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Physiologically based pharmacokinetic modeling and occupational exposure assessment studies often use minute ventilation rates (VE), alveolar ventilation rates (VA) and cardiac outputs (Q) that are not reflective of the physiological variations encountered during the aggregate daytime activities of individuals from childhood to adulthood. These variations of VE, VA and Q values were determined for healthy normal-weight individuals aged 5–96 years by using two types of published individual data that were measured in the same subjects (n = 902), namely indirect calorimetry measurements and the disappearance rates of oral doses of deuterium (2H) and heavy-oxygen (18O) in urine monitored by gas-isotope-ratio mass spectrometry. Arteriovenous oxygen content differences (0.051–0.082 ml of O2 consumed ml−1 of blood) and ratios of the physiological dead space to the tidal volume (0.232–0.419) were determined for oxygen consumption rates (0.157–0.806 l min−1) required by minute energy expenditures ranging from 0.76 to 3.91 kcal min−1. Generally higher values for the 2.5th up to the 99th percentile for VE (0.132–0.774 l kg−1 min−1, 4.42–21.69 l m−2 min−1), VA (0.093–0.553 l kg−1 min−1, 3.09–15.53 l m−2 min−1), Q (0.065–0.330 l kg−1 min−1, 2.17 to 9.46 l m−2 min−1) and ventilation–perfusion ratios (1.12–2.16) were found in children and teenagers aged 5–

The methodology developed in our previous studies ( Brochu et al. 2006a-c ) for the determination of physiological daily inhalation rates was improved by integrating into the calculation process, both nighttime and daytime respiratory parameters, namely oxygen uptake factors (H) and ventilatory equivalents (VQ). H values during fasting (0.2057±0.0018 L of O2/kcal; mean±SD) and postprandial phases (0.2059±0.0019 L of O2/kcal) as well as VQ values for subjects at rest (27.4±4.8 to 32.2±3.1, unitless) and during the aggregate daytime activities (29.9±4.2 to 33.7±7.2) were determined and combined with published doubly labeled water measurements for the calculation of daily inhalation rates in normal-weight males and females aged 0.22-96 years (n=1235). Depending upon the unit value chosen, the highest 99th percentiles for inhalation data were found in males aged 35 to

The distribution of physiological daily inhalation rates for pregnant and lactating females aged 11 to 55 years was determined according to total daily energy expenditures, energy costs for growth, pregnancy and lactation (maternal milk-energy synthesis and breast-energy output) in free-living females. Such published data were obtained using a methodology based on the disappearance rates of predetermined doses of doubly labeled water (2H2O and H2 18O) in urine from non-pregnant and non-lactating females (n = 357), as well as saliva from gravid and breastfeeding females (n = 91), monitored by gas-isotope-ratio mass spectrometry over an aggregate period of about 6,000 days. Monte Carlo simulations were necessary to integrate total daily energy requirements of non-pregnant and non-lactating females into energy costs and weight changes at the 9th, 22nd, and 36th week of pregnancy and at the 6th and 27th, postpartum week: 540,000 data were simulated. The present article confirms that physiological dai...

This study compared two toxicokinetic approaches for determining correction factors to be applied to occupational exposure limits (ELs) for unusual exposure scenarios: a classic one-compartment toxicokinetic approach and the physiologically based toxicokinetic (PBTK) approach. The approaches were applied to three typical unusual exposure scenarios: four consecutive 10-hour workdays followed by 3 days of recovery; three consecutive 12-hour workdays followed by 4 days of recovery; and a 4/3 work schedule. Results indicate that use of an adjustment method for ELs based on contaminant toxicokinetics generates less protective correction factors (i.e., a smaller adjustment) than those obtained using the U.S. Occupational Safety and Health Administration approach, which is based on Haber's law. Among all scenarios tested, the highest adjustment required, resulting from the use of a toxicokinetic approach (PBTK or one-compartment), was for the 4/3 work schedule and for a contaminant with a half-life equal to 18 hours. In that case the ELs would need to be reduced by 26%. Based on previous work, the authors believe an adjustment based on a toxicokinetic approach is more realistic from a toxicological standpoint. Given the value of a substance's half-life, the use of the graphs of Hickey and Reist (developed from a one-compartment toxicokinetic model) is a rapid and reliable means of establishing the correction factor. However, this approach is limited to simple and repetitive scenarios. For more complex exposure scenarios, such as that corresponding to a 4/3 work schedule, a one-compartment model also can be developed for each of the needs. Finally, the use of PBTK models allows greater flexibility for adjusting ELs for novel work schedules.

Research activities sought development of a method to adjust exposure limits for 694 substances for unusual work schedules. A consensus was established on the basic toxicological principle for adjustment; criteria for adjustment were selected by a panel of scientists coordinated by a committee of international experts and supported by toxicokinetic modeling; and a group of toxicologists attributed primary health effects and related adjustment category to each substance. A consensus among scientists and employers' and workers' representatives was established on the protocol of the application, in the field, of the adjusted exposure limits. The guiding toxicological principle for adjusting exposure standards to unusual work schedules is to guarantee an equivalent degree of protection for workers with unusual schedules as for workers with a conventional schedule of 8 hours per day, 5 days per week. The process of the adjustment is inspired from the Occupational Safety and Health Administration logic for attribution of primary health effects and adjustment categories ranging from no adjustment to daily or weekly adjustments. The adjusted exposure limits are calculated according to Haber's rule. Decisions on attribution of adjustment categories for the following toxicological effects were reached: respiratory sensitizers (asthma); skin sensitizers; tissue irritants versus tissue toxicants; methemoglobinenia-causing agents; cholinesterase inhibitors; and reproductive system toxicants and teratogens. A simple procedure is presented to facilitate the calculation, application, and interpretation of the adjusted exposure limits.

The objective of the present study was to develop a physiologically based pharmacokinetic (PBPK) model for a ternary mixture of alkyl benzenes [toluene (TOL), m-xylene (XYL), and ethylbenzene (EBZ)] in rats and humans. The approach involved the development of the mixture PBPK model in the rat and extrapolation to humans by substituting rat physiological parameters and blood:air partition coefficients in the model with those of humans, scaling maximal velocity for metabolism on the basis of body weight0.75 and keeping all other model parameters species-invariant. The development of the PBPK model for the ternary mixture in the rat was accomplished by initially validating or refining the existing PBPK models for TOL, XYL, and EBZ and linking the individual chemical models via the hepatic metabolism term. Accordingly, the Michaelis-Menten equation for each solvent was modified to test four possible mechanisms of metabolic interaction (i.e., no interaction, competitive inhibition, noncompetitive inhibition, and uncompetitive inhibition). The metabolic inhibition constant (Ki) for each binary pair of alkyl benzenes was estimated by fitting the binary chemical PBPK model simulations to previously published data on blood concentrations of TOL, XYL, and EBZ in rats exposed for 4 hr to a binary combination of 100 or 200 ppm of each of these solvents. Competitive metabolic inhibition appeared to be the most plausible mechanism of interaction at relevant exposure concentrations for all binary mixtures of alkyl benzenes in the rat (Ki,TOL-XYL = 0.17; Ki,TOL-EBZ = 0.79; Ki,XYL-TOL = 0.77; Ki,XYL-EBZ = 1.50; Ki,EBZ-TOL = 0.33; Ki,EBZ-XYL = 0.23 mg/L). Incorporating the Ki values obtained with the binary chemical mixtures, the PBPK model for the ternary mixture simulated adequately the time course of the venous blood concentrations of TOL, XYL, and EBZ in rats exposed to a mixture containing 100 ppm each of these solvents. Following the validation of the ternary mixture model in the rat, it was scaled to predict the kinetics of TOL, XYL, and EBZ in blood and alveolar air of human volunteers exposed for 7 hr to a combination of 17, 33, and 33 ppm, respectively, of these solvents. Model simulations and experimental data obtained in humans indicated that exposure to atmospheric concentrations of TOL, XYL, and EBZ that remain within the permissible concentrations for a mixture would not result in biologically significant modifications of their pharmacokinetics. Overall, this study demonstrates the utility of PBPK models in the prediction of the kinetics of components of chemical mixtures, by accounting for mechanisms of binary chemical interactions.

