Mixture effects of oxygenated PAHs and benzo[a]pyrene on cardiovascular development and function in zebrafish embryos

Polycyclic aromatic compounds (PACs), including polycyclic aromatic hydrocarbons (PAHs) and oxygenated PAHs (oxy-PAHs), are common environmental pollutants known to cause health effects in humans and wild-life. In particular, vertebrate cardiovascular development and function are sensitive to PACs. However, the interactive effects of PAHs and oxy-PAHs on cardiovascular endpoints have not been well studied. In this study, we used zebrafish embryos (ZFEs) as a model to examine developmental and cardiovascular toxicities induced by the three environmental oxy-PAHs benzo[a]fluorenone (BFLO), 4H-cyclopenta[def]phenanthren-4-one (4H-CPO) and, 6H-benzo[cd]pyren-6-one (6H-BPO), and the PAH benzo[a]pyrene (BaP) either as single exposures or binary oxy-PAH + PAH mixtures. 6H-BPO induced developmental and cardiovascular toxicity, including reduced heartbeat rate and blood flow, at lower doses compared to the other compounds. Exposure to binary mixtures generally caused enhanced toxicity and induction of aryl hydrocarbon receptor (AhR)-regulated gene expression (ahr2 and cyp1a) compared to single compound exposure. This was associated with differential expression of genes involved in cardiovascular development and function including atp2a2, myh6, tbx5 and zerg. AhR-knock-down significantly reduced the cardiovascular toxicity of 6H-BPO and its binary mixture with BaP indicating a significant AhR-dependence of the effects. Measurements of internal concentrations showed that the toxicokinetics of BaP and 6H-BPO were altered in the binary mixture compared to the single compound exposure, and most likely due to CYP1 inhibition by 6H-BPO. Altogether, these data support that similar to interactions between PAHs, mixtures of PAHs and oxy-PAHs may cause increased developmental and cardiovascular toxicity in ZFEs through an AhR-dependent mechanism

MGST1, a GSH transferase/peroxidase essential for development and hematopoietic stem cell differentiation.

We show for the first time that, in contrast to other glutathione transferases and peroxidases, deletion of microsomal glutathione transferase 1 (MGST1) in mice is embryonic lethal. To elucidate why, we used zebrafish development as a model system and found that knockdown of MGST1 produced impaired hematopoiesis. We show that MGST1 is expressed early during zebrafish development and plays an important role in hematopoiesis. High expression of MGST1 was detected in regions of active hematopoiesis and co-expressed with markers for hematopoietic stem cells. Further, morpholino-mediated knock-down of MGST1 led to a significant reduction of differentiated hematopoietic cells both from the myeloid and the lymphoid lineages. In fact, hemoglobin was virtually absent in the knock-down fish as revealed by diaminofluorene staining. The impact of MGST1 on hematopoiesis was also shown in hematopoietic stem/progenitor cells (HSPC) isolated from mice, where it was expressed at high levels. Upon promoting HSPC differentiation, lentiviral shRNA MGST1 knockdown significantly reduced differentiated, dedicated cells of the hematopoietic system. Further, MGST1 knockdown resulted in a significant lowering of mitochondrial metabolism and an induction of glycolytic enzymes, energetic states closely coupled to HSPC dynamics. Thus, the non-selenium, glutathione dependent redox regulatory enzyme MGST1 is crucial for embryonic development and for hematopoiesis in vertebrates

Interactions between polycyclic aromatic hydrocarbons in complex mixtures and implications for cancer risk assessment.

