Brain neuronal chromatin responses in acute soman intoxicated rats

Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD₅₀ (1 LD₅₀=135 g/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity; conversely, increases in F-DNA yield and chromophore area signify enhanced neuroexcitation. At 1 hr post-soman there was a dose-dependent inactivation of AChE with a moderate increase in chromatin activation, i.e., nuclear hypertrophy and chromatin dispersion. At 24 hr post-soman there was a partial restoration of AChE activity, notably in striatal neurons, with a suppression in chromatin template activity. These data indicate that actions of soman on neuronal functioning are time-dependent. The absence of any dose-related neuronal chromatin changes may signify existence of non-cholinergic mediated events.     

Cytophotometric analysis of neuronal chromatin and RNA changes in oxotremorine-treated rats

Neuronal nucleic acid responses were examined within the rat striatum and sensorimotor cortex (layer V) following single intraperitoneal injections of the central cholinergic-muscarinic agonist oxotremorine (0.1, 0.7, or 1.0 mg/kg). After stoichiometric Feulgen and azure B staining of brain sections, scanning-integrating microdensitometry was used to quantify Feulgen-deoxyribonucleic acid levels, changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA yield) and azure B-ribonucleic acid (RNA) content of neurons on an individual basis. Changes in neuronal nuclear and nucleolar volumes were also determined histometrically. Within the striatum and sensorimotor cortex, oxotremorine produced marked dose-dependent elevations in both F-DNA yield and RNA content. These metabolic increases were typically paralleled by elevations in nuclear and nucleolar volumes. The data demonstrate that the oxotremorine-induced central muscarinic activation is associated with dose-related enhancements in neuronal chromatin template activity, RNA content, and protein synthetic capacity.     

Cytophotometric assessment of granulosa cell protein and nucleic acid levels during the estrous cycle in rats treated with T-2 toxin

Female Sprague-Dawley rats (160-180g.) with normal estrous cyclicity established by vaginal smears, were injected intraperitoneally with 0.45 mg/kg (low dose) or 0.68 mg/kg (high dose) of T-2 toxin, a trichothecene with potent protein inhibitory abilities. Control animals were injected with only the 100% ethanol vehicle. All animals were decapitated at 8 hours post-exposure, and their ovaries removed and processed for paraffin sectioning. Coomassie, Feulgen, and azure B/DNase staining procedures were used to show granulosa cell protein levels, F-DNA stainability, and basophilia/RNA levels, respectively. Quantification of these parameters was accomplished using scanning-integrating microdensitometry. T-2 toxin treatment groups had granulosa cell protein levels significantly lower than those of the control animals. However, rats exposed to the lower dose of T-2 toxin generally showed a more marked suppression of protein levels than the high dose group, regardless of the stage of the estrous cycle. In addition, rats that received lower doses of T-2 toxin had impaired translation and template activity in response to injury, when compared with the rats in the high dose group. These results are attributed to the lesser degree of circulatory impairment in the low T-2 toxin dosage group, which allows a higher amount of T-2 toxin to interact with the cells.     

Scanning cytophotometric analysis of myocardial nucleic acid and chromatin changes in soman toxicated rabbits

Myocardial nucleic acid responses were analysed in New Zealand White rabbits 20 min-1 h and 6-8 h following single subcutaneous injections of soman (20, 30, or 40 micrograms kg-1). Scanning-integrating microdensitometry was used to quantify Azure B-RNA and Feulgen-DNA (F-DNA) levels, and changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA reactivity) of individual ventricular myocardial cells. With a dosage of 20 micrograms kg-1 soman, no RNA alterations were evidenced at 1 h whereas at 6-8 h myocardial cells exhibited higher RNA levels and an increase in F-DNA reactivity of chromatin. With dosages of 30 and 40 micrograms kg-1 soman there was an augmentation in RNA levels and in the acid hydrolysability of nuclear chromatin at both 20 min-1 h and 6-8 h. It is postulated that the observed cellular transformations represent a compensatory augmentation in myocardial metabolic functioning presumably in response to an increased functional demand on the ventricular myocardium. The absence of cytopathic or cytochemical evidence of impairment in nucleic acid metabolism is inconsistent with the premise that soman exerts direct cytotoxic effects on rabbit myocardium.     

