Stage-specific exposure of Caenorhabditis elegans to cadmium identifies unique transcriptomic response cascades and an uncharacterized cadmium responsive transcript.

Age/stage sensitivity is considered a significant factor in toxicity assessments. Previous studies investigated cadmium (Cd) toxicosis in Caenorhabditis elegans, and a plethora of metal-responsive genes/proteins have been identified and characterized in fine detail; however, most of these studies neglected age sensitivity and stage-specific response to toxicants at the molecular level. This present study compared the transcriptome response between C. elegans L3 vs L4 larvae exposed to 20 µM Cd to explore the transcriptional hallmarks of stage sensitivity. The results showed that the transcriptome of the L3 stage, despite being exposed to Cd for a shorter period, was more affected than the L4 stage, as demonstrated by differences in transcriptional changes and magnitude of induction. Additionally, T08G5.1, a hitherto uncharacterized gene located upstream of metallothionein (mtl-2), was transcriptionally hyperresponsive to Cd exposure. Deletion of one or both metallothioneins (mtl-1 and/or mtl-2) increased T08G5.1 expression, suggesting that its expression is linked to the loss of metallothionein. The generation of an extrachromosomal transgene (PT08G5.1:: GFP) revealed that T08G5.1 is constitutively expressed in the head neurons and induced in gut cells upon Cd exposure, not unlike mtl-1 and mtl-2. The low abundance of cysteine residues in T08G5.1 suggests, however, that it may not be involved directly in Cd sequestration to limit its toxicity like metallothionein, but might be associated with a parallel pathway, possibly an oxidative stress response.

Juggling cadmium detoxification and zinc homeostasis: A division of labour between the two C. elegans metallothioneins.

The chemical properties of toxic cadmium and essential zinc are very similar, and organisms require intricate mechanisms that drive selective handling of metals. Previously regarded as unspecific "metal sponges", metallothioneins (MTLs) are emerging as metal selectivity filters. By utilizing C. elegans mtl-1 and mtl-2 knockout strains, metal accumulation in single worms, single copy fluorescent-tagged transgenes, isoform specific qPCR and lifespan studies it was possible to demonstrate that the handling of cadmium and zinc by the two C. elegans metallothioneins differs fundamentally: the MTL-2 protein can handle both zinc and cadmium, but when it becomes unavailable, either via a knockout or by elevated cadmium exposure, MTL-1 takes over zinc handling, leaving MTL-2 to sequester cadmium. This division of labour is reflected in the folding behaviour of the proteins: MTL-1 folded well in presence of zinc but not cadmium, the reverse was the case for MTL-2. These differences are in part mediated by a zinc-specific mononuclear His3Cys site in the C-terminal insertion of MTL-1; its removal affected the entire C-terminal domain and may shift its metal selectivity towards zinc. Overall, we uncover how metallothionein isoform-specific responses and protein properties allow C. elegans to differentiate between toxic cadmium and essential zinc.

Environmental carcinogen benzo[a]pyrene alters neutral lipid storage via a cyp-35A2 mediated pathway in Caenorhabditis elegans.

Polycyclic aromatic hydrocarbons (PAHs), in particular benzo [a]pyrene (BaP), have been identified as carcinogenic components of tobacco smoke. In mammals, the toxicological response to BaP-diol-epoxide is driven by cytochrome P450 (CYP1A1), a pathway which is absent in Caenorhabditis elegans. In contrast, in worms prominently the CYP-35 enzyme family seems to be induced after BaP exposure. In C. elegans, BaP exposure reduces the accumulation of lysosomal neutral lipids in a dose dependent manner and the deletion of cyp-35A2 results in a significant elevation of neutral lipid metabolism. A cyp-35A2:mCherry;unc-47:GFP dual-labelled reporter strain facilitated the identification of three potential upstream regulators that drive BaP metabolism in worms, namely elt-2, nhr-49 and fos-1. This newly described reporter line is a powerful resource for future large-scale RNAi regarding toxicology and lipid metabolism screens.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Benzo[a]pyrene and Caenorhabditis elegans: defining the genotoxic potential in an organism lacking the classical CYP1A1 pathway.

