevis with control 22 C[120]. comprehensive study; they did not suggest any correlation of variations in biomarkers level with other parameters. They also reported that mussels of both sites have specific chemical and PCBs (90.2 mg/g dw) as La Fourcade. The seasonal contamination profiles but having a similar range of values. contamination trend at Carteau showed a sixfold higher level For example, both sites were highly contaminated by heavy of pyrolytic pollutants in winter. It showed that seasonal metals (201 and 258.4 mg/kg dw, resp.) and considered as variation in contamination levels was a man-made activity moderatelyimpactedforpolychlorinatedbiphenylsandpoly- whichcorrelatedwellwiththeirdailylifeneeds[110]. Another cyclic aromatic hydrocarbons; nevertheless, contamination studybyHameretal.[111] investigated the concentrations levels at Carteau were twice as high for PAHs (101.5 mg/g dw) of HSP70 in the gills of the mussel Mytilus galloprovincialis BioMed Research International 9

100000 combination with other environmental factors, pollutants can 80000 contribute to the weakening of defense and regulatory mechanisms of studied organisms. Hence, the biomarkers of expo- 60000 sure related to these mechanisms of early physiological regu- 40000 lation are subject to variations that make it difficult to detect

(relative density) (relative the specific effects of chemical pollutants. The interference of

70 20000 natural environmental factors in the expression of biomark-

Hsp 0 ers is an important issue with respect to the use of biomarkers 4.1 6.1 8.1 12.5 in monitoring the biological effects of pollutants in their Station natural environments, making field interpretations difficult. Therefore, the effects of environmental factors should also be Figure 6: HSP70 levels in Potamocorbula amurensis (means ± SD, considered in sampling strategies for monitoring programs to 𝑛=90)measuredeachmonth(𝑛=6–8 per station) from 07/1996 prevent false interpretation of results. Furthermore, certain to 01/1998 (except 10/96, 1–3/97, and 10/97). One-way ANOVA and field studies have also shown that stress response can occur Tukey analysis revealed two major groups of sites: group A with sites even at minute pollutant concentrations that are usually 𝑃 < 0.001 4.1 and 6.1 and group B with sites 8.1 and 12.5 ( )[106]. prevalent in the environment. Increasing knowledge on the kinetics and persistence of the stress response to complex 100 environmental mixtures (the influence of both physiological and environmental parameters), the constitutive levels of 80 HSPs and the acquisition of tolerance are required before safe 60 application of HSPs to assess onsite pollution. 40 4.7. Detection of HSPs through Northern and Western Blot. Mortality (%) Mortality 20 Normally, both techniques are used for HSPs detection to 0 quantify HSPs as biomarkers of toxicity. Some researchers 012345678910 emphasized that northern blot is a highly sensitive and Exposed toxicity initial step in the detection of environmental stress on gene expression. Other scientists emphasized on western blot by 5 15 the fact that changes in mRNA expression do not necessarily 10 25 correspond to changes in protein levels [7]orthatpolyribo- Figure 7: The concentration-response curve of Neomysis integer for some may be involved in protein synthesis under certain cir- a mixture of six metals at different salinities (5, 10, 15, and 25‰). cumstances.AsimplemeasureofmRNAmayyieldadoubtful The toxicity decreases with increasing salinity and higher salinities reliability.Whichisreallymorereliableremainsquestionable. above 25‰ had no further influence on the 96 h LC50 of the mixture For some HSPs, a correlation was noted between mRNA ofsixmetals(Hg,/Cd,/Cu,/Zn,Ni,/Pb)whichissituatedatavalue induction and its proteins (HSP60, HSP68, and HSP84) between 4.4 and 4.6 T.U [104]. [114]. Some others did not find any correlation between mRNA levels and protein synthesis, for example, HSP68 [115].SomecaseshavealsobeenseenwheremRNAlevels seasonally collected from different sites of the Rovinj coastal remained constant after exposure to heavy metals (ZnCl2, area (Croatia). They observed maximal levels of HSP72 and 0–330 𝜇M), while protein levels significantly increased in a HSP70 in summer (September) and minimal concentrations dose-dependent manner [116]. Hence, conclusions cannot be in winter (December). HSP70 showed significant correlation drawn only on the basis of either one, and both aspects should with the sea temperature (𝑟 = +0.822, 𝑃 < 0.05)only.Simi- be explored in proposed model organisms. larly, a significant seasonal (March and September) difference We do not intend to imply a lack of quality of the work in HSP70 content has also been found in centipedes collected in the studies validating HSPs as biomarkers. In fact, all such from unpolluted areas [112]. Female fishes collected from two studies are valid and we acknowledge them for their valuable ∘ ∘ different localities during spring (26.5 C) and winter (4.8 C) findings. However, in our opinion, there are some logical also displayed a similar trend in the ovarian and liver tissue of questions that must be addressed with reference to certain the black bullhead Lepomis macrochirus, in head and kidney studies. For example, Arts et al. [52]narratedthatHSP60 of the bluegill sunfish Ameiurus melas, andinthegilltissue response in the nematode Plectus acuminatus had an indica- of both species [113]. tivevaluerelatedtoHSP70responseinisopodsandcould Thusseasonalvariationsactthroughtemperatureand be a suitable biomarker for less heavily contaminated soils. nutritional regimes, as well as through quantity and quality of Such reasoning is ambiguous. If we need to check the extent pollutants dumped into environments according to seasonal of soil contamination prior to concluding the significance activities of human beings. As a result, complex molecular of a biomarker response then what is the advantage of such interactions in actual environmental habitats are operating biomarkers? on biological structures, and in the case of chronic pollution Most of the studies validated HSPs as biomarker of toxic- theactionofthetoxicsubstancesmaynotbepredominant ity on the basis of 2–5 toxicants and even some on the basis of but is associated with many other environmental stressors. In only a single toxicant [58, 59, 78]. Just a very few investigators 10 BioMed Research International considered synergistic effects.¨ Aıt-A¨ıssa et al. [13] investigated HSPs [119]. These studies suggest the application of the maximum number of toxicants (3 metals and 15 organic such biomarkers in combination as a set for ERA. chemicals) in this context. We were unable to locate any (c) Toxicity responses broadly vary among individuals of reference for the studies with analysis of significantly broad different species. Some examples exist where toxicity range of toxicants or studies containing all possible heavy stress alters the HSP levels in some organs (e.g., in gills metals or all organic pollutants. If an initial study used Cu, andliversincaseoftrout)andinsomecaseswholeof Cd, and Hg for gill and hepatopancreas in fishes, for example, the organism as in gammarids [105]. Such studies and a more useful continuation of such research would be to thosethathavecontradictedthevalidityofHSPsas continue testing different metals on the same organs of same biomarker for ERA can still help us in the selection of animal rather than using the same metals for a different organisms. animal. In this latter case it is very difficult if not impossible to validate HSPs as a biomarker of toxicity exposure or of (d) The expression pattern of HSPs is not only tissue- adverse effects. For example, if we are testing the organism for specific [34, 120] but also species-specific, as revealed environmental risk assessment in which expression of HSP70 by decrease, moderately increase, and overexpression or HSP60 was considered as biomarker in response to Cu, of HSP73 in COS-7 cells (African green monkey Cd,Zn,andHg,theenvironmentmayalsohaveCrand/or kidney cells), A549 cells (human lung tumor cells), organic toxicant in addition to electromagnetic waves; then andratskidneycells,inexposureto100,200,or400M how can we interpret the results, as HSPs respond to a variety NiCl2,respectively,for4days[121]. Such studies of toxicants in suppressive as well as in overexpressive ways suggest the selection of organisms as a combination depending on the concentration of toxicant and duration set for ERA. of exposure? To broaden the testing in a range of potential toxicants and environmental factors for a single species would To determine the variability of sublethal effects of pol- advance the field to a far greater extent. lutants, only a few studies have been conducted up to the third point. For example, Downs et al. [122, 123]developed 4.8. Application of Multiple Biomarkers as a Set. The rela- a molecular biomarker system (MBS) based on 9 specific tionship of one biomarker to other potential biomarkers cellular parameters to assess the physiological status of the on exposure to particular contaminants in different species grass shrimp Palaemonetes pugio (exposed to Cd, atrazine, and in different organs or tissues of the same species is a and bunker fuel) and mud snails Ilyanassa obsoleta (exposed key consideration prior to their widespread application in to Cd, atrazine, bunker fuel, endosulfan, and heat stresses). environmental management. Though more complex, such a They assayed HSP60, HSP70, alpha B-crystallin homologue, technique may enable the use of biomarkers to give a much lipid peroxide, total glutathione level, ubiquitin, mitochon- clearer picture of the environmental situation. Certain studies drial manganese superoxide dismutase, metallothionein, and have already begun to consider this area. Their systematic cytochrome P-450 2E homologue. They reported that MBS approach is being considered here as follows. was distinguishable among responses to each stressor and to nonstressed control conditions; that is, the biomarkers (a) Certain studies have used various xenobiotics as a tool metallothionein and cytochrome P450 2E homologue distin- to study stress protein synthesis in target organs in guished between metal and nonmetal stresses. A¨ıt-A¨ıssa et al. order to evaluate the target tissue-specificity of the [74] confirmed by multivariate analyses that some correlation toxicant. For example, the kidney is a target tissue exists between these biomarkers and concluded the use for chronic Cd exposure, so HSP expression in it can of complementary biomarkers as necessary to discriminate be used as biomarker [117] and even some metals between different treatments and to highlight interactive like Hg induce regional and cell-specific stress protein effects. In addition to animals, HSP70 could potentially be expressions in rat kidneys [86]. Similarly, tissue- appliedtothedetectionofstressinaquaticplantslikeFucus specific differences in the accumulation of HSP70 and serratus and Lemna minor.Butitwouldbemosteffective HSP60 in Mytilus edulis exposed to a range of copper when used in conjunction with other measurements to pro- concentrations have also been reported by Sanders et vide a stressor-specific biomarker profile or fingerprint [49]. al. [38]. Such studies could help in the selection of a In the case of soil, Arts et al. [52]reportedthatHSP60 combination of target tissues/organs for evaluation of response in the nematode Plectus acuminatus had an indica- HSPs as a biomarker for ERA. tive value when related to HSP70 response in isopods and could be a suitable biomarker for less heavily contaminated (b) Some stressors have quite opposite effects on the soils. Similarly, for analysis of HSP70 in coelomocytes, Homa expression of HSP70 in different cases. For example, et al. [54] also reinforced the notion given by earlier studies Ni concentration of 600 M has no effect on HSP70 that the value of biomarkers is higher when they are employed expression at the transcriptional level in HeLa cells in combination as suite rather than individually [93, 124]. [118], whereas it has shown sufficient expression in Previous researchers tested only a limited number of toxi- black sea bream fibroblast cell line at concentration cants and, in some cases, only heavy metals were under inves- of 0.01 M [10]. Arsenite is a much lesser inducer tigation. The application of these suits is possible in an already of MT but a more effective inducer of HSPs, while tested environment/locality with similar levels of toxi- nickelisagoodinducerofMTbutpoorinducerof cants and conditions. In any other region with a different BioMed Research International 11

nature or extent of contamination results may remain con- be considered; it seems that anionic conjugates of metal (SO4, founded. However, such an approach can yield better results Cl2,etc.)mayberesponsibleforspecificinductionofHSP if applied like a taxonomic key. genes. This can be observed from the fact that metal ions Gupta et al. [62] considered HSPs as suitable early conjugated with SO4 were able to modulate the transcription warning bioindicator of cellular hazard. It was further argued ofHSP70;howeverthiswasnotthecaseformetalsconjugated that despite having enormous use in toxicology, the current with Cl2 [126]. state of knowledge in defining a mechanism of action or TranscriptionprofileofHSP70wasalsostudiedinMytilus accurately predicting toxicity based on stress gene expression coruscus in response to fuel and heavy metals stress [127]. In warrants further investigation. The properties of heat shock all the treatments HSP expression was induced with varying proteins as (i) part of the cellular protective machinery, (ii) degree of magnitude. HSP70 expression steadily increased inducible nature against a wide range of chemicals, and (iii) with the passage of time and reached to the maximum (about higher conservation across the taxa have proven them to be 6-fold increase) after 25 days of the treatment. However, valuable as a first tier biomarker in risk assessment. Although expression started decreasing after gaining the peak level, stress gene expression has proved promising to understand andatday30,expressionwasabouttwofoldofthecontrol. 2+ 2+ the toxicity of chemicals, literature linking activation of stress Similar pattern was observed for heavy metals Cu and Cd genes to mechanism of toxicity is limited [62]. but with more pronounced increase in the expression level 2+ 2+ In a recent study, HSP expression profile was used as of HSP70. Both Cu and Cd enhanced expression to 10- biomarker in the fish for monitoring the water quality of fold and 11-fold, respectively. However, time of maximum 2+ ariver[125]. Four of HSP genes, namely, HSP30, HSP60, expression was different; in the case of Cu , peak expression 2+ HSP70, and HSP90, were amplified and sequenced by using wasachievedatday15whileinthecaseofCd it was at day degenerate primers. Later on, gene specific primers were 9[127]. Although authors have advocated HSP70 as potential developed and subsequently used to monitor the expression biomarker for heavy metals and fuels, this notion is ques- of above mentioned four genes. Compared to the fish at tionable due to the fact that HSPs expression is responsive reference site, up to 10-fold difference in expression of HSP to other environmental factors like increase in temperature. 70 was observed, in the liver. More profound differences Moreover,itseems,atleast,inthisstudybyLiuetal.[127] were observed in expression of HSP30 in the kidneys of fish. that HSPs expression showed a delayed response to heavy However, as far as HSP60 and HSP90 are concerned, no metals stress, as compared to hydrocarbons. In the latter case, difference in expression level was observed. These differences expression was modulated in matter of hours while for heavy in HSP expression correlated well with the quality of water; metals, it happened in days. In real environmental conditions more profound differences were observed in downstream it will become almost impossible to correlate the change in water as compared to upstream. Authors were of the opinion expression of HSPs to a particular stressor. that HSP30 and HSP70 expression can be used as biomarker HSP70 expression was taken as one of the biomarkers for for evaluation of water quality [125]. studying the effects of heavy metal accumulation in milk fish Transcription profiles of two of HSP genes, that is, (Chanos chanos) collected from polluted sites of Kattupalli HSP70 and HSP90, were used as biomarkers against metals Island, India [128]. Through immune fluorescence scanning and organic compounds’ stress in marine diatom Ditylum and western blotting, HSP70 accumulation was observed in brightwellii (Db) [126]. D. brightwellii cultures were exposed gills and liver of the milk fish. Expression of HSP70 was more 4, NiSO4,CuCl2, to various metal compounds, namely, CuSO in the gills of fish collected from polluted site as compared and NiCl2, and HSPs gene expression was monitored by to that from less polluted site. Similar expression pattern of using real-time PCR. It was observed that all tested metal HSP70 was observed in the liver tissue of the fish [128]. The compounds induced the expression of HSP90 gene; however induction pattern was different according to the tested metal authors have shown more realistic approach in drawing the conclusion based on this study. In spite of showing optimism compound. All concentrations of CuSO4 effectively induced the transcription of HSP90 while only higher concentrations in taking HSPs as a biomarker, they have stressed on more of CuCl2 and NiSO4 were able to cause a significant increase integratedapproachforassessmentofmetalcontaminationin in expression. NiCl2 initially increased the expression of ecosystem. To summarize, HSP70 can be taken as biomarker, HSP90 in a concentration-dependent manner. However, at however not in isolation. Instead it could be more logical to much elevated level opposite effect was observed; that is, use it as biomarker along with other parameters like oxidative expression decreased with further increase in NiCl2 level. stress biomarkers and ultrastructural changes. HSP70 expression followed a different pattern; expression Few reports have also shown the downregulation of was induced by CuSO4 and NiSO4 but not by CuCl2 and HSP70 gene in response to heavy metal stress. In a recent NiCl2. Moreover, the expression of former two genes was not study, Luo et al. [129] have observed the effect of long-term concentration dependent. In the same study, effect of thermal heavy metals stress on Crassostrea hongkongensis,througha stress and organic pollutants was also analyzed. Thermal proteomic approach. Differentially expressed proteins were stress induced expression of both genes; however, tested identified in oyster exposed to heavy metals such as zinc, organic pollutants had no significant effect on expression copper, manganese, and lead. One of differentially regulated of HSP genes. This data shows that HSPs are differentially proteins was identified to be HSP70. In contrast to usu- involved in defense against various stressors. Based on find- ally reported upregulation, HSP70 was found to be down- ings of above mentioned study, an important point needs to regulated in Crassostrea hongkongensis [129]. This unusual 12 BioMed Research International transcription pattern was attributed to prolonged exposure the focus upon broadening the testing of a range of potential to heavy metals. This raises further question mark on the toxicants and environmental factors for a limited number of validity of HSPs as a universal biomarker of stress. key target species. Furthermore, there is need of search for target organs specific for a particular toxicant. With such a 5. Closing Remarks systematic and focused approach such biomarkers could yet possibly be elevated, when applied in sets, from the current All studies, whether supporting or contradicting the validity general indications they provide to becoming techniques of HSPs as biomarker of effect or exposure, are useful for which yield much more specific, useful, and accurate data establishing the biological exposure limits of toxicants and readily applicable for environmental management. in creating awareness about their biological effects. Though some contradictory studies reject the application of HSPs as Conflict of Interests biomarkers in various fields and situations, these actually, from one perspective, also aid the more appropriate appli- The authors do not have any conflict of interests or financial cation of HSPs as biomarkers elsewhere by suggesting the gain from this paper. types or organisms or conditions where HSPs may be less suitable or useful. Furthermore, these studies also suggest certain new fields of research in proposed model organisms Acknowledgments for ERA. For example, the search for self- and cross-tolerance The authors thankfully acknowledge the permission granted and seasonal, individual, sex, and gender based variations in by Elsevier Publisher, Oxford University Press, Allen Press thelevelsofHSP70/HSP60aloneaswellasincombination Inc., and The American Society for Biochemistry and Molec- with various environmental factors (temperature, salinity, ular Biology for the use of certain illustrations and a table for pH, oxygen/hypoxia, etc.) with emphasis on toxicant uptake, the compilation of present paper. accumulation, detoxification, synergistic effects, threshold levels, and induction kinetics of HSPs in proposed models are all areas of valid further examination. Further, “suit of References biomarkers in a set of organisms” should also be investigated under these guide lines before suggestion of the application [1] S. Wicherek and J. P.Oudinet, “Environment and health-studies of such for environmental risk assessment. 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Research Article Protective Role of Omega-3 Polyunsaturated Fatty Acid against Lead Acetate-Induced Toxicity in Liver and Kidney of Female Rats

Heba M. Abdou1 and Mohamed A. Hassan2 1 Zoology Department, Faculty of Science, Alexandria University, Moharram Bey, Alexandria 21511, Egypt 2 Protein Research Department, Genetic Engineering and Biotechnology Research Institute (GEBRI), City of Scientific Research and Technological Applications (SRTA-City), New Borg El-Arab, Alexandria 21934, Egypt

Correspondence should be addressed to Heba M. Abdou; dr.heba [email protected]

Received 11 February 2014; Revised 30 April 2014; Accepted 21 May 2014; Published 18 June 2014

Academic Editor: Anilava Kaviraj

Copyright © 2014 H. M. Abdou and M. A. Hassan. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The present study was conducted to investigate the protective role of Omega-3 polyunsaturated fatty acids against lead acetate- induced toxicity in liver and kidney of female rats. Animals were divided into four equal groups; group 1 served as control while groups 2 and 3 were treated orally with Omega-3 fatty acids at doses of 125 and 260 mg/kg body weight, respectively, for 10 days. These groups were also injected with lead acetate (25 mg/kg body weight) during the last 5 days. Group 4 was treated only with lead acetate for 5 days and served as positive control group. Lead acetate increased oxidative stress through an elevation in MDA associated with depletion in antioxidant enzymes activities in the tissues. Moreover, the elevation of serum enzymes activities (ALT, AST, ALP, and LDH) and the levels of urea and creatinine were estimated but total proteins were decreased. Also, lead acetate-treatment induced hyperlipidemia via increasing of lipid profiles associated with decline in HDL-c level. Significant changes of Hb, PCV, RBCs, PLT, and WBCs in group 4 were recorded. The biochemical alterations of lead acetate were confirmed by histopathological changes and DNA damage. The administration of Omega-3 provided significant protection against lead acetate toxicity.

1. Introduction oxidative damage in the kidney as evidenced by enhancement of lipid peroxidation [6, 7]. Lead is a highly poisonous Lead is one of mankind’s oldest environmental and occu- environmental pollutant and is known to affect organs like pational toxins [1].Theexposuretoleadcanoccurfroma liver, kidney, blood, and central nervous system of mammals multitudeofsourcessuchassoil,air,water,andindustrial [8, 9]. Several reports have indicated that lead can cause pollutants. There are, worldwide, six categories of products neurological, hematological, gastrointestinal, reproductive, considered as source of lead exposure, that is, gasoline circulatory, and immunological pathologies [10, 11]. Omega-3 additives, food can soldering, lead based paints, ceramic fatty acids (Omega-3 FAs) are considered as strong antioxi- glazes, drinking water systems, and folk remedies [2]. Health dants and their role as anticancer agent has been extensively hazards from increased lead exposure as a result of industrial confirmed in most of the human malignancies [12, 13]. and environmental pollution are recognized. It has been Furthermore, the anti-inflammatory potential of long chain found to cause a wide range of biochemical and physiological Omega-3 FAs in many chronic diseases has been suggested dysfunctions [3]. Moreover, the long term lead exposure [14, 15]. The role of Omega-3 FAs in inhibiting proliferation, generates reactive oxygen species and different free radi- inducing apoptosis, and promoting differentiation in many cals. Also, it inhibits antioxidant enzymes activities, such cancers has been recently studied [16, 17]. In addition, another as superoxide dismutase (SOD) and catalase (CAT), while finding indicates that Omega-3 FAs act synergistically with it decreases the level of glutathione [4, 5]. Lead induced certain chemotherapeutic agents [18]. Omega-3 FAs were 2 BioMed Research International found to play protective roles in the liver, cardiovascular Table1:PCRprimersusedinRAPD-PCR,GC%,andannealing system,andkidneyandtheyhavebeenwidelyusedinclinical temperature. peroperative total parenteral nutrition [19, 20]. Therefore, the 󸀠 󸀠 ∘ Primers Sequence 5 →3 GC% Annealing temperature ( C/sec) present study was carried out to investigate the protective effects of Omega-3 FAs against lead acetate-induced oxidative 1GTCCATGCCA6030/60 stress, biochemical changes, and DNA damage. 2 ACA TCG CCCA 60 30/60 3 ATG CCC CTG T 60 30/60 2. Materials and Methods and separately homogenized (10% w/v) using a homoge- 2.1. Chemicals. Lead acetate was purchased from Merck nizer (Potter-Elvehjem) in ice-cold sodium potassium phos- (Germany). phate buffer (0.01 M, pH 7.4) containing 1.15% of KCl. The Omega-3 was purchased from Efamol Ltd., 14 The Mole ∘ homogenates were centrifuged at 10,000 ×gfor20minat4C Business Park Leatherhead, Surrey, KT227BA, UK in the and the supernatant was used for assaying of the enzymes South East of England. All other chemical materials that were activities. used in this study were purchased from Sigma Chemical Co., (St. Louis, MO, USA). 2.4. Biochemical Analysis. Stored serum samples were analyzed for the activities of aspartate aminotransferase (AST; 2.2. Animals and Experimental Design. Twenty-eight adult EC 2.6.1.1), alanine aminotransferase (ALT; EC 2.6.1.2), alka- Wistar albino female rats (weighting 170–200 gm) were line phosphatase (AlP; EC 3.1.3.1), and lactate dehydroge- obtained from the animal house of Faculty of Medicine, nase (LDH; EC 1.1.1.27) which were determined using kits Alexandria University, Egypt. The local committee approved from Sentinel Ch. (via principle Eugenio 5-20155 Milan, the design of the experiments and the protocols were carried Italy). Also, serum total protein, albumin, urea, creatinine, out according to the guidelines of the National Institutes cholesterol, total lipids, triglycerides, HDLC, and low den- of Health (NIH). Rats were housed in stainless steel cages sity lipoprotein (LDLC) were determined using kits from placed in a well-ventilated rat house, maintained for two Sentinel Ch. (via principle Eugenio 5-20155 Milan, Italy). weeksasacclimatizationperiodunderstandardlaboratory The lipid peroxidation end product, MDA, was measured conditions on free supply of food and water provided ad as thiobarbituric acid reactive substance. Also, the levels of libitum, and subjected to natural light for 12 hrs and dark for GSH and the activities of antioxidant enzymes including the 12 hrs cycles. After the period of acclimatization, rats were catalase enzyme (CAT; EC 1.11.1.6), superoxide dismutase divided randomly into four groups, 7 animals in each. The (SOD, EC.1.15.1.1), and glutathione peroxidase (GPx; EC. animal experiments were conducted for 10 days. Group 1 was 1.1.1.9) were assayed using commercial assay kits according injecteddailywith0.5mLofsalinesolution(0.9%NaCl)i.p. to the manufacturer’s instructions. for 10 days and was used as negative control (−ve). Groups 2 and 3 were administrated orally with two doses of Omega-3 2.5. Histopathology. Specimens of liver tissues were immedi- (125 and 260 mg/kg body weight, resp.) by gavage for the first atelyfixedin10%formalin,treatedwithconventionalgradeof fivedaysasprotectiveagent[21]. These groups were injected alcohol and xylol, embedded in paraffin, and sectioned at 4– (i.p.) by 0.5 mL of lead acetate at a dose of 25 mg/kg body 6 𝜇 thickness. The sections were stained with Haematoxylin weight/day for the other 5 days in combination with Omega- and Eosin (H&E) stain for studying the histopathological 3. Group 4 was injected (i.p.) by 0.5 mL lead acetate only at changes [23]. a dose of 25 mg/kg body weight/day for the last 5 days of the experiment and was used as positive control (+ve) according to Ponce-Canchihuaman´ et al. [22]. 2.6. Random Amplified Polymorphic DNA Technique (RAPD) 2.6.1. Extraction of DNA. DNA was extracted from livers of 2.3. Blood Collection and Tissue Preparation. At the end of the four groups following the method described by Bardakci treatment, rats fasted for 12 hrs before being anesthetized and and Skibinski [24]. sacrificed by cervical dislocation. Blood samples were col- lectedfromthesacrificedanimalsandleftinrefrigeratorfor 30 min before centrifugation. The clear nonhemolyzed sera 2.6.2. Polymerase Chain Reaction (PCR) Primers. In the ∘ were stored at −20 Ctillmeasurements.However,heparin present work, ten-base long oligonucleotides primers were was used as an anticoagulant and noncoagulated blood was used to initiate the PCR amplifications. Primers were ran- tested shortly after collection for applying in determination domlyselectedonthebasisofGCcontentandannealing of hemoglobin (Hb), packed cells volume (PCV), red blood temperature for RAPD-PCR amplification as in Table 1. cells (RBCs) count, white blood cells (WBCs), and platelets (PLT) count by particle counter (ERMA Inc., Tokyo, model 2.6.3. PCR Amplification and Agarose Gel Electrophoresis. PCE-210). PCR amplifications were performed according to the proce- Liver and kidney were immediately removed and washed dure described by Williams et al. [25]usingtheisolatedDNA using chilled saline solution. These tissues were minced from three hepatic samples of each group. BioMed Research International 3

Table 2: The activities of AST, ALT, ALP, LDH, levels of MDA, total protein, albumin, urea, and creatinine after lead exposure and Omega-3 treatment in serum of female rats. Experimental groups (Group 2) (Group 3) Parameters (Group 1) (Group 4) Omega-3 Omega-3 −ve control +ve control (125 mg/kg b.w.) + lead acetate (260 mg/kg b.w.) + lead acetate AST (U/L) 56.69 ± 0.565a 58.63 ± 1.279a 51.35 ± 0.373a 104.27 ± 0.677b ALT (U/L) 36.32 ± 0.276a 40.53 ± 0.260a 38.69 ± 0.416a 90.07 ± 0.612b ALP (U/L) 42.43 ± 0.358a 48.74 ± 0.475a 41.44 ± 0.279a 87.77 ± 0.534b LDH (U/L) 149.29 ± 0.703a 159.07 ± 0.845a 148.81 ± 0.845a 219.88 ± 1.082b Total protein (gm/dL) 7. 1 0 ± 0.115a 6.11 ± 0.244a 7. 6 1 ± 0.217a 4.88 ± 0.103b Albumin (g/dL) 3.96 ± 0.095a 3.75 ± 0.071a 4.05 ± 0.102a 2.66 ± 0.083b Urea (mg/dL) 31.09 ± 0.225a 40.04 ± 0.715a 31.59 ± 0.450a 63.56 ± 0.962b Creatinine (mg/dL) 0.38 ± 0.004a 0.47 ± 0.011a 0.38 ± 0.004a 0.96 ± 0.026b MDA (nmol/mL) 17.08 ± 0.296a 18.81 ± 0.538a 17.47 ± 0.349a 51.99 ± 0.620b Values are expressed as means ± SE; 𝑛=7for each treatment group. a, b indicate the significant results statistically; 𝑃 < 0.05.

