Enhancement of benzo[a]pyrene diol epoxide mutagenicity by sulfite in a mammalian test system

Sulfur dioxide, a ubiquitous air pollutant, is a co-carcinogenfor benzo[a]pyrene (BP). We have demonstrated previously thatthe interaction between sulfite, the physiological form of sulfurdioxide, and (±)-7r, 8t-dihydroxy-9t, 10t-epoxy-7, 8,9, 10-tetrahydrobenzo[a]pyrene (anti-BPDE), the ultimate carcinogenicform of BP, results in an enhanced mutagenic effect in Salmonellatyphimurium strains TA98 and TA100. We report here that thissame co-mutagenic effect of sulfite occurs in a mammalian cellline. Treatment of Chinese hamster V79 cells with 50 nM anti-BPDE,a concentration on the linear portion of the dose-response,resulted in a four-fold increase in mutations at the hprt locusrelative to the spontaneous rate. When V79 cells were exposedto 1 or 10 mM sulfite immediately prior to the addition of anti-BPDE,the mutation rate increased by 73% and 210%, respectively, overthat elicited by anti-BPDE alone. Sulfite itself was moderatelycytotoxic, but caused no increase in mutation over the spontaneousrate. Characterization of the dose- and time-dependance of thisenhancement of diol epoxide mutagenicity by sulfite closelyresembled the effects seen previously in the bacterial system.In particular, enhancement by sulfite was evident when sulfitewas added to the cells between 60 min and 1 min prior to theaddition of the diol epoxide. Concurrent addition of sulfiteand the diol epoxide attenuated the enhancement, and the effectwas lost altogether when sulfite was added 10 min after thediol epoxide. The specificity of this effect of sulfite wasshown by comparison with sulfate, which at concentrations ofeither 1 or 10 mM exhibited modest cytotoxicity, but neitherwas directly mutagenic nor able to enhance the mutagenic effectof anti-BPDE. Binding studies with labeled anti-BPDE showedthat the addition of 10 mM sulfite increased binding of anti-BPDEto DNA by over 43%, corresponding to the observed increase inmutant frequency. Interestingly, this difference in level ofDNA modification was not apparent after 30 min to 2 h exposures,but only emerged at the 4 h time point. The 4 h point was routinelyused for all mutagenicity studies. Binding of anti-BPDE-derivedmaterials to cellular RNA was not altered by 10 mM sulfite.The emergence of increased DNA modification at the latest timepoint suggests either a more prolonged period of active DNAbinding than would occur with diol epoxide, or a differencein the ability to recognize and clear specific DNA adducts.Both possibilities are discussed in regard to the observed formationof 7r,8t,9t-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene- lOc-sulfonate(BPT-10-sulfonate) in those incubations. BPT-10-sulfonate isa relatively stable BP derivative which retains the abilityto covalently modify DNA. The role of this derivative in theenhancement of diol epoxide mutagenicity by sulfite is stronglysuggested by these data.     

Mutability of p53 hotspot codons to benzo(a)pyrene diol epoxide (BPDE) and the frequency of p53 mutations in nontumorous human lung

p53 mutations are common in lung cancer. In smoking-associated lung cancer,the occurrence of G:C to T:A transversions at hotspot codons, e.g., 157, 248, 249,and 273, has been linked to the presence of carcinogenic chemicalsin tobacco smoke including polycyclic aromatic hydrocarbons suchas benzo(a)pyrene (BP). In the present study, we have used a highly sensitive mutation assay to determine the p53 mutation load in nontumorous human lung and to study the mutability of p53 codons 157, 248, 249, and 250 to benzo(a)pyrene-diol-epoxide (BPDE), an active metabolite of BP in human bronchial epithelial BEAS-2B cells. We determined the p53 mutational load at codons 157, 248, 249, and 250 in nontumorous peripheral lung tissue either from lung cancer cases among smokers or noncancer controls among smokers and nonsmokers. A 5-25-fold higher frequency of GTC(val) to TTC(phe) transversions at codon 157 was found in nontumorous samples (57%) from cancer cases (n = 14) when compared with noncancer controls (n = 8; P < 0.01). Fifty percent (7/14) of the nontumorous samples from lung cancer cases showed a high frequency of codon 249 AGG(arg) to AGT(ser) mutations (P < 0.02). Four of these seven samples with AGT(ser) mutations also showed a high frequency of codon 249 AGG(arg) to ATG(met) mutations, whereas only one sample showed a codon 250 CCC to ACC transversion. Tumor tissue from these lung cancer cases (38%) contained p53 mutations but were different from the above mutations found in the nontumorous pair. Noncancer control samples from smokers or nonsmokers did not contain any detectable mutations at codons 248, 249, or 250. BEAS-2B bronchial epithelial cells exposed to doses of 0.125, 0.5, and 1.0 microM BPDE, showed G:C to T:A transversions at codon 157 at a frequency of 3.5 x 10(-7), 4.4 x 10(-7), and 8.9 x 10(-7), respectively. No mutations at codon 157 were found in the DMSO-treated controls. These doses of BPDE induced higher frequencies, ranging from 4-12-fold, of G:C to T:A transversions at codon 248, G:C to T:A transversions and G:C to A:T transitions at codon 249, and C:G to T:A transitions at codon 250 when compared with the DMSO-treated controls. These data are consistent with the hypothesis that chemical carcinogens such as BP in cigarette smoke cause G:C to T:A transversions at p53 codons 157, 248, and 249 and that nontumorous lung tissues from smokers with lung cancer carry a high p53 mutational load at these codons.     

