ERK1/2 modulation in the central nervous system of male and female thiamine-deficient mice with amprolium.

Thiamine deficiency experimental models focus on using the pyrithiamine analog in male rodents, making the thiamine deficiency effects in females and the use of other thiamine antagonists, such as amprolium, unknown. We investigated the impact of thiamine deficiency with amprolium in the cerebral cortex and thalamus of male and female mice by evaluating the modulation of ERK1/2 phosphorylation. The animals were exposed for 20 days to thiamine-deficient chow with different doses of amprolium (20, 40, 60 and 80 mg/kg) and at different treatment periods (five, 10, 15 or 20 days) at a dose of 60 mg/kg. After treatments, ERK1/2 phosphorylation was analyzed by western blot. In male mice, we observed a progressive increase in ERK1/2 phosphorylation in both the cerebral cortex and thalamus in response to the dose of amprolium. In females, ERK1/2 phosphorylation did not progressively increase in response to the amprolium dosage. However, an increase in phosphorylation at the higher doses of 60 and 80 mg/kg was observed. We observed a more intense increase in ERK1/2 phosphorylation in males' cerebral cortex and thalamus from 10 days onwards. In females, the ERK1/2 modulation profiles were similar. The results show that thiamine deficiency induction with amprolium is efficient, compatible with other recognized models that use pyrithiamine, showing changes in cell signaling in the nervous system. The study showed differences in response to thiamine deficiency with amprolium between male and female mice in relation to ERK1/2 phosphorylation and demonstrated that females respond positively to the method and can also be used as model animals.

Anticoccidial activity of Aloe Vera Leafs' aqueous extract and vaccination against Eimeria tenella: pathological study in broilers.

This study aimed to assess the efficacy of an anticoccidial vaccine and the anticoccidial activity of Aloe vera in broiler chickens infected with Eimeria tenella (E. tenella). A total of 225 healthy, sexless, one-day-old broiler chicks (avian48) from a commercial broiler company were randomized into nine experimental groups of 25 chicks. The groups were as follows: Group 1 (control, vaccinated, non-infected), Group 2 (vaccinated and infected with 5 × 104 sporulated oocysts), Group 3 (vaccinated, infected with 5 × 104 sporulated oocysts, and treated with Aloe vera), Group 4 (infected with 5 × 104 sporulated oocysts and treated with Aloe vera), Group 5 (positive control, infected with 5 × 104 sporulated oocysts), Group 6 (challenged with 5 × 104 sporulated oocysts and then treated with amprolium), Group 7 (treated with amprolium), Group 8 (blank control negative group), and Group 9 (treated with Aloe vera gel).Various parameters were evaluated, including clinical signs, growth performance, oocyst shedding, hematological and immunological parameters, and pathological lesion scoring. The results demonstrated that Aloe vera improved growth performance, reduced oocyst shedding, and decreased caecal lesion scores in E. Tenella-infected broiler chicks. The use of Aloe vera in combination with either amprolium or anticoccidial vaccines provided a potential solution to the issues of drug resistance and drug residues.In conclusion, this study provides valuable insights regarding the control of coccidiosis in broilers. Supplementing the chicken diet with Aloe vera had beneficial effects on the pathogenicity and infectivity of E. tenella, making it a cost-effective alternative as an herbal extract with no adverse side effects for coccidiosis control. These findings suggest that Aloe vera can be considered a potential candidate for inclusion in broiler diets for effective coccidiosis control.

Persea americana extract protects intestinal tissue from Eimeria papillata-induced murine Infection.

