The expression of intermediate filament protein nestin as related to vimentin and desmin in regenerating skeletal muscle.

Intermediate filament (IF) proteins show specific spatial and temporal expression during development of skeletal muscle. Nestin, the least known muscle IF, has an important role in neuronal regeneration. Therefore, we analyzed the expression pattern of nestin as related to that of vimentin and desmin during skeletal muscle regeneration. Nestin and vimentin appear at 6 h post-injury in myoblasts, with maximum expression around day 3-5 post-injury. Thereafter, vimentin expression ceases completely, whereas that of nestin is downregulated to remain only in the sarcoplasm next to neuromuscular and myotendinous junctions. Desmin appears at 6-12 h post-injury and becomes the predominant IF in myofibers simultaneously with the appearance of cross-striations. The expression pattern and colocalization of nestin and vimentin, known to form heteropolymers, suggests that they are essential during the early dynamic phase of the myofiber regeneration when migration, fusion, and structural modeling of myogenic cells occurs, whereas desmin is responsible for keeping myofibrils in register in mature myofibers. In conclusion, the expression of nestin is dynamically orchestrated with that of vimentin and desmin during skeletal muscle regeneration and recapitulates that seen during myogenesis, i.e. these IFs have key functional roles in the construction and restoration of skeletal myofibers.

trkB-receptor activation contributes to the kainate-induced increase in BDNF mRNA synthesis.

1. The expression of brain-derived neurotrophic factor (BDNF) mRNA is induced by neuronal activity through increased intracellular calcium. As BDNF also increases intracellular calcium levels through trkB activation, we have examined here whether BDNF also regulates the synthesis of its own mRNA. 2. Neurotrophin mRNA expression was induced with kainic acid administration in transgenic mice overexpressing the dominant-negative form of BDNF receptor trkB and wild-type littermates. 3. Kainate strongly induced BDNF mRNA expression in both genotypes, but the up-regulation was significantly lower in transgenic mice. 4. These data suggest that the synthesis of BDNF mRNA is at least partly mediated by BDNF release and the activation of trkB receptors. The present findings further suggest that the BDNF signaling system in brain is regulated by positive feedback.

Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle.

The intermediate filament proteins nestin, vimentin, and desmin show a specific temporal expression pattern during the development of myofibers from myogenic precursor cells. Nestin and vimentin are actively expressed during early developmental stages to be later down-regulated, vimentin completely and nestin to minimal levels, whereas desmin expression begins later and is maintained in mature myofibers, in which desmin participates in maintaining structural integrity. In this study we have analyzed the expression levels and distribution pattern of nestin in intact and denervated muscle in rat and in human. Nestin immunoreactivity was specifically and focally localized in the sarcoplasm underneath neuromuscular junctions (NMJs) and in the vicinity of the myotendinous junctions (MTJs), ie, in regions associated with acetylcholine receptors (AChRs). This association prompted us to analyze nestin in neurogenically and myogenically denervated muscle. Immunoblot analysis disclosed a marked overall increase of accumulated nestin protein. Similar to the extrajunctional redistribution of AChRs in denervated myofibers, nestin immunoreactivity extended widely beyond the NMJ region. Re-innervation caused complete reversion of these changes. Our study demonstrates that the expression levels and distribution pattern of nestin are regulated by innervation, ie, signal transduction into myofibers.

Carcinogenicity of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in the rat.

