Evaluation of the ToxRTool's ability to rate the reliability of toxicological data for human health hazard assessments.

Regulatory agencies often utilize results from peer reviewed publications for hazard assessments. A problem in doing so is the lack of well-accepted tools to objectively, efficiently and systematically assess the quality of published toxicological studies. Herein, we evaluated the publicly available software-based ToxRTool (Toxicological data Reliability assessment Tool) for use in human health hazard assessments. The ToxRTool was developed by the European Commission's Joint Research Center in 2009. It builds on Klimisch categories, a rating system established in 1997, by providing additional criteria and guidance for assessing the reliability of toxicological studies. It also transparently documents the study-selection process. Eight scientists used the ToxRTool to rate the same 20 journal articles on thyroid toxicants. Results were then compared using the Finn coefficient and "AC1" to determine inter-rater consistency. Ratings were most consistent for high-quality journal articles, but less consistent as study quality decreased. Primary reasons for inconsistencies were that some criteria were subjective and some were not clearly described. It was concluded, however, that the ToxRTool has potential and, with refinement, could provide a more objective approach for screening published toxicology studies for use in health risk evaluations, although the ToxRTool ratings are primarily based on study reporting quality.

A qualitative retrospective analysis of positive control data in developmental neurotoxicity studies.

Testing for neurodevelopmental effects commonly involves both functional and neuropathological assessments in offspring during and following maternal exposure. The use of positive controls in neurotoxicity screening has been advocated by numerous expert groups. Evaluation of positive control data allows evaluation of laboratory proficiency in detecting changes in the structure and function of the developing nervous system and comparison of the sensitivity of assessments in different studies and laboratories. This project surveyed approaches taken in contract and industrial laboratories in generating and providing these data. Positive control data submitted in support of 34 developmental neurotoxicity (DNT) studies from 16 different laboratories were summarized by test method for information on the following: age relevance of test subjects, the presence of a dose-response relationship, gender, group size, statistics, report quality, quality assurance, and the year the study was conducted. Endpoints included the following: developmental landmarks, clinical observations (CO), motor activity, startle response, learning and memory, qualitative neuropathology, and quantitative brain morphometry (linear measurements of selected brain regions). Results ranged from no positive control data for three laboratories, to one laboratory that submitted 17 separate positive control reports. The qualitative range was similarly broad, from excellent to poor. Various problems were identified, including the following: inappropriate report structure (e.g., copies of poster presentations), lack of individual data, inadequate methodological details, submission of very old data (>10 years) or data from completely different laboratories, use of inappropriate positive control chemicals or doses that were without effect, lack of statistical analysis, use of only one sex, and use of incompatibly aged animals. Analyses revealed that there were only 3 out of 16 laboratories that had submitted positive control data adequate for proficiency purposes for all of the major endpoints in the DNT study. Adequate positive control data are very useful in a weight-of-evidence approach to help determine the biological significance of results, and also to increase the confidence in negative results from DNT studies.

Neuroendocrine responses to intravenous infusion of physostigmine in patients with Alzheimer disease.

We have reported that physostigmine, a reversible cholinesterase inhibitor, enhances verbal memory in patients with Alzheimer disease (AD). To elucidate the mechanism of cognition enhancement, plasma hormones were measured during high-dose acute and low-dose chronic steady-state intravenous infusions of physostigmine in nine subjects with AD. High-dose hormone responses were measured during and for 24 h after the infusion of physostigmine 1-1.5 mg over 45-60 min. Chronic responses were measured during continuous intravenous infusions of physostigmine at doses (0.5-25 mg/day) that escalated over 2 weeks, and then during 1 week infusion of the dose that optimized cognition (2-12 mg/day) or placebo administered in a randomized, double-blind, cross-over design. A replicable improvement in verbal memory was found in five subjects. High-dose physostigmine infusion that produced noxious side effects resulted in significant elevation above baseline in plasma levels of adrenocorticotrophic hormone (ACTH) (p = 0.0001), cortisol (p = 0.0001), and beta-endorphin (p = 0.0001). Chronic physostigmine administration, in the absence of adverse effects, produced no significant elevation in ACTH (p = 0.08), cortisol (p = 0.70), or beta-endorphin (p = 0.82). These results indicate that high-dose physostigmine activates the hypothalamic-pituitary-adrenal (HPA) axis, likely representing a "stress response." In contrast, cognition-enhancing doses do not produce a peripheral corticosteroid response. Thus, physostigmine-induced memory improvement is independent of the activation of the HPA axis.

Food and Drug Administration Proposed Guidelines for Neurotoxicological Testing of Food Chemicals.

