Effects of perfluorinated compounds on hepatic fatty acid oxidation in avian embryos using a tritium release assay

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SCHOOL OF SCIENCE AND TECHNOLOGY Master program in Molecular Medical Biology

MASTER’S THESIS IN

MOLECULAR MEDICAL BIOLOGY

45 hp

HT2008-VT2009

Effects of perfluorinated

compounds on hepatic fatty acid

oxidation in avian embryos using a

tritium release assay

Results from a pilot study

Ola Westman

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INSTITUTIONEN FÖR NATURVETENSKAP

Abstract

The large use of perfluorinated compounds (PFCs) to produce fluoropolymers in consumer and industrial applications, including insecticides, plastics, non-stick surfaces and fire fighting foams has led to a well known widespread occurrence and high concentrations are found in wild life including avian species. For instance, concentrations of perfluorooctane sulfonate (PFOS) in eggs from the common guillemot in the Baltic Sea are among the highest in the Nordic environment. In our laboratory studies, PFOS has caused early mortality in chicken at doses close to concentrations found in eggs of the Baltic guillemot. The mechanisms behind the avian toxicity are unclear but many studies suggest mechanisms including lipid homeostasis. We have designed a method in which hepatic embryonic tissue from chicken (Gallus domesticus) is used to investigate the effects of PFCs on the β-oxidation of fatty acids. The purpose of this project was to assess the effects of PFOS, perfluorooctanoic acid (PFOA) or perfluorobutane sulfonate (PFBS) on the hepatic fatty acid oxidation using an egg injection technique followed by the use of a tritium release assay with palmitate (16:0) as substrate. The embryos were exposed in ovo and on day 10 of incubation embryo livers are incubated in vitro with tritiated fatty acids. The β-oxidation was significantly induced after exposed to 1 mg/kg PFOS (p = 0.003) and 10 mg/kg PFOS (p = 0.04), and difference in oxidation values was 39% and 34% respectively compared to control. The oxidation effect was not significant (p > 0.05) in samples exposed to PFOA (4 mg/kg) or PFBS (20 mg/kg), however noted, the difference in oxidation values was 18% and 30.5% respectively, compared to control calculated on current average. The results show that in ovo exposure in combination with an in vitro method, using a tritium release assay to detect effects on the β-oxidation of fatty acids in avian embryo hepatic tissue could be a useful method to elucidate possible mechanisms behind avian developmental toxicity.

Keywords: Chicken (Gallus domesticus), Hepatocytes, Perfluorooctane sulfonate

(PFOS), Perfluorooctanoic acid (PFOA), Perfluorobutane sulfonate (PFBS). Fatty acid oxidation, Tritium release assay.

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Introduction

The large use of perfluorinated compounds (PFCs) to produce fluoropolymers in consumer and industrial applications, including insecticides, plastics, non-stick surfaces and fire fighting foams has led to a well known widespread occurrence, from areas of use and production to remote parts of the world. The PFCs have recently been classified as new persistent organic pollutants (Giesy and Kannan, 2002; Renner, 2001; Renner, 2003; Kelly et al., 2007). Two widely distributed PFCs and commonly detected globally in a wide range of species and food webs the last decades are perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA). PFOS and PFOS-related products are found in more then 200 different applications that range from paints and waxes, to aviation hydraulic fluids etc. PFOS-related substances are compounds that belong to a fluorinated sulphonate group and they can break down to give what is known as PFOS in the environment. PFOA is being used in hundreds of applications as processing aid, for example, in the making of water-resistant clothing and non-stick surfaces on cookware and other man-made products (Hekster et al., 2003; Lau et al., 2004; de Vos et al., 2008). PFOS and PFOA are both members of the chemical group that goes by the name perfluouoalkylated substances (PFAS) and they have the same eight-carbon chain molecule structure. The PFAS belongs to a family of chemicals (fluorinated surfactants) containing fluorine in the structure that has replaced at least one hydrogen atom in the hydrophobic part of the molecule. If all hydrogens in the structure has been replaced by fluorine then we have a fully fluorinated compound a.k.a. perfluorinated surfactants. The fully flouorinated hydrocarbons are in many ways quit unique in their structure; they are, for example: non-flammable, hydrophobic, very stable in air at high temperature, have low surface tension, not subject for photolysis, and do not readily degrade by oxidizing agents, alkalis or even strong acids etc. Together, this makes them highly persistent and non-biodegradable in the environment (Lau et al., 2004; Nakayama et al., 2005).

