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.

3D 3He diffusion MRI as a local in vivo morphometric tool to evaluate emphysematous rat lungs.

In this work, we investigate (3)He magnetic resonance imaging as a noninvasive morphometric tool to assess emphysematous disease state on a local level. Emphysema was induced intratracheally in rats with 25 U/100 g body wt of porcine pancreatic elastase dissolved in 200 microl saline. Rats were then paired with saline-dosed controls. Nine three-dimensional (3D) (3)He diffusion-weighted images were acquired at 1, 2, or 3 wk postdose, after which the lungs were harvested and prepared for histological analysis. Recently introduced indexes sensitive to the heterogeneity of the air space size distribution were calculated. These indexes, D(1) and D(2), were derived from the moments of the mean equivalent airway diameters. Averaged over the entire lung, it is shown that the average (3)He diffusivity (D(ave)) correlates well with histology (R = 0.85, P < 0.0001). By matching small (0.046 cm(2)) regions in (3)He images with corresponding regions in histological slices, D(ave) correlates significantly with both D(1) and D(2) (R = 0.88 and R = 0.90, respectively, with P < 0.0001). It is concluded that (3)He MRI is a viable noninvasive morphometric tool for localized in vivo emphysema assessment.

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.

Disposition of lead (Pb) in saliva and blood of Sprague-Dawley rats following a single or repeated oral exposure to Pb-acetate.

Biological monitoring for lead (Pb) is usually based upon a determination of blood Pb concentration; however, saliva has been suggested as a non-invasive biological matrix for assessing exposure. To further evaluate the potential utility of saliva for biomonitoring, the disposition of Pb was evaluated in whole blood (WB), red blood cells (RBC), plasma, parotid gland, bone, and saliva following either a single oral dose of 100mg Pb-acetate/kg body weight in rats or approximately 1-week after 5 sequential daily oral gavage doses of 1, 10, or 100mg Pb-acetate/kg/day. Saliva volume, pH, total saliva protein, and alpha-amylase activity were also determined. At specified times post-dosing groups of animals were anesthetized and administered pilocarpine to induce salivation. Saliva was collected, the animals were humanely sacrificed, and tissue samples were likewise collected, weighed, and processed for Pb analysis. Following a single dose exposure to Pb-acetate, Pb was detectable in all samples by 30 min post-dosing. For both the single and repeated dose treatments the concentration of Pb was highest in WB and RBC relative to plasma and saliva. However, the Pb rapidly redistributed (within 5-days post-treatment) from the blood into the bone compartment based on the substantial decrease in WB and RBC Pb concentration, and the concurrent increase in bone Pb following repeated exposure at all dose levels. Although there is clear variability in the observed Pb concentrations in plasma and saliva, there was a reasonable correlation (r(2)=0.922) between the average Pb concentrations in these biological matrices, which was consistent with previous observations. The single oral dose of Pb-acetate resulted in a decrease in salivary pH which recovered by 24h post-dosing and a decrease in alpha-amylase enzyme activity which did recover within 5-days of ceasing exposure. It is currently unclear what impact these slight functional changes may or may not have on Pb salivary clearance rates. These results demonstrate a feasibility to rapidly detect Pb in saliva and suggest that saliva may correlate best with plasma Pb concentration.

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.

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.

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.

A Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the organophosphate insecticide chlorpyrifos in rats and humans.

A PBPK/PD model was developed for the organophosphate insecticide chlorpyrifos (CPF) (O,O-diethyl-O-[3,5,6-trichloro-2-pyridyl]-phosphorothioate), and the major metabolites CPF-oxon and 3,5,6-trichloro-2-pyridinol (TCP) in rats and humans. This model integrates target tissue dosimetry and dynamic response (i.e., esterase inhibition) describing uptake, metabolism, and disposition of CPF, CPF-oxon, and TCP and the associated cholinesterase (ChE) inhibition kinetics in blood and tissues following acute and chronic oral and dermal exposure. To facilitate model development, single oral-dose pharmacokinetic studies were conducted in rats (0.5-100 mg/kg) and humans (0.5-2 mg/kg), and the kinetics of CPF, CPF-oxon, and TCP were determined, as well as the extent of blood (plasma/RBC) and brain (rats only) ChE inhibition. In blood, the concentration of analytes followed the order TCP >> CPF >> CPF-oxon; in humans CPF-oxon was not quantifiable. Simulations were compared against experimental data and previously published studies in rats and humans. The model was utilized to quantitatively compare dosimetry and dynamic response between rats and humans over a range of CPF doses. The time course of CPF and TCP in both species was linear over the dose range evaluated, and the model reasonably simulated the dose-dependent inhibition of plasma ChE, RBC acetylcholinesterase (AChE), and brain (rat only) AChE. Model simulations suggest that rats exhibit greater metabolism of CPF to CPF-oxon than humans do, and that the depletion of nontarget B-esterase is associated with a nonlinear, dose-dependent increase in CPF-oxon blood and brain concentration. This CPF PBPK/PD model quantitatively estimates target tissue dosimetry and AChE inhibition and is a strong framework for further organophosphate (OP) model development and for refining a biologically based risk assessment for exposure to CPF under a variety of scenarios.

