Department of Chemistry, Faculty of Sciences, University of Indonesia, Jakarta, Indonesia and
1 Department of Toxicology, University of Würzburg, Würzburg, Germany
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbreviations: CrotdGp, crotonaldehydedGp adduct; dAp, deoxyadenosine 3'-monophosphate; dCp, deoxycytidine 3'-monophosphate; dG, deoxyguanosine; dGp, deoxyguanosine 3'-monophosphate; dTp, deoxythymidine 3'-monophosphate; MN, micrococcus nuclease; NER, nucleotide excision repair; NP1, nuclease P1; PEI, polyethyleneimine; RAL, relative adduct labeling; SPDE, spleen phosphodiesterase.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Crotonaldehyde induced hepatocellular carcinoma in Fischer 344 rats (12) and according to an epidemiological study in the workplace the compound is considered to lead to lung cancer in humans (13). It is mutagenic in Salmonella typhimurium strains TA100 (14,15) and TA104 (16), induces the sfiA gene in the SOS chromotest (17), causes DNA strand breaks in L1210 cells (18) and is genotoxic and mutagenic in the normal human lymphoblast cell line GM0621 (19). In a mutation analysis we observed that a total of 82% of the point mutations were at G:C base pairs with a hot-spot at base pair 133 in the supF gene of plasmid pZ189 (19). We found that crotonaldehyde has highest reactivity with guanine bases and forms a pair of diastereomers of a 1,N2-propanodeoxyguanosine adduct regioisomer, in which the OH group in the newly formed ring is adjacent to the N-1 atom of the guanine moiety, as the major adducts and to a lesser extent also 7,8-cyclic propanodeoxyguanosine adducts and biscyclic 1,N2,7,8-dipropanodeoxyguanosine adducts (20). The chemical structures of the deoxyguanosine adducts were ascertained from spectroscopic data (20,21).
Detection of the 1,N2-propanodeoxyguanosine adducts of crotonaldehyde in animals and human tissue is an appropriate measure of exposure to this substance (target dose) and also of the initiation of cancer cells. Detection of crotonaldehydeDNA adducts can help to better estimate the variant individual exposure and to improve cancer risk assessment for crotonaldehyde.
We have therefore developed, optimized and adapted a 32P-post-labeling technique for 1,N2-propanodeoxyguanosine adducts of crotonaldehyde based on nuclease P1 (NP1) enrichment and polyethyleneimine (PEI)cellulose TLC of the labeled adducts for in vivo detection. Here we want to show the results obtained in different organs of Fischer 344 rats after gavage of single high doses and after multiple gavage of low doses.
Recently Nath and Chung published a different 32P-post-labeling technique and reported that they found background adduct levels in untreated animals (22,23). In contrast, we could not find any adducts in untreated animals. The varying exposure described above or differences in the method could account for this discrepancy (see Discussion).
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Instrumentation
Proton NMR spectra were recorded on a Bruker AC 600 and mass spectra were measured on a Finigan MAT-Trio 2000 quadrupole mass spectrometer with a Finigan electrospray interface.
Synthesis of the crotonaldehyde adduct (CrotdGp) standards
Aliquots of 80 mg (0.21 mmol) dGp and 126 mg (1.8 mmol) crotonaldehyde were dissolved in 40 ml of 100 mM phosphate buffer, pH 8.5, and reacted at 80°C. For analysis HPLC system 1 was used and for isolation of the adducts HPLC system 2. Maximum yield was obtained after 20 h at 80°C and pH 8.5.
System 1.
Hewlett Packard HP 1050 pump with Hewlett Packard photodiode array detector (detection at 260 nm) and a Rheodyne injector on a RP 18 Nucleosil column (length 250 mm, i.d. 4 mm), with a linear gradient from 10 mM ammonium formate buffer, pH 4.7, to methanol at a rate of 1 ml/min over 40 min.
System 2.
