Mutational specificity of the anti 1,2-dihydrodiol 3,4-epoxide of 5-methylchrysene

An SV40-based pS189 shuttle vector, which contained a supF target gene and was replicated in human cells (Ad293), was used to determine the mutational specificity of anti 5-methylchrysene 1,2-dihydrodiol 3,4-epoxide, the active metabolite of the environmentally prevalent carcinogen 5-methylchrysene. The frequency of supF mutants containing point mutations increased with dose to approximately 40 times the spontaneous frequency. The induced mutations were not randomly distributed but occurred preferentially at mutagenic hotspots, which were not all identical to those reported by others for benzo[a]pyrene dihydrodiol epoxide, a metabolite with similar chemistry.     

Preferential mutagenesis at G.C base pairs by the anti 3,4-dihydrodiol 1,2-epoxide of 7-methylbenz[a]anthracene

The racemic anti-dihydrodiol epoxide of 7-methylbenz[a]anthracene preferentially induced mutations at G.C base pairs in the pS189 shuttle vector. Mutations were not randomly distributed throughout the supF target gene, but were concentrated at five hotspots. The hotspots for this agent did not correspond exactly to those produced by any other dihydrodiol epoxide examined to date, indicating that dihydrodiol epoxide structure and reactivity play a major role in determining mutagenic hotspots.     

Inhibition by a peri-fluorine atom of 1,2-dihydrodiol formation as a basis for the lower tumorigenicity of 12-fluoro-5-methylchrysene than of 5-methylchrysene

12-Fluoro-5-methylchrysene, which has a fluorine atom at a peri position, is less carcinogenic toward mouse skin than is 5-methylchrysene. To determine the basis for this observation, we identified metabolites of 12-fluoro-5-methylchrysene formed by rat liver in vitro, and used these as standards to study the metabolism of [3H]-12-fluoro-5-methylchrysene in mouse liver in vitro and in mouse epidermis in vivo. Metabolites were identified by their ultraviolet, mass, and nuclear magnetic resonance spectra, by comparison to synthetic standards, and by chemical transformations. Dihydrodiols, phenols, and hydroxymethyl derivatives of 12-fluoro-5-methylchrysene were characterized. The extents of formation of 1,2-dihydro-1,2-dihydroxy-12-fluoro-5-methylchrysene and 7,8-dihydro-7,8-dihydroxy-12-fluoro-5-methylchrysene in mouse liver in vitro were strongly influenced by pretreatment with 3-methylcholanthrene, but the ratio of 1,2-dihydrodiol to 7,8-dihydrodiol was lower than in the metabolism of 5-methylchrysene carried out under identical conditions. The major metabolites of [3H]-12-fluoro-5-methylchrysene formed in mouse epidermis, 0.33 to 4 hr after topical application, were 7,8-dihydro-7,8-dihydroxy-12-fluoro-5-methylchrysene and 7-hydroxy-12-fluoro-5-methylchrysene. The ratio of 7,8-dihydro-7,8-dihydroxy-12-fluoro-5-methylchrysene to 1,2-dihydro-1,2-dihydroxy-12-fluoro-5-methylchrysene in mouse epidermis, 2 hr after application of [3H]-12-fluoro-5-methylchrysene, was 68:1, compared to 1:1 for the corresponding dihydrodiols of 5-methylchrysene. These results show that fluorine substitution at the 12-peri position of 5-methylchrysene inhibits formation of the 1,2-dihydrodiol in the adjacent ring. Since the 1,2-dihydrodiol is a major proximate carcinogen of 5-methylchrysene, the results provide an explanation for the relatively low carcinogenicity of 12-fluoro-5-methylchrysene.     

1,2-dihydro-1,2-dihydroxy-5-methylchrysene, a major activated metabolite of the environmental carcinogen 5-methylchrysene.

