Benzo[a]pyrene Exposure Research Articles

Risk of breast cancer associated with long-term exposure to benzo[a]pyrene (BaP) air pollution: Evidence from the French E3N cohort study

BackgroundBenzo[a]pyrene (BaP) is an endocrine-disrupting pollutant formed during incomplete combustion of organic materials. It has been recognized as a reproductive and developmental toxicant, however epidemiological evidence of the long-term effect of ambient air BaP on breast cancer (BC) is limited. Thus we evaluated associations between ambient air BaP exposure and risk of BC, overall and according to menopausal status and molecular subtypes (estrogen receptor negative/positive (ER−/ER+) and progesterone receptor negative/positive (PR−/PR+)), stage and grade of differentiation of BC in the French E3N cohort study. MethodsWithin a nested case-control study of 5222 incident BC cases and 5222 matched controls, annual BaP exposure was estimated using a chemistry-transport model (CHIMERE) and was assigned to the geocoded residential addresses of participants for each year during the 1990–2011 follow-up period. Multivariable conditional logistic regression models were used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). ResultsOverall, cumulative airborne BaP exposure was significantly associated with the overall risk of BC, for each 1 interquartile range (IQR) increase in the concentration levels of BaP (1.42 ng/m3), the OR = 1.15 (95% CI: 1.04–1.27). However, by menopausal status, the significant positive association remained only in women who underwent menopausal transition (i.e. premenopausal women at inclusion who became postmenopausal at diagnosis), OR per 1 IQR = 1.20 (95% CI: 1.03–1.40). By hormone receptor status, positive associations were observed for ER+, PR + and ER + PR + BC, with ORs = 1.17 (95% CI: 1.04–1.32), 1.16 (95% CI: 1.01–1.33), and 1.17 (95% CI: 1.01–1.36) per 1 IQR, respectively. There was also a borderline positive association between BaP and grade 3 BC (OR per 1 IQR = 1.15 (95% CI: 0.99–1.34). ConclusionsWe provide evidence of increased risk of BC associated with cumulative BaP exposure, which varied according to menopausal status, hormone receptor status, and grade of differentiation of BC. Our results add further epidemiological evidence to the previous experimental studies suggesting the adverse effects of BaP.

Benzo[a]pyrene and Caenorhabditis elegans: defining the genotoxic potential in an organism lacking the classical CYP1A1 pathway

Benzo[a]pyrene (BaP) is bioactivated in most organisms by the cytochrome P450 (CYP) enzymes, mainly CYP1A1, ultimately resulting in the reactive metabolite BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) capable of covalently binding to DNA and forming adducts. This step has been defined as the key process in cancer initiation in humans. However, limited knowledge is available about the consequences of BaP exposure in organisms lacking this classical CYP1A1 pathway, one example is the model nematode Caenorhabditis elegans. The aim of this study was to define the genotoxic potential of BaP in C. elegans and to advance our understanding of xenobiotic processing in the absence of the CYP1A1 pathway. Exposure to high concentrations of BaP (0–40 µM) significantly affected life cycle endpoints of C. elegans, which were manifested by a reduced reproductive output and shortened life span. An optimised comet assay revealed that DNA damage increased in a dose-dependent manner; however, no bulky DNA adducts (dG-N2-BPDE) were observed by 32P-postlabelling. Global transcriptomic analysis by RNA-Seq identified responsive transcript families, most prominently members of the cyp-35 and UDP-glucuronosyltransferases (UGTs) enzyme families, both of which are linked to xenobiotic metabolism. Strains harbouring mutations in the cyp-35A2 and cyp-35A3 genes were notably less prone to BaP-mediated toxicity, and BaP led to longevity in cyp-35A5 mutants. In summary, BaP induces transcriptional, genotoxic and phenotypic responses in C. elegans, despite the absence of the classical CYP1A1 bioactivation pathway. This provides first evidence that parallel pathways are implicated in BaP metabolism in C. elegans and this seems to be mediated via the cyp-35 pathway.

Benzo(a)pyrene-induced cytotoxicity, cell proliferation, DNA damage, and altered gene expression profiles in HT-29 human colon cancer cells.

