* Battelle, Pacific Northwest Division, and
Battelle, Columbus Operations, P.O. Box 999, Richland, Washington 99352
Received August 23, 2002; accepted November 12, 2002
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ABSTRACT |
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Key Words: glycol ethers; 2-butoxyethanol; butoxyacetic acid; forestomach, dosimetry.
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INTRODUCTION |
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Hemolysis appears to be a root cause for the induction of liver tumors in male B6C3F1 mice (progression of hepatocellular adenomas to carcinomas and hemangiosarcomas). Chronic hemolysis and the accumulation of hemosiderin in the Kupffer cells results in oxidative stress in the livers of male mice, which are particularly sensitive due to an inherently low antioxidant capacity compared with female mice, rats, and humans (Kamendulis et al., 1999; Klaunig et al., 1998
; Park et al., 2002
). Vascular endothelial cells are also continually exposed to hemolysis byproducts, which may represent an additional stress on the hepatic vascular endothelial cells. Since hemolysis is unlikely to occur in humans (Ghanayem et al., 1987
; Klaunig et al., 1998
; Udden, 1994
; Udden and Patton, 1994
) and Park et al.(2002)
have shown that neither BE nor BAA affect the liver cells directly, it is questionable whether hepatic tumor production will be a factor for humans.
Although chronic hemolysis may conceivably contribute to forestomach lesions and subsequent tumor production observed in the NTP (2000) study in mice (Table 1
) via localized blockage of capillaries, followed by ischemia reperfusion injuries, the majority of the available evidence points towards a chronic irritation (cytotoxicity) compensatory hyperplasia mechanism caused by localized high concentrations of BE or its major metabolite, BAA. Many glycol ethers, including BE, are known to irritate mucous membranes (Boatman and Knaak, 2001
). The major alkoxyacetic acid metabolite, BAA, is likely to cause similar effects. Thus, chronic contact irritation from either consumption of BE deposited on the fur as a result of grooming, dissolution of BE in the mucous layers of the respiratory tract followed by mucociliary clearance and oral consumption, partitioning from systemic blood circulation, or local forestomach metabolism of BE to BAA are likely candidates for further study.
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The tissue disposition of radio-labeled BE in mice was investigated in a series of autoradiographic studies by Green et al. (2001a,b
; 2002)
and Bennette, et al. (2001)
. In these studies, high local concentrations of radioactivity were present in the buccal cavity, esophagus, and stomach tissues following either intravenous or short-term inhalation exposures. In the subchronic and chronic inhalation studies conducted by NTP (2000)
, treatment-related lesions were limited to the forestomach; no lesions were noted in the esophagus or buccal cavity. Radioactivity was also detected on the fur of mice exposed via inhalation, supporting earlier conclusions from the NTP chronic inhalation study that oral consumption via grooming of the fur may be an important factor in forestomach dosimetry, chronic irritation, and subsequent tumor promotion. In support of this hypothesis, Green et al.(2001a)
also demonstrated that forestomach lesions, similar to those seen in the subchronic inhalation study, could be produced in female B6C3F1 mice given single oral doses of BE at dose levels
150 mg/kg. However, Green et al. (2001b)
also detected 14C in mouse forestomachs following intravenous injection of 2-butoxyethanol, indicating that the mouse forestomach tissue may have a propensity for accumulating 14C, although the chemical form and the source(s) for the accumulation was not known.
The purpose of this study was, therefore, to investigate the pharmacokinetics of butoxyethanol and its major metabolite, butoxyacetic acid, in female B6C3F1 mice, with an emphasis on target tissue dosimetry and forestomach toxicity. This study was divided into five experiments as outlined in Table 2. The first experiment was to establish whether oral administration of undiluted, neat 2-butoxyethanol, to mimic grooming of BE deposited on the fur during inhalation exposures, is required to produce forestomach irritation and toxicity in male and female B6C3F1 mice or whether other routes of exposure can also result in comparable toxicity. The second experiment was to determine the target organ dosimetry and kinetics of BE and its major metabolite BAA in female mice, following oral and parenteral routes of exposure. The remaining three experiments were all designed to investigate several possible sources of BE and BAA in the forestomach and glandular stomach tissues of female mice, including deposition of BE on fur during inhalation studies and ingestion via grooming, the presence of BE and BAA in saliva, and the partitioning of BE and BAA in stomach tissues and stomach contents.
