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Trace elements (TEs) remain of significant toxicological concern as many are critical for global decarbonisation. TEs accumulate in sediments so benthic polychaetes (e.g. Hediste diversicolor and Alitta virens) are highly relevant for ecotoxicology. However, ecological/biological differences could influence TE accumulation and biomonitoring suitability. Exploiting multiple sympatric populations (Solent, UK), we measure sediment and tissue concentrations generating EFs (enrichment factors), AEIs (Adverse Effects Indexes) and tissue bioaccumulation factors. We also assess stable isotope compositions to elucidate diet influences. Despite diverse anthropogenic activity in the Solent, the majority of TEs present low levels of sediment contamination at the sites. For Ni, Pb and As, a combination of mean AEIs >1 and some sediment concentrations exceeding SQVs (Sediment Quality Values) indicate a slight toxicological risk. For Cu and Hg, high EFs and AEI scores confirm they are the greatest risk, thus requiring source identification/control. However, only mean As tissue concentrations reflect contaminated sites, therefore, identifying the As-source(s) is also a priority. Sediment and tissue concentration relationships were generally negative and not significant for both species. Although a significant negative relationship for Cd for A. virens requires further investigation, the lack of evidence for TE bioaccumulation from sediment may limit both species' biomonitoring suitability for low-contamination sites. Species differences in tissue concentration were also TE specific: H. diversicolor had significantly higher concentrations for Ag, Cu, Hg, Ni and Zn, whilst the reverse was true for Cd, Fe, Cr and As. Whilst ecological differences and that feeding sources are site and species-specific (as evidenced by C, N and S stable isotopes analysis) cannot be ignored, the diverse tissue concentrations strongly suggest different TE regulation strategies per species. Together these data will be important for ecotoxicologists and regulators to select the 'best' polychaete biomonitor and assess TE toxicity under future global decarbonisation trajectories for TE inputs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Gordon Watson reports financial support was provided by European Union. Gordon Watson reports a relationship with European Union that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Globally, 6 million coral reef fishers provide ~25% of emergent countries' catch, but species have low value. The marine aquarium trade (MAT) targets high-value biodiversity, but missing data amplify draconian governance and demand for international prohibition. To stimulate sustainability and reef conservation investment, we generate a fiscal baseline using the first global analysis of numbers, diversity, and biomass of MAT-traded organisms. Each year, ~55 million organisms worth US$2.15 billion at retail are traded comparable with major fisheries, e.g., tuna. A sustainable MAT also requires overexploitation assessments. We identify 25 species/genera with "Extremely High" risk ratios and place the Indonesian and Sulu-Celebes Seas in the highest exploitation category. Despite predicted hobbyist number increases, unabated reef degradation and low governance will transform the MAT into an aquaculture-dominated industry decoupled from communities (i.e., culture located in importing countries). A "MAT-positive" future requires evidence-based management/governance, consumer education, and sustainable practice incentivization but can address the biodiversity and social and economic inequality crises.

