Immunological evaluation of the mycotoxin patulin in female b6C3F1 mice

Patulin is a mycotoxin produced by many fungal species of the genera Penicillium, Aspergillus and Bryssochamys. Previous literature reports have suggested that patulin is toxic to the immune system. The studies presented were conducted to provide a comprehensive assessment of the effects of patulin on the immune system. Unlike previous reports, the doses of patulin used (0.08, 0.16, 0.32, 0.64, 1.28 and 2.56 mg/kg) were based on predicted human exposure levels. Female B6C3F₁ mice were exposed orally to patulin for 28 days. Effects were not observed on final body weight or body weight gain. Relative weight of the liver, spleen, thymus, kidneys with adrenals, and lungs was not affected. Peripheral blood leucocyte and lymphocyte counts were decreased by approximately 30% in the two highest dose groups. The leucocyte differential was not altered. Total spleen cell, total T-cell (CD3⁺), helper T-cell (CD4⁺CD8⁻), B-cell (surface immunoglobulin⁺) and monocyte (MAC-3⁺) counts were not changed. Cytotoxic T-cell (CD8⁺CD4⁻) counts were increased 50% only by the highest dose. Natural killer cell (NK1.1⁺CD3⁻) and monocyte (MAC-1⁺) counts were increased 30% and 24%, respectively, only in the 0.08 mg/kg group. Humoral immune function as assessed by antibody-forming cell response and serum IgM titre to sheep erythrocytes, and cell-mediated immune function evaluated utilizing natural killer cell activity and the mixed lymphocyte reaction were not altered. Oral exposure to patulin for 28 days did not alter the ability of female B6C3F₁ mice to mount either a cell-mediated or humoral immune response.     

Cadmium-induced postaxial forelimb ectrodactyly: association with altered sonic hedgehog signaling

Administration of CdSO₄ to C57BL/6 mice at day 9.5 of gestation induces a high incidence of postaxial forelimb ectrodactyly in the offspring. We propose that Cd²⁺ exposure impairs the process of anterior/posterior formation in the limb bud, a process that is directed by Sonic hedgehog (Shh) signaling. We show that exposure of the mouse embryo to Cd²⁺ disrupts Shh signaling as measured by polarizing activity of mouse limb bud ZPA grafted to a host chick wing, and activity of a Gli:luciferase reporter exposed to limb bud lysates. Yet the expression of Shh and its translation are not affected by Cd²⁺ exposure. We propose that teratogen exposure affects the processing of Shh in the cells in which it is made.     

Effects of ochratoxin a on the mouse immune system after subchronic exposure

The effects on the immune system of oral, subchronic exposure to ochratoxin A (OA) at 6, 250 or 2600 μg/kg diet were studied in female Balb/c mice. After 28 days of exposure, antibody production to sheep red blood cells, as measured in the plaque-forming cell assay and expressed as number of plaque-forming cells/spleen, was suppressed in a dose-dependent manner which was significant in the two highest exposure groups. In addition, a decrease in thymocyte cell counts was seen in the 250-μg/kg group. After 90 days of exposure, flow cytometry analysis of thymic lymphocyte subpopulations revealed a decreased proportion of mature (CD4⁺ or CD8⁺) cells. Furthermore, the mitogenic responsiveness of thymocytes and splenocytes to concanavalin A (Con A) was significantly decreased. This effect was observed in all three treatment groups. Interleukin-2 production of Con A-stimulated lymphocytes, natural killer cell activity, and humoral antibody titres to a viral antigen were not affected by OA treatment. The present results indicate that subchronic, oral exposure to OA affects certain immune functions in mice at exposure levels that may be found in contaminated food products.     

