A novel water delivery system for administering volatile chemicals while minimizing chemical waste in rodent toxicity studies.

Rodent toxicity studies typically use water bottles to administer test chemicals via drinking water. However, water bottles provide inconsistent exposure of volatile chemicals due to varying headspace, and lead to excessive waste of test material. To refine drinking water toxicity studies in rodents by enhancing sample quality and consistency, and minimizing waste, we designed and implemented a novel water delivery system that keeps the water chilled, headspace free and protected from light. Materials used were resistant to chemical interaction. In this gravity-fed system, a 6-L Teflon water bag, stored in a polystyrene cooler on the cage rack, was connected to a stainless steel manifold delivering water to five cages via specialized drinking valves. Due to the absence of headspace in the water bag, this system allows consistent exposure of volatile chemicals. In addition, small diameter tubing throughout the system reduces the amount of test material residing in the system and minimizes chemical waste.

A unifying concept for assessing toxicological interactions: changes in slope.

Robust statistical methods are important to the evaluation of toxicological interactions (i.e., departures from additivity) among chemicals in a mixture. However, different concepts of joint toxic action as applied to the statistical analysis of chemical mixture toxicology data or as used in environmental risk assessment often appear to conflict with one another. A unifying approach for application of statistical methodology in chemical mixture toxicology research is based on consideration of change(s) in slope. If the slope of the dose-response curve of one chemical does not change in the presence of other chemicals, then there is no interaction between the first chemical and the others. Conversely, if the rate of change in the response with respect to dose of the first chemical changes in the presence of the other chemicals, then an interaction is said to exist. This concept of zero interaction is equivalent to the usual approach taken in additivity models in the statistical literature. In these additivity models, the rate of change in the response as a function of the i(th) chemical does not change in the presence of other chemicals in a mixture. It is important to note that Berenbaum's (1985, J. Theor. Biol. 114, 413-431) general and fundamental definition of additivity does not require the chemicals in the mixture to have a common toxic mode of action nor to have similarly shaped dose response curves. We show an algebraic equivalence between these statistical additivity models and the definition of additivity given by Berenbaum.

Neurotoxicological and statistical analyses of a mixture of five organophosphorus pesticides using a ray design.

Environmental exposures generally involve chemical mixtures instead of single chemicals. Statistical models such as the fixed-ratio ray design, wherein the mixing ratio (proportions) of the chemicals is fixed across increasing mixture doses, allows for the detection and characterization of interactions among the chemicals. In this study, we tested for interaction(s) in a mixture of five organophosphorus (OP) pesticides (chlorpyrifos, diazinon, dimethoate, acephate, and malathion). The ratio of the five pesticides (full ray) reflected the relative dietary exposure estimates of the general population as projected by the US EPA Dietary Exposure Evaluation Model (DEEM). A second mixture was tested using the same dose levels of all pesticides, but excluding malathion (reduced ray). The experimental approach first required characterization of dose-response curves for the individual OPs to build a dose-additivity model. A series of behavioral measures were evaluated in adult male Long-Evans rats at the time of peak effect following a single oral dose, and then tissues were collected for measurement of cholinesterase (ChE) activity. Neurochemical (blood and brain cholinesterase [ChE] activity) and behavioral (motor activity, gait score, tail-pinch response score) endpoints were evaluated statistically for evidence of additivity. The additivity model constructed from the single chemical data was used to predict the effects of the pesticide mixture along the full ray (10-450 mg/kg) and the reduced ray (1.75-78.8 mg/kg). The experimental mixture data were also modeled and statistically compared to the additivity models. Analysis of the 5-OP mixture (the full ray) revealed significant deviation from additivity for all endpoints except tail-pinch response. Greater-than-additive responses (synergism) were observed at the lower doses of the 5-OP mixture, which contained non-effective dose levels of each of the components. The predicted effective doses (ED20, ED50) were about half that predicted by additivity, and for brain ChE and motor activity, there was a threshold shift in the dose-response curves. For the brain ChE and motor activity, there was no difference between the full (5-OP mixture) and reduced (4-OP mixture) rays, indicating that malathion did not influence the non-additivity. While the reduced ray for blood ChE showed greater deviation from additivity without malathion in the mixture, the non-additivity observed for the gait score was reversed when malathion was removed. Thus, greater-than-additive interactions were detected for both the full and reduced ray mixtures, and the role of malathion in the interactions varied depending on the endpoint. In all cases, the deviations from additivity occurred at the lower end of the dose-response curves.

