Fluorescence HPLC methods for detecting benzo[a]pyrene-7,8-dihydrodiol 9,10-oxide-deoxyadenosine adducts in enzyme-digests of modified DNA: improved sensitivity

The fluorescence of mononucleoside adducts derived from thebinding of anti-7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetra-hydrobenzo[a]pyrene(BPDE I) to N⁶-deoxyadenosine (BPDE-dA adducts) is 10–100times stronger (depending on the methanol/water solvent composition)than the fluorescence of adducts derived from the binding ofthis diol epoxide derivative to N²-deoxyguanosine. It is shownhere that these fluorescence characteristics can be used toquantitate the relatively low yields of BPDE-dA adducts by fluorescencedetection when BPDE–modified DNA is subjected to enzymaticdegradation to the mononucleoside levels, followed by HPLC analysisof the digests.     

Characterization of common CYP1B1 variants with different capacity for benzo[a]pyrene-7,8-dihydrodiol epoxide formation from benzo[a]pyrene

Cytochrome P450 1B1 (CYP1B1), an extrahepatic enzyme inducible by smoking, is overexpressed in many tumors and catalyzes the metabolic activation of procarcinogens such as polycyclic aromatic hydrocarbons. In human, CYP1B1 is genetically polymorphic and five common missense mutations causing amino acid substitution have been identified. In this study, we have investigated CYP1B1 haplotypes present in a Spanish population and carried out functional analyses of the corresponding enzymes in yeast using benzo[a]pyrene as a substrate. CYP1B1*1, CYP1B1*2, CYP1B1*3, CYP1B1*4, CYP1B1*6, and CYP1B1*7, encoding combinations of the Arg48Gly, Ala119Ser, Leu432Val, Asn453Ser, and Ala443Gly amino acid substitutions, were present at frequencies of 14.3%, 25.5%, 38.8%, 18.1%, 0.4%, and 2.6%, respectively. The variant CYP1B1 forms were heterologously expressed with human reductase in Saccharomyces cerevisiae and kinetic analyses of benzo[a]pyrene metabolism were carried out. CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, Leu432Val, and Ala443Gly, exhibited a significantly decreased capacity (P < 0.001) for the formation of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol from benzo[a]pyrene as indicated by lower intrinsic clearance (Vmax/Km). A somewhat decreased clearance was observed for CYP1B1.4, whereas no significant differences in kinetic properties among the remaining variant enzymes were observed as compared with CYP1B1.1. Thus, genetic polymorphism in the CYP1B1 gene, as defined by the haplotypes investigated, might cause interindividual differences in susceptibility (e.g., to lung cancer induced by smoking). The results indicate the necessity to make molecular epidemiologic investigations regarding the association of the specific CYP1B1 haplotypes and cancer risk.     

Identification of benzo[a]pyrene-7,8-dione as an authentic metabolite of ({+/-})-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene in isolated rat hepatocytes

Dihydrodiol dehydrogenase (DD) has been shown to catalyze theoxidation of (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-diol) to yield benzo[a]pyrene-7,8-dione (BPQ) in uninducedfortified rat liver S100 fractions but the formation of BPQhas not been observed in whole cells. In these studies [³H]BP-diolwas incubated with isolated hepatocytes from uninduced ratsfor 0–20 min at 37°C. Organic-extractable radioactivityin the cell media accounted for 20% of the total [³H]BP-dioladded. Reverse phase (RP)-HPLC analysis of this fraction revealedthe formation of an unknown metabolite that co-chromatographedwith an authentic synthetic standard of BPQ. The identity ofthe unknown metabolite was further established by: (i) co-chromatographywith synthetic BPQ under both RP- and normal phase-HPLC conditionsusing diode array detection, which indicated that the metaboliteshared UV/ vis spectral identity with standard BPQ; and by (ii)electron impact mass spectrometry of the unknown metabolitewhich gave the same parent and fragment ions as the syntheticstandard. The formation of BPQ by isolated hepatocytes was foundto be 0.50 nmol/3x10⁶ cells/10 min, and represented 7% of thetotal organic-soluble metabolites in the extracellular media.Its formation was abolished by the addition of indomethacin,a competitive inhibitor of DD, indicating that this enzyme wasresponsible for BPQ formation. Other organic-soluble metabolitesformed corresponded to BP-tetraols (hydrolysis products of theanti-and syn-diol epoxides). Examination of the aqueous phaseof the extracellular media indicated that a large portion ofBP-diol was converted to glucuronide and sulfate conjugates.Under the conditions employed BP-tetraols and BPQ were formedto an equal extent implying that in hepatocytes isolated fromuninduced rats, DD and CYP1A1 contributed equally to the metabolismof BP-diol.     