In this review we discuss the effects of exposure to complex PAH mixtures in vitro and in vivo on mechanisms related to carcinogenesis. Of particular concern regarding exposure to complex PAH mixtures is how interactions between different constituents can affect the carcinogenic response and how these might be included in risk assessment. Overall the findings suggest that the responses resulting from exposure to complex PAH mixtures is varied and complicated. More- and less-than additive effects on bioactivation and DNA damage formation have been observed depending on the various mixtures studied, and equally dependent on the different test systems that are used. Furthermore, the findings show that the commonly used biological end-point of DNA damage formation is insufficient for studying mixture effects. At present the assessment of the risk of exposure to complex PAH mixtures involves comparison to individual compounds using either a surrogate or a component-based potency approach. We discuss how future risk assessment strategies for complex PAH mixtures should be based around whole mixture assessment in order to account for interaction effects. Inherent to this is the need to incorporate different experimental approaches using robust and sensitive biological endpoints. Furthermore, the emphasis on future research should be placed on studying real life mixtures that better represent the complex PAH mixtures that humans are exposed to.FormasAccepte

Persistent activation of DNA damage signaling in response to complex mixtures of PAHs in air particulate matter

Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are present in air particulate matter (PM) and have been associated with many adverse human health effects including cancer and respiratory disease. However, due to their complexity, the risk of exposure to mixtures is difficult to estimate. In the present study the effects of binary mixtures of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) and complex mixtures of PAHs in urban air PM extracts on DNA damage signaling was investigated. Applying a statistical model to the data we observed a more than additive response for binary mixtures of BP and DBP on activation of DNA damage signaling. Persistent activation of checkpoint kinase 1 (Chk1) was observed at significantly lower concentrations of air PM extracts than BP alone. Activation of DNA damage signaling was also more persistent in air PM fractions containing PAHs with more than four aromatic rings suggesting larger PAHs contribute a greater risk to human health. Altogether our data suggests that human health risk assessment based on additivity such as toxicity equivalency factor scales may significantly underestimate the risk of exposure to complex mixtures of PAHs. The data confirms our previous findings with PAHcontaminated soil (Niziolek-Kierecka et al. 2012) and suggests a possible role for Chk1 Ser317 phosphorylation as a biological marker for future analyses of complex mixtures of PAHsFormasCancer- och AllergifondenStockholm UniversityEU/FP7 Marie Curie IRG fellowshipAccepte

Benzo[a]pyrene-specific online high-performance liquid chromatography fractionation of air particulate extracts : a tool for evaluating biological interactions.

Benzo[a]pyrene (B[a]P) is a known human carcinogen and is commonly used as a surrogate for assessing the carcinogenic risk posed by complex mixtures of polycyclic aromatic hydrocarbons (PAHs) present in air particulate matter (PM). However, studies have shown that using B[a]P as a surrogate may underestimate the carcinogenic potential of PAH mixtures, as the risk assessment approach does not consider interaction effects. Thus, toxicological studies using B[a]P to assess its carcinogenic potential in environmentally derived complex mixtures, as opposed to single compound experiments, could improve risk assessment. The intention of the present study was to develop an online HPLC fractionation system for the selective removal of B[a]P from air PM extracts. Two serial pyrenylethyl (PYE) columns enabled selective separation of B[a]P from its isomers and other PAHs as well as a short fractionation cycle of 30min. One run consisted of three collection steps: the first fraction contained PAHs eluting earlier than B[a]P, the second contained B[a]P and the last contained later-eluting PAHs. The selectivity and recovery of the system was investigated using extracts of Stockholm air PM samples. The overall recovery for all PAHs was approximately 80%, and the system proved to be selective, as it removed 94% of B[a]P and less than 3% of benzo[b]fluoranthene from the complex PAH mixture. Exposing human cells to blanks generated by the fractionation system did not induce cytotoxicity or DNA damage signalling. In conclusion, the online HPLC system was selective for B[a]P fractionation whilst minimising run-to-run variation and allowing repeated fractionations for larger samples due to its relatively short run time.FormasAccepte