Coccidiosis is the most prevalent disease-causing widespread economic loss among farm and domestic animals. Currently, several drugs are available for the control of this disease but resistance has been confirmed for all of them. There is an urgent need, therefore, for the identification of new sources as alternative treatments to control coccidiosis. The present work aimed to study the effect of the Persea americana extract (PAE) as an anti-coccidial, anti-oxidant, and anti-apoptotic modulator during murine intestinal Eimeria papillata infection. A total of 25 male mice were divided into five groups, as follows: Group1: Non-infected-non-treated (negative control), Group2: Non-infected-treated group with PAE (500 mg/kg b.w). Group3: Infected-non-treated (positive control), Group4: Infected-treated group with PAE (500 mg/kg b.w.), and Group5: Infected-treated group with Amprolium (120 mg/kg b.w.). Groups (3-5) were orally inoculated with 1 × 103 sporulated E. papillata oocysts. After 60 min of infection, groups (4 and 5) were treated for 5 consecutive days with the recommended doses of PAE and amprolium. The fact that PAE has an anti-coccidial efficacy against intestinal E. papillata infection in mice has been clarified by the reduction of fecal oocyst output on the 5th day post-infection by about 85.41%. Moreover, there is a significant reduction in the size of each parasite stage in the jejunal tissues of the infected-treated group with PAE. PAE counteracted the E. papillata-induced loss of glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (TCA). E. papillata infection also induced an increase in the apoptotic cells expressed by caspase-3 which modulated after PAE treatment. Moreover, the mRNA expression of the goblet cell response gene, mucin (MUC2), was upregulated from 0.50 to 1.20-fold after treatment with PAE. Based on our results, PAE is a promising medicinal plant with anti-coccidial, anti-oxidant, and anti-apoptotic activities and could be used as a food additive.

Comparative efficacy of allopathic and herbal drugs in sheep naturally infected with coccidiosis.

Development of anticoccidial resistance and concerns of drug residues have prompted the evaluation of alternatives to allopathic drugs. In current study, anticoccidial effect of amprolium was compared with that of Curcuma longa and Zingiber officinale. Ninety (90) sheep, naturally infected with Eimeria spp. and having a minimum oocyst per gram (OPG) count of faeces above 5000 were randomly selected and divided into six groups of 15 animals each. Animals were supplemented with amprolium @ 62.50 mg/kg body weight (bw) (GI), turmeric @ 200 and 300 mg/kg bw (GII and GIII) and ginger @ 200 and 300 mg/kg bw (GIV and GV), orally for 7 days and GVI animals were kept as untreated infected control. Faecal samples were collected on '0' day before treatment and on 8th, 14th, 21st and 28th day after starting treatment and evaluated using Faecal oocyst count reduction test (FOCRT). The efficacy of amprolium was 93.18%, 96.82%, 95.56% and 95.80% on 8th, 14th, 21st and 28th day, after starting treatment. Turmeric @200 mg/kg b.w. showed efficacy of 41.49%, 52.37%, 61.47% and 60.08% and turmeric @ 300 mg/kg bw was 44.92%, 54.32%, 64.21% and 61.95% effective on 8th, 14th, 21st and 28th day, respectively. Ginger @200 mg/kg bw showed efficacy of 38.51%, 53.48%, 55.38% and 55.53% and ginger @ 300 mg/kg bw was 39.65%, 54.81%, 57.18% and 58.22% effective on 8th,14th, 21st and 28th day, respectively. The results justify use of amprolium for clinical coccidiosis while Curcuma longa and Gingiber officinale could be used as natural prophylactic alternatives.

Dietary tea tree (Melaleuca alternifolia) essential oil as alternative to antibiotics alleviates experimentally induced Eimeria tenella challenge in Japanese quails.

