Rat Erythrocytes Research Articles

Different Protocols of Physical Training: Effect on Markers of Oxidative Stress in Erythrocytes in Rats

The imbalance between oxidant molecules and antioxidant agents is characterized as oxidative stress (OS) and may lead to severe damage to the organism. In contrast, the physical training of aerobic and resistive character promotes increases of the antioxidant response, resulting in a balance and/or minimizing damage. Therefore, the objective of the study is to verify the effect of aerobic training, resistive training and concurrent training on oxidative stress of Wistar rats. Methods: To perform the experiment, 40 Wistar rats were subjected to eight weeks of training. The animals were distributed into 4 different groups: control, aerobic, resistive and concurrent (combination of aerobic and resistive protocols in the same section) an h/day, five days/week, for eight weeks. At the end of the experimental period: the antioxidant enzymes (catalase, glutathione peroxidase and superoxide dismutase), biomarkers of oxidative stress (TBARs and H2O2) as well as the activity of reduced and oxidized glutathione were analyzed. Results: Regarding the markers, TBARs and H2O2 trainings were not effective in promoting significant reduction. In contrast, the three training protocols caused an increase in SOD and GSH-GPx enzymes. Conclusion: Therefore, we concluded that both training protocols promoted benefit to the organism, because they caused the increase of antioxidant enzymes without disturbance in the pro-oxidant markers.

Influence of chlorpromazine on the resistance of erythrocytes of rats of different ages to hypertonic conditions

The osmotic stability of native and modified with chlopromazine (CPR) erythrocytes of 1- and 12-month rats to hypertonic conditions in sucrose solutions and hypertonic shock (4.0 M NaCl) has been studied. It has been shown that 2-min incubation of rat erythrocytes of different ages in hypertonic sucrose media does not reveal any differences in the osmotic stability of these cells. In this case, CPR does not affect cell hemolysis. An increase of the incubation time in hypertonic sucrose solutions to 30 minutes allowed detecting a greater osmotic sensitivity of erythrocytes of 1-month animals to the action. In this case, the protective effect of CPR for older age rat erythrocytes (12 months) has been established. It has been found that in 4.0 M NaCl the hemolysis level of animal erythrocytes of both age groups increases with preliminary incubation (2 min) in a sucrose medium with a concentration of 0.7 M and above. With increasing exposure time (30 min) in sucrose hypertonic solutions, the sensibilization of animal erythrocytes of both age groups to the action of hypertonic shock is also intensified. In this study it has been shown that the influence of CPR on the sensitivity of 1-month-old animal erythrocytes to the transfer in 4.0 M NaCl depends on the tonicity and duration of the cell initial incubation in sucrose solutions. Thus, CPR increases the osmotic resistance of erythrocytes, which were preexposed in sucrose solutions at a concentration of 0.6–0.8 M for 2 and 30 min. Some increase of hemolysis level of these animals erythrocytes modified with CPR in 4.0 M NaCl has been observed after 2 min of incubation in sucrose solutions at a concentration of 0.27–0.5 M. The exclusively protective influence of CPR on 12-month-old animal erythrocytes in conditions of hypertonic shock has been revealed. A quantitative estimation of the efficiency of CPR at hypertonic shock (4.0 M NaCl) of different age animal erythrocytes has been carried out by calculation of the antihemolytic activity value (AG). Short-term incubation (2 min) in a sucrose media does not reveal any differences in the values of AG of CPR for erythrocytes of both age groups. For the cells of young rats, increase of AG of CPR is observed at incubation in sucrose medium to 30 min and for erythrocytes of the older group – to 10 min. With an increase in incubation time of up to 60 min the CPR efficiency in hypertonic saline media is reduced for rat cells in both age groups but in varying degrees.

