Using physiologically based pharmacokinetic modeling and benchmark dose methods to derive an occupational exposure limit for N-methylpyrrolidone.

The developmental effects of NMP are well studied in Sprague-Dawley rats following oral, inhalation, and dermal routes of exposure. Short-term and chronic occupational exposure limit (OEL) values were derived using an updated physiologically based pharmacokinetic (PBPK) model for NMP, along with benchmark dose modeling. Two suitable developmental endpoints were evaluated for human health risk assessment: (1) for acute exposures, the increased incidence of skeletal malformations, an effect noted only at oral doses that were toxic to the dam and fetus; and (2) for repeated exposures to NMP, changes in fetal/pup body weight. Where possible, data from multiple studies were pooled to increase the predictive power of the dose-response data sets. For the purposes of internal dose estimation, the window of susceptibility was estimated for each endpoint, and was used in the dose-response modeling. A point of departure value of 390 mg/L (in terms of peak NMP in blood) was calculated for skeletal malformations based on pooled data from oral and inhalation studies. Acceptable dose-response model fits were not obtained using the pooled data for fetal/pup body weight changes. These data sets were also assessed individually, from which the geometric mean value obtained from the inhalation studies (470 mg*hr/L), was used to derive the chronic OEL. A PBPK model for NMP in humans was used to calculate human equivalent concentrations corresponding to the internal dose point of departure values. Application of a net uncertainty factor of 20-21, which incorporates data-derived extrapolation factors, to the point of departure values yields short-term and chronic occupational exposure limit values of 86 and 24 ppm, respectively.

Methyl isobutyl ketone exposure-related increases in specific measures of α2u-globulin (α2u) nephropathy in male rats along with in vitro evidence of reversible protein binding.

Chronic exposure to methyl isobutyl ketone (MIBK) resulted in an increase in the incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female Fischer 344 rats. Since a number of chemicals have been shown to cause male rat renal tumors through the α2u nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of α2u nephropathy including renal cell proliferation in male and female F344 rats following exposure to the same inhalation concentrations used in the National Toxicology Program (NTP) cancer bioassay (0, 450, 900, or 1800ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2u staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2u concentration. There was an exposure-related increase in all measures of α2u nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2u. The changes in HDA and α2u concentration were comparable to d-limonene, an acknowledged inducer of α2u nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2u, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to α2u and cause nephropathy. Together, the exposure-related increase in measures of α2u nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2u, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2u nephropathy-mediated mode of action (MoA).

Impact of repeated nicotine and alcohol coexposure on in vitro and in vivo chlorpyrifos dosimetry and cholinesterase inhibition.

Chlorpyrifos (CPF) is an organophosphorus insecticide, and neurotoxicity results from inhibition of acetylcholinesterase (AChE) by its metabolite, chlorpyrifos-oxon. Routine consumption of alcohol and tobacco modifies metabolic and physiological processes impacting the metabolism and pharmacokinetics of other xenobiotics, including pesticides. This study evaluated the influence of repeated ethanol and nicotine coexposure on in vivo CPF dosimetry and cholinesterase (ChE) response (ChE- includes AChE and/or butyrylcholinesterase (BuChE)). Hepatic microsomes were prepared from groups of naive, ethanol-only (1 g/kg/d, 7 d, po), and ethanol + nicotine (1 mg/kg/d 7 d, sc)-treated rats, and the in vitro metabolism of CPF was evaluated. For in vivo studies, rats were treated with saline or ethanol (1 g/kg/d, po) + nicotine (1 mg/kg/d, sc) in addition to CPF (1 or 5 mg/kg/d, po) for 7 d. The major CPF metabolite, 3,5,6-trichloro-2-pyridinol (TCPy), in blood and urine and the plasma ChE and brain acetylcholinesterase (AChE) activities were measured in rats. There were differences in pharmacokinetics, with higher TCPy peak concentrations and increased blood TCPy AUC in ethanol + nicotine groups compared to CPF only (approximately 1.8- and 3.8-fold at 1 and 5 mg CPF doses, respectively). Brain AChE activities after ethanol + nicotine treatments showed significantly less inhibition following repeated 5 mg CPF/kg dosing compared to CPF only (96 ± 13 and 66 ± 7% of naive at 4 h post last CPF dosing, respectively). Although brain AChE activity was minimal inhibited for the 1-mg CPF/kg/d groups, the ethanol + nicotine pretreatment resulted in a similar trend (i.e., slightly less inhibition). No marked differences were observed in plasma ChE activities due to the alcohol + nicotine treatments. In vitro, CPF metabolism was not markedly affected by repeated ethanol or both ethanol + nicotine exposures. Compared with a previous study of nicotine and CPF exposure, there were no apparent additional exacerbating effects due to ethanol coexposure.