Waters system Millenium 2010 software, Waters Model 510 pumps, Waters 486 UV detector (detection at 360 nm), Rheodyne 7125, Knauer RP 18 column (length 300 mm, i.d. 8 mm), with a linear gradient from 10 mM ammonium formate buffer, pH 4.7, to methanol at a flow rate of 4 ml/min over 40 min (Figure 1A).
|
1H NMR (Bruker 600 MHz, D2O): 1.2 (d, J11,6 = 6.3 Hz, 3H, H-11), 1.6 (m, J7a,7b = 13.0 Hz, 1H, H-7a), 2.2 (pseudo-td, J7a,7b = 13.0 Hz, J7b,6 = 13 Hz, J7b,8 = 2.3Hz, 1H, H-7b), 2.6 (ddd, J2'a2'b = 13.8 Hz, J2'a,1 = 6.3 Hz, J2'a,3' = 3.3Hz, 1H, H-2'a), 2.8 (pseudo-td, J2'b,2'a = 13.0 Hz, J2b',1' = 7.0 Hz, J2'b,3' = 7.0 Hz, 1H-2'b), 3.7 (m, 1H, H-6), 3.75 (dd, J5'a,5'b = 12.0Hz, J5'a,4' = 4.7 Hz, H-5'a), 3.8 (dd, J5'b,5'a = 12.4 Hz, J5'a,4' = 3.4 Hz, 1H, H-5'b), 4.2 (pseudo-q, J4',3' = 3.6 Hz, J4',5'a = 3.6 Hz, 1H, H-4'), 6.25 (pseudo-t, J1',2'a = 7.0 Hz, J1',2'b = 7.0 Hz, 1H, H-1'), 6.28 (pseudo-T, J8,7a = 2.6 Hz, J8,7b = 2.6 Hz, 1H, H-8), 7.9 (s, 1H, H-2).
13C NMR (Bruker 150.9 MHz, D2O): (d.p.m.) 22.2 (C-11), 37.9 (C-7), 39.9 (C-2'), 42.0 (C-6), 64.0 (C-5'), 73.2 (C-8), 77 (C-3'), 87 (C-1), 89 (C-4'), 110 (C-10b), 140 (C-2), 153 (C-3b), 154 (C-4b)', 161 (C-10).
Electrospray MS (70 keV) (Finigan MAT-Trio 2000 quadrupole): m/z 439 (M+Na)+ (base peak, 100%), 462 (M+2Na)+ (45%) and 418 (M+H)+ (78%).
Dependence of the stability of the Crot-dGp standards on pH value
Samples of 48 nmol of the standard were incubated in 200 µl of 0.1 M phosphate buffer of varying pH values from pH 2 to pH 13 at 37°C for 72 h. Aliquots were analyzed with HPLC sytem 1.
Stability of the adducts against NP1
The stability assay was performed as described recently (25). The mixtures were analyzed with HPLC system 1. As a control for comparison, 8 nmol dGp was also investigated.
Binding studies with calf thymus DNA
Aliquots of 20 mg of calf thymus DNA were dissolved in 5 ml of 0.1 M sodium phosphate buffer, pH 7.2, and reacted with 90 µl of crotonaldehyde at either 37 or 60°C for 8, 16 or 48 h. To determine the detection sensitivity a reaction temperature of 37°C and reaction time of 16 h were used. The mixtures were filtered through an elution filter, then 100 µl of 0.1 M sodium chloride solution were added and the DNA on the filter was precipitated with 70% ethanol and washed three times with absolute ethanol.
Animal treatments
Single oral doses of either 300 or 200 mg crotonaldehyde/kg body wt dissolved in 0.2 ml of corn oil were administered by gavage to 8-week-old female Fischer 344 rats (200230 g) purchased from Harlan Winkelmann (Borchem, Germany). The rats were killed after either 12 or 20 h. Four rats were used for each group and three post-labeling determinations were carried out for each organ.
Multiple gavage.
Oral doses of either 10 or of 1 mg/kg body wt in 0.1 ml of corn oil were administered to 8-week-old female Fischer 344 rats five times a week for 6 weeks. The rats were killed 20 h after the last gavage. Four rats were used for each group and two determinations were carried out.
Persistence of the adducts.
Oral doses of 10 mg/kg body wt were administered to 8-week-old female Fischer 344 rats five days a week for 4 weeks. The first group of animals was killed 24 h after the last gavage (i.e. 4 weeks and 1 day after the start of application), the second group was killed 1 week after the last gavage (5 weeks after the beginning of application) and the third group was killed 2 weeks after the last gavage (6 weeks after the start).
DNA isolation
The standard method described by Gupta (24) provided the best results and was used throughout these investigations.