Metabolism of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and glutathione conjugates in isolated rat hepatocytes

Isolated hepatocytes from 3-methylcholanthrene (MC)-treatedrats metabolized trans-7, 8-dihydroxy-7, 8-dihydrobenzo[a]pyrene(BP-7, 8-diol) and (± )-7ß, 8-dihydroxy-9,10-oxy-7, 8, 9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE) to watersoluble conjugates including glutathione (GSH) conjugates. Underthe conditions employed 35% of total water soluble productsderived from BP-7, 8-diol could be accounted for by GSH conjugates.The corresponding figure for anti-BPDE was estimated to be >80%.Isolated hepatocytes metabolized BP-7, 8-diol and anti-BPDEto GSH conjugates at maximal rates of 0.5 and 9 nmol per 106cells per min, respectively. Thus, identifying the rate limitingstep in the reaction sequence as the metabolism of BP-7, 8-diolto the GSH conjugating intermediates. In addition to the directconjugation of anti-BPDE with GSH, anti-BPDE but not the correspondingBP-tetraols, was further metabolized to reactive intermediatesthat subsequently bound to cellular proteins or reacted withGSH forming water soluble conjugates. The identity or identitiesof these novel reactive intermediates is discussed.     

Metabolic activation of benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide to protein-binding products and the inhibitory effect of glutathione and cysteine

Trans-7, 8-dihydroxy-7, 8-dihydrobenzo(a)pyrene (BP-7, 8-diol)and the anti-isomer of trans-7, 8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene(BPDE) were found to be activated by microsomes isolated from3-methylcholanthrene (MC)-treated rats to reactive intermediatesthat bound covalently to microsomal proteins. The extent ofbinding was markedly reduced by the presence of reduced glutathione(GSH) or cysteine. Fluorescence spectroscopic studies on theproducts derived from BP-7,8-diol and BPDE after microsomalactivation in presence of GSH or cysteine revealed the formationof a common reactive intermediate with unique fluorescence properties.The involvement of cytochrome P-448 in the activation of BP-7,8-dioland BPDE to protein-binding products was inferred by the requirementfor NADPH and almost complete inhibition by -naphtho-flavone.Furthermore, microsomes from MC-treated rats could be replacedby a reconstituted system containing purified cytochrome P-448,NADPH-cytochrome reductase and co-factors. The conjugation ofthe reactive intermediates from BP-7,8-diol and BPDE with GSHor cysteine did not require the presence of either microsomesor cyutosol, thus indicating a non-catalytic reaction. Theseresults emphasize the importance of cellular nucleophiles suchas GSH and cysteine in the deactivation of reactive benzo(a)pyrene(BP) intermediates and also provides evidence for the furtheractivation of the ultimate carcinogen BPDE to more reactivedectro-philes and may thus have relevance concerning the regulationof BP-induced carcinogenesis.     

Peroxidase-mediated glutathione conjugation of benzo[a]pyrene-7,8-dihydrodiol is enhanced by benzo[a]pyrene phenols in vitro.