2.6.4. Agarose Gel Electrophoresis. The amplified DNA frag- MDA level in both liver and kidney extracts compared to the ments were separated on 1.5% agarose gel and stained with control group (Table 3). Pretreatment of rats with Omega- ethidium bromide. DNA ladder in range (100 bp–3000 bp) 3 (125 or 260 mg/kg body weight) prior to and during the wasusedinthisstudyasmarkerforamplifiedpattern.The injection with lead acetate ameliorated these parameters to amplified pattern was visualized and photographed by gel reach the normal level. Furthermore, the dose of 260 mg/kg documentation system. body weight was more effective than the dose of 125 mg/kg body weight in increasing the activities of SOD and GPx in the extracts of the liver and kidney. 2.7. Statistical Analysis. The data entry was done into a binary data matrix as discrete variables and was analyzed The present data indicated that the serum total lipids, 𝑃< according to Steel and Torrie [26]. Statistical significance of cholesterol, triglycerides, and LDL-c were significantly ( 0.05 the difference in values of control and treated animals was ) increased by lead acetate treatment, while HDL-c levels calculated by (𝐹) test at 5% significance level. Data of the were decreased (Table 4). The other striking finding in the present study were statistically analyzed by using Duncan’s present study is that Omega-3 at both doses (125 or 260 mg/kg multiple range test (SAS, 1986). All RAPD profiles were body weight) nearly normalized the lipid profiles in the serum analyzed using stat program which showed the similarity of rats and became similar to the control values (Table 4). between the amplified PCR products. The best amplified PCR product of each group was selected to compare between them 3.2. Hematological Analysis. Hematological parameters at genetic levels. revealed that the Hb and PCV values and the RBCs and PLT counts were significantly decreased𝑃 ( < 0.05)inlead acetate treated group compared to the negative control group 3. Results (Table 5). However, the results exhibited that Omega-3 at 3.1. Biochemical Parameters. The results showed that the both doses (125 or 260 mg/kg body weight) nearly normalized treatment with lead acetate significantly𝑃 ( < 0.05)increased the hematological parameters to become similar to the serumAST,ALT,AlP,andLDHcomparedtothecontrol normal values (Table 5). On the other hand, WBCs count in 𝑃 < 0.05 (Table 2). On the other hand, data indicated that the serum lead acetate treated rats were significantly ( )elevated total proteins and albumin were significantly (𝑃 < 0.05) as compared with the control group. However, treatment decreased after lead acetate treatment compared to the con- with Omega-3 at a dose of 260 mg/kg body weight was more trol group. Meanwhile, serum AST, ALT, AlP, total proteins, effective than the other dose in normalizing the WBCs count. and albumin were normalized after treatment with either of the two doses of Omega-3 (125 mg/kg or 260 mg/kg body 3.3. Histopathological Investigations. The histopathological weight) in combination with lead acetate, compared to the studies of rats’ livers are represented in (Figure 1). The light lead acetate treated group (Table 2). Also, Table 2 indicated micrographs of liver tissues demonstrated normal hepato- that the levels of serum urea, creatinine, and MDA were cytes in the control group showed normal hepatic archi- significantly𝑃 ( < 0.05) increased in the lead acetate tecture with distinct hepatic cells, sinusoidal spaces, and a treated rats compared to the control ones reflecting renal central vein (Figure 1(a)), while livers of rats treated with impairment. On the other hand, treatment with lead acetate lead acetate (group 4) showed loss of cellular architecture significantly𝑃 ( < 0.05) decreased the activities of GPx, CAT, with dilatation of blood sinusoids, hemorrhage in the portal and SOD and the level of reduced GSH while it increased vein, degenerated hepatocytes with pyknotic nuclei, and 4 BioMed Research International

Table 3: The effect of Omega-3 and lead acetate on specific activity of liver and kidney antioxidant enzymes in female rats.

Experimental groups (Group 2) (Group 3) Parameters (Group 1) (Group 4) Omega-3 Omega-3 −ve control +ve control (125 mg/kg b.w.) + lead acetate (260 mg/kg b.w.) + lead acetate Liver a a a b GPX (U/mg protein) 40.53 ± 0.240 36.54 ± 0.909 48.50 ± 0.341 22.44 ± 0.487 CAT (U/mg protein) 27.53 ± 0.554a 23.92 ± 0.583a 34.46 ± 0.743a 16.67 ± 0.489b SOD (U/mg protein) 11.34 ± 0.355a 10.40 ± 0.346a 18.11 ± 0.495b 7. 6 4 ± 0.303c GSH (U/g tissue) 27.84 ± 0.312a 26.08 ± 0.446a 29.16 ± 0.271a 12.72 ± 0.303b MDA (nmol/g tissue) 50.34 ± 0.259a 54.62 ± 0.288a 51.41 ± 0.282a 100.54 ± 0.322b Kidney a a b c GPX (U/mg protein) 30.64 ± 0.521 27.43 ± 0.597 39.89 ± 0.479 21.54 ± 0.360 CAT (U/mg protein) 53.17 ± 0.477a 46.97 ± 0.557a 63.30 ± 0.583b 20.41 ± 0.562c SOD (U/mg protein) 98.92 ± 0.457a 81.99 ± 0.693a 98.60 ± 0.285a 41.75 ± 0.451b GSH (U/g tissue) 53.81 ± 0.682a 50.39 ± 0.350a 55.01 ± 0.724a 28.51 ± 0.334b MDA (nmol/g tissue) 22.19 ± 0.320a 37.60 ± 0.382b 22.94 ± 0.515a 51.82 ± 0.313c Values are expressed as means ± SE; 𝑛=7for each treatment group. a, b, and c indicate the significant results statistically; 𝑃 < 0.05.

Table 4: Serum lipid and lipoprotein profiles of female rats after lead exposure and Omega-3 treatment.

Experimental groups (Group 2) (Group 3) Parameters (Group 1) (Group 4) Omega-3 Omega-3 −ve control +ve control (125 mg/kg b.w.) + lead acetate (260 mg/kg b.w.) + lead acetate TL (mg/dL) 201.59 ± 2.215a 179.85 ± 1.879a 150.19 ± 0.745b 404.00 ± 9.398c TG (mg/dL) 96.58 ± 0.706a 100.35 ± 0.671a 95.23 ± 0.686a 131.41 ± 0.541b Cholesterol (mg/dL) 54.96 ± 1.665a 61.29 ± 0.854a 54.43 ± 1.269a 84.05 ± 2.194b LDLC (mg/dL) 25.83 ± 0.438a 28.77 ± 0.431a 19.84 ± 0.399a 41.73 ± 0.470b HDLC (mg/dL) 29.14 ± 0.688a 28.75 ± 0.573a 30.19 ± 0.531a 18.17 ± 0.472b Values are expressed as means ± SE; 𝑛=7for each treatment group. a, b, and c indicate the significant results statistically; 𝑃 < 0.05. vacuolated cytoplasm lymphocytes aggregation inside the as well as, presence of some glomeruli with the capsular space hepatic tissue as in Figures 1(d1) and 1(d2). However, livers (Figures 2(b) and 2(c)). of rats treated with lead acetate plus Omega-3, 125 mg/kg (group 2), and rats treated with lead acetate plus Omega- 3.4. Genetic Analysis Using RAPD-PCR. Three of 10-mer. 3, 260 mg/kg (group 3), revealed that most of the histolog- primers were used for investigating the significant changes of ical alterations induced in lead acetate treated groups were theDNAisolatedfromlivertissues.Thethreeprimerspro- markedly reduced (Figures 1(b) and 1(c)). Meanwhile, lead duced clear, sharp, monomorphic, and polymorphic bands acetate treatment induced severe histopathological changes as in Figures 3, 4,and5. Primer 1 gave band patterns of in the kidney tissues (Figure 2(d))suchasswellingofcon- almostthesameprofilebetweenthethreeamplifiedsamples voluted tubules, disruption of Bowman’s capsule, shrunken of each group so it did not clarify any differenceFigure ( 3). glomeruli with the capsular space, cytoplasmic pyknosis In contrast, the other primers (primer 2 and primer 3) were of some nuclei, destruction of the epithelium lining the most informative and they produced reproducible and the tubules, hemorrhagic area in renal tubules, and dilation in most distinguishable banding profiles between the amplified therenaltubulescomparedtonormalhistologicalstructure. samples of each group after RAPD assays as in Figures 4 and 5. On the other hand, the histopathological studies of the The amplified fragments of PCR products were summarized kidneys of the control rats revealed normal glomerulus as in Table 6. Primers 2 and 3 produced highly similar RAPD surrounded by the Bowman’s capsule and proximal and fingerprints for negative control group (group 1) and groups distal convoluted tubules without any inflammatory changes 2 and 3 while they detected some changes in hepatic DNA (Figure 2(a)). Treatment with Omega-3 at both doses before of lead acetate treated group (group 4). We observed similar and in combination with lead acetate slightly improved the RAPD-PCR fingerprint using primer 2 in all 12 samples from kidney histology but extravasation of blood element with the different groups as in Figure 3.InFigure 4,theamplified dilation of some proximal and distal tubules was still present RAPD products of group 4 using primer 2 lost some bands BioMed Research International 5

Table 5: Changes in hematological parameters of female rats treated after lead exposure and Omega-3 treatment.

Experimental groups (Group 2) (Group 3) Parameters (Group 1) (Group 4) Omega-3 Omega-3 −ve control +ve control (125 mg/kg b.w.) + lead acetate (260 mg/kg b.w.) + lead acetate Hb (g/dL) 14.74 ± 0.070a 13.70 ± 0.155 a 14.31 ± 0.107a 9.67 ± 0.153b PCV (%) 41.14 ± 0.388a 40.25 ± 0.503a 42.84 ± 0.618a 31.86 ± 0.346b −1 RBCs (×1012 L ) 5.67 ± 0.133a 4.97 ± 0.174a 5.96 ± 0.162a 3.74 ± 0.056b −1 WBCs (×109 L ) 3.97 ± 0.196a 4.98 ± 0.084ab 3.90 ± 0.076a 5.96 ± 0.130b −1 PLT (×1012 L ) 255.14 ± 8.250a 217.57 ± 3.518a 268.29 ± 9.551a 116.57 ± 8.352b Values are expressed as means ± SE; 𝑛=7for each treatment group. a, b indicate the significant resultstatistically, s while ab may be significant or not significant; 𝑃 < 0.05.

C.V H S

(a)

L S H C.V S H

(b) (c) L

C.V

(d1) (d2)

Figure 1: Paraffin sections stained by haematoxylin and eosin (H&E, ×200) for histopathological examination of liver tissues of rats as follows: control group (a) showing normal hepatocytes architecture (H), central vein (C.V), and normal blood sinusoids (S); (b) liver tissue of group 2 (lead acetate + Omega-3, 125 mg/kg body weight); (c) liver tissue of group 3 (lead acetate + Omega-3, 260 mg/kg body weight) showing histological alterations induced by lead acetate that were markedly reduced in groups 2 and 3. Liver tissue of group 4 (lead acetate treated rats) (d1 and d2) showing distended and hemorrhage in the portal vein ( ), loss of the normal architecture, degenerated hepatocytes with pyknotic nuclei (◻), and degenerated hepatocytes with vacuolated cytoplasm ( ). Condensed nuclei ( ) and lymphocytes aggregation (L) inside the hepatic tissue. 6 BioMed Research International

S D P

D S G G

P D

(a) (b)

P D

S D G G P P S

Figure 2: Paraffin sections stained by haematoxylin and eosin (H&E, ×200) for histopathological examination of the kidney tissue of rats treated as follows: control group (a); lead acetate plus Omega-3, 125 mg/kg body weight group (b); lead acetate plus Omega-3, 260 mg/kg body weight group (c). Kidney tissue of lead acetate treated rats (d) showing disruption of Bowman’s capsule, shrunken glomeruli G with the capsular space S, and cytoplasmic pyknosis of some nuclei ( ), The degenerative changes in the epithelial cells lining the renal tubules (◻), hemorrhagic area ( )inrenaltubules,anddilationintherenaltubulesarecomparedtonormalhistologicalstructureoftheglomerulus and tubules in control group (a). Histological alterations induced in lead acetate at both doses of Omega-3 groups (b) and (c) were markedly reduced.

(1) (2)(3) (1) (2)(3) (1) (2)(3) (1) (2)(3) of their three samples compared to the samples of negative M gp1 gp1 gp1 gp2 gp2 gp2 gp3 gp3 gp3 gp4 gp4 gp4 control group and treated groups with Omega-3. Moreover the pattern showed a smear in the beginning of lanes 1 and 3 of group 4. The PCR products using primer 2 gave obvious 3000 results through the bands profile which were begun to appear

1500 at about 900 bp while the patterns of other groups were 1000 started at about 1350 bp. In addition, the RAPD-PCR using 900 800 primer 3 did not amplify the first sample of group 4 (lane 1 700 (bp) 600 500 of group 4) while lanes 2 and 3 of group 4 lost their bands at 400 approximately 1650 bp compared to other groups as shown in 300 200 Figure 5.Theamplificationproductsobtainedbythismethod showedthepresenceofnumerousbandsfrom250to1300bp 100 with primer 1, 300 to 1500 bp with primer 2, and 250 to 1700 bp with primer 3, respectively (Figures 3, 4,and5). The RAPD products were scored as present (1) or Figure 3: Pattern RAPD-PCR (primer 1) of hepatic DNA samples absent (0) for each primer-genotype combination. The results exposed to lead acetate and treated with two doses of Omega-3 using primer 1. The DNA ladder is in lane (M); lanes (gp1) represent group of RAPD patterns of the 3 primers were summarized as 1(−ve control), lanes (gp2) group 2 (Omega-3 with dose 125 mg/kg in Table 6. Thirty-six bands were scored where 33 were body weight and lead acetate), lanes (gp3) group 3 (Omega-3 with polymorphic and 3 of them were monomorphic. Jaccard’s dose 260 mg/kg body weight and lead acetate), and lanes (gp4) coefficient of similarity was measured and a dendrogram group 4 (+ve group). (Figure 6) based on similarity coefficients was generated BioMed Research International 7

Table 6: Random primers showing polymorphism of DNA from liver of the four groups.

Number of Number of Nucleotide Total number of Fragments size Primer code 󸀠 󸀠 monomorphic polymorphic sequence 5 →3 amplified fragments range (bp) fragments fragments 1 GTC CAT GCCA 13 2 11 250–1300 2 ACA TCG CCCA 12 1 11 300–1500 3 ATG CCC CTG T 11 0 11 250–1700 Total 36 3 33

(1) (2)(3) (1) (2)(3) (1) (2)(3) (1) (2)(3) Tree diagram for 4 variables M gp1 gp1 gp1 gp2 gp2 gp2 gp3 gp3 gp3 gp4 gp4 gp4 unweighted pair group average percent disagreement 0.30

3000 0.25

1500 0.20 1000 900 800 (bp) 700 600 0.15 500 400 distance Linkage 300 0.10 200

100 0.05 gp4 gp3 gp2 gp1

Figure 4: Pattern RAPD-PCR (primer 2) of hepatic DNA samples Figure 6: Dendrogram of the four applied groups generated by exposed to lead acetate and treated with two doses of Omega-3 using UPGMA based on 3 RAPD primers, where gp1 is group 1, gp2 is primer 2. The DNA ladder is in lane (M); lanes (gp1) represent group group 2, gp3 is group 3, and gp4 is group 4. 1(−ve control), lanes (gp2) group 2 (Omega-3 with dose 125 mg/kg body weight and lead acetate), lanes (gp3) group 3 (Omega-3 with dose 260 mg/kg body weight and lead acetate), and lanes (gp4) Table 7: Agreement percentage of RAPD profile. group 4 (+ve group). Arrows indicate loss of some amplification products of different groups. gp1 gp2 gp3 gp4 gp1 100 81 75 64 gp2 81 100 78 72 (1) (2)(3) (1) (2)(3) (1) (2)(3) (1) (2)(3) gp3 75 78 100 89 1 1 1 2 2 2 3 3 3 4 4 4 M gp gp gp gp gp gp gp gp gp gp gp gp gp4 64 72 89 100

by using unweighted pair group method with arithmetic 3000 mean (UPGMA). The best amplified PCR was selected from 1500 each group to compare between them using stat software.

1000 The analysis of the results described the similarity between (bp) 900 800 different samples of liver tissues (Table 7). The similarity of 700 600 positive control (group 4) and treated group with Omega-3 500 400 (group 2) was about 64% and 81%, respectively, compared 300 to negative control (group 1). The variations of the RAPD 200 profiles of treated Omega-3 groups were compared to the 100 negative and positive control groups.

Figure 5: Pattern RAPD-PCR (primer 3) of hepatic DNA samples 4. Discussion exposed to lead acetate and treated with two doses of Omega-3 using primer 3. The DNA ladder is in lane (M); lanes (gp1) represent group Lead has been known to be an environmental pollutant and 1(−ve control), lanes (gp2) group 2 (Omega-3 with dose 125 mg/kg its toxicity has also been associated with health hazards [8]. body weight and lead acetate), lanes (gp3) group 3 (Omega-3 with The liver acts as chief player in detoxification process andis dose 260 mg/kg body weight and lead acetate), and lanes (gp4) one of the target organs affected by lead toxicity owing to group 4 (+ve group). Arrows indicate loss of some amplification its storage in the liver. Data shown in Table 2 demonstrated products of different groups. that treatment with lead acetate caused a significant elevation 8 BioMed Research International in the activities of liver enzymes AST, ALT, ALP, and LDH wasobservedinthecurrentstudyduetodecreaseofSOD in serum confirming the histological damage shown in the and CAT activities [10]. Enzymes, such as GPx, CAT, and liver (Figure 1). The present results revealed a significant SOD may contribute to the explanation of the mechanisms increase in ALT, AST, and ALP in serum of lead acetate responsibleforthedecreaseinGSHconcentrationinliver treatedratscomparedwithnegativecontrolgroup.These and kidney due to the exposure to this heavy metal [22]. results are in agreement with the results of Herman et al. The chemoprotective effect of Omega-3 on liver tissue [27], Ibrahim et al. [28], and Mehana et al. [29]. However, was confirmed by the attenuation of the activities of serum the activities of LDH were significantly elevated in serum ALT, AST, ALP, and LDH in addition to the normalization of of lead acetate treated rats and this result is similar to that serum protein and albumin contents (Table 2). These results of Ibrahim et al. [28]. The increasing of LDH in serum of areconsistentwiththeresultsofAttaiaetal.[35]. The mode lead acetate treated group may be due to spill out of this of action of Omega-3 can be intercepted pharmacologically at enzyme from the liver cytosol into the blood stream and/or different levels with agents that scavenge free reactive oxygen, liver dysfunction and disturbance in the biosynthesis of this block their generation, or enhance endogenous antioxidant enzyme with alteration in the permeability of liver membrane capabilities [35]. according to Yousef [30]. Also, Gaskill et al. [31]reportedthat The current results also indicated that treatment with releasing of AST, ALT, and LDH from the cell cytosol can Omega-3 decreased the level of MDA associated with an occur as secondary changes to cellular necrosis. In addition, elevation in SOD and CAT activities, as well as in GSH significant decrease in the total proteins and albumin in the content, in groups 2 and 3. The decrease in the MDA serum of lead acetate treated group was compared to control level by Omega-3 may be due to its antioxidant properties group. Liver synthesizes proteins, among which is albumin, that inhibited lipid peroxidation [36] and this action helps and the decrease in the total proteins and albumin levels in stabilize the reactive radicals, preserve the cellular integrity, liver could be attributed to changes in protein and free amino and restrain the severity of lead acetate. GSH plays a key acids metabolism and their synthesis in the liver [31]. This role in many cellular processes involving protection of cells adverse effect might be caused by the interference of lead with against oxidative stress, xenobiotics, and radiation and it is protein synthesis or by the binding of lead to some metal- abundant with low molecular weight intracellular thiol [37]. binding proteins and their removal through detoxification In our study, Omega-3 prevented the decrement of GSH processes [30]. In an attempt to clarify the mechanism level suggesting that Omega-3 may protect the SH group of involved, it has been reported that lead caused a disruption in GSH from the reactive radicals that are produced from lead protein and RNA synthesis. Also, the observed decrease in the acetate toxicity. Similarly, Attaia and Nasr [38]foundthat total proteins and albumin in the liver could be attributed to Omega-3 could maintain normal levels of SOD and CAT the damaging effect of lead acetate on liver cells as confirmed activities. The antioxidant and anti-inflammatory effects of by increasing in the activities of serum AST and ALT (Table 2) Omega-3 through scavenging of free radicals and inhibiting after treatment of rats with lead acetate32 [ ]. In the current lipidperoxidationhavebeenreportedpreviouslybyPauwels study, the induced elevation of albumin, urea, and creatinine and Kostkiewicz [36]. This oxidant/antioxidant theory may duetoleadacetateadministrationindicatedthatthekidney explain the protective role of Omega-3 fatty acids against the function was affected. In addition, lead acetate caused a hepatotoxicity and nephrotoxicity of lead acetate. significant elevation in serum urea and creatinine reflecting In the present study, a significant increase in serum total renalimpairmentthatiscoincidingwithhistologicaldamage lipids, cholesterol, triglycerides, and LDL-c and a significant of the kidney as shown in Figure 2 [33, 34]. It can be decrease of HDL-c of the rats treated with lead acetate were concluded that oxidative damages may be the primary cause estimated (Table 4). HDL-c helps to scavenge cholesterol of lead toxicity leading to lipid peroxidation and cellular from extrahepatic tissues and the decrease of HDL-c concen- damage.Thus,theobviouschangeinliverandkidney trationasinthisstudycontributedtoincreasingcholesterol functions is related to the intensity of cellular damage. It has levels. There is evidence linking increased serum cholesterol been shown that lead acetate undergoes metabolism in liver and LDL-c levels to a higher risk for developing coronary via esoteric and oxidative pathways generating elevated MDA levels that lead to hepatic necrosis [31]. The increased levels heart diseases [39]. The present results exhibited that there of MDA in the present study are associated with a reduced was a significant decrease in serum total lipids, cholesterol, level of GSH and increased activities of serum enzymes which triglycerides, and LDL-c and a significant increase of HDL-c indicated the occurrence of an oxidative insult that caused of the rats treated animals with Omega-3 in groups 2 hepatic and renal damage. Moreover, the toxicity with lead and 3 compared to lead acetate treated group (Table 4). acetate in rats of group 4 leads to depletion of GPx, CAT, and Devasagayametal.[40] suggested that oxidative modification SOD enzymes activities in liver and kidney (Tables 2 and 3) of low-density lipoproteins (LDL-c) caused by reactive oxy- and these results are matching with the results which were gen species results in the formation of foam cells which is the achieved in a previous study [10]. The possible explanation initial lesion of atherosclerosis. They also reported that LDL-c couldberelatedtotheproposedroleofGSHintheactive oxidation and atherogenesis can be inhibited by nutritional excretion of lead through bile by binding to the thiol group antioxidants. There are also epidemiological evidences and of GSH and then being excreted. A decrease in GSH levels interventional studies to correlate higher level of antioxidant- could lead to oxidative stress and a consequent increase in rich food uptake with lower incidence of coronary heart lipid peroxidation [7]. The presence of lipid peroxidation disease [40]. BioMed Research International 9

The results of the present study demonstrated that lead changes in RAPD profiles, disappearance of bands and acetate administration to female rats resulted in significant appearance of new PCR products which occurred in the decrease of Hb, PCV, RBCs, and platelet count (PLT) of profiles generated by exposed rats to lead acetate. The present the rats treated with lead acetate in contrast to those in the data showed that the RAPD-PCR method is useful for the negative control rats (Table 5). On the other hand, WBCs screening and characterization of genomic regions that have count of lead acetate treated rats was elevated compared to undergone alterations as the result of lead acetate exposure. the negative control group, and these results are in agreement Several similar findings have been reported by Castano˜ with those described by Kim et al. [41]andSimseketal. and Becerril [51]andLiuetal.[52]thatusedRAPD-PCR [42]. However, Topashka-Ancheva et al. [43]showedthat to analyze the induced DNA damage. However, random lead could damage the erythrocytes membrane resulting amplified polymorphism of DNA (RAPD) showed distinct in hemolysis or decrease of blood iron level which may differences in animal groups exposed to lead acetate (group be the reason of decreasing the concentration of Hb and 4) and treated with Omega-3 (groups 2 and 3) at both doses PCV.Thesehematologicalalterationsmightbealsodueto (125 and 260 mg/kg body weight). Also, RAPD reflected the the effect of lead on the activity of 𝛿-aminolevulinic acid protective effect of Omega-3 on DNA. These results were dehydrogenase which acts as key enzyme of heme synthesis. consistent with those obtained by Elelaimy et al. [53]who Previous study reported that lead inhibits the conversion reported that Omega-3 pre-/posttreatment to azathioprine of coproporphyrinogen III to protoporphyrin IX leading to showed high significance in reducing the percentage of DNA reduction in Hb production and shortening of life span of fragmentation compared to azathioprine treated mice. erythrocytes [44]. The results obtained in this study indicated that Omega-3 successfully maintained normal haematological parameters against the toxicity induced by lead acetate 5. Conclusion in female rats. Our data are in accordance with previous In this study, the effect of lead acetate as one of the haz- results which reported that supplementing rats with different ardous heavy metals was studied using biochemical tests; doses of Omega-3 showed appreciable improvement in the histopathological study and genomic analysis showed the haematological indices as evidenced by significant increase high risk of lead toxicity through the exposure to lead inHb,PCV,andRBCcountsanddecreaseinWBCcounts acetate. Omega-3 acts as antioxidant compound and has [45]. protective and treatment effect versus lead toxicity, so it Figures 1(a), 1(b), and 1(c) showed normal cellular archi- should be tested on other heavy metals and environmental tecture with distinct hepatic cells, sinusoidal spaces, and a toxic compounds. The biochemical analysis confirmed the centralveinthatwereobservedinthenegativecontrolgroup free radical scavenging properties of Omega-3 as antioxidant and Omega-3 at both doses-treated groups. Figure 1((d1) compoundsaswellastheabilityofOmega-3toimprove and (d2)) showed that lead acetate treatment induced severe liver and kidney functions and haematological parameters. histopathological alterations in liver; most of the intrahepatic RAPD-PCR technique proved that it is a useful and effective blood vessels, especially the central veins, were dilated and techniquetostudytheDNAdamageduetoleadtoxicity congested. In addition, the hepatocytes lost their normal through the mutation of DNA which can be studied through architecture and vacuolization with pyknotic nuclei appeared the absence or intensity of different pattern bands. So, the inthecytoplasm.Theseresultsareinagreementwiththe present results indicated that coadministration of Omega- results of Abdel-Moneim et al. [46]. Our histological inves- 3 had protective role against hepatotoxicity, renal toxicity, tigations of renal tissue revealed that Pb-acetate treatment haematotoxicity, and genotoxicity induced by lead acetate. results in progressive glomerular and tubular alterations. These findings are in agreement with the results of Abdel- Moneim et al. [34]. Omega-3 treatment caused a significant Conflict of Interests decrease in the histopathological changes induced by lead acetateintheliverandthekidney(Figures1 and 2)and The authors declare that there is no conflict of interests partially restored these changes in lead acetate plus Omega-3 regarding the publication of this paper. treated groups. RAPD and arbitrarily primed polymerase chain reaction References technique (AP-PCR) are powerful tools for gene mapping, population, pedigree analysis, phylogenetic studies, and [1] M. N. Chatterjee and S. 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Research Article Assessment of DNA Damage and Telomerase Activity in Exfoliated Urinary Cells as Sensitive and Noninvasive Biomarkers for Early Diagnosis of Bladder Cancer in Ex-Workers of a Rubber Tyres Industry

Delia Cavallo,1 Valentina Casadio,2 Sara Bravaccini,2 Sergio Iavicoli,1 Enrico Pira,3 Canzio Romano,3 Anna Maria Fresegna,1 Raffaele Maiello,1 Aureliano Ciervo,1 Giuliana Buresti,1 Wainer Zoli,2 and Daniele Calistri2

1 Department of Occupational Medicine, INAIL-Italian Workers’ Compensation Authority, Research Area, Monteporzio Catone, 00040 Rome, Italy 2 Bioscience Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, 47014` Forlı-Cesena, Italy 3 Department of Traumatology, Orthopaedics and Occupational Medicine, University of Turin, 10126 Turin, Italy

Correspondence should be addressed to Delia Cavallo; [email protected]

Received 15 November 2013; Accepted 22 March 2014; Published 30 April 2014

Academic Editor: Ahmed El Nemr

Copyright © 2014 Delia Cavallo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of the present study was to identify sensitive and noninvasive biomarkers of early carcinogenic effect at target organ touse in biomonitoring studies of workers at risk for previous occupational exposure to potential carcinogens. Standard urine cytology (Papanicolaou staining test), comet assay, and quantitative telomerase repeat amplification protocol (TRAP) assay were performed in 159 ex-rubber workers employed in tyres production and 97 unexposed subjects. In TRAP positive cases, a second level analysis using FISH (Urovysion) was done. Cystoscopy results were available for 11 individuals whose 6 FISH/TRAP/comet positive showed in 3 cases a dysplastic condition confirmed by biopsy, 1 comet positive resulted in infiltrating UBC to the biopsy and with hyperplasia and slight dysplasia to the urinary cytology, 1 comet positive resulted in papillary superficial UBC to the biopsy, 1 FISH/TRAP positive showed a normal condition, and 2 TRAP positive showed in one case a phlogosis condition. The results evidenced good concordance of TRAP, comet, and FISH assays as early biomarkers of procarcinogenic effect confirmed by the dysplastic condition and UBC found by cystoscopy-biopsy analysis. The analysis of these markers in urine cells could be potentially more accurate than conventional cytology in monitoring workers exposed to mixture of bladder potential carcinogens.

