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In order to categorize the distribution, source, and effects of polycyclic aromatic hydrocarbons (PAHs) in aquatic systems of southern India, chemical and toxicological analyses were performed on surface and core sediments, collected from Adyar river, Cooum river, Ennore estuary, and Pulicat lake near Chennai city. The total PAH concentration in surface sediment ranged from 13 to 31,425ng/g with a mean value of 4320ng/g; the concentration was markedly higher in Cooum river compared to that at other sites. The historical PAH dissemination in core samples in the Cooum river, Ennore estuary, and Pulicat lake ranged from 30 to 31,425ng/g, from 8.6 to 910ng/g, and from 62 to 546ng/g, respectively. Surface sediments were predominantly contaminated with low molecular weight (LMW) PAHs. Historical profiles suggest that PAH contamination in the area is now greater than it had been in the past. PAH accumulation in Pulicat lake was distinct from that at other locations where high molecular weight (HMW) PAHs were predominant. DNA damage in HepG2 cells treated with sediment extracts from different locations showed a good correlation with their respective total PAH levels. Statistical analysis revealed that 3-ring and 4-ring PAHs may synergistically contribute to the genotoxic potency compared to others in sediments. The study also showed that a majority of PAHs in the study area indicated a petrogenic origin. Based on the enrichment and toxicological assessment of PAHs in sediments, Cooum river was shown to suffer the highest biological impairment among the studied water bodies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7-10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4-5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

We recently developed a novel procedure to obtain liver-macrophages in sufficient number and purity using a mixed primary culture of rat and bovine hepatocytes. In this study, we aim to apply this method to the neonatal swine liver. Swine parenchymal hepatocytes were isolated by a two-step collagenase perfusion method and cultured in T75 culture flasks. Similar to the rat and bovine cells, the swine hepatocytes retained an epithelial cell morphology for only a few days and progressively changed into fibroblastic cells. After 5-13 days of culture, macrophage-like cells actively proliferated on the mixed fibroblastic cell sheet. Gentle shaking of the culture flask followed by the transfer and brief incubation of the culture supernatant resulted in a quick and selective adhesion of macrophage-like cells to a plastic dish surface. After rinsing dishes with saline, the attached macrophage-like cells were collected at a yield of 10(6) cells per T75 culture flask at 2-3 day intervals for more than 3 weeks. The isolated cells displayed a typical macrophage morphology and were strongly positive for macrophage markers, such as CD172a, Iba-1 and KT022, but negative for cytokeratin, desmin and α-smooth muscle actin, indicating a highly purified macrophage population. The isolated cells exhibited phagocytosis of polystyrene microbeads and a release of inflammatory cytokines upon lipopolysaccharide stimulation. This shaking and attachment method is applicable to the swine liver and provides a sufficient number of macrophages without any need of complex laboratory equipments.

We report a simple and efficient method to obtain macrophage-like cells from the mixed primary cultures of adult rat liver cells. A parenchymal hepatocyte enriched fraction was prepared from adult rat livers and seeded into culture flasks. After 7 to 10 days of culture, when most hepatocytes were degenerated or transformed into fibroblastic cells, macrophage-like cells vigorously proliferated on the cell sheet. By shaking the flasks, macrophage-like cells were readily detached. Subsequent transfer and incubation in plastic dishes resulted in quick and selective adhesion of macrophage-like cells, while other contaminating cells remained suspended in the medium. After rinsing with saline, attached macrophage-like cells were harvested with 95 to 99% purity, as evaluated by flow cytometry or immunocytochemistry. These cells showed typical macrophage morphology and were strongly positive for markers of rat macrophages, such as ED-1, ED-3, and OX-41, but negative for cytokeratins and alpha-smooth muscle actin. They possessed functional properties of typical macrophages, including active phagocytosis of latex beads, proliferative response to recombinant GM-CSF, secretion of inflammatory and anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells. As more than 10(6) cells can be recovered repeatedly from a T75 culture flask at two to three day intervals for more than two weeks, our procedure might implicate a novel alternative to obtain Kupffer cells in sufficient number and purity without complex equipment and skills., (2010 Elsevier B.V. All rights reserved.)

Prion diseases are fatal neurodegenerative disorders that are caused by infectious agents known as prions. Prions are composed primarily of the pathogenic prion protein isoform, PrP(Sc). Because significant levels of infectivity have been detected in excrement from animals infected with scrapie and chronic wasting disease, studies on the dynamics of PrP(Sc) levels in contaminated soil are needed to assess the possible horizontal transmission of prion diseases. Using protein misfolding cyclic amplification, we developed a sensitive detection method for scrapie PrP(Sc) that is mixed with soil. Our detection method has the advantage of not requiring extraction of PrP(Sc) from soil and could provide a sensitivity 1000 to 10,000 times higher than that obtained with an extraction-based method. In addition, we found that PrP(Sc) levels in experimentally contaminated agricultural soils declined to different extents over the course of a 6-month incubation period. Our method appears to be a very useful technique for monitoring PrP(Sc) levels in soil., (Copyright 2010 Elsevier Inc. All rights reserved.)

