Genotoxicity of nitrosulfonic acids, nitrobenzoic acids, and nitrobenzylalcohols, pollutants commonly found in ground water near ammunition facilities

2-Amino-4,6-dinitrobenzoic acid (2-A-4,6-DNBA), 4-amino-2,6-dinitrobenzoic acid (4-A-2,6-DNBA), 2,4,6-trinitrobenzoic acid (2,4,6-TNBA), 2-amino-4, 6-dinitrobenzylalcohol (2-A-4,6-DNBAlc), 4-amino-2,6-dinitrobenzylalcohol (4-A-2,6-DNBAlc), 2,4-dinitrotoluol-5-sulfonic acid (2,4-DNT-5-SA), 2,4-dinitrotoluol-3-sulfonic acid (2,4-DNT-3-SA), and 2, 4-dinitrobenzoic acid (2,4-DNBA) are derivatives of nitro-explosives that have been detected in groundwater close to munitions facilities. In the present study, the genotoxicity of these compounds was evaluated in Salmonella/microsome assays (in strains TA100 and TA98, with and without S9 and in TA98NR without S9), in chromosomal aberration (CA) tests with Chinese hamster fibroblasts (V79), and in micronucleus (MN) assays with human hepatoma (HepG2) cells. All compounds except the sulfonic acids were positive in the bacterial mutagenicity tests, with 2,4,6-TNBA producing the strongest response (8023 revertants/micromol in TA98 without S9 activation). 2-A-4,6-DNBA was a direct acting mutagen in TA98, but negative in TA100. The other positive compounds were approximately 1-3 orders of magnitude less mutagenic than 2,4,6-TNBA in TA98 and in TA100; relatively strong effects ( approximately 50-400 revertants/micromol) were produced by the benzylacohols in the two indicator strains. With the exception of 2,4-DNBA, the mutagenic responses were lower in the nitroreductase-deficient strain TA98NR than in the parental strain. 2,4-DNBA produced a marginally positive response in the V79-cell CA assay; the other substances were devoid of activity. Only the benzoic acids were tested for MN induction in HepG2 cells, and all produced positive responses. As in the bacterial assays, the strongest effect was seen with 2,4,6-TNBA (significant induction at >or=1.9 microM). 4-A-2,6-DNBA was positive at 432 microM; the weakest effect was observed with 2,4,-DNBA (positive at >or=920 microM). The differences in the sensitivity of the indicator cells to these agents can be explained by differences in the activities of enzymes involved in the activation of the compounds. The strong responses produced by some of the compounds in the human-derived cells suggest that environmental exposure to these breakdown products of nitro-explosives may pose a cancer risk in man.     

Effects of excitatory amino acids and neuropeptide Y on the discharge activity of suprachiasmatic neurons in rat brain slices

Effects of

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-glutamate, AMPA, NMDA and NPY on the discharge activity of neurons located in the ventral subdivision of the suprachiasmatic nucleus were examined in submerged coronal slices of the rat hypothalamus. All substances were bath applied. Application of

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-glutamate (14 neurons examined) induced an excitatory response in 8 suprachiasmatic neurons (+248.9±122.24%, mean±S.E.M.; P<0.001). A biphasic response, i.e. an initial transient excitation (+54.3±8.21%; P<0.001) succeeded by an inhibition (−66.2±9.31%; P<0.001), was observed in 6 neurons. Application of AMPA (36 neurons examined) resulted in an excitation of 31 neurons (+209.2±58.58%; P<0.0001). Application of NMDA (57 neurons examined) induced an excitation in 34 neurons (+253.8±91.18%; P<0.0001), but an inhibition in 8 neurons (−75.7±6.52; P<0.0001). Biphasic effects of NMDA with an excitatory component (+58.7±9.94%; P<0.0001) succeeded by an inhibitory component (−62.0±8.07%; P<0.0001) were observed in 13 neurons. In 5 of 13 examined cases, the inhibitory component of neuronal responses to NMDA was significantly attenuated by the simultaneous application of strychnine (attenuation was 56%; P<0.05). The application of NPY (40 neurons examined) induced significant effects on the discharge rate of 29 suprachiasmatic neurons. 18 of these neurons were inhibited (−59.3±6.39%; P<0.0001) whereas 11 neurons were excited (+156.6±107.22%; P<0.001) by NPY. In 8 of 11 neurons examined, the NPY-induced inhibition was significantly attenuated by 92% during simultaneous application of strychnine (P<0.001). In 23 NPY-sensitive neurons, the discharge activity was also affected by NMDA. Neurons excited by NPY were also excited by NMDA (8 cells). In neurons inhibited by NPY, application of NMDA induced either an inhibition (3 cells) an excitation (5 cells) or a biphasic effect (7 cells). Results suggest a direct excitatory effect of AMPA, NMDA and NPY on suprachiasmatic neurons. In contrast, inhibitory actions of NMDA and NPY are considered induced by an activation of inhibitory interneurons. Antagonistic effects of strychnine suggest an involvement of glycinergic interneurons in a subpopulation of neurons inhibited by NMDA and in most neurons inhibited by NPY. The involvement of inhibitory mechanisms in photic entrainment of the circadian system is discussed. An integrative model of excitatory and inhibitory actions of EAA and NPY on suprachiasmatic neurons is proposed.     