Coastal management requires cost-effective, yet accurate, assessments of habitat condition, especially in areas protected by statutory conservation measures. Unmanned Aerial Vehicles (UAVs) provide alternatives to manned aircraft and walk-over (WO) surveys. To support coastal managers with method selection, we compare the costs and benefits of the three techniques using the extent of bait collection (sediment scarring from manual digging) on intertidal mudflats from three UK sites. UAV and WO surveys were conducted in parallel and aerial photography was downloaded from the Channel Coastal Observatory (CCO). Digging was digitised from estimations on foot (WO) or by manually labelling imagery with confidence assigned (UAV/CCO). Method efficacy is compared with respect to spatial coverage, control over survey time/location, spatial resolution, positioning accuracy, and area of digging detected. Personnel hours and up-front costs (e.g. training/equipment), costs for personnel time standardised by shore area, personnel risk, and environmental impact are also compared. Regarding efficacy, CCO imagery had extensive shore coverage compared to UAV and WO, however, assessments are restricted to times/locations with available imagery. Each method's resolution was sufficient to detect digging. WO achieved the highest resolution (on foot), but the lowest positioning accuracy, in contrast to accurate feature delineation on aerial imagery. An additive two-way ANOVA revealed a significantly higher percent area of 'dug' sediment (all confidence levels) recorded by UAV than WO. CCO was the most cost-effective with no fieldwork/equipment costs. UAV had the highest up-front costs, but WO was more costly for personnel hours/km 2 for survey time and digitisation. For all methods, digitisation was the most time-consuming aspect. Compared to WO, UAV achieved rapid shore surveys and the CCO and UAV methods minimise personnel risks. UAV and WO both cause wildlife disturbance, with trampling an additional WO impact. With each method suited to sediment disturbance assessment, selection will depend on resources and objectives and will be aided by this holistic cost-benefit analysis. Cost-effectiveness will improve with evolving regulations that facilitate UAV use and technological developments (e.g. machine learning for disturbance detection) that could significantly expedite imagery analysis and enable broadscale assessments from CCO or satellite imagery., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The invasive Asian date mussel (Arcuatula senhousia) inhabits diverse global coastal environments, in some circumstances posing significant ecological and economic risks. Recently recorded in the Greater North Sea ecoregion, an established population has not previously been confirmed. Combining historical and field data, we provided baseline information from the UK and recorded colonisation in a variety of habitats. Gonadal development was assessed using the gonadosomatic index (GSI) to determine if an intertidal soft-sediment population is self-sustaining. Arcuatula senhousia records from subtidal muddy/mixed-sediment within a major estuarine system from 2007 to 2016 were also analysed. First detected in 2011, spatial distribution was variable across the years within the subtidal, with individuals found at 4-9 out of 25 sites, and densities per site varying from 10 to 290 individuals per m 2 . The intertidal population was, in part, associated with seagrass (Zostera spp.) and attached to bivalves. In marinas, individuals were attached to concrete tiles, associated with live Mytilus edulis, and to dead Ostrea edulis. Mean GSI from the intertidal population differed across months, peaking in July before declining in September/October, but with high inter-individual variability. Arcuatula senhousia is reproducing and maintaining viable populations. Using a natural capital approach, we identify the potential impacts on Europe's functionally important habitats, fisheries and aquaculture if its spread continues.

Trace elements (TEs) frequently contaminate coastal marine sediments with many included in priority chemical lists or control legislation. These, improved waste treatment and increased recycling have fostered the belief that TE pollution is declining. Nevertheless, there is a paucity of long-term robust datasets to support this confidence. By mining UK datasets (100s of sites, 31 years), we assess sediment concentrations of arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn) and use indices (PI [Pollution], TEPI [Trace Element Pollution] and I geo [Geoaccumulation]) to assess TE pollution evolution. PI and TEPI show reductions of overall TE pollution in the 1980s then incremental improvements followed by a distinct increase (2010-13). Zn, As and Pb I geo scores show low pollution, whilst Cd and Hg are moderate, but with all remaining temporally stable. I geo scores are low for Ni, Fe and Cr, but increasing for Ni and Fe. A moderate pollution I geo score for Cu has also steadily increased since the mid-1990s. Increasing site trends are not universal and, conversely, minimal temporal change masks some site-specific increases and decreases. To capture this variability we strongly advocate embedding sufficient sentinel sites within observation networks. Decreasing sediment pollution levels (e.g. Pb and Hg) have been achieved, but stabilizing I geo and recently increasing TEPI and PI scores require continued global vigilance. Increasing Ni and Fe I geo scores necessitate source identification, but this is a priority for Cu. Local, regional and world analyses indicate substantial 'hidden' inputs from anti-fouling paints (Cu, Zn), ship scrubbers (Cu, Zn, Ni) and sacrificial anodes (Zn) that are also predicted to increase markedly. Accurate TE input assessments and targeted legislation are, therefore, urgently required, especially in the context of rapid blue economic growth (e.g. shipping)., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present Data In Brief methodological paper details the acquisition, mining and pre-processing of elemental concentration data in marine sediments (coastal and open sea) of Southern England, presented and discussed in the co-submitted Environment International paper entitled: "Three decades of trace element sediment contamination: the mining of governmental databases and the need to address hidden sources for clean and healthy seas" [1]. Elemental sediment concentration data were obtained from the two main UK environmental sources, i.e. the Environment Agency (EA) and the Marine Environment Monitoring and Assessment National (MERMAN) database managed by the British Oceanographic Data Centre (BODC). The merged database is the result of a rigorous data selection-validation process and provides spatially and temporally extensive records of arsenic (As), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), mercury (Hg), nickel (Ni), lead (Pb) and zinc (Zn) concentrations for hundreds of sites over 31 years (1983-2013). Additional records of manganese (Mn), aluminium (Al), lithium (Li), tin (Sn) [and tributyltin (TBT)], barium (Ba), antimony (Sb), boron (B), calcium (Ca), molybdenum (Mo), cobalt (Co), selenium (Se), potassium (K), magnesium (Mg), beryllium (Be), vanadium (V), titanium (Ti), sodium (Na), silver (Ag), thallium (Tl) and strontium (Sr) are also included. The full secondary database is hosted in the Mendeley Data repository and the geo-spatial information to map sites is given in supplementary files to the paper. To provide end-users with the relevant context on spatial and temporal coverage, monitoring statistics are given for the nine trace elements (TEs). Site-specific statistics include: the first and last year of sediment monitoring, the number of years monitored, and minimum, maximum, mean and median numbers of years monitored. Also given are summary data on the number of sites monitored each year, from the first records from 1983 to 2013. For the nine TEs (total and strong acid digestion techniques are considered separately for Cr and Fe), monitoring statistics are presented separately for coastal and open sea sites. Data are relevant to diverse end-users to assess the local and regional contaminant loads and to contextualize anthropogenic threats to benthic systems in multiple locations from the French/English Channel, southern North and Celtic Seas., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have or could be perceived to have influenced the work reported in this article., (© 2021 The Authors.)

