Episodic ozone exposure in adult and senescent Brown Norway rats: acute and delayed effect on heart rate, core temperature and motor activity.

Setting exposure standards for environmental pollutants may consider the aged as a susceptible population but the few published studies assessing susceptibility of the aged to air pollutants are inconsistent. Episodic ozone (O3) is more reflective of potential exposures occurring in human populations and could be more harmful to the aged. This study used radiotelemetry to monitor heart rate (HR), core temperature (T(c)) and motor activity (MA) in adult (9-12 months) and senescent (20-24 months) male, Brown Norway rats exposed to episodic O3 (6 h/day of 1 ppm O3 for 2 consecutive days/week for 13 weeks). Acute O3 initially led to marked drops in HR and T(c). As exposures progressed each week, there was diminution in the hypothermic and bradycardic effects of O3. Senescent rats were less affected than adults. Acute responses were exacerbated on the second day of O3 exposure with adults exhibiting greater sensitivity. During recovery following 2 d of O3, adult and senescent rats exhibited an elevated T(c) and HR during the day but not at night, an effect that persisted for at least 48 h after O3 exposure. MA was elevated in adults but not senescent rats during recovery from O3. Overall, acute effects of O3, including reductions in HR and T(c), were attenuated in senescent rats. Autonomic responses during recovery, included an elevation in T(c) with a pattern akin to that of a fever and rise in HR that were independent of age. An attenuated inflammatory response to O3 in senescent rats may explain the relatively heightened physiological response to O3 in younger rats.

Ozone induces glucose intolerance and systemic metabolic effects in young and aged Brown Norway rats.

Air pollutants have been associated with increased diabetes in humans. We hypothesized that ozone would impair glucose homeostasis by altering insulin signaling and/or endoplasmic reticular (ER) stress in young and aged rats. One, 4, 12, and 24 month old Brown Norway (BN) rats were exposed to air or ozone, 0.25 or 1.0 ppm, 6 h/day for 2 days (acute) or 2 d/week for 13 weeks (subchronic). Additionally, 4 month old rats were exposed to air or 1.0 ppm ozone, 6 h/day for 1 or 2 days (time-course). Glucose tolerance tests (GTT) were performed immediately after exposure. Serum and tissue biomarkers were analyzed 18 h after final ozone for acute and subchronic studies, and immediately after each day of exposure in the time-course study. Age-related glucose intolerance and increases in metabolic biomarkers were apparent at baseline. Acute ozone caused hyperglycemia and glucose intolerance in rats of all ages. Ozone-induced glucose intolerance was reduced in rats exposed for 13 weeks. Acute, but not subchronic ozone increased α2-macroglobulin, adiponectin and osteopontin. Time-course analysis indicated glucose intolerance at days 1 and 2 (2>1), and a recovery 18 h post ozone. Leptin increased day 1 and epinephrine at all times after ozone. Ozone tended to decrease phosphorylated insulin receptor substrate-1 in liver and adipose tissues. ER stress appeared to be the consequence of ozone induced acute metabolic impairment since transcriptional markers of ER stress increased only after 2 days of ozone. In conclusion, acute ozone exposure induces marked systemic metabolic impairments in BN rats of all ages, likely through sympathetic stimulation.

Susceptibility of adult and senescent Brown Norway rats to repeated ozone exposure: an assessment of behavior, serum biochemistry and cardiopulmonary function.

