Dermatotoxicokinetic Modeling of p-Nitrophenol and Its Conjugation Metabolite in Swine following Topical and Intravenous Administration

G. L. Qiao*,1, S. K. Chang{dagger}, J. D. Brooks{ddagger} and J. E. Riviere{ddagger}

* National Institute for Occupational Safety and Health (NIOSH), CDC, Morgantown, West Virginia 26505; {dagger} Department of Veterinary Medicine, National Taiwan University, Taipei, Taiwan, Republic of China; and {ddagger} Center for Cutaneous Toxicology and Residue Pharmacology, North Carolina State University, Raleigh, North Carolina 27606

Received September 3, 1999; accepted November 12, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of a dermatotoxicokinetic (dTK) model for p-nitrophenol (PNP), a common metabolite from a variety of compounds and a biomarker of organophosphate (OP) insecticide exposure, may facilitate the kinetic modeling and risk assessment strategy for its parent compounds. In order to quantify and then clarify in vivo-in vitro correlation of PNP disposition, multicompartment kinetic models were formulated. Female weanling pigs were dosed with [14C]PNP intravenously (150 µg in ethanol, n = 4) or topically onto non-occluded abdominal skin (300 µg/7.5cm2 in ethanol, n = 4). PNP and p-nitrophenyl-ß-D-glucuronide (PNP-G) profiles were determined in plasma and urine in addition to total 14C quantitation in many other samples. Disposition parameters (rate constants, Ftop, T1/2, T1/2Ka, AUC, Vss, Clp, MAT, and MRT) and the simulated chemical mass-time profiles on the dosed skin surface and in the local, systemic, and excretory compartments were also determined. Total recoveries of 97.17 ± 4.18% and 99.80 ± 2.41% were obtained from topical and intravenous experiments, respectively. Ninety-six hours after topical and intravenous application, 70.92 ± 9.72% and 98.65 ± 2.43% of the dose were excreted via urine, and 0.55 ± 0.16% and 0.51 ± 0.10% via the fecal route, respectively. Peak excretion rate and time were also determined. It was suggested by experimental observation and modeling that urinary 14C excretion correlates with the systemic tissue depletion profile well and may be used as a biomarker of PNP exposure. This study also supports the strategy of using urinary PNP as a biomonitoring tool for OP pesticide exposure, although some precautions have to be taken. The strategy used in this study will be useful in comprehensive dTK modeling in dermal risk assessment and transdermal drug delivery.

Key Words: p-nitrophenol (PNP); toxicokinetics; skin absorption; metabolism; pig.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The use of metabolic biomarkers to quantify systemic exposure to environmental chemicals receives increasing attention. Dermal exposure has been demonstrated to be a primary route for systemic exposure to many environmental chemicals (Honeycutt et al., 1985Go; Wang et al., 1989Go; Wester and Maibach, 1989Go). p-Nitrophenol (4-nitrophenol, PNP) is often used as a biomarker to quantitate dermal (Qiao et al., 1994Go) or even oral exposure (Pena-Egido et al., 1988Go) to many organophosphate (OP) pesticides such as parathion and methyl parathion. Although OP pesticide usage has been reduced in the United States since 1992, PNP dermal exposure hazard is still a concern due to PNP's direct use and its occupational or environmental appearance via pesticide degradation and waste disposal. Urinary excretion of PNP parallels urinary excretion of parathion and paraoxon after dermal parathion exposure in the pig (Qiao et al., 1994Go). Additionally, a linear relationship exists between the plasma concentration of orally dosed parathion and the urinary excretion rate of PNP in the rabbit (Pena-Egido et al., 1988Go). In order to use PNP as a biomarker for parent compound exposure, we should be prudent before an understanding of PNP's disposition after topical and systemic exposures independent of exposure to its parent pesticides can be established. Similarly, such an understanding is required to properly integrate in vitro dermal absorption data into in vivo risk assessment. This can best be accomplished by developing a comprehensive dermatotoxicokinetic (dTK) model of PNP disposition that can then be used to facilitate dTK modeling of its parent pesticides.

PNP is a common metabolite or degradation product of OP pesticides, occurring both in vivo in animals and humans and in the environment. According to a national survey, PNP is the fourth most commonly detected contaminant found in human urine screening in the United States (Kutz et al., 1992Go). PNP is used mainly in the manufacture of drugs, fungicides, OP pesticides, and dyes, in addition to its direct use as a fungicide for leather products and as a leather-darkening agent. It can degrade in water and surface soil, but the breakdown takes longer in deeper soil and in groundwater. PNP has been detected in urine samples of people who did not have any known exposure to PNP. Of environmental concern, PNP often comes from the in vivo breakdown of common OP pesticides (e.g., parathion, methyl parathion) used in agriculture and forestry. PNP was selected as the sole biomarker used in a recent public health incident with methyl parathion house spray [Agency for Toxic Substance And Disease Registry (ATSDR, 1996Go)]. However, due to PNP's coexposure potential with its parent compounds and PNP's dermal absorption-enhancing effect, proper usage of urine PNP as a biomarker for dermal exposure of OP pesticides and PNP (as a fungicide) requires careful kinetic evaluation of PNP dermatodisposition. Additionally, PNP has often served as a model substrate in conjugation studies with different in vitro and in vivo systems (Antoine et al., 1993Go; Berry et al., 1975Go; Bock et al., 1973Go; Gessner, 1974Go; Hamada and Gessner, 1975Go; Machida et al., 1982Go; Minck et al., 1973Go; Moldeus et al., 1976Go; Vessey and Zakim, 1973Go; Vessey et al., 1973Go) and has even been used in the purification of enzyme isoforms (Antoine et al., 1993Go).