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Due to the high persistence and bioaccumulation, PFOS and PFOA are found globally in various types of wildlife samples (Bossi et al., 2005; Beach et al., 2006; Giesy and Kannan, 2001; Hoff et al., 2005a; Holmström et al., 2005; Van de Vijer et al., 2005; Houde et al., 2006; Tao et al., 2006; Smithwick et al., 2006) and also globally in human samples, including breast milk, serum and plasma etc. (Apelberg et al., 2007; Calafat et al., 2007; Nakata et al., 2007; Kärrman et al., 2007; Ericson et al., 2008; Hölzer et al., 2008). Being the dominant PFC in the environment, high concentrations of PFOS, greater than other perfluorinated compounds, have been found in the liver tissue of top predators including polar bears (Ursus maritimus), bottlenose dolphins (Tursiops

truncates) and double-crested cormorant (Phalacrocorax auritus) (Giesy and Kannan,

2001; Kannan et al., 2005b; Houde et al., 2005; Smithwick et al., 2005), then for example PFOA which has been shown to be less bio accumulative compared with PFOS (Kannan et al., 2005). The PFCs, due to their amphiphilic properties, binds to proteins compared to other contaminants for instance polychlorinated biphenyls (PCBs) or polycyclic aromatic hydrocarbons (PAHs), which, in contrast, accumulate in fat tissue. One example of such protein, that PFCs bind to, is albumin, a protein mainly found in eggs, blood and liver (Jones et al., 2003; Han et al., 2003; Chen and Guo, 2009; Zhang et al., 2009).

Although the big PFC producers, a.k.a. the 3M Companies, started to phase out PFOS and other PFOS-related compounds that has the possibility to degrade to PFOS in the beginning of 2002 and instead began to use a alternatives to PFOS-compounds and PFOA, one known as perfluorobutane sulfonate (PFBS), the production of PFOS and other PFOS-related compounds are still being produced by manufactures outside the USA (Butenhoff et al., 2006). PFBS is an example of a more environmentally friendly perflouoroalkylated substance that contains a short setup with only a four carbon chain molecule structure which makes it impossible for PFBS to degrade into PFOS or PFOA and thereby considered to be less toxic and not so bio-accumulative in the environment (Newsted et al., 2008). PFOA and other PFOA-related chemicals that can degrade to PFOS in the environment are also still being produced, both in the USA and in other

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countries, although some the major companies have committed to reduce the production, no later then the year 2015, by 95% (Lau et al., 2004; Holmström et al., 2005).

The toxicity of PFCs has been studied for several years examining physiological actions, for example, exposure of PFOS and PFOA in mice and rats (Lau et al., 2003; Lau et al., 2006), toxicity of PFOS in monkeys (Seacat et al., 2002) and also in rabbits (Case et al., 2001), however, its effects on avian species, including laboratory studies and wild birds, are still less clear. A few avian studies have been presented over the years, addressing questions concerning the health impact of the PFCs in birds, most of them reporting reduced hatchability, induced liver abnormalities, decreases in body weight, increased mortality and accumulation sites of PFOS and PFOA (Giesy and Jones, 2001; Giesy and Jones, 2004; Newsted et al., 2005; Newsted et al., 2006; Molina et al., 2006; Yoo et al., 2009). In studies of wild birds, concentrations of PFOS have been reported in many species including bald eagles (Haliacctus leucocephalus) (Kannan et al., 2001), in songbirds near a fluorochemical plant (Danwe et al., 2007), in egg yolk samples from top predator birds in the area of Lake Winnipeg and Lake Huron (Giesy and Kannan, 2001) and also in plasma measured in galucous gulls (Larus

hyperboreus) from Svalbard (Verreault et al., 2005). Studies from the Baltic Sea have

reported very high concentrations of PFOS (1.023 mg/kg) measured in eggs from the common guillemot (Uria aalge) (Holmström et al., 2005). In our laboratory studies PFOS has caused early mortality in chicken at doses close to concentrations found in eggs of the Baltic guillemot (Engwall et al., 2006) and environmentally relevant concentrations has also been reported to cause brain asymmetry and immune alterations in chicken following in ovo exposure to PFOS (Peden-Adams et al., 2009). The mechanisms behind the avian toxicity are not well known, however, some laboratory studies suggest mechanisms including lipid homeostasis, disturbance of fatty acid metabolism or changes in the expression of genes associated with fatty acid oxidation (Kennedy et al., 2004; Yeung et al., 2007; Cwinn et al., 2008). The avian embryo metabolizes yolk fatty acids during incubation to obtain energy for development and

