RESUMO
Human rotavirus (HRV) is a major cause of childhood diarrhea in developing countries where widespread malnutrition contributes to the decreased oral vaccine efficacy and increased prevalence of other enteric infections, which are major concerns for global health. Neonatal gnotobiotic (Gn) piglets closely resemble human infants in their anatomy, physiology, and outbred status, providing a unique model to investigate malnutrition, supplementations, and HRV infection. To understand the molecular signatures associated with immune enhancement and reduced diarrheal severity by Escherichia coli Nissle 1917 (EcN) and tryptophan (TRP), immunological responses and global nontargeted metabolomics and lipidomics approaches were investigated on the plasma and fecal contents of malnourished pigs transplanted with human infant fecal microbiota and infected with virulent (Vir) HRV. Overall, EcN + TRP combined (rather than individual supplement action) promoted greater and balanced immunoregulatory/immunostimulatory responses associated with greater protection against HRV infection and disease in malnourished humanized piglets. Moreover, EcN + TRP treatment upregulated the production of several metabolites with immunoregulatory/immunostimulatory properties: amino acids (N-acetylserotonin, methylacetoacetyl-CoA), lipids (gamma-butyrobetaine, eicosanoids, cholesterol-sulfate, sphinganine/phytosphingosine, leukotriene), organic compound (biliverdin), benzenoids (gentisic acid, aminobenzoic acid), and nucleotides (hypoxathine/inosine/xanthine, cytidine-5'-monophosphate). Additionally, the levels of several proinflammatory metabolites of organic compounds (adenosylhomocysteine, phenylacetylglycine, urobilinogen/coproporphyrinogen) and amino acid (phenylalanine) were reduced following EcN + TRP treatment. These results suggest that the EcN + TRP effects on reducing HRV diarrhea in neonatal Gn pigs were at least in part due to altered metabolites, those involved in lipid, amino acid, benzenoids, organic compounds, and nucleotide metabolism. Identification of these important mechanisms of EcN/TRP prevention of HRV diarrhea provides novel targets for therapeutics development. IMPORTANCE Human rotavirus (HRV) is the most common cause of viral gastroenteritis in children, especially in developing countries, where the efficacy of oral HRV vaccines is reduced. Escherichia coli Nissle 1917 (EcN) is used to treat enteric infections and ulcerative colitis while tryptophan (TRP) is a biomarker of malnutrition, and its supplementation can alleviate intestinal inflammation and normalize intestinal microbiota in malnourished hosts. Supplementation of EcN + TRP to malnourished humanized gnotobiotic piglets enhanced immune responses and resulted in greater protection against HRV infection and diarrhea. Moreover, EcN + TRP supplementation increased the levels of immunoregulatory/immunostimulatory metabolites while decreasing the production of proinflammatory metabolites in plasma and fecal samples. Profiling of immunoregulatory and proinflammatory biomarkers associated with HRV perturbations will aid in the identification of treatments against HRV and other enteric diseases in malnourished children.
Assuntos
Infecções por Escherichia coli , Transplante de Microbiota Fecal , Desnutrição , Infecções por Rotavirus , Triptofano , Animais , Humanos , Lactente , Aminobenzoatos , Biliverdina/metabolismo , Colesterol , Coenzima A/metabolismo , Coproporfirinogênios , Citidina/metabolismo , Diarreia , Escherichia coli/metabolismo , Vida Livre de Germes , Inosina/metabolismo , Lipídeos , Desnutrição/terapia , Desnutrição/complicações , Metaboloma , Microbiota , Nucleotídeos/metabolismo , Fenilalanina/metabolismo , Rotavirus , Sulfatos , Suínos , Triptofano/farmacologia , Urobilinogênio/metabolismo , XantinasRESUMO
Bilirubin, a natural intermediate in heme degradation, is a valuable Chinese medicine used in more than 50 traditional Chinese medicine (TCM) preparations. At present, bilirubin is mainly produced by extraction from pig bile, but a shortage of the raw material has increased the price, to about US$10,000/kg in the Chinese market. Biliverdin, the precursor of bilirubin, is more abundant and less expensive than bilirubin, but it is not used in TCM. Thus, the biotransformation of biliverdin by biliverdin reductase (BvdR) may be a practical way to produce bilirubin. In this study, the codon-optimized gene of biliverdin reductase (mbvdR) from the cyanobacterium Synechocystis was cloned into Escherichia coli BL21(DE3), and the conditions for BL21-mBvdR expressing BvdR were optimized. Resting BL21-mBvdR cells were employed as biocatalysts to biotransform biliverdin to bilirubin. At a concentration of biliverdin substrate of 450 mg/L in the reaction mixture, the bilirubin content in dry cells reached 20.8 ± 0.8 mg/g, with a conversion yield of 72.3%. Therefore, recombinant E. coli expressing BvdR can be applied to biotransform biliverdin to bilirubin, providing a potential alternative process for bilirubin production.
