Effects of short-chain fatty acids on intestinal function in an enteroid model of hypoxia.

The healthy GI tract is physiologically hypoxic, but this may be perturbed by certain acute and chronic stressors that reduce oxygen availability systemically. Short-chain fatty acids have been shown to have beneficial effects on intestinal barrier function and inflammation. Therefore, our objective was to see whether short-chain fatty acids (SCFA) would improve GI barrier function, reduce production of pro-inflammatory cytokines, and increase the expression of genes regulating GI barrier function in enteroids exposed to hypoxia. Human duodenal enteroid monolayers were placed under hypoxia (1.0% O2) for 72 h with either 24, or 48 h pre-treatment with a high acetate ratio of SCFA's or high butyrate ratio or placed under hypoxia concurrently. Transepithelial electrical resistance (TEER) increased with SCFA pre-treatment, especially 48 h of pre-treatment and this was maintained through the first 48 h of hypoxia while cells saw barrier function dramatically decrease by 72 h of hypoxia exposure. Inflammatory protein secretion largely decreased with exposure to hypoxia, regardless of SCFA pre-treatment. Gene expression of several genes related to barrier function were decreased with exposure to hypoxia, and with concurrent and 24 h SCFA pre-treatment. However, 48 h SCFA pre-treatment with a high butyrate ratio increased expression of several metabolic and differentiation related genes. Overall, pre-treatment or concurrent treatment with SCFA mixtures were not able to overcome the negative impacts of hypoxia on intestinal function and cells ultimately still cannot be sustained under hypoxia for 72 h. However, 48 h pre-treatment maintains TEER for up to 48 h of hypoxia while upregulating several metabolic genes.

Human iPSC colon organoid function is improved by exposure to fecal fermentates.

The host-microbe interaction is critical for intestinal homeostasis. By-products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by-product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human-induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter-1, (6.2-fold, 2.1-fold, and 1.8-fold, respectively; p < 0.05). Mucin production (mucin-2, mucin-4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin-3) was noted by immunofluorescence in 25% supernatant- treated cells. A dose-response increase in barrier function was observed over the 72-h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL-1ß, 6.15 pg/ml; IL-6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC-treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by-products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Thrombospondin-1 mediates Drp-1 signaling following ischemia reperfusion in the aging heart.

BACKGROUND: Ischemia reperfusion (IR) injury leads to activation of dynamin-related protein (Drp-1), causing mitochondrial fission and generation of reactive oxygen species (ROS), but the molecular mechanisms that activate Drp-1 are not known. The purpose of this study was to establish a link between Thbs-1 and fission protein (Drp-1) through Pgc-1α following IR in advancing age. METHODS: Female Fischer-344 rats were divided into four groups: Young Control, Young + IR, Old Control, and Old + IR. Heart function and coronary flow were evaluated at baseline and 72 hours after IR, hearts were explanted and mitochondrial ROS generation was measured using MitoPY1, as well as protein levels of Thbs-1, Pgc-1α, and Drp-1. In vitro, rat aortic endothelial cells (RAEC) were treated with siRNA or plasmid for Pgc-1α to evaluate Pgc-1α effect on Drp-1. RESULTS: Mitochondrial ROS generation in heart tissue increased in both age groups following IR. Old animals exhibited diastolic dysfunction at baseline; after IR they displayed reduced systolic function and exacerbated diastolic dysfunction compared to young controls. IR increased Thbs-1 and Drp-1 expression in young and old hearts compared to control. siRNA to Pgc-1α enhanced levels of Drp-1 in RAECs and increased ROS generation after hypoxia, while Pgc-1α plasmid ameliorates Drp-1 expression in the presence of exogenous Thbs-1. CONCLUSION: These results highlight a novel signaling pathway by which Thbs-1 regulates mitochondrial fission protein (Drp-1) and ROS generation during hypoxia, and presumably, following IR. Inhibiting Thbs-1 immediately after IR may prevent Drp-1-mediated mitochondrial fission and is likely to improve the diastolic function of the heart by reducing ROS-mediated cardiomyocyte damage in the aged population.