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growth (Sato et al., 2006; Moran, 2007). In avian, as in all animals, the liver is the main organ where fatty acids are being oxidized to produce ATP, and as presented previously in this text, the liver is also the organ in which high concentrations of PFCs are being found. Fatty acids consist of a chain of carbon atoms and the length of the chain varies between different groups, there are over a 1000 known fatty acids and a large range of them are present in organism, one of the most common ones is known as palmitic acid which has 16 carbons (16:0) in its chain. The fatty acids are transported in plasma bound to serum albumin and the metabolic system is highly conserved among the many different species, including; fatty acid β-oxidation and genes involved in metabolism and transportation of fatty acids etc (Eaton et al., 1996; Meng et al., 2005).

The catabolism of fatty acids to produce energy is achieved by enzymes that are present in the mitochondrial matrix initiated by the linking of coenzyme A to the carboxyl end of the fatty acid. The coenzyme-A derivate is then proceeding through a series of reactions that will split off a molecule a acetyl coenzyme A from the end of the fatty acid and then transfer two pairs of hydrogen atoms so that one pair is transferred to FAD and the other one to NAD. This reaction is known as β-oxidation and in this spiral of four catalyzing steps different overlapping chain length specific enzymes are active, for example, the medium long chain-acyl-CoA dehydrogenase (MCAD) responsible for chains of 4 to 12 in length and the long chain-acyl-CoA dehydrogenase (LCAD) which is responsible for taking care off carbon chains length between 8 to 18 etc. Most fatty acids are oxidized in mitochondria; however, some long chain and also the ones called very long chain fatty acids are oxidized in peroxisomes. In the final step the end products are primarily acetyl-CoA which then goes to the tricarboxylic (TCA) acid cycle to produce ATP, however if there remains any parts of acetyl-CoA then it goes right back into another spin in the β-oxidation spiral (Kunau et al., 1995; Coates and Tanaka, 1992; Wanders et al., 2001). The metabolic pathway is a sensitive mechanism that require very precise homeostatic control, especially during the embryonic period hormones associated with growth and gene expression are under production. For example, glucagon have a close relation to energy metabolism in the avian, but the most

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important hormones that are associated to the development and growth in the avian embryo are insulin-like growth factors I and II (IGF-I and IGF-II) and thyroxine (T4) (Lu et al., 2007; Zhou et al., 2007). In summary, fatty acid metabolism in order to obtain energy is a delicate system where even small changes of condition during embryo development could result in a state of disadvantageous outcome for the organism. Accumulating evidence collected recent years has demonstrated that PFOS and PFOA are affecting lipid metabolism, disturbing of fatty acid metabolism and changes in the expression of genes associated with fatty acid oxidation and fatty acid β-oxidation (Kennedy et al., 2004; Hu et al., 2005; Guruge et al., 2006). Overall, studies presenting effects on the fatty acid metabolism in avian due to exposure of PFCs and also possible mechanisms behind the avian developmental toxicity of PFCs are limited.

We have designed a method in which hepatic embryonic tissue from chicken (Gallus

domesticus) is used to investigate the effects of PFCs on the β-oxidation of fatty acids.

The PFCs used (mg/kg) in this pilot study were; perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) or perfluorobutane sulfonate (PFBS). The purpose of this project is to assess the effects of PFOS, PFOA or PFBS on the hepatic fatty acid oxidation using an egg injection technique followed by the use of a tritium release assay with palmitate (16:0) as substrate in this pilot study; however, other substrates, for example, myristate (14:0), octanoate (8:0) or other fatty acids of choice, can also be directly applied to investigate the spiral of fatty acid β-oxidation in avian or other species. Using this tritium release assay the effects of PFCs on avian hepatic fatty acid metabolism is studied in vitro, which can be a practical method in elucidating one of the possible mechanisms behind the avian developmental toxicity of PFCs or for other research purposes. In this method, the fatty acid undergoes oxidation and repetitive cleavage of carbons, which then enters the tricarboxylic pathway. It is then further metabolized and after successive cycles of β-oxidation the radioactive part of the fatty acid is released as tritiated water. The radioactive sample is collected and measured in a scintilator counter. The measured data from the tritiated water, e.g. converted β-particles into visible light, is then analysed and calculated with the help of standard formula and,