A physiologically based pharmacokinetic model for trichloroethylene in the male long-evans rat.

A physiologically based pharmacokinetic (PBPK) model for trichloroethylene (TCE) in the male Long-Evans (LE) rat was needed to aid in evaluation of neurotoxicity data collected in this rodent stock. The purpose of this study was to develop such a model with the greatest possible specificity for the LE rat. The PBPK model consisted of 5 compartments: brain, fat, slowly perfused tissue, rapidly perfused viscera, and liver. Partition coefficients (blood, fat, muscle, brain, liver) were determined for LE rats. The volumes of the brain, liver, and fat compartments were estimated for each rat, with tissue-specific regression equations developed from measurements made in LE rats. Vapor uptake data from LE rats were used for estimation of Vmaxc. As blood flow values for LE rats were not available, values from Sprague-Dawley (SD) and Fischer-344 (F344) rats were used in separate simulations. The resulting values of Vmaxc were used to simulate tissue (blood, liver, brain, fat) TCE concentrations, which were measured during (5, 20, 60 min) and after (60 min of TCE followed by 60 min of air) flow-through inhalation exposures of LE rats to 200, 2000, or 4000 ppm TCE. Simulation of the experimental data was improved by use of F-344 blood-flow values and the corresponding Vmaxc (8.68 mg/h/kg) compared to use of SD flows and the associated Vmaxc (7.34 mg/h/kg). Sensitivity analysis was used to determine those input parameters with the greatest influence on TCE tissue concentrations. Alveolar ventilation consistently (across exposure concentration, exposure duration, and target tissue) had the greatest impact on TCE tissue concentration. The PBPK model described here is being used to explore the relationship between measures of internal dose of TCE and neurotoxic outcome.

A comparison of Haber's rule at different ages using a physiologically based pharmacokinetic (PBPK) model for chloroform in rats.

Haber's rule as commonly interpreted in inhalation toxicology, can be stated as exposure concentration times duration equals a constant biological effect, or C x t=k. In other words, identical products of concentration and duration lead to the same effect. The goals of this paper are to develop a biological and pharmacokinetic modeling approach for chloroform, and to evaluate Haber's rule for different ages by taking into account the physiological changes due to growth and aging in rats. Three-dimensional dose-response surfaces for liver toxicity were generated for each age group of interest: adolescent, adult, and senescent rats. The three-dimensional surfaces were then characterized with a generalized description of Haber's rule for each age group. The simulations suggest that adolescent rats need higher exposure levels in order to achieve similar levels of liver damage compared to adults or senescent rats, if the comparison is made using the same exposure length. In summary, a pharmacokinetic modeling approach with a biological framework including the chemical's mode of action, was used to relate concentration, exposure duration and effect. Major advantages of this approach include: the potential ability to extrapolate to humans, the inclusion of aging in the simulations, and the ability to summarize the results using a generalized form of Haber's rule.

Building social relationships through valued roles: three older adults and the community membership project.

The experiences of three older adults with developmental disabilities highlight ways in which social relationships can be nourished through the establishment of community roles. Members of the Community Membership Project of the Center on Aging and Community used paid community builders working individually with older persons who had developmental disabilities in order to help them obtain positive, valued social roles and relationships with nondisabled community members. As a means to this end, participants were supported to engage in community activities corresponding to the interests and talents discovered in an initial period of exploration. Stories of these community builders' work illustrate the intentional strategies and concerted effort necessary to create community connections and meaningful relationships.

Neurotoxic and pharmacokinetic responses to trichloroethylene as a function of exposure scenario.