The present study was undertaken to investigate the mechanism of toxicokinetic interaction between toluene (TOL) and m-xylene (XYL) in vivo in the male Sprague-Dawley rat by physiologically based toxicokinetic (PBTK) modeling. First, the metabolic constants (Vmax and Km) were determined for TOL and XYL individually by conducting a series of closed-chamber inhalation exposures of three rats to starting concentrations of 500 to 4000 ppm. The values of Km (TOL, 0.55 mg/liter; XYL, 0.20 mg/liter) and Vmax (TOL, 4.8 mg/hr/kg; XYL, 8.4 mg/hr/kg) were obtained following best visual fit of PBTK model simulations to experimental data. Then using the same experimental set-up, rats were exposed to three different mixtures of both solvents (500 ppm TOL + 1000 ppm XYL; 1000 ppm TOL + 1000 ppm XYL; 1000 ppm TOL + 500 ppm XYL). The data from the time course of chamber solvent concentrations were analyzed with a binary chemical mixture PBTK model that had four mechanistic hypotheses of metabolic interaction (i.e., no interaction, competitive inhibition, noncompetitive inhibition, and uncompetitive inhibition) quantitatively defined in the liver compartment. The validity of the various model descriptions was verified with open-chamber inhalation exposure data on toxicokinetics of TOL and XYL. Overall, the results of this combined experimental and modeling approach are consistent with a competitive metabolic inhibition between XYL and TOL in the rat.

The present study was undertaken to investigate the influence of different exposure scenarios on the elimination of toluene and m-xylene in alveolar air and other biological fluids in human volunteers. The study was also aimed at establishing the effectiveness of physiologically based toxicokinetic models in predicting the value of biological monitoring data after exposure to toluene and m-xylene. Two adult male and two adult female white volunteers were exposed by inhalation, in a dynamic, controlled-environment exposure chamber, to various concentrations of toluene (21-66 ppm) or m-xylene (25-50 ppm) in order to establish the influence of exposure concentration, duration of exposure, variation of concentration within day, and work load on respective biological exposure indices. The concentrations of unchanged solvents in end-exhaled air and in blood as well as the urinary excretion of hippuric acid and m-methyl-hippuric acid were determined. The results show that doubling the exposure concentration for both solvents led to a proportional increase in the concentrations of unchanged solvents in alveolar air and blood at the end of a 7-h exposure period. Cumulative urinary excretion of the respective metabolites exhibited a nearly proportional increase. Adjustment of exposure concentration to account for a prolongation of the duration of exposure resulted in essentially identical cumulative urinary excretion of the metabolites. Induced within-day variations in the exposure concentration led to corresponding but not proportional changes in alveolar concentration for both solvents, depending on whether or not sampling preceded or followed peak exposure to solvent. At the end of repeated 10-min periods of physical exercise at 50 W, alveolar air concentrations of both solvents were increased by 40%. Experimental data collected during the present study were adequately simulated by physiologically based toxicokinetic modeling. These results suggest that alveolar air solvent concentration is a reliable index of exposure to both toluene and m-xylene under various experimental exposure scenarios. For clinical situations likely to be encountered in the workplace, physiologically based toxicokinetic modeling appears to be a useful tool both for developing strategies of biological monitoring of exposure to volatile organic solvents and for predicting alveolar air concentrations under a given set of exposure conditions.

Physiologically based pharmacokinetic modeling and occupational exposure assessment studies often use minute ventilation rates (VE), alveolar ventilation rates (VA) and cardiac outputs (Q) that are not reflective of the physiological variations encountered during the aggregate daytime activities of individuals from childhood to adulthood. These variations of VE, VA and Q values were determined for healthy normal-weight individuals aged 5-96 years by using two types of published individual data that were measured in the same subjects (n = 902), namely indirect calorimetry measurements and the disappearance rates of oral doses of deuterium (²H) and heavy-oxygen (¹⁸O) in urine monitored by gas-isotope-ratio mass spectrometry. Arteriovenous oxygen content differences (0.051-0.082 ml of O₂ consumed ml⁻¹ of blood) and ratios of the physiological dead space to the tidal volume (0.232-0.419) were determined for oxygen consumption rates (0.157-0.806 l min⁻¹) required by minute energy expenditures ranging from 0.76 to 3.91 kcal min⁻¹. Generally higher values for the 2.5th up to the 99th percentile for VE (0.132-0.774 l kg⁻¹ min⁻¹, 4.42-21.69 l m⁻² min⁻¹), VA (0.093-0.553 l kg⁻¹ min⁻¹, 3.09-15.53 l m⁻² min⁻¹), Q (0.065-0.330 l kg⁻¹ min⁻¹, 2.17 to 9.46 l m⁻² min⁻¹) and ventilation-perfusion ratios (1.12-2.16) were found in children and teenagers aged 5-16.5 years compared with older individuals. The distributions of cardiopulmonary parameters developed in this study should be useful in facilitating a scientifically sound characterization of the inter-individual differences in the uptake and health risks of lipophilic air pollutants, particularly as they relate to younger children.

This review is a critical survey and evaluation of recent literature on solvent interactions for the assessment of health risk. It addresses the implications of multiple solvent exposures 1) by examining the influence of solvent-solvent and ethanol-solvent interactions on the biological indices of chemical exposure, and 2) by indicating how the eventual modifying effects can be considered in the biological monitoring of mixed exposure. Reviewed studies reveal the effects of toxicokinetic interactions on the biological parameters, and the gaps in our knowledge. The measurement of potentially toxic molecular species is suggested for the biological monitoring of multiple chemical exposure. This approach appears to be important for drawing better quantitative conclusions on the internal exposure to biologically active chemical species. Finally, research needs arising from the critical analysis of the literature are briefly described.

