Center for Nutrition and Toxicology, Department of Biosciences, Karolinska Institute, Novum, S-141 57 Huddinge and
1 Department of Genetic and Cellular Toxicology, Wallenberg Laboratory, Stockholm University, S-106 91 Stockholm, Sweden
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: ESI-MS, electrospray ionization mass spectrometry; 1-HP-adenine, 1-(2-hydroxypropyl)adenine; N6-HP-adenine, N6-(2-hydroxypropyl)adenine; 3-HP-cytosine, 3-(2-hydroxypropyl)cytosine; 7-HP-guanine, 7-(2-hydroxypropyl)guanine; 3-HP-uracil, 3-(2-hydroxypropyl)uracil; PO, propylene oxide.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several studies have shown that PO is genotoxic and a rodent carcinogen (13). The mutagenic activity of PO has been evaluated in bacterial and mammalian systems (47). At high doses PO induced sex-linked recessive lethal mutations in Drosophila and the mutagenic potency was enhanced in repair-deficient females (3). In rodents PO induces, at high exposure levels, tumours in the forestomach after oral administration (8) and nasal tumours upon inhalation (9,10). No epidemiological data concerning cancer risks of PO are available, but based on extrapolation from experimental data it can be assumed to have at most only a weak carcinogenic potential for humans (11,12).
Alkylation of DNA bases by PO has been the subject of several in vitro studies (1317). High nucleophilic selectivity of PO as well as of other related epoxides results in predominantly ring nitrogen alkylation of DNA bases with substitution at the ß-carbon (16). In DNA treated in vitro with PO the major product was at the 7-position of guanine, followed by the 3-position of adenine, the 3-position of uracil and the N6-position of adenine (16). The formation of haemoglobin and DNA adducts of PO have been used for molecular dosimetry studies in laboratory animals (12,18,19) and 7-substituted guanine is the only specific adduct of PO so far measured in vivo (2,12,18,20).
Ring nitrogen-substituted DNA adducts are generally unstable and their biological effects may, therefore, depend on chemical transformations secondary to the initial site of DNA alkylation. Examples of such labile lesions are 7-substituted guanine as well as 3-substituted adenine, both of which spontaneously depurinate and form apurinic sites (13,21). Other examples are adducts at the 3-position of cytosine, which have been shown to deaminate to the corresponding 3-substituted uracil, and 1-substituted adenine, which are converted to N6-substituted products in a Dimroth rearrangement (16,22; Figure 1). Direct alkylation at N6 of adenine can occur, but primarily after substitution at the
-carbon of epoxides containing electron-withdrawing substituents, such as styrene oxide (23,24).
|
The aim of the present study was to investigate the presence of 1-(2-hydroxypropyl)adenine (1-HP-adenine) and 3-HP-cytosine as well as their secondary products N6-(2-hydroxypropyl)adenine (N6-HP-adenine) and 3-HP-uracil in the nasal epithelium, lung and lymphocytes of PO-exposed rats using the 32P-postlabelling method (28). The initially formed DNA adducts of PO were not expected to be chemically stable and they, or their rearrangement products, might be removed by enzymatic repair. For this reason, the stability of these adducts in DNA in vitro as well as in vivo was studied. Data from analysis of 7-(2-hydroxypropyl)guanine (7-HP-guanine) in the respiratory nasal epithelium, olfactory nasal epithelium, lung, lymphocytes, spleen, liver and testis of the same rats have been published elsewhere (20).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Reaction with nucleotides
3'- or 5'-dAMP, 3'- or 5'-dCMP and 3'- or 5'-dUMP (18 mg) were reacted with a 510 times molar excess of PO in 50 mM TrisHCl buffer, pH 7.4, at room temperature for 2472 h. The modified nucleotides were isolated by HPLC separation. The adducts were characterized by UV spectrophotometry, mass spectrometry and rearrangement experiments.
