1 Curriculum in Toxicology, Campus Box 7431, Rosenau Hall 253c, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
2 Department of Environmental Sciences and Engineering, Campus Box 7431, Rosenau Hall 253c, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
3 Institute of Toxicology, GSF National Research Center for Environment and Health, D-85764 Neuherberg, Germany
5 To whom correspondence should be addressed Email: james_swenberg{at}unc.edu
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Abstract |
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Abbreviations: AP, apurinic/apyrimidinic; ddH2O, double-distilled water; GCHRMS, gas chromatographyhigh-resolution mass spectrometry; N6-HPdAdo, N6-(2-hydroxypropyl)deoxyadenosine; 7-HPG, N7-(2-hydroxypropyl)guanine; NRE, nasal respiratory epithelium; PO, propylene oxide
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Introduction |
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The genotoxicity and carcinogenicity of PO in bacterial and mammalian systems has been reviewed elsewhere (25). In summary, PO has been shown to be mutagenic in bacterial and mammalian systems in vitro (610), but has not been shown to be mutagenic in vivo by exposure routes relevant to humans (6,11).
Long-term carcinogenicity studies of PO demonstrated that for all routes investigated, increased incidences of tumors occurred at the site of contact in exposed rats. Intragastric administration of PO to SpragueDawley rats resulted in a dose-dependent increase in squamous cell carcinomas of the forestomach (12) whereas s.c. injection of PO to female NMRI mice led to the appearance of tumors (sarcomas) at the injection site at high doses only (13). F344 rats exposed their lifetime to PO by inhalation developed papillary adenomas of the nasal respiratory epithelium, whereas exposure to B6C3F1 mice led to the development of hemangiomas, hemangiosarcomas and adenocarcinomas within the nasal tissues (1416). Tumors were observed at high concentrations only (300 p.p.m.). Inhalation exposure of Wistar rats to PO (0, 30, 100 and 300 p.p.m. for 124 weeks) resulted in an increase in non-neoplastic lesions in the nasal cavity of rats exposed to 100 or 300 p.p.m. (17). One squamous cell carcinoma of the nose was diagnosed in a 30 p.p.m. male (1/61) and another in a 300 p.p.m. male (1/63).
IARC (1994) classified PO as a possible human carcinogen based on positive rodent cancer bioassays and insufficient epidemiological data concerning its carcinogenicity in humans. Current estimates of PO cancer risk in humans [EPA, 1994, Integrated Risk Information System (IRIS) Propylene Oxide File] were obtained by linear extrapolation of tumor data. In the case of PO, rodent tumor data were obtained at concentrations that are 15 times higher than those potentially occurring in the workplace.
The mechanism by which PO causes cancer is not clear. PO directly reacts with DNA to form mainly the N7-(2-hydroxypropyl)guanine (7-HPG) adduct and a minor amount of cytosine and adenine adducts (18,19). Studies in vivo (5,20,21) demonstrated a marked difference in DNA alkylation between tissues, with a much greater number of 7-HPG adducts in nasal tissue than in systemic organs after inhalation exposure of rats to 500 p.p.m. PO for 20 days. Molecular dosimetry data for PODNA adducts may provide insights into its mode of action.
The objectives of this research were: (i) to quantify the exposure-dependent accumulation of 7-HPG in tissues of rats exposed to PO by the inhalation route; (ii) to examine the differences in DNA alkylation between tissues at different concentrations and lengths of exposure; and (iii) to examine the relationship between the exposureresponse in 7-HPG accumulation and the tumor incidence curve. The long-term goals of this research are to use molecular dosimetry data and cell proliferation data to understand better the mechanism of PO carcinogenesis, and thus to improve the current PO cancer risk assessments.
