Mortality of horn fly (Diptera: Muscidae) larvae in bovine dung supplemented with loline alkaloids from tall fescue

Larvae of arthropod ectoparasites of livestock, such as the horn fly, Haematobia irritans (L.), may be exposed to acyl-loline alkaloids in dung of ruminant livestock ingesting herbage of the tall fescue (Festuca arundinacea Schreb.)-endophyte association [Neotyphodium coenophialum (Morgan-Jones & W. Gams) Glenn, Bacon & Hanlin comb. nov.]. Biological activity of alkaloid-supplemented bovine dung was assayed by growth, development, and survival of 1st instars of horn fly. An extract from tall fescue seed, containing N-formyl loline (NFL), N-acetyl loline (NAL), and loline (59:21:20 by mass, respectively) caused 100% mortality of horn fly larvae when dung was supplemented at > or = 100 micrograms/g. Probit analysis of data corrected for natural mortality indicated a LD50 of 30 micrograms/g (95% fidicial limits: 20-49 micrograms/g). When horn fly larvae were introduced to dung supplemented with up to 50 microM of acyl-loline derivatives, mortality of larvae varied significantly between alkaloids (P < 0.0001). Probit analysis indicated that NFL [LD50: 34 microM (95% fidicial limits: 3-53 microM)] was more toxic than NAL [LD50: 46 microM (0-83 microM)], and that loline hydrochloride was not toxic.     

Asking for the indicator function of bioassays evaluating soil contamination: are bioassay results reasonable surrogates of effects on soil microflora?

In evaluating the biological effect of solid materials like soil a bacterial contact assay often shows higher sensitivity than elutriate testing. Results of the Bacillus cereus contact assay for some environmental important toxicants are presented in this article. A comparison with another heterotrophic soil bacterium, Arthrobacter globiformis, shows comparable sensitivity. In a bioassay approach organisms at the level of individuals or populations are exposed to soil material to determine the significance of contaminants. An investigation that incorporates community level processes in comparison with toxicity test results provides a better understanding of the indicator function of bioassays. Comparison of soil bioassays (aqueous and solid phase) with ecological parameters demonstrates the problems in predicting ecological effects of soil contamination.     

Lack of transforming activity of fumonisin B1 in BALB/3T3 A31-1-1 mouse embryo cells

The capacity of fumonisin B1 (FB1) to induce morphological transformation of cultured mammalian cells was assessed using BALB/3T3 A31-1-1 mouse embryo cells. FB1 with 90% purity was prepared from Fusarium proliferatum grown on whole corn. Cell growth was not inhibited by 48 hr of exposure at concentrations up to 1000 micrograms/ml. Moderate inhibition was induced by 6 days of exposure. In transformation assays with a 48-hr exposure, increases in transformed foci were observed at some concentrations; however, the responses were not reproducible. Prolonged exposure for up to 4 wk at 10, 100 and 500 micrograms/ml failed to induce increases in transformed foci. Analysis of combined results showed that only the increase induced by a 48-hr exposure at 500 micrograms/ml was significant. A trend test indicated the lack of a dose response for concentrations of 10-1000 micrograms/ml. FB1 seems to lack in vitro transforming activity.     

Primary gastric Burkitt''s lymphoma presenting with c-myc gene rearrangement

A polymerase chain reaction (PCR)-based rapid screening procedure was developed to test individual horn flies, Haematobia irritans irritans (L.), for the presence of a specific nucleotide substitution in the sodium channel gene sequence that has been associated with pyrethroid resistance. By a systematic optimization of reaction conditions and judicious choice of PCR primers differing in DNA sequence by a single nucleotide, we identified pyrethroid-susceptible or resistant sodium channel alleles in individual flies. Laboratory and field populations were examined by both the PCR assay and conventional filter paper bioassays with the pyrethroid cyhalothrin to verify that populations containing greater proportions of individuals with the resistant sodium channel allele DNA sequence also had higher bioassay LC50 values. The PCR assay for resistance alleles gave definitive information on the genotype of an individual fly and detected the presence of heterozygous individuals that might serve as reservoirs of resistance genes in field populations.     