Coccidiosis is one of the most common infectious diseases seen in Japanese quails. The current study was conducted to evaluate the impact of tea tree essential oil (TTEO) on growth performance and intestinal health of quails in response to Eimeria tenella challenge. A total of 250 Japanese quails were divided into five treatments: untreated uninfected (negative control); untreated infected (positive control); infected + Amprolium; infected and 1% TTEO; infected and 2% TTEO. Except negative control, all groups were orally dosed with 5 × 104 sporulated oocysts of E. tenella. The results revealed that supplementation of 1% TTEO and treatment of amprolium improved feed intake, weight gain and feed conversion ratio in infected quails compared to the positive control. Similarly, lesion score and mortality was significantly (p < 0.01) reduced in quails supplemented with 2% TTEO and amprolium treated birds. Moreover, oocysts counts and histological features of caecum in infected birds were reversed in 1% TTEO and amprolium treatment. The histological findings of amprolium and 1% TTEO supplemented quails showed intact intestinal villi with mild sloughed epithelium. In conclusion, 1% TTEO can be safely used to control coccidiosis in Japanese quails as natural effective compound.

Influence of Eimeria spp. Infection on Chicken Jejunal Microbiota and the Efficacy of Two Alternative Products Against the Infection.

Eimeria spp. are important intestinal pathogens of chickens (Gallus gallus domesticus). Anticoccidial feed additives, chemicals, and ionophores have traditionally been used to control Eimeria infections in broiler production. Thus, the trend toward antibiotic-free and organic production requires new approaches to coccidiosis prevention. Two not mutually exclusive methods are the use of plant extracts with antiparasitic activity and manipulation of the intestinal microbiota by pre- and probiotics. In the present study, birds were inoculated with a combination of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. We profiled the jejunal microbiome at multiple time points postinfection to investigate the changes in jejunum microbiota and to identify the time point of the maximum difference between infected and noninfected birds. Additionally, we assessed the anticoccidial effects of two anecdotal treatment methods, green tea and apple cider vinegar, as well as amprolium. Green tea and apple cider vinegar had no effect on oocyst shedding, but green tea reduced the mild unspecific lesions in coccidia-infected birds; there was no influence on unspecific lesions in uninfected controls. Jejunal contents were collected on the day of the infection and 1, 2, 4, 6, 10, and 14 days postinfection (dpi) for investigation of the intestinal microbiota by 16S ribosomal (r)RNA gene sequencing. Comparison of the untreated-uninfected and the untreated-infected groups showed a maximum community dissimilarity of 10 dpi. From 4 days after infection, Clostridiales were significantly enriched at the expense of Lactobacillales in infected compared with uninfected birds. Interestingly, treatment with green tea prevented proliferation of Clostridiales induced by the coccidia and increased the relative abundance of Melainabacteria.

Influencia de la infección de Eimeria spp. en el microbiota yeyunal del pollo y en la eficacia de dos productos alternativos contra la infección. Los protozoarios del género Eimeria spp. son importantes patógenos intestinales de los pollos (Gallus gallus domesticus). Los aditivos anticoccidiales en el alimento, los productos químicos y los ionóforos se han utilizado tradicionalmente para controlar las infecciones por Eimeria en la producción de pollos de engorde. Por lo tanto, la tendencia hacia la producción orgánica y libre de antibióticos requiere nuevos enfoques para la prevención de la coccidiosis. Dos métodos no mutuamente excluyentes son el uso de extractos de plantas con actividad antiparasitaria y la manipulación de la microbiota intestinal por prebióticos y probióticos. En el presente estudio, las aves fueron inoculadas con una combinación de Eimeria acervulina, Eimeria maxima y Eimeria tenella. Se determinó el perfil del microbioma yeyunal en múltiples puntos de tiempo después de la infección, para investigar los cambios en la microbiota del yeyuno e identificar el momento donde se observa la diferencia máxima entre las aves infectadas y no infectadas. Además, se evaluaron los efectos anticoccidiales de dos métodos de tratamiento basados por datos anecdóticos, el té verde y el vinagre de sidra de manzana, así como el amprolium. El té verde y el vinagre de sidra de manzana no tuvieron ningún efecto sobre el desprendimiento de oocistos, pero el té verde redujo las lesiones leves inespecíficas en aves infectadas con coccidia; no hubo influencia en lesiones inespecíficas en controles no infectados. Los contenidos yeyunales se recogieron el día de la infección y a los días 1, 2, 4, 6, 10 y 14 después de la infección (dpi) para la investigación de la microbiota intestinal mediante secuenciación del gene del ARN ribosómico 16S. La comparación de los grupos no tratados-no infectados y no tratados-infectados mostró una diferencia máxima en la comunidad a los diez días después de la inoculación. A partir de los cuatro días después de la infección, los agentes Clostridiales se enriquecieron significativamente a expensas de los Lactobacillales en aves infectadas en comparación con las aves no infectadas. Curiosamente, el tratamiento con té verde evitó la proliferación de Clostridiales inducida por los coccidios y aumentó la abundancia relativa de melainabacterias.

