PAH bioremediation with Rhodococcus rhodochrous ATCC 21198: Impact of cell immobilization and surfactant use on PAH treatment and post-remediation toxicity.

Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental contaminants that are harmful to ecological and human health. Bioremediation is a promising technique for remediating PAHs in the environment, however bioremediation often results in the accumulation of toxic PAH metabolites. The objectives of this research were to demonstrate the cometabolic treatment of a mixture of PAHs by a pure bacterial culture, Rhodococcus rhodochrous ATCC 21198, and investigate PAH metabolites and toxicity. Additionally, the surfactant Tween ® 80 and cell immobilization techniques were used to enhance bioremediation. Total PAH removal ranged from 70-95% for fluorene, 44-89% for phenanthrene, 86-97% for anthracene, and 6.5-78% for pyrene. Maximum removal was achieved with immobilized cells in the presence of Tween ® 80. Investigation of PAH metabolites produced by 21198 revealed a complex mixture of hydroxylated compounds, quinones, and ring-fission products. Toxicity appeared to increase after bioremediation, manifesting as mortality and developmental effects in embryonic zebrafish. 21198's ability to rapidly transform PAHs of a variety of molecular structures and sizes suggests that 21198 can be a valuable microorganism for catalyzing PAH remediation. However, implementing further treatment processes to address toxic PAH metabolites should be pursued to help lower post-remediation toxicity in future studies.

Review of the zebrafish as a model to investigate per- and polyfluoroalkyl substance toxicity.

The existence of thousands of per- and polyfluoroalkyl substances (PFAS) and evidence that some cause adverse health effects has created immense need to better understand PFAS toxicity and to move beyond one-chemical-at-a-time approaches to hazard assessment for this chemical class. The zebrafish model enables rapid assessment of large libraries of PFAS, powerful comparison of compounds in a single in vivo system, and evaluation across life stages and generations, and has led to significant advances in PFAS research in recent years. The focus of this review is to assess contemporary findings regarding PFAS toxicokinetics, toxicity and apical adverse health outcomes, and potential modes of action using the zebrafish model. Much of the peer-reviewed literature has focused on a small subset of PFAS structural subclasses, such as the perfluoroalkyl sulfonic acids and perfluoroalkyl carboxylic acids. However, recent data on more diverse PFAS structures are enabling prioritization of compounds of concern. Structure-activity comparisons and the utilization of modeling and 'omics technologies in zebrafish have greatly contributed to our understanding of the hazard potential for a growing number of PFAS and will surely inform our understanding and predictive capabilities for many more PFAS in the future.

Concentration-response gene expression analysis in zebrafish reveals phenotypically-anchored transcriptional responses to retene.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants and are associated with human disease. Canonically, many PAHs induce toxicity via activation of the aryl hydrocarbon receptor (AHR) pathway. While the interaction between PAHs and the AHR is well-established, understanding which AHR-regulated transcriptional effects directly result in observable phenotypes and which are adaptive or benign is important to better understand PAH toxicity. Retene is a frequently detected PAH in environmental sampling and has been associated with AHR2-dependent developmental toxicity in zebrafish, though its mechanism of toxicity has not been fully elucidated. To interrogate transcriptional changes causally associated with retene toxicity, we conducted whole-animal RNA sequencing at 48 h post-fertilization after exposure to eight retene concentrations. We aimed to identify the most sensitive transcriptomic responses and to determine whether this approach could uncover gene sets uniquely differentially expressed at concentrations which induce a phenotype. We identified a concentration-response relationship for differential gene expression in both number of differentially expressed genes (DEGs) and magnitude of expression change. Elevated expression of cyp1a at retene concentrations below the threshold for teratogenicity suggested that while cyp1a expression is a sensitive biomarker of AHR activation, it may be too sensitive to serve as a biomarker of teratogenicity. Genes differentially expressed at only non-teratogenic concentrations were enriched for transforming growth factor-ß (TGF-ß) signaling pathway disruption while DEGs identified at only teratogenic concentrations were significantly enriched for response to xenobiotic stimulus and reduction-oxidation reaction activity. DEGs which spanned both non-teratogenic and teratogenic concentrations showed similar disrupted biological processes to those unique to teratogenic concentrations, indicating these processes were disrupted at low exposure concentrations. Gene co-expression network analysis identified several gene modules, including those associated with PAHs and AHR2 activation. One, Module 7, was strongly enriched for AHR2-associated genes and contained the strongest responses to retene. Benchmark concentration (BMC) of Module seven genes identified a median BMC of 7.5 µM, nearly the highest retene concentration with no associated teratogenicity, supporting the hypothesis that Module seven genes are largely responsible for retene toxicity.

