* Institute of Toxicology, GSF National Research Center for Environment and Health, D-85764 Neuherberg, Germany; Institut für Toxikologie und Umwelthygiene, Technische Universität München, München, Germany;
Present address: STTV-National Product Control Agency for Welfare and Health, Chemicals Department, P.O. Box 210, FIN-00531 Helsinki, Finland;
Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA; ¶ Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674, USA; || Department of Biosciences, Karolinska Institute, Novum, S-14157 Huddinge, Sweden; ||| Department of Molecular Biology and Functional Genomics, Stockholm University, S-10691 Stockholm, Sweden
1 To whom correspondence should be addressed at GSF-Institute of Toxicology, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany. Fax: + 49-8931873449. E-mail: johannes.filser{at}gsf.de
Received August 25, 2004; accepted October 6, 2004
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ABSTRACT |
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Key Words: propylene oxide; glutathione; lung; liver; blood; inhalation; respiratory nasal mucosa; rat.
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INTRODUCTION |
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PO alkylates nucleophilic sites of macromolecules introducing a hydroxypropyl-group. Following inhalation or intraperitoneal administration of PO to rats and mice, adducts to DNA have been detected in all tissues investigated (Osterman-Golkar et al., 2003; Ríos-Blanco et al., 1997
, 2003a
; Segerbäck et al., 1994
, 1998
; Snyder and Solomon, 1993
; Svensson et al., 1991
). N7-(2-hydroxypropyl)guanine (7-HPG) showed the highest adduct levels as determined in vitro (Solomon et al., 1988
) and in vivo. DNA adducts have also been detected in a new study carried out in PO-exposed workers (Czène et al., 2002
). Following exposure of mice, rats, and humans to PO, adducts to amino acids of hemoglobin (cysteine, histidine, or N-terminal valine) have been described by several groups (Boogaard et al., 1999
; Czène et al., 2002
; Farmer et al., 1982
; Högstedt et al., 1990
; Osterman-Golkar et al., 2003
; Pero et al., 1985
; Ríos-Blanco et al., 2002
; Segerbäck et al., 1994
; Svensson et al., 1991
).
PO was mutagenic in Drosophila and in microorganisms, and clastogenic in mammalian cells in vitro (reviewed in Giri, 1992; IARC, 1994
), and at very high doses (300 and 450 mg/kg, intraperitoneal administration) in mice in vivo (Bootman et al., 1979
; Farooqi et al., 1993
). However, after oral PO administration to mice, neither micronucleus formation in polychromatic erythrocytes in bone marrow (2 x 500 mg/kg) nor dominant lethal mutations had been detected following repeated PO administrations of 250 mg/kg/day for 14 days (Bootman et al., 1979
). Also, an inhalation study in rats (300 ppm; 7 h/day, 5 days) did not result in dominant lethal mutations (Hardin et al., 1983b
). Furthermore, sperm abnormalities were not detected in mice, which were exposed to 300 ppm (7 h/day, 5 days; Hardin et al., 1983a
). Moreover, there were no increases in the sister chromatid exchanges (SCEs) nor in chromosomal aberrations in Cynomolgous monkeys that were exposed via inhalation for over 2 years to 0, 100, and 300 ppm PO (Lynch et al., 1984b
). In vitro studies in which the genotoxic potencies of PO, ethylene oxide, and epichlorohydrin were compared demonstrated that PO was a much less potent genotoxicant than the two other epoxides (Agurell et al., 1991
; Kolman and Dusinská, 1995
; Kolman et al., 1997
).
In long-term studies with rats and mice, PO induced tumors at the application site. In some rat studies, an increase in tumors has also been detected in a few glands in which tumors developed spontaneously. Upon intragastric administration of PO to Sprague-Dawley rats (0, 15, 60 mg/kg; 2 doses per week; 109.5 weeks) tumors developed dose-dependently in the forestomach (Dunkelberg, 1982). Repeated subcutaneous injections to NMRI mice of up to 2.5 mg PO per mouse (1 dose/week; 95 weeks) led to local tumors at the injection site, in a dose-dependent manner (Dunkelberg, 1981
). Two long-term inhalation studies have been conducted in Fischer 344 rats and one in B6C3F1 mice. The most striking findings reported in these studies were exposure-related increases in the incidences of inflammatory lesions and hyperplastic changes and a low incidence of nasal tumor formation in the high exposure groups (
300 ppm) only. Adenomas (37%) occurred in the nasal cavity of Fischer 344 rats (Lynch et al., 1984a
, exposure concentrations 0, 100, and 300 ppm) and (Renne et al., 1986
, exposure concentrations 0, 200, and 400 ppm). Combined incidences of hemangiomas and hemangiosarcomas of 20% (males) and 10% (females) and of adenocarcinomas of 4% (females) were reported in nasal cavity of B6C3F1 mice exposed to 400 ppm PO (Renne et al., 1986
, exposure concentrations 0, 200, and 400 ppm). One study was conducted with Wistar rats that were exposed to 0, 30, 100, and 300 ppm PO (6 h/day, 5 days/week, 124 weeks). In the nasal mucosa, increased degenerative and hyperplastic changes were reported for all treatment groups; three male animals developed nasal tumors: one ameloblastic fibrosarcoma and one squamous cell carcinoma at the low dose, and one squamous cell carcinoma at the high dose. In four males of the high-dose group a carcinoma was detected in the larynx or pharynx, trachea, or lungs (Kuper et al., 1988
).