Single-dose topical exposure to the pyrethroid insecticide, permethrin in C57BL/6N mice: effects on thymus and spleen

Immunomodulatory effects of single topical exposure to permethrin were evaluated in 5-week-old female C57BL/6N mice. Mice exposed to 5–25 μl permethrin (equivalent to 220–1100 mg/kg body weight) on shaved interscapular skin were evaluated for altered body weight; splenic and thymic organ weight and cellularity; thymocyte cell surface expression, cellular apoptosis; splenic macrophage phagocytosis and hydrogen peroxide production; splenic B cell antibody production and T cell cytolytic activity; and mitogen-induced proliferation of splenocytes and thymocytes after in vivo or in vitro permethrin exposure. Topical permethrin application (25 μl) caused 32% inhibition of splenic T cell proliferation; in vitro exposure to permethrin also diminished splenocyte proliferation by 72% at 25 μ and 86% at 100 μ . permethrin did not appear to affect other leukocyte functional assays. Dose-related decreases in thymic cellularity of 52 and 80% were seen in mice exposed to 15 and 25 μl permethrin, respectively. Apoptosis was significantly increased in CD4⁻8⁻ and CD4⁻8⁺ thymocytes, and the CD4⁺CD8⁺ thymocyte subpopulation was most severely diminished, suggesting possible chemical-induced apoptotic mechanism of thymic atrophy. Permethrin also caused splenic hypocellularity by 31% at 15 μl, and by 50% at 25 μl, an effect that may relate to inhibited proliferation or reduced seeding from the hypocellular thymus.     

Thiols in Scenedesmus vacuolatus upon exposure to metals and metalloids

Phytochelatins are intracellular metal ligands produced by algae when exposed to elevated metal concentrations. In freshwater ecosystems, algae are exposed to a wide range of metals and metalloids. The aim of this study was thus to investigate phytochelatin induction in freshwater algae upon metal and metalloid exposure. To that purpose, the unicellular green alga Scenedesmus vacuolatus, was exposed to Cu, Zn, Ni, Pb and Ag, as well as to As(III), As(V), Sb(III) and Sb(V), and examined for its thiol content (gamma-glutamylcysteine, glutathione and phytochelatins). Glutathione content was found to decrease upon the exposure to Zn and to increase upon the exposure to Pb and Ag. Phytochelatins were only induced by Cu (at [Cu²⁺] = 8 × 10⁻¹¹ M) and Pb (at [Pb²⁺] = 8 × 10⁻¹¹ to 8 × 10⁻¹⁰ M), where [Cu²⁺] and [Pb²⁺] are computed free metal ion concentrations. Glutathione content also decreased upon the exposure to Sb(V) whereas an increase was observed as a result as the exposure to As(III) and As(V). The metalloids As(III), As(V) and Sb(III) in the concentration range from 8 × 10⁻⁶ to 2 × 10⁻⁴ M (total concentrations of oxyanions) were inducing phytochelatins. Glutathione and phytochelatin content in S. vacuolatus do thus sensitively respond to exposure to a number of metals and metalloids.     

Enhancement of antigen-induced eosinophilic inflammation in the airways of mast-cell deficient mice by diesel exhaust particles

The present study was conducted to clarify the involvement of mast cells in the exacerbating effect of diesel exhaust particles (DEP) toward allergic airway inflammation and airway hyperresponsiveness (AHR). Airway inflammation by the infiltration of cosinophils with goblet cell proliferation and AHR, as well as by the production of antigen-specific IgG1 and IgE, in plasma were examined using mast cell-deficient mice (W/W^v) and normal mice (W/W⁺). Both groups of mice received ovalbumin (OVA) or OVA+DEP intratracheally. The eosinophilic airway inflammation and goblet cell proliferation promoted by OVA were significantly greater in W/W⁺ than in W/W^v. A similar result was observed in AHR, but was not significant among both groups of mice. DEP enhanced OVA induced-allergic airway inflammation, goblet cell proliferation, and development of AHR in W/W^v, but not in W/W⁺. DEP decreased production of antigen-specific IgG1 and IgE in both groups of mice. Mast cells were observed in the submucosal layer of the main bronchus in W/W^v. The number of mast cells was significantly decreased by OVA treatment. The results indicate that mast cells are not necessary to enhance airway damage and development of AHR in W/W^v by DEP. However, mast cells may be required for the OVA-induced cosinophilic inflammation, airway damage with goblet cell proliferation, and AHR in W/W⁺.     

