Jeremy K. Larson

Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish

Abstract Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs]) and neurobehavior (i.e., visual startle and spontaneous locomotion) in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM) for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation) and transgenerational actions (F2 generation) were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation) of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans.

Engineered Nanomaterials: An Emerging Class of Novel Endocrine Disruptors

ABSTRACT Over the past decade, engineered nanomaterials (ENMs) have garnered great attention for their potentially beneficial applications in medicine, industry, and consumer products due to their advantageous physicochemical properties and inherent size. However, studies have shown that these sophisticated molecules can initiate toxicity at the subcellular, cellular, and/or tissue/organ level in diverse experimental models. Investigators have also demonstrated that, upon exposure to ENMs, the physicochemical properties that are exploited for public benefit may mediate adverse endocrine-disrupting effects on several endpoints of mammalian reproductive physiology (e.g., steroidogenesis, spermatogenesis, pregnancy). Elucidating these complex interactions within reproductive cells and tissues will significantly advance our understanding of ENMs as an emerging class of novel endocrine disruptors and reproductive toxicants. Herein we reviewed the recent developments in reproductive nanotoxicology and identified the gaps in our knowledge that may serve as future research directions to foster continued advancement in this evolving field of study.

Low-dose gold nanoparticles exert subtle endocrine-modulating effects on the ovarian steroidogenic pathway ex vivo independent of oxidative stress

Abstract Gold nanoparticles (GNPs) have gained considerable attention for application in science and industry. However, the untoward effects of such particles on female fertility remain unclear. The objectives of this study were to (1) examine the effects of 10-nm GNPs on progesterone and estradiol-17β accumulation by rat ovaries ex vivo and (2) to identify the locus/loci whereby GNPs modulate steroidogenesis via multiple-reference gene quantitative real-time RT-PCR. Regression analyses indicated a positive relationship between both Star (p < 0.05, r2 = 0.278) and Cyp11a1 (p < 0.001, r2 = 0.366) expression and P4 accumulation upon exposure to 1.43 × 106 GNPs/mL. Additional analyses showed that E2 accumulation was positively associated with Hsd3b1 (p < 0.05, r2 = 0.181) and Cyp17a1 (p < 0.01, r2 = 0.301) expression upon exposure to 1.43 × 13 and 1.43 × 109 GNPs/mL, respectively. These results suggest a subtle treatment-dependent impact of low-dose GNPs on the relationship between progesterone or estradiol-17β and specific steroidogenic target genes, independent of oxidative stress or inhibin.

Developmental Methylmercury Exposure Affects Swimming Behavior and Foraging Efficiency of Yellow Perch (Perca flavescens) Larvae

Methylmercury (MeHg) is a pervasive and ubiquitous environmental neurotoxicant within aquatic ecosystems, known to alter behavior in fish and other vertebrates. This study sought to assess the behavioral effects of developmental MeHg exposure on larval yellow perch (Perca flavescens)—a nonmodel fish species native to the Great Lakes. Embryos were exposed to MeHg (0, 30, 100, 300, and 1000 nM) for 20 h and then reared to 25 days post fertilization (dpf) for analyses of spontaneous swimming, visual motor response (VMR), and foraging efficiency. MeHg exposures rendered total mercury (THg) body burdens of 0.02, 0.21, 0.95, 3.14, and 14.93 μg/g (wet weight). Organisms exposed to 1000 nM exhibited high mortality; thus, they were excluded from downstream behavioral analyses. All MeHg exposures tested were associated with a reduction in spontaneous swimming at 17 and 25 dpf. Exposure to 30 and 100 nM MeHg caused altered locomotor output during the VMR assay at 21 dpf, whereas exposure to 100 nM MeHg was associated with decreased foraging efficiency at 25 dpf. For the sake of comparison, the second-lowest exposure tested here rendered a THg burden that represents the permissible level of consumable fish in the United States. Moreover, this dose is reported in roughly two-thirds of consumable fish species monitored in the United States, according to the Food and Drug Administration. Although the THg body burdens reported here were higher than expected in the environment, our study is the first to analyze the effects of MeHg exposure on fundamental survival behaviors of yellow perch larvae and advances in the exploration of the ecological relevance of behavioral end points.

Female Reproductive Impacts of Dietary Methylmercury in Yellow Perch (Perca flavescens) and Zebrafish (Danio rerio)

The purpose of this study was to evaluate the effects of environmentally relevant dietary MeHg exposures on adult female yellow perch (Perca flavescens) and female zebrafish (Danio rerio) ovarian development and reproduction. Yellow perch were used in the study for their socioeconomic and ecological importance within the Great Lakes basin, and the use of zebrafish allowed for a detailed analysis of the molecular effects of MeHg following a whole life-cycle exposure. Chronic whole life dietary exposure of F1 zebrafish to MeHg mimics realistic wildlife exposure scenarios, and the twenty-week adult yellow perch exposure (where whole life-cycle exposures are difficult) captures early seasonal ovarian development. For both species, target dietary accumulation values were achieved prior to analyses. In zebrafish, several genes involved in reproductive processes were shown to be dysregulated by RNA-sequencing and quantitative real-time polymerase chain reaction (QPCR), but no significant phenotypic changes were observed regarding ovarian staging, fecundity, or embryo mortality. Yellow perch were exposed to dietary MeHg for 12, 16, or 20 weeks. In this species, a set of eight genes were assessed by QPCR in the pituitary, liver, and ovary, and no exposure-related changes were observed. The lack of genomic resources in yellow perch hinders the characterization of subtle molecular impacts. The ovarian somatic index, circulating estradiol and testosterone, and ovarian staging were not significantly altered by MeHg exposure in yellow perch. These results suggest that environmentally relevant MeHg exposures do not drastically reduce the reproductively important endpoints in these fish, but to capture realistic exposure scenarios, whole life-cycle yellow perch exposures are needed.

Neurobehavioral Analysis Methods for Adverse Outcome Pathway (AOP) Models and Risk Assessment

The emerging use of neurobehavioral analysis techniques in toxicology promotes the implementation of neurobehavior, a powerful integrator of molecular, physiological, and environmental stimuli, in the development of Adverse Outcome Pathway (AOP) models. In recent years, zebrafish have been extensively investigated for their potential as a model organism in behavioral toxicology due to their low maintenance cost and similarities with rodent behavior and physiology. This chapter will review: (1) the beneficial role of neurobehavioral assays in the development of AOPs; (2) the diverse neurobehavioral endpoints to be considered in the evaluation of neurotoxicity and; (3) the challenges of integrating neurobehavioral outcomes into AOP development. Discussion of the many neurobehavioral screening assays that have been adapted from rodents to zebrafish is included. Furthermore, this chapter will review studies in which behavioral phenotypes and neurophysiological outcomes have been anchored to specific molecular initiating events induced by a chemical exposure. Although the study of the genetic and physiological basis of behavior is still nascent, there are many noteworthy studies that have enabled the creation of AOP models for the prediction of how chemical exposure affects the behavior of individuals in a population and, in turn, how these alterations can affect population dynamics.

Superovulation and Ovulation Induction

Superovulation and ovulation induction are technologies that use various preparations of hormones to stimulate the ovaries, thereby producing follicles and oocytes in numbers greater than normal (i.e., superovulation); in anovulatory women, these techniques can be used in theory to produce a single preovulatory follicle (usually) and a single oocyte (i.e., ovulation induction).