Intestinal enteroids recapitulate the effects of short-chain fatty acids on the intestinal epithelium.

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore, the objective was to study the effects of SCFAs on biomarkers of ISC activity, differentiation, barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET), propionate (PROP), or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments, but increased with ACET. As a result of BUT and PROP treatments, there was an increase in differentiation markers for enterocyte, Paneth, goblet, and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3ß, Reg3γ, and Defb1 were stimulated by BUT and PROP, but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures, although tight junctions were influenced by BUT and PROP, but not ACET in monolayer format. Furthermore, BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall, individual SCFAs are potent stimulators of cellular gene expression, however, PROP and especially BUT show great efficacy for driving cell differentiation and gene expression.

Cardioprotective effects of high-intensity interval training are mediated through microRNA regulation of mitochondrial and oxidative stress pathways.

Human studies have shown high-intensity interval training (HIIT) has beneficial cardiovascular effects and is typically more time-efficient compared with traditional endurance exercise. The main goal of this study is to show the potential molecular and functional cardiovascular benefits of HIIT compared with endurance training (ET). Three groups of mice were used including sedentary-control, ET mice, and HIIT mice groups. Results indicated ejection fraction was increased in HIIT compared with ET while fractional shortening was increased in the HIIT group compared with both groups. Blood flow of the abdominal aorta was increased in both exercise groups compared with control. Increases in cross-sectional area and mitochondrial and antioxidative markers in HIIT compared with control were observed, along with several microRNAs. These findings indicate HIIT has specific cardiac-protective effects and may be a viable alternative to traditional ET as a cardiovascular preventative medicine intervention.

TFAM overexpression diminishes skeletal muscle atrophy after hindlimb suspension in mice.

The present study aims to investigate if overexpressing the mitochondrial transcription factor A (TFAM) gene in a transgenic mouse model diminishes soleus and gastrocnemius atrophy occurring during hindlimb suspension (HLS). Additionally, we aim to observe if combining exercise training in TFAM transgenic mice prior to HLS has a synergistic effect in preventing skeletal muscle atrophy. Male C57BL/6J-based transgenic mice (12-14 weeks old) overexpressing TFAM were assigned to a control (T-Control), 7-day HLS (T-HLS), and 2-week exercise training prior to 7-day HLS (T-Ex + HLS) groups. These groups were compared to male C57BL/6J wild-type (WT) mice (12-14 weeks old) assigned to Control, 7-day HLS (HLS), 2-week exercise training prior to 7-day HLS (Ex + HLS), and 2-week exercise training (Ex). Overexpressing TFAM results in a decrease of 8.3% in soleus and 2.6% in gastrocnemius muscle weight to bodyweight ratio after only HLS compared to wild-type mice incurring a loss of 27.1% in soleus and 21.5% in gastrocnemius muscle after HLS. Our data indicates TFAM may play a critical role in protecting skeletal muscle from disuse atrophy and is correlated with increased expression of antioxidants (SOD-2) and potential redox balance. TFAM may be an attractive molecule of interest for potential, future therapeutic development. NEW AND NOTEWORTHY: To the best of our knowledge, this is the first time a TFAM overexpression transgenic mouse model is being used in the analysis of disuse-induced skeletal muscle atrophy. Here we provide evidence of a potential role for TFAM in diminishing skeletal muscle atrophy.

Hypertension exaggerates renovascular resistance via miR-122-associated stress response in aging.

