The effect of lead incorporation on the elemental composition of earthworm (Annelida, Oligochaeta) chloragosome granules

The elemental compositions of chloragosome 'granules' in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes. An in vivo investigation using fully quantitative electron probe X-ray micro-analysis

The elemental compositions of chloragosome "granules" in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1 = uncharacterised; Pool 2 = P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the "control" chloragosomes. In Zn-contaminated chloragosomes, most (approximately 70%) was bound by P-containing ligand(s) but some (less than 1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

The effect of lead incorporation on the elemental composition of earthworm (Annelida,Oligochaeta) chloragosome granules

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted (Dinas Powys) and heavily Pb-polluted (Wemyss) soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. High Pb concentrations were found in chloragosomes of Wemyss animals; Pb was not detected in chloragosomes of Dinas Powys animals. Partial correlation and regression analysis indicated that the in vivo accumulation of Pb by chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Pb is bound by P-containing ligand(s) in the chloragosome matrix. The sequestration of Pb by chloragosomes results in the detoxification of the metal by accumulative immobilization.     

Subcellular localization of polluting metals in roadside earthworms exposed to traffic exhaust gases.

The aim was to make a subcellular localization of metals in tissue from lumbricid earthworms exposed to environmental pollution. Scanning transmission electron microscopy in combination with energy dispersive X-ray microanalysis of tissue fixed in glutaraldehyde only, and with no electron staining, was used. Zinc was registered in the metachromatic mucous granules of the epidermal cells, and zinc, iron and lead in the chloragosomes of the chloragocytes suggesting that metal may be excreted together with the slime or stored in chloragosomes. Relatively few metal nuclear inclusions were encountered probably due to the fact that some metal leaks out during the preparation process. A comparison is made with a chemical analysis of cellular fractions (Talberg, 1977).     

The accumulation and intracellular compartmentation of cadmium,lead, zinc and calcium in two earthworm species (Dendrobaena rubida and Lumbricus rubellus) living in highly contaminated soil

Summary The earthworm Lumbricus rubellus contained more Ca and Zn, and less Pb and Cd, than Dendrobaena rubida living in the same contaminated disused-mine soil. Differences in the kinetics of Ca turnover may account for some of the inter-specific differences in heavy metal burdens, although the calciferous glands do not seem to be directly involved in heavy metal excretion. A comparison of the present findings with published data indicated that the concentration of soil Ca and the bioavailability of heavy metals, both factors being allied to soil pH, are important exogenous determinants of heavy metal accumulation by different earthworm populations. Electron microprobe X-ray analysis of air-dried smears of chloragogenous tissue showed that the metals were fairly specifically compartmentalized into two distinct organelles in both worms: Ca, Pb and Zn were found (associated with P) in the chloragosomes; Cd was found (with S and probably in stoichiometric association) in a more electron-lucent vesicular component, designated the cadmosome, but which may be identical with the debris vesicles which are characteristic inclusions in conventionally-fixed chloragocytes. The in vivo incorporation of Pb by the chloragosomes of D. rubida was accompanied by the loss of Ca, Zn and P.     

Uptake and long-time storage of natural and synthetic dyes by earthworm chloragocytes. In vivo and in vitro investigations

Cationic or anionic dyes adsorbed onto cellulose granulate were transported across the gut wall, bound to blood proteins, and accumulated by the chloragocytes. Solubility in water promoted accumulation. The dyes ended up mainly in the chloragosomes. Down to 20 μmol dye per litre soil water resulted in visible accumulation. Worms which after dye-exposure were kept dye-free for 5 months retained substantial amounts of dye in the chloragosomes. In vitro experiments indicate that the binding to chloragosomes of synthetic and natural phenolics is by ion exchange with calcium phosphate and with an uncharacterized matrix-bound calcium chelator, aided by hydrophobic interactions between the dye and constituents of the chloragosome matrix. The findings are relevant for the evaluation of the effects of constant or periodic soil contamination with industrial or agricultural organochemicals.     

