Cytotoxic Homo- and Hetero-Dimers of o-toluidine, o-anisidine, and Aniline Formed by In Vitro Metabolism.

Several aromatic amine compounds are urinary bladder carcinogens. Activated metabolites and DNA adducts of polycyclic aromatic amines, such as 4-aminobiphenyl, have been identified, whereas those of monocyclic aromatic amines, such as o-toluidine (o-Tol), o-anisidine (o-Ans), and aniline (Ani), have not been completely determined. We have recently reported that o-Tol and o-Ans are metabolically converted in vitro and in vivo to cytotoxic and mutagenic p-semidine-type dimers, namely 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD) and 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), respectively, suggesting their roles in urinary bladder carcinogenesis. In this study, we found that when o-Tol and o-Ans were incubated with S9 mix, MMBD and MxMxBD as well as two isomeric heterodimers, MMxBD and MxMBD, were formed. Therefore, any two of o-Tol, o-Ans, and Ani (10 mM each) were incubated with the S9 mix for up to 24 h and then subjected to LC-MS to investigate their metabolic kinetics. Metabolic conversions to all nine kinds of p-semidine-type homo- and hetero-dimers were observed, peaking at 6 h of incubation with the S9 mix; MxMxBD reached the peak at 6.1 ± 1.4 µM. Homo- and hetero-dimers containing the o-Ans moiety in the diamine structure showed a faster dimerization ratio, whereas levels of these dimers, such as MxMxBD, markedly declined with further incubation. Dimers containing o-Tol and Ani were relatively stable, even after incubation for 24 h. The electron-donating group of the o-Ans moiety may be involved in rapid metabolic conversion. In the cytotoxic assay, dimers with an o-Ans moiety in the diamine structure and MMBD showed approximately two- to four-fold higher cytotoxicity than other dimers in human bladder cancer T24 cells. These chemical and biological properties of homo- and hetero-dimers of monocyclic aromatic amines may be important when considering the combined exposure risk for bladder carcinogenesis.

New horizons of DNA adductome for exploring environmental causes of cancer.

Chemical carcinogenesis is focused on the formation of DNA adducts, a form of DNA damage caused by covalent binding of a chemical moiety to DNA. The detection of carcinogen-DNA adducts in human tissues, along with demonstration of mutagenicity/carcinogenicity in experimental systems, and validation of adducts as biomarkers of environmental exposure and indicators of cancer risk in molecular epidemiological studies suggests a pivotal role of DNA adducts in cancer development. However, accurate measurement of DNA adducts in varied biological samples is challenging. Advances in mass spectrometry have prompted the development of DNA adductome analysis, an emerging method that simultaneously screens for multiple DNA adducts and provides relevant structural information. In this review, we summarize the basic principle and applications of DNA adductome analysis that would contribute to the elucidation of the environmental causes of cancer. Based on parallel developments in several fields, including next-generation sequencing, we describe a new approach used to explore cancer etiology, which integrates analyses of DNA adductome data and mutational signatures derived from whole-genome/exome sequencing.

o-Anisidine Dimer, 2-Methoxy-N4-(2-methoxyphenyl) Benzene-1,4-diamine, in Rat Urine Associated with Urinary bladder Carcinogenesis.

Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

Multifocal origin of occupational cholangiocarcinoma revealed by comparison of multilesion mutational profiles.

Recently identified occupational cholangiocarcinoma among printing workers is characterized by chronic bile duct injuries and precancerous or early cancerous lesions at multiple sites of the bile ducts. These observations suggested the potential multifocal carcinogenesis of the disease. We performed whole-exome analysis of multiple lesions, including the invasive carcinomas and precancerous lesions of four occupational cholangiocarcinoma cases. A much higher mutation burden was observed in both the invasive carcinomas (mean 76.3/Mb) and precancerous lesions (mean 71.8/Mb) than in non-occupational cholangiocarcinomas (mean 1.6/Mb). Most somatic mutations identified in 11 of 16 lesions did not overlap with each other. In contrast, a unique trinucleotide mutational signature of GpCpY to GpTpY was shared among the lesions. These results suggest that most of these lesions are multiclonal in origin and that common mutagenic processes, which may be induced by exposure to haloalkanes or their metabolites, generated somatic mutations at different sites of the bile ducts. A similarly high mutation rate had already been identified in the precancerous lesions, implying an increased potential for carcinogenesis throughout the biliary tree. These genomic features support the importance of ongoing close follow-up of the patients as a group at high risk of recurrence.