Induction of apoptosis by fungal culture materials containing cyclopiazonic acid and T-2 toxin in primary lymphoid organs of broiler chickens

Thirty-six, twenty-eight-day-old broiler chicks were randomly distributed into three groups of 12 birds each. Two groups were fed diets containing 10 ppm cyclopiazonic acid (CPA) and 1ppm T-2 toxin, respectively, to determine the mechanism of cell death in spleen and thymus at 6, 12, 24, and 36 h of post-treatment. The other group served as control. T-2 toxin treated group showed significant (P < 0.01) induction of apoptosis in thymus with peak induction at 24 h post-treatment where as, no significant differences were observed between the control and CPA groups. The CPA toxin treated group showed significant (P < 0.01) induction of apoptosis in spleen with peak induction at 24 h post-treatment. No significant differences were observed between the control and T-2 toxin group even though the latter showed a slight increase in the quantity of apoptotic cells at 36 h post-treatment in spleen. The semi-thin sections stained with toluidine blue from the spleen of CPA treated group exhibited crescent margination of chromatin against the nuclear envelope and shrinkage of lymphoid cells without any surrounding inflammation, the characteristics of apoptosis. The apoptotic thymocytes from T-2 fed birds appeared shrunken with condensed nucleus and showed crescent margination of chromatin against the nuclear envelope without any surrounding inflammation when compared with well-defined nuclei with dispersed chromatin in normal thymocytes. Ultrastructurally, splenocytes of the CPA treated group and thymocytes of the T-2 toxin treated birds showed apoptotic bodies characterized by crescent margination of the chromatin against the nuclear envelope. The study indicates that one route of the CPA and T-2 toxin induced cell death in lymphoid organs of broiler chicken is by apoptosis.Forms part of M.V.Sc. thesis of the first author approved by the Tamil Nadu Veterinary and Animal Sciences University, Chennai 600 051, India.     

The enzymes of the metabolism of exogenous compounds in the comparative assessment of the toxicity of trichothecene mycotoxins

Moderate clinical, biochemical and hematologic signs of intoxication were observed in mice after single administration of HT-2 toxin (deacetylated derivative of T-2 toxin) in LD50 of 12.75 mg/kg or in 1/5 of LD50 for 7 days. The toxin produced no toxic effect when 1/10 and 1/50 of LD50 were administered for 14 days. With the dose exceeding 1/50 of LD50 a reduction in cytochrome P-450 content, carboxylesterase activity and increased activity of UDP-glucuronosyltransferase and glutathione transferase were recorded. T-2 toxin produced a more pronounced toxic effect, inhibited UDP-glucuronosyltransferase and insignificantly stimulated glutathione transferase activity. Lower HT-2 toxin toxicity is believed to depend on its ability to activate conjugation reactions to a greater extent than T-2 toxin.     

In vitro metabolism of T-2 toxin in rats.

T-2 toxin was rapidly converted in the 9,000 X g supernatant fraction of rat liver homogenate into HT-2 toxin, T-2 tetraol, and two unknown metabolites designated as TMR-1 and TMR-2. TMR-1 was characterized as 4-deacetylneosolaniol (15-acetoxy-3 alpha, 4 beta, 8 alpha-trihydroxy-12,13-epoxytrichothec-9-ene) by spectroscopic analyses. Since the same metabolites were also obtained from HT-2 toxin used as substrate, it was concluded that T-2 toxin was hydrolyzed preferentially at the C-4 position to give HT-2 toxin, which was then metabolized to T-2 tetraol via 4-deacetylneosolaniol. In addition to HT-2 toxin, 4-deacetylneosolaniol and T-2 tetraol, a trace amount of neosolaniol was transformed from T-2 toxin by rat intestinal strips. In vitro metabolic pathways for T-2 toxin in rats are proposed.     

Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats

3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described.     

Structures of deepoxytrichothecene metabolites from 3'-hydroxy HT-2 toxin and T-2 tetraol in rats.

3'-Hydroxy HT-2 toxin and T-2 tetraol, in vivo metabolites of T-2 toxin, were orally administered to Wistar rats, and four metabolites having a trichothec-9,12-diene nucleus, which were termed deepoxytrichothecenes, were newly found in the excreta. Their structures were confirmed as 3'-hydroxy-deepoxy HT-2, 3'-hydroxy-deepoxy T-2 triol, 15-acetyl-deepoxy T-2 tetraol, and deepoxy T-2 tetraol on the basis of mass and nuclear magnetic resonance spectroscopy. Resolution of T-2 metabolites and corresponding deepoxytrichothecenes by gas-liquid and thin-layer chromatography was also described.     