Ozone (O3) is a pervasive air pollutant that produces pulmonary and cardiovascular dysfunction and possible neurological dysfunction. Young and old individuals are recognized as being susceptible to O3; however, remarkably little is known about susceptibility with senescence. This study explored the pulmonary, cardiovascular and neurological effects of O3 exposure in adult (4 m) and senescent (20 m) Brown Norway rats exposed to 0 or 0.8 ppm O3 for 6 h, 1 d/week, for 17 weeks. Ventilatory function was assessed 1 and 7 d after each exposure (Buxco). Heart rate, blood pressure (tail cuff) and motor activity were measured biweekly. Blood, aorta and bronchoalveolar lavage fluid (BALF) were analyzed 24 h after the last exposure for pulmonary inflammation, serum biomarkers and aorta mRNA markers of vascular disease. Measures of normal ventilatory function declined following each O3 exposure in both adult and senescent rats, however, senescent rats took weeks to exhibit a decline. Evidence for residual respiratory effects of O3 7 d after exposure in both age groups was observed. O3 had no effect on either heart rate or blood pressure, but decreased motor activity in both age groups. BALF indicated mild neutrophilic inflammation and protein leakage in adults. Age affected 17/58 serum analytes, O3 affected 6/58; 2/58 showed an age-O3 interaction. Leptin, adiponectin, lipocalin and insulin were increased in senescent rats. Overall, adult rats exhibited more immediate effects of episodic O3 than senescent rats. Residual effects were, however, obtained in both ages of rat, especially for ventilatory endpoints.

Acute administration of dopaminergic drugs has differential effects on locomotion in larval zebrafish.

Altered dopaminergic signaling causes behavioral changes in mammals. In general, dopaminergic receptor agonists increase locomotor activity, while antagonists decrease locomotor activity. In order to determine if zebrafish (a model organism becoming popular in pharmacology and toxicology) respond similarly, the acute effects of drugs known to target dopaminergic receptors in mammals were assessed in zebrafish larvae. Larvae were maintained in 96-well microtiter plates (1 larva/well). Non-lethal concentrations (0.2-50 µM) of dopaminergic agonists (apomorphine, SKF-38393, and quinpirole) and antagonists (butaclamol, SCH-23390, and haloperidol) were administered at 6 days post-fertilization (dpf). An initial experiment identified the time of peak effect of each drug (20-260 min post-dosing, depending on the drug). Locomotor activity was then assessed for 70 min in alternating light and dark at the time of peak effect for each drug to delineate dose-dependent effects. All drugs altered larval locomotion in a dose-dependent manner. Both the D1- and D2-like selective agonists (SKF-38393 and quinpirole, respectively) increased activity, while the selective antagonists (SCH-23390 and haloperidol, respectively) decreased activity. Both selective antagonists also blunted the response of the larvae to changes in lighting conditions at higher doses. The nonselective drugs had biphasic effects on locomotor activity: apomorphine increased activity at the low dose and at high doses, while butaclamol increased activity at low to intermediate doses, and decreased activity at high doses. This study demonstrates that (1) larval zebrafish locomotion can be altered by dopamine receptor agonists and antagonists, (2) receptor agonists and antagonists generally have opposite effects, and (3) drugs that target dopaminergic receptors in mammals appear, in general, to elicit similar locomotor responses in zebrafish larvae.

Toluene effects on the motor activity of adolescent, young-adult, middle-age and senescent male Brown Norway rats.

Life stage is an important risk factor for toxicity. Children and aging adults, for example, are more susceptible to certain chemicals than are young adults. In comparison to children, relatively little is known about susceptibility in older adults. Additionally, few studies have compared toxicant susceptibility across a broad range of life stages. Results are presented for behavioral evaluations of male Brown Norway rats obtained as adolescents (1 month), or young (4 months), middle-age (12 months) and senescent (24 months) adults. Motor activity was evaluated in photocell devices during 30-min sessions. Age-related baseline characteristics and sensitivity to toluene (0, 300, 650, or 1000mg/kg, p.o.) were determined. In Experiment 1, young-adult, middle-age and senescent rats were treated with corn-oil vehicle before five weekly test sessions. Baselines of horizontal and vertical activity decreased with age, but each age-group's averages remained stable across weeks of testing. Baseline activity of older rats was more variable than that of the young adults; older rats were also more variable individually from week to week. Toluene (1000mg/kg) increased horizontal activity proportionately more in senescent rats (ca. 300% of control) than in middle-age or young-adult rats (ca.145-175% of control). Experiment 2 established toluene dose-effect functions in individual adolescent, young-adult, middle-age and senescent rats; each rat received all treatments, counterbalanced across four weekly sessions. Toluene produced dose-related increases in horizontal activity that increased proportionately with age. Experiment 3 replicated the effects of toluene (1000mg/kg) in Experiment 1, showing that toluene-induced increases in horizontal activity were greatest in the oldest rats. Collectively, the results show that aging increased susceptibility to toluene and also increased variability in toluene response. Given the rapid growth of the aged population, further research is needed on aging-related susceptibility to environmental contaminants.

Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables.

The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental toxicity. We are exploring methods to detect developmentally neurotoxic chemicals using zebrafish behavior at 6 days of age. The behavioral paradigm simultaneously tests individual larval zebrafish under both light and dark conditions in a 96-well plate using a video tracking system. We have found that many variables affect the level or pattern of locomotor activity, including age of the larvae, size of the well, and the presence of malformations. Some other variables, however, do not appear to affect larval behavior including type of rearing solution (10% Hank's vs. 1:3 Danieau vs 60 mg/kg Instant Ocean vs 1× and 1:10× EPA Moderately Hard Water). Zebrafish larval behavior using a microtiter plate format may be an ideal endpoint for screening developmentally neurotoxic chemicals, but it is imperative that many test variables be carefully specified and controlled.

Rearing conditions differentially affect the locomotor behavior of larval zebrafish, but not their response to valproate-induced developmental neurotoxicity.

Zebrafish (Danio rerio) are widely used in developmental research, but still not much is known about the role of the environment in their development. Zebrafish are a highly social organism; thus exposure to, or isolation from, social environments may have profound developmental effects. Details of rearing conditions are often sparse in the zebrafish literature. This study compared (1) the activity of larval zebrafish that were raised individually vs in groups, and (2) the effect of the developmental neurotoxicant valproate. We randomly assigned embryos to single- or group-reared social environments from 0 to 5days post fertilization (dpf), while treating them with or without valproate (final concentration 48µM) from 0 to 2dpf, resulting in a total of four groups (group control, group treated, single control, single treated). At 5dpf all embryos were transferred to singly-housed environments where they remained for locomotor activity testing (alternating periods of light and dark) conducted on day 6. Larvae that had been reared in groups had higher levels of activity in the dark period compared to larvae that had been raised individually. Valproate increased activity in both the singly-reared and group-reared larvae during periods of darkness but not light. Further analyses of dark activity indicated that rearing condition did not differentially affect larval responses to valproate. These results indicate that rearing conditions affected the locomotion of zebrafish larvae, but did not alter the effect of the developmental neurotoxicant valproate.

Acute neuroactive drug exposures alter locomotor activity in larval zebrafish.

As part of the development of a rapid in vivo screen for prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae by assessing the acute effects of prototypic drugs that act on the central nervous system. Initially, we chose ethanol, d-amphetamine, and cocaine, which are known, in mammals, to increase locomotion at low doses and decrease locomotion at higher doses. Wild-type larvae were individually maintained in 96-well microtiter plates at 26 degrees C, under a 14:10 h light:dark cycle, with lights on at 0830 h. At 6 days post-fertilization, ethanol (1-4% v/v), d-amphetamine sulfate (0.1-20.0 microM) or cocaine hydrochloride (0.2-50.0 microM) were administered to the larvae by immersion. Beginning 20 min into the exposure, locomotion was assessed for each animal for 70 min using 10-minute, alternating light (visible light) and dark (infrared light) periods. Low concentrations of ethanol and d-amphetamine increased activity, while higher concentrations of all three drugs decreased activity. Because ethanol effects occurred predominately during the light periods, whereas the d-amphetamine and cocaine effects occurred during the dark periods, alternating lighting conditions proved to be advantageous. These results indicate that zebrafish larvae are sensitive to neuroactive drugs, and their locomotor response is similar to that of mammals.

The dynamics of successive induction in larval zebrafish.

