The potential of recycled materials used in agriculture to contaminate food through uptake by livestock.

The potential for contaminant uptake from recycled materials used in livestock farming, to animal tissues and organs, was investigated in three practical modular studies involving broiler chickens, laying chickens and pigs. Six types of commercially available recycled materials were used either as bedding material for chickens or as fertilizer for cropland that later housed outdoor reared pigs. The contaminants studied included regulated contaminants e.g. polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs, dioxins) and polychlorinated biphenyls (PCBs), but related contaminants such as polybrominated diphenylethers (PBDEs), hexabrominated cyclododecane (HBCDD), polychlorinated naphthalenes (PCNs), polybrominated dioxins (PBDD/Fs) and perfluoroalkyl substances (PFAS) were also investigated. Contaminant occurrence in the recycled materials was verified prior to the studies and the relationship to tissue and egg concentrations in market ready animals was investigated using a weights of evidence approach. Contaminant uptake to animal tissues and eggs was observed in all the studies but the extent varied depending on the species and the recycled material. PCBs, PBDEs, PCDD/Fs, PCNs and PFAS showed the highest potential to transfer, with laying chickens showing the most pronounced effects. PBDD/Fs showed low concentrations in the recycled materials, making it difficult to evaluate potential transfer. Higher resulting occurrence levels in laying chickens relative to broilers suggests that period of contact with the materials may influence the extent of uptake in chickens. Bio-transfer factors (BTFs) estimated for PCDD/F and PCBs showed a greater magnitude for chicken muscle tissue relative to pigs with the highest values observed for PCBs in laying chickens. There were no significant differences between BTFs for the different chicken tissues which contrasted with the high BTF values for pigs liver relative to muscle. The study raises further questions which require investigation such as the effects of repeated or yearly application of recycled materials as fertilizers, and the batch homogeneity/consistency of available recycled materials.

Transfer and uptake of polychlorinated dibenzo-p-dioxins and furans (PCDD/Fs) and polychlorinated biphenyls (PCBs) into meat and organs of indoor and outdoor reared pigs.

This study was designed to investigate how and to what extent PCDD/Fs and PCBs are transmitted from exposure sources to porcine muscle and other tissues derived from pigs. The experimental approach involved two longitudinal studies in which indoor and outdoor pigs were reared to market readiness using typical animal husbandry practices; closely matched samples of soil, feed, bedding, meat, etc. were collected and analysed for PCDD/Fs and PCBs. The total PCDD/F + PCB WHO-TEQs in pig liver were much higher than in meat and kidney samples from the same animals and exceeded the current relevant European Union maximum limits (6 ng PCDD/F-TEQ kg⁻¹ fat). Liver samples were also characterised by much lower PCB contributions to the total TEQ than for the corresponding meat and kidney samples, and by a predominance of many of the hepta- and octa-substituted PCDD/Fs. At ages approaching market readiness, TEQ values in meat samples from outdoor pigs tended to be slightly higher than those from comparable ages in the indoor programme, possibly due to additional intake from soil. Biotransfer factors (BTFs) were derived for each of the 39 PCDD/F and PCB congeners measured. Interpretation of the findings focused particularly on trends in four selected congeners, namely: 2,3,7,8-TCDD, 2,3,4,7,8-PeCDF, PCB 153 and PCB 169. Increases in the BTF for PCB 169 in the pig-rearing programmes were noticed when the diet changed from being dominated by sow's milk to feed. Much higher transfer factors for many of the more heavily chlorinated PCDD/Fs (e.g. 2,3,4,7,8-PeCDF) were found in liver compared with meat or kidney samples from the same animals. Soil consistently accounted for at least 30% of input for many hexa- or higher chlorinated PCDD/Fs, while it rarely representing more than 10% of the total intake.

The assimilation of dioxins and PCBs in conventionally reared farm animals: occurrence and biotransfer factors.

The assimilation of PCDD/Fs and PCBs in chickens, pigs and sheep was investigated in studies using conventional animal husbandry practices. Closely matched samples of muscle (meat), liver, kidneys, eggs, milk, feed, soil and grass were collected of which 105 were analysed. The data obtained were consistent with the PCB and PCDD/F TEQ concentrations to be expected in rural background locations. A slight decline in TEQ values in meat with increasing age was evident in pigs, sheep and broiler chickens. Higher TEQ values in meat from outdoor pigs compared to those raised indoors, and an increase in TEQs in eggs as a result of free-range conditions might be attributable to additional contaminant intakes from soil. TEQ values in samples of sheep meat were slightly higher than those for pigs and chickens and market ready lowland sheep showed higher meat TEQs than the highland species. PCDD/F TEQs were considerably higher in the liver than meat. Contaminant transfer from dietary sources was investigated using biotransfer factors (BTFs) which tended to be higher in chickens than in sheep or pigs. BTFs for all animals declined in magnitude with age, but on average, BTFs for pigs and chickens showed a sharper initial decline than for sheep. The relative magnitude of the BTFs usually followed the order: (highest first) PCB 153, PCB 169, PCB 126, 1,2,3,7,8-PeCDD/2,3,4,7,8-PeCDF and 2,3,7,8-TCDD. This may suggest that higher chlorinated congeners accumulate more readily in meat tissues. Congener-specific BTF variations were found to be associated with variables such as dietary composition during rearing, differences between feed and animal species.

