1 Curriculum in Toxicology and
2 Department of Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA and
3 Institute of Toxicology, GSF National Research Center for Environment and Health, D-85764 Neuherberg, Germany
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Abstract |
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Abbreviations: AP, apurinic/apyrimidinic; ARP, aldehyde-reactive probe; ddH2O, doubly distilled water; EO, ethylene oxide; GC-HRMS, gas chromatographyhigh resolution mass spectrometry; Hb, hemoglobin; MMS, methylmethanesulfonate; 7-HEG, N7-(hydroxyethyl)guanine; N6-HPdAdo, N6-(2-hydroxypropyl)deoxyadenosine; 1-HPdAdo, N1-(2-hydroxypropyl)deoxyadenosine; 3-HPdCyd, N3-(2-hydroxypropyl)deoxycytidine; 3-HPdUrd, N3-(2-hydroxypropyl)deoxyuridine; 7-HPG, N7-(2-hydroxypropyl)guanine; 7-MG, N7-methylguanine; N-DL-HPVal-Leu-anilide, N-DL-2-hydroxypropyl-Val-Leu-anilide; N-HPVal, N-(2-hydroxypropyl)valine; PBS, phosphatebuffered saline; PO, propylene oxide.
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Introduction |
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Long-term carcinogenicity studies of PO demonstrated that the formation of tumors occurred mainly at the entry site or site of contact in exposed rodents. Intragastric administration of PO to SpragueDawley rats resulted in an increased incidence of squamous cell carcinomas of the forestomach in a dose-dependent manner (15). Subcutaneous injection of PO to female NMRI mice led to the appearance of tumors (sarcomas) at the injection site in the high dose groups (16). Long-term exposure of male and female F344 rats to PO by the inhalation route resulted in tumor formation (papillary adenomas, 35%) in the nasal passages of animals in the high exposure groups (300 p.p.m.) (1719). Kuper et al. (20) evaluated the carcinogenic effects of PO in Wistar rats following inhalation exposure. One squamous cell carcinoma of the nose was found in a 30 p.p.m. male (1/61) and another in a 300 p.p.m. male (1/63). None were present in females of any group or in the male controls. In mice, exposure to PO (400 p.p.m.) by the inhalation route resulted in the formation of hemangiomas (8%), hemangiosarcomas (7%) and adenocarcinomas (2%) within the nasal passages (18,19). The formation of tumors at the entry site or site of contact suggests that the route of administration affects the distribution of PO in the tissues and determines which organs are going to be exposed to higher concentrations of the compound and are, therefore, more likely to show a cytotoxic response.
If the route of administration affects the distribution of PO among tissues, then accumulation of DNA damage (e.g. DNA adducts) will also depend on exposure route. PO directly reacts with DNA to form mainly the N7-(2-hydroxypropyl)guanine (7-HPG) adduct (21). Exposure of rats to [14C]PO by i.p. injection or inhalation (22) showed an equal distribution of 7-HPG between liver and lung in rodents exposed to the compound i.p. However, a greater accumulation of DNA adducts was observed in the lung (0.13 pmol/µmol guanine) compared with liver (0.03 pmol/µmol guanine) after inhalation exposure. The authors concluded that this difference was probably due to more efficient DNA repair in the liver. Then again, it is likely that the lung received greater exposure than the liver when the inhalation route was employed. Published preliminary results on DNA alkylation in liver and nasal tissues of male rats exposed to PO by the inhalation route demonstrated a marked difference in alkylation between tissues with a greater level of alkylation in the nose (7).
While 7-HPG is not considered to be a pro-mutagenic DNA adduct, it is known that an increase in the number of N7-alkylguanine adducts increases the rate of depurination in DNA and it has been speculated that this leads to the formation of abasic sites that can be mutagenic. A highly sensitive assay for the measurement of spontaneous and chemically induced depurination in DNA has been developed in our laboratory (23) and used for the measurement of endogenous apurinic/apyrimidinic (AP) sites in tissues (24). In this study, we utilized this method to measure the number of AP sites in nasal respiratory epithelium, lung, liver and testis of control and PO-exposed rats (500 p.p.m. for 20 days).
PO also reacts directly with hemoglobin (Hb). The primary targets for alkylation of Hb by PO are cysteine, histidine and the N-terminal valine (1,25,26). Hemoglobin adduct measurements have been used for biological monitoring of exposure to alkylating compounds (2,26).