Acetone potentiation of haloalkane-induced liver injury is a well-known phenomenon. Acetone-treated rats challenged with a trichloroethylene–CCl4 mixture exhibit a more severe liver injury than that predicted by the addition of the single potentiating effects of each. The purpose of the present study was to determine if acetone exerted similar interactions with other haloalkane mixtures. The testing protocol used was designed and performed to allow categorization of interactions occurring among two or three agents. Rats were treated (p.o.) with corn oil or acetone (10.2 mmol/kg) and were administered (i.p.) 18 h later 1,1-dichloroethylene (0.6 mmol/kg), trichloroethylene (5.6 mmol/kg), tetrachloroethylene (19.6 mmol/kg), 1,1,1-trichloroethane (10.0 mmol/kg), 1,1,2-trichloroethane (1.1 mmol/kg), 1,1,2,2-tetrachloroethane (1.0 mmol/kg), CHCl3 (6.2 mmol/kg), CCl4 (1.0 mmol/kg), or a mixture of two haloalkanes (all 28 combinations were tested). Liver injury was assessed 24 h later using plasma alanine aminotransferase activity and a quantitative histological evaluation. In corn oil pretreated rats, the hepatotoxic responses observed for the 28 mixtures were additive for 26 of 28 mixtures and supra-additive for 2 of 28, whereas in acetone-pretreated rats the responses observed were additive for 17 of 28, infra-additive for 10 of 28, and supra-additive for 1 of 28. Mixtures containing 1,1,1-trichloroethane or tetrachloroethylene resulted only in no change in toxicity or infra-additivity. Increased toxic responses (additivity and supra-additivity) were observed with certain binary mixtures containing CCl4, CHCl3, 1,1,2-trichloroethane, or 1,1-dichloroethylene. Tetrachloroethylene yielded infra-additive responses when combined in a binary mixture with other haloalkanes, CCl4 in particular. The effect exerted by acetone on the liver injury induced by haloalkane mixtures appears to be predictable on the basis of the particular haloalkanes present in the mixture. Halogenated methanes, chemicals with minimal use in the industrial workplace, showed the greatest potential for additive and supra-additive interactions with other haloalkanes. The approach described is a useful method for examining the potential toxicity of mixtures.Key words: acetone, haloalkanes, liver injury, mixtures, interactions.

Hexachlorobenzene (HCB) induces hepatic porphyria in rats. Various protocols of repeated cumulative and daily doses of HCB administered for several weeks until porphyria develops have been traditionally used. In order to undertake studies on early biochemical events occurring in HCB-induced porphyria, we have designed an experimental model involving the administration of a minimal amount of HCB inducing a fully developed porphyria in a well defined and predictable time frame. Groups of Sprague-Dawley rats were given (po, in 10 ml/kg of corn oil) a cumulative dose of 1500 mg HCB/kg as 50 mg/kg for 6 weeks (5 days/week) or 100 mg/kg for 3 weeks (5 days/week). In female, but not male, rats treated for 6 weeks, HCB caused a porphyria as measured by urinary uroporphyrin and hepatic porphyrin levels; this total dose given to female rats in 3 weeks was not, however, porphyrinogenic. Female rats were given 12 consecutive daily doses of 50 mg HCB/kg followed by a no-treatment period of 30 days; this cumulative dose of 600 mg HCB/kg induced a porphyria after 6 weeks. The approximate minimally effective cumulative dose inducing porphyria was determined to be 400 mg HCB/kg, regardless of the magnitude of the daily dose (25, 50, or 100 mg/kg). Finally, the administration of a cumulative dose of 500 mg HCB/kg (50 mg/kg, 5 days/week for 2 weeks or 100 mg/kg/day for 5 days) induced after 5 to 6 weeks a porphyria that persisted for more than 500 to 600 days. These results suggest that induction of porphyria is dependent upon the administration of a threshold cumulative dose of HCB during an initial phase followed by a delay period with no further treatment during which full development of biochemical alterations in the heme synthesis cycle occurs.

Male rats are more sensitive to the nephrocarcinogenic effect of hexachlorobenzene (HCB) than are female rats. The purpose of this study was to shed light on this phenomenon by investigating mechanisms of subchronic nephrotoxicity of HCB. Groups of rats were administered HCB in corn oil (po) at 100 mg/kg, 5 days per week for 15 days or at 50 mg/kg, 5 days per week for 50 days. Urine was collected on Days 1, 8, and 15 for the 15-day treatment and on Day 50 for the 50-day treatment. Glucosuria, proteinuria, and enzymuria (gamma-glutamyl transpeptidase) were measured to assess renal function. Twenty-four hours after the last HCB treatment, the animals were killed and kidneys were removed for histopathological evaluation. Urine analyses showed no indication of renal dysfunction in treated animals compared to controls during the 15-day treatment. However, histology of male rat kidneys revealed degenerative and regenerative cellular foci accompanied by an increased accumulation of protein droplets in epithelial cells of the proximal tubules. The same histological observations were also made in male rats after a 50-day HCB treatment but this time they were accompanied by renal function alterations. In female rats, no such renal functional or histological alterations were observed. The histopathological observations in male rats correspond well with the protein droplet nephropathy; the latter is characteristic of the accumulation in kidney cells of alpha 2u-globulin probably caused by the reversible binding of a chemical to alpha 2u that renders the protein indigestible to kidney proteases. alpha 2u-Globulin was measured in the cytosol of male rats and was found to be increased 11-fold compared to controls. Also, HCB was found to be bound reversibly to alpha 2u. These results suggest that HCB induces a male rat specific nephropathy that could explain the higher incidence of kidney tumors in male rats compared to female rats.

This study was undertaken to characterize the mechanism of toxicokinetic interaction between toluene (TOL) and m-xylene (XYL) in the rat using physiologically-based toxicokinetic (PBTK) modeling approach. First, the metabolic rate constants were determined by conducting closed-chamber inhalation exposures with individual solvents (Vmax: TOL = 4.8, XYL = 8.4 mg/hr/kg; Km: TOL = 0.55, XYL = 0.2 mg/l). Then, using the same experimental set-up, rats were exposed to different binary mixtures of TOL and XYL. PBTK analysis of the data showed competitive inhibition as the plausible mechanism of TOL/XYL interaction. This mechanistic modeling study suggests that the interaction between TOL and XYL is likely to be observed when the exposure concentration exceeds 50 ppm of each solvent.

A sensitive and reproducible gas chromatographic procedure for the quantitative determination of urinary o-cresol is described. The first step involves acid hydrolysis (2N HCl, 100 degrees C, 10 min), which yields unconjugated o-cresol. After extraction (methylene chloride, pH 2), the organic layer is concentrated by evaporation and samples are analyzed by gas chromatography-flame ionization detection with a DB-5 column (30 m x 0.25 mm, 0.25 microns). Initial oven temperature is set at 30 degrees C for 12 min and the increased 2 degrees C/min to 93 decrees C. 3,4-Dimethylphenol is used as the internal standard. The detection limit of the method is 0.36 mumol/L (1 microL injection). Additional validation data were obtained by analysis of urine samples collected from volunteers exposed to various concentrations of toluene: 10, 20, 30, 50, and 100 ppm over 7 h. Urinary o-cresol concentrations (0-3, 3-7, 7-24, and 0-24 h) were highly correlated with toluene exposure.