Novel o-Toluidine Metabolite in Rat Urine Associated with Urinary Bladder Carcinogenesis.

o-Toluidine (o-Tol), a monocyclic aromatic amine, causes bladder cancer in humans and experimental animals and is therefore classified as a Group 1 carcinogen (IARC) in which the carcinogenicity of o-Tol is involved in metabolic activation, DNA damage, and DNA adduct formation. In the DNA adduct formation mechanism, o-Tol is metabolized by N-hydroxylation, N-acetoxylation, and then deacetoxylation to produce an electrophilic nitrenium ion, which is able to bind to a DNA base, such as dG-C8. Therefore, dG-C8-o-Tol is thought to be a plausible DNA adduct of o-Tol exposure. However, direct detection of dG-C8-o-Tol in biological samples has not been reported yet. Here, we show that a novel o-Tol metabolite, 2-methyl-N1-(2-methylphenyl)benzene-1,4-diamine (MMBD), a dimer by head-to-tail binding, was identified for the first time in o-Tol-exposed rat urine. MMBD was also detected in a reaction of o-Tol and S9 mix, indicating the formation was catalyzed by an enzymatic reaction. Moreover, MMBD showed a potent stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains,and cytotoxicity in human bladder carcinoma T24 cells and human spleen lymphoblastoid TK6 cells compared with o-Tol. Furthermore, a DNA adduct (m/z 478.1) corresponding to dG-MMBD was detected in the reaction of calf thymus DNA with rat urine containing MMBD, and also in hepatic DNA of rats treated with o-Tol. These results therefore suggested that o-Tol-induced bladder carcinogenesis could be at least partly attributed to MMBD formation. The possible dimerization of monocyclic aromatic amines should be considered in the evaluation of the risk of bladder carcinogenesis after exposure.

Comprehensive analysis of DNA adducts (DNA adductome analysis) in the liver of rats treated with 1,4-dioxane.

1,4-Dioxane is a genotoxic carcinogen, and its mutagenic properties were recently observed in the liver of guanine phosphoribosyl transferase (gpt) delta transgenic rats. However, the mechanisms of its genotoxicity remain unclear. We analyzed DNA adduct formation in rat livers following 1,4-dioxane treatment. After administering 1,4-dioxane in drinking water at doses of 0, 20, 200, and 5,000 ppm, liver adduct formation was analyzed by DNA adductome analysis. Adducts in treated rat livers were dose-dependently increased compared with those in the control group. Principal component analysis-discriminant analysis (PCA-DA) clearly revealed two clusters of DNA adducts, associated with 0 ppm and low-dose (20 ppm) 1,4-dioxane-treatment versus middle- and high-dose (200, 5,000 ppm)-treated rats. After confirming the intensity of each adduct, three adducts were screened as characteristic of 1,4-dioxane treatment. Two of the three candidates contained thymine or cytidine/uracil moieties. Another candidate was identified as 8-oxo-dG based on mass fragmentation together with high-resolution accurate-mass (HRAM) mass spectrometry data. Oxidative stress responses may partly explain the mechanisms of increased mutations in the liver of gpt delta rats following 1,4-dioxane treatment.

DNA Adductome Analysis Identifies N-Nitrosopiperidine Involved in the Etiology of Esophageal Cancer in Cixian, China.

Esophageal cancer is prevalent in Cixian, China, but the etiology of this disease remains largely unknown. Therefore, we explored this by conducting a DNA adductome analysis. Both tumorous and nontumorous tissues were collected from patients who underwent surgical procedures at Cixian Cancer Hospital and the Fourth Hospital of Hebei Medical University, which is in a low-incidence area. N2-(3,4,5,6-Tetrahydro-2H-pyran-2-yl)deoxyguanosine (THP-dG) was the major adduct detected in samples from esophageal cancer patients in Cixian. The precursor of THP-dG, N-nitrosopiperidine (NPIP), exhibited a strong mutagenic activity under metabolic activation in the Ames test and a significant dose-dependent increase in mutation frequency during an in vivo mutagenicity test with guanine phosphoribosyltransferase (gpt) delta rats. The NPIP-induced mutation was dominated by A:T to C:G transversions, followed by G:C to A:T and A:T to G:C transitions, in the liver and esophagus of animal samples. A similar mutational pattern was observed in the mutational signature of esophageal cancer patients that demonstrated weak correlation with THP-dG levels. These findings suggested that NPIP exposure is partly involved in the development of esophageal cancer in Cixian residents.

