An evaluation of contaminated estuarine sites using sediment quality guidelines and ecological assessment methodologies.

Toxic contaminants may enter estuarine ecosystems through a variety of pathways. When sediment contaminant levels become sufficiently high, they may impact resident biota. One approach to predict sediment-associated toxicity in estuarine ecosystems involves the use of sediment quality guidelines (ERMs, ERLs) and site-specific contaminant chemistry while a second approach utilizes site-specific ecological sampling to assess impacts at the population or community level. The goal of this study was to utilize an integrated approach including chemical contaminant analysis, sediment quality guidelines and grass shrimp population monitoring to evaluate the impact of contaminants from industrial sources. Three impacted sites and one reference site were selected for study. Grass shrimp populations were sampled using a push-netting approach. Sediment samples were collected at each site and analyzed for metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Contaminant levels were then compared to sediment quality guidelines. In general, grass shrimp population densities at the sites decreased as the ERM quotients increased. Grass shrimp densities were significantly reduced at the impacted site that had an ERM exceedance for chromium and the highest Mean ERM quotient. Regression analysis indicated that sediment chromium concentrations were negatively correlated with grass shrimp density. Grass shrimp size was reduced at two sites with intermediate levels of contamination. These findings support the use of both sediment quality guidelines and site-specific population monitoring to evaluate the impacts of sediment-associated contaminants in estuarine systems.

An evaluation of polycyclic aromatic hydrocarbon (PAH) runoff from highways into estuarine wetlands of South Carolina.

This study investigated the concentrations and potential toxicity of polycyclic aromatic hydrocarbons (PAHs) associated with highway runoff into adjacent estuarine wetlands from road segments representing three levels of average daily traffic (ADT): low (<5,000 ADT), moderate (10,000-15,000 ADT), and high use (>25,000 ADT) based on SC Department of Transportation data. Sediments from three estuarine wetland habitats (tidal creeks, Spartina marsh, and mud flats) adjacent to these road segments were sampled to represent nine highway use class/habitat type combinations. Surficial sediments were collected at 3, 25, and 50 meters from the upland/wetland interface along transects established perpendicular to the road at each site, with additional samples taken from the road berm. Average PAH concentrations, representing 25 compounds, ranged from 3.9 to 11,000 ng/g dry weight. Berm samples had significantly greater total PAH concentrations than samples taken in any of the wetland habitats. Average total PAH concentrations decreased with increasing distance from the road berm within the wetland habitats sampled, but the differences were not statistically significant. Average total PAH concentrations also were not significantly different among the wetland habitats compared. Analysis of PAH profiles indicated that the PAH source was dominated by pyrogenic combustion products rather than from petrogenic sources. This, combined with the presence of dibenzothio-phene, which is a tire oxidation product, indicated that the primary source of PAHs was related to vehicles. Two sites with total PAH concentrations exceeding published bioeffects levels were resampled for bioassay tests using the amphipod Ampelisca verrilli, the polychaete Streblospio benedicti, and the clam, Mercenaria mercenaria, with the first two assays conducted under UV lighting since previous studies had demonstrated enhanced UV toxicity of PAHs for these species. No toxicity was observed in the amphipod or polychaete assays. Toxicity was observed in the juvenile clam assay at one site, possibly due to the combined effects of PAHs and other contaminants present.

The effects of the contemporary-use insecticide (fipronil) in an estuarine mesocosm.

To examine the effects of environmentally realistic fipronil concentrations on estuarine ecosystems, replicated mesocosms containing intact marsh plots and seawater were exposed to three treatments of fipronil (150, 355, and 5000 ng/L) and a Control. Juvenile fish (Cyprinidon variegatus), juvenile clams (Mercenaria mercenaria), oysters (Crassostrea virginica), and grass shrimp (Palaemonetes pugio) were added prior to fipronil in an effort to quantify survival, growth, and the persistence of toxicity during the planned 28-day exposure. Results indicated that there were no fipronil-associated effects on the clams, oysters, or fish. Shrimp were sensitive to the highest two concentrations (40% survival at 355 ng/L and 0% survival at 5000 ng/L). Additionally, the highest fipronil treatment (5000 ng/L) was toxic to shrimp for 6 weeks post dose. These results suggest that fipronil may impact shrimp populations at low concentrations and further use in coastal areas should be carefully assessed.

Origin and distribution of polycyclic aromatic hydrocarbons in surficial sediments from the savannah river.