Administration of anethol dithiolthione (ADT) to rodents can afford protection against some chemically induced toxicities. The aim of the present study was to assess the effects of ADT on hexachloro-1,3-butadiene (HCBD)-induced nephrotoxicity in the rat and to determine the mechanism of its action. Renal integrity was evaluated by measuring urinary excretion of glucose, protein, and gamma-glutamyl transpeptidase and by histological evaluation. A 3-day pretreatment with ADT (300 mg/kg/day) protected against the toxicity of various doses of HCBD (ranging from 15.6 to 62.5 mg/kg). The pretreatment increased (1.4-fold) the nonprotein sulfhydryl content (NPSH) of the liver. However, it did not modify the biliary excretion of radiolabeled materials in [14C]HCBD- treated (20 mg/kg) rats, nor that of the bioactivated HCBD metabolite, S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-glutathione (PCBG). Moreover, ADT pretreatment protected rats against the nephrotoxicity induced by PCBG (20 mg/kg) itself. The extent of covalent binding to kidney proteins of [14C]HCBD-derived metabolites was not modified by pretreatment with ADT. Incubation of rat kidney cortical slices in a medium containing 0.1 mM of the nephrotoxic glutathione (PCBG) or cysteine (PCBC, S-(1,2,3,4,4-pentachloro-1,3-butadienyl)-L-cysteine) conjugates of HCBD for 30 min resulted in a 75% reduction in the slice/medium ratio of p-aminohipurate (PAH) compared to that seen in controls. When the cortical slices were incubated with ADT (30 min, 0.2 mM) prior to incubation with the nephrotoxic conjugates, the reduction was only 33%. Neither the in vitro nor the in vivo treatments did modify the activity of renal cytosolic beta-lyase; however, the latter treatment caused an increase in NPSH content. A 15-min incubation of kidney cortical slices with glutathione (10 mM) resulted in a 5-fold increase of NPSH, but failed to prevent the reduction in PAH uptake caused by PCBG and PCBC. Altogether, the in vivo and renal slice data suggest that ADT protects rats against HCBD-induced nephrotoxicity by a mechanism that does not involve the modulation of HCBD conjugation with liver GSH, nor the modulation of the kidney NPSH level and beta-lyase activity. The mechanism of protection conferred to rats by an ADT pretreatment against HCBD-induced nephrotoxicity appears to take place in the kidney at a step beyond the generation of ultimate toxic metabolites derived from PCBC.

The objective of the present study was to compare the influence of various binary mixtures containing ethylbenzene (EBZ), toluene (TOL) or xylene (XYL) administered by inhalation, with the influence exerted by a ternary mixture, on the kinetics of these solvents in blood. Groups of four rats were exposed for 4 h to TOL, XYL and EBZ, singly or in combination. The concentration of TOL, XYL and EBZ in blood was measured at various times (5, 30, 60, 90 and 120 min) following the end of exposure and the areas under the blood concentration curves (AUC) were calculated. Results showed that exposures to binary and ternary mixtures resulted in significantly higher (P < 0.05) blood concentrations of unchanged solvents as a result of metabolic interaction between these solvents. When the comparison was based on individual solvents, there was no difference between effect exerted by the ternary mixture and the binary mixtures, except for one. However, a comparison based on the total concentration of unchanged solvents disclosed that exposure to the ternary mixture resulted in greater interactive effects (3.17-fold increase) than exposures to binary mixtures (1.97-fold increase), whereas four out of six binary mixtures produced higher total levels of unchanged solvents in blood compared to the ternary mixture. This study shows that the greater risk of toxicity often thought to be associated with exposures to complex mixtures should not only be related to the magnitude of interactive effects among components (i.e., degree of mutual metabolic interaction) resulting from combined exposures, but also should take into account, as is universally recognized, the internal total dose of toxic chemicals in target organs/tissues.

A physiologically-based pharmacokinetic (PBPK) model for a mixture of toluene (TOL) and xylene (XYL), developed and validated in the rat, was used to predict the uptake and disposition kinetics of TOL/XYL mixture in humans. This was accomplished by substituting the rat physiological parameters and the blood:air partition coefficient with those of humans, scaling the maximal velocity for hepatic metabolism on the basis of body weight0.75, and keeping all other model parameters species-invariant. The human TOL/XYL mixture PBPK model, developed based on the quantitative biochemical mechanism of interaction elucidated in the rat (i.e., competitive metabolic inhibition), simulated adequately the kinetics of TOL and XYL during combined exposures in humans. The simulations with this PBPK model indicate that an eight hour co-exposure to concentrations that remain within the current threshold limit values of TOL (50 ppm) and XYL (100 ppm) would not result in significant pharmacokinetic interferences, thus implying that data on biological monitoring of worker exposure to these solvents would be unaffected during co-exposures.

To confirm the relationship between exposure to coal tar pitch volatiles and bladder cancer among primary aluminum production workers, we carried out a case-control study among blue-collar workers who had worked more than 1 year between 1950-1979 in a major plant using mostly the Soderberg process in the Province of Quebec. Cases of bladder cancer (ICD code 188) diagnosed between 1970-1979 (n = 69) were mostly included in a previously reported study. To these were added cases diagnosed between 1980-1988 (n = 69). Each case was matched to three controls on date of birth, date of hiring, and length of service at the company. Smoking habits were assessed from the medical records at the company. Benzene-soluble matter (BSM) and benzo(a)pyrene (BaP) were used as indicators of environmental exposure to coal tar pitch volatiles in the workplace. The estimated risk for current smokers was 2.63 (95% C.I. 1.29-5.37). Estimates of risk by occupational exposure were adjusted for smoking. Men who had worked in the Soderberg potrooms were at higher risk of developing the disease, the risk increasing with the time spent in these departments. Similarly, a strong association between risk and cumulative exposure to BSM or to BaP was observed. The risks associated with cumulative exposure to BSM (mg/m3-years) and to BaP (microgram/m3-years) were described with mathematical models. Using a linear model (1 + bx) and lagging 10 years before the diagnosis, BaP cumulative exposure was a better indicator of risk than BSM cumulative exposure. The risk for each year of exposure to BaP at a concentration of 1 microgram/m3 increased by 1.7% (0.8%-3.2%). Using the same model for BSM, a worker exposed to the current threshold limit value of 0.2 mg/m3 for 40 years will sustain a risk of 2.22 (1.56-3.48). Comparison of risks according to different periods of diagnosis (1970-1979 vs. 1980-1988) did not reveal any significant temporal changes on risk estimates.

The purpose of the present study was to investigate the influence of different exposure scenarios on the elimination of trichloroethylene (TRI) and 1,1,1-trichloroethane (1,1,1-TRI) in alveolar air and other biological fluids in human volunteers. In addition, it was sought to establish an interactive process between experimental data gathering and simulation modeling in an attempt to predict the influence of the different scenarios of exposure to TRI and 1,1,1-TRI on their respective biological monitoring indices and thus to establish the flexibility and validity of simulation models. Two adult male and two adult female Caucasian volunteers were exposed by inhalation, in a dynamic controlled exposure chamber, to various concentrations of TRI (12.5-25 ppm) or 1,1,1-TRI (87.5-175 ppm) in order to establish the influence of exposure concentration, duration of exposure, variation of concentration within day, and work load on biological exposure indices. The concentrations of unchanged solvents in end-exhaled air and in blood as well as the urinary excretion of trichloroethanol (TCE) and trichloroacetic acid (TCA) were determined. The results show that doubling the exposure concentration for both solvents led to a proportional increase in the concentrations of unchanged solvents in alveolar air and blood at the end of a 7-h exposure period; this proportionality was still observable in 1,1,1-TRI expired air samples 16 h after the end of the third exposure day. In the case of urinary excretion of TCE and TCA, the proportionality between excretion and exposure concentration was not as good. It was once again observed that the slow excretion of both metabolites leads to progressive cumulation and that their urinary determination is subject to considerable interindividual variations. After adjustment (lowering) of the exposure concentration to account for a prolongation of the duration of exposure (from 8 to 12 h) it was observed that the concentrations of TRI or 1,1,1-TRI towards the end of both exposure periods are more a reflection of the actual exposure concentration than of the exposure duration. Despite important interindividual variations, these adjusted and nonadjusted exposures led to almost identical average total urinary excretion over 24 h) of TCE and TCA after exposure to 1,1,1-TRI, as was also the case for TCE but not for TCA after exposure to TRI. Induced within-day variations in the exposure concentration led to corresponding but not proportional changes in alveolar air concentrations for both solvents. After exposure to peak concentrations there was a lag period before alveolar air concentrations returned to prepeak levels. At the end of repeated 10-min periods of physical exercise at 50 W, alveolar air concentrations of TRI were increased by 50% while those of 1,1,1-TRI increased by only 12%. After optimization of the physiologically based toxicokinetic model parameters with experimental data collected during the first exposure scenario, results pertaining to the three other scenarios were adequately simulated by the optimized models. Overall, the results of the present study suggest that alveolar air solvent concentration is a reliable index of exposure to both TRI and 1,1,1-TRI under various experimental exposure scenarios. These results also suggest that urinary excretion of TCE and TCA must be interpreted with caution when assessing exposure to either solvents. For exposure situations likely to be encountered in the workplace, physiologically based toxicokinetic modeling appears to be a useful tool both for developing strategies of biological monitoring of exposure to volatile organic solvents and for predicting alveolar air concentrations under a given set of exposure conditions.