From the findings that nasal tumors had developed only when inflammatory lesions were present, one could conclude that nasal tumorigenicity of PO did not result solely from its genotoxicity. Other mechanisms are probably involved, too. Detailed quantitative knowledge of such mechanisms is required as a prerequisite for a scientifically sound estimate of the tumor risk of PO to humans.
Metabolic elimination of PO is mediated by epoxide hydrolase (EH, Faller et al., 2001; Guengerich and Manson, 1980
) and by glutathione S-transferase (GST, Faller et al., 2001
; Fjellstedt et al., 1973
). The latter enzyme catalyzes the conjugation of PO with glutathione (GSH). This process could cause a decrease in the cytosolic nonprotein sulfhydryl (NPSH) content of which GSH is the major contributor. GSH is involved in a multitude of cellular functions (reviewed in, e.g., Pompella et al., 2003
). Severe perturbations of GSH status can lead to cytotoxicity (reviewed in, e.g., Comporti, 1989
), apoptosis (reviewed in, e.g., Cotgreave and Gerdes, 1998
; Slater et al., 1995
), and cell proliferation (reviewed in, e.g., Burdon, 1995
). As a consequence of apoptotic or necrotic cell death, a regenerative proliferation of cells might be expected too. Following repeated PO inhalation exposure, induction of cell proliferation was observed in respiratory nasal mucosa (RNM) of rats (Eldridge et al., 1995
; Ríos-Blanco et al., 2003b
). Also, PO adducts to DNA were detected in all tissues studied; by far the highest levels were found in RNM, with up to 25-fold higher adduct levels than other, systemically exposed tissues (Osterman-Golkar et al., 2003
; Ríos-Blanco et al., 1997
, 2003a
; Segerbäck et al., 1998
). However, there are no data from in vivo studies on the relationship between internal PO, GSH, and external PO exposure. Consequently, the goal of the present work was to investigate PO concentrations in blood and NPSH levels in tissues of male Fischer 344/N rats exposed over different exposure durations to diverse concentrations of atmospheric PO. Air concentration of single PO exposures ranged up to 750 ppm PO; the highest concentration of repeated exposures lasting up to 4 weeks was 500 ppm. PO was determined in venous blood and NPSH in the sites-of-entrance RNM and lung, in the main metabolizing organ, liver, and in blood by which PO is distributed systemically.
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MATERIAL AND METHODS |
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Animals and housing conditions. Male Fischer 344/N rats were obtained from Charles River Wiga (Deutschland) GmbH, (Sulzfeld, Germany). Body weights were in the range of 180250 g at the start of the exposures. Incoming animals were briefly anesthetized using diethyl ether and marked with ear tags. Thereafter, animals were housed at the GSF-Institute of Toxicology in stainless-steel wire cages. The housing conditions have been described in detail in an earlier study (Ríos-Blanco et al., 1997). Treatment of animals were done in agreement with the German Animal Welfare Law.
Exposure to propylene oxide. Three exposure studies were carried out, in which a total of 222 rats were exposed to constant atmospheric PO concentrations (Table 1). The mean exposure concentrations varied on average 2.2% of the target concentrations (n = 29). The maximum mean deviation from the target concentration was 5% in the 1-day exposure to 10 ppm. Rats were assigned to exposure groups on the basis of their body weight, resulting in comparable average body weights and standard deviations in all groups. All exposures were started between 9 and 11 A.M. and lasted for precisely 6 h.
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For the 1- to 5-day exposure study, 50 rats were distributed into four groups. At the first day 15 rats were exposed to 50 and another 15 to 100 ppm PO, 10 air-exposed rats served as controls for the 50 ppm and 10 for the 100 ppm exposures. Every day, each exposure group was reduced by three animals and each control group by two animals, which were sacrificed immediately at the end of the 6-h daily exposure. Animals were used to determine NPSH in selected tissues.
