An aqueous extract from Artemisia capillaris inhibits acute gastric injury through mucosal stabilization.

BACKGROUND: Artemisia capillaris is among the most abundantly used traditional medicines, utilized in East Asia to treat diverse illnesses, including gastrointestinal tract diseases. We previously reported that an aqueous extract of A. capillaris (AEAC) inhibited gastric inflammation induced by HCl/ethanol via reactive oxygen species scavenging and NF-κB downregulation. To date, the pharmacological potential of AEAC for promoting mucosal integrity has not been studied. RESULTS: Here, we report that a single treatment with AEAC increased mucus production, and repeated administration of AEAC abolished HCl/ethanol-induced mucosal injury in vivo. Single- and multiple-dose AEAC treatments measurably increased the expression of mucosal stabilizing factors in vivo, including mucin (MUC) 5 AC, MUC6, and trefoil factor (TFF) 1 and TFF2 (but not TFF3). AEAC also induced mucosal stabilizing factors in both SNU-601 cells and RGM cells through phosphorylation of extracellular signal-regulated kinases. CONCLUSION: Taken together, our results suggest that AEAC protects against HCl/ethanol-induced gastritis by upregulating MUCs and TFFs and stabilizing the mucosal epithelium. © 2021 Society of Chemical Industry.

The aqueous extract from Artemisia capillaris inhibits acute gastric mucosal injury by inhibition of ROS and NF-kB.

Artemisia capillaris, also called "InJin" in Korean, has been used as traditional oriental medicine in Korea because of its various pharmacological activities. These include hepatoprotective, analgesic, and antipyretic activities. The present study was designed to validate the beneficial effects of the aqueous extract of A. capillaris (AEAC) against acute gastric mucosal injury and investigate the underlying molecular mechanisms. The pharmacological efficacy of AEAC was evaluated using the gastric ulcer index and histological examination. AEAC decreased gastric mucosal lesions mediated by HCl/ethanol in vivo in a dose-dependent manner. Interestingly, the mucosal damage was almost prevented by pretreatment with 200 or 400?mg/kg AEAC. However, AEAC did not have acid-neutralizing activity in vitro and did not prevent histamine secretion in HMC-1 mast cells. In the gastric mucosa, AEAC also significantly inhibited lipid peroxide formation through superoxide dismutase (SOD) activation. Moreover, AEAC strongly reduced the generation of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and interleukin-1? (IL-1?), through nuclear factor kappa B (NF-?B) downregulation. Taken together, our findings suggest that AEAC inhibits inflammation and maintains oxidant/antioxidant homeostasis, resulting in a gastro-protective effect against HCl/ethanol-induced gastric damage. Therefore, AEAC might be a promising drug or useful neutraceutical for treatment of gastritis and gastric ulcer.

Long-Term Clinical Outcomes of Transient and Persistent No Reflow Phenomena following Percutaneous Coronary Intervention in Patients with Acute Myocardial Infarction.

BACKGROUND AND OBJECTIVES: There is limited information on the transient or persistent no reflow phenomenon in patients with acute myocardial infarction (AMI) undergoing percutaneous coronary intervention (PCI). SUBJECTS AND METHODS: The study analyzed 4329 patients with AMI from a Korean multicenter registry who underwent PCI using coronary stents (2668 ST-elevation and 1661 non-ST-elevation myocardial infarction [MI] patients): 4071 patients without any no reflow, 213 with transient no reflow (no reflow with final thrombolysis in myocardial infarction [TIMI] flow grade 3), and 45 with persistent no reflow (no reflow with final TIMI flow grade≤2). The primary endpoint was all-cause mortality during 3-year follow-up. We also analyzed the incidence of cardiac mortality, non-fatal MI, re-hospitalization due to heart failure, target vessel revascularization, and stent thrombosis. RESULTS: The persistent no reflow group was associated with higher all-cause mortality (hazard ratio [HR] 1.98, 95% confidence interval [CI] 1.08-3.65, p=0.028) and cardiac mortality (HR 3.28, 95% CI 1.54-6.95, p=0.002) compared with the normal reflow group. Transient no reflow increased all-cause mortality only when compared with normal reflow group (HR 1.58, 95% CI 1.11-2.24, p=0.010). When comparing transient and persistent no reflow, persistent no reflow was associated with increased all-cause mortality (46.7 vs. 24.4%, log rank p=0.033). CONCLUSION: The persistent no reflow phenomenon was associated with a poor in-hospital outcome and increased long-term mortality mainly driven by increased cardiac mortality compared to the transient no reflow phenomenon or normal reflow.