1. Introduction factors [4]. Of particular aetiological importance is a history of exposure to chemical substances, which, as carcinogens Bladder cancer is a frequent and very aggressive malignant or cocarcinogens, may lead to the development of carcinoma tumor representing during 2011 the fourth most common with a latency of up to 30 years [2]. malignancy in men and the ninth in women [1]. More than Workersemployedinrubberindustryappeartohavea 90% of bladder malignancies are urothelial bladder carcino- mas (UBC) characterized by proliferation of inner superficial significant excess cancer risk of the urinary bladder asso- layer of urinary bladder (urothelium) that is constantly ciated with occupational exposure to complex mixture of exposed to metabolites and other chemicals through contact chemicals [5–9]. The International Agency for Research on with urine [2, 3]. Smoking is the most common risk factor Cancer (IARC) classified “rubber manufacturing” as carcino- for UBCs and occupational exposure to aromatic amines and genic for humans (group 1) on the basis of epidemiological polycyclic aromatic hydrocarbons are other important risk reports indicating excess of cancer risk at several sites (bla- 2 BioMed Research International dder, lung, stomach, and blood) [10, 11]. The bladder cancer biomonitoring studies of workers at risk for previous occu- risk in rubber workers has been ascribed principally to pational exposure to potential carcinogens. 2-naphthylamine present as contaminant of the phenyl-2- naphthylamine, used in the past as antioxidant, but also 2. Materials and Methods to numerous other chemicals including other antioxidant aromatic amines, acrylonitrile, 1,3-butadiene, styrene, poly- 2.1. Subjects. The study was carried out on 159 ex-rubber cyclic aromatic hydrocarbons (PAHs) used as reinforcing workersaged31to81years,withmeanage(±SD) 60 ± 12 agents, organic accelerators, activators, vulcanization agents, years, employed in tyres production, at variable times in and substances produced during high temperature processes the period between 1962 and 2006, potentially exposed in (mixing and vulcanization) of rubber production many of the period 1962–1980 to the potential carcinogen phenyl-2- whichareknowntobegenotoxic[12]. naphthylamine, in the period 1981–1995 to aromatic amines Early detection of bladder cancer in this job category at (IPPD, 6-PPD), and in entire period to other chemicals such risk for previous exposure to bladder potential carcinogens is as carbon black, 1,3-butadiene, styrene, polycyclic aromatic particularly important for improved prognosis and long-term hydrocarbons (PAHs) used as reinforcing agents, organic survival. accelerators, activators, and vulcanization agents. A group of Detection and monitoring of urinary bladder cancer are 97unexposedhealthysubjectsaged30to83years(mean usually done by urine cytology, cystoscopy, and histology [13, age 57 ± 12 years) were used as controls. The Local Ethics 14]. However these methods are highly subjective, expensive, Committee approved the study protocol and all subjects and invasive and often are not able to reveal low-grade UBC gave written informed consent to take part in the study. at the first stage of transformation (urine cytology) or flat Anagraphic, clinical, working information and lifestyle habits bladder cancer in situ and bladder cancer which remain (smoking, dietary habit, and alcohol consumption) were below the mucosa surface (cystoscopy) [15]. obtained from a questionnaire administered by specialized Noninvasive methods which are able to compete with medical personnel. cystoscopy and to implement cytology diagnostic accuracy Onthebasisoftheperformedactivities,theworkingcycle are still needed. has been divided into 4 stages (Figure 1): stage 1 (preparation In recent years, some noninvasive tests performed on and weighing of raw materials), stage 2 (mixing raw materials voided urine have been developed, approved by FDA [16], in Bambury, cutting the compound/immersion cooling tanks and are commercially available: nuclear matrix protein 22 andrubberizedchassisproduction),stage3(pneumatic (NMP22) levels, Fluorescent In Situ Hybridization (FISH) assembly and installation), stage 4 (vulcanization), and phase UroVysion assay and immunocyt [2, 14, 16, 17]. Other urinary 5 (checking, storage, and transportation of the finished biomarkers such telomerase activity (TA) levels by Telomeric product). The steps 1, 2, and 4 of the production cycle repeat amplification protocol (TRAP) assay18 [ , 19], DNA are at greater risk of exposure to the presence of carbon damage in exfoliate urinary cells by comet assay [20], and black, antioxidants such as aromatic amines, IPA, additives, DNA methylation markers are being investigated [16]and curatives, accelerants, and activators. seem to be good candidates for early detection of bladder cancer. However these biomarkers require being validated in Since quantitative measurements of past exposure to further studies on subjects at risk. amines and other chemicals were not available, we recon- The use of noninvasive and highly sensitive test in com- structed the past exposure of ex-workers using matrix bination with standard assays can improve early diagnosis job/exposure. In particular for each worker, we considered of new early stages bladder cancer and could be particularly the following working information: department, task and useful to screen professionally high-risk groups like rubber seniority in specific work stage, year of the beginning, and workers. end of exposure. On the basis of these information, we It has been demonstrated that comet assay is able to sen- identified five classes of risk for previous exposure to amines sitively reveal early, still repairable, DNA damage and furnish and other potentially carcinogenic chemicals (considering useful information on early effects induced by exposure to theperiod1962–1980atgreaterriskforthepresenceof genotoxicsubstances[21–23]. aromatic amines such as phenyl-2-naphthylamine and other Many recent studies have also showed that telomerase chemicals no longer allowed and for the probable lack of activity quantification by TRAP assay in urine sediments, personal or environmental protective measures) (Table 1). alone or in combination with a second level analysis, has an important role in distinguishing cancer patients from healthy 2.2. Urine Collection. The urine samples from both workers individuals and also symptomatic patients [15, 18]. and control subjects were collected freshly in the morning of In our study, we investigated in exfoliated urothelial the first two days of week and divided in three aliquots. One cells of ex rubber workers employed in tyre production, the aliquot was immediately processed for cytological analysis, presence of DNA damage by comet assay, and the telomerase while the other were treated to be sent to the laboratories activity levels by TRAP assay, combining them with FISH where comet assay and TRAP analysis were performed. analysis. The results are compared with the commonly used In particular for TRAP analysis, 3 mL of urine samples ∘ cytologyandcystoscopyusedasgoldstandard. wascentrifuged(5000rpmfor10minat4C) within 1 to 3 The aim is to identify sensitive and noninvasive biomark- hours of urine sample collection, washed once in PBS buffer, ∘ ers of early carcinogenic effect at target organ to use in resedimented by centrifugation (5000rpm for 10min at 4 C), BioMed Research International 3

Table 1: Distribution of the studied population in different classes of risk on the basis of working information.

Tasks/working stage Exposure time (years) Exposure period Class of risk Number of subjects Cabine electrician <20 Not relevant Machines maintenance-greasing <10 Not relevant 1 6 Tubes checking-inflating <10 Not relevant Cabine electrician >20 Machines maintenance-greasing 10–15 Worker stages 1/2/4 (higher exposure risk) <31962–19802 20 Worker stages 1/2/4 (higher exposure risk) <51981–2006 Tubes checking-inflating >10 Machines maintenance-greasing >15 Worker stages 1/2/4 (higher exposure risk) 3–5 1962–1980 3 29 Worker stages 1/2/4 (higher exposure risk) 5–10 1981–2006 Worker stages 1/2/4 (higher exposure risk) 5–10 1962–1980 4 30 Worker stages 1/2/4 (higher exposure risk) >10 1981–2006 Worker stages 1/2/4 (higher exposure risk) >10 1962–1980 5 74

∘ frozen at −80 C, and sent to the Laboratory where the analysis Electrophoresiswasdoneinthesamebufferat25Vand was performed. For comet assay, one 50 mL aliquot of each 300mAfor20mintoallowthedamagedDNAorfragments freshly voided urine sample was centrifuged (1500 rpm for to migrate towards the anode. The slides were then washed 10 min), suspended in 20 mL of PBS buffer, and transferred three times with Tris HCl 0.4 M for 5 min and stained with ∘ within 24 hours, at 4 C and in the dark, to the laboratory 50 𝜇Lethidiumbromide(10𝜇g/mL). Slides were examined by where the analysis was performed. In the cases analysed also eye at 200x magnification under a fluorescence microscope. by FISH, in accordance with the manufacturer’s instruction Anundamagedcellappearedasanucleoidandacellwith (UroVysion; Abbott/Vysis, Downers Grove, IL), one aliquot damaged DNA as a comet. About 1000 cells from each case ofatleast33mLofurinewasusedandsenttothelaboratory were examined for presence of comets by an experienced where the FISH was performed. observer and the percentage of comets on total cells was calculated. 2.3. Comet Assay. Comet assay with appropriate modifica- To evaluate the extent of DNA damage, images of 50 tions, due to the specific cell type used, was performed to randomly selected comets were acquired and analyzed from evaluate early DNA damage in exfoliated urinary cells. Briefly, each sample with specific image analyzer software (Delta immediately before performing comet assay, exfoliated uri- Sistemi, Italy) that allows measuring specific parameters at nary cells were washed in the PBS buffer solution and then level of single comet image. suspended in about 100 𝜇Lofthesamebuffer. Measurements of comet parameters were % DNA in the Ninety microliters of normal-melting agarose (NMA) 1% tail, tail length, and tail moment (the product of relative tail ∘ in PBS at 50 C was layered onto bond gel film (Sigma), intensityandlength,usedasameasureofDNAdamage).The immediately covered with a coverslip, and allowed to solidify mean tail moment (Tm) and % DNA in the tail of 50 comets ∘ at 4 Cfor5min.Thecoverslipwasthenremovedandabout hadbeencalculatedforeachsubject. 80 𝜇L of exfoliated cells suspension was mixed with 80 𝜇L ∘ The mean values Tm = 30 and tail DNA% = 26 were of low-melting agarose (LMA) 0.7% in PBS at 37 Cand selected as cut off normal values. For each subject the layered on top of the film. A coverslip was added and the ≥ ∘ presence of mean values of Tm and tail DNA% than cut off film was left to solidify at4 Cfor10min.Thecoverslipswere normal values were considered indicative of DNA damage. taken off and the films were layered onto glasses slides and treated with 70 𝜇L/slide of Proteinase K (1 mg/mL) for 2 h at room temperature, in order to digest proteins and inactivate 2.4. TRAP Assay. Pelleted cells were resuspended in 200 𝜇L nucleases responsible for degrading DNA. Then the slides oficecoldTRAPlysisreagent[24] and left on ice for 30 min. were bathed in freshly prepared lysis solution (2.5 M NaCl, Cell lysates were centrifuged at 10000 ×gfor20minat ∘ ∘ 100 mM Na2EDTA, 10 mM Tris with 1% Triton X-100 and 4 C and the supernatant was stored at −20 C. Urine sample ∘ 10%DMSOaddedfresh)overnight,inthedarkat4C. The aliquots containing 1 𝜇g of protein lysate were used for the slides were removed from the lysing solution and placed in a TRAP assay. As previously described [25], telomerase prod- horizontal gel electrophoresis tank filled with fresh alkaline ucts were evaluated on fluorescent electropherograms and the buffer (1 mM Na2EDTA and 300 mM NaOH, pH 13) for area underlying the different peaks was calculated. To obtain ∘ 40 min at 4 C to allow denaturing and unwinding the DNA semiquantitative levels of telomerase activity, an internal and the expression of alkaline-labile sites. telomerase assay standard (ITAS; 25 attograms), amplified by 4 BioMed Research International

thesame2primersusedforthetelomeraseactivityassay,was included in the TRAP buffer. For the correct evaluation of telomerase activity values of each sample, a standard curve was used. In particular, protein concentrations corresponding to 10, 30, 100, 300, 1000, and 3000 cells of a human bladder Rubber Process oils Carbon black cancer line (MCR) were analyzed in each assay and used as the reference curve. To obtain quantitative evaluations Stage 1: preparation and weighing of materials of telomerase activity, the areas of each sample were also normalizedtothe150-basepairITASpeak.Therelative telomerase activity per cell for each sample is presented as the Banbury mixer: percentage of the ratio of TRAP ladder/ITAS per cell versus high temperature the value of MCR and expressed in arbitrary enzymatic units and pression (AEUs). All experiments were performed in duplicate, and when variations were greater than 15%, observed in about 10% Closed mixing of cases, a third analysis was performed. Telomerase activity was expressed as a continuous variable in all analyses.

2.5. FISH Analysis. Exfoliated urothelial cells were collected from each urine aliquot by cytospin centrifugation (1200 ×g for 5 min). Cells were fixed in Carnoy’s fixative for 20 min and dehydrated for 2 min each in ethanol scalar dilutions (70%, 85%, and 100%) after which the slides were heated ∘ ∘ Open mixing Addition of vulcanizing agents at 45 Cfor2-3minandstoredat4Cforamaximum (accelerators, activators, sulfur) of 1 month. Hybridization was carried out according to Stage 2: mixing the manufacturer’s instructions (UroVysion; Abbott/Vysis). The probe mixture consisted of fluorescently labeled probes targeted on the pericentromeric regions of chromosomes 3 (CEP3 Spectrum Red), 7 (CEP7 Spectrum Green), and 17 (CEP17 spectrum Aqua) and on band 9p21 locus (LSI 9p21 Spectrum Gold) over each target area; 3 𝜇Loftheprobe solution was applied. Target DNA and FISH probe were code- Addition of fabric (polyester Addition of steel wire ∘ or nylon) and blending carcass and tread natured at 73 C for 2 min using a commercial hybridization Assembling and automatic system (HYBrite; Abbott/Vysis) and subsequently incubated ∘ 󸀠 roller pressing at 39 Covernight.4,6-Diamidino-2-phenylindole was used Stage 3: fabric and wire addition and assembling for counterstaining and cells were viewed using a fluorescent microscope (Axioscope, Zeiss, Gottingen,¨ Germany). All slides were scanned and the number of CEP3, CEP7, CEP17, and 9p21 signals in the cells was determined. The presence of at least 25 cells per slide was required to consider a sample Vulcanization furnace: high temperature and pression as evaluable, and two signals for each chromosome were required to consider a cell as normal. The presence of four or more cells with gains for two or more chromosomes (3, 7,and17)inthesamecellor12cellsormorewithzero9p21 Stage 4: curing (vulcanization and press) signals was the criterion to consider a specimen as positive. Slides were evaluated independently by two experts blinded to the patient’s history and histological findings.

2.6. Cytology. Standard urine cytology (Papanicolaou staining test) for the assessment of markers indicative of bladder cancer was performed. Briefly, urinary cells were prefixed Stage 5: finish, inspection, storage, and delivery in ethanol, harvested by cytocentrifugation, spread on slide, Figure 1: Working stages of tyre production process. The process dried, stained with Papanicolaou staining, and then analyzed starts with weighing rubber, process oils, carbon black, and other by an experienced observer, in order to identify cells mor- additives. In the mixing stage, the raw materials are mixed together phology and presence of infective agents. and heated. Then assembling with reinforcements occurs and finally the tyre undergoes vulcanization in curing presses. The higher risk in 2.7. Statistical Analysis. Statistical analysis was performed developing bladder cancer in stages 1, 2, and 4 is due to the presence with SPSS 16.0 package (SPSS Inc., IL, USA). We analysed of potentially carcinogenic compounds. The last stage comprises inspection, testing, and delivery. preliminarily the presence of possible significant differences BioMed Research International 5

Table 2: Distribution of studied subjects for age, smoking habits, alcohol intake, drug assumptions, dietary, and statistical analysis of differences. Statistical significance of Ex-exposed group Control group Variable Modality differences among groups (𝑛 = 159) (𝑛=97) 𝑃 value 60 ± 12 57 ± 12 Mann Whitney test Age (years) Mean ± SD Mean ± SD 𝑃 = 0.003 Smoking Nonsmokers 79 52 (8 missing exposed) Ex-smokers 37 17 Chi square test 𝑃 = 0.441 2missingcontrols Smokers 35 26 Recent drugs intake No 127 88 Chi square test 𝑃 = 0.1 (1 missing exposed) Yes 31 9 Diagnostic radiation exposure in the last six No 120 65 Chi square test 𝑃 = 0.338 months Yes 39 32 Alcohol assumption No 510 (15 missing exposed) Sometime 24 46 Chi square test 𝑃 < 0.001 6missingcontrols Yes 115 35 Dietary habit (fruits and vegetables intake) Sometime 77 34 (1 missing exposed) Often 53 49 Chi square test 𝑃 = 0.014 3missingcontrols Usually 28 11 Dietary habit (grilled and smoked food intake) Sometime 24 43 (3 missing exposed) Often 78 39 Chi square test 𝑃 < 0.001 3missingcontrols Usually 54 12

between exposed subjects and controls for confounding dietary habit (grilled and smoked food intake, alcohol intake) factors: by nonparametric Mann-Whitney 𝑈-test for age (Table 2). and by chi square test for smoking habits, alcohol intake, For comet assay, tail moment (Tm) indicative of DNA drug assumptions, diagnostic radiation exposure, and dietary damagewasevaluatedandsubjectswithmeanvalues≥ than habit. Nonparametric Mann-Whitney 𝑈-test was used to cutoff normal value (Tm = 30) were considered positive. The test the significance of differences in the studied biological mean Tm values were slightly higher in rubber workers than parameters (tail moment and TRAP mean values) between in controls (16.81 versus 14.11) and DNA damage was present exposed and unexposed subjects, Kruskal-Wallis with post in 10% (16/159) workers in respect to 2% (2/97) of controls. hoc Bonferroni test was used to test the significance of Tm values were significantly higher in the subjects included in differences in the studied biological parameters among the the highest class of risk compared with the other classes of risk different classes of risk, and 𝑃 ≤ 0.05 was considered sig- and with the controls (𝑃 = 0.023). The rubber workers with nificant. Chi square test followed by exact Fisher test when DNA damage were included mainly (13/16) in the class at the expected frequencies were <5wasusedtoanalyzethe highest risk showing a significant association with exposure significance of association between positivity frequency at (exact Fisher test 𝑃 value = 0.020 in the highest class of risk 2 cometassayorTRAPandexposurealsoinrelationtothe and 𝜒 𝑃 value = 0.015 in exposed group versus controls) class of risk. The contingency table was used to analyze the (Table 3). differences of positive/negative comet test and TRAP results TRAP analysis showed results ranging from 5 to 123 both in exposed subjects and controls and the statistical arbitrary enzymatic units (AEU), with median values of 37 significance has been assessed with chi square test. AEU in ex-exposed and 34 AEU in controls. Considering Pearson’s correlation coefficients (𝑟)werecalculatedto positive TRAP values ≥50 AEU, the subjects with TRAP evaluate the relationship between TRAP and comet results positive levels were 26/159 (16%) in exposed group and 15/97 both in exposed subjects and controls. (15%) in controls. The rubber workers with TRAP positive levels were included mainly (18/26) in the class at the highest risk (Table 3). Concordant comet and TRAP results were 3. Results observed in 139/159 (87%) ex-exposed group with positivity in 11 workers belonging prevalently (10/11) to the classes (4 No statistically significant differences were evidenced for con- and 5) with at least 10 years in mixing and curing activities, founding factors such as smoking habits, diagnostic radiation at higher risk for previous exposure to carcinogens, showing exposure, and drug assumptions between rubber workers and significant concordance between positive/negative comet and controls while significant differences were found for age and TRAP results with a chi square 𝑃 value <0.001. In addition, 6 BioMed Research International

Table 3: Comet test, TRAP, and FISH results on exfoliated urothelial cells of ex-workers distributed for class of risk and total ex-exposed group and controls.

Number of subjects Number of subjects Number of subjects Class of risk Tail moment TRAP 𝑁umber of subjects with DNA damage with TRAP with FISH and ex-exposed workers Mean ± SD Median + SD (%) positive (%) TRAP positive (%) 1 6 13.30 ± 10.72 33.50 ± 9.01 2 20 14.32 ± 7. 1 8 33 . 0 0 ± 13.21 1/26 (3.8%) 3 29 11.58 ± 8.11 1/16 (6.2%) 35.83 ± 15.49 4/26 (15.4%) 1/14 (7.1%) 4 30 14.81 ± 10.97 2/16 (12.5%) 35.41 ± 13.08 3/26 (11.5%) 2/14 (14.3%) 5 74 20.39 ± 15.96 13/16 (81.3%) 41.00 ± 21.83 18/26 (69.3%) 11/14 (78.6%) 𝑃 value 159 0.023 0.743 Kruskal -Wallis Post hoc Bonferroni 5 > 3 (0.022) 𝑃 𝜒2 value 159 0.054 0.111 𝑃 Exact Fisher test 0.020 0.065 value 14/19 TRAP+ Totalex-exposedones 159 16.81 ± 13.23 16/159 (10%) 37 ± 18 26/159 (16%) (74%) Total controls 97 14.11 ± 7.18 2/97 (2%) 34 ± 20 15/97 (15%) 𝑃 value 0.584 0.917 Mann Whitney 𝑃 value 𝜒2 0.015 0.850 correlation analysis between comet and TRAP results showed particularly for cancer at first stages); comet test is able to statistical significance between Tm and AEU values only in identify early DNA damage that could increase the risk of workers group (Pearson’s correlation index 𝑟 = 0.225, 𝑃 value cancer and TRAP analysis seems to be able to sensitively =0.005). detect telomerase activity levels in superficial urothelial cell FISH analysis was performed on 19 TRAP positive cases carcinoma [15, 18, 26]. andshowedpositiveconcordantresultsin14/19(74%)cases Comet assay on the ex-rubber workers employed in tyre (Table 3), 9 of which were positive also for comet assay. production, exposed in the past (1962 to the early 90) to Cytological analysis showed hyperdysplastic features only aromatic amines (phenyl-2-naphthylamine, IPPD, 6-PPD, in one rubber worker, included in the class at highest risk, DBPPD), showed that tail moment values are significantly resulted positive also at comet test with confirmed diagnosis higher for the subjects included in the highest class of risk of infiltrating UBC by biopsyTable ( 4), while negative results compared with the other classes of risk and controls. More- for atypical cells were observed in the remaining cases. over we found the presence of DNA damage (established Among subjects with FISH, TRAP, or comet posi- on the basis of cutoff normal values for TM) in the 10% of tive results, 11 subjects agree to perform cystoscopy. The exposed workers (16/159 subjects) in respect to the 2% of cystoscopy-biopsy identified in 3 subjects (with FISH, TRAP control group (2/97 subjects). and Comet positive results) a dysplastic condition; in 1 comet We found DNA damage in workers employed for 25–30 positive subject infiltrating UBC (resulted with hyperdispla- years in work stages 1-2-4, corresponding to the production sia/slight dysplasia to the urinary cytology) and in 1 comet functions “preparation of materials,” “mixing,” and “vulcan- positive subject a papillary superficial UBC. The remaining ization,” areas with potential hazard for exposure to a wide subjects (3 FISH/TRAP/comet positive and 2 TRAP positive) range of chemical compounds used in rubber manufacturing. showed a normal or phlogosis condition (Table 4). The presence of higher DNA adduct levels in exfoliated bladder cells of rubber workers involved in the production func- 4. Discussion tions “mixing” and “curing” was demonstrated by Vermeulen et al. [27] suggesting for these work stages potential exposure This study aims at identifying a set of sensitive and nonin- to compounds that can interact with DNA in urothelial cells. vasive techniques for early diagnosis of bladder lesions. Here Our comet results seem to support the above study showing a genotoxicity test, such as comet assay, and the quantitative a relationship between past exposure in specific work stages TRAP assay are combined with FISH analysis. The results are of rubber production and genotoxic effects. On 8 ex-exposed compared with the commonly used cytology and cystoscopy with positive comet assay (6 of which resulted in being used as gold standard. positive also at TRAP and FISH analyses), the cystoscopic Cytology test is very specific (a positive result is highly examination was performed showing in 3 cases a dysplastic indicative of bladder cancer) but suffers from low sensitivity condition, in 2 cases the presence of urothelial cancer, and in (a negative result does not exclude the diagnosis of cancer, 3 cases a normal condition. Therefore, 5/8 individuals, sub- BioMed Research International 7

Table 4: Comparison of cystoscopy-biopsy with cytology, TRAP, FISH, and comet test results on exfoliated urothelial cells of ex-exposed workersdistributedforclassofrisk. Urinary Subject code Class of risk Cystoscopy-biopsy Cytology TRAP FISH Comet 35 5 Dysplastic condition Negative Positive Positive Positive 44 5 Negative Negative Positive Positive Positive 65 5 Papillary UBC pTaG2 Negative Negative Not performed Positive 86 5 Dysplastic condition Negative Positive Positive Positive 91 5 Negative Negative Positive Positive Negative 110 4 Negative Negative Positive Positive Positive 117 4 Negative Negative Positive Not performed Negative 148 5 Papillary infiltrating UBC pT3bN1G3 Hyper-dysplasia Negative Not performed Positive 152 5 Phlogosis Negative Positive Negative Negative 178 5 Negative Negative Positive Positive Positive 207 5 Dysplastic condition Negative Positive Positive Positive

mitted to cystoscopy, showed conditions predisposing to cell Acknowledgment transformations. These findings demonstrated the suitability of urinary This work has been supported by Funds of the Italian cells, representing the target tissue of exposure to potential Ministry of Health for the project “Noninvasive molecular bladder carcinogens used in rubber industry, and the higher approaches for early diagnosis of bladder cancer in subjects sensitivity of comet assay in respect to standard cytology at risk (2005).” in identifying early genotoxic effect in agreement with the study of Fracasso et al. 2004 [20] performed on patients with References clinical suspicion of bladder cancer or urological disorders. Comet assay on exfoliated urinary cells could represent a [1] A. Lamarca and J. Barriuso, “Urine telomerase for diagnosis and sensitive and noninvasive biomarker of DNA damage to use surveillance of bladder cancer,” Advances in Urology,vol.2012, jointly to other more specific tests, in biomonitoring workers ArticleID693631,7pages,2012. exposed to mixture of bladder potential carcinogens such as [2] S. Bravaccini, V. Casadio, D. Amadori, D. Calistri, and R. Sil- rubber workers. vestrini, “The current role of telomerase in the diagnosis of It could be important to monitor those patients who had bladder cancer,” Indian Journal of Urology,vol.25,no.1,pp.40– 46, 2009. positive results for at least two of the three tests (COMET, TRAP, and FISH), even if at the present the cystoscopy has [3] M. Lazzeri, “The physiological function of the urothelium— more than a simple barrier,” Urologia Internationalis,vol.76,no. not evidence of malignant pathologies, because the three tests 4, pp. 289–295, 2006. may have identified very early steps of the disease, that is not [4] M. Burger, J. W. Catto, G. Dalbagni et al., “Epidemiology and yet detectable with the cystoscopic examination. risk factors of urothelial bladder cancer,” European Urology,vol. 63,no.2,pp.234–241,2013. [5] M. Kogevinas, M. Sala, P.Boffetta, N. Kazerouni, H. Kromhout, 5. Conclusions and S. Hoar-Zahm, “Cancer risk in the rubber industry: a review of recent epidemiological evidence,” Occupational and Altogether, the findings show that the analysis of these bio- Environmental Medicine,vol.55,no.9,pp.1–12,1998. molecular markers in urine cells could be potentially more [6]K.Golka,A.Wiese,G.Assennato,andH.M.Bolt,“Occupat- accurate than conventional cytology in monitoring workers ional exposure and urological cancer,” World Journal of Urology, exposed to mixture of bladder potential carcinogens demon- vol.21,no.6,pp.382–391,2004. strating the efficacy of a new generation noninvasive tests, [7] S.M.Olfert,S.A.Felknor,andG.L.Delclos,“Anupdatedreview to be used for early diagnosis of bladder cancer in subjects of the literature: risk factors for bladder cancer with focus on that were potentially in the past exposed to occupational occupational exposures,” Southern Medical Journal,vol.99,no. carcinogenic agents in tires manufacturing. 11,pp.1256–1263,2006. [8] T. Sorahan, “Bladder cancer risks in workers manufacturing chemicals for the rubber industry,” Occupational Medicine,vol. 58,no.7,pp.496–501,2008. Conflict of Interests [9] R. C. Reulen, E. Kellen, F. Buntinx, M. Brinkman, and M. P. Zeegers, “A meta-analysis on the association between bladder The authors declare that there is no conflict of interests cancer and occupation,” Scandinavian Journal of Urology and regarding the publication of this paper. Nephrology, vol. 42, supplement 218, pp. 64–78, 2008. 8 BioMed Research International

[10] “IARC Summary & Evaluation: The Rubber Industry,” vol. 28, [27] R. Vermeulen, G. Talaska, B. Schumann, R. P.Bos, N. Rothman, 1982. and H. Kromhout, “Urothelial cell DNA adducts in rubber [11] “IARC Monographs on the Evaluation of Carcinogenic Risks to workers,” Environmental and Molecular Mutagenesis,vol.39,no. Humans—Overall Evaluations of Carcinogenicity: An Updat- 4, pp. 306–313, 2002. ing of IARC Monographs,” 1987. [12] G. Talaska, B. Gaultney, S. Peters, P. Succop, and R. Vermeulen, “2-Naphthol levels and genotoxicity in rubber workers,” Toxi- cology Letters, vol. 213, no. 1, pp. 45–48, 2012. [13]I.Proctor,K.Stoeber,andG.H.Williams,“Biomarkersinblad- der cancer,” Histopathology,vol.57,no.1,pp.1–13,2010. [14]R.Srivastava,V.K.Arora,S.Aggarwal,A.Bhatia,N.Singh,and V. Agrawal, “Cytokeratin-20 immunocytochemistry in voided urine cytology and its comparison with nuclear matrix protein- 22 and urine cytology in the detection of urothelial carcinoma,” Diagnostic Cytopathology,vol.40,no.9,pp.755–759,2012. [15] S. Bravaccini, V.Casadio, R. Gunelli et al., “Combining cytology, TRAP assay, and FISH analysis for the detection of bladder cancer in symptomatic patients,” Annals of Oncology,vol.22,no. 10, pp. 2294–2298, 2011. [16] T. Reinert, “Methylation markers for urine-based detection of bladder cancer: the next generation of urinary markers for diagnosis and surveillance of bladder cancer,” Advances in Urology, vol.2012,ArticleID503271,11pages,2012. [17] D. Calistri, V. Casadio, S. Bravaccini, W. Zoli, and D. Amadori, “Urinary biomarkers of non-muscle-invasive bladder cancer: current status and future potential,” Expert Review of Anticancer Therapy, vol. 12, no. 6, pp. 743–752, 2012. [18] V. Casadio, S. Bravaccini, R. Gunelli et al., “Accuracy of urine telomerase activity to detect bladder cancer in symptomatic patients,” The International Journal of Biological Markers,vol.24, no. 4, pp. 253–257, 2009. [19] M. A. Sanchini, S. Bravaccini, L. Medri et al., “Urine telomerase: an important marker in the diagnosis of bladder cancer,” Neo- plasia,vol.6,no.3,pp.234–239,2004. [20] M. E. Fracasso, P. Franceschetti, D. Doria, G. Talamini, and F. Bonetti, “DNA breaks as measured by the alkaline Comet assay inexfoliatedcellsascomparedtovoidedurinecytologyinthe diagnosis of bladder cancer: a study of 105 subjects,” Mutation Research,vol.564,no.1,pp.57–64,2004. [21] D. Cavallo, P. Tomao, A. Marinaccio et al., “Evaluation of DNA damage in flight personnel by Comet assay,” Mutation Research, vol. 516, no. 1-2, pp. 148–152, 2002. [22] D. Cavallo, I. Iavicoli, A. Setini et al., “Genotoxic risk and oxidative DNA damage in workers exposed to antimony trioxide,” Environmental and Molecular Mutagenesis,vol.40,no.3,pp. 184–189, 2002. [23] D. Cavallo, C. L. Ursini, B. Rondinone, and S. Iavicoli, “Evalua- tion of a suitable DNA damage biomarker for human biomonitoring of exposed workers,” Environmental and Molecular Mutagenesis,vol.50,no.9,pp.781–790,2009. [24] R. Fedriga, R. Gunelli, O. Nanni, F. Bacci, D. Amadori, and D. Calistri, “Telomerase activity detected by quantitative assay in bladder carcinoma and exfoliated cells in urine,” Neoplasia,vol. 3, no. 5, pp. 446–450, 2001. [25] M. A. Sanchini, R. Gunelli, O. Nanni et al., “Relevance of urine telomerase in the diagnosis of bladder cancer,” TheJournalofthe American Medical Association,vol.294,no.16,pp.2052–2056, 2005. [26] Y. Lin, H. Miyamoto, K. Fujinami et al., “Telomerase activity in human bladder cancer,” Clinical Cancer Research,vol.2,no.6, pp. 929–932, 1996. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 795481, 11 pages http://dx.doi.org/10.1155/2014/795481

Research Article Effects of Freshwater Pollution on the Genetics of Zebra Mussels (Dreissena polymorpha) at the Molecular and Population Level

Emilia G. Thomas,1 Maja Šrut,2 Anamaria Štambuk,2 Göran I. V. KlobuIar,2 Alfred Seitz,1 and Eva Maria Griebeler1

1 Department of Ecology, Zoological Institute, University of Mainz, P.O. Box 3980, 55099 Mainz, Germany 2 Department of Zoology, Faculty of Science, University of Zagreb, Rooseveltov Trg 6, 10000 Zagreb, Croatia

Correspondence should be addressed to Eva Maria Griebeler; [email protected]

Received 15 November 2013; Accepted 22 March 2014; Published 27 April 2014

Academic Editor: Ahmed El Nemr

Copyright © 2014 Emilia G. Thomas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp) and reference sites (Cl) at the river Drava, Croatia, and applied microsatellite and DNA damage analyses (Comet assay, micronucleus test (MNT)). Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dpdueto genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat.

