Cytotoxicity Bioassay Research Articles

Identification of norharman as the cytotoxic compound produced by the sponge (Hymeniacidon perleve)‐associated marine bacterium Pseudoalteromonas piscicida and its apoptotic effect on cancer cells

Marine invertebrates harbour a wealth of micro-organisms in their bodies. Most of these micro-organisms can catabolize antibiotic compounds as chemical-defence compounds. These compounds not only play an important protective role for their producer and for their hosts, but also have high potential in medicinal applications. In order to discover natural anticancer products, 29 marine bacterial strains have been isolated from the sponge Hymeniacidon perleve, samples of which were collected from the intertidal zone during low tide off Nanji island in Eastern China. By means of a cytotoxicity bioassay, one strain, NJ6-3-1, with significant cytotoxic activity, was selected for culture in a 30-litre fermentation tank. The major cytotoxic compound in the metabolites of NJ6-3-1, separated by means of a bioassay-guided fractionation process, has been identified as norharman (a beta-carboline alkaloid) by electron-impact MS and NMR analyses. Norharman showed cytotoxicity towards both the HeLa cervical-cancer cell line and the BGC-823 stomach-cancer cell line, with an IC(50) of 5 microg/ml. Several methods were used to study the mechanism by which norharman is cytotoxic to HeLa cells. By means of an Acridine Orange/ethidium bromide dual-staining assay, condensation of chromatin was observed. A TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) assay showed degradation of DNA. Flow-cytometric analysis indicated that norharman could arrest cells at the G(2)/M phase of the cell cycle. These results demonstrate the cytotoxic mechanism of norharman involves the induction of apoptosis in HeLa cells.

Antitumor Effects of Zerumbone fromZingiber zerumbetin P-388D1Cellsin Vitroandin Vivo

The fresh rhizome of Zingiber zerumbet (L.) Roscoe ex Smith (Zingiberaceae) is widely used as a folk medicine in Taiwan. In this study, the fresh rhizome was extracted with 95 % EtOH and partitioned with diethyl ether. The antitumor effects of the diethyl ether extract were measured in cultured P-388D (1) cells and in an animal model of P-388D (1)-bearing CDF (1) mice. The results indicated that the extract could induce DNA fragmentation in P-388D (1) cells in vitro, and significantly prolong the life of P-388D (1)-bearing CDF (1) mice (ILS% = 127.8) at a dosage of 5 mg/kg body weight. After column chromatography combined with an MTT cytotoxicity bioassay, zerumbone, a cyclic sesquiterpene was isolated from the diethyl ether extract. Zerumbone inhibited the growth of P-388D (1) cells, induced DNA fragmentation in culture, and significantly prolonged the life of P-388D (1)-bearing CDF (1) mice (ILS% = 120.5) at a dosage of 2 mg/kg. Furthermore, zerumbone inhibited the growth of a human leukemia cell line, HL-60 cells, in a time- and concentration-dependent manner, with IC (50) values of 22.29, 9.12, and 2.27 microg/mL for 6, 12, and 18 h, respectively. The cell cycle of HL-60 cells was observed after treatment with zerumbone, which induced G (2)/M cell cycle arrest in HL-60 cells in a time- and concentration-dependent manner, and decreased the cyclin B1/cdk 1 protein level. These results suggest that zerumbone is an active principal of Z. zerumbet and is potentially a lead compound for the development of anticancer drugs.

Novel Antibacterial Diterpenoids from Larix chinensis Beissn

Two novel labdane-type diterpenoids, butanedioic acid mono[(13S)-13-hydroxylabda-8(17),14-dien-19-yl] ester (1) and butanedioic acid bis[(13S)-13-hydroxylabda-8(17),14-dien-19-yl] ester (2), along with the eleven known diterpenoids 3-13 and three other known compounds, were isolated from the bark of Larix chinensis. Their structures were elucidated both spectroscopically and chemically. The major diterpenoids 1-9, 11, and 13, as well as the very abundant phenolic compound 16, were subjected to antibacterial and cytotoxic bioassays. Compounds 7 and 9 showed remarkable in vitro inhibition of Staphylococcus aureus and S. epidermidis (MIC = 37-73 microM).

Antitubercular Sesterterpenes from the Thai Sponge Brachiaster sp.