Mass spectrometric analysis
Electrospray ionization mass spectrometry (ESI-MS) was performed using a Finnigan LCQ LC/MSn system equipped with an ion trap mass analyser. The spectra were obtained in the negative ion mode. The samples, dissolved in the eluent of water/acetonitrile/NH3 (49%:50%:1%), were applied by loop injection (10 µl) into the running solvent from the syringe pump at a flow rate of 0.1 ml/min. For MS/MS experiments precurser ions were selected in the ion trap analyser and fragmented. The full scan data were acquired for 701000 m/z and for MS/MS data 70500 m/z. Electrospray voltage was 5.0 kV and capillary temperature 250°C.
For 1-substituted dAMP the ESI-MS showed a pseodomolecular ion [M-H] at m/z 388. This corresponds to a molecular weight of 390, since in the solvent used the exocyclic N6 amino group is in the imino form and the molecule is then further deprotonated in the ESI system before entering the mass analyser. The MS/MS experiment on the [M-H] ion showed products at m/z 192, which corresponds to an alkylated adenine after cleavage of the glycosylic bond, and m/z 195, which corresponds to deoxyribose with a phosphate group. For N6-substituted dAMP the same [M-H] of m/z 388 was observed with the same MS/MS fragments.
For 3-substituted dCMP, a pseuodomolecular ion [M-H] at m/z 364 was observed, which corresponds to a molecular weight of 366, because the exocyclic amino group of cytosine is in the imino form, similarly to in the case of 1-substituted dAMP. MS/MS fragments were observed at m/z 168, which was interpreted as an alkylated cytosine base, and at m/z 195, which corresponds to deoxyribose with a phosphate. For 3-substituted dUMP, a deprotonated molecular ion [M-H] was observed at m/z 365, showing a difference of 1 mass unit from the corresponding 3-substituted dCMP, as expected. The MS/MS fragment of the alkylated uracil base was detected at m/z 169 as well as the fragment of m/z 195, as above.
In vitro reaction with DNA
Two samples were prepared by reaction of DNA in vitro with PO. For one sample (PO-DNA-1), salmon testis DNA (2.2 mg/ml 50 mM TrisHCl, pH 7.4) was reacted with 300 mM PO at room temperature for 16 h. The reaction mixture was extracted twice with ethyl acetate and DNA precipitated with ethanol. For another DNA sample ([14C]PO-DNA-2), 1 mCi of 2-14C-labelled PO was dissolved in 2.5 ml of methanol. Calf thymus DNA (0.6 ml, 4 mg/ml 40 mM TrisHCl, pH 7.4) was mixed with [14C]PO (0.5 ml) and allowed to react at 37°C for 48 h. After incubation, the sample was extracted with ethyl acetate and DNA precipitated with ethanol. 7-HP-guanine and 3-HP-adenine were released from [14C]PO-DNA-2 by selective depurination (10 mM phosphate buffer, pH 6.0, 100°C, 30 min). The residual DNA was used for analysis of formed adducts utilizing 14C radiochromatography.
14C radiochromatography analysis
[14C]PO-DNA-2 was digested with micrococcal nuclease (80 mU/µg DNA in 3 mM bicine, 0.5 mM CaCl2, pH 9.0) at 37°C for 2 h, followed by incubation with spleen phosphodiesterase (1.6 mU/µg DNA in 20 mM ammonium acetate, pH 5.0) for another 2 h and the formed nucleotides separated by HPLC (system B). Fractions of 1.4 ml (2 min) were collected from the column and radioactivity was measured by liquid scintillation counting. Concentrations of PO adducts were calculated from the specific activity of [14C]PO.
Animal treatments and preparation of DNA
Male F344 rats were exposed to 500 p.p.m. PO for 4 weeks (6 h/day, 5 days/week) as described previously (2,20). The animals were killed directly after treatment or 3 days later. Nasal epithelia, lungs and lymphocytes were frozen at 80°C. DNA was isolated as described previously (29). The DNA concentration was determined from the UV absorption at 260 nm. RNA contamination of enzymatically digested DNA (30) was analysed by HPLC with UV detection and found to be <1%.
