Enhanced DNA adduct formation by benzo[a]pyrene in human liver cells lacking cytochrome P450 oxidoreductase.

Diet is a major source of human exposure to polycyclic aromatic hydrocarbons (PAHs), of which benzo[a]pyrene (BaP) is the most commonly studied and measured. BaP has been considered to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes whose activity can be modulated by cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes. Previous studies showed that BaP-DNA adduct formation was greater in the livers of Hepatic Reductase Null (HRN) mice, in which POR is deleted specifically in hepatocytes, than in wild-type (WT) mice. In the present study we used human hepatoma HepG2 cells carrying a knockout (KO) in the POR gene as a human in vitro model that can mimic the HRN mouse model. Treatment to BaP for up to 48 h caused similar cytotoxicity in POR KO and WT HepG2 cells. However, levels of BaP activation (i.e. BaP-7,8-dihydrodiol formation) were higher in POR KO HepG2 cells than in WT HepG2 cells after 48 h. This also resulted in substantially higher BaP-DNA adduct formation in POR KO HepG2 cells indicating that BaP metabolism is delayed in POR KO HepG2 cells thereby prolonging the effective exposure of cells to unmetabolized BaP. As was seen in the HRN mouse model, these results suggest that cytochrome b5, another component of the mixed-function oxidase system, which can also serve as electron donor to CYP enzymes along with NADH:cytochrome b5 redutase, contributes to the bioactivation of BaP in POR KO HepG2 cells. Collectively, these findings indicate that CYPs play a more important role in BaP detoxication as opposed to activation.

Deletion of cytochrome P450 oxidoreductase enhances metabolism and DNA adduct formation of benzo[a]pyrene in Hepa1c1c7 cells.

The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.

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.

Genotoxicity of fine and coarse fraction ambient particulate matter in immortalised normal (TT1) and cancer-derived (A549) alveolar epithelial cells.

Human exposure to airborne particulate matter (PM) is associated with adverse cardiopulmonary health effects, including lung cancer. Ambient PM represents a heterogeneous mixture of chemical classes including transition metals, polycyclic aromatic hydrocarbons (PAHs) and their derivatives such as nitro-PAHs, many of which are classified as putative carcinogens. As the primary site of human exposure to PM is the lungs, we investigated the response of two alveolar epithelial cell lines, the tumour-derived A549 and newly described TT1 cells, to fine and coarse PM collected from background and roadside locations. We show that coarse PM elicits a genotoxic response in the TT1 cells, with the strongest signal associated with the background sample. This response could be recapitulated using the organic extract derived from this sample. No responses were observed in PM-challenged A549 cells. Fine PM failed to elicit a genotoxic response in either cell line despite the higher PAH concentrations within this fraction. Consistent with the lack of a simplistic association between PM PAH content and the observed genotoxic response, TT1 cells treated with benzo[a]pyrene (BaP) demonstrated no increase in the selected markers. In contrast, a pattern of response was observed in TT1 cells challenged with 3-nitrobenzanthrone (3-NBA) similar to that with coarse PM. Together, these data illustrated the suitability of the TT1 cell line for assessing PM-induced genotoxicity and challenge the contention that fine roadside PM poses the higher cancer risk. Furthermore, the response to 3-NBA and not BaP suggests a major contribution of nitro-PAHs to the overall toxicity of PM. Environ. Mol. Mutagen. 59:290-301, 2018. © 2018 The Authors Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

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.

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 γH2AX, 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 γH2AX, 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.

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.

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.

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 BP equivalent concentrations in 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 PAH-contaminated 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 PAHs.

Therapy-induced carboplatin-DNA adduct levels in human ovarian tumours in relation to assessment of adduct measurement in mouse tissues.

Despite an increasing understanding of the molecular mechanisms by which platinum drug DNA adducts interact with cellular processes, the relationship between adduct formation in tumours and clinical response remains unclear. We have determined carboplatin-DNA adduct levels in biopsies removed from ovarian cancer patients following treatment. Reliability of DNA adduct measurements in tissues samples were assessed using experimental animals. Platinum-DNA adduct levels were measured using inductively coupled plasma mass spectrometry (ICP-MS) and plasma drug concentrations determined by atomic absorption spectrometry (AAS). Adduct levels in tissues and plasma pharmacokinetics were determined in Balb/c mice exposed to platinum drugs. Comparisons of adduct levels in tumour and normal tissue were made in nu/nu mice carrying human neuroblastoma xenografts. At 30 min post-cisplatin administration, adduct levels in DNA from kidney and liver were approximately 10- and 6-fold higher than spleen or tumour. By 60 min, levels in liver and kidney, but not spleen or tumour, had fallen considerably. Carboplatin showed high adduct levels only in kidney. Adduct levels in tumour xenografts were comparable to those induced in vitro with similar drug exposures. In clinical samples removed 6h after drug administration, adduct levels ranged from 1.9 to 4.3 and 0.2 to 3.6 nmol Pt/g DNA for tumour biopsies and peripheral blood mononuclear cells, respectively. No correlation was apparent between these two data sets. The present results demonstrate that reliable measurements of adducts in clinical tumours are feasible. Future results should provide insight into drug resistance.