Direct and indirect photoreactions of chromophoric dissolved organic matter : roles of reactive oxygen species and iron

Thesis (Ph. D.)--Joint Program in Oceanography (Massachusetts Institute of Technology, Dept. of Civil and Environmental Engineering, and the Woods Hole Oceanographic Institution), 2002.Vita.Includes bibliographical references.Photochemical transformations of chromophoric dissolved organic matter (CDOM) are one of the principal processes controlling its fate in coastal waters. The photochemical decomposition of CDOM leads to the formation of a variety of biologically available carbon substrates. Photomineralization of CDOM to dissolved inorganic carbon may constitute a significant flux in the global carbon cycle. Photoreactions ultimately lead to the destruction of the chromophores and hence to the loss of absorption and fluorescence (bleaching), thus acting as a sink for CDOM. Photodecomposition may proceed both via direct photochemical reactions, following absorption of photons by CDOM, or via indirect processes, involving DOM reactions with photochemically generated intermediates such as reactive oxygen species (ROS). The reactions of CDOM with two important ROS, superoxide (02-) and hydroxyl radical (OH), have different consequences. Superoxide reactions with CDOM did not appear to degrade the CDOM. Instead, CDOM catalysed the dismutation of 02- to 02 and HOOH. This reactivity has the effect of limiting the steady-state concentration of 02- in most coastal waters. In contrast, reactions of CDOM with radiolytically produced OH formed CO2 and several low molecular weight carboxylic acids, as well as bleached both the absorption and fluorescence at slow rates. These reactions did not increase the bioavailability of this material to a microbial consortium. Both direct and indirect photochemical processes are expected to be accelerated by the presence of iron.(cont.) However, addition of iron to several coastal seawater samples neither increased the rate of photobleaching nor the apparent quantum yield (AQY) of CO. Similarly, the addition of the siderophore desferrioxamine B did not change the photobleaching rates or the CO AQYs. The addition of 2[mu]M Fe to solutions of Suwannee River Fulvic Acid did not increase the photobleaching rates. In combination with prior results, these findings suggest that indirect photoreactions do not increase the photobleaching rates of CDOM in coastal systems. A model of CDOM photobleaching based on the assumption of negligible indirect photobleaching processes and multiple non-interacting chromophores was created utilizing photobleaching data produced with monochromatic light to calculate the spectra and exponential decay rates of independent components. These components were then used to calculate bleaching spectra for broadband light and compared with actual bleaching spectra.by Jared Verrill Goldstone.Ph.D

Isolation and phylogeny of novel cytochrome P450 genes from tunicates (Ciona spp.) : a CYP3 line in early deuterostomes?

Author Posting. © Elsevier B.V., 2006. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Molecular Phylogenetics and Evolution 40 (2006): 760-771, doi:10.1016/j.ympev.2006.04.017.Cytochromes P450 (CYPs) form a gene superfamily involved in the biotransformation of numerous endogenous and exogenous natural and synthetic compounds. In humans, CYP3A4 is regarded as one of the most important CYPs due to its abundance in liver and its capacity to metabolize more than 50% of all clinically used drugs. It has been suggested that all CYP3s arose from a common ancestral gene lineage that diverged between 800 and 1100 million years ago, before the deuterostome-protostome split. While CYP3s are well known in mammals and have been described in lower vertebrates, they have not been reported in non-vertebrate deuterostomes. Members of the genus Ciona belong to the tunicates, whose lineage is thought to be the most basal among the chordates, and from which the vertebrate line diverged. Here we describe the cloning, exon-intron structure, phylogeny, and estimated expression of four novel genes from Ciona intestinalis. We also describe the gene structure and phylogeny of homologous genes in Ciona savignyi. Comparing these genes with other members of the CYP clan 3, show that the Ciona sequences bear remarkable similarity to vertebrate CYP3A genes, and may be an early deuterostome CYP3 line.These studies were supported in part by NIH grant 2-P42-ES07381 to J.J. Stegeman. Tim Verslycke was supported by the Postdoctoral Scholar Program at the Woods Hole Oceanographic Institution, with funding from the Ocean Life Institute and by a Fellowship of the Belgian American Educational Foundation. Jared Goldstone was supported by a Ruth Kirschstein National Research Service Award (NIH 5F32ES 012794)

