Structure-function relationship of T-2 toxin and its metabolites in inducing thymic apoptosis in vivo in mice

Recently we found that a single administration of T-2 toxin (T-2), a trichothecene mycotoxin, into mice induced DNA fragmentation, a biochemical hallmark of apoptosis, in the thymus. In this study, we investigated the effective chemical structure(s) of T-2-derived metabolites capable of inducing thymic apoptosis in vivo in mice. Metabolic conversion of T-2 to 3'-hydroxy-T-2 toxin (3'-OH-T-2) did not diminish the apoptosis-inducing activity, since essentially the same level of fragmented DNA was detected in the thymus taken from mice injected with either T-2 or 3'-OH-T-2. In contrast, hydrolysis of T-2 and 3'-OH-T-2 at the carbon-4 (C-4) position to HT-2 toxin (HT-2) and 3'-hydroxy-HT-2 toxin (3'-OH-HT-2), respectively, greatly decreased the level of DNA fragmentation. Similarly, hydrolysis of T-2 at the carbon-8 (C-8) position to neosolaniol strongly diminished its ability to induce DNA fragmentation. T-2 tetraol, having no ester groups, was unable to induce apoptosis. Based on the data presented in this study, we concluded that both the acetyl group at the C-4 position and the isovaleryl or 3'-hydroxyisovaleryl group at the C-8 position of the T-2 molecule are important for inducing cell death through apoptosis in the thymus.     

Changes in dopamine, serotonin and their metabolites in discrete brain areas of rat offspring after in utero exposure to cocaine or related drugs

The concentrations of dopamine (DA), serotonin (5HT) and their metabolites were quantified in 5 brain areas of rats exposed to saline, cocaine (15 mg/kg b.i.d.), amitriptyline (10 mg/kg), or amfonelic acid (AFA, 1.5 mg/kg) throughout gestation. Male pups from 3 similarly treated dams were fostered to 2 surrogate dams. The process of breeding and rearing was repeated 4 times with new dams to build the groups to 4-12, since only one pup per litter was used for any one measurement. AFA was used to mimic the dopamine (DA) uptake blockade and stimulant properties of cocaine and amitriptyline was used to mimic the other pharmacological effects of cocaine. At postnatal days (PND) 30, 60, and 180, one pup per litter was removed for HPLC analysis of monoamines. A second pup received 0.3 mg/kg haloperidol, catalepsy assessed after 1 hr, and the brain used for analysis. The cataleptic response to haloperidol was unaffected by any prenatal treatment. The striatum from PND 30 cocaine rats had decreased levels of DA without a decrease in DA metabolites. At PND 60 in cocaine exposed rats, DA and DOPAC concentrations were increased, and 5HT levels were decreased in the striatum. The amitriptyline-exposed group exhibited decreased 5HT and 5-HIAA levels in the striatum. The hypothalamus of the cocaine group had lower levels of 5-HIAA, and other brain areas had a trend for lower levels of 5HT and 5-HIAA. At PND 180, DOPAC was increased in the striatum and prefrontal cortex of the cocaine group. Haloperidol-induced altered monoamine metabolism was unaffected by any prenatal treatment at any age. These data suggest that age-related changes in the DA and 5HT neurotransmission systems occur in rats exposed prenatally to cocaine. However, the ability of the dopaminergic system to respond to a challenge by a DA receptor blocker is unaltered by these in utero treatments.     

Effects of clozapine metabolites and chronic clozapine treatment on rat brain GABAA receptors

Computer science can assist the physician or the surgeon in defining an optimal strategy for an intervention, and then in performing it. This implies introducing new sensors, then processing all the data gathered about the patient, and finally operating systems for guidance of the very act of intervention.     

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.     