We reported previously that glutathione (GSH) is oxidized by peroxidases to a thiyl radical that can react with a number of chemicals, including the penultimate carcinogenic metabolite benzo[a]pyrene-7,8-dihydrodiol (7,8-B[a]PD), to give GSH conjugates. Here, we report that phenolic metabolites of benzo[a]pyrene (B[a]P) enhance the peroxidase-mediated formation of glutathione conjugates of 7,8-B[a]PD. The GSH conjugation of 7,8-B[a]PD in a horseradish peroxidase/peroxide system was increased over control values as follows: 9-OH-B[a]P by 4-fold, 7-OH-B[a]P by 3-fold, 1-OH-B[a]P by 2-fold. In contrast 3-OH-B[a]P was ineffective. A phenolic derivative of another polycyclic aromatic hydrocarbon (PAH), benz[a]anthracene, also enhanced GSH conjugation of 7,8-B[a]PD. The enhancement was dependent upon the presence of the phenol, horseradish peroxidase and peroxide. The phenolic compounds, including 3-OH-B[a]P, were also efficient reducing cofactors for the peroxidase. With the exception of 3-OH-B[a]P, the phenolic metabolites of PAH enhanced peroxidase-mediated formation of thiyl radical as detected by electron spin resonance spectrometry. Since both phenols and dihydrodiols are metabolites of B[a]P catalyzed by the cytochromes P450 system, enhancement of peroxidase-dependent 7,8-B[a]PD-GSH conjugation by phenols suggests a possible interaction between peroxidases and cytochromes P450 systems. This interaction may contribute to the detoxication of the penultimate carcinogenic PAH-dihydrodiols and other chemicals.     

p53-dependent global genomic repair of benzo[a]pyrene-7,8-diol-9,10-epoxide adducts in human cells

The global genomic repair of DNA adducts formed by the human carcinogen (+/-)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) has been studied by 32P-postlabeling in human fibroblasts in which p53 expression can be regulated. At low BPDE adduct levels (10-50 adducts/10(8) nucleotides), repair was rapid and essentially complete within 24 h in p53+ cells, whereas no repair was detected within 72 h in similarly treated p53- cells. At 10-fold higher BPDE adduct levels, repair under both conditions was rapid up to 8 h, after which a low level of adducts persisted only in p53- cells. These results demonstrate a dependence on p53 for the efficient repair of BPDE adducts at levels that are relevant to human environmental exposure and, thus, have significant implications for human carcinogenesis.     

Evidence for substantial formation of r-7,t-8-dihydroxy-c-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene- deoxyguanosine in human lymphocytes treated in vitro with benzo[a]pyrene

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).     

Inactivation of DNA-binding metabolites of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by glutathione and glutathione S-transferases

The binding to DNA of reactive metabolites of trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene(BP-7, 8-diol) was studied following the incubation of tritiatedbenzo[a]pyrene (BP) and BP-7, 8-diol with nuclei from liversof 3-methyl-cholanthrene-treated rats. Binding was inhibitedto a small extent by glutathione (GSH) alone and to a much greaterextent by GSH and cytosol or purified GSH-transferases B andE. In this respect GSH-transferases A and C were also active,but less so. Inhibition of binding of BP-7,8-diol metabolitesto DNA mediated by GSH-transferases was associated with theformation of GSH conjugates. The extent of inhibition of bindingwas similar in incubations of nuclei alone, nuclei and rat livermicrosomes, and calf thymus DNA and rat liver microsomes. Thisindicates that reactive metabolites of BP-7, 8-diol, formedeither by nuclei or microsomes, are readily accessible to solubleGSH-transferases. GSH and cytosol were also active in inhibitingDNA-binding of reactive metabolites from 9-hydroxybenzo[a]pyrene(9-OH-BP). Thus, in the rat hepatocyte GSH and GSH-transferasesmay be important in protecting DNA from electrophilic attackby reactive BP-7, 8-diol and 9-OH-BP species.     

Differential metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol by human CYP1A1 variants