Effects of nicotine exposure on in vitro metabolism of chlorpyrifos in male Sprague-Dawley rats.

The routine use of tobacco products may modify key metabolizing systems, which will further impact the metabolism of environmental contaminants. The objective of this study was to quantify the effect of repeated in vivo exposures to nicotine, a major pharmacologically active component of cigarette smoke, on in vitro metabolism of chlorpyrifos (CPF). CPF is an organophosphorus (OP) insecticide that is metabolized by cytochrome P-450 (CYP450) to its major metabolites, chlorpyrifos-oxon (CPF-oxon) and 3,5,6-trichloro-2-pyridinol (TCP). Male Sprague-Dawley rats were dosed subcutaneously with 1 mg nicotine/kg for 1, 7, or 10 d. Rats were sacrificed 4 or 24 h after the last nicotine treatment, and liver microsomes were prepared. The microsomes were incubated with varying concentrations of CPF and the production of the metabolites CPF-oxon and TCP were measured. The metabolism of CPF to the active oxon metabolite did not show significant changes following repeated nicotine treatments, evidenced by the unchanged pseudo first-order clearance rate of V(max)/K(mapp). The V(max) describing the metabolism of CPF to the inactive metabolite, TCP was increased in 24-h postdosing groups, after both single and repeated treatments of nicotine. In contrast, the metabolism to TCP was unchanged in groups evaluated at 4 h (single or repeated) post nicotine dosing. Some basic marker substrate activities were also investigated to ensure that nicotine exerted effects on CYP450 activities. Total P450 reduced spectra were not altered by nicotine treatment, but marker substrate activities for CYP1A and CYP2E1 were increased at 24 h after the single treatment, and marker substrate activity for CYP2B was decreased 4 h after 7 d of treatment. Results of this in vitro study suggest that repeated nicotine exposure may result in altered metabolism of CPF. Future in vivo experiments based on these results need to be conducted to ascertain the impact of in vivo nicotine exposures on CPF metabolism in rats.

Development of a physiologically based pharmacokinetic and pharmacodynamic model to determine dosimetry and cholinesterase inhibition for a binary mixture of chlorpyrifos and diazinon in the rat.

Physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models have been developed for the organophosphorus (OP) insecticides chlorpyrifos (CPF) and diazinon (DZN). It is anticipated that these OPs could interact at a number of important metabolic steps including: CYP450 mediated activation/detoxification, B-esterases [carboxylesterase (CaE), butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE)] or PON-1 (A-esterase) oxon detoxification. We developed a binary PBPK/PD model for CPF, DZN and their metabolites based on previously published models for the individual insecticides. The metabolic interactions (CYP450) between CPF and DZN were evaluated in vitro and suggests that CPF is more substantially metabolized to its oxon metabolite than DZN, which is consistent with observed in vivo potency (CPF>DZN). Each insecticide inhibited the other's in vitro metabolism in a concentration-dependent manner. The PBPK model code used to describe the metabolism of CPF and DZN was modified to reflect the type of CYP450 inhibition kinetics (i.e. competitive vs. non-competitive), while B-esterase metabolism was described as dose-additive, and no PON-1 interactions were assumed between CPF- and DZN-oxon with the enzyme. The binary model was then evaluated against previously published rodent dosimetry and cholinesterase (ChE) inhibition data for the mixture. The PBPK/PD model simulations of the acute oral exposure to single-mixtures (15 mg/kg) vs. binary-mixtures (15+15 mg/kg) of CFP and DZN resulted in no differences in the predicted pharmacokinetics of either the parent OPs or their respective metabolites, while cholinesterase inhibition was reasonably described using the dose-additive model. A binary oral dose of CPF+DZN (60+60 mg/kg) did result in observable changes in the DZN pharmacokinetics where C(max) was more reasonably fit by modifying the absorption parameters. It is anticipated that at low environmentally relevant binary doses, most likely to be encountered in occupational or environmental related exposures, that the pharmacokinetics are expected to be linear, and ChE inhibition dose-additive.