The metabolic activation of the environmental carcinogen 5-methylchrysene was studied by combining high-pressure liquid chromatographic analysis of metabolites formed in vitro with assays of these metabolites for mutagenic activity toward Salmonella typhimurium. Metabolites were formed by incubation of 5-methylchrysene with the 9000 x g supernatant from Aroclor-treated rat livers. With the use of reverse-phase columns, the metabolites were resolved into nine peaks, A to I. Each peak was collected and tested for mutagenicity with activiation. Significant mutagenic activity was observed primarily in peak E and to a lesser extent in peak D. None of the other metabolites showed significant mutagenic activity. The major mutagenic metabolite (peak E) was identified as 1,2-dihydro-1,2-dihydroxy-5-methylchrysene (7.0% from 5-methylchrysene); Peak D was 7,8-dihydro-7,8-dihydroxy-5-methylchrysene (2.6% from 5-methylchrysene). Other metabolites included 9,10-dihydro-9,10-dihydroxy-5-methylchrysene, 9-hydroxy-5-methylchrysene, 7-hydroxy-5-methylchrysene, 1-hydroxy-5-methylchrysene, and 5-hydroxymethylchrysene. These results indicate that 1,2-dihydro-1,2-dihydroxy-5-methylchrysene is a major proximate mutagen of 5-methylchrysene.     

Reaction mechanisms of trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydro-5-methylchrysene with DNA in aqueous solutions

Reactions of trans-l,2-dihydroxy-anti-3,4-epoxy-l,2,3,4-tetrahydro-5-methylchrysene(anti-5-MeCDF) with DNA and the effects of ionic strength onthe reaction were studied in aqueous buffer solution (5 mM sodiumphosphate, pH 7) by means of absorption and fluorescence spectroscopy.The results are compared with those obtained with the widelystudied metabolite model compound trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene(BaPDE5.The rate constant of hydrolysis of 5-MeCDE is k = 1.0 ? 10⁴s,which is {small tilde}35 times smaller than the value of k forBaPDE under similar conditions. As in the case of BaPDE, therate of reaction of 5-MeCDE is accelerated in the presence ofDNA. This effect is attributed to the rapid formation of physicalassociation complexes (binding constant K) and the subsequentslower formation (rate constant k₃) of carbocations at DNA bindingsites, which in turn decay rapidly via hydrolysis to tetraols(l,2,3,4-tetrahydroxytetrahydro-5-methylchrysene, 5-MeCT) andto covalent adducts. The values of K and k₃ are 2800 ? 300/Mand 8.7 ? 10^–3/s respectively, and are reduced to 450? 100/M and 1.8 ? 10^–3/s in the presence of 0.1 M NaCl.The fraction of 5-MeCDE molecules which bind covalently to DNAis, on the other hand, constant under these conditions and liesin the range of 5–8%. Similar values for the covalentbinding are observed for BaPDE, even though the physical associationconstant K is {small tilde}10 times larger than for 5-MeCDEunder similar conditions. This difference in the values of Kare attributed to the larger aromatic ring system in BaPDE whichallows for a higher interaction of this molecule with the basesof DNA. Finally, the tetraol derived from the hydrolysis of5-MeCDE also binds non-covalently to DNA, but the value of Kis {small tilde}3 times smaller than for the diol epoxide.     

Mutational specificity of 2-amino-3-methylimidazo-[4,5-f]quinoline in the hprt locus of CHO-K1 cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a food carcinogen formed in cooked meats, can induce gene mutation at the hprt locus of CHO-K1 cells in the presence of hepatic 59 mix. To elucidate the molecular nature of IQ-induced mutation, we characterized the entire coding region of the hypoxanthine phosphoribosyl-transferase gene of 23 independent mutants derived from IQ-treated CHO cells by direct sequencing of polymerase chain reaction-amplified cDNA. Ten of the 23 IQ-induced mutants examined contained single base substitutions; one mutant had three single-base substitutions. Among the base substitutions, G·C→CG (six of 13) and A·T→CG (three of 13) transversions predominated. Most of the base-substitution mutations occurred preferentially at a middle G and had a dA in their 3′ ends. Of the 13 other mutations (56.5%), 12 missing one or more complete exons were splice-site mutations, and one mutant had a partial deletion of an exon. A high frequency of complete exon deletion (11 of 12) in exons 2–5 was observed. Interestingly, 75% of the mutants (nine of 12) with splice-site mutations were induced by IQ only at higher concentrations (300–500 μM). This was probably due to the occurrence of GC base-substitution mutations that affected hprt mRNA splicing, especially at the intron-exon boundaries. © 1995 Wiley-Liss, Inc.     