Cytochrome P450 1A1 (CYP1A1) plays a key role in the metabolism of carcinogens, such as benzo[a]pyrene (B[a]P) and metabolites to ultimate carcinogens. Three human allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were coexpressed by coinfection of baculovirus-infected insect cells with human NADPH-P450 reductase. These recombinant enzymes (in microsomal membranes) were used to analyze whether CYP1A1 polymorphisms affect catalytic activities towards B[a]P and B[a]P-7,8-dihydrodiol. The complete spectrum of phase I metabolites, including the tetrahydrotetrols resulting from hydrolysis of the ultimate carcinogen, B[a]P-7,8-dihydrodiol-9,10-epoxide, was examined by HPLC. Wild-type enzyme showed the highest total metabolism of B[a]P, CYP1A1.2 was approximately 50%, and CYP1A1.4 approximately 70%. Km values for all metabolites with CYP1A1.2 were generally significantly lower than with wild-type enzyme (e.g. B[a]P-7,8-diol formation: 13.8 microM for wild-type, 3.5 microM for CYP1A1.2 and 7.7 microM for CYP1A1.4). Addition of epoxide hydrolase markedly increases the relative diol-to-phenol activities by all three variants. However, CYP1A1.4 exhibits the greatest efficiency to produce diol species. Each variant produced the diol epoxides from B[a]P-7,8-dihydrodiol. CYP1A1.1 exhibited with 10.4 pmol/min/pmol CYP1A1 the greatest total rate for 7,8-diol metabolites followed by CYP1A1.2 (7.2 pmol/min/pmol CYP1A1) and CYP1A1.4 (5.5 pmol/min/pmol CYP1A1). All enzyme variants produced about three times more diol epoxide 2-derived metabolites than diol epoxide 1-derived ones, whereby both rare allelic variants exhibited statistically significantly increased formation of diol epoxide 2. This study showed that the three CYP1A1 variants had different enzyme kinetics properties to produce both the diol metabolites from B[a]P and the ultimate mutagenic species diol epoxide 2 from B[a]P-7,8-dihydrodiol, which must be considered in the evaluation of individual susceptibility to cancer.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-diol by human cytochrome P450 1B1

Benzo[a]pyrene (B[a]P), a ubiquitous environmental, tobacco and dietary carcinogen, has been implicated in human cancer etiology. The role of human cytochrome P450 1B1 in the metabolism of B[a]P is poorly understood. Using microsomal preparations of human P450 1A1, 1A2 and 1B1 expressed in baculovirus-infected insect cells, as well as human and rat P450 1B1 expressed in yeast, we have determined the metabolism of B[a]P, with and without the addition of exogenous epoxide hydrolase, and B[a]P-7,8-dihydrodiol (7,8-diol), each substrate at a concentration of 10 microM. HPLC analysis detected eight major metabolites of B[a]P and four metabolites of the 7,8-diol. The results of these studies indicate that cytochrome P450 1B1 carries out metabolism of B[a]P along the pathway to the postulated ultimate carcinogen, the diol epoxide 2, at rates much higher than P450 1A2 but less than P450 1A1. The rates of formation of the 7,8-diol metabolite in incubations with epoxide hydrolase are 0.17 and 0.38 nmol/min/nmol P450 for human P450 1B1 and 1A1, respectively, and undetectable for 1A2. The rates of total tetrol metabolite formation from the 7,8-diol, which are indicative of diol epoxide formation, are 0.60, 0.43 and 2.58 nmol/min/nmol P450 for 1B1, 1A2 and 1A1 respectively. In agreement with other reports of rat P450 1B1 activity, our data show this rat enzyme to be very active for B[a]P and 7,8-diol, with rates higher than human P450 1B1. In addition to the established role of P450 1A1 in B[a]P metabolism, P450 1B1 may significantly contribute to B[a]P and 7,8-diol metabolism and carcinogenesis in rodent tumor models and in humans.      

Separation and characterization of post-labeled DNA adducts of stereoisomers of benzo[a]pyrene-7,8-diol-9,10-epoxide by immobilized boronate chromatography and HPLC analysis

