The neurotoxic effects of triethyltin (TET) sulfate on myelinating cultures of mouse spinal cord.

Well myelinated cultures of mouse spinal cord, exposed to varying concentrations of triethyltin sulfate, were examined at different time intervals by light and electron microscopy. At a high concentration of TET (10(-6)M) for 1-2 hours, the myelin sheaths and cells showed marked degenerative changes. At lower concentrations of TET (10(-7)-10(-9) M) intramyelinic and other types of vacuole formation were seen; however, these lesions were always accompanied by changes in the perikarya, dendrites and axons of neurons and in the astrocytes. Also, there was an apparent increased number of neurofilaments and neurotubules in many axons. The findings are discussed in relation to the lesions induced in the experimental animal by alkyl tin intoxication.

Cellular and molecular effects of trimethyltin and triethyltin: relevance to organotin neurotoxicity.

Many of the neurotoxic aspects of organotin exposure have been described. Organotin exposure culminates in its accumulation in the CNS and PNS. The clinical picture is dominated by neurological disturbances; yet, the primary basis for their neurotoxicity is unknown. Trimethyltin (TMT) is primarily a CNS neurotoxin affecting neurons within the hippocampal pyramidal band and the fascia dentata. Triethyltin (TET) is a neurotoxin that produces a pathological picture dominated by brain and spinal cord edema. The first part of this review summarizes the current understanding of the interaction of TMT and TET with biologically active sites in the induction of neurotoxicity. In the second part, several hypotheses for the differential neurotoxic effects of these organotins and their shortcomings are discussed.

The response of oligodendrocytes to chemical injury.

Oligodendrocytes establish relationships with axons at myelination which commit the cell to make and then maintain certain volumes of myelin. As a result of this oligodendrocytes are a heterogenous population of cells. At one extreme, large cells support a single internode on large diameter axons while at the other, small cells support many internodes on small diameter axons. Although it is common practice to separate chemicals which cause vacuolation of myelin sheaths from those which bring about cell death and thus demyelination, many compounds produce vacuolation and/or cell degeneration depending on concentration; an observation which suggests that myelin sheath-associated vacuolation reflects oligodendrocyte toxicity rather than a specific myelinopathy. The restoration of myelin sheath-axon relationships following chemically induced demyelination requires a complex sequence of cell-cell interactions to occur in an orderly manner if new myelin sheaths are to be formed. Recruitment of new oligodendrocytes can be separated from the interaction of oligodendrocytes with axons which results in the laying down of a myelin sheath. The latter event can only take place in the absence of demyelinating agents and in the presence of astrocytes.

Neurotoxic effects of neonatal triethyltin (TET) exposure are exacerbated with aging.

Neonatal Long-Evans rats dosed with TET (5 mg/kg; IP) or saline on postnatal day (PND) 10 were examined across the life span for neural damage and performance on spatial learning tasks. A subset of rats were sacrificed to assess early damage with Nissl-staining, Timm's histochemistry, and glial fibrillary acidic protein (GFAP) immunohistochemistry 2, 7, or 14 days after dosing. Littermates were tested behaviorally in a T-maze spatial delayed alternation task on PND 23 or PND 90, and in a Morris water maze place learning task at 3, 12, or 24 months postdosing and then sacrificed for histological analysis. In neonatal rats, histological analysis indicated gliosis in discrete cortical regions, loss of Nissl-stained neurons in the hippocampal formation, entorhinal cortex and piriform cortex, and loss of Timm's staining in the entorhinal cortex. The behavioral assessment at PND 23 indicated a significant impairment in the T-maze. However, no significant impairments were observed in the T-maze at 3 months or the water maze at 3 or 12 months postdosing. At 24 months, TET-treated rats showed significant deficits in acquisition and retention of the water maze task compared with age-matched controls. Both groups of 24 months old rats were significantly impaired compared with young controls. At 24 months, there was a general age-related decrease in the optical density of Timm's staining in cortical regions (9%), compounded by a further decrease in the entorhinal cortex and outer molecular layer of the dentate gyrus of the hippocampus in TET treated rats (30%). These data indicate that early developmental exposure to an organometal resulted in morphological damage that was apparent behaviorally only during early postnatal development and with advanced aging.

