High frequency of deletion mutations in p53 gene from squamous cell lung cancer patients in Taiwan.

Lung cancer is the leading and second-leading cause of cancer deaths among women and men in Taiwan, respectively. However, the molecular mechanisms involved in lung tumorigenesis in Taiwan remain poorly defined. A study that analyzed the mutation spectrum of the p53 tumor suppressor gene in 35 female lung cancer patients in Hong Kong showed that a high proportion of the mutations observed were deletions, suggesting the possible involvement of a distinct mutagenic factor(s) in Chinese female lung cancer patients (Y. Takagi et al., Cancer Res., 55: 5354-5357, 1995). Therefore, to gain insight into the role of the p53 tumor suppressor gene and possible etiological factors in lung tumorigenesis in Taiwan, we investigated the mutation spectra of exons 4-11 in the p53 tumor suppressor gene of 60 lung cancer patients in Taiwan. These data were also correlated with clinical pathological characteristics of patients. Lung tumors were surgically resected, genomic DNA was isolated, and their mutation spectra were examined using PCR/single-strand conformational polymorphism analysis and direct sequencing. The frequency of p53 gene mutation was 18% (11 of 60). However, distinct patterns of p53 gene mutation were observed. Seven of 11 mutations detected (64%) were deletions of 1-12 bp at G:C bp or at bp in the immediate vicinity of repetitive sequences and/or tandem repeat sequences. In addition, two patients (2 of 11, 18%) exhibited nonsense mutations. In contrast to the frequent occurrence of missense mutations in the p53 gene reported in the literature, the majority (82%) of the mutations in lung cancer patients in Taiwan were nonmissense mutations, ie., deletions and nonsense mutations. Immunohistochemical staining indicated that p53 mutations including non-in-frame deletions and nonsense mutations all resulted in no expression of p53 protein. Notably, mutations occurred more frequently in patients suffering from squamous cell carcinoma (SQ). Nine of 31 SQ patients (29%) exhibited deletions or nonsense mutations, suggesting that deletions and nonsense mutations in the p53 gene are involved in the formation of SQ in Taiwan. In addition, mutations occurred more frequently in patients with stage III or IV lung cancer. However, mutations were not correlated with patients' smoking habits. Our data suggest that p53 gene mutation involved in the formation of SQ and distinct environmental factor(s) and/or genetic factor(s) that induced specific short deletions in repeat sequences may be involved in lung tumorigenesis in Taiwan.

Bacterial mutagenicity, metabolism, and DNA adduct formation by binary mixtures of benzo[a]pyrene and 1-nitropyrene.

Air pollutants are a complex mixture containing polycyclic organic compounds. Among these are 1-NP and B[a]P, which are important contributors to the mutagenicity of diesel exhaust and airborne particulate matters. To investigate the interaction of a complex mixture of airborne mutagens, the mutagenicity of 1-NP was examined with S. typhimurium TA98 and TA98NR in the presence and absence of B[aP. B[a]P exhibited a more antagonistic effect on the mutagenicity of 1-NP in strain TA98 than in strain TA98NR. Also studied were (1) the inhibitory effects of B[a]P on the nitroreductive metabolism of 1-NP and (2) DNA adduct formation by 1-NP. Nitroreductase was associated with the metabolism of 1-NP, and was reduced in a dose-dependent manner in a binary mixture of 1-NP and B[a]P. HPLC analysis showed that the amounts of 1-AP and NAAP, the metabolites of 1-NP, were significantly decreased by the addition of B[a]P in mixtures. The results indicate that the antagonistic effect of B[a]P on the mutagenicity of 1-NP is mediated through altering its nitroreductive metabolism.

Modulatory effects of polycyclic aromatic hydrocarbons on the mutagenicity of 1-nitropyrene: a structure-activity relationship study.

Benzo[a]pyrene (B[a]P) is able to inhibit the mutagenicity of 1-nitropyrene (1-NP) through the reduction of nitroreductase activity and formation of adducts with DNA. The relationships between the chemical structure of 9 polycyclic aromatic hydrocarbons (PAHs) and antagonistic effects on the 1-NP-induced mutation were evaluated by the binary mixtures of 1-NP and PAHs with Salmonella typhimurium TA98 in the absence of S9 mix. Remarkably different antagonistic effects of 9 PAHs on the mutagenicity of 1-NP were observed. Among the tested PAHs, coronene demonstrates the most antagonistic potential followed by benzo[g,h,i]perylene (B[g,h,i]P), benzo[e]pyrene (B[e]P), dibenzo[a,h]pyrene (DB[a,h]P), benzo[a]pyrene (B[a]P) and pyrene. Naphthalene, anthracene, and chrysene had only minor inhibitory activity on the 1-NP mutagenicity. The modifying effects of PAHs on the nitroreductase activity of TA98 strains in the presence of 1-NP were further examined from the production of 1-AP. The statistical analytical data showed that the inhibitory effect of PAHs on the mutagenicity of 1-NP significantly correlated with their effects on the nitroreductase activity (r = -0.69, p < 0.05). In addition, the formation of 1-NP-DNA adducts of the binary mixtures of 1-NP and PAH was determined by the 32P-postlabeling method. The results indicated that the modulatory effects of PAHs on the formation of 1-NP-DNA adducts were correlated well with their antagonistic activity (r = -0.91, P < 0.01). From the above results, the relationships between the chemical structure of PAHs and the antagonistic effects on the 1-NP mutagenicity were revealed by the surface area and electronic parameters of PAHs. The planar molecular area of PAHs was more convincingly correlated with the antagonistic effect on the mutagenicity of 1-NP (r = -0.81, p < 0.01) than that with the difference in energy, delta E, between EHOMO and ELUMO (r = 0.69, p < 0.05). According to the above, two possible mechanisms are involved in the interactive effect of the binary mixtures: (1) a higher binding affinity with nitroreductase for PAHs having a large planar surface area; and (2) a high energy of interaction between 1-NP and PAHs with a low delta E might decrease the nitroreductive capability.

