Application of bioassays in toxicological hazard, risk and impact assessments of dredged sediments.

Given the potential environmental consequences of dumped dredged harbour sediments it is vital to establish the potential risks from exposure before disposal at sea. Currently, European legislation for disposal of contaminated sediments at sea is based on chemical analysis of a limited number of well-known contaminants for which maximum acceptable concentrations, action levels (ALs), have been set. The present paper addresses the issue of the applicability of in vitro and in vivo bioassays for hazard, risk and local impact assessment of dredged polluted sediments to be disposed of at sea. It discusses how and to what extent selected bioassays can fill in the gaps left open by chemical analysis and the way in which the bioassays may contribute to the present licensing system for disposal. Three different purposes for application were distinguished: the most basic application (A) is a rapid determination of the hazard (potential toxicity) of dredged sediments which is then compared to ALs in a licensing system. As with chemical analysis on whole sediment extracts, the bioavailability of the chemicals is not taken into account. As in vitro assays with sediment extracts are not sensitive to matrix effects, a selection of specific in vitro bioassays can be suitable fast and standardized additions for the licensing system. When the outcome of (A) does not convincingly demonstrate whether the sediment is clean enough or too polluted, further bioanalysis can help the decision making process (B). More aspects of the mostly unknown complex chemical mixtures are taken into account, including the bioavailability and chronic toxicity focusing on ecologically relevant endpoints. The ecotoxicological pressure imposed by the dredged sediments can be quantified as the potentially affected fraction (PAF) based on chemical or biological analysis of levels of contaminants in sediment or biota. To validate the predicted risk, the actual impact of dumped harbour sediments on local ecosystems (C) can be determined using a dedicated set of in vitro and in vivo bioassays as well as bio-indicators selected based on the information obtained from (A) and (B) and on the characteristics of the local ecosystem. Conversely, the local sediment impact assessment (C) can direct fine-tuning of the selection of chemical and bioassay analyses and for setting safe levels in the licensing system. It is concluded that in vitro and in vivo bioassays and biological indicators are useful tools in the process of hazard, ecotoxicological risk and impact assessment of dredged harbour sediments, provided they are consciously chosen and quality criteria for assay performance are defined.

Confounding factors in bioassays with freshwater and marine organisms.

The use of bioassays in ecological risk assessments often raises questions about the causative factors, and insight into the possibility that confounding factors, such as pH or increased ammonia concentrations, might be responsible for the observed toxicity is needed. It was decided to develop a practical approach for the Dutch situation, in which a first screening is carried out based on provisional criteria. In collecting the required data, dozens of experiments were performed, while the scientific literature was searched for additional information. It is concluded that the provisional criteria specified are at present useful tools in interpreting results of bioassays. Depending on the outcome and the aim of the research, it might be necessary to further reduce uncertainties in the interpretation. This might require some additional experiments, using alternative controls or test procedures or altering the composition of the original sample.

Organic contaminants and trace metals in flounder liver and sediment from the Amsterdam and Rotterdam harbours and off the Dutch coast.

Organic contaminants [polychlorinated biphenyls (PCBs), organochlorine pesticides (OCPs), polybrominated diphenylethers (PBDEs), nonylphenols], organotin compounds and trace metals (cadmium, chromium, mercury and zinc) were determined in flounder (Platichthys flesus) liver and sediment from the Amsterdam harbour (North Sea Canal) and Rotterdam harbour (Euromonding) and off the Dutch coast between the Amsterdam and Rotterdam harbour mouths in order to assess the level of contamination in these harbours and to study contamination gradients.

Analysis of a genomic segment of white spot syndrome virus of shrimp containing ribonucleotide reductase genes and repeat regions.

White spot syndrome is a worldwide disease of penaeid shrimp. The disease agent is a bacilliform, enveloped virus, white spot syndrome virus (WSSV), with a double-stranded DNA genome that probably contains well over 200 kb. Analysis of a 12.3 kb segment of WSSV DNA revealed eight open reading frames (ORFs), including the genes for the large (RR1) and small (RR2) subunits of ribonucleotide reductase. The rr1 and rr2 genes were separated by 5760 bp, containing several putative ORFs and two domains with multiple sequence repeats. The first domain contained six direct repeats of 54 bp and is part of a coding region. The second domain had one partial and two complete direct repeats of 253 bp at an intergenic location. This repeat, located immediately upstream of rr1, has homologues at several other locations on the WSSV genome. Phylogenetic analysis of RR1 and RR2 indicated that WSSV belongs to the eukaryotic branch of an unrooted parsimonious tree and, further, seems to suggest that WSSV and baculoviruses probably do not share an immediate common ancestor. The present analysis of WSSV favours the view that this virus is either a member of a new genus (Whispovirus) within the Baculoviridae or a member of an entirely new virus family.

Neisseria gonorrhoeae mutants altered in toxicity to human fallopian tubes and molecular characterization of the genetic locus involved.