A new scalarane-type sesterterpene, 12-deacetoxyscalarin 19-acetate (2), and two naturally new derivatives of manoalide-type sesterterpenes, (E)- and (Z)-neomanoalide 24,25-diacetates (3 and 4), were isolated from the Thai sponge Brachiaster sp., along with five other known sesterterpenes: heteronemin (1), heteronemin acetate (5), 12-epi-19-deoxyscalarin (6), 12-deacetyl-12-epi-19-deoxyscalarin (7), and manoalide 25-acetate (8). The antitubercular and cytotoxic activities of all eight compounds were evaluated to reveal the potent activity of compounds 1, 2, 5, and 8. Among these, compound 2 showed an interesting bioactivity profile, in possessing potent antitubercular activity and being practically inactive in the cytotoxicity bioassay.

Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease

Background & Aims: Steroid-refractory Crohn’s disease responds to therapy with the chimeric anti-tumor necrosis factor (TNF)-α antibody infliximab. Etanercept, a recombinant TNF receptor/immunoglobulin G fusion protein, is highly effective in rheumatoid arthritis but not in Crohn’s disease. Because both infliximab and etanercept are TNF-α-neutralizing drugs, we investigated the differences in TNF-α-neutralizing capacity and human lymphocyte binding and apoptosis-inducing capacity of both molecules. Methods: We used a nuclear factor κB reporter assay and a cytotoxicity bioassay to study TNF-α neutralization by infliximab and etanercept. Lymphocyte binding and apoptosis-inducing capacity was investigated using fluorescence-activated cell sorter analysis, annexin V staining, and cleaved caspase-3 immunoblotting using mixed lymphocyte reaction-stimulated peripheral blood lymphocytes (PBL) from healthy volunteers and lamina propria T cells from patients with Crohn’s disease. Results: Both infliximab and etanercept neutralized TNF-α effectively. Infliximab bound to activated PBL and lamina propria T cells, whereas binding of etanercept was equal to a nonspecific control antibody. Infliximab but not etanercept induced peripheral and lamina propria lymphocyte apoptosis when compared with a control antibody. Infliximab activated caspase 3 in a time-dependent manner, whereas etanercept did not. Conclusions: Although both infliximab and etanercept showed powerful TNF-α neutralization, only infliximab was able to bind to PBL and lamina propria T cells and subsequently to induce apoptosis of activated lymphocytes. These data may provide a biological basis for the difference in efficacy of the 2 TNF-α-neutralizing drugs.

Oral administration of type II collagen suppresses pro-inflammatory mediator production by synoviocytes in rats with adjuvant arthritis.

The objective of this study was to investigate the effect of the oral administration of type II collagen (CII) on pro-inflammatory mediator production by synoviocytes in rats with adjuvant arthritis (AA). Sprague-Dawley rats were fed with bovine CII either before immunization with Complete Freund's adjuvant (CFA) or after initiation of arthritis. Hind paw secondary swelling was measured and synoviocytes were harvested. Sera from portal vein of oral tolerized rats were collected and in vitro synoviocytes culture or synoviocytes-Peyer's Patches (PP) cells coculture system were developed. Interleukin (IL)-1 activity was measured by a mouse thymocyte activation assayed by MTT dye reduction and tumour necrosis factor (TNF) activity was measured by an L929 cytotoxicity bioassay. Nitric oxide (NO) and malondialdehyde (MDA) levels were measured by biochemical methods. We found that feeding with CII (5, 50 and 500 micro g/kg) for 7 days before immunization significantly suppressed hind paw secondary swelling measured at day 16, 20, 24 and 28 (all P < 0.01) and pro-inflammatory mediator (IL-1, TNF, NO and MDA) production by synoviocytes (all P < 0.01) in rats with AA. Feeding with CII (5, 50 and 500 micro g/kg) for 7 days after initiation of arthritis had a similar effect. CII (1, 10, 100 micro g/ml) had no effect on IL-1 and TNF production by synoviocytes in vitro, but CII 10 micro g/ml suppressed IL-1 and TNF production by synoviocytes-PP cells coculture system (P < 0.01), which was antagonized by anti-TGF-beta antibody (10 micro g/ml) (P < 0.01). Portal serum (1 : 10) from oral tolerized rats suppressed IL-1 and TNF production by synoviocytes (P < 0.01), which was also antagonized by anti-TGF-beta antibody (10 micro g/ml) (P < 0.01). We conclude that oral administration of CII had prophylactic and therapeutic effects on AA and over-production of IL-1, TNF, NO and MDA by synoviocytes was suppressed. Bystander active suppression may be the main mechanism of oral CII in the suppression of synoviocyte function.