Strategies are needed for assessing the risks of exposures to airborne toxicants that vary over concentrations and durations. The goal of this project was to describe the relationship between the concentration and duration of exposure to inhaled trichloroethylene (TCE), a representative volatile organic chemical, tissue dose as predicted by a physiologically based pharmacokinetic model, and neurotoxicity. Three measures of neurotoxicity were studied: hearing loss, signal detection behavior, and visual function. The null hypothesis was that exposure scenarios having an equivalent product of concentration and duration would produce equal toxic effects, according to the classic linear form of Haber's Rule ((italic)C(/italic) times t = k), where C represents the concentration, t, the time (duration) of exposure, and k, a constant toxic effect. All experiments used adult male, Long-Evans rats. Acute and repeated exposure to TCE increased hearing thresholds, and acute exposure to TCE impaired signal detection behavior and visual function. Examination of all three measures of neurotoxicity showed that if Haber's Rule were used to predict outcomes across exposure durations, the risk would be overestimated when extrapolating from shorter to longer duration exposures, and underestimated when extrapolating from longer to shorter duration exposures. For the acute effects of TCE on behavior and visual function, the estimated concentration of TCE in blood at the time of testing correlated well with outcomes, whereas cumulative exposure, measured as the area under the blood TCE concentration curve, did not. We conclude that models incorporating dosimetry can account for differing exposure scenarios and will therefore improve risk assessments over models considering only parameters of external exposure.

A multiple-purpose design approach to the evaluation of risks from mixtures of disinfection by-products.

Drinking water disinfection has effectively eliminated much of the morbidity and mortality associated with waterborne infectious diseases in the United States. Various disinfection processes, however, produce certain types and amounts of disinfection by-products (DBPs), including trihalomethanes (THM), haloacetic acids, haloacetonitriles, and bromate, among others. Human health risks from the ubiquitous exposure to complex mixtures of DBPs are of concern because existing epidemiologic and toxicologic studies suggest the existence of systemic or carcinogenic effects. Researchers from several organizations have developed a multiple-purpose design approach to this problem that combines efficient laboratory experimental designs with statistical models to provide data on critical research issues (e.g., estimation of human health risk from low-level DBP exposures, evaluation of additivity assumptions as useful for risk characterization, estimation of health risks from different drinking water treatment options). A series of THM experiments have been designed to study embryonic development, mortality and cancer in Japanese medaka (Oryzias latipes) and liver and kidney endpoints in female CD-1 mice. The studies are to provide dose-response data for specific mixtures of the 4 THMs, for the single chemicals, and for binary combinations. The dose-levels and mixing ratios for these experiments were selected to be useful for development and refinement of three different statistical methods: testing for departures from dose-additivity; development of an interactions-based hazard index; and use of proportional-response addition as a risk characterization method. Preliminary results suggest that dose-additivity is a reasonable risk assessment assumption for DBPs. The future of mixtures research will depend on such collaborative efforts that maximize the use of resources and focus on issues of high relevance to the risk assessment of human health.

Organ weights and fat volume in rats as a function of strain and age.

The Fischer 344 (F344) rat and the Sprague-Dawley (SD) rat are used commonly to evaluate potential adverse health effects resulting from environmental exposure to chemicals. They are also the most common rat strain/stock used in physiologically based pharmacokinetic (PBPK) modeling. Accurate characterization of model input parameters will improve the usefulness of PBPK model predictions. Thus, organ (i.e., liver, kidneys, spleen, stomach, small intestine, large intestine, heart, lungs, brain) weights and body fat were measured in male SD rats of different ages (4 to 40 wk) and in young (9 to 10 wk) and old (22 to 23 mo) male F344 rats. Comparison of age-matched (9 to 10 wk) F344 and SD rats revealed that the SD rats weighed significantly more and had significantly higher absolute organ weights. These significant differences usually disappeared when organ weights were expressed as a percentage of body weight (relative organ weight). Percent body fat was significantly lower in the age-matched SD rats (6.48%) than in their F344 counterparts (8.67%). As expected, both body weight and absolute organ weights were significantly higher in old than in young F344 rats. However, these differences were largely reversed when relative organ weights were considered, with most relative organ weights significantly lower in the old F344 rats. Body fat as a percentage of body weight was 14.02% in the old F344 rats. When SD rats of various ages were examined, relative organ weights declined between the ages of 4 and 14 wk. In contrast, significant differences in percent body fat were not detected among the SD rats of different ages and weights examined in this study (4 to 40 wk, approximately 75 to approximately 450 g). In summary, values for physiological input parameters are provided that should prove useful in development and implementation of more accurate PBPK models.

NOAEL and LOAEL determinations of acute hepatotoxicity for chloroform and bromodichloromethane delivered in an aqueous vehicle to F344 rats.

