Wastewater Surveillance Data as a Complement to Emergency Department Visit Data for Tracking Incidence of Influenza A and Respiratory Syncytial Virus - Wisconsin, August 2022-March 2023.

Wastewater surveillance has been used to assist public health authorities in tracking local transmission of SARS-CoV-2. The usefulness of wastewater surveillance to track community spread of other respiratory pathogens, including influenza virus and respiratory syncytial virus (RSV), is less clear. During the 2022-23 respiratory diseases season, concentrations of influenza A virus and RSV in wastewater samples in three major Wisconsin cities were compared with emergency department (ED) visits associated with these pathogens. In all three cities, higher concentrations of influenza A virus and RSV in wastewater were associated with higher numbers of associated ED visits (Kendall's tau range = 0.50-0.63 for influenza-associated illness and 0.30-0.49 for RSV-associated illness). Detections of both influenza A virus and RSV in wastewater often preceded a rise in associated ED visits for each pathogen, and virus material remained detectable in wastewater for up to 3 months after pathogen-specific ED visits declined. These results demonstrate that wastewater surveillance has the potential to complement conventional methods of influenza and RSV surveillance, detecting viral signals earlier and for a longer duration than do clinical data. Continued use of wastewater surveillance as a supplement to established surveillance systems such as ED visits might improve local understanding and response to seasonal respiratory virus outbreaks.

Emissions from a flex fuel GDI vehicle operating on ethanol fuels show marked contrasts in chemical, physical and toxicological characteristics as a function of ethanol content.

This study assessed the gaseous and particulate emissions, as well as the toxicological properties of particulate matter (PM) from a flex fuel vehicle equipped with a wall-guided gasoline direct injection engine over triplicates cold-start and hot-start LA92 cycles. The vehicle was operated on a Tier 3 E10 fuel, an E10 fuel with higher levels of aromatics than the Tier 3 E10, an E30, and an E78 blend. Total hydrocarbon (THC), non-methane hydrocarbon (NMHC), carbon monoxide (CO), particulate emissions, and gaseous toxics (of benzene, toluene, ethylbenzene, xylenes (BTEX), and 1,3-butadiene) reduced for E30 and E78 blends compared to both E10 fuels. Formaldehyde and acetaldehyde emissions substantially increased with the higher ethanol blends. The high aromatic E10 fuel increased the emissions of THC, NMHC, particulates, and BTEX compared to the Tier 3 E10 fuel and the higher ethanol blends, as well as showed higher concentrations of accumulation mode particles. The GDI PM did not exhibit any measurable mutagenicity at the PM concentrations tested. Cytotoxicity varied only within a small range and concentrations of PM, eliciting a cytotoxic response similar to those by ambient aerosol. The outcomes of our two measures of PM oxidative potential (macrophage ROS and DTT) were significantly correlated, with the E78 blend exhibiting the least oxidative potential and the E30 the greatest. Gene expression analysis at both the mRNA and protein level indicates that there is the potential for GDI PM emissions to contribute to inflammation and etiology of disease such as asthma, and in contrast to the ROS and DTT outcomes, the E78 fuel PM exhibited the greatest potential to elicit pro-inflammatory cytokine (TNFα) production. Overall, the trends in toxicity emission rates (activity/mi) across the ethanol blends was driven primarily by PM mass emission rate contrasts and only secondarily by the differences in intrinsic toxicity of the PM.

Chemical composition and health risk indices associated with size-resolved particulate matter in Pearl River Delta (PRD) region, China.