OBJECTIVE: Hypertension at advanced age damages microvasculature and initiates many disease conditions including chronic kidney disease (CKD). In the present study, we sought to determine molecular alterations occurring in angiotensin-II (Ang-II)-induced aged kidney. METHODS: Old (75-80 weeks) and young (12-14 weeks) wild-type mice (C57BL/6J) were infused with Ang-II (1000 ng/kg per min) for 4 weeks using osmotic minipumps to induce hypertension. Blood pressure, renovascular density, and renal vascular resistance were measured by telemetry, barium angiography, and renal ultrasound, respectively. Molecular analysis was performed by RT-PCR, western blotting, and immunostaining. RESULTS: Aged hypertensive mice showed significant increase in blood pressure, increased resistive index, and reduced vasculature compared with young mice with Ang-II. The cytoprotective and anti-inflammatory molecule hemeoxygenase-1 (Ho-1) was found to be downregulated in the hypertensive aged mice whereas its putative regulator Bach-1 was increased. Antagonistically, an increase in inflammatory chemokine Mcp-1 was observed in the same mice group along with an increase in extracellular matrix protein, collagen. In addition, DNA damage marker γH2AX was found to be high in hypertensive kidney, especially in aged hypertensive animals along with increased miR-122. Transfection with a mimic of miR-122 into mesangial cells showed an increase of Bach-1 expression and concomitant decease in Ho-1. CONCLUSION: Our findings suggest that aged animals fail to counteract hypertensive condition resulting in upregulation of miR-122 and subsequently Bach-1, leading to decreased levels of Ho-1 and an increase in DNA damage and tissue inflammation. Together, these lead to increased collagen deposition thereby causing reduced vascular density and increased renal resistive index.

Exercise preconditioning diminishes skeletal muscle atrophy after hindlimb suspension in mice.

The aim of the present study was to investigate whether short-term, concurrent exercise training before hindlimb suspension (HLS) prevents or diminishes both soleus and gastrocnemius atrophy and to analyze whether changes in mitochondrial molecular markers were associated. Male C57BL/6 mice were assigned to control at 13 ± 1 wk of age, 7-day HLS at 12 ± 1 wk of age (HLS), 2 wk of exercise training before 7-day HLS at 10 ± 1 wk of age (Ex+HLS), and 2 wk of exercise training at 11 ± 1 wk of age (Ex) groups. HLS resulted in a 27.1% and 21.5% decrease in soleus and gastrocnemius muscle weight-to-body weight ratio, respectively. Exercise training before HLS resulted in a 5.6% and 8.1% decrease in soleus and gastrocnemius weight-to-body weight ratio, respectively. Exercise increased mitochondrial biogenesis- and function-associated markers and slow myosin heavy chain (SMHC) expression, and reduced fiber-type transitioning marker myosin heavy chain 4 (Myh4). Ex+HLS revealed decreased reactive oxygen species (ROS) and oxidative stress compared with HLS. Our data indicated the time before an atrophic setting, particularly caused by muscle unloading, may be a useful period to intervene short-term, progressive exercise training to prevent skeletal muscle atrophy and is associated with mitochondrial biogenesis, function, and redox balance. NEW & NOTEWORTHY Mitochondrial dysfunction is associated with disuse-induced skeletal muscle atrophy, whereas exercise is known to increase mitochondrial biogenesis and function. Here we provide evidence of short-term concurrent exercise training before an atrophic event protecting skeletal muscle from atrophy in two separate muscles with different, dominant fiber-types, and we reveal an association with the adaptive changes of mitochondrial molecular markers to exercise.

Embryonic atrazine exposure elicits proteomic, behavioral, and brain abnormalities with developmental time specific gene expression signatures.

Atrazine (ATZ), the second most commonly used herbicide in the United States, is an endocrine disrupting chemical linked to cancer and a common drinking water contaminant. This study further investigates ATZ-related developmental toxicity by testing the following hypotheses in zebrafish: the effects of embryonic ATZ exposure are dependent on timing of exposure; embryonic ATZ exposure alters brain development and function; and embryonic ATZ exposure changes protein abundance in carcinogenesis-related pathways. After exposing embryos to 0, 0.3, 3, or 30 parts per billion (ppb) ATZ, we monitored the expression of cytochrome P450 family 17 subfamily A member 1 (cyp17a1), glyoxalase I (glo1), ring finger protein 14 (rnf14), salt inducible kinase 2 (sik2), tetratricopeptide domain 3 (ttc3), and tumor protein D52 like 1 (tpd52l1) at multiple embryonic time points to determine normal expression and if ATZ exposure altered expression. Only cyp17a1 had normal dynamic expression, but ttc3 and tpd52l1 had ATZ-related expression changes before 72 h. Larvae exposed to 0.3 ppb ATZ had increased brain length, while larvae exposed to 30 ppb ATZ were hypoactive. Proteomic analysis identified altered protein abundance in pathways related to cellular function, neurodevelopment, and genital-tract cancer. The results indicate embryonic ATZ toxicity involves interactions of multiple pathways. SIGNIFICANCE: This is the first report of proteomic alterations following embryonic exposure to atrazine, an environmentally persistent pesticide and common water contaminant. Although the transcriptomic alterations in larval zebrafish with embryonic atrazine exposure have been reported, neither the time at which gene expression changes occur nor the resulting proteomic changes have been investigated. This study seeks to address these knowledge gaps by evaluating atrazine's effect on gene expression through multiple time points during embryogenesis, and correlating changes in gene expression to pathological alterations in brain length and functional changes in behavior. Finally, pathway analysis of the proteomic alterations identifies connections between the molecular changes and functional outcomes associated with embryonic atrazine exposure.