The objectives of this research were to measure the molecular dose and distribution of 7-HPG in tissues of rats exposed to PO by the inhalation route, to evaluate the persistence of 7-HPG in DNA, to investigate whether accumulation of 7-HPG increases the number of abasic sites and to quantitate valine alkylation in Hb in the same group of exposed animals. The long-term goals of this research are to better understand the mechanism of PO carcinogenesis and to use molecular dosimetry data to improve current risk assessment for PO.
This project is part of a collaborative effort between research groups to develop sensitive and specific methods for the measurement of PO DNA and Hb adducts. The independently developed methods provide the opportunity for cross-validation of the data. In this paper we report the use of gas chromatographyhigh resolution mass spectrometry (GC-HRMS) for the quantitation of 7-HPG in DNA and of N-(2-hydroxypropyl)valine (N-HPVal) in globin. 32P-post-labeling and gas chromatographytandem mass spectrometry methods have been utilized for the measurement of 7-HPG and N-HPVal, respectively, in the same group of exposed animals (27,28).
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Materials and methods |
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Animal exposures
Forty male F344 rats divided into four exposure chambers were exposed at GSF, Neuherberg, Germany, according to Filser et al. (29) for 4 weeks (6 h/day, 5 days/week) to PO vapor, which was generated simultaneously using one vaporization system. The animals, animal maintenance and exposure conditions are described in more detail elsewhere (7). The concentrations of PO vapor determined in the exposure chambers were calculated as mean p.p.m. per chamber and ranged between 502 and 503 p.p.m. with a maximum coefficient of variation of 2.4%. In the two control chambers, with a total of 20 control animals, no PO was detected.
Tissue and blood collection
Exposed and control animals were killed with carbon dioxide within 7 h after the end of the final exposure period or 3 days after the final exposure. Liver, lung, spleen and testis of each animal were harvested and placed separately in small vials. The tubes were immediately frozen in liquid nitrogen and stored at 80°C. Nasal respiratory and olfactory tissues were either processed on the same day after cooling the heads to 4°C or the heads were frozen at 80°C and processed on the following day immediately after thawing. Nasal tissues from animals killed 3 days post-exposure were processed on the same day after the heads were cooled to 4°C. Separation and preparation of respiratory and olfactory nasal tissues were conducted according to Casanova-Schmitz et al. (30). Separated nasal tissue samples of each animal were placed in tared ice-cold Eppendorf cups containing 0.5 ml of 20 mM TrisHCl, 1 mM MgCl2, pH 8.0, buffer and the tissue weight was determined. The cups were frozen in liquid nitrogen and stored at 80°C until shipment, for which dry ice was used. Blood samples (4.56.5 ml) were obtained by puncture of the aorta using disposable syringes (Braun, Melsungen, Germany). The syringes were heparinized with Liquemin N 25000 (Hoffmann-La Roche, Grenzach-Wyhlen, Germany). Lymphocytes were isolated from blood using Ficoll-Paque (Pharmacia, Uppsala, Sweden). The isolated cells were stored at 80°C. The erythrocytes were washed with cold saline and centrifuged (three times). The packed erythrocytes were stored at 80°C until shipment, for which dry ice was used.
Samples available for DNA adduct analysis
DNA samples from tissues (nasal respiratory, nasal olfactory, lung, spleen, liver and testis) and cells (lymphocytes) collected from individual animals were obtained from Dr Dan Segerbäck (Center for Nutrition and Toxicology, Karolinska Institute, Stockholm, Sweden). DNA was extracted from cellular nuclei of tissue homogenates and lymphocytes as described by Gupta (31). Ethanol-precipitated DNA was dissolved in water and RNA contamination (<2%) checked by HPLC after digestion to nucleosides. The values of the ratio A260:A280 were all between 1.6 and 1.9. These samples were utilized for quantitation of 7-HPG by GC-HRMS.
Samples available for Hb adduct analysis
Globin samples from red blood cells of exposed and control rats were isolated by Dr Siv Osterman-Golkar (Stockholm University Stockholm, Sweden). Globin was precipitated according to Mowrer et al. (32).
