ToxSci Advance Access originally published online on March 6, 2007
Toxicological Sciences 2007 97(2):336-347; doi:10.1093/toxsci/kfm038
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Evaluation of Effects from Repeated Inhalation Exposure of F344 Rats to High Concentrations of Propylene
* Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674
Haskell Laboratory for Health and Environmental Sciences, E.I. du Pont de Nemours and Company, Newark, Delaware 19714
Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Lovelace Respiratory Research Institute, Albuquerque, New Mexico 87108
¶ Food Safety and Toxicology, Michigan State University, East Lansing, Michigan 48824
|| Lyondell Chemical Company, Houston, Texas 77010
1 To whom correspondence should be addressed at Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Washington Street, Midland, MI 48674. Fax: (989) 638-9863. E-mail: lpottenger{at}dow.com.
Received December 3, 2006; accepted February 16, 2007
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Chronic exposure to propylene does not result in any increased incidence of tumors, yet does increase N7-hydroxypropylguanine (N7-HPGua) adducts in tissue DNA. To investigate any potential for genotoxicity (mutagenicity or clastogenicity), male F344 rats were exposed via inhalation to up to 10,000 ppm propylene for 1, 3, or 20 days (6 h/day, 5 days/week). The endpoints examined included gene (Hprt, splenocytes) and chromosomal (bone marrow micronucleus [MN]) mutations, hemoglobin (hydroxypropylvaline, HPVal) adducts in systemic blood, and DNA adducts (N7-HPGua) in several tissues. Similarly exposed female and male F344 rats, implanted with bromodeoxyuridine (BrdU) minipumps, were evaluated for nasal effects (irritation via histopathology and cell proliferation via BrdU). Internal dose measures provided clear evidence for propylene exposure, with HPVal increased for all exposures; N7-HPGua was increased in all tissues from rats exposed for more than 1 day (except lymphocytes). Saturation of propylene conversion to propylene oxide was apparent from the adduct dose-response curves. There were no biologically significant genotoxic effects demonstrated at any exposure level, with no increase in Hprt mutant frequency or in bone marrow MN formation. In addition, no histopathological changes were noted in rodent nasal tissues nor any induction of cell proliferation in nasal tissues. These results demonstrate that repeated exposure of rats to high concentrations of propylene (
10,000 ppm) does not produce evidence of local nasal cavity toxicity or evidence of systemic genotoxicity to hematopoietic tissue, despite the formation of N7-HPGua adducts. In addition, these data indicate that formation of N7-HPGua does not correlate with any measure of genotoxic effect, neither mutagenic nor clastogenic. Key Words: propylene; rat; in vivo mutation; biomarkers; DNA adducts.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Propylene (CASRN 115-07-01) is an important industrial chemical used as an intermediate and raw material in the production of a large range of chemicals and products, such as polypropylene, acrylonitrile, oxo alcohols, propylene oxide (PO), cumene, isopropyl alcohol, and other chemicals (Lundberg, 1996
; Schoenberg et al., 1985). Also, it is used widely as an alkylation feedstock for octane improvement. Propylene occurs in refined petroleum products and is produced in the petroleum cracking process. In 2001, over 37,000 kilotonnes were produced (OECD, 2003). Environmental propylene results from natural and anthropogenic sources (IARC, 1994), as it is both produced by vegetation and released as a product from incomplete combustion (e.g., automotive exhaust, cigarette smoke). Propylene was reviewed in 2003 by the Organization for Economic and Co-operative Development (OECD) Screening Information Data Set Initial Assessment Meeting 18 as part of the International Chemistry Council Association/High Production Volume Chemicals effort, where a conclusion of "low priority for further work" was agreed based on the available toxicity (environment and mammalian) and exposure database (OECD, 2003). More recently, the American Council of Governmental and Industrial Hygienists finalized a Threshold Limit Value 8-h Time-Weighted Average of 500 ppm propylene (ACGIH, 2006) based on nasal irritation effects at high exposure levels.
Propylene has been tested extensively for toxicity (OECD, 2003). It has been shown to elicit narcosis at high exposure levels (46,000 ppm; Drummond, 1993) and is estimated to cause asphyxiation due to displacement of oxygen at 236,000 ppm or above (OECD, 2003), both of which are considerably higher than the lower explosivity level for propylene of 20,000 ppm. All standard and nonstandard tests have demonstrated no evidence of in vivo toxic effects other than mild rhinitis (nasal inflammation) and associated epithelial alterations suggesting chronic, low-grade nasal irritation in rodents following chronic (2 years, lifetime) exposure to 5000 or 10,000 ppm, with no obvious dose-response relationship. The data set includes four chronic bioassays, one pair in Sprague-Dawley rats and Swiss mice (Ciliberti et al., 1988) and the other pair in F344 rats and B6C3F1 mice (NTP, 1985a), none of which resulted in any increased incidence in tumors for exposures up to 5000 and 10,000 ppm, respectively. In addition, a standard in vivo test conducted in rats for developmental (prenatal) toxicity (OECD 414) did not indicate any toxic effects from exposures up to 10,000 ppm propylene (Gamer, unpublished data; OECD, 2003). Work conducted in vitro, such as bacterial and/or mammalian cell mutagenicity tests, have mostly given negative results, with no increases in mutagenic response following propylene exposure (Hughes et al., 1984; McGregor et al., 1991; Victorin and Stahlberg, 1979). There are some in vitro results where one bacterial strain (out of five tested) showed slight increases in mutagenicity following high-level exposures to propylene with metabolic activation compared with control cultures; however, the increases never reached twofold over background (Inveresk, unpublished data; OECD, 2003), thus were considered of little biological relevance. National Toxicology Program (NTP) also conducted Ames bacterial mutagenicity tests on propylene, and the results are listed as a positive in a table on the NTP internet Web site (entitled "Positive in in vitro [Salmonella] tests and negative in both rodent species in the 2-year Carcinogenesis Study"). However, additional detail provided by NTP (NTP, unpublished results) indicated that this was again for a single bacterial strain (out of four tested), with activation. NTP also tested propylene with Drosophila melanogaster (sex-linked recessive lethal) and in the mouse lymphoma forward mutation assay, both of which were considered negative for induction of mutagenic effects (NTP, unpublished results).
The pharmacokinetics of propylene have been studied in rodents (Filser et al., 2000; Golka et al., 1989) and in humans (Filser, unpublished data). Uptake/absorption of inhaled propylene by rodents and humans is limited, in part due to its low blood-air partition coefficient. Rats metabolized about 16% of inhaled propylene, while data from a human volunteer pharmacokinetics study demonstrated that 92% of inhaled propylene (20 ppm) was exhaled unchanged (n = 5 subjects; Filser, unpublished data). The latter finding indicates that only 8% of the inhaled propylene is available for metabolism in humans via cytochromes P450, mainly P4502E1, to PO, an epoxide intermediate. Formation of PO from propylene is a saturable reaction, with maximum rates reached at exposure levels of about 1000 ppm propylene in mice and rats (Filser et al., 2000). The PO formed can either react with cellular macromolecules, such as hemoglobin (Hb) or DNA, or be further biotransformed, either via epoxide hydrolase (EH) to propylene glycol or via glutathione-S-transferase (GST)–mediated conjugation with glutathione (GSH) (Faller et al., 2000, 2001; Lee et al., 2005) to form a GSH conjugate. Presence of PO-induced Hb and DNA adducts has been reported in rodents exposed via inhalation to propylene (Eide et al., 1995; Svensson and Osterman-Golkar, 1984; Svensson et al., 1991).
