Biomedical subjects

Shantu Amin

Publications and source records attributed to Shantu Amin.

At least 19 recordsLinked to original sources

PubMed2006

Investigation of the genotoxicity of dibenzo[c,p]chrysene in human carcinoma MCF-7 cells in culture.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants that have been linked to certain human cancers. The fjord region PAH dibenzo[a,l]pyrene exhibits the highest levels of carcinogenic activity of all PAH as yet tested in rodent tumor models. Another hexacyclic aromatic hydrocarbon, dibenzo[c,p]chrysene (DBC), is a unique PAH that possesses one bay region and two fjord regions within the same molecule. Due to its structure, which is a merger of the fjord region PAHs benzo[c]phenanthrene, benzo[c]chrysene, and benzo[g]chrysene, DBC is of considerable research interest. In order to investigate the pathway of regioselective metabolism we have studied the cytotoxicity, metabolic activation and DNA adduct formation of DBC in human mammary carcinoma MCF-7 cells in culture. The cytotoxicity assay indicated undisturbed cell proliferation even at concentrations as high as 4.5 microM (1.5 micro g/ml) DBC. Concurrently, DNA adducts were detected in MCF-7 cells treated with DBC only in low amounts (0.6 pmol adducts/mg DNA). On the contrary, exposure to anti-DBC-1,2-diol-3,4-epoxide and anti-DBC-11,12-diol-13,14-epoxide, two putatively genotoxic metabolites of DBC, resulted in high levels of DNA adducts (33 and 51 pmol adducts/mg DNA, respectively). Although DBC was not efficiently transformed into DNA-reactive metabolites in MCF-7 cells in culture, the results from our study indicate that the two fjord region diol-epoxide derivatives of DBC may serve as ultimate genotoxic metabolites once they are enzymatically generated under certain circumstances in vitro or in vivo.

Cell Line, Tumor↗

PubMed2006

Targeting mitogen-activated protein kinase/extracellular signal-regulated kinase kinase in the mutant (V600E) B-Raf signaling cascade effectively inhibits melanoma lung metastases.

Malignant melanoma has a high propensity for metastatic spread, making it the most deadly form of skin cancer. B-RAF has been identified as the most mutated gene in these invasive cells and therefore an attractive therapeutic target. However, for uncertain reasons, chemotherapy inhibiting B-Raf has not been clinically effective. This has raised questions whether this pathway is important in melanoma metastasis or whether targeting a protein other than B-Raf in the signaling cascade could more effectively inhibit this pathway to reduce lung metastases. Here, we investigated the role played by (V600E)B-Raf in melanoma metastasis and showed that targeting this signaling cascade significantly reduces lung metastases. Small interfering RNA (siRNA)-mediated inhibition was used in mice to reduce expression (activity) of each member of the signaling cascade and effects on metastasis development were measured. Targeting any member of the signaling cascade reduced metastasis but inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (Mek) 1 and Mek 2 almost completely prevented lung tumor development. Mechanistically, metastatic inhibition was mediated through reduction of melanoma cell extravasation through the endothelium and decreased proliferative capacity. Targeting B-Raf with the pharmacologic inhibitor BAY 43-9006, which was found ineffective in clinical trials and seems to act primarily as an angiogenesis inhibitor, did not decrease metastasis, whereas inhibition of Mek using U0126 decreased cellular proliferative capacity, thereby effectively reducing number and size of lung metastases. In summary, this study provides a mechanistic basis for targeting Mek and not B-Raf in the mutant (V600E)B-Raf signaling cascade to inhibit melanoma metastases.

Amino Acid Substitution↗

PubMed2006

Characterization of naphtho[1,2-a]pyrene and naphtho[1,2-e]pyrene DNA adducts in C3H10T1/2 fibroblasts.

