Changes in axonally transported proteins in the mature and developing rat nervous system during early stages of methyl mercury exposure

It was established by means of SDS polyacrylamide gel electrophoresis that direct injections of methyl mercury (10 micrograms Hg) into the mature rat vitreous body of the eye decrease protein synthesis in the retina and optic nerve at 4 hours after injection. Although the global spectrum of polypeptides did not change, a specific decrease in the volume of polypeptides of 20-23 K daltons molecular weight was evident. Conversely, systemic exposure to methyl mercury resulted in increased protein synthesis of polypeptides of 20-23 K molecular weight both in adult (8 mgHg/kg/day for 8 days) and neonatal rats (2 mgHg/kg/day for 10 days). In addition, specific changes in the volume of polypeptides 75-90 K molecular weight were noted in sciatic nerves of neonatal rats. These data are consistent with a bimodal response in protein synthesis following MeHg treatment. Local presence of MeHg following direct injection into the eye causes a reduction in protein synthesis, while chronic systemic exposure results in increased synthesis and transport of proteins in both mature and developing optic nerves and neonatal sciatic nerves. Thus, these systems possess the capacity to attempt regenerative processes through induction of a small subset of proteins known as GAPs (Growth-Associated Proteins) during the early stages of systemic methyl mercury exposure. These wide spread and system-specific changes are consistent with growth-specific functions during the early stages of methyl mercury exposure.     

Effects of systemic methyl mercury-adulterated water consumption on fast axonal transport in the rat visual system

The present study was designed in an effort to determine whether changes in fast axonal transport in the mature rat visual system can be directly correlated with the onset of neurological dysfunction. Methyl mercury was administered in the drinking water at a concentration of 54 micrograms Hg/ml. Fast axonal transport of proteins in the optic nerve and tract was quantified by scintillation spectrometry of protein-bound radioactivity along the visual pathway after an intraocular injection of 3H-proline. At 8 hours after injection the labeled protein had reached the lateral geniculate body both in controls and treated animals. However, two-way analysis of variance revealed a significant decrease in the volume of transported protein-bound radioactivity along the visual pathway. Thus, while the rate of fast axonal transport does not seem to be correlated with the onset of motor dysfunction, the onset of neurological symptoms may be associated with abnormal transport capacity. Treatment lowered body weight to the same extent in males and females. Hind limb cross-over occurred after 25.6 +/- 0.8 days and was followed quickly by hind limb paralysis (32 +/- 0.6 days). The cerebellum revealed pyknotic nuclei throughout the internal granular layer. Purkinje cells appeared normal. No pathological changes were noted in the kidneys.     

The use of astrocytes in culture as model systems for evaluating neurotoxic-induced-injury.

The prevailing thought that astrocytes function predominantly as passive metabolic or even physical support for neurons has faded over the last 20 years. Today these stellar shaped cells are credited with an expanded role, playing key functions in CNS development, homeostasis, and pathology. In probing their expanded roles, primary astrocyte culture systems have proven to be an indispensable tool. Astrocytes have been implicated in both a defensive and facilitatory capacity for many toxic injuries. Evidence for a protective role of astrocytes in modulating CNS toxicity is afforded by observations that the toxicity of glutamate to cortical neurons is diminished upon astrocytic enrichment of the cell culture (Rosenberg and Aizenman, 1989). In cultures of rat cerebral cortex in which astrocyte proliferation is stringently suppressed, glutamate neurotoxicity occurs at low glutamate concentrations similar to those which are normally found in the extracellular space in the hippocampus. In the presence of excess astrocytes, concentrations of glutamate one-hundred fold higher are required to produce equivalent neurotoxicity (Rosenberg and Aizenman, 1989). Astrocytes can facilitate the action of neurotoxins via a modulating process which takes place within the astrocyte or by a direct cytotoxic effect. Whereas primary astrocyte cultures remain unaffected by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; Marini et al., 1989), they function prominently in the selective destruction of dopaminergic neurons of the nigrostriatal pathway in humans, other primates and rodents (Davis et al. 1979; Langston et al., 1983; Burns et al., 1983; Langston et al., 1984; Heikkila et al., 1984; Jarvis and Wagner, 1985). Thus, while MPTP by itself is not toxic to cerebellar cells in co-culture with cerebellar astrocytes, MPTP is toxic to the granule cells (Marini et al, 1989). This is thought to be due to an astrocyte-mediated conversion of MPTP to its highly polar and toxic metabolite, 1-methyl-4-phenylpyridinium ion (MPP+; Chiba et al. 1984). There is compelling evidence that astrocytes respond directly or indirectly to a number of other neurotoxins. Direct cytotoxic effects on astrocytes constitute the major morphologic feature in hyperammonemia (Norenberg, 1981), a condition implicated as an etiologic factor in several CNS disorders. In addition, a predisposition of astrocytes for methylmercury uptake (Aschner et al., 1990 a,b) offers a possible explanation for the observed neurotoxicity of this heavy metal, since a direct toxic effect on astrocytes would result in failure of astrocyte homeostatic functions, indirectly resulting in neuronal impairment, injury and death.     

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.     

