Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Biological activities associated with the volatile compound 2,5-bis(1-methylethyl)-pyrazine
    Janssens, Thierry K.S. ; Tyc, Olaf ; Besselink, Harrie ; Boer, Wietse de; Garbeva, Paolina - \ 2019
    FEMS Microbiology Letters 366 (2019)3. - ISSN 0378-1097
    Burkholderia - Paenibacillus - mode of action - pyrazine - toxicity - transcriptional reporter assays - volatiles

    Pyrazines are 1,4-diazabenzene-based volatile organic compounds and known for their broad-spectrum antimicrobial activity. In the present study, we assessed the antimicrobial activity of 2,5-bis(1-methylethyl)-pyrazine, produced by Paenibacillus sp. AD87 during co-culture with Burkholderia sp. AD24. In addition, we were using transcriptional reporter assays in E. coli and mammalian cells to decipher the possible mode of action. Bacterial and mammalian luciferase reporter strains were deployed to elucidate antimicrobial and toxicological effects of 2,5-bis(1-methylethyl)-pyrazine. At high levels of exposure, 2,5-bis(1-methylethyl)-pyrazine exerted strong DNA damage response. At lower concentrations, cell-wall damage response was observed. The activity was corroborated by a general toxicity reporter assay in E. coli ΔampD, defective in peptidoglycan turnover. The maximum E. coli cell-wall stress activity was measured at a concentration close to the onset of the mammalian cytotoxicity, while other adverse outcome pathways, such as the activation of aryl hydrocarbon and estrogenic receptor, the p53 tumour suppressor and the oxidative stress-related Nrf2 transcription factor, were induced at elevated concentrations compared to the response of mammalian cells. Because of its broad-spectrum antimicrobial activity at lower concentrations and the relatively low mammalian toxicity, 2,5-bis(1-methylethyl)-pyrazine is a potential bio-based fumigant with possible applications in food industry, agriculture or logistics.

    Use of proteomics to detect sex-related differences in effects of toxicants : implications for using proteomics in toxicology
    Rietjens, Ivonne M.C.M. ; Vervoort, Jacques ; Maslowska-Górnicz, Anna ; Brink, Nico Van den; Beekmann, Karsten - \ 2018
    Critical Reviews in Toxicology 48 (2018)8. - ISSN 1040-8444 - p. 666 - 681.
    mode of action - proteomics - Sex related differences - state-of-the-art - toxicants

    This review provides an overview of results obtained when using proteome analysis for detecting sex-based differences in response to toxicants. It reveals implications to be taken into account when considering the use of proteomics in toxicological studies. It appears that results may differ when studying the same chemical in the same species in different target tissues. Another result of interest is the limited dose-response behavior of differential abundance patterns observed in studies where more than one dose level is tested. It is concluded that use of proteomics to study differences in modes of action of toxic compounds is an active area of research. The examples from use of proteomics to study sex-dependent differences also reveal that further studies are needed to provide reliable insight in modes of action, novel biomarkers or even novel therapies. To eventually reach this aim for this and other toxicological endpoints, it is essential to consider background variability, consequences of timing of toxicant administration, dose-response behavior, relevant species and target organ, species and organ variability and the presence of proteoforms.

    Importance of release location on the mode of action of butyrate derivatives in the avian gastrointestinal tract
    Moquet, P.C.A. ; Onrust, L. ; Immerseel, F. Van; Ducatelle, R. ; Hendriks, W.H. ; Kwakkel, R.P. - \ 2016
    Worlds Poultry Science Journal (2016). - ISSN 0043-9339 - p. 61 - 80.
    broiler - butyrate - mode of action - release location

    In the field of animal nutrition, butyrate is used as a zootechnical ingredient and can be used as an unprotected salt or in the form of protected derivatives such as butyrate glycerides or butyrate-loaded matrices. Dietary butyrate supplementation has been shown to improve growth performance and resilience of broiler chickens through distinct mechanisms, operating on both eukaryotic and prokaryotic cells. Firstly, butyrate influences endogenous avian cells in multiple ways: it is an agonist of free-fatty acid receptors, an inhibitor of pro-inflammatory pathways, an epigenetic modulating agent and acts as an energy source. Secondly, butyrate influences the microbiota residing in the avian gastrointestinal tract (GIT) as a result of its bacteriostatic properties. The responses, e.g. changes in growth performance, gut morphology, carcass traits or nutrient digestibility of chickens, to dietary butyrate supplementation are inconsistent with factors such as additive inclusion level, diet composition, age and health status of the bird modulating the effects of butyrate and its derivatives. For many derivatives, the precise GIT segment wherein butyrate is released is unclear. Release location may affect the observed responses to butyrate given the diversity of cell types and pH conditions encountered throughout the gastrointestinal tract of poultry, and the differences in microbiota composition in the different gut segments. As a consequence, our understanding of the mode of action of butyrate is hampered. Characterisation of existing derivatives and development of targeted-release formulations are, therefore, important to gain insight in the different physiological effects butyrate can elicit in broiler chickens.

