Nik Veldhoen

Toxicity of glyphosate‐based pesticides to four North American frog species

Glyphosate-based herbicides are among the most widely used pesticides in the world. We compared the acute toxicity of the glyphosate end-use formulation Roundup Original to four North American amphibian species (Rana clamitans, R. pipiens, R. sylvatica, and Bufo americanus) and the toxicity of glyphosate technical, the polyethoxylated tallowamine surfactant (POEA) commonly used in glyphosate-based herbicides, and five newer glyphosate formulations to R. clamitans. For R. clamitans, acute toxicity values in order of decreasing toxicity were POEA > Roundup Original > Roundup Transorb > Glyfos AU; no significant acute toxicity was observed with glyphosate technical material or the glyphosate formulations Roundup Biactive, Touchdown, or Glyfos BIO. Comparisons between the four amphibian species showed that the toxicity of Roundup Original varied with species and developmental stage. Rana pipiens tadpoles chronically exposed to environmentally relevant concentrations of POEA or glyphosate formulations containing POEA showed decreased snout-vent length at metamorphosis and increased time to metamorphosis, tail damage, and gonadal abnormalities. These effects may be caused, in some part, by disruption of hormone signaling, because thyroid hormone receptor beta mRNA transcript levels were elevated by exposure to formulations containing glyphosate and POEA. Taken together, the data suggest that surfactant composition must be considered in the evaluation of toxicity of glyphosate-based herbicides.

PCB-related alteration of thyroid hormones and thyroid hormone receptor gene expression in free-ranging harbor seals (Phoca vitulina)

Persistent organic pollutants are environmental contaminants that, because of their lipophilic properties and long half-lives, bioaccumulate within aquatic food webs and often reach high concentrations in marine mammals, such as harbor seals (Phoca vitulina). Exposure to these contaminants has been associated with developmental abnormalities, immunotoxicity, and reproductive impairment in marine mammals and other high-trophic-level wildlife, mediated via a disruption of endocrine processes. The highly conserved thyroid hormones (THs) represent one vulnerable endocrine end point that is critical for metabolism, growth, and development in vertebrates. We characterized the relationship between contaminants and specific TH receptor (TR ) gene expression in skin/blubber biopsy samples, as well as serum THs, from free-ranging harbor seal pups (n = 39) in British Columbia, Canada, and Washington State, USA. We observed a contaminant-related increase in blubber TR-α gene expression [total polychlorinated biphenyls (∑PCBs); r = 0.679; p < 0.001] and a concomitant decrease in circulating total thyroxine concentrations (∑PCBs; r = −0.711; p < 0.001). Consistent with results observed in carefully controlled laboratory and captive feeding studies, our findings suggest that the TH system in harbor seals is highly sensitive to disruption by environmental contaminants. Such a disruption not only may lead to adverse effects on growth and development but also could have important ramifications for lipid metabolism and energetics in marine mammals.

Characterization of the histone H2A.Z-1 and H2A.Z-2 isoforms in vertebrates.

BackgroundWithin chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues.ResultsWe report that H2A.Z-1 and H2A.Z-2 are expressed across a wide range of human tissues, they are both acetylated at lysine residues within the N-terminal region and they exhibit similar, but nonidentical, distributions within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1. The phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution.ConclusionsOur biochemical, gene expression, and phylogenetic data suggest that the H2A.Z-1 and H2A.Z-2 variants function similarly yet they may have acquired a degree of functional independence.

Distinctive gene profiles occur at key points during natural metamorphosis in the Xenopus laevis tadpole tail.

Thyroid hormones (THs) are essential for tadpole metamorphosis into a juvenile frog; however, a complex interplay between additional hormones and signaling events also contributes to this dramatic developmental phase. A major mechanism of TH action is the nuclear receptor‐mediated regulation of gene transcription of responsive genes. By using the precocious metamorphic model, several genes have been identified as TH responsive in the regressing tail. Many of these genes also exhibit altered expression during natural metamorphosis. Although identification of these genes provides insight into the mechanism whereby TH acts, complex interplay between TH and other hormones and the developmental stage‐dependency of tissue responses contribute to the timing and coordination of metamorphic events. We investigated the temporal gene expression profile in Xenopus laevis tadpole tails from premetamorphosis through metamorphic climax by using a combination of a novel frog cDNA array containing 420 genes and quantitative real‐time PCR. Seventy‐nine genes were identified whose steady‐state mRNA expression levels were altered in the tadpole tail during natural metamorphosis, of which 34 have previously been identified to be TH responsive in frogs or mammals. Of these genes, 75 clustered into 13 groups that displayed distinct developmental expression profiles. The levels of 28 transcripts were altered during premetamorphosis, 31 during prometamorphosis, and 43 with the onset of tail regression. This work establishes an important baseline for determining the mechanisms whereby tissues undergo differing metamorphic fates.