Size-resolved particulate matter (PM) was collected at the Heshan Super-Station in the Pearl River Delta (PRD) region, China, to evaluate their chemical characteristics and potential health risks. The chemical mass closures illustrate that the dominant fraction in coarse (2.5 µm < Dp < 10 µm) PM was dust, while organic matter made up a substantial portion of both fine (0.1 < Dp < 2.5 µm) and ultra-fine (Dp < 0.10 µm) PM fractions. The size distribution of most PM components varied substantially. PM, NO3-, K+, Cl-, Na+ and most of the transition/redox metals displayed bimodal size distributions with the dominant peak at 0.32-0.56 µm plus a small peak at 1.8-3.2 µm. In contrast, unimodal size distributions were found for the rest of the species, such as water-soluble organic carbon (WSOC), NH4+, and SO42- and the majority of oxyanion metals with a single peak at 0.32-0.56 µm, and Mg2+, Ca2+, and dust tracer elements which mainly accumulated in coarse particles. Based on the crustal enrichment factor (CEF) analysis, Cd, Zn, Sb, Sn, As, Pb, Mo, Cu, and Cr primarily originated from anthropogenic activities, while Ti in all size fractions and Sr, Mg, Na, and Fe in fine and ultra-fine particles were mainly emitted from natural sources. The potential health risk assessment of trace metals was performed using the hazard quotient (HQ) and excess lifetime cancer risk (ELCR) indices. Although the adverse health effects of most metals were limited, significant potential carcinogenic risks were found for As and Cr in both fine and coarse particle size fractions, which contributed more than 95% of total ELCR. Therefore, considering that these two elements were mainly emitted from industrial processes, improvements in air quality and health risks in the PRD region can be largely achieved by reducing the emissions of local industrial sources.

Seasonal variations in the oxidative stress and inflammatory potential of PM2.5 in Tehran using an alveolar macrophage model; The role of chemical composition and sources.

The current study was designed to assess the association between temporal variations in urban PM2.5 chemical composition, sources, and the oxidative stress and inflammatory response in an alveolar macrophage (AM) model. A year-long sampling campaign collected PM2.5 samples at the Sharif University in Tehran, Iran. PM-induced reactive oxygen species (ROS) production was measured both with an acellular dithiothreitol consumption assay (DTT-ROS; ranged from 2.1 to 9.3 nmoles min-1 m-3) and an in vitro macrophage-mediated ROS production assay (AM-ROS; ranged from 125 to 1213 µg Zymosan equivalents m-3). The production of tumor necrosis factor alpha (TNF-α; ranged from ~60 to 518 pg TNF-α m-3) was quantified as a marker of the inflammatory potential of the PM. PM-induced DTT-ROS and AM-ROS were substantially higher for the colder months' PM (1.5-fold & 3-fold, respectively) compared with warm season. Vehicular emission tracers, aliphatic diacids, and hopanes exhibited moderate correlation with ROS measures. TNF-α secretion exhibited a markedly different pattern than ROS activity with a 2-fold increase in the warm months compared to the rest of the year. Gasoline vehicles and residual oil combustion were moderately associated with both ROS measures (R ≥ 0.67, p < 0.05), while diesel vehicles exhibited a strong correlation with secreted TNF-α in the cold season (R = 0.89, p < 0.05). mRNA expression of fourteen genes including antioxidant response and pro-inflammatory markers were found to be differentially modulated in our AM model. HMOX1, an antioxidant response gene, was up-regulated throughout the year. Pro-inflammatory genes (e.g. TNF-α and IL1ß) were down-regulated in the cold season and displayed moderate to weak correlation with crustal elements (R > 0.5, p < 0.05). AM-ROS activity showed an inverse relationship with genes including SOD2, TNF, IL1ß and IL6 (R ≥ -0.66, p < 0.01). Our findings indicate that Tehran's PM2.5 has the potential to induce oxidative stress and inflammation responses in vitro. In the current study, these responses included NRF2, NF-κB and MAPK pathways.

Physical, chemical, and toxicological characteristics of particulate emissions from current technology gasoline direct injection vehicles.

We assessed the physical, chemical and toxicological characteristics of particulate emissions from four light-duty gasoline direct injection vehicles when operated over the LA92 driving cycle. Our results showed that particle mass and number emissions increased markedly during accelerations. For three of the four vehicles tested, particulate matter (PM) mass and particle number emissions were markedly higher during cold-start and the first few accelerations following the cold-start period than during the hot running and hot-start segments of the LA92 cycle. For one vehicle (which had the highest emissions overall) the hot-start and cold-start PM emissions were similar. Black carbon emissions were also much higher during the cold-start conditions, indicating severe fuel wetting leading to slow evaporation and pool burning, and subsequent soot formation. Particle number concentrations and black carbon emissions showed large reductions during the urban and hot-start phases of the test cycle. The oxidative potential of PM was quantified with both a chemical and a biological assay, and the gene expression impacts of the PM in a macrophage model with PCR (polymerase chain reaction) and ELISA (enzyme-linked immunosorbent assay) analyses. Inter- and intra-vehicle variability in oxidative potential per milligram of PM emitted was relatively low for both oxidative assays, suggesting that real-world emissions and exposure can be estimated with distance-normalized emission factors. The PCR response from signaling markers for oxidative stress (e.g., NOX1) was greater than from inflammatory, AhR (aryl hydrocarbon receptor), or MAPK (mitogen-activated protein kinase) signaling. Protein production associated with inflammation (tumor necrosis factor alpha-TNFα) and oxidative stress (HMOX-1) were quantified and displayed relatively high inter-vehicle variability, suggesting that these pathways may be activated by different PM components. Correlation of trace metal concentrations and oxidative potential suggests a role for small, insoluble particles in inducing oxidative stress.

