Monoclonal antibodies to a benzo[a]pyrene diolepoxide modified protein

Monoclonal antibodies have been developed which specificallyrecognize protein modified by 7ß,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE-I). These antibodies have been characterized as to sensitivityand specificity by an enzyme-linked immunosorbent assay. Theyreact with BPDE-I-modified proteins and do not cross-react withnon-modified protein. All the antibodies have higher sensitivitytoward BPDE-I-tetraols than to BPDE-I-modified protein. Theycross-react with BPDE-I-deoxyguanosine, BPDE-I-DNA and BPDE-II-rabbitserum albumin. Because of the high antibody sensitivity fortetraols, an immunoassay was developed for quantitating DNAand protein adducts based on release of tetraols from modifiedsamples. This approach was validated using radiolabeled BPDE-Imodified DNA and protein.     

Post-mortem stability of benzo[a]pyrene diolepoxide-DNA adducts in rat organs

In order to evaluate the stability of benzo[a]pyrene diolepoxide-DNAadducts two separate studies were carried out in rats, eithertreated i.p. with benzo[a]pyrene (100 mg/kg body wt) or sham-exposed.The measurement of DNA adducts in 155 samples of liver, lungor heart, each of them tested in duplicate, was performed bysynchronous fluorescence spectrophotometry. In the first studyfragments of rat liver or lung were stored for varying timesat varying temperatures. No decrease of adduct levels occurredat 4°C for at least 72 h, whereas a significant decreasewas recorded in both liver and lung after 48 h at 20°C or24 h at 37°C. In the second study liver, lungs and heartwere collected from rats either immediately after killing orafter storage of cadavers for 16 h at 20°C or 16 h at 20°Cplus 24 h at 4°C, thereby mimicking typical storage conditionsof human cadavers before autopsy. Under these conditions nosignificant variation of fluorescent adducts was observed inany organ. In conclusion, at least for this kind of adduct,the use of autopsy samples following proper storage of cadaversseems to be acceptable.     

Spectroscopic characteristics and site I/site II classification of cis and trans benzo[a]pyrene diolepoxide enantiomer-guanosine adducts in oligonucleotides and polynucleotides

The highly tumorigenic isomer (+)-7,8-dihydroxy-anti-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE] and its non-tumorigenic enantiomer (-)-anti-BPDE are known to react predominantly with the exocyclic amino group (N2) of deoxyguanine in DNA and to form adducts of different conformations. The spectroscopic characteristics (UV absorbance, fluorescence and circular dichroism) of stereochemically defined (+)-trans, (-)-trans, (+)-cis and (-)-cis d(5'-CACATGBPDETACAC) adducts in the single-stranded form, or complexed with the complementary strand d(5'-GTGTACATGTG) in aqueous solution, were investigated. The spectroscopic characteristics of the double-stranded d(5'-CACATGBPDETACAC).d(5'-GTGTACATGTG) adducts can be interpreted in terms of two types of conformations. In site I-type conformations, there is an approximately 10 nm red shift in the absorption maxima, which is attributed to significant pyrenyl residue-base interactions; in site II-type adducts, the red shift is only approximately 2-3 nm, and the pyrene ring system is located at external, solvent-exposed binding sites. The spectroscopic characteristics of the BPDE-modified duplexes are of the site II type for the (+)- and (-)-trans, and of the site I type for the (+)- and (-)-cis adducts. In adducts derived from the binding of (+)-anti-BPDE to poly(dG-dC).(dG-dC) and poly(dG).(dC), the trans/cis BPDE-N2-dG adduct ratio is 6 +/- 1; in the case of (-)-anti-BPDE this ratio is only 0.4 +/- 0.1 and 0.6 +/- 0.15 in poly(dG-dC).(dG-dC) and poly(dG).(dC) respectively. The spectroscopic properties of these BPDE-modified polynucleotide adducts are consistent with those of the BPDE-modified oligonucleotide complexes; the cis adducts are correlated with site I adduct conformations, while the trans adducts are of the site II type. The correlations between adduct characteristics and biological activities of the two BPDE enantiomers are discussed.     