Four new bioactive polybrominated diphenyl ethers of the sponge Dysidea herbacea from West Sumatra, Indonesia

The marine sponge Dysidea herbacea collected from Indonesia yielded four new polybrominated diphenyl ether congeners 2-5 and the known derivatives 1, 6, and 7. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (1H, 13C, COSY, 1H-detected direct and long-range 13C-1H correlations) and mass spectrometric (EIMS) data. All of the compounds were active against the Gram-positive bacteria Bacillus subtilis and the phytopathogenic fungus Cladosporium cucumerinum. The isolated polybrominated compounds were also active in the brine shrimp lethality test. In the latter bioassay, compounds 1 and 6 were the most active with LC50's of 0.96 [SE +/- 0.19] and 0.94 [SE +/- 0.70] microg/mL, respectively.     

Effects of age on cardiovascular responses to adrenaline in man

Sensitivity to toxicants is a major criterion for selecting organisms for bioassay testing. If a sensitive species is also abundant and occupies a role as prey for many other species within a community, then the species become a valuable tool in environmental monitoring. These features apply to larval midge Chironomus petiolatus in freshwater environments of central Chile. The youngest larval instar is the most sensitive and presents the additional feature of lower survival within control arenas, making it more difficult to discern toxicant-related mortality from background mortality. In this work, we perform acute bioassays with the three larval stages of C. petiolatus and K2Cr2O7 as reference toxicant, with the goal of selecting a particular instar as the best bioassay tool using two criteria: sensitivity and background mortality. Sensitivity is evaluated through Monte Carlo estimation of LC50 and background mortality through bootstrap resampling, and a final Bioassay Performance Index as the product of LC50 and background mortality. For this task we developed a new computationally intensive statistical algorithm. Results show that the best bioassay tool is not the youngest and most sensitive instar but an intermediate one.     

Pediatric ureteral ureteral outlet obstruction and obstructive megaureter: observation or operation?

Thirty-nine extracts of 13 plants used traditionally as medicinal by the Ese'ejas were studied in order to determine their cytotoxic effect in the brine shrimp. Infusions showed no toxicity. Those plants that tested positive for methanolic and dichloromethane extracts were assayed for DNA-binding activity. Cytotoxicity was not due to the presence of compounds that interact with DNA. Antimicrobial activity of plants used to treat infectious diseases was also performed for the decoctions. These proved to be active against some of the test microrganisms used in the assay.     

Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia

The increased frequency and distribution of red tides requires the development of high-throughput detection methods for paralytic shellfish toxins (PST). Community ethics also requires that there be a reduced reliance upon the standard mouse bioassay. A biomolecular assay such as the sodium channel 3H-saxitoxin binding assay can satisfy both of these requirements but may be compromised by cross-reactivity with the structurally unrelated tetrodotoxins (TTX). This study utilised the sodium channel assay but also an alternative 3H-saxitoxin binding assay based upon a saxiphilin isoform from the centipede Ethmostigmus rubripes to screen for PSTs. Saxiphilin is a novel transferrin which binds saxitoxin (STX) but differs from the sodium channel in not having any measurable affinity for TTX. A detailed analysis of toxin composition was achieved by high performance liquid chromatography (HPLC). Various crustaceans and molluscs accumulate PSTs and TTX, thus proving useful biomarkers for these toxins in their immediate environment and an ideal challenge to the detection and analysis of PSTs in this presumptive screening program. Also, there has been little investigation of PSTs in invertebrates from the Indian Ocean so this region was selected to extend our knowledge of the distribution of these toxins. 190 crabs and shellfish encompassing 31 species were collected from reefs along the North-West Australian coast and tested for PSTs and TTX by sodium channel and saxiphilin bioassays as well as HPLC. PSTs were detected in 18 species of crabs and shellfish of the 31 species tested. Eight of these species have not been previously described as toxic, these being the crabs Euzanthus exsculptus, Lophozozymus octodentatus, Metopograpsus frontalis, Pilumnus pulcher, Platypodia pseudogranulosa and Portunus pelagicus, and the molluscs Tectus fenestratus and Trochus hanleyanus. By HPLC, only one or both of STX and decarbamoyl-STX was detected in any extract. Some extracts markedly inhibited 3H-saxitoxin binding by the sodium channel but not by saxiphilin. The close agreement between toxin quantification by the PST specific methods of HPLC and the saxiphilin bioassay is indicative that the additional toxicity detected by the sodium channel assay is TTX.     