Assuntos
Biliverdina , Cianobactérias , Animais , Bilirrubina/metabolismo , Biliverdina/genética , Biliverdina/metabolismo , Biotransformação , Cianobactérias/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , SuínosRESUMO
OBJECTIVE: Heme oxygenase-1 (HO-1) plays a critical protective role in various insults-induced acute lung injury (ALI) through its strong anti-inflammatory, anti-oxidant, and anti-apoptotic properties, but its protective role and mechanism on seawater aspiration-induced acute lung injury remains unclear. This study aimed to explore the therapeutic potential and mechanism of HO-1 to attenuate seawater aspiration-induced ALI in vivo and in vitro. METHODS: The viability and invasion of A549 cell were analyzed through cell counting kit-8 and lactate dehydrogenase release assay; the transcriptional level of inflammatory cytokines (TNF-α, IL-6, IL-8 and MCP-1) and cell proliferation-related cytokines (FoxM1, Ccnb1 and Cdc25C) in seawater-treated A549 cell were tested by qPCR; apoptotic cells were analyzed by flow cytometryd; HO-1mRNA and protein were determined by qPCR and western blotting; the fluorescent indicators (DCFH-DA, dihydroethidium, MitoSox Red and Fluo-4) were used to monitor generation of ROS and mitochondrial function. The lung wet/dry weight radio and lactate dehydrogenase activity, Sirius red staining, TUNEL assay and immunohistochemical staining with anti-pan Cytokeratin antibody were analyzed in seawater-drowning mice. The role of HO-1 on seawater-drowning pulmonary injury was explored via HO-1 activity inhibitors (Zinc protoporphyrin) in vitro and in vivo. RESULTS: Seawater exposure decreased the cellular viability, increased the production of pro-inflammatory cytokines (IL-6, IL-8 and TNF-α), induced cellular apoptosis and inhibited the expression of cell proliferation-related cytokines (FoxM1, Ccnb1 and Cdc25C). Moreover, seawater exposure led to mitochondrial dysfunction in A549 cells. Supplement of HO-1 sepcific inducer (heme) or its catalytic product (biliverdin) significantly attenuated seawater-induced A549 damage and promoted cell proliferation. However, Zinc protoporphyrin abolished the beneficial effects of HO-1 on seawater drowning-induced pulmonary tissue injury. CONCLUSION: HO-1 attenuates seawater drowning-induced lung injury by its anti-inflammatory, anti-oxidative, and anti-apoptosis function.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Afogamento/metabolismo , Heme Oxigenase-1/metabolismo , Células A549 , Animais , Biliverdina/metabolismo , Proliferação de Células , Citocinas/genética , Humanos , Inflamação/metabolismo , Masculino , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Água do MarRESUMO
Photothermal nanomaterials that integrate multimodal imaging and therapeutic functions provide promising opportunities for noninvasive and targeted diagnosis and treatment in precision medicine. However, the clinical translation of existing photothermal nanoagents is severely hindered by their unclear physiological metabolism, which makes them a strong concern for biosafety. Here, the utilization of biliverdin (BV), an endogenic near-infrared (NIR)-absorbing pigment with well-studied metabolic pathways, to develop photothermal nanoagents with the aim of providing efficient and metabolizable candidates for tumor diagnosis and therapy, is demonstrated. It is shown that BV nanoagents with intense NIR absorption, long-term photostability and colloidal stability, and high photothermal conversion efficiency can be readily constructed by the supramolecular multicomponent self-assembly of BV, metal-binding short peptides, and metal ions through the reciprocity and synergy of coordination and multiple noncovalent interactions. In vivo data reveal that the BV nanoagents selectively accumulate in tumors, locally elevate tumor temperature under mild NIR irradiation, and consequently induce efficient photothermal tumor ablation with promising biocompatibility. Furthermore, the BV nanoagents can serve as a multimodal contrast for tumor visualization through both photoacoustic and magnetic resonance imaging. BV has no biosafety concerns, and thereby offers a great potential in precision medicine by integrating multiple theranostic functions.
