Analysis of tetrahydroxylated benzo[a]pyrene isomers in hair as biomarkers of exposure to benzo[a]pyrene.

A first gas chromatography-tandem mass spectrometry (GC-MS/MS) method was designed for analysis of four tetrahydroxylated benzo[a]pyrene metabolites (benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol, benzo[a]pyrene-r-7,t-8,t-9,t-10-tetrahydrotetrol, benzo[a]pyrene-r-7,t-8,c-9,c-10-tetrahydrotetrol, and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol) in hair. Hair powder extract was submitted to liquid-solid extraction, followed by C18 solid-phase purification. The analytes were derivatized with use of N-methyl-N-(trimethylsilyl)trifluoroacetamide and then analyzed by GC-MS/MS in negative chemical ionization mode. The calibration curve was linear from the limit of quantification (LOQ) to 20 pg/mg in hair. The coefficient of determination of the calibration curve was more than 0.975 for all the analytes investigated. The LOQs ranged from 0.075 to 0.2 pg/mg in hair. The method was afterward applied to the analysis of hair of 16 rats randomly allocated to experimental groups receiving 16 polycyclic aromatic hydrocarbons solubilized in oil at 0 or 0.8 mg/kg body weight by oral administration three times per week for 90 days. The analysis of monohydroxylated and dihydroxylated benzo[a]pyrenes was conducted in parallel by GC-MS/MS on the same samples. All tetrahydroxylated benzo[a]pyrene isomers were detected in hair samples collected from rats exposed to polycyclic aromatic hydrocarbons. Benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol, the most abundant isomer in hair of treated rats, was also the principal isomer released in DNA adduct hydrolysis in humans. Moreover, the benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol concentrations in hair were significantly greater than those of 2-hydroxybenzo[a]pyrene, 1-hydroxybenzo[a]pyrene, 7-hydroxybenzo[a]pyrene, and 4-hydroxybenzo[a]pyrene and similar to those of 9-hydroxybenzo[a]pyrene and 3-hydroxybenzo[a]pyrene. The method was also sufficiently sensitive to monitor environmental levels of exposure because two hair specimens in the eight analyzed from smokers were above the LOQ for benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol and benzo[a]pyrene-r-7,t-8,c-9,t-10-tetrahydrotetrol. This study therefore demonstrated that tetrahydroxylated benzo[a]pyrenes in hair might be a useful biomarker for the assessment of both the general population and occupationally exposed workers.

In vivo examination of the genotoxicity of the urban air and surface soil pollutant, 3,6-dinitrobenzo[e]pyrene, with intraperitoneal and intratracheal administration.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP) was identified as a new potent mutagen toward Salmonella strains in surface soil and airborne particles. Because data of in vivo examination of the genotoxicity of 3,6-DNBeP are limited, micronucleus test was performed in peripheral blood and bone marrow, and comet assay in the lungs of mice treated with 3,6-DNBeP. In male ICR mice intraperitoneally (i.p.) injected with 3,6-DNBeP, the frequency of micronuclated polychromatic erythrocytes (MNPCEs) was increased in the peripheral blood and bone marrow after 24 h in a dose-dependent manner. Compared to controls, the highest dose of 3,6-DNBeP (40 mg/kg B.W.) induced 7.3- and 8.7-fold increases of MNPCE frequency in the peripheral blood and bone marrow, respectively. Furthermore, when 3,6-DNBeP was intratracheally (i.t.) instilled to male ICR mice, 3,6-DNBeP at the highest dose of 0.1 mg/kg body exhibited 3.1-fold increase of DNA tail moment in the lungs at 3 h after the instillation compared to controls. The values of DNA tail moment at 9 and 24 h after the instillation were increased up to 3.5 and 4.2-fold, respectively. These data indicate that 3,6-DNBeP is genotoxic to mammalians in in vivo and suggest that 3,6-DNBeP may be a carcinogenic compound present in the human environment.

Characterisation of B(a)P metabolites formed in an ex vivo pig skin model using three complementary analytical methods.

