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Chem. Res. Toxicol. 2006, 19, 392-398
Epimerization and Stability of Two New cis-Benzo[a]pyrene Tetrols by the Use of Liquid Chromatography-Fluorescence and Mass Spectrometry Carlos Sagredo,*,†,‡ Raymond Olsen,†,§ Tyge Greibrokk,§ Pa˚l Molander,†,§ and Steinar Øvrebø†,‡ The National Institute of Occupational Health, P.O. Box 8149 Dep., N-0033 Oslo, Department of Biology, UniVersity of Oslo, P.O. Box 1066 Blindern, N-0316 Oslo, and Department of Chemistry, Section of Analytical Chemistry, UniVersity of Oslo, P.O. Box 1033 Blindern, N-0315 Oslo, Norway ReceiVed September 28, 2005
Quantitative determination of the hydrolysis products from proteins and DNA gives valuable information regarding the reactive metabolite that forms the protein and DNA adduct. Quantification of proteinbenzo[a]pyrene (BP) adducts represents a more sensitive method than quantification of BP-DNA adducts. The aim of the present study was to identify two hydrolysis products from BP-derived protein adducts found in vitro and in vivo in a previous study. Male Wistar rats were injected i.p. with BP, and serum albumin was isolated and subjected to acid hydrolysis at 70 °C for 3 h. The hydrolysate was subjected to LC separation, and fractions of the two unknown compounds were collected. The molecular masses of the two unknown compounds were in accordance with being tetrols as judged by LC electrospray mass spectrometry. The fragmentation patterns were characteristic of tetrols with formation of the molecular ion and the loss of water molecules. In addition, the compounds were subjected to acid hydrolysis at 70 °C with 0.1 M HCl for 3 h. We observed that two of the known tetrols epimerized to the two unknown tetrols and vice versa. This is probably a characteristic epimerization involving not only position C10OH but also another site like position C7-OH. The in vivo findings of the two unknown adducts are probably the result of the formation of BPDE III in the metabolism of BP. These two tetrols must then have the C7-OH and C8-OH groups in a cis position. Introduction Polycyclic aromatic hydrocarbons (PAHs) constitute a large class of compounds formed during incomplete combustion of organic matter and fossil fuels in industrial processes, automobile exhaust, cigarette smoke, and charbroiled food (1, 2). Exposure to PAHs is high in occupational environments where workers are exposed to PAHs mostly through inhalation and skin uptake. Several PAHs are classified as carcinogens (1, 2). Among the carcinogenic PAHs, benzo[a]pyrene (BP) is wellstudied and has served as a model for the carcinogenic and mutagenic effects of PAHs (3-7). BP is metabolically activated to bay region diol epoxides, such as (()-anti- and (()-syn-7,8dihydrodiol 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, i.e., (()-BPDE I and (()-BPDE II. The (+)-BPDE I isomer, in particular, has been shown to have the highest mutagenic and carcinogenic activity and hence is considered to be the ultimate carcinogen (8-10). The biotransformation of BP may result in other reactive diol epoxides such as 9,10-dihydrodiol 7,8-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene, syn-BPDE III, and antiBPDE III with the epoxide groups in a nonbay position (11, 12). Protein and DNA adducts from these diol epoxides have been identified both in vivo and in vitro. These epoxides were found to be less mutagenic in bacterial cells and less carcinogenic in mammalian cells but with an increased cytotoxic effect * To whom the correspondence should be addressed. Tel: +47 23 19 53 00. Fax: +47 23 19 52 00. E-mail:
[email protected]. † The National Institute of Occupational Health. ‡ Department of Biology, University of Oslo. § Department of Chemistry, Section of Analytical Chemistry, University of Oslo.
