Oslo, June Ingebjørg Haukeli - PDF

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Acknowledgement The work in this thesis was carried out from August 2013 to June 2014, at the Center of Reproduction and Reproductive Toxicology (CRRT), Department of Production Animal Clinical Sciences

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Acknowledgement The work in this thesis was carried out from August 2013 to June 2014, at the Center of Reproduction and Reproductive Toxicology (CRRT), Department of Production Animal Clinical Sciences (ProdMed), campus Adamstuen, for the Master`s degree in Biotechnology at the Norwegian University of Life Sciences (NMBU). The thesis was part of a 5 year project funded by the Research Council of Norway (NRF); Does environmental pollutants interact with stress responses. First of all I want to express great gratitude to my supervisor, Professor Erik Ropstad for encouragement and support throughout the year. You were always available for motivating talks and gave constructive feedback and comments on my writing process. I thank you also for the statistical help. I am extraordinary grateful to my co- supervisor Alexandra Hudecova for providing training in the laboratory and for giving me constructive feedback on my writing. You were always positive and kept me going when times were tough. Thank you for always being there for me and supporting me, it has been invaluable. I would also like to thank you for all the great talks, both relevant and irrelevant to my thesis. I also want to thank Morten Sørlie for taking on the role as internal supervisor. Thank to Hanne Friis Berntsen and Dr. Karin Zimmer, for kindly providing the test mixtures. Kristine Eraker Aasland Hansen and Christer Wiggen, thank for helping me collecting blood for the experiments on lymphocytes. I am also grateful to thank Professor Maria Dusinska, Berit Modalen and Dr. Naouale El Yamani at the Norwegian Institute for Air Research (NILU) for kindly letting me use your microscope for scoring my comet assay experiments. Inger A. Hagen, your optimism and support is highly appreciated. Thank you for reading through my thesis; you are truly a good friend. Additionally, I would like to acknowledge all associates at ProdMed and members of the environmental and reproductive toxicology (ERT) group. Special thanks to my fellow master students Henriette P.B. Samuelsen and Stine E. B. Aurbakken for great talks and a good time. I And last, but definitely not least, I am deeply grateful to my dearest family, friends and boyfriend. You have encourage, motivated and supported me throughout the time consuming work with this thesis. Without your loving support this work could not have been completed. Thank you. Oslo, June 2014 Ingebjørg Haukeli II Abstract A wide range of synthetic chemicals have been used extensively for decades. They are ubiquitous in the environment, and can therefore pose adverse effects on humans and wildlife. Mixtures of persistent organic pollutants (POPs) that are relevant to real life exposure, are of emerging concern when it comes to their potentially adverse effects and the ability to interact with other substances. The main objective of this study was to investigate exposure relevant mixture of POPs, based on levels recently measured in blood and breast milk in Scandinavia, for the in vitro study. The in vivo study based the mixture on estimated daily intake measured in food. DNA damage was assessed by the method of choice; comet assay and used on the human adrenocortical carcinoma cell line H295R and isolated lymphocytes from mice. The effect of a mixture containing brominated, chlorinated and perfluorinated compounds (total mixture), and sub mixtures containing respectively, brominated, chlorinated and perfluorinated compounds, were used on the cytotoxic potential, characterized by means of Alamar Blue viability assay. The total mixture and the sub - mixtures were used to assess the percent of DNA damage in the adrenal cell line H295R. Isolated lymphocytes from mice were used to assess the percent of DNA damage, both in vitro and in vivo, using the total mixture. The in vitro exposure of the mice lymphocytes revealed a small, but significant increase in the percent of DNA damage lymphocytes for the exposure dose 10-8 when compared to the solvent control, and there were an indication that each mouse reacted differently to exposure and exposure dose. Furthermore, the in vivo exposure on mice lymphocytes did not show any significant change in DNA damage when compared to the unexposed control group, but the high group and the low group were significantly different from each other. The in vitro exposure of the H295R cells gave a significant dose response relationship for the perfluorinated sub mixture and the total mix. Furthermore, exposure to the mixtures did not reveal any significant differences at the concentrations corresponding to the approximate levels of relevant POPs measured in human blood and breast milk. However, the highest dilution of the perfluorinated sub