A pharmacological and behavioural study on beta, beta'-iminodipropionitrile (IDPN)-treated brine shrimp Artemia salina L

The neurotoxic substance beta, beta-iminodipropionitrile (IDPN), which induces hyperkinesia in some mammals, was found to be neurotoxic to the brine shrimp Artemia salina L. (Crustacea, Anostraca) inducing discoordination of limb-movement and sedation of motility. Up to 9 metabolities of IDPN were detected in Artemia, including beta-alanine, cyanoacetic acid, and beta-aminopropionitrile (BAPN). Brine shrimp, especially nauplius (1 day), may be a useful test animal for the biological assay of IDPN.     

Alternatives to the 2-species bioassay for the identification of potential human carcinogens

It is proposed that the standard 2-species rodent cancer bioassay protocol, as perfected by the US National Toxicology Program (NTP), has already fulfilled its most useful role by providing an unequalled carcinogenicity database by which to re-assess the type of carcinogen worthy of definition. Continued use of this resource and time consuming protocol can no longer be justified, except in rare circumstances of high and protracted human exposure to a chemical of unknown carcinogenicity. In those rare instances an enlarged bioassay of three or four test species should perhaps be considered, there being nothing fundamental about the rat/mouse combination. In the large majority of cases, however, a practical estimation of the carcinogenic potential of a chemical can be formed in the absence of lifetime carcinogenicity bioassay data. This can be achieved by its sequential study, starting with an appreciation of its chemical structure and anticipated reactivity and mammalian metabolism. After the shortterm evaluation of a range of additional properties of the agent, including its genetic toxicity, rodent toxicity and tissue-specific toxicity, confident predictions of the genotoxic and/or non-genotoxic carcinogenic potential of the agent can be made. In most situations these predictions will be suitable for framing hazard reduction measures among exposed humans. In some situations it may be necessary to evaluate these predicted activities using limited bioassays, a range of which are considered. Extensions of these limited carcinogenicity bioassays to a standard 2-year/2-species bioassay can only be supported in cases where the non-carcinogenicity of the agent becomes the important thing to define. The US NTP have evaluated the carcinogenicity of approximately 400 chemicals over the past 20 years, at a cost of hundreds of millions of US dollars. The experience gained by that and related initiatives, worldwide, can now be harnessed to classify thousands of priority chemicals as being either probable carcinogens or probable noncarcinogens. That can now be achieved using a fraction of the earlier resources and in a fraction of the time that would be required for the conduct of 2-species bioassays. The comfort factor for one group of people of the order of the present system, coupled to the comfort factor for another group of the delay in carcinogenicity assessment enforced by the present council of perfection, are the two main factors delaying transfer to a streamlined system for assessing the carcinogenic potential of chemicals to humans. A third delaying factor in the need for new and focused test data. Coordinated acquisition of such data could rapidly remove the first two obstacles.     