Metabolism of the bay-region trans-1,2-dihydrodiol of benz[a]anthracene in rat liver microsomes occurs primarily at the 3,4-double bond

The bay-region hydroxyl groups of benz[a]anthracene (+/-)trans-1,2-dihydrodiol are known to be exclusively in quasi-axial conformations. This dihydrodiol was stereoselectively metabolized in rat liver microsomes to form two optically active 1,2,3,4-tetrahydrotetrols as the predominant products. Thus, the bay-region axial hydroxyl groups of benz[a]anthracene trans-1,2-dihydrodiol do not direct metabolism away from the vicinal 3,4-double bond.     

Malignant transformation of the liver tumour precursor cell line OC/CDE 22 by the four stereoisomeric fjord region 3,4-dihydrodiol 1,2-epoxides of benzo[c]phenanthrene

In previous work we established the rat liver oval cell lineOC/CDE 22 in order to study in vitro mechanisms of liver celltransformation. We have now exposed OC/CDE 22 cells to eachof the four optically active fjord region dihydrodiol epoxidesof benzo[c]phenanthrene to investigate their capacity for malignanttransformation of liver cells. All four configurational isomers,which are among the most potent carcinogenic metabolites ofpolycyclic aromatic hydrocarbons tested in murine tumour models,malignantly transform OC/CDE 22 cells at a 2 µM dose level,resulting in a similar colony-forming efficiency in soft agar.Inoculation of the transformed cells into newborn syngeneicrats produced an extremely high incidence of carcinomas witha short latency period. The induced carcinomas displayed cholangiocellular,adenoid and solid growing structures. Neither cell growth insoft agar nor induction of tumor formation in newborn rats wereachieved if confluent OC/CDE 22 cell cultures were exposed toeach of the four stereoisomers and left in the confluent statefor 4 weeks. In contrast, if confluent cells were exposed tothe four stereoisomers, immediately split and then subculturedas usual, full transformation was accomplished. Our resultsindicate that the fjord region dihydrodiol epoxides of benzo[c]phenanthreneare highly efficient transforming agents for rat liver cellsand that proliferation plays a pivotal role in the liver celltransformation process induced by polycyclic aromatic hydrocarbons.     

Mutational specificity of 2-nitro-3,4-dimethylimidazo[4,5-f]quinoline in the lacI gene of Escherichia coli

2-Nitro-3,4-dimethylimidazo[4,5-f]quinoline (NO₂-MeIQ) is thenitro derivative of 2-amino-3,4-dimethylimi-dazo[4,5-f]quinoline(MeIQ), one of the food pyrolysis product mutagens and carcinogens.The mutational specificity of NO₂-MeIQ was determined usingthe lacI system in an Escherichia coli strain, EE125, harbouringthe plasmid pKM101. NO₂-MeIQ acts as a direct-acting mutagentowards this strain, and induces a broad range of mutationalalterations. At higher doses; G:C     