Antibody-induced conformational changes in the Torpedo nicotinic acetylcholine receptor: a fluorescence study.

Quantitative fluorescence spectroscopy was used to develop a structural picture of the effects of two monoclonal antibodies (mAbs) on the conformation of the Torpedo nicotinic acetylcholine receptor (nAChR). The two mAbs (A6 and B1) examined selectively blocked ligand binding to either the high-affinity (A) or the low-affinity (B) binding sites for agonists/competitive antagonists. The distances between dansyl-C6-choline bound to the unblocked agonist/competitive antagonist binding site and one of two lipophilic probes (C12-eosin or C18-rhodamine) partitioned into the lipid membrane were estimated by using fluorescence resonance energy transfer. Control experiments demonstrated that both mAbs decreased the affinity and fluorescence lifetime of receptor-bound dansyl-C6-choline. The binding of the B1 mAb to the B site did not significantly change the calculated distance between the unblocked A binding site and the membrane surface. However, the binding of the A6 mAb to the A site induced the B site to move into close proximity to the lipid membrane. This conformational change was confirmed by a 45-fold increase in the paramagnetic quenching of the B-site-bound dansyl-C6-choline fluorescence by lipid-intercalated 5-doxylstearate. The results indicate that these mAbs not only selectively block ligand binding to either the A or the B acetylcholine sites but also, in the case of the A6 mAb, induce global conformational changes of the receptor, which appear to involve a movement of the B binding site into close proximity of the lipid membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocytes and macrophages outnumber oligodendroglia in normal fish spinal cord.

As shown by staining with a monoclonal antibody against fish CD45, leukocytes are present in very large numbers in the fish central nervous system. Their subtypes were distinguished by electron microscopy and found to include all major hematogenous forms except thrombocytes, the most numerous being tissue macrophages and lymphocytes. As a population, they differ fundamentally from ramified microglia, the restricted form of myeloid cells present in the central nervous system in mammals. They are rare in most grey matter regions but are concentrated in myelinated fiber tracts as well as in certain strata of the radial glial network. The macrophages engulf discarded myelin and outnumber the oligodendrocytes in normal spinal cord white matter, where the density of lymphocytes is > 5000-fold greater than reported in rat.

Diversity amongst the microglia in growing and regenerating fish CNS: immunohistochemical characterization using FL.1, an anti-macrophage monoclonal antibody.

We have immunohistochemically characterized the forms and distribution of microglia--the macrophages of the CNS--in fish, using a new monoclonal antibody (mAb), FL.1. This mAb specifically reacts with resident macrophages throughout the body in Oreochromine fish, including Kuppfer cells, gut-associated myeloid cells, and peritoneal macrophages, as well as with microglia, but circulating monocytes are not labelled with FL.1. The FL.1-epitope, which is lost following treatment with reducing agents, has an extracellular location and is associated with three integral membrane glycoprotein variants. FL.1-staining shows that microglia are extremely abundant throughout the fish CNS. For example, they comprise a third of the glia in the optic nerve, and 30% of all cells, including neurons, in the spinal cord, i.e., fish have about tenfold more microglia than mammals. Two forms of FL.1-positive microglia are predominant in fish, one resembling their mammalian counterparts, but less ramified, and the other comprising smaller rounded cells with very little cytoplasm, which are most numerous in the ependymal region of the optic tectum. Apart from the conventional microglia, the optic nerves also contain large lipid-laden macrophages which comprise a third form of FL.1-positive cell in the CNS. Fish optic nerves contain astrocytes of a distinct type which form reticular networks, but lack connections to capillaries (Maggs and Scholes, J. Neurosci. 1990;10:1600-1614). The co-distribution of foamy macrophages may have a metabolic role that is performed by ordinary astrocytes elsewhere in the CNS. An antiserum against the beta 2 subunit of the human leukocyte integrins (Kishimoto et al., Cell 1987a; 50:193-202) was found selectively to recognize the foamy macrophages in Oreochromis. Following lesion to the optic nerve, FL.1-labelling shows that microglia proliferate throughout the visual pathway. In the optic tectum, the additional FL.1-positive cells are concentrated in the vicinity of degenerating retinal axons and their terminals. Most of the microglia in the injured optic nerve have amoeboid morphologies, and the foamy macrophages become depleted.

Monoclonal antibodies directed against a synthetic peptide corresponding to the alpha-bungarotoxin binding region of the acetylcholine receptor.

Murine monoclonal antibodies have been produced against a 32 amino acid synthetic peptide corresponding to residues 173-204 on the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica. All of the monoclonal antibodies were of the IgM subtype and most cross-reacted with the purified native receptor. None of the antibodies were effective in blocking alpha-bungarotoxin binding to the receptor nor, conversely, did alpha-bungarotoxin interfere with antibody binding. However, two monoclonal antibodies, previously shown to bind near the ligand binding site on the native receptor, did compete partially (50%) with the binding of one of the IgM monoclonal antibodies.