Polycyclic aromatic hydrocarbons (PAHs) are a class of carcinogenic chemicals that are ubiquitous in the environment. Fjord-region naphthopyrene isomers are structurally similar to the potent fjord-region PAH carcinogen dibenzo[a,l]pyrene and thus have the potential to be potent carcinogens. Naphtho[1,2-a]pyrene (N[1,2-a]P) exhibited similar bacterial mutagenicity and morphological cell transforming activity when compared to benzo[a]pyrene (B[a]P), whereas the structural isomer, naphtho[1,2-e]pyrene (N[1,2-e]P) was inactive is these bioassays. In this study, we examined the formation of DNA adducts in C3H10T1/2Cl8 (C3H10T1/2) mouse embryo fibroblasts exposed to N[1,2-a]P or N[1,2-e]P and their respective dihydrodiols. The DNA adducts were characterized by co-chromatography with reaction products from anti-N[1,2-a]P diol epoxide (DE) or anti-N[1,2-e]PDE and polydeoxyadenosine (dAdo) or oligodeoxyguanosine (dGuo). C3H10T1/2 fibroblasts exposed to N[1,2-a]P or N[1,2-a]P-9,10-diol produced both anti-N[1,2-a]P-DE-dAdo and -dGuo adducts with total DNA adduction levels of 22.2 to 33.3 pmol DNA adducts/mug DNA. C3H10T1/2 fibroblasts exposed to N[1,2-e]P produced 2 major and 1 minor adducts. C3H10T1/2 fibroblasts exposed to N[1,2-e]P-11,12-diol produced 2 major adducts. All of the identified adducts were anti-N[1,2-e]PDE-dGuo and -dAdo adducts. While the total DNA adduct level in N[1,2-e]P-11,12-diol-treated fibroblasts was extremely high, 105.9 pmol DNA adducts/mug DNA, the level in N[1,2-e]P-treated fibroblasts was 1.47 pmol DNA adducts/microg DNA. We conclude that lack of biological activity of N[1,2-e]P may be related to its inability to form sufficient amounts of N[1,2-e]P-11,12-diol, which would then be metabolized to sufficient amounts of anti-N[1,2-e]PDE needed to transform these fibroblasts.

Animals↗

PubMed2006

Effects of polycyclic aromatic hydrocarbons (PAHs) on vascular endothelial growth factor induction through phosphatidylinositol 3-kinase/AP-1-dependent, HIF-1alpha-independent pathway.

Previous studies have demonstrated that exposure to polycyclic aromatic hydrocarbons (PAHs) and its derivatives is associated with an increased risk of skin cancers, and the carcinogenic effect of PAHs is thought to involve both tumor initiation and promotion. Whereas PAH tumor initiation is well characterized, the mechanisms involved in the tumor promotion of PAHs remain elusive. In the present study, we investigated the effects of PAHs on vascular endothelial growth factor (VEGF) expression by comparison of its induction between the active metabolite and its parent compound (B[a]PDE versus B[a]P) or between active compound and its relatively inactive analog (5-MCDE versus CDE). We found that exposure of cells to (+/-)-anti-benzo-[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) or (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) led to marked induction of VEGF in Cl41 cells, whereas benzo[a]pyrene (B[a]P) or chrysene-1,2-diol-3,4-epoxide (CDE) did not exhibit significant inductive effects. Exposure of cells to B[a]PDE and 5-MCDE did not induce HIF-1alpha activation, whereas AP-1 was significantly activated. Moreover, overexpression of TAM67 (a dominant-negative mutant c-Jun) dramatically blocked that VEGF induction. Electrophoretic mobility shift assay showed that AP-1 was only able to specifically recognize and bind to its AP-1 potential binding site within -1136 and -1115 of the VEGF promoter region. Site-directed mutation of this AP-1 binding site eliminated the VEGF transcriptional activity induced by B[a]PDE, suggesting that the AP-1 binding site between -1136 and -1115 in the VEGF promoter region is critical for VEGF induction by B[a]PDE. In addition, overexpression of Deltap85 (a dominant-negative mutant PI-3K) impaired B[a]PDE- and 5-MCDE-induced VEGF induction. Considering our previous findings that PI-3K is an upstream mediator for c-Jun/AP-1 activation, we conclude that the VEGF induction by B[a]PDE and 5-MCDE is through PI-3K/AP-1-dependent and HIF-1alpha-independent pathways. These findings may help us to understand the mechanisms involved in PAH carcinogenic effects.

