Project description:Background and Methods: High mercury (Hg) levels are a significant concern in many aquatic environments, and adverse effects of Hg exposure in fish can include altered behavior, reproduction, growth, and increased stress responses. In addition, human health concerns over Hg continue to warrant new research on its mechanisms of action. The objectives of this study were to investigate the impacts of Hg on the female largemouth bass (LMB) central nervous system transcriptome by conducting a laboratory injection experiment with 2.5 M-BM-5g/g body weight in addition to sampling LMB from an environment that contains high levels of Hg (St. MaryM-bM-^@M-^Ys River, Florida, USA). These animals were compared to LMB that inhibit river systems with lower Hg (Big Wekiva, Santa Fe and St. JohnM-bM-^@M-^Ys Rivers Florida, USA). A LMB 8 x 15 K microarray was used for hypothalamic (laboratory) and whole brain (field) transcriptomics analysis. Results: Mercury in the blood of LMB collected from St MaryM-bM-^@M-^Ys River was significantly elevated 3-fold (~ 0.1 ng Hg/ml blood) compared to LMB collected from the other three sites (~ 0.03 ng Hg/ml blood). In the hypothalamus of Hg injected LMB, there were 937 genes that showed differential expression (raw p = 0.05) but there were no probes that passed an FDR for multiple hypothesis correction. In contrast, in field collected LMB there were 2909 genes that that showed differential expression (raw p = 0.05) of which 38 were significant after correction for multiple hypothesis testing (FDR = 0.05). These included Hsp90 co-chaperone Cdc37, multicopper oxidase, mismatch repair protein Msh6, and 16 kDa heat shock protein A. Functional enrichment in whole brains of LMB collected from the field sites revealed that genes involved in the biological processes of protein folding and targeting, regulation of protein metabolic process, and protein degradation (i.e. ubiquitin cycle) were over-represented. Interestingly, gene set enrichment analysis identifed expression targets that were in common between the laboratory experiment (MeHg injected) and field collected LMB and these consisted of mainly homeobox transcription factors (PROP paired-like homeobox 1, LIM homeobox 3 and paired-like homeodomain 1 and 2). Lastly, prevalent themes emerged in the SNEA expression targets that included neuropeptide receptor signaling, steroid signaling, and structural components, such as beta-actin, integrins, and stress fibres. Conclusions: This study characterizes novel cell signaling pathways that underlie Hg toxicity in the teleostean central nervous system. field study with methyl mercury

Project description:Background and Methods: High mercury (Hg) levels are a significant concern in many aquatic environments, and adverse effects of Hg exposure in fish can include altered behavior, reproduction, growth, and increased stress responses. In addition, human health concerns over Hg continue to warrant new research on its mechanisms of action. The objectives of this study were to investigate the impacts of Hg on the female largemouth bass (LMB) central nervous system transcriptome by conducting a laboratory injection experiment with 2.5 µg/g body weight in addition to sampling LMB from an environment that contains high levels of Hg (St. Mary’s River, Florida, USA). These animals were compared to LMB that inhibit river systems with lower Hg (Big Wekiva, Santa Fe and St. John’s Rivers Florida, USA). A LMB 8 x 15 K microarray was used for hypothalamic (laboratory) and whole brain (field) transcriptomics analysis. Results: Mercury in the blood of LMB collected from St Mary’s River was significantly elevated 3-fold (~ 0.1 ng Hg/ml blood) compared to LMB collected from the other three sites (~ 0.03 ng Hg/ml blood). In the hypothalamus of Hg injected LMB, there were 937 genes that showed differential expression (raw p = 0.05) but there were no probes that passed an FDR for multiple hypothesis correction. In contrast, in field collected LMB there were 2909 genes that that showed differential expression (raw p = 0.05) of which 38 were significant after correction for multiple hypothesis testing (FDR = 0.05). These included Hsp90 co-chaperone Cdc37, multicopper oxidase, mismatch repair protein Msh6, and 16 kDa heat shock protein A. Functional enrichment in whole brains of LMB collected from the field sites revealed that genes involved in the biological processes of protein folding and targeting, regulation of protein metabolic process, and protein degradation (i.e. ubiquitin cycle) were over-represented. Interestingly, gene set enrichment analysis identifed expression targets that were in common between the laboratory experiment (MeHg injected) and field collected LMB and these consisted of mainly homeobox transcription factors (PROP paired-like homeobox 1, LIM homeobox 3 and paired-like homeodomain 1 and 2). Lastly, prevalent themes emerged in the SNEA expression targets that included neuropeptide receptor signaling, steroid signaling, and structural components, such as beta-actin, integrins, and stress fibres. Conclusions: This study characterizes novel cell signaling pathways that underlie Hg toxicity in the teleostean central nervous system.

