Laurence Dinan

Phytoecdysteroids: biological aspects

Phytoecdysteroids are a family of about 200 plant steroids related in structure to the invertebrate steroid hormone 20-hydroxyecdysone. Typically, they are C27, C28 or C29 compounds possessing a 14alpha-hydroxy-7-en-6-one chromophore and A/B-cis ring fusion (5beta-H). In the present review, the distribution, biosynthesis, biological significance and potential applications of phytoecdysteroids are summarised.

Assessment of a microplate-based bioassay for the detection of ecdysteroid-like or antiecdysteroid activities

Abstract We previously devised a simple and rapid bioassay for the detection of ecdysteroid agonistic and antagonistic activities based on the ecdysteroid-specific responses of the BII (l[2]mbn) tumorous blood cell line of Drosophila melanogaster. We have now assessed the bioassays reproducibility, specificity and robustness. A characteristic response is obtained with a variety of ecdysteroids, but no response occurs in the presence of any of eight vertebrate steroids. The bioassay is robust enough to cope with methanol extracts of freeze-dried plant samples after hexane partition to remove plant pigments. Thus, the bioassay has all the required properties for receptor-based phytochemical screening of large numbers of plant samples for the purpose of identifying new potent phytoecdysteroids, non-steroidal ecdysteroid analogues and anti-ecdysteroids.

Chromatographic procedures for the isolation of plant steroids

In this review, we consider the general principles and specific methods for the purification of different classes of phytosteroids which have been isolated from plant sources: brassinosteroids, bufadienolides, cardenolides, cucurbitacins, ecdysteroids, steroidal saponins, steroidal alkaloids, vertebrate-type steroids and withanolides. For each class we give a brief summary of the characteristic structural features, their distribution in the plant world and their biological effects and applications. Most classes are associated with one or a few plant families, e.g., the withanolides with the Solanaceae, but others, e.g., the saponins, are very widespread. Where a compound class has been extensively studied, a large number of analogues are present across a range of species. We discuss the general principles for the isolation of plant steroids. The predominant methods for isolation are solvent extraction/partition followed by column chromatography and thin-layer chromatography/HPLC.

An extensive ecdysteroid CoMFA.

The ecdysteroid agonist activity of 71 HPLC-purified ecdysteroids was measured in the Drosophila melanogaster BII tumorous blood cell line assay. The resultant log(ED50) values, spanning almost 6 orders of magnitude, were used to construct a comparative molecular field analysis (CoMFA) model in which conformations were selected by homology to the crystal structure of ecdysone. Model A was constructed by utilization of the region- focused electrostatic indicator field (q2=0.631, r2=0.903, 5 components, 4 outliers). Model B made use of region-focused electrostatic and steric indicator fields along with MlogP (q2=0.694, r2=0.892, 5 components, 4 outliers). The model and its underlying bioassay data support a pharmacophore hypothesis in which ecdysteroid binding is understood to be due principally to the summation of localized interactions from approximately six specific loci. This is in contrast to previous structure-activity relationship hypotheses which are formulated in terms of the presence or absence of essential functional groups, without which ecdysteroid receptor affinity would be completely absent. The present CoMFA model is utilized to predict the activities of heretofore unknown ecdysteroids.

Effects of four synthetic musks on the life cycle of the harpacticoid copepod Nitocra spinipes

A full life-cycle (</=26 days exposure) toxicity test with the harpacticoid copepod Nitocra spinipes was used to study the effects of one nitro musk (musk ketone) as well as three polycyclic musks (Tonalide, Celestolide and Galaxolide). A subchronic individual life-table endpoint, the larval development rate, was recorded after 7-8 days exposure of juveniles and was significantly decreased in copepods exposed to sublethal concentrations of musk ketone, Celestolide and Galaxolide. However, none of the Tonalide concentrations had any effect on larval development. The lowest Galaxolide concentration (0.02 mg/l), which affected juvenile development, was about 100 times below the adult 96-h-LC(50)-value of 1.9 mg/l (95% confidence interval: 1.4-2.7 mg/l). However, none of the four musks had any agonistic or antagonistic activity in the ecdysteroid-sensitive Drosophila melanogaster B(II)-cell line. This indicates that the decrease in larval development rate was due to pharmacological effects rather than steroid receptor-mediated endocrine disruption. A modified Euler-Lotka equation was used to calculate a population-level endpoint, the intrinsic rate of natural increase (r(m)), from individual life-table endpoints, i.e. mortality rate, time of release of first brood, sex ratio, the fraction of ovigerous females among all females as well as the number of nauplii per ovigerous female. The second highest musk ketone concentration (0.1 mg/l) was the only treatment, which significantly affected r(m) (***P<0.001). At the highest musk ketone (0.3 mg/l) and Celestolide (0.3 mg/l) concentrations, all copepods were dead at the end of the exposures. This shows that a sensitive individual life-table endpoint is protective over the population-level endpoint r(m). Though we think that it is necessary to obtain population-level endpoints from standardised toxicity test, for ecologically successful risk characterisation of synthetic musks as well as other chemicals. The results from the present study show that it is possible to obtain population-level data from the full life-cycle test with N. spinipes. However, there seems to be little risk that synthetic musks are harmful to copepods at present environmental concentrations.