Haplotypes of DNMT1 and DNMT3B are associated with mutagen sensitivity induced by benzo[a]pyrene diol epoxide among smokers

The mutagen sensitivity assay is an in vitro measure of DNA repair capacity used to evaluate intrinsic susceptibility for cancer. The high heritability of mutagen sensitivity to different mutagens validates the use of this phenotype to predict cancer susceptibility. However, genetic determinants of mutagen sensitivity have not been fully characterized. Recently, several studies found that three major cytosine DNA methyltransferases (DNMTs), especially DNMT1, have a direct role in the DNA damage response, independent of their methyltransferase activity. This study evaluated the hypothesis that sequence variants in DNMT1, DNMT3A and DNMT3B are associated with mutagen sensitivity induced by the tobacco carcinogen benzo[a]pyrene diol epoxide (BPDE) in 278 cancer-free smokers. Single-nucleotide polymorphisms (n = 134) dispersed over the entire gene and regulatory regions of these DNMTs were genotyped by the Illumina Golden Gate Assay. DNA sequence variation in the DNMT1 and DNMT3B loci was globally associated with breaks per cell (P < 0.04 for both). No global association between DNMT3A and breaks per cell was seen (P = 0.09). Two haplotypes in block1 of DNMT1 (H284) and 3B (H70) were associated with 16 and 24% increase in breaks per cell, respectively. Subjects with three or four adverse haplotypes of both DNMT1 and 3B had a 50% elevation in mean level of breaks per cell compared with persons without adverse alleles (P = 0.004). The association between sequence variants of DNMT1 and 3B and mutagen sensitivity induced by BPDE supports the involvement of these DNMTs in protecting the cell from DNA damage.     

Inhibition of DNA synthesis, independent of DNA adduct formation, by benzo[a]pyrene diol epoxide in mammalian cells

Treatment of SV40-infected CV-1 cells with the ultimate carcinogenanti-benzo[a]pyrene diol epoxide (BPDE) at 1 x 10 ^–4 mg/mlor higher reduced the rate of viral DNA synthesis to an extentdependent on the BPDE concentration; similar reductions in cellularDNA synthesis were produced in infected and uninfected cells.Treatment of cells with BPDE, followed by removal of BPDE, atvarious times before infection with SV40 gave the same results.Recovery or partial recovery of DNA synthesis occurred whenthe BPDE concentration was below 6 x 10 ^–4 mg/ml; at higherconcentrations the cells were killed. Simultaneously replicatingviral DNA's from viruses infecting the same cells before andafter BPDE treatment of the cells exhibited the same reducedrate of synthesis. The evidence indicates that covalent adductsin viral DNA are not responsible for its reduced replicationrate; moreover, it is probable that an insignificant numberof adducts is produced in intracellular viral DNA at BPDE concentrationsthat do not kill CV-1 cells. Rather, it appears likely thatBPDE inhibits viral DNA synthesis by attacking cellular DNAor non-DNA targets. Caution is therefore required in relatingthe effects of BPDE and other carcinogens to DNA adduct formation.     