Transcriptomic and Long-Term Behavioral Deficits Associated with Developmental 3.5 GHz Radiofrequency Radiation Exposures in Zebrafish.

The rapid deployment of the fifth-generation (5G) spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, concerns among the public about the safety and health effects of radiofrequency radiations (RFRs) emitted from the newer-generation cell phone frequencies remain, partly due to the lack of robust scientific data. Previously, we used developmental zebrafish to model the bioactivity of 3.5 GHz RFR, a frequency used by 5G-enabled cell phones, in a novel RFR exposure chamber. With RFR exposures from 6 h post-fertilization (hpf) to 48 hpf, we observed that, despite no teratogenic effects, embryos showed subtle hypoactivity in a startle response behavior assay, suggesting abnormal sensorimotor behavior. This study builds upon the previous one by investigating the transcriptomic basis of RFR-associated behavior effects and their persistence into adulthood. Using mRNA sequencing, we found a modest transcriptomic disruption at 48 hpf, with 28 differentially expressed genes. KEGG pathway analysis showed that biochemical pathways related to metabolism were significantly perturbed. Embryos were grown to adulthood, and then a battery of behavioral assays suggested subtle but significant abnormal responses in RFR-exposed fish across the different assays evaluated that suggest potential long-term behavioral effects. Overall, our study suggests the impacts of RFRs on the developing brain, behavior, and the metabolome should be further explored.

Zebrafish Behavioral Assays in Toxicology.

Zebrafish behavioral assays are commonly used to identify and study environmental stressors that elicit adverse effects on neurobehavior. Behavioral assay platforms are available for multiple life stages (embryonic, juvenile, and adults) and are robust in detecting stressor-induced acute effects on neurodevelopment as well as long term deficits in sensory mechanisms, social behavior, learning, memory, and neurodegenerative diseases. Within this chapter, we present an overview of zebrafish behavioral assays that are commonly used to study environmental neurotoxicants.

Systematic developmental toxicity assessment of a structurally diverse library of PFAS in zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are a class of widely used chemicals with limited human health effects data relative to the diversity of structures manufactured. To help fill this data gap, an extensive in vivo developmental toxicity screen was performed on 139 PFAS provided by the US EPA. Dechorionated embryonic zebrafish were exposed to 10 nominal water concentrations of PFAS (0.015-100 µM) from 6 to 120 h post-fertilization (hpf). The embryos were assayed for embryonic photomotor response (EPR), larval photomotor response (LPR), and 13 morphological endpoints. A total of 49 PFAS (35%) were bioactive in one or more assays (11 altered EPR, 25 altered LPR, and 31 altered morphology). Perfluorooctanesulfonamide (FOSA) was the only structure that was bioactive in all 3 assays, while Perfluorodecanoic acid (PFDA) was the most potent teratogen. Low PFAS volatility was associated with developmental toxicity (p < 0.01), but no association was detected between bioactivity and five other physicochemical parameters. The bioactive PFAS were enriched for 6 supergroup chemotypes. The results illustrate the power of a multi-dimensional in vivo platform to assess the developmental (neuro)toxicity of diverse PFAS and in the acceleration of PFAS safety research.