Combined exposures to multiple chemicals may result in interactions leading to a significant increase or decrease in the overall toxicity of the mixture compared to the summation of the toxicity of the components. A large number of chemical interactions have been described in animal studies by administering high doses of chemicals by routes and scenarios often different from anticipated human exposures. Though limited, there is some evidence for the occurrence of several supra-additive (the combined effects are greater than the simple summation of the individual effects) and infra-additive (the combined effects are smaller than the simple summation of the individual effects) chemical interactions in humans. For example, toxicokinetic interactions between several solvents have been found to occur in the workplace, whereas those involving pesticides have been reported less frequently, especially during accidental exposures. Toxic interactions involving nutritionally important metals and metalloids appear to occur more frequently, since several of them have an important role in a variety of physiological and biochemical processes. On the contrary, there is not much evidence to confirm the occurrence of toxic interactions among the commonly encountered inorganic gaseous pollutants in humans. Overall, the majority of chemical interactions observed in animal studies have neither been investigated in humans nor been extrapolated to humans based on appropriate mechanistic considerations. Future research efforts in the chemical interactions arena should address these issues by focusing on the development of mechanistically and biologically based models that allow predictions of the extent of interactions likely to be observed in humans.

This study was undertaken to determine whether previous subacute treatment with ethanol could modify the kinetics of m-xylene in humans. A group of six volunteers was exposed twice to either 100 or 400 ppm of m-xylene during two hours (between 0800 and 1000). Ethanol was given orally in the early evening on each of two consecutive days before exposures (total ethanol intake of 137 g). Such ethanol pretreatment affected the kinetics of m-xylene but only at the high exposure (400 ppm). The modifications were: (1) decreased concentration of m-xylene in blood and alveolar air during and after exposure; (2) increased urinary excretion of m-methylhippuric acid at the end of exposure. Ethanol treatment also enhanced the elimination of antipyrine in saliva. Overall, this study showed that the effect of enzyme induction on the metabolism of m-xylene, after ethanol ingestion, depends on the exposure concentration and is not likely to occur as long as the exposure concentrations remain under the current maximum allowable concentration (100 ppm) in the workplace.

Potential toxic interaction between hexachlorobenzene (HCB) and methyl isobutyl ketone (MiBK) was investigated using two different schedules of toxicant administration. The first schedule involved simultaneous administration of HCB (50 mg/kg/d p.o. in 10 ml/kg corn oil at 10.00 a.m. for 5 d/wk) and MiBK (7.5 mmol/kg/d p.o. in 10 ml/kg corn oil at 4.00 p.m. for 3 d/wk) for 6 weeks. The second schedule involved an initial dosing of 25 or 50 mg HCB/kg/d for 12 consecutive days, followed by the administration of 7.5 mmol MiBK/kg every other day for 27 days. When administered simultaneously, MiBK reduced the severity of HCB-induced porphyria, but when given sequentially after HCB accumulation, it enhanced the porphyrinogenic response. These results suggest that the effect of combined exposure to HCB and MiBK on hepatic porphyria depends on the sequence of the administration of both chemicals, and that the mechanism involved in this interaction may invoke both the induction and inhibition of specific hepatic isoenzymes by MiBK.

The present study was undertaken to describe the influence of simultaneous exposure by inhalation to toluene and xylene on some aspects of their respective metabolic disposition. Adult male rats were exposed acutely (5 h) to 75, 150, and 225 ppm of toluene or xylene and to various mixtures of these solvents: toluene (75 ppm) and xylene (225 ppm), toluene (150 ppm) and xylene (150 ppm), toluene (225 ppm) and xylene (75 ppm). Compared with single exposure, simultaneous exposure resulted in lower amounts of excreted hippuric acid (20–30%) and methylhippuric acids (4–40%) in urine over a period of 24 h, even though significant differences were seen only with the toluene (150 ppm) and xylene (150 ppm) combination. In addition, increased concentrations of solvents in blood (toluene, 230%; xylene, 500%) and in brain (toluene, 230%; xylene, 320%) were found during the immediate post-exposure period. Simultaneous exposure also enhanced the pulmonary elimination of both solvents (toluene, 190–240%; xylene, 340–650%). Influence of repeated simultaneous exposure (9 days) was investigated for the toluene (150 ppm) and xylene (150 ppm) combination and the results compared with those of repeated exposure to each solvent administered singly. Under these conditions, repeated simultaneous exposure decreased the excretion of urinary metabolites, but only after the first exposure. On the other hand, simultaneous exposure resulted in significantly higher concentrations of toluene (210%) and xylene (240%) in blood throughout the entire 9-day exposure period. These results strongly suggest mutual metabolic interactions (inhibition) between toluene and xylene that affect the metabolic disposition of both solvents and ultimately the biological monitoring of data of exposure to a combination of solvents in rats.Key words: toluene, xylene, simultaneous exposure, biological monitoring, metabolic interaction, inhalation.