Chloroform (CHCl3) and bromodichloromethane (BDCM) are generally the two most prevalent disinfection by-products formed during chlorination of drinking water, and both have been shown to be hepatotoxic, nephrotoxic, and carcinogenic in rodents. As the toxicity of these trihalomethanes (THMs) has most often been studied with corn oil as the vehicle of administration, the objectives of this study were to assess hepatotoxicity after exposure to single, low dosages of CHCl3 and BDCM given orally in an aqueous vehicle to estimate a lowest-observed-adverse-effect level (LOAEL) and a no-observed-adverse-effect level (NOAEL) and to compare toxic potency. Ninety-day-old male Fischer 344 rats were gavaged with either 0.125, 0.1875, 0.25, 0.5, 0.75, 1.0, or 1.5 mmol CHCl3 or BDCM/kg body weight in 10% Alkamuls EL-620 (5 ml/kg body weight). At 24 h postgavage, serum was collected for analysis of clinical chemistry indicators of liver damage. Both CHCl3 and BDCM induced dose-dependent hepatotoxicity; serum alanine aminotransferase, aspartate aminotransferase, and sorbitol dehydrogenase were elevated significantly over control at 1.5, 1.0, and 0.5 mmol/kg. At these dose levels after 24 h, the two THMs appeared to be equipotent hepatotoxicants. Additional assessments at later time points demonstrated that BDCM causes more persistent liver damage than CHCl3 (Lilly et al., 1997). At 0.25, 0. 1875, and 0. 125 mmol of either THM/kg, significant increases over control were not detected for any measured endpoint. Therefore, these data indicate that the acute, oral NOAELs and LOAELs for liver toxicity are 0.25 and 0.5 mmol/kg, respectively, for both CHCl3 and BDCM. These determinations should provide a basis to establish new exposure limits for One-Day Health Advisories for these prevalent THMs.

The hepatic interaction of aliphatic alcohols with halogenated hydrocarbons.

As has been noted, advancement in understanding of chemical interactions requires an integrated approach. Given the large number of binary mixtures of aliphatic alcohols and halogenated hydrocarbons that can be formulated, and because limitations of time and resources make it impossible to test them all, careful thought should be given to selection of pairs for laboratory experimentation. For any given pair of chemicals, the type of interaction (addition, synergism, antagonism, potentiation) should be determined and described by appropriate experimental designs and statistical methodology. This has been done for various alcohol-halocarbon mixtures. Work to expand our understanding of the mechanism(s) underlying the interaction of aliphatic alcohols and halogenated hydrocarbons would be particularly useful, as an improved mechanistic understanding would improve our ability to extrapolate across dose levels (from high laboratory exposure concentrations to typical human environmental exposure concentrations) and across species (from laboratory animals to humans).

Application of physiologically based pharmacokinetic modelling to combination toxicology.

Non-additive toxicity has been demonstrated in laboratory animals for a large number of temporally separated or concurrent multiple chemical exposures. These exposures are typically at concentrations higher than those found in the environment, leading to the question of the applicability of the results to the human situation. Physiologically based pharmacokinetic (PBPK) modelling has been applied successfully to single chemicals; its utility for extrapolation across species and dose has been demonstrated. Use of PBPK modelling in the study of chemical mixtures is increasing although still limited. The use of PBPK modelling by various investigators in the field of combination toxicology is reviewed. PBPK modelling has been used to examine: the role of increased metabolism in non-additive toxicity resulting from temporally separated exposures; the influence of the time interval separating two chemical exposures; and the role of inhibition of metabolism in concurrent exposure to two chemicals. In summary, development of a PBPK or PBPK/pharmacodynamic model for a combined exposure provides a basis for extrapolation across species, route and dose, and a useful tool for risk assessment.

Experimental designs, statistics and interpretation.

The Working Group on Experimental Designs, Statistics and Interpretation considered the use of statistics in combination toxicology, the terminology used to describe the interaction(s) of chemicals, the use of efficient experimental designs to minimize animal use, the diverse interests and goals covered by combination toxicology and approaches useful for complex mixtures. The importance of the use of appropriate experimental designs and statistical methodology was recognized. Given the present lack of consensus on terminology and methodology, it is recommended that investigators provide in their publications the definition of additivity and the mathematical model being used.