Altered microRNA regulation of short chain fatty acid receptors in the hypertensive kidney is normalized with hydrogen sulfide supplementation.

Hypertension affects nearly one third of the adult US population and is a significant risk factor for chronic kidney disease (CKD). An expanding body of recent studies indicates that gut microbiome has crucial roles in regulating physiological processes through, among other mechanisms, one mode of short chain fatty acids (SCFA) and their target receptors. In addition, these SCFA receptors are potential targets of regulation by host miRNAs, however, the mechanisms through which this occurs is not clearly defined. Hydrogen sulfide (H2S) is an important gasotransmitter involved in multiple physiological processes and is known to alleviate adverse effects of hypertension such as reducing inflammation in the kidney. To determine the role of host microRNAs in regulating short chain fatty acid receptors in the kidney as well as the gut, C57BL/6J wild-type mice were treated with or without Ang-II and H2S donor GYY4137 (GYY) for 4 weeks to assess whether GYY would normalize adverse effects observed in hypertensive mice and whether this was in part due to altered gut microbiome composition. We observed several changes of SCFA receptors, including Olfr78, Gpr41/43 and predicted microRNA regulators in the kidney among the different treatments. Increased expression of inflammatory markers Il6 and Rorc2, along with Tgfß, were found in the hypertensive kidney. The glomerular filtration rate (GFR) was improved in mice treated with Ang-II + GYY compared with Ang-II only, indicating improved kidney function. The Erysipelotrichia class of bacteria, linked with high fat diets, was enriched in hypertensive animals but reduced with GYY supplementation. These data point towards a role for miRNA regulation of SCFA receptors in hypertensive kidney and are normalized by H2S supplementation.

Ablation of toll-like receptor 4 mitigates cardiac mitochondrial dysfunction in hyperhomocysteinemia.

Hyperhomocysteinemia (HHcy) is a risk factor for adverse cardiovascular events; however, the mechanism for development of this disease is still unknown. Toll-like receptor 4 (TRL4) is a molecule involved in the immune response pathway and is quickly becoming a receptor of interest in the field of hypertension. In this study, we hypothesized that ablation of TLR4 mitigates cardiac mitochondrial dysfunction in a model of HHcy. Five strains of mice (C57BL/6J, CBS+/-, C3H, CBS+/-/C3H, and C3H/HeOuJ) 10-12 weeks old were utilized. We found that HHcy causes heart hypertrophy and promotes oxidative stress while mice with HHcy and inactivated TLR4 showed significant improvement in examined parameters. A dominance of endothelial cell mitochondrial fission over mitochondrial fusion in HHcy and oxidative stress was observed, which may explain the endothelial cell loss and dysfunction that contributes to inward cardiac remodeling.

Ablation of Toll-like receptor 4 mitigates central blood pressure response during hyperhomocysteinemia.