Brominated flame retardants: Activities in a crustacean development test and in an ecdysteroid screening assay

Brominated flame retardants (BFRs) were investigated for toxic effects both in vivo and in vitro in two invertebrate bioassays. Subchronic effects of tetrabromobisphenol A (TBBPA), tribromophenol (TBP), and four polybrominated diphenyl ethers ([PBDEs]; BDE-28, BDE-47, BDE-99, and BDE-100) on larval development of the marine copepod Acartia tonsa were studied. For TBBPA and TBP 5-d effective median concentration (EC50) values for inhibition of the larval development rate were 125 and 810 microg/L, respectively, whereas the PBDEs were much more potent with 5-d EC50 in the low microg/L range (1.2 microg/L for BDE-100; 4.2 microg/L for BDE-99; 13 microg/L for BDE-28; and 13 microg/L for BDE-47). These concentrations were up to two orders of magnitude below the 48-h LC50 for acute adult toxicity (108 microg/L for BDE-28; 400 microg/L for TBBPA; 520 microg/L for BDE-100; 705 microg/L for BDE-99; 1,500 microg/L for TBP; and 2,370 microg/L for BDE-47). To distinguish between general toxicological and endocrine-mediated toxic effects, the BFRs were assessed in vitro for ecdysteroid agonistic/antagonistic activity with the ecdysteroid-responsive Drosophila melanogaster B(II)-cell line. The pentabrominated diphenyl ethers BDE-99 and BDE-100 showed weak ecdysteroid antagonistic activity. Thus, these PBDEs may be regarded as potential endocrine disrupters in invertebrates. The combination of in vitro assays and subchronic biotests with ecologically important crustacean species is a rapid and cost-effective tool when screening for sublethal effects of BFRs and other chemicals.

Identification and ecdysteroid antagonist activity of three resveratrol trimers (suffruticosols A, B and C) from Paeonia suffruticosa

Abstract Bioassay-guided HPLC analysis of the seeds of Paeonia suffruticosa has afforded three novel resveratrol trimers (suffruticosol A, suffruticosol B and suffruticosol C), together with cis-resveratrol and paeoniflorin. The structures of these new compounds have been elucidated mainly by comprehensive 1D- and 2D-NMR experiments. Resveratrol and its oligomers are active as ecdysteroid antagonists (ED50 values = 10 to 50 μM vs. 5 × 10−8M 20-hydroxyecdysone) in the Drosophila melanogaster BII bioassay. The activities of other “pseudo-oestrogens” in this bioassay have also been assessed.

The Effects of Ingested 20-Hydroxyecdysone on the Larvae of Aglais urticae, Inachis io, Cynthia cardui (Lepidoptera: Nymphalidae) and Tyria jacobaeae (Lepidoptera: Arctiidae).

A comparative survey was carried out to investigate the effects, distribution and metabolism of ingested 20-hydroxyecdysone in four species of lepidopteran larvae in relation to the phytoecdysteroid content of the insects host plants. Analysis of the leaves of the host plants of each of the species revealed a strong relationship between the levels of phytoecdysteroids and the relative tolerance of the larvae to ingested 20-hydroxyecdysone. Monophagous or oligophagous species (Aglais urticae, Inachis io) feeding on ecdysteroid-negative host plants were either deterred from feeding or showed marked abnormalities in growth and development after incorporation of 20-hydroxyecdysone in their diets. Oligophagous or polyphagous species (Tyria jacobaeae, Cynthia cardui) which feed on host plants from families which are known to contain phytoecdysteroid-positive species, were able to tolerate low levels of 20-hydroxyecdysone in their diets, but exhibited developmental defects at high concentrations. These species were termed semi-tolerant. In each of the species, ingested [3H]20-hydroxyecdysone appeared to follow the same fate as injected [3H]20-hydroxyecdysone. The data are compared to those obtained in previous studies, where truly polyphagous species were shown to tolerate very high concentrations of 20-hydroxyecdysone in their diets by the production of ecdysteroid 22-fatty acyl esters. Copyright 1997 Elsevier Science Ltd. All rights reserved

Insect cell lines as tools for studying ecdysteroid action

Summary Recent research concerning ecdysteroid-responsive and ecdysteroid-producing cell lines is reviewed. The advantages and limitations of cell lines of defined and undefined origin are considered with regard to their suitability for studies on molecular, physiological, morphological and developmental aspects of ecdysteroid action. The considerable potential for future studies involving insect cell lines is indicated.

MOSCHAMIN, CIS-MOSCHAMINE, MOSCHAMINDOLE AND MOSCHAMINDOLOL: FOUR NOVEL INDOLE ALKALOIDS FROM CENTAUREA MOSCHATA

Abstract Four novel indole alkaloids: (E) N-(3-methoxy-4-hydroxycinnamoyl)-5-hydroxytryptamine (moschamine, 1), (Z) N-(3-methoxy-4-hydroxycinnamoyl)-5-hydroxytryptamine (cis-moschamine, 2), {2-(3-methoxy-4-hydroxyphenyl)}-dihydrofuro[kl]-1H-pyrrolo[[fg]-2-oxo-1,2,3,4,5,6-hexahydro-3-benzazocine (moschamindole, 3) and 1-(3-methoxy-4,7-dihydroxybenzyl)-10-hydroxy-1H-Pyrrolo[fg]-2-oxo-1,2,3,4,5,6-hexahydro-3-benzazocine (moschamindolol, 4) have been isolated from the seeds of Centaurea moschata. The structures of these compounds have been determined primarily on the basis of extensive 1D and 2D NMR experiments.