New cytochalasin from Rosellinia sanctae-cruciana, an endophytic fungus of Albizia lebbeck.

AIM: To explore the potential of Rosellinia sanctae-cruciana an endophytic fungus associated with Albizia lebbeck for pharmaceutically important cytotoxic compounds. METHODS AND RESULTS: One novel cytochalasin, named jammosporin A (1) and four known analogues (2-5) were isolated from the culture of the endophytic fungus R. sanctae-cruciana, harboured from the leaves of the medicinal plant A. lebbeck. Their structures were elucidated by extensive spectroscopic analyses including one-dimensional and two-dimensional nuclear magnetic resonance data along with MS data and by comparison with literature reports. In preliminary screening the ethyl acetate extract of the fungal culture was tested for cytotoxic activity against a panel of four cancer cell lines (MOLT-4, A549, MIA PaCa-2 and MDA-MB-231), and found to be active against MOLT-4 with an IC50 value of 10 µg ml-1 . Owing to the remarkable cytotoxic activity of the extract the isolated compounds (1-5) were evaluated for their cytototoxicity against the MOLT-4 cell line by MTT assay. Interestingly, compounds 1-2, 4 and 5 showed considerable cytotoxic potential against the human leukaemia cancer cell line (MOLT-4) with IC50 values of 20·0, 10·0, 8·0 and 6·0 µmol l-1 , respectively, while compound 3 showed an IC50 value of 25 µmol l-1 . This is the first report of the existence of this class of secondary metabolites in R. sanctae-cruciana fungus. CONCLUSION: This study discovered a novel compound, named jammosporin A, isolated for the first time from R. sanctae-cruciana, an endophytic fungus of A. lebbeck with anticancer activity against the MOLT-4 cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: Rosellinia sanctae-cruciana represents an interesting source of a new compound with bioactive potential as a therapeutic agent against a human leukaemia cancer cell line (MOLT-4).

Lethality of cytochalasin B and other compounds isolated from fungus Aspergillus sp. (Trichocomaceae) endophyte of Bauhinia guianensis (Fabaceae).

Endophytic fungi are fungi that colonize internal tissues of plants; several biologically active compounds have been isolated from these fungi. There are few studies of compounds isolated from endophytic fungi of Amazon plants. Thus, this study aimed the isolation and structural identification of ergosterol (1), ergosterol peroxide (2), mevalonolactone (3), cytochalasin B (4) and cytochalasin H (5) from Aspergillus sp. EJC 04, an endophytic fungus from Bauhinia guianensis. The cytochalasin B (4) and the diacetate derivative of cytochalasin B (4a) showed high lethality in the brine shrimp assay. This is the first occurrence of cytochalasins in Amazonian endophytic fungi from B. guianensis.

Tolerated doses in zebrafish of cytochalasins and jasplakinolide for comparison with tolerated doses in mice in the evaluation of pre-clinical activity of microfilament-directed agents in tumor model systems in vivo.