Animals↗

PubMed2006

Array CGH analysis reveals chromosomal aberrations in mouse lung adenocarcinomas induced by the human lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Exposure to genotoxic carcinogens in tobacco smoke is a major cause of lung cancer. However, the effect this has on DNA copy number and genomic stability during lung carcinogenesis is unclear. Here we used bacterial artificial chromosome array-based comparative genomic hybridization to examine the effect of NNK, a potent human lung carcinogen present in tobacco smoke, on the major genomic changes occurring during mouse lung adenocarcinogenesis. Observed were significantly more gross chromosomal changes in NNK-induced tumors compared with the spontaneous tumors. An average of 5.6 chromosomes were affected by large-scale changes in DNA copy number per NNK-induced tumor compared with only 2.0 in spontaneous lung tumors (p = 0.017). Further analysis showed that gains on chromosomes 6 and 8, and losses on chromosomes 11 and 14 were more common in NNK-induced tumors (p <or= 0.002). The changes on chromosomes 8, 11, and 14 were positively (p <or= 0.001) associated with the degree of chromosome instability (CIN). This association was less significant for gains on chromosomes 6 and 12 (p = 0.035). In contrast, loss of distal chromosome 4 was very frequent (71%) in both NNK-induced and spontaneous tumors, and showed no association with CIN phenotype. Each of these chromosomes contains sites of orthology with those altered in human lung adenocarcinomas, suggesting similar roles in human lung cancer. We next investigated the mechanism of NNK-induced CIN and found that a genotoxic metabolite of this carcinogen causes numerous karyotypic changes in lung epithelial cells. With similar changes evident in NNK-induced tumors, this suggests that the persistence of such damage in exposed lung epithelia may initiate CIN during lung carcinogenesis. These results show for the first time that NNK potently induces gross chromosomal alterations in lung adenocarcinogenesis and modulates the evolutionary pathway of these tumors through the recruitment of genes with altered DNA copy number.

Adenocarcinoma↗

PubMed2006

Molecular targets of the chemopreventive agent 1,4-phenylenebis (methylene)-selenocyanate in human non-small cell lung cancer.

Clinical chemoprevention trials of lung cancer have been somewhat disappointing and the development of highly effective chemopreventive agents is urgently needed. We previously showed that the organoselenium 1,4-phenylenebis(methylene)selenocyanate (p-XSC) is a potent chemopreventive agent in numerous preclinical animal models including a lung tumor model that employs carcinogens found in tobacco smoke. The goal of this study is to define molecular targets that will be highly promising in the design of future chemoprevention trials of non-small cell lung cancer (NSCLC), which is by far the most common type of lung cancer cases. In the present investigation, we showed that p-XSC at several doses (2.5, 5, 10 and 20 microM) including physiological levels (2.5-5.0 microM) of selenium is capable of inhibiting cell growth in a dose-dependent manner and inducing apoptosis in three NSCLC cells (NCI-H460, NCI-1299 and A549). To clarify the mechanism involved at the molecular level, we focused only on NCI-460 cells and examined the effects of p-XSC on markers that are known to be critical in the development of NSCLC. Using western blot analysis, we showed that p-XSC reduced the expression of cyclooxygenase-2 (COX-2) and phospholipase A2 (PLA2); although p-XSC inhibited both Akt and p-Akt but its effect was not significant. Using cDNA microarray approach (3800 genes per array) we found that p-XSC upregulates 22 genes by > or = 2-fold while downregulates 13 genes by < or = 0.5-fold; these altered genes include transcriptional factors, growth factors and those involved in xenobiotic metabolism as well as pro- and anti-apoptotic genes. Expression of selected genes was confirmed by RT-PCR; p-XSC reduced the levels of COX-2, PLA2, NF-kappaB and Cyclin D1 but enhanced the levels of glutathione peroxidase-5. Collectively, the results of this study showed that p-XSC alters several molecular markers in a manner that can account for its inhibitory effect of cell growth and induction of apoptosis; therefore, p-XSC may be considered a promising candidate for clinical chemoprevention of NSCLC.