An original method was developed to separate, identify and quantify the different benzo(a)pyrene (B(a)P) metabolites formed through oxidative and conjugative pathways. All B(a)P metabolites were separated by an improved high-performance liquid chromatography method, then detected and quantified relatively by online radioactivity detection. At the same time, metabolite structures were characterised by tandem mass spectrometry using two complementary ionisation modes: electrospray ionisation in the negative mode and atmospheric pressure chemical ionisation in the positive mode. This method was successfully applied to the analysis of B(a)P metabolites, produced by incubation of B(a)P with the ex vivo pig ear skin model. These include glucuronic acid and sulphate conjugates of B(a)P-OHs and B(a)P-diols, as well as direct phase I metabolites: B(a)P-tetrol, B(a)P-diones, B(a)P-catechols, B(a)P-diols and B(a)P-OHs.

Genotoxicity of 3,6-dinitrobenzo[e]pyrene, a novel mutagen in ambient air and surface soil, in mammalian cells in vitro and in vivo.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP), newly identified in airborne particles and surface soil, is a potent mutagen in Salmonella typhimurium. The present study investigated the genotoxic potency of 3,6-DNBeP in vitro and in vivo using mammalian cell strains (Chinese hamster CHL/IU and human HepG2) and ICR mice, respectively. In the hprt gene mutation assay using HepG2 cells, the spontaneous mutant frequency was 61.1 per 10(5) clonable cells, which increased to 229 per 10(5) clonable cells after treatment with 1.0 microg/ml (3 microM) 3,6-DNBeP. Notably, in HepG2 cells with increased N-acetyltransferase 2 activity, the mutant frequency increased to 648 per 10(5) clonable cells by treatment of 1.0 microg/ml (3 microM) 3,6-DNBeP. The sister chromatid exchange frequency increased approximately three times the control level in HepG2 cells treated with 3,6-DNBeP at a concentration of 1.0 microg/ml (3 microM). In HepG2 and CHL/IU cells, the frequency of the cells with micronuclei was 0.9 and 1.2%, and the frequencies increased to 2.3 and 7.6% after 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment, respectively. The H2AX phosphorylation level increased 8-fold compared with the background level with 1.0 microg/ml (3 microM) 3,6-DNBeP-treatment in HepG2 cells. Moreover, the comet assay showed that 3,6-DNBeP produced DNA damage in the cells of liver, kidney, lung and bone marrow in ICR mice 3 h after intraperitoneal injection at 40 mg/kg (0.12 mmol/kg) body weight. These data indicate that 3,6-DNBeP is genotoxic to mammalian cells in vitro and in vivo.

The role of electronic properties to the mutagenic activity of 1,6- and 3,6-dinitrobenzo[a]pyrene isomers.

Equilibrium geometries, infrared spectra, vertical first ionization potential (IP), electronic affinity (EA), dipole moment (mu) and electronic dipole polarizability (alpha) of 1,6- and 3,6-dinitrobenzo[a]pyrene isomers (1,6-DNBaP and 3,6-DNBaP) were evaluated by means of Density Functional Theory (DFT) and recent semiempirical PM6 method. Structural, energetic and vibrational properties of DNBaP isomers are substantially similar to each other. Calculated IP, EA and alpha values of these isomers are practically identical, while mu of 3,6-DNBaP (8.2 D at DFT level) is predicted to be ca. 4 times the value of 1,6-DNBaP isomer (1.9 D at DFT level), owing to favorable mutual orientation of the individual nitro group vectors. Higher direct-mutagenic activities of 3,6-DNBaP with respect to 1,6-DNBaP isomer by 1-2 orders of magnitude might be determined by its peculiar electronic charge distribution, which through stronger electrostatic and inductive interactions, can promote much more effectively binding to active-site of enzymes involved in mutagenic pathways. On the other hand, orientation of the nitro substituents relatively to the plane of the aromatic moiety, molecular sizes, as well as nitroreduction and oxidation reactions seem not to have a key role in the determination of the different mutagenic behaviour of these isomers.

Mutagenicity of surface soil from residential areas in Kyoto city, Japan, and identification of major mutagens.