PNP can cause reversible blood disorders in both animals and humans, such as a decreased ability to carry O2 to tissues (ATSDR, 1992Go) via methemoglobinemia, in addition to its CNS effects, mutagen activity, and direct burns on skin and eyes (Cooper et al., 1997Go). Cutaneous irritation reactions such as erythema, edema, moderate to severe corneal cloudiness, blistered conjunctival tissue, and corneal neovascularization were observed in rats and rabbits after large-dose dermal exposure. Considerable ingestion of PNP also led to death of rats, mice, and rabbits. There is no evidence of carcinogenic activity in male and female Swiss-Webster mice receiving PNP topical doses of up to 160 mg/kg, three times/week for 78 weeks (ATSDR, 1992Go), although PNP is considered a mutagen (Cooper et al., 1997Go).

The pig is a well-accepted animal model for studying human percutaneous absorption (Bartek et al., 1972Go; Hawkins and Reifenrath, 1984Go; Meyer et al., 1978Go; Monteiro-Riviere and Riviere, 1996Go; Qiao et al., 1993Go; Reifenrath and Hawkins, 1986Go; Reifenrath et al., 1984Go; Wester et al., 1998Go). Topical absorption of PNP in vitro and ex vivo in porcine models have been examined (Brooks and Riviere, 1996Go; Chang et al., 1994aGo,bGo). Dermal absorption and dTK modeling were also conducted following intravenous (iv) and topical application of parathion, one of the PNP parent compounds (Qiao et al., 1993Go; 1994Go; Qiao and Riviere, 1995Go). The parathion disposition dTK model included the parathion-derived PNP components, which provided a tool to explore the differences between the kinetic disposition of directly applied PNP and that of metabolically derived PNP in the in vivo porcine model. PNP disposition is relatively simple, as PNP is not significantly metabolized in the skin, and therefore the in vivo and in vitro disposition differences are minimized.

This study was designed to a) quantify the cutaneous and systemic disposition fate of PNP in vivo in swine by formulating a dTK model after iv and topical application; b) explore the similarity/difference of PNP disposition in the skin with direct topical PNP dose and with metabolically derived PNP; and c) facilitate the dTK modeling strategy for its parent compounds and in transdermal drug delivery and human dermal risk assessment studies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Eight- to ten-week-old female weanling Yorkshire pigs (~20 kg, T-Sharp, Rocky Mount, NC) were acclimated for 1 week. The animals were individually housed in metabolic cages (72°F and 12:12 h light:dark cycle), given 15% (protein) pig and sow pellets (~2 lbs/pig/day) and given free access to water. Eight pigs were randomly assigned to receive either topical or iv exposure (n = 4/exposure). Throughout the experiments, all pigs were humanely handled according to preapproved North Carolina State University animal use and care protocols.

Drugs and Chemicals
[14C-ring-2,6]PNP (11.7 mCi/mmol) was purchased from Sigma Chemical Co. (St. Louis, MO). Ethanol (absolute, Aaper Alcohol and Chemical Co., Shelbyville, KY) was used as the dosing vehicle. Cold PNP (10 µg/µl) and p-nitrophenyl-ß-D-glucuronide (PNP-G) (Sigma) were used in topical dose formulation and high performance liquid chromatography (HPLC) methodology development. Other chemicals were HPLC or gas chromatography (GC) grade.

Dosing
Topical application.
Pigs were anesthetized with halothane following ketamine (11 mg/kg) + xylazine (1.5 mg/kg) intramuscular (im) injection. The ear vein and jugular veins were catheterized. Hair in the selected abdominal dosing site was clipped carefully 6 h prior to PNP dosing (Qiao et al., 1993Go, 1997Go). Approximately 300 µg of PNP (150 µg cold PNP + 150 µg [14C]PNP with 10 µCi total radioactivity) in 100 µl ethanol vehicle was evenly applied to a 7.5 cm2 circular dosing zone in the abdominal area of pigs. This dose provided a surface concentration of 40 µg/cm2. The dosed skin was non-occlusively protected by a Hill Top® chamber with holes, covered by nylon screening, and positioned by Elasticon® wrapping tape.

Intravenous dosing.
To avoid any potential dose-dependent kinetics of PNP disposition in the pig, a smaller iv dosage (half of the total topical dose, i.e., 150 µg) was selected based on the estimated dermal PNP bioavailability of 50%. This pure labeled PNP (~10 µCi, no cold PNP) in ethanol was injected into the bloodstream through an ear vein cannula followed by a 10-ml physiologic saline flush to provide an instant and complete bolus dose.