The fact that some chemicals may adversely affect the nervous system is certainly not a new concept in regulatory toxicology. In 1982, the FDA issued testing guidelines for the safety evaluation of proposed direct food and color additives which included the assessment of nervous system toxicity as part of the general toxicological profile. However, these guidelines provide only broad and nonspecific recommendations as to how this assessment may best be carried out. The information derived from toxicity screening studies conducted according to these guidelines enable little more than the detection of clearly evident adult nervous system toxicity associated with general neuropathology and overt neurological dysfunction. Little consistent or systematically documented information is typically developed about other equally important types of neurotoxic effects including, for example, behavioral dysfunction and developmental neurotoxicity. Concerns about these more subtle types of neurotoxic effects have become a prominent public health issue and have resulted in demands for an increasing level of assurance that efforts are being made to minimize even further the risks of neurotoxicity from human exposure to chemical substances. In an effort to address these concerns, the FDA is including specific attention to neurotoxicity in a proposed revision of its toxicity testing guidelines for food additives. These proposed guidelines focus on a more careful evaluation of structural and functional measures of neurotoxicity as a routine component of safety assessment. This focus will enable the development of the type of information needed for a more effective assessment of the full spectrum of neurotoxic hazards. The revised guidelines for neurotoxicity testing will be discussed in terms of the FDA's overall approach to safety assessment.

Clinical pharmacokinetics of arecoline in subjects with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetics and pharmacodynamics of intravenously administered arecoline in subjects with Alzheimer's disease. METHODS: Plasma arecoline concentrations were measured during and after high-dose (i.e., 5 mg intravenously over 30 minutes) and up to 2 weeks of continuous multiple-dose steady-state intravenous infusions of arecoline in 15 subjects with mild to moderate Alzheimer's disease. During multiple-dose infusions, the dose of arecoline was escalated from 0.5 to 40 mg/day. Psychometric tests were administered at baseline and every other dose to determine an "optimal dose" for each subject. This dose then was administered for 1 week using a randomized, placebo-controlled, double blind, crossover design. Plasma drug concentrations were measured by GC-MS. RESULTS: The optimal dose of arecoline varied fourfold across subjects (4 mg/day, n = 6; 16 mg/day, n = 3) with mean plasma half-lives of 0.95 +/- 0.54 and 9.3 +/- 4.5 (SD) minutes. Clearance and volume of distribution were 13.6 +/- 5.8 L/min and 205 +/- 170 (SD) L, respectively. At the dose that optimized memory, the mean plasma level was 0.31 +/- 0.14 (SD) ng/ml, and it predicted the optimal dose in all subjects. CONCLUSIONS: Because optimal dose variation is due to differing plasma kinetics, the plasma arecoline level measured at a single infusion rate can be used to choose the optimal dose for memory enhancement in patients with Alzheimer's disease.

Differential response to the cholinergic agonist arecoline among different cognitive modalities in Alzheimer's disease.

Nine patients with possible or probable dementia of the Alzheimer type were tested on nine cognitive tests prior to (two times) and during continuous intravenous administration of five different doses of the muscarinic cholinergic agonist arecoline (1, 4, 16, 28, and 40 mg/day). The present analysis examined whether improvement on cognitive testing for each patient during arecoline treatment was most likely to occur at the same dose for all tests or whether different test scores improved at different doses of arecoline. Results indicated there were significant differences among tests in the dose at which most patients showed improved cognitive performance. These differences may have therapeutic significance, as verbal ability tended to improve at low doses of arecoline, whereas attention and visuospatial ability tended to improve at higher doses of arecoline.

Clinical pharmacokinetics of physostigmine in patients with Alzheimer's disease.

OBJECTIVE: To study the pharmacokinetic and pharmacodynamic properties of physostigmine in subjects with Alzheimer's disease. METHODS: Plasma physostigmine concentration and butyrylcholinesterase inhibition were measured in blood samples collected during and after a single high-dose (1 to 1.5 mg for 45 to 60 minutes) and a sustained low-dose steady-state intravenous infusion in nine subjects with Alzheimer's disease. Escalating doses (0.5 to 25 mg/day) were administered during a 2-week period. A dose (2 to 12 mg/day) that optimized cognition in each subject was identified and then administered in a randomized, double-blind, placebo-controlled crossover design for 1 week. RESULTS: The elimination half-life of physostigmine was 16.4 +/- 3.2 (SE) minutes. Clearance and volume of distribution were 7.7 +/- 0.9 (SE) L/min and 2.4 +/- 0.6 (SE) L/kg, respectively. Butyrylcholinesterase inhibition half-life was 83.7 +/- 5.2 (SE) minutes. During sustained steady-state infusion, plasma physostigmine concentration (r = 0.95) and butyrylcholinesterase inhibition (r = 0.99) were linearly correlated with the dose. In five cognitive responders, the memory enhancement was significantly correlated (r = 0.86; p < 0.05) with butyrylcholinesterase inhibition. CONCLUSIONS: These results showed that, in cognitive responders, memory enhancement by physostigmine in Alzheimer's disease is correlated directly to the magnitude of plasma cholinesterase inhibition. Furthermore, during single-dose conditions, the dynamic half-life is five-fold longer than the kinetic half-life.