BACKGROUND: Several epidemiologic studies have suggested that the consumption of chlorinated drinking water may be associated with the development of certain cancers in humans. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a byproduct of the chemical reactions that occur in chlorinated drinking water, has been found to be mutagenic in bacteria and mammalian cells; however, its potential to cause tumors in animals has not been tested previously. PURPOSE: The objective of this study was to evaluate the carcinogenicity of MX in rats given MX in their drinking water. METHODS: MX was administered to male and female Wistar rats (50 rats per dose group) in drinking water for 104 weeks at concentrations yielding the average daily doses of MX of 0.4 mg/kg of animal weight (low dose), 1.3 mg/kg (mid dose), and 5.0 mg/kg (high dose) for males and 0.6 mg/kg, 1.9 mg/kg, and 6.6 mg/kg for females, respectively. Control rats received water from the same source used for preparation of the MX dose formulations (after its adjustment to the same pH range). Body weight, clinical signs, and food and water consumption were recorded regularly. At the end of the treatment period, the animals were killed and full histopathologic analysis was performed on 47 tissues and all lesions. RESULTS: Dose-dependent increases in tumor incidence were observed in rats given MX-containing drinking water; the same MX doses had no obvious toxic effects on animals. MX consumption increased most drastically the prevalence of follicular adenoma (up to 43% and 72% in high-dose males and females, a test [one-sided] for positive trend in all dose groups P = .0045 and P = .0000, respectively) and carcinoma (55% [P = .0000] and 44% [P = .0000], respectively) in thyroid glands and cholangioma in the liver (8% [P = .0009] and 66% [P = .0000] in the high-dose males and females, respectively). Among rats given the higher doses of MX in their drinking water, cortical adenomas of the adrenal glands were increased in both sexes, alveolar and bronchiolar adenomas of the lungs and Langerhans' cell adenomas of the pancreas were increased in males, and lymphomas, leukemias, and adenocarcinomas and fibroadenomas of the mammary glands were increased in females. Even the lowest MX dose studied was carcinogenic. CONCLUSION: MX is a potent carcinogen in both male and female rats, and it causes tumors at doses that are not overtly toxic to rats. IMPLICATIONS: Although these findings cannot be extrapolated to humans, MX should be studied as a candidate risk factor in the possible association between consumption of chlorinated drinking water and cancer in humans.

Subchronic toxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in Wistar rats.

The subchronic (14-18 wk) toxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a mutagenic by-product in chlorinated drinking water, was evaluated in Wistar rats. In a range-finding study, MX was administered daily for 14 days by gavage in deionized water to male rats (five animals per group) at doses of 12.5, 25, 50, 100 or 200 mg/kg body weight. The doses above 50 mg/kg were lethal and three out of five animals also died during treatment at 50 mg/kg. The range-finding study was repeated with doses of 5, 10 and 20 mg MX/kg, given on 5 days a week, to both males and females (10 animals per group). These doses were not overtly toxic but caused several changes in plasma clinical chemistry at 10 and 20 mg MX/kg in comparison with the controls. These included increased urea, creatinine and bilirubin and decreased inorganic phosphate and potassium in females and increased cholesterol in males. In the subchronic toxicity study, rats (15 per group, were given MX by gavage, on 5 days a week, at doses of 0 (controls) or 30 md/kg (low dose) for 18 wk, or, in the high-dose group, at doses increasing from 45 to 75 mg/kg over 14 wk. The high dose was finally lethal (two males and one female died) and caused hypersalivation, wheezing respiration, emaciation and tangled fur in animals. The body weights of the high-dose males decreased by 15%, and food consumption was decreased by 15 to 20%, but the water consumption increased by 15% to 60%. Plasma cholesterol and triglycerides were elevated and urine excretion was increased. Urine specific gravity was decreased and the relative weights of the liver and kidneys were increased in both sexes at both doses in comparison with the controls. At both doses, duodenal hyperplasia occurred in males and females, and slight focal epithelial hyperplasia in the forestomach was observed in males. Splenic atrophy and haemosiderosis were seen in two high-dose females, and epithelial cell atypia in the urinary bladder of one high-dose male and female. The frequency of bone marrow polychromatic erythrocytes with micronuclei was slightly increased in low-dose males. The results indicate that repeated administration of MX disturbs the fluid-electrolyte balance and induces diuresis, causes mucosal hyperplasia in the gastro-intestinal tract as a local effect, and affects lipid metabolism.

Altered enzyme activities of xenobiotic biotransformation in kidneys after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) to rats.

Activities of the xenobiotic metabolizing enzymes were measured in the liver, kidney, duodenum and lung microsomes and cytosol fractions of Wistar rats after subchronic administration of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a potent bacterial mutagen in chlorinated drinking water. MX was administered by gavage at the dose level of 30 mg/kg for 18 weeks (low dose), or at the dose level which was raised gradually from 45 mg/kg for 7 weeks via 60 mg/kg for 2 weeks to a clearly toxic dose of 75 mg/kg for 5 weeks (high dose). Microsomal and cytosolic preparations were made and the activities of 7-ethoxyresorufin-O-deethylase (EROD), pentoxyresorufin-O-dealkylase (PROD), NADPH-cytochrome-c-reductase, UDP-glucuronosyltransferase (UDPGT) and glutathione-S-transferase (GST) were measured. Kidneys were affected most. A dose-dependent decrease was observed in EROD (90% in males, 80% in females at the high dose) and in PROD (58% in females at the high dose) in kidneys. An increase was, however, detected in kidney NADPH-cytochrome-c-reductase (66% in females at high dose), UDPGT (89% in males and 97% in females at high dose) and GST activities (56% in males and 50% in females at high dose). MX caused only a few changes in the enzyme activities of the liver. The EROD activity was decreased 25% to 37%, both in the livers of males and females, but the total content of P450s was not altered. Hepatic GST activity was elevated in females in a dose-dependent manner (31% and 44%). GST activity was elevated in duodenum in females (59%) at the high dose. There were no marked changes in the enzyme activities in the lungs. MX was a weak inhibitor of EROD activity both in the liver and kidney microsomes in vitro, decreasing the EROD activity by 53% and 43%, respectively at the concentration of 0.9 mM. The results indicate that MX decreases the activity of phase I metabolism enzymes, but induces phase II conjugation enzyme activities, particularly in kidneys in vivo. It is possible that these changes contribute to metabolism of MX in kidneys and renders them susceptible to MX in the course of repeated exposure.