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in this study, expressed as pmol palmitate oxidized per mg chicken embryo hepatic tissue per hour (pmol/mg tissue/hour), which is proportional to the β-oxidation of the fatty acid used in the assay. Preliminary screening using cultured fibroblasts to detect defects in the fatty acid oxidation, measuring the production of tritiated water from myristate, palmitate or oleate radiolabelled with tritium at the 9,10 position have been widely used over the years (Manning et al., 1990; Olpin et al., 1992; Pollitt, 1995). Some initial work was performed before this current study in order to establish the conditions for this modified tritium release technique, with respect to, for example; incubation time, exposure time and other adjustments (data not shown). The method that was adapted as a platform in this new designed version of a tritium release assay was first described by N. Venizelos et al using fibroblasts in order to detect β-oxidation defects in patients (Venizelos et al., 1998) and this was in turn a refined version of the original method described by A. Moon and W. J. Rhead (Moon and Rhead, 1987). The method used in this pilot study is performed to detect enzyme effects in the chain of fatty acid oxidation of the avian embryo primary hepatocytes exposed in ovo to PFOS, PFOA or PFBS, using the entire embryonic liver in this new version of a tritium release assay. To the best of our knowledge, this is the first time such study has been performed.

Results

The results from the two separate series shows that the fatty acid oxidation in the chicken embryo primary hepatocytes was induced in some of the tested treatment groups compared to controls (Fig.1 and Fig.2). The values in this section are shown as mean and standard derivation (SD) in pmol/mg tissue/hour. The information is also presented in table (See Table 1). In the starting round, the oxidation of palmitic acid in the treated group of eggs injected with 10 mg/kg perfluorooctane sulfonate (PFOS) was

1.39 (0.13) pmol/mg tissue/hour, statistically significant (p = 0.003), and the group of

eggs injected with 4 mg/kg perfluorooctanoic acid (PFOA) that had an oxidation value of 1.18 (0.23) pmol/mg tissue/hour, not significant (p > 0.05), compared to the control group injected with 5% dimethyl sulfoxide (DMSO) which had an oxidation value of

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1.00 (0.16) pmol/mg tissue/hour. In the second round, the oxidation in the first group of

the eggs injected with 1 mg/kg PFOS was 0.79 (0.12) pmol/mg tissue/hour, statistically significant (p = 0.04), and the eggs injected with 20 mg/kg perfluorobutane sulfonate (PFBS) had an oxidation value of 0.77 (0.14) pmol/mg tissue/hour, not significant (p > 0.05), compared to the control group injected with milli-Q water, which had an oxidation value of 0.59 (0.13) pmol/mg tissue/hour.

Discussion

The purpose of this pilot study was to assess the effects of perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA) or perfluorobutane sulfonate (PFBS) on the hepatic fatty acid oxidation using an egg injection technique followed by the use of a tritium release assay with palmitate (16:0) as substrate. The results from this current pilot showed that the metabolism was induced in some of the treatment groups compared to the controls (Fig. 1 and Fig. 2). Important to point out is that the two series in this current study the controls are completely different. The control used in the first of the two series was 5% dimethyl sulfoxide (DMSO) in Milli-Q water since DMSO was being used as carrier. DMSO is commonly used in research studies to dissolve drugs or chemicals that do not dissolve well in water and in this current study we used 5% DMSO in sterile water (Milli-Q water) as carrier in the first set. In other related studies, 100% DMSO or 10% DMSO in safflower oil has been used in different ways (Molina et al., 2006; Peden-Adams et al., 2009). However, DMSO may also cause unexpected effects, including; increased effects of steroids, increased absorption of drugs in tissue and up/down regulation of many genes (Marks and Breslow, 2007). During initial studies performed before this current pilot study we included a control group of unexposed (control) samples in the test and the results (data not shown) showed higher oxidation values for the unexposed group compared to DMSO control suggesting that the actual oxidation values in this current study might in fact be even higher then the ones presented. Further studies should be executed to find out more details about the effects of DMSO on hepatic fatty acid oxidation.

