Nutrient limitation of phytoplankton in three tributaries of Chesapeake Bay: Detecting responses following nutrient reductions.

Many coastal ecosystems suffer from eutrophication, algal blooms, and dead zones due to excessive anthropogenic inputs of nitrogen (N) and phosphorus (P). This has led to regional restoration efforts that focus on managing watershed loads of N and P. In Chesapeake Bay, the largest estuary in the United States, dual nutrient reductions of N and P have been pursued since the 1980s. However, it remains unclear whether nutrient limitation - an indicator of restriction of algal growth by supplies of N and P - has changed in the tributaries of Chesapeake Bay following decades of reduction efforts. Toward that end, we analyzed historical data from nutrient-addition bioassay experiments and data from the Chesapeake Bay long-term water-quality monitoring program for six stations in three tidal tributaries (i.e., Patuxent, Potomac, and Choptank Rivers). Classification and regression tree (CART) models were developed using concurrent collections of water-quality parameters for each bioassay monitoring location during 1990-2003, which satisfactorily predicted the bioassay-based measures of nutrient limitation (classification accuracy = 96%). Predictions from the CART models using water-quality monitoring data showed enhanced nutrient limitation over the period of 1985-2020 at four of the six stations, including the downstream station in each of these three tributaries. These results indicate detectable, long-term water-quality improvements in the tidal tributaries. Overall, this research provides a new analytical tool for detecting signs of ecosystem recovery following nutrient reductions. More broadly, the approach can be adapted to other waterbodies with long-term bioassays and water-quality data sets to detect ecosystem recovery.

Nutrient limitation of phytoplankton in Chesapeake Bay: Development of an empirical approach for water-quality management.

Understanding the temporal and spatial roles of nutrient limitation on phytoplankton growth is necessary for developing successful management strategies. Chesapeake Bay has well-documented seasonal and spatial variations in nutrient limitation, but it remains unknown whether these patterns of nutrient limitation have changed in response to nutrient management efforts. We analyzed historical data from nutrient bioassay experiments (1992-2002) and data from long-term, fixed-site water-quality monitoring program (1990-2017) to develop empirical approaches for predicting nutrient limitation in the surface waters of the mainstem Bay. Results from classification and regression trees (CART) matched the seasonal and spatial patterns of bioassay-based nutrient limitation in the 1992-2002 period much better than two simpler, non-statistical approaches. An ensemble approach of three selected CART models satisfactorily reproduced the bioassay-based results (classification rate = 99%). This empirical approach can be used to characterize nutrient limitation from long-term water-quality monitoring data on much broader geographic and temporal scales than would be feasible using bioassays, providing a new tool for informing water-quality management. Results from our application of the approach to 21 tidal monitoring stations for the period of 2007-2017 showed modest changes in nutrient limitation patterns, with expanded areas of nitrogen-limitation and contracted areas of nutrient saturation (i.e., not limited by nitrogen or phosphorus). These changes imply that long-term reductions in nitrogen load have led to expanded areas with nutrient-limited phytoplankton growth in the Bay, reflecting long-term water-quality improvements in the context of nutrient enrichment. However, nutrient limitation patterns remain unchanged in the majority of the mainstem, suggesting that nutrient loads should be further reduced to achieve a less nutrient-saturated ecosystem.

Fishing for teratogens: a consortium effort for a harmonized zebrafish developmental toxicology assay.