For the third studyrepeated 6-h exposures over 3 days and 4 weeks (5 days/week)groups of eight animals each were exposed to constant atmospheric PO concentrations of 5, 25, 50, 300, and 500 ppm. Three animals of each group were used to determine PO in blood and NPSH in selected tissues. Blood and tissues of the remaining five rats/group were used to quantify DNA and hemoglobin adducts (Osterman-Golkar et al., 2003; Ríos-Blanco et al., 2002
, 2003a
). Per exposure concentration and time point (3 days and 4 weeks) eight air-exposed rats served as controls for the exposures to 5 and 500 ppm and four animals for those to 25, 50, and 300 ppm. Of the 56 control animals, 30 were used as NPSH controls, and most of the residual ones were used for the determination of background adducts and to validate zero PO exposure by PO measurement in blood.
Most of the exposures were carried out in the dynamic chambers described below. Those animals that were used for the determination of PO in blood and NPSH in tissues were exposed on the last exposure day in a closed all-glass chamber. This was because the blood samples had to be collected immediately after stopping the PO exposure in order to avoid loss of PO due to metabolism and exhalation. The large dynamic chambers could not be purged quickly enough following the end of exposures.
Exposure in the dynamic chambers. The exposure system consisted of six chambers as described above. PO vapor was generated using a vaporization technique. By means of infusion pumps (Precidor Type 5003, Infors, Bottmingen, Swiss) connected to gastight glass syringes (1-, 5-, and 10-ml syringes with PTFE plunger tip from Hamilton, Darmstadt, Germany, and Unimetrics 100-ml glass syringes with PTFE plunger tip from Machery-Nagel, Düren, Germany) liquid PO was injected at room temperature into vaporization glass chambers of 60 ml each (in-house construction) supplied with pressured ambient conditioned air precleaned by charcoal and particle filters (about 10 l/min; generated with an oil-free membrane pump). The resulting PO vapors were passed through PTFE and stainless steel tubing into the air supply of 70 l/min (resulting in a total air flow of 80 l/min, see above) of the respective chambers. Through a septum at the outlet of each chamber, air samples were taken in 530 min time intervals using disposable syringes (B. Braun, Melsungen, Germany) and were analyzed by method B as described below. Within 15 min from starting the exposure, 80% of the target PO concentrations were achieved in each chamber. No PO could be detected in the control chamber atmosphere, which was analyzed hourly for PO by method A (see below; detection limit 0.02 ppm).
Exposure in a closed chamber. On the very last exposure day, animals destined for determination of PO in blood were exposed in a closed all glass chamber under atmospheric pressure and room temperature. The system consisted of a glass-sphere (63 l), equipped with an 8 cm long neck (inner diameter 15 cm) closed by a round lid with three ports. Two ports were covered by Teflon-coated synthetic rubber septa; the third one was connected to a passive oxygen supply system (Filser, 1992). The sphere contained a circular, foldable floor plate of solvent-inert polyvinylidene fluoride (PVDF) with a diameter of 40 cm. Exhaled CO2 was trapped with 30 g Drägersorb 800 (Drägerwerk, Lübeck, Germany) which was below the floor plate. The concentration of PO in the chamber was determined by gas chromatography (see below, GC method B) and kept constant by injecting liquid PO repeatedly through one of the septa into the chamber air in order to compensate for the loss of PO by metabolism. Three (most exposures) or two (single exposures) rats were placed onto the floor plate at intervals of 40 min. Each animal was exposed for exactly 6 h. About 15 min before sacrifice of each rat, it was removed from the chamber and immediately anesthetized. In the 1-day exposure experiments, Ketavet® was injected intraperitonially (120 mg/kg). In all other experiments a mixture containing 90 mg Ketavet®, 20 mg Rompun®, and 0.05 mg Atropin sulfate per kg body weight was used. Directly thereafter, the anesthetized, lightly breathing animal was returned to the chamber. Opening the chamber in order to place or remove an animal resulted in a maximum decrease of the PO concentration in the chamber air of 20% (data not shown). Within 5 min, the target concentration was readjusted by compensating for the amount of PO being lost. At the end of the exposure the anesthetized animal was removed from the chamber and immediately sacrificed in order to prevent PO exhalation. Blood was collected within 2 min.
Collection of blood and tissue samples. Blood samples (4.56.5 ml) were obtained by puncture of the vena cava caudalis immediately before entering the heart (mixed venous blood) using disposable 5- or 10-ml syringes made of polypropylene and sterilized with ethylene oxide (Braun, Melsungen, Germany). The syringes were heparinized with Liquemin® N 25000. About 0.5 ml blood was taken for NPSH measurements and frozen in Eppendorf tubes (2 ml, Eppendorf, Hamburg, Germany) using liquid nitrogen. The remaining blood was used to determine PO concentrations as described below.
Liver and lung were removed, frozen using liquid nitrogen, and stored at 80°C.
RNM was isolated according to Casanova-Schmitz et al. (1984), Mery et al. (1994)
, and Faller et al. (2001)
. The isolated tissues were placed in 1.5-ml Eppendorf tubes containing 0.9 ml potassium phosphate puffer (pH 7.4; 10 mmol/l) and were frozen using liquid nitrogen and stored at 80°C.