Understanding metal toxicity to benthic systems is still an ecotoxicological priority and, although numerous biomarkers exist, a multi-biomarker and endpoint approach with sediment as the delivery matrix combined with life-history relevant exposure timescales is missing. Here we assess potential toxicity by measuring a suite of biomarkers and endpoints after exposing the ecologically important polychaete Alitta(Nereis)virens to sediment spiked with environmentally relevant concentrations of copper and zinc (and in combination) for 3, 6 and 9 months. We compared biomarker and endpoint sensitivity providing a guide to select the appropriate endpoints for the chosen time frame (exposure period) and concentration (relevant to Sediment Quality Guidelines) needed to identify effects for benthic polychaetes such as A. virens. Target bioavailable sediment and subsequent porewater concentrations reflect the global contamination range, whilst tissue concentrations, although elevated, were comparable with other polychaetes. Survival reduced as concentrations increased, but growth was not significantly different between treatments. Metabolic changes were restricted to significant reductions in protein after 9 months exposure across all copper concentrations, and reductions in lipid at high copper concentrations (3 months). Significant changes in feeding behaviour and increases in metallothionein-like protein concentration were limited to the medium and high copper and zinc concentrations, respectively, both after 6 months exposure. Despite data highlighting A. virens' metal tolerance, DNA damage and protein concentrations are the most sensitive biomarkers. Copper and zinc cause biomarker responses at concentrations routinely found in coastal sediments that are characterised as low contamination, suggesting a reappraisal of the current input sources (especially copper) is required., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Copper and zinc are metals that have been traditionally thought of as past contamination legacies. However, their industrial use is still extensive and current applications (e.g. nanoparticles and antifouling paints) have become additional marine environment delivery routes. Determining a pollutant's genotoxicity is an ecotoxicological priority, but in marine benthic systems putative substances responsible for sediment genotoxicity have rarely been identified. Studies that use sediment as the delivery matrix combined with exposures over life-history relevant timescales are also missing for metals. Here we assess copper and zinc's genotoxicity by exposing the ecologically important polychaete Alitta virens to sediment spiked with environmentally relevant concentrations for 9 months. Target bioavailable sediment and subsequent porewater concentrations reflect the global contamination range for coasts, whilst tissue concentrations, although elevated, were comparable with other polychaetes. Survival generally reduced as concentrations increased, but monthly analyses show that growth was not significantly different between treatments. The differential treatment mortality may have enabled the surviving worms in the high concentration treatments to capture more food thus removing any concentration treatment effects for biomass. Using the alkaline comet assay we confirm that both metals via the sediment are genotoxic at concentrations routinely found in coastal regions and this is supported by elevated DNA damage in worms from field sites. However, combined with the growth data it also highlights the tolerance of A. virens to DNA damage. Finally, using long term (decadal) monitoring data we show stable or increasing sediment concentrations of these metals for many areas. This will potentially mean coastal sediment is a significant mutagenic hazard to the benthic community for decades to come. An urgent reappraisal of the current input sources for these 'old pollutants' is, therefore, required., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