Nanomolar levels of PAHs in extracts from urban air induce MAPK signaling in HepG2 cells.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that occur naturally in complex mixtures. Many of the adverse health effects of PAHs including cancer are linked to the activation of intracellular stress response signaling. This study has investigated intracellular MAPK signaling in response to PAHs in extracts from urban air collected in Stockholm, Sweden and Limeira, Brazil, in comparison to BP in HepG2 cells. Nanomolar concentrations of PAHs in the extracts induced activation of MEK4 signaling with down-stream increased gene expression of several important stress response mediators. Involvement of the MEK4/JNK pathway was confirmed using siRNA and an inhibitor of JNK signaling resulting in significantly reduced MAPK signaling transactivated by the AP-1 transcription factors ATF2 and c-Jun. ATF2 was also identified as a sensitive stress responsive protein with activation observed at extract concentrations equivalent to 0.1 nM BP. We show that exposure to low levels of environmental PAH mixtures more strongly activates these signaling pathways compared to BP alone suggesting effects due to interactions. Taken together, this is the first study showing the involvement of MEK4/JNK/AP-1 pathway in regulating the intracellular stress response after exposure to nanomolar levels of PAHs in environmentalFormasAccepte

Detection of benz[j]aceanthrylene in urban air and evaluation of its genotoxic potential.

Benz[j]aceanthrylene (B[j]A) is a cyclopenta-fused polycyclic aromatic hydrocarbon with strong mutagenic and carcinogenic effects. We have identified B[j]A in air particulate matter (PM) in samples collected in Stockholm, Sweden and in Limeira, Brazil using LC-GC/MS analysis. Determined concentrations ranged between 1.57 and 12.7 and 19.6-30.2 pg/m(3) in Stockholm and Limeira, respectively, which was 11-30 times less than benzo[a]pyrene (B[a]P) concentrations. Activation of the DNA damage response was evaluated after exposure to B[j]A in HepG2 cells in comparison to B[a]P. We found that significantly lower concentrations of B[j]A were needed for an effect on cell viability compared to B[a]P, and equimolar exposure resulted in significant more DNA damage with B[j]A. Additionally, levels of gammaH2AX, pChk1, p53, pp53, and p21 proteins were higher in response to B[j]A than B[a]P. On the basis of dose response induction of pChk1 and gammaH2AX, B[j]A potency was 12.5- and 33.3-fold higher than B[a]P, respectively. Although B[j]A levels in air were low, including B[j]A in the estimation of excess lifetime cancer risk increased the risk up to 2-fold depending on which potency factor for B[j]A was applied. Together, our results show that B[j]A could be an important contributor to the cancer risk of air PM.FormasAccepte

Relatório de estágio profissional

O presente Relatório foi realizado no âmbito do Estágio Profissional I e II. Trata-se de um trabalho elaborado com base na observação de aulas e na experimentação didática; os materiais obtidos (como, por exemplo, horários, dados sobre as turmas, fichas, etc.) foram submetidos a uma análise documental

A Method for Efficient Calculation of Diffusion and Reactions of Lipophilic Compounds in Complex Cell Geometry

A general description of effects of toxic compounds in mammalian cells is facing several problems. Firstly, most toxic compounds are hydrophobic and partition phenomena strongly influence their behaviour. Secondly, cells display considerable heterogeneity regarding the presence, activity and distribution of enzymes participating in the metabolism of foreign compounds i.e. bioactivation/biotransformation. Thirdly, cellular architecture varies greatly. Taken together, complexity at several levels has to be addressed to arrive at efficient in silico modelling based on physicochemical properties, metabolic preferences and cell characteristics. In order to understand the cellular behaviour of toxic foreign compounds we have developed a mathematical model that addresses these issues. In order to make the system numerically treatable, methods motivated by homogenization techniques have been applied. These tools reduce the complexity of mathematical models of cell dynamics considerably thus allowing to solve efficiently the partial differential equations in the model numerically on a personal computer. Compared to a compartment model with well-stirred compartments, our model affords a more realistic representation. Numerical results concerning metabolism and chemical solvolysis of a polycyclic aromatic hydrocarbon carcinogen show good agreement with results from measurements in V79 cell culture. The model can easily be extended and refined to include more reactants, and/or more complex reaction chains, enzyme distribution etc, and is therefore suitable for modelling cellular metabolism involving membrane partitioning also at higher levels of complexity