In the US alone, around 60,000 lives/year are lost to colon cancer. In order to study the mechanisms of colon carcinogenesis, in vitro model systems are required in addition to in vivo models. Towards this end, we have used the HT-29 colon cancer cells, cultured in Dulbecco's Modified Eagle Medium (DMEM), which were exposed to benzo(a)pyrene (BaP), a ubiquitous and prototypical environmental and dietary toxicant at 1, 10, 100nM and 1, 5, 10, and 25μM concentrations for 96h. Post-BaP exposure, growth, cytotoxicity, apoptosis, and cell cycle changes were determined. The BaP metabolite concentrations in colon cells were identified and measured. Furthermore, the BaP biotransformation enzymes were studied at the protein and mRNA levels. The BaP exposure-induced damage to DNA was assessed by measuring the oxidative damage to DNA and the concentrations of BaP-DNA adducts. To determine the whole repertoire of genes that are up- or downregulated by BaP exposure, mRNA transcriptome analysis was conducted. There was a BaP exposure concentration (dose)-dependent decrease in cell growth, cytotoxicity, and modulation of the cell cycle in the treatment groups compared to untreated or dimethylsulfoxide (DMSO: vehicle for BaP)-treated categories. The phase I biotransformation enzymes, CYP1A1 and 1B1, showed BaP concentration-dependent expression. On the other hand, phase II enzymes did not exhibit any marked variation. Consistent with the expression of phase I enzymes, elevated concentrations of BaP metabolites were generated, contributing to the formation of DNA lesions and stable DNA adducts, which were also BaP concentration-dependent. In summary, our studies established that biotransformation of BaP contributes to cytotoxicity, proliferation of tumor cells, and alteration of gene expression by BaP. • Benzo(a)pyrene (BaP) is an environmental and dietary toxicant. • BaP causes cytotoxicity in cultured HT-29 colon cancer cells. • mRNA transcriptome analyses revealed that BaP impacts cell growth, cell cycle, biotransformation, and DNA damage.

Effects of BαP and TBBPA on multixenobiotic resistance (MXR) related efflux transporter activity and gene expressions in gill cells of scallop Chlamys farreri.

The multixenobiotic resistance mechanism (MXR) provides aquatic organisms with the capacity to adapt to polluted environments, which can be inhibited by chemosensitizers. In the present study, the effect of two typical marine persistent organic pollutants, benzo(a)pyrene (BaP) and tetrabromobisphenol A (TBBPA), on the most relevant ABC transporters, ABCB1, ABCC1, and ABCG2 of scallop Chlamys farreri was tested. MXR transporter efflux activity of cultured gill cells of the scallops was evaluated by measuring the intracellular fluorescent intensity of Calcein-AM and rhodamine 123 with flow cytometry. The results showed that ABCB1 and ABCC1 transporters demonstrated increased activity compared with ABCG2 in mediating MXR efflux activity. BaP and TBBPA were able to suppress the efflux transporter activity of ABC transporters significantly, of which BaP revealed block effects by acting on the ABCB1 transporter. Additionally, exposure of BaP and TBBPA only significantly upregulated the expression level of ABCC1 gene.This study demonstrated the promising utility of efflux transporter activity in conjunction with biomarkers such as mRNA levels in identification of chemosensitizer.

Co-exposure of Bi2O3 nanoparticles and bezo[a]pyrene-enhanced in vitro cytotoxicity of mouse spermatogonia cells.

Recent attention has been focused on reproductive toxicity of nanoscale materials in combination with pre-existing environmental pollutants. Due to its unique characteristics, bismuth (III) oxide (Bi2O3) nanoparticles (BONPs) are being used in diverse fields including cosmetics and biomedicine. Benzo[a]pyrene (BaP) is a known endocrine disruptor that most common sources of BaP exposure to humans are cigarette smoke and well-cooked barbecued meat. Hence, joint exposure of BONPs and BaP in humans is common. There is scarcity of information on toxicity of BONPs in combination with BaP in human reproductive system. In this work, combined effects of BONPs and BaP in mouse spermatogonia (GC-1 spg) cells were assessed. Results showed that combined exposure of BONPs and BaP synergistically induced cell viability reduction, lactate dehydrogenase leakage, induction of caspases (-3 and -9) and mitochondrial membrane potential loss in GC-1 spg cells. Co-exposure of BONPs and BaP also synergistically induced production of pro-oxidants (reactive oxygen species and hydrogen peroxide) and reduction of antioxidants (glutathione and several antioxidant enzymes). Experiments with N-acetyl-cysteine (NAC, a reactive oxygen species scavenger) indicated that oxidative stress was a plausible mechanism of synergistic toxicity of BONPs and BaP in GC-1 spg cells. Present data could be helpful for future in vivo research and risk assessment of human reproductive system co-exposed to BONPs and BaP.