To clarify the mutagenic potential of surface soil in residential areas in Kyoto city, surface soil samples were collected twice or three times from 12 sites, and their organic extracts were examined by the Ames/Salmonella assay. Almost all (>92%) samples showed mutagenicity in TA98 without and with S9 mix, and 8/25 (32%) samples showed high (1000-10,000 revertants/g of soil) or extreme (>10,000 revertants/g of soil) activity. Moreover, to identify the major mutagens in surface soil in Kyoto, a soil sample was collected at a site where soil contamination with mutagens was severe and continual. The soil extract, which showed potent mutagenicity in TA98 without S9 mix, was fractionated by diverse column chromatography methods. Five major mutagenic constituents were isolated and identified to be 1,6-dinitropyrene (DNP), 1,8-DNP, 1,3,6-trinitropyrene (TNP), 3,9-dinitrofluoranthene (DNF), and 3,6-dinitrobenzo[e]pyrene (DNBeP) by co-chromatography using high performance liquid chromatography and spectral analysis. Contribution ratios of 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP to total mutagenicity of the soil extract in TA98 without S9 mix were 3, 10, 10, 10, and 6%, respectively. These nitroarenes were detected in surface soil samples collected from four different residential sites in other prefectures, and their contribution ratios to soil mutagenicity were from 0.7 to 22%. These results suggest that surface soil in residential areas in Kyoto was widely contaminated with mutagens and there were some sites where surface soils were heavily polluted. 1,6-DNP, 1,8-DNP, 1,3,6-TNP, 3,9-DNF, and 3,6-DNBeP may be major mutagenic constituents that contaminate surface soil in Kyoto and other residential areas.

Determination of 3,6-dinitrobenzo[e]pyrene in surface soil and airborne particles by high-performance liquid chromatography with fluorescence detection.

We developed a sensitive analytical method and an efficient clean-up method to quantify 3,6-dinitrobenzo[e]pyrene (3,6-DNBeP) in surface soil and airborne particles. After purification using a silica gel column and two reversed-phase columns, 3,6-DNBeP was reduced to 3,6-diaminobenzo[e]pyrene by a catalyst column and analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector. 3,6-DNBeP was detected in all of the soil samples and airborne particles examined. The concentration of 3,6-DNBeP in surface soil and airborne particles was determined in the ranges of 347-5007 pg/g of soil and 137-1238 fg/m3, respectively.

Glucuronidation of carcinogen metabolites by complementary DNA-expressed uridine 5'-diphosphate glucuronosyltransferases.

Five UDP glucuronosyltransferases (UGT) were synthesized from complementary DNAs expressed in COS 7 cells and were tested for their capacities to glucuronidate a range of 2-acetylaminofluorene and benzo(a)pyrene-hydroxylated metabolites. Three forms, UGT1*06, UGT2B1, and UGT2B2 [names of UGT forms follow recommended nomenclature (B. B. Burchell et al., DNA Cell Biol., 10: 487-494, 1991)], had similar capacities to glucuronidate the reactive metabolite, N-hydroxy-2-acetylaminofluorene. The less reactive 1-, 3-, 5-, and 8-hydroxy derivatives of this aromatic amine were glucuronidated by UGT1*06 and UGT2B2 to varying degrees, but these were not substrates of UGT2B1. The three isozymes also glucuronidated phenolic metabolites of benzo(a)pyrene. UGT1*06 was more active toward 2- and 5-hydroxybenzo(a)pyrene, whereas UGT2B1 preferentially glucuronidated the 4- and 11-hydroxy derivatives and UGT2B2 preferentially glucuronidated the 1-, 2-, 8-, and 9-hydroxy metabolites. Two other UDP glucuronosyltransferases, UGT2B3 and UGT2B6, that glucuronidated testosterone when expressed in COS 7 cells were both inactive toward all the carcinogen metabolites tested. These results demonstrate that the glucuronidation of metabolites of 2-acetylaminofluorene and benzo(a)pyrene is mediated by at least three UDP glucuronosyltransferases and that each form glucuronidates a unique spectrum of metabolites.

Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in ras codon 61 mutational hot spots.

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbons display stronger tumorigenic activities in rodent studies than comparable bay region diol-epoxides, but the molecular basis for this difference between fjord and bay region derivatives is not understood. Here we tested whether the variable effects of these genotoxic metabolites of polycyclic aromatic hydrocarbons may result from different DNA repair reactions. In particular, we compared the repairability of DNA adducts formed by bay region benzo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but significantly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phenanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyclic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-induced proto-oncogene activation. Synthetic DNA substrates were assembled using a portion of human N-ras or H-ras that includes codon 61, and stereospecific B[a]P or B[c]Ph adducts were synthesized on adenine N6 at the second position of these two ras codon 61 sequences. DNA repair was determined by incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision products. These cell-free assays showed that all tested bay region B[a]P-N6-dA adducts are removed by the human nucleotide excision repair system, although excision efficiency varied with the particular stereochemical configuration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N6-dA adducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractory to the nucleotide excision repair process. These findings indicate that the exceptional tumorigenic potency of B[c]Ph or related fjord region diol-epoxides may be attributed, at least in part, to slow repair of the stable base adducts deriving from the reaction of these compounds with DNA.