Standards
The 7-HPG and 7-HP[13C4]G standards were prepared as described previously (7). N-DL-2-hydroxypropyl-Val-Leu-anilide (N-DL-HPVal-Leu-anilide) (>95%) was purchased from Bachem Bioscience (King of Prussia, PA). The internal standard globin (~3 nmol N-[2H6]HPVal/mg globin) was kindly provided by Dr Siv Osterman-Golkar. The concentration reflects the total for both diastereomers. The internal standard globin was prepared by incubation of human erythrocytes with [2H6]PO (28).
Preparation and quantitation of the N-DL-HPVal-Leu-anilide standard solution
Based on weight, a stock solution of N-DL-HPVal-Leu-anilide was prepared in doubly distilled water (ddH2O) (0.402 mg/ml) and stored at 80°C. An aliquot of this solution was freeze dried and quantitated by NMR using t-butanol as the reference standard. A deviation of 2.7% was found between the concentration of N-DL-HPVal-Leu-anilide based on weight and that obtained by NMR analysis. The concentration calculated by NMR was used throughout the analysis for the preparation of diluted standard solutions.
Analysis of 7-HPG by GC-HRMS
The method used for the analysis of 7-HPG has been described elsewhere (7). Briefly, 7-HPG was released from DNA by neutral thermal hydrolysis (100°C, 20 min in H2O) converted to N7-(2-hydroxypropyl)xanthine, derivatized twice with pentafluorobenzyl bromide and analyzed by GC-HRMS. 7-HP[13C4]G was used as an internal standard and was added to the DNA sample solutions prior to hydrolysis. Samples were dissolved in toluene (50 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 the internal standard. 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 level 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 7-HP[13C4]G added. The calibration curve standard solutions were analyzed as described above. One additional sample (method blank) to which no internal standard or analyte standard was added was analyzed at the same time. 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 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 = 0.9996) with a coefficient of variability of <10% from between four and six injections. Guanine concentrations were determined from aliquots obtained from each DNA sample prior to addition of the internal standard. Guanine was released from DNA by mild acid hydrolysis (70°C, 30 min in 0.1 N HCl) and separated from DNA hydrolysates by HPLC. A strong cation exchange column (250x4.6 mm i.d., Partisil SCX, 10 µm particle size; Alltech Associates, Deerfield, IL) was used with isocratic elution (100 mM ammonium formate, 10% methanol, pH 3.2) at a flow rate of 2.0 ml/min. The area of the peak corresponding to guanine was determined from UV absorption at 254 nm and compared with a standard curve. The amount of guanine in the sample was estimated by multiplying the amount of guanine obtained in the aliquot by a conversion factor which led to the total amount of guanine in the amount of sample used for GC-HRMS analysis.
Analysis of 7-HPG in [14C]PO-modified salmon testis DNA: method validation
The level of 7-HPG in a sample of salmon testis DNA modified in vitro with 14C-labeled PO (43.3 mCi/mol) (kindly provided by Dr Dan Segerbäck) was determined by GC-HRMS, HPLC-UV absorbance and radiochromatography in order to validate the mass spectrometry method. For radiochromatography, DNA samples (0.5 mg) in solution were heated for 30 min at 100°C to release 7-HPG followed by cold acid precipitation (33). Adducts in the supernatant fractions were separated by SCX-HPLC at a flow rate of 1.5 ml/min using gradient elution from 100% 0.02 M ammonium formate, 4% methanol, pH 4.0, to 100% 0.2 M ammonium formate, 4% methanol, pH 4.0, over 30 min. Eluate from the column was collected every 30 s and radioactivity in each fraction measured by liquid scintillation counting. The level of 7-HPG in these samples was also determined by UV absorption of the corresponding peak at 280 nm. The peak area was compared with a standard calibration curve. The concentration of guanine was measured in aliquots obtained from the sample before hydrolysis and analyzed as described above (Analysis of 7-HPG by GC-HRMS). For GC-HRMS analysis, DNA samples (0.3 µg) were hydrolyzed (100°C, 30 min) and derivatized as described above (Analysis of 7-HPG by GC-HRMS). The concentration of guanine was obtained from aliquots taken from the sample prior to addition of the internal standard. Dilutions of the in vitro modified salmon testis DNA sample (100x, 300x and 1000x) were also prepared and analyzed for 7-HPG concentration. The actual dilutions were 99x, 301x and 1082x. The concentration of 7-HPG in the in vitro modified salmon testis DNA obtained by GC-HRMS was used as the reference value from which expected levels of 7-HPG in the dilutions were calculated.