Age-dependent pharmacokinetic and pharmacodynamic response in preweanling rats following oral exposure to the organophosphorus insecticide chlorpyrifos.

Previous studies have indicated that juvenile rats are more susceptible than adults to the acute toxicity from exposure to the organophosphorus insecticide chlorpyrifos (CPF) and age-dependent differences in metabolism and sensitivity to cholinesterase (ChE) inhibition may be responsible. Metabolism involves CYP450 activation and detoxification of CPF to CPF-oxon and 3,5,6-trichloro-2-pyridinol (TCP), as well as cholinesterase (acetyl- and butyrylcholinesterase), carboxylesterase (CaE), and A-esterase (PON-1) detoxification of CPF-oxon to TCP. The pharmacokinetics of CPF, TCP, and the extent of blood (plasma/RBC), and brain ChE inhibition in rats were determined on postnatal days (PND)-5, -12, and -17 following oral gavage administration of 1 and 10mg CPF/kg of body weight. As has been seen in adult animals, for all preweanling ages the blood TCP exceeded the CPF concentration, and within each age group there was no evidence of non-linear kinetics over the dose range evaluated. Consistent with previous results, younger animals demonstrated a greater sensitivity to ChE inhibition as evident by the age-dependent inhibition of plasma, RBC, and brain ChE. The brain may be particularly sensitive in younger animals (i.e. PND-5) due to substantially lower levels of ChE activity relative to later preweanling stages and adults. Of particular importance was the observation that even in rats as young as PND-5, the CYP450 metabolic capacity was adequate to metabolize CPF to both TCP and CPF-oxon based on the detection of TCP in blood and extensive ChE inhibition (biomarker of CPF-oxon) at all ages. In addition, the increase in the blood TCP concentration ( approximately 3-fold) in PND-17 rats relative to the response in the younger rats, are consistent with an increase in CYP450 metabolic capacity with age. This is the first reported study that evaluated both the pharmacokinetics of the parent pesticide, the major metabolite, and the extent of ChE inhibition as a function of preweanling age. The results suggest that in the preweanling rat, CPF was rapidly absorbed and metabolized, and the extent of metabolism and ChE inhibition was age-dependent.

Pharmacokinetic and pharmacodynamic interaction for a binary mixture of chlorpyrifos and diazinon in the rat.