Decreased adenosine cyclic 3',5'-monophosphate phosphodiesterase activity in rat brain following triethyltin intoxication.

The possible involvement of cerebral cAMP-phosphodiesterase (PDE) in the intoxication and brain edema formation after exposure to triethyltin (TET) has been studied in vitro and in vivo in the rat. In vitro studies showed an irreversible inhibition of the particulate and soluble phosphodiesterase activities. In vivo, both high i.v. single dose and repeated oral administration of low TET doses led to a significant decrease of the particulate activities. Phosphodiesterase inhibition preceded edema formation. The soluble activities were less influenced and their inhibition could be a consequence of the edema formed in the brain tissue rather than of a TET direct action upon the enzyme. Kinetic analysis of the brain particulate enzyme from the TET-treated rats showed a significant decrease in the Vmax of the substrate high affinity enzyme form when compared to controls.

Toxicity of organotin compounds for polymorphonuclear leukocytes: the effect on phagocytosis and exocytosis.

Phagocytosis and concomitant release of enzymes by rabbit polymorphonuclear leukocytes (PMNs) are inhibited by micromolar concentrations of triphenyltin and tributyltin; inhibition by triethyltin occurs at higher concentrations. Chemotactic peptide-induced exocytosis is inhibited at the same concentrations as phagocytosis. Tributyltin causes cell lysis at slightly higher concentrations as required for inhibition of phagocytosis and exocytosis. The organotin compounds have little effect on ATP level in PMNs, which makes an effect on metabolic energy providing processes unlikely. The increase of Ca2+-permeability of the plasma membrane, induced by chemotactic peptide, is inhibited by the organotin compounds. Inhibition of exocytosis by triphenyltin can be counteracted by a number of sulfhydryl compounds. The results suggest that the organotin compounds interfere with PMN function in an early phase of cell activation, where all functions have a common pathway, and where vulnerable sulfhydryl groups play a pivotal role.

Cognitive and neuroanatomical effects of triethyltin in developing rats: role of age of exposure.

Long-Evans rat pups were injected i.p. on postnatal day 5 (PND5) or 12 with 0, 3, or 5 mg/kg triethyltin sulfate (TET) and then tested on T-maze delayed alternation on PND21 or 28. Delayed alternation learning was impaired on PND21 and 28 in pups given 5 mg/kg TET. Pups given 5 mg/kg TET on PND5 were more impaired on delayed alternation than pups given 5 mg/kg TET on PND12. Pups given 3 mg/kg TET on PND5 or 12 were unimpaired at either age of testing. On the day following training, pups were sacrificed for histological assessment employing Nissl- or immunohistochemical staining for glial fibrillary acidic protein (GFAP), a putative marker of gliosis. Pups given 5 mg/kg TET on PND5 showed increases in GFAP immunoreactivity (IR) in subiculum, amygdala, hippocampus, piriform cortex, and entorhinal cortex with concomitant decreases in Nissl-stained cells in these regions. Pups given 5 mg/kg TET on PND12 showed increases in GFAP IR in piriform cortex, amygdala and dorsal hippocampus with concomitant decreases in Nissl-stained cells in these regions. Exposure to 3 mg/kg TET on PND5 and PND12 produced a mild increase in GFAP IR in piriform cortex and amygdala but no discernible loss of Nissl-staining in these respective regions. TET-induced behavioral deficits appear related to damage of structural correlates of the human temporal lobe and not piriform cortical pathology. These results demonstrate that the day of exposure greatly influences the magnitude of the cognitive deficits and neuropathology associated with exposure to TET. There appears to be a critical period during postnatal development for the developmental neurotoxicity of this compound.