Sulfonamide functional head on short-chain perfluorinated substance drives developmental toxicity.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitously detected in environmental and biological samples and cause adverse health effects. Studies have predominately focused on long-chain PFAS, with far fewer addressing short-chain alternatives. This study leveraged embryonic zebrafish to investigate developmental toxicity of a short-chain series: perfluorobutane sulfonate (PFBS), perfluoropentanoic acid (PFPeA), perfluorobutane sulfonamide (FBSA), and 4:2 fluorotelomer sulfonic acid (4:2 FTS). Following static exposures at 8 h postfertilization (hpf) to each chemical (1-100 µM), morphological and behavioral endpoints were assessed at 24 and 120 hpf. Only FBSA induced abnormal morphology, while exposure to all chemicals caused aberrant larval behavior. RNA sequencing at 48 hpf following 47 µM exposures revealed only FBSA significantly disrupted normal gene expression. Measured tissue concentrations were FBSA > PFBS > 4:2 FTS > PFPeA. This study demonstrates functional head groups impact bioactivity and bioconcentration.

Behavior Effects of Structurally Diverse Per- and Polyfluoroalkyl Substances in Zebrafish.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitously detected in the environment, and some pose significant human and environmental health concerns globally. While some PFAS induce adverse health effects, relatively few toxicological studies adequately address the broad structural diversity of this chemical class. In the current study, we evaluated 58 individual PFAS spanning 14 structural subclasses and 2 mixtures at single concentrations for developmental toxicity in zebrafish using highly sensitive behavior endpoints. Following developmental exposure to PFAS, zebrafish were assessed for mortality and challenged with an embryonic photomotor response (EPR) assay at 24 h postfertilization (hpf) and with larval photomotor response (LPR) and larval startle response assays at 120 hpf. We found that none of the tested PFAS exposures elicited significant mortality or aberrant EPR; however, exposure to 21 individual PFAS from multiple structural subclasses and 1 mixture induced aberrant larval behavior. We then evaluated developmental toxicity across a concentration range of 0-100 µM for 10 perfluoroalkyl carboxylic acids (PFCAs; 4-carbon perfluorobutanoic acid through the 13-carbon perfluorotridecanoic acid). Exposure to the PFCAs did not cause significant mortality or morphological effects, with the exception of perfluorooctanoic acid and perfluorononanoic acid, and did not induce aberrant EPR. All PFCAs, except for longer-chain perfluorododecanoic acid caused abnormal LPR following exposure to at least one concentration. In this study, we evaluated a broad set of PFAS not previously assessed for in vivo sublethal behavior endpoints and confirmed previous findings that exposure to some PFAS induces abnormal behavior in developing zebrafish. The data from this study will guide the selection of PFAS for which to investigate modes of toxic action.

Phenotypically Anchored mRNA and miRNA Expression Profiling in Zebrafish Reveals Flame Retardant Chemical Toxicity Networks.