ВЛИЯНИЕ ОБЩЕГО ОХЛАЖДЕНИЯ ГОМОЙОТЕРМНОГО ОРГАНИЗМА НА ЭРИТРО- И ЛЕЙКОЦИТАРНЫЕ ПОКАЗАТЕЛИ КРОВИ

The objectives of the study were the investigation of reactions of the erythrocyte and leukocyte links of rat’s blood under general immersion hypothermia (IH). Methods. The IH was simulated by “forced swimming” test in cold water (0°C, 5 min, down to 27°C). The dynamics of erythrocyte transformation and distribution of their shapes were studied by a small-angle light scattering method. The types and number of leukocytes were determined in blood smears. The transformation indices of erythrocytes (ITE) and integral leukocyte indices (ILI) were calculated. Results. Under IH a decrease in the number of discocytes and an increase in erythrocytes altered forms were observed; the values of transformation indices, reversible and irreversible transformations were increased. The development of IH was accompanied by leukocytosis and a decrease in the number of segmented neutrophils on the background of the stab cells increase. The young, plasmatic and polychromatophilic cells as well as cytoplasm plasmatization in single leukocytes were noted. Calculation of ILI revealed activation in tissue destruction processes, immediate type of hypersensitivity and an increase in the Garkavi adaptation index. On the contrary, 24 hrs after the IH, the proportion of the discocytes increased on the expense of highly resistant flattened discocytes appearance, and the proportion of altered cells decreased due to their elimination from the blood. The values of the indices of transformation, reversibility and reversible transformation were decreased, but the index of irreversible transformation has increased by an order of magnitude. Twenty four hrs after the IH, the leukocytosis and the increase in the stab neutrophils against the background of lymphopenia as well as the appearance of young cell forms, polychromatophils and plasmatic cells were noted. Changes in ILI indicated the activation of the cellular link of the immune system, specific defense cells, delayed type hypersensitivity processes and macrophage system components, tissue decay processes, intoxication and allergisation. Conclusions. General immersion hypothermia significantly altered the quantitative and qualitative ratios of the shapes and types of blood cells in rats, affected the processes of erythrocyte transformation and the activation of distinct immunity links (according to the calculation of ITE and ILI, respectively). The direction and intensity of these changes depend on the terms of observation (immediately and 24 hrs after cooling).

Antidiabetic effects and erythrocyte stabilization by red cabbage extract in streptozotocin-treated rats.

The protective effect of red cabbage extract (RCE) was evaluated in rats with streptozotocin-induced diabetes, assessing a probable role of this extract in the prevention of erythrocyte impairments associated with a high risk of vascular complications in diabetes. RCE was analyzed by ultrahigh performance liquid chromatography and mass spectrometry, and 11 anthocyanins, 3 hydroxybenzoic acids and 9 hydroxycinnamic acids were identified. Type 1 diabetes was induced by streptozotocin (60 mg kg-1) in Wistar male rats (n = 8 per group). After 7 days of acclimatization, streptozotocin-treated rats were given RCE (800 mg kg-1) or vehicle by intragastric administration for 4 weeks. The RCE treatment lowered blood glucose, and glycated and fetal hemoglobin concentrations and improved glucose tolerance as well as considerably raised serum insulin, proinsulin and C-peptide levels in streptozotocin-treated rats. Simultaneously, RCE improved pancreatic islet morphology, increasing the amount of pancreatic β-cells in diabetic animals. The RCE administration prevented anemia in rats with streptozotocin-induced diabetes, enhanced erythrocyte resistance to acid hemolysis, and normalized reticulocyte production as well as sialic acid content in erythrocyte membranes. The enhanced lectin-induced erythrocyte aggregation in diabetic rats was significantly lowered after the RCE treatment. RCE demonstrated a significant antioxidant effect, decreasing MDA and protein carbonyl contents and increasing catalase and glutathione peroxidase activities in erythrocytes. These results indicate that RCE can be considered as a promising candidate for use as a drug or a food supplement to alleviate diabetes and its vascular complications.

Automated synthesis of [68Ga]oxine, improved preparation of 68Ga-labeled erythrocytes for blood-pool imaging, and preclinical evaluation in rodents.