Sampling and 14C Assay
Blood samples (14 topical and 30 iv per animal, 5 ml each) were withdrawn via the jugular vein cannula from 0 to 96 h. Plasma was routinely separated. Urine was collected immediately after voiding. Feces samples were collected once a day, weighed, and homogenized with distilled water (~50:50, w/w). At 96 h postdosing, pigs were sacrificed and the dosing materials/devices were collected and extracted with ethyl acetate: Elasticon tape and screen 2 x 45 ml; dosing chamber 5 x 15 ml; cotton swabs for skin surface wash [10% dishwashing liquid (three times, Dove®, Lever Bros. Co., New York, NY) and water (three times)] 2 x 45 ml. The stratum corneum was isolated by tape stripping (10 times) and digested in ethyl acetate (15 ml per stripping). Full-thickness skin, subcutaneous fat, and muscle samples at the dosed site were carefully excised and assayed. All samples of tissues and excreta with detectable 14C were also assayed for total 14C recovery in our mass balance study. For more details, our original in vivo dermal absorption work should be consulted (Qiao et al., 1993Go).

All the samples were stored at –20°C until analysis. An aliquot of 250–500 µl liquid or 100–700 mg solid sample was completely burned in a tissue oxidizer followed by Liquid Scintillation Counting (LSC, TriCarb 1900TR liquid scintillation analyzer, Packard Instrument Co., Downers Grove, IL) for total 14C assay. HPLC separation was performed to study PNP metabolism as described below.

HPLC Analysis
Sample preparation.
Three-milliliter urine (or 1-ml plasma) samples were extracted twice with 5 ml (4 ml for plasma) ethyl acetate, acidified with 0.6 ml (0.2 ml for plasma) 1N HCl followed by additional three ethyl acetate extractions. During each extraction, the samples were shaken at 100 (60 for plasma) OSC/min for 15 min and then stood until phase separation. All five organic extracts were pooled in a clean vial and brought to dryness in a gentle N2 stream for HPLC analysis. The dried samples were stored at –20°C until HPLC separation.

HPLC separation of PNP and PNP-G in plasma and urine.
Separations were performed with a Waters chromatographic system equipped with a 600E solvent delivery system, 717+ autosampler, column temperature control unit, 996 Photodiode Array Detector (PDA), ChemStation with Millennium 2010 software (Waters Corp., Milford, MA), and an automatic fraction collector (Foxy 200, ISCO, Inc., Lincoln, NE). A C18 reverse-phase column (µ-Bondapack, 3.9 x 300 mm), operated at 25°C, was eluted with a mobile phase (0.7ml/min) of acetonitrile/0.0085M KH2PO4 (65:35, v/v, pH = 5.0). An aliquot of 50 µl of each processed sample was injected to separate PNP and PNP-G to quantify these two compounds via LSC.

HPLC separation methodology was developed using cold standards of PNP and PNP-G in methanol chromatographed and detected at 254 nm. The chromatogram of the nonlabeled standards is shown in Figure 1Go. The extraction efficiency ranged from 102 to 106% for PNP and 82 to 91%, for PNP-G as calculated by applying a published method (Qiao et al., 1994Go).



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FIG. 1. Chromatogram of p-nitrophenol (PNP) and p-nitrophenyl-ß-D-glucuronide (PNP-G): HPLC conditions: column = µ-Bondapack C18 reverse-phase, mobile phase = acetonitrile/0.0085M KH2PO4 (65:35, v/v, pH 5.0, flow rate of 0.7 ml/min), and detection wavelength = 254 nm.

 
HPLC-LSC analysis.
The dried samples were dissolved in methanol (300 µl for urine or 200 µl for plasma), from which 50-µl aliquots were injected for HPLC separation. The peak fractions were automatically collected applying the time windows of 3'30"–5'12" for PNP-G (peaking at 3.44 min), and 5'12"–6'42" for PNP (peaking at 5.46 min). Fifteen milliliters of EcolumeTM liquid scintillation cocktail (ICN Biomedicals, Inc., Irvine, CA) was added to each fraction collection, mixed, and stood for at least 4 h before LSC analysis. The fractional amount of compound j in sample i assayed by HPLC is

This fraction was multiplied by the total burned DPM in sample i (DPMi,burned), and the result was converted to fraction dose of compound j in sample i (Aj,i, % dose):

DPM is disintegrations per minute in the LSC analysis; n = 2 refers to the total number of compounds in question (PNP and PNP-G) in the HPLC analysis. The chemical amount-time (Aj,i-Ti) profiles of PNP and PNP-G in urine and plasma over time and the total 14C amount-time (Ai-Ti) profiles in the ith assay of other samples (termination samples) were used in dTK model formulation.

dTK Modeling and Parameter Estimation
This proposed comprehensive dTK model for topical and iv PNP disposition (Fig. 2Go) was constructed based on preexisting PNP dermal absorption, metabolism, and modeling data (Chang et al., 1994aGo; Qiao et al., 1994Go; Qiao and Riviere, 1995Go; Williams et al., 1994Go). Because PNP-G was not identified in perfusate samples from topically dosed isolated perfused porcine skin flap (IPPSF), we excluded a local skin PNP-G compartment in this model, although phase II conjugation reactions (glucuronide and sulfate formation) may occur in pig skin for some other substrates (Qiao et al., 1994Go; Qiao and Riviere, 1995Go) or in human skin epidermal cells for PNP (Rugstad and Dybing, 1975Go). This model has 12 individual compartments (Comps. 1–12) and 3 pooled compartments (Comps. 21–23: dosed tissue pool, central plasma pool, and urine pool, respectively) to predict either the flux of individual chemicals (PNP and PNP-G) or the total radiolabel profiles for comparison with the existing total 14C absorption/disposition data.