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.

Potential technology for studying dosimetry and response to airborne chemical and biological pollutants.

Advances in computational, and imaging techniques have enabled the rapid development of three-dimensional (3-D) models of biological systems in unprecedented detail. Using these advances, 3-D models of the lungs and nasal passages of the rat and human are being developed to ultimately improve predictions of airborne pollutant dosimetry. Techniques for imaging the respiratory tract by magnetic resonance imaging (MRI) were developed to improve the speed and accuracy of geometric data collection for mesh reconstruction. The MRI resolution is comparable to that obtained by manual measurements but at much greater speed and accuracy. Newly developed software (NWGrid) was utilized to translate imaging data from MR into 3-D mesh structures. Together, these approaches significantly reduced the time to develop a 3-D model. This more robust airway structure will ultimately facilitate modeling gas or vapor exchange between the respiratory tract and vasculature as well as enable linkages of dosimetry with cell response models. The 3-D, finite volume, viscoelastic mesh structures form the geometric basis for computational fluid dynamics modeling of inhalation, exhalation and the delivery of individual particles (or concentrations of gas or vapors) to discrete regions of the respiratory tract. The ability of these 3-D models to resolve dosimetry at such a high level of detail will require new techniques to measure regional airflows and particulate deposition for model validation.

The pharmacokinetics and metabolism of 14C/13C-labeled ortho-phenylphenol formation following dermal application to human volunteers.

1. The pharmacokinetics and metabolism of uniformly labeled 14C/13C-ortho-phenylphenol (OPP) were followed in six human male volunteers given a single 8 h dermal dose of 6 microg OPP/kg body weight formulated as a 0.4% (w/v) solution in isopropyl alcohol. The application site was covered with a non-occlusive dome allowing free movement of air, but preventing the loss of radioactivity due to physical contact. At 8 h post-exposure the non-occlusive dome was removed, the dose site was wiped with isopropyl alcohol containing swabs and the skin surface repeatedly stripped with tape. Blood specimens, urine, and feces were collected from each volunteer over a 5 day post-exposure period and were analyzed for radioactivity and metabolites (urine only). 2. Following dermal application, peak plasma levels of radioactivity were obtained within 4 h post-exposure and rapidly declined with virtually all of the absorbed dose rapidly excreted into the urine within 24 h post-exposure. A one-compartment pharmacokinetic model was used to describe the time-course of OPP absorption and clearance in male human volunteers. Approximately 43% of the dermally applied dose was absorbed through the skin with an average absorption half-life of 10 h. Once absorbed the renal clearance of OPP was rapid with an average half-life of 0.8 h. The rate limiting step for renal clearance was the relatively slower rate of dermal absorption; therefore the pharmacokinetics of OPP in humans was described by a 'flip-flop' single compartment model. Overall, the pharmacokinetics were similar between individuals, and the model parameters were in excellent agreement with the experimental data. 3. Approximately 73% of the total urinary radioactivity was accounted for as free OPP, OPP-sulfate and OPP-glucuronide conjugates. The sulfate conjugate was the major metabolite (approximately 69%). Therefore, total urinary OPP equivalents (acid-labile conjugates+free OPP) can be used to estimate the systemically absorbed dose of OPP. 4. The rapid excretion of OPP and metabolites into the urine following dermal exposure indicates that OPP is unlikely to accumulate in humans upon repeated exposure. Based on these data, blood and/or urinary OPP concentration (acid-labile conjugates) could be utilized to quantify the amount of OPP absorbed by humans under actual use conditions.

Comparative metabolism of ortho-phenylphenol in mouse, rat and man.