Radiolabeled erythrocytes have multiple applications in nuclear medicine, including blood pool imaging. Historically they have been labeled with SPECT radionuclides. A PET blood pool imaging agent is highly desirable as it would improve clinical applications with better image quality and resolution, higher sensitivity, and dynamic scanning capabilities. With the coming of age of modern 68Ge/68Ga generator systems, gallium-68 is now widely accessible. In this paper we describe an updated method for the preparation of 68Ga-labeled erythrocytes and their preliminary use in rodent blood pool imaging. A novel automated synthesis of [68Ga]oxine using a 68Ga/68Ge generator and automated synthesis module is reported. [68Ga]Oxine was synthesized in 50 ± 5% (n = 3) non-decay corrected radiochemical yield and >99% radiochemical purity. Rat and human erythrocytes were successfully labeled with the complex in 42% RCY, and the 68Ga-labeled erythrocytes have been shown to clearly image the blood pool in a healthy rat. Human erythrocytes labelled with [68Ga]oxine were shown to be viable up to 2 hours post-labelling, and washout of the radiolabel was minimal up to 1 hour post-labelling. Further optimization of the labeling method to translate for use in human cardiac and oncologic blood pool PET imaging studies, is underway.

Haemato-protective influence of dietary fenugreek (Trigonella foenum-graecum L.) seeds is potentiated by onion (Allium cepa L.) in streptozotocin-induced diabetic rats

We have recently reported the beneficial modulation of metabolic abnormalities and oxidative stress in diabetic rats by dietary fenugreek seeds and onion. This investigation evaluated the protective influence of dietary fenugreek seeds (100 g kg−1) and onion (30 g kg−1) on erythrocytes of streptozotocin-induced diabetic rats, through modulation of reduced haematological indices and antisickling potency. This study also evaluated the altered erythrocyte membrane lipid profile and beneficial countering of increased lipid peroxidation, osmotic fragility, along with reduced membrane fluidity and deformability, nitric oxide production and echinocyte formation. Dietary fenugreek seeds and onion appeared to counter the deformity and fragility of erythrocytes partially in diabetic rats by their antioxidant potential and hypocholesterolemic property. The antisickling potency of these spices was accomplished by a substantial decrease in echinocyte population and AGEs in diabetic rats. Further insight into the factors that might have reduced the fluidity of erythrocytes in diabetic rats revealed changes in the cholesterol: phospholipid ratio, fatty acid profile, and activities of membrane-bound enzymes. Dietary fenugreek seeds and onion offered a beneficial protective effect to the red blood cells, the effect being higher with fenugreek + onion. This is the first report on the hemato-protective influence of a nutraceutical food component in diabetic situation.

A composition of medicinal plants with an enhanced ability to suppress microsomal lipid peroxidation and a protective activity against carbon tetrachloride-induced hepatotoxicity

Treatment of liver injury induced by various toxicants represents a serious clinical challenge. Here, we utilized the ability of natural agents to inhibit microsomal lipid peroxidation (LPO) as the in-vitro screening paradigm for selecting efficacious tissue-protective combinations of cooperatively acting medicinal plants. Based on screening of 70 water-ethanol extracts obtained from different parts of 65 plants we prepared a highly active phytocomposition (PC-1) containing oregano (Origanum vulgare), wild thyme (Thymus serpyllum) and coltsfoot (Tussilago farfara) aerial parts, valerian (Valeriana officinalis) leaves and little-leaf linden (Tilia cordata) flowers. PC-1 extract exhibited the strongest anti-PLO and antihemolytic effects in vitro compared to those of the individual plants and other compositions tested. Using luciferase reporter assay and Western blotting in HepG2 human hepatocellular carcinoma cells, we found that PC-1 extract activated the Nrf2/antioxidant response element signaling pathway more effectively than the extracts of other phytocompositions. Importantly, oral administration of PC-1 extract (100–200 mg/kg) markedly ameliorated liver injury in rats acutely or chronically intoxicated by carbon tetrachloride. This was evidenced by improved liver histology, blood chemistry parameters, and microsomal LPO status and superoxide dismutase activity. In addition, treatment with PC-1 extract salvaged the osmotic resistance of erythrocytes in carbon tetrachloride-intoxicated rats. Collectively, these data support the strategy of in-vitro plant selection for developing efficacious tissue-protective phytocompositions.

Carnosine and N-acetyl cysteine protect against sodium nitrite-induced oxidative stress in rat blood.

