Antagonistic effects of a COX1/2 inhibitor drug in human HepG2 cells exposed to an environmental carcinogen.

Understanding interactions between legacy and emerging environmental contaminants has important implications for risk assessment, especially when mutagens and carcinogens are involved, whose critical effects are chronic and therefore difficult to predict. The current work aimed to investigate potential interactions between benzo[a]pyrene (B[a]P), a carcinogenic polycyclic aromatic hydrocarbon and legacy pollutant, and diclofenac (DFC), a non-steroidal anti-inflammatory drug and pollutant of emerging concern, and how DFC affects B[a]P toxicity. Exposure to binary mixtures of these chemicals resulted in substantially reduced cytotoxicity in human HepG2 cells compared to single-chemical exposures. Significant antagonistic effects were observed in response to high concentrations of B[a]P in combination with DFC at IC50 and ⅕ IC50. While additive effects were found for levels of intracellular reactive oxygen species, antagonistic mixture effects were observed for genotoxicity. B[a]P induced DNA strand breaks, γH2AX activation, and micronuclei formation at ½ IC50 concentrations or lower, whereas DFC induced only low levels of DNA strand breaks. Their mixture caused significantly lower levels of genotoxicity by all three endpoints compared to those expected based on concentration additivity. In addition, antagonistic mixture effects on CYP1 enzyme activity suggested that the observed reduced genotoxicity of B[a]P was due to its reduced metabolic activation as a result of enzymatic inhibition by DFC. Overall, the findings further support the growing concern that co-exposure to environmental toxicants and their non-additive interactions may be a confounding factor that should not be neglected in environmental and human health risk assessment.

Human Pesticide Exposure in Bolivia: A Scoping Review of Current Knowledge, Future Challenges and Research Needs.

Numerous studies have shown that pesticide exposure is linked to adverse health outcomes. Nevertheless, in Bolivia, where there is an increasing use of pesticides, the literature is sparse. To address knowledge gaps and guide future research in Bolivia, we conducted a scoping review spanning 22 years (January 2000 to December 2022). Our search identified 39 peer-reviewed articles, 27 reports/documents on Bolivian regulations, and 12 other documents. Most studies focused on farmers and revealed high pesticide exposure levels, assessed through biomarkers of exposure, susceptibility, and effect. The literature explored a range of health effects due to pesticide exposure, spanning from acute to chronic conditions. Many studies highlighted the correlation between pesticide exposure and genotoxic damage, measured as DNA strand breaks and/or micronuclei formation. This was particularly observed in farmers without personal protection equipment (PPE), which increases the risk of developing chronic diseases, including cancer. Recent findings also showed the alarming use of banned or restricted pesticides in Bolivian crops. Despite existing Bolivian regulations, the uncontrolled use of pesticides persists, leading to harmful health effects on the population and increasing land and water pollution. This review underscores the need for the stringent enforcement of regulations and continued research efforts, and it provides a scientific foundation for decision-making by relevant authorities.

Reactive oxygen species-dependent transient induction of genotoxicity by retene in human liver HepG2 cells.

Retene is a polycyclic aromatic hydrocarbon (PAH) emitted mainly by biomass combustion, and despite its ubiquity in atmospheric particulate matter (PM), studies concerning its potential hazard to human health are still incipient. In this study, the cytotoxicity and genotoxicity of retene were investigated in human HepG2 liver cells. Our data showed that retene had minimal effect on cell viability, but induced DNA strand breaks, micronuclei formation, and reactive oxygen species (ROS) formation in a dose- and time-dependent manner. Stronger effects were observed at earlier time points than at longer, indicating transient genotoxicity. Retene activated phosphorylation of Checkpoint kinase 1 (Chk1), an indicator of replication stress and chromosomal instability, which was in accordance with increased formation of micronuclei. A protective effect of the antioxidant N-acetylcysteine (NAC) towards ROS generation and DNA damage signaling was observed, suggesting oxidative stress as a key mechanism of the observed genotoxic effects of retene in HepG2 cells. Altogether our results suggest that retene may contribute to the harmful effects caused by biomass burning PM and represent a potential hazard to human health.

A yearlong monitoring campaign of polycyclic aromatic compounds and other air pollutants at three sites in Sweden: Source identification, in vitro toxicity and human health risk assessment.