An improved fluorometric assay for dosimetry of benzo(a)pyrene diol-epoxide-DNA adducts in smokers' lung: comparisons with total bulky adducts and aryl hydrocarbon hydroxylase activity.

An improved high-performance liquid chromatography/fluorometric assay has been established to quantitate the benzo(a)pyrene (BP) tetrols released after acid hydrolysis of lung DNA from lung cancer patients, so that the formation of benzo(a)pyrene diol-epoxide-DNA adducts can be measured. The r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydro-BP isolated by high-performance liquid chromatography was determined by chromatography in two different solvent systems and fluorescence spectroscopy. This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy- 7,8,9,10-tetrahydro-BP, requires 100-500 micrograms of DNA, and can measure 1 adduct/10(8) unmodified nucleotides. As this assay does not use immunoaffinity chromatography or solvent extraction, it allows a > 90% recovery of benzo(a)pyrene diol-epoxide-DNA adducts. This procedure has been tested on 13 DNA samples prepared from nontumorous lung parenchyma taken from lung cancer patients at surgery and revealed the presence of DNA adducts of the anti-benzo(a)pyrene diol-epoxide in 9 of 11 samples from smokers and in 2 of 2 ex-smokers. In only two samples from smokers the formation of adducts derived from syn-benzo(a)pyrene diol-epoxide was detected. A 15-fold variation in DNA adduct level was found in 11 of 13 DNA samples, with a range of 0.6-9.9 adducts of benzo(a)pyrene diol-epoxide/10(8) nucleotides. In samples containing both anti- and syn-benzo(a)pyrene diolepoxide-DNA adducts, the anti/syn adduct ratio is 2:1. A highly significant correlation was found between pulmonary microsomal aryl hydrocarbon hydroxylase activity and the level of benzo(a)pyrene diolepoxide-DNA adduct (r = 0.91; P < 0.001; n = 13). A crude linear correlation between the amounts of these adducts and those of bulky DNA adducts determined by 32P-postlabeling assay was observed in the same samples (r = 0.78; P < 0.02; n = 13). Thus this highly sensitive and specific procedure is suitable for measuring benzo(a)pyrene diolepoxide-DNA adducts in human tissues from environmentally exposed subjects and could be adapted to measure polycyclic aromatic hydrocarbons other than BP.

A general role for c-Fos in cellular protection against DNA-damaging carcinogens and cytostatic drugs.

One of the earliest responses of cells upon exposure to DNA-damaging agents is the induction of c-fos. To elucidate the biological role of Fos expression, we analyzed cells deficient in c-Fos upon treatment with different DNA-damaging agents, including carcinogens and antineoplastic drugs. We show that cells lacking c-Fos are hypersensitive with regard to reproductive cell death, apoptosis, and chromosomal breakage after treatment with agents inducing methylation lesions, bulky adducts, or crosslinks in DNA. They were not significantly hypersensitive to ionizing radiation. The activities of various repair enzymes and glutathione S-transferase and the level of proliferating cell nuclear antigen were not altered in c-fos-/- fibroblasts. Furthermore, the cells were able to remove the main methylation lesions from DNA. c-Fos-deficient cells exhibited a more severe mutagen-induced block to DNA replication and were compromised in the abolition of replication blockage. The data provide compelling evidence that c-Fos/activator protein-1 plays a decisive and general role in cellular defense against genotoxic agents, which require DNA replication to induce chromosomal instability. They are consistent with the hypothesis that impaired recovery from DNA replication inhibition upon mutagen exposure is causally involved in c-fos-/- hypersensitivity.

Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase transcription elongation past benzo[a]pyrenediol epoxide-modified DNA.

