Benzo[A]Pyrene and Benzo[E]Pyrene: Photoreactivity and Phototoxicity toward Human Keratinocytes.

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds derived mostly from the incomplete combustion of fossil fuels and biomass. Human skin can absorb PAHs and the uptake increases with their molar mass and lipophilicity. Benzopyrene is high molecular weight PAH frequently appearing in ambient pollution. It exists in two isomeric forms: benzo[a]pyrene (BaP) and benzo[e]pyrene (BeP), which exhibit different biological activity. Although certain properties of benzopyrenes suggested photoreactivity of the compounds, no direct measurements were previously conducted to characterize their photochemical activity. In this study, quantum yield and action spectra of singlet oxygen photogeneration by BaP and BeP were measured by time-resolved near-infrared phosphorescence, and the ability of both compounds to photogenerate superoxide anion was assessed by electron paramagnetic resonance (EPR) spin-trapping. The measurements revealed high efficiency of benzopyrenes to photogenerate singlet oxygen and their ability to photogenerate superoxide anion. Using HaCaT cells as single-layer skin model, we demonstrated concentration-dependent and light-dependent cytotoxicity of BaP and BeP. The compounds induced damage to the cell mitochondria and elevated the levels of intracellular reactive oxygen species.

Benzopyrene induces oxidative stress and increases expression and activities of antioxidant enzymes, and CYP450 and GST metabolizing enzymes in Ulva lactuca (Chlorophyta).

MAIN CONCLUSION: Benzopyrene is rapidly incorporated and metabolized, and induces oxidative stress and activation of antioxidant enzymes, and CYP450 and GST metabolizing enzymes in Ulva lactuca. To analyze absorption and metabolism of benzo[a]pyrene (BaP) in Ulva lactuca, the alga was cultivated with 5 µM of BaP for 72 h. In the culture medium, BaP level rapidly decreased reaching a minimal level at 12 h and, in the alga, BaP level increased until 6 h, remained stable until 24 h, and decreased until 72 h indicating that BaP is being metabolized in U. lactuca. In addition, BaP induced an initial increase in hydrogen peroxide decreasing until 24 h, superoxide anions level that remained high until 72 h, and lipoperoxides that initially increased and decreased until 72 h, showing that BaP induced oxidative stress. Activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (AP), glutathione reductase (GR) and glutathione peroxidase (GP) were increased, whereas dehydroascorbate reductase (DHAR) activity was unchanged. The level of transcripts encoding these antioxidant enzymes was increased, but transcripts encoding DHAR remained unchanged. Interestingly, the activity of glutathione-S-transferase (GST) was also increased, and inhibitors of cytochrome P450 (CYP450) and GST activities enhanced the level of BaP in algal tissue, suggesting that these enzymes participate in BaP metabolism.

Marine Actinomycetes-Derived Secondary Metabolites Overcome TRAIL-Resistance via the Intrinsic Pathway through Downregulation of Survivin and XIAP.

Resistance of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis represents the major hurdle to the clinical use of TRAIL or its derivatives. The discovery and development of lead compounds able to sensitize tumor cells to TRAIL-induced cell death is thus likely to overcome this limitation. We recently reported that marine actinomycetes' crude extracts could restore TRAIL sensitivity of the MDA-MB-231 resistant triple negative breast cancer cell line. We demonstrate in this study, that purified secondary metabolites originating from distinct marine actinomycetes (sharkquinone (1), resistomycin (2), undecylprodigiosin (3), butylcyclopentylprodigiosin (4), elloxizanone A (5) and B (6), carboxyexfoliazone (7), and exfoliazone (8)), alone, and in a concentration-dependent manner, induce killing in both MDA-MB-231 and HCT116 cell lines. Combined with TRAIL, these compounds displayed additive to synergistic apoptotic activity in the Jurkat, HCT116 and MDA-MB-231 cell lines. Mechanistically, these secondary metabolites induced and enhanced procaspase-10, -8, -9 and -3 activation leading to an increase in PARP and lamin A/C cleavage. Apoptosis induced by these compounds was blocked by the pan-caspase inhibitor QvD, but not by a deficiency in caspase-8, FADD or TRAIL agonist receptors. Activation of the intrinsic pathway, on the other hand, is likely to explain both their ability to trigger cell death and to restore sensitivity to TRAIL, as it was evidenced that these compounds could induce the downregulation of XIAP and survivin. Our data further highlight that compounds derived from marine sources may lead to novel anti-cancer drug discovery.