Air pollution is a complex mixture of gases and particulate matter (PM) with local and non-local emission sources, resulting in spatiotemporal variability in concentrations and composition, and thus associated health risks. To study this in the greater Stockholm area, a yearlong monitoring campaign with in situ measurements of PM10, PM1, black carbon, NOx, O3, and PM10-sampling was performed. The locations included an Urban and a Rural background site and a Highway site. Chemical analysis of PM10 was performed to quantify monthly levels of polycyclic aromatic compounds (PACs), which together with other air pollution data were used for source apportionment and health risk assessment. Organic extracts from PM10 were tested for oxidative potential in human bronchial epithelial cells. Strong seasonal patterns were found for most air pollutants including PACs, with higher levels during the winter months than summer e.g., highest levels of PM10 were detected in March at the Highway site (33.2 µg/m3) and lowest in May at the Rural site (3.6 µg/m3). In general, air pollutant levels at the sites were in the order Highway > Urban > Rural. Multivariate analysis identified several polar PACs, including 6H-Benzo[cd]pyren-6-one, as possible discriminatory markers for these sites. The main sources of particulate pollution for all sites were vehicle exhaust and biomass burning emissions, although diesel exhaust was an important source at the Highway site. In vitro results agreed with air pollutant levels, with higher oxidative potential from the winter samples. Estimated lung cancer cases were in the order PM10 > NO2 > PACs for all sites, and with less evident seasonal differences than in vitro results. In conclusion, our study presents novel seasonal data for many PACs together with air pollutants more traditionally included in air quality monitoring. Moreover, seasonal differences in air pollutant levels correlated with differences in toxicity in vitro.

Non-additive mixture effects of benzo[a]pyrene and pesticides in vitro and in vivo: Role of AhR signaling.

Polycyclic aromatic hydrocarbons (PAHs) and pesticides are two major groups of environmental contaminants which humans are simultaneously exposed to. However, potential mixture interactions of these groups of chemicals are not well-studied. In this study, the effects of binary mixtures of the PAH benzo[a]pyrene (B[a]P) and the commonly used pesticides chlorpyrifos, paraquat and tebuconazole on human liver HepG2 cells were investigated. The results showed that binary mixtures of B[a]P and paraquat or tebuconazole mainly caused additive effects on cell viability and cytochrome P4501a1 (CYP1A1) expression compared to single compound exposures. In contrast, the binary mixture with chlorpyrifos interacted antagonistically on cell viability and ROS production, whereas synergistic effects were observed for induction of CYP1A1 expression. B[a]P and chlorpyrifos also inhibited the activity of recombinant human CYP1A1 enzyme. To verify the synergistic in vitro results, zebrafish (Danio rerio) embryos were exposed to binary mixtures of B[a]P and chlorpyrifos. The mixtures caused synergistic induction of CYP1A expression, as well as synergistic developmental toxicity on multiple endpoints including non-inflated swim bladder, yolk-sac and pericardial edema, and spinal deformation. The effects were reduced upon morpholino-mediated knockdown of the aryl hydrocarbon receptor (AhR), indicating an AhR-dependence of the synergistic toxicity. Altogether, these data suggest that the combination of AhR activation and CYP1A1 inhibition is responsible for the underlying non-additive interaction between B[a]P and chlorpyrifos in vitro and in vivo.

Determination of whole mixture-based potency factors for cancer risk assessment of complex environmental mixtures by in vitro testing of standard reference materials.

Whole mixture-based testing using in vitro new approach methodologies (NAMs) has been suggested to facilitate the hazard and risk assessment of complex environmental mixtures. Previous studies have shown that phosphorylation of DNA damage signaling proteins checkpoint kinase 1 (pChk1) and histone 2AX (γH2AX) are sensitive markers that can be used for estimating carcinogenicity potencies in vitro. Here, and with the aim to better validate the applicability, in vitro-based Mixture Potency Factors (MPFs) of Standard Reference Materials (SRMs) from environmental polycyclic aromatic hydrocarbon (PAH)-containing mixtures were determined and compared to published mutagenicity and tumorigenicity data. Also, genotoxicity was assessed by a flow cytometry-based micronucleus (MN) assay which showed that only benzo[a]pyrene (B[a]P) and coal tar SRM (SRM1597a) caused dose-dependent increases of MN formation, while extracts of diesel particulate matter (SRM1650b), diesel particulate extract (SRM1975), and urban dust (SRM1649b) did not. However, a dose-dependent activation of DNA damage signaling was observed for all PAHs and SRMs. The results demonstrated that all SRMs were more potent than B[a]P, at B[a]P-equivalent concentrations, to induce pChk1 and γH2AX, and that western blot was more sensitive than the In-Cell Western assay in detecting their activation in response to these complex mixtures. Relative MPFs, based on dose-response modelling of pChk1 and γH2AX, ranged 113 - 5270 for the SRMs, indicating several orders of magnitude higher genotoxic potential than B[a]P. Moreover, these MPFs were in good agreement with potency values based on published data from Salmonella mutagenicity and in vivo carcinogenicity studies. In conclusion, these comparisons further validate the feasibility of applying in vitro NAMs, such as whole-mixture based MPFs, in cancer risk assessment of complex mixtures.

