David Freestone

Antibiotics pollution in Jiulong River estuary: Source, distribution and bacterial resistance

To gain insight into the antibiotic pollution in the Jiulong River estuary and the pollutant sources, we analyzed the concentration of 22 widely-used antibiotics in water samples collected from the river and estuary, 17 and 18 sampling sites, respectively. Contamination with sulfonamides, quinolones and chloramphenicols was frequently detected and the distribution pattern of antibiotics suggested that most of the pollutants are from the Jiulong River, especially from the downstream watersheds. To reveal the ecological effects, we isolated 35 bacterial strains from the estuary and analyzed their antibiotic resistance to the eight most frequently detected antibiotics. The bacteria were subsequently classified into seven different genera by 16SrDNA sequencing. Up to 97.1% of the bacteria showed resistance and 70.6% of strains showed multi-resistance to these antibiotics, especially to sulfonamides. This study demonstrated a pattern of antibiotic contamination in the Jiulong River and its estuary and illustrated high bacterial antibiotic resistance which was significantly correlated with the average antibiotics concentrations and detected frequencies in the estuary.

ATP7B Expression in Human Breast Epithelial Cells Is Mediated by Lactational Hormones

A role for the copper transporter, ATP7B, in secretion of copper from the human breast into milk has previously not been reported, although it is known that the murine ortholog of ATP7B facilitates copper secretion in the mouse mammary gland. We show here that ATP7B is expressed in luminal epithelial cells in both the resting and lactating human breast, where it has a perinuclear localization in resting epithelial cells and a diffuse location in lactating tissue. ATP7B protein was present in a different subset of vesicles from those containing milk proteins and did not overlap with Menkes ATPase, ATP-7A, except in the perinuclear region of cells. In the cultured human mammary line, PMC42-LA, treatment with lactational hormones induced a redistribution of ATP7B from a perinuclear region to a region adjacent, but not coincident with, the apical plasma membrane. Trafficking of ATP7B was copper dependent, suggesting that the hormone-induced redistribution of ATP7A was mediated through an increase in intracellular copper. Radioactive copper (64Cu) studies using polarized PMC42-LA cells that overexpressed mAtp7B protein showed that this transporter facilitates copper efflux from the apical surface of the cells. In summary, our results are consistent with an important function of ATP7B in the secretion of copper from the human mammary gland.

The omega-3 fatty acid, DHA, decreases neuronal cell death in association with altered zinc transport

Docosahexaenoic acid (DHA) is the major polyunsaturated fatty acid in neuronal cell membranes. We hypothesize that DHA induces a decrease in neuronal cell death through reduced ZnT3 expression and zinc uptake. Exposure of M17 cells to DHA‐deficient medium increased the levels of active caspase‐3, relative to levels in DHA‐replete cells, confirming the adverse effects of DHA deficiency in promoting neuronal cell death. In DHA‐treated M17 cells, zinc uptake was 65% less and ZnT3 mRNA and protein levels were reduced in comparison with DHA‐depleted cells. We propose that the neuroprotective function of DHA is exerted through a reduction in cellular zinc levels that in turn inhibits apoptosis.

Diesel exhaust particulate matter induces multinucleate cells and zinc transporter-dependent apoptosis in human airway cells

The cellular effects of biodiesel emissions particulate matter (BDEP) and petroleum diesel emissions particulate matter (PDEP) were compared using a human airway cell line, A549. At concentrations of 25 μg/ml, diesel particulate matter induced the formation of multinucleate cells. In cells treated with a mixture of 80% PDEP:20% BDEP, 52% of cells were multinucleate cells compared with only 16% of cells treated with 20% PDEP:80% BDEP with a background multinucleate rate of 7%. These results demonstrate a causal relation between the formation of multinucleate cells and exposure to exhaust particulate matter, in particular diesel exhaust. Exposure of A549 cells to PDEP induced apoptosis, seen by active caspase‐3 expression and the presence of cleaved pancytokeratin. PDEP exhaust was a much stronger inducer of cellular death through apoptosis than BDEP. There was an eightfold increase in the expression of SLC30A3 (zinc transporter‐3 or ZnT3) in cells exposed to 80% PDEP:20% BDEP compared to untreated cells. The increase in ZnT3 expression seen in apoptotic cells following PDEP suggests a role for this zinc transporter in the apoptotic pathway, possibly through controlling zinc fluxes. As exposure to diesel exhaust particles is associated with asthma and apoptosis in airway cells, diesel exhaust particles may directly contribute to asthma by inducing epithelial cell death through apoptotic pathway.

