High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of $\text{Salmonella}\text{typhimurium}$ and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of $\text{S.}\text{typhimurium}$ and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of $\text{S.}\text{typhimurium}$. The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.     

An analysis of DNA replication in synchronized CHO cells treated with benzo[a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (+/-)7 beta,8 alpha- dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-dihydrobenzo[a]pyrene (BP diol epoxide I) were used to test the 'block-gap' model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 microM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the 'block-gap' model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the 'block-gap' model of replicative bypass repair in carcinogen damaged mammalian cells.     

Caffeine enhancement of the initiation of DNA replication in benzo[a]pyrene diol epoxide damaged cells

We have used a newly developed pH stepwise alkaline elution method to show that caffeine enhances DNA initiation (DNA replication in sub-replicon size nascent strands) in (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDEI) damaged mouse primary epidermal cells. Caffeine alone caused a dose-dependent increase in DNA initiation without an effect on DNA elongation (joining of replicon-sized nascent DNA). BPDEI alone inhibited DNA elongation as shown by a relative increase in sub-replicon size nascent DNA. When BPDEI treated cells were incubated with caffeine, there was a dose-dependent increase in sub-replicon size nascent DNA without a significant effect on the proportion of joined replicons. Therefore, caffeine can enhance DNA initiation in mammalian cells damaged with a reactive form of the carcinogen benzo[a]pyrene and this may account for the biological interaction between caffeine and the ultimate carcinogenic form of benzo[a]pyrene.     

Stabilization of a reactive, electrophilic carcinogen, benzo[a]pyrene diol epoxide, by mammalian cells

We have studied several features of the interactions of 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) with a DNA repair-proficient derivative of Chinese hamster ovary cells (CHO), AT3-2, and with a UV-light sensitive mutant, UVL-10, derived from AT3-2. Methods were developed for quantitating the amount of unhydrolysed BPDE-I associated with cells and for purifying DNA from cells under conditions where artificial labeling during preparation is minimized. In both cell types, about 30% of the BPDE-I added to a cell culture is rapidly taken up by the cells and is maintained in a cellular compartment in which the half-life of BPDE-I is about 10-fold longer than in aqueous medium. The kinetics of covalent binding to DNA were measured in both cell types and found to be described well by a single exponential process with a half-life of about 60 min. This is virtually identical to the half-life for intracellular hydrolysis of BPDE-I (57 min), consistent with the suggestion that this intracellular, relatively stable BPDE-I is responsible for binding.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Differential efficiency of mutagenesis at three genetic loci in CHO cells by a benzo[a]pyrene diol epoxide

The formation of DNA adducts by the ultimate carcinogen 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (BPDE-I) has been implicated in the process of carcinogenesis. In a line of Chinese hamster ovary (CHO) cells designated AT3-2 and in two derivative mutant lines, UVL-1 and UVL-10, originally selected for hypersensitivity to UV-irradiation, we have measured the formation of BPDE-I: DNA adducts and the production of biological damage. The quantity and quality of BPDE-I: DNA adducts formed initially in the 3 cell lines are identical over a wide range of BPDE-I doses. However, the UVL lines are unable to remove adducts from their DNA, while the AT3-2 cells remove about 50% of the BPDE-I: DNA adducts in a 24-h incubation. Correlated with this, the UVL lines are more sensitive to the lethal effects of BPDE-I than are the AT3-2 cells. Mutant frequencies were measured at the aprt, hprt and oua loci and were found to increase linearly with BPDE-I: DNA adduct formation at doses which gave greater than 50% survival. At the hprt and oua loci, the efficiency of mutation induction was similar for AT3-2 and UVL-10 cells. UVL-1 cells showed slightly higher (within a factor of 2-3) mutant frequencies in response to BPDE-I compared to AT3-2 at these two loci. However, at the aprt locus the repair-deficient cells were much more highly mutable (9-15-fold) than the repair-proficient AT3-2 cells. Based on the measured average level of adduct formation, it is calculated that 15% of the BPDE-I: DNA adducts in the aprt gene are converted into mutations. However, the possibility exists that the aprt locus is subject to higher levels of modification by BPDE-I than is the bulk DNA, which would lead to an artifactually high apparent conversion frequency.     