Assuntos
Biliverdina/metabolismo , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Animais , Biliverdina/química , Complexos de Coordenação/química , Xenoenxertos , Humanos , Raios Infravermelhos , Células MCF-7 , Manganês/química , Camundongos , Imagem Multimodal , Imagem Óptica , Técnicas Fotoacústicas , Fototerapia , Nanomedicina TeranósticaRESUMO
The eukaryotic heme oxygenases (HOs) (E.C. 1.14.99.3) convert heme to biliverdin, iron, and carbon monoxide (CO) in three successive oxygenation steps. Pathogenic bacteria require iron for survival and infection. Extracellular heme uptake from the host plays a critical role in iron acquisition and virulence. In the past decade, several HOs required for the release of iron from extracellular heme have been identified in pathogenic bacteria, including Corynebacterium diphtheriae, Neisseriae meningitides, and Pseudomonas aeruginosa. The bacterial enzymes were shown to be structurally and mechanistically similar to those of the canonical eukaryotic HO enzymes. However, the recent discovery of the structurally and mechanistically distinct noncanonical heme oxygenases of Staphylococcus aureus and Mycobacterium tuberculosis has expanded the reaction manifold of heme degradation. The distinct ferredoxin-like structural fold and extreme heme ruffling are proposed to give rise to the alternate heme degradation products in the S. aureus and M. tuberculosis enzymes. In addition, several "heme-degrading factors" with no structural homology to either class of HOs have recently been reported. The identification of these "heme-degrading proteins" has largely been determined on the basis of in vitro heme degradation assays. Many of these proteins were reported to produce biliverdin, although no extensive characterization of the products was performed. Prior to the characterization of the canonical HO enzymes, the nonenzymatic degradation of heme and heme proteins in the presence of a reductant such as ascorbate or hydrazine, a reaction termed "coupled oxidation", served as a model for biological heme degradation. However, it was recognized that there were important mechanistic differences between the so-called coupled oxidation of heme proteins and enzymatic heme oxygenation. In the coupled oxidation reaction, the final product, verdoheme, can readily be converted to biliverdin under hydrolytic conditions. The differences between heme oxygenation by the canonical and noncanonical HOs and coupled oxidation will be discussed in the context of the stabilization of the reactive Fe(III)-OOH intermediate and regioselective heme hydroxylation. Thus, in the determination of heme oxygenase activity in vitro, it is important to ensure that the reaction proceeds through successive oxygenation steps. We further suggest that when bacterial heme degradation is being characterized, a systems biology approach combining genetics, mechanistic enzymology, and metabolite profiling should be undertaken.
Assuntos
Bactérias/enzimologia , Biliverdina/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Biliverdina/química , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Corynebacterium diphtheriae/enzimologia , Enzimas , Heme/química , Heme Oxigenase (Desciclizante)/química , Hidroxilação , Ferro/química , Ferro/metabolismo , Neisseriaceae/enzimologia , Oxirredução , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/enzimologia , Yin-YangRESUMO
Immune function can be modulated by multiple physiological factors, including nutrition and reproductive state. Because these factors can vary throughout an individual's lifetime as a result of environmental conditions (affecting nutrition) or life-history stage (e.g. entering the adult reproduction stage), we must carefully examine the degree to which developmental versus adult conditions shape performance of the immune system. We investigated how variation in dietary access to carotenoid pigments - a class of molecules with immunostimulatory properties that females deposit into egg yolks - during three different developmental time points affected adult immunological and reproductive traits in female mallard ducks (Anas platyrhynchos). In males and females of other avian species, carotenoid access during development affects carotenoid assimilation ability, adult sexual ornamentation and immune function, while carotenoid access during adulthood can increase immune response and reproductive investment (e.g. egg-laying capacity, biliverdin deposition in eggshells). We failed to detect effects of developmental carotenoid supplementation on adult immune function [phytohemagglutinin-induced cutaneous immune response, antibody production in response to the novel antigen keyhole limpet hemocyanin (KLH), or oxidative burst, assessed by changes in circulating nitric oxide levels], carotenoid-pigmented beak coloration, ovarian development, circulating carotenoid levels or concentration of bile pigments in the gall bladder. However, we did uncover positive relationships between circulating carotenoid levels during adulthood and KLH-specific antibody production, and a negative relationship between biliverdin concentration in bile and KLH-specific antibody production. These results are consistent with the view that adult physiological parameters better predict current immune function than do developmental conditions, and highlight a possible, previously unstudied relationship between biliverdin and immune system performance.