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MATERIALS AND METHODS |
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Animals.
A number of experiments were designed to address target tissue dosimetry and potential forestomach lesions as outlined in Table 2. Adult male and female B6C3F1 mice used in the target tissue histopathology and fur deposition (inhalation) studies were purchased from Taconic Laboratory Animals and Services (Germantown, NY), the same source that was used in the NTP inhalation studies. For the inhalation exposures, slightly older (10-week-old) mice were used, in order to be of sufficient size to fit in the nose-only exposure system. Female B6C3F1 mice appeared to be slightly more sensitive to the induction of forestomach lesions than males in the subchronic and chronic NTP inhalation studies (Table 1
; NTP, 2000
). Therefore, adult female B6C3F1 mice approximately 8 weeks of age, purchased from Charles River Laboratories (Raleigh, NC), were used in the tissue dosimetry and pharmacokinetics studies.
The rooms in which the animals were housed were maintained on a 12-h light/dark cycle and designed to maintain adequate temperatures, relative humidity, and airflows. Deionized water (reverse osmosis) and Purina Certified Rodent Chow #5002 (Purina Mills, Inc., St. Louis, MO) were provided ad libitum. The animals were acclimated to the laboratory for at least 5 days prior to dosing or exposure. During the acclimation period, animals were uniquely marked with a tail tattoo, weighed, and randomly assigned to subgroups, based upon targeted sacrifice times.
Experiment 1: Target tissue histology.
Previous studies using drinking water as a route of administration failed to produce forestomach toxicity (NTP, 1993). This lack of forestomach irritation could have been the result of either a lower dose rate or a dilution of BE in water to concentrations that were not irritating to the mucosa. Therefore, no dosing vehicle was used in this study in order to determine if contact between the forestomach and undiluted (neat) BE, as would occur following grooming of BE condensed on the fur following whole body inhalation exposures, could cause forestomach lesions similar to those observed in the NTP studies.
Preliminary studies were conducted in groups of male and female mice, 5/sex/dose, at dose levels of 0 (saline control), 100, 400, or 800 mg/kg/day for 5 consecutive days, to determine if animals would tolerate the treatment regimen and to set the dose levels in the definitive study. Since no overt clinical signs were observed at 800 mg/kg/day, a few remaining animals (5/sex) were administered 1200 mg/kg/day for three consecutive days. Only these highest-dose animals demonstrated a significantly decreased hematocrit (15% in males and 28% in females). Based upon results in the preliminary study, male and female B6C3F1 mice, up to 16/sex/dose, were treated by oral gavage at initial dose levels of 0 (saline control), 400, 800, or 1200 mg/kg/day of neat (undiluted) 2-butoxyethanol. Half the animals (8/sex/dose) were targeted for forestomach toxicity evaluations, with the remainder reserved for evaluations of liver effects. Feed was withheld from the mice for approximately 4 h prior to dosing and returned approximately 2 h after dosing, to simulate the brief fasting conditions associated with inhalation studies. After two days of dosing, mortality was unexpectedly observed at all dose levels. Therefore, dose levels for each group were arbitrarily reduced by half (i.e., to 200, 400, or 600 mg/kg/day) in an effort to improve survival. Dosing was discontinued when, after two additional doses (four doses total), mortality did not improve.
Each surviving animal was weighed and necropsied the day after the final dose. Stomach, liver, and spleen weights were recorded and blood was drawn by cardiac puncture for determination of the hematocrit. Slices of forestomach and glandular stomach tissues, as well as portions of each liver, were fixed in neutral buffered formalin for histopathological evaluations. Since the liver and hemolytic effects were used to design more definitive studies on the oxidative stress mechanisms (Park, et al., 2002), only the forestomach histopathology methods and results are presented.