Exposure to high levels of PO is known to induce site-of-contact tumors in rodents by inhalation (Kuper et al., 1988; Lynch et al., 1984a; NTP, 1985b), oral gavage (Dunkelberg, 1982), or sc injection (Dunkelberg, 1981). The PO inhalation bioassays had no-observable-effect-levels (NOELs) for tumors of 100 and 200 ppm for Lynch et al. (1984a) and NTP (1985b), respectively, while results for chronic exposure to 300 and 400 ppm demonstrated an increase in nasal tumors in rats and mice. The extensive genotoxicity data available for PO have recently been reviewed (Albertini and Sweeney, 2007). PO demonstrates mutagenic and clastogenic effects in vitro (Agurell et al., 1991; Bootman et al., 1979; McGregor et al., 1991; Zamora et al., 1983), but in vivo exposures to PO by relevant routes (oral and inhalation) have not resulted in measurable genotoxic effects, not micronucleus (MN) induction (Bootman et al., 1979), sister chromatid exchanges (SCE), chromosomal aberrations (CA) following chronic exposures in monkeys (Lynch et al., 1984b), nor dominant lethal effects in mice (repeated dose and oral gavage; Bootman et al., 1979) or rats (repeated dose and inhalation; Hardin et al., 1983). In contrast, repeated ip injection of very high doses of PO into mice resulted in quantifiable increases of MN, SCE, and CA (Farooqi et al., 1993).
Given the saturable pharmacokinetics of propylene metabolism, it is unlikely that metabolically formed PO will reach in vivo levels capable of causing toxicity. The present study was conducted to determine if repeated exposure to high levels of propylene resulted in quantifiable increases in mutagenicity endpoints measured systemically, in the presence of clear evidence of internal exposure (PO-induced Hb and DNA adducts). In addition, potential elicitation of nasal effects was investigated based on the recognized target tissue for PO, which is known to induce irritation and cell proliferation in nasal respiratory mucosa (Eldridge et al., 1995; Ríos-Blanco et al., 2003), and to induce tumors in nasal mucosa following chronic exposure (Lynch et al., 1984a; NTP, 1985b).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several laboratories contributed to this study, providing the following aspects of conduct and data analysis and interpretation:
- DuPont Haskell Laboratory for Health and Environmental Sciences (Newark, DE): Good Laboratory Practice (GLP) inhalation exposures, animal in-life phase including bromodeoxyuridine (BrdU) pump implantation, MN, animal and tissue collection and preparation, and cell proliferation analyses.
- The Lovelace Respiratory Research Institute (LRRI) (Albuquerque, NM): In vivo splenocyte hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene mutation assay, including expression time animal in-life phase and assay conduct, analysis, and interpretation.
- Michigan State University (MSU) (East Lansing, MI): Review and interpretation of nasal histopathology.
- University of North Carolina (UNC) (Chapel Hill, NC): Hb and DNA purification and analyses and interpretation of hydroxypropylvaline (HPVal) and N7-hydroxypropylguanine (N7-HPGua) adduct data.
- The Lovelace Respiratory Research Institute (LRRI) (Albuquerque, NM): In vivo splenocyte hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene mutation assay, including expression time animal in-life phase and assay conduct, analysis, and interpretation.
Study Overview
Groups of 16–24 male and 8 female F344 rats (total of 288 rats) were exposed via whole-body inhalation to 0, 200, 2000, or 10,000 ppm propylene for 1, 3, or 20 days (4 weeks, 5 days/week, 6 h/day). Experiments were designed such that all animals were about 9 weeks of age at the end of exposures. Prior to the final three exposures, Alzet 3-day minipumps were implanted in rats destined for cell proliferation analysis. Following the final exposure period, selected tissues were collected and either fixed in formalin or frozen and used to investigate the following endpoints: nasal respiratory epithelium (NRE) for histopathological effects and for induction of cell proliferation; MN induction in bone marrow erythrocytes; N-terminal HPVal Hb adducts in systemic blood; and N7-HPGua DNA adducts in several tissues. In addition, Hprt gene mutation induction in splenocytes was evaluated in rats after exposure. Several laboratories were involved in this collaborative effort, so frozen tissues (Hb and DNA adducts), slides (histopathology), or postexposure rats (Hprt mutant frequency analysis) were shipped to participating investigators. The work conducted at the inhalation exposure facility was done in accordance with U.S. Environmental Protection Agency's GLP Standards (EPA, 1989) in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International: DuPont Haskell Laboratory for Health and Environmental Sciences, Newark, DE. Other participating laboratories underwent review of in-life phase, data, and final reports by a Quality Assurance expert. Data from some analyses have been published elsewhere (Walker et al., 2004) and so will be described only briefly here for continuity.
Chemicals and Reagents
Propylene (CASRN 115-07-01) was supplied by Matheson Tri Gas (Montgomery, PA) at 99.5% purity, which was confirmed to be 99.75% pure by gas chromatography in a GLP characterization conducted by Exygen Research (State College, PA). Reanalysis after the final exposure demonstrated that the test material was stable over the study's duration. Cyclophosphamide monohydrate (CP, Sigma-Aldrich, St Louis, MO) was used as the positive control for MN and Hprt determinations and was dissolved in Milli-Q water. Conditioned filtered air served as the vehicle and vehicle control (0 ppm). BrdU was obtained from Sigma-Aldrich. All other chemicals were from standard commercial suppliers as indicated. Proteinase K and 70% phenol/chloroform (water saturated) were from Applied Biosystems (Foster City, CA), chloroform was from J.T. Baker (Phillipsburg, NJ), Microcon-3 filters were from Millipore (Billerica, MA), HPLC-grade water and methanol were from Fisher Scientific (Fair Lawn, NJ), and HCl was from Mallinckrodt (Paris, KY). The analytical high-performance liquid chromatography (HPLC) column was an Aquasil C-18 (150 x 2 mm, 5 µ) from Keystone Scientific (ThermoElectron, Bellefonte, PA). N7-HPGua and its stable isotope internal standard, [13C4]-N7-HPGua, were synthesized in Dr J. A. Swenberg's laboratory (UNC), as described elsewhere (Rios-Blanco et al., 1997).
Animals and Husbandry
Male and female F344 rats (approximately 4 weeks old upon arrival) were obtained from Charles Rivers Laboratories, Inc. (Raleigh, NC) and housed in a facility accredited by AAALAC International (DuPont Haskell Laboratory). All animal use activities were reviewed and approved by the Institutional Animal Care and Use Committee. Animals were individually identified via tail tattoo and individually housed in temperature-controlled (20 ± 2°C) and humidity-controlled (50 ± 10%) rooms; PMI Nutrition International, LLC Certified Rodent LabDiet 5002, and tap water were available ad libitum, except during inhalation exposures. After a minimum of 1-week acclimation, healthy animals were assigned to groups of eight for various endpoints, using a computer-generated randomization program based on body weight such that the mean body weights for each group were not statistically different across groups within a sex.