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FIG. 2. Comprehensive dermatotoxicokinetic (dTK) model for p-nitrophenol (PNP) and p-nitrophenyl-ß-D-glucuronide (PNP-G) disposition following intravenous and topical exposure in vivo in swine (DD = dosing device; Evap. = evaporation)

 
To more accurately formulate the dTK model for PNP, we implicitly assumed that the systemic disposition of PNP is independent of application route (topical vs iv), that is, the biologic system handles PNP disposition identically once PNP is introduced into the general circulation, regardless of exposure route. The systemic and excretory parameter estimation was based mainly on the PNP iv bolus data. The topical experiment data were used to estimate the cutaneous (epidermis and above) parameters and also to confirm both the compartmental structure and parameters for the iv model. Model parameters were primarily estimated according to direct analysis of PNP, PNP-G, and total 14C profiles in the central systemic (Comps. 7 and 8) and urinary excretion subunits (Comps. 11 and 12). Observed and calculated plasma profiles were given to facilitate data comparison (this report with other published blood profiles). Liver and other highly perfused organs were not separated from the central systemic (plasma) compartments where the systemic metabolism of PNP occurred. Topical experiments allowed for additional parametric constraints, characterizing local cutaneous disposition by providing some terminal experimental observations of the skin surface (Comp. 1, swab), stratum corneum (Comp. 4, tape strips), and dosing device (DD, Comp. 2, dosing chamber + wrapping elastic tape + window screening). Nonrecovered 14C was assigned to evaporative loss (Comp. 3) in our topical dTK modeling effort. Because there was no PNP vapor recondensation seen in previous ex vivo PNP evaporation and kinetic modeling studies (Brooks and Riviere, 1996Go; Williams et al., 1994Go), a one-way connection between compartments 1 (skin surface) and 3 (evaporation) was proposed in this model (Fig. 2Go). A net PNP transfer from the dosed skin surface (Comp. 1) to dosing device for binding (Comp. 2) was represented by another one-way connection as illustrated. The sum of radiolabel left in the skin compartments (Comps. 4 and 5) below the stratum corneum (the "pool compartment" representing epidermis and other cutaneous tissues, Comp. 21) corresponded to the skin 14C measure.

The 6-compartment iv model (Comps. 7–12) was first formulated by simultaneously fitting the iv urine and plasma Aj,i-Ti data, employing a numerical least-squares minimization algorithm (KINETICA, North Carolina State University, Raleigh, NC). Both the structure and the mean parameters of the iv model were then fixed (although subject to minor adjustment to best fit the data sets from each individual animals) and incorporated into the 12-compartment topical model (Fig. 2Go), which was fitted to the topical Aj,i-Ti and Ai-Ti data from plasma, urine, and other samples assayed. Rate constants, peaks, and peak times were estimated. In the modeling and data simulation, we used fractional dose in each of the anatomical regions or kinetic compartments, as the volumes of many compartments (e.g., dosing device binding–Comp 2; evaporation loss–Comp. 3; and urine–Comps. 11, 12, 23) were not definable/measurable or were extremely variable across individuals. This is the reason for not using concentration profiles, which require a volume term. In fact, we measured concentrations in all the samples and had to convert the concentration results to fractional dose in whole organ/blood by using whole organ weight or volume. For absorption data conversion, the total blood volume (BV, ml/100g) in a pig (< 25 kg) was estimated based on body weight (W, kg) using BV = 9.5W–0.68 (Engelhardt, 1966Go). About 65% (v/v) of the total blood volume was plasma in young pigs, assuming a PCV (packed cell volume) of ~35%. Area under the (zero moment) curve (AUC, % dose x h), area under the first moment curve (AUMC, % dose x h2), mean residence time (MRT, h), mean absorption time (MAT, h), topical bioavailability (Ftop, %), T1/2Ka (PNP absorption or PNP-G metabolic formation half-life), T1/2 (elimination half-life), distribution volume at steady state (Vss), and clearance (Cl) were calculated according to the following equations (Williams, 1999Go):









Data Analysis
Total percutaneous absorption was determined as the percentage of the applied radioactive dose appearing in the urine and feces over the 96-h observation, because no detectable 14C tissue residue was found at the end of experiment (complete excretion). However, total dermal penetration was calculated as the sum of absorption (see above) plus local dosed tissue label residues (Comp. 21 and 4). ANOVA with LSD multicomparison test was conducted wherever applicable at {alpha} = 0.05 significance level (SAS, Inc., Cary, NC).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The result of complete HPLC separation of cold PNP standard from its conjugation product of PNP-G is illustrated in Figure 1Go. Retention times (tR) for PNP-G and PNP peaks were determined as 3.4 and 5.5 min, respectively. This degree of HPLC separation allowed precise peak fraction collection using programmed time windows for further LSC analysis of the biologic samples with low concentration of 14C radiolabel.