On the nature of the mutations induced by the diolepoxide of benzo[a]pyrene in mammalian cells

We have previously described the induction by r-7,t-8-di-hydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BPDE) of 8-azaguanine resistant (ACr) Chinese hamster V79 cellmutants, 40% of which were found to contain material which cross-reacted(CRM) with antiserum to hypoxanthine-guanine phosphoribosyltransferase(HPRT) and whose AG^r phenotype we ascribed to missense mutation(Brookes et al., 1982). We now report that we have been unableto demonstrate by Southern blotting any change in the HPRT genein 11 CRM-negative mutants. We have, moreover, found HPRT mRNAof normal size and amount in most of these mutants. Examinationof the revertants of one mutant indicates the probable occurrenceof changes within an amino acid codon in the genesis of mutantand revertant. Our results suggest that BPDE functions primarilyas a point mutagen.     

Synthesis of a novel fluorinated benzo[a]pyrene: 4,5-difluorobenzo[a]pyrene

The synthesis of 4,5-difluorobenzo[a]pyrene, as a fluorinated probe to investigate the involvement of the K-region in the further metabolic activation of benzo[a]pyrene metabolites, is described. Benzo[a]pyrene-4,5-dione obtained from 2,3-dichloro-5,6-dicyano-1,4-benzoquinone oxidation of cis-4,5-dihydro-4,5-dihydroxybenzo[a]pyrene was fluorinated with dimethylaminosulfur trifluoride to give 4H,5H,4,4,5,5,-tetra-fluorobenzo[a]pyrene. Defluorination using lithium aluminum hydride in tetrahydrofuran gave 4,5,-difluorobenzo[a]pyrene.     

Biochemical and immunological characterisation of mutants induced in V79 Chinese hamster cells by a benzo[a]pyrene diolepoxide

A series of 8-azaguanine resistant mutants was induced by treatmentof V79 Chinese hamster cells with either r-7,t-8-di-hydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene(anti-BPDE) or methyinitrosourea (MNU). Hypoxanthine phos-phoribosyitransferase(HPRT) activity in the mutants was determined for both hypoxanthineand azaguanine as substrates. With antiserum to purified brainHPRT, cross-reacting material was also determined and analysedby two dimensional polyacrylamide gel electrophoresis. By thesecriteria mutants induced by anti-EPDE or MNU did not differappreciably and the data obtained was consistent with the inductionof point mutations by both carcinogens. The relevance of theseresults to the correlation of carcinogencity with mutagenicityin V79 cells, but not in bacteria, is discussed.     

Detection of benzo[a]pyrene-DNA adducts in cultured cells treated with benzo[a]pyrene diol-epoxide by quantitative immunofluorescence microscopy and 32P-postlabelling; immunofluorescence analysis of benzo[a]pyrene-DNA adducts in bronchial cells from smoking individuals

Monoclonal antibodies were raised against the reaction product of benzo[a]pyrene diol-epoxide (BPDE) and deoxyguanosine-5'-monophosphate. The antibodies were used for detection of DNA adducts in situ in BPDE-treated cultured human fibroblasts by immunofluorescence microscopy. Analogue-digital conversion of the fluorescence signal and further image processing allowed measurement of the immunospecific fluorescence in the nuclei of these cells. The results are compared with the adduct levels measured in isolated DNA by 32P-postlabelling. Preliminary results are shown of the application of the immunofluorescence method to the analysis of DNA adducts in bronchial cells obtained from smoking individuals.     

Properties of covalent benzo[a] pyrene diol eporide-DNA adducts investigated by fluorescence techniques

The spectroscopic absorption and fluorescence properties ofadducts derived from the covalent binding of (±)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,l0-tetrahydrobenzo[a]pymne(BPDE) to DNA are re-examined in view of conflicting interpretationsregarding the Coaformations of these adducts which currentlyexist in the literature. The fluorescence decay profiles wereaccurately determined utilizing synchrotron-pulsed light sourceexcitation and the time-correlated single photon counting technique.The coaformational properties of the adducts were probed bydetermining their accessibilities to acrylamide, a known fluorescencequencher, and by comparing the accessibilities of the BPDE-DNAadducts with those of known model systems with intercalative,partially intercalative and minor groove binding conformations.In contrast toanyofthesemodelsystems,tkfhmesmmofthearomahicpyrenyl residues in the covalent BPDE - DNA ad- exhibit significantsensitivity to acrylamide, suggesting that these residues arelocated at binding sites with significant solvent exposure.A quantitative analysis of the acrylamide fluorescence quenchingaccording to a dynamic Stem—Volmer quenching model suggeststhe following characteristics: the major (65%) component (1.4ns lifetime) is characterized by significant exposure to thesotvent environment; the second component (6–7 ns lifetime)can be subdivided into a solvent-accessible and a solvent-inaccessiblecomponent, the inaccessible fraction being attributed to minoradducts, possibly with a quasi-intercalative conformation. Theamplitude of the third, long-lived (200-ns) component is variable;it arises from the photochemical decomposition of the adductswhich gives rise to tetraols (7,8,9,1O-tetrahydro-tetrahydroxybenzo[a]pyrene).The variable content of these degradation products accountsfor most discrepancies in the fluorescence properties of thecovalent BPDE-DNA adducts previously reported.     