Inhalation Exposure Chamber Conditions
Whole-body exposures were conducted in Rochester-type 1.4-m3 stainless steel/glass chambers, with animals individually housed in wire mesh cages during the exposure period. Chamber atmospheres were generated by dilution of propylene with houseline air. Propylene was metered from the cylinder with Brooks model MF50x Mass Flow Controllers via stainless steel or Teflon lines to the inhalation chamber air supply. The controllers were regulated and monitored by the Camile Inhalation Toxicology Automated Data System (CITADS). Chamber concentrations were controlled by varying the feed rate of propylene to the exposure chamber. During exposures, chamber concentration was monitored repeatedly (every 30 min during the exposure period) by injecting a sample of chamber air onto a Hewlett Packard (Palo Alto, CA) model 6890 Plus Series Gas Chromatograph equipped with a flame ionization detector. An Agilent Technologies (Palo Alto, CA) HP-5 column was used for isothermal (75°C) separation, and the chamber concentration of propylene was determined from a standard curve derived from gas standards prepared fresh daily. Chamber airflow, temperature, and relative humidity were monitored continually with the CITADS; mean values for all exposures ranged from 270 to 300 l/min, 21°C to 23°C, and 34 to 50%, respectively. Chamber oxygen concentration was monitored and maintained at 19% or greater.
Inhalation Exposure Details
Groups of eight male rats were exposed to 0, 200, 2000, or 10,000 ppm propylene vapors for 1, 3, or 20 days (5 days/week, 6 h/day), on a staggered schedule. A parallel set of female rats (eight per group) were exposed to identical concentrations for 20 days only. Animals were observed daily for clinical signs. An auditory alerting response was observed prior to exposure, three times during each exposure period, and after the exposure ended. Body weights were collected prior to the initial exposure and weekly thereafter until terminal sacrifice, when final body weights were determined.
Positive Control Treatment
A 2-mg/ml solution of CP (in Milli-Q water) was prepared immediately prior to dosing. CP was administered to eight male rats (MN-positive control) by a single oral intubation using a dose volume of 10 ml/kg, yielding a dosage of 20 mg/kg. On inhalation exposure day 19, a separate group of eight untreated, male rats (Hprt-positive control) received a single oral intubation of the positive control agent CP (20 mg/kg in water).
BrdU Treatment
Animals designated for cell proliferation induction studies were each fitted with an Alza 2 ML1 osmotic minipump (Alza Corp., Palo Alto, CA) containing 20 mg/ml BrdU in 0.5 N sodium bicarbonate. Minipumps were implanted sc in the back of designated rats while under light pentobarbital anesthesia immediately following the end of exposure on the day prior to the last three scheduled exposures, approximately 18 h before the third from last exposure began. Thus, the BrdU was present during the final three 6-h inhalation exposures for these groups.
Terminal Sacrifice and Tissue Collection
Animals designated for Hb and DNA adduct analyses were euthanized via CO2 asphyxiation within 2 h after exposure, and a maximum of blood was collected from the vena cava and placed into Becton-Dickinson 8-ml Cell Preparation Tubes containing sodium citrate anticoagulant. Blood samples were centrifuged to separate lymphocytes from red blood cells. Cells were washed twice in isotonic saline. Lymphocytes and red blood cells from individual animals were frozen separately in liquid nitrogen and stored at – 80°C until they were shipped on dry ice. Spleen, lung, and liver were removed, weighed, and frozen as individual animal samples in liquid nitrogen. Respiratory epithelium was collected from the nasal tissue by splitting the skull sagitally along the major suture line and exposing the tissues lining the nasal cavity. NRE tissue was collected from the naso- and maxillo-turbinates, lateral wall, and cartilaginous portion of the nasal septum. Tissues from each individual animal were collected into labeled, plastic, sealable tubes and snap-frozen immediately in liquid nitrogen. All these tissues were stored at – 80°C until they were shipped on dry ice to UNC.
Animals designated for cell proliferation studies were euthanized by sodium pentobarbital overdose and exsanguination. Nasal cavities, liver, and duodenum were removed and fixed in 10% neutral-buffered formalin for 18–25 h and transferred to 70% ethanol. Nasal cavities were then decalcified in formic acid/sodium citrate solution, trimmed, embedded in paraffin, and cut at a 5-µm thickness. After decalcification, the nasal passages were transversely sectioned at the following four specific anatomic locations according to Young (1981): levels 1, 2, 3, and 4 (see Fig. 1). A total of four blocks were obtained and further processed to paraffin, namely those with the anterior face at sections 1, 2, 3, and 4. These blocks were oriented in the paraffin block with the anterior face down. Liver slices from the median lobe were also embedded in paraffin, and a piece of duodenum was included in each tissue block to confirm delivery of BrdU to all tissues after staining. Tissue sections (5 µm thick) were cut from the anterior face of each nasal tissue block and from the liver, were placed on glass slides, and histochemically stained with hematoxylin and eosin (H&E). H&E slides were shipped to MSU for histopathological evaluation. An additional slide was prepared from each block for BrdU immunohistochemistry. Microscopic analysis of BrdU incorporation was expressed as BrdU-labeled cells per unit length basement membrane (Unit Length Labeling Index, [ULLI]) for left or right maxilloturbinate.
|
The evaluation of propylene's potential for induction of MN formation was conducted in accordance with OECD Guideline 474, under GLP conditions. Inhalation-exposed animals (male rats only) designated for MN analysis were euthanized by CO2 asphyxiation within 2 h after exposure, and the marrow from one femur of each rat was aspirated into a syringe containing fetal bovine serum (FBS) and transferred to a centrifuge tube containing FBS. Positive control (CP treated) animals were treated similarly. Erythrocytes were collected by centrifugation, after which most of the supernatant was removed. The pellet was suspended in FBS, and a small drop was placed on a precleaned microscope slide. Slide smears were made using a Mini Prep blood-smearing instrument (Geometric Data, Wayne, PA); at least three slides per animal were prepared. The slides were air dried and fixed using absolute methanol. Bone marrow smears were stained with acridine orange, a DNA/RNA-specific fluorochrome, labeled using a computerized code, and analyzed coded. Two thousand polychromatic erythrocytes (PCEs) per rat were scored for the presence of MNs (round, bright yellow-green fluorescing bodies). Color was used to distinguish PCEs (red-orange) from normochromatic erythrocytes (NCEs) (gray-green). Inclusions in PCEs which were improperly shaped or stained or were not in the focal plane of the cell were considered artifacts and not scored as MNs. Cells containing more than one MN were scored as a single micronucleated polychromatic erythrocyte. The proportion of PCEs among 1000 total erythrocytes (expressed as the PCE/NCE ratio) was determined for each animal. Criteria for a valid test, and for negative and positive results, were established in the study protocol and applied to the results.
Sample Preparation and Analysis for Hb Adducts
Globin isolation.