Paraffin-embedded forestomach tissues were serially sectioned and stained with hematoxylin and eosin (H&E) or antibodies against proliferating cell nuclear antigen (PCNA). Histological evaluations of H&E sections were conducted to determine evidence of necrosis and/or irritation of the gastric mucosa. The primary determination of cells in S-phase was accomplished by analyses of PCNA incorporation, according to Eldridge and Goldsworthy (1996).
To investigate if parenteral administration of BE could also produce forestomach lesions in mice, additional animals were administered saline solutions of BE by either intraperitoneal (ip) injection, which results in first-pass metabolism in the liver, or subcutaneous (sc) injection, which bypasses the first-pass liver metabolism. Dose levels were 0 (saline control) and 400 mg/kg/day for five consecutive days.
Experiment 2: Tissue dosimetry and pharmacokinetics.
Since forestomach irritation, a precursor to tumor formation, was induced following multiple routes of exposure (see Results), ip injection and oral gavage were chosen to facilitate accurate dosing and to compare oral vs. parenteral administration on forestomach tissue dosimetry. Groups of 30 female mice were administered target doses of 50 or 250 mg BE/kg BW by ip injection or 250 mg BE/kg BW by oral gavage. Each dose solution was prepared in physiologically buffered saline (pH 7.4) and analyzed for BE to confirm the target dose level. Each mouse was weighed and administered dose solutions at a rate of 10 ml/kg by gavage or at a rate of 5 ml/kg body weight by ip injection. The dosing syringe was weighed before and after dosing to determine the actual volume delivered to each animal, and the dosing solutions were analyzed by gas chromatography/flame ionization detection to confirm the actual dosage.
At each scheduled sacrifice time (0.5, 1, 3, 6, 9, 12, and 24 h), animals were anesthetized in an 80% CO2 atmosphere and blood samples were collected by closed-chest cardiac puncture into Vacutainers® containing heparin, sealed and frozen on dry ice. The time of death was recorded at the completion of the blood draw and all animals were rapidly dissected to remove and weigh the kidney, liver, and stomach tissues. Stomach tissue was rinsed of its contents with ice-cold PBS, separated into forestomach (esophageal sphincter to limiting ridge) and glandular stomach (limiting ridge to pyloric sphincter) sections (each maintained as full thickness), and weighed. The tissues were flash-frozen in 4-ml amber vials and stored along with the blood samples at -80°C until analysis. The mice scheduled for sacrifice after 24 h were individually housed in glass metabolism cages, and urine was collected on dry ice from the 012 and 1224-h intervals. These animals had food and water ad libitum during this time. The cages were rinsed with a minimal amount of deionized water (20 ml) after the collection of each urine sample, and the cage wash sample was frozen until analysis. Blood, tissues, and urine were analyzed for BE and BAA by gas chromatography/flame ionization detection (GC/FID) or gas chromatography/mass spectrometry (GC/MS). Urine and cage wash samples were also acid-hydrolyzed to quantify potential conjugates of BE and BAA.
The areas under the curve (AUC) for both BE and BAA were calculated by the trapezoidal rule from 024 h (AUC024) and extrapolated to infinity (AUC) by addition of the quantity equal to the last measurable concentration divided by the elimination rate constant (Kel). The apparent first-order elimination rate constant (Kel) was calculated from a log-linear plot of the last three (or more) nonzero concentrations in the blood-concentration vs. time curve. The terminal phase blood half-life was obtained by dividing 0.693 by the elimination rate constant (Kel).
Experiment 3: Fur deposition following inhalation exposure.
Female B6C3F1 mice were exposed, either by whole-body or nose-only inhalation, to a single 6-h exposure to a target-concentration of 250 ppm BE, the highest concentration used in the NTP inhalation bioassay (2000). BE vapor was generated on a heated (
105°C) glass surface and a metered air stream flowed through the generator (
20 l/min) and into the exposure chamber (whole-body or nose-only). BE chamber concentrations reached 90% of the stable final concentration within 10 min for the whole-body chamber and 3 min for nose-only chambers. Exposure duration was 6 h to the time 90% of the final concentrations were reached.
