Stability of thioTEPA and its metabolites, TEPA, monochloroTEPA and thioTEPA-mercapturate, in plasma and urine

The degradation of N,N′,N′′-triethylenethiophosphoramide (thioTEPA) and its metabolites N,N′,N′′-triethylenephosphoramide (TEPA), N,N′-diethylene,N′′-2-chloroethylphosphoramide (monochloroTEPA) and thioTEPA-mercapturate in plasma and urine has been investigated. ThioTEPA, TEPA and monochloroTEPA were analyzed using a gas chromatographic (GC) system with selective nitrogen/phosphorous detection; thioTEPA-mercapturate was analyzed on a liquid chromatography-mass spectrometric (LC-MS) system. The influences of pH and temperature on the stability of thioTEPA and its metabolites were studied. An increase in degradation rate was observed with decreasing pH as measured for all studied metabolites. In urine the rate of degradation at 37°C was approximately 2.5±1 times higher than at 22°C. At 37°C thioTEPA and TEPA were more stable in plasma than in urine, with half lives ranging from 9–20 h for urine and 13–34 h for plasma at pH 6. Mono- and dichloro derivatives of thioTEPA were formed in urine and the monochloro derivative was found in plasma. Degradation of TEPA in plasma and urine resulted in the formation of monochloroTEPA. During the degradation of TEPA in plasma also the methoxy derivative of TEPA was formed as a consequence of the applied procedure. The monochloro derivative of thioTEPA-mercapturate was formed in urine, whereas for monochloroTEPA no degradation products could be detected.     

Effects of 1-beta-D-arabinofuranosylcytosine and phorbol ester on differentiation of human K562 erythroleukemia cells

We have previously demonstrated that 1-beta-D-arabinofuranosylcytosine (ara-C) induces hemoglobin synthesis in human K562 erythroleukemia cells. The present study extends these findings by demonstrating that ara-C treatment of K562 cells results in both increased heme synthesis and accumulation of alpha-, gamma-, epsilon-, and zeta-globin RNA. The results also demonstrate that ara-C enhances K562 cell surface expression of glycophorin. Furthermore, we demonstrate that phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) inhibits the effects of ara-C on heme production, accumulation of globin RNA, and glycophorin expression. The inhibitory effect occurs maximally when K562 cells are treated with TPA before undergoing ara-C-induced commitment to erythroid differentiation. These findings suggest that TPA inhibits an early step in the process required for ara-C to enhance expression of genes involved in the erythroid program.     

Nonylphenol-induced oxidative stress and cytotoxicity in testicular Sertoli cells

Nonylphenol (NP) as an environment contaminant has been demonstrated to adversely affect male reproduction. The main objective of this study was to evaluate NP-induced oxidative stress and toxicity in testicular Sertoli cells. Sertoli cells were exposed to 10–40 μM NP for 24 h. Cell death and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay, while fluorescein diacetate (FDA) and propidium iodide (PI) staining was used to examine the morphological changes following NP exposure. Subsequently, we found that short-term treatment (2 h) of NP caused intracellular accumulation of reactive oxygen species (ROS), which was evaluated by loading of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) without visible morphological changes. Loss of mitochondrial membrane potential (MMP) was detected following 12 and 24 h treatment of NP and assessment by Rhodamine 123 (Rh123) staining. In addition, incubation with NP for 12 h also increased lipid peroxidation of Sertoli cells. These results indicated that low micromolar concentrations of NP induce an adverse oxidative stress in rat Sertoli cells.     

Implication of alterations in intracellular calcium ion homoeostasis in the advent of paracetamol-induced cytotoxicity in primary mouse hepatocyte monolayer cultures

We have examined the fluctuation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fluorescent probe quin-2 during the cytotoxic response induced by low concentrations (100–250 μ ) of the model hepatotoxin paracetamol (APAP) in primary mouse hepatocyte cultures over 5 days. APAP-associated increases in [Ca²⁺]_i were recorded prior to APAP-associated cytotoxicity, and correlated with the subsequent loss of cell viability as measured by intracellular lactate dehydrogenase and K⁺ efflux. Co-incubation with promethazine (1 μ ) or ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N′,N′-tetraacetic 0215 acid (4 m ) attenuated both the APAP-associated [Ca²⁺]_i changes and cytotoxicity. These results support the hypothesis that mobilization of intracellular Ca²⁺ may be an important early event in APAP-induced hepatotoxicity.     

