Joanna Homa

Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica

Earthworms are sensitive bioindicators of soil pollution. The aim of present investigations was to study the effects of heavy metals on earthworms and on their coelomocytes involved in the defence reactions. Adult individuals of Allolobophora chlorotica collected in Krakow (K) soil were kept in the laboratory either in the K soil, or were transferred to unpolluted soil from the rural area Sierbowice (S) or to the heavily polluted (Zn>Pb>Cd>Cu) soil from the industrial area, Bukowno (B). They were kept there at 22 °C for up to 8 weeks. Cocoons and juveniles appeared in S and K soil samples. The number and activity of the coelomocytes of worms maintained in S and K soils were unaffected despite some accumulation of heavy metals in the earthworm tissues. In contrast, in the B soil samples, bioaccumulation of metals was strongest, high mortality of adults was recorded, body mass was reduced, and reproduction completely inhibited. Coelomocytes retrieved from the B soil survivors exhibited significant impairment of pinocytosis and plastic adherence. Perhaps impairment of immune functions contributed to the poor survival under conditions of heavily polluted B soil samples.

Metal-specific effects on metallothionein gene induction and riboflavin content in coelomocytes of Allolobophora chlorotica.

Metal pollution affects earthworm coelomocytes, including their differential counts, riboflavin content and metallothioneins (MT) involved in metal homoeostasis and detoxification. The present work shows effects of Ni, Cu, Zn, Cd, and Pb at the same molarity (1mM) on coelomocytes of Allolobophora chlorotica after 2-day worm dermal exposure to metal chlorides. Numbers of coelomocytes/eleocytes extruded by electric shock and amounts of riboflavin in coelomocyte lysates were significantly decreased in Cu-exposed worms, less diminished in response to Ni, Zn, Cd, and unaffected by Pb. In sharp contrast, real-time PCR revealed a very strong (272 fold) MT-mRNA induction in response to Cd only. The induction was very low in response to Zn, Cu, Pb, and Ni ions (2.6, 2.1, 1.4, and 1.3-fold, respectively). In conclusion, decreased cell counts and riboflavin content are molecular biomarkers of Cu exposure while induction of MT-mRNA is a molecular biomarker of worm Cd exposure.

Dermal exposure of Eisenia andrei earthworms: Effects of heavy metals on metallothionein and phytochelatin synthase gene expressions in coelomocytes

Parameters such as total number of coelomocytes, riboflavin content in coelomocytes, expression of genes implied in metal homeostasis, and detoxification mechanisms can be used as biomarkers to assess the impact of metals on annelids. Defense biomarkers (detoxification gene expressions and coelomocyte parameters) were investigated in the ecotoxicologically important species Eisenia andrei following in vivo exposure to 5 different metals (zinc, copper, nickel, lead, and cadmium) at known concentrations. Coelomocyte numbers and riboflavin content were not affected by metallic exposure, but metal-specific gene expression variations were evidenced.

Effective activation of antioxidant system by immune-relevant factors reversely correlates with apoptosis of Eisenia andrei coelomocytes

Oxidative stress is harmful to the microbes but also to the host, and may result in bystander damage or death. Because of this, respiratory burst triggered in phagocytes by pathogens is counteracted by production of antioxidative factors. The aim of this work was to examine effectiveness of the latter system in earthworms Eisenia andrei by induction of reactive oxygen species, lipofuscin and phenoloxidase by natural (LPS, zymosan, Micrococus luteus) and synthetic (phorbol ester, PMA) stimulants. The compounds impaired numbers, viability (increased apoptosis) and composition of coelomocytes, and triggered the antioxidant activity of catalase and selenium-dependent glutathione peroxidase. The natural pathogenic compounds, unlike PMA, strongly activated antioxidative responses that diminished cell apoptosis. Moreover, repeated exposure to the same or different pathogenic compounds did not induce respiratory burst exhausted phenotype showing that coelomocytes are constantly at bay to withstand numerous infections. The current study reveals importance and efficiency of the oxidative–antioxidative systems in annelids but also confirms its evolutionary conservatism and complexity even in lower taxa of the animal kingdom.

Conservative Mechanisms of Extracellular Trap Formation by Annelida Eisenia andrei: Serine Protease Activity Requirement.

Formation of extracellular traps (ETs) capturing and immobilizing pathogens is now a well-established defense mechanism added to the repertoire of vertebrate phagocytes. These ETs are composed of extracellular DNA (extDNA), histones and antimicrobial proteins. Formation of mouse and human ETs depends on enzymes (i) facilitating decondensation of chromatin by citrullination of histones, and (ii) serine proteases degrading histones. In invertebrates, initial reports revealed existence of ETs composed of extDNA and histones, and here we document for the first time that also coelomocytes, immunocompetent cells of an earthworm Eisenia andrei, cast ETs which successfully trap bacteria in a reactive oxygen species (ROS)-dependent and -independent manner. Importantly, the formation of ETs was observed not only when coelomocytes were studied ex vivo, but also in vivo, directly in the earthworm coelom. These ETs were composed of extDNA, heat shock proteins (HSP27) and H3 histones. Furthermore, the formation of E. andrei ETs depended on activity of serine proteases, including elastase-like activity. Moreover, ETs interconnected and hold together aggregating coelomocytes, a processes proceeding encapsulation. In conclusion, the study confirms ET formation by earthworms, and unravels mechanisms leading to ET formation and encapsulation in invertebrates.

β1,6-branched complex-type N-glycans affect FAK signaling in metastatic melanoma cells.

ABSTRACT Integrin-dependent binding of the cell to extracellular matrix (ECM) is a key activator of the focal adhesion kinase (FAK) signaling pathway. N-glycosylation of integrins affects their interactions with ECM proteins. Using WM266-4 cells with overexpression of β1,6-acetylglucosaminyltransferase V, we showed that β1,6-branched N-glycans increased tyrosine phosphorylation of FAK in metastatic melanoma cells, resulting in enhanced migration on vitronectin (VN). The co-localization of αvβ3 integrin and FAK in focal adhesions of melanoma cells growing on VN indicates their interaction in signal transduction. Melanoma cell migration on VN was mediated by αvβ3 caring overexpressed β1,6-branched structures, important for FAK upregulation.

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.

Earthworm coelomocyte extracellular traps : structural and functional similarities with neutrophil NETs

Invertebrate immunity is associated with natural mechanisms that include cellular and humoral elements, similar to those that play a role in vertebrate innate immune responses. Formation of extracellular traps (ETs) is a newly discovered mechanism to combat pathogens, operating not only in vertebrate leucocytes but also in invertebrate immune cells. The ET components include extracellular DNA (exDNA), antimicrobial proteins and histones. Formation of mammalian ETs depends on enzymes such as neutrophil elastase, myeloperoxidase, the citrullination of histones and protease activity. It was confirmed that coelomocytes—immunocompetent cells of the earthworm Eisenia andrei—are also able to release ETs in a protease-dependent manner, dependent or independent of the formation of reactive oxygen species and rearrangement of the cell cytoskeleton. Similar to vertebrate leukocytes (e.g., neutrophil), coelomocytes are responsible for many immune functions like phagocytosis, cytotoxicity and secretion of humoral factors. ETs formed by coelomocyte analogues to neutrophil ETs consist of exDNA, histone H3 and attached to these structures proteins, e.g., heat shock proteins HSP27. The latter fact confirms that mechanisms of ET release are conserved in evolution. The study on Annelida adds this animal group to the list of invertebrates capable of ET release, but most importantly provides insides into innate mechanisms of ET formation in lower animal taxa.