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Materials and methods |
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Animal exposures
The study was conducted largely in compliance with good laboratory practice regulations as promulgated by the US Environmental Protection Agency and the Organization for Economic Cooperation and Development. Exposures were carried out at the GSF Institute of Toxicology, Neuherberg, Germany. Nine-week-old male F344 rats were purchased from Charles River Deutschland (Sulzfeld, Germany) and were marked with ear tags. The rats were assigned to one of six exposure groups per exposure period (n = 5/group), based on their body weight. The group assignments were adjusted to result in mean group body weights that were not significantly different from one another. Treated and control animals were provided with food (Standard Chow 1324, Altromin, Lage, Germany) and water ad libitum except during the inhalation exposure periods. The animals were exposed to nominal PO concentrations of 0, 5, 25, 50, 300 and 500 p.p.m. for 6 h/day, 5 days/week for 3 or 20 days. PO vapor was generated by a single vaporization system (22) using a separate glass chamber for each concentration. Animals were observed daily for mortality and overt clinical signs of toxicity. Detailed descriptions of analytical techniques and inhalation chambers are found elsewhere (5,22). Animals were killed with carbon dioxide within 5 h after the end of the final exposure period. Nasal respiratory epithelium, lung and liver were collected, placed in plastic vials and frozen in liquid nitrogen. Tissue samples were stored at -80°C until analysis.
DNA isolation
DNA was extracted from nasal respiratory epithelium (NRE), lung and liver of control and exposed rats by phenolchloroform extraction. Briefly, lung and liver tissue were thawed and homogenized in cold PBS with a Tehran homogenizer (Wheaton Instruments, Millville, NJ). Samples were kept on ice throughout the whole procedure. After centrifugation (2000 g, 15 min), the nuclear pellets were resuspended in cell lysis buffer overnight at 4°C with proteinase K. The suspensions were extracted twice with a mixture of phenol: chloroform:water and once with Sevag (24:1, chloroform:isopentyl alcohol), followed by ethanol precipitation. The resulting DNA was incubated in PBS with a mixture of RNase T1 and RNase A for 30 min at 37°C. After a second precipitation of DNA with cold ethanol, the DNA pellet was re-suspended in double distilled water (ddH2O) and DNA concentration measured by absorbance at 260 nm of an aliquot in 20 mM Tris buffer, pH 7.4 (20 A260 = 1 mg/ml). Measurements of the ratio A260/A280 were between 1.6 and 1.9 indicating minimal protein contamination. The homogenization step was not performed for NRE. Nasal tissues were immediately incubated in cell lysis buffer. The rest of the DNA isolation procedure was as given for the other tissues. The quality of DNA from NRE samples was not appropriate according to the A260/A280 ratio, possibly due to protein or salt contamination. For further purification, cell lysis buffer from Puregene (Gentra Puregene) was added to the DNA samples (7.6:1 buffer:solution) from NRE along with protein precipitation solution (3:1 lysis buffer:precipitation solution). Samples were mixed and centrifuged (>8000 g) for 10 min. DNA was precipitated from the supernatant by alcohol addition. DNA pellets were resuspended in ddH2O and DNA concentration measured by absorbance as explained for the rest of the tissues. No difference in 7-HPG number was found between NRE DNA from samples prior to or after purification by the Gentra Puregene kit. However, the quality of the NRE DNA improved as noted in the appearance of the UV spectrum (data not shown).