mixture was cytotoxic to the H295R cells. The III perfluorinated sub mixture caused an abrupt decline in viability, at the highest dilution (10-3 ), while the other mixtures were stable at about 95 %. IV Sammendrag Et bredt spekter av syntetiske kjemikalier har vært brukt i flere tiår, de er allestedsnærværende i miljøet, og kan dermed utgjøre skadelige effekter på mennesker og dyreliv. Mikser av persistente organiske miljøgifter (POPs) som er relevante for virkelighetsnær eksponering, er av voksende bekymring når det gjelder deres potensielle negative effekter og mulighet for interaksjon seg i mellom. Hovedmålet med denne studien var å undersøke eksponerings-relevante blandinger av POPs, basert på nivåer som er målt i blod og morsmelk i Skandinavia, for in vitro studiene. In vivo studiene baserte miksen på estimert daglig inntak fra mat. DNA-skade ble detektert ved metoden; Comet assay og anvendt på den humane binyrebark cellemodellen -H295R og isolerte lymfocytter fra mus, både in vitro og in vivo. Effekter av miksen som inneholder bromerte, klorerte og perfluorerte forbindelser (total miksen), og sub-miksene som inneholder henholdsvis bromerte, klorerte og perfluorerte forbindelser, ble brukt for å studere cytotoksisitet, karakterisert ved Alamar Blue levedyktighets assay. De samme miksene ble brukt til å studere prosent DNA-skade i binyrebark cellelinje-h295r. Isolerte lymfocytter fra mus ble brukt til å studere prosent DNA-skade, både in vitro og in vivo, med den totale miksen. In vitro-eksponering av isolerte lymfocytter fra mus viste en liten, men signifikant økning i prosenten av DNA-skade, for eksponeringsdose 10-8 når den ble sammenlignet med løsningsmiddelkontrollen. Det var også en indikasjon på at hver mus reagerer forskjellig på eksponering og eksponerings doser. In vivo eksponeringen på lymphocytter isolert fra mus viste ingen signifikant endring i DNA-skade sammenlignet med den ikke-eksponerte kontroll gruppen, men den høye gruppen og den lave gruppen var signifikant forskjellige fra hverandre. In vitro-eksponering av H295R cellene ga et signifikant dose respon forhold for den perfluorerte sub miksen og den totale miksen. Videre, eksponering til miksene viste ingen signifikant forskjell på de konsentrasjonene som tilsvarer de omtrentlige nivåene av relevante miljøgifter, målt i humant blod og brystmelk. Den høyeste fortynningen av den perfluorerte sub-miksen var cytotoksisk for H295R cellene. Den perfluorerte sub-miksen forårsaket en V drastisk reduksjon i levedyktighet, ved den høyeste fortynning (10-3 ), mens de andre miksene var stabile over 95 %. VI Abbreviations C: µl: µm: AB Assay: BFR: CO₂: DAPI: DDT: DMEM: DMSO: DNA: DNA: ECD: EDI: EDTA: ELFO: FBS: H295R: H₂O₂: HBCD: HCB: Degrees Celsius Microliter Micromolar Alamar Blue Assay Brominated flame retardant Carbon dioxide 4`.6-diamidino- 2- phenyllindole Dichlorodiphenyltrichloroethane Dulbecco`s Modifies Eagle Medium Dimethyl Sulfoxide Deoxyribonucleic acid Deoxyribonucleic acid Endocrine Disrupting chemical Estimated daily intake Ethylene Diamine Tetra acetic Acid Electrophoresis solution Fetal bovine serum Human adrenocortical carcinoma cell line Hydrogen peroxide Hexabromocyclododecane Hexachlorobenzene VII HCH: HepG2 cells: LMP: mg: ml: mm: NaCl: NaOH: NMP: OCP: p,p` -DDE: PBDE: PBS: PCB: PFC: PFDA: PFHxS: PFNA: PFOA: PFOS: PFUnDA: POP: SCGE: Hexachlorocyclohexane Liver hepatocellular cells Low melting point Milligram Milliliter millimolar Sodium chloride Sodium hydroxide Normal melting point Organochlorine pesticide Dichlorodiphenyltrichloroethane Polybrominated Diphenyl Ethers Phosphate buffered saline Polychlorinated biphenyls Perfluorinated compounds Perfluorodecanoic acid Perfluorohexanesulfonic acid Perfluorononaoic acid Perfluorooctanic acid Perfluorooctane sulfonate Perfluoroundecanoic acid Persistent organic pollutant Single cell gel electrophoresis VIII SD: SEM: Tris Base: Triton X-100: UNEP: Standard error Standard error mean Tris (2, 3-dibromopropyl) phosphate Polyethylene glycol p-(1, 1, 3, 3-tetramethylbutyl)-phenyl ether United Nations Environment Programme IX Contents Acknowledgement... I Abstract... III Sammendrag... V Abbreviations... VII 1. Introduction Persistent organic pollutants Perfluorinated compounds Brominated Compounds Polybrominated diohenyl ethers (PBDEs) Hexabromocyclododecane (HCBD) Organochlorinated Compounds Polychlorinated biphenyls (PCBs) Organochlorine pesticides Mixed exposure Endocrine disrupting compounds Assessment of cytotoxicity Genotoxicity Cell models Aim of study Materials and methods Chemicals mixtures In vitro mixture In vivo mixture Human adrenocortical carcinoma cell line H295R Expansion and plating of the H295R cells In vitro exposure of the H295R cells Isolation of lymphocytes from mice Alamar Blue assay Preparation of the H295R cells for AB assay Comet assay (Single- cell gel electrophoresis) Preparation of solutions Preparation of the H295R cells Comet assay on mice lymphocytes In vitro Comet assay X In vivo comet assay Scoring the comets Visual scoring