Proton nuclear magnetic resonance studies on brain edema.

The water in normal and edematous brain tissues of rats was studied by the pulse nuclear magnetic resonance (NMR) technique, measuring the longitudinal relaxation time (T1) and the transverse relaxation time (T2). In the normal brain, T1 and T2 were single components, both shorter than in pure water. Prolongation and separation of T2 into two components, one fast and one slow, were the characteristic findings in brain edema induced by both cold injury and triethyl tin (TET), although some differences between the two types of edema existed in the content of the lesion and in the degree of changes in T1 and T2 values. Quantitative analysis of T1 and T2 values in their time course relating to water content demonstrated that prolongation of T1 referred to the volume of increased water in tissues examined, and that two phases of T2 reflected the distribution and the content of the edema fluid. From the analysis of the slow component of T2 versus water content during edema formation, it was demonstrated that the increase in edema fluid was steady, and its content was constant during formation of TET-induced edema. On the contrary, during the formation of cold-injury edema, water-rich edema fluid increased during the initial few hours, and protein-rich edema fluid increased thereafter. It was concluded that proton NMR relaxation time measurements may provide new understanding in the field of brain edema research.

Triethyltin-induced neuronal damage in neonatally exposed rats.

Neuropathological and biochemical effects of neonatal exposure to the alkyl metal triethyltin were examined in juvenile male Long Evans rats. Rats were injected intraperitoneally on postnatal day 5 with 6 mg/kg of triethyltin bromide and sampled on day 20. The brains of tin-treated animals weighed significantly less than either saline or starved controls and exhibited a marked caviation of the ventrolateral surfaces. Histologically, neuronal necrosis was noted in the entorhinal and transitional cortex, an observation confirmed by immunocytochemical staining of astrocytes. Hippocampal involvement was further evidenced by a protrusion of the molecular layer of the dentate gyrus, and an abnormal histochemical staining pattern of acetylcholinesterase in this layer. Sections stained by the Timm's method for the deposition of heavy metals showed a marked reduction in the staining of the hippocampal CA4,3,2 sectors and an absence of stained laminae in the outer molecular layer of the dentate gyrus. Receptor binding assays indicated a selective depression of the benzodiazepine receptor in the hippocampus of tin-treated pups compared to starved controls. Taken in concert, these data indicate that neonatal exposure to triethyltin produces severe neuronal damage in the posterior cortex and a derangement of hippocampal afferent circuitry.

Developmental neuropathology of organotin compounds.

The literature concerning the developmental neuropathology of organotins is reviewed. To date, neuropathological effects have been convincingly demonstrated for trimethyltin (TMT) and triethyltin (TET). Both compounds may damage the developing CNS; however they have different cellular targets. TMT is a neuronotoxin which damages areas of the limbic system, cerebral cortex, and brainstem. TET is a myelinotoxin which causes massive myelinic edema by yet undetermined mechanisms. Neuronal death is also seen following TET intoxication during the neonatal period, possibly as a result of elevated intracranial pressure. The neuropathological features of TMT and TET intoxication during early life are presented. Neurochemical and behavioral alterations resulting from congenital and/or neonatal exposure to organotins are also discussed.

Neurobehavioral toxicity of triethyltin in rats as a function of age at postnatal exposure.

Triethyltin (TET) has been shown to be neurotoxic when injected on postnatal day (PND) 5. In the present experiment we examined the toxicity of a single exposure to TET at several postnatal ages. Rat pups were injected ip with 0 (saline), 1.5, 3.0, or 6.0 mg/kg TET bromide on PND 1, 5, 10 or 15. In agreement with our previous data, PND-5 exposure to 6 mg/kg TET produced behavioral toxicity and decreased adult brain weight. High dose pups were less successful in descending on a rope at 20 and 21 days of age, and were hyperactive in figure-eight mazes at 29-30 and 57-58 days of age. The spatial distribution of activity was also altered: photocell counts were increased primarily in the figure-eight area of the maze. The size of the milk bands was reduced in 6 mg/kg pups injected on either PND 1 or PND 5. Preweaning growth was decreased following all injection ages; this reduction was most pronounced for pups exposed to TET on PND 1 and PND 5. Mating behavior was disrupted in 6 mg/kg males irrespective of age at exposure. These data demonstrate a differential sensitivity to the toxicity of TET during postnatal life, with maximal susceptibility on PND 5.