Effects of oxidation defense system exposure to Benzo(a)pyrene on CYP450 gene expression and EROD activity in Crassostrea gigas and Mytilus coruscus

ABSTRACT Benzo[a]pyrene (BaP) is widely used as a reference compound when studying the toxicity of Polycyclic aromatic hydrocarbons (PAHs) to natural communities. In this study, 15d after exposure to BaP at the nominal concentrations of 5, 1 and 0.2 μg/L, liver, gill and mantle tissues of Crassostrea gigas and Mytilus coruscuswere taken for BaP uptake assessment, and Cytochrome P450 (CYP450) gene expression level and 7-ethoxyresorufifififin O-deethylase (EROD) activity testing, respectively. CYP450 gene expression level and EROD activity increased significantly with the increase of BaP exposure concentrations. The Significant correlations between BaP uptake and both CYP450 gene expression level and EROD activity indicates that they can be used as a suitable tools for the study of comprehensive levels of biomarker exposed to PAH. There were significant differences in the measurement results of these tissue antioxidant defenses parameters between different treatments.

Diallyl Sulfide-Mediated Modulation of the Fatty Acid Synthase (FASN) Leads to Cancer Cell Death in BaP-Induced Lung Carcinogenesis in Swiss Mice.

PurposeDiallyl sulfide (DAS), one of the organo-sulfur secondary metabolites in garlic, has been shown to inhibit the proliferation of cancer cells. The present study aimed to evaluate the mechanism of DAS in the prevention of benzo[a]pyrene (BaP)-induced lung cancer in a murine model.Materials and MethodsThe mice were exposed to 50 mg/kg of BaP twice a week for 4 weeks in order to induce lung carcinoma. Pretreatment of mice with DAS (100 mg/kg) was started 2 weeks before BaP exposure and further continued for 21 weeks. The effect of DAS and BaP was evaluated by studying various parameters in the serum and tissues of the treated or untreated BaP-exposed mice.ResultsThe histopathological findings demonstrated that DAS prevented the progression of malignant lung cancer and metastasis in the liver. A significant drop was observed in BaP-induced tumor marker enzymes (ADA, AHH, γ-GT, LDH) in the serum of the mice treated with DAS. Moreover, DAS treatment resulted in the recovery of antioxidant enzymes, SOD and CAT, in BaP-exposed mice. The induction of apoptosis and the destruction of cellular ROS were detected in cancer cells from the mice pre-treated with DAS. The immunohistochemical analysis revealed the up-regulation of fatty acid synthase (FASN) in the lungs and liver tissues of BaP-exposed mice and the treatment with DAS inhibited FASN expression.ConclusionThe findings of the present study indicated that DAS-induced apoptosis is strongly associated with the downregulation of FASN in tumor tissues. To the best of our knowledge, this is the first study that describes the role of FASN in BaP-induced lung carcinogenesis.

Mutational signature modelling in vitro recapitulates bladder cancer initiation

Smoking is the best established risk factor for bladder cancer and direct mutagenesis by smoke carcinogens on the cellular genome can be described in clonal populations as mutational signatures. A mutational signature summarises a lifetime of DNA-damage (caused by interaction between mutagenic processes, tissue-specific gene transcription, and the DNA-repair machinery) as mutational classes in the context of their local genomic chemistry. Our aim was to establish a novel experimental model for understanding carcinogenic initiation in normal human urothelium. Finite normal human urothelial cell cultures were established in vitro and differentiated to form functional barrier epithelia capable of activating the smoke-derived procarcinogen benzo[a]pyrene (BaP), via CYP1A1, into mutagenic metabolites. New in vitro approaches were developed allowing chronic BaP-exposure, subsequent clonal expansion of NHU cells for whole genome DNAseq and mutational signature derivation. This approach retains tissue heterogeneity during the carcinogenic exposure, modelling the early selection pressures in urothelium that lead to clonal expansion and cancer. The single-base (SBS) and double-base signatures (DBS) derived from BaP-exposed urothelial tissues were dominated by C>A and CC>AA transversions, respectively. The InDel (ID) signature described cytosine deletions in homopolymer regions. Comparison to the Catalogue of Somatic Mutations in Cancer (COSMIC) using the Signal pipeline showed homology with SBS4, DBS2 and ID3, respectively; which have been detected by others in bladder cancer patients. Mutation enrichment in specific genes (eg KMT2D and CDKN1A) mirrored in situ observations by others; effectively modelling the competitive advantage mutations convey in the carcinogenic tissue environment. BaP is detected in urine, metabolically activated by urothelial CYP1A1, and therefore may be a bladder mutagen. However, C>A transversions contribute a minor fraction of all mutations recorded in bladder tumours. Widespread DNA damage caused by chronic BaP exposure did not trigger any of the APOBEC-like mutational processes (SBS2/13) that dominate bladder tumour genomes, indicating a missing factor that the new model system is well positioned to identify.