as compared to (+)-BPDE I (13). The importance of the BPDE III diol epoxides in the BP metabolism is evident from the fact that one of the major metabolites characterized in urine from BP-exposed germ-free rats was the acetylcysteine conjugate of BPDE III (14). The identification of DNA and protein adducts is important for risk assessment of both the cytotoxic effects and the carcinogenic potential (15, 16). Different methods have been used to study DNA and protein adducts, like postlabeling, immunoassay, and determination of hydrolysis products, mainly BP tetrols after acid or enzymatic hydrolysis of protein and DNA (17-21). Unfortunately, the hydrolysis conditions necessary to release the tetrols affect the ratio of released tetrols due to an isomerization between the molecules. This stability difference has been recognized to be a result of an isomerization in particular at the C10-OH sites in the tetrol molecule (22-24). Nevertheless, these tetrols are well-characterized and are the result of the adduct formation of (()-BPDE I and (()-BPDE II with DNA and proteins. Hence, the tetrol measurement gives valuable information of the in vivo metabolism of BP where the tetrols reflect the formation of specific reactive diol epoxides. However, because of the possible formation of BPDE III addcuts, the hydrolysis of adducted DNA and protein could give rise to four additional tetrols. These BP tetrols would have in common the C7-OH and C8-OH groups in a cis position. The existence of these four stereoisomers is still uncertain. Islam et al. have reported the finding of two possible new BP tetrols, found in BP-exposed rats and compared to the hydrolysis of syn-BPDE III and anti-BPDE III (24, 25). The two unknown compounds were shown to have similar chromatographic properties and fluorescence characteristics as the standard tetrols.
10.1021/tx0502746 CCC: $33.50 © 2006 American Chemical Society Published on Web 01/28/2006
Epimerization and Stability of cis-Benzo[a]pyrene Tetrols
In addition, they behaved similarly to the four standard tetrols in both dose-response experiments and time-course experiments. In the present study, we have isolated BP tetrols after hydrolysis of plasma proteins from BP-exposed rats. In addition, we have determined the molecular masses and the stability and epimerization of two new tetrols and found evidence for an additional site for epimerization.
Materials and Methods Chemicals. (()-anti-BPDE III (benzo[a]pyrene-r-10,t-9-dihydrodiol,t-7,8-epoxide) and (()-syn-BPDE III (benzo[a]pyrene-r10,t-9-dihydrodiol,c-7,8-epoxide) were from Dr. R. G. Harveys’s laboratory. (()-Benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol (BPtetrol I-1), (()-benzo[a]pyrene-r-7,t-8,t-9,10-tetrahydrotetrol (BPtetrol I-2), (()-benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol (BPtetrol II-1), and (()-benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol (BP-tetrol II-2) were purchased from the National Cancer Institute, Chemical Carcinogen Repository (Midwest Research Institute, Kansas City, MO). HPLC grade methanol was obtained from Fluka (Buchs, Switzerland). HCl (37% concentrated) was obtained from Merck (Darmstadt, Germany). Water was obtained from a Milli-Q ultrapure water purification system (Millipore, Bedford, MA). Hydrolysis of syn- and anti-BPDE III. Two 50 µL solutions of, respectively, 40 µg/mL anti-BPDE III in THF and 40 µg/mL syn-BPDE III in THF were mixed with water to a final volume of 500 µL. Both solutions were incubated overnight in the dark at room temperature. BP Exposure of Wistar Rats. Wistar male rats weighing about 230 g were fed a standard diet (B&K Universal A/S, Norway) and given water ad libitum. BP was solubilized in corn oil (25 mg/mL) and injected intraperitoneally with a final dose of 100 mg/kg body weight. After 3 days, the rats were anesthetized (2 mL Hyponorm/ Dornicum/kg body weight, subcutaneous) and drained for blood. Plasma Protein Isolation. The blood from the animals was collected with Na-heparin, and plasma was separated by centrifugation at 1200g for 15 min. Plasma was withdrawn and precipitated with two volumes of cold acetone and left for 30 min followed by centrifugation at 1200g. The precipitated albumin was washed with 4 mL of acetone:ethyl acetate (1:1) to remove unbound BP metabolites. The precipitate was air-dried at room temperature and solubilized in 900 µL of 10 mM Tris-HCl/1 mM EDTA, pH 8.0. Adduct Purification. To 900 µL of albumin solution was added 100 µL of 1 M HCl, and this solution was incubated at 70 °C for 3 h. Water and methanol were added to a final volume of 5 mL with 10% methanol. This solution was applied to preconditioned Sep-Pak C18 cartridges (Millipore, Milford, MA) followed by 10 mL of washing with water and elution by 5 mL of methanol. The eluate was evaporated at 45 °C under a nitrogen stream and resolubilized in 500 µL of 10% methanol. The samples were stored at -20 °C. Epimerization. Fifty microliters of each tetrol isomer standard and the unknown BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 were dried under a stream of N2 gass and redissolved in 50 µL of THF. The solution was then added to 450 µL of 0.1 M HCl and acid hydrolyzed for 0, 1, 3, 6, and 24 h at 4, room temperature, 37, 50, 60,70, 80, and 90 °C. Each compound was acid hydrolyzed separately. Adduct Quantification and LC Instrumentation. The LCfluorescence analysis was performed on an Agilent 1100 LC system using a Zorbax C8 stationary phase with 5 µm particles and a 3.9 mm × 150 mm column (Agilent) attached with an Agilent 1100 fluorescence detector. The injection volume was typically 20 µL, and the samples were separated by a linear gradient of water and methanol by increasing the methanol content from 30 to 100% in 40 min. The column temperature was 40 °C, and the flow rate was set at 1 mL/min. The excitation and emission wavelengths were 341 and 381 nm, respectively.