OBJECTIVE: The objective of this study was to define the mechanisms of homocysteine-induced effects on the aortic wall that promote vascular remodeling and hypertension as well as explore the role of Toll-like receptor 4 in homocysteine-induced effects. METHOD: Five strains of mice were utilized in this study: C57BL/6J, C3H/HeOuJ, CBS+/-, C3H/HeJ and CBS+/-/C3H. Aorta, heart and blood were collected at the end of the experiments. Blood pressure (BP) was recorded using noninvasive tail cuff method. To determinate effects of vasoactive agent and endothelial-dependent vasodilator on aorta contractility, we performed vascular function measurements. In addition, the expression of mitochondrial fusion and fission proteins, antioxidant markers and collagen fragments were assessed. RESULTS: BP measurements demonstrated a significant increase in SBP and DBPs in CBS+/- mice compared with other groups. CBS+/- mice aorta had lower response to phenylephrine and acetylcholine compared with other groups; however, CBS+/-/C3H mice response was improved. Dynamin-related protein 1 protein expression was significantly upregulated in CBS+/- mice, whereas C3H mice showed downregulation. In addition, CBS+/- mice showed increased oxidative stress, inflammation and decreased nitric oxide. These effects were normalized in CBS+/-/C3H mice. CONCLUSION: Our findings demonstrate the dominance of endothelial cell mitochondrial fission over mitochondrial fusion in hyperhomocysteinemia and oxidative stress. This may explain the endothelial cell loss and dysfunction that follows collagen deposition, which contributes to inward aorta remodeling in hypertension.

Hydrogen sulfide alleviates hypertensive kidney dysfunction through an epigenetic mechanism.

Hypertension is a major risk factor for chronic kidney disease (CKD), and renal inflammation is an integral part in this pathology. Hydrogen sulfide (H2S) has been shown to mitigate renal damage through reduction in blood pressure and ROS; however, the exact mechanisms are not clear. While several studies have underlined the role of epigenetics in renal inflammation and dysfunction, the mechanisms through which epigenetic regulators play a role in hypertension are not well defined. In this study, we sought to identify whether microRNAs are dysregulated in response to angiotensin II (ANG II)-induced hypertension in the kidney and whether a H2S donor, GYY4137, could reverse the microRNA alteration and kidney function. Wild-type (C57BL/6J) mice were treated without or with ANG II and GYY4137 for 4 wk. Blood pressure, renal blood flow, and resistive index (RI) were measured. MicroRNA microarrays were conducted and subsequent target prediction revealed genes associated with a proinflammatory response. ANG II treatment significantly increased blood pressure, decreased blood flow in the renal cortex, increased RI, and reduced renal function. These effects were ameliorated in mice treated with GYY4137. Microarray analysis revealed downregulation of miR-129 in ANG II-treated mice and upregulation after GYY4137 treatment. Quantitation of proteins involved in the inflammatory response and DNA methylation revealed upregulation of IL-17A and DNA methyltransferase 3a, whereas H2S production enzymes and anti-inflammatory IL-10 were reduced. Taken together, our data suggest that downregulation of miR-129 plays a significant role in ANG II-induced renal inflammation and functional outcomes and that GYY4137 improves renal function by reversing miR-129 expression.NEW & NOTEWORTHY We investigated epigenetic changes that occur in the hypertensive kidney and how H2S supplementation reverses adverse effects. Inflammation, aberrant methylation, and dysfunction were observed in the hypertensive kidney, and these effects were alleviated with H2S supplementation. We identify miR-129 as a potential regulator of blood pressure and H2S regulation.

Homocysteine and hydrogen sulfide in epigenetic, metabolic and microbiota related renovascular hypertension.

Over the past several years, hydrogen sulfide (H2S) has been shown to be an important player in a variety of physiological functions, including neuromodulation, vasodilation, oxidant regulation, inflammation, and angiogenesis. H2S is synthesized primarily through metabolic processes from the amino acid cysteine and homocysteine in various organ systems including neuronal, cardiovascular, gastrointestinal, and kidney. Derangement of cysteine and homocysteine metabolism and clearance, particularly in the renal vasculature, leads to H2S biosynthesis deregulation causing or contributing to existing high blood pressure. While a variety of environmental influences, such as diet can have an effect on H2S regulation and function, genetic factors, and more recently epigenetics, also have a vital role in H2S regulation and function, and therefore disease initiation and progression. In addition, new research into the role of gut microbiota in the development of hypertension has highlighted the need to further explore these microorganisms and how they influence the levels of H2S throughout the body and possibly exploiting microbiota for use of hypertension treatment. In this review, we summarize recent advances in the field of hypertension research emphasizing renal contribution and how H2S physiology can be exploited as a possible therapeutic strategy to ameliorate kidney dysfunction as well as to control blood pressure.