BACKGROUND/AIM: Chemotherapeutic approaches involving microtubule-directed agents such as the vinca alkaloids and taxanes are used extensively and effectively in clinical cancer therapy. There is abundant evidence of critical cytoskeletal differences involving microfilaments between normal and neoplastic cells, and a variety of natural products and semi-synthetic derivatives are available to exploit these differences in vitro. In spite of the availability of such potential anti-neoplastic agents, there has yet to be an effective microfilament-directed agent approved for clinical use. Cytochalasins are mycogenic toxins derived from a variety of fungal sources that have shown promising in vitro efficacy in disrupting microfilaments and producing remarkable cell enlargement and multi-nucleation in cancer cells without producing enlargement and multi-nucleation in normal blood cells. Jasplakinolide is a sponge toxin that stabilizes and rigidifies microfilaments. Insufficient in vivo data has been acquired to determine whether any of the microfilament-directed agents have valuable preferential anticancer activity in pre-clinical tumor model systems. This is partly because the limited availability of these agents precludes their initial use in large-scale mammalian pre-clinical studies. Therefore, the present study sought to determine the tolerated in vivo doses of cytochalasins and jasplakinolide in zebrafish (Danio rerio), a well-studied fish cancer model that is 1.5% the size of mice. We also determined the tolerated levels of a variety of clinically active anti-neoplastic agents in zebrafish for comparison with tolerated murine doses as a means to allow comparison of toxicities in zebrafish expressed as µM concentrations with toxicities in mice expressed in mg/kg. MATERIALS AND METHODS: Tolerated doses in zebrafish with various cytochalasins or jasplakinolide were determined by adding the solubilized test agent to water in which the fish were maintained for 24 h, then restored to their normal tanks and monitored for a total of 96 h. RESULTS: Cytochalasin D at 0.2 µM gave an approximate LD50 in zebrafish, while cytochalasin B was fully-tolerated at 5 µM, and gave an LD50 of 10 µM. 21,22-dihydrocytochalasin B was fully-tolerated at 10 µM. Cytochalasin C was tolerated fully at 1 µM, ten-fold higher than the level for cytochalasin D that was tolerated. Jasplakinolide at 0.5 µM did not exhibit any apparent acute toxicity or affect fish behavior for four days, but delayed toxicity was evident at days 4 and 6 when the fish died. Further, the addition of 5 µM glutathione (GSH) at the time of treatment substantially decreased the toxicity of 10 µM cytochalasin B, a level of cytochalasin B that not otherwise tolerated in vivo. Such observations were likely due to GSH-mediated alkylation of C-20 in cytochalasin B, thereby reducing the rate of oxidation to the highly toxic congener, cytochalasin A, and reacting with any cytochalazin A formed. The protective effects of GSH are further supported by its ability to react with α, ß-unsaturated ketone moieties, as is found in cytochalasin A. GSH at 0.8 uM was able to reduce the toxicity of 0.8 µM cytochalasin D, but it took 20 µM GSH to fully protect against the toxicity of 0.8 µM cytochalasin D. CONCLUSION: Pre-clinical evaluation of rare natural products such as microfilamented-directed agents for efficacy in vivo in tumor-bearing zebrafish is a feasible prospect. Dose-limiting toxicities in zebrafish expressed as µM concentrations in water can be used to estimate in vivo toxicities in mice expressed as mg/kg.

A new cytochalasin from endophytic Phomopsis sp. IFB-E060.

AIM: To study the chemical constituents of the solid culture of the endophyte Phomopsis sp. IFB-E060 in Vatica mangachapoi. METHOD: Isolation and purification were performed through silica gel column chromatography, gel filtration over Sephadex LH-20, ODS column chromatography, and HPLC. Structures of the isolated compounds were elucidated by a combination of spectroscopic analyses (UV, CD, IR, MS, 1D, and 2D NMR). The cytotoxicity of the isolates was evaluated in vitro by the MTT method against the human hepatocarcinoma cell line SMMC-7721. RESULTS: Five compounds were isolated from the solid culture of the endophyte Phomopsis sp. IFB-E060 and their structures were identified as 18-methoxy cytochalasin J (1), cytochalasin H (2), (22E, 24S)-cerevisterol (3), ergosterol (4), and nicotinic acid (5). Compound 1 had an inhibition rate of 24.4% at 10 µg·mL(-1) and 2 had an IC50 value of 15.0 µg·mL(-1), while a positive control 5-fluorouracil had an inhibition rate of 28.7% at 10 µg·mL(-1). CONCLUSION: 18-Methoxy cytochalasin J (1), produced by endophytic Phomopsis sp. IFB-E060, is a new cytochalasin with weak cytotoxicity to the human hepatocarcinoma cell line SMMC-7721.

Cytotoxic cytochalasin metabolites of endophytic Endothia gyrosa.

In addition to the known metabolites cytochalasin H (1), cytochalasin J (2), and epoxycytochalasin H (3), two new 10-phenyl-(11)-cytochalasans, named cytochalasin Z10 and Z11 (4 and 5, resp.) were isolated from the solid substrate culture of Endothia gyrosa IFB-E023, an endophytic fungus residing inside the healthy leaf of Vatica mangachapo (Dipterocarpaceae). The structure determination of 4 and 5 was accomplished through correlative analyses of their spectral data (UV, ESI-MS, IR, (1)H- and (13)C-NMR, COSY, NOESY, HMQC, and HMBC). Metabolites 1-5 were demonstrated to be substantially cytotoxic to the human leukaemia K562 cell line with the IC(50) values of 10.1, 1.5, 24.5, 28.3, and 24.4 microM, respectively, which are comparable to that of 5-fluorouracil (33.0 microM), co-assayed as the positive reference.