Hydrolysis, derivatization and quantification of 7-HPG by gas chromatographyhigh-resolution mass spectrometry (GC-HRMS)
The method for the derivatization of 7-HPG in DNA samples has been described in detail elsewhere (5,21). The same standard solutions of 7-HPG and 7-HP [13C4]G used for the analysis of 7-HPG in different tissues after exposure to 500 p.p.m. PO for 20 days (5) were used for the analysis of 7-HPG in the samples described in the present study. DNA samples (2300 µg) were spiked with 0.8 pmol of 7-HP[13C4]G, dried under vacuum and rehydrated in water (0.5 µg/µl) at 4°C overnight. 7-HPG was released from DNA by neutral thermal hydrolysis (100°C, 20 min in ddH2O), converted to the N7-(2-hydroxypropyl)xanthine, derivatized twice with PFBBr and analyzed by GCHRMS. Samples were dissolved in toluene (3050 µl) from which 11.5 µl samples were injected into the GC column. The molecular ion minus one pentafluorobenzyl group was measured by selected ion monitoring at m/z 569.0671 for 7-HPG and m/z 573.0806 for 7-HP[13C4]G. The peak area ratio between 7-HPG and 7-HP[13C4]G was obtained from each sample and compared with the ratio obtained with standard calibration curves. The amount of 7-HPG in each sample was then derived from calibration curves where known amounts of the 7-HPG standard were derivatized with a known amount of the internal standard, which was equivalent to the amount of internal standard added to each sample analyzed. The calibration curve solutions prepared contained 02.0 pmol 7-HPG with 0.8 pmol of internal standard added. The calibration curve standard solutions were analyzed as described above. Calibration curves were obtained by linear regression analysis of peak area ratio (7-HPG: 7-HP[13C4]G) against the amount of 7-HPG (fmol) in the standard calibration solutions. Equations for the calibration curves were then used to calculate the amount of 7-HPG in the samples from the peak area ratio (7-HPG: 7-HP[13C4]G). The calibration curves showed a linear relationship with 7-HPG concentration (r2 = 0.999) with a coefficient of variability of <15% from four to eight injections. A sample (method blank) to which no internal standard or analyte standard was added was analyzed each time to check for contamination of reagents. Samples of [14C]PO-modified salmon testis DNA, with a known amount of 7-HPG, were run repeatedly to check for the system performance and the method accuracy. The preparation of the [14C]PO-modified salmon testis DNA solutions is described elsewhere (21). In previous reports (5,21), the concentration of 7-HPG in DNA was expressed as pmol adduct/µmol guanine. Guanine measurements were not obtained for all samples in this study due to the limited amount of DNA in the nasal respiratory tissue. Therefore, the concentration of adducts in this study is uniformly expressed as pmol adduct/mg DNA. The guanine content in rat DNA samples can be estimated from the following relationship: µmol guanine = µg DNA (0.22)/320.
GCHRMS analysis of 7-HPG adducts
GCHRMS chromatograms were obtained on a HP 5890 GC interfaced to a VG70-250 SEQ GC/hybrid mass spectrometer in the electron capture negative ion chemical ionization mode. The mass resolving power was 10 K. Direct injections using a press-fit liner were made onto a DB-5 fused silica capillary column (15 m x 0.32 mm). Helium head pressure was 10 psi. The ion source temperature was 250°C. Methane (3 x 10-5 mbar) was used as the reagent gas. The emission current was 0.5 mA. The injector temperature was 290°C. The temperature program was: 1 min at 70°C, 20°C/min to 290°C followed by an increment of 50°C/min to reach 300°C.
Statistical analysis
Linear regression analyses of the 7-HPG accumulation in each tissue were obtained using Excel. Differences in DNA adduct accumulation in the same tissue at different exposure levels were determined by unpaired sample t-tests.
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Results |
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Analysis of 7-HPG in tissues
Accumulation of 7-HPG was measured in NRE, lung and liver from male F344 rats exposed to PO (0, 5, 25, 50, 300 or 500 p.p.m) for 3 or 20 days (6 h/day, 5 days/week). Results from the analysis of 7-HPG in different tissues of rats exposed to PO by the inhalation route are summarized in Tables I and II. Analysis of 7-HPG in tissues of control animals showed no evidence of endogenous formation of PO DNA adducts. The limit of detection of the method was 50 fmol/sample based on measurement of standards. Analysis of 7-HPG in NRE, lung and liver of rats exposed to PO by inhalation showed a dose-dependent increase in adduct accumulation for both exposure periods. The nose had the highest concentration of adducts followed by lung and liver, respectively, for both exposure periods. The accumulation of 7-HPG adducts in each tissue at 5 p.p.m PO was significantly different (P < 0.05) from the accumulation of adducts in the same tissue at 25 or 50 p.p.m. PO after 3 or 20 days of exposure. Significant differences (P < 0.001) were observed between high and low levels of exposure in each tissue for both lengths of exposure. The amount of 7-HPG in NRE was significantly different (P < 0.001) from the rest of the tissues at all concentrations and lengths of exposure. The amount of 7-HPG in lung was significantly higher than in liver (P < 0.05) between 25 and 500 p.p.m. for both lengths of exposure. The amount of 7-HPG in lung was not significantly different from the amount found in liver after exposure to 5 p.p.m PO for 20 days. The number of 7-HPG adducts in liver DNA of rats exposed to 5 p.p.m. PO for 3 days was below the limit of detection.