Sodium nitrite (NaNO2 ) is widely used in the food industry as a preservative and colorant in meat and fish products. Industrialization and improper agricultural practices have greatly increased human exposure to high nitrite levels, mainly through contaminated drinking water, causing various health disorders. We have investigated the protective effect of carnosine (CAR) and N-acetyl cysteine (NAC) on NaNO2 -induced toxicity in rat blood. CAR is a bioactive dipeptide found in mammalian muscle while NAC is a synthetic sulfhydryl amino acid and an important precursor of glutathione. Animals were given a single acute oral dose of NaNO2 at 60 mg/kg body weight with or without prior administration of either CAR or NAC. Rats were sacrificed after 24 h, blood was withdrawn and plasma and erythrocytes were isolated. Administration of NaNO2 alone increased methemoglobin levels and methemoglobin reductase activity, decreased the activities of antioxidant defense and metabolic enzymes and significantly weakened the total antioxidant capacity of rat erythrocytes. Similar effects were seen in plasma of NaNO2 -treated rats. In contrast, administration of CAR or NAC, prior to NaNO2 treatment, markedly attenuated the NaNO2 -elicited deleterious effects. Thus, CAR and NAC can mitigate nitrite-induced metabolic alterations and oxidative damage probably due to their intrinsic biochemical antioxidant properties. This study suggests that CAR and NAC can be potentially used as therapeutic/protective agents against NaNO2 toxicity.

In vivo assessment of polydatin, a natural polyphenol compound, on arsenic-induced free radical overproduction, gene expression, and genotoxicity.

Arsenic (As) is a well-known contaminant of global groundwater. Its exposure causes several hazardous effects on animals and human via oxidative stress. The present study examined the effect of polydatin (PD) on free radical overproduction in rats exposed to As. Thirty-five male rats randomly allocated into five equal groups. To the control group, physiological saline was given orally and to the second group only 100mg/L As was given by drinking water for 60days. The other groups were treated with As (100mg/L) and PD orally at 50, 100, and 200mg/kg/day, respectively. Treatment with As enhanced malondialdehyde level but decreased glutathione level in blood, liver, kidney, brain, lung, and heart of rats. Also, As decreased superoxide dismutase and catalase activities of erythrocyte, liver, kidney, brain, lung, and heart in rats. Furthermore, As treatment gave rise to increased DNA damage and gene expressions of interleukin 1 beta (IL-1β), nuclear factor kappa beta (NFκB), p53, and tumor necrosis factor-α (TNF-α) in the lung, brain, kidney, and liver. However, treatment of PD ameliorated As-exposed lipid peroxidation, antioxidant enzymes activities, DNA damage, gene expressions, and histopathological changes in tissues. In conclusion, PD has a dose-dependent protective effect on lipid peroxidation and antioxidant defense mechanism in rats against As exposure.

Morphofunctional State of Erythrocytes in Rats with Walker-256 Carcinoma Treated with Docetaxel and Xymedon.

We compared the effect of xymedon (100 mg/kg) and Mexidol (50 mg/kg) on morphometric parameters of erythrocytes and oxygen-transporting function of hemoglobin in rats with Walker-256 carcinoma treated with docetaxel (15 mg/kg once intraperitoneally on day 11 after tumor cells transplantation). Xymedon and Mexidol were injected intramuscularly for 10 days starting from day 11 of the experiment. The studied parameters were evaluated on experimental days 14 and 22. Similar to Mexidol, xymedon prevented changes in the erythrocyte geometric parameters induced by docetaxel and neoplastic process, but increased hemoglobin packing density in erythrocytes (by 32%) more effectively than Mexidol. Optimization of oxygen-transporting function of hemoglobin and normalization of its structural and functional parameters changed by docetaxel treatment (content of oxyhemoglobin, hemoglobin affinity for oxygen and its ability to bind and release oxygen, intensity of symmetric and asymmetric vibrations of pyrrole rings) in rats receiving xymedon took longer time than in case of Mexidol treatment, but the effects were similar.