Assuntos
Fenômenos Fisiológicos da Nutrição Animal/imunologia , Biliverdina/metabolismo , Carotenoides/metabolismo , Dieta , Patos/imunologia , Fatores Etários , Análise de Variância , Animais , Carotenoides/administração & dosagem , Feminino , Hemocianinas/imunologia , Óxido Nítrico/metabolismo , Fotoperíodo , Explosão Respiratória/imunologia , Pele/imunologiaRESUMO
OBJECTIVES: Heme oxygenase-1 (HO-1) which degrades Heme to free iron, biliverdin and carbon monoxide (CO) plays an important role in inflammation. There are, however, conflicting data concerning the role of HO-1 in rheumatoid arthritis (RA) and the therapeutic potential of individual heme degradation products remains to be determined. We therefore investigated the effect of CO and biliverdin upon therapeutic administration in the murine collagen induced arthritis (CIA) model of RA. METHODS: CIA was induced in DBA/1 mice. Anti-CII antibody levels were determined by ELISA. Mice were scored for paw swelling and grip strength. After the first clinical signs of arthritis one group of animals was treated with biliverdin, the second group was treated with CO. After 60 days all animals were sacrificed and analysed for histomorphological signs of arthritis. RESULTS: All animals immunised with CII developed serum anti-CII antibodies. Antibody levels were decreased in the CO-treated group. Both, Biliverdin and the CO-treated animals, showed an improvement in clinical disease activity. Histological analysis revealed significantly less inflammation, erosion and reduced numbers of osteoclasts in CO-treated animals only, whereas cartilage degradation was prevented in both biliverdin and CO-treated animals. CONCLUSIONS: Our data demonstrate a beneficial effect of CO, in particular, and biliverdin, on inflammation and bone destruction in the CIA mouse model.
Assuntos
Artrite Experimental/tratamento farmacológico , Biliverdina/uso terapêutico , Monóxido de Carbono/uso terapêutico , Heme Oxigenase-1/metabolismo , Articulações/efeitos dos fármacos , Administração por Inalação , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Biliverdina/administração & dosagem , Biliverdina/metabolismo , Monóxido de Carbono/administração & dosagem , Monóxido de Carbono/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Articulações/metabolismo , Articulações/patologia , CamundongosRESUMO
Despite a century of research, several clinically relevant areas of bilirubin biochemistry remain controversial, poorly understood, or unrecognized. These include: (i) The structure and molecularity of bilirubin under physiological environments such as membranes, brain tissue and when bound to proteins. Related to this is the large number of structurally different bilirubin species that may occur in blood under pathological conditions and their potential effects on measurements of bilirubin and free bilirubin. (ii) The mechanism of phototherapy, the neurotoxicity of the photoisomers produced and their influence on measurements of bilirubin and free bilirubin. (iii) The role of membrane transporters in the passage of unconjugated bilirubin across the placenta, intestine, vascular epithelium, blood-brain barrier, and into the liver. (iv) Biochemical mechanisms of bilirubin toxicity, pharmacologic prevention of kernicterus, the contribution of bilirubin to antioxidant defenses, and the practical value of free bilirubin measurements for identifying infants at most risk of kernicterus.
Assuntos
Bilirrubina/química , Bilirrubina/metabolismo , Biliverdina/química , Biliverdina/metabolismo , Hiperbilirrubinemia Neonatal/metabolismo , Animais , Bilirrubina/sangue , Biliverdina/sangue , Humanos , Hiperbilirrubinemia Neonatal/prevenção & controle , Recém-Nascido , Fígado/metabolismo , Modelos Moleculares , FototerapiaRESUMO
Glutathione (GSH) and bilirubin are prominent endogenous antioxidant cytoprotectants. Despite tissue levels that are thousands of times lower than GSH, bilirubin is effective because of the biosynthetic cycle wherein it is generated from biliverdin by biliverdin reductase (BVR). When bilirubin acts as an antioxidant, it is oxidized to biliverdin, which is immediately reduced by BVR to bilirubin. Why does the body employ both of these 2 distinct antioxidant systems? We show that the water-soluble GSH primarily protects water soluble proteins, whereas the lipophilic bilirubin protects lipids from oxidation. Mice with deletion of heme oxygenase-2, which generates biliverdin, display greater lipid than protein oxidation, while the reverse holds for GSH depletion. RNA interference depletion of BVR increases oxidation of lipids more than protein. Depletion of BVR or GSH augments cell death in an oxidant-specific fashion.