Cellular Metabolism in High-Throughput In Vitro Reporter Gene Assays and Implications for the Quantitative In Vitro-In Vivo Extrapolation.

High-throughput in vitro reporter gene assays are increasingly applied to assess the potency of chemicals to alter specific cellular signaling pathways. Genetically modified reporter gene cell lines provide stable readouts of the activation of cellular receptors or transcription factors of interest, but such reporter gene assays have been criticized for not capturing cellular metabolism. We characterized the metabolic activity of the widely applied AREc32 (human breast cancer MCF-7), ARE-bla (human liver cancer HepG2), and GR-bla (human embryonic kidney HEK293) reporter gene cells in the absence and in the presence of benzo[a]pyrene (BaP), an AhR ligand known to upregulate cytochrome P450 in vitro and in vivo. We combined fluorescence microscopy with chemical analysis, real-time PCR, and ethoxyresorufin-O-deethylase activity measurements to track temporal changes in BaP and its metabolites in the cells and surrounding medium over time in relation to the expression and activity of metabolic enzymes. Decreasing BaP concentrations and formation of metabolites agreed with the high basal CYP1 activity of ARE-bla and the strong CYP1A1 mRNA induction in AREc32, whereas BaP concentrations were constant in GR-bla, in which neither metabolites nor CYP1 induction was detected. The study emphasizes that differences in sensitivity between reporter gene assays may be caused not only by different reporter constructs but also by a varying biotransformation rate of the evaluated parent chemical. The basal metabolic capacity of reporter gene cells in the absence of chemicals is not a clear indication because we demonstrated that the metabolic activity can be upregulated by AhR ligands during the assay. The combination of methods presented here is suitable to characterize the metabolic activity of cells in vitro and can improve the interpretation of in vitro reporter gene effect data and extrapolation to in vivo human exposure.

Synergistic effects of sulfur dioxide and polycyclic aromatic hydrocarbons on pulmonary pro-fibrosis via mir-30c-1-3p/ transforming growth factor ß type II receptor axis.

SO2 and PAHs are well-known pollutants of coal burning and significant contributors to haze episodes. The purpose of the study is to determine whether the combined effects of SO2 and BaP are synergetic and to investigate the pro-fibrotic influences and possible mechanism from the aspect of microRNAs. In the present study cellular metabolic activity of BEAS-2B was assessed using MTT probe. C57BL/6 mice were exposed to BaP (40 mg/kg b.w.) for 5 days or SO2 (7 mg/m3) inhalation for 4 weeks alone or together. Lung tissues were processed for histology to assess pulmonary fibrosis. The protein level of pulmonary pro-fibrotic genes (Col1a1, Col3a1, alpha-SMA, fibronectin) and TGFßR2 were analyzed by Western blot and immunofluorescence in vivo and in vitro. Furthermore, we clarified that the microRNA expression of mir-30c-1-3p by real-time RT-PCR. The luciferase reporter assay was used to determine the binding sites of mir-30c-1-3p in the 3'-UTR of TGFßR2. It was confirmed that SO2 and BaP acted together to produce synergistic effects in cellular metabolic activity. Coexisting of SO2 and BaP increased the protein expression of pro-fibrotic genes and TGFßR2 and decreased mir-30c-1-3p in vivo and in vitro. Dual-luciferase reporter gene assays showed that TGFßR2 was a validated target of mir-30c-1-3p. All above results demonstrated that mir-30c-1-3p was involved in the synergistic pro-fibrotic effects of SO2 and BaP in lung via targeting TGFßR2. This work implies the potential risk of pulmonary fibrosis from the co-existence of SO2 and PAHs and provides new insights into the molecular markers for relevant diseases.

Benzopyrone represents a privilege scaffold to identify novel adenosine A1/A2A receptor antagonists.

