Teratogenic and Embryotoxic Effects of Polycyclic Aromatic Compounds Report

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Teratogenic and Embryotoxic Effects of

Polycyclic Aromatic Compounds

Report

Svenja Wagner, B.Sc. Örebro, February 2016

Course: Environmental Science, Independent Project for the

Degree of Bachelor, MX107G, G2E, 15 Credits

Supervisor: Steffen Keiter

Examinator: Magnus Engwall

ECT grade: B

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1. Introduction

Pollutants are ubiquitously distributed in the environment (Shimada et al. 2003; Upham et al.1996). Consequently, humans and animals are continuously exposed to contaminants, occurring in natural forms as well as in synthetic processed manner as heavy metals or polycyclic aromatic compounds (PACs). The present study is focusing on the teratogenic and embryotoxic effects of certain polycyclic aromatic compounds as they are abundant in the terrestrial and aquatic environment (Braga et al. 2000, Vendrame et al. 2000).

PACs not only appear naturally in soils or accrue through forest fires and ocean oil seeps, but are also existent in urban air, street dust due to automobile exhaust and besides also in soot, cigarette smoke or industrial processed food (Billiard et al. 2006; Braga et al. 2000; Machala et al. 2001; Shuttleworth and Cerniglia 1995; Tuyen et al. 2014; Upham et al. 1996). Other industrial products containing PACs are crude oil and coal tar (Trilecová et al. 2011; Upham et al. 1996; Weis et al. 1998). According to this, sediment concentrations of PACs in urban and industrialized centers are up to two-thirds higher than in rural areas (Dabestani et al. 1999).

1.1 Polycyclic Aromatic Compounds (PAC)

PACs are a class of planar organic molecules, consisting of two or more fused benzene rings, inducing chemical carcinogenesis and generating developmental malformations due to their teratogenicity and embryotoxicity (Braga et al. 2000; Machala et al. 2008; Rhodes et al. 2004; Vendrame et al. 2000). PACs are formed during incomplete combustion for instance of fossil fuels or pyrolysis of other organic materials, as well as from chemical reactions of PACs in the atmosphere and metabolic reactions in organisms (Billiard et al. 2006; Larsson et al. 2014; Upham et al.1996; Weis et al. 1998).

The carcinogenicity of PACs varies from very strong carcinogens to inactive ones Braga et al. 2000). Chemical substitution in PAC molecules, for instance methylation, can drastically affect the carcinogenic activity of PACs depending on the site of substitution and on the number of substituted groups (Braga et al. 2000; Marvanová et al. 2007). The presence of methyl groups breaks the electron-hole symmetry of non-methylated molecules (Vendrame et al. 2000). As a result, active molecules can become inactive or the other way around, so that the carcinogenicity of PACs can be increased or decreased through the structural alteration of the molecules (Braga et al. 2000).

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1.2 Effect of Polycyclic Aromatic Compounds on fishes

The aquatic environment is a collecting pond for many chemicals such as PACs and therefore has high pollution levels. Thus, for instance contaminated marine or river sediments may affect aquatic organisms as fishes (Larsson et al. 2014; Machala et al. 2001). Fishes are an important food resource for human beings and major sentinels for quality of the aquatic environment (Lammer et al. 2008). Polycyclic aromatic compounds can cause diverse damaging effects in early-life stages of fishes as pericardial and yolk sac edemas, reduced growth, craniofacial deformities as well as cardiovascular dysfunction and death after the chronic exposition during the embryonic state (Barron et al. 2003; Billiard et al. 2006). The embryonic toxicity is not only attributed to the sensitivity of the early life-stage in general and the exposure during critical developmental periods, but also to the high bioaccumulation and limited biotransformation of polycyclic aromatic compounds (Petersen and Kristensen 1998).

1.3 Aim of the study

Methylated and hydroxylated PACs are ubiquitously distributed pollutants in the environment but are not well analyzed yet (Tuyen et al. 2014). Therefore, the aim of the present study is to determine particular properties of selected methylated and hydroxylated PACs regarding their embryotoxicity and teratogenicity, using the Fish embryo toxicity test (FET) with Danio rerio as test organism. Specifically we aim on the determination of the LC50-values for the tested PACs. The zebrafish (Danio rerio) has been selected as it has evolved to one of the most prominent in vivo model organisms in environmental science to investigate the potential health risk of pollutants towards humans and wildlife. Moreover, the zebrafish has achieved high popularity as model organism based on specific properties as its small size, high fecundity and rapid development on the one hand, and its amenability to genetic and chemicals screens and the extensive literature base, on the other hand. (Braunbeck et al. 2014; Laale et al.1977).The identification of LC50-values is useful for the risk assessment of the selected PACs.

Furthermore, more specifically the purpose of the study is the measurement of length of the zebrafish tail applying the Tail length test (TLT) to receive information about the embryotoxic and teratogenic effect of 7,12-dimethylbenzo[a]anthracene on aquatic organisms.