Since variant Creutzfeldt-Jakob disease (vCJD) has been suspected to be attributable to the infectious agents associated with bovine spongiform encephalopathy (BSE), it is important to prevent the transmission of pathogenic forms of prion protein (PrP(Sc)) through contaminated feeding materials such as meat and bone meal (MBM). Here, we demonstrate that the Maillard reaction employing a formulation of glucose in combination with sodium hydrogen carbonates effectively reduced the infectivity (approximately 5.9-log reduction) of a scrapie-infected hamster brain homogenate. In addition to a bioassay, a protein misfolding cyclic amplification (PMCA) technique, in which PrP(Sc) can be amplified in vitro, was used as a rapid test for assessing PrP(Sc) inactivation. The PMCA analysis also indicated that the PrP(Sc) level in the infected material significantly decreased following the Maillard reaction. Therefore, the Maillard reaction can be employed for the decontamination of large amounts of byproducts such as MBM.

Abnormal isoform of prion proteins (PrP(Sc)), which are infectious agents associated with prion diseases, retain infectivity after undergoing routine sterilization processes. A sensitive method to detect the infectivity is a bioassay, and it has been used for assessing prion inactivation. However, the result is obtained after several hundred days. Here, protein misfolding cyclic amplification (PMCA) in which PrP(Sc) can be amplified in vitro was applied for assessing prion inactivation by dry heating and autoclaving. Scrapie-infected hamster brains were inactivated under various conditions, and residual infectivity and PrP(Sc) were detected by the bioassay and PMCA, respectively. The PMCA results were in good agreement with those of the bioassay. In samples autoclaved at temperatures below 150 degrees C, while infected mice died in the bioassay, protease-resistant PrP (PrP(res)) signals were detected in the second or third round of PMCA. Three rounds of PMCA require only 6 days, which means that the PMCA method is much faster than the bioassay.

Previous studies in various species have demonstrated that cadmium levels in milk are low and that cadmium is retained in the mammary glands of lactating rodents. The objective of the present study was to examine mammary glands following cadmium exposure during peak lactation. Mice were given 5 microg, 100 microg or 2000 microg cadmium/kg body weight subcutaneously on lactation days 8-10 and mammary glands were dissected on lactation day 11 for histology, quantitative gene expression and mineral analyses. Cadmium exposure induced morphological changes in the lactating mammary gland. A remodelling of the lactating mammary tissue including an increase in fat content, a less active feature of the mammary alveolar epithelial cells and more condensed appearance of the milk alveoli were observed. Although these changes were most prominent in the animals exposed to the highest cadmium dose similar morphological alterations were indicated at the two lower doses. A significant negative dose-response relationship between beta-casein gene expression and exposure of cadmium was demonstrated, while mRNA levels of alpha-lactalbumin were not affected by cadmium treatment. Furthermore, reduced levels of calcium in the mammary glands of the dams exposed to the highest cadmium dose and a positive correlation between calcium and beta-casein were demonstrated. No other differences were detected among the cadmium dose groups in mammary levels of calcium, zinc, iron or copper. In conclusion, our results indicate that cadmium disturbs the function of the lactating mammary gland, which consequently may impair the development of the suckling offspring.

The presence of maize intrinsic and recombinant cry1Ab genes in the gastrointestinal (GI) contents, peripheral blood mononuclear cells (PBMC), and visceral organs of calves fed genetically modified Bt11 maize was examined by PCR in a subchronic 90-day performance study. Samples were collected from six Japanese Black/Holstein calves fed Bt11 maize and from six calves fed non-Bt maize. Fragments of maize zein (Ze1), invertase, chloroplast, and cry1Ab were detected inconsistently in the rumen fluid and rectal contents 5 and 18 h after feeding. The chloroplast DNA fragments of ribulose-1,5-bisphosphate carboxylase/oxygenase and tRNA were detected inconsistently in the PBMC, the visceral organs, and the longissimus muscle, while the cry1Ab gene was never detected in PBMC or in the visceral organs. These results suggest that feed-derived maize DNA was mostly degraded in the GI tract but that fragmented DNA was detectable in the GI contents as a possible source of transfer to calf tissues. These results also suggest that the recombinant cry1Ab genes were not transferred to the PBMC and tissues of calves fed Bt11 maize.