Antigens, Human Platelet↗

PubMed2005

A solid-phase extraction/high-performance liquid chromatography-based (32)P-postlabeling method for detection of cyclic 1,N(2)-propanodeoxyguanosine adducts derived from enals.

The cyclic 1,N(2)-propanodeoxyguanosine (PdG) adducts are Michael addition products from reactions of deoxyguanosine (dG) with enals, including acrolein (Acr), crotonaldehyde (Cro), pentenal (Pen), heptenal (Hep), and 4-hydroxy-2-nonenal (HNE). Although this is a general reaction, only the PdG adducts derived from Acr, Cro, and HNE have been detected in vivo as endogenous DNA lesions. Our previous in vitro study demonstrated that PdG adducts of Acr, Cro, and Pen are predominantly derived from oxidation of omega-3 polyunsaturated fatty acids (PUFAs), whereas the long-chain Hep and HNE adducts are from omega-6 PUFAs. PdG adducts are important because they represent a new class of endogenous promutagenic DNA lesions with potential roles in carcinogenesis. Earlier, we developed a (32)P-postlabeling method for detecting PdG adducts from Acr and Cro and a modified method for the long-chain HNE adducts. Both methods require multiple high-performance liquid chromatography steps and, in some cases, time-consuming thin-layer chromatography for purification. There is a lack of a single, versatile, and efficient method for simultaneous detection of all five enal-derived PdG adducts. In this paper, we report an improved (32)P-postlabeling method which permits detection of Acr, Cro, Pen, Hep, and HNE adducts in a single DNA sample. This method relies on solid-phase extraction for adduct enrichment before and after (32)P-labeling; all five PdG adducts were converted to the ring-opened derivatives for confirmation of identities and quantification. The method was validated using the synthetic adducts and enal-modified DNA and was finally applied to rat liver DNA and rat liver DNA samples spiked with different amount of standards. The detection limit was determined to be as low as 0.5 fmol in 80 microg DNA, corresponding to 9 adducts/10(9) dG.

Aldehydes↗

PubMed2005

Convenient syntheses of dibenzo[c,p]chrysene and its possible proximate and ultimate carcinogens: in vitro metabolism and DNA adduction studies.

Dibenzo[c,p]chrysene (DB[c,p]C) is the only hexacyclic polycyclic aromatic hydrocarbon having two fjord regions, both in different chemical environments. Its environmental presence and relative tumorigenic potency are not known due to the lack of synthetic standards. We report here the synthesis of dibenzo[c,p]chrysene (1), its proximate carcinogens, i.e., trans-1,2-dihydroxy-1,2-dihydro-DB[c,p]C (2) and trans-11,12-dihydroxy-11,12-dihydro-DB[c,p]C (3), and possible ultimate carcinogens, i.e., anti-trans-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-DB[c,p]C (4) and anti-trans-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydro-DB[c,p]C (5). The syntheses of 1 and the appropriately methoxy-substituted DB[c,p]C (12 and 27), key intermediates for the synthesis of its proximate and ultimate metabolites, were tried first using a Suzuki cross-coupling reaction. However, the cyclization of olefins (10 and 11) gave poor yields of the desired products. An alternate method was thus developed employing a photochemical approach. The in vitro metabolism of DB[c,p]C was established with the S9 fraction of liver homogenate from phenobarbital/beta-naphthoflavone-induced Sprague-Dawley rats. The major dihydrodiol formed was identified as the fjord region 11,12-dihydroxy-11,12-dihydro-DB[c,p]C, while the major and minor phenols were identified as 11-hydroxy-DB[c,p]C and 12-hydroxy-DB[c,p]C, respectively. Further, the DNA adduction studies with the calf thymus DNA led to a mixture of dA and dG adducts for both fjord region diol epoxides (4 and 5). Interestingly, the dA to dG ratio for 1,2-dihydroxy-3,4-epoxide was much higher (3.2) compared to that of 11,12-dihydroxy-13,14-epoxide (0.5).