Adenosine receptor antagonists are under investigation as potential drug candidates for the treatment of certain cancers, neurological disorders, depression and potentially improve tumour immunotherapy. The benzo-γ-pyrone scaffold is well-known in medicinal chemistry with diverse pharmacological activities attributed to them, however, their therapeutic potential as adenosine receptor antagonists have not been investigated in detail. To expand on the structure-activity relationships, the present study explored the adenosine A1 and A2A receptor binding affinities of a selected series of benzo-γ-pyrone analogues. In vitro evaluation led to the identification of 5-hydroxy-2-(3-hydroxyphenyl)-4H-1-benzopyran-4-one with the best adenosine A2A receptor affinity among the test compounds and was found to be non-selective (A1Ki = 0.956 µM; A2AKi = 1.44 µM). Hydroxy substitution on ring A and/or B play a key role in modulating the binding affinity at adenosine A1 and A2A receptors. Adenosine A1 receptor affinity was increased to the nanomolar range with hydroxy substitution on C6 (ring A), while meta-hydroxy substitution on ring B governed adenosine A2A receptor affinity. The double bond between C2 and C3 of ring C as well as C2 phenyl substitution was shown to be imperative for both adenosine A1 and A2A receptor affinity. Selected benzo-γ-pyrone derivatives behaved as adenosine A1 receptor antagonists in the performed GTP shift assays. It may be concluded that benzo-γ-pyrone based derivatives are suitable leads for designing and identifying adenosine receptor antagonists as treatment of various disorders.

Benzo(e)pyrene Inhibits Endothelium-Dependent NO-Mediated Dilation of Retinal Arterioles via Superoxide Production and Endoplasmic Reticulum Stress.

Purpose: To investigate whether benzo(e)pyrene (B(e)P), a toxicant in cigarette smoke, affects the endothelium-dependent nitric oxide (NO)-induced vasodilation of the retinal arterioles, and whether oxidative stress, distinct protein kinase signaling pathways, and endoplasmic reticulum (ER) stress are associated with the B(e)P-induced effect on the retinal arterioles. Methods: In this in vitro study, porcine retinal arterioles were isolated, cannulated, and pressurized without flow. These vessels were treated with intraluminal administration of B(e)P or B(e)P plus blockers for 180 minutes. Diametric changes to agonists were recorded by videomicroscopy. Results: Intraluminal treatment with 100 µM B(e)P for 180 minutes significantly reduced the arteriolar vasodilation caused by the endothelium-dependent NO-mediated agonists bradykinin and A23187 but not that caused by endothelium-independent NO donor sodium nitroprusside. The adverse effects of B(e)P on the vasodilatory action of bradykinin were prevented by the superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) inhibitor apocynin, the c-Jun N-terminal kinase (JNK) inhibitor SP600125, the p38 mitogen-activated protein kinase inhibitor SB203580, genistein, resveratrol (RSV), and the ER stress inhibitor 4-phenylbutyrate (4-PBA). The xanthine oxidase inhibitor allopurinol did not alter the effect of B(e)P on the vasodilatory action induced by bradykinin. Conclusions: B(e)P decreases the endothelium-dependent NO-induced vasodilation in the retinal arterioles through the production of superoxide from NADPH oxidase, which is linked to JNK and p38 kinase. The results suggested that ER stress is instrumental in B(e)P-induced endothelial dysfunction and that genistein and RSV might preserve endothelial function.

Effects of Black Raspberry Extract and Berry Compounds on Repair of DNA Damage and Mutagenesis Induced by Chemical and Physical Agents in Human Oral Leukoplakia and Rat Oral Fibroblasts.