Oxidative potential of size-fractionated atmospheric aerosol in urban and rural sites across Europe.

In this study we applied several assays, an in vitro rat alveolar macrophage model, a chemical ROS probe (DTT, dithiothreitol), and cytokine induction (TNFα) to examine relationships between PM-induced generation of reactive oxygen species (ROS) and PM composition, using a unique set of size-resolved PM samples obtained from urban and rural environments across Europe. From April-July 2012, we collected PM from roadside canyon, roadside motorway, and background urban sites in each of six European cities and from three rural sites spanning the continent. A Hi-Vol sampler was used to collect PM in three size classes (PM>7, PM7-3, PM3) and PM was characterized for total elements, and oxidative activity quantified in unfiltered and filtered PM extracts. We measured a remarkable uniformity in air concentrations of ROS and especially DTT activity across the continent. Only a 4-fold difference was documented for DTT across the urban sites and a similar variance was documented for ROS, implying that chemical drivers of oxidative activity are relatively similar between sites. The ROS and DTT specific activity was greater at urban background sites (and also rural sites) than at urban canyon locations. PM3 dominated the size distribution of both ROS activity (86% of total) and DTT activity (76% of total), reflecting both the large contribution of PM3 to total PM mass levels and importantly the higher specific oxidative activity of the PM3 in comparison with the larger particles. The soluble fraction of total activity was very high for DTT (94%) as well as for ROS (64%) in the PM3. However in the larger PM size fractions the contributions of the insoluble components became increasingly significant. The dominance of the insoluble PM drivers of activity was particularly evident in the TNFα data, where the insoluble contribution to cytokine production could be 100-fold greater than that from soluble components. ROS and DTT activity were strongly correlated in the PM3 (r = 0.93), however oxidative activity was not correlated with any measured inorganic element in this size cut. In contrast, significant correlations of both ROS and DTT oxidative activity with specific groups of chemical elements were documented in the larger PM size fractions.

ROS-generating/ARE-activating capacity of metals in roadway particulate matter deposited in urban environment.

In this study we investigated the possible causal role for soluble metal species extracted from roadway traffic emissions in promoting particulate matter (PM)-induced reactive oxygen species (ROS) production and antioxidant response element (ARE) promoter activation. To this end, these responses have been evaluated in alveolar macrophage and epithelial lung cells that have been exposed to 'Unfiltered', 'Filtered' and 'Filtered+Chelexed' water extracts of PM samples collected from the roadway urban environments of Thessaloniki, Milan and London. Except for Thessaloniki, our results demonstrate that filtration resulted in a minor decrease in ROS activity of the fine PM fraction, suggesting that ROS activity is attributed mainly to water-soluble PM species. In contrast to ROS, ARE activity was mediated predominantly by the water-soluble component of PM present in both the fine and coarse extracts. Further removal of metals by Chelex treatment from filtered water extracts showed that soluble metal species are the major factors mediating ROS and ARE activities of the soluble fraction, especially in the London PM extracts. Finally, utilizing step-wise multiple-regression analysis, we show that 87% and 78% of the total variance observed in ROS and ARE assays, respectively, is accounted for by changes in soluble metal concentration. Using a statistical analysis we find that As, Zn and Fe best predict the ROS-generating/ARE-activating capacity of the near roadway particulate matter in the pulmonary cells studied. Collectively, our findings imply that soluble metals present in roadside PM are potential drivers of both pro- and anti-oxidative effects of PM in respiratory tract.

ROS production and gene expression in alveolar macrophages exposed to PM(2.5) from Baghdad, Iraq: Seasonal trends and impact of chemical composition.