Some key parameters of iv and topical PNP are listed in Tables 1–3GoGoGo. The chemical amount-time profiles of iv and topical PNP are plotted in Figures 3–5GoGoGo and Figures 6–11GoGoGoGoGoGo, respectively.


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TABLE 1 14C Disposition Parameters following Topical (10 µCi, 300 µg/7.5cm2) and Intravenous (10 µCi, 150 µg) Applications of [14C-Ring-2,6]PNP in Female Weanling Pigs
 

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TABLE 2 PNP Pharmacokinetic Modeling Parameters (h–1, mean ± SEM) following Topical (10 µCi, 300 µg/7.5cm2) and Intravenous (10 µCi, 150 µg) Applications of [14C-Ring-2,6]PNP in Female Weanling Pigs
 

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TABLE 3 PNP Disposition Kinetic Parameters (mean ± SEM) following Topical (10 µCi, 300 µg/7.5cm2) and Intravenous (10 µCi, 150 µg) Applications of [14C-Ring-2,6]PNP in Female Weanling Pigs
 


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FIG. 3. The observed (symbols) against computer-predicted (lines) mean (± SEM) amount-time profiles of [14C]p-nitrophenol (PNP), p-nitrophenyl-ß-D-glucuronide (PNP-G), and total 14C in the plasma (central compartments 7, 8, and 22) following iv injection of [14C]PNP at a dose of 150 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 4. The dTK model-prediction of [14C]p-nitrophenol (PNP) profiles in the peripheral and deep systemic tissues (compartments 9 and 10) following iv injection of [14C]PNP at a dose of 150 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 5. The observed (symbols) against computer-predicted (lines) mean (± SEM) cumulative urinary excretion profiles of [14C]p-nitrophenol (PNP), p-nitrophenyl-ß-D-glucuronide (PNP-G), and total 14C (compartments 12, 11, and 23) following iv injection of [14C]PNP at a dose of 150 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 6. The observed (symbols) against computer-predicted (lines) mean (± SEM) dosing device binding and evaporative loss profiles of [14C]p-nitrophenol (PNP, compartments 2 and 3) following topical exposure of [14C]PNP at a dose of 300 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 7. The observed (symbols) against computer-predicted (lines) mean (± SEM) profiles of [14C]p-nitrophenol (PNP)on the dosed skin surface and in the stratum corneum (compartments 1 and 4) following topical exposure of [14C]PNP at a dose of 300 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 8. Mean (± SEM) [14C]p-nitrophenol (PNP) profiles in the epidermis (compartment 5) and local cutaneous tissues (compartment 6) and the observed (open circle) against computer-predicted (line) total 14C in the local dosed tissues following topical exposure of [14C]PNP at a dose of 300 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 9. The observed (open circle) against computer-predicted (lines) mean (± SEM) amount-time profiles of [14C]p-nitrophenol (PNP), p-nitrophenyl-ß-D-glucuronide (PNP-G), and total 14C in the plasma (central compartments 7, 8, and 22) following topical exposure of [14C]PNP at a dose of 300 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 10. The dTK model-prediction of [14C]p-nitrophenol (PNP) profiles in the peripheral and deep systemic tissues (compartments 9 and 10) following topical exposure of [14C]PNP at a dose of 300 µg in ethanol in female weanling pigs (n = 4).

 


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FIG. 11. The observed (symbols) and computer-predicted (lines) mean (± SEM) cumulative urinary excretion profiles of [14C]p-nitrophenol (PNP), p-nitrophenyl-ß-D-glucuronide (PNP-G), and total 14C (compartments 12, 11, and 23) following topical exposure of [14C]PNP at a dose of 300 µg in ethanol in female weanling pigs (n = 4).

 
In the iv study, total recovery was 99.8% (Table 1Go). Because this complete recovery was predominately from urine (98.7%), the absolute percutaneous absorption was calculated without any correction/normalization for incomplete recovery or for significant tissue residues after non-occlusive topical application. Only 0.5% of the iv dose was eliminated via the fecal route. Less than 0.04% of the initial iv dose remained in the body by 96 h, indicating no tissue accumulation of PNP or its metabolite in the pig.

With this non-occlusive topical application of PNP, a complete recovery of 97% was determined (Table 1Go). About 71% of the applied PNP dose was excreted via urinary routes and 0.6% via fecal routes within 96 h (Table 1Go). Less than 1% of the dose was found in the stratum corneum (SC) layer and 17% remained on the dosed skin surface. Approximately 6% and 2% of the dose were found in the dosing device and in the dosed tissues at 96 h, respectively. PNP did not accumulate in the body after dermal absorption. More label was found in the urine after iv than that following topical [14C]PNP application, although no exposure route effect on fecal excretion was observed (Table 1Go).