The ubiquitous use of flame retardant chemicals (FRCs) in the manufacture of many consumer products leads to inevitable environmental releases and human exposures. Studying toxic effects of FRCs as a group is challenging since they widely differ in physicochemical properties. We previously used zebrafish as a model to screen 61 representative FRCs and showed that many induced behavioral and teratogenic effects, with aryl phosphates identified as the most active. In this study, we selected 10 FRCs belonging to diverse physicochemical classes and zebrafish toxicity profiles to identify the gene expression responses following exposures. For each FRC, we executed paired mRNA-micro-RNA (miR) sequencing, which enabled us to study mRNA expression patterns and investigate the role of miRs as posttranscriptional regulators of gene expression. We found widespread disruption of mRNA and miR expression across several FRCs. Neurodevelopment was a key disrupted biological process across multiple FRCs and was corroborated by behavioral deficits. Several mRNAs (e.g., osbpl2a) and miRs (e.g., mir-125b-5p), showed differential expression common to multiple FRCs (10 and 7 respectively). These common miRs were also predicted to regulate a network of differentially expressed genes with diverse functions, including apoptosis, neurodevelopment, lipid regulation and inflammation. Commonly disrupted transcription factors (TFs) such as retinoic acid receptor, retinoid X receptor, and vitamin D regulator were predicted to regulate a wide network of differentially expressed mRNAs across a majority of the FRCs. Many of the differential mRNA-TF and mRNA-miR pairs were predicted to play important roles in development as well as cancer signaling. Specific comparisons between TBBPA and its derivative TBBPA-DBPE showed contrasting gene expression patterns that corroborated with their phenotypic profiles. The newer generation FRCs such as IPP and TCEP produced distinct gene expression changes compared to the legacy FRC BDE-47. Our study is the first to establish a mRNA-miR-TF regulatory network across a large group of structurally diverse FRCs and diverse phenotypic responses. The purpose was to discover common and unique biological targets that will help us understand mechanisms of action for these important chemicals and establish this approach as an important tool for better understanding toxic effects of environmental contaminants.

The chemistry and toxicology of vaping.

Vaping is the process of inhaling and exhaling an aerosol produced by an e-cigarette, vape pen, or personal aerosolizer. When the device contains nicotine, the Food and Drug Administration (FDA) lists the product as an electronic nicotine delivery system or ENDS device. Similar electronic devices can be used to vape cannabis extracts. Over the past decade, the vaping market has increased exponentially, raising health concerns over the number of people exposed and a nationwide outbreak of cases of severe, sometimes fatal, lung dysfunction that arose suddenly in otherwise healthy individuals. In this review, we discuss the various vaping technologies, which are remarkably diverse, and summarize the use prevalence in the U.S. over time by youths and adults. We examine the complex chemistry of vape carrier solvents, flavoring chemicals, and transformation products. We review the health effects from epidemiological and laboratory studies and, finally, discuss the proposed mechanisms underlying some of these health effects. We conclude that since much of the research in this area is recent and vaping technologies are dynamic, our understanding of the health effects is insufficient. With the rapid growth of ENDS use, consumers and regulatory bodies need a better understanding of constituent-dependent toxicity to guide product use and regulatory decisions.

Systematic Assessment of Exposure Variations on Observed Bioactivity in Zebrafish Chemical Screening.

The embryonic zebrafish is a powerful tool for high-throughput screening of chemicals. While this model has significant potential for use in safety assessments and chemical prioritization, a lack of exposure protocol harmonized across laboratories has limited full model adoption. To assess the potential that exposure protocols alter chemical bioactivity, we screened a set of eight chemicals and one 2D nanomaterial across four different regimens: (1) the current Tanguay laboratory's standard protocol of dechorionated embryos and static exposure in darkness; (2) exposure with chorion intact; (3) exposure under a 14 h light: 10 h dark cycle; and (4) exposure with daily chemical renewal. The latter three regimens altered the concentrations, resulting in bioactivity of the test agents compared to that observed with the Tanguay laboratory's standard regimen, though not directionally the same for each chemical. The results of this study indicate that with the exception for the 2D nanomaterial, the screening design did not change the conclusion regarding chemical bioactivity, just the nominal concentrations producing the observed activity. Since the goal of tier one chemical screening often is to differentiate active from non-active chemicals, researchers could consider the trade-offs regarding cost, labor, and sensitivity in their study design without altering hit rates. Taken further, these results suggest that it is reasonably feasible to reach agreement on a standardized exposure regiment, which will promote data sharing without sacrificing data content.

The multi-dimensional embryonic zebrafish platform predicts flame retardant bioactivity.