Chlorpyrifos (CPF) and diazinon (DZN) are two commonly used organophosphorus (OP) insecticides and a potential exists for concurrent exposures. The primary neurotoxic effects from OP pesticide exposures result from the inhibition of acetylcholinesterase (AChE). The pharmacokinetic and pharmacodynamic impact of acute binary exposures of rats to CPF and DZN was evaluated in this study. Rats were orally administered CPF, DZN, or a CPF/DZN mixture (0, 15, 30, or 60 mg/kg) and blood (plasma and RBC), and brain were collected at 0, 3, 6, 12, and 24 h postdosing, urine was also collected at 24 h. Chlorpyrifos, DZN, and their respective metabolites, 3,5,6-trichloro-2-pyridinol (TCP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP), were quantified in blood and/or urine and cholinesterase (ChE) inhibition was measured in brain, RBC, and plasma. Coexposure to CPF/DZN at the low dose of 15/15 mg/kg did not alter the pharmacokinetics of CPF, DZN, or their metabolites in blood. A high binary dose of 60/60 mg/kg increased the C(max) and AUC and decreased the clearance for both parent compounds, likely due to competition between CPF and DZN for CYP450 metabolism. At lower doses, most likely to be encountered in occupational or environmental exposures, the pharmacokinetics were linear. A dose-dependent inhibition of ChE was noted in tissues for both the single and coexposures, and the extent of inhibition was plasma > RBC > or = brain. The overall relative potency for ChE inhibition was CPF/DZN > CPF > DZN. A comparison of the ChE response at the low binary dose (15/15 mg/kg), where there were no apparent pharmacokinetic interactions, suggested that the overall ChE response was additive. These experiments represent important data concerning the potential pharmacokinetic and pharmacodynamic interactions for pesticide mixtures and will provide needed insight for assessing the potential cumulative risk associated with occupational or environmental exposures to these insecticides.

Derivation of a human equivalent concentration for n-butanol using a physiologically based pharmacokinetic model for n-butyl acetate and metabolites n-butanol and n-butyric acid.

The metabolic series approach for risk assessment uses a dosimetry-based analysis to develop toxicity information for a group of metabolically linked compounds using pharmacokinetic (PK) data for each compound and toxicity data for the parent compound. The metabolic series approach for n-butyl acetate and its subsequent metabolites, n-butanol and n-butyric acid (the butyl series), was first demonstrated using a provisional physiologically based pharmacokinetic (PBPK) model for the butyl series. The objective of this work was to complete development of the PBPK model for the butyl series. Rats were administered test compounds by iv bolus dose, iv infusion, or by inhalation in a recirculating closed chamber. Hepatic, vascular, and extravascular metabolic constants for metabolism were estimated by fitting the model to the blood time course data from these experiments. The respiratory bioavailability of n-butyl acetate (100% of alveolar ventilation) and n-butanol (50% of alveolar ventilation) was estimated from closed chamber inhalation studies and measured ventilation rates. The resulting butyl series PBPK model successfully reproduces the blood time course of these compounds following iv administration and inhalation exposure to n-butyl acetate and n-butanol in rats and arterial blood n-butanol kinetics following inhalation exposure to n-butanol in humans. These validated inhalation route models can be used to support species and dose-route extrapolations required for risk assessment of butyl series family of compounds. Human equivalent concentrations of 169 ppm and 1066 ppm n-butanol corresponding to the rat n-butyl acetate NOAELs of 500 and 3000 ppm were derived using the models.

Determination of age and gender differences in biochemical processes affecting the disposition of 2-butoxyethanol and its metabolites in mice and rats to improve PBPK modeling.

2-Butoxyethanol (BE) is the most widely used glycol ether solvent. BEs major metabolite, butoxyacetic acid (BAA), causes hemolysis with significant species differences in sensitivity. Several PBPK models have been developed over the past two decades to describe the disposition of BE and BAA in male rats and humans to refine health risk assessments. More recent efforts by Lee et al. [Lee, K.M., Dill, J.A., Chou, B.J., Roycroft, J.H., 1998. Physiologically based pharmacokinetic model for chronic inhalation of 2-butoxyethanol. Toxicol. Appl. Pharmacol. 153, 211-226] to describe the kinetics of BE and BAA in the National Toxicology Program (NTP) chronic inhalation studies required the use of several assumptions to extrapolate model parameters from earlier PBPK models developed for young male rats to include female F344 and both sexes of B6C3F1 mice and the effects of aging. To replace these assumptions, studies were conducted to determine the impact of age, gender and species on the metabolism of BE, and the tissue partitioning, renal acid transport and plasma protein binding of BAA. In the current study, the Lee et al. PBPK model was updated and expanded to include the further metabolism of BAA and the salivary excretion of BE and BAA which may contribute to the forestomach irritation observed in mice in the NTP study. The revised model predicted that peak blood concentrations of BAA achieved following 6 h inhalation exposures are greatest in young adult female rats at concentrations up to 300 ppm. This is not the case predicted for old (> or =18 months) animals, where peak blood concentrations of BAA in male and female mice were similar to or greater than female rats. The revised model serves as a quantitative tool for integrating an extensive pharmacokinetic and mechanistic database into a format that can readily be used to compare internal dosimetry across dose, route of exposure and species.