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Now, when we take a look at the results from this current study we see that in the starting round we have significant effect (p = 0.003) in the treated group of eggs injected with 10 mg/kg PFOS, showing an oxidation value of 1.39 (0.13) pmol/mg tissue/hour, which means that we have a difference of 39% compared to the control. The second treated group of eggs injected with 4 mg/kg PFOA showed an oxidation value of 1.18 (0.23) pmol/mg tissue/hour, the result here was not statistically significant (p > 0.05) however, oxidation value 18% in difference compared to the control, which had oxidation value of 1.00 (0.16) pmol/mg tissue/hour. In the second test we had injected the first group of eggs with 1 mg/kg PFOS and the result from this set showed significant effect (p = 0.04), the oxidation value was 0.79 (0.12) pmol/mg tissue/hour, which means that the oxidation difference was 34%. This environmentally relevant dose is close to the concentration that has been reported in eggs (1.023 mg/kg) from the common guillemot living in the Baltic Sea area (Uria aalge) (Holmström et al., 2005). Approximate dose has also been reported in a laboratory study to cause brain asymmetry and immune alterations in chicken following in ovo exposure (Peden-Adams et al., 2009) and in our laboratory studies same concentration has caused early mortality in chicken (Engwall et al., 2006). The eggs injected with 20 mg/kg PFBS had an oxidation value of 0.77 (0.14) pmol/mg tissue/hour. The value was not significant (p > 0.05) however, the difference in oxidation value was 30.5% compared to the control, which had an oxidation value of 0.59 (0.13) pmol/mg/hour. This might be of interest in the context of PFBS being a perfluorinated compound used as an environmentally safer alternative compared to PFOS and PFOA, suggesting that future studies should be executed in order to clarify these findings. The dose 20 mg/kg PFBS used in the current study did not result in any early mortality; however, during initial studies eggs injected with 40 mg/kg resulted in 100% mortality (data not shown), suggesting that LD50 in

chicken exposed to PFBS in ovo being approximately 30 mg/kg. Further studies should be performed to investigate the action and toxicity of PFBS in avian species. During incubation the eggs were examined to control viability and remove any dead embryos (Table 1), no deceased embryos were removed in the second test. However, the dose of PFOS used in the first set was 10 mg/kg egg, weight which is LD50 level for in ovo

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injection in chicken seen in our previous laboratory studies (data not shown), had an mortality of 53%, followed by a mortality of 26% in the treated group injected with 4 mg/kg PFOA, however in this group the LD50 level is still a bit unclear when it comes

to the in ovo treatment in chicken and should be investigated further.

The purpose of this project was to assess the effects of PFOS, PFOA or PFBS on the hepatic fatty acid oxidation and the results shows that our method using a tritium release assay appears to be a very useful tool elucidating possible mechanisms behind avian developmental toxicity. Different types of disorders in the metabolic machinery causes differences in released amount of tritium, a difference in released amount of tritium greater than 20% compared to control in 3 experiments is needed to claim a statistical significance (Brivet et al, 1999). The explanation of the observed increased metabolism of fatty acid in this current pilot study is not clear. As we mentioned in the introduction, collected evidence has demonstrated that PFCs are affecting lipid metabolism and, for example, causing changes in gene expression and disturbing fatty acid metabolism etc (Kennedy et al., 2004; Hu et al., 2005; Guruge et al., 2006). Our method is clearly useful as an early step for measurement of fatty acid oxidation following exposure to PFCs; however, to get more detailed information one will have to use other research techniques. Now, in this current study we have reported increased metabolism due to PFC exposure, what is the explanation to this result and how can that affect the organism? Well, some possible explanations have been proposed by researchers resent years. One simple explanation, suggested by Hu et al. (2005) and Guruge et al. (2006), is that the PFCs in some way are mistaken as a degradable substrate by the enzymes in the β-oxidation spiral due to the structural similarity between fatty acids and therefore are able to enter the metabolic machinery. The fact that these perfluorinated compounds not are degradable will lead to a situation were β-oxidation fails to metabolize the PFCs which means that the machinery then will be forced to try to compensate this by activating more enzymes to take care of the load (Hu et al., 2005; Guruge et al., 2006). Hu et al. (2005) also suggests another explanation, namely, that PFOS alters the permeability of the peroxisomal membrane in a way so that the fatty acid influx