    Samen leren anders te snoeien : Praktijknetwerk, een samenwerking tussen Combinatie Mauritz BV en Huverba BV en de netwerkpartners Damcon BV en Praktijkonderzoek Plant & Omgeving (WUR-PPO-Boomkwekerij)
    Baltissen, A.H.M.C. ; Sluis, B.J. van der - \ 2013
    Lisse : Praktijkonderzoek Plant & Omgeving BBF - 30
    boomteelt - straatbomen - snoeien - mechanische methoden - proeven op proefstations - werkwijze - cultuurmethoden - kwekers - inventarisaties - vergelijkend onderzoek - automatisering - arboriculture - street trees - pruning - mechanical methods - station tests - mode of action - cultural methods - growers - inventories - comparative research - automation
    Snoeien bepaalt de kwaliteit van jonge laanbomen en is daarmee van belang voor de marktafzet. Deskundige snoei is belangrijk om evenwicht tussen de boven- en ondergrondse delen van jonge laanbomen tijdens de teelt op peil te houden. Ook bij het uitplanten en tijdens de verzorging daarna is goede snoei essentieel. Snoeien is nu een handmatige arbeidsintensieve teeltmaatregel. Voortgaande mechanisering en automatisering is van belang om de kosten laag te houden en voldoende rendement te halen. Ook de schaarste aan aanbod van arbeid op de agrarische arbeidsmarkt is een knelpunt. Het wordt steeds moeilijker om personeel te vinden voor deze noodzakelijke teeltmaatregel. Het snoeien mechaniseren of op termijn zelfs robotiseren om het arbeidsprobleem op te lossen, is dus een mogelijke oplossingsrichting om de genoemde knelpunten op te lossen. Uit de inventarisatie over mechanisering/automatisering van het snoeien blijkt dat de wijze van snoeien verschilt per ondernemer, boom, grondsoort, klimaat, functie, mogelijke klant. Op deze wijze is mechanisering van het snoeien een lastig traject, onmogelijk zelfs. Dat was aanleiding om anders tegen snoeien te gaan aankijken. Kan het snoeien anders zodat mechanisering wel mogelijk is?
    Adviesbasis voor de bemesting van akkerbouwgewassen
    Haan, J.J. de; Geel, W.C.A. van - \ 2013
    Kennisakker.nl 2013 (2013)20 maart.
    akkerbouw - gewassen - mest - organische meststoffen - samenstelling - werkwijze - stikstof - fosfaat - kalium - bemesting - arable farming - crops - manures - organic fertilizers - composition - mode of action - nitrogen - phosphate - potassium - fertilizer application
    De Adviesbasis voor de bemesting van akkerbouwgewassen bevat de meest actuele bemestingsadviezen. Deze adviezen zijn vastgesteld door de Commissie Bemesting Akkerbouw/Vollegrondsgroenteteelt, die bestaat uit vertegenwoordigers van bedrijfsleven, onderzoek en voorlichting. De adviezen hebben een landbouwkundige grondslag, dat wil zeggen gebruik van het advies leidt tot een economisch optimaal resultaat. De vermelde adviezen gelden voor een gemiddelde situatie. Op basis van eigen ervaringen en kennis kunnen ze aan de eigen specifieke situatie worden aangepast. De Adviesbasis geeft hiervoor handvaten in de vorm van voetnoten en aanvullende opmerkingen.
    Development and hazard assessment of nanoparticles
    Bhattacharjee, S. - \ 2012
    Wageningen University. Promotor(en): Ivonne Rietjens; Han Zuilhof, co-promotor(en): Gerrit Alink; Ton Marcelis. - S.l. : s.n. - ISBN 9789461733481 - 240
    nanotechnologie - deeltjes - gezondheidsgevaren - risicoschatting - cytotoxiciteit - ratten - biologische beschikbaarheid - werkwijze - nanotechnology - particles - health hazards - risk assessment - cytotoxicity - rats - bioavailability - mode of action

    A series of highly monodisperse silicon nanoparticles (Si NP) with either
    positively (amine), neutral (azide) or negatively (carboxylic acid) charged
    covalently attached organic monolayers were synthesized and investigated for
    their cytotoxicity. Infrared data confirmed the presence of these covalently
    attached surface groups. The Si NP were characterized by absorption and
    fluorescence spectroscopy. The cytotoxicity was investigated in Caco -2 cells by
    determining the cell viability and proliferation. The EC50 values for the Si NP
    ranged from 20 μg/l for the amine-terminated Si NP, via 550–850 μg/l for the
    azide-terminated Si NP to non-toxic (no measureable EC50) for the carboxylic acid -
    terminated Si NP. These results indicated a trend in cytotoxicity, depending on
    surface charge, i.e. that positively charged Si NP we re more cytotoxic than
    negatively charged Si NP. Interestingly, it appeared that the cytotoxicity of the
    amine terminated Si NP (Si NP-NH2) depended strongly on the presence of fetal calf
    serum (FCS) in the medium.

    Onkruidbestrijding met Basta in zomerbloemen
    Bulle, A.A.E. ; Slootweg, G. ; Trompert, J.P.T. - \ 2010
    onkruidbestrijding - onkruiden - bloementeelt - teeltsystemen - werkwijze - schade - herbiciden - zomerbloemen - weed control - weeds - floriculture - cropping systems - mode of action - damage - herbicides - summer flowers
    Poster over een onderzoek naar onkruidbestrijding met Basta in zomerbloemen.
    Mechanisms of toxic action of the flavonoid quercetin and its phase II metabolites
    Woude, H. van der - \ 2006
    Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): Gerrit Alink. - Wageningen : Ponsen & Looijen - ISBN 9789085043492 - 229
    quercetine - werkwijze - metabolische detoxificatie - risicoschatting - quercetin - mode of action - metabolic detoxification - risk assessment
    During and after absorption in the intestine, quercetin is extensively metabolised by the phase II biotransformation system. Because the biological activity of flavonoids is dependent on the number and position of free hydroxyl groups, a first objective of this thesis was to investigate the consequences of phase II metabolism of quercetin for its biological activity. For this purpose, a set of analysis methods comprising HPLC-DAD, LC-MS and 1 H NMR proved to be a useful tool in the identification of the phase II metabolite pattern of quercetin in various biological systems. These studies showed that the 3'- and 4'-hydroxyl groups of quercetin, (catechol hydroxyl groups) were important targets for methylation, sulfation and glucuronidation. Methylation of a catechol hydroxyl group of quercetin proved to decrease the pH-dependent radical scavenging capacity of the compound, both by increasing its pK a for deprotonation and by decreasing its electron-donating properties. Methylation of a catechol hydroxyl group had a similar effect as replacement of the hydroxyl group by a hydrogen atom. Regarding the pro-oxidant properties of quercetin, methylation of a catechol hydroxyl group of quercetin did not eliminate the pro-oxidant chemistry of quercetin, reflected in the formation of covalent adducts with glutathione upon oxidation of quercetin by horseradish peroxidase. However, methylated quercetin proved to form only 42% of the level of DNA adducts in exposed cells as compared to a similar amount of unconjugated quercetin, indicating that methylation of quercetin attenuates also this biological reactivity towards DNA.

