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Polycyclic aromatic hydrocarbons (PAHs) comprise an important class of environmental pollutants that are known to cause lung cancer in animals and are suspected lung carcinogens in humans. Moreover, evidence from cell-based studies points to PAHs as modulators of the epigenome. The objective of this work was to assess patterns of genome-wide DNA methylation in lung tissues of adult offspring initiated in utero with the transplacental PAH carcinogens dibenzo [def,p]chrysene (DBC) or benzo [a]pyrene (BaP). Genome-wide methylation patterns for normal (not exposed), normal adjacent and lung tumor tissues obtained from adult offspring were determined using methylated DNA immunoprecipitation (MeDIP) with the NimbleGen mouse DNA methylation CpG island array. Lung tumor incidence in 45-week old mice initiated with BaP was 32%, much lower than that of the DBC-exposed offspring at 96%. Also, male offspring appeared more susceptible to BaP as compared to females. Distinct patterns of DNA methylation were associated with non-exposed, normal adjacent and adenocarcinoma lung tissues, as determined by principal components, hierarchical clustering and gene ontology analyses. From these methylation profiles, a set of genes of interest was identified that includes potential important targets for epigenetic modification during the process of lung tumorigenesis in animals exposed to environmental PAHs.

OBJECTIVES: Inhalation of crystalline silica (Si) has been linked to the pathogenesis of human autoimmunity such as lupus. Dietary lipid intervention can delay progression of Si-induced autoimmunity in the lupus-prone female NZBWF1 mice. However, the role of gut microbiome relative to dietary lipids and Si-induced autoimmunity is unknown. We seek to ask whether Si-induced pathogenicity and supplementation of a Western diet with DHA can alter gut microbial populations. METHODS: Four experimental diets were used in this study: modified Total Western Diet (MTWD), MTWD supplemented with DHA (↑DHA), MTWD with reduced saturated and ω-6 fatty acids (↓SFω6), and MTWD with ↓SFω6 and ↑DHA (↓SFω6/↑DHA). Groups of mice (n = 8/gp) were fed one of the four isocaloric experimental diets beginning at 6 wks of age. After 2 wks, mice were anesthetized and intranasally instilled with phosphate-buffered saline (PBS) or Si in PBS. DNA obtained from fecal pellets for gut microbiome analysis was collected from an individual mouse at 13 (early-phase) and 22 (late-phase) wks of age. DNA was extracted and sequenced on the Illumina MiSeq platform. The relative abundance of microbial taxa was differentiated by pairwise comparisons using ANalysis of COmposition of Microbiomes (ANCOM), Linear Mixed model (LM), and LDA Effect Size (LEfSe). RESULTS: Lupus triggering by Si with dietary lipid intervention caused a significant difference in Lachnospiraceae bacterium 609 (Lb609) abundance depending on the stringency of analysis frameworks. Under early-phase data, Lb609 was significantly found to be higher in the group of Si-exposed MTWD (MTWD/Si) than all other groups in LefSe analysis (LDA score >3). LM showed the same results except the comparison between MTWD/Si and ↓SFω6 (FDR-adjust P > 0.05). In the results of ANCOM, Lb609 in MTWD/Si was only significantly different from the groups of PBS-exposed MTWD (MTWD/PBS) and ↓SFω6+↑DHA. Although Lb609 was identified discriminatively between MTWD/Si and MTWD/PBS in all analyses from late-phase data, it was not differential in other comparisons. CONCLUSIONS: Lupus triggering altered microbial composition reflected in increased Lb609 and decreased when supplementation was applied. Further, the studies revealed that interventions on microbial populations were dependent on time. FUNDING SOURCES: The USDA National Institute of Food and Federal Appropriations.