Chem. Res. Toxicol., Vol. 19, No. 3, 2006 393 Fractionation of BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 was performed on a Waters 625 LC System (Waters, Milford, MA) using a Nova-Pak C18 stationary phase with 5 µm particles and a 3.9 mm × 150 mm column (Waters) equipped with a LC 240 (Perkin-Elmer, Ltd., Beaconsfield, United Kingdom) fluorescence detector. The excitation and emission wavelengths were 341 and 381 nm, respectively. The injection volume was 100 µL, and the compounds were separated by isocratic elution with 40% methanol and 60% water. The fractions of BP-7,8-cis-tetrol 1 and BP-7,8cis-tetrol 2 from both in vivo and in vitro hydrolysis were collected, dried under a stream of N2, and redissolved in ethanol. The samples were stored at -20 °C. Standard and sample solutions were made by dissolving appropriate amounts of BP-7,8-cis-tetrol 1 or BP7,8-cis-tetrol 2 ethanol solution in 10% (v/v) methanol-water solution or in 10% (v/v) 10 mM ammonium acetate buffer (pH 5)-acetonitrile solution when used in LC-MS determination of the tetrols. Fluorescence Analysis of BP-7,8-cis-tetrol 1 and BP-7,8-cistetrol 2. The BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 fractions from both in vivo and in vitro were analyzed with synchronous fluorescence analysis with a RF-5000 fluorescence spectrometer (Shimzau, Kyoto, Japan). The excitation was varied from 200 to 600 nm with a fixed emission wavelength at 379 and 399 nm, respectively. The emission was varied from 200 to 600 nm with a fixed excitation wavelength at either 245, 266, 277, 312, 326, or 343 nm, respectively. Mass Spectrometric Determination of BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2. LC-MS was performed on a Waters capLC system using a microbore Kromasil C18 column with 3.5 µm particles (1 mm × 150 mm) coupled with a UV-diode array detector and a Quatro LC on-line tandem quadrupole mass spectrometer from Micromass (Manchester, United Kingdom). The mass spectrometer was equipped with a Z-spray atmospheric pressure ionization ion source prepared for electrospray ionization (ESI). The flow rate was set at 40 µL/min with a linear gradient consisting of a 10 mM ammonium-acetate buffer and acetonitrile by increasing the acetonitrile content from 10 to 45% in 20 min prior to a hold time of 6 min. The ESI source was operated in negative mode with a capillary voltage of -3 kV, a sample cone voltage of -25 V, and an extraction cone voltage of -4 V. The nebulizer and desolvation gas flow rates were 90 and 360 L/h, respectively. The desolvation temperature and the source temperature were 300 and 110 °C, respectively. The injection volume was 2.5 µL.