The frozen, washed erythrocyte samples were thawed and diluted with an equal volume of deionized, distilled water, and globin was isolated according to the method of Mower et al. (1986). Briefly, the diluted red blood cells were lysed with 50mM HCl in 2-propanol and centrifuged at 1500 x g for 30 min to remove cell membranes. Globin was precipitated from the supernatant with ethyl acetate and centrifuged at 1500 x g for 5 min. The precipitate was washed with ethyl acetate until no color remained in the supernatant. The globin was washed once more with pentane and centrifuged at 1500 x g for 5 min; the supernatant was discarded and the pellet dried under a gentle stream of nitrogen and was then dried further under vacuum. The derivatization was performed according to the modified Edman degradation of Törnqvist et al. (1986) for the specific cleavage of N-terminal alkylated valines of the four chains of Hb using pentafluorophenyl isothiocyanate (PFPITC). Briefly, 50–100 mg globin was dissolved in 1.5 ml formamide with 20 µl 1 N NaOH/1.5 ml formamide, 50 pmol [2H6]HPVal internal standard, and 20 µl pentafluorophenyl isothiocyanate (Fluka, Buchs, Switzerland). Samples were incubated overnight with vigorous shaking. After 90 min with vigorous shaking at 45°C, the samples were then extracted three times with diethyl ether. The extracts were combined and dried. The residue was resuspended in 1 ml fresh 0.1M Na2CO3 and loaded onto C18 minicolumns (200 mg, Alltech, Deerfield, IL) that were preconditioned with 2 ml methanol and 2 ml 50% formamide/water. The columns were rinsed with 1 ml distilled, deionized water and dried. Acetonitrile (3 ml, HPLC grade; Mallinckrodt, Hazelwood, MO) was used to elute the columns. Samples were dried in a speed-vac (Savant, Holbrook, NY). The residue was reconstituted in 50–100 µl toluene and placed in GC vials. Samples were stored at – 20°C until assayed.
Gas chromatography–tandem mass spectrometry analysis of HPVal.
The analysis of HPVal-PFPTH was carried out using a Finnigan Trace GC 2000 attached to a Finnigan TSQ7000 mass spectrometer (San Jose, CA). The settings for the gas chromatograph were helium as a carrier gas at a constant pressure of 25 psi; temperature programing, 1 min at 80°C, followed by a ramp of 10°C/min to 210°C, an increase in temperature of 4°C/min to 240°C, followed by a sharp increase in temperature of 80°C/min to 320°C, which is held for 1 min before cooling back to 80°C. The column used for analysis was a 30-m Alltech EC-5 (0.25 mm ID, 0.25 µm film thickness; Deerfield, IL). The operating procedures for the mass spectrometer were methane reagent gas at an ion source pressure of 3.5 torr (467 Pa); ion source temperature, 110°C; emission current, 0.3 mA; electron energy, 200 eV; and collision energy, 7 eV. Argon was used as a collision gas at a pressure of 2.5 mtorr (0.33 Pa). Aliquots of 2 µl of sample, in toluene, were injected on column. HPVal eluted as a double peak with a retention time of 16.4 min (peak 1) and 16.5 min (peak 2). The detection limit of the assay was 
5 pmol/g globin. A plot of HPVal:[2H6]HPVal ratio (where labeled HPVal is from labeled globin), showed a linear relationship (R2 = 0.995) over a range of 1–1000 pmol. The mass filter in the first quadrupole was set to allow only ions formed by loss of HF (M-20) from either the internal standard or the adduct into the second mass filter. Product ions corresponding to m/z 318 for the analyte and m/z 320 for the internal standard were selected in the third quadrupole.
Sample Preparation and Analysis for DNA Adducts, N7-HPGua
DNA isolation.
The frozen tissues were thawed, homogenized in phosphate-buffered saline (PBS) (pH 7.4) and centrifuged to obtain a nuclear pellet, and DNA extraction was carried out as described previously in Nakamura et al. (2000). Briefly, the nuclear pellets were incubated in lysis buffer (Applied Biosystems) overnight at 4°C with proteinase K (500 mg/ml). The resulting DNA was extracted twice with a mixture of 70% phenol/chloroform (water saturated; Applied Biosystems) and once with chloroform/isoamyl alcohol (50:1), followed by cold ethanol precipitation. The extracted DNA was incubated in PBS (pH 7.4) with RNase A, followed by DNA precipitation with cold ethanol. Then, the DNA pellet was resuspended in sterilized distilled water and the resulting DNA solution was stored at – 80°C until assayed.
Neutral thermal hydrolysis.
The volume of the DNA solution (300 µg) was adjusted to 400 or 500 µl by adding double distilled water prior to neutral thermal hydrolysis (NTH). Each DNA sample was spiked with 321 fmol of the internal standard, N7-HP[13C4]guanine. Then the samples were subjected to NTH by immersing the 1.5-ml centrifuge tube containing samples into a boiling water bath for 30 min. The hydrolysate, containing the adducts released by NTH, was separated from the DNA backbone by rapidly cooling samples in ice and transferring them into Microcon-3 filters that were prewashed with double distilled water. The filters were centrifuged for 30 min, then washed with an additional 20 µl double distilled water to recover the maximum amount of depurinated adduct, and they were centrifuged for an additional 15 min. The filtrates were combined, and the final volume of the sample injected onto liquid chromatography–mass spectrometry (LC-MS) was reduced by centrifugal evaporation and reconstituted in 10% acetonitrile to 50 µl; samples were stored at – 70°C until analyzed.
LC-MS/MS methodology for quantitation of N7-HPGua.
The LC-MS/MS system consisted of a Surveyor HPLC unit and a TSQQUANTUM triple quadrupole mass spectrometer from Thermo Finnigan (San Jose, CA). The samples (10 µl) were injected onto an Aquasil C-18 column (150 x 2.0 mm, 5 µm; Alltech) using the Surveyor autosampler. HPLC mobile phases consisted of water with 0.1% acetic acid (A) and acetonitrile with 0.1% acetic acid (B). The initial gradient started with 100% of A and was held for 1 min. From 1 to 10 min, the amount of B was linearly increased to 15%, and then increased to 80% over the next 5 min; column reequilibration time was 10 min. The LC pump flow rate was 200 µl/min; the HPLC column was maintained at 30°C using the column oven in the autosampler. The HPLC effluent from the first 3 min was directed to waste using a Rheodyne 77505 valve (Rohnert Park, CA) in order to reduce contamination of the electrospray source and to improve performance. Diversion at the beginning of the run served mainly as an online desalting step to lessen suppression of the electrospray ionization due to presence of salts in the sample. The HPLC effluent from 3 to 15 min was directed to the mass spectrometer for the detection and quantitation of N7-HPGua.
Electrospray ionization was done in positive ion mode, using nitrogen as sheath. A spray voltage of 4500 V was applied to the tip of the electrospray nozzle. The tandem mass spectrometric detection was done in the selected reaction monitoring (SRM) mode. The SRM transitions used were m/z 210–152 (parent ion losing the hydroxypropyl moiety with the guanine ion remaining) and m/z 214–156 for N7-HPGua and its [13C4]-labeled internal standard, respectively, using argon for collision-induced dissociation at 1.5 x 10–3 mtorr and a collision energy of 22 V. Retention time of N7-HPGua was 9.05 min. Electrospray ionization and SRM parameters were optimized for maximum sensitivity by making 5-µl loop injections of N7-HPGua standard and using the automated optimization feature of the XCalibur software. Calibration curves were generated by using the standard solutions prepared by spiking varying amounts of N7-HPGua into the solutions that contained a constant amount of internal standard. The amount of N7-HPGua in the sample was determined by relating the amount of N7-HPGua to the relative response factor, the ratio of peak area of the analyte to that of the internal standard. In order to ensure the instrument performance, both sensitivity and reliability, quality control (QC) samples prepared for propylene-exposed samples were analyzed prior to injection of a batch of samples. These QC samples were prepared by spiking control DNA with the analyte at the lowest concentration expected for a particular set of samples. Besides the QC samples, blanks and standards were inserted in appropriate positions in the sample queue to monitor any possible carryover and the sensitivity. The limit of detection of the assay was determined to be 0.05 fmol/µg DNA when 300 µg DNA was analyzed. The range of linearity of the standard curve was 2.5–50 fmol standard injected on column with the R2 value at 0.99. Extensive inside and outside rinsing of the autosampler needle was employed to avoid any possible carryover. Data acquisition and processing were performed using XCalibur version 1.3 software (Thermo Finnigan) running under the Microsoft 2000 operating system. Peak integration, calibration (using the internal standard), and quantitation were carried out using the QuanBrowser feature of the software. Analysis reports generated by the software were examined manually to eliminate false peak identification and incorrect integration.