Study on the interaction of amprolium HCl and dinitolmide in animal-derived food products with BSA by multiple spectroscopies and molecular modeling techniques.

Communicated by Ramaswamy H. Sarma.

In vitro effect of two commercial anti-coccidial drugs against myxospores of Kudoa septempunctata genotype ST3 (Myxozoa, Multivalvulida).

Kudoa septempunctata (Myxozoa: Multivalvulida) myxospores infect the trunk muscles of olive flounder (Paralichthys olivaceus). In this study, two popular commercially formulated anti-coccidial drugs (amprolium hydrochloride and toltrazuril) were serially diluted and incubated with purified mature Kudoa septempunctata myxospores. The viability of K. septempunctata spores was determined after a 2-day incubation followed by Hoechst 33342 and propidium iodide staining, and scanning electron microscopy. Amprolium hydrochloride significantly decreased spore viability (18% of control) at a concentration of 920 µg/mL, whereas toltrazuril showed almost no effect (83% of control). Viability of the control (untreated spores) was 90%. In vivo studies are required to confirm the efficacy of amprolium hydrochloride in fish infected with K. septempunctata myxospores on their growth and immune system performance.

Thiamine is a substrate of organic cation transporters in Caco-2 cells.

The aim of this study was to characterize the intestinal absorption of thiamine, by investigating the hypothesis of an involvement of Organic Cation Transporter (OCT) family members in this process. [(3)H]-T(+) uptake was found to be: 1) time-dependent, 2) Na(+)- and Cl(-)-dependent, 3) pH-dependent, with uptake increasing with a decrease in extracellular pH and decreasing with a decrease in intracellular pH, 4) inhibited by amiloride, 5) inhibited by the thiamine structural analogues oxythiamine and amprolium, 6) inhibited by the unrelated organic cations MPP(+), clonidine, dopamine, serotonin, 7) inhibited by the OCT inhibitors decynium22 and progesterone. Moreover, the dependence of [(3)H]-T(+) uptake on phosphorylation/dephosphorylation mechanisms was also investigated and [(3)H]-T(+) uptake was found to be reduced by PKA activation and protein tyrosine phosphatase and alkaline phosphatase inhibition. In conclusion, our results are compatible with the possibility of thiamine being transported not only by ThTr1 and/or ThTr2, but also by members of the OCT family of transporters (most probably OCT1 and/or OCT3), thus sharing the same transporters with several other organic cations at the small intestinal level.

Ethanol promotes thiamine deficiency-induced neuronal death: involvement of double-stranded RNA-activated protein kinase.