Black raspberries (BRB) have been shown to inhibit carcinogenesis in a number of systems, with most studies focusing on progression. Previously we reported that an anthocyanin-enriched black raspberry extract (BE) enhanced repair of dibenzo-[a,l]-pyrene dihydrodiol (DBP-diol)-induced DNA adducts and inhibited DBP-diol and DBP-diolepoxide (DBPDE)-induced mutagenesis in a lacI rat oral fibroblast cell line, suggesting a role for BRB in the inhibition of initiation of carcinogenesis. Here we extend this work to protection by BE against DNA adduct formation induced by dibenzo-[a,l]-pyrene (DBP) in a human oral leukoplakia cell line (MSK) and to a second carcinogen, UV light. Treatment of MSK cells with DBP and DBPDE led to a dose-dependent increase in DBP-DNA adducts. Treatment of MSK cells with BE after addition of DBP reduced levels of adducts relative to cells treated with DBP alone, and treatment of rat oral fibroblasts with BE after addition of DBPDE inhibited mutagenesis. These observations showed that BE affected repair of DNA adducts and not metabolism of DBP. As a proof of principle we also tested aglycones of two anthocyanins commonly found in berries, delphinidin chloride and pelargonidin chloride. Delphinidin chloride reduced DBP-DNA adduct levels in MSK cells, while PGA did not. These results suggested that certain anthocyanins can enhance repair of bulky DNA adducts. As DBP and its metabolites induced formation of bulky DNA adducts, we investigated the effects of BE on genotoxic effects of a second carcinogen that induces bulky DNA damage, UV light. UV irradiation produced a dose-dependent increase in cyclobutanepyrimidine dimer levels in MSK cells, and post-UV treatment with BE resulted in lower cyclobutanepyrimidine dimer levels. Post-UV treatment of the rat lacI cells with BE reduced UV-induced mutagenesis. Taken together, the results demonstrate that BE extract reduces bulky DNA damage and mutagenesis and support a role for BRB in the inhibition of initiation of carcinogenesis.

Genome-wide identification of 52 cytochrome P450 (CYP) genes in the copepod Tigriopus japonicus and their B[α]P-induced expression patterns.

Cytochrome P450s (CYPs) are enzymes with a heme-binding domain that are found in all living organisms. CYP enzymes have important roles associated with detoxification of xenobiotics and endogenous compounds (e.g. steroids, fatty acids, and hormones). Although CYP enzymes have been reported in several invertebrates, including insects, little is known about copepod CYPs. Here, we identified the entire repertoire of CYP genes (n=52) from whole genome and transcriptome sequences of the benthic copepod Tigriopus japonicus, including a tandem duplication (CYP3026A3, CYP3026A4, CYP3026A5), and examined patterns of gene expression over various developmental stages and in response to benzo[α]pyrene (B[α]P) exposure. Through phylogenetic analysis, the 52 T. japonicus CYP genes were assigned to five distinct clans: CYP2 (22 genes), CYP3 (19 genes), CYP4 (two genes), CYP20 (one gene), and mitochondrial (eight genes). Developmental stage and gender-specific expression patterns of the 52 T. japonicus CYPs were analyzed. CYP3022A1 was constitutively expressed during all developmental stages. CYP genes in clans 2 and 3 were induced in response to B[α]P, suggesting that these differentially modulated CYP transcripts are likely involved in defense against exposure to B[α]P and other pollutants. This study enhances our understanding of the repertoire of CYP genes in copepods and of their potential role in development and detoxification in copepods.

Identification of 28 cytochrome P450 genes from the transcriptome of the marine rotifer Brachionus plicatilis and analysis of their expression.

Whole transcriptomes of the rotifer Brachionus plicatilis were analyzed using an Illumina sequencer. De novo assembly was performed with 49,122,780 raw reads using Trinity software. Among the assembled 42,820 contigs, 27,437 putative open reading frame contigs were identified (average length 1235bp; N50=1707bp). Functional gene annotation with Gene Ontology and InterProScan, in addition to Kyoto Encyclopedia of Genes and Genomes pathway analysis, highlighted the metabolism of xenobiotics by cytochrome P450 (CYP). In addition, 28 CYP genes were identified, and their transcriptional responses to benzo[α]pyrene (B[α]P) were investigated. Most of the CYPs were significantly upregulated or downregulated (P<0.05) in response to B[α]P, suggesting that Bp-CYP genes play a crucial role in detoxification mechanisms in response to xenobiotics. This study sheds light on the molecular defense mechanisms of the rotifer B. plicatilis in response to exposure to various chemicals.

Anti-inflammatory Effects of Phyllanthus emblica L on Benzopyrene-Induced Precancerous Lung Lesion by Regulating the IL-1ß/miR-101/Lin28B Signaling Pathway.