Surface sediments collected from the Savannah River, located in the southeastern state of Georgia, USA, in June-July 1994 were analyzed for individual polycyclic aromatic hydrocarbons (PAHs). Three subdivisions of the river were identified for the study: upstream from, adjacent to, and downstream from the city of Savannah. There was high spatial variability in the total PAH (SigmaPAH) concentrations that ranged from 29 to 5,375 ng/g with an average concentration of 1,216 +/- 1,161 (SD). Of the three subdivisions, the highest SigmaPAH concentrations were in the middle segment, which was adjacent to urban and industrial areas. To elucidate sources, molecular indices based on indices among phenanthrene versus anthracene and fluoranthene versus pyrene were used to determine pyrogenic and petrogenic sources, respectively. These indices have been used by other authors to differentiate sources. In most cases, PAHs in sediments nearest the city of Savannah were of high temperature and pyrogenic origin. These pyrogenic PAHs were highly associated with toxicity to benthic organisms. The two-ringed naphthalene and substituted naphthalenes, which are petroleum-related PAHs, were significantly higher in the lower section of the river relative to the subdivisions. This river segment receives inputs primarily from shipping and boating traffic. Perylene, which is indicative of nonanthropogenic terrestrial inputs of carbon, had the highest concentration among the individual PAHs measured. High perylene concentrations were found at stations located upstream and adjacent to forested terrain and where salinity level was low. To discriminate pattern differences and similarities of individual PAHs among samples, principal component analysis (PCA) was performed on the more hydrophobic and persistent nonalkylated PAHs. These differences and similarities were used to infer perylene origin. PCA was performed on 14 nonalkylated PAHs that was normalized to the sum of nonalkylated PAHs, using a correlation matrix. Generally, the PAHs were separated into group patterns according to chemical and physical properties associated with log K(OW), except perylene. Perylene, a five-ringed PAH, was distinctly separated from the other five-ringed PAHs. The sources for perylene are likely from biogenic, terrestrial precursors. The collected data show that pyrogenic PAHs were highly associated with biological effects on benthic organisms, based on bioassay results. Perylene, a nonanthropogenic PAH, was found throughout the river and constituted a large percentage of total PAHs in the upper river.

Toxicological studies in tropical ecosystems: an ecotoxicological risk assessment of pesticide runoff in South Florida estuarine ecosystems.

A multiyear study in the C-111 canal system and associated sites in Florida Bay was undertaken to determine the potential pesticide risk that exists in South Florida. After the examination of extensive pesticide concentration data in surface water, tissues, and semipermeable membrane devices (SPMDs), canal contamination seems to be derived from the extensive agricultural production that drains into the C-111 canal. The results of this study indicate that runoff from agricultural processes led to quantifiable pesticide residues in both canal and bay surface water, which occasionally exceeded current water quality criteria. The major pesticide of concern was endosulfan, which was detected at 100% of the sites sampled. Endosulfan exposure did not cause any acute effects in fish and crustaceans deployed in field bioassays. Chronic effects were observed in copepods, clams, and oysters but could not be attributed to endosulfan exposure. The decision to alter the C-111 canal flow and allow increased freshwater flow into the adjacent Everglades National Park may result in discharges of pesticides into the Everglades. Continued monitoring in this area is needed during this change in flow regime.

Current progress in isolation and characterization of toxins isolated from Pfiesteria piscicida.

The isolation and partial purification of toxic substances derived from Pfiesteria piscicida Steidinger & Burkholder extracts is described. Four distinct bioassay systems were used to monitor bioactivity of the P. piscicida extracts, including a high throughput cell cytotoxicity assay and a reporter gene assay as well as assays using brine shrimp and fish. Using these bioassays to guide fractionation, we have isolated two distinct, active fractions from Pfiesteria culture medium and cell mass extracts on the basis of their solubility characteristics. We have identified and characterized a bioactive lipophilic substance from Pfiesteria-derived extracts as di(2-ethylhexyl)phthalate, a commonly used plasticizer. The source of this typically man-made substance has been identified as originating from Instant Ocean (Aquarium Systems, Mentor, OH, USA), a commercially available seawater salt mixture used to prepare our mass culture growth medium. We have developed chromatographic methodology to isolate a bioactive polar compound isolated from extracts of Pfiesteria culture and presently report the characterization of the activity of this substance. The molecular structural analysis of the polar active component(s) using mass spectrometry and nuclear magnetic resonance spectroscopy is currently under way.

The impact of participation in a study of medical treatment of lower urinary tract symptoms on the incidence of prostate surgery.

Is the incidence of prostate surgery influenced by participation in a study of medical treatment of lower urinary tract symptoms (LUTS)? In order to research the possible answers to this question, we established a retrospective, observational study of the 127 patients from Norway who participated in the Scandinavian Reduction of the Prostate Study (SCARP), which was a multicenter, double-blind, placebo-controlled 2-year study to assess the clinical efficacy of 5 mg finasteride in patients with an enlarged prostate and moderate LUTS. In the present study, the ten Norwegian urologists who were involved in SCARP, performed a chart review and recorded all cases of prostate surgery that had taken place during the 2-year SCARP period and the year following SCARP. The incidence of prostate surgery was 19.8 per 1,000 person-years during the SCARP period, versus 127 per 1,000 person-years the following year (P < 0.001). Among the Norwegian participants in SCARP, prostate surgery was performed more frequently after completion of SCARP. Several possible explanations are discussed.