Gas-liquid chromatographic determination of T-2 toxin and diacetoxyscirpenol in corn and mixed feeds

A gas-liquid chromatographic method has been developed for the simultaneous determination of the T-2 toxin and diacetoxyscirpenol (DAS) in corn and mixed feeds. The analytes are extracted with aqueous methanol. Ammonium sulfate is used to denature and precipitate proteinaceous compounds and to salt out low polar compounds. The analytes are selectively concentrated into chloroform, which is washed with aqueous potassium hydroxide to remove acidic compounds. Residual interferences are removed by silica gel column chromatography. Heptafluorobutrylimidazole (HFBI) is added to form esters of the analytes. The HFB-esters are separated on an SE-30 glass column and measured witha 63Ni electron capture detector, using methoxychlor as an internal standard. The method has been applied to corn, livestock feeds, and pet food. The limit of detection is 100 ng T-2 toxin and 25 ng DAS/G. Average recoveries of 105 and 97% and coefficients of variation of 17 and 10% were obtained for T-2 toxin and DAS, respectively.     

Influence of morphine on protein synthesis in discrete subcellular fractions of the rat brain

An attempt was made to induce conflict behavior in rabbits by conditioning under hypothalamic electrical stimulation. Behavioral analysis was performed using our newly devised technique which employs the use of a vibration apparatus fixed to head and waist, and a recording of various behavior. Conditioning was carried out by sensory stimuli flight behavior. Reinforcement was as follows: the CS used was flickering light photic (10 Hz, 10 musec.,) and acoustic (250 Hz, 15 dB) and UCS electrical stimulation (100 Hz, 1 msec., not exceeding 2 v) in the medial hypothalamic area and peri-fornix. The restrained animals were placed on a table in an acoustically isolated room with a discrete monotonous background noise. CRs were obtained within a short time as were responses: (1) During the autonomicsomatic responses, (2) continuous run and (3) non-continuous run. Data obtained during the non-continuous run represent a conflict-induced type of behavior which may be applied in studies related to the psychopharmacological actions of drugs.     

Effect of ketamine anaesthesia on the content of monoamines and their metabolites in the rat brain

The effects of ketamine anaesthesia (100 mg/kg i.p.) on the content of brain 5-hydroxytryptamine (5HT), 5-hydroxyindoleacetic acid (5HIAA), noradrenaline (NA), dopamine (DA) and homovanillic acid (HVA) were studied in male Wistar rats. Fifteen min after ketamine injection, when the rats were deeply anaesthetized, the 5HT content in many brain regions tended to be increased. An opposite tendency was found in the brain 5HIAA content. In rats treated with probenecid, which markedly lengthened ketamine anaesthesia, the accumulation of 5HIAA was significantly reduced by ketamine. In addition to ketamine anaesthesia, probenecid was found to lengthen thiopental anaesthesia. One hour after the ketamine administration, when the rats were no longer anaesthetized but were excited, the brain NA concentration was increased by 17% (P less than 0.02). The brain DA content was unchanged, but at 15 min and 1 hour after ketamine administration the striatal HVA content was increased by about 55% (P less than 0.05), suggesting an increased turnover of DA. The results suggest that during recovery from ketamine anaesthesia the increased NA content and the increased DA turnover may be associated with the postanaesthetic excitement of the rat, whereas the decreasamine anaesthesia.     

Effect of adrenal steroids on preproneurokinin-A gene expression in discrete regions of the rat brain

Glycogen phosphorylase b has been purified to homogeneity from the fat body of larval Manduca sexta. The purification procedure involved ammonium sulfate precipitation, and chromatography of DEAE-cellulose, 5'-AMP-Sepharose and Q-Sepharose. The final product, which showed a single band on SDS-PAGE with a M(r) = 92,500, was purified 50-fold from the original homogenate in a yield of about 3%. The molecular mass of the native purified phosphorylase b was estimated to be 186,000 Da from gel filtration, suggesting that the native enzyme is a dimer. The apparent Km values for glycogen, phosphate and 5'-AMP were 1.4 mM, 82 mM and 1.1 mM, respectively. The enzyme had a pH optimum of 7.05, and was inhibited by ATP, ADP and glucose, but not by trehalose, even at high concentration. Conversion of phosphorylase b into the a form was achieved by incubation with rabbit phosphorylase kinase and Mg(2+)-ATP. The molecular mass of phosphorylase a was estimated to be 250,000 Da by gel filtration chromatography. The specific activity of the a form in the presence of 5'-AMP was 1.6-1.7-fold higher than the specific activity of the b form under the same conditions. Thus, 5'-AMP activates the a form by about 20%, whereas ATP has no effect on the phosphorylase a activity.     