Quantitative analysis of mutagenicity and carcinogenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in F344 gpt delta transgenic rats.

Quantitative analysis of the mutagenicity and carcinogenicity of the low doses of genotoxic carcinogens present in food is of pressing concern. The purpose of the present study was to determine the mutagenicity and carcinogenicity of low doses of the dietary genotoxic carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Male F344 gpt delta transgenic rats were fed diets supplemented with 0, 0.1, 1, 10 or 100 ppm IQ for 4 weeks. The frequencies of gpt transgene mutations in the liver were significantly increased in the 10 and 100 ppm groups. In addition, the mutation spectra was altered in the 1, 10 and 100 ppm groups: frequencies of G:C to T:A transversion were significantly increased in groups administered 1, 10 and 100 ppm IQ in a dose-dependent manner, and the frequencies of G:C to A:T transitions, A:T to T:A transversions and A:T to C:G transversions were significantly increased in the 100 ppm group. Increased frequencies of single base pair deletions and Spi- mutants in the liver, and an increase in glutathione S-transferase placental form (GST-P)-positive foci, a preneoplastic lesion of the liver in rats, was also observed in the 100 ppm group. In contrast, neither mutations nor mutation spectra or GST-P-positive foci were statistically altered by administration of IQ at 0.1 ppm. We estimated the point of departure for the mutagenicity and carcinogenicity of IQ using the no-observed-effect level approach and the Benchmark dose approach to characterise the dose-response relationship of low doses of IQ. Our findings demonstrate the existence of no effect levels of IQ for both in vivo mutagenicity and hepatocarcinogenicity. The findings of the present study will facilitate an understanding of the carcinogenic effects of low doses of IQ and help to determine a margin of exposure that may be useful for practical human risk assessment.

Establishment of an in vivo simulating co-culture assay platform for genotoxicity of multi-walled carbon nanotubes.

Engineered nanomaterials (ENM) are now used in a wide variety of fields, and, thus, their safety should urgently be assessed and secured. It has been suggested that inflammatory responses via the phagocytosis of ENM by macrophages is a key mechanism for their genotoxicity. The present study was conducted to establish a mechanism-based assay to evaluate the genotoxicity of ENM under conditions simulating an in vivo situation, featuring a co-culture system of murine lung resident cells (GDL1) and immune cells (RAW264.7). GDL1 were cultured with or without RAW264.7, exposed to a multi-walled carbon nanotube (MWCNT), and then analyzed for mutagenicity and underlying mechanisms. Mutation frequencies induced in GDL1 by the MWCNT were significantly greater with the co-existence of RAW264.7 than in its absence. Mutation spectra observed in GDL1 co-cultured with RAW264.7 were different from those seen in GDL1 cultured alone, but similar to those observed in the lungs of mice exposed to the MWCNT in vivo. Inflammatory cytokines, such as IL-1ß and TNF-α, were produced from RAW264.7 cells treated with the MWCNT. The generation of reactive oxygen species and the formation of 8-oxodeoxyguanosine in GDL1 exposed to the MWCNT were greater in the co-culture conditions than in the single culture conditions. Based on these findings, it is indicated that inflammatory responses are involved in the genotoxicity of MWCNT, and that the presently established, novel in vitro assay featuring a co-culture system of tissue resident cells with immune cells is suitable to evaluate the genotoxicity of ENM.

Influence of GSH S-transferase on the mutagenicity induced by dichloromethane and 1,2-dichloropropane.