BACKGROUND: Heavy alcohol consumption causes cerebellar degeneration, and the underlying mechanism is unclear. Chronic alcoholism is usually associated with thiamine deficiency (TD) which is known to induce selective neurodegeneration in the brain. However, the role of TD in alcohol-induced cerebellar degeneration remains to be elucidated. The double-stranded RNA-activated protein kinase (PKR) is a potent antiviral protein. Viral infection or binding to dsRNA causes PKR autophosphorylation and subsequent phosphorylation of the alpha-subunit of eukaryotic translation factor-2alpha, leading to inhibition of translation or apoptosis. PKR can also be activated by cellular stresses. METHODS: In this study, we used an in vitro model, cultured cerebellar granule neurons (CGNs), to investigate the interaction between TD and ethanol and evaluate the contribution of their interaction to neuronal loss. TD was induced by treatment with amprolium in association with ethanol. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assay. PKR expression/phosphorylation and subcellular distribution was analyzed with immunoblotting and immunocytochemistry. RESULTS: Thiamine deficiency caused death of CGNs but ethanol did not. However, TD plus ethanol induced a much greater cell loss than TD alone. TD-induced PKR phosphorylation and ethanol exposure significantly promoted TD-induced PKR phosphorylation as well as its nuclear translocation. A selective PKR inhibitor not only protected CGNs against TD toxicity, but also abolished ethanol potentiation of TD-induced loss of CGNs. CONCLUSIONS: Ethanol promoted TD-induced PKR activation and neuronal death. PKR may be a convergent protein that mediates the interaction between TD and ethanol.

Anticoccidial effects of Ageratum conyzoides.

Ageratum conyzoides (Asteraceae) is folklorically used to treat diseases associated with bleeding. A study was conducted to determine the efficacy of Ageratum conyzoides in treating caecal coccidiosis of broilers. Acute toxicity test was done using thirty 28 days old broiler chicks which were divided into six groups of five birds each. The birds were given 250-3000 mg of extract/kg bodyweight orally. Group VI received equal volumes of distilled water. The birds were observed for 24h for signs of toxicity. Twenty-five growing broilers were divided into five groups of five birds each. At 35 days of age, birds in groups A to D were each infected orally with 8000 oocysts of Eimeria tenella. Fifteen days post-infection, groups A and B were treated with 500 and 1000 mg/kg of the extract, respectively. Group C received Amprolium in drinking water as 0.012 and 0.006% solutions for 7 days, respectively. Groups D and E were the negative and positive controls. No signs of toxicity were observed during the acute toxicity test. The faecal oocyst per gram of faeces decreased steadily in all the treatment groups until it became zero. The packed cell volumes, weight and red blood cell counts of the treated birds were significantly (P<0.05) higher than those of the infected untreated control. This confirms its ethnoveterinary use in the treatment of coccidiosis.

Comparative characteristic of thiamine antagonists on apoptosis induction in different types of nerve cell lines.

Abnormalities in oxidative metabolism and inflammation accompany many neurodegenerative diseases. The mechanisms of neurodegeneration induced by thiamine deficiency remain incompletely elucidated. The susceptibility of various types of nerve cells to thiamine (vitamin B) antagonists--oxythiamine (OT), pyrithiamine (PT) and amprolium (Am) was investigated. Four cell lines (neuronally differentiated rat PC-12, rat astrocytes DITNC, neuronally differentiated human SH-SY5Y and human astrocytic cells 1321N1) were used for experiments as neural cell models. When different cell types were cultivated with thiamine antagonists, a significant decrease of viability was detected in a time- and dose-dependent manner as demonstrated by the WST-1 colorimetric assay. These data were similar to those of caspase 3 activity and DNA fragmentation induced by thiamine antagonists. All tested cell lines were more vulnerable to OT and PT than to Am. Am displayed a pronounced damaging action on neuronal cells and had a modest influence on astrocytes. The last observation gives the basis to suppose, that neuronal cells need external arrival of thiamine more than astrocytes. Thus, the results testify that various types of nerve cells have different susceptibility to the thiamine antagonists and this relates to extent of apoptosis development.

Thiamine deficiency caused by thiamine antagonists triggers upregulation of apoptosis inducing factor gene expression and leads to caspase 3-mediated apoptosis in neuronally differentiated rat PC-12 cells.