Aryl hydrocarbon receptor mediates benzo[a]pyrene-induced metabolic reprogramming in human lung epithelial BEAS-2B cells

Polycyclic aromatic hydrocarbon exposure accelerates the initiation and progression of lung cancer through aryl hydrocarbon receptor (AHR) signaling. Metabolic reprogramming is a hallmark of cancer. However, how AHR reprograms metabolism related to the malignant transformation in of benzo[a]pyrene (BaP)-exposed lung cells remains unclear. After confirming that BaP exposure activated AHR signaling and relevant downstream factors and then promoted epithelial-mesenchymal transition, an untargeted metabolomics approach was employed to discover AHR-mediated metabolic reprogramming and potential therapeutic targets in BaP-exposed BEAS-2B cells. We found that 52 metabolites were significantly altered in BaP-exposed BEAS-2B cells and responsive to resveratrol (RSV) intervention. Pathway analysis revealed that 28 and 30 metabolic pathways were significantly altered in response to BaP exposure and RSV intervention, respectively. Notably, levels of most amino acids were significantly decreased, while those of most fatty acids were significantly increased in BaP-exposed BEAS-2B cells, and above changes were abolished by RSV intervention. Besides, levels of amino acids and fatty acids were highly correlated with those of many metabolites and AHR signaling upon BaP exposure and RSV intervention (the absolute values of Pearson correlation coefficients above 0.8). We further discovered a decrease in peroxisome proliferator-activated receptor (PPAR) A/G signaling and an increase in fatty acid import by the transporter FATP1 in BaP-exposed BEAS-2B cells. Furthermore, inhibition of AHR signaling by CH-223191 abolished BaP-induced repression of PPARA/G signaling and activation of FATP1 in BEAS-2B cells, demonstrating the regulatory role of AHR signaling in fatty acid accumulation via mediating PPARA/G-FATP1 signaling. These data suggested amino acid and fatty acid metabolism, AHR and PPAR-FATP1 signaling as potential therapeutic targets for intervening BaP-induced toxicity and related diseases. As far as we known, fatty acid accumulation and high correlations of AHR signaling with amino acid and fatty acid metabolism are novel phenomena discovered in BaP-exposed lung epithelial cells.

Global H3K79 di-methylation mediates DNA damage response to PAH exposure in Chinese coke oven workers.

Polycyclic aromatic hydrocarbons (PAHs) are the main contaminants of coke oven emissions which can induce serious genetic damage in coke oven workers. Epigenetic alternations play essential roles in the regulation of DNA damage effect of PAHs. Previous studies indicate that H3K79 di-methylation (H3K79me2) is integral in DNA damage repair. However, the potential role of H3K79me2 in DNA damage response (DDR) following PAHs exposure is still unclear. In this study, we recruited 256 male coke oven workers and control workers, and examined H3K79me2 and DNA damage in their peripheral blood lymphocytes (PBLCs). The results showed that global H3K79me2 of coke oven workers was 29.3% less than that of the controls (P<0.001). The H3K79me2 was negatively correlated with the concentration of urinary 1-hydroxypyrene (1-OHP) (β=-0.235, P<0.001) and level of genetic damage evaluated by comet assay (βTail DNA %=-0.313, P<0.001; βOTM=-0.251, P=0.008). Consistently, we found that benzo(a)pyrene (BaP) inhibited H3K79me2 in immortalized human bronchial epithelial (HBE) cells in a time-dependent manner. In order to explore the function of H3K79me2 in PAHs DDR, we established histone 3.1/3.3 K79A mutant cells (H3K79A) to suppress H3K79me2. H3K79A cells showed more serious DNA damage and decreased cell viability than control cells after BaP treatment. In addition, we also found that the expression of DOT1L, the only methyltransferase in H3K79, was repressed by BaP dose-dependently. DOT1L knockdown resulted in decreased H3K79me2 level and aggravated DNA damage after BaP exposure. This suggests that BaP induces H3K79me2 repression via inhibiting DOT1L expression. In conclusion, these findings indicate that PAH exposure decreases the level of global H3K79me2, which is integral for DNA damage response regulation of PAHs.