A consortium of biopharmaceutical companies previously developed an optimized Zebrafish developmental toxicity assay (ZEDTA) where chorionated embryos were exposed to non-proprietary test compounds from 5 to 6 h post fertilization and assessed for morphological integrity at 5 days post fertilization. With the original 20 test compounds, this achieved an overall predictive value for teratogenicity of 88% of mammalian in vivo outcome [Gustafson, A. L., Stedman, D. B., Ball, J., Hillegass, J. M., Flood, A., Zhang, C. X., Panzica-Kelly, J., Cao, J., Coburn, A., Enright, B. P., et al. (2012). Interlaboratory assessment of a harmonized Zebrafish developmental toxicology assay-Progress report on phase I. Reprod. Toxicol. 33, 155-164]. In the second phase of this project, 38 proprietary pharmaceutical compounds from four consortium members were evaluated in two laboratories using the optimized method using either pond-derived or cultivated-strain wild-type Zebrafish embryos at concentrations up to 100µM. Embryo uptake of all compounds was assessed using liquid chromatography-tandem mass spectrometry. Twenty eight of 38 compounds had a confirmed embryo uptake of >5%, and with these compounds the ZEDTA achieved an overall predictive value of 82% and 65% at the two respective laboratories. When low-uptake compounds (≤ 5%) were retested with logarithmic concentrations up to 1000µM, the overall predictivity across all 38 compounds was 79% and 62% respectively, with the first laboratory achieving 74% sensitivity (teratogen detection) and 82% specificity (non-teratogen detection) and the second laboratory achieving 63% sensitivity (teratogen detection) and 62% specificity (non-teratogen detection). Subsequent data analyses showed that technical differences rather than strain differences were the primary contributor to interlaboratory differences in predictivity. Based on these results, the ZEDTA harmonized methodology is currently being used for compound assessment at lead optimization stage of development by 4/5 of the consortium companies.

Comparative effects of sodium channel blockers in short term rat whole embryo culture.

This study was undertaken to examine the effect on the rat embryonic heart of two experimental drugs (AZA and AZB) which are known to block the sodium channel Nav1.5, the hERG potassium channel and the l-type calcium channel. The sodium channel blockers bupivacaine, lidocaine, and the l-type calcium channel blocker nifedipine were used as reference substances. The experimental model was the gestational day (GD) 13 rat embryo cultured in vitro. In this model the embryonic heart activity can be directly observed, recorded and analyzed using computer assisted image analysis as it responds to the addition of test drugs. The effect on the heart was studied for a range of concentrations and for a duration up to 3h. The results showed that AZA and AZB caused a concentration-dependent bradycardia of the embryonic heart and at high concentrations heart block. These effects were reversible on washout. In terms of potency to cause bradycardia the compounds were ranked AZB>bupivacaine>AZA>lidocaine>nifedipine. Comparison with results from previous studies with more specific ion channel blockers suggests that the primary effect of AZA and AZB was sodium channel blockage. The study shows that the short-term rat whole embryo culture (WEC) is a suitable system to detect substances hazardous to the embryonic heart.

Relating nutrient and herbicide fate with landscape features and characteristics of 15 subwatersheds in the Choptank River watershed.

Excess nutrients and agrochemicals from non-point sources contribute to water quality impairment in the Chesapeake Bay watershed and their loading rates are related to land use, agricultural practices, hydrology, and pollutant fate and transport processes. In this study, monthly baseflow stream samples from 15 agricultural subwatersheds of the Choptank River in Maryland USA (2005 to 2007) were characterized for nutrients, herbicides, and herbicide transformation products. High-resolution digital maps of land use and forested wetlands were derived from remote sensing imagery. Examination of landscape metrics and water quality data, partitioned according to hydrogeomorphic class, provided insight into the fate, delivery, and transport mechanisms associated with agricultural pollutants. Mean Nitrate-N concentrations (4.9 mg/L) were correlated positively with percent agriculture (R(2)=0.56) and negatively with percent forest (R(2)=0.60). Concentrations were greater (p=0.0001) in the well-drained upland (WDU) hydrogeomorphic region than in poorly drained upland (PDU), reflecting increased denitrification and reduced agricultural land use intensity in the PDU landscape due to the prevalence of hydric soils. Atrazine and metolachlor concentrations (mean 0.29 µg/L and 0.19 µg/L) were also greater (p=0.0001) in WDU subwatersheds than in PDU subwatersheds. Springtime herbicide concentrations exhibited a strong, positive correlation (R(2)=0.90) with percent forest in the WDU subwatersheds but not in the PDU subwatersheds. In addition, forested riparian stream buffers in the WDU were more prevalent than in the PDU where forested patches are typically not located near streams, suggesting an alternative delivery mechanism whereby volatilized herbicides are captured by the riparian forest canopy and subsequently washed off during rainfall. Orthophosphate, CIAT (6-chloro-N-(1-methylethyl)-1,3,5-triazine-2,4-diamine), CEAT (6-chloro-N-ethyl-1,3,5-triazine-2,4-diamine), and MESA (2-[(2-ethyl-6-methylphenyl) (2-methoxy-1-methylethyl)amino]-2-oxoethanesulfonic acid) were also analyzed. These findings will assist efforts in targeting implementation of conservation practices to the most environmentally-critical areas within watersheds to achieve water quality improvements in a cost-effective manner.