Software scoring Ethics Statistical analysis Results Alamar Blue assay H295R cells and DNA damage Lymphocytes and DNA damage In vitro exposure In vivo exposure Discussion Cell viability H295R cells and DNA damage Lymphocytes and DNA damage In vitro In vivo Conclusion Future perspectives References XI 1. Introduction 1.1 Persistent organic pollutants The Second World War gave rise to an industry that led to production and use of synthetic chemical compounds. Scientists started to recognize their potential hazard on wildlife and humans (El-Shahawi et al., 2010). The book Silent spring by Rachel Carson (Carson 1962) was one of the first influential publications that raised awareness towards the persistent organic pollutants (POPs) (Wu et al., 2008). Persistent organic pollutants (POPs) are according to the United Nations Environment Program (UNEP), organic chemical substances that possess a particular combination of physical and chemical properties such that, once released into the environment, they; (i) remain intact for exceptionally long periods of time (many years), (ii)become widely distributed throughout the environment as a result of natural processes involving soil, water and most notably, air, (iii) accumulate in the fatty tissue of living organisms including humans, and are found at higher concentrations at higher levels in the food chain, (iv) are toxic to both humans and wildlife (UNEP, 2013a). The sources of emission are determined by where and how they are used, and the POPs may be released intentionally or unintentionally. Unintentionally released POPs are typically industrial chemicals or by products that are released by volatilization or leakage. Pesticides are intentionally released POPs, and example of that is dichlorodiphenyltrichloroethane (DDT), which are released at their point of application (Vallack et al., 1998). POPs are organic compounds which have the ability to migrate in air, soil and sediments. Furthermore, another major pathway for POPs is the atmospheric transport, which contributes to the global spread and distributions as well as the river and ocean current (Hardell et al., 2010b). They have the ability to accumulate in the food chains and can therefore be harmful for the health of humans and wildlife (El-Shahawi et al., 2010). POPs can travel long distances due to their stability in the atmosphere, and they tend to migrate towards colder areas, where they descend because of the cold temperature (El- Shahawi et al., 2010). Even in arctic areas have POPs been detected, where no such substances are used or produced (Vallack et al., 1998). Most documented effects have been in birds and marine mammals (Jones and de Voogt, 1999). There are many concerns regarding 1 POPs, especially their ability to bioaccumulate in certain organisms and biomagnification can occur in top predators (Mackay and Fraser, 2000). Declines in marine population have been reported related to DDT and PCBs (Vasseur and Cossu-Leguille, 2006a). Thousands of POP chemicals exist and they are typically hydrophobic and lipophilic and therefore stored in fatty tissue. POPs is a wide group of chemicals including organochlorine pesticides, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) among others (Jones and de Voogt, 1999) and several of them are listed in the Stockholm convention, to protect human health and the environment (Hardell et al., 2010a) and to reduce the production and release of POPs (Lammel and Lohmann, 2012). Dietary intake via especially products like fish and meat, are the main exposure source to POPs for humans (Hardell et al., 2010a). Risk assessment of POPs has traditionally focused on the effect of single compounds, but in real life we are exposed to multiple compounds. Investigation of the effects of mixtures reflecting environmental contaminants is considered a key issue to modern toxicology (Carpenter et al., 2002) (Kortenkamp, 2007). This study focus on mixture effects of POPs derived from respectively levels measured in human blood and breast milk or food, in Scandinavia Perfluorinated compounds. PFCs are man-made chemicals and do not occur naturally in the environment. They have been produced since the 1950s; because of their unique properties, such as anti-wetting or surfactant they are much used in industry and consumer products (Florentin et al., 2011). A fully fluorinated hydrophobic linear carbon chain attached to various hydrophilic heads, is a typical characterization for the perfluorinated compounds (PFCs) (Florentin et al., 2011). They are typically 4-14 atoms in length with a charged moiety, typically carboxylate or sulfonate (Eriksen et al., 2010). PFCs have an extreme resistance due to the carbon fluorine (C F) bond, this makes them resistant to degradation by heat, reactions with strong acids or bases and oxidizing agents or photolysis (Florentin et al., 2011). 