New cytotoxic indolic metabolites from a marine Streptomyces.

Three new cytotoxic 3,6-disubstituted indoles (1-3) were isolated from the mycelium of a strain identified as Streptomyces sp. (BL-49-58-005), which was separated from a Mexican marine invertebrate, and their structures established by analysis of NMR and mass spectral data. GI(50) values for 1 and 2 in cytotoxic bioassays against a panel of 14 different tumor cell lines were estimated at micromolar range, while compound 3 showed no activity in the same assays.

Expression of tumor necrosis factor alpha and its mRNA in the spinal cord following a weight-drop injury.

The effect of traumatic spinal cord injury (SCI) on the expression of tumor necrosis factor (TNFalpha) and its mRNA was examined. Quantitative reverse transcription polymerase chain reaction assay showed TNFalpha mRNA level increased > 20-fold at the lesion site by 1 h after the injury compared to that in uninjured controls. The TNFalpha mRNA level was still significantly higher than in the controls 72 h after the injury. TNFalpha mRNA in the samples collected immediately caudal to the lesion site was also increased. Levels of TNFalpha protein, determined by a cytotoxic bioassay, were also significantly increased at the lesion site. The TNFalpha protein level was maximal 1 and 8 h after the injury and still significantly higher than in the controls 24 h post-injury. The present results demonstrate that TNFalpha is rapidly induced in the spinal cord after the injury, and it may play a significant role in secondary events in SCI.

Cytotoxic Anticancer Candidates from Natural Resources

Natural products have been regarded as important sources that could produce potential chemotherapeutic agents. Over 50% of anticancer drugs approved by United States Food and Drug Administration since 1960 were originated from the natural resources, especially from terrestrial plants. Based on cytotoxicity bioassay, over 400 compounds have been isolated from plants, marine organisms and microorganisms from the period of 1996 to 2000. Recently, interest of natural product research has slowly moved to marine organisms. As a result, almost 50% of reported cytotoxic compounds were isolated from marine organisms such as sponges and corals. Also, traditional cytotoxic compounds of acetogenins, alkaloids and terpene skeletons have been reported continuously. In this review, we will present the cytotoxic compounds obtained from natural sources from 1996 to 2000, and the structures and cytotoxic activity of natural compounds isolated from territorial, marine and microorganism resources.

Expression of neuron-associated tumor necrosis factor alpha in the brain is increased during persistent pain

Background and Objectives: Evidence implicates the pleiotropic cytokine tumor necrosis factor alpha (TNFα) in the pathogenesis of persistent pain. The present study employs a chronic constriction injury (CCI) model of neuropathic pain to examine TNFα production in the central nervous system (CNS) and in the periphery in this pain model. Methods: CCI-induced hyperalgesia is assessed by measuring the nociceptive threshold using the hot-plate test. The development of hyperalgesia is correlated to levels of TNFα by assessing: bioactive TNFα in homogenates of sciatic nerves, cervical spinal cord, thoracolumbar spinal cord, as well as in plasma using the WEHI-13 variant cytotoxicity bioassay; and mRNA for TNFα in sections of locus coeruleus by in situ hybridization. Results: We have previously demonstrated that TNFα bioactivity in the region of the brainstem containing the locus coeruleus is increased concurrent with the development of hyperalgesia, returning to baseline values by day 14, when hyperalgesia has ceased. Constitutive levels of TNFα are demonstrated in the plasma, sciatic nerves, and cervical and thoracolumbar spinal cord of control rats, sham-operated rats, and rats undergoing CCI. Levels of TNFα are significantly elevated in the injured sciatic nerve by day 8 postligature placement, concurrent with maximal hyperalgesia, and remain elevated when hyperalgesia has abated at day 14 postligature placement. Additionally, TNFα activity is increased in the thoracolumbar region of the spinal cord by day 4 postligature placement and remains elevated during hyperalgesia (day 8), as well as after hyperalgesia has dissipated (day 14). The increase in TNFα expression is specific to discrete regions of the CNS, rather than being the result of a systemic inflammatory response, since TNFα bioactivity in plasma is, in fact, decreased in rats undergoing CCI. Additionally, accumulation of mRNA specific for TNFα is significantly increased in neurons within a region of the brain containing the locus coeruleus at days 2, 8, and 14 postligature placement, contemporaneous with the development of hyperalgesia. Conclusions: The increases in TNFα within regions of the brain and spinal cord that are associated with adrenergic neuron function, as well as with modulation of pain perception, and the time course and distribution of the increases in TNFα accumulation support a neuromodulatory role for TNFα within the CNS in the development and maintenance of neuropathic pain. Reg Anesth Pain Med 2002;27:357-366.