1. Ortho-phenylphenol (OPP) was well absorbed in the male B6C3F1 mouse, with 84 and 98% of the administered radioactivity recovered in the 0-48-h urine of animals administered a single oral dose of 15 or 800 mg/kg respectively. High absorption and rapid elimination were also seen in the female and male F344 rat with 86 and 89% respectively of a single oral dose (27-28 mg/kg) found in the urine in 24 h. OPP was also rapidly eliminated from human volunteers following dermal exposure for 8 h (0.006 mg/kg), with 99% of the absorbed dose in the urine in 48 h. 2. Sulphation of OPP was found to be the major metabolic pathway at low doses in all three species, accounting for 57, 82 and 69% of the urinary radioactivity in the male mouse (15 mg/kg, p.o.), male rat (28 mg/kg, p.o.) and male human volunteers (0.006 mg/kg, dermal). OPP-glucuronide was also present in all species, representing 29, 7 and 4% of the total urinary metabolites in the low dose groups of mouse, rat and human volunteers respectively. 3. Conjugates of 2-phenylhydroquinone (PHQ) in these single-dose studies accounted for 12, 5 and 15% of the dose in mouse, rat and human, respectively. Little or no free OPP was found in any species. No free PHQ or PBQ was found in the mouse, rat or human (LOD = 0.1-0.6%). 4. A novel metabolite, the sulphate conjugate of 2,4'-dihydroxybiphenyl, was identified in rat and man, comprising 3 and 13% of the low dose respectively. 5. Dose-dependent shifts in metabolism were seen in the mouse for conjugation of parent OPP, indicating saturation of the sulphation pathway. Dose-dependent increases in total PHQ were also observed in mouse. 6. This study was initiated to elucidate a mechanistic basis for the difference in carcinogenic potential for OPP between rat and mouse. However, the minor differences seen in the metabolism of OPP in these two species do not appear to account for the differences in urinary bladder toxicity and tumour response between mouse and rat.

Pharmacokinetics of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) in the beagle dog and rhesus monkey: perspective on the reduced capacity of dogs to excrete this organic acid relative to the rat, monkey, and human.

The pharmacokinetics of triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid) were measured in the beagle dog and rhesus monkey and compared with the kinetics observed in rats and humans. In addition, studies were conducted in anesthetized dogs to better understand the mechanism by which [14C]triclopyr is eliminated in this species. Triclopyr was dissolved in distilled water, and administered as a single oral dose of 0.5, 5, or 20 mg/kg to three male dogs. A single male rhesus monkey was given an intravenous dose of 30 mg [14C]triclopyr/kg body wt on two occasions separated by 10 days. Anesthetized male dogs, were implanted with venous, arterial, and urethral catheters and given increasing amounts of triclopyr to produce plasma triclopyr levels ranging from 0.3 to 27 microg eq/mL. In the monkey, triclopyr was rapidly eliminated from the plasma (t1/2 = 6.3 hr) with >95% of the urinary 14C activity excreted within 24 hr postdosing. In the dog, orally administered triclopyr was rapidly and effectively absorbed at every dose level with virtually all of it excreted in the urine by 72 hr postdosing. However, the kinetics were slightly nonlinear, and the fraction of the dose excreted in the urine decreased with increasing dose. Several nonlinear processes may collectively contribute to the modest nonlinear pharmacokinetics in the dog. Plasma protein binding of triclopyr in the dog ranged from 94 to 99%, was nonlinear, and was an important determinant in the renal clearance of triclopyr. The nonlinear plasma protein binding indicates that glomerular filtration became disproportionately more important as plasma triclopyr concentration increased. There was good evidence for a high-affinity low-capacity active-secretory process that was saturated by low plasma triclopyr concentrations. As plasma triclopyr concentrations increased, tubular reabsorption begins to exceed secretion, resulting in decreased renal clearance. The volume of distribution, normalized for body weight, was constant across all species. While clearance and half-life could be allometrically scaled to body weight for the rat, monkey, and human, the dog had a much slower clearance and longer half-life for triclopyr elimination than predicted allometrically. These data demonstrate that the pharmacokinetics of triclopyr in the dog are markedly different than in rat, monkey, and human.

Evaluation of renal function in rhesus monkeys and comparison to beagle dogs following oral administration of the organic acid triclopyr (3,5,6-trichloro-2-pyridinyloxyacetic acid).