In an effort to identify potential cytotoxins expressed by Neisseria gonorrhoeae, we have identified a locus that, when mutated in the gonococcus, results in a significant increase in toxicity of the strain to human fallopian tube organ cultures (HFTOC). This locus, gly1, contains two open reading frames (ORFs) which are likely cotranscribed. ORF1 encodes a polypeptide of 17.8 kDa with a signal sequence that is recognized and processed in Escherichia coli and N. gonorrhoeae. The 15.6-kDa processed polypeptide has been observed in membrane fractions and filtered spent media from cultures of E. coli expressing gly1 and in outer membrane preparations of wild-type N. gonorrhoeae. The gly1 locus is not essential for bacterial survival, and it does not play a detectable role in epithelial cell adhesion, invasion, or intracellular survival. However, a gly1 null mutant causes much more damage to fallopian tube tissues than its isogenic wild-type parent. A strain complemented in trans for the gly1 mutation showed a level of toxicity to HFTOC similar to the level elicited by the wild-type parent. Taken together, these results indicate an involvement of the gly1 locus in the toxicity of N. gonorrhoeae to human fallopian tubes.

High-performance liquid chromatographic determination of the imino acids (opines) meso-alanopine and D-strombine in muscle extract of invertebrates.

A sensitive HPLC method is presented for the determination of the imino acids alanopine and strombine, anaerobic metabolites that are formed in muscle tissue of several species of invertebrates. The separation of alanopine and strombine was achieved using the Alltech OA 2000 cation-exchange column. The analysis of the two opines does not require any complicated derivatization and can be performed in a pH neutralized sulphuric acid solution. The sensitivity of this method is in the range of 100 pmol to at least 10 nmol for both investigated opines. For the first time opines were demonstrated in the bivalves Macoma balthica and Cerastoderma edule.

In vitro invasiveness of CTL clones and in vivo dissemination of CTL hybridomas.

Activated spleen T cells are invasive in hepatocyte and fibroblast cultures, and this property is dominantly expressed in T cell hybridomas. The invasive potential of the hybrids correlates with their capacity to disseminate in vivo. We have used this model to study the invasive and migratory properties of cytotoxic T lymphocytes (CTLs). Two murine CTL clones were highly invasive, independent of their state of activation. CTL hybridomas, derived from one of the clones, were similarly invasive. In vivo, CTL hybridoma cells disseminated to extravascular sites in the liver, kidneys, lungs, ovaria, tubae, uterus, and lymphoid, mesenchymal, and fat tissues. Within 7 to 14 days, 10(6) cells were lethal in 100% of mice. The adhesion molecules CD2, CD8, CD54, L-selectin, and CD49d (VLA-4 and LPAM-1 alpha-chain) were not expressed by all CTL hybridomas and therefore not indispensable for invasion in vitro and dissemination in vivo. In contrast, LFA-1 (CD11a/CD18), CD44, and VLA-6 (CD49f/CD29) were expressed on all hybrids. LFA-1 antibodies inhibited CTL hybridoma invasion in vitro, but antibodies inhibiting CD44-hyaluronate and VLA-6-laminin interaction had no effect. These results suggest that migration of cytotoxic T cells into noninflamed tissues is independent of their activation state and does not require L-selectin, LPAM-1, CD2, and VLA-4.

Putative invasion-specific proteins in mouse T-cell hybridomas that differ in invasive and metastatic potential.

Fusion of invasive, activated T-lymphocytes with non-invasive BW5147 T-lymphoma cells mainly yields highly invasive (HI), highly metastatic T-cell hybridomas. In addition, several non-invasive (NI), non-metastatic hybrids have been obtained, probably due to loss of involved gene(s) by chromosome segregation. Here we have compared a panel of HI and NI hybrids in a search for proteins specifically expressed by either cell type. MAbs were raised against HI hybrids, but out of more than 1,000 none bound exclusively to HI cells. Furthermore, polyclonal rat, rabbit and chicken antisera did not immunoprecipitate specific proteins from total lysates, and the expression of 18 (T-cell) surface markers did not correlate with invasiveness. These results indicated that the number of differences between HI and NI hybridomas was surprisingly small. This notion was confirmed by 2-dimensional gel electrophoresis. Among 1,000 detectable spots, we found only 2 clear-cut differences between HI and NI T-cell hybridomas, whereas multiple differences were found between individual hybrids. One protein (p130) was expressed at much higher levels by HI than by NI hybrids in this panel, whereas the other (p15) was only seen in NI hybrids. These proteins are primary candidates for a role in invasion.

Tumour necrosis factor-alpha stimulates invasiveness of T-cell hybridomas and cytotoxic T-cell clones by a pertussis toxin-insensitive mechanism.