Monoclonal antibodies specific for each of the two toxin-binding sites of Torpedo acetylcholine receptor.

We have isolated and characterized 12 monoclonal antibodies (mAbs) that block the binding of alpha-bungarotoxin (alpha-BuTx) to the acetylcholine receptor (AChR) of Torpedo californica. Two of the mAbs block alpha-BuTx binding completely; the other 10 inhibit only about 50% of the binding. The mAbs that partially inhibit alpha-BuTx binding can be divided into two groups by examination of the additive effect of pairs of mAbs on toxin binding, and by analysis of competition between mAbs for binding to the AChR. These two groups of mAbs, which we have termed A and B, appear to recognize different toxin-binding sites on the same receptor. A and B mAbs were used to determine the kinetic and pharmacological properties of the two sites. The site recognized by A mAbs binds alpha-BuTx with a forward rate constant of 0.98 X 10(5) M-1 s-1, d-tubocurarine (dTC) with a KD of (6.8 +/- 0.3) X 10(-8) M, and pancuronium with a KD of (1.9 +/- 1.0) X 10(-9) M. The site recognized by B mAbs binds alpha-BuTx with a forward rate constant of 9.3 X 10(5) M-1 s-1, dTC with a KD of (4.6 +/- 0.3) X 10(-6) M, and pancuronium with a KD of (9.3 +/- 0.8) X 10(-6) M. Binding of A and B mAbs to the AChR was variably inhibited by nicotinic cholinergic agonists and antagonists, and by alpha-conotoxin. The observed pattern of inhibition is consistent with the relative affinity of the two sites for antagonists as given above but also indicates that the mAbs recognize a diversity of epitopes within each site.

Monoclonal antibodies against plant proteins recognise animal intermediate filaments.

Four monoclonal antibodies were raised against polypeptides present in a high-salt detergent-insoluble fraction from cells of Chlamydomonas reinhardtii. Indirect immunofluorescence microscopy of fibroblasts and epithelial cells grown in culture using these plant antibodies revealed staining arrays identical to those obtained with well characterised antibodies to animal intermediate filaments. Immunofluorescence microscopy of Chlamydomonas with these monoclonal antibodies and a monoclonal antibody that recognises all animal intermediate filaments (anti-IFA) gave a diffuse, patchy cytoplasmic staining pattern. Both the plant antibodies and anti-IFA stained interphase onion root tip cells in a diffuse perinuclear pattern. In metaphase through to telophase, the labelling patterns colocalised with those of microtubules. Labelling of the phragmoplast was also detected but not staining of the preprophase band. On Western blots of various animal cell lines and tissues, all the antibodies labelled known intermediate filament proteins. On Western blots of whole Chlamydomonas proteins, all the antibodies labelled a broad band in the 57,000 Mr range, and three antibodies labelled bands around 66,000 and 140,000 Mr but with variable intensities. On Western blots of whole onion root tip proteins, all the antibodies labelled 50,000 Mr (two to three bands) polypeptides and a diffuse band around 60,000 Mr and three of the antibodies also labelled several polypeptides in the 90,000-200,000 Mr range. The consistent labelling of these different bands by several different monoclonal antibodies recognising animal intermediate filaments makes these polypeptides putative plant intermediate filament proteins.

Chromatin proteins share antigenic determinants with neurofilaments.

Antigenic determinants common to distinct proteins may be unambiguously identified by the use of monoclonal antibodies. Some monoclonal antibodies to mammalian neurofilaments have recently been shown to cross-react with the neurofibrillary tangles found at high density in the brains of senile dements with Alzheimers disease (SDAT). Here, we show that these antibodies also cross-react with chromatin proteins, including the linker histones H1 and H1(0). Elevated levels of histone H1(0) have also been reported in SDAT brains.

All classes of intermediate filaments share a common antigenic determinant defined by a monoclonal antibody.

We have produced a monoclonal antibody that reacts with all classes of intermediate filaments in immunofluorescence assays, including glial filaments in astrocytes, neurofilaments in axons, tonofilaments in epithelial PtK2 cells and intermediate filaments in fibroblasts. It also binds to Z lines in skeletal muscle. In SDS-polyacrylamide gels, the antibody binds to most and perhaps all of the major intermediate filament proteins that have been previously defined, including glial fibrillary acidic protein, the three vertebrate neurofilament proteins (the "neurofilament triplet"), vimentin, desmin, several cytokeratins and the neurofilament proteins of squid and the marine worm Myxicola. In addition, the antibody binds to a protein with an approximate molecular weight of 66,000 that may be a component of all intermediate filaments. These findings suggest that all vertebrate and invertebrate intermediate filament proteins share a common antigenic determinant and raise the possibility that all intermediate filaments contain a 66,000 molecular weight protein.