It is widely accepted that if well managed, the marine aquarium trade could provide socio-economic stability to local communities while incentivising the maintenance of coral reefs. However, the trade has also been implicated as having potentially widespread environmental impacts that has in part driven developments in aquaculture to relieve wild collection pressures. This study investigates the biodiversity in hobbyist aquaria (using an online survey) and those species currently available from an aquaculture source (commercial data and hobbyist initiatives) in the context of a traffic light system to highlight gaps in aquaculture effort and identify groups that require fisheries assessments. Two hundred and sixty nine species including clown fish, damsels, dotty backs, angelfish, gobies, sea horses and blennies, have reported breeding successes by hobbyists, a pattern mirrored by the European and US commercial organisations. However, there is a mismatch (high demand and low/non-existent aquaculture) for a number of groups including tangs, starfish, anemones and hermit crabs, which we recommend are priority candidates for local stock assessments. Hobbyist perception towards the concept of a sustainable aquarium trade is also explored with results demonstrating that only 40% of respondents were in agreement with industry and scientists who believe the trade could be an exemplar of a sustainable use of coral reefs. We believe that a more transparent evidence base, including the publication of the species collected and cultured, will go some way to align the concept of a sustainable trade across industry stakeholders and better inform the hobbyist when purchasing their aquaria stock. We conclude by proposing that a certification scheme established with government support is the most effective way to move towards a self-regulating industry. It would prevent industry "greenwashing" from multiple certification schemes, alleviate conservation concerns, and, ultimately, support aquaculture initiatives alongside well managed ornamental fisheries.

Late gametogenic Nereis virens were incubated for up to 2.5 months in environmentally relevant concentrations of copper-spiked sediment. Sequential extraction confirmed that much more labile copper (in actual and percentage terms) was present as spiked concentrations increased, although the residual fractions contained similar amounts across concentrations. This is also reflected in the tissue concentration of the worms which increased in line with the sediment concentrations. Adult mortality was not dependent on the exposure time, but higher concentrations usually induced greater mortality for both sexes. Oocytes were significantly smaller at higher concentrations although pairwise comparisons did not show specific differences. Spawning of males occurred a number of days earlier in the higher concentrations. Differences in the number of embryos developing normally after in vitro fertilizations of oocytes fertilized with sperm from exposed males and non-exposed males showed that sperm were more susceptible to toxicity, but oocytes were also affected at the highest concentration. These results show that there are direct and indirect reproductive consequences of parental exposure to copper with implications for recruitment and subsequent colonization of polluted sediments for this ecologically and commercially important species., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The marine aquarium industry has great potential to generate jobs in low-income coastal communities creating incentives for the maintenance of a healthy coral reef, if effectively managed. In the absence of current monitoring or legislation to govern the trade, baseline information regarding the species, number and source location of animals traded is missing despite being critical for its successful management and sustainability. An industry assessment to establish the number and provenance of species of ornamental polychaetes (sabellids and serpulids) traded was undertaken across UK wholesalers and retailers. Six geographical regions exporting fan worms were identified. Singapore contributed the highest percentage of imports, but of only one worm "type" whereas Bali, the second largest source, supplied five different worm "types". Over 50% of UK retailers were supplied by one wholesaler while the remainder were stocked by a mixture of one other wholesaler and/or direct imports from the source country. We estimate that up to 18,500 ornamental polychaetes (16,980 sabellids and 1,018 serpulids) are sold annually in the UK revealing a drastic underestimation of currently accepted trade figures. Incorrect identification (based on exporting region or visual characteristics) of traded animals exacerbates the inaccuracy in market quantification, although identification of preserved sabellids using published keys proved just as inconclusive with high within-species variability and the potential for new or cryptic species. A re-description of the polychaete groups traded using a combination of molecular and morphological techniques is necessary for effective identification and market quantification. This study provides the first assessment of ornamental polychaetes but more importantly highlights the issues surrounding the collection of baseline information necessary to manage the aquarium trade. We recommend that future management should be community based and site-specific with financial and educational support from NGOs, local governments and industry members.