Analysis of N-terminal valine adducts by GC-HRMS
All the glassware utilized in the analysis of N-HPVal in globin was silanized in a solution of 5% dimethyldichlorosilane in pentane, rinsed twice in methanol (reagent grade) and air dried. Globin samples were derivatized according to the N-alkyl Edman degradation method (32). Samples of 58 mg globin from exposed rats and 0.3 mg internal standard ([2H6]PO-treated human globin) were dissolved in 1.5 ml formamide in silanized borosilicate glass culture tubes (13x100 mm) followed by addition of 40 µl of 1 M NaOH and 10 µl of pentafluorophenyl isothiocyanate. The sample tubes were placed in a multi-mixer (Lab-line Instruments, IL) and left to react at room temperature overnight (1518 h) followed by a 1.5 h incubation period at 45°C. The sample solutions were transferred to Centricon-30 cartridges (Amicon, Beverley, MA) and centrifuged at 6300 r.p.m. for ~24 h. The filtrates were extracted three times with 3 ml of ethyl ether and the ether fractions dried under a stream of N2 at 40°C. The residues were dissolved in toluene (2 ml) and washed sequentially with 2x2 ml of ddH2O, 2x2 ml of freshly prepared 0.1 M Na2CO3 and 3x2 ml of ddH2O. The toluene layer was dried under a stream of N2 at 60°C. The dried residues were dissolved in toluene (50 µl) and analyzed by GC-HRMS by selected ion monitoring at m/z 362.0712 for N-HPVal and m/z 368.1083 for N-[2H6]HPVal. The calibration curve samples were prepared by the addition of different amounts of N-HPVal-Leu-anilide (0, 100, 250, 500, 600, 800 and 1000 pmol) to 10 mg globin solutions (globin from control rats) followed by addition of 0.3 mg of internal standard ([2H6]PO globin). The calibration curve samples were derivatized as described above. The peak area ratio N-HPVal:N-[2H6]HPVal was used for quantitation of N-HPVal using standard calibration curves. A plot of N-HPVal:N-[2H6]HPVal ratio as a function of N-HPVal-Leu-anilide (pmol) showed a linear relationship with N-HPVal-Leu-anilide concentration (r2 = 0.992) with a coefficient of variability of <10% from 23 injections.
GC-HRMS analysis of DNA adducts
GC-HRMS 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 10K. Direct injections using a press-fit liner were made onto a DB-5 fused silica capillary column (15 mx0.32 mm). Helium head pressure was 10 p.s.i. The ion source temperature was 250°C. Methane (3x105 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 50°C/min to 300°C.
GC-HRMS analysis of Hb adducts
The same instrument as used for DNA adduct analysis was utilized for Hb adduct analysis. The GC separation was performed on an Alltech EC-5 column (30 mx0.32 mm, 1.0 µm film thickness) with 10 p.s.i. He head pressure and 220°C injector temperature. The source temperature was set at 175°C but reached 250°C during N-HPVal analysis. The temperature program in the GC was 1 min at 100°C, 20°C/min to 240°C, followed by 10°C/min to 275°C. Perfluorokerosene was used for tuning and calibration of the mass spectrometer.
DNA extraction for AP site measurement
DNA from nasal respiratory epithelium, lung, liver and testis of control and exposed rats was extracted by a procedure slightly modified from the method reported by Nakamura et al. (23). Briefly, frozen lung, liver and testis tissues were thawed and homogenized in 1x phosphate-buffered saline (PBS) with a Tehran homogenizer (Wheaton Instruments, Millville, NJ). After centrifugation, the nuclear pellets were incubated in cell lysis buffer (Applied Biosystems) overnight at 4°C with proteinase K (Applied Biosystems). The buffer solution was then extracted twice with a mixture of 70:20:10 phenol/chloroform/water (Applied Biosystems) and once with Sevag (24:1 chloroform/isopentyl alcohol), followed by ethanol precipitation. The extracted DNA was incubated in 1x PBS with a mixture of RNase T1 (Roche Biochemicals) and RNase A (Sigma Chemical Co.). After DNA precipitation with cold ethanol, the DNA pellet was resuspended in ddH2O and DNA concentration measured by absorbance at 260 nm. For nasal respiratory epithelium the tissue was suspended in cell lysis buffer without prior homogenization in PBS. The rest of the DNA isolation procedure was as for the other tissues.