In vitro antiprotozoal,antimicrobial and antitumor activity of Pavetta crassipes K. Schum leaf extracts

Aim of the study

To study the potential benefit of the traditional medicinal plant Pavetta crassipes K. Schum (Rubiaceae), which is widely distributed throughout West Africa, the methanol and dichloromethane extracts were isolated from the plant leaves to determine if they exhibited antiprotozoal, antibacterial, antifungal or antitumor activity in vitro.

Materials and methods

The methanol and dichloromethane extracts and their specific fractions were obtained using bioassay-guided fractionation and investigated for antiproliferative activity in vitro in microorganisms (Staphylococcus aureus, Escherichia coli and Candida albicans), protozoans (Trypanosoma cruzi, Trypanosoma brucei, Leishmania infantum and Plasmodium falciparum), and cancer (U373, PC3, MXT and A549) and normal cell lines (NHDF and MRC-5).

Results

Most of the alkaloid fractions investigated exhibited antiproliferative activity in all the cancer cell lines, microorganisms and protozoans studied.

Conclusions

The benefit of Pavetta crassipes as a traditional medicinal remedy was confirmed using antiprotozoal and cytotoxicity assays in vitro. These analyses revealed that the components present in the alkaloid extract of Pavetta crassipes are responsible for its antiprotozoal and cytotoxic efficacy.     

Post-ischemic administration of nimodipine following focal cerebral ischemic-reperfusion injury in rats alleviated excitotoxicity, neurobehavioural alterations and partially the bioenergetics

The present study focuses on the temporal calcium significance in middle cerebral artery occluded (2 h ischemia)-reperfused (70 h reperfusion) rats treated with nimodipine (NM) through concurrent measurements of excitotoxicity, bioenergetics and neurobehavioural paradigms. Further, the suitable therapeutic time window of calcium channel antagonism in stroke was also ascertained. NM (5 mg/kg, i.p.) was administered at pre (30 min before the induction of ischemia), during (1 h following occlusion of MCA) and post-ischemic (3 h after begin of reperfusion) states. The magnitude of neuroprotection in terms of excitotoxicity (glutamate, glutamine synthetase, Na⁺K⁺ATPase), bioenergetics (ATP, NAD⁺) and neurobehavioural paradigms (neurological score and open field exploratory behaviour) were measured and compared to ensure the therapeutic time-window of NM in stroke. Middle cerebral artery occlusion-reperfusion (MCAO/R) was found to elevate glutamate, glutamine synthetase levels and deplete Na⁺K⁺ATPase activity in the vehicle treated group (IR group). Significant decrease in bioenergetics such as ATP and NAD⁺ levels was also observed. Further, IR group demonstrated grievous oxidative stress (increase in lipid peroxidation, protein carbonyl content, nitrite/nitrate levels and decrease in superoxide dismutase and glutathione levels) along with anxiogenic behaviour, neurological deficits and neuronal damage and decreased nuclear to cytoplasm ratio in CA1 hippocampal region. Post-ischemic NM administration reversed the excitotoxicity, neurobehavioural and histopathological alterations significantly, but it restored bioenergetics level in MCAO/R rats only partially.These findings were further confirmed with the combination treatment (CT) of post-ischemic NM and pre-ischemic memantine (MN) administration, since MN showed protective effect in the pre-ischemic administration (Babu and Ramanathan, 2009). The failure of NM to forefend the neurodegeneration on pre- and during-ischemic administration suggests that the initial phase damages in ischemic-reperfusion (IR) might be mediated through other mechanism(s) such as glutamergic overstimulation or reverse operation of glutamate transporters. From the present study, it is concluded that calcium plays a crucial role in post-ischemic status and the suitable therapeutic time window of calcium antagonism is the post-ischemic state.     