It has been suggested that dichloromethane (DCM) and 1,2-dichloropropane (DCP) are responsible for occupational cholangiocarcinoma. Dihaloalkanes are metabolically activated by GSH S-transferase theta1 (GSTT1) to yield products such as episulfonium ions. However, whether the GSTT1-mediated step of these dihaloalkanes is related to occupational cholangiocarcinoma is not known. In the present study, we investigated the influence of GSTT1 activation on the mutagenicity of DCM and 1,2-DCP using GSTT1-expressing Salmonella typhimurium TA100 (TA100-GST). Since the mutagenicity of DCM was significantly increased in TA100-GST compared with mock control (TA100-pCTC), GSTT1 is thought to be involved in the mutagenicity of DCM. Mutation spectrum analysis on the hisG gene revealed that C:G to A:T transversions were the predominant form observed in DCM-treated TA100-pCTC. However, C:G to T:A transitions were dramatically increased in TA100-GST. We also analysed the DCM-DNA adduct, N2-GSH-Me-dG, and formation of N2-GSH-Me-dG was increased in TA100-GST compared with TA100-pCTC. On the other hand, 1,2-DCP did not increase the numbers of revertants in TA100-GSTT1. In mutation spectrum analysis, C:G to T:A transitions was predominant in both TA100-pCTC and TA100-GSTT1. These findings suggest that GSTT1 has little involvement in DCP mutagenicity, and other mechanisms might be more important for bioactivation and consequent genotoxicity. Clarification of the mechanisms underlying the development of DCM- and/or 1,2-DCP-related human cholangiocarcinoma may help establish risk assessment and prevention strategies against occupational cancer.

Hypermutation and unique mutational signatures of occupational cholangiocarcinoma in printing workers exposed to haloalkanes.

Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers' cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers' cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers' cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.

Major Anaerobic Bacteria Responsible for the Production of Carcinogenic Acetaldehyde from Ethanol in the Colon and Rectum.

AIMS: The importance of ethanol oxidation by intestinal aerobes and facultative anaerobes under aerobic conditions in the pathogenesis of ethanol-related colorectal cancer has been proposed. However, the role of obligate anaerobes therein remains to be established, and it is still unclear which bacterial species, if any, are most important in the production and/or elimination of carcinogenic acetaldehyde under such conditions. This study was undertaken to address these issues. METHODS: More than 500 bacterial strains were isolated from the faeces of Japanese alcoholics and phylogenetically characterized, and their aerobic ethanol metabolism was studied in vitro to examine their ability to accumulate acetaldehyde beyond the minimum mutagenic concentration (MMC, 50 µM). RESULTS: Bacterial strains that were considered to potentially accumulate acetaldehyde beyond the MMC under aerobic conditions in the colon and rectum were identified and referred to as 'potential acetaldehyde accumulators' (PAAs). Ruminococcus, an obligate anaerobe, was identified as a genus that includes a large number of PAAs. Other obligate anaerobes were also found to include PAAs. The accumulation of acetaldehyde by PAAs colonizing the colorectal mucosal surface could be described, at least in part, as the response of PAAs to oxidative stress. CONCLUSION: Ethanol oxidation by intestinal obligate anaerobes under aerobic conditions in the colon and rectum could also play an important role in the pathogenesis of ethanol-related colorectal cancer.

Suppressive effects of the NADPH oxidase inhibitor apocynin on intestinal tumorigenesis in obese KK-A(y) and Apc mutant Min mice.

Obesity is a risk factor for colorectal cancer. The accumulation of abdominal fat tissue causes abundant reactive oxygen species production through the activation of NADPH oxidase due to excessive insulin stimulation. The enzyme NADPH oxidase catalyzes the production of reactive oxygen species and evokes the initiation and progression of tumorigenesis. Apocynin is an NADPH oxidase inhibitor that blocks the formation of the NADPH oxidase complex (active form). In this study, we investigated the effects of apocynin on the development of azoxymethane-induced colonic aberrant crypt foci in obese KK-A(y) mice and on the development of intestinal polyps in Apc mutant Min mice. Six-week-old KK-A(y) mice were injected with azoxymethane (200 µg/mouse once per week for 3 weeks) and given 250 mg/L apocynin or 500 mg/L apocynin in their drinking water for 7 weeks. Six-week-old Min mice were also treated with 500 mg/L apocynin for 6 weeks. Treatment with apocynin reduced the number of colorectal aberrant crypt foci in KK-A(y) mice by 21% and the number of intestinal polyps in Min mice by 40% compared with untreated mice. Both groups of mice tended to show improved oxidation of serum low-density lipoprotein and 8-oxo-2'-deoxyguanosine adducts in their adipose tissues. In addition, the inducible nitric oxide synthase mRNA levels in polyp tissues decreased. Moreover, apocynin was shown to suppress nuclear factor-κB transcriptional activity in vitro. These results suggest that apocynin and other NADPH oxidase inhibitors may be effective colorectal cancer chemopreventive agents.

Comprehensive DNA adduct analysis reveals pulmonary inflammatory response contributes to genotoxic action of magnetite nanoparticles.

Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields; however, its toxicological properties are not well documented. In our previous report, MGT showed genotoxicity in both in vitro and in vivo assay systems, and it was suggested that inflammatory responses exist behind the genotoxicity. To further clarify mechanisms underlying the genotoxicity, a comprehensive DNA adduct (DNA adductome) analysis was conducted using DNA samples derived from the lungs of mice exposed to MGT. In total, 30 and 42 types of DNA adducts were detected in the vehicle control and MGT-treated groups, respectively. Principal component analysis (PCA) against a subset of DNA adducts was applied and several adducts, which are deduced to be formed by inflammation or oxidative stress, as the case of etheno-deoxycytidine (εdC), revealed higher contributions to MGT exposure. By quantitative-LC-MS/MS analysis, εdC levels were significantly higher in MGT-treated mice than those of the vehicle control. Taken together with our previous data, it is suggested that inflammatory responses might be involved in the genotoxicity induced by MGT in the lungs of mice.

Validity of a self-administered food frequency questionnaire in the estimation of heterocyclic aromatic amines.

BACKGROUND: Clarification of the putative etiologic role of heterocyclic aromatic amines (HAAs) in the development of cancer requires a validated assessment tool for dietary HAAs. This study primarily aimed to evaluate the validity of a food frequency questionnaire (FFQ) in estimating HAA intake, using 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) level in human hair as the reference method. METHODS: We first updated analytical methods of PhIP using liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS) and measured 44 fur samples from nine rats from a feeding study as part-verification of the quantitative performance of LC-ESI/MS/MS. We next measured PhIP level in human hair samples from a validation study of the FFQ (n = 65). HAA intake from the FFQ was estimated using information on intake from six fish items and seven meat items and data on HAA content in each food item. Correlation coefficients between PhIP level in human hair and HAA intake from the FFQ were calculated. RESULTS: The animal feeding study of PhIP found a significant dose-response relationship between dosage and PhIP in rat fur. Mean level was 53.8 pg/g hair among subjects with values over the limit of detection (LOD) (n = 57). We found significant positive correlation coefficients between PhIP in human hair and HAA intake from the FFQ, with Spearman rank correlation coefficients of 0.35 for all subjects, 0.21 for subjects with over LOD values, and 0.34 for subjects with over limit of quantification. CONCLUSION: Findings from the validation study suggest that the FFQ is reasonably valid for the assessment of HAA intake.

In vivo genotoxicity of a novel heterocyclic amine, aminobenzoazepinoquinolinone-derivative (ABAQ), produced by the Maillard reaction between glucose and l-tryptophan.

We recently demonstrated that a novel heterocyclic amine, 5-amino-6-hydroxy-8H-benzo[6,7]azepino[5,4,3-de]quinolin-7-one (ABAQ), is produced from glucose and l-tryptophan by the Maillard reaction at physiological temperature and pH, and that ABAQ was strongly mutagenic for Salmonella strains in the presence of S9 mix. Here, we present the results of three in vivo genotoxicity assays of ABAQ. The comet assay revealed that DNA damage was significantly increased in the livers, kidneys, lungs, and bone marrows of ICR mice, 3h after i.p. injection of ABAQ (50mg/kg body weight (bw)). To evaluate clastogenicity, the peripheral blood micronucleus test was performed, also in ICR mice. ABAQ induced micronucleated reticulocytes (MNRETs) in a dose-dependent manner; the frequency of MNRETs was significantly elevated at all i.p. doses (12.5, 25, and 50mg/kg bw) after 48h. To investigate the mutagenicity of ABAQ in vivo, gpt delta transgenic mice were treated with five consecutive administrations of ABAQ by gavage at doses of 25 or 50mg/kg per week for 3 weeks. The frequencies of gpt mutations (MF) in the liver of mice increased significantly compared with controls, in a dose-dependent manner. No significant increase of gpt MF was detected in the kidneys. Base substitutions predominated; both G:C→A:T and A:T→C:G mutations were significantly increased by ABAQ. The Spi(-) MF was also significantly increased in the liver after ABAQ treatment. If formed in vivo, ABAQ may give rise to adverse genotoxic effects.

Pierisin, Cytotoxic and Apoptosis-Inducing DNA ADP-Ribosylating Protein in Cabbage Butterfly.