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Discussion |
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Results from the present research: (i) demonstrated a much higher level of DNA alkylation in the NRE than in lung and liver, respectively, at all PO concentrations and exposure periods; (ii) showed a linear concentration-dependent increase in 7-HPG accumulation in NRE, lung and liver after 3 days and in NRE and lung after 20 days of exposure; and (iii) demonstrated that NRE 7-HPG adduct accumulation is not sufficient to induce tumor formation as it had a linear response, while nasal tumors were clearly non-linear (Figure 3).
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The linear concentration-dependent increase in 7-HPG accumulation in all tissues after 3 days of exposure and in NRE and lung after 20 days of exposure suggests that saturation of detoxication or DNA repair does not occur in these tissues under these exposure conditions. Detoxication of PO in rat tissues occurs mainly by glutathione S-transferase (GST)-mediated glutathione (GSH) conjugation and by epoxide hydrolase (EH)-mediated hydrolysis (23). Recent in vitro studies have demonstrated that the GST and EH activities per mg of tissue are higher in rat nasal mucosa than in lung and liver (23). GSH measurements done in the same rats used in the present study revealed a concentration-dependent GSH depletion being most pronounced in the nasal tissue at the highest exposure concentration (24). In the liver, the GSH concentration following a 20 day exposure to 500 p.p.m. PO was much lower than that in rats exposed to 300 p.p.m., suggesting reduced detoxication of PO via GST. Blood concentrations (unpublished data) and hemoglobin adducts (25) of PO were also measured in these animals, with both exhibiting a sublinear response after 20 days exposure to 500 p.p.m. PO. When liver 7-HPG and hydroxypropylvaline adducts were plotted against blood PO concentration, a linear response was evident for both. Thus, it appears that the sublinear response in 7-HPG accumulation in liver is secondary to saturation of systemic detoxication following 20 days exposure to 500 p.p.m. PO. The lack of a sublinear response in lung and NRE reflects the direct exposure to PO in the air. The reduction in the ratio of NRE:liver 7-HPG at 20 days of exposure reflects saturation of detoxication and its resultant increase in hepatic 7-HPG, rather than a decrease in NRE alkylation.
DNA repair by methylpurine DNA glycosylase is thought to be the principal pathway for glycosylase-mediated removal of N7-alkylguanines from DNA. In vivo studies (21) demonstrated a half-life of 57 days for 7-HPG in rat tissues. This value compares well with the half-life of 7-HPG (5 days) obtained in vitro (20) suggesting that 7-HPG is lost from DNA primarily by chemical depurination, rather than by base excision repair. It has been speculated that N7-alkylguanine adducts are promutagenic due to an increase in spontaneous depurination of N7-alkylated bases from DNA leading to formation of abasic sites. A highly sensitive slot blot method for the measurement of apurinic/apyrimidinic (AP) sites was used to measure AP sites in tissues (NRE, lung, liver and testis) of control and exposed (500 p.p.m. for 20 days) male F344 rats (21). There was clear evidence of 7-HPG accumulation in DNA in all tissues under the aforementioned exposure conditions. The number of abasic sites was not increased in NRE, in spite of a 17-fold increase in the number of depurinations compared with that of normal DNA, suggesting that DNA repair of AP sites is highly efficient and that AP sites are not likely to be a source of PO mutations.
PO alkylation of other DNA bases may be responsible for the low mutagenic potential of PO. Other DNA adducts formed from the reaction of PO with DNA are N3-(2-hydroxypropyl) adenine, N3-(2-hydroxypropyl)deoxyuridine (3-HPdUrd), N6-(2-hydroxypropyl)deoxyadenosine (N6-HPdAdo), N1-(2-hydroxypropyl)deoxyadenosine (18, 19, 26, 27) and possibly N1-(2-hydroxypropyl)deoxyinosine.