Contact allergy to common ingredients in hair dyes

Background. It is widely accepted that there is a molecular weight (MW) cut‐off of 500, such that single chemicals with MWs higher than 500 cannot be skin sensitizers. If true, this could serve as a useful principle for designing non‐sensitizing chemicals. Objectives. To assess whether the 500 MW cut‐off is a myth or a reality. Methods. A database of 699 chemicals tested for skin sensitization in guinea pigs or mice was analysed to establish the number of tested chemicals with MW > 500, and to establish whether any of these were sensitizers. Results. Only 13 (2%) of the 699 chemicals in the database have MW > 500. Of the 13 tested compounds with MW > 500 in the database, five are sensitizers and eight are non‐sensitizers. Conclusions. The 500 MW cut‐off for skin sensitization is a myth, probably derived from the widespread misconception that ability to efficiently penetrate the stratum corneum is a key determinant of sensitization potency. The scarcity of sensitizers with MW > 500 simply reflects the general scarcity of chemicals with MW > 500.     

阻断一氧化氮形成对大鼠海马和大脑皮层由行为训练诱发的生长抑素mRNA表达的影响(英文)

用水迷宫和一次性被动回避反应两种行为学训练方法 ,结合脑室内注射一氧化氮合酶 (NOS)抑制剂 NAME(Nω-nitro-L -arginine)和原位杂交技术 ,观察了阻断 NOS前后大鼠海马和大脑皮层前额叶等区域由行为学训练诱发的生长抑素 (somatostatin,SOM) m RNA阳性细胞数量的改变。结果显示 :(1)同未经训练的对照组相比 ,两种行为学训练都引起海马和大脑皮层中 SOMm RNA阳性细胞的显著增加 ;(2 )在脑室中注射了 NAME的实验组动物 ,两种行为学训练都不能再诱发上述阳性细胞的增加 ,同时NAME也阻止了训练组出现的学习和记忆的形成。以上结果提示 ,一氧化氮参与了作为脑内学习和记忆神经化学基础之一的生长抑素表达增加的调控     

A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon

We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide. This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers. It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.     

Ca channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats

Whole-cell patch-clamp recordings were used to study voltage-gated Ca²⁺ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O₂). Currents activated at potentials of −40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca²⁺ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 μM) always partially inhibited currents and Bay K 8644 (2–5 μM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker ω-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca²⁺ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca²⁺ channels in type I cells.     

Oxidative stress in hepatocytes and stimulatory state of Kupffer cells after reperfusion differ between warm and cold ischemia in rats

Rat liver was kept at 4°C or 37°C in MEM, and reperfused through a closed circulation from the hepatic vein to the portal vein at 37°C with the same solution. Although purine nucleoside phosphorylase and ALT activities were increased in the perfusate, depending on the duration of ischemia at both 4°C and 37°C, the ratio of the latter to the former was significantly higher after 37°C-ischemia than after 4°C-ischemia. The stimulation stage of Kupffer cells evaluated in situ by formazan deposition after liver perfusion with nitro blue tetrazolium and phorbol myristate acetate was elevated after 4°C-ischemia longer than 1 h, but not after 37°C-ischemia. In contrast, the degree of oxidative stress in hepatocytes assessed by formazan deposition after liver perfusion with nitro blue tetrazolium alone was greater after 37°C-ischemia than after 4°C-ischemia. These results suggest that oxidative stress in hepatocytes and the stimulatory state of Kupffer cells after ischemia-reperfusion may differ between 4°C-ischemia and 37°C-ischemia, probably leading to different development of liver damage.