Results The identification of the molecular mass by ESI-MS and the fragmentation pattern support the evidence of two LC peaks being BP tetrols. The isomers were isolated from plasma from rats exposed to BP and produced in vitro by hydrolysis of synand anti-BPDE III. An LC-fluorescence chromatogram with the two unknown and the four standard tetrols is shown in Figure 1. The degree of fragmentation in ESI is very soft as compared to other ionization techniques in MS, providing the necessary information to identify the molecular masses of, e.g., BP-7,8cis-tetrol 1 and BP-7,8-cis-tetrol 2. The most important fragments are the molecular mass m/z 319 and to some extent the fragments m/z 301 and m/z 283, which are presumably the result of water loss (26, 27). We also identified an ion at m/z 379, which is the acetate adduct of the molecular ion, from acetate buffer in the mobile phase. The mass spectrometer was tuned using BP-tetrol I-1, and the experimental parameters were found satisfactory to identify the tetrols. The limit of detection of the mass spectrometer was 80 fmol of BP-tetrol I-1. A typical MS fragmentation of in vitro BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 is presented in Figure 2a,b, respectively. The formation of adducts in vivo is small; hence, it was necessary to use single ion reaction (SIR) with the molecular
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Figure 1. LC chromatogram of a standard mixture of tetrols from the Chemical Carcinogen Repository and BP-7,8-cis-tetrol 1 and BP-7,8cis-tetrol 2 isolated from the hydrolysis of albumin.
Figure 3. (a) Characteristic SIR chromatogram of in vivo BP-7,8-cistetrol 1 from BP-exposed rats. (b) Characteristic SIR chromatogram of in vivo BP-7,8-cis-tetrol 2 from BP-exposed rats.
Figure 2. Characteristic MS fragmentation pattern of in vitro (a) BP7,8-cis-tetrol 1 and (b) BP-7,8-cis-tetrol 2.
ion m/z 319 and the two fragments m/z 301 and m/z 283 to confirm the molecular mass of the BP-7,8-cis-tetrol 1 and BP7,8-cis-tetrol 2. The simultaneous detection of BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 by SIR and the UV-diode array is presented in Figure 3. The fluorescence spectra of the in vivo BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 were characteristic of the tetrols with fluorescence maximum at approximately 245 and 342 nm, respectively. The excitation and emission spectra of BP-7,8cis-tetrol 1 and BP-7,8-cis-tetrol 2 with BP-tetrol I-1 as a reference are illustrated in Figure 4.
The hydrolysis conditions necessary to release the tetrols from the adducted protein and DNA will also affect the tetrol ratio due to an epimerization between the molecules. We have therefore hydrolyzed each tetrol isomer and the two unknown compounds separately. We observed an isomerization between BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 and BP-tetrol II-1 and BP-tetrol II-2. The isomerization resulted in a constant ratio between BP-7,8cis-tetrol 1 and BP-7,8-cis-tetrol 2 and between BP-tetrol II-1 and BP-tetrol II-2. When BP-7,8-cis-tetrol 2 was treated with acid, a major part was first rapidly transformed to BP-7,8-cis-tetrol 1 and the ratio between the tetrols remained constant. Increasing the temperature leads then to the formation of both BP-tetrol II-1 and BPtetrol II-2. The amounts formed are small but detectable; see Figure 5. The epimerization was similar for BP-7,8-cis-tetrol 1, with a part first transformed to BP-7,8-cis-tetrol 2. Then, the temperature increase formed small but detectable amounts of BP-tetrol II-1 and BP-tetrol II-2 (data not shown). Acid treatment of BP-tetrol II-2 gives rise to the formation of BPtetrol II-1, while increasing the temperature leads to small but detectable amounts of BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2; see Figure 6. The epimerization of BP-tetrol II-1 was similar with the formation of BP-tetrol II-2. When the temperature was increased, small amounts of BP-7,8-cis-terol 1 and BP-7,8-cistetrol-2 were observed (data not shown).
Epimerization and Stability of cis-Benzo[a]pyrene Tetrols
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Figure 4. Emission scan of BP-tetrol I-1, in vivo BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 using (a) excitation maximum at 245 nm and (b) emission of 379 nm.
A similar hydrolysis reaction was carried out for the two remaining tetrols BP-tetrol I-1 and BP-tetrol I-2, but the formation of BP-7,8-cis-tetrols 1 and 2 was not observed. The epimerization step at C10-OH appears to be fast and reversible. The second epimerization step appears to be slower and was observed only for temperatures above 70 °C and for a prolonged period of hydrolysis time generally above 3 h. Such hydrolysis conditions will generally decompose and reduce the total tetrol content.