Whole body exposures were conducted in an 85-liter polycarbonate chamber. Mouse cages were suspended in the center of the chamber above a stainless-steel catch pan. BE vapor was introduced from multiple ports along the top and face of the chamber and was exhausted from ports below the catch pan. The generator airflow (20 l/min) resulted in
14 air changes per h. The distribution of BE vapor in the chamber was determined prior to the start of the study by comparing the concentrations from 3 different chamber positions.
The nose-only exposure unit was a flow-past design consisting of two concentric stainless-steel manifolds; one supplied fresh vapor to the breathing zone of each animal and one removed the expired and excess vapor. Airflow through the unit was 20 l/min from the generator (inlet) with an exhaust flow of
18 l/min, to prevent the animals from rebreathing their expired air. For concentration determination, three of the ports (one from each tier) were used to collect samples of the breathing atmosphere; samples were also collected from the inlet manifold using an additional port from the lowest tier. Uniformity of the test-article concentration in the exposure unit was determined prior to the study.
The exposure concentrations of BE were determined by gas chromatographic analysis of atmosphere samples collected on charcoal sampling tubes (ORBO-101) during the exposure. Known volumes of the chamber atmosphere were collected using a calibrated critical-orifice-controlled sampler at a constant flow rate of 0.2 l/min for 3 min each. Toluene containing 1-phenylhexane internal standard was added to the primary charcoal bed after sample collection, to desorb the test chemical from the charcoal. The sample extracts were analyzed using an HP-5890 gas chromatograph (GC), as described below. Nominal exposure concentrations were calculated using the mass of test article pumped, the total time during which the metering pump was running, and the dilution airflow into the chamber. This value was compared to the average exposure concentrations obtained by the charcoal sampling tubes.
At the end of each exposure (nose-only and whole-body), five mice were sacrificed using CO2, transferred to individual beakers, and washed 2x in 50 ml of hot water. Each fur rinse was stored frozen (-80°C) until analyzed. This fur washing procedure was similar to that used by Tyl, et al.(1995) to determine the deposition of ethylene glycol on the fur of rats and mice exposed to ethylene glycol aerosols.
An additional 5 mice/exposure were anesthetized immediately after inhalation exposures, using 70% CO2, and samples of blood were collected via the retro-orbital sinus. Each blood sample was stored frozen (-80°C) until analysis. The final groups of 5 mice/exposure were placed in individual metabolism cages (Lab Products, Seaford, Delaware) for an 18-h urine collection. The urine samples were collected over dry ice. Following urine sample collection, each metabolism cage was rinsed with 25 ml of distilled water. Each urine sample and cage rinse was stored frozen (-80°C) until analysis.
Experiment 4: Excretion into saliva.
To determine the kinetics of BE and BAA in blood vs. saliva, groups of mice were administered a target dose of 250 mg BE/kg body weight (bw), either by ip injection or oral gavage. Salivation was induced by injecting the cholinergic agonist, pilocarpine, a few min before beginning to collect saliva. Groups of mice (4 animals/dose/time) were anesthetized with an ip injection of ketamine:xylazine (87:13; 100 mg/kg) at specified times up to 2.5 h postdosing. Five min after the animal was anesthetized, pilocarpine (1 mg/kg) was injected and a glass capillary tube was used to collect saliva from the oral cavity for up to 45 min. Aliquots of saliva were collected every 15 to 30 min (100200 µl). Blood was collected from the retro-orbital sinus (
100 µl) at the midpoint of each saliva collection interval and by cardiac puncture at the end of saliva collection.
Experiment 5: Retention in stomach contents.
To determine if stomach contents could act as a sink for BE or BAA and thereby provide a prolonged exposure to stomach tissues, an additional group of animals was dosed with a target of 250 mg BE/kg bw by ip injection. The same methods of dosing and tissue collection used for the tissue dosimetry and pharmacokinetics studies (Experiment 2) were used for this additional group of animals, and blood, BE, and BAA were quantified in stomach tissue and stomach contents. The sacrifice times for this group were 3, 6, and 9 h and were chosen based on the results from the previous saliva study. In particular, forestomach tissue concentrations of BE were found to be significantly higher than other tissues by 6 to 9 h after dosing.
Analytical methods.