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2. Material and Methods

2.1 Chemicals

Table 2.1: List of used chemicals

Chemicals Abbreviation Purity CAS-Number Supplier

9-(10H) Acridone 9-Acr 99% 578-95-0 Sigma-Aldrich

9-methylacridine 9-MA 99% 611-64-3 Chiron AS

7-methyl

benzo[a]anthracene 7-MBA 99% 56-55-3 Sigma-Aldrich

7,12-dimethyl

benzo[a]anthracene DMBA ≥99% 57-97-6 Sigma-Aldrich

7-methyl

benzo[a]pyrene 7-MBaP 98% 63041-77-0 Sigma-Aldrich

3,4 dichloroaniline 3,4-DCA 98% 95-76-1 Sigma-Aldrich

Dimethylsulfoxide DMSO ≥99% 67-68-5 Sigma-Aldrich

Different methylated and hydroxylated PACs are tested on their acute toxicity. 9(10H)-acridone (9-Acr), a derivate of acridine, which alkaloids are used as cancer and anti-malaria drugs and which as well show antimicrobial activity against bacteria such as Bacillus subtilis (Basco et al. 1994; Guilbaud et al. 2002; Lwande et al 1983). 9-methylacridine (9-MA) is also a derivate of acridine with a methyl group at position 9. 9-Acr is mildly basic solid and naturally occurs in coal tar (Pereira et al. 1983). 7-methylbenzo[a]anthracene (7-MBA) the methylated derivative of benzo[a]anthracene, exhibits only a weak tumor initiating activity compared to 9-MA (Norpoth et al. 1984). Particularly, 7-MBA has the ability to induce DNA damage in form of DNA adducts, whose accumulation is an approximate value for the exposition with carcinogenes (Marvanová et al. 2008). 7-MBA can induce the expression of Cytochrome P450 monoxygenase enzymes (Tuyen et al. 2014).

7,12-dimethylbenzo[a]anthracene (DMBA), the double methylated derivative of anthracene, is the most potent mutagenic and carcinogenic PAC. DMBA is used as an experimental tumor inducer in skin and mammary tissue and as an immunosuppressor (Higginbotham et al. 1993).

7-methylbenzo[a]pyrene (7-MBaP), the single methylated derivate of benzo[a]pyrene, is a potent genotoxic PAC. 7-MBaP is classified as a human carcinogen, especially causing skin cancer (Trilecová et al. 2011). It mainly occurs in coal tar, soot and exhausted gas of automobiles, but also in cigarette smoke and through roasting of coffee beans.

2.2 Test organism

Zebrafish (Danio rerio) has been selected as a test organism to analyze the teratogenic and developmental effects of the PACs. This tropical freshwater fish belonging to the Cyprinidae family lives naturally in slow flowing or stagnant water in the Ganges River System in South and Southeast-Asia. The silver and blue striped coloured fishes can reach a length of 4.5 cm

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and have short generation cycle of 2-3 month. Females are able to spawn the whole year around every 2 to 3 days under ideal conditions, whereas each female produce 200 until 300 eggs with a transparent chorion (Scholz et al.2008).

Danio rerio is a common used test organism, because of several reasons. The transparency of the egg encasing chorion enables the observation of the extraordinarily fast larval development. Another advantage, in using Danio rerio as a test organism is its high fecundity (Braunbeck et al. 2014). In addition, the maintenance of zebrafishes and the harvest of the eggs is easy achievable and keen (Laale 1977). Moreover, the use of zebrafish eggs for toxicity testing is a good alternative to fish acute toxicity testing and reduces the total amount of animal testing in concern for animal welfare and to apply the principles of the 3R’s (Replacement, Reduction and Refinement) established by Russell and Burch 1959 (Fraysse et al. 2006; Lammer et al. 2008). Both embryo in its pre-hatching stage, and eleuthero-embryo before the onset of exogenous feed, are considered as substitutes, since the relationship between both is very strong (Lammer et al. 2008). The embryo is regarded as most vulnerable stage in the developmental process and due to that is a clear indicator of chemical toxicity (Fraysse et al. 2006; Yang et al. 2009). The cells in developing embryos, particularly at early stages, are very sensitive to chemicals and toxicants, since they do not have an active immune system (Ikegami et al. 1997). The embryonic life stage is also highly predictive of later life stages and biological and molecular mechanisms during the embryogenesis could be influenced and besides impaired by chemicals and toxicants (Fraysse et al. 2006; Lammer et al. 2008).

For toxicity testing the fish embryo toxicity test (FET) in accordance to OECD guideline 236 has been applied, which uses definite morphological criteria as mortality endpoints for the calculation of LC50 of the tested chemical compounds(Tab.2.2).

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Table 2.2: Morphological criteria in accordance to OECD guideline 236. The morphological criteria are distinguished in lethal endpoints and sub-lethal malformations. The development of the fish embryo is controlled after 24, 48, 72 and 96 hours post fertilization (hpf).

Endpoint 24 hpf 48 hpf 72 hpf 96 hpf Lethal endpoints Coagulated embryo + + + + Lack of somite formation + + + + Non-detachment of the tail + + + + Lack of heartbeat + + + Sub-lethal malformations Underdevelopment of the eyes + + + + Lack of blood circulation + + + Unfertilized egg + + + + Abnormal heartbeat/abnormal blood circulation + + +

Hatched too early + + +

Hatch length + Edema (pericard, yolk) + + + Lack of pigmentation + + + Deformation of the embryo + + + Deformation of the tail + + + Underdeveloped embryo + + + +

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8 2.3 Fish maintenance and egg retrieval

Zebrafishes of the Strain AB from Karolinska Institute Zebrafish Core Facility, Stockholm (Stock no. 1407) are kept in glass aquaria at a water temperature of 26 +/- 1 °C, with a pH value of 7.2 and a water hardness of 3-6 °dH. A constant day/ night (14 / 10 hours) rhythm is maintained. The fishes are fed 7 days a week twice a day. In the morning, the food consists out of dry flakes, whereas in the afternoon the fishes get live Artemia nauplii at least four times a week, preferably the day before spawning to enhance the egg production. The size of the spawning group amounts to one third male and two-thirds female per spawning tank. Once or twice a week spawning vessels, consisting of a flat basin which is covered by a synthetic net provided with artificial plants, are placed in the aquaria tanks before the onset of light in the morning. The artificial plants display a breeding stimulus, whereas the net prevents the fishes from feeding the own breed. Each mating, spawning and fertilization proceeds 30min after the onset of light in the morning. The spawning vessels are carefully removed and the eggs are gathered in beakers no more than two hours subsequently to the onset of light.