Procedures for Hprt Analysis
Approximately 3 days following the final 20-day exposure, animals designated for Hprt mutation analysis (control, propylene-exposed, and CP-positive control groups) were assigned coded identifiers and were shipped to Dr V.E.W. at the LRRI (Albuquerque, NM) to be held for an appropriate expression time (8-week postexposure) until euthanized. The spleens were collected and prepared for splenocyte culture and quantitation of mutations in the Hprt gene. In brief, isolated splenic lymphocytes were cultured in medium containing a growth factor (IL-2) and a mitogen (Concanavalin A) and subsequently scored for colony growth in the presence or absence of a selection agent (6-thioguanine) (Walker et al., 2004). After 8 and 9 days of culture, respectively, Hprt and cloning efficiency (CE) plates were scored blind by two different readers. Mutant frequencies were calculated as the ratio of the mean CE in selective medium to that in nonselective medium. Once analyses were completed, the code was broken and statistical analyses were conducted.
Data Handling and Statistics
Descriptive statistics (e.g., mean ± SD) were conducted on exposure concentration and environmental conditions data and CE determinations. Generally, data were evaluated with the individual rat as the experimental unit of analysis. Body weight and cell proliferation (expressed as ULLI) data were tested for lack of trend, and if that were not significant, then sequential application of the Jonckheere-Terpstra trend test or one-way ANOVA followed with Dunnett's test was done. As described in Walker et al. (2004), the results were statistically analyzed for significance using the Pearson product-moment correlation for CE data and the Mann-Whitney rank sum test for Hprt mutation frequency data. For all analyses, a p value 0.05 was considered to be statistically significant. Sample size estimates based upon published Hprt mutant frequency data indicate that a sample size of eight rats per group provides 80% power for detecting a significant difference of 80% between a test group and a control group (based upon a background mutant frequency of 4.5 ± 1.6 x 10–6, mean ± SD).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Exposures
Overall, the mean daily propylene exposure levels ranged from 100 to 104% of target, with the following ranges for mean daily propylene levels for each exposure group: 203–208, 2000–2020, and 10,000–10,100 ppm.
Clinical Signs and Animal Observations
There were no treatment-related mortalities or clinical signs of toxicity observed prior, during, or following the exposure periods over the 4 weeks. There were a few incidental clinical observations recorded, including one female (2000 ppm) that was euthanized in extremis on day 7 due to a swollen face. Necropsy did not reveal anything of clinical significance. Body weights were comparable with respective controls for all complete exposure intervals for all treatment groups. One subset of eight male rats exposed to 10,000 ppm (destined for the Hprt study) demonstrated a statistically significantly lower body weight gain for exposure days 7–14 and a statistically significantly increased body weight gain for exposure days 14–21. The decrement was small compared to controls (6%), transient, and not present in other subsets of 10,000 ppm exposure groups, and did not result in any statistically significant differences in overall body weight gain for that subset over the entire 20-day exposure period. Thus, these body weight effects were considered to be due to biological variability and not treatment related.
Histopathology and Cell Proliferation
Microscopic analysis was conducted on H &E-stained slides from nasal regions 1, 2, 3, and 4 prepared from male and female rats exposed to propylene via whole-body inhalation for either 3 days (males only) or 20 days (males and females). The analysis did not demonstrate any propylene-specific nasal lesions or other histopathological changes in male or female F344 rats following 4 week of inhalation exposure to up to 10,000 ppm propylene as compared with air-exposed rats. In particular, no evidence of exposure-related inflammation (rhinitis) or alterations (e.g., degeneration, necrosis, hyperplasia, metaplasia) in the squamous, transitional, respiratory, or olfactory epithelium lining the nasal airways was found in any of the sections examined from these propylene-exposed rats. In addition, there were no apparent exposure-related changes, compared to that of controls, in the density of BrdU labeling in the four specific nasal epithelial populations. Quantitative results for analysis of ULLI for BrdU-labeled nasal epithelium are shown in Table 1. No statistically significant changes (increases or decreases) in ULLI were observed in nasal epithelium from any of the exposure groups, including 4 weeks of exposure to 10,000 ppm propylene. Table 2 summarizes the lack of increase in BrdU incorporation by liver, measured as a nontarget tissue. As expected, duodenum did demonstrate incorporation of BrdU (data not shown).
|
|
Hb and DNA Adducts
Inhalation exposure of F344 rats to propylene resulted in clear evidence of internal exposure, with quantifiable numbers of HPVal adducts in systemic blood from all exposed animals and of N7-HPGua adducts in DNA from all exposed tissues evaluated, except following single exposures to 200 ppm or in lymphocytes following 3-day exposure to 200 ppm. The number of adducts increased with increasing propylene levels up to 2000 ppm propylene, as shown in Tables 3 and 4 and in Figure 2, and then reached a plateau such that the numbers of HPVal and N7-HPGua adducts for tissues from 10,000 ppm exposures (1-, 3-, and 20-day exposures) were similar to the ones for 2000-ppm exposures. The SD for the HPVal data from the 3-day/10,000 ppm group was large (
50%); this is due to a single sample with a very low value, which did not meet the outlier criteria for exclusion. The number of N7-HPGua adducts was similar across the tissues evaluated (e.g., 1.95–2.57 fmol/µg DNA following 20 days at 10,000 ppm), suggesting that the epoxide formed through metabolism was stable enough to circulate in blood. Although the SD values were overlapping, the mean level of N7-HPGua in lung (20-day/10,000 ppm) was slightly higher than liver, which may reflect the high capacity of liver for detoxification or the role of lung as a portal of entry for an inhalation exposure. No Hb or DNA adducts were detectable in control blood or in control DNA from any tissues evaluated. The lack of dose proportionality for both HPVal and N7-HPGua, observed up to 10,000 ppm propylene, is likely due to the saturation of P450-mediated conversion of propylene to PO, the epoxide metabolite.
|
|
|
Micronucleus
Inhalation exposure of male rats to up to 10,000 ppm propylene for 4 weeks had no effect on the proportion of PCE per 1000 erythrocytes scored per animal or on the frequency of micronucleated erythrocytes per 2000 PCEs scored per animal, as is shown in Table 5. The positive control, CP, clearly induced an increased frequency of MNs and a decreased ratio of PCEs, indicating adequacy of the assay conditions to detect genotoxicity. Therefore, inhalation exposure of F344 male rats to up to 10,000 ppm propylene did not cause any in vivo genotoxicity/clastogenicity as measured by increases in formation of MNs in bone marrow.
|
Hypoxanthine-Guanine Phosphoribosyl Transferase
As was described in Walker et al. (2004)
, inhalation exposure of F344 rats to propylene up to 10,000 ppm for 4 weeks did not result in any quantifiable increases in mutant frequency at the Hprt gene (Table 6). Treatment of F344 rats with CP, however, did induce a twofold increase in Hprt mutant frequency, demonstrating that the assay was capable of detecting a mutagenic effect.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The results presented here provide clear evidence that inhalation exposure of rats to propylene results in quantifiable internal exposure to its epoxide metabolite, PO, with demonstrated increases in both PO-specific Hb and DNA adducts. At the same time, these results demonstrate no biologically significant effects from this exposure, including no induction of MN formation in bone marrow erythrocytes, no increase in Hprt mutant frequency in splenocytes, and no evidence of nasal irritation or induction of cell proliferation in nasal respiratory tissue.