Cloning a new cytochrome P450 isoform (CYP356A1) from oyster Crassostrea gigas

Author Posting. © Elsevier B.V., 2008. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Marine Environmental Research 66 (2008): 15-18, doi:10.1016/j.marenvres.2008.02.010.We have cloned the full-length cDNA of the first member of a new cytochrome P450 (CYP) family from the Pacific oyster Crassostrea gigas. This new CYP gene was obtained based on an initial 331 bp fragment previously identified among the list of the differentially expressed genes in oysters exposed to untreated domestic sewage. The full-length CYP has an open reading frame of 1500 bp and based on its deduced aminoacid sequence was classified as a member of a new subfamily, CYP356A1. A phylogenetic analysis showed that CYP356A1 is closely related to members of the CYP17 and CYP1 subfamilies. Semiquantitative RT-PCR was performed to analyze the CYP356A1 expression in different tissues of the oyster (digestive gland, gill, mantle and adductor muscle). Results showed slightly higher CYP356A1 expression in digestive gland and mantle, than the other tissues, indicating a possible role of the CYP356A1 in the xenobiotic biotransformation and/or steroid metabolism.This work was supported by CNPq-Universal to ACDB. ACDB is recipient of Productivity Fellowship from CNPq

Cytochrome P450 diversity and induction by gorgonian allelochemicals in the marine gastropod Cyphoma gibbosum

© The Authors, 2010. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in BMC Ecology 10 (2010): 24, doi:10.1186/1472-6785-10-24.Intense consumer pressure strongly affects the structural organization and function of marine ecosystems, while also having a profound effect on the phenotype of both predator and prey. Allelochemicals produced by prey often render their tissues unpalatable or toxic to a majority of potential consumers, yet some marine consumers have evolved resistance to host chemical defenses. A key challenge facing marine ecologists seeking to explain the vast differences in consumer tolerance of dietary allelochemicals is understanding the biochemical and molecular mechanisms underlying diet choice. The ability of marine consumers to tolerate toxin-laden prey may involve the cooperative action of biotransformation enzymes, including the inducible cytochrome P450s (CYPs), which have received little attention in marine invertebrates despite the importance of allelochemicals in their evolution. Here, we investigated the diversity, transcriptional response, and enzymatic activity of CYPs possibly involved in allelochemical detoxification in the generalist gastropod Cyphoma gibbosum, which feeds exclusively on chemically defended gorgonians. Twelve new genes in CYP family 4 were identified from the digestive gland of C. gibbosum. Laboratory-based feeding studies demonstrated a 2.7- to 5.1-fold induction of Cyphoma CYP4BK and CYP4BL transcripts following dietary exposure to the gorgonian Plexaura homomalla, which contains high concentrations of anti-predatory prostaglandins. Phylogenetic analysis revealed that C. gibbosum CYP4BK and CYP4BL were most closely related to vertebrate CYP4A and CYP4F, which metabolize pathophysiologically important fatty acids, including prostaglandins. Experiments involving heterologous expression of selected allelochemically-responsive C. gibbosum CYP4s indicated a possible role of one or more CYP4BL forms in eicosanoid metabolism. Sequence analysis further demonstrated that Cyphoma CYP4BK/4BL and vertebrate CYP4A/4F forms share identical amino acid residues at key positions within fatty acid substrate recognition sites. These results demonstrate differential regulation of CYP transcripts in a marine consumer feeding on an allelochemical-rich diet, and significantly advance our understanding of both the adaptive molecular mechanisms that marine consumers use to cope with environmental chemical pressures and the evolutionary history of allelochemical-metabolizing enzymes in the CYP superfamily.Financial support for this work was provided by the Ocean Life Institute Tropical Research Initiative Grant (WHOI) to KEW and MEH; the Robert H. Cole Endowed Ocean Ventures Fund (WHOI) to KEW; the National Undersea Research Center - Program Development Proposal (CMRC-03PRMN0103A) to KEW and a National Science Foundation Graduate Research Fellowship to KEW

Caenorhabditis elegans generates biologically relevant levels of genotoxic metabolites from aflatoxin B1 but not benzo[a]pyrene in vivo