Short-time gene expression response to valproic acid and valproic acid analogs in mouse embryonic stem cells.

Prediction of developmental toxicity in vitro could be based on short-time toxicogenomic endpoints in embryo-derived cell lines. Microarray studies in P19 mouse embryocarcinoma cells and mouse embryos have indicated that valproic acid (VPA), an inducer of neural tube defects, deregulates the expression of many genes, including those critically involved in neural tube development. In this study, we exposed undifferentiated R1 mouse embryonic stem cells to VPA and VPA analogs for 6 h and used CodeLink whole-genome expression microarrays to define VPA-responsive genes correlating with teratogenicity. Compared with the nonteratogenic analog 2-ethyl-4-methylpentanoic acid, VPA and the teratogenic VPA analog (S)-2-pentyl-4-pentynoic acid deregulated a much larger number of genes. Five genes (of ∼2500 array probes correlating with the separation) were sufficient to effectively separate teratogens from nonteratogens. A large fraction of the target genes correlating with teratogenicity are functionally related to embryonic development and morphogenesis, including neural tube formation and closure. Similar responses in R1 were found for most genes previously identified as VPA responsive in P19 and embryos. A subset of target genes was evaluated as candidate markers predictive of potential teratogenicity against a range of known teratogens using TaqMan expression arrays. These marker genes showed a positive predictive value for the teratogens butyrate and trichostatin A, which like VPA and (S)-2-pentyl-4-pentynoic acid are known histone deacetylase (HDAC) inhibitors but not for compounds that are likely to act by other mechanisms. This indicates that HDAC inhibition may be a major mechanism by which VPA induces gene deregulation and possibly teratogenicity.

Early transcriptional responses in mouse embryos as a basis for selection of molecular markers predictive of valproic acid teratogenicity.

Cell-based in vitro assays would potentially reduce animal testing in preclinical drug development. Mouse embryos exposed to the teratogenic drug valproic acid (VPA) in utero for 1.5, 3 or 6h on gestational day 8 were analyzed using microarrays. Significant effects on gene expression were observed already at 1.5h, and 85 probes were deregulated across all time points. To find transcriptional markers of VPA-induced developmental toxicity, the in vivo data were compared to previous in vitro data on embryonal carcinoma P19 cells exposed to VPA for 1.5, 6 or 24h. Maximal concordance between embryos and cells was at the 6-h time points, with 163 genes showing similar deregulation. Developmentally important Gene Ontology terms, such as "organ morphogenesis" and "tube development" were overrepresented among putative VPA target genes. The genes Gja1, Hap1, Sall2, H1f0,Cyp26a1, Fgf15, Otx2, and Lin7b emerged as candidate in vitro markers of potential VPA-induced teratogenicity.

Pollutant fate and spatio-temporal variability in the choptank river estuary: factors influencing water quality.

