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The DNA adduct formation of (±)--7, 8-dihydroxy-7, 8-dihydrobenzo(a)pyrene (BPD) and (±)-7β, 8α-dihydroxy-9α, 10α-epoxy-7, 8, 9, 10-tetrahydrobenzo(a)pyrene (-BPDE) were compared and the persistence and disappearance of the adducts in both mouse and rat epidermis determined. BPD (100 nmol/mouse in 150 μl acetone and 200 nmol/rat in 300μl acetone) and -BPDE (77 nmol/mouse in 150 μJ tetrahydrofuran) and 154 nmol/rat in 300 μ tetra-hydrofuran) were topically applied to 50-day-old male Swiss mice and 35-day-old Wistar rats. To improve the identification of the DNA adducts formed, an acid hydrolysis technique was used to convert the BPD- and -BPDE- de-oxyribonucleoside adducts formed in mouse and rat skin to BP tetrols. The modified deoxyribonucleosides and BP tetrols obtained by hydrolysis of adducts were isolated by reverse-phase h.p.l.c. At approximately similar doses per unit area of treated skin, the initial total binding of these compounds to epidermal DNA and the level of modified deox-yribonucleosides was ∼ 6-fold lower in rat skin epidermis than in mouse skin epidermis. Similar ratios of (±)--BPDE-deoxyguanosine (dGuo) to (±)--BPDE-dGuo adducts (5.7 and 6.1, determined by h.p.l.c. analysis of BP tetrols obtained by hydrolysis of modified dGuo) were found in both mouse and rat epidermis a short time (6 h)after topical application of (±)--BPD. Three hours after topical application of (±)--BPDE, the ratios of BP-7, 10/8, 9-tetrol to 7/8, 9, 10-tetrol were 9: 1 in mouse epidermal DNA and 6: 1 in rat epidermal DNA. One and three weeks after application of these two compounds, only (+)--BPDE-dGuo was detected in mouse epidermis; 2 and 0.2% of the initial (+)--BPDE-dGuo level was found to persist in the epidermal DNA from BPD- and -BPDE-treated mice respectively. No DNA adducts were detected in rat epidermis 3 weeks after BPD and -BPDE treatment. Thus, 3 weeks after topical application of BPD and -BPDE to mouse and rat skin, the DNA adducts completely disappeared form rat epidermis while they persisted in mouse epidermis. The results suggest that: (i) the persistence of (+)--BPDE-dGuo may be related to carcinogenesis in mouse epidermis by BPD and anti-BPDE; (ii) the complete disappearance of the -BPDE-dGuo adduct may also account in part for the relative resistance of tissue from this species to the carcinogenic action of benzo(a) pyrene. [ABSTRACT FROM PUBLISHER]

The objective of the present study was to compare DNA and protein adduct formation of benzo[a]pyrene-4, 5-oxide (BPO) and to determine the persistence of the adducts in both mouse and rat epidermis. (±)BPO at a dose of 100 nmol/mouse and 200 nmol/rat was topically applied to male Swiss mice and Wistar rats. Three hours after application, there was 3-fold less binding of BPO to mouse epidermal DNA than to rat epidermal DNA; inversely, the amount of BPO bound to mouse skin protein was 3.6 times higher than in rat skin protein. One and three weeks after application of BPO, persistence of 17–20% of the initial amount of BPO-DNA adducts and 2–4% of initial amount of BPO bound to protein was detected in both mouse and rat skin epidermis. H.p.l.c. analysis of the enzymatic hydrolysates of DNA from mouse and rat epidermis 3 h after application of BPO showed five distinct products: one early-eluting, two BPO-deoxyguanosine (dGuo) (ratio 1.5:1) and two BPO-deoxyadenosine (dAdo) adducts (ratio 2:1). The ratio of the total modified dGuo to the total modified dAdo was 2:1. The amount of total BPO-dGuo and BPO-dAdo adducts was 3.5 times greater in rat than in mouse epidermis. Persistence of the major BPO-dAdo adduct was observed in mouse and rat epidermal DNA, and 1 and 3 weeks after topical application of BPO there was a 6-fold greater amount of the persisting BPO-dAdo adduct in rat skin epidermis than in mouse skin epidermis (4.1 and 0.66 pmol/mg DNA, respectively). Minor amounts of the BPO-dGuo were found to persist in rat skin epidermis DNA. [ABSTRACT FROM PUBLISHER]