Carcinogenic polycyclic aromatic hydrocarbons : Theoretical, molecular, in vitro and cellular characterization of biotransformation and DNA damage

Polycyclic aromatic hydrocarbons (PAHs) are widespread mutagenic and carcinogenic environmental pollutants, which require metabolic activation to electrophilic intermediates and subsequent covalent binding to critical targets in DNA to elicit their biological activity. Bayand fjord-region diol epoxides (DEs) have been identified as the ultimate mutagenic and carcinogenic metabolites of PAHs. The balance between metabolic activation and detoxification routes influences the extent of DE-DNA adduct-formation. The most important detoxification pathway of DEs is glutathione transferase (GST) catalyzed conjugation with glutathione (GSH). Human GSTs of Alpha class have been assayed with the ultimate carcinogenic ( )-anti- and (+)-syn-DEs derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-DE of the planar benzo[a]pyrene [(+)-anti-BPDE]. In general, the activities were in the order: (+)-syn- DBPDE > ( )-anti-DBPDE > (+)-anti-BPDE. GSTA1-1 was found to be the most efficient enzyme and demonstrated a remarkable catalytic efficiency (kcat/Km) of 464 mM-1s-1 with (+)- syn-DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to ( )-anti-DBPDE was explained by molecular modeling showing the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The results showed that the spatial orientation of the hydroxyl groups are important determinants for the catalytic efficiency and thus responsible for the observed difference in catalytic activity. Investigating preferences in structure of (+)-syn- and ( )-anti-DBPDE using DFT showed several levels of flexibility. Because of the distorted structure, the molecule can readily flip its DE moiety relative to the aromatic ring system ( in and out ). Furthermore, the hydroxyl groups on the saturated DE ring were found to be either in a diequatorial or in a diaxial conformation. Our results showed a lower energy profile and thus a preference for the in-diequatorial conformation for both DEs. The possibility of transversions on different levels might have biological consequences, both in detoxication and in DNA adduct formation, adapting to DNA and thus escape recognition/repair by the DNA repair machinery. To study individual GSTs in a more biologically relevant system, V79 cells stably overexpressing different human GSTs were constructed and characterized. Factors governing the accessibility of lipophilic DE substrates for GSTs in the cell were investigated. With the highly reactive (+)-anti-BPDE, 1-2 % of the expected activity was observed, whereas the corresponding values for the less reactive ( )-anti-DBPDE were up to 13 %. Furthermore, the protective effect of individual GSTs against DE induced DNA adduct formation was determined. In general, an increase in GST activity was concomitant with a decrease in DNA adduct formation. DBPDE showed the highest DNA binding capacity among the DEs tested. GSTA1-1 showed the highest GSH conjugating capacity and offered best protection against DBPDE induced DNA adduct formation. With BPDE, GSTP1-1 was most active in GSH conjugate formation whereas GSTM1-1 displayed the most effective protection against formation of DNA adducts. Overall, this demonstrates the difficulty in extrapolating data obtained with pure enzymes to the complex situation in the intact cell. We have compared the formation and removal of adducts as a function of time formed by ( )- anti-DBPDE and (+)-anti-BPDE in A549 human epithelial lung carcinoma cells. The treatment with DBPDE resulted in an initial increase of adducts to a maximal level of adducts after 1 hr of incubation. This was followed by an apparent, although not statistically significant, slow removal of adducts. In cells treated with BPDE the maximal level adducts was reached within 20 min of exposure. The formation was followed by an initial rapid decline in the adduct level and a later statistically significant 10-fold slower rate of adduct removal. Comparing the rate of removal of adducts derived from BPDE with those of DBPDE, the latter are obviously more refractory to the NER coupled repair than the former. Moreover, we observe a significant increase in ratio of dA/dG adducts for DBPDE, indicating that dA adducts are especially refractory to repair. The apparent resistance of adducts from DBPDE to be eliminated may reflect the ability of such adducts to escape recognition and/or the subsequent removal by the NER machinery