Maternal occupational benzo(a)pyrene exposure and risk of cancer in children

Background/Aim: The International Agency for Research on Cancer has classified benzo(a)pyrene (BaP) as carcinogenic to humans, based on sufficient evidence of carcinogenicity in experimental animals and supportive data on mechanistic pathways in humans. BaP is also a developmental toxicant. Yet, few studies assessed whether parental exposure to BaP is related to an increased risk for childhood cancers. Previous studies reported possible associations between environmental BaP exposure with risk increases in neuroblastoma and acute lymphoblastic leukemia. The aim of our study was to examine the relation between maternal occupational BaP exposure with risk of cancer in offspring.Methods: In this case-control study of Danish children (8339 cases, 208475 controls), we ascertained cases from the Cancer Registry and controls from the Central Population Register. Jobs during pregnancy were ascertained from the Supplemental Pension Fund, a mandatory supplement to the state pension. BaP exposure was determined via a job-exposure matrix developed for the Danish workforce. We used conditional logistic regression to determine associations with childhood cancer.Results: Ever occupational exposure to BaP during pregnancy was related to an increase in risk of neuroblastoma (Odds Ratio=1.65, 95% Confidence Interval 1.22, 2.23), while there was no increased risk for ALL (OR=1.02). We did not estimate any compelling risk increase for any other type of cancer, with effect estimates close to the null.Conclusions: This is the second study to report possible associations between BaP and neuroblastoma. Occupational exposures tend to be higher than ambient environmental exposures likely leading to greater fetal loss, which may in part explain variation in leukemia results compared to other studies.

Environmental Pollutant Benzo[a]pyrene Induces Recurrent Pregnancy Loss through Promoting Apoptosis and Suppressing Migration of Extravillous Trophoblast

Methods The implantation sites, fetus resorption, and abnormal fetuses were studied in pregnant mice treated with different doses of BaP by oral gavage from day 1 to day 10 of gestation. Additionally, apoptosis and related signaling pathway, and the migration and invasion of trophoblasts, were assessed before and after exposure of BPDE in Swan 71 trophoblast cell. Besides, the migration and invasion, and its related signaling pathway, were assessed in villi obtained from women. Results We observed a concentration-dependent incidence of abnormal murine fetuses, beginning with 0.1 mg/kg BaP; with a BaP concentration of 2 mg/kg, no fetuses developed. Correspondingly, a BPDE concentration-dependent apoptosis of human trophoblasts. Beginning with 0.5 μM BPDE exposure, Bax/Caspase-3 were increased and Bcl-2 decreased. Furthermore, BPDE also inhibited, in a dose-dependent manner, the migration of villous explants from elective abortion women, consistent with the reduced migration of villous explants from women with recurrent pregnancy loss (RPL), and reduced the cell immigration in Swan 71 trophoblasts, in a dose-dependent manner measured by transwell assays. Conclusions Our study results provide mechanistic insight to the effect of BPDE on trophoblast dysfunction through enhanced cell apoptosis and inhibited migration, providing further experimental evidence to the causative links between BaP exposure and PRL.

Benzo(a)pyrene exposure induced neuronal loss, plaque deposition, and cognitive decline in APP/PS1 mice