BACKGROUND: Phyllanthus emblica L (PEL), a well-known medical plant, has been used in Asian countries for a long time. Increasing evidence suggests that it can prevent the tumorigenesis of cancer associated with nonresolving inflammation. However, the possible anti-inflammatory mechanism responsible for preventing tumorigenesis of precancerous lung lesions is not well elucidated. MATERIALS AND METHODS: Male A/J mice were randomly divided into 5 groups with 10 mice in each group: (1) blank group (saline), (2) benzo(a)pyrene [B(a)P] group, (3) and (4) B(a)P + PEL (5 g/kg/d, 10 g/kg/d, administered by gavage), (5) B(a)P + celecoxib (30 mg/kg/d, administered by gavage). Nodes on the lung surface were observed and calculated. The levels of macrophage inflammatory protein (MIP-2), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1ß were detected by enzyme-linked immunosorbent assay (ELISA) kits. Cyclo-oxygenase-2 (COX-2), hypoxia-inducible factor-1 (HIF-α), IL-1ß, miR-101, and Lin28B protein levels were evaluated by immunohistochemistry and Western blotting. RESULTS: PEL extract treatment significantly reduced the number of nodes on the lung surface and attenuated B(a)P-induced levels of proinflammatory cytokines MIP-2, TNF-α, IL-6, and IL-1ß in lung tissue. The protein expressions of COX-2 and HIF-α were significantly decreased by the treatment of PEL. In addition, both PEL extract and celecoxib markedly upregulate the expression of miR-101 while downregulating IL-1ß and Lin28B levels. CONCLUSION: Our study indicated that treatment with PEL extract can not only protect the lung from inflammatory injury but effectively prevent precancerous lung lesions through regulating the IL-1ß/miR-i101/Lin28B signaling pathway.

Sensitivity of human cells expressing low-fidelity or weak-catalytic-activity variants of DNA polymerase ζ to genotoxic stresses.

Translesion DNA polymerases (TLS pols) play critical roles in defense mechanisms against genotoxic agents. The defects or mutations of TLS pols are predicted to result in hypersensitivity of cells to environmental mutagens. In this study, human cells expressing DNA polymerase ζ (Pol ζ) variants with low fidelity or weak catalytic activity have been established with Nalm-6-MSH+ cells and their sensitivity to mutagenicity and cytotoxicity of benzo[a]pyrene diol epoxide (BPDE) and ultraviolet-C light (UV-C) was examined. The low-fidelity mutants were engineered by knocking-in DNA sequences that direct changes of leucine 2618 to either phenylalanine (L2618F) or methionine (L2618M) of Pol ζ. The weak-catalytic-activity mutants were generated by knocking-in DNA sequences that direct changes of either tyrosine 2779 to phenylalanine (Y2779F) or aspartate 2781 to asparagine (D2781N). In addition, a +1 frameshift mutation, i.e., CCC to CCCC, was introduced in the coding region of the TK1 gene to measure the mutant frequencies. Doubling time and spontaneous TK mutant frequencies of the established cell lines were similar to those of the wild-type cells. The low-fidelity mutants displayed, however, higher sensitivity to the mutagenicity of BPDE and UV-C than the wild-type cells although their cytotoxic sensitivity was not changed. In contrast, the weak-catalytic-activity mutants were more sensitive to the cytotoxicity of BPDE and UV-C than the wild-type cells, and displayed much higher sensitivity to the clastogenicity of BPDE than the wild-type cells in an in vitro micronucleus assay. These results indicate that human Pol ζ is involved in TLS across DNA lesions induced by BPDE and UV-C and also that the TLS plays important roles in induction of mutations, clastogenicity and in cellular survival of the damaged human cells. Similarities and differences in in vivo roles of yeast and human Pol ζ in genome integrity are discussed.

Benzopyrene promotes lung cancer A549 cell migration and invasion through up-regulating cytokine IL8 and chemokines CCL2 and CCL3 expression.

Tobacco-sourced carcinogen including benzopyrene (B[a]P) in lung cancer metastasis has not been fully reported. In this study, lung carcinoma A549 cell line was used to investigate the potential roles of tobacco-sourced B[a]P on cell metastasis and invasion and to assess its underlying mechanism. Effects of tobacco-sourced carcinogen on A549 cell proliferation, metastasis, and invasion were analyzed using MTT assay, Transwell assay, and scratch method, respectively. The effects of tobacco-sourced carcinogen on cytokines and chemokines secretion were detected using enzyme-linked immunosorbent assay. Moreover, correlation between inflammatory factor expression and cancer cell migration and invasion was assessed using siRNA-mediated gene silencing. Data showed that both B[a]P and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone either at high or low dose performed no significant difference on A549 cell proliferation with time increasing. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone performed no significant difference on A549 cell migration and invasion while B[a]P significantly increased A549 cell migration and invasion compared to the control group (P < 0.05). Consequently, except for IL-6, IL-8, CCL-2, and CCL-3, secretions were significantly increased by B[a]P treatment compared to the control (P < 0.05). Furthermore, when CCL-2 and CCL-3 were silenced, the migrated and invasive A549 cells were significantly decreased compared to the control, respectively (P < 0.05), while silenced IL-8 drastically decreased the migrated and invasive cells compared to the control (P < 0.01). Taken together, this study illustrated that there may be significant correlation between smoking and lung cancer metastasis. B[a]P maybe an excellent contributor for lung cancer metastasis through up-regulating IL-8, CCL-2, and CCL-3 expression.