2.4 Selection of fish eggs

The fertilized eggs are selected visually using a binocular microscope. Freshly spawned eggs are characterized by a fully transparent perivitelline space, containing the yolk with the germinal disc forming the animal pole, and surrounded by the egg membrane (Lammer et al. 2008). For the following Fish Embryo Toxicity and Tail Length Tests, only normal developed eggs at 8-cell stage at the least were picked. The eggs were transferred into oxygen saturated ISO water with a hardness of 100-300mg/l CaCO3 and a temperature of 26 +/- 1°C (OECD 2013).

2.5 Test design- Fish Embryo Toxicity Test (FET) with Danio rerio

The eggs were transferred in the appropriate testing solution, covered with parafilm to prevent evaporation, to start the exposure as soon as possible, but no later than 90 minutes after the fertilization or rather the 16 cell stage.

Artificial ISO water in the absence of a chemical compound was used as negative control (mortality <15%), whereas 3,4-dichloroaniline (3,4-DCA) with a concentration of 4mg/l dissolved in millipore water was utilized as positive control (mortality>80%). Additionally, a solvent control composed of 0.4% dimethyl sulfoxid (DMSO) (mortality<15%) was prepared to prove that the solvent has neither significant effects on the hatching success or the mortality rate nor on the normal healthy development of the embryo without phenotypic defects (OECD 2013). Each chemical was tested in five different concentrations in two or

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three independent technical replicates with 20 biological replicates of Danio rerio eggs per test concentration and per control.

Table 2.3: Concentrations of test solutions for each of the tested methylated PACs in FET. Concentrations are given in µM chemical compound per L solvent solution.

C1 [µM/L] C2 [µM/L] C3 [µM/L] C4 [µM/L] C5 [µM/L] 9-Acr 10 5 2.5 1.25 0.625 9-MA 4 2 1 0.5 0.25 7-MBA 10 5 2.5 1.25 0.625 DMBA 10 5 2.5 1.25 0.625 7-MBaP 10 5 2.5 1.25 0.625

The concentrations were prepared as dilutions of the DMSO stock solution with artificial water. Consecutively, the selected eggs were transferred into 96-well plates filled with 250ml freshly prepared test solutions for each concentration and controls per well. Concluding, the well plates were covered with a gas-permeable foil sheet and incubated at 26+/- 1°C for 96h exposure. The test solutions and controls were changed daily. Every 24h the development of the zebra fish embryos was controlled with an inverted microscope at magnification of 4x (Inverted Microscope, Olympus CKX41). Thereby, both teratogenic effects on the development as the tail curvature scoliosis, and embryotoxic effects as mortality by means of lethal endpoints were documented with the Microscope Imaging Software Olympus cellSensTm. The coagulation of the embryo, lack of somite formation and non-detachment of the tail after 24h as well as lack of heartbeat after 48h were considered as lethal endpoints. After 96h exposure, the test was terminated by anaesthetizing the embryos with 99% pure ethanol.

2.6 Test design- Tail Length Test (TLT) with Danio rerio

After selecting the fertilized eggs, in each case 15 of them were transferred into 96-well plates, containing 250ml of freshly prepared test solution of 7,12 dimethylbenzo(a)anthracene with a concentration of 5µM/L respectively ISO water as negative control and 0,4% DMSO solution as solvent control. The plate was covered with foil sheet and stored in the incubator at 26+/- 1°C for 96h exposure. The development of the embryos was examined every day and kept records of with the Microscope Imaging Software Olympus cellSensTm. Also the solution and controls were changed from day-to-day. After 96h exposure, the zebrafish embryos were severally successively converted into a 24-well plate, comprising 2ml saturated benzocaine solution, to measure the tail length in a tranquilized condition. The tail length was measured from the beginning of the first somite (anterior part)

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to the end of the most posterior somite (Fraysse et al. 2006). Concluding, the embryos were anaesthetized with 99%pure ethanol.

2.7 Data analysis

All spreadsheet calculations were conducted with Microsoft ExcelTM 2007. Graphs were plotted either with Microsoft ExcelTM 2007 or with GraphPad PrismTM 5. Statistical analyses were performed with GraphPad PrismTM 5. The datasets of the FET test were analyzed with non-linear regression to determine the LC50 values, whereas the datasets of the TLT test were analyzed using the unpaired two-tailed t-test. Passing this one with p<0.05, the TLT data sets were evaluated statistically with one-way ANOVA on ranks or non-parametric Kruskal-Wallis-Test and Dunn’s post-hoc test, subsequently.

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11 9-Acr (24 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-Acr PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 9-Acr (48 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-Acr PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 9-Acr (72 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-Acr PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 9-Acr (96 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-Acr PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ]

3. Results

3.1 Fish Embryo Toxicity Test with Danio rerio

The mortality rate of zebrafish embryos was evaluated for five different concentrations for each of the tested PACs as well as for the negative (NC), solvent (DMSO) and positive (PC) control after 24, 48, 72 and 96 hour exposure (Figure 3.1; 3.2; 3.3; 3.4; 3.5).

Figure 3.1.: Mortality rate of zebrafish embryos in % after 24, 48, 72 and 96 hour exposure with five different concentrations of 9(10H)-Acridone (9-Acr) analyzed with non-linear regression. Black dots represent 9-Acr, red squares depict the positive control (PC), and whereas the green triangles show the negative (NC) and the blue triangles show the solvent control (DMSO).