Bioinformatic and Expression Analyses of Genes Mediating Zinc Homeostasis in Nostoc punctiforme

ABSTRACT Zinc homeostasis was investigated in Nostoc punctiforme. Cell tolerance to Zn2+ over 14 days showed that ZnCl2 levels above 22 μM significantly reduced cell viability. After 3 days in 22 μM ZnCl2, ca. 12% of the Zn2+ was in an EDTA-resistant component, suggesting an intracellular localization. Zinquin fluorescence was detected within cells exposed to concentrations up to 37 μM relative to 0 μM treatment. Radiolabeled 65Zn showed Zn2+ uptake increased over a 3-day period, while efflux occurred more rapidly within a 3-h time period. Four putative genes involved in Zn2+ uptake and efflux in N. punctiforme were identified: (i) the predicted Co/Zn/Cd cation transporter, putative CDF; (ii) the predicted divalent heavy-metal cation transporter, putative Zip; (iii) the ATPase component and Fe/Zn uptake regulation protein, putative Fur; and (iv) an ABC-type Mn/Zn transport system, putative zinc ZnuC, ZnuABC system component. Quantitative real-time PCR indicated the responsiveness of all four genes to 22 μM ZnCl2 within 3 h, followed by a reduction to below basal levels after 24 h by putative ZIP, ZnuC, and Fur and a reduction to below basal level after 72 h by putative CDF efflux gene. These results demonstrate differential regulation of zinc transporters over time, indicating a role for them in zinc homeostasis in N. punctiforme.

Copper transport during lactation in transgenic mice expressing the human ATP7A protein

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.

Reproductive toxic effects of sublethal cadmium on the marine polychaete Perinereis nuntia

To gain insight into the reproductive toxicity of sublethal cadmium on marine polychaetes, Perinereis nuntia sandworms were exposed to cadmium via artificially contaminated seawater. Cadmium influence on sexual maturation, egg laying, fertilization, zygote hatching and vitellogenin expression levels were analyzed. Results indicated that 23.05 and 563.87 microg L(-1) cadmium significantly delayed sexual maturation. Fertilization rate was significantly inhibited by 563.87 microg L(-1) cadmium while lower concentrations showed no significant effect. Zygote hatching was significantly inhibited by cadmium concentrations greater than 1.12 microg L(-1). We identified a vitellogenin gene sequence in P. nuntia and found that mRNA transcription was significantly upregulated by cadmium. These results indicate that sublethal cadmium levels cause dose-dependent reproductive toxicity on P. nuntia by inhibiting sexual maturation, fertilization and zygote hatching, and the increased expression of vitellogenin suggesting cadmium has strong feminization effects on polychaetes.

Comparative analyses of cadmium and zinc uptake correlated with changes in natural resistance-associated macrophage protein (NRAMP) expression in Solanum nigrum L. and Brassica rapa

Environmental context Soils contaminated with metals can pose both environmental and human health risks. This study showed that a common crop vegetable grown in the presence of cadmium and zinc readily accumulated these metals, and thus could be a source of toxicity when eaten. The work highlights potential health risks from consuming crops grown on contaminated soils. Abstract Ingestion of plants grown in heavy metal contaminated soils can cause toxicity because of metal accumulation. We compared Cd and Zn levels in Brassica rapa, a widely grown crop vegetable, with that of the hyperaccumulator Solanum nigrum L. Solanum nigrum contained 4 times more Zn and 12 times more Cd than B. rapa, relative to dry mass. In S. nigrum Cd and Zn preferentially accumulated in the roots whereas in B. rapa Cd and Zn were concentrated more in the shoots than in the roots. The different distribution of Cd and Zn in B. rapa and S. nigrum suggests the presence of distinct metal uptake mechanisms. We correlated plant metal content with the expression of a conserved putative natural resistance-associated macrophage protein (NRAMP) metal transporter in both plants. Treatment of both plants with either Cd or Zn increased expression of the NRAMP, with expression levels being higher in the roots than in the shoots. These findings provide insights into the molecular mechanisms of heavy metal processing by S. nigrum L. and the crop vegetable B. rapa that could assist in application of these plants for phytoremediation. These investigations also highlight potential health risks associated with the consumption of crops grown on contaminated soils.