Effect of continuous darkness on circadian morphological rhythms in pinealocytes of the Chinese hamster,Cricetulus griseus

Summary Effects of a short-term exposure to continuous darkness on 24-h morphological variations in pinealocytes in the superficial pineal of the Chinese hamster (Cricetulus griseus) were examined. Pinealocytes contained type-1, -2 and -3 synaptic ribbons (SR), which had a central dense structure showing rod-like, various and ring-like profiles, respectively, and the quantity of each type of SR was expressed by SR index. 24-h changes in the type-1 and type-3 SR indices persisted in darkness and thus may be endogenous in nature. As under alternating light and dark (LD) conditions, the type-2 SR indices were almost constant over a 24-h period under continuous darkness, but the indices were larger in animals under darkness than in those under LD conditions. The 24-h variations in the nuclear and cytoplasmic volumes were abolished after exposing animals to darkness for 7 days, suggesting that these rhythms may be regulated exogenously. The amount of condensed chromatin exhibited a circadian change; this rhythm persisted under darkness. The results suggest that 24-h variations in the nuclear and cytoplasmic volumes in pinealocytes of the Chinese hamster are regulated by mechanisms different from those controlling the rhythms in SR and chromatin, and that the changes in the nuclear and cytoplasmic volumes and chromatin are related to the change in synthetic activity of pinealocytes.     

Subclinic effect of the administration of T-2 Toxin and Nivalenol in mice

Four experiments using T-2 toxin and nivalenol at different dosage, which represented the 25% and 40% of the LD50 (experiment A: 1.04 mg of T-2 toxin per kilogram of body weight, experiment B: 2.34 mg of T-2 toxin/kg b.w., experiment C: 1.04 mg of T-2 toxin/kg b. w. and 2.34 mg of T-2 toxin/kg b.w.; experiment D: 0.82 mg of nivalenol/kg b.w. and 1.845 mg of nivalenol/kg b.w.) were conducted on 400 mice. Both toxins were administered to mice of different ages (experiments A and B were adults, experiment C and D were young) by intraperitoneal single injection, and the clinical signs, hematological variables and histoanatomo pathological changes were studied. All animals survived. No changes anatomo-histopathological nor significative differences in weight gain were observed. Different behaviors were found for nivalenol and T-2 toxin. The most significant change was the increase in the level of monocytes in old animals, so this could be a biological indicator for T-2 toxin subclinical intoxication.     

Intranuclear differences in the response of Purkinje cell DNA of the rat cerebellum to bleomycin

Summary A single dose of the DNA-binding cytostatic agent bleomycin (100 g/g body weight, subcutaneously) was given to 10-day-old rats to study unscheduled repair DNA synthesis in nucleolar and in bulk nuclear chromatin of postmitotic Purkinje neurons. The Feulgen reaction and Hoechst 33342 staining were used for quantitative evaluation of nuclear DNA content and chromatin structure. The repair synthesis of DNA was detected by ³H-thymidine autoradiography.The data showed a lesser staining of Purkinje as well as granule cell DNA by Hoechst 33342 in bleomycin-treated animals than in controls, but there was no difference in staining with the Feulgen reation. The mechanisms of DNA staining by both cytochemical methods suggest that bleomycin reacted preferentially with AT-rich and single stranded DNA in cerebellar cells in vivo. Weak ³H-thymidine labelling was found in Purkinje cells of both control and treated rats, but in the latter group the labelling was more pronounced near or over the nucleolus. The enhanced unscheduled DNA synthesis in the nucleolar region of Purkinje cells of treated animals may be due to greater damage of DNA in this region or may indicate a greater ability of the nucleolar chromatin to repair its DNA.Dedicated to Professor Dr. Z. Lojda, Dr. Sc., on the occasion of his 60th birthday.     

Effect of pertussis toxin on water metabolism in the rat

Pertussis toxin administered to rats resulted in a polyuric state that was due to enhanced renal water excretion. Pertussis toxin also induced a defect in renal water conservation. These abnormalities in renal water excretion could not be attributed to polydipsia, impaired synthesis and/or release of arginine vasopressin or renal tubular dysfunction with solute diuresis. No evidence of pertussis toxin-induced renal tubular damage was present. These results indicate that pertussis toxin induces nephrogenic diabetes insipidus and this defect occurs at the level of the renal collecting tubule.     