Arachidonic and palmitic acid utilization in aged rat brain areas

We have previously demonstrated that the arachidonic acid (20:4) incorporation into brain lipids differs according to the age of the animals used and the experimental conditions adopted. These differences led to a further investigation of arachidonic acid uptake in both aged and adult rat brains, its transformation into CoA derivatives, its incorporation into diacyl-glycerols and polar lipids, and finally its oxidation to CO2. These metabolic parameters were then compared with those obtained after using the saturated fatty acid palmitate (16:0). In both cases slices or mitochondria from different brain areas of 24-month-old and 4-month-old rats were examined. The results obtained indicate that the uptake of the fatty acids into cells is not modified by age. However, the successive metabolic transformations of the acids are altered to a considerable extent. In particular, in 24-month-old animals (compared with 4-month-old rats) there is a significant decrease of 20:4 in its incorporation into lipids as well as its oxidation to CO2, while arachidonoyl-CoA content increases by about 50%. This increased amount of CoA derivative, which has a potent detergent effect, may interfere with membrane structure and affect membrane physiological functions. Furthermore, because the free arachidonate pool is maintained in a dynamic equilibrium with its esterified forms, the final result may be a perturbation of this equilibrium.     

Topical application of T-2 toxin inhibits the contact hypersensitivity response in BALB/c mice

T-2 toxin, a trichothecene mycotoxin, has previously been shown to alter immune functions and promote skin tumors. We demonstrate that topically applied T-2 toxin reduces the ear swelling response to oxazolone challenge in BALB/c mice. For this reduction in ear swelling to occur, toxin application must be at, or within, 1 h after challenge. Dose-response studies showed a 44% reduction in ear swelling with 30 ng of T-2 toxin as compared with a similar reduction with 300 ng of dexamethasone. T-2 toxin did not affect Ag transport from the challenge site to the draining lymph nodes as measured by FITC transport. However, T-2 toxin significantly reduced both MHC class II (Ia) expression and Ag presentation at the same concentrations. Because T-2 toxin, a known protein synthesis inhibitor, was found to inhibit protein synthesis in epidermal cell cultures as measured by [3H]-leucine incorporation, cycloheximide was also examined. Cycloheximide reduced both oxazolone-induced ear swelling and Ag presentation in a similar manner to T-2 toxin. One mechanism of action for T-2 toxin in reducing the contact hypersensitivity response is via inhibition of protein synthesis and effective Ag presentation by epidermal Langerhans cells. This may involve alterations in Ia Ag expression, although a role for class II in the induction phase of the contact hypersensitivity response has not been established definitively.     

Efficacy of various inorganic sorbents to reduce the toxicity of aflatoxin and T-2 toxin in broiler chickens

Experiments were conducted to determine the efficacy of three inorganic sorbents, S1, S2, and S3, to reduce the toxicity of aflatoxins (AF) and T-2 toxin in male broiler chickens from day of hatch to 21 d of age. The compounds had been reported to bind to AF and T-2 toxin in vitro. S1 and S2 were the same basic compound that had been stored for different lengths of time following activation. In Experiments 1, 2, and 3, the appropriate diets were produced to contain no mycotoxins, the specific adsorbent at 0.5% of diet, AF alone at 5 mg/kg of diet, T-2 alone at 8 mg/kg of diet, AF at 5 mg/kg of diet plus the specific sorbent at 0.5% of diet, or T-2 at 8 mg/kg of diet plus the specific sorbent at 0.5% of diet. The specific sorbents used were: 1) Experiment 1, S1; 2) Experiment 2, S1 and S2; and 3) Experiment 3, S3. In Experiments 1 and 3, S1 and S3, respectively, showed no protection against AF or T-2 toxin as measured by BW gain, when compared to AF alone group. In Experiment 2, S1 showed no protection; however S2 reduced the effects of AF on BW gain by 25% as compared to AF alone diet. The data demonstrate that under the conditions of our experiment: 1) one of the sorbents provided some protection against aflatoxicosis; 2) there was variability in protection against aflatoxicosis between two different samples of the same sorbent that had been stored for different lengths of time following activation; 3) protection by the sorbents against the effects of T-2 toxin was not observed.     

