ToxSci Advance Access originally published online on January 4, 2008
Toxicological Sciences 2008 102(2):219-231; doi:10.1093/toxsci/kfm311
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Concentrations of the Propylene Metabolite Propylene Oxide in Blood of Propylene-Exposed Rats and Humans—a Basis for Risk Assessment
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* Institute of Toxicology, Helmholtz Zentrum München, D-85764 Neuherberg, Germany
Institut für Toxikologie und Umwelthygiene, Technische Universität München, München, Germany
Experimental Toxicology and Ecology, BASF AG, Ludwigshafen, Germany
1 To whom correspondence should be addressed at Institute of Toxicology, Helmholtz Zentrum München, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany. Fax: +49-89-3187-3449. E-mail: johannes.filser{at}helmholtz-munchen.de.
Received September 4, 2007; accepted December 22, 2007
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ABSTRACT |
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Propylene (PE) was not carcinogenic in long-term studies in rodents. However, its biotransformation to propylene oxide (PO) raises questions about a carcinogenic risk. PO alkylates macromolecules, is a direct mutagen, and caused tumors in rodents at high concentrations. In order to acquire knowledge on the species-specific PO concentrations in blood resulting from PE exposure, we exposed male Fischer 344/N rats in closed exposure chambers to constant PE concentrations, between 20.1 and 3000 ppm (7 h at least), and four male volunteers to mean constant PE concentrations of 9.82 and 23.4 ppm (180 min) in inhaled air. In the animal experiments, PE and PO were measured in the chamber atmosphere, PE by gas chromatography with flame ionization detection (GC/FID), PO by GC/FID or GC with mass-selective detection (GC/MSD). In the human studies, PE was measured in inhaled and exhaled air by GC/FID. PO was quantified by GC/MSD from exhaled breath collected in gasbags. Blood concentrations of PO were calculated based on the measured PO concentrations in air using the blood-to-air partition coefficients of 60 (rat) and 66 (human). In rats, PO blood concentrations ranged from 53 nmol/l at 20.1 ppm PE to 1750 nmol/l at 3000 ppm PE. In humans, mean blood concentrations of PO were 0.44 and 0.92 nmol/l at mean PE concentrations of 9.82 and 23.4 ppm, respectively. These findings should be taken into consideration when estimating the carcinogenic risk of PE to humans based on carcinogenicity studies in PE- or PO-exposed rats.
Key Words: propylene; propylene oxide; rat; human; metabolism.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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The unsaturated gaseous hydrocarbon propylene (CAS No. 115-07-01; PE) is produced by thermal cracking of heavy hydrocarbons or as a byproduct of petroleum refinery operations. In 2002, a total of about 60 million metric tons was produced in Asia, Europe, and North America (The Dow Chemical Company, 2006
). This major industrial chemical is mainly used in the production of polypropylene and other plastics. Very low atmospheric concentrations of PE in rural areas (0.01–4.8 ppb) and in cities (typically from 0.3 to 32 ppb) may result from incomplete combustion processes (automobile exhaust, cigarette smoke, forest fires), from natural formation in plants, and from PE release during production and use (summarized in e.g., IARC, 1994).
In four long-term carcinogenicity studies with two rat and two mouse strains, inhaled PE was not tumorigenic even at exposure concentrations of up to 5000 ppm (Sprague–Dawley rats and Swiss mice: Ciliberti et al., 1988) and up to 10,000 ppm (F3474/N rats and B6C3F1 mice: Quest et al., 1984; U.S. National Toxicology Program, 1985a). Also, in male F344 rats, exposed for 4 weeks to 0, 200, 2000, or 10,000 ppm, Hprt mutant frequencies in splenic T lymphocytes were not significantly increased over the background (Walker et al., 2004). In Salmonella typhimurium PE did not induce mutations (Victorin and Ståhlberg, 1988). In mouse lymphoma cells the results were inconclusive (McGregor et al., 1991). However, in PE-exposed rodents alkylation of hemoglobin and DNA (Svensson and Osterman-Golkar, 1984 (only hemoglobin); Eide et al., 1995; Pottenger et al., 2007; Svensson et al., 1991) was detected and was related to propylene oxide (CAS No. 75-56-9; PO) because of the formation of hydroxypropyl adducts with hemoglobin and DNA that were also found in animals following PO exposure (Nivard et al., 2003; Osterman-Golkar et al., 1999, 2003; Plná et al., 1999; Ríos-Blanco et al., 1997, 2000, 2002, 2003; Segerbäck et al., 1994, 1998; Svensson et al., 1991). Also in PO-exposed humans, adducts with hemoglobin (Ball et al., 2005; Boogaard et al., 1999; Czène et al., 2002; Jones et al., 2005; Pero et al., 1985) and DNA (Czène et al., 2002) have been measured. Recently, a physiological toxicokinetic (PT) model for inhaled PO was published in which PO adducts with hemoglobin and DNA in rats and with hemoglobin in humans were simulated (Csanády and Filser, 2007).
The actual metabolic formation of PO from PE was demonstrated in vitro by means of a cytochrome P450, cytochrome P450 reductase, and reduced nicotinamide adenine dinucleotide phosphate (NADPH) containing system (Groves et al., 1986) and by using liver microsomes together with an NADPH-generating system (Wistuba et al., 1989). In vivo, PO formation was shown in PE-exposed rats (Maples and Dahl, 1991; Rampf et al., 1996) and in one volunteer, who was exposed to 25 ppm PE (Filser et al., 1997).