Physiologically based pharmacokinetic estimated metabolic constants and hepatotoxicity of carbon tetrachloride after methanol pretreatment in rats.

A single 6-hr exposure to inhaled methanol (MeOH) has been shown to enhance carbon tetrachloride (CCl4) hepatotoxicity. The objective of the present study was to use gas uptake data and the development of a physiologically based pharmacokinetic model (PBPK) to determine in vivo changes in CCl4 metabolism resulting from MeOH pretreatment. Adult male F344 rats (167-197 g) were exposed to 10,000 ppm MeOH (constant concentration) via inhalation for 6 hr. Individual rats were exposed using gas uptake techniques to CCl4 alone or to CCl4 either 24 or 48 hr after initiation of MeOH pretreatment. The following initial concentrations were used for CCl4: 0, 25, 100, 250, and 1000 ppm with exposures lasting 6 hr. Vmax (metabolic rate) was estimated from gas uptake data and Km (Michaelis constant) was assumed constant after methanol pretreatment. For CCl4 alone, Vmax was 0.11 mg/hr (Vmaxc = 0.37 mg/hr/kg) and Km was 1.3 mg/liter. Vmax was 0.48 mg/hr (Vmaxc = 1.6 mg/hr/kg) for the 24-hr MeOH + CCl4 group and Vmax was 0.18 mg/hr (Vmaxc = 0.6 mg/hr/kg) for the 48-hr MeOH + CCl4 group. For CCl4 alone, serum markers of hepatotoxicity alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) were increased significantly only at 1000 ppm CCl4. Both serum markers of hepatotoxicity in the 24-hr MeOH + CCl4 group increased as a function of CCl4 concentration when compared with 0 ppm CCl4 controls. The maximum increase occurred at 1000 ppm CCl4, where ALT and SDH increased by 392- and 286-fold, respectively. At 100, 250, and 1000 ppm CCl4, ALT and SDH values for the 24-hr MeOH + CCl4 groups were significantly increased relative to control (0 ppm CCl4), CCl4 alone, and 48-hr MeOH + CCl4. ALT and SDH levels in the 48-hr MeOH + CCl4 groups were not statistically different from the respective CCl4 alone groups.

Effect of subchronic corn oil gavage on the acute toxicity of orally administered bromodichloromethane.

Bromodichloromethane (BDCM) is a by-product of water chlorination and is the second most common trihalomethane (THM) in finished drinking water. It has been reported that delivery of THMs in corn oil can influence the site and magnitude of toxic and carcinogenic responses in rodents, perhaps by inducing metabolizing enzymes or altering tissue composition. To determine if corn oil influences the acute toxicity of BDCM, adult male F-344 rats were pretreated 5 days/week for 6 weeks with oral doses of corn oil or water at a volume of 5 ml/kg. Following pretreatment, animals were gavaged with a single dose of 0, 200 or 400 mg BDCM/kg in 10% Emulphor. Urine was collected at timed intervals over a 48-h period following BDCM administration. Rats were sacrificed at this time and organs and blood removed. Urine and serum were analyzed for indicators of toxicity. Corn oil pretreatment did not enhance the acute hepato- or nephrotoxicity of BDCM, suggesting that vehicle effects noted in previous THM toxicity and carcinogenicity studies are more likely due to pharmacokinetic differences between administration in corn oil and aqueous gavage vehicles than to altered tissue composition or physiological changes.

Methanol potentiation of carbon tetrachloride hepatotoxicity: the central role of cytochrome P450.

Evidence to explain the enhanced hepatotoxicity of carbon tetrachloride (CCl4) following methanol exposure by inhalation is presented. Hepatic microsomes prepared from male F344 rats exposed to methanol at concentrations up to 10,000 ppm showed increased p-nitrophenol hydroxylase activity but no increase in pentoxyresorufin-O-dealkylase or ethoxyresorufin-O-deethylase activities. Hepatic antioxidant levels, glutathione levels and glutathione-S-transferase activity in methanol-treated animals were not different from controls. In vitro metabolism of CCl4 was also increased in microsomes from methanol-treated animals. Pretreatment with allyl sulfone, a specific chemical inhibitor of cytochrome P450 2E1, abolished the difference in microsomal metabolism between exposed and control animals. This study shows that methanol exposure induces cytochrome P450 2E1, which appears to be the principal toxicokinetic mechanism responsible for the increased metabolism and thus the increased hepatotoxicity of CCl4.