Effects of food restriction on the periodicity of corticosteroids in plasma and on monoamine concentrations in discrete brain nuclei

The concentrations of plasma corticosteroids and of norepinephrine, dopamine and serotonin in microdissected brain regions were measured at 08.00, 12.00 and 20.00 h in male rats fed ad libitum and in rats whose food intake was restricted to 09.30-11.30 h. In ad libitum fed animals, plasma corticosteroids were lowest at 08.00 and highest at 20.00 h. As demonstrated previously, restriction of food availability was associated with appearance of a peak in corticosteroids at 08.00 h. In ad libitum fed animals, serotonin and dopamine concentrations in the median eminence were higher at 20.00 than at 08.00 h. Restriction of food availability significantly decreased the levels of these neurotransmitters at 20.00 h. In the paraventricular nucleus, amygdala, and hippocampus of ad libitum fed animals, serotonin levels were lower at 20.00 than at 08.00 or 12.00 h. In food-shifted animals, this pattern was reversed so that lowest levels of serotonin occured at 08.00 and markedly elevated levels were observed at 12.00 and 20.00 h. No changes were noted in norepinephrine content of the median eminence or paraventricular nucleus of ad libitum fed or food restricted animals. These results indicate that the shift in the periodicity of corticosteroid secretion produced by a restricted feeding regime is accompanied by changes in the periodicity of neurotransmitter concentrations in specific regions of the brain, and that such patterns are dissimilar in different regions.     

Synaptic organization and neurotransmitters in the rat accessory olfactory bulb

The accessory olfactory bulb (AOB) is the first relay station in the vomeronasal system and may play a critical role in processing pheromone signals. The AOB shows similar but less distinct lamination compared with the main olfactory bulb (MOB). In this study, synaptic organization of the AOB was analyzed in slice preparations from adult rats by using both field potential and patch-clamp recordings. Stimulation of the vomeronasal nerve (VN) evoked field potentials that showed characteristic patterns in different layers of the AOB. Current source density (CSD) analysis of the field potentials revealed spatiotemporally separated loci of inward current (sinks) that represented sequential activation of different neuronal components: VN activity (period I), synaptic excitation of mitral cell apical dendrites (period II), and activation of granule cells by mitral cell basal dendrites (period III). Stimulation of the lateral olfactory tract also evoked field potentials in the AOB, which indicated antidromic activation of the mitral cells (period I and II) followed by activation of granule cells (period III). Whole cell patch recordings from mitral and granule cells of the AOB supported that mitral cells are excited by VN terminals and subsequently activate granule cells through dendrodendritic synapses. Both CSD analysis and patch recordings provided evidence that glutamate is the neurotransmitter at the vomeronasal receptor neuron; mitral cell synapses and both NMDA and non-NMDA receptors are involved. We also demonstrated electrophysiologically that reciprocal interaction between mitral and granule cells in the AOB is through the dendrodendritic reciprocal synapses. The neurotransmitter at the mitral-to-granule synapses is glutamate and at the granule-to-mitral synapse is gamma-aminobutyric acid. The synaptic interactions among receptor cell terminals, mitral cells, and granule cells in the AOB are therefore similar to those in the MOB, suggesting that processing of chemosensory information in the AOB shares similarities with that in the MOB.     