Neonatal triethyltin exposure alters adult electrophysiology in rats.

In adults, triethyltin (TET) produces degeneration of white matter, edema, vacuolization on myelin and histotoxic hypoxia. To determine the functional consequences of perinatal exposure to TET, albino rats were administered either 0, 3, 6, or 9 mg/kg TET on postnatal day 5. Upon reaching adulthood, the rats were implanted with electrodes for recording visual evoked potentials (VEPs) and hippocampal afterdischarges (ADs). In addition to these tests, 17 days of kindling trials were administered to the rats followed by testing with pentylenetetrazol and picrotoxin for seizure susceptibility. TET increased latencies of P2, P3, and N3 of the VEP in a dose dependent fashion. TET also decreases N1P2 amplitudes and produced gender-specific alterations in both P1, N1, and N2 latencies and N2P3 amplitudes. TET produced alterations in duration of the AD recorded from cortex during kindling, but did not produce significant alterations in any of the other variables tested. The results support previous studies, since they show that the adult VEP is sensitive to perinatal toxicant exposure.

Neuromuscular function and organotin compounds.

Mammalian exposure to toxic levels of the trialkyltin compounds, triethyltin (TET) and trimethyltin, results in pathological manifestations largely restricted to the nervous system. The remarkable features of TET toxicity are cerebral edema and muscular weakness. Rats exposed orally to TET (10-30mg TET Br/l of drinking water) progress through an increasingly compromised state beginning with mild ataxia and hindlimb weakness after one week, spastic paresis of the hindlimbs by two weeks; and hindlimb paraplegia and sensory changes by 3 weeks. Histopathological studies of chronic TET-exposed rats report minimal ultrastructural damage to distal peripheral nerves, myelin, and muscle. Chromatolytic reactions are observed in some alpha motor neurons; intramyelinic vacuolization in the ventral roots and horn is substantial by 3 weeks. Myelin vacuolization and degeneration are observed to a lesser extent in the dorsal roots of the spinal cord. Wet weights and myofiber diameters of EDL and soleus muscles are reduced during chronic TET intoxication, but no histopathology is evident using light microscopy. Conduction of compound action potentials in vivo along distal sensory fibers, ventral roots and distal motor fibers (in sciatic n.) is normal in 3 week TET rats as compared to control; however, nerve conduction velocity is decreased in the segment of the H-reflex arc involving the dorsal roots. Earlier studies by Stoner and coworkers led to suggestions that the neuromuscular junction may be preferentially affected by TET and could contribute, in part, to the symptoms of muscular weakness. In support of this hypothesis preliminary studies from our laboratory and others indicate that neurotransmission is functionally depressed at the myoneural junction following chronic TET treatment in vivo or when applied in vitro to isolated muscle preparations. Stimulated, but not unstimulated, release of acetylcholine from the vascular perfused rat phrenic nerve-hemidiaphragm preparation is decreased by TET especially at higher stimulation rates (20 Hz). In vitro administration of TET Br (10(-6)M) results in an irreversible decrease in the amplitude of evoked endplate potentials; chronic in vivo exposure to TET causes a decrease in the resting membrane potential of soleus muscle (in situ recordings) and provokes a peculiar post-stimulus (200Hz bursts) elevation of spontaneous miniature endplate potentials in isolated cut diaphragm preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Myelin basic protein-messenger RNA (MBP-mRNA) expression during triethyltin-induced myelin edema.