DNA replication and transcription are affected adversely by the presence of bulky adducts that are generated by the covalent binding of a variety of metabolically activated environmental pollutants to cellular DNA. When these lesions are not cleared by cellular repair enzymes prior to replication, mutations and ultimately tumor initiation can occur. Transcription and DNA repair appear to be intimately connected, since certain adducts are more efficiently removed from the transcribed strands of active loci than from non-transcribed strands and other quiescent domains in the genome. The mechanism by which RNA polymerases deal with bulky adducts during DNA transcription is therefore of great interest. The availability of site-specifically modified and stereochemically defined oligodeoxyribonucleotides derived from the covalent reaction of 7r, 8t-dihydroxy-9, 10t-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) with guanine residues prompted us to study the efficiencies of transcription past these lesions using bacteriophage T7 RNA polymerase. We show here that T7 RNA polymerase can bypass such lesions in a DNA template, providing that a cytosine residue is incorporated opposite anti-BPDE-modified guanine. However, when an incorrect base (most frequently a purine) is inserted opposite the modified site, the RNA polymerase stalls, and the complex dissociates, resulting in a truncated transcript. The ability of the T7 RNA polymerase to discriminate between a correct and an incorrect inserted base and, accordingly, to continue or terminate transcription, might constitute an important mechanism that ensures the fidelity of transcription past a modified base present on the transcribed strand of the DNA template.

Individual values of excretion of benzo[a]pyrene metabolites and susceptibility to its carcinogenic effect in rats.

In white outbred LIO rats exposed to multiple intraperitoneal (i.p.) doses (10 mg/kg) of benzo[a]pyrene (BP) in the form of a water-lipid emulsion, individual peculiarities of the excretion of its metabolites, BP-7,8-diol and 3-hydroxy-BP (3-OH-BP) in urine and feces were detected and compared with the carcinogenic effect. Parameters of BP metabolite excretion differed from those found in our previous experiments with rats exposed to single high i.p. doses of BP (100 and 200 mg/kg), dissolved in sunflower oil [11,12]. In comparison with our previous observation, in the present study, the carcinogenic effect was considerably weaker (5/22 versus 10/19). The rats that developed tumours of internal tissues (four peritoneal malignant histiocytomas and one lung lymphosarcoma), excreted higher quantities of BP-7,8-diol in the urine than other rats. The possible implication of monitoring excretion of BP metabolites for predicting individual susceptibility to its carcinogenic effect is discussed.

Morphological transformation of C3H10T1/2CL8 cells by cyclopenta-fused derivatives of benzo[a]pyrene and benzo[e]pyrene.

Cyclopenta-fused homologs of polycyclic aromatic hydrocarbons (PAH) have proven to be more genotoxic and tumorigenic than their parent PAHs. In an effort to uncover their mechanisms of metabolic activation, the morphological transforming activities of dibenzo[k,mno]acephenanthrylene (CP(3,4)B[a]P), dibenzo[j,mno]acephenanthrylene (CP(1,12)B[a]P) and naphtho[1,2,3,4-mno]acephenanthrylene (CPB[e]P) were studied in C3H10T1/2CL8 mouse embryo fibroblasts. CP(3,4)B[a]P, a PAH with a blocked K region and unblocked bay region, was highly active inducing an average of 1.1 Type II and III foci/dish at 5 micrograms/ml with an average of 67% of the dishes containing foci. This activity was similar to that of benzo[a]pyrene. CP(1,12)B[a]P and CPB[e]P were inactive. The relative positions of the cyclopenta-ring and bay region may play an essential role in the metabolic activation of these PAHs and their biological activities.

Tumorigenicity of 6-halogenated derivatives of benzo[a]pyrene in mouse skin and rat mammary gland.

Studies of the tumorigenicity of 6-halogenated derivatives of benzo[a]pyrene (BP) can provide evidence about the role of the 6 position in the carcinogenic activation of BP. Female Swiss and A-strain mice were treated on the skin with BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-chlorobenzo[a]pyrene (6-C1BP), 6-bromobenzo[a]pyrene (6-BrBP) and 6-iodobenzo[a]pyrene (6-IBP) by repeated application, and in some cases by initiation-promotion. While BP was more potent than 6-FBP, only these two compounds exhibits tumor-initiating and carcinogenic activity in mouse skin. Female Sprague-Dawley rats were treated with BP, 6-FBP, 6-ClBP, and 6-BrBP by intramammillary injection. BP and 6-FBP induced high levels of mammary epithelial tumors and fibrosarcomas. 6-ClBP elicited only a high percentage of fibrosarcomas, whereas 6-BrBP induced a few adenocarcinomas. These results indicate that chloro or bromo substitution at C-6 in BP reduces or eliminates carcinogenic activity. Conversely, 6-FBP, from which the fluoro substituent has been chemically and metabolically removed by one-electron oxidation, displays a moderate carcinogenic activity which is consistent with activation by either one-electron oxidation or monooxygenation.

Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of fluorinated derivatives of benzo[a]pyrene and 3-methylcholanthrene.

Comparative studies of tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland were conducted with benzo[a]pyrene (BP) and 3-methylcholanthrene (MC) derivatives. SENCAR mice were initiated with BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-methylBP, 7-FBP, 8-FBP, 9-FBP, 10-FBP, or 10-azaBP and promoted with tetradecanoyl phorbol acetate. The same compounds plus BP 7,8-dihydrodiol were tested by intramammillary injection in female Sprague-Dawley rats. Tumor-initiating activity in mice and/or carcinogenicity in rats were observed for BP, 6-methylBP, 6-, 7-, 8-, and 10-FBP, whereas 9-FBP was inactive in both experiments and 10-azaBP was only marginally active in the mammary gland. BP 7,8-dihydrodiol was carcinogenic in rat mammary gland, although it was less potent than BP. MC, 8-FMC, 10-FMC, and 3-methylcholanthrylene were also tested in Sprague-Dawley rats by intramammillary injection. All compounds were carcinogenic, with MC displaying the most potent activity. The less potent carcinogenic activity of BP 7,8-dihydrodiol in the mammary gland, compared with BP, and the moderate-to-weak tumor-initiating and/or carcinogenic activity of 7-, 8-, and 10-FBP suggest that the bay-region diol-epoxide pathway does not play a significant role in the activation of BP in these two target tissues. Similarly, the carcinogenic activity of 8-FMC and 10-FMC, in which the bay-region diol-epoxide pathway is blocked, suggests that this mechanism of activation is not important in the carcinogenicity of MC in rat mammary gland.

Molecular characterization of hprt mutations from Chinese hamster ovary cells treated with 1-, 3-, and 6-nitrosobenzo[a]pyrene.

1-, 3-, and 6-nitrobenzo[a]pyrene (nitro-BaP) are environmental contaminants that can be metabolized to genotoxic derivatives by either nitroreduction or ring-oxidation. In this study, we examined the types of mutations produced by the primary nitroreduced metabolites, 1-, 3-, and 6-nitroso-BaP (NO-BaP) in the hprt gene of Chinese hamster ovary cells. RNA from 6-thioguanine-resistant mutants was reverse-transcribed to cDNA and the hprt coding sequence was amplified and sequenced. The mutational patterns produced by the three compounds exhibited extensive similarities: 1) base pair substitutions accounted for 67% (28/42) of 1-NO-BaP, 51% (26/51) of 3-NO-BaP, and 50% (11/22) of 6-NO-BaP mutations; 19-36% of the mutations were exon deletions and 14-18% were frameshifts; 2) most (64-84%) of the simple base pair substitutions occurred at G:C, mainly G:C-->T:A and G:C-->C:G transversions; 3) 98% (46/47) of the simple base pair substitutions at G:C had the mutated dG on the non-transcribed strand and 81% (38/47) were located with the mutated dG flanked 3' by at least one purine; and 4) most simple base pair substitutions (48/62, 77%) occurred in exons 2, 3, and 8 of the hprt gene. Although there were no significant differences among the mutation profiles of the NO-BaPs, a significant difference did exist between the mutation pattern produced by 3-NO-BaP and the mutation pattern previously determined for the ring-oxidized product of 3-nitro-BaP metabolism, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9, 10-tetrahydro-3-nitrobenzo[a]pyrene. This observation indicates that differences in the structures of closely related adducts can be important enough to have an effect on mutation profiles.

Fluorescence line-narrowing studies of antibody-benzo[a]pyrene tetrol complexes.

Benzo[a]pyrene tetrol (BPT) was used as a fluorescent probe to investigate the nature of antigen binding by two different monoclonal antibodies (MAb) that recognize a variety of derivatives of anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrenes (BPDE). Fluorescence line-narrowed spectra of the physical complexes of BPT formed with antibodies 8E11 and 3C3 were recorded at 4 K by employing vibronic excitation into the S1 electronic state. The frequencies of the vibrational modes of the S1 state were only marginally affected, though changes in relative intensities of some bands were observed. Fluorescence spectra recorded at 77 K by excitation into the S2 state showed that the (0,0) fluorescence emission of BPT was shifted to red on complex formation. Intensity ratios of the (0,0) band and the main vibrational band at 1300 cm-1 were used to assess the degree of interior binding of the chromophore. Quenching studies with acrylamide were employed to designate the complexes as type I, solvent inaccessible, or type II, solvent accessible. These studies also indicated that antibody 3C3 complexes tend to be more heterogeneous compared to the 8E11 complex. Deuterated BPT-d-12 also formed complexes with both antibodies, however, with different quenching behavior.