Heptachlor: toxicity to and uptake by several estuarine organisms

Technical-grade heptachlor (65% heptachlor, 22% trans-chlordane, 2% cis-chlordane, and 2% nonachlor) was tested in 96-hr bioassays to determine its toxicity to estuarine animals. The test organisms and the 96-hr LC50 or EC50s based on measured concentrations in water) are as follows: American oyster (Crassostrea virginica), 1.5 mug/liter; pink shrimp (Penaeus duorarum), 0.11 mug/liter; grass shrimp (Palaemonetes vulgaris), 1.06 mug/liter; sheepshead minnow (Cyprinodon variegatus), 3.68 mug/liter; pinfish (Lagodon rhomboides), 3.77 mug/liter; and spot (Leiostomus xanthurus), 0.85 mug/liter. Analytical-grade heptachlor (99.8% heptachlor) and heptachlor epoxide (99%) were also studied. The analytical-grade heptachlor 96-hr LC50 for pink shrimp and spot was 0.03 mug/liter and 0.86 mug/liter, respectively, while that for pink shrimp exposed to heptachlor epoxide was 0.04 mug/liter. Heptachlor was accumulated and some metabolized to its epoxide by all animals tested. Fish and oysters accumulated heptachlor in their tissues 2,800-21,300 times the measured concentration in water; shrimp, only 200-700 times.     

Detection of an anatoxin-a(s)-like anticholinesterase in natural blooms and cultures of cyanobacteria/blue-green algae from Danish lakes and in the stomach contents of poisoned birds

Ten natural bloom samples of cyanobacteria from the Danish lakes Knud s? (5), Ravn s? (4), and Salten Langs? (1) collected during 1993-1995 were assayed for toxicity by mouse bioassay, for acetylcholinesterase inhibiting activity by a colorimetric method, and for microcystins by enzyme-linked immunosorbent assay. In the mouse bioassay, seven samples were neurotoxic, two were non-toxic and one gave a protracted toxic response. One of the non-toxic and the single protracted toxic sample both contained anticholinesterase activity equivalent to 4 micrograms anatoxin-a(s) g-1. The neurotoxic samples contained equivalents to 20-3300 micrograms anatoxin-a(s) g-1. The highest anticholinesterase activities (equivalent to 2300 and 3300 micrograms anatoxin-a(s) g-1, respectively) were found in samples collected from Lake Knud s? in connection with bird-kills in 1993 and 1994. Small amounts of microcystins (0.1-0.9 microgram g-1) were detected in all samples but one. All Lake Knud s? and Lake Ravn s? samples were dominated by Anabaena lemmermannii, and the Lake Salten Langs? sample by several species of Anabaena. Gel filtration profiles indicated similarity between the toxic component from the Lake Knud s? 1994 bloom with registered bird-kills and anatoxin-a(s) isolated from Anabaena flos-aquae NRC-525-17. Anticholinesterase-producing cultures of A. lemmermannii were isolated from the Lake Knud s? 1993 bloom. These laboratory cultures produced anatoxin-a(s) equivalents of 29-743 micrograms g-1. Other cultures of A. lemmermannii isolated from Lake Knud s? and Lake Ravn s? were hepatotoxic or non-toxic. Dead birds collected from Lake Knud s? during the neurotoxic 1993 Anabaena bloom possibly died from cyanobacterial toxicosis. The stomach contents contained colonies and single trichomes of Anabaena, and anticholinesterase activities equivalent to 2.1-89.7 micrograms anatoxin-a(s) kg-1 body weight and microcystins (53-95 ng kg-1) were also detected.     

Preclinical drug development paradigms for chemopreventives

Preclinical screening studies and animal efficacy testing models currently are used by the National Cancer Institute's chemoprevention drug discovery program to assess and identify chemical agents and natural products that may have the potential to prevent human cancer. Identification of potential cancer preventing agents begins by subjecting each compound to a sequential series of short-term, in vitro prescreens of mechanistic, biochemical assays to provide quantitative data to help establish an early indication of chemopreventive efficacy and to assist in prioritizing agents for further evaluation in longer-term, in vitro transformation bioassays and whole animal models. Promising chemical agents or combinations of agents that work through different inhibitory mechanisms subsequently are tested in well-established, chemically induced, animal tumor models, which include models of the lung, bladder, mammaries, prostate, and skin. These preclinical bioassays afford a strategic framework for evaluating agents according to defined criteria, and not only provide evidence of agent efficacy, but also serve to generate valuable dose-response, toxicity, and pharmacokinetic data required prior to phase I clinical safety testing. Based on preclinical efficacy and toxicity screening studies, only the most successful agents considered to have potential as human chemopreventives progress into clinical chemoprevention trials.     