Embryonic Atrazine Exposure Elicits Alterations in Genes Associated with Neuroendocrine Function in Adult Male Zebrafish.

The developmental origins of health and disease (DOHaD) hypothesis states that exposure to environmental stressors early in life can elicit genome and epigenome changes resulting in an increased susceptibility of a disease state during adulthood. Atrazine, a common agricultural herbicide used throughout the Midwestern United States, frequently contaminates potable water supplies and is a suspected endocrine disrupting chemical. In our previous studies, zebrafish was exposed to 0, 0.3, 3, or 30 parts per billion (µg/l) atrazine through embryogenesis, rinsed, and allowed to mature to adulthood. A decrease in spawning was observed with morphological alterations in offspring. In addition, adult females displayed an increase in ovarian progesterone and follicular atresia, alterations in levels of a serotonin metabolite and serotonin turnover in brain tissue, and transcriptome changes in brain and ovarian tissue supporting neuroendocrine alterations. As reproductive dysfunction is also influenced by males, this study assessed testes histology, hormone levels, and transcriptomic profiles of testes and brain tissue in the adult males. The embryonic atrazine exposure resulted in no alterations in body or testes weight, gonadosomatic index, testes histology, or levels of 11-ketotestosterone or testosterone. To further investigate potential alterations, transcriptomic profiles of adult male testes and brain tissue was completed. This analysis demonstrated alterations in genes associated with abnormal cell and neuronal growth and morphology; molecular transport, quantity, and production of steroid hormones; and neurotransmission with an emphasis on the hypothalamus-pituitary-adrenal and hypothalamus-pituitary-thyroid axes. Overall, this data indicate future studies should focus on additional neuroendocrine endpoints to determine potential functional impairments.

Embryonic atrazine exposure alters zebrafish and human miRNAs associated with angiogenesis, cancer, and neurodevelopment.

MicroRNAs (miRNAs) are short, single-stranded RNA that regulate post-transcriptional control of mRNA translation. Knowledge on the role of these critical regulators in toxicological responses in increasing, but is still limited. Atrazine is a herbicide used throughout the Midwestern US that is reported to frequently contaminate potable water supplies above the maximum contaminant level of 3 parts per billion. Atrazine is a suspected endocrine disrupting chemical and studies have begun to investigate the genetic mechanisms of toxicity; however, studies investigating epigenetic mechanisms are limited. In this study both zebrafish and human miRNAs were significantly altered in response to an embryonic atrazine exposure of 0.3, 3, or 30 ppb in zebrafish. Altered miRNAs are known to play a role in angiogenesis, cancer, or neuronal development, differentiation, and maturation. Targeted analysis of altered human miRNAs with genes previously identified to be altered by atrazine exposure revealed several targets linked to cell cycle and cell signaling. Further analysis of hsa-miRNA-126-3p, which had altered expression in all three atrazine treatments at 72 hpf, revealed alterations also occurred at 60 hpf in the 30 ppb treatment group. Results from this study indicate miRNA deregulation in zebrafish and human miRNAs following an embryonic atrazine exposure in zebrafish.

An embryonic atrazine exposure results in reproductive dysfunction in adult zebrafish and morphological alterations in their offspring.

