Caenorhabditis elegans stress related gene responses to selected pesticides

Pesticides are used widely and more than 2 million tons are released in the environment annually (data for 2000-2001, Kiely 2004). Details of their toxicity towards non-target organisms, are not complete for many of these pesticides and serious environmental issues have previously arisen as a result (e.g. effects of DDT on reproduction of wild birds). Unfavourable conditions, including the presence of toxicants, can induce the stress response pathways through which an organism attempts to metabolise the harmful chemicals or counter their effects. The stress response network contains a number of gene pathways controlled by transcriptional regulators which control expression of genes in one or several groups. In their attempt to counter stress, stress-response genes act in an interactive manner and should therefore be studied together as a network, rather than individually. This study examines the effect of a number of commonly used pesticides on C. elegans, a non-target organism. We focus on stress-response gene expression patterns and in some cases perform assays for physiological effects. We also present supplementary qPCR experiments to confirm previous results on the effect of dichlorvos on C. elegans gene expression. Our results show that some tested pesticides are not toxic to the nematode, whereas rotenone proves highly toxic and chlorpyriphos, endosulfan, DDT and carbendazim are moderately toxic with DDT showing significant inhibition of feeding as well. Our data partly confirm the gene array results previously obtained for dichlorvos. Our study provides information on how a number of pesticides affect stress-response gene expression. Together with previous data on the effect of heavy metals and parallel data from Drosophila, these findings will inform the development of a dynamic mathematical model of the stress-response network (SRN)

Caenorhabditis elegans stress related gene responses to selected pesticides

Pesticides are used widely and more than 2 million tons are released in the environment annually (data for 2000-2001, Kiely 2004). Details of their toxicity towards non-target organisms, are not complete for many of these pesticides and serious environmental issues have previously arisen as a result (e.g. effects of DDT on reproduction of wild birds). Unfavourable conditions, including the presence of toxicants, can induce the stress response pathways through which an organism attempts to metabolise the harmful chemicals or counter their effects. The stress response network contains a number of gene pathways controlled by transcriptional regulators which control expression of genes in one or several groups. In their attempt to counter stress, stress-response genes act in an interactive manner and should therefore be studied together as a network, rather than individually. This study examines the effect of a number of commonly used pesticides on C. elegans, a non-target organism. We focus on stress-response gene expression patterns and in some cases perform assays for physiological effects. We also present supplementary qPCR experiments to confirm previous results on the effect of dichlorvos on C. elegans gene expression. Our results show that some tested pesticides are not toxic to the nematode, whereas rotenone proves highly toxic and chlorpyriphos, endosulfan, DDT and carbendazim are moderately toxic with DDT showing significant inhibition of feeding as well. Our data partly confirm the gene array results previously obtained for dichlorvos. Our study provides information on how a number of pesticides affect stress-response gene expression. Together with previous data on the effect of heavy metals and parallel data from Drosophila, these findings will inform the development of a dynamic mathematical model of the stress-response network (SRN)

The Impact of the C-Terminal Region on the Interaction of Topoisomerase II Alpha with Mitotic Chromatin.

Type II topoisomerase enzymes are essential for resolving DNA topology problems arising through various aspects of DNA metabolism. In vertebrates two isoforms are present, one of which (TOP2A) accumulates on chromatin during mitosis. Moreover, TOP2A targets the mitotic centromere during prophase, persisting there until anaphase onset. It is the catalytically-dispensable C-terminal domain of TOP2 that is crucial in determining this isoform-specific behaviour. In this study we show that, in addition to the recently identified chromatin tether domain, several other features of the alpha-C-Terminal Domain (CTD). influence the mitotic localisation of TOP2A. Lysine 1240 is a major SUMOylation target in cycling human cells and the efficiency of this modification appears to be influenced by T1244 and S1247 phosphorylation. Replacement of K1240 by arginine results in fewer cells displaying centromeric TOP2A accumulation during prometaphase-metaphase. The same phenotype is displayed by cells expressing TOP2A in which either of the mitotic phosphorylation sites S1213 or S1247 has been substituted by alanine. Conversely, constitutive modification of TOP2A by fusion to SUMO2 exerts the opposite effect. FRAP analysis of protein mobility indicates that post-translational modification of TOP2A can influence the enzyme's residence time on mitotic chromatin, as well as its subcellular localisation

Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present

Transgenic nematodes as biosensors for metal stress in soil pore water samples

Caenorhabditis elegans strains carrying stress-reporter green fluorescent protein transgenes were used to explore patterns of response to metals. Multiple stress pathways were induced at high doses by most metals tested, including members of the heat shock, oxidative stress, metallothionein (mtl) and xenobiotic response gene families. A mathematical model (to be published separately) of the gene regulatory circuit controlling mtl production predicted that chemically similar divalent metals (classic inducers) should show additive effects on mtl gene induction, whereas chemically dissimilar metals should show interference. These predictions were verified experimentally; thus cadmium and mercury showed additive effects, whereas ferric iron (a weak inducer) significantly reduced the effect of mercury. We applied a similar battery of tests to diluted samples of soil pore water extracted centrifugally after mixing 20% w/w ultrapure water with air-dried soil from an abandoned lead/zinc mine in the Murcia region of Spain. In addition, metal contents of both soil and soil pore water were determined by ICP-MS, and simplified mixtures of soluble metal salts were tested at equivalent final concentrations. The effects of extracted soil pore water (after tenfold dilution) were closely mimicked by mixtures of its principal component ions, and even by the single most prevalent contaminant (zinc) alone, though other metals modulated its effects both positively and negatively. In general, mixtures containing similar (divalent) metal ions exhibited mainly additive effects, whereas admixture of dissimilar (e.g. trivalent) ions often resulted in interference, reducing overall levels of stress-gene induction. These findings were also consistent with model predictions

The α isoform of topoisomerase II is required for hypercompaction of mitotic chromosomes in human cells.

As proliferating cells transit from interphase into M-phase, chromatin undergoes extensive reorganization, and topoisomerase (topo) IIα, the major isoform of this enzyme present in cycling vertebrate cells, plays a key role in this process. In this study, a human cell line conditional null mutant for topo IIα and a derivative expressing an auxin-inducible degron (AID)-tagged version of the protein have been used to distinguish real mitotic chromosome functions of topo IIα from its more general role in DNA metabolism and to investigate whether topo IIβ makes any contribution to mitotic chromosome formation. We show that topo IIβ does contribute, with endogenous levels being sufficient for the initial stages of axial shortening. However, a significant effect of topo IIα depletion, seen with or without the co-depletion of topo IIβ, is the failure of chromosomes to hypercompact when delayed in M-phase. This requires much higher levels of topo II protein and is impaired by drugs or mutations that affect enzyme activity. A prolonged delay at the G2/M border results in hyperefficient axial shortening, a process that is topo IIα-dependent. Rapid depletion of topo IIα has allowed us to show that its function during late G2 and M-phase is truly required for shaping mitotic chromosomes

Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present