Hexachlorobenzene (HCB) induces hepatic porphyria in rats. Various protocols of repeated cumulative and daily doses of HCB administered for several weeks until porphyria develops have been traditionally used. In order to undertake studies on early biochemical events occurring in HCB-induced porphyria, we have designed an experimental model involving the administration of a minimal amount of HCB inducing a fully developed porphyria in a well defined and predictable time frame. Groups of Sprague-Dawley rats were given (po, in 10 ml/kg of corn oil) a cumulative dose of 1,500 mg HCB/kg as 50 mg/kg for 6 weeks (5 days/week) or 100 mg/kg for 3 weeks (5 days/week). In female, but not male, rats treated for 6 weeks, HCB caused a porphyria as measured by urinary uroporphyrin and hepatic porphyrin levels; this total dose given to female rats in 3 weeks was not, however, porphyrinogenic. Female rats were given 12 consecutive daily doses of 50 mg HCB/kg followed by a no-treatment period of 30 days; this cumulative dose of 600 mg HCB/kg induced a porphyria after 6 weeks. The approximate minimally effective cumulative dose inducing porphyria was determined to be 400 mg HCB/kg, regardless of the magnitude of the daily dose (25, 50, or 100 mg/kg). Finally, the administration of a cumulative dose of 500 mg HCB/kg (50 mg/kg, 5 days/week for 2 weeks or 100 mg/kg/day for 5 days) induced after 5 to 6 weeks a porphyria that persisted for more than 500 to 600 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies that specifically address the influence of controlled human exposure to a combination of solvents on the biological monitoring of exposure are limited in number. The present study was undertaken to investigate whether simultaneous exposure of human volunteers to toluene and xylene could modify the respective metabolic disposition of these solvents. Five adult Caucasian men were exposed for 7 consecutive h/day over 3 consecutive days to 50 ppm toluene and 40 ppm xylene either separately or in combination in a dynamic, controlled exposure chamber (low-level exposure). The experiment was repeated three times at intervals of 2 weeks. In another experiment, three subjects were exposed to 95 ppm toluene and 80 ppm xylene or a combination of both for 4h (high-level exposure). The concentration of unchanged solvents in blood (B) and in end-exhaled air (EA) as well as the urinary excretion of hippuric acid (HA) and methylhippuric acids (MHAs) were determined. Simultaneous exposure to the lowest level of solvents did not alter the concentration of unchanged solvents in blood or in exhaled air (average of 3-weekly means; single vs mixed exposure at 6.5 h exposure): B-toluene, 77.1 vs 78.1 μg/100 ml; B-xylene, 67.6 vs 77.8 μg/100 ml; EA-toluene, 9.9 vs 9.5 ppm; EA-xylene, 5.3 vs 4.8 ppm. Similarly, mixed exposure did not modify the excretion of urinary metabolites during the 3- to 7-h exposure period: HA, 1.11 vs 1.11 g/g creatinine; MHAs, 0.9 vs 0.87 g/g creatinine. However, simultaneous exposure to higher levels did affect the concentration of unchanged solvents in blood and in exhaled air as measured at 3.5 h exposure (mean value for three subjects ± SD): B-toluene, 135.7 ± 26.7 vs 215.7 ± 34.9 μg/100 ml; B-xylene, 114 ± 19 vs 127.6 ± 22.1 μg/100 ml; EA-toluene, 16.6 ± 0.4 vs 20.5 ± 2.8 ppm; EA-xylene, 9.9 ± 0.6 vs 12.3 ± 1.2 ppm. Such effects were accompanied by a significant delay in the urinary excretion of HA but not of MHAs. These data suggest that there is a threshold level below which metabolic interaction between toluene and xylene is not likely to occur in humans.

We have developed a relatively simple and reproducible HPLC procedure for the determination of urinary metabolites of styrene. Urine samples (pH 2) are extracted with ethyl acetate, the organic layer is evaporated to dryness, and the residues are dissolved in water-methanol (1:1). Samples are analyzed by HPLC with a C18 reversed-phase column and a water (pH 6, 5mM tetrabutylammonium dihydrogen phosphate)-acetonitrile gradient and ultraviolet detection at 225 nm. Using this method, it is possible to determine simultaneously mandelic and phenylglyoxylic acids, N-acetyl-S-(1-phenyl-2-hydroxyethyl)-L-cysteine (M1), and N-acetyl-S-(2-phenyl-2-hydroxyethyl)-L-cysteine (M2). The internal standard is p-hydroxybenzoic acid. Additional validation data are obtained by analysis of urine samples obtained from rats treated with a wide range of styrene doses.

Evidence previously reported suggest that 1,3-butanediol (BD) enhances the hepatotoxic effect of a single small dose of carbon tetrachloride (CCl4) in a dose-related manner. The present study provides additional information concerning the quantitative relationship between the severity of the ketotic state produced by BD and the magnitude of the potentiation observed and emphasizes the use of ketone bodies (KB) to predict the potential hazard of the BD-CCl4 interaction. Liver damage was modulated in male Sprague-Dawley rats by varying the concentration of the BD solutions ingested prior to a CCl4 challenge (0.1 ml/kg, i.p.). These data were compared to ketone bodies in plasma, hepatic tissue and urine. BD produced a dose-dependent metabolic ketosis observable at dosages between 1.1 and 9.9 g/kg per day given for 7 days. Plasma and liver data correlated well together. Concomitantly, potentiation of the CCl4-induced liver injury was also dose-related for the same dosage range; the minimum effective dosage of BD for potentiation was estimated as 1.1 g/kg per day. The linear correlations between hepatic or plasma KB values and the indices of hepatic dysfunction (ALT, OCT) were highly significant. Using a semiquantitative method, a correlation was also found for the urinary KB data. These results suggest that plasma KB concentrations might be useful for predicting possible potentiation of the hepatonecrotic effect of CCl4 by BD.

Norethandrolone (NE) and other androgenic steroids have been shown to be renotropic in various species and have also been reported to have salutary effects in patients with diminished renal function. Renal cortical slices prepared from rats pretreated with NE showed an increased capability to concentrate p-aminohippuric acid (PAH). Pretreatment with NE failed to stimulate the transport of the organic base tetraethylammonium and the organic acid benzylpenicillin. Stimulation of PAH transport was observed after eight daily subcutaneous injections of NE. No stimulation was observed with shorter pretreatment intervals. When NE was given subcutaneously for 14 days at doses of 2, 6, or 20 mg kg−1 day−1, significant stimulation of PAH transport was seen at all three dose levels but no dose–effect relationship was apparent. Stimulation of PAH transport was seen in female rats as well as castrated and intact males. In addition to its general anabolic properties, NE induces the synthesis of hepatic microsomal drug-metabolizing enzymes. For comparative purposes, therefore, the effect of pregnenolone-16α-carbonitrile (PCN) was also investigated. This agent is a potent inducer of drug metabolism but is neither anabolic nor renotropic. When rats were pretreated with an inducing dose of PCN (75 mg kg−1 day−1 for 3 days), there was no significant stimulation of PAH transport. It would seem, then, that the stimulatory effect of NE on PAH transport is more closely associated with its generalized anabolic effect than with its ability to induce hepatic microsomal enzymes.

We describe a fluorometric method for determining δ-aminolaevulinate dehydratase (EC 4.2.1.24) activity in human erythrocytes and compare it with the existing colorimetric method. Incubation conditions are identical. In the proposed method, the porphobilinogen formed during incubation is converted to a stable and highly fluorescent uroporphyrin by heating for 20 min at 93 °C in an acidic medium in the presence of air. The correlation between results by the two methods was good (r = 0.89). We found a good negative correlation between the activity of the enzyme and the concentration of circulating lead with both methods (r = -0.61 for present method and -0.59 for colorimetric method) for groups of persons subjected to various degrees of lead exposure. Our method is more sensitive and accurate, requires less sample, and the fluorescence is more stable than is the color in the colorimetric method.

When male Sprague-Dawley rats were treated with 1 mg mercuric chloride (HgCl2)/kg, sc 6 hr prior to or simultaneously with a single 2.5-mmole/kg ip dose of bromobenzene and sacrificed 48 hr after the bromobenzene dose, the activities of serum transaminases (SGOT and SGPT) were found to be significantly reduced when compared with those obtained in bromobenzene-alone-treated rats. Similar phenomena were observed when rats were treated simultaneously with 1 or 2 mg HgCl2/kg and 1 mmole bromobenzene/kg, but not when bromobenzene was given 6 hr prior to HgCl2 injection. When 5 mmole bromobenzene/kg and 1 mg HgCl2/kg were given simultaneously to the animals, such an apparent reduction in bromobenzene toxicity was again observed. In each case, HgCl2 alone had no effect on the transaminase activities. HgCl2 (1 mg/kg, sc) treatment reduced the hepatic microsomal cytochrome P-450 content. Treatment with 1 mg HgCl2/kg 6 hr prior to bromobenzene injection (2.5 mmole/kg) significantly reduced the urinary excretion of para- and metabromophenols, and parabromocatechol during 0 to 24-hr period without affecting the urinary thioethers. These data suggest a possible reduction in the rate of formation of bromobenzene epoxide intermediate due to mercury pretreatment, resulting in a lowering of the steady state level of this epoxide so that an inhibition of hepatotoxicity due to bromobenzene could occur. However, simultaneous treatments of HgCl2 and bromobenzene failed to modify the urinary metabolic excretion pattern of bromobenzene. When rats were given 10, 50, and 100 ppm of HgCl2 in drinking water daily for 4 weeks prior to an ip injection of 2.5 mmole bromobenzene/kg and were sacrificed 48 hr after the dose, no changes in SGOT and SGPT activities were observed. These results indicate that changes in the metabolism and hepatotoxicity of bromobenzene due to mercury depend on (a) the dose and time of mercury administration, and (b) the mode of administration of mercury, acute or chronic.