Nonenolides and cytochalasins with phytotoxic activity against Cirsium arvense and Sonchus arvensis: a structure-activity relationships study.

A structure-activity relationships study was conducted assaying 15 natural analogues and derivatives belonging to two groups of organic compounds, nonenolides and cytochalasins, for their toxicity against the composite perennial weeds Cirsium arvense and Sonchus arvensis occurring through the temperate region of world. The toxic nonenolides (stagonolide, putaminoxin, pinolidoxin) and cytochalasins (deoxaphomin, cytochalasins A, B, F, T, Z2 and Z3) were isolated from phytopathogenic Stagonospora, Phoma and Ascochyta spp. The pinolidoxin (7,8-O,O'-diacetyl- and 7,8-O,O'-isopropylidene-pinolidoxin) and cytochalasins B (21,22-dihydro-, 7-O-acetyl- and 7,20-O,O'-diacetyl-cytochalasin B) derivatives were obtained by chemical modifications of the corresponding toxins. Among the 15 compounds tested, stagonolide and deoxaphomin proved to be the most phytotoxic to C. arvense and S. arvensis leaves, respectively. The tested phytotoxic nonenolides were stronger inhibitors of photosynthesis in C. arvense leaves than cytochalasines A and B. Stagonolide had less effect on membrane permeability in C. arvense leaves than cytochalasin B. Significant changes of light absorption by C. arvense leaves in visible and infrared spectra were caused by stagonolide. The functional groups and the conformational freedom of the ring, appear to be important structural features for the nonenolides toxicity, whereas and the presence of the hydroxy group at C-7, the functional group at C-20 and the conformational freedom of the macrocyclic ring are important for the cytochalasins toxicity.

Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture.

Birds are uricotelic, and because they excrete urate by renal tubular secretion, they provide a convenient model for examination of this process. Primary monolayer cultures of the isolated renal proximal tubule epithelium from the domestic chicken, Gallus gallus L., were mounted in Ussing chambers where several substrates/inhibitors of renal organic anion transporters were tested for the sidedness and specificity of their effects on transepithelial urate transport. Transepithelial electrical resistance, electrical potential and sodium-dependent glucose current were monitored to detect nonspecific effects. Under control short-circuited conditions the ratio of unidirectional fluxes of [(14)C]urate was found to be 3:1. Active net secretion was specifically inhibited by 1 mmol l(-1) probenecid and 10 mmol l(-1) para-aminohippuric acid (PAH). Bromocresol Green, cimetidine, nocodozole, cytochalasin D and ouabain also inhibited secretion but were toxic. Interstitial-side lithium (5 mmol l(-1)) and glutarate (1 mmol l(-1)) specifically blocked transport, but 10-100 micromol l(-1) glutarate had no effect. Interstitial estrone sulfate (ES) stimulated urate secretion at 10 micromol l(-1) but was inhibitory at 500 micromol l(-1). Active PAH secretion (5:1 flux ratio) was inhibited 34% by 330 micromol l(-1) urate. ES (500 micromol l(-1)) blocked the remainder. From the lumen side, glucose-free, Cl(-)-free and high K(+) (30 mmol l(-1)) solutions, or an alkaline pH of 7.7 had no effect on urate transport and neither did several compounds known to be uricosuric. Lumen-side methotrexate (500 micromol l(-1)) and MK571 (20 micromol l(-1)) strongly inhibited urate secretion. MK571 had no effect from the interstitial side. RT-PCR revealed mRNA for OAT1-, OAT3-, MRP2- and MRP4-like organic anion transporters in chicken proximal epithelium.

Cytochalasins Z4, Z5, and Z6, three new 24-Oxa[14]cytochalasans produced by Phoma exigua var. heteromorpha.