Benzo[a]pyrene (BaP) is bioactivated in most organisms by the cytochrome P450 (CYP) enzymes, mainly CYP1A1, ultimately resulting in the reactive metabolite BaP-7,8-dihydrodiol-9,10-epoxide (BPDE) capable of covalently binding to DNA and forming adducts. This step has been defined as the key process in cancer initiation in humans. However, limited knowledge is available about the consequences of BaP exposure in organisms lacking this classical CYP1A1 pathway, one example is the model nematode Caenorhabditis elegans. The aim of this study was to define the genotoxic potential of BaP in C. elegans and to advance our understanding of xenobiotic processing in the absence of the CYP1A1 pathway. Exposure to high concentrations of BaP (0-40 µM) significantly affected life cycle endpoints of C. elegans, which were manifested by a reduced reproductive output and shortened life span. An optimised comet assay revealed that DNA damage increased in a dose-dependent manner; however, no bulky DNA adducts (dG-N2-BPDE) were observed by 32P-postlabelling. Global transcriptomic analysis by RNA-Seq identified responsive transcript families, most prominently members of the cyp-35 and UDP-glucuronosyltransferases (UGTs) enzyme families, both of which are linked to xenobiotic metabolism. Strains harbouring mutations in the cyp-35A2 and cyp-35A3 genes were notably less prone to BaP-mediated toxicity, and BaP led to longevity in cyp-35A5 mutants. In summary, BaP induces transcriptional, genotoxic and phenotypic responses in C. elegans, despite the absence of the classical CYP1A1 bioactivation pathway. This provides first evidence that parallel pathways are implicated in BaP metabolism in C. elegans and this seems to be mediated via the cyp-35 pathway.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Proanthocyanidins of Natural Origin: Molecular Mechanisms and Implications for Lipid Disorder and Aging-Associated Diseases.

Proanthocyanidins are phytonutrients formed by oligomerization or polymerization of subunits catechin, epicatechin, and their gallic acid esters. Proanthocyanidins are a component of many plants and thus form an integral part of the human diet. Oligomeric proanthocyanidins are currently marketed as medicinal products that target vascular disorders and chronic pathological conditions, many of which are age-associated. Proanthocyanidins are also characterized by their effects on energy homeostasis. Not dissimilar to their chemically synthesized counterparts, naturally extracted proanthocyanidins act via inhibition of lipases, stimulation of energy expenditure, or suppression of appetite. Here we review the current knowledge-base and highlight challenges and future impacts regarding involvement of proanthocyanidins in global lipid metabolism, with a focus on the molecular mechanisms and pathological conditions that progress with aging.

Molecular genetic and biochemical characterization of a putative family of zinc metalloproteins in Caenorhabditis elegans.

Four highly similar genes (W08E12.2, W08E12.3, W08E12.4 and W08E12.5) which are consecutively aligned on chromosome IV of the C. elegans genome are predicted to code for small (120-141aa) yet cysteine rich (18-19 cysteines) proteins. Cloning and sequencing of the genomic regions of the isoforms confirmed the presence and order of all genes. The generation of transgenic worms strains with an integrated single copy or extrachromosomal multi-copy PW08E12.3;W08E12.4::GFP uncovered that W08E12.3 and W08E12.4 are constitutively expressed in the pharynx and significantly induced in worms exposed to 100 µM Zn. Knockdown by RNAi did not have a marked consequence on reproductive performance nor was a Zn-dependent effect on nematode growth observed. However, RNAi of these genes led to an accumulation of Zn in the intestinal cells. W08E12.3 was recombinantly expressed in E. coli and the purified protein was shown to be able to bind up to 6.5 Zn molecules at neutral pH. Zn-binding was acid-labile and the apo protein was observed at pH < 4.3. This characterization suggests W08E12.2, W08E12.3, W08E12.4 and W08E12.5 belong to a family of putative Metalloproteins which, akin to metallothioneins, may play an important role in Zn-sensing, homeostasis and/or detoxification.

Metallothionein from Wild Populations of the African Catfish Clarias gariepinus: From Sequence, Protein Expression and Metal Binding Properties to Transcriptional Biomarker of Metal Pollution.

Anthropogenic pollution with heavy metals is an on-going concern throughout the world, and methods to monitor release and impact of heavy metals are of high importance. With a view to probe its suitability as molecular biomarker of metal pollution, this study has determined a coding sequence for metallothionein of the African sharptooth catfish Clarias gariepinus. The gene product was recombinantly expressed in Escherichia coli in presence of Zn(II), Cd(II), or Cu, and characterised by Electrospray Ionisation Mass Spectrometry and elemental analysis. C. gariepinus MT displays typical features of fish MTs, including 20 conserved cysteines, and seven bound divalent cations (Zn(II) or Cd(II)) when saturated. Livers from wild C. gariepinus fish collected in all three seasons from four different sites on the Kafue River of Zambia were analysed for their metal contents and for MT expression levels by quantitative PCR. Significant correlations were found between Zn and Cu levels and MT expression in livers, with MT expression clearly highest at the most polluted site, Chililabombwe, which is situated in the Copperbelt region. Based on our findings, hepatic expression of MT from C. gariepinus may be further developed as a major molecular biomarker of heavy metal pollution resulting from mining activities in this region.