Charles Sherrington identified the properties of the synapse by purely behavioral means-the study of reflexes-more than 100 years ago. They were subsequently confirmed neurophysiologically. Studying reflex interaction, he also showed that activating one reflex often facilitates another, antagonistic one: successive induction, which has since been demonstrated in a wide range of species, from aphids to locusts to dogs and humans. We show a particularly orderly example in zebrafish (Danio rerio) larvae; the behavior (locomotion) of larvae is low in dark and intermediate in light, but low in light and substantially higher in dark when dark followed light. A quantitative model of a simple dynamic process is described that readily captures the behavior pattern and the effects of a number of manipulations of lighting conditions.

Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol.

The increasing use of zebrafish (Danio rerio) in developmental research highlights the need for a detailed understanding of their behavior. We studied the locomotion of individual zebrafish larva (6 days post-fertilization) in 96-well microtiter plates. Movement was recorded using a video-tracking system. Time of day results indicated locomotion, tested in darkness (infrared), decreased gradually from early morning to a stable level between 13:00 and 15:30 h. All further studies were conducted in early-to-late afternoon and lasted approximately 1 h. Each study also began with a period of darkness to minimize any unintended stimulation caused by transferring the plates to the recording platform. Locomotion in darkness increased initially to a maximum at 4 min, then decreased steadily to a low level by 20 min. Locomotion during light was initially low and then gradually increased to a stable level after 20 min. When 10-min periods of light and dark were alternated, activity was low in light and high in dark; curiously, activity during alternating dark periods was markedly higher than originally obtained during either extended dark or light. Further experiments explored the variables influencing this alternating pattern of activity. Varying the duration of the initial dark period (10-20 min) did not affect subsequent activity in either light or dark. The activity increase on return to dark was, however, greater following 15 min than 5 min of light. Acute ethanol increased activity at 1 and 2% and severely decreased activity at 4%. One-percent ethanol retarded the transition in activity from dark to light, and the habituation of activity in dark, while 2% ethanol increased activity regardless of lighting condition. Collectively, these results show that locomotion in larval zebrafish can be reliably measured in a 96-well microtiter plate format, and is sensitive to time of day, lighting conditions, and ethanol.

Effects of weekly exposure to anatoxin-a and nicotine on operant performance of rats.

This study examined the effects of acute and weekly administration of anatoxin-a and nicotine on operant performance. Anatoxin-a is a potent nicotinic receptor agonist produced by cyanobacteria, which are found in fresh waters throughout the world. Anatoxin-a is a potential human health hazard and has been responsible for numerous deaths of wildlife, livestock and domestic animals. Remarkably little is known, however, about the effects of anatoxin-a on behavior. Nicotine, the psychomotor stimulant in tobacco, has many well-documented behavioral effects, which often diminish (i.e. tolerance develops) when it is given daily. Male Long Evans rats initially were trained to respond under a multiple variable-ratio 30-response variable-interval 60-s (mult VR-30 VI 60-s) schedule of food reinforcement. They were then divided into 12 groups of 8 that received four weekly subcutaneous injections of anatoxin-a (0.05-0.2 mg/kg), nicotine (0.125-1.8 mg/kg), or vehicle 5-min prior to testing. When initially administered, each compound decreased response rates and reinforcement rates in both components of the multiple schedule. Substantial tolerance developed to the disruptive effects of nicotine with weekly administration. Tolerance also developed to the effects of anatoxin-a, although to a lesser degree; the highest dose severely decreased performance with little evidence of recovery. In conjunction with prior findings, these results suggest the behavioral effects of anatoxin-a and nicotine are similar, but not identical, and that relatively infrequent (episodic) administration can produce tolerance.

Effects of acute and weekly episodic exposures to anatoxin-a on the motor activity of rats: comparison with nicotine.

