Drug discrimination learning in lead-exposed rats.

Lead acetate (0.02 or 0.5 percent) was administered to dams throughout the lactation period with half of the litters continuing on lead after weaning. Drug thresholds for d-amphetamine were determined by using the drug-discrimination learning paradigm. All the offspring that had been exposed to lead were less sensitive to the stimulus properties of d-amphetamine irrespective of whether or not they had continued on lead after weaning.

The role of specimen banking in risk assessment.

The risk assessment process is described with a focus on the hazard identification and dose-response components. Many of the scientific questions and uncertainties associated with these components are discussed, and the role for biomarkers and specimen banking in supporting these activities are assessed. Under hazard identification, the use of biomarkers in defining and predicting a) biologically adverse events; b) the progression of those events towards disease; and c) the potential for reversibility are explored. Biomarker applications to address high-to-low dose extrapolation and interindividual variability are covered under dose-response assessment. Several potential applications for specimen banking are proposed.

Reproductive toxicology of disinfection by-products.

The chronic exposure of large segments of the population to disinfected drinking water has necessitated an evaluation of the health effects of the by-products of the chlorination process. This paper reviews the available information concerning the reproductive consequences associated with exposure to disinfection by-products. Four groups of compounds are discussed: the trihalomethanes, in particular chloroform; the chlorinated phenols; chlorinated humic substances; and the haloacetonitriles. In the pregnant female, chloroform and the 2- and 2,4-chlorophenols produced low levels of embryo- and fetotoxicity. Chloroform induced terata when administered by inhalation. The chlorinated humic substances and 2,4,6-trichlorophenol were without significant reproductive effects. The haloacetonitriles showed in utero toxicity, becoming more severe with increasing halogen substitution.

Spermatotoxicity associated with acute and subchronic ethoxyethanol treatment.

Investigations of the male reproductive toxicity of ethoxyethanol (ethylene glycol monoethyl ether) have been restricted exclusively to histopathological assessments of the testes. The present study consisted of two experiments designed to document the spermatotoxicity of ethoxyethanol (EE) as reflected in evaluations of ejaculated rat semen. The basic strategy involved the evaluation of individual ejaculates recovered from the genital tract of a female rat prior to exposure of the males to EE. Repeated assessments of the ejaculate were made during the experimental phase according to specified protocols. Adult Long-Evans hooded male rats received 0, 936, 1872 or 2808 mg/kg (PO) of EE for five consecutive days. Semen evaluations were then conducted at weeks 0, 1, 4, 7, 10 and 14 after exposure. Males in the highest two dose groups showed declines in sperm counts by week 4 and were essentially azoospermic by week 7. At this time, males receiving the lowest dose of EE also exhibited decreases in ejaculated sperm counts. An increase in abnormal sperm shapes was also observed. Over the ensuing weeks all males exhibited varying degrees of recovery as reflected by increasing ejaculated sperm counts. In a second experiment, males were treated with 0 or 936 mg/kg of EE daily (5 days/week) for 6 weeks with semen evaluations conducted weekly. By week 5, sperm counts were significantly depressed and there was an increase in the number of abnormal shapes. Sperm motility was depressed by week 6. The temporal trends seen in these experiments suggested a differential sensitivity of the spermatocyte stage to EE toxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

Statistical issues in risk assessment of reproductive outcomes with chemical mixtures.

Establishing the relationship between a given chemical exposure and human reproductive health risk is complicated by exposures or other concomitant factors that may vary from pregnancy to pregnancy. Moreover, when exposures are to complex mixtures of chemicals, varying with time in number of components, doses of individual components, and constancy of exposure, the picture becomes even more complicated. A pilot study of risk of adverse reproductive outcomes among male wastewater treatment workers and their wives is described here. The wives of 231 workers were interviewed to evaluate retrospectively the outcomes of spontaneous early fetal loss and infertility. In addition, 87 workers participated in a cross-sectional evaluation of sperm/semen parameters. Due to the ever-changing nature of the exposure and the lack of quantification of specific exposures, six dichotomous variables were used for each specific job description to give a surrogate measure of exposure. Hence, no quantitative exposure-response relationships could be modeled. These six variables were independently assigned by two environmental hygienists, and their interrater reliability was assessed. Results are presented and further innovations in statistical methodology are proposed for further applications.