Recent evidence suggests that alterations in oxidative metabolism induced by thiamine deficiency lead to neuronal cell death. However, the molecular mechanisms underlying this process are still under extensive investigation. Here, we report that rat pheochromocytoma PC-12 cells differentiated in the presence of NGF into neurons undergo apoptosis due to thiamine deficiency caused by antagonists of thiamine - amprolium, pyrithiamine and oxythiamine. Confocal laser scanning fluorescence microscopy revealed that annexin V binds to PC-12 cells in presence of thiamine antagonists after 72 h incubation. Results also show that thiamine antagonists trigger upregulation of gene expression of mitochondrial-derived apoptosis inducing factor, DNA fragmentation, cleavage of caspase 3 and translocation of active product to the nucleus. We therefore propose that apoptosis induced by amprolium, pyrithiamine or oxythiamine occurs via the mitochondria-dependent caspase 3-mediated signaling pathway. In addition, our data indicate that pyrithiamine and oxythiamine are more potent inducers of apoptosis than amprolium.

Antiparasitic and immunomodulatory effect of innovative treatments against Myxobolus sp. infection in Diplodus puntazzo.

The potential antiparasitic and immunomodulatory effect of three treatments against myxosporean parasites on the innate immune system of sharpsnout sea bream (Diplodus puntazzo) was investigated. Fish naturally infected with Myxobolus sp. (Bivalvulida/Platysporina), a histozoic parasite mainly affecting the renal interstitial tissue, were treated by oral administration of a combination of salinomycin with amprolium, Origanum essential oil or fumagillin in a small-scale field trial. Various leucocyte functions influenced by myxosporean infection were examined in order to determine treatment effects on leucocyte immunocompetence of treated fish. One month post treatment all drugs caused a significant decrease in prevalence and intensity of infection in comparison to untreated, infected fish. The effect was most prominent in salinomycin with amprolium treated fish, which 1-month post treatment contained either no cysts at all or a few spores free in melanomacrophage centres revealing almost total elimination of the parasite and the antiparasitic action of the treatment. There was no histopathological evidence of drug toxicity. Antiparasitic action was accompanied by a significant enhancement of phagocytic activity demonstrated by ingestion of large numbers of latex beads and the secretion of high levels of reactive nitrogen intermediates by phagocytes in vitro. Complete restoration of the diminished mitogenic responses and serum lysozyme secretion was also detected in salinomycin with amprolium-treated fish compared to untreated, infected fish. These data suggest that salilomycin with amprolium may be a promising treatment for myxosporean infections in intensively cultured warm-water fish, exhibiting action partially via the enhancement of host, innate immune functions and leading to parasite elimination.

Three thiamine analogues differently alter thiamine transport and metabolism in nervous tissue: an in vivo kinetic study using rats.

Thiamine (T) analogues pyrithiamine, oxythiamine or amprolium in amounts 10-1000 times higher than labelled T, were i.p. injected into rats together with 14C-T (30 microg; 46.25 KBq). The radioactivity associated with T and its phosphoesters in the plasma and cerebral cortex, brainstem, cerebellum, and sciatic nerve were determined at time intervals from 0.25 to 240 h from injection. The experimental data obtained were processed with a mathematical compartmental model that calculated the fractional rate constants. These are the amount of content in a given compartment that is replaced in 1 h and expressed in per hour. The results showed that all three analogues inhibited thiamine entry from plasma. Instead, oxythiamine enhanced T phosphorylation to T pyrophosphate (TPP); amprolium and oxythiamine enhanced TPP dephosphorylation to monophosphate (TMP); pyrithiamine reduced TPP dephosphorylation and TMP formation, while none of the analogues modified TMP dephosphorylation to T. In conclusion, in living rats, the action of T analogues was much more complex than could be expected from their structure and action in vitro.

Residual activity of anticoccidial drugs in chickens after withdrawal of medicated feeds.