Insulin-enhanced antitumor effect of 5-fluorouracil in vivo

Objective  To determine if insulin treatment can enhance the antitumor effect of 5-fluorouracil (5-FU), and to explore the mechanism of the enhancement of insulin. Methods  S180 sarcoma, H22 liver cancer and human Eca-109 esophageal cancer cells were transplanted into nude mice to evaluate the inhibitory effect on tumor growth of insulin alone or in combination with 5-FU. The levels of serum insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) were determined. Results  Compared with 5-FU treatment alone, the tumor weight of H22 liver cancer and S180 sarcoma was reduced further with high, medium and low-dose insulin (0.09, 0.06, 0.03 U/20 g) + 5-FU treatment. When a high dosage of insulin + 5-FU was administered, tumor weight was significantly reduced (P < 0.05). The inhibitory rate of growth of S180 sarcoma and H22 liver cancer reached 50.2% and 51.4%, respectively, which was significantly higher than 24.9% and 27.9% in the group receiving 5-FU alone (P < 0.05). High, medium and low-dose insulin combined with 5-FU significantly inhibited the growth of Eca-109 cancer cells (P < 0.05). Compared with the control group, the level of serum IGF-1 decreased (P < 0.05), whereas the level of serum IGFBP-3 slightly increased in the 5-FU ± insulin groups (P > 0.05). In mice with H22 liver cancer and S180 sarcoma the IGF-1 level with high-dose insulin + 5-FU treatment was significantly lower compared to treatment with 5-FU alone (P < 0.05), but the difference was not significant in mice transplanted with esophageal cancer cells. Conclusion  Insulin can enhance the anti-tumor effect of 5-FU without significantly increasing 5-FU toxicity. Although changes in the serum IGF-1 or IGFBP-3 level do not explain the mechanism of the insulin-induced enhancement on 5-FU on growth, a decrease in the level of serum IGF-1 and an increase in serum IGFBP-3 may be important in the chemotherapeutic response. This work was supported by a grant from the Project for Science and Technology Creative Talents of Henan Province, China (No. 2005026).     

Tumorigenicity of the diastereomeric benz[a]anthracene 3,4-diol-1,2-epoxides and the (+)- and (-)-enantiomers of benz[a]anthracene 3,4-dihydrodiol in newborn mice.

The tumorigenic activity of benz[a]anthracene (BA), the (+)- and (-)-enantiomers of trans-3,4-dihydroxy-3,4-dihydrobenz[a]anthracene (BA 3,4-dihydrodiol), and the racemic diastereomers of the BA 3,4-diol-1,2-epoxides [i.e., either or both of the diastereomeric 1,2-epoxides derived from BA 3,4-dihydrodiol in which the epoxide oxygen is cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 4-hydroxyl group) was examined in newborn Swiss-Webster mice. The mice were administered ip a total dose of 280 nmoles of compound in divided doses consisting of 40 nmoles within 24 hours of birth, 80 nmoles at 8 days of age, and 160 nmoles at 15 days of age. The experiment was terminated when the animals were 26 weeks of age. BA 3,4-diol-1,2-epoxide-2 was the most potent compound tested. All animals treated with BA 3,4-diol-1,2-epoxide-2 developed pulmonary tumors with an average of 13.3 tumors per mouse. BA 3,4-diol-1,2-epoxide-1 produced pulmonary tumors in 42% of the mice with an average of only 0.56 tumors per mouse. The (-)-enantiomer of BA 3,4-dihydrodiol with [3R,4R] absolute stereochemistry was the second most tumorigenic derivative of BA tested; it produced pulmonary tumors in 71% of the mice with an average of 1.88 tumors per mouse. BA and the (+)-enantiomer of BA 3,4-dihydrodiol had little or no tumorigenic activity at the dose tested. A comparison of the average number of pulmonary tumors per mouse revealed that BA 3,4-diol-1,2-epoxide-2 was about 30-fold more tumorigenic than was BA 3,4-diol-1,2-epoxide-1, 8-fold more tumorigenic than was (-)-BA 3,4-dihydrodiol, and greater than 85-fold more tumorigenic than was BA. These data indicate that in newborn mice BA 3,4-dihydrodiol and a BA 3,4-diol-1,2-epoxide are proximate and ultimate carcinogenic metabolites of BA, respectively.     

Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli. III: The cyclic nitrosamine N-nitrosopyrrolidine is a complex mutagen

The mutational specificity of the cyclic nitrosamine N-nitrosopyrrolidine (NPYR) was determined through the DNA sequence characterization of 33 lacl-d mutations of Escherichia coli. Base substitution was the predominant class of mutation induced (91%). The majority of these (64%) occurred at GC base pairs, in accordance with the predicted significance of NPYR-derived guanine adducts. In addition, this nitrosamine efficiently produced other kinds of base substitution events as 11 of the 33 mutations occurred at AT base pairs. Deletion, frameshift, and duplication events were also recovered. The complexity of the NPYR mutational spectrum appears to be consistent with the suggestion that this compound acts through both direct and indirect mutational pathways.     