32P-postlabelling
PO adducts were analysed by 32P-postlabelling as described by Randerath et al. (28). DNA (110 µg) was hydrolysed with prostatic acid phosphatase (20 mU/µg DNA) and nuclease P1 (0.2 µg/µg DNA) at pH 5.2. After incubation (37°C, 45 min) cold ethanol (100 µl) was added to the sample and proteins were precipitated at 20°C for 20 min. After centrifugation for 10 min the supernatant was transferred to a fresh tube and evaporated to dryness. Adducted dinucleotides were 32P-labelled with 14 µCi of [32P]ATP and 6 U of polynucleotide kinase in a total volume of 2 µl of the labelling buffer (20 mM CHES, 10 mM MgCl2, 10 mM dithiothreitol, 1 mM spermidine, pH 9.6) by incubation at 37°C for 1 h. Snake venom phosphodiesterase (0.5 mU/µg DNA) was added and the sample incubated for 40 min. Synthesized UV markers (1-HP-5'-dAMP, N6-HP-5'-dAMP, 3-HP-5'-dCMP and 3-HP-5'-dUMP) were added to all samples prior to HPLC analysis. The UV traces of these are not shown in the figures. The reason is that the presence of other UV peaks (e.g. from normal deoxyribonucleosides) together with the radioactivity peaks would complicate interpretation. The amounts of radioactivity present in detected peaks were estimated by injecting known amounts of [32P]ATP.
Recoveries of PO adducts in the postlabelling assay were estimated from analysis of [14C]PO-DNA-2 in which the level of PO adducts had been determined by 14C radiochromatography analysis (see above). A 10 times diluted sample of this DNA standard, with a known concentration of PO adducts, was labelled in parallel to each set of DNA samples from PO-treated rats and used as an external standard for correction of determined adduct levels.
Stability of 1-HP-adenine and 3-HP-cytosine in DNA
PO-DNA-1 (0.5 mg/ml) was incubated in 50 mM TrisHCl, pH 7.4, at 37°C. Aliquots of 10 µg of DNA were removed after 0, 6, 19, 48, 90, 117 and 240 h. DNA was precipitated with ethanol, washed and analysed by 32P-postlabelling in duplicate samples.
In vitro repair study
Triplicate samples of PO-DNA-1 (10 µg) were incubated with uracil-DNA glycosylase (1 U/µg DNA) in 20 mM TrisHCl, pH 8.2, at 37°C for 1 h. Aliquots of the same DNA were incubated without enzyme or with enzyme preheated at 95°C for 5 min. Samples were precipitated with ethanol and analysed by 32P-postlabelling.
Triplicate samples of PO-DNA-1 (1020 µg) or untreated DNA were also incubated with 20 µl of protein extract from human HeLa or HaCaT cells (a generous gift from Dr Hans Krokan) in 40 µl of buffer (50 mM TrisHCl, 20 mM NaCl, 1 mM EDTA and 0.8 mM dithiothreitol, pH 7.5) at 37°C for 2 h. Other PO-DNA-1 samples were incubated under the same conditions, but without addition of protein extract. Samples were then purified by incubation with RNase A, RNase T1 and proteinase K, followed by phenol and chloroform/isoamyl alcohol extraction and precipitation with ethanol. Each DNA sample (12 µg) was analysed twice by 32P-postlabelling.
[14C]PO-DNA-2 (100 µg) was incubated with the protein extract from HeLa cells in the same manner as described for PO-DNA-1. The DNA was then precipitated with ethanol and the supernatant evaporated. The dry residue was dissolved and analysed (together with UV standard 3-HP-cytosine) by 14C radiochromatography as described above.
Preparation of protein extract from HeLa and HaCaT cells
Briefly, cells (38x106) were trypsinized, cold phosphate-buffered saline with 10% fetal calf serum was added and cells were centrifuged at 4°C. The cell pellet was washed with phosphate-buffered saline and resuspended in 0.71.0 ml of the same buffer. The cells were lysed by sonication and debris separated by centrifugation. The supernatant was transferred to a new tube, checked for uracil-DNA glycosylase activity and frozen under liquid nitrogen.