The mortality rate of zebrafish embryos exposed to five different 9-Acr concentrations is approximately 0% after 24h and does not increase during the remaining period of time. According to this, 9(10H)-acridone shows hardly any acute toxic effect on the embryonal development of Danio rerio. Both the negative and solvent controls have a similar mortality rate around 0% during the whole exposure period as expected. The positive control shows a rising mortality rate from 24h to 96h and achieves a value of 100% mortality after 96h as anticipated (Figure 3.1). Figure 7.1 in the supplementary material displays the mortality rate of each concentration of 9-Acr of both replicates separately.

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12 9-MA (24 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-MA PC NC DMSO LC50 = 2.517M/L log concentration [µM/L] M o rt a lit y [ % ] 9-MA (48 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-MA PC NC DMSO LC50 = 2.413M/L log concentration [µM/L] M o rt a lit y [ % ] 9-MA (72 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-MA PC NC DMSO LC50 = 2.341M/L log concentration [µM/L] M o rt a lit y [ % ] 9-MA (96 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 9-MA PC NC DMSO LC50 = 2.341M/L log concentration [µM/L] M o rt a lit y [ % ]

Figure 3.2: Mortality rate of zebrafish embryos in % after 24, 48, 72 and 96 hour exposure with the five different concentrations of 9-methylacridine (9-MA) analyzed with non-linear regression. Black dots represent 9-MA, red squares depict the positive control (PC), and whereas the green triangles show the negative (NC) and the blue triangles show the solvent control (DMSO).

In contrast to 9-Acr, 9-methylacridine indicates a strong acute toxic effect on the embryonal development of the zebrafish. The mortality rate of the zebrafish embryos exposed to five different 9-MA concentrations is approximately 95% after 24h and does not rise during the residual term. The LC50-value, thus the lethal concentration for half the amount of the tested organisms, is 2.3 µM/L. Both the negative and solvent controls have a similar mortality rate around 0% during the whole exposure period as expected. The positive control shows an ascending mortality rate from 24h to 96h and attains a value of 100% mortality after 96h as anticipated (Figure 3.2). In order to compare the mortality rate of each concentration of 9-MA of all three replicates separately, examine figure 7.2 in the supplementary material.

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13 7-MBA (24 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 7-MBA (48 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 7-MBA (72 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 7-MBA (96 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ]

Figure 3.3: Mortality rate of zebrafish embryos in % after 24, 48, 72 and 96 hour exposure with the five different concentrations of 7-methylbenzo[a]anthracene (7-MBA) analyzed with non-linear regression. Black dots represent 7-MBA, red squares depict the positive control (PC), and whereas the green triangles show the negative (NC) and the blue triangles show the solvent control (DMSO).

The mortality rate of zebrafish embryos exposed to five different 7-methylbenzo[a]anthracene concentrations is approximately 0% after 24h and does not increase during the remaining period of exposure time. Accordingly, 7-MBA shows almost no acute toxic effect on the embryonal development of Danio rerio. Both the negative and solvent controls show a similar mortality rate around 0% during the whole exposure period as supposed. The positive control exhibits a rising mortality rate from 24h to 96h and reaches a value of 100% mortality after 96h as expected (Figure 3.3). The figure 7.3 in the supplementary material depicts the mortality rate of each concentration of 7-MBA of two replicates to compare them particularly.

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14 DMBA (24 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 DMBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] DMBA (48 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 DMBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] DMBA (72 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 DMBA PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] DMBA (96 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 DMBA PC NC DMSO LC50 = 9.031M/L log concentration [µM/L] M o rt a lit y [ % ]

Figure 3.4: Mortality rate of zebrafish embryos in % after 24, 48, 72 and 96 hour exposure with the five different concentrations of 7,12-dimethylbenzo[a]anthracene (DMBA) analyzed with non-linear regression. Black dots represent DMBA, red squares depict the positive control (PC), and whereas the green triangles show the negative (NC) and the blue triangles show the solvent control (DMSO).

The dimethylated PAC 7,12 dimethylbenzo[a]anthracene indicates an increased mortality rate, thus an acute toxic effect on the embryonal development of the zebrafish. However, the mortality rate of the exposed zebrafish embryos is not as high as that one of 9-MA. In contrast to that one of 9-MA, the mortality rate of also ascend during the remaining exposure time from approximately 30% after 24h to about 60% after 96h. The LC50-value of DMBA amounts to 9.031µm/L. The mortality rate of both the negative and solvent controls is nearly 0% during the whole observed time period as it is supposed to be. In contrast, the positive control shows a rising mortality rate and achieves 100% mortality after 96h as expected (Figure 3.4). For comparison only, the figure 7.4 in the supplementary material illustrates the mortality rate of each concentration of all three replicates separately.

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15 7-MBaP (96 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBaP PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 7-MBaP (24 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBaP PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 7-MBaP (48 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBaP PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ] 7-MBaP (72 h) -1 0 1 2 3 4 5 0 20 40 60 80 100 7-MBaP PC NC DMSO log concentration [µM/L] M o rt a lit y [ % ]

Figure 3.5: Mortality rate of zebrafish embryos in % after 24, 48, 72 and 96 hour exposure with the five different concentrations of 7-methylbenzo[a]pyrene (7-MBaP) analyzed with non-linear regression. Black dots represent 7-MBaP, red squares depict the positive control (PC), and whereas the green triangles show the negative (NC) and the blue triangles show the solvent control (DMSO).