Author Posting. © The Authors, 2010. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Toxicological Sciences 118 (2010): 444-453, doi:10.1093/toxsci/kfq295.There is relatively little information regarding the critical xenobiotic-metabolizing cytochrome P450 (CYP) enzymes in Caenorhabditis elegans, despite this organism’s increasing use as a model in toxicology and pharmacology. We carried out experiments to elucidate the capacity of C. elegans to metabolically activate important promutagens via CYPs. Phylogenetic comparisons confirmed an earlier report indicating a lack of CYP1 family enzymes in C. elegans. Exposure to aflatoxin B1 (AFB1), which is metabolized in mammals by CYP1, CYP2, and CYP3 family enzymes, resulted in significant DNA damage in C. elegans. However, exposure to benzo[a]pyrene (BaP), which is metabolized in mammals by CYP1 family enzymes only, produced no detectable damage. To further test whether BaP exposure caused DNA damage, the toxicities of AFB1 and BaP were compared in nucleotide excision repair-deficient (xpa-1) and - proficient (N2) strains of C. elegans. Exposure to AFB1 inhibited growth more in xpa-1 than N2 nematodes, but the growth-inhibitory effects of BaP were indistinguishable in the two strains. Finally, a CYP-NADPH reductase- deficient strain (emb-8) of C. elegans was found to be more resistant to the growth inhibitory effect of AFB1 exposure than N2, confirming that the AFB1- mediated growth inhibition resulted from CYP-mediated metabolism. Together, these results indicate that C. elegans lacks biologically significant CYP1 family-mediated enzymatic metabolism of xenobiotics. Interestingly, we also found that xpa-1 nematodes were slightly more sensitive to chlorpyrifos than were wild-type. Our results highlight the importance of considering differences between xenobiotic metabolism in C. elegans and mammals when using this alternative model in pharmaceutical and toxicological research.This work was supported in part by NIH R21 NS065468 (JNM); the National Toxicology Program Z01ES102046 (WAB), the Intramural Research Program of the National Institute of Environmental Health Sciences Z01ES102045 (JHF). JVG was supported by NIH Grants to John Stegeman (R01-ES015912, and the Superfund Basic Research Program at Boston University 5- P42-ES007381)

The chemical defensome of five model teleost fish

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Eide, M., Zhang, X., Karlsen, O. A., Goldstone, J., Stegeman, J., Jonassen, I., & Goksoyr, A. The chemical defensome of five model teleost fish. Scientific Reports, 11(1), (2021): 10546, https://doi.org/10.1038/s41598-021-89948-0.How an organism copes with chemicals is largely determined by the genes and proteins that collectively function to defend against, detoxify and eliminate chemical stressors. This integrative network includes receptors and transcription factors, biotransformation enzymes, transporters, antioxidants, and metal- and heat-responsive genes, and is collectively known as the chemical defensome. Teleost fish is the largest group of vertebrate species and can provide valuable insights into the evolution and functional diversity of defensome genes. We have previously shown that the xenosensing pregnane x receptor (pxr, nr1i2) is lost in many teleost species, including Atlantic cod (Gadus morhua) and three-spined stickleback (Gasterosteus aculeatus), but it is not known if compensatory mechanisms or signaling pathways have evolved in its absence. In this study, we compared the genes comprising the chemical defensome of five fish species that span the teleosteii evolutionary branch often used as model species in toxicological studies and environmental monitoring programs: zebrafish (Danio rerio), medaka (Oryzias latipes), Atlantic killifish (Fundulus heteroclitus), Atlantic cod, and three-spined stickleback. Genome mining revealed evolved differences in the number and composition of defensome genes that can have implication for how these species sense and respond to environmental pollutants, but we did not observe any candidates of compensatory mechanisms or pathways in cod and stickleback in the absence of pxr. The results indicate that knowledge regarding the diversity and function of the defensome will be important for toxicological testing and risk assessment studies.The work was supported by the Norwegian Research Council as part of the iCOD and iCOD 2.0 projects (Grant Nos. 192441/I30 and 244654/E40), and the dCod 1.0 project (Grant No. 248840) which is part of Centre for Digital Life Norway. The American collaborators were funded by the National Institute of Health (USA) NIH P42ES007381 (Boston University Superfund Center to JJS and JVG), NIH R21HD073805 (JVG) and NHI R01ES029917 (JVG) grants. The Ocean Outlook exchange program funded the trans-Atlantic collaboration

The cytochrome P450 (CYP) superfamily in cnidarians

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Pankov, K., McArthur, A. G., Gold, D. A., Nelson, D. R., Goldstone, J., & Wilson, J. Y. The cytochrome P450 (CYP) superfamily in cnidarians. Scientific Reports, 11(1), (2021): 9834, https://doi.org/10.1038/s41598-021-88700-y.The cytochrome P450 (CYP) superfamily is a diverse and important enzyme family, playing a central role in chemical defense and in synthesis and metabolism of major biological signaling molecules. The CYPomes of four cnidarian genomes (Hydra vulgaris, Acropora digitifera, Aurelia aurita, Nematostella vectensis) were annotated; phylogenetic analyses determined the evolutionary relationships amongst the sequences and with existing metazoan CYPs. 155 functional CYPs were identified and 90 fragments. Genes were from 24 new CYP families and several new subfamilies; genes were in 9 of the 12 established metazoan CYP clans. All species had large expansions of clan 2 diversity, with H. vulgaris having reduced diversity for both clan 3 and mitochondrial clan. We identified potential candidates for xenobiotic metabolism and steroidogenesis. That each genome contained multiple, novel CYP families may reflect the large evolutionary distance within the cnidarians, unique physiology in the cnidarian classes, and/or different ecology of the individual species.This study was supported by grants from the Natural Science Engineering Research Council of Canada in the form of a Discovery Grant (RGPIN-328204-2011 and RGPIN-05767-2016) to J.Y.W. A.G.M. held a Cisco Research Chair in Bioinformatics, supported by Cisco Systems Canada, Inc. K.V.P. was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Undergraduate Student Research Award (USRA). Computer resources were supplied by the McMaster Service Lab and Repository computing cluster, funded in part by grants to A.G.M. from the Canadian Foundation for Innovation (34531 to A.G.M.)