Anatoxin-a is a naturally occurring nicotinic agonist produced by cyanobacterial blooms; exposures are likely to occur episodically when the blooms repeatedly form and dissipate. Tolerance and sensitization to nicotine's effects on the motor activity of rats can occur when administered episodically at weekly intervals. It was therefore of interest to compare the effects of anatoxin-a and nicotine when given weekly. Adult male Long Evans rats were tested daily (M-F) in a photocell device, that recorded both horizontal and vertical motor activity, during 30-min sessions. Anatoxin-a and nicotine were given s.c. once a week for 4 weeks, just prior to a test session. Anatoxin-a was given as the (+) isomer and as the racemate. Dose ranges were: (+)anatoxin-a, 0.075-0.225 mg/kg; (+/-)anatoxin-a, 0.2-0.95 mg/kg; and (-)-nicotine, 0.3-1.8 mg/kg. Each experiment also included a saline-control group. Nicotine initially decreased both horizontal activity and, to a greater extent, vertical activity. Tolerance developed to nicotine's effects with weekly administration. Both forms of anatoxin-a also initially decreased horizontal and vertical activity, and to roughly equivalent degrees. Neither form of anatoxin-a, however, induced tolerance with weekly administration. Thus, anatoxin-a and nicotine can be distinguished by their effects on motor activity with episodic treatment, suggesting different sites of action for the compounds in the nervous system.

Sensitization and tolerance with episodic (weekly) nicotine on motor activity in rats.

Nicotine's effects on motor activity have been studied extensively. Sensitization or tolerance can develop to nicotine's acute effects with daily exposure. Limited data indicate that sensitization can also develop when nicotine is given less frequently than daily. The present experiments were designed to extend this finding and to more fully characterize the effects of nicotine on motor activity when given at weekly intervals. In both experiments, the horizontal and vertical activity of adult female Long-Evans (LE) rats was recorded in photocell chambers. In Experiment 1, either saline or nicotine hydrogen tartrate (0.3, 0.6, 1.2 or 1.8 mg of salt/kg BW, s.c.) was administered once each week to rats that were tested daily (M-F). Acute nicotine administration produced no significant effect on horizontal activity at lower doses, while the highest dose produced a decrease (ca. 30%). Substantial and significant dose-related decreases in vertical activity were also obtained initially. Weekly dosing produced tolerance to nicotine's decreasing effects on vertical activity and increases (i.e., sensitization) in horizontal activity at all doses, and these effects persisted for at least 3 weeks. Experiment 2 partially replicated the results of Experiment 1 and indicated further that small sequential dose variations generally had little influence on nicotine tolerance and sensitization. The present results on horizontal activity extend prior findings of sensitization to weekly nicotine to include a broad range of doses. Results also showed that tolerance, but not sensitization, occurred to nicotine's effects on vertical activity over a comparable dose range. Further research is warranted on the importance of episodic, or recurring intermittent exposures in determining nicotine's effects, and those of other nicotinic agents, on behavior.

Prospects on behavioral studies of marine and freshwater toxins.

While there is a long-standing tradition of using behavioral methods to study the effects of manufactured drugs and environmental chemicals, comparatively little attention has focused until recently on the behavioral effects of marine or freshwater toxins. A vast array of microorganisms, found in a variety of waters, are known to occasionally "bloom" and produce toxins that can cause either blatant toxicity (i.e., lethality) or damage to a number of organ systems. The nervous system is a known target for many of the toxins. Considerable research has in the past been carried out to determine toxin effects on the survivability of laboratory rodents (typically mice) following acute exposures. Newer research has shown, however, prominent toxin-induced alterations in motor, sensory, autonomic and cognitive functions at sublethal exposure concentrations. Future toxin research can capitalize upon a wealth of behavioral paradigms already available in toxicology, pharmacology and neuroscience.

Nicotine effects on the activity of mice exposed prenatally to the nicotinic agonist anatoxin-a.

