Novel form of phosphate activated glutaminase in cultured astrocytes and human neuroblastoma cells, PAG in brain pathology and localization in the mitochondria.

A novel form of phosphate activated glutaminase (PAG), catalyzing the synthesis of glutamate from glutamine, has been detected in cultured astrocytes and SH-SY5Y neuroblastoma cells. This enzyme form is different from that of the kidney and liver isozymes. In these cells we found high enzyme activity, but no or very weak immunoreactivity against the kidney type of PAG, and no immunoreactivity against the liver type. PAG was also investigated in brain under pathological conditions. In patients with Down's syndrome the immunoreactivity in the frontoparietal cortex was significantly reduced. The findings leading to our conclusion of a functionally active PAG on the outer face of the inner mitochondrial membrane are discussed, and a model is presented.

Regulating and assessing risks of cholinesterase-inhibiting pesticides: divergent approaches and interpretations.

This document presents a revised framework for conducting worker and dietary risk assessments for less-than-lifetime exposures to organophosphate or carbamate pesticides based on red blood cell (RBC) or brain acetylcholinesterase (AChE) inhibition or the presence of clinical signs and symptoms. The proposals for appropriate uncertainty factors are based on the biological significance of the cholinesterase (ChE) inhibition noted at the lowest-observed-effect level (LOEL) and the degree of uncertainty in the extrapolation between human and animal data. An extensive evaluation of industry data, not previously summarized, and the available literature indicate that the following risk assessment principles are supportable and protective of human health: Plasma ChE inhibition is not an adverse effect, and therefore should not be utilized in risk assessments. Red blood cell AChE is not associated with the nervous system and inhibition is not per se an adverse (neurotoxic) effect. When available, cholinergic effects or brain AChE inhibition data should take precedence over RBC AChE for determining no-observed-effect levels (NOELs). When available, human RBC AChE inhibition or cholinergic effects data should take precedence over animal data for determining NOELs. Due to the lack of adversity associated with inhibition of RBC AChE, the use of a 10-fold (10x) uncertainty factor from the NOEL is adequate when RBC AChE inhibition data from either animal or human studies are used to assess human risk. Due to greater potential for adversity, NOELs for brain AChE inhibition and cholinergic effects identified in animal studies should receive a default uncertainty factor of 100x; lower uncertainty factors may be used on a case-by-case basis. NOELs based on cholinergic effects noted in human studies should only require a 10x uncertainty factor, since an interspecies extrapolation factor from animals to humans is unnecessary. For RBC and brain AChE activity the threshold for defining a NOEL should be less than or equal to 20% difference from control activity in all species. For risk assessment purposes, duration and route of the study should reflect the expected duration and route of exposure for humans (i.e., a 21-d or 28-d dermal study for subchronic occupational dermal exposure assessment). When dermal data are not available, a subchronic oral toxicity study and an appropriate dermal penetration factor should be used. A general default of 10% absorption should be used, analogous to the United Kingdom and German exposure models that are widely used in Europe. The recommendations in this document are generally consistent with current risk assessment procedures used by Canada, the European Community (EC), and the United Kingdom (UK).

Alterations in gait following ethanol exposure during the brain growth spurt in rats.

Walking patterns were assessed in rats that had been exposed to alcohol neonatally during a period encompassing the brain growth spurt. Rat pups were exposed via an artificial rearing technique to either a 2.50% (w/v) or 2.15% (w/v) EtOH-milk formula on Days 26-32 postconception. An artificially reared control group and a suckle-control group were also included in the experiment. Gait patterns were assessed in animals from each of the neonatal treatment groups at 43, 67, and 87 days postconception. No differences in gait patterns were evident on Day 43 postconception; however, on Days 67 and 87 animals exposed to alcohol during the neonatal period displayed an abnormal gait. These animals had a shortened stride length and an increased angle of placement of the hindfeet relative to artificially reared and suckle-control animals. The altered gait pattern may be the result of alcohol-induced hippocampal and cerebellar damage during the brain growth spurt.

Neonatal ethanol exposure: functional alterations associated with cerebellar growth retardation.

The effects of alcohol exposure during the brain growth spurt on development and on behavioral assessments of functional alterations in the cerebellum were examined in the rat. Rat pups were exposed via an artificial rearing technique to either a 2.50% w/v or 2.15% w/v EtOH-milk formula during a period encompassing the brain growth spurt. An artificially reared control group and a suckle control group were also included. Peak blood alcohol concentrations for animals in the high and low dose alcohol exposure groups were approximately 300 mg/dl and 180 mg/dl, respectively. Reductions in brain minus cerebellum to body weight (BR-C/BD) and cerebellum to body weight (C/BD) ratios were noted in animals from each of the alcohol-treated groups. Some catch-up growth in terms of brain mass was noted in animals from each of the alcohol-exposed groups. Animals exposed to alcohol during the neonatal period displayed deficits on several tests of balance and motor ability. Alcohol-exposed animals performed more poorly than controls when traversing two parallel horizontal rods and on tests of hindlimb and head elevation. No differences were noted in the ability to remain on a rotating drum. These results suggest that some of the behavioral consequences of neonatal ethanol exposure might be due to ethanol's actions on the cerebellum.