Assuntos
Bilirrubina/fisiologia , Glutationa/fisiologia , Antioxidantes , Bilirrubina/deficiência , Bilirrubina/metabolismo , Biliverdina/metabolismo , Citoproteção , Glutationa/deficiência , Glutationa/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Peroxidação de Lipídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/deficiência , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Proteínas/metabolismoRESUMO
BACKGROUND: Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function. METHODS/RESULTS: Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1. CONCLUSIONS: We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.
Assuntos
Antioxidantes/farmacologia , Citoproteção , Células Endoteliais/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Ácidos Heptanoicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fatores de Transcrição Kruppel-Like/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Pirróis/farmacologia , Atorvastatina , Bilirrubina/metabolismo , Biliverdina/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/enzimologia , Células Endoteliais/metabolismo , Indução Enzimática , Inibidores Enzimáticos/farmacologia , Ferritinas/genética , Ferritinas/metabolismo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Quelantes de Ferro/farmacologia , Fatores de Transcrição Kruppel-Like/genética , Ácido Mevalônico/farmacologia , Oxidantes/farmacologia , Prenilação , Regiões Promotoras Genéticas/efeitos dos fármacos , Protoporfirinas/farmacologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Terpenos/farmacologiaRESUMO
Heme oxygenase (HO) is the rate limiting enzyme in the breakdown of heme into carbon monoxide (CO), iron and biliverdin. To date three mammalian HO isozymes have been identified, and only one of them (HO-1) is inducible. The products of HO-catalyzed reaction, particularly CO and biliverdin/bilirubin have been shown to exert protective effects in the liver against different noxious stimuli. The results of experimental data show that induction of HO-1 expression contributes to protection against liver damage induced by several chemical compounds such as acetaminophen.
Assuntos
Heme Oxigenase-1/metabolismo , Heme/metabolismo , Hepatopatias/enzimologia , Hepatopatias/prevenção & controle , Fígado/enzimologia , Acetaminofen/efeitos adversos , Animais , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Catálise/efeitos dos fármacos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas , Citoproteção/efeitos dos fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Indução Enzimática , Alho/química , Heme/química , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/efeitos dos fármacos , Humanos , Isoenzimas/genética , Fígado/efeitos dos fármacos , Estresse Oxidativo , Substâncias Protetoras/farmacologiaRESUMO
The oxidative cleavage of heme to release iron is a mechanism by which some bacterial pathogens can utilize heme as an iron source. The pigA gene of Pseudomonas aeruginosa is shown to encode a heme oxygenase protein, which was identified in the genome sequence by its significant homology (37%) with HemO of Neisseria meningitidis. When the gene encoding the neisserial heme oxygenase, hemO, was replaced with pigA, we demonstrated that pigA could functionally replace hemO and allow for heme utilization by neisseriae. Furthermore, when pigA was disrupted by cassette mutagenesis in P. aeruginosa, heme utilization was defective in iron-poor media supplemented with heme. This defect could be restored both by the addition of exogenous FeSO4, indicating that the mutant did not have a defect in iron metabolism, and by in trans complementation with pigA from a plasmid with an inducible promoter. The PigA protein was purified by ion-exchange chromotography. The UV-visible spectrum of PigA reconstituted with heme showed characteristics previously reported for other bacterial and mammalian heme oxygenases. The heme-PigA complex could be converted to ferric biliverdin in the presence of ascorbate, demonstrating the need for an exogenous reductant. Acidification and high-performance liquid chromatography analysis of the ascorbate reduction products identified a major product of biliverdin IX-beta. This differs from the previously characterized heme oxygenases in which biliverdin IX-alpha is the typical product. We conclude that PigA is a heme oxygenase and may represent a class of these enzymes with novel regiospecificity.