The pharmacological interaction between acutely administered ethanol and methaqualone was investigated in mice. Data were submitted to analysis of variance according to a 3 × 3 factorial design. When associated at hypnotic doses (ethanol [Formula: see text], orally, and methaqualone [Formula: see text], i.p.), both drugs lengthened sleeping time in a supraadditive manner. At lethal or near lethal doses (ethanol [Formula: see text] and methaqualone [Formula: see text]), complex interactions were observed: an antagonistic effect of ethanol (2 g/kg) against methaqualone toxicity and at least additive effects of both drugs when ethanol was given at 6 g/kg. When methaqualone was replaced by sodium barbital (725 mg/kg, i.p.) ethanol (2 g/kg) failed to exhibit any antagonistic effect. Because methaqualone at near lethal doses possesses convulsant properties, the protection afforded by ethanol (2 g/kg) might be due to its mild anticonvulsant effects. At 6 g/kg the central depressant properties of ethanol appear to be contributory to the lethality of methaqualone; in addition, at high doses, ethanol facilitates the distribution of methaqualone to the brain. This investigation suggests that the ethanol–methaqualone association induces a rather complex set of pharmacological interactions; it especially demonstrates the antagonistic properties of subhypnotic doses of ethanol against the toxicity of methaqualone.

Measurements of aniline hydroxylation, aminopyrine N -demethylation and cytochrome P-450 content after a 3-week treatment with phenobarbital (PB), diphenylhydantoin (DPH) or a combination of the two drugs were undertaken during normal vitamin D status (D +) and vitamin D deficiency (D −) with or without vitamin D 3 (D 3 ) supplementation. Serum calcium concentrations were reduced after D deprivation but responded by a significant increase toward normal values to a single pharmacological dose of D 3 . Serum phosphorus concentrations were also slightly raised by the supplementation. Even in the presence of higher cytochrome P-450 content in D − rats, aniline hydroxylase and aminopyrine N -demethylase activities were lower in D − than in D + animals. These two enzymatic parameters, as well as cytochrome P-450 content, were increased by anticonvulsant (ACV) drug treatment regardless of the D nutritional status. The in vivo hexobarbital sleeping time was shortened by ACV drugs but the sleeping time tended to be longer in D − than in D + rats. Supplementation with 1000 I.U. of D 3 , lowered aniline hydroxylase activity both in D + and D− animals; the supplementation had no effect on aminopyrine N -demethylase activity in D + animals but had an inhibitory effect after PB and a stimulatory effect after DPH treatment in D− animals. Cholecalciferol supplementation lowered cytochrome P-450 content toward normal values in D− rats while it had no effect in D + animals. These observations suggest that (1) PB and DPH pretreatment do not alter the normal response of serum calcium and phosphorus to a single pharmacological dose of D 3 ; (2) in a state of vitamin D deficiency accompanied by hypocalcemia, the inducing capacity of PB and DPH on the liver mixed function oxidase system is not lost; (3) under certain circumstances, vitamin D 3 , can influence the catalytic activity of the mono-oxygenase complex; (4) cytochrome P-450 is influenced by vitamin D deficiency and/or changes in extracellular calcium but the forms induced by PB and DPH may not necessarily be the ones specifically involved in vitamin D metabolism.

Adult male rats were injected s.c. with 0.4 or 4 mg/kg of nickel chloride, sodium selenite, sodium arsenite, antimony chloride, cadmium chloride or mercuric chloride (0.5 or 4 mg/kg) before being given sodium warfarin s.c. (0.75 mg/kg). Only mercuric and cadmium chlorides were found to significantly prolong the prothrombin time. With mercuric chloride, the effect was dose-dependent. The prolongation of anticoagulant response was present when the administration of mercuric chloride preceded (48, 24 and 12 h), was concomitant with, or followed (6 and 12 h), that of warfarin. When mercuric chloride was given at 18, 21 or 24 h after warfarin, prolongation of the prothrombin time was no longer observed.

A high-performance liquid chromatographic assay for the simultaneous analysis of hippuric and methylhippuric acids in urine is described. Compounds are first extracted from acidified urines after addition of o-methylbenzoylalanine (internal standard), using methyl-t-butyl ether. The organic phase is evaporated under nitrogen flow and the residue dissolved in the mobile phase which consisted of 91% potassium phosphate buffer (12.0mM, pH: 2.0), 4.5% methanol, and 4.5% tetrahydrofuran. The method described allows complete separation of meta- and para-methylhippuric acids in less than 20 min using a stainless steel column packed with octadecyl-dimethysilyl silica. The authors used this method to determine the urinary excretion of hippuric and methylhippuric acids in rabbits following exposure to a mixture of toluene and isomers of xylene. Data indicated that under the actual experimental conditions of exposure o-xylene is excreted to a lesser extent as a methylhippuric acid than the meta- and para-xylenes.

This study was undertaken to investigate the influence of ethanol given either acutely or chronically on the pharmacological response to methaqualone. In order to define the mechanism(s) responsible for the interaction between ethanol and methaqualone, a study was also conducted on the influence of ethanol on microsomal activity, as well as on the disappearance curves of methaqualone in blood and brain. Although acute administration of ethanol (4 g/kg, orally) leads to a progressive inhibition of microsomal enzyme activity towards methaqualone, this is not accompanied by a prolongation of the sleeping time to the hypnotic. Under these conditions animals given both ethanol and methaqualone regain their righting reflex at higher brain concentrations of methaqualone than those given methaqualone only; this can be interpreted as being an acutely acquired tolerance phenomenon resulting from exposure to ethanol. Chronic ingestion (7 and 14 days) of 6% ethanol dissolved in an aqueous solution of 10% dextrose shortens the sleeping time to methaqualone provided the animals have access to ethanol until the administration of methaqualone. Once again, under these conditions, the concentrations of methaqualone in blood and brain at awakening are more elevated in pretreated animals than in control ones. These results suggest the development of a functional tolerance state as a result of ethanol pretreatment. Chronic ingestion of dextrose leads to inhibition of microsomal enzyme activity, yet this is not sufficient to prevent the appearance of a tolerance phenomenon.