Physiologically based pharmacokinetic/pharmacodynamic model for the organophosphorus pesticide diazinon.

Diazinon (DZN) is an organophosphorus pesticide with the possibility for widespread exposures. The toxicological effects of DZN are primarily mediated through the effects of its toxic metabolite, DZN-oxon on acetylcholinesterases, which results in accumulation of acetylcholine at neuronal junctions. A physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model was developed to quantitatively assess the kinetics of DZN and its metabolites in blood and the inhibition of cholinesterases in plasma, RBC, brain, and diaphragm. Focused in vivo pharmacokinetic studies were conducted in male Sprague-Dawley rats and the data were used to refine the model. No overt toxicity was noted following doses up to 100mg/kg. However, cholinesterases in plasma, RBC, brain and diaphragm were substantially inhibited at doses of 50 mg/kg. In plasma, total cholinesterase was inhibited to less than 20% of control by 6 h post dosing with 100 mg/kg. Inhibition of brain acetylcholinesterase (AChE) following 100 mg/kg exposures was approximately 30% of control by 6 h. Diaphragm butyrylcholinesterase (BuChE) inhibition following 100 mg/kg dosing was to less than 20% of control by 6 h. The PBPK/PD model was used to describe the concentrations of DZN and its major, inactive metabolite, 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP) in plasma and urinary elimination of IMHP. The fit of the model to plasma, RBC, brain, and diaphragm total cholinesterase and BuChE activity was also assessed and the model was further validated by fitting data from the open literature for intraperitoneal, intravenous, and oral exposures to DZN. The model was shown to quantitatively estimate target tissue dosimetry and cholinesterase inhibition following several routes of exposures. This model further confirms the usefulness of the model structure previously validated for chlorpyrifos and shows the potential utility of the model framework for other related organophosphate pesticides.

Comparison of chlorpyrifos-oxon and paraoxon acetylcholinesterase inhibition dynamics: potential role of a peripheral binding site.

The primary mechanism of action for organophosphorus (OP) insecticides, like chlorpyrifos and parathion, is to inhibit acetylcholinesterase (AChE) by their oxygenated metabolites (oxons), due to the phosphorylation of the serine hydroxyl group located in the active site of the molecule. The rate of phosphorylation is described by the bimolecular inhibitory rate constant (k(i)), which has been used for quantification of OP inhibitory capacity. It has been proposed that a peripheral binding site exists on the AChE molecule, which, when occupied, reduces the capacity of additional oxon molecules to phosphorylate the active site. The aim of this study was to evaluate the interaction of chlorpyrifos oxon (CPO) and paraoxon (PO) with rat brain AChE to assess the dynamics of AChE inhibition and the potential role of a peripheral binding site. The k(i) values for AChE inhibition determined at oxon concentrations of 1-100 nM were 0.206 +/- 0.018 and 0.0216 nM(-1)h(-1) for CPO and PO, respectively. The spontaneous reactivation rates of the inhibited AChE for CPO and PO were 0.084-0.087 (two determinations) and 0.091 +/- 0.023 h(-1), respectively. In contrast, the k(i) values estimated at a low oxon concentration (1 pM) were approximately 1,000- and 10,000-fold higher than those determined at high CPO and PO concentrations, respectively. At low concentrations, the k(i) estimates were approximately similar for both CPO and PO (150-180 [two determinations] and 300 +/- 180 nM(-1)h(-1), respectively). This implies that, at low concentrations, both oxons exhibited similar inhibitory potency in contrast to the marked difference exhibited at higher concentrations. These results support the potential importance of a secondary peripheral binding site associated with AChE kinetics, particularly at low, environmentally relevant concentrations.