荆芥汤中荆芥最佳煎煮时间的实验研究

目的：通过研究荆芥后下对荆芥汤中胡薄荷酮含量及镇咳、镇痛、免疫功能的影响，探讨荆芥的最佳煎煮时间，为荆芥特殊煎煮方法的临床应用提供理论依据。方法：按照《中国药典》（2015年版）中荆芥含量测定要求测定胡薄荷酮含量；采用致咳、致痛法观察荆芥后下对小鼠镇咳、镇痛作用的影响，通过分析小鼠CD4⁺、CD8⁺、IL-1、IL-6水平，探讨荆芥后下对小鼠免疫功能的影响。结果：荆芥煎煮7.5 min时，荆芥汤中胡薄荷酮含量最高，与空白组比较其能够明显改善小鼠痛阈值、咳嗽潜伏期及咳嗽次数（P<0.01），提高小鼠CD4⁺、CD8⁺、IL-1、IL-6水平（P<0.05）。结论：荆芥后下影响荆芥汤中胡薄荷酮含量及治疗效果，荆芥入解表类汤剂宜采用"后下"，最佳时间为5~10 min。     

Stimulus-evoked glutamate release is diminished by acute exposure to uranium in vitro

Uranium is used in civilian applications, in the manufacture of nuclear fuel, and by the military for munitions and armament, but little information is available on its neurotoxicity. Neurological dysfunctions have been observed after chronic exposure in both animals and humans, but the actions of acute exposure on amino acid neurotransmission have not been investigated. The following study was performed to examine the effects of uranyl ion (UO₂^+ 2) on hippocampal glutamatergic and GABAergic function as possible bases for the neurotoxicity and to assess the direct effects on the exocytotic process. Nominal UO₂^+ 2 concentrations were applied to superfused hippocampal synaptosomes to permit estimation of the metal's potency on endogenous transmitter release in the presence and absence of Ca^+ 2. K⁺-evoked glutamate release was diminished in the range of 10 nM–316 μM UO₂^+ 2, resulting in an IC₅₀ of 1.92 μM. In contrast, the potency of UO₂^+ 2 to decrease stimulated GABA release was reduced, producing an IC₅₀ ≈ 2.6 mM. In the absence of Ca^+ 2 in the superfusion medium there was no systematic change in the magnitude of glutamate or GABA release, suggesting that UO₂^+ 2 does not possess Ca^+ 2-mimetic properties. The inhibitory potency of UO₂^+ 2 on glutamate release is similar to the potencies of other multivalent metal ions, suggesting by inference an action exerted on voltage-sensitive Ca^+ 2 channels. The bases for the reduced potency to inhibit GABA release is not known, but differential sensitivity to other heavy metals has been reported for glutamate and GABA neurotransmission. These findings indicate a profile of neurotoxicity not unlike that of other metal ions, and indicate the importance of extending subsequent studies to chronic exposure models.     

Effect of a phospholipase A2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents

, , , and . Effect of a phospholipase A₂ with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon 27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A₂ with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca²⁺-activated K⁺current; the sensitivity for voltage activation of the K⁺ current was enhanced by increasing free Ca²⁺ in the recording pipette from 10⁻⁸ M to 2 × 10⁻⁶ M. In contrast, peripheral blood lymphocytes possessed voltage-activated K⁺ currents that were inhibited by increasing intracellular Ca²⁺.

Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10⁻⁶ M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K⁺ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K⁺ currents in human lymphocytes, suggesting a specific increase in intracellular Ca²⁺ levels in both cell types.

The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca²⁺ levels. Exposure of either lymphocytes or epithelial cells to toxin (10⁻⁶ M) resulted in a transient increase in Ca²⁺. However, while the Ca²⁺ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca²⁺-regulated K⁺ channels may reflect an irreversible action of the B.fasciatus phospholipase A₂ on a Ca²⁺-dependent regulatory process.     