Cytochalasins Z4, Z5, and Z6, three new cytochalasans, were isolated from the wheat culture of Phoma exigua var. heteromorpha together with the known cytochalasins A, B, F, T, Z2, and Z3, 7-O-acetylcytochalasin B, and deoxaphomin. Z2 and Z3, together with Z1, have recently been described as new 24-oxa[14]cytochalasans produced by Pyrenophora semeniperda, a fungus proposed for biological control of grass weeds. All three new cytochalasins were characterized as 24-oxa[14]cytochalasans by extensive use of NMR and MS techniques. Cytochalasins Z4 and Z5 proved to be structurally related to cytochalasin B, whereas Z6 was related to cytochalasin F. When assayed on tomato seedlings at 10(-4) M, Z6 caused a very low inhibition of rootlet elongation (30%), whereas Z4 and Z5 were almost inactive. On brine shrimp, at the same concentration, only Z5 had a minor effect, with 20% mortality, whereas the other two metabolites proved to be inactive.

Cytochalasins Z1, Z2 and Z3, three 24-oxa[14]cytochalasans produced by Pyrenophora semeniperda.

Three new cytochalasans, named cytochalasins Z1, Z2 and Z3, were isolated from the wheat culture of Pyrenophora semeniperda, a fungus proposed to biologically control grass weeds. Other cytochalasins isolated from the same organic extract were identified as the already known cytochalasins F, T, deoxaphomin and cytochalasins B, the latter being produced in very large amounts. All three new cytochalasins were characterized as 24-oxa[14]cytochalasans by extensive use of NMR and MS techniques. Cytochalasins Z1 and Z2 proved to be structurally related to cytochalasin T, whereas cytochalasin Z3 was related to cytochalasin B. When assayed on wheat and tomato seedlings, cytochalasin Z3, in comparison to the new cytochalasins, cytochalasin B, its 21,22-dihydroderivative, cytochalasin F and deoxaphomin showed a remarkable ability to inhibit root elongation. The possibility of using these metabolites in biological control strategies is discussed.

[Changes in the topography of various proteins in cultured neurons after selective injury of various cytoskeletal elements with neurotoxins]. / Izmeneniia topografii nekotorykh belkov vneshnei membrany kul'tiviruemykh neironov pri izbiratel'nom povrezhdenii razlichnykh élementov tsitoskeleta neirotoksinami.

The spinal cord and hippocampal primary cultures were incubated with three neurotoxins (separately) known to impair the main components of the cytoplasmic cytoskeleton: 1) colchicine blocking the repolymerization of microtubules, 2) cytochalasin preventing elongation of actin filaments, and 3) beta, beta'-iminodipropionitrile (IDPN), causing disorganisation of neurofilaments. The distribution of surface membrane molecules on the surface of the neurons was evaluated in the ultrastructural study after treatment with the neurotoxins on the 5th, 12th, and 15th days in vitro (DIV). On the 12 DIV, the density of immunogold labelled neural cell adhesion molecules (NCAM) on IDPN-treated hippocampal neurons increased 1.45 times comparing to the controls. On the 5 DIV, the density of WGA (wheat germ agglutinin)-binding membrane glycoproteins increased 2.09 times on colchicine-treated neurons, and 3.98 times on cytochalasin-treated ones, whereas on the 12 DIV, the increase was 3.28 and 2.72 times, respectively, as compared to the control cultures of the same age. These data provide insights into the mechanisms of neurodegenerative changes in the nerve cells and into the relationship between the cytoskeletal elements and the surface molecules on the neuronal plasmatic membrane.

Apoptosis pattern elicited by several apoptogenic agents on the seminiferous epithelium of the adult rat testis.

Spontaneous germ cell death during spermatogenesis is an important event, and the usefulness of the seminiferous epithelium as an in vivo model to study apoptosis has been evidenced. Nevertheless, the response of the testis to apoptogenic agents has not been analyzed. This study was designed to determine germ cell sensitivity to induction of apoptosis and to provide baseline data on the testis response to several apoptogenic agents. Induced apoptosis was assessed by in situ DNA 3'-end labeling and quantified at every stage of the spermatogenic cycle. The shortest response time for every agent was established based on morphological and quantitative criteria. Our results show significantly increased incidence of germ cell deaths after all treatments, mainly at stages I, XII, and XIV. These specific stages coincide with those at which the greatest numbers of spontaneous germ cell deaths occur in control animals. Moreover, the rapid and highly specific response of germ cells to all the apoptogenic agents used in the present study indicate that apoptosis must be tightly regulated at these stages of the seminiferous epithelium. As a consequence, we propose that the disruption of apoptosis control might be an important determinant for idiopathic male infertility.