BackgroundExposure to benzo(a)pyrene (BaP) was associated with cognitive impairments and some Alzheimer’s disease (AD)-like pathological changes. However, it is largely unknown whether BaP exposure participates in the disease progression of AD.ObjectivesTo investigate the effect of BaP exposure on AD progression and its underlying mechanisms.MethodsBaP or vehicle was administered to 4-month-old APPswe/PS1dE9 transgenic (APP/PS1) mice and wildtype (WT) mice for 2 months. Learning and memory ability and exploratory behaviors were evaluated 1 month after the initiation/termination of BaP exposure. AD-like pathological and biochemical alterations were examined 1 month after 2-month BaP exposure. Levels of soluble beta-amyloid (Aβ) oligomers and the number of Aβ plaques in the cortex and the hippocampus were quantified. Gene expression profiling was used to evaluate alternation of genes/pathways associated with AD onset and progression. Immunohistochemistry and Western blot were used to demonstrate neuronal loss and neuroinflammation in the cortex and the hippocampus. Treatment of primary neuron-glia cultures with aged Aβ (a mixture of monomers, oligomers, and fibrils) and/or BaP was used to investigate mechanisms by which BaP enhanced Aβ-induced neurodegeneration.ResultsBaP exposure induced progressive decline in spatial learning/memory and exploratory behaviors in APP/PS1 mice and WT mice, and APP/PS1 mice showed severer behavioral deficits than WT mice. Moreover, BaP exposure promoted neuronal loss, Aβ burden and Aβ plaque formation in APP/PS1 mice, but not in WT mice. Gene expression profiling showed most robust alteration in genes and pathways related to inflammation and immunoregulatory process, Aβ secretion and degradation, and synaptic formation in WT and APP/PS1 mice after BaP exposure. Consistently, the cortex and the hippocampus of WT and APP/PS1 mice displayed activation of microglia and astroglia and upregulation of inducible nitric oxide synthase (iNOS), glial fibrillary acidic protein (GFAP), and NADPH oxidase (three widely used neuroinflammatory markers) after BaP exposure. Furthermore, BaP exposure aggravated neurodegeneration induced by aged Aβ peptide in primary neuron-glia cultures through enhancing NADPH oxidase-derived oxidative stress.ConclusionOur study showed that chronic exposure to environmental pollutant BaP induced, accelerated, and exacerbated the progression of AD, in which elevated neuroinflammation and NADPH oxidase-derived oxidative insults were key pathogenic events.

Multigenerational Impacts of Benzo[a]pyrene on Bone Modeling and Remodeling in Medaka (Oryzias latipes).

Ancestral benzo[a]pyrene (BaP) (1 μg/L, 21 days) exposure has previously been shown to cause skeletal deformities in medaka (Oryzias latipes) larvae in the F1-F3 generation. However, when and how this deformity is induced during bone development remain to be elucidated. The col10a1:nlGFP/osx:mCherry double transgenic medaka model was employed to determine the temporal and spatial changes of col10a1:nlGFP- positive osteochondral progenitor cells (OPCs) and osx:mCherry-positive premature osteoblasts (POBs) [8 days postfertilization (dpf)-31 dpf] in combination with changes in bone mineralization at the tissue level. Ancestral BaP exposure delayed the development of col10a1:nlGFP- and osx:mCherry-positive osteoblasts and reduced the abundance of col10a1:nlGFP-positive osteoblast progenitors and col10a1:nlGFP/osx:mCherry double-positive premature osteoblasts during critical windows of early vertebral bone formation, associated with reduced bone mineralization in embryos (14 dpf) and larvae (31 dpf), compressed vertebral segments in larvae (31 dpf), and reduced bone thickness in adult male medaka (6 months old) of the F1-F3 generations. Both Col10a1:nlGFP and osx:mCherry were identified as potential targets of epigenetic modifications underlying the transgenerational inheritance of BaP bone toxicity. The present study provides novel knowledge of the underlying mechanisms of transgenerational toxicity of BaP at the cellular level.

Exposure to benzo(a)pyrene from juvenile period to peripubertal impairs male reproductive parameters in adult rats

Benzo(a)pyrene (BaP) is a persistent organic pollutant and endocrine disruptor that can compromise the steroidogenesis process by interacting with the StAR protein, causing adverse effects on male reproduction. However, consequences of prepubertal BaP exposure and its impacts on adult life are yet unknown. This study investigated the effects of BaP exposure from the juvenile period to peripubertal on reproductive parameters in adult male rats. Males were exposed to 0; 0.1; 1 or 10 μg/kg/day of BaP from post-natal (PND) 23 to PND 53 (by gavage). The lowest dose of BaP was able to compromise the male copulatory behavior, as evidenced by the delay in the first mount, intromission and ejaculation. Furthermore, BaP-treated groups showed lower sperm quality (disrupted motility and morphology) and quantity, reduced relative weights of the thyroid and seminal gland. Serum testosterone levels and the Leydig cells nuclei volume were decreased by BaP exposure whereas the StAR expression was increased. Histopathological changes in the testis also were detected in the males exposed to BaP. These results showed that prepubertal BaP-exposure adversely influenced the male reproductive system in the adult life, indicating that a comprehensive risk assessment of BaP-exposure on prepubertal period is necessary.