Human tissue monitoring and specimen banking: opportunities for exposure assessment, risk assessment, and epidemiologic research.

A symposium on Human Tissue Monitoring and Specimen Banking: Opportunities for Exposure Assessment, Risk Assessment, and Epidemiologic Research was held from 30 March to 1 April 1993 in Research Triangle Park, North Carolina. There were 117 registered participants from 18 states and 5 foreign countries. The first 2 days featured 21 invited speakers from the U.S. Environmental Protection Agency, the Centers for Disease Control and Prevention, the National Institute of Environmental Health Sciences, various other government agencies, and universities in the United States, Canada, Germany, and Norway. The speakers provided a state-of-the-art overview of human exposure assessment techniques (especially applications of biological markers) and their relevance to human tissue specimen banking. Issues relevant to large-scale specimen banking were discussed, including program design, sample design, data collection, tissue collection, and ethical ramifications. The final group of presentations concerned practical experiences of major specimen banking and human tissue monitoring programs in the United States and Europe. The symposium addressed the utility and research opportunities afforded by specimen banking programs for future research needs in the areas of human exposure assessment, risk assessment, and environmental epidemiology. The third day of the symposium consisted of a small workshop convened to discuss and develop recommendations to the U.S. Environmental Protection Agency regarding applications and utility of large-scale specimen banking, biological monitoring, and biological markers for risk assessment activities.

The automated analysis of rat sperm motility following subchronic epichlorohydrin administration: methodologic and statistical considerations.

The automated analysis of sperm motion endpoints is potentially useful in identifying male reproductive toxicants and ultimately in predicting fertility in humans. The present study was designed to evaluate the automated analysis of rat sperm motility characteristics following subchronic administration of epichlorohydrin. This type of validation is a prerequisite for inclusion of sperm motion measurements in the process of reproductive risk assessment. In the present studies videotapes were made of cauda epididymal spermatozoa from Long-Evans rats, both untreated and treated with epichlorohydrin. From analysis of videotapes of control epididymal spermatozoa, the relationship of various sperm motion endpoints and settings of the CellSoft computer-assisted sperm motion analysis system (Cryo Resources, Ltd., New York, NY) is described. Optimal settings of the system for analysis of rat spermatozoa are detailed. Employing data from both control and epichlorohydrin-treated animals, a statistical methodology is described that evaluates: (1) the distributions of CellSoft generated sperm motion endpoints, (2) the correlations between these endpoints, and (3) techniques for detection of dose-related effects.

Correlation of sperm motion parameters with fertility in rats treated subchronically with epichlorohydrin.

We investigated the relationship between fertility and sperm motin endpoints in rats treated subchronically with the male reproductive toxicant, epichlorohydrin (ECH). Male rats were given ECH orally for 23 days at dosages of 0, 6.25, 12.5, or 25 mg/kg/day. They were mated twice (at 19 and 22 days) to estimate fertility by (1) detection of fertilized ova (presence of sperm head and tail or two pronuclei) 18 hours after mating and by (2) counting implants on day 14 of gestation. Both indices showed dose-related reductions (P less than 0.001). Motion parameters of cauda epididymal sperm were assessed using the CellSoft computer-assisted sperm motion analysis (CASA) system after the rats were asphyxiated on day 25. Curvilinear velocity, straight-line velocity, linearity, and amplitude of lateral head displacement were reduced in a dose-related manner. The fertility indices, percent fertilized ova, and percent implantation on day 14 of gestation were correlated significantly (r = 0.68; P = 0.0001). The following motion parameters were also correlated significantly with fertility (P less than 0.0003; r1 = percent fertilized ova and r2 = percent implantation): linearity (r1 = 0.42; r2 = 0.40), amplitude of lateral head displacement (r1 = 0.54; r2 = 0.48), curvilinear velocity (r1 = 0.53; r2 = 0.50), straight-line velocity (r1 = 0.55; r2 = 0.50), and percent motile sperm (r1 = 0.42; r2 = 0.32). These results suggest a relationship between toxicant-induced reductions in sperm motion and fertility.

The effects of carbon disulfide on the reproductive system of the male rat.