    A second objective of this thesis was to obtain more insight into the possible toxic effects of quercetin by studying various mechanisms that might be relevant in the context of carcinogenesis. Quercetin appeared to have a biphasic effect on the proliferation of cancer cell lines expressing the estrogen receptor (ER). The stimulation of cancer cell proliferation was ER-dependent and appeared to occur at concentrations that are physiologically relevant in humans. With respect to the pro-oxidant activity of quercetin, peroxidase- and tyrosinase-type oxidative enzyme activity did not play a major role in the intracellular formation of covalent adducts of quercetin with DNA and protein, indicating that the formation of covalent adducts of quercetin with cellular macromolecules might also be relevant in cell types lacking oxidative enzyme activity. Furthermore, the covalent quercetin DNA adducts were of transient nature, which may either eliminate or attenuate the adverse effects of covalent DNA adduct formation. The studies presented in this thesis provided indications for the dualistic character of quercetin, regarding its role in the process of cancer development.
    Werkwijze en methode binnen de Bioveem-aanpak
    Baars, T. ; Iepema, G. ; Eekeren, N.J.M. van; Baars, E. - \ 2005
    Lelystad : Animal Sciences Group (Rapport / Bioveem 11) - 43 p.
    melkveehouderij - biologische landbouw - methodologie - werkwijze - onderzoeksprojecten - nederland - kennis - dairy farming - organic farming - methodology - mode of action - research projects - netherlands - knowledge
    Dit rapport geeft een overzicht van wat de Bioveem-aanpak is en hoe deze tot stand is gekomen. De Bioveem-aanpak is ontwikkeld in het project Bioveem en gefundeerd in de ervaringswetenschap. Bij de start van Bioveem is bewust gekozen voor het werken met ervaringskennis, omdat deze werkwijze goed aansluit bij de biologische landbouw die zeer divers is qua stijlen, intensiteit en regionaliteit. Generieke oplossingen en adviezen zijn hierbij niet wenselijk, er wordt juist gezocht naar systemische en geïndividualiseerde oplossingen. De omslag naar een werkwijze waarin ervaringskennis centraal wordt gesteld, was tevens nodig om de kloof tussen onderzoek en praktijk te dichten. De verwachting is dat via deze werkwijze innovaties boven komen drijven, waarmee knelpunten binnen de biologische en de gangbare melkveehouderijsector kunnen worden opgelost. Binnen de Bioveem-aanpak vindt deze zoektocht plaats samen met innoverende ondernemers, dat wil zeggen: veehouders die een eigen en herkenbaar doel hebben met hun bedrijf, die zelf bezig zijn nieuwe kennis te ontwikkelen en die (op onderdelen) voorlopen op andere bedrijven.
    Onderzoek naar de werking van biologische meststoffen in de containerteelt
    Aendekerk, T.G.L. - \ 2004
    Boskoop : Praktijkonderzoek Plant & Omgeving, Sector Bomen - 27
    organische meststoffen - werkwijze - containerplanten - sierplanten - organic fertilizers - mode of action - container grown plants - ornamental plants
    Het aantal biologische organische meststoffen dat reeds op de markt is gebracht is vrij groot. De werkingsduur en welke voedingsstoffen vrijkomen is echter niet duidelijk. De fabrikanten geven bij hun producten aanbevelingen over de te gebruiken hoeveelheden , doch deze geadviseerde hoeveelheden voor deze organische meststoffen zijn vooral gebaseerd op de te verwachte mestbehoefte van het gewas. De teeltduur en de meststofbehoefte van siergewassen is sterk variabel. De keuze van het type meststof is afhankelijk van de werking van de meststof en de meststofbehoefte van het gewas. De boomkwekerijgewassen zijn ingedeeld naar meststofbehoefte. Deze zijn voor de teelt in de containers aangegeven in de reeds ontwikkelde adviesbasis. Voor korte teelten is de toepassing van de organische meststoffen meestal eenvoudiger. Uitgevoerd onderzoek met enkele De Ceuster Meststoffen (DCM) heeft dit reeds bewezen. Voor de langere teelten in potten en containers voor het uitgebreide aanbod van deze biologische organische meststoffen is meer inzicht noodzakelijk. Het doel van dit uit te voeren onderzoek is inzicht te krijgen in de werking van deze meststoffen die als leverantie van voedingsstoffen voor de plant kunnen dienen. Door dit inzicht kan het type meststof, de gewenste hoeveelheid en het tijdstip van aanwenden beter worden vastgesteld. In herfst en winter 2002/2003 werden de organische biologische meststoffen via incubatie onderzocht op hun werkingsduur en leverantie aan voedingsstoffen. In het laboratorium werden deze organische biologische meststoffen met veen geïncubeerd om het vrijkomen van de voedingsstoffen vast te leggen. Ook werd vastgesteld of door veen nutriënten werden geïmmobiliseerd of vastgelegd. In 2003 werd een vervolgonderzoek als één jarige teeltproef in containers uitgevoerd. Hierbij werd gestreefd naar optimalisatie van het meststoffen aanbod aan de behoefte van het gewas. Trappenproeven met doseringen van organische meststoffen werden ingezet. Analyses van de potgrond en gewas werden tijdens de teelt uitgevoerd. Tijdens de teelt werd aan de hand van de potgrondanalyses vastgesteld, met hoeveel biologische meststof moet worden gemest om een “goede groei “ van het gewas te verkrijgen
    De bodem onder de zorgboerderij : naar een onderbouwing van de heilzame eigenschappen van een zorgboerderij
    Hassink, J. ; Ketelaars, D. - \ 2003
    In: Handboek Dagbesteding Wageningen : Plant Research International - p. 1 - 25.
    zorgboerderijen - werkwijze - effecten - sociale structuur - persoonlijke ontwikkeling - stimulansen - agrarische bedrijfsvoering - sociale zorg - social care farms - mode of action - effects - social structure - personal development - incentives - farm management - social care
    Onderzoek naar de werking van zorgboerderijen. Geconcludeerd wordt dat een zorgboerderij een kansrijke plek is om persoonlijke groei en ontwikkeling van cliënten mogelijk te maken. Het is een omgeving die prikkelt en stimuleert en daarnaast voldoende structuur en veiligheid kan bieden opdat cliënten de uitdaging ook aan durven gaan. Een zorgboerderij is ook een omgeving met voldoende ruimte om elkaar niet in de weg te zitten en voldoende variatie om werk of dagbesteding op-maat te bieden. De diversiteit in werkzaamheden en de activering van alle zintuigen, de structuur en het ritme dat een zorgboerderij kan bieden, het levert allemaal een bijdrage aan de gelegenheid voor cliënten om veiligheid, uitdaging en verbinding te ervaren
    Ozonides: intermediates in ozone-induced toxicity : a study on their mechanism of toxic action and detoxification by antioxidants
    Hempenius, R.A. - \ 2000
    Agricultural University. Promotor(en): J.H. Koeman; I.M.C.M. Rietjens; G.M. Alink. - S.l. : S.n. - ISBN 9789058083524 - 123
    ozon - toxiciteit - werkwijze - antioxidanten - ontgifting - longfunctie - ratten - ozone - toxicity - mode of action - antioxidants - detoxification - lung function - rats