In vitro cytotoxicity and genotoxicity of single and combined pesticides used by Bolivian farmers.

We previously showed that farmers in Bolivia are exposed to many pesticides, some at elevated levels, and that this was associated with increased risk of genetic damage. To improve the understanding of possible mixture effects, the cytotoxicity and genotoxicity of pesticides were studied in vitro using human liver HepG2 cells. The studied pesticides were 2,4-D, chlorpyrifos, cypermethrin, glyphosate, methamidophos, paraquat, profenofos, and tebuconazole. Three mixtures (U1, U2, and U3) were based on profiles of urinary pesticide metabolites and one mixture on the most frequently used pesticides (S1). The results showed that paraquat and methamidophos were the most cytotoxic pesticides (EC50 ≤0.3 mM). Paraquat, chlorpyrifos, tebuconazole, and the U1, U2, and U3 mixtures, which contained a large proportion of either chlorpyrifos or tebuconazole, significantly increased intracellular ROS levels. Most pesticides activated DNA damage signaling through proteins Chk1 and H2AX. Strongest responses were elicited by paraquat, profenofos, chlorpyrifos, cypermethrin, and the S1 mixture, which contained 25% paraquat. Comet assay revealed significant increases of DNA damage in response to paraquat, cypermethrin, and U2 and S1 mixtures, which contained high levels of cypermethrin and paraquat, respectively. In summary, we showed that the tested pesticides, alone or in mixtures, in general induced oxidative stress and that most pesticides, and especially paraquat and cypermethrin, were genotoxic in HepG2 cells. We could also show that mixtures dominated by these two pesticides displayed a marked genotoxic potency, which agreed with our previous population studies.

Application of Text Mining in Risk Assessment of Chemical Mixtures: A Case Study of Polycyclic Aromatic Hydrocarbons (PAHs).

BACKGROUND: Cancer risk assessment of complex exposures, such as exposure to mixtures of polycyclic aromatic hydrocarbons (PAHs), is challenging due to the diverse biological activities of these compounds. With the help of text mining (TM), we have developed TM tools-the latest iteration of the Cancer Risk Assessment using Biomedical literature tool (CRAB3) and a Cancer Hallmarks Analytics Tool (CHAT)-that could be useful for automatic literature analyses in cancer risk assessment and research. Although CRAB3 analyses are based on carcinogenic modes of action (MOAs) and cover almost all the key characteristics of carcinogens, CHAT evaluates literature according to the hallmarks of cancer referring to the alterations in cellular behavior that characterize the cancer cell. OBJECTIVES: The objective was to evaluate the usefulness of these tools to support cancer risk assessment by performing a case study of 22 European Union and U.S. Environmental Protection Agency priority PAHs and diesel exhaust and a case study of PAH interactions with silica. METHODS: We analyzed PubMed literature, comprising 57,498 references concerning priority PAHs and complex PAH mixtures, using CRAB3 and CHAT. RESULTS: CRAB3 analyses correctly identified similarities and differences in genotoxic and nongenotoxic MOAs of the 22 priority PAHs and grouped them according to their known carcinogenic potential. CHAT had the same capacity and complemented the CRAB output when comparing, for example, benzo[a]pyrene and dibenzo[a,l]pyrene. Both CRAB3 and CHAT analyses highlighted potentially interacting mechanisms within and across complex PAH mixtures and mechanisms of possible importance for interactions with silica. CONCLUSION: These data suggest that our TM approach can be useful in the hazard identification of PAHs and mixtures including PAHs. The tools can assist in grouping chemicals and identifying similarities and differences in carcinogenic MOAs and their interactions. https://doi.org/10.1289/EHP6702.