OBJECTIVES: A Western type dietary pattern is a major risk factor for colitis-associated colorectal cancer (CAC). Observations from transgenerational studies suggest that epimutations may be inherited, resulting in persistent aberrant gene expression in offspring. Previously, our group reported that ancestral exposure to the total Western diet (TWD) markedly increased colon cancer incidence and disease severity in F(3) offspring that were not fed this diet directly. Moreover, exposure to TWD over multiple generations markedly exacerbated the disease in F(3) offspring as compared to those fed TWD directly. For the present work, we hypothesized that ancestral or multiple generation exposure to the TWD may result in differential expression of cancer critical genes in such a way that explains the greater tumor abundance and burden observed in these mice. METHODS: C57BL/6 J mice were bred for three generations, during which they were fed a standard diet (AIN93G) for all generations or the total Western diet for rodents (TWD) during only the F(0) generation (ancestral), the F(0) through F(3) generations (multi-generation), or only the F(3) generation (direct). The azoxymethane and dextran sodium sulfate (AOM/DSS) model of CAC was employed in F(3) offspring, from which colon mucosa RNA was isolated and used for Illumina RNAseq with EdgeR for differential expression analysis. RESULTS: About 700 to 4500 differentially expressed genes (DEGs) were identified for colon mucosa from AOM/DSS-initiated offspring compared to their sham controls. For AOM/DSS-initiated mice, >100 DEGs were identified comparing multi-generation TWD-fed mice to their AIN93G-fed counterparts, and 36 genes were different from those direct TWD-fed. Of note, in sham-initiated mice, 101 DEGs were identified comparing direct TWD-fed mice to multi-generation TWD-exposed offspring. Interestingly, these DEGs were associated with defense response, immune response, and response to interferon biological process ontology terms. CONCLUSIONS: Exposure to the TWD over multiple generations caused significant changes in genes involved in immune response in third generation offspring. Assessment of genome-wide patterns of DNA methylation in these colon tissue samples is ongoing. FUNDING SOURCES: USDA NIFA AFRI Grant No. 2014-67017-21755; Utah Agricultural Experiment Station Project UTA-1178.

Cancer risk assessment utilizing rodents requires extrapolation across five orders of magnitude to estimate the Virtually Safe Dose (VSD). Regulatory agencies rely upon the Linear Extrapolated Dose (LED) except when sufficient information on mechanism of action justifies alternative models. Rainbow trout (Oncorhynchus mykiss) has been utilized at Oregon State University as a model for human cancer for forty years. Low cost and high capacity, made possible by our unique facility, along with low spontaneous background and high sensitivity, allow design and conduct of statistically challenging studies not possible in rodents. Utilization of custom microarrays demonstrates similarities in gene expression in trout and human hepatocellular carcinoma (HCC). We have completed one study employing over 42,000 trout with dibenzo[a,l]pyrene (DBP) and determined the dose resulting in 1 additional cancer in 5000 animals, a 50-fold enhancement over the mouse ED(01) study. Liver tumor incidence at low dose deviated significantly from linearity (concave down), whereas, DBP-DNA adductions deviated slightly (convex up). A second study is underway with aflatoxin B(1) (AFB(1)). Results to date indicate AFB(1) at low dose, in contrast to DBP, elicits a linear dose-response function on the log-log scale which falls below the LED with a slope slightly greater than 1.0. Such studies demonstrate the statistical power of the trout cancer model and strengthen the case for incorporation of these data-sets into risk assessment for these environmental human carcinogens.