Successful validation of genomic biomarkers for human immunotoxicity in Jurkat T cells in vitro.

Previously, we identified 25 classifier genes that were able to assess immunotoxicity using human Jurkat T cells. The present study aimed to validate these classifiers. For that purpose, Jurkat cells were exposed for 6 h to subcytotoxic doses of nine immunotoxicants, five non-immunotoxicants and four compounds for which human immunotoxicity has not yet been fully established. RNA was isolated and subjected to Fluidigm quantitative real time (qRT)-PCR analysis. The sensitivity, specificity and accuracy of the screening assay as based on the nine immunotoxicants and five non-immunotoxicants used in this study were 100%, 80% and 93%, respectively, which is better than the performance in our previous study. Only one compound was classified as false positive (benzo-e-pyrene). Of the four potential (non-)immunotoxicants, chlorantraniliprole and Hidrasec were classified immunotoxic and Sunset yellow and imidacloprid as non-immunotoxic. ToxPi analysis of the PCR data provided insight in the molecular pathways that were affected by the compounds. The immunotoxicants 2,3-dichloro-propanol and cypermethrin, although structurally different, affected protein metabolism and cholesterol biosynthesis and transport. In addition, four compounds, i.e. chlorpyrifos, aldicarb, benzo-e-pyrene and anti-CD3, affected genes in cholesterol metabolism and transport, protein metabolism and transcription regulation. qRT-PCR on eight additional genes coding for similar processes as defined in ToxPi analyzes, supported these results. In conclusion, the 25 immunotoxic classifiers performed very well in a screening with new non-immunotoxic and immunotoxic compounds. Therefore, the Jurkat screening assay has great promise to be applied within a tiered approach for animal free testing of human immunotoxicity.

Enhanced aggressiveness of benzopyrene-induced squamous carcinomas in transgenic mice overexpressing the proprotein convertase PACE4 (PCSK6).

PACE4 (PCSK6) is a proprotein convertase (PC) capable of processing numerous substrates involved in tumor growth, invasion, and metastasis. Because of the human relevancy of the tobacco-associated carcinogen benzo[a]pyrene (B(a)P) we investigated whether transgenic mice in which this PC is targeted to the epidermis (K5-PACE4) may be more susceptible to B(a)P complete carcinogenesis than wild type (WT) mice. In an in vitro experiment, using cell lines derived from skin tumors obtained after B(a)P treatment, we observed that PACE4 overexpression and activity accounts for an increased proliferation rate, exaggerated sensitivity to the PC inhibitor CMK, and interference with IGF-1R autophosphorylation. Squamous cell carcinomas, obtained from K5-PACE4 mice subjected to complete chemical carcinogenesis, were characterized by a 50% increase in cell proliferation, when compared with similar tumors from WT mice. In addition, tumors from K5-PACE4 mice showed deeper invasion into the underlying dermis. Thus, mice overexpressing PACE4 exhibited tumors of increased growth rate and invasive potential when exposed to the human carcinogen B(a)P, further supporting the significance of PCs in tumor growth and progression.

Antimutagenic effects of aqueous fraction of Myristica fragrans (Houtt.) leaves on Salmonella typhimurium and Mus musculus.

