Evolution to pollution in Gulf killifish (Fundulus grandis) from Galveston Bay, TX, USA.

Anthropogenic contamination associated with industrial activity is a widespread and active threat to the stability of organisms. The Houston Ship Channel (HSC) is one example of a heavily impacted environment, where industrial activity has contributed to extreme levels of pollution with various classes of contaminants, such as polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs), polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). This dissertation studies the impacts of chronic multi-generational exposure to industrial contamination on the population structure, resistance and demography in a keystone coastal species – Gulf killifish (Fundulus grandis). We have characterized their sensitivity to contaminants in populations from 12 locations across Galveston Bay, as well as the contamination levels at those sites. We found a gradient of resistance that was positively correlated with contaminant concentrations. This resistance was also correlated to a suppression of the aryl hydrocarbon receptor pathway (AHR), as estimated via the activity of a down-stream regulated enzyme – cytochrome P450 1A (CYP1A). To better understand the impacts of this adaptation, we evaluated the cross-resistance and fitness cost of populations to mechanistically and environmentally relevant stressors, but we were unable to confirm any fitness costs. We showed that the heritability of this resistance is biparental and multi-generational, which suggest that this is a genetic trait. Finally, we performed a full genome resequencing of seven populations along this gradient of resistance and discovered that genomic regions under selection in adapted populations included the AHR pathway. To determine if regulatory benchmarks on the compounds driving this adaptation would protect from such events, we performed a meta-analysis of all evolutionary events to contamination with PCBs and PAHs and found several locations, in which populations have adapted at values below regulation. Here we show that Gulf killifish populations have undergone a rapid evolutionary adaptation to a gradient of anthropogenic contaminants in Galveston Bay. In addition, we suggest that evolutionary toxicology studies, as described here, can be informative for regulatory purposes for compounds that may drive population-wide change

Evolutionary toxicology : meta-analysis of evolutionary events in response to chemical stressors

The regulatory decision-making process regarding chemical safety is most often informed by evidence based on ecotoxicity tests that consider growth, reproduction and survival as end-points, which can be quantitatively linked to short-term population outcomes. Changes in these end-points resulting from chemical exposure can cause alterations in micro-evolutionary forces (mutation, drift, selection and gene flow) that control the genetic composition of populations. With multi-generation exposures, anthropogenic contamination can lead to a population with an altered genetic composition, which may respond differently to future stressors. These evolutionary changes are rarely discussed in regulatory or risk assessment frameworks, but the growing body of literature that documents their existence suggests that these important population-level impacts should be considered. In this meta-analysis we have compared existing contamination levels of polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) that have been documented to be associated with evolutionary changes in resident aquatic organisms to regulatory benchmarks for these contaminants. The original intent of this project was to perform a meta-analysis on evolutionary events associated with PCB and PAH contamination. However, this effort was hindered by a lack of consistency in congener selection for "total" PCB or PAH measurements. We expanded this manuscript to include a discussion of methods used to determine PCB and PAH total contamination in addition to comparing regulatory guidelines and contamination that has caused evolutionary effects. Micro-evolutionary responses often lead populations onto unique and unpredictable trajectories. Therefore, to better understand the risk of population-wide alterations occurring, we need to improve comparisons of chemical contamination between affected locations. In this manuscript we offer several possibilities to unify chemical comparisons for PCBs and PAHs that would improve comparability among evolutionary toxicology investigations, and with regulatory guidelines. In addition, we identify studies documenting evolutionary change in the presence of PCB and PAH contamination levels below applicable regulatory benchmarks

Differential Expression of the Activator Protein 1 Transcription Factor Regulates Interleukin-1ß Induction of Interleukin 6 in the Developing Enterocyte.

The innate immune response is characterized by activation of transcription factors, nuclear factor kappa B and activator protein-1 and their downstream targets, the pro-inflammatory cytokines including interleukin 1β and interleukin 6. Normal development of this response in the intestine is critical to survival of the human neonate and delays can cause the onset of devastating inflammatory diseases such as necrotizing enterocolitis. Previous studies have addressed the role of nuclear factor kappa B in the development of the innate immune response in the enterocyte, however despite its central role in the control of multiple pro-inflammatory cytokine genes, little is known on the role of Activator Protein 1 in this response in the enterocyte. Here we show that the canonical Activator Protein 1 members, cJun and cFos and their upstream kinases JNK and p38 play an essential role in the regulation of interleukin 6 in the immature enterocyte. Our data supports a model whereby the cFos/cJun heterodimer and the more potent cJun homodimer downstream of JNK are replaced by less efficient JunD containing dimers, contributing to the decreased responsiveness to interleukin 1β and decreased interleukin 6 secretion observed in the mature enterocyte. The tissue specific expression of JunB in colonocytes and colon derived tissues together with its ability to repress Interleukin-1β induction of an Interleukin-6 gene reporter in the NCM-460 colonocyte suggests that induction of JunB containing dimers may offer an attractive therapeutic strategy for the control of IL-6 secretion during inflammatory episodes in this area of the intestine

Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies

Interleukin 1β induction of IL-6 is mediated by the Stress Activated Protein Kinases, JNK and p38 in immature H4 enterocytes.