Polycyclic aromatic compounds in particulate matter and indoor dust at preschools in Stockholm, Sweden: Occurrence, sources and genotoxic potential in vitro.

Children spend a significant amount of their day in preschool; thus, environmental quality at preschools may have an impact on children's health. In the present study, we analyzed polycyclic aromatic compounds (PACs), including PAHs, alkylated PAHs and oxygenated PAHs (OPAHs), in indoor and outdoor air particulate matter (PM10) and indoor dust at preschools in Stockholm, Sweden. There were significant correlations between PAC levels in outdoor and indoor PM10, with in general higher PAC levels outdoors. Fluoranthene and pyrene were detected at highest levels in all sample types, although phenanthrene and methylated phenanthrene derivatives also were found at high levels in indoor dust. In addition, the highly carcinogenic PAHs 7H-benzo[c]fluorene, 7,12-dimethylbenz[a]anthracene, benz[j]aceanthrylene, and dibenzo[a,l]pyrene were detected in some samples. Benzanthrone was the most prevalent OPAH in PM10 samples and 9,10-anthraquinone in indoor dust. Based on diagnostic ratios and Positive Matrix Factorization we identified vehicle emission and biomass burning as important PAC sources for all samples analyzed. However, poor correlation between PAC levels in indoor PM10 and indoor dust suggested additional sources for the latter. Measuring activation of DNA damage signaling in human cells exposed to organic extracts of the samples indicated substantial genotoxic potential of outdoor PM10 and indoor dust. Determination of benzo[a]pyrene equivalents demonstrated that the highly potent PAHs benz[j]aceanthrylene and dibenz[a,h]anthracene contributed more than 20% to the total carcinogenic potency of the samples. We conclude that PAC levels at Stockholm preschools are relatively low but that outdoor air quality may impact on the indoor environment.

Expression of Interleukin 6 signaling receptors in carotid atherosclerosis.

Interleukin (IL) 6 contributes to atherosclerotic plaque development through IL6 membrane-bound (IL6R and gp130) and soluble (sIL6R and sgp130) receptors. We investigated IL6 receptor expression in carotid plaques and its correlation with circulating IL6 and soluble receptor levels. Plasma samples and carotid plaques were obtained from 78 patients in the Biobank of Karolinska Endarterectomies study. IL6, sIL6R, and sgp130 were measured in plasma and IL6, IL6R, sIL6R, GP130, and sGP130-RAPS (sGP130) gene expression assessed in carotid plaques. Correlations between plaque IL6 signaling gene expression and plasma levels were determined by Spearman's correlation. Differences in plasma and gene expression levels between patients with (n = 53) and without (n = 25) a history of a cerebral event and statin-treated (n = 65) and non-treated (n = 11), were estimated by Kruskal-Wallis. IL6 and its receptors were all expressed in carotid plaques. There was a positive, borderline significant, moderate correlation between plasma IL6 and sIL6R and the respective gene expression levels (rho 0.23 and 0.22, both p = 0.05). IL6R expression was higher in patients with a history of a cerebrovascular event compared to those without (p = 0.007). Statin-treated had higher IL6R, sIL6R, and sGP130 expression levels and plasma sIL6R compared to non-treated patients (all p < 0.05). In conclusion, all components of the IL6 signaling pathways are expressed in carotid artery plaques and IL6 and sIL6R plasma levels correlate moderately with IL6 and sIL6R. Our data suggest that IL6 signaling in the circulation might mirror the system activity in the plaque, thus adding novel perspectives to the role of IL6 signaling in atherosclerosis.

Transcriptional mutagenesis dramatically alters genome-wide p53 transactivation landscape.

The transcriptional error rate can be significantly increased by the presence of DNA lesions that instruct mis-insertion during transcription; a process referred to as transcriptional mutagenesis (TM) that can result in altered protein function. Herein, we determined the effect of O6-methylguanine (O6-meG) on transcription and subsequent transactivation activity of p53 in human lung H1299 cells. Levels of TM and effects on transactivation were determined genome wide by RNA-seq. Results showed that 47% of all p53 transcripts contained an uridine misincorporation opposite the lesion at 6 h post transfection, which was decreased to 18% at 24 h. TM at these levels reduced DNA binding activity of p53 to 21% and 80% compared to wild type p53, respectively. Gene expression data were analysed to identify differentially expressed genes due to TM of p53. We show a temporal repression of transactivation of > 100 high confidence p53 target genes including regulators of the cell cycle, DNA damage response and apoptosis. In addition, TM repressed the transcriptional downregulation by p53 of several negative regulators of proliferation and differentiation. Our work demonstrates that TM, even when restricting its effect to an individual transcription factor, has the potential to alter gene expression programs and diversify cellular phenotypes.