Anatoxin-a is a nicotinic agonist produced by several genera of cyanobacteria, and has caused numerous deaths of wildlife, livestock and domestic animals world-wide. Several studies in the literature have shown that exposure of mice and rats to nicotine early in development alters its effects when the rodents are subsequently challenged with nicotine. We therefore determined the effect of nicotine on the motor activity of adult mice that had been exposed prenatally to anatoxin-a. Pregnant CD-1 mice received either saline vehicle or one of two doses of (+/-) anatoxin-a (125, 200 microg/kg), i.p., on GD13-17. As adults (8 months), control mice of both genders were used to determine the effect of nicotine (0, 0.1, 0.3, 1.0 or 3.0 mg/kg, s.c.) on motor activity measured for 30-min in a photocell device. Under these conditions, nicotine produced dose-related decreases in both horizontal and vertical activity, with an ED50 estimated to be 0.65 mg/kg. Next, additional control mice and mice exposed prenatally to anatoxin-a received the nicotine ED50 and saline vehicle, in a counterbalanced fashion, with one week separating treatments. Nicotine decreased both horizontal and vertical activity in all mice, regardless of prenatal anatoxin-a treatment. Thus, no enduring effects of prenatal anatoxin-a were obtained in adult mice following nicotine challenge.

Acquisition, steady-state performance, and the effects of trimethyltin on the operant behavior and hippocampal GFAP of Long-Evans and Fischer 344 rats.

Strain differences represent an overlooked variable that may play an important role in neurotoxic outcomes that can impact regulatory decision making. Here, we examined the strain-dependent effects of trimethyltin (TMT), a compound used as a positive control for behavioral and neurochemical assessments of neurotoxicity. Adult male Long-Evans (LE) and Fischer 344 (F344) rats (n=12 each) were trained to respond under a multiple, fixed-interval 3-min fixed-ratio 10-response (multi FI 3-min FR10) schedule of milk reinforcement. Acquisition was characterized by time-dependent changes in several behavioral endpoints in both strains, although rate of acquisition of the fixed-interval pattern of responding was slower in F344 rats. Steady-state (baseline) performance was characterized by slower overall rates of responding in F344 rats. There was little evidence of strain differences in many of the other baseline performance measures. Rats of each strain were then divided into two equal groups that received either 1 ml/kg saline or 8.0 mg/kg iv TMT approximately 18 h before the next test session. TMT produced transient changes in the performance of LE and F344 rats that lasted for several sessions. For many behavioral measures, F344 rats were more affected by TMT than were LE rats. TMT-induced reactive gliosis, as assessed by assaying glial fibrillary acidic protein (GFAP), was also greater in F344 rats than in LE rats. These results suggest F344 rats may be more susceptible to TMT-induced neurotoxicity than are LE rats.

Neurobehavioral evaluations of mixtures of trichloroethylene, heptachlor, and di(2-ethylhexyl)phthalate in a full-factorial design.

One approach to the toxicological evaluation of chemical mixtures is to construct full dose-response curves for each compound in the presence of a range of doses of each of the other compounds, i.e., a factorial design. This study was undertaken as part of an interdisciplinary project to evaluate a mixture of three environmental pollutants. A full-factorial design was undertaken to determine the neurobehavioral consequences of short-term repeated exposure to five dose levels each of three chemicals, in order to characterize potential two- and three-way interactions. Adult female F344 rats received (p.o.) for 10 days either one of five doses of trichloroethylene, di(2-ethylhexyl)phthalate, or heptachlor, or else one of all possible chemical combinations. Neurobehavioral evaluations were conducted using motor activity and an abbreviated functional observational battery. Response-surface analysis was applied to each of the endpoints. Hypotheses were tested based on the estimated model parameters; of primary interest was the overall test for interaction among the three chemicals. In addition, an abbreviated design was created by fitting only a subset of the data to the model. In general, significant overall interactions that deviated from response additivity were detected for most endpoints (11 of 14). All of the interactions on the neurobehavioral endpoints showed either antagonism, or else an interaction that could not be fully characterized. Often the results of the abbreviated dataset analysis were not the same as for the full-factorial design. This study was extremely intensive, in terms of the number of rats and time required for conduct of the study as well as the data analysis. These results underscore the need for more economical approaches to evaluate the toxic effects of mixtures of chemicals.