    Ozone is a major constituent of photochemical smog. The toxicity of ozone is well documented and has been related to its strong oxidative potential. The principal target organ for ozone toxicity is the respiratory system. Unsaturated fatty acids, which are present in both the lipids of the lung lining fluid and the cell membranes of the cells that line the airways, are thought to be primary target molecules for ozone. Ozone reacts with unsaturated fatty acids via the so-called Criegee mechanism. Along with aldehydes and hydroxyhydroperoxides, Criegee ozonides are the main products of the reaction of ozone with unsaturated fatty acids. It is generally assumed that these lipid ozonation products act as secondary toxins in ozone-induced toxicity. However, very little is known about the reactivity and fate of ozonides and the role they play in ozone toxicity. Therefore, the aim of the present study was to gain insight into the mechanism of the toxic action of ozonides in relation to ozone-induced lung toxicity.For the investigations the ozonide from the polyunsaturated fatty acid methyl linoleate was used. On ozonation of methyl linoleate the major product formed appeared to be the trans -9,10-methyl linoleate ozonide (MLO).It has been suggested that ozonides are a kind of peroxide, and it is, therefore, believed that ozonides are a source of free radicals. This implies that ozonides might be capable of initiating the chain autoxidation of other non-ozonated polyunsaturated fatty acids in the membrane bilayer, and consequently of producing a cascade of damage. This hypothesis was investigated by comparing the in vitro cellular toxic action of MLO with a model peroxidative agent, i.e., cumene hydroperoxide. The ozonide was shown to be three times more toxic towards alveolar macrophages than the peroxide. On the basis of the cellular protection of antioxidants it was clearly shown that the ozonide and the hydroperoxide exert their toxic effects using different mechanisms. Whereas the results with the model hydroperoxide, cumene hydroperoxide, confirmed the mechanism by which peroxides exert their toxic effects, namely by lipid peroxidation and/or depletion of intracellular glutathione (GSH) levels, the investigations regarding the potency of MLO to induce lipid peroxidation revealed that the main toxic mechanism of MLO did not proceed via a radical-mediated mechanism. Nevertheless, suppletion of cells with the lipid-soluble radical scavengerα-tocopherol resulted in a significant protection towards ozonide exposure. In addition, preincubation of MLO withα-tocopherol resulted in a detoxification of the ozonide. This suggests thatα-tocopherol is able to interact directly with the ozonides themselves, thus scavenging these reactive intermediates.Investigations regarding the chemical characteristics of the detoxification of MLO byα-tocopherol revealed that the main products formed were the aldehyde nonanoic 9-oxo methyl ester and the acid nonanedioic acid monomethyl ester. This finding is in agreement with the general opinion that the decomposition of ozonides results in the formation of aldehydes and acids. In general, it is believed that thermal decomposition of ozonides proceeds via homolytic cleavage of the peroxide bond to yield the oxy bi-radical followed by a rearrangement. A modification on this mechanism includes concerted homolysis and intramolecular hydrogen atom abstraction. In contrast to the results obtained at elevated temperatures (≥50 °C), in previous studies ozonides have been shown to be stable compounds at 37 °C. No radical formation could be detected when MLO was incubated for 30 min at 37 °C using spin traps and electron spin resonance (ESR). Taking into account the fact that peroxide bond homolysis is an essential part of ozonide decomposition, one might postulate thatα-tocopherol, being an efficient hydrogen atom donor, facilitates the process of O-O bond homolysis, thereby inducing aldehyde and acid formation already at relatively low (i.e.≤37 °C) temperatures.An additional interesting finding was that the degradation products of MLO, i.e., nonanoic 9-oxo methyl ester and nonanedioic acid monomethyl ester (azelaic acid monomethyl ester), were not toxic towards alveolar macrophages at concentrations where MLO showed complete loss of cell viability. This observation is especially of importance because it is often suggested that Criegee ozonides will be formed in relatively small amounts (ca. 10%) when ozone reacts with unsaturated fatty acids in the lung lining fluids and may, therefore, play a minor role in ozone-induced toxicity. However, the results of the present study clearly indicate that ozonides are far more toxic than their aldehyde and acid type degradation products, which are generally observed as major products resulting from the reaction of ozone with fatty acids under physiological conditions.In addition, the mechanism underlying the reaction of another cellular antioxidant, namely glutathione, with polyunsaturated fatty acid ozonides was investigated. In previous studies it has been shown that preincubation of MLO with glutathione caused a significant detoxification of the ozonide. Furthermore, the detoxification reaction was shown to be catalysed by glutathione S -transferases leading to the formation of oxidised glutathione and aldehydes. The reaction of ozonides and peroxides with glutathione was investigated using molecular orbital calculations and the frontier orbital theory. In addition to the results obtained in the comparative study on the toxicity of ozonides and peroxides, the reaction of ozonides with the nucleophilic agent glutathione appeared also to be different when compared with the reaction with peroxides. On the basis of semi-empirical molecular computer calculations the nucleophilic attack by glutathione on the ozonide is expected to occur at one of the carbon atoms of the ozonide ring instead of at one of the peroxidic oxygen atoms as in the case of peroxides. A mechanism for the glutathione S -transferase-mediated detoxification of ozonides, different from that of the reaction with hydroperoxides has been proposed.Furthermore, the in vivo toxicity of ozonides was investigated. Methyl linoleate ozonide (MLO) (0.07 mmol/100 g body wt) was administered to female Wistar rats either intravenously or intraperitoneally. After 24 h the rats were killed and the effects were examined. MLO was found to be toxic only after intravenous administration. The major effects were observed in the lungs. The lungs became enlarged from edema and showed severe haemorrhages. Furthermore, the total thiol level was depleted in serum and lung tissue, accompanied by a decrease in the activity of thiol-dependent enzymes. The vitamin E levels in serum and lung tissue were also reduced. The malondialdehyde (MDA) concentrations in serum and lung tissue were elevated suggesting that in vivo oxidation had occurred. On intraperitoneal administration of MLO, no effects on enzyme activities, thiol and vitamin E content in lung tissue were observed. In serum, however, as on intravenous administration, an increase in the MDA levels and decreases in total thiol and vitamin E levels were found. In view of the route of administration it is to be expected that the ozonide is partially cleared by the liver, and the ozonide and its potentially toxic products are further detoxicated by vitamin E and thiols in the serum before they reach the lung. The above data show that the main target organ for ozonides is the lung, and that the effects caused by MLO in vivo are in many respects similar to the effects found after acute ozone exposure.In short, the most important conclusions of the present studies are:

  • Ozonides exert their toxic effects by a mechanism different from that from the structurally related peroxides.
  • The protective action ofα-tocopherol against ozonides proceeds via a direct interaction by whichα-tocopherol facilitates ozonide degradation through peroxide bond homolysis already at 37 °C.
  • The involvement of ozonides in ozone-induced toxicity may be more important than currently suggested, since they are far more toxic than their aldehyde-type degradation products, which are generally observed as major products resulting from the reaction of ozone with unsaturated fatty acids under physiological conditions.
  • Overall, the results presented in the thesis provide new insights into the toxic mechanism of ozonides and their implications for ozone-induced lung toxicity.

    Molecular and biochemical studies on the Ah receptor pathway in flounder (Platichthys flesus)
    Besselink, H. - \ 1998
    Agricultural University. Promotor(en): J.H. Koeman; A. Brouwer. - S.l. : Besselink - ISBN 9789054857648 - 136
    visziekten - polycyclische koolwaterstoffen - werkwijze - polychloorbifenylen - chemoreceptoren - platichthys flesus - bot (vissen) - metabolisme - retinoïden - cytochroom p-450 - fish diseases - polycyclic hydrocarbons - mode of action - polychlorinated biphenyls - chemoreceptors - platichthys flesus - flounder - metabolism - retinoids - cytochrome p-450

    The research presented in this thesis focused on the mechanistic aspects of the toxic and biochemical effects of PCBs in flounder (Platichthys flesus), with the aim to provide a scientific basis for the suggested involvement of PCBs in the aetiology of diseases observed in flounder. Therefore, the first goal was to study the biochemical effects of PCBs in flounder by investigating the inducibility of cytochrome P4501A and associated EROD activity and glutathione-S-transferase activity upon administration to PCBs and TCDD in vivo. Secondly, the mechanism of action of PCBs in flounder was investigated by identifyingg the hepatic microsomal Ali receptor pathway. In addition, the potency of individual PCB congeners and a commercial PCB mixture to specifically inhibit the catalytic CYP1A activity was studied using in vitro techniques. Finally, the endocrine disrupting effect of PCBs was studied by analysing the retinoid and thyroid hormone levels in flounder after acute and chronic exposure to either a commercial PCB mixture or contaminated harbour sludge.
    summary of results

    Short-time exposure of flounder to the technical PCB mixture Clophen A50 showed minor effects on total hepatic cytochrome P450 concentrations and only a relatively slight induction in EROD activity, indicating that flounder is not very sensitive towards i.p. and oral administration of Clophen A50 ( chapter 3 and 5). The most potent PCB congener (CB-126), in terms of mammalian derived TCDD toxic equivalents (TEQ), was found to be more potent as inducer of CYP1A protein and activity in flounder, but not to the extend as was expected based on TCDD TEQs ( chapter 5). On the other hand, high induction of the hepatic microsomal CYP1A content and associated EROD activity were observed in flounder orally exposed to TCDD ( chapter 4). Interestingly, TCDD induced EROD activity in flounder could be inhibited by co-treatment with Clophen A50. In contrast, administration of flounder with a combination of TCDD and CB-126 resulted in an additive effect on EROD activity ( chapter 5). With respect to induction of hepatic CYP1A protein content, administration of combinations of both Clophen A50 and CB-126 with TCDD resulted in an additive effect compared to exposure of flounder to the individual compounds. These results indicated a direct inhibition of CYP1A catalytic activity by residual PCBs present in the microsomal suspension, rather than inhibition of transcription of the CYP1A gene or translation of CYP1A mRNA. Evidence for direct inhibition of EROD activity by individual PCB congeners and the technical PCB mixture Clophen A50 is presented in chapter 6.