N3-(2-hydroxypropyl)adenine is formed in much lower amounts than 7-HPG and is also lost rapidly by chemical depurination. The lack of an increase in AP sites confirms that this is not a likely mechanism for mutagenesis. N6-HPdAdo results from the rearrangement (Dimroth rearrangement) of N1-(2-hydroxypropyl)deoxyadenosine, whereas N1-(2-hydroxypropyl)deoxyinosine can form by the deamination of this initial adduct. Recent studies suggested that N6-HPdAdo is not efficiently repaired and thus, it can accumulate in DNA (19,27). The amount of N6-HPdAdo (measured after Dimroth rearrangement) in NRE after exposure of rats to 500 p.p.m. PO for 20 days was 2% the concentration of 7-HPG adducts in the same tissue. While the N6-HPdAdo adduct occupies a WatsonCrick hydrogen bond position, recent studies on the related adduct of the butadiene monoepoxide demonstrated that it was only weakly mutagenic (28).
No similar studies have been done on N1-(2-hydroxypropyl)deoxyadenosine or N1-(2-hydroxypropyl)deoxyinosine. Future studies should aim to investigate the potential role of these adducts in PO carcinogenesis.
3-HPdUrd results from the hydrolytic deamination of N3-(2-hydroxypropyl)deoxycytidine. Alkylation at N3 of cytosine followed by hydrolytic deamination and formation of the uracil derivative could lead to CT transitions and C
A transversions (2931). The amount of 3-HPdUrd in the NRE of rats exposed to 500 p.p.m. PO for 20 days was 0.02% of the concentration of 7-HPG (19). Results from in vitro DNA repair studies and in vivo molecular dosimetry (19) suggest that enzymatic repair of N3-(2-hydroxypropyl)deoxycytidine occurs in vivo prior to conversion to the more stable 3-HPdUrd adduct. In vivo exposureresponse studies can provide information on the importance of N3-(2-hydroxypropyl)deoxyuridine accumulation to tumor formation.
The linear exposureresponse for 7-HPG accumulation in NRE cannot explain the tumor formation threshold observed at exposures below 300 p.p.m. in the rodent bioassay (Figure 3). Interestingly, increases in tissue inflammation (rhinitis) and cell proliferation were observed in the nasal passages of rodents exposed to concentrations of PO (300 p.p.m.) that were carcinogenic in the bioassays (15,16). Nasal tissue damage and increases in nasal respiratory cell proliferation have also been observed after exposure of rats to PO by the inhalation route for 1 or 4 weeks (32). In companion studies of the present research, significant increases in cell proliferation were observed only at high concentrations (
300 p.p.m.) in the nasal respiratory epithelium of rats exposed to PO (0, 5, 25, 50, 300 or 500 p.p.m.) for 3 or 20 days (unpublished data). 7-HPG adducts were quantifiable in the nasal respiratory epithelium at concentrations (550 p.p.m.) that were non-carcinogenic in rodent bioassays. The exposure concentrations below 300 p.p.m. PO did not induce nasal tissue damage or increases in nasal respiratory cell proliferation in short-term inhalation exposure studies (32). These observations suggest that the formation and accumulation of 7-HPG adducts in the nasal mucosa are not a sufficient factor in PO carcinogenesis and that PO-induced cell proliferation, that is only present at high concentrations of PO (
300 p.p.m.), may be a critical factor for tumorigenesis in this tissue.
The linear exposureresponse for 7-HPG accumulation in NRE would support the use of the linearized multistage model for PO cancer risk. However, cell proliferation and DNA repair findings suggest that the accumulation of this adduct does not play a critical role in PO carcinogenesis. In addition, the amount of 7-HPG in NRE at the lowest concentration tested (5 p.p.m.) under steady-state conditions was comparable with the reported amount of other endogenously formed adducts (2 per 106 normal nucleotides) in humans and unexposed animals (33,34). If we assume a linear response in 7-HPG accumulation at levels relevant to workplace exposure regulations we will obtain very low amounts of 7-HPG in NRE that are unlikely to destabilize the normal level of background mutations in rodents. Estimates of PO cancer risk at low levels obtained by extrapolation of tumor data do not take into account these findings.
Data on PO DNA adduct molecular dosimetry and DNA replication in target and non-target tissues for carcinogenesis along with data on toxicokinetic parameters such as detoxication, distribution and DNA repair, should improve the accuracy of current PO cancer risk assessments.
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Notes |
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Acknowledgments |
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References |
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