Previous investigations in Atlantic croaker ovaries and primary co-cultured theca and granulosa cells have identified multiple signal transduction pathways involved in the control of gonadotropin-induced steroidogenesis, including adenylyl cyclase- and calcium-dependent signaling pathways. In the present study, evidence was obtained for an involvement of a third signal transduction pathway, a mitogen-activated protein kinase (MAP kinase) signaling cascade, in the regulation of gonadal steroidogenesis in this lower vertebrate teleost model. Gonadotropin-stimulated testosterone synthesis was markedly attenuated by two antagonists of mitogen-activated protein kinase kinases 1/2 (MEK1/2, also known as Map2k1/Map2k2). Moreover, treatment with gonadotropin-induced MEK1/2-dependent phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERK1/2, also known as Mapk3/Mapk1) in a concentration- and time-dependent manner in co-cultured croaker theca and granulosa cells. Active MEK1/2 was required for a complete steroidogenic response to activators of the adenylyl cyclase pathway, including forskolin and dbcAMP, suggesting that the target(s) of MAP kinase signaling are distal to cAMP generation and activation of cAMP-dependent protein kinase (PKA). Interestingly, dbcAMP caused a similar increase of ERK1/2 phosphorylation as was observed with gonadotropin treatment, although an inhibitor of PKA did not attenuate this response. Finally, there was no evidence of cross-talk between calcium-dependent signaling pathways and this MAP kinase cascade. While drugs that block calcium-dependent signal transduction, including inhibitors of voltage-sensitive calcium channels, calmodulin, and calcium/calmodulin-dependent kinases, significantly reduced gonadotropin-induced testosterone accumulation, these drugs had no apparent effect on hCG-induced ERK1/2 phosphorylation.

The identities of the membrane receptors mediating the majority of rapid, cell surface-initiated, nongenomic (i.e. nonclassical) steroid actions described to date are unclear. Two novel 7-transmembrane spanning proteins, representing two distinct classes of steroid membrane receptors, membrane progestin receptor alpha (mPRalpha) and a membrane estrogen receptor (mER), GPR30, have recently been identified in several vertebrate species. Evidence that both receptors activate G-proteins and function as G-protein coupled receptors (GPCRs) is briefly reviewed. New data on progestin actions on fish gametes suggest a widespread involvement of mPRalpha in oocyte maturation and sperm hyperactivity in this vertebrate group. Information on the second messenger pathways activated upon estrogen binding to a membrane estrogen receptor in croaker gonads and preliminary evidence for the presence of a GPR30-like protein in fish gonads are discussed. Finally, initial characterization of the ligand binding, G-protein activation and molecular size of a membrane androgen receptor (mAR) in croaker ovaries suggests the presence of a third unique steroid receptor in fish gonads that also may function as a GPCR.

The involvement of calcium-dependent signal transduction pathways in the regulation of ovarian steroidogenesis was investigated in Atlantic croaker. Treatment with the calcium ionophores A23187 and ionomycin caused a 2- to 5-fold increase in basal steroid accumulation by croaker ovarian tissue in vitro. A23187 potentiated human chorionic gonadotropin (hCG)-induced testosterone (T) accumulation, whereas it inhibited accumulation of estradiol-17beta (E(2)) and the conversion of T to E(2), suggesting that intracellular calcium modulates aromatase enzyme activity. Gonadotropin stimulation of ovarian steroidogenesis was decreased in the presence of EGTA and inhibitors of voltage-sensitive calcium channels (VSCCs) and inositol-1,4,5-triphosphate-receptors (IP(3)Rs), indicating that releases of calcium from both intracellular and extracellular stores are components of the signal transduction pathways initiated by gonadotropin. Calmodulin is also involved in the regulation of ovarian steroidogenesis in croaker, since the calmodulin inhibitors W-7 and trifluoperazine (TFP) attenuated hCG-stimulated T and E(2) accumulation. These results are broadly similar to those reported previously in goldfish and suggest that the major calcium-dependent signaling pathways involved in gonadotropin stimulation of ovarian steroidogenesis in tetrapods are also present in teleosts. In addition, the involvement of calcium in the regulation of aromatase activity was demonstrated for the first time in a vertebrate ovary. Finally, acute exposure to 0.001-1 ppm Aroclor 1254 induced up to a 5-fold increase in hCG-stimulated E(2) accumulation, and this effect was attenuated by co-treatment with inhibitors of VSCCs and calmodulin, suggesting the existence of a novel mechanism of endocrine disruption by an environmental contaminant involving alteration of calcium-dependent signaling pathways regulating steroidogenesis.