Project description:Lake Apopka (Fl, USA) experienced heavy uses of organochlorine pesticides (OCPs) in the 1950s-1970 during an intense agricultural period. As a result, the region remains heavily contaminated and was declared a Superfund site by the US Environmental Protection Agency. Aquatic organisms inhabit the freshwater systems in and around Lake Apopka, however the impacts of sub chronic exposure to OCPs in this natural environment are not known. The objectives of this study were to determine the effects of the contamination on the reproductive axis of largemouth bass (LMB) (Micropterus salmoides). In October (2007), healthy LMB were stocked into natural mesocosms and remained in mesocosms before being sampled in January 2008 (at early oogenesis). Additional LMB were placed into mesocosms for 2 months in February (2008) and sampled in April (2008) (oocyte maturation). LMB placed in these mesocosms for four months had a 2-20X higher contaminant load for OCPs (e.g. DDE, dieldrin, methoxychlor) than LMB collected from reference sites. Gonadosomatic index for LMB collected in April from the mesocosms were not different that LMB from reference sites. Vitellogenin levels in LMB collected from the mesocosms in January and April were not significantly different than fish collected from Welaka in late vitellogenin or early maturation respectively. Steroids were depressed in mesocosm fish. Microarray analysis revealed that the expression profiles of genes in the LMB ovary were unique compared to LMB collected form reference sites. Transcripts that showed altered abundance in LMB from the mesocosms were insulin-like growth factor I and steroidogenic acute regulatory protein. Interestingly, differentially expressed transcripts showed a significant and positive correlation for LMB sampled in January and April despite the 3 month period in between samplings. Sub-network enrichment analysis for cellular processes showed that retinoic acid metabolism and germ-cell development were decreased in mesocosm-exposed fish but processes such as vitellogenesis, amino acid catabolism, granulosa cell function, vitamin D metabolism, and hormone biosynthesis were increased in mesocosm-exposed fish. These data suggest that (1) LMB from the mesocosms are exhibiting unique gene profiles that may impair normal reproduction and that (2) microarray analysis in the field can provide site specific information by discriminating LMB from reference and polluted sites. 12 microarrays on LMB collected from the wild in January. These include St John River (n=4), DeLeon Springs (n = 4), and animals placed in Apopka mesocosms (n=4).

Project description:Lake Apopka (Fl, USA) experienced heavy uses of organochlorine pesticides (OCPs) in the 1950s-1970 during an intense agricultural period. As a result, the region remains heavily contaminated and was declared a Superfund site by the US Environmental Protection Agency. Aquatic organisms inhabit the freshwater systems in and around Lake Apopka, however the impacts of sub chronic exposure to OCPs in this natural environment are not known. The objectives of this study were to determine the effects of the contamination on the reproductive axis of largemouth bass (LMB) (Micropterus salmoides). In October (2007), healthy LMB were stocked into natural mesocosms and remained in mesocosms before being sampled in January 2008 (at early oogenesis). Additional LMB were placed into mesocosms for 2 months in February (2008) and sampled in April (2008) (oocyte maturation). LMB placed in these mesocosms for four months had a 2-20X higher contaminant load for OCPs (e.g. DDE, dieldrin, methoxychlor) than LMB collected from reference sites. Gonadosomatic index for LMB collected in April from the mesocosms were not different that LMB from reference sites. Vitellogenin levels in LMB collected from the mesocosms in January and April were not significantly different than fish collected from Welaka in late vitellogenin or early maturation respectively. Steroids were depressed in mesocosm fish. Microarray analysis revealed that the expression profiles of genes in the LMB ovary were unique compared to LMB collected form reference sites. Transcripts that showed altered abundance in LMB from the mesocosms were insulin-like growth factor I and steroidogenic acute regulatory protein. Interestingly, differentially expressed transcripts showed a significant and positive correlation for LMB sampled in January and April despite the 3 month period in between samplings. Sub-network enrichment analysis for cellular processes showed that retinoic acid metabolism and germ-cell development were decreased in mesocosm-exposed fish but processes such as vitellogenesis, amino acid catabolism, granulosa cell function, vitamin D metabolism, and hormone biosynthesis were increased in mesocosm-exposed fish. These data suggest that (1) LMB from the mesocosms are exhibiting unique gene profiles that may impair normal reproduction and that (2) microarray analysis in the field can provide site specific information by discriminating LMB from reference and polluted sites. 8 microarrays on LMB collected from the wild in April. These include St John River (n=4) and animals placed in Apopka mesocosms (n=4).

