A novel assay for the detection of anthelmintic activity mediated by cuticular damage to nematodes: validation on Caenorhabditis elegans exposed to cysteine proteinases.

Plant cysteine proteinases (CPs) from Carica papaya kill parasitic and free-living nematodes in vitro by hydrolysis of the worm cuticle, a mechanism that is different to all commercially available synthetic anthelmintics. We have developed a cheap and effective, rapid-throughput Caenorhabditis elegans-based assay for screening plant CP extracts for anthelmintic activity targeting cuticular integrity. The assay exploits colorimetric methodology for assessment of cuticular damage, and is based on the ability of viable cells to incorporate and bind Neutral red dye within lysosomes and to release the dye when damaged. Living worms are pre-stained with the dye, exposed to CPs and then leakage of the dye through the damaged cuticle is quantified by spectrophotometry. In contrast to motility assays and semi-subjective interpretation of microscopical images, this colorimetric assay is independent of observer bias. Our assay was applied to a series of C. elegans bus mutant strains with leaky cuticles and to cystatin knockout mutants. At ambient temperature and over 0.5-24 h, both bus mutants and the cystatin knockouts were highly susceptible to CPs, whereas wild-type Bristol N2 worms were essentially unstained by Neutral red and unaffected by CPs, providing validation for the utility of this assay.

Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity.

Toxicity of the dithiocarbamate fungicide mancozeb to the nontarget soil nematode, Caenorhabditis elegans.

We have previously shown that the dithiocarbamate fungicide, Mancozeb, strongly induces lacZ reporter expression from an endogenous heat-shock promoter (hsp16) in the PC72 transgenic strain of the nematode Caenorhabditis elegans. Such evidence of organismal stress, in a nontarget species at subapplication concentrations, was much less apparent for the related fungicide, Maneb, which only weakly induced reporter expression. We now show that reporter induction by Mancozeb is marginal (<60%) after a few hours' exposure, but increases substantially (to almost 10-fold) after overnight exposure. In conjunction with our previous results using intermediate exposure periods, this suggests that the factor limiting reporter responses is likely to be a slow rate of uptake and/or metabolism of the fungicide. We confirm that a potentially toxic metabolite of dithiocarbamate fungicides, namely ethylenethiourea (ETU), has minimal toxicity toward C. elegans, even after prolonged exposure at high concentrations. We demonstrate that exposure to Mancozeb (but not ETU) significantly inhibits larval growth in C. elegans, although this parameter is not markedly more sensitive than reporter induction as a toxicological endpoint. Finally, we have used two-dimensional electrophoresis to show that high concentrations of both Maneb and Mancozeb drastically simplify the protein spot profile compared with controls. However, only in the latter case is there evidence of novel proteins being induced. Both fungicides appear toxic to C. elegans, but only Mancozeb induces a strong heat-shock response.

Caenorhabditis elegans as a biomonitor for immunological stress in nematodes.

An experimental system has been developed using Caenorhabditis elegans (Secernentea: Rhabditida), to monitor immunological stress in nematodes. The transgenic C. elegans strain PC72 carries a lacZ reporter gene fused to a C. elegans hsp16-1 gene, which is inducible for beta-galactosidase activity at the heat stress temperature of 26 degrees C. The investigate the possibility of using PC72 to monitor immunological stress, its surface coat was targeted, to mimic immune attack, by raising immune sera against surface coat components selectively removed by the cationic detergent cetyltrimethylammoniunm bromide. Initially, a highly significant induction of beta-galactosidase activity was seen in PC72 incubated in either surface-reactive or naïve rabbit serum. Complement (C3) was detected over the entire surface of adult PC72 and was thought to be responsible for stress-induction with naïve sera. When the immunoglobulin (Ig)G fraction of naïve sera was used in isolation, no stress-induction was seen. In contrast, a two-fold increase in beta-galactosidase activity was seen in the presence of surface-reactive IgG (SR-IgG) which recognised surface components of between 6 and 40 kDa in western blot. The belief that surface reactive IgG could induce a stress response was reinforced by analysis of hsp-16 protein expression. Cationised ferritin was then used to assess whether stress-induction was truly a surface reactive event; binding of cationised ferritin to the nematode surface also resulted in two-fold induction of beta-galactosidase activity. To investigate the downstream biological effects of stress induction, worm growth and fecundity were measured in the presence of IgG preparations. A significant reduction was seen in both worm length and fecundity only when larvae were incubated in surface-reactive IgG, compared to both naïve IgG and K-medium controls. In conclusion, it would appear that C. elegans is a suitable model to monitor the induction of immunological stress at the level of the nematode surface coat. Given the ability of nematode surface antigens to protect the vaccinated host in animal model systems, and the close phylogenetic relationships which exist between C. elegans and nematodes of medical and veterinary importance, it is conceivable that the immunological targets in or on the surface of C. elegans warrant rapid identification.