Treatment of Alzheimer disease by continuous intravenous infusion of physostigmine.

Physostigmine, a reversible and nonselective cholinesterase inhibitor, administered by steady-state, continuous intravenous infusion to carefully selected subjects with mild-moderate Alzheimer disease, produced significant but modest improvement in memory in five of nine subjects. Drug dosing was limited by the occurrence of adverse effects. Apparent tolerance to adverse effects was observed in two subjects when the dose of physostigmine was escalated slowly over at least 2 weeks. Steady-state cholinesterase inhibition by physostigmine appears to produce sustained cognitive improvement in some subjects with Alzheimer disease without substantially altering its therapeutic index.

Neuroendocrine responses to intravenous infusion of arecoline in patients with Alzheimer's disease.

We have reported that arecoline, a muscarinic receptor agonist replicably enhanced verbal memory in five of nine subjects with Alzheimer's disease (AD). To investigate the mechanism of cognitive improvement, circulating hormone measurements were made during high-dose acute and low-dose chronic intravenous (i.v.) arecoline administration to AD patients. Acute hormone responses were measured during, and for 6 h after, infusion of arecoline 5 mg i.v. over 30 min. Chronic responses were measured in cognitive responders during continuous i.v. infusion of arecoline escalating over 2 weeks (0.5-40 mg/day) and then during a 1 week infusion of the dose optimizing cognition (4-16 mg/day). Acute arecoline administered to 14 subjects produced unpleasant side-effects (e.g. nausea, vomiting), mean adrenocorticotrophic hormone (p = .0006), cortisol (p = .0001) and beta-endorphin (p = .0001) levels were elevated. During chronic arecoline treatment, no side-effects occurred and plasma cortisol, adrenocorticotrophic hormone and beta-endorphin levels were unchanged in nine subjects overall and in five cognitive responders. Thus, high-dose arecoline activates the hypothalamic-pituitary-adrenal (HPA) axis and may increase other anterior pituitary hormone levels, likely representing a 'stress response', but cognition-enhancing, low doses of arecoline do not produce a glucocorticoid response. Hence, arecoline-induced memory improvement is not due to the induction of 'stress' nor to the elevation of peripheral corticosteroid levels.

Memory improvement without toxicity during chronic, low dose intravenous arecoline in Alzheimer's disease.

Arecoline, a cholinergic agonist, administered at low doses by continuous intravenous infusion for up to 2 weeks, significantly and replicably improved memory in five of nine subjects with mild-moderate Alzheimer's disease. During dose finding, performance on a verbal memory task improved with an inverted U-shaped relation to dose. Six of nine subjects were classified as responders. During blinded, placebo-controlled, individualized optimal dosing for 5 days, verbal memory again improved in five of six responders but not in any non-responder. No adverse drug effects occurred. Arecoline, and possibly other cholinergic agonists, can safely improve memory in Alzheimer's disease at doses much lower than previously studied.

Individual trajectories of cognitive decline in patients with dementia of the Alzheimer type.

The course of decline was studied in 16 patients with probable or definite dementia of the Alzheimer type (DAT) over 2.7 to 6.8 years from first to last evaluation. Overall severity of dementia was measured with the Wechsler Adult Intelligence Scale (WAIS), the Dementia Rating Scale (DRS), and the Mini-Mental State Examination (MMSE), at approximately annual intervals. An initial plateau phase, during which language and cognitive functions did not change for periods of 9 to 35 months, was observed in 5 patients who initially had an isolated memory impairment without significant impairment of nonmemory language or visuospatial function. Once nonmemory functions began to decline, the rate of decline was remarkably steady in most individual patients but varied markedly among patients. The initial rate of decline after the plateau phase, as measured with the WAIS and DRS, was a significant predictor of subsequent rate in individual patients (r = .66, p less than .01, and r = .67, p less than .01, for the WAIS and DRS, respectively). The MMSE was a less reliable measure of longitudinal change in dementia severity and did not predict future rates of decline (r = .29). These results demonstrate a biphasic trajectory of decline in patients with DAT. Stable interindividual differences in rate of decline may provide a basis for designing more sensitive studies of treatments intended to slow or halt the progress of DAT.