Anti-diol epoxide of benzo[a]pyrene induces transient Mdm2 and p53 Ser15 phosphorylation, while anti-diol epoxide of dibenzo[a,l]pyrene induces a nontransient p53 Ser15 phosphorylation

The polycyclic aromatic hydrocarbons (PAHs) dibenzo[a,l]pyrene (DBP) and benzo[a]pyrene (BP) are environmental contaminants and potent carcinogens. DBP is several orders of magnitude more mutagenic/carcinogenic than BP. This can be ascribed to differences in DNA binding efficiency of their ultimate carcinogenic bay- and fjord-region diol epoxide (DE) intermediates, differences in structural features of the DNA adducts and differences in DNA adduct recognition and the subsequent downstream signaling. In this study, we have characterized the effect of the ultimate carcinogenic DEs, (+)-anti-BPDE and (-)-anti-DBPDE following short exposure times, on Mdm2 and p53 pathway in A549 human lung epithelial carcinoma cells. In contrast to (-)-anti-DBPDE, (+)-anti-BPDE induces stabilization of phosphorylated Mdm2. (+)-anti-BPDE-induced effects on Mdm2 were transient and correlated with transient p53 Ser15 phosphorylation. DNA adducts of (-)-anti-DBPDE are more refractory to removal by nucleotide excision repair (NER) than adducts of (+)-anti-BPDE and do not induce Mdm2 phosphorylation. This suggests a role of phosphorylated Mdm2 in the repair process. In addition, (-)-anti-DBPDE, in contrast to (+)-anti-BPDE, induced prolonged p53 Ser15 phosphorylation as well as phosphorylation of p53 at Ser46, a phosphorylation site associated with apoptosis. It is also concluded that p53 Ser15 phosphorylation and antibody 2A10-site specific Mdm2 alterations are induced by nonidentical signaling pathways by the bay- and fjord-region DE. These differences may reflect the different carcinogenic potential of these compounds.     

Use of UvrABC nuclease to quantify benzo[a]pyrene diol epoxide-DNA adduct formation at methylated versus unmethylated CpG sites in the p53 gene.

We have used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction (LMPCR) techniques to map and quantify (+/-)anti-7beta, 8alpha-dihydroxy-9alpha, 10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (BPDE) adduct formation in the p53 gene of human cells. We found that BPDE adduct formation, as revealed by UvrABC incision, preferentially occurred at methylated CpG sites that correspond to the mutational hotspots observed in human lung cancers. Our hypothesis is that it is this methylated CpG sequence-dependent preferential adduct formation, rather than selective growth advantage, that is the major determinant of the p53 mutation pattern in human cancers. Given the far reaching ramifications of such conclusions for cancer etiology, a legitimate question is raised regarding the reliability of using the UvrABC incision method for quantifying and determining the sequence-dependency of adduct formation. Is the higher frequency of UvrABC cutting at methylated versus unmethylated CpG sites due to the preference of the nuclease for cutting at those sites or due to the preferential formation of BPDE adducts at those sites? In order to distinguish between these two possibilities, we have analyzed the kinetics of UvrABC incision at BPDE adducts formed at either methylated CpG sites versus other sequences, or unmethylated CpG sites versus other sequences in exon 5 of the p53 gene. We have found that the UvrABC cutting kinetics are identical for both cases. On the basis of these results we conclude that under proper cutting conditions, UvrABC nuclease reacts with and incises with equal efficiency, BPDE adducts formed at methylated or unmethylated CpG sites as well as other sequences, and that the extent of UvrABC incision accurately reflects the extent of BPDE-DNA adduct formation. These conclusions were further supported by results obtained using a DNA synthesis blockage assay.     

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.     

Presence of benzo[a]pyrene diol epoxide adducts in target DNA leads to an increase in UV-induced DNA single strand breaks and supF gene mutations

