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.

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Genetic Components of Root Architecture Remodeling in Response to Salt Stress
Julkowska, M.M. ; Koevoets, I.T. ; Mol, S. ; Hoefsloot, H. ; Feron, R. ; Tester, M.A. ; Keurentjes, J.J.B. ; Korte, A. ; Haring, M.A. ; Boer, G.J. de; Testerink, C. - \ 2017
The Plant Cell 29 (2017)12. - ISSN 1040-4651 - p. 3198 - 3213.
Salinity of the soil is highly detrimental to plant growth. Plants respond by a redistribution of root mass between main and lateral roots, yet the genetic machinery underlying this process is still largely unknown. Here, we describe the natural variation among 347 Arabidopsis thaliana accessions in root system architecture (RSA) and identify the traits with highest natural variation in their response to salt. Salt-induced changes in RSA were associated with 100 genetic loci using genome-wide association studies. Two candidate loci associated with lateral root development were validated and further investigated. Changes in CYP79B2 expression in salt stress positively correlated with lateral root development in accessions, and cyp79b2 cyp79b3 double mutants developed fewer and shorter lateral roots under salt stress, but not in control conditions. By contrast, high HKT1 expression in the root repressed lateral root development, which could be partially rescued by addition of potassium. The collected data and multivariate analysis of multiple RSA traits, available through the Salt_NV_Root App, capture root responses to salinity. Together, our results provide a better understanding of effective RSA remodeling responses, and the genetic components involved, for plant performance in stress conditions.
The FEMA GRAS assessment of aliphatic and aromatic terpene hydrocarbons used as flavor ingredients
Adams, T.B. ; Lucas Gavin, C. ; McGowen, M.M. ; Waddell, W.J. ; Cohen, S.M. ; Feron, V.J. ; Marnett, L.J. ; Munro, I.C. ; Porthogese, P.S. ; Rietjens, I. ; Smith, R.L. - \ 2011
Food and Chemical Toxicology 49 (2011)10. - ISSN 0278-6915 - p. 2471 - 2494.
food-additives amendment - chronic progressive nephropathy - hyaline droplet nephropathy - d-limonene para-mentha-1,8-diene - sister-chromatid exchanges - organic anion transporters - human risk-assessment - 3 continental sites - beta-myrcene - alpha-pinene
This publication is the thirteenth in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2600 substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, pharmacokinetics and toxicology. Flavor ingredients are evaluated individually and in the context of the available scientific information on the group of structurally related substances. Scientific data relevant to the safety evaluation of the use of aliphatic and aromatic terpene hydrocarbons as flavoring ingredients are evaluated. The group of aliphatic and aromatic terpene hydrocarbons was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their rapid absorption, metabolic detoxication, and excretion in humans and other animals; their low level of flavor use; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies and the lack of significant genotoxic potential.
Identification of a resistance gene Rpi-dlc1 to Phytophthora infestans in European accessions of Solanum dulcamara
Golas, T.M. ; Sikkema, A. ; Gros, J. ; Feron, R.M.C. ; Berg, R.G. van den; Weerden, G.M. van der; Mariani, C. ; Allefs, J.J.H.M. - \ 2010
Theoretical and Applied Genetics 120 (2010)4. - ISSN 0040-5752 - p. 797 - 808.
late-blight resistance - broad-spectrum resistance - race-specific resistance - potato late blight - r-gene - disease-resistance - chromosome-ix - bulbocastanum - tomato - locus
Initial screening of 14 Solanum dulcamara accessions enabled the identification of individuals resistant and susceptible to Phytophthora infestans. Crosses between contrasting genotypes resulted in three F2–BC1 populations segregating for resistance to late blight in a laboratory assay and under field conditions. Genetic profiling of one of these populations using 128 AFLP primers generated three markers linked to the resistant phenotype. Blast analysis of the sequenced markers resulted in a plausible gene position on the distal end of the long arm of chromosome 9 that could be confirmed by CAPS markers. Thus, we describe a first resistant gene, named Rpi-dlc1, from S. dulcamara, a Solanum species native to Europe. In addition, one population was tested for broadness of resistance responses using a set of seven additional P. infestans isolates, varying in virulence. This indicated the possible presence of additional Rpi genes.
Genetic structure of European accessions of Solanum dulcamara L. (Solanaceae)
Golas, T.M. ; Feron, R.M.C. ; Berg, R.G. van den; Weerden, G.M. van der; Mariani, C. ; Allefs, J.J.H.M. - \ 2010
Plant Systematics and Evolution 285 (2010)1-2. - ISSN 0378-2697 - p. 103 - 110.
phytophthora-infestans - aflp - populations - nigrum
Solanum dulcamara (bittersweet) is one of the few native species of Solanum present in Europe. It is a common weed that occupies a wide range of habitats and is often found in the direct vicinity of cultivated potatoes (Solanum tuberosum), where it could transmit diseases. A broad sampling of European S. dulcamara accessions was carried out to gain insight into the population structure and crossing preferences of this species. Three amplified fragment length polymorphism (AFLP®) primer combinations generating 288 polymorphic fragments were used to analyze 79 bittersweet accessions (245 individuals). Dendrograms revealed a low level of genetic polymorphism in the bittersweet populations, caused partially by the out-crossing nature of this species
GRAS Flavoring Substances 24
Smith, R.L. ; Waddell, W.J. ; Cohen, S.M. ; Feron, V.J. ; Marnett, L.J. ; Portoghese, P.S. ; Rietjens, I. ; Adams, T.B. ; Lucas Gavin, C. ; McGowen, M.M. ; Taylor, S.V. ; Williams, M.C. - \ 2009
Food Technology 63 (2009)6. - ISSN 0015-6639 - p. 46 - 105.