Effect of single and paired metal inputs in soil on a stress-inducible transgenic nematode.

A toxicity test using a transgenic strain of the free-living soil nematode Caenorhabditis elegans carrying a stress-inducible beta-galactosidase reporter has been adapted for use in soil biomonitoring. High concentrations (250 microg. g(-1)) of cadmium are required to induce the stress response in worms exposed to Lufa 2.2 soil. Even at relatively high concentrations, the response to copper and zinc additions alone is minimal, yet combinations of cadmium and copper in the test soil induce a larger response than with cadmium alone at the same concentration. In contrast, the addition of both zinc and cadmium induces a lower response than cadmium additions alone. Analysis of the interstitial water suggests that there is preferential occupation by copper of sorption sites in the soil, allowing more cadmium to remain in solution. Conversely, cadmium and zinc would appear to interact similarly with the soil constituents, resulting in an increase of both metals in solution with increased additions to the soil. Aquatic tests mimic the results of the soil test, so it is not increased cadmium availability alone that causes an increased stress response when both cadmium and copper are present. The presence of other metals could reduce the amount of cadmium available, which may be one factor in the zinc moderation of the stress response to cadmium. Intracellular mechanisms may also contribute to the copper enhancement of the stress response to cadmium.http://link. springer-ny.com/link/service/journals/00244/bibs/37n4p503.++ +html

The toxicity of dithiocarbamate fungicides to soil nematodes, assessed using a stress-inducible transgenic strain of Caenorhabditis elegans.

The dithiocarbamate fungicides maneb and mancozeb induce a short-term stress response in a transgenic Caenorhabditis elegans strain (PC72) carrying a reporter lacZ gene under the control of a homologous heat shock (hsp16) promoter. This response can be readily monitored as induced beta-galactosidase activity, either by in situ staining or by a quantitative fluorometric enzyme assay. Particularly strong responses are induced by mancozeb (three- to fivefold above controls at 500 microg mL(-1)), causing acute toxicity at concentrations comparable to those recommended for field application (2 mg mL(-1)). Although much of this fungicide is adsorbed by soil, sufficient (ca. 6%) enters the soil water compartment to cause mild stress in the transgenic worm assay. Among possible metabolites from mancozeb breakdown, neither Mn2+ nor ethylenethiourea (ETU) is particularly toxic even at 10% of the optimum mancozeb dosage. Stress responses to a range of other pesticides are also reported, and in several cases it is clear that a nontarget soil species (here, transgenic C. elegans) may be sensitive to low-level contamination.

The accumulation and histological effects of organometallic fungicides propineb and maneb in the livers of pregnant rats and their offspring.

Dithiocarbamate propineb and maneb are organometal fungicides, which are widely used for the control of diseases in plants. Female Wistar rats were exposed orally to 200 and 400 ppm propineb (Zn-containing dithiocarbamate) and 250 ppm maneb (Mn-containing dithiocarbamate), from the 6th day of gestation up to birth. We found that the body weights of both newborn litters and their fungicide-treated mothers were lower than those of controls. Histological examination of the livers of fungicide-treated pregnant females and the offspring showed a variety of histopathological effects. Moreover, the analysis of Zn and Mn concentrations in the livers of pregnant females exposed to organometallic fungicides during pregnancy demonstrated that the metal concentrations in the liver were higher than those of controls. Similarly, the hepatic metal concentrations were significantly increased in the litters, indicating the transplacental passage of the organometallic fungicides.

The accumulation and histological effects of organometallic fungicides Propineb and Maneb in the kidneys of fetus and female rats during pregnancy.