Blood, liver, forestomach tissue, glandular stomach tissue, cage washes, fur washes, urine, and saliva were all analyzed by acidification of a weighed quantity of sample (100 mg each) with 0.9 ml of 0.9 M H2SO4. Approximately 0.25g of Na2SO4 (to improve extraction efficiencies) was added and the samples were extracted with an equal volume of ethyl acetate containing known concentrations of the internal standards: ethoxyethanol and ethoxyacetic acid. H2SO4, and ethyl acetate volumes were adjusted based on sample weights.
To determine the extent of conjugation of BE and BAA, aliquots (100 µl) of urine and cage wash samples were hydrolyzed by the addition of HCl (
200 µl 6.0 N HCl) and incubated for 1 h at 110°C. Following incubation, 0.25g Na2SO4 was added to each sample and extracted with 200 µl ethyl acetate containing known concentrations of ethoxyethanol and ethoxyacetic acid internal standards.
Quantitation of BE and BAA in blood, glandular stomach tissue, cage wash, urine, fur washes, and saliva were performed using a Hewlett Packard 6890 Gas Chromatograph with flame ionization detection (Hewlett Packard, Avondale, PA). Separation and quantification were achieved with a 30 m x 0.32 mmid x 1.0 µm-film thickness Stabilwax DA capillary column (Restek, Bellefonte, PA). Splitless injections of 2.0 µl of extract were conducted at an injector temperature of 210°C. The initial oven temperature was 80°C for 1 min, increased at 20°C/min to 240°C, and held for 1 min. Hydrogen was used as the carrier gas at 25 psi for 5 min, then rapidly increased to 30 psi. The FID temperature was 275°C. Under these conditions, ethoxyethanol, butoxyethanol, ethoxyacetic acid, and butoxyacetic acid had retention times of 2.4, 3.8, 6.9, and 7.9 min, respectively. Slight variations in retentions times were observed for each matrix.
Due to the presence of interferences by GC/FID, quantitation of BE and BAA in liver, forestomach tissue, and stomach contents was performed on a Hewlett Packard 6890 gas chromatograph equipped with a Hewlett Packard 5973 Mass Selective Detector. Sample injections, columns, and oven conditions were similar to those described for GC/FID analysis. For GC/MS analysis, helium was used as the carrier gas, rather than hydrogen, at an initial pressure of 25 psi for 5 min, and rapidly increased to 30 psi. Quantification of butoxyethanol and butoxyacetic acid in liver was achieved by selected ion monitoring of m/z 31, 57, or 73 for BE at 3.8 and of m/z 31 or 58 for BAA at 7.9 min retention time. The ions monitored for any given matrices varied due to detection limits, linearity of response, and background interferences. The appropriate ions were determined using matrix standards.
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RESULTS |
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Initial efforts to obtain a unit-length labeling index (ULLI) by counting the number of cells in S-phase per mm of the mucosa within the forestomach in at least four 0.25 mm random sections per animal proved inappropriate, due to the focal nature of the lesions. Therefore, analyses of PCNA labeling was limited to a qualitative assessment of the relative amount of S-phase cells in histologically normal forestomach epithelium versus the epithelium surrounding histological lesions. There was evidence of an increase in PCNA staining intensity in regions surrounding focal areas of hyperplasia and inflammation, as compared to either the forestomach epithelium of control mice or to histologically normal regions within the forestomach of BE-exposed mice. Lesions with focal regions of inflammation and thickening of the epithelium showed greater overall PCNA staining as well as clearer evidence of S-phase cells.
Minimal lesions, similar to those produced in the lower-dose (200400 mg/kg/day) oral gavage groups, were observed in 1/6 of the ip-dosed mice and 2/6 of the sc-dosed mice following 4 days of dosing with 400 mg/kg/day. No lesions were observed in the controls. Representative pictures of forestomach lesions following each route of exposure are shown in Figure 1.
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Elimination half lives (T1/2) and areas under the curve (AUC) were determined for both BE and BAA (Tables 4 and 5
). For BE, both T1/2 and AUC were higher for gavage dosing than ip dosing. Regardless of route, the T1/2 and AUC were higher in forestomach than any other tissue. Blood T1/2 and AUC for BE could not be determined from data in the pharmacokinetic experiment since T1/2 was shorter than the earliest time points collected. The T1/2 for BAA in blood ranged from 1 h following ip injection to 2.1 h following a gavage dose.