Animals↗

PubMed2005

Effects of suberoylanilide hydroxamic acid and trichostatin A on induction of cytochrome P450 enzymes and benzo[a]pyrene DNA adduct formation in human cells.

In this study, we investigated the effects of histone deacetylase (HDAC) inhibitors suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) on the metabolism of polycyclic aromatic hydrocarbons (PAH) in human mammary carcinoma derived MCF-7 cells in culture. Benzo[a]pyrene (B[a]P) induces cytochrome P450 (CYP) 1A1, CYP1B1 and other xenobiotic metabolizing enzymes. Results from our study indicated a significant increase in CYP activity in comparison to vehicle control in cells treated with SAHA or TSA as measured by ethoxyresorufin-O-deethylase assay. However, co-treatment with 1.0 microM SAHA and BP, reduced the mRNA levels of CYP1B1 relative to B[a]P alone. When co-treated with 1.0 microM TSA and BP, a reduction in the mRNA levels of both CYP1A1 and CYP1B1 was observed relative to BP alone. We further investigated to ascertain if the differential expression and activity of CYP1A1 and CYP1B1 influenced levels of B[a]P DNA adduct formation. MCF-7 cells co-treated with B[a]P and SAHA or TSA formed DNA adducts, although no significant differences in levels of DNA binding were revealed. These results suggest that while CYP enzyme activity and gene expression were affected by the HDAC inhibitors SAHA and TSA, they had no apparent influence on B[a]P DNA binding.

Antineoplastic Agents↗

PubMed2005

Glutathione depletion enhances the formation of endogenous cyclic DNA adducts derived from t-4-hydroxy-2-nonenal in rat liver.

Earlier, we detected the cyclic adducts of deoxyguanosine (dG) derived from t-4-hydroxy-2-nonenal (HNE), a long chain alpha,beta-unsaturated aldehyde (enal) product from oxidation of omega-6 polyunsaturated fatty acids, in tissue DNA of rats and humans as endogenous DNA damage. Recent evidence implicates the cyclic HNE adducts in human liver carcinogenesis. Because glutathione (GSH) protects against oxidative stress, we undertook a study to examine the effect of GSH depletion on the HNE-derived cyclic adducts in vivo. Four F344 rats were administered L-buthionine-(S,R)-sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at 10 mM in drinking water for 2 weeks. Rats in the control group were given water only. Livers were harvested, and each liver was divided into portions for GSH and DNA adduct analyses. The BSO treatment depleted hepatic GSH by 77%; the GSH levels were reduced from 6.3 +/- 0.3 in the control rats to 1.5 +/- 0.1 micromol/g tissues in the treated group. The formation of HNE-dG adducts, analyzed by an HPLC-based 32P-postlabeling assay, was increased by 4-fold, from 6.2 +/- 2.2 nmol/mol dG in liver DNA of control rats to 28.5 +/- 16.1 nmol/mol dG in the rats treated with BSO (p <0.05). The formation of 8-oxodG in liver DNA was also increased as a result of BSO treatment, although the increase was not statistically significant. These results further support the endogenous origin of HNE-dG adducts and, more importantly, indicate a critical role that GSH plays in protecting against in vivo formation of the promutagenic cyclic DNA adducts derived from HNE.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2005

Synthesis and identification of major metabolites of environmental pollutant dibenzo[c,mno]chrysene.