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increases which then would lead to higher enzyme activity (Hu et al., 2005). One possible way that this increase can affect the organism, suggested by Hu et al. (2005) and Guruge et al. (2006), is that potentially toxic hydrogen peroxide may be produced from acyl-CoA as a result of significant induction and this situation can cause oxidative stress which is a potential threat to the organism (Hu et al., 2005; Guruge et al., 2006). However, further studies are needed to confirm all these suggestions. Future studies using this method should also include other fatty acids with different chain length than palmitate, for example, myristic acid (14:0) or oleic acid (18:0), to put some more stress on other enzymes in the β-oxidation, in order to see if this changes the oxidation value. We also suggest that in the next step include protein determination of tissue by using the Bradford method as a complement to the system now in use. Further studies assessing fatty acid metabolism in avian should also include cell cultures and microarray techniques to study gene expression patterns, to see if these induced effects following exposure to PFOS, PFOA or PFBS are consistent or vary among other avian species. Finally, future studies should continue to provide research results assessing perfluorinated compounds and related chemicals introduced by the industry as new alternatives to old ones, in order to obtain information about the effects if used and to guard the environment from health hazards.

To summarize, in this current study we have reported of induced metabolic effects on the hepatic β-oxidation system following exposure in ovo to PFOS, PFOA and PFBS. The metabolism was clearly induced in some of the treatment groups compared to the controls calculated on current average. A significant increase of oxidation value was seen in the embryo primary hepatocytes following exposure to PFOS 1 mg/kg (p = 0.003) and 10 mg/kg (p = 0.04), difference in oxidation values was 39% and 34% respectively, compared to control. The oxidation values were not significant (p > 0.05) in samples exposed to PFOA (4 mg/kg) and PFBS (20 mg/kg), however noted, the oxidation values were 18% and 30.5% in difference, respectively, compared to control.

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Conclusions

Our studies suggest a small but significant increase in β-oxidation of fatty acids in chicken embryonic liver tissue in vitro after in ovo exposure to PFOS. The mechanisms behind the avian toxicity of PFOS are unclear but many studies in mammals suggest mechanisms including lipid homeostasis. We have designed a method in which hepatic embryonic tissue from chicken (Gallus domesticus) is used to investigate the effects of PFCs on the β-oxidation of fatty acids. The results show that in ovo exposure in combination with an in vitro method, using a tritium release assay to detect effects on the β-oxidation of fatty acids in avian embryo hepatic tissue could be a useful method to elucidate possible mechanisms behind avian developmental toxicity.

Methods

On day 0, performed in two separate series, fertilized, un-incubated chicken eggs (tot. n

= 114) were obtained from Ova Production, Vittinge, Sweden and then placed in a circulated air incubator with rotating bars (J. Hemel Brutgeräte, A420) with a maintained temperature at 37.5 C and 60% relative humidity. The eggs were candled the following days to assess viability and eggs that did not develop (tot. n = 24) were excluded from the study.

On day 4 of incubation the eggs were randomly assigned into different treatment

groups and marked for identification. The eggs were candled to check viability, then weighed (Denver Instrument, DL-501), and cleaned with 70% alcohol followed by marking the air cells with pencil. Following drilling (Proxxon GG12) the eggs were then exposed in ovo by injections of l/g per egg into the air cell with sterile pipette and the holes then sealed with melted paraffin. In the starting round, the treated groups were injected with 10 mg PFOS/kg (CAS: 2795-39-3; LOT: 77282) (n = 15, mean egg wt = 53.3 g), 4 mg PFOA/kg (CAS: 335-67-1; LOT: 17,146-8) (n = 15, mean egg wt = 50 g) or 5% dimethyl sulfoxide (DMSO) carrier control (n = 9, mean egg wt = 44.2 g). In the second round, the eggs were injected with 1 mg PFOS/kg (n = 12, mean egg wt = 58.8

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g), 20 mg PFBS/kg (CAS: 29,420-49-3; LOT: S41139-148) (n = 12, mean egg wt = 56.6 g) or milli-Q water (Millipore®) control (n = 10, mean egg wt = 51.49 g). The eggs were then again placed in the incubator with a maintained temperature at 37.5 C and 60% relative humidity. The following days the eggs were candled to control viability and any dead embryos were removed.

On day 10 the incubation ended and the eggs were examined to control viability. From

the remaining number, six eggs from each group were randomly chosen and the embryos were dissected to remove livers followed by weight control (MyCal, SI-114) of the livers. The livers were quickly transferred into a 24 cell well plate containing serum free medium (MEM Alpha Medium, LOT: 447390) placed in a incubator (Termaks, B8133) with a maintained temperature at 37.5°C and 60% relative humidity and slowly rocked on a horizontal shaker (LIC Instruments, 440) for 2 hours.