The objective of this study was to assess the impact of changes in atmospheric particulate matter (PM) composition on oxidative stress markers in an in-vitro alveolar macrophage (AM) model. Fifty-three PM2.5 samples were collected during a year-long PM sampling campaign in Baghdad, Iraq, a semi-arid region of the country. Monthly composites were analyzed for chemical composition and for biological activity using in-vitro measurements of ROS production and gene expression in the AM model. Twelve genes that were differentially expressed upon PM exposure were identified and their co-associations with the composition of PM2.5 were examined. Ten of those genes were up-regulated in January and April composites; samples which also exhibited high ROS activity and relatively high PM mass concentration. ROS production was statistically correlated with total PM2.5 mass, levoglucosan (a wood burning tracer) and several trace elements of the PM (especially V and Ni, which are associated with oil combustion). The expression of several cytokine genes was found to be moderately associated with PM mass, crustal materials (indication of dusty days or dust storms) and certain metals (e.g. V, Fe and Ni) in the PM. Thus, the ROS activity association with PM2.5, may, in part, be driven by redox-active metals. The antioxidant response genes (Nqo1 and Hmox1) were moderately associated with polyaromatic hydrocarbons (PAHs) and showed a good correlation (r-Pearson of >0.7) with metals linked to vehicle-related emissions (i.e. Cu, Zn and Sb). Examining these associations in a larger sample pool (e.g. daily samples) would improve the power of the analysis and may strengthen the implication of these chemicals in the oxidative stress of biological systems, which could aid in the development of new metrics of PM toxicity.

Assessing the role of chemical components in cellular responses to atmospheric particle matter (PM) through chemical fractionation of PM extracts.

In order to further our understanding of the influence of chemical components and ultimately specific sources of atmospheric particulate matter (PM) on pro-inflammatory and other adverse cellular responses, we promulgate and apply a suite of chemical fractionation tools to aqueous aerosol extracts of PM samples for analysis in toxicity assays. We illustrate the approach with a study that used water extracts of quasi-ultrafine PM (PM0.25) collected in the Los Angeles Basin. Filtered PM extracts were fractionated using Chelex, a weak anion exchanger diethylaminoethyl (DEAE), a strong anion exchanger (SAX), and a hydrophobic C18 resin, as well as by desferrioxamine (DFO) complexation that binds iron. The fractionated extracts were then analyzed using high-resolution sector field inductively coupled plasma mass spectrometry (SF-ICPMS) to determine elemental composition. Cellular responses to the fractionated extracts were probed in an in vitro rat alveolar macrophages model with measurement of reactive oxygen species (ROS) production and the cytokine tumor necrosis factor-α (TNF-α). The DFO treatment that chelates iron was very effective at reducing the cellular ROS activity but had only a small impact on the TNF-α production. In contrast, the hydrophobic C18 resin treatment had a small impact on the cellular ROS activity but significantly reduced the TNF-α production. The use of statistical methods to integrate the results across all treatments led to the conclusion that sufficient iron must be present to participate in the chemistry needed for ROS activity, but the amount of ROS activity is not proportional to the iron solution concentration. ROS activity was found to be most related to cationic mono- and divalent metals (i.e., Mn and Ni) and oxyanions (i.e., Mo and V). Although the TNF-α production was not significantly affected by the chelexation of iron, it was greatly impacted by the removal of organics with the C18 resin and all other metal removal methods, suggesting that iron is not a critical pathway leading to TNF-α production, but a wide range of soluble metals and organic compounds in particulate matter play a role. Although the results are specific to the Los Angeles Basin, where the samples used in the study were collected, the method employed in the study can be widely employed to study the role of components of particulate matter in in vitro or in vivo assays.

An in vitro alveolar macrophage assay for the assessment of inflammatory cytokine expression induced by atmospheric particulate matter.