Effects of traumatic brain injury on the cholinergic system in the rat

Rats subjected to a mild to moderate fluid percussion injury exhibit memory deficits that are similar to rats that have received lesions of the septohippocampal system. Because the cholinergic system plays a major role in septohippocampal function, we studied the kinetics of the synthetic enzyme for acetylcholine, choline acetyltransferase (ChAT), at 1 h, 24 h, or 5 days after a fluid percussion injury. Decreases in ChAT activity were found in the dorsal hippocampus (25%), frontal (32%), and temporal (23%) cortices 1 h after injury. In the parietal cortex, a greater than 50% increase in ChAT activity was observed at all time intervals assessed. At 5 days after TBI, there was an 18% increase in ChAT activity in the medial septal area. These data provide evidence that a mild to moderate fluid percussion injury produces changes in the cholinergic system in brain areas related to memory.     

Effect of repeated immobilization on serotonin metabolism in different rat brain areas and on serum corticosterone

The effect of daily repeated 10 min immobilization on the serotoninergic neurotransmission and serum corticosterone levels was studied. Male Lewis rats were immobilized for a 10 min period daily once or on 5 consecutive days. Serotoninergic neurotransmission was followed using differential in vivo pulse voltammetry with carbon fibre electrodes measuring extracellular 5-hydroxyindoleacetic acid (5-HIAA) levels. Recordings were performed in brain areas involved in the control of behaviour, mood, and stress response such as the frontal cortex, the hippocampal CA-3 and dentate gyrus, the striatum, and the raphe nuclei dorsalis (NRD) and medialis (MRN). The first immobilization resulted in an increase of the extracellular 5-HIAA levels in all areas under study, except the striatum where no reaction was observed. The major effect was recorded in the frontal cortex, showing an increase of about 400% as compared to control, which lasted for 3h after the end of the immobilization period. Beginning on day 2 in all areas, except the striatum, a consecutive habituation to the stressor seemed to occur, since the stress-induced increase in the voltammetric signal was found to be reduced after consecutive immobilization. Serum corticosterone levels were measured directly after a single and after 5 daily immobilization periods. After single immobilization the serum corticosterone level was found to be about 270 ng/ml. After the 5th immobilization about 300 ng/ml were detected. These differences were not found to be significant. In summary, our data indicate that the serotonin metabolism shows habituation in nearly all brain areas after repeated immobilization, though the corticosterone level at the end of the immobilization period was comparable after single and repeated immobilization.     

Influence of fumonisin B1, present in Fusarium moniliforme culture material, and T-2 toxin on turkey poults

Diets containing 300 mg fumonisin B1 (FB1)/kg of feed and 5 mg T-2 toxin/kg of feed singly or in combination were fed to female turkey poults (Nicholas Large White) from day of hatch to 21 d of age. When compared with controls, 21-d body weight gains were reduced 21% by FB1, 26% by T-2, and 47% by the combination. the efficiency of feed utilization was adversely affected by FB1 and the combination of FB1 and T-2. Relative weights (grams/100 g BW) of the liver and gizzard were increased in poults fed the FB1 and the combination diets; whereas, the relative weight of the pancreas was increased in all treated groups. All poults were scored for oral lesions using a scale of 1 to 4 (1 = no visible lesions, 4 = severe lesions). Oral lesions were present in all poults fed the T-2 diet (average score of 3.29) or the combination diet (average score of 3.54). Serum concentration of cholesterol was decreased and lactate dehydrogenase activity was increased in poults fed the FB1 and combination diets. The activity of aspartate aminotransferase and the values for red blood cells, hemoglobin, and hematocrit were increased only in poults fed the combination diet. Inorganic phosphorus concentration was decreased only in poults fed the combination diet. The increased toxicity in poults fed the combination diet for most variables can best be described as additive, although some variables not altered by FB1 or T-2 singly were significantly affected by the combination, indicating that the combination may pose a potentially greater problem to the turkey industry than either of the mycotoxins individually.