Project description:Lake Apopka (Fl, USA) experienced heavy uses of organochlorine pesticides (OCPs) in the 1950s-1970 during an intense agricultural period. As a result, the region remains heavily contaminated and was declared a Superfund site by the US Environmental Protection Agency. Aquatic organisms inhabit the freshwater systems in and around Lake Apopka, however the impacts of sub chronic exposure to OCPs in this natural environment are not known. The objectives of this study were to determine the effects of the contamination on the reproductive axis of largemouth bass (LMB) (Micropterus salmoides). In October (2007), healthy LMB were stocked into natural mesocosms and remained in mesocosms before being sampled in January 2008 (at early oogenesis). Additional LMB were placed into mesocosms for 2 months in February (2008) and sampled in April (2008) (oocyte maturation). LMB placed in these mesocosms for four months had a 2-20X higher contaminant load for OCPs (e.g. DDE, dieldrin, methoxychlor) than LMB collected from reference sites. Gonadosomatic index for LMB collected in April from the mesocosms were not different that LMB from reference sites. Vitellogenin levels in LMB collected from the mesocosms in January and April were not significantly different than fish collected from Welaka in late vitellogenin or early maturation respectively. Steroids were depressed in mesocosm fish. Microarray analysis revealed that the expression profiles of genes in the LMB ovary were unique compared to LMB collected form reference sites. Transcripts that showed altered abundance in LMB from the mesocosms were insulin-like growth factor I and steroidogenic acute regulatory protein. Interestingly, differentially expressed transcripts showed a significant and positive correlation for LMB sampled in January and April despite the 3 month period in between samplings. Sub-network enrichment analysis for cellular processes showed that retinoic acid metabolism and germ-cell development were decreased in mesocosm-exposed fish but processes such as vitellogenesis, amino acid catabolism, granulosa cell function, vitamin D metabolism, and hormone biosynthesis were increased in mesocosm-exposed fish. These data suggest that (1) LMB from the mesocosms are exhibiting unique gene profiles that may impair normal reproduction and that (2) microarray analysis in the field can provide site specific information by discriminating LMB from reference and polluted sites.

Project description:Today, many contaminants of emerging concern can be measured in waters across the United States, including the tributaries of the Great Lakes. However, just because the chemicals can be measured does not mean that they necessarily result in harm to fish and other aquatic species. Complicating risk assessment in these waters is the fact that aquatic species are encountering the chemicals as mixtures, which may have additive or synergistic risks that cannot be calculated using single chemical hazard and concentration-response information. We developed an in vitro effects-based screening approach to help us predict potential liver toxicity and cancer in aquatic organisms using water from specific Great Lakes tributaries: St. Louis River (MN), Bad River (WI), Fox River (WI), Manitowoc River (WI), Milwaukee River (WI), Indiana Harbor Canal (IN), St. Joseph River (MI), Grand River (MI), Clinton River (MI), River Rouge (MI), Maumee River (OH), Vermilion River (OH), Cuyahoga River (OH), Genesee River (NY), and Oswego River (NY). We exposed HepG2 cells for 48hrs to medium spiked with either field collected water (final concentration of environmental samples in the exposure medium were 75% of the field-collected water samples) or purified water. Using a deep neural network we clustered our collection sites from each tributary based on water chemistry. We also performed high throughput transcriptomics on the RNA obtained from the HepG2 cells. We used the transcriptomics data with our Bayesian Inferene for Sustance and Chemical Toxicity (BISCT) Bayesian Network for Steatosis to predict the probability of the field samples yielding a gene expression pattern consistent with predicting steatosis as an outcome. Surprisingly, we found that the probability of steatosis did not correspond to the surface water chemistry clustering. Our analysis suggests that chemical signatures are not informative in predicting biological effects. Furthermore, recent reports published after we obtained our samples, suggest that chemical levels in the sediment may be more relevant for predicting potential biological effects in the fish species developing tumors in the Great Lakes basin.

Dataset Information

Characterizing gene regulatory networks in the brain of largemouth bass inhabiting rivers containing high levels of methyl-mercury (lab study)

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