Dithiocarbamate propineb and maneb are organometal fungicides, which are widely used for the control of diseases in plants. Female Wistar rats were exposed orally to 200 and 400 ppm propineb and 250 ppm maneb, from the sixth day of gestation up to birth. We found that the body weights of both one-day old litters and their fungicide-treated mothers were lower than those of controls. Histological examination of the kidneys of fetus and fungicide-treated pregnant females showed a variety of histopathological effects. Moreover, the analysis of zinc (Zn) and manganese (Mn) concentrations (using inductively coupled plasma-atomic emission spectrometry) in the kidneys of pregnant females exposed to organometallic fungicides during pregnancy demonstrated that the metal concentrations in the kidney were higher than those of controls. However, the renal metal concentrations were significantly increased in the litters subjected to the fungicides during gestation, indicating that high levels of the trace metals in the organ of fetus may well be due to the fungicides easily passing the placental barrier.

Transgenic nematodes as biomonitors of microwave-induced stress.

Transgenic nematodes (Caenorhabditis elegans strain PC72), carrying a stress-inducible reporter gene (Escherichia coli beta-galactosidase) under the control of a C. elegans hsp16 heat-shock promoter, have been used to monitor toxicant responses both in water and soil. Because these transgenic nematodes respond both to heat and toxic chemicals by synthesising an easily detectable reporter product, they afford a useful preliminary screen for stress responses (whether thermal or non-thermal) induced by microwave radiation or other electromagnetic fields. We have used a transverse electromagnetic (TEM) cell fed from one end by a source and terminated at the other end by a matched load. Most studies were conducted using a frequency of 750 MHz, at a nominal power setting of 27 dBm. The TEM cell was held in an incubator at 25 degrees C inside a shielded room; corresponding controls were shielded and placed in the same 25 degrees C incubator; additional baseline controls were held at 15 degrees C (worm growth temperature). Stress responses were measured in terms of beta-galactosidase (reporter) induction above control levels. The time-course of response to continuous microwave radiation showed significant differences from 25 degrees C controls both at 2 and 16 h, but not at 4 or 8 h. Using a 5 x 5 multiwell plate array exposed for 2 h, the 25 microwaved samples showed highly significant responses compared with a similar control array. The wells most strongly affected were those in the rows closest to the source, whereas the most distant row did not rise above control levels, suggesting a shadow effect. These differential responses are difficult to reconcile with general heating effects, although localised power absorption affords a possible explanation. Experiments in which the frequency and/or power settings were varied suggested a greater response at 21 than at 27 dBm, both at 750 and 300 MHz, although extremely variable responses were observed at 24 dBm and 750 MHz. Thus, lower power levels tended, if anything, to induce larger responses (with the above-mentioned exception), which is opposite to the trend anticipated for any simple heating effect. These results are reproducible and data acquisition is both rapid and simple. The evidence accrued to date suggests that microwave radiation causes measurable stress to transgenic nematodes, presumably reflecting increased levels of protein damage within cells (the common signal thought to trigger hsp gene induction). The response levels observed are comparable to those observed with moderate concentrations (ppm) of metal ions such as Zn2+ and Cu2+. We conclude that this approach deserves further and more detailed investigation, but that it has already demonstrated clear biological effects of microwave radiation in terms of the activation of cellular stress responses (hsp gene induction).

Use of stress-inducible transgenic nematodes as biomarkers of heavy metal pollution in water samples from an english river system.

Transgenic strains of the nematode Caenorhabditiselegans, which carry stress-inducible lacZ reporter genes, aremeasurably stressed by exposure to heavy metals in aqueous solution. Thisstress response can be quantified, using enzymatic assays for the reportergene-product (Escherichia coli beta-galactosidase), or estimatedapproximately by in situ staining for beta-galactosidase in exposedworms. Stress responses to heavy metals have been demonstrated both inlaboratory tests using Cd2+ or Hg2+, and also in watersamples taken from a metal-polluted river system in southwest England. TheRiver Carnon flows through an area with an ancient mining history,principally for Sn, but also for Cu and other metals; As, Cd, Al, Mn, and Zn,as well as large amounts of Fe, are all present in these ore bodies. Foursites in the Carnon river basin were compared with respect to theirmacroinvertebrate diversity, physical and chemical characteristics (includingthe concentrations of As, Cd, Al, Cu, Mn, Zn, and Fe). Transgenic worms wereexposed to water samples from these four sites, and also to a 0.33%(v/v) dilution of metal-laden minewater from the principal local mine (WhealJane). Transgene expression was induced in all five cases, though markedlyless so for the least polluted of the sites (which also supported a richermacroinvertebrate fauna). Two different transgenic strains were tested inthis study; strain PC72 (using a homologous hsp16 promoter) isslightly more sensitive to most metal-containing water samples than strainCB4027 (using a heterologous Drosophila hsp70 promoter). Bothtransgenic strains and two different assay methods gave essentially similarresults. These findings demonstrate that transgenic nematodes could provide arapid and simple assessment of aquatic pollution, in that the transgeneresponse is inducible by mixtures of dissolved metals at concentrationsactually encountered in metal-polluted watercourses.