    The relative sensitivity of flounder towards TCDD exposure was established by comparing the flounder computed no effect level (CNEL) for EROD activity to -lowest observed -adverse - effect levels (LOAELs) from other fish species and the rat (chapter 4). The rat was clearly more sensitive towards TCDD exposure, but other fish species, such as carp and rainbow trout, were only slightly more sensitive than flounder.

    A number of biotransformation enzymes other then CYP1A are also found or suggested to be modulated by the Ali receptor signal transduction pathway. Among them are certain forms of the phase 11 enzyme system such as glutathione-S-transferase (GST), In contrast to some other fish species and mammals, hepatic cytosolic GST activity in flounder was not altered upon exposure to TCDD (chapter 4). Moreover, no effect on GST activity was observed in flounder after Clophen A50 exposure, whereas a slight inhibition was observed upon CB-126 administration ( chapter 5). In contrast, a strong inhibition of GST activity was observed when flounder were treated with combinations of TCDD and either CB-126 or Clophen A50 ( chapter 5). An explanation for these observations was not found, but it was suggetsed that GST substrate inhibition by PCB-metabolite residues could have occurred.

    To better understand the contradiction in induction pattern of the flounder CYP1A enzyme system towards exposure to either PCBs or TCDD, the hepatic Ah receptor pathway in flounder was characterised ( chapter 6). In addition, the potency of a number of PCB congeners and Clophen A50 to inhibit the CYP1A catalytic activity in vitro was studied. In chapter 6, evidence for the presence of low levels of Ali receptor (1-7 fmol/mg protein) in flounder hepatic cytosol was presented using protamine sulphate and hydroxylapatite analysis and velocity sedimentation on sucrose gradient. The level of Ali receptor in flounder was similar to levels of receptor reported in some other fish species, but much lower than Ali receptor levels reported in mammals. Additional evidence for the presence of the cytosolic Ali receptor in flounder liver was provided by first-strand cDNA synthesis and subsequent amplification of flounder poly A+ RNA using RT-PCR. The specificity of the 690 bp RT-PCR reaction product was established by southern blotting and hybridisation. Subsequent sequencing of the RT-PCR product showed that its deduced amino acid sequence was 75% identical to the killifish species Fundulus heteroclitus AhR-2 sequence and 61% identical to the Fundulus AhR-1 sequence, which is more similar to mammalian AhRs. Binding of the liganded Ah receptor to the DRE using a complementary pair of synthetic oligonucleotides, corresponding to wild type Ali receptor site of DRE3, could be demonstrated using rat and guinea pig derived cytosol. In contrast, DRE binding of liganded Ali receptor could not be demonstrated using flounder cytosol. These data show that the hepatic AhR pathway is only marginally present in flounder But since a good induction of CYP1A was observed in flounder exposed to TCDD, the flounder AhR pathway is functional and the apparent low responsiveness of flounder CYP1A towards PCB exposure can not be attributed to a non-functional Ah receptor pathway.

    A more plausible explanation for the low CYP1A inducibility upon PCB exposure in flounder is direct inhibition of CYP1A catalytic activity by residual PCBs present in the microsomal suspension. Evidence for substrate inhibition by PCB congeners was provided in chapter 6. All of the PCB congeners tested, as well as Clophen A50, were capable of in vitro inhibition of the flounder CYP1A catalytic activity in a competitive way. The competitive inhibition of CYP1A activity by PCBs occurred at similar PCB concentrations in flounder as in rat. CB- 126 was found to be the most potent inhibitor of EROD activity whereas CB- 153 was least potent. The inhibition constants (K i ) of the tested PCBs were close to the Michaelis constant (K m ) for ethoxyresorufin. These studies also suggested a higher catabolic efficiency of the flounder CYP1A enzyme system towards ethoxyresorufin as compared to the rat CYP1A system.

    Short-term exposure of flounder towards TCDD did not result in either retinoid or thyroid alterations ( chapter 4). In contrast alterations in retinoid and thyroid hormone levels were observed in flounder exposed to Clophen A50. But such changes were not dependent on the dose of PCB administered ( chapter 3). These results would suggest that PCBs are less endocrine disrupting in flounder as they are in mammals. However, in a chronic exposure experiment in which flounder were exposed to contaminated harbour sludge for three years, retinol levels in both plasma and liver were reduced (chapter 7). In addition, a negative correlation between hepatic retinol concentrations and CYP1A protein levels was observed, indicating involvement of PHAH inducible enzymes.

    Conclusions

    From these studies, the following main conclusions can be drawn:

    1 . The commercial PCB mixture Clophen A50 only cause minor effects on hepatic microsomal CYP1A protein levels and associated EROD activity in flounder, even at Clophen A50 concentrations as high as 500 mg/kg body weight. This low responsiveness towards Clophen A50 exposure is observed despite a functional hepatic Ah receptor pathway in flounder.

    2. The commercial PCB mixture Clophen A50 inhibit the TCDD induced CYP1A activity. This finding can, at least to some extend, be attributed to competitive inhibition of the CYP1A activity by residual PCB congeners present in the reaction mixture.

    3. In contrast to rodents, short-term exposure of flounder towards PCBs or TCDD does not induce meaningful retinoid and thyroid hormone disrupting effects.

    4. Long-term exposure of flounder towards contaminated harbour sludge causes a marked decline of plasma and hepatic retinoid levels.

    5. Exposure up to 3 weeks to PCBs and TCDD does not induce any gross pathological effects in flounder, even at high doses of PHAHs administered.