The rate constants of the iv and topical model are given in Table 2Go. Some key kinetic parameters are also given in Tables 3 and 4GoGo. As assumed according to pharmacokinetic theory, most systemic mass transfer rates governing postabsorption processes (distribution, metabolism, and elimination) proved to be identical for topical and iv application (p > 0.05), demonstrating route-independent systemic disposition of PNP. Only k7-12 for iv was smaller than that for topical (p < 0.05), possibly suggesting the existence of a concentration-dependent or saturable blood-to-urine transfer process. The opposite was found for peripheral tissue PNP returning to blood, that is, a smaller k9-7 was determined with a topical application than with iv. This may indicate that the peripheral tissue compartment for PNP (Comp. 9) is only able to hold a constant amount of PNP. If more PNP is in the peripheral tissue, as seen with the iv dose, the tissue may not be able to retain the compound and will instead release extra PNP back to the general circulation for systemic elimination. This also explains insignificant PNP tissue accumulation in the pig, as we experimentally observed in this study. Similar AUCs for Comp. 9 were determined for iv and topical exposure, although a larger k9-7 was determined with the iv dose.


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TABLE 4 PNP Disposition Kinetic Parameters Derived from Plasma Data Topical (10 µCi, 300 µg/7.5cm2) and Intravenous (10 µCi, 150 µg) Applications of [14C-Ring-2,6]PNP in Female Weanling Pigs
 
MRTs for appropriate compartments after topical dose were generally longer than iv. The model-based calculation of topical PNP bioavailability of 57.4% favorably reflects the observed total dermal absorption of ~70%. Similar results can also be obtained by using AUC ratio () either for 14C plasma (56%, Comp. 22) or for PNP in plasma (57%, Comp. 7) (Table 4Go). No dose adjustment was necessary in the dermal bioavailability calculation, as the AUC unit was the fractional dose-based unit of % dose x h instead of the chemical weight-based unit of µg/ml x h. The dermal absorption half-life of PNP was 18 h, which is similar to the PNP-G metabolic formation half-life (T1/2ka of PNP-G) from PNP conjugation (Table 4Go). Plasma clearance of PNP was faster than 14C, but not necessarily indicating a slower clearance of PNP-G because the metabolism from PNP to PNP-G occurred in the pooled central compartment of Comp. 22 (Table 4Go). PNP and 14C elimination half-lives were identical and less than 1 h. Fast distribution half-life was graphically estimated as 5 min (Fig. 3Go) for PNP, but was slower for pooled 14C. From the computer modeling, we found that equilibrium of dosing device binding and the evaporation processes of PNP were well established around 24 h after dermal exposure (Fig. 6Go). PNP concentration changes over time on skin surface, in the SC, and dosed tissues were predicted by the model (Figs. 7 and 8GoGo). All processes prior to penetration through cutaneous vasculature (absorption) had passed their peaks by the first 24 h after topical application. Plasma and blood profiles of PNP, PNP-G, and total 14C are given in Figure 9Go. Profiles in the systemic peripheral (Comp. 9) and deep tissue (Comp. 10) compartments are illustrated in Figure 10Go. As shown in Figure 11Go, slightly greater than one-half of the total 14C found in urine by 96 h was confirmed to be PNP-G, with the remainder being PNP. More PNP-G was formed in the body during the early stage of the topical experiments.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
After iv application, plasma PNP profiles (Fig. 3Go) indicated disposition features for a classical 2- or 3-compartment TK model as seen in a semilogarithmic drug amount-time plot. The distribution half-lives are about 5 min for PNP but 6–7 min for total label. Elimination half-lives of PNP and 14C label were about 50 min (Fig. 3Go and Table 4Go). Approximately 95% of the radioactive PNP dose was eliminated from blood within 1 h after iv application. This plot also revealed that PNP contributed much more to total plasma 14C counts than PNP-G during the first 30–60 min. Parallel decline of PNP and PNP-G profiles in plasma was determined thereafter (Fig. 3Go). Intravenous PNP disposition kinetics reported here were very similar to those of parathion previously determined after iv application in the pig (Qiao et al., 1994Go).

Computer simulation of the PNP residue profiles over time in the systemic tissue compartments (Fig. 4Go) is useful in estimating overall body burden and time for maximal tissue exposure of PNP. For example, we can predict that by 1–1.5 days post PNP iv application, all the tissues/organs of the animals will be cleared of PNP. This was verified by the fact that urine excretion of label was completed by about 1 day (Fig. 5Go) and by complete recovery of the iv PNP dose (Table 1Go). The highest modeled tissue concentration was observed around 2–3 h after iv dose. Accordingly, the highest possibility of tissue toxicity could be expected during this time frame. For the shallow tissue compartment (Comp. 9, relatively quicker equilibration with blood), a peak time was determined at about 2 h, while a deeper tissue compartment (Comp. 10, relatively slower equilibration with blood) may hold the largest PNP burden at 3 h (Fig. 4Go). This provides useful information for PNP tissue kinetics study design and drug residue prediction in tissues.