After incubation of PO with salmon sperm DNA (Lawley and Jarman, 1972) or calf thymus DNA (Djuric et al., 1986; Randerath et al., 1981; Solomon et al., 1988), different DNA adducts were detected. PO was mutagenic in microorganisms and in Drosophila, mutagenic and clastogenic in mammalian cells in vitro (reviewed in Giri, 1992; IARC, 1994; Kolman et al., 2002), and clastogenic at very high doses in vivo in mice (300 and 450 mg/kg, intraperitoneal administration: Bootman et al., 1979; Farooqi et al., 1993) and in Drosophila (
1000 ppm, inhalation: Vogel and Nivard, 1998). Its genotoxic potency was generally several times lower than that of ethylene oxide, both in vivo (Lynch et al., 1984a [no PO induced effects at all in peripheral lymphocytes of monkeys in contrast to ethylene oxide]; Högstedt et al., 1990; Farooqi et al., 1993; Vogel and Nivard, 1997, 1998) and in vitro (Agurell et al., 1991), or epichlorohydrin (Kolman et al., 1997). PO was positive in neoplastic cell transformation tests inducing less transformation frequencies in the same dose range than ethylene oxide (Kolman and Du
inská, 1995). The genotoxic profile of PO has been reviewed recently (Albertini and Sweeney, 2007).
In long-term studies with rats and mice, PO induced tumors mainly at the application site. Upon intragastric administration to Sprague–Dawley rats tumors developed in the forestomach (Dunkelberg, 1982). Subcutaneous injections to rats (Walpole, 1958) or to NMRI mice (Dunkelberg, 1981) resulted in tumors at the injection site. The predominant findings in three inhalation studies (Fischer 344 rats: Lynch et al., 1984b (only males) and Renne et al., 1986; U.S. National Toxicology Program, 1985b; B6C3F1 mice: Renne et al., 1986; U.S. National Toxicology Program, 1985b) consisted in low incidences of nasal tumors in the high exposure groups ( 300 ppm). In an inhalation study with Wistar rats exposed to 0, 30, 100, and 300 ppm PO, three male animals developed nasal tumors: 2 of 61 examined at the low concentration (30 ppm) and 1 of 63 examined at the highest concentration (300 ppm), while increased degenerative and hyperplastic changes in the nasal mucosa were reported for all treatment groups (both genders). Additionally, in 4 of 63 examined males of the 300 ppm group a carcinoma was detected in the larynx or pharynx, trachea, or lungs (Kuper et al., 1988). In the PO rat studies, increases in tumor incidences were also found in other organs: adrenal pheochromocytomas at 100 and 300 ppm (but not dose-dependent: Lynch et al., 1984b), mammary gland tumors in females at 300 ppm (relevance doubted because within the historical control incidence in this laboratory: Kuper et al., 1988), and endometrial stromal tumors of the uterus at 200 and 400 ppm PO as well as thyroid tumors in females at 400 ppm (both not considered as being treatment related because of low incidence relative to that in historical controls or relatively common: Renne et al., 1986; U.S. National Toxicology Program, 1985b).
The findings that, in long-term animal studies, PE did not induce tumor formation, in contrast to its alkylating metabolite PO, was explained by Golka et al. (1989), who found that metabolism of PE in rats displayed saturation kinetics, an observation since confirmed also for mice (Filser et al., 2000). From comparing theoretically possible PO concentrations in the rat body resulting from PE exposure with calculated PO body concentrations resulting from PO exposure, Golka et al. (1989) concluded that the possible body burden by the metabolically formed PO would not be adequate to lead to an increase in tumors in the PE study with rats. In spite of the relevance of this conclusion for a PE risk assessment, PO was only quantified in blood of rats exposed up to four weeks to various PO concentrations (Lee et al., 2005) but PO measurements in rats or humans exposed to PE are missing with the exception of a preliminary study in one volunteer (Filser et al., 1997). To provide a dosimetric basis for extrapolating the carcinogenic risk of PO from rats to PE-exposed humans and to verify the conclusion from Golka et al. (1989), it is important to acquire knowledge on the PO concentrations in blood resulting from PE exposure of rats and humans. Therefore, the objective of the present work was to investigate the burden of the metabolic intermediate PO in blood of rats and volunteers exposed to PE.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Chemicals
All commercial chemicals were purchased with the highest purity available from Ridel-de Haën, Seelze, Germany, and Merck, Darmstadt, Germany. Soda lime "Drägersorb 800" was from Drägerwerk, Lübeck, Germany, and Heparin-Na (Liquemin, 25,000 I.E./5 ml) from Hoffmann-La Roche, Grenzach-Wyhlen, Germany. Helium 5.0, oxygen (
99.5), nitrogen 5.0, synthetic air 5.0, and hydrogen 5.0 were from Linde (Unterschleißheim, Germany). PE 3.5 ( 99.95%) was obtained from Messer Griesheim (Krefeld, Germany). PO (rac. 99.995%) was a gift from Arco Chemie, Rotterdam, The Netherlands. [2H6]-PO (PO-d6, 99.7 atom% D) was from Isotec (Miamisburg, OH). Handling of all chemicals during sample preparations was carried out under a chemical exhaust hood.