1. Introduction These genetic processes can reduce overall population fitness and accelerate population extinction [8–10]. The fast growing human population has altered freshwater To obtain an integrated assessment of the impact of ecosystems profoundly [1]. Hazardous substances present pollution on populations, the use of multiple biomarkers at in the wastewater from agriculture, industry, and human different levels of biological organization has been strongly settlements end up in aquatic ecosystems [2]. Effluents of suggested [2, 7, 11]. If chemical contamination is responsible wastewater treatment plants (WWTP) are one of the major for an emergent effect at the population level, responses at sources of genotoxicants in surface waters [3, 4]. Evolutionary lower levels of biological organization should also be apparent toxicology investigates the effects of chemical pollutants on [11]. The establishment of this causal relationship is essential the genetics of natural populations [5–7]. Toxicants may to support the conclusion that an emerging population effect induce DNA damage along with long-term DNA changes in isduetocontaminationexposure[7]. freshwater organisms. It has been suggested that the resulting The zebra mussel Dreissena polymorpha has been applied genomic instability plays an important role in decreasing as bioindicator for passive as well as active biomonitoring [12– fitness of populations and hence may have significant conse- 14], for example, for genotoxicity monitoring in freshwater quences for short- and long-term survival of populations [2]. ecosystems [15]. Moreover, pollution induces stochastic effects on population Inparticular,wepresentacasestudyoftwomussel genetics causing a decline in population size and conse- populations from the river Drava (Croatia). These two pop- quently inbreeding and the overall loss of genetic diversity. ulations are considered to have the same genetic background 2 BioMed Research International but are exposed to different environmental conditions. The cadmium, zinc, manganese, twelve polycyclic aromatic hy- first population (Dp) was collected at a polluted site where drocarbons (PAHs), and polychlorinated biphenyls (PCBs) mussels were constantly exposed to the effluent of a municipal were measured. The pH was measured with a pH meter WWTP, whereas the second (Cl) was collected at a reference (type WTW pH 526). Dissolved oxygen was measured with site. an oximeter (type WTW OXI 730). The concentrations of We first studied the genetic constitution of both popula- metals(Cr,Cu,Ni,Pb,Fe,Cd,Zn,andMn)inthewater tions applying microsatellite analysis at the population level were determined using inductively coupled plasma-optical and DNA damage analysis at the molecular and cellular level. emission spectrometry (ICP-OES, IRIS Intrepid II XSP, For the population level we hypothesized to detect a genetic Thermo)accordingtotheHRNENISO11885[17]stand- bottleneck in population Dp, caused by increased mortality ard method. PAHs were determined by a high perform- resulting from the exposure of the population to the WWTP ance liquid chromatography (HPLC system Agilent 1100 effluent (hypothesis 1.1). This bottleneck could be a result Series-thermostatted autosampler G1329A, binary pump of selection or of random genetic drift due to a decrease G1312A) with fluorimetric detection (fluorescence detector in population size. At the molecular and cellular level, we G1321A by Agilent Technologies) (HPLC-FLD). The 12 PAHs expected increased genotoxic effects in Dp compared to Cl (naphthalene, fluorene, phenanthrene, anthracene, fluoran- (hypothesis 1.2). thene, pyrene, chrysene, benzo(b)fluoranthene, benzo(k) Second, in a laboratory experiment, we exposed ran- fluoranthene, benzo(a)pyrene, benzo(g,h,i)perylene, and domly selected individuals from both populations to polluted indeno(1,2,3-𝑐𝑑)pyrene) were analyzed according to the EPA municipal wastewater. For this experiment, we expected that 550 [18] standard method. The total PAH concentration the two populations would react differently to these altered corresponded to the sum of the concentrations of the 12 environmental conditions. (i) Under the assumption that PAHs analyzed. For PCBs, water samples were extracted selective processes have changed the genetic constitution of by dichloromethane. The extracts were evaporated ona population Dp in the source habitat, Dp could be more rotary evaporator (Rotavapor R-210 with Heating Bath resistant to the wastewater and show less DNA damage than B-491, Vacuum Pump V-700, and Vacuum Controller V-850, Cl (hypothesis 2.1). (ii) If alternatively, Dp had a decreased Buchi),¨ purged, and then concentrated in a stream of nitrogen genetic diversity due to random genetic drift leading to a gas. PCBs were quantified by a gas chromatography with a 63 decreased fitness under altered environmental conditions10 [ , Ni Electron capture detector (GC-17A-ECD, Shimadzu) 16], we expected that Dp would be more severely affected by accordingtotheHRNENISO6468[19]standardmethod. the wastewater and exhibit a higher level of genotoxic effects than the control population Cl (hypothesis 2.2). 2.2.2. Sediment Analyses. Only one sediment sample could becollectedintheWWTPeffluentchannel(W.C.,Figure 1), 2. Materials and Methods since the bottom at Cl2 (Figure 1)andDpconsistedofsmall pebbles. To evaluate the degree of pollution of this sediment 2.1. Sampling Sites. The zebra mussels were sampled in sample we measured the concentrations of heavy metals JulyandAugust2009innorthernCroatia(Figure 1). The ∘ ∘ (chromium, copper, nickel, lead, cadmium, zinc, cobalt, and reference site Cl (46.3143678 N, 16.4154166 E) was situated mercury), arsenic, molybdenum, mineral-oil hydrocarbons, in the Cakovecˇ lake. Due to the high water level at the time PAHs, and PCBs. point of water sampling, water samples and mussels for the The methods used for determining heavy metal concen- laboratory experiment were taken at the second reference trations (Cr, Cu, Ni, Pb, Cd, Zn, Co, and Hg), arsenic, mineral site situated just behind the dam of the Cakovecˇ lake, about ∘ ∘ oil hydrocarbons, and PAHs have been previously described 600 m upstream from Dp (46.306274 N, 16.471809 E). The ∘ ∘ in detail [20]. The concentration of Mo was determined in the contaminated sampling site (Dp: 46.303452 N, 16.478352 E) same way as the heavy metals. For PCB detection, the oven- was about 750 m downstream from Cl. Approximately 10 m dried samples (5 g) were transferred to a Soxhlet apparatus upstream from the Dp site, a municipal WWTP effluent flows and extracted with 100 mL of n-hexane. The extracts were into the river Drava. evaporated on a rotary evaporator (Rotavapor R-210 with HeatingBathB-491,VacuumPumpV-700,andVacuum 2.2. Water and Sediment Analyses. Allchemicalanalysesof Controller V-850, Buchi)¨ and concentrated in a stream of water and sediment were performed by the Institute of Public nitrogen gas. They were then transferred into a centrifuge ˇ Health “Dr. Andrija Stampar,”Zagreb, Croatia, except for pH tube and cleaned up with concentrated sulphuric acid (min. and oxygen, which were directly measured in the field. 96%). If the samples were highly contaminated repeated acid clean-up was employed. PCBs were quantified by a gas 2.2.1. Water Analyses. Water samples were collected at the 63 chromatography with a Ni Electron capture detector (GC- Dp site, at the WWTP effluent channel, approximately 20 m 17A-ECD, Shimadzu) according to EPA 8082 [21]standard upstream from the outlet into the river Drava, and at a method. reference site Cl2 (Figure 1). For the laboratory experiment, we additionally sampled water in a wastewater channel leading to the main city collector of municipal wastewater 2.3. Microsatellite Analysis. For population genetic analyses, in Zagreb. In the water samples, pH and the concentrations thesamplesizewas48individualsforeachofthepopulations. of dissolved oxygen, chromium, copper, nickel, lead, iron, Genomic DNA was extracted from each of the individuals BioMed Research International 3

Cl2 W.C.Dp Drava

Figure 1: Location of the sampling sites in Croatia. Cl: Cakovecˇ lake (control population); Cl2: reference site for water analyses and reference sampling site of mussels used in the laboratory experiment; Dp: contaminated population located downstream from the mouth of the wastewater treatment plant effluent channel (W.C.). For sediment analyses a sample was taken in the W.C. from the posterior adductor muscle of ethanol-preserved Table 1: Primer set used for microsatellite analysis. samplesusingtheHighPurePCRTemplatePreparationKit 󸀠 󸀠 (Roche Diagnostics GmbH, Mannheim, Germany). The six Locus Primer sequence (5 -3 ) F: GTT TCT TTG CCG GTC TAA TAA TAG AGT TAA C primers used for microsatellite analysis and the respective A6 PCR protocols are based on Naish and Boulding [22], Astanei R: GTG ATT GTG TAT CTG CTA TAA ACC F: GTT TCT TCG TGT GCT CAT GTT TCC TCC et al. [23], and Thomas et al. [24]. The primers A6, B6, B9, B6 and C5 from Naish and Boulding [22] and Astanei et al. [23] R: CGT TGT TCA AGC AAT AAG AAA GAC were partially modified in the Institute of Zoology, University F: GTT TCT TTT GAC AAT ATC CTG TCT AAT G B9 of Mainz (Table 1) using the program PRIMER3PLUS [25], R: GCG TGT GTT TTT GAA ACG TG or by PIG-tailing [26]. The primers Dpol9 and Dpol19 were F: GTT TCT TGC ACT GTC AAC GTC ACA CTT TTG C5 those from Thomas et al.24 [ ]. The genotype data were R: CCT TGC TAA CAG CTC GGT TGT ATC generatedonABI3130xlDNAAnalyzer.Foreachofthe F: M13(-18)TGG TTG ATG CAG TGA CCC TA Dpol9 individuals the allele fragment size calls were made using R: the GENEMAPPER 4.0 software (Applied Biosystems, Foster TGT CGC TTG ATC CAT GTT TT F: M13(-18)GCA TTC CAT CAA AAA CAC AGA T City, CA, USA). One individual of population Dp provided a Dpol19 low DNA quality and was therefore removed from the genetic R: GAT CAA CAC CAA AGT TCG TTT C data set. The primers are based on Naish and Boulding [22], Astanei et al. [23], and The microsatellite data set was checked for scoring errors Thomas et al. [24]. We slightly modified the primer pairs of the loci A6, B6, due to stuttering, large allele dropouts, and null alleles with B9, and C5 (Naish and Boulding [22]; Astanei et al. [23]). the program Microchecker [27]. We tested the potential influence of scoring errors on Hardy-Weinberg Equilibrium (HWE) with the jackknife procedure described by Morin cells was examined, and the extent of DNA migration was et al. [28] and executed by the program “R” [29]. Linkage determined as a percentage of the tail DNA using an image disequilibria between pairs of loci were tested using the analysis system Komet 5, Kinetic Ltd. program GENEPOP, version 4.0.10 [30].

2.5. Micronucleus Test. Aliquots of 0.1 mL hemolymph mixed 2.4. Comet Assay. MusselscollectedfromthetwositesCl with 0.1 mL phosphate-buffered saline (PBS) in 10 mM and Dp were transported within 90 min to the Department ethylenediaminetetraacetic acid (EDTA) were placed on of Zoology, Faculty of Science in Zagreb. Hemolymph was slides and left for 15 min in a humidified chamber at room withdrawn from the posterior adductor muscle sinus with a temperature allowing hemocytes to settle down. The subse- hypodermic syringe for subsequent Comet assay and MNT. quent procedure was conducted as described previously [31]. The Comet assay was conducted as described in detail by Micronuclei were identified according to the criteria given in Stambukˇ et al. [31]. For each individual, one slide with 50 Majone et al. [32] and Kirsch-Volders et al. [33]. 4 BioMed Research International

2.6. Statistical Analyses zero, using a nonparametric permutation approach described by Excoffier et al. [41]. Furthermore, the age-class-specific 2.6.1. Field Populations—Genetic Bottlenecks. We checked inbreeding coefficients (𝐹 ) were estimated and also tested both field populations for possible bottlenecks using the IS for significance. program BOTTLENECK, version 1.2.02 [34]. With this The distance method applied in the locus-by-locus program, the population sample is tested for an excess of AMOVA was the number of different alleles. With the help heterozygosity in comparison to the heterozygosity expected (𝐻 >𝐻 ) of this method, the locus-specific inbreeding coefficients at mutation-drift equilibrium 𝐸 EQ .Inabottlenecked (𝐹 ) 𝐹 IS , variances among age classes within populations ( SC), population, the number of alleles is reduced faster than 𝐹 variances among age classes among populations ( CT), and the allele frequencies by a strong reduction in population 𝐹 the overall fixation indices ( IT)werecalculatedandtestedfor density. As 𝐻𝐸 is calculated from allele frequencies and 𝐻 EQ significant deviation from zero. All AMOVA analyses were is calculated from allele numbers, a significant excess of 𝐻 >𝐻 computed with the program Arlequin, version 3.5.1.2 [42]. heterozygosity ( 𝐸 EQ) is evidence of a recent bottleneck in a population [35]. We chose the two-phase mutation 2.6.5. Comet Assay and MNT. For each group (field and labo- model (TPM) in this analysis, since it has been described as ratory populations, see below), mean values of DNA damage the most appropriate model for microsatellite loci [35, 36]. were calculated based on the mean of each individual within The Wilcoxon test was used to test statistical significance of a group. The data are presented as mean ± SEM (standard bottlenecks. error), for both Comet assay and MNT. To assess differences between groups, we performed the Mann-Whitney 𝑈 test. 2.6.2. Field Populations—Genetic Diversity and Hardy- Weinberg Equilibrium (HWE). The overall observed and 2.7.Laboratory Exposure. Approximately 200 mussels per site expected heterozygosity (genetic diversity) of the two were acclimatized to laboratory conditions for two weeks populations and respective heterozygosities for each of the in glass aquaria. The aquaria contained dechlorinated, well- loci were estimated with the program GENEPOP, version aerated water, which was renewed every other day, but not 4.0.10 [30]. The same program was applied for testing any sediment. Every other day the mussels were fed with algae overall HWE by the exact “HW test” for each population 𝑈 Chlorella sp. and by the statistically more powerful score test ( test) After this acclimatization period, 50 mussels of each pop- differentiating between heterozygote deficiency𝐻 ( 𝑂 <𝐻𝐸) 𝐻 >𝐻 ulation were exposed to the polluted municipal wastewater and excess ( 𝑂 𝐸)[37]. In case of overall deficiency of Zagreb in aquaria for three days. The wastewater in the of heterozygotes the score test of heterozygote deficiency aquariawasrenewedeveryday.Atthesametime,other was also applied to each of the six loci to detect which of samples of 50 mussels of each population were exposed to the loci had caused a deficiency of heterozygotes in the dechlorinated tap water and were used as a control. At the populations Cl and Dp. In all score tests conducted, Markov end of the experiment, seven to twelve individuals were taken chain parameters were 10,000 for dememorization, with 500 from each treatment to assess genotoxicity by the Comet assay batches and 5,000 iterations per batch. and MNT.

2.6.3. Field Populations—Population Differentiation and Inbreeding. To assess overall differences in gene pools of 3. Results thepopulationsClandDp,wecalculatedthedifferentiation 3.1. Water and Sediment Analysis. Results of the water 𝐹 between the two populations ( ST) and the inbreeding and sediment chemical analysis are presented in Table 2. 𝐹 𝐹 coefficients ( IS)usingthe -statistics by Weir and Very slight differences were observed between the water Cockerham [38]asimplementedintheprogramFSTAT parameters at the sampling sites in the WWTP effluent [39]. Weir and Cockerham [38] weight allele frequencies channel (WcW) and Dp in comparison to the clean site accordingtosamplesizes,aswellastheestimatesofthese Cl2 (Figure 1). Only decreased oxygen concentration clearly statistics, are not influenced by differences in sample sizes of indicated increased water pollution at these two polluted 𝐹 𝐹 populations. The ST and IS estimates were calculated over sites. all loci, as well as separately for each of the loci. The sediment analysis of the WWTP effluent channel clearly showed that the water running through this channel 2.6.4. Field Populations—Analysis of Molecular Variance has been heavily polluted, at least from time to time. (AMOVA). To rate changes in the genetic composition of the field populations between generations, the individuals of both 3.2. Field Populations—Review of Microsatellite Data. populations were divided into three distinct age classes. We The analysis of the genetic data set with the program determined the age of individuals by counting the annual Microchecker [27] did not detect any scoring errors due rings on the surface of their shells [40]. The first age class tostutteringorlargealleledropouts.Italsodidnotreveal consisted of one- and two-year-old zebra mussels, the second any hint for existing null alleles, except for locus C5 in Dp. age class contained three- and four-year-old mussels, and the This locus had a significant excess of homozygotes, which third age class comprised the five-and-six-year-old mussels. could indicate the presence of null alleles. The estimated The 𝐹-statistics of the age classes (across all six loci) rate of possible null alleles of locus C5 in Dp was low were estimated and tested for significant deviation from (0.0723). The six individuals that were suspected to have null BioMed Research International 5

Table 2: Results of water and sediment chemical analyses.

Water parameters Cl2 Dp WcW ZaW Sediment parameters WcS pH 8.07 7.74 7.87 7.20 Co (mg/kg) 10.4 < O2 (mg/L) 8.68 4.62 4.27 NA As (mg/kg) 0.05 Cr (𝜇g/L) <2 <2 <219Cr(mg/kg)804 Cu (𝜇g/L) <20 <20 <20 36 Cu (mg/kg) 242 Zn (𝜇g/L) <20 <20 <20 88 Zn (mg/kg) 1300 Ni (𝜇g/L) <5 <5 <517Ni(mg/kg)44.8 Fe (𝜇g/L) 314 126 99.2 1110 Hg (mg/kg) 1.36 Pb (𝜇g/L) <5 <5 <57 Pb(mg/kg)218 Cd (𝜇g/L) <1 <1 <1 <1Cd(mg/kg)<3 Mn (𝜇g/L) 24.9 38.8 58.5 84 Mo (mg/kg) 42.4 PCB (𝜇g/L) <0.02 <0.02 <0.02 <2.0 PCB (mg/kg) <5 Total PAH (𝜇g/L) <0.005 <0.005 <0.005 0.255 Total PAH (mg/kg) 27.8 Mineral-oil hydrocarbons (mg/kg) 176.6 Concentrations of abiotic parameters and contaminants of four water samples and one sediment sample are depicted. Cl2: reference site for water analyses in river Drava (Figure 1),Dp:contaminatedsiteinriverDrava(Figure 1), WcW: water sample of the wastewater treatment plant effluent channel that flows into the river Drava, ZaW: water sample of municipal wastewater in Zagreb used in the laboratory experiment, and WcS: sediment sample of wastewater treatment plant effluent channel that flows into the river Drava (W.C., Figure 1).

Table 3: Locus-specific and overall genetic characteristics of field populations. 𝐹 𝐹 𝐻 𝐻 𝐻 𝐻 𝐹 𝐴 𝐴 Locus IS (Cl) IS (Dp) 𝑂/ 𝐸 (Cl) 𝑂/ 𝐸 (Dp) ST (Cl/Dp) (Cl) (Dp) A6 −0.032 0.014 0.917/0.889 0.896/0.906 0.007 17 15 B6 0.012 0.064 0.917/0.927 0.830/0.886 0.002 18 13 B9 0.106 0.016 0.771/0.862 0.809/0.822 0.006 10 9 C5 0.043 0.167 0.750/0.784 0.638/0.765 −0.010 8 6 Dpol9 −0.102 0.092 0.708/0.643 0.600/0.660 −0.008 7 7 Dpol19 −0.102 0.004 0.792/0.784 0.787/0.791 −0.006 9 9 Across all loci 0.007 0.057 0.809/0.815 0.760/0.807 −0.001 69 59 𝐹 𝐻 𝐻 𝐹 Inbreeding coefficients ( IS), observed and expected heterozygosity ( 𝑂/ 𝐸) of population Cl and Dp, genetic differentiation ( ST) between Cl and Dp, and 𝐴 𝐹 number of alleles ( ) for Cl and Dp. None of the ST values was significantly different from zero. Significant deficits of heterozygotes from Hardy-Weinberg 𝐹 equilibrium are given in bold. Note: small negative ST values are statistical artefacts and are observed in populations with very low genetic differentiation.

alleles by the software Microchecker were not considered observed heterozygosity 𝐻𝑂 was 0.809 in Cl and 0.760 in to be influential on HWE estimates for the population Dp. 𝐻𝑂 deviated significantly from 𝐻𝐸 only for Dp, but basedonthejackknifeprocedure[28]. Therefore, we did not for Cl (Table 3). 𝐻𝑂 was lower in Dp than in Cl for not remove these individuals from the data set. Moreover, all six analyzed loci. For Dp, locus C5 showed the strongest low null allele frequencies have a negligible impact even difference between observed and expected heterozygosity in parentage analysis, whereupon parentage analysis is (𝐻𝑂 = 0.638, 𝐻𝐸 = 0.765) while no difference was observed expected to have a higher bias from null alleles than analysis for this locus in Cl (𝐻𝑂 = 0.750, 𝐻𝐸 = 0.784)(Table 3). of population structure [43]. We detected no significant The exact tests implemented in GENEPOP did not show linkage disequilibrium between all pairs of the six loci any significant deviation from HWE for both populations 𝑃 = 0.443 𝑃 = 0.237 studied applying sequential Bonferroni correction. ( Cl , Dp ). The score tests of heterozygote 𝑃 = 0.586 𝑃 = 0.967 excess also corroborated HWE ( Cl , Dp ), whereas the score tests of heterozygote deficiency revealed 3.3. Field Populations—Genetic Bottlenecks. The Wilcoxon 𝑃 = 0.033 𝐻 > a significant deficit of heterozygotes in Dp( ), but test revealed a significant excess of heterozygosity ( 𝐸 𝑃 = 0.414 𝐻 𝑃 = 0.023 𝑃 = 0.219 not in Cl ( ). Score tests for heterozygote deficiency EQ)inDp( ), but not in Cl ( ). This individually carried out for each of the six loci and each of result suggests that the Dp population has undergone a recent thetwopopulationsrevealedthatalllociconformedtoHWE, genetic bottleneck [35]. Furthermore, there was a loss of rare except for locus C5 in Dp, which had a significant deficit of alleles in population Dp at four out of the six analyzed loci. heterozygotes (𝑃 = 0.013, Table 3).

3.4. Field Populations—Population-Specific Genetic Diversity 3.5. Field Populations—Population Differentiation. The score and HWE. The genetic diversity computed over all loci by test indicated a significant deficit of heterozygotes in Dp. The 𝐻 = 0.815 𝐹 GENEPOP was similar for both populations with 𝐸 largest IS value was observed for locus C5 in population Dp 𝐻 = 0.807 𝐹 = 0.167 𝐹 = −0.001 in Cl and 𝐸 in Dp. Based on all loci, the ( IS , Table 3). The fixation index ( ST ) 6 BioMed Research International

Table 4: Analysis of molecular variance (AMOVA) over all six microsatellite loci for field populations.

Source of variation Sum of squares Variance component % of total variation Fixation index 𝑃 value − − 𝐹 = −0.001 Among groups 2.320 0.001 0.060 CT 0.556 − − 𝐹 = −0.001 Among age classes within groups 9.843 0.002 0.069 SC 0.542 𝐹 = 0.032 Among individuals within age classes 222.574 0.077 3.172 IS 0.044 𝐹 = 0.030 Within individuals 223.000 2.354 96.957 IT 0.043 Total 457.737 2.428 The results are weighted averages over all six loci for 85 individuals comprising two populations and divided into three age classes. For the groupl, leve the individuals of the three age classes belonging to the same population were pooled. Listed are the source of variation, sum of squared deviations, the variance component estimates, the percentage of total variance, the fixation indices, and the significance of the variance components and of the fixation indicesestimated by performing 1023 permutations for the analyses. Note: small negative variance components; % of total variation and fixation indices are statisticalartefacts and are observed among entities with very low genetic differentiation (here groups and age classes). of the two populations did not differ from zero and thus indi- 40 cated no genetic differentiation between the two populations 35 b (Table 3). 30 ) 25 % 20 c 3.6. Field Populations—AMOVA of the Different Age Classes in 15

tDNA ( tDNA a Cl and Dp and Locus-by-Locus AMOVA. AMOVA indicated 10 a a a that the major proportion of genetic variation was due to 5 genetic differences within individuals (96.96%). The overall 0 𝐹 = 0.032 𝑃 = 0.044 Cl Cl Cl Dp Dp Dp inbreeding coefficient ( IS , )andthe 𝐹 = 0.030 𝑃 = 0.043 Site overall fixation index ( IT , )bothdeviated significantly from zero. In contrast, the variances among age 𝐹 = −0.001 𝑃 = 0.542 Figure2:DNAdamagemeasuredbytheCometassayinthe classes within the populations ( SC , ) 𝐹 = −0.001 𝑃 = 0.556 hemocytes of zebra mussels of the Cl and Dp populations, river andamongthepopulations( CT , ) Drava. Light grey bars correspond to field populations, dark grey werenotsignificant(Table 4). The AMOVA analysis for the bars to population samples exposed to dechlorinated tap water in age classes of each of the two populations revealed that the the laboratory experiment, and black bars to population samples second and third age classes of population Dp had significant exposed to wastewater in the laboratory experiment. Different letters 𝐹 𝑃 < 0.05 IS values above zero, whereas all other age classes had indicate statistically different DNA damage ( ). 𝐹 insignificant IS values close to zero. This observation across 𝐹 = 0.073 loci demonstrated that the second age class ( IS , 12 𝑃 = 0.036 𝐹 = 0.098 𝑃 = 0.035 d )andthirdageclass( IS , )ofDp c, d had an increased proportion of homozygotes in comparison 10 to the first age class of Dp and to all three age classes of Cl. 8

Locus-by-locus AMOVA carried out over all age classes ‰ 6 showed that only locus C5 was significantly different from a a zero in the 𝐹-statistics. The overall fixation index was signifi- MN ( ) 4 𝐹 = 0.094 𝑃 = 0.047 a, b cant ( IT , )forlocusC5(Table 5). 2 b 0 3.7. Field Populations—Comet Assay and Micronucleus Test. Cl Cl Cl Dp Dp Dp ResultsoftheCometassaycarriedoutforfieldpopulations Site are shown in Figure 2. The percentages of tail DNA did not differ significantly between the Cl and Dp sites. The frequency Figure 3: Number (per mill) of micronuclei (MN) in hemocytes of zebra mussels of the Cl and Dp populations, river Drava. Light grey of cells exceeding 50% of tail DNA was 0.29% for the control bars indicate field populations, dark grey bars population samples population Cl and 0.30% for population Dp. exposed to dechlorinated tap water in the laboratory experiment, DNAdamageasassessedbytheMNTshowedasignifi- and black bars population samples exposed to wastewater in the lab- cantly increased frequency of micronuclei in hemocytes for oratory experiment. Different letters indicate statistically different field mussels collected at the Dp site in comparison to the numbers of micronuclei (𝑃 < 0.05). mussels originating from the Cl site (𝑃 = 0.008, Figure 3).