Fatty acid preparation

The livers were then to be exposure to the palmitic mixture (palmitate 16:0) performed in a period of 2 hours. The pre-prepared mixture contained 75 μl (0.84 nmol) radioactive palmitic acid [9,10 (n)-3H] [1 mCi Amersham TRK 909] with a specificity of 47,7 Ci/mmol and 810 μl (108 nmol) unmarked palmitic acid (Sigma P 0500, CAS 57-10-3) suspended in 40 μl 1M NaOH 96% ethanol (Kemetyl, CAS: 64-17-5). The mixture was then evaporated under of gentle stream of nitrogen gas at 30-40 C until completely dry and the palmitic salt was then re-suspended with 1 ml Hank's buffered salt solution (HBSS) (SVA, Batch 5512, Art.nr: 991761) containing 5 g/L bovine serum albumin (BSA) (Sigma, Fraction V 96%, CAS: 9048-46-8) mixed (BioSan, Vortex VI Plus, V100876) and then stirred on a orbital shaker for 1 hour at 400 rpm (IKA®, Taquara, MS1 Minishaker, RJ 22713-000) and then suspended with 9 ml HBSS. The final concentration of total fatty acid was then up to a total of 21 mol/L.

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Tritium release assay

The medium was suspended from the 24 well plates and then the livers were washed with 400 μL HBSS two times. A total number of three tissue samples were then randomly picked, one from each of the three groups, and treated with 1 mL methanol (Merck M1.06007, CAS: 67-56 1) for 10 minutes to obtain negative controls. The hepatic tissue were then exposed to 500 μL palmitic mixture and placed ones more in the incubator with a maintained temperature at 37.5°C and 60% relative humidity and slowly rocked on the horizontal shaker for 2 hours. The reaction was aborted by placing the well plate on ice.

Dowex ion exchange

On day 11 400 μL sample was extracted from each well into micro tubes containing

400 μL 10% trichloroacetic acid (TCA) (Merck M1.00807, CAS: 76-03-09) making a total volume of 800 μL sample in each micro tube and the tubes were then centrifuged at 8500g for 8 minutes (Eppendorf AG, Centrifuge 5415D). The hepatic tissue were saved and stored in freezer. The supernatants were then directly extracted into new micro tubes and mixed with 140 μL 6mol/L NaOH (Merck M1.06498, CAS: 1310-73-2) and then a final sample volume of 900 μl was transferred into pre-prepared Pasteur pipettes columns (Bilbate, Pasteur pipette, short form, 150 mm) loaded with 1 mL Dowex ion exchange (200-400 Mesh Fluka 44340, CAS: 60267-37-0), that binds un-metabolized fatty acids and exchanges them for chloride ions, letting the tritium water pass. The Dowex had been pre-prepared, e.g. mixing 12 g Dowex with 40 mL milli-Q water and left to swell for 3 hours, then washed twice more with same volume milli-Q water, then transferred into the columns and finally eluted three times more with 1 mL milli-Q water.

Scintillation and analysis

Ones the samples were transferred into the pipette columns each were then eluted with 1 mL milli-Q water and collected in scintillation vials, and then 600 μl sample were taken

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from each vial and transferred into new scintillation vials followed by adding 8 mL scintillation cocktail (OptiPhase, Hisafe 3,Wallac SC/9195/21, PerkinElmer) into each vial. The vials were then placed in the scintillation counter (Wallac Winspectral™ 1414 Liquid Scintillation Counter, PerkinElmer) together with two other scintillation vials each containing 50 μL palmitic mixture and 8 mL scintillation cocktail and two vials containing only 8 mL scintillation cocktail. Two randomly chosen scintillation vials containing palmitic mixture were recounted after added 50 μL 3H-Toluen standard (DuPont/NEN, NES-004) with a radioactive specificity of 2.45*106 disintegrations per minute (DPM). The measured data from the tritiated water e.g. converted β-particles into visible light, was then analysed, calculated with the help of standard excel formula (calculations not shown) and expressed as pmol palmitate oxidized per mg hepatic embryonic tissue per hour (pmol/mg tissue/hour), proportional to the β-oxidation of the fatty acid used in the assay. The test series contained five samples in each group from which mean value and standard derivation (SD) were calculated and compared to controls. A t-test was used to compare the difference of oxidized palmitate acid and the results from that measured data were presented as oxidation value in the report. Statistical significance was set at p < 0.05.

