1 Department of Toxicology, University of Würzburg, Versbacher Str. 9, D-97078 Würzburg, Germany
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Abstract |
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Abbreviations: 3'-dGp, deoxyguanosine-3'-monophosphate; CBI, covalent binding index; dG, deoxyguanosine; F344 rats, Fischer 344 rats; Hex-dGp, deoxyguanosine 3'-monophosphate adduct of 2-hexenal (for structure see Figure 1c for IUPAC nomenclature and structural features see Results); Hex-pdGp, deoxyguanosine 3'-,5'-bisphosphate adduct of 2-hexenal; PEI, polyethylene imine; RP, reverse phase.
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Introduction |
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2-Hexenal is a common component of plants (1824) and the main intake for humans is considered to result from consumption of fruit, vegetables and fruit juices, but also from beverages like tea (25 p.p.m.) (22). In bananas, for instance, the 2-hexenal concentration is 40 p.p.m. (20) and in beans it is 34 p.p.m. (21). 2-Hexenal is considered to act as a natural fungicide and protects plants not only from penetration by fungi (19,25) but also by other micro-organisms (26) after being injured. Evidently, the local 2-hexenal concentration can be very high at the site of injury and the concentration given above represents only a mean concentration over all plant parts. De Lumen et al. (21) and Stone et al. (27) provided mechanisms for the formation of 2-hexenal from unsaturated fatty acids, e.g. linolenic acid by lipoxygenase, under air. Furthermore 2-hexenal is a plant flavouring component responsible for the typical aroma of some fruits and herbs (2127). The utilization of 2-hexenal as `natural identical' flavouring or as `natural' fungicide was taken into consideration (28). Furthermore, breeding of plants resistant against fungi or genetic manipulation could also lead to increased 2-hexenal concentrations in those plants.
All data available suggest that humans are permanently exposed to 2-hexenal and that 2-hexenal plays a role in human carcinogenicity according to its mutagenic/genotoxic activities. Unfortunately, no data from cancer studies are available on 2-hexenal. Therefore, cancer risk assessment for this compound was hitherto not possible.
In our previous in vitro studies (1) we have investigated the interaction of this compound with DNA components in detail and have shown that a pair of diastereomers of the regioisomer of exocyclic 1,N2-propanodeoxyguanosine adducts is formed as the major stable adduct in which the OH-group is adjacent to the N1-atom of the guanine moiety. In general, this type of DNA adduct is considered as a promutagenic lesion (see above) and can lead to initiation of cancer cells. Detection and quantitation of these adducts as markers for the initiation of cancer could decisively improve the cancer risk assessment. The in vivo formation of the exocyclic DNA adducts of 2-hexenal had not previously been demonstrated (29). We have developed a new 32P-post-labeling technique based on nuclease P1 enrichment and TLC separation of the DNA adduct spots in order to detect and quantitate the respective DNA adducts of 2-hexenal in vivo and to allow improved cancer risk assessment.
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Materials and methods |
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Synthesis of Hex-dGp adducts
Aliquots of 30 mg (0.077 mM) 3'-dGp were reacted with 50 µl (0.43 mM) 2-hexenal in 1 ml 0.1 M sodium phosphate buffer (pH 10.7) at room temperature. Maximum yield was obtained after 8 days. The Hex-3'-dGp adducts obtained were separated by HPLC performed on a Hewlett Packard 1050 pump, a Rheodyne injector and a Hewlett Packard photodiode array detector. The reaction mixture was separated on a Nucleosil C18 column (length 250 mm, i.d. 8 mm, particle size 10 µm) with a linear gradient from 10 mM ammonium formate buffer pH 4.7 to methanol with a flow rate of 3.0 ml/min in 80 min (HPLC system 1). The 1H-NMR and mass spectra of the two diastereomers were practically identical. The HPLC-chromatogram, the UV-spectrum and the chemical structures of the diastereomers are shown in Figure 1a, b and c.
1H NMR (Bruker 600 MHz, D2O) d = 0.92 (t, J13,12 = 7.4 Hz, 3H, H-13); 1.351.48 (m, 2H, H-12); 1.511.69 (m, 3H, H-11, H-7a); 2.24 (pseudo-td, J7b,7a = 13.9 Hz, J7b,6 = J7b,8 = 3.1 Hz, 1H, H-7b); 2.61 (ddd, J2'a,2'b = 13.9 Hz, J2'a,1' = 6.4 Hz, J2'a,3' = 3.2 Hz, 1H, H-2'a); 2.78 (pseudo-td, J2'b,2'a = 13.9 Hz, J2'b,1' = J2'b,3' = 7.0 Hz, 1H, H-2'b); 3.643,72 (m, 1H, H-6); 3.76 (dd, J5'a,5'b = 12.5 Hz, J5'a,4' = 4.7 Hz, 1H, H-5'a); 3.77 (dd, J5'b,5'a = 12.5 Hz, J5'a,4' = 3.6 Hz, 1H, H-5'b); 4.20 (pseudo-q; J4',3' = J4',5'a = J4',5'b = 3.8 Hz, 1H, H-4'); 4.784.84 (m, 1H, H-3'); 6.24 (pseudo-t, J1',2'a = J1',2'b = 7.0 Hz, 1H, H-1'); 6.29 (pseudo-t, J8,7a = J8,7b = 2.6 Hz, 1H, H-8); 7.92 (s, 1H, H-2).