The elemental composition of the chloragosomes of two earthworm species (Lumbricus terrestris and Allolobophora longa) determined by electron probe X-ray microanalysis of freeze-dired cryosections

Summary The elemental composition of the chloragosomes of the two earthworm species, one with complex highly active Ca-secreting calciferous glands (L. terrestris) and the other with non-secretory glands (A. longa), was determined by the electron probe X-ray microanalysis of unfixed freeze-dried cryosections. The predominant constituents of the chloragosomes of both species were P, Ca, Zn and S, with lesser quantities of K, Cl and Fe. The most striking species differences in chloragosomal chemistry were the higher concentrations (expressed as relative mass fractions) of P(×2.1), Ca(×1.5), and Zn(×2.3) in L. terrestris, and the much higher S(×19.6) in A. longa. These differences were discussed in relation to the general ecophysiology of the two species, and more specifically in relation to heavy metal uptake and binding.     

The lysosomes of earthworm chloragocytes: biochemical and morphological characterization

Nine latent and sedimentable acid hydrolases have been detected in the homogenates of earthworm chloragocytes. Their full activity was revealed by treatment with Triton X-100, a Waring blender treatment, freezing and thawing, hypotonic media or incubation at pH 5 and 25 degrees C. Solubilization paralleled the activation of the enzymes. Together with kinetic studies, these results indicate that the acid hydrolases of the chloragocytes are inside typical lysosome-like particles whose membrane is impermeable to their substrates. It could be shown by density equilibration centrifugation that the lysosomes of those cells constitute a heterogeneous population of subcellular particles distinct from the chloragosomes. Moreover, their digestive function has been directly demonstrated by the capture and degradation of serum albumin. The lysosomes of the chloragocytes have been clearly identified as polyvesicular bodies by electron microscopic analysis of the fractions obtained by density equilibration centrifugation and by examination of the whole tissue, as such or after endocytosis of serum albumin or ferritin. Finally, our results do not support a possible relationship between the lysosomes and the chloragosomes of the chloragocytes.     

Determination of some enzymes and macro- and microelements in stallion seminal plasma and their correlations to semen quality

Seminal plasma is very important for sperm metabolism as well as sperm function and survival and transport in the female genital tract. Analysis of enzyme activities and concentrations of elements can estimate integrity and function of sperm cell membranes. In man much data are available about biochemical analyses of seminal plasma. However, not many studies have been conducted in horses yet. We collected ejaculates from 72 stallions, measured the volume, obtained seminal plasma by centrifugation and examined spermatozoa with light microscopy for motility, concentration, for dead sperm and morphology. Of seminal plasma fluid, we measured activities of aspartate-amino-transferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AlP), acid phosphatase (AcP) and lactate-dehydrogenase (LDH) as well as concentrations of sodium (Na(+)), potassium (K(+)), total and ionised calcium (Ca(TOTAL)/Ca(2+)), magnesium (Mg(2+)), phosphate (P), chloride (Cl), copper (Cu), iron (Fe) and zinc (Zn). In addition, correlations among different parameters in light microscopy and seminal plasma were statistically examined by using the Spearman rank correlation coefficient. Median enzyme activities for AST, GGT, AlP, AcP and LDH were 80.0, 7,500, 30,200, 20.0, 81.0 IU/L, respectively. Concentrations of Na(+), K(+), Ca(TOTAL), Ca(2+), Mg(2+), P, Cl were 110.5, 22.1, 2.9, 1.7, 3.1, 1.1 and 114.5 mmol/L, and of microelements Cu, Fe and Zn were 17.8, 1.9 and 13.2 micromol/L, respectively. Furthermore, we found significant correlations between semen volume as well as sperm concentration and AST, GGT, AlP, AcP and LDH as well as Fe and Zn. This made us propose a primary testicular and epididymal origin of these parameters. Significant correlation between GGT and motility may be a sign for its function for cell protection against free radicals. LDH activity significantly correlates with motility and progressive motility, live:dead-ratio and pathomorphology. In our study, LDH seems to be the most predictive enzyme for semen quality. This is the first report about GGT, AcP and LDH activities as well as iron in equine seminal plasma.     