Product inhibition of benzo[a]pyrene metabolism in uninduced rat liver microsomes: Effect of diol epoxide formation

Conversion of benzo[a]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770–13776). Competition between BP and 7,8-dihydrodiol for P-450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4–6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2–25 pmol/mg) of the very active cytochrome P-450_c, which is the predominant form in MC-microsomes. The major effect of elevated levels of P-450_c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of K_m (7.9–2.6 μM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5–15% of total BP metabolites). The formation of DE was directly correlated with the content of P-450_c (r = 0.94). The presence of increased levels of P-450_c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.     

Induction of vaginal Lactobacillus phages by the cigarette smoke chemical benzo[a]pyrene diol epoxide

Because smoking increases a woman's risk of contracting bacterial vaginosis (BV), which is manifested by a reduction of vaginal lactobacilli and an overgrowth of anaerobic bacteria, chemicals contained in cigarette smoke were analyzed in vitro to determine their role in reducing lactobacilli. The result showed that trace amounts of benzo[a]pyrene diol epoxide (BPDE), which can be found in vaginal secretion of women who smoke, significantly increased phage induction in lactobacilli. This finding implies that smoking may reduce vaginal lactobacilli by promoting phage induction.     

Base-sequence dependence of noncovalent complex formation and reactivity of benzo[a]pyrene diol epoxide with polynucleotides

The base-sequence selectivity of the noncovalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene (BPDE) to a series of synthetic polynucleotides in aqueous solutions (5 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.0, 22 degrees C) was investigated. The magnitude of a red-shifted absorbance at 353 nm, attributed to intercalative complex formation, was utilized to determine values of the association constant Kic. Intercalation in the alternating pyridine-purine polymers poly(dA-dT).(dA-dT) (Kic = 20,000 M-1), poly(dG-dC).(dG-dC) (4200 M-1), and poly(dA-dC).(dG-dT) (9600 M-1) is distinctly favored over intercalation in their nonalternating counterparts poly(dA).(dT) (780 M-1), poly(dG).(dC) (1800 M-1), and poly(dA-dG).(dT-dC) (5400 M-1). Methylation at the 5-position of cytosine gives rise to a significant enhancement of intercalative binding, and Kic is 22,000 M-1 in poly(dG-m5dG).(dG-m5dC). In a number of these polynucleotides, values of Kic for pyrene qualitatively follow those exhibited by BPDE, suggesting that the pyrenyl residue in BPDE is a primary factor in determining the extent of intercalation. Both BPDE and pyrene exhibit a distinct preference for intercalating within dA-dT and dG-m5dC sequences. The catalysis of the chemical reactions of BPDE (hydrolysis to tetrols and covalent adduct formation) is enhanced significantly in the presence of each of the polynucleotides studied, particularly in the dG-containing polymers. A model in which catalysis is mediated by physical complex formation accounts well for the experimentally observed enhancement in reaction rates of BPDE in the alternating polynucleotides; however, in the nonalternating polymers a different or more complex catalysis mechanism may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Translesion replication of benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyadenosine and deoxyguanosine by human DNA polymerase iota