Flame retardant chemicals (FRCs) commonly added to many consumer products present a human exposure burden associated with adverse health effects. Under pressure from consumers, FRC manufacturers have adopted some purportedly safer replacements for first-generation brominated diphenyl ethers (BDEs). In contrast, second and third-generation organophosphates and other alternative chemistries have limited bioactivity data available to estimate their hazard potential. In order to evaluate the toxicity of existing and potential replacement FRCs, we need efficient screening methods. We built a 61-FRC library in which we systemically assessed developmental toxicity and potential neurotoxicity effects in the embryonic zebrafish model. Data were compared to publicly available data generated in a battery of cell-based in vitro assays from ToxCast, Tox21, and other alternative models. Of the 61 FRCs, 19 of 45 that were tested in the ToxCast assays were bioactive in our zebrafish model. The zebrafish assays detected bioactivity for 10 of the 12 previously classified developmental neurotoxic FRCs. Developmental zebrafish were sufficiently sensitive at detecting FRC structure-bioactivity impacts that we were able to build a classification model using 13 physicochemical properties and 3 embryonic zebrafish assays that achieved a balanced accuracy of 91.7%. This work illustrates the power of a multi-dimensional in vivo platform to expand our ability to predict the hazard potential of new compounds based on structural relatedness, ultimately leading to reliable toxicity predictions based on chemical structure.

Impacts of high dose 3.5 GHz cellphone radiofrequency on zebrafish embryonic development.

The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. In this study, we used the embryonic zebrafish model to assess the impacts of a 3.5 GHz RFR on biology- a frequency typically used by 5G-enabled cell phones and lies within the 4G and 5G bandwidth. We established a plate-based exposure setup for RFRs, exposed developing zebrafish to 3.5 GHz RFR, specific absorption rate (SAR) ≈ 8.27 W/Kg from 6 h post fertilization (hpf) to 48 hpf, and measured a battery of morphological and behavioral endpoints at 120 hpf. Our results revealed no significant impacts on mortality, morphology or photomotor response and a modest inhibition of startle response suggesting some levels of sensorimotor disruptions. This suggests that the cell phone radiations at low GHz-level frequencies are likely benign, with subtle sensorimotor effects. Through this assessment, we have established a robust setup for zebrafish RFR exposures readily amenable to testing various powers and frequencies. Future developmental exposure studies in zebrafish will evaluate a wider portion of the radio frequency spectrum to discover the bioactive regions, the potential molecular targets of RFR and the potential long-term effects on adult behavior.

Rapid well-plate assays for motor and social behaviors in larval zebrafish.

Behavior phenotypes are a powerful means of uncovering subtle xenobiotic chemical impacts on vertebrate nervous system development. Rodents manifest complex and informative behavior phenotypes but are generally not practical models in which to screen large numbers of chemicals. Zebrafish recapitulate much of the behavioral complexity of higher vertebrates, develop externally and are amenable to assay automation. Short duration automated assays can be leveraged to screen large numbers of chemicals or comprehensive dose-response for fewer chemicals. Here we describe a series of mostly automated assays including larval photomotor response, strobe light response, blue color avoidance, shoaling and mirror stimulus-response performed on the ZebraBox (ViewPoint Behavior Technologies) instrument platform. To explore the sensitivity and uniqueness of each assay endpoint, larval cohorts from 5 to 28 days post fertilization were acutely exposed to several chemicals broadly understood to impact different neuro-activities. We highlight the throughput advantages of using the same instrument platform for multiple assays and the ability of different assays to detect unique phenotypes among different chemicals.

Assessing the hazard of E-Cigarette flavor mixtures using zebrafish.