Restoration of the Chesapeake Bay, the largest estuary in the United States, is a national priority. Documentation of progress of this restoration effort is needed. A study was conducted to examine water quality in the Choptank River estuary, a tributary of the Chesapeake Bay that since 1998 has been classified as impaired waters under the Federal Clean Water Act. Multiple water quality parameters (salinity, temperature, dissolved oxygen, chlorophyll a) and analyte concentrations (nutrients, herbicide and herbicide degradation products, arsenic, and copper) were measured at seven sampling stations in the Choptank River estuary. Samples were collected under base flow conditions in the basin on thirteen dates between March 2005 and April 2008. As commonly observed, results indicate that agriculture is a primary source of nitrate in the estuary and that both agriculture and wastewater treatment plants are important sources of phosphorus. Concentrations of copper in the lower estuary consistently exceeded both chronic and acute water quality criteria, possibly due to use of copper in antifouling boat paint. Concentrations of copper in the upstream watersheds were low, indicating that agriculture is not a significant source of copper loading to the estuary. Concentrations of herbicides (atrazine, simazine, and metolachlor) peaked during early-summer, indicating a rapid surface-transport delivery pathway from agricultural areas, while their degradation products (CIAT, CEAT, MESA, and MOA) appeared to be delivered via groundwater transport. Some in-river processing of CEAT occurred, whereas MESA was conservative. Observed concentrations of herbicide residues did not approach established levels of concern for aquatic organisms. Results of this study highlight the importance of continued implementation of best management practices to improve water quality in the estuary. This work provides a baseline against which to compare future changes in water quality and may be used to design future monitoring programs needed to assess restoration strategy efficacy.

Valproic acid-induced deregulation in vitro of genes associated in vivo with neural tube defects.

The utility of an in vitro system to search for molecular targets and markers of developmental toxicity was explored, using microarrays to detect genes susceptible to deregulation by the teratogen valproic acid (VPA) in the pluripotent mouse embryonal carcinoma cell line P19. Total RNA extracted from P19 cells cultured in the absence or presence of 1, 2.5, or 10mM VPA for 1.5, 6, or 24 h was subjected to replicated microarray analysis, using CodeLink UniSet I Mouse 20K Expression Bioarrays. A moderated F-test revealed a significant VPA response for 2972 (p < 10(-3)) array probes (19.4% of the filtered gene list), 421 of which were significant across all time points. In a core subset of VPA target genes whose expression was downregulated (68 genes) or upregulated (125 genes) with high probability (p < 10(-7)) after both 1.5 and 6 h of VPA exposure, there was a significant enrichment of the biological process Gene Ontology term transcriptional regulation among downregulated genes, and apoptosis among upregulated, and two of the downregulated genes (Folr1 and Gtf2i) have a knockout phenotype comprising exencephaly, the major malformation induced by VPA in mice. The VPA-induced gene expression response in P19 cells indicated that approximately 30% of the approximately 200 genes known from genetic mouse models to be associated with neural tube defects may be potential VPA targets, suggestive of a combined deregulation of multiple genes as a possible mechanism of VPA teratogenicity. Gene expression responses related to other known effects of VPA (histone deacetylase inhibition, G(1)-phase cell cycle arrest, induction of apoptosis) were also identified. This study indicates that toxicogenomic responses to a teratogenic compound in vitro may correlate with known in vitro and in vivo effects, and that short-time (< or =6 h) exposures in such an in vitro system could provide a useful component in mechanistic studies and screening tests in developmental toxicology.

Molecular targets and early response biomarkers for the prediction of developmental toxicity in vitro.

There is an urgent need for new in vitro methods to predict the potential developmental toxicity of candidate drugs in the early lead identification and optimisation process. This would lead to a reduction in the total number of animals required in full-scale developmental toxicology studies, and would improve the efficiency of drug development. However, suitable in vitro systems permitting robust high-throughput screening for this purpose, for the most part, remain to be designed. An understanding of the mechanisms involved in developmental toxicity may be essential for the validation of in vitro tests. Early response biomarkers - even a single one - could contribute to reducing assay time and facilitating automation. The use of toxicogenomics approaches to study in vitro and in vivo models in parallel may be a powerful tool in defining such mechanisms of action and the molecular targets of toxicity, and also for use in finding possible biomarkers of early response. Using valproic acid as a model substance, the use of DNA microarrays to identify teratogen-responsive genes in cell models is discussed. It is concluded that gene expression in P19 mouse embryocarcinoma cells represents a potentially suitable assay system, which could be readily used in a tiered testing system for developmental toxicity testing.