Animals used for adduct determination were sacrificed with carbon dioxide. Blood samples and tissues were collected as described elsewhere (Ríos-Blanco et al., 2003a; Osterman-Golkar et al., 2003
).
Determination of propylene oxide in air. PO was measured in air samples using gas chromatographs (GC-8A, Shimadzu, Duisburg, Germany or Packard 437A, Chrompack, Frankfurt/M., Germany) equipped with packed columns and flame ionization detectors (FID). Different analytical tasks required different methods, as follows. Method A was developed to determine gaseous PO in headspace vials containing blood samples and in air samples collected from those chambers that contained the nonexposed control animals. Gas samples of 2 ml were taken by disposable syringes (Braun, Melsungen, Germany) and were injected via a 1-ml gas sample loop onto a Porapack Q column (5080 mesh, 1/8 in x 2.5 m). With this column a baseline separation of PO from acetone was achieved. Method B was used to monitor PO in the atmosphere of exposure chambers by injecting gas probes via a 1-ml or 0.5-ml gas sample loop on a Porapack Q column (5080 mesh, 0.751.5 m) or on a Tenax TA column (5080 mesh, 1/8 in x 1.75 m). For method B, a baseline separation of PO from acetone was not necessary, since the air concentration of exhaled acetone was negligible compared to that of the PO concentration. The shorter columns permitted shorter time intervals between the measurements. This was required for the quasi-continuous monitoring of the exposure chambers. For both methods, separation was done isothermally at 160°C using N2 as carrier gas. Injector and detector temperatures were kept at 210°C. Using method A, the retention times of PO and acetone were about 3.4 and 4.3 min, respectively. The retention time for PO ranged from 1 to 1.7 min when method B was used. Peaks were recorded by either a Shimadzu C-R5A or by an HP 3394A (Hewlett Packard, Waldbronn, Germany) integrator. From time to time, calibration curves were constructed for each method by injecting pure PO or defined mixtures of PO and air into desiccators of known volumes. Calibration curves were linear in the range of 0.02553 ppm for method A and 0.31000 ppm for method B. The coefficients of variation were 7% and 5% for methods A and B, respectively. The limit of detection, defined as three times the signal-to-noise ratio, was 0.02 ppm for method A and 0.1 ppm for method B. For daily determinations a one-point calibration was performed in the range of the expected PO concentration.
Determination of propylene oxide in blood. A headspace method was developed for the determination of PO in blood. In order to monitor the decline of PO in blood over time, PO vapor was measured in the air of several headspace vials (7.9 ml) by sampling a single gas probe from each vial at different time points after the addition of blood from PO-exposed animals. Between 3 and 6 preweighed headspace vials were sealed with Teflon-coated septa and alumina crimp seal caps. Immediately before blood collection from the PO-exposed animal, a cannula (0.45 x 12 mm, B. Braun, Melsungen, Germany) was inserted in each headspace vial for pressure equilibration. Then, a blood sample from the PO-exposed animal was distributed among the headspace vials, with about 0.51 ml blood injected via a second cannula into each vial. Immediately after injection, both cannulae were removed from each vial. Thereafter, the vials were kept in a shaking water bath at 37°C. After 510 min equilibration, about 12 ml gas sample was taken from the headspace of the first vial using a disposable syringe, and a cannula (0.45 x 12 mm) was instantly inserted for pressure equilibration. The gas sample was analyzed for PO as described above (Method A). From the other vials, headspace samples were collected by the same procedure within time intervals of 510 min. The volume of blood (VB) present in each vial was determined by weighing the vials before and after the experiment. The density of blood was assumed to be 1 g/ml. The concentration of PO in blood (CB) from each headspace sample was calculated using the PO concentration measured in the gas phase (CG), the partition coefficient blood:air (Pba = 60; Schmidbauer, 1997), the blood volume (VB), the volume of the headspace (VG), and the recovery coefficient (fR):
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The initial PO concentration in blood was determined by extrapolating the concentration decline of PO to time point zero (time of blood sampling) by fitting an exponential decline through the data obtained for each blood sample series (see below).
The recovery coefficient fR was determined in independent experiments. Blood of naïve rats was added to headspace vials. Thereafter, 9 µl of aqueous PO solutions containing defined amounts of PO were injected, establishing PO blood concentrations between 0.820 µmol/l. Concentrationtime courses were monitored up to 55 min by the headspace method described above. The PO concentrations decreased following first-order kinetics with a half-life of 53.7 ± 6.0 min (n = 8). By comparing the known PO concentrations at time point zero to those extrapolated from the obtained data points, the recovery coefficient was calculated to be 0.70 ± 0.05 (n = 8). This was independent of the PO concentrations.