Human DNA polymerase ι (polι) is a Y-family polymerase whose cellular function is presently unknown. Here, we report on the ability of polι to bypass various stereoisomers of benzo[a]pyrene (BaP) diol epoxide (DE) and benzo[c]phenanthrene (BcPh) DE adducts at deoxyadenosine (dA) or deoxyguanosine (dG) bases in four different template sequence contexts in vitro. We find that the BaP DE dG adducts pose a strong block to polι-dependent replication and result in a high frequency of base misincorporations. In contrast, misincorporations opposite BaP DE and BcPh DE dA adducts generally occurred with a frequency ranging between 2 × 10^–3 and 6 × 10^–4. Although dTMP was inserted efficiently opposite all dA adducts, further extension was relatively poor, with one exception (a cis opened adduct derived from BcPh DE) where up to 58% extension past the lesion was observed. Interestingly, another human Y-family polymerase, polκ, was able to extend dTMP inserted opposite a BaP DE dA adduct. We suggest that polι might therefore participate in the error-free bypass of DE-adducted dA in vivo by predominantly incorporating dTMP opposite the damaged base. In many cases, elongation would, however, require the participation of another polymerase more specialized in extension, such as polκ.     

Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine

Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N²-deoxyguanosine (dG) and N⁶-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol γ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol γ opposite the BaP DE–dG adducts resembles that observed with the Y family pol η. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.     

Binding geometries of benzo[a]pyrene diol epoxide isomers covalently bound to DNA. Orientational distribution

Flow linear dichroism (LD) of different benzo[a]pyrene diol epoxide (BPDE) isomers covalently bound to calf thymus DNA or poly(dG-dC) provides information about binding geometry and DNA perturbation. With anti-BPDE the apparent angle between the long axis (z) of the pyrene chromophore and the DNA helix axis is approximately 30 degrees as evidenced from the LD of z-polarized absorption bands in the pyrenyl chromophore at 252 and 346 nm. The corresponding angle for the in-plane short axis (y) is determined to be approximately 70 degrees from a y-polarized band at 275 nm. The binding of (+)-anti-BPDE to DNA is found to cause a considerable reduction of the DNA orientation. This is ascribed to a decreased persistence length of DNA, owing either to increased flexibility ("flexible joints") or to permanent kinks at the points of binding. The reduced linear dichroism (LDr), i.e., the ratio between LD and isotropic absorbance, of the long-wavelength absorption band system of BPDE bound to DNA exhibits a wavelength dependence that indicates a relatively wide orientational distribution of the z axis of pyrene. Fluorescence data support the conclusion of a heterogeneous distribution, and a very low polarization anisotropy indicates a mobility between the different orientational states, which is rapid compared to the fluorescence lifetime (nanosecond time scale). Attempts are made to simulate the observed LDr features of the (+)-anti-BPDE-poly(dG-dC) complex using different distribution models on the assumption that the angular dependence of the spectral perturbation is due to dispersive interactions with DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA polymerase eta participates in the mutagenic bypass of adducts induced by benzo[a]pyrene diol epoxide in mammalian cells

Y-family DNA-polymerases have larger active sites that can accommodate bulky DNA adducts allowing them to bypass these lesions during replication. One member, polymerase eta (pol eta), is specialized for the bypass of UV-induced thymidine-thymidine dimers, correctly inserting two adenines. Loss of pol eta function is the molecular basis for xeroderma pigmentosum (XP) variant where the accumulation of mutations results in a dramatic increase in UV-induced skin cancers. Less is known about the role of pol eta in the bypass of other DNA adducts. A commonly encountered DNA adduct is that caused by benzo[a]pyrene diol epoxide (BPDE), the ultimate carcinogenic metabolite of the environmental chemical benzo[a]pyrene. Here, treatment of pol eta-deficient fibroblasts from humans and mice with BPDE resulted in a significant decrease in Hprt gene mutations. These studies in mammalian cells support a number of in vitro reports that purified pol eta has error-prone activity on plasmids with site-directed BPDE adducts. Sequencing the Hprt gene from this work shows that the majority of mutations are G>T transversions. These data suggest that pol eta has error-prone activity when bypassing BPDE-adducts. Understanding the basis of environmental carcinogen-derived mutations may enable prevention strategies to reduce such mutations with the intent to reduce the number of environmentally relevant cancers.     