Toxicogenomics of iron oxide nanoparticles in the nematode C. elegans.

We present a mechanistic study of the effect of iron oxide nanoparticles (SPIONs) in Caenorhabditis elegans combining a genome-wide analysis with the investigation of specific molecular markers frequently linked to nanotoxicity. The effects of two different coatings were explored: citrate, an anionic stabilizer, and bovine serum albumin, as a pre-formed protein corona. The transcriptomic study identified differentially expressed genes following an exposure to SPIONs. The expression of genes involved in oxidative stress, metal detoxification response, endocytosis, intestinal integrity and iron homeostasis was quantitatively evaluated. The role of oxidative stress was confirmed by gene expression analysis and by synchrotron Fourier Transform infrared microscopy based on the higher tissue oxidation of NP-treated animals. The observed transcriptional modulation of key signaling pathways such as MAPK and Wnt suggests that SPIONs might be endocytosed by clathrin-mediated processes, a putative mechanism of nanotoxicity which deserves further mechanistic investigations.

Metallothionein 2 and Heat Shock Protein 72 Protect Allolobophora chlorotica from Cadmium But Not Nickel or Copper Exposure: Body Malformation and Coelomocyte Functioning.

Earthworms serve as good indicators of heavy metal contamination due to their innate sensitivity towards soil pollution. However, to date, not many studies have focused on endogeic earthworms, such as the omnipresent Allolobophora chlorotica. The current study was designed to verify whether this earthworm could serve as a novel distinctively susceptible species for environmental contamination studies. We show that the dermal exposure to Cu, Ni, and Cd affected the mortality and morphology of A. chlorotica, and the number and functioning of coelomocytes. These features particularly were pronounced in animals treated with Ni and Cu and interestingly to a lesser extend with Cd. In contrast, Cd induced a strong expression of metallothioneins (MT-2) and heat shock proteins (HSP72). The presence of MT-2 was detected not only in coelomocytes but also in the intestine, blood vessels, and epidermis. In conclusion, Allolobophora chlorotica coelomocytes are adopted to respond differentially to various heavy metals, generating powerful response towards potentially most dangerous exogenous non-essential elements.

Deletion of Phytochelatin Synthase Modulates the Metal Accumulation Pattern of Cadmium Exposed C. elegans.

Environmental metal pollution is a growing health risk to flora and fauna. It is therefore important to fully elucidate metal detoxification pathways. Phytochelatin synthase (PCS), an enzyme involved in the biosynthesis of phytochelatins (PCs), plays an important role in cadmium detoxification. The PCS and PCs are however not restricted to plants, but are also present in some lower metazoans. The model nematode Caenorhabditis elegans, for example, contains a fully functional phytochelatin synthase and phytochelatin pathway. By means of a transgenic nematode strain expressing a pcs-1 promoter-tagged GFP (pcs-1::GFP) and a pcs-1 specific qPCR assay, further evidence is presented that the expression of the C. elegans phytochelatin synthase gene (pcs-1) is transcriptionally non-responsive to a chronic (48 h) insult of high levels of zinc (500 µM) or acute (3 h) exposures to high levels of cadmium (300 µM). However, the accumulation of cadmium, but not zinc, is dependent on the pcs-1 status of the nematode. Synchrotron based X-ray fluorescence imaging uncovered that the cadmium body burden increased significantly in the pcs-1(tm1748) knockout allele. Taken together, this suggests that whilst the transcription of pcs-1 may not be mediated by an exposure zinc or cadmium, it is nevertheless an integral part of the cadmium detoxification pathway in C. elegans.

Earthworm Lumbricus rubellus MT-2: Metal Binding and Protein Folding of a True Cadmium-MT.

Earthworms express, as most animals, metallothioneins (MTs)-small, cysteine-rich proteins that bind d(10) metal ions (Zn(II), Cd(II), or Cu(I)) in clusters. Three MT homologues are known for Lumbricus rubellus, the common red earthworm, one of which, wMT-2, is strongly induced by exposure of worms to cadmium. This study concerns composition, metal binding affinity and metal-dependent protein folding of wMT-2 expressed recombinantly and purified in the presence of Cd(II) and Zn(II). Crucially, whilst a single Cd7wMT-2 species was isolated from wMT-2-expressing E. coli cultures supplemented with Cd(II), expressions in the presence of Zn(II) yielded mixtures. The average affinities of wMT-2 determined for either Cd(II) or Zn(II) are both within normal ranges for MTs; hence, differential behaviour cannot be explained on the basis of overall affinity. Therefore, the protein folding properties of Cd- and Zn-wMT-2 were compared by ¹H NMR spectroscopy. This comparison revealed that the protein fold is better defined in the presence of cadmium than in the presence of zinc. These differences in folding and dynamics may be at the root of the differential behaviour of the cadmium- and zinc-bound protein in vitro, and may ultimately also help in distinguishing zinc and cadmium in the earthworm in vivo.