MS (electrospray +4 kV): m/z = 446 (M+ + H).
13C-NMR (150.0 MHz, D2O): (p.p.m.) = 15.8 (C-13), 22.5 (C-12), 31.1 (C-11), 34.3 (C-7), 40.6 (C-2'), 51.8 (C-6), 65.0 (C-5'), 72.0 (C-8), 73.2 (C-3'), 86.4 (C-4'), 89.1 (C-1'), 111.3 (C-10a), 117.6 (C-2), 151.8 (C-3a), 153.6 (C-4a), 160.5 (C-10).
Stability of the adducts
A sample of 7.5 nmol of the Hex-dGp adducts was incubated in 0.1 M phosphate buffer at 37°C for 5 days. The pH value was varied from pH 2 to pH 13. Aliquots were analyzed by the same HPLC setup as described above using a Nucleosil C18 column (length 250 mm, i.d. 4 mm, particle size 5 µm) with a linear gradient from 10 mM ammonium formate buffer pH 4.7 to methanol with a flow rate of 1.0 ml/min in 20 min (HPLC-System 2).
Studies on the nuclease P1 stability of the nucleoside-3'-monophosphates
Aliquots of 8 nmol of the different nucleoside 3'-phosphates (equivalent to 10 µg DNA) were incubated with 6 µl nuclease P1 mixture in a total volume of 18 µl for 10 min at 37°C. The nuclease P1 mixture consisted of 1.2 µl (0.6 µg, 10% of the regular amount used for post-labeling) nuclease P1 solution, 1.8 µl 0.3 mM ZnCl2 and 3.0 µl buffer (0.25 M sodium acetate, 40 mM sodium succinate, 20 mM CaCl2). After 10 min the reaction was stopped by adding 2.4 µl of 0.5 M Tris base. An aliquot (10 µl) of the mixture was analyzed by HPLC System 2. The pH value of the sodium acetate buffer was varied for the determination of optimum pH.
In order to determine the MichaelisMenten kinetics, different amounts of the substrate were used at pH 4.0 as described above. The evaluation was performed according to LineweaverBurk using the equation: 1/v = 1/vmax + (KM/vmax)x1/S, where KM is the MichaelisMenten constant.
Using Hex-dGp, the incubation was performed with 6 µg nuclease P1 for 180 min.
Treatment of animals
The animals were fed with standard diet from Altromin (Lage, Germany). Oral doses of 500, 200 or 50 mg 2-hexenal/kg body wt dissolved in corn oil were administered to 8-week-old male Fischer 344 (F344) rats (190210 g) (four rats per group) by gavage. Control animals were treated with corn oil only. The rats were sacrificed at different times ranging from 8 h to 4 days. The following organs were taken: duodenum, esophagus, forestomach, glandular stomach, kidney, liver, lung, rectum, urinary bladder. The liver was perfused with 0.9% sodium chloride solution and the other organ tissues were washed with sodium chloride solution. The organs were either worked up directly or frozen with liquid nitrogen and stored at 70°C. DNA was isolated according to the phenol extraction method as described by Gupta (30). The concentration of the DNA was quantitated using the absorption at 260 nm.
DNA hydrolysis and nuclease P1 treatment
DNA (10 µg) was incubated for 4 h at 37°C with 3 µl enzyme mixture containing 0.2 U/µl (1 µg/µl) micrococcus nuclease, 0.002 U/µl (1 µg/µl) spleen phosphodiesterase and 4 µl DNA digestion buffer (25 mM CaCl2, 50 mM sodium succinate pH 6.0) in a total volume of 20 µl. Next, 6 µl nuclease P1 mixture was added which consisted of 1.2 µl (6 µg) nuclease P1 solution, 1.8 µl 0.3 mM ZnCl2 and 3 µl 250 mM sodium acetate pH 4.0. The reactant mixture was incubated for 1 h at 37°C and the reaction stopped by adding 2.4 µl 0.5 M Tris base. The solution was desiccated to dryness and redissolved in 10 µl water.