Sephadex LH-20 chromatography of DNA digests of C3H/10T1/2 cells treated with [H]9-hydroxybenzo[a]pyrene (9-OH-BaP) and [C]BaP-7, 8-diol showed: (1) the presence only of uncharacterized H radioactivity eluted in the early portion of the gradient; [H]9-OH-BaP-4,5-oxide-modified deoxyribonucleosides were not observed. (ii) The total amount of [C]-labelled radioactivity corresponded to nucleoside moieties which were modified by - and -benzo[a]pyrene diol-epoxide. The absence of nucleosides modified by 9-OH-BaP-4,5-oxide in C3H/10T1/2 cells observed in this study may account in part for the relative resistance of these cells to the mutagenic and transforming action of 9-OH-BaP. [ABSTRACT FROM PUBLISHER]

Male Swiss mice and Wistar rats, susceptible and resistant, respectively, to the carcinogenic effects of benzo[a]pyrene, were treated topically with 250 nmol/mouse and 1000 nmol/rat of tritium labelled benzo[a]pyrene (BaP). The initial formation of BaP diol-epoxide deoxyguanosine adduct was approximately similar in the skin epidermis of the two species. After 3 weeks, the persistence of some 6.5% of initial BaP diol-epoxide deoxyguanosine was observed in mouse skin DNA, while this adduct was completely removed from DNA of rat skin. The total excision of BaP diol-epoxide deoxyguanosine adduct in rat epidermis may contribute in part for the relative resistence of rat skin to the carcinogenic actions of BaP. [ABSTRACT FROM PUBLISHER]

Benzo[a]pyrene diol epoxide (BPDE)-DNA adducts are involved in the induction of p53 mutations and probably in the causation of human lung cancer associated with cigarette smoking. The ratio between CYP1A1 and GST enzyme activities is a critical determinant of the target dose of carcinogenic BPDE and other DNA-reactive PAH metabolites. In this review, we summarize the published data on modulation of (+)-anti-BPDE-DNA adduct levels in smokers' lungs by CYP1A1*2 genotypes alone or in combination with GSTM1 polymorphism and compare these results with those reported for aromatic/hydrophobic (bulky) DNA adducts. The data published so far show only a trend for a non-significant increase in bulky DNA adduct levels in subjects with GSTM1*0 or the CYP1A1*2-GSTM1*0 genotype combination. In contrast, a clear dependence of (+)-anti-BPDE-DNA adduct levels was found as a function of the CYP1A1 and GSTM1 genotypes: In lung parenchyma, this adduct was more pronounced in persons with the GSTM1*0 genotype, and CYP1A1*2-GSTM1*0 carriers had higher (+)-anti-BPDE-DNA adduct levels than those with CYP1A1*1/*1-GSTM1*0. The homozygous CYP1A1*2/*2 carriers in the GSTM1*0 group had the highest (+)-anti-BPDE-DNA adduct levels. Our analysis leads to the conclusion that the risk-modifying effects of metabolic genotypes and of gene interactions might be more easily identifiable if specific markers of structurally defined adducts were used, such as the (+)-anti-BPDE-DNA adduct. These results are also consistent with the hypothesis that BP (PAH) induce G:C to T:A transversion mutations in the hotspot codons of the p53 tumor suppressor gene and are thus involved in malignant transformation of the lung tissue of smokers.

The level of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39 coke oven workers exposed to polycyclic aromatic hydrocarbons (PAH) and 39 non-exposed persons (controls) were investigated, each of the groups consisting of smokers and non-smokers. The adduct level was measured by an improved HPLC/fluorescence method (Rojas, M., Alexandrov, K., van Schooten, F. J., Hillebrand, M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557-560, 1994) through the release of the corresponding benzo[a]pyrene (B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51% of coke oven workers exposed to PAH and in 18% of the non-exposed (control) subjects. The mean level of anti-BPDE-DNA adducts/10(8) nucleotides in coke oven workers (15.7 +/- 37.8) was approximately 8 times higher than in non-exposed subjects (2.0 +/- 8.7). The interindividual variation of adduct levels was approximately 100-fold in coke oven workers and approximately 50-fold in controls respectively. Smokers in the exposed group had 3.5 times more DNA adducts than non-smokers. With the exception of one non-smoker with very high adduct levels (52.8 adducts/10(8)), the control subjects showed the presence of barely detectable adducts in only 16% of the samples examined. The increased in vivo formation in some smokers and high variability of anti-BPDE-DNA adducts in coke oven workers suggests variations in genetically controlled activation/inactivation reactions of PAH metabolism.