Since 2007, electronic cigarette (e-cigarette) sales in the U.S. have surpassed those of tobacco cigarettes. This is due, in part, to manufacturer's claims that they are a safer alternative to tobacco cigarettes. However, formaldehyde, acrolein, and diacetyl have been detected in e-cigarettes and public knowledge of e-cigarette composition and ingredient bioactivity is conspicuously lacking. We evaluated the toxicity of nine e-cigarette flavor mixtures and their constituents in the developmental zebrafish, an excellent whole animal biosensor of chemical hazard. Seven of the nine flavors (78%) elicited adverse developmental responses at 1% by volume. The number of toxic endpoints varied greatly between flavors. Two flavors, Grape and Bubble Gum, had similar chemical compositions, but different toxicity profiles. We hypothesized that the toxicity was driven by a constituent present only in the Bubble Gum flavor, cinnamaldehyde. To replicate this toxicity, we built our own defined mixture. The addition of varying concentrations of cinnamaldehyde suggested that it drove the toxicity of these mixtures and that e-cigarette hazard can be flavor dependent.

Biodegradability and toxicity of monorhamnolipid biosurfactant diastereomers.

Synthetic monorhamnolipids differ from biologically produced material because they are produced as single congeners, depending on the ß-hydroxyalkanoic acid used during synthesis. Each congener is produced as one of four possible diastereomers resulting from two chiral centers at the carbinols of the lipid tails [(R,R), (R,S), (S,R) and (S,S)]. We compare the biodegradability (CO2 respirometry), acute toxicity (Microtox assay), embryo toxicity (Zebrafish assay), and cytotoxicity (xCELLigence and MTS assays) of synthetic rhamnosyl-ß-hydroxydecanoyl-ß-hydroxydecanoate (Rha-C10-C10) monorhamnolipids against biosynthesized monorhamnolipid mixtures (bio-mRL). All Rha-C10-C10 diastereomers and bio-mRL were inherently biodegradable ranging from 34 to 92% mineralized. The Microtox assay showed all Rha-C10-C10 diastereomers and bio-mRL are slightly toxic according to the US EPA ecotoxicity categories with 5 min EC50 values ranging from 39.6 to 87.5 µM. The zebrafish assay showed that of 22 developmental endpoints tested, only mortality was observed at 120 h post fertilization; all Rha-C10-C10 diastereomers and bio-mRL caused significant mortality at 640 µM, except the Rha-C10-C10 (R,R) which showed no developmental effects. xCELLigence and MTS showed IC50 values ranging from 103.4 to 191.1 µM for human lung cell line H1299 after 72 h exposure. These data provide key information regarding Rha-C10-C10 diastereomers that is pertinent when considering potential applications.

Ecotoxicity of the insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) and its reduced metabolite 3-amino-1,2,4-triazol-5-one (ATO).

The insensitive munitions compound 3-nitro-1,2,4-triazol-5-one (NTO) was recently approved by the U.S. Army to replace cyclotrimethylene trinitramine (RDX) in conventional explosives. As its use becomes widespread, concern about the potential toxicity of NTO increases. NTO can undergo microbial reduction to 3-amino-1,2,4-triazol-5-one (ATO), which is recalcitrant in waterlogged soils. In this study, the acute toxicity of NTO and ATO towards various organisms, including microorganisms (i.e., methanogenic archaea, aerobic heterotrophs, and Aliivibrio fischeri (Microtox assay)), the microcrustacean Daphnia magna (ATO only), and zebrafish embryos (Danio rerio), was assessed. NTO was notably more inhibitory to methanogens than ATO (IC50=1.2mM,>62.8mM, respectively). NTO and ATO did not cause noteworthy inhibition on aerobic heterotrophs even at the highest concentrations tested (32.0mM). High concentrations of both NTO and ATO were required to inhibit A. fischeri (IC20=19.2, 22.4mM, respectively). D. magna was sensitive to ATO (LC50=0.27mM). Exposure of zebrafish embryos to NTO or ATO (750µM) did not cause lethal or developmental effects (22 endpoints tested). However, both compounds led to swimming behavior abnormalities at low concentrations (7.5µM). The results indicate that the reductive biotransformation of NTO could enhance or lower its toxicity according to the target organism.