Mutational specificities of environmental carcinogens in the lacl gene of Escherichia coli. II: A host-mediated approach to N-nitroso-N,N-dimethylamine and endogenous mutagenesis in vivo

An intrasanguineous host-mediated assay was used to determine the mutational specificity of the hepatocarinogen N-nitroso-N,N-dimethylamine metabolized in vivo. A total of 114 forward mutations in the lacl gene of Escherichia coli reisolated from the livers of treated Swiss albino mice were characterized at the DNA sequence level. Consistent with the methylating ability of this compound and the demonstrated mutagenic specificity of O6-methylguanine, the predominant mutation was the G:C----A:T transition. These were recovered, on average, seven times more frequently at guanines flanked (5') by a purine residue than at those preceded by a pyrimidine residue--a specificity similar to that reported for many direct-acting SN1 alkylating agents. This nitrosamine appears to be distinguished from related N-nitroso methylating compounds by the induction of additional mutational events. Here, the exceptions consisted of four A:T----G:C transitions, four A:T site transversions, and a single G:C----T:A transversion. In addition, the DNA sequence alterations of 34 I- mutants of E. coli reisolated from otherwise untreated mice were identified. The predominant mutation was the G:C----A:T transition, which accounted for almost half of all background mutations. The sites at which these mutations were recovered appear to indicate that some of these mutations may have arisen as a result of an accelerated rate of cytosine deamination. These data suggest that many of the additional "spontaneous" mutations observed under in vivo conditions resulted from genotoxic events occurring during the host-defense (immune) reaction.     

Mutational specificities of N-nitrosamines in a host-mediated assay: comparison with direct-acting N-nitroso compounds in vitro and an approach to deducing the nature of ultimate mutagens in vivo.

The mutational activities and specificities of several N-nitrosamines in Salmonella recovered from mouse liver in the host-mediated assay (HMA) were compared with the specificities of related direct-acting N-nitroso compounds in vitro. The specificities of the direct-acting methyl, ethyl, propyl, and 2-hydroxypropyl compounds were all different and presumably are attributable to the DNA adducts resulting from the corresponding alkyldiazonium or carbonium ions. Introduction of a 2-hydroxyl group greatly influenced the mutational specificity. The 2-oxopropyl compound showed the same specificity as the methyl compound. This result is consistent with one of the known breakdown pathways of the oxopropyl diazonium ion (or related reactive species), which leads to a methyl diazonium ion. The N-nitrosodialkylnitrosamines N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosodipropylamine (NDPA), which all require metabolic activation, showed specificities in the HMA similar to those of their direct-acting counterparts. The cyclic nitrosamine N-nitrosopyrrolidine was weakly active in the HMA, although its direct-acting derivative was a potent mutagen in vitro. The results for NDMA and NDEA were consistent with most previous studies of the metabolism of these compounds in vivo. However, NDPA can yield methylating, and probably hydroxypropylating, species in addition to propyldiazonium ion. As the specificity of NDPA was similar to that of a propylating agent, NDPA appears to lead to genotoxic products in the mouse liver mainly by direct alpha-hydroxylation. The initial results described here indicate that mutational specificity in the HMA can be used to deduce metabolic pathways leading to genotoxic products when the appropriate proximate mutagens are available as standards. Furthermore, we observed a reasonable correlation between potency in the HMA and hepatocarcinogenesis.     