Inhibition of the MAP kinase cascade blocks heregulin-induced cell cycle progression in T-47D human breast cancer cells.

Members of the erbB family of receptor tyrosine kinases are commonly overexpressed in human breast cancer. However, the relative contribution of particular signalling pathways activated downstream of these receptors to the mitogenic response of transformed breast epithelial cells remains poorly characterized. Administration of heregulin-beta2 (HRG), a ligand for erbB3 and erbB4, to growth arrested T-47D human breast cancer cells leads to activation of both the PI3-kinase and MAP kinase signalling pathways and potent stimulation of cell cycle progression. Specific inhibitors were used to assess the role of these pathways in HRG-induced mitogenesis and to identify underlying mechanisms in terms of regulation of gene expression. Treatment with the MEK inhibitor PD98059 led to a complete block of HRG-induced entry into S-phase, whilst administration of the PI3-kinase inhibitor wortmannin resulted in only modest inhibition. In addition, administration of PD98059 8 h after HRG was equipotent with simultaneous administration in inhibiting entry into S-phase. However, delaying addition for 14-16 h after HRG, when the cells were entering S-phase, was without effect. HRG stimulation led to sequential induction of c-myc, cyclin D1, cyclin E and cyclin A gene expression and hyperphosphorylation of the retinoblastoma protein pRB. p21 (WAF1/CIP1/SDI1) gene expression was rapidly induced by HRG, but significant changes in p27 (KIP1) protein levels were not detected. Preincubation with PD98059 blocked the HRG-dependent induction of cyclin D1 mRNA, p21 and c-Myc protein and pRB phosphorylation. These findings demonstrate that MEK activation is critical to HRG-induced S-phase entry in these cells whilst PI3-kinase plays a minor role. Moreover, these data are compatible with HRG-induced activation of MEK being critical for a mid-G1 transition point and implicate c-myc and cyclin D1 as key targets of the MAP kinase pathway involved in this response.

Analysis of Grb7 recruitment by heregulin-activated erbB receptors reveals a novel target selectivity for erbB3.

Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Co-expression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at +2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at +3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.

D-penicillamine causes free radical-dependent inactivation of activator protein-1 DNA binding.

D-Penicillamine (beta, beta-dimethyl cysteine) is an antirheumatic thiol drug with a poorly understood mechanism of action. On the basis that gold(I) thiolates and D-penicillamine are both capable of forming stable bonds with endogenous thiols, we sought a common target of action. Cysteine residues in the basic DNA binding domains of Jun and Fos, members of the activator protein-1 (AP-1) transcription factor family, have been identified as likely targets for the therapeutic action of antirheumatic gold(I) thiolates. The current study demonstrates that AP-1 DNA binding is inhibited by D-penicillamine in the presence of Fenton reagents (Fe2+/EDTA and H2O2) but not with either agent alone. The effect is biphasic, with maximum inhibition in the concentration range of approximately 100-250 microM. Cysteine has qualitatively similar properties, although the effect is less pronounced. In contrast, glutathione and thiomalate do not inhibit AP-1 DNA binding, even in the presence of Fenton reagents. Mutant proteins were used to identify the cysteine residues within the DNA binding domains of Jun and Fos that are essential for the inhibitory action of D-penicillamine. The results suggest that D-penicillamine is distinguished from other thiols by its formation of sulfur-containing radicals capable of inhibiting AP-1 DNA binding by a mechanism involving the cysteine residues of Jun and Fos.

Heregulin (HRG)-induced mitogenic signaling and cytotoxic activity of a HRG/PE40 ligand toxin in human breast cancer cells.