Background: Melanoma is an aggressive tumor with a propensity to rapidly metastasize. The PTEN gene encodes a phosphatase with an unusual dual specificity for proteins and lipids. Mutations of PTEN have been found in various human cancers, including glioblastoma, prostate, breast, lung, and melanoma. Here we investigate in vitro the effects of blocking PI3K signaling using adenoviral-delivered PTEN (Ad-PTEN) in cell lines derived from both early- and late-stage melanoma., Materials and Methods: Ad-PTEN transduced melanoma cell lines or normal cells were assayed for cell death, apoptosis, gene expression, invasion and migration, and regulation of angiogenesis., Results: The PTEN locus from RGP and metastatic melanoma cell lines was sequenced; no coding region mutations were found. Adenoviral transfer of PTEN into melanoma cells containing wild-type PTEN alleles led to tumor-specific apoptosis and growth inhibition, with coordinate inhibition of AKT phosphorylation. Ad-PTEN suppressed cell migration by metastatic melanoma cells with concomitant increase in the level of cell surface E-cadherin. Immunohistochemical and confocal analyses localized PTEN to the cytoplasm and demonstrated enrichment at the cell membrane. Ad-PTEN inhibited angiogenesis as demonstrated by the tube formation assay using human vascular endothelial cells., Conclusions: These studies indicate that Ad-PTEN can inhibit tumor cells via multiple mechanisms and has pro-apoptotic, anti-metastatic, and anti-angiogenic properties. Thus, PI3K blockade via Ad-PTEN may be a promising approach for the treatment of early- and late-stage melanoma, even in tumors that do not harbor PTEN mutations.

Background: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines., Materials and Methods: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis)., Results: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers., Conclusions: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

PTEN, a putative tumour suppressor gene associated with prostate and other cancers, is known to be located within the chromosomal region 10q23.3. Transcription of the PTEN gives rise to multiple mRNA species. Analyses by Northern blots, using cell lines which express PTEN together with cell lines which have lost the PTEN or carry a truncated version of the gene, has allowed us to demonstrate that the pseudogene is not transcribed. In addition, 3' RACE studies confirmed that the multiple mRNA species arising from the gene probably result from the use of alternative polyadenylation sites. No evidence for tissue- or cell-specific patterns of transcription was found. Analysis by 5' RACE placed the putative site for the start of transcription around 830 bp upstream of the start codon. A map of the location of the PTEN gene with a series of overlapping YAC, BAC and PACs has been constructed and the relative position of eight microsatellite markers sited. Two known and one novel marker have been positioned within the gene, the others are in flanking regions. The more accurate location of these markers should help in future studies of the extent of gene loss. Several polymorphisms were also identified, all were within introns. Four of the common polymorphisms appear to be linked. In blood, DNA from 200 individuals, including normal, BPH and prostate cancer patients, confirmed this link. Only two samples of 200 did not carry the linked haplotype, both were patients with advanced prostate cancer. It is possible that such rearrangements within PTEN could be evidence of predisposition to prostate cancer in this small number of cases.