The herbicide atrazine, a suspected endocrine disrupting chemical (EDC), frequently contaminates potable water supplies. Studies suggest alterations in the neuroendocrine system along the hypothalamus-pituitary-gonadal axis; however, most studies address either developmental, pubertal, or adulthood exposures, with few investigations regarding a developmental origins hypothesis. In this study, zebrafish were exposed to 0, 0.3, 3, or 30 parts per billion (ppb) atrazine through embryogenesis and then allowed to mature with no additional chemical exposure. Reproductive function, histopathology, hormone levels, offspring morphology, and the ovarian transcriptome were assessed. Embryonic atrazine exposure resulted in a significant increase in progesterone levels in the 3 and 30 ppb groups. A significant decrease in spawning and a significant increase in follicular atresia in the 30 ppb group were observed. In offspring, a decrease in the head length to body ratio in the 30 ppb group, along with a significant increase in head width to body ratio in the 0.3 and 3 ppb groups occurred. Transcriptomic alterations involved genes associated with endocrine system development and function, tissue development, and behavior. This study provides evidence to support atrazine as an EDC causing reproductive dysfunction and molecular alterations in adults exposed only during embryogenesis and morphological alterations in their offspring.

Developmental origins of neurotransmitter and transcriptome alterations in adult female zebrafish exposed to atrazine during embryogenesis.

Atrazine is an herbicide applied to agricultural crops and is indicated to be an endocrine disruptor. Atrazine is frequently found to contaminate potable water supplies above the maximum contaminant level of 3µg/L as defined by the U.S. Environmental Protection Agency. The developmental origin of adult disease hypothesis suggests that toxicant exposure during development can increase the risk of certain diseases during adulthood. However, the molecular mechanisms underlying disease progression are still unknown. In this study, zebrafish embryos were exposed to 0, 0.3, 3, or 30µg/L atrazine throughout embryogenesis. Larvae were then allowed to mature under normal laboratory conditions with no further chemical treatment until 7 days post fertilization (dpf) or adulthood and neurotransmitter analysis completed. No significant alterations in neurotransmitter levels was observed at 7dpf or in adult males, but a significant decrease in 5-hydroxyindoleacetic acid (5-HIAA) and serotonin turnover was seen in adult female brain tissue. Transcriptomic analysis was completed on adult female brain tissue to identify molecular pathways underlying the observed neurological alterations. Altered expression of 1928, 89, and 435 genes in the females exposed to 0.3, 3, or 30µg/L atrazine during embryogenesis were identified, respectively. There was a high level of overlap between the biological processes and molecular pathways in which the altered genes were associated. Moreover, a subset of genes was down regulated throughout the serotonergic pathway. These results provide support of the developmental origins of neurological alterations observed in adult female zebrafish exposed to atrazine during embryogenesis.

Embryonic ionizing radiation exposure results in expression alterations of genes associated with cardiovascular and neurological development, function, and disease and modified cardiovascular function in zebrafish.

The relationship between ionizing radiation (IR) and carcinogenesis is long established, but recently the association between IR and other diseases is starting to be recognized. Currently, there is limited information on the genetic mechanisms governing the role of IR in non-cancer related adverse health effects and in regards to an early developmental exposure. In this study, zebrafish embryos were exposed to a range of IR doses (0, 1, 2, 5, 10 Gy) at 26 h post fertilization (hpf). No significant increase in mortality or hatching rate was observed, but a significant decrease in total larval length, head length, and eye diameter was observed in the 10 Gy dose. Transcriptomic analysis was conducted at 120 hpf to compare gene expression profiles between the control and highest IR dose at which no significant differences were observed in morphological measurements (5 Gy). 253 genes with well-established function or orthology to human genes were significantly altered. Gene ontology and molecular network analysis revealed enrichment of genes associated with cardiovascular and neurological development, function, and disease. Expression of a subset of genetic targets with an emphasis on those associated with the cardiovascular system was assessed using Quantitative PCR (qPCR) to confirm altered expression at 5 Gy and then to investigate alterations at lower doses (1 and 2 Gy). Strong correlation between microarray and qPCR expression values was observed, but zebrafish exposed to 1 or 2 Gy resulted in a significant expression alteration in only one of these genes (LIN7B). Moreover, heart rate was analyzed through 120 hpf following IR dosing at 26 hpf. A significant decrease in heart rate was observed at 10 Gy, while a significant increase in heart rate was observed at 1, 2, and 5 Gy. Overall these findings indicate IR exposure at doses below those that induce gross morphological changes alters heart rate and expression of genes associated with cardiovascular and neurological functions.