TNFα Production in Whole Blood Cultures from Healthy Individuals

Tumor necrosis factor alpha (TNFα) is a major mediator of inflammatory responses and also plays a prominent role in bridging the innate and adaptive phases of immunity. In the present work we attempted to study TNFα production in endotoxin-stimulated blood of healthy individuals, and the inter-individual variability in TNFα production. For this study, we used diluted whole blood stimulated with lipopolysaccharide (LPS). The levels of the pro-inflammatory cytokine TNFα were measured by ELISA and by the L929 cytotoxicity bioassay in 16 and 18 healthy donors, respectively. There were highly significant inter-individual variations in the induced TNFα production. It is worth noting that there was no difference in sensitivity between ELISA and the cytotoxicity L929 bioassay. We concluded that whole blood culture is a sensitive method to determine the pro-inflammatory cytokine production in response to endotoxin stimuli in a relevant physiologic milieu. Our data indicate that this method provides appropriate information about the state of cellular immunity of the individual.

IgE enhances FcεRI expression and IgE-dependent TNF-α release from canine skin mast cells

The role of IgE on mast cell (MC) activation is well known. Recent studies have demonstrated that IgE also has the ability to up-regulate the high affinity IgE receptor (FcεRI) on the surface of human and murine MC, leading to an increased production of cytokines and chemokines. In the present study, we have examined the influence of IgE levels on FcεRI expression, and its consequences on TNF-α production from canine skin MC. Mature MC were enzymatically dispersed from the skin biopsies of 6–8 dogs and were cultured for up to 5 days in medium supplemented with recombinant canine stem cell factor (SCF) (6 ng/ml), in the presence of increasing serum IgE concentrations (ranging from 0 to 80 μg/ml). Subsequently, skin MC were activated with anti-IgE, and TNF-α concentration was assessed 5 h post-activation by a cytotoxic bioassay. FcεRI receptors were identified in MC surface by flow cytometry. MC cultured for up to 5 days in the presence of high serum IgE concentration (8 μg/ml) produced twice the quantity of TNF-α than MC cultured in the absence of serum IgE, in response to stimulation with anti-IgE. Moreover, the percentage of FcεRI-positive skin cells was found to be approximately double in cells cultured with serum IgE compared to that cultured in the absence of IgE, following saturation of IgE receptors. These results suggest that, as found in human and murine MC, IgE may induce an up-regulation of the FcεRI density and an enhancement in the secretory activity of canine skin MC. This study could be of great interest in designing new therapeutic strategies for controlling MC activation in inflammatory and allergic processes.

Antibacterial activity against resistant bacteria and cytotoxicity of four alkaloid toxins isolated from the marine sponge Arenosclera brasiliensis

Arenosclerins A–C and haliclonacyclamine E, new tetracyclic alkylpiperidine alkaloids isolated from the marine sponge Arenosclera brasiliensis, were subjected to antimicrobial and cytotoxic bioassays. Fourteen samples of microorganisms were used: Candida albicans, Staphylococcus aureus, Escherichia coli, and 12 antibiotic-resistant bacteria isolated from hospital environment. The minimum inhibitory concentration activity of each alkaloid was determined. The four compounds displayed antibacterial activity, but no antifungal activity against C. albicans. Haliclonacyclamine E and arenosclerins A and C were active against a larger number of bacteria strains than arenosclerin B. However, arenosclerins B and C presented more potent antibacterial activity. The alkaloids displayed inhibitory activity against both Gram positive and Gram negative bacteria. Cytotoxicity bioassays using the MTT method showed that these compounds present cytotoxic activity against human HL-60 (leukemia), L929 (fibrosarcoma), B16 (melanoma) and U138 (colon) cancer cell lines at concentrations between 1.5 and 7.0μg/ml. The results obtained indicated that A. brasiliensis alkaloids have a potent toxic activity. The broad cytotoxic and antimicrobial activities presented by A. brasiliensis alkaloids suggest a defensive role of arenosclerins and haliclonacyclamine E against microbial infection and/or the action of potential predators at the sponge's natural habitat.

