环磷酰胺处理雄小鼠对受精卵和二细胞期胚胎DNA甲基化和组蛋白H3K23乙酰化的影响

目的探讨环磷酰胺处理雄性小鼠对受精卵和早期胚胎DNA甲基化修饰和组蛋白H3第23位赖氨酸(H3K23)乙酰化修饰的影响。方法 20只性成熟的ICR雄小鼠饲喂环磷酰胺6 mg.kg-1 4周。雄鼠与雌鼠交配后收集原核期受精卵和二细胞期胚胎。间接免疫荧光染色技术结合激光共聚焦扫描显微镜技术检测胚胎细胞中DNA甲基化和组蛋白H3K23乙酰化修饰的分布和水平。结果与正常对照组相比,环磷酰胺组原核期受精卵和二细胞期胚胎的DNA甲基化修饰水平和分布没有明显差异;但与正常对照组(3%)相比,环磷酰胺组的二细胞期胚胎微核现象(20%)明显升高(P<0.01);与正常对照组相比,环磷酰胺组受精卵雄原核和二细胞期胚胎细胞核的组蛋白H3K23乙酰化水平明显降低(P<0.05),且胚胎间的变化差异明显升高,其中部分原核期受精卵(58%)和二细胞期胚胎(44%)染色水平明显偏低。结论长期低剂量环磷酰胺处理雄鼠可以导致部分受精卵和二细胞期胚胎组蛋白H3K23乙酰化异常,并且显著增加微核发生率。     

Melatonin-mediated regulation of human MT(1) melatonin receptors expressed in mammalian cells

In mammals, the pineal hormone melatonin activates G protein-coupled MT(1) and MT(2) melatonin receptors. Acute exposure of recombinant MT(1) and MT(2) melatonin receptors to supraphysiological concentrations of melatonin differentially regulates these two receptors with the MT(2), but not the MT(1), exhibiting rapid desensitization and internalization. In the present study, we sought to determine whether prolonged exposure to supraphysiological and physiological concentrations of melatonin desensitized and/or internalized the MT(1) melatonin receptor. Using a Chinese hamster ovary (CHO) cell line stably expressing MT(1)-FLAG or transiently expressing MT(1)-green fluorescent protein (GFP) melatonin receptors, we found that prolonged exposure (8h) to supraphysiological concentrations of melatonin (100 nM) significantly increased the number of MT(1) melatonin receptors and decreased the affinity (K(i)) of melatonin for competition for 2-[125]iodomelatonin. A similar treatment also desensitized the MT(1) melatonin receptor-mediated stimulation of [(35)S]GTPgammaS binding, but did not internalize the receptor. In contrast, prolonged exposure to a concentration of melatonin mimicking nocturnal levels (400 pM) did not affect the number of MT(1) melatonin receptors, the affinity for melatonin, or the functional sensitivity of the receptor. We conclude that in vivo endogenous melatonin does not significantly affect the functional sensitivity of MT(1) melatonin receptors, however, exogenous melatonin taken therapeutically at doses above physiological levels could desensitize the receptor thereby affecting physiological responses mediated following activation of MT(1) melatonin receptors.     

醋酸铅体外染毒对移植后小鼠胚泡发育的影响

目的　用胚胎移植技术评价醋酸铅对啮齿类动物的生殖毒性。方法　采用 2 0和 40 μg mL浓度的醋酸铅对小鼠着床前胚胎进行体外染毒 ,将染毒后可胚泡化的胚胎移植于假孕母鼠体内继续生长发育 ,观察出生前胎鼠形态学改变和出生后子鼠的行为改变 ,并对子鼠大脑进行病理检查。结果　 ( 1)在实验剂量下 ,各染毒组出生前胎鼠形态学检查未见异常。 ( 2 ) 40 μg mL醋酸铅染毒组子鼠出现行为异常 ;回旋运动、阴性趋地运动、翻正反射和断崖回避阳性日均迟于阴性对照组 (P <0 0 5、P <0 0 1、P <0 0 5、P <0 0 5 ) ;悬挂时间明显短于阴性对照组 (P <0 0 5 ) ;10日龄游泳积分低于阴性对照组 (P <0 0 1)。 ( 3 )电镜超微结构显示各染毒组子鼠大脑中毒性损伤。结论　小鼠植入前胚泡体外醋酸铅染毒影响移植子鼠的行为功能 ,并引起中枢神经组织病理损伤 ;胚胎移植技术用于检测化学物胚胎毒性 ,排除母体因素的干扰 ,是一重要研究手段     