Two experimental protocols were employed to determine the effects of carbon disulfide (CS2) on the reproductive system of the male rat. In the first experiment, adult Long Evans hooded rats were exposed to 0, 350 or 600 ppm CS2 vapor for 10 weeks (5 h/day, 5 days/week). CS2 exposure caused no change in reproductive organ weights nor in plasma gonadotropin levels. However, animals exposed to 600 ppm CS2 had slightly lower epididymal sperm counts and significantly reduced plasma testosterone levels. In order to determine if monitoring hormone levels and sperm status in the same male over time might increase the sensitivity of detecting a toxic reaction, the second protocol was employed. Male rats were exposed to 0 or 600 ppm CS2. After 0, 1, 4, 7 and 10 weeks of exposure, males were observed for mating behavior, and ejaculated sperm count and plasma hormone levels were determined. Animals exposed to 600 ppm CS2 had significantly shorter times to mount and to ejaculate and decreased ejaculated sperm counts. Plasma gonadotropin levels were similar in both groups while plasma testosterone levels were marginally depressed in CS2-exposed animals in the early weeks. These data indicate that CS2 is a toxin of the male reproductive system resulting in abnormal coital behavior and decreased sperm counts. The second experimental protocol proves to be a sensitive method for assessing adverse effects in the male reproductive system.

Factors associated with reduced fertility and implantation rates in females mated to acrylamide-treated rats.

A series of studies was conducted to examine the role of copulatory dysfunction, spermatotoxicity, and/or impaired fertilization in the reduced rates of fertility and implantation observed in females mated to acrylamide-treated male rats. In initial experiments, males were gavaged with 0, 5, 15, 30, 45, or 60 mg/kg acrylamide (ACR) for 5 days and then mated serially to naive females. ACR treatment reduced fertility and increased pre- and post-implantation loss, primarily over the first 3 weeks post-treatment. The effects at Week 1 appeared to result from an interference in sperm transport as demonstrated by the absence of sperm in the uteri of females following a single ejaculation by ACR-treated male rats. The effect however was transient, with recovery of fertility in all but the 60 mg/kg group by Week 2. Attempts to explain the reduced rate of implantation concentrated on characterizing changes in measures of ejaculated sperm count and various motility parameters and evaluating sperm fertilizing ability. Males were again dosed acutely with ACR (p.o.). ACR produced statistically significant, but modest, alterations in sperm motility at Week 3. More prominent was the marked decrease in the number of fertilized ova recovered from females mated to ACR-treated males at Week 3. These data suggest that events critical to the fertilizing ability of the sperm appear to play a major role in the reduced reproductive competence associated with ACR treatment in male rats.

Research to strengthen the scientific basis for health risk assessment: a survey of the context and rationale for mechanistically based methods and models.

Assessment of health risks is an integral part of regulatory decision-making that occurs at the interface between science (e.g. facts) and policy (e.g. values). Because existing scientific knowledge and understanding are often inadequate to answer the most critical risk-related questions, regulatory agencies have developed sets of formalized 'science policies' to extrapolate from existing data to real-life events and situations. These science policies, as, for example, the use of default assumptions or exposure scenarios, can introduce significant uncertainties into the final risk estimate. We survey the rationale for research to reduce extrapolation-related uncertainties, focusing specifically on the need to develop mechanistically based methods and models, including test methods to identify and characterize health effects, integrated human exposure models, physiologically based pharmacokinetic (PBPK) models and biologically based dose-response (BBDR) models.

Effects of trichloroethylene exposure on male reproductive function in rats.

The present study was designed to evaluate the influences of trichloroethylene (TCE) on the reproductive system of male rats. In addition, information was obtained on the distribution and metabolism of TCE. At 100 days of age, male rats were allowed to copulate with ovariectomized, hormonally primed females and copulatory behaviors scored. Fifteen minutes post-ejaculation, females were sacrificed and ejaculate and semen plug recovered from the uterus and vagina for evaluation. These data served as a pre-exposure baseline for each animal. TCE exposure was then initiated with animals intubated with either 0, 10, 100, or 1000 mg/kg of TCE (10 males/group) for 5 days/week for 6 weeks. Copulatory behaviors and semen evaluations were conducted at Weeks 1 and 5 as well as 4 weeks post-exposure. Three males/group were sacrificed at the end of the sixth week of exposure and levels of TCE and its metabolites measured in various organs and blood. The remaining animals were sacrificed at the end of Week 10. TCE-related effects were seen primarily in the 1000 mg/kg group as reduced body weight gain, elevated liver/body weight ratios, and impaired copulatory behavior. However, the copulatory performance of the "affected" males had returned to normal by the fifth week of exposure. Although TCE and its metabolites concentrated to a significant extent in the male reproductive organs, semen evaluations failed to reveal any indices of spermatotoxicity. The initial alterations in copulatory behavior may be attributed to the narcotic properties of TCE. Tolerance to this pharmacological effect may explain the absence of these effects by the fifth week of exposure.

