Biomedical subjects

A John Morgan

Publications and source records attributed to A John Morgan.

10 recordsLinked to original sources

PubMed2006

Autofluorescence in eleocytes of some earthworm species.

Immunocompetent cells of earthworms, coelomocytes, comprise adherent amoebocytes and granular eleocytes (chloragocytes). Both cell populations can be expelled via dorsal pores of adult earthworms by exposure to an electric current (4.5 V) for 1 min. Analysis by phase contrast/fluorescence microscopy and flow cytometry demonstrated that eleocyte population of several species exhibits a strong autofluorescence. A high percentage (11-35%) of autofluorescent eleocytes was recorded in Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion sp. (O. cyaneum, O. tyrtaeum tyrtaeum and O. tyrtaeum lacteum). In contrast, autofluorescent coelomocytes were exceptionally scarce (less than 1%) in representative Aporrectodea sp. (A. caliginosa and A. longa) and Lumbricus sp. (L. castaneus, L. festivus, L. rubellus, L. terrestris). Thus, this paper for the first time describes profound intrinsic fluorescence of eleocytes in some--but not all--earthworm species. The function (if any) and inter-species differences of the autofluorescent coelomocytes still remain elusive.

Animals↗

PubMed2006

Riboflavin content in autofluorescent earthworm coelomocytes is species-specific.

We have recently shown that a large proproportion of earthworm coelomocytes exhibit strong autofluorescence in some species (Dendrobaena veneta, Allolobophora chlorotica, Dendrodrilus rubidus, Eisenia fetida, and Octolasion spp.), while autofluorescent coelomocytes are very scarce in representatives of Lumbricus spp. and Aporrectodea spp. Riboflavin (vitamin B2) was identified as a major fluorophore in Eisenia jetida coelomocytes. The main aim of the present experiments was to quantify riboflavin content in autofluorescent coelomocytes (eleocytes) from several earthworm species through a combination of flow cytometric and spectrofluorometric measurements. Spectrofluorometry of coelomocyte lysates showed that riboflavin was non-detectable in the coelomocytes of Aporrectodea spp. and Lumbricus spp., but was a prominent constituent of lysates from species with autofluorescent eleocytes. In the latter case, riboflavin content was the highest in E. fetida, followed by Octolasion spp. > A. chlorotica > D. rubidus. The riboflavin content of coelomocytes correlates positively with eleocyte autofluorescence intensity measured by flow cytometry and visible with fluorescence microscopy.

Animals↗

PubMed2005

Heavy metals affect the coelomocyte-bacteria balance in earthworms: environmental interactions between abiotic and biotic stressors.

Three-day dermal exposure of Dendrobaena veneta to metal ions differentially disrupts the immunocompetence/pathogen balance. Zn does not accumulate in the earthworm body, Cu accumulation is temperature-independent while Cd accumulation is stronger at 22 degrees C than at 10 degrees C. During in vitro incubation with metal ions at 22 degrees C, growth of coelom-derived bacteria is enhanced by Zn, but significantly or almost completely inhibited by Cu or Cd. In contrast, under in vivo conditions at 22 degrees C, bacterial load is decreased only after Cd exposure, but increased after Zn and Cu exposures. At 10 degrees C bacteria growth is almost completely inhibited in all groups except Cu-treated animals. Coelomocyte number is unaffected in animals exposed to Zn, but significantly decreased after exposure to Cd (at 22 degrees C) and Cu (at 22 degrees C and 10 degrees C) with concomitant changes of amoebocyte-to-eleocyte ratio in favour of amoebocytes. Metal exposure up-regulates expression of metallothioneins in coelomocytes, mainly amoebocytes.

Amoeba↗

PubMed2005

Metal compartmentation and speciation in a soil sentinel: the earthworm, Dendrodrilus rubidus.