3.8. Laboratory Exposure—Comet Assay and Micronucleus of mussels after the exposure to municipal wastewater than Test. After two weeks of acclimatization and three days of in the mussels of the control populations exposed to the 𝑃 = 0.0002 𝑃 = 0.0002 exposure to dechlorinated tap water DNA damage reached dechlorinated tap water ( Cl , Dp ). basal levels that were very similar between the two popu- The genotoxic response measured by the Comet assay in the lations,inbothCometassayandMNT.TheCometassay individuals from the Cl population exposed to the wastewater revealed a significantly higher DNA damage in hemocytes was significantly higher in comparison to the response of BioMed Research International 7

Table 5: Locus-by-locus analysis of molecular variance (AMOVA) across both field populations. 𝐹 𝑃 𝐹 𝑃 𝐹 𝑃 𝐹 𝑃 Locus IS value SC value CT value IT value A6 −0.014 0.720 0.008 0.154 0.005 0.309 −0.002 0.601 B6 0.032 0.211 0.007 0.146 −0.0003 0.535 0.039 0.161 B9 0.076 0.054 −0.022 0.994 0.013 0.106 0.068 0.085 C5 0.092 0.058 0.019 0.069 −0.017 1.000 0.0943 0.047 Dpol9 0.006 0.523 −0.019 0.970 −0.002 0.805 −0.015 0.625 Dpol19 −0.001 0.568 −0.003 0.531 −0.005 0.698 −0.009 0.571 𝑃 𝐹 𝐹 𝐹 Estimates and values of IS (locus-specific inbreeding coefficients), SC (variances among age classes within populations), CT (variances among age classes 𝐹 𝑃 < 0.05 𝑃 among populations), and IT (overall fixation indices) for each of the six microsatellite loci. Significant ( ) values are in bold. mussels from the Dp population (𝑃 = 0.012, Figure 2). The by water analysis is greatly confounded by sampling strategy frequency of cells exceeding 50% of tail DNA for the Cl and frequency and provides very little information on pol- population was 0% for the mussels in dechlorinated tap water lution pressure occurring through the extended periods of and10.99%forthemusselsexposedtothewastewater.Forthe time. Dp population it was 0% for the mussels in dechlorinated tap water and 1.77% for the mussels exposed to the wastewater. 4.1.2. Genetic Diversity and Population Differentiation. The A significant increase in frequency of micronucleated populations Cl and Dp had nearly identically high levels of hemocytes was detected in the mussels from the Dp popu- genetic diversity (Cl: 0.8164, Dp: 0.8065). Similarly high levels lation after the exposure to wastewater in comparison to the 𝑃 = 0.0002 of genetic diversity (0.79–0.94) assessed by microsatellite mussels in dechlorinated tap water ( , Figure 3), analysis have been reported for other populations of D. andalsoincomparisontothemusselsfromtheClpopulation 𝑃 = 0.023 polymorpha intheliterature[23, 48]. The fact that the genetic and exposed to the wastewater ( ). The frequency of diversity of the contaminated population was not altered by micronucleiinmusselsfromtheClpopulationexposedtothe the WWTP effluent is coherent with the conclusions derived wastewater was not significantly different from the Cl mussels 𝑃 = 0.089 from a meta-analysis across different taxa (plants, inverte- in tap water ( ). brates, and vertebrates) by DiBattista [49]. He found that There was no mortality of the mussels exposed to dechlo- pollution could both decrease and increase genetic variation rinated tap water during the two weeks of acclimatization or in populations (defined as genetic diversity or mean number during the three days of exposure to the tap water. After the of alleles per locus) through genetic drift and directional third day of exposure to municipal wastewater, the mortality selectionontheonehandandincreasedmutationrateand rate was 77% for population Cl and 84% for population Dp. selection for heterozygotes on the other hand. Dreissena DNAdamagemeasuredbytheCometassayandMNTdid polymorpha larvae remain in the plankton up to three weeks not differ significantly between the control laboratory popu- or even longer and consequently can be drifted over 300 km lationCl(intapwater)andthecontrolfieldpopulationCl 𝑃 = 0.118 𝑃 = 0.076 before settlement [50, 51]. This specific life history trait makes (Comet assay: ,MNT: ,Figures2 and 3). mutational load as a cause of high genetic diversity highly unlikely.Thus,thehighgeneticdiversityseeninpopulation 4. Discussion Dpcouldhaveresultedfromvariousselectiveandstochastic genetic processes. Moreover, decreases in genetic diversity 4.1. Field Populations—Population Level can be diluted by recent migration events, as it was sug- 4.1.1. Genetic Bottleneck. Consistent with hypothesis 1.1 we gested for contamination exposed populations of the arctic found that Dp had undergone a significant genetic bottleneck amphipod Orchomenella pinguis [52] and the Mediterranean which reflects a strong reduction in number of individuals mussel Mytilus galloprovincialis inhabiting polluted marine in the past. The genetic bottleneck in Dp was accompanied ecosystems [53]. The latter study observed higher genetic byalossofrareallelesinfouroutofsixoftheanalyzed diversity of mussel populations exposed to environmental microsatellite loci. This suggests that nonselective random pollution, what is in high concordance with our results. genetic drift had affected population Dp44 [ , 45]. Based on There was no significant genetic differentiation between the observed high values of several analyzed heavy metals, the two populations Cl and Dp. A high gene flow between PAHs, and mineral oil hydrocarbons in the sediment, we can the two populations is consistent with their high genetic conclude that this site is exposed to contamination inflow, similarity. In this part of the river Drava, gene flow in the and that the bottleneck in population Dp had probably D. polymorpha mussels could only result from the down- resulted from the WWTP effluent contamination [46]. stream swamping of free-swimming larvae in high densities Genetic bottlenecks have also been observed for other [54]. This passive dispersal mode of larvae strongly pre- populations of bivalves, for example, for razor clams (Ensis vents genetic population differentiation55 [ ]. As the distance siliqua) after an oil spillage47 [ ]. between the two sampling sites Cl and Dp is only about The water analysis at the Dp site and the effluent channel 750m,itisverylikelythatthemusselsoftheClandDpsite revealedonlyadecreaseinoxygenconcentration.However, areoffspringofthesamemusselpopulationsthatinhabited in high flow systems like rivers, assessment of pollution status the Cakovecˇ lake or even a location more upstream the 8 BioMed Research International river Drava. High levels of gene flow preventing population than for Cl. This result indicates pollution at the Dp site. differentiation are also consistent with the similar high levels The levels of micronucleated hemocytes found for zebra of genetic diversity observed in the two populations. mussels from Dp site are comparable to the levels reported for other polluted sites in the literature [15, 60, 61]. The MNTresultiscontrarytotheCometassay,inwhichthe 4.1.3. Selection and Genetic Drift. We found a significant Cl and Dp populations did not show significantly different deviation from Hardy-Weinberg equilibrium (HWE) for the responses. Several studies have also described that a high Dp population, whereas population Cl was in HWE. This response seen in MNT is not necessarily reflected at the deviation observed in Dp was caused by locus C5, which same intensity in the Comet assay [62, 63]. The following only deviated significantly from HWE and showed a deficit reasons are given for this inconsistency: first, MNT and of heterozygotes. While Astanei et al. [23]alsoreporteda Comet assay assess different aspects of DNA damage. MNT deficit of heterozygotes in D. polymorpha populations based detects clastogenic and aneugenic effects, while Comet assay on microsatellite loci and concluded that it was most likely measures single- and double-strand breaks, alkali labile sites, caused by null alleles, other studies did not find deviations DNA-DNA and DNA-protein crosslinks [63, 64]. Second, from HWE [48, 56]. Our analyses did not indicate a potential thelevelofDNAdamagemeasuredbytheCometassaycan influence of null alleles and suggest that the deviations belowerthantheinitiallevel,asitispossiblethatsome fromHWEatlocusC5musthavebeencausedbyanother ofthedamagehasalreadybeenrepaired[65]. The MNT genetic process. Alternatively, it is possible that a locus- only assesses nonrepairable DNA damage and chromosome specific analysis of population genetic structure reveals novel loss that will persist until apoptosis of the damaged cells. candidatelocithataretheobjectofselectionduetogenetic Third (and in connection to the second), acclimatization of hitchhiking [57, 58]. A linkage of the neutral microsatellite mussels inhabiting a contaminated site through activation of locus C5 to a gene undergoing selection could explain the the DNA repair mechanism is possible, what could also be observed deviation from HWE. We hypothesize that locus C5 facilitated through differential regulation of genes coding for is located near a coding gene that is under selection only in DNA repair, and even genetic selection could have acted in the contaminated Dp population, but not in the noncontam- the favor of better adopted genotypes [65–67]. For example, inated Cl population. A possible candidate gene could be a Black et al. [65]carriedoutaCometassayforthefreshwater gene that is involved in DNA repair mechanisms and/or in bivalve Anodonta grandis inhabiting a site contaminated with antioxidative or detoxification processes (see molecular and lead,cadmium,andzinc.Theydidnotfindanyincreased cellular level). Our interpretation is corroborated by another DNA damage in the mussels originating from this site. D. polymorpha population studied by us which inhabits a Mussels of the same species from a noncontaminated site heavilypollutedsiteintheriverSio,Hungary.Thispopulation kept in laboratory showed DNA breakage even at low also showed a deviation from HWE at locus C5 (unpublished concentrations of lead. The authors concluded that this data). Other population genetic studies also found a selective discrepancy in response in the Comet assay was due to an effect on a single microsatellite locus that was explained by enhanced DNA repair mechanism of the mussels from the the genetic hitchhiking model [58, 59]. A genetic linkage contaminated area. mapping of the microsatellite loci for D. polymorpha could As the mussels from the Dp site had a low DNA dam- clarify which coding gene is linked to C5. age in the Comet assay in combination with an increased The results obtained for age classes also indicated an rate of micronuclei, we hypothesize that they had adapted indirect selection process affecting locus C5. For population to the polluted environment by an enhanced DNA repair Dp, the inbreeding coefficients (𝐹 ) increased with the age IS mechanism and/or antioxidative defense. Inducement of any ofthemusselsandsignificantlydifferedfromzeroforthe of these mechanisms is expected to affect the results of the age classes 2 and 3. This suggests that the proportion of Comet assay stronger than the results of the MNT. homozygotes increased with the age of the mussels indicating ongoing selection. The increase in homozygosity is reflected by the overall deficit of heterozygotes observed in Dp. 4.3. Laboratory Exposure—Molecular and Cellular Level. In total, our results derived from microsatellite analysis After exposure to municipal wastewater, the mussels of the Cl support hypothesis 1.1 that a genetic bottleneck had affected population showed a significantly higher level of DNA dam- the Dp population, but not the Cl population. This bottleneck age in the Comet assay than the mussels of the Dp population. caused random genetic drift. Moreover, we also detected In contrast, we observed a significantly higher frequency of a strong indirect selective effect on locus C5 in the Dp micronucleated hemocytes in the mussels belonging to the population. The selective process causing this indirect effect Dp population. These results coincide with our expectation on C5 has obviously been acting on the zebra mussels for that the two populations Cl and Dp would react differently years, which we demonstrated by the age class analysis. under altered environmental conditions due to their different genetic constitution (hypothesis 2.1 or 2.2). 4.2. Field Populations—Molecular and Cellular Level. Based on the results of the population genetic analysis, Hypothesis 1.2 which assumes that the field population Dp the Comet assay, and the MNT carried out for the field should have a higher level of genotoxic effects than the field populations, we have hypothesized that the mussels of the population Cl was confirmed only by MNT. We observed contaminated Dp site had been acclimatized to the local a significantly higher percentage of micronuclei for Dp WWTP effluent discharge by an enhanced DNA repair BioMed Research International 9 mechanism and/or enhanced antioxidative mechanism. This (molecular, cellular, and population level) with a time- and possible acclimatization was apparently reflected only in the cost-effective system owing to the three well-established successful repair of DNA damage revealed by the Comet techniques. We believe that this research is a case study assay and not in MNT. A more efficient DNA repair sys- reinforcing the often proposed need to integrate population tem and/or antioxidative mechanisms in the Dp population genetic measures into ecotoxicological investigations [6, 7, resulting from acclimatization processes could also explain 16]. the differences in responses of the two populations seen in the pollution treatment in the Comet assay. Acclimatiza- Conflict of Interests tion through the DNA repair system and/or antioxidative mechanisms could be concordant with the low level of tail The authors declare that there is no conflict of interests DNA found in the field population Dp, and the significantly regarding publication of this paper. lowerpercentageoftailDNAfoundinthelaboratoryDp population in comparison to the laboratory Cl population in the pollution treatment. This explanation is consistent with Acknowledgments our hypothesis 2.1 for the laboratory experiment, where we The authors’ thanks are due to Dr. Dana Berens for her helpful assumed that population Dp was more resistant to wastewater comments on the paper. This research was funded by the duetopreviouslyactingselectionprocessesinthesource International Office of the Johannes Gutenberg University of habitat. 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Review Article Biomarkers of Type II Synthetic Pyrethroid Pesticides in Freshwater Fish

Anilava Kaviraj1 and Abhik Gupta2

1 Department of Zoology, University of Kalyani, Kalyani, West Bengal 741235, India 2 Department of Ecology & Environmental Science, Assam University, Silchar, Assam 788011, India

Correspondence should be addressed to Anilava Kaviraj; [email protected]

Received 16 February 2014; Accepted 31 March 2014; Published 23 April 2014

Academic Editor: Erhan Unlu

Copyright © 2014 A. Kaviraj and A. Gupta. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Type II synthetic pyrethroids contain an alpha-cyano group which renders them more neurotoxic than their noncyano type I counterparts. A wide array of biomarkers have been employed to delineate the toxic responses of freshwater fish to various type II synthetic pyrethroids. These include hematological, enzymatic, cytological, genetic, omic and other types of biomarkers. This review puts together the applications of different biomarkers in freshwater fish species in response to the toxicity of the major type II pyrethroid pesticides and assesses their present status, while speculating on the possible future directions.

1. Introduction (Table 1). Type II (𝛼-cyano) pyrethroids are more potent neurotoxicants than type I (noncyano) pyrethroids, with Pyrethroids are synthetic derivatives of pyrethrins, the toxic 𝛼 component contained in the flower of the plant Chrysan- toxicity being solely attributed to -cyano substituent [2, 3]. themum cinerariaefolium [1]. The insecticidal properties of Synthetic pyrethroid pesticides, especially the type II pyrethrins are derived from ketoalcoholic esters of chrysan- compounds, are extensively used in pest management all over themic and pyrethroic acids, which are strongly lipophilic in the world because of their relatively low toxicity to birds and natureandhavetheabilitytopenetraterapidlyintothebody mammals. However, they are known to have pronounced of insect pests to produce toxicosis. However, the naturally toxic effects on bees, freshwater fish, and other aquatic occurring pyrethrins are extremely sensitive to light and organisms [4, 5]. It is, therefore, necessary to evaluate the breakdowninafewhours,beforeadequatequantityof current status of the various biomarkers used for delineating pyrethrinsisaccumulatedinthebodyofinsectstokillthem. the toxic effects of type II synthetic pyrethroids on freshwater Interesting developments were held during the last twenty fish and envisage the directions that the biomarker research years in modifying structure, stereochemistry, and formu- may take in near future. A biomarker is defined as “a change lations to develop thousands of modern pyrethroids with in biological response which can be related to exposure to or chemical properties and biological performance suitable for toxic effects of environmental chemicals”6 [ ]. The level of the agriculture field. These modifications include halogenations response could range from those generated at molecular lev- of the cyclopropane side chain of the pyrethrin molecule, els to those at the community or even the ecosystem level [6]. addition of cyano group in the alcohol moiety [2], mixing Against this broad background, the present review attempts of geometric and optical isomers, and formulations of the to provide a synthesis of the knowledge base available on technical grade (containing 85% or more of the active ingre- the biochemical and physiological biomarkers of the type dients) compound with carriers and solvents. The pyrethroids II pyrethroids in freshwater fish identifying the parameters containing alpha-cyano group are grouped as type II and which can be established as unequivocal biomarkers of these noncyano pyrethroids are grouped as type I pyrethroids pesticides. 2 BioMed Research International

∗ Table 1: Pyrethrins, type I pyrethroids, and type II pyrethroids .

Pyrethrins: constituents of natural pyrethrum extracts Synthetic derivatives of pyrethrin: pyrethroids Active ingredient Type I Type II Esters of Esters of Pyrethric Esters without Esters with Chrysanthemic acid acid alpha-cyano group alpha-cyano group Allethrin Fenvalerate Pyrethrin-I Pyrethrin-II Types of 1st generation 3rd generation pyrethrins/pyrethroids Resmethrin Cyhalothrin Cinerin-I Cinerin-II gradually developed 2nd generation 4th generation Permethrin Cypermethrin Jasmolin-I Jasmolin-II 3rd generation 4th generation Bifenthrin Deltamethrin 4th generation 4th generation ∗ Based on [1, 2].

2. Hematological Biomarkers [17]. Depletion of hepatic glycogen due to cypermethrin treatment has been observed in Tilapia mossambica [18] Although somewhat simple and largely nonspecific, vari- and Clarias batrachus [19]. Glycogen depletion is a regula- ous properties of blood and serum have long been used tory step against xenobiotic insult. It increases intermediary as biomarkers of xenobiotic toxicity, including pesticides, metabolism resulting in the protection of the hepatocytes. in fish. The most common blood parameters monitored Increase in the activity of lactate dehydrogenase (LDH), an include total erythrocyte count, total leucocyte count, and enzyme responsible for conversion of pyruvate to lactate haemoglobin and haematocrit content, which were found to in fish exposed to sublethal concentration of cypermethrin decrease on cypermethrin exposure in the Indian major carps [16, 20, 21]and𝜆-cyhalothrin [22], is an indication of such Labeo rohita and Catla catla [7]. However, several workers [8– metabolism. Hyperglycaemia, though not specific, is a quick 11] recorded increased lymphocyte, leucocyte, and erythro- response of the adverse effects of the toxic doses of type II cyte counts, packed cell volume, and hemoglobin in several pyrethroids. species of fishes challenged with cypermethrin, deltamethrin, and fenvalerate. Jayaprakash and Shettu [12]observeda decrease in hemoglobin content, total erythrocyte count, 4. Enzymes of Energy packed cell volume, mean corpuscular volume, and mean Metabolism as Biomarkers corpuscular hemoglobin concentration and an increase in total leukocyte count, mean corpuscular volume, erythrocyte The enzymes of energy metabolism have also been found sedimentation rate, and clotting time values in the snakehead, to have the potential to be used as biomarkers of type II Channa punctatus, exposed to deltamethrin. Other hemato- pyrethroid toxicity in fish. Total adenosine triphosphatase, logical variables such as total serum protein, albumin, and sodium-potassium adenosine triphosphatase, and magne- globulin contents, albumin-globulin ratio, plasma glucose, sium adenosine triphosphatase activities in the gills of Catla alanine aminotransferase, and cholinesterase were reduced catla were significantly reduced when exposed to sublethal in fishes exposed to cypermethrin and deltamethrin7 [ , concentrations of cypermethrin [7]. Cypermethrin-challe- 13]. On the contrary, significantly higher erythrocyte count, nged Clarias batrachus showed significant inhibition of the +2 + + haemoglobin, haematocrit, plasma total protein, albumins, activities of total, Mg ,andNa-K ATPase enzymes, calcium, and ammonia contents, and activities of aspartate while it increased the levels of glycogen phosphorylase (a), aminotransferase and creatine kinase were observed in the thereby suggesting that these enzymes had the potential to deltamethrin-treated group compared to the control group be used as biomarkers of cypermethrin exposure in fish [23]. + + [13]. Thus the hematological biomarkers, though relatively Deltamethrin also inhibited the activities of Na -K ATPase simple to measure and fairly useful in detecting xenobiotic in the gill and heart tissues of the freshwater fish Ancistrus effects, are of rather limited utility and often somewhat multispinis [5]. Liver alkaline phosphatase is also known to contradictory in their response. playaroleinglycogenmetabolism.Theenzymeiscapableof inactivating phosphorylase enzymes and promotes glycogen 3. Hyperglycemia as a Biomarker synthesis. Inhibition of alkaline phosphatase activity in the liver is thus related to the breakdown of glycogen to meet Reduction in hepatic glycogen accompanied by a rise in blood the energy demand under stress or decrease in the rate glucose is a common reaction of fish against xenobiotic insult. of transphosphorylation or uncoupling of oxidative phos- Cypermethrin induced hyperglycaemia has been recorded in phorylation. Saha and Kaviraj [17]observedthatalkaline Indian major carp Labeo rohita [14], Korean rock fish Sebastes phosphatase activity in the liver of H. fossilis was reduced after −1 schlegeli (Hilgendorf) [15], rainbow trout Oncorhynchus exposure to 0.3–0.5 𝜇gL cypermethrin. Das and Mukherjee mykiss [16], and Asian air breathing fish Heteropneustes fossilis [14] also observed a reduction of alkaline phosphatase in the BioMed Research International 3 braintissueofLabeo rohita following exposure to cyperme- [45]. Saha and Kaviraj [17]observedthatascorbicacidlevel thrin. But there are reports that alkaline phosphatase activity depleted in liver and kidney of H. fossilis in response to of fish increases after exposure to cypermethrin8 [ , 15, 20, 24]. 0.5 𝜇g/Lofcypermethrin,probablybecauseofinvolvement Higher activities of lactate dehydrogenase (LDH) and acid of ascorbic acid in counteracting the stress of cypermethrin. and alkaline phosphatases were also observed in Channa When fish were fed ascorbic acid through diet, ascorbic acid punctatus exposed to cypermethrin and 𝜆-cyhalothrin [22]. level increased in liver and kidney but decreased in blood of Necrosis of liver and subsequent leakage of alkaline phos- cypermethrin-treated fish as compared to control. Liver and phataseintobloodstreammightberesponsibleforan kidney probably accumulated ascorbic acid from the blood increaseoftheenzymeinblood[25]. stream as a defense mechanism against cypermethrin.

5. Oxidative Stress Biomarkers 6. Enzymes of Nitrogen Metabolism as Biomarkers Lipid peroxidation, induced by reactive oxygen species (ROS), is a common oxidative stress biomarker of toxicants. Increased activities of enzymes of nitrogen metabolism such Lipid peroxides are capable of modifying the properties of as aspartate aminotransferase (AST), alanine aminotrans- biological membranes, eventually resulting in damage of ferase (ALT), glutamate dehydrogenase, and glutamine syn- cellular membrane [26–28]. The most widely used assay for thetase in response to cypermethrin [46]and𝜆-cyhalothrin lipid peroxidation is malondialdehyde (MDA), a secondary [40] exposure served as useful biomarkers in fish. Jee et al. productoflipidperoxidationcausedbyfreeradicals[22, 29]. [15] found an increase in the levels of serum glutamic-acid Fish, like other organisms, have evolved defense systems oxaloacetic-acid-transaminase and glutamic-acid pyruvic- comprising both enzymatic and nonenzymatic processes to acid-transaminase in Korean rockfish (Sebastes schlegeli) minimize cell damage by lipid peroxides. Catalase (CAT), an exposed to cypermethrin. Velisek et al. [16]foundanincrease enzymatic antioxidant, scavenges ROS and converts them to in the activity of aspartate aminotransferase (AST), creatine less reactive species thereby preventing lipid peroxidation. kinase (CK), and lactate (LACT) in rainbow trout after Fish exposed to deltamethrin showed a tendency towards exposure to cypermethrin. Increase in the activities of ALT, increase in the level of MDA and reduction in the activity AST, and transaminases appeared as specific reaction of of CAT, although cadmium pre-exposure at low concentra- type II pyrethroids. Alanine aminotransferase (ALT) and tions appeared to modulate deltamethrin-induced oxidative aspartate aminotransferase (AST) activities increased in stress [30, 31]. Channa punctatus exposed to alphamethrin response to cypermethrin in Orecochromis niloticus [20]. also showed a decrease in the activity of CAT [32]. Other Marked increase in the activities of transaminases in Clarias type II synthetic pyrethroids such as 𝜆-cyhalothrin [33], batrachus [47] and an increase in the activities of aspartate cypermethrin [34, 35], cyfluthrin36 [ ], deltamethrin [37, 38], and alanine aminotransferase in Prochilodus lineatus have fenvalerate [39], cypermethrin, and 𝜆-cyhalothrin [40]also also been reported in response to cypermethrin, a type II increased lipid peroxidation and/or reduced CAT activity in pyrethroid [24]. different species of freshwater fish. Dinu et al. [41]observed that superoxide dismutase (SOD) and catalase (CAT) activities were reduced in the liver along with activation of 7. Acetylcholinesterase (AChE) glutathioneperoxidase(GPX),glutathionetransferase(GST), Activity as Biomarker and glutathione reductase (GR), while an increase in CAT, GST, and GR was recorded in the intestine. Since liver is AChE activity has been established as a potent biomarker for involved in detoxification of xenobiotics, while intestine is organophosphorus and carbamate pesticides, while unequiv- engaged in absorption, this difference could be linked to ocal evidence is lacking for organochlorines. The activity of their specific metabolic pathways. This finding, therefore, the AChE was not inhibited by deltamethrin in three species calls for tissue-specific understanding and application of the of fishes [5, 48–50]. Similarly, esfenvalerate exposure had no biomarker approach, rather than adopting a more generalized effectonAChEactivity.Onthecontrarybothk-cyhalothrin one common for all tissues and organisms. However, several and cypermethrin exhibited tissue-specific as well as dose studieshavealsoreportedincreasedCATactivityinfish dependent decrease in the activity of AChE, with a greater exposed to type II pyrethroids such as cypermethrin [42]. inhibition of AChE activity in brain when compared to that in Cytochrome P450 and its numerous isomers comprise a muscle and gills [51]. Thus the evidence for AChE inhibition very important set of enzymatic oxidants that are actively by type II pyrethroids is not unequivocal and needs further involved in the bioelimination of xenobiotics entering the investigations in different species and a broad range of these body [43]. Bhutia et al. [44] found that several cytochrome pesticides. P450 isozymes were induced in the hepatic microsomes of Heteropneustes fossilis exposed to cypermethrin, while 8. Gene Expression as Biomarker some isozymes were inhibited when compared to that in the control. The total cytochrome P450 content was significantly Esfenvalerate exposure invoked alterations in the expres- induced upon cypermethrin exposure. sion of genes associated with immune responses, along Ascorbic acid serves as a nonenzymatic antioxidant with apoptosis, redox, osmotic stress, detoxification, and biomarker of oxidative stress of pyrethroid pesticides to fish growth and development. Swimming impairment correlated 4 BioMed Research International significantly with expression of the enzyme aspartoacylase be a useful biomarker in fish exposed to type II synthetic (ASPA), which is involved in brain cell function. Selected pyrethroids. genes were investigated for their use as molecular biomarkers, and strong links were determined between measured down- 10. Omic Biomarkers regulation in ASPA and observed behavioral responses in fish exposed to environmentally relevant pyrethroid concentra- The emerging field of “omics” that comprises genomics, tions. Microarray technology proved to be a useful approach proteomics, metabolomics, and others is expected to have in screening for and generation of molecular biomarkers in increasing applications in the field of ecotoxicology by estab- endangered, nonmodel organisms. It was especially valuable lishing newer biomarkers of environmental contaminants for identifying specific genes that could be directly linked [59]. Metabolomics is emerging as a new and robust approach with sublethal toxicological endpoints such as changes in towards the study of xenobiotic insult. The applicability and expression levels of neuromuscular genes resulting in mea- potential of the metabolomics approach for the elucidation of surable swimming impairments in larval delta smelt [52]. toxicological effects of pesticides and the underlying mech- 1 Shietal.[42] found that cypermethrin downregulated ogg1 anisms was examined in a study, which used a Hnuclear and increased p53 gene expression as well as the caspase-3 magnetic resonance (NMR) based metabolomics approach to activity in zebra fish embryos, resulting in oxidative stress investigate the toxicity of 𝜆-cyhalothrin in goldfishCarassius ( and apoptosis. Thus gene expression holds promise as a more auratus). The 𝜆-cyhalothrin exposure influenced levels of specific biomarker of type II pyrethroid toxicity. However, many metabolites, such as leucine, isoleucine, and valine in further researches are required to explore its application brain and kidney; lactate in brain, heart, and kidney; alanine under different situations and in a wide range of species. in brain and kidney; choline in brain, heart, and kidney; tau- rine in brain, heart, and kidney; N-acetylaspartate in brain; 9. Genotoxic Effects as Biomarkers myo-inositol in brain; phosphocreatine in brain and heart; 2-oxoglutarate in brain; cis-aconitate in brain, and broke Chromosomal aberrations (CA), nuclear abnormalities (NA), the balance of neurotransmitters and osmoregulators. It also and the micronucleus test (MN) are the most commonly caused oxidative stress and disturbed metabolisms of energy employed measures of genotoxicity. Campana et al. [53]pro- and amino acids. The implication of glutamate—glutamine— vided one of the early validations of the MN test in fishes for gamma-aminobutyric axis in 𝜆-cyhalothrin induced toxicity the assessment of genotoxic pollutants, when they studied the was demonstrated. Thus the study was a positive step towards genotoxicity of the pyrethroid 𝜆-cyhalothrin in erythrocytes the discovery of precise biomarkers for pesticide pollution of Cheirodon interruptus interruptus. The results obtained in aquatic environment [60]. Besides, proteomic approaches demonstrated the genotoxic effects of this pyrethroid in the have been utilized to assess the effects of zinc, cadmium, fish.¸avas C ¸ and Ergene-Goz¨ ukara¨ [54]showedthatnuclear and various cyanotoxin exposures in fish [59]. It is expected (micronucleus frequency) and nucleolar (changes in quanti- that similar approaches would be increasingly explored for tative characteristics of nucleoli) biomarkers could be gain- their adoption and incorporation into the diverse biomarker fullyusedtoevaluatethefunctionalandstructuralgenotoxic repertoire for fish. effects of the type II synthetic pyrethroid 𝜆-cyhalothrin on the fish Garra rufa. The frequency of micronucleated ery- 11. Specificity in Sodium Channel Interactions throcytes was significantly increased in exposed fish while the nucleolar parameters were repressed, thereby demonstrating Several researchers have attempted to elucidate molecular the genotoxic potential of 𝜆-cyhalothrin. These findings also mechanism of action of type I and II pyrethroids and suggestedthattheuseofnucleolarbiomarkersonfishfin specificity in their interactions with voltage-sensitive sodium cells, in addition to micronucleus test, could provide valuable channels [2, 61, 62]. Type II pyrethroids are known to inhibit information in aquatic genotoxicity studies. Velmurugan et the deactivation of sodium channels more than that by their al. [55] observed that different types of chromosomal aberra- type I counterparts. Insect sodium channels are large trans- tions such as chromatid breaks, centromeric fusion, acentric membrane proteins composed of four homologous domains fragments, chromosomal gaps, sticky plates, aneuploidy, and (I to IV). While studying point (amino acid) mutations at ring chromosomes could serve as useful biomarkers of 𝜆- various regions of insect sodium channels of pyrethroid- cyhalothrin in the fish Mystus gulio exposed to 6.4 ppb of this resistantinsectpopulationitwasobservedthatthelackof 1111 pyrethroid. glycine at position 1111 (G ) and charge neutralization or In subsequent studies, it was shown that deltamethrin reversal of two positively charged lysines immediately down- readily induced MN and NAs along with increased lipid stream in the intracellular linker connecting domains II and peroxidation [56], while cypermethrin also showed increased III of the sodium channels of cockroach turned them more 1111 frequencies of CA and MN in a concentration-dependent resistant to type II but not to type I pyrethroids [61]. G manner [57]. Exposure to 𝜆-cyhalothrin induced MN and and two downstream lysines are thus assumed to be involved NAs in the mosquito fish Gambusia affinis,withtheNAs selectively in the response of sodium channels to type II comprising blebbed, lobed, and notched nuclei. The ratios pyrethroids due to their direct or indirect interaction with of polychromatic to normochromatic erythrocytes were also the 𝛼-cyano group. Interactions of type II pyrethroids with found to decrease in pesticide-challenged mosquito fish voltage-sensitive sodium channel are also well documented [58]. These results suggest that genotoxic damage could in mammals [2]. But hardly is there any information on BioMed Research International 5

pyrethroid-sodium channel interaction in fish and there is with special reference to the clinical, biochemical and haema- a need to concentrate researches in this field for identifying tological effects,” Environmental Toxicology and Pharmacology, specific biomarkers of type II pyrethroid toxicity. vol.24,no.3,pp.212–217,2007. [11] A. Hedayati and R. Tarkhani, “Hematological and gill histopa- 12. Conclusions thological changes in iridescent shark, Pangasius hypoph- thalmus (Sauvage, 1878) exposed to sublethal diazinon and Biomarkers of oxidative stress appear to have been the most deltamethrin concentrations,” Fish Physiology and Biochemistry, widely used category of biomarkers of type II synthetic 2013. pyrethroids in the last decade. The accentuated generation [12] C. Jayaprakash and N. 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Research Article Metallothionein Induction in the Coelomic Fluid of the Earthworm Lumbricus terrestris following Heavy Metal Exposure: A Short Report

A. Calisi, M. G. Lionetto, E. De Lorenzis, A. Leomanni, and T. Schettino

Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Via Provinciale Lecce-Monteroni, Monteroni, 73100 Lecce, Italy

Correspondence should be addressed to M. G. Lionetto; [email protected]

Received 19 November 2013; Accepted 25 February 2014; Published 3 April 2014

Academic Editor: Anilava Kaviraj

Copyright © 2014 A. Calisi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Earthworms are useful bioindicator organisms for soil biomonitoring. Recently the use of pollution biomarkers in earthworms has been increasingly investigated for soil monitoring and assessment. Earthworm coelomic fluid is particularly interesting from a toxicological perspective, because it is responsible for pollutant disposition and tissue distribution to the whole organism. The aim of the present work was to study the effect of heavy metal exposure on metallothionein (Mt) induction in the coelomic fluid of Lumbricus terrestris in view of future use as sensitive biomarker suitable for application to metal polluted soil monitoring and assessment. L. terrestris coelomic fluid showed a detectable Mt concentration of about 4.0 ± 0.6 𝜇g/mL (mean ± SEM, 𝑛=10)in basal physiological condition. When the animals were exposed to CuSO4 or CdCl2 or to a mixture of the two metals in OECD soils for 72 h, the Mt specific concentration significantly (𝑃 < 0.001) increased. The Mt response in the coelomic fluid perfectly reflected the commonly used Mt response in the whole organism when the two responses were compared on the same specimens. These findings indicate the suitability of Mt determination in L. terrestris coelomic fluid as a sensitive biomarker for application to metal polluted soil monitoring and assessment.