Excitatory amino acid transporters expressed by synovial fibroblasts in rats with collagen-induced arthritis

Although previous studies have demonstrated increased levels of the brain neurotransmitter glutamate (Glu) in the synovial fluid from patients with arthritis, not much attention has been paid to the possible role of Glu in joint synovial tissues to date. Constitutive expression of mRNA was for the first time shown with glutamate aspartate transporter, glutamate transporter-1 and excitatory amino acid carrier-1 (EAAC1), in addition to with particular ionotropic and metabotropic Glu receptors, in cultured synovial fibroblasts prepared from knee joints of male Lewis rats. Immunohistochemical analysis revealed high localization of immunoreactive EAAC1 at synovial tissues. The accumulation of [³H]Glu occurred in a temperature- and sodium-dependent manner in cultured synovial fibroblasts, with a K_m of 23.1 ± 1.1 μM and a V_max of 237.1 ± 31.1 pmol/(mg protein min), respectively. In rats with arthritis induced by immunization to type-II collagen, marked increases were seen in hind paw volume, cytokine mRNA expression and Glu levels in synovial tissues, in addition to histological erosion. In cultured synovial fibroblasts prepared from these arthritic rats, [³H]Glu accumulation was drastically increased with biochemical and pharmacological profiles similar to those seen in normal synovial fibroblasts. The exposure to Glu at 500 μM doubled the incorporation of 5-bromo-2′-deoxyuridine in cultured synovial fibroblasts of arthritic but not normal rats, without significantly affecting mRNA expression of different cytokines in both synovial fibroblasts. These results suggest that Glu may at least in part play a role in mechanisms associated with cellular proliferation through particular transporters functionally expressed by synovium in rheumatoid arthritis.     

Comparison of chlordecone effects on autoimmunity in (NZBxNZW) F(1) and BALB/c mice

Arecoline, the main areca alkaloid in betel quid (BQ), is reported to have cytotoxic, genotoxic, and mutagenic effects in various cells. It shows strong correlation to the incidence of oral submucous fibrosis, leukoplakia, and oral cancer. To clarify the role of arecoline in BQ-induced carcinogenesis, primary human gingival keratinocyes (GK) and human KB epithelial cells were used for studying the molecular mechanisms of arecoline-mediated cell cycle deregulation for comparison. After 24 h of exposure, arecoline (0.2–0.8 mM) inhibited KB cell growth in a dose- and time-dependent manner with a reduction in cell number by 27–37 and 37–58%, respectively, as determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) and sulforhodamine B (SRB) assays. Incubation of KB cells with arecoline (0.1–0.4 mM) caused late-S and G2/M phases’ cell cycle arrest. Western blot analysis revealed that arecoline induced cyclin Bl, Wee 1, and phosphorylated cdc2 protein levels whereas it declined p21 protein expression in KB cancer cells. Nevertheless, arecoline induced p21, but decreased cdc2 and cyclin B1 protein levels in GK. We demonstrated that higher concentrations of arecoline (0.2–1.2 mM) induced both cell necrosis and apoptosis as detected by DNA fragmentation and Annexin V–PI staining after long-term (48 h) treatment. Our results suggest that differential regulation of S and/or G2/M cell cycle-related proteins in the GK and KB cells play a crucial role in different stages of BQ-mediated carcinogenesis.     

Are beta-adrenoreceptors involved in the intestinal absorption and tissue distribution of iron in the rat?

⁵⁹Fe was incorporated in vivo into intestinal sacs prepared in iron deficient rats and ⁵⁹Fe counts were detected in blood, spleen, liver, femur and intestine. The i.v. injection of isoproterenol (i.v. 2 μg/rat) or N′,O′-dibutyryladenosine 3′,5′-cyclic monophosphate (10 μM/100 g b.w.) enhanced significantly the iron counts in blood, spleen, liver and femur but not in intestine. (−)-Propranolol (2 mg · kg⁻¹ b.w.i.v.) antagonized the stimulatory effect of isoproterenol. It is suggested that isoproterenol increases iron absorption via the stimulation of β-adrenoreceptors of the intestinal mucosa.     