Animals
Male Fischer 344/N rats (mean body weight [bw] ± SD: 264.5 ± 32.5 g, n = 50) were purchased from Charles River WIGA (Sulzfeld, Germany). All experimental procedures with animals were performed in conformity with the "Guide for the Care and Use of Laboratory Animals" (National Research Council, 1996) under the auspices of the authorized representative for animal welfare of Helmholtz Zentrum München. Up to at least 4 days before use, rats were housed (two per cage) in the Institute of Toxicology in a macrolon type III cage placed in an individually ventilated cage top-flow system (Tecniplast, Buguggiate, Italy). This system provided the animals with filtered room air. A constant 12-h light/dark cycle was maintained in the climate-controlled chamber room at 22–24°C and 55–65% relative humidity. Animals had free access to standard pellet chow (Nr. 1324 from Altromin, Lage, Germany) and tap water.
Humans
Four male, healthy, and nonsmoking volunteers (A, B, C, D), all professional toxicologists, participated in the PE exposure study. Ages and bw were A, 55 years, 94 kg; B, 44 years, 58 kg; C, 33 years, 76 kg; D, 39 years, 79 kg. The human study protocol was reviewed and accepted by the Ethics Committee of the Medical Faculty of the Technical University Munich.
Respiratory parameters of each volunteer were established immediately after each exposure by determining average breathing frequency, tidal volume, and the product of both parameters, the average pulmonary ventilation, over a time span of 10 min using a spirometer ("Spirotest Junior," Jaeger, Würzburg, Germany). Mean parameters ± SD are given in Table 1. Breathing frequencies, additionally measured at least five times during each exposure, agreed with the values presented in Table 1.
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Experiments with Rats
The exposures of the Fischer 344/N rats to constant PE concentrations, measurement of atmospheric PE, and determination of rate of PE metabolism in relation to the PE exposure concentration at steady state had been described in detail previously (Filser et al., 2000
). Briefly, two naïve rats were exposed together for at least 7 h at an average temperature of 23°C in closed all-glass exposure chambers of about 6.6 l to constant atmospheric PE concentrations ranging from 20 to 3000 ppm. Mean PE concentrations in the present work (Table 2) differ somewhat from those of 12.3, 62, 104, 253, 373, 496, 1000, and 3000 ppm given in Filser et al. (2000), because only those PE exposures are considered in which metabolically produced PO was determined. In addition, means and SD were calculated from areas under the concentration–time courses, whereas in the former publication the time spans between the measurements had not been taken into account. PE concentrations were maintained constant (coefficient of variation less than 3% of the desired atmospheric concentration) by repeatedly re-injecting defined amounts of PE gas into the chamber atmosphere in order to replenish the PE losses resulting from PE uptake and metabolism by the exposed animals. The small amount of soda lime (about 40 g) used to scrub exhaled CO2 from the chamber air did not lead to a measurable loss of the PE concentration as shown previously (Schmidbauer, 1997). Atmospheric PE concentrations within the exposure chambers were monitored from air samples that were collected periodically and analyzed for PE using a gas chromatograph equipped with a flame ionization detector (GC/FID method A). During these experiments, metabolically produced, exhaled PO was monitored in the atmosphere of the closed chamber using a GC equipped with a mass-selective detector (GC/MSD method A) at PE exposure concentrations of 20.1, 63, 101, 251, and 495 ppm and using GC/FID method A at PE exposure concentrations of 101, 251, 378, 459, 1010, and 3000 ppm.
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Under such conditions of exposure to a constant concentration of a parent compound (here PE), the atmospheric concentration of a volatile biotransformation product (here PO) generally increases with time until a plateau concentration in the chamber atmosphere is reached. At plateau, the formation rate of the biotransformation product equals its elimination rate. If elimination reflects metabolism exclusively, the atmospheric concentration of the biotransformation product is in thermodynamic equilibrium with its concentration in blood (Filser, 1992
; Filser et al., 2007).
In the case of PO, the elimination represented both the metabolic elimination by the exposed animals plus the reaction of atmospheric PO with soda lime. The latter had been quantified in previous work in an exposure chamber containing PO vapor and fresh or used (moistened) soda lime (30 g), because soda lime can hydrolyze the epoxide with different rates depending on its H2O content and on the amount of bound CO2. The shortest elimination half-life (12.9 h) was obtained with soda lime that was pre-exposed over 8 h in a closed all-glass chamber (6.6 l) to the exhaled air and the urine produced by two naïve rats during this time span. The elimination rate constant (0.054 h–1) of this soda lime-catalyzed PO hydrolysis was less than 5% of the elimination rate constant of PO in the air of closed chambers of 6.4 l containing two male Fischer 344/N rats exposed in gas uptake studies to initial PO concentrations between 30 and 3300 ppm (Csanády and Filser, 2007; Schmidbauer, 1997). Considering this small percentage, the influence of soda lime on the PO loss from the chamber atmosphere was not taken into account, and the PO concentrations in air and in blood were considered to be in equilibrium when the plateau was reached. Consequently, the PO concentration in rat blood (nmol/l), which corresponded to the PO air concentration (nmol/l) at plateau, was calculated from the product of the latter with the blood-to-air partition coefficient of PO (60 [mean] ± 1.3 [SD], n = 6, Schmidbauer, 1997). For converting the PO air concentration from ppb to nmol/l, the air concentration in ppb was divided by 25, the molar volume (l) of an ideal gas at 23°C and 740 Torr. Except for the two highest PE concentrations (1000 and 3000 ppm), these calculated steady-state blood concentrations of PO equaled the time-weighted average PO blood concentrations defined by the area under the concentration–time curve of PO in blood from zero to infinity (
[nmol·h/l]) for an exposure time of 8 h divided by this exposure time. For each of the two highest PE concentrations, the time-weighted average PO concentration in blood was calculated from the sum of the rectangular area [nmol·h/l] from t = 0 to t = 8 h under the PO plateau concentration in air (nmol/l) (reached after about 7 h of exposure) and the area that exceeded this plateau under the initial peak (after about 90 min; compare Fig. 2). Dividing this sum through the exposure time of 8 h and multiplying the result by the blood-to-air partition coefficient of PO yielded the corresponding time-weighted average PO concentration in blood.