Excision of benzo[a]pyrene diol epoxide I adducts from nucleosomal DNA of confluent normal human fibroblasts

The formation and removal of covalent adducts of racemic 7 beta, 8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) was studied in nucleosomal DNA of confluent cultures of normal human fibroblasts (NF). For this purpose NF were prelabeled in their DNA with [14C]-thymidine and treated with [3H]BPDE I. The adducts were composed of 77% (7R)-N2-(7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl)deoxyguanosine, 12% of the corresponding 7S-enantiomer and of minor amounts of adducts to cytosine and adenine. The adduct composition did not change significantly in 24-h post treatment incubation. Bulk mononucleosomes were prepared from micrococcal nuclease digested nuclei and their DNA analyzed by gel electrophoresis. The adduct concentrations were determined in 145 base pair (b.p.) nucleosomal core-DNA, 165 b.p. chromatosomal DNA and in total nuclear DNA. From these data the concentration in nucleosomal linker-DNA was calculated. The initial adduct distribution was non-random and 6.3 times higher in 47 b.p. linker-DNA relative to 145 b.p. core-DNA and 9.2 times higher in 27 b.p. linker-DNA relative to 165 b.p. chromatosomal DNA. Adduct removal was very rapid during the first 8 h and more efficient from linker-DNA than from core-DNA. After this early phase the adducts located in 145 b.p. core-DNA became refractory to further excision and represent a major fraction of the adducts persisting in DNA of NF over a prolonged period. In contrast, further adduct removal was observed from nucleosomal linker-DNA.     

Inhibition of SV40 DNA replication by benzo[a]pyrene diol epoxide adducts: two recovery modes

Anti-benzo[a]pyrene diol epoxide (BPDE) adducts produced in vitro in SV40 initially inhibit SV40 DNA replication in vivo, in cells unexposed to BPDE. A single adduct in a replicon is probably sufficient to block DNA replication. The recovery process appears to begin immediately after infection. The rate of recovery of replicative capacity is inversely related to the initial adduct number. Holding the infected cells temporarily under conditions that prevent viral DNA replication results subsequently in increased recovery, proportional to the holding time. The mechanism of recovery appears to be constitutive and prereplicative. In addition, there is a second mode of recovery which is induced by pretreatment of the host cells with BPDE before infection. The effect of pretreatment is similar to that of extending the holding time before replication: the first molecules begin to replicate earlier but the subsequent rate of recovery is unchanged. The induced mechanism may be either a limited stoichiometric repair process or a slow replicative bypass.     

Modification of deoxyribonucleic acid by a diol epoxide of benzo[a]pyrene. Relation to deoxyribonucleic acid structure and conformation and effects on transfectional activity.

The effects of secondary structure on DNA modification by (+/-)-7 beta, 9 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+/-)BPDE I] were investigated. No differences in the total extent of (+/-) BPDE I binding to double- and single-stranded calf thymus DNA were found. High-performance liquid chromatography (LC) of the nucleoside adducts obtained from hydrolysates of native and denatured calf thymus, as well as from superhelical and linear plasmid DNA, indicated that in all cases the major adduct (60--80% of total adducts) was formed by reaction of the (+) enantiomer of BPDE I with the N-2 position of dG residues in the DNA. A minor adduct formed from the reaction of the (-) enantiomer with dG residues was also detected and was present in greater amounts in denautred DNA than in native DNA. Small amounts of BPDE I--dA and BPDE I--dC adducts were also detected in both the single- and double-stranded DNAs. Restriction enzyme analysis of BPDE I modified SV40 and phage lambda DNA provided evidence that the modification of DNA by this carcinogen is fairly random with respect to nucleotide sequence. Partial hydrolysis of modified plasmid DNA by the single-strand-specific S1 nuclease and LC analysis of the nucleoside adducts in the digested and undigested fractions of the DNA revealed no preferential excision by the S1 nuclease of the different BPDE I--deoxynucleoside adducts. Functional changes in BPDE I modified DNA were demonstrated. With increasing extents of modification, there was a decrease in the ability of plasmid DNA to transfect a receptive Escherichia coli strain to antibiotic resistance.