Antonio Planchart

Perturbation of Defense Pathways by Low-Dose Arsenic Exposure in Zebrafish Embryos

Background Exposure to arsenic is a critical risk factor in the complex interplay among genetics, the environment, and human disease. Despite the potential for in utero exposure, the mechanism of arsenic action on vertebrate development and disease is unknown. Objectives The objective of this study was to identify genes and gene networks perturbed by arsenic during development in order to enhance understanding of the molecular mechanisms of arsenic action. Methods We exposed zebrafish embryos at 0.25–1.25 hr postfertilization to 10 or 100 ppb arsenic for 24 or 48 hr. We then used total RNA to interrogate genome microarrays and to test levels of gene expression changes by quantitative real-time polymerase chain reaction (QPCR). Computational analysis was used to identify gene expression networks perturbed by arsenic during vertebrate development. Results We identified a set of 99 genes that responded to low levels of arsenic. Nineteen of these genes were predicted to function in a common regulatory network that was significantly associated with immune response and cancer (p < 10−41). Arsenic-mediated expression changes were validated by QPCR. Conclusions In this study we demonstrated that arsenic significantly down-regulates expression levels of multiple genes potentially critical for regulating the establishment of an immune response. The data also provide molecular evidence consistent with phenotypic observations reported in other model systems. Additional mechanistic studies will help explain molecular events regulating early stages of the immune system and long-term consequences of arsenic-mediated perturbation of this system during development.

Oral-aboral axis specification in the sea urchin embryo III. Role of mitochondrial redox signaling via H2O2

In sea urchin embryos, specification of the secondary (oral-aboral) axis occurs via nodal, expression of which is entirely zygotic and localized to prospective oral ectoderm at blastula stage. The initial source of this spatial anisotropy is not known. Previous studies have shown that oral-aboral (OA) polarity correlates with a mitochondrial gradient, and that nodal activity is dependent both on mitochondrial respiration and p38 stress-activated protein kinase. Here we show that the spatial pattern of nodal activity also correlates with the mitochondrial gradient, and that the latter correlates with inhomogeneous levels of intracellular reactive oxygen species. To test whether mitochondrial H(2)O(2) functions as a redox signal to activate nodal, zygotes were injected with mRNA encoding either mitochondrially-targeted catalase, which quenches mitochondrial H(2)O(2) and down-regulates p38, or superoxide dismutase, which augments mitochondrial H(2)O(2) and up-regulates p38. Whereas the former treatment inhibits the initial activation of nodal and entrains OA polarity toward aboral when confined to half of the embryo via 2-cell stage blastomere injections, the latter does not produce the opposite effects. We conclude that mitochondrial H(2)O(2) is rate-limiting for the initial activation of nodal, but that additional rate-limiting factors, likely also involving mitochondria, contribute to the asymmetry in nodal expression.

Caspar carboxylates: the structural basis of tobamovirus disassembly.

Carboxylate groups have been known for many years to drive the disassembly of simple viruses, including tobacco mosaic virus (TMV). The identities of the carboxylate groups involved and the mechanism by which they initiate disassembly have not, however, been clear. Structures have been determined at resolutions between 2.9 and 3.5 A for five tobamoviruses by fiber diffraction methods. Site-directed mutagenesis has also been used to change numerous carboxylate side chains in TMV to the corresponding amides. Comparison of the stabilities of the various mutant viruses shows that disassembly is driven by a much more complex set of carboxylate interactions than had previously been postulated. Despite the importance of the carboxylate interactions, they are not conserved during viral evolution. Instead, it appears that during evolution, patches of electrostatic interaction drift across viral subunit interfaces. The flexibility of these interactions confers a considerable advantage on the virus, enabling it to change its surface structure rapidly and thus evade host defenses.

2,3,7,8-Tetrachlorodibenzo-p-dioxin upregulates FoxQ1b in zebrafish jaw primordium.