The urine excretion of the iv PNP and its conjugate PNP-G could be completed by about 1 day, and more than 90% of the iv dose was excreted in urine within the first 12 h (Fig. 5Go). PNP-G and PNP contribute about two-thirds and one-third of the total urine label, respectively (Fig. 5Go). This was consistent with the time frame of modeled tissue residue profiles (Fig. 4Go). Very similar results were obtained with urine PNP excretion following iv dose of its parent compound parathion (Qiao et al., 1994Go). Therefore, urine PNP monitoring can serve as an easy and reliable tool to assess the exposure and resultant tissue residue of PNP and likely its parent compound (parathion) exposure in the pig model. Obviously, this study supports the strategic development of risk assessment by ATSDR using urine PNP as a biomarker of methyl parathion exposure due to the illegal house spraying with this pesticide in the United States (ATSDR, 1996Go). However, this biomonitoring is appropriate only if methyl parathion shows a quick and complete biotransformation to PNP in the human. Otherwise, PNP urine level monitoring may not be able to serve as an ideal tool for methyl parathion risk assessment if the disposition processes of methyl parathion or its active metabolite methyl paraoxon are largely different from that of PNP. A much faster urine excretion rate was determined for PNP-G than for PNP, as a 3–7 times faster blood-to-urine chemical transfer rate of PNP-G (k8-11) than PNP (k7-12) was predicted (Table 2Go). Similar rate ratio (~2) and absolute values of PNP-G (5.01 h–1) and PNP (3.19 h–1) blood-to-urine transfer rates were also determined after parathion exposure (Qiao et al., 1994Go). This is consistent with the principle of chemical detoxification in the body and the fact that PNP-G is more water soluble and therefore more readily excreted via the renal route.

As shown in Figure 6Go, dosing device PNP binding was expressed as the net transfer of PNP from the topical dose (vapor, liquid, or solid) to the dosing device and occurred only during the first 24 h. The binding was saturated or a zero net transfer between the dose and the device was reached at 24 h. Dose evaporation only happened during the first 24 h after exposure (Fig. 6Go) with evaporation rate constant of 0.009 h–1 (Table 2Go) under non-occlusive conditions. Very low in vivo PNP evaporation loss of 3% was estimated using the dose-recovery difference approach (Qiao et al, 1994Go, Qiao and Riviere, 1995Go) in this study.

It seems that this dTK model could underestimate the skin surface residue profile, as a much higher surface residue was experimentally determined when compared to the computer simulation at the end of the experiments (Fig. 7Go). This may be due partially to the back-and-forth direct contact of the skin surface against the contaminated dosing chamber. It is possible to reload some 14C-PNP to the surface from the dosing device right before the termination of the experiments. This would surely make an unreasonably higher assayed surface 14C residue than the computer simulation result. In addition, math modeling limitations (e.g., use of simplest model, more weight for more important or more reliable sample data sets, etc.) may also contribute to this and other differences between the observed and calculated results, although effort was made to get the best overall data fitness.

Theoretically, postabsorption disposition kinetics (i.e., distribution, metabolism, and excretion) of a drug should be intrinsic and route independent. Therefore, most dTK model parameters (transfer rates) governing postabsorption processes should be similar for a compound either directly dosed via various exposure routes (topical, oral, inhalation, iv, etc.) or metabolically generated in the skin during penetration. Only some mass transfer rates, especially the absorption/metabolic formations rates, may be affected by the compound dosed (PNP dosed directly or a dose of its parent compound such as parathion or methyl parathion) or by the dosing method. This study demonstrated that PNP local tissue distribution, PNP metabolism to PNP-G, and PNP blood-to-urine transfer rates were similar for both directly-dosed PNP, as reported in this work, and for parathion-derived PNP, as in our previous study in the pig (Qiao et al., 1994Go). The results presented here demonstrate that the model rate constants with an identical dTK model structure were similar for iv and topical PNP application (Table 2Go). Therefore, via this study it was suggested that in complex in vivo dTK modeling, toxicants should be intravenously dosed and the iv data were modeled initially to simplify the more complex topical modeling task.PNP absorption has been demonstrated to be as significant as 6–22% within 8 h after in vitro, ex vivo, or in vivo dermal exposure in pig models in a dose-dependent fashion (Brooks and Riviere, 1996Go; Chang et al., 1994aGo; Qiao et al., 1996Go). Similarly, other researchers reported 35% and 11% of topical PNP could be absorbed in rabbits and dogs, respectively (ATSDR, 1996Go). If the experiment was extended to 4 days as with this in vivo pig model, over 70% of the topical PNP dose could be absorbed. Tissue residues were also found to be insignificant (ATSDR, 1996Go; Qiao et al., 1996Go). PNP dermal absorption has also been studied in other in vitro static and flow-though diffusion cell systems with animal or human skin (Hinz et al., 1991Go; Hotchkiss et al., 1992Go). PNP absorption after oral and inhalation exposure was found to be complete and quick. More than 80–90% of an oral PNP dose was absorbed in rabbits, and the blood Tmax was only several minutes in monkeys (ATSDR, 1996Go).

Quite often, the absolute amount (mass) of dermally absorbed penetrant can be increased with dose, although the fractional dose absorbed may be decreased. Interestingly, 2- to 4-fold increases in fractional PNP dermal absorption were observed with dose increases from 4 to 400 µg PNP/cm2 in our in vitro (Chang et al., 1994aGo) and ex vivo IPPSF (Brooks and Riviere, 1996Go) dermal absorption studies. Additionally, larger PNP doses also gave a quicker absorption peak (Tmax), deeper skin penetration, and a larger local tissue residue in our ex vivo study (Brooks and Riviere, 1996Go). This suggests that PNP enhances its own dermal absorption in terms of both rate and extent. PNP also enhances dermal absorption of its parent compound parathion (Chang et al., 1994aGo).