Exposure to DNA damaging agents and mutagens often occurs as combinations of agents, or as complex mixtures of chemicals. We found that plasmid DNA adducted with benzo[a]pyrene diol epoxide (BPDE) was more susceptible to UV-induced single strand breaks than was control DNA. To determine whether the increase in DNA damage also applied to mutagenic lesions, the supF gene forward mutation assay was used to compare mutations induced by BPDE alone, UVB, UVC, BPDE followed by UVB and BPDE followed by UVC. It was found that the mutation frequency for BPDE + UVB (1167 in 10(4) transformants) was higher than BPDE alone (12 in 10(4) transformants) or UVB alone (446 in 10(4) transformants), and the mutation frequency for BPDE + UVC (197 in 10(4) transformants) was higher than BPDE alone or UVC alone (26 in 10(4) transformants). For BPDE + UVB and BPDE + UVC there was a significant increase in plasmids with multiple mutations. Whilst these indicate error prone repair due to the single strand breaks, the different mutation frequencies in plasmids treated to give similar levels of strand breaks suggest other mechanisms for the mutations in plasmids with single mutation events. The spectrum of non-multiple mutations in the two combined treatments included both UV signature mutations (GC-->AT as the most common mutation) and BPDE signature mutations (GC-->TA and GC-->CG as the most common mutations). However, the increase in absolute mutation frequency of BPDE signature mutations between BPDE treatment and BPDE + UV treatment was greater than the increase in absolute mutation frequency of UV signature mutations, even though the level of BPDE adducts was identical in each case. These results suggest two possibilities: (i) the BPDE adducts are photoactivated to a more mutagenic lesion, or (ii) the presence of UV lesions lead to the BPDE adducts becoming more mutagenic.     

High mobility group 1 and 2 proteins bind preferentially to DNA that contains bulky adducts induced by benzo[a]pyrene diol epoxide and N-acetoxy-acetylaminofluorene

High mobility group (HMG) proteins 1 and 2 are abundant non-histone chromosomal proteins that bind preferentially DNA that is bent or underwound. Previous studies have shown that these proteins preferentially bind to DNA damaged by the crosslinking agents cis-diammine-dichloro-platinum(II), chromium(III) and UV-C radiation. Here we have studied the binding of HMG-1/2 proteins to a duplex oligonucleotide damaged by benzo(a)pyrene diol epoxide or N-acetoxy-acetylaminofluorene using an electrophoretic mobility shift assay. Both chemicals induce monoadducts that are known to distort DNA structure. The affinities of HMG-1/2 for DNA damaged by benzo[a]pyrene diol epoxide or N-acetoxy-acetylaminofluorene were similar to that for UV-irradiated DNA, which were an order of magnitude higher than for undamaged DNA. In contrast, DNA modified by dimethyl sulfate was not preferentially recognised by HMG-1/2.     

Frameshift mutation in codon 176 of the p53 gene in rat esophageal epithelial cells transformed by benzo[a]pyrene dihydrodiol

Mutations in the p53 tumor suppressor gene have been associated with exposure to environmental chemical carcinogens. Cultured rat esophageal epithelial cells were transformed in vitro by treatment with benzo[a]pyrene dihydrodiol (BP-DHD). A BP-DHD-transformed cell line and control cell lines were analyzed for mutations in the p53 gene and in the Ha-ras gene by single-strand conformation polymorphism analysis of polymerase chain reaction-amplified products and direct DNA sequencing. The deletion of one cytosine in codons 174–176 (TGCCCCCAC → TGCCCCAC) of the p53 gene was found only in the BP-DHD-transformed cell line. The BP-DHD-transformed cells were highly invasive and tumorigenic when transplanted into syngeneic rats, whereas control lines either were nontumorigenic or formed epithelial cysts. BP-DHD-transformed cells and control lines were negative for mutations in the Ha-ras gene. Our results suggest that the tumorigenic potential of the BP-DHD-transformed cell line is associated with a frameshift mutation in codon 176 of the p53 gene but not with mutations in the Ha-ras gene. The G/C-rich codons 174–176 in the ratp53 gene may be specific targets for BP-DHD. ©1995 Wiley-Liss, Inc.     

Suppressive activities of OGG1 and MYH proteins against G:C to T:A mutations caused by 8-hydroxyguanine but not by benzo[a]pyrene diol epoxide in human cells in vivo