Similar polycyclic aromatic hydrocarbon and genotoxicity profiles of atmospheric particulate matter from cities on three different continents.

The extractable organic material (EOM) from atmospheric total suspended particles (TSP) contains several organic compounds including non-substituted polycyclic aromatic hydrocarbons (PAHs), alkyl-PAHs, and nitro-PAHs. These chemicals seem to be among the key drivers of TSP genotoxicity. We have shown previously that the mutagenic potencies of the EOM from Limeira, Stockholm, and Kyoto, cities with markedly different meteorological conditions and pollution sources are similar. Here we compare the profiles of non-substituted PAHs (27 congeners), alkyl-PAHs (15 congeners), and nitro-PAHs (7 congeners) from the same EOM samples from these cities. We also compared the genotoxicity profiles using comet and micronucleus assays in human bronchial epithelial cells. The profiles of PAHs, as well as the cytotoxic and genotoxic potencies when expressed in EOM, were quite similar among the studied cities. It seems that despite the differences in meteorological conditions and pollution sources of the cities, removal, mixing, and different atmospheric transformation processes may be contributing to the similarity of the PAHs composition and genotoxicity profiles. More studies are required to verify if this would be a general rule applicable to other cities. Although these profiles were similar for all three cities, the EOM concentration in the atmospheres is markedly different. Thus, the population of Limeira (∼10-fold more EOM/m3 than Stockholm and ∼6-fold more than Kyoto) is exposed to higher concentrations of genotoxic pollutants, and Kyoto's population is 1.5-fold more exposed than Stockholm's. Therefore, to reduce the risk of human exposure to TSP genotoxins, the volume of emissions needs to be reduced.

Genotoxicity and DNA damage signaling in response to complex mixtures of PAHs in biomass burning particulate matter from cashew nut roasting.

Approximately 3 billion people world-wide are exposed to air pollution from biomass burning. Herein, particulate matter (PM) emitted from artisanal cashew nut roasting, an important economic activity worldwide, was investigated. This study focused on: i) chemical characterization of polycyclic aromatic hydrocarbons (PAHs) and oxygenated (oxy-) PAHs; ii) intracellular levels of reactive oxygen species (ROS); iii) genotoxic effects and time- and dose-dependent activation of DNA damage signaling, and iv) differential expression of genes involved in xenobiotic metabolism, inflammation, cell cycle arrest and DNA repair, using A549 lung cells. Among the PAHs, chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene, and benz[a]anthracene showed the highest concentrations (7.8-10 ng/m3), while benzanthrone and 9,10-anthraquinone were the most abundant oxy-PAHs. Testing of PM extracts was based on B[a]P equivalent doses (B[a]Peq). IC50 values for viability were 5.7 and 3.0 nM B[a]Peq at 24 h and 48 h, respectively. At these low doses, we observed a time- and dose-dependent increase in intracellular levels of ROS, genotoxicity (DNA strand breaks) and DNA damage signaling (phosphorylation of the protein checkpoint kinase 1 - Chk1). In comparison, effects of B[a]P alone was observed at micromolar range. To our knowledge, no previous study has demonstrated an activation of pChk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro, in lung cells exposed to cashew nut roasting extracts. Sustained induction of expression of several important stress response mediators of xenobiotic metabolism (CYP1A1, CYP1B1), ROS and pro-inflammatory response (IL-8, TNF-α, IL-2, COX2), and DNA damage response (CDKN1A and DDB2) was also identified. In conclusion, our data show high potency of cashew nut roasting PM to induce cellular stress including genotoxicity, and more potently when compared to B[a]P alone. Our study provides new data that will help elucidate the toxic effects of low-levels of PAH mixtures from air PM generated by cashew nut roasting.

Polycyclic aromatic compounds in urban soils of Stockholm City: Occurrence, sources and human health risk assessment.