Dibenzo[c,mno]chrysene commonly known as naphtho[1,2-a]pyrene (N[1,2-a]P) is an environmental pollutant, recently identified in coal tar extract, in air-borne particulate matter, in marine sediment, and in cigarette-smoke condensate. We recently reported an efficient synthesis of N[1,2-a]P and examined its in vitro metabolism by male Sprague Dawley rat liver S9 fraction, which resulted in a number of dihydrodiol and phenolic metabolites. The synthesis of 10-hydroxy-N[1,2-a]P and fjord region N[1,2-a]P trans-9,10-dihydrodiol, which were identified among the various metabolites, was assigned earlier by comparing with the synthetic standards. The other major metabolites were separated by HPLC and, based on the 1H NMR analysis, were tentatively suggested to be the two K-region dihydrodiols, that is, N[1,2-a]P trans-4,5-dihydrodiol (6) and N[1,2-a]P trans-7,8-dihydrodiol (7), and the hydroxy derivatives of N[1,2-a]P. To unequivocally assign the structure to each of the peaks and to have them in larger amounts for toxicological studies, we have now synthesized the two K-region dihydrodiols and the 1-/3-hydroxy-N[1,2-a]P, short-listed based on the proton NMR of the collected peaks. The K-region dihydrodiols 6 and 7 were prepared by the treatment of N[1,2-a]P with OsO(4) to give a mixture of cis dihydrodiols 2 and 3, followed by pyridinium chlorochromate-assisted oxidation to quinones 4 and 5, and finally reduction with NaBH(4) to afford the dihydrodiols 6 and 7 with the desired trans stereochemistry. The 1-hydroxy-N[1,2-a]P (22) and 3-hydroxy-N[1,2-a]P (23) were synthesized using a photochemical approach. As expected, all the synthesized dihydrodiol and phenolic derivatives of N[1,2-a]P identified with those obtained from in vitro metabolism enabling the assignment of all the major metabolites.

Chromatography, High Pressure Liquid↗

PubMed2004

Methylation of cytosine at C5 in a CpG sequence context causes a conformational switch of a benzo[a]pyrene diol epoxide-N2-guanine adduct in DNA from a minor groove alignment to intercalation with base displacement.

It is well known that CpG dinucleotide steps in DNA, which are highly methylated at the 5-position of cytosine (meC) in human tissues, exhibit a disproportionate number of mutations within certain codons of the p53 gene. There is ample published evidence indicating that the reactivity of guanine with anti-B[a]PDE (a metabolite of the environmental carcinogen benzo[a]pyrene) at CpG mutation hot spots is enhanced by the methylation of the cytosine residue flanking the target guanine residue on the 5'-side. In this work we demonstrate that such a methylation can also dramatically affect the conformational characteristics of an adduct derived from the reaction of one of the two enantiomers of anti-B[a]PDE with the exocyclic amino group of guanine ([BP]G adduct). A detailed NMR study indicates that the 10R (-)-trans-anti-[BP]G adduct undergoes a transition from a minor groove-binding alignment of the aromatic BP ring system in the unmethylated C-[BP]G sequence context, to an intercalative BP alignment with a concomitant displacement of the modified guanine residue into the minor groove in the methylated meC-[BP]G sequence context. By contrast, a minor groove-binding alignment was observed for the stereoisomeric 10S (+)-trans-anti-[BP]G adduct in both the C-[BP]G and meC-[BP]G sequence contexts. This remarkable conformational switch resulting from the presence of a single methyl group at the 5-position of the cytosine residue flanking the lesion on the 5'-side, is attributed to the hydrophobic effect of the methyl group that can stabilize intercalated adduct conformations in an adduct stereochemistry-dependent manner. Such conformational differences in methylated and unmethylated CpG sequences may be significant because of potential alterations in the cellular processing of the [BP]G adducts by DNA transcription, replication, and repair enzymes.

Base Sequence↗

PubMed2004

DNA damage, repair, and mutation induction by (+)-Syn and (-)-anti-dibenzo[a,l]pyrene-11,12-diol-13,14-epoxides in mouse cells.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (-)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10(-5). This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (-)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (-)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (-)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (-)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (-)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (-)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (-)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (-)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis.

Animals↗

PubMed2004

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide.

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2004

Repair kinetics of trans-4-hydroxynonenal-induced cyclic 1,N2-propanodeoxyguanine DNA adducts by human cell nuclear extracts.

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [(32)P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were approximately 1200/10(6) dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of alpha[(32)P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [(32)P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 degrees C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.

Aldehydes↗

PubMed2004

A Highly Abbreviated Synthesis of Dibenzo[def,p]chrysene and Its 12-Methoxy Derivative, a Key Precursor for the Synthesis of the Proximate and Ultimate Carcinogens of Dibenzo[def,p]chrysene.