The concentrations of HPVal in blood and of N7-HPGua in liver and lung in rats exposed to 200, 2000, or 10,000 ppm propylene for 4 weeks were compared with the data obtained for these same adducts formed in rats following 4-week inhalation exposure to 50 ppm PO (Swenberg, unpublished data) and measured using the same analytical methods. Table 7 summarizes the predicted internal dose of PO in the propylene-exposed tissues based on adduct concentrations. It is clear from these comparisons that the metabolic conversion of propylene to PO is nearly saturated at the 2000-ppm exposure, given the close equivalence of predicted internal PO from both 2000 and 10,000 ppm propylene. This is true for HPVal (systemic blood) and N7-HPGua adduct levels in the lung and liver. In addition, the level of HPVal and N7-HPGua adducts measured for blood and lung, respectively, predicted very similar PO internal dose across exposure levels, while internal dose of PO predicted for liver is clearly higher. This supports the hypothesis that the main site of propylene conversion is in the liver, with the systemic blood and lung dose mostly arising from the circulating reactive metabolite. This is despite the lung representing a portal-of-entry tissue, thus typically receiving a higher exposure than the liver, and despite the liver's greater capacity for detoxification. It is possible that the slightly higher internal dose for the lung versus systemic blood may stem from the known metabolic capability present in lung type II epithelial and Clara cells.
|
It is not clear why the histopathological results demonstrating lack of nasal irritation reported here are different from those reported by NTP, where nondose-related signs of nasal irritation were described in F344 rats following chronic (104 weeks) exposure to the same high levels of propylene (NTP, 1985a). An expert review of the nasal slides from the NTP study confirmed the presence of nasal irritation and the lack of dose response (Harkema, unpublished data). The chronic study conducted by Ciliberti et al. (1988)
in Sprague-Dawley rats and Swiss mice did not evaluate nasal tissue histopathology. However, the lung was examined, and no effects were identified for chronic exposure up to 5000 ppm propylene (Ciliberti et al., 1988). If propylene gas has irritant properties, usually a repeated exposure over 4 weeks would be considered adequate to induce a typical irritant response in rodent nasal tissues. Thus, if the NTP results are due to any irritant properties of propylene, the results reported here would strongly suggest that those properties must be extremely weak. Alternatively, it is possible that other factors contributed to the reported observations of nasal irritation, such as dryness of mucous membranes due to chronic inhalation exposure.
The negative results for mutagenicity and clastogenicity endpoints described in this report support the published data showing a lack of induction of tumorigenesis following chronic (2 years) inhalation exposure of rats and mice to high levels of propylene, up to 10,000 ppm, or one-half the lower explosion limit (Ciliberti et al., 1988; NTP, 1985a).
There is a strong parallel with the available data on the analogous chemical, ethylene. Ethylene also is metabolized to an epoxide intermediate, ethylene oxide, which is detoxified via conjugation with GSH by GST or hydrolysis by EH (Brown et al., 1996; Filser et al., 1993, 2000; Golka et al., 1989). The reactive intermediate can react with cellular macromolecules, and published data demonstrate formation of Hb (hydroxyethylvaline) and DNA (N7-hydroxyethylguanine, N7-HEGua) adducts following exposure of rats and mice to ethylene (Eide et al., 1995; Rusyn et al., 2005; Segerbäck et al., 1983; Walker et al., 2000; Wu et al., 1999; Zhao et al., 1997). Exposure to high concentrations of ethylene also did not result in any biologically significant effects, including no induction of MN formation in bone marrow erythrocytes (Vergnes and Pritts, 1994), no increase in Hprt mutant frequency in splenocytes (Walker et al., 2000), no increases in apurinic (AP) sites (Rusyn et al., 2005), and no increases in tumor incidence in rats following a 2-year exposure to 3000 ppm ethylene (Hamm et al., 1984).
The data for ethylene and propylene support the hypothesis that N7-HEGua and N7-HPGua adducts are unlikely to result in genotoxic effects, as these adducts were formed but no evidence of mutagenic effects was observed, neither in vivo as reported here and elsewhere or in vitro for almost all assays, whether bacterial, mammalian, or Drosophila (NTP, unpublished results; OECD, 2003; Riley et al., 1996; Vergnes and Pritts, 1994; Victorin and Stahlberg, 1979; Walker et al., 2004).
As discussed in a recent review by Albertini and Sweeney (2007), N7-alkylguanine adducts formed from small epoxides such as PO or ethylene oxide apparently do not cause distortion of the DNA double helix or do they interfere with base pair hydrogen bonding. This may explain their lack of mutagenic effects. They are hypothesized to result in mutation only via the following mechanism/mode of action: the loss of N7-alkylguanine adducts via depurination of the adducted base, leaving an AP site in the DNA. Lesions such as AP sites are known to be mutagenic if DNA replication occurs in their presence, as they present a noninformational site (Cabral-Neto et al., 1992). Identical AP sites form due to spontaneous depurination of normal bases, as well as for the labile N7-alkylguanine bases. In addition, AP sites are present during intermediate steps of base excision repair (BER), following cleavage of the glycosidic bond by glycosylases. During BER, the AP site is then removed by cleavage of the DNA backbone by endonucleases, followed by removal of the noninformational site with 5' cleavage of the backbone and replacement of the missing sequence by DNA polymerase. The data from Rusyn et al. (2005) do not support mutagenesis from ethylene oxide (EO) via formation of AP sites. These authors suggested that the mutagenic effects seen with EO were likely to be the result of minor promutagenic adducts, such as O6-HEGua, N1-HEAdenine, or possibly ring-opened N7-HEGua. This study did not try to identify any of these minor adducts in propylene-exposed tissues.