Simultaneous determination of flavonoids in Ixeridium gracile by micellar electrokinetic chromatography

A micellar electrokinetic chromatography (MEKC) method has been developed for the quantitative analysis of five flavonoids: luteolin 7-O-glucoside (LG), 2′,4′-dihydroxy-dihydrochalcone (DD), 2′,4′-dihydroxy-chalcone (DC), 7-hydroxy-flavanone (HF) and quercetin 3-O-galactoside (QG) in Ixeridium gracile with UV detection at 275 nm. The applied voltage was 25 kV and the capillary temperature was kept constant at 25 °C. The effects of buffer pH, the concentration of electrolyte and organic modifier on migration behavior were studied. Optimum separation condition was achieved with 15 mM borate, 30 mM sodium dodecyl sulfate (SDS) and 10% (v/v) ethanol at pH 10.5. Regression equations showed good linear relationships (correlation coefficients: 0.9984, 0.9991, 0.9994, 0.9995 and 0.9997) between the peak area of each compound and their concentrations. The relative standard deviations (R.S.D.) of the migration time and peak area were less 1.67 and 3.53% (intra-day), and 1.82 and 3.73% (inter-day), respectively, under the optimum separation conditions. The contents of the five active compounds in I. gracile were determined with satisfactory repeatability and recovery.     

Influence of dichlorodiphenylchloroethylene on vascular endothelial growth factor and insulin-like growth factor in human and rat ovarian cells

1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (p,p′-DDE, DDE), a metabolite of DDT is a persistent hormonally active environmental toxicant present in human serum and follicular fluid. The objective of this study was to investigate the effects of DDE on the expression of the ovarian vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF-1) in primary cultures of human granulosa cells and in the rat ovary. Granulosa cells were obtained at the time of oocyte retrieval for in vitro fertilization and cultured with environmentally relevant concentrations of DDE. Immature female rats were treated with 100 μg DDE/kg body weight or vehicle at 28 and 31 days of age and then euthanized at 50 days of age for collection of ovarian tissue. Expression of VEGF, the VEGF receptor fetal liver kinase (Flk-1) and IGF-1 were determined by Western blotting analysis of protein lysates from granulosa cell cultures and by immunohistochemistry in the rat ovary. DDE at concentrations of 100–1000 ng/mL increased the expression of VEGF, Flk-1 and IGF-1 in vitro in primary cultures of human granulosa cells, with the highest expression occurring at 1000 ng/mL. Similarly, acute administration of DDE resulted in a significant increase in immunoreactive VEGF, Flk-1 and IGF-1 in the rat ovary. We conclude that DDE, at levels, which have been detected in humans, alters the expression of the ovarian growth factors VEGF and IGF-1 both in vivo and in vitro. This alteration in expression of growth factors may lead to altered ovarian function as seen in polycystic ovaries and impaired fertility.     

Clonidine accumulation in human neuronal cells

After transport across several epithelial barriers including the blood–brain barrier, clonidine interacts with ₂-adrenergic receptors and imidazoline binding sites in the brain. We hypothesized that neuronal cells take up clonidine thereby removing the drug from the extracellular fluid compartment. Uptake of [³H]clonidine into SH-SY5Y neuroblastoma cells was linear for up to 1 min, unaffected by inside directed Na⁺ or Cl⁻ gradients but strongly inhibited by an outside pH of 6.0. The cells accumulated [³H]clonidine 50–70-fold uphill against a concentration gradient. Unlabeled clonidine, guanabenz, imipramine, diphenhydramine, maprotiline, quinine and the endogenous monoamine phenylethylamine (2 mM) strongly inhibited the [³H]clonidine uptake by 60–95%. Tetraethylammonium, choline and N-methyl-4-phenylpyridinium had no effect. The accumulation at pH 7.5 was saturable with an apparent Michaelis–Menten constant (K_t) of 0.7 mM. We conclude that SH-SY5Y cells not only bind clonidine to extracellular receptors but also take up the drug rapidly by a specific and concentrative mechanism.     