Assuntos
Proteínas de Bactérias/genética , Heme Oxigenase (Desciclizante)/fisiologia , Heme/metabolismo , Neisseria meningitidis/enzimologia , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Biliverdina/metabolismo , Teste de Complementação Genética , Heme Oxigenase (Desciclizante)/genética , Peróxido de Hidrogênio/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Mutação , Pseudomonas putida/enzimologia , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
In order to perform environmental risk assessments with regard to oil contamination in the sea, it is important to obtain knowledge about threshold levels for possible adverse effects in marine organisms. With this objective in mind, selected biomarkers were studied in Atlantic cod (Gadus morhua) chronically exposed to mechanically dispersed crude oil. The fish were exposed for 30 days in a continuous flow system to nominal concentrations of 0.06, 0.25 and 1 ppm North Sea crude oil. Fish were sampled five times during the exposure period. In addition, the 1 ppm group and the control group were sampled 1 week after the end of exposure. Polyaromatic hydrocarbon (PAH) concentrations in the seawater were analysed regularly by direct fluorescence and, at one occasion, by gas chromatography with mass spectrographic detection (GC/MS) measurements. Liver samples were analysed for parent PAH levels by means of GC/MS measurements, and PAH metabolites in bile were analysed by means of fixed wavelength fluorescence. Cytochrome P450 induction in liver was estimated by ethoxyresorufin-O-deethylase (EROD) activity, and hepatic DNA adducts were analysed by the 32P-postlabelling assay. The parent PAH concentrations in liver showed peak levels 3 days after the start of exposure, followed by a reduction towards the end of the experiment. In contrast, the PAH metabolites in bile and EROD activity showed generally increasing levels throughout the whole exposure period, indicating an increased biotransformation efficiency. The level of DNA adducts in the 1 ppm group showed a stable increase during the entire exposure period. Only a slight, non-significant decrease in DNA adduct levels was observed after 7 days of recovery in clean water. Exposure-dependent responses were observed for all three biomarkers. The lowest nominal concentration of dispersed oil in water, 0.06 ppm, corresponded to a measured total PAH concentration in the water of 0.3 ppb. Atlantic cod exposed to this concentration showed increased levels of PAH metabolites in bile and a slight induction of CYP1A, as well as formation of DNA adducts when compared with control fish. Particularly noteworthy is the detection of DNA adducts at such a low exposure concentration of oil in water, which, to our knowledge, is a novel finding. These dose-response data may serve as useful contributions when assessing environmental risk with regard to marine oil pollution.
Assuntos
Bile/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Adutos de DNA/metabolismo , Peixes/metabolismo , Petróleo/toxicidade , Poluentes Químicos da Água/toxicidade , Ácido p-Aminoipúrico/metabolismo , Animais , Bile/efeitos dos fármacos , Biliverdina/análise , Biliverdina/metabolismo , Biomarcadores , Citocromo P-450 CYP1A1/metabolismo , Água Doce/análise , Cromatografia Gasosa-Espectrometria de Massas , Fígado/química , Fígado/metabolismo , Controle de Qualidade , Fatores de Risco , Espectrometria de FluorescênciaRESUMO
The cDNAs encoding full-length type A and B phytochromes (phyA and phyB, respectively) from potato were expressed in inducible yeast systems (Saccharomyces cerevisiae and Pichia pastoris). In addition, a deletion mutant of phyB (delta 1-74) was expressed. The apoproteins were reconstituted into chromoproteins by incorporation of the native chromophore, phytochromobilin (P phi B), and of phycocyanobilin (PCB). The incorporation of P phi B yielded chromoproteins with difference absorptions lambda max at 660 and 712 nm (Pr and Pfr, respectively) for phyA, and at 665 and 723 nm for phyB. All difference maxima of PCB phytochromes are blue-shifted by several nanometers with respect to those obtained with the P phi B chromophore. The deletion construct with PCB shows difference absorption maxima at 652 and 705 nm with the Pfr absorbance considerably reduced. Time-resolved kinetic analysis of a phyB-type phytochrome by nanosecond flash photolysis was performed for the first time. Recombinant full-length phyB afforded transient absorbance changes similar (but not identical) to those of phyA from Avena, whereas the kinetic behavior of these intermediates was very different. Contrary to phyA from Avena, the I700 intermediate from phyB reconstituted with either PCB or P phi B decayed following single exponential kinetics with a lifetime of 87 or 84 microseconds, respectively, at 10 degrees C. The formation of Pfr of PCB-containing recombinant phyB (phyB-PCB) could be fitted with three lifetimes of 9, 127, and 728 ms. The corresponding lifetimes of phyB-P phi B are 22.5, 343, and 2083 ms. Whereas for phyB-PCB all three millisecond lifetimes are related to the formation of Pfr, the 2 s component of phyB-P phi B is concomitant with a rapid recovery of Pr. For recombinant potato phyA and delta 1-74 phyB, no time-resolved data could be obtained due to the limited quantities available. As described for phytochromes of other dicotelydons, the Pfr forms of full-length phyA and PhyB of potato underwent rapid dark conversion to Pr.