Earthworms are well-studied organisms in ecotoxicology because of their keystone ecological status and metal-accumulating capacity. However, the direct estimation of the bioreactive fractions of accumulated metal burdens remains technically elusive. In this study we exploited two physical techniques, electron probe X-ray microanalysis (EPXMA) and X-ray absorption spectroscopy (XAS), to improve understanding of the subcellular spatial distributions, ligand affinities, and coordination chemistries of Cd, Pb and Zn in a field population of the epigeic earthworm, Dendrodrilus rubidus. EPXMA and XAS analyses were performed on cryopreparations to maintain compositional fidelity; EPXMA data were analyzed by multivariate statistics. XAS provided whole-worm insights; EPXMA provided in situ, subcellular data from the major metal-sequestering tissue, the chloragog. Both techniques showed that Cd is coordinated with S; the measured Cd-S bond distance in XAS suggests a metallothionein-type ligand. The mean Cd:S molar ratio (EPXMA) of 0.36 is higher than the ratio of 0.29 estimated from published biochemical data. EPXMA and XAS data also found that Ca, Pb, and Zn are predominantly bound to one or more O-donating, probably phosphate-rich, ligands. X-ray distribution maps (EPXMA) of the hepatocyte-resembling chloragocytes revealed that the O-seeking (Ca, Pb, Zn) metals and S-seeking Cd bioaccumulate in distinct organelles. Extended X-ray absorption fine structure showed that the Pb complex is not biogenic pyromorphite, although X-ray absorption near edge structure did not eliminate the possibility. XAS provided no evidence of Pb spillage from the "sequestration compartment" within D. rubidus. However, the correspondence of Pb with Ca and P in EPXMA is not as strong as that of Zn. This is indicative either of spillover or of a second, hitherto unidentified, sequestered-Pb pool. By exploiting the complimentary techniques of EPXMA and XAS,we are closer to describing the mechanistic link between equilibrated body burdens and biomarker responses in earthworms.

Animals↗

PubMed2005

Hierarchical responses of soil invertebrates (earthworms) to toxic metal stress.

The concept of a hierarchical cascade of biological responses to stress occurring across different levels of biological organization is an underlying principle of both theoretical and regulatory ecology/ecotoxicology. This study investigates the reality of this cascade for earthworms exposed to toxic metal stress. Gene expression was the most sensitive endpoint (EC50 = 616 microg Zn g(-1)) followed by the integrity of coelomocyte lysosomal membrane (EC50 = 645 microg Zn g(-1)). This confirms that, in accordance with the cascade concept, suborganism level endpoints respond at lower metal concentrations than higher organization endpoints. The relative sensitivity of the higher organization parameters was not as predicted by the cascade. Organic material removal was more sensitive (EC50 = 997 microg Zn g(-1)) than L. rubellus reproduction (EC50 = 3236 microg Zn g(-1)), L. rubellus population size (EC50 = 5000-11500 microg Zn g(-1)), and earthworm community diversity (EC50 = 1737 microg Zn g(-1)). This can be attributed to (1) the relative insensitivity of L. rubellus to metals and (2) general toxic effects of metals on the earthworm energy budget (and thus feeding). On the basis of these results, it can be concluded that predictive assessments of the consequences of environmental stressors needs to include approaches that respect the relative sensitivities of different taxa, while retrospective appraisals should exploit the sensitivity of low organization level responses.

Animals↗

PubMed2005

Cu and Cd effects on the earthworm Lumbricus rubellus in the laboratory: multivariate statistical analysis of relationships between exposure, biomarkers, and ecologically relevant parameters.

This study sets out to examine the potential of a suite of novel molecular biomarkers as early warning indicators of environmental state and damage. Transcriptional responses of four genes, metallothionein 1 and 2, amine oxidase, and the lysosomal associated glycoprotein, were measured in the earthworm Lumbricus rubellus exposed to increasing concentrations of cadmium and copper in OECD soil. These responses were compared to metal body concentrations and lifecycle parameters: survival, cocoon production, and growth. Adverse physiological effects were observed at concentrations 1/3rd to 1/10th those of the artificial soil LC50. Multivariate statistics, principal component analysis (PCA), was used to investigate the correlations between the different variables. Three key components were derived explaining 77.6% of the variance, with component 1 contributing 32.4%, component 2 contributing 26.7%, and component 3 contributing 18.5%. These components were interpreted in terms of population health, pollutant exposure, and detoxification pathways, respectively. It is proposed that the use of such a suite of biomarkers could serve as indicators of the "health" of the soil environment and provide early warning signals of potential danger to the biota or as a means of monitoring soil remediation.

Animals↗

PubMed2005

Early-phase immunodetection of metallothionein and heat shock proteins in extruded earthworm coelomocytes after dermal exposure to metal ions.

This paper provides direct evidence that earthworm immune cells, coelomocytes, are exposed to bio-reactive quantities of metals within 3 days after dermal exposure, and that they respond by upregulating metallothionein (MT) and heat shock protein (HSP70, HSP72) expression. Indirect support for the hypothesis that coelomocytes are capable of trafficking metals was also obtained. Coelomocytes were expelled from adult individuals of Eisenia fetida after 3-day exposure either to metal ions (Zn, Cu, Pb, Cd) or to distilled water (controls) via filter papers. The number of coelomocytes was significantly decreased after Cu, Pb, or Cd treatment. Cytospin preparations of coelomocytes were subjected to immunoperoxidase staining with monoclonal antibodies against human heat shock proteins (HSP70 or HSP72), or rabbit polyclonal antibodies raised against metallothionein 2 (w-MT2) of Lumbricus rubellus. Applied antibodies detected the respective proteins of E. fetida and revealed that the expression of HSP70, HSP72 and w-MT2 proteins was either induced or significantly enhanced in coelomocytes from metal-exposed animals. In conclusion, stress protein expression in earthworm coelomocytes may be used as sensitive biomarkers of metal contaminations. Further experimentation is needed for quantitative analysis of kinetics of metal-induced stress protein expression in earthworm coelomocytes.