A mixed formulation of lactic acid bacteria inhibits trinitrobenzene-sulfonic-acid-induced inflammatory changes of the colon tissue in mice.

Lactic acid bacteria (LAB) are probiotics that provide numerous beneficial effects on the host body, especially on the intestine. Combining several strains of LAB, we prepared a formulation containing four different LAB and studied its anti-inflammatory activity both in vitro and in vivo. The formulation significantly reduced NO production from RAW 264.7 cells treated with bacterial lipopolysaccharide, indicating that the formulation might include antiinflammatory activity. The formulation also suppressed inflammatory change induced by trinitrobenzene sulfonic acid (TNBS) in mice, where oral or rectal administration of the formulation protected the colon tissue from the damage by TNBS. Expressions of the IL-6 and FasL genes appeared to be down-regulated by the formulation in TNBS-treated colon tissues, suggesting that the suppression of those genes may be involved in the anti-inflammatory activity of the formulation.

Four-Week Repeated Oral Toxicity Study of AIP1, a Water-soluble Carbohydrate Fraction from Artemisia iwayomogi in Mice.

Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found in Korea and used as a traditional medicine to treat liver disease. AIP1, a water-soluble carbohydrate fraction from Artemisia iwayomogi, showed anti-tumor and immuno-modulating activities in animal studies. A subacute toxicological evaluation of AIP1 was performed for 4 weeks in ICR mice. After administration of AIP1 (0, 20, 100, 500 mg/kg/day), the clinical signs, mortalities, body weight changes, hematology, blood clinical biochemistry, urinalysis, organ histopathology, organ weights and gross finding were examined. The results showed that there were no significant differences in body weight changes, food intakes, water consumptions, or organ weights among different dose groups. Also we observed no death and abnormal clinical signs during the experimental period. Between the groups orally treated with AIP1 and the control group, there was no statistical significance in hematological test or serum biochemical values. Histopathological examination showed no abnormal changes in AIP1 groups. These results suggest that no observed adverse effect level (NOAEL) of the oral administration of AIP1 for 4 weeks was considered to be more than 500 mg/kg/ day in mice under the condition investigated in current study.

Amelioration of oxidative stress by dandelion extract through CYP2E1 suppression against acute liver injury induced by carbon tetrachloride in Sprague-Dawley rats.

The protective effects of common dandelion leaf water extract (DLWE) were investigated by carbon tetrachloride (CCl4) induced hepatitis in Sprague-Dawley rats. The animals were divided into five groups: normal control, DLWE control, CCl4 control, and two DLWE groups (0.5 and 2 g/kg bw). After 1 week of administering corresponding vehicle or DLWE, a single dose of CCl4 (50% CCl4/olive oil; 0.5 mL/kg bw) was administered 24 h before killing in order to produce acute liver injury. The DLWE treatment significantly decreased CCl4-induced hepatic enzyme activities (AST, ALT and LDH) in a dose dependent manner. Also, the obstructed release of TG and cholesterol into the serum was repaired by DLWE administration. Hepatic lipid peroxidation was elevated while the GSH content and antioxidative enzyme activities were reduced in the liver as a result of CCl4 administration, which were counteracted by DLWE administration. Furthermore, the hepatocytotoxic effects of CCl4 were confirmed by significantly elevated Fas and TNF-α mRNA expression levels, but DLWE down-regulated these expressions to the levels of the normal control. Highly up-regulated cytochrome P450 2E1 was also lowered significantly in the DLWE groups. These results indicate that DLWE has a protective effect against CCl4-induced hepatic damage with at least part of its effect being attributable to the attenuation of oxidative stress and inflammatory processes resulting from cytochrome P450 activation by CCl4.

A Carbohydrate Fraction, AIP1, from Artemisia Iwayomogi Reduces the Action Potential Duration by Activation of Rapidly Activating Delayed Rectifier K Channels in Rabbit Ventricular Myocytes.