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The applied procedure for determining blood concentrations of an internally produced exhaled gaseous compound from its plateau concentrations in the atmosphere of a closed chamber can be used when the blood-to-air partition coefficient of the substance is higher than 50 (see Filser et al., 2007
). For safety reasons, all exposures were conducted under a chemical exhaust hood.
Studies with Volunteers
Using the open exposure system (Fig. 1), each volunteer was exposed twice over exactly 3 h to a constant PE concentration: once to about 10 ppm and once to about 23 ppm PE in inhaled air. The time intervals between both exposures lasted from several weeks to several months, for volunteers A, B, and C. Volunteer D was exposed twice within 3 days, first to the low and 2 days later to the higher PE concentration.
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The open exposure system was described in detail in Filser et al. (2000)
. Briefly, a mixture of PE gas from an all-glass storage container with room air (150 l/min) passed through an all-glass mixing chamber and a polytetrafluoroethene (PTFE) tube (Polytetra, Mönchengladbach, Germany) into a chemical exhaust hood. The PE concentrations in this airflow (about 10 or 23 ppm) were maintained constant with a maximum coefficient of variation of ± 10%. The volunteer inhaled PE through an inhalation valve of a two-valve breathing mask, which was connected by a T-pipe to the PTFE tube transporting the PE gas. Two minutes after starting the exposures and subsequently every 10–15 min, air samples of 500 µl were taken via a septum in front of the inhalation valve, using a gastight syringe (500 µl, Hamilton, Bonaduz, Switzerland), and were analyzed for PE by GC/FID method B. Before and at specified time points during the exposures, a fresh gasbag of 2.5 l (Plastigas from Linde, Unterschleißheim, Germany), containing CaSO4 (2 or 4 g), was mounted gastight to the exhalation valve of the mask and a breath of mixed-exhaled air was collected. After closing the gasbag, it was taken away from the valve, and an air sample of 500 µl was collected from the bag via its septum by means of a gastight syringe and analyzed by GC/FID method B for PE concentration in the exhaled air.
The administration of CaSO4 to the gasbags did not show any influence on the half-life of PO in the gasbags, which had been determined in 24-h experiments at room temperature to be about 31 h. In all but one experiment (exposure of volunteer A to 10 ppm PE) PO PO-d6 was used as internal standard for PO. Before starting the 10 ppm PE exposure experiment with volunteer A, a calibration gasbag was prepared which contained exhaled air, CaSO4, and a defined amount of PO yielding a concentration of about 1 ppb. Air samples of this calibration gasbag were analyzed three times for PO by GC/MSD method B. The mean signal value was used to calibrate the PO concentrations measured during the exposure in the exhaled air in the gasbag. In all other experiments, 2 ml of a calibration gas containing 1 ppm PO-d6 in air were injected as internal standard into the closed gasbag via its septum, immediately after taking the air samples for the analysis of exhaled PE. Prior to each experiment, a one-point calibration was prepared in a gasbag containing a defined volume of exhaled air of the respective volunteer, CaSO4, and defined amounts of PO and PO-d6 resulting for each epoxide in a concentration of about 1 ppb. The mean value of the signal ratios of PO to PO-d6, determined by at least two measurements, was divided by the volume of the air in the calibration gasbag. The resulting value was used as calibration factor.
Each gasbag was stored for at least 3 h at room temperature in order to reduce the water content of its air by forming a hydrate with the added CaSO4 before an air sample of 10 ml was withdrawn and analyzed for PO (exposure of volunteer A to 10 ppm PE) or for PO plus PO-d6 (all other exposures) by GC/MSD method B. For the latter exposures, the PO concentration in the sample of exhaled air was obtained from the ratio of the measured PO-to-PO-d6 signals, which was divided by the corresponding volume of exhaled air and then divided by the calibration factor.
Following the PO and PO-d6 measurements, the volume of exhaled air in each gasbag was determined by displacement of the air by water in a graduated cylinder. Knowledge of this air volume enabled to calculate the exact concentrations of added internal standard (PO-d6) and of exhaled PO in the corresponding gasbag.
PO concentrations in blood at steady state were calculated by multiplying the mean concentration of PO in the exhaled air collected in the gasbags by 3/2 (for obtaining the corresponding, approximated alveolar air concentration: see Fiserova-Bergerova, 1983) and by the partition coefficient human blood-to-air (66, Schmidbauer, 1997), considering the molar volume of an ideal gas (25 l) at 23°C and 740 Torr: PO in blood (nmol/l) = PO in exhaled air (ppb) x 3/2 x 66/25 (l/mol).
The rate of PE metabolism (µmol/h) was calculated for each volunteer using his pulmonary ventilation, the difference between the PE mean concentrations in inhaled and exhaled air, and the molar volume of an ideal gas (25 l) at 23°C and 740 Torr: rate of PE metabolism (µmol/h) = pulmonary ventilation (l/min) x 60(min/h) x difference between mean inhaled and exhaled PE concentrations (ppm)/25 (l/mol). This procedure results in some overestimation of the rate of metabolism because steady state of PE is not achieved during the exposure time. However, about 90% of the steady-state concentration of PE is reached in human blood after about 30 min as has been demonstrated and confirmed by a PT model (Filser et al., 2000). After this exposure time, an estimation by the PT model yields a maximum rate of PE uptake into the adipose tissue of about 0.2 µmol/h per ppm atmospheric PE for a human with a bw of 70 kg. This rate represents the maximum overestimation of the rates of metabolism calculated by the presently used method.