The intergenerational toxic effects on offspring of medaka fish Oryzias melastigma from parental benzo[a]pyrene exposure via interference of the circadian rhythm

Benzo[a]pyrene (BaP), a widely existed polycyclic aromatic hydrocarbon pollutant in aquatic environment, has toxic effects on marine animals and their generations, but the intergenerational immunotoxic mechanism underlying has not been clearly understood. In the study, the offspring of marine medaka (oryzias melastigma) which were exposed to 0.5 μg L−1 BaP suffered from circadian rhythm oscillation disorders and severe DNA damage. Many clock-associated genes like per1 were significantly modulated in offspring, both per1 and p53 were significantly inhibited that altered the progression of cell cycle and inhibited DNA repair, which possibly resulted in the increased mortality of offspring. The hypermethylation of the per1 promotor and abnormal levels of N6-methyladenosine (m6A) suggested that the underlying mechanism was probably related to the epigenetic modification. Moreover, the offspring from paternal BaP exposure had more severe DNA damage and a higher degree of hypermethylation than those from maternal exposure. F1 larvae from BaP-exposed parents were more sensitive to BaP exposure, showing that the expression of immune and metabolism-related genes were significantly up-regulated. Taken together, the parental toxicity induced by BaP could be passed to F1 generation and the mechanism underlying was probably associated with a characteristic circadian rhythm disorder.

Calliandra portoricensis ameliorates ovarian and uterine oxido-inflammatory responses in N-methyl-N-nitrosourea and benzo[a]pyrene-treated rats.

Infertility resulting from reproductive impairment is traumatic in families. Exposure to chemicals may play insidious roles not easily connected to infertility. We examined benzo[a]pyrene (BaP), and N-methyl nitrosourea (NMU)-induced ovarian and uterine toxicity and the role of Calliandra portoricensis in mitigating toxicity. In a bid to illuminate folk medical claims cloaked in mystery, unearthing lost knowledge, advance natural chemopreventive agents, and report new evidence lacking in the literature attributed to CP. Although CP is known to exhibit anticonvulsant, antidiarrheal, antipyretic, antirheumatic, and analgesic effects in humans, its possible roles for mitigating toxicity stemming from inadvertent chemical exposures are reported here. Our findings affirm and further show that CP abates toxic response incumbent on oxidative damage and inflammatory responses associated with NMU and BaP exposure. Development of phytochemical derived from CP may serve as a potential natural therapy against chemical toxicities in individuals inadvertently exposed, and promote human health and reproductive satiety.

Exposure to benzo[a]pyrene triggers distinct patterns of microRNA transcriptional profiles in aquatic firefly Aquatica wuhana (Coleoptera: Lampyridae)

Larval aquatic fireflies in fresh water are adversely affected by water pollutants such as benzo(a)pyrene (BaP). However, their response to BaP stress at the microRNA (miRNA)-regulatory level remains unknown. Here, transcriptomes containing 31,872 genes and six miRNA transcriptional profiles were obtained for Aquatica wuhana larvae, and comparative analysis was performed between larvae exposed to BaP (0.01 mg/L) and unexposed controls. Fifteen of 114 miRNAs identified via bioinformatics were detected as differentially expressed (DEMs) upon BaP exposure. Analysis results of predicted target genes of DEM suggests that BaP exposure primarily triggered transcriptional changes of miRNA associated with five major regulatory categories: 1) osmotic balance, 2) energy metabolic efficiency, 3) development, 4) xenobiotic metabolism (oxidative stress), and 5) innate immune response. Based on six innate immune- and xenobiotic metabolism-related pathways enriched by the predicted DEM targets, 11 key BaP-responsive DEMs were further screened to investigate dynamic changes of expression in response to BaP stress at five time points, and also to validate the miRNA sequencing data using quantitative real-time PCR. This study provides valuable information for the protection of firefly resources and supplements the understanding of miRNA regulatory mechanisms in response to water deterioration.