We investigated the effects of a hot-water extract of Artemisia iwayomogi, a plant belonging to family Compositae, on cardiac ventricular delayed rectifier K(+) current (I(K)) using the patch clamp technique. The carbohydrate fraction AIP1 dose-dependently increased the heart rate with an apparent EC(50) value of 56.1+/-5.5 microg/ml. Application of AIP1 reduced the action potential duration (APD) in concentration-dependent fashion by activating I(K) without significantly altering the resting membrane potential (IC(50) value of APD(50): 54.80+/-2.24, IC(50) value of APD(90): 57.45+/-3.47 microg/ml). Based on the results, all experiments were performed with 50 microg/ml of AIP1. Pre-treatment with the rapidly activating delayed rectifier K(+) current (I(Kr)) inhibitor, E-4031 prolonged APD. However, additional application of AIP1 did not reduce APD. The inhibition of slowly activating delayed rectifier K(+) current (I(Ks)) by chromanol 293B did not change the effect of AIP1. AIP1 did not significantly affect coronary arterial tone or ion channels, even at the highest concentration of AIP1. In summary, AIP1 reduces APD by activating I(Kr) but not I(Ks). These results suggest that the natural product AIP1 may provide an adjunctive therapy of long QT syndrome.

Constituents of Limonia acidissima inhibit LPS-induced nitric oxide production in BV-2 microglia.

The ethyl acetate (EtOAc) soluble fraction of the 85% ethanol (EtOH) extract of the dried bark of Limonia acidissima potently inhibited nitric oxide (NO) production in lipopolysaccharide (LPS) activated BV-2 cells, a microglial cell line. Bioassay-guided column chromatography separation afforded a new stereoisomer of neolignan, (7'E)-(7R,8S)-4-hydroxy-3,5'-dimethoxy-4',7-epoxy-8,3'-neolig-7'-en-9,9'-diyil diacetate (1), together with two known lignans, (+)-yangambin (2) and (+)-syringaresinol (3), three known triterpenoids, hederatriol (4), basic acid methyl ester (5), and 3ß-hydroxyolean-12-en-11-one (6), and four known fatty acid derivatives, cascarillic acid (7), (+)-α-dimorphecolic acid (8), 8(R)-hydroxylinoleic acid (9), and (6Z,9Z,12Z)-pentadecatrienoic acid (10). The structure of the new compound 1 was elucidated by detailed analysis of spectroscopic data and circular dichroism (CD) spectroscopy. Compounds 1, 3-6, and 8-10 isolated from L. acidissima significantly reduced NO production in LPS-stimulated BV-2 microglia cells.

TOP1 and 2, polysaccharides from Taraxacum officinale, attenuate CCl(4)-induced hepatic damage through the modulation of NF-kappaB and its regulatory mediators.

In this work, we estimate the inhibitory effect of two polysaccharides from Taraxacum officinale (TOP) on CCl(4)-induced oxidative stress and inflammation in Sprague-Dawley rats. TOP1 and 2 (304, 92 mg/kg bw) were administered for 7 days via a stomach sonde, and hepatitis was induced by a single dose of CCl(4) (50% CCl(4)/olive oil; 0.5 mL/kg bw) administration. CCl(4) significantly elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. Histopathological observation further revealed that CCl(4)-induced moderate levels of inflammatory cell infiltration, centrilobular fatty change, apoptosis, and necrosis. However, TOPs pretreatment markedly decreased AST and ALT activities as well as hepatic lesions. TOPs also increased free radical scavenging activity, as exhibited by a lowered TBARS concentration. TOPs pretreatment also reversed other hepatitis-associated symptoms, including GSH depletion, inhibited anti-oxidative enzyme activities, up-regulation of NF-kappaB and increased expression of its regulatory inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1beta. These results suggest that TOPs have a hepatoprotective effect by modulating inflammatory responses and ameliorating oxidative stress.