Determination of soluble nonprotein thiols. Generally, frozen tissues were thawed on ice and homogenized while ice-cold. Then, the concentration of soluble nonprotein thiols (NPSH) was determined using Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid), DTNB) according to Riddles et al. (1979) and Eyer and Podhradsky (1986)
. The concentration of NPSH in rat tissues is mostly comprised of GSH (Potter et al., 1995
; Potter and Tran, 1993
).
Liver tissue samples of 0.1 g were placed in an ice-cooled 15-ml Teflon-glass homogenizer (B. Braun, Melsungen, Germany) and were homogenized with 10 ml ice-cold potassium phosphate buffer (pH 7.4; 10 mmol/l). A 1-ml aliquot of the homogenate was used for protein determination. An additional 1-ml aliquot was mixed with 1 ml protein precipitation solution (PPS; 1.67% meta-phosphoric acid, 0.02% disodium ethylenediaminetetraacetate dihydrate, and 30% NaCl in water) and centrifuged (0°C, 16000 x g, 5 min). The pH of the supernatant (1 ml) was adjusted to 6.0 using 80 µl NaOH (1 mol/l), and 0.5 ml Ellman's reagent (1 mmol/l DTNB and 0.034 mmol/l trisodium citrate dihydrate) was added.
Lung tissue was weighed and cut in small pieces. Adequate amount of buffer was added before homogenization to obtain a tissue-to-potassium phosphate buffer (pH 7.4; 10 mmol/l) ratio of about 1:15 (see liver). A 1-ml aliquot of homogenate was retained for protein determination. A further aliquot of 0.3 ml was mixed with 0.7 ml PPS and then centrifuged. The supernatant was filtered (microfilter Minisart, pore size 0.45 µm, Sartorius, Göttingen, Germany) and the pH of the filtrate (0.5 ml) was adjusted to 6.0 using 60 µl NaOH (1 mol/l). Thereafter Ellman's reagent was added.
RNM tissue was weighed immediately after tissue isolation and then frozen in 0.9 ml potassium phosphate buffer (pH 7.4; 10 mmol/l). After thawing on ice, it was directly homogenized in an ice-cooled 2-ml Teflon-glass homogenizer. An aliquot of 0.3 ml homogenate was mixed with 0.7 ml PPS and centrifuged. The pH of the supernatant (0.5 ml) was adjusted to 6.0 using 60 µl NaOH (1 mol/l). Thereafter Ellman's reagent was added. For protein determination, the remaining freshly prepared homogenate was used.
The heparinized blood samples were thawed on ice. Blood samples (50 µl) were diluted with 0.45 ml potassium phosphate buffer (pH 7.4; 10 mmol/l). An aliquot of 0.3 ml was mixed with 0.7 ml PPS and centrifuged. Thereafter the same steps were followed as given for lung samples.
After adding Ellman's reagent to the aliquots, they were incubated in the dark for 15 min at room temperature (2225°C). Thereafter, the extinction at = 412 nm was measured against a blank sample using a UV/VIS photometer (Lambda 16, Perkin Elmer, Überlingen, Germany). The blank samples were prepared identically to the tissue samples with the exception that potassium phosphate buffer (pH 7.4; 10 mmol/l) was added instead of tissue homogenate. NPSH determination was carried out at least in triplicate for liver, lung, and blood and in duplicate for RNM. Calibration samples were also prepared in phosphate buffer by adding GSH to yield concentrations of 0.0050.2 mmol/l; this concentration range resulted in linear calibration curves.
Determination of soluble proteins. The concentration of soluble proteins in tissue homogenates was determined by the Biuret method (Gornall et al., 1949).
An aliquot of the tissue homogenate (40 µl) was mixed with 1 ml Biuret reagent (0.1 mol/l NaOH, 16 mmol/l sodium potassium tartrate x 4 H2O, 6 mmol/l copper sulfate x 5 H2O, 15 mmol/l potassium iodide). The sample was incubated in the dark for at least 30 min at room temperature. Thereafter, the extinction was determined at = 546 nm using a UV/VIS photometer. The color complex was destroyed by adding 50 µl KCN solution (6 mol/l). Following a further incubation of 5 min, the extinction was measured again. The resulting extinction difference was used to calculate the protein concentration. Protein determination was carried out in triplicate. Calibration samples were also prepared in phosphate buffer by adding bovine serum albumin resulting in concentrations of 1.2520 mg/ml; this concentration range resulted in linear calibration curves.