Characterization of the in vitro kinetic interaction of chlorpyrifos-oxon with rat salivary cholinesterase: a potential biomonitoring matrix.

The primary mechanism of action for organophosphorus (OP) insecticides such as chlorpyrifos (CPF) involves the inhibition of acetylcholinesterase (AChE) by their active oxon metabolites resulting in a wide range of neurotoxic effects. These oxons also inhibit other cholinesterases (ChE) such as butyrylcholinesterase (BuChE), which represents a detoxification mechanism and a potential biomarker for OP insecticide exposure/response. Salivary biomonitoring has recently been explored as a practical method for examination of chemical exposure, however, there are few studies exploring the use of saliva for OP insecticides. To evaluate the use of salivary ChE as a biological monitor for OP insecticide exposure, a modified Ellman assay in conjunction with a pharmacodynamic model was used to characterize salivary ChE in adult male Sprague-Dawley rats. Comparison of rat saliva, brain, and plasma ChE activity in the presence of selective inhibitors of AChE and BuChE (BW284C51 and iso-OMPA, respectively) with different ChE substrates indicated that rat salivary ChE activity is primarily associated with BuChE (>95%). Further characterization of rat salivary BuChE kinetics yielded an average total BuChE active site concentration of 1.20+/-0.13 fmol ml(-1) saliva, an average reactivation rate constant (Kr) of 0.070+/-0.008 h(-1), and an inhibitory rate constant (Ki) of approximately 9 nM(-1) h(-1). The pharmacodynamic model successfully described the in vitro BuChE activity profile as well as the kinetic parameters. These results support the potential utility of saliva as a biomonitoring matrix for evaluating occupational and environmental exposure to CPF and other OP insecticides.

In vitro rat hepatic and intestinal metabolism of the organophosphate pesticides chlorpyrifos and diazinon.

Chlorpyrifos (CPF) and diazinon (DZN) are thionophosphorus organophosphate (OP) insecticides; their toxicity is mediated through CYP metabolism to CPF-oxon and DZN-oxon, respectively. Conversely, CYPs also detoxify these OPs to trichloropyridinol (TCP) and 2-isopropyl-4-methyl-6-hydroxypyrimidine (IMHP), respectively. In addition, A-esterase (PON1) metabolism of CPF- and DZN-oxon also forms TCP and IMHP. This study evaluated the role intestinal and hepatic metabolism may play in both the activation and detoxification of CPF and DZN in Sprague-Dawley rats. Similar CYP- and PON1-mediated metabolic profiles were demonstrated in microsomes from liver or isolated intestinal enterocytes. The metabolic efficiency was estimated by calculating the psuedo-1st order rate constant from the metabolic constants by dividing Vmax/Km. In enterocyte microsomes, the CYP metabolic efficiency for metabolism to the oxon metabolites was approximately 28-fold greater for CPF than DZN. Compared on a per nmol P450 basis, the Vmax for CPF in enterocytes was approximately 2-3 times higher than in liver microsomes for the production of CPF-oxon and TCP. The Michaelis-Menten rate constant (Km) for the metabolism of CPF to CPF-oxon was comparable in liver and enterocyte microsomes; however, the enterocyte Km for TCP production was higher (indicating a lower affinity). The smaller volume of intestine, lower amount of CYP, and higher Km for TCP in the enterocyte microsomes, resulted in a lower catalytic efficiency (2 and 62 times) than in liver for oxon and TCP. PON1-mediated metabolism of CPF- and DZN-oxon was also demonstrated in liver and enterocyte microsomes. Although PON1 affinity for the substrates was comparable in hepatic and enterocytic microsomes, the Vmax were 48- to 275-fold higher, in the liver. These results suggest that intestinal metabolism may impact the metabolism of CPF and DZN, especially following low-dose oral exposures.