AIP1, a carbohydrate fraction from Artemisia iwayomogi, modulates the functional differentiation of bone marrow-derived dendritic cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells (APC) particularly important in the initiation of primary T cell-mediated immune responses. Thus, inhibition of the differentiation and function of DC could lead to the suppression of immunological hyperresponsiveness. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found in Korea and has been used as a traditional anti-inflammatory medicine. We investigated suppressive effects of carbohydrate fraction 1 from the water extracts of A. iwayomogi (AIP1) on the differentiation and function of bone marrow-derived dendritic cells. Bone marrow cells were cultured in the presence of granulocyte monocyte-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 6-7 days. Then, non-adherent cells were harvested for subsequent analyses. Percentage(s) of CD11c+ MHC II+ cell population(s) mostly composed of immature or mature DC and the allogeneic T cell stimulating ability of the cells were reduced by AIP1. Proteomic analyses along with RT-PCR revealed that expressions of several proteins including TNF receptor-associated factor (TRAF) 5-like protein, pyruvate kinase M2 (PKM2), and coactosin-like protein 1 (CLP1) were down-regulated upon AIP1 treatment.

A carbohydrate fraction, AIP1 from Artemisia iwayomogi suppresses pulmonary eosinophilia and Th2-type cytokine production in an ovalbumin-induced allergic asthma. Down-regulation of TNF-alpha expression in the lung.

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. The incidence of asthma is on the rise despite ongoing intensive asthma research. Artemisia iwayomogi, a member of the Compositae, is a perennial herb easily found around Korea and has been used as a traditional anti-inflammatory medicine in liver diseases. We investigated suppressive effects of AIP1, a water-soluble carbohydrate fraction from A. iwayomogi on ovalbumin-induced allergic asthma in BALB/c mice and studied the possible mechanisms of its anti-allergic action. AIP1 significantly reduced pulmonary eosinophilia and Th2 cytokine expression in the lungs as well as serum IgE levels. Flow cytometric analysis of lung-infiltrating cells showed that the surface levels of CD11c and MHC II in CD11c+MHC II+ cells, potent dendritic cells, decreased in animals treated with AIP1. Expression of TNF-alpha, one of several proinflammatory cytokines released into the airway during episodes of asthma, was down-regulated by AIP1 injection, suggesting that reduced expression of TNF-alpha could account for the suppression of pulmonary eosinophilia and Th2-type cytokine production by AIP1.

2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates functional differentiation of mouse bone marrow-derived dendritic cells Downregulation of RelB by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

We have previously shown that benzo(a)pyrene inhibits the growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DCs) [Hwang, J.A., Lee, J.A., Cheong, S.W., Youn, H.J., Park, J.H., 2007. Benzo(a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo(a)pyrene. Toxicol. Lett. 169, 82-90]. Since the toxic effects of benzo(a)pyrene are aryl hydrocarbon receptor (AhR)-dependent, we examined the effects of the very potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the growth and functional differentiation of mouse BM-derived DCs. Ten nanomolars of TCDD had significant effects on functional differentiation of mouse DCs derived from BM cultured in the presence of GM-CSF and IL-4. The yields of DCs, flow-cytometrically analyzed for co-expression of CD11c/MHCII or CD11c/CD86, were reduced for TCDD-treated cultures, but TCDD itself had no effect on the growth of BM. DCs from TCDD-treated cultures expressed higher levels of MHCII and CD86, whereas expression of CD11c was reduced, compared with vehicle-treated cultures. Production of IL-10, but not IL-12, by the DCs from TCDD-treated cultures was decreased. Allogeneic T-cell stimulating ability of TCDD-treated DCs was increased compared to control DCs. The effects of TCDD were dependent on aryl hydrocarbon receptor (AhR), because alpha-naphthoflavone, an AhR antagonist, suppressed the effects of TCDD on IL-10 production and T-cell stimulating ability. RT-PCR revealed the downregulation of RelB, a transcription factor necessary for DCs differentiation and function. Taken together, although benzo(a)pyrene and TCDD exert their effects via binding to AhR, their effects on the growth and functional differentiation of bone marrow-derived DCs are different.

Ethanol extract of fermented soybean, Chungkookjang, inhibits the apoptosis of mouse spleen, and thymus cells.