In studies of acetone-potentiated liver injury induced by haloalkanes, acetone is usually given by gavage, whereas industrial exposure to acetone normally occurs by inhalation. It was of interest to verify if the route of administration influences the potentiation. Male Sprague-Dawley rats were exposed for 4 hr to acetone vapors or treated orally with acetone; the minimal effective dose (MED) levels for potentiating CCl4-induced liver injury were estimated to be 2500 ppm and 0.25 ml/kg, respectively. Groups were treated with acetone using 0.4, 1, 2, 4, or 6 times the MED. Half of each group was killed at various time intervals after treatment for blood acetone measurements by gas chromatography; the other half was challenged with CCl4 (0.1 ml/kg, ip) 18 hr after acetone, and killed 24 hr later. Plasma alanine aminotransferase (ALT) activity and bilirubin concentrations were measured. Inhalation and oral administration of acetone both potentiated CCl4 toxicity. Rats exposed repetitively to acetone vapors (10 daily exposures) and subsequently challenged with CCl4 exhibited liver toxicity that was not significantly different from that of rats subjected to a single exposure. Correlations between ALT activities and maximal blood acetone concentrations were found to be linear (positive) and significant for both routes. For a given blood acetone concentration, however, toxicity was least severe following acetone exposure by inhalation. When the concept of threshold concentrations was applied to the data, the severity of the toxic response was dependent on the blood acetone concentration above the threshold, irrespective of the route of administration.

Administration of acetone to rats in amounts larger than or equal to a minimal effective dosage (MED) is known to potentiate the severity of the liver damage produced by CCl 4 alone. It has been reported that CCl 4 -induced hepatotoxicity is also enhanced by the previous administration of trichloroethylene (TCE). In addition, TCE-CCl 4 mixture-induced liver injury is potentiated by acetone. The present study was undertaken to determine if the acetone MED is decreased when the haloalkane challenge is a mixture of TCE-CCl 4 instead of CCl 4 alone. The effect of varying mixture compositions was also evaluated. In a first series of experiments, male Sprague-Dawley rats received corn oil or acetone (0.05−0.25 ml/kg, po); 18 hr later, they received an ip injection of either CCl 4 (0.1 ml/kg) or [TCE (0.25 ml/kg)-CCl 4 (0.1 ml/kg)]. In a second series, rats received corn oil or acetone (0.75 ml/kg), and were challenged with TCE (1.5 ml/kg), CCl 4 (0.25 ml/kg), or a mixture of TCE-CCl 4 , where TCE and CCl 4 dosages were equal to 25−75%, 50−50%, and 75-25%, respectively, of those used for the administration of the solvents alone. In both series, rats were killed 24 hr after the haloalkane challenge. Liver injury was assessed biochemically (plasma ALT activities and bilirubin concentrations) and morphologically. When TCE was added to a solution of CCl 4 , smaller doses of CCl 4 were required to produce equally severe liver injury. The MED of acetone required to potentiate CCl 4 -induced liver injury was at least five times smaller when TCE (0.25 ml/kg) was added to the challenge solution of CCl 4 . The severity of the injury produced by TCE-CCl 4 mixtures administered in different proportions was constant, and potentiated to the same extent by a given dose of acetone. These observations suggest that the presence of haloalkane mixtures can result in severe liver injury with lower doses of hepatotoxicants. They further illustrate that prior exposure to acetone may markedly affect the response elicited by the haloalkane mixture.

While the biotransformation of methyl n-butyl ketone (MnBK) in animals is well characterized, little is known about the quantitative relationship between hepatic and plasma MnBK concentrations. This study provides such information and emphasizes the usefulness of MnBK metabolite quantification, as well as MnBK-induced metabolic ketosis for the biological monitoring of MnBK exposure in rats. Elimination of MnBK was followed 24 hr after oral administration (0.95, 1.90, and 5.70 mmol/kg in corn oil) to male Sprague-Dawley rats. Two metabolites [2-hexanol (2HOL), and 2,5-hexanedione (2,5HD)] were also monitored and their kinetics determined. These data were compared to ketone body (KB) concentrations found in plasma and liver during the same period. Plasma concentrations of MnBK and 2,5HD correlated well with those in the liver. This was not the case for 2HOL. MnBK, 2HOL, and 2,5HD were no longer detected in plasma and liver 18 hr after dosing. Meanwhile, a marked ketosis was observed from 12 to 24 hr. This ketotic state was due to an increase in beta-hydroxybutyrate (BOHB), while acetoacetate remained at its basal levels. These data indicate that MnBK can induce ketosis in rats and suggest that the resulting BOHB might be used as an alternative biological monitor of MnBK exposures at high concentrations.

The effects of the concentration of long-chain free fatty acids and bovine serum albumin on the binding characteristics of warfarin with albumin have been studied by adsorption and fluorescence spectroscopy using the method of ultrafiltration. The binding affinity of warfarin is higher in a concentrated than in a dilute albumin solution. In a dilute solution of serum albumin (55 μM or less), the binding constant of the warfarin-albumin complex is increased in the presence of 100 μM (or less) of either lauric or oleic acid or a mixture of these two acids, but higher concentrations of the fatty acids decrease such binding. A mixture of saturated and unsaturated fatty acids shows the same effects as those by either fatty acid. Simultaneous measurements of the fluorescence peak intensity and polarization of fluorescence of warfarin bound to albumin in a dilute solution indicate that low and high concentrations of free fatty acids induce different conformational states of the same warfarin-binding sites on the albumin molecule. Such a dualistic behavior of free fatty acids could be best interpreted in terms of allosteric interactions involving heterotropic effects. On the other hand, in a concentrated solution of serum albumin, the binding constant of warfarin is decreased in the presence of both low and high concentrations of free fatty acids. Such a decrease in warfarin binding with concentrated albumin in the presence of free fatty acids is not due simply to a displacement of warfarin by free fatty acids from the warfarin-binding sites, but also could result simultaneously from a further conformational change of warfarin-binding sites caused by the interaction of warfarin and free fatty acids (a negative heterotropic effect).

Adult male Fischer 344 rats were used to investigate the effect of phenobarbital on methoxyflurane- and sodium fluoride-induced nephropathy. Pretreatment consisted of the ip administration of phenobarbital sodium at a dose of 50 mg/kg/day for 5 days; control animals were given saline. Pretreated animals were exposed to anesthetic concentrations of methoxyflurane during a period of 2 or 4 hr, whereas other pretreated animals were exposed only to oxygen, the methoxyflurane carrier gas. For comparative purposes, other pretreated animals received sodium fluoride in saline at doses of 0, 4.2 and 8.4 mg/300g. The results show that phenobarbital pretreatment enhances the nephrotoxicity of methoxy-flurance as evidenced by a significant interaction between phenobarbital and methoxyflurane on plasma concentrations of sodium and potassium, urinary volume, urinary excretion of urea, and urinary concentration test. In contrast, the nephrotoxicity of sodium fluoride was not affected by phenobarbital. These results provide good evidence that the urinary concentration test is a reliable monitor of the potentiating effect of phenobarbital on methoxyflurane nephrotoxicity.

Postnatal administration of lead to young rats via maternal milk increases the permeability of the blood–brain barrier to trypan blue. However, since this regimen delays postnatal maturation, the question arises whether growth retardation per se could explain the altered permeability to the dye. The present investigation was undertaken to examine this possibility. A first group of newborn rats were fed by dams having free access to normal food; a second group was fed by dams receiving 3% lead as the acetate salt in their food; a third group was fed by dams whose normal diet was restricted to the amount taken daily by dams of the second group. Signs of encephalopathy as shown by urinary incompetence and hind-limb paralysis were observed only in young pups exposed to lead; similarly, only the latter showed increased brain permeability to trypan blue. These results suggest that the altered permeability of the blood–brain barrier in young rats is the direct consequence of lead toxicity and not of growth retardation secondary to a nutritional effect of lead.