Aldehyde-reactive probe (ARP) slot blot assay
The AP site assay was performed by a procedure slightly modified from the method reported by Nakamura et al. (23). Briefly, 58 µg DNA in 150 µl of PBS were incubated with 1 mM ARP (Dojindo Molecular Technologies, Gaithersburg, MD) at 37°C for 10 min. After precipitation using cold ethanol, DNA was resuspended in TrisEDTA buffer. The DNA concentration was measured with a spectrophotometer and the DNA solution was then prepared at 1.1 or 0.55 µg/220 µl TrisEDTA buffer. Heat-denatured DNA was then immobilized on a nitrocellulose membrane. The nitrocellulose membrane was soaked in 5x SSC and then baked in a vacuum oven at 80°C for 30 min. The membrane was pre-incubated with 20 ml of TrisHCl buffer containing bovine serum albumin at room temperature for 2030 min. The nitrocellulose membrane was then incubated in the same solution, after addition of streptavidin-conjugated horseradish peroxidase (BioGenex), at room temperature for 45 min. After the nitrocellulose membrane was rinsed, the enzymatic activity on the membrane was visualized by use of enhanced chemiluminescence reagents (Amersham Corp.). The nitrocellulose filter was then exposed to imaging film and the developed film was analyzed using an Ultrascan XL scanning densitometer.
Statistical analysis
Calibration curves were obtained by linear regression analysis. Statistical analysis of the difference between AP site numbers between tissues of control and exposed rats was performed with the Student t-test for unpaired samples. Statistics software (Microsoft Excel 97) was used to calculate the correlation coefficient between the GC-HRMS and 32P-post-labeling methodologies for the quantitation of 7-HPG adducts.
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Results |
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In order to validate the accuracy of the GC-HRMS method, quantification of 7-HPG in an in vitro [14C]PO-modified salmon testis DNA sample was performed by three different methods: HPLC-UV, 14C radiochromatography and GC-HRMS. The amount of 7-HPG obtained by radiochromatography was calculated from the specific activity of the [14C]PO (43.3 mCi/mol) and the radioactivity counts obtained for the peak corresponding to 7-HPG in each sample. The results are shown in Table I. Analysis of 7-HPG by GC-HRMS and radiochromatography led to comparable results. The concentration obtained by HPLC with UV detection was greater than with the other two methods. This difference is probably due to the low specificity of the UV method compared with the other two methods. Measurements of 7-HPG in serial dilutions of the in vitro [14C]PO-modified DNA sample are found in Table II
. The greatest deviation (15%) from the expected value was observed at the low concentration level. The concentrations of 7-HPG in the first two dilutions (99x and 301x) were in agreement with the expected value (3% deviation). These dilutions will be used in future analyses for quality control for the accuracy of the method.
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Measurement of AP sites in tissues of control and PO-exposed (500 p.p.m.) rats
The number of AP sites measured in nasal respiratory epithelium, lung, liver and testis of control (0 p.p.m.) and PO-exposed (500 p.p.m.) rats are found in Table IV. There was no significant difference in the number of AP sites between the exposed and control groups for any of the tissues.
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Discussion |
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Analysis of tissues of rats exposed to 500 p.p.m. PO (6 h/day, 5 days/week for 4 weeks) demonstrated that 7-HPG was quantifiable in all tissues examined by GC-HRMS. The highest level of 7-HPG was found in nasal respiratory tissue, which was the target tissue for tumor formation in long-term inhalation studies in rodents. The amount of 7-HPG in nasal respiratory tissue was twice the amount in olfactory tissue, probably due to a higher concentration and flow of PO in the anterior portion of the nasal passages. A greater concentration of PO in the anterior nasal passage correlates with previous findings where a greater extent of tissue damage was seen in the respiratory tissue of rats exposed to high concentrations of PO for 4 weeks (34).
Accumulation of 7-HPG in liver, lymphocytes and spleen was similar, suggesting a uniform distribution of PO among systemic organs in accordance with the similar values of partition coefficients for the tissue:air distribution of PO measured for various tissues (35). The concentration of 7-HPG in lung was about nine times lower than in the nose and about three times greater than the concentration in liver, suggesting direct and systemic exposure of lung tissue to PO during inhalation. However, 7-HPG adduct levels in testis were ~2-fold lower than the other systemic tissue levels. The lower amount of DNA adducts in testis could be the result of a smaller concentration of the compound reaching this tissue due to low blood flow and limited diffusion as a result of the distance from blood vessels to developing spermatids.