Abstract A25: Arsenic and benzo(a)pyrene coexposure synergizes in inducing cancer stem cell-like property and lung tumorigenesis

Abstract Arsenic and benzo(a)pyrene (BaP) are among the most common environmental pollutants that humans are exposed to. Both arsenic and BaP are well-recognized human carcinogens that cause lung cancer. Since millions of people are exposed to arsenic through contaminated drinking water and high levels of BaP are found in cigarette smoke and barbecue cooked meat, arsenic and BaP coexposure is thus very common in humans. However, the combined effect and the underlying mechanism of arsenic and BaP coexposure are poorly understood. Using cell culture and mouse models, we investigated the combined effect of arsenic and BaP coexposure. It was found that arsenic and BaP coexposure acts synergistically in inducing cell malignant transformation as evidenced by forming more than an additive number of soft agar clones compared to arsenic or BaP exposure alone in BEAS-2B cells for 30 weeks. We also determined the combined effect of arsenic and BaP coexposure in inducing cancer stem cell (CSC)-like property by using a well-established assay known as suspension culture sphere formation assay. It was found that BEAS-2B cells transformed by arsenic or BaP exposure alone are capable of forming small numbers of spheres. However, BEAS-2B cells transformed by arsenic and BaP coexposure form more than an additive number of spheres compared to cells transformed by arsenic or BaP exposure alone, indicating that arsenic and BaP coexposure also acts synergistically in inducing CSC-like property. Since CSCs or CSC-like cells are considered tumor-initiating cells, we first compared the tumorigenic effect of BEAS-2B cells transformed by arsenic, BaP alone, or arsenic and BaP coexposure by subcutaneous inoculation of 0.25 × 106 cells into the right flank of 6-week old nu/nu nude mice. It was found that 0 of 5 mice injected with BEAS-2B-Control cells, 0 of 5 mice injected with arsenic exposure alone-transformed cells, 2 of 5 mice injected with BaP exposure alone-transformed cells develop tumors; however, all 5 mice injected with arsenic and BaP coexposure transformed cells develop tumors. These results indicate that cells transformed by arsenic and BaP coexposure have significantly stronger tumor-initiating capability than cells transformed by arsenic or BaP exposure alone. To further investigate whether arsenic and BaP coexposure acts synergistically in inducing mouse lung tumors, male A/J mice were exposed to vehicle control, arsenic, BaP, or arsenic plus BaP for 34 weeks. No lung tumors were found in control and arsenic exposure alone mice. All mice with BaP exposure developed lung tumors. However, mice exposed to arsenic plus BaP developed significantly more (tumor multiplicity) and bigger (tumor burden) lung tumors compared to mice exposed to BaP alone. Together, these results demonstrate a synergism of arsenic and BaP coexposure in inducing lung tumorigenesis. Further mechanistic studies revealed that arsenic and BaP coexposure synergizes in causing epigenetics deregulation to enhance their carcinogenicity. Citation Format: Zhishan Wang, Ping Yang, Yunfei Li, Drew Maddy, Chengfeng Yang. Arsenic and benzo(a)pyrene coexposure synergizes in inducing cancer stem cell-like property and lung tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A25.

Benzo[a]pyrene induces microbiome dysbiosis and inflammation in the intestinal tracts of western mosquitofish (Gambusia affinis) and zebrafish (Danio rerio)

Benzo[a]pyrene (BaP) is one of the most well studied carcinogenic polycyclic aromatic hydrocarbons (PAHs) that has been associated with a wide range of toxic effects in aquatic organisms. In the present study, the mosquitofish and zebrafish were exposed to BaP (100 μg/L) for 15 days. We analyzed the intestinal microbial community of mosquitofish and zebrafish using 16S rRNA gene amplicon sequencing and also performed transcriptional profiling of the inflammation pathway related genes in the intestinal tissues. Our results showed that BaP exposure induced similar changes to the composition of microbial community in mosquitofish and zebrafish. At the phylum level, the abundance of Proteobacteria decreased while the abundance of Firmicutes increased following BaP exposure. At the genus level, a common pathogenic genus staphylococcus significantly increased in the BaP treatment groups, compared to the control (DMSO, ~0.001% v/v). In addition, it was observed that BaP significantly increased the mRNA level of il1β in both mosquitofish and zebrafish. The transcript levels of il6, il8, il10 and ifnphi1 were significantly increased in zebrafish, however not in mosquitofish, following Bap exposure. Our findings suggest that BaP could induce microbiota dysbiosis and inflammation in the intestine of mosquitofish and zebrafish.