The mortality rate of the zebrafish embryos exposed to five different 7-methylbenzo[a]pyrene concentrations is approximately 0% after 24h and does not rise during the residual exposure time. Correspondingly, 7-MBaP shows barely any acute toxic effect on the embryonal development of Danio rerio. Both the negative and solvent controls show a similar mortality rate around 0% during the whole exposure period as anticipated. The positive control exhibits an increasing mortality rate from 24h to 96h and attains a value of 100% mortality after 96h as expected (Figure 3.5). Figure 7.5 in the supplementary material compares the mortality rate of each concentration of 7-MBaP of two replicates individually.

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16 3.2 Tail length Test (TLT) with Danio rerio

The measurement of the tail length of the zebrafish embryo has the purpose to receive specific information about the teratogenic effect of 7,12-dimethylbenzo[a]anthracene on the embryonal development of Danio rerio as a model for aquatic organisms.

DMBA causes different sub-lethal malformations during the embryonal development. Figure 3.6 illustrates some of the teratogenic effects of DMBA, such as the shortened tail length, tail curvature as scoliosis, a deformity of the tail tip as well as edema on the yolk and on the pericard. Abnormal heartbeat and blood circulation were also observed.

Figure 3.6: Sub-lethal malformation of zebrafish embryos exposed in DMBA. Besides the shortened tail length the figure pictures tail curvatures such as scoliosis, deformations of the tail tip and edemas on the pericard and on the yolk. For a better comparability, a scale was added.

The statistical analysis of the tail length test datasets was first performed with the unpaired two-tailed t-test (Figure 3.7). After passing the t-test with p ≤ 0.05, the datasets were analyzed statistically with one-way ANOVA on ranks and the post-hoc Dunn’s test afterwards. The analysis of the pooled datasets of the measured tail length from the zebrafish embryos exposed to DMBA of all five replicates in total is shown in figure 3.8. For the individual statistical analysis of the measured tail length of each replicate, see figure 7.6 in the supplementary material.

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Figure 3.7: Comparison of the measured average tail length of zebrafish embryos exposed to DMBA or the negative (NC) respectively solvent (DMSO) control in fivefold repetition. Asterisks indicate significant differences between negative/solvent controls and exposed samples. The approximate p-value for the unpaired two-tailed t-test for the first, second and third and fifth TLT is P< 0.001. It is not possible to determine the p-value for the forth TLT.

The tail length is statistically different for the exposed sample and for the control groups (negative and solvent control). It was a significantly reduced tail length of the zebrafish embryos exposed to DMBA compared to the control groups detected. Thus, DMBA caused the larvae to emerge significantly smaller than the negative respectively solvent controls, consequently, it also induced significant reductions in larval hatch length. The approximate p-value of the first, second, third and fifth tail length test is under 0.001 indicated by asterisks in figure 3.7. It is not possible to determine the p-value for the forth tail length test because there was just one test organism measurable due to the high mortality rate.

0 1000 2000 3000 DMBA NC DMSO 1.Tail length test

*** ta il l e n g th (i n µ m )

2. Tail length test

0 1000 2000 3000 DMBA NC DMSO *** ta il l e n g th (i n µ m )

4.Tail length test

0 1000 2000 3000 DMBA NC DMSO ta il l e n g th (i n µ m )

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Figure 3.8: Statistical analysis of the pooled datasets of the measured tail length from exposed zebrafish embryos and control groups (negative controls (NC) and solvent controls (DMSO)) with ANOVA on ranks and post-hoc Dunn’s test. Asterisks indicate that the tail length of the exposed sample group is significantly shorter than of the control groups (P<0.001).

The size of the pooled amount of measured zebrafish tails of the exposed sample and of the control groups differ due to relatively high concentration of 5 µM/L of DMBA, which is close to the acute toxicity border and thus, causes a high mortality rate.

The average measured tail length of zebrafish embryos exposed to DMBA is 1701.62 µm. In contrast to that, the average measured tail length of the NC is 2770.79 µm, thus quite similar to the average measured tail length of the SC with 2781.56 µm. According to this, the tail length of the exposed sample group is significantly shorter than of the control groups. The p-value is under 0.001.

ANOVA on ranks and Dunn's Test

0 1000 2000 3000 4000 NC

all tail length tests:DMBA DMSO *** n=26 n=50 n=50 ta il l e n g th (i n µ m )

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4. Discussion

4.1 Embryotoxic and teratogenic effects of PACs

Some of the tested PACs showed embryotoxic as well as teratogenic effects. 9(10H)-Acridone (9-Acr), 7-methylbenzo[a]anthracene MBA) and 7-methylbenzo[a]pyrene (7-MBaP) did not shown any acute toxic effect on the embryonal development of the test organism Danio rerio. However, embryotoxic and teratogenic effects were shown for 9-methylacridine (9-MA). Specific teratogenic effects of 7,12-dimethylbenzo[a]anthracene (DMBA) were characterized by sub-lethal malformations and proven by measured tail lengths using the Tail Length Test (TLT).

The embryotoxic effect of 9-MA was evidenced through the high mortality rate of the exposed zebrafish embryos compared to the control group.

Moreover, the mortality rate of the exposed sample group stayed nearly the same during the whole exposure time; thus, it is obvious that the chorion has no protective function against 9-MA and its effect did not increase after longer exposure times.

Sub-lethal effects in zebrafish embryos caused by exposure to PACs were already characterized by Barron et al. 2003 and include precardial and yolk sac edema, disruption of the cardiac function, reduced growth as well as spinal deformities. The most sensitive and informative sublethal malformations among them are the tail length and tail curvature frequency(Fraysse et al. 2006).

Dimethylated PACs such as DMBA have also been identified as inducers of increased blue sac disease in Japanese medaka and affect also its embryonic development (Rhodes et al.2005). They are also known for their negative impact on the hatching success of embryonic fish and also the reduced hatch length (Rhodes et al. 2004).