32P-post-labeling
To the hydrolyzed and nuclease P1-treated solution 6.5 µl labeling mixture was added. It was made as a mixture of 1 µl kinase buffer (100 mM dithiothreitol, 100 mM MgCl2, 10 mM spermidine, 200 mM bicine/NaOH, pH 9.5), 0.5 µl 800 mM bicine, 5 µl 2.2 µmol [-32P]ATP (167 TBq/mmol, 4500 Ci/mmol; 1.9 MBq, 50 µCi) and 0.25 µl (7.5 U) T4 polynucleotide kinase. The sample was incubated for 45 min at 37°C. Finally, 4 µl (40 µU) apyrase was added and the solution further incubated for 30 min at the same temperature.
Chromatography
For the detection of the Hex-pdGp adducts in vivo a contact transfer method was used. The different samples were applied in portions of ~5 µl to an RP18 TLC sheet (length: 10 cm) to which a wick was attached (7 cm, Whatman #17). The chromatography was performed with 0.4 M ammonium formate/formic acid pH 6.0 overnight. The origins were cut out (1x1 cm2, no washing) and attached with clothes pegs to PEIcellulose sheets (10x10 cm2). The transfer to the PEIcellulose material was carried out by developing the sheets with n-propanol:water:nonidet-P40 (50:50:1, v/v/v) at 50°C. The RP18 chips were removed and the TLC plates were washed thoroughly with water. After drying, the chromatography was performed in the first direction (D1) with 1.2 M ammonium formate/formic acid pH 3.5 after attaching wicks (4 cm, Whatman #1). The wicks were removed and the TLC sheets were washed in water and air-dried. Another wick (4 cm, Whatman #1) was attached for the chromatography in the second direction (D2) with 0.3 M ammonium sulphate (adjusted to pH 7.5 with disodium hydrogenphosphate). The chromatograms were analyzed by a PhosphorImagerTM (Molecular Dynamics).
Quantitation of DNA adducts
Quantitation of the adduct levels was based on the recovery of the amount of adducts for the reference compound Hex-dGp in an extra sample with DNA of a control animal. The number of nucleotides was calculated via the UV absorption of the DNA solution. The recovery of the adducts was determined in the same manner: the respective amounts of the Hex-dGp standards were added to DNA, the whole post-labeling procedure was performed and quantitated as described above, and the mean values and standard deviations of 35 independent determinations of the recoveries were calculated.
Calculation of the covalent binding index (CBI)
The CBI was calculated according to Lutz (31), dividing the adduct levels measured in adducts/106 nucleotides by the dose applied in mmol substance/kg body wt.
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Results |
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32P-post-labeling technique
The detection of the respective adducts Hex-dGp 1 and 2 is based on a 32P-post-labeling nuclease P1 technique and on the separation of the labeled adducts with PEIcellulose TLC. In addition, the contact transfer method with RP18 TLC as described in Materials and methods was used in our new method in order to minimize the radioactive background on the TLC sheet, in particular, in the region of Hex-dGp spots. With this TLC method, the diastereomers Hex-dGp 1 and 2 appear in one spot and are clearly separated from normal nucleotides and other radioactive impurities (see also Figure 3b).
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Under our conditions the amount of enzyme (m) is 6 µg nuclease P1 and the reaction volume (V) is 18 µl.
With this method we received a labeling efficiency of 35% and a recovery of 10%. The limit of quantification is in the range of 0.03 adducts/106 nucleotides. The relative standard deviation at the limit of quantification is ~40%; in comparison, the relative standard deviation at a level of 1 adduct/106 nucleotides is 27% with our method (see Materials and methods, `Quantification of DNA adducts').
Detection of Hex-dGp adducts in male F344 rats
Figure 3a represents the post-labeling TLC of liver DNA of untreated rats as control. Figure 3b
shows the 32P-post-labeling TLC analysis of liver DNA of male F344 rats 2 days after p.o. application of 500 mg 2-hexenal/kg body wt (right hand side). No adducts could be found in the liver DNA or in other organs of untreated F344 rats used throughout our experiments, who were kept under our standard conditions and fed with our standard diet (see Materials and methods). Figure 4
demonstrates the time dependence of adduct formation with 2-hexenal. Highest adduct levels were obtained 2 days after the gavage. Four days after gavage the level is even higher than after 1 day. No adducts could be detected 8 h after gavage.