Electrokinetic and Biochemical Changes in Erythrocytes under the Action of Terahertz Range Electromagnetic Waves

The effects of electromagnetic radiation of a gyrotron with an operating frequency of 263 GHz on rat erythrocyte electrophoretic mobility, malonic dialdehyde, ATP, and 2,3-diphosphoglycerate concentration have been studied in vitro. The blood was irradiated under continuous and pulsed gyrotron emission modes with the estimated incident energy flux on an object from 0.1 to 20 mW/cm2. It has been established that the studied erythrocyte characteristics changed in different directions depending on the mode and the incident energy flux: the erythrocyte electrophoretic mobility rate and ATP concentration decreased, while the malonic dialdehyde and 2,3-diphosphoglycerate concentration increased at a low incident energy flux and continuous exposure mode; all studied parameters increased under the pulsed exposure mode. An incident energy flux increase caused a reduction of these effects.

A regenerative erythropoietic response does not increase the frequency of Pig-a mutant reticulocytes and erythrocytes in Sprague-Dawley rats.

The in vivo rodent Pig-a mutation assay is a sensitive test to identify exposure to mutagenic substances, and has been proposed as an assay for the identification of impurities for pharmaceuticals. Red blood cells (RBCs) and reticulocytes (RETs) are analyzed by flow cytometry after exposure to potentially mutagenic chemicals for cells deficient in the cell surface anchored protein CD59, representing mutation in the X-linked Pig-a gene. The full potential of the assay as well as its limitations are currently being explored. The current study investigated the effects of regenerative erythropoietic bone marrow responses on the frequency of Pig-a mutated reticulocytes (RETCD59- ) and erythrocytes (RBCCD59- ). We hypothesized that a robust regenerative erythropoietic response would not increase the basal frequency of RETCD59- or RBCCD59- cells. Two groups of six male Sprague-Dawley rats either had 2 mL of blood sampled each day via an indwelling catheter over a period of 5 days or were minimally sampled for hematology and used as controls. Blood was also then collected and evaluated 5, 18, and 49 days after the initial bleed period for the number of Pig-a mutant cells in either the RET or RBC population. Despite the expected decrease in hematocrit and the correlative increase in reticulocytes after bleeding, no increase in the number of Pig-a mutant cells was observed in male Sprague-Dawley rats that were bled for five consecutive days. These results indicate that changes in erythropoiesis and hematology parameters in rats appear to have no effect on the background levels of Pig-a mutated RETs and RBCs. Environ. Mol. Mutagen. 59:91-95, 2018. © 2017 Wiley Periodicals, Inc.

Antibacterial activity against cariogenic bacteria and cytotoxic and genotoxic potential of Anacardium occidentale L. and Anadenanthera macrocarpa (Benth.) Brenan extracts

ObjectivesThe present study aimed to assess the antibacterial activity against bacteria with cariogenic relevance, toxic and genotoxic potential of the plants Anacardium occidentale L. and Anadenanthera macrocarpa (Benth.) Bernam. DesignUsing a microdilution technique, the extracts were submitted to minimum inhibitory concentration (MIC) testing against Streptococcus mitis (ATCC 903), Streptococcus mutans (ATCC 25175), Streptococcus oralis (ATCC 10557), Streptococcus salivarius (ATCC 7073), Streptococcus sanguinis (ATCC 15300) and Streptococcus sobrinus (ATCC 27609). The toxicity of the extracts was then verified against eukaryotic cells. Additionally, a micronucleus assay was performed to investigate the potential mutagenic effects of the extracts on rat erythrocytes. The Student’s t-test, Bonferroni test, and one-way ANOVA followed by Tukey’s tests were used for statistical analysis, at a significance level of 5%. ResultsWhile the A. occidentale extract was able to inhibit all of the tested strains, with S. mutans and S. mitis being the most susceptible to that extract́s action, the A. macrocarpa did not show antimicrobial activity. Interestingly, the hemolytic, oxidant and antioxidant activities were slightly observed for either extract, even at high concentrations (1000mg/mL). The micronucleus assay showed no significant changes in the cells exposed to the extracts. ConclusionThe A. occidentale extract has potential as an antimicrobial agent with low eukaryotic cell toxicity or mutagenic activity. The A. macrocarpa extract, although absent of antibacterial activity might as well be a safe and effective phytotherapeutic alternative.