Dibenzo[def,p]chrysene (DBC) (1), is by far the most mutagenic and toxic polycyclic aromatic hydrocarbon identified. Its metabolic activation leads to trans-11,12-dihydroxy-11,12-dihydro-DBC (2), which is further metabolized to the ultimate metabolite, anti-trans-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydro-DBC (3), that binds to DNA causing mutations and ultimately tumor induction. We report a facile route for the syntheses of DBC (1) and its 12-methoxy derivative (12-methoxy-DBC) (13), a key intermediate for the synthesis of 2 and 3, using a Suzuki cross-coupling approach.

Benzopyrenes↗

PubMed2004

PI-3K and Akt are mediators of AP-1 induction by 5-MCDE in mouse epidermal Cl41 cells.

5-Methylchrysene has been found to be a complete carcinogen in laboratory animals. However, the tumor promotion effects of (+/-)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) remain unclear. In the present work, we found that 5-MCDE induced marked activator protein-1 (AP-1) activation in Cl41 cells. 5-MCDE also induced a marked activation of phosphatidylinositol 3-kinase (PI-3K). Inhibition of PI-3K impaired 5-MCDE-induced AP-1 transactivation, suggesting that PI-3K is an upstream kinase involved in AP-1 activation by 5-MCDE. Furthermore, we found that Akt is a PI-3K downstream mediator for 5-MCDE-induced AP-1 transactivation, whereas another PI-3K downstream kinase, p70(S6K), was not involved in AP-1 activation by 5-MCDE. Moreover, inhibition of Akt activation blocked 5-MCDE-induced activation of extracellular signal-regulated protein kinases (ERKs) and c-Jun NH(2)-terminal kinases (JNKs), whereas it did not affect p38K activation. Consistently, overexpression of a dominant-negative mutant of ERK2 or JNK1 blocked the AP-1 activation by 5-MCDE. These results demonstrate that 5-MCDE is able to induce AP-1 activation, and the AP-1 induction is specifically through a PI-3K/Akt-dependent and p70(S6K)-independent pathway.

Animals↗

PubMed2004

Mutagenicity of benzo[b]phenanthro[2,3-d]thiophene (BPT) and its metabolites in TA100 and base-specific tester strains (TA7001-TA7006) of Salmonella typhimurium: evidence of multiple pathways for the bioactivation of BPT.

Benzo[b]phenanthro[2,3-d]thiophene (BPT), and a number of its metabolites, including BPT-3,4-diol, BPT sulfoxide, BPT sulfone, and 3-hydroxyBPT were assessed for their mutagenic activity in Salmonella typhimurium strain TA100, and S. typhimurium base-specific strains TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006. Among the compounds tested in strain TA100, BPT, BPT sulfone, and 3-hydroxyBPT did not show any significant mutagenic response in the presence of S9. In contrast BPT sulfoxide and BPT-3,4-diol (a precursor to the bay-region diol epoxide of BPT) showed significant mutagenic activity in the presence of S9. Surprisingly, BPT sulfoxide was nearly 3.3-fold more mutagenic than BPT-3,4-diol in the presence of S9. BPT sulfoxide also displayed intrinsic mutagenic activity, which was nearly 1.5-fold less than that displayed by BPT-3,4-diol in the presence of S9. In base specific tester strains, BPT sulfoxide was the most active metabolite in strains TA7002, TA7004, and TA7005 with S9 activation. In these strains, BPT-3,4-diol was 2- to 7-fold less mutagenic than BPT sulfoxide in the presence of S9. Only in strain TA7006, BPT-3,4-diol was four-fold more mutagenic than BPT sulfoxide. The fact that BPT sulfoxide is significantly more mutagenic than BPT-3,4-diol in S. typhimurium strain TA100 suggests that the formation of sulfoxide may be the principal pathway for the metabolic activation of BPT to mutagenic products. Based on the results from Tester Strain TA7005, it indicate that BPT and its most mutagenic metabolite BPT sulfoxide induce predominantly CG --> AT transversion, which is observed as the most frequent base substitution mutation of p53 tumor-suppressor gene in human lung cancer.

Animals↗