The heregulins (HRGs) are a family of growth factors that bind direction to erbB3 and erbB4 and induce tyrosine phosphorylation of erbB2 via receptor heterodimerization. Since erbB2, erbB3, and erbB4 (erbB2-4) are often overexpressed in human breast cancer cells, we produced recombinant HRGs and a HRG-based ligand toxin to investigate the signaling events triggered by HRGs and the ability of these ligands to specifically target such cells. Recombinant HRG beta 2 stimulated the tyrosine phosphorylation of erbB2-4 in ZR-75-1 human breast cancer cells. This was accompanied by the tyrosine phosphorylation of Shc and the formation of complexes between Shc and the adapter protein Grb2. Complexes were also detected between Shc and erbB2-4. However, GRb2 was detected in erbB2 and erbB4 but not erbB3 immunoprecipitates. Thus, these receptors exhibit mechanistic differences in their coupling to Ras signaling, and HRG beta 2 administration triggers multiple inputs into the Ras signaling pathway, involving receptor-Grb2, receptor-Shc, and Shc-Grb2 complexes. HRG beta 2 addition also stimulated the association of erbB3 with phosphatidylinositol-3-kinase. In accordance with the activation of key mitogenic signaling pathways, HRG beta 2 stimulated the proliferation of MCF-7 and T-47D human breast cancer cells. Moreover, when tested for the ability to stimulate cell cycle re-entry of T-47D cells arrested under serum-free conditions, HRG beta 2 was more effective than insulin, previously the most potent mitogen identified using this system. Finally, a HRG beta 2 PE40 ligand toxin was constructed and found to exhibit cytotoxic activity against human breast cancer cells overexpressing erbB3 alone or in combination with erbB4 and/or erbB2.

Comparative effects of gold on the interactions of transcription factors with DNA.

We have previously hypothesized that a mode of action of the anti-rheumatic gold salt, aurothiomalate (AuTM), is the inhibition of DNA binding by transcription factors. Studies of the progesterone receptor (PR), which has a zinc finger structure in the DNA binding domain, were consistent with this hypothesis (1). Here we show that AuTM also markedly inhibits DNA binding by the transcription factor AP-1 and has less potent effects for AP-2, NF-1 and TFIID.

Breath methane and colorectal cancer.

The association between colorectal cancer and breath methane is controversial. We compared a group of 59 patients with unresected colorectal cancer with a group of control subjects matched for age and sex. We also studied 43 of the cancer patients before and 3-6 months after resection. Sixty-three per cent of the patients with unresected carcinoma and 56% of the control subjects were methane excretors (NS). We found no significant change in methane excretion status after resection. Because recent colonic cleansing has been shown to influence methane production in the colon, 15 breath methane excretors were studied immediately before the start of cleansing, on the day of colonoscopy, and on the 7th day thereafter. Forty per cent became breath-methane-negative on the day of colonoscopy, but all 15 were excretors with a median of 60% of the precleansing concentration on the 7th day. The present study does not confirm an association between breath methane and colorectal cancer. It is unlikely that colonic cleansing procedures influenced the results of this study.

[The role of proteases in the growth, invasion and spread of cancer cells]. / Proteasers betydning for cancercellers vekst, invasivitet og spredning.

Cancer cells show a greater capability than normal cells do to break down proteins in the surrounding tissue. This tissue destruction involving proteolytic enzymes is probably essential for the invasion and metastatic spread of malignant cells. The process takes place through an interplay of proteolytic enzyme systems where plasmin-mediated proteolysis plays an important role. Plasminogen activator activity and receptors for plasminogen activators have been discovered in tumors, mainly in areas with invasive growth and tissue degradation. Patients with malignant diseases often demonstrate abnormalities in their blood coagulation, including hyperaggregability of platelets. Experimental research has shown that therapy with antiplatelet drugs, and prophylaxis with protease inhibitors, can limit spread of tumors.

Studies on components of the contact phase system in patients with advanced gastrointestinal cancer.

The authors have studied components of the contact system in plasma obtained from patients with advanced gastrointestinal cancer. Plasma samples from 118 healthy blood donors served as controls. Plasma prekallikrein (PKK) values, evaluated by chromogenic peptide substrate technique, were significantly decreased in patients with cancer compared with healthy blood donors. High molecular weight kininogen (HMwK) and Hageman factor (FXII) values, assayed by immunochemical techniques, were also decreased in the patients with cancer. The changes of contact factors were most pronounced in patients with liver metastasis. The most striking observation in our study, however, was the elevated inhibitor values in patients with cancer. Alpha-2-macroglobulin (alpha 2-M) and C1 inhibitor (C1INH) values, determined both by functional and immunochemical techniques, were markedly increased in patients with cancer. In conclusion, this study shows that patients with intestinal cancer have reduced values of contact factors and markedly elevated inhibitor values which indicate that development of malignant tumors in the gastrointestinal tract is associated with changes in the contact system of plasma.

A randomized study on hormone-resistant prostatic cancer: estramustine phosphate versus low dose epirubicin with or without medroxyprogesterone acetate. A Norwegian multicenter study.

A prospective randomized study has been carried out in order to compare three different treatment modalities for symptomatic metastatic hormone-resistant prostatic cancer. A total of 79 patients were included. One group was treated with estramustine phosphate, another with Epirubicin plus Medroxyprogesterone acetate (MPA), while the third arm consisted of Epirubicin plus placebo. Best palliation was obtained by the combination of Epirubicin and MPA. This combination also seemed to be associated with the longest response duration.