Immunoaffinity chromatography of carcinogen DNA adducts with polyclonal antibodies directed against benzo[a]pyrene diol-epoxide-DNA

Polyclonal antibodies specific for (+/-)-trans-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(BPDE) CAS: 58917-67-2]-DNA adducts were obtained from the sera of New Zealand White rabbits immunized with BPDE-DNA. These antibodies did not recognize benzo[a]pyrene [(BP) CAS: 50-32-8] or DNA alone or other carcinogen adducts, such as aflatoxin (CAS: 1402-68-2)-DNA or aminopyrene (CAS: 58-15-1)-DNA, up to the concentrations used. In a competitive enzyme-linked immunosorbent assay, 0.18 microgram BPDE-DNA/ml (single stranded), equivalent to 7 pmol BPDE adduct, caused 50% inhibition with this antibody. (When referring to the DNA content of BPDE-DNA, the authors gave the concentration in microgram/ml; when referring to the BPDE content of BPDE-DNA, the authors gave the concentration as pmol/ml.) Chemical and enzymic modifications of the BPDE-DNA substrate suggested that the epitope for the antibody is greater than that represented by a BPDE-nucleoside adduct. The specific BPDE-DNA antibodies were covalently bound to cyanogen bromide-activated Sepharose 4B, and the extent of ligand binding to the immunoaffinity column was measured with the use of [3H]BPDE-DNA as substrate. Maximum binding to the immunoaffinity column was obtained after DNase 1 digestion of [3H]BPDE-DNA: The bound adducts could be readily eluted from the column with 50 mM NaOH. The binding of DNase 1-digested [3H]BPDE-DNA to the immunoaffinity column was dose related and not affected by the addition of unmodified DNA. The columns have proven to be reusable. Samples of [3H]BP-DNA isolated from the skin of mice treated topically with either 0.75 mumol [3H]BP/mouse or 1.5 mumol [3H]BP/mouse were examined by immunoaffinity chromatography. Binding values of 6.0 and 12.2 pmol BP/mg DNA were obtained; these values from immunoaffinity chromatography were slightly lower than those determined by high-pressure liquid chromatography analysis (9 and 17 pmol BP/mg DNA). With chemically reacted BPDE-DNA, around 70% of that applied was retained by immunoaffinity chromatography, whereas with [3H]BP-DNA isolated from the in vivo treatment of mouse skin, only 40% was retained--a possible reflection of the greater heterogeneity of the in vivo BP-DNA adducts. This immunoaffinity chromatography technique should prove useful in the selective examination of levels of BPDE-DNA adducts present in biological samples.     

Topical treatment of mice with benzo[a]pyrene or parenteral administration of benzo[a]pyrene diol epoxide--DNA to rats results in faecal excretion of a putative benzo[a]pyrene diol epoxide--deoxyguanosine adduct

The administration of [³H]BPDE-DNA, whether by i.p. or i.v.injection, to male Wistar rats resulted in the majority of theradioactivity being recovered in the faeces. Excretion was rapid:within 24 h post-injection, 45% of the applied dose was recoveredin the faeces. H.p.l.c. analysis of radioactive material extractedfrom the faeces by methanol showed that it contained a singlecomponent which co-chromatographed with [³H]BPDE-dGuo and whichwas not affected by treatment with alkaline phosphatase, arylsulphatase or ß-glucuronidase. To determine if thisphenomenon occurs after topical application of BP to a targettissue, such as mouse skin, animals were treated with [³H]BPand their faeces collected. After an extensive extraction procedureinvolving differential solubility in organic solvents, SephadexLH-20 chromatography and h.p.l.c, a product was isolated frommice faeces which had characteristics consistent with a [³H]BPDE-dGuoadduct. These findings are discussed in relation to detectionof BPDE adducts in human populations.     

Radioimmunoassay for benzo[a]pyrene.