美沙拉嗪、柳氮磺胺吡啶对葡聚糖硫酸钠诱导的Balb/c小鼠急性溃疡性结肠炎的治疗和免疫影响

目的：探讨美沙拉嗪和柳氮磺胺吡啶对葡聚糖硫酸钠（DSS）致Balb/c小鼠急性溃疡性结肠炎的作用效果和对急性溃疡性结肠炎免疫功能的影响。方法：Balb/c小鼠给予3.5% DSS水溶液自由饮用7 d建立溃疡性结肠炎模型,通过测定小鼠DAI指数、脏器指数、血清IL-4、结肠组织匀浆液IL-8的表达水平和小鼠体内CD4⁺细胞、CD8⁺细胞、CD4⁺CD25⁺细胞的变化,来评价两种药物对小鼠急性溃疡性结肠炎的作用。结果：给药治疗后,美沙拉嗪组和柳氮磺胺吡啶组的DAI评分和CMDI评分均降低（P<0.01或者P<0.05）,小鼠症状缓解。结肠黏膜充血水肿减轻,且两种药物均能\\有效抑制病灶部位炎细胞的浸润。与模型组相比,美沙拉嗪组和SASP组CD3⁺细胞数增加,CD8⁺细胞数明显上升,CD4⁺CD25⁺细胞占CD4⁺细胞比例增加,血清中IL-4含量显著上升（P<0.01）,组织中IL-8含量显著下降（P<0.01）;而美沙拉嗪组CD4⁺细胞数目略有增加,SASP组CD4⁺细胞数增加比较明显。结论：美沙拉嗪和柳氮磺胺吡啶可减轻DSS诱导的UC小鼠稀便、血便症状;两种药物对UC疾病的治疗可能与提高CD4⁺细胞水平和IL-4含量及上调CD8⁺细胞水平有关。     

Action of the Na ionophore monensin on vascular smooth muscle of guinea-pig aorta

The effects of the Na⁺ ionophore monensin on contractile responses were investigated in guinea-pig aorta in normal and high K⁺ solutions. In normal K⁺ (5.4 mM) solution, monensin (2 × 10⁻⁵ M) produced a rapid increase in tension followed by slow relaxation. This contraction was markedly inhibited by phentolamine (10⁻⁵ M) or prazosin (10⁻⁶ M) and was accompanied by an increase in tritium efflux from tissue preloaded with [³H]norepinephrine. In the presence of phentolamine, monensin (1–2 × 10⁻⁵ M) or ouabain (1−2 × 10⁻⁵ M) caused only a small and slowly developing contraction. Simultaneous application of these agents caused a more rapid and greater contraction. Either monensin or ouabain gradually increased cellular Na⁺ and decreased cellular K⁺ content. When monensin was applied simultaneously with ouabain, there was a rapid increase in cellular Na⁺ and loss of cellular K⁺. In high K⁺ (65.4 mM) solution, monensin (10⁻⁶ M) slightly reduced the increased tension level but when external glucose was omitted monensin markedly inhibited the contraction. A significant decrease in tissue ATP content was observed only when monensin was applied in glucose-free solution. Similarly, hypoxia (N₂ bubbling) markedly inhibited the high K⁺ contraction and decreased the tissue ATP content only in the absence of glucose. These results suggest that monensin produces a neurogenic contraction due to the release of endogenous catecholamines and also produces a myogenic contraction by a decrease in transmembrane Na⁺ and K⁺ gradients when the Na⁺ and -K⁺ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.     