The current study evaluated the effects of triclopyr (3,5, 6-trichloro-2-pyridinyloxyacetic acid) on renal function following oral administration in the beagle dog and rhesus monkey. Male rhesus monkeys were orally administered triclopyr by gavage at a dose of 5 mg/kg/day, 7 days/week for 28 days, after which the dosage was increased to 20 mg/kg/day for 102 consecutive days. Groups of male dogs were administered either a single oral dose of 5 mg/kg triclopyr or were fed a diet spiked with triclopyr at a dose of 5 mg/kg/day for 47 consecutive days. The following functional and clinical chemistry parameters were evaluated: exogenous phenolsulfonphthalein (PSP) excretion, inulin and para-aminohippurate (PAH) clearance (monkeys only), endogenous serum creatinine, and blood urea nitrogen (BUN) at multiple time points during the study. Creatinine, BUN, and inulin clearance were within the normal range from both species following triclopyr administration which indicates that repeated administration of triclopyr in the dog and monkey had no effect on glomerular filtration rate (GFR). In monkeys, the percentage excretion of PSP and PAH appeared to increase following triclopyr administration (20 mg/kg/day), suggesting that these weak organic acids may be competing for the same plasma protein-binding site enhancing their clearance. More importantly, these data strongly suggest that triclopyr is not competing with PSP or PAH for the active secretory site within the monkey kidney proximal tubules. In contrast, PSP clearance studies in dogs clearly demonstrated that triclopyr administration (5 mg/kg) can significantly decrease the percentage PSP excretion even following a single dose administration. The decrease in percentage PSP was reversible and inversely related to the plasma triclopyr concentration. Overall, these data clearly indicate that triclopyr effectively competes with PSP for the active secretory site within the dog kidney proximal tubules. In contrast, the monkey was insensitive to the effects of triclopyr on the active secretory process even at doses fourfold higher (20 mg/kg/day) than the effective dose in the dog (5 mg/kg/day). These findings suggest that the effect observed on PSP and PAH excretion in the dog represent a physiological competition for excretion and not toxicity.

Comparative pharmacokinetics of [14C]metosulam (N-[2,6-dichloro-3-methylphenyl]-5,7-dimethoxy-1,2,4- triazolo[1,5a]-pyrimidine-2-sulfonamide) in rats, mice and dogs.

This study was conducted to provide data on the pharmacokinetics of [14C]metosulam (N-[2,6-dichloro-3-methylphenyl]-5,7-dimethoxy-1,2,4-triazolo-[1,5a]- pyrimidine-2-sulfonamide). Groups of male Sprague-Dawley rats, CD-1 mice and Beagle dogs were given a single oral gavage dose of 100 mg [14C]metosulam kg-1 body weight and blood, urine, feces and selected tissue specimens were collected up to 168 h for rats and mice and 216 h post-dosing for dogs. Two of these dogs received a second oral dose of 100 mg kg-1 and were humanely euthanized at 12 h post-dosing and selected tissues were collected. The third dog was administered an intravenous dose of 1 mg kg-1 and plasma, urine and feces were collected for 72 h post-dosing. Specified tissue specimens were analyzed for 14C activity and selected tissues were evaluated for localization of 14C activity by histoautoradiography. Selected urine and plasma specimens were also profiled for metabolites by high-performance liquid chromatography. [14C]Metosulam was absorbed rapidly (t1/2 < 1 h) in all three species. Mice and dogs absorbed ca. 20% of the orally administered dose of [14C]metosulam, compared to > 70% absorption in the rat. Analysis of 14C activity and histoautoradiography of the dog eyes indicated that the retina, a target for toxicity in the dog, did exhibit affinity for the radiotracer. There was no evidence of 14C localization in the kidneys of dogs or in the eyes of rats. In rats and mice the 14C plasma time-course was fit to a two-compartment pharmacokinetic model, whereas the dog was fit to a one-compartment model. The half-lives for the rapid initial (alpha) and slower terminal phases (beta) were 9 h and 60 h for the rat and 20 h and 155 h for mice, respectively. The dog had an elimination t1/2 of 73 h. In all three species, [14C]metosulam and metabolites were excreted in the urine and quantitatively the relative amount of [14C]metosulam metabolism followed the pattern of mice > rats > dogs. These data suggest that the observed ocular lesion in dogs is due to metosulam and may in part be due to its selective affinity for the dog retina.