Apoptosis is a step of the cell cycle which is important in the regulation of immune cell populations. Chungkookjang is a Korean traditional fermented soybean containing microorganisms, enzymes, and bioactive compounds which was used in the treatment of mouse spleen as well as thymus cells (CH1-fermented soybean containing barley, wormwood, and sea tangle; CH2-fermented soybean) and was found to exhibit substantially reduced small DNA fragmentation. An MTT assay showed that the treatment of CH1 and CH2 into the mouse splenocytes and thymocytes sharply increased their survival. Moreover, a FACS analysis also showed that CH1 and CH2 are effective at suppressing the apoptosis of splenocytes and thymocytes. The fermented soybean isoflavone concentrations, which are implicated in lowering breast and prostate cancers, lowering the risk of cardiovascular diseases, and improving bone health, were determined using Capillary Electrophoresis-Electrochemical Detection (CE-ED). The amount of Daidzein in fermented soybean significantly increased by 44-fold dramatically, compared with those in unfermented soybean. In this study, we demonstrated that ethanol extracts of Chungkookjang promote the survival of the mouse spleen and thymus cells in culture by suppressing their apoptotic death. Future studies should investigate which genes are related to apoptosis of the immune cells.

Benzo(a)pyrene inhibits growth and functional differentiation of mouse bone marrow-derived dendritic cells. Downregulation of RelB and eIF3 p170 by benzo(a)pyrene.

In this study, we have investigated effects of benzo(a)pyrene (BP) on growth and functional differentiation of mouse bone marrow (BM)-derived dendritic cells (DC). 1 microM BP dramatically inhibited growth of BM cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although little alterations in surface expression of CD11c, major histocompatibility complex (MHC II), and CD86 molecules characteristic of mature DC were induced by BP, production of cytokines including IL-12, IL-10, and TNF-alpha, and allogeneic T cell stimulating ability were severely impaired. Some of the effects of BP were dependent on arylhydrocarbon receptor (AhR), because alpha-naphthoflavone, an AhR antagonist, suppressed the effects of BP on IL-12 production and T cell stimulating ability, but not on DC proliferation. Expression of RelB, a transcription factor necessary for DC differentiation and function, and eIF3 p170, a subunit of eukaryotic translation initiation factor (eIF)3, was reduced upon BP treatment.

AIP1, a water-soluble fraction from Artemisia iwayomogi, suppresses thymocyte apoptosis in vitro and down-regulates the expression of Fas gene.

The AIP1 fraction, a small water-soluble fraction purified from Artemisia iwayomogi, was shown to increase antibody production and suppress transplanted tumor cell growth in mice. In order to understand its immuno-modulating activity, we have examined the effect of the AIP1 on mouse thymocytes in vitro. Treatment of mouse thymocytes in culture with the fraction resulted in the suppression of the cell death and the extension of the cell survival. A mouse gene array provided a profile of gene expression change showing the pattern of up- and down-regulated genes by the AIP1 treatment, suggesting that the Fas/FasL-dependent apoptosis pathway might be modulated by the fraction.

A carbohydrate fraction, AIP1, from Artemisia iwayomogi down-regulates Fas gene expression and suppresses apoptotic death of the thymocytes induced by 2,3,7,8-tectrachlorodibenzo-p-dioxin.

Apoptotic death of mouse thymocytes in vitro, as induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), involves the up-regulation of Fas gene expression, while a carbohydrate fraction, AIP1, from Artemisia iwayomogi suppresses the death of thymocytes in culture along with the down-regulation of Fas gene expression. We have now investigated whether the AIP1 fraction modulates TCDD-induced thymocyte death. When treated with TCDD and AIP1 fraction together, the thymocytes do not show apoptosis induced by the TCDD treatment. The AIP1 supplementation to the TCDD treatment also down-regulates the TCDD-induced Fas gene up-regulation. These findings indicate that the AIP1 fraction suppresses TCDD-induced thymocyte apoptosis through the modulation of Fas gene expression.

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-beta-(1-->3),(1-->4)-glucanase useful for the preparation of oligosaccharides from barley beta-glucan.

An endo-beta-(1-->3),(1-->4)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo-beta-(1-->3),(1-->4)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis , transformed with the recombinant plasmid pBLC771, produced an extracellular endo-beta-(1-->3),(1-->4)-glucanase that was 130 times (7176 mU ml(-1)) more active than that of the gene donor cells (55 mU ml(-1)), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml(-1)) more active than that of the gene donor cells. M(r) of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble beta-glucan hydrolyzed by over-produced endo-beta-(1-->3),(1-->4)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.