Vertebrate jaw development can be disrupted by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-a potent activator of the aryl hydrocarbon receptor (AHR) transcription factor required for transducing the toxic effects of TCDD. We used zebrafish (Danio rerio) embryos to investigate transcriptional responses to TCDD with the goal of discovering novel, jaw-specific genes affected by TCDD exposure. Our results uncovered a novel target of TCDD-activated Ahr belonging to the evolutionarily conserved family of forkhead box transcription factors. Quantitative real-time polymerase chain reaction analysis demonstrated that FoxQ1b was upregulated by TCDD 7- and 10-fold at 24 and 48 h postfertilization (hpf), respectively. The rate of TCDD-induced FoxQ1b expression was more rapid than that of Cyp1a, a known direct target of TCDD-activated Ahr. TCDD-mediated induction of FoxQ1b was suppressed in the presence of an Ahr antagonist, alpha-naphthoflavone, as well as following knockdown of Ahr2 expression using an Ahr2-specific morpholino antisense oligonucleotide. In situ hybridization analysis of FoxQ1b expression at 48 hpf demonstrated that FoxQ1b is specifically expressed in the jaw primordium where it discretely outlines a developing jaw structure known as Meckels cartilage--a conserved structure in all jawed vertebrates that develops abnormally in the presence of TCDD. These results identify a novel target of TCDD-activated Ahr and suggest that FoxQ1b may play a role in craniofacial abnormalities induced by developmental exposure to TCDD.

Zebrafish: A Marvel of High-Throughput Biology for 21st Century Toxicology

The evolutionary conservation of genomic, biochemical, and developmental features between zebrafish and humans is gradually coming into focus, with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing.

Advancing toxicology research using in vivo high throughput toxicology with small fish models.

Summary Small freshwater fish models, especially zebrafish, offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5-6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We also review many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health.

Aquatic models, genomics and chemical risk management

The 5th Aquatic Animal Models for Human Disease meeting follows four previous meetings (Nairn et al., 2001; Schmale, 2004; Schmale et al., 2007; Hinton et al., 2009) in which advances in aquatic animal models for human disease research were reported, and community discussion of future direction was pursued. At this meeting, discussion at a workshop entitled Bioinformatics and Computational Biology with Web-based Resources (20 September 2010) led to an important conclusion: Aquatic model research using feral and experimental fish, in combination with web-based access to annotated anatomical atlases and toxicological databases, yields data that advance our understanding of human gene function, and can be used to facilitate environmental management and drug development. We propose here that the effects of genes and environment are best appreciated within an anatomical context - the specifically affected cells and organs in the whole animal. We envision the use of automated, whole-animal imaging at cellular resolution and computational morphometry facilitated by high-performance computing and automated entry into toxicological databases, as anchors for genetic and toxicological data, and as connectors between human and model system data. These principles should be applied to both laboratory and feral fish populations, which have been virtually irreplaceable sentinals for environmental contamination that results in human morbidity and mortality. We conclude that automation, database generation, and web-based accessibility, facilitated by genomic/transcriptomic data and high-performance and cloud computing, will potentiate the unique and potentially key roles that aquatic models play in advancing systems biology, drug development, and environmental risk management.

Machine learning reveals sex-specific 17β-estradiol-responsive expression patterns in white perch (Morone americana) plasma proteins

With growing abundance and awareness of endocrine disrupting compounds (EDCs) in the environment, there is a need for accurate and reliable detection of EDC exposure. Our objective in the present study was to observe differences within and between the global plasma proteomes of sexually mature male and female white perch (Morone americana) before (Initial Control, IC) and after 17β‐estradiol (E2) induction. Semiquantitative nanoLC‐MS/MS data were analyzed by machine learning support vector machines (SVMs) and by two‐way ANOVA. By ANOVA, the expression levels of 44, 77, and 57 proteins varied significantly by gender, treatment, and the interaction of gender and treatment, respectively. SVMs perfectly classified male and female perch IC and E2‐induced plasma samples using the protein expression data. E2‐induced male and female perch plasma proteomes contained significantly higher levels of the yolk precursors vitellogenin Aa and Ab (VtgAa, VtgAb), as well as latrophilin and seven transmembrane domain‐containing protein 1 (Eltd1) and kininogen 1 (Kng1). This is the first report that Eltd1 and Kng1 may be E2‐responsive proteins in fishes and therefore may be useful indicators of estrogen induction.