Inhibitors of angiogenesis hold potential in the treatment of cancer and other diseases where the disease is caused or maintained by the inappropriate growth of blood vessels. In the present study, a novel inhibitor of angiogenesis was synthesized by covalently linking a nonanticoagulating derivative of heparin, heparin adipic hydrazide (HAH), by an acid-labile bond to the antiangiogenic steroid, cortisol. The rationale was that the heparin derivative, which binds to sulfated polyanion receptors on endothelial cells, should concentrate the steroid on the surface of vascular endothelial cells. Endocytosis of the conjugate and decomposition of the acid-labile linkage inside lysosomes and other acidic intracellular compartments should then lead to release of the cortisol and expression of its antiproliferative activity. Analysis of the stability of HAH-cortisol showed that it was stable at pH 7.4 and broke down rapidly (t1/2 15 min) at pH 4.8 at 37 degrees C. Treatment of murine pulmonary capillary endothelial cells with HAH-cortisol at 10(-5) M (with respect to cortisol) suppressed their DNA synthesis by 50% and inhibited their migration into wounded areas of confluent monolayers. HAH-cortisol at 10(-4) M (with respect to cortisol) did not suppress the DNA synthesis of Lewis lung carcinoma cells. Daily i.p. injections of HAH-cortisol into mice bearing s.c. sponge implants retarded vascularization of the sponge, and injections directly into the sponge abolished vascularization for as long as the injections were continued. Daily i.v. injections of HAH-cortisol at doses causing no apparent toxicity retarded the growth of solid s.c. Lewis lung carcinomas in mice by up to 65%. In all of these assays, equivalent treatments with a mixture of the HAH plus cortisol was significantly less effective. The antiproliferative effect of HAH-cortisol on endothelial cells appeared independent of the glucocorticoid activity of the steroid since HAH conjugated to 5 beta-pregnane-3 alpha,17 alpha,21-triol-20-one, a steroid lacking glucocorticoid or mineralocorticoid activity, was even more effective at inhibiting DNA synthesis by murine pulmonary capillary endothelial cells than was HAH-cortisol. In conclusion, HAH-cortisol represents the prototype of a new class of angiogenesis inhibitors for the treatment of cancer and other angiogenic diseases.

An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [3H]leucine by CEM cells was inhibited by 50% at an M-T151--ricin-A-chain concentration (IC50) of 4.6 nM compared with an IC50 of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC50 values of 20-30 pM. The addition of ricin B chain to CEM cells treated with M-T151--ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly. Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151--ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [3H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.

Biliary radioactivity excretion was studied in 10 patients with postcholecystectomy T-tube drainage after intravenous administration of 3H-1,25-dihydroxyvitamin D3. The mean +/- SD radioactivity excreted in T-tube bile expressed as a percentage of the administered dose was 18.9 +/- 10.7% per 24 hours. After correction for incomplete bile collection the value obtained was 28.8 +/- 12.8%. The mean chloroform solubility of the biliary radioactivity increased from 17.0 +/- 8.4% to 69.4 +/- 15.1% after incubation with beta-glucuronidase. High performance liquid chromatography of chloroform extracts of bile revealed that most of the eluted radioactivity was more polar than 1,25(OH)2D3. The percentage radioactivity eluting as 3H-1,25(OH)2D3 increased from approximately 2.4 +/- 1.9 to 16.2 +/- 8.0 after incubation with beta-glucuronidase. We conclude that significant amounts of intravenously administered 3H-1,25(OH)2D3 are excreted in bile, mostly as more polar metabolites. The increase in free 3H-1,25(OH)2D3 after incubation with beta-glucuronidase indicates that glucuronides of 1,25(OH)2D3 are present in bile.

A monoclonal anti-Thy-1.1 antibody (OX7) was coupled to either native or chemically deglycosylated ricin A-chain (dgA) using one of two different cross-linking agents. One cross-linker, N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), generates a sterically hindered disulfide bond which is relatively resistant to reduction, whereas the other, 2-iminothiolane hydrochloride, generates an unhindered disulfide bond with greater lability. A two-compartment pharmacokinetic model was used to analyze the blood levels of each immunotoxin and its breakdown product (free antibody) after i.v. injection into mice. Immunotoxins prepared with SMPT broke down in vivo 6.3-fold more slowly than those prepared with 2-iminothiolane hydrochloride, and immunotoxins containing native A-chain were cleared 2- to 3-fold more rapidly from the bloodstream than those containing dgA. As a result, 24 h after injection, 16% of the OX7-SMPT-dgA remained in the blood as compared with 0.4 to 2.5% of the other immunotoxins. Immunotoxins prepared with dgA were about 3-fold more toxic to mice than those prepared with native A-chain, whereas immunotoxins prepared with SMPT were only slightly more toxic than those prepared with 2-iminothiolane hydrochloride. When equivalent toxic doses of the immunotoxins were administered i.v. to mice which had been given injections of Thy-1.1+ AKR-A/2 lymphoma cells, the OX7-SMPT-dgA gave the best antitumor effect. A dose equivalent to one-seventh of the median lethal dose extended the survival time of the animals by the extent expected if 99.999% of the tumor cells had been eradicated. Furthermore, the tumors that did develop in the mice treated with OX7-SMPT-dgA were mutants which were resistant to all the immunotoxins. Some of the mutants were deficient in Thy-1.1 whereas others were not. In conclusion, both the use of the SMPT cross-linker and deglycosylation of the A-chain significantly improve the therapeutic index of the immunotoxins in AKR-A/2 tumor-bearing mice.