The values reported in this paper for nasal respiratory epithelium, nasal olfactory epithelium and liver are lower than previously reported (7). The 7-HPG measurements in the nasal and liver tissue for the set of samples reported in this paper were done using the same standard solution as used for the analysis of 7-HPG in the rest of the tissues. The ratio between 7-HPG and 7-HP[13C4]G for dilutions from the stock solutions was obtained by electrospray mass spectrometry analysis and it was within ±5% from the expected value. The levels reported previously (7) were based on a different standard calibration solution. The standard solutions used previously were stored in plastic vials at 20°C. We found that the solutions were not stable when stored in plastic vials at 20°C. This may have decreased the concentration of the previous standard solution, leading to a smaller analyte to internal standard peak area ratio. The best alternative is to store the solutions in glass vials at 80°C.
Analysis of 7-HPG in tissues of animals killed 3 days after cessation of PO exposure demonstrated a similar percentage of adduct disappearance in all tissues, which suggests that no major differences in DNA repair were present between tissues. A half-life of 57 days was estimated based on the GC-HRMS results assuming a first order rate of elimination of 7-HPG from DNA. This value compares well with the half-life of 7-HPG (5 days) obtained in vitro (27). These results suggest that 7-HPG is lost from DNA primarily by chemical depurination, rather than by enzymatic repair, when rodents are exposed to high concentrations of PO.
However, the ionic strength of the buffer used for DNA depurination studies is known to affect the depurination rate. Schumaker (36) demonstrated a slow but progressive breakdown of DNA after heating DNA solutions of various ionic strengths (H2O or 0.001, 0.01 or 0.1 M NaCl) at 100°C for 14 h. The rate of degradation was slower in 0.1 M NaCl (2x when compared with H2O). Similar studies (37,38) have shown that depurination of native DNA progresses more rapidly in buffers of low ionic strength. Salt concentrations of 100150 mM are believed to resemble intracellular salt concentrations (39). According to this, the buffer used for the 7-HPG depurination studies (20 mM TrisHCl, pH 7.4) was not representative of intracellular ionic conditions. If this is the case, the rate of spontaneous depurination of 7-HPG obtained in vitro is faster than expected under physiological conditions. A slower rate of depurination of 7-HPG in vitro would suggest a greater role for in vivo DNA repair.
The concentration of 7-HPG in lung and spleen from rats exposed to 500 p.p.m. PO was about half the number of N7-(2-hydroxyethyl)guanine (7-HEG) adducts found in lung and spleen of F344 rats exposed to 100 p.p.m. EO for the same period of time (40). The difference in alkylation between epoxides could be partially attributed to different rates of alkylation with nucleophilic sites in DNA and Hb. The second order rate constants [1 (mol DNA nucleotide h)1] for the reaction of EO and PO with the N7 position in guanine (in human blood DNA) in vitro are 16x103 and 5.1x103, respectively (41). The corresponding second order rate constants for the reaction of EO and PO with N-terminal valine in human blood in vitro are 2.7 and 1.0. A slower rate of reaction of PO with electron-rich groups in macromolecules accompanied by better detoxification of PO could account for the differences in DNA and Hb adduct formation in mammalian systems in vivo.
The DNA samples analyzed for this report were also analyzed by 32P-post-labeling (27) as part of a collaborative effort for cross-validation of DNA adduct quantitation. The results using GC-HRMS reported here demonstrated an excellent linear correlation with the results from 32P-post-labeling, with correlation coefficients of 0.99 for the immediate post-exposure and the 3-day post-exposure tissues and absolute values within 20% of those obtained by post-labeling.
Unlike 7-HEG, no endogenous formation of 7-HPG was shown by GC-HRMS analysis of DNA from control rats. The limit of detection of the method is 50 fmol per sample based on measurement of standards. This method is not limited by the amount of DNA, which enabled us to increase the amount of DNA (up to 400 µg) used in the analysis when necessary. Analysis of DNA from control rats by 32P-post-labeling also did not show evidence of endogenous formation of 7-HPG. If endogenously formed 7-HPG was present in DNA from control animals, the amount was below the limit of detection of both techniques.