Bioactive metabolites from Sicilian marine fennel, Crithmum maritimum

Bioactivity-guided fractionation of the lipid extract of Crithmum maritimum using the brine shrimp lethality assay led to the isolation of three bioactive compounds. Two of these are known C17 polyacetylenic metabolites, falcarinol [1] and falcarindiol [2], previously isolated from several species of the Umbelliferae and Araliaceae. The third active principle was identified as O-geranylvanillin [3], an aromatic ether described in the literature as a synthetic compound but unknown as a natural product. Cytotoxic activity of the pure compounds was significant for 1 and 2, much less intense for 3.     

Stability of fumonisins in thermally processed corn products

Little is known about the stability of fumonisins in corn-based foods during heating. This study investigated the effects of canning, baking, and roasting (dry heating) processes on the stability of fumonisins in artificially contaminated and naturally contaminated corn-based foods. All samples were analyzed for fumonisin levels by both a commercial enzyme-linked immunosorbent assay (ELISA) and a high-performance liquid chromatographic (HPLC) method. Canned whole-kernel corn showed a significant (P < or = 0.05) decrease in fumonisins by both ELISA (15%) and HPLC (11%) analyses. Canned cream-style corn and baked corn bread showed significant (P < or = 0.05) decreases in fumonisin levels at an average rate of 9% and 48%, respectively, as analyzed by ELISA. Corn-muffin mix artificially contaminated with 5 micrograms of fumonisin B1 (FB1) per g and naturally contaminated corn-muffin mix showed no significant (P < or = 0.05) losses of fumonisins upon baking. Roasting cornmeal samples artificially contaminated with 5 micrograms of FB1 per g and naturally contaminated cornmeal samples at 218 degrees C for 15 min resulted in almost complete loss of fumonisins.     

Byssotoxin A, a secondary metabolite of Byssochlamys fulva

Byssochlamys fulva, isolated from corn, was grown on nutrient-amended shredded wheat medium for 14 days at 25 C. Crude solvent extract from these cultures was toxic to brine shrimp, chicken embryos, and rats. The extract was slightly inhibitory to the germination of of pea seeds, but was nontoxic to ten species of bacteria and one of yeast. One metabolite was isolated, given the trivial name byssotoxin A, and partially characterized chemically and physically.     

Bioactivity of human growth hormone in serum: validation of an in vitro bioassay

GH, in clinical practice, is determined by RIA, but RIA estimates may not accurately reflect serum GH bioactivity. The available measures of GH bioactivity lack either sensitivity, specificity, or a physiologically relevant end point. The objective of this research was to develop a physiologically relevant GH bioassay which would not only measure the bioactivity of purified GH preparations, but would also have sufficient sensitivity to measure GH bioactivity in human serum. The method consisted of incubating murine 3T3-F442A adipocytes in serum-free medium containing BSA, 14C-glucose, and increasing concentrations of GH or test materials for 24 h, followed by measurement of conversion of glucose to lipid. Interference by nonspecific serum factors was reduced by the addition of 10 micrograms/liter insulin, 25 nM dexamethasone, and 37 nM estradiol to the medium. In the presence of 10 micrograms/liter insulin, 50 micrograms/liter insulin-like growth factor-1 did not alter the ability of GH to suppress lipid accumulation. Epinephrine and glucagon could suppress lipid accumulation but only at concentrations greatly in excess of the physiological range in serum. Twenty two thousand dalton hGH produced dose-dependent suppression of lipid accumulation which was linear between 0.625 and 10 micrograms/liter (r = 0.926; P = 0.0001) with a half-maximal response of 3.0 +/- 0.2 micrograms/liter (n = six experiments). The intra- and interassay coefficients of variation were 7% and 19%, respectively. The assay was specific for GH since addition of human PRL produced suppression of lipid accumulation only at concentrations where contamination of the preparation by GH became a significant factor. ACTH also suppressed lipid accumulation but only at doses of 1000 micrograms/liter or greater. Human placental lactogen and hLH, hFSH, and hTSH did not cross-react with GH in this assay. Addition of human serum did not alter the slope of ED50 of the GH dose-response curve. Pools of serum from prepubertal and pubertal boys and girls, subjects treated with arginine or insulin, a diabetic girl, and a boy with gigantism who had a serum GH content of 80 micrograms/liter by RIA and 40 micrograms/liter by bioassay, produced dose response curves parallel to that of the GH standard curve. Serum from patients with hypopituitarism did not produce significant suppression of lipid accumulation in any assay. Recovery of 5 micrograms/liter GH added to human serum was 94%. Twenty thousand dalton GH also suppressed lipid accumulation in this assay, but was 2-fold less potent than 22,000 dalton GH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Development of a luminescent bioassay for thyroid stimulating antibodies