Developmental exposure to non-dioxin-like polychlorinated biphenyls promotes sensory deficits and disrupts dopaminergic and GABAergic signaling in zebrafish

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Brun, N. R., Panlilio, J. M., Zhang, K., Zhao, Y., Ivashkin, E., Stegeman, J. J., & Goldstone, J. Developmental exposure to non-dioxin-like polychlorinated biphenyls promotes sensory deficits and disrupts dopaminergic and GABAergic signaling in zebrafish. Communications Biology, 4(1), (2021): 1129, https://doi.org/10.1038/s42003-021-02626-9.The most abundant polychlorinated biphenyl (PCB) congeners found in the environment and in humans are neurotoxic. This is of particular concern for early life stages because the exposure of the more vulnerable developing nervous system to neurotoxic chemicals can result in neurobehavioral disorders. In this study, we uncover currently unknown links between PCB target mechanisms and neurobehavioral deficits using zebrafish as a vertebrate model. We investigated the effects of the abundant non-dioxin-like (NDL) congener PCB153 on neuronal morphology and synaptic transmission linked to the proper execution of a sensorimotor response. Zebrafish that were exposed during development to concentrations similar to those found in human cord blood and PCB contaminated sites showed a delay in startle response. Morphological and biochemical data demonstrate that even though PCB153-induced swelling of afferent sensory neurons, the disruption of dopaminergic and GABAergic signaling appears to contribute to PCB-induced motor deficits. A similar delay was observed for other NDL congeners but not for the potent dioxin-like congener PCB126. The effects on important and broadly conserved signaling mechanisms in vertebrates suggest that NDL PCBs may contribute to neurodevelopmental abnormalities in humans and increased selection pressures in vertebrate wildlife.This work was supported by the Swiss National Science Foundation P2EZP2_165200 (NRB), the Boston University Superfund Research Program NIH 5P42ES007381 (J.J.S. and J.V.G.), the Woods Hole Center for Oceans and Human Health (NIH: P01ES021923 and P01ES028938; NSF: OCE-1314642 and OCE-1840381) (N.R.B., J.M.P., and J.J.S.), and the National Natural Science Foundation of China 22006099 (K.Z. and Y.Z.) and the Shanghai Pujiang Program 19PJ1404900 (K.Z. and Y.Z.)

The new vertebrate CYP1C family : cloning of new subfamily members and phylogenetic analysis

Author Posting. © The Authors, 2005. This is the author's version of the work. It is posted here by permission of Elsevier B. V. for personal use, not for redistribution. The definitive version was published in Biochemical and Biophysical Research Communications 331 (2005): 1016-1024, doi:10.1016/j.bbrc.2005.03.231.Two novel CYP1 genes from teleost fish constituting a new subfamily have been cloned. These paralogous sequences are designated CYP1C1 and CYP1C2. Both genes were initially obtained from untreated scup Stenotomus chrysops tissues by RT-PCR and RACE. Scup CYP1C1 and CYP1C2 code for 524 and 525 amino acids, respectively, and share 80-81% identity at the nucleotide and amino acid levels. Orthologues of CYP1C1 and CYP1C2 were identified in genome databases for other fish species, and both CYP1B1 and CYP1C1 were cloned from zebrafish (Danio rerio). Phylogenetic analysis shows that CYP1Cs and CYP1Bs constitute a sister clade to the CYP1As. Analysis of sequence domains likely to have functional significance suggests the two CYP1Cs in scup may have catalytic functions and/or substrate specificity that differ from each other and from those of mammalian CYP1Bs or CYP1As. RT-PCR results indicate that CYP1C1 and CYP1C2 are variously expressed in several scup organs.This work was supported by EPA grant R 827102-01-0 and NIH grants 5 P42-ES07381 and ES04696. JVG is supported by a Ruth L. Kirschstein NRSA Fellowship (F32 ES012794)