Effects of cytosolic ATP and other nucleotides on Ca-activated K channels in cultured bovine adrenal chromaffin cells

The effects of cytosolic ATP on Ca²⁺-dependent K⁺ (K_Ca) channel activation in cultured bovine adrenal chromaffin cells were investigated by using single-channel recording patch-clamp techniques. Application of ATP to the intracellular surface of excised inside-out patches activated K_Ca channels in a dose-dependent manner at 30 μM to 10 mM. The K_Ca channels also were activated by 3 mM of adenosine 5′-O-(3′-thiotriphosphate) (ATPγS), a non-hydrolyzable analogue of ATP, but not by 5′-adenylylimidodiphosphate (AMP-PNP) (from 300 μM to 3 mM). Furthermore, other nucleotides also activated K_Ca channels in inside-out patches. This modulation took place without addition of exogenous protein kinase and was dependent on the presence of Mg²⁺ in the bathing solution. Staurosporine, a non-specific kinase inhibitor, or H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein kinase inhibitor, was unable to alter ATP-mediated K_Ca channel activation. Following complete removal of Mg²⁺, a higher concentration of ATP (10 mM) and other nucleotides was required to activate K_Ca channels; however, Mg²⁺ was ineffective in altering the activation of K_Ca channels by itself. It is concluded that intracellular ATP and other nucleotides activate K_Ca channels directly. These nucleotides may regulate catecholamine release by changing the cell membrane potential in adrenal chromaffin cells.     

Acute renal toxicity of thiabendazole (TBZ) in ICR mice

The acute toxic effects of thiabendazole [2-(4′-thiazolyl)benzimidazole; TBZ] on the kidneys of ICR mice were investigated. The mice were given 0, 250, 500 or 1000 mg TBZ/kg body weight by gavage (using olive oil as a vehicle), and the kidneys were subjected to pathological examination at 1, 3, 5 or 24 hr after dosing. Gross findings were slight enlargement and the presence of whitish areas (white maculae) in kidneys of treated mice at 3, 5 or 24 hr after dosing. Histological findings were desquamation of degenerated cells in proximal tubules of treated mice at 1 hr. Dilation of proximal, distal and collecting tubules was apparent in treated mice at 3, 5 and 24 hr. TBZ-induced renal injury was reduced by pretreatment with inducers of the microsomal monooxygenase system (sodium phenobarbital, β-naphthoflavone and 3-methylcholanthrene) and were enhanced by pretreatment with inhibitors of that system (2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride and piperonyl butoxide). The concentration of TBZ in blood at 1 or 5 hr after dosing was lower in mice pretreated with microsomal monooxygenase system inducers and was higher in those pretreated with the inhibitors, than in those given TBZ alone. These results suggest that TBZ-induced renal injury may be attributable to the parent compound rather than its metabolites. Measurement of organic ion uptake into renal slices revealed significant depression of [1-¹⁴C]tetraethylammonium bromide (TEA) uptake in treated mice at 1 or 5 hr, whereas uptake of p-[glycyl-2-³H]aminohippurate (PAH) was not depressed at 1 or 5 hr after dosing. The reduction in uptake of TEA is interpreted as the result of competitive suppression of the tubular transport of TEA by TBZ. TBZ-induced renal injury was reduced by organic cation transport inhibitors [N′-methylnicotinamide (NMN) or thiamine] but not by organic anion transport inhibitor [p-(dipropylsulphamyl)benzoic acid probenecid], suggesting that the reduction of TBZ-induced renal injury is the result of competitive suppression of the tubular transport of TBZ by NMN or thiamine.     

Different effects of two cyclic chalcone analogues on cell cycle of Jurkat T cells

It has been previously shown that the cyclic chalcone analogues E-2-(4′-methoxybenzylidene)- (2) and E-2-(4′-methylbenzylidene)-1-benzusuberone (3) inhibited proliferation of various murine and human tumor cells. In order to gain new insights into the cytotoxic mechanism of the two compounds detection of apoptosis and necrosis of Jurkat T cells exposed to 2 and 3 were performed by flow cytometry using the Annexin V-FITC and propidium iodide double staining method. Analysis of the DNA histograms at 8, 24 and 48 h exposure times showed that near equitoxic doses of 2 and 3 had different effects on the cell cycle of the exposed cells. The immediate (8 h) effect of 2 was a remarkable decrease of cells in the G₀/G₁ phase and increase in the G₂/M phase. This effect could also be seen in the histogram of cells at the 24 h time point. On the contrary, such an effect of 3 could not be observed. At the 24 and 48 h time points accumulation of sub-G₀ (apoptotic and necrotic) and hyperdiploid cells could be detected after both treatments. Incubation of 2 and 3 with reduced glutathione under cell-free conditions indicated spontaneous conjugation (non-redox) reaction at pH 7.4 and pH 9.0. Analyzing the mechanism of action the total thiol content of the cells exposed to compounds 2 and 3 was determined. Compound 2 showed to reduce the total cellular thiol level both under nutrient-free and nutrient-supplemented conditions. Under the latter conditions an increase of the total thiol level of the cells exposed to 3 for 4 h could be observed. The different effect of the two compounds on the cellular thiol status might contribute to the different tumor cytotoxicity of the cyclic chalcone analogues 2 and 3. Investigation of antioxidant capacity of the compounds by monitoring time course of the Fenton-reaction initiated in vitro degradation of 2-deoxyribose indicated that both compounds displayed hydroxyl radical scavenger activity. The experiments provide further details of dual – cytotoxic and cytoprotective (chemopreventive) – effects of the compounds.     