Scanning electron-microscopic study of the involvement of coelomic cells in earthworm antibacterial defense

Summary The first steps of an antibacterial reaction in the earthworm Eisenia fetida andrei were investigated. The main cellular mediators of this activity are the chloragocytes, a class of free coelomocytes existing only in annelids. Our observations using scanning electron microscopy have shown that chloragocytes were able to agglutinate and perhaps to destroy pathogenic bacteria such as Bacillus megaterium in the same way that they agglutinate and lyse vertebrate erythrocytes. Bacteria known to be non-pathogenic for the worm, such as Acinetobacter, were not agglutinated but slowly eliminated by segregation into brown bodies. Chloragocytes maintained in vitro, lost their chloragosomes and exhibited stronger agglutination activity against pathogenic bacteria than chloragocytes in situ. From this increased efficiency of chloragocytes in vitro, we infer that, in normal living conditions, chloragocytes probably intervene in antibacterial defense mainly after their extrusion from the coelomic cavity and their spreading and degranulation at the surface of the integument.     

The composition of pyrophosphate heavy metal detoxification granules in barnacles

A major detoxification mechanism involving the binding of excess metals to inorganic, insoluble metalliferous granules occurs in many invertebrates. X-ray microanalysis of granules extracted from the barnacles Elminius modestus Darwin and Semibalanus balanoides (L.) (Crustacea: Cirripedia) indicated that one granule type was predominant. Examples of this granule type usually contained Fe and P, or Zn and S, or a combination of all four elements as major constituents. The predominant form of P in the granules was pyrophosphate, P₂O⁴⁻₇, with oprthophosphate and other polyphosphates present in lower amounts. The S was present as organic S. Biochemical analysis showed high levels of Ca, Fe, Mg, K and Zn in the granules with Cu and Ag present in smaller quantities. Considerable variation was shown between granules.     

The occurrence and localisation of heavy metals and glycogen in the earthwormsLumbricus rubellus andDendrobaena rubida from a heavy metal site

Summary The lead content of whole earthworms, highest in contaminated site specimens, was significantly higher inDendrobaena rubida thanLubricus rubellus and a species difference in zinc was also recorded. Selective feeding and differential absorption are discounted and a species difference in maximum tolerance to body lead is suggested. Copper was low in all specimens. Chloragocytes and intestinal tissue showed significantly higher lead levels in contaminated earthworms than in control material. Ultrastructurally, chlorgocytes from contaminated earthworms showed electron dense flecks associated with the chroragosome peripheries and within the debis vesicles. Very fine flecks occurred in the nuclei, but mitochondria and Golgi were indistinguishable from control material. Preliminary X-ray microanalysis of contaminated chloragocytes revealed lead and phosphorus. The deposits within the chloragocytes might represent unbound lead precipitated by phosphate buffer; flecks being absent from contaminated, citrate buffered material and from control material. The chloragosomes are proposed as possible sites for sequestered lead.Chloragocyte and intestinal glycogen levels were significantly higher in control material where the chloragocyte cytoplasm was rich in -glycogen rosettes, these being absent from lead contaminated cells. The glycogen-lead correlation suggests that the metabolism of contaminated chloragocytes is directed towards lead sequestration, though differing nutritional states cannot be ignored.The assistance of Mr. P.M. Webster, University of Keele, is gratefully acknowledged.     

Quantitative assessment of lysosomal size, number and enzyme activity in mouse kidney during maturational development.

Image and cytochemical analyses were undertaken to determine possible correlation between the number and size of acid phosphatase-positive granules (lysosomes), and variation in acid phosphatase (AcP) activity in the proximal tubule cells of mouse-kidney during growth and development. Eighteen ddY strain mice ages: 1 day, 1 and 2 weeks, and 1, 2 and 10 months were used. The lanthanide-based method for the ultrastructural localization of AcP-activity was employed. The number and size of AcP positive granules were quantitatively analyzed by image analysis, and AcP activity by X-ray microanalysis. Significance was evaluated by 2-tailed-Student's t-test for the difference between means. AcP activity was observed in the lysosomes and the reaction product appeared dense and heterogeneous. In some cells, it appeared apparently homogeneous. The results showed that the number and size of AcP Positive granules (lysosomes) increased significantly from the first day after birth, recorded a peak in one week time and thereafter, it gradually declined until the 10th month. The result of X-ray microanalysis demonstrated a variation in accordance with the degree of AcP activity at different ages of the animals studied. The AcP activity decreased significantly from day one and progressively until the 10th month. From the results of the present work, it could be inferred that the changes in size and number of AcP positive granules, at least, at the early stage, and/or the variation in AcP activity are related to the growth and development of the animal.     