Polycyclic aromatic compounds (PACs) are ubiquitous pollutants that are found everywhere in our environment, including air, soil and water. The aim of this study was to determine concentrations, distribution, sources and potential health risk of 43 PACs in soils collected from 25 urban parks in Stockholm City, Sweden. These PACs included 21 PAHs, 11 oxygenated PAHs, 7 methylated PAHs, and 4 azaarenes whose concentrations ranged between 190 and 54 500, 30.5-5 300, 14.9-680, and 4.17-590 ng/g soil, respectively. Fluoranthene was found at the highest levels ranging between 17.7 and 9800 ng/g, benzo[a]pyrene between 9.64 and 4600 ng/g, and the highly potent carcinogen dibenzo[a,l]pyrene up to 740 ng/g. The most abundant oxy-PAH was 6H-benzo[cd]pyren-6-one (2.09-2300 ng/g). Primary sources of PAHs were identified by use of diagnostic ratios and Positive Matrix Factorization modelling and found to be pyrogenic including vehicle emissions and combustion of biomass. Estimating the incremental lifetime cancer risks (ILCRS) associated with exposure to PAHs in these soils indicated that the PAH levels in some parks constitute a considerable increased risk level for adults and children (total ILCR > 1 × 10-4). Compared to worldwide urban parks contamination, we conclude that the PAC soil levels in parks of Stockholm City in general are low, but that some parks are more heavily contaminated and should be considered for clean-up actions to limit human health risks.

Increased levels of genotoxic damage in a Bolivian agricultural population exposed to mixtures of pesticides.

During the past decades, farmers in low to middle-income countries have increased their use of pesticides, and thereby the risk of being exposed to potentially genotoxic chemicals that can cause adverse health effects. Here, the aim was to investigate the correlation between exposure to pesticides and genotoxic damage in a Bolivian agricultural population. Genotoxic effects were assessed in peripheral blood samples by comet and micronucleus (MN) assays, and exposure levels by measurements of 10 urinary pesticide metabolites. Genetic susceptibility was assessed by determination of null frequency of GSTM1 and GSTT1 genotypes. The results showed higher MN frequency in women and farmers active ≥8 years compared to their counterpart (P < 0.05). In addition, age, GST genotype, alcohol consumption, and type of water source influenced levels of genotoxic damage. Individuals with high exposure to tebuconazole, 2,4-D, or cyfluthrin displayed increased levels of genotoxic damage (P < 0.05-0.001). Logistic regression was conducted to evaluate associations between pesticide exposure and risk of genotoxic damage. After adjustment for confounders, a significant increased risk of DNA strand breaks was found for high exposure to 2,4-D, odds ratio (OR) = 1.99 (P < 0.05). In contrast, high exposure to pyrethroids was associated with a reduced risk of DNA strand breaks, OR = 0.49 (P < 0.05). It was also found that high exposure to certain mixtures of pesticides (containing mainly 2,4-D or cyfluthrin) was significantly associated with increased level and risk of genotoxic damage (P < 0.05). In conclusion, our data show that high exposure levels to some pesticides is associated with an increased risk of genotoxic damage among Bolivian farmers, suggesting that their use should be better controlled or limited.

In vitro and in vivo genotoxicity of oxygenated polycyclic aromatic hydrocarbons.

Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of environmental pollutants found in complex mixtures together with PAHs. In contrast to the extensively studied PAHs, which have been established to have mutagenic and carcinogenic properties, much less is known about the effects of oxy-PAHs. The present work aimed to investigate the genotoxic potency of a set of environmentally relevant oxy-PAHs along with environmental soil samples in human bronchial epithelial cells (HBEC). We found that all oxy-PAHs tested induced DNA strand breaks in a dose-dependent manner and some of the oxy-PAHs further induced micronuclei formation. Our results showed weak effects in response to the oxy-PAH containing subfraction of the soil sample. The genotoxic potency was confirmed in both HBEC and HepG2 cells following exposure to oxy-PAHs by an increased level of phospho-Chk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro. We further exposed zebrafish embryos to single oxy-PAHs or a binary mixture with PAH benzo[a]pyrene (B[a]P) and found the mixture to induce comparable or greater effects on the induction of DNA strand breaks compared to the sum of that induced by B[a]P and oxy-PAHs alone. In conclusion, oxy-PAHs were found to elicit genotoxic effects at similar or higher levels to that of B[a]P which indicates that oxy-PAHs may contribute significantly to the total carcinogenic potency of environmental PAH mixtures. This emphasizes further investigations of these compounds as well as the need to include oxy-PAHs in environmental monitoring programs in order to improve health risk assessment.