In order to calculate as a percentage the fraction of inhaled PE, which was metabolized at steady state, the difference of the mean inhaled and exhaled PE concentrations was multiplied by 100 and then divided by the mean concentration of PE in inhaled air.
Gas Chromatography
GC/FID method A.
PE and PO concentrations in the atmospheres of the animal exposure chambers were determined using a GC (GC-8A Shimadzu, Duisburg, Germany) equipped with an FID. Air samples of 10 ml were taken from the atmosphere of the exposure systems by means of disposable syringes (free of PO; Braun, Melsungen, Germany) equipped with stainless steel needles. Of these samples, 5 ml were injected onto the column via a gas sample loop at room temperature. Separations were done isothermally (above 300 ppm at 130°C, below 300 ppm at 140°C) on a stainless steel column (2.5 m x 1/8'' x 2 mm) packed with Porapak Q (50–80 mesh; Chrompack, Frankfurt, Germany). Nitrogen was used as carrier gas with a pressure of 3.0 kg/cm2. The detector was kept at 200°C and was supplied with hydrogen (0.6 kg/cm2) and synthetic air (0.6 kg/cm2). Areas under the peaks (and peak heights for PO at concentrations below 300 ppb, respectively) were determined using the integrator C-R6A Chromatopac of Shimadzu. Retention times of PE were 1.6 min at 130°C and 1.4 min 140°C. Those of PO were 7.6–8.0 min and 5.9–6.1 min at 130°C and 140°C, respectively. Chromatography runs were stopped after the elution of endogenously formed acetone (about 12 min at 130°C and 10 min at 140°C). For quantification, four calibration curves were constructed, two for PE, one from 0 to 300 ppm and another one from 300 to 3700 ppm, and two for PO, one from 0 to 125 ppb and the second one from 0 to 1000 ppb. For atmospheric PE, 4 concentrations including 0 ppm were chosen as calibration points for both concentration ranges. The calibration curves for atmospheric PO relied on 6 concentrations for the first and five concentrations including 0 ppb for the second curve. Each calibration concentration was the mean of nine replicates except the calibration concentrations for the second PO calibration curve from 0 to 1000 ppb, which were based on triplicate. Linear regression analyses revealed correlation coefficients of at least 0.9999 for PE. Those for PO were 0.9996 and 0.998 for the concentration ranges between 0 and 125 ppb and between 0 and 1000 ppb, respectively. The limit of detection, defined as three times the background noise, was 25 ppb for PO. For PE, it was not investigated since PE concentrations were at least two orders of magnitude above this limit. For quantification in the exposure experiments, one-point calibrations with PE and PO were conducted in the concentration range of the corresponding experiment.
GC/MSD method A.
PO concentrations in the atmospheres of the animal exposure chambers were determined using a GC (HP 5890 series II Plus, Hewlett-Packard, Böblingen, Germany) equipped with an MSD (HP MSD 5972, Hewlett-Packard, Böblingen, Germany) and a Thermal Desorption Cold Trap Injector (CP-4010 with the trap CPSil 5 CB, 30 cm x 0.53 mm, Chrompack, Frankfurt, Germany). Gas samples of 5 ml were collected by means of a 10-ml gastight syringe (Series 1000, Hamilton, Bonaduz, Switzerland) and injected within 1 min into the injector system containing the precooled trap (–100°C; helium flow through the trap: 20 ml/min). After further 4 min, the trap was heated within a few seconds to 200°C and maintained at this temperature for 1 min. Separation of the gas mixture was done on a capillary column (PoraPlot U, 25 m length, 0.32 mm i.d., 10 µm film, with particle trap 2.5 m, Chrompack, Frankfurt, Germany) using helium as carrier gas with a system pressure of 80 kPa. The temperature program of the column oven was started in parallel with the heating of the trap. The initial temperature of 70°C was held for 1 min. Then it increased with a rate of 8°C/min to 140°C, remained constant for 5 min, and decreased again to 70°C. The temperature of the transfer line was kept at 300°C. For ionization, the MSD was used in electron impact ionization mode (70 eV). Detection was done in two modes: the "scan mode" was used at PE exposure concentrations of 180 ppm and above, the "selected ion monitoring" (SIM) mode for the PO analysis at lower PE concentrations. In the SIM mode, the molecular ion (M+) with m/z 58 was monitored. Areas under the peaks were determined manually using the ChemStation software (Hewlett-Packard, Böblingen, Germany). For atmospheric PO, linear calibration curves through the origin were constructed with six concentrations between 0.7 and 95 ppb (SIM mode) and with five concentrations between 180 and 1100 ppb (Scan mode). Linear regression analyses revealed correlation coefficients of at least 0.999. The retention time of PO was about 9.3 min when the PO-containing atmosphere was free of vaporous H2O, and was 9.2 min when PO was collected from an atmosphere (25°C) saturated with water vapor. The limit of detection, defined as three times the background noise, was 70 ppb PO in the Scan mode and 0.5 ppb in the SIM mode. For quantification in the exposure experiments, one-point calibrations were conducted in the PO concentration range of the corresponding experiment. These calibrations were done repeatedly during the exposure experiments to balance the changing sensitivity of the detector resulting from the water vapor in the atmospheres of the exposure chambers that increased with the exposure time.
GC/FID method B.