Emodin inhibits the mutagenicity and DNA adducts induced by 1-nitropyrene.

Polygonum cuspidatum S. (PC) is frequently used as a laxative and an anticancer drug in Chinese medicine. The inhibitory effect of this herb and its component, emodin, on the direct-acting mutagenicity of 1-nitropyrene (1-NP) was examined using the Ames/microsomal test with Salmonella typhimurium TA98 and the genotoxicity of 1-NP was evaluated using the SOS chromotest with E. coli PQ37. Emodin and water extracts of PC markedly decreased the mutagenicity of 1-NP in a dose-dependent manner in both assay systems. Furthermore, emodin and the extracts of PC significantly inhibited the formation of 1-NP DNA adducts in S. typhimurium TA98 in the 32P-postlabeling study. The results suggest that PC extracts and emodin act as blocking and/or suppressing agents to reduce the direct-acting mutagenicity of 1-NP.

Benzo[g,h,i]perylene synergistically transactivates benzo[a]pyrene-induced CYP1A1 gene expression by aryl hydrocarbon receptor pathway.

Although benzo[g,h,i]perylene (BghiP) has been found to promote the carcinogenesis of benzo[a]pyrene (BaP) in animal models, not much is known about this cocarcinogenic mechanism. In this study, human hepatoma HepG2 cells cotreated with BaP and BghiP were used as a model to investigate the cocarcinogenic mechanism of BghiP in BaP-induced carcinogenesis. DNA adduct formation is thought to initiate carcinogenesis, so the effect of BghiP on BaP-DNA adduct formation was evaluated using a (32)P-postlabeling assay. The BaP-DNA adduct levels increased following the addition of BghiP, in a dose-dependent manner. However, no adducts were formed with BghiP alone. Our previous report showed that cytochrome P450 1A1 (CYP1A1) is responsible for the metabolic activation of BaP and the formation of B[a]P adduct in HepG2 cells. Western blot and Northern blot analyses were used to evaluate whether BaP-induced CYP1A1 protein and mRNA levels increased following the addition of BghiP. Our data showed that BghiP enhanced BaP-induced CYP1A1 protein and its mRNA levels. To understand whether BghiP enhances BaP-induced CYP1A1 gene expression through the aryl hydrocarbon receptor (AhR) signaling pathway, a gel retardation assay was performed to elucidate the synergistic mechanism of BghiP in BaP-induced CYP1A1 gene expression. The results showed that BghiP causes an increase in the nuclear accumulation of AhR in cells and/or activation of AhR to a DNA-binding form. There was a concordant increase in the transcription activation of CYP1A1 gene and the induction of AhR signal pathway. Our findings demonstrated that BghiP enhances BaP-induced CYP1A1 transcription by AhR activation and suggested that the induction mechanism of CYP1A1 contributes to the cocarcinogenic potential of BghiP in BaP-induced carcinogenesis.

Suppressive effects of methyl methacrylate on the mutagenicity and DNA adduct formation induced by 1-nitropyrene and benzo[a]pyrene.

Methyl methacrylate (MMA) is widely used as a cement in dentistry, orthopaedic surgery and ophthalmology. Studies based on short-term genotoxicity tests have produced conflicting results in the last two decades. In the present study, the effects of MMA on the mutagenicity of 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P) were evaluated with the Salmonella typhimurium TA98 strain in the absence and presence of S9 mix. The direct-acting mutagenicity of 1-NP was markedly decreased by MMA in a dose-dependent manner. However, a low inhibitory effect of MMA on the metabolic-acting mutagenicity of B[a]P was observed. MMA did not show mutagenicity within the concentrations of 4.7-37.6 microM either with or without S9 mix. The inhibitory effect of MMA was not due to its cytotoxicity because very low and/or no cytotoxicity of MMA to S. typhimurium TA98 was observed. To confirm the antimutagenicity of MMA against 1-NP and B[a]P, a 32P-postlabelling method was used to determine whether MMA modified DNA adduct formation produced by both compounds in calf thymus DNA. MMA inhibits the formation of 1-NP- and B[a]P-DNA adducts in a dose-dependent manner. The DNA adduct of 1-NP reduced by MMA was greater than that of B[a]P. Thus, we suggested that MMA was possibly acting as an inhibitor of chemical carcinogenesis.