Kinetic characteristics of acetylcholinesterase and structural-functional state of rat erythrocyte membranes at moderate hypothermia

Artificial hypothermic state of homeothermic animals contributes to the stimulation of free radical processes in red blood cells. In order to understand what are the consequences of oxidative damage of erythrocyte membrane, we examined the dependence of the kinetic characteristics of integral membrane enzyme acetylcholinesterase (AChE) and structural and functional state of the membrane on the duration of mild hypothermia. For this purpose we reduced body temperature of adult Wistar rats by external cooling to 30°C (short-term moderate hypothermia) and then prolonged hypothermia up to 1.5 and 3 h. A short-term hypothermia was followed with an increase in V max and a decrease in K m, promoting an increase in the catalysis effectiveness.The optimum point on the graph of the concentration dependence was shifted to the area of lower concentrations, and the character of enzyme–substrate interactions at high concentrations of the enzyme changed. Upon prolongation of hypothermia, changes in the AChE kinetic characteristics favored normalization of the enzyme activity and concentration dependence. To test the hypothesis of a possible influence of the lipid matrix on the kinetic characteristics of AchE, we studied structural properties of the erythrocyte membranes using fluorescent probe pyrene. The observed changes in the structural and dynamic characteristics of erythrocyte membranes after a 1.5-h hypothermia suggested a reduction in microviscosity of both total and annular lipids. Prolongation of hypothermia up to 3 h favored normalization of this parameter. It was found that the indicators of the structural state of erythrocyte membranes at different durations of hypothermia correlate with certain kinetic characteristics of AChE. The data indicate that the prolongation of mild hypothermia up to 3 h triggers adaptive mechanisms directed to normalization of the erythrocytes membrane functioning.

Synergistic Effect of Rapamycin and Metformin Against Age-Dependent Oxidative Stress in Rat Erythrocytes.

Erythrocytes are particularly vulnerable toward age-dependent oxidative stress-mediated damage. Caloric restriction mimetics (CRMs) may provide a novel strategy for the maintenance of redox balance as well as effective treatment of age-associated diseases. Herein, we have investigated the beneficial effect of cotreatment with CRM-candidate drugs, rapamycin (an immunosuppressant drug and inhibitor of mammalian target of rapamycin) and metformin (an antidiabetic biguanide and activator of adenosine monophosphate kinase), against aging-induced oxidative stress in erythrocytes and plasma of aging rats. Male Wistar rats of age 4 (young) and 24 months (old) were coexposed to rapamycin (0.5 mg/kg body weight [b.w.]) and metformin (300 mg/kg b.w.), and data were compared with the response of rats receiving an independent exposure to these chemicals at similar doses. The exposure of individual candidate drugs significantly reversed the age-dependent alterations in the endpoints associated with oxidative stress such as reactive oxygen species, ferric reducing ability of plasma, malondialdehyde, reduced glutathione, plasma membrane redox system, plasma protein carbonyl, and acetyl cholinesterase in erythrocytes and plasma of aging rats. However, the cotreatment with rapamycin and metformin showed a significant augmented effect compared with individual drug interventions on reversal of these age-dependent biomarkers of oxidative stress, suggesting a synergistic response. Thus, the findings open up further possibilities for the design of new combinatorial therapies to prevent oxidative stress- and age-associated health problems.

Protective effects of Mentha spicata against nicotine-induced toxicity in liver and erythrocytes of Wistar rats.

The aim of this study was to investigate the protective effect of Mentha spicata supplementation against nicotine-induced oxidative damage in the liver and erythrocytes of Wistar rats. Bioactive substances were determined by liquid chromatography - electrospray ionization - tandem mass spectrometry analysis. Animals were divided into 4 groups of 6 rats each: a normal control group, a nicotine-treated group (1 mg/kg), a group receiving M. spicata extract (100 mg/kg), and a group receiving both M. spicata extract (100 mg/kg) and nicotine (1 mg/kg). Many phenolic acids were identified in the M. spicata aqueous extract. After 2 months of treatment, nicotine induced an increase in the level of white blood cells and a marked decrease in erythrocytes, hemoglobin, and haematocrit. Aspartate transaminase, alanine transaminase, alkaline phosphatase, and lactate dehydrogenase activities were also found to be higher in nicotine-treated group than those of the control group. Furthermore, nicotine-treated rats exhibited oxidative stress, as evidenced by a decrease in antioxidant enzymes activities and an increase in lipid peroxidation level in liver and erythrocytes. Interestingly, the oral administration of M. spicata extract by nicotine-treated rats alleviated such disturbances. M. spicata contained bioactive compounds that possess important antioxidant potential and protected liver and erythrocytes against nicotine-induced damage.