N7-HPGua adducts are known to form following inhalation exposure to PO (Ríos-Blanco et al., 2000, 2003; Segerbäck et al., 1998). Their formation demonstrated a dose response that was less than dose proportionate but linear over a range of 5–500 ppm PO for 3 days or 4 weeks (20 days) of exposure, although the slope was different for each tissue examined. The number of N7-HPGua measured in rat tissues following propylene exposure reached a plateau, whereas following PO exposure N7-HPGua increased linearly from 5 to 500 ppm PO. The maximum number of N7-HPGua reached in lung and liver following 20-day repeated exposure to 10,000 ppm propylene was equivalent to between 25 and 80 ppm PO, based on the data from Ríos-Blanco et al. (2003) and 35 and 70 ppm PO based on the more recent data (Table 7). These findings demonstrate that the dose of PO to lung and liver from 10,000 ppm propylene is below the lowest NOEL for PO-induced nasal tumorigenesis in rodents, 100 ppm, and well below the lowest LOEL, 300 ppm (Lynch et al., 1984a). This difference is explained by the saturation of metabolic conversion of propylene to PO, presumably due to the saturation of propylene metabolism via cytochrome P450. Data from Plná et al. (1999) indicate that the N7-HPGua adducts formed in rats following 20-day exposure to PO are about 70% removed by 3 days after the final exposure; it seems reasonable to assume similar kinetics for the N7-HPGua formed following exposure to propylene.
There are published data demonstrating that, despite very heavy loads of N7-HPGua found in rat nasal mucosa following 4 weeks (20 days) of exposure to 500 ppm PO, the level of AP sites does not change compared to control animals (Ríos-Blanco et al., 2000). This lack of induction of AP sites in PO-exposed target tissue has since been confirmed by additional work in rats and mice (Swenberg, unpublished data). Thus, even with a high load of N7-HPGua adducts, there was no difference in the number of AP sites. These data suggest that repair of AP sites is extremely effective and is well maintained by the cell in vivo, even under extreme exposure conditions. This conclusion is also supported by the studies of Rusyn et al. (2005) with ethylene and ethylene oxide.
The negative mutagenicity and clastogenicity data described in the present study are similar to what had been seen with ethylene (Rusyn et al., 2005; Vergnes and Pritchard, 1994; Walker et al., 2000). This phenomenon is also consistent with a lack of increase in AP sites and the predicted minimal (if any) formation of minor promutagenic DNA adducts, although these endpoints (AP sites, minor promutagenic adducts) have not been specifically evaluated following propylene exposure. The number of such promutagenic adducts, if present, does not reach the threshold needed for mutations following propylene exposures of up to 10,000 ppm for 4 weeks (20 days), as was evidenced by the lack of Hprt mutation induction.
Overall, the results presented here indicate that repeated exposure (3 or 20 days) of rats to high concentrations of propylene (up to 10,000 ppm) does not produce evidence of local toxicity to the nasal cavity or evidence of systemic genotoxicity to hematopoietic tissue in the rat, despite the formation of N7-HPGua adducts. This evidence for no systemic genotoxicity from propylene exposure supports the two sets of negative propylene chronic carcinogenicity bioassays, each conducted in rats and mice. In addition, these data indicate that formation of N7-HPGua does not correlate with any measure of genotoxic effect, neither mutagenic nor clastogenic.
|
ACKNOWLEDGMENTS |
|---|
The quality assurance reviews of the data and conducting laboratories performed by Dr J. Baldwin are greatly appreciated. The stimulating and insightful discussions with Dr B.B. Gollapudi are gratefully acknowledged by L.H.P. This study was sponsored by the Olefins Panel of the American Chemistry Council and utilized the Biomarker Facility Core of National Institutes of Health grant P30 ES10126. Authors L.H.P. and M.I.B. are employed by companies that manufacture propylene.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Agurell E, Cederberg H, Ehrenberg L, Lindahl-Kiessling K, Rannug U, Törnqvist M. Genotoxic effects of ethylene oxide and propylene oxide: A comparative study. Mutat. Res. (1991) 250:229–237.[ISI][Medline]
Albertini RA, Sweeney LM. Propylene Oxide: Genotoxicity Profile of a Rodent Nasal Carcinogen. CRC Toxicology (2007) (in press).
ACGIH. Final Propylene Support Document. Threshold Limit Values for Chemical Substances and Physical Agents and Biological Exposure Indices (2006) Cincinnati, OH: ACGIH.
Bootman J, Lodge DC, Whalley HE. Mutagenic activity of propylene oxide in bacterial and mammalian systems. Mutat. Res. (1979) 67:101–112.[CrossRef][ISI][Medline]
Brown CD, Wong BA, Fennell TR. In vivo and in vitro kinetics of ethylene oxide metabolism in rats and mice. Toxicol. Appl. Pharmacol. (1996) 136:8–19.[CrossRef][ISI][Medline]
Cabral-Neto JB, Gentil A, Cabral RE, Sarasin A. Mutation spectrum of heat-induced abasic sites on a single-stranded shuttle vector replicated in mammalian cells. J. Biol. Chem. (1992) 267:19718–19723.
Ciliberti A, Maltoni C, Perino G. Long-term carcinogenicity bioassays on propylene administered by inhalation to Sprague–Dawley rats and Swiss mice. Ann. N. Y. Acad. Sci. (1988) 534:235–245.[Abstract]
Drummond I. Light hydrocarbon gases: A narcotic, asphyxiant, or flammable hazard? Appl. Occup. Environ. Hyg. (1993) 8:120–125.
Dunkelberg H. Carcinogenic activity of ethylene oxide and its reaction products 2-chloroethanol, 2-bromoethanol, ethylene glycol and diethylene glycol. I. Carcinogenicity of ethylene oxide in comparison with 1,2-propylene oxide after subcutaneous administration in mice. Zentralbl. Bakteriol. Mikrobiol. Hyg. b (1981) 174:383–404.[Medline]
Dunkelberg H. Carcinogenicity of ethylene oxide and 1,2-propylene oxide upon intragastric administration to rats. Br. J. Cancer (1982) 4:924–933.
Eide I, Hagemann R, Zahlsen K, Tareke E, Törnqvist M, Kumar R, Vodicka P, Hemminki K. Uptake, distribution, and formation of hemoglobin and DNA adducts after inhalation of C2–C8 1-alkenes (olefins) in the rat. Carcinogenesis (1995) 16:1603–1609.
Eldridge SR, Bogdanffy MS, Jokinen MP, Andrews LS. Effects of propylene oxide on nasal epithelial cell proliferation in F344 rats. Fundam. Appl. Toxicol. (1995) 27:25–32.[CrossRef][ISI][Medline]
Faller TH, Csanády GA, Kreuzer PE, Baur CM, Filser JG. Kinetics of propylene oxide metabolism in microsomes and cytosol of different organs from mouse, rat, and humans. Toxicol. Appl. Pharmacol. (2001) 172:62–74.[CrossRef][ISI][Medline]
Farooqi Z, Törnqvist M, Ehrenberg L, Natarajan AT. Genotoxic effects of ethylene oxide and propylene oxide in mouse bone marrow cells. Mutat. Res. (1993) 288:223–228.[ISI][Medline]
Filser JG, Denk B, Törnqvist M, Kessler W, Ehrenberg L. Pharmacokinetics of ethylene in man: Body burden with ethylene oxide and hydroxyethylation of hemoglobin due to endogenous and environmental ethylene. Arch. Toxicol. (1993) 66:157–163.[CrossRef][ISI]
Filser JG, Schmidbauer R, Rampf F, Baur CM, Putz C, Csanády GA. Toxicokinetics of inhaled propylene in mouse, rat, and human. Toxicol. Appl. Pharmacol. (2000) 160:40–51.