Involvement of the extracellular signal-regulated protein kinase (ERK) pathway in the induction of apoptosis by cadmium chloride in CCRF-CEM cells

When CCRF-CEM cells were incubated with 5–40 μM CdCl_2, apoptosis was observed most clearly at 10 μM. Prior to the development of apoptosis, mitogen-activated protein kinases (MAPKs), i.e. extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK, were activated with different sensitivity to CdCl₂ exposure. ERK and p38 MAPK were phosphorylated with incubation of 1 μM CdCl_2, but higher than 20 μM CdCl₂ was required for the clear phosphorylation of JNK. In the time–course study, ERK and p38 MAPK were phosphorylated earlier than JNK after CdCl₂ exposure. The in vitro activities of MAPKs also increased in response to CdCl₂ exposure. Pretreatment with an intracellular Ca²⁺ chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA/AM), suppressed almost completely CdCl₂-induced phosphorylation of JNK and p38 MAPK, but not ERK phosphorylation, indicating that the activation of JNK and p38 MAPK depends on the intracellular Ca²⁺ but that of ERK does not. On the other hand, treatment with a MAPK/ERK kinase (MEK) inhibitor, U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), suppressed CdCl₂-induced ERK activation and the apoptosis as well. The inhibition of p38 MAPK activity with SB203580 (4-[4-fluorophenyl]-2-[4-methylsulfinylphenyl]-5-[4-pyridyl]1H-imidazole) did not protect cells from apoptosis. The present results showed that the activation of ERK, JNK, and p38 MAPK is differently regulated in CCRF-CEM cells exposed to CdCl_2, and that the ERK pathway seems to be responsible for the induction of apoptosis by CdCl₂ exposure in this human T cell line.     

Chemical change of chlorinated bisphenol A by ultraviolet irradiation and cytotoxicity of their products on Jurkat cells

Chlorinated derivatives of bisphenol A (ClBPAs) have been detected in wastewater from waste paper recycling plants. BPA and ClBPAs are always exposed to ultraviolet (UV) radiation in the environment and consequently various photoproducts might be produced. Acute cytotoxicity of photoproducts of BPA and ClBPAs are not known. In this study, we investigated the cytotoxicity and chemical structure of photoproducts of BPA and ClBPAs (3-chlorobisphenol A (3-ClBPA), 3,3′-dichlorobisphenol A (3,3′-diClBPA) and 3,3′,5-trichlorobisphenol A (3,3′,5-triClBPA)) after UV irradiation (UVA, UVB and UVC). The toxicities of photoproducts on Jurkat cells were determined by Alamar Blue assay, and the chemical structures of the photoproducts were identified using GC/MS. The cytotoxicities of 3-ClBPA and 3,3′-diClBPA were higher than that of BPA and 3,3′,5-triClBPA. In addition, the toxicities of ClBPAs were increased by the irradiation of UVB and UVC at 100 J/cm² and decreased at 1000 J/cm² in comparison with those at 100 J/cm², indicating that their structures had changed. 3-Hydroxybisphenol A (3-OHBPA) was detected in the photoproducts of 3-ClBPA irradiated with UVB and UVC at 100 J/cm², and 3-OHBPA and 3-chloro-3′-hydroxybisphenol A (3-Cl-3′-OHBPA) were detected in those of 3,3′-diClBPA. However, these hydroxylated BPAs were not detected in the photoproducts exposed to 1000 J/cm². The cytotoxicity of 3-OHBPA was the almost same as ClBPAs after UVB and UVC irradiation. These results indicate that the formation of hydroxylated BPAs might contribute to the increase in toxicity caused by UV irradiation.     