A benzo[a]pyrene (BP)-bovine serum albumin conjugate was synthesized and used to immunize 2.5- to 3.0-kg New Zealand White rabbits. The resulting antisera to BP bound trace amounts of [3H] (55 pg; 6,000 counts/min). A radioimmunoassay (RIA) to BP was developed by the antiserum first being titered with the [3H]BP and then a standard curve being constructed from the addition of unlabeled BP (0.4-15.8 pmoles; 0.1-4.0 ng/assay tube). The RIA could reliably detect 0.4 pmoles (0.1 ng) BP. The specificity with respect to structurally related polycyclic aromatic hydrocarbons was examined by means of competitive binding. Here concentrations of 4.0-400 pmoles of the compound were used, and the resulting competitive curves were compared for relative cross-reactivity at 50% B/B0 (counts per minute labeled BP bound to antiserum in the presence of corresponding concentrations of unlabeled BP/counts per minute labeled BP bound to antiserum in the absence of unlabeled BP). The antiserum B4-3 was specific to BP with the closest cross-reacting substance being 3-hydroxybenzo[a]pyrene (20% relative cross-reactivity). BP added to pooled human serum was measurable by the RIA at 1 ng/ml. A BP RIA may have potential use for the quantification of the absorbed dose of this carcinogen in man.     

Molecular dosimetry of DNA adducts and sister chromatid exchanges in human lymphocytes treated with benzo[a]pyrene

We examined the relationship between benzo[a]pyrene-DNA adducts and sister chromatid exchanges (SCEs) in human lymphocytes. Cultures of isolated phytohemagglutinin (PHA)-stimulated lymphocytes from two normal donors were treated with 0.01-5.0 microM B[a]P from 24 to 72 h of culture. Using the highly sensitive 32P-postlabeling assay, we identified seven B[a]P-DNA adducts, one of which accounted for greater than 90% of the total DNA modifications. This adduct comigrated on polyethylenimine plates with the adduct produced by (+)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydro-benzo[a]pyrene. B[a]P-DNA adduct levels ranged from 0.02 to 8 adducts/10(7) nucleotides. SCE frequencies measured in parallel cultures ranged from 8 to 46 SCEs/cell. At the same B[a]P concentrations, B[a]P-induced SCE frequencies and B[a]P-DNA adduct levels were higher in lymphocytes from donor 1 than in lymphocytes from donor 2. There was a linear correlation between the number of B[a]P-DNA adducts and the number of SCEs induced; slopes of the linear regressions of induced SCEs on B[a]P-DNA adducts were similar for both donors. Our data suggest that SCE induction by B[a]P in human lymphocytes results from covalent DNA modification.     

Binding of benzo[a]pyrene and intracellular transport of a bound electrophilic benzo[a]pyrene metabolite by lipoproteins

Human serum lipoproteins were isolated by means of size exclusionh.p.l.c. Non-covalent uptake of [³H]benzo[a]pyrene was quantitatedfor fractions collected from the effluent of a liquid chromatographicseparation of human serum, and was found to directly correlatewith the lipoprotein concentration. An electrophilic benzo[a]pyrenemetabolite, [³H]trans 7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene,non-covalently associated with low density lipoproteins wastransferred to human lymphocytes in vitro and bound acid-precipitablenucleic acids of the lymphocytes as a function of time. Benzo[a]-pyrenemetabolite binding to lymphocyte DNA was demonstrated by meansof CaCl density gradient analysis. Non-mitogen-stimulated lymphocytesexposed to very low concentrations of carcinogen in the presenceof low density lipo-protein demonstrated [³H]thymidine incorporation;without the concomitant addition of low density lipoproteinthe low concentrations of carcinogen did not stimulate [³H]thymidineincorporation.     

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.     