Monte Carlo analysis of the human chlorpyrifos-oxonase (PON1) polymorphism using a physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model.

Susceptibility to organophosphate (OP) insecticides, like chlorpyrifos (CPF), may result from differences in the extent of metabolic detoxification of the active metabolite, CPF-oxon. A genetic polymorphism in the arylesterase (PON1; CPF-oxonase) detoxification of OPs, results in the expression of a range of enzyme activities within humans. This study utilized Monte Carlo analysis and physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) modeling to investigate the impact of human CPF-oxonase status on the theoretical concentration of CPF-oxon in the brain. At low doses ( approximately 5 microg/kg) the model is insensitive to changes in CPF-oxonase. However, with increasing dose (>0.5 mg/kg) the model suggests a dose-dependent non-linear increase in the brain CPF-oxon concentration, which is associated with CPF-oxonase activity. Following repeated high dose exposure, the model predicted brain CPF-oxon concentration was approximately 8x higher (5 mg/kg) versus a single exposure, whereas, at low doses (5 microg/kg), the brain concentrations were comparable regardless of exposure duration. This suggests that at low environmentally relevant exposures other esterase detoxification pathways may compensate for lower CPF-oxonase activity.

Development of an integrated microanalytical system for analysis of lead in saliva and linkage to a physiologically based pharmacokinetic model describing lead saliva secretion.

There is a need to develop reliable portable analytical systems for biomonitoring lead (Pb) in noninvasively collected saliva samples. In addition, appropriate pharmacokinetic analyses are used to quantitate systemic dosimetry based on the saliva Pb concentrations. A portable microfluidics/electrochemical device was developed for the rapid analysis of Pb based on square wave anodic stripping voltammetry, in which a saliva sample flows over an electrode surface, Pb2+ is chemically reduced and accumulated, and the electric potential of the electrode scanned. The system demonstrates a good linear response over a broad Pb concentration range (1-2000 ppb). To evaluate the relationship between saliva and blood Pb, rats were treated with single oral doses ranging from 20 to 500 mg Pb/kg of body weight, and 24 hours later were administered pilocarpine, a muscarinic agonist to induce salivation. To correlate saliva levels with internal dose, blood and saliva were collected and quantitated for Pb by inductively coupled plasma-mass spectrometry (ICP-MS) and by the microanalytical system. The quantitation with the microanalytical system was slightly less (approximately 75-85%) than with ICP-MS; however, the response was linear, with concentration suggesting that it can be used for the quantitation of salivary Pb. To facilitate modeling, a physiologically based pharmacokinetic (PBPK) model for Pb was modified to incorporate a salivary gland compartment. The model was capable of predicting blood and saliva Pb concentration based on a limited data set. These results are encouraging, suggesting that once fully developed the microanalytical system coupled with PBPK modeling can be used as important tools for real-time biomonitoring of Pb for both occupational and environmental exposures.

Assessing dermal absorption.

The article highlighted in this issue is "Comparative in Vitro-in Vivo Percutaneous Absorption of the Pesticide Propoxur" by Johannes J. M. van de Sandt, Wim J. A. Meuling, Graham R. Elliott, Nicole H. P. Cnubben, and Betty C. Hakkert (pp 15-22).

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling.

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.

Determination of biokinetic interactions in chemical mixtures using real-time breath analysis and physiologically based pharmacokinetic modeling.