Cadmium-induced gene expression changes in the mouse embryo, and the influence of pretreatment with zinc.

Cadmium (Cd) administered to female C57BL/6 mice on gestation day 8 induces a high incidence of anterior neural tube defects (exencephaly). This adverse effect can be attenuated by maternal pretreatment with zinc (Zn). In this study we used replicated microarray analysis and real-time PCR to investigate gene expression changes induced in the embryo 5 and 10h after maternal Cd exposure in the absence or presence of Zn pretreatment. We report nine genes with a transcriptional response induced by Cd, none of which was influenced by Zn pretreatment, and two genes induced only by combined maternal Cd exposure and Zn pretreatment. We discuss the results in relation to the possibility that Cd is largely excluded from the embryo, that the teratogenic effects of Cd may be secondary to toxicity in extraembryonic tissues, and that the primary protective role of Zn may not be to reverse Cd-induced transcription in the embryo.

Valproic acid teratogenicity: a toxicogenomics approach.

Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell-based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo.

Disturbing endoderm signaling to anterior neural plate of vertebrates by the teratogen cadmium.

Cadmium accumulation in the mouse gut endoderm occurs until the closure of the vitelline duct (day 9 post-coitus; p.c.), producing anterior neural tube defects (NTD). The anterior part of the primitive endoderm, designated as the primary signaling center for anterior patterning, expresses several transcription factors of importance for head formation. Here, we studied the expression levels of some of these transcription factors (Hesx1, HNF3beta, Cerl, Otx2 and Sox2), and cell death induced after single cadmium administration to dams on days 7, 8 and 9 p.c. Stage specific down-regulation of Hesx1, Cerl, and Sox2, and an up-regulation of HNF3beta were observed. No effect was seen in Otx2 expression levels. Cell death was increased in the neuroepithelium of the cranial neural folds, and in areas where neural crest cells migrate, but not in the gut endoderm. It is proposed that cadmium-induced NTD is due to interference with head-inductive signals from the endoderm to the adjacent layers.

Cadmium-induced changes in apoptotic gene expression levels and DNA damage in mouse embryos are blocked by zinc.

Cadmium is a potent teratogen in laboratory animals, causing exencephaly when administered at early stages of development. Due to its heterogenicity with respect to molecular targets, the mechanisms behind cadmium toxicity are not well understood. In the present study, C57BL/6 pregnant mice were treated with saline, cadmium, or zinc plus cadmium at 8 days post-coitus and studied 24 h after exposure. Cadmium induced significant DNA damage in the embryonic cells. Cadmium also induced embryonic growth retardation, as well as a significant upregulation of p53, p21, and Bax transcription levels. At the same time, there was a downregulation of Bcl-2, shifting the equilibrium Bcl-2/Bax toward the apoptotic pathway. There was an increase in apoptotically stained cells in the cadmium-treated embryos, and pro-caspase-3 was significantly activated. Zinc pretreatment maintained DNA damage at the control levels. It also prevented cadmium-induced effects on the expression levels of p53 and p21. The cadmium-induced decrease in Bcl-2 was inhibited, whereas the Bax levels were maintained closer to the control values. The Bad transcripts did not change at any experimental condition. Morphologically, zinc could maintain the embryological development, where apoptotic areas were as in the controls, and decrease por-caspase-3 activation. In summary, cadmium administered to pregnant mice increased primary DNA damage and activated the apoptotic pathway. These effects could be ameliorated by zinc pretreatment, and, because of that, it is possible that the mechanisms of cadmium-induced teratogenicity are related to zinc metabolism.