PCB-associated changes in mRNA expression in killer whales (Orcinus orca) from the NE Pacific Ocean.

Killer whales in the NE Pacific Ocean are among the worlds most PCB-contaminated marine mammals, raising concerns about implications for their health. Sixteen health-related killer whale mRNA transcripts were analyzed in blubber biopsies collected from 35 free-ranging killer whales in British Columbia using real-time quantitative polymerase chain reaction. We observed PCB-related increases in the expression of five gene targets, including the aryl hydrocarbon receptor (AhR; r(2) = 0.83; p < 0.001), thyroid hormone α receptor (TRα; r(2) = 0.64; p < 0.001), estrogen α receptor (ERα; r(2) = 0.70; p < 0.001), interleukin 10 (IL-10; r(2) = 0.74 and 0.68, males and females, respectively; p < 0.001), and metallothionein 1 (MT1; r(2) = 0.58; p < 0.001). Best-fit models indicated that population (dietary preference), age, and sex were not confounding factors, except for IL-10, where males differed from females. While the population-level consequences are unclear, the PCB-associated alterations in mRNA abundance of such pivotal end points provide compelling evidence of adverse physiological effects of persistent environmental contaminants in these endangered killer whales.

Molecular profiling of marine fauna: integration of omics with environmental assessment of the world's oceans.

Many species that contribute to the commercial and ecological richness of our marine ecosystems are harbingers of environmental change. The ability of organisms to rapidly detect and respond to changes in the surrounding environment represents the foundation for application of molecular profiling technologies towards marine sentinel species in an attempt to identify signature profiles that may reside within the transcriptome, proteome, or metabolome and that are indicative of a particular environmental exposure event. The current review highlights recent examples of the biological information obtained for marine sentinel teleosts, mammals, and invertebrates. While in its infancy, such basal information can provide a systems biology framework in the detection and evaluation of environmental chemical contaminant effects on marine fauna. Repeated evaluation across different seasons and local marine environs will lead to discrimination between signature profiles representing normal variation within the complex milieu of environmental factors that trigger biological response in a given sentinel species and permit a greater understanding of normal versus anthropogenic-associated modulation of biological pathways, which prove detrimental to marine fauna. It is anticipated that incorporation of contaminant-specific molecular signatures into current risk assessment paradigms will lead to enhanced wildlife management strategies that minimize the impacts of our industrialized society on marine ecosystems.

Spatio-temporal characterization of retinal opsin gene expression during thyroid hormone-induced and natural development of rainbow trout

The abundance and spatial distribution of retinal cone photoreceptors change during thyroid hormone (TH)-induced and natural development of rainbow trout (Oncorhynchus mykiss). These changes are thought to allow the fish to adapt to different photic environments throughout its life history. To date, the ontogeny of rainbow trout cone photoreceptors has been examined using physiological and morphological approaches. In this study, we extended these observations by measuring opsin gene expression in retinal quadrants during natural and TH-induced development. Gene expression during natural development was investigated in retinae from fish at both parr and smolt stages. The role of TH in modulating opsin gene expression was determined in TH-treated parr and control fish sampled after two, nine, and 22 days of treatment. Total RNA was isolated from each retinal quadrant and steady-state opsin mRNA levels were measured using reverse transcriptase real-time quantitative polymerase chain reaction (QPCR) analysis. Expression of ultraviolet-sensitive opsin (SWS1), rod opsin (RH1), middle wavelength-sensitive opsin (RH2), and long wavelength-sensitive opsin (LWS) transcripts vary spatially in the parr retina. Smolts, compared to parr, had downregulated SWS1 expression in all quadrants, lower LWS expression dorsally, higher RH1 expression nasally, and higher RH2 expression dorsally. In TH-treated parr, SWS1 opsin expression was downregulated in the nasal quadrants by two days. SWS1 displayed the greatest degree of downregulation in all quadrants after nine days of treatment, with an increase in short wavelength-sensitive (SWS2) and RH2 opsin mRNA expression in the temporal quadrants. This study reveals that opsin genes display spatially significant differences within rainbow trout retina in their level of mRNA expression, and that regulation of opsin expression is a dynamic process that is influenced by TH. This is particularly evident for SWS1 gene expression in parr following TH-induced and natural development.