HPLC separations
The previously described (20) Beckman HPLC System Gold (Fullerton, CA) connected to a 168 diode array detector was used for all separations. For preparation of standards a 4µ Genesis 4.6x250 mm C18 reversed phase column (Jones Chromatography, Hengoed, UK) was used. For separation of 32P-labelled samples, a 171 radioisotope detector with a modified flow cell (20) was also connected to the HPLC system and the 4.6 mm column was replaced by a 5µ Luna 2.0x250 mm C18 reversed phase column (Phenomenex, Torrance, CA). A pre-column filter and a 5µ Kromasil 2.0x50 mm C18 pre-column (Phenomenex) were installed in front of the analytical column. Inorganic phosphate and residual ATP as well as labelled residual dCMP and dGMP were separated from adducts on the pre-column by diverting the first 0.5 ml (2.5 min) to waste using a four-port switching valve (Valco Instruments, Houston, TX).
The Genesis column was run with a linear gradient of 100% 50 mM ammonium formate, pH 4.6, to 30% methanol over 55 min for separation of reactions with 3'- or 5'-dAMP (system A) and to 15% methanol over 40 min for separation of reactions with 3'- or 5'-dCMP (system B). The flow rate was 0.7 ml/min.
The Luna column (system C) was run isochratically with 100% 0.5 M ammonium formate containing 20 mM phosphoric acid, pH 4.6, for 15 min, followed by 1% methanol for 20 min, 5% methanol for 25 min, a linear gradient to 15% methanol over 10 min, isochratically with 15% methanol for 15 min and finally a linear gradient to 100% methanol over 5 min. The flow rate through the analytical column was 0.2 ml/min and was maintained using a split flow system (20).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
In all reactions of nucleotides with PO, two additional peaks were observed (Figure 2). These were of the same amplitude and had UV spectra identical to the original nucleotide. It was therefore assumed that they represented phosphate alkylations. Supported by the recorded UV and mass spectra, literature data, including our own work with allyl glycidyl ether (34), as well as the conducted rearrangement experiments, we conclude that the prepared standards were indeed 1-HP-dAMP, N6-HP-dAMP, 3-HP-dCMP and 3-HP-dUMP.
14C radiochromatography analysis of in vitro modified DNA
When [14C]PO-DNA was digested with micrococcal nuclease and spleen phosphodiesterase to 3'-nucleotides and analysed by HPLC, radioactivity peaks corresponding to the UV trace of the prepared standards (1-HP-3'-dAMP, N6-HP-3'-dAMP, 3-HP-3'-dCMP and 3-HP-3'-dUMP) were observed (not shown). Radioactivity measurement of triplicate samples indicated the presence of 20 ± 1 (1-HP-3'-dAMP), 2.0 ± 0.2 (N6-HP-3'-dAMP), 2.4 ± 0.2 mol (3-HP-3'-dCMP) and 4.8 ± 0.4 mol (3-HP-3'-dUMP) per 106 mol of normal nucleotides.
32P-postlabelling of in vitro modified DNA
32P-postlabelling of PO-DNA followed by HPLC with on-line radioisotope detection (system C) showed that 1- and N6-HP-5'-dAMP, as well as 3-HP-5'dCMP and 3-HP-dUMP, could be analysed simultaneously (Figure 6A). Depending upon the incubation time with PO, the relative amounts of these adducts varied, but 1-HP-5'-dAMP was always found to be the major product. Based on known adduct levels in [14C]PO-DNA-2, the recoveries through all steps of the postlabelling procedure were 5070% for 1-HP-5'dAMP, 3-HP-dCMP and 3-HP-dUMP and 20% for N6-HP-dAMP.
|
|
32P-postlabelling of DNA from rat tissues
DNA samples from nasal tissues, lung and lymphocytes from rats exposed to PO were analysed by HPLC (system C) following nuclease P1 enrichment and postlabelling. Radioactivity peaks, showing co-chromatography with the UV trace of each of the two diastereomers of 1-HP-5'-dAMP, were detected in all three tissues (Figure 4B), whereas these peaks were not present in unexposed rats (Figure 4A
). In addition, a minor peak showing co-chromatography with the UV trace of N6-HP-5'-dAMP (amounting to ~20% of 1-HP-adenine) was detected, but only in the respiratory nasal epithelium (data not shown). The identification of the major product as 1-HP-5'-dAMP was further strengthened by incubation of a postlabelled sample from nasal epithelium at pH 13 (80°C, 0.5 h), representing conditions under which 1-HP-5'-dAMP is completely converted to N6-HP-5'-dAMP. When this sample was analysed, no peak corresponding to 1-HP-5'-dAMP was observed and a new peak appeared that co-chromatographed with N6-HP-5'-dAMP (Figure 4C
). No radioactivity was present in the position of N6-HP-5'-dAMP when samples from nasal tissues of unexposed rats were incubated under the same conditions (data not shown).