黄芪多糖对阿霉素治疗小鼠乳腺癌的增效减毒机制研究

目的：探讨黄芪多糖对阿霉素治疗乳腺癌的增效减毒作用机制,为乳腺癌的治疗提供帮助。方法：建立小鼠乳腺癌皮下移植肿瘤模型,44只荷瘤小鼠随机平分为4组,即对照组（生理盐水治疗）、DOX组（阿霉素治疗）、APS组（黄芪多糖治疗）和APS+DOX组（黄芪多糖和阿霉素联合治疗）;观察治疗后荷瘤小鼠肿瘤体积大小、体质量变化以及平均存活时间、各器官组织学变化;检测荷瘤小鼠治疗前后T淋巴细胞、应激反应因子以及肝肾功能变化。结果：经过联合治疗后,APS+DOX组荷瘤小鼠肿瘤体积比显著低于对照组、APS组和DOX组;同时APS+DOX组荷瘤小鼠平均存活时间明显长于对照组、APS组和DOX组;HE染色表明荷瘤小鼠经过黄芪多糖和阿霉素联合治疗后,各器官损伤程度低于DOX组和对照组;治疗后APS+DOX组荷瘤小鼠肝功能和肾功能各指标、GSH、SOD以及T淋巴细胞（CD3⁺、CD4⁺、CD4⁺/CD8⁺和NK）水平显著高于DOX组但显著低于APS组和对照组,APS+DOX组CD8⁺、MDA水平显著低于DOX组但显著高于APS组、对照组,数据比较差异存在统计学意义（P<0.05）。结论：黄芪多糖能够有效促进阿霉素对乳腺癌皮下移植肿瘤小鼠的治疗和平均存活时间的延长,同时能够提高荷瘤小鼠免疫功能、抑制氧化应激反应和改善荷瘤小鼠的肝肾功能,有利于促进乳腺癌的治疗。     

Subacute toxicity of ammonia to Atlantic salmon (Salmo salar L.) in seawater: effects on water and salt balance, plasma cortisol and plasma ammonia levels

Atlantic salmon (Salmo salar L.) postsmolts weighing about 300 g were exposed to two replicates of six ammonia levels in running seawater at 4–9°C, 34–35‰ salinity, pH 7.7–7.8 and 5–9 mg/l O₂. Mean water ammonia levels ranged from 0.07 (control tanks) to 12.84 mg/l TA-N (< 1–100 μg/l NH₃-N). Blood and skeletal muscle tissue samples were taken after 2 and 5 weeks of exposure, and plasma osmolality, Na⁺, Cl⁻, Ca²⁺, Mg²⁺, TA (total ammonia) and cortisol levels and muscle tissue water content measured. Plasma cortisol was significantly increased at all water ammonia levels above control after 2 weeks of exposure, but the cortisol levels were low and did not increase with increasing water ammonia level. After 5 weeks of exposure, plasma cortisol was only significantly increased in fish from two single tanks with low and intermediate ammonia levels. No effects were found on muscle tissue water content or plasma Na⁺ or Mg²⁺ levels. Plasma osmolality and Cl⁻ levels were significantly increased with a LOEC (Lowest Observed Effect Concentration) of 10.59 mg/l TA-N (81 μg/l NH₃-N) after 2 weeks and 12.84 mg/l TA-N (100 μg/l NH₃-N) after 5 weeks of exposure. The increases in plasma osmolality and Cl⁻ were small, however, and all values were within the normal range. Plasma TA levels increased linearly with increasing water TA level, and the LOEC was 3.59 mg/l TA-N (28 μg/l NH₃-N) after 2 weeks and 0.82 mg/l TA-N (6 μg/l NH₃-N) after 5 weeks of exposure.     