The Content Levels of Chemical Elements of Plants in the Castanozems Typical Steppe Zone of Nei Monggol

The contents of N, P, K, Ca, S, Fe, Al, Si, Na, of 122 plant species in the XiLin River valley in Nei Monggol were studied. These results have shown as follows: 1. The contents of N, P. K, Ca, S, Fe, Al, Si, Na of 122 plants belong to three levels: N, K, Ca are 101-100(%) P, S, Si, Na are 100-10-1(%); Fe, A1 are 10-1-10-2(%). 2. The contents of chemical elements of 122 plants were divided into three levels (high, middle and low content). The range of the content and the number of plants in each level were obtained.     

Intracellular inclusions of uncultured magnetotactic bacteria.

Magnetotactic bacteria produce magnetic crystals in organelles called magnetosomes. The bacterial cells may also have phosphorus-containing granules, sulfur globules, or polyhydroxyalkanoate inclusions. In the present study, the ultrastructure and elemental composition of intracellular inclusions from uncultured magnetotactic bacteria collected in a marine environment are described. Magnetosomes contained mainly defect-free, single magnetite crystals with prismatic morphologies. Two types of phosphorus-containing granules were found in magnetotactic cocci. The most common consisted of phosphorus-rich granules containing P, O, and Mg; and sometimes also C, Na, Al, K, Ca, Mn, Fe, Zn, and small amounts of S and Cl were also found. In phosphorus-sulfur-iron granules, P, O, S, Na, Mg, Ca, Fe, and frequently Cl, K, and Zn, were detected. Most cells had two phosphorus-rich granules, which were very similar in elemental composition. In rod-shaped bacteria, these granules were positioned at a specific location in the cell, suggesting a high level of intracellular organization. Polyhydroxyalkanoate granules and sulfur globules were less commonly seen in the cells and had no fixed number or specific location. The presence and composition of these intracellular structures provide clues regarding the physiology of the bacteria that harbor them and the characteristics of the microenvironments where they thrive.     

Zinc sequestration by earthworm (Annelida: Oligochaeta) chloragocytes

Summary The elemental compositions of chloragosome granules in the earthworm Lumbricus rubellus living in non-polluted and heavily Zn-polluted soils were determined by fully quantitative electron probe X-ray microanalysis. P, Ca, S and Zn were the major elemental components of the chloragosomes. The in vivo accumulation of Zn by the chloragosomes was accompanied by diminished chloragosomal Ca concentrations. Zn was apparently bound by at least two ligand pools (Pool 1=uncharacterised; Pool 2=P-containing ligands, binding approximately 45% and 55% of the Zn, respectively) in the control chloragosomes. In Zn-contaminated chloragosomes, most (70%) was bound by P-containing ligand(s) but some (<1%) was also bound by S-containing ligands. It is suggested that the sequestration of Zn in chloragosomes results in the detoxification of the metal by accumulative immobilisation.     

Studies on the Characteristic of Element Contents in the Dominant Plant Species of the Three gorges Region in China

The characteristics of the contents of 20 elements (Al, Fe, Mn, Ca, Mg, K, S, Si, P, Cd, Cu, Zn, Ti, Ni, Sr, Mo, Na, B, Cr, V) in 16 plant species collected from the Three Gorges Region in China were investigated. The average contents of Ca, K and Mg were higher than 1 000 μg·g-1, that of Al, P, Si, Fe, S and Mn ranged between 100—1 000 μg·g-1 and Ti, Cu, Ni, Cr, Mo, Cd and V were less than 10 μg·g- 1. The level of Na content was less than that of the reported. The main character of the element contents was of the Ca> K type. The contents of P, S, Ca and K in different plant samples showed a normal distribution pattern, while Al and Mn showed a elements lognormal distribution pattern. Plant species differed greatly in the element contents. On analyzing the coefficient of variation (C. V., % ), Al, Mn, Mg, Ni, Sr and Fe had higher C.V., while the C.V. of K, S, P, Cr, Cd and Cu was less than 60%, and Cu had the lowest C.V. The correlations between Al and Fe, Al and Ti, Al and Cr, A1 and V, Cd and Sr, Cd and Mo, Fe and V, Zn and Cr, Ni and Sr, Mg and Ni, Mo and Sr, Ca and Sr, Cr and Mo, Na and Mg, Na and P, P and S were statistically significant in different plant species. The classification of the 16 plant species and 20 dements by two-way indicator species analysis (TWINSPAN) method may suggest the difference in dement contents of the different plant species.     