|
|
|
In vitro repair study
When PO-DNA-1 was treated with uracil-DNA glycosylase under physiological conditions for 1 h (conditions sufficient for a more or less complete hydrolysis of uracilglycosidic bonds in uracil-containing single- or double-stranded DNA) and analysed by the 32P-postlabelling assay, the concentration of 3-HP-5'-dUMP and 3-HP-5'-dCMP was not reduced compared with samples incubated without enzyme or with inactivated enzyme, respectively.
When the same DNA was incubated with protein extract from HeLa cells for 2 h and analysed by the 32P-postlabelling assay, the concentration of 3-HP-5'-dCMP remaining in DNA was reduced to 40% compared with samples incubated without protein extract (Figure 6 and Table II
). The levels of other PO adducts were not altered. Similar results were found for HaCaT cells, i.e. ~50% of the 3-HP-5'-dCMP was eliminated from DNA during incubation (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It has earlier been shown that other alkylation products of PO with DNA in vitro are at the 3-position of adenine, the 3-position of uracil and the N6-position of adenine (16). In agreement with studies of adducts from other simple epoxides (16,32,35), it is assumed that N6-HP-adenine and 3-HP-uracil are formed via base catalysed rearrangements from 1-HP-adenine and 3-HP-cytosine, respectively. The rates of these transformations are often faster for adducts to free deoxyribonucleosides and deoxyribonucleotides than for adducts to DNA (32,36). Previous studies suggested that these spontaneous rearrangements are also fast for PO under physiological conditions (16,17). However, we found the rearrangement of 1HP-adenine to N6-HP-adenine in vitro to be slow. The half-life of 1-HP-adenine under physiological conditions was 56 days as free 3'- or 5'-nucleotides and 9.2 days in DNA. Solomon et al. (16), on the other hand, reported only N6-HP-adenine (no 1-HP-adenine) after 10 h of reaction of PO with DNA.
We could also show that deamination of 3-HP-cytosine to 3-HP-uracil was faster than the rearrangement of 1-HP-adenine under physiological conditions in vitro, but not as fast as previously indicated (16,17). This was true for the adducted nucleotide as well as for this adduct in native DNA. In PO-DNA the half-life for deamination of 3-HP-cytosine was ~2.2 days, whereas only 3-HP-uracil (no 3-HP-cytosine) was found after 10 h of reaction with PO. After treating DNA in vitro with [14C]PO for 48 h we found the sum of 1- and N6-HP-adenine to be 3.5% of 7-HP-guanine and the sum of N-3-cytosine/uracil to be 1.7%, respectively. Solomon et al. (16) reported 0.8 and 10% for those products after 10 h exposure. The reasons for the discrepancies between our results and the previous study (16) are unknown, but it has been well established that 1-alkyladenine as well as 3-alkylcytosine are preferentially formed in single-stranded DNA (34,36). The quality of the presumably double-stranded commercial DNA as well as differences in reaction conditions, DNA digestion procedures (37) and analytical methods are, therefore, likely to have been contributing factors.
Low molecular weight adducts are normally not resistant towards the dephosphorylating activity of nuclease P1. However, we have previously shown that this enzyme could be used for adduct enrichment of adenine and cytosine adducts of allyl glycidyl ether (38). This observation was confirmed in the present study, i.e. 1-HP-adenine, 3-HP-cytosine and 3-HP-uracil were very resistant towards nuclease P1 (total recoveries 5070%); N6-HP-adenine was partly dephosphorylated by this enzyme and therefore recovery of this adduct was lower (20%). In this study we used a new variant of the 32P-postlabelling method, where adducts are labelled as dinucleotides and then converted to 5'-mononucleotides by snake venom phosphodiesterase (28). This procedure increased the yield of all PO adducts ~3-fold compared with the standard P1 method, where adducts were labelled as 3'-monophosphates and dephosphorylated to 5'-monophosphate by nuclease P1 (38). The lower yield when using the original P1 assay was most likely due to incomplete dephosphorylation of the labelled bisphosphate adducts by nuclease P1.
