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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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Biological activity of Pakistani isolate SpltNPV-Pak-BNG in second, third and fourth instar larvae of the leafworm Spodoptera litura
Ali, Ghulam ; Vlak, Just M. ; Werf, Wopke van der - \ 2018
Biocontrol Science and Technology 28 (2018)5. - ISSN 0958-3157 - p. 521 - 527.
biological activity - larval instars - potency - S. litura - Spodoptera litura nucleopolyhedroviruses
Recently, a novel isolate of the baculovirus Spodoptera litura nucleopolyhedrovirus (SpltNPV) has been isolated from Pakistan, which is distinct from the type species SpltNPV-G2 (ICTV). Here, we examined the biological activity of this isolate (SpltNPV-Pak-BNG) in second (L2), third (L3) and fourth instar (L4) larvae of the leafworm S. litura, more specifically to measure biological properties that are relevant for use of this virus for pest control under field conditions. The median lethal dose for L2 and L3 instar larvae was similar, but significantly lower than for L4 larvae. Likewise, the survival time was similar for L2 and L3 larvae (84 h), but was significantly longer for L4 instar larvae (108 h). Thus, in terms of efficacy, S. litura L2 and L3 instar larvae are the preferred targets for S. litura control with SpltNPV-Pak-BNG in field crops in Pakistan. On the basis of our data spray regimes can be designed to control the leafworm in cotton and vegetable crops targeting L2 and L3 larvae.
Biological and genetic characterization of a Pakistani isolate of Spodoptera litura nucleopolyhedrovirus
Ali, Ghulam ; Werf, Wopke van der; Vlak, Just M. - \ 2018
Biocontrol Science and Technology 28 (2018)1. - ISSN 0958-3157 - p. 20 - 33.
biological activity - genetic characterization - Spodoptera litura nucleopolyhedroviruses
Spodoptera litura is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to Bacillus thuringiensis toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of S. litura nucleopolyhedrovirus (SpltNPV, Baculoviridae), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD50) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT50) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes polyhedrin, DNApol, pif-2 and lef-8 confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of S. litura in Pakistan.
The influence of phase II conjugation on the biological activity of flavonoids
Beekmann, K. - \ 2016
University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): L. Actis-Goretta. - Wageningen : Wageningen University - ISBN 9789462577640 - 171 p.
flavonoids - biological activity - in vitro - biosynthesis - peroxisomes - microarrays - daidzein - genistein - oestrogen receptors - isoflavones - quercetin - kaempferol - serine proteinases - threonine - flavonoïden - biologische activiteit - biosynthese - peroxisomen - daidzin - genisteïne - oestrogeenreceptoren - isoflavonen - quercetine - serine proteïnasen

Flavonoid consumption is often correlated with a wide range of health effects, such as the prevention of cardiovascular diseases, neurodegenerative diseases, and diabetes. These effects are usually ascribed to the activity of the parent flavonoid aglycones, even though these forms of the flavonoids generally have a low systemic bioavailability. During uptake, flavonoids undergo phase II metabolism and are present in the systemic circulation nearly exclusively as conjugated metabolites. The aim of this thesis was to study the effect of conjugation on the biological activity of selected flavonoids towards different endpoints relevant for human health. To this end, conjugation with glucuronic acid was taken as the model type of conjugation because this modification is generally observed to be the most important metabolic conjugation reaction for flavonoids in man.

A review of scientific literature published until early 2012 reveals that metabolic conjugation can affect the biological activity of flavonoids in different ways. Conjugation can increase, decrease, inverse or not affect the biological activity, depending on the flavonoid, the type and position of conjugation, the endpoint studied, and the assay system used. Based on the literature reviewed it is concluded that the effect of conjugation has to be studied on a case-by-case basis.

As the research on the biological activity of biologically relevant flavonoid conjugates is often hampered by the generally low commercial availability and high prices of these conjugates, a simple and versatile method for the biosynthesis of metabolically relevant flavonoid conjugates is described. Using this method, relevant conjugates can be prepared from different flavonoid substrates in sufficient quantities for in vitro bioassays. Further, an efficient strategy for the identification of these flavonoid conjugates by LC-MS and 1H-NMR using MetIDB (Metabolite Identification Database), a publicly accessible database of predicted and experimental 1H-NMR spectra of flavonoids, is presented.

To study the effect of conjugation on the biological activities of flavonoids, several different assay systems and endpoints were used to study the activity of different flavonoids and their conjugates. The effects of quercetin, kaempferol, and their main plasma conjugates quercetin-3-O-glucuronide and kaempferol-3-O-glucuronide (K-3G) on different endpoints related to peroxisome proliferator-activated receptor (PPAR)-γ were studied. PPAR-γ activation is reported to have positive health effects related to adipogenesis, insulin resistance and inflammation. The presented results show that the flavonoid aglycones increased PPAR-γ mediated gene expression in a stably transfected reporter gene cell line, and that glucuronidation diminished this effect. These observed increases in reporter gene expression were accompanied by increased PPAR-γ receptor-mRNA expression upon exposure to kaempferol, an effect that was also reduced by glucuronidation. Using the cell-free Microarray Assay for Real-time Coregulator-Nuclear receptor Interaction (MARCoNI) it was demonstrated that, unlike the known PPAR-γ agonist rosiglitazone, neither the flavonoid aglycones nor the conjugates are agonistic ligands of the PPAR-γ receptor. Supporting the hypothesis that the tested compounds have a different mode of action from normal LBD agonism, quercetin appeared to synergistically increase the effect of rosiglitazone in the reporter gene assay. The modes of action behind the observed effects remain to be elucidated and might include effects on protein kinase activities affecting expression of the PPAR-γ receptor, or posttranscriptional modifications of PPAR-γ.

Another type of nuclear receptor known to be targeted by certain flavonoids are the estrogen receptor (ER)α- and ERβ. ERs are the main targets of estrogenic compounds, and upon their activation different transcriptional responses with opposite effects on cell proliferation and apoptosis are elicited; ERα activation stimulates cell proliferation, while ERβ activation causes apoptosis and reduces ERα mediated induction of cell proliferation. Using the MARCoNI assay, the intrinsic estrogenic effects of the two main dietary isoflavones daidzein and genistein, and their plasma conjugates daidzein-7-O-glucuronide and genistein-7-O-glucuronide on the ligand induced coregulator binding of ERα- and ERβ-LBD were studied and compared to the effect of the positive control 17β-estradiol (E2). The results show that the tested isoflavone compounds are less potent agonists of ERα- and ERβ-LBD than E2, although they modulate the LBD-coregulator interactions in a manner similar to E2. Genistein is shown to be a more potent agonist than daidzein for both receptor subtypes. While in the MARCoNI assay genistein had a strong preference for ERβ-LBD activation over ERα-LBD activation, daidzein had a slight preference for ERα-LBD activation over ERβ-LBD activation. Glucuronidation reduced the intrinsic agonistic activities of both daidzein and genistein to induce ERα-LBD and ERβ-LBD - coregulator interactions and increased their average half maximal effective concentrations (EC50s) by 8 to 4,400 times. The results presented further show that glucuronidation changed the preferential activation of genistein from ERβ-LBD to ERα-LBD and increased the preferential activation of daidzein for ERα-LBD; this is of special interest given that ERβ activation, which is counteracting the possible adverse effects of ERα activation, is considered one of the supposedly beneficial modes of action of isoflavones.

Many flavonoids are reported to be inhibitors of protein kinases. To study the effect of conjugation on the inhibition of serine/threonine protein kinases by flavonoids, kaempferol and its main plasma conjugate K-3G were selected as model compounds. Protein kinases are involved in a wide range of physiological processes by controlling signaling cascades and regulating protein functions; modulation of their activities can have a wide range of biological effects. The inhibitory effects of kaempferol, K-3G, and the broad-specificity protein kinase inhibitor staurosporine on the phosphorylation activity of recombinant protein kinase A (PKA) and of a lysate prepared from the hepatocellular carcinoma cell line HepG2 were studied using a microarray platform that determines the phosphorylation of 141 putative serine/threonine phosphorylation sites derived from human proteins. The results reveal that glucuronidation reduces the intrinsic potency of kaempferol to inhibit the phosphorylation activity of PKA and HepG2 lysate on average about 16 and 3.5 times, respectively. It is shown that the inhibitory activity of K-3G in the experiments conducted was not caused by deconjugation to the aglycone. Furthermore, the results show that kaempferol and K-3G, unlike the broad-specificity protein kinase inhibitor staurosporine, did not appear to inhibit all protein kinases present in the HepG2 lysate to a similar extent, indicating that kaempferol selectively targets protein kinases, a characteristic that appeared not to be affected by glucuronidation. The fact that K-3G appeared to be only a few times less potent than kaempferol implies that K-3G does not necessarily need to be deconjugated to the aglycone to exert potential inhibitory effects on protein kinases.

The results obtained in the present thesis support the conclusion that glucuronidation of flavonoids does not necessarily abolish their activity and that flavonoid glucuronides may be biologically active themselves, albeit at higher concentrations than the parent aglycones. In line with the conclusions from the earlier literature review, an updated literature review on the effect of conjugation on the biological activity of flavonoids concludes that that the effect of conjugation on the biological activity of flavonoids depends on the type and position of conjugation, the endpoint studied and the assay system used. Based on the results described and the literature reviewed in this thesis, several recommendations and perspectives for future research are formulated. Several methodological considerations are formulated that need to be taken into account when studying the biological activity of flavonoids and their conjugates to avoid confounding results. Further, the relevance of the gut microbiome for flavonoid bioactivity is highlighted, and considerations regarding the pharmacokinetics and pharmacodynamics of flavonoids in vivo are formulated. Altogether, it can be concluded that circulating flavonoid conjugates may exert biological activities themselves, and that understanding these is a prerequisite to successfully elucidate the mechanisms of action behind the biological activities linked to flavonoid consumption.

The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. - \ 2015
University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
fungi contain very large numbers of genes related to SM production. Yet these genes are typically
silent under laboratory conditions. There are now many tools and strategies available to activate
or clone silent SM genes. This thesis details our efforts to apply various methods to define and
then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
containing many silent SM genes and gene clusters.

In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
of growth conditions. Heterologous expression of silent SM genes in a production host is also
discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
source of novel biosynthetic enzymes.

In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
of these SM genes or gene clusters were speculated, based on their similarity to those characterized
in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
fungal species. Remarkably, two predicted functional gene clusters were linked to the production
of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
findings, the majority of SM genes remained silent and only cladofulvin was detected. During
growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
another mechanism associated with a biotrophic lifestyle.

In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
of discovering new compounds. Many Ascomycete-specific global regulators of SM production
and morphological development in other fungi were identified in C. fulvum. We investigated
three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
This suggests that cladofulvin production is not required for asexual reproduction. The main
finding of this chapter is that global regulator manipulation cannot considered to be a universal
tool to discover new fungal natural products.

In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
is produced by many fungi and plants. Once synonymous only with constipation, this former
laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
forming novel dimers from existing monomers.

In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
active during early and late stages of infection of tomato, but are down-regulated during biotrophic
growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
inoculation (dpi). At each time point the infections were inspected microscopically to detect any
phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
(UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
results suggested that cladofulvin confers resistance to abiotic stress.

In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
enable discovery of homologous genes encoding enzymes with different specificities. Combining
bioinformatic and functional analyses should prove to be a powerful strategy for discovering
compounds with new biological activities, or enzymes relevant to metabolic engineering.

Plant Biotechnology meets Immunology : plant-based expression of immunologically relevant proteins
Wilbers, R.H.P. - \ 2015
University. Promotor(en): Jaap Bakker, co-promotor(en): Arjen Schots; Geert Smant. - Wageningen : Wageningen University - ISBN 9789462574335 - 229
plantenbiotechnologie - immunologie - planten - eiwitten - farmaceutische eiwitten - interleukine 10 - ontstekingsremmers - biologische activiteit - cytokinen - genexpressie - transforming growth factor - wormen - recombinant eiwitten - glycoproteïnen - plant biotechnology - immunology - plants - proteins - pharmaceutical proteins - interleukin 10 - antiinflammatory agents - biological activity - cytokines - gene expression - helminths - recombinant proteins - glycoproteins

The incidence of inflammatory disorders in industrialized countries has dramatically increased over the last decennia, which is believed to result from a change in life-style. Treatment of these inflammatory disorders relies on the intervention in immune responses thereby restoring homeostasis. For now, many inflammatory disorders are treated with broad-acting immunosuppressive drugs or monoclonal antibodies that specifically target pro-inflammatory molecules of the immune system. An alternative therapeutic approach would be to use immunomodulatory proteins that are naturally involved in re-establishing immune homeostasis. This thesis describes the plant-based expression of a variety of immunomodulatory cytokines that may be used as biopharmaceutical proteins in the future. Furthermore, this thesis contains a pioneering chapter on the plant-based expression of immunomodulatory helminth-secreted glycoproteins.

In Chapter 2 we describe the plant-based expression of the immune-regulatory cytokine human transforming growth factor β1 (TGF-β1). By co-expressing human furin with latent TGF-β1 we were able to engineer the post-translational proteolytic processing of TGF-β1, which enabled the production of biologically active TGF-β1. In Chapter 3 we reveal that aggregation is a major production bottleneck for the anti-inflammatory cytokine interleukin-10 (IL-10). By protein engineering we were able to prevent aggregation and created a biologically active fusion protein of IL-10. In Chapter 4 we express biologically active IL-22 in plants. We reveal that, in contrast to current literature, its activity is independent of the presence of N-glycans or their composition. This chapter further reveals that plants offer a powerful tool to allow investigation into the role of N-glycans in protein folding and biological activity of glycoproteins. In Chapter 5 we further explore the potential of glyco-engineering in plants by engineering helminth-like N-glycans. We produce large quantities of two major egg antigens from Schistosoma mansoni and successfully engineer Lewis X, LDN and LDNF N-glycan structures. These plant biotechnological research lines are a showcase for the potential of engineering proteins as well as post-translational modifications in plants with special emphasis on N-glycan engineering. Altogether, the results presented in the first four chapters reveal the remarkable flexibility of plants as a production platform for recombinant proteins. It showcases the potential of engineering proteins as well as post-translational modifications in plants, but it especially highlights the engineering of tailor made N-glycans in plants. This, combined with the speed of transient expression by means of agroinfiltration, makes transient expression in Nicotiana benthamiana a powerful tool to study the role of N-glycans on glycoprotein function.

In parallel to these plant biotechnological research lines, we also developed an in vitro model system based on mouse bone marrow-derived cells to study immunological responses. We used this model to obtain clues on why IL-10 therapy has not been as successful as previously anticipated. In Chapter 6 we have set-up biological activity assays based on bone marrow-derived cells and reveal that IL-10 activity is dependent on both IL-10R1 and IL-10R2, but not IL-10R2-associated signalling via Tyk2. We also show that interactions between IL-10R1 and IL-10R2 (both intracellular and extracellular) reduce cellular binding of IL-10, but are crucial to initiate IL-10 mediated signalling. Furthermore, we observed that macrophages and dendritic cells respond differently to IL-10. This was further investigated in Chapter 7 where we reveal that GM-CSF (the cytokine used to differentiate dendritic cells) is responsible for negatively regulating early IL-10-mediated responses. Strikingly, GM-CSF does not strongly affect the IL-10-induced activation of the transcription factor STAT3. Instead, GM-CSF induces strong constitutive phosphorylation of GSK-3β, a signalling component downstream of the PI3K/Akt pathway. These immunological chapters give novel insights on the mechanism of initiating IL-10-induced signalling and on the possible integration of signal transduction pathways elicited by different cytokines. Ultimately this knowledge could provide us with new therapeutic strategies to treat inflammatory disorders.

Physiologically based biokinetic (PBBK) modeling and validation of dose-, species-, interindividual- and matrix dependent effects on the bioactivation and detoxification of safrole
Martati, E. - \ 2013
University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): Ans Punt. - S.l. : s.n. - ISBN 9789461737458 - 202
safrol - biologische activiteit - ontgifting - modelleren - fysiologie - wiskundige modellen - safrole - biological activity - detoxification - modeling - physiology - mathematical models

Keywords: safrole, PBBK model, DNA adduct, mace

Safrole has been demonstrated to be carcinogenic in rodent studies at high doses of the pure compound. The use of pure safrole in foodshas already been prohibited. As a result, the main exposure to safrole occurs through the use of herbs and spices containing low levels of safrole, such as nutmeg, mace, star anise, pimento, cinnamon, and black pepper, and food products containing these herbs and spices or their essential oils.

The Scientific Committee on Food of the European Union concluded in their evaluation that safrole is genotoxic and carcinogenic and that reductions in the exposure and restriction in the use levels are indicated. This opinion is based on carcinogenicity data from rodent studies as adequate human data were not available. Therefore, translation from animal bioassays at high dose levels of the pure compound to the risk for the human population exposed to safrole at relatively low levels via dietary intake within the complex food matrix is obviously needed. The aim of this thesis was to obtain insight into the dose-, species-, interindividual- and matrix dependent effects on the bioactivation and detoxification of safrole using physiologically based biokinetic (PBBK) modeling.

PBBK models for safrole in male rats and humans were developed based on in vitro metabolic parameters determined, in silico derived partition coefficients, and physiological parameter values taken from literature. The performance of the PBBK model for rats was evaluated by comparison of predicted levels of 1,2-dihydroxy-4-allylbenzene, 1′-hydroxysafrole glucuronide and total urinary safrole metabolites to the reported levels of these metabolites in urine of rats exposed to safrole. This evaluation revealed that the predictions adequately matched observed experimental values. The PBBK model for humans was evaluated by comparison of the PBBK predicted and the reported experimental data on the level of total safrole metabolites detected in the urine of human volunteers exposed to safrole whichshowed an adequately match. The comparison of the PBBK model for rats and humans revealed that the predicted level of formation of 1ʹ-hydroxysafrole in human liver is fourfold higher than that for rat liver and the predicted formation of 1ʹ-sulfooxysafrole is about fivefold higher than that for rat liver. This indicates that the interspecies differences in toxicokinetics for bioactivation of safrole between rat an human are in line with the uncertainty factor normally taken into account for interspecies differences in toxicokinetics of 4. Species differences between humans and rats in the nature of the detoxification pathways of 1ʹ-hydroxysafrole were larger, with the formation of 1ʹ-oxosafrole being the main detoxification pathway in humans but a minor pathway in rats and glucuronidation of 1ʹ-hydroxysafrole being less important in humans than in rats. Monte Carlo simulations revealed that the formation of 1′-sulfooxysafrole was predicted to vary 4- to 17-fold in the population (fold-difference between the 95th and median, and 95th and 5th percentile, respectively).

Risk assessment of safrole resulting from consumption of herbs and spices containing safrole should be performed taking into account the possible modulating effect of other compounds present in these herbs or spices. In this study, mace was chosen as the model spice of interest because it contains significant levels of safrole. Mace fraction with the highest SULT inhibiting activity was identified as malabaricone C. Studies using human HepG2 cells exposed to 1ʹ-hydroxysafrole and in the presence of mace extract showed that formation of the DNA adduct N2-(trans-isosafrol-3′-yl)-2′-deoxyguanosine was inhibited. To investigate the possible effects on safrole bioactivation to 1′-sulfooxysafrole by malabaricone C-containing mace extract could also be expected in vivo, the SULT inhibition was integrated into the PBBK model. The PBBK models predicted that at a dose of 50 mg/kg bw safrole and a ratio of malabaricone C-containing mace extract to safrole similar to the level of these constituents in mace, inhibition of 1′-sulfooxysafrole formation by malabaricone C-containing mace extract for rats and humans amounts to 90 and 100%, respectively. To see whether the inhibition of safrole DNA adduct formation by malabaricone C-containing mace extract is also observed in in vivo, to the end, Sprague-Dawley rats were orally exposed to mace extract and safrole. The results demonstrated that safrole DNA adduct formation in the liver of Sprague-Dawley rats by the mace extract was reduced by 55%.

The results of the in vitro and in vivo studies that demonstrated inhibition of the formation of safrole DNA adducts by mace extract, support that combination effects should be taken into account in the risk assessment when safrole is tested in the presence of a relevant food matrix. To integrate thefood matrix dependent modulation of safrole bioactivation in the risk assessment of safrole, the so-called Margin of Exposure (MOE) approach can be used. This revealed that when safrole would be tested in rodent bioassays in the presence of a matrix containing SULT inhibitors the MOE values would be higher and the need for risk management actions would be lower.

In vitro reporter gene assays for assessment of PPAR- and Nrf2-mediated health effects of tomato and its bioactive constituents
Gijsbers, L. - \ 2013
University. Promotor(en): Ivonne Rietjens; Jaap Keijer, co-promotor(en): Jac Aarts. - S.l. : s.n. - ISBN 9789461735096 - 159
solanum lycopersicum - tomaten - assays - bioactieve verbindingen - biologische activiteit - reporter-genen - genexpressie - tomatoes - bioactive compounds - biological activity - reporter genes - gene expression

The consumption of food products with health-promoting properties, such as for example margarines with plant sterols, fruit juice enriched with calcium and cereals with (soluble) fibre, has increased rapidly during the last years. The present thesis provides proof-of-principle that reporter gene assays are effective tools to investigate the effects of functional foods and food compounds on gene expression pathways.

In order to test fruits and vegetables for selected functions in reporter gene assays, extraction methods for vegetables and fruits are needed which result in extracts suitable for testing in cell-based assays. In this thesis, methods are described to prepare three different tomato extracts, containing 1) apolar compounds such as isoprenoids, 2) semi-polar compounds, such as flavonoid glycosides and 3) deglycosylated semi-polar compounds such as flavonoid aglycones. All three tomato extracts were compatible with cell-physiological and cell culture conditions.

Next, the three tomato extracts and individual tomato compounds were tested for their capacity to induce EpRE-mediated, PPARγ-mediated and PPARα-mediated gene expression using three reporter gene assays; the previously described EpRE-LUX assay and the newly developed PPARγ CALUX and PPARα CALUX assays. Both flavonoid glycosides and the tomato extract containing semi-polar compounds were unable to induce EpRE-mediated, PPARγ-mediated and PPARα-mediated gene expression, while the tomato extract containing deglycosylated semi-polar compounds, as well as individual flavonoid aglycones were able to induce EpRE-mediated, PPARγ-mediated and PPARα-mediated gene expression. Some individual isoprenoids and the tomato extract containing apolar compounds were also inducing PPARγ-mediated and PPARα-mediated gene expression, but not EpRE-mediated gene expression.

To assess differences between tomato varieties, extracts containing deglycosylated semi-polar compounds were generated from fruits of different tomato varieties. These were then tested for their capacity to induce EpRE-mediated (97 varieties), PPARγ-mediated (7 varieties) and PPARα-mediated (7 varieties) gene expression. The extracts of these varieties were all able to induce both EpRE-mediated, PPARγ-mediated and PPARα-mediated gene expression. For EpRE-mediated gene expression, a 3-fold difference was found between the least potent and the most potent variety; for PPARγ-mediated gene expression, this difference was 1.6-fold, and for PPARα-mediated gene expression this was 1.7-fold.

Metabolomics profiles of the 97 extracts were generated in order to identify phytochemicals responsible for the differences in potency of the 97 tomato varieties to induce EpRE-mediated gene expression. The flavonoid aglycone quercetin was identified as one of the main compounds responsible for the ability of tomato extracts to induce EpRE-mediated gene expression using multivariate analysis that combined the reporter gene assay data with metabolite profiles of the same tomato extracts. In addition, yet unidentified compounds correlated with the response in the EpRE-LUX assay and these compounds may also contribute to the observed induction of EpRE-mediated gene expression.

In conclusion, the work presented in this thesis provides proof-of-principle that reporter gene assays can be implemented for screening bioactive food compounds as well as whole fruit and vegetable extracts for their capacity to induce gene expression through specific health-related transcription factor.It was shown that reporter gene assays are able to pick up differences in activation of these transcription factors induced by different varieties of tomato. Furthermore, this thesis provides proof-of-principle that active ingredients contributing to the activity of the whole tomato extracts could be identified by combining reporter gene assays and metabolite profiling. Our results indicate that combining multiple reporter gene assays and metabolomics profiling, is useful in fast screening of larger numbers of food items and food compounds. Therefore, the use of reporter gene assays as a first tier in a tiered approach aiming at supporting health claims will limit the number of animal studies and human studies needed, by enabling the ranking and selection of highly promising food items or constituents for further in vivo testing.

Physiologically based kinetic (PBK) models to give insight into dose-, species-, matrix- and interindividual human variation-dependent effects on bioactivation and detoxification of methyleugenol
Al-Subeihi, A.A. - \ 2013
University. Promotor(en): Ivonne Rietjens; Peter van Bladeren, co-promotor(en): Ans Punt. - S.l. : s.n. - ISBN 9789461734297 - 225
methyleugenol - biologische activiteit - ontgifting - carcinogenen - methyl eugenol - biological activity - detoxification - carcinogens

Methyleugenol, which occurs naturally in various herbs such as tarragon, basil, nutmeg and allspice, is added to food either directly as a flavoring substance or as a constituent of added essential oils (Smith et al., 2002). The interest in the risk of methyleugenol as a food constituent came from its widespread use in a variety of foods and beverages as well as its structural resemblance to the known carcinogen safrole (Johnson et al., 2000). In addition, methyleugenol has been reported to be DNA reactive and carcinogenic, inducing malignant tumors in multiple tissues of rats and mice as well as inducing unscheduled DNA synthesis in rat liver (Ding et al., 2011; NTP, 2000; Smith et al., 2002). The safety of human exposure to methyleugenol at low dietary intake levels has been assessed several times (Hall and Oser, 1965; NTP, 2000; SCF, 2001; Smith et al., 2002) without reaching a scientific agreement on how to translate the carcinogenicity data of rodent animal experiments obtained at high levels of exposure to the relevant human situation. A recent evaluation, performed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008, has indicated that although evidence of carcinogenicity to rodents given high doses of methyleugenol exists, further research is needed to assess the potential risk to human health at relevant dietary exposure resulting from the presence of methyleugenol in foods and essential oils and its use as flavoring agent (JECFA, 2008). Predicting the cancer risk in humans at relevant dietary intake levels requires extrapolation of the animal carcinogenicity data taking in consideration dose, species, and interindividual variation. Furthermore, it implies extrapolation from rat or mouse studies with high dose levels of the pure compound to the human situation in which exposure at low dose levels occurs within the context of a complex food matrix. The aim of the present PhD project was to obtain quantitative insight into the consequences of dose- and species-dependent effects and of interindividual differences and matrix effects for the bioactivation and detoxification of methyleugenol by using physiologically based kinetic (PBK) modeling. The first chapter of this thesis presents background information to the topic. In chapter 2, a physiologically based kinetic (PBK) model for the alkenylbenzene methyleugenol in rat was defined based on in vitro metabolic parameters determined using relevant tissue fractions, in silico derived partition coefficients (Payne and Kenny, 2002 and reference therin), and physiological parameters (Brown et al., 1997) derived from the literature. The model was based on the model previously developed for the related alkenylbenzene estragole and consists of eight compartments including liver, lung, and kidney as metabolizing compartments, and separate compartments for fat, arterial blood, venous blood, richly perfused and slowly perfused tissues (Punt et al., 2008). Evaluation of the model was performed by comparing the PBK predicted concentration of methyleugenol in the venous compartment to methyleugenol plasma levels reported in the literature, by comparing the PBK predicted dose-dependent % of formation of 2-hydroxy-4,5-dimethoxyallylbenzene, 3-hydroxy-4-methoxyallylbenzene, and 1′- hydroxymethyleugenol glucuronide to the corresponding % of metabolites excreted in urine reported in the literature, which were demonstrated to be in the same order of magnitude (Solheim and Scheline, 1976). With the model obtained the relative extent of bioactivation and detoxification of methyleugenol at different oral doses was examined. At low doses, formation of3-(3,4-dimethoxyphenyl)-2-propen-1-olandmethyleugenol-2′,3′-oxideleadingto detoxification appear to be the major metabolic pathways, occurring in the liver. At high doses, the model reveals a relative increase in the formation of the proximate carcinogenic metabolite 1′- hydroxymethyleugenol, occurring in the liver. This relative increase in formation of 1′- hydroxymethyleugenol leads to a relative increase in formation of 1′-hydroxymethyleugenol glucuronide, 1′-oxomethyleugenol, and 1′-sulfooxymethyleugenol the latter being the ultimate carcinogenic metabolite of methyleugenol. These results indicate that the relative importance of different metabolic pathways of methyleugenol may vary in a dose-dependent way, leading to a relative increase in bioactiviation of methyleugenol at higher doses. In subsequent studies described in chapter 3 a physiologically based kinetic (PBK) model for methyleugenol in human based on in vitro and in silico derived parameters was identified based on the model previously developed for the related alkenylbenzene estragole. The model consists of six compartments including liver as metabolizing compartment, and separate compartments for fat, arterial blood, venous blood, richly perfused and slowly perfused tissues (Punt et al., 2009). With the model obtained, bioactivation and detoxification of methyleugenol at different dose levels could be investigated. The outcomes of this human model were compared with those of the PBK model for methyleugenol in male rat. The results obtained reveal that formation of 1′-hydroxymethyleugenol glucuronide, a major metabolic pathway in male rat liver, appears to represent a minor metabolic pathway in human liver whereas in human liver a significantly higher formation of 1′-oxomethyleugenol compared with male rat liver is observed. Furthermore, formation of 1′-sulfooxymethyleugenol, which readily undergoes desulfonation to a reactive carbo-cation that can form DNA or protein adducts, is predicted to be the same in the liver of both human and male rat at oral doses of 0.0034 up to 300 mg/(kg bw). Altogether it was concluded that despite a significant difference in especially the metabolic pathways of the proximate carcinogenic metabolite 1′-hydroxymethyleugenol between human and male rat, the influence of species differences on the ultimate overall bioactivation of methyleugenol to 1′-sulfooxymethyleugenol appears to be negligible. Moreover, the PBK model predicted the formation of 1′-sulfooxymethyleugenol in the liver of human and rat to be linear from doses as high as the benchmark dose (BMD10) down to as low as the virtual safe dose (VSD). This shows that kinetic data do not provide a reason to argue against linear extrapolation from the rat tumor data to the human situation. Another aim of the present PhD study was to study the effect of the basil constituent nevadensin on the bioactivation and genotoxicity of herb based methyleugenol. The results presented in chapter 4 show that nevadensin is able to inhibit DNA adduct formation in HepG2 cells exposed to the proximate carcinogen 1′-hydroxymethyleugenol in the presence of this flavonoid. This inhibition occurs at the level of sulfotransferase (SULT)-mediated bioactivation of 1′-hydroxymethyleugenol. In order to investigate possible in vivo implications the SULT inhibition by nevadensin was integrated into the male rat and human PBK models for bioactivation and detoxification of methyleugenol. The results thus obtained reveal that coadministration of methyleugenol with nevadensin may reduce the levels of bioactivation of 1′- hydroxymethyleugenol to the DNA reactive metabolite, without reducing its detoxification via glucuronidation or oxidation. This effect may be significant even at realistic low dose human exposure levels. The results obtained point at a potential reduction of the cancer risk when methyleugenol exposure occurs by oral intake within a relevant food matrix containing SULT inhibitors compared to what is observed in rodent bioassays upon exposure to pure methyleugenol dosed by gavage. Besides dose-dependent effects, species differences effects, and matrix effects on the bioactivation of methyleugenol the effect of interindividual variation on methyleugenol detoxification and bioactivation was investigated in chapter 5. To this end we predicted the level of formation of the ultimate carcinogenic metabolite 1′-sulfooxymethyleugenol in the human population by taking the variability in key bioactivation and detoxification reactions into account using Monte Carlo simulations. Insight in the variation in relevant metabolic routes was obtained by determining kinetic constants for the metabolic reactions by specific isoenzymes or by measuring the kinetic constants in incubations with a range of individual human liver fractions. The results of the study indicate that formation of 1′-sulfooxymethyleugenol is predominantly affected by i) P450 1A2 catalyzed bioactivation of methyleugenol to 1′- hydroxymethyleugenol ii) P450 2B6 catalyzed epoxidation of methyleugenol and iii) the apparent kinetic constants for detoxification of 1′-hydroxymethyleugenol via oxidation and iv) the apparent kinetic constants for bioactivation of 1′-hydroxymethyleugenol to 1′- sulfooxymethyleugenol. Based on the Monte Carlo simulation a chemical-specific adjustment factor (CSAF) for intraspecies variation could be derived which is defined as the 95th or 99th percentile divided by the 50th percentile of the predicted distribution of the formation of 1′- sulfooxymethyleugenol in the liver. The obtained CSAF value at the 95th percentile was 3.7 indicating that the default uncertainty factor of 3.16 for human variability in kinetics (WHO, 1999) may adequately protect 95% of the population. While protecting 99% of the population requires a larger uncertainty factor of 5.8. Altogether, the results shown in this thesis reveal that integrating in vitro metabolic parameters within a framework of a PBK model provides a good method to evaluate the occurrence of dose-dependent effects, species differences, and human variability in detoxification and bioactivation of a genotoxic carcinogen. Moreover, the results presented in this thesis show the possible protective effect of the basil constituent nevadensin on SULT catalysed bioactivation and DNA adduct formation of methyleugenol in vitro. Upon validation of these effects in vivo, it may turn out that rodent carcinogenicity data on methyleugenol substantially overestimate the risks posed when humans are exposed to methyleugenol within a nevadensin containing food matrix.

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