Data handling and review. Time-weighted mean atmospheric exposure concentration () was calculated by using the atmospheric concentrations (Ci) determined successively during the given exposure period (T).
was obtained by dividing the area under the concentration-time curve by T. The standard deviation (SDtw) from
was calculated according to Sachs (1997)
:
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RESULTS |
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Propylene Oxide in Blood
PO concentrations measured in blood at the end of a single 6 h exposure increased linearly with the atmospheric PO concentration up to 750 ppm (Fig. 1A) showing a slope of 0.058 ± 0.001 (µmol/L)ppm. Likewise, after repeated daily 6-h exposures over 3 days and 4 weeks PO blood concentrations increased linearly (Figs. 1B and 1C) up to exposure concentrations of 300 ppm with slopes of 0.040 ± 0.002 and 0.046 ± 0.004 (µmol/L)ppm, respectively. The slopes between 300 ppm and 500 ppm were steeper than for the lower concentrations: 0.085 (µmol/L)ppm after 3 days and 0.122 (µmol/L)ppm after 4 weeks.
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NPSH in Tissues
For each study (single, 1- to 5-days, 3-days, 4-weeks exposure), tissue NPSH values from the control rats were pooled because no influence of the experimental condition was observed (Table 2). The NPSH levels in the control tissues were similar when based on mg protein. However, distinct differences in the tissue NPSH levels were found, when based on wet tissue weight. Clearly, the protein content is tissue-specific.
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DISCUSSION |
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Following 3 days of exposure to 500 ppm PO, the mean PO concentration in blood (29 µmol/l) was almost the same as after the single exposure. After 4 weeks, the PO blood concentration was 31% higher (mean: 38 µmol/l). Concerning these repeated exposures, the PO concentrations in blood were relatively higher at 500 than at 300 ppm if related to the PO concentration in air. In those of the 3-days and 4-weeks exposed rats that had been selected for the determination of PO adducts, similar pictures were obtained with respect to the adduct levels to hemoglobin (N-(2-hydroxypropyl)valine; HOPrVal; Osterman-Golkar et al., 2003; Ríos-Blanco et al., 2002
) and to liver DNA (N7-(2-hydroxypropyl)guanine; 7HPG; Osterman-Golkar et al., 2003
; Ríos-Blanco et al., 2003a
). After 3 days, increases of hemoglobin and DNA adducts correlated linearly with the atmospheric PO exposure concentration up to 500 ppm, but after 4 weeks, the adduct levels at 500 ppm were clearly elevated. Plotting these adduct levels versus the corresponding PO concentrations in blood, linear relationships are obtained for both the 3-days and the 4-weeks exposures (Figs. 5A and 5B). The flatter curves after 3 days compared to those after 4 weeks result from the difference in the duration of the exposures, since it needs several days (DNA) and weeks (hemoglobin) until the rates of adduct elimination become as high as those of adduct formation. The linearity of the hemoglobin adduct curve indicates the direct dependence of the adduct levels from the PO blood burden (Fig. 5A). From the linearity of the DNA adduct curve it follows that the PO concentration in blood parallels the PO burden in the liver at steady state (Fig. 5B).
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The half-life of PO of 59 min was determined in the blood samples that had been collected at the end of the inhalation exposures. It is substantially less than the half-lives in water at 37°C (87 h; according to Ross, 1950) or in 0.15 mmol/l phosphate buffer at pH 7.4 and 37°C (16 h; Faller et al., 2001
). For spontaneous conjugation with 1 mmol/l GSH in phosphate buffer, a half-life of about 25 h can be calculated from data presented in Faller et al. (2001)
. Thus, the elimination of PO from blood is primarily enzyme-mediated. After adding PO to rat blood pretreated with diethyl maleate in order to deplete GSH, we determined a considerably longer half-life of PO elimination (about 6.5 h; data not shown). By comparing the rate constants derived from the half-lives of spontaneous hydrolysis (khydr = ln2/16), of GSH depletion (kdepl = ln2/6.5), and of total PO elimination from blood (ktot = 60 x ln2/59), the share of probably EH dependent PO elimination is obtained to be 9% (100 x (kdepl khydr)/ktot) from the total elimination. Similarly, the share of GST catalyzed PO elimination in blood is obtained to be 81% (100 x (ktot kGSH kdepl)/ktot with kGSH = ln2/25). Consequently, in rat blood, about 80% of the PO elimination is catalyzed by GST, and only about 10% could be metabolized by EH (valid for PO concentrations up to 40 µmol/l; see Fig. 1). GST is well known to occur in erythrocytes of mammals (al-Turk et al., 1987
; Shimizu et al., 1991
). The existence of EH was proven in lymphocytes of mice, rats, and humans (Sotnichenko et al., 1985
). Likely, the EH level in blood is much less than that of GST, because in different rat tissues the activities of both enzymes to PO were similar (Faller et al., 2001
).