Transgenesis in animal systems: a view from within.

The introduction of 'foreign' or altered genes into animals arouses both scientific and public concern. So too does any extension of this practice to human beings at the somatic (tissue) level, let alone interference in the human germ line (genetic material passed on to future generations). The range of possible genetic modifications, both those in progress and those likely in the near future, is so vast as to make a uniform Christian response untenable. As with other technologies, there are both laudable and dubious uses for genetic engineering in animal systems; it is neither an unmixed blessing nor an unmitigated curse. It is argued that Christians should engage with these issues on a case-by-case basis, giving due weight to the possible risks and concomitant suffering for the animals used, but basing our ultimate approval or disapproval on the congruence of means and aims with those evinced by Jesus in his ministry of healing, teaching and reconciliation.

Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans.

In a transgenic strain of Caenorhabditis elegans carrying a stress-inducible lacZ reporter gene, short-term sublethal exposure to heavy metals activates transgene expression. The transgene response to Cd2+ is strongly inhibited by Ca2+ ions; furthermore, Ca2+ reduces the net accumulation of Cd2+ by worms. Both Ca2+ and a variety of calcium uptake inhibitors (nifedipine, La3+, verapamil) depress the dose response of the transgene to Cd2+. Calcium ionophore (A23187) slightly increases transgene activity in control and Cd2+ treated worms, but has a much larger effect in the case of Mn2+, reflecting its much greater affinity for this ion.

A choice between glycogen and delta-crystallin accumulation is made in glial cells and not influenced by overlying neurons.

Chick-embryo neuroretinal cells convert extensively into lens under low-glucose conditions, but this transdifferentiation process is blocked by high-glucose media. We have previously observed an inverse relationship between the levels of glycogen (a marker of normal retinoglial differentiation) and of delta-crystallin (a lens marker) in such cultures. However, most of the glycogen accumulated under high-glucose conditions is apparently localized in those glial (G) cells underlying clusters of neurons (N cells). We here show that glial-enriched cultures (largely depleted of N cells) both accumulate glycogen and fail to transdifferentiate in high-glucose media. Moreover, glycogen localization in groups of glial cells is unaffected by the absence of N cells. Thus the choice between normal and foreign differentiation pathways is made autonomously within the retinoglial-cell population and is not influenced significantly by the presence or absence of N cells.

Expression in non-lens tissues of an enzyme activity related to the 'lens-specific' protein, delta crystallin.

When chick embryo neutral retina (NR) cells are cultured for long periods in vitro, they undergo extensive transdifferentiation into lens and express the lens protein, delta crystallin. We now demonstrate that this process is accompanied by a change in the chromatin conformation of the delta-gene locus from DNAase1-resistant to DNAase1-sensitive in the nuclei of most cells. Transcripts hybridising to a delta probe are also much more prevalent among the in vitro transcription products from lens or transdifferentiated NR culture nuclei, as compared to nuclei from fresh NR tissue. Published evidence indicates that the chick delta 1 crystallin gene encodes the major structural protein of embryonic lens fibres, whereas the closely related delta 2 gene may encode the urea-cycle enzyme argininosuccinate lyase (ASL). Our present data lends further support to this view. Both immunodetectable delta-related protein(s) and ASL activity are present in fresh embryonic NR tissue, as well as in mouse and Rana liver, and in Rana lens. Our polyclonal anti-delta antibody also cross-reacts with a major constituent of commercial bovine ASL, of the same molecular size as chick delta crystallin. Immunoselection studies suggest that the ASL activity in chick embryonic NR is conferred mainly by the delta-related protein band. So-called 'ectopic' expression of delta crystallin in embryonic NR (and other tissues) may thus involve the delta 2/ASL gene, and could reflect some metabolic requirement for ASL activity.

Effects of Pluronic F-68 on 2-deoxyglucose uptake and amino acid incorporation into chick embryonic fibroblasts in vitro.