Mutational specificities of environmental carcinogens in the lacl Gene of Escherichia coli VII: The host-mediated assay and its comparison with in vitro mutagenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

To investigate the influence of different types of metabolic activation (9,000 x g supernatant (S9) activation vs. a host-mediated approach) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mutational specificity, we determined by DNA sequencing the distribution of forward mutations recovered in the N-terminal region of the lacl gene of Escherichia coli. After activation with the S9 liver fraction from rats treated with Aroclor 1254, a diverse spectrum of mutations was recovered, with 55% of the events being G:C→A:T transitions. In contrast, after the host-mediated assay in mice, G:C→A:T transitions accounted for over 94% of the mutations recovered. Generally, NNK metabolism can proceed through two distinct pathways, involving either α-methyl or methylene hydroxylation. These two pathways produce different distributions of DNA damage. The difference in the mutational spectra we observed thus likely reflects the difference in the contributions of each pathway under the two different treatment conditions.     

Mutational specificity of the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]-indole in mammalian cells

We have used the pZipHprtNeo shuttle vector to determine the types of DNA sequence alterations induced by a potent carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P2). The shuttle vector contains a human cDNA hprt as the target gene and is stably integrated into a chromosome of the mouse cell line VH12. After Trp-P2 treatment, 59 independent HPRT- mutant clones of VH12 were isolated and altered sequences of the mutant hprt- cDNA genes were determined. Mutations induced by Trp-P2 comprised a variety of events; base substitutions, frameshifts, deletions and complex. Frameshifts were the most frequent mutational events (51%), and base substitutions were the next most frequent (30%) followed by deletions (14%). Examination of the DNA sequence context in the mutant genes revealed that approximately 70% of mutations induced by Trp-P2 occurred at G:C sites and thymine residues were the suggested target for the remainder of mutations. The results seem consistent with the previously reported finding that in vivo, metabolically activated Trp-P2 specifically binds to the C8 position of guanine residues in DNA to form C8G-Trp-P2 adducts (Hashimoto et al., Mutat, Res., 105, 9-13, 1982). As for molecular mechanisms, we showed that slippage and slippage misalignment could predict the generation of a large portion of Trp-P2-induced mutations found in the cDNA gene.     

二乙酰去水卫矛醇联合p53基因转染对肝癌细胞生长的影响

目的：观察野生型p53基因转染联合二乙酰去水卫矛醇(1，2：5，6-dianhydro-3，4-(liacetylgalactitol，DADAG)对肝癌细胞生长的影响。方法：将克隆有野生型p53基因的pUHD10-3质粒，通过脂质体(lipofectamine)介导转染人肝癌细胞，同时，在培养液中加入DADAG，用四甲基偶氮唑(MTT)法分析细胞的生长情况。结果：人肝癌细胞HLE转染野生型p53基因后，其生长作用明显受到抑制，细胞生长抑制率在第4天可达67．68％。如果HLE细胞中只加入含DADAG的培养液后，其细胞生长也受到一定的抑制，生长抑制率最高可达到53．09％。肝癌细胞HLE转染野生型p53基因后联合应用DADAG时，细胞的生长抑制率在第4天可达到85．37％，实验结果显示DAD-AG联合野生型p53基因增强对肝癌细胞的生长抑制作用。结论：DADAG能加强野生型p53基因对肝癌细胞的生长抑制作用。     

Down-regulation of KLF5 in cancer-associated fibroblasts inhibit gastric cancer cells progression by CCL5/CCR5 axis

It was well known that cancer-associated fibroblasts (CAFs) were an essential factor in tumor progression. However, the actual mechanism of stromal fibroblasts activation and tumor promoting effects remain unclear. Here, we showed that KLF5 expression was more frequently observed in gastric cancer-associated fibroblasts compared with normal mucosal fibroblasts. Moreover, KLF5 expression in tumor stroma was closely associated with clinicopathological features such as tumor size, invasion depth, cell grade and lymph node metastasis, as well as poor prognosis in patients with gastric cancer. In addition, we further demonstrated that KLF5-regulating CAFs affect gastric cancer cells progression by CCL5 secretion and activation of CCR5. Taken together, we concluded that KLF5 expression in gastric cancer-associated fibroblasts contribute to poor survival and promote cancer cells progression by activation of CCL5/CCR5 axis, which suggesting that KLF5 in CAFs might be considered as a promising target for the treatment of gastric cancer.