Seven anticoccidial drugs commonly used in poultry (diclazuri), monensin, salinomycin, halofuginone, nicarbazin, robenidine, amprolium, and lasalocid) were tested for residual activity after withdrawal. In each test, the products were given at the recommended level to cages of 10 broiler chickens. Oral inoculation with coccidia was given after withdrawal of medication. Birds pretreated with 1 ppm of diclazuril and inoculated with Eimeria tenella after drug withdrawal had normal weight gain and very low lesion scores. Residual activity depleted gradually over several days, as shown by higher lesion scores when medication was withdrawn for up to 3 days before inoculation. Similar results were observed when young birds were inoculated with a mixture of E. tenella, E. maxima and E. acervulina, and also when birds were given diclazuril to market weight (6 weeks of age) and inoculated with a mixture of six species of Eiméria (The above species plus E. brunetti, E. mitis, and E. necatrix) after withdrawal of medication for 2 days. In contrast, there was no evidence of residual anticoccidial activity with nicarbazin, halofuginone, lasalocid, amprolium, salinomycin or monensin. Overall, the residual activity was unique to diclazuril.

Thiamine deficiency--induced partial necrosis and mitochondrial uncoupling in neuroblastoma cells are rapidly reversed by addition of thiamine.

Culture of neuroblastoma cells in a medium of low-thiamine concentration (6 nM) and in the presence of the transport inhibitor amprolium leads to the appearance of overt signs of necrosis; i.e., the chromatin condenses in dark patches, the oxygen consumption decreases, mitochondria are uncoupled, and their cristae are disorganized. Glutamate formed from glutamine is no longer oxidized and accumulates, suggesting that the thiamine diphosphate-dependent alpha-ketoglutarate dehydrogenase activity is impaired. When thiamine (10 microM) is added to the cells, the O2 consumption increases, respiratory control is restored, and normal cell and mitochondrial morphology is recovered within 1 h. Succinate, which is oxidized via the thiamine diphosphate-independent succinate dehydrogenase, is also able to restore a normal O2 consumption (with respiratory control) in digitonin-permeabilized thiamine-deficient cells. Our results therefore suggest that the slowing of the citric acid cycle is the main cause of the biochemical lesion induced by thiamine deficiency as observed in Wernicke's encephalopathy.

Thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors.

When neuroblastoma cells were transferred to a medium of low (6 nM) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [3H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity (KD = 1.4 +/- 0.2 nM). Thiamine deficiency leads to an increase in both Bmax and KD. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.

Thiamin and derivatives as modulators of rat brain chloride channels.

Several membrane fractions were prepared from rat brain by differential and sucrose density gradient centrifugation. Most fractions took up 36Cl- rapidly at a rate linear with time during the first 30-60 s, then the rate progressively slowed down. The lowest rate of uptake was found in the mitochondrial fraction. Oxythiamin partially inhibited 36Cl- uptake in all fractions. In P2 (crude synaptosomal fraction), oxythiamin decreased the initial rate of uptake by 32%, the apparent Ki being 1.5 mM. Thiamin and amprolium were less effective as inhibitors. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (0.1-1 mM) inhibited 36Cl- uptake by 40-50%. In the presence of this compound at a concentration > or = 5 x 10(-4) M, oxythiamin became ineffective. 36Cl- uptake was increased by GABA (0.1 mM) and this effect was antagonized by picrotoxin as expected, but not by oxythiamin. The rate of 36Cl- uptake did not appreciably depend on the external chloride concentration and was unaffected by bumetanide or by replacement of external Na+ by choline. Taken together, these data suggest that the oxythiamin-sensitive 36Cl- influx is essentially diffusional and is not related to the GABA receptor or the Na:K:2Cl co-transport. Partial replacement of external Na+ by K+ or treatment with 0.1 mM veratridine (which should both result in membrane depolarization) increased 36Cl- uptake by 50 and 30% respectively; the inhibitory effect of oxythiamin was enhanced to the same proportion.(ABSTRACT TRUNCATED AT 250 WORDS)