川芎嗪联用环孢素A抗皮肤移植排斥反应的作用机制研究

目的：探讨川芎嗪（TMPZ）联用亚剂量环孢素A（CsA）对同种异体小鼠皮肤移植的抗排斥反应作用。方法：将BALB/c小鼠作为供体,C57BL/6小鼠作为受体,行背-背皮肤移植术。随机分为模型组、TMPZ组（50 mg·kg^-1）、CsA组（10 mg·kg^-1）、TMPZ+亚剂量CsA组（TMPZ 50 mg·kg^-1+CsA 5 mg·kg^-1）、假手术组,腹腔注射给药10 d。观察受体鼠移植皮片的存活情况,酶联免疫吸附法（ELISA）检测血浆中白介素-2（IL-2）、IL-4、IL-10、γ-干扰素（IFN-γ）含量,流式细胞术分析脾脏CD4⁺、CD25⁺T淋巴细胞亚群的比例。结果：各给药组小鼠移植皮片的存活天数均有不同程度的延长,与模型组相比差异均有显著性（P<0.01）。TMPZ+CsA组的移植皮片存活天数比TMPZ组或CsA组均有显著延长（P<0.05或P<0.01）;与模型组相比,TMPZ+CsA组以及TMPZ单用组均可明显减少血浆Th1类细胞因子（IL-2、IFN-γ）的表达（P<0.01）,明显增高Th2类细胞因子（IL-4、IL-10）的表达（P<0.01）。与模型组相比,各给药组均可明显增高脾脏CD4⁺ CD25⁺T淋巴细胞的比例（P<0.05或P<0.01）。结论：TMPZ具有抑制同种异体小鼠皮肤移植排斥反应的效果,与CsA联合应用可能具有协同作用,其作用机制可能与影响Th1/Th2细胞因子、刺激CD4⁺、CD25⁺T淋巴细胞的表达等有关。     

Effect of atropine on cyanide-induced acute lethality in mice

The effects of atropine on acute lethality induced by cyanide were investigated in mice. The LD₅₀ value of cyanide (s.c. injection) was 8.4 (7.6–9.3) mg/kg. However, the LD₅₀ value of cyanide (s.c.) was significantly increased by 1.5-fold when atropine (32 mg/kg) was injected s.c. in mice. Furthermore, the combined administration of atropine (32 mg/kg). Ca²⁺ (500 mg/kg) and sodium thiosulfate (1 g/kg) tremendously increased the LD₅₀ value by 5.6-fold in mice although sodium thiosulfate or Ca²⁺ alone increased the LD₅₀ 2.5- or 1.5-fold. On the other hand, although the LD₅₀ value of cyanide (intracerebroventricular injection (i.v.t.)) was 52.0 (47.4–57.0) μ/brain, the LD₅₀ value of cyanide (i.v.t.) was significantly increased by 1.3- or 1.61-fold in mice 10 min after s.c. injection of atropine (32 mg/kg) or Ca²⁺ (500 mg/kg). Furthermore, the combined administration of atropine and Ca²⁺ increased the LD₅₀ value of cyanide by 2.1-fold. These results suggest that atropine inhibits cyanide-induced acute lethality and promotes the antagonistic effect of thiosulfate and Ca²⁺ in mice.     

Effects of [Na] and [Ca] on the responses to milrinone in rat cardiac preparations

In the present investigation we have studied the influence of changing the [Ca²⁺] and [Na⁺] on the cardiac responses to milrinone in various preparations of rat heart. Milrinone (5 × 10⁻⁵ to 8 × 10⁻⁴ M) produced a dose-dependent positive chronotropic effect on right atrium and a positive inotropic effect on left atrium and papillary muscle of the rat. A decrease in [Ca²⁺] (from 2.2 to 1.1 mM) or an increase in [Na⁺] (from 120 to 60 mM) increased the milrinone-induced inotropic effect in left atrium and papillary muscle. However, in right atrium the chronotropic effect of milrinone was significantly decreased under these conditions. Opposite changes to milrinone-induced responses were observed when [Ca²⁺] was increased (to 3.3 mM) or when the [Na⁺] was decreased to 60 mM. Nifedipine (3 × 10⁻³ M), a selective Ca²⁺ channel blocker, significantly inhibited the chronotropic response to milrinone in right atrium. However, the inotropic response to milrinone was found to be significantly greater in the presence of nifedipine. A veratridine-induced positive inotropic effect in the left atrium was also significantly increased in the presence of nifedipine. Tetrodotoxin (TTX, 1 × 10⁻⁶ M), a fast sodium channel blocker, significantly reduced the inotropic response to milrinone in left atrium and papillary muscle. A milrinone-induced dose-dependent increase in the baseline tension was observed in the right atrium which was abolished in low [Ca²⁺] and significantly increased in high [Ca²⁺]. Our data suggest the possibility that milrinone increases Ca²⁺ influx in the right atrium to cause the chronotropic effect. Milrinone also may possess an action like veratridine, involving an increased influx of Na⁺ through fast Na⁺ channels in left atrium and papillary muscle, and this action is possibly involved in the positive inotropic effect.     