Effect of nickel chloride and cadmium acetate on the development of preimplantation mouse embryos in vitro

Preimplantation mouse embryos were used to investigate the toxic effect of nickel chloride and cadmium acetate on early embryo development in vitro.Embryos at the 2- and 4–8 cell stage were cultured in approximately 0.05 ml of mouse embryo culture medium (No. 16), overlaid with paraffin oil and incubated in a humidified atmosphere of 5% CO₂ in air for 48 h. NiCl₂ · 6H₂O was added to the culture medium at concentrations of 10–1000 μM, Cd(CH₃COO)₂ · 2H₂O at concentrations of 10–50 μM. Morphological criteria were used to check embryonic development.Ten micromolars of nickel chloride affected adversely the development of Day 2 embryos (2-cell stage), whereas 300 μM was needed to affect Day 3 embryos (8-cell stage). Toxic effect of cadmium acetate on Day 2 embryos was observed at a concentration of 10 μM.     

Monitoring of organ formation in rat embryos after in vitro exposure to azathioprine, mercaptopurine, methotrexate or cyclosporin A

Rat embryos in the organ formation phase (days 9.5-11.5 post coitum) were cultivated in pure rat serum in the presence of an Aroclor 1254 pretreated liver microsomal preparation (S9-mix). Various concentrations of the immunosuppressive drugs azathioprine (AZ), 6-mercaptopurine (MP), methotrexate (MTX) or cyclosporin A (CS-A) were added at the beginning of the culture period. Forty-eight hours later, malformations were observed in the AZ, MP and MTX treated embryos at concentrations as low as 1 microgram/ml, 1.8 micrograms/ml and 0.05 microgram/ml, respectively. This indicates that these drugs have a direct effect on embryonic development. They selectively affected the rhombencephalic and telencephalic brain regions. Other malformations were seen in the caudal trunk, the heart and forelimb regions, and in the vesicular structures. It is suggested that the similarity of the pharmacological action of these drugs, that is, the DNA de novo synthesis inhibition, was the cause of the comparable types of malformations observed. Higher AZ, MP and MTX concentrations caused concentration-dependent increases in the types and incidences of malformations, as well as inhibited overall growth and differentiation. CS-A, a new type of immunosuppressant agent, had no effect on the morphogenetic events at the concentrations tested. These results are generally in agreement with the literature data, indicating that AZ, MP and MTX induce malformations in whole-animal systems, whereas CY-A does not. When AZ and MTX were assayed in the rat species in vivo, on the other hand, embryolethalities and retardations, but few malformations, were observed. The possibility of controlled exposure in vitro may, therefore, offer the advantage that clearer distinctions between embryolethal and teratogenic effects can be made.     

Elucidating the mechanisms of nickel compound uptake: a review of particulate and nano-nickel endocytosis and toxicity

Nickel (Ni) is a worldwide pollutant and contaminant that humans are exposed to through various avenues resulting in multiple toxic responses — most alarming is its clear carcinogenic nature. A variety of particulate Ni compounds persist in the environment and can be distinguished by characteristics such as solubility, structure, and surface charge. These characteristics influence cellular uptake and toxicity. Some particulate forms of Ni are carcinogenic and are directly and rapidly endocytized by cells. A series of studies conducted in the 1980s observed this process, and we have reanalyzed the results of these studies to help elucidate the molecular mechanism of particulate Ni uptake. Originally the process of uptake observed was described as phagocytosis, however in the context of recent research we hypothesize that the process is macropinocytosis and/or clathrin mediated endocytosis. Primary considerations in determining the route of uptake here include calcium dependence, particle size, and inhibition through temperature and pharmacological approaches. Particle characteristics that influenced uptake include size, charge, surface characteristics, and structure. This discussion is relevant in the context of nanoparticle studies and the emerging interest in nano-nickel (nano-Ni), where toxicity assessments require a clear understanding of the parameters of particulate uptake and where establishment of such parameters is often obscured through inconsistencies across experimental systems. In this regard, this review aims to carefully document one system (particulate nickel compound uptake) and characterize its properties.     

Cytosolic beta-glycosidases for activation of glycoside prodrugs of daunorubicin

Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.     