Targeting of mycotoxins to reticuloendothelial system of mice with carrier erythrocytes

The tricothecene mycotoxin, T-2 toxin, was encapsulated in bovine erythrocytes for in vivo delivery of T-2 toxin to macrophages. Intraperitoneal injection of bovine carrier erythrocytes (5 X 10(8) cells) containing T-2 toxin saturated mouse liver uptake of erythrocytes by 6 h postinjection. At 24 h postinjection, 20% of the injected carrier cells containing toxin were localized in the liver of mice. Saturation of the liver uptake of bovine carrier cells was independent of encapsulated or free T-2 toxin. A dose of T-2 toxin sufficient to inhibit 50% of the macrophage protein synthesis was targeted to the liver via the carrier erythrocytes. A methodology for delivery of highly toxic molecules to liver macrophages is described.     

大鼠老化过程大脑皮层及小脑神经元染色质构象分析

 本文在前文~[2]的基础上进一步以MCN和DNaseⅠ为探针研究大鼠脑神经元终末分化后不同生理时期染色质构象,结果表明:MCN酶解DNA产物PAGE显示脑老化过程大脑皮层及小脑神经元染色质核小体单体DNA分别保持在176bp和215bp水平,核小体连接DNA长度存在组织差异,但不受老化影响;<2>DNaseⅠ酶解DNA产物PAGE显示各年龄组大脑皮层及小脑神经元染色质DNA存在10bp间隔重复结构和相同的泳动区带分布特征,提示脑老化中染色质具有稳定的B型双螺旋结构和一致的螺线管卷曲形式。染色质DNaseⅠ降解率随年龄增加而降低,提示老化导致活性染色质区域减少,老化过程脑神经元染色质构象改变成为其转录功能减退的结构基础。     

Interaction of T-2 toxin and murine lymphocytes and the demonstration of a threshold effect on macromolecular synthesis

Although T-2 toxin intoxications have been described as radiomimetic, we find that T-2 toxin does not preferentially affect multiplying cells. Among the targets of T-2 toxin toxicity, DNA, RNA and protein synthesis inhibition are analysed. All three types of macromolecular syntheses are affected by a threshold dose of T-2 toxin which corresponds to the interaction of approx. 1 X 10(5) T-2 toxin molecules with the same number of T-2 toxin receptors (Gyongyossy-Issa, M.I.C. and Kachatourians, G.G. (1984) Biochim. Biophys. Acta 803, 197-202). Since toxic effects occur faster at higher toxin concentrations than at lower levels, the time-toxic effect relationship may be defined by a constant. Based on these observations, we hypothesize that complete receptor-occupation is the critical first step in the course of T-2 toxin toxicity events.     

Modification of in vitro metabolism of T-2 toxin by esterase inhibitors

In vitro metabolism of T-2 toxin with S-9 fraction obtained from livers of phenobarbital-treated pigs and rats in the presence of different esterase inhibitors, including NaF, p-hydroxymercuribenzoate, phenylmethylsulfonyl fluoride, eserine sulfate, diisopropylfluorophosphate, and diethyl p-nitrophenyl phosphate, was studied. The metabolism was completely shifted to the hydroxylation at the C-3' position in the T-2 toxin molecule when esterase inhibitors were present. Diethyl p-nitrophenyl phosphate was found to be the most potent among six esterase inhibitors tested. In the presence of 10(-4) M diethyl p-nitrophenyl phosphate, 3'-hydroxy-T-2 toxin was the only metabolite detected. Similar results were obtained when other T-2-related metabolites were tested. The yield of conversion of T-2 toxin, acetyl T-2 toxin, HT-2 toxin and T-2 triol to their respective 3'-hydroxyl derivatives were 82, 73, 72, and 75%, respectively.     