A new fluorometric assay was validated for quantification of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in white blood cells (WBC) from humans exposed to polycyclic aromatic hydrocarbons (PAH). This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene derived from acid hydrolysis of BPDE-DNA, and can measure 1 BPDE adduct per 10(8) unmodified nucleotides. The quantity of WBC DNA required depends on the modification level and varies between 5 and 500 micrograms. The assay was applied to seven WBC DNA samples from lung cancer patients, six of whom were heavy smokers, and to three WBC DNA samples from healthy subjects employed in an aluminum production plant. High levels of BPDE-DNA adducts, ranging from 62 to 533 adducts/10(8) nucleotides were found in six out of seven DNA samples from the lung cancer patients. In WBC DNA from healthy persons BPDE-DNA adducts were detected only in two non-smokers, but at a much lower level than in lung cancer patients (4-10 adducts/10(8) nucleotides). Using coded WBC DNA samples, BPDE-DNA adduct levels measured by fluorometry of the B[a]P-tetrols, were compared with the results obtained by 32P-postlabeling (nuclease P1 enrichment) and ELISA measurements. A good correlation and proportionality was found between the levels of BPDE-DNA adducts measured by fluorometry and 32P-postlabeling (r = 0.95, P < 0.001, n = 8). The correlation between fluorometry and ELISA was much lower and not significant (r = 0.61, P = 0.1, n = 6). Moreover, the ELISA grossly overestimated BPDE-DNA adduct levels measured by the other two methods. The results demonstrate that the highly sensitive and specific fluorometric assay is suitable for measuring BPDE-DNA adducts in WBC from humans exposed to benzo[a]pyrene.

Benzo[a]pyrene (B[a]P)-tetrols formed after stereoselective cytochrome P450-dependent metabolism from (-)-trans-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene [(-)-B[a]P-7, 8-diol] by lung microsomes (n = 19) and peripheral blood lymphocytes (n = 13) from lung cancer patients were measured, and the effect of smoking explored. B[a]P-tetrols were quantified by an HPLC/fluorescence assay with a detection limit of approximately 300 attomol, after incubation with peripheral blood lymphocytes or microsomes from lung cancer patients who were current cigarette smokers, ex-smokers and non-smokers. In lymphocytes from these subjects, high, medium and low metabolic activities respectively for (-)-B[a]P-7,8-diol to tetrol conversion were found, but there was no statistically significant difference between smokers, ex-smokers and non-smokers. When the B[a]P-tetrol formation by human lung microsomes was measured, recent smokers had 4- to 7-fold higher (P = 0.04) metabolic activity than ex-smokers and non-smokers. The mean lung microsomal arylhydrocarbon hydroxylase (AHH) activity was three times higher in smokers than in non-smokers and was undetectable in ex-smokers. AHH activity was correlated with tetrol formation in the same lung microsomal samples (r = 0.62, P less than 0.01 in smokers; and r = 0.67, P less than 0.01 in all subjects). When subjects were grouped according to smoking habits, however, no correlation was detected between mean tetrol formation by lung microsomes and that of lymphocytes. Thus, lymphocytes cannot serve as a surrogate for lung microsomes concerning the pulmonary metabolism of (-)-B[a]P-7,8-diol. The much higher B[a]P-tetrol formation observed in lung microsomes from smokers is in accord with a reported higher pulmonary AHH activity, cytochrome P450IA level, and CYP1A1 gene expression in recent tobacco smokers.

A new sensitive fluorometric assay was established to measure the stereospecific formation of benzo[alpha]pyrene tetrols formed after cytochrome P450-dependent metabolism of (--)-7,8-dihydroxy-7,8- dihydrobenzo[a]pyrene by human hair follicles. This simple assay requires three human hair follicles and a low (0.5-2.0 microM) substrate concentration and has a limit of detection of approximately 0.3 fmol of tetrols. Freshly isolated human hair follicles from 20 adult volunteers (10 non-smokers and 10 smokers) were assayed. While intersubject and seasonal variations were observed, the assay was found to be reproducible for a given subject. This rapid and non-invasive assay provides a new means for metabolic phenotyping of human subjects for their capacity to metabolize (--)-7,8-dihydroxy-7,8-dihydrobenzo[alpha]pyrene to its carcinogenic form (+)anti-benzo[a]pyrene diolepoxide.