In the present study, the main effects observed for DMBA are reduced tail length, tail curvature such as scoliosis and deformation of the tail tip. The twisted axis could be a result of uncontrolled contractions of the axis musculature, muscular tissue disorganization and a neuromuscular system defect due to acetylcholine accumulation induced by the acetylcholine esterase inhibitor DMBA (Aberlin 1981; Nguyen et al. 1997; Fraysse et al. 2006).

Biologically smaller fish are more vulnerable and susceptible to predation and starvation than larger fishes (Tonn et al. 1986). Larger larvaes are probably more active at an earlier age, thus, can swim longer distances as well as swim with a greater speed, and therefore have probably more food resources at their disposal (Miller et al. 1986). Even relatively minor initial larval size differences at hatch could have substantial effects on the survival (Miller et al.1988). The reduction in hatch length is not attributed to an early emergence of the chorion, but to the impact of DMBA, decreasing or retarding available resources for embryonic growth and development (Rhodes et al. 2004).

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4.2 Effects of PACs on the Aryl hydrocarbon Receptor pathway

The Aryl hydrocarbon Receptor (AhR) activation is one of the main effects of many carcinogenic polycyclic aromatic compounds (Machala et al. 2001). The ligand-activated transcription factor AhR is related to upregulation of expression of particular xenobiotic metabolizing monoxygenase enzymes embroiled in metabolic activation of PACs such as DMBA or 9-MA as well as tumor promotion (Denison et al. 2002; Hahn 1997; Machala et al. 2008; Nebert and Dalton 2006; Sjögren et al. 1996). Thus, many PACs enhance their own activation by binding to Ah-receptor (Nebert and Dalton 2006). The activation of Cytochrome P450 monoxygenase enzymes such as CYP1A and CYP1B through the AhR is a decisive determinant of carcinogenic or co/carcinogenic potency of PACs (Cheung et al. 1993; Shimada et al. 2003).

The unliganded Ah-receptor is a complex consisting of two molecules of the 90-kDa heat-shock protein (hsp90) and after that forms a complex with the AhR-Interacting protein AIP (also termed ARA9 and XAP2) and the co-chaperone p23 (Kazlauskas et al. 2001; Meyer and Perdew 1999). Binding of ligand to the AhR leads to dissociation of the co-factors and the activation of the Ah-receptor (Hahn 1998).

The AhR-complex affects the expression of genes influencing basic cellular processes and survival mechanisms as growth and the programmed cell death (Brouwer et al. 1995). Therefore, the activation of AhR raises interruption of cell signaling pathways regulating cell proliferation, differentiation and apoptosis, hence, contributes to carcinogenicity of PACs (Bock et al. 2005; Marlowe et al. 2005; Trilecová et al. 2011).

The induction of the xenobiotic monoxygenase enzyme P450 CYP1A and CYP1B by Aryl hydrocarbon receptors are important pathways for the metabolism and removal of PACs from cells (Denison et al. 2002). Consequently, AhR is conductive to the detoxification process of exogenous compounds as PACs (Denison et al. 1995; Hahn et al. 2006; Nebert and Dalton 2006). CYP1A and CYP1B have indeed similar but not identical substrate specificity towards PACs (Shimada et al. 2003).

Cytochrome P450 dependent monooxgenase enzymes catalyze hydroxylation reactions, reduction and oxidative transformation, which results in activation or inactivation of PACs (Behnisch et al. 2001; Bernhardt 1996). It has been considered that CYP1A and CYP1B are also responsible for the activation of most carcinogenic polycyclic aromatic compounds to epoxide intermediates and then further to more reactive diol-epoxides (Shimada et al. 1996). In contrast to unsubstituted PACs, the oxidation of methylated PACs seems to increase their AhR mediated potency (Larsson et al. 2014).

The AhR pathway also seems to have a protective or adaptive role against cardiovascular defects caused by exposure of zebrafish embryos to high concentrations of low molecular weight tricyclic PACs (Incardona et al. 2005). PACs can act not only as AhR agonists and

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CYP1A inducer but also as inhibitors of CYP1A (Billiard et al. 2006). Nevertheless, the induction of Cytochrome P450-dependent monooxygenase CYP1A, part of the diverse supergene family CYP, which can be found in most organisms, also makes a contribution to toxicity of PACs (Bernhardt 1996; Marlowe et al. 2005).

In general, Aryl hydrocarbon receptor-pathways are similar among mammals and lower vertebrates as fishes (Hahn 1998). However, teleosts as the Atlantic Killifish (Fundulus heteroclitus) have at least two Ah-receptors (AhR1 and AhR2), in contrast to mammals in general and humans specifically, which possess only a single AhR. Other fishes have even more Ah-receptors such as the pufferfish (Takifugu rubripes) which owns five AhR, or the spiny dogfish (Squalus acantias) and the zebrafish (Danio rerio) each with three Aryl hydrocarbon receptors (AhR1A, AhR2, AhR1B) (Andreasen et al. 2002; Tanguay et al. 2009). The three Ah-receptors in zebrafish are located on different chromosomes. The AhR1A maps to chromosome 16, whereas AhR2 is located on chromosome 22. Thereby, the AhR1A on chromosome 16 shares a conserved synteny with human chromosome 7, where the single human Ah-receptor is located (Andreasen et al., 2002). The third Ah-receptor AhR1B is adjacent to AhR2 on chromosome 22 (Karchner et al. 2005). AhR1A/B and AhR2 exhibit different expression patterns. AhR1A/B is primarily expressed in brain, gonad and heart, whereas Ahr2 most expression patterns are found in tissues (Hahn et al. 2006).