Fungitoxic activity of some cytochalasins and their derivatives on Phytophthora species.

The activity of cytochalasin B was tested on 8 Phytophthora species, while the same toxin, some of its derivatives and natural analogues, namely cytochalasin F and deoxaphomin, were assayed at 2 x 10(-5) - 2 x 10(-4) M on the most sensitive species, P. cactorum. A significant inhibitory activity on P. cactorum was shown by cytochalasin B, its 7-monoacetyl derivative, and deoxaphomin. The hydroxy group at C-20 and the conformational freedom of the macrocyclic ring proved to be important structural features for this activity. The 7-hydroxy group at C-7 appeared to have no influence on this toxicity, while a size reduction associated with the carbocyclic nature of the macrocycle seems to lightly increase the activity. The 7-O-acetylcytochalasin B showed selective toxic activity on P. cactorum at the tested concentration, thus suggesting a possible use as a fungicide for this compound.

Effects of multidrug resistance-related ATP-binding-cassette transporter proteins on the cytoskeletal activity of cytochalasins.

Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research. In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins. Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents. This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis. The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ([3H]cytochalasin B accumulation) which was also restorable by P-gp modulators. Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ([3H]-azidopine) suggested cytochalasins as P-gp-binding agents. In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity. In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP. Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage. Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.

Cytochalasin W, a new 24-oxa[14]cytochalasan from Phoma exigua var. heteromorpha.

A further cytochalasin was isolated from liquid culture filtrates of Phoma exigua var. heteromorpha together with cytochalasins U and V and other well-known cytochalasins. The metabolite, named cytochalasin W, was characterized by spectroscopic and chemical methods as a new 24-oxa[14]cytochalasan, bearing a formyl group on the macrocyclic ring. Cytochalasin W showed toxic activity in the brine shrimp assay.

Differential interactions of cytochalasins with P-glycoprotein.

Cytochalasins are a family of structurally related natural product cytotoxins that selectively depolymerize microfilaments. In this study, the interaction between several cytochalasins and the drug transporter P-glycoprotein was investigated. Dihydrocytochalasin B and cytochalasin E consistently sensitized P-glycoprotein-overexpressing human breast carcinoma cells (MCF-7/ADR) to daunomycin, vinblastine, and actinomycin D without affecting the cytotoxicity of cisplatin. These compounds did not affect the sensitivities of the parental MCF-7 cells to anticancer drugs, indicating that their effects are due to P-glycoprotein inhibition. Effects of dihydrocytochalasin B and cytochalasin E were observed at concentrations as low as 2.5 and 5 microM, respectively. In contrast, cytochalasins A, B, C, D, H, and J did not sensitize MCF-7/ADR cells to any of the drugs. The accumulation of [3H]-vinblastine by MCF-7/ADR cells and by drug-resistant human ovarian carcinoma cells (SKVLB1) was increased to the greatest extent by verapamil, followed by dihydrocytochalasin B > cytochalasin E > cytochalasin B, whereas cytochalasins A, C, D, H, and J did not alter intracellular accumulation of the drug. Similarly to verapamil, dihydrocytochalasin B significantly stimulated the ATPase activity of P-glycoprotein, while other cytochalasins were ineffective. These results demonstrate that very closely related compounds can differentially interact with P-glycoprotein. For example, the only difference between cytochalasin B and dihydrocytochalasin B is the saturation of a carbon-carbon double bond in dihydrocytochalasin B. These structural differences may provide important insight into chemical determinants for drug interaction with P-glycoprotein.

Cytochalasin B induces cellular DNA fragmentation.

Cellular DNA fragmentation can be induced in many biological instances without plasma membrane damage. The fungal metabolite, cytochalasin B, is capable of modifying numerous cellular functions related to DNA synthesis. In this work it is demonstrated that cytochalasin B is capable of inducing DNA fragmentation in a number of cells lines. This DNA fragmentation occurs before plasma membrane lysis and over a period of hours. Cytochalasin E and villin, agents that act on the microfilaments, also induce DNA fragmentation. Phorbol dibutyrate, a diacylglyceral analog, is able to inhibit cytochalasin B-induced DNA fragmentation in a dose-dependent fashion. These findings support the interpretation that cytochalasin B is inducing DNA fragmentation via its effect on the actin filaments.