DNA hydrolysis and NP1 treatment
DNA hydrolysis and NP1 treatment were performed as recently described (25). Aliquots of 10 mg DNA were used for each hydrolysis and the final enzyme concentrations were 6.2x103 U/µl MN and 6.2x104 U/µl SPDE in a total volume of 16.3 µl. An aliquot of 12 µl (6 µg) of NP1 solution was used for the NP1 treatment.
32P-post-labeling
To the digested and NP1-enriched solution was added 8.3 µl of a labeling mixture. The labeling mixture consisted of 4 µl of kinase buffer (200 mM bicine/NaOH, pH 8.7, 100 mM dithiothreitol, 10 mM spermidine, 25 mM MgCl2), 4 µl [-32P]ATP (167 TBq/4500 Ci/mmol) and 0.25 µl (7.5 U) T4 polynucleotide kinase. The sample was incubated for 55 min at 37°C. Then, 4 µl (40 mU) of apyrase was added and the solution was incubated for 40 min at 37°C.
PEIcellulose TLC
A 3.5 cm wick (Whatman no. 17) was attached to a 13.5x20 cm prewashed PEIcellulose sheet from Macherey and Nagel (Düren, Germany). The eluent in the first dimension was 0.7 M ammonium formate, pH 3.5. After development for 3.5 h the wick was cut off, the sheet was dried and another 3 cm wick (Whatman no. 1) was attached in the second dimension. The eluent in the second dimension was 0.3 M ammonium sulfate in 10 mM sodium dihydrogen phosphate buffer, pH 7.5.
Autoradiography and quantitation of the adducts
The adduct spots were visualized on a 20x25 cm X-Omat X-ray film from Kodak in an autoradiography cassette equipped with intensifying screens. Using these autoradiograms the adduct spots on the TLC sheet were exactly marked with a pencil and cut out and the radioactivity was measured by Cerenkov counting. The background radiation on the TLC sheets was measured by the same method. The relative adduct labeling (RAL) of the NP1 method was calculated by the equation RAL = [c.p.m. (adducts)]/(AxZxM), where A is the specific activity of the [-32P]ATP in d.p.m./pmol, Z is the counting efficiency of the counter and M is the the amount of DNA in pmol. The labeling efficiency (%) was calculated according to labeling efficiency = [c.p.m. (adducts)]x100/(AxZxP), where P is the amount of the adduct standard in fmol.
Detection sensitivity
Aliquots of from 1 to 40 nmol of calf thymus DNA treated with crotonaldehyde (see above) were labeled as described after NP1 enrichment and the labeled adducts were chromatographed by PEIcellulose chromatography. The lowest radioactivity at which the adduct spots could be clearly detected after subtraction of the background was 260 c.p.m. The absolute detectable amount of DNA adduct was 92 amol in a total of 0.92 nmol of DNA according to a calculated RAL of 107. This means that a total of 92 amol of adduct could be detected. When using 10 µg of DNA the detection limit was 92 amol adduct/32.4 nmol DNA, or ~3 amol adduct/109 amol DNA. i.e. 3 adducts/109 nucleotides.
Recovery
The recovery was determined by adding 35 fmol of adduct standard to 10 µg of untreated DNA either from calf thymus or isolated from Fischer 344 rats (RAL 109x108). Then the complete labeling procedure was carried out.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
32P-post-labeling technique
The CrotdGp adducts can be readily detected by the 32P-post-labeling technique. The CrotdGp standards are stable within the pH range 49 over 72 h. This means that appropriate pH values can be chosen for the different steps of the post-labeling procedure within pH 49. Both adduct diastereomers are also stable against NP1 up to nuclease concentrations of 15 µg/µl. For comparison, the normal nucleotide dGp is completely dephosphorylated at a concentration of 5 µg/µl and at a concentration of 1 µg/µl 80% of the initial amount (0.8 nmol/ml) of the unmodified dGp is decomposed. The CrotdGp adducts are therefore eminently suitable for the NP1 enrichment procedure. Moreover, during development of the method, not shown here in detail, we found that there is no significant difference in the labeling efficiencies between the two diastereomers. In order to determine whether DNA is completely hydrolyzed by our method, 10 mg of calf thymus DNA were enzymatically hydrolyzed as described in Materials and methods and the amounts of the unmodified 3'-monophosphate nucleotides dAp, dCp, dGp and dTp were measured at different incubation times from 1 to 20 h. Maximum hydrolysis was obtained after only 4 h and a constant ratio of ~20% dCp, 20% dGp, 30% dAp and 30% dTp was measured, demonstrating that the DNA was completely hydrolyzed under the experimental conditions applied.