Exposures to air pollution in the form of particulate matter (PM) can result in excess production of reactive oxygen species (ROS) in the respiratory system, potentially causing both localized cellular injury and triggering a systemic inflammatory response. PM-induced inflammation in the lung is modulated in large part by alveolar macrophages and their biochemical signaling, including production of inflammatory cytokines, the primary mechanism via which inflammation is initiated and sustained. We developed a robust, relevant, and flexible method employing a rat alveolar macrophage cell line (NR8383) which can be applied to routine samples of PM from air quality monitoring sites to gain insight into the drivers of PM toxicity that lead to oxidative stress and inflammation. Method performance was characterized using extracts of ambient and vehicular engine exhaust PM samples. Our results indicate that the reproducibility and the sensitivity of the method are satisfactory and comparisons between PM samples can be made with good precision. The average relative percent difference for all genes detected during 10 different exposures was 17.1%. Our analysis demonstrated that 71% of genes had an average signal to noise ratio (SNR) ≥ 3. Our time course study suggests that 4 h may be an optimal in vitro exposure time for observing short-term effects of PM and capturing the initial steps of inflammatory signaling. The 4 h exposure resulted in the detection of 57 genes (out of 84 total), of which 86% had altered expression. Similarities and conserved gene signaling regulation among the PM samples were demonstrated through hierarchical clustering and other analyses. Overlying the core congruent patterns were differentially regulated genes that resulted in distinct sample-specific gene expression "fingerprints." Consistent upregulation of Il1f5 and downregulation of Ccr7 was observed across all samples, while TNFα was upregulated in half of the samples and downregulated in the other half. Overall, this PM-induced cytokine expression assay could be effectively integrated into health studies and air quality monitoring programs to better understand relationships between specific PM components, oxidative stress activity and inflammatory signaling potential.

Effects of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas).

High concentrations (375 ng/L) of the steroid hormone progesterone (P4) were measured in snowmelt runoff associated with large livestock-feeding operations in Wisconsin. To gain insight into the potential endocrine-disrupting effects of P4 in fish, experiments were conducted to evaluate the effects of short-term exposure to environmentally relevant concentrations of P4 on reproduction and embryonic development in the fathead minnow (Pimephales promelas). For the reproduction assay, groups of reproductively mature fish were exposed for 21 d to nominal concentrations of 0, 10, 100, and 1,000 ng/L P4 in a flow-through system, and various key reproductive endpoints (e.g., egg number, fertilization success) were quantified throughout the exposure period. The embryonic development assay consisted of incubating fathead minnow eggs in static culture to quantify the effects of P4 on early development and hatching success. Progesterone caused dose-dependent decreases in fecundity and fertility and significantly reduced gonadosomatic index and vitellogenin gene expression in females. There were no effects of P4 on early embryonic development or hatching success. Progesterone may be a significant endocrine-disrupting chemical in fish.

Reproductive and developmental toxicity of dioxin in fish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD or dioxin) is a global environmental contaminant and the prototypical ligand for investigating aryl hydrocarbon receptor (AHR)-mediated toxicity. Environmental exposure to TCDD results in developmental and reproductive toxicity in fish, birds and mammals. To resolve the ecotoxicological relevance and human health risks posed by exposure to dioxin-like AHR agonists, a vertebrate model is needed that allows for toxicity studies at various levels of biological organization, assesses adverse reproductive and developmental effects and establishes appropriate integrative correlations between different levels of effects. Here we describe the reproductive and developmental toxicity of TCDD in feral fish species and summarize how using the zebrafish model to investigate TCDD toxicity has enabled us to characterize the AHR signaling in fish and to better understand how dioxin-like chemicals induce toxicity. We propose that such studies can be used to predict the risks that AHR ligands pose to feral fish populations and provide a platform for integrating risk assessments for both ecologically relevant organisms and humans.

Reactive oxygen species activity and chemical speciation of size-fractionated atmospheric particulate matter from Lahore, Pakistan: an important role for transition metals.

In this study a sensitive macrophage-based in vitro reactive oxygen species (ROS) assay was coupled with chemical fractionation tools and a year-long sampling program to further our understanding of the role of water-soluble metals in aerosol toxicity. The location is the polluted urban environment of Lahore, Pakistan, where we collected 24 h PM10 and PM2.5 samples every 6(th) day from January 2007 through January 2008. The intrinsic (i.e. particulate matter (PM) mass-normalized) toxicity of the Lahore aerosol, representative of highly polluted developing nations, is compared with toxicity of aerosols from several urban environments in the USA. The monthly patterns of PM2.5 and PM10 water-soluble aerosol ROS-activity were similar with maxima in fall and mid-late winter, and minima over the period April through August and in early winter. Coarse PM ROS-activity was a consistent and significant component (42 +/- 13%) of total activity. The intrinsic activity of the PM2.5 and coarse PM was remarkably similar in a given month. Chelex treatment of the Lahore PM extracts removed a very large and consistent fraction of the water-soluble ROS-activity (96.5 +/- 2.8% for the PM10). Desferrioxamine (DFO) treatment of these extracts also removed a large and relatively consistent fraction of the water-soluble ROS-activity (87.8 +/- 5.3%). Taken together, the DFO and Chelex data imply that transition metals, particularly iron, are major factors mediating ROS-activity of water extracts of the Lahore PM. Statistical modeling (step-wise linear regression and cluster analysis) identified a small subset of metals (Mn, Co, Fe, Ni) as the potential ROS-active species. Several water-soluble "trace" metals present at very high concentrations in the PM extracts (Zn, Pb, Cd), that were effectively removed on Chelex, but are not redox-active, exhibited relatively poor correlations with ROS. The median intrinsic water-soluble ROS-activity measured in the Lahore PM was more than an order-of-magnitude greater than that measured in aerosols from the Long Beach/Los Angeles region and approximately 4-fold greater than the activity of Denver area PM.