in-vitro - genotoxicity tests - prooxidant action - mammalian-cells - ethyl maltol - hinokitiol - apoptosis - carcinogenicity - identification - ingredients
The 24th publication by the FEMA Expert Panel presents safety and usage data on 236 new generally recognized as safe flavoring ingredients
The FEMA GRAS assessment of a,b-unsaturated aldehydes and related substances used as flavor ingredients
Adams, T.B. ; Lucas-Gavin, C. ; Taylor, S.V. ; Waddell, W.J. ; Cohen, S.M. ; Feron, V.J. ; Goodman, J. ; Rietjens, I.M.C.M. ; Marnett, L.J. ; Portoghese, P.S. ; Smith, R.L. - \ 2008
Food and Chemical Toxicology 46 (2008)9. - ISSN 0278-6915 - p. 2935 - 2967.
sister-chromatid exchanges - glutathione s-transferases - acid nitrite invivo - long-term toxicity - sorbic acid - lipid-peroxidation - cytochrome-c - dna-damage - allyl alcohol - 1,n(2)-propanodeoxyguanosine adducts
This publication is the 12th in a series of safety evaluations performed by the Expert Panel of the Flavor and Extract Manufacturers Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavoring substances under conditions of intended use. Since then, the number of flavoring substances has grown to more than 2200 chemically-defined substances. Elements that are fundamental to the safety evaluation of flavor ingredients include exposure, structural analogy, metabolism, toxicodynamics and toxicology. Scientific data relevant to the safety evaluation for the use of aliphatic, linear ¿,ß-unsaturated aldehydes and structurally related substances as flavoring ingredients are evaluated. The group of substances was reaffirmed as GRAS (GRASr) based, in part, on their self-limiting properties as flavoring substances in food; their low level of flavor use; the rapid absorption and metabolism of low in vivo concentrations by well-recognized biochemical pathways; adequate metabolic detoxication at much higher levels of exposure in humans and animals; the wide margins of safety between the conservative estimates of intake and the no-observed-adverse effect levels determined from subchronic and chronic studies. While some of the compounds described here have exhibited positive in vitro genotoxicity results, evidence of in vivo genotoxicity and carcinogenicity occurs only under conditions in which animals are repeatedly and directly exposed to high irritating concentrations of the aldehyde. These conditions are not relevant to humans who consume ¿,ß-unsaturated aldehydes as flavor ingredients at low concentrations distributed in a food or beverage matrix.
Genetic diversity of the African hexaploid species Solanum scabrum Mill. and S. nigrum L. (Solanaceae)
Manoko, M.L.K. ; Berg, R.G. van den; Feron, R.M.C. ; Weerden, G.M. van der; Mariani, C. - \ 2008
Genetic Resources and Crop Evolution 55 (2008)3. - ISSN 0925-9864 - p. 409 - 418.
fragment length polymorphism - aflp analysis - wild relatives
Two hexaploid species of Solanum sect. Solanum are present in Africa: Solanum scabrum and S. nigrum. Solanum scabrum is a widely cultivated species and is used as a leafy vegetable, as a source of medicine and as a source of ink dye. In previous studies a wide range of morphological diversity has been reported in this species and in some studies subspecies have been proposed. Subspecies are also recognized in S. nigrum. However, it has not been established whether or not the morphological differences are reflected at the genomic level. The present study applies AFLPs to study the genetic diversity in S. scabrum and its relationship to geographical provenance, morphological differences and the possible existence of subspecies within S. scabrum and S. nigrum. The data obtained were analyzed with cluster analysis (using UPGMA and NJ). The results indicate that the genetic variation within S. scabrum was higher within accessions than between accessions. Accessions did not cluster according to their geographical provenance, indicating that accessions from different geographical areas were not significantly different genetically. The clustering reflected neither morphological differences nor domestication status (cultivated or wild). The morphological differences exhibited by S. scabrum could be due to selection by farmers for different plant types. The AFLP derived clustering pattern did not segregate the subspecies recognized in S. scabrum and S. nigrum into separate subclusters.
GRAS Flavoring substances 23 - The 23rd publication by the FEMA Expert Panel presents safety and usage data on 174 new generally recognized as safe flavoring ingredients.
Waddell, W.J. ; Cohen, S.M. ; Feron, V.J. ; Goodman, J.I. ; Marnett, L.J. ; Portoghese, P.S. ; Rietjens, I.M.C.M. ; Smith, R.L. ; Adams, T.B. ; Gavin, C.L. ; McGowen, M.M. ; Williams, M.C. - \ 2007
Food Technology 61 (2007)8. - ISSN 0015-6639 - p. 22 - 28.
AFLP markers support separation of Solanum nodiflorum from Solanum americanum sensu strictio (Solanaceae)
Manoko, M.L.K. ; Berg, R.G. van den; Feron, R.M.C. ; Weerden, G.M. van der; Mariani, C. - \ 2007
Plant Systematics and Evolution 267 (2007)1-4. - ISSN 0378-2697 - p. 1 - 11.
genetic-relationships - l. - biosystematics - relatives - homology - barley - wild
This study was aimed at examining the relationships between the African material of Solanum americanum (also designated as S. nodiflorum), accessions of this taxon from other geographical areas, and American S. americanum using AFLP markers. 96 individuals representing 39 accessions of S. americanum sensu lato and related diploid species from the widest possible geographical range, and one accession of S. dulcamara (as outgroup) were used. The AFLP results suggested that American S. americanum differs from S. nodiflorum and that the material investigated in this study can be assigned to three different species: S. americanum sensu stricto, S. nodiflorum and a Solanum species from Brazil. These species can be differentiated based on a combination of floral and fruit characteristics.
Characterization of Toxoplasma Gondii-specific T cells recovered from vitreous fluid of patients with ocular toxoplasmosis
Feron, E.J. ; Klaren, V.N.A. ; Wierenga, E.A. ; Verjans, G.M.G.M. ; Kijlstra, A. - \ 2001
Investigative ophthalmology and visual science (2001). - ISSN 0146-0404 - p. 3228 - 3232.
Health Council of the Netherlands. Deoxynivalenol (DON).
Koeman, J.H. ; Feron, V.J. ; Fink-Gremmels, J. ; Lowik, M.R.H. ; Mulder, G.J. ; Sauer, P.J.J. ; Waard, M.A. de; Stoppelaar, J.M. de; Theelen, R.M.C. ; Pijls, L.T.J. - \ 2001
The Hague, the Netherlands : Health Council of the Netherlands - ISBN 9789055493944 - 70
fusarium - toxinogene schimmels - risicoschatting - tarwe - vomitoxine - toxinogenic fungi - risk assessment - wheat - vomitoxin
Using interleukin 10 to interleukin 6 ratio to distinguish primacy introcular lymphoma and uveitis - Response
Ongkosuwito, J.V. ; Feron, E.J. ; Doornik, C.E.M. van; Lelij, A. van der; Hoyng, C.B. ; Heij, E. La; Kijlstra, A. - \ 1999
Investigative ophthalmology and visual science 40 (1999). - ISSN 0146-0404 - p. 2462 - 2463.
Endotoxins: health-based recommended occupational exposure limit.
Feron, V.J. ; Beems, R.B. ; Brokamp, J.J.A.M. ; Heederik, D.J.J. ; Henderson, P.T. ; Hontelez, L.C.M.P. ; Jong, G. de; Mik, G. de; Molier-Bloot, J. - \ 1998
Unknown Publisher
Interactions of [alpha,beta]-unsaturated carbonyl compounds with the glutathione-related biotransformation system
Iersel, M.L.P.S. van - \ 1998
Agricultural University. Promotor(en): Peter van Bladeren; J.H. Koeman. - S.l. : Van Iersel - ISBN 9789054859086 - 120
meervoudige resistentie tegen geneesmiddelen - multiple drug resistance
<p><strong>Introduction</strong><br/>Modulation of glutathione-related biotransformation steps may play a role in important phenomena as anticarcinogenicity and multidrug resistance. Glutathione-related biotransformation comprises three main aspects i.e. glutathione, the glutathione S-transferases and the multidrug resistance associated protein pump. In Figure 6.1 is shown how the levels and relative activities of these three entities interact.</p><p>The research presented in this thesis focused on the effects of the ubiquitous class ofα,β-unsaturated carbonyl compounds on these glutathione-related processes, especially glutathione S-transferase P1-1, while a secondary aim was to provide insight in the metabolism of these compounds.</p><p><CENTRE><img src="/wda/abstracts/i2521.gif" width="310" height="330" HSPACE=6 PACE=6/></CENTRE><br/><strong>Figure 6.1 Interactions between the three aspects of the glutathione-related biotransformation system.</strong></p><p>Firstly, studies were conducted to expand understanding of the mechanisms of both GST inhibition and glutathione conjugation.</p><p>Secondly, the effects of a series of exogenousα,β-unsaturated carbonyl compounds on the glutathione-related biotransformation were studied in a cellular system, as all three aspects are integrated in such a system and the relative importance of the various steps can be estimated.</p><p>Finally, an important endogenousα,β-unsaturated ketone, prostaglandin A <sub>2</sub> , was selected and its metabolism and effects were studied to emphasise the significance of the glutathione-related metabolism for endogenous compounds and obtain insight into the possible role of GST inhibition in regulation of physiological processes.</p><p><strong>Summary</strong><br/>To elucidate mechanistic features of the covalent interaction betweenα,β-unsaturated carbonyl compounds and GSTP1-1 and to study the involvement of the cysteine residues in this interaction, investigations were performed with mutants of GSTP1-1 ( <em>chapter 2</em> ). In these mutants cysteine 47 and/or cysteine 101 were mutated into a serine. Theα,β-unsaturated carbonyl compounds, used in this study, inhibited GSTP1-1 activity, but when both cysteine residues were mutated, almost no inhibition of GSTP1-1 could be observed. Mutation of only the cysteine 47 residue already had the same effect. However, especially high concentrations of theα,β-unsaturated compounds still inhibited the double mutant GSTP1-1 to a certain extent, suggesting that lower reactivity sites in the enzyme can be modified as well. From the compounds studied, ethacrynic acid, acrolein, curcumin, and 4-hydroxy-2-nonenal were the most potent covalent inhibitors. As the compounds used are Michael acceptors, reversal of inhibition of activity by an excess of glutathione was investigated. Only for ethacrynic acid and crotonaldehyde, inhibition could be totally reversed. But inhibition by, for instance, acrolein could not be reversed; experiments using matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) showed that covalent cross linking between subunits occurred by acrolein (results not shown), as was demonstrated for eugenol previously (Rompelberg <em>et al</em> ., 1996). For the other compounds used, partial restoration of GSTP1-1 activity was observed, again indicating that reactions with other amino acids or binding places play a role as well.</p><p>The results of this chapter indicate thatα,β-unsaturated carbonyl derivatives inhibit GSTP1-1 irreversibly mainly by binding to the cysteine residues, especially cysteine 47. This covalent inactivation might in particular be important, when glutathione concentrations are low.</p><p>The potential relevance of stereoselective formation of glutathione conjugates ofα,β-unsaturated carbonyl compounds for the actual effects of these compounds led to the investigation of the relative formation of the two diastereoisomers of model compound ethacrynic acid. Ethacrynic acid has become a thoroughly and widely studied compound, lately especially with regard to multidrug resistance (Schultz <em>et al</em> ., 1997; Shen <em>et al</em> ., 1997). Glutathione conjugation of ethacrynic acid leads to the formation of two diastereoisomers, chemically in almost equal amounts (48:52). Although it has been shown that rat GST mu did not catalyse glutathione conjugation stereoselectively (Ploemen <em>et al</em> ., 1993b), human GSTP1-1 is stereospecific for the formation of one diastereoisomer, in this thesis designated as diastereoisomer A. GSTA1-1, but not GSTA1-2 and GSTA2-2 is stereoselective for the same diastereoisomer. No significant deconjugation of the diastereoisomeric mixture or of diastereoisomer A alone could be detected, chemically or upon addition of GSTP1-1; the latter probably due to inhibition of the enzyme. Clearly the equilibrium for glutathione conjugation of ethacrynic acid is strongly in favour of product formation. As a first step to study the role of conjugation in relation to the other glutathione-related aspects, IGR-39 human melanoma cells, containing high levels of GSTP1-1, were exposed to ethacrynic acid. Diastereoisomer A was preferentially produced in the medium. Although this has not yet been proven definitively, this was probably due to the stereoselective formation of diastereoisomer A by GSTP1-1 catalysis rather than stereoselective transport of the conjugates ( <em>chapter 3</em> ).</p><p>From this chapter it is clear that the chemical and enzyme catalysed equilibria for the reaction between ethacrynic acid and glutathione are strongly in favour of product formation. GSTP1-1 stereospecifically catalyses the glutathione conjugation of ethacrynic acid GSTA1-1 is stereoselective for the same diastereoisomer. Furthermore it was shown that stereoselectivity plays a role in cellular systems.</p><p>As glutathione conjugation and inhibition of GST activity have mainly been studied using cytosol or purified enzymes, a method was developed to investigate modulation of glutathione conjugation in intact IGR-39 human melanoma cells by the quantification of the excretion of S-(2,4-dinitrophenyl)glutathione (DNPSG), the glutathione conjugate of the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB). By investigating intracellular glutathione levels, GST activity and intra- and extracellular DNPSG concentration, some determinants involved in the mechanisms of inhibition of DNPSG excretion could be identified ( <em>chapter 4</em> ). These mechanisms include depletion of glutathione levels, reversible and irreversible inhibition of glutathione S-transferase activity, and modulation of the efflux of glutathione conjugates by an effect on the multidrug resistance associated protein (MRP) pump.</p><p>Using this assay, a series ofα,β-unsaturated carbonyl compounds were tested for their inhibiting properties of DNPSG excretion. Curcumin, an antioxidant and anticarcinogenic compound, was the most potent inhibitor of DNPSG excretion in these cells, followed by ethacrynic acid. Citral did not show any effect of DNPSG excretion up to 100 mM and acrolein was too toxic to get any effect ( <em>chapter 4</em> ). The mechanisms of inhibition differed between the variousα,β-unsaturated carbonyl compounds. For curcumin and ethacrynic acid, glutathione depletion, reversible inhibition of GSTs and covalent modification of GSTP1-1 all three play a role in the inhibition of DNPSG excretion. However for <em>trans</em> -2-hexenal and cinnamaldehyde, reversible GST inhibition seems to be the major determinant for its effect. Crotonaldehyde mainly inhibits DNPSG excretion by depleting glutathione, but reversible inhibition does presumably plays a role as well. Curcumin and ethacrynic acid also inhibit the efflux of DNPSG by an effect on the transport of the glutathione conjugate out of the cells, probably by the multidrug resistance associated protein (MRP) pump. Indeed, it has been shown that the glutathione conjugate of EA has an equal efficiency for transport by the MRP pump compared to DNPSG (Zaman <em>et al.</em> , 1996).</p><p>Some of theα,β-unsaturated carbonyl compounds, used in this study, thus inhibit GST avtivity in human melanoma cells. They modulate the glutathione related biotransformation system in these cells in different ways, i.e. glutathione depletion, reversible and irreversible inhibition of GST activity and/or influence on the efflux of glutathione conjugates.</p><p>For the endogenousα,β-unsaturated ketone prostaglandin A <sub>2</sub> a more complete picture of its metabolism and the influence on glutathione-related biotransformation could be obtained, as it was also possible to analyse PGA <sub>2</sub> -glutathione conjugate excretion into the medium during exposure of the cells to PGA <sub>2</sub> . After loading IGR-39 human melanoma cells with [ <sup>3</SUP>H] glycine and subsequent exposure to PGA <sub>2</sub> , both diastereoisomers of the PGA <sub>2</sub> -glutathione conjugate are excreted into the medium, however with a clear excess of the S-form. Previous work indicates that this is the result of the preferential formation of the S-form by GSTP1-1 that is present in the cells and not from a stereoselectivity in transport (Bogaards <em>et al</em> ., 1997; Evers <em>et al</em> ., 1997). Incubation of IGR-39 human melanoma cells with PGA <sub>2</sub> during 1 or 4 hours clearly influenced the glutathione-related metabolism. After 1 hour exposure, DNPSG excretion was reduced mainly due to inhibition of the efflux of the conjugate. Indeed, it has recently been shown that the glutathione conjugates of PGA <sub>2</sub> have a higher affinity for MRP compared to DNPSG (Evers <em>et al</em> ., 1997). After 4 hours, total DNPSG formation was reduced markedly, resulting from depletion of glutathione and reversible and irreversible inhibition of GSTs; inhibition of efflux then only played a minor role. Although irreversible inhibition already accounted for about 25% of the GST inhibition by PGA <sub>2</sub> , depletion of intracellular GSH with BSO resulted in an even higher level of covalent inactivation. Experiments with purified GSTP1-1 and mutants missing one or two cysteine residues, revealed that this covalent inhibition of GSTP1-1 resulted from the binding of PGA <sub>2</sub> to mainly the cysteine 47 moiety of the enzyme. This inactivation could be totally reversed by an excess of glutathione, indicative of a retro-Michael reaction ( <em>chapter 5</em> ).</p><p>The results of this chapter again prove that GSTP1-1 might play a role in scavenging alkylating agents especially when GSH concentrations are low, or conversely might serve as a storing or transport protein for physiologically important compounds such as PGA <sub>2</sub> .</p><p><strong>Perspectives</strong><br/>Both endogenous and exogenousα,β-unsaturated carbonyl compounds thus appear to influence several aspects of the glutathione-related biotransformation system. They are conjugated to glutathione, thereby depleting glutathione and thus influencing the redox status of the cells, which plays a role in regulation of Phase II enzymes (Talalay <em>et al</em> ., 1995; Primiano <em>et al</em> ., 1997). These conjugates usually are less toxic than their parent compounds, but it is also possible that they undergo retro-Michael cleavage, releasing the reactive compound under different circumstances, then also influencing the redox status. The glutathione conjugates themselves are inhibitors of GST activity (product binding) and probably by binding the enzyme, they are transported to efflux pumps such as MRP. Stereoselectivity in the formation of GSH conjugates might influence function and toxicity ofα,β-unsaturated carbonyl compounds. Future studies should be performed to further elucidate the conjugation mechanism and the relevance of stereoselectivity <em>in vivo</em> . One can only speculate about the physiological importance of this phenomenon. Studies with the recently developed GST pi knock-out mice (Henderson <em>et al.</em> , 1996) and MRP knock-out mice (Wijnholds <em>et al</em> ., 1997) could give some useful information.α,β-Unsaturated carbonyl compounds can inhibit GST activity both by competitive inhibition on the active site as well as covalent inactivation on the cysteine residues of the enzyme. This covalent binding is reversible due to retro-Michael reaction and most likely constitutes a functional role as well.</p><p>Man is exposed to substantial amounts of theseα,β-unsaturated carbonyl compounds every day, dependent on life style factors, such as diet, smoking, contact with traffic exhaust. Therefore these findings are of considerable relevance. Especially when considering the total daily exposure to the variousα,β-unsaturated carbonyl compounds, concentrations reached in the body might equal the concentrations used in this study. For instance: acrolein is present in wine up to about 3.8 ppm (70 mM) (Feron <em>et al</em> .,1991); curcumin, the major component of the spice curry, is widely used and consumption for adult Indians is estimated on about 125 mg/day (Opdyke and Letizia, 1983); cinnamaldehyde is present in food up to 700ppm (4.7 mM) (Feron <em>et al</em> ., 1991); adding the additional endogenously producedα,β-unsaturated carbonyl compounds, the combined exposure very likely influences the glutathione-related biotransformation system.</p><p>The fact that endogenousα,β-unsaturated carbonyl compounds as 4-hydroxy-2-nonenal, <em>trans</em> -2-hexenal and prostaglandin A <sub>2</sub> are good covalent inhibitors of GSTP1-1 and that covalent modification occurs intracellularly, supports the assumption, that GSTP1-1 might not only play a role in glutathione conjugation but also has other cellular functions. In this respect one can think of GSTP1-1 as a transport or storage protein for endogenous compounds and/or as a general intracellular scavenging protein for electrophilic agents.</p><p>The suggestion that GSTP1-1 might function as a storage for endogenous compounds, is a commonly accepted function of GSTs in general (Listowsky, 1988). GSTP1-1 is known to have a hydrophobic pocket, which binds fatty acids (Nishihira <em>et al</em> ., 1992). Experiments with fatty acids and human GSTP1-1 revealed that linolenic acid is capable of inhibiting GSTP1-1 activity thereby not affecting covalent modification of GSTP1-1 by ethacrynic acid (unpublished results). This means that it should be possible to bind covalently modified GSTP1-1 on the fatty acid binding site. Interesting possibilities arise when this would be possible with regard to biomonitoring exposure to electrophilic compounds as GSTP1-1 is a major GST present in erythrocytes.</p><p>Closely linked to this storage function, is the possible function of GSTP1-1 as a transport protein. As indicated for prostaglandin A <sub>2</sub> in <em>chapter 5</em> , GSTP1-1 can transport this compound intracellularly to the nucleus by binding it. Localization of GSTP1-1 in human tissue, using immunohistochemical techniques, indeed show the presence of this isoenzyme in the nucleus (Terrier <em>et al</em> ., 1990). Compounds that are delivered in the nucleus can for instance change thiols, from GST to a transcription factor or other protein and accordingly trigger all sorts of events. It becomes now more and more accepted that genes, involved in protection against carcinogens are regulated by the redox status of cells (Talalay <em>et al</em> ., 1995; Primiano <em>et al</em> ., 1997; Itoh <em>et al</em> ., 1997). Another aspect in the role of GSTP1-1 as a transport protein is the capability of the enzyme to bind products. For instance the glutathione conjugate of ethacrynic acid is an even better inhibitor of GSTP1-1 than the parent compound. One could clearly think of a role of GSTP1-1 in transporting glutathione conjugates from the site of formation to efflux pumps in the plasma membrane, for instance MRP.</p><p>The third notion, that GSTP1-1 might function as a general scavenging protein, might especially be apparent when glutathione levels are low. Lipid peroxidation products as HNE and reactive oxygen species can thus be neutralized, but also other electrophilic compounds. The inactivation of GSTP1-1 by 4-hydroxy-2-nonenal (HNE) and the only partial recovery of activity after incubation with a molar excess of glutathione ( <em>chapter 2</em> ) are in line with previous findings with H <sub>2</sub> O <sub>2</sub> (Sluis-Cremer <em>et al</em> ., 1996). The ability of otherα,β-unsaturated carbonyl compounds to inactivate purified GSTP1-1 as well as GSTP1-1 in cells ( <em>chapter 2, 4 and 5</em> ), together with previous results (Berhane and Mannervik, 1990; Terada <em>et al</em> ., 1995) also support a general scavenging role of GSTP1-1.</p><p>The significance of MRP in the maintenance of intracellular concentrations of both functional and toxic or carcinogenic agents is under current investigation. However,α,β-unsaturated carbonyl compounds may influence its transport activity, for a start by depletion of glutathione, which seems to be essential for MRP. Furthermore, as the glutathione conjugates of both EA and PGA <sub>2</sub> are substrates, the glutathione conjugates of otherα,β-unsaturated carbonyl derivates might be substrates as well. Future research should focus on structure activity relationships for MRP substrates. The importance of stereoselectivity in the transport of these conjugates by MRP also merits further investigation (Evers <em>et al</em> ., 1997, Loe <em>et al</em> ., 1997).</p><p><strong>Conclusion</strong><br/>In conclusion, the results in this thesis demonstrate for the first time that GST activity is inhibited in cells exposed toα,β-unsaturated carbonyl compounds. It also became clear that GST activity should not be studied on its own, but, as it is a part of a glutathione-mediated biotransformation system, it should be investigated in conjunction with glutathione levels and the multidrug resistance associated protein (MRP). Moreover, the apparent involvement of GSTP1-1 in the metabolism of the endogenous compound prostaglandin A <sub>2</sub> , indicates a possible role of this isoenzyme in regulation of cell proliferation. Mostα,β-unsaturated carbonyl compounds, studied in this thesis, interact with the glutathione-related biotransformation system (i.e. glutathione conjugation, glutathione depletion, both reversible and irreversible inhibition of GST activity, modulation of MRP); some with three aspects, some only with one or two. In view of the multiple roles of this system in cellular physiology, cell proliferation, gene regulation, anticarcinogenicity and multidrug resistance,α,β-unsaturated carbonyl compounds indeed seem very important, especially as man is exposed to this class of compounds in everyday life. The results open further perspectives for the development of therapeutic agents regarding multidrug resistance and anticarcinogenicity. The potential effect of these compounds on vital processes emphasise the need for future research on the total exposure of people to these compounds, especially via diet and environment.</p>
Exposure of humans to complex chemical mixtures: hazard identification and risk assessment.
Feron, V.J. ; Groten, J.P. ; Bladeren, P.J. van - \ 1998
Archives of Toxicology 20 (1998). - ISSN 0340-5761
Toxicological evaluation and risk assessment of chemical mixtures.
Cassee, F.R. ; Groten, J.P. ; Bladeren, P.J. van; Feron, V.J. - \ 1998
Critical Reviews in Toxicology 28 (1998). - ISSN 1040-8444 - p. 73 - 101.
Subacute toxicity of a combination of nine chemicals in rats: detecting interactive effects with a two level factorial design.
Groten, J.P. ; Schoen, E.D. ; Bladeren, P.J. van; Kuper, F.C.F. ; Zorge, J.A. van; Feron, V. - \ 1997
Fundamental and Applied Toxicology 36 (1997). - ISSN 0272-0590 - p. 15 - 29.
Influence of growth factors and medium composition on benzo[a]pyrene- and vitamin A-induced cell proliferation and differentiation in hamster tracheal epithelium in organ culture.
Wolterbeek, A.P.M. ; Ciotti, M.A.L.T. ; Schoevers, E.J. ; Roggeband, R. ; Baan, R.A. ; Feron, V.J. ; Rutten, A.A.J.J.L. - \ 1996
Toxicology in Vitro 10 (1996). - ISSN 0887-2333 - p. 359 - 369.
Effects of vitamin A and [beta]-carotene on respiratory tract carcinogenesis in hamsters : in vivo and in vitro studies
Wolterbeek, A.P.M. - \ 1995
Agricultural University. Promotor(en): J.H. Koeman; V.J. Feron; A.A.J.J.L. Rutten. - S.l. : Wolterbeek - ISBN 9789054853749 - 158
carcinoom - neoplasma's - ademhalingsziekten - retinol - carotenen - provitaminen - carotenoïden - muridae - muizen - ratten - carcinoma - neoplasms - respiratory diseases - carotenes - provitamins - carotenoids - mice - rats
<strong>Summary</strong><br/>Respiratory tract cancer is the leading cause of death by cancer in 'Western' countries. The greater part of lung cancers are caused by smoking. Furthermore, environmental air pollution and occupational exposure contribute to the high incidence of lung cancer. Because it seems to be an almost impossible task to eliminate exposure of man to all these factors, considerable effort has been focused on identifying naturally occurring or synthetic compounds which can prevent the formation of respiratory tract cancer. In this regard, (pro)vitamin A (vitamin A and β-carotene) have been shown very promising. In a large number of epidemiological and experimental studies it has been shown that (pro)vitamin A inhibits the formation of respiratory tract cancer. However, the results of these studies are not always consistent and some studies even showed that (pro)vitamin A increases the incidence of lung cancer. Although the effect of (pro)vitamin A on the formation of respiratory tract cancer has been studied extensively, the mechanisms by which (pro)vitamin A influences the process of respiratory tract carcinogenesis are still not fully understood. In the studies described in this thesis, using both an <em>in vitro</em> and an <em>in</em> vivo approach, the effects of vitamin A and β-carotene on various stages of the process of chemically-induced respiratory tract carcinogenesis were investigated (Figure 1). The emphasis was on the effects of vitamin A and β-carotene on benzo[a]pyrene (B[a]P)-induced DNA-adduct formation, DNA-repair activities, cell proliferation and histomorphological changes in the hamster respiratory tract epithelium. Furthermore, the relationships between DNA-adduct formation, DNA-repair activities, cell proliferation and the expression of the tumour suppressor gene p53 were investigated.<br/> <br/><img src="/wda/abstracts/i1924_1.gif" height="830" width="600"/><br/><em>In vitro studies</em><br/>In the first <em>in vitro</em> experiments, the formation and repair of B[a]P-DNA adducts in hamster and rat tracheal epithelial cells was studied (Chapters 3 and 4). It was shown that <em>in vitro</em> the main DNA adduct formed in hamster tracheal epithelial cells was the trans-addition product of deoxyguanosine and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). This is the same DNA adduct as formed <em>in vivo</em> in tracheal epithelial cells of hamsters intratracheally treated with B[a]P. Furthermore, it is the same adduct as has been frequently observed in human respiratory tract cells. In rat tracheal epithelial cells two major DNA adducts were found <em>in vitro</em> : the BPDE-dG adduct and an adduct that is probably derived from interaction of <em>syn</em> -BPDE and deoxyadeno- s-ine. Both the formation of B[a]P-DNA adducts and the B[a]P-induced DNA-repair activities in hamster tracheal epithelial cells were time- and concentration-dependent. In rat tracheal epithelial cells, the formation of B[a]P-DNA adducts was 10 times lower than in hamster tracheas. Furthermore, unlike in hamster tracheal epithelial cells, B[a]P did not induce DNA-repair activities in rat tracheal epithelial cells. In the studies described in Chapter 5, the effect of vitamin A and β-carotene on the formation and repair of B[a]P-DNA adducts in hamster tracheal epithelial cells was investigated. It was shown that both vitamin A and β-carotene slightly inhibited the formation of B[a]P-DNA adducts. In addition, vitamin A and β-carotene increased B[a]P-induced DNA-repair activities. This suggests that the observed decrease in B[a]P-DNA adducts is a positive effect of vitamin A and β-carotene, probably also partly caused by an increase in DNA-repair activities. The effect of vitamin A on DNA-adduct formation and DNA-repair activities depended on the concentration of B[a]P versus the concentration of vitamin A. At a low B[a]P concentration relative to the concentration of vitamin A the formation of B[a]P-DNA adducts was inhibited by vitamin A, whereas at a relatively high concentration of B[a]P the formation of DNA adducts was enhanced by vitamin A.<p>The role of B[a]P and vitamin A in cell proliferation in hamster tracheal epithelium in organ culture is described in Chapter 6. It was shown that the effects of B[a]P and vitamin A on cell proliferation strongly depended on the culture medium used; in tracheas cultured in Ham's F12 medium cell proliferation was decreased by B[a]P treatment compared to control tracheas, while cell proliferation in tracheas treated with vitamin A in combination with B[a]P was increased compared to tracheas treated with B[a]P alone. In tracheas cultured in CMRL-1066 medium, the effects of B[a]P and vitamin A on cell proliferation were opposite to those observed in tracheas cultured in Ham's F12 medium: cell proliferation in tracheas cultured in CMRL-1066 medium and treated with B[a]P was increased compared to control tracheas, while vitamin A decreased B[a]P-induced cell proliferation. To explain these opposite effects of B[a]P and vitamin A on cell proliferation, various medium components and growth factors were investigated. The concentration of CaCl <sub><font size="-2">2</font></sub><strong>.</strong> 2H <sub><font size="-2">2</font></sub> O revealed to be the most important factor: supplementation of CaCl <sub><font size="-2">2</font></sub><strong>.</strong> 2H <sub><font size="-2">2</font></sub> O to the Ham's F12 culture medium mimicted the effects of B[a]P and vitamin A on cell proliferation in CMRL-1066 medium. These results clearly indicate that Ca <sup><font size="-2">2+</font></SUP>is an important regulator of proliferation of hamster tracheal epithelial cells. Furthermore, the results of these experiments showed that the level of B[a]P-DNA adducts was inversely related to cell proliferation in tracheas cultured in Ham's F12 medium. Although these results suggest that the tumour suppressor gene p53 might be involved by inhibiting cell proliferation as a consequence of DNA damage, we were unable to show a direct relationship between the level of B[a]P-DNA adducts, cell proliferation and expression of the p53 tumour suppressor protein in hamster tracheal epithelium in organ culture. <em></em><p><em>In vivo studies</em><br/>The most widely applied <em>in vivo</em> model to study the aetiology and pathogenesis of respiratory tract cancer in experimental animals is based on repeated intratracheal instillations of a saline suspension of fine crystalline B[a]P particles attached to ferric oxide as a carrier. Various aspects of this method are discussed in Chapter 2, showing that the dose of B[a]P and the size of the B[a]P particles are the most important variables influencing the tumour response. In a first <em>in vivo</em> experiment into the effect of vitamin A and β-carotene on B[a]P-induced (pre)neoplastic changes in the respiratory tract of hamsters, the response of the respiratory tract epithelium was too low. This might be due to an insuficiently high B[a]P dose, possibly in combination with a relatively insensitive strain of hamsters used. The low response hampered studying potential effects of vitamin A or P-carotene on the (pre)neoplastic response (Chapter 7). An interesting observation in this experiment was an exceptionally low mortality of hamsters fed a high-β-carotene diet. Although we were unable to establish the exact cause of death of hamsters not receiving β-carotene, the most conspicuous difference between hamsters in the high-β-carotene group and hamsters in other groups was a decrease in lipid peroxidation in the livers of hamsters in the former group. Probably, this effect was not only due to the high concentration of β-carotene in the diet, but was also related to a high dietary level of α-tocopherol and ascorbyl palmitate (also present in the β-carotene beads and used to protect β-carotene from oxidation). To obtain a higher tumour response, hamsters were treated in a second experiment with a higher total dose of B[a]P (Chapter 8 and 9). In this study, a clear relationship appeared to exist between the extent of B[a]P-DNA adduct formation, the induction of cell proliferation and the immunocytochemically detected expression of the p53 protein in hamster tracheal epithelial cells. However, in this experiment the formation of B[a]P-DNA adducts was not found to be affected by a high dietary level of β-carotene, probably due to the high B[a]P dose. Furthermore, β-carotene did not affect B[a]P-induced cell proliferation and expression of the p53 protein in tracheal epithelial cells. Chapter 9 describes the histomorphological aspects of this hamster study, using conventional histopathology and immunohistochemical techniques for the detection of various cytokeratins and glutathione <em>S</em> -transferase (GST)-isoenzyme Pi. From this study, it appeared that B[a]P influenced both the expression of cytokeratins and the expression of the GST-isoenzym Pi. However, in accordance with the results described in Chapter 8, β-carotene did not inhibit B[a]P-induced lesions in the respiratory tract epithelium of hamsters.<p><strong>Concluding remarks</strong><br/>Finally, the studies described in this thesis allow the following conclusions:<p>- In vitro, vitamin A and β-carotene decrease slightly but consistently the formation of B[a]P-DNA adducts, probably due to an increase in DNA-repair activities. The effect of vitamin A on the formation of B[a]P-DNA adducts depends on the concentration of B[a]P versus the concentration of vitamin A.<p>- The effects of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelial cells in organ culture strongly depend on the tissue-culture medium used, in particular on the concentration of Ca <sup><font size="-2">2+</font></SUP>in the medium. The effects of B[a]P and vitamin A on cell proliferation observed in tracheas cultured in CMRL-1066 medium are similar to the effects generally observed <em>in vivo</em> .<p>- The hamster tracheal organ culture model is very suitable to study the B[a]P-induced formation of DNA adducts and DNA-repair activities. Both the formation and repair of B[a]P-DNA adducts is dose and time dependent. Furthermore, the main adduct formed <em>in vitro</em> is similar to the adduct formed <em>in vivo</em> after intratracheal instillation of B[a]P, and moreover, this adduct is frequently observed in man.<p>- A high dietary dose of β-carotene, possibly in combination with a high level of et-tocopherol and ascorbyl palmitate, strongly increases the survival of hamsters.<p>- In tracheal epithelia] cells of hamsters treated intratracheally with B[a]P, a relationship between the level of B[a]P-DNA adducts, cell proliferation and p53 expression is observed.<p>- The effect of vitamin A on B[a]P-induced DNA-adduct formation and cell proliferation, as observed in the <em>in vitro</em> experiments, was not found in <em>in vivo</em> experiments, probably due to the high B[a]P dose applied.<p>- β-carotene did not affect the formation of (pre)neoplastic changes in the respiratory tract epithelium of hamsters intratracheally treated with B[a]P as evaluated by conventional histopathology, cytokeratin expression, and glutathione S-transferase isoenzyme Pi expression.<p>- Although intratracheal instillation of B[a]P to Syrian golden hamsters is one of the most widely applied models to study respiratory tract cancer in experimental animals, the tumour response is difficult to control due to a large number of variables affecting the response. The most important variables influencing the tumour response are the dose of B[a]P and the size of the B[a]P particles.<p>In conclusion, although the <em>in vitro</em> experiments described in this thesis show that vitamin A and β-carotene may influence the process of respiratory tract carcinogenesis, <em>in vivo</em> it was not possible to show a modulating effect of vitamin A and β-carotene on B[a]P-induced respiratory tract cancer in hamsters. To explain the inconsistencies in the effect of vitamin A and β-carotene on respiratory tract cancer, further in-depth research should he focused on the molecular mechanisms underlying this effect. The concentration of vitamin A and β-carotene, in particular the concentration of the active metabolite retinoic acid, in target cells should be measured in relation to the action of these molecules on the genomic level.
Benzo[_]pyrene-induced respiratory tract cancer in hamsters fed a diet rich in ß-carotene. A histomorphological study.
Wolterbeek, A.P.M. ; Schoever, E.J. ; Bruyntjes, J.P. ; Rutten, A.A.J.J.L. ; Feron, V.J. - \ 1995
Journal of Environmental Pathology, Toxicology and Oncology 14 (1995). - p. 35 - 43.
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