Ontogenetic changes in photoreceptor opsin gene expression in coho salmon(Oncorhynchus kisutch, Walbaum)

SUMMARY Pacific salmonids start life in fresh water then migrate to the sea, after a metamorphic event called smoltification, later returning to their natal freshwater streams to spawn and die. To accommodate changes in visual environments throughout life history, salmon may adjust their spectral sensitivity. We investigated this possibility by examining ontogenetic and thyroid hormone (TH)-induced changes in visual pigments in coho salmon (Oncorhynchus kisutch, Walbaum). Using microspectrophotometry, we measured the spectral absorbance (quantified by λmax) of rods, and middle and long wavelength-sensitive (MWS and LWS) cones in three age classes of coho, representing both freshwater and marine phases. Theλ max of MWS and LWS cones differed among freshwater (alevin and parr) and ocean (smolt) phases. The λmax of rods, on the other hand, did not vary, which is evidence that vitamin A1/A2 visual pigment chromophore ratios were similar among freshwater and ocean phases when sampled at the same time of year. Exogenous TH treatment long wavelength shifted the λmax of rods, consistent with an increase in A2. However, shifts in cones were greater than predicted for a change in chromophore ratio. Real-time quantitative RT-PCR demonstrated that at least two RH2 opsin subtypes were expressed in MWS cones, and these were differentially expressed among alevin, parr and TH-treated alevin groups. Combined with changes in A1/A2 ratio, differential expression of opsin subtypes allows coho to alter the spectral absorbance of their MWS and LWS cones by as much as 60 and 90 nm, respectively. To our knowledge, this is the largest spectral shift reported in a vertebrate photoreceptor.

Triclosan Affects Thyroid Hormone–Dependent Metamorphosis in Anurans

Our original work on Rana catesbeiana (bullfrog) tadpoles exposed to triclosan (TCS) during induction of precocious metamorphosis that was presented in Veldhoen et al. (2006) suggested that frog tadpoles exposed to the antibacterial agent exhibit changes through prometamorphosis. In a recent study, Fort et al. (2010) exposed premetamorphic Xenopus laevis tadpoles to triclosan for 21 days into prometamorphosis. This work, published in Toxological Sciences (Fort et al., 2010), stated that environmentally relevant TCS concentrations do not alter the normal course of thyroid-mediated metamorphosis in this standard anuran model. Herein, we describe the apparent discrepancy between these two studies, and on examination of the data presented in the study of Fort et al. (2010), we do not come to the same conclusion that triclosan had no effects on metamorphosis. Fort et al. (2010) stated that ‘‘larval developmental stage at exposure day 21 was not significantly different from controls based on observed parameters.’’ A major indicator of exposure effect is progression through the developmental stages as defined by Nieuwkoop and Faber (NF; Nieuwkoop and Faber, 1994). The relevant stages in the study are between NF stages 54 and 66; the range of stages attained by NF stage 51 tadpoles after 21 days of exposure. Even though these are numerical stages, they are categorical, defined by distinctive morphological characteristics. The number of tadpoles that were at these stages was reported in Table 3 of the study of Fort et al. (2010), and chi-square analysis was performed. The authors stated that ‘‘Overall, the frequency of each developmental stage in each of the treatment concentrations following 21 days of exposure to TCS was not significantly different from each other (chi-square, p 1⁄4 0.319).’’ It was not clear how the analysis was done, but using the same data as presented in Table 3 in the study of Fort et al. (2010) in a 9 3 5 contingency table with NF 54–56, 63–64, and 65–66 stages grouped and all other stage categories kept separate gave a chi-square 1⁄4 48.7, p 1⁄4 0.029, degrees of freedom 1⁄4 32. In addition, a pairwise 7 3 2 contingency table comparison of the control with each of the TCS treatments individually gave the results shown in Table 1. Therefore, the data presented by Fort et al. (2010) do indeed show that TCS at the three lower exposure concentrations had an effect on the postembryonic development of X. laevis tadpoles. This is consistent with our observations with the bullfrog (Veldhoen et al., 2006.). Moreover, the authors observed that tails from stagematched NF 60 tadpoles showed significantly elevated thyroid hormone receptor b (TRb) transcript levels in the two middle concentrations examined. The 1.5-fold increase observed relative to the control animals was interpreted as ‘‘likely not biologically significant,’’ and the authors point to a lack of concentration dependence of the TCS exposure on altered transcript abundance. Putting this information in context, TRb transcript levels increase in the tadpole tail by approximately sevenfold between NF stages 54 and 60 (Wagner and Helbing,

Use of heterologous cDNA arrays and organ culture in the detection of thyroid hormone-dependent responses in a sentinel frog, Rana catesbeiana.

Despite the extensive use of wildlife species in elucidating important biological processes, very few gene expression tools are available. For example, many frog species with different sensitivities and ecological niches are used as sentinel species for environmental contaminants and as developmental models. However, gene expression analyses have been essentially limited to one laboratory species. In an attempt to extend gene expression analyses to relevant indigenous species, we have developed a frog cDNA array with probes designed against conserved protein-encoding sequences. Changes in gene expression profiles were identified in cultured tail tips of Rana catesbeiana tadpoles during induction of tail regression by exogenous thyroid hormone and are associated with a transition from active cell proliferation to increased apoptotic activity. The expression profiles of selected genes representative of different response patterns were further characterized in tails of tadpoles undergoing natural metamorphosis using de novo designed biomarker probes and quantitative real-time polymerase chain reaction analysis. The results support the cross-species application of cDNA arrays that can direct the development of gene expression biomarkers for indigenous wildlife species.