H2S: A New Approach to Lifespan Enhancement and Healthy Ageing?

Ageing, a progressive structural and functional decline, is considered to be a major risk factor for virtually all ageing-associated pathologies and disabilities, including Alzheimer's disease, Parkinson's disease, stroke, diabetes, atherosclerosis and certain cancers. Biogerontology research has now been largely directed towards finding novel drug targets to decelerate the ageing process and attain healthy ageing in order to delay the onset of all ageing-related diseases. H2S has been reported to exert vasodilatory, antioxidant, antiapoptotic and anti-inflammatory actions and has been shown to act as a signalling molecule, neuromodulator and cytoprotectant. Intriguingly, H2S has been reported to regulate cell cycle and survival in healthy cells which suggests that it may regulate cell fate and hence the ageing process. This chapter sets out to provide an overview of the current knowledge regarding the involvement of H2S in ageing, with a specific focus on the invertebrate model nematode C. elegans.

The double mutation of cytochrome P450's and fatty acid desaturases affect lipid regulation and longevity in C. elegans.

An imbalance between energy uptake and energy expenditure can lead to obesity and increase the risk of coronary heart disease, high blood pressure, stroke, type II diabetes and some cancers. Given that key elements of the energy pathway are evolutionary conserved, invertebrate research is an attractive alternative that overcomes the many legislative, financial and experimental hurdles typical of research with higher metazoan animals. Recent studies have suggested that some members of the cytochrome P450 superfamily are involved in lipid metabolism in addition to the traditional xenobiotic activity. To investigate this notion in more detail, the present study aimed to pinpoint phenotypic, genetic and genomic-level responses of Caenorhabditis elegans using selected deletion mutants including fat-5 (a member of the Δ9 desaturases) and cyp-35A2 (a member of the cytochrome P450 family). The creation of a fat-5(tm420);cyp-35A2(gk317) mutant uncovered that the deletion of both genes resulted in a strain which is marked by an extended lifespan. Furthermore, it diminished the overall level of Nile Red positive compartments, which is indicative of a change in lipid metabolism. Comprehensive transcriptomics revealed that several genes involved in aging and lipid transport/homeostasis were modulated following the double deletion of fat-5 and cyp-35A2. Taken together, the results suggest the presence of a putative correlation between longevity and lipid regulation and given that both genes have human homologs, this finding may offer a new lead to investigate in higher organisms.

Hydrogen sulfide is an endogenous regulator of aging in Caenorhabditis elegans.

AIMS: To investigate the role of endogenous hydrogen sulfide (H2S) in the control of aging and healthspan of Caenorhabditis elegans. RESULTS: We show that the model organism, C. elegans, synthesizes H2S. Three H2S-synthesizing enzymes are present in C. elegans, namely cystathionine γ lyase (CSE), cystathionine ß synthetase, and 3-mercaptopyruvate transferase (MPST or 3-MST). Genetic deficiency of mpst-1 (3-MST orthologue 1), but not cth-2 (CSE orthologue), reduced the lifespan of C. elegans. This effect was reversed by a pharmacological H2S donor (GYY4137). GYY4137 also reduced detrimental age-dependent changes in a range of physiological indices, including pharyngeal contraction and defecation. Treatment of C. elegans with GYY4137 increased the expression of several age-related, stress response, and antioxidant genes, whereas MitoSOX Red fluorescence, indicative of reactive oxygen species generation, was increased in mpst-1 knockouts and decreased by GYY4137 treatment. GYY4137 additionally increased the lifespan in short-lived mev-1 mutants with elevated oxidative stress and protected wild-type C. elegans against paraquat poisoning. The lifespan-prolonging and health-promoting effects of H2S in C. elegans are likely due to the antioxidant action of this highly cell-permeable gas. INNOVATION: The possibility that novel pharmacological agents based on the principle of H2S donation may be able to retard the onset of age-related disease by slowing the aging process warrants further study. CONCLUSION: Our results show that H2S is an endogenous regulator of oxidative damage, metabolism, and aging in C. elegans and provide new insight into the mechanisms, which control aging in this model organism.