Lrrc10 is required for early heart development and function in zebrafish.

Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease.

Blocking expression of AHR2 and ARNT1 in zebrafish larvae protects against cardiac toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which xenobiotic compounds perturb normal development. Embryos treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization exhibit a range of adverse effects on the heart: an early reduction in cardiac myocyte number, followed by a change in heart looping and morphology, with an apparent compaction of the ventricle and overall decrease in heart size. These changes are accompanied by impaired cardiac function including a decrease in cardiac output and eventually irreversible ventricular standstill. The mechanisms involved in mediating effects of TCDD on the heart remain unknown. However, it is widely accepted that aryl hydrocarbon receptor (AHR) activation mediates endpoints of TCDD toxicity in vertebrates. In zebrafish, there are multiple forms of AHR and AHR nuclear translocator protein (ARNT) raising the question about whether different endpoints of TCDD toxicity are mediated by different components of the AHR/ARNT pathway. To address this question we used morpholino oligonucleotide technology to specifically block the expression of zfAHR2, zfARNT1, zfARNT2, and zfCYP1A, and assessed the previously described effects of TCDD on heart morphology, size, and function in the developing morphants. We report that blocking zfAHR2 and zfARNT1 expression provided protection against the TCDD-mediated alteration in heart morphology, reduced cardiac myocyte number, decreased cardiac output and ventricular standstill in zebrafish larvae, while the zfarnt2 and zfcyp1a morpholinos did not block the TCDD-induced cardiac toxicity.

Zebrafish and cardiac toxicology.

Model systems are a mainstay in toxicological research. Zebrafish are rapidly becoming an important model organism for studying vertebrate development. The advantages of zebrafish: short reproductive cycle, production of numerous transparent, synchronously developing embryos, low cost, and standardization make zebrafish an attractive model for toxicologists as well. The use of these fish to study heart development has moved forward very rapidly, laying the groundwork for studying the effects of chemicals on cardiac development and function. Here we describe approaches that can be used to study cardiac toxicity in developing zebrafish, focusing on examples where zebrafish embryos have been especially useful in understanding the impact of specific toxicants on heart development and function.

Heart malformation is an early response to TCDD in embryonic zebrafish.

The zebrafish (Danio rerio) has become an attractive vertebrate model for studying developmental processes, and is emerging as a model system for studying the mechanisms by which toxic compounds perturb normal development. When exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) shortly after fertilization, zebrafish embryos exhibit pericardial edema and reduced blood flow by 72 h post fertilization (hpf). To better understand the progression of dioxin toxicity in zebrafish, we have examined the effects of TCDD on heart development. At 72 hpf, TCDD-treated embryos exhibited altered looping, with the atria positioned distinctly posterior to the ventricles, contrary to the looping of control hearts, where the two chambers lied side by side. Moreover, the ventricles in dioxin-exposed hearts became more compact, and the atria elongated in comparison to controls. These defects are not secondary to pericardial edema because they were observed when edema formation was suppressed with osmotic support. In addition to morphological changes, TCDD produced functional deficits in the developing hearts, including blood regurgitation and a striking ventricular standstill that became prevalent by 120 hpf. We also assessed the effect of TCDD on the heart size using stereological measurements, which demonstrated significant reduction in heart tissue volume at 72 hpf. Perhaps our most significant finding was a decrease in the total number of cardiomyocytes in TCDD-exposed embryos by 48 hpf, one day prior to observable effects on peripheral blood flow. We conclude that the developing heart is an important target for TCDD in zebrafish.