1. Introduction significantly affected by pollutants reaching the soil system. On the other hand, they can influence pollutant availability Soil pollution has enormously increased during the last in the soil with their burrowing activity [4]. Earthworms can decades due to industrial activities, urban waste, intensive be exposed to contaminants in two principal ways. Firstly, use of biocides and fertilizers, and atmospheric deposition. living in the soil they are in direct contact with soil pore The most diffusive pollutants occurring in soil are heavy water and therefore with pollutants therein dissolved. The metals, which can enter the soil from different sources, such as pesticides, fertilizers, organic and inorganic amendments, earthworm skin is extremely permeable to water [5]andit mining,wastes,andsludgeresidues[1]. In contrast to harmful represents a main route for contaminant uptake [6, 7]. Sec- organic compounds, heavy metals do not decompose and do ondly, these organisms ingest large amounts of soil; therefore, not disappear from soil even if their release to the environ- they are continuously exposed to contaminants adsorbed to ment can be restricted [2]. Therefore, the effects of heavy solid particles through their alimentary tract [8]. Previous metal contamination on soil organisms and decomposition studies under both field and laboratory conditions have processes persist for many years. demonstrated that earthworms bioaccumulate certain metals Earthworms are very important organisms for soil for- (suchasCd,Cu,Zn,Pb,andMo)fromsoil[9–12]. They mation and organic matter breakdown in most terrestrial tolerate high tissue metal concentrations [12–14]duetowell- environments and they have been considered to be con- developed trafficking and storage pathways for heavy metals, venientindicatorsoflanduseimpactandsoilfertility[3]. including metallothioneins (Mt) [15]. Mts are low-molecular- Because of their particular interactions with soil, they are weight cysteine-rich metal-binding proteins that are involved 2 BioMed Research International in regulation of metabolism of trace metals and protection surface but also in the soil deeper layer. It has a large size against heavy metal toxicity and oxidative stress [16, 17] which permits handling and collection of enough amounts in a wide range of phylogenetic groups [18–20], including of coelomic fluid without compromising the survival of earthworms [21–23]. Earthworms express two distinct MT the animal. L. terrestris specimens were exposed to copper isoforms (MT-1 and MT-2) characterized by a high cysteine sulphate or cadmium chloride or a mixture of the two metals content and no significant aromatic residues [24]. Their in OECD soil as described in the Section 2. metal-responsiveness was confirmed by determining Mt- specific expression profiles in earthworms exposed to soils of differing heavy metal concentrations. The two isoforms have 2. Methods different subcellular distributions and functions, namely, the 2.1. Experimental Design. Ahomogeneousgroupofearth- homeostasis and detoxification of essential and nonessential worms (L. terrestris)(𝑛 = 130) of the same size (body weight metals, respectively [13, 24, 25]. of depurated—void gut—earthworms was 2.14 ± 0.18 g) was Although the amino acid sequences of more than 50 acclimated for 48 h in 11 boxes (10 animals per box) each invertebrate MT and MT-like proteins have already been ∘ containing 1 kg of soil at 18±1 C and 16 : 8 h light/dark regime determined, little is known about the biochemical properties and located in a climatized room. of earthworm MTs. So far, only 5 MT genes of earthworms The OECD soil utilized was a mixture of 10% sphagnum have been cloned from Lumbricus castaneus, Eisenia fetida, peat,20%kaolinclay,and70%air-driedsandwithmorethan Lumbricus rubellus,andLumbricus terrestris [26, 27]whose 50% of the particles between 50 and 200 microns according to expression is differentially regulated by different heavy metals the OECD guidelines [42]. The dry constituents were blended [13, 24, 28]. The utility of Mt induction in earthworms in the correct proportions and mixed thoroughly. The soil as reliable biomarker in monitoring and assessment soil moisture content was adjusted to 45% of the water holding metal pollution has been demonstrated by several studies on capacity with deionized water. The pH was 5.6 adjusted to 6.0 different earthworm species [28–30]. with calcium carbonate [43]. The constructed soil represented Previous studies demonstrated a significant induction of the drilosphere soil. Mt proteins in different earthworm species such as Lumbricus After this acclimation period 20 specimens were utilized rubellus, Eisenia fetida,andEisenia andrei exposed to cad- for the determination of Mt concentration in the coelomic mium [31–33]orinLumbricus mauritii exposed to Pb and fluidandinthewholeorganisminbasalphysiological Zn contaminated soil [27]andinLumbricus terrestris exposed conditions. The remaining 110 specimens were utilized for the to cadmium, copper, and mercury [30, 34, 35]. Morgan et al. exposure experiment. They were divided into five subgroups: [8] previously described the immunoperoxidase localization the first𝑛=30 ( ) was utilized as initial control (at time 0), of Mt in the major organs and tissues of the earthworm the second (𝑛=20) was exposed to copper sulphate, the Lumbricus rubellus sampled from a mine soil heavily polluted third (𝑛=20) was exposed to cadmium chloride, the fourth with Pb, Zn, and Cd. MT expression was strongly detected in (𝑛=20) was exposed to a mixture of the two metals, and the chloragogenous tissue and also in the apical cytoplasm the fifth was utilized as final control to be analyzed at theend of intestinal epithelial cells, within the narrow tubular region of the exposure experiment. The exposure time was 72 h.As of nephridia, in the secretory epithelia of the calciferous previously demonstrated [35], this time is sufficient to induce gland, and in coelomocytes contiguous with chloragocytes a significant response to heavy metal exposure in L. terrestris. attached to the gut. Homa et al. [36, 37] described Mt gene The toxicant concentrations utilized were 45 mg/kg of soil for induction in coelomocytes of the earthworms Eisenia fetida copper sulphate and 1 mg/kg of soil for cadmium chloride, and Allolobophora chlorotica after three days of dermal respectively. They are far below the EC50 values reported exposure to metals. for copper and cadmium in earthworms [44]. These con- Earthwormcoelomicfluidisparticularlyinterestingfrom centrations represent copper sulphate and cadmium chloride a toxicological perspective, because it is responsible for intervention values, which are generic soil quality standards pollutant disposition and tissue distribution to the whole basedonpotentialriskstohumanhealthandecosystems organism. Its cells (coelomocytes) are involved in the internal asindicatedinDMnumber47125October1999[45]. The defense system [38–40] and any impairment of coelomo- toxicants were dissolved in water and added to the terrain cyte functioning can compromise the health of the entire at the start of the exposure experiment. The exposure to the organism. Therefore, this physiological fluid is very interest- mixture of the two metals was performed by adding each ing for the development of novel nondestructive pollution metal to the soil at the same concentration used for the single biomarkers. exposure. The aim of the present work was to study the effect of At time 0 and 72 hours coelomic fluid was sampled metal exposure on Mt induction in the coelomic fluid of from each specimen by puncturing postclitellum segments the earthworm L. terrestris in view of future use as sensitive of the coelomic cavity by a sterilized hypodermic syringe nondestructive biomarker suitable for metal polluted soil (1 mL syringe, needle 27G). Coelomic fluid sampling did not monitoring and assessment. compromise the animal survival. We selected L. terrestris because of its ecological habits in the soil. It is an anecic species that forms temporary deep burrows and comes to the surface to feed [41]. Therefore, 2.2. Mt Measurement. Mt coelomic concentration was it can be exposed to pollutants present not only in the soil determined by the spectrophotometric method previously BioMed Research International 3 described [46, 47] in either the whole organism or the 5 coelomic fluid. Briefly, tissues were homogenized (1 : 3 wt/vol for whole organism or 1 : 3 vol/vol for coelomic fluid) in the following buffer: 0.5 M sucrose, 20 mM Tris-HCl ∗∗∗ buffer, pH 8.6, added with 0.006 mM leupeptin, 0.5mM 4 phenylmethylsulphonilfluoride as antiproteolytic agents, and 0.01% 𝛽-mercaptoethanol as a reducing agent. Then, thehomogenatewastreatedtoobtainapartiallypurified Mt fraction by ethanol/chloroform precipitation. Mt concen- 3 tration in the samples was quantified by spectrophotometric titration of the sulphydryl residues using the Ellman’s 󸀠 reagent (5,5 -dithiobis-2-nitrobenzoic acid) using reduced glutathione (GSH) as a standard. Data were expressed as 𝜇g protein) g Mt/mg 2 MT/mg of proteins. (𝜇 Protein concentration was quantified by the Bradford assay [48] using NanoDrop ND-1000 UV-Vis (Thermo Sci- entific, Waltham, MA, U.S.A.). 1

2.3. Electrophoresis. Coelomic fluid homogenate (see above) of control and heavy metal exposed animals following 72 0 hours of exposure was analyzed by sodium-dodecyl-sulfate Coelomic ﬂuid Whole organism polyacrylamide gel electrophoresis on 1 mm thick slab gels −1 according to the method of Laemmli [49]withreducing Figure 1: Mt specific concentration (expressed as 𝜇g⋅mg proteins) conditions (5% (V/V) 2-mercaptoethanol) using Protean II in the coelomic fluid and in the whole organism determined in ∗∗∗ Xi cell (Bio-Rad Laboratories, Marnes La Coquette, France) unexposed L. terrestris. 𝑃 < 0.001 (student’s t-test). and acrylamide/bisacrylamide slab gels (15% T, 2.67% C, 375 mM Tris-HCl, pH 8.8 containing 0.1% (W/V) SDS). Pure When the animals were exposed for 72 h to CuSO4 or rabbit Mt (SIGMA, St. Louis, USA) was analyzed in parallel. ∘ CdCl2 or to a mixture of the two metals in OECD soils, the The separation was performed at 15 C using Laemmli buffer Mt specific concentration significantly𝑃 ( < 0.001)increased (25mMTris,192mMglycine,and0.1%(W/V)SDS,pH8.3 both in the coelomic fluid or in the whole organism with from Bio-Rad) for 5 min at 200 V, then for 3.5 h at 350 V. respect to control group, as indicated in Figure 2.Onthe The gel was stained with Coomassie Brilliant Blue for protein other hand, control animals did not show any significant detection. changes with respect to time 0 following 72 h of exposure under the same conditions of test organisms except for 2.4. Statistical Analysis. Data were analyzed by two-way the absence of metals in the soil. In all the cases, the Mt ANOVA and Newman Kewls posttest using a two factor specific concentration was significantly𝑃 ( < 0.001)higher orthogonal experimental design: factor (A) “metal exposure” inthecoelomicfluidwithrespecttothewholeorganism.As which included four levels (“not exposed” or control animals, demonstrated by the two-way ANOVA, the two variability “Cd exposed,” “Cu exposed,” and “Cd + Cu exposed”) and factors “metal exposure” and “tissue” (see Section 2), exerted factor (B) “tissue” which included two levels (“coelomic fluid” an extremely significant effect, accounting for 44.17% and and “whole organism”). WinGmav 5 software (designed, 20.37% of the total variance, respectively. There was no coded, and compiled by A. J. Underwood and M.G. Chap- significant interaction between the two variability factors, man,InstituteofMarineEcology,UniversityofSydney,Aus- suggesting that metal exposure induced a detectable increase tralia) was used. The homogeneity of variance was previously in the Mt expression in the same way on both coelomic fluid tested by Cochran’s test. Data are expressed as mean ± S.E.M. andthewholeorganism.ThepercentageofMtinduction in the whole organism or coelomic fluid did not vary 3. Results significantly between the metal exposure conditions (CuSO4, CdCl2, and mix) (Table 1). The electrophoretic analysis of L. terrestris coelomic fluid showed a detectable Mt concentra- thecoelomicfluid(Figure 3) confirmed the results obtained tion of about 4.0 ± 0.6 𝜇g/mL (mean ± SE, 𝑛=10)inbasal spectrophotometrically, showing a significant increase in physiological condition. intensity of the bands corresponding to Mt in samples With the aim to compare the MT concentration in the exposed to heavy metals. coelomic fluid with the Mt concentration determined in the whole organism, the specific MT concentration, referred 4. Discussion to the protein content of the sample, was calculated. As presented in Figure 1, the Mt specific concentration in the Earthworm biomarkers are becoming increasingly important coelomic fluid appeared significantly higher with respect to in the recent years in the evaluation of the effects of contam- the MT concentration determined in the whole organism. inants on soil organisms [50–52]. They are considered useful 4 BioMed Research International

16 Mt ControlCd Cu Mix rabbitST kDa 14 ∗∗

12 ∗∗ 10 ∗∗ Figure 3: SDS gel electrophoresis of L. terrestris coelomic fluid. ∗∗ Line 1: coelomic fluid from control specimens; line 2: coelomic fluid 8 ∗∗ ∗∗ from copper sulphate exposed specimens; line 3: coelomic fluid from cadmium chloride exposed specimens; line 4: coelomic fluid from specimens exposed to a mixture of the two metals; line 5: pure rabbit

g Mt/mg protein) g Mt/mg 6

(𝜇 Mt;line6:molecularmassmarkers.

0 the coelomic fluid from the chloragogen tissue surrounding Control Control Cu Cd Mix the intestine and blood vessels [55]. As demonstrated by Morgan et al. [8], chloragocytes, as well as the chloragogenous Time 0 Time 72 h tissuefromwhichtheyderive,areabletosynthetizeMt.Mt Coelomic ﬂuid concentration detected in the coelomic fluid in the present Whole organism study probably represented the Mt concentration of the above −1 Figure 2: Mt specific concentration (expressed as 𝜇g⋅mg proteins) cells. in the coelomic fluid and in the whole organism determined in When the animals were exposed to heavy metals such control and metal exposed L. terrestris (see Section 2). The statistical as copper sulphate or cadmium chloride in OECD soil significance of data was analyzed by two-way ANOVAand Newman for 72 h the coelomic fluid, MT concentration significantly ∗∗ Kewls posttest (see Section 2). 𝑃 < 0.01. increased, as indicated by both the electrophoretic profile of thecoelomicfluidandthespectrophotometricanalysisofthe coelomic fluid Mt enriched fraction. This result is in agree- Table 1: Percentage variation of metallothionein concentration in L. ment with results found by Homa et al. [36, 37]whofound terrestris coelomic fluid or whole organism following 72 h exposure Mt gene induction in coelomocytes of the earthworms Eisenia to CdCl2, CuSO4 or a mixture of the two metals in OECD soil (for the concentration used see Section 2). The percentage variation was fetida and Allolobophora chlorotica after dermal exposure to calculated according to the formula: (treated − control/control) × metals for three days. The responses observed in L. terrestris 100. following exposure to 45 mg/kg of soil for copper sulphate or 1 mg/kg of soil for cadmium chloride respectively were almost Coelomic fluid Whole organism the same, suggesting the higher sensitivity of L. terrestris to 99.0 ± 6.5 106.4 ± 9.4 CuSO4 cadmium with respect to copper. 80.4 ± 9.4 93.8 ± 11.5 CdCl2 In order to compare the Mt response in the coelomic 149.0 ± 6.5 130.5 ± 7.0 CdCl2 + CuSO4 fluid with the most utilized destructive measure of the Mt Data are reported as mean ± S.E.M. concentration in the whole organism, the two determinations were performed in parallel on the same specimens. The obtained results demonstrated that the Mt response in the tools in soil monitoring and assessment as an early warning coelomic fluid perfectly reflects the Mt response in the whole of adverse ecological effects [52, 53]. This has increased the organisms both qualitatively and quantitatively. interest in the study of earthworm biological responses to These findings indicate the suitability of Mt determi- pollutants. Earthworm physiological fluids, such as coelomic nation in L. terrestris coelomic fluid as nondestructive fluids and blood, offer an interesting field for exploitation biomarker for application to metal polluted soil monitoring of novel sensitive nondestructive biomarkers. In long-term and assessment. biomonitoring of native populations inhabiting polluted TheuseofcoelomicfluidforMtdetectioninLumbri- areas, the use of nondestructive biomarkers as markers of cus terrestris offers many advantages with respect to the condition becomes of relevance. commonly used whole-body measure, particularly for in- In the present study, we assessed the suitability of the field application. In fact, coelomic fluid can be easily sam- Mt determination in the coelomic fluid of the earthwork L. pled in native animals in field without compromising the terrestris as sensitive biomarker of exposure to heavy metals. animal survival, avoiding the stressful translocation to the In physiological conditions L. terrestris coelomic fluid laboratory. Moreover, considering the recent development of showed a significant basal concentration of Mt. It is known novel biomarkers in the earthworm coelomic fluids [22], the that earthworm coelomocyte population is comprised of standardization of the Mt determination contributes to the amoebocytes originating from mesenchymal lining of the development of multiple assays on this important earthworm coelom [54] and eleocytes (chloragocytes) sloughed into tissue. BioMed Research International 5

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Research Article Reduced Bone and Body Mass in Young Male Rats Exposed to Lead

Fellipe Augusto Tocchini de Figueiredo,1 Raquel Fernanda Gerlach,1 Márcia Andreia Mesquita Silva da Veiga,2 Flavio Venancio Nakadi,2 Junia Ramos,1 Erika Reiko Kawakita,1 Carolina de Souza Guerra,1 and João Paulo Mardegan Issa1

1 Faculdade de Odontologia de Ribeirao Preto, Universidade de Sao Paulo, Avenida do Cafe S/N, 14040-904 Ribeirao Preto, SP, Brazil 2 Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto, Universidade de Sao Paulo, Avenida do Cafe S/N, 14040-904 Ribeirao Preto, SP, Brazil

Correspondence should be addressed to Joao˜ Paulo Mardegan Issa; [email protected]

Received 16 January 2014; Revised 10 February 2014; Accepted 18 February 2014; Published 30 March 2014

Academic Editor: Anilava Kaviraj

Copyright © 2014 Fellipe Augusto Tocchini de Figueiredo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The aim of this study was to see whether there would be differences in whole blood versus tibia lead concentrations over timein growing rats prenatally. Lead was given in the drinking water at 30 mg/L from the time the dams were pregnant until offspring was 28- or 60-day-old. Concentrations of lead were measured in whole blood and in tibia after 28 (28D) and 60 days (60D) in control (C) and in lead-exposed animals (Pb). Lead measurements were made by GF-AAS. There was no significant difference (𝑃 > 0.05) in the concentration of whole blood lead between Pb-28D (8.0 ± 1.1 𝜇g/dL) and Pb-60D (7.2 ± 0.89 𝜇g/dL), while both significantly varied (𝑃 < 0.01) from controls (0.2 𝜇g/dL). Bone lead concentrations significantly varied between the Pb-28D (8.02 ± 1.12 𝜇g/g) and the Pb-60D (43.3 ± 13.26 𝜇g/g) lead-exposed groups (𝑃 < 0.01), while those exposed groups were also significantly higher (𝑃 < 0.0001) than the 28D and 60D control groups (Pb < 1 𝜇g/g). The Pb-60D group showed a 25% decrease in tibia mass as compared to the respective control. The five times higher amount of lead found in the bone of older animals (Pb-60D versus Pb- 28D), which reinforces the importance of using bone lead as an exposure biomarker.

1. Introduction lead levels, which do not cause symptoms of acute poisoning, are associated with cognitive and neurological disorders [4]. Lead is a highly toxic metal and has a wide distribution for its Bone is considered the best marker for lead exposure [5], great usefulness. Several studies have linked health problems but it cannot be used for lead determinations in humans with high concentrations of industrial minerals, elements yet. Nonetheless, in animals bone lead determinations are coming from contaminated rivers, and carelessness of the relatively easy to perform so far as sacrifice of animals is con- public service of water [1, 2]. According to a US study from cerned.Thedeterminationsofleadinbonedohavesomeana- 2003, data provided by the 1999-2000 NHANES study esti- lytical challenges, for instance, the lack of reference materials mated that approximately 0.45 million children under 6 years forleadinbone.Ontheotherhand,boneleaddeterminations of age showed whole blood lead levels exceeding or equal to also have some advantages—even from an analytical point 10 𝜇g/dL Pb, which were considered high blood lead levels for of view. For instance, when planning lead determinations by children [3]. Even though lead levels have decreased in many ion-coupled plasma mass spectrometry, the bone acid extract countries in the last decades, it is now known that even low is more convenient than the analysis of whole blood. 2 BioMed Research International

∘ We have recently exposed female rats to both lead and completely freed of soft tissue, maintained at 40 Cfor48 fluoride, before they got pregnant, and we have determined hours, and weighed on an analytical balance. The level of lead and fluoride in the female offspring when these animals significance for the differences accepted in this study was were 81-day-old [6].Forthatstudy,wecomparedleadin 𝑃 < 0.05. different mineralized tissues and lead in whole bone, and there was high consistency among measurements. When 2.2. Equipment and Materials. Lead was determined by discussing those data, we have found only a few studies in Graphite Furnace Atomic Absorption Spectrometry (GF- theliteraturethatshowedtheamountofleadstoredinbone AAS) on AA600 model (Perkin Elmer, USA). The inert when animals were chronically exposed to low lead levels, protective gas purge was argon with 99.999% of purity (White in comparison to dozens of studies that determined lead in Martins, Sao˜ Paulo, Brazil). All glassware and plastics used whole blood. were properly cleaned with nitric acid to avoid contamina- Sinceexposuretorelativelylowleadlevelsisknownto tion. For this study, the following limits of detection (LOD) have consequences in many physiological and psychological and quantification (LOQ) were obtained: LOD: 0.4 mg/L and aspects for both humans and animals, we have wondered LOQ: 1.2 mg/L. whether the lead determinations in whole blood and in bone would agree after a short exposure time (28 and 60 days with 2.3. Whole Blood Sample Preparation. Blood was collected exposure starting at pregnancy) in young rats exposed to low in plastic syringes that were previously decontaminated with lead levels. To our knowledge, the comparison of whole blood nitric acid. Needles, syringe, and metal free falcon were rinsed versus bone lead levels has not been made in animals exposed with sodium heparin 5000 mg/ml, and, after collection prepa- torelativelylowleadlevels,suchasthe30mg/Ldose(given ration,thebloodwascollected(anaverageof1ml).Thereafter ∘ in the drinking water) used in this study. blood samples were stored at −20 C,beinganalyzedbyGF- Doing a literature search using the words “rat,” “lead” AAS. (which actually introduces many unrelated studies), and “bone,”the number of published studies that determined lead 2.4. Whole Blood Lead Determination. Lead was deter- in whole blood and bone is not large, and those studies are mined in whole blood following the method described by presented in Table 1. In most of those studies, lead is provided Parsons and Slavin [7], where the modifier comprises in the drinking water, and the concentrations of lead given 0.2% NH4H2PO4,0.5%TritonX-100,and0.2%HNO3,which vary from 50 to 1000 mg/L. was used as the sample diluent. NIST 955c was used as a To our knowledge, the comparison of whole blood versus standard with known amount of lead after every 15 samples. bone lead levels has not been made in animals exposed to Samples and standard, prepared as described above, were diluted 1 : 10 into 1 ml cups and placed in the sampler AS relativelylowleadlevels,suchasthe30mg/Ldoseusedin 𝜇 this study. Therefore, this study aimed at determining lead in 800 (Perkin Elmer, USA). Twelve l aliquots were then whole blood and in tibia bone of 28- and 60-day-old male automaticallypipettedintotheZeeman-typegraphitetube. rats, as well as assessing growth by measuring body weight and tibia weight at 60 days. 3. Results and Discussion The lead concentrations found in whole blood are shown 2. Materials and Methods in Table 2,being1.2𝜇g/dL (±0.7) in the C-28D, 1.6 𝜇g/dL (±1.5) in the C-60D, 8.0 𝜇g/dL (±1.1) in the group Pb-28D, 2.1. Animals. This study was approved by the Ethics Com- and 7.2 𝜇g/dL (±0.89) in the group Pb-60D, with significant mittee for Experimentation with Animals, University of Sao˜ differences when groups of animals exposed to lead were Paulo, Campus of Ribeirao˜ Preto, Brazil, under protocol compared with the controls (𝑃 < 0.001), but with no signif- number 07.1.346.53.3 and complies with the international icant differences found between the groups exposed to lead guidelines for the use of animals in experimentation. Forty- for 28 and 60 days. nine male Wistar rats were used, divided in the following In contrast, the lead concentrations found in the bone groups: 28-day-old controls (C-28D, 𝑛=10)and28-day- samples (Figure 1)were8.2𝜇g/g (±1.1) in the Pb-28D group old lead-exposed rats (Pb-28D, 𝑛=10) and 60-day-old and 43.3 𝜇g/g (±13.2) in the Pb-60D (𝑃 < 0.0001), while the controls (C-60D, 𝑛=12) and 60-day-old lead-exposed C-28D group showed 1.5 𝜇g(±1.1 𝜇g/g) and the C-60D group rats (Pb-60D, 𝑛=17). Water was provided ad libitum. showed 2.3 𝜇g(±1.8 𝜇g/g) (the difference for those groups Animals exposed to lead received water containing lead versus the respective lead-exposed groups was significant at since birth up to 28 or 60 days. Lead was provided in the 𝑃 < 0.001 and 𝑃 < 0.0001, resp.). The number of samples for drinking water at 30 mg/L of lead in the form of lead acetate those groups was C-28D (𝑛=10), C-60D (𝑛=9), Pb-28D (CH3COO(Pb)2.3H2O) per 1 liter of deionized water. At the (𝑛=12), and Pb-60D (𝑛=12). end of the experiment, the animals were anesthetized by In this study, we also compared whether this relatively low ketamine 100 mg/kg and xylazine 10 mg/kg intraperitoneally. dose of lead had consequences for bone mass development One milliliter of whole blood was collected via cardiac and animal’s weight gain (body weight). Figure 2 shows puncture with a heparinized syringe, whose preparation body weight of 60-day-old animals in grams (g). Sample is described below. The animals were then sacrificed by number was 𝑛=12in the 60-day control group (C-60D) anesthetic overdose, and tibiae were collected. Tibiae were and 𝑛=16in the Pb-60D (𝑛=16). Data distribution BioMed Research International 3 Main outcome worsened condition ossification Vit. E/Pb hippocampus damage lead/hypoxia group Lead interference with chondrogenesis is more formation in growing rats delayed growth and altered pronounced than with bone Ratsexposedtoleadshowed Chronic lead exposure causes increases between 2 and 3x the similar to osteoporotic diseases Coexposure to lead and fluoride Lead intoxication induces effects bone properties was found in the such as dentin, enamel, and bone. amount of lead in calcified tissues, Negative effect on bone growth and g/dL) 𝜇 6.8 ± 1.7 14.2 ± 2.6 21-day group—50 49-day group—93.4 whole blood ( Lead concentration in g/g) 𝜇 — 22.6 ± 8.5 76.7 ± 11.0 in bone ( 700.03 ± 78.52 43.78 ± 7.20 632.29 ± 94.23 48.28 ± 8.54 Lead concentration 18 weeks) 30.99 9.16 12 weeks) 12 weeks) water (days) ∼ ∼ ∼ 84 ( 84 ( Exposure period 20 (gestation time) — — 21 days of treatment exposure to unleaded followed by 27 days of groups +Vit. E control 30 mg/L 30 mg/L 250 mg/L 250 mg/L 500 mg/L 500 mg/L Treatment 1000 mg/L Table 1: Summary of rat studies with lead determinations in whole blood and bone. Gender 81 Female — Female 50 mg/L 126 ( 70 Female 111 Female 1000 mg/L 91 110 Male 500 mg/L 84 32.23 to 93.43 11.01 to 23.21 20 in uterus Fetuses ended (days) Age when study Pb Special Hypoxic condition Normoxic 111 Female 1000 mg/L 91 Pb + fluoride ] ] ] ] ] ] 8 9 10 11 12 6 Authors [ [ [ [ [ [ 4 BioMed Research International

Table 2: Lead concentrations found in whole blood of controls (C- 800 28D and C-60D) and animals exposed to lead (30 mg/L) in the drinking water since pregnancy to day 28 (Pb-28D) and day 60 of ∗ life (Pb-60D). 600

C-28D C-60D Pb-28D Pb-60D 𝜇 ∗ ∗ 400 Lead ( g/dL) in 1.2 (±0.7) 1.6 (±1.5) 8.0 (±1.1) 7. 2 ( ±0.89) whole blood Weight (g) Weight ∗ 𝑃 < 0.001 for differences with age-matched controls. 200

60 ∗ 0

C--60D Pb 60D 40 Groups

g/g) Figure 2: Body weight (g) of control and Pb-exposed rats after 60 𝜇 days.Bodyweightat60days.