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Discussion |
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The new post-labeling technique we have developed offers the opportunity to detect 2-hexenal adducts in vivo at a relatively low detection limit. Optimum conditions for the single steps of the labeling procedures could be worked out and it was demonstrated that the respective Hex-dGp adducts are chemically stable over a wide pH range and against the enzyme nuclease P1. Both properties are prerequisites for a sensitive 32P-post-labeling detection of these adducts because no enrichment method other than the nuclease P1 method proved to be effective for the adducts in our experiments and because the optimum pH values for the various steps of the labeling procedure differed over a wide range. With this 32P-post-labeling technique we could demonstrate in vivo DNA adduct formation of 2-hexenal for the first time. Recently Gölzer et al. (29) reported that they could not find 2-hexenal adducts in the stomach of rats treated with 320 mg/kg body wt 2-hexenal by gavage. The authors looked, however, for adducts as early as 12 h after gavage. As we have shown in Figure 4, we could not find adducts 8 h after gavage and think it likely that adducts would still not have been found 12 h after gavage. Thus, the data of Gölzer et al. are in line with ours in that 2-hexenal adducts appear relatively late after gavage. This effect is surprising at first glance since 2-hexenal is a directly alkylating compound which requires no bioactivation for adduct formation. Recently, Witz (39) pointed out that conjugation of
,ß-unsaturated carbonyl compounds with glutathione must not necessarily be a detoxication mechanism because the conjugation is reversible in equilibrium and that the conjugate may dissociate in cell compartments of low glutathione content, e.g. in the cell nucleus where DNA is located. Glutathione addition to the double bond of 2-hexenal (Michael addition) is considered to be one of the major and fastest biological reactions which these compounds undergo (7,39). On the other hand, 2-hexenal does not possess high chemical reactivity and the reaction rate of 2-hexenal with DNA components is relatively low in vitro (32) (see also `Synthesis of Hex-dGp adducts' in Material and methods). Evidently it also takes some time until detectable amounts of adducts are formed in vivo. Figure 4
also indicates that the Hex-dGp adducts might be repaired to a certain extent since the adduct level 4 days after gavage is only one third that detected 2 days after gavage, however, the decrease in the adduct level can also be explained, at least in part, by cell turnover.
The organ distribution of the adducts (Figure 5a and b) demonstrates a tendency for site of first contact binding, despite the fact that adducts are formed rather late. Evidently, 2-hexenal is directly absorbed after gavage by the tissues of the forestomach, esophagus and duodenum but to a lesser degree in the glandular stomach. Systemic distribution evidently also occurs to a certain extent because a high adduct level is also found in the kidney. A first pass effect should be expected in the liver and, most likely, the systemic distribution of 2-hexenal runs partly via the stabilized transport form of the 2-hexenalglutathione conjugate which may prevent 2-hexenal from further biotransformation such as epoxidation of the double bond. Glutathione conjugation and Michael addition of other SH-group-containing biological molecules such as certain amino acids is probably also responsible for the dependence of the adduct levels on the doses. High doses of 2-hexenal lead to a glutathione depletion and also to a decrease of other SH-group-containing compounds and therefore a higher amount of the dose is available for DNA binding. Thus, the binding at higher doses is disproportionate if compared with lower doses (see Figure 6a
). This effect is observed, in particular, if considering the relationship between the CBI and the dose (Figure 6b
). According to its definition (see Materials and methods, `Calculation of the CBI') the CBI is, in general, independent of the dose. The CBI can be utilized for a cancer risk estimation (31). The adduct levels, or the CBI as found in the case of higher 2-hexenal doses, may be considered as irrelevant for cancer risk assessment since such high doses of 2-hexenal do not occur in the normal human diet, however, glutathione depletion may also occur in humans under certain circumstances, e.g. intake of certain medication, independently of the 2-hexenal intake and glutathione depletion results in increased CBIs as pointed out above. Therefore, higher CBIs than that calculated for the lowest dose should also be taken into consideration if performing a cancer risk assessment. In every case the CBI of 2-hexenal is extremely low and according to Lutz (31) the carcinogenic potency of substances with such low CBIs is also extremely low. If considering that the intake of 2-hexenal via food is very low, according to the literature data shown in the Introduction, one can estimate that the cancer risk arising from 2-hexenal intake via consumption of fruit and vegetables is also very low. The dose versus DNA adduct formation relationship shown here demonstrates that relatively high single doses are required to detect DNA adducts with the detection limit of our method. According to refs 1928 a daily 2-hexenal intake of ~0.15 mg/kg body wt/day can be estimated. As a single dose this intake would lead to very low and unmeasurable DNA adduct levels considering our dose versus DNA adduct level relationship. Thus, we can estimate, on the basis of both the expected low DNA adduct levels and the low CBI, that the benefit from eating fruit and vegetables is much higher than the low, unavoidable risk from 2-hexenal intake. However, nothing can be said about a steady state DNA adduct level after a permanent low intake of 2-hexenal via food and beverages. The situation deserves a somewhat more precise assessment on the basis of the TD50, which can be derived from the CBIs according to the correlation of Lutz (31), and on the basis of a more exact evaluation of the daily 2-hexenal intake. This more precise assessment will be performed and discussed in a forthcoming paper.
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Acknowledgments |
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Notes |
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References |
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