Golka K, Peter H, Denk B, Filser JG. Pharmacokinetics of propylene and its reactive metabolite propylene oxide in Sprague-Dawley rats. Arch. Toxicol. Suppl. (1989) 13:240–242.[Medline]
Hamm TE, Guest D, Dent JG. Chronic toxicity and oncogenicity bioassay of inhaled ethylene in Fischer-344 rats. Fundam. Appl. Toxicol. (1984) 4:473–478.[CrossRef][ISI][Medline]
Hardin BD, Schuler RL, McGinnis PM, Niemeier RW, Smith RJ. Evaluation of propylene oxide for mutagenic activity in 3 in vivo test systems. Mutat. Res. (1983) 117:337–344.[CrossRef][ISI][Medline]
Hughes TJ, Sparacino C, Frazier S. Validation of Chemical and Biological Techniques for Evaluation of Vapors in Ambient Air/Mutagenicity Testing of Twelve (12) Vapor-Phase Compounds (1984) Report of EPA (No. EPA-600/1-84-005).
IARC. IARC Monographs: Working Group on the Evaluation of Carcinogenic Risks to Humans: Some Industrial Chemicals (1994) Vol. 60. Lyon: IARC Scientific Publications, IARC. 1–560.
Kuper C, Reuzel P, Feron V, Verschuuren H. Chronic inhalation toxicity and carcinogenicity study of propylene oxide in Wistar rats. Food Chem. Toxicol. (1988) 26:159–167.[CrossRef][ISI][Medline]
Lee MS, Faller TH, Kreuzer PE, Kessler W, Csanády GA, Putz C, Ríos-Blanco MN, Pottenger LH, Segerbäck D, Osterman-Golkar S, et al. Propylene oxide in blood and soluble nonprotein thiols in nasal mucosa and other tissues of male Fischer 344/N rats exposed to propylene oxide vapor—Relevance of glutathione depletion for propylene oxide-induced rat nasal tumors. Toxicol. Sci. (2005) 83:177–189.
Lundberg P. Scientific Basis for Swedish Occupational Standards XVII: Consensus Report for Propene (1996) Soma, Sweden: National Institute for Working Life.
Lynch DW, Lewis TR, Moorman WJ, Burg JR, Groth D, Khan A, Ackerman L, Cockrell B. Carcinogenic and toxicologic effects of inhaled ethylene oxide and propylene oxide in F344 rats. Toxicol. Appl. Pharmacol. (1984a) 76:69–84.[CrossRef][ISI][Medline]
Lynch DW, Lewis TR, Moorman WJ, Burg JR, Gulati DK, Kaur P, Sabharwal PS. Sister-chromatid exchanges and chromosome aberrations in lymphocytes from monkeys exposed to ethylene oxide and propylene oxide by inhalation. Toxicol. Appl. Pharmacol. (1984b) 76:85–95.[CrossRef][ISI][Medline]
McGregor D, Brown AG, Cattanack P, Edwards I, McBride D, Riach C, Shepherd W, Caspary WJ. Responses of the L5178Y mouse lymphoma forward mutation assay: V. Gases and vapors. Environ. Mol. Mutagen. (1991) 17:122–129.[ISI][Medline]
Mower J, Törnqvist M, Jensen S, Ehrenberg L. Modified Edman degradation applied to hemoglobin for monitoring occupational exposure to alkylating agents. Toxicol. Environ. Chem. (1986) 11:215–231.
Nakamura J, La DK, Swenberg JA. 5'-Nicked apurinic/apyrimidinic sites are resistant to beta-elimination by beta-polymerase and are persistent in human cultured cells after oxidative stress. J. Biol. Chem. (2000) 275:5323–5328.
National Toxicology Program (NTP). Toxicology and Carcinogenesis Studies of Propylene (CAS No. 115-07-1) in F344/N Rats and B6C3F1 Mice (Inhalation Studies) (1985a) Report NTP TR 272. National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC.
NTP. Toxicology and Carcinogenesis Studies of Propylene Oxide (CAS No. 75-56-9) in F344/N Rats and B6C3F1 Mice (Inhalation Studies) (1985b) Report NTP TR 267, National Toxicology Program, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. Research Triangle Park, NC.
Organization for Economic and Co-operative Development (OECD). ICCA/HPV SIDS Dossier (SIAR, SIAP, IUCLID) for propylene (2003) Available at http://www.jetoc.or.jp/HP_SIDS/pdffiles/115-07-1.pdf.
Plná K, Nilsson R, Koskinen M, Segerbäck D. 32P-postlabelling of propylene oxide 1- and N6-substituted adenine and 3-substituted cytosine/uracil: Formation and persistence in vitro and in vivo. Carcinogenesis (1999) 20:2025–2032.
Riley S. Unpublished Corning Hazleton (1996) Report No. 1458/2-1050. Cited in: SIDS OECD Dossier on the HPV Phase 4 Chemical Ethylene, June 1997. Available at http://portalserver.unepchemicals.ch/Publications/Screening%20Infornation%20Data%20Set.htm.
Ríos-Blanco MN, Plná K, Faller T, Kessler W, Hakansson K, Kreuzer PE, Ranasinghe A, Filser JG, Segerbäck D, Swenberg JA. Propylene oxide: Mutagenesis, carcinogenesis and molecular dose. Mutat. Res. (1997) 380:179–197.[ISI][Medline]
Ríos-Blanco MN, Faller TH, Nakamura J, Kessler W, Kreuzer PE, Ranasinghe A, Filser JG, Swenberg JA. Quantitation of DNA and hemoglobin adducts and apurinic/apyrimidinic sites in tissues of F344 rats exposed to propylene oxide by inhalation. Carcinogenesis (2000) 21:2011–2018.
Ríos-Blanco MN, Ranasinghe A, Lee MS, Faller T, Filser JG, Swenberg JA. Molecular dosimetry of N7-(2-hydroxypropyl)guanine in tissues of F344 rats after inhalation exposure to propylene oxide. Carcinogenesis (2003) 24:1233–1238.
Rusyn I, Asakura S, Li Y, Kosyk O, Koc H, Nakamura J, Upton PB, Swenberg JA. Effects of ethylene oxide and ethylene inhalation on DNA adducts, apurinic/apyrimidinic sites and expression of base excision DNA repair genes in rat brain, spleen, and liver. DNA Repair (2005) 4:1099–1110.[Medline]
Segerbäck D. Alkylation of DNA and hemoglobin in the mouse following exposure to ethene and ethene oxide. Chem. Biol. Interact. (1983) 45:139–151.[CrossRef][ISI][Medline]
Segerbäck D, Plná K, Faller T, Kreuzer PE, Hakansson K, Filser JG, Nilsson R. Tissue distribution of DNA adducts in male Fischer rats exposed to 500 ppm of propylene oxide–quantitative analysis of 7-(2-hydroxypropyl)guanine by 32P-postlabeling. Chem. Biol. Interact. (1998) 115:229–246.[CrossRef][ISI][Medline]
Schoenberg M, Blieszner J, Papadopoulos C. Propylene. In: Kirk-Othemer Concise Encyclopedia of Chemical Technology—Grayson M, Eckroth D, eds. (1985) New York: J. Wiley & Sons. 906–961.
Svensson K, Osterman-Golkar S. Kinetics of metabolism of propene, and covalent binding to macromolecules in the mouse. Toxicol. Appl. Pharmacol. (1984) 73:363–372.[CrossRef][ISI]