The objective of the present study was to compare DNA and protein adduct formation of benzo[a]pyrene-4,5-oxide (BPO) in vivo and to determine the persistence of the adducts in both mouse and rat epidermis. (+/-)BPO at a dose of 100 nmol/mouse and 200 nmol/rat was topically applied to male Swiss mice and Wistar rats. Three hours after application, there was 3-fold less binding of BPO to mouse epidermal DNA than to rat epidermal DNA; inversely, the amount of BPO bound to mouse skin protein was 3.6 times higher than in rat skin protein. One and three weeks after application of BPO, persistence of 17-20% of the initial amount of BPO-DNA adducts and 2-4% of initial amount of BPO bound to protein was detected in both mouse and rat skin epidermis. H.p.l.c. analysis of the enzymatic hydrolysates of DNA from mouse and rat epidermis 3 h after application of BPO showed five distinct products: one early-eluting, two BPO-deoxyguanosine (dGuo) (ratio 1.5:1) and two BPO-deoxyadenosine (dAdo) adducts (ratio 2:1). The ratio of the total modified dGuo to the total modified dAdo was 2:1. The amount of total BPO-dGuo and BPO-dAdo adducts was 3.5 times greater in rat than in mouse epidermis. Persistence of the major BPO-dAdo adduct was observed in mouse and rat epidermal DNA, and 1 and 3 weeks after topical application of BPO there was a 6-fold greater amount of the persisting BPO-dAdo adduct in rat skin epidermis than in mouse skin epidermis (4.1 and 0.66 pmol/mg DNA, respectively). Minor amounts of the BPO-dGuo were found to persist in rat skin epidermis DNA.

The possibility that the amounts of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene- deoxyguanosine (anti-BaP diol epoxide-dGuo) and r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene- deoxyguanosine (syn-BaP diol epoxide-dGuo) may vary in human lymphocyte cultures from different donors was investigated by comparing DNA adducts formed after treatment with [G-3H]benzo[a]-pyrene (4 microM) for 24 h. In most cases, greater than 50% of the DNA adducts were derived from r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (syn-BaP diol epoxide).

The in vivo DNA adduct formation of (+/-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (BPD) and (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) were compared and the persistence and disappearance of the adducts in both mouse and rat epidermis determined. BPD (100 nmol/mouse in 150 microliter acetone and 200 nmol/rat in 300 microliter acetone) and anti-BPDE (77 nmol/mouse in 150 microliter tetrahydrofuran and 154 nmol/rat in 300 microliter tetrahydrofuran) were topically applied to 50-day-old male Swiss mice and 35-day-old Wistar rats. To improve the identification of the DNA adducts formed, an acid hydrolysis technique was used to convert the BPD- and anti-BPDE-deoxyribonucleoside adducts formed in mouse and rat skin to BP tetrols. The modified deoxyribonucleosides and BP tetrols obtained by hydrolysis of adducts were isolated by reverse-phase h.p.l.c. At approximately similar doses per unit area of treated skin, the initial total binding of these compounds to epidermal DNA and the level of modified deoxyribonucleosides was approximately 6-fold lower in rat skin epidermis than in mouse skin epidermis. Similar ratios of (+/-)-anti-BPDE-deoxyguanosine (dGuo) to (+/-)-syn-BPDE-dGuo adducts (5.7 and 6.1, determined by h.p.l.c. analysis of BP tetrols obtained by hydrolysis of modified dGuo) were found in both mouse and rat epidermis a short time (6 h) after topical application of (+/-)-trans-BPD. Three hours after topical application of (+/-)-anti-BPDE, the ratios of BP-7,10/8,9-tetrol to 7/8,9,10-tetrol were 9:1 in mouse epidermal DNA and 6:1 in rat epidermal DNA. One and three weeks after application of these two compounds, only (+)-anti-BPDE-dGuo was detected in mouse epidermis; 2 and 0.2% of the initial (+)-anti-BPDE-dGuo level was found to persist in the epidermal DNA from BPD- and anti-BPDE-treated mice respectively. No DNA adducts were detected in rat epidermis 3 weeks after BPD and anti-BPDE treatment. Thus, 3 weeks after topical application of BPD and anti-BPDE to mouse and rat skin, the DNA adducts completely disappeared from rat epidermis while they persisted in mouse epidermis. The results suggest that: the persistence of (+)-anti-BPDE-dGuo may be related to carcinogenesis in mouse epidermis by BPD and anti-BPDE; the complete disappearance of the anti-BPDE-dGuo adduct may also account in part for the relative resistance of tissue from this species to the carcinogenic action of benzo[a]pyrene.