The effects of prenatal alcohol exposure on odor associative learning in rats.

Alcohol was administered to pregnant females via a liquid diet that contained either 35% ethanol-derived calories (35% EDC) or 0% EDC on gestation days 6-20. An ad lib lab chow group (LC) was also included. In Experiment 1, odor-aversion learning was examined in 10-day-old offspring. While both the 0% EDC and LC groups displayed odor aversions, the 35% EDC offspring did not. In Experiment 2, learning was assessed in an appetitive paradigm in three-day-old offspring. Once again, the 35% EDC offspring showed no evidence of learning. Experiment 3 examined odor-aversion learning in adults. Both alcohol-exposed offspring and controls learned the odor association equally well. These findings suggest that odor associative learning is a sensitive indicator for alcohol-related learning deficits in rat pups although these deficits may dissipate as the offspring matures. Since odor associations play a critical role in neonatal behaviors, these deficits may help explain other behavioral anomalies noted following prenatal alcohol exposure.

Behavioral and developmental effects of prenatal exposure to pentazocine and tripelennamine combinations.

Long-Evans, hooded rats were intubated with one of four dose combinations of pentazocine and tripelennamine on Days 7-20 of pregnancy: 0:0 (mg/kg pentazocine:tripelennamine), 20:10, 40:0, or 40:20. An additional group had free access to lab chow and water throughout pregnancy. At birth, reduced body weights were evident in all drug-treated offspring. Growth deficits were not noted at 5, 10, 15 or 20 days of age. Three measures of activity were collected at 18 days of age, however, none of these measures were differentially affected as a function of prenatal treatment. At 85 days of age, offspring were tested in a two-way shuttle avoidance paradigm. Although the number of avoidances was not significantly affected by prenatal treatment, offspring exposed to these drugs in combination had shorter response latencies than controls with increased training and made more intertrial crossing responses. Additional offspring were tested for seizure susceptibility at 100 days of age. None of the parameters of seizure activity were significantly affected by prenatal drug treatment. Prenatal exposure to pentazocine and tripelennamine resulted in prenatal growth deficits, increased activity during the intertrial interval and decreased response latencies in a shuttle avoidance learning task, suggesting that this polydrug combination may be associated with some long-term behavioral teratogenic risks.

Social play in juvenile rats prenatally exposed to alcohol.

Offspring of rat dams that consumed isocaloric liquid diets containing either 35% or 0% ethanol-derived calories (EDC) from gestation days 6-20 were tested for play-fighting behavior as juveniles. Offspring from a group of dams maintained on standard lab chow and water throughout gestation were also included. Animals were tested in pairs, with offspring from each of the three prenatal treatment conditions (35% EDC, 0% EDC, and lab chow) being paired with another same-sex animal from one of these three prenatal treatment groups. Although play-fighting in juveniles is normally sexually dimorphic, this normal pattern was absent in juveniles prenatally exposed to alcohol. Male alcohol-exposed offspring displayed feminized behavior while female alcohol-exposed offspring showed masculinized behavior. This reversal of the normal sexually dimorphic aspects of play suggests that some of the behavioral disturbances associated with prenatal alcohol exposure may result, in part, from an alcohol-induced disruption of the hormonal environment in which the fetus develops.

Delayed taste aversion learning in preweanling rats exposed to alcohol prenatally.

Parallels between the behavioral profiles of rats exposed to alcohol prenatally and those with hippocampal damage suggest that hippocampal dysfunction may underlie some of the behavioral abnormalities resulting from prenatal alcohol exposure. Because of possible hippocampal involvement in the acquisition of a delayed conditioned taste aversion, this task was assessed in rat pups exposed to alcohol prenatally. Long-Evans rats were maintained on liquid diets containing either 35% or 0% ethanol derived calories from Days 6-20 of gestation. Pair-feeding procedures and an ad lib Lab Chow (LC) group were included. Fifteen-day-old offspring from these three groups were given access to saccharin and then injected immediately (0 hr) or 2 hr later with either lithium chloride (LiCl) or sodium chloride (NaCl). Immediate pairing of saccharin with LiCl produced a marked taste aversion, although this effect was less pronounced in 35% EDC pups. In the 2 hr condition, weaker aversions were exhibited and again the 35% EDC group showed the least aversion. However, prenatal treatment did not interact with the injection interval.

Considerations for the design, implementation, and interpretation of animal models of fetal alcohol effects.

Animal models have provided clear insights into the clinical manifestations of fetal alcohol syndrome (FAS) and fetal alcohol effects (FAE). The value of such models depends upon the strengths of the experimental procedures. Researchers interested in fetal alcohol effects must be aware of issues and considerations which strengthen their investigations and those issues which place limitations upon interpretations. Methodological considerations associated with the use of animal models, choice of a test animal, method of ethanol administration, control groups, and data analysis are discussed.