Comparison of the solid-matrix luminescence properties and photophysical parameters of two products from benzo(a)pyrene-DNA adducts.

The solid-matrix luminescence properties and several calculated photophysical parameters of two important products from the benzo(a)pyrene-DNA adducts were compared. The products were benzo(a)pyrene-r-7,t-8,9,c-10-tetrahydrotetrol (I-1) and 7R,8S,9S-trihydroxy-10R-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10- tetrahydrobenzo(a)pyrene (BPDE-dG). The solid-matrix luminescence data were obtained for I-1 and BPDE-dG adsorbed on two different solid matrices, namely, 1% alpha-cyclodextrin (CD)/NaCl and 25% trehalose/NaCl and at two different temperatures (93 K and 296 K). The 25% trehalose/NaCl gave higher fluorescence and phosphorescence quantum yields from both I-1 and BPDE-dG in contrast to the 1% alpha-CD/NaCl matrix. The BPDE-dG showed lower fluorescence quantum yields on the solid matrices compared to I-1. The lower fluorescence quantum yields for BPDE-dG were attributed to a photoinduced electron transfer mechanism. In contrast to the room-temperature solution fluorescence of BPDE-dG, BPDE-dG gave rather high fluorescence quantum yields at room temperature when adsorbed on the two solid matrices. From solid-matrix luminescence quantum yields and solid-matrix luminescence lifetimes, many photophysical parameters were calculated and compared. Several differences among the rate constants were noted with the two solid matrices for BPDE-dG and I-1. For example, BPDE-dG showed internal conversion at 296 K with 25% trehalose/NaCl, but no internal conversion was observed at 93 K with this solid matrix. Also, for BPDE-dG the phosphorescence rate constants at 296 K and 93 K were very small with 25% trehalose/NaCl, but the intersystem crossing rate constants from the triplet state to the ground state were very large.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative tumorigenicity of 1- and 3-nitrobenzo[a]pyrenes, and 3,6- and 1,6-dinitrobenzo[a]pyrenes in F344/DuCrj rats.

Our earlier study revealed that 1- and 3-nitrobenzo[a]pyrene (NBP), 1,6- and 3,6-dinitrobenzo[a]pyrene (DNBP), nitrated derivatives of benzo[a]BP (BP), are present in the environment. These derivatives are potent mutagens for Salmonella tester strains and we have preliminarily reported them to be carcinogenic in F344/DuCrj rats. In this study, the tumorigenic action of 1- and 3-NBP, 1.6- and 3,6-DNBP, and BP induced by subcutaneous injection into rats was found to differ according to the NO2-substitution in the BP structure. The chemicals were suspended in equal volumes of beeswax and tricaprylin, and rats were subcutaneously injected with single doses of 500, 1000, and 2000 microg for 1- and 3-NBP, and of 8, 40, 200, and 1000 microg for 3,6- and 1,6-DNBP, and BP as a positive control. 3,6-DNBP and BP induced tumors in a dose-dependent manner at the injection site. Rats given 1000 microg of 3,6-DNBP (2924 nmol) and BP (3968 nmol) developed subcutaneous tumors at the rate of 70 and 80%, respectively, and those given a minimum dose of 23 nmol for 3.6-DNBP and 32 nmol for BP per rat developed tumors at a rate of 4.8 and 18.2%, respectively. However, rats given 500 and 1000 microg of 1- and 3-NBP did not develop any tumors while those given a high dose, 2000 microg, of each chemical developed tumors at only one of ten animals used. It was concluded, therefore, that these chemicals are weak carcinogens. Histologically, most of the tumors were malignant fibrous histiocytomas. Rats given various doses of 1,6-DNBP did not develop any tumors at the injection site. The failure of 1,6-DNBP to induce tumors may involve its metabolites because of the lower mutagenicity of its reduction products, 1-nitroso-6-NBP and 1-amino-6-NBP. It is suggested, therefore, that tumorigenicities of NBPs and DNBPs differ according to the NO2-substitution on the chemical structure, which may be due to the possible nitroreduction of the chemicals.