Schedule-controlled behavior in rats exposed perinatally to the PCB mixture Aroclor 1254.

Exposure to polychlorinated biphenyls (PCBs) has been shown to detrimentally affect learning and memory in children as well as schedule-controlled behavior in experimental animals. The objective of the present series of experiments was to extend research into the effects of PCBs on behavior maintained under both short (30 s) and long (5 min) fixed-interval (FI) schedules as well as an FI 3-min with reinforcement omission. Long-Evans rats were exposed to 0 or 6 mg/kg/day Aroclor 1254 (A1254) via oral gavage from Gestation Day 6 (GD 6) through Postnatal Day 21 (PND 21). At approximately PND 90, acquisition and steady-state performance were assessed under a series of FI reinforcement schedules consisting of FI 30-s, FI 5-min, and FI 3-min with 33% of the scheduled reinforcers omitted. Performance measures included index of curvature (IOC), response rate, and postreinforcement pause (PRP). There were no effects of A1254 on the acquisition of behavior under the FI 30-s schedule. Subsequently, there was an initial decrease in response rate and IOC and an increase in PRP following the transition from FI 30-s to the FI 5-min; there were, however, no treatment-related effects on any measure. During the reinforcement-omission procedure, there was an increase in the rate of responding and a decrease in IOC and PRP following omission intervals irrespective of treatment. These data are inconsistent with previous findings and suggest that perinatal A1254 exposure in the rat does not disrupt temporally organized behavior.

A randomization test-based method for risk assessment in neurotoxicology.

A current trend in risk assessment for systemic toxicity (noncancer) endpoints is to utilize the observable range of the dose-effect curve in order to estimate the likelihood of obtaining effects at lower concentrations. Methods to accomplish this endeavor are typically based on variability in either the effects of fixed doses (benchmark approaches), or on variability in the doses producing a fixed effect (probabilistic or tolerance-distribution approaches). The latter method may be particularly desirable because it can be used to determine variability in the effect of an agent in a population, which is an important goal of risk assessment. This method of analysis, however, has typically been accomplished using dose-effect data from individual subjects, which can be impractical in toxicology. A new method is therefore presented that can use traditional groups-design data to generate a set of dose-effect functions. Population tolerances for a specific effect can then be estimated from these model dose-effect functions. It is based on the randomization test, which assesses the generality of a data set by comparing it to a data set constructed from randomized combinations of single point estimates. The present article describes an iterative line-fitting program that generates such a data set and then uses it to provide risk assessments for two pesticides, triadimefon and carbaryl. The effects of these pesticides were studied on the locomotor activity of laboratory rats, a common neurobehavioral end point. Triadimefon produced dose-dependent increases in activity, while carbaryl produced dose-dependent decreases in activity. Risk figures derived from the empirical distribution of individual dose-effect functions were compared to those from the iterative line-fitting program. The results indicate that the method generates comparable risk figures, although potential limitations are also described.

Episodic exposures to chemicals: what relevance to chemical intolerance?

Episodic exposures refer to intermittent acute exposures to chemicals that ordinarily have a rapid onset and short duration of effect. There has been a long tradition in preclinical behavioral pharmacology of using episodic-exposure paradigms in order to establish dose-response functions in individual organisms. In these experiments, stable baselines of behavior are first established and then followed by administering varying doses of a drug intermittently, for example, once or twice a week. The power of this approach is well established; the within-subjects design reduces error variance, allows exploration of the entire range of effective doses, and can be used to identify individual differences in drug sensitivity. Of course, the approach is only applicable to reversibly acting compounds, and checks need to be included to insure effects of one dose are not influenced by prior exposure to another dose. We have used baseline approaches to evaluate the effects of pesticides and solvents on the behavior of adult male rats and mice. Moreover, a novel probabilistic dose-tolerance analysis applied to the data suggests substantial individual differences in chemical sensitivity, often spanning orders of magnitude. These results suggest that individual differences in chemical sensitivity may be much greater than previously acknowledged.