NPSH in Tissues of Control Animals
The NPSH levels (mean ± SD, n) in the control animals were in RNM 26.1 ± 2.9 nmol/mg protein, n = 52 corresponding to 3.8 ± 0.3 µmol/g; in lungs 24.7 ± 2.6 nmol/mg protein, n = 65 corresponding to 1.6 ± 0.2 µmol/g; in livers 32.3 ± 2.9 nmol/mg protein, n = 52 corresponding to 6.6 ± 0.5 µmol/g; and in blood 1.04 ± 0.07 µmol/g, n = 49. Previously published NPSH or GSH concentrations determined in control rats are very similar: respiratory epithelium 4.2 ± 0.6 µmol/g (Potter et al. 1995); lungs 1.01.8 µmol/g (Deutschmann and Laib 1989
; McKelvey et al. 1986
; Moron et al. 1979
; Potter and Tran 1993
); liver 47 µmol/g (Deutschmann and Laib 1989
; McKelvey et al. 1986
; Moron et al. 1979
; Potter and Tran 1993
); blood 1.08 ± 0.16 µmol/g (Potter and Tran 1993
). The good agreement of the present control values with published ones indicates that neither anesthesia nor the freezing-and-thawing procedure of the tissues had any significant effect on the NPSH concentrations.
NPSH in Tissues of Exposed Animals
NPSH levels in blood determined over five consecutive days of exposure to 50 and 100 ppm PO showed a minimum at the second day of exposure to 100 ppm. A similar picture was obtained for the liver. These findings reflect the observation that the blood level of GSH is mainly driven by the GSH efflux from the liver, which itself depends on the hepatic GSH content (summarized in Ookhtens and Kaplowitz, 1998). After repeated exposures to 500 ppm PO, NPSH in blood was only marginally decreased, whereas in liver a substantial NPSH loss was observed. The difference might suggest an increased GSH efflux from the liver to the blood, in order to compensate for the GSH consumption in other organs and tissues. Investigations in rats have shown an increased GSH efflux due to GSH demand from other organs, which occurs at the expense of the hepatic GSH concentration (Lauterburg et al., 1984
; Lew et al., 1985
).
The liver is quantitatively the most relevant organ for the elimination of PO, which is metabolized by EH and GST (Faller et al., 2001). GSH will deplete if more GSH-conjugates are formed than GSH is resynthesized. Such an effect has been reported with several compounds as ethylene oxide, 1,2-epoxybutene-3 (Johanson and Filser, 1993
), styrene-7,8-oxide (Csanády et al., 1994
), bromobenzene, iodobenzene, diethylmaleate (Casini et al., 1985
), t-butylhydroxyanisole, (Eaton and Hamel, 1994
), and acetaminophen (Henderson et al., 2000
). Following a single exposure, NPSH depletion became clearly evident at 100 ppm PO. Repeated exposures to this concentration induced the greatest NPSH loss after the second exposure day. Thereafter NPSH levels were in the range of the control values. When exposing male rats of the same strain repeatedly (6 h/day, 5 days) to 500 ppm PO, Faller (1997)
observed after 2 days also an increase in hepatic NPSH levels, measured immediately at the end of the daily exposures (less NPSH depletion) as well as 18 h thereafter (higher morning levels). This effect resulted probably from a compensatory induction of GSH resynthesis. The linkage of the rebound of GSH above control levels with the increase of gamma-glutamylcysteine synthetase has been demonstrated in mice treated separately with the GSH-depleting chemicals diethylmaleate, phorone (2,6-dimethyl-2,5-heptadien-4-one), or tbutylhydroxyanisole (Borroz et al., 1994
). In the present study, hepatic NPSH concentrations measured after 3 days or 4 weeks of exposures were dependent on the PO concentration but not on the duration of exposure. The GSH production seems to have reached its maximum already on the third exposure day, resulting in effective adaptation of the liver to the PO burden.
In lungs, NPSH levels decreased linearly with increasing PO concentrations following the single exposures. Clear differences from the control values became evident at 150 ppm and higher. It is noticeable that the NPSH depletion was less marked than in the liver, although, based on DNA adduct data (Osterman-Golkar et al., 2003; Ríos-Blanco et al., 2003a
), the PO burden was higher in the lung, a portal-of-entry organ. This finding becomes understandable if one considers the in vitro data obtained by Faller (1997)
indicating that GST activity in lung was 4-fold lower than that in liver, based on tissue wet weight. The average pulmonary NPSH concentrations of about 70% (3 days and 4 weeks of exposure; 300 ppm) and 50% (4 weeks; 500 ppm) of the corresponding controls after repeated PO exposures were somewhat higher than after single exposures to the same concentrations. A pulmonary NPSH decrease to less than about 60% of the control value went along with lung toxicity in Wistar rats that had been repeatedly (30 days) intraperitoneally treated with the GSH biosynthesis inhibitor buthionine sulfoximine (Thanislass et al., 1995
). Lung toxicity was apparent through reduced activities of superoxide dismutase, catalase, and glutathione peroxidase, enhanced lipid peroxidation, and histopathological inflammatory changes.