Therapeutic elastic tape reduces morbidity after wisdom teeth removal--a clinical trial.

OBJECTIVES: Although the extraction of an impacted third molar (3M) is a routine procedure, postoperative morbidities typically include swelling, pain, and trismus. The aim of the present study was to investigate whether the application of kinesiologic tape can improve the postoperative morbidities associated with 3M surgery, thereby improving the postoperative well-being of patients. MATERIALS AND METHODS: Forty patients assigned for prospective 3M removal were randomized into two treatment groups (with/without kinesiologic tape). Facial swelling was quantified using a five-line measurement at six specific time points. Pain scores were assessed using a visual analog scale, and mouth opening range was assessed by means of standard calipers. In addition, all patients were asked to evaluate overall satisfaction and swelling (both groups) and the effect of the tape on movement and comfort (taped group only). RESULTS: The postoperational application of kinesiologic tape reduced significantly all investigated parameters: swelling, pain, and trismus. Furthermore, patients with kinesiologic tape reported a significantly lower morbidity rate. CONCLUSION: The application of kinesiologic tape following a 3M surgery is a simple and economical, yet medically relevant approach. CLINICAL RELEVANCE: Kinesiologic tape offers patients a less traumatic postoperational experience and therefore holds promise to enhance the quality of life of a large cohort of the population.

Transcript expression patterns illuminate the mechanistic background of hormesis in caenorhabditis elegans maupas.

The animal model Caenorhabditis elegans was employed to study polyphenol- and humic substances-induced hormetic changes in lifespan. A detailed insight into the underlying mechanism of hormesis was uncovered by applying whole genome DNA microarray experimentation over a range of quercetin (Q), tannic acid (TA), and humic substances (HuminFeed(®), HF) concentrations. The transcriptional response to all exposures followed a non-linear mode which highlighted differential signaling and metabolic pathways. While low Q concentrations regulated processes improving the health of the nematodes, higher concentrations extended lifespan and modulated substantially the global transcriptional response. Over-represented transcripts were notably part of the biotransformation process: enhanced catabolism of toxic intermediates possibly contributes to the lifespan extension. The regulation of transcription, Dauer entry, and nucleosome suggests the presence of distinct exposure dependent differences in transcription and signaling pathways. TA- and HF-mediated transcript expression patterns were overall similar to each other, but changed across the concentration range indicating that their transcriptional dynamics are complex and cannot be attributed to a simple adaptive response. In contrast, Q-mediated hormesis was well aligned to fit the definition of an adaptive response. Simple molecules are more likely to induce an adaptive response than more complex molecules.

C. elegans aging is modulated by hydrogen sulfide and the sulfhydrylase/cysteine synthase cysl-2.

Exogenous hydrogen sulfide (H2S) administration and endogenous H2S metabolism were explored in the nematode C. elegans. Chronic treatment with a slow-releasing H2S donor, GYY4137, extended median survival by 17-23% and increased tolerance towards oxidative and endoplasmic reticulum (ER) stress. Also, cysl-2, a sulfhydrylase/cysteine synthase in C. elegans, was transcriptionally upregulated by GYY4137 treatment and the deletion of cysl-2 resulted in a significant reduction in lifespan which was partially recovered by the supplementation of GYY4137. Likewise, a mammalian cell culture system, GYY4137 was able to protect bovine aortic endothelial cells (BAECs) from oxidative stress and (H2O2)-induced cell death. Taken together, this provides further support that H2S exerts a protective function which is consistent with the longevity dividend theory. Overall, this study underlines the therapeutic potential of a slow-releasing H2S donor as regulators of the aging and cellular stress pathways.

Hormesis and longevity with tannins: free of charge or cost-intensive?

Hormetic lifespan extension is, for obvious reasons, beneficial to an individual. But is this effect really cost-neutral? To answer this question, four tannic polyphenols were tested on the nematode Caenorhabditis elegans. All were able to extend the lifespan, but only some in a hormetic fashion. Additional life trait variables including stress resistance, reproductive behavior, growth, and physical fitness were observed during the exposure to the most life extending concentrations. These traits represent the quality of life and the population fitness, being the most important parameters of a hormetic treatment besides lifespan. Indeed, it emerged that each life-extension is accompanied by a constraining effect in at least one other endpoint, for example growth, mobility, stress resistance, or reproduction. Thus, in this context, longevity could not be considered to be attained for free and therefore it is likely that other hormetic benefits may also incur cost-intensive and unpredictable side-effects.