8-Hydroxyguanine (8OHG), an oxidatively damaged base, and benzo[a]pyrene-diol-epoxide (BPDE), a metabolite of benzo[a]pyrene found in cigarette smoke, are thought to be major causes for G:C to T:A transversions in DNA of human cells. In this study, we assessed the abilities of OGG1, MYH and APE1 proteins, which are components of a base excision repair pathway, to suppress G:C to T:A transversions caused by 8OHG or BPDE by a bacterial suppressor tRNA (supF) forward mutation assay using a shuttle plasmid, pMY189. The introduction of a single 8OHG residue at position 159 of the supF gene and treatment with BPDE led to a 65- and 34-fold increase in mutation frequencies of the pMY189 plasmid, respectively, after replication in the NCI-H1299 human lung cancer cell line. G:C to T:A transversions were predominantly induced in these plasmids. Both the mutation frequency of the 8OHG-containing plasmid in NCI-H1299 cells and the occurrence of G:C to T:A transversions at position 159 in the supF gene were significantly reduced by overexpression of OGG1 and MYH proteins, but not by that of APE1 protein. In contrast, neither mutation frequency nor the occurrence of G:C to T:A transversion of the BPDE-treated plasmid was reduced by overexpression of OGG1, MYH and APE1 proteins. These results indicate that OGG1 and MYH function as suppressors for G:C to T:A transversions by 8OHG but not by BPDE in human cells.     

Combined subcarcinogenic benzo[a]pyrene and UVA synergistically caused high tumor incidence and mutations in H-ras gene, but not p53, in SKH-1 hairless mouse skin

Combined subcarcinogenic doses of benzo[a]pyrene (BaP) and UVA induced H-ras, but not p53, gene mutations 8 weeks before tumor emergence in SKH-1 mice. Neither UVA (40 kJ/m2) nor BaP (8 nmol) induced any tumors after mice were topically treated 3 times/week for 25 weeks. However, combined BaP-UVA treatment synergistically increased tumor incidence and multiplicity. All tumors induced by BaP-UVA were malignant. The epidermis was collected from mice treated for 2, 6 and 10 weeks. DNA from UVB- (0.3 kJ/m2) or BaP-UVA-(8 nmol and 40 kJ/m2-induced tumors was isolated and screened for H-ras and p53 mutations. Four types of point mutation, GGC-->GAC, GCC, GTC and CGC, occurred in UVB-induced tumors at H-ras codon 13; and one type of point mutation, GGA-->GAA, at codon 12. Treatment with either BaP alone or BaP-UVA for 10 weeks caused GGA-->GAA mutation at codon 12 or GGC-->GAC mutation at codon 13 in nontumor skin, respectively, as well as in tumors induced by BaP-UVA. All of the 10-week samples treated with either BaP or BaP-UVA showed detectable mutations at codons 12 and 13, but the genetic load was significantly higher in BaP-UVA-treated mice than in those exposed only to BaP. UVA alone induced mutations at codon 12 in only one-third of samples. G-->A mutations induced by BaP or BaP-UVA at position 38 of codon 13 have not been reported previously. C-->T transitions were detected in p53 hot spots of exon 8 in 2 of 19 BaP-UVA-induced tumors but were not found in nontumor skin.     

Murine squamous cell carcinoma cell lines produced by a complete carcinogenesis protocol with benzo[a]pyrene exhibit characteristic p53 mutations and the absence of H-ras and cyl 1/cyclin D1 abnormalities

In this report, we describe the isolation and characterizationof six murine squamous cell carcinoma cell lines (BPCC) derivedfrom carcinomas produced by a complete carcinogenesis protocolwith benzo[a]pyrene (B[a]P). All six cell lines were tumorigenicto varying degrees in nude mice, and several were spontaneouslymetastatic to the lungs. The in vivo invasive potential of eachBPCC cell line was determined using de-epithelialized trachealxenotransplants into which cells were inoculated. This assayrevealed positive association of tumor grade with in vivo invasiveness,yet no clear relationship to the spontaneous metastatic potentialof the cell lines, suggestive that invasive potential is onlyone determinant of the overall metastatic phenotype. At themolecular level, all six BPCC cell lines revealed the absenceof mutations in the H-ras oncogene and no amplification or rearrangementin the cyl l/cyclin Dl putative oncogene. Analysis of the p53tumor suppressor gene revealed a direct correlation betweenpositive nuclear immunohistochemical staining of the p53 proteinin four BPCC cell lines and the presence ofp53 mutations identifiedby direct sequence analysis. The localization of mutations toexons 7 and 8 of the p53 gene and the detection of G to T transversionsin two of the four cell lines bearing p53 mutations are in agreementwith previous analyses of a large series of primary B[a]P-inducedmurine skin tumors. In addition, frameshift mutations were identifiedin two cell lines. The correlation of the biological and molecularproperties of these BPCC cell lines with the known characteristicsof primary squamous cell carcinomas induced by B[a]P indicatesthat these cell lines could be useful tools in elucidating themechanisms of tumorigenesis of this important chemical carcinogen.