It is not clear which PO-induced DNA lesions are responsible for the mutagenic action of PO. It has been speculated that N7-alkylguanine adducts are potentially mutagenic due to an increase in the rate of depurination of alkylated bases leading to the formation of abasic sites. An increase in the formation of abasic sites may cause miscoding if DNA replication occurs before repair of the damage takes place. A highly sensitive slot blot method for the measurement of AP sites has been developed in our laboratory and used for measurement of the steady-state number of AP sites in rat tissues, as well as human liver (23,24). This method was used to measure AP sites in tissues (nasal respiratory epithelium, lung, liver and testis) of control (0 p.p.m.) and exposed male F344 rats (500 p.p.m. PO for 20 days). While there was clear evidence that under these exposure conditions there was accumulation of 7-HPG adducts in DNA from these tissues, AP site measurements demonstrated that there were no significant differences in AP site numbers between control and exposed groups among the tissues analyzed. These data suggest that there is efficient repair of AP sites in DNA of nasal respiratory epithelium, lung, liver and testis under the described exposure conditions.
Data published by Nakamura et al. (23) showed a significant increase in AP site number in DNA of H2E1 cells exposed to 0.5 and 1.5 mM methylmethanesulfonate (MMS) for 24 h. No increases were seen at lower concentrations. The number of AP sites in cells exposed to 0.5 mM MMS was twice as high as in control cells. The corresponding N7-methylguanine (N7-MG) adduct measurement at 0.5 mM MMS was ~950 pmol N7-MG/µmol guanine (unpublished data). The levels of N7-MG adducts in cells exposed to 0.5 mM MMS for 24 h, where significant increases in AP site numbers were seen, were only 36% greater than those measured in nasal tissue of rats exposed to 500 p.p.m. PO by inhalation for 20 days. However, results of AP site measurements demonstrated that there was no significant increase in AP site numbers in the nose, even when the number of adducts was similar to the amount of N7-MG in cells exposed to MMS concentrations that increased AP site number in DNA. It is possible that induction of DNA repair occurred during the 20 day exposure period, while no induction occurred in the single exposure cell culture model.
The absence of increased numbers of AP sites due to exposure to a high concentration of PO correlates with the weak carcinogenicity (35% tumor incidence) found in rodents exposed to PO by the inhalation route for 2 years (1719) and the lack of chromosomal damage in lymphocytes of monkeys exposed to 300 p.p.m. for >1 year (14). The low tumor incidence in the nasal passages at high levels of PO exposure could be the result of fixation of minor lesions during cell proliferation. Increases in cell proliferation were observed in the nasal passages of rodents exposed to high concentrations of PO that were positive in the cancer bioassays (1719). Increases in cell proliferation have also been observed after 1 and 4 weeks exposure of rats to PO by the inhalation route (34).
Alkylation of other DNA bases may also play a role in PO mutagenesis by directly altering normal base pairing. Other products formed from the in vitro reaction of PO with DNA are N3-(2-hydroxypropyl)adenine, N3-(2-hydroxypropyl) deoxyuridine (3-HPdUrd) and N6-(2-hydroxypropyl)deoxyadenosine (N6-HPdAdo) (21). 3-HPdUrd results from the hydrolytic deamination of N3-(2-hydroxypropyl)deoxycytidine (3-HPdCyd). Studies by Snow et al. (42) indicated that DNA adducts induced by PO at template cytosine residues are mutagenic in Escherichia coli. Also, site-modified replication studies with the analogous EO lesion, N3-(2-hydroxyethyl)deoxyuridine, demonstrated that in the absence of proofreading this lesion could be mutagenically bypassed and that dA and dT could be incorporated opposite the lesion (4344). According to these results, if this adduct is not repaired it can lead to CT transitions and C
A transversions. A small amount of 3-HPdUrd (0.02% the concentration of 7-HPG) was found in nasal respiratory epithelium from the same group of rodents exposed to 500 p.p.m. PO for 20 days described in this study as analyzed by 32P-post-labeling (45). The relative amount of 3-HPdUrd found in vivo was considerably lower (100 times) than the concentration of 3-HPdUrd found in in vitro PO-modified DNA. Incubation of PO-modified DNA with a protein extract from mammalian cells led to the release of 3-HPdCyd (~62%), but not of 3-HPdUrd, from DNA (45). Results from the in vitro DNA repair studies along with in vivo dosimetry studies suggest enzymatic repair of 3-HPdCyd from DNA prior to conversion to the more stable 3-HPdUrd.