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Acknowledgements

This degree project was performed at the MTM Research Centre, Department of Natural Science, Örebro University with laboratory support from the School of Health and Medical Science, Department of Clinical Medicine, Örebro University.

First of all I wish to sincerely thank my head supervisor Professor Magnus Engwall, Department of Natural Science, for the opportunity, ideas, guidance and supporting my degree project. I also wish to express my sincere gratitude to Associate Professor Nikolaos Venizelos, Department of Clinical Medicine, for the opportunity, advices, laboratory support and nice talks. Special thanks to Ph.D. Sdt. Marcus Nordén, for suggestions, corrections, support in the lab and being a good friend. Also big thanks to Teaching Assistant Jessica Johansson and Ph.D. Sdt. Ravi Vumma for laboratory support. Finally, extra special thanks to my parents Hans Svedlund and Karin Westman for their love and never ending support.

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Figures and tables Figure 1. -0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60

PFOS 10 m g/kg PFOA 4 m g/kg Control

p m o l/ h m g t is s u e

Figure 1. Showing oxidation values from the induced metabolic effects on

the hepatic β-oxidation system following exposure in ovo to PFOS and PFOA in the current study. The metabolism was induced in some of the treatment groups compared to the control calculated on current average. A significant increase of oxidation value was seen in the embryo primary hepatocytes (n = 5) following exposure to 10 mg/kg egg weight PFOS,

1.39 (0.13) pmol/mg tissue/hour (p = 0.003), the difference in oxidation

compared to control was 39%. The oxidation value 1.18 (0.23) pmol/mg tissue/hour were not significant (p > 0.05) in PFOA samples (n = 5), 4 mg/kg egg weight, however, and the differences in oxidation was 18% compared to control which had an oxidation value of 1.00 (0.16) pmol/mg tissue/hour.

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INSTITUTIONEN FÖR NATURVETENSKAP Figure 2. -0,20 0,40 0,60 0,80 1,00 1,20 1,40 1,60 PFOS 1 m g/kg PFBS 20 m g/kg Control p m o l/ m g t is s u e / h

Figure 2. The metabolism was also induced in some of the treatment

groups of the second round compared to the control calculated on current average. A significant increase of oxidation value was seen in the embryo primary hepatocytes (n = 5) following exposure to 1 mg/kg egg weight PFOS, 0.79 (0.12) pmol/mg tissue/hour, (p = 0.04), and oxidation difference was 34% compared to control. The group (n = 5) exposed to 20 mg/kg egg weight perfluorobutane sulfonate (PFBS) had an oxidation value of 0.77 (0.14) pmol/mg tissue/hour, not significant (p > 0.05), compared to the control, however, the difference in oxidation was 30.5%. Oxidation value for the control (n = 5) was 0.59 (0.13) pmol/mg tissue/hour.

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Table 1. Results from the current pilot study showing mean values and

standard derivation (SD) of the palmitic acid oxidation rate in the chicken embryo primary hepatocytes and mean weight of the hepatic (liver) tissue in the treatment groups.

1st Test Tissue weight Oxidation value

Mean Mean SD

Treatment Numbers (n) (mg) (pmol/mg tissue/hour) P-value PFOS 10 mg/kg: (n=5) 42.76 1.39 0.13 0.003 PFOA 4 mg/kg: (n=5) 37.20 1.18 0.23 0.20 Control (DMSO): (n=5) 30.12 1.00 0.16

2nd Test Tissue weight Oxidation value

Mean Mean SD

Treatment Numbers (n) (mg) (pmol/mg tissue/hour) P-value PFOS 1 mg/kg: (n=5) 32.40 0.79 0.12 0.04

PFBS 20 mg/kg: (n=5) 33.44 0.77 0.14 0.08 Control (Milli-Q): (n=5) 37.12 0.59 0.13

Figure 3.

Figure 3. Showing mortality. During incubation 53% of the embryos

exposed to 10 mg/kg egg weight perfluorooctane sulphonate (PFOS), 26% of the embryos exposed to 4 mg/kg egg weight perfluorooctanoic acid (PFOA) were removed from the first round in the current study and also one (11%) from the control (DMSO) group. No deceased embryos were found in the second round.

0 0,5 1 1,5 2 2,5 3 3,5 4 4,5

Day 1 Day 2 Day 3 Day 4 Day 5

Number of deceased embryos:

PFOS 10 mg/kg

PFOA 4 mg/kg

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