In humans, changes in the GSH concentrations from bronchio-alveolar lavage samples are often observed with pulmonary diseases, which are associated with chronic inflammation (Cantin and Begin, 1991). An association between GSH depletion and activation of transcription factors regulating the genes for pro-inflammatory mediators has been reported (summarized in Rahman and MacNee, 2000
).
RNM showed the most severe NPSH depletion. This effect might result from the high PO burden of the nasal mucosa being in direct contact with PO in the inhaled air and the high GST activity, which is 1.6 times higher in RNM than in the liver if normalized for tissue wet weight (Faller, 1997). NPSH depletions were observed already 1 h after the begin of single exposures to
50 ppm PO, as has been demonstrated by Morris et al. (2004)
in the isolated upper respiratory tract of male F344 rats. Repeated exposures over 5 days to 50 and 100 ppm PO did not lead to an adaptation of the RNM to PO, since the NPSH levels were about 40% of the control levels at the end of each exposure day. An adaptation was seen following repeated exposures to 300 and 500 ppm, where NPSH levels after 3 days and 4 weeks of repeated exposures were higher than after a single 6-h exposure. This reduced depletion in NPSH levels following repeated exposures was probably due to a rebound effect, with an increased rate of GSH resynthesis. Accordingly, from the second day of repeated exposures (6 h/day, 15 days) of male Fischer 344/N rats to 500 ppm PO, the morning NPSH levels in RNM (measured 18 h after the end of the previous exposure) were up to 260% above the corresponding control levels, evidence for a pronounced rebound effect (Faller, 1997
). At the end of each of these repeated exposures, NPSH had declined to 28% of the control values. Obviously, the PO tissue burden in RNM was so high that a considerable share of NPSH was consumed by reaction with PO.
GSH Depletion, Cytotoxicity, Cell Proliferation, and Tumorigenesis
From in vivo findings in mice, rats, and humans, it has been concluded that hepatic GSH depletion to about 1030% of the control value is required for liver toxicity (Mitchell et al., 1973; Uhlig and Wendel, 1992
; Younes and Siegers, 1981
). After repeated exposure of rats to 300 and 500 ppm PO, NPSH concentrations were far above such a critical range in liver and blood. Consequently, no cytotoxicity was expected for either of these tissues, and there were no exposure-related changes found in cell proliferation in the liver (Ríos-Blanco et al., 2003b
; study done independently of the present one). In RNM however, after repeated exposures, NPSH was depleted to about 50% of control values at 50 ppm PO and 30% at 300 and 500 ppm PO. A comparison of the NPSH content in RNM with the proliferation determined in the same tissueexpressed as unit length labeling index (ULLI) by incorporation of bromodeoxyuridine (BrdU) in rats exposed to 0, 5, 25, 50, 300, or 500 ppmPO for 3 days or 4 weeks (Ríos-Blanco et al., 2003b
)demonstrates reciprocal behavior (Fig. 6). With increasing atmospheric concentrations of PO, NPSH levels in RNM decreased, approaching a plateau at about 300 ppm. In contrast, cell proliferation was comparable to the control value up to 50 ppm and was significantly induced at 300 and 500 ppm PO. The regression line constructed for the ULLI in dependence of the PO concentration between 50 and 500 ppm PO shows at about 180 ppm PO a doubling of the ULLI-values that had been obtained below 50 ppm PO. This expected doubling is supported by data from Eldridge et al. (1995)
. These authors investigated in repeatedly PO-exposed (0, 10, 20, 50, 150, 525 ppm; 6 h/day, 5 days/week; 1 and 4 weeks) male Fischer 344 rats PO-induced respiratory epithelium hyperplasia, and, by means of ULLI, nasal cell proliferation. They reported respiratory epithelium hyperplasia to be most common in animals from the 525-ppm group and to a lesser extent in those rats exposed to 150 ppm PO. Minimal respiratory epithelium hyperplasia was also detected in a few animals in the other concentration groups, including the control group. Although ULLI did not yield a significantly increased cell proliferation in RNM at 150 ppm PO, the authors, considering a twofold increase over controls to be biologically significant, plotted a curve suggesting a doubling of cell proliferation in RNM to be reached at about 190 ppm PO after 1week of exposure. Figure 6 shows that the NPSH level, estimated from the linear curve linking the NPSH content at 50 and 300 ppm PO, decreases to about 12 nmol/mg protein at 180 ppm PO. Assuming cell proliferation in RNM to be a consequence of the repeated and severe perturbation of the GSH levels in RNM, a daily NPSH loss of about 60% of the control value seems to be required for an about twofold increase in cell proliferation over controls.
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ACKNOWLEDGMENTS |
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The data were presented in part at the 37th Annual Meeting of the Society of Toxicology, Seattle, WA (Lee et al., 1998b); 39th Spring Meeting of the Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie, Mainz, Germany (Lee et al., 1998a
); 41st Spring Meeting of the Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie, Mainz, Germany (Lee et al., 2000
).
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