<p>Immature H4 cells (A, B) and mature NCM-460 cells (C, D) were plated in 24 well plates overnight, incubated in low serum media for 3h and treated with IL-1β (0.5ng/ml) in the absence or presence of the JNK inhibitor SP600125 and the p38 inhibitor SB203580 (triplicate treatments) as indicated. Tissue culture media was harvested after 6 h and assayed for IL-6 by ELISA. Mean +/-S.E (n = 3) from 2 experiments are presented. Control un induced compared to IL-1 and IL-1 induced compared to IL-1 in the presence of inhibitors, ***p<0.001, **p<0.01 *p<0.05) analyzed by Student T-test.</p

The IL-6 promoter is repressed by JunB in the mature NCM-460 enterocyte.

<p>The IL-6-651 luciferase promoter construct was transfected into NCM-460 cells alone or co-transfected with expression plasmids for c Jun, Tam67 or JunB as indicated. A Renilla luciferase plasmid was co-transfected for estimation of transfection efficiency. After overnight culture, transfectants were either untreated (A) or treated with IL-1β (B). Transfectants (n = 3) were harvested and assayed for luciferase. Normalized data, presented as % basal promoter activation (A) or % IL-1β induced promoter activation (B), in the presence of empty plasmid control (-) or in the presence of the indicated plasmids. Data is presented as Means +/-SE (n = 3), un transfected compared to transfected **p<0.01, *p<0.05. A representative experiment of three separate experiments is presented with consistent results.</p

The IL-6 promoter AP-1 and NFĸB sites are equipotent in IL-1β induction of the IL-6 reporter in the immature H4 enterocyte.

<p><b>(</b>A) Linear map of the IL-6 promoter (IL-6 651 Luc) reporter showing the AP-1 site (-283), the NFkB site at (-72) and the C/EBP site at -154 from the transcription start site. <b>(B)</b> Effect of mutation of the AP-1 and NFκB binding sites on IL-1β induction of the IL-6 promoter. H4 cells were transfected with the IL-6 promoter reporter plasmids, IL-6-651 wild type, (WT) and (IL-6 Mt AP-1) mutated in the AP-1 site and (IL-6Mt NFκB) mutated in the NFκB site (<b>C)</b> Effect of IL-1β stimulation on NFκB and (D) AP-1 Luciferase reporter plasmids in immature H4 cells. Reporter plasmids were transfected into H4 cells together with a Renilla luciferase plasmid for estimation of transfection efficiency. After overnight culture, transfectants (n = 3) were either untreated (open bars) or treated with IL-1b (black bars) for 18h and were harvested and assayed for firefly and renilla luciferase. Data is normalized to renilla luciferase and expressed as Means +/-SE (n = 3), control un induced compared to IL-1 induced, ***p<0.001, *p<0.05. A representative experiment of three separate experiments is presented with consistent results.</p

The canonical AP-1 family members, cJun and cFos are highly expressed in the immature ileal xenografts and decrease during maturation.

<p>Whole tissue lysates (15ug) from immature xenografts (16 weeks) compared to more mature ileal xenografts (23, 28, 44 weeks) 3 individual mice per developmental stage, 12 mice total. Organ cultures from xenografts were equilibrated in organ culture media for 3h and incubated for 18h in the presence or absence of IL-1b. Lysates were western blotted for the presence of cJun (A) and cFos (B) as well as other members, including JunD, Fra1 and FosB and loading control GAPDH. In A (lower panel) densitometry analysis of the developed bands normalized to βActin is presented. Results are expressed as Means+/-SE (n = 3). In C lysates derived from mature colon and ileum were compared for the presence of JunB and in D immature ileal lysates were blotted for the presence of phosphorylated JNK and p38 (D) with loading control, βActin.</p

Interleukin 1β induction of IL-6 promoter function is enhanced by cJun in the immature H4 enterocyte.

<p><b>In A</b> The IL-6-651 wild type Luciferase plasmid was transfected into H4 cells alone or co-transfected with expression plasmids for cJun, Tam67, JunB JunD, and JunD DN, dominant negative as indicated. A renilla expression plasmid was co-transfected for estimation of transfection efficiency. After overnight culture, serum starved transfectants (n = 3) were either untreated (A) or treated with IL-1b (B) for 18h and were harvested and assayed for firefly and renilla luciferase. Normalized data presented as basal promoter activation (A) or IL-1β induced promoter activation (B) in the presence of empty plasmid control (-) or in the presence of the indicated plasmids. Means +/-SE (n = 3), control un transfected compared to transfected ***p<0.001, **p<0.01, *p<0.05. A representative experiment of three separate experiments is presented with consistent results</p

IL-1β induction of IL-6 gene transcription <i>via</i> AP-1 in the developing enterocyte.

<p>Interleukin 1 (IL-1) binding to the IL-1 receptor (IL-1R1) and co-receptor (IL-1RAc) and formation of the signaling module containing MyD88, IRAK (IL-1 receptor associated kinase) and TRAF6 (TNF receptor associated factor). The Map kinase signaling module is activated, upstream of the Map kinase kinases (MKKs) and Stress Activated Protein kinases (SAPKs) JNK and p38. We have previously shown MyD88 and TRAF-6 mRNA is up regulated in the immature enterocyte [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0145184#pone.0145184.ref008" target="_blank">8</a>] Constitutive and IL-1β induced JNK and p38 phosphorylation, together with high expression levels of their target transcription factors, cJun and cFos results in increased IL-6 gene transcription in the immature enterocyte. Expression of JunD later in enterocyte development facilitates formation of the less potent JunD/cJun or JunD/cFos dimers which replace the activating cJun/cJun or cJun/cFos dimers resulting in lower IL-6 gene transcription. A JNK independent pathway involving JunB in the colon represses IL-6 gene transcription.</p