Foreign metallothionein-I expression by transient transfection in MT-I and MT-II null astrocytes confers increased protection against acute methylmercury cytotoxicity

The mechanisms associated with metallothionein (MT) gene regulation are complex and poorly understood. Only a modest increase in brain MT expression levels is attained by exposure to metals, MT gene transfection, and MT gene knock-in techniques. Accordingly, in the present study, MT null astrocytes isolated from transgenic mice deficient in MT-I and MT-II genes were introduced as a zero background model of MT expression. MT protein levels were determined by western blot analysis. MT proteins in MT-I and MT-II null astrocytes were undetectable. Transient MT-I gene transfection increased the levels of foreign MT expression in MT-I and MT-II null astrocytes by 2.3-fold above basal levels in wild-type astrocytes. Intracellular Na(2)51CrO(4) efflux and D-[2,3-3H]aspartate uptake were studied as indices of acute methylmercury (MeHg) (5 microM) cytotoxicity. In MT-I and MT-II knockout astrocytes MeHg led to significant (p<0.01) increase in Na(2)51CrO(4) efflux and a significant (p<0.05) decrease in the initial rate (1 min) of D-[2, 3-3H]aspartate uptake compared to MT-I and MT-II knockout controls. Transfection of the MT-I gene in MT-I and MT-II null mice significantly (p<0.01) decreased the effect of MeHg on Na(2)51CrO(4) efflux in MT null, as well as wild-type astrocytes. MT-I gene transfection in MT-I and MT-II null astrocytes reversed the inhibitory effect of MeHg on D-[2,3-3H]aspartate uptake, such that initial rates of uptake in MT-I transfected cells in the presence and absence of MeHg (5 microM) were indistinguishable. These results demonstrate that: (1) astrocytes lacking MTs are more sensitive to MeHg than those with basal MT protein levels, (2) the MT-I gene can be overexpressed in MT-I and MT-II null astrocytes by transient MT-I gene transfection, and (3) that foreign MT expression endows astrocytes with increased resistance to MeHg.     

Long-term outcomes: what should the focus be?

The neonatal intervention trials of the 1980s and early 1990s focused primarily on short-term outcomes. Contemporary clinical trials have recognized the importance of longer-term outcomes but have rarely been powered to achieve that aim. This review discusses important and clinically relevant outcomes that future trials should be powered to address and identifies the challenges facing the neonatal clinical trials community. These challenges include consensus definitions of relevant outcomes that are objective and validated, variability among centers in populations and practices, and the need for predictive surrogate markers of long-term outcomes. Future trials must be designed and powered to address the potential for harm as well as the prospect of benefit.     

Metabolic memory for vascular disease in diabetes

Although the terms "metabolic memory" and "legacy effect" have been used to describe the prolonged benefits of good blood glucose control, the former is now recognized as a phenomenon related to the prolonged harm produced mainly by hyperglycemia. At least three randomized clinical trials (Diabetes Control and Complications Trial in type 1 diabetes, United Kingdom Prospective Diabetes Study and Steno-2 in type 2 diabetes) have demonstrated that patients treated intensively for a period of time have a lower risk of micro- and macrovascular complications that persists during subsequent follow-up, even after their tight control has relented and the levels of glycated hemoglobin in the conventionally treated group improve. The mechanisms are not fully understood but most probably relate to the physiopathology of vascular complications of diabetes, and in recent years a unifying theory has been emerging to understand them. The excess superoxide anion produced by the mitochondria in response to hyperglycemia leads through disturbances at the nuclear level to the accumulation of potentially harmful substances such as advanced glycated end-products, protein kinase C, and nuclear factor κB, which are directly implicated in the development of vascular complications in diabetes. These adverse effects are not reversed when the high blood glucose is corrected, and some may be permanent because of epigenetic changes. Some antidiabetes drugs and antioxidant substances have produced partial reversibility of the mechanisms involved in the metabolic memory at the experimental level, but probably the best strategy is to optimize the metabolic control as early as possible, even before diabetes is diagnosed.     

Increased axonal transport in the rat optic system after systemic exposure to methylmercury: differential effects in local vs systemic exposure conditions

Axonal transport was studied by several techniques in the optic system of adult female Long-Evans rats following systemic exposure to methylmercury in 5 mM Na2CO3. Control rats were treated with the buffer alone. Four mg Hg/kg body weight for 4-6 days, or for 12 days, induced significant changes in the rate of protein synthesis in the retinal cells and in the rate of propagation of protein-bound radioactivity along the ganglion cell axons. Axonal transport of particulate material in both groups treated with methylmercury increased to a rate of 147 mm/day compared to 93 mm/day in controls. Methylmercury was distributed evenly throughout the retinogeniculate system. No clinical neuropathy was evident in either mercury-treated group. It is proposed that the increased rates of transport may represent an adaptive compensatory response to distal axonopathy caused by methylmercury. To investigate why systemic dosing produced effects opposite to those observed with local application of MeHg, various doses of MeHg were tested in the local and systemic paradigms, including doses which yielded equal concentrations of Hg in the retina. The results indicate that the differential response between the two treatment conditions is not a function of local dose, per se. Local and systemic application produce different dose-effect curves, which do not coincide at any dose.