Zebrafish embryo toxicity of 15 chlorinated, brominated, and iodinated disinfection by-products.

Disinfection to protect human health occurs at drinking water and wastewater facilities through application of non-selective oxidants including chlorine. Oxidants also transform organic material and form disinfection by-products (DBPs), many of which are halogenated and cyto- and genotoxic. Only a handful of assays have been used to compare DBP toxicity, and researchers are unsure which DBP(s) drive the increased cancer risk associated with drinking chlorinated water. The most extensive data set employs an in vitro model cell, Chinese hamster ovary cells. Traditionally, most DBP research focuses on the threat to human health, but the effects on aquatic species exposed to DBPs in wastewater effluents remain ill defined. We present the developmental toxicity for 15 DBPs and a chlorinated wastewater to a model aquatic vertebrate, zebrafish. Mono-halogenated DBPs followed the in vivo toxicity rank order: acetamides>acetic acids>acetonitriles~nitrosamines, which agrees well with previously published mammalian in vitro data. Di- and tri-halogenated acetonitriles were more toxic than their mono-halogenated analogues, and bromine- and iodine-substituted DBPs tended to be more toxic than chlorinated analogues. No zebrafish development effects were observed after exposure to undiluted or non-concentrated, chlorinated wastewater. We find zebrafish development to be a viable in vivo alternative or confirmatory assay to mammalian in vitro cell assays.

Colloidal Gold Nanoparticles Induce Changes in Cellular and Subcellular Morphology.

Exposure of cells to colloidal nanoparticles (NPs) can have concentration-dependent harmful effects. Mostly, such effects are monitored with biochemical assays or probes from molecular biology, i.e., viability assays, gene expression profiles, etc., neglecting that the presence of NPs can also drastically affect cellular morphology. In the case of polymer-coated Au NPs, we demonstrate that upon NP internalization, cells undergo lysosomal swelling, alterations in mitochondrial morphology, disturbances in actin and tubulin cytoskeleton and associated signaling, and reduction of focal adhesion contact area and number of filopodia. Appropriate imaging and data treatment techniques allow for quantitative analyses of these concentration-dependent changes. Abnormalities in morphology occur at similar (or even lower) NP concentrations as the onset of reduced cellular viability. Cellular morphology is thus an important quantitative indicator to verify harmful effects of NPs to cells, without requiring biochemical assays, but relying on appropriate staining and imaging techniques.

Transgenerational inheritance of neurobehavioral and physiological deficits from developmental exposure to benzo[a]pyrene in zebrafish.

Benzo[a]pyrene (B[a]P) is a well-known genotoxic polycylic aromatic compound whose toxicity is dependent on signaling via the aryl hydrocarbon receptor (AHR). It is unclear to what extent detrimental effects of B[a]P exposures might impact future generations and whether transgenerational effects might be AHR-dependent. This study examined the effects of developmental B[a]P exposure on 3 generations of zebrafish. Zebrafish embryos were exposed from 6 to 120h post fertilization (hpf) to 5 and 10µM B[a]P and raised in chemical-free water until adulthood (F0). Two generations were raised from F0 fish to evaluate transgenerational inheritance. Morphological, physiological and neurobehavioral parameters were measured at two life stages. Juveniles of the F0 and F2 exhibited hyper locomotor activity, decreased heartbeat and mitochondrial function. B[a]P exposure during development resulted in decreased global DNA methylation levels and generally reduced expression of DNA methyltransferases in wild type zebrafish, with the latter effect largely reversed in an AHR2-null background. Adults from the F0 B[a]P exposed lineage displayed social anxiety-like behavior. Adults in the F2 transgeneration manifested gender-specific increased body mass index (BMI), increased oxygen consumption and hyper-avoidance behavior. Exposure to benzo[a]pyrene during development resulted in transgenerational inheritance of neurobehavioral and physiological deficiencies. Indirect evidence suggested the potential for an AHR2-dependent epigenetic route.