Comparison of four different treatment conditions of extended exposure in the in vitro micronucleus assay using TK6 lymphoblastoid cells

In the OECD Guideline 487, a total of four extended exposure treatment conditions are proposed for the in vitro micronucleus (MNvit) assay in the presence and absence of a cytokinesis block and with or without a recovery period. This guideline also states that rodent cell lines and human lymphocytes can be used as shown by many validated studies but that human cell lines such as TK6 and HepG2 are not yet validated. In this present study each extended exposure condition was characterized by investigation using TK6 cells and nine chemicals known to be able to induce micronucleus (MN) in rodent cell lines. The results revealed two concerns: six chemicals did not show significant MN induction in the ‘cytokinesis block without recovery period’; two aneugens showed no dose-dependent cytotoxicity in the ‘cytokinesis block with recovery period’. Further investigation revealed that 3–4 times higher spontaneous MN frequency than that in the other conditions is a possible reason for the low sensitivity, and this high spontaneous MN frequency was not observed in Chinese hamster lung cells under the identical treatment condition. With regard to the two conditions without cytokinesis block, two negative substances were evaluated and found to be negative, suggesting the validity of the TK6 test system for these conditions. Although our findings showed a few concerns for the treatment with cytokinesis block, the TK6 cells were considered to be a reliable cell line to be used for detecting potential inducers of MN in the in vitro micronucleus assay based on the overall results.     

Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y(12)-receptor

The P2Y(12)-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y(12)-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC(50) concentrations of about 0.3nM. In cells expressing R256A constructs, the IC(50) concentration amounted to 25nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent pK(B)-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent pK(B)-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y(12)-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.     

Effects of dietary flavonoids on major signal transduction pathways in human epithelial cells

Flavonoids (FVs) are an important class of plant compounds postulated to be one of the constituents responsible for the beneficial effects of fruits and vegetables on health, including heart disease and cancer. At pharmacological levels, various naturally-occurring flavonoids have been shown to be cancer-protective in a variety of animal models and flavonoid derivatives, such as flavopyridol, are being assessed as chemotherapy drugs in clinical trials. This report has investigated the effects of the most common dietary FVs on several major signalling pathways in biopsies of human epithelial cells using primary cultures freshly isolated from biopsies and has obtained evidence for the previously unrecognised importance of stress kinase responses induced by kaempferol (KF), apigenin (AP) and luteolin (LU). KF, AP and LU all activated ATM/ATR (mutated in ataxia-telangiectasia and related) kinases and the p38 stress kinase and this was associated with induction of GADD45 and cell cycle arrest in G2, but not induction of apoptosis. These effects were not due to general toxicity since they were reversible on removal of FV. The inductions of ATM/ATR and p38 were functionally important since caffeine, an inhibitor of ATM/ATR, and the p38-specific inhibitor, SB203580, prevented induction of GADD45 and growth arrest by these three flavonoids. In contrast, although quercetin (QU) activated ATM (but not ATR), it did not activate p38 kinase, GADD45 or p53. QU may interfere with one of the lipoxygenase (LOX) pathways since the growth inhibitory effects of QU (but not the other three flavonoids) could be reversed by addition of LOX metabolites, particularly 12- and 15-hydroxyeicostetraenic acids.     

Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: comparison with human and rat receptor

Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48 h. Radioligand diprenorphine and [D-AIa(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-stimulated GTP gamma S binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTP gamma S binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

Dopamine D(2) receptor-induced COX-2-mediated production of prostaglandin E(2) in D(2)-transfected Chinese hamster ovary cells without simultaneous administration of a Ca(2+)-mobilizing agent

We have earlier demonstrated that dopamine stimulates the liberation of the prostaglandin E(2) (PGE(2)) precursor, arachidonic acid, in Chinese hamster ovary cells transfected with the rat dopamine D(2) receptor (long isoform), also without concomitant administration of a Ca(2+)-releasing agent [Nilsson et al., Br J Pharmacol 1998;124:1651-8]. In the present report, we show that dopamine, under the same conditions, also induces a concentration-dependent increase in the production of PGE(2), with a maximal effect of 235% at approximately 100 microM, and with an EC(50) of 794 nM. The effect was counteracted by the D(2) antagonist eticlopride, pertussis toxin, the inhibitor of intracellular Ca(2+) release TMB-8, incubation in Ca(2+)-free experimental medium, and PKC desensitization obtained by chronic pretreatment with the phorbol ester TPA. It was also antagonized by the non-specific cyclooxygenase (COX) inhibitor, indomethacin, and by the selective COX-2 inhibitor, NS-398, but not by the specific COX-1 inhibitor, valeryl salicylate. Both the non-specific phospholipase A(2) inhibitor, quinacrine, and an inhibitor of cPLA(2) and iPLA(2), AACOF3, counteracted the effect; in contrast, a selective iPLA(2) inhibitor, BEL, and a selective sPLA(2) inhibitor, TAPC, were ineffective. No effects of dopamine were obtained in control cells mock-transfected with the p3C vector only. The results reinforce previous assumptions that dopamine may interact with eicosanoid metabolism by means of D(2) receptor activation, and implicate an involvement of cPLA(2) and COX-2 in this effect. It is suggested that measurement of dopamine-induced PGE(2) production may serve as a convenient way to study D(2) receptor function in vitro.     