For mammals, endogenous physiological functions of CYP1 are already well known (Nebert et al. 2006). In zebrafish (Danio rerio), 95 CYP genes, part of 51 CYP gene families, have been identified (Goldstone et al. 2010). Especially, the Cytochrome P450-dependent monooxygenase enzymes CYP1 are regulated by Ah-receptors. Five CYP1 genes (cyp1a, cypb1, cyp1c1, cyp1c2, and cyp1d1) in four subfamilies have been detected in Danio rerio (Goldstone et al. 2010; Hahn et al. 1997). CYP1A is playing an important role in mediating toxicity of PACs in early life stages of fishes (Billiard et al. 2006). In contrast to carcinogenic mechanisms of toxicity in mammals, cancer does not seem to be the cause of toxicity of PACs in early life stages of fish (Billiard et al. 2006).

Also other AhR-responsive genes such as CYP1B1 play a key role in embryotoxicity and teratogenicity of PACs through their metabolic activation to toxic metabolites (Shimada et al. 2004). Thus, the observed sub-lethal malformations as edema on the yolk sac and pericard composed after exposure of the zebrafish embryo with the AhR-agonist DMBA as well as the high mortality rate of MA could be induced through the activation of DMBA respectively 9-MA to toxic and teratogenic metabolites due to their ligation to the Ah-receptor.

CYP1B1 overlaps in function with CYP1A. However, it has a greater tendency to metabolize substrates to more toxic products than CYP1A (Billiard et al. 2006).

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In zebrafish, ahr1a is a nonfunctional pseudogene, whereas ahr1b encodes a functional, transcriptionally active AhR-protein, early expressed in zebrafish development (Andreasen et al. 2002; Karchner et al. 2005).

The AhR mediated toxicity arising after activation through bonded both unsubstituted and methylated PAC molecules, as well as induction of AhR-related genes and subsequent transformation to toxic and teratogenic metabolites such in case of DMBA, should be considered in risk assessment of PACs (Barron et al. 2004; Behnisch et al. 2001).

4.3 Influence of PACs on Gap Junctional Intercellular Communication

Tumor promoting effects of PACs involve the inhibition of Gap Junctional Intercellular Communication (GJIC) (Machala et al. 2008; Marvanová et al. 2008; Upham et al. 1996). The mechanism of down-regulation and inhibition of GJIC is a posttranslational modification of gap junction proteins (Upham et al. 1996). The down-regulation of Gap Junctional Intercellular Communication leads to the release of genotoxically damaged cells as well as uncontrolled cellular growth causing the disruption of both homeostasis and cell-cell communication and eventually the development of tumors (Machala et al. 2008; Trosko et al. 1990; Trosko and Upham 2005). It is obvious that inhibited GJIC is related to carcinogenesis, since most cancer cells have dysfunctional GJIC, and tumor promoting agents reversibly inhibits GJIC as well as oncogenes down-regulate GJIC (Trosko et al. 1988).

PACs containing bay regions formed by fused rings or bay-like regions molded from methyl substitution at the top of benzene rings inhibit GJIC more than linear PACs are able to (Weis et al. 1998). Thus, the position of the substituted methyl group is a crucial determinant of mutagenic and genotoxic effects as the GJIC inhibitory activity of PACs (Machala et al. 2008). Acute inhibition of GJIC is propably not dependent on AhR-activation (Machala et al. 2008).

Methylated anthracenes such as 9-MA and DMBA play a potent role in tumor promotion and have inhibiting effects on Gap Junctional Intercellular Communication (GJIC) depending on the position of the methylated group (Upham et al. 1996). The PACs inhibiting GJIC are less efficient AhR inducers (Marvanová et al. 2008).

4.4 Regulation of tail development in Danio rerio

The tail organizer in Danio rerio embryo is located at the ventral margin and is responsible for the formation of non-axial tail tissue (Agathon et al. 2003). It is active since the late gastrula stage and is independent of the dorsal Spemann organizer, which regulates the development of the body axes and the induction of the nervous system (Agathon et al. 2003). At the end of gastrulation the tail organizer, called tailbud, is established of aggregated marginal cells from the dorsal Spemann organizer and the ventral margin (Agathon et al.

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2003; Hammerschmidt et al. 1996). According to studies in Xenopus, the tail formation is a prosecution of the gastrulation (Gont et al.1993).

After gastrulation, additional morphogenetic processes are initiated, necessary for the posterior extension of the body axis and the outgrowth of the tail (Hammerschmidt et al. 1996). In zebrafish, these incipient processes are the ventral movement of the tailbud on the yolk sac, the constriction of the posterior part of the yolk sac to the yolk tube and eventually the detachment of the tail tip from the yolk sac (Hammerschmidt et al.1996).

The induction of the tail organizer deriving from the ventral margin is due to the endogenous BMP, Nodal and Wnt8 signaling pathways, which stimulates the marginal cells (Agathon et al. 2003). The triple stimulation of these signaling pathways is the essential determinant for tail development in Danio rerio (Agathon et al. 2003). Moreover, the loss of just one of these signaling pathways impedes development of the tail (Agathon et al. 2003). In the process, Nodal induces both Wnt8 and BMP, but is not able to induce the tail on its own since it also induces the strong expression of BMP inhibitors such as Noggin and Chordin (Agathon et al. 2003). The zebrafish features a rapidly growing amount of mutations with impact on the early development such as the mutation no tail, leading to a lack of posterior structures and the failure of the differentiation of the notochord in zebrafish (Driever et al. 1994; Griffin et al. 1995; Mullins et al. 1994).

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5. Summary

Polycyclic aromatic compounds are ubiquitously distributed pollutants in the aquatic and terrestrial environment containing harmful properties on creatures such as carcinogenicity, teratogenicity and toxicity, but are not so well analyzed yet.