The hyperthyroidism of Graves Disease (GD) is due to thyroid stimulating antibodies (TSAb) which are thyrotropin (TSH) agonists. They are detected routinely by measuring their ability to inhibit TSH binding to the receptor (TBII), which does not reflect their true biological activity. Current bioassays which measure cAMP by RIA, are not suitable for routine use. We have developed a luminescent bioassay for TSAb, by introducing a cAMP responsive luciferase construct into CHO cells stably expressing the human TSH receptor (TSHR). Clone lulul displays dose dependent TSH response detectable from 10 microU/ml and maximal at 10 mU/ml when a >25 fold increase in light output is obtained. 34 euthyroid sera were tested to determine a reference range, with values >1.5 relative light units (R.L.U.) being considered positive. An international TSAb standard responded in a dose dependent manner with 10 mIU/ml giving an R.L.U. of >10. The assay was adapted to a 96 well format for automatic readout and 100 treated GD samples (50 TBII negative and 50 TBII positive) were tested, 73% being positive. In contrast only 4% of 79 control sera from individuals with Hashimoto's, non-thyroid autoimmunity or multinodular goitre produced R.L.U. >1.5. When 44 of the GD sera were compared in a traditional salt-free bioassay, 61% were positive compared with 75% in the new luminescent assay. In conclusion, we have developed a luminescent bioassay for TSAb, using unfractionated serum which is capable of high throughput suitable for routine use.     

Gibbilimbols A-D, cytotoxic and antibacterial alkenylphenols from Piper gibbilimbum

Fractionation of the petroleum ether extract from the leaves of Piper gibbilimbum collected in Papua New Guinea afforded four new alkenylphenols, gibbilimbols A-D (1-4). The structures of the isolates were elucidated by spectroscopic methods, mainly 1D- and 2D-NMR spectroscopy. Gibbilimbols A-D were found to be toxic to brine shrimp with an LC50 of approximately 5 microg/mL. Gibbilimbols A-D were further found to be cytotoxic toward KB nasopharyngal carcinoma cells (ED50 7.8-2.1 microg/mL). All isolates also showed antibacterial activity toward Staphylococcus epidermidis and Bacillus cereus.     

Scientific rationale for the replacement of an in vivo bioassay for protein drugs with a physico-chemical assay

Recent progress in both protein drug production technologies including biotechnology and protein characterization methodologies has enabled us to mass-produce and extensively characterize highly purified protein drugs. The quality control of these protein drugs, being based on such a background, should be carried out using various modern protein analytical methodologies. One of the points to be considered regarding control strategies for these protein drugs should be the development and standardization of more specific, precise, simple and economical assay methodology. The replacement of existing in vivo bioassays with certain in vitro assays, including physico-chemical assays, is one possible directions along this line. This paper describes strategies for the replacement of an in vivo bioassay for protein drugs with a physico-chemical assay. Such approaches have been applied for recombinant human growth hormone and recombinant human insulin, the potencies of which have been estimated by in vivo bioassay. Scientific rationale for such approaches are also discussed.