Role of Fas apoptosis and MHC genes in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunotoxicity of T cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is well known for its immunotoxic effects particularly on the thymus as well as on T and B lymphocyte functions. Previous studies have suggested that TCDD may induce apoptosis in thymocytes although its demonstration in vivo has met with limited success. TCDD has also been shown to alter the major histocompatiblity complex- (MHC) encoded molecules, however, its role in immunotoxicity is not clear. In the current study, we investigated the role of Fas (CD95), an important molecule involved in the induction of apoptosis, on TCDD-mediated immunotoxicity using mice bearing homozygous lpr mutation which leads to failure of expression of Fas. When TCDD was administered orally at 0, 0.1, 1.0, or 5.0 μg/kg body weight for 11 days, it was found to be less toxic to the thymocytes from C57BL/6 lpr/lpr mice (Ah-responsive, Fas⁻) when compared to C57BL/6 +/+ mice (Ah-responsive, Fas⁺). Similar results were obtained when peripheral T cell responsiveness to antigenic challenge with conalbumin was studied in these mice. When mice that differed only at the MHC were compared for immunotoxic effects of TCDD, it was noted that B10.D2 (Ah-responsive, H-2^d) were more sensitive to TCDD-mediated thymic atrophy and peripheral T cell dysfunction when compared to B10 mice (Ah-responsive, H-2^b). In all TCDD-sensitive strains tested, the thymic atrophy was accompanied by a uniform depletion of all four subset of T cells (CD4⁺, CD4⁺CD8⁺ , CD4⁻CD8⁻, and CD8⁺) and the percentage of these subsets was not altered. Furthermore, in these strains, TCDD suppressed the antigen-specific peripheral T cell responsiveness but not the responsiveness of naive resting T cells to polyclonal mitogens. Lastly, using cell-mixing experiments, it was demonstrated that TCDD directly affected the T cells responding to conalbumin but not the antigen presenting cells (APCs). Together, our studies demonstrate that although Ah locus plays the primary role, determining the toxicity of TCDD on the T cells, there are secondary factors such as expression of Fas or the Abbreviations: Ah, aryl hydrocarbon; APC, antigen presenting cell; ConA, concanavalin A; FITC, fluorescein isothiocyanate; LN, lymph node; mAb, monoclonal antibody; MHC, major histocompatability complex; PE, phycoerythrin; PHA, phytohemagglutinin; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCR, T cell receptor; TNF, tumor necrosis factor.     

8-isoprostaglandin E(2) activates Ca(2+)-dependent K(+) current via cyclic AMP signaling pathway in murine renal artery

The inhibitory pathway of 8-isoprostaglandin E₂ was investigated in murine renal arterial smooth muscle. K⁺ current was augmented in a concentration-dependent fashion, with an average increase of 123 ± 28% (n = 6) following application of 10^− 5 M 8-isoPGE₂. This augmentation was observed in the presence of 4-aminopyridine (4-AP, 10^− 3 M) but not that of charybdotoxin (ChTx, 10^− 7 M). Fluorimetric recordings showed marked concentration-dependent increase of cytosolic Ca²⁺ levels by 8-isoPGE₂, while an enzyme-linked immunosorbent assay (ELISA)-based cyclic AMP assay showed increased cAMP levels by 10^− 7 M 8-isoPGE₂ challenge. The isoprostane-induced augmentation was prevented by the ryanodine receptor blocker ruthenium red (10^− 5 M) or the adenylate cyclase blocker SQ 22536 (10^− 4 M). The protein kinase A (PKA) inhibitor H89 (10^− 5 M) inhibited resting K⁺ currents (78 ± 5%, n = 5) but did not prevent 8-isoPGE₂ from augmenting the remaining K⁺ current. We conclude that 8-isoPGE₂ enhances Ca²⁺-dependent K⁺ currents in murine renal artery through a cAMP-dependent pathway which may involve internally sequestered Ca²⁺.     