Pierisin-1 was serendipitously discovered as a strong cytotoxic and apoptosis-inducing protein from pupae of the cabbage butterfly Pieris rapae against cancer cell lines. This 98-kDa protein consists of the N-terminal region (27 kDa) and C-terminal region (71 kDa), and analysis of their biological function revealed that pierisin-1 binds to cell surface glycosphingolipids on the C-terminal side, is taken up into the cell, and is cleaved to N- and C-terminal portions, where the N-terminal portion mono-ADP-ribosylates the guanine base of DNA in the presence of NAD to induce cellular genetic mutation and apoptosis. Unlike other ADP-ribosyltransferases, pieisin-1 was first found to exhibit DNA mono-ADP-ribosylating activity and show anti-cancer activity in vitro and in vivo against various cancer cell lines. Pierisin-1 was most abundantly produced during the transition from the final larval stage to the pupal stage of the cabbage butterfly, and this production was regulated by ecdysteroid hormones. This suggests that pierisn-1 might play a pivotal role in the process of metamorphosis. Moreover, pierisin-1 could contribute as a defense factor against parasitization and microbial infections in the cabbage butterfly. Pierisin-like proteins in butterflies were shown to be present not only among the subtribe Pierina but also among the subtribes Aporiina and Appiadina, and pierisin-2, -3, and -4 were identified in these butterflies. Furthermore, DNA ADP-ribosylating activities were found in six different edible clams. Understanding of the biological nature of pierisin-1 with DNA mono-ADP-ribosylating activity could open up exciting avenues for research and potential therapeutic applications, making it a subject of great interest in the field of molecular biology and biotechnology.

Epidemiology of esophageal cancer in Japan and China.

In preparation for a collaborative multidisciplinary study of the pathogenesis of esophageal cancer, the authors reviewed the published literature to identify similarities and differences between Japan and China in esophageal cancer epidemiology. Esophageal squamous cell carcinoma (ESCC) is the predominant histologic type, while the incidence of esophageal adenocarcinoma remains extremely low in both countries. Numerous epidemiologic studies in both countries show that alcohol consumption and cigarette smoking are contributing risk factors for ESCC. There are differences, however, in many aspects of esophageal cancer between Japan and China, including cancer burden, patterns of incidence and mortality, sex ratio of mortality, risk factor profiles, and genetic variants. Overall incidence and mortality rates are higher in China than in Japan, and variation in mortality and incidence patterns is greater in China than in Japan. During the study period (1987-2000), the decline in age-adjusted mortality rates was more apparent in China than in Japan. Risk factor profiles differed between high- and low-incidence areas within China, but not in Japan. The association of smoking and drinking with ESCC risk appears to be weaker in China than in Japan. Genome-wide association studies in China showed that variants in several chromosome regions conferred increased risk, but only genetic variants in alcohol-metabolizing genes were significantly associated with ESCC risk in Japan. A well-designed multidisciplinary epidemiologic study is needed to examine the role of diet and eating habits in ESCC risk.

In vivo examination of the genotoxicity of the urban air and surface soil pollutant, 3,6-dinitrobenzo[e]pyrene, with intraperitoneal and intratracheal administration.

3,6-Dinitrobenzo[e]pyrene (3,6-DNBeP) was identified as a new potent mutagen toward Salmonella strains in surface soil and airborne particles. Because data of in vivo examination of the genotoxicity of 3,6-DNBeP are limited, micronucleus test was performed in peripheral blood and bone marrow, and comet assay in the lungs of mice treated with 3,6-DNBeP. In male ICR mice intraperitoneally (i.p.) injected with 3,6-DNBeP, the frequency of micronuclated polychromatic erythrocytes (MNPCEs) was increased in the peripheral blood and bone marrow after 24 h in a dose-dependent manner. Compared to controls, the highest dose of 3,6-DNBeP (40 mg/kg B.W.) induced 7.3- and 8.7-fold increases of MNPCE frequency in the peripheral blood and bone marrow, respectively. Furthermore, when 3,6-DNBeP was intratracheally (i.t.) instilled to male ICR mice, 3,6-DNBeP at the highest dose of 0.1 mg/kg body exhibited 3.1-fold increase of DNA tail moment in the lungs at 3 h after the instillation compared to controls. The values of DNA tail moment at 9 and 24 h after the instillation were increased up to 3.5 and 4.2-fold, respectively. These data indicate that 3,6-DNBeP is genotoxic to mammalians in in vivo and suggest that 3,6-DNBeP may be a carcinogenic compound present in the human environment.