Spread of malignant lymphoid cells into rat central nervous system with intact and disrupted blood-brain barrier.

The pathways of spread of malignant lymphoid cells into the central nervous system (CNS) were studied using a T lymphoblastic leukaemia/lymphoma model of inbred PVG rats. The effects of intraperitoneal, intracarotid, intravenous, intrathecal and intracerebral routes of transplantation were analysed, and the significance of the blood-brain barrier (BBB) in preventing neoplastic cell invasion was studied by disrupting the BBB with focal cold injury. Extraneurally transplanted cells appeared first in the dura and subarachnoid space. From the latter they spread further into the perivascular space of penetrating cortical vessels. Parenchymal tumour cell foci were only seen in terminally ill rats, usually associated with damage to the vessel wall. Intrathecal transplantation did not accelerate the progression of the disease. Intracerebrally transplanted cells readily produced parenchymal infiltrates with diffuse invasion into the white matter, perivascular spreading into the cortex, and contralateral extension along the corpus callosum. Parenchymal invasion did not occur immediately after disruption of the BBB, but in the chronic phase neoplastic cells infiltrated the injured area. In conclusion, the model closely resembles human CNS leukaemia. Malignant cells appeared to enter the CNS through the deficient BBB of the subarachnoid vessels, whereas the BBB of the intracerebral vessels and perivascular glia limitans were very resistant to leukaemic cell invasion. This underlines the difference between the subarachnoid and perivascular v. intraparenchymal compartments. Preceding BBB damage may predispose to brain metastases. The parenchymal dissemination of malignant cells was similar to that in primary CNS lymphoma and it followed the same spreading pathways as the extracellular fluid.

Cytogenetic effects of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in rat peripheral lymphocytes in vitro and in vivo.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting Salmonella mutagen found in wood pulp chlorination effluents and chlorinated drinking water. In cultured rat peripheral lymphocytes, MX induced significant dose-related increases in sister-chromatid exchanges (SCEs) and chromosome aberrations at doses of 20-60 micrograms/ml and of 60-80 micrograms/ml, respectively. MX produced primarily chromatid-type as opposed to chromosome-type aberrations. The peripheral lymphocytes of male and female rats exposed to MX by gavage 5 days a week for 14-18 weeks showed significant dose-related increases in SCEs at both levels of exposure (30 and 45-75 mg/kg) in both sexes. The present results demonstrate for the first time that MX is genotoxic in vivo.

Pharmacokinetics in rat of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a drinking water mutagen, after a single dose.

The pharmacokinetics of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) was evaluated after a single oral or intravenous administration in the rats using 14C-labelled compound. Twenty to 35% of the dose was absorbed into circulation from the gastrointestinal tract as assessed from the excretion in urine. The mean elimination half-life of the radioactivity in blood (T1/2 k10) was 3.8 hr. Traces of radioactivity remained in the blood for several days. The tissues lining the gastrointestinal and urinary tract, kidneys, stomach, small intestines and urinary bladder contained the highest radioactivity. The activity declined slowest in the kidneys. Urine was the main excretion route. Seventy-seven % of the total amount excreted appeared in urine in 12 hr and 90% in 24 hr. No radioactivity was exhaled in air suggesting that elimination through respiration did not occur. After an intravenous administration of 14C-MX, the T1/2 k10, was much longer, 22.9 hr, and the total elimination half-life (T1/2 beta), 42.1 hr. The results indicate that MX is absorbed from the gastrointestinal tract to a considerable degree and it is excreted in urine very rapidly. A fraction of MX or its metabolites is retained in blood for a longer period of time. The pharmacokinetics of MX does not suggest extensive cumulation of MX in tissues after continuous exposure.