Regulatory agencies are challenged to conduct risk assessments on chemical mixtures without full information on toxicological interactions that may occur at real-world, low-dose exposure levels. The present study was undertaken to investigate the pharmacokinetic impact of low-dose coexposures to toluene and trichloroethylene in vivo in male F344 rats using a real-time breath analysis system coupled with physiologically based pharmacokinetic (PBPK) modeling. Rats were exposed to compounds alone or as a binary mixture, at low (5 to 25 mg/kg) or high (240 to 800 mg/kg) dose levels. Exhaled breath from the exposed animals was monitored for the parent compounds and a PBPK model was used to analyze the data. At low doses, exhaled breath kinetics from the binary mixture exposure compared with those obtained during single exposures, thus indicating that no metabolic interaction occurred with these low doses. In contract, at higher doses the binary PBPK model simulating independent metabolism was found to underpredict the exhaled breath concentration, suggesting an inhibition of metabolism. Therefore the binary mixture PBPK model was used to compare the measured exhaled breath levels from high- and low-dose exposures with the predicted levels under various metabolic interaction simulations (competitive, noncompetitive, or uncompetitive inhibition). Of these simulations, the optimized competitive metabolic interaction description yielded a Ki value closest to the Km of the inhibitor solvent, indicating that competitive inhibition is the most plausible type of metabolic interaction between these two solvents.

Assessment of the percutaneous absorption of trichloroethylene in rats and humans using MS/MS real-time breath analysis and physiologically based pharmacokinetic modeling.

The development and validation of noninvasive techniques for estimating the dermal bioavailability of solvents in contaminated soil and water can facilitate the overall understanding of human health risk. To assess the dermal bioavailability of trichloroethylene (TCE), exhaled breath was monitored in real time using an ion trap mass spectrometer (MS/MS) to track the uptake and elimination of TCE from dermal exposures in rats and humans. A physiologically based pharmacokinetic (PBPK) model was used to estimate total bioavailability. Male F344 rats were exposed to TCE in water or soil under occluded or nonoccluded conditions by applying a patch to a clipper-shaved area of the back. Rats were placed in off-gassing chambers and chamber air TCE concentration was quantified for 3-5 h postdosing using the MS/MS. Human volunteers were exposed either by whole-hand immersion or by attaching patches containing TCE in soil or water on each forearm. Volunteers were provided breathing air via a face mask to eliminate inhalation exposure, and exhaled breath was analyzed using the MS/MS. The total TCE absorbed and the dermal permeability coefficient (K(P)) were estimated for each individual by optimization of the PBPK model to the exhaled breath data and the changing media and/or dermal patch concentrations. Rat skin was significantly more permeable than human skin. Estimates for K(P) in a water matrix were 0.31 +/- 0.01 cm/h and 0.015 +/- 0.003 cm/h in rats and humans, respectively. K(P) estimates were more than three times higher from water than soil matrices in both species. K(P) values calculated using the standard Fick's Law equation were strongly affected by exposure length and volatilization of TCE. In comparison, K(P) values estimated using noninvasive real-time breath analysis coupled with the PBPK model were consistent, regardless of volatilization, exposure concentration, or duration.

A real-time in-vivo method for studying the percutaneous absorption of volatile chemicals.

Realistic estimates of percutaneous absorption following exposures to solvents in the workplace, or through contaminated soil and water, are critical to understanding human health risks. A method was developed to determine dermal uptake of solvents under non-steady-state conditions using real-time breath analysis in rats, monkeys, and humans. The exhaled breath was analyzed using an ion-trap mass spectrometer, which can quantitate chemicals in the exhaled breath stream in the 1-5 ppb range. The resulting data were evaluated using physiologically-based pharmacokinetic (PBPK) models to estimate dermal permeability constants (Kp) under various exposure conditions. The effects of exposure matrix (soil versus water), occlusion versus non-occlusion, and species differences on the absorption of methyl chloroform, trichloroethylene, and benzene were compared. Exposure concentrations were analyzed before and at 0.5-hour intervals throughout the exposures. The percentage of each chemical absorbed and the corresponding Kp were estimated by optimization of the PBPK model to the medium concentration and the exhaled-breath data. The method was found to be sufficiently sensitive for animal and human dermal studies at low exposure concentrations over small body surface areas, for short periods, using non-steady-state exposure conditions.