Discussion LC coupled with negative electrospray mass spectrometry made it possible to identify the molecular masses of BP-7,8cis-tetrol 1 and BP-7,8-cis-tetrol 2. Wang et al. (26, 27) used LC-MS to analyze BP metabolites including tetrols from mice that were exposed to asphalt fumes. Characteristic fragmentation
patterns were found with the loss of two water molecules. Although Wang et al. used the positive detection mode, the fragmentation patterns using the negative mode as in our study were nearly identical with the loss of water and the formation of the fragments m/z 301 and 283 in addition to the molecular mass m/z 319. We also identified an additional fragment at m/z 379, which is the acetate adduct of the molecular ion. Fluorescence and MS data support the evidence of BP-7,8cis-tetrol 1 and BP-7,8-cis-tetrol 2 as two tetrols from hydrolysis of protein adducts. Furthermore, the observed epimerization during acid treatment at various temperatures between BP-tetrol II-1, BP-tetrol II-2, BP-7,8-cis-tetrol 1, and BP-7,8-cis-tetrol 2 reveals epimerization at both C10-OH and probably at C7OH. This is shown in Figure 7 and hence may illustrate a possible structure for the two unknown tetrols, although we cannot infer the cis/trans of positions C9-OH and C10-OH. Similar hydrolysis reactions were carried out for the two
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Figure 5. (a) In vitro BP-7,8-cis-tetrol 2 with a minor peak of BP7,8-cis-tetrol 1 before epimerization. (b) The epimerization at C10OH results in a constant ratio between BP-7,8-cis-tetrol 1 and BP-7,8cis-tetrol 2. (c) Increasing the temperature from 60 to 70 °C leads to the formation of BP-tetrol II-2. (d) Increasing the temperature from 70 to 80 °C results in the formation of both BP-tetrol II-1 and BP-tetrol II-2.
remaining tetrols BP-tetrol I-1 and BP-tetrol I-2, but the only epimerization observed was at C10-OH. Although these two tetrols may epimerize at C7-OH under different conditions, it would lead to the formation of the two remaining BP-7,8-cistetrol 3 and BP-7,8-cis-tetrol 4. One of these tetrols would have four hydroxyl groups at the same side of the ring and likely be very unstable due to steric hindrance between the hydroxyl groups. These tetrols have not yet been observed, neither in vivo nor in vitro as far as we know. Careful considerations regarding the hydrolysis conditions should be taken in order to avoid tetrol decomposition and C7-OH epimerization, i.e., avoid a temperature above 80 °C. The hydrolysis conditions and the stability of the tetrols have been investigated before by Jansen et al.22 and Melikian et al.23 They observed a series of epimerization paths between the four standard tetrols where the epimerization at C10 was the most important. The tetrol BPtetrol I-2 was found unstable epimerizing to the tetrol BP-tetrol I-1 under hydrolysis conditions. The same was observed for BP-tetrol II-2 epimerizing to BP-tetrol II-1. The instability was believed to be due to steric interaction between C10-OH and C8-OH in the half-chair conformations of both BP-tetrol I-1 and BP-tetrol II-1. The 1-3 transannular interaction is believed to be relieved upon epimerization (8). In another report, Islam
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Figure 6. (a) BP-tetrol II-2 before epimerization. (b) The epimerization at C10-OH results in a constant ratio between BP-tetrol II-1 and BPtetrol II-2. (c) Increasing the temperature from 60 to 70 °C leads to the formation of BP-7,8-cis-tetrol 1. (d) Increasing the temperature from 70 to 80 °C results in the formation of both BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2.
Figure 7. Relation between BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 and BP-tetrol II-1 and BP-tetrol II-2. The horizontal reaction illustrates the epimerization at position C10, and the vertical reaction illustrates the epimerization at position C7. The epimerization at C10-OH appears to be fast as compared to the epimerization at C7-OH.