For the determination of atmospheric PE in the volunteer studies, the same GC system was used as for GC/FID method A. Gas samples of 500 µl were taken by means of a gastight syringe (number 1750, Hamilton, Bonaduz, Switzerland) and injected into the heated injector (200°C). Separations were done isothermally (100°C) on a stainless steel column (3.5 m x 1/8'' x 2 mm) packed with Tenax TA (60–80 mesh; from Chrompack). Gases, gas pressures and detector temperature were as described for GC/FID method A. For quantification, areas under the peaks (retention time 1.85 min) were determined. To verify proportionality between detector signal and PE concentration, calibration samples with concentrations of 1–100 ppm PE were prepared. Calibration samples were analyzed in triplicates. The calibration curve obtained by linear regression analysis through the origin had a correlation coefficient of 0.9997. The limit of detection, defined as a signal-to-noise ratio of 3, was calculated to be 0.1 ppm. For quantification in the exposure experiments, one-point calibrations were conducted in the concentration range of the exposure concentration.
GC/MSD method B.
PO and the internal standard PO-d6 in the atmospheres of the gasbags that contained the air exhaled by a volunteer were determined using a GC (HP 6890A, Agilent Technologies, Waldbronn, Germany) equipped with a MSD (HP MSD 5973 Network, Agilent Technologies) and the above-described Thermal Desorption Cold Trap Injector. Gas samples of 10 ml were collected and injected slowly (about 1.8 ml/min) into the injector system before it was heated within seconds from –100°C up to 200°C. For separation, the same column and the same carrier gas (system pressure 52 kPa) were used as in GC/MSD method A. The temperature program was revised in order to remove water from the column rapidly: after holding the initial temperature of 70°C for 1 min, it increased with a rate of 8°C/min to 140°C, remained constant for 1 min, and increased further with a rate of 20°C/min to a final temperature of 170°C, which was held for 10 min before the oven was cooled again to 70°C. MSD conditions were identical with those of GC/MSD method A. In SIM mode, molecular ions (M+) of both PO (m/z 58) and PO-d6 (m/z 64) were monitored. Retention times of PO-d6 and PO were about 10.1 and 10.2 min and were dependent on the water content of the column. Areas under the peaks were determined manually. To verify the proportionality between detector signal and PO concentration, calibration samples with concentrations between 0.2 ppb and 1 ppm PO were prepared in the atmosphere of gasbags containing exhaled air and CaSO4. Linear regression analysis through the origin revealed a correlation coefficient of 0.99997. The limit of detection, defined as a signal-to-noise ratio of 3, was 0.05 ppb in the SIM mode. This 10-fold higher sensitivity compared to the GC/MSD method A resulted from three sources: the doubled injection volume, reduced impact of water on the column by predrying of the exhaled air with CaSO4, and the use of a newer, more sensitive MSD.
Data Handling
Experiments as well as recording of all data and results were conducted in accordance with Good Laboratory Practice (GLP) requirements.
Statistics
Regression lines, means ± SD (standard deviation) of measured data, and comparison of means by the unpaired two-tailed t-test were calculated using Prism 4 for Macintosh (GraphPad Software, San Diego, CA). Time-weighted means and corresponding SD were calculated as described in Lee et al. (2005). Calculations were carried out using Microsoft Excel 2004 for Mac (Microsoft Deutschland, Unterschleißheim).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Experiments with Rats
The PO concentrations measured in the atmospheres of closed exposure chambers, each containing two rats exposed to constant PE concentrations between 20 and 3000 ppm, are depicted in Figure 2. Up to 500 ppm PE, the PO concentrations increased with the exposure time finally reaching characteristic plateaus. At the two highest PE exposure concentrations of 1000 and 3000 ppm, concentrations of exhaled PO in the chamber atmosphere quickly reached maximum values of 830 and 900 ppb, respectively, followed by decreases to almost identical plateau concentrations of 600 and 620 ppb, respectively, that were reached after about 7 h of exposure. The PE exposure concentrations and the PO plateau concentrations measured in the atmospheres of the exposure systems are given in Table 2. The Table shows also the corresponding calculated PO concentrations in blood at steady state or the time-weighted average PO concentrations in blood. The latter were used to construct Figure 3 in which these and the rates of PE metabolism are plotted versus the corresponding PE concentrations in air. Obviously, the saturation kinetics of the metabolism of PE, already demonstrated and discussed using the same and additional PE data (Filser et al., 2000
), is reflected by the PO concentrations in blood. In other words, following a single exposure of rats to PE, the PO blood concentrations at steady state are not expected to increase substantially above the value reached at 3000 ppm PE.
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Studies with Volunteers
Concentrations of PE in inhaled air as well as of PE and PO in exhaled breath, collected in the gasbags, are plotted in dependence of the exposure time in Figure 4 for volunteers A and B, and in Figure 5 for volunteers C and D. The variation observed in the inhaled air resulted from the decrease of the PE concentration in the storage container (see Fig. 1) and from the periodical re-injection of PE into its atmosphere. Mean concentrations of PE were somewhat lower in the exhaled than in the inhaled air; this difference resulted predominantly from the metabolism of PE. It was statistically significant (two-tailed t-test; p
0.05) for all but two experiments (volunteers A and D; 10 ppm).