Sulfite-dependent mutagenicity of benzo[a]pyrene derivatives

Benzo[a]pyrene (BP) and sulfur dioxide (SO₂) are ubiquitousair pollutants and are also components of tobacco smoke. AlthoughSO₂ itself is not carcinogenic, concurrent administration withBP results in enhancement of respiratory tract tumorigenesis.In biological systems, SO₂ exists as its hydrated form, sulfite(SO₃^2–). Sulfite readily undergoes autoxidation, generatingpotent oxidant species. When 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene(BP-7,8-diol) is included in sulfite autoxidation mixtures itis converted to more polar products, most notably 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrenes(BP tetraols). This implies the intermediacy of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetra-hydro-benzo[a]pyrenes (BPDE). We report herethe sulfite-dependent conversion of BP-7,8-diol to forms highlymutagenic to Salmonella typhimurium strain TA 98. This activationis observed at BP-7,8-diol concentrations of from 2 to 40 µMand at sulfite concentrations of from 0.5 to 10 mM. In the presenceof 10 µM BP-7,8-diol, half-maximal activation is observedat 1.6 mM sulfite. Sulfite itself is neither toxic nor mutagenicto the bacteria under these conditions. The time course of theactivation of BP-7,8-diol and its sensitivity to inhibitionby antioxidants indicate a requirement for sulfite autoxidation.These data further support the sulfite-dependent epoxidationof BP-7,8-diol. Not only does sulfite convert this promutagento its active mutagenic form, sulfite also enhances the mutagenicactivity of BP diolepoxides toward the tester strain. The reversionfrequency in response to 0.1–0.5 µM anti-EPDE isincreased by up to 33% in the presence of 1 mM sulfite, andby up to 270% with 10 mM sulfite. The mechanism of this enhancementof anti-BPDE activity is not known, but could be related toinhibition of the glutathione-S-transferase system which hasbeen previously reported for sulfite. These results are discussedin regard to the noted cocarcinogenicity of sulfur dioxide forBP.     

Mutagenicity of benzo[a]pyrene bay-region sulfonates

The interaction between the sulfite anion and specific benzo[a]pyrene (B[a]P) derivatives produces a novel class of benzo[a]pyrene sulfonates. (+/-)-7,8,9-Trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-sulfonate (B[a]PT-10-sulfonate) is formed in high yields in incubations containing (+/-)-7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (anti-BPDE) and sulfite, and sulfite strongly enhances the mutagenicity of the diolepoxide toward Salmonella typhimurium under those conditions. Although B[a]PT-10-sulfonate itself shows little direct mutagenicity over a 1-20 microM concentration range, this reactive bay-region intermediate does enhance the mutagenicity of anti-BPDE in strains TA98 and TA100 by up to 280%. No significant enhancement was seen when up to 20 microM B[a]PT-10-sulfonate was used in concert with another direct-acting mutagen, N-acetoxy-acetylaminofluorene (N-AcO-AAF). The isomeric product derived from sulfite and (+/-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-diol) is (+/-)-7,8,10-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-9-sulfonate (B[a]PT-9-sulfonate). Like B[a]PT-10-sulfonate, B[a]PT-9-sulfonate is not mutagenic to strains TA97, TA98 and TA100. This sulfonate exhibited little enhancing activity with anti-BPDE over a 1-20 microM concentration range, but did enhance the mutagenic response of strain TA98 to 0.2 microM N-Aco-AAF by up to 128%. Sulfite, anti-BPDE and B[a]PT-sulfonates were also examined for the ability to induce a forward mutation at the hgprt locus (8-azaguanine resistance) in strains of S.typhimurium. Sulfite caused a marked enhancement of forward mutation due to anti-BPDE in both TA98 and TA100. Surprisingly, concurrent administration of B[a]PT-10-sulfonate with anti-BPDE did not increase the number of mutant colonies. The extensive conversion of anti-BPDE to B[a]PT-10-sulfonate under conditions where sulfite enhances diolepoxide mutagenicity, when coupled with this enhancement of diolepoxide mutagenicity by B[a]PT-10-sulfonate in the reverse mutation assay, supports this novel B[a]P derivative as a mediator of the sulfite-dependent enhancement of B[a]P genotoxicity. Determining why this enhancing effect was not seen when selecting for mutation at the hgprt locus of S.typhimurium will require further study.     

Possible role of 6-hydroxymethylbenzo[a]pyrene as a proximate carcinogen of benzo[a]pyrene and 6-methylbenzo[a]pyrene

Benzo[a]pyrene (B[a]P) and 6-methylbenzo[a]pyrene ( 6-MeB[a]P) are metabolized by fortified rat-liver homogenates to a number of metabolites including one which is indistinguishable from 6-hydroxymethylbenzo[a]pyrene (6–OHMeB[a]P) by either thin-layer chromatography or ultra-violet absorption spectra. This compound is a potent carcinogen when administered by subcutaneous injection to rats. These observations are in accord with the hypothesis that methylation or hydroxymethylation is one of the first steps in metabolic activation of carcinogenic polycyclic hydrocarbons.