Оценка взаимодействия окисленного графена с мембранами эритроцитов и белками плазмы крови крыс методом спиновых зондов

Оценка взаимодействия окисленного графена с мембранами эритроцитов и белками плазмы крови крыс методом спиновых зондов

Modulation of P-glycoprotein by Stemona alkaloids in human multidrug resistance leukemic cells and structural relationships

BackgroundMultidrug resistance (MDR) is a major reason for the failure of chemotherapy in the treatment of cancer patients. P-gp over-expression in MDR cancer cells is a multifactorial phenomenon with biochemical resistance mechanisms. Stemofoline (STF), isolated from Stemona bukillii, has been reported to be an MDR reversing compound. PurposeThis study investigated whether other Stemona alkaloids that had been purified from Stemonaceae plants exerted MDR modulation activity. MethodsMTT assay was performed to determine the MDR reversing property of the alkaloids. Modulation of P-gp function by these compounds was investigated using cell cycle analysis and P-gp fluorescent substrate accumulation assays. P-gp expression was determined by Western blot analysis. We preliminarily examined the safety of these compounds in normal human fibroblasts and human peripheral blood mononuclear cells (PBMCs) using the MTT assay, and in red blood cells (human and rat) through in vitro hemolysis assays. ResultsThree of the eight alkaloids tested, isostemofoline (ISTF), 11Z -didehydrostemofoline (11Z-DSTF) and 11E-didehydrostemofoline (11E-DSTF), enhanced the chemotherapeutic sensitivity of MDR leukemic K562/Adr cells, which overexpressed P-gp. The P-gp functional studies showed that these three alkaloids increased the accumulation of P-gp substrates, calcein-AM (C-AM) and rhodamine123 (Rho 123) in K562/Adr cells, while this effect was not seen in drug sensitive parental K562 cells. Whereas, the alkaloids did not alter P-gp expression as was determined by Western blotting analysis. ConclusionThe alkaloids reversed MDR via the inhibition of P-gp function. For pharmaceutical safety testing, the alkaloids were found to be not toxic to normal human fibroblasts and PBMCs. Moreover, the effective compounds did not induce hemolysis in either human or rat erythrocytes. These compounds may be introduced as potential candidate molecules for treating cancers exhibiting P-gp-mediated MDR.

Acetylcholinesterase measurement in various brain regions and muscles of juvenile, adolescent, and adult rats

Several insecticides, chemicals, and drugs are known to inhibit acetylcholinesterase (AChE) activity, responsible for the cleavage of the neurotransmitter acetylcholine. The administration of AChE inhibitors leads to typical parasympathomimetic (toxic) symptoms in rats. In order to differentiate between compounds acting in various regions of the brain or in peripheral nerves, regulatory authorities demand the measurement of AChE activity in different compartments and the study of potential toxicity at different developmental stages. In the present paper, instructions are given for the necropsy of various regions of the brain depending on rat age. Furthermore, a method validation procedure is described for measuring AChE in these parts of the brain as well as peripheral nerves, serum, and erythrocytes in juvenile, adolescent, and adult rats according to the US EPA method. All investigations were performed within the frame of a regulatory extended one-generation reproductive study (EOGRTS, OECD TG 443). AChE activity increases age dependently in parts of the forebrain (cortex, hippocampus, striatum, but decreases in the mid- and hindbrain (cerebellum, brain stem, medulla oblongata) as well as in peripheral nerves (heart, diaphragm, gastrocnemius muscle). Sex-dependent differences of the AChE activity occur after an age of 11 weeks. The implication of AChE measurement in different brain regions of various age groups is discussed regarding the assessment of AChE inhibitors.

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats.

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2',5'-ADP-Sepharose 4B affinity gel. The effects of Ast and Al3+ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al3+ inhibited the enzyme activity noncompetitively (IC50 values; 0.679mM, Ki values 1.32mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al+Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P<0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al+Ast groups (P<0.05).