Copper and lactational hormones influence the CTR1 copper transporter in PMC42-LA mammary epithelial cell culture models

Adequate amounts of copper in milk are critical for normal neonatal development, however the mechanisms regulating copper supply to milk have not been clearly defined. PMC42-LA cell cultures representative of resting, lactating and suckled mammary epithelia were used to investigate the regulation of the copper uptake protein, CTR1. Both the degree of mammary epithelial differentiation (functionality) and extracellular copper concentration greatly impacted upon CTR1 expression and its plasma membrane association. In all three models (resting, lactating and suckling) there was an inverse correlation between extracellular copper concentration and the level of CTR1. Cell surface biotinylation studies demonstrated that as extracellular copper concentration increased membrane associated CTR1 was reduced. There was a significant increase in CTR1 expression (total and membrane associated) in the suckled gland model in comparison to the resting gland model, across all copper concentrations investigated (0-50 μM). Regulation of CTR1 expression was entirely post-translational, as quantitative real-time PCR analyses showed no change to CTR1 mRNA between all models and culture conditions. X-ray fluorescence microscopy on the differentiated PMC42-LA models revealed that organoid structures distinctively accumulated copper. Furthermore, as PMC42-LA cell cultures became progressively more specialised, successively more copper accumulated in organoids (resting<lactating<suckling), indicating a link between function and copper requirement. Based on previous data showing a function for CTR1 in copper uptake, we have concluded that under the influence of hormones and increased extracellular copper levels, CTR1 participates in uptake of copper by mammary epithelial cells, as a prerequisite for secretion of copper into milk.

Copper Levels in Buccal Cells of Vineyard Workers Engaged in Various Activities

OBJECTIVES Copper-based compounds have been used as agricultural fungicides for many years. Their use in Australia is escalating with increase in the scale of planting and associated pest problems. The objective of this study was to identify viticulture activities associated with high exposure to foliage sprays. It would be determined if occupational exposure of vineyard workers to copper-based sprays was associated with raised body copper levels through analysis of saliva and buccal cells. METHODS The activities of six vineyard workers from four vineyards in the Yarra Valley Victoria, Australia, were monitored over a period of 2 years. During this period, workers carried out seasonal activities, including fungicide spraying, canopy management, and tractor operation. Saliva and buccal cells from workers were collected and analysed for copper levels that were then correlated with the different types of vineyard activity. RESULTS The buccal cells of vineyard workers exposed to copper through seasonal activities including fungicide spraying, canopy management, and tractor operation contained copper levels of 0.87, 1.24, and 0.95 ng Cu per 10(6) cells, respectively. This was up to 10-fold higher than the copper levels in buccal cells from the control subjects (0.1 ng Cu per 10(6)). Copper levels in buccal cells from workers participating in other viticulture activities such as shoot thinning, bunch counting, and disbudding were not significantly different from those of control subjects. The levels of copper in saliva samples of both workers undertaking any vineyard activity and control subjects were below the level of detection. CONCLUSIONS Seasonal activities undertaken in vineyards that involved direct contact with copper, in particular canopy management, fungicidal spraying, and tractor operation were associated with high copper levels in buccal cells of workers. This indicates that copper derived from copper-based fungicidal compounds is accumulated within body cells. The lack of detectable copper levels in saliva suggests that the route of transport of copper into buccal cells is not through saliva. The results indicate potential adverse health risks associated with use of copper fungicide. Recommendations are made in relation to the precautions that should be taken in relation to use of copper sprays and to validate buccal cells as an indicator of body copper status.