Analysis of the inhibition of commitment of murine erythroleukemia (MEL) cells to terminal maturation by N6-methyladenosine.

Treatment of cultured murine erythroleukemia (MEL or Friend) cells with N6-methylated derivatives of adenosine inhibited erythroid cell differentiation induced by various agents. N6-Methyladenosine (N6mAdo) inhibited initiation of commitment to terminal maturation and prevented accumulation of hemoglobin in a concentration-dependent manner. Treatment with N6mAdo slowed cell growth without causing substantial inhibition in the rate of DNA synthesis and a marked decrease in viability and clonogenic potential of MEL cells. Furthermore, N6mAdo decreased the cytoplasmic accumulation of beta(major) globin mRNA and affected its structural integrity in MEL cells. Cells pre-exposed to N6mAdo failed to initiate commitment as early as control cells upon challenge with the inducer dimethyl sulfoxide. N6mAdo-induced inhibition of commitment was not reversed but rather was potentiated by the presence of adenine, L-homocysteine and/or L-methionine, agents involved in the active methylation cycle. To this respect, N6mAdo-induced inhibition of commitment was found to be different from that caused by cordycepin (3'-deoxyadenosine, an inhibitor of RNA methylation and mRNA polyadenylation). The latter inhibition was fully reversed by the addition of L-methionine. These findings indicate that N6-methyladenosine: (a) blocks a central process that is required for initiation of commitment; and (b) decreases accumulation of beta (major) globin mRNA, causes mRNA degradation and prevents hemoglobin synthesis. Due to the differential sensitivity of N6mAdo- and cordycepin-induced blockade of commitment to L-methionine, these agents inhibit commitment by acting via two different mechanisms impinging on the final pathway of MEL erythroid cell maturation.     

Basil extract inhibits the sulfotransferase mediated formation of DNA adducts of the procarcinogen 1′-hydroxyestragole by rat and human liver S9 homogenates and in HepG2 human hepatoma cells

The effects of a basil extract on the sulfation and concomitant DNA adduct formation of the proximate carcinogen 1′-hydroxyestragole were studied using rat and human liver S9 homogenates and the human hepatoma cell line HepG2. Basil was chosen since it contains the procarcinogen estragole that can be metabolized to 1′-hydroxyestragole by cytochrome P450 enzymes. Basil extract addition to incubations of rat and human liver S9 homogenates with 1′-hydroxyestragole, the sulfotransferase cofactor PAPS, and 2′-deoxyguanosine resulted in a dose-dependent inhibition of N²-(trans-isoestragol-3′-yl)-2′-deoxyguanosine formation. Because the inhibition resembled the inhibition by the sulfotransferase inhibitor pentachlorophenol and since the inhibition was not observed in incubations with the direct electrophile 1′-acetoxyestragole it is concluded that the inhibition occurs at the level of the sulfotransferase mediated bioactivation step. Additional experiments in HepG2 cells revealed the same protective effect of basil extract in intact cells, demonstrating that the inhibitors are able to enter the cells.

The results of this study suggest that bioactivation and subsequent adverse effects of 1′-hydroxyestragole might be lower in a matrix of other basil ingredients than what would be expected on the basis of experiments using 1′-hydroxyestragole as a single compound.     

Neural crest cell motility in valproic acid

Neural crest cells (NCCs) exit the dorsal neural tube and migrate to sites where they form diverse tissues. Valproic acid (VPA) is an anticonvulsant drug that induces neural tube and related defects. Altered NCC migration and proliferation have been proposed as mechanisms of teratogenicity. We cultured neural tube segments from chick embryos in 0.75–3.0 mM VPA. We used image analysis, proliferation assays, and fluorescence localization to investigate NCCs during VPA exposure. VPA inhibited attachment of explants and the number that produced migrating cells. VPA markedly decreased the proportion of cells migrating individually, promoting migration as epithelial sheets. VPA at 3 mM decreased cellular spreading. Area and perimeter change per minute were reduced, but migration velocity was not. VPA at 2 mM reduced proliferation 11% and 3 mM arrested proliferation. Immunostaining of VPA-exposed explants revealed N-cadherin-positive cell boundaries within sheets, but independent NCCs did not stain. F-actin staining was reduced in independent NCCs. The data support a VPA mechanism involving interference with epithelial–mesenchymal transition.