    Comparing the present results with the outcome of studies on mammals and a range of other fish species, the conclusion can be drawn that flounder is relatively insensitive to PCB exposure regarding the parameters studied. Hence this study does not provide evidence for the involvement of PCBs in the development of the lesions observed in flounder from Dutch coastal and estuarine areas.

    One may speculate about the nature of other factors involved in the diseases concerned, like pollutants other than PCBs. PAHs for example, are known to bind covalently to DNA after metabolisation, thus possibly initiating carcinogenesis. One may also assume that synergistic actions may occur between certain pollutants. However, the present studies showed that both individual PCB congeners and complex mixtures of PCBs are competitive inhibitors of EROD activity in vitro. If such effects also occur in the wild, PCBs may even antagonise the toxic potential of other pollutants. For instance, inhibition of CYP1A activity by residual PCB congeners might result in reduced formation of reactive PAH metabolites and thus reducing the risk for covalent DNA binding and tumour formation. Hence, this would also decrease the likelihood that PAH initiate carcinogenesis. Furthermore, non-chemical background stressors may also be involved, such as physical disturbance and pathogenic micro-organisms in the case of infectious diseases.

    A remark should be made about the usefulness of EROD activity as biomarker for monitoring exposure of fish to environmental pollutants such as PHAHs and PAHs. As a spinoff of the observed competitive inhibition of EROD activity by PCBs, one should reconsider the value of EROD activity as biomarker. On the other hand, induction CYP1A protein levels is not inhibited by PCBs. Therefore, CYP1A protein levels might be a better biomarker than EROD activity for exposure assessment of fish to aromatic compounds.

    Finally, in view of the recent developments in science, more emphasis should be put on developing appropriate tools to study the role of environmental pollutants, such as PCBs or related compounds in carcinogenesis. For example, the RAS gene sequence has been identified in flounder (see appendix). Whereas in humans expression of the oncogen is characteristic for a number of tumors, this relation has yet to be elucidated in fish. Nevertheless, the study of alterations in the expression of onco- and suppressor genes might be a useful approach in further studies.

    De werking van beta-agonisten als groeibevorderaars
    Vlieger, J.F. de - \ 1992
    Wageningen : RIKILT-DLO (Rapport / DLO-Rijks-Kwaliteitsinstituut voor Land- en Tuinbouwprodukten (RIKILT-DLO) 92.49) - 63
    bèta-adrenerge agonisten - kalveren - werkwijze - groeibevorderaars - beta-adrenergic agonists - calves - mode of action - growth promoters
    Ozone and nitrogen dioxide : a study on mechanisms of toxic action and cellular defense
    Rietjens, I.M.C.M. - \ 1986
    Landbouwhogeschool Wageningen. Promotor(en): J.H. Koeman; G.M. Alink. - Wageningen : Rietjens - 200
    toxicologie - ozon - stikstofdioxide - atmosfeer - aërosolen - samenstelling - stof - luchtverontreiniging - luchtkwaliteit - werkwijze - toxicology - ozone - nitrogen dioxide - atmosphere - aerosols - composition - dust - air pollution - air quality - mode of action

    Ozone and nitrogen dioxide are major toxic components of photochemical smog. They arise from the combustion of fossil fuels (traffic, industrial processes) and from solar radiation-catalyzed reactions in polluted atmospheres.

    The morphological, physiological and biochemical effects of ozone and nitrogen dioxide on the respiratory system of man and experimental animals have been investigated over the last decades.

    More recently the development of i) isolation and cell culture procedures for different types of lung cells and ii) model systems for invitro exposure of cells to gaseous compounds, offers new possibilities to study the mechanism of toxic action of ozone and nitrogen dioxide. An advantage of the cell model is that changes in homogeneous cell populations can be studied, although it is recognized that effects found in isolated cell cultures always need invivo validation.

    The experiments described in this thesis were undertaken to further elucidate i) the mechanisms of action of ozone and nitrogen dioxide, as well as ii) the mechanisms of cellular protection against both gaseous compounds.

    Invitro exposure of cells: was achieved by using a system in which cells are grown on a thin teflon membrane and exposed by means cif gas diffusion through this membrane.

    Experiments were carried out using either cells from the A549 cell line or primary cultures of alveolar macrophages or alveolar type II pneumocytes, isolated from the lungs of control and in some cases (chapter 6a + b) from ozone or nitrogen dioxide exposed rats.

    Part I of this thesis starts with a review of literature data on historical backgrounds, physical characteristics, concentrations encountered in the environment and toxic effects of ozone and nitrogen dioxide (chapter 1).

    This is followed by a review on the current theories with regard to the mode of toxic action of ozone and nitrogen dioxide as well as to the mechanisms of cellular protection against these compounds (chapter 2).

    Finally a review of invitro exposure models is presented, including a description of the gas diffusion mediated exposure model applied in this thesis (chapter 3).

    Part II of this thesis deals with the experiments carried out to obtain additional insight into i) the mode of toxic action of ozone and nitrogen dioxide and ii) the mechanisms providing protection against both gases in an intact cell system.

    First experiments are described in which the mechanisms of toxic action of ozone and nitrogen dioxide were compared (chapter 4). In the invitro exposure model applied ozone appeared to be 10 times more toxic than nitrogen dioxide. This difference is comparable with the difference in toxicity reported for invivo exposures.

    In addition it was demonstrated that the cellular antioxidant compounds, vitamin E ( α-tocopherol), vitamin C (ascorbic acid) and glutathione, are all involved in the protection of cells against ozone or nitrogen dioxide.