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Prior to starting the PE exposures, PO was not detected in the exhaled air. However, after start of exposure to PE, PO was detected in all samples of collected exhaled air. Except for the 10 ppm PE exposure of volunteer B (Fig. 4), the concentrations of PO in exhaled breath were constant and did not increase with exposure time. The PO concentrations measured during the exposure of volunteer B to 10 ppm PE suggest about a twofold increase in exhaled PO over the 3-h exposure. However, when averaging these PO concentrations over the exposure period, the standard deviation was similar to the standard deviations of the other exposures, where no indication of a time-dependent increase in exhaled PO was found. Therefore, exhaled PO was expressed as mean also for the exposure to 10 ppm PE of volunteer B.
For every single PE exposure, Table 3 shows the measured concentrations of PE in the inhaled air and of both exhaled PE and exhaled PO in the gasbags. In addition, the calculated fraction of inhaled PE that was metabolized, the rates of metabolism of PE, and the corresponding concentrations of PO in blood at steady state are given. Table 4 presents, for an average male volunteer (mean ± SD; n = 4) of 76.8 ± 14.8 kg, the overall means of the PE exposure concentrations, the fractions metabolized, the rates of metabolism, and the PO concentrations in blood at steady state representing also the time-weighted average PO concentration in blood. The small value for metabolized fraction at both concentrations signifies that the majority of inhaled PE was exhaled unchanged (mean 91.5 ± 1.8%). Exhalation of unchanged PE had previously been identified as a major route of elimination based on results from one human subject (93%: Filser et al., 2000).
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In Figure 6A, the rates of PE metabolism at steady state determined in the four individuals and mean rates ± SD are plotted versus the atmospheric exposure concentration of PE. A straight line through the origin was fitted to the data (slope 1.22 ± 0.12 µmol PE per h per ppm PE). At an exposure concentration of 25 ppm PE, the mean rate of metabolism is 30 µmol/h. Considering the standard deviations, this extrapolated value agrees with the rate of 20.5 µmol/h previously calculated for a 70 kg man from data obtained only in one volunteer (Filser et al., 2000
).
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In Figure 6B, the time-weighted average PO concentrations in blood of the 4 individuals and mean PO concentrations ± SD are plotted versus the exposure concentration of PE. Again, a straight line through the origin (slope 0.040 ± 0.0029 nmol PO/l blood/ppm PE) was constructed because no PO (detection limit 0.05 ppb equivalent to 0.13 nmol/l blood) could be detected in the initial breath samples, taken before starting the exposures. As Figures 6A and 6B demonstrate, the dependence of the rate of PE metabolism on the PE exposure concentration is paralleled by the PO concentrations in blood. This behavior reflects the observation made in rats (Fig. 3).
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Rats
PO concentrations in the chamber atmosphere peaked after about 90 min at the two highest PE concentrations with maximum rates of PE metabolism (1000 and 3000 ppm; Fig. 2). The subsequent decreases point at a rapid inactivation of a PO-producing CYP species, a conclusion made also by Maples and Dahl (1991)
when measuring PO in blood of PE-exposed rats. Such an effect was shown by Kunze et al. (1983), who incubated liver microsomes from phenobarbital-pretreated rats with PE and NADPH. PE inactivated CYP by N-alkylation of the pyrrole ring D of the prosthetic heme group. It was likely that CYP2E1 was alkylated because PE metabolism in rats could be inhibited by 85–96% following pretreatment of the animals with the CYP2E1 inhibitor sodium diethyldithiocarbamate (Filser et al., 2000). At lower PE concentrations, no peak of PO was identified in the chamber atmosphere, probably because PO formation was too slow to inactivate the metabolizing CYP species, perhaps due to resynthesis of this isozyme.
PO distributes almost uniformly in the organism as follows from its tissue-to-blood partition coefficients that are very similar (between 0.83, liver-to blood and 1.06, fat-to-blood; Csanády and Filser, 2007), and its biological half-life (4.7 min in a male Fischer 344/N rat of 250 g; Schmidbauer, 1997), which is 21 times longer than the blood circulation time (0.22 min). The latter is the product of the blood volume (18.5 ml, the sum of the volumes of arterial, venous, and lung blood in a 250 g rat; Csanády and Filser, 2007) with the inverse of the cardiac output (83 ml/min; Csanády and Filser, 2007).
The relation of PO blood concentrations resulting from 1-day exposures (7–8 h) to the PE exposure concentrations (Fig. 3) resembles that obtained by Pottenger et al. (2007), who plotted hydroxypropyl adducts with the N-terminal valine of hemoglobin (HPVal) determined in male F344 rats following whole body inhalation exposures to PE over 3 days (6 h/day) and 4 weeks (6 h/day, 5 days/week) versus the PE exposure concentrations of 0, 200, 2000, and 10,000 ppm PE. At 2000 ppm PE, almost maximum PO blood concentrations (this work, Fig. 3) and HPVal levels (Pottenger et al., 2007) were reached. Furthermore, PO concentrations in blood and HPVal levels were about 3 times higher at 2000 ppm than at 200 ppm PE. Considering these similarities, it appears that the results obtained in 1-day PE-exposed rats hold also for longer periods of repeated PE exposures.