Developmental toxicity of halogenated acetonitriles: drinking water by-products of chlorine disinfection.

The developmental toxicity of acetonitrile and 5 halogenated derivatives was examined with an in vivo teratology screen adapted for use in the Long-Evans rat. The screen was extended to an evaluation of growth till postnatal Days 41-42, and weight of several organs at sacrifice. Acetonitrile was without developmental effects even at doses toxic to the dam. Of the halogenated compounds, treatment with trichloroacetonitrile (TCAN) and dichloroacetonitrile (DCAN) resulted in reduced fertility and increased early implantation failure. There was no effect on litter size in females bearing live litters, but pup birth weight was reduced in all litters exposed to halogenated compounds. Perinatal survival of the pups was adversely impacted by DCAN and TCAN. Postnatal growth till Day 4 was reduced by DCAN and bromochloroacetonitrile (BCAN) while growth till Day 42 was consistently affected only by TCAN. Some general observations were made on the usefulness of the criteria used in the screen, and TCAN, the most toxic of the halogenated compounds, was selected for further in-depth evaluation.

Toxicological considerations in the assessment of lead exposure.

Many uncertainties remain today concerning effects of lead on the nervous system, particularly in regard to levels of lead exposure causing minimal brain dysfunction in children. Past studies in children have failed to define adequately the nature of the effect, the critical period and duration of exposure and the role of other factors in rendering the developing organism susceptible to lead. Prospective studies now underway should contribute greatly to the resolution of these issues. The studies propose to monitor both lead exposure and behavior at fixed intervals from birth for up to five years. The index of exposure in all studies is blood lead concentration. Test batteries vary considerably among studies, but most propose to include the Bayley Scales and the McCarthy Scales. These design features should greatly facilitate comparison of results across the studies. In the design of animal studies certain standardized features across studies also seem desirable, particularly concerning the characterization of lead exposure. Repeat measures of blood lead concentration in rats receiving lead via dams' milk and via water after weaning indicate that the response of individual animals is extremely variable, both as to the pattern of lead build-up and as to the degree of exposure. It is to be expected that biological responses would vary accordingly. It also follows that dose-response and dose-effect relationships in animal studies should be analyzed taking into account this problem. Data analysis should consider the dose and response of each animal individually. In the design of dosage regimens the profile of blood lead levels in adults and young children reported in the literature are useful guidelines in the design of exposure regimens. Many studies are concerned with non-behavioral aspects of lead toxicity e.g. disposition of neurotransmitters, morphological features of brain maturation and alterations in blood-brain barrier function. It would be extremely useful to relate the findings to some fairly standardized behavioral endpoint as well as to well-defined blood lead profiles.

Fertility of workers chronically exposed to chemically contaminated sewer wastes.

Few studies have investigated the reproductive effects of exposure to chemical mixtures. The purpose of this study was to assess fertility in males exposed to mixed industrial and domestic wastes. A detailed reproductive history was obtained from the wives of 231 employees in order to evaluate fertility. Daily work records were used to define exposure status. To ascertain problems of infertility, the ratios of observed live births to expected live births (generated from U.S. birth probabilities) for exposed and nonexposed groups were calculated, and the ratios of these Standardized Fertility ratios (SRFs) were compared. Other analyses considered the couples' contraceptive history and preexposure versus postexposure experience. Though multiple statistical approaches were used to examine the data, the conclusion of this study was that exposure to chemical mixtures was not associated with a decrease in the couples' fertility.

Dominant lethal effects of subchronic acrylamide administration in the male Long-Evans rat.