Mass spectrometry imaging (MSI) of metals in mouse spinal cord by laser ablation ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been developed as a powerful MS imaging (MSI) tool for the direct investigation of element distributions in biological tissues. Here, this technique was adapted for the analysis of native mouse spinal cord cryosections of 3.1 mm × 1.7 mm by implementing a new conventional ablation system (NWR-213) and improving the spatial resolution from 120 μm to 65 μm in routine mode. Element images of the spinal cord are provided for the first time and the metalloarchitecture was established using a multimodal atlas approach. Furthermore, the spatial distribution of Rb was mapped for the first time in biological tissue. Metal concentrations were quantified using matrix-matched laboratory standards and normalization of the respective ion intensities to the average (13)C ion intensity of standards and samples as a surrogate of slice thickness. The "butterfly" shape of the central spinal grey matter was visualized in positive contrast by the distributions of Fe, Mn, Cu and Zn and in negative contrast by C and P. Mg, Na, K, S and Rb showed a more homogenous distribution. The concentrations averaged throughout grey matter and white matter were 8 and 4 μg g(-1) of Fe, 3 and 2 μg g(-1) of Cu, 8 and 5 μg g(-1) of Zn, 0.4 and 0.2 μg g(-1) of Mn. The carbon concentration in white matter exceeded that of grey matter by a factor of 1.44. Zn and Cu at 9 and 4 μg g(-1), respectively, were particularly enriched in the laminae I and II, in line with the high synaptic and cellular density there. Surprisingly Zn but not Cu was enriched in the central channel. Rb occurred at 0.3 μg g(-1) with a distribution pattern congruent to that of K. The coefficients of variation were 6%, 5%, 8% and 10% for Fe, Cu, Zn and Mn, respectively, throughout three different animals measured on different days. These MSI analyses of healthy wild type spinal cords demonstrate the suitability of the established techniques for investigating diseased or transgenic states in future imaging studies.     

Isolation,purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris

Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 m size range. Hydrolase measurements showed that -D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as -D-galactosidase and -D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.     

Vertical distribution of elements in cells and matrix of epiphyseal growth plate cartilage determined by quantitative electron probe analysis

Quantitative electron probe analysis was performed on chick epiphyseal growth cartilage prepared by two anhydrous methods, ultrathin cryosections and freeze-dried epoxy-embedded tissue. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm, mitochondria, extracellular matrix, matrix vesicles, and mineral nodules in four zones of the cartilage--proliferative, prehypertrophic, early hypertrophic, and early calcification. The exceptionally high levels of Na and K (up to 550 and 200 mmol/kg wet wt, respectively) found in the matrix are believed to be largely bound to fixed anions. Within cells, Na was higher than K (140 versus 20-34 mmol/kg wet wt), a condition that may reflect hypoxia. Ca and P were low in cells and unmineralized matrix. Ca and P were high in mitochondrial granules of the early hypertrophic zone and diminished in amount in the calcifying zone; the converse occurred in matrix vesicles. Mg was low to undetectable except in heavily mineralized structures (i.e., mitochondrial granules, matrix vesicles, and mineral nodules). S levels were high in matrix (approximately 400 mmol/kg wet wt) and increased slightly with maturation. The amount of S present greatly exceeds Ca levels and implies that sulfate, the predominant form of sulfur in proteoglycans, may serve as an ion-exchange mechanism for the passage of Ca through the matrix to sites where Ca and phosphate are precipitated.