Conjugation, especially with glucuronide, is the major PNP metabolic pathway in animals. PNP glucuronidation proceeded in kidney, lung, and liver, but sulfation occurred almost exclusively in liver (Machida et al., 1982Go). It was demonstrated that human skin epithelial cells have PNP conjugation activity (Rugstad and Dybing, 1975Go). PNP was rapidly conjugated up to 70% of an ip dose by 3 h and up to 95% by 12 h, and PNP-G was the dominant metabolite completely excreted in urine (Gessner, 1974Go). It was demonstrated that PNP-G accounted for 70% of the oral PNP dose in rabbits. Similar results were obtained from a rat study after PNP iv dose, with a very quick formation of PNP conjugation products within 1 min (Machida et al., 1982Go). This demonstrated that PNP-G also accounted for two-thirds of the total excretion in the pig (Fig. 5Go), which is similar to PNP metabolic disposition in other species via different application routes. PNP conjugation is not sex specific but may be saturable/dose-dependent. PNP conjugation reactions seemed to be limited by the hepatic blood perfusion rate, i.e., the hepatic extraction ratio of PNP can be as high as 1.0 (Machida et al., 1982Go). It was also found that extrahepatic conjugation metabolism of PNP was considerable (46% of the entire glucuronidation capacity) in animals (Machida et al., 1982Go). At least three isoenzymes are responsible for PNP conjugation in the rat (Antoine et al., 1993Go). Up to 1992, no study was conducted regarding excretion in humans following dermal exposure (ATSDR, 1992Go).

About 78% of the absorbed PNP dermal dose in the rabbit and 92% of an iv dose in the dog appeared in the urine within 1 day. Fecal elimination is very minor (< 1%) in both cases (ATSDR, 1996Go). In this pig study, urine excretion of PNP (one-third) and PNP-G (two-thirds) was completed within 1 day after iv application of PNP (Fig. 5Go). However, a continuous urine excretion was observed following dermal exposure. This was due mainly to the prolonged dermal exposure (no skin wash during the 4-day study) and continuous percutaneous absorption (Fig. 11Go) but not due to PNP/PNP-G release from tissues; label excretion is fast and tissue residue is insignificant after both iv and topical exposure of PNP. PNP-G is more polar than the parent PNP and thus easier to excrete via the renal route. Similar to the dog, rabbit (ATSDR, 1996Go), and rat (Gessner, 1974Go) studies, we found that fecal excretion was very minor (~0.5%) following both iv and dermal exposure of PNP in the pig (Table 1Go).

Urinary PNP, as a sole biomarker of human pesticide exposure, was used as a key decision-making criterion for a remediation strategy related to the illegal indoor application of methyl parathion in several states in the United States (ATSDR, 1996Go; Grissom et al., 1998Go). Urine PNP can be from sources other than methyl parathion exposure such as direct PNP exposure from methyl parathion environmental degradation, although methyl parathion imposes the major health concern via its active metabolite of methyl paraoxon. Additionally, PNP can greatly enhance dermal absorption of compounds like parathion and itself (Chang et al., 1994aGo,bGo). Due to all of those, the urine PNP monitoring strategy for methyl parathion dermal risk assessment should be carefully implemented. When aged methyl parathion containing PNP (e.g., methyl parathion degradation) is exposed to skin, the resultant urine PNP should represent two sources—direct dermal absorption of PNP and metabolic generation of PNP from systemic/cutaneous metabolism of methyl parathion. Therefore, environmental methyl parathion levels and urinary PNP levels alone might not be adequate for assessing human health threats associated with indoor methyl parathion exposure. A more integrated exposure assessment strategy, based on the better understanding of mixed exposure and more importantly metabolic and environmental degradation kinetics of methyl parathion, becomes essential for risk analysis and management of the methyl parathion house spray incident.

In conclusion, the PNP TK modeling strategy developed here will be useful in the comprehensive dTK modeling of its parent compounds for human dermal risk assessment as well as in transdermal drug delivery studies. PNP postabsorption disposition kinetics were exposure independent. This model is structurally identical to the model used for published parathion modeling work. PNP was one of the most effective percutaneous penetrants, with percutaneous absorption of over 70% after dermal exposure in swine. PNP and its conjugation metabolite PNP-G were rapidly and completely transported from blood into urine with very minor fecal excretion and insignificant tissue residues in swine. Urinary excretion is the primary elimination route for PNP and its metabolite, with about one-third as PNP and two-thirds as PNP-G after iv, but equally as PNP and PNP-G following topical PNP exposure. The results support the strategy of applying urine PNP as a biomarker of OP pesticide exposure assessment, although some precautions have to be taken.


    ACKNOWLEDGMENTS
 
This work was supported mainly by NCSU/CCTRP funds. The authors thank Drs. N. A. Monteiro-Riviere in NCSU, S. C. Soderholm, F. H. Frasch in NIOSH, and P. L. Williams at Polytheoretics, Inc. for their assistance in the preparation of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed at NIOSH, M/S 3030, 1095 Willowdale Rd., Morgantown, WV 26505–2888. Fax: (304) 285–6041. E-mail: gaq1{at}cdc.gov. Back


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 RESULTS
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