Acrylamide, a widely used vinyl monomer, is well known as a neurotoxin but inactive as a mutagen in bacterial test systems. The experiments reported demonstrate that after subchronic oral dosing in the male rat, acrylamide induced significant elevations in both pre- and post-implantation loss following dominant lethal testing. These effects were seen at doses which failed to produce clinical or pathological evidence of neurotoxicity. In an accompanying cytogenetic study, no increase in chromosome aberrations was observed in spermatogonia or spermatocytes of treated animals. When spermatocytes from treated spermatogonial stem cells were analyzed, reciprocal translocations (4) were observed in the treated animals and not in the control, but the significance of this result still needs to be established.

Evoked potential alterations in methylmercury chloride toxicity.

The present study was designed to assess the sensitivity of evoked potential techniques to detect alterations in offspring exposed to methylmercury chloride (MMC) at different developmental periods. Recordings were obtained from the visual cortex (VEP) and lateral geniculate (LGP) in 30-day old offspring. Results revealed decreased VEP latencies for peaks N1, P1, and P2 in offspring from mothers exposed either during gestation, or nursing and in offspring exposed directly to MMC for 9 days after weaning. Although not significant, a similar trend was observed in the LPG. It is suggested that the decreased latencies may be a result of compressed brain development.

The effects of developmental and/or direct lead exposure on FR behavior in the rat.

In the present study, 21-day old female rats were exposed to daily doeses of 750 mg/kg of lead acetate via restricted water intake regimen for 70-80 days prior to mating. Treatment was then continued throughout gesation and nursing. At weaning, litters from half of the treated and control mothers were placed on treatment for the remainder of the experiment. This manipulation yielded four groups for testing: Group Pb/Pb, developmental and direct, postweaning exposure; Group Pb/C, developmental exposure only; group C/Pb, direct exposure only; and Group C/C, no exposure to lead acetate treatment. Beginning at 42--49 days of age, offspring were shaped to bar press on a Fixed Ratio 20 (FR20) schedule of reinforcement and then received 20 sessions each 20 min in length. Analyses revealed that Group Pb/Pb received significantly fewer reinforcements/min across sessions that the other three groups and also took significantly longer to emit each 20 response block. Contrary to previous reports in the literature, it is suggested that rats may not be impervious to postweaning lead exposure, particularly when there is a history of developmental exposure.

Influence of parental lead exposure on subsequent learning ability of offspring.

This study was designed to assess the learning ability of rat offspring following the exposure of one or both parents to lead acetate (Pb) from 30-90 days of age. At that time, parents were mated to yield four groups: Group Pb-Pb, both parents had received Pb; Group Pb-N, only the mother had received Pb; Group N-Pb, only the father had received Pb; Group N-N, the control parents. Mothers were continued on their respective treatments throughout gestation and nursing. Testing of offspring began at 30 days of age, employing a black-white discrimination water T maze. Analysis of results revealed that the three Pb groups made more errors than the controls, but did not differ from one another. However, offspring in Group Pb-Pb had longer swimming times than those in Groups Pb-N and N-Pb, who, in turn, had longer swimming times than Group N-N. Thus dual parental exposure was more severe than single parental exposure, which, however, still exerted a detrimental effect compared to control performance.

Effects of methaqualone on learning: a failure to observe tolerance.

The study was designed to assess the effects of Methaqualone (MTQ) on learning, as well as the effects of 15 days of pretask drug exposure on performance. Forty CFE rats were randomly divided into four groups: Group I received daily IP injections of MTQ for 15 days prior to and throughout the duration of the task; Group II served as a control for Group I, receiving injections of the carrier vehicle; Group III received MTO at the start and throughout the duration of the task; Group IV served as the control for Group III. The task consisted of two pretraining days of swimming down a straight, all-white alleyway, followed by 5 trials/day for 4 days on a black-white discrimination task in a water T maze, with the white side being reinforced for all animals. Analyses revealed that MTQ animals made significantly more errors than controls. Pretask drug exposure did not significantly alter Group I performance versus Group III. Furthermore, continued injections for half of the animals in Group I and II for eight days following the last learning day did not improve Group I performance when retested on the same task. Thus MTQ adversely affected learning with prolonged drug exposure failing to reverse such debilitation.