Tumour necrosis factor-alpha (TNF-alpha) stimulated invasion by mouse T-cell hybridomas and cytotoxic T-lymphocyte clones into rat embryo fibroblast monolayers. The effect on these highly invasive cells was limited: invasion was stimulated maximally to 130% of controls. However, when cells were pretreated with pertussis toxin (PT), which inhibits invasion to +/- 20% of controls, a clearcut effect was observed: 400 U TNF-alpha per ml stimulated invasion usually two- to threefold, and sometimes even up to 10-fold. Therefore, experiments were done with PT-pretreated cells. Stimulation was dose dependent and maximal at 200-400 U TNF-alpha per ml. An anti-TNF-alpha monoclonal antibody completely abolished TNF-alpha-induced invasion. The effect was maximal 30 min after addition of cells and TNF-alpha to the monolayer and then declined. TNF-alpha preincubation of T-cell hybridoma cells, but not of fibroblasts, had a similar stimulatory effect, which was also maximal after 30 min. This shows that TNF-alpha acts directly on the T-cell hybridoma cells. Invasive T-cell hybridomas colonize many tissues from the blood similarly as normal T cells. Our data thus suggest that TNF-alpha can stimulate migration of normal T lymphocytes into inflamed tissues and can promote metastasis of malignant T lymphomas. The signals involved are transmitted via a pertussis toxin-insensitive pathway.

Invasiveness in hepatocyte and fibroblast monolayers and metastatic potential of T-cell hybridomas in mice.

Fusion of noninvasive, nonmetastatic BW5147 T-lymphoma cells with normal T-lymphocytes usually resulted in highly invasive and metastatic T-cell hybridomas, apparently due to properties derived from the normal T-cell. Occasionally hybrids arose that were non- or low invasive, probably by loss of relevant genes upon chromosome segregation, since these cells contained much less DNA than highly invasive hybrids. The metastatic potential of 20 representative T-cell hybridomas was tested by tail vein injection in syngeneic mice and cells were found to be either nonmetastatic (NM), low metastatic (LM), or high metastatic (HM). NM hybrids were tumorigenic but did not form metastases and HM hybridomas caused wide-spread metastasis. LM cells formed metastases in a limited number of mice and predominantly in lymphoid tissues. In hepatocyte cultures, NM cell lines were found to be the least invasive, HM cells the most, whereas LM hybrids exhibited intermediate levels. Invasiveness was not only measured in rat hepatocyte cultures but also in rat embryo fibroblast monolayers, and the relative invasive capacity in both model systems correlated well. Pertussis toxin inhibited invasion in both systems to 20-30% of control values. This suggests that the mechanisms of invasion into hepatocyte and fibroblast cultures are at least partially similar and that the fibroblast invasion assay is a relevant model to study aspects of lymphoma metastasis. We conclude that invasive potential is a prerequisite for T-cell hybridomas to colonize tissues from the bloodstream and that a minimum level of invasiveness is necessary for extensive and wide-spread metastasis formation.

Role of mineralizing cartilage in osteoclast and osteoblast recruitment.

Periost-free, live and/or devitalized cartilaginous long bone rudiments of fetal mice were transplanted under the renal capsule of adult syngeneic mice to study the role of cells and intercellular matrix in the recruitment and formation of osteoclasts and osteoblasts, both identified by means of enzyme- and immunohistochemical methods. Live bone rudiments recruited host-derived osteoclasts within 5 days after transplantation. Osteoblasts developed as rapidly as osteoclasts and participated in the modeling of the rudiments into hemopoietic bone marrow containing ossicles. Devitalized bone rudiments, killed before osteoclastic invasion had occurred, did not recruit osteoclasts or osteoblasts, and were not resorbed up till 35 days after transplantation. Co-transplantation of live and devitalized bone rudiments however resulted in osteoclastic resorption of the killed rudiments, starting 9 days after transplantation. Again the live rudiments were modeled into ossicles. Devitalized bone rudiments which had been invaded by osteoclasts before killing and transplantation, did recruit host osteoclasts, but at a slower rate than live rudiments, and depending on the number of resorption sites at the time of transplantation. Osteoblasts were not formed. These data suggest that in developing long bones chondrocyte activity is involved in the recruitment of osteoclasts as well as osteoblasts. Matrix components diffusing from resorbing surfaces seem to be involved in osteoclast recruitment.

Cartilage response to mechanical force in high-density chondrocyte cultures.

High-density cultures of chick embryonic chondrocytes were exposed to intermittent compressive force (ICF) of physiologic magnitude for 24 hours. Proteoglycan synthesis was significantly increased in chondrocyte cultures exposed to ICF as compared with control cultures. Similar effects were found in explants of epiphyseal cartilage. Proteoglycans extracted with guanidine-HCl from cultures exposed to ICF aggregated better with hyaluronic acid than did control cultures, as shown by Sepharose 2B gel chromatography. In addition, the amount of non-extractable proteoglycans was increased in ICF cultures. We conclude that ICF not only increases the synthesis of proteoglycans but also improves the aggregating capacity of proteoglycans and the coherence of proteoglycans with other matrix components. High-density cultures of epiphyseal chondrocytes provide a suitable model to study the processes involved in the perception of and the subsequent cellular response to compressive force by cartilage.