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

Sensitivity of Salmonella YG5161 for detecting PAH-associated mutagenicity in air particulate matter.

The Salmonella/microsome assay is the most used assay for the evaluation of air particulate matter (PM) mutagenicity and a positive correlation between strain TA98 responses and benzo[a]pyrene (B[a]P) levels in PM has been found. However, it seems that the major causes of PM mutagenicity in this assay are the nitro and oxy-PAHs. Salmonella YG5161, a 30-times more responsive strain to B[a]P has been developed. To verify if YG5161 strain was sufficiently sensitive to detect mutagenicity associated with B[a]P mutagenicity, PM samples were collected in Brazil and Sweden, extracted with toluene and tested in the Salmonella/microsome microsuspension assay. PAHs and B[a]P were determined and the extracts were tested with YG5161 and its parental strain TA1538. The extracts were also tested with YG1041 and its parental strain TA98. For sensitivity comparisons, we tested B[a]P and 1-nitropyrene (1-NP) using the same conditions. The minimal effective dose of B[a]P was 155 ng/plate for TA1538 and 7 ng/plate for YG5161. Although the maximum tested dose, 10 m(3) /plate containing 9 ng of B[a]P in the case of Brazilian sample, was sufficient to elicit a response in YG5161, mutagenicity was detected at a dose as low as 1 m(3) /plate (0.9 ng). This is probably caused by nitro-compounds that have been shown to be even more potent than B[a]P for YG5161. It seems that the mutagenicity of B[a]P present in PM is not detectable even with the use of YG5161 unless more efficient separation to remove the nitro-compounds from the PAH extract is performed.FormasAccepte

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