Triethyltin (TET) is a neurotoxicant that produces severe but transient cerebral edema, characterized ultrastructurally by vacuolation of the intraperiod line of central nervous system (CNS) myelin. TET has been reported to depress levels of myelin basic protein (MBP), a protein thought to play a critical role in myelin compaction. In the present study, the genomic expression (i.e., mRNA) of MBP was monitored throughout the pathogenesis of TET-induced myelin edema and recovery in Sprague-Dawley rats given a single injection of a neuropathic (8.0 mg/kg) or non-neuropathic (0.8 mg/kg) dose of TET-bromide. Levels of MBP-mRNA from the anterior and posterior brain were collected 1 hr, 3 hr, 2d, and 7d, postexposure. The optic nerve and caudal brainstem, representing anterior and posterior brain sites, respectively, were examined at the same time-points for ultrastructural evidence of edema and recovery. Our data indicate that neuropathic doses (8.0 mg/kg) of TET significantly stimulated MBP transcript throughout the brain at all exposure time-points. The magnitude and time-course of this stimulation differed in the anterior and posterior brain, with the latter region showing higher levels of MBP-mRNA. In the posterior brain, the highest levels of mRNA correlated with the appearance of edema in the caudal brainstem. In the anterior brain, MBP-mRNA levels were only marginally increased over controls. Ultrastructural evidence of myelin edema was confined to the brainstem in rats treated with neuropathic dose of TET. Intralamellar vacuolation appeared at 3 hr and 2d postexposure and could be correlated with peak levels of MBP transcript, whereas, recompacted myelin, which appeared by 7d postexposure, was associated with declining levels of the mRNA. Ultrastructural changes in the oligodendroglia were suggestive of metabolic stimulation and correlated with high MBP-mRNA levels. In summary, these data indicate that an initial genomic event in TET-induced myelin edema is stimulation of MBP transcript.

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells.

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.

The effects of postpartum exposure to triethyl tin on the neurobehavioral functioning of rats.

Male and female Fischer rat pups received sc injections of 1.5 or 3 mg/kg of triethyl tin bromide (TET), ethanol vehicle or distilled water on day 5 postpartum. At 21, 28, 60, and 90 days of age, the rats were weighed and assessed in a battery of tests which included fore- and hindlimb grip strength, negative geotaxis, startle responsiveness and spontaneous motor activity. Animals at 60 days of age were also tested in a two-way shuttle box avoidance procedure. Exposure to TET did not affect body weight at any time during testing, nor did it affect negative geotaxis of grip strength, Startle response to an air puff stimulus was decreased in males and females at 21 and 60 days of age, while no effects were observed at 28 or 90 days of age. Responsiveness to an acoustic stimulus was decreased at 60 days of age only. Spontaneous motor activity of males was increased at 21, and 28 and 60 days of age, while females had elevated activity at 28 and 60 days of age. Relative to ethanol controls, rats of both sexes were significantly hypoactive at 90 days of age. In a two-way shuttle box procedure, latencies to emerge prior to the first trial were elevated in TET exposed male and female rats. The number of discriminated avoidance responses made during a 60 trial session was decreased in females receiving the 3 mg/kg dose. Activity during the intertrial interval was not affected. A single postnatal exposure to TET can produce long-term alterations in the sensorimotor capabilities of rats; the effect appears to occur equally in both sexes and is independent of toxicant-induced body weight changes.

Behavioral toxicity of acute and subacute exposure to triethyltin in the rat.

The behavioral effects of both acute and subacute triethyltin (TET) exposure were examined in the rat. Animals acutely exposed to TET at doses of 0, 1.5 and 3.0 mg/kg showed a dose-related decrease in motor activity when tested between 2 and 4 hr following exposure. Subacute (3 week) exposure to TET in the drinking water (5 or 10 ppm) resulted in performance decrements in the following: maze activity, open field behavior, acoustic startle response and landing foot-spread. Early signs of behavioral deficits were observed 2 weeks after 10 ppm TET. These effects were reversible within one month after termination of exposure.