et al. (24) found a reversible epimerization for the tetrols, including the two unknown tetrols at C10-OH under acidic conditions. They observed a constant ratio between isomers only
Epimerization and Stability of cis-Benzo[a]pyrene Tetrols
differing by the C10-OH orientation and did not find evidence for epimerization at other C-OH sites, although a possible epimerization at C9-OH was not rejected. The possible finding of two new tetrols raises the question of the nature of the metabolic precursor of such species. Our result indicates that these adducts are tetrols, based from both the MS data and the finding that other tetrols epimerize to our unknown compounds. They probably arise from some diol epoxides other than the bay region epoxides BPDE I and BPDE II. The biotransformation of BP forms other reactive electrophiles such as 9,10-dihydrodiol 7,8-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, syn-BPDE III, and anti-BPDE III with the epoxide groups in a nonbay position. Protein and DNA adducts with these diol epoxides have certainly been identified both in vitro and in vivo. The epoxides were found to be less toxic in bacterial cells and less mutagenic in mammalian cells (11, 12). Three major adducts were identified by the use of NMR, which included the N7 and the N2 position of deoxyguanosine. Another adduct was postulated to be a C8-substituted deoxyguanosine. The conformation of these adducts differed from adducts derived from bay region diol epoxide, which primarily involves the exocyclic amino group of either deoxyguanosine or deoxyadenosine (13). The BPDE III adducts were labile, in particular the N7 adducts in DNA were found unstable at 37 °C (13). The metabolism of BP is complex with the formation of a wide range of adducts and metabolites (14, 28, 29). Some highly reactive BP metabolites, which recently have been identified, are the triol epoxides of BP, namely, 3-hydroxy-7,8-dihydrodiol 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, syn-BPTE, and anti-BPTE. These two metabolites are bay region trihydrotriol epoxides that may be formed through the intermediacy of BPDE I and BPDE II or 3-hydroxy-7,8-dihydrodiol 7,8-dihydrobenzo[a]pyrene (22). The epoxides formed a series of adducts in vitro with DNA and individual mononucleotides that were identified by 32P-postlabellig. anti-BPTE produced three major and several minor adducts with DNA while syn-BPTE produced two major adducts with DNA. Another interesting metabolite, which has been shown to form DNA adducts, is the 9-OH-BP (29). This metabolite requires a metabolic activitation to a reactive electrophile like 9-OH-BP-4,5-epoxide. Single DNA adducts at the N2 position of dG have been identified by MS (29). These newly identified triol epoxides and the 9-OH-BP-4,5-epoxide are not likely to be the precursors of our adducts since the MS data show clearly that the molecular mass of our metabolites is of tetrols and neither triols nor pentols. The BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 are formed both in vivo in BP-exposed rats and in vitro by the hydrolysis of syn- and anti-BPDE III. The biological formation of these adducts illustrates the existence of a significant metabolic pathway through the diol epoxides syn- and anti-BPDE III. The identification of conjugates of BPDE III in BP-exposed rat urine illustrates the importance of BPDE III in the BP metabolism (14). Although BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2 do not reflect the formation of the ultimate carcinogen, BPDE I, they do reflect the formation of another highly reactive and cytotoxic epoxide, i.e., BPDE III. As a biomarker, these tetrols could be interesting when dealing with the effects of the BP exposure other than the carcinogenic effect. An interesting observation is that we only found BP-7,8-cis-tetrol 1 and BP7,8-cis-tetrol 2 as adducts in protein and DNA and not as free metabolites. The hydrolysis conditions necessary to release the tetrols affect the ratio of the released tetrols due to tetrol epimerization. This epimerization is fast and reversible at position C10-OH in the tetrol molecule. When the temperature
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increases above 70 °C over a prolonged period of time, an epimerization at position C7-OH in the tetrol molecule is observed, which seems to be very slow. The biologically relevant tetrols should, nevertheless, be the sum between BPtetrol I-1 and BP-tetrol I-2 and between BP-tetrol II-1 and BPtetrol II-2. In addition, it must be necessary to account for the sum of BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2. In the present study, we have isolated two new BP adducts in plasma proteins from BP-exposed rats and determined the molecular masses and the stability and epimerization of these two new compounds. For practical reasons, they have been named BP-7,8-cis-tetrol 1 and BP-7,8-cis-tetrol 2. These results give supporting evidence of BP-7,8-cis-tetrol 1 and BP-7,8-cistetrol 2 as two new tetrols with the C7-OH and C8-OH groups in a cis position. Acknowledgment. Thanks to Dr. R. G. Harvey for providing (()-anti-BPDE III and (()-syn-BPDE III. Dr. Einar Eilertsen’s professional advice and help in animal treatment are greatly acknowledged.
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