Lee et al. (2005) determined at the end of 6-h exposures PO in blood of male Fischer 344/N rats that were exposed up to 4 weeks (6 h/day, 5 days/week) to constant concentrations of gaseous PO of 5, 25, 50, 300, and 500 ppm. After 3 days and 4 weeks, the PO concentrations in blood resulting from a given PO exposure concentration were very similar. After 4 weeks, the slope, representing the PO concentration in blood as a linear function of the PO exposure concentration up to 300 ppm, was 0.046 µmol PO/l blood/ppm atmospheric PO. Using this relation between the PO blood concentration and the atmospheric PO exposure concentration as well as the relation between the time-weighted average PO blood concentration and the atmospheric PE exposure concentration (represented by the curve that shows the rate of PE metabolism in Fig. 3), PO and PE exposures can be compared based on the PO concentration in blood. By this procedure, exposure concentrations of 20, 200, 2000, and 3000 ppm PE are shown to be equivalent with exposure concentrations of 1.1, 13, 36, and 38 ppm PO concerning the resulting PO blood concentration. Interestingly, similar values for PE exposure concentrations of 200, 2000, and 10,000 ppm were published by Pottenger et al. (2007), although they were based on far more complex experimental methods than the values presented here, which are founded on direct determinations of PO in blood and in chamber atmosphere.
Humans and Species Comparison
In one male volunteer, exposed for up to 190 min to 5.1 and 24.6 ppm PE, and in rats, exposed in closed systems to various initial PE concentrations from 10 to 10,000 ppm, inhalation toxicokinetics, that is, PE absorption, distribution, and rates of metabolism, was already studied by means of a PT model (Filser et al., 2000). It has been shown in both species that inhaled PE is barely absorbed and greater than 90% is exhaled unchanged. The rates of absorption of atmospheric PE into blood have been discussed to be limited by the cardiac output. It was demonstrated that PE concentrations in blood at steady state are very similar in rats and humans for equal conditions of exposure to PE of up to 25 ppm. At low PE concentrations, the rates of metabolism have been discussed to be limited by transport through the metabolizing organs, for PE concentrations below 25 ppm leading to a sevenfold higher rate of PE metabolism per kg bw in rats than in humans (Fig. 8 in Filser et al., 2000). The present data on PE exhalation and metabolism obtained in four volunteers are in agreement with the previous results and conclusions.
To the best of our knowledge, this is the first time that metabolically formed PO was determined in PE-exposed humans in extension of an earlier report on a volunteer who had exhaled PO (0.2 ppb) during exposure to 25 ppm PE (Filser et al., 1997). So far, no measured adducts with hemoglobin or DNA have been reported in PE-exposed volunteers or workers that could be used for comparison with our results.
A considerable species difference was found between humans and rats concerning the concentration of the metabolite PO calculated in blood at similar PE exposure concentrations of 23.4 ppm PE (humans) and 20.1 ppm (rats). The PO concentration in human blood was 58-fold lower than in rat blood. One could argue that the average calculated blood concentrations in humans could still increase within the time frame from 3 to 8 h. However, except from the 10 ppm exposure of volunteer B, the PO concentrations in exhaled air gave no indication for a further increase in PO. But even a twofold increase in the PO blood concentrations in humans from 3 to 8 h would not affect substantially the large species difference in the PO blood concentrations. It remains to be elucidated whether this difference can be explained by a PT model that will combine both the model developed for PE (Filser et al., 2000) and that for PO (Csanády and Filser, 2007). Distinct species differences were found in the activities of PO metabolizing enzymes of liver and lung cell fractions (Faller et al., 2001). Human microsomal epoxide hydrolase activities toward PO (expressed as Vmax/Km) were two to four times (liver) and six to eight times (lung) higher than those of rats. Glutathione S-transferase activities toward the same substrate were in human lung cytosol two to four times higher than in rat lung cytosol and were identical in liver cytosol of both species. A similar remarkable difference between rats and humans has been concluded for the blood burden of the 1,3-butadiene-metabolite 1,2-epoxy-3-butene, from comparing in both species binding indices of N-(2-hydroxy-3-butenyl)-valine, the adduct of 1,2-epoxy-3-butene with the N-terminal valine of hemoglobin (Osterman-Golkar and Bond, 1996). The authors estimated for this 1,2-epoxy-3-butene surrogate an about 100-fold less binding index for humans than for rats, considering an exposure to a median BD concentration of 1 ppm. There could also exist a species difference in an intrahepatic first pass metabolism for epoxides formed metabolically from olefins. Such an effect has been postulated for several olefins that are biotransformed to epoxides (1,3-butadiene: Filser and Bolt, 1984; Johanson and Filser, 1993; styrene: Csanády et al., 1994, 2003; Filser et al., 2002). However, further work is required in order to provide a mechanistic or toxicokinetic explanation for the very low PO concentrations found in PE-exposed humans.
Considering PO to be the ultimate toxicant, our results confirm our earlier conclusion (Golka et al., 1989) making clear why PE was in rats neither tumorigenic after chronic exposure (Ciliberti et al., 1988; Quest et al., 1984; U.S. National Toxicology Program, 1985a) nor mutagenic following inhalation exposure for four weeks (Walker et al., 2004) to PE concentrations up to 10,000 ppm. As shown in Figure 3 and discussed above, the maximum time-weighted average PO concentration in blood of PE-exposed Fischer 344/N rats is reached at about 3000 ppm PE and does not exceed 2000 nmol/l. This PO blood concentration is about half an order of magnitude lower than that of about 9000 nmol/l resulting from exposure to 200 ppm PO (Lee et al., 2005), an exposure concentration that was too low for resulting in PO treatment–related tumors in a long-term study (Renne et al., 1986; U.S. National Toxicology Program, 1985b).
In the four male volunteers exposed to a mean PE concentration of 23.4 ppm, the calculated mean PO blood concentration of 0.92 nmol/l is four orders of magnitude lower than that in rats exposed to 200 ppm PO. When taking into consideration the present data for risk estimation, the extrapolated health risk of PE to humans from the metabolically produced PO can be expected to be extremely low.
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Cefic Lower Olefins Sector Group.
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