The effects of the nonionic surfactant Pluronic F-68 on 2-deoxyglucose uptake and amino acid incorporation in vitro into chick embryonic fibroblasts prepared from day 7 embryos have been studied. During early culture growth (3-10 days), Pluronic supplementation at 0.05% (w/v), but not at 0.1%, stimulated 2-deoxyglucose uptake by 20%-30% above control levels. Pluronic also markedly increased amino acid incorporation, maximally at 3 days of culture (200%-300% above control levels with 0.1% and 0.05% Pluronic, respectively), but to a lesser extent thereafter. These effects of Pluronic were seen only when freshly-prepared solutions were used; 'aged' solutions produced no comparable stimulation of 2-deoxy-glucose uptake or amino acid incorporation.

Stimulatory effects of retinal extract and fibroblast growth factor on lentoidogenesis in cultures of chick embryo neuroretinal cells.

A crude extract prepared from embryonic chick retina stimulates growth and particularly transdifferentiation into lens when added as a supplement to neuroretinal (NR) cultures in vitro. This effect is especially marked when using a medium (H) containing 5% horse serum, where growth factors are likely to be limiting. The level of delta-crystallin (lens marker) production in such cultures increases with the concentration of extract. Using extracts from earlier and later stages of retinal development, there is an age-dependent decline in the extent to which transdifferentiation is stimulated. However, such extracts have little effect on the activity of CAT, a neuronal marker enzyme. These effects are most probably mediated by growth factors present in the retinal extract acting upon Müller glial cells or their precursors in the NR cultures. In support of this suggestion, we show that purified fibroblast growth factor (but not epidermal growth factor) exerts similar effects on both culture growth and delta-crystallin accumulation.

pp60c-src expression in transdifferentiating cultures of embryonic chick neural retina cells.

Chick embryo neural retinal cells transdifferentiate extensively into lens cells when cultured in Eagle's MEM containing horse and fetal calf sera (FHMEM). Such cultures express elevated levels of pp60c-src-associated tyrosine kinase activity relative to parallel cultures prevented from transdifferentiating by the addition of supplementary glucose (FHGMEM) or replacement of MEM by medium 199 (F199). Northern blotting and in vitro translation studies suggest that c-src mRNA levels are only slightly higher in late transdifferentiating (FHMEM) cultures as compared to parallel blocked (FHGMEM or F199) cultures. By immunocytochemical staining, we show that pp60c-src protein is largely localized in cell groups undergoing conversion into lens (i.e. expressing delta crystallin) in late FHMEM cultures. Initial studies of pp60c-src in chick lens tissues during development indicate that higher kinase activity is found in the epithelial cells relative to mature lens fibres. Thus pp60c-src may be expressed both during the differentiation of lens cells in vivo and during the transdifferentiation of neural retina cells into lens in vitro.

Glucose metabolism in transdifferentiating and glucose-blocked cultures of chick embryo neuroretinal cells: an inverse relationship between glycogen and delta-crystallin accumulation.

Chick embryo neuroretinal (NR) cells transdifferentiate extensively into lens when cultured for several weeks in low-glucose (FH) medium, but fail to do so when high levels of supplementary glucose (FHG) are present. We show here that most aspects of glucose metabolism are promoted in high-glucose cultures, including lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities, 2-deoxyglucose uptake, pentose shunt activity and lactate production. Continuous supplementation of high-glucose cultures with low levels of ouabain (FHGO) significantly lowers 2-deoxyglucose uptake, from FHG levels down towards FH levels, especially during the early stages of NR culture. Much later, extensive transdifferentiation into lentoids (with concomitant delta-crystallin accumulation) occurs in these FHGO cultures, which thus resemble FH rather than FHG controls. Another parameter strongly affected by ambient glucose levels is the accumulation of glycogen. Both glycogen itself and glycogen synthetase activity increase steadily in FHG cultures, but decrease slightly under FH conditions. Glycogen accumulation in FHG cultures is largely confined to glial-like cells, particularly those underlying clusters of neurones. Other studies have shown that glial differentiation in vitro is promoted by histotypic interactions with retinal neurones. Thus high glucose may act in concert with neuronal influences to stimulate or stabilize the normal differentiation of retinal glial cells, whose characteristic features in vivo include glycogen synthesis and storage. Furthermore, we show that supplementation of high-glucose cultures with forskolin or dibutyryl cyclic AMP (both of which promote glycogenolysis) results in a slower rate of glycogen accumulation and in enhanced transdifferentiation into lens. In both respects, the forskolin- and dibutyryl cAMP-supplemented FHG cultures are intermediate between FH and FHG controls. Thus the enhancement of normal glial differentiation in NR cultures by high glucose may inhibit or preclude subsequent transdifferentiation into lens.