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The concentration of BE in the blood of five female mice at the end of each exposure averaged 3.0 and 3.9 mg/l for whole-body and nose-only exposures, respectively. The concentrations of the major metabolite, BAA, in blood were 235 and 390 mg/l for the whole-body and nose-only exposures, respectively. These concentrations of BE and BAA were similar to the levels reported in the NTP chronic toxicokinetic study for equivalent exposure concentrations (Dill et al., 1998).
Low levels of unmetabolized BE (67.668.7 µg) were found in the urine of mice collected 018 h after both whole-body and nose-only exposures. The BE in urine was presumed to have come from the fur of the mice, since very little BE is expected to be excreted in urine unconjugated, based upon results from the Tissue Dosimetry and Pharmacokinetics study (Experiment 2) discussed above. Comparable amounts of free BAA were found in the urine following nose-only (2021 ± 537 µg) vs. whole-body (1783 ± 645 µg) exposure methods and were similar to levels reported in an NTP chronic toxicokinetic study (Dill et al., 1998).
Experiment 4: Excretion into saliva.
The actual doses for the salivary clearance studies were 258.0 ± 65.6 and 265.4 ± 41.8 mg/kg for the ip or oral gavage doses, respectively. Since the results from the tissue dosimetry and pharmacokinetics experiment (Experiment 2) indicated rapid blood-elimination kinetics and saliva and blood collections were carried out from 7 min to 2 h following ip injection and for up to 3 h following oral gavage. Peak blood and saliva BE concentrations were detected at the first time point (15 min for blood,
7.5 min for saliva) post-BE injection, regardless of route. Concentrations of BE in blood and saliva were nearly identical at all times and were below the level of detection after 1.3 h (Figs. 4a
and 5a
). BAA levels, however, did increase over the first 1530 min and declined thereafter. Saliva BAA levels mirrored blood levels at all time points, albeit at approximately 4-fold lower levels (Figs. 4b
and 5b
).
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Experiment 5: Retention in stomach contents.
The actual dose administered, via ip injection, for the stomach contents experiment was 193.3 ± 23.1 mg/kg. Figures 6 and 7
show the stomach-content levels of BE and BAA at 3, 6, and 9 h post-ip dosing. The concentration of BE was higher in the stomach contents than in the forestomach tissue; no BE was detected in either blood or glandular stomach tissue at any time point in these animals, although it was present in the tissue dosimetry and pharmacokinetics experiment (Experiment 2) at a 26% higher dose.
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DISCUSSION |
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Oral dosing of neat BE to male and female mice resulted in dose-response irritation to the forestomach that was similar to the lesions observed in the NTP subchronic and chronic inhalation studies (Fig. 1). Forestomach lesions consisted of focal areas of irritation and epithelial hyperplasia. The proliferative responses were combined with increased PCNA immunohistochemistry of epithelial cells surrounding the lesions vs. normal cells in the forestomach. This is in contrast to earlier 2-week and 13-week drinking water studies conducted by the NTP (NTP, 1993
), where no forestomach irritation was observed even at dose levels as high as 1400 mg/kg/day. These results indicate that high dose rates or at least locally high concentrations of BE or BAA may be required to produce forestomach irritation and toxicity.
Forestomach lesions were also observed in female mice following ip and sc injections of 400 mg/kg/day for 4 days, indicating that oral dosing is not required to produce toxicity (Fig. 1). These data suggest that systemic delivery of BE or its metabolites may cause forestomach lesions in the mouse similar to those observed in the NTP chronic inhalation bioassay. Thus, oral consumption via grooming of 2-butoxyethanol condensed on the fur of mice may not be the only explanation for forestomach irritation in the inhalation studies. Since forestomach irritation, a precursor to tumor formation, was induced following multiple routes of exposure, ip injection and oral gavage were chosen to compare oral vs. parenteral administration on forestomach tissue dosimetry.