2 Since PFCs have been used in the industry and as consumer products and because of their resistant to degradation, they are found several places in environment and wildlife (Haug et al., 2010). PFCs have a global occurrence and, they are found in water, sediment, fish, birds, marine mammals as well as blood and milk of humans (Hu and Hu, 2009), but they mainly distribute to liver and blood (Karrman et al., 2006). PFCs are widespread and their distribution and degradation in the environment is complex, the major exposure pathway for human is through food, but inhalation of dust may also be a potential source of exposure (D'Hollander et al., 2010). PFOA and PFOS are the PFCs with the highest concentration found in human serum in Norway (Haug et al., 2010). They are also the most studied PFCs (Florentin et al., 2011), PFOS consists of 8 carbon atoms and a sulfonic acid group (Figure 1,A), while PFOA has 8 carbon atoms and a carboxylic acid group (Figure 1,B) (Olsen et al., 2007). Figure 1. Chemical structure of PFOS (A) and PFOA (B) Brominated Compounds Brominated flame retardants (BFRs) contain a diversity of chemicals, polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) are some of them. During the last two decades they have drawn attention due to their environmental and human concerns. The main routs of release into the environment are via effluents from factories producing BFRs and plastic products. Hazardous waste, facilities that recycle plastic, metal from electronic equipment and accidental fires are other possible ways of release. The fate and behavior of the BFRs depend on their degree of bromination, higher brominated compounds are less mobile in the environment and often tend to end up in sediment near the emission source (Watanabe and Sakai, 2003). 3 Polybrominated diohenyl ethers (PBDEs) Polybrominated diphenyl ethers (PBDEs) are organic chemicals; they all have a common structure and are widely used as flame retardants in various industries. The compounds are halogenated and the structure is characterized with two benzene rings (Hites, 2004) and different compositions of bromine atoms (Figure 2). There are 209 PBDEs and they are distinguished by the number of bromine atoms (1-10) and their position (ortho, meta, para position). PBDEs are found in water, soil, sediment and living organisms, reasons for this is that they are volatile and are insoluble in water (Yue and Li, 2013). Figure 2. General chemical formula to PBDEs Polybrominated diphenyl ethers (PBDEs) have been widely used since the 1970s. PBDEs are polymer additives and therefore not chemically bound to materials, they tend to leach into the surrounding environment. They are considered contaminants in the environment, due to their high production, lipophilicity and persistence. Over the past 30 years levels of PBDEs in humans and the environment have increased (He et al., 2008). PBDEs are used as flame retardants in a variety of construction materials, textiles and polymers for electronic equipment. PBDEs and polychlorinated biphenyls (PCBs) have a similar chemical structure (Song et al., 2009). The variation in the roots of exposure is due to the variation of chemicals and their variation because of physiochemical properties and molecular weight. Diet appears as the main rout of exposure for the general human exposure, this is particularly from the lower brominated congeners. Fatty fish is the most important food group. Release from consumer products that 4 are treated with these compounds could also be a rout of exposure, due to inhalation of air. Work environment is also a place where people may be exposed (Watanabe and Sakai, 2003) Hexabromocyclododecane (HCBD) In addition to the PBDEs; Hexabromocyclododecane (HBCD) is also included in the brominated sub mixture. HBCD is a brominated flame retardant with 16 possible stereoisomers, used for plastics and textiles. HBCD is highly lipophilic and accumulates in biota and is one of the most used BRFs (Heeb et al., 2005). The chemical structure of HBCD is shown in Figure 3 (Yamada-Okabe et al., 2005). HCBD is added as an additive or reactive component in a variety of polymers and over the past decades the demand of HBCD has increased (Wu et al., 2013). Figure 3. Chemical structure of HBCD Organochlorinated Compounds The organochlorines consist of a broad family of synthetic organic compounds, with chlorine substitutes. Most organochlorines are highly lipophilic (Mrema et al., 2013), and due to their persistence and bioaccumulation properties, some of them are grouped under Persistent Organic pollutants (POPs), The main exposure route for humans is via consumption of meat, fish and dairy products (Strom et al., 2014). Exposure can also occur via the placenta and primarily via breast milk (Klincic et al., 2014). 5 Polychlorinated biphenyls (PCBs) Polychlorinated biphenyls (PCBs) are synthetic organochlorine chemicals and they have been produced since the 1920s (Robertson and Ludewig, 2011). There are 209 different PCB compounds (congeners) and they all have different numbers and the composition of the chlorine substitutes in the molecule varies. Two six carbon rings, benzene rings, are linked to
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