The labeled adducts are clearly separated from unmodified nucleotides and other impurities by the TLC method used and both adduct diastereomers, CrotdGp 1 and CrotdGp 2, appear as one spot (Figure 2). The 32P-post-labeling procedure developed provides a labeling efficiency of 8090%, a recovery of 38 ± 4.3% and a detection limit of 3 adducts/109 nucleotides. The absolute detection limit is 92 amol.
|
Animal studies
Adduct levels after a single gavage of high doses.
The adduct levels in liver DNA were 3.4 ± 0.5 adducts/108 nucleotides 20 h after gavage of 300 mg/kg body wt and 2.9 ± 0.15 adducts/108 nucleotides 20 h after gavage of 200 mg/kg body wt. The adduct levels after application of 200 mg/kg body wt are clearly higher 20 h after gavage (2.9 ± 0.15 adducts/108 nucleotides) than 12 h after gavage (1.7 ± 0.6 adducts/108 nucleotides). No adducts could be found in the liver DNA of untreated Fischer 344 rats at our limit of detection (Figure 2A). Figure 3
shows the adduct levels in four selected organs of Fischer 344 rats. The highest levels were detected in the liver, followed by lung, kidney and large intestine.
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Furthermore, we have shown that the enzymatic hydrolysis used in this study leads to total digestion of the DNA and complete release of the four deoxynucleoside 3'-monophosphates. The identity of the monophosphates was ascertained by retention times, co-chromatography and UV spectroscopy. The results are in accord with those of Reddy et al. (27) and Gupta (28). Both studies also found a ratio of ~20% dCp, 20% dGp, 30% dAp and 30% dTp after hydrolyzing DNA to the nucleotide 3'-monophosphates.
In our in vivo studies with Fischer 344 rats we have demonstrated that DNA adducts of crotonaldehyde can be detected after a single per os administration of high doses and after multiple gavage of low doses by the described 32P-post-labeling method. There is a clear dose dependence in adduct levels in the rat liver, however, the difference in adduct levels between the 300 and 200 mg/kg body wt doses is low. The reason for this is at present not clear. The adduct level is significantly higher 20 h after gavage than 12 h after gavage of 200 mg/kg body wt. This effect is in line with other observations with ,ß-unsaturated carbonyl compounds. With 2-hexenal, for instance, a maximum in adduct levels was also observed relatively late after administration (25,29). This effect is surprising at first glance since crotonaldehyde is a direct-acting alkylating compound which forms the DNA adducts CrotdGp 1 and 2 without the need for metabolic activation. Metabolism of crotonaldehyde is expected to lead to lower adduct levels since the main metabolic pathway, conjugation with glutathione (30), would result in a decrease in crotonaldehyde concentration. On the other hand, Witz (30) has recently shown that glutathione conjugation may not necessarily result in detoxification. The conjugates are unstable and can dissociate in equilibrium in cell compartments with low glutathione concentration, e.g. the cell nucleus, in which DNA is located. This effect could account for the relatively long time lapse after application until a maximum in adducts is found, since crotonaldehyde can be readily conjugated and then it will take some time until the conjugate is distributed, transported to the cell nucleus, where the conjugate dissociates, and the liberated crotonaldehyde can bind to DNA. It must, however, be stated that the reactivity of crotonaldehyde with DNA components is quite low, as shown in the in vitro binding studies with calf thymus DNA, where we found higher adduct levels after 48 h incubation than after 8 h incubation. Furthermore, the adduct levels are relatively low considering that crotonaldehyde reacts directly with DNA in vitro. These low adduct levels, in addition, underline the relatively low reactivity of crotonaldehyde with DNA components.
There is a clear difference in adduct levels in the four different rat organs investigated. Adduct levels were highest in the liver, followed by the lung, and are lower in the kidney and large intestine. Interestingly, tumors were induced by crotonaldehyde in those organs with the highest adduct levels, i.e. in rat liver in a long-term animal assay after administering crotonaldehyde in the drinking water (12) and in human lung according to an epidemiological study after exposure in the workplace (13).