The kinetics of BE in female mice showed a rapid conversion to BAA, with peak BE concentrations having occurred prior to the initial blood samples collected after 15 min (from the saliva study). Concentrations of BAA, as measured in the saliva study, indicated peak blood levels occurred at approximately 30 min postdosing. Analysis of tissue levels of BE and BAA indicated that both chemicals are retained in the stomach tissues, especially the forestomach region, to a greater extent than any other tissue. After oral gavage, initial BE levels in the mouse stomachs were much greater than any other tissue. These levels remained elevated for up to 12 h postdosing. Likewise, BAA levels in the forestomach following oral gavage did not parallel the other tissues and appeared to decline more slowly.
While stomach tissue concentrations of BE and BAA are expected to be elevated following oral gavage, remarkably similar trends in stomach tissue concentrations were observed after ip dosing. By 1 h postdosing, BE levels in the forestomach were greater than for any other tissue. This higher level of BE in the stomach (both forestomach and glandular stomach) was maintained throughout the 24-h tissue-sampling period. Following the 50 mg/kg ip dose, BE levels were still detected in the forestomach after 24 h, but were nondetectable in any other tissue, including blood, after 30 min. Forestomach BAA concentrations paralleled other tissues for up to 3 to 6 h, then showed an apparent decline in the elimination rate. It is possible that the relative increase in BAA in stomach tissue was a consequence of the retention or secretion of BE into the stomach followed by metabolism to BAA. A comparison of the alcohol and aldehyde dehydrogenase-mediated metabolism of BE in the mouse and rat indicates that forestomach tissues from the mouse may metabolize BE to BAA at a faster rate than in the rat (Green et al. 2001a).
Several studies, including those described herein, have indicated that the forestomach BE deposition is derived from a combination of several means: (1) grooming; (2) swallowing of mucous containing material; (3) salivary excretions and re-ingestion; and (4) grooming of BE present on the fur. High local concentrations of radioactivity were detected in the buccal cavity, salivary glands, esophagus, and stomach tissue following either intravenous (Bennette et al., 2001) or short-term inhalation (Green et al., 2001a
,b
) exposures to 14C-BE. While the amounts of BE recovered from the fur of mice exposed by whole-body or nose-only inhalation exposures were minimal in the present study (Experiment 3), the specific disposition of BE and BAA in the forestomach of female B6C3F1 mice following ip or oral gavage dosing (Experiment 2) supports these various methods for delivery of BE to the forestomach tissues.
The mouse stomach is anatomically very different from the human stomach. Unlike the human, the mouse stomach has two different regions. Food is initially deposited from the centrally located esophagus into the forestomach before it moves to the glandular stomach. The forestomach is a site of bacterial digestion with no counterpart in the human stomach. The mouse glandular stomach, like the human stomach, is secretory.
The design of the rodent forestomach allows for a slower rate of emptying of food from the stomach and enhanced digestion (DeSesso and Jacobsen, 2001). Both BE and BAA were present in the stomach contents at greater concentrations and total amounts than in the stomach tissues themselves (Figs. 6
and 7
). It is likely that small amounts of food remain in the forestomach and act as a sink for BE, which is swallowed or partitioned across the stomach mucosa. This sink, in turn, may act as a continual source for BE to forestomach tissues, which may further metabolize BE to BAA and maintain a localized tissue concentration long after BE and BAA have been cleared from the rest of the body (Fig. 8
). In fact, locally high BE and BAA concentrations could have occurred in the tissues if these materials were associated with the epithelial cells and not distributed throughout the full thickness of the forestomach and glandular stomach as they were collected and analyzed. It is unlikely that BE and BAA preferentially bind to stomach tissue macromolecules, since the partition coefficients for BE and BAA are not significantly different in the stomach vs. any other tissue of rats or mice (Corley et al., 1998; Farris, 1998
; Lee et al., 1998
). A PBPK model for BE is currently being modified to include the forestomach and to further investigate the potential mechanisms leading to the forestomach disposition of BE and its metabolite, BAA.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Chemical Dosimetry, Battelle, Pacific Northwest Division, P.O. Box 999, MSIN P7-59, Richland, WA 99352. Fax: (509) 376-9064. E-mail: torka.poet{at}pnl.gov.
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