Binding and cytotoxic effects of Clostridium botulinum type A, C1 and E toxins in primary neuron cultures from foetal mouse brains

Binding of purified Clostridium botulinum type A, C1 and E toxins to cultured cells was studied by an immunocytochemical method. Type A and C1 toxins bound strongly to neuron cultures prepared from brains of foetal mice, but binding of type E toxin was weak. None of the toxin types bound to the feeder layer, composed of non-neuronal cells. The heavy-chain component of the type C1 toxin bound to neurons, but the light chain component did not. Type C1 toxin also bound only to cell lines of neuronal origin. When type C1 toxin [final concentration 4 x 10(2) LD50 (10 ng) per well] was added to primary neuron cultures in 96-well plates, degeneration of neuronal processes and rounding of neuronal somas were observed, but type A and E toxins did not produce such changes. The binding and cytotoxic activities of type C1 toxin were blocked by heat treatment (80 degrees C for 30 min) or by preincubation of the toxin with polyclonal anti-C1 IgG and some of the monoclonal antibodies which neutralized the toxin activity in mice. In the neuronal processes treated with C1 toxin, many degenerated mitochondria, membranous dense bodies and vesicles were observed by electron microscopy; these ultrastructural changes were similar to those of Wallerian degeneration in vivo.     

In vitro formation of 3'-hydroxy T-2 and 3'-hydroxy HT-2 toxins from T-2 toxin by liver homogenates from mice and monkeys.

In vitro metabolism of T-2 toxin was studied in homogenates of mouse and monkey livers. In addition to several hydrolyzed products, including HT-2 toxin, neosolaniol, 4-deacetylneosolaniol, 15-deacetylneosolaniol, and T-2 tetraol, two metabolic products were isolated from the incubation mixture. Their structures were confirmed as 3'-hydroxy T-2 toxin and 3'-hydroxy HT-2 toxin on the basis of mass and nuclear magnetic resonance spectroscopy. The formation of these hydroxylated metabolites was found in the microsomes in the presence of NADPH, and the hydroxylation reaction was enhanced by treating mice with phenobarbital. The results suggest that a cytochrome P-450 is catalyzing the hydroxylation at the C-3' position of T-2 and HT-2 toxins. An in vitro metabolic pathway of T-2 toxin in the hepatic homogenates containing the NADPH-generating system is proposed.     

The effect of vitamin E supplementation on reduction of lymphocyte DNA damage induced by T-2 toxin and deoxynivalenol in weaned pigs

The objective of the present study was to establish the effect of deoxynivalenol (DON) and T-2 toxin on lipid peroxidation, lymphocyte DNA fragmentation and immunoglobulin production in weaned pigs, and furthermore, to evaluate the potential of vitamin E (α-tocopheryl acetate) in prevention of toxin mediated changes. Forty-eight weaned castrated male crossbred pigs (mean live weight at the beginning of the experimental period was 11.7 kg) were randomly assigned to five experimental groups: control (without toxin and vitamin E), T-2 (3 mg/kg T-2 toxin), T-2 + E (3 mg/kg T-2 toxin + 100 mg/kg vitamin E), DON (4 mg/kg DON) and DON + E (4 mg/kg DON + 100 mg/kg vitamin E). After 14 days of treatment blood was collected for analysis. Lipid peroxidation was studied by assays of malondialdehyde (MDA), total antioxidant status (TAS) of plasma and erythrocyte glutathione peroxidase (GPx). DNA damage in lymphocytes was measured by comet assay. Serum immunoglobulin levels were evaluated by enzyme-linked immunosorbent assay (ELISA) and the hepatotoxicity was studied by measuring plasma liver enzyme levels (alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyl-transferase (GGT). Production parameters of both DON groups were significantly impaired in comparison to the control. DON significantly increased the amount of DNA damage in lymphocytes by 28%. Moreover, the levels of TAS were lowered by addition of DON. T-2 toxin significantly impaired daily live weight gain and feed conversion, increased the amount of DNA damage in lymphocytes by 27%, decreased total serum IgG and did not alter plasma TAS. Plasma and 24-h urinary malondialdehyde (MDA) excretion rate and erythrocyte Gpx levels did not differ among the groups. Supplementation with vitamin E did not improve production parameters impaired by DON and T-2 toxin and only partially protected lymphocyte DNA from toxin impact. To our knowledge, these are the first data on genotoxic effects of moderate doses of DON and T-2 toxin on pig lymphocytes. The effect of DON and T-2 toxin on the immune system was reflected as a change in immunoglobulin synthesis, which might be toxin and species specific. According to other results no major induction of oxidative stress could be proven. Enhancement of antioxidant status with vitamin E in the case of DON and T-2 toxin intoxication can be beneficial for remaining the lymphocyte DNA integrity.