Natural plant extracts offer a promising hope in the prevention/treatment of cancer arising from genetic mutations. This study evaluated in vitro and in vivo mutagenic and antimutagenic effects of aqueous fraction of Myristica fragrans (AFMF) leaves on TA100 strain of Salmonella typhimurium and Mus musculus (Male Swiss albino mice), respectively. The antioxidant activity of AFMF against 2,2-diphenyl-1-picrylhydrazyl (DPPH), total phenolic and flavonoid contents were determined, followed by its phytochemical elucidation using the Ultra Performance Liquid Chromatography technique (UPLC). The mutagenicity of AFMF at 4, 20, 50, 100, 200, 500, and 1000 µg/well was <2.0 in S. typhimurium and the induced micronucleated polychromatic and normochromatic erythrocytes at 500, 1000, 2000, and 4000 mg/kg were not significantly different from the negative control (p≥0.05). The mutagenic activity of benzo[a]pyrene and cyclophosphamide was significantly suppressed above 50.0% throughout the tested concentrations. Fifty percent of the free radicals from DPPH were scavenged by AFMF at 0.11 mg/ml. Total phenolic and flavonoid contents of AFMF were 51.0 mg GAE/g and 27 mg QE/g, respectively. Rutin was elucidated by the UPLC technique, and thereby suspected to be the phytochemical responsible for the observed antimutagenic activity. Thus far, AFMF seems to contain a promising chemotherapeutic agent for the prevention of genetic damage that is crucial for cancer development.

The relationships between XPC binding to conformationally diverse DNA adducts and their excision by the human NER system: is there a correlation?

The first eukaryotic NER factor that recognizes NER substrates is the heterodimeric XPC-RAD23B protein. The currently accepted hypothesis is that this protein recognizes the distortions/destabilization caused by DNA lesions rather than the lesions themselves. The resulting XPC-RAD23B-DNA complexes serve as scaffolds for the recruitment of subsequent NER factors that lead to the excision of the oligonucleotide sequences containing the lesions. Based on several well-known examples of DNA lesions like the UV radiation-induced CPD and 6-4 photodimers, as well as cisplatin-derived intrastrand cross-linked lesions, it is generally believed that the differences in excision activities in human cell extracts is correlated with the binding affinities of XPC-RAD23B to these DNA lesions. However, using electrophoretic mobility shift assays, we have found that XPC-RAD23B binding affinities of certain bulky lesions derived from metabolically activated polycyclic aromatic hydrocarbon compounds such as benzo[a]pyrene and dibenzo[a,l]pyrene, are not directly, or necessarily correlated with NER excision activities observed in cell-free extracts. These findings point to features of XPC-RAD23B-bulky DNA adduct complexes that may involve the formation of NER-productive or unproductive forms of binding that depend on the structural and stereochemical properties of the DNA adducts studied. The pronounced differences in NER cleavage efficiencies observed in cell-free extracts may be due to differences in the successful recruitment of subsequent NER factors by the XPC-RAD23B-DNA adduct complexes, and/or in the verification step. These phenomena appear to depend on the structural and conformational properties of the class of bulky DNA adducts studied.

Benzo-[a]-pyrene induces FAK activation and cell migration in MDA-MB-231 breast cancer cells.

Benzo-[a]-pyrene (B[a]P) is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant. It is a mammary carcinogen in rodents and contributes to the development of human breast cancer. However, the signal transduction pathways induced by B[a]P and its role in breast cancer progression have not been studied in detail. Here, we demonstrate that B[a]P induces cell migration through a lipoxygenase- and Src-dependent pathway, as well as the activation of focal adhesion kinase, Src, and the extracellular signal-regulated kinase 2 in MDA-MB-231 breast cancer cells. However, B[a]P is not able to promote migration in the mammary nontumorigenic epithelial cells MCF12A. Moreover, B[a]P promotes an increase of αvß3 integrin-cell surface levels and an increase of metalloproteinase (MMP)-2 and MMP-9 secretions. In summary, our findings demonstrate that B[a]P induces the activation of signal transduction pathways and biological processes involved in the invasion/metastasis process in MDA-MB-231 breast cancer cells.

Adenine-DNA adducts derived from the highly tumorigenic Dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not.

The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N(2)-dG) and adenine (N(6)-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N(2)-dG linkage site is ∼35 times more susceptible to NER dual incisions than the stereochemically identical N(6)-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar but somewhat smaller effect (factor of ∼15) is observed. The striking resistance of the bulky N(6)-dA in contrast to the modest to good susceptibilities of the N(2)-dG adducts to NER is interpreted in terms of the balance between lesion-induced DNA distorting and DNA stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA.