Menzel, Ralph (2009) International Worm Meeting "Gene silencing based functional analysis of C. elegans' cytochromes P450: PUFA metabolism, biotransformation and malfunction in fat storage."

Menzel, Ralph

[International Worm Meeting, 2009]

Cytochromes P450 (CYP) are heme-thiolate monooxygenases and form one of the largest superfamilies of enzyme proteins. They are found in virtually all organisms and contribute to diverse vital processes, such as carbon source assimilation, biosynthesis of hormones and structural components; other forms can degrade xenobiotics and interact with carcinogens. Although 75 full length CYP genes have been identified in the genome of C. elegans, the individual biological function of the vast majority is mostly unknown yet. Our investigations started with the validation that C. elegans possesses spectrally active CYP proteins, as demonstrated by CO-difference spectrum from microsomal fractions. To complete the monooxygenase system CYP enzymes need a carrier protein used for conveying reducing equivalents coming typically from NADPH. A genomic screen suggested emb-8 as the only C. elegans gene in question to encode a protein that resembles mammalian NADPH-CYP reductases (CPR). Indeed, the CPR component was easily detectable by measuring the microsomal NADPH-cytochrome c reductase activity and it abolished upon prevention of functional emb-8 expression. Moreover, CPR expression was found essential for all CYP activities measured in this study. To identify CYP components of specific monooxygenase systems, a systematic gene silencing by RNAi as well as by using cyp-mutant strains was performed. Several pre-selected enzymatic reactions, proved as CYP dependent in C. elegans, were used as screening systems. By doing this we identified CYP-29A3 and CYP-33E2 as the major isoforms contributing to the metabolism of eicosapentaenoic acid (EPA) and arachidonic acid (AA) to specific sets of regioisomeric epoxy- and w-/(w-1)-hydroxy-derivatives. Heterologous expression of both CYP-forms in SF9 insect cells is in progress. Moreover, CYP34A6 and CYP14A isoforms were found to be required for hydroxylation of an ortho-substituted, non-coplanar tetrachlorbiphenyl (PCB52) to C3-, C4- and/or C6-hydroxy-PCB52. This finding shows for the first time the so far only predicted involvement of C. elegans'' CYP forms in biotransformation. Further CYP-forms of subfamily 35A, which were under suspicion to be part of biotransformation, too, could be assigned to the fat storage pathway. Both individual gene silencing of cyp-35A2, 3, 4, and 5 resulted in a dramatic decrease of intestinal fat content of well fed young adults, measured by Nile red vital staining. Our investigation may facilitate future studies on C. elegans'' CYP enzymes, which are much less investigated than its status as model organism lets expect.

Menzel, Ralph et al. (2017) International Worm Meeting "Nematodes as important source for omega-3 long-chain fatty acids in the soil food web and the impact in nutrition for higher trophic levels."

Menzel, Ralph, Ruess, Liliane, Chrzanowski, Henrik, Geweiler, Diana

[International Worm Meeting, 2017]

PUFA (Polyunsaturated fatty acids) content of food resources, in particular omega-3 long-chain (LC) PUFAs with three or more double bonds, are considered essential for growth, reproduction, and neural development of higher animals. Surprisingly, and in contrast to aquatic ecosystems, omega-3 LC-PUFAs seem not widely available in terrestrial food webs. Far-reaching ideas indeed proclaim aquatic ecosystems as the principal source of these LC-PUFAs in the whole biosphere, including inhabitants of terrestrial ecosystems. Interestingly, de novo synthesis of omega-3 LC-PUFAs, which requires the presence of ?12 (fat-2) and omega-3 (fat-1) desaturases absent in vertebrates, has been observed in Caenorhabditis elegans and other nematodes. This calls for studies to assign how this remarkable metabolic capability enhances or even determines the availability of omega-3 LC-PUFAs in the soil food web. Our project addresses the following questions: How common is the ability for de novo synthesis of omega-3 LC-PUFAs across different functional groups of nematodes, whether or not is this capability related to their diet, and how efficiently is the transfer of these PUFAs to higher trophic level, such as microarthropods? Using a genetic approach by doing PCR with degenerated primers we identified so far two novel fat-2 like genes in the free-living soil nematodes Acrobeloides buetschlii and Oscheius dolichuroides. Sequence alignments with already known nematode fat-2 genes supports the idea that already an ancestral nematode did contain such gene rather than a horizontal gene transfer to only a subgroup of nematodes. By using GC/MS analyses we observed the trophic transfer of nematode LC-PUFAs to nematophagous Collembola, as Folsomia candida and Protaphorura fimata. In fact, Collembola reflected the PUFA-richness or -poorness of their nematode prey, achieved by feeding C. elegans mutants with defects in the PUFA biosynthesis, in their neutral lipid fraction. In contrast, Collembolas PUFA-content of the phospholipid fraction remained unchanged, suggesting that these invertebrates also possess the metabolic capability to de novo synthesize PUFAs, including omega-3 LC-PUFAs.

Ralph Menzel et al. (2006) European Worm Meeting "PCB52 Induced Gene Expression in the Nematode Caenorhabditis elegans"

Jana Kulas, Stephen Sturzenbaum, Shuang Li, Hui Ling Yeo, Ralph Menzel

[European Worm Meeting, 2006]

Ralph Menzel1, Jana Kulas1, Hui Ling Yeo1, Shuang Li1, and Stephen Strzenbaum2. Polychlorinated biphenyls (PCBs) are ubiquitous organic chemicals which can affect numerous biological responses. Because PCBs are toxic to man, recalcitrance to degradation and persist in the environment, they are listed in the inventory of priority pollutants compiled by numerous Environmental Protection Agencies.. Here we present an investigation of PCB52, an ortho-substituted, non-coplanar 2,2,5,5-tetrachlorbiphenyl. PCB52 affected the reproductive capacity of C. elegans in a strict concentration dependent manner, thereby highlighting its toxicological significance. Based on the calculated EC20 (5 mg/L), whole genomic DNA microarray experiments were performed to identify differentially expressed genes. Using a two-fold increase/decrease cut-off, 1158 genes were up-regulated and 560 down-regulated by PCB52. Up-regulated genes constituted family members of small heat shock proteins, chemoreceptors, nuclear hormone receptors, lipases as well as cytochromes P450 (CYP) and short-chain dehydrogenases/reductases (SDR), which are known to be involved in the biotransformation of persistent organic pollutants. Significant enriched classes of down-regulated genes are involved in organelle biogenesis, cell integrity and further general physiological processes, such as macromolecule metabolism and nucleotide binding.. The induction of five highly differentially regulated gene classes was confirmed by semi-quantitative RT-PCR experiments. Using a multiple RNAi approach the consequences of gene knockdown of single genes as well as entire gene subfamilies was determined. The efficiency of RNAi was proven by RT-PCR. Whilst GST and SDR gene expression knockdown intensify the toxicity of PCB52 (reflected in a reduced reproductive output) the knockdown of several CYP forms increased the reproductive capacity. This may indicate that the lack of CYP gene expression prevents the generation of toxic metabolites.

Ralph Menzel et al. (2002) European Worm Meeting "Xenobiotically induced cytochrome P450 gene expression in Caenorhabditis elegans."

Matthias Roedel, Rudolf Achazi, Ralph Menzel

[European Worm Meeting, 2002]

Cytochromes P450 (CYP) are a super family of heme containing NADPH dependent monooxygenases. These enzymes play important roles in the biotransformation of many drugs, carcinogens, steroid hormones and environmental chemicals. CYP gene expression can be induced by the presence of these compounds in the environment. At high concentrations many of the inducers produce a wide range of biological effects on animals and other organisms, e.g. mortality or reproductive failure.

Ralph Menzel et al. (2002) European Worm Meeting "Xenobiotically induced cytochrome P450 gene expression in Caenorhabditis elegans."

Rudolf Achazi, Ralph Menzel, Matthias Roedel

[European Worm Meeting, 2002]

Cytochromes P450 (CYP) are a super family of heme containing NADPH dependent monooxygenases. These enzymes play important roles in the biotransformation of many drugs, carcinogens, steroid hormones and environmental chemicals. CYP gene expression can be induced by the presence of these compounds in the environment. At high concentrations many of the inducers produce a wide range of biological effects on animals and other organisms, e.g. mortality or reproductive failure.

Ralph Menzel et al. (2001) International C. elegans Meeting "A systematical gene expression screen of the Caenorhabditis elegans cytochrome P450 genes."

Rudolf Achazi, Ralph Menzel, Thierry Bogaert

[International C. elegans Meeting, 2001]

Cytochromes P450 (CYP) are a super family of heme containing NADPH dependent monooxygenases which catalyze the oxidative metabolism of many exogenous and endogenous compounds. These proteins have been particularly implicated in the biotransformation of many drugs and other xenobiotics. Moreover, P450 gene expression can be induced by the presence of these compounds in the medium. The soil nematode Caenorhabditis elegans is probably the simplest animal having the status of a laboratory model. Its genome contains the remarkable number of 80 cytochrome P450 genes. In order to study CYP gene expression worms were exposed to 18 different cytochrome P450 inducers. Using subfamily-specific primers we could amplify a pooled set of exon-rich CYP fragments to create a P450 subfamily specific gene filter. In that way we were able to check systematically the influence of the different inducers to CYP expression at the same time. Interestingly, the well known CYP1A inducers b -naphtoflavone, PCB52 and Lansoprazol were the most active ones, especially in case of family CYP35. Taking advantage of the C. elegans whole genome DNA microarray created in Stuart Kim's lab (Stanford University, USA) we could confirm and extend our results significantly. Differentially expressed CYP genes were further analyzed by a semi-quantitative RT-PCR. The strongest expression were observed for the genes CYP35A1-4 and 35C1. In addition, a transgenic C. elegans line expressing GFP under control of the CYP35A2 promoter showed a strong induction of the fusion by b -naphthoflavone in the intestine. Altogether, xenobiotic inducible gene expression of C. elegans could be a useful tool to develop a biomonitoring screening system.

Ralph Menzel et al. (2005) International Worm Meeting "Chemoattraction and whole genome transcriptional responses in the nematode Caenorhabditis elegans exposed to humic material"

Jana Kulas, Stephen Strzenbaum, Ralph Menzel, Anne Brenwaldt, Christian EW Steinberg

[International Worm Meeting, 2005]

Humic substances (HS) are complex organic molecules constituting the most abundant source of dissolved organic matter (DOM) in freshwater and soil ecosystems. To investigate whether HS display a potential to exert chemical cues, the nematode model organism Caenorhabditis elegans was chosen for clearly defined reasons. Firstly, C. elegans satisfies all typical criteria to function as an ecotoxicological test organism, secondly, exquisite molecular genetic tools are available, and, finally, the entire genome of C. elegans has been decoded. Here we present three approaches to investigate the effects of a natural DOM isolate from the Lake Fuchskuhle and a synthetic HS (HS1500) on the nematode. In detail they encompass: (i) a behavioral chemoattractant bioassay, (ii) a reproduction assay, and (iii) the analysis of whole genome transcriptional expression patterns. A simple attractant test was devised. Petri dishes were prepared containing alternating piles of bacteria around the peripheral region of the plate, lacking HS or supplemented with HS, respectively. Nematodes were placed in the centre of the petri dish and subsequent movements scored. As a result, the adults, but not the juveniles, preferentially moved to the food bacteria with HS. This phenomenological experiment provided evidence that HS exert distinct chemical cues. The reproduction test utilized age-synchronized L1 larvae. Brood size was calculated from the total number of second-generation juveniles exposed to control conditions (HS devoid) or either of four different concentrations of each HS. Only the highest concentration (100 mg/L DOC) resulted in a decrease in brood size, highlighting an overall intrinsic tolerance to HS. Finally, an oligonucleotide-based whole genome microarray was performed with Cy3/Cy5 labeled cDNAs, derived from RNA samples isolated from HS treated and non-treated worms. The cluster analysis (cut-off set to two-fold increase/decrease, p<0.05, n=5) revealed that 84 genes were significantly induced and 235 genes were significantly repressed. Notable transcriptional changes were identified in chemosensors, olfactory receptors, as well as enzymes of the biotransformation system (CYP, UGT and GST), thereby confirming that HS1500 as well as the natural HS Fuchskuhle are recognized as environmental signaling chemicals.

Ralph Menzel et al. (2003) International Worm Meeting "Xenobiotically induced cytochrome P450 genes in Caenorhabditis elegans: GFP-fusions and embryonic lethality by RNAi."

Ralph Menzel, Ruth Boose, Sarah Forster

[International Worm Meeting, 2003]