An oligosaccharide fraction from Korean mugwort herb suppresses death of the mouse thymocytes in culture by down-regulating the Fas death receptor gene.

Korean mugwort herb is a preparation of dried leaves from Artemisia species and has been used as a traditional medicine in Asia. An oligosaccharide fraction, AVF3, purified from the preparation promoted survival of the mouse thymocytes in culture. A mouse gene array study suggests that the AVF3 may modulate Fas/FasL dependent apoptotic cell death and thus has influence on the survival of the thymocytes in culture. RT-PCR analysis confirmed the down-regulation of the Fas gene by the AVF3 treatment, supporting that the AVF3 modulated thymocyte death by suppressing the Fas gene expression.

TCDD-induced apoptosis in EL-4 cells deficient of the aryl hydrocarbon receptor and down-regulation of IGFBP-6 prevented the apoptotic cell death.

Although the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been well known for its immunosuppressive activity, the mechanisms of its action have been difficult to elucidate, partly because of its inability of exerting its effects in vitro. We previously reported that insulin-like growth factor-binding protein-6 (IGFBP-6) expression in the thymus was increased by TCDD treatment of mice and that the TCDD-up-regulation of the IGFBP-6 gene was also observed with EL-4 mouse thymoma cells. In the present study, we examined the effects of IGFBP-6 on the TCDD-mediated cytotoxicity in EL-4 cells. By stably expressing IGFBP-6 sense or anti-sense mRNA in the EL-4 line of mouse thymoma cells, it was possible to isolate clones in which IGFBP-6 expression was increased or decreased. Clones expressing IGFBP-6 sense mRNA displayed increased sensitivity to cytotoxicity mediated by TCDD, whereas clones expressing IGFBP-6 anti-sense mRNA displayed reduced sensitivity. TCDD-induced DNA fragmentation was less pronounced in clones expressing IGFBP-6 anti-sense mRNA than clones expressing IGFBP-6 sense mRNA or the empty vector. Caspase 3 was activated by TCDD and anti-sense IGFBP-6 expression reduced its activity. Interestingly, the effects of TCDD were exerted without aromatic hydrocarbon (Ah) receptor (AhR). Taken together, the results have shown that IGFBP-6 mediates the immunotoxic effects of TCDD in EL-4 cells in an AhR-independent pathway.

The fungicide benomyl inhibits differentiation of neural tissue in the Xenopus embryo and animal cap explants.

The toxic effect of benomyl on the embryogenesis of Xenopus laevis was investigated, and the tissues most affected by benomyl were identified. The toxicity of benomyl at various concentrations (5-20 microM) was tested with the Xenopus frog embryo teratogenesis assay (FETAX), used with slight modification. All test embryos subjected to 20 microM of benomyl died, and exposure to 10 and 15 microM benomyl produced growth inhibition and 11 types of severe external malformations. Histological examination of the test embryos showed dysplasia of the brain, eyes, intestine, otic vesicle, and muscle and swelling of the pronephric ducts and integuments. Among the tissues and organs affected, malformation of neural tissue was the most severe. The presumptive ectoderm isolated from st. 9 embryo was cultured in 10 ng/mL of activin A to induce neural tissue and mesoderm. When it was cultured with 10 ng/mL of activin A in the presence of 1 and 10 microM of benomyl, neural tissue induction was inhibited more severely than that of any other tissue. The gene expression of cultivated explants was investigated by reverse transcription-polymerase chain reaction (RT-PCR) assay in order to study the inhibition of neural tissue by benomyl. The results showed that with increasing benomyl concentration, the expression of the neural-specific marker NCAM (neural cell adhesion molecule), was more strongly inhibited than the muscle-specific marker muscle actin. Electron micrographs of test explants showed many residual yolk platelets and mitochondrial degeneration. In the present investigation the most severe toxic effects of benomyl were seen in the nerve tissues of the Xenopus embryo. This inhibition of neural development may have been caused by the inhibition of the assembly of neural microtubules and by the effect of benomyl on neuronal proliferation and migration.