Effects of acute infusion of the muscarinic cholinergic agonist arecoline on verbal memory and visuo-spatial function in dementia of the Alzheimer type.

1. Treatment of patients with dementia of the Alzheimer type (DAT) with arecoline, a muscarinic cholinergic receptor agonist, reportedly improves performance on a picture recognition memory task, but not on other memory measures. To examine further possible performance improvements following arecoline treatment, patients with DAT were treated with a 30 min intravenous infusion of arecoline (5 mg). 2. Psychometric testing was done at five time points (two before and three following the infusion). Patients were tested on a memory task (Buschke selective reminding) and a test of visuo-spatial performance (figure copying). 3. No net change from baseline was seen in mean scores following arecoline infusion. However, the changes in performance on the two tasks were correlated (p less than 0.02) over subjects at 10 min but not at 1.5 or 5.5 hr following the infusion. 4. This result suggests that although individual patients vary in their response to a given dose of arecoline, their responses are consistent across types of tasks. Thus the lack of a mean drug effect may be due to individual differences in response rather than to a lack of response.

Effects of long-term continuous infusion of the muscarinic cholinergic agonist arecoline on verbal memory in dementia of the Alzheimer type.

Alzheimer's disease (AD) is accompanied by depletion of cholinergic markers in the central nervous system. In an attempt to improve cognitive function in AD, arecoline (a muscarinic cholinergic receptor agonist) was given to patients with probable or possible AD in a two-phase design, and verbal memory function was examined. First, escalating doses of arecoline, range .5-40 mg/day, were administered by continuous intravenous infusion over a 2-week period. Based on neuropsychological test performance, an optimal dose of arecoline was identified, and this dose then was infused continuously for 5 days in a double-blind, placebo-controlled, counter-balanced trial. Long-term recall on a selective reminding task was significantly improved (from 11.8 to 20.1 words, p less than .05) during dose finding by arecoline at 4 mg/day. Two of eight patients were nonresponders; that is, they showed no improvement at any dose during the dose-finding study. Responders demonstrated significant improvement on total recall (p less than .05) during the double-blind study. These results indicate that some patients demonstrate reliable improvements of verbal memory during arecoline treatment.

Long-term effects of cholinergic agonists on memory.

Animal models provide important information about strategies that can be used to assess the effects of chronic exposure to drugs or other compounds. Furthermore, when used in conjunction with analyses designed to utilize the many advantages of animal models to examine learning and memory, these kinds of experiments can have significant ramifications for assessing the mnemonic effects of chronic drug use in people. Finally, given that aging is accompanied by significant deterioration of neurochemical systems, which may compromise the functions of those systems, the results from the experiments with DFP predict that changes in learning and memory abilities that are not apparent in young individuals may begin to surface as those individuals age or may appear following any other kind of insult to their nervous system.

Neurotoxicology dose/response assessment for several cholinesterase inhibitors: use of uncertainty factors.

Dose/response assessment for non-carcinogenic endpoints typically uses the Acceptable Daily Intake (ADI) or Reference Dose (RfD) approach, in which a dose believed to cause no toxic effect is divided by a number of safety or uncertainty factors (e.g., to control for variability and cross-species extrapolation), in order to estimate a dose presumed to have no adverse effects in humans. With the establishment of neurotoxicology testing guidelines, routine use of uncertainty factors to undertake neurotoxicity dose/response assessment procedures is likely. This approach to dose/response assessment has not yet been widely applied to neurotoxicity data. The purpose of this study was to assess the use of uncertainty factors and the assumptions underlying the use of the uncertainty factor method in assessing risk for several known human neurotoxicants. Because of the availability of a large neurotoxicity data base which included human exposure data, parathion, diisopropylfluorophosphate, physostigmine and acrylamide were chosen for this analysis. Literature searches were conducted for both human and animal data. The resulting data were assigned to one of five end point categories: Neurochemistry/neuropathology, physiology/consummatory behavior, sensory/motor, electrophysiology, and learning/memory behavior. No-observed-adverse-effect levels (NOAELs) and/or lowest-observed-adverse-effect levels (LOAELs) were determined when possible for each end point and for several species. Reference doses (RfDs) were calculated and compared across species. A number of issues and critical assumptions were identified.