DNA encoding ricin A chain was derived from preproricin cDNA and ligated into the expression vector pDS5/3. Transcription is controlled from the coliphage promoter PN25 fused with the lac operator of E.coli. When induced, E.coli 71.18 cells transformed with the recombinant plasmid express ricin A chain which is soluble and has full biological activity.

The carbohydrate present on ricin A chain causes ricin A chain immunotoxins to be cleared rapidly in animals by the reticuloendothelial system. In an effort to overcome this problem we destroyed the carbohydrate on ricin A chain by treating it with a mixture of sodium metaperiodate and sodium cyanoborohydride and then linked the "deglycosylated" A chain to monoclonal anti-Thy 1.1 antibody. The deglycosylation procedure did not affect the ability of the A chain component of the immunotoxin to inhibit protein synthesis in a cell-free system or the capacity of the immunotoxin to inhibit protein synthesis in Thy-1.1 positive lymphoma cells in vitro. Immunotoxins prepared with deglycosylated A chain were cleared from the bloodstream of mice more slowly than native ricin A chain immunotoxins. The difference in the blood clearance rates of the two immunotoxins could be accounted for by a decreased entrapment of the deglycosylated ricin A chain immunotoxin by the liver. Both immunotoxins broke down in vivo with the appearance of free antibody in the bloodstream. The site of cleavage of the immunotoxin was possibly the liver because immunotoxins taken up by it rapidly became unreactive with antiricin but retained reactivity with anti-mouse immunoglobulin G suggesting that dissociation of the A chain from the antibody had occurred. The immunotoxins taken up by the liver were metabolized further and the acid insoluble radioactive metabolites gradually accumulated in the stomach, thyroid, and salivary gland. The deglycosylated ricin A chain immunotoxin should be a more effective antitumor agent in vivo because it is cleared from the blood more slowly and so has greater opportunity to localize within the tumor target.

Immunotoxins containing the ribosome-inactivating protein, saporin, are very effective antitumor agents but are highly toxic to mice. They induce severe necrotic lesions in the liver parenchyma of the recipients. Such extensive damage to the liver parenchyma is not observed with ricin A-chain immunotoxins even at 5-fold higher dosage. The hepatotoxicity of the saporin immunotoxins was found in the present study to arise from a combination of two effects. First, saporin and saporin immunotoxins were 30- and 6-fold more toxic to primary cultures of mouse liver parenchymal cells than were ricin A-chain and ricin A-chain immunotoxins, respectively. This was despite the fact that the cells bound 4- to 5-fold less saporin or saporin immunotoxins than ricin A-chain or ricin A-chain immunotoxins. The binding of ricin A-chain and its immunotoxin to the cells was mediated through the carbohydrate residues present on the A-chain whereas saporin is not glycosylated and thus must bind to other sites on the cell surface which result in transport of saporin relatively efficiently to the cytosol. The second reason for the hepatotoxic action of the saporin immunotoxin was that it had a longer blood half-life (t 1/2 alpha = 1.1 h; t 1/2 beta = 17.1 h) than the ricin A-chain immunotoxin (t 1/2 = 0.52 h; t 1/2 beta = 9.7 h). Analyses using a two-compartment pharmacokinetic model showed that the two immunotoxins broke down in vivo to give free antibody at a similar rate (t 1/2 = 10-12 h) but that the ricin A-chain immunotoxin was eliminated 11 times more rapidly than the saporin immunotoxin by routes other than breakdown. It was calculated that, in mice given a median lethal dose of saporin immunotoxin, the blood levels of immunotoxin remained above the concentration that killed 50% of parenchymal cells in vitro for more than 48 h. In mice given a median lethal dose of ricin A-chain immunotoxin, the blood levels fell below the concentration that was toxic to parenchymal cells in vitro within 4 h. The longer blood half-life of the saporin immunotoxin may also explain our previous finding that it had antitumor activity superior to that of a ricin A-chain immunotoxin in mice.

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)tolue ne (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-iminothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.