Animals↗

PubMed2004

Cadmium detoxification in earthworms: from genes to cells.

Cadmium pollution has clear ecotoxicological consequences as it is readily bio-available and has a reported tendency to bio-accumulate in soil biota. Understanding the mechanisms of cadmium trafficking pathways within sentinel terrestrial invertebrates, such as the earthworm, is therefore considered to be of importance. Using X-ray microanalysis, quantitative polymerase chain reaction, and immunohistochemical techniques, we were able to demonstrate that the earthworm has the intrinsic capacity to efficiently sequester and compartmentalize cadmium via a metallothionein-mediated trafficking pathway. There is evidence that wMT-2, rather than wMT-1, is the major isoform implicated in the detoxification of cadmium and the identification of three independent wMT-2 loci (totalling over 25 kb of genomic sequence) has revealed a complex genomic organization. Complementary in silico analysis of over 6500 expressed sequence tags has identified a third metallothionein isoform, wMT-3, found to be highly enriched in embryonic tissue. In summary, this paper provides a detailed dissection of the genetic, molecular, and cellular basis of a sophisticated pathway that facilitates the uptake, accumulation, transport, and excretion of cadmium.

Amino Acid Sequence↗

PubMed2004

Toxicological, cellular and gene expression responses in earthworms exposed to copper and cadmium.

This study correlates sub-organismal changes with toxicological effects in earthworms (Lumbricus rubellus) exposed to copper and cadmium. Both metals reduced survival and reproduction at the highest concentration (LC50 5.11 microM Cu g(-1) and 4.04 microM Cd g(-1); cocoon production EC50s 5.17 microM Cu g(-1) and 1.86 microM Cd g(-1), all values as dry mass soil). Cadmium significantly reduced lysosomal membrane stability (at 1.86 microM Cd g(-1) and higher), upregulated metallothionein gene expression (at least sevenfold in all treatments) and reduced lysosome-associated-glycoprotein gene expression. Copper did not lower lysosomal membrane stability, but did upregulate metallothionein gene expression (at 2.5 microM Cu g(-1)), reduce lysosome-associated-glycoprotein gene expression and gave a nonlinear pattern for mitochondrial ribosomal subunit transcript expression (reduced at 0.35 and 0.811 microM Cu g(-1); higher at 2.5 microM Cu g(-1)). Correlation of metal body residue concentrations and cellular and molecular genetic responses with juvenile production rate confirmed a relationship for metallothionein expression, lysosomal membrane stability and cadmium tissue concentration in cadmium-exposed worms. Relationships between responses were also found for both metals. These suggested mechanisms for the interaction of cadmium and copper with specific gene products and with organelle (mitochondrial, lysosomal) functioning.

Animals↗

PubMed2002

Morphological plasticity in metal-sequestering earthworm chloragocytes: morphometric electron microscopy provides a biomarker. of exposure in field populations.

Morphometric measurements of the volume fractions (Vv) of two prominent metal-sequestering compartments, chloragosome granules and debris vesicles, were made on transmission electron micrographs of chloragocytes in Dendrodrilus rubidus (Annelida: Oligochaeta: Lumbricidae) from one uncontaminated site and three qualitatively and quantitatively different metalliferous soils associated with disused Pb/Zn mines. We also examined the relative volume fractions of the alimentary wall and attached chloragogenous tissue by light microscopy. Several conclusions relevant to environmental diagnostics were reached: Metal identity and degree of soil pollution exert strong effects on chloragocyte ultrastructure; morphometry by light microscopy indicated that the metal-induced structural effects are characterized by intracellular alterations not accompanied by a significant expansion of the tissue volume; elevated metal burdens can reduce chloragosome Vv and, concomitantly, increase the Vv of their autophagic derivatives, the debris vesicles; the measured cellular alterations were more closely correlated with integrated tissue metal burdens than they were with integrated soil metal burdens; and estimates of tissue metal distribution, derived by combining morphometric data and published whole-worm and chloragosome metal concentrations, suggested that the alimentary wall of earthworms may sequester Pb, Zn, and Cd. Our study shows that cellular changes, directly or indirectly, may yield quantifiable expressions of metal loads bioaccumulated by earthworms. However, the practical use of cellular changes as biomarkers of soil quality probably will require automated light-microscopic morphometry in combination with organelle-specific molecular probes to be cost effective.

Animals↗