We also found adducts after multiple gavage of low crotonaldehyde doses. The adduct levels observed in the study with multiple gavage are higher when compared with the single gavage experiments. After a 6 week gavage of 1 mg/kg body wt, for instance, the adduct levels were in the same range as those obtained with a single gavage of 200 mg/kg body wt, although the total dose by multiple gavage was only 30 mg/kg body wt. These experimental conditions leading to quasi-steady-state levels are closer to and more comparable with the situation of permanent intake of crotonaldehyde via the diet or by inhalation of tobacco smoke and indicate that permanent intake of low doses leads to more effective DNA adduct formation than occasional intake of high doses. As noted in the Introduction, the highest crotonaldehyde exposures are considered to occur via the diet and tobacco smoke. Dietary intakes of 10100 µg/kg body wt in rats, depending on the type of food, are conceivable. Similar high dietary intakes are also conceivable for humans under certain circumstances and excessive tobacco smoking could lead to intakes in the same range (see Introduction). The question is whether permanent intake of such doses would lead to detectable adduct levels in the range 110 adducts/109 nucleotides. The adducts also persist to a certain extent in liver DNA (Figure 5). The decrease in adduct levels 1 and 2 weeks after the last gavage (Figure 5
) indicates that the adducts are also repaired to a certain extent; the effect can, however, also be partly explained by cell turnover. Little is known about the repair of exocyclic 1,N2-propanodeoxyguanosine adducts. Recently, nucleotide excision repair (NER) of 1,N2-propadienodeoxyguanosine adducts (adducts of malondialdehyde) and for comparison also that of 1,N2-propano adducts were investigated (31). Only 1% of the 1,N2-propanodeoxyguanosine lesions were repaired by NER in vitro. The half-life of the malondialdehyde adduct in mice was 12.5 days (32). This result is in line with our persistence study of propano adducts in rat livers from which we could estimate a half-life of ~10 days (Figure 5
). Similar exocyclic adducts, 1,N6-ethenoadenine and 3,N4-ethenocytosine, are evidently readily repaired by specific DNA glycosylases, the mammalian alkylpurine-DNA N-glycosylases and human G/T(U) mismatch-DNA glycosylases, respectively (33). It is at present not entirely clear whether ethenodeoxyguanosine adducts, which in their chemical structure are closer to 1,N2-propanodeoxyguanosine adducts than ethenodeoxyadenosine adducts and ethenodeoxycytosine adducts, are also repaired by base excision repair (33). On the other hand, it was recently shown that another exocyclic adduct, p-1,N2-benzoquinone deoxyguanosine, which is also structurally close to the propeno adducts, is repaired by human apurinic/apyridimidinic endonucleases and by bacterial endonucleases and exonucleases (33).
The relative persistence of the adducts supports the hypothesis that high intakes of crotonaldehyde, as described above, could lead to background levels of adducts. The low reactivity of crotonaldehyde with nucleotides speaks against this hypothesis. We could not find any adducts in tissues of untreated rats fed and maintained under our conditions up to a detection limit of 3 adducts/109 nucleotides. This is in contrast to the results of Nath and Chung (22,23), who found adducts in untreated Fischer 344 rats, approximately in the range of adducts/109108 nucleotides. As shown above, dietary intake and also environmental exposure, via the air, animal housing, disinfectants, etc., could lead to detectable levels of adducts in animals not directly treated with crotonaldehyde. This, as already addressed in the Introduction, may account for the fact that Chung et al. found adducts in untreated animals and we did not. Furthermore, one cannot exclude the possibility that during the work up procedures crotonaldehyde could be formed, e.g. by oxidation of lipids, and bind to DNA, although crotonaldehyde is not considered as a major product of lipid peroxidation (34). The question of whether and to what extent crotonaldehyde adducts are really formed in tissues of untreated rats is of importance for the estimation of the role of crotonaldehyde in carcinogenesis. Extrapolation of exposure and intake of crotonaldehyde via different routes and estimation of adduct levels on the basis of doseadduct level relationships worked out in animal models could allow a better cancer risk assessment of crotonaldehyde. The 32P-post-labeling procedure presented here offers the possibility of a relatively fast and reliable quantitation of the main adducts of crotonaldehyde with high detection sensitivity and it is appropriate to determine doseadduct level relationships for cancer risk assessment.
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|