Cytotoxicity in vitro and preliminary antitumor activity in vivo of a novel organotin compound.

The cytotoxic effect and antitumor activity induced by the novel organotin compound triethyltin(IV)lupinyisulfide hydrochloride, have been investigated. Different patterns of antiproliferative effects have been observed in a panel of human tumor cell lines in vitro. Toxicity studies in mice reported acute toxicity at the doses of 21 and 17.5 mg/kg which progressively disappeared at lower concentrations. On this basis, the doses of 3.5, 7 and 14 mg/kg were selected to assess the antitumor activity in vivo against the P388 leukemic cells xenografted in mice. This compound was able to induce a dose-dependent significant reduction of tumor volume, up to 46%, at the highest concentration (p = 0.0062) without important toxicity, as also confirmed by histological analysis of the main organ tissues. This preliminary study seems to hold interest for further investigations in different tumor models as well as for the evaluation of optimal drug route and schedule.

Neurotoxic effects of trimethyltin and triethyltin on human fetal neuron and astrocyte cultures: a comparative study with rat neuronal cultures and human cell lines.

Trimethyltin (TMT) and triethyltin (TET) caused cell death in cultures of primary human neurons and astrocytes, rat neurons and human neuroblastoma cell lines. Human neurons and astrocytes showed a delayed response to TMT cytotoxicity. After 24h of TMT exposure, LC50 values were 148.1, 335.5 and 609.7 microM for SK-N-MC neuroblastoma cell line, neurons and astrocytes, respectively. Over 5 days of exposure, the cytotoxic potency of TMT increased about 70-fold in human cortical neurons. Rat hippocampal neurons were the most vulnerable cells to TMT cytotoxicity, exhibiting an LC50 value 30-fold lower (1.4 microM) than that of rat cerebellar granule cells (44.28 microM). With the exception of rat hippocampal neurons, TET was more potent than TMT in inducing cell death (LC50 values of 3.5-16.9 microM). Moreover, TET was more effective than TMT in increasing intracellular free Ca2+ concentration in human and rat neurons. This work shows that human fetal neuron and astrocyte cultures are a useful model for studying the neurotoxic effects of these environmental contaminants and, thus, predicting their impact on human health.

Na+/K(+)-ATPase in rat brain and erythrocytes as a possible target and marker, respectively, for neurotoxicity: studies with chlordecone, organotins and mercury compounds.

Due to the inaccessibility of human nerve tissue for direct biochemical evaluation, there appears to be a need to identify peripheral markers which will reflect toxicity to the central nervous system by relatively non-invasive means. The aim of this study was to investigate whether the enzyme Na+/K(+)-ATPase in erythrocytes could be used as a marker for effects on the same enzyme in brain tissue. The compounds chosen to test this hypothesis were the pesticide chlordecone, the organotin compounds triethyltin and tributyltin, mercuric chloride and methyl mercury. All compounds were found to inhibit in vitro Na+/K(+)-ATPase activity in rat brain (IC50s = 0.9-56 microM) and in rat erythrocytes (IC50s = 1.2-66 microM) with similar potencies. However, administration of these compounds in vivo at high doses produced no significant inhibition of either brain or erythrocyte Na+/K(+)-ATPase activity, despite observed symptoms of neurotoxicity. Dialysis experiments indicated that dissociation of the compounds by dilution during tissue preparation was not responsible for the lack of detectable in vivo inhibition. Measurements of metal concentrations in brain by atomic absorption spectrometry after in vivo administration of triethyltin, mercuric chloride and methyl mercury indicated that levels of these compounds were too low to inhibit significantly NA+/K(+)-ATPase activity. These results suggest that inhibition of Na+/K(+)-ATPase activity might not represent the mechanism responsible for the neurotoxicity of these compounds, and that erythrocyte Na+/K(+)-ATPase activity is not a useful marker for neurotoxicity following acute exposures.