Modulation of the hyperpolarisation-activated current, Ih, in rat facial motoneurones in vitro by ZD-7288

ZD-7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] and Cs⁺ have been used to distinguish the currents contributing to inward rectification in neonatal rat facial motoneurones (FMs). ZD-7288 (0.1–10 μM) inhibited a current that reversed at −43.7±3.7 mV in artificial cerebrospinal fluid (ACSF) containing 3 mM K⁺ (n=9), and displayed the time and voltage dependence of the hyperpolarisation-activated current, I_h. Depolarisation-activated transient (I_K(A)) and sustained outward currents were unaffected by ZD-7288. The IC₅₀ for block of I_h by ZD-7288 was around 0.2 μM. Onset of inhibition was slow and no recovery was seen after washing in ZD-7288-free ACSF for up to 4 h. In the presence of ZD-7288, Ba²⁺ and Rb⁺ blocked an inwardly rectifying potassium (K⁺) current, confirming both the presence of I_K(IR) and its insensitivity to ZD-7288. Cs⁺ rapidly and reversibly blocked both I_h and I_K(IR). Inhibition of I_h by ZD-7288 showed no use dependence, internally applied ZD-7288 also blocked I_h, and tail current analysis indicated inhibition to be voltage-independent. In the presence of internal guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) and after previous exposure to ZD-7288, 5-hydroxytryptamine (5-HT), but not noradrenaline, promoted a recovery of I_h that was not observed if ZD-7288 was present throughout the recording period. This interaction between ZD-7288 and irreversible 5-HT-receptor activation may be related to the mechanism underlying ZD-7288-mediated block of these channels.     

Cadmium-induced nephrotoxicity in rhesus monkeys (Macaca, mulatta) in relation to protein calorie malnutrition

In this study, we compared results obtained in protein calorie malnourished (PCM) monkeys and controls given Cd²⁺ (5 mg Cd²⁺/kg body wt./day) orally for 24 weeks. After 16 weeks of Cd²⁺ exposure, an indolent renal failure develops in PCM monkeys which resulted in significant increase in urinary excretion of total protein, Cd²⁺, Zn²⁺ and Ca²⁺ as compared to corresponding Cd²⁺-treated control group. In isolated proximal tubule brush border membrane vesicles (BBMV), Cd²⁺, Zn²⁺ and Ca²⁺ transport were significantly impaired in Cd²⁺-exposed PCM monkeys as compared to Cd²⁺-treated controls. The mechanism of higher urinary excretion of Cd²⁺, Zn²⁺ and Ca²⁺ was examined by analyzing the kinetic parameters of transport systems. The kinetic studies of Cd²⁺, Zn²⁺ and Ca²⁺ transport systems in the BBMV preparations of Cd²⁺-exposed PCM monkeys exhibited a significant decrease in V_max and an appreciable increase in K_m as compared to Cd²⁺-treated controls. These findings suggested that Cd²⁺ treatment of PCM monkeys caused either a decrease in the number of transporters in the brush border membrane or an increase in the number of less active transporters for Cd²⁺, Zn²⁺ and Ca²⁺. Furthermore, brush border membrane-bound enzymes, viz. alkaline phosphatase and leucine aminopeptidase, activities were significantly impaired in Cd²⁺-exposed PCM monkeys. Cadmium content in kidney cortex of Cd²⁺-exposed PCM monkeys was 3.34-fold higher than Cd²⁺-exposed controls. These findings also established that Cd²⁺ not bound to metallothionein (MT) was significantly higher in Cd-exposed PCM monkeys, which may be an important determinant in renal toxicity by interacting with sensitive sites in the renal cells and causing renal damage in Cd-exposed PCM monkeys.