The carcinogenic polycydlic aromatic hydrocarbon (PAH) benzo[a]pyrene(BaP) is enzymatically activated in cells to an ultimate carcinogenicmetabolite, benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BaPDE),which reacts with DNA to form covalent adducts involved in theinitiation of cancer. Previously, a post-labeling procedurethat uses adenosne-5'-O-(3'-[³⁵S]-thiotriphosphate) was developedto facilitate adduct analysis by HPLC. The much greater carcinogenicpotency of (+)-anti-BaPDE makes it essential to be able to separateand identify the adducts formed by all four BaPDE enantiomersin DNA of cells exposed to BaP. Reversed-phase HPLC (RPHPLC)resolved the major (+)-anti-BaPDE-N²-deoxyguanosine [(+)-anti-BaPDE-N²-dG]adduct from the (+)-syn-BaPDE-N²-dG adduct. However, anti-BaPDE-N²-dGadducts formed by (+)-and (–)-anti-BaPDE were not resolved.By using ion-pair RPHPLC (IP-RPHPLC) with tetrabutylammoniumphosphate, the [³⁵S]post-labeled (–)-anti-BaPDE-N²-dGadduct eluted 3 min prior to the [³⁵S]labeled (+)-anti-BaPDE-N²-dGadduct. In contrast, the major syn-BaPDE-N²-dG adducts wereresolved better by RPHPLC than by IP-RPHPLC. The differencein conditions required for optimal separation of anti- and syn-BaPDE-DNAadducts necessitated the development of an immobilized boronatechromatography technique for the separation of anti- from syn-BaPDE-DNAadducts prior to analytical HPLC analysis. At 4°C and withelution buffers containing high salt concentrations, the [³⁵S]post-labeledanti-BaPDE-DNA adducts were selectively retained by a boronatecolumn whereas the [³⁵S]labeled syn- BaPDE-DNA adducts werenot. Analysis of the multiple BaP-DNA adducts formed in BaP-treatedhamster embryo cells by these techniques gave results comparableto those obtained by other methods. The major BaP-DNA adductswere anti-BaPDE-N²-dG 14% from (–)- and 86% from (+)-anti-BaPDE.The ability of these techniques to detect low levels of PAH-DNAadducts because of the high specific radioactivity of ³⁵S andto separate the DNA adducts formed by stereolsomeric PAN diolepoxides adducts by boronate chromatography and HPLC will facilitatestudies of the role of individual PAH-DNA adducts in the inductionof biological effects such as toxicity and carcinogenesis.     

DNA polymerase action on benzo[a]pyrene-DNA adducts.

A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.     

Role of glutathione conjugate efflux in cellular protection against benzo[a]pyrene-7,8-diol-9,10-epoxide-induced DNA damage

Glutathione (GSH) conjugation of (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE], the activated metabolite of benzo[a]pyrene, is believed to be an important mechanism in detoxification of this environmental and dietary carcinogen. Here, we demonstrate that the intracellular accumulation of GSH conjugate of (+)-anti-BPDE (BPD-SG) caused a statistically significant increase in (+)-anti-BPDE-induced DNA adduction. The relationship between intracellular accumulation of BPD-SG and (+)-anti-BPDE-induced DNA adduction was studied using a canine kidney epithelial cell line (MDCKII) and its variants overexpressing multidrug resistance transporter (MDR1) or canalicular multispecific organic anion transporter (cMOAT; also known as multidrug resistance protein 2). MDR1 and cMOAT are implicated in ATP-dependent efflux of anticancer drugs or GSH-xenobiotic conjugates, or both. The GST activity toward (+)-anti-BPDE in parental MDCKII cells did not differ from that in subline overexpressing MDR1 (MDCKII-MDR1) or cMOAT (MDCKII-cMOAT). Intracellular accumulation of BPD-SG, after a 5- or 10-min incubation with 1 microM (+)-anti-BPDE, was significantly higher in parental (41- to 67-fold) and MDCK II-MDR1 cells (31- to 43-fold) than in the MDCKII-cMOAT cells. Interestingly, the levels of DNA adducts of (+)-anti-BPDE, after a 30-min incubation with 0.1 or 0.5 microM [(3)H](+)-anti-BPDE, were significantly higher (about 2.1- and 1.7-fold, respectively) in parental cells than in the MDCKII-cMOAT cells. The results of the present study indicate that in addition to GSH conjugation, the efflux of BPD-SG may be essential for cellular protection against (+)-anti-BPDE-induced DNA damage.     

Metabolism of benzo[a]pyrene and benzo[a]pyrene-7,8-dihydrodiol in human mammary epithelial cells: feedback inhibition by 7-hydroxybenzo[a]pyrene