Experimental and computational approaches yield a high-resolution, 1-Mb physical map of the region harboring the mouse t haplotype sterility factor, tcs1.

The loci responsible for the phenomena of t haplotype-specific male sterility and high transmission ratio distortion (TRD) map to the inversions that characterize the mouse t complex of Chromosome (Chr) 17 (reviewed by Lyon 2000). These loci are referred to as Tcd1, tcs1, Tcd2 (tcs2), Tcd3 (tcs3), and Tcr1, respectively (Fig. 1). The exact nature of the Tcd/tcs (t complex distorter/t complex sterility) factors is unknown, whereas Tcr1 (t complex responder) has been shown to encode a novel spermatogenesis-specific kinase, SMOK1, in wild-type that is present as a mutant fusion protein involving the N terminus of SMOK1 and the C terminus of a ribosomal kinase encoded by Rps6ka2, in t (Herrmann et al. 1999). The mechanism by which these factors interact to bring about sterility or TRD remains a mystery. However, genetic evidence suggests that during spermatogenesis in a t/+ mouse, the mutant TCD/TCS factors negatively affect the wild-type TCR1, thus inactivating wild-type sperm. Consequently, the t sperm proceed to fertilize the eggs, thus explaining the high transmission observed for t. However, in viable yet sterile t/t males, the double dose of mutant TCD/TCS factors is thought to overcome the otherwise refractory mutant TCR1, thus interfering with its activity and therefore inactivating all t sperm. Lyon (1986) proved that t-mediated male sterility was due to homozygosity of the Tcd/tcs-encoded factors. Experiments using a spontaneous deletion, Del(17)T, which deletes the region harboring Tcd1/tcs1 in trans to either a complete t or a partial t (in which only the mutant Tcd2/tcs2, Tcd3/tcs3, and Tcr1 loci were present) demonstrated that the deletion mimicked sterility and TRD effects, respectively. Thus, the deletion behaved as if it were an allele of the mutant t-associated Tcd1/tcs1 and suggested that Tcd1/tcs1 must be a loss-of-function mutation (Lyon 1992). Since tcs and Tcd factors at each of the three loci were genetically inseparable, Lyon hypothesized that tcs and Tcd factors are the same within each locus. However, Planchart et al. (2000) were able to separate the two phenomena at the proximal locus (Tcd1/tcs1) by using a deletion panel having much higher resolving power (You et al. 1997). They showed that certain deletions in trans to complete t haplotypes induced sterility; however, when in trans to partial t haplotypes, the deletions had no effect on the transmission of the partial t. It was possible to narrow the region wherein tcs1 is located to between D17Aus9 and Tctex1. A single 1-Mb YAC spanned this region. On the other hand, they found that within the proximal region of the t complex, several loci affected TRD and did not co-map with tcs1 (Planchart et al. 2000), consistent with similar results reported by Lyon et al. (2000). In order to dissect the sterility-critical region with the aim of discovering the gene encoding tcs1, we began to build a contig of mouse BACs, using the RPCI-23 library. Furthermore, we searched the databases at NCBI, primarily the high throughput genomic sequence (htgs) database, for sequenced mouse BACs from this library that map to the sterility-critical region. We began by searching the htgs database, using the sequence of D17Leh48 and Tctex1 as the query sequences. We found two BACs positive for D17Leh48, 147G23 and 248C20, and two BACs positive for Tctex1, 103I10 and 103I20. All four were from the RPCI-23 library. Since the data in htgs are in phase I of sequencing, none of the files were annotated, and all consisted of many unordered pieces. Each file, 193 kb on average, was split into multiple 30-kb files and then submitted to BLAST at the National Center for Biotechnology Information (NCBI), using the blastx program and searching the SwissProt database. To avoid using a large amount of time to perform the BLAST search by manual input, we wrote a Unix C-shell script that took advantage of the Wisconsin GCG package to perform the tasks automatically in batch mode (script is freely available by contacting authors). 147G23 and 248C20 were found to contain a large fragment of Synj2 (synaptojanin2) (Fig. 2), which encodes a phosphatidylinositol polyphosphate 5-phosphatese initially described by Nemoto et al. (1997). Whereas the two BACs overlap extensively, 147G23 extends more proximally than 248C20, and 248C20 extends more distally than 147G23. Furthermore, 248C20 harbors the first 13 exons of Synj2 (representing only 589 of the approximately 1250 amino acids of the protein), but 147G23 contains exons 1 through 3 only. In addition to Synj2, we also identified Tlm, the mouse T lymphoma oncogene, in both BACs (Fig. 2). 103I10 and 103I20 were found to harbor the B form of Tctex1 (Lader et al. 1989), as well as the Vil2 gene encoding villin2 (ezrin), a member of a family of actin-binding cytoskeletal proteins that includes RADIXIN and MOESIN. In addition, a putative protein phosphatase 2 inhibitor was also found. Interestingly, these BACs were reported to be from Chr 15. However, multiple studies have clearly established Tctex1 to reside in the proximal region of Chr 17. Furthermore, analysis of the sequence by the electronic PCR feature of NCBI revealed numerous Chr 17-specific STS markers. Therefore, we conclude that these BACs are from Chr 17 and that Vil2 maps to this chromosome as well. A fifth BAC, 335F22, was shown to contain D17Leh48 as well as D17Aus9 (Y. You, personal communication), thus providing us with BACs that anchored the ends of the tcs1-critical region. Since the average size of the sequenced BACs was 193 kb, we still lacked 600 kb of BAC coverage. In order to close the contig, we resorted to the HindIII fingerprint data actively generated by the Vancouver Genome Sequence Centre. The data were analyzed with the program FPC (Soderlund et al. 1997). We were able to select BACs within the RPCI-23 library that would close the contig and provide us with a minimum tiling path based on selecting BACs that had a minimum of overlap in their HindIII restriction fragment patterns. These BACs are shown in Fig. 2. End sequence, if available, from each of the BACs was obtained from either the Correspondence to: A. Planchart; E-mail: ajp@jax.org Mammalian Genome 12, 668–670 (2001). DOI: 10.1007/s00335-001-1002-9