Cytochromes P450 (CYP) are a super family of heme containing and NADPH dependent monooxygenases which catalyze the oxidative metabolism of many exogenous and endogenous compounds. While the investigation of CYP induction pathways in mammals has made considerable progress, very little is known about P450s from invertebrates, such as the nematode Caenorhabditis elegans. C. elegans possesses, however, a remarkably high number of 80 different cytochrome P450 genes. The functions of the encoded proteins are almost unknown. In a previous study it could be demonstrated that the five different CYP forms 35A1-A5 are strongly inducible by PAHs, PCBs and different drugs [1]. In order to study this xenobiotically induced gene expression different transgenic C. elegans lines expressing GFP under control of the CYP35A1-5 promoter regions were constructed. With this tool it was possible to follow the GFP production in vivo depending on the applied inducer. Induction with -naphthoflavone resulted in all cases in an expression of the green fluorescent protein exclusively in the worm intestine, identifying this compartment as the likely tissue of P450 35A localization. For the CYP35A2 and A5 forms promoter-deletion experiments were performed revealing a 400 bp and a 250 bp fragment, respectively, to be sufficient to induce GFP production. While the 35A2 promoter caused GFP production in all developmental stages, the 35A5 based gene expression was mainly detectable in eggs and in L1/L2 larvae. In particular in case of the 35A2 promoter region a striking correspondence to mammalian xenobiotic response elements (XRE) has been found. To obtain more information about the biological functions of the five different CYP35A forms dsRNA producing E. coli strains for RNAi by feeding experiments were produced. Surprisingly, embryonic lethality was observed as the loss of function phenotype for all CYP35A forms with exception of 35A2. This might indicate that at least one or more CYP35A forms play an essential role in the embryogenesis of C. elegans. Using semi-quantitative RT-PCRs the cross-reactivity level of the individual RNAi constructs was determined. Future efforts will be focused to characterize the blocked embryonic development more in detail.[1] Menzel R., Bogaert T. and Achazi A. (2001) A systematical gene expression screen of the Caenorhabditis elegans cytochrome P450 genes revealed CYP35 as strong xenobiotic inducible. Arch. Biochem. Biophys. 395, 158-168.

Ralph Menzel et al. (2008) European Worm Meeting "Humic material induces behavioral and transcriptional responses in the nematode Caenorhabditis elegans and extends its lifespan"

Kerstin Pietsch, Heike Staudacher, Stefanie Rienau, Ralph Menzel, Christian E W Steinberg, Nadine Saul, Waltraud Wild

[European Worm Meeting, 2008]

Humic substances (HS) are complex organic molecules constituting the most. abundant source of natural organic matter (NOM) in freshwater and soil. ecosystems. They are taken up by organisms and interact on various. molecular and biochemical levels. Recent advances have identified that they. interfere with biological systems, via the induction of biotransformation. enzymes and the production of internal oxidative stress.. The nematode model organism Caenorhabditis elegans was chosen to. investigate whether natural and synthetic HS induce (i) a behavioral. attraction, (ii) the reproduction, (iii) an increased lifespan, and (iv) a. response in transcriptional expression. The phenomenological attractant. experiments provided evidence that HS attract the worm and exert distinct. chemical cues. In the reproduction assay, only the highest concentration. (30 mg/L dissolved organic carbon) resulted in a slight decrease in brood. size, highlighting an overall intrinsic tolerance towards humic material.. Lifespan experiments showed that at least one HS increases the lifespan of. Caenorhabditis elegans. Finally, DNA array experiments were performed from. control and HS treated worms. Significant transcriptional changes. (exceeding a two-fold in- or decrease) were identified and in chemosensors,. olfactory receptors, as well as enzymes of the biotransformation system. (glutathione S-transferases, short-chain dehydrogenases) and. vitellogenines. The results were proved by quantitative real-time RT-PCR,. thereby confirming that humic material is recognized as an environmental. signaling chemical.

Zhou, Yiwen et al. (2017) International Worm Meeting "Stereospecific bioactivity of the eicosanoid 17,18-epoxyeicosatetraenoic acid on the pharyngeal pumping of Caenorhabditis elegans."

Zhou, Yiwen, Menzel, Ralph

[International Worm Meeting, 2017]

17,18-epoxyeicosatetraenic acid (17,18-EEQ), the most abundant cytochrome P450 (CYP)-eicosanoid in C. elegans, is a potential signaling molecule in the regulation of pharyngeal pumping activity. Here we tested the hypothesis that stereo-discrimination of this chiral molecule may hint the existence of a receptor in the pharyngeal muscle and/or neuronal cells. We analyzed the effect of short-term incubation in both well fed and starved condition and found that the stereoisomer 17(R),18(S)-EEQ, clearly rescued impairments of pharyngeal pumping in D-6 fatty acid desaturase mutant strain fat-3(wa22). After treatment with the 17(S),18(R)-EEQ stereoisomer, however, we found no effect on pumping activity. In addition, we will analyze the total endogenous eicosanoid stereoisomer content of C. elegans by chiral LC/MS-MS to see whether or not only specific stereoisomers are endogenous resources. 17(R),18(S)-EEQ was more active in ceeh-1(ok3153) mutant background in comparison to other stains. We suspect that ceeh-1(ok3153), failing to produce a functional active soluble hydrolase (sEH), is unable to efficiently catalyze the hydrolysis of 17,18-EEQ to the corresponding diol (17,18-DHEQ), which we found correlated with a loss of function of this signaling molecule. 17,18-DHEQ treatment was inactive in a C. elegans' pumping assay.