Repeated exposure to diisopropylfluorophosphate (DFP) produces increased sensitivity to cholinergic antagonists in discrimination retention and reversal.

This experiment examined the effects of repeated exposure to diisopropylfluorophosphate (DFP), an organophosphate anticholinesterase, on the retention and reversal of a visual discrimination and on the number of muscarinic receptors in the brain. Rats were trained in a serial reversal procedure. After achieving stable performance, the rats were divided into two groups. One group received repeated injections of DFP, the other group received injections. To determine whether DFP-treated rats would be more sensitive than normal rats to stresses on the cholinergic system, each rat was injected with saline or one of three doses of scopolamine, a muscarinic receptor blocker, prior to testing on every 6th day. DFP alone caused no impairment in performance. Scopolamine produced a greater impairment in DFP-treated rats than in control rats. Similar results were obtained in a second behavioral task, match-to-sample in a water maze, using the same DFP treatment protocol and only one dose of scopolamine. The number of muscarinic receptors and acetylcholinesterase activity levels were reduced on the 2nd and 15th day after the end of DFP treatment. These results demonstrate that although repeated exposure to organophosphate anticholinesterases may not alter discrimination behavior directly, it may compromise the central nervous system so that it cannot react normally when challenged.

Hippocampal and amygdaloid involvement in working memory for nonspatial stimuli.

Hippocampal lesions in rats lead to an impairment of performance in spatial delayed conditional discriminations. The effect of such lesions on nonspatial tasks is controversial. In monkeys, both the hippocampus and the amygdala are involved in nonspatial delayed conditional discriminations. The effect of amygdaloid lesions in rats on this type of task has not been studied. To clarify the role of hippocampus and amygdala in a cue-relevant/space-irrelevant delayed conditional discrimination, rats were trained on a delayed match-to-sample task with visual and tactile cues as discriminative stimuli. Rats were then given one of five lesions: control, complete fimbria-fornix, partial fimbria-fornix, complete amygdala, or partial amygdala. Amygdaloid lesions, partial or complete, did not impair choice accuracy. Fimbria-fornix lesions did impair choice accuracy, and the magnitude and duration of the impairment was a function of the size of the lesion. Partial fimbria-fornix lesions produced a slight impairment that disappeared with continued testing. Complete fimbria-fornix lesions produced chance performance throughout postoperative testing. These results indicate that the fimbria-fornix, but not the amygdala, is involved in nonspatial delayed match-to-sample.

Long-term behavioral changes in rats following organophosphonate exposure.

The organophosphorus compound soman irreversibly inhibits cholinesterase in both the central and peripheral nervous systems. High doses of this compound produce seizures and death in animals. Surviving animals exhibit neural lesions and behavioral abnormalities. The behavioral effects of a single exposure to soman were evaluated in rats injected with 50 micrograms/kg or 85 micrograms/kg soman or with saline. Each rat was tested for either activity in an open field or performance in a 14 choice point multiple T-maze. All rats were then tested for reactivity to tactile stimuli. Some rats exposed to soman showed increased activity in the open field, learning deficits in the Stone maze, and increased reactivity to tactile stimuli, while others showed behavior similar to that of controls. An increase in reactivity was correlated with increased open field activity and with poor performance in the Stone maze. Rats which had received soman and were abnormal in behavioral tests were more likely to have abnormal brain pathology than rats which had received soman and were normal in behavioral tests.

Behavioral consequences of intraperitoneal carbon monoxide administration in rats.

Blood carboxyhemoglobin (HbCO) was determined 15, 30, 45, 60, 90, and 120 min following ip injection of 2.5, 5, 10, 20, and 40 ml pure carbon monoxide (CO)/kg body wt in rats. These CO doses produced HbCO concentrations of 12, 24, 35, 45, and 60%, respectively, at 30 min postinjection. Once these normative data were obtained, a group of eight naive rats were trained to produce a rapid sequence of responses in a stimulus-tracking task, then they were exposed to each of the doses of CO. The 10-ml/kg dose produced a slight decrement in performance, the 20-ml/kg dose reduced correct responses by nearly half, and the 40-ml/kg dose resulted in virtually complete cessation of responding. CO exposure resulted in longer pauses in responding with increasing dose, but the distribution of errors produced in the stimulus-tracking task remained relatively parallel across the range of CO exposures. Thus CO exposure impaired tracking performance but had relatively little effect on the pattern of errors rats produced during the stimulus-tracking test. Taken together these results contradict previous reports purporting to show that CO by ip administration has no behavioral effects; instead, the results indicate that CO administration via the ip route has very similar effects to inhaled CO on behavior.