Morphological and toxicological variability of Prorocentrum lima clones isolated from four locations in the south-west Indian Ocean

Eight clones of the toxic dinoflagellate Prorocentrum lima (Ehrenberg) Dodge from four sites (two clones per site) on the coral reef of La Réunion, Mayotte, Europa, and Mauritius Islands in the SW Indian Ocean were isolated and cultivated under the same conditions. Morphological features of each clone, including cell size and valve and marginal pore numbers, were examined by scanning electron microscopy. The toxic potential of each clone was determined by protein phosphatase type 2A (PP2A) inhibition test and fibroblast cell line FR3T3 bioassay. Scanning electron microscopy showed that variation in morphological features of clones within and between sites was minimal and not significant. However, equivalent okadaic acid content, determined by PP2A assay, was different within and between clones isolated from the four islands. Cytotoxicity bioassay with the FR3T3 cell line confirmed the variation on global toxic potential within and between the eight P. lima clones. This test also suggested the presence of other toxic compounds without PP2A inhibiting activity in crude extracts of some clones.

Toxic evaluation of organic extracts from airborne particulate matter in Puerto Rico.

In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.

Toxic Evaluation of Organic Extracts from Airborne Particulate Matter in Puerto Rico

In recent years, several hypotheses have emerged to explain the toxicologic activity of particulate matter. Organic compounds, ultrafine particles, biologic components, and transition metals are some of the constituents that reportedly exert some type of adverse effect on human health. A considerable fraction of the urban particulate matter consists of carbon compounds, which originate mostly from anthropogenic sources. The toxicity of organic fractions from particulate matter have been mainly evaluated by considering their mutagenic activity. This research expands on the toxicologic profile of organic compounds adsorbed to particulate matter, specifically in Puerto Rico, by using the cytotoxic neutral red bioassay (NRB). The NRB uses normal human epidermal keratinocytes or other types of cells to measure the effect on cell viability when exposed to organic compounds associated to the particles in the air. We validated the NRB for particulate matter by using a standard reference material (SRM 1649). We used the NRB to determine toxicologic differences of extracts between an urban industrialized site with anthropogenic activity versus a coastal region with less human activity. The cytotoxicity associated with organic compounds in particulate matter collected at the urban industrialized site was detected in both the particulate matter (3/4) 10 microm in aerodynamic diameter (PM(10)) and particulate matter (3/4) 100 microm in aerodynamic diameter (PM(100)). Greater toxic effects were observed in PM(10) extracts than in PM(100) extracts, but PM(10) toxic effects were not significantly different from those in PM(100). The extracts from the industrialized site were more cytotoxic than the extracts from coastal reference site, although in the summer, extracts from both sites were significantly cytotoxic to normal human epidermal keratinocytes. In addition, the nonpolar extracts of both PM(10) and PM(100) exerted the greatest cytotoxicity, followed by the polar, and, finally, the moderately polar extract. This study demonstrates that extracts from the Guaynabo industrialized site were more toxic than similar extracts obtained from a reference coastal site in Fajardo, Puerto Rico.

Cytotoxic and DNA interaction activities of extracts from medicinal plants used in Argentina

Eight crude extracts from seven Argentine plants with cancer-related ethnobotanical uses have been subjected to a bioscreening study to detect cytotoxic activity. The plants studied were: Aristolochia triangularis, Baccharis grisebachii, Bolax gummifera, Eupatorium hecatanthum, Erythrina crista-galli, Pterocaulon polystachium and Salpichroa origanifolia. Crown gall tumour inhibition, DNA interaction and cytotoxicity towards KB cells were assayed using the potato disc, the DNA-methyl green (DNA-MG) and the KB cells cytotoxicity bioassays respectively. The results obtained indicate that A. triangularis (ED50=47 μg/ml), B. gummifera (ED50=32 μg/ml) and E. hecatanthum (ED50=35 μg/ml) contained cytotoxic compounds against KB cells. All of the plants studied inhibited the growth of crown gall tumours, showing correlation between the experimental data and the uses reported for these plants. Moreover, the results obtained for the extracts of E. hecatanthum and P. polystachium indicate the presence of compounds that interact with DNA (48 and 22% of absorbance decrease, respectively). The results obtained suggest that cytotoxicity could play an important role in the activities claimed for the plants under study.

Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts

In October of 1996, a Gymnodinium breve bloom occurred in shellfish harvesting waters of Alabama, Mississippi and Louisiana, Gulf of Mexico, USA. Bloom densities reached 5.6 × 105 cells liter−1 and bloom residence at shellfish sampling stations ranged from 3 to 28 days. Brevetoxin-2 dominated G. breve toxin profiles in bloom seawater extracts. Shellfish toxicity, assessed by mouse bioassay, exceeded the guidance level for up to 75 days after the bloom had dissipated. Cytotoxicity assays and mouse bioassays showed similar temporal patterns of shellfish toxicity, but the two methods differed in estimations of brevetoxin-3 equivalent toxicity by a factor of 93 to 1. LC-ESI-MS showed the temporal patterns in shellfish toxicity reflected metabolism of G. breve toxins. The molecular ions m/z 1004, 1017 and 1033 dominated LC-ESI-MS spectra of toxic chromatographic fractions from the extracts and were identified as brevetoxin metabolites on the basis of LC-APCI-MS-MS. The discrepancy between cytotoxicity and mouse bioassay estimates of brevetoxin-3 equivalent toxicity resulted from the difference in extraction efficiency of solvents used in the respective methods and the relative sensitivity of the assays to toxin metabolite mixtures present in the extracts. The normalized cytotoxicity assay showed 75 % agreement with mouse bioassay positive test samples and 64 % agreement with mouse bioassay negative test samples. Published in 1999 by John Wiley & Sons, Ltd.

Monitoring brevetoxins during a Gymnodinium breve red tide: comparison of sodium channel specific cytotoxicity assay and mouse bioassay for determination of neurotoxic shellfish toxins in shellfish extracts

In October of 1996, a Gymnodinium breve bloom occurred in shellfish harvesting waters of Alabama, Mississippi and Louisiana, Gulf of Mexico, USA. Bloom densities reached 5.6x10(5) cells liter(-1) and bloom residence at shellfish sampling stations ranged from 3 to 28 days. Brevetoxin-2 dominated G. breve toxin profiles in bloom seawater extracts. Shellfish toxicity, assessed by mouse bioassay, exceeded the guidance level for up to 75 days after the bloom had dissipated. Cytotoxicity assays and mouse bioassays showed similar temporal patterns of shellfish toxicity, but the two methods differed in estimations of brevetoxin-3 equivalent toxicity by a factor of 93 to 1. LC-ESI-MS showed the temporal patterns in shellfish toxicity reflected metabolism of G. breve toxins. The molecular ions m/z 1004, 1017 and 1033 dominated LC-ESI-MS spectra of toxic chromatographic fractions from the extracts and were identified as brevetoxin metabolites on the basis of LC-APCI-MS-MS. The discrepancy between cytotoxicity and mouse bioassay estimates of brevetoxin-3 equivalent toxicity resulted from the difference in extraction efficiency of solvents used in the respective methods and the relative sensitivity of the assays to toxin metabolite mixtures present in the extracts. The normalized cytotoxicity assay showed 75% agreement with mouse bioassay positive test samples and 64% agreement with mouse bioassay negative test samples. Published in 1999 by John Wiley & Sons, Ltd.

Elevated serum tumor necrosis factor-alpha concentrations and bioactivity in Type 2 diabetics and patients with android type obesity

The role of tumor necrosis factor-alpha in insulin resistance has been studied in 59 patients with Type 2 diabetes, 28 with android type obesity and 35 healthy lean controls. Immunoreactive concentrations and bioactivity of serum tumor necrosis factor-alpha have repeatedly been determined in 8 weeks intervals for 12 months, five times per patients, by using ELISA and L929 cell cytotoxicity bioassay. Significantly higher immunoreactive tumor necrosis factor-alpha concentrations and bioactivity have been found in both, the Type 2 diabetic and obese groups as compared to the healthy persons. Tumor necrosis factor-alpha concentrations and bioactivity have showed a significant positive linear correlation with the elevated basal serum C-peptide levels and body mass indexes in both groups of patients. According to these data the cytokine might play a role in insulin resistance in obesity as well in Type 2 diabetes.