A pharmacokinetic model of anaerobic in vitro carbon tetrachloride metabolism.

Carbon tetrachloride (CCl4) is a potent hepatotoxic agent whose toxicity is mediated through cytochome P450-dependent metabolism. Results from anaerobic in vitro experiments with hepatic microsomes isolated from male F-344 rats indicate that chlorofom (CHCl3) formation from CCl4 is nonlinear with dose. Dose is traditionally expressed as the amount of CCl4 added to the vial. In this study, a pharmacokinetic model has been developed to calculate the concentration of CCl4 in the microsomal suspension. Hepatic microsomes prepared from fed and fasted animals were incubated with CCl4 under anaerobic conditions and formation of CHCl3 over a 5-min incubation period was monitored by headspace gas chromatography. Dose-response curves, based on total amount of CCl4 added to the microsomes, revealed a nonlinear, biphasic appearance of CHCl3, with fasting slightly increasing CHCl3 production in microsomes prepared from fasted rats. Microsomes were also pretreated with the CYP2E1 inhibitor, diallyl sulfone (DAS), before addition of CCl4. In uninhibited microsomes, there appeared to be a high-affinity saturable phase of metabolism occurring at lower concentrations followed by a linear phase at higher CCl4 concentrations. Following DAS pretreatment, the saturable portion of the dose-response curve was inhibited more than the linear phase with the biphasic CHCl3 production becoming more linear. DAS inhibition eliminated the effect of fasting on CHCl3 formation. The best fit kinetic constants for the saturable phase resulted in an estimate of V(max) of 0.017 mg/h/mg protein (V(maxc) = 7.61 mg/h/kg) and Km of 2.3 mg/l (15 microM). The linear phase rate constant (kf) was determined to be 0.046 h-1) (kfc = 0.03 h-1). In conclusion, a pharmacokinetic model has been developed for anaerobic in vitro metabolism of CCl4 to CHCl3 that estimates metabolic rates based on CHCl3 formation and actual CCl4 concentration in the microsomal suspension.

Chemical mixtures: challenge for toxicology and risk assessment.

It is now well-recognized that human environmental exposures are not to single chemicals. Rather, humans are exposed, either concurrently or sequentially, to multiple chemicals. Challenges that chemical mixtures pose for risk assessment and toxicology are presented. Challenge areas include increasing the peer-reviewed publication of human studies, improving access to peer-reviewed data and examining multiple target organs. Two difficult challenges are development of a common, consistent language and the use of appropriate and innovative experimental designs and analyses. The challenge of elucidation of mechanism(s) offers a rational basis for extrapolation across dose levels, exposure durations and exposure routes as well as to other species and to other similar chemicals. Of particular importance is focusing effort on those areas of investigation where answers have the greatest potential for reducing uncertainty in risk assessments for chemical mixtures and on those chemical mixtures and multiple chemical exposures that have the greatest potential impact on human health. A particularly fruitful area for future investigation is determination of the likelihood of nonadditive interactions in humans exposed to multiple chemicals at environmental exposure levels.

Fasting for less than 24 h induces cytochrome P450 2E1 and 2B1/2 activities in rats.

Cytochrome P450 (CYP) 2E1 activity is induced after 24 h of fasting but no information is available for shorter fasting periods. We investigate the induction of CYP 2E1, 2B1/2 and 1A1 in young adult male F344 rats after 8, 16 and 24 h of fasting compared to control. Liver microsomes were analyzed for the following enzyme activities: p-nitrophenol hydroxylase (PNP) for CYP 2E1, pentoxyresorufin-O-dealkylase (PROD) for CYP 2B1/2 and ethoxyresorufin-O-deethylase (EROD) for CYP 1A1. After each fasting interval, the activities per mg microsomal protein for PNP and PROD increased but the activity of EROD remained unchanged. Western blots for CYP 2E1 and CYP 2B1 showed increases comparable to the PNP and PROD activities, respectively. On a whole organ basis, increases were found for PNP and PROD activities, while decreases were found for EROD activity and total microsomal protein. The results are consistent with an induction of CYP 2E1 and CYP 2B1/2 activities after as little as 8 h of fasting.