    The protection of α-tocopherol was shown to be dependent on its 6-hydroxyl group and not to be mediated by a structural stabilizing membrane effect of the compound arising from its strong physicochemical association with for example sensitive arachidonyl fatty acid residues. This could be concluded from the observations that i) α-tocopherol, at concentrations that provided optimal protection against the oxidative compounds, did not influence cell membrane fluidity and that ii) phytol and the methyl ether of α-tocopherol, two structural α-tocopherol analogues, could not provide protection against ozone or nitrogen dioxide comparable to the α-tocopherol protection.

    Furthermore chapter 4 presents invitro data which clearly demonstrate that the reaction pathways involved in ozone and nitrogen dioxide induced cell damage must be different. This conclusion is based on the observations that i) vitamin C enrichment of cells provided significantly better protection against nitrogen dioxide than against an equally toxic amount of ozone, that ii) glutathione depletion increased the cellular sensitivity towards ozone to a greater extent than the sensitivity towards nitrogen dioxide and that iii) α-tocopherol dependent protection of the cells was accompanied by a significantly greater reduction in cellular α-tocopherol upon nitrogen dioxide than upon ozone exposure.

    The observations referred to above are compatible with the hypothesis that ozone damage proceeds by the ionalr mechanism for ozonide formation, whereas nitrogen dioxide induced cell damage is induced by a radical mediated lipid peroxidative pathway.

    This hypothesis includes the involvement of oxidation of unsaturated membrane lipids in the mechanisms of ozone or nitrogen dioxide induced cell damage.

    Evidence for the involvement of lipid oxidation in the mechanism of ozone or nitrogen dioxide induced cell damage is presented in the next chapter, (chapter 5), in which experiments are described that investigated the influence of polyunsaturated fatty acid (PUFA) supplementation on the sensitivity of cells towards ozone and nitrogen dioxide.

    The results showed that cells enriched in their PUFA content demonstrate an increased sensitivity towards both ozone and nitrogen dioxide. It was also shown that this increased sensitivity was not caused by an increased membrane fluidity but really by the increased number of unsaturated fatty acids. Therefore these results clearly point to the involvement of lipid oxidation in the mechanism of action of both ozone and nitrogen dioxide.

    In addition to the evidence for a difference in the mechanisms of toxic action of ozone and nitrogen dioxide observed in the invitro experiments, evidence suggesting a difference in their toxicity was also obtained in invivo experiments (chapter 6a + b). Exposure of rats to doses of both gaseous compounds that equally induced glucose-6-phosphate dehydrogenase activity in whole lung homogenates and in isolated alveolar macrophages and type II pneumocytes, resulted in a greater increase in the activity of glutathione peroxidase in cell material derived from ozone than from nitrogen dioxide exposed rats.

    In addition it was shown that the increased activities of the enzymes of the glutathione peroxidase system, observed in lung homogenates of ozone or nitrogen dioxide exposed rats were caused by cell proliferation as well as by the increase of enzyme activities within individual cells.

    In the literature these inductions have often been coupled to a glutathione dependent mechanism of cellular defense. To test this hypothesis alveolar macrophages and type II pneumocytes isolated from exposed rats were exposed to ozone or nitrogen dioxide invitro . From these studies it appeared that cells derived from exposed animals revealed no increased resistance to the oxidative compounds as compared to the cells isolated from non-exposed animals. This in spite of a significantly increased glutathione peroxidase activity in the cells derived from exposed animals. Hence it can be concluded that an increased cellular glutathione peroxidase activity is not related to an increased cellular resistance towards ozone or nitrogen dioxide.

    Data presented in the next chapter (chapter 7) demonstrated even more clearly that the glutathione dependent protection of cells against ozone is not mediated by the glutathione peroxidase catalyzed detoxification of fatty acid hydroperoxides. A549 cells showed a significantly increased sensitivity towards ozone upon depletion of their cellular glutathione content, which clearly points to a glutathione dependent mechanism of cellular protection, although these cells do not contain a detectable glutathione peroxidase activity. This observation obviously excludes the glutathione peroxidase catalyzed detoxification of lipid hydroperoxides as a main mechanism for glutathione dependent cellular protection against ozone.

    Additional results demonstrated the loss of glutathione from the cytoplasm of ozone exposed cells. This loss of glutathione from the cytoplasm of exposed cells was caused by leakage and/or active transport of glutathione out of exposed cells to the surrounding medium. The loss could not be ascribed to incorporation of a substantial amount of glutathione into mixed disulfides. This observation excludes a second hypothesis for the glutathione dependent protection of cells against ozone, viz. its incorporation into mixed disulfides, thus protecting cellular thiol groups from irreversible oxidation by ozone or its reactive intermediates.

    The results presented in the next chapter (chapter 8), indicate that the increased ozone sensitivity of glutathione depleted cells is not caused by an impaired regeneration of α-tocopherol in these cells. This follows from the observations that i) glutathione depleted, ozone exposed cells did not contain decreased levels of α-tocopherol and that ii) vitamin E supplementation could not diminish the increased ozone sensitivity of the glutathione depleted cells.

    So far all three hypotheses mentioned in the literature for a glutathione dependent cellular protection against ozone, had been excluded. From this, and from the observation that vitamin C supplementation abolished part of the increased ozone sensitivity of glutathione depleted cells, it is concluded that the most likely mechanism for a glutathione dependent protection of cells against ozone is provided by its action as a direct scavenger of reactive initial and/or intermediate species.

    Experiments described in the final chapter of part II (chapter 9), actually demonstrate the ability of glutathione to detoxify a possible ozone intermediate; the ozonide derived from methyl linoleate. Methyl linoleate ozonide appeared to be toxic towards alveolar macrophages at concentrations between 10 to 100 μM, and showed characteristics with regard to cellular antioxidant protection that were similar to those for ozone itself.

    In addition the detoxification of methyl linoleate ozonide by glutathione appeared to be even more pronounced when c atalyzed by glutathione S-transferase.

    Hence the glutathione S-transferase catalyzed detoxification of fatty acid ozonides provides a new point of view on the protective role of glutathione in ozone exposed cells.

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