Lead ( 20 1.5

0 ∗ 1.0 C--28DC60DPb --28DPb60D Groups

Figure 1: Lead in tibia bone after 28 and 60 days of exposure to lead. (g) Weight Lead concentrations in tibias of 28- and 60-day-old rats. 0.5

was normal. Unpaired Student’s t-test shows a statistically 0.0 ∗ significant difference ( 𝑃 = 0.0006) between controls C--60D Pb 60D and lead-exposed animals. In Figure 3,tibiaweightwas Groups expressed in grams (g), and sample number was 𝑛=12 in the C-60D group, and 𝑛=17in the Pb-60D. Differences Figure 3: Weight (g) of tibia of control and Pb-exposed rat after 60 were statistically significant at 𝑃 = 0.0004 (Mann-Whitney days. Tibia weight of rat aged 60 days. test). Adecreaseinbodyweight(orbodymass)ofthePb-60D animals was observed (𝑃 < 0.01 for comparison with age- Regardingtheeffectofleadonadecreaseinbonemass,there matched controls and with the Pb-28D animals). The weight have been already descriptions of lead leading to decreased decrease was 17% of the C-60D animals’ weight (Figure 2). bone mass, but with much higher doses. Female rats with 21 Tibia weight was also decreased in the Pb-60D group, days of age were exposed to 1000 mg/L of lead acetate for 90 alargerdecrease(∼25%) when the Pb-60D was compared days, showing an average of 632.29 mg/g (±94.23) of lead in with the age-matched control (𝑃 < 0.0004)(Figure 3). This bone ash [8]. In another study, male rats were exposed to suggests that the changes in bone biology are to some extent 500 mg/L of lead acetate during 12 weeks, reaching 58.16 ± independent of the metabolic changes that affect body mass. 15.57 𝜇g/gofleadinbone[9]. Results of a decreased total body mass are in accordance with Many inconsistencies found in the concentration of lead other studies on the effects of lead in rodents. The study of found in the literature are probably related to the fact that Conti et al. (2012) [8]showeda10%decreaseinbodymass wholebloodisthemostwidelyusedbiomarkerofexposure. of lead-exposed animals in comparison to controls, but lead When different animal studies are compared and only whole doses in that study were much higher, as described below. bloodleadismeasuredanddisplayed,resultswilllikelynotbe Delayed growth with decreased body mass for age was consistentwiththetrueexposure.Asknownforquitesome also observed with doses of 50 mg/L and 250 mg/L of lead time now, lead in bones is the best direct measure related to in the drinking water for 270 and 180 days, respectively [13]. the degree of exposure to lead [15, 16]. In a study on the effect The timing between 60 and 90 days has been described to of lead on rat fetuses [10], significant differences were found be particularly important for lead incorporation, since this in size and body weight between the following groups that time window is characterized by rapid growth in rodents [14]. were exposed to lead acetate in the mother’s drinking water This is the probable reason why we only observed differences 0.250 mg/L and 500 mg/L. The authors concluded that there in total body mass and tibia mass in the Pb-60D group. was an inverse relationship between the amount of lead to BioMed Research International 5 which the fetuses were exposed and the size and weight of the [3] P.A.Meyer,T.Pivetz,T.A.Dignam,D.M.Homa,J.Schoonover, animals, and that lead interferes with normal development and D. Brody, “Surveillance for elevated blood lead levels among since the very early stages, leading to a delay in bone devel- children—United States, 1997–2001,” MMWR Surveillance Sum- opment. It must be mentioned that in the real environment maries,vol.52,no.10,pp.1–21,2003. coexposures are certainly not an exception but are many [4] CDC, Low Level Lead Exposure Harms Children: A Renewed times the case. And some toxicants are bone-seeking agents, Call for Primary Prevention, CDC, 2012. such as lead, and could also be easily determined in bone. A [5]D.J.Bellis,A.C.Todd,andP.J.Parsons,“Aninterlaboratory case in point is lead and fluoride, both found in industrial comparison of bone lead measurements via K-shell X-ray areas, and whose coexposures increase by 2-3 times the fluorescence spectrometry: validation against inductively cou- concentrations of lead in calcified tissues and whole blood, pled plasma mass spectrometry,” JournalofAnalyticalAtomic not changing the fluoride concentrations. Interestingly, the Spectrometry,vol.27,no.4,pp.595–603,2012. enamel defects normally found in rats under conditions of [6]R.M.Sawan,G.A.S.Leite,M.C.P.Saraiva,F.Barbosa,J. high fluoride exposure are much worsened in the presence of E. Tanus-Santos, and R. F. Gerlach, “Fluoride increases lead concentrations in whole blood and in calcified tissues from lead [17]. Therefore, the careful measurement of lead in bone lead-exposed rats,” Toxicology,vol.271,no.1-2,pp.21–26,2010. is not only very important for the better understanding of [7]P.J.ParsonsandW.Slavin,“ArapidZeemangraphitefurnace theleadamounttowhichtheanimalwasexposedbutisalso atomic absorption spectrometric method for the determination important to understand possible outcomes such as function of lead in blood,” Spectrochimica Acta B: Atomic Spectroscopy, of organs and cells. vol.48,no.6-7,pp.925–939,1993. Sinceinanimalstheboneiseasilyobtained,thisstudy [8]M.I.Conti,A.R.Terrizzi,C.M.Leeetal.,“Effectsof supplies the details of how to obtain accurate measures and lead exposure on growth and bone biology in growing rats supports the need to use bone as the best marker of internal exposed to simulated high altitude,” Bulletin of Environmental dose. Contamination and Toxicology, vol. 88, no. 6, pp. 1033–1037, 2012. 4. Conclusions [9]H.W.Li,J.G.Deng,Z.C.Duetal.,“Protectiveeffectsof mangiferin in subchronic developmental lead-exposed rats,” The first finding of this study shows a 5 times increase inthe Biological Trace Element Research ,vol.152,no.2,pp.233–242, concentration of lead in the bone found in the 60-day-old 2013. rats in comparison with the 28-day-old rats, with no changes [10] H. A. Saleh, G. A. El-Aziz, M. M. El-Fark, and M. El-Gohary, found in the whole blood lead concentrations of these ani- “Effect of maternal lead exposure on craniofacial ossification mals. This finding lends additional support to the importance in rat fetuses and the role of antioxidant therapy,” Anatomia ofboneasatissuethatstoresleadduringtheremodeling Histologia Embryologia,vol.38,no.5,pp.392–399,2009. process characteristic of growth. Lead determinations in bone [11] J. D. Hamilton and E. J. O’Flaherty, “Effects of lead exposure should be a requirement when growing animals exposed on skeletal development in rats,” Fundamental and Applied to lead are analyzed. So far, most studies in animals only Toxicology,vol.22,no.4,pp.594–604,1994. determine lead in whole blood as a way to characterize [12] E. E. Beier, J. R. Maher, T. J. Sheu et al., “Heavy metal lead the exposure to lead. This study shows differences in lead exposure, osteoporotic-like phenotype in an animal model, and concentrations in bone and whole blood in animals exposed depression of Wnt signaling,” Environmental Health Perspec- to lead for 28 and 60 days. Finally the differences in body tives,vol.121,no.1,pp.97–104,2013. weight in 60-day animals were also presented. [13] L. D. Grant, C. A. Kimmel, and G. L. West, “Chronic low-level lead toxicity in the rat. II. Effects on postnatal physical and behavioral development,” Toxicology and Applied Pharmacol- Conflict of Interests ogy,vol.56,no.1,pp.42–58,1980. The authors declare that there is no conflict of interests [14]M.I.Luster,R.E.Faith,andC.A.Kimmel,“Depressionof regarding the publication of this paper. humoral immunity in rats following chronic developmental lead exposure,” Journal of Environmental Pathology and Toxicol- ogy, vol. 1, no. 4, pp. 397–402, 1978. Acknowledgments [15]E.J.O’Flaherty,“PhysiologicallybasedmodelsforBone- This study was supported by FAPESP (the State of Sao Paulo Seeking elements. V. Lead absorption and disposition in childhood,” Toxicology and Applied Pharmacology,vol.131,no.2,pp. Research Foundation) and CNPQ (the Brazilian Research 297–308, 1995. Council). [16] K. R. Mahaffey, R. Goyer, and J. K. Haseman, “Dose response to lead ingestion in rats fed low dietary calcium,” Journal of References Laboratory and Clinical Medicine,vol.82,no.1,pp.92–100,1973. [1] E. K. Atibu, N. Devarajan, F. Thevenon et al., “Concentra- [17] G. A. S. Leite, R. M. M. Sawan, J. M. Teofilo,I.M.Porto,F.B.´ tion of metals in surface water and sediment of Luilu and Sousa, and R. F. Gerlach, “Exposure to lead exacerbates dental Musonoie Rivers, Kolwezi-Katanga, Democratic Republic of fluorosis,” Archives of Oral Biology,vol.56,no.7,pp.695–702, Congo,” Applied Geochemistry,vol.39,pp.26–32,2013. 2011. [2] EPA, Summary of The Reduction of Lead In Drinking Water Act and Frequently Asked Questions E.U.S.E.P. Agency, Editor 2013,EPA:EPAwebsite. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 945963, 9 pages http://dx.doi.org/10.1155/2014/945963

Research Article Mercury Speciation in Hair of Children in Three Communities of the Amazon, Brazil

Jamile Salim Marinho,1,2 Marcelo Oliveira Lima,1 Elisabeth Conceição de Oliveira Santos,1 Iracina Maura de Jesus,1 Maria da Conceição N. Pinheiro,2 Cláudio Nahum Alves,2 and Regina Celi Sarkis Muller2

1 Evandro Chagas Institute, 316 Km, 07 Road, Levilandia,ˆ 67030-000 Ananindeua, PA, Brazil 2 Federal University of Para,´ 01 Augusto Correaˆ Street, Guama,´ 66075-110 Belem,´ PA, Brazil

Correspondence should be addressed to Jamile Salim Marinho; [email protected]

Received 21 October 2013; Accepted 27 January 2014; Published 11 March 2014

Academic Editor: Erhan Unlu

Copyright © 2014 Jamile Salim Marinho et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Children from riverside communities located downstream of gold mining areas may be chronically exposed to relatively high levels of MeHg through the consumption of fish of this region. The objective of this study was to evaluate and compare levels of THg and MeHg in hair of children less than 12 years in communities near mines in the municipality of Itaituba and in communities far from prospecting areas in the city of Abaetetuba. The communities of Itaituba (Barreiras and Sao˜ Luiz do Tapajos)´ had THg mean levels −1 −1 of 5.64±5.55 𝜇g⋅g (0.43–27.82) and 11.41±7.16 𝜇g.g (1.08–28.17), respectively, and an average count of MeHg relative to THg of −1 92.20% and 90.27%, respectively. In the Maranhao˜ community, the THg average concentrations results were 2.27±2.11 𝜇g⋅g (0.13– 9.54) and the average values were 93.17% for MeHg. Children of Itaituba had average levels of mercury above the limit established −1 by the World Health Organization (10 𝜇g⋅g ) and the strong correlation coefficient between the communities (𝑅 = 0.968 and 𝑃 = 0.0001) suggests the hair as an excellent biomarker of human exposure to organic mercury in riverside populations of the Tapajos,´ which has the intake of fish daily as main source of protein dietary.

1. Introduction Previous studies indicate that the bioaccumulation of Hg is harmful to human being, with increments of risk In the Amazon, there are records that the industrial and in vulnerable groups such as that, for example, children artisanal mining of gold and, possibly, other natural and can receive burden of Hg from mothers infected during anthropogenic sources, have increased the concentration pregnancy [11]. By the experimental studies, there are reports of mercury (Hg) in the environment, resulting, through on the harmful effects of Hg to the development of human the trophic chain, in process of bioaccumulation in fish biological systems, with evidence of alterations on the car- consumed by riverside communities. Previous studies related diovascular, neurological, immune, and respiratory systems; elevated levels of Hg in fish consumption in these communi- effects that may persist for entire life12 [ , 13]. ties [1–5]. Children are more vulnerable to mercury exposure The mercury is toxic in its various physical and chemical [14]. Therefore, children from riverside communities located forms, being identified as one of the most dangerous pol- downstream from gold mining areas, may be chronically lutants in the environment [6–8]. This toxicity is amplified exposed to relatively high levels of MeHg by fish consump- in the organomercury forms, particularly methylmercury tion [15]. From previous studies, it is estimated that MeHg (MeHg), a substance of high volatility and easily absorbed represents approximately 70–90% of the total mercury (THg) across biological membranes, with records of efficient bioac- consumed by the fish [15–17]. Thus, in these communities, the cumulation and long-term attachment to the tissues [9, 10]. environmental monitoring of THg and MeHg levels in hair 2 BioMed Research International

∘ 󳰀 󳰀󳰀 ∘ 󳰀 󳰀󳰀 56 0 0 W 50 50 0 W

W E Pará

S Brazil

Maranhão

∘ 󳰀 󳰀󳰀 2 10 0 S Abaetetuba City

Barreiras

São Luiz do Tapajós

∘ 󳰀 󳰀󳰀 7 20 0 S Itaituba City

(km) 200100 0 200

South America Communities Brazil Drainage Pará State Sampling stations

Figure 1: Study area encompassing the communities of Barreiras, Sao˜ Luiz do Tapajos,´ and Maranhao.˜

associated with the epidemiological information represents River, near the town of Itaituba in southwestern Para.´ In this important steps to ensure the quality of life of these popula- region, considered as the Hg exposure, there are records of tions. more than four decades of gold mining [18]. The Maranhao˜ ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 The objective of this study was to perform a quantitative community (Lat (S): 1 39 56 and Long (W): 48 49 10 ), with comparison of THg and MeHg levels in children younger approximately 500 inhabitants, is located on the banks of the than 12 years on populations in near and distant prospecting Guajara´ of Beja, a tributary of the right bank of the ParaRiver,´ areas of the Amazon, associating these results with epidemi- inthenortheasternregionoftheStateofPara,´ an area where ological data obtained through interviews. there are no records of exposure to Hg.

2. Material and Methods 2.2. Epidemiology. Individuals who reside in the communi- 2.1. Study Area. For this study, children were selected in three ties selected for this study have similar lifestyles, highlighting Amazonian communities: Sao˜ Luiz do Tapajos,´ Barreiras, and the continuous intake of fish as the main protein source. Maranhao˜ (Figure 1). The localities of Sao˜ Luiz do Tapajos´ However, these populations are differentiated based on the ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 (Lat (S): 4 28 11 and Long (W): 56 15 20 ) and Barreiras influences exerted by gold mining activities whose environ- ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 (Lat (S): 4 5 34 and Long (W): 55 41 11 ), with 460 and 740 mentalexposuretoHgishigherinthosecommunitieslocated people,respectively,arelocatedonthebanksoftheTapajos´ on the banks of the Tapajos´ River. BioMed Research International 3

Barreiras Maranhão 30 30 ∗

∗ ) ) 20 −1 20 −1 g · g · g g (𝜇 (𝜇

10 THg ∗ THg 10 ∗ ∗

0 0

Female Male Female Male Gender Gender Gender Gender Female Female Male Male (a) (b)

São Luiz do Tapajós 30

) 20 −1 g · g (𝜇

10 THg

Female Male Gender Gender Female Male (c)

Figure 2: Box plot average levels of THg by gender.

The recruitment of individuals was performed by invita- one (1) cm from the scalp of children with stainless steel tion in the communities. The research project was explained dissecting scissors, and stored in paper envelopes properly to the people responsible for these children, who gave permis- identified. These samples were then sent to the Division of sion by signing the Free and Clarified Consent Term (FCCT). Mercury in the Laboratory of Toxicology, Section of Envi- Between2007and2009,atotalof195childrenaged0–12years ronment (SAMAM) of the Evandro Chagas Institute (ECI), oldwereselectedtoparticipateinthisstudy.Ofthisgroup, located in the town of Ananindeua, State of Para,´ Brazil. In the 37 children are residents of the community of Barreiras, 40 laboratory, the samples were washed with neutral detergent are from Sao˜ Luiz do Tapajos,´ and 118 are from the Maranhao˜ solution (Extran-Merck MA 0-alkaline) (diluted 100 times) community. to eliminate possible exogenous contamination, deionized Epidemiological data, such as age, sex, and length of stay water (Milli-Q) to remove sediments, and acetone (Merck) to attheplace,wereobtainedthroughinterviewswithparen- enable water evaporation, reducing the humidification. After tal or legal guardian monitoring. Approval for the study pro- the washing step and subsequent drying, the samples were cedures was obtained by the Ethics Committee in Research transferred to 20 mL amber vials and perforated with stainless of the Evandro Chagas Institute, by registration number steel dissecting scissors for greater homogenization [19]. 0013/2008, approved on 09/06/2009. 2.4. Total Mercury (THg). In analysis of THg in hair, the 2.3. Sampling and Sample Management. The hair samples methodology developed by [19]wasappliedthatinvolves were collected from the head occipital area, approximately the steps of wet digestion, reduction with stannous chloride 4 BioMed Research International

30 30

N = 37; R = 0,993; P = 0,001 ) ) 20 20 −1 −1 g g · · N = 118; R = 0,990 ; P = 0,0001 g g (𝜇 (𝜇 THg THg 10 10

0 0

0 6 12 18 24 0 6 12 18 24 −1 −1 MeHg (𝜇g·g ) MeHg (𝜇g·g ) Communities Communities Barreiras Maranhão (a) (b) 30 N=40; R = 0,933 ; P = 0,0001

) 20 −1 g · g (𝜇

THg 10

0 6 12 18 24 −1 MeHg (𝜇g·g ) Communities São Luiz do Tapajós (c)

Figure 3: Correlation between levels of THg and MeHg in Barreiras, Sao˜ Luiz do Tapajos,´ and Maranhao.˜ Confidence interval is 95%.

(SnCl2), and quantification by cold vapor atomic absorption absorbance of 253.7 mm. The reading is performed within spectrometry (CVAAS). In this method, about 0.01 g of each oneminuteandcanbemeasureduntil0.1ngofHgwithhigh sample was weighed in a 50 mL volumetric flask (Pyrex), and accuracy [19]. 1 mL of deionized water was added plus 2 mL of HNO3 + IntheanalysisofTHg,calibrationcurveswereused:0,20, HClO4 (1 : 1) and 5 mL of H2SO4 andheatedfor30minutes ∘ 50, and 100 ng of Hg prepared starting from standard solution in a hot plate at a temperature of 230 C. After cooling, the of 0.1 ppm MeHg-cysteine. solution was measured with deionized water obtained by the water purification system (Millipore, Academic Milli-Q 2.5. Methylmercury (MeHg). IntheanalysisofMeHginhair, model) to a final volume of 50 mL. the simplified method proposed by Akagi19 [ ]wasapplied, After digestion, 5 mL of sample was added in the reactor which involves leaching with hydrochloric acid, extraction vial of the Hg analyzer, Automatic Mercury Analyzer Hg- with toluene, and quantification by gas chromatography with 201 (Sanso Company), with the addition of 1 mL of stannous electron capture detector (GC-ECD). In this method, approx- 2+ 0 chloride (SnCl2-10%) to reduce Hg to Hg .Then,theHg imately 0.02 g of each sample was weighed and transferred vapor generated is conducted into a flask containing NaOH to a round bottom centrifuge tube of 10 mL. Subsequently, at 5 N, that neutralizes the acid vapors, and a flask containing 2 drops of ethanol and a small amount of cotton to moisten an ice bath, which condenses water vapor. Thereupon, the Hg and cover the sample were added, respectively. For leaching vapors are transported to the photoabsorption cell to measure from MeHg, samples were immersed in 3 mL of HCl at 2 N BioMed Research International 5

100,00 in other words, in the research, there was the prevalence of 90,00 children at school phase. Regarding gender, in the Barreiras, 80,00 the male children (54%) are of larger numbers than the 70,00 females (46%). On the other hand, in Sao˜ Luiz do Tapajos,´ this 60,00 predominance was higher in female children (60%) than male 50,00 ones (40%). This inversion was also observed in Maranhao˜ 40,00 community, in which the number of female children (52.5%) 30,00 was also higher than male children (47.5%). 20,00 Children evaluated (%) evaluated Children Children from Barreiras and Sao˜ Luiz do Tapajos´ were 10,00 also divided into another two groups, in relation to paternal 0,00 São Luiz do Tapajós Barreiras Maranhão occupation, in order to analyze the importance of familial Communities evaluated influences on exposure to Hg: (y) children whose fathers were −1 fisherman and (x) children whose fathers were not fisherman. Until the NL ( ≤2𝜇g·g ) −1 In Barreiras, the children (x) are of higher amounts (60%) Above the NL (>2 𝜇g·g ) −1 than the children (y) (40%). In contrast, in Sao˜ Luiz do Above the LBT (>10 𝜇g·g ) Tapajos,´ this profile was higher in children (y) (55%) than Figure 4: Percentage number of children with levels of THg until in (x) (45%). As regards the duration of breast-feeding, in normality limit (NL), above the normality limit (NL) and above Barreiras, the average time was about 6 months, while, in Sao˜ limits of biological tolerance (LBT). Luiz do Tapajos,´ this mean increased to 10 months.

∘ 3.3. Results of HG. The average results of THg and MeHg, and heated at 100 Cfor5minutes.Aftercooling,thesamples standard deviation, and the mean percentage of MeHg in were centrifuged at 1200 rpm for 3 minutes. Then, 1 mL of children of the communities investigated are presented in −1 the supernatant was transferred to a conical tube centrifuge Table 2. The highest average levels of THg (11.41 𝜇g⋅g )and −1 of 10 mL and 2 mL of toluene was added with stirring for MeHg (10.30 𝜇g⋅g ) in hair of children were found in the 3 minutes to extract the MeHg in the phase of HCl to the community of Sao˜ Luiz do Tapajos.´ These values are twice phaseoftoluene.Afterdigestion,2𝜇Lofsamplewasinjected −1 −1 the average of THg (5.64 𝜇g⋅g ) and MeHg (5.20 𝜇g⋅g ) into the GC-ECD (Yanaco), series G-6800. Glass column of × found in Barreiras and four times higher than the results of 3.0 mm 0.75 m was used and packed with 10% KOCl-Hg on 𝜇 ⋅ −1 𝜇 ⋅ −1 Chromosorb W (AW-DMCS, 60-80, Ltd., Kyoto, Japan). THg (2.27 g g ) and MeHg (2.11 g g )intheMaranhao˜ community. These results confirm previous comparative studies with riverside populations of areas exposed and not 2.6. Quality Control. As quality criteria for the analysis of THg and MeHg, Certified Reference Material (CRM) IAEA exposed to Hg, which detect higher levels of THg in the 86 and IAEA 085 were used. community of Sao˜ Luiz do Tapajos´ [15, 18, 22, 23], possibly because of higher proximity of the community with the gold mining area of the Tapajos River Basin [5]. 2.7.Statistics. For statistical analyses, the data were submitted to the program Microsoft Excel 2007 and MINITAB 14 Regarding gender, in the communities of Barreiras and 𝑃≤ Sao˜ Luiz do Tapajos,´ the female children had average levels software20 [ ]. For results with statistical significance, 𝜇 ⋅ −1 𝜇 ⋅ −1 0.05 was used. In correlation analyses, 𝑅=1for perfect of THg higher than 4.27 g g (2.29–4.60) and 5.12 g g 𝑅 ±0.9 𝑅 (6.12–19.41), relatively higher than the levels found in correlation; next to ,forstrongcorrelation; next to −1 ±0.1 𝑅=0 male children, which values were 4.1 𝜇g⋅g (2.12–7.61) and ,forweakcorrelation;and ,forzerocorrelation −1 were considered [21]. 7. 7 3 𝜇g⋅g (4.3–13.2), respectively. In the Maranhao˜ community, the THg highest mean levels were found in males, −1 1.78 𝜇g⋅g (1.04–2.13), compared to THg levels found in 3. Results and Discussion −1 females, 1.50 𝜇g⋅g (1.01–2.61). THg mean levels by gender 3.1. Quality Control. The average results of the analysis of are shown in Figure 2. In contrast, studies have shown higher THg and MeHg in the samples from CRM and their reference levels of mercury in male children from the communities of values are shown in Table 1. The great analytical recovery Barreiras and Sao˜ Luiz dos Tapajos´ [18]. Also, the occurrence forTHg(99.21%)andMeHg(94.35%)showedthatthe of outliers in the community of Barreiras is noteworthy, methodology was effective. where there was registration of two children; both are sons −1 of fisherman father, with levels of 21.96 𝜇g⋅g (female) and −1 3.2. Epidemiology. From the epidemiological information, 27.82 𝜇g⋅g (male). In this case, the female child deserves the children were grouped into different age groups according more detailed attention, whose epidemiological surveys show to the stages of life [18]: (A) breast-feeding (0-1 years), (B) delays in the development of the motor system. prekindergarten or preschool (2–6 years), and (C) childhood In the analysis of correlation, a strong correlation between or school (7–12 years). In the three communities, despite the the levels of THg and MeHg in hair of children of Barreiras different sample sizes, a similar pattern of distribution of (𝑅 = 0.99 and 𝑃 = 0.0001), Sao˜ Luiz do Tapajos´ (𝑅= individuals between stages of life evaluated was observed; 0.93 and 𝑃 = 0.0001), and Maranhao˜ (𝑅 = 0.99 and 6 BioMed Research International

Barreiras Maranhão 30 30 ∗

∗ ) 20 ) 20 −1 −1 g·g g·g 𝜇 𝜇 THg ( 10 THg ( 10 ∗ ∗ ∗ ∗ ∗

0 0

A (0-1 years) B (2–6 years) C (7–12 years) A (0-1 years) B (2–6 years) C (7–12 years) Age groups Age groups A (0-1 years) A (0-1 years) B (2–6 years) B (2–6 years) C (7–12 years) C (7–12 years) (a) (b)

São Luiz do Tapajós 30

) 20 −1 g · g (𝜇

THg 10

A (0-1 years) B (2–6 years) C (7–12 years) Age groups A (0-1 years) B (2–6 years) C (7–12 years) (c)

Figure 5: Box plot average levels of THg in hair of children by age groups. The horizontal line represents the limits of biological tolerance −1 (LBT) by WHO (10 𝜇g⋅g ).

Table 1: Analytical recovery of THg and MeHg in Certificate Reference Material (CRM).

∗ −1 −1 CRM Analyte 𝑁 Mean ± SD (𝜇g⋅g ) Reference values (𝜇g⋅g )Recovery(%) IAEA 086 THg 38 0.568 ± 0.084 0.573 99.21 IAEA 085 MeHg 5 23.2 ± 5.35 20.73 94.35 ∗ Number of samples.

Table 2: The average results of THg and MeHg and the mean percentage of MeHg.

−1 −1 ∗ HgT (𝜇g⋅g )MeHg(𝜇g⋅g ) ∗∗ Communities 𝑁 %MeHg Mean ± SD Range Mean ± SD Range Maranhao˜ 139 2.27 ± 1.84 0.13–9.54 2.11 ± 1.82 0.12–9.54 93.17 S.L. do Tapajos´ 40 11.41 ± 7.16 1.08–28.17 10.30 ± 7.67 0.96–25.74 90.27 Barreiras 37 5.64 ± 5.55 0.43–27.82 5.20 ± 5.06 0.43–23.20 92.20 ∗ Number of samples ∗∗ Mean percentage of methylmercury (MeHg/THg × 100). BioMed Research International 7

Barreiras São Luiz do Tapajós

25 25 ∗

20 20 ) ) −1 −1 15 15 g g · · g g (𝜇 (𝜇

10 10 MeHg MeHg

5 5

0 0

x y x y y: Children with fisherman father y: Children with fisherman father x: Children without fisherman father x: Children without fisherman father (a) (b)

Figure6:BoxplotlevelsofMeHginBarreirasandSao˜ Luiz do Tapajos´ versus paternal occupation.