Heavy Metal Exposure and Metabolic Syndrome: Evidence from Human and Model System Studies

Purpose of ReviewMetabolic syndrome (MS) describes the co-occurrence of conditions that increase one’s risk for heart disease and other disorders such as diabetes and stroke. The worldwide increase in the prevalence of MS cannot be fully explained by lifestyle factors such as sedentary behavior and caloric intake alone. Environmental exposures, such as heavy metals, have been implicated, but results are conflicting and possible mechanisms remain unclear. To assess recent progress in determining a possible role between heavy metal exposure and MS, we reviewed epidemiological and model system data for cadmium (Cd), lead (Pb), and mercury (Hg) from the last decade.Recent FindingsData from 36 epidemiological studies involving 17 unique countries/regions and 13 studies leveraging model systems are included in this review. Epidemiological and model system studies support a possible association between heavy metal exposure and MS or comorbid conditions; however, results remain conflicting. Epidemiological studies were predominantly cross-sectional and collectively, they highlight a global interest in this question and reveal evidence of differential susceptibility by sex and age to heavy metal exposures. In vivo studies in rats and mice and in vitro cell-based assays provide insights into potential mechanisms of action relevant to MS including altered regulation of lipid and glucose homeostasis, adipogenesis, and oxidative stress.SummaryHeavy metal exposure may contribute to MS or comorbid conditions; however, available data are conflicting. Causal inference remains challenging as epidemiological data are largely cross-sectional; and variation in study design, including samples used for heavy metal measurements, age of subjects at which MS outcomes are measured; the scope and treatment of confounding factors; and the population demographics vary widely. Prospective studies, standardization or increased consistency across study designs and reporting, and consideration of molecular mechanisms informed by model system studies are needed to better assess potential causal links between heavy metal exposure and MS.