Utility of frozen cell lines in medium throughput electrophysiology screening of hERG and NaV1.5 blockade

Introduction

The development of drug candidates must take into account that many compounds have off-target activity against voltage-gated ion channels (VGIC) which may prevent their progression to market. Of particular concern are hERG and hNa_V1.5. Screening against these ion channels is necessary but expensive, partially due to maintenance of constantly cultured cell lines. Here, we show that frozen HEK-293 cells can be maintained indefinitely, reducing variability in cell performance, time and expense of cell culture.

Methods

Cells, constantly cultured or frozen, were assayed on the PatchXpress 7000A using tool compounds.

Results

Amitriptyline, quinidine, compound A, fluoxetine and imipramine inhibited hERG with IC₅₀s (paired values denote constantly cultured and frozen, respectively) of 4.8 ± 0.4 and 5.1 ± 0.4, 1.4 ± 0.1 and 1.1 ± 0.1, 24.4 ± 2.4 and 21.9 ± 1.8, 2.1 ± 0.4 and 2.1 ± 0.1, 5.2 ± 0.4 and 4.0 ± 0.2 μM. Quinidine, flecainide, mexiletine and amitriptyline inhibited hNa_V1.5 with IC₅₀s of 46.6 ± 4.3 and 28.0 ± 2.3, 7.6 ± 0.7 and 6.2 ± 0.5, 153.5 ± 13.0 and 106.0 ± 4.7, 5.5 ± 0.5 and 4.8 ± 0.2 μM. Voltage dependences of activation (V_1/2) for hERG were statistically identical, 0.4 ± 0.8 mV and 2.5 ± 0.5 mV. In hNa_V1.5, the V_1/2 of inactivation and activation were statistically identical, −82.7 ± 0.1 mV versus − 84.9 ± 0.3 mV, −47.5 ± 0.3 mV versus − 45.0 ± 0.6 mV. Current density in both conditions in hERG experiments was similar, 47.0 ± 4.1 pA versus 42.3 ± 6.0 pA/pF.

Discussion

hERG and hNa_V1.5 screens run using frozen cells have statistically identical IC₅₀s, voltage dependence of activation, IV relationships and current density to screens using continuously cultured cells. Frozen cells have more constant performance and allow rapid switching between experiments on several cell lines without sacrificing data quality.     

Pro-oxidative toxicity of cells in suspension does not point to a cell cultural artefact as an explanation of the increased susceptibility of alveolar epithelial-like cells after glucocorticoid pretreatment

The influence of cell numbers on peroxide-(tertiary butylhydroperoxide (tBHP) or hydrogen peroxide-(HP)) or zinc-(zinc chloride) induced oxidative stress was assessed in alveolar epithelial-like cell lines in this work. Differences in cell numbers change the cellular glutathione and glutathione reductase activity as well as the amount of exported glutathione and therefore might influence susceptibility against oxidative stress.Toxicity due to zinc decreased, toxicity due to HP increased, while tBHP-mediated toxicity was unchanged in our experiments when cells were exposed in suspension as compared to monolayers. Toxicity of HP correlated to the glutathione content in monolayers and in cell suspensions, while zinc- or tBHP-mediated toxicity did not correlate towards glutathione.Decreasing cellular glutathione and the activity of some antioxidative enzymes by glucocorticoid pretreatment had no effect on toxicity of zinc or tBHP in L2 cells in suspensions, while toxicity in monolayers was increased. Glucocorticoid pretreatment seems to increase toxicity of HP in A549 monolayers according to the lowered protein content, while toxicity might be changed by a different way when cells are incubated as cell suspensions. No explanation as a cell culture artificial effect was observed, therefore we assume the increased toxicity after glucocorticoid pretreatment occurs in vivo as well.