1-HP-adenine was detected in respiratory nasal epithelium, lung and lymphocytes of PO-exposed rats, but N6-HP-adenine was only detected in the respiratory epithelium. The highest level of 1-HP-adenine was found in the respiratory nasal epithelium (Table I), which is also the major target tissue for tumour induction in the rat following inhalation of PO (10,39). 7-HP-guanine has previously been analysed in different tissues of PO-exposed rats used in this study (20) and for the purpose of comparison some of the latter data are included in Table I
. The tissue distribution of 7-HP-guanine as well as of the adenine adducts was very similar. For 7-HP-guanine the levels in lung and lymphocytes were 17 and 10% and for the adenine adducts 15 and 9%, respectively, of that in the respiratory nasal epithelium.
In rats killed 3 days after cessation of exposure, practically no decrease in 1-HP-adenine (measured after rearrangement to N6) was observed in the nasal tissue and the lung (Table I), indicating no or very slow repair of this adduct. In comparison, the levels of 7-HP-guanine decreased by about one-quarter from their initial concentrations, most likely due to chemical depurination of the adducts (20).
Because we found 3-HP-cytosine/uracil to be present at a level of ~2% of that of 7-HP-guanine in the in vitro experiment, we expected to find these adducts in tissues of PO-exposed rats. However, only 3-HP-uracil could be detected and only in the nasal epithelium at a concentration that was 0.02% of 7-HP-guanine, i.e. 100 times lower than found in vitro. After excluding methodological sources of error and since 3-HP-uracil was chemically stable in DNA, we tentatively concluded that this adduct (or its precursor 3-HP-cytosine) was repaired in vivo. Since it has been suggested that these adducts are repaired by uracil glycosylase (25), we incubated PO-modified DNA with the bacterial version of this enzyme. However, neither 3-HP-uracil nor 3-HP-cytosine was released from PO-DNA by this enzyme. It has been shown that uracil glycosylase from different species have somewhat different substrate specificities and it cannot, therefore, be excluded that this enzyme could be involved in the repair of these adducts. After incubation of PO-modified DNA with protein extracts from mammalian cells, 3-HP-cytosine, but not 3-HP-uracil, levels in DNA decreased (Figure 6 and Table II
) and the release of this adduct from DNA to supernatant was demonstrated using 14C radiochromatography. These data suggest an enzymatic removal of 3-HP-cytosine in the cell-free system. Although this remains to be demonstrated to occur in the rat in vivo, it is likely that the very low levels of 3-HP-cytosine/uracil found in the PO-exposed rats is due to fast enzymatic release of 3-HP-cytosine from DNA before this adduct has the chance to undergo deamination to the more stable 3-HP-uracil.
The results from the present as well as our previous study (20) demonstrate that except for 3-HP-adenine, all adducts known to be formed in vitro are also formed in vivo and can be detected by the postlabelling assay. For the first time 1- and N6-HP-adenine and 3-HP-uracil have been shown to be present in PO-exposed animals. Because very low levels of the presumably potent mutagenic 3-HP-cytosine/uracil were detected in vivo, the biological role of 1-HP-adenine and the apurinic sites from 7-HP-guanine and 3-HP-adenine might be more important than indicated by results from in vitro test systems (25,26). 1-HP-adenine was the second most frequent adduct of PO in vivo, but it amounted to only 2% of 7-HP-guanine in the tissues of the PO-exposed rats. On the other hand, this adduct seemed to be stable in vivo. 1-HP-adenine has the advantage over 7-HP-guanine that it is more conveniently analysed and has a higher chemical stability and recovery, which results in high sensitivity (detection limit 0.3 mol/109 mol of normal nucleotides using 10 µg DNA). Moreover, using the Dimroth rearrangement of 1-HP-adenine to N6-HP-adenine, HPLC analysis can be carried out in two steps and the level of detection can be increased by using larger amounts of DNA. The specificity and sensitivity of the analysis of 1-HP-adenine is very high and therefore this adduct represents a feasible alternative to 7-HP-guanine for monitoring PO exposures.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|