Kinetic and morphological changes induced in human blood leucocytes by cytochalasin D and E.

The effect of increasing concentrations of cytochalasin D and E, up to toxicity, on the velocity of blood leucocytes from normal subjects was measured in vitro using a high-resolution objective and phase-contrast time-lapse photography. The dose-response effect for the two different cytochalasins differed in accordance with the different cell specificity of their membrane binding. The average velocity of granulocytes was reduced at cytochalasin D concentrations above 5 x 10(-7)M and cytochalasin E concentrations above 5 x 10(-5)M. The effect on monocytes and eosinophils was similar. In contrast the velocity of lymphocytes was not affected until cytotoxic concentrations were reached. The concentration ranges which inhibited locomotion corresponded well with the concentration ranges of the cytochalasins which have an in vitro effect on microfilaments. The concentrations which induced additional morphological changes in lymphocytes also correlate well with the concentrations found to inhibit cross-linking in vitro, as well as those known to induce morphological changes in, for example, fibroblasts in vivo. Cytotoxic effects were first observed with ten-fold higher concentrations of cytochalasin E than of cytochalasin D.

Glutathione status and the incidence of neural tube defects elicited by direct acting teratogens in vitro.

Valproic acid (VPA), cytochalasin D (CD) and 7-hydroxy-2-acetylaminofluorene (7-OH-AAF) each caused abnormal closure of the anterior neuropore in rat embryos cultured in vitro in the absence of an exogenous bioactivation system. Morphological comparisons showed that although all three compounds prevented normal neural tube closure, each did so in a distinctive manner. Modulation of GSH in cultured rat conceptuses was evaluated to determine whether common responses occurred relative to the ability of different chemicals to elicit neural tube defects. Malformation incidence in embryos (10-14 somites) varied widely following exposure to CD (44%), 7-OH-AAF (29%) or VPA (17%). The incidence of CD-elicited malformations was increased by 50% following GSH depletion by L-buthionine-S, R-sulfoximine (BSO) and was decreased by nearly 60% when the cysteine pro-drug 2-oxothiazolidine-4-carboxylate (OTC) was added to the culture medium. GSH modulation also exerted significant effects on the incidence of abnormal neurulation caused by VPA or 7-OH-AAF. A relatively low incidence of open neural tubes produced by VPA or 7-OH-AAF alone was shown to be a function of the state of maturation in the embryos. Conceptuses cultured in the presence of VPA or 7-OH-AAF from an earlier gestational age (6-10 somites) showed 2-3 fold increases in the number of embryos with open neural tubes. Differential alterations in protein and DNA content were observed among embryos and yolk sacs after various treatments indicating possible differences in the site of embryotoxicity. These results demonstrate the role of GSH status on the capacity of three chemically diverse compounds to elicit abnormal neurulation in cultured rat embryos and suggest some possible mechanisms by which normal neurulation may be compromised.

Effects of protein deficiency on the teratogenicity of cytochalasins in mice.

The developmental toxicity of cytochalasins B (CB) and D (CD) was evaluated in protein-deprived mice. Pregnant CD-1 mice were assigned to control (26%), 16%, 8%, or 4% dietary protein groups on gestation day 1 and dosed by gavage with 0 or 1.5 mg/kg CB or CD on gestation day 8 (plug = day 1). They were killed and subjected to teratological examination on day 18. CD, but not CB, increased prenatal mortality but failed to interact significantly with dietary protein level. Fetal weights were decreased in the 4% and 8% dietary protein groups, but cytochalasin treatment did not exacerbate this effect. Cytochalasin treatment was associated with gross fetal malformations, primarily neural tube defects. Although CB and CD did not significantly increase the percentage of grossly malformed fetuses per litter, the data was suggestive of such an effect, and the incidence of affected litters was increased by cytochalasin treatment in all but the 4% protein group. Skeletal defects, such as jaw malformations, rib or sternebrae variations, and unossified skull bones appeared to be increased by both cytochalasin treatment and dietary protein deficiency. The differences from control values were nonsignificant, however, except for some cases of cytochalasin effects on skull ossification. These results show a general lack of effect of protein deprivation on the developmental toxicity of cytochalasins.