N6-HPdAdo is formed after rearrangement of N1-(2-hydroxy-propyl)deoxyadenosine (1-HPdAdo). The amount of N6-HPdAdo (measured after rearrangement from 1-HPdAdo) in nasal respiratory epithelium from rats after 20 days exposure to 500 p.p.m. PO was 2% the concentration of 7-HPG (45). No decrease in the concentration of N6-HPdAdo was observed in rats killed after 3 days of recovery, suggesting slow repair of this adduct. Incubation of in vitro PO-modified DNA with a protein extract from mammalian cells did not alter the concentration of 1-HPdAdo or N6-HPdAdo in DNA. This adenine adduct occupies a WatsonCrick hydrogen bond position and therefore accumulation of this adduct together with increased cell proliferation at high PO concentrations would be expected to increase the probability of developing mutations. According to in vitro studies done by Solomon et al. (21), the ratio of formation of N6-HPdAdo with respect to 7-HPG is 0.7%, which suggests accumulation of the adduct in vivo, where a ratio of 2% was observed. Solomon et al. (21) did not demonstrate formation of 1-HPdAdo in DNA in vitro. More recent studies (45) have demonstrated that N6-HPdAdo (total of N6-HPdAdo and 1-HPdAdo) formed at a level of 3.5% with respect to 7-HPG in DNA in vitro. Based on this, the in vivo studies suggest that some repair occurred. Additional research will be needed to clarify this issue.
The N-HPVal levels in globin from exposed rats reported here, quantified by GC-HRMS, were in good agreement with those reported previously by Osterman-Golkar et al. (28), for which the same set of globin samples was analyzed by gas chromatographytandem mass spectrometry. As expected, analysis of PO Hb adducts in globin of exposed rats showed no differences in adduct concentration between animals killed directly after the end of exposure and animals killed 3 days after. Propylene oxide Hb adducts can be used as systemic dosimeters of exposure, as the present study and others (22,27) have shown comparable concentrations of PO DNA adducts among rodent systemic tissues. It is important to recognize that this relationship occurs when identical daily exposures are employed. Recently, Boogaard et al. (46) demonstrated a good correlation between low airborne concentrations of EO and PO and formation of the corresponding N-terminal valine adducts in blood of occupationally exposed workers. The airborne concentrations of the epoxides were monitored by personal air monitoring devices during the entire work shift. The authors estimated the concentration of EO and PO N-terminal valine adducts that correspond to the current occupational exposure limits and suggested the use of these values as biological exposure limits for EO and PO.
It has been suggested that Hb adducts can be used as surrogates of molecular dose in DNA because of proportional reaction rates for the binding of electrophilic compounds to DNA and Hb (4749). Based on this assumption, Hb adducts are an indirect measure of the reaction of the electrophilic compound with DNA and can be used to determine the in vivo dose of direct acting carcinogens. This assumption may not apply for many alkylating compounds, including PO, where we have shown marked differences in PO DNA adduct accumulation among tissues after inhalation exposure. Such differences would be difficult to predict from Hb adduct measurements. Accumulation of DNA adducts depends not only on the reaction rates of the alkylating agent with DNA, but also on how much of the compound is deposited in the tissues under different exposure conditions, the extent of detoxification and DNA repair. Information on the relationship of DNA adduct formation and Hb adduct formation after exposure of rodents to EO over a range of doses and times have demonstrated that the relationship is tissue and species dependent and not necessarily directly proportional (40,50). Exposure route also plays an important role in the regional deposition of the compound. Previous studies (22) showed equal accumulation of DNA adducts in lung and liver after i.p. administration of PO, however, we have demonstrated that there is a large difference in DNA adduct accumulation among tissues after inhalation exposure.
The use of Hb adducts as molecular monitors of target tissue dose for PO risk assessment does not accurately predict cancer risk due to the large differences in molecular dose among tissues. A 43-fold difference in DNA adducts between nasal respiratory epithelium and testis was shown for a single molecular dose of Hb adducts. Thus, it is clear that tissue-specific data are required for improved risk assessment. Doseresponse studies on DNA and Hb adduct formation in rats after PO exposure and their relationship to biochemical processes like detoxification, DNA replication and repair will reduce the uncertainty associated with extrapolating risk to humans from data obtained in experimental animals.
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