The metabolism of benzo[a]pyrene (B[a]P) and (-)-transbenzo[a]pyrene-7,8-dihydrodiol (B[a]P-diol) was compared in human mammary epithelial cells (HMEC) grown in serum-free medium, MCDB-170. Conversion of B[a]P-diol to the carcinogen (+)-benzo[a]pyrene-7,8-dihydroxy-9,10-epoxide (BPDE), as measured by analysis of their tetraol hydrolysis products, occurred much more efficiently in cultures incubated with [3H]B[a]P-diol than in cultures incubated with [3H]B[a]P. In cultures pretreated with unlabeled B[a]P (24 h, 400 nM), the conversion of [3H]-B[a]P-diol to [3H]tetraols is inhibited 49%, while the conversion of [3H]B[a]P to [3H]B[a]P-diol- is not affected. These observations led to the identification of a major B[a]P-derived metabolite as 7-hydroxybenzo[a]pyrene (B[a]P-7-ol), which was found to be an extremely potent and selective inhibitor of the conversion of B[a]P-diol to BPDE, with a KI estimated at 3-12 nM. Thus B[a]P activation in HMEC appears to be significantly limited by a feedback inhibition pathway induced by B[a]P-7-ol. The potency and selectivity of the B[a]P-7-ol-induced inhibition suggests that the diol to diolepoxide conversion is affected by a selective oxygenase in HMEC, rather than a non-enzymatic, peroxy radical-induced mechanism. B[a]P-7-ol should prove to be a valuable tool in the study of B[a]P carcinogenesis.     

Chinese medicinal herbs modulate mutagenesis, DNA binding and metabolism of benzo[a]pyrene 7,8-dihydrodiol and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide.

Oldenlandia diffusa(OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumors. In this study, the effects of aqueous extracts of these two herbs on benzo[a]pyrene 7,8-dihydrodiol. (BaP 7,8-DHD) and benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE)-induced mutagenesis using Salmonella typhimurium TA100 as the bacterial tester strain and rat liver 9000 x g supernatant (S9) as the metabolic activation system were assessed. We also determined the effects of these two herbs on BaP 7,8-DHD and BPDE binding to calf thymus DNA. Organosoluble metabolites of BaP 7,8-DHD and water-soluble conjugates of BaP 7,8-DHD and BPDE were analyzed by high-performance liquid chromatography (HPLC) and alumina column liquid chromatography. Mutagenesis assays revealed that these two herbs produced a significant concentration-dependent inhibition of histidine-independent (His+) revertants induced by BaP 7,8-DHD and BPDE. OD and SB also inhibited BPDE-induced mutagenesis in a concentration-dependent manner in the absence of S9. SB had a greater inhibitory effect than OD. SB significantly inhibited BaP 7,8-DHD and BPDE binding to DNA while OD significantly enhanced DNA binding of both compounds. OD and SB inhibited the formation of organosoluble metabolites of BaP 7,8-DHD and decreased the formation of water-soluble conjugates of BaP 7,8-DHD and BPDE. However, the fraction of the total radioactivity in the water-soluble conjugates present as sulfate and glutathione was increased by OD and SB. Glucuronide fraction was decreased. The results of this study affirm our previous work suggesting that these two Chinese medicinal herbs possess antimutagenic properties and further suggest that they act as blocking agents through a scavenging mechanism.     

Benzo[a]pyrene-7,8-diol-9,10-epoxide-DNA adducts and increased p53 protein in mouse skin

p53 protein expression has been shown to increase in responseto DNA damage in cell culture systems. We have Studied p53 expressionand benzo[a]pyrene (B[a]P) -induced DNA-damage in the form ofbenzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts as measuredby synchronous fluorescence spectrophotometry (SFS) in B[a]P-treatedC57BL/6 mouse skin. Polyclonal murine antibody CM5, which iscomparable to human CM1, detecting both wild-type and mutatedprotein, was used. BPDE-DNA adducts reached their maximum at24 h after all dosage regimens, but were very well detectablealso at 12 and 48 h after the treatment, while no adducts weremeasurable at 1 week and thereafter. P53 expression was seenin 9/17(53%) skin samples from mouse treated with 500 µgof B[a]P 12–48 h after the treatment, while all 25 (100%)cases of similarly treated mouse skins were negative after 30weeks of the treatment. Only one positive sample of total 11was found among mice treated with repeated 62.5 µg dosesand this was 24 h after the last treatment. After one 62.5 µgdose all mice were negative. This is the first report of anassociation of p53 protein with DNA damage in vivo and givessupport for the putative function of p53 in cellular defensemachinery towards chemical damage.