To elucidate the involvement of specific ultraviolet (UV) wavelengths in solar mutagenesis, we used a laser system to investigate the induction of DNA damage, both in the overall genome and at the nucleotide resolution level, in the genomic DNA of transgenic Big Blue mouse fibroblasts irradiated with a series of UV wavelengths, inclusive of UVC (λ<280 nm), UVB (λ=280-320 nm), and UVA (λ>320 nm). Subsequently, we sought correlation between the locations of UV-induced DNA lesions in the cII transgene of irradiated DNA samples and the frequency distribution and codon position of the induced cII mutations in counterpart mouse cells irradiated with simulated sunlight. Using a combination of enzymatic digestion assays coupled with gel electrophoresis, immunodot blot assays, and DNA footprinting assays, we demonstrated a unique wavelength-dependent formation of photodimeric lesions, i.e., cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts [(6-4)PPs], based on direct UV absorption of DNA, in irradiated mouse genomic DNA, which could partially explain the induction of mutations in mouse cells irradiated with simulated sunlight. Most notably, there was a divergence of CPD and (6-4)PP formation at an irradiation wavelength of 296 nm in mouse genomic DNA. Whereas substantial formation of (6-4)PPs was detectable in samples irradiated at this wavelength, which intensified as the irradiation wavelength decreased, only small quantities of these lesions were found in samples irradiated at wavelengths of 300-305 nm, with no detectable level of (6-4)PPs in samples irradiated with longer wavelengths. Although CPD formation followed the same pattern of increase with decreasing wavelengths of irradiation, there were substantial levels of CPDs in samples irradiated with UVB wavelengths borderlined with UVA, and small but detectable levels of these lesions in samples irradiated with longer wavelengths. Because the terrestrial sunlight spectrum rolls off sharply at wavelengths ~300 nm, our findings suggest that CPDs are the principal lesion responsible for most DNA damage-dependent biological effects of sunlight.

Aromatic amines are a widespread class of environmental contaminants present in various occupational settings and tobacco smoke. Exposure to aromatic amines is a major risk factor for bladder cancer development. The etiologic involvement of aromatic amines in the genesis of bladder cancer is attributable to their ability to form DNA adducts, which upon eluding repair and causing mispairing during replication, may initiate mutagenesis. We have investigated the induction of DNA adducts in relation to mutagenesis in bladder and various nontarget organs of transgenic Big Blue mice treated weekly (i.p.) with a representative aromatic amine compound, 4-aminobiphenyl (4-ABP), for six weeks, followed by a six-week recovery period. We show an organ-specificity of 4-ABP in inducing repair-resistant DNA adducts in bladder, kidney, and liver of carcinogen-treated animals, which accords with the bioactivation pathway of this chemical in the respective organs. In confirmation, we show a predominant and sustained mutagenic effect of 4-ABP in bladder, and much weaker but significant mutagenicity of 4-ABP in the kidney and liver of carcinogentreated mice, as reflected by the elevation of background cII mutant frequency in the respective organs. The spectrum of mutations produced in bladder of 4-ABP-treated mice matches the known mutagenic properties of 4-ABP-DNA adducts, as verified by the preponderance of induced mutations occurring at G:C base pairs (82.9%), with the vast majority being G:C!T:A transversions (47.1%). Our data support a possible etiologic role of 4-ABP in bladder carcinogenesis and provide a mechanistic view on how DNA adduct-driven mutagenesis, specifically targeted to bladder urothelium, may account for organ-specific tumorigenicity of this chemical. Cancer Prev Res; 5(2); 299-308. Ó2011 AACR.

Secondhand smoke (SHS) exposure is a known risk factor for lung cancer development in lifelong nonsmokers; however, the mechanistic involvement of SHS in the genesis of this malignancy remains elusive. The present study is the first comprehensive investigation of SHS mutagenicity in vivo, in which we have established the mutagenic effects of SHS in transgenic Big Blue mice, and subsequently found correlations between our experimental findings and those obtained from our analysis of the largest database of mutations in human TP53, which is the most frequently mutated gene in human lung cancer. We demonstrate that whole-body SHS exposure of mice for 5 h/d, 5 d/wk for a duration of 2 or 4 mo elicits a significant mutagenic response in the lung, trachea, and bladder of exposed animals, as reflected by the elevation of background cII mutant frequency in the respective organs. The organ-specific mutagenicity of SHS is most pronounced in the lung and remains persistent both in the lung and bladder of SHS-exposed animals after a 1-mo recovery in clean air. The induced cII mutagenesis in the lung of SHS-exposed mice perfectly recapitulates our analysis of the TP53 mutations in human lung cancer in nonsmokers. Remarkably, the relative frequencies of all types of mutations in the TP53 gene of nonsmokers' lung tumors and in the cII transgene of lung cellular DNA from SHS-exposed mice are indistinguishable from one another. We provide the first verification of a mechanistic mode of action for SHS of relevance for carcinogenesis and the first experimental evidence linking SHS exposure to lung cancer in nonsmokers.

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Secondhand smoke (SHS) exposure is a known risk factor for lung cancer in lifelong nonsmokers. However, the underlying mechanism of action of SHS in lung carcinogenesis remains elusive. We have investigated, using the (32)P-postlabeling assay, the genotoxic potential of SHS in vivo by determining the formation and kinetics of repair of DNA adducts in the lungs of mice exposed whole body to SHS for 2 or 4 months (5h/day, 5 days/week), and an ensuing one-month recovery period. We demonstrate that exposure of mice to SHS elicits a significant genotoxic response as reflected by the elevation of DNA adduct levels in the lungs of SHS-exposed animals. The increases in DNA adduct levels in the lungs of SHS-exposed mice are dose-dependent as they are related to the intensity and duration of SHS exposure. After one month of recovery in clean air, the levels of lung DNA adducts in the mice exposed for 4 months remain significantly higher than those in the mice exposed for 2 months (P<0.0005), levels in both groups being significantly elevated relative to controls (P<0.00001). Our experimental findings accord with the epidemiological data showing that exposure to smoke-derived carcinogens is a risk factor for lung cancer; not only does the magnitude of risk depend upon carcinogen dose, but it also becomes more irreversible with prolonged exposure. The confirmation of epidemiologic data by our experimental findings is of significance because it strengthens the case for the etiologic involvement of SHS in nonsmokers' lung cancer. Identifying the etiologic factors involved in the pathogenesis of lung cancer can help define future strategies for prevention, early detection, and treatment of this highly lethal malignancy.