In the present study, the embryotoxic and teratogenic effects of selected hydroxylated and methylated PACs on the embryonal development of Danio rerio as an aquatic model organism were analyzed with the Fish Embryo Toxicity Test (FET) and the Tail Length Test (TLT) to obtain information on the toxic and teratogenic impact of the tested PACs on the environment.

Two of the five tested PACs, 9-MA and DMBA, showed embryotoxic respectively teratogenic effects on the embryonal development of the zebrafish. The embryotoxicity of 9-MA was indicated in the high mortality rate of the exposed zebrafish embryos, whereas the teratogenic effect of DMBA was revealed in the emergence of sub-lethal malformations during the embryonal development such as a shortened tail length, tail curvatures, tail tip deformity or the formation of edema on the yolk sac and pericard as well as abnormal heartbeat and blood circulation.

The high mortality rate of the zebrafish embryos exposed to 9-MA did not increase over the exposure time of 96h, which suggests that the chorion of the zebrafish egg could not protect the embryo at all against the strong embryotoxic effect of 9-MA.

The sub-lethal malformations of the zebrafish embryos exposed to DMBA could be induced to the metabolic activation of AhR-agonist DMBA through the AhR-pathway or the accumulation of the neurotransmitter Acetylcholine due to the inhibitory function of DMBA on the ACh-Esterase, which caused a neuromuscular system defect or uncontrolled contractions of the axis musculature.

Further research, may focus on the mode of action of PACs such as 9-MA and DMBA and their impact on organisms in order to take reasonable precautions to avoid or to diminish the uptake of PACs from the environment.

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7. Supplementary material

Figure 7.1: Mortality rate in % for five different tested concentrations of 9(10H)-acridone (9-Acr) as well as the negative (NC), solvent (SC) and positive (PC) control in two replicates.

Figure 7.2: Mortality rate in % for five different tested concentrations of 9-methylacridine (9-MA) as well as the negative (NC), solvent (SC) and positive (PC) control in three replicates.

0 10 20 30 40 50 60 70 80 90 100 NC SC C1 C2 C3 C4 C5 PC M o rtali ty ( in % ) Replicate 1 Replicate 2 C1= 0,625µM/l C2=1,25µM/l C3=2,5µM/l C4= 5µM/l C5=10µM/l NC=negative Control SC= 0,4% DMSO Control PC= positive Control

9-Acr

0 10 20 30 40 50 60 70 80 90 100 NC SC C1 C2 C3 C4 C5 PC M o rtali ty ( in % ) Replicate 1 Replicate 2 Replicate 3 C1= 0,25µM/l C2= 0,5µM/l C3= 1µM/l C4= 2µM/l C5= 4µM/l NC=negative Control SC= 0,4% DMSO Control PC= positive Control

9-MA

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Figure 7.3: Mortality rate in % for five different tested concentrations of 7-methylbenzo[a]anthracene (7-MBA) as well as the negative (NC), solvent (SC) and positive (PC) control in two replicates.

7-MBA

0 10 20 30 40 50 60 70 80 90 100 NC SC C1 C2 C3 C4 C5 PC M o rtali ty ( in % ) Replicate 1 Replicate 2 Replicate 3 C1= 0,625µM/l C2=1,25µM/l C3=2,5µM/l C4= 5µM/l C5=10µM/l NC=negative Control SC= 0,4% DMSO Control PC= positive Control

DMBA

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Figure 7.4: Mortality rate in % for five different tested concentrations of 7,12-dimethylbenzo[a]anthracene (DMBA) as well as the negative (NC), solvent (SC) and positive (PC) control in three replicates.

Figure 7.5: Mortality rate in % for five different tested concentrations of 7-methylbenzo[a]pyrene (7-MBaP) as well as the negative (NC), solvent (SC) and positive (PC) control in two replicates.

Figure 7.6: Statistical analysis of the tail length in µm after hatching for each of the five replicates with different sample sizes of measured zebrafish embryo tails (1. Tail Length Test (TLT) n=6; 2. TLT n=10; 3. TLT n=6; 4. TLT n=1; 5.TLT n=3) as well as for the combined amount of measured zebrafish embryo tails of the negative (NC) and the solvent (DMSO) control with one-way ANOVA on ranks and post-hoc Dunn’s test. Asterisks indicate significant differences between negative/solvent controls and

7-MBaP

ANOVA on ranks and Dunn's Test

0 500 1000 1500 2000 2500 3000 n=6

1.Tail length test: DMBA 2.Tail length test: DMBA 3.Tail length test: DMBA 4.Tail length test: DMBA 5.Tail length test: DMBA NC DMSO n=6 n=10 n=6 n=1 n=3 n=50 n=50 *** _*** *** ** ta il l e n g th (i n µ m )

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exposed samples. The approximate P-value for the ANOVA on ranks and post-hoc Dunn’s test for the first, second and third TLT is P< 0.001, whereas it is P< 0.01 for the fifth TLT. It is not possible to determine the P-value for the fourth TLT.

8.Acknowledgement

I would like to thank Steffen Keiter for the option to work at MTM and for his careful and instructive supervision during the project work and besides.

I would like to thank Magnus Engwall as well for the opportunity to complete the practical course at MTM and to appraise this report.

Also Martha deserves my gratitude for her kind and great support in the lab, her patience, the possibility to stay and work at her place for the last weeks of my stay in Örebro.

Finally, I would like to thank my family, my boyfriend and friends who encouraged and supported me from afar during my stay abroad at Örebro University.

Teratogenic and Embryotoxic Effects of Polycyclic Aromatic Compounds Report