𝑃 = 0.0001), Figure 3, was observed. The strong correlation do Tapajos,´ the highest levels of THg were found in children −1 coefficient found between THg and MeHg in hair and the of the C group (5.5 and 12.5 𝜇g⋅g , resp.); these results may be high percentage of MeHg (>90%) suggest that the hair can related to the time of exposure of children of this group, that be considered as an excellent biomarker of MeHg exposure is larger compared with A and B groups. In the Maranhao˜ in children, especially in communities where there is the community, the highest levels of THg were found in children −1 record of high fish consumption by these individuals at all of the B group (1.7 𝜇g⋅g ). The communities of Barreiras, stages of life [1, 24, 25]. Consequently, the measurement Sao˜ Luiz do Tapajos,´ and Maranhao˜ showed different levels of of Hg levels in hair is considered the main indicator of mercury in different age groups, confirming previous reports exposure in populations exposed to MeHg and is used to that different mercury levels can be found in different age define international guidelines [26]. groups [18, 23, 27]. In Barreiras, despite the fact that the found THg average Regarding the time (in months) of breast-feeding, there levels were below the limits of biological tolerance (LBT = was no significant correlation with MeHg in the communities 𝜇 ⋅ −1 10 g g ) established by World Health Organization (WHO), of Barreiras (𝑅=−0.01, 𝑃 = 0.971)andSao˜ Luiz dos Tapajos´ about 10% of children of all age groups had THg levels (𝑅 = −0.05, 𝑃 = 0.763). In the Maranhao˜ community, above this limit and 86.48% of children had shown THg −1 information was not collected on duration of breast-feeding levels above the normality limit (NL = 2 𝜇g⋅g )proposedby −1 of children. This can be explained by the fact that inorganic WHO, reaching a maximum concentration of 27.82 𝜇g⋅g , mercuryismoreeasilytransmittedfrombloodtomilkthan THg level almost three times higher than LBT. In Sao˜ Luiz the methylmercury [11, 28–30]. The preferential inorganic Hg do Tapajos,´ 92.5% of children had THg levels above the distribution to breast milk is consistent with the association NLand57.5%reachedconcentrationsaboveLBT,reaching between Hg in plasma, since MeHg is preferably partitioned −1 the highest concentration of 28.17 𝜇g⋅g .IntheMaranhao˜ to erythrocytes [31].Studiesalsoshowedthatthemercuryof community, despite being a candidate region to control area, the breastfed children’s hair was not significantly correlated −1 about 43% of children had THg levels above the NL (2 𝜇g⋅g ), with levels of mercury in breast milk [32]. but there were no children with levels above the LBT of In the community of Barreiras, there was a significant −1 10 𝜇g⋅g (Figure 4). These results are strong indication that, correlation (𝑅 = 0.67, 𝑃 = 0.0001) between the levels even today, the children of the communities located on the of MeHg found with the current weight of children. The banks of the Tapajos´ River are more exposed to MeHg mainly consumption of food, including fish, is directly proportional than those of the community of Sao˜ Luiz do Tapajos.´ to the weight of the children. Then, this may result in The average of THg concentrations per age groups is elevated caloric and protein intake, which can increase the provided in box plot in Figure 5. In Barreiras and Sao˜ Luiz levels of childhood exposure to mercury [11]. Furthermore, 8 BioMed Research International children may have not yet fully developed the metabolic The results showed that the decrease in craft production excretion pathways, resulting in inefficiency of detoxification in the region begins to be reflected in decreased levels of and physiological elimination of contaminants [11]. This mercury in the children of the region. This decrease, although result was not found for the community of Sao˜ Luiz do still small in scale, represents a decrease of environmental Tapajos´ (𝑅 = −0.02, 𝑃 = 0.892). exposure to mercury in the Amazon. In relation to family life, children were divided into Children of the Tapajos´ had higher average levels of two groups (x and y), namely, y represented children with mercury compared to children of the community Maranhao˜ fisherman father, and x was related to children whose fathers (area without the influence of mining). These data alerts were not fisherman. The children of the (y) group had higher us about the importance of comparative studies in order to average levels of MeHg in relation to the children of (x) obtain accurate assessments and help to establish reference group in both communities (Figure 6). In the community of levels of mercury in exposed and not exposed children in the Barreiras, the children of (y) group showed average levels Amazon. It also becomes relevant an clinical, epidemiological −1 of MeHg of 7.33 𝜇g⋅g (3.38–11 : 27), while, in the (x) group and laboratory study more refined in children from Barreiras 𝜇 ⋅ −1 community, who presented high levels of Hg and recorded ones, it was 3.75 g g (2.68–4.81). In Sao˜ Luiz do Tapajos,´ problems in the development of the motor system. although the (y) group had showed higher average levels of −1 MeHg, 10.54 𝜇g⋅g (4.88–10.67), compared with the values −1 of (x) group, 6.44 𝜇g⋅g (1.73–16.11), this group had children Conflict of Interests with mercury levels above the ones established by WHO 𝜇 ⋅ −1 The authors declare that there is no conflict of interests (10 g g ). Knowing that the aquatic biota is the main regarding the publication of this paper. mercury transfer form from contaminated environment to humans in riverside communities [4, 9, 18, 27, 33], this result is justified, since it has been found, through epidemiological Acknowledgments questionnaire, that 76.62% of the children of this region have fishasmainsourceofdietaryprotein. The authors thank the Evandro Chagas Institute and the Federal University of Para´ for supporting the searches. In studies in humans in the Tapajos´ River Basin, possible reductionofHglevelsinrecentyears[34]hasbeenreported. −1 In 2001, the Hg levels obtained average of 20 𝜇g⋅g [1, 25, 35, References 36]. However, further studies in the same populations showed −1 [1] M. Harada, J. Nakanishi, E. Yasoda et al., “Mercury pollution lower levels, approximately 15 𝜇g⋅g [5, 8, 13, 18, 22, 37], 𝜇 ⋅ −1 intheTapajosRiverbasin,Amazonmercurylevelofheadhair buttheselevelsarestillaboveLBT(10 g g )establishedby and health effects,” Environment International,vol.27,no.4,pp. WHO. In children, this tendency is also observed. In a study 285–290, 2001. in the community of Sao˜ Luiz do Tapajos´ in 1999, the THg −1 [2]R.C.Marques,J.G.Dorea,W.R.Bastos,M.deFreitas´ levels found in children were 15 𝜇g⋅g [36], and, in 2007,such −1 Rebelo,M.deFreitasFonseca,andO.Malm,“Maternalmercury levels were 13.39 𝜇g⋅g [18]. Our results confirm a possible exposure and neuro-motor development in breastfed infants reduction of Hg levels in children from the communities from Porto Velho (Amazon), Brazil,” International Journal of −1 of Sao˜ Luiz do Tapajos´ and Barreiras (11.41 and 5.64 𝜇g⋅g , Hygiene and Environmental Health,vol.210,no.1,pp.51–60, resp.). In Sao˜ Luiz do Tapajos,´ despite having average levels of 2007. THg lower than those found in previous studies, this average [3] E. C. De Oliveira Santos, I. M. De Jesus, E. Da Silva Brabo et al., is still above LBT. “Mercury exposures in riverside amazon communities in Para, Brazil,” Environmental Research,vol.84,no.2,pp.100–107,2000. [4] M. Gochfeld, “Cases of mercury exposure, bioavailability, and 4. Conclusions absorption,” Ecotoxicology and Environmental Safety,vol.56,no. 1,pp.174–179,2003. Mercury exposure represents a growing health problem, mainly for riverside children, who are particularly susceptible [5]M.C.N.Pinheiro,B.M.Macchi,J.L.F.Vieiraetal.,“Mercury to the effects of mercury exposure during specific periods exposure and antioxidant defenses in women: a comparative study in the Amazon,” Environmental Research,vol.107,no.1, of rapid growth and development [11]. The riverside com- pp. 53–59, 2008. munities of the Tapajos´ Valley are exposed to mercury for decades and despite the fact that recent studies show a trend [6] D. Mergler, H. A. Anderson, L. H. M. Chan et al., “Methylmer- cury exposure and health effects in humans: a worldwide of decrease of mercury levels in these populations [34], in concern,” Ambio,vol.36,no.1,pp.3–11,2007. this research these concentrations in the children hair still meet the above biological tolerance limit established by WHO [7] P. Holmes, K. A. F. James, and L. S. Levy, “Is low-level envi- 𝜇 ⋅ −1 ronmental mercury exposure of concern to human health?” (10 g g ). Science of the Total Environment,vol.408,no.2,pp.171–182, The strong correlation coefficient (𝑅 = 0.96 and 𝑃= 2009. 0.0001 ) confirms hair as an excellent biomarker of human [8] D. Grotto, J. Valentini, M. Fillion et al., “Mercury exposure exposure to the organic mercury in riverside populations of and oxidative stress in communities of the Brazilian Amazon,” the Tapajos,whichhastheconsumptionoffishdailyasmain´ Science of the Total Environment,vol.408,no.4,pp.806–811, feed protein source. 2010. BioMed Research International 9

[9] M. E. Crespo-Lopez,A.M.Herculano,T.C.Corvelo,andJ.´ [27] A. L. De Sa,´ A. M. Herculano, M. C. Pinheiro, L. C. L. Silveira, L. Do Nascimento, “Mercury and neurotoxicity,” Revista de J. L. M. Do nascimento, and M. E. Crespo-Lopez,´ “Human Neurologia,vol.40,no.7,pp.441–447,2005. exposure to mercury in the west region of ParaState,”´ Revista [10] T. W. Clarkson and L. Magos, “The toxicology of mercury and Paraense de Medicina,vol.20,no.1,pp.19–25,2006. its chemical compounds,” Critical Reviews in Toxicology,vol.36, [28] M. Yoshida, C. Watanabe, H. Satoh, T. Kishimoto, and Y. no. 8, pp. 609–662, 2006. Yamamura, “Milk transfer and tissue uptake of mercury in [11] WHO, Children’s Exposure to Mercury Compounds,World suckling offspring after exposure of lactating maternal guinea Health organization, Geneva, Switzerland, 2010. pigs to inorganic or methylmercury,” Archives of Toxicology,vol. [12] “JECFA (United Nations Food and Agriculture Organization 68,no.3,pp.174–178,1994. and World Health Organization Joint Expert Committee on [29] A. Oskarsson, A. Schutz,¨ S. Skerfving, I. P.Hallen,´ B. Ohlin, and Food Additives),” 2007, http://whqlibdoc.who.int/publications/ B. J. Lagerkvist, “Total and inorganic mercury in breast milk and 2007/9789241660587 eng.pdf. blood in relation to fish consumption and amalgam fillings in [13] M. Fillion, D. Mergler, C. J. Sousa Passos, F. Larribe, M. lactating women,” Archives of Environmental Health,vol.51,no. Lemire, and J. R. D. Guimaraes,˜ “A preliminary study of 3,pp.234–241,1996. mercury exposure and blood pressure in the Brazilian Amazon,” [30] M. Sakamoto, M. Kubota, S.-I. Matsumoto, A. Nakano, and H. Environmental Health: A Global Access Science Source,vol.5, Akagi, “Declining risk of methylmercury exposure to infants article 29, 2006. during lactation,” Environmental Research,vol.90,no.3,pp. [14] US EPA, ADecadeofChildren’sEnvironmentalHealth 185–189, 2002. Research, Environmental Protection Agency, Washington, DC, [31] S. Skerfving, “Mercury in women exposed to methylmercury USA, 2007, http://www.epa.gov/ncer/publications/research through fish consumption, and in their newborn babies and results synthesis/ceh report 508.pdf. breast milk,” Bulletin of Environmental Contamination and [15]M.C.N.Pinheiro,M.Harada,E.Yasodaetal.,“Toxicological Toxicology, vol. 41, no. 4, pp. 475–482, 1988. and epidemiological data on human exposure to mercury in the [32] A. C. Barbosa and J. G. Dorea,´ “Indices of mercury contamina- Tapajos River basin: 1994–1998,” Environmental Science,vol.10, tion during breast feeding in the Amazon Basin,” Environmental no.2,pp.99–105,2003. Toxicology and Pharmacology,vol.6,no.2,pp.71–79,1998. [16] M.D.C.N.Pinheiro,J.Nakanishi,T.Oikawaetal.,“Methylmer- [33] M. E. Crespo-lopez,G.L.Mac´ edo,ˆ S. I. D. Pereira, G. P. F. cury human exposure in riverine villages of Tapajos basin, Arrifano,D.L.W.Picanc¸o-diniz, J. L. M. Do Nascimento et al., Para State, Brazil,” Revista da Sociedade Brasileira de Medicina “Mercury and human genotoxicity: critical considerations and Tropical, vol. 33, no. 3, pp. 265–269, 2000. possible molecular mechanisms,” Pharmacological Research, [17] A. C. Barbosa, W. Jardim, J. G. Dorea,´ B. Fosberg, and J. vol. 60, pp. 212–220, 2009. Souza, “Hair mercury speciation as a function of gender, age, [34] J. J. B. Nevado, R. C. Rodr´ıguez Mart´ın-Doimeadios, F. J. and body mass index in inhabitants of the negro river basin, Guzman´ Bernardo et al., “Mercury in the Tapajos´ River basin, amazon, brazil,” Archives of Environmental Contamination and Brazilian Amazon: a review,” Environment International,vol.36, Toxicology,vol.40,no.3,pp.439–444,2001. no. 6, pp. 593–608, 2010. [18] M. C. N. Pinheiro, M. E. Crespo-Lopez,´ J. L. F. Vieira et [35]O.Malm,F.J.P.Branches,H.Akagietal.,“Mercuryand al., “Mercury pollution and childhood in Amazon riverside methylmercury in fish and human hair from the Tapajos river villages,” Environment International,vol.33,no.1,pp.56–61, basin, Brazil,” Science of the Total Environment,vol.175,no.2, 2007. pp. 141–150, 1995. [19] H. Akagi, Mercury Analysis Manual, Ministry of the Environ- [36] P. Grandjean, R. F. White, A. Nielsen, D. Cleary, and E. C. De mental,Tokyo,Japan,2004. Oliveira Santos, “Methylmercury neurotoxicity in Amazonian [20] MINITAB Statistical Software, Release 14 for Windows,State children downstream from gold mining,” Environmental Health College, Pa, US, 2003. Perspectives,vol.107,no.7,pp.587–591,1999. [21] S. F. Costa, Introduc¸ao˜ ilustrada aestat´ ´ıstica,harbra,Sao˜ Paulo, [37] C. J. S. Passos and D. Mergler, “Human mercury exposure and Brazil, 3rd edition, 1998. adverse health effects in the Amazon: a review,” Cadernos de [22] M. C. N. Pinheiro, R. C. S. Muller,¨ J. E. Sarkis et al., “Mercury Saude Publica, vol. 24, no. 4, pp. S503–S520, 2008. and selenium concentrations in hair samples of women in fertile age from Amazon riverside communities,” Science of the Total Environment, vol. 349, no. 1-3, pp. 284–288, 2005. [23] M. C. N. Pinheiro, T. Oikawa, J. L. F. 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Research Article Involvement of Oxidative Stress and Inflammation in Liver Injury Caused by Perfluorooctanoic Acid Exposure in Mice

Bei Yang,1 Weiying Zou,1 Zhenzhen Hu,2 Fangming Liu,3 Ling Zhou,4 Shulong Yang,1 Haibin Kuang,1 Lei Wu,1 Jie Wei,1 Jinglei Wang,1 Ting Zou,1 and Dalei Zhang1

1 Department of Physiology, Medical College of Nanchang University, Nanchang 330006, China 2 Department of Pathophysiology, Medical College of Nanchang University, Nanchang 330006, China 3 Library of Nanchang University, Nanchang 330006, China 4 Nanchang Medical School, Nanchang 330006, China

Correspondence should be addressed to Dalei Zhang; [email protected]

Received 25 October 2013; Accepted 19 January 2014; Published 2 March 2014

Academic Editor: Erhan Unlu

Copyright © 2014 Bei Yang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Perfluorooctanoic acid (PFOA) is widely present in the environment and has been reported to induce hepatic toxicity in animals and humans. In this study, mice were orally administered different concentrations of PFOA (2.5, 5, or 10 mg/kg/day). Histological examination showed that the exposure to PFOA for 14 consecutive days led to serious hepatocellular injury and obvious inflammatory cell infiltration. In addition, malondialdehyde formation and hydrogen peroxide generation, indicators of oxidative stress, were significantly induced by PFOA treatment in the liver of mice. Furthermore, hepatic levels of interleukin- 6, cyclooxygenase-2, and C-reactive protein, markers of inflammatory response, were markedly increased by exposure to PFOA in mice. These results demonstrated that PFOA-induced hepatic toxicity may be involved in oxidative stress and inflammatory response in mice.

1. Introduction that geometric mean PFOA concentrations in serum were 5.2, 3.95, 3.92, and 4.13 ng/mL in the US population, respectively Perfluorooctanoic acid (PFOA), a member of the perfluo- [7]. roalkyl acid (PFAA) family of compounds, is a strong sur- In recent years, there has been increasing concern regard- factant that is widely used in the manufacture of lubricants, ing potential adverse effects of PFOA on animal and human medical equipment, paper and textile coatings, oil repellents, health. Laboratory studies have shown that PFOA elicits a upholstery, polishes, food packaging, and fire fighting foams variety of toxicities, including hepatotoxicity [8], carcinogen- [1]. Due to the presence of strong carbon-fluorine bonds, icity [9], neurotoxicity [10], mutagenicity [11], developmental it is practically nonbiodegradable and highly persistent in toxicity [12], immunotoxicity [13], and genotoxicity [14]. the environment [2].PFOA,aswellasotherPFAAs,has Epidemiologic studies have also demonstrated that PFOA been detected in a variety of environmental matrices from exposure is positively associated with hyperuricemia [15], around the globe, including surface waters, air, sludge, soils, cardiovascular disease [16], chronic kidney disease [17], sediments, and polar ice caps [2]. Furthermore, detectable thyroid disease [18], and hepatocellular damage [19]. levels of PFOA have been found in wildlife and humans [3– In the body, PFOA is distributed predominantly to the 5]. In particular, the presence of PFOA and PFOS has been liver and plasma in humans and animals [20]. The liver serves identified in human tissue samples, including liver, kidney, as the main target organ for PFOA, which causes an incre- adipose tissue, brain, basal ganglia, hypophysis, thyroid, ased liver weight, hepatocytic hypertrophy, hepatic triglyc- gonads, pancreas, lung, skeletal muscle, and blood from eride accumulation, multifocal coagulation, and liquefaction nonoccupationally exposed subjects [6]. Data from NHANES necrosis in rodents [8, 21, 22]. In addition, PFOA expo- 1999-2000, 2003-2004, 2005-2006, and 2007-2008 showed sure increases the incidence of malignant hepatocellular 2 BioMed Research International carcinoma in rats [23]. Although considerable numbers of 16 studies have reported the adverse effects of PFOA exposure a on the liver, the underlying mechanisms have not yet been fully elucidated. Many environmental contaminants have 12 b been reported to induce oxidative stress and to result in hep- c atic injury in experimental animals [24–26]. Moreover, severe environmental pollutants have been implicated to induce 8 hepatic inflammation27 [ –29]. Therefore, the present study d was designed to determine whether PFOA-induced hepatic toxicity was involved in oxidative stress and inflammatory 4 response. Relative liver weight (% of body weight) (% of weight liver Relative 0 2. Materials and Methods 0 2.5 5 10 PFOA (mg/kg) 2.1. Animals. Male Kunming (KM) mice weighing 20–22 g were purchased from the Laboratory Animal Center of Figure 1: Relative liver weight after exposure to different concentra- ∘ Nanchang University. Mice were maintained at 22 ± 2 Cand tions of PFOA. Values are expressed as mean ± SEM (𝑛=4). Bars relative humidity (50% ± 10%) with a 12 h light/dark cycle and with different letters are statistically different (𝑃 < 0.05). acclimatized for 1 week prior to the start of the experiment. All animal procedures were performed in accordance with theGuidelinesforCareandUseofLaboratoryAnimalsof 2.6. Measurement of Interleukin 6 (IL-6), Cyclooxygenase-2 Nanchang University and approved by the Animal Ethics (COX-2), and C-Reactive Protein (CRP). The frozen liver tis- Committee of Nanchang University. sue was homogenized with ice-cold saline. The levels of IL-6, COX-2, and CRP in liver tissue homogenates were deter- 2.2. Treatments. PFOA (96% purity, Sigma-Aldrich, USA) mined using commercially available ELISA kits, in accor- was dissolved in dimethyl sulfoxide (DMSO). Mice were dance with the manufacturers’ instructions (Xitang Biotech- orally administered different concentrations of PFOA (2.5, 5, nology, Shanghai, China). or 10 mg/kg/day) once daily for 14 consecutive days. Controls received an equivalent volume of DMSO. At the end of 2.7. Statistical Analysis. Data were presented as the mean ± treatment period, the mice were sacrificed after anesthesia SEM and evaluated by one-way analysis of variance with sodium pentobarbital. Blood samples were collected and (ANOVA) and Duncan’s multiple-range tests using the GLM livers were aseptically excised and weighed. Liver tissues were procedure of SAS 8.1 software. 𝑃 < 0.05 was considered fixed in 4% paraformaldehyde for histological examination ∘ statistically significant. or frozen in liquid nitrogen and then stored at −80 Cfor biochemical analyses. 3. Results 2.3. Measurement of Serum Enzymes. The blood samples ∘ 3.1. Effect of PFOA on Liver Weight and Morphology. Oral were centrifuged at 13,000 rpm at 4 Cfor30mintoseparate administration of PFOA (2.5–10 mg/kg/day) for 14 consecu- serum. The activities of serum alanine aminotransferase tive days caused obvious hepatic hypertrophy and induced (ALT), aspartate aminotransferase (AST), alkaline phos- a significant increase in the relative liver weight in a dose- phatase (ALP), lactate dehydrogenase (LDH), and total bile dependent manner (𝑃 < 0.05)(Figure 1). Histological exami- acids (TBA) were determined with a biochemical analyzer nation of liver sections showed deranged liver architecture, (7180, HITACHI, Japan). severe edema, vacuolar degeneration, focal necrosis, and obvious infiltration of inflammatory cells in mice exposed to 2.4. Histology. The fixed liver samples were dehydrated in PFOA. The maximal effect was observed at the highest ethanol gradient solutions, embedded in paraffin, and sec- concentration (10 mg/kg/day) (Figure 2(d)) and intermediate tioned at 5 𝜇m. The sections were stained with hematoxylin effects were found at the doses of 2.5 and 5 mg/kg/day and eosin and observed under an optical microscope (IX71 (Figures 2(b) and 2(c)). These adverse histological changes Olympus, Japan). were absent in the liver of control mice (Figure 2(a)).

2.5. Measurement of Malondialdehyde (MDA) and Hydrogen 3.2. Effect of PFOA on Serum AST, ALT, ALP, LDH, and TBA Peroxide (H2O2). The levels of MDA and2 H O2 in liver tissue Levels. PFOA administration induced an obvious increase homogenates were measured using commercial kits (Jian- in serum ALT levels in a dose-dependent manner in mice cheng Institute of Biotechnology, Nanjing, China), in accor- (𝑃 < 0.05)(Figure 3(a)). Compared with the control, serum dance with the manufacturers’ instructions. The analyses AST, ALP, LDH, and TBA levels were significantly increased were performed with a UV 1800 spectrophotometer (Shi- by treatment with PFOA (5–10 mg/kg/day) (Figures 3(b)– madzu, Japan). 3(e)). There was no significant reduction in these biochemical BioMed Research International 3

(a) (b)

Figure 2: Liver histopathology after exposure to PFOA 0 (a), 2.5 (b), 5 (c), or 10 (d) mg/kg/day for 14 days. Sections of liver were stained with hematoxylin and eosin and then were visualized under an IX71 Olympus microscope. Magnification: 100x.

markers of liver function in the lowest exposure group shown that PFOA exposure can increase the size and relative (2.5 mg/kg/day) compared with the control group (Figure 3). weight of the liver in mice [8, 22]. In the present study, oral exposure to PFOA for 14 consecutive days caused obvious hepatomegaly and induced a significant increase in liver 3.3. Effect of PFOA on Liver MDA Formation and2 H O2 Gen- eration. To explore whether PFOA exposure led to oxidative weight in a dose-dependent manner. The observation was stress in the mouse liver, two indexes of oxidative stress, consistent with the previous studies. In the histopathological evaluation, the liver of PFOA-treated mice showed morpho- MDA and H2O2, were determined. After PFOA exposure logical changes, including structure damage, hepatocellular for14days,thelevelsofMDAandH2O2 in the liver tissue significantly increased compared with the control𝑃< ( necrosis, edema, and inflammatory cell infiltration. More- 0.05) (Figures 4(a) and 4(b)). The lowest dose of PFOA had over, biochemical evaluation indicated that PFOA treatment led to a significant increase in serum enzymes, including AST, no effect on H2O2 generation compared with the control (Figure 4(b)). ALT, ALP, LDH, and TBA. The leakage of large quantities of serum enzymes into the blood stream was associated with centrilobular necrosis, ballooning degeneration, and cellular 3.4. Effect of PFOA on Liver CRP, IL-6, and COX-2 Levels. infiltration of liver30 [ ]. Previous reports have suggested a To investigate whether PFOA exposure-induced liver injury positive association between PFOA exposure and serum ALT was associated with inflammatory process, three markers of and AST levels [8, 19]. Our results confirmed the hepatic inflammatory response, CRP,IL-6, and COX-2 were detected toxicity of PFOA in mice. in liver tissue. After exposure for 14 days, the moderate Oxidative stress is considered a critical pathophysiolog- dose of PFOA (5 mg/kg/day) caused a significant reduction ical mechanism in different pathologies, including cardio- in the hepatic levels of COX-2 compared with the control vascular diseases, cancer, diabetes, rheumatoid arthritis, or 𝑃 < 0.05 ( ). However, the high concentration of PFOA neurological disorders [31]. Numerous studies have demon- (10 mg/kg/day) significantly increased hepatic CRP,IL-6, and strated that oxidative stress was an important causative factor 𝑃 < 0.05 COX-2 levels compared with control group ( ). The in the mechanism of action of environmental contaminants low-dose exposure to PFOA (2.5 mg/kg/day) did not alter the [24–26]. The balance between prooxidant endogenous and 𝑃 > 0.05 hepatic levels of the three cytokines ( )(Figure 5). exogenous factors and antioxidant defenses in biological systems can be used to assess toxic effects under stress- 4. Discussion ful environmental conditions, especially oxidative damage induced by chemical pollutants [32]. Perfluorinated compounds are emerging environmental con- Exposure to PFOA has been demonstrated to generate taminants of public health concern. Previous studies have reactive oxygen species (ROS) and cause oxidative DNA 4 BioMed Research International

700 500 a a a 600 400 500 300 400 b b b 300

ALT (U/L) ALT 200 AST (U/L) AST 200 c 100 100 d

0 0 0 2.5 5 10 0 2.5 5 10 PFOA(mg/kg) PFOA (mg/kg) (a) (b) 700 a 3000 a a 600 2500 500 b 2000 400 1500 300 b c c ALP (U/L) LDH (U/L) LDH 1000 200 b

100 500

0 0 0 2.5 5 10 0 2.5 5 10 PFOA (mg/kg) PFOA (mg/kg) (c) (d) 14 a 12

mmol/L) b 𝜇 6

TBA ( TBA 4 c c 2

0 0 2.5 5 10 PFOA (mg/kg) (e)

Figure 3: Serum levels of AST (a), ALT (b), ALP (c), LDH (d), and TBA (e) after exposure to different concentrations of PFOA. Values are expressed as mean ± SEM (𝑛=4). Bars with different letters are statistically different (𝑃 < 0.05).

damage in HepG2 cells [14].However,theincreasein the liver of wild-type mice [35]. In addition, exposure to ROS production was not concentration-dependent [33]. In perfluorononanoic acid (PFNA) and perfluorododecanoic cultured tilapia hepatocytes, exposure to PFOA induced a acid (PFDoA) significantly increased the levels of2 H O2 and dose-dependent decrease in cell viability accompanied by MDA but inhibited the activities of superoxide dismutase and an increase in MDA formation [34]. In vivo evaluation, catalase in the liver of rats [36, 37]. MDA and H2O2 can PFOA increased the levels of 8-hydroxydeoxyguanosine (8- be used as indirect measurements of lipid peroxidation and OHdG), an indicator of oxidative DNA damage, in the liver cellular injury. In the present study, PFOA treatment induced of Ppar𝛼-nullmicebutdidnotelevate8-OHdGlevelsin an elevation in MDA formation and H2O2 generation in BioMed Research International 5

0.5 200 a a 0.4 150 b b 0.3 c 100 0.2 b b

CRP (ng/mg protein) CRP (ng/mg 50 b

MDA (nmol/mg protein) (nmol/mg MDA 0.1

0 0 0 2.5 5 10 0 2.5 5 10 PFOA (mg/kg) PFOA (mg/kg) (a) (a) 16 30 a

a a 25 12 b b 20 8 15 b (mmol/g protein) (mmol/g

2 10 IL-6 (pg/mg protein) (pg/mg IL-6 4

O b

2 b H 5 0 0 0 2.5 5 10 0 2.5 5 10 PFOA (mg/kg) PFOA (mg/kg) (b) (b) 25

Figure 4: Hepatic levels of MDA (a) and H2O2 (b) after exposure a to different concentrations of PFOA. Values are expressed as mean 20 ± SEM (𝑛=4). Bars with different letters are statistically different (𝑃 < 0.05). 15

b 10 b

COX-2 (ng/mg protein) (ng/mg COX-2 5 the liver of mice, suggesting that PFOA-induced hepatic c toxicity was related to oxidative stress, which caused lipid 0 peroxidation and hepatocyte injury. 0 2.5 5 10 Inflammation is a local immune response to infection PFOA (mg/kg) and injury. PFOA has been known to induce inflammation by elevating the expression of proinflammatory cytokines (c) 𝛼 𝛽 tumor necrosis factor and interleukin-1 andIL-6inthe Figure 5: Levels of CRP (a), IL-6 (b), and COX-2 (c) in liver spleen and mast cells [38, 39]. In the liver, proinflammatory tissue after exposure to different concentrations of PFOA. Values are cytokines produced by hepatocytes participate in hepatotoxic expressed as mean ± SEM (𝑛=4). Bars with different letters are responses [40]. A previous report showed that exposure to statistically different𝑃 ( < 0.05). PFOA might sensitize hepatic parenchymal cells to other toxicants and thereby aggravate liver injury during acute inflammation41 [ ]. As markers of inflammation, IL-6, CRP, and COX-2 are widely used for estimation of various inflam- 5. Conclusion matory states. In the present study, exposure to a high dose of PFOA (10 mg/kg/day) significantly increased the levels In this study, we showed that oral exposure to PFOA for of IL-6, CRP, and COX-2 in the liver tissue of mice. Our 14consecutivedayscausedanincreaseinserumAST,ALT, results indicated a possible role of PFOA in inflammation and ALP, LDH, and TBA levels and induced hepatocellular hepatic injury. necrosis, edema, and inflammatory cell infiltration in mice. 6 BioMed Research International

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