TGF beta promotes repair of bulky DNA damage through increased ERCC1/XPF and ERCC1/XPA interaction.

Transforming growth factor beta (TGFß) is multifunctional cytokine that is involved in the coordination and regulation of many cellular homeostatic processes. Compromised TGFß activity has been attributed to promotion of human cancers. Recent studies have identified a role for TGFß in response to radiation-induced DNA damage, suggesting a link between TGFß and the DNA damage response with implications for cancer development. In this study, the effects of TGFß on promoting the repair of bulky DNA damage, through modulation of nucleotide excision repair (NER), were investigated. We show that treatment of cells with exogenous TGFß leads to enhanced repair of DNA damage formed by polycyclic aromatic hydrocarbons and ultraviolet-C radiation; similarly, cells with constitutively activated endogenous TGFß signaling show comparable responses. This effect of TGFß is independent of the cell cycle. The response to TGFß is decreased in cells that have compromised TGFß signaling through RNA interference of Smad4 and is decreased in NER-deficient cells and cells with compromised NER through RNA interference of excision repair cross-complementing group 1 (ERCC1). Increased interaction and nuclear localization of ERCC1/xeroderma pigmentosum (XP) F and ERCC1/XPA proteins is observed after TGFß treatment. Our study represents the first experimental evidence of a role for TGFß in the repair of bulky DNA damage resulting from promotion of the interaction and localization of repair protein complexes involved in the incision step of NER.

O6-methylguanine-induced transcriptional mutagenesis reduces p53 tumor-suppressor function.

Altered protein function due to mutagenesis plays an important role in disease development. This is perhaps most evident in tumorigenesis and the associated loss or gain of function of tumor-suppressor genes and oncogenes. The extent to which lesion-induced transcriptional mutagenesis (TM) influences protein function and its contribution to the development of disease is not well understood. In this study, the impact of O6-methylguanine on the transcription fidelity of p53 and the subsequent effects on the protein's function as a regulator of cell death and cell-cycle arrest were examined in human cells. Levels of TM were determined by RNA-sequencing. In cells with active DNA repair, misincorporation of uridine opposite the lesion occurred in 0.14% of the transcripts and increased to 14.7% when repair by alkylguanine-DNA alkyltransferase was compromised. Expression of the dominant-negative p53 R248W mutant due to TM significantly reduced the transactivation of several established p53 target genes that mediate the tumor-suppressor function, including CDKN1A (p21) and BBC3 (PUMA). This resulted in deregulated signaling through the retinoblastoma protein and loss of G1/S cell-cycle checkpoint function. In addition, we observed impaired activation of apoptosis coupled to the reduction of the tumor-suppressor functions of p53. Taking these findings together, this work provides evidence that TM can induce phenotypic changes in mammalian cells that have important implications for the role of TM in tumorigenesis.

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.

Cancer Risk Assessment of Airborne PAHs Based on in Vitro Mixture Potency Factors.

Complex mixtures of polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants associated with adverse human health effects including cancer. However, the risk of exposure to mixtures is difficult to estimate, and risk assessment by whole mixture potency evaluations has been suggested. To facilitate this, reliable in vitro based testing systems are necessary. Here, we investigated if activation of DNA damage signaling in vitro could be an endpoint for developing whole mixture potency factors (MPFs) for airborne PAHs. Activation of DNA damage signaling was assessed by phosphorylation of Chk1 and H2AX using Western blotting. To validate the in vitro approach, potency factors were determined for seven individual PAHs which were in very good agreement with established potency factors based on cancer data in vivo. Applying the method using Stockholm air PAH samples indicated MPFs with orders of magnitude higher carcinogenic potency than predicted by established in vivo-based potency factors. Applying the MPFs in cancer risk assessment suggested that 45.4 (6% of all) cancer cases per year in Stockholm are due to airborne PAHs. Applying established models resulted in <1 cancer case per year, which is far from expected levels. We conclude that our in vitro based approach for establishing MPFs could be a novel method to assess whole mixture samples of airborne PAHs to improve health risk assessment.